ISNI 2006 Abstracts

Journal of Neuroimmunology 178 (2006) 1 – 271 www.elsevier.com/locate/jneuroim

ISNI 2006 Abstracts Congress Schedule Sunday, October 15th pm Pre-congress Program Open-to-Public Seminar (Language: Japanese) Treatment of neuroimmunological diseases: Recent progress Distinguished Review Talks Part I: Chairs: R. Liblau, France/Chang-Sung Koh, Japan “Tolerance” mechanisms for natural killer cells: Licensing by self-MHC class I W. M. Yokoyama, USA The role of NK cells in MS T. Yamamura, Japan Heat shock protein-60 and autoimmunity I. R. Cohen, Israel Viral infection and neurological disease R. S. Fujinami, USA Part II: Chairs: H. Wekerle, Germany/T. Saida, Japan Mechanisms of immune-mediated demyelination in the CNS J. P. Antel, Canada Neuropathology of multiple sclerosis H. Lassmann, Austria Inflammatory myopathies R. Hohlfeld, Germany Immune mediated neuropathies H.-P. Hartung, Germany

Monday, October 16th am Opening and JSNI Lecture Chairs: M. Osame, Japan/Y. Itoyama, Japan T. Tabira, Japan Welcome Address: J. P. Antel, Canada/A. Igata, Japan T. Tabira, Japan The Dale E. McFarlin Lecture Chair: T. Tabira, Japan C. S. Raine, USA Plenary symposium: Understanding MS: Basic research opens the door Chairs: R. Hohlfeld, Germany/S. S. Zamvil, USA H. Lassmann, Austria L. Steinman, USA H. Wekerle, Germany pm Luncheon Seminar 1: MS sponsored by Nihon Schering K.K. doi:10.1016/j.jneuroim.2006.07.002

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Workshops, Oral Sessions and Posters Workshop 1 Workshop 2 Workshop 3 Workshop 4 Workshop 5 Oral Session 1A Oral Session 1B Oral Session 2A Oral Session 2B Oral Session 3A Oral Session 3B Oral Session 4A Oral Session 4B Poster Session 5 Poster Session 6 Poster Session 7 Poster Session 9 Poster Session 10 Poster Session 11 Poster Session 12 Poster Session 13 Poster Session 14 Poster Session 15 Evening Seminar 1: MG sponsored by Astellas Pharma Inc.

Tuesday, October 17th am Special Lecture Chair: H. Nishitani, Japan T. Honjo, Japan Concurrent Symposia International symposium: Alzheimer's disease, neurodegeneration and immunity Opening Remarks: F. Takaku, Japan Session I: Alzheimer's disease: Immune responses to Aβ and vaccination therapy Chairs: T. Tabira, Japan/H. L. Weiner, USA T. Tabira, Japan P. L. McGeer, Canada A. Monsonego, Israel H. Hara, Japan H. L. Weiner, USA pm Luncheon Seminar 2: ALZ sponsored by Eisai Co., Ltd. / Pfizer Japan Inc. Session II: Microglia, immune responses and neurodegeneration Chairs: S. H. Appel, USA/G. Sobue, Japan S. H. Appel, USA D. W. Cleveland, USA

Genetics of neuroimmunological disorders Chairs: G. C. Ebers, UK/T. Olsson, Sweden Chemokine in neuroimmunology Chairs: R. M. Ransohoff, USA/E. Meinl, Germany Glial reactions in neuroinflammation Chairs: M. J. Carson, USA/Y. Iwasaki, Japan Gene therapy and immunotherapy for brain tumors Chairs: J. C. Glorioso, USA/K. Itoh, Japan EAE: Pathogenesis & regulation Chairs: Y. Matsumoto, Japan/D. C. Wraith, UK BBB Chairs: T. Kanda, Japan/Elga de Vries, The Netherlands CNS inflammation and regulatory mechanism Chairs: Y. Ohara, Japan/T. Abo, Japan Immune-mediated pathology and neuroprotection (1) Chairs: K. Nomura, Japan/J. Kipnis, USA Immune-mediated pathology and neuroprotection (2) Chairs: S. Amor, The Netherlands/K. Sakai, Japan NMO and MS in Japan Chairs: K. Fujihara, Japan/J. Correale, Argentina Inflammatory myopathies Chairs: R. Hohlfeld, Germany/M. Nishizawa, Japan Immunological studies of MS (1) Chairs: M. Matsui, Japan/M. Pender, Australia Immunological studies of MS (2) Chairs: K. Ota, Japan/P. Stinissen, Belgium Immunology EAE-pathogenesis EAE-therapy Psychoneuroimmunology and cytokine effect on the CNS MS-genetics and etiology MS-pathology and experimental models MS-immunological studies MS-biomarker and diagnosis MS-therapy MS-clinical studies

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U.-K. Hanisch, Germany A. Suzumura, Japan D. Baker, UK H. Wakita, Japan Session III: Stem cells, neurogenesis, neuroregeneration and immunity Chairs: T. Yamamura, Japan/ M. Schwartz, Israel S. J. Khoury, USA G. Martino, Italy M. Schwartz, Israel J. Kipnis, USA J. Satoh, Japan Closing Remarks: I. Kanazawa, Japan Proteomics, transcriptomes and disease markers in MS Chairs: L. Steinman, USA /R. Martin, Spain R. Martin, Spain M. Comabella, Spain M. H. Han, USA N. Schaeren-Wiemers, Switzerland Neuro-immuno-endocrine network Chairs: H. Offner, USA/G. Matarese, Italy C. C. Whitacre, USA G. Matarese, Italy R. H. Straub, Germany H. Offner, USA Myasthenia gravis and myasthenic syndrome Chairs: J. Newsom-Davis, UK/M. Takamori, Japan M. Motomura, Japan H. J. Kaminski, USA N. Willcox, UK M. Takamori, Japan Emerging molecules in autoimmunity and allergy Chairs: V. Kuchroo, USA/R. H. DeKruyff, USA V. Kuchroo, USA R. H. DeKruyff, USA L. P. Kane, USA H. Kikutani, Japan Workshops Workshop 6 Workshop 7

Workshop 8 Workshop 9

Evening Seminar 2: NEURO sponsored by Teijin Pharma Limited

Wednesday, October 18th am Special Lecture Chair: J. P. Antel, Canada S. Akira, Japan Plenary Symposium: Glycolipid, NKT cells and immune regulation Chairs: T. Yamamura, Japan/O. Lantz, France M. Taniguchi, Japan S. Miyake, Japan O. Lantz, France G. De Libero, Switzerland

MS therapy Chairs: K. W. Selmaj, Poland/J. Kira, Japan Experimental studies on autoimmune neuropathies Chairs: S. Kusunoki, Japan/N. Latov, USA Autoantibodies in CNS diseases Chairs: A. Vincent, UK/K. Tanaka, Japan EAE: Regulatory mechanisms Chairs: M. K. Racke, USA/T. Shin, Korea

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pm Luncheon Seminar 3: CIDP sponsored by Takeda Pharmaceutical Company Limited / Nihon Pharmaceutical Co., Ltd. Concurrent Symposia Neural regeneration and neuroprotection Chairs: G. Martino, Italy and H. Neumann, Germany T. D. Palmer, USA A. Mackay-Sim, Australia M. Dezawa, Japan H. Neumann, Germany Human immunology and translational research Chairs: J. Zhang, USA/R. Gold, Germany B. Hemmer, Germany S. S. Zamvil, USA R. Gold, Germany J. Zhang, USA Innate immune recognition in the CNS Chairs: T. Owens, Denmark/F. Aloisi, Italy T. Owens, Denmark J. M. van Noort, The Netherlands F. Aloisi, Italy S. Rivest, Canada Autoimmune neuropathies Chairs: N. Yuki, Japan/H. J. Willison, UK S. Kusunoki, Japan N. Yuki, Japan K. Sheikh, USA H. J. Willison, UK Central and peripheral tolerance to prevent autoimmunity Chairs: S. Sakaguchi, Japan/J. Goverman, USA J. Goverman, USA S. M. Anderton, UK C. M. Beacher-Allan, USA S. Sakaguchi, Japan Workshops and Posters Workshop 10 Workshop 11 Workshop 12

Workshop 13 Workshop 14

Poster Session 1 Poster Session 2 Poster Session 3 Poster Session 4 Poster Session 8 Poster Session 16 Poster Session 17 Poster Session 18 Poster Session 19 Poster Session 20 Poster Session 21 Poster Session 22 Poster Session 23

IVIG treatment and the role of B cells in MS Chairs: A. Bar-Or, Canada/N. Shibuya, Japan Studies in neuroimmune pharmacology Chairs: R. M. Donahoe, USA/T. J. Rogers, USA Virus–host immune interactions associated with diseases of the nervous system Chairs: S. Jacobson, USA/C. R. Bangham, UK Stem cell-mediated immunomodulation of EAE Chairs: A. Monsonego, Israel/S. Miyake, Japan Pathogenesis of MS Chairs: B. Hemmer, Germany/Z. Illes, Hungary

Cell death and survival Glia BBB/Homing Chemokine Neuro–endocrine–immune interaction PNS diseases MG and inflammatory myopathies Paraneoplastic and antibody-mediated diseases Neurodegenetive diseases and dementia Ischemia, injury and brain tumor Infection Other disease Therapy

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Thursday, October 19th am Plenary Symposium: MS genetics Cairs: D. A. Hafler, USA/A. Compston, UK D. A. Hafler, USA A. Compston, UK G. J. Stewart, Australia S. Kikuchi, Japan pm Luncheon Seminar 4: Apheresis sponsored by Asahi Kasei Medical CO., LTD. Concurrent Symposia The blood–brain barrier in neuroinflammation Chairs: B. Engelhardt, Switzerland/J. Greenwood, UK B. Engelhardt, Switzerland A. Prat, Canada H. de Vries, The Netherlands A. V. Andjelkovic-Zochowska, USA J. Greenwood, UK Cytokine network and neuroinflammation Chairs: B. Becher, Switzerland/D. J. Cua, USA B. Becher, Switzerland R. Ransohoff, USA S. J. Khoury, USA D. J. Cua, USA Viral infection and autoimmune diseases Chairs: M. G. von Herrath, USA/S. D. Miller, USA M. G. von Herrath, USA S. D. Miller, USA H. Cantor, USA M. W. Cunningham, USA Psychoneuroimmunology Chairs: F. S. Dhabhar, USA/N. Kawamura, Japan F. S. Dhabhar, USA C. L. Coe, USA S. Kanba, Japan N. Kawamura, Japan Demyelination, remyelination and axonal pathology in MS Chairs: B. D. Trapp, USA/C. S. Raine, USA B. D. Trapp, USA K.-A. Nave, Germany M. T. Filbin, USA R. J. M. Franklin, UK Oral Sessions Oral Session 5A Oral Session 5B Oral Session 6A Oral Session 6B Oral Session 7A Oral Session 7B Oral Session 8A Oral Session 8B

EAE regulation Chairs: C. C. Whitacre, USA/S. Sakoda, Japan Mechanisms of neuroimmune crosstalk Chairs: J. L. Croxford, Japan/D. Sun, USA Immunological regulation of CNS homeostasis Chairs: D. Baker, UK/H. Matsuo, Japan Viral infection and prion Chairs: K. Usuku, Japan/Y. Kuroda, Japan Immunotherapy (1) Chairs: H. Onodera, Japan/R. Liblau, France Immunotherapy (2) Chairs: S. Kikuchi, Japan/A. Uccelli, Italy Myasthenia gravis Chairs: Y. Nomura, Japan/N. Willcox, UK PNS diseases Chairs: S. Araga, Japan/H.-P. Hartung, Germany

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Poster Prize Award Ceremony and Closing Chairs: T. Yamamoto, Japan/K. Arimura, Japan Oral Presentation by Prize Awardees Prize Award Ceremony Closing Remarks: J. P. Antel, Canada/T. Tabira, Japan/M. K. Racke, USA International Society of Neuroimmunology Officers • President • J. P. Antel, Canada

Secretary/Treasurer T. Owens, Denmark

• Vice-President • H. Wekerle, Germany • International Advisory Board of the ISNI • F. Aloisi, Italy • E. Benveniste, USA • C. F. Brosnan, USA • C. D. Dijkstra, NL • D. Hafler, USA • H. P. Hartung, Germany • S. L. Hauser, USA • W. Hickey, USA • R. Hohlfeld, Germany • V. Kuchroo, USA

R. Martin, Spain G. Martino, Italy Y. Matsumoto, Japan S. Miller, USA T. Olsson, Sweden H. Perry, UK K. W. Selmaj, Poland T. Tabira, Japan C. Whitacre, USA B. Zalc, France

• Honorary Officer • C. Raine, USA • A. Vincent, UK • Organization of the Eighth International Congress • Local Organizing Committee • Honorary President • A. Igata • H. Nishitani • M. Takamori • President • T. Tabira • Secretary General • J. Kira • Japanese Advisory Committee • T. Abo • S. Araga • K. Arimura • K. Asakura • K. Fujihara • H. Fukaura • T. Fukazawa • H. Hara • T. Hattori • K. Hisanaga • T. Hosokawa • T. Inuzuka • Y. Itoyama • Y. Iwasaki • T. Kanda • N. Kawamura

T. Nakamura M. Nishizawa K. Nomura Y. Nomura Y. Ohara T. Ohashi R. Ohno H. Onodera M. Osame K. Ota K. Ozawa T. Saida T. Saito K. Sakai S. Sakoda J. Satoh

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• S. Kikuchi • C. Koh • T. Kondo • Y. Kuroda • S. Kusunoki • M. Matsui • Y. Matsumoto • H. Matsuo • S. Miyake • H. Mizusawa • H. Murai • M. Nakagawa

N. Shibuya K. Shimizu G. Sobue A. Suzumura K. Tanaka M. Tanaka K. Tashiro S. Tsuji K. Usuku T. Yamamoto T. Yamamura N. Yuki

• International Advisory Committee • F. Aloisi, Italy • J. P. Antel, Canada • C. Brosnan, USA • D. Hafler, USA • H. Hartung, Germany • S. L. Hauser, USA • R. Hohlfeld, Germany • V. Kuchroo, USA • K. Lucas, The Netherlands • R. Martin, Spain • G. Martino, Italy • Y. Matsumoto, Japan

T. Olsson, Sweden V. Perry, UK C. Raine, USA R. Ransohoff, USA M. Schwartz, Israel K. W. Selmaj, Poland L. Steinman, USA A. Vincent, UK H. Weiner, USA H. Wekerle, Germany T. Yamamura, Japan

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Plenary Lectures

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Messages to neuroimmunologists from Asia

Molecular strategy to cure autoimmune diseases

T. Tabira

Tasuku Honjo

National Institute for Longevity Sciences, NCGG, Aichi, Japan

Department of Immunology and Genomic Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan

Unfortunate incidents and regional peculiarities have frequently made a big progress in understanding diseases. The semple type rabies vaccine induced large demyelinating plaques in certain Japanese (Shiraki). We could demonstrate myelin antigens including proteolipid protein (PLP) in the vaccine and successfully induced autoimmune encephalomyelitis in animals with this vaccine. Since the use of a new type vaccine, we have never encountered such cases. Optic-spinal MS (OS-MS) is an Asian type MS characterized by severe optic and spinal lesions, higher age of onset, much higher female preponderance, lower incidence of oligoclonal bands, long spinal cord lesions in MRI and others. For some reasons, OS-MS is decreasing and classical MS is increasing in Japan. Recently, certain antibodies were demonstrated with a high frequency and aquaporin 4 is demonstrated as a possible antigen (Lennon). Concentric sclerosis (Baló) was prevailing in the Philippines, and some cases were also found in Japan and China. It is mostly acute monophasic, CSF changes are relatively subtle, severe brain edema is common and corticosteroid is effective. It is interesting to note that ischemia-induced proteins are expressed in the lesion without vascular lesions (Lassmann). The disease abruptly disappeared in the Philippines. The high incidence of acute motor axonal neuropathy (AMAN) is also unique in Asia. The molecular mimicry of lipooligosaccharide in Campylobacter jejuni and GM1 ganglioside is well demonstrated (Yuki). HAM/ TSP (Osame, Igata), Fisher syndrome (Kusunoki), and others are also memorable messages from Asia. Here I review these messages accompanied by my personal history.

Multiple sclerosis: How far have we come because of Dale?

PD-1 belongs to the CD28/CTLA-4 co-receptor family and negatively regulates immune responses upon interaction with its ligands, PD-L1 and PD-L2. PD-1−/− mice develop various autoimmune diseases depending on their genetic background. On BALB/c background, PD-1−/− mice develop autoimmune dilated cardiomyopathy (DCM) with high-titer autoantibodies against 30 kDa cardiac antigen. We have recently identified the 30 kDa antigen as cardiac troponin I (cTnI) and showed that the deregulated production of anti-cTnI autoantibody is the main cause of DCM in PD-1−/− mice. More recently we analyzed the function of FcγRIIB in the DCM of BALB/c-PD-1−/− mice, because FcγRIIB has been known to play pivotal roles in the removal and inhibition of autoreactive B cells. DCM of BALB/cPD-1−/− mice was not affected by the disruption of FcγRIIB, however BALB/c-FcγRIIB−/−PD-1−/− mice developed autoimmune hydronephrosis with concomitant production of autoantibodies against urothelium. Massive inflammation was observed around the pelvis of the kidney and at the junction between the ureter and the bladder, which were considered to be the cause of the occlusion and the subsequent development of hydronephrosis. BALB/c-FcγRIIB−/−PD-1+/− mice developed hydronephrosis with the similar frequency as BALB/c-FcγRIIB−/−PD-1−/− mice, whereas BALB/cFcγRIIB−/− mice did not show any autoimmune symptoms. Therefore the autoimmunity to urothelial cells seems to be controlled primarily by FcγRIIB and exaggerated by PD-1 deficiency. These observations suggest the collaborative role of FcγRIIB and PD-1 in the regulation of autoimmunity against urinary tract but not heart and may help understand the multi-genic nature of the autoimmune diseases.

Cedric S. Raine

Viral recognition by innate immunity

Albert Einstein College of Medicine, NY, USA

Shizuo Akira

This presentation will compare the status of Neuroimmunology at the time of Dale McFarlin's passing in 1992, with where we stand today, particularly as it relates to multiple sclerosis (MS), an area to which Dale's Neuroimmunology Branch at NIH made seminal contributions. Indeed, the origins of much of what we know in MS from the standpoint of immunogenetics, TCR/MHC mapping and usage, myelin epitopes, mouse models, Th1- and Th2-type cytokine patterns, among others, can be traced directly to works emanating from these NIH laboratories. Since 1992 and based in considerable part on Dale's contributions, investigators have developed molecular probes to uncover and dissect a variety of novel pathogenetic mechanisms of relevance to MS, which, depending upon lesion stage, have been shown to display heterogeneity. It has also become clear that the understanding of lesion progression in MS involves the regulation of events at the margins of established lesions where apparently normal white matter abuts totally demyelinated, gliotic parenchyma, the two separated by a few microns only and no ongoing structural pathology. Exploration of molecular pathways at this interphase may reveal mechanisms which function to restrain lesion expansion on the one hand, and to restrict repair, on the other. In addition, mechanisms like the re-expression of developmental pathways at the lesion edge, the proliferative and regenerative capacity of oligodendroglial precursor cells, factors deleterious to axonal health, and the innate capacity of glial cells in and around the lesion to interact with the immune system, have became major topics of research in recent years. This lecture will attempt to put the above mechanisms in context at the lesion margin and to show how much Dale's work has influenced our present day thinking. Supported in part by NS 08952, NS 11920 and NS 07098; NMSS 1001K-11; and the Wollowick Family Foundation.

Research Institute for Microbial Diseases, Osaka University, and Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Suita 565-0871, Japan Mammalian Toll-like receptors play a critical role in detection of invading pathogens as well as triggering of subsequent inflammatory and immune responses. Individual TLRs recognize different microbial components. Adaptor MyD88 is essential for the responses to all TLR ligands except for TLR3 ligand, and is involved in induction of inflammatory cytokines through activation of IRAKs and TRAF6. In addition, the TLR3- and TLR4-mediated signaling possesses a MyD88independent pathway, which is activated by another adapter molecule, TRIF. The TRIF-dependent pathway activates IRF3 through TBK1, which results in induction of type 1 interferon. Plasmacytoid dendritic cells (pDC) rapidly produce IFN alpha in response to viral infection or in response to TLR7-(imidazoquinolines, sRNA) or TLR9-ligand (CpG DNA). Such IFN alpha induction by pDC involves formation of a complex consisting of MyD88, TRAF6, IRAK-1 and IRF7, in which IRAK-1 acts as IRF7 kinase. Besides TLRs, recent findings have shown the presence of cytosolic detector system of invading pathogens. Two DExD/H box RNA helicases, retinoic acid inducible protein-I (RIG-I) and melanoma differentiation-associated gene 5 (mda-5) are involved in antiviral responses by recognizing dRNA in the cytoplasm. RIG-I and mda-5 recognize distinct RNA viruses. We identified a novel molecule named IPS-1that bridges between RIG-I/mda-5 and TBK-1. Recently, we have generated IPS-1 knockout mice, and shown the essential role in RIG-I and mda-5 dependent signaling pathways. In this symposium I will discuss recent progress in the TLR-dependent and independent recognition of viral infections.

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Distinguished Review Talks

Abstracts DR01-01

“Tolerance” mechanisms for natural killer cells: Licensing by self-MHC class I Weyne M. Yokoyama Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110 USA Natural killer (NK) cells express MHC class I-specific receptors that inhibit their reactions against cellular targets. These receptors explain the “missing-self” hypothesis in which NK cells survey tissues for normal expression of MHC class I that prevent NK cell activation. However, it has been difficult to reconcile fundamental observations such as the lack of autoreactive NK cells in MHC class I-deficient hosts. Moreover, NK cell receptor genes are highly polymorphic and segregate independently from the genes for the extremely polymorphic MHC alleles, indicating that NK cells must possess a mechanism to permit the appropriate pairing of receptors with self-MHC. Recent observations indicate a novel mechanism by which NK cells develop functional competence as a result of a productive interaction between their MHC-specific receptors and self-MHC. We have termed this process, “licensing” of NK cells by self-MHC. Licensing may be more broadly relevant to autoimmunity since there are many leukocytes with similar receptors.

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Heat shock proteins (HSP) were initially identified as a family of stress-induced proteins characterized by their chaperone activity. However, they were later identified as self-antigens recognized by host T cell and autoantibodies in many autoimmune diseases. Recently, the role of HSP as endogenous activators of the innate immune system has been unveiled. Here, I shall discuss the relevance of HSP60 for the regulation of inflammation and autoimmune disease. HSP60 both initiates and resolves inflammation. Its pro-inflammatory activity derives from its innate TLR4-dependent activation of macrophages and dendritic cells; microgram/ml concentrations are required. Its anti-inflammatory activity results from 3 signaling functions: (1) Epitopes of HSP60, complexed to MHC-II, are expressed by activated effector T cells; these complexes activate anti-ergotypic regulatory T cells. (2) HSP60 is an innate activator (via TLR4) of antigen-presenting B cells which then secrete IL10 and polarize responding T cells towards Th2. (3) HSP60, nanograms/ml suffice, acts as a co-stimulator of CD4+CD25+ Tregs and enhances the ability of relatively low concentrations of the Tregs to down-regulate CD4+CD25− or CD8+ target T cells, detected as inhibition of target T-cell proliferation and IFN-γ and TNF-α secretion. The anti-inflammatory effects of HSP60 on T and B cells can account for its success in down-regulation of autoimmune diseases such as human or mouse Type 1 diabetes mellitus and rat adjuvant arthritis. A peptide of HSP60 is currently in phase 3 trials in new-onset human diabetes. DR01-04

DR01-02

Viral infection and neurological disease The role of NK cells in MS R. S. Fujinamia, L. L. McCoya, I. Tsunodaa and J. L. Whittonb Takashi Yamamura University of Utah School of Medicine, Salt Lake City, Utah, USA; bThe Scripps Research Institute, La Jolla, California, USA

a

Department of Immunology, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan Compared with autoimmune T cells or B cells, much less attention has been paid onto the role of NK cells in multiple sclerosis (MS). Although speculative, this could be explained by limited understanding of NK cell biology some time ago, or possibly due to some controversies in the literature. However, as NK cells are a major source of regulatory cytokines such as IFN-γ and because they would functionally be polarized towards NK1 or NK2, their involvement in the pathogenesis of autoimmune diseases like MS is thought to be most likely. In fact, recent works from our laboratory have showed that NK cells in the peripheral blood of MS are remarkably altered regarding cytokine secretion profile and surface phenotypes (Takahashi et al. JCI 2001; Takahashi et al. Brain 2004). Our initial key observation was that NK cells in remission of MS would exhibit the properties of NK2 cells, with some similarities to Th2 cells. This change is likely to be protective against MS and can be interpreted as an adaptive change to maintain the state of remission. Further analysis has supported this idea and also convinced us that “Peripheral NK cell is a window into the disease status of MS.” For example, we have recently found that a higher expression of CD11c on NK cells predicts an occurrence of relapse within a few months, whereas absence of CD11c and CD95 on NK cells would define the most stable remission (Aranami et al. submitted). In terms of the mechanism of how NK cells would mirror the disease status, I will present our hypothetical model and discuss on the implications and the future direction of research.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) where environmental factors are implicated. Migration studies suggest that some event(s) early in life primes genetically susceptible individuals for MS later in life. Also different viral infections are associated with exacerbations of MS. However, no single virus has been identified as the causative agent of MS. We have developed a model where infection of mice with a virus having molecular mimicry can prime these mice for later disease induced by a different virus. Young female SJL/J mice are infected with a recombinant vaccinia virus (VV) encoding myelin proteolipid protein (PLP). After infection when recombinant VVPLP has been cleared, mice have no clinical signs of experimental autoimmune encephalomyelitis (EAE). When VVPLP primed mice are then challenged with murine cytomegalovirus, mice develop exacerbations of EAE. These data indicate that viruses having molecular mimicry could prime genetically susceptible animals for disease early in life that can be induced by a different virus later in life. These data could also explain why no single agent has been isolated as the causative MS agent. Our data indicate that infections need not occur within the CNS for these events to take place. This work was supported by NIH. DR02-01

Mechanisms of immune-mediated demyelination in the CNS J.P. Antel, MD

DR01-03 McGill University, Montreal, Canada

Heat shock protein-60 and autoimmunity Irun R. Cohen Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

The capacity of immune mediated mechanisms to mediate demyelination within the CNS has been recognized since the description of cases of acute disseminated encephalomyelitis (ADEM) that followed the introduction of the neural tissue containing Pasteur rabies vaccine in

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the 1880s and the subsequent development of its animal model experimental auto-immune encephalomyelitis (EAE). These disorders indicate the requirement for auto-reactive CD4 T cells to initiate an inflammatory response within the CNS. The immune cells and molecules that actually mediate injury of myelin or its cell of origin the oligodendrocyte (OLG) likely extend beyond these inflammation initiating cells and include constituents of the adaptive and innate immune systems. Selective neural target injury could reflect the properties of the immune effectors and/or the target cells. Selective immune effectors would include OLG/myelin specific MHC class 1 restricted αβ CD8 T cells and antibody. Injury mediated by members of the tumor necrosis factor (TNF) superfamily (fas, TRAIL, TNF) provide examples of target-conferred selectivity dependent on expression of the requisite receptor. A chronic inflammatory environment can contribute both to enhanced production of such effector molecules and upregulation of their receptors. Such an environment can also enhance expression of molecular partners (CD56, NKG2D and their ligands) involved in non MHC restricted cytotoxic responses mediated by innate immune cells (NK cells, γδ T cells) and even induce such molecules on αβ T cells. Neuro-protective strategies can be directed at the immuneneural molecules directly involved in the injury process or at modulating the CNS environment that regulates their expression and/ or activity.

including MHC and co-stimulatory molecules. Furthermore, muscle cells can secrete many cytokines, chemokines and cell adhesion molecules, as well as receptors of the innate immune system. Conversely, elements of the immune system affect muscle cell properties. Therefore, muscle cells should be considered as active modulators rather than passive targets of immune reactions. This is relevant to the immunopathogenesis of the different inflammatory myopathies. In polymyositis and inclusion body myositis, clonally expanded CD8-positive T cells invade muscle fibers that express MHC class I antigens. The causative antigen(s) have not yet been defined. Recently, however, it has become possible to “revive” putatively pathogenic, autoimmune T cells from frozen specimens of human muscle biopsy tissue. Using CDR3 spectratyping, clonally expanded T cell receptor (TCR) β-chains can be identified in muscle sections of patients with inflammatory myopathy. The putatively pathogenic T cells can be isolated by laser-microdissection, and co-expressed pairs of TCR alpha- and beta-chains identified by a multiplex PCR protocol which allows the concomitant amplification of both chains from single cells. The paired TCR alpha- and beta-chains are functionally expressed in T-hybridoma cells, which can be used to search for the target antigen(s). Refs.: Hohlfeld and Engel, Immunol. Today 15: 269–274, 1994; Wiendl, Hohlfeld, and Kieseier, Trends Immunol. 26: 373–380, 2005; Dalakas and Hohlfeld, Lancet 362: 971–982, 2003.

DR02-02 DR02-04

Neuropathology of multiple sclerosis H. Lassmann

Immune mediated neuropathies

Center for Brain Research, Medical University of Vienna, Vienna, Austria

Hans-Peter Hartung

Multiple sclerosis (MS) has initially been defined as a chronic inflammatory disease of the central nervous system, which gives rise to focal demyelinated plaques in the white matter. Although such demyelinated plaques are the diagnostic hallmark of the pathology, the disease is much more complex. Focal white matter plaques are highly heterogeneous regarding the immunopathological profile, the patterns of tissue destruction and their capacity for repair. This heterogeneity is in part stage dependent and in part reflects a true diversity between patients or patient subgroups. In addition, pathological alterations are not restricted to focal white matter plaques, but the CNS is affected in a global sense. This is particularly the case in patients with primary or secondary progressive MS. In such patients extensive demyelination is also found in the cerebral cortex and in the deep brain stem nuclei. Furthermore, there is massive diffuse axonal injury and damage in the normal appearing white matter, which is associated with persistent inflammation by T-cells, B-cells and Plasma Cells as well as by global and diffuse microglia activation. Only part of the spectrum of pathological alterations within the MS brain can so far be detected by magnetic resonance imaging. In addition, the pathogenesis of the diverse components of MS pathology is apparently different. The complex pathology of MS suggests that therapeutic strategies should be tailored to cope with these stage and patient dependent variability.

Department of Neurology, Heinrich-Heine-University of Duesseldorf, Germany

DR02-03

Inflammatory myopathies R. Hohlfeld Institute for Clinical Neuroimmunology, Ludwig Maximilians University of Munich, and Department of Neuroimmunology, Max-Planck Institute of Neurobiology, Martinsried, Germany There is an intense cross-talk between muscle and immune cells. Muscle cells can express many immunologically important molecules,

The peripheral nerve is targeted by aberrant immune responses in a number of acute and chronic neuropathies including Guillain–Barre Syndrome, CIDP, paraproteinemic neuropathies, vasculitic and paraneoplastic neuropathies. Local immune circuitry is made up of endoneurial macrophages and Schwann cells. The PNS is separated from the systemic immune compartment and blood by the blood–nerve barrier which is however absent or deficient at its most proximal and distal parts where therefore immune cells, antibodies and mediators can readily access neural structures. Principles of normal immune reactivity in nerve and responses to infections and danger signals will be reviewed. These concepts will be illustrated with reference to Guillain–Barre Syndrome and CIDP. In both predominantly the myelin sheath is attacked by disturbed immunity. Glycolipid antibodies have been implicated in dysfunction and structural damage in GBS and its variants whereas myelin proteins have been identified in a number of patients with CIDP as target autoantigens. The concept of molecular mimicry has been elaborated in depth in GBS which is often antedated by infections with Campylobacter jejuni or cytomegalovirus. Molecular structures contained in the LPS fraction of C. jejuni are shared with the peripheral nerve, both in myelin and the axolemma. Current treatments will be reviewed. They encompass plasma exchange and IVIG for GBS and CIDP, immunosuppressants and immunomodulatory agents and monoclonal antibodies to B and T cell antigens. References Kieseier BC et al.: Advances in understanding and treatment of immunemediated disorders of the peripheral nervous system. Muscle Nerve 30: 131–156, 2004. Köller H et al.: Chronic inflammatory demyelinating polyradiculoneuropathy. New Engl J Med 352: 1343–1356, 2005. Kieseier B, Hartung HP, Wiendl H: Immune circuitry in the peripheral nervous system. Current Opinion Neurology 2006, in press.

Abstracts

Plenary Symposia

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Abstracts

PS01-01

Mechanisms of tissue injury in multiple sclerosis H. Lassmann Center for Brain Research, Medical University of Vienna, Vienna, Austria Focal demyelinated plaques in the white matter, closely resembling those seen in multiple sclerosis (MS), can be induced in certain models of experimental autoimmune encephalomyelitis (EAE). Thus most current pathogenetic concepts and most therapeutic strategies in MS are based on data, obtained in EAE models. However, there are some major differences between EAE and MS, which may in part explain the limited success to transfer experimentally developed therapeutic strategies into the human setting. In contrast to most EAE models, inflammation in MS is driven dominantly by Class I MHC restricted T-cells. In addition, the patterns of microglia activation suggest an important role of innate immunity in triggering initial focal white matter lesions and diffuse injury of the normal appearing white matter. In such lesions the production of reactive oxygen and nitrogen intermediates appears to trigger a hypoxia-like tissue injury through radical mediated mitochondrial injury. In addition, auto-antibodies are involved in tissue injury through complement activation at least in a subset of patients. However, as shown in the paradigm of Devic's type of neuromyelitis optica, primary demyelination is even achieved, when the respective auto-antibodies are directed against an astrocytic antigen. Finally, axonal injury, a major cause of permanent neurological deficit, is a regular feature of MS pathology and the mechanisms of axonal injury differ from those responsible for demyelination. Based on this complexity it seems naïve to cure the disease by interfering with a single mechanism. PS01-02

The challenges for antigen specific therapy of MS Lawrence Steinman Department of Neurology and Neurological Sciences, Stanford, USA

This presentation will review EAE variants with regard to their usability as models for human MS. There will be a discussion of new transgenic models representing mechanisms determining spontaneous initiation, the course of CNS autoimmunity, the distribution of lesions within the CNS and the cellular composition of the inflammatory infiltrate. Then I shall review aspects of the early, inflammatory phase of MS plaque generation, in particular concerning the dynamics of immune cell invasion into the central nervous system (CNS). PS02-01

Regulatory function of NKT cells in the protection of autoimmune disease development Masaru Taniguchi, Ryusuke Nakagawa, Satoshi Kojo, and Hiroshi Watarai RIKEN Research Center for Allergy and Immunology, Yokohama, Japan NKT cells produce large amounts of both IFN-γ and IL-10 at the same time and thus mediate dual functional activities, including protective and regulatory functions. When NKT cells are in vivo activated with a single injection of α-galactosylceramide (α-GalCer), splenic DCs undergo a rapid maturation and acquire a potential to produce high IL-12, which induces Th1-type response. By contrast, repeated α-GalCer-stimulation activates NKT cells to keep producing IL-10 but stop producing IFN-γ, resulting in generation of regulatory DCs, which produce high IL-10 and low IL-12. In fact, IL-10 from NKT cells in the absence of IFN-γ keeps maintaining regulatory activity of DCs to produce IL-10 for a long time (at least 30 days), and also generates IL-10-producing regulatory T cells. The high IL-10 and low IL-12 production in the regulatory DCs are due to the upregulation of phosphorylation of p44/42 extracellular signal-regulated kinase (Erk-1/2) and the augmented production of IκB induced suppression of NFκB activation, respectively. When experimental allergic encephalomyelitis (EAE) is induced, EAE development is significantly suppressed by generating regulatory CD4+ T cells which are induced by the transfer of myelin oligodendrocyte glycoprotein-pulsed DCs obtained by repeated αGalCer-stimulation. These findings indicate that NKT cells play important roles in inducing regulatory T cells suppressing EAE development. PS02-02

The challenges facing the development of antigen specific therapy for MS are formidable: There is abundant evidence of widespread immune responses to constitutively expressed and inducible molecules within the nervous system. Proteins and their peptide constituents, carbohydrates and lipids are all targeted by the adaptive immune response. Nevertheless strategies exist for antigen specific therapy of MS. Recent progress in the development of DNA vaccines and tolerizing lipids for MS will be discussed. Effective adjuvants to bolster antigen specific therapy, including adjunctive cytokines, and drugs like the statins will be described. PS01-03

Modelling Multiple Sclerosis: Experimental Autoimmune Encephalomyelitis variants representing distinct aspects of human disease Hartmut Wekerle Max-Planck-Institute for Neurobiology, D-82152 Martinsried, Germany Experimental Autoimmune Encephalomyelitis (EAE) is often termed “the” model of human Multiple Sclerosis (MS). This is, however an oversimplification. First of all, MS has a very multifaceted disorder, with no single experimental model representing the entire complexity of the human disease. On the other hand, EAE comes in numerous, distinct variants, which may reflect individual aspects of MS.

Invariant NKT cells in autoimmunity Sachiko Miyake Department of Immunology, National Institute of Neuroscience, NCNP, Japan CD1d-restricted invariant Vα14 (Vα14i) NKT cells are a unique lymphocyte subset implicated in a variety of immunoregulation. Vα14i NKT cells inhibit disease in some autoimmune disease models such as experimental autoimmune encephalomyelitis (EAE) and type I diabetes whereas they exacerbate disease in other models such as arthritis and allergic airway inflammation. Even though the role of Vα14i NKT cells in the pathogenesis is reciprocal in these models, activation of Vα14i NKT cells with synthetic glycolipid ligands suppresses these diseases. OCH, sphingosine truncated derivatives of α-galactosylceramide (α-GalCer), stimulate Vα14i NKT cells to selectively produce Th2 cytokines and prevent Th1-mediated autoimmune disease models. The other derivatives of α-GC suppressed antibody-induced arthritis and allergic airway inflammation. These results highlight the therapeutic potential to target Vα14i NKT cells in the treatment of these diseases. Recently an invariant Vα19-Jα33 T cell receptor α-chain (Vα19i) expressing, MR1-dependent cell population emerged as an second invariant NKT cell population. Vα19i NKT cells, however, have an unidentified role in autoimmunity. We demonstrate that over-expression or adoptive transfer of Vα19i NKT cells inhibited EAE. Conversely, MR1deficient mice, lacking Vα19i NKT cells, exhibited exacerbated disease.

Abstracts The mechanisms of Vα19i NKT cell-mediated suppression of disease will also be discussed. PS02-03

Mucosal associated invariant T cells: An evolutionarily conserved T cell subset restricted by MR1 O. Lantza, b, L. Dubana, b, I. Cruz-Mouraa, b and E. Treinerc

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PS03-00

The Admixture Multiple Sclerosis Susceptibility Locus on Chromosome 1 is refined to a 4 Mb Interval in an African-American Population D. Hafler2,3,4, P. De Jager2,3,4, N. Patterson2, J. Oksenberg5, B. Cree5, S. Hauser5, D Reich1,2 Department of Genetics, Harvard Medical School, Boston, MA; 2Program in Medical and Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA; 3Department of Neurology, Harvard Medical School, Boston, MA; 4Division of Molecular Immunology, Center for Neurologic Disease, Brigham and Women’s Hospital, Boston, MA; 5Department of Neurology, University of California, San Francisco, CA

1

Laboratoire d'Immunologie, Institut Curie, Paris, France; bInserm U653, Paris, France; cInserm E0351, Amiens, France

a

There are only two T cell subsets displaying a conserved invariant repertoire among species, NK-T cells and the recently discovered Mucosal Associated Invariant T (MAIT) cells both of which express highly conserved TCRα chains (iVα24/14-Jα18 and iVα7.2/19-Jα33, respectively) and recognize related ligands (CD1d and MR1, respectively) in human and mouse. MAIT cells are – Highly abundant in cattle blood, human blood and gut and less so in mouse gut, – Selected by B cells and not by the thymic epithelium, – Restricted by MR1, a strikingly phylogenetically conserved MHC class Ib molecule. – TAP and Ii chain independent. – Dependent upon the commensal microbial flora (they are absent from germ free mice). – Secrete high levels of TH2 cytokines (IL-4, IL-5 and IL-13). The function of MAIT cells is unknown but the high conservation of both the TCRα chain and the MHC ligand between species suggests an important one, such as gut epithelial homeostasis or control of the class of the immune response in the gut. Alternatively, MAIT cells could be involved in the defense against ubiquitous pathogens. Importantly, accumulation of MAIT cells have been found in the lesions of multiple sclerosis (MS) patients as well as in nerve biopsy samples from chronic inflammatory demyelinating polyneuropathy (CIDP) patient.

The HLA DRB1*1501 haplotype is the only well-validated genetic risk for MS. However, we recently published a genome-wide admixture scan that identified a second locus associated susceptibility for MS in 582 African-American patients (LOD score 5.2). The degree of European ancestry in these patients at this chromosome 1 locus rises to 27% from a genome-wide baseline of 21%, suggesting the presence of a risk allele of European origin in this region. Control subjects showed no significant difference in the level of European ancestry at this locus. We are expanding our collection of African-American subjects with MS and have now studied 654 subjects using our admixture mapping strategy. We are also performing fine-mapping analyses by comparing our affected subjects with over 500 African-American control subjects. Association to susceptibility for MS is the primary outcome of the study. The addition of subjects and markers to our published dataset has improved the maximal association to a LOD score of 6.9 and has refined our locus from an 8 Mb to a 6 Mb segment. Efforts are ongoing to further refine this locus; an additional 200 affected subjects and 500 markers are currently being genotyped. We are rapidly refining the location of this second MS susceptibility allele; 50 genes lie in the current interval of 6 Mb. Finding this risk allele will be particularly informative for AfricanAmerican patients with MS as it may identify a new molecule or pathway involved in the pathophysiology of MS. This work may therefore generate new targets for therapy and may be broadly applicable to all patients with MS.

PS02-04 PS03-01

The response to self lipids in autoimmune diseases

Whole genome association screen in multiple sclerosis

G. De Libero Experimental Immunology, Department of Research, University Hospital, Basel, Switzerland Recognition of self is essential for repertoire selection, immune regulation and autoimmunity. It may be a consequence of infection, representing the escape strategy adopted by pathogens but also inciting autoimmunity. Immune response to self lipid antigens is a novel mechanism that may contribute to autoimmune diseases. T cell recognition of self lipids is influenced by the repertoire of immunogenic self lipids, the rules of lipid presentation by CD1 isoforms and by the regulation of lipid metabolism. The modifications occurring in lipid structure during ontogeny, synthesis in different organs and in inflammation may lead to generation of highly immunogenic self lipids and to the efficient activation of autoreactive T cells. Bacterial infection may also have dramatic effects on lipid metabolism and on lipid antigen presentation. This is illustrated by enhanced de novo synthesis of immunogenic self lipids in infected cells which thus become stimulatory for self lipid-reactive T cells. The mechanisms regulating T cell response to self lipids will be discussed in the context of the autoimmune response in Multiple Sclerosis.

DAS Compston on behalf of The International Multiple Sclerosis Genetics Consortium (IMSGC)⁎ Early candidate gene studies quickly identified the importance of the extended human leukocyte antigen (HLA) DR15 haplotype in conferring susceptibility to multiple sclerosis. But subsequent whole genome linkage screens have failed conclusively to identify any other susceptibility factors. Results from the most recent high-density linkage screen indicate that non-HLA loci exert only modest effects individually, and will require testing for association in large datasets if they are to be identified with any statistical confidence. Results from the human genome variation project have confirmed that linkage disequilibrium (LD), the correlation between closely mapped loci, is a highly variable but common feature of the human genome. One often considered consequence of LD is the possibility effectively to screen very nearly all the common variations in the human genome by typing just a proportion of these polymorphisms, especially if the typed markers are carefully chosen to capture as much of the available variation as possible (e.g. tagging). The technology required to undertake such “indirect” whole genome association screening is now a reality. In our indirect association screen for multiple sclerosis the International Multiple Sclerosis Genetics Consortium (IMSGC) is genotyping the Affymetrix 500k chip in 1000 trio families (an affected

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individual and both parents). Based on data from the International HapMap project we anticipate that this chip will allow us to interrogate more than 80% of the common variation in the human genome. The completed dataset will comprise approximately 1.5 billion genotypes. Preliminary results will be presented.

Australian data presented on interleukin 7 receptor, chemokine genes including fractalkine, and vitamin D receptor. PS03-03

MS genetics in Japan PS03-02 Seiji Kikuchia and Toshiyuki Fukazawab

Immunologic candidate gene studies in multiple sclerosis a

G J Stewart Institute for Immunology and Allergy Research, Westmead Millennium Institute, Australia Genetic susceptibility studies are aimed at furthering knowledge of pathogenesis and, through this, identifying targets for new drug development. Apart from the well replicated associations with the major histocompatibility complex, candidate gene studies in diseases of complex genetics, including multiple sclerosis, have yielded few replicated associations despite intense international efforts. These difficulties include small effects of individual alleles leading to the need for large studies, probable epistatic interactions between alleles of multiple genes, disease heterogeneity, possible variation in interaction with environment variables, chance associations, allelic heterogeneity, population stratification, sizeable gaps in knowledge of pathogenesis and the need for international consortia for large scale replication studies. International focus has moved towards genome screens with eventual candidate association studies in chromosomal regions identified through screening. Nonetheless, candidate gene studies continue to produce useful information particularly the genes reside in from genomic regions associated with MS, are combined with gene expression analyses, involve polymorphisms with known effects on function and are carried out with planned replication. In view of the likely involvement of the peripheral immune system in MS, our studies have focussed on key immune molecules including those involved in antigen presentation, T cell activation, cytokines and chemokines. Interleukin 7 is a key molecule in T cell homeostasis and genetic variation in the IL-7 receptor has been shown to influence clinical outcomes in several diseases. Progress will be reviewed and current

Department of Neurology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; b Department of Neurology, Nishimaruyama Hospital, Sapporo, Japan In Japan, the low prevalence rate of MS and the relatively high frequency of unique clinical subtype of MS (opticspinal MS; OSMS) have been emphasized. Ethnic differences may affect not only the susceptibility, but also the phenotypic expression of MS, including the clinical manifestations, site of lesions, disease course, and prognosis. Here, we review the results of recent studies and hypotheses concerning genes related to MS in Japan. An association between DRB1⁎1501 and “conventional MS (CMS)” has also been confirmed in Japanese populations, suggesting that DRB1⁎1501 is associated with MS worldwide. In OSMS, on the other hand, DPB1⁎0501 allele has been shown to be a disease-susceptibility gene. Oligoclonal IgG bands (OCB) are present in most patients with MS in Western countries; however, in Japanese MS patients, the OCB-positive rate is not as high. HLA-DR15 association with OCB-positive MS, and DR4 association with OCB-negative MS were confirmed. From our candidate gene studies other than HLA, gene polymorphisms of Vitamin D receptor, estrogen receptor, osteopontin, and TRAIL were associated with susceptibility of CMS in Japanese. MS patients with fulminant attacks were clinically and immunogenetically distinct from those free of such attacks. HLA profiles were similar among patients with fulminant attacks irrespective of the lesion distributions while the CTLA4 gene polymorphism was associated with the fulminancy. An approach to treatment by genetic engineering may be possible once an understanding of the genetic influences and mechanisms involved in the causation of MS is established.

Abstracts

International Symposium: Alzheimers disease, neurodegeneration and immunity

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Abstracts

IS01-01

Alzheimer's disease and immune-mediated therapy: An overview T. Tabira National Institute for Longevity Sciences, NCGG, Aichi, Japan Alzheimer's disease (AD) is a neurodegenerative disease characterized by senile plaques, neurofibrillary tangles and neuronal death. It is now widely accepted that amyloid beta protein (Aβ) has a central role in the pathological mechanism of AD. Aβ itself, particularly an oligomeric form is neurotoxic. Further, aggregated Aβ forms amyloid fibrils and are deposited in the senile plaque, where innate immune responses to β amyloid also induce neural damage. Thus, prevention of Aβ production and clearance of β amyloid seem to be effective for prevention and treatment of AD. Schenk D. et al. first described that active and passive immunization with Aβ cleared and prevented β amyloid deposits in amyloid precursor protein transgenic mice. Intramuscular immunization with Aβ and adjuvant was tried in AD patients in US and Europe. However, because of a serious side effect subacute meningoencephalitis, the clinical trial was halted. Although the trial was halted, it gave us important information. Autopsied brain of patients who had received the vaccine suggested effective clearance of β amyloid in the human brain, too. Those patients who had elevated titers of tissue amyloid plaque immunoreactive antibodies showed slower decline of cognitive functions than those who did not have such antibodies. Thus, development of a safer and more effective vaccine is now a world-wide topic. Here we discuss the mechanism of the meningoencephalitis and development of safer strategies in this session.

Alzheimer's disease (AD) is an age-related progressive neurodegenerative disorder characterized by memory loss and severe cognitive decline. These clinical features are manifested morphologically by excessive accumulation of extracellular amyloid β-peptide (A-beta) in the brain parenchyma, particularly in the hippocampus and cerebral cortex, leading to neuronal loss. In addition, aggregates of A-beta that forms neuritic plaques in the brain become toxic in that they trigger chronic glial activation and thereby interfere with normal brain function. Investigating how the immune system is involved in the disease is based on new emerging characteristics of three biological systems: autoimmunity, brain–immune interactions, and neurogenesis. Our data demonstrate that A-beta specific T cells are induced in AD and that the specificity and magnitude of these responses depend primarily on HLA-DR alleles. Limited expression of IFN-γ in the brain, as observed during normal brain aging, is essential to promote migration of these A-beta reactive T cells to the parenchymal tissue in the hippocampus and subsequently interaction with local antigen presenting cells. In contrast to the injurious effects induced by chronic innate immune mechanisms involved in AD, T-cell-mediated immunomodulation may support tissue recovery by regulating glial activation and differentiation, clearance of A-beta, and neuronal survival. The translation of these results to safe immunotherapeutic approaches will be discussed. IS01-04

Mucosal immunotherapy for Alzheimer's disease with viral vectors H. Haraa, M. Inoueb, M. Hasegawab, Y. Yonemitsuc, T. Nabeshimad and T. Tabiraa National Center for Geriatrics and Gerontology, Obu, Japan; bDNAVEC corporation, Tsukuba, Japan; cKyushu University, Fukuoka, Japan; d Nagoya University, Nagoya, Japan

a

IS01-02

The pivotal role of complement in chronic neurodegenerative disease Patrick L McGeer Kinsmen Laboratory of Neurological Research, University of British Columbia, Vancouver BC, Canada Complement is a phylogenetically ancient host protective system. It identifies targets, opsonizes them, recruits attacking cells through its anaphylotoxins, and kills invaders through its membrane attack complex. Unfortunately it is not perfect in distinguishing friend from foe, and the host is armed with a series of regulators that normally provide self protection. In a number of chronic neurodegenerative disorders, autodestruction by the membrane attack complex has been identified in post mortem tissue. These disorders include Alzheimer disease, Parkinson disease, the fronto-temporal dementias, the Parkinson–dementia complex of Guam, atherosclerotic vascular disease, age-related macular degeneration and diabetes type 2. These are not autoimmune diseases. They all involve complement generation by local tissues and complement activation by factors other than antibodies. The most damaging component is the membrane attack complex. Inhibitors of this complex should have broad application in a range of age-related degenerative disorders. Conversely, those Japanese who have inherited a defective C9 gene should be relatively spared many of these disorders and may fall into the super healthy category. IS01-03

Peripheral and brain autoimmune mechanisms in Alzheimer's disease Alon Monsonego The Department of Microbiology and Immunology and National Institute of Biotechnology, Ben-Gurion University, Beer-Sheva, Israel

Background: Alzheimer's disease is characterized by progressive loss of cognitive function due to amyloid-β (Aβ) deposits in the central nervous system. Based on the amyloid cascade theory, many reports indicated that immunotherapy is effective for the treatment of Alzheimer's disease. We developed the mucosal immunotherapy for Alzheimer's disease by nasal administration of recombinant sendaivirus vector expressing Aβ 1–43/IL10. Method: 24–25-months old APP transgenic mouse were nasally administered once with recombinant sendaivirus vector expressing Aβ 1– 43/IL-10. After 8 weeks of treatment, mice were sacrificed and investigated the amyloid burdens in the brains by immunohistochemistry and ELISA. Results: Serum antibody levels for Aβ were elevated after four weeks of treatment. Immunohistochemistry of the mouse brain tissue showed that the extra-cellular amyloid deposits were clearly decreased compared to the nontreated mouse. The amount of Aβ measured by ELISA was also decreased in both soluble and insoluble fractions of the brain tissues. The inflammation and lymphocytes infiltration were not recognized in each organ including brain. Conclusion: Nasal administration of recombinant sendaivirus vector expressing Aβ 1–43/IL-10 is advantageous in that no cellular immune response was elicited and only antibody production was induced, furthermore, no adjuvant is required. This new mucosal immunotherapy does not induce the strong immune reactions and can reduce the side effects of encephalitis as found in Aβ peptides vaccination. IS01-05

Antibody independent clearance of A-beta in a mouse model of Alzheimer's disease by activation of microglia via nasal vaccination H.L. Weinera, S. Petrovica, W. Xiaa, M. Goldberga, J. Vegaa, D.S. Burtb, and D. Frenkela a

Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA USA; bGlaxo Smith Kline, Laval, PQ, Canada

Abstracts Methods: Age- and sex-matched littermates (APP-J20 mice) received a weekly nasal treatment with Protollin (a proteosome-based adjuvant used in human influenza vaccination) 1 μg/treatment, plus glatiramer acetate (GA, an approved drug for MS) 25 μg/treatment, beginning at age 5 months and were treated for 8 months. Results: We found a significant reduction in amyloid fibril (∼ 80%) in GA + Protollin treated mice versus controls (P < 0.001). There was reduction of both (50%) soluble Aβ and (49%) insoluble form of Aβ (p < 0.02) as measured by ELISA and elevation of total serum Aβ levels. The treated animals exhibited no toxicity as measured by body weight, eating habits, tail tone, or mobility. Clearance of Aβ was also observed with nasal Protollin alone and direct intrahippocampal injection of Protollin activated microglia and reduced Aβ plaques. We also found reduction in astrocytosis following nasal vaccination with GA + Protollin or Protollin alone. We previously reported that nasal vaccination with GA + Protollin, decreased Aβ plaques in 14 month old APP Tg mice, vaccinated animals developed activated microglia (CD11b+ cells) that co-localized with Aβ fibrils, and the extent of microglial activation correlated with the decrease in Aβ fibrils (JCI: 115:2423, 2005). In this study, after chronic treatment for 8 months we observed no difference in microglial activation, suggesting that once Aβ is cleared there is downregulation of microglial activation. Conclusion: Our results demonstrate an antibody-independent therapeutic approach for the treatment of AD which targets microglial cells and is also effective, without toxicity, when given chronically to young mice as prevention.

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a

The Ludwig Institute, University of California at San Diego, bCenter for Neurologic Study, and the cSalk Institute, La Jolla CA USA; dThe Ohio State University, Columbus, OH USA; eRiken Center for Neuroscience, Tokyo, Japan The primary characteristic of Amyotrophic Lateral Sclerosis (ALS) is the premature death of motor neurons. An inherited form is caused by mutation in superoxide dismutase (SOD1). Toxicity is non-cell autonomous, requiring mutant SOD1 acting within motor neurons, neighboring astrocytes and microglia. Silencing mutant expression selectively within motor neurons slows disease onset but with little effect on progression. Reducing mutant SOD1 synthesis in microglia by selective gene excision has little effect on disease onset, but strikingly extends survival after onset, demonstrating that damaged microglia play an essential role in disease progression. Further, using primary motor neuron and microglial cells, only conditioned medium from mutant SOD1 microglial cells produces neurotoxicity on mutant expressing motor neurons. Exploiting these findings, a gene therapy has been developed for targeting almost any gene using continuous infusion to deliver antisense oligonucleotides to neurons and non-neuronal neighbors throughout the nervous system. Oligonucleotide infusion to promote catalytic degradation of the mRNA encoding mutant SOD1 slows disease progression. Thus, antisense oligonucleotide therapy can be an effective means of treating neurodegenerative diseases, including ALS, Alzheimer's, Parkinson's, and Huntington's, for which appropriate target genes have been identified. IS02-03

IS02-01

Phenotypic diversity of microglia and macrophages Microglia: The double-edged sword in motor neuron injury Uwe-Karsten Hanisch S.H. Appel, J.S. Henkel, D.R. Beers, Q. Xiao, and W. Zhao Institute of Neuropathology, University of Göttingen, Germany Methodist Neurological Institute, Houston, USA Immune/inflammatory factors have been implicated in the pathogenesis of motoneuron degeneration in amyotrophic lateral sclerosis with the presence in spinal cord tissue of activated microglia, dendritic cells, IgG deposits, lymphocytic infiltrates, and marked increases in MCP-1, the chemokine involved in immune cell recruitment. Transgenic mice overexpressing mutant human superoxide dismutase (mSOD1) provide a useful model to investigate mechanisms of motoneuron injury. In such mice, activated microglia and increased MCP-1 are present prior to onset of weakness; and dendritic cells subsequently appear. To define the potential role of microglia in disease pathogenesis, we used a mouse model unable to develop myeloid cells or lymphoid cells (PU.1−/−). Bone marrow transplants of these mice result in donor-derived microglia in spinal cord parenchyma. Transplantation of mSOD1 bone marrow alone did not induce disease. However, PU.1−/− mice with mSOD1 expressed in motor neurons and transplanted with wild type mSOD1 donor cells had greater motoneuron preservation and prolonged disease duration and survival compared with mice receiving mSOD1 donor cells. In vitro studies confirmed that wildtype microglia were less neurotoxic than mSOD1 microglia. Transplant of these mSOD1/PU.1−/− mice with bone marrow lacking the receptor for MCP-1 (CCR2−/−) removed the neuroprotective effects of wildtype microglia. When mSOD1 was expressed in RAG2−/− mice, which lack functional T- and B-cells, disease progression was actually accelerated. These studies suggest that CNS parenchymal microglia can be neuroprotective or toxic, with the specific effects modulated by communication with peripheral immune cells.

Several findings reveal that treatment with certain cytokines, confrontations with microbial agents or exposure to tumor material and apoptotic cells trigger macrophages to acquire distinct reactive phenotypes. Phenotypes are thereby not defined by a single release product or receptor expression, but comprise timed, coordinated, often even reciprocal induction patterns of mediators for cellular communication and profiles of effector molecules (e.g. cytokines, metabolic or extracellular matrix-modelling enzymes, surface structures for phagocytosis or antigen presentation). Adaptations of the executive behavior come with functional consequences, including opposite effects on inflammation, skewing for types of immune responses and orientation for either defense or repair. Although microglia, the CNS tissue macrophage equivalent, is challenged by virtually any neuropathological scenario, less is known concerning the phenotypic diversity and dynamics, associated molecular characteristics and functional consequences. Conceivably, these cells also choose rather variable response options for an adequate coping with an exogenous or endogenous threat and to decide on a recruitment of adaptive immune support. Yet microglia and macrophages of non-CNS origin may differ in reactive profiles as imposed by the specific biochemical milieu, structural vulnerability and control of defence mechanisms in neural tissues. Most notably, distinct reactive phenotypes may not only occur as alternatives, but develop (interchange) stage-specifically during an activation process. Highly organized, context-determined, situation-adapted switches in anti/inflammatory messenger production, timed recruitment vs. decline of immune infiltrates, phagocytotic clearance and tissue rebuilding attempts could be elements of an open, (usually) selflimiting and (mostly) efficient program for protection and restoration. Findings supporting this view are discussed.

IS02-02 IS02-04

Neuroinflammation and non-cell autonomous death of motor neurons in ALS Don W. Clevelanda, Severine Boilleea, Richard A. Smitha,b, Timothy M. Millera, Brian K. Kaspard, Fred H. Gagec, and Koji Yamanakaa,e

Neurotoxicity by microglia: Mechanisms and potential therapeutic strategy Akio Suzumura

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Department of Neuroimmunology, RIEM, Nagoya University, Nagoya, Japan Accumulation of activated microglia are observed in or around degenerating neurons. These cells may play a critical role on both neurotoxicity and neuroprotection. We have shown recently that the most toxic agent from activated microglia is glutamate which disturb mitochondrial respiratory chain to cause neuronal dysfunctions. Glutamate and TNF-α from activated microglia differentially contribute to neuronal damage in neurodegenerative diseases. However, the clinical trials to block these factors failed in serious adverse effects. Here, we show that TNF-α stimulates extensive microglial glutamate release to cause excito-neurotoxicity, by activating glutaminase. In addition, glutamate is released from microglia through the hemichannels of gap junction, but not through glutamate transporters. Either a hemichannel blocker or glutaminase inhibitors effectively suppressed microglial glutamate release and subsequent neuronal cell death. The unique mechanisms of microglial glutamate release will give us a new strategy against neurodegenerative diseases without perturbation of physiological signals of glutamate. IS02-05

Role of immunity in neurodegeneration in autoimmune encephalomyelitis

a

Department of Vascular Dementia Research, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu City, Japan; bStroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA The subcortical ischemic form of vascular dementia is a common type of vascular dementia, and one of the major causes of cognitive decline in elderly people. Ischemic white matter lesions are the characteristic pathological changes in subcortical ischemic vascular dementia. Ischemic white matter lesions and impairment of memory function have been reported in the rat under chronic cerebral hypoperfusion. Using this model, we investigated the effects of mucosal tolerance to E-selectin on white matter damage and memory impairment. Wistar rats were subjected to the permanent occlusion of both common carotid arteries. Mucosal tolerance was induced by repetitive intranasal administration of E-selectin. The control group received PBS. Rarefaction of white matter was evaluated by luxol fast blue stain. Activation of vessels was examined with immunohistochemistry for tumor necrosis factor (TNF)-α and E-selectin. Memory impairment was evaluated by object recognition, T-maze spontaneous alternation, and T-maze memory retention tests. Mucosal tolerance to E-selectin had a potent protective effect against white matter rarefaction. In addition, mucosal tolerance to E-selectin had a suppressive effect on activation of brain vessels. Mucosal tolerance to E-selectin also protected against memory impairment. These results indicate that the mucosal tolerance to E-selectin suppresses vessel activation support of local immunological and inflammatory processes and protects against ischemic white matter damage. These results can provide a new therapeutic strategy using a mucosal tolerance to E-selectin against subcortical ischemic vascular dementia.

D. Baker Institute of Neurology, University College London, London, United Kingdom For many years multiple sclerosis (MS) has been thought to result from autoimmune attack of the central nervous system (CNS). As such, experimental autoimmune encephalomyelitis (EAE) — an autoimmune model of MS has greatly influenced the development of immunotherapeutic paradigms aimed at treating MS. Despite some limited success in inhibiting relapsing disease, there has been poor translation of animal studies into the clinic and progressive MS has largely been unresponsive to immunotherapy. However, it has become increasingly recognized that neurodegenerative elements are responsible for disease progression in MS. Axonal loss increases with increasing numbers of neurological attacks and results in the development of permanent residual paresis. As active clinical disease in MS and EAE are associated with mononuclear cell infiltration of the CNS infiltration, demyelination and axonal loss, inhibition of inflammation is one route to neuroprotection and if relapsing disease is inhibited sufficiently early in the disease course, it will slow the rate of atrophy of the CNS and axonal loss. This is readily apparent in EAE and has been more recently shown in MS. However, when immunotherapy is initiated late into chronic-relapsing disease course, whilst relapsing disease can still be eliminated such as using antigen-specific tolerance induction, there appears to be a slow deterioration in mobility, an increase in neurological signs and ongoing axonal damage that is (auto)immuneindependent. Therefore just as appears to be the case in MS once sufficient damage has been accumulated then inhibition of (auto) immunity via neuroimmunological approaches may be insufficient to control progressive disease elements.

IS03-01

Immune regulation of neural stem cell programs in EAE S. J. Khoury Brigham and Women's Hospital, and Harvard Medical School, Boston, MA, USA Multiple sclerosis is characterized by a progressive pathology and alteration in remyelination capacity. Neural stem cells and oligodendrocyte progenitors can generate myelinating oligodendrocytes during development and during normal adult turnover, but endogenous progenitors cells in the MS appear to fail with chronic progression and the responses of endogenous cells in EAE appear to be limited. We hypothesize that inflammatory mediators may act upon the genetic program of NSCs and initiate the process of repair, but in the chronic phase inflammatory mediators may decrease the intrinsic properties of NSCs, resulting in failure of repair. NSCs express immune-related genes that include chemokines and cytokines receptors, and we have shown that some of these molecules such as the chemokine SDF-1 are permissive for the repair function of NSCs during injury. EAE lesions express signaling molecules for neural stem cells, yet there is a lack of neurogenesis and lack of oligodendrogenesis in the chronic phase, and this lack of function is correlated with microglia activation in vivo and in vitro. In order to identify potential mechanisms, we have focused in the molecular biology of candidate genes that may mediate such interactions, we will present data regarding the molecular effects of prototypical Th1 cytokines in the molecular programs of NSCs. Conclusion: Identifying the molecular mechanism for the responses of NSCs to injury in EAE, will help to increase the efficacy of exogenous NSC transplantation and increase the activation of the intrinsic properties of endogenous stem cells in models of multiple sclerosis.

IS02-06 IS03-02

Protective effect of mucosal tolerance to E-selectin against memory impairment and white matter injury in a rat model of vascular dementia a

Hideaki Wakita and bJohn M. Hallenbeck

Neural stem cell transplantation: Cell replacement vs. bystander neuroprotection G. Martino

Abstracts Neuroimmunology Unit, DIBIT — San Raffaele Scientific Institute, Milan, Italy Recent evidence challenges the sole view that neural stem/precursor cells (NPCs) achieve their therapeutic efficacy exclusively by a cellreplacement mechanism. In fact, NPCs may also promote central nervous system (CNS) repair by their intrinsic neuroprotective ability, which are mainly exerted by undifferentiated cells releasing, at the site of tissue damage, a milieu of neuroprotective molecules temporally and spatially orchestrated by environmental needs. This milieu contains molecules (such as immunomodulatory substances, neurotrophic growth factors and stem cell regulators) that are constitutively expressed by NPCs for maintaining tissue homeostasis both during development and in adulthood. The intrinsic nature (pleiotropism and redundancy) of these molecules as well as their ‘constitutive’ expression by different types of stem cells (such as mesenchymal and haematopoietic stem cells) may represent a stem cell signature. This might also reconcile data showing that other sources of somatic stem cells (such as mesenchymal stem cells and haematopoietic stem cells), with very low potential of neural (trans) differentiation, may efficiently promote CNS repair. Thus, cell plasticity can also be viewed as the capacity of somatic stem cells to adapt their fate and function to specific environmental needs, which arise as a result of pathological conditions — this is the notion of therapeutic plasticity. The ability of transplanted NPCs to protect the brain from several types of injuries using different and/or multifaceted bystander strategies is of significant importance for the future of stem cell-based therapeutic approaches. IS03-03

T-cell based therapy operates in synergy with adult neural stem cells for neuronal survival and renewal: Implications for Alzheimer's disease Michal Schwartz Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel [email protected] Several years ago we proposed, contrary to the prevailing wisdom, that maintenance and repair of the central nervous system (CNS) needs the assistance of the immune system. Because both innate and adaptive immunity were known to be implicated in the pathologies of inflammatory and non-inflammatory neurodegenerative diseases (such as multiple sclerosis in the first instance and Alzheimer's disease in the second), the question arose: under what conditions does immune activity operate as part of the repair process and thus need to be boosted, and when does it become part of the pathology and hence require modulation? Our early findings led us to formulate the concept of “protective autoimmunity”. According to this concept, T cells that recognize CNSrelated autoantigens (“autoimmune” T cells) protect neural tissue and induce neural cell renewal from endogenous stem cells by locally providing growth factors and causing microglia to adopt supportive phenotypes. Using an animal model of Alzheimer's disease, we demonstrated that T-cell-based vaccination with a weak agonist of autoantigens can shift the default microglial phenotype towards one that enables microglia to engulf aggregated β-amyloid and produce growth factors, leading to arrest of disease progression and induction of functional rescue, tissue repair, and even neurogenesis. A similar T cellbased approach was shown by our group to be applicable in acute brain injury, stroke, and other neurodegenerative conditions. Our studies argue in favor of autoimmunity as a physiological system of defense and maintenance against the neurodegenerative diseases or age-related cognitive losses that result, at least in part, from aging of the immune system.

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T cells — New and unexpected players in cognition Jonathan Kipnis Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska 68198-5800, USA Recent results obtained in rodents suggest that T cells have an important role in several aspects of neuronal plasticity, such as hippocampal neurogenesis and spatial learning and memory. Cognitive performance, tested in learning and memory tasks, was impaired in immune-deficient mice compared to their wild-type counterparts. Superior performance of wild type mice was correlated with activation of GFAP-positive astrocytes in the hippocampus. Restoration of a T cell pool in immune deficient mice, which resulted in improved cognitive abilities, was also accompanied by significant increase in GFAP positive cells in their hippocampi. Our in vitro results provide evidence that astrocytes, major non-neuronal cells that actively participate in neuronal networking, activated with T cells possess a phenotype supportive of neuronal functioning and establishment of neuronal network. Therefore, T cell-mediated improvement of cognitive functions might encounter, at least in part, on an enhanced neuronal plasticity supported by astrocytes as a result of interaction with T cells. Based on this hypothesis, we boosted a T cell response by immunization with a synthetic Copolymer-1 (glatiramer acetate), which gives rise to T cells with partial recognition of brain-specific antigens. Such immunization significantly improved the performance of wild type mice in cognitive task. These findings suggest that the decline in immune activity known to be associated with several mental and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease, might account, at least in part, for the cognitive dysfunction observed in these patients. Such new understandings could lead to the identification of new therapeutic targets and the development of new therapeutic approaches. IS03-05

Nogo, Nogo receptor, and TROY expression in demyelinating lesions of multiple sclerosis Jun-ichi Satoha a

Department of Bioinformatics, Meiji Pharmaceutical University, Tokyo, Japan

Objective: To study Nogo and its receptor (NgR) expression in MS lesions. Background: Nogo is a family of myelin-associated neurite outgrowth inhibitors, composed of three distinct isoforms (A, B and C), all of which share a small segment Nogo-66. Nogo-A is expressed on oligodendrocytes, where Nogo-66 binds to NgR expressed constitutively on a subset of neurons and axons. NgR signaling requires a coreceptor p75NTR or TROY, and an adaptor LINGO-1. Because axonal injury is a pathological hallmark of MS, we investigated Nogo and NgR expression in MS lesions. Methods: MS and non-MS brains were studied by immunohistochemistry using anti-Nogo-A (sc25600), NgR (AB5615), p75 (ME20.4), TROY (sc13711), and LINGO-1 (UBI07678) antibodies. Results: Nogo-A was markedly upregulated in surviving oligodendrocytes (OL), while NgR was enhanced in reactive astrocytes (AS) and microglia/ macrophages (MP) in active demyelinating lesions of MS. Nogo-A and NgR were also identified in a subpopulation of neurons (NE), while Nogo-A was undetectable in AS or MP, and NgR was not expressed on OL. p75 was restricted in limited regions, while TROY and LINGO-1 expression was widespread in the CNS in MP, AS and a subpopulation of NE, but not in OL. TROY protein levels were upregulated, while LINGO-1 was downregulated in MS brains. Conclusions: Nogo-A expressed on OL, by interacting with the NgR/ LINGO-1/TROY complex on AS, MP and NE, might regulate glial– neuronal interactions in active demyelinating lesions of MS.

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Concurrent Symposia

Abstracts CS01-01

Understanding disease pathogenesis of multiple sclerosis by genomics and proteomics approaches Roland Martin Catalan Institute of Research and Advanced Studies (ICREA), Unitat de Neuroimmunologia Clinica, Institut de Recerca Hospital Universitari Vall D'Hebron, Barcelona, Spain The etiology of multiple sclerosis (MS) remains incompletely understood, but a complex genetic background with multiple, weakly diseaseassociated susceptibility genes and probably also environmental factors contribute to disease. As a result, the pathogenetic processes that underlie the clinical manifestation and disease courses are heterogeneous and may vary from patient to patient. The main components that cause tissue damage in MS are an autoimmune inflammatory process, which leads to demyelination, axonal loss and glial activation, but at least in some patients, there appears to be an increased vulnerability of the CNS tissue to injury as well. Currently, we have no clinical signs, MRI imaging measures or biomarkers that allow us a clear separation of patients according to the underlying disease pathogenesis. A better understanding of disease heterogeneity could lead to earlier diagnosis, more precise prognosis and the identification of more effective treatments. Attempts to identify single markers that capture the pathogenesis of MS and other complex diseases have not been very successful, most likely because a multitude of molecules are involved that form parts of complex pathways. Hypothesis-oriented research that investigates each factor in isolation needs to be complemented by methods that allow us to study simultaneously large numbers of genes, their transcription and the resulting protein patterns. Recent advances in the areas of mapping single nucleotide polymorphism (SNP) haplotypes and gene expression at the level of almost the entire genome offer clear advantages in deciphering disease heterogeneity of complex diseases, but they are not without problems. The presentation will summarize important methodological aspects, consider how to design such studies, and also present recent data.

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1

Departments of Neurology, Medicine and Pathology, Stanford University School of Medicine, Stanford, USA; 2Department of Physiology, University of Connecticut Health Center, Farmington, USA and 3Department of Pathology, Albert Einstein College of Medicine, Bronx, USA Demyelinating lesions or “plaques” in multiple sclerosis (MS) have distinct histological features that reflect their stage of evolution. To identify key molecules involved at different stages during lesion progression, we performed proteomic profiling of three types of MS plaques using mass spectrometry and bio-informatics. Fresh frozen autopsy brain samples from six MS patients and age-matched controls were classified as (1) acute plaque (AP, characterized by presence of inflammatory cells, ongoing demyelination), (2) chronic active plaque (CAP, characterized by chronic demyelination, astrogliosis with active rim) or (3) chronic plaque (CP, characterized by lack of inflammatory cells, dense astrogliosis). Samples from the plaques were isolated by laser capture microdissection and global protein identification was carried out by SDS-PAGE, in-gel protease digestion, liquid chromatography and mass spectrometry. Identified proteins were analyzed by bio-informatics software (SEQUEST, INTERACT, INTERSECT and emPAI) for identification of unique proteins and quantitation. A total of 3894 proteins were identified. Analysis revealed proteins unique to AP (N = 172), CAP (N = 248) and CP (N = 252), and proteins common to all three types (N = 89). There were 77 proteins associated with active inflammation and 102 unique to chronic lesions. Finally, we assigned protein complexes unique to each type of plaque which may be of biological importance. These results provide the first and most comprehensive information on global protein expression in MS plaques, enhance our understanding of the molecular pathogenesis of MS lesions and identify potential key molecules involved in disease progression that offer possibilities for therapeutic targets. CS01-04

Endogenous neuroprotective mechanisms in multiple sclerosis T. Zeisa, U. Graunmana, A.J. Stecka, W. Brückb and N. Schaeren-Wiemersa a

CS01-02

Biomarker discovery in MS Combining genome-wide SNP mapping and microarray studies M. Comabella Institut de Recerca, Unitat de Neuroimmunologia Clínica, Hospital Universitari Vall d'Hebron, Barcelona, Spain A significant body of data on gene expression has been generated in multiple sclerosis (MS) using microarrays. Although results are very promising, microarray-based studies still receive much criticism mainly due to the multiple potential sources of variability that often make these studies difficult to reproduce and interpret. This talk focuses on the current status and future directions of the use of DNA microarrays in MS: a) microarray studies in peripheral blood cells comparing gene expression signatures between MS patients and healthy controls; b) microarray studies in MS brain lesions; c) gene expression profiling studies in the animal model of MS or experimental autoimmune encephalomyelitis (EAE); d) treatment related studies with microarrays in MS. CS01-03

Proteomic profiling of multiple sclerosis plaques May H. Han1, Sunil Hwang2, William H. Robinson1, Cedric S. Raine3, Raymond Sobel1, David K. Han2 and Lawrence Steinman1

Neurobiology, Department of Research, University Hospital Basel, Pharmacenter, Switzerland; bInstitute of Neuropathology, Georg-AugustUniversity Göttingen, Germany MS is a chronic inflammatory disease of the CNS leading to focal destruction of myelin. However, the earliest changes that lead to lesion formation are not known. Our recent microarray analysis of normalappearing white matter (NAWM) revealed upregulation of a number of functionally related genes known to be involved in endogenous neuroprotection as well as in maintenance of cellular homeostasis (Brain Pathology 2003). Our results introduced a novel concept for the molecular pathogenesis of MS with ischemic preconditioning as an important mechanism for neuroprotection. The question arises, which of these alterations occur in the early stage of MS, and which are the consequences after long disease progression. Biopsy tissues are routinely embedded in paraffin, and therefore, we have established a protocol for successful isolation of total RNA allowing quantitative RT-PCR analysis, and detection of low abundant genes. In an ongoing study, we have characterized NAWM from a needle biopsy of an 18-year old female patient. Immunopathological examination revealed pathology, which is in agreement with Pattern III lesions after the classification by Lucchinetti and Brück (2002). We isolated total RNA from NAWM tissue samples of this patient and performed qRT-PCR. Strikingly, we could detect very high NOS expression levels in NAWM of this biopsy, which was much less evident in NAWM of post-mortem tissues. Based on these data, we believe that these alterations reflect very early changes in MS. We speculate that depending on the regulation of the endogenous neuroprotective mechanisms, lesion formation might either be prevented or sustained.

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Abstracts

CS02-01

CS02-03

Hormonal effects on autoimmune disease

The dual role of the sympathetic nervous system in chronic inflammation

C.C. Whitacre, M.A. McClain, T.L. Papenfuss, N.N. Gatson, N. Damico, I.E. Gienapp, F. Song and T. Shawler The Ohio State University, Columbus, Ohio, USA Women with multiple sclerosis (MS) experience a decreased relapse rate during late pregnancy, with a flare of disease 3–6 months postpartum. We examined the effect of pregnancy on experimental autoimmune encephalomyelitis (EAE) in the SJL mouse and the impact of pregnancy on ongoing disease. Pregnant animals showed a reduced incidence of EAE and a decrease in clinical severity, while mice induced for EAE during the post-partum period showed more severe disease. The most striking results were in mice who became pregnant after the acute disease period, showing complete protection during mid and late pregnancy. Cells from protected mice produced less TNF-α and more IL-10 than non-pregnant controls. CD4/CD25+ cells were not increased in mice immunized during pregnancy and the primary source of IL-10 was CD11b+ cells. We explored the role of dendritic cells (DC) in mediating pregnancy-induced protection from EAE. In mice immunized during late pregnancy, we found significant increases in costimulatory molecule expression (CD80, CD86, PDL1 and PDL2) on DCs. Similar increases in costimulatory molecules, PDL1 and PDL2, were noted on DCs from mice treated with estriol, the pregnancy estrogen. Transfer of DCs from estrioltreated mice protected recipients from EAE. These results suggest that during pregnancy, an immunoregulatory environment promotes DC upregulation of negative costimulatory molecules and production of IL10. (Supported by NIH NS48316, T32 AI 55411 and National MS Society RG3272.)

R. H. Straub Laboratory of Neuroendocrino-Immunology, Department of Internal Medicine I, University Hospital Regensburg, Germany In this presentation, we demonstrate the dual pro- and anti-inflammatory role of the SNS (SNS) in inflammatory joint disease via distinct adrenoceptors. The dual role of the SNS depends on involved compartments, on timing of distinct effector mechanisms during the inflammatory process, on availability of respective adrenoceptors on target cells, and on an intricate shift from beta- to alpha-adrenergic signaling in the progressing course of the inflammatory disease (beta- to alpha-adrenergic shift). A further critical point for the dual role of the SNS in inflammation is the underlying change of immune effector mechanisms during the process of disease progression and also the behavior of sympathetic nerve fibers in inflamed tissue (nerve fiber loss). This is accompanied by a relative lack of anti-inflammatory glucocorticoids in relation to inflammation. In quintessence, in very early stages of inflammatory joint disease, the SNS plays a predominant proinflammatory role whereas in late stages of the disease the SNS most probably exerts anti-inflammatory effects. Since patients with rheumatoid arthritis most often present themselves in the chronic phase of the disease, support of anti-inflammatory sympathetic pathways can be a promising therapeutic option in chronic inflammatory diseases. CS02-04

Experimental stroke causes early immune activation followed by systemic immunosuppression

CS02-02 H. Offner

The intricate interface between immune and metabolic regulation: A role for leptin in the pathogenesis of multiple sclerosis? Giuseppe Matarese Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, via S. Pansini 5 — 80131 — Napoli, Italy; email: [email protected] Over the last few years a series of molecules known to play a function in the metabolic function have also been shown to play an important role in the regulation of the immune response. In this context, the adipocytederived hormone leptin has been shown to regulate the immune response both in normal as well as in pathological conditions. More specifically, it has been shown that conditions of reduced leptin production (i.e. genetic leptin deficiency, anorexia nervosa, malnutrition) are associated with increased susceptibility to infections. Conversely, immune-mediated disorders such as autoimmune disorders are associated with increased secretion of leptin and production of pro-inflammatory pathogenic cytokines. Leptin could represent the “missing link” between immune response, metabolic function and nutritional status. Indeed more recently, leptin-deficient mice have been shown to be resistant to a series of experimentally-induced autoimmune disorders including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Normal wild-type mice show increased secretion of leptin in serum upon EAE induction, and brain inflammatory infiltrates stain positive for leptin. Finally, leptin neutralization with anti-leptin antibodies, improves EAE course and progression and profoundly alters intracellular signalling of myelin-reactive T cells. These data suggest that leptin can be considered as a major link between immune and metabolic regulation and that strategies aimed at interfering with the leptin axis could represent innovative therapeutic tools for autoimmune disorders.

Oregon Health and Science University, Portland, OR, USA Induction of stroke not only produces local ischemia and brain damage, but also has profound effects on peripheral immune responses. Our general hypothesis is that focal cerebral ischemia alters immunocyte function through brain-to-spleen signaling via the sympathetic nervous system. We observed an initial rapid intra-splenic activation of immunocytes, including T-lymphocytes. Some of these T-cells appeared to be released from the “post-stroke” spleen within hours, homed to the brain and secreted cytokines that amplified peri-infarct inflammation. Subsequently, there was loss of immune competence, characterized by splenic atrophy and increased splenocyte apoptosis. This was accompanied by reduced T cell proliferation and secretion of inflammatory cytokines. These changes produced a drastic reduction in B cell numbers, a novel increase in CD4+FoxP3+ regulatory Tcells, and an increase in the percentage of non-apoptotic macrophages/ monocytes. Immunosuppression in response to brain injury may account for the reduction of inflammatory factors in the stroke affected brain, but also potentially could curtail protective immune responses in the periphery. These findings provide new evidence to support the contention that damage to the brain caused by cerebral ischemia produces immunosuppression caused by cell death as well as an increased presence of CD4+FoxP3+ Treg cells. The net effect for a stroke survivor would be enhanced risk for crippling systemic infections and septicemia. CS03-01

Clinical characteristics and pathophysiology of the biopsied neuromuscular junction in Japanese MuSK antibody-positive myasthenic patients Masakatsu Motomuraa, Hirokazu Shiraishia, Toshiro Yoshimurab

Abstracts a

First Department of Internal Medicine, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan; b Health Sciences, Nagasaki University, Nagasaki, Japan Myasthenia gravis (MG) is an antibody (Ab)-mediated autoimmune disease of the neuromuscular junction. In approximately 80% of patients, autoantibodies to the muscle nicotinic acetylcholine receptor (AChR) are present, but the remaining 20% of patients are classified as seronegative. In 2001, Hoch et al., first showed that 70.8% of AChR Ab-seronegative MG patients, but not AChR-Ab-seropositive MG patients, have serum autoantibodies against the muscle-specific receptor tyrosine kinase (MuSK), which mediates agrin-induced clustering of AChRs during synapse formation, and is also expressed at the mature neuromuscular junction. Subsequent studies of AChR Ab-seronegative MG patients reported seropositivity from 3.8% to 63.5% and our seropositivity of MuSK Abs was 24.6%. Our clinical aspects of MuSK Ab-positive myasthenic patients (n = 70) are the following: (1) female-predominant (1:3), with onset between ages 2 to 70 years, (2) most patients had prominent neck or respiratory muscle weakness, (3) response to cholinesterase inhibitors was variable, (4) most patients with ocular type are negative for MuSK antibodies, (5) absence of thymoma, and no improvement after thymectomy, (6) most patients had a good response to plasma exchange and steroid therapy, but a proportion of MuSK patients who present muscle atrophy had a poor prognosis, and (7) there was no reduction of AChRs at the motor endplates of biopsied biceps brachii muscle in MuSK Ab-positive MG patients. Our results indicate that the pathogenesis of MuSK Ab-positive MG is not explained by complementmediated destruction of the motor endplate. In conclusion, MuSK Abs status should help diagnose MG with atypical presentations and ensure appropriate patient treatment. Further examinations to elucidate the pathomechanism of the MuSK Abs are needed. CS03-02

Preferential involvement of extraocular muscle by myasthenia gravis H.J. Kaminskia,b, L.L. Kusnera, Y. Zhoua, and C. Richmondsa

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CS03-03

Autoimmunising mechanisms in thymus and thymoma in myasthenia gravis (MG) patients N. Willcoxa, M.I. Leitea, Y. Kadotaa, H. Shionoa, Y. Roxanisa, B.P. Morganb and A. Vincenta a

Neuroscience Group, Weatherall Institute for Molecular Medicine, University of Oxford, UK; bDepartment of Medical Biochemistry and Immunology, School of Medicine, University of Cardiff, UK

Mechanisms of autoimmunisation are hard to study, especially in humans. We have focussed on ‘hyperplastic’ and neoplastic changes in the MG thymus. Patients with early-onset MG (EOMG; age < 40) or thymomas have pathogenic antibodies against the native muscle acetylcholine receptor (AChR). Autoantibodies have not yet been detected in standard assays (against AChR or MuSK) in 5–10% of patients with typical generalised MG (‘anti-AChR−’), despite their mild thymic hyperplasia. In >80% of EOMG patients, infiltrates of T-cell areas and germinal centres (GC) compress the hyperplastic medullary thymic epithelial cells (mTEC). Some mTEC express HLA-class II and unfolded AChR subunits, and could prime helper T-cells (Th) against AChR in the abnormal adjacent professional lymphoid tissue. We hypothesise that: (1) the primed Th then evoke ‘early autoantibodies’; these attack nearby thymic myoid cells expressing intact AChR — which frequently label for activated C3c and even C9 complement components. The similar labelling in >50% of the anti-AChR− patients suggests that they too belong to the EOMG spectrum; (2) autoantibodies diversify in the GC to recognise native AChR, including the fetal isoform expressed by myoid cells. Thymomas are neoplasms of TEC, which again express isolated AChR subunits. In addition to anti-AChR, ∼ 70% of patients have high titre neutralising antibodies against IFN-α (all 12 subtypes), and >50% against IL-12, at diagnosis. These latter increase when thymomas recur or metastasise; they are also produced spontaneously by mature plasma cells in the thymomas, implying stimulation by some cell type(s) there. Attempts to identify these will be summarised. CS03-04

Case Western Reserve University, Cleveland, Ohio, USA; bLouis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio, USA

a

Investigation of the unique susceptibility of extraocular muscle (EOM) by myasthenia gravis (MG) has led to novel insights into the disease and neuromuscular transmission. The reasons for their susceptibility are multiple, based on the molecular and physiological properties of EOM, the requirements of the ocular motor system, and the nature of the autoimmune pathology. Even minor EOM weakness will sufficiently misalign the visual axes to produce dramatic symptoms and proprioceptive feedback is limited to overcome such a deficit. The EOM neuromuscular junctions are subject to high neuronal stimulation, which would make them more susceptible to fatigue. EOM synapses have structural features that would make them susceptible to neuromuscular blockade; secondary synaptic folds are simplified and certain fibers contract in a tonic fashion such that any compromise of transmission would lead to a reduction contraction force. The combined features of high neuronal stimulation and junctional features that make transmission failure more likely suggests these neuromuscular junctions behave more like central nervous system synapses. EOM junctions have lower levels of intrinsic complement regulatory factors, which would make them more prone to damage by acetylcholine receptor (AChR) antibodies. Among ocular MG patients, serum AChR antibody levels are low, which would support the concept that EOM junctions are more susceptible to antibody injury than other junctions. Specific inhibition of the complement system could be of particular utility in treatment of ocular myasthenia. Improvement in understanding of the EOM susceptibility to MG may continue to provide insights into MG pathogenesis and neuromuscular transmission.

Antibodies to presynaptic receptors acting for synaptic plasticity in myasthenias M. Takamori and K. Otsuka Neurological Center, Kanazawa-Nishi Hospital and Kanazawa University, Japan In myasthenia gravis (MG), acetylcholine (ACh) release from the nerve terminal is upregulated when synaptic transmission is postsynaptically impaired by anti-acetylcholine receptor (AChR) antibodies. The mechanism of this synaptic plasticity acts via intraterminal second messengers. This depends on a cytoplasmic free Ca2+-dependent components, but is not caused via regulatory effects on the presynaptic voltage-gated Ca2+ channels (VGCCs). Independently of membrane depolarization, the presynaptic Ca2+ influx is triggered via the phospholipase C activation and phosphatidylinositide (PI) pathway which is not affected by intracellular Ca2+ stores. This focused our attention onto two mechanisms. The one is the membraneassociated Ca2+ influx channels, transient receptor potential classical subfamily-3 (TRPC3), which is activated through the pathway including brain-derived neurotrophic factor and receptor tyrosine kinase B. The other is the G-protein-coupled M1 muscarinic AChR (M1 mAChR), which is efficient to P/Q-type VGCC-mediated Ca2+ influx via the PI pathway and, in a different manner, physiologically associated with TRPC-mediated Ca2+ influx via the activation by diacylglycerol. Forty MG patients, 26 positive and 14 negative for anti-AChR antibodies, were studied for antibodies to TRPC3 peptide

26

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(extracellularly exposed segment) by ELISA, and to M1 mAChR by immunoblots using a recombinant protein. Anti-TRPC3 antibodies were detected in 36% of tested sera, and anti-M1 mAChR antibodies were found in 63% of tested sera; some positive sera were from anti-AChR-negative MG patients. The synaptic plasticity in MG is potentially disturbed by immunemediated presynaptic receptor dysfunctions. Also, our finding could provide a clue to understanding presynaptic effects of anti-AChR-negative (anti-MuSKpositive or -negative) MG plasma. CS04-01

Tim family of genes: Role in T cell differentiation, autoimmunity and tolerance Vijay K. Kuchroo Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA USA. [email protected]

expressed on TH2 cells. In the mouse, TIM-1 costimulates T cell activation and prevents the development of respiratory tolerance. In humans, TIM-1 functions as the Hepatitis A virus (HAV) cellular receptor, and infection with HAV is strongly associated with protection against atopy. However, this association occurs only in individuals who have certain polymorphic variants of TIM-1. Binding of TIM-1 by HAV or its natural ligand may affect T cell differentiation and the development of Th2 responses. Since HAV is associated with a reduced risk of developing atopy and the incidence of HAV infection has been significantly reduced in industrialized countries over the past 30 years, the discovery of a genetic interaction between HAV and TIM-1 provides the first molecular genetic evidence of the hygiene hypothesis. CS04-03

Signaling by TIM family proteins L.P. Kane and A.J. de Souza

CD4+ T helper lymphocytes can be divided into two subsets of effector cells, known as Th1 and Th2 cells, based upon their functional properties and the cytokines they produce. While much is known about the functional properties of these subsets, there are few known surface molecules that distinguish between them. We have recently identified and characterized a novel transmembrane protein, TIM-3, which contains an immunoglobulin (Ig)like and a mucin-like domain and is expressed only on differentiated Th1 cells. In vivo administration of soluble Tim3.Ig fusion protein induces hyperproliferation of Th1 responses, abrogates tolerance induction and enhances the clinical and pathologic severity of experimental autoimmune encephalomyelitis (EAE), a Th1-dependent autoimmune disease. These data suggest that Tim-3 may be an inhibitory molecule and blockade of Tim-3: Tim3L pathway by Tim3.Ig fusion protein results in the enhancement of Th1 response. Emerging data suggest that Tim.3 ligand (Tim.3L) is a critical molecule which regulates expansion and function of Th1 cells. Using the Tim3.Ig fusion protein, we have biochemically purified the Tim3L and identified it as galectin-9. Galectin-9 induces death in Th1 cells in Tim-3 dependent manner. In vivo administration of Tim3.L during an ongoing immune response, results in selective loss of IFN-g but not IL-4 or IL-10 producing cells. This data demonstrates that Tim3: TIM-3.L pathway has evolved to ensure effective termination of effector proinflammatory Th1 immunity. The role of this gene family in regulating effector functions of Th1 and Th2 cells and regulating autoimmune and allergic diseases will be discussed.

Department of Immunology, University of Pittsburgh, Pittsburgh, PA USA

CS04-02

A role of the class IV semaphorin, Sema4A in Th1/Th2 regulation

Recent studies have begun to elucidate the functional properties of the proteins of the T-cell immunoglobulin and mucin domain (TIM) family. However, little is currently known about how these proteins transmit signals to the inside of responding cells to affect function. We have been working to understand the biochemical mechanisms behind the functions of the closely related murine forms of TIM-1 and TIM-2. We have found that expression of TIM-1 provides a co-stimulatory signal that increases the activation of NFAT- and AP-1-dependent transcription. Our results suggest that tyrosine phosphorylation of TIM-1 is necessary for this effect. We are currently attempting to determine how TIM-1 affects intracellular signaling pathways that regulate NFAT and AP-1, as well as how phosphorylation of TIM-1 allows it to couple to these pathways. We have also been studying TIM-2, for which there is no apparent orthologue in humans, and found – in contrast to our results with TIM-1 – that TIM-2 expression in T cell lines leads to impaired activation of NFAT and AP-1. This effect appears to occur relatively far downstream in the TCR signaling pathway, since NFAT induction by PMA/ionomycin is also inhibited by TIM-2. We are currently working to localize this inhibitory effect more precisely. CS04-04

Role of TIM-1 in regulating allergy and tolerance R. H. DeKruyffa, J. J. McIntireb, S. E. Umetsuc and D. T. Umetsua a

Division of Immunology, Children's Hospital, Boston and Department of Pediatrics, Harvard Medical School, Boston, MA, USA; bDepartment of Pediatrics, Stanford University, Stanford, CA, USA; cDepartment of Microbiology–Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Atopic diseases, including asthma, allergic rhinitis, and atopic dermatitis, are caused by environmental factors in genetically predisposed individuals. The prevalence of these diseases has risen dramatically over the past two decades, and epidemiological studies suggest that this increase in asthma prevalence may result from improved hygiene and a reduction in the frequency of infections. Using a congenic mouse model of asthma, we simplified the analysis of this complex trait and identified the T cell immunoglobulin domain, mucin-like domain (TIM) gene family that encodes cell-surface glycoproteins. Recent studies demonstrate that the TIM family, particularly TIM-1, plays a critical role in immune responses that regulate the development of atopic disease. In the mouse, TIM-1 is expressed on CD4+ T cells after activation and is preferentially

Hitoshi Kikutani Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan Semaphorins have been originally identified as chemorepulsive cues for axon guidance during neuronal development. Several semaphorins have been recently shown to play important roles in the immune system. The class IV transmembrane semaphorin Sema4A is expressed constitutively on DCs and induced on Th1-polarized cells but not on Th2-polarized cells. Sema4AFc binds to Tim-2, a member of the Tim protein family, whose expression is induced on activated T cells, and enhances in vitro activation and differentiation of T cells. DCs derived from Sema4A−/− mice show impaired allostimulatory activities in mixed lymphocyte cultures although they can express normal levels of co-stimulatory molecules and produce various cytokines in vitro. Sema4A−/− T cells also fail to differentiate into Th1 cells when cultured with anti-CD3 and anti-CD28 in Th1-inducing conditions, although they can differentiate into Th2 cells in Th2-inducing conditions. Transfer experiments using antigen-pulsed DCs from Sema4A−/− or wildtype mice demonstrate that both DC-derived Sema4A and T cell-derived Sema4A are necessary for T cell activation and differentiation; the former is

Abstracts required for T cell priming and the latter for Th1 differentiation. The findings suggest that Sema4A can be a potential therapeutic target for Th1 and Th2 diseases. Indeed, anti-Sema4A mAb can suppress a relapse of experimental autoimmune encephalomyelitis (EAE) in SJL mice even when administered after an acute EAE has been established. In vivo administration of Sema4AFc can also suppress picrylchloride-induced atopic dermatitis (AD)-like skin lesion in not only Sema4A−/− BALB/c mice but also AD prone NC/nga mice.

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CS05-03

A challenge to auto-cell transplantation: Based on specific induction system of bone marrow stromal cells Mari Dezawa Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Kyoto, Japan

CS05-01

Olfactory ensheathing cells and spinal cord repair

Bone marrow stromal cells (MSCs) have the capability under specific conditions to differentiate into various cell types. Here we demonstrate a highly efficient and specific induction of Schwann cells (peripheral glial cells), neurons and skeletal muscle cells from both rat and human MSCs. Schwann cells, which constitute peripheral nervous system, are known to support axonal elongation. MSCs were treated with beta-mercaptoethanol followed by retinoic acid (RA) and cytokines expressed markers of Schwann cells. These cells promoted axonal regeneration, and resulted in functional recovery when transplanted into the spinal cord injury model. Neuronal induction could be achieved by NICD transfection followed by trophic factor administration of bFGF+CNTF+ forskolin. MSCs expressed markers related to neural stem cells after transfection with NICD, and subsequent administration of trophic factors induced post-mitotic neurons without differentiation of glial cells. Some of the induced cells showed action potentials compatible with characteristics of functional neurons. Further treatment of the neurons with GDNF increased the proportion of dopamineproducing cells and transplantation of these neurons showed improvement in rat model of Parkinson's disease. Highly efficient skeletal muscle induction could also be achieved by the administration of a combination of cytokines followed by NICD transfection. Induced cells differentiate into muscle fibers upon transplantation into degenerated muscles. Since induced cells contained satellite-like cells, they contributed to subsequent regeneration upon repetitive damage without additional transplantation of cells. Our MSC differentiation system may contribute substantially to a major advance toward eventual cell-based therapies for neurodegenerative and muscle degenerative diseases.

Alan Mackay-Sim

CS05-04

Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Qld, Australia

Clearance of tissue debris by TREM2-transduced myeloid cells promotes recovery of experimental autoimmune encephalomyelitis

Altered neurogenesis in response to injury or disease T. D. Palmer Department of Neurosurgery, Stanford University, USA Both in development and in the adult, neural stem/progenitor cells play active roles in forming and maintaining central nervous system function. In the adult brain, the hippocampus and olfactory bulb continue to produce neurons throughout life and as these new neural elements become functionally integrated into the pre-existing circuitry, they contribute to select cognitive functions. Both injury and disease have a significant impact on the natural process of neurogenesis in the adult hippocampus and we are utilizing both in vivo and in vitro stem cell models to determine how different microenvironments of the brain influence endogenous or transplanted stem cell activity and fate. Recent work has shown that immune system activation and inflammation critically influences neurogenesis within the hippocampus and ongoing work explores pharmaceutical immunomodulation and its ability to normalize neurogenesis and function within the pro-inflammatory context of the injured or diseased brain. CS05-02

Olfactory ensheathing cells are specialized glial cells surrounding the olfactory nerve. There is abundant evidence in animals that transplantation of olfactory ensheathing cells promote functional recovery after transplantation into the injured spinal cord including regrowth of axons across the injury site and recovery of climbing, walking, reaching, and breathing. Olfactory ensheathing cells are accessible by biopsy from the olfactory mucosa in the nose and can be grown in large numbers in vitro. We recently initiated a single-blind, Phase I clinical trial of transplantation of autologous olfactory ensheathing cells into the human paraplegic spinal cord. Participants are six male paraplegics, 18–55 years of age, with stable, complete thoracic injuries 6–32 months previously, with stable spinal column, no implanted prostheses, and no syrinx. Three participants were transplanted with autologous olfactory ensheathing cells grown from small nasal biopsies. Three participants are a control for the assessors, who are blind to treatment status. Regular assessments include MRI, medical, neurological and psychosocial assessments, and standard American Spinal Injury Association and Functional Independence Measure assessments. At one year after cell implantation, there were no indications that the procedure is unsafe, no evidence of spinal cord damage, no reported neuropathic pain, and no evidence of cyst, syrinx or tumour formation. Participants will be followed for 3 years to confirm long-term safety and to compare neurological, functional and psychosocial outcomes with the control group. We conclude transplantation of autologous olfactory ensheathing cells into the injured spinal cord is feasible and is safe up to one year postimplantation.

Kazuya Takahashi1, Marco Prinz2, and Harald Neumann1 1

Neural Regeneration Unit, Institute of Reconstructive Neurobiology, University Bonn LIFE and BRAIN Center and Hertie-Foundation, Sigmund-Freud-Str. 25, 53127 Bonn, Germany; 2Department of Neuropathology, University Hospital Göttingen, 37075 Göttingen, Germany Background: Inflammation can successfully be prevented in multiple sclerosis, but tissue repair is still a major challenge for therapy. Bone marrow or blood-derived hematopoietic myeloid precursors are suitable and accessible sources for cell-based repair therapies. Efficient removal of apoptotic cells and cellular debris without inflammation during degenerative central nervous system (CNS) diseases is essential for repair of the injured tissue and disease recovery. Methods: Bone marrow-derived myeloid precursors were expanded in culture and lentivirally transduced with the microglial innate immune receptor triggering receptor expressed on myeloid cells-2 (TREM2). TREM2-transduced myeloid cells were intravenously applied in mice afflicted by experimental autoimmune encephalomyelitis (EAE). Results: TREM2-transduced myeloid precursors showed increased phagocytic activity and anti-inflammatory cytokine production after stimulation. Intravenous application of TREM2-transduced myeloid cells at the peak of EAE led to a significant amelioration of clinical symptoms, reduction in

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axonal damage and prevention of demyelination. The TREM2-transduced myeloid cells migrated into the inflammatory spinal cord lesions within a few hours after i.v. application and locally created an anti-inflammatory cytokine milieu within the spinal cord. The TREM2 expressing myeloid cells resolved inflammation by removing cellular debris and creating a milieu supportive for repair of injured tissue. Conclusion: Data demonstrate that TREM2 is involved in tissue debris clearance and resolution of inflammation in EAE. Furthermore, we identified TREM2 as a novel target for the therapy of inflammatory autoimmune brain diseases.

differentiation by inhibiting ERK signaling. However, while oral simvastatin inhibits T cell proliferation, it is less effective in inducing Th1/2 modulation. These individual differences may be important when choosing a statin for testing in human autoimmune conditions. Based primarily upon the beneficial immune modulation of atorvastatin in EAE, we are conducting a multi-center, randomized, placebocontrolled study to determine whether oral atorvastin (Lipitor®) (80 mg) will reduce the risk of conversion to clinically definite MS in patients that have experienced a clinically isolated syndrome (CIS). This trial, which is funded by the Immune Tolerance Network (ITN) of the NIH is actively recruiting patients.

CS06-01 CS06-03

B cells and antibodies in multiple sclerosis Bernhard Hemmer Neuroimmunology Group, Department of Neurology, Heinrich HeineUniversity, Düsseldorf, Germany Multiple sclerosis (MS) is a chronic disease of the CNS that is characterized by inflammation, demyelination and axonal injury. Although the etiology of MS is still unknown, many findings point to a central role for the immune system in the pathogenesis of the disease. The occurrence of local antibody synthesis in CSF demonstrated by the presence oligoclonal bands or intrathecal IgG production is still the only diagnostic laboratory marker in MS. Paradoxically, however, B cells were neglected in MS research for decades owing to their dispensable role in EAE and the lack of suitable technology, and it is only recently that their impact in MS pathogenesis has been thoroughly studied. B cells from MS lesions or CSF comprise dominant clonotypes containing replacement mutations in their Bcell receptor genes, compatible with an antigen-driven selection process. The same B-cell clonotypes are found in the CSF during the course of the disease, implying that they are periodically recruited, or that they persist in the CNS compartment. This model is compatible with other CSF parameters. Immunoglobulin (predominantly IgG1) is released in CSF and lesions of MS patients. B-cell cytokines such as BAFF are also detected in MS lesions. Plasma blasts and plasma cells – terminally differentiated B cells that are usually only found in acute infectious diseases – can be identified in the CSF compartment of MS patients. Their number correlates not only with local IgG synthesis, but also with the extent of CNS inflammation. Although these findings strongly support a role for the humoral immune response in MS, the specificity and function of this response remains to be determined. CS06-02

Immunomodulation by statins and their potential role in MS therapy Scott S. Zamvil

Recruitment of regulatory T-cells to treat EAE Ralf Gold1, Shin-Young Na2, Fred Lühder1, Thomas Hünig2 1

Institute for MS Research, University of Göttingen, D-37073 Göttingen, Germany; 2Institute for Virology and Immunobiology, University of Würzburg, Germany

CD4+CD25+ regulatory T cells (Treg cells) play a key role in controlling autoimmunity and inflammation. Therefore, therapeutic agents capable of elevating numbers and/or increasing effector functions of this T cell subset are highly desirable. In a previous report we showed that a superagonistic monoclonal antibody specific for rat-CD28 (JJ316) expands and activates FoxP3+ Treg cells in vivo. Single intravenous administration of a low dose of the CD28 superagonist into either DA rats or Lewis rats suffering from experimental autoimmune encephalomyelitis (EAE) proved to be highly efficacious. In further experiments a novel, spontaneous mouse EAE mediated by CD8− T cells was treated with Treg cells. In transgenic mice expressing the model antigen ovalbumin (OVA) in oligodendrocytes (ODC), CD8, but not CD4 T-cells with transgenic OVA-specific T-cell receptors (TCR) caused fulminant EAE. Spontaneous development of severe EAE mainly affected cerebellum and brainstem, suggesting the name ‘Charcot mouse’ for this specific phenotype. Transfer of polyclonal Treg cells activated by a novel mouse CD28-specific superagonistic monoclonal antibody (D665) protected all animals from lethal EAE. In surviving mice, transferred regulatory T-cells were found in the affected regions of the CNS. brain and spinal cord. Our data indicate that polyclonal Treg cells have therapeutic efficacy in CD4 and CD8 mediated rodent EAE. CS06-04

Regulatory T cell responses induced by T cell vaccination in patients with multiple sclerosis J. Zhanga,b, J. Honga,b and Y. C. Q. Zangb a

Department of Neurology and Program in Immunology, University of California, San Francisco, San Francisco, CA, USA Statins, which are widely prescribed for cholesterol reduction, have immunomodulatory properties that may be beneficial in treatment of MS and other Th1 mediated autoimmune diseases. Data indicate that immunomodulation by statins occurs in a cholesterol-independent manner. Recently, we have connected isoprenoid intermediates of the mevalonate pathway with signaling pathways in Th1/2 cell differentiation. In vivo depletion of the isoprenoid intermediates geranylgeranyl-pyrophosphate (GGPP) and farnesyl-PP by oral atorvastatin inhibited Th1 responses by decreasing geranylgeranylated RhoA and farnesylated Ras at the plasma membrane, which was associated with reduced phosphorylation of ERK and p38, and reduced DNA binding of their co-target, c-fos. Our recent data indicate that oral atorvastatin and rosuvastatin are highly potent in inducing Th2

Joint Immunology Laboratory of Institute of Health Sciences and Shanghai Institute of Immunology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences and Shanghai JiaoTong University School of Medicine, Shanghai, China; bDepartment of Neurology, Baylor College of Medicine, Houston, USA

Immunization with irradiated autologous autoreactive T cells (T cell vaccination) is shown to induce regulatory T cell responses in patients with multiple sclerosis (MS). The regulatory T cell reactivity is thought to contribute to suppression and depletion of circulating myelin-reactive T cells used for vaccination. Here, the characteristics and regulatory mechanism of CD8+ anti-idiotypic T cells and CD4+ regulatory T cells induced by T cell vaccination will be reviewed, with an emphasis on newly characterized CD4+ regulatory T cells (PNAS, 103, 5024–5029, 2006). These CD4+ regulatory T cell lines were generated from patients with MS that received immunization with irradiated autologous myelin basic protein (MBP)-

Abstracts reactive T cells. CD4+ regulatory T cell lines had marked inhibition on autologous MBP-reactive T cells and displayed two distinctive patterns distinguishable by the expression of transcription factor Foxp3 and cytokine profile. The majority of the T cell lines had high Foxp3 expression and secreted both interferon-γ and IL-10 as compared to the other pattern characteristic of low Foxp3 expression and predominant production of IL-10 but not interferon-γ. CD4+ regulatory T cell lines of both patterns expressed CD25 and reacted with activated autologous T cells but not resting T cells, irrespective of antigen specificity of the target T cells. It was evident that they recognized preferentially a synthetic peptide corresponding to residues 61–73 of the IL-2 receptor alpha-chain. T cell vaccination correlated with increased Foxp3 expression and T cell reactivity to peptide 61–73. The findings have important implication in the understanding of the role of CD4+ regulatory T cell response induced by T cell vaccination. CS07-01

Innate signaling for CNS glial responses Trevor Owens Medical Biotechnology Center, University of Southern Denmark, Odense, Denmark Signals from both the central nervous system (CNS) and immune system contribute to pathology in inflammatory demyelinating diseases such as multiple sclerosis. We study innate signals for glial response, that are both exogenouslyand endogenously-derived. This presentation will describe the activation of glial cells in the mouse brain, in response to axonal injury, cuprizone demyelination and cytokine-expressing adenovirus. Glial activation in response to these experimental stimuli is initiated independently of immune involvement, and results in chemokine and cytokine secretion. Injury-induced microglial Toll-like receptor-2 signals act as upstream on-switches for glial cytokine and chemokine responses, and microglial proliferative expansion. Whereas injury- or cuprizone demyelination-induced glial cytokines and chemokines appear sufficient for entry of macrophages and T cells to the CNS, leukocyte entry in response to transgenic or adenoviral interferon gamma-induced chemokine production requires additional signals, that can be provided through recognition of the pathogen-derived innate trigger, pertussis toxin. Glial cross-talk is mediated by cytokines, with characteristic intracellular signals. Activated microglia and macrophages show antigen-presenting cell surface phenotypes, and can activate T cells in culture. However, the T cells that enter the CNS, in the absence of deliberate immunization, require signals additional to chemokines and cytokines for functional activation. Our findings identify a complex of signaling events that must combine for immune pathology in the CNS. CS07-02

Toll-like receptor 3 on astrocytes mediates neuroprotective responses and can be activated by an endogenous protein agonist J.M. van Noort, M. Bsibsi, E. van Duijvenvoorden, C. Persoon-Deen, M. Vogt, A. Baghat TNO Quality of Life, Leiden, The Netherlands Toll-like receptors (TLRs) are expressed by a variety of cell types and function as sensors of microbial structures such as endotoxins, peptidoglycans and nucleic acids. In response to such molecules TLRs generally activate pro-inflammatory host defense responses. Human astrocytes express a limited repertoire of TLRs that are only found on the cell surface. In particular TLR3 appears to be special to astrocytes since it is strongly and preferentially upregulated in response to a variety of stimuli including cytokines, oxidative stress and TLR agonists themselves. Gene profiling of the response of astrocytes to TLR3 activation revealed a powerful response dominated by anti-inflammatory cytokines,

29

neuroprotective molecules, remyelination signals and several chemokines. Functional tests confirmed the ability of the collection of TLR3-induced astrocyte products to enhance survival of neurons in organotypic human brain slice cultures. Given this apparent tissue repair function of surfaceexposed TLR3 on astrocytes we examined the possible presence of an endogenous TLR3 agonist in the central nervous system. Indeed, genomescale screening of a brain tumor-derived cDNA bank revealed a protein that is preferentially expressed in the human CNS and can activate a TLR3-dependent response by astrocytes. Details of this protein and its possible role in TLR3-mediated control of CNS inflammation and repair will be presented and discussed. CS07-03

Dendritic cells and lymphoid microenvironments in the inflamed CNS Francesca Aloisi1, Barbara Serafini1, Roberta Magliozzi1,2, Abhilash Vora, Richard Reynolds2 1

Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy; 2Department of Cellular and Molecular Neuroscience, Imperial College London, London, UK Chronic inflammatory diseases, such as autoimmune and infectious diseases, are associated with repeated recruitment and persistence of immune cells in the target tissue. Evidence is accumulating that, even within an immune privileged site such as the central nervous system (CNS), chronic inflammation leads to the organization of lymphoid microenvironments that may contribute to sustain a local immune response against microbial or self antigens. Dendritic cells (DC) recruited to the inflamed CNS are thought to play a key role in this process. In multiple sclerosis and its animal model EAE, the perivascular space of inflamed blood vessels in the white matter is the preferential site where dendritic cell accumulate, take up myelin components and interact with CNS-infiltrating lymphocytes. The inflamed meninges provide an even more supportive environment for the generation of ectopic lymphoid-like tissue. Here, myeloid and plasmacytoid DC can be detected and B-cell follicles with follicular dendritic cells (FDC) develop in a substantial proportion of patients with secondary progressive MS. FDC, which are of mesenchymal origin, have a central role in the selection of memory B cells during germinal center reactions. Remarkably, development of ectopic lymphoid tissue in MS is associated with extensive cortical demyelination and an aggressive clinical course. It is proposed that ‘seeding’ of the brain by lymphocytes and antigen presenting cells and their organization in lymphoid-like structures exacerbates the inflammatory process as a consequence of an increased availability of pathogenic, presumably autoreactive, lymphocytes and their products. Prevention or eradication of lymphoid microenvironments nested within the CNS should be considered as an important goal for therapeutic intervention. CS07-04

Innate immunity and bone marrow stem cells in brain diseases and repair Serge Rivest Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University 2705 Laurier Boulevard, Québec, Canada G1V 4G2 An elegant innate immune response takes place in an organized fashion in the CNS during systemic bacterial/viral infection. This inflammatory response, characterized by the expression of toll-like

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receptors (TLR), cytokines, chemokines and proteins of the complement system in the circumventricular organs and other structures devoid of blood–brain barrier, diffuses progressively into microglia across the brain parenchyma and may lead to an adaptive immune response. The cerebral innate immunity is likely to be an essential player in the etiology of inflammatory CNS disorders resulting from infection as well as those assumed to have an immune etiology. On the other hand, molecules of the innate immunity have been found to trigger the production of neurotrophic factors and promote neurorepair and remyelination in response to brain injuries, trauma and toxin-induced demyelination. Exciting new data will also be presented in regard to the role of newly recruited microglia from the bone marrow stem cells in CNS diseases, such as Alzheimer's Disease (AD). This disease is characterized by the deposition of aggregated beta-amyloids that form amyloid plaques. These senile plaques are composed of microglia, which infiltrate the core of amyloid bodies and these cells have different origins. Those deriving from the bone marrow stem cells have the ability to eliminate the plaques and inhibition of this process was found to aggravate the disease. These data provide novel insights for the development of new therapeutic strategies using bone marrow stem cells as vehicle for gene therapy and the elimination of toxic senile plaques. Our work on this subject is currently funded by the Canadian Institutes of Health Research (CIHR). CS08-01

Going solo or in need of a friend: Ganglioside and lipid clusters form novel antigenic determinations in GBS S. Kusunoki Kinki Univeristy School of Medicine, Osaka, Japan

Department of Neurology, Dokkyo Medical University School of Medicine, Tochigi, Japan Ganglioside mimicry of Campylobacter jejuni lipo-oligosaccharide (LOS) is a cause of Guillain–Barré syndrome (GBS). GM1-like and GD1alike LOSs are synthesized by Campylobacter sialyltransferase Cst-II, Nacetylgalactosaminyl-transferase CgtA, and galactosyltransferase CgtB. C. jejuni is grouped into several LOS locus classes based on the organization of the LOS biosynthesis genes. Isolates from GBS patients belonged to classes containing cgtA, B, and cst-II genes, which are essential for the biosynthesis of ganglioside-like LOS. Now we have identified the C. jejuni genes responsible for the development of GBS. Cst-II consists of 291 amino acids. The fifty-first of the amino acid determines its enzymatic activity. Cst-II (Thr51) can make GM1-like and GD1a-like LOSs, whereas Cst-II (Asn51) can make GT1a-like LOS. Neuropathic strains with Cst-II (Thr51) expressed the GM1 and GD1a epitopes, whereas the strains with Cst-II (Thr51) expressed the GQ1b epitope. Patients infected with Cst-II (Thr51) strains had anti-GM1 or anti-GD1a antibodies, and the patients showed limb weakness. Patients infected with Cst-II (Asn51) strains had anti-GQ1b antibodies, and the patients showed ophthalmoplegia and ataxia. In summary, Cst-II (Thr51) strains can make GM1-like and GD1a-like LOSs, and induce the production of anti-GM1 or anti-GD1a antibodies. Then the patients develop limb weakness. Cst-II (Asn51) strains can make GT1a-like LOS, and induce the production of anti-GQ1b antibodies. Then the patients develop ophthalmoplegia and ataxia. In other words, the Cst-II polymorphism determines the clinical presentation, GBS or Fisher syndrome. Now we have proposed a new paradigm that microbial genetic polymorphism can determine the clinical presentation of human autoimmune disease. CS08-03

Modeling GBS from man to mouse: How pathology informs pathogenesis

Anti-ganglioside antibody titers are frequently elevated in the acute phase sera from patients with Guillain–Barré syndrome (GBS). They are useful diagnostic markers as well as possible pathogenetic factors of GBS. Antibody assay has been usually performed by ELISA, in which each purified ganglioside is used as an antigen. However, because each ganglioside is present in the plasma membrane, where it is colocalized with phospholipids and other gangliosides to form lipid rafts, we should pay attention on the effects of such other lipid molecules. We recently performed ELISA by the use of a mixture antigen composed of a ganglioside and a phospholipid. As a result, anti-GM1-positive sera had higher antibody titers against a mixture of GM1 and several phospholipids as phosphatidic acid than against GM1 alone. In contrast, no phospholipid provided significant enhancement on the anti-GQ1b antibody titers. Sphingomyelin provided decrease of the activity for both anti-GM1 and Anti-GQ1b antibodies. We next used a mixture antigen composed of two different gangliosides and found that some GBS sera had antibodies specific for a ganglioside complex (GSC), which is a conformational epitope formed by two gangliosides such as GM1 and GD1a (GM1/GD1a), GD1a/GD1b, GD1b/GT1b and GM1/GT1b. AntiGSC-positive GBS were featured by antecedent gastrointestinal infection and lower cranial nerve deficits. Among the anti-GSC antibodies, antiGD1a/GD1b and/or anti-GD1b/GT1b antibodies were associated with severe disability and a requirement of mechanical ventilation. They may be predictors of severe GBS. We should consider clustered antigenic epitopes to determine the pathogenetic role of antiganglioside antibodies in GBS.

K.A. Sheikh

CS08-02

Neuroimmunology Group, University Department of Neurology, Institute of Neurological Sciences, Southern General Hospital, Glasgow, Scotland G51 4TF

It's all in the genes: Campylobacter polymorphisms direct the clinical features of GBS N. Yuki

Department of Neurology, Johns Hopkins University, Baltimore, USA Guillain–Barré syndrome (GBS) comprises a group of clinically and pathophysiologically related, acute monophasic neuropathic disorders of autoimmune origin. Anti-ganglioside antibodies (Abs) are most frequently recognized autoimmune responses in this group of neuropathies. Abs with GM1 and GD1a specificity are reported to associate with motor axonal variant of GBS and poor recovery in both axonal and demyelinating forms of the disease. Acute motor axonal neuropathy (AMAN) variant of GBS is the most frequent form in northern China, and it has a high frequency in the developing world and in Japan. Physiological, pathological, and immunopathological studies in the AMAN form indicate Ab-mediated axonal injury or dysfunction. Our laboratory is developing mouse models of this human disease. The pathobiologic effects of anti-ganglioside Abs on nerve fibers will be discussed. These translational studies may help to elucidate pathogenetic mechanisms involved in immune antibody-mediated axonal injury. Acknowledgements: Research supported by NINDS and GBS Foundation. CS08-04

Changing the shape of GBS therapy: Translating information from models to clinical practice H. J. Willison

Guillain Barré syndrome (GBS) is the prototypic autoimmune neuropathy and the foremost cause of neuromuscular paralysis worldwide with a global

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incidence of ∼1.5/105. Onset can be rapid, leading to total paralysis within 48 h. The patients left severely disabled or dead represent a major social and economic burden. Recent clinical–serological studies have identified serum anti-ganglioside antibodies in GBS cases and chronic neuropathies. Gangliosides are sialic acid-containing glycosphingolipids highly enriched in the nervous system. Anti-GM1 and -GD1a antibodies characterise the motor axonal form of GBS and anti-GQ1b antibodies characterise Miller Fisher syndrome (MFS). In murine models, we have shown that anti-ganglioside antibodies target gangliosides in neuronal and glial membranes, including motor nerve terminals. Morphologically, dense antibody and complement deposits are found in the synaptic cleft and on perisynaptic Schwann cells (pSCs), alongside a loss of neurofilament with destruction of the nerve terminal, and pSC death. Manipulating ganglioside levels in GM2/GD2 synthase and GD3 synthase null-mutant can enhance or attenuate disease. The terminal attack complex of complement is critical to development of acute neural injury by allowing calcium ingress, activation of calpain and degradation of the nerve terminal. Both calcium depletion and calpain inhibition can protect the cytoskeleton from degradation. Human immunoglobulin therapy also protects against nerve injury through anti-idiotypic effects. Inhibition of complement activation is also able to attenuate injury. These studies show that nerve terminal gangliosides can act as targets for human disease-associated autoantibodies and that this site provides a useful model system for testing new pathogenesis paradigms and therapeutic strategies.

The development of new therapeutics for autoimmune disorders will benefit from an understanding of the natural regulatory mechanisms that can promote resolution of disease. With this in mind, it is notable the multiple sclerosis (MS) often follows a benign “relapsing remitting” course. We are investigating the regulation of the mouse model of MS — experimental autoimmune encephalomyelitis (EAE). The disease in C57BL/6 mice peaks in the second week after immunization with the CNS autoantigenic peptide, MOG(35–55). A recovery phase follows, correlating with a specific accumulation of CD4+CD25+ cells within the CNS. These CNS-derived Tregs preferentially produce IL-10, express FoxP3, are anergic, act as potent suppressors of responder CD25− cells in vitro, and their transfer in vivo accelerates recovery from EAE in recipient mice. CD25− depletion exacerbates disease, and delays recovery. Moreover, CD25− depletion after recovery also renders mice fully susceptible to re-induction of EAE. Data will be presented relating to origins, mode of action and specificity of the CNS Treg cells. Our data highlight the key role of Tregs in the natural recovery from an ongoing inflammatory autoimmune disease and in subsequent resistance, and that their effects appear to be within the target organ. Thus there is real potential for Treg-directed therapies to boost natural regulation of active autoimmune disease.

CS09-01

CS09-03

Regulating myelin basic protein-specific T cell responses in vivo a

a

a,b

a

c

J. Goverman , S. Cabbage , E.S. Huseby , B. Sather and T. Brabb a

Department of Immunology, University of Washington, Seattle, WA, USA; bPresent address: Howard Hughes Medical Institute, Department of Immunology, National Jewish Medical Center, Denver, CO, USA; c Department of Comparative Medicine, University of Washington, Seattle, WA, USA

A loss of tolerance in myelin-specific T cells is believed to contribute to the pathogenesis of multiple sclerosis. Our previous studies showed that CD4+ T cells specific for myelin basic protein (MBP)121–140, a highly immunogenic MBP epitope in B10.PL mice, are largely tolerized by clonal deletion in the thymus. However, the developmentally regulated expression of MBP allows MBP121–140-specific T cells to escape central tolerance in young mice when little MBP is synthesized. We defined the peripheral tolerance mechanisms that prevent these T cells from mediating autoimmunity later in life when they encounter MBP in the periphery. Regulatory T cells are essential to prevent MBP121–140-specific T cell mediated autoimmunity. Regulatory T cells do not prevent the proliferation, expansion or trafficking of MBP-specific T cells transferred into wild-type mice. Instead, Th1 cytokine production by MBP-specific T cells was strongly suppressed. Suppression of Th1 cytokines and protection from autoimmunity was abrogated if endogenous antigen-presenting cells (APCs) were activated during the first week after transfer of MBP-specific T cells into recipients. Interestingly, activating APCs thirty days after transfer of the MBP-specific T cells no longer triggered autoimmunity. Instead, the MBPspecific T cells that persisted in the periphery of wild-type mice responded to this stimulus by further suppressing their Th1 cytokine responses. The tolerant phenotype of MBP-specific T cells persisting in wild-type mice was dependent on both T cell intrinsic and host factors, and did not represent a terminally differentiated state as re-transfer of the regulated MBP-specific T cells into T cell-deficient recipients caused autoimmune disease. CS09-02

Natural regulation of CNS autoimmune disease Richard O'Connor, Katy Malpass, Mandy J. McGeachy and Stephen M. Anderton

Institute for Immunology and Infection Research, University of Edinburgh, King's Buildings, Edinburgh, UK, EH9 3JT UK

Human natural Tregs are composed of two functionally distinct subsets of cells based on expression of MHC Class II ex vivo C. M. Baecher-Allan, C. Ashley, C. Costantino, G. Beriou, and H. A. Hafler Harvard Medical School and Brigham and Women's Hospital, Boston, MA, USA Natural CD25high regulatory (Treg) populations, isolated from patients with multiple sclerosis (MS), have been shown to be decreased in their suppressive ability as compared to Tregs isolated from healthy donors. We have subsequently found that CD25high Treg population consists of two functionally distinct suppressive subsets that can be identified by direct ex vivo expression of HLA Class II (HLA DR). In these studies, the Tregs that express HLA DR (DR+ Tregs) exhibit a strong and swift suppression while the DRneg Tregs induce a delayed inhibition accompanied by suppression of Th1 but increases in Th2 cytokines. The DR+ and DRneg Tregs also exhibit striking differences in their production and utilization of IL-10. While both DR+ and DRneg Tregs express high levels of FoxP3 and exhibit a strong cell contact-dependent suppression, DRneg Tregs can also suppress via the production of IL-10, which is not produced by the DR+ Tregs. In contrast, the DR+ Tregs express the highest levels of IL-10R, and target cell production of IL-10 inhibits DR+ Treg regulatory function, resulting in increased co-culture proliferation. Whether IL-10 inhibition of DR+ Treg suppression occurs by reducing the suppressive ability of the DR+ Treg or by increasing the resistance of responder T cell to suppression is under investigation. Additional studies are also underway to assess the activity and IL-10 sensitivity of the individual DR+ and DRneg Treg subsets isolated from patients with MS to elucidate the mechanism for the deficiency in suppression by the total Treg population when isolated from patients with MS. CS09-04

Foxp3-expressing regulatory T cells in self-tolerance and autoimmune disease S. Sakaguchi, N. Sugimoto, M. Ono, T. Oida, K. Hirota, T. Yamaguchi, and T. Nomura Kyoto University, Institute for Frontier Medical Sciences, Kyoto, Japan

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Abstracts

Naturally occurring CD25+CD4+ regulatory T cells (Treg) actively engage in the maintenance of immunologic self-tolerance and immunoregulation. They specifically express the transcription factor Foxp3 as a master control molecule for their development and function. Although several cell surface molecules have been reported as Treg-specific markers, such as CD25, GITR (glucocorticoid-induced TNF receptor family-related gene/protein), and CTLA-4, they are also expressed on activated T cells derived from CD25−CD4+ naïve T cells. To identify Treg-specific molecules controlled by Foxp3, we performed DNA microarray analysis and found that several genes, including Gpr83, Ecm1, and helios, are specifically expressed by natural Tregs, some of which are Foxp3-dependent. We will discuss the roles of these gene products in natural Tregs and how Foxp3 controls their expression.

responses. Immunohistochemistry studies performed on brain sections of MS patients also reveal the presence of CD83high, HLA-DRhigh, CD123high and DCSIGNhigh expressing cells in acute lesions. Finally, we show that the in vitro differentiation of monocytes into DCs can be inhibited by blocking HBECs production of GM-CSF and TGF beta. Our data support the notion that unique populations of immature and mature DCs arise from migration of peripheral blood CD14 monocytes across the human BBB through the concerted actions of MCP-1, TGF beta and GM-CSF.

CS10-01

Elga de Vries1, Gerty Schreibelt1, Gijs Kooij1, J. Hendriks1, Christien Dijkstra1, Eric Ronken2

Molecular mechanisms of T cell migration across the BBB in vivo Britta Engelhardt Theodor Kocher Institute, University of Bern, Bern, Switzerland In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood–brain barrier (BBB) and gain access to the central nervous system (CNS). It is well established that α4-integrins are actively involved in leukocyte recruitment across the BBB. In contrast, the involvement of the selectins, L-selectin, E- and P-selectin and their respective carbohydrate ligands such as PSGL-1 in this process has been controversially discussed. We have demonstrated that antibodies directed against E- and P-selectin or their major leukocyte ligand PSGL-1 do not interfere with the recruitment of inflammatory cells across the BBB or the development of clinical EAE. We have now extended these studies and demonstrate that lack of E- and Pselectin in E/P-selectin-deficient mice or lack of L-selectin in L-selectindeficient mice does not prevent leukocyte migration across the BBB and the development of clinical EAE. Taken together, our findings suggest that selectins are not required for leukocyte recruitment across the BBB during EAE and point to a predominant role of α4-integrins in this process. Our findings will be discussed in light of the potential risks versus benefits of anti-α4-integrin therapy of multiple sclerosis. CS10-02

Transmigration of human CD14 monocytes across the blood brain barrier induces their differentiation into dendritic cells

CS10-03

Reactive oxygen species induce alterations in blood–brain barrier integrity

1

Department of Molecular Cell Biology and Immunology, VU University Medical Center Amsterdam; 2Solvay Pharmaceutical Research Laboratories, Weesp, The Netherlands In multiple sclerosis (MS), migration of monocytes across the blood– brain barrier (BBB) is a crucial step in the formation of lesions in the central nervous system (CNS). To enter the CNS, monocytes have to cross the BBB, during which reactive oxygen species (ROS) play an essential role. Previously we showed in the animal model for MS, experimental allergic encephalomyelitis (EAE) that the anti-oxidant lipoic acid (LA) and the flavonoid luteolin suppressed the development of clinical signs and prevented new relapses, which was coupled to reduced cellular infiltration, axonal damage and oxidative stress. Our recent data demonstrate that monocyte adhesion to cultures of brain endothelium induces production of ROS by monocytes within minutes and monocyte adhesion to a model for the BBB in vitro leads to an enhanced permeability. Exogenously administered ROS specifically activates the signalling pathway of PI-3 kinase within the brain endothelium, subsequently inducing cytoskeleton and tight junction changes. Using life cell imaging techniques, we were able to visualize that superoxide in time induces bundles of F-actin and alterations in the integrity of the tight junction. Besides its modulating effect on the integrity of the brain endothelium, superoxide also induces specific alteration in brain endothelial gene and protein expression, revealing the upregulation of markers of oxidative stress and inflammation. Our results indicate that anti-oxidants may be suitable as a new therapy for MS not only to limit cellular infiltration into the brain but also by stabilization of the BBB to resist an inflammatory attack. CS10-04

Igal Ifergan and Alexandre Prat

Chemokines and BBB function University of Montreal, Neuroimmunology Laboratory, CHUM-Notre-Dame Hospital, Montreal, Quebec, Canada The blood–brain barrier (BBB) plays an important role in regulating transmigration of immune cells into the central nervous system (CNS). Disruption of the BBB and trafficking of auto-reactive T cells and antigen presenting cells into the CNS are seen as important and early events in lesion development in Multiple Sclerosis (MS). However, to date there is still much controversy surrounding the presence and recruitment of dendritic cells (DCs) within the human brain. We elected to demonstrate that human peripheral blood CD14 monocytes cross the BBB and, by doing so, differentiate into a DC-like phenotype. Using our in vitro model of trans-human brain endothelial cells (HBECs) migration, we identified chemokine MCP-1 as the main chemoattractant for monocytes migration across the BBB. We further demonstrate that the presence of HBECs during the inflammatory process leads to differentiation of monocytes into immature (HLA-DRlow, CD80low, CD86low and CD83neg) and mature (HLA-DRhigh, CD80high, CD86high, DC-SIGNhigh and CD83high) DC phenotypes. Such DCs have the capacity to initiate CD4 T cell immune

A.V. Andjelkovic1,2, S.M.Stamatovic1, O.B. Dimitrijevic1, R.F. Keep2,3, S.L. Kunkel1 Department of Pathology, Neurosurgery and Integrative Physiology, University of Michigan, Ann Arbor, MI USA Blood–brain barrier (BBB) disruption occurs in various inflammationassociated neuropathological conditions such as stroke, multiple sclerosis and Alzheimer's disease. The central events that take place during BBB disruption are remodeling of the brain endothelial cell surface, vascular endothelial cell contraction, altered distribution of endothelial intercellular junction proteins and increased BBB permeability with consequent extravasations of leukocytes and other blood components. Chemokines play a critical role in regulation of inflammatory process at BBB mostly by mediating the leukocytes trafficking. More recently, using in vitro and in vivo models of brain ischemia–reperfusion injury, we have demonstrated that chemokines (particularly monocyte chemoattractant protein-1 (MCP-1/

Abstracts CCL2), can also cause BBB ‘opening’ by inducing interendothelial junction protein redistribution and endothelial actin cytoskeleton rearrangement. The effect of CCL2 on BBB integrity is mediated by at least two distinct signaling pathways: RhoGTPase (RhoA) and PKCs (PKCα and PKCζ). Further examination revealed that endocytotic internalization of transmembrane tight junction proteins (occludin, claudin-5 and JAM-1) stimulated by CCL2 is important for disassembly of complex endothelial tight junctions as well as paracellular route formation. However, the fate of such internalized proteins is recycling back to the plasma membrane. Collectively, these data provide new information on the underlying mechanisms through which chemokines induce BBB disruption that is relevant to multiple disease states and hopefully, will provide insight about methods to preventing such a disruption. CS10-05

Effect of statins on lymphocyte migration across the blood– brain barrier J. Greenwood Institute of Ophthalmology, London, UK Lymphocyte adhesion to brain endothelial cells (EC) is known to initiate a number of signaling cascades, the physiological significance of which remains poorly defined. However, signaling generated through engagement of CNS EC surface adhesion molecules, such as ICAM-1 and VCAM-1, or through an unidentified G-protein coupled receptor, has been shown to be essential for successful lymphocyte transendothelial migration. The identification of key components of these EC pathways raises the possibility that targeted pharmacological intervention may provide us with novel antiinflammatory strategies for the treatment of diseases such as multiple sclerosis (MS). Efficient transduction of ICAM-1 mediated signaling responses in CNS EC, and subsequent transendothelial migration of Tlymphocytes, is critically dependent on the activation of the small GTPase Rho. Pharmacological inhibition of Rho proteins can be achieved by preventing their posttranslational prenylation through inhibition of the cholesterol synthesis pathway that provides isoprenoid pyrophosphates. We have explored the effect of statins, potent inhibitors of the enzyme HMGCoA reductase which is the rate limiting step of cholesterol synthesis, upon ICAM-1-mediated signaling pathways and transendothelial lymphocyte migration in vitro and neuroinflammatory disease in vivo. We have shown that statin treatment of CNS EC inhibits lymphocyte migration and attenuates experimental CNS autoimmune disease. Data presented will further support the pleiotropic action of statins and their potential use in treating MS. CS11-01

Identification of a novel cytokine produced by encephalitogenic T cells and induced by IL-23 Burkhard Becher Division of Neuroimmunology, University of Zurich, Winterthurer Strasse 190 CH-8057 Zürich, Switzerland Tissue-directed autoimmune diseases were widely held to be mediated by the conduct of self-reactive TH cells. We and others have previously shown that neither Th1 nor Th2 gene-expression serves as a reliable marker for encephalo-pathogenicity. In contrast to the APC-derived cytokine IL-12, IL-23 was shown to be absolutely critical for the induction of autoimmune disease. It becomes increasingly evident that IL23 supports the expansion of a distinct lineage of CD4+ cells that produce IL-17 (ThIL-17 cells) and appear play a major role in autoimmune inflammation. Using high density transcriptomics, we could identify an additional gene specifically induced by IL-23 in memory T cells. We

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discovered an IL-10 related cytokine whose expression profile is identical with that of the disease associated IL-17. Its expression correlates exquisitely with that of IL17 pointing towards a major role of this illdescribed cytokine in inflammatory disease. Taken together, this molecule will help to define the encephalopathogenic phenotype of autoaggressive effector TH cells in the context of Neuroimmune disease. CS11-02

Chemokines in the nervous system: Beyond chemoattraction R.M. Ransohoff Neuroinflammation Research Center, Cleveland Clinic, Cleveland, OH, USA Inflammation of the central nervous system (CNS) entails the activation of resident microglia and recruitment of hematogenous leukocytes. Thus defined, inflammation accompanies most neurological disorders, including multiple sclerosis (MS), stroke, neoplasia, trauma and HIV-1-associated dementia, as well as Alzheimer's disease and other primary neurodegenerations. Chemokines comprise a family of peptides that act through G proteincoupled receptors (GPCRs) to regulate leukocyte migration throughout all tissues, in an exquisitely specific and flexible fashion. Initial studies asked how chemokines and chemokine receptors governed inflammatory cell recruitment to the CNS during immune-mediated or virus-induced inflammation. More recently, it has become clear that the CNS complement of constitutive chemokines supports developmental and neurophysiological functions as well as regulating the activation of microglia. Because GPCRs can serve as drug targets, these results have implications for the understanding and treatment of disease by neurologists. Supported by the US National Institutes of Health (NS38667; NS32151; NS54100), by the National Multiple Sclerosis Society (USA); by the Charles A. Dana Foundation; by the Robert Packard Center for ALS Research; and by the Nancy Davis Center Without Walls. CS11-03

Transferable tolerance by modified dendritic cells in experimental autoimmune encephalomyelitis S. J. Khoury Brigham and Women's Hospital, and Harvard Medical School, Boston, MA, USA Dendritic cells are potent antigen presenting cells (APCs) that can mediate tolerance or immunity depending on their state of maturation. Ex-vivo incubation of DCs with CTLA4Ig and MOG peptide alters the biological function of these cells rendering them tolerogenic. Ex-vivo modified DCs have the characteristics of immature DCs, with decreased expression of MHC II, CD80 and CD86, and cytokine production. We show that B7 molecules expressed by DCs are required for the tolerogenic effect, but not Fc-gamma receptors, and the mechanism of protection is independent of indoleamine 2,3dioxygenase. Tolerogenic DCs exhibit decreased expression of surface CD80, CD86, MHC II molecules, and cytokine production. Interestingly, protection from EAE is transferable by APCs but not T cells from the original recipients. Conclusion: Ex-vivo exposure of DCs to tolerizing regimen such as CTLA4Ig may provide a novel therapeutic option with fewer systemic side effects for autoimmune diseases. CS11-04

Transcriptional regulation of the IL-23/IL-17 immune axis during CNS autoimmune inflammation Daniel J. Cua

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Abstracts

Schering-Plough Biopharma, DNAX Research, 901 California Avenue, Palo Alto, CA 94304-1104, USA TH17 is a novel T cell subset driven by TGF-β, IL-6 and IL-23 that has potent pro-inflammatory activities. While TGF-β and IL-6 are sufficient for differentiation of naïve T cells into IL-17 producing cells, IL-23 is required for their expansion, survival and pro-encephalitogenic functions. The hypothesis that TH17 is a distinct T cell lineage predicts that TGF-β and/or IL-6 must promote the expression of a transcription regulator that controls the immune repertoire of the IL-23/IL-17 immune axis. Using Affymetrix gene-expression analysis, we found a number of TH17 cell-specific novel genes encoding putative proteins with potential DNA binding activities. Genetic depletion experiments confirmed that one of these DNA binding proteins, orphan nuclear receptor-like factor, is necessary for the differentiation and function of TH17 cells. Mice lacking this orphan nuclear receptor are EAE resistant and deficient in TH17 development. The identification of this transcription regulator provided further evidence that TH17 is a distinct T cell lineage that has unique roles in immunity. The implications of these findings for IL-23/IL-17 immune axis regulation of CNS autoimmune inflammation will be discussed. CS12-01

Viral infections, Tregs, autoimmunity and immunotherapy Matthias G. von Herrath, Christophe Filippi, Urs Christen, Tom Wolfe and Damien Bresson

We have developed an infection-induced molecular mimicry model of CNS autoimmune disease induction by engineering a non-pathogenic variant of Theiler's virus (TMEV) to encode 30–39mer peptides encompassing the immunodominant myelin PLP139–151 epitope or mimics of this epitope. SJL mice infected with TMEV encoding either the native PLP139–151 determinant or molecular mimics, e.g. H. influenzae protease IV (HI574–586) which shares 6/13 amino acids or mouse hepatitis virus papain-like proteinase (MHV3821–3832) which shares only 3/13 amino acids with PLP139–151, develop an early-onset demyelinating disease mediated by PLP139–151-specific Th1 cells. Relative to the importance of innate immune signals in disease induction, we have found that disease is only induced when the mimics are delivered by genetically engineered TMEV, not by immunization with the synthetic peptide mimics in CFA. Proline at the secondary MHC class II contact residue is required for effective cross-reactivity as its addition to the native MHV sequence increases its ability to cross-activate PLP139–151-specific autoreactive T cells, while substitution of proline in the HI mimic peptide has the opposite effect. Early onset demyelinating disease and PLP139–151-specific cross-reactive responses are also induced by infection with viruses engineered to express 30–39 residues entirely encompassing the H. influenzae (HI39-TMEV) or MHV (MHV30-TMEV) epitopes indicating that the mimics are capable of being processed from their endogenous flanking residues. Our results thus describe a structural requirement for autoreactive T cell activation by potential PLP139–151 mimic peptides, and provide further support for infection-induced molecular mimicry in the pathogenesis of autoimmune disease. CS12-03

La Jolla Institute for Allergy and immunology, Immune regulation lab, 10355 Science Center Drive, San Diego, CA 921221 USA ([email protected]) We have been interested in defining the role of viral infections in autoimmunity. During my presentation I will discuss the following aspects: 1. Our earlier studies have shown that viral infections can do both, enhance or abrogate an ongoing autoimmune process. The outcome depends on the timing of the infection in relation to the autoimmune process and the nature of the virus. If molecular mimicry occurs, enhancement can be expected. In contrast, too strong cross-reactivities can lead to apoptosis of autoreactive lymphocytes and their demise and prevention of autoimmunity. 2. Viral infections can be essential to create an inflammatory milieu to precipitate autoimmune diabetes. This occurs through IFN-gamma, MHC class I and IP-10 upregulation in the islets. We will discuss the finding that precipitating autoimmunity without viral infection of the targeted organ is much more difficult illustrating the natural safeguards preventing accidental induction of autoreactive processes. 3. Induction of Tregs can occur with viral and autoantigens. Our data indicate that the efficacy of Treg induction after virus infection depends on the epitope presented. In general, virally-induced Tregs are of poorer ability to prevent autoimmunity than Tregs induced to autoantigens. 4. Autoantigen specific Tregs can be used in immunotherapy and act as bystander suppressors. Novel mechanistic insights will be presented and discussed.

Innate immune mechanisms that promote development of effector and regulatory CD4 lineages in EAE (Experimental Autoimmune Encephalomyelitis) M. L. Shinoharaa and H. Cantora a

Department of Cancer Immunology and AIDS, Dana–Farber Cancer Institute; Department of Pathology, Harvard Medical School, Boston MA 02115 USA

We have previously shown that HSV-1 viral infection can provoke autoimmunity through interactions with both the innate and adaptive immune responses. Epitopes expressed by HSV-1 that cross-react with self antigen can trigger expansion of autoreactive T cells and consequent autoimmune disease. A second set of interactions between HSV-1 and the innate immune system also plays an essential role in effective expansion of pathogenic T cells. We will summarize our recent experimental analysis of interactions between viruses and the innate immune system that regulate development of autoimmune disease in EAE (Experimental Autoimmune Encephalomyelitis). These findings suggest that expression of the innate mediators Osteopontin (Opn) and TGF-β by dendritic cell subsets regulates Th17 and T-reg development and may play a key role in the development of EAE. CS12-04

Molecular mimicry, autoimmunity, and infection CS12-02

Innate and adaptive immune requirements for the induction of CNS autoimmunity via virus-induced molecular mimicry S. D. Millera, A. E. Ercolinia, J. L. Croxfordb and J. K. Olsonc Northwestern University Medical School, Chicago, IL, USA; bNational Institute of Neuroscience, Tokyo, Japan; cUniversity of Wisconsin, Madison, WI, USA

a

M.W. Cunninghama, C. J. Coxa, S. E. Swedob and C.A. Kirvana a

University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; National Institute of Mental Health, Bethesda, MD USA

b

Molecular mimicry is the central theme of autoimmune sequelae following group A streptococcal infection in humans. Immune responses against the group A streptococcal bacterium are directed against host tissues and lead to the major manifestations of rheumatic fever including rheumatic heart disease and Sydenham chorea. Sydenham chorea, a

Abstracts movement disorder, is the major neurological manifestation of acute rheumatic fever. Human monoclonal antibodies were derived from Sydenham chorea and used to investigate the molecular mechanisms of immune pathogenesis. Human chorea monoclonal antibodies demonstrated specificity for streptococcal membranes and the group A streptococcal carbohydrate epitope, N-acetyl-beta-D-glucosamine, as well as the brain antigen lysoganglioside on the surface of neuronal cells in culture. Monoclonal autoantibodies (Mab), acute sera (IgG), and cerebrospinal fluid (IgG) recognized lysoganglioside and the intracellular protein tubulin which was identified as a 55 kd protein with the N terminal sequence MREIVHLQ. Nucleotide sequence analysis of VH and VL genes of chorea mAbs revealed sequence homology with a V gene encoding anti-tubulin antibody. The nucleotide sequences and reactivities of the chorea mAbs were distinctly different from mAbs derived from rheumatic heart disease. Reactivity of mAbs with caudate putamen tissue was blocked by lysoganglioside. When mixed with neuronal cells, chorea mAb 24.3.1, acute chorea serum IgG or CSF IgG demonstrated antibody-mediated cell signaling in neuronal cells by activation of calcium/calmodulin dependent protein kinase II activity and tritiated dopamine release. In summary, the data suggest mimicry and an antibody-mediated cell signaling mechanism in the brain which promotes dopamine release and chorea in humans. CS13-01

Effects of stress on immune function: The good, the bad and the beautiful F. S. Dhabhar Stanford University School of Medicine, Stanford, California, USA Stress is known to suppress immunity and increase susceptibility to infections and cancer. Paradoxically, stress exacerbates autoimmune diseases like multiple sclerosis, suggesting that stress may also enhance immune responses. Therefore, it is important to elucidate the conditions and mechanisms mediating these bidirectional effects of stress on immune function. It has been shown that acute stress experienced during primary antigen exposure increases memory T cell formation and induces a longlasting increase in immunity (International Immunology, 17:1059; American J Physiology, 289:R738). Acute stress experienced during antigen re-exposure enhances a secondary immune response (J Immunology, 156:2608). Stress-induced changes in leukocyte distribution (PNAS, 102:5808), epinephrine, and corticosterone (PNAS, 96:1059), are systemic mediators, and gamma-interferon is one local mediator (PNAS, 97:2846) of immunoenhancement. In contrast, chronic stress is immunosuppressive (BBI, 11:286). For example, chronic stress increases susceptibility to skin cancer by suppressing Type 1 cytokines and protective T cells while increasing suppressor T cell numbers (J National Cancer Institute, 97:1760). We suggest that the adaptive function of a stress response is to promote survival, i.e., stress hormones prepare the cardiovascular, musculoskeletal and immune systems for oncoming challenges (e.g. attacker) detected by the brain. However, stress exacerbates disease when immunoenhancement is directed against innocuous or self antigens, or when immunosuppression/ dysregulation occurs during chronic stress. In view of the ubiquitous nature of stress and its significant immunoprotective and immunopathological effects, it is important to elucidate the mechanisms mediating stress– immune interactions and to meaningfully translate findings from bench to bedside. (Support: NIH-AI48995, NIH-CA107498, and The Dana Foundation.) CS13-02

Prenatal influences on the development of immunity C.L. Coe and G. R. Lubach

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Harlow Center for Biological Psychology, University of Wisconsin, Madison, WI, USA Background: The prenatal and early rearing environments play a critical role in initiating normal ontogeny. As a consequence, maternal disturbances can affect the maturational trajectory of the fetus and chronically affect functioning postpartum. Like the brain, the immature immune system is designed to ‘learn’ from the environment, which includes many aspects of maternal care. Methods: Pregnancy conditions of over 150 rhesus monkeys were manipulated, including by psychological disturbance of the gravid female, administration of dexamethasone, or viral infection. The impact on the infant's behavioral, immune and brain development was assessed. Results: Maternal stress, antenatal corticosteroids, and influenza exposure significantly affected neurological development. The offspring evinced immature neuromotor reflexes at birth, greater emotionality during the first year of life, and a smaller hippocampus as juveniles. Smaller hippocampal size was associated with a lower neurogenesis and altered pituitary–adrenal activity. Immune alterations were also evident, including a reduced capacity of neonatal lymphocytes to recognize ‘self’ from ‘other ‘antigen’, decreased proliferative responses, and lower cytokine production in cultured cells. Several mediation pathways were identified including a predisposition for iron deficiency anemia in the fast-growing infant. In addition, prenatally stressed infants established a different profile of gut microbiota, with lower levels of protective Lactobacilli and Bifidobacteria. Conclusion: Adverse events during fetal life can affect health postpartum by impacting the brain and immune systems at vulnerable points and changing regulatory set points. In addition to maternal cortisol, disturbance of micronutrient transmission and establishment of an abnormal profile of gut bacteria were identified as important factors. CS13-03

Mechanism of interferon-induced depression: Involvement of hippocampal neurogenesis S. Kanbaa, N. Kanekob, K. Kudoc, K. Takemotod and H. Watid a

Department of Neuropsychiatry, Kyushu University, Fukuoka, Japan; bKeio University, cTokyo University, dYamanashi University The therapeutic use of human interferon-alpha (hIFN-α) is known to cause various neuropsychiatric side effects. In particular, depression occurs in 30–45% of patients, frequently interrupting treatment. hIFN-α treated animals also show depression-like behaviors. However, mechanisms underlying the depression caused by hIFN-α remain to be defined. Recently, a decrease in adult hippocampal neurogenesis was revealed as a possible neuropathological mechanism of depression. Neurogenesis is known to be regulated by various cytokines. Therefore, we investigated the effect of subchronic hIFN-α treatment (5000–50,000 IU/kg/day) on neurogenesis in the adult rat dentate gyrus (DG). The doses correspond to clinical doses and have been shown to induce depressive behavior in rodents without reducing voluntary activity. Immediately after a one-week treatment with hIFN-α, BrdU-labeled proliferating cells were decreased in the DG, but we did not detect an effect on cell differentiation into mature granule cells until three weeks after the treatment. After hIFN-α treatment, interleukin-1beta (IL-1β), a major proinflammatory cytokine, increased in the hippocampus. Co-administration of an IL-1 receptor antagonist (IL-1Ra) completely blocked the hIFN-α-induced suppression of cell proliferation in the DG. Our results demonstrated that hIFN-α suppressed cell proliferation but not cell differentiation in the DG, and that IL-1β plays an essential role in the suppression. The decreased cell proliferation caused by hIFN-α-induced IL-1β may be partly responsible for hIFN-α induced depression.

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Abstracts

CS13-04

CS14-02

Stress, immunity and cancer incidence

Oligodendrocytes and Schwann cells support axon function and survival independent of their role in myelination: Implications for multiple sclerosis

Noriyuki Kawamuraa, Toshio Ishikawaa, and Norito Kawakamib National Center of Neurology and Psychiatry, Japan; bTokyo University, Japan

a

Chronic stressors and depressive symptoms are associated with suppression of both cellular and humoral immunity. Recently low NK cell activity was shown to be associated with increased cancer risk. In addition, it is reported that critical life events and depressive symptoms are the risk factors for cancer incidence. Thus, one can hypothesize that stressors and/or stress symptoms are one of the major risk factors via stress-induced immuno-suppression. In 1997, we have started an epidemiological cohort study in order to elucidate the relationship between stress, immunity and cancer. Following approval of the ethical aspects of this study by the institutional review boards, we administered questionnaires to 4392 workers, aged 20 to 60 years old, working at a company manufacturing motor vehicle parts and collected blood samples from the 3790 (86.3%) responders who gave written informed consent. Information on the questionnaires included gender, birth date, lifestyle habits, marital status, education., family history of cancer, past and current history of diseases and job stress. At baseline, we measured lymphocyte subpopulations for all the participants, whereas cytokine and NK cytotoxity assays for 247 of participants randomly selected from the total subjects. According to the follow-up data, we have found that depressive symptoms were associated with increased cancer incidence, lower NK cell activities and lower cytokine productions, indicating that the relevance of hypothesis. CS14-01

Loss of axonal Na+/K+ ATPase in chronic lesions of multiple sclerosis Christine Fowler, Elizabeth A.Young, Graham Kidd, Ansi Chang, Bruce D. Trapp

K.-A. Navea, J. Edgarb, I.R. Griffithsb, K. Jansena, C.M. Kassmanna, M. Klugmanna, C. Lappe-Siefkea, G. Meyer-zu-Hörstea, W. Möbiusa, M.H. Seredaa, H. Wernera a

Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen, Germany; bApplied Neurobiology Group, Institute of Comparative Medicine, University of Glasgow, UK

Oligodendrocytes and Schwann cells are well known for their common function in faciliting the rapid impulse propagation in the nervous system, by enwrapping axons with myelin. Experimental genetic data suggest there are additional functions of ensheathing glia that may even precede myelination (in evolutionary terms) and are relevant for disease. We have previously shown that null mutations of two glial genes, Plp1 and Cnp1, do not impair the functional myelination of the CNS but cause progressive axonal degeneration in the absence of inflammation (Griffiths et al., Science, 1998; Lappe-Siefke et al., Nat Genet., 2003). Axon loss is preceded by swellings and slowing of retrograde transport (Edgar et al., JCB, 2004), but the underlying cause is not understood. Recently, a proteom analysis of myelin membranes, purified from brains of PLP null mutants, revealed the virtual absence of an NAD+ dependent regulatory enzyme (Jansen et al., unpublished). This suggests that some oligodendrocyte functions are coupled to the energy state of the white matter. In support, we find extensive axonal loss and demyelination in Pex5 conditional mouse mutants that lack functional peroxisomes, which are known for their role in fatty acid β-oxydation. Surprisingly, the oligodendrocyte-specific loss of Pex5 shares many pathological features with human X-linked ALD, including the invasion of B- and T-cells into the brain (Kassmann et al., unpublished). This novel mouse mutant proves experimentally that glial peroxisomes are important for axonal maintenance and that a primary oligodendrocyte defect is sufficient to cause a secondary inflammatory response, a finding relevant to the etiology of multiple sclerosis. CS14-03

Mechanisms to promote axonal regeneration in vivo

Department of Neurosciences, Cleveland Clinic, Cleveland, OH, USA

Marie T. Filbin

Neurodegeneration is the major cause of permanent neurological disability in individuals with the demyelinating disease, multiple sclerosis. The continuous and irreversible neurological decline that occurs during the latter stages of MS is thought to result from degeneration of chronically demyelinated axons. The molecular mechanisms responsible for the degeneration or dysfunction of chronically demyelinated axons are poorly understood. One proposed mechanism involves dysfunction of the Na+/K+ ATPase, which is essential for nerve transmission. In this study, we determined the distribution of Na+/K+ ATPase subunits in normal human white matter and demyelinated lesions in multiple sclerosis. Na+/K+ ATPase subunits α1, α3 and β1 were detected in the internodal axolemma and absent from the nodal axolemma of myelinated fibers in the adult human brain. Following demyelination, all three subunits were initially detectable on demyelinated axolemma. In contract, over 50% of demyelinated axons in chronic inactive lesions of multiple sclerosis contained little or no Na+/K+ ATPase immunoreactivity. As a result, these chronically demyelinated axons cannot exchange axoplasmic Na+ for extracellular K+ and their axolemma remains in a depolarized state and incapable of nerve transmission. We propose that loss of axonal Na+/K+ ATPase is a major contributor to axonal degeneration and continuous neurological decline in multiple sclerosis patients.

Biology Department, Hunter College, 695 Park Avenue, New York, NY 10024, USA One of the major impediments to axonal regeneration after injury is inhibitors in myelin. Three myelin inhibitors have been identified in NogoA, MAG and OMgp. One approach to overcome these inhibitors to encourage regeneration is to change the intrinsic state of the axon such that it no longer recognizes these molecules as inhibitory. We have shown that if the neuronal cAMP levels are elevated either artificially with an analogue such as db cAMP or by priming neurons with a variety of neurotrophins (NGF, BDNF) before exposure to the inhibitor, MAG and myelin in general no longer inhibit axonal growth. Furthermore, we have also shown that the longrecognized ability of spinal dorsal column axons to regenerate if the peripheral branch of the same neuron is lesioned beforehand is a consequence of a transient increase in endogenous cAMP levels in the DRG cell bodies. Importantly, injection of db cAMP directly into the DRG in the absence of a conditioning lesion, is sufficient to induce regeneration of subsequently lesioned dorsal column axons. This cAMP effect is transcription dependent and we have identified 4 very different genes that are up-regulated in response to either elevation of cAMP or a conditioning lesion. All 4 of these proteins block inhibition by myelin and they are currently being tested for their ability to promote regeneration in vivo.

Abstracts CS14-04

Remyelination and axon protection Robin J.M. Franklin Cambridge Centre for Brain Repair, University of Cambridge, Cambridge CB3 OES, UK Remyelination, the process by which new myelin sheaths are restored to demyelinated axons, represents one of the most compelling examples of adult multipotent progenitor cells contributing to regeneration of the injured CNS. This process can occur with remarkable efficiency in both clinical

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disease, such as multiple sclerosis, and in experimental models, revealing an impressive ability of the adult CNS to repair itself. However, the inconsistency of remyelination in multiple sclerosis, and the loss of axonal integrity that results from its failure, makes enhancement of remyelination an important therapeutic objective. Identifying potential targets will depend on a detailed understanding of the cellular and molecular mechanisms of remyelination. This talk will review (1) the nature of the cell or cells that respond to demyelination and generate new oligodendrocytes, identifying current areas of uncertainty and addressing the role of adult CNS stem and progenitor cells, (2) intrinsic factors regulating precursor differentiation and (4) how an environment favourable to remyelination is generated, and will introduce the concept of a matrix of signalling events critical for the successful completion of remyelination.

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Abstracts

Workshop 1: Genetics of neuroimmunological disorders

Abstracts

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WS01-K1

WS01-02

Genetics of neuroimmunological disorders

PRKCA and multiple sclerosis: Association in independent populations

T. Olssona and George Ebersb a

Neuroimmunology Unit, Karolinska Hospital, Stockholm, Sweden; University of Oxford, UK

J. Saarelaa, S. P. Kallioa, D. Chenb, A. Montpetitc, M. R. Lincolnd, A. Palotiee,f, G. C. Ebersd, T. J. Hudsonc and L. Peltonena,e,f

b

National Public Health Institute, Helsinki, Finland; bDavid Geffen School of Medicine at UCLA, Los Angeles, USA; cMcGill University and Genome Quebec Innovation Centre, Montreal, Canada; dUniversity of Oxford, Oxford, UK; eThe Broad Institute of MIT and Harvard, Cambridge, USA; f University of Helsinki, Helsinki, Finland

a

There is solid evidence for the role of genes regulating MS. Their deciphering may allow definition of mechanisms central for disease and thereby for more precise and selective therapy. The MHC/HLA complex is well established as a risk gene region. Studies in experimental autoimmune encephalomyelitis (EAE) have shown allele specific disease protective and promoting actions from both the class I and class II genes, possibly to be conveyed by critical peptide–MHC molecule interactions. Similar data now accumulate for human MS as well. Linkage analyses have failed to show any non-HLA region and the paradigm generally used to justify genome searches may be incorrect or the effect of any individual locus would be small. Indeed, the few examples of discrete non-MHC genes with suggested or proven influence display odds ratios in the order of 1.5, such as the PKRCA, IL7R and MHCIITA. There is some interest in whole genome SNP typing with association analysis of large case-control materials but there is no assurance that this approach will be fruitful. Gene mappings in EAE may reveal genes and/or mechanisms which are shared between species with options for experimental studies of function. Collectively, there are now around 50 quatitative traits loci (QTLs) described in different rodent crosses. Upon fine dissection each of these often resolves into two to five subQTLs, showing that linkage peaks mostly depend on more than one gene and by extrapolation that the number of genes which potentially has a role in MS approaches hundreds. The experimental QTLs can be fine dissected. Albeit cumbersome, definite disease relevant variants of genes result and the first few positioned genes start to appear in the literature. WS01-01

HLA associations to “fringe phenotypes” in multiple sclerosis K. Imrella, A.-K. Hedströmb, J. Hillerta, A.-M. Landtblomc, T. Mastermana

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS. Complex diseases such as MS likely result from problems in networks of interactions between several genes and environmental factors. HLA-DRB1⁎15 is the only consistent locus observed in most populations, but numerous genome screens also provide evidence for a MS locus on 17q. Sixty-three Finnish MS families were ascertained to identify a susceptibility gene within the previously established 3.4-Mb region on 17q24, which is flanked by segmental duplications. Such a complex genome structure may predispose to copy-number or other structural variation. Initial association implicated PRKCA, which was further analyzed in 211 Finnish and 554 Canadian MS families, utilizing a dense SNP set. The original PRKCA association was replicated in 148 independent Finnish MS families (rs887797, p = 0.0004), while a flanking SNP showed association in Canadian population (p = 0.0097). SNP haplotype analysis revealed two allelic variants of PRKCA to be over-represented in Finnish or Canadian MS cases (odds ratios: 1.34 and 1.64). An analysis of 200 MS families from Sweden, Denmark and Norway suggested a role for the Finnish risk haplotype in MS susceptibility also in other Scandinavian populations. Transcript levels of PRKCA correlated with the copy number of the risk alleles in 20 Finnish and 11 CEPH individuals of European origin in an initial expression analysis. These results imply involvement of PRKCA in the etiology of MS. Moreover, function of PRKCA in a signal transduction pathway altering blood–brain barrier permeability supports its role in chronic inflammation of CNS and myelin destruction.

a

Karolinska Institutet, Stockholm, Sweden; bKarolinska University Hospital, Stockholm, Sweden; cLinköping University Hospital, Linköping, Sweden

In Western countries, frequencies of cases of opticospinal (OS) multiple sclerosis (MS), and of MS cases lacking oligoclonal immunoglobulin G bands (OCB) in the cerebrospinal fluid, are very low. From a database of 2199 patients with MS or possible MS, we identified 85 patients with OCB-negative MS and 58 patients with OSMS. (OSMS was defined broadly as clinical evidence of involvement of the optic nerve, brainstem or spinal cord, without clinical or radiological evidence of disease in the cerebrum or cerebellum.) Carriage rates of HLA class II alleles in these “fringe phenotypes” were compared with rates in clinically and paraclinically more typical MS. HLA genotyping was performed in subsets of patients as well as in healthy controls. Carriage of DRB1⁎04, but not of DRB1⁎15, was associated with OCB-negative MS (odds ratio [OR], 2.1; 95% confidence interval [CI], 1.2 to 3.8). High-resolution genotyping of HLA-DRB1⁎04 uncovered a highly significant riskconferring association between the DRB1⁎0404 allele and OCB-negative MS (OR, 4.3; 95% CI, 1.9 to 9.7). The frequency of DRB1⁎15 was slightly (yet non-significantly) higher in OSMS than in conventional MS. In Japan and Sardinia, DRB1⁎04 is also more common in MS patients without OCB than in patients with OCB; yet, in Japan, it is DRB1⁎0405 that is elevated in OCB-negative MS. In conclusion, in Sweden, the fringe phenotype OSMS is not uniquely associated with any HLA-DRB1 allele; OCB-negative MS, however, appears to be immunogenetically distinct from OCB-positive MS.

WS01-03

Protective influence on multiple sclerosis by HLA-A⁎02: Implications for a two step model of pathogenesis A. Martins Silva2, A. Bettencourt1, C. Pereira1, C. Carvalho3, P. P. Costa3, L. Monteiro2, B. Martins Silva1,3 1

Immunogenetics Laboratory—ICBAS/University of Porto, Porto, Portugal; Neurology Service—HGSA, Porto, Portugal; 3Immunogenetics Laboratory —INSA Dr. Ricardo Jorge-Porto, Portugal 2

Multiple sclerosis (MS) was traditionally thought to be a CD4+ T-cells mediated disease, associated with HLA-DRB1⁎15. However, a two-step model, recently proposed by Friese (2005), implicates CD4+ T cells in the beginning of MS and CD8+ T-cells in the chronic phase of the disease. This model suggests that HLA-class I may also contribute to MS, as already described for HLA-A⁎03 and HLA-A⁎02. The aim of this study was to investigate the role of HLA-A⁎02 and HLA-A⁎03 alleles on disease susceptibility and severity in a Portuguese MS population. We studied 242 unrelated patients with clinically definitive MS according McDonald criteria, attending the outpatient neurological clinic of HGSA and 142 healthy controls from North of Portugal. To evaluate disease severity, patients were divided in 3 groups: 44 have

40

Abstracts

Benign MS (EDSS ≤ 3, ≥ 10 years after onset), 36 Non-Benign MS (EDSS ≥ 3, at same stage) and 41 Aggressive MS (EDSS ≥ 6, ≤15 years after onset). HLA-A⁎03 frequency was similar in patients and controls. HLA-A⁎02 was negatively associated with MS (35.1% vs. 52.7%, OR = 0.486, p = 0.0006). This difference was even higher in DR15 negative individuals (32.9% vs. 57.0%, OR = 0.370, p = 0.00006). When disease severity was considered this association was observed only in Benign group (34.1% vs. 52.7%, OR = 0.464, p = 0.030). Our results are in accordance with Fogdell-Hahn (2000) establishing HLA-A⁎02 allele as a protective genetic marker to MS in this Portuguese population. This observation supports the Friese model for MS, highlighting the importance of HLA-Class I in CD8 T-cells function and its role in disease outcome. Work supported by Serono Portugal.

the region encompassing Idd5.1, Idd5.2 and Idd5.3 were as susceptible to EAE as the NOD strain, mice having the entire Idd5 region including Idd5.4, the most distal Idd5 subregions, were resistant to EAE. As compared to NOD-derived T cells, T cells from the EAE-resistant congenic mice produce lower amounts of Th1 and higher amounts of Th2 cytokines. This cytokine alteration may be responsible for disease protection in the EAE-resistant congenic mice. Genetic elements present in the region for the disease resistance have not been characterized. The identity of the gene causing EAE resistance will require the development of additional congenic strains to reduce the size of the subregion and thereby the number of candidate genes, one of which encodes the T cell inhibitory molecule PD-1. Whether a polymorphism in PD-1 impart genetic resistance and affects Th1/Th2 differentiation is being studied.

WS01-04

Evidence the Y-chromosome influences experimental allergic encephalomyelitis in female mice

Idd5.4 on chromosome 1 mediates resistance to experimental autoimmune encephalomyelitis Manabu Araki1, Denise Chung1, Lalitha Vijaykrishnan1, Raymond A. Sobel2, Kara Hunter3, Laurence B. Peterson4, Linda S. Wicker3 and Vijay K. Kuchroo1

WS01-05

C. Teuschera, R. Noubadea, K. Spacha, B. McElvanya, J. Y. Bunna, P. D. Fillmoreb, J. F. Zacharyb and E. P. Blankenhornc University of Vermont, Burlington, VT, USA; bUniversity of Illinois UrbanaChampaign, Champaign, IL, USA; cDrexel University College of Medicine, Philadelphia, PA, USA

a

1

Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA; 2Laboratory Service, Veterans Affairs Medical Center, Palo Alto, CA 94304, USA; 2Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA; 3Juvenile Diabetes Research Foundation/Wellcome Trust, Diabetes and Inflammation Laboratory, Cambridge Institute of Medical Research, University of Cambridge, Cambridge, UK; and 4Department of Pharmacology, Merck Research Laboratories Rahway, NJ 07065, USA Idd5, a type 1 diabetes (T1D) susceptibility region, is located on chromosome 1. Introgression of DNA from T1D-resistant strain onto the NOD background protects from not only T1D but also experimental autoimmune encephalomyelitis (EAE). To study the Idd5 region for disease susceptibility, congenic mice having the entire locus and subcongenic lines having defined portions of the region were generated. Our previous study showed that Idd5.1, which is the most proximal Idd5 and contains the Ctla4 and Icos, enhanced EAE severity and regulated ICOS expression and IL-10 production. Here we show the effect on EAE of three additional subregions: Idd5.2, Idd5.3 and Idd5.4. Whereas congenic mice having B10-derived DNA introgressed throughout

Experimental allergic encephalomyelitis (EAE), an autoimmune model of multiple sclerosis, is a complex disease influenced by genetic, intrinsic and environmental factors. In this study, we questioned whether parent-oforigin effects influence EAE, using reciprocal F2 intercross progeny generated between EAE susceptible SJL/J (S) and EAE resistant B10.S/ SgMcdJ (B) mice. EAE susceptibility and severity was found to be different in female BS × BS intercross mice as compared to females from the three other birth crosses (BS × SB, SB × SB and SB × BS), and in fact, both traits in female mice resembled those of their male siblings. This masculinization is associated with transmission of the SJL/J Y-chromosome and an increased ♂:♀ sex ratio. Related studies using C57BL/6J Ychromosome substitution strains demonstrate that the Y-chromosome again influences EAE in both male and female mice, and that the disease course and MOG35–55 specific immune response in females resembles that of their male littermates. Importantly, this is the first experimental evidence demonstrating the existence of a Y-chromosome polymorphism capable of modifying autoimmune disease susceptibility in both males and females.

Abstracts

Workshop 2: Chemokine in neuroimmunology

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WS02-K1

WS02-02

Chemokines in physiology and pathology of the CNS

CXCL10 but not CXCL9 deficient mice are highly sensitive to herpes simplex virus type 1 infection

E. Meinla,b and R. Ransohoffc,d

D. J. J. Carra, T. Wuesta, R. Welnerb, R. Pelayob, J. Farberc, A. Lusterd, and P. Kincadeb

Max-Planck-Institute of Neurobiology, Martinsried, Germany; bInstitute of Clinical Nueroimmunology, University of Munich, Germany; c Neuroinflammation Research Center, Lerner Research Institute, Cleveland, OH, USA; dMellen Center for MS treatment and Research, Cleveland Clinic Foundation, Cleveland, OH, USA

a

Chemokines comprise a family of about 50 small proteins targeting at least 18 receptors. Chemokine functions greatly exceed the regulation of immune cell trafficking. During development of the CNS the chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors by arresting their migration. CXCR4 is essential for forming the normal cerebellar cytoarchitecture by regulating proliferation and migration cerebellar granule cells. In the healthy adult brain certain chemokines like CXCL12 (on astrocytes, endothelial cells) and CX3CL1 (on neurons) are displayed. Neuronal derived CX3CL1 can be shed and regulates the state of activation of microglia. A production of chemokines inside of the CNS by astrocytes and microglia is induced in autoimmune inflammation, infection and also after neurotrauma. Chemokine production is stimulated via T cell derived cytokines and via innate immunity. The role of chemokines in CNS infections is indicated by the observation that human subjects with a loss-offunction mutation of CCR5 have a higher risk of developing encephalitis after infection with West Nile virus. In EAE models and in MS the inflammatory cytokines CXCL10/IP10, CCL2/MCP-1, CCL5/RANTES, but also the homeostatic chemokines CXCL12/SDF-1, CXCL13/BCA-1, and CCL19/Mip3b are involved in lesion formation. The crucial role of chemokines in neuroinflammation provides the possibility to develop novel chemokine-directed therapeutic strategies.

The role of CXCR3 ligands, CXCL9 and CXCL10, in herpes simplex virus type 1 (HSV-1) infection was investigated. C57BL/6 wild type (WT), CXCL9 deficient (CXCL9−/−), and CXCL10 deficient (CXCL10−/−) mice were infected with HSV-1 (1000 plaque forming units/cornea) and assessed for virus titer as well as the host response to infection in the trigeminal ganglion (TG) and brain stem (BS) during acute infection (days 3–7 post infection, pi). HSV-1 titers recovered in the TG and BS of CXCL10−/− mice were 1–2 logs higher in comparison to CXCL9−/− or WT mice day 3–day 7 pi. Likewise, there was a significant loss in thymocytes in the CXCL10−/− mice following infection relative to the other two groups which did not correlate with changes in corticosterone levels but rather, the incidence of detectable virus within the thymus. Bone marrow progenitor cell commitment was noticeably modified with a strong change toward myeloid and away from lymphoid progenitors. In evaluating T cell infiltration into the TG and BS of infected mice, CXCL9−/− mice possessed more CD4 and CD8 T cells in the TG compared to the WT mice day 7 pi. In contrast, CXCL10−/− mice possessed more CD4 and CD8 T cells in the BS compared to the WT mice day 7 pi. The absolute number of NK cells (NK1.1+CD3−) was reduced in the TG and BS of CXCL10−/− mice compared to the WT or CXCL9−/− mice. Collectively, the results indicate the disparity between CXCL9 and CXCL10 relative to a clinically significant neurotropic virus infection.

a

The University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; bOklahoma Medical Research Foundation, Oklahoma City, OK, USA; c NIAID/NIH, Bethesda, MD, USA; dMassachusetts General Hospital, Charlestown, MA, USA

WS02-03 WS02-01

The chemokine CXCL13 is a key molecule in autoimmune Myasthenia Gravis A. Meraouna1, G. Cizeron-Clairac1, R. Le Panse1, J. Bismuth1, F. Truffault1, C. Tallaksen2 and S. Berrih-Aknin1 1

CNRS–UMR 8162, IPSC, Le Plessis-Robinson, France (sonia. [email protected]); 2Department of Neurology, Ulleval University Hospital, N-0407 Oslo, Norway

Myasthenia Gravis (MG) is associated with ectopic germinal centres in the thymus. Thymectomy and glucocorticoids are the main treatments but they induce operative risks and side effects, respectively. The aim of this study was to propose new therapies more efficient for MG. We hypothesized that molecules dysregulated in MG thymus and normalized by glucocorticoids may play a key role in thymic pathogenesis. Using gene chip analysis, we identified 88 genes complying with these criteria, the most remarkable being the B cell chemoattractant (CXCL13). Its expression was increased in thymus and sera of glucocorticoid untreated patients and decreased in response to treatment in correlation with clinical improvement. Normal B cells were actively chemoattracted by thymic extracts from glucocorticoid untreated patients, an effect inhibited by anti-CXCL13 antibodies. In the thymus, CXCL13 was preferentially produced by epithelial cells and overproduced by epithelial cells from MG patients. Altogether, our results suggest that a high CXCL13 production by epithelial cells could be responsible for germinal centre formation in MG thymus. Furthermore, they show that this gene is a main target of corticotherapy. Thus, new therapies targeting CXCL13 could be of interest for MG and other autoimmune diseases characterized by ectopic germinal centre formation.

Differential effects of decoy chemokine (7ND) gene therapy on acute, biphasic and chronic autoimmune encephalomyelitis: Implication for the pathomechanisms of lesion formation Il-Kwon Park, Kuniko Kohyama, Mie Nakajima and Yoh Matsumoto Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), exhibit several clinical subtypes such as relapsing–remitting and secondary progressive forms. Although clinical signs are similar among disease subtypes, it becomes evident that the lesion formation in the central nervous system (CNS) may take place by different mechanisms. In the present study, we induced three different types of EAE, i.e. acute, biphasic and chronic EAE, in rats and examined the effects of decoy chemokine (7ND) gene therapy, which inhibit the migration of macrophages. Interestingly, it was demonstrated that the clinical course of acute EAE was not affected, whereas chronic EAE was completely suppressed with the treatment. Furthermore, the relapse, but not the first attack, of biphasic EAE was inhibited by gene administration. Histopathological examinations revealed that the number of infiltrating macrophages was reduced in all the stages of three types of EAE. Real time PCR analysis showed that the levels of cytokine and chemokine mRNA paralleled with the macrophage behavior in the CNS. These findings suggest that in acute EAE and the first attack of biphasic EAE where anti-macrophage therapy was almost ineffective, pathogenic T cells play a critical role. In contrast, the relapse of biphasic EAE and chronic EAE were thought to be produced mainly by macrophages. Thus, the mechanisms of the lesion formation are not uniform and immunotherapy

Abstracts

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should be performed on the basis of information about the pathomechanisms of the lesion formation in autoimmune diseases.

contribute to neuronal development in early life. Supported by NIH grant R01NS041862.

WS02-04

WS02-05

SDF-1α modulates the innate rhythm of network-driven GABA-mediated giant depolarizing potential in the developing nervous system

The role of CX3CR1 in aminoglycoside ototoxicity

A. Kasiyanov1, H.Tamamura2, N. Fujii3, H. Xiong1

a

Eisuke Satoa, Richard M. Ransohoffa,b and Keiko Hirosec,a

University of Nebraska Medical Center, Omaha, USA; 2Tokyo Medical and Dental University, Tokyo, Japan; 3Kyoto University, Kyoto, Japan

Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, USA; bMellen Center, Department of Neurology, Cleveland Clinic, Cleveland, USA; cHead and Neck Institute, Cleveland Clinic, Cleveland, USA

The chemoattractant stromal cell-derived factor-1 alpha (SDF-1α) and its receptor, the CXC chemokine receptor 4 (CXCR4), play an important role in the formation of hippocampal circuitry and synaptic plasticity in addition to their involvement in brain inflammatory processes. To understand the role that CXCR4 might play in hippocampal circuitry development, we studied the effects of SDF-1α, the only natural ligand for CXCR4 receptors, on network-driven, GABA-mediated giant depolarizing potentials (GDPs) in immature rat hippocampal CA3 neurons using whole-cell patch clamp technique. We demonstrated that SDF-1α down-regulates the GDP firing frequency in CA3 neurons without significant change of neuronal passive membrane properties, suggesting that the site of action for SDF-1α is on interneurons and/or glial cells, instead of the recorded neuron per se. The SDF-1α-induced down-regulation of GDP firing was blocked by T140, a CXCR4 receptor antagonist. Application of T140 alone enhanced GDP firing frequency, indicating an involvement of endogenous SDF-1α in modulating GDP activity. Taken together, our results showed that exogenous and endogenous SDF-1α modulates GDP firing via CXCR4 receptors in developing hippocampus. As GDPs are the innate rhythm of network-driven GABA-mediated synaptic events, the modulation of GDPs by SDF-1α may

Our recent studies demonstrated the accumulation of CD45+, CD68+, CX3CR1+ and Iba-1+ macrophages in the cochlea, following acoustic trauma. Drug-induced ototoxicity is a distinct and clinically-relevant cause of sensorineural hearing loss. It is unknown whether inflammatory cells play a role in ototoxic injury. This study was designed to investigate what role CX3CR1 plays in hearing loss and hair cell death after aminoglycoside exposure. CX3CR1+/+, CX3CR1+/− and CX3CR1−/− mice at age 8 weeks were exposed to 900 mg/kg kanamycin intraperitoneally (IP) delivered twice daily for 15 days. Hearing thresholds were recorded 14 days after the final dose of kanamycin (age 12 weeks), and cochleas were harvested for histologic analysis. Hearing thresholds in CX3CR1−/− mice were profoundly elevated when compared to either the wild type or the CX3CR1+/− mice. In addition, hair cell damage was greatest in the CX3CR1−/− mice when compared to their wild type and heterozygous littermates. Neither hair cell numbers nor hearing thresholds were abnormal in control CX3CR1−/− mice which were not exposed to aminoglycoside. Our data indicate a protective role for CX3CR1 in aminoglycoside ototoxicity, possibly acting through cochlear macrophages, to reduce hair cell death and lessen hearing impairment.

1

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Abstracts

Workshop 3: Glial reactions in neuroinflammation

Abstracts WS03-K1

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a

Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Montreal, Canada

Glial inflammation: The good, the bad and the ugly Monica J. Carson1 and Yuzo Iwasaki2, 1

University of California, Riverside, USA; 2Tohoku University Postgraduate School of Medicine, Miyagi, Japan Over the last 20 years, there has been a dramatic re-evaluation of the type of cellular immune responses that can and do occur within the central nervous system (CNS). For the better part of the last century, the CNS was believed to be both immunologically inert and immunologically separated from the peripheral immune system. It was viewed primarily as a tissue of post-mitotic cells that were highly vulnerable to the onslaught of activated immune cells if and when these immune cells infiltrated the CNS. In this view, the only elements protecting the CNS from immune-mediated harm was the presence of an intact blood–brain barrier (BBB) and the absence of a fully immunologically population of competent tissue macrophages. Current data now indicates that the CNS is immunologically competent and is actively interactive with the peripheral immune system. Inflammation is also now recognized to be a prominent feature of many classic neurodegenerative disorders even in the absence of substantial infiltration of peripheral immune cells. Furthermore, in stark contrast to the older view, neuroinflammation is now realized to have both neuroprotective as well as neurotoxic aspects. Here we will discuss new classes of receptors (TREMs, TLTs and LR8) with the potential to trigger and modify glial inflammatory responses.

We previously reported that Toll-like receptor (TLR) ligation tailors the activation profile of human microglia; TLR3, in particular, induces the production of both IL-12 and IFN-beta (â). The functional consequences to adaptive immune responses downstream of such TLR-mediated polarization remain undefined in human cells. We compared the capacity for adult human microglia, once activated in vitro with ligands for TLR2, TLR3, or TLR4, to regulate CD4+ T cell responses, specifically Th1-priming. We tested the ability for these ligands (poly(inosinic):poly(cytidylic) acid for TLR3, lipopolysaccharide for TLR4, palmitoyl-3-cysteine-serine-lysine-4 for TLR2) to induce the maturation of microglia in terms of MHC and costimulatory molecule expression using flow cytometry. The proliferation and interferon-gamma (IFNã) secretion of allogeneic CD4+ lymphocytes was determined following culture with microglia pre-activated via TLR2, TLR3 or TLR4 for 24 h. We found that TLR signaling increased microglial MHC class I and II, CD80 and CD86 surface expression downstream of TLR3 and TLR4. While TLR-mediated activation of adult microglia did not result in any significant increase in overall CD4+ T cell proliferation, the ligation of TLR3 by microglia led to a significant boosting of IFNã secretion by T cells. TLR-signaling can thus lead to the maturation of human microglia as antigen presenting cells, and TLR3mediated activation can specifically bridge innate and adaptive immune responses by driving Th1 T cell polarization. WS03-03

WS03-01

Identification of soluble CD14 as an endogenous agonist for Toll-like receptor 2 on human astrocytes by genome-scale functional screening of glial cell derived proteins M. Bsibsia, E. van Duijvenvoordena, C. Persoon-Deena, J.J. Bajramovicb, J. M. van Noorta and M.H.J. Vogtc TNO Quality of Life, Leiden, The Netherlands; bBPRC, Rijswijk, The Netherlands; cVU University Medical Center, The Netherlands

a

Human astrocytes express a very limited repertoire of Toll-like receptor (TLR) family members including TLR1–4, which are expressed on the cell surface. Recent data indicated that TLRs on astrocytes not only play a role in host defense but also in tissue repair responses. This prompted us to examine the possibility that endogenous TLR agonists could be expressed in the human central nervous system to regulate the apparently dual astrocyte functions during trauma or inflammation. We developed a high throughput, genome-scale functional screening method to identify these endogenous TLR agonists. The method is based on the pooled transfection of brain-derived genes in eukaryotic cells and the evaluation of functional effects of translated proteins on primary human astrocytes. IL-8 release was used as read-out, since this cytokine is highly produced by astrocytes upon activation via TLR. Using this strategy, we identified an alternatively spliced variant of soluble CD14. Using a panel of TLR transfected HEK293 cells, we found that soluble CD14 only induced IL-8 release by TLR2 transfected HEK293 cells, and not TLR3 or TLR4 transfected HEK293 cells. These data indicate that apart from its well-known ability to act as a coreceptor for TLR-dependent signaling by peptidoglycans or LPS, soluble CD14 itself also acts as a direct agonist for TLR2. Currently, we are further exploring the functional effects of soluble CD14 on human primary astrocytes. The genome-scale screening method will be used to identify proteins based on their functional properties. WS03-02

TLR-mediated activation of human microglia can regulate CD4+ Th1 polarization C. S. Jacka, N. Arboura, M. Blaina, J. Antela

A critical role of toll-like receptor 2 in nerve injury-induced spinal cord glial cell activation and pain hypersensitivity D. Kima, M.A. Kimb, M.S. Kima, I.-H. Choa, S. Leec, E.-K. Joc, S.-Y. Choia, K. Parka, J.S. Kima, S. Akirad, H.S. Nab, S.B. Oha and S.J. Leea a

Program in Molecular and Cellular Neuroscience, Department of Oral Physiology, School of Dentistry, Seoul National University, Seoul, Republic of Korea; bDepartment of Physiology, Medical Science Research Center, Korea University College of Medicine, Seoul, Republic of Korea; c Department of Microbiology, Choong-Nam National University, Daejeon, Republic of Korea; dDepartment of Host Defense, Osaka University, Osaka, Japan The activation of spinal cord glial cells has been implicated in the development of neuropathic pain upon peripheral nerve injury. The molecular entity that activates glial cells, however, has not been elucidated. To address this lack of information, primary spinal cord glial cells were stimulated with various molecules that are putatively released upon peripheral nerve injury in the spinal cord dorsal horn. Our in vitro data indicate that spinal cord glial cells are relatively unresponsive to the stimulation of glutamate, substance P, ATP, lysophosphatidic acid (LPA), and fractalkine. Interestingly, necrotic neuronal cell extract (NNCE), however, induced significant upregulation of the TNF-α, IL1β, IL-6, and iNOS genes that are implicated in the development of neuropathic pain. Studies using primary glial cells isolated from TLR2 knockout mice indicate that NNCE activate glial cells via TLR2. In addition, behavioral studies using TLR2 knockout mice demonstrate that the expression of TLR2 is required for the induction of mechanical allodynia and thermal hyperalgesia due to spinal nerve axotomy. The nerve injury-induced spinal cord microglia and astrocyte activation is reduced in the TLR2 knockout mice. Similarly, the nerve injury-induced proinflammatory gene expression in the spinal cord is also reduced in the TLR2 knockout mice. These data demonstrate that TLR2 plays a critical role in nerve injury-induced spinal cord glial cell activation and subsequent pain hypersensitivity. In addition, our data imply that endogenous TLR2 agonist, released from the damaged sensory neurons, may activate spinal cord glial cells via TLR2 and thereby induce neuropathic pain.

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WS03-04

WS03-05

Microglial activation is correlated with decreased expression of EAAT-2 in the cerebral cortex of HIV-1 infected patients: A neuroprotective role of microglia in AIDS encephalopathy

TrkB is physiologically expressed on astrocytes and is upregulated in multiple sclerosis lesions

Izumo Sa, Xing HQa, Kuboda Ra, Hayakawa Ha,b, Gelpi Eb, Budka Hb a

Center for Chronic Viral Diseases, Kagoshima University, Kagoshima, Japan; bInstitute of Neurology, Vienna University, Vienna, Austria Pathogenesis of AIDS encephalopathy has been discussed in correlation with microglial activation and infiltration of HIV-1-infected macrophages. Glutamate transporter-1 (EAAT-2) was primarily expressed on astrocytes and keeps extracellular glutamate concentration low in the brain by taking up glutamate, which prevent neurons from excitotoxic cell death. Gray and Gras reported that both microglia and brain macrophages expressed EAAT-2 and have a neuroprotective role in AIDS encephalopathy. In order to clarify precise roles of microglia in AIDS encephalopathy, we examined autopsy brains of twenty patients with HIV-1 infection by immunohistochemistry. We demonstrated decrease of EAAT-2 expression, diffuse activation of microglia, and expression of EAAT-2 by activated microglia in the frontal cortex. Expression of IL-1β and TNF-α were detected only in glial nodules, but not in diffusely activated microglia. These cortical changes were neither correlated with severity of HIV encephalitis nor presence of HIV-1-infected cells. A quantitative analysis of EAAT-2 expression and microglial activation demonstrated that the number of Iba1-positive activated microglia was increased in 12 cases and the area of EAAT-2 expression was declining in 12 cases. There was a significant negative correlation between areas of EAAT-2 expression and numbers of Iba1-positive activated microglia (P < 0.01) among the cases with decreased EAAT-2 expression. These data indicate that activation of microglia occurs according with reduction of EAAT-2 expression on astrocytes in the cerebral cortex of AIDS patients. Expression of EAAT-2 by activated microglia suggests its compensatory effect to prevent neurons from glutamate neurotoxicity.

C. Farinaa,b, C. K. Ayatab, M. Krumbholzc, H. Lassmannd, F. Aloisie, R. Hohlfeldb,c and E. Meinlb,c a

Department of Neuroimmunology, Neurological Institute C. Besta, I-20133 Milan, Italy; bDepartment of Neuroimmunology, Max-Planck-Institute of Neurobiology, D-82152 Martinsried, Germany; cInstitute for Clinical Neuroimmunology, Ludwig-Maximilians-University, D-81377 Munich, Germany; dDivision of Neuroimmunology, Institute for Brain Research, University of Vienna, Vienna, Austria; eDepartment of Cell Biology and Neuroscience, Istituto Superiore Sanitá, I-00161 Rome, Italy Neurotrophins play a fundamental role in regulating proliferation and differentiation of neurons. They exert their actions through the p75 receptor or the different Trk receptors. Little is known about their potential effects on glia cells. Published data indicate that astrocytes express neurotrophin receptors (NTR) mainly after activation or neoplastic transformation. The aim of the present study was to analyse the NTR profile in human astrocytes in vitro and in situ in normal and in multiple sclerosis (MS) brain samples. Molecular analyses showed that resting foetal astrocytes transcribed mRNA for p75, TrkB and TrkC, but not TrkA. When TrkB and TrkC isoforms were analysed by real time PCR, mainly the truncated isoforms were detectable in resting astrocytes. FACS analysis demonstrated protein expression of p75, TrkB and TrkC. Exposure of the cells to IFN-gamma enhanced p75 levels at the mRNA and at the protein levels, whereas TrkB and TrkC were unchanged. Immunofluorescence showed membrane staining for all three receptors. Furthermore immunohistochemical analysis showed constitutive expression of TrkB in situ on astrocytes in normal brain specimens and enhanced levels within MS lesions. Together, these data suggest a role of neurotrophins in regulation of glia cell reactivity.

Abstracts

Workshop 4: Gene therapy and immunotherapy for brain tumors

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Abstracts

WS04-K1

Gene therapy and immunotherapy for brain tumors Kyogo Itoha,b and Joseph C. Gloriosob Immunology, Kurume University School of Medicine, Japan; bMolecular Genetics and Biochemistry, University of Pittsburgh Medical Center, USA

a

Development of new treatment modality is needed to improve the prognosis of brain tumors. Recent progress of basic and clinical research in the field of neuron oncology suggests that both gene therapy and immunotherapy could have promising features as new treatment modalities. This workshop takes five distinguished presentations related on gene therapy and immunotherapy for brain tumors. The first presentation reports new findings on glioma-infiltrating dendritic cells, while the second one shows that the invasion promoting effect of microglia on glioblastoma cells is suppressed by cyclosporine A. The third paper presents findings supporting a paradigm in which galectin-9 expression on low grade gliomas inactivates tumor-infiltrating T cells, which in turn results in promotion of tumor cell proliferation and invasiveness, using a CD4+ T cell clone established from glioblastoma tissue. The last two papers reports new data related to gene therapy. The fourth paper shows new findings on HSV vector-mediated co-delivery of mutant IkBa and HSV thymidine kinase against glioblastoma, while the last presentation indicates that suicide gene therapy using high-titer retrovirus vector is cytotoxic to the cancer stem cells derived from malignant gliomas. We hope active discussion on each of the five distinguished presentations to better understand their potential for development of clinically effective treatment modalities to glioblastoma multiforme. WS04-K2

HSV gene vectors and treatment of chronic pain and cancer J. C. Glorioso, P. Grandi, D. Krisky, W. F. Goins, J. Goss and D. F. Fink Department of Molecular Genetics and Biochemistry, University of Pittsburgh, School of Medicine, Pittsburg, USA Herpes simplex virus is a promising vehicle for delivery of genes to the nervous system. Our laboratory has exploited replication defective vectors for treatment of peripheral nerve diseases that include chronic pain and peripheral neuropathy and for treatment of central nervous system diseases that include cancer and Parkinson's disease. Experiments will be presented that describe current vector engineering for these applications and specific studies related to the treatment of chronic pain using enkephalin gene vectors. In addition, given the interest in the use of oncolytic HSV vectors for treatment of cancer, I will describe new vectors that rely on mutations affecting ICP0 and ICP22 for virus attenuation. These vectors have a highly improved profile for both safety and efficacy in brain tumor therapies. WS04-01

Functional and morphological characterization of tumorinfiltrating dendritic cells in malignant gliomas M. El-Beltagya, H.L. Khova, H.-P. Eugsterb, A. Fontanab, Y. Yonekawaa, and K. Freia Departments of aNeurosurgery and bInternal Medicine, University Hospital Zurich, Zurich, Switzerland Objective: Although many mechanisms are responsible for aggressive behavior of glioblastomas, the role of systemic immunsuppression by this tumors appears to be a critical component of the pathogenesis. Dendritic cells (DCs) play a critical role in orchestrating anti-tumor immunity.

Previous work implicates defects and/or impaired maturation/migration of DCs as a critical defect in the immune response against cancer. The aim of this study was to identify and characterize glioma infiltrating DCs by immunohistochemistry (IHC), FACS and functional assays. Methods: IHC was performed on 24 gliomas using DC-specific antibodies and the infiltrating mononuclear cell populations derived from six tumors were evaluated by three-color-FACS analysis. In addition, the stimulatory capacity of monocyte-derived DCs from tumor patients were compared with controls in an allogenic MLR and the levels of IL-12 and IFN-α production were assessed by ELISA. Results: DCs were found to be present in high grade gliomas at very low numbers and mainly in an immature state. The origin of glioma-derived DCs was mainly myeloid and the T cell stimulatory capacity was strongly reduced in allogenic MLR. Interestingly, the capacity of monocyte-derived DCs of patients was also reduced albeit not being statistically significant to controls. Conclusion: Taken together, these results suggest that the inhibition of DC development/function represents an additional mechanism by which gliomas can escape immune recognition. WS04-02

The invasion promoting effect of microglia on glioblastoma cells is inhibited by cyclosporine A Marcin Sliwa1, Darko Markovic2, Michael Synowitz2,3, Rainer Glass2, Aleksandra Wesolowska1, Malgorzata Zawadzka1, Helmut Kettenmann2, Bozena Kaminska1 1

Laboratory of Transcription Regulation, Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Str., 02-093 Warsaw, Poland; 2Cellular Neuroscience Group, Max Delbrück Center for Molecular Medicine (MDC), Robert Rössle Str. 10, 13092 Berlin, Germany; 3 Department of Neurosurgery, Helios Hospital Berlin, Hobrechtsfelder Chaussee 56, 13125 Berlin, Germany Tumor cells seem to recruit stromal and inflammatory cells to the tumor site and transform into tumor-supportive cells. Invasion of glioma cells into brain tissue is a hallmark of glioblastomas and contributes to the failure of current therapeutic treatments. Activated microglia are abundant in brain tumors and may support tumor growth and invasiveness. However, defense functions of microglial cells against glioma are compromised by the tumor, e.g. through impaired surface expression of MHC class II, the immune-suppressed microglia may still display tumorpromoting activity. We found that Cyclosporine A (CsA) can affect growth of glioma cells in vitro by inhibiting signaling pathways, which are essential for tumor proliferation and invasiveness. In this work, we demonstrate that migration of EGFP-transfected glioblastoma cells in organotypic brain slices was significantly inhibited by 1–30 μM CsA. This inhibitory effect was lost when glioblastoma cells were injected into microglia-depleted brain slices. In in vitro studies we demonstrate that microglia-derived factors increase glioma invasiveness in Matrigel assays which is associated with activation the PI-3K/Akt signaling pathway. CsA abolishes the invasion promoting effect of microglia. On the other hand, glioma-derived soluble factors induce morphological transformation of microglia into ameboid microglia, activate MAPK signaling, although production of pro-inflammatory factors such as nitric oxide or IL-6 was not observed. We found that CsA may interfere with glioma-associated microglia stimulation, turning them into more resting state. Our finding that CsA may impair invasive growth of glioma cells, at clinically relevant concentrations, provides a novel therapeutic strategy. WS04-03

The Galectin-9/TIM3 pathway and human glioma pathogenesis Lisa Bregolia, Mitsuomi Hirashimab, Vijay K. Kuchrooa, and David E. Andersona

Abstracts a

Brigham and Women's Hospital and Harvard Medical School, Boston, USA; bKagawa University Medical School, Kagawa, Japan

Approximately 15,000 patients die each year in the United States from GBMs. Two well-documented aspects of human malignant gliomas are that they are highly invasive and that immunity directed against them is ineffective. We hypothesize that glioma expression of Gal-9 regulates both of these aspects of glioma pathogenesis. Gal-9 expression by peripheral human tumors has a favorable clinical prognosis and appears to limit tumor cell metastasis/invasiveness. Thus, one prediction is that glioma expression of Gal-9 limits tumor cell proliferation and/or invasiveness. Indeed, we have found that siRNA-mediated reduction of Gal-9 in a primary GBM cell line enhances tumor cell proliferation. Moreover, culture of this cell line in TGF-β, which is expressed at higher levels in high-grade versus low-grade gliomas, reduces tumor cell expression of Gal-9. In addition, quantitative RT-PCR analysis of Gal-9 levels expressed by GBM tumor cells versus reactive astrocytes indicates that GBM tumor cells express lowers levels of Gal-9 in situ. On the other hand, Gal-9 has recently been identified as a ligand of TIM-3, and been shown to kill IFN-γ-secreting Th1 cells. To this end, we have cloned CD4+ T cells that infiltrate GBMs and found that exposure to recombinant Gal-9 inhibits IFN-γ secretion from activated Th1 cells, which can be blocked with antiTIM3 monoclonal antibody. Collectively, the data suggest a paradigm in which Gal-9 expression by low-grade gliomas inactivates infiltrating TIM3+CD4+ and CD8+ T cells, and that with increasing stages of malignancy, down-modulation of Gal-9 on gliomas promotes tumor cell proliferation and invasiveness. WS04-04

Combination gene therapy for glioblastoma involving herpes simplex virus (HSV) vector-mediated co-delivery of mutant IκBα and HSV thymidine kinase against glioblastoma Shusuke Moriuchia, Joseph C. Gloriosob, Motohiko Marunoc, Shuichi Izumotoc, and Toshiki Yoshiminec a

Department of Neurosurgery, Osaka National Hospital, Osaka, Japan; Department of Molecular Genetics and Biochemistry, University of Pittsburgh, USA; cDepartment of Neurosurgery, Osaka University School of Medicine, Osaka, Japan

b

To improve the effectiveness of HSV thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication defective HSV vector TOIκB expressing both HSV-TK and a mutant form of the NF-κB inhibitor IκBα (IκBαM) was developed. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF-κB was already translocated to the nucleus without receptor-dependent signaling by TNF-α. Following infection with TOIκB,

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nuclear translocation of NF-κB in U-87 MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOIκB for U-87 MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOIκB infection killed 85% of the cells compared to 20% killed by TOZ.1 infection. In the presence of GCV, these numbers increased to 95–100% for TOIκB and 80–85% for TOZ.1. The survival of nude mice implanted into the brain with U-87MG tumor cells was markedly prolonged by intratumoral TOIκB injection and GCV administration. Survival of TOIκB + GCV group was significantly longer (P < 0.02, Wilcoxon test) than for the control groups (TOZ.1 or TOIκB only, PBS or PBS + GCV). These results suggest that IκBαM expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo. WS04-05

Efficacy of suicide gene therapy using high-titer retrovirus vector for cancer stem cells derived from malignant glioma T. Yawataa, K. C. Parka, S. Toyonagaa, T. Chiharaa, N. Masahiraa, K. Ikenakab, K. Shimizua a

Department of Neurosugery, Kochi Medical School, Nankoku, Japan; Division of Neurobiology and Bioinformatics, National Institute for Physiological Science, Okazaki, Japan

b

Despite many efforts to develop effective therapy, the outcome of malignant glioma remains poor. Gene therapy for this disease using retroviral vector is attractive, because the virus can infect only mitotic cells. We established the packaging cell line PAMP51 producing high-titer retrovirus expressing suicide gene HSV-TK and concentrated the virus with titers as high as 1–1011–12 c.f.u./ml by low speed centrifugation. Using this retroviral vector, mouse glioma model was cured completely by administration of ganciclovir (GCV). Recently, several investigators identified a small population of cancer stem cells in brain tumor tissues and cell lines. These cancer stem cells forms spheres and maintains self-renewal capacity, tumorigenecity and multiple drug resistance. The existence of the cancer stem cells is thought to be a cause for recurrence of the tumors after treatment with chemotherapy, radiotherapy and surgical resection. Therefore, we investigated the therapeutic efficacy of suicide gene therapy for the cancer stem cells derived from glioma cells. A high transduction rate and gap junction mediated bystander effect allowing killing of non-transduced cells enhance the efficacy of this suicide gene therapy. A lower efficiency of transduction with retrovirus vector and the expression of connexin 43, a component of gap junction were observed in cancer stem cells. This result suggests that non-transduced cancer stem cells can be killed via bystander effect. To gain insight about bystander effect in cancer stem cells, we are currently performing immunohistochemistry studies in mouse glioma model.

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Abstracts

Workshop 5: EAE: Pathogenesis & regulation

Abstracts WS05-K1

Immune regulation in CNS autoimmune disease David C. Wraith⁎ and Yoh Matsumoto$ ⁎Department of Cellular and Molecular Medicine, University of Bristol, United Kingdom, $Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Japan Studies undertaken in the speaker's laboratory have shown that a distinct population of regulatory cells can be induced by injection of soluble peptides. Such peptide treatment led to the generation of regulatory T (PI-Treg) cells that were anergic, failed to produce IL-2 but responded to antigen by secreting IL-10. The cells were predominantly CD25−and CTLA-4+ and their anergic state was reversed by addition of IL-2 in vitro. PI-Treg cells were able to suppress both proliferation and IL-2 production from naïve T cells in vivo, suppression being abrogated by neutralisation of IL-10. Depletion of CD25+ cells did not affect the suppressive properties of PI-Treg cells. Furthermore, PI-Treg cells could be generated in mice that do not spontaneously generate CD25+ regulatory cells. These results suggest that ‘natural’ and ‘induced’ regulatory cells fall into distinct subsets. This distinction has recently been confirmed by demonstrating that PI-Treg cells do not express FoxP3, a member of the forkhead-winged helix family of transcription factors that controls differentiation of ‘natural’ CD25+ Treg cells. Recent work has led to the identification of a novel set of genes upregulated in T cells rendered tolerant by peptide therapy. Some of these genes control cell division and/or cytokine production and thereby control tolerance. Clarification of the mechanisms controlling antigenspecific tolerance will lead to the development of safer and more effective vaccines for the treatment of allergic and autoimmune diseases. WS05-01

A new model of experimental autoimmune encephalomyelitis (EAE) mediated by memory CD4 cells: Differential functions of co-stimulatory pathways in regulating autoreactive memory CD4+ T cells in vivo W. Elyamana, P. Kivisäkka, Vijay K. Kuchrooa, Mohamed H. Sayeghb and Samia J. Khourya

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10 and reduced IFN-γ secretion was observed in effector cell recipients after CTLA4-Ig treatment, while ICOS-L blockade resulted in a similar shift in memory cell recipients. Our data suggest the presence of specific mechanisms of disease mediated by memory CD4+ cells and indicate a differential role of co-stimulatory pathways in regulating effector versus memory T cells. WS05-02

Differential regulation of IL-12/IFN-γ and IL-23/IL-17 axes might account for discrepancy in susceptibility towards experimental autoimmune encephalomyelitis between Albino Oxford and Dark Agouti rats M. Markovica, Dj. Miljkovicb, M. Momcilovicb, S. Stosic-Grujicicb, Z. Ramica, M. Mostarica-Stojkovica a

Institute for Microbiology and Immunology, School of Medicine, University of Belgrade, Serbia; bInstitute for Biological Research “Sinisa Stankovic”, Belgrade, Serbia

We have previously shown that Albino Oxford (AO) rats are resistant to induction of experimental autoimmune encephalomyelitis (EAE). In this study we compared the production of cytokines with the presumed role in the pathogenesis of EAE, namely interferon γ (IFN-γ) and interleukins (IL) 12, 17 and 23, in AO rats and EAE susceptible Dark Agouti (DA) rats. To this end, draining (popliteal) lymph node cells (DLNC) were collected 6 days post-immunization with spinal cord homogenate and carbonyl iron as an adjuvant. The cells were stimulated with concanavalin A (ConA) or specific antigen myelin basic protein (MBP) and the production of cytokines was measured on mRNA and protein level using RT-PCR and ELISA, respectively. DLNC of DA rats expressed higher levels of mRNA and produced more IFN-γ and IL-17 than AO rats. Since IL-12 and IL-23 are potent inducers of IFN-γ and IL-17 synthesis, respectively, we further analyzed mRNA expression of their subunits: p40 (common for IL-12 and IL-23), p35 (IL-12) and p19 (IL-23). Interestingly, while the expression of p35 and p19, similarly to other cytokines, was higher in DA animals, AO rats expressed more p40 mRNA. Nevertheless, DA rats made more p40 protein as well as IL-12. Taken together, our data suggest that the differential regulation of IL-12/IFN-γ and IL-23/IL-17 axes, and p40 in particular, in the inductive phase of EAE could be responsible for the discrepancy in susceptibility to EAE between these two strains. WS05-03

a

Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; bTransplantation Center, Brigham and Women's Hospital and Children's Hospital, Boston, MA, USA

A limitation of many experimental models of autoimmune disease is that they focus on targeting prevention of disease or on therapy of early disease triggered by naïve T cells. Memory T cells are less dependent on positive co-stimulatory signals than naïve cells and also less susceptible to tolerance induction. An important clinical goal is to develop strategies that suppress the function of chronically activated memory T cells. We developed a new model of EAE that is induced by antigen-specific memory CD4+ cells generated from mice immunized with MOG > 100 days prior to transfer. Memory CD4+ cells induced more severe EAE than effector T cells, both clinically and pathologically. T cells from memory cell recipients proliferated more in response to MOG and secreted higher levels of IL-2, IL-17 and IFN-γ compared to recipients of recently generated effector cells. Percentages of CD4+CD25+Foxp3+ cells were not affected, indicating that differences in clinical disease were not due to differential expansion of regulatory T cells. Effector cell-mediated EAE was suppressed by CTLA4-Ig, but aggravated by anti-ICOS-L treatment. By contrast, memory cellmediated disease was suppressed by ICOS-L blockade, while CTLA4Ig had no effect. A shift in the cytokine balance towards increased IL-

IL-18-independent IL-18Ra engagement is required for the development of autoimmunity I. Gutchera, E. Uricha, M. Prinzb and B. Bechera University Hospital Zurich, Zurich, Switzerland; bUniversity of Göttingen, Göttingen, Germany

a

IL-18 is considered to be a vital cofactor, together with IL-12, for the polarization of TH1 cells. TH1 cells have long been suspected to be the major pathogenic cell population in autoimmune diseases such as rheumatoid arthritis or multiple sclerosis. However, we and others could recently, firmly establish that in the animal models for RA and MS, IL-12 is not a disease promoting factor and that TH1 polarized cells are not a prerequisite for disease development. We therefore wanted to determine the role of another TH1-polarizing cytokine namely IL-18 and its receptor in the context of EAE. In line with the IL-12 findings, we found that IL-18-deficient mice are fully susceptible to EAE induced by immunization with MOG peptide. Surprisingly however, we discovered that mice deficient in IL-18Ra are EAE-resistant, suggesting the presence of an alternative ligand with encephalitogenic properties. We could establish that loss of IL-18Ra lesions an accessory leukocyte and not lymphocytes directly. We further

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Abstracts

demonstrate here that IL-18Ra signaling promotes the generation of IL-17producing CD4+ T cells, which are now considered to be the major pathogenic population in EAE as well as other autoimmune diseases. Our results establish that IL-18 and TH1 development are dispensible for EAE pathogenesis, while IL-18Ra signaling by an as yet unidentified ligand (IL18RL) is absolutely essential for the generation of encephalitogenic TH-17 cells. WS05-04

Antibodies to native myelin oligodendrocyte glycoprotein are critical for severe chronic experimental autoimmune encephalomyelitis and demyelination in mice and marmosets a

a

a

b

c

N. Heijmans , A. Jagessar , P. Smith , T. Menge , J. M. van Noort , E. Blezerd, B. 't Harta, D. Pham-Dinhe, J. Bauerf and S. Amora a

Department of Immunobiology, BPRC Rijswijk, The Netherlands; Neuroimmunology Laboratories, Department of Neurology, University of California, San Francisco, USA; cDivision of Biomedical Research, TNO Pharma, Leiden, The Netherlands; dImage Sciences Institute, University Medical Centre Utrecht, Utrecht, The Netherlands; eInstitut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie, Hôpital de la Salpêtrière, Paris, France; fBrain Research Institute, University of Vienna, Austria b

Myelin oligodendrocyte glycoprotein (MOG) is a powerful encephalitogen for experimental autoimmune demyelination. However, the use of MOG peptides or recombinant proteins representing part of the protein, fail to address the possible pathogenic role of the full-length myelin-derived protein expressing post-translational modifications. Immunisation with central nervous system (CNS) tissues from wild-type (WT) and MOGdeficient (MOG−/−) mice demonstrates that MOG in myelin is necessary for the development of chronic demyelinating experimental autoimmune encephalomyelitis (EAE) in mice and marmosets since myelin from WT mice induces chronic EAE while from MOG−/− myelin induced a mild selflimiting monophasic disease. Following immunisation all animals developed T-cell responses to recombinant mouse MOG (rmMOG) indicating that MOG released from myelin is antigenic. The lack of chronic disease following immunisation with MOG−/− myelin indicated that such responses were not pathogenic. Antibodies in WT but not MOG−/− myelin immunised animals recognised rmMOG and a MOG transfected fibroblast cell line but not MOG

peptides. In all animals antibodies to myelin basic protein, oligodendrocyte specific protein, proteolipid protein and galactocerebroside were observed but did not correlate with disease severity. The data indicate that the conformational form of MOG is crucial for generation of pathogenic antibodies while glycosylation of MOG protein is not required for such antibody recognition. These data reveal that immunisation with the post-translational modified form of MOG in myelin promotes chronic autoimmune demyelinating neurological disease probably due to pathogenic antibodies to unmodified protein rather than linear epitopes or T cells. WS05-05

Real time imaging of autoreactive effector T cells in CNS lesions of Autoimmune Encephalomyelitis N. Kawakamia, A.S. Fariasa, C. Cordiglieria, F. Odoardia, H. Wekerlea, and A. Flügela a

Department of Neuroimmunology, Max Planck Institute for Neurobiology, Martinsried, Germany

Experimental Autoimmune Encephalomyelitis (EAE) is induced by brain antigen-specific CD4+ T cells. Upon transfer to healthy recipients the cells induce massive CNS inflammation and severe paralytic disease. Using two-photon microscopy we visualize the behavior of retrovirally labeled MBP-specific T cells in the CNS of Lewis rats during different phases of clinical EAE. Imaging of intact animals and of acute spinal cord slices enabled us to track autoreactive effector T cells in meningeal/ perivasular areas as well as deep within the CNS parenchyma. We report here on two major findings: effector T cells redistribute in the course of EAE. In the primary clinical phase (days 2.5 after T cell transfer) the effector cells were restricted to perivascular locations. Beginning day 3 after transfer, however, they spread into the meninges and penetrated deeply into the CNS parenchyma. Their motility behavior profoundly changed over the course of EAE. T cell in the perivascular cuffs moved predominantly along the vessel walls. Cells within the CNS parenchyma moved seemingly non-directed. The cells displayed two distinct motility patterns: “motile” cells rapidly moved through the compact white matter, while “stationary” cells were attached and moved around a fixed anchor point. The ratio between these two fractions changed significantly over time. Whereas in early EAE the number of motile cells exceeded the one of the stationary cells this ratio became inverted during late EAE.

Abstracts

Workshop 6: MS therapy

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Abstracts

WS06-K1

Immunointervention in MS — How appropriate is it? Krzysztof W. Selmaja, Jun-ichi Kirab a

Department of Neurology, Medical University of Lodz, Poland; Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University

b

During the last decade, we have witnessed unprecedented progress in the therapy of multiple sclerosis (MS). Despite the obvious benefit for clinical outcome, the new therapies have extensively tested the autoimmune concept of MS. Although evidence for autoimmune mechanisms in MS is largely indirect, proof can only be achieved by the demonstration that therapy based on the correction of immune deviation is able to restore neurological function. How do the drugs currently used in MS address this aim? Interferon beta and Glatiramer, the two most widely used immunomodulating drugs in MS, are effective in reducing disease activity only by 30%. Does it mean that the autoimmune concept is challenged or do immune mechanisms in MS require additional points of interception? The clinical heterogeneity of MS may imply that several independent mechanisms exist and these might need to be separately targeted. In this regard, the use of nonantigen specific immune therapies aimed at a common pathway during the generation of the immune response might overcome some difficulties related to the claimed heterogeneity of MS. However, the recent history of side effects induced by Natalizumab have shown that antigen non-specific therapy can culminate in the severe loss of immune surveillance. Therefore, more selective immune therapies based upon the antigen reactivity of specifically-defined cell subpopulations or an effector immune mediator, may offer more promise. An additional point of complexity in MS therapy has been raised by the demonstration that neurodegeneration and brain atrophy represent important pathologic events of this disease. Currently, several immune-related molecules are being tested in clinical trials in MS and in the near future we should become more knowledgeable on how a correction of the immune response at different levels influences the course of the disease. WS06-01

Immune surveillance of the central nervous system in multiple sclerosis patients treated with natalizumab Stuve, Olaf1; Marra, Christina M.2; Jerome, Keith R.3; Cook, Linda3; Cravens, Petra D.1; Cepok, Sabine4; Frohman, Elliot1; Phillips, Theodore5; Arendt, Gabriele4; Hemmer, Bernhard4; Monson, Nancy1; Racke, Michael1 1

Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA; 2Neurology, University of Washington, Seattle, WA, USA; 3 Laboratory Medicine, University of Washington, Seattle, WA, USA; 4 Neurology, Heinrich Heine University, Dusseldorf, Germany; 5Multiple Sclerosis Center at Texas Neurology, Baylor University Medical Center, Dallas, WA, USA; 6Center for Immunology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA

significantly lower in natalizumab-treated MS patients compared with OND patients and untreated MS patients. Natalizumab therapy decreased the CD4:CD8 ratio in the CSF to levels similar to that of HIV-infected patients. JCV DNA was not detected in natalizumab-treated patients. Six months after cessation of therapy, low lymphocyte counts in the CSF persisted, whereas the CD4:CD8 ratio normalized. The patient with the highest total leukocyte, CD4+ and CD8+ T cell counts in the CSF experienced a clinical relapse. Interpretation: Our data suggest that a low CSF CD4:CD8 ratio in natalizumab-treated patients may confer an increased risk of developing PML. WS06-02

Successful therapy of multiple sclerosis (MS) targeting highaffinity IL-2 receptor reveals regulatory role of CD56bright NK cells on T cell responses in humans B. Bielekovaa,b, M. Catalfamoc, S. Reichert-Scrivnera, A. Packera, M. Cernaa, T. A. Waldmannd, H. McFarlanda, P. Henkartc and R. Martina,e Neuroimmunology Branch, NINDS, NIH, MD, USA; bWaddell Center for MS, Department of Neurology, University of Cincinnati, OH, USA; c Experimental Immunology Branch and dMetabolism Branch, NCI, NIH, MD, USA; eCatalan Insttitute for Research and Advanced Studies, Unidad de Neuroimmunologia Clinica, Hospital Universitari Vall D-Hebron, Barcelona, Spain

a

Administration of Daclizumab, a humanized monoclonal antibody directed against the IL-2 receptor α-chain, strongly reduces brain inflammation in multiple sclerosis (MS) patients. The purpose of this study was to elucidate the mechanism of action of Daclizumab in MS. The percentages and absolute numbers of immune cells were tracked by ex-vivo flow cytometry. Functional assays included flow-cytometry based proliferation and intracellular cytokine production; transwell T cell-survival assays and chromium-release cytotoxicity assays. Contrary to expectations, Daclizumab treatment lead to only a mild functional blockade of CD4+ T cells, the major candidate in MS pathogenesis. Instead, Daclizumab therapy was associated with a gradual decline in circulating CD4+ and CD8+ T cells and significant expansion of CD56bright natural killer (NK) cells in vivo, and this effect correlated highly with the treatment response. In vitro studies showed that NK cells inhibited T cell survival in activated PBMC cultures by a contact-dependent mechanism. Moreover, CD56bright NK cells isolated from Daclizumabtreated patients were directly cytotoxic to activated, but not resting autologous T cells in perforin-dependent manner. Positive correlations between expansion of CD56bright NK cells and contractions of CD4+ and CD8+ T cell numbers in individual patients in vivo provides supporting evidence for NK-cell mediated negative immunoregulation of activated T cells during Daclizumab therapy. Our data support existence of an immunoregulatory pathway wherein activated CD56bright NK cells inhibit T cell survival. This immunoregulation has potential importance for the treatment of autoimmune diseases, transplant rejection and toward modification of tumor immunity. WS06-03

Objective: To evaluate whether treatment of multiple sclerosis (MS) patients with natalizumab, an antibody against VLA-4, interferes with immune surveillance of cerebrospinal fluid (CSF) and peripheral blood (PB). Background: Natalizumab therapy was recently associated with development of progressive multifocal leukoencephalopathy (PML), a demyelinating disorder of the CNS caused by JC virus (JCV) infection. Design/Methods: Cell numbers and cellular phenotypes in CSF and PB were analyzed by flow cytometry in MS patients treated with natalizumab, untreated MS patients, patients with other neurological disease (OND), and HIV-infected patients. JCV DNA in the CSF and PB of these patient cohorts was quantified by kinetic PCR. Results: CSF total leukocyte counts, CD4+ and CD8+ T cells, CD19+ B cells and CD138+ plasma cells were

Increased apoptotic elimination of activated T-cells following Campath-1H as a treatment of multiple sclerosis J.L. Jones, S.A.J. Thompson, J. Deans, V.H. Robertson, D.A.S. Compston and A.J. Coles Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Multiple sclerosis (MS) is believed to be an autoimmune disease in which aberrant immune responses lead to demyelination, loss of

Abstracts oligodendrocytes and acute axonal transection. Suggested mechanisms for the breakdown in immune self-tolerance include impaired CD4+CD25hi function and the dysregulated apoptotic elimination of autoreactive Tcells. Campath-1H is a monoclonal antibody that induces T-cell lymphopaenia and reduces the MS relapse rate by 90%. By flow cytometry we investigated the effect of Campath-1H on the apoptotic death of T-cells emerging into the lymphopaenic environment. We observed a significant increase in spontaneous T-cell death and anti-Fas induced T-cell death (5% to 30%, and 10% to 50% respectively). Pancaspase inhibition largely reversed this, confirming cell death was predominantly apoptotic. Increased T-cell death was also observed following stimulation with myelin basic protein (MBP) and other specific antigens. Following treatment, however, stimulation with MBP and other recall antigens led to a reduction in cell death at day 3, when compared to unstimulated cells or to those exposed to neo-antigen (keyhole limpet haemocyanin). This effect disappeared at day 8. This implies that factors released early in the memory response of peripheral blood mononuclear cells extracted from a lymphopaenic environment protect against apoptosis. Mechanisms leading to the altered apoptotic state of T-cells and their altered response to MBP following Campath1H are discussed. Elimination of activated T-cells is important in immune homeostasis and in the avoidance of immunopathology. Our findings suggest that Campath1H's efficacy may, in part, be due to increased apoptotic elimination of activated T-cells. WS06-04

Frequency of CD4+Foxp3+ T regulatory cells (Tregs) in peripheral blood of multiple sclerosis: Comparison to studies identifying Tregs as CD4+CD25high cells Takayuki Kondo, M.D.1, Takamitsu Mizota, M.D.1, Takahiko Saida, M.D.2, Hiromi Yokoyama, M.D.1, Mika Komori, M.D.2, Makoto Matsui, M.D.3, Noritoshi Shibuya, M.D.1, Hidenori Matsuo, M.D.1 1

National Hospital Organization, Nagasaki Medical Center of Neurology, Nagasaki, Japan; 2National Hospital Organization–Utano National Hospital, Kyoto, Japan; 3 Department of Neurology, Kanazawa Medical University, Kanazawa, Japan

Multiple sclerosis (MS) is assumed to result from the breakdown of peripheral tolerance. Since CD4+Foxp3+ T regulatory cells (Tregs) play roles in maintaining peripheral tolerance, it is important to determine if dysregulation of Tregs contributes to the onset and/or progression of MS. Although previous studies identified human Treg as CD4+CD25high cells, activated T cells also express CD25 molecule. To specifically assess the frequency of Tregs, we analyzed expression of Foxp3 by CD4+CD25+ cells. Methods: PBMC were obtained from 19 consenting patients with MS and 13 healthy controls (HC). Among the patients enrolled, 11 patients were receiving IFN-beta therapy. Cells were stained with anti-human CD4 PECy5 (RPA-T4) and anti-human CD25 PE (BC9), and then anti-Foxp3 FITC

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(PCH101) intracellularly. Expression of these molecules was determined by FACSCalibur. Results: CD4+CD25+ cells were divided into CD25low and CD25high fractions. In HC samples, an average of 85% of CD4+CD25high expressed Foxp3, but a considerable number of Foxp3+ cells were also found among CD4+CD25low. In contrast, Foxp3-negative non-Tregs constituted more than 50% of CD4+CD25high in five patients on IFN-beta therapy and in one not receiving IFN-beta therapy. The proportion of Tregs in CD4 T cells in MSPB was significantly decreased compared with that of HC-PB (2.83% vs. 5.51%, p < 0.05, t-test). Interestingly, IFN-beta therapy significantly increased the Treg frequency in MS-PB up to an average of 4.40% (p < 0.05, t-test). Discussion: We show a decrease in the frequency of circulating Tregs in MS. Of note, IFN-beta may partially reverse the deficit of Tregs in MS. WS06-05

Prognostic relevance of antimyelin antibodies for the progression to multiple sclerosis after a first demyelinating event: Results of the BENEFIT trial J. Kuhlea, M. Mehlinga, H.-P. Hartungb, M. S. Freedmanc, C. H. Polmand, G. Edane, D. H. Millerf, X. Montalbang, L. Bauerh, S. Dahmsh, C. Pohlh, R. Sandbrinkh, R. Lindberga and L. Kapposa University Hospital, Basel, Switzerland; bHeinrich-Heine-Universität, Düsseldorf, Germany; cThe Ottawa Hospital, Ottawa, Ontario, Canada; d Vrije Universiteit Medical Center, Amsterdam, The Netherlands; eClinique Neurologique, Rennes, France; fThe National Hospital for Neurology and Neurosurgery, London, UK; gHospitals Vall d'Hebron, Barcelona, Spain; h Schering AG, Berlin, Germany

a

Patients with a clinically isolated syndrome (CIS) have a high risk of progression to clinically definite multiple sclerosis (CDMS). However, individual prognosis is unpredictable. Berger et al. reported a significantly increased risk of conversion to CDMS in patients with antibodies against myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) (NEJM 2003; 349:139–145). In the BEtaferon(r) in Newly Emerging multiple sclerosis For Initial Treatment study (BENEFIT), a total of 468 patients with a CIS and at least two clinically silent brain MRI lesions were investigated. In this multicentre, double-blind trial, patients were randomised to IFNB-1b (n = 292) or placebo (n = 176) and treated for 24 months or until CDMS was diagnosed. Regular visits were scheduled for the collection of data on neurological disability and MRI before treatment and at study month 3, 6, 9, 12, 18 and 24. We measured serum anti-MOG and anti-MBP antibodies at baseline by Western blot. At month 24, 28% (IFNB-1b) and 45% (placebo) of the patients had reached CDMS. Correspondingly, 69% and 85% had reached “McDonald MS”. Data on the relation of antibodies to conversion to CDMS and “McDonald MS” will be presented. These analyses will allow an evaluation of the clinical relevance of antiMOG/MBP antibody testing in a representative CIS population thoroughly characterized clinically and by MRI in the setting of a clinical trial.

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Workshop 7: Experimental studies on autoimmune neuropathies

Abstracts

57

WS07-K1

WS07-02

Autoimmunity to glycolipids in neuropathy; Role of T-cells and of CD1 dependent antigen presentation

Campylobacter jejuni cell body antigen induced experimental autoimmune neuritis

N. Latov⁎ and S. Kusunoki⁎⁎ ⁎Deparment of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, USA, and ⁎⁎Department of Neurology, Kinki University School of Medicine, Osaka, Japan Autoimmunity to glycolipids is implicated in the pathogenesis of several neuropathic syndromes, in association with IgM monoclonal gammopathies, or following exposure to such bacteria as Campylobacter jejuni (Cj), that has cross-reactive lipopolysaccharides (LPS). A great deal has been learned about the anti-glycolipid antibodies, but relatively little is known about the role of T-cells in the associated neuropathies. Systemic transfer of IgG, but not of IgM antibodies, without T-cells, has been demonstrated to cause neurological dysfunction in experimental adult animals. IgMs might require the presence of T-cell reactivity, against the same or another nerve antigen, to exert their effects. Inflammatory T-cells could provide chemotactic factors or activate the monoclonal B-cells, facilitate IgM penetration through the blood nerve barrier, expose cryptic glycolipid antigens, or create a pro-inflammatory environment that amplifies the response. High titers of IgG anti-ganglioside antibodies are closely associated with variants of the Guillain–Barre syndrome. The antibodies are T-cell dependent as they are of the IgG1 and IgG3 isotypes. The T-cells might additionally damage the nerves via T-cell dependent effector mechanisms. LPS activates B-cells via T-cell independent mechanisms, so that the development of a T-cell response following Cj infection, is unexplained. However, the CD1 pathway of lipid antigen presentation has recently been linked to both GBS and CIDP, so that if Cj LPS is presented to T-cells via the same pathway, it might also induce a T-cell dependent anti-glycolipid response. The role of T-cells and of CD1 mediated antigen presentation warrant further investigation. WS07-01

C.-S. Koha, S. Miyoshia, K. Oanaa, K. Nakayamaa, T. Eharaa, M. Kyogashimab and T. Shinc Shinshu University, Matsumoto, Japan; bAichi Cancer Center Research Institute, Nagoya, Japan; cCheju National University, Jeju, South Korea

a

The etiology of the Guillain–Barré syndrome (GBS) still remains elusive. GBS is currently viewed as a group of syndromes with several distinctive subtypes. 15–40% of the patients with GBS develop the syndrome after being infected by the Gram-negative bacterium Campylobacter jejuni (C. jejuni), a leading cause of acute gastroenteritis in humans. The recognition of C. jejuni infection as a frequent antecedent event in GBS has raised many questions about specific disease mechanisms involved. Experimental autoimmune neuritis (EAN) is considered the in vivo model of GBS. There has been, however, no animal model of EAN which is induced by C. jejuni cell body antigen. We here report that a new form of EAN was successfully induced for the first time in animals by using C. jejuni cell body antigen. C. jejuni, Penner serotypes O:19, was isolated from the patient with GBS and cultured. EAN could be induced in Th1 bearing animals such as Lewis rats, SJL/J mice, and DBA/2 mice, but not be induced in Th2 bearing animals such as C57BL/6 mice by a single sensitization of C. jejuni cell body antigen in complete Freund's adjuvant. Animals with EAN develop limp tail to paraparesis. By histology inflammatory cell infiltration was observed in sciatic nerves, especially in perineurium, and cauda equine. Immunohistological study showed that infiltrated cells were ED1, a marker of macrophage, positive. Taken together, these findings suggest that Th1 immune responses may play an important role in the pathogenesis of the new form of EAN by using C. jejuni cell body antigen. WS07-03

Effects of Dps from Campylobacter jejuni on nerve conduction and nodal sodium channel in vivo

The effect of rho-kinase inhibitor fasudil in experimental autoimmune neuritis

M. Minoharaa, H. Piaoa, Y. Mizunoeb, M. Osoegawaa, H. Muraia, J. Kiraa

Arnold Angelo M. Pineda, MD1, Motozumi Minohara, MD, PhD1, Xiaojia Sun, MD1, Takaaki Ishizu, MD1, Hiroaki Shimokawa, MD2, Jun-ichi Kira, MD, PhD1

a

Department of Neurology, Kyushu University, Fukuoka, Japan; Department of Bacteriology, Kyushu University, Fukuoka, Japan

b

1

Objective: Campylobacter jejuni (C. jejuni) is now the most recognized antecedent cause of Guillain–Barre syndrome (GBS). Recently, we identified and characterized the iron-binding protein Dps from C. jejuni (C-Dps). The aim of this study was to determine whether C-Dps contributes to the pathogenesis of GBS. Methods: We tried to evaluate the effect of exposure to C-Dps on nerve conduction after intraneural injection. Binding studies of C-Dps to rat neuronal tissues were performed. Furthermore, we evaluated the clustering of sodium channels at the node of Ranvier and the direct effect of C-Dps to sodium channels using the whole-cell patch-clamp method. Results: We found that (a) C-Dps, but neither PBS nor heat-denatured CDps, when injected into the rat sciatic nerve significantly decreased proximal-compound muscle action potential (CMAP), while motor conduction velocity did not change significantly, (b) C-Dps binds to the nodes of Ranvier and the outer surfaces of myelin sheath, (c) in the C-Dps injected sciatic nerve, immunostaining of the sodium channel was markedly decreased at the node of Ranvier, (d) C-Dps had no direct effect on sodium channels in the rat hippocampal CA1 neuron. Conclusions: C-Dps has direct neuro-toxicity mediated by the downregulation of sodium channels at node of Ranvier. These mechanisms may contribute to the axonal damage in GBS with preceding C. jejuni infection.

Department of Neurology Neurological Institute, Graduate School of Medical Sciences, Kyushu University,Fukuoka, Japan; 2Department of Cardiovascular Medicine, Tohoku University, Sendai, Japan

Objective/methods: To study the potential role of rho-kinase inhibitor fasudil in the course of EAN (Experimental autoimmune neuritis), we induced EAN in Lewis rats using P0 peptide 180–199. In the preventive group, PBS (n = 14) and fasudil 100 mg/kg/day (n = 13), rats were immunized and PBS or fasudil were delivered via Alzet minipump starting day-2 until day 30 post-immunization. On Day 10, splenocytes were harvested, cultured and assessed for incorporation of [3H] thymidine for proliferation assays. Cytokine assays (IFN-γ and IL-4) were also done. Weight and clinical scores were recorded daily. The treatment group was divided into two: control, PBS (n = 6) and fasudil 100 mg/kg/day (n = 7) respectively. Fasudil was started at the onset of clinical signs (days 12–13) delivered continuously also via Alzet miniosmotic pump for 28 days. Results: In the preventive study, the incidence of EAN was 78% (11/14) in the PBS group while it was 38% (5/13) for fasudil and significantly different using Fisher's exact test (p = 0.054). The mean clinical scores were also significantly lower in the fasudil-preventive group than the PBS group using Mann–Whitney U-test (p < 0.05) on days 14–23 with

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days 21–22 (p value < 0.001) of EAN. Proliferation assays on Day 10 also showed significantly higher [3H] thymidine incorporation in the PBS group than the fasudil-preventive group. While the IFN-γ of the culture supernatant also showed higher levels in the PBS group as well as higher IFN-γ/IL-4 ratio also for PBS. In the treatment study, mean clinical scores were also significantly lower in the fasudil-treatment group than the PBS group on days 21–25 post-immunization with a p value <0.05. Conclusion: Fasudil hydrochloride shows promise of effectiveness in promoting and probably hastening neurological recovery during the course of EAN.

reactive splenocyte medium induced a transient PDR reduction, accompanied by ARP accumulation. Intra-neural injection of synthetic ARP induced a similar reduction in PDR. Furthermore, systemic treatment with an antisense oligonucleotide, EN101, which suppresses AChE-R expression attenuated conduction block formation following intra-neural reactive splenocyte medium injection. Thus AChE-R and ARP may play a key role in inflammation-associated neuropathies, and EN101 might emerge as a novel treatment for such neuropathies. WS07-05

WS07-04

The role of membrane attack complex (MAC) in complement component C6 deficient Lewis rat

“Readthrough” acetylcholinesterase facilitates inflammation associated neuropathies in an antisense suppressible manner

Giang T. Tran, Nicole Carter, Murray Killingsworth, Masaru Nomura, Bruce M. Hall, and Suzanne J. Hodgkinson

A. Doria, T. Saara, G. Iferganea, H. Soreqb and I. Wirguina

Research Laboratory, University of New South Wales, Department of Medicine, Liverpool Hospital, Liverpool NSW 2170 Australia

a

Neuroimmunology Laboratory, Department of Neurology, Soroka Medical Center, Ben-Gurion University, Beer-Sheva, Israel; b Department of Biological Chemistry, Hebrew University of Jerusalem, Jerusalem, Israel Inflammatory factors that penetrate through a disrupted blood–nerve barrier may play a common pathophysiological role in various neuropathies. Disruption of the blood–nerve barrier concomitant to induction of systemic inflammation by lipopolysaccharide (LPS) administration caused a transient conduction block in rats. LPS administration leads to proteolytic cleavage of the stress-induced “readthrough” acetylcholinesterase variant (AChE-R), and accumulation of its distinct cleavable C-terminal peptide (ARP). We therefore examined the involvement of AChE-R and ARP in inflammation-associated nerve conduction impairments. Dissociated rat splenocytes were reacted in vitro with Campylobacter jejuni (Cj) or E. coli LPS. Cell-free reactive splenocyte medium was injected intra-neuronally to rat sciatic nerves. Medium from untreated splenocytes served for control. Compound muscle action potentials recorded from the intrinsic foot muscles following stimulation proximal- and distal to the injection site were recorded, and the proximal-to-distal amplitude ratio (PDR) was calculated, representing conduction efficiency. Nerve segments that include the injection site were collected for analyses. AChE-R mRNA was identified by in situ hybridization within the cytoplasm of Schwann cell bodies, and myelin sheaths. Immunohistochemistry identified ARP within the cytoplasm of Schwann cells, and additionally within axons. Intra-neural injection of

The role of antibody deposition and complement activation, especially the role of membrane attack complex (MAC) in the mediation of injury in experimental allergic neuritis (EAN) is thought to be critical. In previous studies, we have examined the course of active experimental allergic encephalomyelitis (EAE) in a strain of PVG rats that are totally deficient in the C6 component of complement (PVG/C6−/−) thus unable to assemble MAC. These PVG/C6−/− rats developed a less severe EAE compared with PVG/C rats following immunization with myelin basic protein (MBP). However, the role of complement in demyelination could not be examined because in the EAE model there is limited central nervous system demyelination. Furthermore, PVG rats are not susceptible to either active or passive EAN. We therefore backcrossed PVG/C6−/− onto the Lewis rat, an EAN susceptible train. After 10 generations we have a congenic Lewis rat that is deficient in C6 (Lewis/C6−/−) that is unable to form MAC. Otherwise, these rats are healthy and immunologically identical to normal Lewis rats. This strain of congenic Lewis rat will allow us to directly examine the role of MAC in EAN. In this study we have shown that Lewis/C6−/− can be susceptible to EAN, but with a lower disease severity as compare to the wild type Lewis rats. Similarly, the level of demyelination in Lewis/C6−/− was significantly less than wild type Lewis. These results suggest that although MAC has a role in demyelination and the overall pathogenesis of the disease, but not essentially a sole factor to these processes.

Abstracts

Workshop 8: Autoantibodies in CNS diseases

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WS08-K1

Antibodies in nervous system diseases

Conclusions: This study demonstrated a high prevalence of serum anti-NAE autoantibodies in patients with HE. Anti-NAE autoantibodies are emphasized, instead of anti-thyroid antibodies, as a useful serological diagnostic marker of HE, and should be included in the diagnostic criteria of HE.

A. Vincenta, and K. Tanakab WS08-02 a

Neurosciences Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK; bDepartment of Neurology, Brain Research Institute, Niigata University, Assahimachi-Dori, Niigata, 9518585 Japan The roles of autoantibodies in central nervous system (CNS) diseases, such as multiple sclerosis, are not clear, but there is increasing evidence for specific autoantibodies to neuronal proteins as markers for other paraneoplastic and non-paraneoplastic neurological diseases. Onconeural antibodies are usually measured by indirect immunohistochemistry/ immunofluorescence but can also be measured by commercial immunoblotting tests. The antibodies (to Hu, Yo, Ri, Ma/Ta, CRMP5/CV2, Amphiphysin, Tr) are highly disease specific, are often found at very high titer, and usually indicate the presence of a particular tumour (e.g. small cell lung cancer, ovarian etc.). However, there are many unanswered questions concerning their relevance; for instance, most of the onconeural antibodies are not thought to be pathogenic and the associated diseases are likely caused by cytotoxic T cells. Whether these are controlled by specific regulatory cells is one question that needs to be answered. There are also several non-paraneoplastic conditions in which there are potentially causative antibodies. Voltage-gated potassium channel antibodies are associated with (usually non-paraneoplastic) peripheral and CNS disorders and are likely to be pathogenic, although this has not been proved for the CNS disorders. Glutamate receptor antibodies can activate the receptors and mimic excess glutamate, leading to neuronal loss. Enolase antibodies are being identified in several diseases (Hashimoto's, retinopathies) and may induce calcium release from internal stores. In addition, there are a number of other antibodies, and techniques employed to identify further specificities in serum and CSF, that should prove useful in the future. WS08-01

Autoantibodies against the amino-terminal of alpha-enolase in Hashimoto's encephalopathy M. Yonedaa, A. Fujiia, A. Itoa, H. Nakagawaa and M. Kuriyamaa a

University of Fukui, Fukui, Japan

Background: Hashimoto's encephalopathy (HE) is an autoimmune encephalopathy distinct from myxoedema encephalopathy, associated with Hashimoto's thyroiditis (HT). HE presents with various neuropsychiatric features. The detection of anti-thyroid antibodies in patient sera is helpful but not sufficient for the diagnosis of HE because of the high prevalence in the normal population. Recently, we reported autoantibodies against the amino (NH2)-terminal region of alpha-enolase (NAE) as a useful diagnostic marker of HE. Methods: We performed immunoblotting analyses of patient's serum against recombinant NAE. We investigated the prevalence of anti-NAE autoantibodies in the sera from 25 patients with HE, and examined their clinical features, laboratory and MRI findings. Results: Patients with HT, who showed encephalopathy with the presence of anti-thyroid antibodies and steroid-responsiveness, demonstrated a high prevalence of anti-NAE autoantibodies in their sera from HE (68%, 17 of 25), compared to a low prevalence in HT without encephalopathy (10%, 2 of 20) (p < 0.001) and absence in other autoimmune conditions. Consciousness disturbance, seizures, cognitive impairment and EEG abnormalities were common. The patients with anti-NAE autoantibodies showed good steroid-responsiveness and a high prevalence of abnormality on EEG (100%).

Examining pathogenicity of anti-enolase antibodies in paraneoplastic and autoimmune retinopathy Thimmappa Anekonda, Landon Karren, Richard G. Weleber, and Grazyna Adamus Neurological Sciences Institute, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon, USA Anti-enolase autoantibodies have been associated with retinal degeneration in patients with autoimmune and paraneoplastic retinopathies. These patients usually suffer from a slow progression of central vision loss and central cone dysfunction, and show a significant reduction in their electroretinograms. There is considerable interest in examining the relationship between anti-enolase antibodies and retinal degeneration since their role in the pathogenicity of retinopathy is not fully understood. We characterized patients' sera for anti-enolase reactivities using Western blotting and ELISA. Investigations were performed according to the guidelines of the “Declaration of Helsinki” and informed consent was obtained at the time of blood collection. Five epitopes in the enolase protein were determined with the major epitope located within the amino acid sequence 36–41. Normal sera without anti-enolase reactivity did not react with enolase peptides. We propose that blocking enolase function by anti-enolase antibodies may lead to a depletion of cellular ATP, a subsequent increase of intracellular calcium, and finally to cell death. We have previously shown that anti-enolase antibodies inhibit enolase function in vitro. In this study, we found that anti-enolase antibodies significantly reduced the cytosolic ATP when retinal neurons were grown with anti-enolase antibody. In addition, by measuring the level of intracellular calcium in retinal cells treated with patients' anti-enolase IgGs, we found that IgGs were potent inducers of intracellular calcium increase. Normal IgG did not induce intracellular calcium or reduce cytosolic ATP. Using calcium blockers, glycolytic inhibition by anti-enolase antibodies led to a sudden release of endoplasmic-stored Ca2+. In conclusion, our results strongly suggest that anti-enolase antibodies have a potential role in the pathogenicity of retinal degeneration by deregulating glucose metabolism in retinal cells. This work was supported by grant from NIH EY13053, FFB, and RPB. WS08-03

Glutamate receptor antibodies induce brain damage and neurobehavioral dysfunction in ‘autoimmune epilepsy’ and ‘neuropsychiatric SLE’ Mia Levite, Yonatan Ganor The Weizmann Institute of Science, Rehovot, Israel Now, after decades of documenting the massive brain pathology caused in numerous human and animal neuronal injuries/diseases, by excess glutamate, the time of glutamate-receptor autoantibodies has come. We can no longer ignore the growing incrementing evidences, showing that two types of glutamate-receptor autoantibodies: antiAMPA subtype 3 (GluR3) and anti-NMDA R2A/B autoantibodies, are present in serum and/or CSF of patients with epilepsy, SLE and stroke, and are highly pathogenic to the CNS, by virtue of killing neurons and glia, inducing various neurobehavioral impairments, and evoking/ augmenting epilepsy.

Abstracts Importantly, in sharp contrast to the blocking autoantibodies playing a pathogenic role in most/all autoimmune diseases, glutamate-receptor antibodies are activating antibodies that can activate their antigen: a key neurotransmitter receptor. As such, glutamate-receptors antibodies can mimic glutamate or excess glutamate, activate ionotropic glutamatereceptors, trigger ion currents, kill neurons, and lead to CNS damage and dysfunction. Herein we wish to summarize the in vivo and in vitro evidences in humans, rabbits, rats and mice, revealed in our lab, as to the presence of glutamate-receptor autoantibodies in patients with epilepsy and SLE, their pathogenic activity, and their unique mechanism of action. Strikingly, the autoimmune-mediated brain damage caused by glutamate-receptor autoantibodies is different in every aspect from that occurring in MS, to date the most studied human autoimmune-mediated CNS pathology. We recommend that glutamate-receptor antibodies should be considered, from now onwards, as a genuine cause for autoimmune-mediated brain damage, unrecognized and underestimated thus far, and that we should develop means to arrest them. WS08-04

Monocloning oligoclonality — or unraveling the secret of oligoclonal bands: Resurrection of expanded CSF plasma clones as recombinant human monoclonal antibodies Sandra Kuenzle, Hans-Christian von Buedingen, Mirjam Meier, Norbert Goebels University Hospital Zurich, Department of Neurology, Clinical Neuroimmunology Unit, Frauenklinikstrasse 26, CH-8091 Zurich, Switzerland Neuroborreliosis is a chronic inflammatory disease of the central nervous system, caused by a tick-borne spirochete, Borrelia burgdorferii (Bb). While in neuroborreliosis and other chronic CNS infections oligoclonal CSF immunoglobulins – which are detectable as so called oligoclonal Ig bands (OCB) after isoelectric focusing – are directed against the causative infectious agent, the specificity and disease relevance of OCB in other chronic inflammatory diseases of the CNS such as multiple sclerosis are still unknown. We have established an experimental system to identify the antigen specificity of OCB, which are the product of oligoclonally expanded CSF plasma cells. Starting from single cell RT PCR of individual CSF plasma cells we identified expanded clones and expressed their heavy and light Ig chain genes as recombinant human monoclonal antibodies (mAb) in a eukaryotic expression system. Using immunoprecipitation of Bb lysate and subsequent MALDI analysis we could identify the antigen specificity of the derived mAb to be directed against the p41 antigen of Bb. We could show that the affinity of this

61

patient derived human mAb is 1000 times higher to native Bb lysate than to denatured recombinant p41. Our results demonstrate on the one hand the validity of our experimental system in resurrecting the original antigen specificity of expanded CSF plasma cell clones, which can be instrumental in the elucidation of the antigen specificity of OCB in MS and related diseases. On the other hand our results imply, that OCB may be directed against conformation dependent epitopes, which might escape identification by currently popular approaches using recombinant antigen expression libraries. WS08-05

Cerebrospinal fluid proteomics profiling predicts cognitive impairment in HIV-1 infected Hispanic women J. Pérez Laspiura, E. R. Andersona, P. Ciborowskib, H. E. Gendelmanb, V. Wojnaa, J. Rodríguez-Orengoa, E. Rodrígueza, J. Andersonb, and L. M. Meléndez-Guerreroa a

University of Puerto Rico Medical Sciences Campus, San Juan, Puerto Rico; bUniversity of Nebraska Medical Center, Omaha, NE, USA

HIV-1 associated cognitive impairment is a diagnosis of exclusion after searches for opportunistic infections and malignancies are eliminated. Thus, the search for disease biomarkers remains timely and relevant. In a step towards achieving this goal an integrated proteomics platform combining surface enhanced laser desorption ionization time of flight (SELDI-TOF), reverse-phase high performance liquid chromatography (RP-HPLC), one dimensional SDS-PAGE electrophoresis, and liquid chromatography tandem mass spectrometry (LC-MS/MS) were employed to determine links between cerebrospinal fluid (CSF) profiling and cognitive impairment. CSF was collected from 20 HIV-1-infected Puerto Rican Hispanic women and fractionated by RP-HPLC. Nine differentially expressed protein peaks were detected on CSF fractions by SELDI-TOF. Electrophoresis and LC MS/MS studies identified 14 common proteins. Among the proteins preferentially identified in cognitively impaired subjects were familial ALS mutant of Cu+ 2, Zn+ 2 superoxide dismutase (mSOD, 15,839 mw), lymphocyte cytosolic protein 1 (L-plastin, 70,289 mw), migration inhibitory factor-related protein (MIF, 10,835 mw), Galectin-7 (GAL7, 14,944 mw), and VGF nerve growth factor (VGF, 67,287 mw). Western blot examination confirmed the preferential protein expression of SOD in cognitively impaired subjects in both Puerto Rican and North American cohorts. This study highlights the utility of proteomics platform profiling towards uncovering unique biomarkers for HIV-1-associated cognitive impairment. Supported by grants NIH-NINDS U54 NS4301, NIH-NCRR-RCMICRC-P20RR11126, and NIH-NCRR-RCMI G12 RR-03051 for the SELDITOF and Beckman 2000 Instruments.

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Workshop 9: EAE: Regulatory mechanisms

Abstracts

63

Transcriptional regulation of encephalitogenic T cells

besides Th2 differentiation, including the inhibitory effect on T cell proliferation and Th1 differentiation reported here significantly contribute to the therapeutic effect of AT in EAE.

Michael K. Rackea, and Taekyun Shinb

WS09-02

The Ohio State University, Columbus, USA; bCheju National University, Jeju, Korea

Oral administration of anti-CD3 antibody suppresses MOG induced autoimmune encephalomyelitis (EAE) in NOD mice, but not in NOD IFN-γ−/− or IL-10−/− mice

WS09-K1

a

Experimental autoimmune encephalomyelitis (EAE) is an animal model of the human disease multiple sclerosis (MS). EAE is thought to be mediated by Th1 cells that secrete proinflammatory cytokines such as IFN-γ and lymphotoxin, although T cells that secrete IL-17 are also thought to play a role in disease pathogenesis. Given their central role in disease pathogenesis, T cells represent an excellent target for regulation and potential therapies. We have been interested in the pathways that regulate the development of inflammation in inflammatory central nervous system (CNS) diseases. In EAE, we have shown that silencing T-bet, a transcription factor important in Th1 cell differentiation, can suppress EAE development. We have also investigated members of the nuclear hormone receptor superfamily, such as peroxisome proliferator activated receptors (PPAR)-α, and their ability to regulate CNS inflammation. Using chromatin immunoprecipitation assays, we show that PPARα can bind the regulatory regions of the IL-4 and IL-5 genes and promote secretion of these cytokines. Using the PPARα agonist gemfibrozil, we can demonstrate receptor-dependent increases in GATA-3, IL-4 and IL-5 and inhibition of T-bet and IFN-γ. These data suggest that targeting the transcriptional regulation of T cell differentiation could be a successful therapeutic strategy in immune-mediated CNS inflammatory diseases. WS09-01

STAT6-mediated Th2 differentiation is not required for prevention and reversal of experimental autoimmune encephalomyelitis by atorvastatin T. Prod´hommea, M.S. Webera, S. Youssefb, C. Rundlea, L. Steinmanb and S. S. Zamvila a

Department of Neurology, University of California, San Francisco, USA; Department of Neurology, Stanford University, USA

b

Objective: To investigate whether experimental autoimmune encephalomyelitis (EAE) protection by atorvastatin (AT) requires STAT6-mediated Th2 differentiation. Background: AT prevents and reverses EAE and is currently tested for multiple sclerosis treatment. Several EAE studies reported a Th2 differentiation of myelin reactive T cells under AT treatment with an upregulation of STAT6 signaling which controls Th2 differentiation. In order to elucidate the clinical significance of AT-mediated Th2 differentiation we evaluated AT-treatment in STAT6-knockout (−/−) mice. Methods: C57BL/6 STAT6−/− mice were fed orally with 1 or 10 mg/kg/d AT starting either 2 days prior to immunization or after MOG p35–55 EAE was established. Mice treated for EAE prevention were evaluated for proliferation and cytokine secretion of MOG p35–55-specific T cells. Naïve T cells from B10.PL IL-4-GFP reporter mice were differentiated into Th2 or Th1 cells as distinguished by FACS (IL4-GFP → Th2). Resting Th1 and Th2 cells were stimulated with anti-CD3/anti-CD28 in the presence of AT. Results: AT treatment could effectively prevent and reverse EAE in STAT6−/− mice comparable to its treatment effect in C57BL/6 wild-type mice. MOG p35–55-specific proliferation and secretion of IFN-g, TNF-a was reduced in T cells from AT-treated STAT6−/− mice. In vitro, AT inhibited proliferation of both Th1 and Th2 cells in a dose-dependent manner. Conclusions: Our data indicate that AT can prevent and reverse EAE independent of STAT6-mediated Th2 differentiation. Other mechanisms

R. Maron, A. Tamvacakis and H.L. Weiner Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115 USA One of the major goals for immunotherapy of autoimmune diseases such as multiple sclerosis (MS) is induction of regulatory T cells. We found that feeding anti-CD3 antibody suppresses PLP induced EAE in SJL mice via induction of regulatory T cells that function in a TGF-β dependent fashion (Nature Medicine, in press). We investigated the effect of oral anti-CD3 in MOG induced EAE in NOD mice which develop a relapsing–remitting followed by a progressive course that occurs in MS. As in SJL mice, oral anti-CD3 (5 μg/feeding) suppressed MOG induced EAE in NOD mice. We then studied oral anti-CD-3 in IFN-γ−/− and IL-10−/− NOD mice. IFN-γ−/− NOD mice developed severe EAE with an exacerbated disease pattern which was not ameliorated by oral anti CD-3. IL-10−/− NOD mice developed a mild relapsing disease which was exacerbated by oral anti-CD3. Wild type NOD mice fed with 5 μg anti-CD3 showed decreased IL-2 and IL-6 secretion and increased TGF-β compared to control IgG. IFN-γ−/− NOD mice secreted increased IL-2 and IL-6 and decreased TGF-β whereas IL10−/− NOD mice had decreased IL-6 and no increase in TGF-β. Oral antiCD3 had no effect on MOG specific IFN-γ secretion in either NOD or IL10−/− NOD mice. These results demonstrate that EAE disease patterns in the NOD mice are affected by deficiencies in IFN and IL-10 and that the inability of oral anti-CD3 to suppress EAE appears linked to the inability to induce regulation by TGF-β in these mice. WS09-03

Therapeutic effect of mouse CD28 superagonist in murine EAE: Activation of pre-existing regulatory T cells F. Lühdera, F. Eliasb, S. Schillinga, T. Hünigb and R. Golda Institute for Multiple Sclerosis Research, Göttingen, Germany; bInstitute for Virology and Immunbiology, Würzburg, Germany

a

CD4+CD25+ regulatory T cells (Treg cells) are crucial for controlling autoimmunity and inflammation. Previously we could show that a rat CD28 specific superagonistic monoclonal antibody is able to activate and expand Treg in vivo. This was considered the major mechanism for its capability to ameliorate EAE in a Lewis rat model. In the present study we demonstrate that a new, analogous superagonistic antibody specific for mouse CD28 significantly delayed disease onset and reduced disease severity of MOGinduced EAE in C57Bl/6 mice. The administration of the mAb ameliorated EAE both in a preventive and therapeutic setting. When CD4+CD25+ Treg cells were depleted in vivo before superagonist administration the protective effect was abolished, demonstrating the importance of these cells as mechanistic link for the protective effect of the CD28 superagonist. In cases where the depletion of Treg was incomplete, however, the CD28 superagonist restored the CD4+CD25+FoxP3+ Treg compartment within 3 days to more than normal levels indicating its potency in Treg activation and expansion in vivo. Adoptive transfer experiments using lymphocytes either from GFP transgenic mice or labeled with CFSE revealed that the expansion of the Treg compartment was not due to conversion of CD4+CD25− T cells into CD4+CD25+ Treg cells but rather the result of rapid proliferation of pre-existing Tregs.

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These results show that even under conditions of a partially depleted Treg compartment, as is associated with human autoimmune diseases, CD28 superagonist therapy can prevent the disease by activating and expanding regulatory T cells.

Conclusions: Modulation of Notch signaling in autoreactive T cells affects their encephalitogenic potential. Notch may be a promising therapeutical target in autoimmune diseases, including MS. WS09-05

WS09-04

The role of T-bet in the regulation of inflammation in the CNS Modulation of Notch signaling affects encephalitogenic potential of autoreactive T cells

Petra D. Cravens, Anne R. Gocke, Sara C. Northrop, Li-Hong Ben, Rehana Z. Hussain, Michael K. Racke and Amy Lovett-Racke

Maciej Jurynczyk1, Anna Jurewicz1, Cedric S. Raine2 and Krzysztof Selmaj1 1

Department of Neurology, Medical University of Lodz, Lodz, Poland; 2 Department of Pathology, Albert Einstein School of Medicine, New York, NY, USA

Background: Notch regulates differentiation of many cell types, including CD4 T cells. Interaction between distinct Notch receptors and their ligands: Jagged and Delta-like effects peripheral T cells responses. Objective: In our study we examined the effect of Notch pathway manipulation on proliferation, cytokine profile and encephalitogenic potential of autoreactive T cells. Methods: Lymph node cells (LNCs) isolated from SJL/J mice preimmunized with PLP139–151 were cultured with gamma-secretase inhibitor (GSI) or with antibodies neutralizing distinct Notch receptors. ELISA and CFSE proliferation assay were used in order to measure cytokine production and proliferation of lymphocyte populations, respectively. The level of Notch protein and expression of Notch genes were assessed by immunoblotting and real-time PCR, respectively. After 3-day culture, LNCs were transferred via tail vein to normal SJL/J mice in order to induce experimental autoimmune encephalomyelis (EAE). The clinical course of EAE in all groups of mice was observed and histopathology of brain and spinal cords was performed. Results: GSI treatment decreases expression of all four Notch genes and reduces production of IFN-γ in PLP-stimulated autoreactive T cells. GSItreated lymph node cells induce milder disease than control LNCs. Histopathology of brains and spinal cords from the ‘GSI-treated’ mice shows diminished inflammation. In the blocking experiments, IFN-γ production was markedly decreased by anti-Notch3 Ab treatment, while it was not affected by selective Notch1 or Notch2 inhibition.

University of Texas Southwestern Medical Center, Department of Neurology and Center for Immunology, USA Our group has recently shown that suppression of the transcription factor T-bet (T-box expressed in T cells), with intravenously administered siRNA directed against T-bet prevented the onset of experimental autoimmune encephalomyelitis (EAE). Since EAE is an immune-mediated, demyelinating disease of the central nervous system that serves as an animal model for multiple sclerosis, it would be appropriate to determine whether silencing of T-bet using siRNA in mice with ongoing EAE would be beneficial. SiRNAT-bet or siRNA-Nonsense (siRNA-NS) were administered intravenously at 2 timepoints to B10.PL mice that had developed EAE after adoptive transfer of encephalitogenic T cells. Splenocytes isolated from mice treated with siRNA T-bet produced less IFN-γ, had reduced IL-23R expression than mice treated with siRNA-NS. CXCR3 expression was not affected. To examine genes that may be trans-activated by T-bet in vivo, chromatin immunoprecipitation assays were performed. T-bet bound to the IL-23R promoter but not the CXCR3 promoter. Engagement of the IL-23R on IL-23R+ T cells results in IL-17 secretion and IL-17 expression in the CNS of siRNA-T-bet mice was decreased. Examination of immune cells present in the CNS of these mice by multi-parameter flow cytometry indicate that both IL-17 and IFN-γ may be produced by CD45+CD4+ CNS cells. The percentage of T cells able to release IFN-γ and IL-17 are reduced in mice treated with siRNA T-bet. These data indicate that amelioration of ongoing EAE via suppression of T-bet may result in anti-inflammatory environment in the CNS due to decreased IL-23R expression and reduced IL-17 and IFN-γ secretion.

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Workshop 10: IVIG treatment and the role of B cells in MS

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WS10-K1

WS10-02

Emerging concepts of humoral and B cell roles in neuroimmune disease

Longterm IV immunoglobulin treatment in chronic inflammatory demyelinating polyneuropathy (CIDP)

A. Bar-Ora and N. Shibuyab

P.A. van Doorna, D.W.J. Dippela, M. Vermeulenb

a

a

Neuroimmunology Unit, Montreal Neurological Institute and McGill University, Montreal Canada; b Department of Neurology, Nagasaki Medical Center of Neurology, Nagasaki, Japan There has been longstanding interest in how B cell responses may contribute to the pathology of neurological diseases. Traditionally, the premise has been that any such contribution relates to the potential of B cells to produce auto-pathogenic antibodies. Targeting autoantibodies continues to be an important therapeutic approach, particularly in disorders where the role of antibodies is well established, such as in myasthenia gravis. In other conditions such as multiple sclerosis, the role of circulating antibodies has been less clear, though pathologic studies continue to implicate CNS-reactive antibodies, as well as B cells within the CNS compartment. Recently, new insights into fundamental properties of B cells have suggested that these cells may contribute in an antibody-independent fashion, both to normal immune responses, as well as in the context of immune mediated diseases. This session and the associated posters will consider the roles of humoral immune responses, as well as antibody-independent B cell involvement in several neurological disorders. Also covered will be consideration of the roles and therapeutic mechanisms of action of existing therapies such as IVIG, as well as emerging approaches in which B cells are targeted more selectively. WS10-01

A disease-specific anti-IgG fraction isolated from pooled human IgG (IVIG) suppresses experimental myastehenia gravis (EAMG) Sara Fuchsa, Tali Fefermana, Roberto Meidlerb, Kai-Yun Zhua, Raanan Margalita, Ningshan Wanga, Orgad Laubb and Miriam C. Souroujona,c

Department of Neurology, Erasmus Medical Center, Rotterdam, The Netherlands; bAcademic Medical Center, Amsterdam, The Netherlands

Objective: CIDP can improve after intravenous immunoglobulin (IVIg) treatment. Most patients need intermittent IVIg to maintain improvement. This study aims to identify factors especially in relation to long-term treatment and prognosis. Methods: Data were collected from all CIDP patients known at the Erasmus Medical Center, treated with IVIg and followed for at least 2 months. 50 clinical and laboratory parameters were analyzed for a possible relation with improvement (Rankin scale). Results: 64 males and 31 females were followed for a period ranging from 2.5 months to 20 years (median 4.0 years). 15 patients received additional treatment to reduce the amount of IVIg. 77/95 (81%) patients improved after IVIg. 66/77 (86%) patients needed at least 2 courses, suggesting that improvement was not due to spontaneous remission. Improvement was related with a relapse in the past (all 12 patients improved), progressive weakness until treatment and no discrepancy in weakness between arms and legs. Mean time on IVIg treatment until remission was 3.5 years (median 2.1 years). Patients with sensory-motor disturbances (p = 0.002 HR 3.2) and a relative short duration of weakness (p = 0.008; HR 2.6) had a higher chance to reach remission after discontinuation of IVIg. 10% needed IVIg for a period over 8.7 years (maximum over 20 years). Severe side-effects were not seen. Conclusion: Most patients need IVIg for a long period of time. Patients with prognostic factors indicating the necessity for long-term treatment may be considered – especially due to the high costs of IVIg – to switch treatment in an early stage of disease. WS10-03

a

Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel; bOmrix Nes-Ziona, Israel and cThe Open University of Israel, Raanana, Israel

Changes in axonal excitability following intravenous immunoglobulin infusions in patients with dysimmune demyelinating neuropathy

Intravenous immunoglobulin (IVIG) administration has been beneficially used in recent years for the treatment of a variety of autoimmune diseases including myasthenia gravis. However, the mechanism of action of IVIG treatment and the fraction responsible for its therapeutic effect in autoimmune diseases are still not established. We have been studying the therapeutic effects of IVIG administration in experimental autoimmune myasthenia gravis (EAMG), a rat model for myasthenia gravis. We showed that IVIG treatment could successfully prevent the induction of EAMG and immunosuppress an ongoing disease. The mechanism by which IVIG modulates EAMG involves suppression of Th1 cells and B cell proliferation but probably does not act via regulatory T cells. After establishing the optimal conditions for EAMG suppression by IVIG administrations we have employed this model in an attempt to isolate a specific suppressive fraction from IVIG preparation and to identify its therapeutic activity. We have demonstrated that chromatography of human IVIG on immobilized rat anti-AChR IgG, isolated from rats with EAMG, results in a complete depletion of the suppressive activity of the IVIG. Moreover, the eluted immunoglobulin fraction that had been adsorbed onto the anti-AChR antibodies and comprises less than 1/100,000 of the IVIG preparation retains the immunosuppressive activity of IVIG. This study supports the notion that the therapeutic effect of IVIG is mediated by an antigen-specific anti-immunoglobulin activity (anti-idiotypic activity) present in pooled human immunoglobulin and raises the possibility that diseasespecific anti-idiotypic activity can be fractionated from IVIG and become a preferential reagent for therapeutic purposes.

Boerio Delphine1,3, Creange Alain2, Hogrel Jean-Yves3, Lefaucheur JeanPascal1 Service de Physiologie — Explorations Fonctionnelles, CHU Henri Mondor, Créteil, France; 2Service de Neurologie, CHU Henri Mondor, Créteil, France; 3Institut de Myologie, GH Pitié-Salpêtrière, Paris, France

1

Background: Dysimmune demyelinating neuropathies are associated with changes in nerve excitability resulting from alterations of axon membrane properties. Objectives: To investigate the effects of intravenous immunoglobulin (IVIgs) on nerve excitability in patients with multifocal motor neuropathy with conduction blocks (MMN) or chronic inflammatory demyelinating polyneuropathies (CIDP). Methods: 16 patients (9 MMN, 7 CIDP) were evaluated before and after IVIgs (0.4 g/kg/day for 5 consecutive days). Absolute (ARP), relative refractory period (RRP) durations and percentages of refractoriness at 2 ms and supernormality at 7 ms interstimuli intervals were determined. Stimulus/response (S/R) and strength/duration (S/D) curves were established. Activity-dependent hyperpolarisation induced by voluntary contraction was appraised on the percentage of conduction block in affected territories. Results: Before IVIgs, patients had longer refractoriness and smaller supernormality than healthy subjects. IVIgs increased refractoriness (78.1 ± 5.8 vs. 69.0 ± 6.7%, p = 0.04) and prolonged RRP (3.1 ± 0.2 vs. 3.8 ± 0.3 ms,

Abstracts p = 0.05) but did not modify ARP duration and supernormality. The SD time constant decreased (242.7 ± 35.6 vs. 352.7 ± 45.2 μs, p = 0.04), while rheobase and S/R curve slope remained unchanged. IVIgs reduced the percentage of activity-dependent conduction block (11.5 ± 0.7 vs.− 5.6 ± 0.6%, p = 0.002). Conclusion: This electrophysiological study provides additional information to usual conduction studies. The refractoriness prolongation is consistent with changes in Na+ conductance or nodal membrane properties, suggesting a reduction in intraaxonal Na+ accumulation, decreasing the occurrence of hyperpolarisation. While the reduction of percentage of conduction block traduced an improvement in Na+/K+ ATPase pump function and a greater conduction. This study revealed potential protective effects of IVIg and improved conduction across conduction blocks in CIDP and MMN.

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Conclusion: When activated, MS patient B cells may abnormally enhance a pro-inflammatory environment by increased release of TNFα and LT. Therapy with mitoxantrone appears to reverse this abnormality, with a pattern suggesting that different B cell populations may contribute distinct cytokines to a local inflammatory environment. These insights are pertinent given the growing interest in B cell roles in MS. WS10-05

Lipid microdomain-mediated signaling: Implications for anti-MOG mediated demyelinating disease SE Pfeiffera, CB Martaa, AR Oliverb, RA Sweetb, R Bansala, NH Ruddleb University of Connecticut Medical School, Farmington, CT, USA; bYale University School of Medicine, New Haven, CT, USA

a

WS10-04

B cells in MS patients exhibit an abnormal effector cytokine profile under Th1 environment M. Niino, M. Duddy, F. Adatia, S. Hebert and A. Bar-Or Montreal Neurological Institute of McGill University, Montreal, Canada Objective: To examine whether (1) a novel B cell regulatory cytokine network is influenced by the local Th1 or Th2 environment in patients with MS and controls (2) such modulation can be targeted therapeutically. Methods: Circulating CD19+ B cells were purified from untreated MS patients (Poser criteria) and matched healthy controls, then stimulated with B cell receptor engagement followed by CD40 stimulation. Modulation of B cell responses by the local T cell environment was assessed by addition of IFNγ (Th1) or IL-4 (Th2). B cell proliferation (thymidine incorporation) and TNFα, LT and IL-10 (ELISA) were subsequently measured. Results: B cell proliferation was no different between MS and controls, under all stimulation conditions. IL-4 addition generally enhanced B cell cytokine production, as expected, with no differences in TNFα production between MS and controls. Upon addition of IFNγ, MS patient B cells produced significantly more TNFα and LT compared to control B cells; IL10 production was not different. Interestingly, following mitoxantrone therapy in MS patients, the addition of IFNγ during MS B cell stimulation was now associated with significantly less TNFα and LT induction compared to baseline data.

Antibodies to myelin components are routinely detected in multiple sclerosis (MS) patients. We find that antibody cross-linking in oligodendrocytes (OLs) of myelin proteins MOG or MAG results in (a) their rapid repartitioning into glycosphingolipid–cholesterol microdomains (‘lipid rafts’1,2, small (nm) membrane entities with few proteins, which upon ligand- or antibody-mediated cross-linking can coalesce into functional signal transduction “activation centers”; high cholesterol/glycosphingolipid content in OLs/myelin suggested that rafts contribute functionally to their physiology.), followed by (b) raft-dependent phosphorylation–dephosphorylation of specific proteins and downstream events, and in the case of MOG, (c) dramatic, rapid loss of myelin-like membrane.3 Immunization of C57BL/ 6 mice with either rat or human MOG produces comparable anti-MOG ELISA titers, however, only human MOG yields B cell dependent EAE and is encephalitogenic in primed B cell deficient mice. Substituting Pro42 with Ser in human MOG (as in rat MOG) eliminates this B cell requirement; nonpathogenic IgGs bind recombinant mouse MOG and deglycosylated MOG in myelin, but only pathogenic IgGs bind glycosylated MOG; only purified IgG to human MOG binds to live rodent OLs in culture and, after crosslinking, induce repartitioning of MOG into lipid rafts and dramatic changes in cell morphology.3 These data provide a strong link between in vivo and in vitro observations regarding demyelinating disease, further suggesting a biochemical mechanism for anti-MOG-induced demyelination, and suggest in vitro tools for determining autoimmune Ab pathogenicity in MS. 1Kim and Pfeiffer (1999); Taylor et al. (2002). 2Marta et al. (2004), Schafer et al. (2004). 3Marta et al. (2003, 2005). NMSS-FG1423A(CM)/RG2394(NHR); NIH-NS10861/NS41078(SP).

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Workshop 11: Studies in neuroimmune pharmacology

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WS11-K1

WS11-02

Studies in the growing inter-discipline of neuroimmune pharmacology

Morphine-dependency modulates progression of simian AIDS R.M. Donahoe1, S. O'Neil2, H.M. McClure3, F. Novembre3, F. Marsteller4 and P. Turner3

R. M. Donahoea and T. J. Rogersb a

University of Utah, Salt Lake City, UT, USA; Philadelphia, PA, USA

b

Temple University,

NeuroImmune Pharmacology is a ‘new” interdisciplinary area of research now represented by the Society of NeuroImmune Pharmacology. This symposium, “Studies in Neuroimmune Pharmacology”, represents some of the broad interests of this ‘new’ interdiscipline. Data on effects of opiates in a monkey model of AIDS show statistically meaningful evidence that opiates modulate AIDS disease progression. These data have considerable implication for opiate addicts infected with HIV1. Data are also presented that show that mu opiate–receptor ligands induce desensitization of the chemokine receptor, CCR5, on macrophages via phoshorylation of protein kinase-C. These fundamental data are important to the role of opiates in neuroimmune function and HIV/AIDS. Similarly, the signal transduction mechanisms involved in stimulation of lymphocytes through delta-opioid T-cell surface receptors are presented, which further elaborates how opioids interact directly with cells of the immune system at the basic molecular level. A human immune-reconstituted mouse model is also used to show that the anti-HIV drug, indinavir, can be delivered effectively via a nanoparticle packaged into bone-marrow derived macrophages as drug carriers. This novel model offers new approaches and new hope for treatment of HIV/AIDS. Finally, structural MRI, magnetic resonance spectroscopy, and positron emission tomography studies were used in humans to show neurological changes in HIV1-infected subjects who also use cocaine and methamphetamines. These studies reveal drugspecific neurologic changes that associate with neurobehavioral, motor and cognitive functions in the patients studied. These combined data show the promise of neuroimmune pharmacology to advance understanding of major medical problems and the means to their resolution. WS11-01

University of Utah, Salt Lake City, UT, USA; 2Harvard University, Cambridge MA, USA; 3Emory University, USA and 4Behavioral Research Consulting, Atlanta, GA, USA

1

Opiate use has been reported to retard or advance AIDS progression, or to have no detectable effect. Human clinical and monkey SIV-model studies have contributed to this equivocation. A small pilot monkey study from our laboratory suggested that opiate-dependency retards AIDS progression. Results of a nearly 5-yr follow-up study confirm this original observation. Male rhesus macaques at around 4 yr of age were adapted over a 1-1/2-yr period to a single-cage holding environment with psychological enrichments. Baseline data were collected, followed by introduction of an opiate-dependency paradigm (4 daily, 6-h apart, sc/im injections of morphine sulphate, 3–2 mg/kg/monkey). Nineteen monkeys received opiate while 17 received equivalent, weight-by-volume, injections of saline as controls. Two weeks after initiation of these injections, all monkeys were injected iv with 10,000 TCID50 of SIVsmm9. Progression of infection and disease was followed for 4 yr thereafter. Morphine retarded progression of AIDS overall (P V 0.05), being largely selective for monkeys that progressed relatively rapidly to AIDS. Opiatedependency also altered cerebral spinal inflammation. Retardation of disease progression corresponded with an ability of opiates to reverse the negative correlation typically seen as viral load increases over time in conjunction with CD4+ T-cell loss. These data represent the most solid evidence to date that opiates alter AIDS progression. In view of conflicting data that opiates exacerbate AIDS progression, current understanding of this issue supports our prior conjecture that the influence of opiates on AIDS is conditional — being dependent on variable drugdependency, viral and immune states, and possibly relating to the influence of these factors over stress. Such findings and conclusions support a need for more thorough understanding of how opiates affect AIDS progression in drug-abusing populations.

Cross-talk between the mu opioid receptor and CCR5 is mediated by protein kinase c zeta (PKCζ)

WS11-03

R.T. Rahim and T. J. Rogers

Dual actions of opioids on signaling in T-cells

Center for Substance Abuse Research, and Fels Institute of Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA

B.M. Sharp and Nahid Shahabi

Previously, our laboratory has shown that heterologous desensitization of CCR5 is induced upon activation of the mu opioid receptor (MOR). The present study examined the signaling events mediating the inactivation of CCR5 by pre-treatment with [D-Ala-N-Me-Phe4-Gly-ol] enkephalin (DAMGO), a MOR-selective agonist. Cell lines, stably-transfected with MOR and CCR5, and human monocyte-derived macrophages were treated with DAMGO (10− 6 M) and used in various assays. Co-immunoprecipitation and Western blot analyses showed that PKCζ associates with CCR5 and that DAMGO induced phosphorylation of PDK-1 (phosphoinositide-dependent kinase-1), a kinase that is known to activate PKCζ. In addition, DAMGO augmented the kinase activity of PKCζ observed upon immuno-precipitation of CCR5 and induced phosphorylation of CCR5. Cells transfected with PKCζ siRNA showed a significant inhibition of PKCζ kinase activity associated with CCR5. In further studies, desensitization of CCR5 led to inhibition of calcium mobilization and decreased chemotactic response to Mip1-beta. Use of a PKCζ pseudosubstrate inhibitor reversed these results. Overall, the data suggest that DAMGO-induced desensitization of CCR5 leads to phosphorylation of CCR5 and that PKCζ mediates this effect. This work was supported by NIDA grants DA16544, DA06654, DA14230, and P30-DA13429.

Department of Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee, United States Opioids modulate an array of functional responses by T-cells, including proliferation, cytokine production, chemotaxis and expression of human immunodeficiency virus-1 (HIV). However, the signaling pathways mediating these effects of opioids have been largely unknown. Recently, we observed that delta opioid ligands exert dual effects on specific T-cell signaling cascades; as such, the opioid itself stimulates, whereas opioid pretreatment attenuates the signaling response to another agent. For example, brief exposure (15 min) to a specific delta opioid agonist (i.e., DADLE) dose-dependently induced phosphorylation of c-jun by activating phosphoinositide 3-kinase (PI3K), protein kinase B (Akt) and the mitogen-activated protein kinase (MAPK) c-jun NH2-terminal kinase (JNK). Moreover, DADLE stimulated the phosphorylation of activating transcription factor-2 (ATF-2; implicated in cytokine gene transcription) and its association with JNK. In contrast to such direct activation of PI3K/Akt-dependent signaling, pretreatment with DADLE inhibited Akt phosphorylation induced by stromal cell-derived factor-1 (SDF-1). Pretreatment of highly purified human peripheral blood T-cells with DADLE for 1 or 3 h, significantly reduced SDF-1-induced Akt

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phosphorylation by approximately 60% (n = 7). In contrast, DADLE failed to stimulate phosphorylation of the MAPKs, ERK1 and 2, and did not affect SDF-1-induced ERK phosphorylation. Recent studies also demonstrate that morphine inhibited Akt phosphorylation stimulated by SDF-1. Together, these studies demonstrate that endogenous and exogenous opioids can exert both direct and indirect modulatory effects on signaling cascades that depend on PI3K/Akt. WS11-04

Pharmacokinetic, immune, and anti-viral responses of nanoparticle indinavir in a murine model of HIV-1 encephalitis Huanyu Dou1,2, Christopher J. Destache1,2,6, Justin R. Morehead1,2, R. Lee Mosley1,2, Michael D. Boska1,2,3, Jeff Kingsley1,2,5, Santhi Gorantla1,2, Larisa Poluektova1,2, Jay A. Nelson1,2,3, Mahesh Chaubal7, Jane Werling7, James Kipp7, Barrett E. Rabinow7, and Howard E. Gendelman1,2,4 Center for Neurovirology and Neurodegenerative Disorders, 2Department of Pharmacology and Experimental Neuroscience, 3Radiology, 4Internal Medicine, and 5Pediatrics, University of Nebraska Medical Center, Omaha, NE 68198-5215, USA; 6Creighton University School of Pharmacy and Health Professions, Omaha, NE 68178, USA and 7Baxter Healthcare Corporation, Round Lake, IL 60073, USA

1

Short title: A nanoparticle delivery system for anti-retroviral therapy Key words: macrophages, nanoparticle, highly active anti-retroviral drugs, human immunodeficiency virus, nonobese servere combined immunodeficient mice, immune reconstitution, indinavir ⁎Corresponding author Howard E. Gendelman, MD; Center for neurovirology and Neurodegenerative Disorders 5880 Nebraska Medical Center; Omaha, Ne 68198-5880; Phone 402-559-8920; FAX 402-559-89922; email: [email protected]. ABSTRACT The introduction of highly active antiretroviral therapy for human immunodeficiency virus infection has all but revolutionized pharmacologic management of human disease. Nonetheless, complex dosing regimens, costs, untoward side effects, limited bio distribution and variable drug pharmacokinetic patterns have affected long-term use and notably, clinical efficacy, within the nervous system. We posit that nanotechnology-derived cell based systems may overcome such limitations. In a first step towards testing this idea, we developed a nanoparticle indinavir (NP-IDV) formulation packaged into bone-marrow-derived-macrophages (BMM) as drug carriers. Drug distribution and disease outcomes were assessed in immune competent and in non-obese diabetic severe combined immunodeficient mice (NOD/SCID) reconstituted with human peripheral blood lymphocytes. In the model, NP-IDV contained within BMM was adoptively transferred into mice. After a single administration, single photon emission computed tomography and histology demonstrated robust lung, liver, and spleen BMM distributions. Tissue and sera IDV levels were z50 nM/ml up to 2 weeks of observation. NP-IDV-BMM administered to virus-challenged immunodeficient humanized mice showed reduced numbers of virusinfected cells in plasma, lymph nodes, spleen, liver, and lung. Numbers of CD4+ T cells were restored after NP-IDV-BMM administration. NOD/SCID mice with active HIV-1 encephalitis showed robust delivery of NP-IDV to affect brain subregions. We conclude that a single dose of NP-IDV using BMM as a carrier is bio-available and effective and warrants consideration for human testing. WS11-05

Basal ganglia abnormalities in HIV patients and chronic psychostimulant drug users L. Chang, T. Ernst University of Hawaii, Honolulu, USA

A variety of in vivo physiological neuroimaging techniques can quantitatively assess brain injury associated with HIV and/or substance abuse. Structural MRI demonstrates atrophy of caudate and putamen in subjects infected with HIV, but enlarged striatal volumes in those with chronic psychostimulant abuse (methamphetamine, cocaine, etc.). Hence HIV and psychostimulant use have opposite effects on brain volumes. Conversely, magnetic resonance spectroscopy (MRS) shows metabolite abnormalities that are additive in HIV patients and methamphetamine users. These spectroscopic abnormalities include decreased neuronal marker N-acetylasparate and elevated glial marker myoinositol, most prominently in the basal ganglia and frontal cortex. Since psychomotor slowing and attention deficits are major features of HIV dementia and psychostimulant abuse, dopamine dysfunction has been postulated. Dopaminergic terminals and synapses have the highest density in the basal ganglia regions. Positron emission tomography studies (with C-11 cocaine) indeed illustrate lower dopamine transporters in HIV patients with dementia, as well as individuals with chronic methamphetamine dependence, compared to seronegative non-drug users. Correlations between cognitive performance and dopamine transporter levels document the functional consequences of dopaminergic deficits. These findings suggest that methamphetamine and cocaine abuse further exacerbate brain injury in HIV patients. Therefore, dopamine augmentation might be beneficial in the treatment of HIV dementia. Collectively, findings from these neuroimaging studies provide insights into the pathophysiological mechanisms of HIV-associated brain injury and drug abuse, and might lead to new therapeutic approaches. Acknowledgments: Studies and resources supported by the NIH (NINDS, NIMH, NIDA, NCRR), the Department of Energy, and the ONDCP. WS11-06

The cholinergic anti-inflammatory pathway L. Ulloa⁎, J. Huston, H. Liao, M. Rosas-Ballina, M. Ochani, K. Ochani and KJ. Tracey Center of Immunology and Inflammation, North Shore University Hospital, 350 Community Drive, Manhasset, New York 11030, USA. [email protected] We recently discovered that the vagus nerve can control peripheral innate immune responses through a mechanism that can be employed for the treatment of inflammatory disorders [Nat Rev DD 4:673, 2005]. Electrical stimulation of the vagus nerve attenuates systemic inflammation in experimental sepsis by inhibiting the production of pro-inflammatory cytokines in the spleen through a mechanism dependent on the common celiac branch. Vagus nerve stimulation fails to attenuate endotoxininduced serum TNF levels in splenectomized animals. Acetylcholine, the principal neurotransmitter of the vagus nerve, inhibits endotoxin-induced TNF and HMGB1 release from human macrophages through a mechanism dependent on the α7-nicotinic acetylcholine receptor (α7nAChr). In vivo, vagus nerve stimulation reduces splenic TNF protein and mRNA levels in wild-type mice, but not in α7nAChr-knockout mice. Nicotine, a more selective cholinergic agonist, abolished HMGB1 release in a dose-dependent manner, and it was a more efficient inhibitor of macrophages. In vivo, treatment with nicotine attenuated serum HMGB1 levels, prevented lethal endotoxemia, and improved survival in established polymicrobial peritonitis, even when the treatment was started after the appearance of the clinical signs of sepsis. Nicotine has already been used in clinical trials for inflammatory disorders such as ulcerative colitis, but its clinical potential is limited by its collateral toxicity. Similar to the development of selective agonists for adrenergic receptors, selective nicotinic agonists for the α7nAChR may represent a promising pharmacological strategy for infectious and inflammatory diseases. This research was supported by the Faculty Awards Program of the NS-LIJHS, NIGMS, and DARPA.

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Workshop 12: Virus-host immune interactions associated with diseases of the nervous system

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WS12-01

Immune containment and cell to cell spread of the human leukemia virus HTLV-1 Charles R. M. Bangham1 and Steven Jacobson2

Thus γδ T-cells, NK/NKT cells, CD4 T-cells, and CD8 T-cells limit HSV 1 infection in the lip and TG but do not restrict the spread of virus in the CNS. In contrast, virus spreads throughout the brain in B6.129P2B2mtm1unc/J mice (CD8 T-cells and NK/NKT cells affected) without significant mortality. This suggests that an interaction between innate and adaptive immunity is required to restrict the spread of HSV 1 to the CNS.

1

Department of Immunology, Wright-Fleming Institute, Imperial College, London, UK; 2 National Institutes of Health, National Institute of Neurological Disorders and Stroke, USA Three broad questions have driven research into the immune response to HTLV-1. First, how does HTLV-1 persist in the individual host? In particular, what is the role of the immune response in controlling or limiting viral persistence? Second, why do some HTLV-1-infected people develop a consequent disease such as HAM/TSP or leukaemia, whereas the majority remain asymptomatic carriers of the virus? Is this difference in the outcome of infection due primarily to variation in the host or variation in the virus? Third, how is the inflammatory lesion in HAM/TSP initiated and maintained, and how can the inflammation be halted? Evidence on the immune response to HTLV-1 will be summarized from recent work in host and viral genetics, DNA expression microarrays, T-cell function and cell biology. This evidence favours two main conclusions: first, that the efficiency of a person's cytotoxic T-lymphocyte (CTL) response to HTLV-1 plays a dominant role in determining that person's proviral load of HTLV-1 and the risk of the associated inflammatory diseases; second, that HTLV-1 is persistently transcriptionally active and spreads directly between lymphocytes by a specialized cell–cell contact known as the virological synapse. Until recently, the inference that HTLV-1 is persistently transcribed rested entirely on evidence from in vitro experiments. However, new evidence from quantification of lymphocyte turnover in vivo strongly supports the view that there is persistent transcription of HTLV-1. These conclusions have implications for therapeutic approaches both in the inflammatory diseases and in the HTLV-1-associated syndrome of adult T-cell leukaemia, and in the immune response to persistent viral infections. WS12-01

Central nervous system (CNS) involvement by herpes simplex virus 1 (HSV 1) is restricted in C57BL/6 (BL/6) mice L.F. Kastrukoffa, A.S. Laua, E. Wilsonb, F. Takeib, and F.R. Carbonec a

Department of Medicine, University of British Columbia, Vancouver, Canada; bTerry Fox Lab, Cancer Research Centre, Vancouver, Canada; c Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia

Following mucosal inoculation with HSV 1, susceptible strains of mice develop recurrent multifocal CNS demyelination preceded by the spread of virus from the lip, through the peripheral nervous system and to the CNS. In contrast, BL/6 mice do not develop CNS demyelination and viral access to the CNS is restricted. The objective of this study is to identify the immune mechanism(s) that restrict HSV 1 entry to the CNS of BL/6 mice. Controls along with BL/6 mice depleted of immune cell subsets and knock out (KO) mice were lip inoculated with HSV 1 strain 2. Viral titers in the lip, trigeminal ganglia (Tg), and brain were determined in serial studies. HSV 1 is restricted to the lip and Tg of controls and BL/6 mice treated with either α-NK1.1 monoclonal antibody (mAb), α-CD4 mAb, α-CD8 mAb, or a combination of α-CD4 and α-CD8 mAbs. Virus is also restricted to the lip and Tg of B6.129P2-Tcrdtm1Mom/J and B6.129-H2dlAbl-Ea/J KO mice. HSV 1 can spread throughout the CNS of B6.129S7-Rag1tm1Mom/J mice but is associated with significant mortality. In contrast, virus can spread throughout the CNS of B6.129P2B2mtm1unc/J mice with infection limited to the first 12 days post infection (PI).

WS12-02

In vivo selection of T-cell receptor junctional region sequences by HLA-DRB1⁎0101 human T-cell lymphotropic virus type 1 (HTLV-1) envelope gp21 peptide complexes in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) H. Nosea, M. Saitob, A.H. Sabouria, K. Usukuc, R. Kubotad, S. Izumod, K. Arimuraa, M. Osamea a

Department of Neurology and Geriatrics, bDepartment of Microbiology, Kanazawa Medical University, Ishikawa, Japan; cMedical Information Technology and Administration Planning, Kumamoto University Hospital, Kumamoto, Japan; dDivision of Molecular Pathology Center for Chronic Viral Diseases, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disease observed only in 1–2% of HTLV-1 infected individuals. We have previously reported that HLA-DRB1⁎0101 was associated with susceptibility to HAM/TSP in two independent HTLV-I infected cohort in Southern Japan (Kagoshima) and Northeastern Iran (Mashhad), whereas HTLV-1 envelope gp21-specific T cells were restricted by DRB1⁎0101. To further characterize the role of DRB1⁎0101 restricted Env specific CD4+ T cells in chronic HTLV-1 infection, we have exploited tetrameric MHC-peptide complexes along with magnetic cell sorting to purify HTLV-1 envelope gp21-specific CD4+ T cells directly from peripheral blood lymphocytes from two HLA-DRB1⁎0101 positive HAM/TSP patients. RT-PCR, spectratyping and sequencing analysis of TCR Vβs revealed that the Vβ-Dβ-Jβ junctional sequences derived from two unrelated HAM/TSP patients shared identical amino acid motifs in CDR3 region. Our data provide strong evidence that several different T cells bearing distinct TCRs are stimulated with HLADRB1⁎0101/envelope gp21 complexes and then proliferate in vivo, i.e. there is selection for certain TCR gene sequences exerted in vivo by the MHC-peptide complex. WS12-03

Safety and efficacy of interferon-α in 167 patients with HTLV1-associated myelopathy (HAM) Nakagawa Ma, Arimura Kb, Izumo Sc, Usuku Kd, Itoyama Ye, Kira Jf, Osame Mb a

Department of Neurology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan; bDepartment of Neurology and Geriatrics, Kagoshima University Graduate School of Medical and Dental Sciences and c Department of Molecular Pathology, Center for Chronic Viral Diseases, Kagoshima University, Kagoshima, Japan; dDepartment of Medical Information Science and Administration Planning, Kumamoto University Hospital, Kumamoto, Japan; eDepartment of Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan; fDepartment of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

Abstracts Objective: A post-marketing surveillance study was undertaken to investigate the safety and efficacy of interferon-α for HAM under routine treatment conditions. Methods: A total of 273 cases from 91 medical institutions were registered into the survey. So far, 167 cases had been evaluated for safety and 152 for efficacy. The efficacy evaluation was rated based on clinical symptoms of HAM. Results: The rate of response to the treatment (rated as improved and fair) at 4 weeks was 67.1%. Factors which significantly affected efficacy at 4 weeks was initial Osame's motor disability score (OMDS) before interferon-α therapy, duration and stage of illness. Sustained improvement of OMDS for at least 5 months after stopping interferon-α was observed in 11 of 30 patients (36.7%). A total of 536 adverse drug reactions (ADRs) occurred in 146 patients, 46 of which were serious. The incidence of ADRs was higher than that of a previously reported controlled trial, although no specific ADRs occurred in patients with HAM. Conclusions: These results indicate that interferon-α treatment is effective for at least patients in the incipient stage of HAM, though the incidence of ADRs is rather high. Long-term administration of interferon-α may have some advantage in maintaining the improved condition during treatment. Thus, the most suitable protocol for interferon-α administration in HAM needs to be defined in the future. WS12-04

What enters the brain stays in the brain Maria Cecilia G. Marcondes1, Sieghart Sopper2, Debbie Watry1, Claudia Flynn1 and Howard Fox1 1

Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, 92037, USA; 2Department of Virology and Immunology, Deutsches Primatenzentrum, Kellnerweg 4, 37077 Göttingen, Germany

Neuro-AIDS is a complication of HIV infection affecting 1/3 of HIV+ individuals, triggered by the early presence of virus in the brain. SIV infection in rhesus macaques parallels human HIV infection, including on development of CNS disease. Neurophysiological alterations allow recognition of early signs of CNS dysfunction. The analysis of alterations on immune-cells in the brain in correlation with CNS dysfunction, show a remarkable 5–15-fold increase of highly activated memory CD3+CD8+ CTLs in infected brains. The specificity of these cells was investigated. We found that >50% of brain CTLs recognize immunodominant epitopes Tat and Gag in monkeys with MamuA⁎01 MHC haplotype. In the blood and CSF, Tat-specific cells peak during the acute phase, and become undetectable thereafter. However, in the brain, these cells remain as a large proportion of accumulating CTLs. We hypothesize that virusspecific cells enter the brain during acute phase, and are maintained to chronic stages. At 28 dpi, the enrichment of Tat and Gag-specific cells in

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the brain was already observed, suggesting an early entry. Factors favoring CD8 survival, such as IL2, IL7 and IL15, were examined. IL2 and IL7 were undetectable, but IL15 increased by 3–5 fold in SIVinfected brains. In vitro, IL15 efficiently supported CD8 survival, increased Ki-67+ cycling cells, and those expressing memory markers. Our results suggest that IL15-rich brain environment supports survival of SIV-specific and bystander memory CTLs. Taken together, increased IL15 levels in infected brains favor activated pro-inflammatory virus-specific CTLs, which enter the brain during the acute phase, and persist, potentially participating on development of inflammatory pathology. WS12-05

Axonal transport of toxic lectin from the PNS targets inflammatory demyelinating lesions to sites of Wallerian degeneration in the CNS in Theiler's virus infection: Lesion development from the axon (inside) to the myelin (outside) I. Tsunodaa, T. Tanakaa, Y. Saijohb, S. E. Doylea, E. J. Terrya, and R. S. Fujinamia Departments of Neurologya, and Neurobiology and Anatomyb, University of Utah School of Medicine, Salt Lake City, Utah, USA In Theiler's murine encephalomyelitis virus (TMEV) infection, an animal model for multiple sclerosis (MS), axonal injury precedes inflammatory demyelination. The distribution of axonal injury during the early phase corresponds to regions where subsequent demyelination occurs during the chronic phase. We hypothesized that axonal injury recruits inflammatory cells into sites of Wallerian degeneration, leading to demyelination. In this scenario, lesions develop from the axon (inside) to the myelin (outside) (Inside-Out model). We have established a method for induction of Wallerian degeneration in the central nervous system (CNS). This approach uses toxic lectin, Ricinus communis agglutinin (RCA) I, and does not lead to a blood–brain barrier breakdown. The lectin is injected into the sciatic nerve and is transported axonally, leading to the death of dorsal root ganglion cells and Wallerian degeneration of the posterior funiculus in the spinal cord. Using a microinjection system, we injected lectin into the sciatic nerve of SJL/J mice, 3 weeks after TMEV infection. Neuropathology was examined, 1 week after lectin injection. Control mice, which received TMEV but no lectin, had inflammatory demyelinating lesions only in the anterior and lateral funiculi. In contrast, lectin-injected infected mice had lesions in the anterior and lateral funiculi as well as in the posterior funiculus, where the lesions were present only ipsilateral to the injection side. We found no differences in anti-TMEV lymphoproliferative responses between groups. This suggests that axonal injury itself could contribute to the recruitment of inflammatory cells into the CNS by altering the local microenvironment, such as activating microglia.

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Abstracts

Workshop 13: Stem cell-mediated immunomodulation of EAE

Abstracts

WS13-01 Intravenous injection of neural precursor cells attenuates experimental autoimmune encephalomyelitis by a peripheral immunosuppressive mechanism O. Einsteina, N. Fainsteina, I. Vakninb, R. Mizrachi-Kola, E. Reihartza, N. Grigoriadisc, I. Lavond, M. Baniyashb, H. Lassmanne and T. Ben-Hura a

Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Hadassah– Hebrew University, Jerusalem, Israel; bThe Lautenberg Center for General and Tumor Immunology, Hadassah–Hebrew University, Jerusalem, Israel; cB′ Department of Neurology, University of Thessaloniki, Thessaloniki, Greece; dLeslie and Michael Gaffin Center for Neuro-Oncology, Hadassah–Hebrew University, Jerusalem, Israel; eBrain Research Institute, University of Vienna, 1090 Vienna, Austria Recent studies showed that neural precursor cells (NPCs) attenuate experimental autoimmune encephalomyelitis (EAE) after intracerebroventricular or intravenous (IV) injection. NPC therapy in EAE was associated with a decrease in brain inflammation and in tissue injury. Here we studied the mechanism by which IV injected NPCs inhibit myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6 mice. IV NPC therapy at 8 days post EAE induction attenuated the clinical course of EAE, and reduced brain inflammation and tissue injury. Microscopical analysis revealed that NPCs did not enter the brain, but were detected in lymph nodes and spleen. The direct interactions between NPCs and lymph node cells (LNC) were examined in vitro. Co-culture experiments showed that neurospheres attenuated significantly the proliferation of EAE-derived T-cells in response to MOG, as well as of naïve T-cells, in response to the non-specific activator ConA. Neurospheres also inhibited MOG-induced production of proinflammatory cytokines in LNCs. Analysis of CFSE emission descent, IL2Rα expression, DNA content and Annexin-V expression indicated that NPCs block T-cell activation and proliferation, rather than induce LNC death. Finally, to examine the relevance of NPC-LNC interactions in vivo, we utilized a proteolipid (PLP)-induced transfer EAE model in SJL/J mice. Transfer of LNCs derived from NPC-treated, PLP-immunized mice into naïve recipients caused a significantly milder disease than transfer of LNCs from non-treated mice. This indicated that NPC therapy reduces the encephalitogenicity of LNCs. Our findings suggest a peripheral immunosuppressive effect of IV-injected NPCs, by a non-specific inhibitory effect on activation and proliferation of lymphocytes in the lymph nodes. WS13-02

Neural stem/precursor cell (NPC) transplantation in experimental autoimmune encephalomyelitis in mice: Unraveling the molecular bases of NPC-dependent immunomodulation Lucia Zanotti, Elena Brambilla, Giuliana Salani, Alessandro Cattalini, Giuseppe Penna, Michela Deleidi, Marco Bacigaluppi, Luciano Adorini, Gianvito Martino, Stefano Pluchino

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subcutaneously with proteolipid protein (PLP)139–151 and i.v. injected with syngenic aNPCs at the onset of the disease. Transplanted aNPCs accumulated and persisted over 100 days after transplantation within major secondary lymphoid organs, where – again – major stem cell regulators (e.g., FGF-II, Notch I, BMPs, etc.) were dynamically (e.g., after 15, 30 and 100 days after the immunization) expressed at protein and mRNA levels. In vitro experiments showed aNPCs efficiently inhibiting antigen-specific proliferation of cocultured PLP-reactive encephalitogenic T cells. Our preliminary data show that systemically-injected aNPCs may survive in vivo even for long periods of time and protect from chronic CNS inflammation owing to their capability to exert immunomodulatory functions within both canonical central (e.g. the CNS) as well as non-canonical peripheral (e.g., lymph nodes) site(s) of accumulation, where stem cell surviving factors are ectopically (re)expressed in response to inflammation. WS13-03

Mesenchymal stem cells treat relapsing–remitting experimental autoimmune encephalomyelitis through a dual effect on inflammation and tissue damage Gerdoni E1, Gallo B2, Casazza S1, Pedemonte E1, Musio S2, Mancardi GL1, Pedotti R2, Uccelli A1 Neuroimmunology Unit — Department of Neurosciences, University of Genoa, Italy; 2Immunology and Muscular Pathology Unit, National Neurological Institute C. Besta, Milan, Italy

1

Mesenchymal stem cells (MSCs), a subset of adult stem cells derived from the bone marrow stroma, represent an appealing tool for regenerative medicine. Recent studies have demonstrated that MSCs modulate immune responses supporting their utilization for the treatment of autoimmunity. Thus, we sought verifying whether MSC can cure mice affected by experimental autoimmune encephalomyelitis induced with the peptide 139–151 of the proteolipid protein (PLP), a myelin antigen capable of inducing a potent pathogenic T and B cells response. MSC treated mice showed a significantly milder disease and fewer relapses compared to controls. In vivo PLP specific antibodies titres and antigen-specific T cell response were significantly lower in MSCs treated mice. The adoptive transfer of encephalitogenic PLP sensitized, preconditioned with MSC, induced a milder disease compared to controls. MSC treated encephalitogenic cells showed decreased production of IFNγ and TNFα and did not proliferate upon antigen recall thus were considered anergic. Upon i.v. injection, Green Fluorescent Protein (GFP) labeled MSCs were detected inside lymph nodes early upon injection and inside the inflamed CNS at later stage. MSC injection resulted in a decreased number of inflammatory infiltrates, reduced demyelination and apoptotic neural cells and, as consequence, sparing of axons and oligodendrocytes. In contrast, no evidence of GFP labeled neural cells were detected, thus not supporting the hypothesis of trans-differentiation. Overall, we propose that MSC may have a dual function that may be effective for the treatment of MS, an autoimmune disease of the CNS where degeneration of neural cells follows inflammation.

1

Neuroimmunology Unit, DiBiT, San Raffaele Scientific Institute, Milan, Italy; 2BioXell, Milano, Italy

Transplantation of undifferentiated adult neural stem/precursor cells (aNPCs) promotes long-lasting neuroprotection in experimental multiple sclerosis by remyelination of injured central nervous system (CSN) axons as well as by immunomodulatory functions. After systemic transplantation, aNPCs enter brain and spinal cord, selectively reach inflamed CNS areas – the “atypical perivascular niches” – where major stem cell regulators are focally (re)expressed – and survive in vivo for up to 120 days after transplantation. To further explore the immunomodulatory properties of adult neural precursor cells (aNPCs) in mice with experimental autoimmune encephalomyelitis (EAE), we have intravenously (i.v.)-injected syngenic aNPCs into mice with the relapsing–remitting form of the disease. SJL female mice were immunized

WS13-04

Treatment of chronic EAE with bone marrow stromal stem cells (MSC): Differentiation of MSCs into cells with neuronal, glial and oligodendrocyte morphology and induction of neuroprotection Karussis, D⁎, Grigoriadis, N#, Kassis, I⁎, Ben-Hur, T⁎, Gouda-Kurkalli, B#, Mizrachi, R⁎, Einstein, O⁎, Abramsky O⁎, Slavin, S# ⁎Neurology and #BMT Department, Hadassah–Hebrew University Hospital, Jerusalem, Israel, and Department of Neurology, AHEPA Hospital— Aristotle University, Thessaloniki, Greece

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Abstracts

Bone marrow (BM) derived mesenchymal stromal stem cells (MSCs) can differentiate under certain circumstances into cells from various tissues. The aim of our study was to investigate the ability of BM-MSCs to migrate into CNS inflamed tissue and differentiate into cells of the neural–glial lineage, in the chronic-EAE model, and to evaluate whether these cells can exert a beneficial clinical effect. MSCs were obtained from GFP-transgenic mice. Cells were cultured with a combination of growth factors (FGF-2/BDNF/FGF-8) and checked for their differentiation properties. MSCs were injected intraventricularly (icv) in C57Bl mice with chronic-EAE at the initial phase of the disease. All animals were followed up clinically for at least two months. In vitro, MSCs showed the ability to differentiate into cells with neural and glial-like morphology and were stained with GFAP+ (∼ 30%), O4+ (∼ 10%), Nestin+ (∼ 10%) and NF160+ (∼20%). Cultured MSCs showed immunomodulatory properties and significantly downregulated lymphocyte proliferations against myelin antigens. In vivo, icv injection of MSCs suppressed the clinical (mortality 0%, vs. 16% in the controls; mean maximal score: 3.08, vs. 3.85) and histopathological manifestations of chronic-EAE and induced significant neuroprotection (>90% of axons unaffected in the treated animals vs. 40% in the controls). MSCs migrated into the inflamed lesions and were found to present immunohistological characteristics of neuronal and glial cells. Our results indicate that autologous BM can provide a source of stem cells which downregulate the clinicopathological signs of chronic-EAE and carry a neuroprotective potential; such treatment protocols may be applicable in diseases like multiple sclerosis. WS13-05

Injection of human neurospheres ameliorates a non-human primate model of multiple sclerosis Stefano Pluchino1, Angela Gritti2, Elena Brambilla1, Alessandro Cattalini1, Hessel Smits3, Giuliana Salani1, Bert t'Hart3, Angelo Vescovi2 and Gianvito Martino1 Neuroimmunology Unit, DiBiT and 2Institute for Stem Cell Research, DiBiT, San Raffaele Scientific Institute, Milan, Italy; 3Department of Immunobiology, Biomedical Primate Research Center, Rijswijk, The Netherlands

1

We have transplanted human foetal NPCs in non-human primates with experimental autoimmune encephalomyelitis (EAE). EAE was induced in 15 marmosets following subcutaneous administration of 100 μg recombinant human myelin-oligodendrocyte glycoprotein (MOG)1–125 in complete Freund's adjuvant. In order to detect also sub clinical EAE, immunized monkeys underwent serial (every 3 weeks after the immunization) brain magnetic resonance (MR). At both clinical or subclinical disease onset, marmosets were injected either intravenously (i.v., n = 5) or intrathecally (i.e., into the cerebrospinal fluid [CSF], n = 4). The remaining six monkeys were used as sham-treated controls. NPCs had been in vitro prelabeled by inserting in them the green fluorescent protein (GFP) gene through 2nd generation lentiviral-base infection. All marmosets received

daily intramuscular Cyclosporine (10 mg/kg) treatment from the time of disease onset. Clinical score and weight were daily monitored along a 90 day-long follow up period. EAE monkeys transplanted i.v. as well as i.c. with hfNPCs showed amelioration from EAE, by means of (i) cumulative daily score; (ii) no. of relapses per day of follow-up; (iii) no. of EAE-related deaths and (iv) cumulative survival. Reduction of demyelination and axonal loss were observed in all transplanted marmosets. At histopathology the great majority of transplanted cells accumulated within the chronicallyinflamed CNS and persisted over time around inflamed CNS perivascular areas without displaying major features of terminal differentiation. These results are of critical and propedeutical importance to envisage the future use of hFNPCs as a safe and efficaciously neuroprotective therapy in MS. WS13-06

Cellular and molecular analysis of SVZ-resident neural stem cells in mice affected by chronic experimental autoimmune encephalomyelitis (EAE) Michela Deleidi1,⁎ , Jaime Imitola2,⁎ , Giuliana Salani1, Elena Brambilla1, Lucia Zanotti1, Luca Muzio1, Andrea Bergamaschi1, Stefano Pluchino1, Samia J. Khoury2,⁎ and Gianvito Martino1,⁎ 1

Neuroimmunology Unit, DiBiT, San Raffaele Scientific Institute, Milan, Italy; 2Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Boston, MA, USA

⁎These authors contributed equally to the work. We have adopted a multidisciplinary approach to analyse kynetics of sub-ventricular zone (SVZ)-resident neural stem/precursor cells (NPCs) during chronic experimental autoimmune encephalomyelitis (EAE) in mice. Adult C57Bl/6 female mice were immunized with myelinoligodendrocyte glycoprotein (MOG)35–55 and combination of in vivo, in situ and ex vivo analyses performed at different timepoints after the immunization. Fast- and slow-cycling SVZ-resident NPCs were studied by intraperitoneal injection with pulsed IddU/BrdU prior to sacrification. The orientation of mitotic cleavage of endogenous dividing NPCs (namely a measure of the (sub)fraction of NPCs undergoing asymmetric vs. symmetric cell division), was also studied. Early (e.g., 20 days after the immunization) increase of BrdU incorporation by both SVZ-resident glial and neuronal progenitors was revealed at confocal microscopy. Parallel in vitro data showed progressive impairment of clonal efficiency within neurosphere cell lines established from EAE mice, starting at 20 dpi, peaking at 30 dpi and then slowly but insufficiently recovering at later time points. No differences of sphere size and in vitro growth rate were found between groups. Our preliminary results show increased cell proliferation within the SVZ neurogenic niche paralleled by striking quantitative impairment of the endogenous neural stem cell compartment. Further studies aiming at the identification of cellular and molecular pathways involved in reactivation/reprogramming of the cell type(s) triggered by the lesion(s) may help solve and/or improve the contribution of endogenous NPCs to repair mechanisms.

Abstracts

Workshop 14: Pathogenesis of MS

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Abstracts

WS14-01

Reciprocal regulation of adenosine receptor expression on foamy macrophages in vitro and in MS lesions L. A. Bovena, K. Assinka, M. van Meursa, J. S. Voermana, Rogier Q. Hintzenb, and J. D. Lamana

Results: NR4A2 and NR4A3 were significantly upregulated in MS T cells, but NR4A2 levels did not correlate with those of OPN. Conclusions: Quantitative real-time RT-PCR confirms upregulation of NR4A2 in MS T cells. Although specific transcriptional targets of NR4A2 in T cells remain unknown, NR4A2 might play a role in immunopathogenetic mechanisms of MS patients. WS14-03

Department of Immunology, bDepartment of Neurology, Erasmus MC, and a,b MS Centre ErasMS, Rotterdam, The Netherlands

a

Adenosine is released at sites of inflammation and tissue damage and activates adenosine receptors. Many of the reported adenosine receptormediated effects are neuroprotective. However, adenosine may also aggravate neuronal injury by promoting inflammation. We have recently demonstrated an anti-inflammatory function for foamy macrophages in multiple sclerosis (MS) lesions and hypothesized that expression of adenosine receptors is altered in these cells thereby affecting their function. Therefore, we determined mRNA expression levels of the adenosine receptors (A1, A2a, A2b, and A3) in human myelin-laden macrophages in vitro and in MS lesions. A3 receptor mRNA expression was significantly downregulated (5-fold) in foamy macrophages as compared to control macrophages. LPS strongly induced A2a mRNA expression in both control and foamy macrophages (100-fold). In contrast, LPS synergistically downregulated A3 receptor mRNA (100-fold) in foamy macrophages compared to control macrophages. To assess whether this potent mRNA regulation also occurs during MS, expression levels were determined in MS brain and brain tissue of non-demented controls. A3 receptor mRNA was significantly upregulated in MS brain and the expression correlated with lesion activity. Immunohistochemistry will reveal which cells are expressing A3 receptors in MS brain. Our data demonstrate that adenosine receptor expression in MS brain and on foamy macrophages in vitro is strongly regulated. The altered balance of the expression levels of the different adenosine receptors is likely to influence the functional response to adenosine. Unraveling the function of these immune-modulatory receptors will allow better understanding of endogenous neuroprotective mechanisms and may thereby open new roads to disease intervention. WS14-02

NR4A2 (Nurr1), an orphan nuclear receptor, is overexpressed in peripheral blood T lymphocytes of multiple sclerosis Yoshimitsu Doi, Shinji Oki, Jun-ichi Satoh, Toshimasa Aranami, Sachiko Miyake, Takashi Yamamura Department of Immunology, National Institute of Neuroscience, NCNP, Tokyo, Japan

Genomic evidence for subtypes of Multiple Sclerosis: Presence of a viral response signature in a subpopulation of patients Lisa G.M. van Baarsena, Tineke C.T.M. van der Pouw Kraana, Jolijn Kragtb, Josefien M.C. Baggena, François Rustenburga, Tineke Hoopera, Jan F. Meilofc, Mike J. Ferod, Christine D. Dijkstraa, Chris H. Polmanb, Cornelis L. Verweija a

Department of Molecular Cell Biology and Immunology, VUMC, Amsterdam, The Netherlands; bDepartment of Neurology, VUMC, Amsterdam, The Netherlands; cDepartment of Neurology, Martini Ziekenhuis, Groningen, The Netherlands; dStanford Functional Genomics Facility, Stanford, USA Multiple Sclerosis (MS) is a heterogeneous disease. Heterogeneity most likely has its origin in the multifactorial nature of the disease, which involves both genetic and environmental factors. Clinical and pathological features of multiple sclerosis (MS) implicate viruses as an environmental factor in the development and or progression of disease. Despite epidemiological support for this hypothesis, the presence and physiological significance of viral infections in MS is still unclear. In this study we analyzed whole blood gene expression profiles of 29 patients with relapsing remitting (RR) MS and 25 age and sex matched healthy controls. Comparative analysis identified a stereotyped gene expression program that appears to reflect a virus response program, in the peripheral blood cells of patients with RRMS. Subsequent analysis revealed that the virus response program was characteristic of not all, but approximately half of the RRMS patients. Thus, the transcriptional signature of the peripheral blood cells defines a subpopulation of RRMS patients who exhibit evidence for a link between viruses and MS. WS14-04

T-cell reactivity to myelin proteolipid protein drives the development of brainstem and cerebellar lesions in multiple sclerosis M.P. Pendera,b, P.A. Csurhesa and J.M. Greera a

Objective: To study a role of the NR4A subfamily of nuclear receptors in the immunopathogenesis of multiple sclerosis (MS). Background: Nurrelated factor-1 (Nurr1; NR4A2), nerve growth factor-induced gene-B (NGFI-B; NR4A1), and neuron-related orphan receptor-1 (NOR-1; NR4A3) are transcription factors of the steroid/thyroid receptor superfamily, rapidly induced by exposure to growth factors and cytokines, suggesting a biological role of cell growth, differentiation and apoptosis (Pei et al. JBC 280:29256–62, 2005). One of transcriptional targets of NR4A2 is Osteopontin (OPN) (Lammi et al. Mol Endocrinol 18:1546–57, 2004). By analyzing a cDNA microarray, we found that NR4A2 is upregulated in CD3+ T cells of MS patients compared with healthy control (CN) subjects (Satoh et al. Neurobiol Dis 18:537–50, 2005). However, an active role of NR4A2 and its transcriptional targets in MS remain unknown. Methods: NR4A1, NR4A2, NR4A3, and OPN mRNA levels were quantitatively analyzed in cDNA of CD3+ T cells of 58 untreated active MS patients and 19 CN subjects by real-time PCR.

Neuroimmunology Research Centre, The University of Queensland, Brisbane, Australia; bRoyal Brisbane and Women's Hospital, Brisbane, Australia

The mechanisms responsible for the localization of lesions in multiple sclerosis (MS) are currently unknown. To investigate this, we performed HLA-DR and HLA-DQ typing and examined T-cell reactivity to myelin proteins in 121 patients with MS not on immunomodulatory therapy, 71 healthy subjects and 47 patients with other diseases of the central nervous system (CNS). We found that 47.0% of MS patients with brainstem or cerebellar lesions, as determined by clinical assessment and/or magnetic resonance imaging, had increased circulating T-cell reactivity to an immunodominant region of myelin proteolipid protein (PLP184–209), compared to 10.0% of MS patients without brainstem or cerebellar lesions, 11.3% of healthy subjects and 19.1% of patients with other CNS diseases (p < 0.0006). We also found that 78.6% of MS patients with brainstem or cerebellar lesions carried HLA-DR4, DR7 or DR13 alleles, whereas only 28.9%

Abstracts of patients without brainstem or cerebellar lesions carried these alleles (p = 0.00000003). Furthermore, 60.9% of MS patients carrying HLA-DR4, DR7 or DR13 alleles had increased T-cell reactivity to PLP184–209, compared to 12.2% of MS patients not carrying these HLA alleles (p = 0.0001). Interestingly, H-2k mice immunized with PLP184–209 also develop inflammatory demyelinating lesions predominantly in the brainstem and cerebellum. Our results indicate that T-cell reactivity to PLP184–209 drives the development of brainstem and cerebellar lesions in MS. In patients carrying HLA-DR4, DR7 or DR13 alleles, therapy with altered peptide ligands based on PLP184–209 might prevent the development of brainstem and cerebellar lesions. WS14-05

A system biology approach to the immune system network reveals functional differences in health and Multiple Sclerosis P. Villoslada, R. Palacios, J. Goñi, N. Velez, J. Sepulcre Neuroimmunology lab, Department of Neurology, University of Navarra, Spain Background: A functional knowledge of the properties of the gene networks that control biological functions might be critical for understanding complex diseases, such as Multiple Sclerosis (MS). Our aim was to model the gene network that controls the T-cell activation–differentiation–suppression process, which is critical for the development of MS, and to assess its functional properties in health and autoimmunity. Methods: We modelled a network with 20 critical immune system genes using scientific text mining and experimental data from 52 healthy controls and 52 patients with MS obtained by real-time PCR by using a Bayesian approach. We compared the functional differences in the network in health and autoimmunity using the Kullback–Leibler (KL) divergence for each pair of nodes. Results: We found a network with 31 links (gene-interactions), 18 already known (expected) and 13 non previously described. We validated 7 out of 9 links: 4 out of 5 expected and 3 out 4 new ones. We found significant differences between the control and MS network, mainly in the regulatory (TGFB1, IL10, JAG1) and proinflammatory pathways (IL-12). In addition we identified the effect of interferon beta therapy in the network, mainly in the interactions between ITGA4–TGFB1 and JAG1–TNF, TGFB1– GATA3, IL10–CD28, TGFB1–ITGB7, ITGA4–ITGB7 and IL10–ITGA4. Conclusion: Our results indicate that assessing the properties of the immune gene network using a system biology approach reveals significant changes in autoimmunity and provides insights in the effect of immunomodulatory therapy.

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WS14-06

Characterization of oligoclonally expanded T-cells derived from multiple sclerosis (MS) lesions M. Montesa, T. Kondob, W. Brückc, V. Jewellsd, D. Armaod, J. Smrtkaa, C. Pinillae, S. Markovic-Plesea a

Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States; bDepartment of Clinical Research, Nagasaki Medical Center of Neurology, Nagasaki, Japan; cDepartment of Neuropathology, Georg-August University, Göttingen, Germany; d Department of Radiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States; eTorrey Pines Institute for Molecular Studies, San Diego, CA, United States Objective: To characterize T-cell infiltrates and in vivo expanded clonotypes derived from MS lesions. Background: Oligoclonally expanded T-lymphocytes perpetuate inflammatory response against CNS myelin antigens. This study addresses their representation in the MS lesions at various stages of development, and their antigen specificity. Methods: Brain and spinal cord tissue was obtained six hours post mortem from a female patient with aggressive relapsing remitting (RR) MS. Lesions were characterized based on the longitudinal CNS MRI, the last one 4 days prior to death, and histopathological and immunohistochemistry studies. Viable cells from six lesions and normal appearing white matter (NAWM) were expanded in vitro. To study in vivo expanded clonotypes and their TCRVb usage in the infiltrates from the lesions and NAWM, we RT-PCRamplified TCR genes and performed single-strand conformational polymorphism (SSCP) pattern analysis. Results: Antigen specificity of MS-lesion derived T-cells was tested against a panel of immunodominant myelin-derived peptides. The highest proliferative response was detected against PLP 190–209 in acute pontine, chronic periventricular and thoracic spine lesions. Cells derived from acute lesion exhibited proliferative response against six myelin peptides, four of which were recognized in chronic thoracic, and three in chronic periventricular lesion. Single-strand conformational polymorphism (SSCP) pattern analysis identified limited number of TCRVB chains expressed correlating to the in vivo expanded clonotypes. It revealed distinct patterns of TCR VB family chains in each MS lesion and NAWM. However, TCRVB4, VB5.1, VB8, and VB13.1 were present in all lesions. Antigen specificity of the clones corresponding to the in vivo expanded clonotypes is presently characterized using synthetic combinatorial peptide libraries. Relevance: This is the first study to characterize post-mortem derived T-cell infiltrates from MS lesions at various stages of development.

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Abstracts

Oral Session 1A: BBB

Abstracts OS1A-01

Endothelial cell Weibel–Palade bodies regulate blood brain barrier function and susceptibility to experimental allergic encephalomyelitis R. Noubadea, J. F. Zacharyb, D. D. Wagnerc, E. P. Blankenhornd, T. Owense, J. Millwarde and C. Teuschera

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cells, and this fast rolling appears to be L-selectin dependent. PSGL-1 is also involved in the rolling of monocytes and Th1 but not Th2 cells, whereas CD44 is involved in the rolling of all activated leukocytes. For transendothelial migration, LFA-1 is crucial for Th1 and Th2 cell diapedesis but not monocytes. In conclusion, reduced shear stress is required for leukocyte to roll at the BRB. Different adhesion molecules are involved in the trafficking of different leukocyte subsets at the BRB. OS1A-03

University of Vermont, Burlington, VT, USA; bUniversity of Illinois at Urbana-Champaign, Champaign, IL, USA; cCBR Institute for Biomedical Research, Boston, MA, USA; dDrexel University College of Medicine, Philadelphia, PA, USA; eMontreal Neurological Institute, Montreal, Canada

a

Pertussis toxin (PTX), an immunopotentiating adjuvant in experimental allergic encephalomyelitis (EAE), also elicits leukocytosis, alters glucose regulation, vascular permeability and blood–tissue barrier functions, and sensitizes the vascular endothelium to vasoactive amines such as histamine (BPHS). The mechanism underlying BPHS is unknown but requires a 2–3 day latency and lasts ∼ 30 days. This suggests that the induction phase is associated with the storage of preformed vasoactive factors that are then released during the effector phase. Weibel–Palade bodies (WPB) within endothelial cells (EC) are secretory granules known to release preformed chemokines, adhesion molecules and von Willebrand Factor (Vwf) following exposure to histamine. Mice with a disrupted Vwf allele (VwfKO) have EC that are deficient in WPB and were used to evaluate the role of this pathway in BPHS and susceptibility to EAE. No significant differences in susceptibility to BPHS between wild-type and VwfKO mice were detected at three days; however, in VwfKO mice sensitivity persisted longer and the LD50-histamine was lower at later time points. Correspondingly, the onset of EAE was earlier, disease more severe and blood brain barrier (BBB) permeability significantly increased in VwfKO mice compared to wild-type mice. Increased BBB permeability in VwfKO mice was not due to increased encephalitogenic T-cell activity since BBB permeability was significantly greater in PTX-treated VwfKO mice compared to mice receiving encephalitogen-complete Freund's adjuvant (CFA) + PTX, CFA + PTX or CFA. Taken together, these data indicate that WPB normally repress BPHS and adjuvant-induced alterations in BBB function associated with actively induced EAE.

Thrombospondin-1, a VLA-4 ligand, plays a critical role in EAE development J. L. Vegaa, S. Maslib, K. Yanga, P. Alcaided, F. W. Luscinskasd, J. Lawlerc, and H. L. Weinera a

Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School., Boston, USA; b Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School., Boston, USA; c Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA; dDepartment of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, USA

Comparison of monocyte and T lymphocyte trafficking at the blood retinal barrier in vivo

Thrombospondin-1 (TSP-1) serves as a ligand for several receptors present on the surface of immune cells, including VLA-4. Because TSP-1 is expressed at high levels by endothelial cells, we investigated its role in the adhesion and traffic of inflammatory cells in experimental allergic encephalomyelitis (EAE). Using in vitro techniques for the study of adhesion and migration under blood flow conditions we show that by virtue of its ability to bind VLA-4, TSP-1 is a potent substrate for the adhesion of Th1 cells and macrophages. This function appears to be critical in vivo, as TSP-1 null mice (C57BL/6 background) are completely resistant to developing MOG-induced EAE, in spite of their ability to generate normal immune responses against MOG [i.e., normal proliferation, antibody and cytokine (IL-2, IFN-γ, IL-4, IL-10, IL-17 and TGF-β), production upon rechallenge with MOG in vitro]. An in vivo role in trafficking for TSP-1 was also suggested by our findings using immunohistochemistry that TSP-1 is expressed constitutively at high levels by perimeningeal blood vessels in the CNS. This was further supported by histological and flow cytometric analyses performed at various intervals post-EAE induction, which showed virtually a complete absence of macrophages and other inflammatory cell infiltrates in the CNS of TSP-1 null mice. Thus our findings suggest that TSP-1 serves as an adhesion molecule, and that its expression by blood vessels is required for the traffic of inflammatory cells into the CNS parenchyma.

H. Xua, A. Manivannanb, R. Dawsona; I.J. Cranea, and J. Liversidgea

OS1A-04

Department of Ophthalmology, bDepartment of Radiology, University of Aberdeen, Scotland, UK

Small G protein RhoA controls lymphocyte recruitment in brain vessels and the induction of experimental autoimmune encephalomyelitis

OS1A-02

a

Neural inflammation is caused by lymphocyte and monocyte infiltration across the tight junctions of blood–brain barrier, but the mechanisms involved in the adhesion and transendothelial migration of different leukocyte subsets are unclear. Using scanning laser ophthalmoscopy we have investigated the trafficking of monocytes and compared this with Th1 and Th2 cell trafficking at the blood–retina barrier (BRB) of the neuroretina, an extended organ of the brain. In normal circumstances, retinal vessels are subjected to high shear stresses and leukocytes do not roll. Activated T cells can migrate into the neuroretina in the absence of rolling and adhesion molecule LFA-1 is critical for the transendothelial migration. In inflamed retina there is a reduced shear stress in both arterioles and venules at the BRB but leukocytes only roll in the venules. Naïve T cells and monocytes roll at similar levels, however primed/activated T cells roll in a higher efficiency. Different adhesion molecules are responsible for the rolling of different leukocyte subsets. In vivo conditioned monocytes roll faster than primed/activated T

L. Ottoboni, B. Rossi, C. Giagulli, M. Martinello, S. Bach, C. Laudanna, G. Constantina Department of Pathology, University of Verona, Verona, Italy We have recently demonstrated that 23–40 domain of small G protein RhoA controls integrin affinity, while 92–119 RhoA domain controls lateral mobility and clustering in naïve T cells, showing that RhoA controls both modalities of integrin activation. The goal of this study was to determine the role of RhoA in the recruitment of autoreactive T lymphocytes in inflamed brain vessels and in the induction of experimental autoimmune encephalomyelitis (EAE). Methods: We used 3 trojan peptides able to block RhoA effector regions, consisting of a sequence of Antennapedia (P1) able to translocate through the cell membranes linked to: (1) 23–40; or (2) 92–119; or (3) 75–92

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domains of RhoA. Intravital microscopy in inflamed mouse brain microcirculation and induction of EAE by adoptive transfer of PLP139– 151 reactive T cells were performed. Results: All P1-derived Trojan peptides did not alter intracellular calcium release, adhesion molecules expression and distribution, and proliferation of PLP139–151 specific T cells. Intravital microscopy studies showed that P1/ 23–40 and P1/92–119 trojan peptides inhibit stable adhesion of encephalitogenic T lymphocytes in inflamed brain venules without affecting rolling, while, in contrast, controls P1 and P1/75–92 peptides were ineffective. Moreover RhoA peptide P1-92–119, but not P1-23–40, was able to block transfer EAE, induced by PLP139–151 specific CD4+ T cells: the incidence of the disease and the mean clinical score were significantly lower in animals treated with RhoA peptide P1-92–119. Conclusion: Selective regions of RhoA control autoreactive T cells migration into the inflamed brain and have a critical role in the induction of EAE. OS1A-05

Signaling through MyD88 regulates leukocyte recruitment after brain injury A. A. Babcocka,b, H. Toft-Hansena,b, S. Akirac and T. Owensa,b a

Medical Biotechnology Institute, University of Southern Denmark, Odense, Denmark; bMontreal Neurological Institute, Montreal, Canada; cOsaka University, Osaka, Japan

Injury to the central nervous system (CNS) provokes an innate inflammatory reaction that engages infiltrating leukocytes with the capacity to repair and/or exacerbate tissue damage. The initial cues that orchestrate leukocyte entry to the injured CNS remain poorly-defined. We have used flow cytometry to investigate whether MyD88, an adaptor protein that transmits signals from Toll-like receptors (TLRs) and receptors for interleukin (IL)-1 and IL-18, regulates leukocyte infiltration to the stabinjured entorhinal cortex (EC) and to sites of axonal degeneration in the denervated hippocampus. We have previously established the kinetics of leukocyte entry to the denervated hippocampus. We now show that significant leukocyte entry occurs within 3 h of stab injury to the EC. Whereas T cells showed small, gradual increases over 5 days, macrophage infiltration was pronounced and peaked within 12–24 h. Infiltration coincided with increased mRNA for matrix-metalloproteinase-12 and a disintegrin and metalloproteinase (ADAM)-12, but not interferon-γ or IL17. MyD88-deficiency reduced macrophage infiltration to the stab-injured EC and the denervated hippocampus by more than 75% at 5 days postinjury. MyD88-deficiency abrogated T cell infiltration to the denervated hippocampus at this time, and reduced T cell entry to the stab-injured EC by 50%. MyD88-deficiency strongly reduced stab-injury induced transcripts for TNFα and CCL2, which were increased several hundred fold at time of peak expression in wildtype mice (3 h). Stab injury-induced leukocyte recruitment and gene expression were unaffected by TLR2 or TLR4 deficiency. These data show that MyD88-dependent signaling is important for proinflammatory gene expression and leukocyte recruitment after CNS injury.

Abstracts

Oral Session 1B: CNS inflammation and regulatory mechanism

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Expression of a dominant-negative chemokine receptor 2 mutant in hematopoietic cells for use in a mouse model of multiple sclerosis

not manifest as histologically detectable inflammatory pathology. These results show that IFNγ induces a characteristic glial chemokine response that by itself is insufficient to promote inflammation, but that CNS chemoattractant signals can synergize with a peripheral infectious stimulus to drive T cell entry into the CNS.

J. Prillera, S. Strickera, A. Mildnerb, H. Schmidtb, H. Prüßa and M. Prinzb

OS1B-03

OS1B-01

a

Molecular Psychiatry and Experimental Neurology, Charité Universitätsmedizin Berlin, Germany; bInstitute of Neuropathology, University of Göttingen, Germany

The chemokine receptor 2 (CCR2) is thought to play an important role in the induction of autoimmune disease in the central nervous system (CNS) by attracting immune effector cells from the periphery into the brain. The aim of this study is to develop a technique that would lead to a loss of function of CCR2 in hematopoietic cells in order to reduce the infiltration of blood borne cells into the brain in experimental autoimmune encephalomyelitis (EAE). Using bone marrow (BM) chimeric mice, we demonstrate that expression of CCR2 on hematopoietic cells, but not on radioresistant CNS-resident cells, determines EAE disease course. Moreover, we find that mixed chimeras with BM from RAG1−/− × CCR2−/− mice are highly susceptible to disease, suggesting that CCR2 expression on T and B lymphocytes is not required for EAE development. We next generated a mutant murine CCR2 (CCR2Y152F) by site-directed mutagenesis. When HEK293 cells were transfected with wildtype CCR2 and stimulated with the CCR2 ligand, monocyte chemoattractant protein-1, a rapid and transient increase in intracellular calcium concentration was observed. This calcium response was completely abolished by cotransfection of HEK293 cells with CCR2Y152F and wildtype CCR2. Transduction of adult hematopoietic stem cells with CCR2Y152F and subsequent generation of BM chimeric mice will be used to determine the contribution of CCR2 on myeloid cells to the inflammatory response in EAE. OS1B-02

Interferon-gamma induced chemokines and pertussis toxin synergize to promote T cell entry to the central nervous system Jason M. Millwarda, Maria Carusoa, Iain L. Campbellb, Jack Gauldiec, Trevor Owensa,d McGill University, Montreal, Canada; bUniversity of Sydney, Sydney, Australia; cMcMaster University, Hamilton, Canada; dUniversity of Southern Denmark, Odense, Denmark

Antigen presentation capacity by glial cell types during CNS infection C. C. Bergmanna, K. Maloneb, L. Hamob, C. Ramakrishnaa, and S. A. Stohlmana Cleveland Clinic Foundation, Cleveland, USA; bUniversity of Southern California, Los Angeles, USA

a

Acute CNS infection by a nonlethal neurotropic coronavirus is controlled by CD8 T lymphocytes. Perforin mediated cytolysis controls virus replication in macrophages/microglia and astrocytes, implicating TcR-class I interactions. By contrast, IFN-gamma controls virus replication in oligodendroglia, suggesting an inherent resistance to cytolysis. Despite these effector functions and T cell retention, virus persists in glia resulting in ongoing demyelination. To investigate the molecular basis of glia-T cell interactions in vivo, microglia, astrocytes and oligodendroglia were isolated from infected mice and analyzed for MHC expression and mRNA encoding antigen processing molecules. Maximal surface class I expression was delayed in astrocytes and oligodendroglia compared to microglia. Enhanced class I expression was consistent with earlier transcription of genes associated with the antigenprocessing machinery, specifically TAP1 and LMP2, in microglia compared to oligodendroglia. Furthermore, the biphasic transcription profile observed in microglia contrasted with the sharp expression peak in oligodendroglia, coinciding with maximal T cell effector function. Oligodendroglia were also distinct from microglia by their strong upregulation of the CD8 T cell inhibitory ligand PDL-1, substantiating strict control of T cell effector function. Contrasting class I expression, class II upregulation was extensive on microglia, but very limited on oligodendroglia and astrocytes. In summary, microglia have enhanced and earlier potential to present antigen compared to oligodendroglia and astrocytes, due to distinct responses to Type I- and Type II-IFN. Nevertheless, prolonged antigen presentation capacity implicated by sustained MHC expression in all glial cell types may be muted by differential expression of modulatory molecules.

a

Inflammation of the central nervous system (CNS), which occurs during multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), is characterized by increased levels of interferon-gamma (IFNγ), a cytokine not normally expressed in the CNS. To investigate the role of IFNγ in CNS, we used intrathecal injection of a replication-defective adenovirus encoding murine IFNγ to IFNγ-deficient SJL/J mice (GKO). This resulted in stable, long-lived expression of ELISA-detectable IFNγ in cerebrospinal fluid. IFNγ induced expression in the CNS of the chemokines CXCL10 and CCL5, to levels comparable to those seen during EAE. Other chemokines (CXCL2, CCL2, CCL3) were not induced. Mice lacking the IFNγ receptor showed no response, and a control viral vector did not induce chemokine expression. Chemokine expression was predominantly localized to meningeal and ependymal cells, and was also seen in astrocytes and microglia. IFNγ-induced chemokine expression did not lead to inflammation. However, when pertussis toxin was given intraperitoneally to mice infected with the IFNγ vector, there was a dramatic increase in the number of T lymphocytes detected in the CNS by flow cytometry. This increase in blood-derived immune cells in the CNS did not occur with pertussis toxin alone, and did

OS1B-04

Immune-mediated loss of neurons secondary to the destruction of supporting astrocytes Peter J. Darlington1,2, Cornelia Podjaski1, Manon Blain1, Katie Horn2, Amit Bar-Or1, Timothy E. Kennedy2, Jack P. Antel1 Neuroimmunology Unit, 2Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, QC, Canada

1

Neuronal injury and loss is an increasingly recognized component of the pathology of multiple sclerosis (MS). Published data indicate that T cells can mediate neuronal damage in an MHC/antigen-restricted as well as an MHC-unrestricted manner, while sparing astrocytes. In contrast, glia-directed neuro-cytotoxicity has been shown to occur in vitro by innate immune-cells such as γ/δ T cells and natural killer (NK) cells. Here, we sought to determine if NK cells could damage human neurons. To do so, we utilized CD56+ NK cells from IL-2-activated, normal adult blood, and primary human neuron–glia cultures derived from fetal brain tissue. Interestingly, we found that although co-incubation of NK cells with neuron–glia cultures resulted in detachment of neurons, the neuron

Abstracts viability as determined by propidium iodide exclusion was normal at all time points examined. In contrast, the underlying astrocytes, which constitute up to 10% of these cultures, were very efficiently killed by the NK cells. Time-lapse video microscopy indicated that NK cells began targeting the glial cells within minutes after co-incubation, whereas neurons were largely ignored. After 24 h, the remaining neurons were found to clump on the few surviving glial cells as determined by confocal microscopy. This data is consistent with a model of indirect neuronal injury mediated by innate immune cells in which neuron loss reflects primary loss of glial, trophic, or substrate support. OS1B-05

Invariant Vα19-Jα33 NKT cells promote ICOS-dependent IL-10 production by B cells which regulate inflammation in a model of multiple sclerosis J. Ludovic Croxforda, Sachiko Miyakea, Michio Shimamurab, and Takashi Yamamuraa a

Department of Immunology, National Institute of Neuroscience, Tokyo Japan; bDevelopmental Immunology Unit, Mitsubishi Kagaku Institute, Tokyo, Japan

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Recently, a new population of CD1-independent NKT cells, restricted by MR1, a non-classical MHC class I molecule, have been described. MR1restricted NKT cells express the invariant Vα19-Jα33 T cell receptor (Vα19+ NKT). However, their function in immunity is unknown. In humans, Vα7.2Jα33 NKT cells (homologous to Vα19+ NKT cells) were detected in multiple sclerosis (MS) inflammatory CNS lesions. Therefore, we determined the role of Vα19-Jα33 NKT cells in experimental autoimmune encephalomyelitis (EAE), a T-cell mediated autoimmune demyelinating disease of the CNS and model of MS. In contrast to humans, mice have extremely low numbers of Vα19 NKT cells. Therefore we used Vα19 TCR Tg mice crossed to CD1 deficient (CD1KO) mice which lack Vα14 NKT cells. We observed that over-expression of Vα19 NKT cells protects mice from EAE, and is associated with a decrease of Th1 cytokines but an increase in IL-10. In addition, adoptive transfer of Vα19 NKT cells to wt EAE mice could also inhibit clinical disease. In contrast, mice deficient in Vα19 NKT cells (MR1KO) exhibited exacerbated EAE. Coculture of Vα19 NKT cells with B cells demonstrated that B cells are induced to produce IL10, an anti-inflammatory cytokine known to inhibit EAE. IL-10 production from B cells was ICOS-dependent but MR1 independent. This study demonstrates that Vα19 NKT cells have a regulatory function in EAE and therefore may be attractive therapeutic targets for the therapy of MS and other autoimmune diseases. Activation of Vα19 NKT cells by novel glycolipids may allow for disease-specific therapy.

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Oral Session 2A: Immune-mediated pathology and neuroprotection (1)

Abstracts OS2A-01

Immunomodulation of microglial phenotypes: Implications for multiple sclerosis and Alzheimer's disease Oleg Butovskya, Maya Koronyo-Hamaouia, Gennady Landab, Gilad Kunisa, Yaniv Ziva, Igor Smirnovc, Steffen Jungd, Hagit Cohene, and Michal Schwartza a

Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel; bKaplan Medical Center, 76100 Rehovot, Israel; c Proneuron Biotechnologies, P.O. Box 277, 74101 Ness Ziona, Israel; d Department of Immunology, The Weizmann Institute of Science, 76100 Rehovot, Israel; eMinistry of Health, Mental Health Center, Anxiety and Stress Research Unit, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84105 Beer Sheva, Israel. Cell renewal in the adult central nervous system (CNS) is limited, and is blocked under inflammatory brain conditions. Microglia play a major role in the cellular immune response associated with neurodegeneration. We show that T-cell-derived cytokines (IFN-γ and IL-4) can induce microglia to become neuroprotective and to induce cell renewal. In contrast, aggregated β-amyloid (Aβ) induces microglia to become cytotoxic and block both neurogenesis and oligodendrogenesis of adult rodent neural stem/progenitor cells (NPCs) In addition, high levels but not low levels of interferon-γ (IFNγ, a cytokine associated with inflammatory autoimmune diseases, confer on rodent microglia a phenotype that impedes oligodendrogenesis from NPCs. IL-4, reversed the impediment, attenuated TNF-α production, and overcame blockage of insulin like growth factor (IGF)-I production.. In rodents with acute or chronic experimental autoimmune encephalomyelitis, injection of IL-4-activated microglia into the cerebral spinal fluid resulted in improved clinical symptoms and increased oligodendrogenesis from the endogenous stem cell pool in the spinal cord, and induced neurogenesis in dentate gyrus spatially associated with microglia expressing MHC-II and IGF-I. Using double-transgenic mice expressing mutant human genes encoding presenilin-1 and amyloid precursor protein (a mouse model of Alzheimer's disease), we show that modulation of microglia into dendritic-like cells, achieved by a T cell-based vaccination with copolymer-1, resulted in reduction of cognitive decline, elimination of plaque formation, and induction of neuronal survival and neurogenesis. These results introduce a new microglia phenotype as necessary players in fighting off neurodegenerative conditions. OS2A-02

β-Amyloid uptake by microglial cells and proinflammatory cytokines S. Kataoka, H. Hara and T. Tabira National Institute for Longevity Sciences, Obu, Japan Alzheimer disease (AD) is characterized by extracellular deposits of fibrillar β-amyloid (Aβ) in the brain, accompanied with abundant phenotypically activated microglia in the brain. Recent studies showed that anti-Aβ immunotherapy reduced amyloid burden in the brains of AD model mice and that Aβ-specific antibodies enhanced Aβ phagocytosis by microglial cells in an IgG receptor (FcR) dependent manner. We addressed whether or not proinflammatory cytokines could influence anti-Aβ antibody-mediated phagocytosis of fAβ by microglia. Using aggregated Aβ and fluorescent-labeled lysosomal probes, we examined phagocytosis of fibrillar Aβ (fAβ) and lysosomal pathway in mouse primary cultured microglial cells and murine Ra2 microglial cell line in the presence or in the absence of anti-Aβ antibodies and proinflammatory cytokines. Anti-Aβ antibodies drastically enhanced fAβ phagocytosis, which anti-CD16/CD32 antibody blocked by 70%. Proinflammatory cytokines such as interleukine-6 (IL-6), tumor necrosis factor α (TNFα) and interferon-γ (IFN-γ) decreased

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Aβ uptake as well as lyososomal vesicle formation. Proinflammatory cytokines did not inhibit Aβ uptake induced by anti-Aβ antibody. However, anti-Aβ antibody treatment in the presence of proinflammatory cytokines caused microglial cell damages characterized by large cytoplasmic vacuoles. Our results show that Aβ-specific antibodies release the proinflammatory cytokine-induced suppression of Aβ phagocytosis by microglial cells, but that Aβ phagocytosis under proinflammatory condition leads to microglial dysfunction. These findings support Aβ vaccination-based therapy combined with anti-inflammatory therapy for clearing Aβ deposits in the AD brain. OS2A-03

New mechanism underlying glatiramer acetate mediated neuroprotection Jianuo Liu, Jamie Lin, Peter Olds, Jason G. Glanzer, Tatiana K. Bronich, Steve Caplan, Yuri Persidsky, Howard E. Gendelman and Jonathan Kipnis University of Nebraska Medical Center, Omaha, USA Glatiramer Acetate (GA) is a synthetic copolymer and has been shown to induce neuroprotection under several acute and chronic neurodegenerative conditions. The mechanism underlying its neuroprotective activities, however, remains obscure. Many studies have focused on the role of T cells induced by GA administration, and particularly on the GA-induced Th2 shift in the profile of the induced T cell response. Since GA can induce rapid neuroprotection following brain trauma or glutamate-intoxication of retinal ganglion cells on a time scale that is insufficient for a T cell mediated response, we hypothesized that GA-induced neuroprotection might be T cell-independent and that a direct effect of GA on neurons might exist. To address this possibility, we treated primary human neurons with staurosporine in the presence or absence of GA. We found that GA significantly increased neuronal survival by up to 70%. This direct neuroprotective effect of GA is associated with its internalization into primary neuronal cells and increase expression of BDNF and activation of PKCα. Moreover, GA enters mononuclear phagocytes via induction of the RhoA pathway and causes an alteration of actin organization, which increases the trans-migratory abilities of monocytes and dendritic cells (DCs) through the blood brain barrier (BBB). DCs loaded with GA (DC vaccination) attenuate degeneration of injured optic nerve fibers as observed in our in vivo optic nerve crush injury model. This novel mechanism of GA-mediated neuroprotection may lead to development of new therapeutic regimens for various neurodegenerative as well as autoimmune diseases. OS2A-04

IL-10-producing CD4+CD25+ T cells play an important role in reduction of neuronal death in a stroke model D. Frenkel1, J. Imitola1, H. Ochi1, A.M.C. Faria1, B.E. Figueroa2, R. M. Friedlander2, and H. L. Weiner1 Center for Neurologic Diseases, Department of Neurology, 2Department of Neurosurgery Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA

1

Inflammation plays an important role in ischemic stroke and in humans IL-10 may have a beneficial effect in stroke. We previously demonstrated reduction of infarct size in a mouse stroke model following transfer of CD4+ T-cells from mice tolerized with myelin oligodendrocyte glycoprotein (MOG) (35–55); CD4+ T cells from nasally tolerized IL-10-deficient mice had no effect. We found less neuronal death and axonal damage as shown by MAP2 staining and SMI-32 staining following nasal tolerization with MOG in wt animals compared to IL-10−/− mice. The cytokine profile of splenocytes from nasally-tolerized IL-10−/− mice revealed a reduction in IFN-γ compared to non tolerized littermates and was associated with

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increased TGF-beta production and elevation in the frequency of LAP+CD4+ T-cells. These results demonstrate the lack of neuroprotection observed in IL-10−/− mice vs. wt mice relates to the absence of IL-10 rather than a lack of tolerance. Flow cytometry analysis showed elevation in the frequency of CD4+CD25+ T-cells following nasal treatment in wt but not in IL-10−/− mice. To further investigate the neuroprotective role of CD4+CD25+ T-cells, we incubated mouse neuronal cultures with CD25-depleted CD4+ T cells. We found increased neuronal cell death following oxidative stress in depleted cultures. Moreover, incubation of mouse neuronal cultures with CD4+CD25+ T cells together with anti-IL-10 antibody significantly reduced the neuroprotective effect of CD4+CD25+ T cells. In conclusion, our data suggest that IL-10-producing CD4+CD25+ T cells play an important role in reduction of neuronal cell death following brain ischemic injury. OS2A-05

EPO mediated neuroprotection against ischemic and excitotoxic injury requires TNFRI signaling E. Taoufik1, M. Mengozzi2, V. Tseveleki1, P. Ghezzi2, A. Cerami3, M. Brines3, L. Probert1 1

Hellenic Pasteur Institute, Athens, Greece; 2Mario Negri Institute for Pharmacological Research, Milan, Italy; 3Kenneth S. Warren Institute, New York, USA

Tumour Necrosis Factor Receptor I (TNFRI) and its ligand TNF are constitutively expressed by CNS neurons and are upregulated after brain injury. TNF has been shown to exert direct neuroprotective effects by maintaining calcium homeostasis, decreasing glutamate-induced currents, and activating a cell protective pathway that involves inhibition of caspase 8, upregulation of FLICE-like inhibitory protein (FLIPL) and induction of p50/ p65 NF-κB (Taoufik et al., under submission). The tissue protective cytokine erythropoietin (EPO) and its receptor EPO-R are expressed by CNS neurons and their expression is upregulated following injury. EPO exerts potent neurotrophic properties and can prevent neuronal apoptosis after metabolic stress, cerebral ischemia, traumatic brain injury, spinal cord injury and kainate induced seizures. In this study we show that TNF and TNFRI are necessary for induction of the EPO/EPO-R neuroprotective pathway following ischemic and excitotoxic injury. Using TNFRI deficient mice and in vivo models of ischemic and excitotoxic injury (permanent middle cerebral artery occlusion (MCAO) and kainate induced seizures) we provide evidence that TNFRI signaling is necessary for EPO-mediated neuroprotection. We further show that TNF, acting specifically through TNFRI, induces the expression of EPO and EPOR in primary cortical neurons after glucose deprivation (GD) and oxygen glucose deprivation (OGD) and this is associated with increased NFκB activation and neuron survival. These results further extend our understanding of the mechanisms of TNF/TNFRI-mediated neuroprotection and provides novel evidence for the beneficial role of brain inflammation in response to injury.

Abstracts

Oral Session 2B: Immune-mediated pathology and neuroprotection (2)

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OS2B-01

OS2B-03

Immunization with neurofilament light protein induces spastic paresis and axonal degeneration in Biozzi ABH mice

Influence of leukaemia inhibitory factor on macrophage function in vitro and during experimental autoimmune encephalomyelitis

R. Huizingaa, N. Heijmansa, P. Schuberta, H. Herrmannb, S. Gschmeissnerc, B. 't Harta and S. Amora a

Department of Immunobiology, Biomedical Primate Research Centre, Rijswijk, The Netherlands; bDivision of Molecular Genetics, German Cancer Research Centre, Heidelberg, Germany, cEM unit, Cancer Research UK, London, UK

Axonal damage is the major cause of irreversible neurological disability in MS patients. While axonal damage correlates with antibodies against neurofilament light (NF-L) protein, a major component of the axonal cytoskeleton, the possible pathogenic role of autoimmunity to axonal antigens such as NF-L has so far been ignored. Here we describe that Biozzi ABH mice immunized with NF-L protein develop neurological disease characterized primarily by spastic paresis concomitant with axonal degeneration and inflammation in the spinal cord and, less so, in the sciatic nerve. In the CNS, F4/80+ macrophages/microglia were abundant while low numbers of CD4+ and CD8+ T-cells were observed. Peripherally, responses of splenocytes to NF-L were characterized by production of the pro-inflammatory cytokines IFN-gamma and TNFalpha. Elevated levels of circulating antibodies recognizing rmNF-L were present in the serum. Immunoglobulin deposits were observed within neuronal cell bodies in the CNS of mice exhibiting clinical disease indicating that antibodies to NF-L may have a pathogenic role in axonal degeneration. We conclude that autoimmunity to NF-L protein induces axonal degeneration and clinical neurological disease in mice. These results indicate that autoimmunity to axonal antigens, as described in MS, may be pathogenic rather than act as merely a surrogate marker for axonal degeneration.

Jerome Hendriks, Niels Hellings, Joris Vanderlocht, Piet Stinissen Hasselt University, Biomedical Research Institute and School of Life Sciences, Transnationale Universiteit Limburg, Diepenbeek, Belgium In the chronic disabling disease multiple sclerosis (MS), infiltration of the central nervous system (CNS) by autoreactive T-cells and macrophages results in damage to glial cells and neurons. Recent findings indicate that inflammatory cells are not merely detrimental but may also have protective effects. We previously showed that leukemia inhibitory factor (LIF), a member of the neurokine family of neurotrophic factors, is secreted by T cells and macrophages, and protects oligodendrocytes from TNF-α induced apoptosis. In experimental autoimmune encephalomyelitis (EAE), the animal model for MS, clinical symptoms are suppressed by LIF treatment. We demonstrate that among immune cells mainly macrophages express neurokine receptors. Thus, neurokines may influence macrophage function and thereby act immunomodulatory. Therefore, we investigated whether the neurokines LIF and oncostatin M affect macrophage function in vitro by studying their effect on myelin phagocytosis, pro-inflammatory cytokine production and oxygen radical production, all important processes in MS lesion formation. We are currently evaluating the in vivo effects of neurokines on the inflammatory response during EAE. Initial results indicate that neurokines can act both pro-inflammatory and anti-inflammatory, depending on their environment. OS2B-04

OS2B-02

Cannabinoid-mediated immunosuppression in experimental allergic encephalomyelitis

Transgenic inactivation of astroglial NF-κB improves functional recovery following spinal cord injury and experimental autoimmune encephalomyelitis

David Baker1, J. Ludovic Croxford1,2, Gareth Pryce1, Samuel M. Jackson1, Catherine Ledent3, Giovanni Marsicano4, Beat Lutz4, Gavin Giovannoni1, Roger G. Pertwee5 and Takashi Yamamura2

Roberta Brambilla1, Kim Esham1, Martin Oudega1, Andres Hurtado1, Scott R. Barnum2, John R. Bethea1

1

The Miami Project to Cure Paralysis; University of Miami, Miami, FL 33136, USA; 2Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA

Institute of Neurology, University College London, UK; 2National Institute of Neuroscience, NCNP, Tokyo, Japan; 3Institut de Recherche Interdisciplinaire en Biologie Humaine et Moleculaire, Free University of Brussels, Belgium; 4Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg-University Mainz, Germany; 5Institute of Medical Sciences, University of Aberdeen, United Kingdom

In the central nervous system (CNS) the transcription factor NF-κB is a key regulator of inflammation and secondary injury processes. Following trauma or disease, the expression of NF-κB-dependent genes is activated, leading to both protective and detrimental effects on CNS recovery. Here we demonstrate that gene-targeted inactivation of astroglial NF-κB in mice (GFAP-IκBα-dn mice) leads to dramatic improvement in locomotor recovery 8 weeks after spinal cord injury (SCI). Histologically, transgenic mice exhibit increased white matter preservation, with selective sparing of reticulospinal and corticospinal tracts. In parallel, GFAP-IκBα-dn mice show reduced expression of proinflammatory chemokines and cytokines (CXCL10, CCL2, TGFβ2), and of chondroitinsulphate proteoglycans forming the glial scar. Similarly, we demonstrate that GFAP-IκBα-dn mice recover significantly better than wild type (WT) following experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. Transgenic mice develop the demyelinating pathology 5 days later than WT and, chronically, recover locomotor function almost to normality. Expression of proinflammatory genes is delayed and reduced. We conclude that inhibition of NF-κB signaling in astrocytes results in protective effects following CNS injury and propose the NF-κB pathway as a new target for the development of therapeutic strategies to treat such pathologies.

Scientific evidence, initially demonstrated in experimental allergic encephalomyelitis (EAE) models, now supports previously anecdotal evidence that cannabinoids may be beneficial in symptom control in multiple sclerosis. Natural cannabinoids such as tetrahydrocannabinol or synthetic cannabinoid (CB) receptor agonists can also induce immunosuppression of EAE. However, the underlying mechanisms and receptor location and subtype involved in cannabinoid-induced immunosuppression remains unclear. We demonstrate that amelioration of EAE by cannabinoids is associated with the suppression of antigen-induced T cell proliferation, pro-inflammatory Th1 (interferon gamma and tumour necrosis factor) cytokine production and inhibition of mononuclear cell infiltration of the central nervous system. Secondly, although leucocytes express CB1 and notably CB2 receptors, cannabinoid-mediated immunomodulation is largely, if not exclusively CB1 receptor-dependent. This was shown using selective agonists and by the loss of cannabinoid activity in CB1-deficient animals. Thirdly, using conditional CB1 knockout mice, we demonstrate that the major pathway of immunosuppression is not due to CB1 receptor stimulation on T cells but more likely the indirect stimulation of CB1 in brain centres. Only high doses of cannabinoids that induced sedative physiological effects and these may not be clinically achievable or relevant. This suggests that

1

Abstracts cannabis use in humans is likely to exhibit no or marginal affects on relapsing disease in multiple sclerosis. OS2B-05

Fibrin/Mac-1 interactions induce microglia activation and regulate relapsing paralysis in central nervous system autoimmune disease Ryan A. Adamsa, Jan Bauerb, Shoana Sikorskia, Matthew J. Flickc, Hans Lassmannb, Jay L. Degenc and Katerina Akassogloua University of California San Diego, La Jolla, CA, USA; bUniversity of Vienna, Vienna, Austria; cChildren's Hospital Research Foundation and the University of Cincinnati, Cincinnati, OH, USA

a

In multiple sclerosis (MS), fibrin deposition temporally and spatially correlates with inflammatory demyelinating plaques. Although the presence of extravascular fibrin(ogen) at sites of inflammatory demyelination in MS has been documented by pathologists for decades, the molecular and cellular mechanisms of fibrin action in CNS pathogenesis have not been investigated. We show that fibrin signals through the Mac-1 (CD11b/

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CD18) integrin receptor to activate microglia and regulate the progression and severity of inflammatory demyelination. Fibrinogen directly activates microglia resulting in dramatic cytoskeletal rearrangements and increased phagocytosis through the activation of Akt and Rho. Inhibition of fibrin/ Mac-1 binding with PI3K inhibitors (LY294002) or blocking antibodies inhibits fibrinogen-induced microglia activation and phagocytosis. Pharmacologic depletion of fibrin using the snake venom ancrod in remitting– relapsing Experimental Autoimmune Encephalomyelitis (EAE) results in reduced activation of CD11b-positive microglia cells and effectively reverses relapsing paralysis. Inhibition of fibrin/Mac-1 interactions in fibrinγ390–396A knock-in mice, as well as after vaccination or intranasal delivery of fibrin γ377–395 peptide attenuates inflammatory demyelination. Our study provides a cellular and molecular definition of the role of fibrin in CNS pathogenesis and shows that fibrinogen-mediated microglia activation regulates the progression and severity of inflammatory demyelination. Since blocking fibrin/Mac-1 interactions affects only the inflammatory and not the coagulation properties of fibrin, targeting the γ377–395 fibrin epitope could represent a potential therapeutic strategy for MS with potential applications for other neuroinflammatory diseases associated with blood–brain barrier disruption and microglia activation. Supported by NIH/NINDS R01 Grant NS052189 and NMSS RG 3782A-2 (KA), and NMSS Postdoctoral Fellowship FG 1582-A-1 (RA).

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Abstracts

Oral Session 3A: NMO and MS in Japan

Abstracts OS3A-01

The fourth nationwide survey of multiple sclerosis in Japan: MRI characteristics of Japanese multiple sclerosis Osoegawafa, M.a; Fukazawa, T.b; Fujihara, K.c; Matsui, M.d; Kohriyama, T.e; Kikuchi, S.f; Doyu, M.g; Yamamura, T.h; Itoyama, Y.c; Saida, T.i; Ochi, H.a; Sakata, K.j and Kira, J.a a

Department of Neurology, Neurological Institute, Graduate school of Medical Sciences, Kyushu University, Fukuoka, Japan; bNishimaruyama Hospital, Sapporo, Japan; cDepartment of Neurology, Tohoku University School of Medicine, Sendai, Japan; dDepartment of Neurology, Kanazawa Medical University, Kanazawa, Japan; eDepartment of Clinical Neuroscience and Therapeutics, Division of Integrated Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; fDepartment of Neurology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; gDepartment of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan; hDepartment of Immunology, National Institute of Neuroscience, NCNP, Tokyo, Japan; iDepartment of Neurology, Center for Neurological Diseases, Utano National Hospital, Kyoto, Japan; jDepartment of Hygiene and Preventive Medicine, Iwate Medical University School of Medicine, Morioka, Japan Background/objective: The aim of the present study was to clarify the characteristics of multiple sclerosis (MS) patients who had the longitudinally extensive spinal cord lesions (LESCL) extending over 3 vertebral segments on MRI using data of the fourth nationwide survey on MS carried out in 2004. Methods: Brain and spinal cord MRIs were analyzed according to the McDonald MRI criteria and were also examined for the presence of LESCL in patients with conventional MS (CMS) and opticospinal MS (OSMS). Moreover, clinical characteristics were compared among OSMS with LESCL, OSMS with spinal cord lesion not extending over 3 vertebral segments (usual SCL), CMS with LESCL and CMS with usual SCL. Results: The frequency of patients who met the McDonald MRI criteria was significantly higher in CMS than in OSMS (45.5% vs. 8.2%, p < 0.0001), while the frequency of LESCL was significantly higher in OSMS than in CMS (41.2% vs. 16.1%, p < 0.0001). Regardless of whether CMS or OSMS, those with LESCL showed female preponderance, higher EDSS, higher frequency of paraplegia and higher CSF cell counts, compared with those having usual SCL. The EDSS of MS subgroups except OSMS with LESCL were significantly correlated with the disease duration. Regardless of whether LESCL or usual SCL, OSMS showed higher age at onset, lower frequency of secondary progression and higher frequency of negative Brain MRI, compared with CMS. Conclusions: The location of the lesions as well as the existence of LESCL is an important factor to decide a clinical feature of MS.

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Method: We retrospectively reviewed the medical records of a total of 23 NMO patients (22 women and one man). We then analyzed the clinical data (age at onset, disease duration and Expanded Disability Status Scale (EDSS) score), CSF findings (cell count, protein concentration and IgG index) and immunological status (NMO-IgG and other autoantibodies, and co-existing autoimmune diseases) in the patients and compared the data of OB-positive patients with those of OB-negative patients. Result: We found two cases of NMO with OB (8.7%) among 23 NMO cases. Both patients were also positive for NMO-IgG. Their common features were long disease durations and co-existing autoimmune diseases (myasthenia gravis and sicca syndrome). There was no difference in age at onset, EDSS score, CSF cell count and CSF protein concentration between the two groups. Conclusion: OB are mostly negative in NMO and helpful for distinguishing it from multiple sclerosis, but they can be positive by long-standing autoimmunity which may not be directly related to NMO. OS3A-03

Anti-aquaporin 4 antibody in Japanese opticospinal multiple sclerosis K. Tanakaa, T. Tania, M. Tanakab, T. Saidab, J. Idezukac, K. Sakimuraa, M. Nishizawaa Brain Research Institute, Niigata University, Niigata, Japan; bUtano National Hospital, Kyoto, Japan; cOjiya Sakura Hospital, Ojiya, Japan

a

Neuromyelitica optica (NMO)-IgG, whose target molecule is probably an aquaporin 4 (AQP4), is reported to be a specific marker for NMO and will be a good tool to distinguish NMO from multiple sclerosis (MS). Opticospinal form of MS (OSMS) which has been categorized in a subtype of MS are prevalent in Asian countries. The differences between OSMS and NMO have long been under discussion. To clarify this argument, we established the immunofluorescence detection system of AQP4 antibody (AQP4-Ab) using AQP4-transfected HEK 293 cells. We examined this antibody in the sera of OSMS with long spinal cord lesions (LCL) with or without cerebral lesions, OSMS without LCL, common form of MS (CMS) and healthy controls. AQP4 was positive in 62% of OSMS with LCL but was negative in any other forms of MS. Among OSMS with LCL, the AQP4-positive group was all women with severe visual disturbance, however other features such as patients' mean age, severity of limb and truncal disabilities, existence of cerebral lesions were not different from AQP4-negative OSMS with LCL. Our results of AQP4-Ab in OSMS with LCL were similar in frequency of NMO-IgG found in NMO patients, indicated that both groups are immunopathologically common and could be the same. OS3A-04

OS3A-02

Clinical and laboratory features of neuromyelitis optica with oligoclonal IgG bands

MRI characteristics of 153 consecutive Japanese patients with clinically definite multiple sclerosis with special emphasis on longitudinally extensive spinal cord and periventricular lesions

M. Nakamuraa, I. Nakashimaa, S. Satob, K. Fujiharaa, Y. Itoyamaa

Matsuoka, T.1; Osoegawa, M.1; Matsushita, T.1; Yoshiura, T.2; Kira, J.1

a

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Objective: Oligoclonal IgG bands (OB) in the celebrospinal fluid (CSF) are an important laboratory finding for the diagnosis of multiple sclerosis (MS), and they are positive in more than 90% of MS patients in Western countries. In contrast, the frequency of OB has been reported to be much lower (about 10%) in neuromyelitis optica (NMO) even when sensitive isoelectric focusing is applied. The present study aimed to reveal the clinical and laboratory features of patients of NMO who were positive for OB.

Background: It is unclear whether or not the McDonald criteria are applicable to multiple sclerosis (MS) in Asians. The present study was designed to clarify the frequency of Japanese MS patients fulfilling the McDonald/Barkhof MRI criteria, and the features of those who had longitudinally extensive spinal cord lesions (LESCL) extending over 3 vertebral segments on MRI. Methods: Subjects were 153 consecutive

Departments of Neurology, Tohoku University School of Medicine, Sendai, Japan; bDepartment of Neurology, Kohnan Hospital, Sendai, Japan

Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Department of Clinical Radiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

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Japanese patients with MS who fulfilled Poser's criteria. All MRI scans were analyzed according to the McDonald/Barkhof's MRI criteria. We pathologically studied periventricular lesions in 3 opticospinal MS (OSMS) and 2 conventional MS (CMS) cases, as well as studying the features of LESCL. Results: The frequency of patients who met the McDonald/ Barkhof's MRI criteria and had periventricular ovoid lesions were significantly higher in CMS than OSMS patients (65.4% vs. 23.7%, p < 0.0001, 88.6% vs. 55.2%, p = 0.0026), while the frequency of LESCL and periventricular rim-like lesions were significantly higher in OSMS than CMS (55.3% vs. 25.0%, p = 0.0016, 62.1% vs. 31.4%, p = 0.0057). Pathologically, OSMS showed periventricular rim-like demyelinating lesions while CMS had demyelinating lesions extending to the deep white matter. LESCL distributed diffusely from the cervical to the thoracic cord, mainly involving the central portion of cord. Gadolinium-enhancing lesions were seen most frequently in the upper thoracic cord. Conclusion: The frequency of Asian MS patients fulfilling McDonald/Barkhof's MRI criteria is low while LESCL and periventricular rim-like lesions are frequently seen in OSMS patients. OS3A-05

Severe brain involvement is a common feature of multiple sclerosis with extensively long spinal cord lesion (LCL-MS) M. Komori, M. Tanaka, Q. Hao, T. Okamoto, M. Matsui, K. Ozawa, N. Kitagawa, T. Oheda, N. Murase, E. Mizuta, O. Komure, H. Sawada, T. Konishi and T. Saida

Multiple Sclerosis Center, Utano National Hospital, Kyoto, Japan Introduction: The McDonald criteria of MS which is largely based on the features of Caucasian patients states the spinal lesion should not be longer than 2 vertebral length and refers to a paper describing such lesions were found in 2–3% of patients. In proposed criteria for neuromyelitis optica by Wingerchuk et al., long cord lesion extending over 3 vertebral segments (LCL) on spinal T2-MRI is the most important characteristic. We report here the frequent brain involvement in Japanese MS patients with LCL (LCLMS). Methods: Among patients examined after 2000, we analyzed the clinical, MRI and laboratory features of 77 consecutive patients with LCL-MS. The average of disease onset age was 36 (2.5–74) years, disease duration 11.4 (1–37) years and the female to male ratio 8 to 1. Results: Site of clinical manifestation at the first attack was in spinal cord 54%, optic nerve 25%, both 6.8%, brainstem/cerebellum 15% and cerebrum 0%. In 98.6% cases, LCL was confirmed within 2 years after the onset. Cerebral symptoms, which were not present at the first attack, later developed in 39%: frequently symptoms were severe and the MRI lesion size was large. Brainstem lesions developed in 60%, 15% of whom required respirator assist. Average of recurrence number was 17.1 (in spinal cord 11.4, optic nerve 4.4, cerebrum 2.0, and brainstem/ cerebellum 1.4). Cerebrospinal fluid IgG oligoclonal bands were mostly negative. Conclusions: We propose LCL-MS as a variant of MS, at least in Japanese population, in which LCL occurs at an early stage and many later develop severe brain lesions.

Abstracts

Oral Session 3B: Inflammatory myopathies

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OS3B-01

Human myoblasts secrete MCP-1 and IL-6 and express ICAM-1 in response to IL-6/sIL-6R complex in vitro

of seven polymyositis muscles. The CCL19,21/CCR7 chemokine system is expressed in inflamed muscles of polymyositis and may be involved in the pathomechanism of polymyositis. OS3B-03

M. Marinoa, F. Scuderia, C. Provenzanoa, Jürgen Schellerb, Stefan RoseJohnb and Emanuela Bartoccionia a

Institute of General Pathology, Università Cattolica, Roma, Italy; Department of Biochemistry, Christian-Albrechts-Universität zu Kiel, Kiel, Germany

b

Mononuclear cell infiltration characterizes the inflammatory reaction in autoimmune polymyositis and rejection of transplanted myoblasts. Monocyte recruitment following neutrophil exudation is regulated by the IL-8/ MCP-1 switch driven by the action of the IL-6/soluble IL-6 receptor (IL-6/ sIL-6R) complex on endothelial/mesothelial cells. The IL-6R consists of a 130 kDa ubiquitously expressed transducing element, and a cytokinespecific 80 kDa receptor subunit; the latter is expressed only on target cells (hepatocytes and subsets of leukocytes) and is released as sIL-6R. The IL-6/ sIL-6R complex can interact with membrane-bound gp130 and activate virtually all cells in the body, through a mechanism called transsignaling. We have previously shown that human myoblasts are able to produce IL-6; the specific object of this study was to verify whether IL-6/sIL-6R complex could drive human myoblasts to substain monocyte recruitment. We used Hyper-IL-6 (a stable analogue of the IL-6/sIL-6R complex) to stimulate human myoblasts and analyzed the expression of MCP-1, IL-8, IL-6, membrane and soluble ICAM-1 and the monocyte chemotactic activity by RT-PCR, ELISA, FACS and chemotaxis assays. We found that human myoblasts do not produce significant levels of IL-8 in response to Hyper-IL-6, while they could use transsignaling to increase IL-6, MCP-1 and ICAM-1 production. The addition of sgp80, binding to IL1β-induced IL-6, greatly increases IL-6 production. Our findings show that muscle cells can use locally secreted IL-6 and transsignaling to start an autoamplificating autocrine IL-6 loop which then can target monocyte chemotaxis and leukocytes trafficking through the formation of an IL-6 and MCP-1 gradient and ICAM-1 modulation. OS3B-02

Expression of CCL19, CCL21/CCR7 chemokine system in muscles of polymyositis M. Tateyama, K. Fujihara, T. Misu, Y. Onodera and Y. Itoyama Department of Neurology, Tohoku University School of Medicine, Sendai, Japan Polymyositis is an autoimmune disorder in which autoaggressive CD8+ T cells are important in the pathogenesis. However, the mechanisms underlying sustained recruitment of these cells in the muscle tissue are still unknown. CCR7 and its ligands CCL19 and CCL21 are a chemokine system related to mononuclear cell migration and antigen presentation, and are suggested to play a key role in several autoimmune disorders. We investigated the expression of CCR7, CCL19 and CCL21 in frozen muscles of polymyositis. In immunohistochemistry, CCR7 was expressed mainly on mononuclear cells that infiltrated in the endomysium of polymyositis. 34.8 ± 9.4% of endomysial mononuclear cells expressed CCR7. By double immunostaining, about 60% of endomysial CD8+ T cells that infiltrated in endomysium and surrounded the nonnecrotic muscle fibers coexpressed CCR7. On the other hand, CCL19 was expressed mainly on muscle fibers in proximity to CCR7+ mononuclear cells, on the endothelium of the vessels and some mononuclear cells. CCL21 immunoreactivities were found on small numbers of mononuclear cells. In some cases, CCL21 immunoreactivities were also found on muscle fibers and the endothelium of vessels. In RT-PCR analysis, transcripts of CCR7 and CCL21 were detected in all the polymyositis muscles examined and that of CCL19 was detected in five out

Molecular interactions between inflammation and β-amyloidassociated degeneration in sporadic Inclusion Body Myositis (sIBM) muscle J. Schmidta,b, R. Rajua, M. Salajegheha, G. Rakocevica and M. C. Dalakasa a

Neuromuscular Diseases Section, National Institutes of Health, Bethesda, MD, USA; bDeparment of Neuroimmunology and Neurology, University of Göttingen, Germany Hallmarks of sIBM are inflammation and β-amyloid-associateddegeneration in the muscle, yet it is unknown whether these pathomechanisms trigger each other. RNA from sIBM-muscle (n = 12), Dermatomyositis (DM, n = 11), or non-myopathic controls (n = 12) was analyzed by real-time-PCR for expression of degeneration-associated-molecules amyloid-precursor-protein (APP), αB-crystallin, ubiquitin; inflammatory markers CXCL9, CCL3, CCL4, IFN-γ, IL-1β, IL-6, TNF-α, TGF-β, inducible-nitric-oxide (NO)synthase (iNOS). Immunohistochemical staining confirmed protein-expression. Magnetic-bead-purified human myotube-cultures were exposed to IFN-γ, TNF-α, IL-1β, or NO-donors, followed by analysis of cytokine- and chemokine-expression by real-time-PCR, immunohistochemistry and sandwich-ELISA. β-amyloid-accumulation was measured by FACS-analysis and immunohistochemistry with grayscale-densitometry. In sIBM-muscle compared to controls, mRNA for all chemokines (≤233-fold) and cytokines was significantly upregulated and half of the sIBM-samples displayed iNOS-expression. In DM, upregulated chemokinemRNA (≤62-fold), TGF-β, and IL-6 remained significantly lower than in sIBM and only few patients expressed IFN-γ, IL-1β or iNOS. Degenerationassociated markers were significantly overexpressed in both disorders, yet only in sIBM, APP significantly correlated with mRNA-levels of chemokines, ubiquitin, IFN-γ, and cellular infiltration. By immunohistochemistry, chemokines and cytokines co-localized to β-amyloid-accumulation in sIBM-myofibers, whereas in DM, they were mainly present in connective tissue and capillaries. Inflammatory responses by myotubecultures were similar to the pattern observed in sIBM-muscle, including striking iNOS-overexpression after IL-1β + IFN-γ. Exposure to NO-donors or IL-1β + IFN-γ caused intracellular β-amyloid-accumulation as revealed by immunhistochemistry and FACS-analysis. In conclusion, the results suggest that a specific interrelationship between degenerative and inflammatory pathomechanisms in sIBM-muscle centers around NO-related cell-stress. The data further our understanding of sIBM-pathology and may help to better comprehend other β-amyloidassociated disorders. OS3B-04

Characterizing T cell receptors from human histological specimens S. Seitzab, H. Wekerlea, R. Hohlfeldab and K. Dornmairab a

Department Neuroimmunology, MPI for Neurobiology, Martinsried, Germany; b Institute for Clinical Neuroimmunology, LMU, Munich, Germany

We describe a strategy to “revive” putatively pathogenic T cells from frozen specimens of human target organs. These T cells may then be used to investigate their yet unknown antigens. To distinguish autoaggressive from irrelevant T cells we focus on cytotoxic effector T cells that are in direct contact with their target cells and are clonally expanded as detected by

Abstracts immunohistochemistry and CDR3-spectratyping. Such cells were isolated by laser-microdissection from frozen biopsy specimens of polymyositis and inclusion body myositis lesions. We identified matching T cell receptor (TCR) α- and β-pairs from several patients by a recently developed multiplex PCR protocol that allows concomitant amplification of both chains even from single T cells. From one patient we isolated 64 T cells which express an expanded Vβ1 chain. In 23 of these cells we identified matching α-chains, 20 of which were identical. The dominance of this clone might indicate an antigen driven clonal expansion and points to its pathogenic relevance. We reconstructed this and several other paired TCR αand β-chains and expressed them functionally in T-hybridoma cells. Presently we are using these “revived” autoaggressive T cells to investigate their target antigens in vitro by cDNA expression and combinatorial synthetic peptide libraries. This protocol should be applicable not only to autoimmune diseases like multiple sclerosis, but also tumors and transplants. OS3B-05

Usefulness of MRI orientated muscle biopsy (MOMB) for diagnoses of inflammatory myopathies S. Matsubaraa, K. Kondoa, K. Sugayaa, K. Miyamotoa, A. Yagishitab, T. Mizutanic and K. Hiramatsud Department of Neurology, bDepartment of Neuroradiology, cDepartment of Neuropathology, Tokyo Metropolitan Neurological Hospital, dDepartment of Rheumatology, Tokyo Metropolitan Fuchu Hospital, Fuchu, Tokyo, Japan

a

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Background: To establish definite diagnoses of inflammatory myopathies including polymyositis (PM), dermatomyositis (DM) and inclusion body myositis (IBM), according to a recent criteria of the diagnoses, it is important to obtain muscle biopsy samples with inflammation. However, inflammatory change is often absent in the tissue obtained by conventional method of biopsy. Objective: To examine usefulness of magnetic resonance imaging (MRI) of skeletal muscles for determination of muscle biopsy sites to make diagnoses of inflammatory myopathies. Materials and methods: Method of MRI orientated muscle biopsy (MOMB) was established. Among 32 cases, excluding those already on steroids, which fulfilled criteria of Bohan and Peter for definite, probable or possible PM or DM and subjected to muscle MRI, 25 showed positive MRI change enabling us to apply MOMB, while 7 were negative on MRI. Muscle biopsy was performed in all these cases. For comparison, biopsies from 48 probable or definite cases of inflammatory myopathies, in which biopsy was performed at the belly of muscles before 2001. Histological change in the muscle biopsy was graded semi-quantitatively. Results: By applying MOMB, number of samples with inflammatory change has increased significantly. By conventional biopsy 75% of specimens showed inflammation, while all samples taken by MOMB did so. On the other hand, in cases without significant change on MRI, chance to observe inflammation from MRI negative case was 29%. Conclusion: MOMB significantly improved the diagnostic value of muscle biopsy for inflammatory myopathies. On the other hand, cases without significant change on muscle MRI emerged as a new issue.

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Abstracts

Oral Session 4A: Immunological studies of MS (1)

Abstracts OS4A-02

Protein separation in cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. Application of SELDI-tof technique to the study of MS I. Bosca1, B. Casanova1, F. Coret2, A. Pascual1, S. Jourdain3, E. Piñero4, V. Esteve4, B. Celda4 Hospital Universitari La Fe, València, Spain; 2Hospital Clínica Universitari de València, Spain; 3Ciphergen Biosystems Inc., France; 4Depto. Química Física, Universitat de Valencia, Spain

1

Objective: To study by means of a novel technique (SELDI-tof — Surface Enhanced Laser Desorption/Ionization Time to Flight Mass Spectroscopy), the CSF proteins distribution and composition in a selected group of MS patients. Method and patients: 30 CSF specimens from a group of healthy controls (HC) (5 samples) and MS patients (25 samples), classified as: Clinically Isolated Syndrome (7 samples), RRMS (7 samples), SPMS (5 samples) and PPMS (6 samples), were studied by means of the SELDI-tof. This technique allows the separation and identification by size and charge of a set of proteins in complex mixtures, as CSF. Results: The intensities of 13 potential differential mass-spectroscopy peaks associated to specific proteins or protein fragments are different between MS patients and healthy controls. The most significant differences were found between CIS and HC and PPMS and HC with 16 and 13 potential bio-markers, respectively. Despite of the reduced number of samples and therefore the limited statistical significance, it seems that two specific resonances assigned at 51,208 Da (p = 0.042) and 167,335 Da (p = 0.067) proteins could be considered as promising potential classifiers among the different MS clinical forms. Conclusion: The preliminary results obtained in this initial study seem to point out that SELDI-tof could be a potential useful technique to be applied in MS diagnosis by an adequate identification of specific proteins directly in CSF as discriminate bio-markers. More extensive studies using SELDI-tof are needed to verify the results obtained so far. OS4A-03

Bacterial peptidoglycan as a cofactor in multiple sclerosis inflammatory activity J. D. Lamana,d, J. S. Voermana,d, L. Vissera,d, L. A. Bovena,d, M. Van Meursa, d , M. J. Meliefa,d, J. J. Bajramovicc,d, B. A. 't Hartc,d, S. Amorc,d, U. Zähringere and R. Q. Hintzenb,d

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lomyelitis models (marmoset and rhesus monkey), the burden of PG in the inflamed brain correlated with severity of pathology and APC subtype in the brain. Leukocytes from monkeys and humans responded to PG challenge by production of IL-12p40. Third, encephalitis could be precipitated in mice by adding PG as the sole microbial compound to incomplete Freund adjuvant plus encephalitogenic peptide. This collective immunopathological and functional evidence from rodent and non-human primate models, as well as from human MS, supports the potential role of PG as a cofactor in MS and warrants further elucidation of receptors and pathways involved. OS4A-04

Identification of novel biomarkers for multiple sclerosis based on the humoral immune response V. Somers, C. Govarts, K. Somers and P. Stinissen Hasselt University, Biomedical Research Institute, and Transnationale Universiteit Limburg, School of Life Sciences, Diepenbeek, Belgium Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system in which both autoreactive T and B cells play a role. To fully explore the complex information present within the autoantibody repertoire of MS patients, we have applied a powerful procedure, called serological antigen selection (SAS), to identify antigens recognized by the humoral immune response in MS. The SAS procedure involves the display of a cDNA expression library from MS brain plaques on filamentous phage and subsequent selection on immunoglobulin G (IgG) present in serum or cerebrospinal fluid (CSF). To enrich for selection of cDNA products binding to IgGs specifically associated with MS, alternative selection strategies on MS patient material and normal samples were performed. Using SAS on pooled CSF (n = 10) from relapsing–remitting (RR) MS patients, we identified a panel of 8 different antigens, which after detailed serological analysis with 161 MS CSF and control CSF, showed exclusive or preferential reactivity to MS patient CSF. Affinity selections on a second pool of CSF from 10 other RR-MS patients resulted in the identification of 3 additional candidate antigens. In addition, selections on two pools of sera from 10 RR-MS patients each resulted in the identification of 20 cDNA clones. These clones are currently being further evaluated with regard to sequence identity and MS-specificity. In conclusion, our findings demonstrate that SAS is useful to identify novel candidate antigens in MS that can be used as diagnostic markers, and can be used to study the humoral immune response in MS. OS4A-05

a

Department of Immunology, Erasmus MC, Rotterdam, The Netherlands; b Department of Neurology, Erasmus MC, Rotterdam, The Netherlands; c Department of Immunobiology, BPRC, Rijswijk, The Netherlands; dMS Centre ErasMS, Rotterdam, The Netherlands; eResearch Center Borstel, Borstel, Germany

IL-15 provided by myeloid cells and astrocytes in the human CNS contributes to the persistence of activated myelin specific CD8 T cells: Implications for multiple sclerosis Nathalie Arbour1,2, Philippe Saikali1, and Jack Antel1

Peptidoglycan (PG) is a major bacterial cell wall compound with strong proinflammatory capacity. Membrane Toll-like receptor 2 is thought to be a signaling receptor for intact PG, while three types of innate receptors recognize small PG motifs intracellularly, namely Nod1/ CARD4, Nod2/CARD15 and NALP3/cryopyrin. Dysregulated function of these molecules is involved in several types of inflammatory disorders, including Crohn's disease and Blau syndrome. We previously proposed that PG is a factor codetermining inflammatory activity in multiple sclerosis (MS), and present three lines of evidence in support. First, in postmortem brain of MS patients, antigen presenting cells (APC) contain PG, presumably resulting from infiltrating cells which acquired PG fragments at the mucosa, and these APC are immunocompetent as evidenced by expression of costimulatory molecules and cytokines. Second, in two non-human primate experimental autoimmune encepha-

1

Neuroimmunology Unit, Montreal Neurological Institute, 3801 University Street, Room 111, Montreal, QC, Canada H3A 2B4; 2Research Center — CHUM, University Of Montreal, Pavilion JA DeSeve, 2099 Alexandre DeSeve, Montreal, QC, Canada, H2L 4M1 CD8 T cells are prominent infiltrating cells in the central nervous system (CNS) during multiple sclerosis (MS). Highly activated memory CD8 T cells have been shown to clonally expand and persist in the CNS of MS patients for up to 5 years. Interleukin-15 (IL-15), a pro-inflammatory cytokine produced by macrophages and dendritic cells, is pivotal in the generation and maintenance of memory CD8 T cells. Higher amounts of IL-15 have been detected in MS patients' blood and cerebrospinal fluid. IL-15 bound to IL15Ralpha on its own cell of origin, activates IL-15Rbeta/gamma bearing CD8

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Abstracts

T cells. We sought to determine whether a local source of IL-15 can play a role in the acquisition of the memory phenotype by myelin specific CD8 T cells, and the persistence of such cells in the CNS of MS patients. Human monocytes/macrophages and astrocytes could provide the local source of IL15/IL-15Ralpha since we observed that these cells express surface bound IL15 up-regulated in response to pro-inflammatory cytokines. Myelin specific

CD8 T cell lines cultured in the presence of IL-15 acquired a memory phenotype (CD45RO, CCR7-, CD62L-) that was not observed in the presence of IL-2. IL-15-cultured CD8 T cell lines contained high amounts of cytolytic enzymes, similar to activated NK cells. These results underline a novel potential role for IL-15 in the survival of memory myelin specific CD8 T cells in the CNS during inflammatory diseases such as MS.

Abstracts

Oral Session 4B: Immunological studies of MS (2)

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Abstracts

OS4B-01

OS4B-03

CD11c on NK cells mirrors the disease activity of multiple sclerosis

Percentage of CD4+CD25+ cells in cerebrospinal fluid: A reliable immune parameter for disease activity in relapsing– remitting multiple sclerosis

Toshimasa Aranami, Sachiko Miyake, Takashi Yamamura National Center of Neurology and Psychiatry, Kodaira, Japan Multiple sclerosis (MS) is an autoimmune disease, showing a great degree of variance in disease activity. We have recently demonstrated that NK cells biased for producing IL-5 (NK2 bias) are associated with remission state of MS. Here we report that upregulation of CD11c on NK cells would characterize a subgroup of MS in remission whose NK cells are lacking NK2 bias. When we compared this group (CD11chigh) with the other group of patients (CD11clow) concerning NK cell expression of IL-5 and GATA-3, we found NK2 bias to be a selective character of CD11clow patients. In contrast, CD11chigh group showed an obvious trend for a larger proportion of HLADR+ cells within NK cells. Since NK cell stimulatory cytokine such as IL-15 was found to upregulate CD11c expression on NK cells in vitro, we postulate that inflammatory signals may play a role in inducing CD11chigh NK cell phenotype. Although there was no clinical difference between CD11chigh and CD11clow at blood sampling, CD11chigh MS developed a clinical relapse significantly earlier than CD11clow during 120 day follow-up. These data point to an interpretation that CD11chigh patients are at a higher risk for developing relapse, which is probably due to the loss of NK2 phenotype. Thus, CD11c on NK cells mirrors the disease activity of MS and may be used as a disease biomarker. OS4B-02

Functional impairment of plasmacytoid dendritic cells in multiple sclerosis M. Stasioleka, A. Bayasb, N. Krusec, A. Wieczarkowieczc, K.V. Toykab, R. Goldb,c, and K. Selmaja Department of Neurology, Medical University of Lodz, Poland; bDepartment of Neurology, University of Wuerzburg, Germany; cInstitute for MS-Research, University of Goettingen, Germany

a

Background: Plasmacytoid DCs (pDCs) represent a subpopulation of DC which exerts divergent functions in innate and adoptive immune response. It is thought that different DC-subtypes may be critically involved in the pathogenesis of multiple sclerosis (MS). Materials and methods: In our study we assessed by flow cytometry the phenotype of peripheral blood DCs from 35 clinically stable, untreated MS patients, 30 healthy controls and 9 patients with pneumonia used as nonspecific inflammatory conditions (NIC). For in vitro functional analyses DCs were isolated by magnetic sorting from leukapheresis material and cultured with trophic factors, unmethylated cytosine–phosphate–guanosine oligodeoxynucleotides (CpG-ODN) or were used as regulators in co-culture system. Results: Ex vivo expression of CD86 and 4-1BBL was significantly lower on pDCs from MS patients than from controls and patients with NIC (22% vs. 47% vs. 41% and 12% vs. 35% vs. 32% respectively). In MS, pDCs stimulated with IL-3 and CD40L showed impaired maturation as demonstrated by significantly lower or delayed up-regulation of CD86, 41BBL, CD40 and CD83. Stimulation of pDCs by CpG-ODN resulted in a significantly lower IFNa secretion in MS than in controls. In MS but not in controls, pDCs failed to up-regulate proliferative responses and IFNg secretion of autologous PBMC in a co-culture system. Moreover depletion of pDCs in MS patients, but not in controls had no effect on generation of CD4+Foxp3+ regulatory T cells. Conclusion: These findings suggest functional abnormalities of pDCs in MS patients, which might help to understand the mechanisms of immune dysregulation in this disease.

M. Matsuia, S. Arayaa, H.-Y. Wangb, K. Ozawab, E. Nishiguchib, M. Matsuia, M. Nakanishia, M. Kaitoa, Y. Gondoa and T. Saidab a

Kanazawa Medical University, Uchinada, Japan; Hospital, Kyoto, Japan

b

Utano National

In a search of reliable immunological parameters that can be used for assessment of disease activity, we examined paired blood and cerebrospinal fluid (CSF) samples obtained from 34 non-treated stable patients with relapsing–remitting multiple sclerosis (RRMS), who had been admitted for confirmation of diagnosis or to assess disease status. The samples were analyzed for humoral immunity as well as cellular immunity using flow cytometric examinations of CD4 and CD8 cells to determine their expression of surface antigens, including CD11a, CD25, CD26, CD29, CD45RA, CCR4, and CXCR3. Thereafter, the patients were followed for at least 1 year at the outpatient clinic, where they underwent neurological examinations as well as gadolinium-enhanced magnetic resonance imaging (MRI) scans, which were performed every 1 to 3 months until relapse became evident. Another group of 18 patients with noninflammatory neurological/psychiatric diseases served as controls. Of 27 RRMS patients who experienced a relapse within 1 year of the initial examinations, 7, 10, 6, and 4 showed relapse within 1, 3, 6, and 12 months, respectively. Those patients could be differentiated by the percentage of CD4+CD25+ cells in the CSF. Patients with a greater than 3.6% ratio of CD4+CD25+ cells in the CSF, which was 2 standard deviations above the mean of the controls, were more likely to relapse within 3 months. These findings suggest that enumeration of CD4+CD25+ cells in the CSF can serve as a reliable measure of disease status in RRMS. However, the immunological relevance (regulatory or inflammatory) of these cells requires additional investigation. 0S4B-04

Altered naïve CD4 T-cell homeostasis in relapsing–remitting multiple sclerosis due to peripheral mechanisms D. A. Duszczyszyna, J. Hackenbrocha, H. Masona, J. P. Antelb, Y. Lapierreb, D. G. Haegerta a

Department of Pathology, McGill University, Montreal, Canada; Department of Neurology, Montreal Neurological Hospital, Montreal, Canada b

Background: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), initiated by CD4 T cells. Our earlier work suggested that relapsing–remitting MS (RRMS) patients have significant alterations in naïve CD4 T cell homeostasis. Objectives: To investigate the involvement of peripheral mechanisms in homeostasis and to confirm our previous novel findings in RRMS. Methods: 19 untreated RRMS patients (mean age = 37.16) were recruited from the Montreal Neurological Hospital MS clinics. All patients had clinically definite MS based on McDonald criteria and were in clinical remission at the time blood was drawn. We recruited an independent set of 22 (mean age = 35) healthy controls, with no history of autoimmune disease. Lymphocytes were obtained using Ficoll-Paque™ and naïve CD4 T-cells were subsequently isolated by Macs® technology. Staining with PE-labeled anti-CD31 (indirect measure of homeostatic proliferation), PE-labeled antiCD127 and PE-labeled anti-Bcl-2 (measures of homeostatic survival) and Facs™ analysis were performed in order to ascertain the percent expression of CD31, CD127 and Bcl-2 respectively. Serum IL-7 levels (measure of homeostatic survival) were determined by ELISA. DNA was extracted from

Abstracts the remaining isolated cells and signal joint T cell receptor excision circles (sjTRECs — a surrogate marker of thymic production) were quantified using real-time PCR. Results: RRMS patients showed a significant decrease in sjTREC levels (p = 0.01), CD31 expression (p = 0.02) and Bcl-2 expression (p = 0.02). Conclusions: Our observations confirm that there are indeed significant naïve CD4 T cell homeostatic abnormalities in RRMS and suggest that nonthymic peripheral mechanisms are involved. 0S4B-05

Identification of a pathogenic antibody response to native myelin oligodendrocyte glycoprotein in multiple sclerosis Dun Zhoua, Rajneesh Srivastavaa, Stefan Nesslera, Verena Grummela, Norbert Sommerb, Wolfgang Brückc, Hans-Peter Hartunga, Christine Stadelmannc, Bernhard Hemmera a

Department of Neurology, Heinrich Heine-University, Moorenstr. 5, 40225 Duesseldorf, Germany; bDepartment of Neurology, Neuroimmunology Group, Philipps-University, Rudolf-Bultmann Stz. 8, 35033 Marburg, Germany; cInstitute of Neuropathology, Georg-August-University, 37099 Göttingen, Germany

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Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Although the cause of MS is still uncertain, many findings point towards an ongoing autoimmune response to myelin antigens. Myelin oligodendrocyte glycoprotein (MOG) is one of the potential diseasecausing self antigens. We determined the serum antibody responses to native full-length MOG protein expressed on a human astroglioma cell line by a cell based assay. Antibodies, mostly IgG, were detected in serum of MS patients that specifically bound the extracelluar domain of MOG. These antibodies were directed against a conformational epitope that elicits a pathogenic antibody response in EAE. IgG but not IgM antibody titers to native MOG were significantly higher in the MS patient group compared to controls. One third of the MS patients, in particular primary progressive patients, had elevated anti-MOG antibody titers. These autoantibodies induced death of MOG-expressing target cells. Sera from MS patients with anti-MOG antibodies stained white matter myelin in rat brain and enhanced demyelination and axonal damage when transferred to EAE animals. In summary, we identified the first pathogenic antibody that may contribute to the inflammatory CNS damage in a subgroup of MS patients.

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Abstracts

Oral Session 5A: EAE regulation

Abstracts

105

0S5A-01

0S5A-03

Unexpected regulatory roles of TLR4 and TLR9 in an animal model for multiple sclerosis

Over-expression of CD24 in the central nervous system leads to enhanced disease progression in experimental autoimmune encephalomyelitis

Monica Marta1, Åsa Andersson1, Magnus Isaksson2 and Anna Lobell1–3

Jin-Qing Liu, Pramod S. Joshi, Joe Carl, Xue-Feng Bai 1

Neuroimmunology Unit, Department of Clinical Neuroscience, Karolinska Institute, Center for Molecular Medicine, Stockholm, Sweden; 2Center for Autoimmunity and Inflammation, Department of Medical Sciences, Uppsala University, Uppsala, Sweden

Department of Pathology, The Ohio State University Medical Center, Columbus, OH 43210, USA

Innate immune mechanisms essential for priming encephalitogenic T cells in autoimmune neuroinflammation are poorly understood. To investigate the role of Toll-like receptors (TLR) and MyD88, the common TLR adaptor molecule, in an animal model of human multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) was induced by immunization with myelin oligodendrocyte glycoprotein (MOG) in complete Freund's adjuvant (CFA) in C57BL/6, TLR- or MyD88-deficient mice. MyD88−/− mice were EAE resistant. However, we expected TLR4−/− to have attenuated disease due to failure to signal the presence of TLR4binding pathogen associated molecular pattern (PAMPs) in mycobacteria, and similarly for TLR9−/− mice due to failure to signal the presence of CpG DNA in mycobacteria. Instead, disease was paradoxically exacerbated in TLR4−/− and TLR9−/− mice. We also expected inhibited disease in TLR6−/− mice due to failure to signal the presence of mycobacterial lipopeptides, but loss of this TLR did not affect the disease severity. MyD88−/− mice failed to express IL-23 and IL-6, and serum and T cell IL-17 were absent. Then, we investigated how TLR signals can be regulatory. mDC IL-23 expression was higher and T cell and serum IL-17 levels were several-fold higher in TLR4−/ − than in WT mice. In TLR9−/− mice serum IL-17 levels were several-fold higher than in WT mice. Our data suggest that MyD88 mediates EAE via induction of mDC IL-23 and IL-6 that drives encephalitogenic TIL-17 cell activation. Data indicates a regulatory role for TLR4 and TLR9 in priming encephalitogenic TIL-17 cells.

Experimental autoimmune encephalomyelitis (EAE) is an experimental model of human disease multiple sclerosis (MS). Our previous studies have shown that CD24, which was originally described as a co-stimulatory molecule, is required for the pathogenesis of EAE. The development of EAE requires CD24 expression on both T cells and local cells of the CNS. In order to understand the role of CD24 expressed on local CNS cells during EAE development, we generated CD24 transgenic mice in which CD24 was expressed in the CNS under the control of the GFAP promotor (AstroCD24TG mice). In AstroCD24TG mice, CD24 was abundantly expressed in astrocytes. Upon immunization, AstroCD24TG mice developed a more progressive disease compared with their non-transgenic littermates (NTG), which was reflected by persistent EAE signs and large areas of demyelination and axonal damage in the spinal cords at later stages of EAE in AstroCD24TG mice. The enhanced EAE progression was associated with higher expression of cytokine genes including IFNγ, IL-17, TNFα and IL-10 but not IL-4 in the spinal cords of AstroCD24TG mice than that of NTG mice. Moreover, expression of a demyelination associated marker P8 was also increased in the spinal cords of AstroCD24TG mice compared with NTG mice. Finally, adoptively transferred 2D2 T cells, which are specific to MOG p35–55, expressed a more mature effector phenotype in the CNS of AstroCD24TG mice than in NTG mice. Thus, over-expression of CD24 in the CNS enhances EAE development via enhanced co-stimulation of encephalitogenic T cells. Since CD24 is drastically increased both on local cells and inflammatory cells in the CNS during EAE, our data have important implications for CD24 targeted therapy of MS.

0S5A-02 0S5A-04

Redefining encephalitogenicity: Identification of a novel pathogenic cytokine induced by IL-23 K. Kreymborga, R. Etzenspergerb, S. Haaka and B. Bechera

Regulatory function of CD4+ and CD8+LAP+ T cells in Experimental Autoimmune Encephalomyelitis (EAE) M.L. Chen, H. Ishikawa, X. Zhang, and H.L. Weiner

a

Department of Neurology, Universitatsspital/University of Zurich, Frauenklinikstrasse 10, CH-8091 Zurich, Switzerland; bWeatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK

Tissue-directed autoimmune diseases were widely held to be mediated by the conduct of self-reactive Th1 cells. However, we have previously shown that neither Th1 nor Th2 gene-expression serves as a reliable marker for pathogenicity. Furthermore, the master-cytokine Interleukin (IL)-23 was shown to be absolutely critical for the induction of autoimmune disease as demonstrated in collagen-induced arthritis (CIA) as well as experimental autoimmune encephalomyelitis (EAE). It becomes increasingly evident that IL-23 supports the expansion of a distinct lineage of CD4+ cells that produce IL-17 (ThIL-17 cells) and play a major role in autoimmune inflammation. Using high density transcriptomics, we could identify an additional gene induced by IL-23. We discovered an IL10 related cytokine whose expression profile is identical with that of the disease associated IL-17 but has – in contrast to IL-17 – so far never been described in the context of EAE. Its expression correlates exquisitely with that of IL17. Our findings point towards a major role of this illdescribed cytokine. Taken together, this molecule will help to define the encephalopathogenic phenotype of autoaggressive effector cells and permits the precise characterization of this novel population of ThIL-17 cells.

Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA EAE is an animal model of multiple sclerosis and regulatory T cells play an important role in modulating EAE and in recovery. We have been studying TGF-β dependent regulatory T cells that are associated with recovery from EAE and that can be induced following oral administration of antigens and more recently oral anti-CD3 monoclonal antibody (Nature Medicine, in press). We have found that the surface expression of latency associated peptide of TGF-β (LAP) marks a T cell with regulatory properties and that CD4+LAP+ T cells increase in the spleen and peripheral blood of animals during the EAE recovery phase. We have now initiated studies of CD8+ LAP+ T cells. CD8+ CD25− LAP+ and CD8+CD25+LAP+ cells from spleens and lymph nodes of SJL mice isolated by high speed cell sorting were anergic when they were stimulated with anti-CD3 in the presence of APC in vitro. Furthermore, these two populations suppress the proliferation of responder T (CD4+CD25−LAP− or CD8+CD25−LAP−) cells in vitro. We also found that CD8+LAP+ T cells produce more TGF-β and significant higher amount of IFNγ compared with CD8+LAP− T cells. Thus, in addition to CD4+LAP+ T cells, CD8+LAP+ T cells may play an important role in the regulation of EAE. They secrete increased amount of IFNγ, which may play a role in EAE as it has been shown than IFNγ deficient animals have

106

Abstracts

a worse form of EAE. The secretion of IFNγ may also suggest a role for CD8+LAP+ T cells as a regulatory T cell in allergy. 0S5A-05

PSGL-1 and Fucosyltransferase-VII are involved in the suppressor activity mediated by CD4+CD25+ regulatory T cells B. Rossia, S. Bacha, M. Martinelloa, L. Ottobonia, S. Angiaria, R. McEverb, J.B. Lowec and G. Constantina a

Department of Pathology, University of Verona, Verona, Italy; Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma USA; cDepartment of Pathology, Case Western Reserve University, Cleveland Ohio, USA b

The mechanisms involved in the regulation of experimental autoimmune encephalomyelitis (EAE) by CD4+CD25+ regulatory T cells (Tregs) are not well understood. The goal of this study was to determine the role of Alpha (1,3) fucosyltransferases (FucT) and P-selectin glycoprotein ligand-1 (PSGL-1) in the suppression of EAE by Tregs. Methods: Active and

transfer EAE models using MOG35–55 peptide were performed in WT/ C57Bl/6, FucT-VII−/−, FucT-IV−/− and PSGL-1−/− mice. Isolation of CD4+CD25+ Tregs, flow cytometry, adhesion and proliferation assays were also performed. Results: FucT-VII−/− and PSGL-1−/− mice developed a more severe active EAE when compared to wt animals. Moreover, encephalitogenic T cells produced from FucT-VII−/− and PSGL-1−/− mice transferred a significantly more severe disease that wt T cells. We observed no significant differences between the expression of adhesion molecules and IL-4 and IFNgamma production of autoreactive T cells from PSGL-1 and FucT-VII deficient mice. However, co-cultures with CD4+CD25+ Tregs and effector cells showed that deficiency of PSGL-1 and FucT-VII leads to a marked decrease of suppression capacity of Tregs. Moreover stimulation of Tregs with anti-CD3 and IL-2 induces an increase of adhesion capacity with an increase expression of PSGL1 and an increase binding of P- and E-selectin chimeras. Importantly, stimulated CD4+CD25+ Tregs display increased expression of functional PSGL-1 and a significantly higher suppressor activity. Conclusion: Our data show that PSGL-1 and FucT-VII are involved in the immune suppression mediated by CD4+CD25+ Tregs in MOG-induced EAE, and suggest that in vitro manipulation of Tregs may lead to increased suppressor activity in inflammatory diseases.

Abstracts

107

Oral Session 5B: Mechanisms of neuroimmune crosstalk

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Abstracts

0S5B-01

Is the immunological synapse glutamatergic? P. Affaticatia, O. Mignena, F. Jamboua, M-C. Potierb, I. Klingel-Schmitta, J. Degrouardc, R. Liblaud, T. Capiode and S. Cohen-Kaminskya a

CNRS UMR8162, Université Paris-Sud, IPSC/IFR13, Hôpital Marie Lannelongue, Le Plessis Robinson, France; bCNRS UMR7637, ESPCI, 75005 Paris, France; cService Commun de Microscopie Electronique, CNRS UMR8080, IFR46, Université Paris-Sud, Orsay, France; dINSERM U563, Hôpital Universitaire Purpan, Toulouse, France; eINSERM U800, IFR118, Université des Sciences et Technologies Lille 1, Villeneuve d'Ascq, France

The immunological synapse forms in the contact zone between two immune system cells and may optimise activation by congregating signalling molecules in specialised regions of the plasma membranes; however, its precise functions remain a matter of debate. It has become clear that L-glutamate, the major excitatory neurotransmitter plays a role in peripheral tissues, including the immune system. Here we show that Lglutamate, a major excitatory amino acid in synaptic transmission in the nervous system, acts as an immunotransmitter at the immunological synapse. Dendritic cells (DCs) – the most potent antigen-presenting cells – contain a vesicular compartment competent for regulated glutamate exocytosis. They also express glutamate-specific vesicular VGLUT transporters conferring a glutamatergic phenotype on neurons and release glutamate through Ca2+-dependent exocytosis. Remarkably, thymocytes express a large repertoire of glutamate receptors (GluRs), mirroring that of neural cells. In particular, they express functional NMDA receptors (NMDARs), which contribute to the Ca2+ signal leading to antigendependent apoptosis in the context of T-DC synapses, mimicking thymic negative selection. We have identified the prototypical post-synaptic PDZ domain protein PSD-95 as a new marker of the immunological synapse. PSD-95 is recruited in the T-DC contact zones where it colocalizes with NMDARs. T cell stimulation, even in the absence of glutamate, also results in the co-localisation of PSD-95 with the NMDAR in the contact zone, suggesting that T-cell receptor triggering is involved in the recruitment of functional NMDARs at the synapse. This work reveals a new aspect of immunological synapse functioning, in which, similar to central nervous synapses, DC-released glutamate elicits focal responses in T cells bearing GluRs. Glutamate should therefore be considered a synaptic transmitter, acting through fine-tuning of Ca2+ mobilization, in both the nervous and immune systems. 0S5B-02

Adrenomedullin (AM) receptor proteins RAMP2 and 3 are expressed on T-cells in addition to the blood–brain barrier (BBB) Liverani, E.1; Mcleod, J.1; Morse, R.1; Bolton, C.2 and Paul, C.1 University of the West of England, Bristol, UK; 2William Harvey Research Institute, London, UK

1

AM is a novel peptide highly expressed by cerebral endothelial cells. In vitro studies show the peptide to regulate key BBB features including transendothelial resistance and permeability. Furthermore, we have shown that AM is elevated during EAE when the BBB is dysfunctional and inflammatory lesions apparent. AM acts primarily through the calcitonin receptor-like receptor (CRLR) in combination with either receptor-activity-modifying-protein (RAMP) 2 or 3. The main cellular target for AM during neuroinflammation is uncertain. We have therefore assessed receptor expression on T-cells plus component cells of the BBB. RAMP2 and 3 and CRLR expression was examined using PCR and FACS techniques. We found activated T-cells predominantly expressed

RAMP3, although some RAMP2 was also present, with a significant proportion sited on the membrane. Conversely, for both ECV304 human endothelial and C6 rat astroglioma cells, the majority of the RAMP2 and 3 was located in the cytoplasm, with predominantly RAMP2 expressed under physiological conditions. CRLR was present in all cell types examined. Interestingly, the response of RAMPs to glucocorticoid exposure (10− 6– 10− 8 M) differed dependent on cell type, indicating that AM could have an important role in management of neuroinflammation. The peptide could therefore be involved in the neuroinflammatory processes evident in human conditions such as multiple sclerosis. 0S5B-03

Role of capsaicin-sensitive sensory nerves and the TRPV1 capsaicin receptor in endotoxin-induced airway inflammation and hyperreactivity in mice Zsuzsanna Helyes1, Krisztián Elekes1, József Németh1, Katalin Sándor1, László Kereskai2, Erika Pintér1, Gábor Pozsgai1, Árpád Szabó1, János Szolcsányi1 Department of Pharmacology and 2Department of Pathology, Faculty of Medicine, University of Pécs, Hungary

1

Capsaicin-sensitive, transient receptor potential vanilloid 1 (TRPV1) receptor-expressing, primary sensory nerves play important regulatory role in the pathomechanism of several inflammatory processes. The aim of the present study was to examine the role of these afferents and TRPV1 receptors in endotoxin-induced airway inflammation and hyperreactivity in mice. Pneumonitis was evoked by intranasal E. coli lipopolysaccharide (LPS) and enhanced pause (Penh), a calculated parameter correlating to airway resistance, was measured by whole body plethysmography. Bronchoconstriction was induced by inhalation of the muscarinic receptor agonist carbachol 24 h after LPS. Histological scoring, measurement of myeloperoxidase activity referring to granulocyte accumulation and determination of the interleukin-1β were performed from the lung. For the inactivation of capsaicin-sensitive afferents resiniferatoxin (RTX) pretreatment was performed, the function of the TRPV1 receptor was examined using gene-deleted (TRPV1−/−) mice. Destroying capsaicin-sensitive afferents by RTX-desensitization markedly enhanced all the inflammatory parameters, but inhibited hyperreactivity supporting the concept that tachykinins directly contribute to bronchoconstriction. Meanwhile, the lack of the TRPV1 receptor increased both inflammation and airway hyperresponsiveness. Specific radioimmunoassay was used to identify the inhibitory mediator released from sensory fibres via TRPV1 activation and it was proved to be somatostatin. LPS increased somatostatin in the lung and plasma in TRPV1+/+, but not in TRPV1−/− mice. The somatostatin receptor antagonist cyclo-somatostatin aggravared inflammatory parameters and hyperresponsiveness pointing out its functional significance. These results provide the first evidence for a novel inhibitory mechanism mediated by somatostatin derived from sensory afferents via TRPV1 receptor activation in the airways. Grants: OTKA F-046635; ETT03543/2003; RET-008/2005. 0S5B-04

Compartmentalized immune response in the barrel field of the mouse primary somatosensory cortex A. Chavarriaa,b, J. Perez-H.a,b, C. Ortiz-Martíneza,b, M. Cerbónb and G. Gutiérrez-Ospinac a

Facultad de Medicina, Universidad Nacional Autónoma de México, México City, México; bFacultad de Química, Universidad Nacional Autónoma de México, México City, México; cInstituto de Investigaciones Biomedicas, Universidad Nacional Autónoma de México, México City, México

Abstracts The brain is constituted by modular units that group nervous cells and connections that process a similar type of neural information. On the other hand, previous observations in patients with neurocysticercosis showed that the immune response differs among brain regions, thus suggesting the existence of immunological compartments in the nervous system. Whether immune compartments and neural modules share their location and function has not been addressed. To explore this possibility we studied the immunological response to lesions carried out within and outside neural modules located in the primary somatosensory cortex of adult mice. These modules are called barrels and they represent collections of mechanosensory receptors. We found that the neural tissue adjacent to lesions placed outside the barrel field showed an intense astrocytic response and MHCII, INFγ, TNFα immunoreactivity in macrophages and activated microglia. These cells also expressed IL4 and IL10. In contrast, lesions placed inside the barrel field displayed MHCII, IFNγ and TNFα immunoreactive microglia and macrophages predominantly in the border of barrels right next to the lesion. There was an extremely reduced response in the barrel center, as it was in barrels close and far from the lesion site. These data support the idea that neural modules may also function as immunological compartments. Our observations open a new perspective for the study and understanding of the physiology and the pathology of the CNS. 0S5B-05

Biological mechanisms underlying the impact of systemic infection on affective behaviour J. Teeling1, L. Felton1, C. Cunningham1, R. Deacon2, J.N.P. Rawlins2 and V.H. Perry1

1

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CNS Inflammation Group, SOBS, University of Southampton, UK; Experimental Psychology, University of Oxford, UK

2

Systemic inflammation, impacts on the brain and gives rise to behavioural changes, often referred to as ‘sickness behaviour’. These symptoms are thought to be mainly mediated by pro-inflammatory cytokines. Here we study communication pathways between the immune system and higher brain function following sub-pyrogenic inflammation. Systemic inflammation was induced in mice using models of bacterial (Lipopolysaccharide (LPS); 1–100 μg/kg) or viral infection (poly I:C; 12 mg/kg), and changes in open-field activity, burrowing behaviour and consumption of glucose solution were assessed. Immune activation was studied in the periphery and brain by measuring cytokine production, and immunohistochemistry. Sub-pyrogenic inflammation resulted in changes in species-typical, untrained behaviours that depend on the integrity of the hippocampus. Increased expression of cytokines was observed in the periphery and selected regions of the brain which coincided with behaviour changes. However, after neutralization of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-a), no significant differences were found when compared to LPSplus-vehicle treated mice. In contrast, pre-treatment of mice with indomethacin completely prevented LPS induced behaviour changes, without affecting cytokine levels. Our work, studying the effect of low grade inflammation on affective behaviour in mice, suggests a key role for prostaglandins, rather than cytokines, in communicating changes in immune status to the brain. This study was sponsored by BBSRC and UCB Celltech.

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Oral Session 6A: Immunological regulation of CNS homeostasis

Abstracts 0S6A-01

Immune cells contribute to adult neurogenesis Y. Ziva, N. Rona, O. Butovskya, A. Finkelsteina, G. Landaa, H. Cohenb, E. Yolesc, J. Kipnisd and M. Schwartza a

Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel; bMinistry of Health Mental Health Center Anxiety and Stress Research Unit, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel; cProneuron Biotechnologies, Weizmann Science Park, 74101 Ness-Ziona, Israel; dPresent address: Departments of Pharmacology and Ophthalmology, Center for Neurovirology and Neurodegenerative Disorders, University of Nebraska Medical Center, Omaha NE 68198-5800, USA Neurogenesis is known to continuously take place in certain ‘neurogenic’ areas of the adult central nervous system (CNS) and can be induced in ‘non-neurogenic’ areas under traumatic or degenerative conditions. Here we introduce T cells and CNS-resident microglia as important players in the regulation of adult neurogenesis. Under normal conditions, immune deficient mice (SCID and nude) and transgenic mice that most of their T-cell pool is specific for an irrelevant antigen (ovalbumin) exhibited impaired hippocampal neurogenesis. In contrast, mice in which the majority of T cells specifically recognize the CNSabundant antigen myelin basic protein showed normal neurogenesis. CNSspecific T cells were also found to be important for spatial learning abilities and for brain-derived neurotrophic factor expression in the dentate gyrus. Environmental enrichment did not evoke enhanced neurogenesis in immune-deficient animals, whereas in wild-type animals it led to enhanced hippocampal neurogenesis coupled with recruitment of T cells and activation of microglia. The clinical implications of these findings were tested using a rat model of cerebral ischemic insult. We demonstrate that Tcell based immune activation following stroke induces a robust elevation of neurogenesis in the hippocampus as well as in the cerebral cortex. Our results suggest that T cells, acting via resident antigen presenting cells, are important regulators of adult neurogenesis under both physiological and pathological conditions. 0S6A-02

Immune cells contribution to hippocampal plasticity is age-dependent, and correlates with local modulation of gene expression Noga Rona , Dvir Netanelya , Jacob Bock Axelsena, Yael Shania , Hagit Cohenb, Eytan Domanya, Gideon Rechavic and Michal Schwartza

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formed neurons generated in the hippocampus was relatively small at infancy, increased at adulthood and decreased for older age. We also compared hippocampal gene expression in wild-type mice and TOVAtransgenic mice, using gene expression screening. We searched for hippocampal expressed genes, which were possibly controlled by immune cells. Although ‘TOVA-transgenic’ mice differ from their controls only in their peripheral T cell repertoire, significant differences in expression of several hippocampal genes were found. Among the differentially expressed genes we found some that were known to be involved in neurogenesis and axonal guidance. Further analysis of such genes will contribute to understanding how adult neurogenesis is controlled. 0S6A-03

Role of T cells in toluene-induced memory-related gene expressions in mouse hippocampus Tin-Tin-Win-Shwe, S. Ahmed, S. Yamamoto, M. Kakeyama, D. Nakajima, S. Goto, H. Fujimaki National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan Recently, it has been demonstrated that lack of mature T cells in mice manifested cognitive deficits and which could be restored by passive T cells transfer. Toluene mainly affects the central nervous system causing memory loss. Toluene may produce some of its effects by directly inhibiting Nmethyl-D-aspartate (NMDA) receptor function in a subunit-selective manner. To investigate the possible involvement of T cells in toluene-induced expression of memory-related gene in mouse hippocampus, we exposed BALB/c and nude mice to toluene at a low dose (9 ppm), and to air control (0 ppm toluene) as well in nose-only exposure chamber for 30 min in 3 consecutive days followed by once a week for 4 weeks. Four hours after the last challenge, we collected the hippocampus and examined the mRNA expression of NMDA receptor, protein kinase and transcriptional factors, which are potentially involved in memory functions, and then chemokines by a quantitative real-time PCR method. Our data clearly demonstrates that NMDA receptor NR2A, Ca2+/calmodulin-dependent kinase, CaMKIV, and transcriptional factor cAMP responsive element-binding protein, CREB-1, CCL2 and CCL3 mRNA expressions increased in the hippocampus of BALB/c mice exposed to 9 ppm toluene compared to that of air control. After immunization with ovalbumin, BALB/c mice showed no difference of NR2A and CaMKIV mRNA expression between air control and toluene (9 ppm). There were no alterations observed in nude mice with or without immunization. These findings indicate that T cells may play a role in memory-related gene expression induced by toluene exposure in BALB/c mice.

Weizmann Institute of Science, Rehovot, Israel; bFaculty of Health and Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel; cSheba Medical Center, Tel Hashomer and Sackler School of Medicine, Tel Aviv, Israel

0S6A-04

Hippocampal neurogenesis is known to take place throughout adult life and is considered necessary for certain types of cognitive abilities. We have lately demonstrated that peripheral immunity contributes to adult hippocampal neurogenesis and spatial learning/memory, and that this is mediated in part by T lymphocytes recognizing brain antigens such as myelin proteins. Here we investigated whether CNS specific T cells contribute to the maintenance of brain plasticity only at adulthood, or whether they are required at earlier stages as well. We examined hippocampal neurogenesis in wild-type mice and in transgenic mice that express a T-cell receptor that recognizes ovalbumin (‘TOVA-transgenic’ mice), and thus bear mainly TOVA cells and no autoimmune cells, at different ages. We found that impaired neurogenesis and learning abilities appeared already at 4 weeks of age. Interestingly, the difference between wild-type and TOVA-transgenic mice in terms of the number of newly

O. Lidmana, M. Abramsb, A. Josephsonb, M. Dieza, L. Olsonb, T. Olssona, F. Piehla

a

Non-MHC genes regulate locomotor behavioral outcome following rat spinal cord contusion injury

a

Department of Clinical Neuroscience, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden; bDepartment of Neuroscience, Karolinska Institutet, Stockholm, Sweden. Secondary degeneration resulting from spinal cord injury is characterized by inflammation and progressive loss of nervous tissue. Recently it has been suggested that injury-induced inflammation contain important elements of autoimmunity, which may be protective or detrimental. In order to dissect this process genetically and to examine potential influence by the MHC locus, known to be of importance for regulation of autoimmune processes, a well-characterized weight-drop contusion model

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was performed in a panel of inbred strains with different susceptibility to neuroinflammation. Included strains were: ACI, DA, LEW and PVG, as well as the MHC congenic strains LEW.1AV1, LEW.1N, LEW.1W and PVG.1AV1. Striking differences in motor behavioral outcome across the strains were observed. Interestingly, the best outcome was demonstrated for the inflammation prone DA rat, whereas the PVG strain, resistant in most inflammatory models, displayed an intermediate response. The different LEW strains, the wild type and three strains congenic for different MHC haplotypes, displayed the same worst outcome. No MHC haplotypedependent effects were observed in a F2 intercross experiment (PVG × PVG.1AV1), providing additional evidence for a dominant influence of non-MHC genes. We conclude that outcome following spinal cord injury is subject to considerable genetic heterogeneity. However, this genetic regulation does not seem to depend on the degree of susceptibility to autoimmune neuroinflammation or, in particular, the MHC complex. These results provide a base for future studies to position genetic variants regulating outcome following spinal cord injury. 0S6A-05

The protective role of autoimmune reaction in the neurodegenerative process caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridine (MPTP) in mice Iwona Kurkowska-Jastrzębskaa, Ilona Joniecb, Agnieszka Ciesielskaa, Ewa Bałkowiec-Iskrab, Andrzej Członkowskib, Anna Członkowska,b

a

Institute of Psychiatry and Neurology, Warsaw, Poland; University of Warsaw, Poland

b

Medical

The response of the immune system during injury of the central nervous system has possible protective role. Here we present the influence of the autoimmune reaction on dopaminergic neurons damage induced by MPTP. C57Bl male mice, 2 (young) and 10 (aged) months old were injected with MPTP (40 mg/kg). One group of each age, 6 days before the injection, had induced autoimmune encephalomyelitis (EAE) by myelin oligodendrocyte glycoprotein (MOG) 35–55 in CFA administration. Mice receiving MOG revealed typical EAE with clinical signs of tail and hind limb paresis. In these mice, MPTP caused less dopaminergic system damage than in MPTP only injected mice. The dopamine level was higher by 10% and 30% and the number of preserved neurons by 16% and 21% in young and aged animals respectively, on the 7th day after MPTP administration. The microglial reaction was diminished and lymphocytic CD8+ infiltration significantly reduced. TGF and GDNF mRNA expressions were significantly higher in MOG receiving mice when compared to MPTP group. We observed also shift in T cells populations, from the preponderance of CD8+ in MPTP group to CD4+ in MOG group. We injected mice with CD4+ anti-MOG T cells a day after MPTP administration and we observed also less damage of the dopaminergic cells when compared to MPTP only injected mice. We showed that immune enhancement is protective in toxic damage of the central nervous system. CD4+ cells are possibly involved in the protective mechanism, but the induction of trophic factors by other factors is also the option.

Abstracts

Oral Session 6B: Viral infection and prion

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0S6B-01

Identification of host cell proteins in purified infectious humanherpesvirus 6A (HHV-6A) viral particles

epidermal nerve fibers also developed in SIV-infected macaques (p = 0.01) but were not strongly correlated with changes in the CV. These findings suggest that primary injury to the neuronal cell body in the DRG mediated by activated macrophages alters functional properties of sensory nerves in HIV-infected individuals.

J. Ahlqvista, M. Hammarstedtb, S. Jacobsonc, H. Garoffb and A. FogdellHahna

0S6B-03

a

Karolinska Institutet, Huddinge, Sweden; bKarolinska Institutet, Novum, Sweden; cNINDS/NIH, Bethesda, USA

Efficacy of intracerebral vaccination against pseudorabies virus in mice

An association between the autoimmune disease multiple sclerosis and HHV-6A has been suggested. In vivo, HHV-6A has been detected in myelin producing cells, i.e. the main cells that are affected in MS. HHV-6A might incorporate host cell proteins into the viral particles during replication. When the virus is detected by the immune system, the incorporated proteins will be presented for the immune system. This could be one mechanism for induction of autoimmunity. Incorporation of host cell proteins have been shown for other viruses, for example the complement proteins CD55 and CD59 that are incorporated into HCMV, HTLV-1 and HIV-1. However, the purity of the virus preparations has been debated since cellular vesicles might contaminate the viral preparations during purification in sucrose gradients. In this study, we used iso-osmotic iodixanol gradients to purify T-cell cultured HHV-6A. In our gradient peak fraction we detect high levels of viral DNA by real-time PCR, expression of gp60/110 by Western blot and intact viral particles by electron microscopy. The purified virus particles were also able to re-infect T cells with good efficiency. We have low cellular contamination in the viral particle containing fractions as seen by analyzing viral and mock preparations by SDS-PAGE. Immunoblotting with anti-CD46 and other anti-host cell protein antibodies gave clear bands in the viral containing fractions compared to the mock containing fraction. In conclusion, this method may be used to study the protein content of viral particles and that may give important clues for how autoimmunity is induced.

Jae-Ho Shin1,2, Yoshihiro Sakoda3, Jae Hoon Kim1,4, Tomohisa Tanaka1, Hiroshi Kida3, Takashi Kimura1, Kenji Ochiai1, Takashi Umemura1 1

Laboratory of Comparative Pathology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan; 2National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul, Korea; 3Laboratory of Microbiology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan; 4Laboratory of Veterinary Pathology, Department of Veterinary Medicine, Cheju National University, Cheju, Korea To evaluate the efficacy of intracerebral (IC) vaccination, mice were immunized with formalin-inactivated pseudorabies virus (PRV) by either subcutaneous (SC) or IC injection, and then 106 plaque-forming units of PRV were introduced into the hindleg of the immunized or non-immunized mice by intramuscular injection. The antibody titer in serum was elevated and boosted by additional vaccination via both the SC and IC route, but was higher after IC vaccination. Intracerebrally immunized mice were completely protected from mortality and neurological signs, whereas all the non-vaccinated and 80% of the subcutaneously vaccinated mice died after developing neurological signs. IC vaccination is more effective at inducing a protective immune response against the transneural spread of virus than a SC vaccination. Keywords: Intracerebral Vaccination; Transneural Spread; Pseudorabies Mouse.

0S6B-02 0S6B-04

SIV-induced functional and pathological changes in the peripheral nervous system J. L. Mankowskia, V. A. Laasta, B. Shima, M. Ringkampa, C. A. Pardoa, P. E. Hauera, P. M. Tarwaterb J. C. McArthura, and M. C. Zinka Johns Hopkins University, Baltimore, USA; bUniversity of Texas, El Paso, USA

a

Peripheral neuropathy is the most common neurological complication associated with HIV-1 infection, affecting over one-third of individuals with AIDS. Studies also have found a high prevalence of HIV sensory neuropathy in HIV-infected patients despite administration of highly active antiretroviral therapy. To study HIV-induced PNS disease, we established an SIV/macaque model in which over 90% of animals developed PNS changes closely resembling those seen in HIV-infected individuals, including inflammation of the dorsal root ganglia with abundant replication of SIV in macrophages and DRG neuronal loss, sural nerve inflammation, and reduction in epidermal nerve fiber density in distal extremities. This constitutes the first primate model of HIVinduced peripheral neuropathy. In this SIV model we examined the relationship between dorsal root ganglia (DRG) changes and sensory nerve function by measuring C-fiber conduction velocities in the sural nerves and epidermal nerve fiber density in the skin of SIV-infected macaques and uninfected control macaques. SIV-infected macaques had significantly lower sensory C-fiber conduction velocity (CV) in sural nerves than uninfected animals (p = 0.01). The extent of conduction velocity decline correlated strongly with extent of macrophage infiltration in the DRG (p = 0.006). Significant declines in density of

Role of dendritic cells in the transmission and induction of neuroinvasion of scrapie following intravenous infection Claudine R Raymond, Moira E Bruce and Neil A Mabbott Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, UK, EH9 3JF. E-mail: claudine.raymond@bbsrc. ac.uk Following peripheral scrapie inoculation, the agent accumulates on follicular dendritic cells (FDCs) in lymphoid tissues before spreading to the brain. In the absence of FDCs disease susceptibility is reduced. How TSE agents reach lymphoid tissues and the nervous system following exposure is not known. Recent evidence suggests possible roles for dendritic cells (DCs) and macrophages, as disease-specific prion protein (PrPSc) has been found associated with each of these cell types following exposure to scrapie. The current study utilized two intravenous infection models of scrapie (ME7 and 139A agent strains) to determine if scrapie-infected mononuclear cells (MNCs), especially DCs and B cells, could deliver infection directly to the nervous system in the absence of FDCs and other lymphoid elements. Scrapie-infected MNCs, B cells and DCs efficiently transferred infection when transfused into wild-type recipients. Indeed, the disease incubation period following transfusion of scrapie-infected DCs was similar to that of the MNCs and B cells even though 10–20× fewer cells were used. However, in the absence of FDCs in TNF-receptor-deficient mice, the ability of the scrapie-infected cells to transmit infection was greatly reduced. These results demonstrate that for both the ME7 and 139A strains of scrapie, efficient neuroinvasion from the periphery requires prior agent replication/

Abstracts accumulation on FDCs. In the absence of this accumulation, DCs and other mononuclear cells capable of delivering the scrapie agent directly to the nervous system but via a substantially less efficient mechanism. Funded by EU grant no. QLRT-2001-01044. 0S6B-05

Regulation of the inflammatory response to Staphylococcus aureus-induced brain abscess by interleukin-10 Werner Stenzel1, Julia Dahm1, Monica Sanchez-Ruiz1, Hrvoje Miletic1, Olaf Utermöhlen2, Dirk Schlüter3, and Martina Deckert1 1

Abteilung für Neuropathologie, Universität zu Köln, Joseph-Stelzmann-Str. 9, D-50931 Köln, Germany; 2Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Universität zu Köln, Joseph-Stelzmann-Str. 9, D50931 Köln, Germany; 3Institut für Medizinische Mikrobiologie, Otto-vonGuericke Universität Magdeburg Leipziger Strasse 44, D-39120 Magdeburg, Germany

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A characteristic of brain abscess is a localized suppurative infection leading to substantial damage of the adjacent CNS tissue. The orchestrated interplay of pro- and anti-inflammatory cytokines released by leukocytes as well as resident cells of the central nervous system is crucial for both an effective host defense, and for limiting tissue damage in brain abscess. To study the regulatory role of interleukin (IL)-10 in brain abscess in vivo, IL-10 deficient (IL-100/0) mice were stereotaxically infected with Staphylococcus (S.) aureus-laden agarose beads. Increased numbers of intracerebral (i.c.) granulocytes, macrophages, CD4+ and CD8+ T cells and higher levels of TNF, IL-1β, and iNOS were observed in IL-100/0 mice than in wild type mice, while chemokines were induced earlier and more pronounced in WT mice. Together with prominent microvascular haemorrhage, necrotic vasculitis, severe brain edema, and markedly increased abscess size, these alterations led to an increased morbidity of IL-100/0 mice. Nevertheless, the hyperinflammatory response of IL-100/0 mice did not improve bacterial elimination. Collectively, these data outline the important role of IL-10 in vivo for the regulation of the i.c. host immune response in experimental S. aureus-induced brain abscess.

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Oral Session 7A: Immunotherapy (1)

Abstracts 0S7A-02

Glatiramer acetate mediated immune modulation is driven by antigen presenting cells and is not T cell antigen specific M.S. Webera, T. Prod´hommea, S. Youssefb, C. Rundlea, O. Stüvec, L. Steinmanb and S. S. Zamvila University of California, San Francisco; bStanford University, Stanford; University of Texas, Southwestern, USA

a

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times, an MRI marker of inflammation. In contrast, in monkeys treated with anti-IL-12p40 mAb changes of the total T2 lesion volume and T2 relaxation times were significantly suppressed. Moreover, the time interval to serious neurological deficit was delayed from 31 ± 10 days to 64 ± 20 days (odds ratio 0.312). These results in a disease model with high similarity to MS are important for ongoing and planned trials of therapies that target IL-12 and/or IL-23. 0S7A-04

c

Objective: To investigate the role of antigen presenting cells (APC) in immunomodulation by glatiramer acetate (GA). Background: GA exerts therapeutic benefit in multiple sclerosis presumably by induction of GA-specific Th2-cells. Recent reports revealed that GAtreatment alters APC function. We investigated (1) whether these APC effects mediate T-cell immunomodulation, (2) whether GA-treated monocytes can adoptively transfer protection from experimental autoimmune encephalomyelitis (EAE) and (3) whether T-cell cross-reactivity between GA and myelin antigen is required for its therapeutic effect. Methods: (PL/JxSJL/J)F1 mice were injected subcutaneously with 150 μg GA/d in PBS prior to or after EAE onset. T-cells were cultured with GA, intact MBP, MOGp35-55 or PLP139-151. Monocytes from GA-treated wild-type or RAG-1-deficient mice were co-cultured with naive T-cells from MBP Ac1-11 TCR transgenic (Tg), MOG35–55 TCR or OVA323–339 TCR Tg mice. GA-treated monocytes were injected into immunized C57BL/6 mice. Results: Daily GA administration without adjuvant prevented or reversed EAE. We did not observe T-cell cross-reactivity between GA and myelin antigens. GA treatment of wild-type and RAG-1-deficient mice, which lack mature T-cells, induced type-II APC, secreting less TNF-a, IL-12 and more IL-10. These APC directed Th2-differentiation of naive myelin- and OVAspecific T cells, induction of regulatory T cells (Treg) and protected recipient mice from EAE. Conclusions: Therapeutic benefit of GA in EAE does not require T-cell crossreactivity between GA and myelin antigen. GA-treatment exerts a direct effect on APC without the requirement of T-cells. GA-mediated APC immunomodulation drives Th2-polarization and Treg-induction independent of T-cell specificity and is sufficient to adoptively transfer EAE protection. 0S7A-03

Suppression of ongoing disease in a non-human primate model of multiple sclerosis by a human-anti-human IL12p40 monoclonal antibody Bert A. 't Harta,b,c, Herbert P.M. Broka, Jacqueline Bensond, George Treacyd, Sandra Amora,c, Rogier Q. Hintzenb,c, Jon D. Lamanb,c, Jan Bauere, Erwin L. A. Blezerf Biomedical Primate Research Centre, Rijswijk, The Netherlands; bErasmus Medical Center, Rotterdam, The Netherlands; cMS Center ErasMS, The Netherlands; dCentocor R&D, Inc., Malvern PA, USA; eCenter for Brain Research, Medical University Vienna, Austria; fUniversity Medical Center, Utrecht, The Netherlands

a

IL-12p40 is a shared subunit of two cytokines with overlapping activities in the induction of autoreactive Th1-cells and therefore a potential target of therapy in Th1-mediated diseases. We have examined whether ongoing disease in a non-human primate model of multiple sclerosis can be suppressed with a new human IgG1κ mAb against human IL-12p40. Lesions developing in the brain white matter were visualized and characterized with standard magnetic resonance imaging (MRI) techniques. To reflect the treatment of MS patients, treatment with the mAb was initiated after active brain white matter lesions were detected in T2-weighted images. In placebo-treated control monkeys we observed the expected progressive increase of the total T2 lesion volume and markedly increased T2 relaxation

TLR3 stimulation suppresses relapsing experimental autoimmune encephalomyelitis (EAE) by inducing endogenous interferon-β Tarik Touil1, Denise Fitzgerald1, Guang-Xian Xhang1, A.M. Rostami1, and Bruno Gran1,2 1

Department of Neurology, Thomas Jefferson University, Philadelphia, USA, and 2Division of Clinical Neurology, University of Nottingham, UK

Objective: We tested the hypothesis that polyinosinic-polycytidylic acid (poly I:C), a Toll-like receptor (TLR)3 agonist and potent type I interferon inducer, can modulate the clinical and pathological expression of EAE, an animal model of multiple sclerosis (MS). Background: Interferon beta (IFNβ) is the most commonly used immunomodulatory treatment for MS and is administered to patients as an exogenous recombinant protein. Microbial components can induce the production of endogenous IFNβ by antigen-presenting cells (APC) through stimulation of TLRs. Poly I:C is a mimic of double-strand RNA that stimulates APC through TLR3. Methods: Female SJL mice were immunized with proteolipid protein (PLP) peptide (PLP)139–151 and treated with poly I:C or PBS injected intraperitoneally during the induction or the relapsing phase of disease. Clinical, pathological, and immunological parameters were assessed. Results: Treatment with poly I:C significantly suppressed clinical and pathological signs of EAE in both treatment protocols. Poly IC induced significantly increased production of the CC-chemokine CCL2 (MCP-1) by spleen cells cultured ex vivo in the presence of PLP139–151. The expression of IFNβ was significantly increased in the spleens of treated mice. Neutralization of either IFNβ or CCL2 in vivo by injection of neutralizing antibodies reversed disease suppression by poly I:C. In vitro neutralization studies in bone marrow-derived dendritic cells showed that the production of CCL2 was IFNβ-dependent. Conclusions: Poly I:C treatment suppresses EAE by mechanisms that involve IFNβ and CCL2. The induction of endogenous IFNβ will be further assessed as a strategy for the treatment of MS. 0S7A-05

Selective COX-2 inhibitor celecoxib prevents experimental autoimmune encephalomyeritis through COX-2 independent pathway Katsuichi Miyamotoa, Sachiko Miyakeb, Susumu Kusunokia, Takashi Yamamurab Department of Neurology, Kinki University, Osaka, Japan; bDepartment of Immunology, National Institute of Neuroscience, NCNP, Tokyo, Japan

a

Cyclooxygenase (COX) is a key enzyme of arachinoic acid metabolism and exist as two distinct isoforms. COX-1 is constitutively expressed in most tissues, whereas COX-2 is inducibly expressed at site of inflammation. Selective inhibitors of COX-2 have been developed and have been used as anti-inflammatory agents. Here we show that a new generation COX-2 inhibitor, celecoxib inhibited experimental autoimmune encephalomyelitis (EAE). Celecoxib but not other COX-2 inhibitors such as nimelsulid prevented myelin oligodendrocyte glycoprotein (MOG)-induced EAE when

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administrated orally on the day of disease induction. Celecoxib also suppressed the severity of EAE even after the initiation of clinical symptoms. Moreover, celecoxib inhibited EAE in COX-2 deficient mice, indicating that celecoxib inhibited EAE in a COX-2-independent manner. In celecoxib-treated mice, IFN-γ production from MOG-specific T cells was

reduced and MOG-specific IgG1 was elevated compared to vehicle-treated mice, suggesting a Th2 bias of MOG reactive T cells in vivo. Infiltration of inflammatory cells was suppressed when treated with celecoxib. These results suggest that celecoxib can be useful as a new additional therapeutic agent for multiple sclerosis.

Abstracts

Oral Session 7B: Immunotherapy (2)

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0S7B-01

Prevention of experimental autoimmune encephalomyelitis via induction of T cells with regulatory function by transfer of ESDC expressing MOG peptide along with TRAIL Shinya Hirata, Satoru Senju, Hidetake Matsuyoshi, Daiki Fukuma and Yasuharu Nishimura The Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Japan Previously, we reported a strategy to prevent experimental autoimmune encephalomyelitis (EAE) by treatment of mice with genetically modified dendritic cells (DC) presenting myelin oligodendrocyte glycoprotein (MOG) peptide in the context of MHC class II molecules and simultaneously expressing TNF-related apoptosis-inducing ligand (TRAIL) or Programmed Death-1 ligand (PD-L1). For genetic modification of DC, we used a recently established method to generate DC from mouse ES cells in vitro (ES-DC). ES cells were sequentially transfected with an expression vector for TRAIL or PD-L1 and a MOG epitope presenting vector. Subsequently, doubletransfectant ES cell clones were induced to differentiate to ES-DC. In this study, we demonstrated that the severity of myelin basic protein (MBP)induced EAE was also reduced by ES-DC-TRAIL/MOG but not by ES-DCPDL1/MOG. This preventive effect diminished, if CD4+CD25+ T cells were depleted by the injection of anti-CD25 mAb into mice before treatment with ES-DC-TRAIL/MOG. In addition, the adoptive transfer of CD4+CD25+ T cells from ES-DC-TRAIL/MOG-treated mice protected the recipient naïve mice from MOG- or MBP-induced EAE. The number of Foxp3+ cells increased in the spinal cord of mice treated with ES-DC-TRAIL/MOG. These results suggest that prevention of EAE by treatment with ES-DCTRAIL/MOG is mediated, at least in part, by MOG-reactive CD4+CD25+ regulatory T cells propagated by ES-DC-TRAIL/MOG. For the treatment of organ specific autoimmune diseases, genetically modified ES-DC presenting some tissue-specific self-antigen along with TRAIL would be effective even when multiple auto-antigens are recognized by autoreactive T cells by the mechanism of epitope spreading. 0S7B-02

Peripheral tolerance induction using ECDI-Fixed APCs for treatment of EAE acts through an indirect mechanisms Danielle M. Turley and Stephen D. Miller Department of Microbiology–Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA Ethylcarbodiimide (ECDI) is a chemical crosslinker that couples antigen to cells delivering a tolerizing signal to T cells. In this study we explored the mechanism of ECDI-antigen (Ag)-coupled cell (Ag-SP) as a treatment of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Ag-SP tolerance has been demonstrated to induce unresponsiveness in autoreactive T cells by presentation of Ag on coupled splenocytes initiating a state of anergy. This tolerance is believed to result from a direct interaction between the host T cell with the fixed donor Agcoupled antigen presenting cell (APC), delivering an Ag specific signal through ligation of the host T cell receptor and peptide:MHC class II complex on the donor APC without costimulation. However, mouse spinal chord homogenate, whole protein, or peptides all function as efficient Ags to induce tolerance suggesting reprocessing of the donor Ag-SP and representation of the dominant epitopes by host APCs. To investigate the mechanism of Ag-SP tolerance we examined donor vs. host MHC class II restriction during Ag-SP tolerance induction. Our data using MHC knockout, allogeneic and/or syngeneic derived donor Ag-SP demonstrate that MHC restriction is not necessary for Ag-SP tolerance induction. It appears that the ECDI-Ag-SP are actively undergoing apoptosis allowing

them to function as an Ag carrier to be reprocessed and represented by host APCs. This further supports the promising use of Ag-SP as an antigen specific therapy for autoimmune and other inflammatory disease. This work was supported by NIH grant NS-30871. 0S7B-03

CD28-superagonist mediated therapy interferes with effector T cell migration in experimental autoimmune encephalomyelitis D. Tischnera, A. Weishauptb, J. van den Brandta, N. Müllera, N. Beyersdorfa, K. V. Toykab, T. Hüniga, R. Goldc, T. Kerkaua and H. M. Reichardta a

Institute for Virology and Immunobiology, University of Würzburg, Germany; bDepartment of Neurology, University of Würzburg, Germany; c Institute for Multiple Sclerosis Research, University of Göttingen, Germany Administration of the superagonistic monoclonal anti-CD28 antibody JJ316 is a highly effective treatment in experimental autoimmune encephalomyelitis (EAE) but the underlying mechanism has remained elusive. Using adoptive transfer EAE in the Lewis rat as our model we could now show that JJ316 interferes with the migration of pathogenic T cells to the CNS and that this is responsible for its great potency in preventing the development and progression of EAE. The inhibitory effect is achieved within the secondary lymphoid organs through the antibody's unique ability to expand and activate FoxP3+ regulatory T cells. Importantly, the presence of regulatory T cells within the CNS is dispensable for the beneficial effect of JJ316, irrespective of whether the antibody is administered prophylactically or therapeutically in overt EAE. Still, regulatory T cells preferentially home to the CNS at later time points after disease initiation, suggesting a potentially important role in the resolution phase or in preventing relapses. As to the mechanism at work, we found that administration of JJ316 prevents the disruption of the blood–brain barrier, favours secretion of Th2 cytokines and abrogates IFN-γ production. The latter results in an impaired expression of CXCR-3, the major chemokine receptor of effector T cells, which presumably accounts for the antibody's ability to inhibit the migration of pathogenic T cells to the CNS. This represents the mechanistic basis of a novel therapeutic concept that, we believe, may be translated into clinical practice once humanized monoclonal antibodies with the same specificity become available. 0S7B-04

Oral administration of anti-CD3 antibody suppresses experimental allergic encephalomyelitis by inducing CD4+CD25−LAP+ regulatory T cells Hirofumi Ochia, Michal Abrahama, Hiroki Ishikawaa, Dan Frenkela, Kaiyong Yanga, Alexandre Bassoa, Henry Wua, Mei-Ling Chena, Roopali Gandhia, Ariel Millerb, Ruth Marona and Howard L. Weinera a

Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School; bCarmel Medical Center, Neuroimmunology Unit, Department of Neurology, USA

Background: Mucosal administration of auto- and allo-antigens induces immunologic tolerance and is effective in treatment of animal models of autoimmunity, inflammation and transplantation. Parenteral anti-CD3 is efficacious in animal models of autoimmunity and in humans, and anti-CD3 is approved for transplant rejection and positive results have been reported in new onset type 1 diabetes. We investigated the effect of oral anti-CD3 in the animal model of multiple sclerosis, experimental allergic encephalomyelitis (EAE). Methods: We orally administered anti-CD3 or isotype control antibody in doses ranging from 5 μg to 500 μg once per day for 5 consecutive days. Results: In the SJL model of PLP induced EAE, oral anti-CD3 suppressed EAE given prior to or at disease peak. Optimal dose was 5 μg; no effect was observed at 50 μg and 500 μg worsened the disease. The PLP-specific immune response showed decreased proliferation, reduced IL-2 secretion

Abstracts and increased IL-10, IL-4 and TGF-β. There was an increase in CD4+CD25−LAP+ (TGF-β latency associated peptide) T cells with enhanced in vitro regulatory activity that was reversed by anti-TGF-β but not anti-IL-10 and that correlated with the dose which suppressed EAE. Adoptive transfer of mesenteric CD4 T cell but not LAP-depleted CD4 T cells suppressed EAE in a TGF-β dependent fashion. Conclusions: Anti-CD3 antibody given orally is immunologically active at mucosal surfaces. Oral anti-CD3 induces CD4+CD25−LAP+ regulatory T cells that function in a TGF-β dependent fashion. These results identify a novel and physiologic mechanism to induce regulatory T cells that is clinically applicable to a variety of immune mediated disorders. 0S7B-05

Glatiramer acetate (Copaxone) therapy restores regulatory, cytotoxic, HLA-E-restricted CD8+ T cells in multiple sclerosis Deepani K. Tennakoon, Riyaz S. Mehta, Sterling B. Ortega, Michael K. Racke, and Nitin J. Karandikar The University of Texas Southwestern Medical Center, Dallas, TX, USA One of the goals of successful therapeutic immune modulation of autoimmune disease is the induction of peripheral tolerance, a large part

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of which is mediated by regulatory/suppressor T cells. We demonstrate here a novel immunomodulatory mechanism by an exogenous peptidebased therapy that incites an HLA Class I-restricted, cytotoxic, suppressor CD8+ T-cell response. We have shown previously that treatment of multiple sclerosis (MS) with glatiramer acetate (GA, Copaxone®) induces differential upregulation of GA-reactive CD8+ Tcell responses. We now show that these GA-induced CD8+ T-cells are regulatory/suppressor in nature. Untreated patients show overall deficit in CD8+ T-cell-mediated suppression, compared to healthy subjects. GA therapy significantly enhances this suppressive ability, which is mediated by cell contact-dependent mechanisms. CD8+ T-cells from GA-treated patients and healthy subjects, but not those from untreated MS patients, exhibit potent, HLA Class I-restricted, GA-specific cytotoxicity. We further show that these GA-induced, cytotoxic CD8+ T-cells can directly kill CD4+ T-cells in a GA-specific manner. Killing is enhanced by preactivation of target CD4+ T-cells and depends on cross-presentation of GA through HLA-E. Thus, we demonstrate that GA therapy induces a suppressor/cytotoxic CD8+ T-cell response, which is capable of modulating in vivo immune responses during ongoing therapy. These studies not only explain several prior observations relating to the mechanism of this drug but also provide important insights into the natural immune interplay underlying this human immune-mediated disease. Supported in part by grants from the NIH and National MS Society.

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Abstracts

Oral Session 8A: Myasthenia gravis

Abstracts 0S8A-01

A sensitive non-isotopic assay for acetylcholine receptor autoantibodies

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over half the patients previously thought to be AChR antibody negative, and that cell based immunofluorescence assays could prove useful in diagnostic testing. 0S8A-03

Rachel Hewer, Ian Matthews, Shu Chen, Vivienne McGrath, Michele Evans, Emma Roberts, Sarah Nute, Jane Sanders, Jadwiga Furmaniak, Bernard Rees Smith

DNA microarray analysis: New drug targets for myasthenia gravis

FIRS Laboratories, RSR Ltd., Parc Ty Glas, Llanishen, Cardiff, CF14 5DU, UK

T. Fefermana, R. Arichaa, P. K. Maitia, G. Clairacb, M. Cuvelierb, S. BerrihAknina,b, S. Fuchsa and M.C. Souroujona,c

Measurement of autoantibodies to the acetylcholine receptor (AChR) is useful in the assessment and management of patients with myasthenia gravis (MG), and development of a new non-isotopic assay for AChRAb which provides specificity and sensitivity at least as high as current isotopic assays is described. In the assay, serum samples (100 μL) were pre-incubated with 25 μL of a mixture of purified foetal and adult-type AChR. AChRAb in 50 μL of the mixture were allowed to compete with three different AChR monoclonal antibodies (MAbs1–3) for binding sites on the AChR: MAb1 was coated onto ELISA plate wells whereas MAb2 and 3 were labelled with biotin and used in liquid phase. MAb-biotin binding was detected by addition of streptavidin peroxidase and substrate. The higher the concentration of AChRAb in the test serum, the greater the inhibition of MAbs binding to the AChR resulting in a reduction in final OD. AChRAb were detected by ELISA in 76/83 MG sera studied (91.6%), whilst 72/83 (86.7%) were positive for AChRAb by immunoprecipitation assay (IPA) based on AChR labelled with 125I-α bungarotoxin. There were 12/83 discrepant samples; 8 sera were positive by ELISA but negative by IPA (0.33– 0.47 nmol/L toxin bound) and 4 sera were negative by ELISA whilst positive by IPA (0.56–2.91 nmol/L toxin bound). All 191 control samples which were negative for AChRAb by IPA, were also negative by ELISA. Overall, results with the AChRAb ELISA and IPA agree well (r = 0.85; n = 83; P < 0.001) and the new assay has good precision and handling characteristics making it suitable for routine use.

a

Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel; bCNRS, Hospital Marie Lannelongue, Le Plessis Robinson, France; c The Open University of Israel, Raanana, Israel Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG) are autoimmune disorders in which the acetylcholine receptor (AChR) is the major autoantigen. DNA microarray technology, supported by quantitative real time PCR, immunohistochemistry and flow cytometry, were used to identify new potential drug targets for MG to delineate genes involved in the pathogenesis of the disease.

M I Leite, J Cossins, D Beeson, N Willcox, A Vincent

1. The chemokine IP-10 and its receptor CXCR3 were found to be overexpressed in LNC and muscles of EAMG rats and in thymuses and muscles of MG patients. CXCR3 was up-regulated in CD4+ T cells of MG patients. 2. The expression of several phosphodiesterase (PDE) subtypes was upregulated in LNC and muscles of EAMG rats. Pentoxifylline (PTX), a general PDE inhibitor, suppressed the progression of EAMG when treatment started at the acute or chronic stages of disease. Suppression was associated with down-regulation of humoral and cellular AChRspecific responses as well as down-regulation of specific PDE subtypes and up-regulation of Foxp3, a transcription factor essential for CD4+CD25+ regulatory T cell function. 3. Data mining of the muscle transcriptome revealed two major groups of deregulated genes common to MG and EAMG: (a) Genes linked to muscle biology including muscle proteins regulating contraction such as myosin polypeptides and myosin binding proteins; (b) Genes coding for the chaperone protein category including several heat shock proteins. There were no inflammation-associated deregulated genes in MG or EAMG.

Neurosciences Group, Department of Clinical Neurology, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK

Our results demonstrate the power of DNA microarray technology to identify novel genes involved in the pathogenesis of myasthenia and other autoimmune diseases.

Antibodies to acetylcholine receptors (AChRs) are present in around 85% of patients with typical generalized myasthenia gravis. A variable proportion of patients without AChR antibodies have antibodies to muscle specific kinase (MuSK). A series of experimental approaches has suggested that some of the remaining AChR antibody negative patients have low affinity antibodies to the AChR; these would not be detected by standard immunoprecipitation tests because solubilised AChR is present in monomeric form and at low concentrations (<1 pM). We, therefore, asked whether we could detect antibodies to AChRs by immunofluorescence of cells expressing high density clusters of AChRs. Human embryonic kidney cells (HEK) were transfected with cDNAs for each of the AChR subunits encoding the adult AChR isoform, and co-transfected with GFP-rapsyn. Rapsyn is a cytoskeletal protein that helps to cluster AChRs at the neuromuscular junction. The cells were incubated with patient or control sera and IgG antibodies detected by Texas-red-anti-human IgG. Control sera did not bind appreciably to the AChR clusters. Four positive controls with high AChR antibodies (>1 nM) showed strong IgG binding to the clusters. Ten of ten sera with low AChR antibodies (0.3–1 nM) showed appreciable IgG binding to the clusters. Ten of 18 sera that had been repeatedly negative (<0.3 nM) by standard immunoprecipitation were positive for IgG binding to the clusters These results show that antibodies to AChRs are present in

0S8A-04

0S8A-02

Antibodies to AChRs in seronegative myasthenia gravis

Altered expression of chemokine receptor CXCR5 on T cells of myasthenia gravis patients H. Onodera1, R. Saito2, H. Tago2, Y. Suzuki2, Y. Itoyama2, Y. Matsumura2, T. Kondo2 National Nishitaga Hospital, Sendai, Japan; 2Tohoku University School of Medicine, Sendai, Japan

1

Myasthenia gravis (MG) is characterized by the T cell-dependent production of anti-acetylcholine receptor antibodies. The chemokine receptor CXCR5 regulates lymphocyte migration and is expressed specifically on a subset of CD4+ T cells named follicular helper T cells (TFH), the key modulators of antibody production by B cells. We studied the frequency of CXCR5-positive lymphocytes in the peripheral blood of MG patients before and after therapy Untreated MG patients showed a significantly higher frequency of CXCR5+CD4+ T cells in the peripheral blood compared with the control group, while no significant difference in the percentages of CXCR5+CD4+ T cells was observed between the

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Abstracts

patients of the hyperplasia group and those of the thymoma group. The CXCR5+CD4+ T cell frequency correlated with the disease severity. The CXCR5+CD4+ T cell frequency of MG patients positive for other autoantibodies together with anti-AChR antibodies was significantly higher than in those having only anti-AChR antibodies. After therapy, the CXCR5+CD4+ T cell percentage decreased gradually to the control level with a significant inverse correlation between the CXCR5+CD4+ T cell frequency and duration after the initiation of MG therapy. These results suggest that CXCR5+CD4+ T cells play an important role in the disease activity of MG and that some MG patients have a systemic abnormality in T cell-dependent antibody production. 0S8A-05

Childhood myasthenia gravis in Japan Y. Nomura, K. Hachimori, Y. Nagao, M. Segawa, K. Kimura and M. Segawa Segawa Neurological Clinic for Children, Tokyo, Japan Objective: The ages at onset of myasthenia gravis (MG) in Japan show two modal patterns, the highest under 3 years of age, which is associated with HLA

DR9/13-DQ6/9, followed by young adult peak. Clinical characteristics, treatments and long term prognosis of the childhood MG are presented. Materials and methods: 270 cases of childhood onset MG who visited this clinic in the past 32 years were evaluated. Clinical types were divided to ocular, latent general (clinically only ocular symptoms, but electrophysiologically myasthenic in the extremity muscles) and general. Treatment consisted with anticholinesterase, steroid, thymetomy or tacrolimus. Results: Clinical types showed general 30%, latent general 50% and ocular 20%. Acetylcholine receptor antibody (AChR Ab) are negative or low in most cases, but some cases with minimum ocular symptoms as sequelae begin to show elevation of AChR Ab at around 20 years of age. Anti-MuSK antibody was studied in 16 cases and showed to be negative. 60% of cases responded to steroid and less than 10% of cases needed thymectomy. Steroid showed best effects when started within 3 years from onset. Onset older than 11 years and general type often needed thymectomy. Tacrolimus was used in 10 cases with good results. Conclusion: Specific immature genetic background seems to play important roles in Japanese childhood MG. Long term prognosis of childhood MG is generally good, but early intervention by steroid or thymectomy is important for the complete remission. Tacrolimus is safely used to switch from steroid.

Abstracts

Oral Session 8B: PNS disease

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0S8B-01

The effect of statin on experimental autoimmune neuritis

rat sciatic nerve induced iNOS gene expression and peripheral nerve degeneration in vivo. Taken together, these data suggest that viral RNA may induce inflammatory Schwann cell activation and peripheral nerve damage in the PNS.

T. Kiyozuka, T. Fujioka, T. Kudeken, K. Murata, H. Ito, and Y. Iwasaki 0S8B-03 Toho University Omori Hospital, Tokyo, Japan

Anti-ganglioside complex antibodies in Miller Fisher syndrome Disease-modulating effect of statin on multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) was unveiled. However, its effect on autoimmune disorders of peripheral nervous system (PNS) remains unclear. To investigate the effect of statin on experimental autoimmune neuritis (EAN), atorvastatin was given orally to EAN rats daily during an entire course. EAN was induced in female Lewis rats by injecting synthetic peptide of bovine myelin P2 protein with CFA. EAN rats fed with vehicle only were used as control. Clinical signs were assessed daily using 8 gradescore system. Cauda equina (CE) were removed at 4, 10, 12, 14, 18, 21, and 24 days post-immunization (DPI). Histology of the CE was studied using frozen sections. mRNA expression of IFN-gamma and IL-10 in the CE was studied using semi-quantitative RT-PCR. Both groups developed EAN at 12 DPI simultaneously although atorvastatin-treated rats showed milder signs and earlier recovery. Mononuclear cell infiltration seen in the CE of atorvastatin-treated rats was also milder than that of control rats. RT-PCR revealed that IFN-gamma mRNA expression at 10 DPI and IL-10 mRNA expression at 12 DPI in atorvastatin-treated rats were decreased compared to the control rats. In EAE, atorvastatin causes shift from Th1 to Th2 cytokine balance resulting in suppression of the neurological deficit. Out data showed that, in EAN, atorvastatin only suppressed Th1 cytokine irrelevant to Th2 cytokine up-regulation as seen in EAE. This difference may reflect different, yet unknown mechanism of modulation of autoimmune inflammation between CNS and PNS. 0S8B-02

Double-stranded RNA induces inflammatory gene expression in Schwann cells, sensory neuronal death, and peripheral nerve demyelination H.K. Leea, C. Parka, I.-H. Choa, S. Leeb, H.-S. Khoc, S.-Y. Choia, S.B. Oha, K. Parka, J.S. Kima, and S.J. Leea a

Program in Molecular and Cellular Neuroscience and Department of Oral Physiology, School of Dentistry, Seoul National University, Seoul, South Korea; bDepartment of Microbiology, Choong-Nam National University, Daejoen, Seoul, South Korea; cDepartment of Oral Medicine and Oral Diagnosis, School of Dentistry, Seoul National University, Seoul, South Korea

Inflammation in the peripheral nervous system (PNS) is one of the characteristics of virus-induced peripheral neuropathy. In this inflammatory response, Schwann cells are actively involved. Recently, toll-like receptor 3 (TLR3) was identified as a receptor for double-stranded RNA (dsRNA) that induces anti-viral and inflammatory responses in cells of the innate immune system. In this study, we investigated the expression of TLR3 and the TLR3-mediated signaling pathways in Schwann cells. TLR3 was constitutively expressed in the primary rat Schwann cells and iSC, an immortalized Schwann cell line. Stimulation with poly(I:C), a synthetic dsRNA, induced the expression of various inflammatory genes such as IP-10, RANTES, TNF-α, and iNOS in Schwann cells. Studies on the intracellular signal transduction pathways using iSC revealed that dsRNA induces the activation of NF-κB, p38, and c-Jun N-terminal kinase (JNK). The activation of NF-κB, p38, JNK, and PKR is required for dsRNA-mediated iNOS gene expression. However, the activation of PI3 kinase and GSK-3β inhibited iNOS gene induction, a process mediated by their inhibitory effects on NF-κB and p38 activation. NO production induced by dsRNA caused neuronal cell death in cultured dorsal root ganglion (DRG). Finally, the introduction of dsRNA into the

K. Kaidaa, M. Kanzakia, D. Moritab, K. Kamakuraa, K. Motoyoshia, M. Hirakawab, S. Kusunokib a

The Third Department of Internal Medicine, National Defense Medical College, Tokorozawa, Japan; bDepartment of Neurology, Kinki University School of Medicine, Osaka-sayama, Japan We recently reported that some ganglioside complexes (GSCs) are target antigens for serum antibodies in patients with Guillain–Barré syndrome (GBS) and that anti-GSC antibodies may be associated with particular clinical features of GBS. Miller Fisher syndrome (MFS), characterized by a clinical triad of ophthalmoplegia, ataxia and areflexia and by the presence of the serum IgG anti-GQ1b antibody, is considered to be a variant of GBS. The purpose of this study is to investigate antibodies to GSCs in sera of patients with MFS. We investigated IgG antibodies to GSCs in sera of 12 consecutive MFS patients using ELISA. Test ganglioside antigens were GM1, GM2, GD1a, GD1b, GT1a, GT1b, GQ1b, and GSCs containing two of the above seven antigens. Seven (58%) of the 12 patients had IgG antibodies to GSCs containing GQ1b or GT1a, five of whom had IgG antibodies to GQ1b–GM1 complex (GQ1b/GM1), GT1a/GM1, GQ1b/GD1b, or GT1a/GD1b and two had antibodies to GQ1b/GD1a, GT1a/GD1a, or GQ1b/GT1b. Four of five patients without sensory symptoms had anti-GQ1b/GM1 antibodies. There were at least three different specificities in MFS-associated antibodies; GQ1b-specific, anti-GQ1b/GM1-positive, and anti-GQ1b/GD1a-positive. Not only GQ1b and GT1a themselves but also clustered epitopes of GSCs including GQ1b or GT1a may be candidates as prime target antigens for serum antibodies in MFS patients. Anti-GQ1b/GM1positive MFS may tend to escape sensory disturbances. 0S8B-04

Complex formation of bacterial GM1- and GD1a-like lipo-oligosaccharides can make GM1b mimicry for autoantibody production M. Kogaa⁎, M. Gilbertb, M. Takahashic, J. Lib, K. Hirataa, and N. Yukia Dokkyo Medical University School of Medicine, Tochigi, Japan; bInstitute for Biological Sciences, National Research Council Canada, Ottawa, Canada; cTokyo Metropolitan Institute of Public Health, Tokyo, Japan; ⁎present affiliation, Yamaguchi University School of Medicine, Yamaguchi, Japan

a

Campylobacter jejuni strains with class A lipo-oligosaccharide (LOS) biosynthesis locus exceptionally carry both GM1- and GD1a-like LOSs and have been found worldwide to be associated with Guillain–Barré syndrome (GBS) development. We assumed that complex formation of these ganglioside epitopes makes the organisms express new structure, as a result generating autoantibody which has different reactivity from antibodies specific for each ganglioside. In this study, we detected IgG autoantibody which specifically reacts with mixture of GM1 and GD1a, not with each ganglioside, in several GBS patients from whom C. jejuni strains carrying both GM1- and GD1a-like LOSs had been isolated. All of these patients had higher titer of anti-GM1b IgG antibody, and absorption study showed that anti-GM1b and anti-GM1/GD1a complex antibodies were absorbed by GM1/GD1a-like LOS. Furthermore, these absorptions were lost when we used LOS as absorber after treatment with neuraminidase from Arthrobacter ureafaciens, which converts GM1/GD1a-like structure to GM1-like one.

Abstracts Immunization with a class A strain (CF90-26; GBS-related), which carries both GM1- and GD1a-like LOSs, induced IgG antibody response against GM1b as well as GM1/GD1a complex, but not individual GM1 or GD1a. These findings strongly suggest that target structure for anti-GM1/GD1a complex antibody mimics GM1b ganglioside, which has structurally different sugar sequence from GM1 and GD1a, but could also be directed for as target antigen for autoantibody in GBS. This is the first example demonstrating that complex formation of bacterial structures is another way to make molecular mimicry in autoantibody production. 0S8B-05

Efficacy of ciclosporin in chronic inflammatory demyelinating polyneuropathy patients who require repeated treatment with intravenous immunoglobulins M. Odakaa, M. Tatsumotoa, K. Hirataa, N. Yukia a

Department of Neurology, Dokkyo Medical University School of Medicine, Kitakobayashi 880, Mibu, Shimotsuga, Tochigi 321-0293, Japan Background: Primary goals of current therapies used for chronic inflammatory demyelinating polyneuropathy (CIDP) are to control symptoms, improve functional ability, and maintain long-term remission. Some

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CIDP patients whom corticosteroids cannot sustain remission, require repeated treatment with intravenous immunoglobulins (IVIG). Objective: To investigate the therapeutic efficacy of ciclosporin for CIDP patients who require repeated IVIG, and to evaluate basic information and target samples size for therapeutic trial with ciclosporin. Methods: Ciclosporin was tried for 14 CIDP patients whom corticosteroids could not sustain remission. An initial dose of ciclosporin was 3 mg/kg/day with plasma trough concentrations between 100 and 150 ng/ml. Frequency of symptomatic relapses and side effects of 11 patients treated with ciclosporin for 12 months were investigated. Results: Eight patients responded to ciclosporin and sustained remission over 12 months; 2 gradually prolonged remission interval after increase of ciclosporin dose; 1 showed no improvement and repeated IVIG were required. Except for 1 patient with renal function disturbance, there have been no serious side effects. The preliminary favorable findings for our patients treated with ciclosporin support target samples size (ciclosporin treatment and extended IVIG treatment groups, each 25 patient) for ciclosporin trial. Conclusions: Ciclosporin is effective therapeutic agent for long-term remission in patients with CIDP who do not show sustained improvement under corticosteroid therapy. Ciclosporin should be tried for patients with CIDP who require repeated IVIG. Randomized parallel group comparison multicenter collaborative research, attending hospitals throughout Japan, will start soon.

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Poster Session 1: Cell death and survival

Abstracts PP01-01

Differential vulnerability of hippocampal neurons: Relationship of tumor necrosis factor and glia in chemical-induced injury G.J. Harrya, C. Lefebvre d'Hellencourta,b, R. Winea, M. Aoyamaa

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Conclusion: These findings indicate that activation of EP1 on neurons contributes to neuronal death and can be influenced by non-neuronal cells. PP01-03

Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina

a

Laboratory of Neurobiology, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC, USA; bUniversité de La Réunion, Réunion-France-DOM

The current study used trimethyltin-induced damage to examine the contribution of tumor necrosis factor (TNF) superfamily in hippocampal dentate granule cell (DG) death and to determine if differential expression of the TNF receptors and relationship to glia would characterize neuronal vulnerability. In the DG, TNFα, TNFR1, TNFR2 mRNA levels (QT-PCR of laser captured material) were elevated; microglia activated; astrocytes hypertrophic. In the protected CA pyramidal layer, TNFα, TNFR1, TNFR2, mRNA levels were elevated; astrocytic processes thickened, and reactive non-phagocytic microglia present. Microglia proliferation was 11, 20, and <1% in the DG, CA, and CA1, respectively. With injury, intracellular distribution of TNFR1 protein in the DG shifted from membrane to cytoplasmic/nuclear, cells expressed active caspase 3 and were in contact with activated microglia. In the CA, intracellular distribution of TNFR2 shifted and cells maintained close contact with GFAP+ astrocytes. Neutralizing antibody to TNFα was neuroprotective; mice null for both TNFR1 and 2 showed damage attenuation; mice null for either TNFR1 or R2 showed exacerbation. TNFR1/2 null mice showed a significant elevation in basal levels and less induction by TMT for TNFα, IL-1α, and TGFβ1 as compared to wildtype. Basal MIP-1α mRNA levels were elevated over wildtype; with similar induction. Higher basal levels and induction of TNFα mRNA occurred in the TNFR1−/− and the elevation in both TNFα and IL1α in the TNFR2−/− mice occurred at the lower dose. These results suggest a sequence of events: proinflammatory cytokines, activation of death receptors, and microglia phenotype determining pattern of neuronal death. PP01-02

Neuroprotection by an EP1 prostanoid receptor antagonist is modulated by non-neuronal cells in culture N.G. Carlsona,b,c, J.D. Blackb, M. Rojasb, K.E. Hillb and J.W. Roseb,c GRECC, bNeurovirology VASLCHCS, cUniversity of Utah, Salt Lake City, UT USA

Y. Inomatab, H. Nakamurac, M. Tanitod, A. Teratanic, T. Kawajib, N. Kondoa, J. Yodoia and H. Taniharab a

Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan; bDepartments of Ophthalmology and Visual Science, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; cThioredoxin Project, Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto, Japan; dDepartment of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma Thioredoxin (TRX), an endogenous redox-activeregulator, plays a cytoprotective role against focal ischemic, excitotoxic, and 1-methyl-4phenylpyridinium-induced brain damage. We investigate whether TRX modulates N-methyl-D-aspartate (NMDA)-induced retinal neurotoxicity. TRX, mutant TRX, or saline were injected together with NMDA intravitreously into rat eyes. Number of retinal ganglion cells (RGCs) was counted by retrograde labeling analysis to evaluate retinal damage. Cellular apoptosis in the retina was measured by TdT-mediated dUTP nick-end labelling (TUNEL) and by measurements of caspase activities. Activation of the mitogen-activated protein kinases (MAPKs), p38 and JNK, and MAPK kinases (MKKs), MKK3/6 and MKK4 were examined by Western blot and immunohistochemistry. Protein carbonylation, nitrosylation and lipid peroxidation were assessed to evaluate antioxidative effects of TRX. RGCs were rescued by TRX, compared with saline, when evaluated by retrograde labelling analysis, 7 days after NMDA-injection. TRX, but not its mutant form, prevented NMDAinduced apoptosis in the retina, as measured by TUNEL. The induction of caspase-3 and caspase-9, but not caspase-8, by NMDA was significantly lower in TRX-treated eyes than in saline-treated eyes. NMDA-induced activation of p38 and JNK after 6 h, and of MKK3/6 and MKK4 after 3 h, was markedly suppressed in RGCs by TRX, but not by the mutant form. NMDA-induced increases in protein carbonylation, nitrosylation, and lipid peroxidation were also suppressed in TRX-treated eyes. TRX effectively attenuates NMDA-induced retinal cell damage, and suppression of oxidative stress and inhibition of apoptotic signalling pathways are involved in this cytoprotective effect.

a

Objective: Our goal was to determine whether the EP1 prostanoid receptor contributed to the excitotoxic death of neurons. Methods: EP1 receptor antagonists (Ono 8711 and SC51089) were examined for neuroprotective effects against an excitotoxic exposure to nmethyl-d-aspartate (NMDA). This was tested in two preparations of primary neuronal cultures containing greater than 90% neurons (PN) or a mixture of neurons and glia. Results: Treatments of Ono 8711 (3, 10 or 30 nM), SC51089 (10 μM) or the COX-2 inhibitor NS398 (30 μM), increased the number of neurons that survived NMDA by a factor of three in PN cultures. In mixed cultures, no neuroprotection was seen with any concentration of Ono 8711, SC51089 or NS398. The percent of neurons that expressed EP1 was 65% in mixed cultures and 90% in PN cultures. NMDA induced a ten-fold increase in PGE2 in both culture preparations, although the net amount of PGE2 was about five-fold higher in the mixed cultures. Combined, these findings indicate that a lack of neuroprotection in mixed cultures by EP1 antagonists was not due to a lack of neuronal EP1 expression nor was it due to an inability to synthesize PGE2.

PP01-04

Trail-induced death of adult human oligodendrocytes is mediated by JNK and mitochondrial activation Mariola Matysiak1, Anna Jurewicz1, Stanislaw Andrzejak2 and Krzysztof Selmaj1 1

Departament of Neurology, Medical University of Lodz, Poland; Department of Neurosurgery, Medical University of Lodz, Poland

2

Background: TRAIL (TNF-related apoptosis inducing ligand) is a member of TNF family that induces death of mainly tumor cells. TRAIL is expressed on autoreactive T cells and induces death of adult human oligodendrocytes by ligation of TRAIL-receptor 1. Intracellular signaling involved in oligodendrocytes injury is still unknown. Methods: In our study we investigated involvement of JNK and mitochondrial pathway in death after TRAIL stimulation. JNK activation was assessed by using antibodies against phosphorylated JNK isoforms and analyzing c-jun phosphorylation by autoradiography. The role of JNK

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activation was estimated by using dominant negative construct of MKK4, kinase upstream of JNK. Changes in mitochondrial membrane permeability was assessed using ApoAlert Mitochondrial Membrane kit. Cell death was analyzed by FACS using Annexin-V and PI staining. Results: Our results shown that intracellular signaling involved in TRAILinduced death of oligodendrocytes is associated with strong, sustained activation of JNK isoform 3. After TRAIL stimulation was observed changes in mitochondrial membrane potential. JNK-3 activation occurred before mitochondrium activation. Dominant negative mutant of MKK4 inhibited TRAIL induced mitochondrial membrane disruption and oligodendrocytes injury. Conclusions: These results indicate that JNK and mitochondrial activation are critically involved in oligodendrocytes death induced by TRAIL and might have importance in protection immune-mediated oligodendrocytes demise. PP01-05

TNF-induced death of human adult oligodendrocytes is apoptosis inducing factor (AIF)-dependent Anna Jurewicz⁎, Mariola Matysiak⁎, Krzysztof Tybor#, Cedric S. Raine^ and Krzysztof Selmaj⁎ ⁎Department of Neurology and #Department of Neurosurgery, Medical University of Lodz, Lodz, Poland, ^Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA Tumor necrosis factor (TNF) induces apoptotic-like cell death of oligodendrocytes, the cell type targeted in MS. The ligation of TNF receptors induces several signal transduction pathways including caspase cascade and mitochondrial derived factors like apoptosis inducing factor (AIF). However, the precise mechanism involved in human oligodendrocyte (hOL) death, is unknown. OLs were prepared from human brain specimen. TNF-induced hOLs death, was detected by flow cytometry with annexinVFITC and PI staining. AIF activity was assessed by hOLs DNA electrophoresis and its nuclei translocation with confocal microscopy. Anti-sense construct (asAIF) was used to prevent AIF activity. Caspase inhibitor, ZVAD.FMK, calpain inhibitor, ZLLY.FMK, serine proteases inhibitor, TPCK, and cathepsin inhibitors, ZFL and ca-074-Me, were used to assessed their involvement in OL death. TNF-induced death of hOLs is noncaspase dependent, as evidenced by: lack of generation of caspase-8, -1 and 3 active subunits; lack of cleavage of caspase-1 and -3 fluorogenic substrates; and lack of hOL death inhibition by ZVAD.FMK. Also calpain, serine proteases, and cathepsin inhibitors were inefficient in prevention of hOLs death. In contrary, TNF-induced hOLs death involved AIF as evidenced by characteristic for AIF-induced cell death large scale DNA fragmentation and AIF translocation to hOLs nuclei upon TNF exposure. Accordingly, TNF-induced hOLs death was prevented by inhibition of AIF with asAIF. In brain sections, within the edge of MS lesion, some oligodendrocytes defined by anti-MBP staining showed nuclei positive for AIF. These results indicate that TNF-induced death of hOLs depends on AIF, information of significance for the design strategies to protect hOLs during immune-mediated demyelination.

Activation of the terminal complement cascade involving C5 to C9 proteins has a beneficial role for oligodendrocytes in experimental allergic encephalomyelitis, an animal model of multiple sclerosis, by protecting them from apoptotic cell death. We have previously shown that sublytic C5b-9 complexes, through posttranslational regulation of Bad, inhibit the mitochondrial pathway of apoptosis induced by serum deprivation. In the present study we examined the possible involvement of the caspase-8 and Fas pathway in oligodendrocyte apoptosis and the role of C5b-9 in this process. In serum-free defined medium oligodendrocytes undergo apoptosis and differentiation concomitantly. Under this condition, we found that caspase-8 processing was increased in association with Bid cleavage and markedly reduced expression of c-FLIPL protein. The caspase-8 inhibitor ZIETD-FMK inhibited cell death associated with differentiation in a dosedependent manner. Exposure to C5b-9 induced an inhibition of caspase-8 activation, Bid cleavage, and a significant increase in c-FLIPL expression. Phosphatidylinositol 3-kinase inhibitor LY294002 reversed these C5b-9 effects. C5b-9 also down-regulated the expression of FasL and the Fas induced apoptosis. These data suggest that C5b-9 through phosphatidylinositol 3-kinase signaling can rescue OLG from Fas-mediated apoptosis by regulating caspase-8 processing. PP01-07

MEK-ERK signaling is involved in interferon-γ-induced death of oligodendroglial progenitor cells T. Itoh, A. Itoh and M. Horiuchi Department of Neurology, University of California Davis/Institute for Pediatric Regenerative Medicine, Shriners Hospital for Children Northern California, Sacramento, California, USA Oligodendrocytes are exposed to various cytokines in inflammatory lesions in the CNS. In this study, we focused on the direct effects of interferon-gamma (IFNG) on purified rat oligodendroglial cultures at different developmental stages. Among the three stages tested, IFNG had direct cytotoxic effects on actively proliferating oligodendrocyte progenitors, but much less on immature oligodendrocytes and none on mature oligodendrocytes. This stage specific susceptibility of progenitors to IFNGinduced cytotoxicity consisted of two components, delay in the cell cycle transition from the G1 phase to the S phase and increased cell death at least partly mediated by apoptosis, suggesting that progression of the cell cycle was tightly linked to this toxic mechanism. As far as could be determined by examining induction of IRF1 mRNA in response to IFNG, there was no functional difference in the signal transducers and activators of transcription (STAT) pathways between progenitors and mature oligodendrocytes. We found that partial inhibition of the ERK pathway, one of mitogen activated protein kinase pathways, by U0126 partially reversed the IFNG-induced cytotoxicity. In addition, ERK activity was quickly downregulated after differentiation of progenitors to immature oligodendrocytes. Therefore, we concluded that simultaneous activation of the STAT pathway by IFNG and the ERK pathway by trophic factors played a role in the stage specific IFNGinduced cytotoxicity in oligodendroglial progenitors. PP01-08

PP01-06

C5b-9 complement complex protects oligodendrocytes from apoptotic cell death by inhibiting caspase-8 processing and upregulating FLIP

SAMSN1, an Akt/protein kinase B (PKB) Substrate that Inhibits Fas-Mediated Apoptosis Giok Kim1, Keith B. Elkon2

H. Rusa, C. Cudricia, F. Niculescub, T. Jansena, E. Zafranskaiaa, M. Fosbrinka, V. Rusb and M. L. Shinc

1

Department of Neurology, bDivision of Rheumatology and Clinical Immunology, Department Medicine, cDepartment of Pathology, University of Maryland School of Medicine, Baltimore, USA

Akt (protein kinase B) is a pivotal regulator of cell growth and survival. Activation of Akt attenuates apoptosis mediated through both the extrinsic death receptors and via the intrinsic mitochondrial pathway. To identify

a

Jeju High-Tech. Industry Development Institute, Jeju, Korea; 2Division of Rheumatology, University of Washington, Washington, USA

Abstracts additional downstream targets of Akt, we screened the human protein database for Akt consensus sequences in proteins of unknown function. A highly conserved protein, SAMSN1, predicted to contain 373 amino acids and three Akt consensus phosphorylation sites was identified. The SAMSN1mRNA arises by alternative splicing of a 10 kb gene on chr.21q11 also encoding the recently described HACS1 protein. SAMSN1 mRNA was predominantly expressed in tissues of hematopoietic origin and was detected in mature B and T lymphocytes and macrophages but not in epithelial lines or primary fibroblasts. Co-expression of SAMSN1 with Akt lead to PI-3 kinase dependent, phosphorylation of SAMSN1 and its interaction with Akt. To explore the cellular localization and function of SAMSN1, wild type SAMSN1and mutants lacking either the Akt phosphorylation sites (Tri-A mutant), nuclear localization signals (ΔNLS), or SAM domain (Δ-SAM) were expressed in 293 T cells. SAMSN1, as well as the Tri-A and Δ-SAM mutants, but not the Δ-NLS mutant, localized to the nucleus indicating that Akt phosphorylation did not regulate cell localization. Jurkat T cells transduced with a retroviral vector expressing WT SAMSN1 were significantly protected from Fas, but not staurosporin, mediated apoptosis and this protection required the putative Akt phosphorylation sites. Together, these data suggest that SAMSN1 as a novel Akt substrate localized to the nucleus of cells of hematopoietic origin that mediates protection from Fas mediated apoptosis. PP01-09

The role of adenosine receptors in IL-6-dependent neuroprotection K. Biber1, A. Pinto-Duarte2, A. M. Dolga3, C. C. Fernandes2, A.P. Ijzerman4, U. Eisel3, A. M. Sebastião2, and H.W.G.M. Boddeke1 1

Department of Medical Physiology, University Medical Center Groningen, University of Groningen, The Netherlands; 2Institute of Pharmacology and Neurosciences, Faculty of Medicine and Institute of Molecular Medicine, University of Lisbon, Portugal; 3Department of Molecular Neurosciences, University of Groningen, The Netherlands; 4Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden, The Netherlands

The immunological response in the brain plays a crucial role in neuropathological conditions. Nowadays, it is consensual that several inflammatory mediators, such as the immunoregulatory cytokine interleukin-6 (IL-6), protect neurons from glutamate-induced neurotoxicity in vitro and in vivo. Despite a large body of experimental evidence, the fundamental mechanisms underlying IL-6-mediated neuroprotection are not understood until today. Our data clearly show that IL-6 is not a neuroprotective agent per se, but that IL-6 instead enhances the expression and function of neuronal adenosine A1 receptor in vitro and in vivo. Accordingly, we provide evidence that IL-6 by

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enhancing neuronal adenosine A1 receptor function (1) efficiently protects hippocampal neurons from hypoxia in acutely prepared brain slices; (2) facilitates neuronal survival in an excitotoxicity paradigm; and (3) protects animals from chemically induced convulsing seizures. It is moreover shown that IL-6-dependent neuroprotection is abolished when the function of neuronal adenosine A1 receptors is blocked. Based on this data we suggested that IL-6 increases neuronal survival in vitro and in vivo by enhancing the function of neuronal adenosine A1 receptors, the brains major protective system. PP01-10

Interferon β counteracts the apoptotic cell death induced by TNF α in primary astrocytes through stimulation of PI-3K and MEK Olga Barca, Jose A. Costoya, Rosa Señarís, Víctor M. Arce Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela, San Francisco 1, 15782, Santiago de Compostela, Spain Although interferon β (INFβ) has been demonstrated to be effective in the treatment of multiple sclerosis (MS), the mechanism(s) underlying its beneficial effects has not been uncovered. In the present work we have investigated the ability of INFβ to counteract the apoptosis induced by TNFα treatment in primary astrocytes. Primary monolayer cultures of rat cortical astrocytes (E15) were treated with different doses of TNFα (1, 5, 25, 50 or 100 ng/mL) for 24 h in the absence or the presence of INFβ (2 ng/mL). Apoptotic cell death was determined by Hoechst 33258 staining. Phosphorylated Akt and ERK were determined by Western blot. TNFα treatment induced a significant increase in astrocyte death starting at the dose of 25 ng/mL. This effect was blocked in the presence of the caspase-8 inhibitor Z-IETD-FMK, indicating that astrocyte death is due to apoptosis. Co-treatment of cultured astrocytes with INFβ significantly reduced TNFα-induced cell death. This antiapoptotic effect of INFβ depends, at least partially, on its ability to stimulate the phosphorylation of Akt. When PI-3K activity was inhibited by means of LY294002 treatment, INFβ failed to promote both Akt phosphorylation and astrocyte survival. In addition INFβ also induced phosphorylation of ERK. Inhibition of MEK activity by means of PD98059 treatment, blocked the ability of INFβ to counteract TNFα-induced apoptosis. On the view of these results it is tempting to speculate that the beneficial effects of interferon treatment on MS patients may depend on its ability to prevent astrocyte death. Supported by FISS grant no. 01/1007.

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Poster Session 2: Glia

Abstracts

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PP02-01

PP02-03

TLR3 triggers an anti-viral response in astrocytes

Impaired cleareance of apoptotic neurons by lack of microglial TREM2 signaling

Mark A. Rivieccio, Hyeon-Sook Suh⁎, Meng-liang Zhao, Sunhee C. Lee and Celia F. Brosnan

Kazuya Takahashi1,2, Christian D.P. Rochford1 and Harald Neumann1,3 1

Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA Toll-like receptors (TLRs) are receptors that mediate innate immunity against pathogens. Toll-like receptor 3 (TLR3) is a viral nucleic acid sentinel activated by dsRNA within intracellular vesicles. TLR3 signals through TRIF (TIR domain-containing adaptor-inducing IFNβ) and TRAF6 to activate interferon regulatory factor 3 leading to the production of IFNβ (primary response), and to a delayed activation of NF-kB leading to the production of proinflammatory cytokines and chemokines. To explore the spectrum of genes induced in human astrocytes by TLR3 we used a microarray approach and the dsRNA mimetic poly I:C (pIC) as ligand. One of the highly induced genes was viperin/cig5, a protein with known anti-viral activity. Viperin/cig5 expression was dependent on IRF3 and NF-kB signaling, and repetitive stimulation with poly(I:C) but not IL1 further increased expression. To determine whether poly(I:C) induced an antiviral state in astrocytes, a pseudotyped HIV viral particle VSVg-envHIV-1 was used. Poly(I:C) significantly abrogated HIV-1 replication whereas IL-1, which also potently activates astrocytes, did not. Viperin induction could also be substantially inhibited by neutralizing antibodies to IFNb, as could HIV-1 replication. To explore a role for viperin in IFNbmediated inhibition of HIV-1 we employed an RNAi approach. RNAi directed against viperin, but not a scrambled RNAi, significantly inhibited viperin expression, and also significantly reversed poly(I:C)-induced inhibition of HIV-1 replication. We conclude that viperin contributes to the antiviral state induced by TLR3 ligation in astrocytes, supporting a role for astrocytes as part of the innate immune response against infection in the CNS.

Neuroimmunology Unit, European Neuroscience Institute, Goettingen, Germany; 2 Department of Neurology and Neurobiology of Aging, Kanazawa University Graduate School of Medical Science, Japan, and 3 Institute of Reconstructive Neurobiology, University Bonn LIFE and BRAIN Center, Bonn, Germany Background: Elimination of apoptotic neurons without inflammation is crucial for brain tissue homeostasis, but the molecular mechanism has not been firmly established. Triggering receptor expressed on myeloid cells-2 (TREM2) is a recently identified innate immune receptor. Mutations in TREM2 and DAP12 have been described in Nasu-Hakola disease patients. We analyzed microglial function in response to TREM2 stimulation or after knock down of TREM2. Methods: Cultured microglia were transduced with Flag-tagged TREM2, short hairpin TREM2 for RNA interference, wild type TREM2 for over-expression, or their control GFP lentiviral vectors. Function in response to TREM2 stimulation, after knock down of TREM2 and co-culture with apoptotic neurons was analyzed by flow cytometry, RT-PCR, and immuno-cytochemistry. Results: TREM2 stimulation increased phagocytic activity, but neither induced inflammatory mediators nor up-regulated surface markers were involved in antigen presentation. Furthermore, TREM2 knock down microglia showed impaired clearance of apoptotic neurons and increased gene transcription of TNFalpha, IL-1beta and NOS2. Over-expression of TREM2 in microglia resulted in increased phagocytosis of apoptotic neurons and decreased gene transcription of inflammatory mediators. Conclusion: TREM2 signaling participates in clearance of apoptotic neurons and down-regulation of inflammation to maintain the immunoprivileged CNS microenvironment. PP02-04

PP02-02

Toll-like receptor profiles and functions in the human brain during multiple sclerosis M. Bsibsia, C. Persoon-Deena, R. Ravid b, J.M. van Noorta TNO Prevention and Health, Leiden, The Netherlands and bNetherlands Brain Bank, Amsterdam, The Netherlands

a

Toll-like receptors (TLRs) are key elements in the control of inflammatory pathways also in the human CNS. In our previous studies, we demonstrated the presence of various TLRs in and on both human microglia and astrocytes. Marked differences, however, separate TLRs on either type of glial cell. In microglia, a rather diverse set of TLRs are expressed and at least TLR3 and TLR4 can be found only inside endosomal vesicles. Astrocytes on the other hand, express a more limited set of TLRs, and only on their cell surface. In our present studies we have found that upon activation astrocytes display a striking preference for expressing elevated levels of TLR3 only. Genomic profiling and functional data indicate that TLR3 activation on astrocytes does not activate a traditional pro-inflammatory response but, instead, a comprehensive neuroprotective, angiogenic and anti-inflammatory response. This suggests that TLR3 on astrocytes may act as a repair mediator. In line with this notion, we recently identified an endogenous CNS protein that activates astrocytes through TLR3. To shed further light on TLR functions in the human CNS during inflammation, TLR profiles in human brain samples including multiple sclerosis lesions of varying stages were evaluated by real-time PCR and protein staining techniques. The results of these analyses and their possible implications will be discussed.

Primate microglia are particularly sensitive to TLR8-mediated activation but do not react to TLR7 and TLR9-mediated activation Jeffrey J. Bajramovica, E.A. Sicka, M. Bsibsib, C. Persoonb, I. Deuzinga, W. de Boera, C. van der Puttena, K.F.H. Assinkc, L.A. Bovenc, J.M. van Noortb, B. 't Hartd, S. Amord a

Alternatives Unit, Biomedical Primate Research Centre, Rijswijk, The Netherlands; bBusiness Unit Biomedical Research, TNO Quality of Life, Leiden, The Netherlands; cDepartment of Immunology, Erasmus MC, Rotterdam, The Netherlands; dDepartment of Immunobiology, Biomedical Primate Research Centre, Rijswijk, The Netherlands Microglia are CNS resident innate immune cells that can become activated upon encountering infectious agents. Toll-like receptors (TLR) are a diverse family of receptors recognizing pathogen-associated molecular patterns that drive activation of the expressing cell. The transformation of resting microglia into activated APC involves multiple steps and the outcome will be determined by the differentiation potential of resting microglia and by the activating stimulus. In order to study this process, we used primary microglia obtained from adult rhesus monkeys. After exposure to different differentiation (M-CSF or GM-CSF) regimes, functional reactivity to TLR-mediated signaling was determined and TLR mRNA expression levels were quantified. Both types of microglia responded to TLR2, 3, 4, 5 and 8-mediated stimulation as measured by the production of TNF-alpha, IL-6, IL-12 and IL-23. Equally important, rhesus microglia were not responsive to TLR7 and 9mediated signaling, this latter in contrast to murine microglia. Both types of microglia were particularly responsive to TLR8-mediated stimulation. However, TLR8-mediated activation evoked a much stronger cytokine response by MCSF-differentiated microglia as compared to GM-CSF-differentiated microglia,

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Abstracts

whereas reactions towards other TLR ligands were comparable. Moreover, MCSF-differentiated microglia expressed higher TLR8 mRNA levels than GMCSF-differentiated microglia. Since TLR8-mediated signaling activates microglia so potently, we are currently screening CNS material of different origin for the presence of – endogenous – TLR8 ligands. Intriguingly, TLR-mediated activation of microglia dramatically enhanced TLR9 mRNA expression levels in GM-CSF-differentiated microglia only, possibly endowing these cells with sensitivity to TLR9-mediated signaling. We aim to present preliminary data on this subject during the meeting. PP02-05

Release of activin A by microglial cells upon stimulation with bacterial TLR-agonists S. Ebert, R. Nau, U. Michel Georg-August-University, Department of Neurology, Göttingen, Germany Objective: Activin A, a member of the transforming growth factor-beta family of growth and differentiation factors, is a multifunctional cytokine, with one of its roles being in the immune system and inflammatory processes. It has neuroprotective properties and can be detected within the central nervous system. We previously demonstrated that concentrations of activin A are elevated in the cerebrospinal fluid (CSF) of patients with meningitis. As the sources of elevated activin A concentrations in CSF during meningitis have not yet been discovered, we examined whether microglial cells release activin A upon stimulation with bacterial Toll-likereceptor (TLR)-agonists. Methods: Primary mouse microglial cells were exposed to agonists of TLR2 [Pam3Cys (Tripalmitoyl-S-glyceryl-cysteine)], TLR4 [LPS (lipopolysaccharide)], and TLR9 [CpG (oligonucleotides containing unmethylated cytosin– guanosin motifs)] for 24 h. Activin A concentrations in the cell culture supernatants were measured by ELISA [median (minimum / maximum)]. Results: While activin A concentrations in the cell culture supernatants were below the detection limit of the ELISA in the control group, they were significantly elevated after treatment with the different TLR-agonists: 279 pg/ ml (196/337) after 1μg/ml LPS (p < 0.0001), 117 pg/ml (0/167) after 1 μg/ml Pam3Cys (p = 0.007), and 77 pg/ml (0/185) after 10 μg/ml CpG (p = 0.02). Discussion: Our results show for the first time that microglial cells release activin A upon activation by bacterial TLR-agonists. This finding provides further evidence for a role of activin A in the innate immune response and suggests that microglial cells are a source of elevated activin A concentrations observed in the CSF during bacterial meningitis. PP02-07

Molecular mechanisms of LPS-induced SOCS-3 gene expression in macrophages and microglia Hongwei Qin, Kevin L. Roberts and Etty N. Benveniste Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Neuroinflammatory events in the Central Nervous System (CNS) are central to the pathogenesis of Multiple Sclerosis, Alzheimer's Disease and HIV-1 Associated Dementia. Immunological activation of macrophages/microglia within the CNS leads to the production of cytokines that ultimately impact on glial and neuronal function. Suppressor Of Cytokine Signaling (SOCS) proteins are important biological regulators in adaptive and innate immune responses. The SOCS-3 protein is critical for regulating signaling by IL-6 cytokine family members, which utilize the STAT-3 protein. Our previous studies demonstrated that SOCS-3 plays an important role in the inhibition of LPS-induced CD40 gene expression. In this study, we describe that LPS is a strong inducer of SOCS-3 expression in macrophages and microglia, which occurs at the transcriptional level. An analysis of the SOCS-3 promoter indicates that AP-1 and two GAS elements are involved in LPS-

induced SOCS-3 transcription in macrophages. LPS-induced SOCS-3 expression involves the endogenous production of IL-6, which contributes to SOCS-3 expression by STAT-3 activation. Blocking IL-6-induced STAT-3 activation by the inclusion of IL-6 neutralizing antibody reduces LPSinduced SOCS-3 gene expression. Furthermore, LPS induces activation of the mitogen-activated protein kinase (MAPK) pathway, which is also involved in LPS-induced SOCS-3 gene expression. These results indicate that LPS-induced MAPK activation and endogenous production of IL-6 that subsequently induces STAT-3 activation play critical roles in the regulation of SOCS-3. We propose that LPS-induced SOCS-3 expression results in a negative feedback loop to attenuate LPS and cytokine-induced immune and inflammatory responses in the CNS. PP02-08

Heterogeneous origin of reactive microglia in injured rat and mouse central nervous system Kate Lykke Lambertsen1⁎, Martin Wirenfeldt1⁎, Rikke Gregersen1, Ishar Dalmau1, Nils Henrik Diemer2, and Bente Finsen1 1

Medical Biotechnology Center, University of Southern Denmark, Odense, Denmark; 2Molecular Neuropathology, University of Copenhagen, Copenhagen, Denmark Microglia serve homeostatic functions in normal CNS, and sense threats to and modulate neuronal survival in injured and diseased CNS. Presently, our understanding of microglial biology and involvement in disease is influenced by observations that exogenous bone marrow (BM)derived cells contribute to reactive microgliosis in the mouse. To determine whether there are species-differences in the recruitment and microglial transformation of BM-derived cells into injured CNS, we analyzed the microglial reaction in radiation BM chimeric mice and rats subjected to classical forms of experimental manipulation, known to elicit predictable, strong microglial reactions with no or minimal involvement of monocytic macrophages. We found that a subpopulation of the reactive, bushy-type microglial cells in the dentate gyrus originated from transformed BM-derived cells after entorhinal cortex lesion in BM chimeric mice. This contrasted BM chimeric rats subjected to global cerebral ischemia, which displayed the characteristic microglial rod cell transformation and microglial hyperplasia in regio superior hippocampus, but in the absence of recruitment and microglial transformation of BM-derived cells. Our results provide evidence that reactive microglia in the rat originate from resident microglia, while having mixed BM-derived and resident origin in the mouse. The data emphasize that there are important species-differences in recruitment of blood-borne microglial precursors to the brain and that speciesdifferences may be an issue when considering using BM-derived cells for gene delivery to the CNS in human neurological disorders. Importantly, BM chimeric rats may provide valuable models to establish conditions for how to attract genetically manipulated BM-derived cells into human CNS. PP02-09

Microarray analysis of microglia versus bone marrow-derived macrophages Vuaillat-savarin Carine1, Wierinckx Anne2, Rey Catherine3, Lachuer Joël1,2, Belin Marie-Françoise1, Nataf Serge1 1

INSERM, U433, Rue Guillaume Paradin, Lyon, F-69008 France; Univ Lyon 1, Villeurbanne, F-69100 France; 2Neuromic, Hôpital Neurologique P. Wertheimer, Bd Pinel, Bron, F-69500 France; 3Neurobiotec Services, Hôpital Neurologique P. Wertheimer, Bd Pinel, Bron, F-69500, France It is now admitted that microglia represent a unique macrophage subpopulation that harbors specific morphology, phenotype and functions.

Abstracts However, the molecular basis for such a specialization is poorly known. In particular, markers which could reliably discriminate microglia from other macrophages, have not been yet identified. In the present work, we used gene array analysis to compare the RNA profiles obtained from microglia versus bone-marrow derived macrophages (BMM). Microglial cells isolated from mixed glial cell cultures were driven toward a “resting” state by growing them for 4 days in the presence of a glial cell conditioned medium. In parallel experiments, BMM were generated by stimulating bone-marrow derived myeloid progenitors with M-CSF, for 4 days. After a control of RNA quality (BioAnalyzer 2100, Agilent), double amplification process was performed before cDNA synthesis. The expression of 20,000 genes was then assessed on a CodeLink plateform from Amersham and data were analyzed with Genespring software from Silicon Genetics. To validate our results, genes of interest were selected and their relative level of expression was evaluated by real-time RT-PCR. A number of immune-related genes were identified including genes coding for MHC class II molecules (up-regulated in macrophages), the IL-10 receptor (up-regulated in microglia,) or the erythropoietin receptor (up-regulated in microglia). However, we also identified genes that were not related to immune functions. In particular, several genes involved in lipid metabolism were found to be specifically expressed by microglia. PP02-10

Characterization of the phagocytic activity of mouse primary microglia towards apoptotic glioma cells D. Nicklesa, A. Abschuetza, R. Geibiga, E. Spiessb, A. Régnier-Vigourouxa a

INSERM U701-DKFZ, Heidelberg, Germany; Germany

b

DKFZ, Heidelberg,

Microglia ingest various pathogens as well as apoptotic T cells or neurons. This potent phagocytic capacity has hardly been analysed in the context of brain tumour where it could serve the purpose of removing dying but also living tumour cells, as described for macrophages. We herein report on quantitative studies, based on flow cytometry assays, and qualitative studies, using time-lapse video microscopy, that we conducted with primary cultures of microglia derived from three mouse strains and three different glioma cell lines. The phagocytic capacity of microglia was evaluated by determining their phagocytic rate (percent of microglia from a total population that phagocytosed tumour cells within a given time). Microglia from either BALB/c, C57BL/6 and VM/Dk strains all phagocytosed etoposide-treated, apoptotic glioma cells. The phagocytic rate varied between 30% and 70% according to the strain but not to the glioma cell line used. No significant uptake of living tumour cells was observed. Beside the known induction of microglial toxic activity, LPS and interferon-gamma stimulation increased the phagocytic rate, save for VM/Dk-derived microglia. As expected, amoeboid microglia present in the unstimulated population were the only phagocytes, whereas upon stimulation this activity was also observed for rod-shaped cells. Immune stimulation enhanced motility and exploratory behaviour. Stimulated microglia not only killed tumour cells but also phagocytosed dead cells present in their close vicinity. Altogether our data indicate that microglia can phagocytose apoptotic but not living tumour cells. Current work focus on the identification of receptors and ligands mediating microglia–glioma cell recognition.

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PP02-11

TIMP-1 regulation in astrocytes during chronic neuroinflammation J. A. Gardner, L. Wu and A. Ghorpade Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA The pathogenesis of many neurodegenerative disorders is exacerbated by an imbalance between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). We previously reported differential TIMP-1 expression in acute versus chronic astrocyte activation, and in brain tissue of patients with HIV-1-associated dementia (HAD). We now investigate possible mechanisms underlying chronic TIMP-1 downregulation in neuroinflammation. We used interleukin (IL)-1β as a model proinflammatory stimulus and measured mRNA stability in U87 astroglioma cells and transcriptional regulation in activated astrocytes. The half-life of TIMP-1 mRNA was quantified using real time PCR to determine the effects of IL-1β stimulation on the stability of TIMP-1 transcripts. TIMP-1 promoter activation was measured using TIMP-1-luciferase reporter constructs in transfected astrocytes. Our results demonstrate that enhanced TIMP-1 mRNA stability may lead to increased TIMP-1 levels following acute activation. However, chronic downregulation of astrocyte-TIMP-1 is likely through promoter regulation and loss of mRNA stabilization. Furthermore, other proinflammatory cytokines including tumor necrosis factor (TNF)-α and interferon (IFN)-γ, along with HIV-1, enhance the effects of IL-1β on astrocyte-TIMP-1 promoter regulation. The minimum TIMP-1 promoter sequence demonstrated the strongest downregulation in promoter activity following activation of transfected astrocytes, suggesting the location of a silencer element. These data are important for unraveling the mechanisms underlying astrocyte responses during chronic inflammation and have broader implications in other inflammatory diseases that involve MMP/TIMP imbalance. PP02-12

Double-stranded RNA induces the expression of retinoic acidinducible gene-I in cultured astrocytes T. Imaiuzmi, H. Yoshida and K. Satoh Department of Vascular Biology, Hirosaki University School of Medicine, Hirosaki, Japan Chemokine generation in astrocytes may be implicated in immune reactions in the central nervous system. Retinoic acid-inducible gene-I (RIGI) is a cytoplasmic signaling molecule that plays a role in innate immunity against viral infections. We have studied the effect of double-stranded RNA on the expression of RIG-I in astrocytes. Polyinosinic-polycitydylic acid (poly IC), authentic double-stranded RNA that mimics viral infection, was found to induce the RIG-I expression in an astrocytic cell line, U373-MG. Poly IC also induced the expression of CCL5, a CC type chemokine, and the inhibition of RIG-I expression by RNA interference resulted in reduced CCL5 expression. We conclude that RIG-I may be involved in innate immunity against viral infection in the central nervous system, in part, by regulating the expression of CCL5 in astrocytes.

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Poster Session 3: BBB/Homing

Abstracts PP03-01

Activation of Peroxisome Proliferator-Activated Receptorgamma (PPARγ) decreases monocyte migration across Blood Brain Barrier (BBB) in a Rho dependent manner 1,2

S. H. Ramirez, Y. Persidsky

1,2

D. Heilman,

1,2

R. Potula,

1,2

J. Haorah and

1,2,3

1

Center for Neurovirology and Neurodegenerative Disorders, Departments of 2Pharmacology/Experimental Neuroscience and 3Pathology/ Microbiology, University of Nebraska Medical Center, Omaha, NE 681985215, USA HIV-1 caused neurological impairment is associated with migration of virus-infected monocytes across BBB and macrophage accumulation in the brain. While PPARγ (the nuclear ligand-responsive transcription factor) is known as a mediator of pleiotropic anti-inflammatory pathways, its contribution to endothelial barrier function remains largely unknown. We tested effects of PPARγ stimulation on monocyte migration across BBB in vitro models (primary human brain microvascular endothelial cells, HMVEC, plated on porous membranes). Application of tumor necrosis alpha (TNF-α) and monocyte chemoattractive protein-1 (MCP-1), a relevant cytokine and chemokine known to increase during HIV-1 infection, augmented monocyte migration by 2.5-fold across BBB. Pretreatment of endothelial monolayers with rosiglitazone, a PPARγ agonist, diminished by ∼ 5-fold the migration of HIV-1 infected monocytes across BBB compared with untreated controls. Similarly, HMVEC pre-treatment with TNF-α increased adhesion of HIV-1 infected monocytes (3-fold), and adhesion was reduced by 42% after simultaneous application of rosiglitazone and TNF-α. Since our previous work indicated that monocyte–HMVEC interactions result in activation of Rho, small GTPase, we tested the idea that increased monocyte migration could be mediated by TNF-α induced Rho activation, and PPARγ agonist will prevent these effects. Indeed, TNF-α increased levels of Rho-GTP (active form), PPARγ activation reduces the levels of Rho-GTP to control levels. Taken together, these results suggest a novel role for PPARγ agonist in modulation of monocyte–endothelial interactions mediated in part through Rho activation. Utilization of PPARγ agonists approved for clinical use may offer new therapeutic approach in preventing BBB dysfunction seen in HIV-1 brain infection. PP03-02

Inhibition of leukocyte recruitment blocks seizures and epilepsy Paolo F. Fabene1, Graciela Navarro Mora1, Marianna Martinello2, Antonio Osculati3, Barbara Rossi2, Flavia Merigo1, Linda Ottoboni2, Ida Cwojdzinski1, Simona Bach2, Donatella Benati1, Pasquina Marzola4, Andrea Sbarbati1, Eugene C. Butcher5, Gabriela Constantin2 1

Department of Morphological–Biomedical Sciences, Section of Anatomy, University of Verona, Strada le Grazie 8, 37134 Verona, Italy; 2Department of Pathology, Section of General Pathology, University of Verona, Strada le Grazie 8, 37134 Verona, Italy; 3Department of Medicine and Public Health, Section of Forensic Medicine, University of Insubria, Via Rossi 9, 21100 Varese, Italy; 4Experimental MRI Center, Faculty of Medicine, University of Verona, Verona, Italy; 5 Department of Pathology (5324), Stanford University School of Medicine, Stanford, CA, 94305-5324, and Palo Alto VA Medical Center, 3801 Miranda Avenue (154B) Palo Alto, CA 94305, USA The pathogenetic link between repeated seizures and epilepsy is completely unknown and this prevents the implementation of successful therapies. The GOAL of this study was to determine a potential role for leukocyte recruitment in the pathogenesis of epilepsy. Methods: Epilepsy was induced by injection of C57Bl mice with pilocarpine. Intravital microscopy was

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performed in brain vessels postseizures. Immunofluorescence, immunohistochemistry, electron microscopy, magnetic resonance imaging, telemetry, behavioral evaluation and cognitive evaluation based on enriched open-field revealed were also performed. Results: In humans we observed granulocytes and T cells accumulation into the epileptic brains. We next verified whether leukocyte recruitment plays a role in the pathogenesis of a widely used experimental model of epilepsy. We found increased permeability, vasodilatation and expression of VCAM-1 after status epilepticus (SE) and repeated seizures. Electron microscopy and magnetic resonance imaging show that granulocytes and T cells migrate into the brain post-seizures. Intravital microscopy shows that blockade of alpha4 integrins and VCAM-1 inhibits granulocytes and Th1 lymphocytes rolling and arrest in brain vessels. Therapeutic treatment of mice with anti-alpha4-integrin mAb leads to a drastic reduction of seizures. Moreover, cognitive evaluation revealed that antialpha4 treated animals present a significant preservation of the behavior compared to epileptic mice. Strikingly, preventive administration of antialpha4integrin therapy, inhibits induction of SE and completely blocks recurrent seizures and development of epilepsy. Conclusion: The results demonstrate an unexpected key role for leukocyte recruitment in the pathogenesis of epilepsy and that anti-leukocyte adhesion therapy has preventive and therapeutic effect in a human model of epilepsy. PP03-03

BBB-crossing T lymphocytes of multiple sclerosis patients at acute stage are Th1 dominant T. Kandaa, S. Ishibashib, Y. Sanoa, M. Yamawakib and H. Mizusawab a

Department of Neurology, Yamaguchi University Graduate School, Ube, Japan; bDepartment of Neurology and Neurological Science, Tokyo Medical and Dental University Graduate School, Tokyo, Japan Background: Although the development of multiple sclerosis (MS) plaques is mainly driven by a Th1 type inflammatory response, the exact mechanism how such restricted populations of T lymphocytes can penetrate into CNS parenchyma is still obscure. Materials and methods: We developed a novel system to evaluate the characters of T lymphocytes attached on and intruded into and beneath human brain microvascular endothelial cell (BMEC) monolayer, using pseudo-3D analysis with confocal laser microscopy. Lymphocytes were obtained from healthy volunteers and MS patients at acute stage. CD4+ lymphocytes were selected and seeded on the TNFα-stimulated human BMEC monolayer. After 6 h, cells were fixed, immonostained, and then the proportion of CD4+CXCR3+ lymphocytes (representing Th1 cells) and CD4+CCR4+ lymphocytes (Th2 cells) (1) attached on the surface of BMEC monolayer and (2) migrated into and beneath BMEC monolayer was quantitatively analyzed using this system. Results: In healthy volunteers, lymphocytes migrated beneath BMEC monolayer were significantly Th2 dominant. Conversely, in MS patients at acute stage, most of the Th2 cells stayed on the surface of monolayer and migrated/intruded lymphocytes were significantly Th1 dominant. Conclusion: Intrusion of T cells into CNS parenchyma might be regulated at the blood–brain barrier and this mechanism is possibly impaired in acute MS patients. PP03-04

Astrocytes maintain blood–brain barrier integrity through the production of Angiotensin II Wosik K1, Devillers-Dodelet A1, Berthelet F2, Prat A1 1

University of Montreal, Neuroimmunology Unit, CHUM Research Center and 2Neuropathology Department, CHUM, Notre-Dame Hospital, Montreal, Canada

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Abstracts

The blood–brain barrier (BBB) restricts the entrance of blood-borne molecules and cells into the central nervous system (CNS), thereby regulating its homeostasis. Astrocytes ensheath brain endothelium and provide crucial factors for the maintenance of the BBB. Multiple sclerosis (MS) is characterized by a breach in BBB integrity. The object of this study was to determine which factors produced by astrocytes contribute to BBB function and integrity. Using human CNS-derived endothelial cells and astrocytes, we have demonstrated that astrocytes express Angiotensinogen (AGT) and produce Angiotensin II, while human brain endothelial cells (HBECs) express angiotensin receptors AT1 and AT2, both in vitro and in situ. The addition of astrocyte conditioned media, Ang II or AT receptor agonist to HBEC cultures induced an important decrease in monolayer permeability as well as specific changes in phosphorylation of the tight junction protein occludin and its concentration into lipid rafts, where tight junctions are assembled. Furthermore, this effect of astrocyte conditioned media could be blocked by the addition of AT1 but not AT2 receptor antagonist. The presence of inflammatory cytokines IFNγ and/or TNFα in the astrocyte cultures blocked their ability to produce Ang II. In addition, using brain sections from normal and MS patients we could demonstrate that blood vessel-proximal human astrocytes in normal brain express AGT and this expression is lost around brain endothelium in active MS lesions. We conclude from these studies that astrocyte-derived angiotensin II, acting via the AT1 receptor, is necessary for BBB integrity. PP03-05

The bioactivity of interferon-beta in Mx-congenic mice L. Dissing-Olesena, M. Wirenfeldta, M. Meldgaarda, M. Thaysen-Andersenb and B. Finsena

Department of Molecular Cell Biology and Immunology, VU Medical Center, Amsterdam, The Netherlands The blood–brain barrier (BBB) controls the entry of circulating molecules and cells into the brain and is characterized by the presence of endothelial cells connected by tight junctions. Inflammatory cell trafficking into the brain complicates several neurological disorders and conceivably depends on mechanisms that regulate tight junction opening. A detailed study of tight junction dynamics during immune cell diapedesis has been lacking. Therefore, we retrovirally expressed the tight junction protein occludin N-terminally tagged to green fluorescent protein (GFP) in rat brain endothelial cells. Confocal microscopy analyses revealed that GFP-occludin colocalized with the intracellular tight junction protein, ZO-1, and was completely absent at cell borders lacking apposing cells. Mutant cells retained endothelial properties including cell morphology, endothelial markers, proliferation rate and adhesive capacity. By means of live cell imaging studies we show that monocytes scroll over the brain endothelial cell surface towards cell–cell contacts, induce gap formation, which is associated with local disappearance of GFP-occludin, and subsequently traverse the endothelium in a paracellular fashion. Immunoblot analyses indicate that loss of occludin was due to protein degradation. Interestingly, occludin contains a putative extracellular MMP cleavage site and using the broad spectrum MMP-inhibitor BB-3103 we show that gap formation, loss of occludin and diapedesis were indeed dependent on the activity of matrix metalloproteinases. Our results provide a novel insight into the mechanism by which leukocytes traverse the BBB and illustrate that therapeutics aimed at the stabilization of the tight junction may be beneficial to resist a neuroinflammatory attack.

a

Medical Biotechnology Center, University of Southern Denmark, Odense, Denmark; bDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark Interferon (IFN)-beta is the first and still leading therapeutic intervention shown to change the cause of relapsing remitting multiple sclerosis. However, the mechanism of action of IFN-beta remains in question. The prevailing view is that the benefit from the treatment can be ascribed to IFNbeta's systemic effects. This study investigates the permeability of the blood–brain barrier (BBB) to IFN-beta and thereby, questioning if IFN-beta potentially has an effect within the CNS between relapses. Clinically, the type I IFN-induced MxA gene is used to monitor IFNbeta treatment. We used the mRNA transcript of the murine-counterpart, the Mx1 gene, as a biomarker for IFN-beta treatment of Mx-congenic mice. Using real-time PCR we showed a peak in the level of Mx1 mRNA in white blood cells 3 h after subcutaneous administration of human IFN-beta-1a compared to the denaturated protein and the constitutive level. Analysis of a dose–response relationship and the biodistribution of IFN-beta showed a clear relationship and increased levels of Mx1 mRNA in various organs including; heart, kidney, liver, lung, and spleen. Finally, the permeability of the BBB to IFN-beta was investigated by examining the level of Mx1 mRNA in the perfused CNS. We demonstrated an induction of high levels of Mx1 mRNA, which indicate that IFN-beta crosses the BBB. Conclusions on these data will be supported by in situ hybridisation. Supported by Warwara Larsen Foundation. PP03-06

Diapedesis of monocytes is associated with matrix metalloproteinase (MMP)-mediated occludin disappearance in brain endothelium Gijs Kooij, Arie Reijerkerk, Susanne M.A. van der Pol, Shadi Khazen, Christine D. Dijkstra, Helga E. de Vries

PP03-07

Highly purified lipoteichoic acid from Gram-positive bacteria induces in vitro blood–brain barrier disruption through glia activation: Role of pro-inflammatory cytokines and nitric oxide A.K. Bal-Pricea, M. Boveria, A. Kinsnera, V. Berezowskib, A. Lenfantb, C. Draingc, R. Cecchellib, M. Dehouckb, T. Hartunga and P. Prietoa a

European Centre for the Validation of Alternative Methods, Institute of Health and Consumer protection, European Commission Joint Research Centre, Via E. Fermi 1, 21020 Ispra (VA), Italy; bBlood Brain Barrier Laboratory, EA 2465, Universite of Artois, Faculté des Sciences Jean Perrin, Lens, France; cDepartment of Biochemical Pharmacology, University of Konstanz, POB MG55, 78457 Konstanz, Germany The co-culture of bovine capillary endothelial cells and rat primary glial cells was established as an in vitro blood–brain barrier (BBB) model to investigate the mechanisms by which the Gram-positive bacterial cell wall components lipoteichoic acid (LTA) and muramyl dipeptide (MDP) induced injury of BBB structure and function. We found that highly purified LTA disrupted BBB integrity in a concentration- and time-dependent manner indirectly, through glia activation. Low trans-endothelial electrical resistance (TEER) and high permeability to fluorescein isothiocyanate–inulin observed in the presence of LTA-activated glial cells were potentiated by MDP and could be reversed only when glial cells were activated with LTA at 10 μg/ml but not with higher LTA concentrations (30 μg/ml). Immunocytochemistry analysis revealed no evident changes in the distribution of the cytoskeleton protein F-actin and tight junction protein occludin and claudin after LTA treatment. However, the tight junction associated protein AHNAK clearly revealed the morphological alteration of the endothelial cells induced by LTA. LTA-activated glial cells produced nitric oxide (NO) and proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin1β (IL-1β) that contributed to LTA-induced BBB disruption, since the direct

Abstracts treatment of the endothelial monolayer with TNF-α and IL-1β increased BBB permeability, whereas the pre-treatment with antibodies against these two cytokines blocked LTA effects. Additionally, NO was also involved in BBB damage, since the NO donor itself (diethylenetriamine-nitric oxide adduct) increased blood–brain barrier permeability and inducible nitric synthase inhibitor (1400 W) partially reversed LTA-induced decrease of trans-endothelial electrical resistance. PP03-08

Altered adrenomedullin and endothelin-1 expression during EAE Barker Sa, Mongru Ra, Bolton Cb, and Paul Cc Queen Mary University of London, bWilliam Harvey Research Institute, London, cUniversity of West England, Bristol; UK

a

Loss of normal blood–brain barrier (BBB) function is an integral feature of the neuroinflammatory process in the human demyelinating disease multiple sclerosis (MS). Adrenomedullin (AM) acts as a vasodilator in many vascular beds including in the cerebral circulation. In vitro work has shown that the peptide can regulate BBB characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation. Indeed, we have shown that AM inhibits endothelial synthesis of the vasoconstrictor, endothelin-1 (ET-1). Furthermore, our preliminary studies demonstrated an elevation of AM during late acute disease in EAE. This study has more fully examined AM expression plus assessed ET-1 changes during EAE. AM and ET-1 levels mRNA were determined in the cerebellum, medulla-pons and spinal cord of Lewis rats inoculated for EAE at height of disease and in early recovery. RT-PCR was carried out on total RNA extracted from rat tissue. All tissues examined showed an increase in AM during acute disease compared to normal levels. AM mRNA remained altered into the recovery phase. The inverse relationship between AM and ET-1 was seen in selected tissues at both timepoints, but was not a feature across all areas, indicating that AM changes may not purely arise from the vascular bed during neuroinflammation. Altered synthesis of AM and ET-1 during neuroinflammatory disease may be of relevance to multiple sclerosis. PP03-09

Development of a physiologic cytokine-activated in vitro blood brain barrier for studying leukocyte diapedesis ex vivo Eroboghene E. Ubogu, M.D.a,c, Melissa K. Callahan, Ph.D.a, Barbara H. Tuckya, Richard M. Ransohoff, M.D.a,b a

Neuroinflammation Research Center, Department of Neurosciences, Cleveland Clinic Foundation, Cleveland, OH, USA; bThe Mellen Center for Multiple Sclerosis Treatment and Research, Department of Neurology, Cleveland Clinic Foundation, Cleveland, OH and cNeurology Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Objective: To develop a physiologic cytokine-activated in vitro blood–brain barrier (aIVBBB) model for studying chemokine-driven leukocyte migration during neuroinflammation. Methods: SV40 T-antigen immortalized human brain microvascular endothelial cells (THBMECs) were cultured to confluence and treated with various concentrations of TNF-α and IFN-γ for 24 h. CCL2 production, a marker of endothelial activation was measured by ELISA, and cell viability ascertained by phase contrast microscopy and Annexin V immunocytochemistry. Transendothelial electrical resistance (TEER) was measured in collagen-coated Transwell™ inserts. Following deduction of physiologic endothelial activation, intercellular adhesion molecule (ICAM)-

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1 and vascular cell adhesion molecule (VCAM)-1 expression were studied by flow cytometry, with fibronectin connecting segment (FN CS)-1 expression deduced by immunocytochemistry. Results: Following endothelial activation, CCL2 production doubled from ∼ 600 pg/mL at rest to 1200 pg/mL. 10 U/mL TNF-α and 20 U/mL IFN-γ (10:20 aTHBMECs) maximally produced CCL2, without compromise to cell viability. There was no change in TEER between the resting and 10:20 aTHBMECs (∼100 Ω cm2), with a progressive decline in resistance with higher cytokine concentrations due to loss of cell–cell contacts secondary to apoptosis-induced cell retraction and death. There was a 450% increase in ICAM-1 and 80% increase in FN CS-1 THBMEC surface expression, with minimal VCAM-1 expression at rest or following physiologic cytokine activation. Conclusions: The 10:20 aTHBMECs form a physiologic aIVBBB that recapitulates the in vivo cerebral microvascular inflammatory milieu. This model can be used to accurately assess chemokine-driven leukocyte transmigration ex vivo as a means of understanding mechanisms and devising potential therapies for neuroinflammation. PP03-10

Establishment of conditionally immortalized in vitro BNB models Yasuteru Sanoa, Fumitaka Shimizua, Toshihiko Maedaa, Masaaki Abea, Hiroto Nakayamaa, Tetsuya Terasakib, Masuo Obinatab, Ri-ichi Takahashic, Masatsugu Uedac and Takashi Kandaa Yamagachi University, Ube, Japan; bTohoku University, Sendai, Japan; YS New Technology Institute Inc., Shimotsuga-gun, Japan

a

c

Purpose: In autoimmune disorders of peripheral nervous system (PNS) such as Guillain–Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy, breakdown of blood–nerve barrier (BNB) has been considered as a key step in the disease process. BNB is also an obstacle for drug delivery in PNS disorders. Hence, it is important to know cellular property of peripheral nerve microvascular endothelial cells (PnMECs) constituting bulk of the BNB. Although many in vitro models of blood– brain barrier (BBB) have been established, very few in vitro BNB models have been reported. So, we established new in vitro BNB models using transgenic rat harboring temperature-sensitive SV40 large T-antigen gene (tsA58 rat) and investigated whether these models posses characteristics as BNB. Method: PnMECs and pericytes were isolated from sciatic nerves of tsA58 rat according to a previously described procedure. PnMECs were identified by the following four criteria: spindle fiber-shaped morphology, immunoreactivity to anti-factor VIII antibody, formation of tight junction and uptake of DiI-Ac-LDL. Pericytes are identified by their peculiar morphology and expression of α-smooth muscle actin. Results: We successfully isolated PnMECs and pericytes from tsA58 rat and these cells are conditionally immortalized nature. PnMECs showed spindle fiber-shaped morphology and expressed uptake of DiI-Ac-LDL, immunoreactivity to anti-factor VIII antibody and barrier properties. Consequently, we built complete in vitro BNB models comprised of PnMECs and pericytes. Conclusions: We established in vitro BNB models from tsA58 rat. These models might facilitate analyses for pathophysiology of autoimmune diseases of PNS, moreover, prediction of in vivo drug transportation into PNS parenchyma. PP03-11

Evidence for differential changes of interendothelial junctions in murine neurocysticerosis dependent upon CNS vasculature J.I. Alvarez1, J.M. Teale2

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Abstracts

1

University of Texas Health Science Center at San Antonio, San Antonio, USA; 2University of Texas at San Antonio, San Antonio, USA

The delicate balance required to maintain homeostasis of the CNS (central nervous system) is controlled by the BBB (blood brain barrier) which impedes influx of most compounds from the blood to the CNS. Upon injury (e.g., infection, inflammation) the BBB is disrupted compromising the CNS. BBB disruption has been represented as a uniform event. However, our group has recently shown in a murine model of neurocysticerosis (NCC) that BBB disruption varies depending upon the anatomical site/vascular bed being analyzed in the CNS. In this study further understanding of the mechanisms of BBB disruption were explored in blood vessels located in leptomeninges (pial vessels) and brain parenchyma (parenchymal vessels) by examining interendothelial junction proteins involved in the integrity of the BBB. Mice were infected intracranially with the parasite Mesocestoides corti and sacrificed at various times post infection. Different anatomical areas of infected brain were analyzed by three color immunofluoresence utilizing antibodies against tight junction (TJ) and adherens junction (AJ) proteins. The results showed increased permeability in vessels with structural alterations, disappearance, or apparent proteolysis of particular TJ and AJ proteins. The extent and timing of these changes differed between pial vessels of the meninges and vessels of the parenchyma (pial vessel disruption within days versus weeks for parenchymal vessels). To approach potential mechanisms, the expression and activity of individual matrix metalloproteinases (MMP) was evaluated by in situ zymography at different times post infection. The results indicated increases in MMP activity at sites of BBB disruption exhibiting leukocyte infiltration. Moreover, the timing of MMP activity in pial and parenchymal vessels correlated with the different kinetic breakdown of these vessels. Thus, BBB disruption is not a homogeneous process but MMP activity is likely to be a key element.

PP03-12

Characterising autoreactive T lymphocyte migration and infiltration — a bird's eye view K.R. Williams1, E.K. Mathey1, A. Silvestri2 , J.V. Forrester2 and C. Linington1 Department of Medicine and Therapeutics1 and Ophthalmology2, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB252ZD, Scotland UK Studies using central nervous system (CNS) antigen specific T cell lines engineered to express GFP have dissected the migratory pathways and activation of lymphocytes during adoptive transfer EAE, an animal model of MS. However these studies are hindered by the difficulty of identifying the initial sites of infiltration in such a complex target organ. To overcome this problem we have utilised another model of autoimmunity, Experimental Autoimmune Uveoretinitis (EAU), in which the target organ is smaller and the location of lymphocyte infiltration is easier to dissect. We generated retinal antigen specific GFP expressing T lymphocytes and tracked their migration to the eye in Lewis rats. We recovered cells from various lymphoid organs and examined their activation status. We characterised the infiltration of these cells into the eye, defining the precise locations of entry and studying macrophage infiltration and antigen presentation. Surgical manipulation and injection of soluble antigen enabled us to dissect the mechanisms further. Our results demonstrate that following transfer of activated GFP T lymphocytes organ specific accumulation was not observed until two days after transfer when the cells reappeared in the spleen in large numbers in a non-activated state. On day three the cells migrated via the blood (still non-activated) and started appearing in the eye where they strongly express activation markers. Activation occurs in the eye at the sites of initial infiltration (iris and ciliary body). This is in concurrence with published observations in EAE and demonstrates that these mechanisms are not specific to particular diseases.

Abstracts

Poster Session 4: Chemokine

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Abstracts

PP04-01

The homeostatic chemokine CCL19 is constitutively expressed in the CNS, up-regulated in multiple sclerosis lesions, and is correlated with intrathecal IgG production Markus Krumbholz1,2, Diethilde Theil3, Sabine Cepok4, Bernhard Hemmer4, Monika Hofbauer2, Cinthia Farina1, Tobias Derfuss1,2, Thomas Arzberger5, Inga Sinicina6, Caroline Hartle7, Jia Newcombe7, Reinhard Hohlfeld1,2, and Edgar Meinl1,2 1

Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, D-82152 Martinsried, Germany; 2Institute for Clinical Neuroimmunology, Ludwig-Maximilians-University, D-81377 Munich, Germany; 3Department of Neurology, Ludwig-Maximilians-University, D-81377 Munich, Germany; 4 Department of Neurology, Heinrich-Heine University, D-40225 Düsseldorf, Germany; 5Institute for Neuropathology, Ludwig-Maximilians-University, D-81377 Munich, Germany; 6Institute of Legal Medicine, LudwigMaximilians-University, D-80337 Munich, Germany; 7NeuroResource, UCL Institute of Neurology, 1 Wakefield Street, London WC1N 1PJ, UK Chemokines are crucial in immunosurveillance and in regulating immune cell trafficking to lymphatic tissue and sites of inflammation. This study focused on the homeostatic chemokines CCL19 and CCL21, which both bind to CCR7 and direct B cells, mature dendritic cells, and a subset of T cells to secondary lymphoid organs. We report that in the normal human brain CCL19 is transcribed, detectable in tissue lysates, and displayed on endothelial cells. In both active and inactive MS lesions CCL19 transcripts were elevated and strong CCL19 immunostaining of astrocytes and blood vessel endothelial cells was seen. In cerebrospinal fluid (CSF) from relapsing remitting and secondary progressive MS patients CCL19 was elevated and correlated with intrathecal IgG production. CCL21, the other CCR7 ligand, was not detected in control or MS CSF samples, and quantitative PCR and immunostaining showed only very low levels of this chemokine in MS lesions. Together, this study suggests a role of CCL19 in immunosurveillance of the CNS and maintenance of immune cells in MS brains. PP04-02

Chemokine-induced in vitro chemotaxis of mononuclear leukocytes in multiple sclerosis M. Jalosinskia and A. Glabinskia

migratory activity of monocytes from MS patients. Our results suggest that in active MS chemokine-induced migratory activity of lymphocytes is increased. This activity may be significantly diminished by treatment with methylprednisolone and mitoxantrone. PP04-03

CXCL1(GRO-alpha) and CXCL6(Granulocyte Chemotactic Protein-2) chemokines — An important factor in stroke neuroinflammation J. Losya, J. Zarembab a

Department of Clinical Neuroimmunology University School of Medicine, Poznan, Poland; bNeuroimmunological Unit Institute of Experimental and Clinical Medicine Polish Academy of Sciences, Poznan, Poland

Objective: Acute cerebral ischaemia induces local inflammatory reaction including expression of chemokines, which precedes relevant leukocyte infiltration contributing to tissue injury. The objective of the study was to test hypothesis that CXCL1 and CXCL6 chemokines, potent neutrophil chemoattractants, play a role in inflammation during early phase of ischaemic stroke. Methods: The CXCL1 and CXCL6 levels in the CSF and serum obtained during 24 h from 23 ischaemic stroke patients aged 72.2 +/− 10.8 years have been measured by ELISA. CSF and blood samples from 15 tension headache patients served as a control group. The neurological stroke severity was estimated with Scandinavian Stroke Scale (SSS) within 24 h of stroke (SSS1) and two weeks later (SSS-2). Results: CXCL1 and CXCL6 levels were significantly elevated in the CSF of patients with ischaemic stroke in comparison with controls (65.6 +/ − 22.3 pg/ml vs. 43.8 +/− 2.3 pg/ml for CXCL1 and 3.1 +/− 0.9 pg/ml vs. 1.8 +/− 0.7 pg/ml for CXCL6). Serum levels of studied chemokines did not differ from control values. CSF CXCL1 and CXCL6 levels correlated significantly with the neurological stroke severity within 24 h and after two weeks from the onset of stroke. This is the first such observation. Conclusions: Our results suggest that CXCL1 and CXCL6 chemokines may play a role in the inflammatory reaction during early phase of ischaemic stroke and CSF CXCL1 and CXCL6 levels are associated with stroke severity and have predictive value for short term stroke outcome. PP04-04

Chemokines and chemokine receptors in a model of central nervous system inflammation

a

Department of Experimental and Clinical Neurology, Medical University of Lodz, Lodz, Poland Active multiple sclerosis (MS) is characterized by the presence of perivascular inflammatory foci localized in the central nervous system (CNS). Inflammatory cells forming those foci migrate from the blood to the CNS. Several studies confirmed that chemokines and their receptors play an important role in that process. The major goal of this study was to analyze the migratory activity of subpopulations of peripheral blood mononuclear cells (PBMC) from the blood of MS patients stimulated by chemokine CCL5/RANTES. Moreover the impact of MS treatment with methylprednisolone (active MS) and mitoxantrone (progressing MS) on CCL5-induced chemotactic activity of PBMC subpopulations was analyzed. Chemotactic activity of mononuclear leukocytes was measured in vitro in Neuroprobe MBA96 chemotaxis chamber using fluorimetric reader. We observed that in active MS before any treatment in vitro chemotactic activity of lymphocytes after stimulation with CCL5 was significantly increased. Spontaneous migration of lymphocytes was similar in all studied groups. Treatment of MS with methylprednisolone and mitoxantrone diminished this activity to the level observed in control groups of patients with other neurological diseases (OND) and healthy controls (HC). This effect was dose dependent. We did not observe any significant changes in spontaneous and stimulated by CCL5

B. Bieleckia, R.M. Ransohoffb, A.R. Glabinskia a

Department of Experimental and Clinical Neurology, Medical University of Lodz, Poland; bNeuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, USA It is well documented that chemokines play an important role in development of inflammatory foci through regulation of cell trafficking within tissues. This multistep process occurs also within the central nervous system (CNS) in many pathological conditions. In a present study we have investigated the role of chemokines and their receptors in the development of CNS inflammation using previously described model of local CNS inflammation. In this model inflammatory focus develops after deposition of BCG (Bacillus Calmette–Guerin) into the brain parenchyma followed by subcutaneous peripheral injection of BCG four weeks later. Accumulation of inflammatory cells around the site of BCG deposition is followed by a subsequent myelin damage. Expression of inflammatory (CCL5) and constitutive (CCL19, CCL21, CX3CL1) chemokines as well as chemokine receptors CCR5, CCR7 and CX3CR1 was analyzed. We performed RNA expression analysis using quantitative RPA assay. For localization of protein

Abstracts expression we used immunohistochemistry. Pathological features of inflammation were confirmed by analysis of expression of many specific markers of inflammatory cell subpopulations. Within 7 days post peripheral challenge we have observed an increase of CCL19 expression in a hemisphere injected with BCG. Interestingly in 10 days post peripheral challenge subsequent increase in contralateral hemisphere was also observed. A similar pattern of expression was also observed for chemokine CCL5. In contralateral hemisphere increase of this expression was observed 2 weeks after peripheral challenge. Our results confirm that some chemokines and their receptors play an important role in the control of cell trafficking during CNS inflammation. PP04-05

Astrocyte-specific CXCL1 overproduction may be neuroprotective during CNS demyelination K. M. Omaria, and C. S. Rainea a

Department of Pathology, Albert Einstein College of Medicine, Bronx, USA

Factors responsible for oligodendrocyte survival, a feature observed around early and active multiple sclerosis (MS) lesions, are not fully elucidated. Existing evidence in rodents demonstrate that the chemokine, CXCL1, induces proliferation and inhibits chemotaxis of oligodendrocyte precursor cells (OPCs). To investigate the effects of CXC chemokines during the course of central nervous system (CNS) inflammatory responses, we generated CXCL1/GFAP double-transgenic (Tg) mice, which overexpress CXCL1 inducibly under the control of the astrocyte specific gene, glial fibrillary acidic protein (GFAP). Double (CXCL1/ GFAP)-Tg, single (CXCL1)-Tg and wildtype (wt) animals were sensitized with myelin oligodendrocyte glycoprotein (MOG) for experimental autoimmune encephalomyelitis (EAE). After onset of clinical signs, CXCL1 production was initiated by intraperitoneal doxycycline injection (500 μg/animal). Preliminary results show that double-Tg animals display much milder clinical disease. Histopathology revealed that although inflammation was present in spinal cord and optic nerve tissue of doubleTg, single-Tg, and wt control animals by 15 days post-immunization (d.p. i.), demyelination and axonal pathology (Wallerian degeneration, WD) were more prominent in the control mice. By 40 d.p.i., inflammation and demyelination were significantly diminished in double-Tg mice, compared to controls, and WD was markedly less. Interestingly, relative to the levels of myelin and axonal pathology, remyelination was greater in the doubleTg group, and there was an associated increase in oligodendrocytes, suggestive of an oligodendroglial response. Ongoing protein expression studies are examining inflammatory infiltrates, OPCs, and proliferation (BrdU incorporation) within the CNS. Thus, preliminary findings suggest a role for CXCL1 in exerting a neuroprotective role during the course of EAE. Supported in part by NMSS RG 1001-K-1, NS 08952, NS 11920, NS 07098 and the Multiple Sclerosis Foundation. PP04-07

Transcriptional regulation of CCR5 in astrocytes and microglia H.F. Kuipersa,b, P.J. Biestaa, H.M. Feenstrab, E.J. Montagneb, E.S. van Haastertb, P. van der Valkb, and P.J. van den Elsena,b a

Leiden University Medical Center, Leiden, The Netherlands; University Medical Center, Amsterdam, The Netherlands

b

VU

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Several chemokines and chemokine receptors, including CCR5, are implicated in the pathogenesis of multiple sclerosis (MS). We show that expression of CCR5 is enhanced in MS lesions of various disease stages. Earlier, we have shown that MS lesions are hallmarked by enhanced expression of “stress-response” transcription factors such as IRF-1, NF-κB and CREB, leading to enhanced expression of both classes of MHC molecules. The expression of these molecules overlaps with the expression of CCR5 in MS lesions. Therefore, we investigated whether these factors are also involved in the transcriptional regulation of CCR5. Using promoter evaluation assays such as electrophoretic mobility shift assay and transient transfection, we determined that neither IRF-1 nor NF-κB is involved in the activation of the CCR5 promoter. Additionally, we show that these factors are not involved in the induction of transcription of endogenous CCR5 in human primary microglia and astrocytes, as determined by RT-PCR. In contrast, we show that CCR5 expression is regulated by the cAMP/CREB pathway. In addition, our data also suggest that epigenetic mechanisms might be implicated in the transcriptional control of CCR5. Taken together, we conclude that expression of CCR5 is regulated by several general, but distinct regulatory mechanisms and that alterations in these mechanisms could account for the aberrant expression of CCR5 in MS lesions. Supported by the Dutch MS Research Foundation (grant 00-407 MS/04543 MS). PP04-08

Differential chemokine expression after neuronal injury E.K de Jong, K. Biber, H.W.G.M. Boddeke Department of Medical Physiology, University Medical Center Groningen, University of Groningen, The Netherlands Chemokines are discussed to be involved in the signalling between damaged neurons and microglia. We have recently shown that the chemokine CCL21 is upregulated in neurons after cellular injury in vitro and in vivo (Biber et al., 2001; de Jong et al., 2005) and attracts microglia via the chemokine receptor CXCR3 (Rappert et al., 2002). In CXCR3 knockout animals microglial activation after injury was almost absent and survival of secondary neurons was much higher when compared to controls (Rappert et al., 2004). Neurons also produce the chemokine CXCL10 which is also a ligand for microglial CXCR3, raising the question why neurons would express two ligands for one receptor. Accordingly, we have investigated and compared the expression and distribution pattern of CXCL10 with that of CCL21 in cultured neurons after glutamate treatment. Both chemokines are packed in vesicles. However, whereas CCL21 protein is induced only after neuronal damage, CXCL10 is expressed constitutively at high levels and is not regulated by neuronal damage. Furthermore, CCL21 is released from damaged neurons, whereas we did not yet find conditions that would induce the release of CXCL10 in damaged neurons. These results show for the first time that two neuronal chemokines that act on the same receptor in microglia are differentially regulated by neurons. Whether both CCL21 and CXCL10 are involved in neuron microglia signaling and whether both chemokines might induce different effects in microglia remains to be established. Biber K, et al., (2001) Glia. 34, 121–33. Rappert A, et al., (2002) J. Immunol. 169: 3221–3226. Rappert A., et al., (2004) J. Neurosci. 24: 8500–8509. de Jong EK, et al., (2005) J. Neurosci. 25:7548–7557.

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Abstracts

Poster Session 5: Immunology

Abstracts PP05-01

A novel role of Class IV semaphorin Sema4D/CD100 in the neuroinflammation Tatsusada Okuno1,2, Yuji Nakatsuji1⁎, Atsushi Kumanogoh2, Masayuki Moriya1, Hitoshi Kikutani2, and Saburo Sakoda1 1

Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan; 2Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Objective: A glial reaction associated with the up-regulation of inducible nitric oxide synthase (iNOS) has been suggested to play an important role in an inflammatory process of neurodegenerative diseases. However, the mechanism how iNOS is induced in glial cells is not clearly understood. To clarify the mechanism of the induction, we focused on class IV semaphorin Sema4D/CD100 which is known to enhance CD40 signaling in B cells and dendritic cells in the immune system. Methods: The expression of Sema4D and its putative receptors, CD72 and Plexin-B1, on primary neural cells was investigated by RT-PCR and Western blot analysis. The effect of Sema4D on the induction of iNOS was investigated by incubating primary astrocytes and microglia with recombinant Sema4D. Results: Sema4D and its receptors were expressed in primary neurons, astrocytes and microglia. CD40 or LPS induced iNOS expression in astrocytes and microglia was increased by the incubation with recombinant Sema4D. This enhancement was also observed in glial cells prepared from mice deficient for CD72. Sema4D enhanced the ERK1/2 activation by CD40 stimulation and an inhibition of ERK1/2 pathway with U0126 partially decreased iNOS expression in microglia. Conclusion: Sema4D/CD100 and its receptors CD72 and Plexin-B1 are expressed on neural cells and could exacerbate neuroinflammatory process by enhancing iNOS expression. Plexin-B1 at least in part participates in this process via ERK1/2 pathway. PP05-02

Temporal genetic pathways in thymic selection reveal shared regulatory networks with neuronal degeneration and apoptosis A. Kauffmannb, F. Jamboua, P. Affaticatia, C. Guerrae, T. Robertc, I. KlingelSchmitta, R. Liblaud, M. Perrièref, P. Dessenb, V. Lazarc, and S. CohenKaminskya a

CNRS UMR8162, IPSC-IFR13, Université Paris-Sud, Hôpital Marie Lannelongue, Le Plessis Robinson, France; bCNRS FRE 2939, Institut Gustave Roussy, Villejuif, France; cUnité de Génomique Fonctionnelle, Institut Gustave Roussy, Villejuif, France; dINSERM U563, CPTP, CHU Purpan, Toulouse, France; eIngenuity Systems Inc., Mountain View, California, 94043, USA; fAgilent Technologies France, 91745 Massy; France Given their shared requirements for the generation of complexity, flexibility, plasticity, memory and specificity, the brain and immune systems might have more in common than first meets the eye. We have recently demonstrated that L-glutamate, a major excitatory amino acid in synaptic transmission in the nervous system, acts as an immunotransmitter at the immunological synapse formed between T and dendritic cells, contributing to calcium mobilization and T cell apoptosis during thymic negative selection. To further explore the compared physiology between the immune and nervous system, we combined genome-wide expression analysis with a new bioinformatics method to identify functional networks involving Nur77 in thymic negative selection. We used Ingenuity Pathway Analysis, a new web-based entry tool, which provides molecular networks with a high degree of connectivity over all genes documented in the

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literature. A systematic temporal expression profiling of thymocytes from TCR transgenic mice undergoing thymic negative selection was generated using Agilent oligonucleotide microarrays. The level of expression of a 44,000 probe set representing around 22,000 mouse known genes was monitored with 9 kinetics time points over a 24 h period, with a focus on 6 early time points over 3 h. Dynamically regulated genes identified using Resolver software were overlaid onto the cellular pathway maps in the Ingenuity Pathway Analysis software application to generate very significant high-scoring networks, including Nur77 in the early time points. In addition to documenting genetic pathways involving Nur77 in thymic negative selection, our findings provide new insights into the regulation of thymocyte apoptosis as it relates to neuronal apoptosis and neurodegeneration. PP05-03

Astrocytes as regulators of human CNS inflammation Alex Kostianovsky and David E. Anderson Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, USA A growing body of literature indicates that resident glial cell populations can modulate immune responses within the central nervous system (CNS). Using primary human astrocytes and ex vivo human monocytes (representative of microglia), we report that astrocytes modulate monocyte activation depending on the nature of the inflammatory stimulus. When monocytes are co-cultured with primary astrocytes in the presence of LPS, monocyte secretion of TNF-α is completely inhibited, whereas there is no inhibition when CD40 ligand is the stimulus. These data suggest that within the human CNS, pathogenmediated inflammation (LPS) is, at least in part, naturally inhibited by astrocytes, whereas T cell-mediated inflammation (CD40 ligand) is not. This observation has relevance to regulation of T cell-mediated inflammation within the CNS, including that associated with multiple sclerosis and responses to brain tumors. Indeed, using a transformed primary malignant glioma (Glioblastoma Multiformae or GBM) cell line, we have found that, in contrast to non-transformed primary human astrocytes, GBM tumor cells acquire the ability to suppress monocyte secretion of TNF-α in response to both LPS and CD40 ligand stimulation. Transwell assays indicate that both soluble and cell-contact dependent mechanisms are responsible for tumor cell-mediated suppression of monocyte activation, and neutralization studies exclude TGF-β, IL-10, COX2 and CD200 as molecules associated with this suppression. A highthroughput screening strategy is planned to identify the relevant pathway (s). Collectively, these results indicate that astrocytes have an inherent regulatory capacity within the CNS that is responsive to nature of the inflammatory stimulus. PP05-04

Neuronal antigen-containing antigen-presenting cells in cervical lymph nodes of multiple sclerosis patients and EAE animals are functionally distinct from myelin antigen-containing cells M. van Zwama,e, R. Huizingab,e, A. F. Wierenga-Wolfa,e, M. J. Meliefa,e, M. van Meursa,e, J. S. Voermana,e, K. P. H. Biberc, H. W. G. M. Boddekec, R. Q. Hintzend,e, B. A. 't Hartb,e, S. Amorb,e, J. D. Lamana,e and L. A. Bovena,e a

Department of Immunology, Erasmus MC, Rotterdam, The Netherlands; Department of Immunobiology, Biomedical Primate Research Centre, Rijswijk, The Netherlands; cDepartment of Medical Physiology, University Medical Centre Groningen, Groningen, The Netherlands; dDepartment of Neurology, Erasmus MC, Rotterdam, The Netherlands; eMS Centre ErasMS, Rotterdam, The Netherlands b

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Abstracts ⁎Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison Medical School, and ‡Department of Anesthesiology, University of Wisconsin, Madison, WI 53706, USA

Multiple sclerosis (MS) is characterized by demyelination and neuronal damage within the central nervous system (CNS). Transfer of CNS antigens to the draining cervical lymph nodes (CLN) likely is crucial in initiating or modulating immune responses against myelin, but also against neurons. Although myelin antigens have been demonstrated in CLN of MS patients, the presence of neuronal antigens in CLN of MS patients has not yet been determined. We here report the presence of neuronal antigens in CLN from MS patients, common marmoset monkeys, rhesus monkeys, and Biozzi ABH mice with EAE. Draining lymph nodes (lumbar lymph nodes and deep CLN) of cuprizone-treated mice, exhibiting extensive CNS demyelination with little neuronal damage, contained abundant myelin antigens but not the neuronal antigen neurofilament light. In human CLN, cells containing the neuronal antigen MAP-2 expressed the APC markers MHC class II and CD40, and the proinflammatory molecules IL-12p40/p70 and TNF-α. In contrast, the majority of cells containing the myelin antigen MOG expressed the anti-inflammatory molecules IL-1 receptor antagonist and TGF-â. 20% of MOG-containing cells expressed the lymph node homing molecule CCR7, whereas CCR7 expression was absent from MAP-2-containing cells. Using an in vitro model, we confirmed increased CCR7 mRNA expression by human monocyte-derived macrophages following human myelin ingestion in vitro. We conclude that neuronal antigen-containing APC are present in CLN during MS and EAE. We hypothesize that the specific immunophenotype of the APC may be caused by the nature of the phagocytozed antigen or the micro-environment at the location of antigen uptake.

The compartments in which critical accessory cell/T cell interactions take place prior to the onset of neuroinflammation have not yet been clarified. In this study we show that naïve T cells can be activated by local resident accessory cells in nervous tissue in the absence of peripheral immune organs. To define CNS-restricted naïve T cell activation, we prepared brain slice cultures from experimental animals and maintained these tissues in artificial cerebral spinal fluid for several days. One hour prior to the preparation of brain slice cultures, mice were injected intracerebrally with ovalbumin antigen. When naïve OT-1 ovalbumin-specific CD8+ T cells were added to these cultures, these T cells become activated, proliferated and migrated into the brain slices which contained cognate antigens. Brain resident dendritic cell-like accessory cells (CD11c+) were also mobile and able to migrate from the brain slices in the presence of inflammatory chemokine, MIP-3β. Proliferation of OT1 antigen-specific T cells and the migration of brain resident accessory cells were abrogated by the deletion of CD11c+ cells from Itgax-DTR/EGFP mice prior to the generation of brain slices. Due to the transgene expression, CD11c expressing cells can be selectively depleted (huDTR) and/or identified (GFP fluorescence) in these mice. Our results suggest that antigen presentation could take place locally in the brain and this event may be important in the initiation of CNS inflammatory diseases. This work was supported by the National Institute of Health (Grant RO1NS 37570-01A2 to Z. Fabry).

PP05-05

PP05-08

Cross-presentation of central nervous system-derived antigen induces robust priming of CD8+T cells in cervical lymph nodes

Metalloproteinases control brain inflammation induced by pertussis toxin in mice overexpressing the chemokine CCL2 in the CNS

Lisa Walter and Matthew L. Albert Institut Pasteur, Paris, France The efficiency of antigen presentation from the central nervous system (CNS) to initiate and induce antigen-specific cytotoxic CD8+ T cell immune responses remains unclear. To determine how presentation of antigen from the brain versus from other tissues differs in inducing a cytotoxic CD8+ T cell response, antigen was administered intracerebrally (IC), subcutaneously (SC), or intravenously (IV) into mice. Mouse splenocytes deficient in major histocompatibility complex class I H-2Kb and expressing the membrane-bound ovalbumin antigen (mOVA/H-2Kb−/ − ), were injected to monitor responses to cross-presentation of the H-2Kb restricted epitope, SIINFEKL. IC-introduced mOVA/H-2Kb−/− splenocytes induce higher in vivo CD8+ T cell killing, interferon gamma production, and proliferation than when administered SC or IV. CD8+ T cell priming from cross-presented IC antigen occurs in the cervical lymph nodes and requires CD4+ T cell help. In addition, CD8+ T cell priming due to crosspresentation, but not direct presentation, requires CD40 expression by the host. Taken together, these data imply that an immune response resulting from cross-presentation of IC antigen, once initiated, is more efficient and therefore more likely to induce immune priming compared with responses initiated elsewhere in the body. Similar to other tissues, antigen crosspresentation from the CNS requires CD40 expression by the host, presumably mediating helper-dependent cross-priming of CD8+ T cells specific to CNS-derived antigen. PP05-06

Antigen specific activation of naïve CD8 T cells by resident antigen presenting cells in the brain: The role of brain antigen presenting cells in the initiation of CNS inflammation Changying Ling⁎, Yakov Verbney‡, Matt Banks‡, Matyas Sandor⁎, and Zsuzsanna Fabry⁎

H. Toft-Hansena, R. Buistb, X.-J. Sunb, A. Schellenbergb, J. Peelingb and T. Owensa a

Medical Biotechnology Center, University of Southern Denmark, Odense, Denmark; bDepartment of Radiology, University of Manitoba, Winnipeg, Canada

Leukocytes accumulate spontaneously in the perivascular space in brains of transgenic (Tg) mice that overexpress CC chemokine ligand 2 (CCL2) under control of a CNS-specific promoter. The Tg mice show no clinical symptoms, even though leukocytes have crossed the endothelial basement membrane. Pertussis toxin (PTx) given intraperitoneally induced encephalopathy and weight loss in Tg mice. We used flow cytometry, USPIOenhanced magnetic resonance imaging and immunofluorescent staining to show that encephalopathy involved leukocyte migration across the glia limitans into the brain parenchyma, identifying this as the critical step in inducing clinical symptoms. Metalloproteinase enzymes (MPs) are implicated in leukocyte infiltration in CNS inflammation. Unmanipulated Tg mice had elevated expression of TIMP-1, MMP-10 and 12 mRNA in the brain. PTx further induced expression of TIMP-1, ADAM-12 and MMPs 8 and 10 in brains of Tg mice. Levels of the microglial-associated MP, MMP15 were not affected in control or PTx-treated Tg mice. PTx also upregulated expression of proinflammatory cytokines IL-1β and TNFα mRNA in Tg CNS. Weight loss and parenchymal infiltration induced by PTx were significantly inhibited by the broad-spectrum MP inhibitor BB-94/Batimastat. Our finding that MPs mediate PTx-induced parenchymal infiltration to the chemokine-overexpressing CNS has relevance for the pathogenesis of human diseases involving CNS inflammation, such as multiple sclerosis. PP05-09

Induction of tolerance for alpha B-crystallin in alpha Bcrystallin knock-out mice Richard Verbeek, Arianne Plomp, Hans van Noort Business Unit Biomedical Research, TNO Quality of Life, The Netherlands

Abstracts Previous data indicate that in humans a strong pro-inflammatory memory T-cell response exists against the CNS myelin antigen alpha Bcrystallin, which qualify it as a potential autoimmune target in multiple sclerosis. In our study we were able to specifically tolerize these proinflammatory memory T-cells for alpha B-crystallin in an animal model. Further efforts are made towards a clinical study. Since in contrast to humans, rodents are naturally tolerant for self alpha B-crystallin, the tolerization studies were performed in alpha Bcrystallin knock-out mice that are highly responsive to the protein antigen and that lack all form of naturally tolerance for alpha B-crystallin. Tolerance-inducing protocols were started 3 weeks after immunization with alpha B-crystallin in CFA. Tolerance was induced by administering intravenous alpha B-crystallin in the tail vein in various dose-timing regiments. Low dose induction of tolerance led to an almost complete abrogation of T-cell responsiveness in lymph node-derived T cells upon subsequent antigenic challenge. Although a limited induction of the T cell response was detected after in vitro re-challenge this was swiftly followed by a state of non-responsiveness. Even a boost with antigen in adjuvant after tolerance induction did not reverse the tolerant state. In contrast to the T-cell response, an increased antibody response was detected. Future efforts will be made to study the tolerance inducing mechanisms such as anergy or regulatory T-cells and the role of antibodies.

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Section of Immunology, Department of Mechanism of Aging, National Institute for Longevity Sciences, Obu, Japan Advancing age is associated with significant alterations in immune functions, including a progressive decline in CD4 T function, in both mice and humans. However, it has been unknown whether regulatory/suppressive CD4 T cells are involved in this decline. Our in vitro analyses revealed that CD4+CD25+ T cells, the well-characterized naturally occurring regulatory/ suppressive CD4 T cells, in aged mice are functionally comparable to those in young mice (i.e., anergic and suppressive), though slightly increased in number. In contrast, functional changes to whole CD4+CD25− T cells were pronounced in aged mice. We separated CD4 T cells into small subpopulations based on the staining with Rhodamine-123 (R123). Cell populations rapidly extruding R123 (referred to as R123lo cells), the majority of aged CD4+CD25− T cells, exhibited a significant hyporesponsiveness, and the remaining cells (CD4+CD25−R123hi T cells) maintained a normal responsiveness. Furthermore, we identified Foxp3 (a transcription factor critical in conferring the regulatory/suppressive function to CD4 T cells)-positive suppressive CD4 T cells among aged hyporesponsive CD4+CD25−R123lo T cells. These results suggest that the age-related decline in T cell-mediated immune responses is ascribable to changes in the CD4+CD25− T cell population, and not to a functional augmentation of suppressive CD4+CD25+ T cells. PP05-12

PP05-10

Immature human dendritic cells express latency-associated peptide (LAP) and inhibit Th1 polarization in a TGF-β dependent fashion R. Gandhi, D. E. Anderson and H. L. Weiner

Lack of VLA-4 expression defines functional immunesuppressive Treg cells in humans Diletta Di Mitri1, Giovanna Borsellino1, Markus Kleinewietfeld2, Adamo Diamantini1, Raffaella Giometto1, Roberta Placido1, Fabiola Puentes2, Luca Battistini1, Olaf Rötzschke2 and Kirsten Falk2 1

Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115 USA Multiple sclerosis (MS) is a chronic Th1 cell mediated inflammatory disease of the CNS. We investigated the role of dendritic cells (DCs) in MS as cells important for initiating an immune response and maintaining peripheral tolerance and found DC activation in the blood of MS patients (J Immunology, in press). The exact mechanism by which DCs are instructing/influencing the generation of regulatory versus effector T cells is not clear. TGF-β is an important immunoregulatory molecule and is implicated in the induction of regulatory T cells. We found that TGF-β is present on the surface of immature human DCs in the form of latency associated peptide (LAP). FACS analysis of human ex-vivo isolated DCs showed membrane bound LAP. The functional importance of surface TGF-β on DCs was assessed by neutralization with recombinant LAP or anti-TGF-β antibody. Such neutralization resulted in enhanced T cell proliferation and IFN-γ secretion by allogeneic CD4+ T cells incubated with ex-vivo isolated human DCs. Furthermore, when DCs were matured by stimulation with LPS so they were no longer tolerogenic, we observed the loss of membrane bound LAP, accompanied by up-modulation of HLAII and co-stimulatory molecules CD80, CD83, CD86 and CD40. Furthermore, initial results demonstrate altered tolerogenic properties of DC subpopulations in MS. Taken together, our results indicate that expression of TGF-β on DCs in the form of LAP may play an important role in the tolerogenic properties of immature DCs, which serve to limit or prevent autoimmune responses and this may be abnormal in MS.

Laboratory of Neuroimmunology, Fondazione Santa Lucia, Rome, Italy; Max-Delbrück-Center for Molecular Medicine, Berlin, Germany

2

Migration and homing is largely controlled by adhesion molecules. We therefore compared expression of various integrins on CD25high regulatory T cells (Treg) and on CD25low effector CD4+T cells. On human PBMC a striking difference was observed for CD49d, the ?-chain of VLA-4 (α4β1). CD49d was highly expressed on the CD25low effector T cells but was down-regulated on most CD25high cells. The differential expression pattern was evident especially on the CD45RO+CCR6+ effector memory-like T cell subsets, where the loss of VLA-4 was compensated on Treg cells by the expression of CLA (cutaneous lymphocyte antigen). CD45RO+CCR6+ Treg cells therefore bind efficiently to the CLA-ligand E-selectin, whereas CD4+ effector T cells preferentially adhere to the VLA-4 ligand VCAM-1. At least in principle, invasion of inflamed tissues can therefore be regulated in a subset-specific way by selective expression of these ligands at the endothelial barrier. Notably, on CCR6+CD25high cells the presence of CD49d correlates with an apparent loss in activity. Foxp3 expressing CD49dhighCD25high cells, present in PBMC of healthy donors as a minor subset, showed only marginal suppression whereas CD49d-CD25high cells efficiently inhibited the proliferation of CD4+CD25− T cells. Thus, the differential expression of VLA-4 and CLA not only allows to selectively recruit effector-memory like Treg (TREM) and conventional effector memory cells (TEM), the absence of CD49d can also be used as key-marker to distinguish functional immune-suppressive Treg cells from other less active Foxp3+ T cells. PP05-13

PP05-11

CD4 CD25 Foxp3 T cells and CD4 CD25 Foxp3 T cells in aged mice

Control of IL-5 production in CD1d-reactive human CD4+ NKT cell clones from MS patients following exogenous IL-2 co-stimulation

T. Nishioka, R. Iida and J. Shimizu

K. Sakuishiab, S. Miyakea, T. Yamamuraa

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a

Department of Immunology, National Institute of Neuroscience, NCNP, Tokyo, Japan; bDepartment of Neurology, University of Tokyo, Tokyo, Japan

PP05-15

CD1d-restricted NKT cells can produce both Th1 and Th2 cytokines. Recently, NKT cells were found to have the potential to suppress the development of experimental allergic encephalomyelitis (EAE), opening up the possibility as a therapeutic target for multiple sclerosis (MS). However, how they naturally regulate immune responses in humans remains largely obscure. We have found that, although human CD4+NKT cell clones cocultured with CD1d+ antigen presenting cells (APCs) do not produce cytokines, by adding exogenous IL-2, a significant number of clones selectively and remarkably produced IL-5. Methods: Human CD4+CD1d-restricted NKT cell clones were established by stimulating fresh PBMC with α-galactosylceramide or OCH, its synthetic analogue, and then sorting for invariant TCR+/Vβ11+/CD4+/CD8− cells. The NKT cell clones were co-cultured with APCs with or without recombinant IL-2, and cytokines in the supernatant were evaluated. Results: When co-cultured with APCs, 8 out of 24 NKT cell clones (including those of MS patients) produced remarkable amounts of IL-5 but little IFNγ in the presence, but not in the absence, of IL-2. CD1d-expressing Hela cells as well as immature DCs served as good APCs, whereas mocktransfected Hela cells did not. CD4+ NKT cells freshly isolated from BALB/ c mice similarly showed a Th2 polarization pattern, supporting the physiological implications of our findings. Conclusion: Taken together, IL-2 plays a key role in triggering the Th2 polarizing potential of CD4+ NKT cells in response to weak endogenous antigen stimulation. We propose a novel IL-2 dependent NKT cell function in immune regulation.

W. Sato, T. Aramami, T. Yamamura

PP05-14

CCR2 as a marker for human T cells producing IL-17

Department of Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira-shi, Tokyo, Japan Recent studies have demonstrated that myelin-reactive IL-17-producing T cells would efficiently mediate the destructive autoimmune pathology of experimental autoimmune encephalomyelitis (EAE). Therefore, the IL-23-IL-17 pathway could be pivotal in the pathogenesis of multiple sclerosis. Although much needs to be learned about the characteristics of IL-17 producing T cells, CCL2 is known to augment the T cell production of IL-17. This allowed us to address if IL-17 producing T cells might express CCR2, the CCL2 receptor. Of interest, CCR2 is highly expressed within MS plaques and CCR2 knockout mice are resistant to EAE. When whole T cells, CCR2+ or CCR2− T cells purified from healthy donor blood were activated by PMA and ionomycin for 1 day, only CCR2+ T cells produced a large amount of IL-17. Both CCR2+ and CCR2− populations produced IFN-γ. In sharp contrast, CCR5+ T cells, but not CCR5-T cells, produced IFN-γ, whereas both of the populations produced IL-17 equally. Notably, most CCR2+ T cells coexpress CCR5, and only a minor population expresses CCR2 alone. We found that the minor CCR2+ CCR5− fraction was the main source of IL17. The CCR2 single positive cells also produced IFN-γ, but it was much less than that from CCR2+CCR5+ fraction. To conclude, we have identified CCR2+CCR5− T cells in the human blood as IL-17-producing T cells, which is consistent with that IL-17-producing T cells would develop via a lineage distinct from the Th1 lineage.

Natural killer T cell ligand OCH as a potential therapeutics for multiple sclerosis: Mechanism for OCH-induced Th2 polarization in vivo

PP05-16

Shinji Oki, Takashi Yamamura and Sachiko Miyake

Marielle Thewissen, Veerle Somers, Koen Venken and Piet Stinissen

Department of Immunology, National Institute of Neuroscience, NCNP, Tokyo, Japan

Hasselt University, Biomedical Research Institute and Transnationale Universiteit Limburg, School of Life Sciences, Diepenbeek, Belgium

Natural killer T (NKT) cells are one of the most potent immune modulators after stimulation with glycolipid ligand such as αgalactosylceramide (αGC) through a massive production of immunoregulatory cytokines including interleukin-4 (IL-4) and interferon-γ (IFN-γ). We have demonstrated that OCH, a sphingosine-truncated analogue of αGC, prevents a variety of experimental autoimmune disease models including experimental autoimmune encephalomyelitis (EAE) by inducing the preferential NKT cell-dependent IL-4 production and subsequent Th2 polarization in vivo. Biochemical and molecular biological analysis revealed that the specific feature of the glycolipid is due to its unstable capture on CD1d and a short-lived stimulation to NKT cells, which is insufficient for effective c-Rel induction followed by the lack of effective IFN-γ production. Intriguingly, OCH induces less IFN-γ production not only by NKT cells but also by natural killer (NK) cells, another major source of IFN-γ after glycolipid administration in vivo. Due to an insufficient primary IFN-γ production and CD40L expression by activated NKT cells, OCH exert marginal IL-12 production by dendritic cells, resulted in lower secondary IFN-γ induction by NKT and NK cells. Accordingly, the availability of IL12 in connection with microbial infection is one of the critical determinants for the cytokine profile after in vivo administration of OCH. Taken together, OCH has a unique property for selective IL-4 production by NKT cells without inducing IFN-γ production by NKT and NK cells. A proper application of OCH in vivo is beneficial for a NKT cell-targeted therapeutic intervention of Th1-mediated autoimmune diseases such as multiple sclerosis (MS).

The loss of CD28 expression on both CD4+ and CD8+ T cells has been described as a good biological indicator of aging of the immune system. Increased percentages of CD4+CD28null T cells have also been associated with a number of pathological conditions. We previously demonstrated elevated percentages of these cells in blood of a subset of healthy controls (HC) and patients with an autoimmune disease. However, the fraction of people with an increased percentage of CD4+CD28null T cells was higher in diseased individuals (48/160) as compared to HC (7/50). To unravel the role of CD4+CD28null T cells in autoimmune diseases some functional characteristics of these cells have been studied. Intracellular FACS staining demonstrated that a large proportion of the CD4+CD28null T cell subset produced IFNγ upon stimulation. The majority of these cells also contained intracellular deposits of cytotoxic molecules. In addition, CD4+CD28null T cells could be detected in cerebrospinal fluid of MS patients and synovial tissue of RA patients. To study antigen reactivity of CD4+CD28null T cells, an in vitro assay was developed. Peripheral blood mononuclear cells are cultured with an array of allo- and autoantigens like myelin basic protein and human collagen type II. Proliferation and cytokine production will demonstrate antigen reactivity. This assay is currently pending. CD4+CD28null T cells represent a cell subset with aberrant functional capacities which can be detected at the site of tissue damage in autoimmune diseases. Unraveling the antigen specificity of CD4+CD28null T cells will provide more insight into their role in the pathogenesis of autoimmune diseases.

The role of CD4+CD28null T cells in autoimmune diseases

Abstracts PP05-17

Endoplasmic reticulum-resident heat shock protein gp96 as an innate sensor of damage induced by tissue remodelling, stress and bacterial peptidoglycan

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chaperoning antigenic self peptides into the immune surveillance pathways, resulting in activation of autoreactive NKT clones with morphogenetic potential (supported by grants from Croatian Ministry of Science). PP05-18

B. Radošević-Stašića, I. Mrakovčić-Šutića, H. Jakovaca, D. Grebića, J. Tomacb and D. Rukavinaa Department of Physiology and Immunology; bDepartment of Histology and Embryology, Medical Faculty, University of Rijeka, Rijeka, Croatia

In vivo modulation of cellular and humoral immune response to tetanus toxoid by mesenchymal stem cells

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Tissue disintegration after injury leads in the endoplasmic reticulum (ER) to activation of adaptive pathways known as the ER stress response. It is directed to correction of unfolded proteins, to the activation of proteasome-dependent ER-associated degradation of the misfolded proteins or to activation of protein translation to modulate the polypeptide traffic into the ER. Since, in these events a crucial role plays gp96, which acts not only as a molecular chaperone but also as an adjuvant, able to induce the specific immune responses against tumours and some bacteria, in this study we analysed its role in conditions of: (1) normal growth, induced by partial hepatectomy, (2) in psychosocial stress without the tissue lesions and (3) after the treatment with bacterial peptidoglycan-monomer (PGM) and PGMZn. Tissue expression of Gp96 protein and mRNA was estimated in liver, thymus and spleen and the data were correlated with phenotype and cytotoxicity of hepatic and splenic mononuclear lymphatic cells (MNLC) against the syngeneic thymocytes, NK and LAK-sensitive targets. All procedures induced fast cytoplasmic overexpression of gp96 staining in hepatocytes, followed by surface expression of gp96 on MNLC and upregulation of gp96 mRNA in the spleen and in the thymus. Simultaneously, in the liver accumulated CD3intermediate/NK1.1+/CD69+ cells, while hepatic and splenic MNLC became highly cytotoxic against syngeneic thymocytes and YAC-1 and P815, implying that during the disturbance of morphostasis gp96 may serve as a natural adjuvant for

S Chiesa1, E Traggiai2 and A Uccelli1 1

Department of Neuroscience Ophthalmology and Genetic, Genoa, Italy; Institute G.Gaslini, Genoa, Italy

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Murine mesenchymal stem cells (MSCs) ameliorate experimental autoimmune encephalomyelitis (EAE), through the induction of peripheral T cell tolerance. Nevertheless, the effect of MSC on the physiological immune response to non self antigens such as tetanus toxoid (TT) is still largely unknown. Thus, we sought analysing the dynamic changes of the humoral and cellular response to TT upon MSCs administration. 1 × 106 MSCs have been injected intravenously in Balb-c mice at different time points following immunization with tetanus toxoid (TT). Thus, we analyzed at different time points the TT specific serum antibody titres and TT specific T cell proliferative response from the peripheral blood of Balb-c mice. Upon sacrifice, the frequency of antigen specific plasma cells, memory B cells and T cells have been analyzed from the bone marrow, lymph nodes and spleen. In the latter, the extent of germinal centres formation has been evaluated. Overall we show that in vivo administration of MSC affect both T and B cell response to a non self antigen such as TT but does not lead to a condition of immunodeficiency. These results are of pivotal importance due to the possible utilization of MSC for the treatment of autoimmune diseases such as multiple sclerosis.

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Poster Session 6: EAE-pathogenesis

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PP06-01

PP06-03

Enhanced pathogenicity of refolded recombinant human MOG in DA rats

Induction of adaptive regulatory T cells during recovery of EAE sensitized with PLP136–150 in SJL/J mice: The presence of suppressor epitope within PLP

K.L. De Graaf1, C. Breithaupt2, R. Weissert1

Youwei Lin, Sachiko Miyake, Takashi Yamamura Hertie Institute for clinical Brain Research, Tübingen, Germany; 2Research Center for Biosciences and Health, University of Salzburg, Austria

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Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system. Antibodies to myelin oligodendrocyte glycoprotein (MOG) are unique in their capacity of inducing demyelination and oligodendrocyte loss in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Although several recent studies highlight the importance of protein conformation for MOG autoantibody binding, most screenings for MOG antibodies in MS patients have not taken MOG conformation into account. We refolded bacterially produced HuMOG and compared pathogenicity of refolded HuMOG versus non-refolded HuMOG in DA rats. Whereas DA rats immunized with non-refolded HuMOG remained perfectly healthy, DA rats immunized with refolded HuMOG developed severe disease. No differences in MOG-directed T-cell responses could be observed between both groups. In contrast, MOG serum antibodies from both groups differed in their capacity of binding native MOG expressed on the surface of eukaryotic cells as measured by FACS. Thus, enhanced pathogenicity of refolded HuMOG in DA rats is probably due to the presence of conformational MOG antibodies upon immunization. These data underscore the importance of using correctly refolded HuMOG for studying MOG antibody responses in MS patients. PP06-02

Divergent function of antibodies, Fc receptors and complement in neuroinflammation E. Urich1, I. Gutcher1, L.M. Howard2, S.D. Miller2, M. Prinz3 and B. Becher1 1

University Hospital of Zuerich, Neuroimmunology Unit, Neurology Clinic, Frauenklinikstrasse 10, 8091 Zuerich, Switzerland; 2Northwestern University, Medical School, Department of Microbiology–Immunology, 303 East Chicago Avenue, Chicago, Illinois 60611, USA; 3University of Goettingen, Department of Neuropathology, Georg-August-University, Robert-Koch-Str.40, 37075 Goettingen, Germany

The precise role of B cells and humoral immunity in both Multiple Sclerosis (MS) as well as Experimental Autoimmune Encephalomyelitis (EAE) remains a subject of intense debate. While intrathecal immunoglobulin (Ig) production and Ig-deposition in inflammatory lesions is a hallmark of MS, mice deficient in B cells and Igs have been shown to develop severe EAE. On the other hand, mice deficient in FcRã, the signaling entity of activating Fc-receptors, are resistant to EAE-induction. To resolve this apparent paradox we generated mice deficient in both B cells and FcRã and induced EAE through immunization with both recombinant MOG1–121 protein or MOG35–55 peptide. The functional expression of FcRã on systemic accessory cells, but not CNS-resident cells, appears to be vital for the development of CNS-inflammation independent of lymphocytes. While these data clearly dismiss any involvement of B cells as mediators in EAE, we found that the injection of Abs directed against MOG drastically worsens EAE severity, inflammation and demyelination in MOG induced EAE. The action of such Abs is again FcR independent and relies entirely on complement activation, as they have no detrimental effect in C1q−/− mice. We conclude that Abs generated during the course of MOG-peptide or protein induced EAE are irrelevant for the pathogenesis of EAE, but that under certain conditions Abs are capable of driving demyelination in an FcRã-independent but complement-dependent fashion.

Department of Immunology, National Institute of Neuroscience, NCNP, Japan SJL/J mice are known to develop chronic, relapsing EAE with epitope spreading after immunization with PLP139–151. Although genetic factors were employed to account for the relapsing–remitting course of this EAE, we found that immunization of SJL/J mice with an overlapping peptide PLP136–150 induced monophasic EAE without relapse, indicating that peptide sequence used for primary immunization is critical for determining the clinical course. Whereas both immunodominant PLP peptides induced cross-reactive T cells, immunization with PLP136–150 tended to generate Th2-biased population, as compared to PLP139–151. More strikingly, mice in the recovery phase of PLP136–150-EAE were completely resistant against re-induction of EAE; whereas PLP139–151EAE mice were fully susceptible to EAE re-induction. Furthermore, we found that after recovery from PLP136–150-induced EAE, suppressor cells would emerge in the draining lymph nodes (LN) that could confer protection against EAE upon cell transfer. These suppressive LN cell populations contained a higher number of adaptive regulatory CD4+CD25+ T cells (Treg) that would co-express CD103 and CD69. Adoptive transfer experiment showed that CD103+CD69+ Treg cells induced by PLP136–150 immunization have the highest potency to suppress EAE in vivo. Our results indicate that the benign nature of PLP136–150 induced EAE may result from the induction of strongly suppressive CD103+CD69+ Treg cells. (Abbreviations: EAE = experimental autoimmune encephalomyelitis, PLP = proteolipid protein) PP06-04

Paralysis of CD4+CD25+ regulatory T cell response in chronic autoimmune encephalomyelitis Yoh Matsumoto, Hiroshi Sakuma, Kuniko Kohyama, Mie Nakajima, Il-Kwon Park Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan To elucidate the pathomechanisms of relapse and chronicity of multiple sclerosis, we examined the role of CD4+CD25+ regulatory T cells (Treg) in its animal model, chronic experimental autoimmune encephalomyelitis (EAE). Chronic EAE was induced in LEW.1AV1 rats by immunization with myelin oligodendrocyte glycoprotein (MOG) and the results obtained were compared with those from acute EAE. In chronic EAE, CD25+ T cells in EAE lesions in the central nervous system (CNS) decreased rapidly at the beginning of the disease, while these cells were maintained at high levels during the recovery from acute EAE. Consistent with this finding, the levels of Foxp3 mRNA in the CNS was significantly lower throughout the course of chronic EAE than that at the recovery of acute EAE. The number of Foxp3+CD4+CD25+ Treg and the levels of Foxp3 mRNA in the lymphoid organ were also significantly lower in chronic EAE. However, there was no difference in the regulatory function of individual CD4+CD25+ Treg cells isolated during the disease course between acute and chronic EAE. Furthermore, the adoptive transfer of CD4+CD25+ Treg that had been activated with anti-CD28 mAb (JJ316) delayed the onset of chronic EAE (after one transfer) and suppressed the development of the disease completely (after two transfers). These findings suggest that impairment of the CD4+CD25+ Treg response, but not the function of individual cells, is

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critical for the development of chronic autoimmune diseases and can be adjustable by autologous Treg transplantation. PP06-05

Natural killer (NK) cells exert cytotoxic effect on autoantigenspecific, encephalitogenic T cells in experimental autoimmune encephalomyelitis

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Traffic of CSF-circulating dendritic cells during experimental auto-immune encephalomyelitis Hatterer Eric, Davoust Nathalie, Belin Marie-Françoise and Nataf Serge INSERM U433, IFR 19, Faculté de Médecine Laennec, Université Claude Bernard Lyon1, France

Wen Xua,b, Hideo Haraa,c and Takeshi Tabiraa,c a

Department of Demyelinating Disease and Aging, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan; b Division of Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; c National Institute for Longevity Sciences, Obu, Japan

The mechanism of natural killer (NK) cell regulatory role in experimental autoimmune encephalomyelitis (EAE) was studied in SJL/J mice. In vivo experiments showed that NK cell depletion by anti-NK1.1 monoclonal antibody treatment enhanced EAE in mice. To investigate the mechanism, we cultured proteolipid protein (PLP)136–150 peptide-specific, encephalitogenic T cell lines, which were used as the NK cell target. Our results show that NK cells exert a direct cytotoxic effect on autoantigenspecific, encephalitogenic T cells. Furthermore, cytotoxicity to PLPspecific, encephalitogenic T line cells was enhanced by using enriched NK cells as effector cells. However, the cytotoxic effect of NK cells to ovalbumin-specific T line cells and ConA-stimulated T cells could also be detected with a lesser efficiency. Our studies indicated that NK cells play a regulatory role in EAE through killing of syngeneic T cells which include myelin antigen-specific, encephalitogenic T cells, and thus ameliorate EAE. PP06-06

Encephalitogenic CNS dendritic cells drive the activation of naïve self-reactive CD4+ T cells

It is now established that dendritic cells (DCs) are able to migrate from the CNS to the cervical lymph nodes, through a yet unidentified pathway. We showed that DCs injected into the CSF of rats target the B-cell follicles of cervical lymph nodes (1). Here, we explored the migratory behavior of CSFcirculating DCs under neuroinflammatory conditions. For this purpose, bone marrow-derived myeloid DCs were labeled with a fluorescent marker (CFSE) then injected stereotaxically into the lateral ventricles of EAE rats. Rats were sacrificed on day 1 or 8 following injections and brains, cervical lymph nodes and axillary lymph nodes were assessed for the presence of CFSE-labeled cells. In addition, intraventricular injections of fluorescent microspheres were performed in order to track endogenous CSF-circulating antigen presenting cells. We found that in the CNS, injected DCs localized in meninges, velums, Virchow–Robin spaces and in several periventricular areas. Similar results were obtained when tracking endogenous MHC class II+ cells with fluorescent microspheres. In the lymph nodes, CFSE-labeled DCs were evidenced in the B-cell zones of cervical but not axillary lymph nodes. Similarly, cells having engulfed fluorescent microspheres were detected in the B-cell zone of cervical but not axillary lymph nodes. Finally, using Western blot analysis, we observed that IgG recovered from cervical versus axillary lymph nodes differ qualitatively and quantitatively in their ability to recognize CNS antigens. Altogether, these results suggest that during EAE, CSF-circulating DCs may be responsible for the development of a preponderant B-cell response, which takes place within the cervical lymph nodes. (1) Hatterer E., Davoust N., Didier Bazès M., Vuaillat C., Malcus C., Belin M.F. and Nataf S. How to drain without lymphatics? Dendritic cells migrate from the cerebrospinal fluid to the B-cell follicules of cervical lymph nodes. Blood, 2006, 107; 806–12.

Samantha L. Bailey, Bettina Schreiner, Stephen D. Miller PP06-09 Department of Microbiology–Immunology, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL, USA Our recent findings show that CD11c+ DCs, but not macrophages (Mϕ) or microglia isolated from the CNS of SJL mice with proteolipid (PLP)178– 191 induced relapsing experimental autoimmune encephalomyelitis (REAE), induced the proliferation of naive (CD62Lhi) transgenic CD4+ T cells specific for the immunodominant myelin peptide PLP139–151 (139TCR) with antigens collected in situ. By laser sorting CNS DC subtypes to high purity at the peak acute phase of R-EAE, we demonstrate CD11b+ DCs induce 139TCR expansion 44 fold, CD11b−B220−CD8α+ DCs (DNDC) and CD11b−B220+ DCs induce 6 and 5 fold expansion, respectively, and Mϕ 2 fold expansion without peptide being added to the cultures. CNS DC driven 139TCR proliferation is MHC class II and B7-1/B7-2 dependant. Levels of IFNγ and IL-2 secreted by 139TCR cells are highest with CD11b+ DCs and DNDCs as activators. CNS Mϕ and CD11b+ DCs induce MIP-1α and RANTES, CD11b+ DCs and DNDCs MCP-1 and all 4 CNS APCs induce IP-10 production in the 139TCR cultures. 139TCR cells closely associate with CD11b+ DCs in CNS inflammatory foci in SJL:139TCR mixed bone marrow chimera (BMC) mice during peak acute R-EAE. 99% of CD11b− DCs and 82% of CD11b+ DCs in the CNS infiltrate from the blood in BMC mice. The 4 CNS APCs internalize PLP in the CNS. The abundance of DCs in the CNS correlates strongly with EAE severity. These data imply that discrete DC populations are recruited to the inflamed CNS that have distinct roles for driving naïve T cell activation in that organ. This work is supported by grants from the National Multiple Sclerosis Society (USA) and the National Institutes of Health.

Vaccination-induced autoimmunity in the absence of secondary lymphoid tissues M. Greter, P. Bargsten, B. Becher Institute of Neurology, Department of Neuroimmunology, University Hospital Zurich, Switzerland Subcutaneous (s.c.) vaccination delivers antigen (Ag) to local dendritic cells (DCs) which subsequently migrate through afferent lymphatics into draining lymph nodes (LNs) where they encounter lymphocytes recognizing their cognate Ag to initiate immunity. Experimental autoimmune encephalomyelitis (EAE) serves as the animal model for multiple sclerosis, a demyelinating autoimmune disease of the central nervous system (CNS). EAE can be induced by s.c. immunization with myelin antigens such as myelin oligodendrocyte glycoprotein (MOG). Secondary lymphoid tissues are widely held to be vital for the initiation of adaptive immunity. Here we demonstrate that alymphoplasia (aly) mice, which are characterized by the complete lack of lymph nodes (LNs) and peyer's patches, are resistant to EAE induced by s.c. immunization with MOG. However, the immunodeficiency results from the impact the aly lesion has on immunity but not from the lack of lymphoreticular structures. This led to the exciting understanding that vaccination induced autoimmunity of the CNS can develop autonomously and without dedicated immune structures. We clearly show that while B cell activation requires a lymphoreticular system and germinal center formation, T cell immunity functions independent of secondary

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lymphoid tissues. We demonstrate that in the absence of secondary lymphoid tissues the liver acts as a site where primary immunity can occur. Inflammation induced tertiary follicles are well described. However this is the first report indicating that vaccination induced fulminant autoimmunity initiated in the liver.

with rat chimeras and Indian ink loaded monocytes showed that the ED1+ cells in DA and Lew were mainly macrophages while in PVG and BN there was massive glial cell activation.

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Blockade of TREM-2 exacerbates experimental autoimmune encephalitis

Role of innate immunity in autoimmune disease: Toll-Like Receptor agonists can trigger autoimmune encephalomyelitis

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C. Buonsantia1, L. Piccioa1, M. Mariania, M. Cellab, S. Gilfillanb, M. Colonnab, P. Panina-Bordignona

N. A. Wolf, T. K. Amouzegar and R. H. Swanborg Bioxell SpA, Milan, Italy; b Department of Pathology, Washington University, St Louis, MO, USA

a

Wayne State University School of Medicine, Detroit, USA Infectious organisms may play a role in human autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), is induced in Lewis rats with rat myelin basic protein (MBP) peptide MBP68–86 in complete Freund's adjuvant (CFA). Although the role of mycobacteria is unclear, it is required for the induction of EAE. To investigate whether Toll-like receptor (TLR) agonists can replace mycobacteria in adjuvant, we immunized rats with MBP68–86 in incomplete adjuvant (lacking mycobacteria), but supplemented with TLR agonists. Rats immunized with either 100 μg MBP68–86 plus 100–300 μg cytosine– guanine dinucleotide-containing oligodeoxynucleotide (CpG), a TLR9 agonist, or MBP68–86 plus 50 μg lipopolysaccharide (LPS), a TLR4 agonist, did not develop EAE, although their T cells proliferated and secreted interferon-gamma when stimulated with MBP68–86. In contrast, rats immunized with 100 μg MBP68–86 plus 100 μg CpG and 50 μg LPS developed clinical EAE. Spleen cells proliferated and secreted interferongamma in response to MBP68–86, and secreted IL-12 in response to CpG or LPS, but not to MBP68–86, suggesting that the TLR agonists promote a Th1 response. Thus, a combination of CpG and LPS can substitute for mycobacteria for induction of EAE. We conclude that a combination of TLR agonists, in the presence of autoantigen, can trigger autoimmune disease via the innate immune system. (Supported by NIH NS06985-37 and NS 048070-02.) PP06-11

The innate immune response to adjuvants dictates the adaptive immune response to autoantigens M. Staykova, D. Linares, S. Fordham, G. Bartell, W. Cowden, D. Willenborg Neurosciences Research Unit, ANU Medical School, Canberra Hospital, Canberra, Australia DA and Lew rats are known as “susceptible”, PVG rats — as “semisusceptible” (30% of the females develop EAE) and BN rats — as “resistant”, based on the development of EAE after immunisation with myelin basic protein or spinal cord homogenate emulsified in complete Freund's adjuvant (MBP-CFA, SCH-CFA). We re-examined EAE susceptibility/resistance in these rat strains based on our knowledge that nitric oxide (NO) can affect generation of T effector cells and/or their transendothelial migratory properties. If another adjuvant is used – carbonyl iron (CI) which does not increase the systemic NO levels – all four rat strains develop acute EAE. There were no significant differences in lymph node and spleen percentages of T lymphocytes (CD4+ and CD8+), B lymphocytes and NK cells when SCH-CFA or SCH-CI were used. There was a significant difference between (DA and Lew) and (PVG and BN) in the macrophage/monocyte numbers, expression of CD80 and CD86 costimulatory molecules and iNOS activity. The cellular composition of CNS inflammatory infiltrates was also different: in DA and Lew the majority of the cells were lymphocytes with some ED1+ cells while in PVG and BN there was massive ED1 positivity. Experiments

1

L.P. and C.B. contributed equally to this work INTRODUCTION: Triggering Receptor Expressed on Myeloid cells-2 (TREM-2) is a stimulatory membrane receptor associated with DAP12, expressed primarily in myeloid cells. Little is known about the function of DAP12/TREM-2 in the central nervous system (CNS). However, a rare autosomal recessive condition, Nasu–Hakola disease (NHD) is associated with loss-of-function mutations in DAP12 and TREM-2. The brain pathology observed in NHD patients suggests that disruption of the TREM-2/DAP12 pathway leads to neurodegeneration with demyelination and axonal loss. The AIM of this study was to determine the role of TREM-2 in experimental autoimmune encephalomyelitis (EAE). Methods: TREM-2 protein expression was investigated using a newly produced monoclonal antibody (MAb) against the mouse TREM-2 receptor. EAE was induced in C57BL/6 mice with the MOG35–55 peptide. Blocking effect of anti-mouse-TREM-2 MAb was assessed using BWZ.36-TREM-2 cell reporter system. Results: TREM-2 protein expression was demonstrated in vitro on macrophages, microglia and dendritic cells. We report that TREM-2 was up-regulated in the spinal cord during both the early inflammatory and chronic phases of EAE. We also demonstrate that TREM-2 was highly expressed on microglial cells in the CNS during EAE. Blockade of TREM-2, obtained treating the mice with the anti-TREM-2 MAb in the effector phase of EAE, results in disease exacerbation with more diffuse CNS inflammatory infiltrates and demyelination in the spinal cord. Conclusions: Our data show a critical role for TREM-2 during inflammatory responses in the CNS and suggest that TREM-2 plays a role in the pathogenesis of autoimmune inflammatory/demyelinating diseases of the CNS. PP06-13

Type 1 interferon receptor (IFNAR)-dependent modulation of myeloid cell activation determines the course of experimental autoimmune encephalomyelitis Marco Prinz1, Hauke Schmidt1, Claudia Detje2, Alexander Mildner1, UweKarsten Hanisch1, Ralf Gold1, Burkhard Becher4, Wolfgang Brück1 and Ulrich Kalinke2 1

Department of Neuropathology, Georg August University, 37075 Göttingen, Germany; 2Department of Immunology, Paul Ehrlich Institute,63225 Langen, Germany; 3Institute for Multiple Sclerosis Research, Georg August University, 37075 Göttingen, Germany; 4 Department of Neurology/ Neuroimmunology Unit, Universitätsspital,8091 Zurich, Switzerland While treatment of MS patients with interferon-b (IFN-b) leads to a marked decrease in the exacerbation rate as well as to delayed sustained disease progression, the precise mechanisms of the beneficial effects of IFNβ are still enigmatic. In this study we show that type 1 interferon receptordeficient mice (IFNAR−/−) were highly susceptible to experimental autoimmune disease (EAE) and developed a more severe disease course with increased CNS inflammation, demyelination and lethality. Since IFNAR is expressed by most in the body, little is known about the actual

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target tissue during disease. To clarify this, we used Cre/loxP-mediated gene targeting to investigate the cell-specific function of IFNAR in vivo. Mice with a specific IFNAR deletion in the central nervous system (NestinCreIFNARflox/flox) revealed no differences in the clinical course and showed compatible tissue damage in the CNS compared to WT controls, indicating that type 1 interferons do not have a direct protective impact on the CNS. Interestingly, neither T cell (CD4−CreIFNARflox/flox) nor B cell (CD19−CreIFNARflox/flox)-specific IFNAR deletion influenced the clinical course and cellular composition of infiltrating cells. However, IFNAR deletion on macrophages/neutrophils (LysM-CreIFNARflox/flox) led to severe disease with an enhanced effector phase and increased disease lethality as seen in IFNAR−/− mice. Deletion of IFNAR in macrophages induced altered MHC class II expression and change of cytokine and chemokine production. In summary, we show that IFNAR triggering, specifically on myeloid cells, but not lymphocytes or CNS cells, is crucial for immunomodulatory effects of type 1 IFN during autoimmune CNS disease. PP06-14

demyelination is still unknown, since conventional BDNF knockout mice die prematurely. We applied the Cre/loxP system to generate mice with a conditional deletion of BDNF in the T-cell lineage (lckCre BDNFflox/− mice) or in myeloid cells (lysMCre BDNFflox/− mice). In these mice, experimental autoimmune encephalomyeltits (EAE) was actively induced with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55. Compared to control mice, MOG-EAE in lckCre BDNFflox/− mice is more severe (mean score on day 43 p.i.: 5.5 +/− 0.5 vs. 3.5 +/− 2.4 in controls). Histological analyses during the chronic disease phase revealed an increased inflammatory infiltration in lckCre BDNFflox/− mice. MOG 35–55 EAE in lysMCre BDNFflox/− mice was of similar severity in the chronic phase of the disease. The immune reaction in lysMCre BDNFflox/− mice was attenuated with a decrease in interferon-gamma production and reduced inflammatory infiltration by at least 60%. Despite less inflammation in chronic MOG-EAE, the extent of axonal damage was not different between lysMCre BDNFflox/− mice and controls. Our findings suggest a novel role of immune-cell derived BDNF in immunoregulation, but also underscore its importance as susceptibility factor for maintaining axonal integrity. These data further support the concept of neuroprotective autoimmunity. Supported by SFB 581 TPA1.

The effects of fibroblast growth factors 1 and 2 on Experimental Autoimmune Encephalomyelitis (EAE)

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Yang Mao-Draayera, Susan C. Buckinghamb, Hillel Panitcha, and Felix Eckensteina,b

Expression of suppressors of cytokine signaling (SOCS) in experimental autoimmune encephalomyelitis (EAE)

a

J. L. Berarda, B. J. Kerra and S. Davida

Department of Neurology, University of Vermont, Burlington, VT, USA; Department of Anatomy and Neurobiology, University of Vermont, Burlington, VT, USA

b

Objective: To investigate the roles of FGF1 and FGF2 in EAE progression. Background: FGFs are potent mitogens and glial cell activators that exhibit broad-spectrum neurotrophic activity. However, it is not known how FGF may affect EAE progression and cytokine profiles in EAE. Design/methods: C57BL6 wt, FGF1/2 double knockout and FGF1/2 heterozygote mice were immunized with MOG35–55 emulsified in CFA. Clinical scores were assessed daily. Serum was taken from day 21 animals immediately preceding euthanasia. Multiplex assays were performed to determine serum cytokine levels in knockouts and heterozygotes. Results: FGF-1/-2 knockouts developed significantly milder clinical symptoms compared to heterozygote and wt mice. Serum levels of IL4, IL10, and KC (CXCL1) levels were significantly higher in FGF1/2 knockout than those in heterozygote mice. Other cytokines including IL1, IL2, IL3, IL5, IL6, GM-CSF, IFN-γ, TNF-α, IL12, IL17, G-CSF, MIP-1α, and RANTES levels showed no significant differences. Conclusions: FGF1/2 knockout mice developed milder EAE than heterozygotes and wt mice, which correlates with serum anti-inflammatory cytokine (TH2) levels. Our findings suggest that Th2 cells are up regulated in the absence of FGF1 and FGF2. Gene expression levels for IL4, IL10, and other cytokines in the CNS are currently being measured.

a

Center for Research in Neuroscience, McGill University Health Center, Montreal, Canada

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system. MS predominantly follows a relapsing– remitting disease course, although a minority of patients develop a primary progressive form of disease. EAE is a widely used animal model of MS. EAE can follow either a relapsing–remitting (RR) or a chronic (CH) disease course. To date, the molecular and pathological differences that underlie these different forms of MS and EAE are not fully understood. We have assessed the expression of SOCS in the spinal cord in RR and CH EAE. Expression of SOCS-1 and SOCS-3 mRNA increases in the early stages of RR disease and decreases during the subsequent remission phase. In contrast, SOCS-1 and SOCS-3 mRNA expression is elevated at all stages of CH disease. As the expression of SOCS is turned on by pro-inflammatory cytokine signaling, these data suggest that the inflammatory response is prolonged in the CH form of EAE. Expression of SOCS at the protein level was assessed in the spinal cord by immunohistochemistry. Expression of SOCS-1 was localized to immune cells within EAE lesions, while SOCS-3 was predominantly expressed in glial cells adjacent to lesions. These results demonstrate that SOCS-1 and SOCS-3 are preferentially upregulated in distinct cell types during the course of EAE.

PP06-15 PP06-17

Neurotrophic factors and neuroinflammation: Experimental autoimmune encephalomyelitis in conditional BDNF-knock out mice R.A. Linker1, D. Lee1, F. Lühder1, N. Kruse1, S.Wiese2, M. Sendtner2, R. Gold1

Regulation of sonic hedgehog mediated neural stem cell differentiation in experimental autoimmune encephalomyelitis Yue Wang, Jaime Imitola, Stine Rasmussen and Samia J. Khoury Center for Neurologic Diseases, Harvard Medical School, Boston, USA

Institute for MS Research, Goettingen, Germany; 2Institute for Clinical Neurobiology, Wuerzburg, Germany

1

Brain derived neurotrophic factor (BDNF) is involved in neuronal and glial development and survival. It is mainly produced by neurons, but also by leukocytes in vitro and in multiple sclerosis (MS) lesions. Yet, the functional relevance of BDNF expression by immune cells in autoimmune

Objectives: To investigate the role of sonic hedgehog (Shh) signaling in neural stem cell (NSC) and precursor cell differentiation, and the effect of inflammation during experimental autoimmune encephalomyelitis (EAE) on this signaling. Methods: Expression of Shh, its receptor smoothened (SMO), and the transcription factor Gli1 was examined in the spinal cords of C57BL/6 mice

Abstracts with MOG-induced EAE by immunohistology; IFN-γ-treated NSCs were examined by real-time PCR and Western blot for Shh and Shh-induced Gli1 expression; effect of Shh on astrocyte-mediated NSCs differentiation was evaluated by anti-Shh antibody or control IgG treated NSCs — adult spinal cord astrocytes co-culture; IFN-γ-treated and untreated NSCs were compared for Shh-mediated differentiation in vitro. Results: Reactive spinal cord astrocytes upregulated Shh and SMO in EAE, especially around perivascular infiltrates and areas of demyelination, while Gli1 was downregulated. In vitro, NSCs treated with IFN-γ also downregulated Gli1. NSCs differentiated into neurons, astrocytes and oligodendrocytes when co-cultured with adult astrocytes, but anti-Shh significantly decreased the number and the maturity of neurons; Shhmediated NSC differentiation was significantly inhibited by IFN-γ, which was independent of cell death. Conclusions: Shh is an important mediator for adult astrocyte-induced NSC differentiation and it is upregulated in reactive astrocytes during EAE. However, IFN-γ may play a critical role in inhibiting Shh-mediated NSC and precursor cell differentiation by downregulating the Shh signaling molecule Gli1 in EAE. PP06-18

Endogenous prion protein regulates the inflammatory response and the extent of CNS damage in a murine model of multiple sclerosis S. Tsutsui, F. R. Jirik Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB Canada T2N 4N1 The role of PrPC in the pathogenesis of neuroinflammatory and neurodegenerative diseases remains elusive. However, a significant number of in vitro experiments have provided evidence that PrPC is neuroprotective, and moreover, PrPC is expressed on lymphocytes, macrophages and myeloid dendritic cells, suggesting that PrPC might potentially be playing a critical role in neuroinflammatory and neurodegenerative diseases. To determine the effects of reduced PrPC expression in a model of MS, experimental allergic encephalomyelitis (EAE), homozygous PrPC knockout (PrP−/−) mice and littermate wild type (PrP+/+) controls were treated with subcutaneous myelin oligodendrocyte glycoprotein (MOG35–55) to induce EAE. Animals were monitored for clinical signs and graded daily. Brain and spinal cord sections from euthanized animals were stained with H–E and silver. PrP−/− mice exhibited an earlier onset and a significantly more severe EAE phenotype than PrP+/+ controls in neurobehavioral score. During the EAE, H–E staining showed marked perivascular infiltration in brain. Silver staining showed the number of axons was reduced in spinal cord of PrP−/− mice compared to PrP+/+ controls. These studies suggest that lack of PrPC expression dysregulates the EAE inflammatory response, and might also render CNS cells more susceptible to injury during. Elucidating the role of PrPC in EAE will lead not only to a better understanding of neurodegenerative disease pathogenesis but also raises the possibility that alterations in prion protein levels or function might be of therapeutic benefit. PP06-19

Critical role for CD5-dependent CK2 activation signals in EAE: Attenuated disease is associated with diminished numbers of γIFN+IL17+ TH-cells in the CNS

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to MOG-peptide induced experimental autoimmune encephalomyelitis (EAE). The attenuated disease was a consequence of decreased ability of activated CD5−/− T-cells to persist. This led to the discovery that CD5 had an engagement-dependent prosurvival activity in T-cells. To test the prediction that the prosurvival activity was mediated by CK2, an anti-apoptotic serine/ threonine kinase that constitutively associates with CD5, we generated mice that expressed CD5 unable bind/activate CK2 using transgenic and “knockin” approaches. Remarkably these CD5-CK2 binding/activation-deficient mice were very resistant to development of EAE. Resistance was not associated with absence of T-cell infiltration in the CNS but rather due to decrease in TH-cells that coexpress both γIFN and IL-17. The newly described TH17 population of T-cells developed with equal efficiency in WT and CK2-binding/activation deficient CD5 mice. We further show that Tcells deficient in CD5–CK2 signaling hyperproliferate following primary stimulation, however, respond very poorly to restimulation compared to WTCD5 T-cells. Our results indicate that the THIFNγ-IL17 is likely to be an important pathogenic effector population in multiple sclerosis. We also show that the CD5–CK2 pathway is an attenuator of T-cell activation and cell death. The coordinate effect of these activities of CD5 is likely to be important in differentiation and persistence of effector T-cells in autoimmunity and immunity to pathogens. Supported by NIH and LRI. PP06-20

NF-kB activation and phosphorylation of IkB is associated with development and progression of the experimental autoimmune encephalomyelitis Insun Hwang, Taekyun Shin, Youngheun Jee Department of Veterinary Medicine, Applied Radiological Science Institute, Cheju National University, Jeju, 690–756, South Korea Development of autoimmune disease requires coordinated expression of a number of immune-related genes which mediate many inflammatory responses. A key player in regulation of inflammatory gene expression is the nuclear factor-kB (NF-kB) activated by phosphorylation and proteolytic degradation of inhibitory protein IkB. However, involvement of NF-kB of transcription factor in various immune-mediated diseases has been proposed but the direct supporting evidence is still lacking. To elucidate the roles of NF-kB and phospho-IkB in the development and progression of EAE, we investigated the expression of nuclear NF-kB (p65) and phospho-IkB in the central nervous system (CNS) of rats during experimental autoimmune encephalomyelitis (EAE). In Western blot analysis, phospho-IkB expressed rapidly at the early stage and increased significantly at the peak stage of disease. In addition, the expression of NF-kB increased at the early and peak stage followed by slight decline at the later stages. The expression of nuclear NF-kB and phospho-IkB was parallel to severity of EAE. Immunohistochemical studies showed that the NF-kB and phospho-IkB immunoreactivity was mainly expressed in inflammatory cells (macrophages, T cells) and glial cells (astrocytes, microglial cells) at the peak stage and disappeared at the recovery stage of disease. These findings suggest that the activation of NF-kB and phosphorylation of IkB is closely associated with autoimmune inflammation in the CNS and plays an important role in the initiation and progression of EAE. PP06-21

Anti-S-nitrosocysteine antibodies are not triggered by nitric oxide production

Chander Raman1, Robert C. Axtell1,2 and Scott R. Barnum2 A.I. Boullerne, A. Sharp, L. Liu and D.L. Feinstein Departments of Medicine1 and Microbiology2, University of Alabama at Birmingham, Birmingham, AL 35294, USA

University of Illinois at Chicago, Chicago, Illinois, USA

We recently reported that mice lacking CD5, a negative regulator of Tcell activation, unexpectedly exhibited delayed onset and decreased severity

An autoimmune antibody response against nitrosylated cysteine (SNOcysteine-BSA) was found elevated in plasma of multiple sclerosis patients

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Abstracts

and correlated with clinical activity (Boullerne et al. 2002 J. Neurosci. 22:123), suggesting that production of nitric oxide (NO) triggered an autoimmune response. To test this hypothesis, mice immunized to develop experimental allergic encephalomyelitis (EAE) were monitored for antibody responses and NO production in plasma. Wild-type C57Bl/6 and NOS-2 null mice were immunized with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG) followed a week later by a MOG booster and treatment with anti-inflammatory PPAR-gamma agonists. The strong increase of NO observed a week after immunization was lessened in low-dose treated mice and abolished in high-dose treated mice with no clinical signs. An increased antibody response was found two weeks after immunization in correlation with clinical signs arising a week later. In the absence of clinical signs there was no antibody response. NOS-2 null mice with EAE did not show increases in NO production over a month follow-up. Their antibody response was reduced by half and showed an opposite fluctuation compared to wild-types: treatment exacerbated the antibody response while clinical signs decreased it. These findings point to a complex role of PPAR-gamma agonists that may promote a Th2-driven antibody response in the absence of NO production which could explain the presence of an antibody response in NOS-2 null mice. PP06-22

Increased phosphorylation of caveolin-1 and p38 mitogenactivated protein kinase in EAE Heechul Kima, Meejung Ahna, Jeeyoung Leea, Changjong Moona, Yoh Matsumotob, Chang Sung Kohc, Taekyun Shina,⁎ a

Department of Veterinary Medicine, Cheju National University, Jeju 690– 756, South Korea; bDepartment of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183, Japan; c Department of Biomedical Laboratory Sciences, Shinshu University School of Health Sciences, 3-1-1 Asahi, Matsumoto 390-8621, Japan

autoimmune encephalomyelitis (EAE). To assess the involvement of MAP kinases in EAE, the expression and localization of phosphorylated form of ERK(p-ERK), JNK(p-JNK) and p38(p-p38) was analyzed in EAE in rats. Western blot analysis showed that all MAP kinases including p-ERK, p-JNK, and p-p38 were significantly increased in the EAE lesions at the peak stage (p < 0.05), and slightly declined in the recovery stage. As well, both MAPK/ ERK kinase 1, an upstream activator, and cAMP responsive element binding protein (CREB), a downstream transcription factor, was activated in EAE affected spinal cords. Immunohistochemistry showed that p-ERK was constitutively expressed in brain cells, including astroglial cells, and showed enhanced immunoreactivity in those cells in EAE, while some T cells and macrophages were positive for p-ERK in EAE lesions. Both p-JNK and p-p38 were intensely immunostained in T cells in EAE lesions. The majority of brain cells and inflammatory cells were positive for p-CREB in EAE lesions. These findings suggest that activation of MAP kinases and its downstream transcription factor play an important role in the initiation of EAE paralysis and subsequent recovery. The functional role of each MAPK will be discussed. PP06-24

Deletion of Pten in antigen-activated T lymphocytes reveals a potential role for Pten in effector populations in the experimental autoimmune encephalomyelitis (EAE) disease model T.A. Johnson, and F.R. Jirik Department of Biochemistry and Molecular Biology, University of Calgary, Calgary AB, Canada

The expression of phospho-specific caveolin-1 (p-caveolin-1) and phosphorylated p38 mitogen-activated protein kinase (p-p38) was analyzed in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis showed that p-caveolin-1 and p-p38 were constitutively expressed in normal spinal cords and that it significantly increased in the spinal cord with EAE at peak stages of EAE (P < 0.05), and decreased slightly at the recovery stage of EAE. Immunohistochemistry showed that p-caveolin-1 was constitutively expressed in few vascular endothelial cells and glial cells of the normal rat spinal cord. In EAE lesions, p-caveolin-1 was intensely immunostained in inflammatory T cells and macrophages. p-p38 was constitutively expressed in neurons, glial cells, and vascular endothelial cells in normal spinal cords and was also expressed in inflammatory cells, as well as increased expression in glial cells in EAE. Taken all into considerations, we postulate that the phosphorylation of caveolin-1 and p38 play an important role in the pathogenesis of EAE. The functional roles of p-caveolin-1 and pp38 will be discussed.

Pten, a phosphoinositol phosphatase, is critical to T lymphocyte ontogeny and self-tolerance via the negative regulation of pathways lying downstream of phosphoinositide 3′ kinase (PI3K). Previous studies using the Cre-loxP system to delete Pten genes in developing thymocytes, have revealed defects in both thymic selection and T cell homeostasis. To examine the functional role of Pten specifically in mature effector T lymphocytes, and in the context of a normal pre-immune T-cell repertoire, we selected a system that allows Cre-mediated gene excisions to occur within CD8+ and CD4+ T cells subsequent to activation. This system, which employs the Granzyme B gene promoter to drive expression of the Cre recombinase upon TCR activation, was used to trigger excisions of exons 4 and 5 of the floxed Pten gene. In mice homozygous for the floxed allele, we were thus able to obtain Pten deletion specifically in a subset of activated T lymphocytes. To characterize this system further, we evaluated the efficiency of Pten gene deletion in mature T cells in vitro, and also studied the consequences of Pten loss in a model of T cell-mediated immunopathology, murine experimental autoimmune encephalomyelitis (EAE). EAE was induced by injection of a synthetic myelin oligodendrocyte glycoprotein peptide (MOG 35–55) in complete Freund's adjuvant and Pertussis toxin. Initial clinical and neuropathological characterization of the MOG-immunized Granzyme B Cre x floxed Pten mice indicates that Pten plays a critical role in regulating not only the severity of EAE, but also the time-course of this disease.

PP06-23

PP06-25

Activation of mitogen-activated protein kinases in experimental autoimmune encephalomyelitis

Exacerbation of experimental autoimmune encephalomyelitis in P2X7R−/− mice

T. Shin

Lanfen Chen and Celia F. Brosnan

Department of Veterinary Medicine, Cheju National University, Jeju, Republic of Korea

Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA

Increasing evidence suggests that mitogen-activated protein (MAP) kinases, including extracellular signal-regulated kinase (ERK), c-Jun NH(2)terminal protein kinase (JNK), and p38, are involved in the pathogenesis of human demyelinating diseases and its animal model experimental

P2X7 receptor (P2X7R), an ATP-gated ionotropic receptor, has been proposed to serve as a regulator of inflammation. In this study we tested whether the development of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, was modified in animals

Abstracts lacking the P2X7R. In P2X7R−/− mice clinical and pathological signs of EAE were more severe than in WT mice. Bone marrow radiation chimeras revealed that greater susceptibility to EAE was detected in chimeric mice of WT host engrafted with P2X7R−/− BM cells, indicating that the genotype of the BM cells regulated disease susceptibility. In vitro, spleen and lymph node cells from P2X7R−/− mice had a higher proliferative activity and less apoptosis in response to MOG peptide. Similarly, significantly fewer Annexin V/PI+ and TUNEL positive lymphocytes were found in the CNS of P2X7R−/− mice early in the disease. Cytokine profiles of the culture supernatants showed significantly decreased expression of IFNγ and IL-6 in MOG-activated spleen cells from P2X7R−/− mice. QPCR and protein assays showed that protein for IL-1β (but not mRNA), as well as mRNA and protein for IL-6 and IFNγ were significantly reduced in spinal cord homogenates. Indoleamine 2, 3-dioxgenase (IDO) production was also decreased in spinal cord homogenates of P2X7R−/− mice. From these data we conclude that the enhanced susceptibility of P2X7R−/− mice to EAE reflects a more vigorous proliferative response to MOG, lower levels of IFNγ, nitric oxide and IDO compared to WT mice, which correlated with loss of apoptotic activity in lymphocytes. PP06-26

Macrophage phenotype during relapsing experimental autoimmune encephalomyelitis can be predicted by MRI at the disease onset J. Mikita, M.S.A. Deloire, T. Touil, M.H. Canron, C. Boiziau, V. Dousset, K. G. Petry, Brochet B. EA2966 Neurobiology of Myelin Disorders Laboratory, University Victor Segalen, Bordeaux, France Macrophage infiltrates in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) can be monitored in vivo by MRI with ultrasmall super paramagnetic iron oxide (USPIO) nanoparticles. In pathological conditions macrophages present at least two main phenotypes: proinflammatory M1 macrophages express inducible Nitric Oxide Synthase (iNOS) while immunomodulatory M2 macrophages express Arginase I. We used a relapsing EAE to study macrophage phenotypes within inflammatory infiltrates at the clinical onset (group I), at the end of 1st attack (group II) and during the 2nd attack (group III). USPIO MRI was performed at the clinical onset and was repeated for animals of group III. Animals with MRI signal changes were considered as MRI+. On serial thin sections of brainstem activated macrophages were quantified by anti ED1, M1 macrophages by anti iNOS labelling using confocal immunofluorescence and M2 by arginase I labelling; axonal damage and loss in brainstem tissue were quantified by classic immunohistochemistry methods. The obtained results were compared with MRI data. MRI+ rats presented more important macrophage recruitment (ED1+ cell number); cells expressing ED1+ and iNOS+ (M1) were also more abundant in MRI+ animals than in MRI− animals. In regard to clinical disease severity and tissue alterations (axonal loss and tissue damage) significant differences between MRI+ and MRI− animals were observed. M2 phenotype analysis is in progress. In conclusion, MRI performed at the onset of EAE predicts the presence of iNOS-expressing macrophages (M1 phenotype) and the extend of nervous tissue lesions in inflamed CNS at different stages of disease course. PP06-27

MRI correlates of lesion pathology in T-cell clone mediated mouse experimental autoimmune encephalomyelitis S.A. Nessler1, S. Boretius2, C. Stadelmann3, A. Bittner4, D. Merkler3, H.P. Hartung1, T. Michaelis2, W. Bruck3, J. Frahm2, N. Sommer4, Bernhard Hemmer1

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1

Heinrich-Heine University, Dusseldorf, Germany; 2Max-Planck-Institute for Biophysical Chemistry, Gottingen, Germany; 3Georg-August-University, Gottingen, Germany; 4Philipps-University, Marburg, Germany Magnetic resonance imaging (MRI) techniques are widely used to investigate multiple sclerosis (MS) lesions. Histopathological correlates of MRI signal alterations are still poorly defined. Here, we compared MRI and histopathology in a murine experimental autoimmune encephalomyelitis (EAE) model. Brainstem lesions were induced by the adoptive transfer of a PLP139–51 specific T-cell clone in SJL/J mice. 25 of 27 histopathologically identified acute EAE lesions were retrieved by T1and T2-weighted high-resolution 3D MRI. Lesions varied with respect to signal intensity on T1- and T2-weighted images. Two dominant MRI lesion patterns could be identified: Lesions with reduced signal intensity on T1- and T2-weighted MRI (type A) and lesions with isointense or slightly reduced signal intensity on T1-weighted MRI and increased signal intensity on T2-weighted MRI (type B). Type A lesions were characterized by significantly denser inflammatory cell infiltrations and more myelin and axonal loss than type B lesions. Comparative analysis revealed that lesional cellularity, myelin loss and axonal density correlated with signal intensities obtained by T1- and T2-weighted images in the lesion center. In perilesional areas, microglia cell numbers and Ig deposition correlated strongly with signal intensities on T2-weighted MRI. Gd-DTPA enhancement correlated with Ig deposition and mainly took place in areas with activated microglia cells. In summary, we established a mouse EAE model suitable for MR imaging. Given the robust correlation between MR signal intensities and histopathological changes the model can be used for MRI evaluation of new therapeutic strategies. PP06-28

Magnetic resonance-guided evaluation of disease burden in mice with chronic experimental autoimmune encephalomyelitis (EAE) Marco Bacigaluppi1, Letterio S. Politi2, Elena Brambilla1, Marcello Cadioli2, Alessandro Cattalini1, Giuseppe Scotti2, Gianvito Martino1, Stefano Pluchino1 1

Neuroimmunology Unit, DiBiT, Scientific Institute, Milan, Italy

2

Neuroradiology Unit, San Raffaele

Magnetic resonance (MR) may help pre-clinical studies in monitoring pathological abnormalities of brain white matter. We investigated whether inflammatory demyelinating lesions affecting the brain of mice with experimental multiple sclerosis (MS) may be detected and monitored over the natural story of the disease model with a 3T human MR apparatus. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice using 200 μg/mouse of myelin-oligodendrocyte glycoprotein (MOG)35–55 in complete Freund's adjuvant, while sex-, strain-, weightand age-matched both non-immunized and CFA-immunized mice were used as controls. Mice (n = 5/group) were evaluated for both basal as well as gadolinium (Gd) contrast enhanced MR images at pre-clinical, acute- and chronic phases of the disease (e.g., 10, 15, and 32 days after immunization [dpi], respectively). Meningeal enhancement and T1 Gd enhancing parenchymal lesions started appearing as early as from 10 days dpi, while peak of “active” demyelinating lesions were recorded at clinical onset (17 dpi). Meningeal enhancement faded over time, progressively disappearing at the 25 dpi. In 50% of EAE mice, new enhancing lesions were detected at 32 dpi, thus indicating a sub-clinical relapse. 74% of examined mice presented at least one demyelinating lesion in the cerebellum, 64% of them had lesions in either the corpus callosum or the external capsule. Neuropathological analysis confirmed the presence of lympho/monocyte infiltrates in meninges and parenchymal T1-weighted Gd-enhanced images. Further studies will be necessary to more carefully evaluate sensibility and specificity of human graded MR in detecting acute and chronic demyelination in EAE mice.

158

Abstracts

PP06-29

MR imaging of monocyte infiltration into the central nervous system in animal models of neuroinflammation

These results elucidate the speed and efficiency of antigen recognition in vivo and add to our understanding of T cell-mediated autoimmunity and its therapy. PP06-31

R.D. Oude Engberinka, E.I. Hoffc, S.M. van der Polb, E.L. Blezera and H. E. de Vriesb a

Image Sciences Institute, University Medical Center Utrecht, The Netherlands; bMolecular Cell Biology and Immunology, VU Medical Center, Amsterdam, The Netherlands; cDepartment of Neurology, Maastricht, The Netherlands

Infiltration of monocytes into the central nervous system is a key event in neuroinflammatory diseases. The development of superparamagnetic particles of iron oxide (SPIO) has made magnetic resonance imaging (MRI) a valuable tool for in vivo cell tracking. It provides the possibility to non-invasively monitor the homing of inflammatory cells towards a site of neural tissue damage. Our aim was to visualize SPIO labeled monocytes entering a brain lesion over time and to compare MR abnormalities with the administration of free label. Cortical lesions were induced in 15 rats by photothrombosis of microvessels. On day 5 after lesion induction, rats received intravenously either no label, free SPIOs or SPIO labeled monocytes. T2⁎W imaging (4.7 T), sensitive for iron, was performed before, 24 h and 72 h after label injection. In addition, three rats were continuously scanned for 8 h starting directly after administration of free SPIOs. Relative increases of MR abnormalities in rats transplanted with SPIOmonocytes were at 72 h larger compared to controls and free SPIO treated rats. The latter displayed MR abnormalities covering the total lesion area at 24 h. Images from the 8 h scans showed MR abnormalities already 1 h after administration of free SPIOs, indicative for leakage of SPIOs over a damaged blood brain barrier. Our results demonstrate that we are able to visualize the infiltration of monocytes and monitor blood brain barrier permeability, using SPIO labeled monocytes and free SPIOs respectively. These imaging strategies are a valuable tool to study the dynamic pattern of monocyte infiltration in neuroinflammation and may contribute to the development of cell-directed therapeutics. PP06-30

Visualization of effector cell activation within autoimmune CNS lesions after soluble antigen treatment F. Odoardi, K. Rune, C. Cordiglieri. N. Kawakami, H. Wekerle, and A. Flügel Department of Neuroimmunology, Max Planck Institute for Neurobiology, Martinsried, Germany Using real time two-photon imaging we visualize the effects of intravenous soluble antigen on retrovirally labeled MBP-specific CD4+ T cells in acute Experimental Autoimmune Encephalomyelitis (EAE) lesions of the Lewis rat. Recently we found that effector T cells in the diseased CNS show two distinct motility patterns: the majority of the cells (65%) moved fast (maximal speed 25 μm/min) and apparently non directed through the compact tissue (motile cells); a second group of effector T cells (35%) was tethered to a fixed point, forming immune synapse-like structures (stationary cells). We now report that intravenous MBP infusion profoundly changes this T cellular locomotion pattern. The velocity of the effector T cells slowed down dramatically and the percentage of stationary cells increased to >75%. These changes were first observed in the meningeal areas starting 1 h after injection, followed by stopping of T cells within the CNS parenchyma 1 h later. Tethering was paralleled by strong up-regulation of pro-inflammatory cytokines and activation markers of the autoaggressive effector T cells. Furthermore, 3–4 h following the infusion of MBP, clinical EAE became worsened with increased weight loss and paralysis.

A plea for proteomics in biomarker research: Application of protein two-dimensional gel electrophoresis, liquid chromatography and mass spectrometry imaging in rat experimental autoimmune encephalomyelitis Debora Dumont1, Jean-Paul Noben1, Kurt Baeten1, Jerome Hendriks1, Ruben Ceuppens2, Stefan Clerens2, Peter Verhaert3, Piet Stinissen1, Lutgarde Arckens2 and Johan Robben1 1

Hasselt University, Biomedical Research Institute (BIOMED), and transnationale Universiteit Limburg, School of Life Sciences, Diepenbeek, Belgium; 2Laboratory of Neuroplasticity and Neuroproteomics, K.U. Leuven, Leuven, Belgium; 3Delft University of Technology, Faculty of Applied Sciences, Analytical Biotechnology Group, Delft, The Netherlands Introduction: At Biomed, research is focused on the autoimmune disorder multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). One of the research topics includes analysis of EAE rat brain by proteomic separation technologies and mass spectrometry. In addition, a mass spectrometry imaging (MSI) initiative was set up. MSI is a newly developed technology that utilizes matrix-assisted laser desorption/ ionisation (MALDI) mass spectrometry to profile and map proteins present in tissue sections. The goal of this study is to identify differentially expressed proteins that can ultimately be used as biomarkers for MS. Methods: Myelin basic protein (MBP)-immunized Lewis rats were sacrificed and brain extracts were subjected to two-dimensional gel electrophoresis (2D-GE) and 2D liquid chromatography (2D-LC), both combined with tandem mass spectrometry. For MSI, cryostat coronal sections of 10 μm were cut, transferred to a stainless steel MALDI target plate and sprayed with matrix. Spectra of 4–25 kDa were acquired using a Bruker Ultraflex II MALDI-TOF/TOF instrument. Results and perspectives: The application of 2D-GE and 2D-LC resulted in the identification of >200 proteins, some of which were found differentially expressed in the brain of EAE animals. Ongoing MSI experiments include the monitoring of selected brain proteins with experimentally determined masses such as myelin basic protein. Future experiments will focus on the identification of differentially distributed proteins in acute EAE as well as in a demyelinating animal model. Differentially expressed proteins found in either proteomic application will be validated for their use as biomarkers in MS. PP06-32

Immunization of mice with the neuronal protein tau induces tauopathy-like pathology and neurological deficits Hanna Rosenmann, PhD1, Nikolas Grigoriadis, MD, PhD2, Dimitrios Karussis, MD, PhD1, Rachel Mizrachi-Koll, BSc1, Moran Boimel, BSc1, Olga Touloumi, BSc2, Haim Ovadia, PhD1, Oded Abramsky, MD, PhD1 1

Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Hadassah University Hospital, Jerusalem, Israel, 2AHEPA University Hospital, Thessaloniki, Greece Objective: To investigate whether immunization with tau-microtubuleassociated protein induces a neurodegenerative disease in animals. The finding may point to a role of autoimmunity in neurodegenerative disorders. Backgroud: A possible role of autoimmunity in Alzheimer's-disease (AD) pathogenesis has lately attracted attention. Immunization with amyloid-beta attenuates amyloid pathology, but also induces neuroinflammation. Very little is known about autoimmunity against the other major component of

Abstracts AD–tau aggregation into neurofibrillary-tangles. To investigate this possibility we actively immunized mice with tau protein and tested whether a neurodegenerative disease is induced. Methods: We immunized C57BL mice with recombinant human tau protein with complete Freund's-adjuvant (CFA) and pertussis. Results: Out of 11 female animals immunized with tau 6 developed neurological signs such as limp tail and limb paralysis, whereas none of the CFA-immunized controls showed any symptoms. In all of the tauimmunized mice we have detected anti-tau Abs in serum, and glial activation as well as tau-related pathology in the spinal cord (and to a lesser extent also in brains). These findings were much more prominent in the clinically affected animals. Mononuclear infiltrates and axonal damage were detected only in the clinically affected tau-immunized animals. Conclusion: We demonstrated a neurodegenerative disease induced by immunization with the tau protein. To our best knowledge this is the first report indicating that tau protein has an immunogenic potential, and can induce neuroinflammation and neurodegeneration. These findings may shed light on the possible involvement of tau autoimmunity in neurodegenerative diseases, and point to the potential danger of therapeutic immunization with tau protein.

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MHC class II molecules play a central role in the control of adaptive immune response through selection of CD4+ T cell repertoire in the thymus and antigen presentation in periphery. Inherited susceptibility to autoimmune disorders such as multiple sclerosis, rheumatoid arthritis and IDDM are associated with particular MHC class II alleles. Advent of HLA transgenic mice has helped us in deciphering role of particular HLA DR and DQ class II molecules in human autoimmune diseases. In mouse, the expression of class II is restricted to professional antigen presenting cells. However in human, class II is also expressed on T cells unlike mouse T cells. We have generated a ‘new’ humanized HLA class II transgenic mice expressing class II molecules on a subset of CD4 T cells. The expression of class II on CD4+ T cells is inducible and classII+CD4+ T cells can present antigen in the absence of APCs. Further, using experimental autoimmune encephalomyelitis (EAE), a well-established animal model of multiple sclerosis, we tested the functional significance of these classII+CD4+ T cells. DR3.AEo transgenic mice were susceptible to PLP91–110 induced EAE and showed CNS pathology accompanied by widespread inflammation and demyelination seen in human MS patients suggesting a role for class II+CD4+ T cells in the pathogenesis. PP06-36

PP06-34

Antigen confirmation contributes to lesion distribution and clinical signs in relapsing remitting MOG-induced EAE 1

1

2

1

E.K. Mathey , K.R.Williams , M.K. Storch , C. Linington

Reinforcement of tolerance to MOG: Targeting CLR on dendritic cells by glycan modified MOG J. J. García Vallejoa, S. Chamorroa, R.Fernandeza B.A. 't Hartb and Y. van Kooyka

1

University of Aberdeen, Aberdeen, United Kingdom; 2Medical University Graz, Graz, Austria

a

Multiple sclerosis (MS) is a chronic disease in which repeated episodes of inflammatory demyelination are associated with an increasing burden of axonal injury, the major cause of chronic disability. To investigate the neuronal response to axonal injury in the context of inflammatory demyelination we have developed a model of relapsing remitting MOGinduced EAE in the Lewis rat characterised by a predictable and focal development of lesions in the cervical spinal cord and cerebellum. Disease is induced by immunisation with refolded recombinant rat MOG in incomplete Freunds adjuvant (IFA) which unlike classical models of disease is characterised by circling and ataxia. Disease course and pathology has now been assessed during the initial episode of disease, relapse and remission. Typically 2 or 3 distinct relapses are observed over a period of 70 days post immunisation with lesions appearing to preferentially target the dorsal column of the cervical spinal cord at early time points with subsequent involvement of cerebellar region of the brain. Inflammation was associated with confluent demyelination and axonal injury. The mechanisms responsible for the selective targeting of specific regions of the CNS are unclear. Preliminary studies indicate local differences in the recruitment and activation of a minimal encephalitogenic T cell response that is amplified by the high titre demyelinating antibody response induced by refolded MOG. This model of EAE will be useful for studying regional differences in the response of the blood brain barrier, axons and defined neuronal cell populations to inflammatory demyelination during relapsing remitting disease.

Dendritic cells (DC) are professional antigen presenting cells that actively initiate adaptive immune responses or control peripheral tolerance to self-antigens. We have demonstrated that in particular signals provided by antigens that target C-type lectin receptors (CLR) function as uptake receptors for glycosylated antigens to maintain homeostasis and induce tolerance. Concomitant DC maturation induced through Toll-like receptors (TLR) triggering are instrumental in inducing auto-immunity. In multiple sclerosis (MS) self-antigens such as myelin/oligodendrocyte glycoprotein (MOG) may induce neuronal demyelination in experimental autoimmune encephalomyelitis (EAE) when combined with adjuvant that activates TLR. To determine whether altered glycosylation of MOG can disrupt this homeostatic control by deranging the CLR/TLR balance, the glycosylation profile of native MOG from human or marmosets was investigated. Common marmoset provides us a unique preclinical model due to its high clinical, pathological and immunological similarities to MS. Our studies demonstrate that MOG may express those glycans that target CLR on DC. CLR are indeed differentially expressed in the healthy and diseased human and marmoset MS brain. Our in-vitro human DC studies will evaluate the mechanism by which glycosylated MOG exhibit their suppression. The suppression by glycosylated MOG on the development of anti-MOG autoimmunity should form the basis for future therapy aimed at tolerance induction in MS.

PP06-35

Role of MHC class II expression CD4+ T cells in PLP91–110 induced EAE in HLA-DR3 transgenic mice Chella S. David, Ashutosh Mangalam, and Moses Rodriguez Mayo Clinic College of Medicine, Rochester, MN, USA

Department of Molecular Cell Biology, VUmc Amsterdam, The Netherlands; bImmunobiology, BPRC, Rijswijk, The Netherlands

160

Abstracts

Poster Session 7: EAE-therapy

Abstracts PP07-01

FTY720, sphingosine 1-phosphate receptor modulator, ameliorates experimental autoimmune encephalomyelitis (EAE). II. Effect of FTY720 on myelin oligodendrocyte glycoprotein-induce EAE in C57BL/6 mice N. Sekia, H. Kataokaa, M. Ohtsukia, N. Satoa, A. Fukunarib and K. Chibaa a

161

compared with interferon-β, and the area of demyelination and the infiltration of CD4+ T cells were decreased in spinal cords of EAE mice. Conclusion: These results indicate that FTY720 exhibits not only a prophylactic but also a therapeutic effect on PLP-induced EAE in SJL/J mice255and that the effect of FTY720 on EAE appears to be due to a reduction of the infiltration of myelin antigen-specific CD4+ T cells into the inflammation site. PP07-03

b

Research Laboratory III, Discovery Technology Laboratory I, Mitsubishi Pharma Corporation, Yokohama, Japan

FTY720 sequesters circulating lymphocytes into secondary lymphoid tissues and thymus by long-term down-regulation of sphingosine 1-phosphate receptor type 1, and is highly effective in myelin proteolipid protein (PLP)induced experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. In this study, we evaluated the therapeutic effect of FTY720 on myelin oligodendrocyte glycoprotein (MOG)-induce EAE in C57BL/6 mice. [Methods] EAE in female C57BL/6 mice was induced by the immunization MOG35–55 at 200 μg/mouse in the presence of Freund's complete adjuvant. [Results] EAE was developed 12 days after immunization of MOG to mice. EAE-established mice were used 17 days after MOG immunization. EAEassociated symptoms were maintained during administration period in the control group. Consistent with PLP-induced EAE in SJL/J mice, MOGinduced EAE was significantly inhibited when FTY720 was administered therapeutically at 0.1 to 0.3 mg/kg orally. The infiltration of CD4+ T cells was more markedly than that of CD8+ T cells in the spinal cords in control EAE mice. In FTY720 groups, the area of demyelination and the infiltration of CD4+ T cells and CD8+ T cells in the spinal cords were decreased compared to control. The elevation of IFN-γ mRNA level in the spinal cord was significantly inhibited in FTY720 groups. In addition, since the elevation of IL-17 mRNA was observed in the spinal cords in EAE mice, we are now analyzing the level of IL-17 mRNA in FTY720 groups. [Conclusion] These results suggest that FTY720 shows a therapeutic effect on MOG-induced EAE in C57BL/6 mice by inhibition of Th1 cell infiltration dominantly. PP07-02

FTY720, sphingosine 1-phosphate receptor modulator, ameliorates experimental autoimmune encephalomyelitis (EAE). I. Effect of FTY720 and FTY720-phosphate on myelin proteolipid protein-induce EAE in SJL/J mice H. Kataokaa, K. Sugaharaa, K. Shimanoa, Y. Maedaa, M. Koyamab, A. Fukunarib, and K. Chibaa Research Laboratory III, bDiscovery Technology Laboratory I, Mitsubishi Pharma Corporation; Yokohama, Japan

Roles of S1P-receptors in the brain: Implications for FTY720 in Multiple Sclerosis A.S. Schubarta, M. Cortes-Crosa, F. Muellershausena, M. Osindea, L. Craveirob, M. Thallmairb, M. Schwabb, R. Sivasankarana, W.L. Wisharta, A. Mira and K. K. Deva a

Novartis Institute for BioMedical Research, Basel, Switzerland; bInstitute for Brain Research, University of Zurich, Switzerland Sphingosine-1-phosphate receptors (S1P-Rs), in particular the S1P1-R is expressed on T-cells and is responsible for T-cell egress from lymphatic tissue. Binding of the S1P-mimick FTY720-P to S1P1-Rs on T-cells causes receptor internalization which in turn prevents the trafficking of T-cells to the blood and CNS. This reduces the accumulation of harmful T-cells in the CNS of Multiple Sclerosis patients and phase II studies with FTY720 have clearly shown protective effects in Multiple Sclerosis. Intriguingly, the five S1P-Rs are also differentially expressed on the four major cell types of the brain, namely Neurons, Oligodendrocytes, Astrocytes, and Microglia. Little is known about the role of S1P-Rs in the brain, but studies using S1P and activation of S1P-Rs indicate that these receptors may modulate all four cell types. FTY720 readily crosses the blood–brain barrier. We therefore suggest that FTY720 works beyond the immune system and plays additional protective roles on CNS cells in Multiple Sclerosis. Here, we present data on the effects of FTY720 on the nervous system using in vitro cell culture assays as well as preliminary experiments with direct brain administration of doses that do not cause peripheral lymphocyte depletion at different stages of relapsing remitting experimental autoimmune encephalomyelitis (EAE) in DA rats. PP07-04

Superagonist peptide-induced T cell tolerance for the treatment of multiple sclerosis Kazuyuki Kawamuraa, Meike Huebenera, Christopher Selfb, Robert Weissertc, and Thomas G. Forsthubera

a

Introduction: FTY720, a new class of immunomodulator, is effectively phosphorylated to FTY720-phosphate (FTY720-P), sequesters circulating lymphocytes into secondary lymphoid tissues and thymus by long-term down-regulation of sphingosine 1-phosphate receptor type 1, and exerts immunosuppressive activity in various allograft and autoimmune disease models. In this study, we evaluated the effect of FTY720 on myelin proteolipid protein (PLP)-induce experimental autoimmune encephalomyelitis (EAE) in mice. Methods: EAE in female SJL/J mice was induced by immunization with PLP139–151 at 50 μg/mouse in Freund's complete adjuvant. and clinical score of EAE was assessed periodically. [Results] EAE was developed 11 days after PLP immunization and relapsed 2 weeks after primary EAE in control group. The development of PLP-induced EAE was almost completely prevented and infiltration of CD4+ T cells into spinal cord was decreased by prophylactic treatment with FTY720 at 0.1 to 1 mg/kg orally and FTY720-P at 0.1 to 1 mg/kg intraperitoneally. When FTY720 or FTY720-P was given after establishment of EAE, the relapse of EAE was markedly inhibited as

University of Texas at San Antonio, San Antonio, USA; b Provid Pharmaceuticals Inc., North Brunswick, NJ, USA; cHertie Institute for Clinical Brain Research, Tübingen, Germany

a

Specific object: Most of the attention has been focused on T cells specific for high-affinity MHC-binding myelin epitopes in MS patients, such as MBP 85–99, but clinical trials targeting these T cells showed only limited success. A recent study showed that myelin-reactive T cells in MS patients are primarily high-avidity T cells specific for low-affinity MHC-binding epitopes. We found that the low-affinity binding MBP 115–126 peptide is a dominant epitope in HLA-DR4 transgenic mice. Studies with B10.PL mice suggested that T cells specific for low-affinity MBP Ac1–9 can be deleted by analogues of this peptide with enhanced binding-affinity for MHC (superagonist). We have therefore investigated whether superagonist peptides can be designed for MBP 115–126 for the treatment of MS. Methods: Superagonist peptide analogues of MBP 115–126 were generated that bound several log-fold better to MHC than the native peptide. Their potential of deleting MBP 115–126-specific T cells was examined in vitro.

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Abstracts

Results: Some of the designed superagonist peptides were as much as 104fold as stimulatory as native MBP 115–126 for MBP 115–126-specific T cells. 70% of the cells stimulated with the superagonist peptides were positively stained for Annexin-V 48 h after activation, indicating induction of apoptosis. Conclusions: Superagonist peptide analogues may be a feasible therapeutic approach to delete encephalitogenic T cells specific for low-affinity HLADR-binding peptides.

MHCs is a natural and chronic process. This possibility was supported by identifying the transfer of MHC to the surface of B cells in ex vivo cell cultures. We hypothesize that the binding and internalization of α1β1 MHCs by cells may trigger an immunomodulatory response to the peptides bound by the α1β1 MHC. PP07-07

PP07-05

Enhancement of regulatory T cells function via epitope specific immunotherapy leads to disease control in EAE

Epicutaneously induced TGF-β-dependent tolerance inhibits experimental autoimmune encephalomyletis

R. Billetta1, M. Omori1, A. Kaspar1, N. Ghahramani1, D. Brigham1, C. Meschter2, P. Lanza1, S. Albani3

Monika Tutaj, Marian Szczepanik

1

Department of Human Developmental Biology, Jagiellonian University, College of Medicine, ul. Kopernika 7, 31-034 Kraków, Poland Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS) with limited treatment modalities. To evaluate the efficacy of epicutaneous (e.c.) tolerance induction in the prevention of CNS autoimmunity, we utilized an animal model of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin basic protein (MBP). We show that application of MBP to the skin prior to the induction of EAE protected mice from developing disease. In addition, e.c. application of MBP at the first signs of EAE, also resulted in suppression of disease. This therapeutic effect was transferable to naïve recipients with lymph node cells from mice treated epicutaneously. These tolerogenic regulatory cells were found to be antigen non-specific, as suppression of EAE also occurred when the antigens such as OVA or TNP were applied e.c. Skin-induced regulatory cells belong to the population of TCRαβ+CD4+CD8+ lymphocytes. The mechanistic basis for the tolerance was found to be the production of TGF-β by this cells population. These data demonstrate that e.c.-induced regulatory T cells are potent inhibitors of antigen-specific T cell responses, and suggest that e.c. tolerization may have potential effectiveness in the treatment of autoimmune disorders. PP07-06

B cells with preferential affinity for partial (α1β1) MHC class II molecules may regulate pathogenic T cells in experimental encephalomyelitis Arthur A. Vandenbark1,2, Yuan K. Chou2, Gregory G. Burrows2, and Jason M. Link1 1

Portland Veterans Affairs Medical Center, 3710 SW US Veterans Hospital Road, Portland, OR 97239, USA; 2Department of Neurology. Oregon Health Sciences University. 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA

Recombinant, two domain major histocompatibility complex (MHC) molecules consisting of the peptide binding (α1β1) domains of MHC Class II have potent therapeutic activity in mice with experimental autoimmune encephalomyelitis (EAE). Following i.v. injection, the α1β1 MHCs were only detectable in the serum and plasma of mice for 15–30 min. This short half-life suggested that cellular adherence was responsible for removing α1β1 MHCs from peripheral blood, and indeed, we found that they adhered preferentially to the cell surface of B cells among murine peripheral blood (PB) and spleen (SP) cells. But neither full length (four domain) recombinant MHC Class II molecules nor MHC Class II tetramers had any detectable affinity for PB or SP cells. The FACS detectable level of α1β1 MHC on the surface of B cells decreased after 1 h of incubation; however, Alexa-488 labeled α1β1 MHCs continued to fluoresce from B cells after overnight incubation indicating that the α1β1 MHC was endocytosed. The binding and cellular internalization of α1β1 MHC molecules suggests that the release and acquisition of α1β1

Androclus Therapeutics, Inc., San Diego, USA; 2Comparative Biosciences, Inc., Sunnyvale, USA; 3UCSD Medical Center, UCSD, San Diego, USA

Present therapeutic approaches to autoimmune disease largely rely on the direct translation to human therapy of the model trigger antigens used in induction of the animal model. We have identified a mechanism, which modulates inflammation independent of the disease-triggering antigen in autoimmunity based on the recognition by T cells of epitopes derived from heat shock proteins (HSP). Our therapeutic approach aims at restoring T cell mediated regulatory mechanisms, impaired in autoimmunity, by tolerizing to the pro-inflammatory HSP epitopes via mucosal treatment. This study aimed at modulating adaptive Tregs using an HSP-derived peptide via mucosal treatment in monotherapy or combination therapy with Copaxone® after EAE induction in Lewis rats. EAE was induced by immunization with myelin basic protein (MBP). Our data show that in MBP-immunized rats, EAE development is suppressed clinically by mucosal administration of a particular HSP-derived peptide. We find that the regulatory mechanism activated involves a change in cytokine expression from inflammatory (IFN-gamma and TNF-alpha) to a regulatory profile (IL-4 and IL-10). Additionally, we observed an upregulation of FoxP3 expressing CD4+CD25+ T cells, indicating a restoration of the regulatory pathway. Histological data were consistent with clinical effect and immunological findings using both mono- and combination therapy. These data suggest the existence of immune regulatory circuits that can be activated by antigens unrelated to the induction of autoimmune disease. The existence of such regulatory pathways suggests that HSP peptidesmediated restoration of Treg function could be of significant therapeutic value for treatment of multiple sclerosis. PP07-08

NK cell-dependent tolerance of experimental autoimmune encephalomyelitis induced by heat shock protein 70-peptide complexes (Hsp70-pc) G. Galazkaa, M. Stasioleka, A. Walczaka, A. Jurewicza, A. Zyliczb, C. F. Brosnanc, C. S. Rainec and K. Selmaja,c a

Department of Neurology, Medical University of Lodz, Lodz, Poland; Department of Molecular Biology, International Institute of Molecular and Cell Biology, Warsaw, Poland; and cDepartment of Pathology (Neuropathology), Neurology and Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA b

Heat shock proteins (hsp) are markedly up-regulated at sites of inflammation during autoimmune diseases like multiple sclerosis (MS). In this study, we have investigated the effect of a pure hsp70 fraction and an hsp70 fraction associated with peptides (hsp-pc), isolated from the CNS of control healthy animals or animals with EAE (experimental autoimmune encephalomyelitis), on the subsequent development of immunization for EAE.

Abstracts The hsp70 and hsp70-pc complexes were isolated from brains of health mice or mice with EAE and purified using affinity chromatography with ATP- or ADP-agarose column (respectively). We have show that hsp70-peptide complexes (hsp70-pc) isolated from brains of mice with EAE prevented the development of EAE clinically and pathologically when administered before proteolipid protein 139–151 (PLP139–151) immunization. In animals in which EAE had been suppressed by hsp70-pc, lymphocytes showed increased cell death in response to PLP139–151 that correlated with elevated IFNγ and NO production. Coculture of spleen cells from hsp70-pc immunized mice with spleen cells from untreated EAE mice, in addition to depletion experiments, showed that NK cells reduced reactivity to PLP139–151. Transfer of NK cells from hsp70-pc immunized mice to recipients sensitized for EAE, abolished disease development. In conclusion, we have demonstrated that peptides derived from inflamed CNS and bound to hsp70 are able to induce a novel regulatory circuit involving NK cells inhibiting autoreactive T cells. These findings might further contribute to our understanding of hsp immunoregulatory function in autoimmune diseases. PP07-09

Neonatal neuroantigen-specific Th1 immunity protects from EAE Harald H. Hofstetter1, Andra Kovalovsky1, Carey L. Shive1, Paul V. Lehmann1, and Thomas G. Forsthuber1,2 1

Case Western Reserve University, Department of Pathology, USA; University of Texas at San Antonio, Department of Biology, San Antonio, TX 78249, USA

163

Multiple Sclerosis (MS) is a chronic inflammatory, presumably autoimmune, demyelinating disease of the central nervous system. In particular there is increasing evidence that auto-antibody production by Blymphocytes which result in targeting the myelin sheath by the complement system as well as Fc-receptor carrying cells may play an important role in the immunopathogenesis of demyelination. Our research is focused on the immunemodulatory potential of antagonists and agonists of Fc-receptors and their influence on the onset and disease course of the widely accepted animal model of MS, the experimental autoimmune encephalomyelitis (EAE). In this animal model disease is induced by immunization with the heterologous myelin oligodendrocyte glycoprotein (MOG). Therefore we administered soluble Fc-receptor (FcR), to diminish the interaction of low affinity FcRs with immune complexes. Additionally an antibody was given targeting the inhibitory FcγRIIb and leading to crosslinking with an opposing activatory FcγR which results in inhibition of the immune response by FcR carrying cells. In this study we investigated the potential effects of soluble FcR and anti-FcγRIIb antibody on the disease course of EAE. Treatment with FcR and anti-FcγRIIb after onset of clinical symptoms resulted in a faster recovery and significant reduction of the severity of EAE symptoms. Histopathological examinations revealed that treatment with soluble FcR as well as with the anti-FcγRIIb antibody strongly reduced the amount of infiltrating cells, especially the number of activated macrophages in the spinal cord. This was associated with a reduction of demyelinated patches and axonal damage. Mice receiving soluble FcR also showed decreased levels of pathogenic antibodies and activated B-cells. Our results support the hypothesis that soluble FcR as well as anti-FcγRIIb are an effective therapeutic approach for B-cell mediated autoimmune diseases.

2

Rationale: The neonatal immune system is believed to be biased towards Th2 immune responses. Consistent with this view, injection of neonatal mice with neuroantigens induces Th2 immunity and protects from induction of experimental autoimmune encephalomyelitis (EAE). In this study we asked whether neuroantigen-specific Th1 immunity could be induced in neonatal mice, and what the outcome would be for autoimmune disease. Methods: Neonatal SJL and B10.PL mice were injected with myelin antigens in CFA and cytokine profiles of the induced T cell responses were measured by ELISPOT assay. Neonatally injected animals were observed for EAE, or the mice were re-injected with myelin antigens as adults to determine protection from EAE. Results: The results show that injection of neonatal SJL and B10.PL mice with PLP139–151 or MBPAc1–11 in CFA resulted in vigorous antigenspecific production of proinflammatory cytokines, including IFN-g and IL17, but not Th2 cytokines, such as IL-5. Importantly, the neonatally injected mice did not develop EAE, despite the abundant neuroantigen-induced production of proinflammatory cytokines, and were protected from induction of the disease upon reinjection with neuroantigen as adults. Conclusions: The data show that a developmental window exists during the neonatal period in which proinflammatory autoreactive T cells that are not pathogenic can be induced. The data suggest that this could be due to a tissue-specific decrease in the propensity to differentiate towards encephalitogenic “TH-17” cells. The results may provide a rationale for the persistence of autoreactive T cells in healthy individuals in the absence of autoimmune pathology. PP07-10

Targeting FcgammaReceptors in the treatment of EAE Pellkofer H.L.1, Schaefer B.2, Breithaupt C.2, Huber R.2, Jacob U.2 Ludwig-Maximilians University of Munich, Munich, Germany; 2MaxPlanck-Institute of Biochemistry, Martinsried, Germany

1

PP07-11

Anti-CD20 B-cell depletion reverses EAE induced by MOG protein, but exacerbates disease induced by its encephalitogenic peptide M.S. Webera, T. Prod´hommea, T. Karnezisb, J.C. Patarroyoa, C. Rundlea, C. Liningtonc, C.C.A. Bernardb, F. Martind and S. S. Zamvila University of California, San Francisco, USA; bMonash University, Melbourne, Australia; cUniversity of Aberdeen, United Kingdom; d Genentech, South San Francisco, USA a

Objective: To investigate anti-CD20 mediated B-cell depletion in experimental autoimmune encephalomyelitis (EAE). Background: B-cells and myelin-specific antibodies (Ab) both play a pathogenic role in CNS autoimmune disease. Recent studies indicate that B cells may also have a regulatory function. We evaluated anti-CD20 mediated B-cell depletion in EAE induced either by MOG p35–55 or by recombinant mouse MOG 1–125 (rMOG). Methods: EAE was induced in human CD20-transgenic C57BL/6 mice in which anti-hCD20-Ab (m2h7) depletes B-cells. For EAE-prevention, mice received weekly i.p. injections of m2h7 or IgG2a-isotype starting 21 days prior to immunization. In reversal-experiments mice were randomized to treatment at onset of paralysis. T cell proliferation, cytokine and Ab production to MOG p35–55 or rMOG as well as histopathological examination was performed in all mice. Results: Anti-CD20 mediated B-cell depletion had beneficial effects in EAE prevention and reversed paralysis in rMOG-induced EAE. B-cell depleted mice revealed decreased titers for anti-rMOG Ab. In contrast, B-cell depletion prior to immunization exacerbated MOG p35–55-induced EAE with enhanced CNS-infiltration and demyelination and a decreased number of infiltrating B-cells. Splenic MOG 35–55 specific responses were shifted towards a proinflammatory Th1-phenotype with a pronounced decrease in IL-10 production. Conclusion: Anti-CD20 B-cell depletion has differential effects on MOGprotein and MOG-peptide induced autoimmune encephalomyelitis. The

164

Abstracts

beneficial effect of anti-CD20 B-cell depletion in rMOG-induced EAE may reflect a decrease in antigen presentation and/or reduced titers for myelinspecific Ab, whereas exacerbation of MOG peptide-induced EAE may relate to a reduction in B-cell regulation. PP07-12

Neuroprotection and neurogenesis induced by peripheral imunomodulatory treatment of experimental autoimmune encephalomyelitis with glatiramer acetate R. Aharoni, R. Eilam and R. Arnon Immunology Department, The Weizmann Institute of Science, Rehovot, 76100, Israel In the autoimmune inflammatory process involved in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), the therapeutic consequences of self neurogenesis and neuroprotection are limited and fail to regenerate functional neurons to compensate the damage. We demonstrate here that the peripheral immunomodulatory treatment for MS/ EAE – glatiramer acetate (GA) – can enhance neurogenesis and generate neuroprotection in the CNS of EAE inflicted mice. EAE was induced by MOG peptide 35–55 in transgenic mice, which selectively express YFP on their neuronal population and in C57BL/6 mice. GA was injected either concurrently with disease induction or after clinical signs appearance. Neurotrophic factor expression and the in situ manifestations of EAE as well as of GA treatment were analyzed immunohistochemically. Neuroprogenitor proliferation was quantified by the expression of the immature neuronal marker DCX and the incorporation of the in vivo proliferation marker BrdU. In EAE inflicted mice neurotrophins expression and neuroproliferation were elevated following disease appearance, but subsequently declined below that of naive mice. In contrast, GA treatment in various disease stages, led to elevation in BDNF, NT3, NT4 and reduction in the neuronal/axonal damage typical to the neurodegenerative disease course. Moreover, GA augmented the three processes characteristic of neurogenesis, namely neuroprogenitor proliferation, migration and differentiation. The neuroprogenitors manifested massive migration through exciting and dormant migration pathways, into injury sites in brain regions which do not normally undergo neurogenesis. They differentiated to mature neuronal phenotype, endorsing a direct linkage between immunomodulation, neuroprotection, neurogenesis and an in situ therapeutic consequence in the CNS. PP07-13

Transcriptional modulation of the immune response by PPARα agonists ameliorates EAE A.R. Gocke⁎, R.Z. Hussain⁎, L. Ben⁎, A.E. Lovett-Racke⁎, and M.K. Racke⁎+

reduced. In vivo administration of siRNA-Pparα inhibited the ability of gemfibrozil to ameliorate EAE and prevented IL-4 and IL-5 secretion, suggesting that the agonist is mediating its effects in a receptor-dependent manner. PPARα was found to directly bind the regulatory regions of the IL-4 and IL-5 genes by chromatin immunoprecipitation and IL-4-deficient mice were more suspectible to EAE, even following gemfibrozil treatment. IL-4deficient mice expressed lower levels of PPARα than controls and T-bet, GATA3, and SRC-1 expression were also found to be altered in gemfibroziltreated mice. Finally, treatment of EAE mice with gemfibrozil and fenofibrate ameliorated disease course significantly. This data suggests that PPARα agonists mediate their anti-inflammatory effects in an IL-4 and receptor-dependent manner and could potentially be used as a treatment for immune-mediated diseases such as MS. PP07-14

The regulation and production of prostaglandins E2 and D2 in acute and chronic-relapsing rodent models of multiple sclerosis S.S. Ayoub, R. Ashrafi, E.G. Wood, C. Bolton Queen Mary, University of London, London, UK Prostaglandins (PGs) are important pathophysiological mediators involved in a wide variety of processes and, in particular, acute and chronic inflammatory reactions. PG synthesis involves the metabolism of arachidonic acid into short-lived molecules through the actions of the cyclooxygenase (COX) enzymes, COX-1, COX-2 and the recently discovered isoform COX-3. The release of PGE2 contributes to the initiation of acute inflammation. In contrast, PGD2 has been shown to aid resolution of inflammatory states (Ayoub et al., 2004). The current study utilised rodent models of experimental autoimmune encephalomyelitis (EAE), the model of the human demyelinating disease multiple sclerosis, to investigate the relationship between PGE2 and PGD2 on the initiation and resolution of neuroinflammation. The levels of PGE2 in central nervous tissues from rodents with EAE were similar to normal values. However, PGE2 concentrations increased significantly during the recovery phase. PGD2 content decreased below control values with the development of EAE but returned to normal levels with the resolution of disease. The study will describe the effects of specific receptor antagonists on the expression of COX enzymes and PG production. In particular, the investigation will provide information on the identity of novel pathways involved in the control and production of the PGs in models of EAE. Reference Ayoub, S.S., Botting, R.M., Goorha, S., Colville-Nash, P.R., Willoughby, D. A. and Ballou, L.R., 2004. Acetominophen-induced hypothermia in mice is mediated by a prostaglandin endoperoxide synthase 1 gene-derived protein. Proc. Nat. Acad. Sci. 101, 11165–11169. PP07-15

Department of Neurology⁎ and Center for Immunology+, University of Texas Southwestern Medical Center, Dallas, TX, USA

Inhibition of CNS inflammation by cholinergic-up-regulation: A new role for acetylcholinesterase inhibitors

Peroxisome Proliferator Activated Receptors (PPARs) are ligandactivated transcription factors belonging to the nuclear hormone receptor superfamily. Rencently, PPARs have been shown to be expressed in macrophages, dendritic cells, T cells, and B cells and play a role in the inflammatory response. Our group has previously shown that the PPARα agonists gemfibrozil and fenofibrate can ameliorate Experimental Autoimmune Encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), by inhibiting IFNγ and promoting IL-4 production. The current study further characterizes the ability of PPARα agonists to induce immune deviation and regulate inflammation in EAE. We found that splenocytes treated with gemfibrozil have altered PPARα, GATA3 and T-bet expression. Using an siRNA specific for PPARα, PPARα and GATA3 levels were

Talma Brenner, Eran Nizri, Camille Sicsic, Iris Lavon and Gabi Amitay Department of Neurology and the Agnes Ginges Center for Human Neurogenetics, Hadassah University Hospital and Hebrew University Medical School, Jerusalem, [email protected] We recently showed that bifunctional compounds comprising nonsteroidal anti-inflammatory (ibuprofen) and cholinergic up-regulation (AChE inhibitor, AChEI; pyridostigmine) moieties ameliorate experimental autoimmune encephalomyelitis (EAE). The presence of cholinergic upregulation moiety increased the anti-inflammatory effect. Consequently, we studied anti-inflammatory effects of acetylcholinesterase inhibitors

Abstracts (AChEI) in CNS inflammation in vivo and in vitro. AChEI suppressed lymphocyte proliferation, pro-inflammatory cytokine production, and extracellular esterase activity. Anti-inflammatory activity was mediated by the alpha-7 nicotinic acetylcholine receptor (neuronal type), the muscarinic receptor had the opposite effect. Treatment of EAE with EN101, an antisense oligodeoxynucleotide, targeted to AChE mRNA, and with conventional AChEI, reduced disease severity and CNS inflammation. Our experimental results suggest that AChEI increases the concentration of extracellular acetylcholine (ACh), rendering it available for interaction with a nicotinic receptor expressed on lymphocytes. These observations point to a novel role for AChEI which may be relevant in CNS inflammatory diseases such as EAE and Multiple Sclerosis. They also emphasize the importance of cholinergic balance in neurological disorders, such as Alzheimer's disease and Myasthenia Gravis, in which these drugs are used. Keywords: acetylcholinesterase, acetylcholinesterase inhibitors, α7 nicotinic receptor, CNS inflammation, experimental autoimmune encephalomyelitis, Alzheimer's disease. PP07-16

Calpain inhibition attenuates immune cell infiltration and neurodegeneration in spinal cord during experimental autoimmune encephalomyelitis (EAE) MK Guytona, A Dasa, DD Matzellea, S Samantaraya, M Azumab, J Inoueb, SK Raya, NL Banika a

b

Medical University of South Carolina, Charleston, SC, USA; Senju Pharmaceutical Company, Kobe, Japan Multiple sclerosis (MS) is a T cell-mediated auto-immune disease of the CNS that leads to demyelination, axonal damage, and neuronal loss. The pathophysiological mechanisms responsible for development of MS and its animal model EAE are not well defined; however, increased expression/ activation of the calcium-activated neural protease calpain may play a role. Thus, we hypothesize that treatment with a calpain inhibitor will attenuate immune cell infiltration and neurodegeneration in EAE spinal cord. To test the hypothesis, EAE was induced in male Lewis and on days 4 and 8 postEAE induction, control and EAE rats were treated with the calpain inhibitor SJA6017 (SJA, 3 mg/kg) or vehicle (DMSO). On day 11 post-EAE induction, animals were sacrificed and lumbar spinal cord segments were collected for immunohistolabeling of cell-specific calpain expression (antimcalapin antibody). Calpain was markedly increased in OX42+ (macrophage/microglia) and IFNγ+ (T cell) cells in EAE animals, as compared to controls. Since very few cells tested positive for OX42 in control tissue, we suspect that these cells are mainly infiltrating macrophages, with some resident microglia. Treatment with SJA blocked calpain expression and migration of OX-42+ and IFNγ+ cells into the CNS. Neuron (NeuN)-specific calpain expression, neuronal death (TUNEL) and axonal damage (de-NFP) were also upregulated in acute EAE spinal cord. Treatment with SJA blocked most neuronal calpain expression, neuronal death, and axonal damage, as compared to vehicle-treated EAE rats. These data suggest that calpain inhibition will attenuate immune cell infiltration and neurodegeneration during EAE and may offer potential therapeutic benefits for MS patients.

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AKT/PKB which is involved in T-cell activation. We showed that clinical symptoms in experimental autoimmune encephalomyelitis (EAE) are reduced by geldanamycin, a naturally occurring HSP90 inhibitor; however whether this involved HSR or CPD was not addressed. We now characterize the effects of novel HSP90 inhibitors on glial and T-cell activation, clinical and histological signs in EAE, and AKT activation. Screening of synthetic HSP90 inhibitors identified several with high (low nM) efficacy to inhibit astrocyte NOS2 and IL1b expression, and T-cell IFNg production. EC72, a geldanamycin derivative, reduced clinical signs of MOG-EAE when given early in disease, and induced recovery when administered to ill mice. Late administration of EC72 also reduced subsequent axonal damage. In vivo treatment with EC72 did not reduce T-cell activation, nor did EC72 inhibit reactivation of primed T-cells in vitro. However, EC72 and other HSP90 inhibitors reduced CD3:CD28 dependent activation of naïve T-cells, which was also reduced by the AKT inhibitor SH5. HSP90 inhibitors induced AKT degradation, and their effects were lessened in cells expressing constitutively active myrAKT. HSP90 inhibitors could block NOS2 induction in HSF1 null cells. These results demonstrate efficacy of novel HSP90 inhibitors in EAE, and suggest that induction of a HSR is not critical for their effects. Supported in part by a grant from Conforma Therapeutics. PP07-18

Fullerene (ABS-75) treatment reduces disease progression and axonal loss in MOG-induced progressive EAE in NOD mice A. S. Bassoa , D. Frenkela , S. Petrovic-Stojkovica , L. Pucketta , A. Monsonegoa, M. Gozinb and H. L. Weinera a

Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, USA; bSchool of Chemistry, Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, Israel

Inflammation-induced oxidative stress can lead to axonal degeneration, which is felt to be a major determinant of progressive neurological disability in Multiple Sclerosis (MS). Water-soluble derivatives of fullerenes are a unique class of allotropic form of carbon compounds with potent antioxidant properties. 10 week old mice were immunized with 150 μg of MOG 35–55 peptide in CFA followed by pertussis toxin. Disease is characterized by an attack followed by a progressive phase with chronic clinical impairment. Following the first attack, animals were distributed into different groups with similar disease courses and treated intraperitoneally either with 200 μl of a 1 μM fullerene solution or vehicle (DMSO 2% in PBS) every day until termination of the experiment on day 63. We tested three different fullerene derivatives (ABS-75, ABS-16, and the C60 fullerene core) and found that ABS-75 treatment initiated after disease onset reduced the clinical progression of chronic EAE in NOD mice (treated vs. control, p < 0.05). Fullerene ABS-75 consists of a C60 carbon fullerene core to which a known NMDA receptor ligand was attached. ABS-75 treatment also reduced axonal loss and demyelination (as evaluated by silver and Luxol fast blue staining, respectively) in the white matter of mice. ABS-75 treated animals also showed reduction in CD11b+ infiltration and astrogliosis as compared to vehicle-treated group. Our data demonstrate a neuroprotective effect of a treatment with a fullerene compound combined with a NMDA receptor ligand that may have applicability in the treatment of progressive MS and other neurodegenerative diseases.

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HSP90 inhibitors reduce EAE D. L. Feinsteina, A. Spagnoloa, and C. Dello Russob

Chronic therapeutic administration of natalizumab reduces clinical severity in a guinea pig experimental autoimmune encephalomyelitis model

University of Illinois, Chicago, IL, USA; bCatholic University, Rome, Italy

a

Constitutively expressed HSP90 forms complexes with transcription factor HSF1, and with other client proteins. HSP90 inhibitors promote HSF1 dissociation which induces a heat shock response (HSR), whereas upon dissociation most client proteins are degraded (CPD), including kinase

Brian T. Wipke, Lilibeth Dofiles, Sally Schroeter, Angie Wadsworth, Eric Peterson, Carlos Lorenzana, Mike Krimm, Ian Gallager and Elizabeth Messersmith Elan Pharmaceuticals, Inc., South San Francisco, CA USA

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Natalizumab is a humanized monoclonal antibody against alpha-4 integrins for use in multiple sclerosis. Its mechanism of action involves blocking binding of lymphocyte-expressed α4β1 and endothelial VCAM-1, thereby inhibiting extravasation of leukocytes into the central nervous system. Natalizumab is cross-reactive for guinea pig α4β1, and can be dosed long-term (>30 days) in guinea pigs without apparent ill-effect or loss of potency. Groups of 15 early chronic stage EAE-afflicted guinea pigs (15 days post-induction, as chronic progression is beginning) were treated subcutaneously every other day for 56 d with 30 mg/kg natalizumab or vehicle. Natalizumab induced peripheral blood lymphocytosis in EAE animals (mean elevation = 4-fold on d28 and 3.5-fold on d56 relative to vehicle EAE animals). Natalizumab induced an immediate reversal in clinical scores, enhanced body weight gain and reduced mortality relative to vehicle, and maintained significant clinical protection throughout completion of the study. Mean paralytic clinical scores of natalizumab-treated animals on d28 and d56 were 0.86 and 0.4 respectively, compared to vehicle EAE clinical scores of 2.23 and 2.62 for d28 and d56. Clinical improvement in natalizumab-treated guinea pigs was paralleled histologically in spinal cord sections. The percentage of lesioned area in white matter was lower in natalizumab-treated animals than in vehicle EAE animals. These results are consistent with human clinical data demonstrating reduced clinical severity (relapse rate and disability score) and reduction in gadolinium-enhancing lesions by MRI. Further characterization of lesions in the guinea pig model will be investigated using cell-type-specific antibodies to identify infiltrating cell types via immunohistochemistry.

Multiple Sclerosis (MS) is a major chronic autoimmune pathology of the central nervous system. Members of the matrix metalloproteinase (MMPs) family are known to be important factors involved at key steps of MS, however little is known of the role of MMP12 in MS. To determine the extent to which MMP12 might be involved in this pathology, we administered orally a MMP12 inhibitor in preventive regimen (3, 10, 30 mg/kg) in the MOG-induced chronic mice as well as in curative regimen (1.5, 5, 15, 30 mg/kg) in the whole spinal cord protractedremitting rat experimental autoimmune encephalomyelitis (EAE) models which are animal models mimicking various aspects of MS. In preventive treatment, the MMP12 inhibitor significantly delayed the disease onset and decreased the severity of the clinical signs over the course of the pathology suggesting the involvement of MMP12 in the induction as well as the following effector phase of the pathology. In the protracted-remitting EAE model, a mixed model of inflammation and early demyelination, oral administration starting at clinical score 1 decreased the severity of the first attack and the following remitting phases at a minimal dose of 5 mg/kg. This effect was paralleled by an elimination of inflammatory infiltrates, demyelination and axonal damage in the spinal cords of treated animals. These results support the potential role of MMP12 in the development of MS-like conditions and strengthen previous findings on MMP12 in MS patients (Vos et al., 2003). PP07-22

PP07-20

TLR4 stimulation delays the onset of chronic experimental autoimmune encephalomyelitis (EAE)

Fc-receptor antagonists are effective in the therapy of autoimmune disease animal models

Denise C. Fitzgeralda, Tarik Touila, Guang-Xian Zhanga, A.M. Rostamia, Bruno Grana,b

Jacob U.1, Pellkofer H.L.2, Schaefer B.3, Breithaupt C.3

a

SuppreMol, Martinsried, Germany; 2Institut für Neuroimmunologie, Klinikum Grosshadern, München, D-81366, Germany; 3 Institut für Strukturforschung, Max Planck Institute of Biochemistry, Martinsried, Germany

1

Fc-receptors of IgG (FcγR) play a crucial role in antibody-mediated autoimmune diseases by linking the humoral to the cellular immune response. Therefore we investigated in animal models whether the administration of soluble Fc-receptors (sFcR) as antagonists of immune complex recognition could influence the course of autoantibody mediated autoimmune disorders. In vitro investigations show that sFcR bind to human and mice pooled IgG, inhibit antibody mediated phagocytosis of red blood cells and are strong antagonists of immune complex binding to B-cells. Finally, in murine models of Multiple Sclerosis (MS), Idiopathic Thrombocytopenic Purpura, Rheumatoid Arthritis and Systemic Lupus Erythematodes treatment with sFcR significantly reduces the severity of symptoms thereby demonstrating the model independency of the approach. Histopathological examinations of the spinal cord of sFcR treated mice in experimental autoimmune encephalomyelitis revealed a decreased amount of infiltrated cells and especially reduced numbers of activated macrophages. Furthermore treatment resulted in decreased demyelinated plaques and axonal damage. Mice receiving the soluble receptor also showed lower levels of pathogenic antibodies and less activated B-cells. PP07-21

Involvement of MMP-12 in various experimental autoimmune encephalomyelitis models of Multiple Sclerosis

Department of Neurology, Thomas Jefferson University, Philadelphia, USA; bDepartment of Neurology, University of Nottingham, Nottingham, United Kingdom The aim of this study was to examine the effect of TLR4 stimulation in EAE, an animal model of multiple sclerosis (MS). Upon recognition of bacterial LPS, TLR4 signaling induces the expression of a range of proand anti-inflammatory mediators. The role of TLR4 in autoimmunity is not well characterized. We induced chronic EAE in female C57BL/6 mice by subcutaneous immunization with myelin oligodendrocyte glycoprotein (MOG35–55) in CFA and intraperitoneal (i.p.) administration of pertussis toxin on days 0 and 2. Mice received LPS (25 μg/day) or PBS i.p. on days 0–5 and were sacrificed at various timepoints. Spleen cells were cultured with or without antigen and assessed for proliferation. Culture supernatants were assayed for cytokine levels by cytometric bead arrays and ELISA. Spinal cord cellular infiltration was characterized by flow cytometry. EAE onset was significantly delayed in LPS treated mice compared to PBS treated groups. Regardless of antigenic stimulus in vitro, the monocyte/macrophage chemoattractant, MCP-1 (CCL2), was elevated in splenocyte culture supernatants of LPS treated mice compared to PBS controls at day 19 post immunization (p.i.). Despite significantly greater T cell proliferation in LPS treated mice at day 6 p.i., there was lower inflammatory infiltration of the spinal cord of these mice at day 19 p.i. compared to PBS treated mice. We conclude that stimulation of TLR4 delays the onset of chronic EAE by mechanisms which include inhibition of lympho-mononuclear cell infiltration into the CNS. This may be due to enhanced chemotactic activity of CCL-2 in the peripheral immune system. PP07-23

B. Grécoa, V. Muzioa, M.C. Magnonea, J.-Y. Gillonb, T. Wellsb, P.F. Zaratina

Argatroban, a direct thrombin inhibitor, suppresses the severity of experimental autoimmune encephalomyelitis

Istituto Di Richerche Biomediche “A. Marxer” — RBM/Serono, Ivrea, Italy; bSerono Pharmaceutical Research Institute, Geneva, Swizerland

S. Miyoshia, M. Kyogashimab, T. Shinc, K. Nakayamaa M. Ichikawaa, Y. Inabaa, S. Ishikawad and C.-S. Koha

a

Abstracts a

Shinshu University, Matsumoto, Japan; bAichi Cancer Center Research Institute, Nagoya, Japan; cCheju National University, Jeju, South Korea; d Shinshu University Hospital, Matsumoto, Japan Experimental autoimmune encephalomyelitis (EAE) is an inflammatory and demyelinating autoimmune disease of the central nervous system (CNS) that provides a model for human multiple sclerosis (MS). Numerous studies have proved the correlation between the coagulation–fibrinolysis system and the pathogenesis of EAE. We have examined the role of coagulation system in EAE by using argatroban, which is a direct inhibitor of thrombin. Argatroban treatment significantly suppressed clinical manifestations of EAE in Lewis rats; the mean cumulative clinical score for argatroban treated rats was 5.0, while saline treated controls scored 9.1 (P < .01). Plasma thrombin– antithrombin Ø complex (TAT) values were significantly lower in argatroban treated rats compared with saline-treated controls (p < 0.01). Immunohistologically, perivascular fibrin depositions were reduced in lumbosacrococcygeal spinal cord of guinea-pig myelin basic protein (GPMBP)-sensitized rats treated with argatroban. RT-PCR revealed that the cytokine levels was reduced within spinal cord of argatroban treated rats compared with control rats, showing cell migration is suppressed by inhibition of fibrin deposition. These results suggest that the activation of coagulation cascade is an important role for development of EAE. Furthermore, our data suggest that fibrin could be a potential therapeutic target in MS. Anticoagulation therapies aiming selectively inhibit the fibrin deposition might be a safer approach than the exogenous administration derived from organisms. PP07-24

Vitamin K2 ameliorates experimental autoimmune encephalomyelitis in Lewis rats M. Moriya, Y. Nakatsuji, T. Okuno, S. Sakoda Department of Neurology, Osaka University Graduate School of Medicine, Suita, Japan Vitamin K2 (VK2) has been widely used for the management of osteoporosis in Japan. It was demonstrated that HMG-coA reductase inhibitors (statins) have beneficial effects on experimental autoimmune encephalomyelitis (EAE), and the inhibition of geranylgeranyl-pyrophosphate (GGPP) synthesis plays a key role in the effects. Since the side chain of VK2 has a common structure to geranylgeranyl-pyrophosphate (GGPP), it could be expected that VK2 has similar effects as seen in statins. Lewis rats were actively immunized with myelin basic protein (MBP). Each rats received intraperitoneal administration of VK2 and clinical scores were evaluated daily. Histological, immunohistochemical, and Western blot analyses of the spinal cords and cultured glial cells were also performed. The clinical severity of EAE was significantly ameliorated by the prophylactic administration of VK2. Histological analysis revealed that inflammatory cellular infiltration was significantly reduced in the spinal cords of VK2treated rats. Immunohistochemical and Western blot analyses revealed that the expression of both MHC class II and inducible nitric oxide synthase (iNOS) were significantly reduced in the spinal cords of VK2-treated rats with EAE. The inhibitory effect of VK2 on the iNOS expression in glial cells was also observed in vitro. Considering the wide use of VK2 without noticeable untoward effects, it may be applicable to the patients with MS. PP07-25

TWEAK and Fn14: Novel molecular targets for the treatment of multiple sclerosis? A. M. Mueller, X. Pedré, I. Kleiter, A. Steinbrecher Department of Neurology, University of Regensburg, 93053 Regensburg, Germany

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Background: The TNF-related weak inducer of apoptosis (TWEAK) mediates proinflammatory effects by its receptor fibroblast-growth-factorinducible-14 (Fn14). We previously showed that TWEAK/Fn14-interactions contribute to lesion development in experimental autoimmune encephalomyelitis (EAE) and that neutralisation of TWEAK or Fn14 ameliorates CNS inflammation in EAE. TWEAK also increased CCL2 release by CNS resident cells. Objective: Revealing the mechanism(s) of TWEAK/Fn14 promoting autoimmune inflammation. Methods: Neutralising TWEAK/Fn14-antibodies were induced by vaccination with the extracellular domain of TWEAK or full length Fn14. Adoptive transfer EAE (AT-EAE) was induced in SJL mice by transfer of PLP-specific lymph node cells (LNCs). C57Bl/6 CCL2+/+ or CCL2−/− mice were immunised with MOG-peptide to induce active EAE. Chemokine release in culture supernatants was measured by ELISA. Results: AT-EAE using vaccination of either donor or recipient mice showed that the primary immune response and T cell encephalitogenicity are not modulated by TWEAK/Fn14. However, Fn14-vaccination of Tcell-recipient mice resulted in significant amelioration of EAE-signs suggesting the blood–brain-barrier as a potential site of action. Vaccination against TWEAK or Fn14 protected both CCL2+/+ and – to a lesser extent – CCL2−/− mice from the development of EAE indicating that the effect of TWEAK/Fn14-blockade on lesion development is only partially dependent on CCL2. Finally, data will be presented showing the influence of TWEAK/Fn14 on migration of mononuclear cells in an in vitro transwell system. Conclusion: TWEAK/Fn14 might promote lesion development via several mechanisms. While TWEAK-induced modulation of CCL2 and other proinflammatory molecules by TWEAK may be involved, TWEAK does not alter the encephalitogenic T cell response. PP07-26

Nucleoside analogues effect on glial response in experimental autoimmune encephalomyelitis Danijela Stojkov1, Irena Lavrnja1, Sanja Pekovic1, Sanja Subasic2, Sasa Jovanovic1, Ivana Nikic2, Ivana Bjelobaba1, Marija Mostarica-Stojkovic3, Stanislava Stosic-Grujicic1, Ljubisav Rakic1, Mirjana Stojiljkovic1 1

Deptartment of Neurobiology and Immunology, Institute for Biological Research “Sinisa Stankovic”, Belgrade, 11000, Serbia and Montenegro; 2 Faculty of Biology, Belgrade, 11000, Serbia and Montenegro; 3Institute for Microbiology and Immunology, School of Medicine, Belgrade, 11000, Serbia and Montenegro

Experimental autoimmune encephalomyelitis (EAE) is an animal model of human disease multiple sclerosis (MS). Clinical signs of EAE are result of an autoaggressive T-cell response against myelin. We have previously shown that combined treatment with nucleoside analogues (ribavirin — R + tiazofurin — T), inosine monophosphate dehydrogenase inhibitors, ameliorates clinical signs and histological lesions of EAE in susceptible rats, when they are given preventatively. The aim of this study was to investigate the effect of combined treatment with R + T, given with the appearance of first EAE clinical sign, on microglia and astrocytes response. These cells of the target tissue also participate in an autoimmune process. The disease was induced in Dark Agouti rats with rat spinal cord homogenate and had acute monophasic course. Ribavirin and tiazofurin were given at a dosage of 30 mg/kg/day and 10 mg/kg every other day, for 15 days, respectively. Control group was immunized and treated with saline. Amelioration of clinical signs and faster recovery was shown in group treated with combination of R and T in comparison to control group. Immunohistochemical analysis of the spinal cord tissue isolated after 15 days of combined therapy revealed decrease in vimentin positive cells and microglia compared to control group. Additionally, morphology of GFAP positive (glial fibrillary acid protein) cells and microglia indicated to reactive type of these cells in control group. Results of this study revealed

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that R and T modulate glial response and have EAE protective effects when they are given from the onset of disease. (This work was supported from Ministry of Science and Environmental Protection, Republic of Serbia, Serbia and Montenegro, Grants 143005, 143029 and 145066) PP07-27

Multiple neuroprotective mechanisms of minocycline in autoimmune CNS inflammation Katharina Maiera, Doron Merklerb, Joachim Gerbera, Naimeh Taheria, Antje V. Kuhnerta, Sarah K. Williamsa, Clemens Neuscha, Mathias Bähra, and Ricarda Diema

limiting factor for remyelination. We have designed a treatment strategy for ongoing EAE that combines γ-secretase inhibition (which blocks Notch signaling in addition to a host of other factors) and anti-CD80(Fab), a wellestablished immunoregulatory strategy. We are additionally designing and testing other myelin repair strategies in combination with immunoregulatory techniques. Preliminary analyses of T cell responses and the extent of lesion repair following combinatorial treatments have yielded promising results for the future of MS therapy. PP07-29

Effect of anti-CD81 antibodies in experimental autoimmune encephalitis S. Dijkstraa, R. Verbeekb, J.M. van Noortb, C.D. Dijkstraa and H.E. de Vriesa

Neurologische Universitätsklinik; bInstitut für Neuropathologie, Göttingen, Germany

a

Axonal destruction and neuronal loss occur early during multiple sclerosis, an autoimmune inflammatory CNS disease that frequently manifests with acute optic neuritis. Available therapies mainly target the inflammatory component of the disease but fail to prevent neurodegeneration. To investigate the effect of minocycline on the survival of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve, we used a rat model of myelin oligodendrocyte glycoprotein (MOG)induced experimental autoimmune encephalomyelitis. Optic neuritis in this model was diagnosed by recording visual evoked potentials and RGC function was monitored by measuring electroretinograms. Functional and histopathological data of RGCs and optic nerves revealed neuronal and axonal protection when minocycline treatment was started on the day of immunization. Furthermore, we demonstrate that minocycline-induced neuroprotection is related to a direct antagonism of multiple mechanisms leading to neuronal cell death such as the induction of anti-apoptotic intracellular signalling pathways and a decrease in glutamate excitotoxicity. From these observations, we conclude that minocycline exerts neuroprotective effects independent of its anti-inflammatory properties. This hypothesis was confirmed in a non-inflammatory disease model leading to degeneration of RGCs, the surgical transection of the optic nerve.

VU University Medical Center, Amsterdam, The Netherlands; bTNOQuality of Life, Leiden, The Netherlands

a

Infiltration of leukocytes into the CNS is a key event in the formation of new lesions in multiple sclerosis (MS). Molecules involved in the interaction between leukocytes and endothelial cells represent prime candidate targets for novel therapeutic approaches. We have previously shown that the tetraspanin CD81, a regulator of the VLA-4 integrin, is involved in the migration of monocytes across the blood brain barrier in vitro. Here we asked whether inhibition of monocyte migration in vivo by use of anti-CD81 antibodies would have a beneficial effect on neurological symptoms in experimental autoimmune encephalitis. Mice were immunized with PLP139–151 and treated with two different hamster anti-mouse CD81 antibodies, isotype control mAb or vehicle (n = 10 per group). Antibodies were injected i.p. at 100 μg per injection every other day for 3 weeks starting 5 days post immunisation. The results showed that clinical symptoms were reduced by 47% in mice treated with Eat2 mAb as compared to vehicle (cumulative clinical scores 25± 7 vs. 47 ± 9; p < 0.05). Interestingly, treatment with the other anti-CD81 mAb 2F7 had no effect on clinical scores, whereas an irrelevant mAb resulted in intermediate scores. Our results provide the first indication that targeting of CD81 in vivo has a beneficial effect on neurological outcome during EAE. We are now validating these results in additional EAE models and studying the pathogenetic mechanisms underlying the treatment effect.

PP07-28 PP07-31

Combining immunotherapy with strategies to promote myelin repair for the treatment of established EAE T. J. Frederick, W.S. Begolka, B. Burnette, M. Degutes and S.D. Miller

Allogeneic versus syngeneic BMT for prevention of EAE: Role of donor chimerism, donor susceptibility to EAE and alloreactivity

Northwestern University, Department of Microbiology and Immunology, Chicago, IL, USA

B. Van Wijmeersch1,2, B. Sprangers1, M. Waer1, A.D. Billiau1,⁎ , B. Dubois2,⁎

Persistent central nervous system (CNS) inflammation and the failure of myelin repair during multiple sclerosis (MS) trigger a progressive deterioration in neurophysiological function and permanent clinical debilitation. Current treatment consists of immunosuppressive therapies targeted against the immune response and have been only moderately successful in ameliorating disease relapses, and have little benefit in slowing disease progression or enhancing remyelination. The goals of the current project are to investigate the cellular and molecular mechanisms regulating the processes of demyelination and remyelination during CNS autoimmune disease and to design immunoregulatory and glial regenerative combinatorial therapies for the treatment of established disease in animal models of MS. Real-time PCR data suggest a correlation between myelin gene expression in the CNS and the distinct phases of relapsing–remitting experimental autoimmune encephalomyelitis (R-EAE). Compared to preclinical stages, we have observed decreased myelin gene expression during acute disease and first relapse and increased Notch signaling molecule expression during R-EAE. Notch inhibits oligodendrocyte differentiation and myelination during development and has been hypothesized as a

1

Laboratory of Experimental Transplantation, 2Laboratory of Neuroimmunology, University of Leuven, Belgium ⁎Contributed equally

Background: Autologous bone marrow transplantation (autoBMT) is explored as a novel treatment for MS. Case reports exist on beneficial effects of allogeneic BMT (alloBMT). However, unresolved questions about the action mechanism warrant investigation in animal models. Aim: To study EAE incidence and disease course after murine BMT and explore the role of donor-EAE-susceptibility, degree of donor-chimerism, and posttransplant alloreactivity. Methods: Allogeneic and syngeneic radiation BM chimeras were prepared using Balb/c (EAE-resistant), C57BL/6 and B6SJLF1 (EAE-susceptible) mice. Some alloBMT groups were given donor-type leukocyte infusions (DLI) to increase donor-chimerism. In all, MOG35–55-EAE was induced on day 28 or 42, followed 14 days later by ex vivo MOG35–55 splenocyte proliferation assays.

Abstracts Results: In C57BL/6 syngeneic chimeras and Balb/c → C57BL/6 low-grade allogeneic chimeras, day-28-induced EAE was prevented in 80% resp. 83%, but day-42-induced EAE was prevented only in 8% resp. 25%. Full donor chimerism (through DLI) in Balb/c → C57BL/6 allogeneic chimeras resulted in complete prevention of day-42-induced EAE, and loss of the MOG35–55proliferative response. In C57BL/6 → B6SJLF1 high-grade allogeneic chimeras, induced after irradiation, neither prevention of EAE nor loss of MOG35–55-specific proliferation were seen. In contrast, non-radiation high-grade C57BL/ 6 → B6SJLF1 chimeras obtained by repetitive DLI (strong in vivo alloreactivity), again resulted in complete prevention of EAE and loss of MOG35–55-specific proliferation. Conclusion: The transient EAE-protective effect of synBMT probably relates to posttransplant aspecific immunosuppression. High-grade donorchimerism from an EAE-non-susceptible donor confers robust EAEresistance, indicating the role of genetic susceptibility. However, equally strong protection was obtained using an EAE-susceptible donor, when a non-irradiation model of DLI-induced chimerism was used, suggesting a role for alloreactivity. PP07-32

Suppression of experimental autoimmune encephalomyelitis by transfer of lymphokine-activated natural killer (NK) cells Shigemi Nagayama, Sachiko Miyake, and Takashi Yamamura

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a

Department of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan; bDepartment of Biochemistry, Graduate School of Medicine, Nagoya University, Nagoya, Japan

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), is a Th1-mediated inflammatory demyelinating disease of the CNS. CD4+CD25+Foxp3+ regulatory T cells (Treg) play crucial roles in the induction of peripheral tolerance by controlling autoreactive T cells. Increasing evidences have shown that Treg cells play key roles on negative regulations the pathogenesis of MS. However, the endogenous factors and mechanisms controlling the peripheral generation of Treg cells are largely unknown. Midkine (MK) is, a 13-KD protein, a member of the heparin-binding growth factors and reportedly exerts pleiotropic activities on cell proliferation, migration, angiogenesis and fibrinolysis in a variety of tissues. In this study, we show that MK-deficient mice are resistant to the induction and progression of EAE. This resistance was closely associated with substantial expansion of Treg cells in peripheral lymph nodes after MOG35–55 immunization as compared with those in wild mice. Administration of MK to MK deficient mice significantly abrogated the resistance of EAE and suppressed the expansion of Treg cells in vivo and in vitro. In addition, the frequency of autoreactive Th1 cells decreased without affecting Th2 population. These results indicate that MK deficiency results in expansion of Treg cells in periphery and control the activation of autoreactive Th1. This is the first demonstration that MK deficiency plays a critical immunomodulatory role in the development of EAE. Target for MK deficiency may be an effective therapeutic strategy for autoimmune diseases, including MS.

Department of Immunology, National Institute of Neuroscience, NCNP, Japan PP07-34 Objective: The purpose of this experiment is to explore if passive transfer of natural killer (NK) cells may suppress the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Methods: EAE was induced in B6 mice by sensitization with myelin oligodendrocyte glycoprotein (MOG). NK cells were isolated from the spleen of non-immunized B6 mice, activated in vitro with IL-2, and were inoculated intravenously into MOG-sensitized mice on the indicated days. Results: At first, we studied whether NK cells are infiltrated within lesions of the central nervous system (CNS) of EAE mice. Immunofluorescence analysis of mononuclear cells isolated from the CNS of MOG-sensitized mice on Days 7, 14 or 21 after sensitization demonstrated the presence of NK cells (CD3-NK1.1+ cells), with the positive correlation of the number of migrated NK cells with the clinical severity of EAE. The intravenous injection of in vitro IL-2-activated NK cells (NK-LAK cells) into MOGsensitized mice on Days 7, 14 and 21 after sensitization clearly suppressed clinical severity of EAE. In order to clarify whether passively transferred NK-LAK cells could infiltrate into the CNS lesion, we transferred NK-LAK cells of green mice into EAE-positive B6 mice. We found that NK-LAK cells of green mice could migrate to the CNS of mice developing EAE. Conclusion: These findings showed that transfer of NK-LAK cells could down-regulate the ongoing inflammation in the CNS lesions of EAE. PP07-33

Expansion of CD4+CD25+Foxp3+ regulatory T cells in Midkine-deficiency mice results in partial resistance to experimental autoimmune encephalomyelitis: A novel immunomodulation by Midkine Jinyan Wanga, Hideyuki Takeuchia, Shijie Jina, Jun Kawanokuchia, Reiko Kunoa, Yoshifumi Sonobea, Izumi Yawataa, Yukiko Doia, Jianfeng Lianga, Guiqin Zhanga, Tetsuya Mizunoa, Hisako Muramatsub, Takashi Muramatsub, and Akio Suzumuraa

RPE cells differentially regulate the activation of uveitogenic CD4 and CD8 T cells Hui Shao, Yan Ke, Ping Zhang, Henry J. Kaplan, Deming Sun Department of Ophthalmology, Kentucky Lion Eye Center and Vision Sciences, University of Louisville, Louisville, KY 40202, USA In previous studies we have shown that RPE (retinal pigment epithelium) were able to express MHC class II if they were exposed to inflammatory cytokines such as IFN-γ. In this study, we determined the in vitro activation of IRBP (Interphotoreceptor Binding Protein) specific CD4 and CD8 T cells in the presence or absence of RPE cells and the factors produced by nonactivated and activated RPE. We also compared the effect of astrocytes, another eye immuno-competent cell, on the in vitro activation of uveitogenic CD4 and CD8 cells. Our results have showed RPE cells inhibited the high-degree-activated, both IRBP-specific CD4 and CD8 T cells, but enhanced low-degreeactivated T cells; differentially, astrocytes inhibited IRBP-CD4 at both highand low-activated degree, but enhanced low-degree-activated CD8 cells. Moreover, factors produced by activated RPE enhanced IRBP-CD4 but inhibited CD8 T cell activation and the factors produced by activated astrocytes inhibited both T cells. Our results suggest that (1) both RPE and astrocytes have regulatory effects on activation of T cells in the eye. At least, a part of the regulatory effect is mediated by the factors they produce, (2): different parenchymal cells of the eye may have distinct regulatory effects on CD4 and CD8 autoreactive T cell. Therefore, studies on the mechanism by which parenchymal cells of the eye regulate the invading autoreactive T cells should provide needed information for therapeutic intervention. Supported in part by NIH grants NEI EY12974 (HS), EY14599 (HS), NEIEY014366 (DS), Vision Research Infrastructure Development (R24 EY015636), grant RG3413A4 from the National Multiple Sclerosis Society, and the Commonwealth of Kentucky Research Challenge Trust Fund.

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Poster Session 8: Neuro-endocrine-immune interaction

Abstracts PP08-01

Estrogen-mediated immunomodulation involves reduced activation of effector T cells, potentiation of Treg cells, and enhanced expression of the PD-1 co-stimulatory pathway Halina Offner1,2,3, Corwyn Hopke3, Arthur A. Vandenbark1,3,4, and Magdalena J. Polanczyk1,3,5 1

Department of Neurology, Oregon Health and Science University, Portland, OR 97239, USA; 2Department of Anesthesiology and Perioperative Medicine, Oregon Health and Science University, Portland, OR 97239, USA; 3Neuroimmunology Research, Veterans Affairs Medical Center, Portland, OR 97239, USA; 4Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239, USA; 5Department of Food Hygiene, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland

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induced Ca+ mobilisation was reduced by selective MOR antagonist betafunaltrexamine suggesting a MOR-mediated action. In short, cells of immature myelo-monocytic HL-60 line express MOR both at the level of mRNA and at the level of a functionally active protein capable of transmitting intracellular signals via Ca+ upon ligation with selective agonists of alkaloid or peptide structure. (1) Gabrilovac J et al., Membrane expression of delta, mu and kappa opioid receptors on HL-60, THP-1 and U937 cell lines. Annual Meeting of the Croatian Immunological Society, Book of Abstracts, Jonjić, S. (ed). Rijeka, 2004, 43. PP08-03

Crosstalk between chemokine, opioid, and vanilloid receptors Ning Zhang1,2 and Joost Oppenheim1 1

Estrogen (E2)-induced immunomodulation involves dual effects on antigen presenting cells (APC) and CD4+CD25+ regulatory T cells (Treg), but not a direct effect on effector T cells. In this report, we further investigated the effects of E2 on APC and Treg cell function. We found that E2 treatment in vivo strongly reduced recovery of APC from the peritoneal cavity and inhibited their ability to induce inflammatory cytokines, IL-12 and IFN-γ, but enhanced secretion of IL-10. Moreover, E2 conditioned bone marrow-derived dendritic cells (BM-DC) could both enhance Treg cell activity and directly inhibit responder T cells in the absence of Treg cells. We examined whether this E2-induced inhibitory activity of BM-DC might involve co-stimulation through the recently described PD-1 pathway. Both E2 and pregnancy markedly enhanced PD-1 expression in several types of APC, including macrophages, B cells, and especially dendritic cells (DC). Similar to E2-induced enhancement of FoxP3 expression and EAE protection, E2-induced enhancement of PD-1+ cells was also mediated through estrogen receptor alpha (Esr1) in DC and macrophages, but not B cells. Based on antibody inhibition studies, PD-1 interaction with its ligands, PDL-1 and especially PDL-2, could mediate either positive or negative regulatory signaling in both mature and immature E2-conditioned DC, depending respectively on a relatively high (10:1) or low (1:1) ratio of T cells:BM-DC. These novel findings indicate that E2-induced immunomodulation is mediated in part through potentiation in BM-DC of the PD-1 costimulatory pathway.

Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, Building 560, Room 21-89A, Frederick, MD 21702-1201, USA; 2Department of Chemical Biology, and State Key Laboratory of Molecular Dynamic and Stable Structures, College of Chemistry, Peking University, Beijing, 100871, China Chemokine receptors serve as a bridge between the immune and neural systems. Neuropeptides, such as opioids, inhibit chemokine receptor function on leukocytes by activating Gi protein and calcium-independent protein kinase C. Conversely, during inflammation, chemokines similarly regulate neuronal sensing in two ways. Activation of chemokine receptors on neurons desensitizes the analgesic μ-opioid receptor and concomitantly enhances the sensitivity of a pain receptor, TRPV1, via protein kinase Cdependent phosphorylation, resulting in hyperalgesic effects. We will present the cellular, biochemical, and in vivo evidence to show that receptor cross-talk provides an important pathway for neuroimmune interactions. PP08-04

The molecular structure basis and receptor mechanisms of interferon-alpha on hypothalamic–pituitary–adrenal axis Yun-Xia Wanga, Feng-Yong Yanga,b, Lei Liua, Hao Yub, Chun-Lei Jianga a

PP08-02

Expression and functional activity of mu-opioid receptors (MOR) on a myelo-monocytic HL-60 cell line J. Gabrilovac, D. Breljak, B. Čupić Ruđer Bošković Institute, Division of Molecular Medicine, Zagreb, Croatia Outside nervous system, opioid receptors were also found on cells of hematopoietic origin in which they participate in modulation of growth, differentiation and functional activity. Based on ligand selectivity there are three main classes of opioid receptors: delta, mu and kappa. Mu-opioid receptors (MOR) are the most important class involved in analgesia mediated by alkaloid opiate morphine. By using polyclonal antibodies to mu-opioid receptors and FACS analysis we have recently reported membrane expression of MOR on a sub-population of HL-60 cells (1). Extending that study we have examined transcriptional expression of MOR by using RT-PCR. The functionality of MOR expressed on HL-60 cells was examined by measuring Ca+ mobilisation triggered by selective MOR agonists. Morphine and endomorphin-2 were used as representative agonists with alkaloid or peptide structure, respectively. Presence of mRNA for MOR was detected in HL-60 cells, which corresponded to that in a neuronal cell line SH-SY5Y. Both morphine and endomorphin-2 triggered Ca+ mobilisation in HL-60 cells in a concentration-dependent manner. Endomorphin-2-

Department of Nautical Medicine, Laboratory of Stress Medicine, Second Military Medical University, Shanghai, PR China; b Department of Pathophysiology, Medical College of Qingdao University, Qingdao, PR China Hypothalamic–pituitary–adrenal (HPA) axis plays a pivotal role in responding to immunological challenges. Cytokines can modulate the function of the HPA axis. Interferon-alpha (IFNα) also has this effect, but the mechanisms are not thoroughly characterized, and the results remain controversial and conflict. This study is to explore the molecular structure basis and receptor mechanisms of IFNα upon the HPA axis in vivo and vitro. Two kinds of IFNα mutants were obtained and used, which are 129Ser-IFNα with analgesic activity and 38Leu-IFNα with antiviral activity. After administration of IFNα and its mutants to ventricle in vivo and to cultured hypothalamic slices, AtT20 cells, and adrenal cortical slices, the contents of CRH, ACTH, and corticosterone in samples were assayed respectively. The results showed that Wild-IFNα and 129Ser-IFNα could decrease CRH, ACTH and corticosterone contents in vivo, and decrease CRH content, stimulate corticosterone secretion in vitro significantly, which could be blocked by opioid receptor antagonist naloxone pretreated. 129SIFNα had no direct effects on ACTH secretion in AtT20 cells without μ opioid receptor expression. 38L-IFNα also had similar effects in vivo and vitro, but with less potency and insensitive to naloxone. It is the first highly suggested that IFNα maybe regulate the HPA axis at least by two different receptor mechanisms: one is opioid receptors and the molecular structure basis is opioid domain of IFNα.

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This work was supported by National Natural Sciences Foundation of China (30300122).

PP08-08

Immunomodulatory effects of SOM 230 PP08-05 Mastrotto C., Casnici C., Lattuada D., Crotta K., Franco P., Marelli O.

Roles of brain IFN-α and 5-HT transporter in immunologically induced fatigue as a model for chronic fatigue syndrome T. Katafuchi Department of Integrative Physiology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan To investigate brain mechanisms of fatigue, the immunologically induced fatigue was conducted in rats by intraperitoneal (IP) injection of a synthetic double-stranded RNAs, polyriboinosinic: polyribocytidylic acid (poly I:C). An IP injection of poly I:C (3 mg/kg) decreased the daily amounts of spontaneous running wheel activity to about 40–60% of the preinjection level until day 9, and then gradually returned to the baseline level by day 14. The amount of mRNA for IFN-α measured by real-time capillary PCR method increased in the hypothalamic nuclei and cortex on day 1 and even day 7. Expression of 5-HT transporter, which is reported be induced by IFNα, also enhanced in the same brain regions where IFN-α increased on day 7. In vivo brain microdialysis revealed a decrease in extracellular concentration of 5-HT in the prefrontal cortex after poly I:C, which was blocked by a selective 5-HT reuptake inhibitor, fluoxetine. Finally, the poly I:C-induced suppression of the running wheel activity was significantly attenuated by a 5-HT1A receptor agonist, but not by 5-HT2, 5-HT3, or dopamine D3 receptor agonists. These findings, taken together, suggest that brain IFN-α and 5HT1A receptors play an important role in the neuronal mechanisms of the poly I:C-induced fatigue. Reference: Katafuchi T, et al.: Neuroscience 120: 837–845, 2003; Katafuchi, T. et al.; Eur. J. Neurosci. 22: 2817–2826, 2005. PP08-07

Department of Pharmacology — University of Milan, Italy The antiproliferative effect of a somatostatin analog SOM 230 and its capacity to regulate cytokine release were evaluated on human peripheral blood lymphocytes (hPBL) activated by phytohemoagglutinin (PHA), pokeweed (PWM) or alloantigens (MLR). PBL proliferation, measured by thymidine uptake, resulted significantly inhibited aside from the stimulus, but alloantigen stimulation were the most affected by the presence of SOM 230 and while mitogen induced proliferation were inhibited by 10− 11 M the alloantigen induced one by 10− 13 M. The profile of cytokine release were evaluated in MLR supernatants by an ELISA test starting at 24 h till five days of culture. IFN gamma, IL2, and IL6 release resulted, inhibited by the presence of SOM 230 while we could not see significant changes in the release of IL4, IL10 and IL12. To evaluate the growth inhibition of lymphocytes, we studied the DNA distribution in hPBL stimulated by PHA or PWM in the presence of SOM-230 10− 11 M by propidium iodure incorporation by flow cytometry analysis. After 48 h of stimulation with PHA or PWM in the presence of SOM 230 the cell cycle analysis demonstrated a modest accumulation of cells in the S-phase. This phenomenon were then analysed by 5-bromo-2′-deoxyuridine (BrdU) incorporation for 30 h with no significative results. We are now studying the cell cycle by a longer pulse of BrdU and, on the other hand, we are evaluating if the antiproliferative effect of SOM 230 is dependent either on a apoptotic (by annexin V test) or autophagic effect (by LC3 immunofluorescence test). PP08-09

Regulation of immune cell function by a non-neuronal cholinergic system

Induction of microglial apoptosis by corticotropin-releasing hormone: A potential regulatory mechanism of neuroinflammation

Y. Moriwaki, K. Yoshikawa, Y. Fujii, and K. Kawashima

K. Suk, J. Ock, S. Kim, and J. Lee

Department of Pharmacology, Kyoritsu College of Pharmacy, Tokyo, Japan

Department of Pharmacology, Kyungpook National University School of Medicine, Daegu, 700-422 Korea; e-mail, [email protected]

Lymphocytes express all the components required to constitute an independent cholinergic system, such as acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and muscarinic and nicotinic ACh receptors (mAChR and nAChR, respectively). Stimulation of mAChRs on T and B cells causes oscillating intracellular Ca2+ signaling and up-regulates c-fos gene expression. Stimulation of nAChRs produces rapid and transient intracellular Ca2+ signaling. We investigated the gene expression of cholinergic components in splenic mononuclear cells (MNLs), bone marrow-derived dendritic cells (BMDCs), and macrophages of C57BL/6J mice. In resting condition, ChAT gene expression was detected only in the brain by RT-PCR, while AChE gene expression was observed in all samples. After ConA or LPS stimulation, ChAT gene expression was detected in MNLs and BMDCs, respectively, suggesting that immunological stimulation induces ACh synthesis. Genes for M1–M5 mAChRs were expressed in all samples. Genes for the α2, α5, α6, α7, α10, and β2 subunits of the nAChR were expressed in all samples, while no expression was detected for the genes for α3 and β3. α4, α9, and β5 gene expressions varied among the samples. The gene for SLURP-1, a recently discovered endogenous peptide allosteric ligand of α7 nAChR, was expressed in all samples. These results suggest that ACh and SLURP-1 play intermediary roles in the dialogue among lymphocytes, BMDCs, and macrophages during immune responses via mAChR- and nAChRmediated pathways in autocrine and/or paracrine fashion, via contact with each other through cell surface molecules such as ICAM-1/LFA and CD4/MHC-II.

Neuropeptides are short-chain peptides found in brain tissue, with some functioning as neurotransmitters and others functioning as hormones. Neuropeptides may directly or indirectly modulate glial functions in the central nervous system (CNS). In the current study, effects of various neuropeptides on the viability and inflammatory activation of cultured microglia have been investigated. While vasoactive intestinal peptide, substance P, cholecystokinin, or neuropeptide Y did not affect microglial cell viability, corticotropin-releasing hormone (CRH) induced a classical apoptosis of mouse microglia in culture as evidenced by nuclear condensation and fragmentation, TUNEL staining, and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP) protein. CRH, however, did not influence nitric oxide production (assessed by Griess reaction) or inflammatory gene expression (assessed by RT-PCR and Western blot analysis) including cytokines and chemokines, indicating that CRH did not affect the inflammatory activation of microglia. The CRH-induced microglial apoptosis appeared to involve a mitochondrial pathway and reactive oxygen species based on mitochondrial membrane potential change, caspase-9 activation, and the sensitivity to antioxidants. Taken together, our results indicate that the stress neuropeptide CRH may regulate neuroinflammation by inducing the apoptosis of microglia, the major cellular source of inflammatory mediators in CNS. Keywords: Apoptosis; Microglia; Inflammation.

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β-Adrenoceptor activation by noradrenaline stimulates production of Interleukin-1 receptor antagonist in primary glial cells E.N. Namee, T.J. Connor Neuroimmunomodulation Research Group, School of Medicine and Trinity College Institute of Neuroscience, University of Dublin, Trinity College, Dublin 2, Ireland

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through second messengers. In T cells, nicotine modulates two distinct signaling pathways: 1. Nicotine increase [Ca2+]i through the Src-like protein tyrosine kinase, Lck that requires the functional integrity of the T cell receptor. 2. Nicotine affects NFκB activation through ERK that is independent of TCR. Thus nicotine modulates T cell and inflammatory responses through the sympathetic autonomic nervous system as well as via direct interaction with a unique α7-nAChR. Thus, nicotine controls the adaptive and inflammatory responses through sans and direct interaction with T cells via two distinct signaling pathways. (Supported by NIDA RO117003, RO1-04208-12, and RO1-04208-7S.) PP08-12

Evidence suggests that inflammation is a significant contributor to pathology in a number of neurodegenerative disease states. In this regard, the pro-inflammatory cytokine interleukin-1β (IL-1β) plays a key role in initiating an immune response within the central nervous system (CNS). The actions of IL-1β can be regulated in vivo by interleukin-1 receptor antagonist (IL-1ra); a molecule that prevents IL-1β from acting on the IL1type I receptor. Consequently, the balance between IL-1ra/IL-1 β is of pathological importance, and pharmacological strategies that tip the balance in favour of IL-1ra may be of therapeutic benefit. Evidence is emerging to suggest that the neurotransmitter noradrenaline elicits anti-inflammatory actions in the CNS, and consequently may play an endogenous neuroprotective role. In this study we report that noradrenaline induces production of secreted IL-1ra from primary rat mixed glial cells, without altering IL-1β concentrations. Noradrenaline-induced IL-1ra production was attenuated by the non-selective β-adrenoceptor antagonist propranolol, and also by the selective β2-adrenoceptor antagonist butoxamine, but not by the selective β1-adrenoceptor antagonist metoprolol. Furthermore, the selective β2-adrenoceptor agonist salbutamol mimicked the ability of noradrenaline to increase IL-1ra production. Taken together these data indicate that the β2-adrenoceptor mediates the ability of noradrenaline to increase IL-1ra production. Consistent with the ability of β-adrenoceptors to activate the cAMP-protein kinase A pathway, the stable cAMP analog dibutryl cAMP mimicked, and the PKA antagonist RP-cAMPs blocked the ability of noradrenaline to increase IL-1ra. Overall, these data further demonstrate that noradrenaline is a neurotransmitter with anti-inflammatory and therefore potential neuroprotective actions within the CNS. The authors acknowledge grant support from Science Foundation Ireland. PP08-11

Nicotine controls T cell function through direct interaction and through the CNS M.L. Sopori1, S. Razani-Boroujerdi1, T. Boyd2, R. Kalra1, J.S. Nandi1, M. Davila-Garcia3, N. Mishra1, R. Langley1, S. Singh1 1

Immunology Division, Lovelace Respiratory Research Institute, Albuquerque, USA; 2Ohio State University, Columbus, USA; 3Howard University, Washington, DC, USA Lymphoid tissues are innervated by the sympathetic autonomic nerve terminals, and the role of this innervation in the immune tissues is not clear. Chronic exposure to nicotine causes immunosuppression and impairs T cell function. When given intracerebroventricularly (ICV), relatively small concentrations of nicotine affect T cell responses, and these effects are not significantly influenced by adrenalectomy, indicating that the HPA axis does not play a major role in the nicotine-induced immunosuppression. However, the immunosuppression is blocked by ganglionic blockers, indicating that under these conditions the effects of nicotine are regulated by the sympathetic autonomic nervous system (sANS). Peripheral administration of nicotine at concentrations seen in smokers, also suppresses the immune system, and the suppression is only partially attenuated by ganglionic blockers. Indeed, T cells express α7-nAChRs; however, in spite of near total homology with the neuronal α7-nAChR, the T cell α7-nAChRs do not form detectable levels of ligand-gated Ca2+ channels and operate essentially

Investigation of sensory neurogenic components in bleomycininduced scleroderma model using TRPV1 receptor and CGRP knockout mice Erika Pintér1, Árpád Szabó1, László Czirják2, Zoltán Sándor1, Zsuzsanna Helyes1, Terézia László3, Krisztián Elekes1, Anna Starr4, János Szolcsányi1, Susan Brain4 1

Department of Pharmacology and Pharmacotherapy, University of Pécs, Pécs, Hungary; 2Department of Pathology, University of Pécs, Pécs, Hungary; 3Department of Immunology and Rheumatology, University of Pécs, Pécs, Hungary; 4Centre of Cardiovascular Biology and Medicine, Kings College, London, UK

Capsaicin-sensitive, transient receptor potential vanilloid 1 (TRPV1) receptor-expressing, sensory nerve terminals exert efferent activities besides the afferent function (nociception). Neuropeptides are released via TRPV1 activation and influence inflammatory processes. This study examined the role of TRPV1 receptors and, calcitonin gene-related peptide (CGRP) in bleomycin-induced scleroderma using transgenic mice. Cutaneous sclerosis of TRPV1 receptor and calcitonin gene-related peptide knockout (TRPV1−/−, CGRP−/−) mice and wild-type (WT) counterparts was induced by daily s.c. injection of bleomycin throughout 30 days. Control groups were treated with the solvent (PBS). The excised skin samples were investigated with histological and biochemical methods. Composite cutaneous sclerosis score was calculated on the basis of thickening, leukocyte infiltration and amount of collagen bundles. The collagen-specific amino-acid, hydroxyproline, from the skin patches was measured with spectrophotometry. Quantitative real-time RT-PCR assay was used to determine type I collagen-alpha mRNA. Bleomycin treatment induced marked cutaneous thickening and fibrosis compared to the PBS-treated group. Composite sclerosis score was 14.5% greater in TRPV1−/− and 43.1% greater in CGRP−/− mice compared to the respective WT groups. Similarly, the amount of hydroxyproline increased by 65.0% in TRPV1−/− and by 25.3% in CGRP−/− animals versus the WT group. Type I collagen-alpha mRNA content was not significantly higher in the skin of bleomycin-treated TRPV1−/− compared to TRPV+/+ mice on the 28th day of the experiment. These results suggest that activation of TRPV1 receptors by inflammatory mediators induces the release of sensory neuropeptides, which exert protective action against fibrosis. Grants: OTKA T046729, ETT-05-598/2003, RET-008/2005, The Wellcome Trust. PP08-13

Immunological and neurological roles of 80-kDa and 100-kDa haemopoietic factors A. Kikuchia,b,c, H. Tomoyasud, M. Kobayashib, N. Sakuragawab and I. Kamob a

Department of Cancer Biology, Division of Oncology, The Institute of Medical Science, The University of Tokyo, Tokyo Japan; bRegenerative

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Medicine, Allied Health Sciences, Kitasato University, Sagamihara Japan; c Institute for Animal Reproduction, Kasumigaura, Japan; dJapan Red Cross Hospital, Tokyo, Japan 80-kDa and 100-kDa factors were originally detected in the conditioned medium of cloned rat thymic myoid cells. Both factors commonly induce the growth and differentiation of monocytic lineage cells from the peritoneal, bone marrow, thymus and brain. Colonies induced by the two factors were different in morphology in an early stage of culture, but showed monocytic cell characteristics in the terminal stage. Immunohistochemical studies indicated that major producers of the two factors were neural cells in the brain and myoid cells in the thymus, suggesting their biological roles other than monocytic cell stimulatory

activity. To examine biological roles, such as immunological and neurological roles, we purified those factors from the brain cells, cultured myoid cells and hyperplastic thymus in which many precursor myoid cells were detected, and tested their immunological and neuro-stimulatory roles. We found that those two factors sustain neuronal growth and stimulate microglial cell growth. Furthermore, the factors stimulate B-cell responses in a synergic fashion with IL-2, without the aid of other T-cell factors such as IL-4 or T-cells themselves and induce immunoglobulin class switches. Those factors also appeared to stimulate T-cells to produce B-cell activating cytokines, leading to B-cell proliferation. These results suggest that the two new haemopoietic factors play important roles not only in monocytic–microglial cell diversification but also in brain physiology and many immunological events.

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Poster Session 9: Psychoneuroimmunology and cytokine effect on the CNS

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PP09-01

Interleukin-1 mediates EAE- and chronic stress-induced behavioral depression and reduced neurogenesis R. Yirmiyaa, I. Goshena, T. Kreisela, O. Ben-Menachema, Y. Pollaka, H. Ovadiab, and T. Ben-Hurb Departments of aPsychology and bNeurology, The Hebrew University of Jerusalem, Israel

challenge, we found that inflammatory cells are not required at the site of challenge in order to trigger neural or behavioral correlates of murine experimental asthma. Altogether, we build a solid ground for understanding neuroimmune interactions during allergic responses that may contribute to the comprehension of psychological disorders associated with allergy. PP09-03

Interleukin-18 induces microglial activation in acute stress S. Sugamaa, M. Hashimotob and B. Contic

The proinflammatory cytokine interleukin-1 (IL-1) is produced both in the periphery and the brain in response to various immune challenges, including autoimmune diseases, as well as following exposure to psychological stressors. Since in humans autoimmune diseases and chronic stress are important risk factors for the development of major depression, and because our previous work implicated IL-1 in behavioral suppression following acute challenges, we sought to examine the role of this cytokine in mediating the depressive-like symptoms induced during episodes of experimental autoimmune encephalomyelitis (EAE) as we well as following exposure to chronic mild stress (CMS), an established model of depression in rodents. We first demonstrated a temporal correlation between the elevation in brain IL-1 and the development of depressive-like symptoms during EAE and CMS exposure. Using mice with deletion of the IL-1 type I receptor or transgenic over-expression of IL-1 receptor antagonist (IL-1ra) in the brain, as well as pharmacological administration of IL-1ra, we demonstrated that the depressive-like effects exhibited by mice with EAE or following exposure to CMS were completely abolished by blockade of IL-1 signaling. Furthermore, we showed that the behavioral effects of CMSinduced elevation of IL-1 are mediated both by adrenocortical activation and via suppression of hippocampal neurogenesis. Finally, chronic administration of IL-1 via osmotic minipumps mimicked the depressive-like behavioral effects of EAE and CMS, and also produced adrenocortical activation and reduced hippocampal neurogenesis. These findings indicate that IL-1 is both necessary and sufficient for the depression associated with autoimmune and chronic stress conditions.

Department of Physiology, Nippon Medical School, Tokyo, Japan; bTokyo Metropolitan Institute for Neuroscience, Tokyo, Japan; cThe Scripps Research Institute, San Diego, USA

a

A growing number of researches report that microglial cells contribute to progress of neurodegeneration, such as Parkinson, Alzheimer, HIV encephalitis and multiple sclerosis. In the present study we demonstrated stressinduced microglial activation in the brain. Based on the finding that circulating IL-18 levels are elevated during the stress, we hypothesized that IL-18 may be involved in the microglial activation in the brain. To test this hypothesis, we administered recombinant IL-18 and monitored microglial activation 6 h after the administration both in-vivo and in-vitro. Under the control conditions as well as 1 ng/g intraperitoneal administration of IL-18, no significant microglial activation was observed. At 5 ng/g of intraperitoneal IL-18 administration, significant microglial activation started to occur. Clear microglial activation was observed following the administration of IL-18 of 10, 20 ng/g as well. In addition, IL-6 and iNOS were induced in microglial cells in response to IL-18 administration. Quantitative real-time PCR study revealed that IL-18 administration induced IL-6, IL-18, iNOS mRNA in cultured microglial cell lines in a dose dependent manner. Furthermore, in IL-18 null mice, microglial activation was reduced in the brain in response to 2 h period of stress. Thus, the present study demonstrates that circulating IL-18 may activate microglial cells in the brain, suggesting that stress may induce microglial activation through stress-induced IL-18.

PP09-02

PP09-04

Neural correlates of IgE-mediated allergy

Age-related bias in function of natural killer T cells and granulocytes after stress: Reciprocal association of steroid hormones and sympathetic nerves

F. A. Costa-Pintoa, A. S. Bassoa, L. C. Sa-Rochaa, L. R. G. Brittob, M. Russoc and J. Palermo-Netoa Department of Pathology — FMVZ–USP, Sao Paulo, Brazil; Department of Physiology and Biophysics — ICB–USP, Sao Paulo, Brazil; cDepartment of Immunology — ICB–USP, Sao Paulo, Brazil

a

b

Although many authors had considered a direct interaction between allergic reactions and behavioral changes, supporting evidence had been elusive. In this series of studies we show that after oral or nasal ovalbumin (OVA) challenge, allergic mice present increased Fos-expression in the paraventricular nucleus of the hypothalamus (PVN) and in the central nucleus of the amygdala (CeA); mice with food allergy display higher levels of anxiety and had increased serum corticosterone levels; and allergyactivated neurons express corticotropin-releasing hormone in the PVN and CeA. OVA-allergic mice develop aversion to an antigen-containing solution, and also avoid a dark compartment previously associated with nebulized OVA. Results on brain Fos expression and behavioral data seem compatible with adaptive responses. Removing IgE by either antibody depletion or the development of oral tolerance precluded every response analyzed here. Csensitive fiber destruction by neonatal capsaicin inhibited the activation in the PVN, but not in the CeA, and decreased the magnitude of food aversion. Cromolyn, a mast cell stabilizer, completely blocked Fos expression in the PVN and CeA, and precluded the development of aversion to the dark compartment associated with nebulized OVA. Employing mice that do not develop an important inflammatory infiltrate following nasal OVA

Abo, T.a; Sagiyama, K.a,b; Tsuchida, M.a; Kawamura, H.a; Wang, S.a; Li, C.a; Bai, X.a; Nagura, T.a and Nozoe, S.b a

Department of Immunology, Niigata University School of Medicine, Niigata, Japan; bDepartment of Psychosomatic Medicine, Kagoshima University Faculty of Medicine, Kagoshima, Japan Stress-associated immune responses were compared between young (8 weeks) and old (56 weeks) mice. Since stress suppresses the conventional immune system (i.e. T and B cells) but inversely activates the primordial immune system (i.e. extrathymic T cells, NKT cells, and granulocytes), these parameters were analysed after restraint stress for 24 h. The thymus became atrophic as a function of age, and an age-related increase in the number of lymphocytes was seen in the liver. Although the number of lymphocytes in both the thymus and liver decreased, the magnitude was much more prominent in the thymus. To determine stress-resistant lymphocyte subsets, two-color immunofluorescence tests were conducted. NKT cells were found to be such cells in the liver of young mice. On the other hand, an infiltration of granulocytes due to stress was more prominent in the liver of old mice than in young mice. Liver injury as a result of stress was prominent in young mice. This age-related bias in the function of NKT cells and granulocytes seemed to be associated with a difference in the responses of catecholamines (high in old mice) and corticosterone (high in young mice) after stress. Indeed, an injection of adrenaline mainly induced the infiltration of granulocytes while that of

Abstracts cortisol activated NKT cells. These results suggest the existence of age-related bias in the function of NKT cells and granulocytes after stress and that such bias might be produced by different responses of sympathetic nerves and steroid hormones between young and old mice. PP09-05

Salivary cortisol and DHEA response to psychosocial stress in socially anxious individuals K. Shirotsukia, S. Izawab, N. Sugayaa, K. C. Yamadac, N. Ogawaa, Y. Oouchia, Y. Naganod and S. Nomurae a

Graduate School of Human Sciences, Waseda University, Saitama, Japan; b Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Tokyo, Japan; c Graduate School of Science and Technology, Nihon University, Tokyo, Japan; dDepartment of Human Studies, Bunkyo Gakuin University, Saitama, Japan; eFaculty of Human Sciences, Waseda University, Saitama, Japan

Social Anxiety Disorder (SAD) is the most common type of anxiety disorders. Kessler et al. (1994) suggested the life time prevalence was more than 13%. SAD often interfere their academic, social, and occupational function. SAD is characterized by the fear of negative evaluation by others, so the patients feel anxiety too much in socially evaluative situation and it impairs psychological and physiological states. In the present study, we conducted Trier Social Stress Test (TSST; Kirschbaum et al., 1993), which produces firm evaluative situation, to investigate salivary cortisol and dehydroepiandrosterone (DHEA) response in socially anxious people. This study was approved by the Ethical Committee of Waseda University. The participants were 17 male students, divided into 9 high socially anxious (SA) group and 8 low SA group by the score of Short Fear of Negative Evaluation Scale (SFNE; Sasagawa et al., 2004). Before experiment, they were informed the purpose of present study and signed the consent form. All experiments were conducted between 15:00 and 19:00. In the results of this study, significant correlations were found between AUC (Area Under the Curve) of cortisol and SFNE (r = − .45, p = .07), and between AUC of cortisol/DHEA ratio and SFNE (r = − 56, p = .02). Further analysis showed DHEA in high SA group rose greater than in low SA group. On the other hand, cortisol in high SA group reacted less in TSST. These results indicated that there was endocrine imbalance in socially anxious people to social stressor and this imbalance might affect the maintenance of SAD. PP09-06

Appraisal about controllability of acute stressor and brain– cardiac–immune association H. Ohiraa, T. Isowab,c, M. Nomurac, N. Ichikawaa, K. Kimuraa, M. Miyakoshia, T. Iidakaa, S. Fukuyamad, T. Nakajimad, J. Yamadad Nagoya University, Nagoya, Japan; bMie Prefectural College of Nursing, Tsu, Japan; cTokai Women's University, Kakamigahara, Japan; dKizawa Memorial Hospital, Minokamo, Japan

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The LC task caused less sense of control for subjects than the HC task. Significant increase of rCBF in the medial and lateral orbitofrontal cortex (OFC) and medial prefrontal cortex (MPFC) was observed in a comparison of the LC minus the HC tasks. More importantly, significant positive correlations between rCBF and the HR, BP, and NK cells were found commonly in the lateral OFC in the LC task, but not in the HC task. The present result showed for the first time that the OFC might be one of pivotal regions for top-down regulation over cardiovascular and immune activity accompanying cognitive appraisal on an acute stressor. PP09-07

Salivary secretory Immunoglobulin A modulation induced by repetitive stressful task and the mathematical model of the modulation S. Nomura Department of Mathematics and Computer Science of Shimane University, Matsue, Japan Recent psychoneuroimmunological and psychoendocrinological studies revealed that the human immune and endocrine secretory substances change its levels sensitively along with participants' subjective mental stress and relaxation. However, the precise modulation of those substances induced by such a mental interventions and its mechanism are still unknown. In this study, one of the major secretory immune substance, secretory Immunoglobulin A (sIgA), was assessed to investigate the normal human secretory response induced by a stressful task of deskwork operations focusing on following two points: (1) the precise changing of salivary sIgA under the repetitive short-term stressful works with beaks procedure, and (2) the mathematical model of sIgA modulation induced by that procedure. The results of the experiment in this study indicated that the changes of sIgA concentration within saliva clearly reflected the participants' subjective stress level with a certain time delay depending on the duration of stressful work. Referring with the results of the experiment and previous sIgA studies, I then propose a mathematical model of sIgA modulation based on the logistic function, which is the nonlinear ordinary differential equation consisting of the first order of exponential increasing term and the second order of nonlinear decreasing term, and in other words, it possesses the homeostatic property of living organisms. The result of the simulation of the model is well consistent with the sIgA modulation profiles of the experiment. Finally, I introduce a blue print of the deskwork stress prediction system constructed via the mathematical model. PP09-08

Oxidation detected by ultra-weak chemiluminescence may be related to neural-immune interactions under acute psychological stress H. Tsuboia, N. Goib, N. Kinaeb, H. Haradac, M. Hiramatsuc, K. Nakamurac, K. Takagib University of Hamamatsu, Hamamatsu, Japan; bUniversity of Shizuoka, Shizuoka, Japan; cHamamatsu Photonics K.K., Hamamatsu, Japan

a

Acute stress elicits multiple responses in autonomic, endocrine, and immune systems. Cognitive appraisal has been thought as one of important modulators of such stress responses. To investigate brain substrates of such crosstalk between the homeostasis systems accompanying cognitive appraisal of an acute stressor, we recorded simultaneously regional cerebral blood flow (rCBF) with 15O-water positron emission tomography, cardiovascular indices (heart rate (HR) and blood pressure (BP)), neuroendocrine indices (concentration of epinephrine, norepinephrine, and adrenocorticotropic hormone (ACTH) in blood), and immune indices (proportions of subsets of lymphocytes (NK cell, helper T cell, cytotoxic T cell, and B cell) in blood) when 11 male subjects conducted a mental arithmetic task with high controllability (HC) and low controllability (LC).

Aim: Stress sensed by the neural system affects the immune system. Previous studies described elevations of secretory immunoglobulin A (sIgA) levels in saliva and increased oxidative status when exposed to acute psychological stress. Human saliva contains peroxidase and the electron donor SCN–. Ultraweak chemiluminescence (UCL) is based mainly on an electronic transfer in the oxidation–reduction reaction and it registers the oxidative reaction. In order to investigate the relationships between immune functions, oxidative stress and neural reactions, the effects of performance anxiety on salivary UCL were examined and the values were compared with the salivary sIgA levels. Participants and methods: Participants were 10 volunteer students. The subjects

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performed a task of a serial aseptic manipulation for cell culture. The task was an examination on accuracy and speed in the aseptic manipulation to evaluate a result of a practical training. Saliva was collected 30 min before, immediately after, 30 min after and 60 min after the performance. Results: One-way repeated measures ANOVA for UCL revealed a significant difference across the four measurement occasions (F =4.45, p<0.05). Post hoc Dunnett's test clarified that UCL levels immediately after the task was significantly greater compared with that 30 min before (p<0.05). Two-way ANOVA with repeated measures indicated that UCL and sIgA exhibited the same change pattern in the four occasions (F=0.28, p=0.84). Conclusion: UCL counts a volume of photons generated during oxidative reactions, which can be an effective monitor of lipid peroxide generation. It was suggested that oxidation may occur during the neural–immune interactions. PP09-09

Therapeutic effects of color on acute psychological stress Kuniaki Takagi1, Nobuhiro Goi1, Yuko Hirai1, Hitoshi Harada1, Akira Ikari1, Kimitsugu Nakamura2, Mitsuo Hiramatsu2, Hirohito Tsuboi3, Reiko Horiki4, Takahiko Ono1 and Naohide Kinae1 University of Shizuoka, Shizuoka, Japan; 2Hamamatsu Photonics K.K., Hamamatsu, Japan; 3University of Hamamatsu, Hamamatsu; 4Color Works, Tokyo, Japan

The results indicated significant (≈ 20%, p < 0.001) differences in NKCC between HR (n = 45) and LR (=45) rats (E:T = 50:1; 33.4 ± 0.8% vs. 27.0 ± 0.8%, E:T = 25:1; 22.4 ± 0.6% vs. 18.0 ± 0.5%, E:T = 12:1; 13.7 ± 0.6% vs. 10.9 ± 0.5%) respectively. Evaluation of a single cell lytic ability on the same effector cells did not reveal any significant effect on the percentage of conjugate formation and on the target lysis. There were no significant differences either in tumor incidence or in tumor growth rate also. The results suggest that HR rat's higher NKCC may not necessarily lead to diminished incidence of carcinogenesis or LR rats posses more effective other anticancer mechanisms. PP09-11

Effect of attraction to the favorite person on innate immune system Masahiro Matsunaga1,2, Taeko Yamauchi2, Toshihiro Konagaya2 and Hideki Ohira1 1

Department of Psychology, Graduate School of Environmental Studies, Nagoya University, Japan; 2Department of Internal Medicine, Aichi Medical University School of Medicine, Japan

1

Aim: Thalassotherapy is used for various diseases from the age of Hippocrates; however, the effective factor and the mechanism are almost unknown. In this study, we focused attention on a blue that was the color of the sea, and examined the influence of the color on acute psychological stress. Participants and methods: Participants were 137 volunteer students. The subjects were divided into eight groups, blue to investigate the color effects. The subjects of four groups had worn color glasses before the Kraepelin test which was acute psychological stress. On the other hand, the subjects of the other groups had worn color glasses after the Kraepelin test. The saliva was collected from the subjects in each stage and the stress markers were measured. Results: In the subjects who had worn color glasses before the test, the salivary IgA (sIgA) and ultraweak Chemiluminescence (UCL) of the subjects in the Red and the Green were higher than that of the group of The Blue. Additionally, in the subjects who had worn color glasses after the test, the sIgA and UCL of the subjects in the Blue and the Green were significantly lower than that of the group of The Red. Conclusion: From these results, we suppose that there is a calming and a recovery effect from an acute psychological stress in blue.

Everybody can “fall in love”. Thus everybody knows that attraction to the favorite person invokes positive feelings and often makes us energetic. The question arising here is whether biological systems, such as autonomic nervous, endocrine, and immune systems, are affected by this positive emotion. To investigate effects of attraction to the favorite person on the biological systems, we recorded various parameters, namely mood states, heart rate (HR), skin conductance level (SCL), serum levels of several hormones (endorphin, oxytocin, vasopressin, norepinephrine, epinephrine, and cortisol), and proportions of T cells and natural killer (NK) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite persons. The mood states were assessed by the profile of mood status (POMS), the serum levels of several hormones were measured by Enzyme-Linked Immunosorbent Assay (ELISA), and the proportions of T cells and NK cells in the lymphocytes were measured by flowcytometer. As results, accompanying self-reported vigorous and better feeling, SCL and the proportion of NK cells significantly increased. No significant changes in HR, the serum levels of various hormones, as well as the proportion of T cells in the lymphocytes were found. These results suggest the possibility that attraction to the favorite person may have a role in activating NK cellrelated innate immune system by means of the activation of SCL-related autonomic nervous system. PP09-12

PP09-10

High motor active rats in novel environment (HR) compared to low motor active (LR) rats are not better against cancer

Temporal redistribution of NK cell subsets during acute psychological stressor K. Kimuraa,b, T. Isowaa, and H. Ohiraa

M.K. Jurkowski, B. Bobek-Billewicz Institute Of Oncology, Gliwice Branch, Poland

Department of Psychology, Nagoya University, Aichi, Japan; bJapan Society for the Promotion of Science

HR and LR rats show a strong correlation between the motility and psychostimulants' self-administration and the differences in their locomotor activity are an index of their behavioral reactivity to the stressful situation. The aim of the study was to asses HR and LR natural killer cell cytotoxicity (NKCC) and, while sometimes significant changes in the concentration and functional activity of immune parameters observed not necessarily lead to higher incidence of illness, we also assessed in vivo HR and LR rats' susceptibility to solid tumor carcinogenesis. The peripheral blood natural killer cell cytotoxicity (NKCC) (standard 4 h 51Cr release assay against YAC-1 target cells, E:T = 50:1; 25:1; 12:1), cytotoxicity of NK lymphocytes at the single cell level (agarose assay) and chemically induced tumor incidence and tumor growth rate were measured.

There were accumulated evidences indicating that an acute psychological stressor could elicit lymphocyte redistribution via sympathetic nervous activation. Furthermore, it was also known that a temporal variation of this redistribution was different between lymphocyte subsets. For instance, under the acute stressor, CD3−CD16+CD56+ natural killer cells (NK cells) were remarkably increased in peripheral blood while CD3+CD4+ helper T cells were slightly decreased or showed no change. One possible explanation about the distinct variations was adhesion molecules expressed on the surface of lymphocytes such as CD62L and CD11a. Recent researches suggested the redistribution of NK cells was occurred differentially based on CD62L expression density. And distinct CD62L expression in NK cell subsets was corresponded to functional differentiations, namely CD56+high

a

Abstracts immunomodulatory function and CD56+low cytotoxic function. Although differential redistributions of these subsets were reported by several studies, temporal variation of these subsets and the relationship with sympathetic nervous activation remained unclear. Thus, present study was conducted to examine these issues in acute stressor paradigm. Ten human participants performed mental arithmetic task as an acute stressor and their blood samples for estimating CD62L+ or CD62L-NK cells, CD62L+ or CD62Lhelper T cells, epinephrine, norepinephrine, and cortisol were taken at each 2 min during the task. Additionally, blood pressure and heart rate were monitored during the experiment. As a result, NK cells showed remarkable increment by the acute stressor and these variations indicated the close relationship with sympathetic nervous activation such as elevated blood pressure. PP09-13

Psycho-educational intervention for perceived social support has an effect on Natural killer cell activity among the elderly in rural Japan

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Recent progress in neuroscience shows that various neurotransmitters, hormones and cytokines play essential roles in neuronal cell survival and death. Various kinds of stress elicit cellular responses, leading to cell death. Sodium fluoride (NaF) is a well-known chemical stressor, and recent reports suggest that NaF induces cell death. In this study, we have investigated the effect of cytokines and neurosteroids on NaF-induced cell death of Neuro2A. Neuro2A cells were cultured in DMEM containing 5% FCS on 96-well microplates. The cells were then maintained without or with sodium fluoride (NaF) and/or other indicated drugs. After the cells were treated with the drugs, they were incubated with WST-8 to measure cell viability. Then, the optical density at 450 nm wavelength was measured with a microtiter plate reader. NaF induced cell death of Neuro2A in its dose and time dependent manners. IL-1alpha, IL-6, TNF-alpha, or LIF had little effect on the cell survival. DHEA, or DHEAS had no effect on the cell survival. These compounds had little effect on NaF-elicited cell death. However, estradiol significantly inhibited the cell death induced by NaF. These results suggest that estradiol has an effect to protect Neuro2A against NaF-induced cell death. PP09-15

Takao Miyazakia,b, Midori Shimuraa, Akihiro Hasegawac, Shotaro Sakamid, Hirofumi Iimorie, Tanji Hoshif, Hirohito Tsuboig, Yukihiro Agouh, Gen Komakia, and Noriyuki Kawamuraa

Cytokines modulate neurotrophin-dependent axon outgrowth of dorsal root ganglia — Dose-dependent effects of IL-4, IL-6, IFNg and TNFa

a

National Institute of Mental Health, National Center of Neurology and Psychiatry, Japan; bJapan Society for the Promotion of Science, cKanazawa Institute of Technology, dGraduate School of Medicine, Public Health and Occupational Medicine, the University of Tokyo, eIimori clinic, fCenter for Urban Studies, Tokyo Metropolitan University, gDepartment of Hygiene, Aich Medical University, hDepartment of Human Science Bunkyo Gakuin University, Japan Background: Relatively little research showed the strategies to change perceived social support to improve health outcomes. The previous intervention study for the elderly women focused on giving enacted social support, and failed to improve perceived social support. So we conducted psycho-educational intervention for the improvement of attitudinal perceived social support among general elderly people and examined the effect on natural killer cell activity. Method: A randomized control cross-over design involved 60 participants 65 years old and over. A ninety-minute intervention was conducted weekly for five successive weeks. The intervention consisted of (1) a lecture about illness and its prevention, (2) relaxation using the ‘Dohsa’ therapy, and (3) the psycho-educational intervention for perceived social support. Each participant was administered questionnaires and taken a blood sample between 9:00 am and 11:00 noon to control for diurnal variations at four times (baseline, pre-intervention, post-intervention, and follow up). NK cell activity was measured against K562 using a standard 4-h51Cr release assay. Result: There was a significant improvement in the perceived social support score and natural killer cell activity only in the intervention group at postintervention period and follow up period. Conclusions: We found that the perceived social support score could be increased by this psycho-educational intervention. It was also suggested that the change of perceived social support had a causal effect on the immunity such as natural killer cell activity.

Sven Hendrix, Greta Golz, Lars Uhlmann and Robert Nitsch Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité — Universitätsmedizin Berlin, Germany Inflammation is part of the wound healing response after mechanical lesion of the peripheral nervous system. However, it is not clear whether inflammatory cytokines exert a beneficial or detrimental effect on neurotrophin (NT)-dependent axonal outgrowth of dorsal root ganglia (DRG). Here, we have analyzed the effects of NTs and cytokines on neurite outgrowth of murine embryonic DRGs in vitro. DRGs were cultured in Matrigel with NGF, NT3 and NT4. All NTs (50 ng/ml) were applied alone, in double or in triple combinations. IL1b, IL4, IL6, IFNg and TNFa (50 or 500 ng/ml) were added to the different NT combinations. Using an image analysis system, the neurite outgrowth zone and axonal density were determined after 48 h. NT3 and NT4 alone and in combination induce a moderate increase in axonal growth, while NGF and NGF-including combinations substantially increase axon growth. IL1b had no effect; IL-4 increased NT4-induced outgrowth; IL6 stimulated NT3+NT4− induced outgrowth. IFNg stimulated neurite extension in NT-free controls as well as in the presence of NT3+NT4− and NT3+NGF. IFNg also stimulated axonal density in the absence of NTs or in the presence of NT4 or NT3+NT4. TNFa inhibited NT3−, NT3+NGF−, NT4+NGF−, and NT3+NT4+ NGF-induced outgrowth. In summary, NTs differentially stimulate DRG axonal outgrowth and density. This NT-induced axon growth is dose-dependently modulated by cytokines. PP09-16

Diazepam effects on blood neutrophil activity and on Ehrlich tumor growth

PP09-14

Effect of cytokines and neurosteroids on neuronal cell death of Neuro2A in response to NaF Ariyuki Kagaya1, Minoru Takebayashi2, Shigeru Morinobu3, and Shigeto Yamawaki3 Hiroshima International University, Higashi-hiroshima, Japan; 2National Hospital Organization Kure Medical Center, Kure, Japan; 3Hiroshima University, Hiroshima, Japan

1

M. Sakai, E. C. Rodrigues-Costa, C. S. Correia, M. L. Z. Dagli and J. Palermo-Neto Department of Pathology — FMVZ–USP, Sao Paulo, Brazil Introduction: Benzodiazepines, such as diazepam, are commonly used for their anxiolytic effects, i.e., by their action on high affinity receptor sites coupled to GABAA receptor complex. Diazepam treatment leads to an increase in serum corticosterone levels, which correlates positively to immunity alterations in laboratory animals.

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Objective: To analyze diazepam effects on innate immunity and on resistance to Ehrlich tumor growth. Methods and results: Swiss Male mice were divided in two groups. Animals from experimental group (E) and control group (C) were treated, per oral, with diazepam (3.0 mg/kg/day) or with control solution (0.1 ml/10 g/day) for 7 days, respectively. Mice from all groups received i.p. 5.0 × 106 Ehrlich tumor cells. Blood neutrophils activity and tumor growth were evaluated after 7 days. Animals treated with diazepam presented: 1 — a decrease on basal neutrophil oxidative burst (C = 36.56 ± 8.60; E = 28.05 ± 7.11); 2 — an increase on neutrophil oxidative burst after an in vitro induction with phorbol-miristate-acetate (C = 83.72 ± 15.88; E = 109.97 ± 22.57) as well as with Zymosan (C = 113.83 ± 21.18; E = 154.01 ± 34.02); 3 — an increase of both neutrophil intensity (C = 25.87 ± 13.31; E = 42.93 ± 11.64) and percent of Zymosan phagocytosis (C = 77.15 ± 9.30; E = 87.40 ± 6.83); 4 — an increase on Ehrlich tumor growth as evaluated by total number of tumor cells of ascitic fluid (C = 58.16 ± 14.00; E = 78.30 ± 19.17 × 107). Conclusion: Diazepam changes neutrophil activity measured in vitro and decreases animals resistance to Ehrlich tumor growth. Grant: CNPq, FAPESP. PP09-17

The effect of atypical antipsychotic drugs on the production of proinflammatory cytokines, nitric oxide and reactive oxygen species by activated microglia Takahiro Katoa, Sadayuki Hashiokaa. Akira Monjia. Yasukawa Keijib and Shigenobu Kanbaa a

Department of Neuropsychiatry, Graduate school of Medical Science, Kyushu University, Fukuoka, Japan; bDepartment of Bio-function Science, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

Microglia has recently been regarded to be a mediator of neuroinflammation by releasing proinflammatory cytokines, nitric oxide and reactive oxygen species (ROS). Microglia thus plays an important role in the pathology of neurodegenerative disease such as Alzheimer's disease (AD) and Parkinson's disease (PD). The pathological mechanisms of schizophrenia remain unclear while some recent neuroimaging studies suggest even schizophrenia may be a kind of neurodegenerative disease like AD and PD. There have so far been only a few studies on the relationship between microglia and schizophrenia. Atypical antipsychotic drugs (APDs) are becoming standard drugs for the treatment of schizophrenia because of their less adverse effects and more effectiveness for the negative symptoms of schizophrenia. We thus investigate the effects of atypical APDs on the production of proinflammatory cytokines, nitric oxide and ROS by activated microglia, and also try to clarify the molecular mechanisms underlying these effects. As compared with haloperidol, a typical conventional APD, several kinds of atypical APDs significantly inhibited the production of proinflammatory cytokines such as IL-1β, IL-6 and tumour necrosis factor α (TNF-α), and nitric oxide by lipopolysaccharide (LPS) + Interferon-γ stimulated microglia. These results suggest that atypical APDs may have more beneficial effects on proinflammatory conditions in the CNS which may play a crucial role in the pathology of schizophrenia. PP09-18

Lithium chloride modulate functions of human monocytederived dendritic cell Sy-Jye Leu1, Ko-Jiunn Liu2, Jia-Zhen He1, Huan-Ching Lin1, Yi-Yuan Yang3, Winston W. Shen4 1

Graduate Institute of Cell and Molecular Biology, Taipei, Taiwan; Division of Cancer Research, National Health Research Institutes, Taipei,

2

Taiwan; 3School of Medical Technology, Taipei Medical University, Taipei, Taiwan; 4Department of Psychiatry, School of Medicine, and Taipei Medical University-Wan Fang Medical Center, Taipei, Taiwan For almost half a century, lithium has been one of the most widely used medications for bipolar depressive illness, although its therapeutic mechanism of action remains obscure. Accumulated preclinical and clinical evidences for the action of lithium in the brain suggest that lithium can affect membrane transport systems, neurotransmitter receptor regulation, second messenger systems, protein kinase C (PKC) regulation, and gene expression. Recent studies showed long term, but not acute, treatment of cultured cerebellar granule cells with LiCl induce a concentration-dependent decrease in proapoptotic p53 and Bax level; and remarkably increased cytoprotective Bcl-2 protein. These results suggest that lithium, in addition to its use in the treatment of bipolar depressive illness, may have an expanded use in improving neurodegeneration. However, regulation of lithium on the dendritic cell was not-well defined. Dendritic cells (DC) are key regulators of immune responses, and were involved in the initiation and maintenance of both innate and the adaptive immune responses. We demonstrate lithium chloride treatment enhanced human monocyte-derived CD86 expression. In addition, lithium chloride increased cytokines, TNF-a IL-8, IL-1b, IL-10 and IL-6 production. However, lithium have no effect on T cell proliferation which assayed by mix lymphocyte reaction. These data suggest that lithium chloride might modulate DC functions via phenotype and cytokines expression. PP09-19

Association of polymorphism in the IL-1ra gene with autism and pervasive developmental disorders N. Yirmiyaa, C. Rosenberga,b, S. Salomona, S. Levia,c, C. Shulmand, R. Yirmiyaa, R. Ebsteina,b a

Department of Psychology, The Hebrew University of Jerusalem, Jerusalem, Israel; bResearch Department, S. Herzog Memorial Hospital, Jerusalem, Israel; cDepartment of Child Psychiatry, The Hebrew University of Jerusalem, Jerusalem, Israel; dSchool of Social Work, The Hebrew University of Jerusalem, Jerusalem, Israel Over the last decade it became evident that pervasive developmental disorders (PDD) in general, and autistic disorder more specifically, are associated with several immune alterations and autoimmune processes. In particular, the innate immune system seems to be activated, accompanied by the production of pro-inflammatory cytokines and neuroinflammatory processes. Viruses, such as measles, and environmental toxins, such as mercury, which are known stimulators of innate immunity have been implicated in the etiology of autism. These stimuli may interact with genetic variables, as there is an increased prevalence of familial autoimmune disorders in parents of probands with PDD. The pro-inflammatory cytokine interleukin-1 (IL-1) may be particularly relevant to PDD because (1) it is involved in emotional, social and cognitive modulation, (2) it is induced by vaccination and toxins, (3) it mediates the immune responses to mercury, and (4) it plays a major role in autoimmunity. Recently, a variable number tandem repeat polymorphism in the second intron of the IL-1 receptor antagonist (IL-1ra) gene has been found to be associated with increased risks of chronic inflammatory and autoimmune diseases. To test the hypothesis that this polymorphism is associated with autism we are currently examining 116 families (128 probands diagnosed with the ADI-R and ADOS-G, and their parents) using a family-based association test (UNPHASED). Furthermore, we are assessing whether the association with IL-1ra is mediated by the role of this gene in specific disturbances in social– emotional and cognitive parameters. The results may implicate genetic alterations in IL-1 signaling in autism and its relationship with autoimmune processes.

Abstracts PP09-20

Study of immuno system alterations in Major Depression Disorder (MDD) R. Farid1, F. Jabari Azad1, F. Farid2, S. Miri1, M. Mohamadi2 1

Bu-Ali Immunology Research Center, Mashad University, Mashad, Iran; Rozbeh Hospital, Tehran University, Tehran, Iran

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CSF, sIL-6R, sIL-2R, IL-4, Gro-a, MCP-3, TNFa, MCP-2, IL-7, IP-10 and GDNF were not detectable in any sample or did not differ significantly between patients and controls in both, serum and cerebrospinal fluid. Conclusion: Our findings indicate a possible role of immunopathological mechanisms in the development of therapy-refractory chronic pain. Our results support immunomodulatory strategies in the management of chronic idiopathic pain syndromes.

2

PP09-22 Major Depression Disorder (MDD) is a common with prevalence of about 15% for men and up to 25% in women. In recent years, the association of immune system alteration and MDD has been reported on the basis of new observations. Therefore, we decided to do more accurate assessment in determining or excluding the relationship between immune system and MDD. Materials and methods: In patients and control group, we determined TNF α, FN γ, IL-4 and IL-10 (by ELISA), serum immunoglobulin, serum proteins, and markers of immune system cells include CD 16–56, CD19, CD 8, CD 4, CD 3. Results: This study was include 37 patients (28/9: F/M) with mean age of 39 years and fifteen number control. In cytokines study were observed no significant differences between the patient and control group. In the patient group (moderate grade), there was significant relationship only between IL10 and IFN γ (p = 0.04). In control group, the relationship between IL-4 and IL-10 was positive and significant (p = 0.04). The study of immunoglobulin in the patients was in the normal range. The amount of total protein and γ in 95% of the patient group were higher than normal range and the number of NK, B, and T cells was in the normal range. Discussion: In the patients with MDD, inhibitory cytokine (IL-10) was increased in severe grade of disease.Total protein of patient group was higher than normal range but immune system cells and Immunoglobulin were in the normal range. PP09-21

Relevance of cytokines, chemokines and other immune mediators for immunomodulatory strategies in patients suffering from chronic idiopathic pain syndromes M. Stocka, H. Vogela, A. M. Waaga-Gasserb, M. Draganb, R. Schedela and G. Sprottea

Increased NK activity in a group of schizophrenic patients M. Vodjgania, H. Matloubia, A. A. Nasehib, M. H. Niknama, A. Kazemnejadc, E. Salehia, T. Aboufazelia and Z. Gheflatia a

Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran; bIran-Helal Institute of Applied Science and Technology, Tehran, Iran; cTarbiat Modarres University, Tehran, Iran

Functional alterations in the immune system of schizophrenic patients have been shown in different studies. The importance of NK cell activity in schizophrenia is due to their role in immune surveillance against tumoral and viral cells. These studies aim to explain the low incidence of cancer in schizophrenic patients. But no consistent findings have emerged. Majority of the inconsistencies must be due to the effects of various confounding factors, such as smoking, medications, etc. The purpose of this study was to evaluate NK activity in schizophrenic patients with eliminating the effect of such factors. In this study, we have chosen 30 medication-free schizophrenic patients and 41 healthy sex, age and smoking status matched individuals. NK cell activity of case and control subjects was measured by MTT (Methyl-Thiazol-Tetrazolium) test. Statistical analysis of the data was obtained using SPSS 11.5. NK activity of patients had a mean of 36.93550 ± 26.152416 (Mean ± SD) and NK activity mean of normal subjects was 22.31171 ± 17.923737. Statistical analysis showed a significant increase in NK activity in schizophrenic patients compared with controls (P = 0.011). Also, NK activity of smoking patients was significantly lower than that of nonsmoking patients (P = 0.02). Other demographic factors did not show any influence on NK activity. The higher activity of NK cells in the schizophrenic patients as compared with the control population could explain the low incidence of cancer in these patients. And decreasing effect of smoking on NK activity in the patients could be one of the responsible factors for the inconsistency in results of different studies.

a

Department of Anaesthesiology, Pain Care Centre, University of Wuerzburg, Germany; bDepartment of Surgery, Molecular Oncology and Immunology, University of Wuerzburg, Germany

PP09-23

Function of T cells in a group of schizophrenic patients This is the first study to give insight to a wide pattern of chemokines, cytokines and their receptors, as well as corticotropin-releasing factor (CRF), substance P (SP) and histamine in serum and cerebrospinal fluid of patients suffering from uncontrollable chronic idiopathic pain syndromes compared to controls. Patients and methods: 10 patients suffering from idiopathic chronic widespread pain syndromes (CWP), such as Fibromyalgia and Myofascial Pain Syndrome (in some cases additionally Complex Regional Pain Syndrome), all of them refractory to conventional pain treatment strategies and 9 controls who underwent spinal anaesthesia for minor lower extremity surgery were enrolled in this study. Serum and cerebrospinal fluid concentrations of above mentioned parameters were determined using high-sensitivity ELISA kits and Luminex-100 technology. Results: In patients' serum SP, histamine, IL-12(p40/p70), Eotaxin, MIP1a, MIP-1b, MCP-1, IL-5, IL-6, MIG and IL-8 were significantly elevated compared to controls. In cerebrospinal fluid SP, CRF, IL-10, IL-1Ra, IFNa, MCP-1, IL-2, MIG and IL-8 were significantly higher compared to controls. G-CSF was detectable in cerebrospinal fluid of 9 patients but in none of the controls. However, IL-1b, RANTES, IL-13, IL-15, IL-7, GM-

H. Matloubia , M. Vodjgania , A. A. Nasehib , M. H. Niknama , A. Kazemnejadc, E. Salehia, T. Aboufazelia and Z. Gheflatia a

Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran; bIran-Helal Institute of Applied Science and Technology, Tehran, Iran; cTarbiat Modarres University, Tehran, Iran For decades, the relationship between the Immune system and schizophrenia has attracted considerable research in Psychoneuroimmunology (PNI). There are a few etiologic hypotheses connected to this proposed relationship, being Viral infections, Autoimmunity and Activation of Tlymphocyte-Macrophage. This Study focuses on a comparative analysis of the immune function of 30 drug-free schizophrenic patients and 42 healthy individuals who are matched in sex and age and Smoking status. In this analysis, proliferation of T cells in response to PHA (PhytoHaemAgglutinin) mitogen, was measured with MethylThiazolTetrazolium Test (MTT). Serum ANA (Anti-Nuclear-Antibody) level and ACA (AntiCytoplasmic-Antibody) presence and Circulating Immune Complexes (CIC) concentration were

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measured with Indirect Immuno-Fluorescence and PEG precipitation tests, respectively. The results of these assays were then analyzed with SPSS 11.5. T cell proliferation of patients had a mean of 1.96417 ± 0.830684 and T cell proliferation mean of normal subjects was 3.72971 ± 1.398237. Statistical analysis showed a significant decrease in T cell proliferation in schizophrenic patients compared with controls (P = 0.000). Serum ANA level and CIC concentration were not significantly different between the two groups (P = 0.661, P = 0.102, respectively). ACA was shown to be present only in the case group (P = 0.004). These results could be evidences of an Autoimmune/Viral hypothesis in at least a group of schizophrenic patients. PP09-24

Association between interleukin-10 promoter polymorphisms and schizophrenia in Iranian population Z. Sabouria, S. N. Bajestanb, H. Takashimab, M. R. Sargolzaeec, M. N. Bajestana, R. Farida, K. Arimurab, A. Sanod, M. Osameb Departments of Allergy and Immunology, cDepartment of Psychiatry Mashhad University of Medical Sciences, Mashhad, Iran; bDepartment of Neurology and Geriatrics, dDepartment of Neuropsychiatry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan

urban pollutants resulting in chronic diseases, in this study we attempted to evaluate the effects of intermittent exposure of mice to gasoline (G) and diesel (D) vapor on brain, lungs and kidney expression of metallothioneins I + II (MTs), which as cysteine-rich proteins with metal binding affinity might provide cytoprotective action on any form of stress or injury. For this purpose C57/BL6 mice were for 1 h/day closed in a small metabolic chamber ventilated with fresh air or with G or D vapors. Protocol was repeated for 10 days and then MTs expression was detected in the brain, lungs and kidney. The findings were compared with data obtained in unstressed mice. It was found that G and D inhalation markedly upregulated the MT expression in ventricular ependyma and astrocytes, and particularly in the hippocampus, where project the stellate and pyramidal cells of lateral olfactory tract and perforant path. High cytoplasmic and nuclear MT expression was found also in alveolar epithelia and in the kidney, implying the protective effects of MT in areas exposed to toxic agents and their products. Simultaneously, both stress and toxic agents increased the cytotoxicity of hepatic and splenic lymphatic cells against the syngeneic thymocytes, implying that autoreactive clones of cells participate in the processes of tissue repair (supported by grants from Croatian Ministry of Science).

a

Several reports show abnormal cytokine levels in psychotic patients and indicate a possible role of the immune response system in the pathogenesis of schizophrenia. Interleukin-10 (IL-10) gene maps on chromosome 1(q31–q32), a locus associated with genetic susceptibility to schizophrenia. To investigate the role of IL-10 on schizophrenia, we analysed the association between four single nucleotide polymorphisms (SNPs) located on IL-10 promoter (−592A/ C, −1082A/G, −2763C/A, −3573T/A) and the risk of schizophrenia in patients (n = 321) and unrelated controls (n = 392), all residing in the city of Mashhad, Iran. The frequencies of IL-10 promoter-3575 T/A SNP T allele and IL-10 promoter −2763C/A SNP C allele were also significantly increased in patients compared with controls (p < 0.05). Furthermore, comparing to controls, the frequency of IL-10 −1082 A allele was significantly increased in all patients (p =0.024), patients with positive family history (n = 63) (p = 0.015), undifferentiated (p = 0.031), non-disorganized (p = 0.011) and nonresidual (p = 0.02) subtypes. IL-10 promoter −1082 AA genotype was significantly increased in family history positive patients (p = 0.021) and nondisorganized groups. Similarly, the frequency of IL-10 −592 A allele was significantly increased in patients with positive family history (p = 0.044) and non-disorganized subtype (p = 0.03). Our preliminary haplotype analyses suggest that the frequency of IL-10 promoter haplotypes, which are associated with high IL-10 production are increased in schizophrenia. Our results suggest that IL-10 promoter SNPs may be associated with the increased risk of schizophrenia. This study shows the different genetics background of schizophrenia subtypes in Iranian population. PP09-25

Chronic exposure to vapors of gasoline and diesel upregulates the metallothionein I and II expression in the olfactory brain of mice D. Grebića, H. Jakovaca, I. Mrakovčić-Šutića, J. Tomacb, A. Bulogc, V. Mićovićc and B. Radošević-Stašića Department of Physiology and Immunology; bDepartment of Histology and Embryology; and c Department of Public Health; Medical Faculty, University of Rijeka, 51000 Rijeka, Croatia

a

Olfactory systems are often used as excellent models for the study of widespread aspects of neural development and neuron–glia interactions after exposure to agents whose route of entry into the CNS might bypass the blood– brain barrier. Since, these pathways might be frequently stimulated by airborne

PP09-26

Modulation of pro-inflammatory and neural activity-related gene expression in the olfactory bulb of mice by nasal inhalation of low-level toluene Sohel Ahmed, Hidekazu Fujimaki, Tin-Tin-Win-Shwe, Shoji Yamamoto, Shinji Tsukahara, Yoshika Kurokawa, Daisuke Nakajima, and Sumio Goto National Institute for Environmental Studies, Ibaraki, Japan Since the induction of multiple chemical sensitivities might be related to volatile organic compounds in houses and offices, it is speculated that there is a positive relationship between immunological and neurological alterations and inhalation of volatile organic compounds. To study the effect of exposure to low-level toluene on the olfactory system neuroimmune responses, male C3H/HeN mice were exposed to filtered air (control), 9, and 90 ppm toluene in nose-only exposure chamber for 30 min per day, on days 0, 1, 2, 7, 14, 21, and 28. These mice were either maintained at non-immunized or immunized condition with ovalbumin (OVA). Using a quantitative real-time PCR method, we investigated the olfactory bulb expression of several genes. Our data demonstrate a significant difference of mRNA-expression of pro-inflammatory genes, interleukin-1β, CCL2 and CCL3 in the OVA-immunized mice exposed to 9 ppm toluene, as compared to that of filtered air control and 90 ppm toluene. In case of the neural activity-related genes, we observed a similar pattern for the glutamatergic NMDA receptor subunits, NR2A and NR2B. However, we did not observe any such pattern for the dopaminergic receptor subtypes, D1 and D2. This is the first study to show in vivo modulation of the olfactory bulb expression of pro-inflammatory and neural activity genes in mice by nasal inhalation exposure to low-level toluene, in concert with antigenic stimulation. PP09-27

Influence of emotion regulation on the immune, autonomic responses, and cognition H. Murakami, H. Ohira Psychology, Nagoya University, Aichi, Japan There are some emotion regulation techniques to decrease facing anxiety and followed physiological responses. Self focused attention is known to increase anxiety state. But there is an adaptive self-focus which decreases anxiety state. The former is maladaptive self-focus includes conceptual, analytical, evaluative way of self-focus. The later is adaptive focusing on direct, intuitive, experiential awareness of experience in the moment.

Abstracts Adaptive self-focus manipulation is known as one of the factor of the mindfulness. Mindfulness is the third wave cognitive behavior therapy which has been developed to treat a range of psychological disorders including depression, anxiety and fibromyalgia. But, the functional mechanism of mindfulness is still unknown. We used adaptive self-focus manipulation as a technique of emotion regulation. We hypothesized that adaptive self-focus manipulation is also effective to decrease autonomic and immune responses. In cognitive theory, there is interaction between emotion and cognition. Previous findings have demonstrated that increasing anxiety state leads to shift to allocate more cognitive resource to negative stimulus. We also demonstrated the influence to cognition which affected by endocrine systems in anxiety state. Our purpose of this study is to demonstrate adaptive self-focus manipulation attenuates anxiety responses in the immune, autonomic responses, and cognitive negativity bias. PP09-28

Magnetic stimulation of left temporo-parieto-occipital (TPO) cerebral cortex: A non-invasive treatment to suppress ongoing human IgE responses YA Moshel, SM Chice, M Nowakowski, R Joks, VE Amassian and HG Durkin

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Center for Allergy and Asthma Research at State University of New York Downstate Medical Center, Brooklyn, NY, USA 11203 We found that magnetic stimulation of left, but not right, TPO cerebral cortex of allergic humans significantly increased their blood CD4+ and CD8+ T cell, but not B cell, numbers at 4–6 h, and strongly suppressed their serum IgE levels at 4–5 days (>90%). In contrast, with right stimulation, the numbers of blood T cells decreased, and serum IgE levels did not change. We previously reported that electrical stimulation of rat left, but not right, TPO cortex increased cell export from the thymus, mediated by sympathetic pathways in the upper spinal cord, resulting in increased blood CD4+ and CD8+ T cells numbers at 4–6 h (Moshel et al. [2005]. J. Neuroimm. 158: 3–13). In contrast, with right stimulation, the numbers of blood T cells decreased. We are studying the ability of left vs. right electrical stimulation of TPO cortex to suppress peak hapten specific IgE responses of BPO-KLH sensitized rats, and the mechanisms involved. Supported by Research Foundation of SUNY.

184

Abstracts

Poster Session 10: MS-genetics and etiology

Abstracts PP10-01

185

a

Hertie Institute for Clinical Brain Research, Tübingen, Germany; Department of Neurology, University of Pecs, Hungary; cDepartment of Neurology, Laboratory of Neuroimmunology, Medical University of Lodz, Poland; dInstitute of Anthropology and Human Genetics, University of Tübingen, Germany; eInstitute of Epidemiology and Social Medicine, University of Münster, Germany b

CD4, a candidate gene for multiple sclerosis? Frida Lundmark1, Hanne Harbo2, Janna Saarela3, Annette Oturai4, Cecilia Lindgren5, Hugh Salter6, Jan Hillert1 1

Division of Neurology, Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden; 2Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway; 3Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland; 4Department of Neurology, Copenhagen University Hospital, Copenhagen, Denmark; 5 Clinical Research Centre, Department of Biosciences at Novum, Karolinska Institutet, Stockholm, Sweden; 6Department of Bioscience, Astrazeneca R&D, Södertälje, Sweden Multiple sclerosis (MS) is a chronic neurological disease affecting the central nervous system. The disease is characterized by demyelination and axonal loss caused by abnormal immunological responses resulting in accumulating neurological disabilities. MS is considered to be a complex disease where both genetic and environmental factors contribute to the pathogenesis. In this study we have investigated the genetic role of the CD4 gene in MS. Nine single nucleotide polymorphisms (SNPs) were selected for genotyping based on the LD pattern in HapMap and to get an even distribution of markers over the gene. 920 Swedish MS patients and 778 Swedish healthy controls were genotyped using the Sequenom method. Three SNPs; rs3782736, rs7957426 and rs10774450 showed a significant pvalue at the 5% significance level. No haplotype association was found and all associated SNPs were located within the same LD block. In order to confirm these results, all three significant markers were genotyped in an independent case control material, consisting of 1720 Nordic (Norway, Denmark and Finland) MS patients and 1416 Nordic healthy controls. None of the associations from the Swedish case/control material did replicate in the Nordic material. We conclude that the CD4 gene is not of major genetic importance in MS in a Nordic population. The study indicates two important factors in performing genetic association studies in complex diseases; the necessity of large case/control materials, and access to independent materials for confirmation of initial associations. PP10-02

Chromosome 19q13 and multiple sclerosis A. Bonetti University of Helsinki, Helsinki, Finland Previous studies have suggested a role of allelic variation on chromosome 19q13 in multiple sclerosis (MS) susceptibility. The object of the study was to test three subregions of 19q13 for association with MS in 440 families using the transmission disequilibrium test. A marker in the 19q13.1 area showed nominally significant association with MS. This marker resides at a suggestive linkage peak, and associated with MS in a previous Italian study. Another association was found with an APOE haplotype on 19q13.2, supported also by a separate case-control analysis. Previous suggestions on ILT6 deficiency or association with D19S585 on 19q13.3–q13.4 were not replicated in this study. These results show that two subregions of chromosome 19q13 are associated with MS warranting further studies on their implication with the disease. PP10-03

Multiple sclerosis and the putative autoimmunity SNP CT60: An association study in patients from Germany, Hungary and Poland B. Grevea, R. Simonenkoa, Z. Illesb, A. Peterfalvib, M.P. Myckoc, K.W. Selmajc, P. Bauerd, K. Bergere, R. Weisserta

Polymorphisms in the CTLA4 gene region have been associated with susceptibility to autoimmune diseases (AID). The recently described single nucleotide polymorphism CT60, located in the 3′ untranslated region of CTLA4 has been shown to be associated with Graves' disease, autoimmune thyroiditis, autoimmune diabetes and other AID. Although one study showed an association of the +49A/G⁎G–CT60⁎G haplotype with Multiple sclerosis (MS), other studies failed to demonstrate association with CT60. Also, there is conflicting data regarding CT60 allele-dependent gene expression. We conducted an association study including so far a total of 630 patients from Hungary, Poland and Germany and 814 control persons. Although the CT60⁎G/G genotype was overrepresented among MS patients, this association did not reach statistical significance. We conclude, that the influence of CT60 on MS susceptibility is – if at all – rather small and might be restricted to subgroups of patients. We will present analysis of MHC stratification and correlation with clinical data. Furthermore, we analyze genotype-dependent expression of CTLA-4 and ICOS on T cells after in vitro stimulation. PP10-04

Pro and anti-inflammatory cytokines gene polymorphism in Multiple Sclerosis patients M. Izad, M. Vodjgani Immunology Department, Tehran University of Medical Sciences, Tehran, Iran The role of inflammatory (IFN-γ, TNF-α, IL-6) and anti-inflammatory (TGF-β, IL-10) cytokines in the pathogenesis of Multiple sclerosis (MS) has been extensively studied. Elevated levels of TNF-α, IL-6, IFN-γ are observed in the blood, CSF and central nervous system (CNS) lesions of MS patients in active phase, while TGF-β and IL-10 are increased in remission or inactive phase. Therefore alteration in cytokine production may influence the susceptibility to MS and this alteration could be due to gene polymorphism. The aim of present study was to analyze whether there was an association between above cytokines' gene polymorphism and susceptibility to MS in the Iranian population. In this study, we screened genomic DNA samples from 98 clinically definite MS patients and 118 healthy controls, using polymerase chain reaction-sequence-specific primers (PCR-SSP). Cytokine gene polymorphisms were determined for TNF-α promoter-308 (G to A), IL-6 promoter-174 (G to C), IL-10 promoter-592 (C to A), -819 (C to T), -1082 (G to A), TGF-β codon 10 (C to A), codon 25 (G to C) and IFN-γ intron 1 + 874. The results indicated that MS patients significantly showed a higher TNF-α GG and TGF-β T/T G/G, T/C G/C genotypes compared to controls. This study also showed that the frequency of genotypes associated with low production of TNF-α increased in MS patients. However, our findings suggest that there could be genetically a dysregulated production of cytokines in Multiple Sclerosis and risk of the disease increase in low producers of TNF-α. PP10-05

A new developed culture method efficiently supports the growth of Chamydia pneumoniae in PBMC and CSF samples from patients with multiple sclerosis C. Continia, S. Seracenia, M. Giuliodoria, V. Biondina, M. Castellazzib, E. Fainardib, E. Granierib Infectious Diseasesa and Neurologyb, University of Ferrara, Italy

186

Abstracts

Chlamydia pneumoniae (Cpn), has been associated with various neurological disorders including multiple sclerosis (MS) on the basis of PCR findings. A question still open concerns the difficulty to cultivate and directly demonstrate Cpn in clinical specimens. Fresh CFS and PBMC samples were obtained from 8 patients with neurological and MRI abnormalities suggestive of MS. Specimens were inoculated on Hep-2 cells in duplicate wells, and incubated in CO2. A part of grown cells were investigated with immunofluorescence (IF) using Cpn-monoclonal antibodies. To increase Cpn inclusions, the resting wells were centrifuged and incubated with additional centrifugations on the 3rd and then on the 4th and 5th culture day, with medium refreshment at 72 h only. Culture supernatants underwent touchdown n-PCR assay targeting 16S rRNA, Momp and HsP 70 gene of Cpn. 3/8 patients were culture positive. CSF n-PCR was negative when DNA was directly extracted, whereas was positive in 1 (16S RNA) and 2 (MOMP) cultured samples when DNA was extracted from the 3rd day culture supernatants. PBMC n-PCR was positive for all genes, in higher positive rates than cultured CSF, when DNA was extracted from culture supernatants at 3 or 6 days. IF, performed before and after culture, was positive in only one PBMC sample. The results obtained confirm the efficacy of this culture system and suggest the presence and viability of Cpn. The combination of additional centrifugation and extension of culture time with PCR, improve the sensitivity of the method and suggest its potential use to detect Cpn. PP10-06

Serological profile of the immune response to microbes in identical twins discordant for multiple sclerosis S. Cannoni1, F. Mengoni2, I. Sauzullo2, S. Santucci1, A. Visconti1, S. Romano1, A. Ghazaryan1, V. Vullo2, M. Salvetti1, G. Ristori1

1

Neurology-Center for Experimental Neurological Therapies (CENTERS), University of Rome “La Sapienza”, Rome, Italy; 2Department of Infectious and Tropical Diseases, University of Rome “La Sapienza”, Rome, Italy

Based on the nationwide series of Italian twins with multiple sclerosis, we performed a co-twin control study on 70 pairs in order to investigate causative factors for MS. Among 80 variables possibly associated with MS etiology, those positevely or negatively associated with predisposition and concordance in twins were mainly linked to infection. This finding prompted us to investigate quantitative serological profiles of immune response against microbes, that are putatively involved in MS etiology, in identical twins discordant for MS. Serum antibodies to EpsteinBarr (EBV) antigens, CMV, Varicella zoster (VZ), red measles, Herpes simplex 1, 2 and 6 viruses and Bordetella pertussis were quantified in 9 twin pairs discordant for disease 1 concordant pair and 1 control unaffected twin pair. The serum antibody titers were compared between affected and unaffected twins. Elevations in affected vs unaffected twins were significant for IgG anti-VCA (RU/ml, 1413.49 vs. 957, p < 0.05), a trend was found for anti-EBNA-1 and 2. Elevations in unaffected vs affected twins was significant for antibodies to VZV (RU/ml, 2980 vs. 1980, p < 0.05). No differences were found for the other microbes. Finally, the concordant twin pair and the unaffected twin pair showed comparable serological profiles. This finding supports the role of infections in the etiology of MS. The serological profiles of discordant twins showed differenced in the reactivity to VZV and EBV. The enhanced immune response to EBV in the affected twins is in keeping with previous work on sporadic patients.

Abstracts

187

Poster Session 11: MS-pathology and experimental models

188

Abstracts

PP11-01

Antioxidant enzyme expression in multiple sclerosis brains J. van Horssena, L. Bob, C.D. Dijkstraa and H.E. de Vriesa Department of Molecular Cell Biology and Immunologya and Pathologyb, VU University Medical Center, Amsterdam, The Netherlands Reactive oxygen species (ROS) play a crucial role in several processes underlying the pathogenesis of multiple sclerosis (MS). They are involved in transendothelial monocyte migration, myelin phagocytosis and breakdown, oligodendrocyte damage and axonal degeneration. Exposure of cells to high levels of ROS induces oxidative stress, which leads to the activation of the nuclear transcription factor Nrf2. Activation of this transcription factor induces expression of antioxidant response elements (ARE)-regulated genes. These genes encode for endogenous enzymes that regulate cellular redox status and offer protection against oxidative stress and inflammation. Thus, expression of Nrf2/ARE proteins may act as a sensitive indicator of oxidative stress. So far, data on the expression ARE-regulated enzymes in brains of MS patients are lacking. Hence, we investigated the distribution of various endogenous antioxidant enzymes, including heme oxygenase, superoxide dismutase, peroxiredoxins, catalase and NAD(P)H:quinone oxidoreductase in different MS lesion types. We showed for the first time that antioxidant enzymes are highly upregulated in active demyelinated MS lesions, particularly in hypertrophic astrocytes and myelin-laden macrophages. We speculate that increased antioxidant enzyme expression may reflect an endogenous defense response and may compensate ROS-mediated cellular toxicity. Compounds that activate the Nrf2-ARE signalling pathway, via expression of detoxifying enzymes, may be a potential therapeutic target for future treatment strategies in MS. PP11-02

Reduced expression of MHC antigen-processing machinery molecules in margins of Multiple Sclerosis lesions: Clues for antiinflammatory countermechanisms at the lesion border? M. Mehlinga, M. Mittelbronnb, P. Simonc, S. Ferroned and H. Wiendle a

Outpatient Clinic Neurology, University Hospitals of Basel, Basel, Switzerland; bDepartment of Brain Research, University of Tübingen, Tübingen, Germany; cMedical Clinic, Department of Sports Medicine, University of Tübingen, Tübingen, Germany; dDepartment of Immunology, Roswell Park Cancer Institute, Buffalo, New York, United States; e Department of Neurology, University of Würzburg, Würzburg, Germany It is assumed that in Multiple Sclerosis (MS) lesions reactivation of infiltrating autoreactive lymphocytes is most likely mediated by local antigen presenting cells (APCs), such as microglia/macrophages. Antigen presentation requires major histocompatibility (MHC) antigen-processing, a complex interplay a several proteins belonging to the antigen-processing machinery (APM). In order to evaulate the regulation of APM molecules in relation to MS lesion formation, we investigated the expression of various MHC APM molecules in human brain specimens from MS patients (n = 4) by means of immunohistochemistry. In relation to MHC class II, microglia/ macrophates exhibited 2 to 3-fold higher expression of MCH APM molecules in active (early-active, late-active and chronic-active) than in inactive (chronic-inactive) MS lesions. Interestingly, albeit higher frequencies of microglia and macrophages in borders of active lesions were observed, the expression of numerous MHC APM components such as delta, low molecular weight protein-2 (LMP-2), LMP-7, LMP-10, transporter associated with antigen zprocessing-1 (TAP1), calnexin and calreticulin was 1.5 to 2 fold higher in lesion centres than in lesion borders or normal appearing white matter (NAWM). In contrast to this, chronic-inactive MS

lesions were characterized by comparable amounts of MHC APM expression in lesion borders and centres. Our data demonstrate microglia cells and macrophages to have the capacity to process and present antigens alike professional APCs. Reduced expression of MHC APM molecules in lesion borders in comparison to lesions centers could suggest antiinflammatory countermechanisms circumventing the broadening of MS-lesions. PP11-03

Both aquaporin-1 (AQP1) and aquaporin-4 (AQP4) are expressed in cultured human astrocytes and non-hypertrophic astrocytes in brains of multiple sclerosis Jun-ichi Satoha, Hiroko Tabunokia, Yusuke Nanrib, Takashi Yamamurab, Kunimasa Arimac and Hidehiko Konnod a

Department of Bioinformatics, Meiji Pharmaceutical University; Department of Immunology, NCNP; cDepartment of Neuropathology, NCNP, Tokyo; dDepartment of Neurology, Nishitaga National Hospital, Sendai, Japan b

Objective: To identify the expression of aquaporins (AQPs) in human neural cells. Background: AQPs constitute an evolutionarily conserved family of integral membrane water transport channel proteins. AQP4 is a target antigen for NMO-IgG in opticospinal multiple sclerosis (OSMS), although the mechanism underlying the highly selective lesion distribution in OSMS remains unknown. Methods: AQP1 and AQP4 expression was studied in cell cultures and brain tissues of four conventional MS cases by RT-PCR, Western blot and immunochemistry. Results: Human astrocytes in culture constitutively expressed both AQP1 and AQP4 on the cell surface. AQP4 protein levels were elevated in astrocytes by exposure to IFNG, but not by TNFA or IL-1B, while AQP1 was unaffected by any cytokines. AQP1 and AQP4 protein levels positively correlated with those of GFAP in MS brain homogenates. Varying numbers of non-hypertrophic astrocytes with elaborated processes contacting blood vessels, but neither macrophages nor neurons, expressed intensely both AQP1 and AQP4 in MS and non-MS brains. Conclusions: AQP1 and AQP4 were expressed predominantly in nonhypertrophic astrocytes widely distributed in the whole CNS, suggesting a redundant role of both AQPs in astrocyte water balance, and an involvement of undefined regional factors in the selective lesion distribution in OSMS. PP11-04

Vα7.2-Jα33 invariant T cells accumulate in multiple sclerosis and CIDP lesions in contrast to a deficiency of Vα24-JαQ NKT cells Zsolt Illesa,b, Takashi Yamamuraa a

Department of Immunology, National Institute of Neuroscience, Tokyo, Japan and bDepartment of Neurology, University of Pecs, Hungary

Deficiency of Vα24-JaQ NKT cells has been described in several autoimmune diseases, and we have observed absence of Vα24-JαQ NKT cells in multiple sclerosis (MS) lesions while conventional Vα24 T cells infiltrated MS plaques. This selective deficiency of invariant Vα24-JαQ NKT cells in MS prompted us to examine mucosal associated invariant T cells (MAIT) in nervous system autoimmunity, considering the proposed complementary functions, similarity and NK1.1 expression in the mouse homologues. Using RT-PCR SSCP method, expression of the reported four invariant TCR chains (Vα4, Vα7.2, Vα19 and Vα24) was examined in 25 MS lesions and 10 inflammatory demyelinating lesions of the peripheral nerves of chronic inflammatory demyelinating polyneuropathy (CIDP). Following

Abstracts

189

Chemokine gradients play a role in lesional expansion of secondary progressive multiple sclerosis

receptors of TNF-related apoptosis-inducing ligand (TRAIL) — DR4, DR5, decoy receptor-1 (DcR1), and DcR2, were expressed in MS lesions and whether expression correlated with disease activity. The results showed that reactivity for DR molecules varied among microglia, astrocytes, and oligodendrocytes in all CNS tissue studied and on inflammatory cells in active MS lesions. DR expression was not specific to MS and was seen at different levels in other neurological diseases (OND), and normal CNS tissue. DcR1 was expressed on oligodendrocytes in the CNS of all groups. To examine the functional implications of DR expression, human oligodendrocytes grown in vitro, were exposed to TRAIL, and apoptosis measured by FACS using the TUNEL technique. The findings both in vivo and in vitro show that although oligodendrocytes in MS, OND and normal CNS express DR5 and display apoptosis when treated with TRAIL in vitro, there was negligible evidence of oligodendrocyte apoptosis in and around MS lesions. These findings suggest that the loss of oligodendrocytes in MS is probably the result of a non-apoptotic mechanism and that there is preservation around and within some lesions that may be linked to the presence of decoy receptors.

Naoyuki Tanumaa,b, Hiroshi Sakumab and Yoh Matsumotob

PP11-07

a

Confocal immunofluorescence microscopy of optic neuritis

RT-PCR with Vα and Cα primers, the amplicons were electrophoresed in non-denaturing SSCP gel. DNA was hybridized with biotinylated internal Cα-, Jα-specific or invariant junctional probes. Our data indicate that invariant Vα7.2-Jα33 MAIT cells infiltrate autoimmune lesions of both the central and peripheral nervous system in contrast to Vα24-JαQ NKT cells, which were only found in CIDP lesions. No other invariant T cells could be identified beside these two subsets in any lesions. In addition, presence of both or one of the Vα24 or Vα7.2 NKT cell subsets was associated with expression of IL-4 in autoimmune demyelinating lesions of the PNS, proposing their anti-inflammatory role. The differential expression of the two subsets in MS lesions suggests that MAIT cells may complement NKT cells and substitute deficiency of invariant Vα24-JαQ T cells in MS. PP11-05

Metropolitan Fuchu Medical Center for Severe Motor and Intellectual Disabilities, Tokyo, Japan; bTokyo Metropolitan Institute for Neuroscience, Tokyo, Japan In secondary progressive (SP) multiple sclerosis (MS), there are slowly expanding demyelinating lesions with only modest inflammatory cell cuffing. Recently, evidence is accumulating that chemokines play an important role, not only in the leukocytes recruitment into the inflammatory site, but also in the recruitment of brain parenchymal cells. To elucidate the pathomechanisms of SPMS in terms of glial activation, we have investigated the expression of chemokines, MCP-1/CCL2 and IP-10/CXCL10, and their receptor, CCR2 and CXCR3 in the demyelinating plaques. Immunohistochemical analysis of autopsy brain tissues provided by UK Multiple Sclerosis Tissue Bank revealed that numerous hypertrophic astrocytes were observed at the rim, but not in the center, of the chronic active lesions. Microglia/macrophages phagocytosing myelin debris were also found at the lesion border. In contrast, T cell infiltration was minimal in these plaques. Characteristically, there were abundant immunoreactivities for MCP-1/ CCL2, IP-10/CXCL10, CCR2 and CXCR3 at the rim of the lesions with ongoing demyelination, whereas these immunoreactivities were relatively weak in the center of the lesions, forming chemokine gradients. Double immunofluorescense staining revealed that both chemokines and their receptors were expressed by hypertrophic astrocytes, while only chemokine receptors were expressed by MHC class II-positive microglia/macrophages. In addition, matrix metalloproteinase-9 (MMP-9) that is reported to be one of final effectors for demyelination was predominantly expressed at the rim of the plaque. These findings suggest that chemokine expression by astrocytes plays an important role in microglia/macrophage activation and expansion of demyelinating lesions in SPMS. Targeting chemokines in SPMS could therefore be a powerful therapeutic approach to inhibit lesional expansion.

John W. Rose1,3, Veda L. Tsoi1, Kenneth E. Hill1,3, Judith Warner4,5, and Noel G. Carlson1–3 Neurovirology Research Laboratory, VASLCHCS; 2GRECC, VASLCHCS; Neurology, University of Utah, Salt Lake City, USA; 4Neuro-ophthalmology, VASLCHCS; 5Neuro-Ophthalmology, Moran Eye Center, University of Utah, Salt Lake City, USA

1

3

Objective: Optic neuritis (ON) is a demyelinating inflammatory disease of the optic nerve which may occur as an isolated disease or related to multiple sclerosis (MS). We have initiated studies to evaluate the immunopathology of ON. Methods: Pathologic optic nerves were obtained at autopsy from a patient with clinical recovery from clinically isolated ON. Normal optic nerves and ON tissues were probed with antibodies to pathologic antigens including myelin basic protein (MBP) fragment, the inducible form of nitric oxide synthase (iNOS), macrophage markers CD14 and CD64, nitrotyrosine, and cyclooxygenase (COX-2). We also examined myelin basic protein (MBP), the oligodendrocyte marker cyclic nucleotide phosphodiesterase (CNPase), and glial fibrillary acidic protein (GFAP). Results: In the affected pathologic nerve, iNOS+ macrophages/microglia, iNOS+ astrocytes, COX-2, and nitrotyrosine were observed. iNOS and COX-2 were occasionally observed in the unaffected nerve. Decreased expression of MBP and CNPase was seen in the pathologic optic nerves, along with evidence of gliosis and ongoing myelin degradation indicated by the presence of MBP fragment. Conclusions: The immunopathology of clinically isolated optic neuritis from this individual resembles that observed in active MS plaques. Despite clinical recovery, there was evidence of ongoing inflammation and demyelination.

PP11-06 PP11-08

Death receptors in the CNS: A role for decoys? B. Cannellaa, S. Gauppa, K. Omaria and C.S. Rainea

CD200–CD200 receptor interaction in the regulation of microglia and macrophages in multiple sclerosis

a

Nathalie Koning1,2, R.M. Hoek2, I. Huitinga1

The mechanism by which myelin and oligodendrocytes are selectively targeted during lesion pathogenesis in multiple sclerosis (MS), is unknown but has been linked to tumor necrosis factor (TNF)-related damage and may be due to expression of TNF-related death-inducing surface receptors. The present study was undertaken to investigate by morphological and biochemical techniques, whether the death receptors DR3, DR6 and four

1

Albert Einstein College of Medicine., New York, USA

Netherlands Institute for Neurosciences, Amsterdam, the Netherlands; Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

2

The membrane glycoprotein CD200 (OX2) is highly expressed on neurons. In mice, CD200 has been shown to provide an inhibitory

190

Abstracts

signal to cells of the myeloid lineage, including macrophages and microglia, via the CD200 receptor (CD200R) (Hoek RM et al. Science, 2000). As activated macrophages and microglia are thought to be pivotal to the development of inflammatory lesions in the central nervous system of patients with MS, we hypothesize that reduced expression of CD200 is involved in activation of these normally quiescent cells. Therefore, we first study the expression of CD200 and CD200R in post-mortem frozen brain tissue from MS patients and controls by immunohistochemistry. Secondly, we use the laser dissection microscope to isolate well defined areas (rim and centre) of chronic active and chronic inactive white matter MS lesions. In these areas, gene expression profiles of CD200 and CD200R in relation to other molecules involved in myeloid cell regulation are determined by real time quantitative PCR, and compared with normal appearing white matter from both MS patients and healthy controls. Preliminary results show upregulation of CD200 in the rim of chronic active lesions, whereas in the rim and centre of chronic inactive lesions, CD200 is downregulated. Grant: Dutch foundation ‘MS Research’. Brain tissue: Netherlands Brain Bank. PP11-10

Remyelination is often incomplete or absent after demyelination in the CNS. Reasons for this attenuated repair might involve inhibitory factors within areas of myelin destruction, failure of demyelinated axons to support myelinating oligodendrocytes, or intrinsic limitations in the capacity of adult oligodendrocyte precursor cells. In the mature CNS, fibres with axonal segments lacking myelin sheaths can be generated experimentally by induction of axonal sprouting. We investigated the ability of axonal sprouting to induce a myelination response in the stratum radiatum and lucidum of the hippocampal CA3 region (srl-CA3) in the adult murine CNS, a region with pure sprouting and no degeneration. Using transection of the entorhino-hippocampal perforant pathway and an oligodendrocyte specific transgenic marker, we demonstrated that axonal sprouting in the srl-CA3 resulted in a significant 28% increase in the number of oligodendrocytes and a marked myelination response with a 41% increase in the length of myelinated fibres. Scattered proliferation of NG2+ oligodendrocyte precursor cells suggested a slow recruitment of myelinating cells. In contrast, in the dentate gyrus where degeneration occurs there was a strong NG2+ cell response but no subsequent change in oligodendrocyte numbers. Our study demonstrates that newly sprouted axons are able to elicit a myelination response in the mature CNS. This points to the healthy axon as a means to stimulate myelin repair in demyelinating disease.

Glutamate excitotoxicity damages demyelinated axons PP11-12 D. Pitt, A. H. Cross and M. P. Goldberg Washington University in St. Louis, St. Louis, USA Glutamate excitotoxicity has recently been implicated in the lesion pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). AMPA receptor antagonists have been shown to protect oligodendrocytes and axons in EAE, without altering the inflammatory response. Only oligodendrocytes, but not axons, are known to express AMPA receptors and axonal damage from excitotoxicity is believed to require the presence of oligodendrocytes [Underhill S.M., Soc. Neurosci. Abstr. 27 (2002), 869.2]. Since axons are lost in chronic demyelinated MS lesions, we examined the hypothesis that axons are vulnerable to glutamate excitotoxicity in the absence of oligodendrocytes. The cuprizone toxicity model was utilized in 4 week-old mice to eliminate oligodendrocytes and myelin from CNS white matter tracts. After 8 weeks of oral cuprizone administration, absence of myelin and oligodendrocytes in the corpus callosum was confirmed by Luxol fast blue and anti-CNPase staining. Microstereotactic infusion of the glutamate receptor agonist, AMPA, but not control vehicle, into the demyelinated corpus callosum resulted in substantial axonal damage as assessed by axon-specific Thy1YFP fluorescent labeling. These results indicate that AMPA toxicity to axons can take place in the absence of oligodendrocytes or myelin. Extracellular glutamate is likely increased in chronic MS lesions due to excess glutamate production and/or decreased glutamate transporter expression. Thus, excitotoxic damage to demyelinated axons is a feasible scenario. Excitotoxicity could therefore contribute to accumulation of axonal loss in MS lesions and may thus represent a target for neuroprotective intervention. D. Pitt is a Fellow of the National MS Society, USA. PP11-11

Axonal sprouting in the adult CNS elicits long-term increases in the number of oligodendrocytes and length of myelinated fibres N. Drojdahl1, H.H. Nielsen1, J. Gardi2, A. Wree3, M. Meldgaard1, A. Peterson4, J.R. Nyengaard2, B. Finsen1 University of Southern Denmark, Odense, Denmark; 2Aarhus University, Aahus, Denmark; 3 Rostock University, Rostock, Germany; 4 McGill University, Montreal, Canada

1

Syncytin-1 mediates endoplasmic reticulum stress in a transgenic mouse model of multiple sclerosis Joseph M. Antony1, Kristofor Ellestad5, Neda Shariat1, Robert Hammond2, Kazunori Imaizumi3, Francois Mallet4 and C. Power1,5 1

Department of Clinical Neurosciences, University of Calgary, Calgary, AB, Canada; 2Department of Pathology, London Health Sciences Centre and the University of Western Ontario, Canada; 3Department of Anatomy, Faculty of Medicine, University of Miyazaki, Japan; 4UMR CNRS-bioMerieux, IFR128 BioSciences Lyon-Gerland, Ecole Normale Superieure de Lyon, Lyon, France, 5Department of Medicine, University of Alberta, Edmonton, AB, Canada

Background: Syncytin-1 is a complex human retroelement envelope protein, which is up-regulated in the CNS of patients with MS and also contributes to oligodendrocyte death and demyelination although the underlying pathogenic mechanisms remain unknown. Methods: We developed a transgenic mouse line that selectively expressed Syncytin-1 in the CNS, together with performing analyses of host responses in brains of MS and non-MS patients and in cultured human neural cells. Results: Implantation of TNFα into the corpus callosum of Syncytin-1 transgenic mice caused neuroinflammation, neurobehavioral deficits and oligodendrocyte injury while also activating the endoplasmic reticulum (ER) stress-related genes, GADD153/CHOP, BiP, PERK, Grp58/ERp57, OASIS and iNOS, compared to wild type littermate controls. Similar findings of ER stress gene and iNOS induction were observed in the brains of MS patients. Syncytin-1 induced both OASIS and iNOS expression in astrocytes in conjunction with the repressive transcription factor, Egr1, which binds the promoter of ASCT1, a putative Syncytin-1 receptor. Likewise treatment of astrocytes with a nitric oxide donor also suppressed ASCT1. Astrocyte dysfunction resulting from ER stress was characterized by diminished levels of ASCT1 and trophic factors, particularly IGF-1. Inhibition of ASCT-1 in astrocytes resulted in reduced oligodendrocyte viability, which was mirrored by reduced ASCT-1 expression in the brains of Syncytin-1 transgenic mice and MS patients compared to respective controls. Discussion: The mechanism of oligodendrocyte damage and demyelination described herein represents a novel pathway by which over-expression of a human endogenous retroelement protein, Syncytin-1, leads to astrocyte

Abstracts dysfunction with adverse consequences in terms of oligodendrocyte viability and function.

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PP11-14

PP11-13

Hypomyelination as a consequence of altered MBP gene transcription

The RR-mouse: A double-transgenic SJL/J mouse model of spontaneous relapsing–remitting Multiple Sclerosis

Stefanie Gauppa, Glaucia Furtadob, Joseph Arezzoa, Sergio Lirab and Cedric S. Rainea

Bernadette Pöllinger1, Gurumoorthy Krishnamoorthy1, Hans Lassmann2, Hartmut Wekerle1, Andreas Holz1

a

1

MPI Neurobiology, Martinsried, Germany; University of Vienna, Austria

2

Institute of Neurology,

We first generated transgenic mice with a TCR specific for MOG peptide 92–106 in context of I-As. The paired TCR genes (Vα8.3 and Vβ4) from an encephalitogenic T cell clone, were injected into fertilized oocytes of FVB origin. The transgenic founders were backcrossed into the SJL/J and B10.S strains. Seven transgenic lines expressed the transgenic TCR with low (<10%), medium (35%) and high (95%) frequency. All transgenic T cells responded vigorously to MOG in vitro, produced IFNγ and TNFα and transferred EAE upon activation. TCRMOG-high transgenic mice, line F1640, were crossed with IgHMOG knock-in mice (IgHMOG) expressing the H chain of an anti-MOG antibody, 8.18C5. Double transgenic mice (SJL/JN3) spontaneously developed EAE in 100% of females and 60% of males within 3 months of age, while >20% of TCRMOG SJL/J mice and none of the IgHMOG mice developed spontaneous RR-EAE. In 89% of females, and 33% of males EAE took a relapsing–remitting course with an initial bout of ataxia and weight loss followed by complete recovery within few days, and, about 2 weeks later, a first relapse, either with ataxia (33%) or classical EAE (67%). Further relapses left neurological residua. The inflammatory lesions, dominated by macrophages and CD4+ T cells, faithfully reflected nature and timing of the clinical disease episodes by location and cellular composition of the lesions.

Albert Einstein College of Medicine, NY, USA; bMt. Sinai Medical School, NY, USA Delayed or immature myelination has been noted in a number of transgenic models in which the transgene in question was under the control of a myelin-related promoter expressed by oligodendrocytes. During a study on mice overexpressing CCL2 (MCP-1) a chemokine involved in monocyte recruitment, in which the chemokine was under control of a myelin basic protein promoter and was produced by oligodendrocytes, we noticed that, widespread evidence of hypomyelination was apparent throughout the CNS in mice up to four months of age, most pronounced in optic nerves and subpial zones of spinal cord. In these areas, oligodendrocytes retained an immature phenotype, structurally and immunocytochemically, the vast majority (>75%) of axons were non-myelinated and of small caliber, the remainder thinly myelinated, and astrocytes and their processes invested the naked axons. Apart from an occasional monocyte within the CNS, breakdown of myelin and oligodendrocyte pathology were not prominent. In older animals, deeper white matter was more normally myelinated but interstitial parenchyma was intensely gliotic and there was some evidence of apoptosis of oligodendrocytes. Electrophysiology for visual evoked potentials showed responses to be delayed and substantially diminished in peak amplitude compared to age-matched, wild-type littermates. Data will be presented on protein levels for the myelination-related gene profile and for developmental pathways associated with oligodendrocyte maturation, which was apparently arrested. These findings underscore the need for the careful analysis of CNS architecture in models involving transgenes expressed by glial cells. Supported in part by NMSS 1001-K-1; NS 08952; NS 11920 and NS 07098.

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Poster Session 12: MS-immunological studies

Abstracts

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PP12-01

PP12-03

Characterization of new Class-I myelin peptides and frequency of responding cells in multiple sclerosis patients and healthy individuals

Dendritic cells from Multiple Sclerosis patients suffering bacterial infections, lower activation threshold and increase survival of autoreactive T cells

L. Berthelot1, D. Laplaud1, C. Ballet1, J. Moynard1, F. Connan2, S. Brouard1, J-G. Guillet2, J. Choppin2, J-P. Soulillou1

M.F. Farez, J. Correale

1

INSERM U643, Nantes, France; 2Institut Cochin, Paris, France

We have shown that CD8 T-cells are the major population exhibiting clonal alterations of T-cell receptor CDR3 length distributions in the blood of multiple sclerosis (MS) patients. We aimed to characterize the epitopes recognized by CD8 T-cells that are derived from myelin antigens (MBP, PLP, MOG). The most frequent Class-I MHC in our patients were HLA-A2, -A3, -B27, -B7 and -B44. Peptides able to bind to these MHC were selected by sequence analysis of antigens, synthesised and tested for their affinity for the corresponding MHC. The recognition of these peptides by MS patients and normal individuals (NI) PBMC was tested by IFNg Elispot. Among the 16 MS patients, 9 exhibited an IFNg response following peptidic stimulation. Among the 8 NI tested, 2 had a similar response. There was a significant increase in the frequency of responding cells in MS patients versus NI (p < 0.05). There was no public recognition of myelin peptides, since each patient had a private response pattern. We identified 22 new epitopes of myelin proteins. Moreover, one patient showed serial positive responses at 4 time points for 3 peptides and responded to new peptides during the follow up. Three patients, tested positive in the first stimulation, were negative in the second, corresponding to a relapse. Collectively, we have characterized new autoreactive peptides. MS patients have a higher frequency of responding cells in blood than NI. Both NI and MS patients respond to various autoreactive peptides, these responses fluctuate in time and may depend on the disease state. PP12-02

Calpain activity correlates with cytokine production in patients with multiple sclerosis (MS) MK Guyton, S Imam, A Das, A Haque, WR Tyor, SK Ray, NL Banik

Institute for Neurological Research Dr. Raúl Carrea (FLENI), Buenos Aires, Argentina Dendritic cells (DC) are potent antigen-presenting cells and are critical for onset of the immune responses generates against infections. Bacteria and viruses have been implicated in autoimmune disease pathogenesis. Indeed, Multiple Sclerosis (MS) relapses are frequently associated with infection, in some instances bacterial. The hypothesis of this study was to establish whether DC from bacteria-infected MS patients modified autoreactive T cells patterns of activation, thus triggering disease exacerbation. CD1+, CD11c+, CD80+, CD86+, CD14−, HLA-DRbright DC were separated from peripheral blood during 11 exacerbations linked to bacterial infections and 11 relapses without infections. DC antigenpresenting capacity was assessed with MBP, MOG, GM1, and GM3 specific autologous T cell clones (TCCs). TCCs stimulated with DC from infected MS patients showed maximal proliferation, and induced the secretion of IL-12, IL-17, and IFN-γ at 10 to 30 times less concentration than after incubation with DC isolated from uninfected individuals. Interestingly, CD1-restricted GM1 and GM3 TCCs incubated with DC from infected MS patients secreted IFN-γ, and IL-12 even in the absence of exogenous antigens. These activation patterns correlate with increased in T cell survival. DC from infected MS patients secreted more IL-12 and IL-18, and showed higher expression of the myeloid differentiation factor 88, as well as molecules CD1, B7-1, B7-2, B7-DC, CD40, CD83 and CCR7, as compared to DC from uninfected individuals. Overall, these results are consistent with the concept that during bacterial infections, DC play a critical role in MS relapse induction. PP12-04

BDNF production by immune cells of patients with relapsing remitting multiple sclerosis is reduced and is not responsive to CD40 stimulation

Medical University of South Carolina, Charleston, SC, USA A. Karni, D Azulay Multiple sclerosis (MS) is a devastating T cell-mediated auto-immune disease of the CNS, The l mechanisms responsible for Th1/Th2 cytokine dysregulation seen in MS are unclear; however, increased expression/ activation of calpain may play are role. Our previous studies indicate that increased calpain activity correlates with production of the Th1 cytokine interleukin (IL)-2 in activated peripheral blood mononuclear cells (PBMCs), we hypothesize that calpain expression/activity will correlate with Th1/Th2 cytokine dysregulation in activated PBMCs from relapsing/remitting MS patients and that treatment with a calpain inhibitor will alter cytokine production. In order to investigate a role of calpain in Th1/Th2 cytokine dysregulation, PBMCs from relapsing/remitting MS patients and age/sexmatched controls were pretreated for 1 h with the calpain inhibitor calpeptin (100 μM) or vehicle (DMSO). Cells were then activated for 24 h with antiCD3 (10 μg/ml) + anti-CD28 (5 μg/ml) and harvested for Western blot analysis of calpain expression and activity. Supernatants were collected for determining levels of Th1 (IL-2) and Th2 (IL-4, IL-10) cytokines. Preliminary data indicate that calpain expression and activity were significantly (p ≤ 0.05) increased in both activated and unactivated PBMCs from remission and relapse patients versus controls. IL-2 production was significantly increased in relapse patients, while IL-4 and IL-10 levels were higher in remission patients. Interestingly, pretreating PBMCs with calpeptin resulted in a decrease in both Th1 and Th2 cytokine levels. These data suggest that calpain plays an important role in the production of cytokines in patients with MS. Supported by NIH-NINDS.

Neuroimmunology Laboratory, Department of Neurology, Tel Aviv Sourasky Medical Center, Sackler's Medical School, Tel Aviv University, Tel Aviv, Israel We studied the production of BDNF and IFN-γ from PBMCs and the immune regulation of neuronal protection in 14 untreated patients with relapsing remitting MS (RR-MS) and 18 healthy controls (HC). Cells were incubated in several conditions: with monoclonal antibodies (mAb) against CD3, CD40 or CD154 or with IL-6 or TNF-α. BDNF and IFN-γ in the supernatants were measured by ELISA. Supernatants of the different conditioned PBMCs were added to neuroblastoma cell lines under starving condition in order to study their neuro-protective potential, as measured by neuronal cell count, neurites count or length. Cellular analysis of BDNF production was done by flow cytometry. PBMCs of RR-MS patients secreted lower BDNF (1044 ± 377 pg/ml) compared to HC (1360 ± 386 pg/ ml, p = 0.046). Monocytes was found to be the dominant BDNF producers (28.0%), compared to T cells (1.0%) and B cells (0.8%). Stimulation of PBMCs from HC with either anti CD3 mAb, anti CD40 mAb or LPS enhanced BDNF secretion (1602 ± 666 pg/ml, p = 0.002; 1483 ± 605 pg/ml, p = 0.01; 2940 ± 1052 pg/ml, p < 0.001, respectively). Only anti CD3 mAb stimulation enhanced IFN-γ secretion (2249 ± 699 pg/ml vs. 31–71 pg/ml in other conditions, p < 0.05). Supernatants of PBMCs that were cultured with anti CD40 or LPS induced protective effect on neuroblastoma cell lines,

194

Abstracts

while supernatants of anti CD3 stimulated PBMCs induced cell death. BDNF secretion from PBMCs of RR-MS patients failed to be up-regulated by anti-CD40. In summary, Lower BDNF production by immune cells in RR-MS is related to dys-regulation of BDNF via CD40 and may contribute the neuronal damage of RR-MS. PP12-05

γδ T cells derived from Multiple Sclerosis patients mediate antibody-dependent cell cytotoxicity via Fcγ receptor III (CD16) Zhihong Chena,b, Iva Farmerb, Mark S. Freedmana,b,c a

Department of Microbiology and Immunology, University of Ottawa, Ottawa, Canada; bOttawa Health Research Institute, Ottawa, Canada; cThe Ottawa Hospital, General Campus, Ottawa, Canada

Multiple Sclerosis (MS) is an autoimmune Central Nervous System (CNS) demyelinating disease characterized by the infiltration of inflammatory cells and soluble immune factors. Though the exact mechanism of immunopathogenesis is still unknown, both innate and adaptive autoimmune responses have been proposed. γδ T cells are involved in innate responses and are concentrated in MS lesions. Activated γδ T cells lyse human oligodendrocytes in vitro but the exact mechanism of lysis is unknown. There are many ways in which γδ T cells could exert cytotoxicity and we have investigated the possibility that they might participate in antibody-dependent cell cytotoxicity (ADCC) via their expression of Fcγ receptor III (FcγRIII, CD16). To investigate if γδ T cells derived from MS patients were able to cause specific injury to target cells via ADCC, we established an in vitro experimental system using a humanized anti-CD20 monoclonal antibody rituximab (Rituxan®) as the bridging antibody and CD20+ B lymphoma cells lines as targets. Cytotoxicity was measured with multiple parameter flow cytometry. Our results show that, with Effector:Target ratio (E:T) at or above 4:1, the ADCC-type specific cytotoxicity of γδ T cells against the Raji cell line was significantly increased by the presence of rituximab (p < 0.02). Our findings suggest that γδ T cells, via Fc receptor binding, could lyse potential targets. Antibodies to myelin proteins are increased in both MS serum and spinal fluid, raising the possibility that if γδ T cells bind these antibodies, they could become effective targeted killers of oligodendrocytes. PP12-06

Human glial cells express NKG2D ligands that trigger immune cytotoxicity: Implications for multiple sclerosis Philippe Saikali1, Jia Newcombe2, Zhihong Chen3, Mark Freedman3, Jack Antel1, and Nathalie Arbour1, 4 1

Neuroimmunology Unit, Montreal Neurological Institute, 3801 University Street, Rm 111, Montreal, QC, Canada H3A 2B4; 2NeuroResource, Institute of Neurology, University College London, 1 Wakefield Street, London WC1N 1PJ, England; 3Ottawa Health Research Institute, University of Ottawa, 501 Smyth Rd, Ottawa, ON, Canada, K1H 8L6; 4Research Center — CHUM, University of Montreal, Pavilion JA DeSeve, 2099 Alexandre DeSeve, Montreal, QC, Canada, H2L 4M1

Immune cells such as CD8, CD4 and gamma delta T cells are identified in lesions within the central nervous system (CNS) during inflammatory diseases such as multiple sclerosis (MS), suggesting that they are actively involved in the oligodendrocyte/neuronal injury. Human NK cells, CD8+ T cells and gamma delta T cells express NKG2D, a stimulating or costimulating receptor recognizing multiple ligands. NKG2D ligands, usually expressed on stressed/damaged cells are aberrantly expressed on targets during autoimmune inflammatory diseases such as celiac disease and

rheumatoid arthritis and can contribute to enhance cytotoxic immune effectors locally. We sought to determine whether NKG2D ligands are expressed on human CNS cells. NKG2D ligands were detected on primary cultures of human adult oligodendrocytes and fetal astrocytes. Proinflammatory cytokines increased their expression on astrocytes, suggesting that under inflammatory conditions, such ligands could be upregulated. In vitro killing assays using activated human gamma delta T cells (or NK cells) as effectors demonstrated that both oligodendrocytes and astrocytes are susceptible to NKG2D-mediated injury since NKG2D blockade consistently reduced the extent of death. Immunohistochemical analyses suggest that NKG2D ligands are selectively detected in MS lesions but are absent from normal appearing white matter. These results imply that the recognition of NKG2D ligands on oligodendrocytes and astrocytes by NKG2D bearing immune effectors could contribute to cytotoxic responses especially in inflammatory conditions such as those occurring in MS. PP12-07

Reduced interferon-type I induced STAT4 activation and interferon type 1 receptor expression on peripheral blood mononuclear cells of interferon-naive relapsing remitting multiple sclerosis patients C.S. Constantinescu, A. Fahey, R.A. Robins University of Nottingham, Nottingham, United Kingdom Multiple sclerosis (MS) is an inflammatory demyelination disease of the central nervous system characterised clinically by neurological deficits which are often relapsing and remitting. Type 1 interferons (IFN), comprising IFN-beta and IFN-alpha, have immunomodulatory effects that can be beneficial in relapsing–remitting (RR) MS. Both IFN-beta and IFNalpha bind the same receptor and activate signalling pathways that include the phosphorylation STAT4 signal transduction molecule. The objective of this study was to compare the IFN activation of STAT4 in patients with MS who were not on treatment with IFN, and normal controls. We assessed STAT4 activation by intracellular staining with a phosphospecific antibody and flow cytometry. We investigated STAT4 activation by IFN-beta and alpha in 30 patients with RR MS and 20 age and sex matched healthy controls. We found that STAT4 activation was significantly reduced in RRMS patients compared to controls. IFN-beta is capable of inducing more STAT4 phosphorylation than IFN-alpha, but both were reduced compared to controls. To determine the mechanisms of reduced STAT4 activation in untreated MS, we quantified interferon type 1 receptor (IFNAR) expression on peripheral blood mononuclear cells of MS compared to controls, by flow cytometry using an antibody against IFNAR and by quantitative reverse transcriptasepolymerase chain reaction. We found that expression of IFNAR is reduced in RRMS patients compared to controls. This study suggests that RRMS patients may have reduced IFN-type 1 responsiveness compared to controls. Since we have shown that IFN-beta induces IFNAR, and MS patients have been shown to be IFN type 1deficient, this deficiency may be explained by reduced IFN-type 1 levels in MS. Current studies will determine whether treatment with IFN-beta enhances IFN responsiveness. Supported by MS Society of UK and Northern Ireland. PP12-08

Detection of NK-cells as putative regulators of multiple sclerosis disease activity during and after pregnancy L. Airasa, M. Sarastea, S. Väisänena, R. Kaajab, M. Färkkiläb and the Finnish Multiple Sclerosis and Pregnancy Study Group a

University of Turku, Turku, Finland and bUniversity of Helsinki, Helsinki, Finland

Abstracts Objective: Multiple sclerosis (MS) typically ameliorates during pregnancy but after the delivery there is an increase in the relapse rate. Our study was conducted as to better understand the immunoregulatory mechanisms behind this phenomenon. Methods: A prospective study including clinical, radiological and/or immunological follow-up of 42 MS-patients during pregnancy and six months into the postpartum period. Groups of healthy pregnant and nonpregnant persons and non-pregnant MS-patients were studied as controls. Laboratory investigations included measurement of intracytoplasmic cytokine production in peripheral blood lymphocytes and subtype analysis of T cells, B cells and NK cells both during and after pregnancy using immunofluorescence staining and FACS analysis. Results: Annualized relapse rate was significantly reduced during third trimester of pregnancy and increased after the delivery. Production of interferon-gamma by peripheral blood mononuclear cells was significantly less during pregnancy than in the postpartum period, resulting with an increased Th2:Th1 ratio during pregnancy in the MS-group but not in the control group. In MS, the Th2:Th1 ratio was 0.36 during pregnancy and 0.24 after the delivery. Diminished MS-disease activity during the last trimester of pregnancy was associated with an expansion of circulating CD56Bright regulatory NK-cells. Simultaneously, the proportion of circulating CD56dim NK-cells was reduced. Conclusions: CD56Bright NK cells are known as efficient producers of the Th2 cytokine IL10 and hence, our observation of in vivo expansion of CD56Bright NK cells during pregnancy may have implications for a potential role of CD56Bright regulatory NK cells in the control of autoimmunity during pregnancy in MS.

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Deptartment of Neurology, Erasmus MC, Rotterdam, The Netherlands; Department of Immunology, Erasmus MC, Rotterdam, The Netherlands; MS Centre ErasMS, Rotterdam, The Netherlands Disease activity in relapsing–remitting multiple sclerosis (MS) is significantly modulated during pregnancy, followed by a clear rebound activity after delivery. The biological mechanisms behind this phenomenon are largely unknown. Better insight in which natural immune and endocrine mechanisms are associated with clinically suppressed disease in MS can be of relevance for future management of the disease. In the Rotterdam Study on Pregnancy in MS, we aim to investigate the immune alterations that can be observed in the peripheral blood. We here investigated gene expression in the different stages before, during and after pregnancy. Blood monocytes were purified using MACS, RNA was extracted and gene expression was probed using Affymetrix RNA-arrays. Samples were assessed in longitudinal series of 4 time points per patient. After correction for multiple testing (22,000 gene transcripts), in a pilot study more than 100 genes were differentially expressed in the monocyte fraction during pregnancy compared with the non-pregnancy timepoint. The majority of genes was downregulated and most were linked to immune functions, such as chemokine family members and proinflammatory cytokines. This lends support to the hypothesis that during pregnancy also innate responses are attenuated. Significant findings will be confirmed with Taqman-PCR and at the protein level using immunoassays. Moreover data will be correlated to serum Il-10, IL-17 and IL-23 levels (measured by ELISA), as well as clinical disease activity, such as exacerbation after delivery.

PP12-09

Differential modulation of the immune response in Multiple Sclerosis by viral and parasite infections J. Correale, M. Fiol, M. Farez, Institute for Neurological Research Dr. Raul Carrea (FLENI), Buenos Aires, Argentina Epidemiological and clinical observations suggest that viral infections may introduce a bias in immune responsiveness in Multiple Sclerosis (MS) patients, which in turn triggers disease exacerbations. Conversely, striking inverse correlation occurs between parasite infections and autoimmune diseases. The immune responses of 23 MS patients during viral infections, and 12 MS patients presenting parasite infections were studied. Numbers of IFN-γ, TNF-α, and IL-12 secreting cells, were higher in PBMC collected during exacerbations associated to viral infections, than during stable disease or exacerbations without infections. In parasite infected MS patients on the other hand, MBP-specific responses showed a significant increase in IL-10 and TGF-β, and a decrease in IL-12 and IFN-γ secreting cells, compared with uninfected MS individuals. In addition, only viral antigen (Ag) stimulation induced maximal myelin-Ag specific T cells effector response, at concentrations 20 to 30 times lower than native Ag alone. Moreover, MBP-specific T cell clones from parasite-infected patients were characterized by a cytokine profile similar to Th3 and Tr1 T cell subsets, and cloning frequency of CD4+CD25+FoxP3+ T cells was substantially increased in parasite infected MS patients compared both to uninfected and viral infected MS individuals. Overall, these observations suggest that whereas viral infections can increase PBMC response to myelin-Ag causing a Th1-like response increasing the relapse risk, parasite infections are able to induce regulatory T cells that on the contrary, can induce remission of the disease.

PP12-11

Functional deficit in CD4+CD25+Foxp3+ regulatory T cells in multiple sclerosis Ying CQ Zang, Jian Hong, Sheri M Skinner, Jingwu Z Zhang Institute of Nutritional Sciences, Shanghai Institutes of Biological Sciences, China Academy of Sciences, China and Department of Neurology, Baylor College of Medicine, Houston, Texas, USA Multiple sclerosis is believed to be a T cell-mediated autoimmune disorder of the central nervous system and may involve impaired immune regulatory mechanism. In this study, we examined the CD4+CD25+T cells isolated from the peripheral blood of MS patients and healthy individuals. We found that the cell number of CD4+CD25+ T cells in MS was slightly higher than in healthy individuals, but the Foxp3 (a Treg specific gene) expression in CD4+CD25+ T cells derived from MS patients was significantly lower than that from healthy controls. Furthermore, CD4+CD25+ T cells isolated from MS patients had less inhibitory effect on T cell activation as compared to CD4+CD25+ T cells derived from healthy controls. Our results suggest that CD4+CD25+ T cells in MS represent mostly in vivo activated inflammatory T cells that lack the expression of Foxp3 and inhibitory function. The functional deficits of CD4+CD25+Foxp3+ regulatory T cells might contribute to hyperactivity of T cells of pro-inflammatory potential in MS. PP12-12

Mobilization of CD34+ myeloid progenitors in the blood of multiple sclerosis patients

PP12-10

Pregnancy as a natural modulator of disease activity in multiple sclerosis

S. Natafa, E. Reynaudc, G. Androdiasb, C. Malcusc. M.F. Belinb, C. Confavreuxa and C. Vuaillatb a

R.F. Neuteboom, K. Assink, J. D. Laman, P.J. van der Spek, L.A. Boven, and R.Q. Hintzen

Neurobiotec, Service de Neurologie A, Hospices Civils de Lyon, Lyon, France; bINSERM U433, Universite Claude Bernard Lyon 1, Lyon, France; c Laboratoire d'Immunologie, Hospices Civils de Lyon, Lyon, France

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Abstracts

Recent data indicate that blood monocytes form a heterogeneous cell population, which comprises a subset of progenitor cells expressing CD34, a surface molecule found on haematopoietic progenitors, haematopoietic stem cells and microglia. These CD14+/CD34+ cells are endowed with a high proliferative potential and display differentiation potential toward macrophages, dendritic cells and possibly microglia. Here, we assessed the behavior of blood-circulating CD34+ myeloid cells in multiple sclerosis (MS) patients. We analyzed by FACS the peripheral blood mononuclear cells (PBMCs) obtained from 10 relapsing–remitting MS (RRMS), 10 primary progressive MS (PPMS) and 10 healthy control subjects. In parallel experiments, we performed a functional assay by culturing PBMC in methylcellulose supplemented with macrophagecolony stimulating factor. The percentage of CD14+ cells, MHC class II+ cells or CD86+ cells was unchanged in MS patients as compared to controls. In contrast, both RRMS patients and RPMS patients showed increased percentages of CD34+ myeloid cells (4.59 ± 0.5% in RRMS patients, 4.57 ± 0.52% in RPMS patients, 2.71 ± 0.23% in controls, ⁎⁎⁎p < 0.005, Student's t test). Such an increase was partly due to the specific expansion of CD14+/CD34+ cells as shown by double-staining experiments. Accordingly, we could generate large colonies of macrophages (at least 50 cells per colony) in MS patients (2 out of 5) but not controls (0 out of 5). Our data show that myeloid progenitors are mobilized in the blood of MS patients. The migratory behavior and fate of such cells under neuro-inflammatory conditions is currently being assessed in mice.

PP12-13

Secondary progressive in contrast to relapsing–remitting multiple sclerosis patients show a normal CD4+CD25+ regulatory T cell function and FOXP3 expression Koen Venken1, Niels Hellings1, Karen Hensen2, Jean-Luc Rummens2, Robert Medaer1, Marie B. D'hooghe3, Bénédicte Dubois4, Jef Raus1 and Piet Stinissen1 1

Hasselt University, Biomedisch Onderzoeksinstituut and Transnational University Limburg, School of Life Sciences, Diepenbeek, Belgium; 2 Clinical Laboratory of Experimental Hematology, Virga Jesse Hospital, Hasselt, Belgium; 3National MS Centre, Melsbroek, Belgium; 4Laboratory for Neuroimmunology, University of Leuven, Leuven, Belgium

In the present study, the phenotypic and functional characteristics of CD4+ CD25+ regulatory T cells (Tregs) isolated from the peripheral blood of patients with relapsing–remitting (RR, n = 31) and secondary progressive (SP, n = 21) multiple sclerosis (MS) were investigated. No significant quantitative abnormalities in CD4+ CD25+ T cells from RRand SP-MS patients were detected. However, the mean suppressive capacity of Tregs towards anti-CD3 induced responder cell proliferation was found to be significantly lower in RR-MS patients (43 ± 7%) as compared to Tregs from SP-MS patients (80 ± 3%). The suppression of MS derived Tregs is correlated with disease duration but not with age indicating that Treg function is more affected in the early phase of the disease process. Tregs from SP-MS patients showed normal levels of FOXP3 mRNA in contrast to Tregs from RR-MS patients that had a reduced FOXP3 expression. Recently, we developed a CFSE based assay to compare myelin specific responses of CD4+ T cells and Treg depleted CD4+ T cells (i.e. CD4+CD25− T cells). CD4+ T cell responses against myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) were increased in Treg depleted CD4+ T cells of healthy controls. We are currently using this indirect approach to analyze Treg suppression of myelin CD4+ T cell reactivity in MS patients. Taken together, these data are the first to demonstrate differences in function and FOXP3 expression between CD4+ CD25+ T cells from patients

with RR- and SP-MS. In addition, Tregs appear to control myelin specific T cell responses in healthy subjects. PP12-14

Enhanced levels of anti-myelin antibodies in multiple sclerosis patients as assessed by flow cytometry E.C.W. Breija, D.A.M. Heijnena, A. van der Goesa, C.E. Teunissena, C.H. Polmanb, C.D. Dijkstraa Department of Molecular Cell Biology and Immunology and bNeurology, VU medical center, Amsterdam, The Netherlands

a

Antibodies are thought to contribute to CNS inflammation and demyelination in a subgroup of multiple sclerosis (MS) patients. Antibodies directed against a number of myelin proteins can be measured in serum of multiple sclerosis (MS) patients, but no single antigen has been associated exclusively with MS. Studies in animal models suggest that antibodies with demyelinating capacity are directed against native conformational and posttranslationally modified epitopes. The current methods to detect anti-myelin antibodies often do not allow recognition of such epitopes, because recombinant or denatured proteins are used as antigens. We have developed a reproducible, flow cytometry-based assay to detect serum antibodies that bind to human whole myelin, allowing detection of antibody binding to conformational and post-translationally modified epitopes. Using this assay, anti-myelin antibodies were measured in serum of 152 MS patients and 40 healthy donors (HD). At the group level, anti-myelin IgM levels in MS were slightly higher than in HD (p = 0.012, Mann– Whitney). In addition, MS patients showed a highly significant increase in anti-myelin IgG (p < 0.0001, Mann–Whitney). Analysis of individual MS patients revealed that approximately 50% of MS patients showed enhanced anti-myelin IgG levels compared to HD. Enhanced anti-myelin IgM or IgG levels were found in all clinical subgroups of MS patients (RR, SP and PP), and the anti-myelin antibody levels did not correlate with disease severity (EDSS, MFSC). In summary, the myelin flow cytometry assay detects increased levels of anti-myelin antibodies in a subpopulation of MS, indicating this assay may provide a valuable biomarker to identify patients with antibody-mediated inflammation. PP12-15

Proteomic analisys of autoantibody reactivity to neural antigens in sera and cerebrospinal fluid of multiple sclerosis patients Bonetti, B.a; Lovato, L.a; Vattemi, G.a; Cianti, R.b; Gini, B.a; Bini, L.b a

Section of Neurology, Department of Neurological Sciences and Vision, University of Verona, Verona, Italy; bDepartment of Molecular Biology, University of Siena, Siena, Italy Several proteins of the central nervous system (CNS) have been investigated as potential targets for antibody-dependent immune response in multiple sclerosis (MS). In spite of these efforts, the precise identity of MS autoantigens has not been clearly defined yet. In most studies, investigators employed recombinant antigens and/or relevant peptides from pre-selected targets. In the present study we overcome such restrictive approach using a large panel of native protein antigens derived from white matter homogenate of human healthy CNS. We compared IgG repertoires in sera and cerebrospinal fluid (CSF) from 17 control subjects and 16 MS patients by immunoblotting after 2D-PAGE resolution. Results: The analysis of seric and CSF IgG reactivity after 2Dimmunoblotting showed altogether 349 spots. Among these, 117 spots were specifically recognized by MS patients; in particular, 73 were identified

Abstracts by seric IgG and 44 by CSF IgG. The majority of these spots was not recognized by corresponding sera, thus indicating a compartimentalized autoimmune response. Noteworthy, 7 spots detected by seric IgG and 7 by CSF IgG were present in >50% of MS cases and absent in control sera. Conclusions: The 2D-PAGE technique allowed us to increase the sensitivity of analysis compared to 1D-PAGE. The study of autoantibody reactivity against CNS antigens with this technique enabled the identification of about 117 spots in the serum and CSF of MS patients, which were not detectable in controls. Their characterization by mass spectrometry will provide important information regarding the target antigens involved in the autoimmune response in each MS subgroup. PP12-16

Autoreactive B-cell mediated immune response as an early fingerprint of neurological disorders associated with autoimmune processes El behi, Mohamed1; Lefranc, Didier1; Zéphir, Hélène1,2; Almeras, Lionel1; Dussart, Patricia1; Dutoit, Virginie1; Drobecq, Hervé3; Launay, David1,4; de Seze, Jérome2; Dubucquoi, Sylvain1; Vermersch, Patrick1,2; Prin, Lionel1 1

Laboratoire d'Immunologie, EA 2686, Faculté de Médecine, Pôle Recheche, 1, Place de Verdun, 59045 Lille Cedex, France; 2Service de Neurologie D, Hôpital Roger-Salengro, CHU Lille, 59037 Lille Cedex, France; 3Institut de biologie de Lille, Lille, France; 4Service de Médecine Interne, Hôpital Claude-Huriez, CHU Lille, 59037 Lille Cedex To identify antigenic targets in autoimmune demyelinating disorders, we have used analysis of serum self-IgG responses associated with a proteomic approach (SERPA) in different clinical and experimental models of neurological disorders associated with autoimmune responses. In a first step, a global analysis, by Western blotting of serum self-IgG responses against healthy and multiple sclerosis (MS) human brain protein antigens allowed to discriminate patterns obtained in MS patients, healthy subjects and in the three clinical forms of MS. A cluster of discriminant antigenic bands were also found by studying sequential changes during the progression of Experimental Autoimmune Encephalomyelitis (EAE), manipulated either by anti-inflammatory drugs or by the enhancement or the depletion of CD4+CD25+ regulatory T cells. To test the significance of such changes as potent hallmark of disease activity, serum self-IgG response was also evaluated in neuropsychiatric systemic lupus erythematosus (NPSLE) and in clinically isolated syndrome patients (CIS), the earliest clinical event in MS. Singular self-IgG patterns were found in NPSLE and prospective studies have shown that a significant number of patients with CIS present autoreactive pattern predictive of the development of clinically definite MS. Subsequently, a proteomic approach, was performed to characterize molecular targets involved. As previously noted in other organ specific autoimmune diseases, ubiquitous proteins appear as major discriminant targets found in MS, EAE and NPSLE. Only in some case, specific brain derived antigenic targets were found. Thus, SERPA provides a promising approach for investigating self candidate antigens involved either in regulatory or in pathogenic processes of autoimmune disorders and for the development of new diagnostic tools. PP12-17

Antigen microarrays identify unique immune repertoires in multiple sclerosis (MS)

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We used antigen microarrays to study the repertoire of serum antibodies in relapsing–remitting (RR), secondary progressive (SP) and primary progressive (PP) MS. Antigen microarrays consisted of 420 antigens including CNS-related autoantigens (e.g., MBP, MOG, PLP), lipids (gangliosides, galactocerebrosides) other autoantigens (e.g., insulin, GAD, collagen, GBM) and viral pathogens (e.g. EBV, herpes, varicella). We randomly assigned 2/3 of the samples to a learning set and 1/3 to a test set. To investigate natural autoantibodies, serum at a 1:10 dilution was assayed on two independent arrays and results analyzed using GeneSpring software. We found that we could distinguish RRMS, SPMS or PPMS from healthy controls by unique antibody patterns, but not by a single antibody reactivity. These antibody patterns were used to analyze the test set and correctly identified 75% of RRMS, 80% of SPMS and 94% of PPMS. The patterns we observed were different from those we found for other neurologic diseases. The discriminating patterns of antibody reactivity demonstrated that IgG was more informative that IgM, however, detailed analysis of IgG1 and IgG4 antibodies did not improve sensitivity. The discriminating antibodies we identified primarily targeted CNSspecific antigens, but also contained antibodies directed against the 60 kDa and the 70 kDa heat shock proteins (HSP60 and HSP70). IgM antibodies to EBV were associated to RRMS and SPMS. Our results demonstrate unique immune repertoires in MS that could be used as biomarkers for the diagnosis and evaluation of MS patients, and potentially to identify healthy individuals at risk for MS. PP12-18

What is the functional relevance of autoantibodies against myelin in people with multiple sclerosis? Shannon J. Beasley, Michael P. Pender and Judith M. Greer Neuroimmunology Research Centre, the University of Queensland, Royal Brisbane and Women's Hospital, Brisbane, Queensland 4029, Australia Many studies have identified antibodies against myelin and axonal proteins and glycolipids in people with multiple sclerosis (MS). In our studies, we have found increased levels of antibodies specific for myelin proteolipid protein (PLP) epitopes in sera of people with MS, compared to healthy controls and patients with other neurological diseases (OND). These studies have primarily used enzyme-linked immunosorbent assays (ELISA) and immunoblotting to detect antibodies. It is possible that the antibodies identified may not be particularly relevant to the disease process, as they may not recognize the antigen in its native configuration, which is likely to be necessary for the antibodies to be of functional relevance in MS. We have therefore been studying whether antibodies from patients with MS can recognize antigen in its native conformation, using human myelin opsonization assays and flow cytometry of stable cell lines expressing human myelin proteins. We find that antibodies from people with MS are significantly better at opsonizing myelin than antibodies from healthy controls or patients with OND. For the flow cytometry assays, stable cell lines have been generated expressing either, PLP and its alternatively spliced isoform, DM20, myelin basic protein (MBP) or oligodendrocyte-specific protein (OSP) attached to a fluorescent marker. The cells are incubated with serum samples from people with MS and controls, a FITC-labelled anti-human antibody is added, and the cells are read by a flow cytometer. This study will show whether the myelin-specific antibodies found in people with MS are likely to be of functional relevance in the disease. PP12-19

F. J. Quintana1, V. Viglietta1, K. O'Connor1, D. A. Hafler1, I. R. Cohen2, S. J. Khoury1, and H. L. Weiner1

Antibodies produced by clonally expanded plasma cells in the cerebrospinal fluid of MS patients display CNS autoreactivity

1

Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, USA; 2The Weizmann Institute of Science, Rehovot, Israel

Hans-Christian von Buedingen, Sandra Kuenzle, Mirjam Meier, Melanie Harrer, Norbert Goebels

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Abstracts

University Hospital Zurich, Department of Neurology, Clinical Neuroimmunology Unit, Frauenklinikstrasse 26, CH-8091 Zurich, Switzerland Oligoclonal IgG, the product of clonally expanded B/plasma cells, are a diagnostic hallmark in the CSF of MS patients. While increasing evidence supports a role of autoantibodies in the pathogenesis of demyelinating MS lesions, the antigenic specificities and pathogenic relevance of oligoclonal CSF IgG remain largely unknown. We performed single cell RT-PCR of expressed immunoglobulin genes on individual CD138+ FACS-sorted CSF-plasma cells from MS patients. Following sequence analysis we expressed paired heavy (H) and light (L) chain genes from clonally expanded plasma cells as whole recombinant human IgG1 in a eukaryotic expression system. Recombinant human monoclonal IgG1 were tested by Western blotting and immunofluorescence staining of brain tissue for their reactivity with CNS antigens. H chain variable region (IGHV) genes of expanded plasma cell clones from the CSF of 4 MS patients mostly utilize IGHV1, 3 and 4 subfamily genes. Some monoclonal recombinant antibodies derived from different patients showed specific reactivity with antigens present in brain tissue. In summary, we find signs of an antigen driven, CNSautoreactive humoral immune response in the CNS compartment of patients with MS. It remains unclear whether this autoantibody response is part of a primary immune response leading to or of a secondary immune response resulting from tissue destruction in MS. The pathogenic relevance of such antibody responses is subject to further investigations.

PP12-20

Diagnostic value of CSF autoantibodies against heterogeneous nuclear ribonucleoprotein A1 and A2/B1 in multiple sclerosis patients Motohiro Yukitakea, Eisaburo Sueokaa, Akemi Satoa, Naoko Sueokaa, Hiromi Ohashia, Mineki Saitob, Shuji Izumoc, Mitsuhiro Osameb, and Yasuo Kurodaa

PP12-21

Prevalence of Helicobacter pylori infection in Japanese patients with multiple sclerosis Wei Li, Motozumi Minohara, Takeshi Matsuoka, Osoegawa Manabu, Murai Hiroyuki, Jun-ichi Kira Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan TEL:+8192-642-5340 [Objective] Helicobacter pylori (HP) infection has been previously reported to be related to various diseases. Neutrophil activating protein (HPNAP) is capable of activating neutrophils and monocytes. The aim of this study was to disclose the prevalence of HP infection and the immune response to HP proinflammatory protein, HP-NAP, in Japanese patients with multiple sclerosis (MS). [Methods] We investigated the presence of anti-HP and anti-HP-NAP reactivity in serum of MS patients (n = 105) and compared them with healthy controls (n = 85). MS patients were clinically classified into two subtypes: opticospinal type (OSMS) or conventional type (CMS). [Results] We found that frequency of HP seropositivity was significantly lower in CMS patients (22.6%), as compared with OSMS patients and healthy controls (p = 0.0019 and p = 0.018, respectively). When analyzed separately by year of birth, for patients born after 1950, HP seropositive rate was significantly lower in CMS (20%) than OSMS patients (40.6%, p = 0.0483). Severe visual impairment (FS 5 or higher than 5) was significantly more common in HP-seropositive patients than seronegative patients in total MS patients (p < 0.05). EDSS score at final follow-up did not differ significantly between HP seropositive and seronegative groups. Anti-HP-NAP antibodies were found to be positive in 4 patients with MS and all of them were OSMS patients (8.2% positive). [Conclusions] Our study indicates a difference in HP seropositivity and the antibody to its proinflammatory protein, HP-NAP, between OSMS and CMS in Japanese. Although further studies are required, these observations may suggest distinction in the childhood environment in sufferers of these conditions. PP12-22

a

Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan; bDepartment of Neurology and Geriatrics and cDivision of Molecular Pathology of Center for Chronic Viral Diseases, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

Objective: The aim of this study is to draw a conclusion on the implication of anti-heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and/or A2/B1 antibodies (Abs) in multiple sclerosis (MS) and human Tcell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Methods: Using recombinant hnRNP A1 and B1 proteins, Western blotting was conducted for detection of anti-hnRNP A1 and A2/B1 Abs in 168 CSF samples in fully masked conditions. Results: All enrolled cases had neurological abnormalities and were tested for CSF anti-HTLV-I Abs. Among the 102 cases with negative anti-HTLV-I Abs, the incidence of CSF anti-hnRNP A2/B1 Abs was the highest in MS (88.0%, n = 25), that was significantly different from the incidence in neurodegenerative diseases (15.5%, n = 20) (p < 0.0001), meningoencephalitis (38.1%, n = 21) (p = 0.0013), and inflammatory polyneuropathies (36.8%, n = 19) (p = 0.0013). Among the 66 cases with positive anti-HTLV-I Abs, the incidence of CSF antihnRNP A1 Abs were far lower in HAM/TSP (35.0%, n = 40) than in the group of other diseases (57.7%, n = 26). Interpretation: The present study confirmed our previous finding regarding the significant association of CSF anti-hnRNP A2/B1 Abs with MS but it failed to demonstrate any diagnostic value of CSF anti-hnRNP A1 Abs in HAM/ TSP. CSF anti-hnRNP A2/B1 Abs may provide a clue in the diagnosis of MS.

Role of chronic infection in the aetiology/pathogenesis of idiopathic parkinsonism S. M. Dobbsab, R. J. Dobbsab, C. Wellera, A. Charlettd, N. Oxladea, I. T. Bjarnasonb, A. B. Pricee, M. Ibrahimc and R.W. Kerwina a

Section of Clinical Neuropharmacology, Institute of Psychiatry; Departments of bGastroenterology, and cImmunology, Guy's, King's, St Thomas' School of Medicine, dStatistics Unit, Health Protection Agency, and e Department of Histopathology, Northwick Park and St. Mark's Hospitals, Imperial College, London Objective: to elucidate causality by systematic exploration of clinical clues, calling on wide-ranging laboratory/clinical expertise. Methods: Simple and reliable objective measurements of facets of parkinsonism underpin statistical modelling, hypothesis generation and efficacy studies. Results: To date, proof-of-principle that infection contributes to idiopathic parkinsonism (IP) has been provided by case studies and interim protocol analyses of an efficacy study contrasting effect, on the time-course of IP facets, of successful Helicobacter pylori (Hp) eradication vs. placebo, and vs. failure (Helicobacter 2005;10:267–297). ‘Malignant’ IP appears converted to ‘benign’, but marked deterioration accompanied failure. Categorisation, according to presence/absence of overt Helicobacter infection, was a useful therapeutic tool even in late IP with cachexia. Although Hp infection is overt in a minority of probands, the predicted probability of having the parkinsonian label depends on the serum

Abstracts immunoblot Hp antibody profile, with clinically-relevant gradients between this “discriminant index” and disease-burden and -progression. Indeed, as we begin to explore urea-breath-test negative IP, we are finding molecular evidence for infection in gastric biopsies, where there is no growth on routine culture (presumably due to low organism density). Moreover, early experience of open intervention suggests that it might prove as beneficial as in culture-positive cases. Conclusion: This points to a direct or surrogate (not necessarily unique) role of a particular infection in the pathogenesis of IP. With eradication-failure, bolus-release of antigen from killed bacteria, could aggravate an effect of ongoing infection. The apparent importance of Helicobacter in the aetiology/pathogenesis of IP is not confined to those with overt infection. PP12-23

Anti-Nogo receptor autoantibody in the serum of multiple sclerosis Hiroyuki Onouea,b, Jun-ichi Satoha,d , Masafumi Ogawac, Takashi Yamamuraa National Institute of Neuroscience, NCNP, Tokyo, Japan; bDepartment of Internal Medicine Division of Neurology, Saga University, Saga, Japan;

a

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c National Center Hospital for MNMD, NCNP Tokyo, Japan; dDepartment of Bioinformatics, Meiji Pharmaceutical University, Tokyo, Japan

Objectives: A myelin-associated neurite outgrowth inhibitor Nogo-A plays a key role in inhibition of axonal regeneration. Axonal damage beginning at the early stage of MS is responsible for permanent neurological deficits, although its molecular mechanism remains unknown. The aim is to study the prevalence of autoantibodies against Nogo-A and Nogo receptor (NgR) in the serum of MS. Methods: The antibodies were identified in the serum of 30 MS patients, 22 patients with non-MS neurological diseases (OND), and 22 healthy control (HC) subjects by Western blot using recombinant human Nogo-A-specific segment (NAS), the shared segment of Nogo-A and -B (NAB), Nogo-66 (N66), the non-glycosylated form of NgR, the glycosylated NgR (NgR-Fc), and myelin oligodendrocyte glycoprotein (MOG). Results: None showed IgG antibodies against NAS or NAB. In contrast, 30% of MS, 23% of OND and 32% of HC subjects exhibited anti-N66 IgG, while 27% of MS, 27% of OND and 18% of HC showed anti-MOG IgG. None of HC but 33% of MS and 14% of OND showed anti-non-glycosylated NgR IgG. Furthermore, 60% of MS, 18% of OND and 14% of HC showed anti-NgR-Fc IgG. Conclusions: Because IgG autoantibodies against N66, NgR and MOG are often detected in the serum of MS and controls, they do not serve as a MSspecific marker.

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Abstracts

Poster Session 13: MS-biomarker and diagnosis

Abstracts PP13-01

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ratio may represent a potential surrogate biomarker for monitoring MS disease duration.

Interleukin-17 expression in multiple sclerosis PP13-03 J. S. Tzartosa, M. A. Frieseb, J. A. Palacea, M. M Esiria, and L. Fuggerb Department of Clinical Neurology, Radcliffe Infirmary, Oxford, UK; bMRC Human Immunology Unit and Department of Clinical Neurology, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK

a

Cell surface adhesion molecules and cytokine profiles in blood and CSF in primary progressive multiple sclerosis I. Elovaaraa,b, M. Ukkonenb, X. Wua and P. Dastidarc a

Multiple sclerosis (MS) is thought to be a predominantly T helper 1 mediated disease. However, recent findings in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), implicate a distinct subset of TH1 and TH2 cells. This novel T helper cell is characterised by the secretion of IL-17 (THIL17). As mice deficient in IL-23, lack the THIL-17 subset IL-23 is thought to drive the differentiation of THIL17. IL-23 knock out mice are resistant to EAE, whereas transferring only THIL-17 cells can induce disease. In addition previous microarray data from MS lesions show highly upregulated IL-17 expression. However, so far it is unclear which cell type and distribution is responsible for IL-17 expression in MS. Using double fluorescent immunohistochemistry techniques and confocal microscopy we observed that IL-17 is present in MS lesions in CD4+ and CD8+ T cells. Interestingly, astrocytes and to a lesser extent oligodendrocytes were also positive for IL-17 staining, whereas macrophages, microglia and neurons were negative. Higher levels of IL-17 were present inside the lesions rather than in normal appearing white matter and it was virtually absent in white matter from control individuals. Further, we identified IL-23 staining in perivascular DCs. These findings are in line with the recently proposed importance for perivascular DCs in EAE pathogenesis and implies a similar role in MS. It is possible that perivascular IL-23 expressing DCs drive the IL-17 expression visualised in MS brains. This interesting new T cell subset rewards further attention to unravel its role in MS pathogenesis. PP13-02

Elevated CSF and serum levels and intrathecal synthesis of active MMP-2 in MS patients with no evidence of MRI disease activity

University of Tampere, Neuroimmunology Unit, Tampere, Finland; Tampere University Hospital, Department of Neurology, Tampere, Finland; c Tampere University Hospital, Department of Radiology, Tampere, Finland b

Objective: We evaluated whether the expressions of adhesion molecules (AMs) and cytokines in blood and CSF could be used as markers of disease activity in primary progressive multiple sclerosis (PPMS). Methods: The expressions of AMs and the levels of 17 cytokines in patients with PPMS were compared with those in SPMS and controls and correlated with the volumes of focal and atrophic changes in brain magnetic resonance imaging (MRI). Results: The expressions of very late activation antigen 4 (VLA-4), lymphocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule1 (ICAM-1) in blood and CSF were higher in PPMS than in controls. Comparison between PPMS and SPMS showed higher levels of ICAM-1 in blood and CSF in PPMS, while the level of vascular adhesion molecule (VCAM-1) was higher only in blood. There was no difference in the levels of cytokines in serum or CSF between PPMS and SPMS or controls, but the evidence for intrathecal synthesis of IL-8 and MCP-1 was found in PPMS. The expressions of CSF VLA-4 in PPMS correlated with the total volume of cerebral lesions and the number of diffuse brain lesions in MRI, while the amount of LFA-1 in CSF correlated with the number of spinal T2 lesions. Conclusions: The upregulated expressions of AMs in blood and CSF and evidence for intrathecal synthesis of MCP-1 and IL-8 in PPMS indicate the importance of inflammatory changes in the pathogenesis of PPMS and suggest the use of these molecules as markers of disease activity in this MS subtype. PP13-04

E. Fainardia, M. Castellazzia, T. Bellinib, M.C. Manfrinatob, E. Baldia, C. Tamborinoa, E. Paolinoa, E. Granieria, F. Dallocchiob Section of Neurology University of Ferrara, Italy, bDepartment of Biochemistry and Molecular Biology, University of Ferrara, Italy

Regulation of monocyte CD163 expression in multiple sclerosis

a

We have measured cerebrospinal fluid (CSF) and serum levels of active matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor TIMP2 by Activity Assay System and ELISA, respectively, in 51 relapsing– remitting (RR) multiple sclerosis (MS) patients, grouped according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity, in 50 patients with other inflammatory neurological disorders (OIND) and in 50 patients with non-inflammatory neurological disorders (NIND). Statistical analysis was performed by Mann–Whitney, with Bonferroni post-hoc correction, and Spearman rank correlation coefficient tests. CSF and serum active MMP-2/TIMP-2 ratios were higher in MS than in NIND (p < 0.05 and p < 0.001, respectively) and in MRI inactive than in MRI active MS patients (p < 0.01 and p < 0.02, respectively) whereas CSF active MMP-2/TIMP-2 ratio was more elevated in MS than in OIND (p < 0.05). An intrathecal synthesis of active MMP-2 and TIMP2 was greater in MS than in OIND and NIND (p < 0.001) and in MRI inactive than in MRI active MS patients (p < 0.05). In addition, a positive correlation (R = 0.303; p < 0.01) was found between serum active MMP-2/ TIMP-2 ratio and MS disease duration. Overall, these results suggest that a shift in MMP-2/TIMP-2 balance towards proteolytic activity of MMP-2 could be relevant in tissue repair of a subset of MS patients with MRI inactive disease and seem to indicate that serum active MMP-2/TIMP-2

Babs O. Fabrieka, Lisa van Winsenb, Rianka Vloeta, Holger J. Mollerc, Bernard M.J. Uitdehaagb, Christine D. Dijkstraa, Timo K. van den Berga a

Department of Molecular Cell Biology, VU Medical centre, Amsterdam, The Netherlands; b Department of Neurology, VU Medical Centre, Amsterdam; cDepartment of Clinical Biochemistry, AAS Aarhus University Hospital, Denmark CD163 is a macrophage scavenger receptor highly expressed on perivascular macrophages in the brain. There are strong indications that CD163 plays a direct active role in the inflammatory response, and proinflammatory and anti-inflammatory mediators tightly regulate CD163. In multiple sclerosis (MS) there also seems to be a regulation of CD163 expression, in the brain at the lesion site the amount of CD163 expressing cells is increased, furthermore we have recently shown that membrane expression of CD163 on monocytes is decreased in MS patients compared to healthy donors, whereas the soluble form (sCD163) is increased in the plasma of the MS patients. To date the mechanism of CD163 shedding remains unclear, however there have been indications that matrix metalloproteinases (MMP) are actively involved. Furthermore, previous studies have shown that in vitro stimulation with dexamethasone (DEX), strongly upregulates CD163 expression on human blood monocytes. High-dose intravenous glucocorticosteroids (GCs) are frequently

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Abstracts

used to treat relapses in multiple sclerosis (MS), but the response to this treatment differs among patients, suggesting differences in sensitivity to GC. Due to the induction of CD163 by GC, we wondered if CD163 might be valuable as a marker to predict glucocorticoid responsiveness. Our results indicate an inducible regulation of CD163 in vitro as well as in vivo. Finally, the in vitro inducibility of CD163 by GC correlated positively with the clinical response to GC, suggesting CD163 as a possible prognostic marker for GC sensitivity. PP13-05

Levels of serum IgM antibody against refolded recombinant human MOG predict responsiveness to corticosteroids for treatment of acute multiple sclerosis relapses Michael Khalil, Bettina Kuenz, Andreas Lutterotti, Rainer Ehling, Claudia Gneiss, Robert Egg, Irmgard Mayringer, Florian Deisenhammer, Markus Reindl and Thomas Berger Clinical Department of Neurology, Innsbruck Medical University, Austria. Correspondence to [email protected] Background: Anti-Myelin-Oligodendrocyte-Glycoprotein (MOG) antibodies (Abs) are discussed as biological marker in multiple sclerosis (MS) patients. High-dose methylprednisolon (HDMP) i.v. therapy has been established for treatment of acute relapses in MS-patients. However, only scant information on the impact of corticosteroid treatment on (auto-) antibodies is available. Objectives: To establish an ELISA test system for detection of serum IgG and IgM Abs directed against refolded recombinant human MOG. To prospectively investigate whether acute relapses and subsequent HDMP therapy have an impact on serum anti-MOG Abs in MS-patients and whether anti-MOG Ab levels might predict the responsiveness to HDMP therapy. Methods: Anti refolded recombinant human MOG ELISA has been evaluated in 184 MS-patients and 188 healthy controls (HC). 25 MSpatients were then prospectively enrolled and serum samples were analyzed in remission before relapse, during acute relapse before HDMP treatment and 6 weeks after HDMP therapy. Results: Anti-MOG IgG (sensitivity 27.7% specificity 89.9%, p < 0.001) and anti-MOG IgM (sensitivity 23.4%, specificity 89.9%, p < 0.001) antibody levels were significantly increased in MS-patients compared to HC. HDMP treatment significantly reduced anti-MOG IgG (p < 0.001) and total IgG (p < 0.01), but not anti-MOG IgM and total IgM Ab levels. Patients with complete remission from acute relapse had a significant increase of antiMOG IgM Ab levels from baseline to acute relapse values. Conclusion: The findings from this prospective study could be important for future decisions in patients with acute relapses, especially decisions regarding either dosing/duration of HDMP or escalating therapies.

We analyzed by flow cytometry CD4+CD8+ apoptotic and CD3+ proliferating cell percentage, by RT PCR the expression of different anti (FLIP, XIAP, Bcl-2) and pro (BID, APAF-1) apoptotic genes in sorted CD4+ and CD8+ T cells stimulated with MBP peptides. Differences were analyzed by t-Student and Mann–Whitney tests. Percentage of apoptotic MBP specific CD4+ and CD8+ T cells decreased in AMS compared to SMS (p < 0.05) and HC (p < 0.05), higher proliferation index of MBP specific T cells was found in AMS and HC compared to SMS (p < 0.05). Higher expression, even thought no statistically significant, of anti and pro apoptotic genes was shown in RRMS compared to HC, however significant increase of anti apoptotic genes FLIP XIAP and Bcl-2, was observed in MBP-specific sorted CD8+cell of AMS compared to HC. The data obtained evidence specific activation of immune system against MBP in patients with AMS and in HC, but the increase of PCD in HC switch off the MBP specific T cell immune response, while, in AMS the decrease of apoptotic MBP specific T cell seems to be involved in the immune mediated destruction of myelin sheath. PP13-07

Differences in TRADD expression indicated by array analysis discriminates between MS disease subtypes Boris-A. Kallmanna, Eva Specha, Antje Kronera, Axel Petzoldb, David Millerb, Alan Thompsonb, Gavin Giovannonib, Peter Rieckmanna Department of Neurology, University of Wuerzburg, Germany; bThe Institute of Neurology, University College London, United Kingdom

a

M. Saresella, aI. Marventano, aR. Mancuso, aR. Mazziotti, bR. Longhi, cR. Cavarretta, cD. Caputo and dP. Ferrante

Relapsing–remitting (RR) and primary progressive (PP) MS differ in their clinical course and most likely also their underlying pathomechanisms. This study aimed to identify biomarkers in association with subtypes of MS, using cDNA array analysis of blood mononuclear cells (PBMC) as an initial approach. Array analysis was performed using pooled RNA derived from PBMC of patients with stable RRMS or PPMS not on immunomodulatory treatment. Using real-time RT-PCR differential expression of candidate genes was quantified in individual MS patients of two independent patient cohorts containing both subgroups (a) 9 RRMS/9 PPMS and (b) 27 RRMS/42 PPMS. Gene expression array analysis identified 51 genes differentially upregulated in RR-MS and 11 in PP-MS. Out of several candidate genes re-analysed individually by quantitative RT-PCR the death domain containing protein TRADD involved in tumor necrosis factor receptor (TNFR) superfamily signalling was consistently expressed at higher levels in PP-MS patients compared to RR-MS and healthy controls. This result was confirmed in two independent cohorts. High expression of TRADD protein associated with infiltrates was detected in the spinal cord of MS patients. Results of Western blots and three-coloured FACS analysis for TRADD in different PBMC populations will be presented. Array analysis is a useful screening approach in pooled MS patient samples to identify differentially expressed genes. TRADD, a member of TNFR superfamily, is one of these candidate genes and may serve as biomarker to discriminate PPMS from RRMS patients. This suggests distinct pathogenic processes in these MS subtypes.

a

PP13-08

PP13-06

Programmed cell death and expression of apoptotic genes in PBMC from Multiple Sclerosis patients a

Laboratory of Molecular Medicine and Biotechnology, Don C. Gnocchi Foundation ONLUS, IRCCS, Milan, Italy; bCNR, cNeurologic Department, Don C. Gnocchi Foundation ONLUS, IRCCS, Milan, Italy; dDepartment of Biomedical Science and Technology, University of Milan, Italy We demonstrated that in Multiple Sclerosis (MS) deregulation of programmed cell death (PCD) play key role in inducing or maintaining autoreactive immune phenomenon leading to demyelinating lesions. In the present study we investigated the PCD of myelin basic protein (MBP)-specific T lymphocytes in 47 relapsing–remitting (RR) MS patients with 29 acute (AMS) or 18 stable MS (SMS) and 30 Healthy Controls (HC).

Inverse association between CSF levels of soluble HLA-G and Fas molecules in MS patients with no evidence of MRI disease activity E. Fainardia, Castellazzia, R. Rizzob, L. Melchiorrib M., E. Granieria, O.R. Baricordib Section of Neurology University of Ferrara, Italy; bDepartment of Medical Genetics, University of Ferrara, Italy

a

Abstracts We have studied by ELISA technique cerebrospinal fluid (CSF) levels of classical soluble HLA-I (sHLA-I), non-classical soluble HLA-G (sHLA-G) and soluble Fas (sFas) molecules in 65 relapsing–remitting (RR) multiple sclerosis (MS) patients, categorized according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity, in 64 patients with other inflammatory neurological disorders (OIND) and in 64 patients with non-inflammatory neurological disorders (NIND). Statistical analysis was performed by Mann–Whitney, with Bonferroni post-hoc correction, and Spearman rank correlation coefficient tests. CSF levels of sHLA-G were higher in MS than in OIND and NIND (p < 0.001 and p < 0.01, respectively) and in MRI inactive than in MRI active MS patients (p < 0.01). CSF sHLA-I concentrations were more elevated in MS than in NIND (p < 0.02), in OIND than in NIND (p < 0.02) and in MRI active than in MRI inactive MS patients (p < 0.01). CSF levels of sFas were lower in MS than in OIND and NIND (p < 0.001 and p < 0.02, respectively) and in MRI inactive than in MRI active MS patients (p < 0.02). In MS patients with no evidence of MRI activity, a trend towards a negative correlation was found between CSF concentrations of sHLA-G and sHLA-I and between CSF levels of sHLA-G and sFas. Our results seems to indicate that CSF sHLA-I/sHLA-G balance could modulate MS activity in opposite directions and suggest a potential immunoregulatory role for sHLA-G in the control of MS disease activity by Fas-mediated apoptosis.

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Results: In acute EAE, USPIO enhanced lesions were detected before Gd enhancement. USPIO were proven to be localized in tissue macrophages. In 7 out of 10 MS patients, about 2/3 of 55 lesions showed both USPIO uptake and gadolinium enhancement. Two patients presented 2 lesions only enhanced by USPIO (negative for Gd), and the remaining lesions were detected by Gd only (Am J Neuroradiol, in press). In acute and relapsing EAE, MRI-USPIO allowed to monitor the efficacy of immunomodulatory therapies (Mult Scler 2004) and to predict, already at clinical EAE onset, the histopathological and handicap severity (Neuroimage, in press). Conclusions: USPIO can be applied safely in inflammatory human diseases. In acute MS, USPIO and Gd provide complementary information about brain lesions. MRI-USPIO is a promising tool to monitor in vivo the dynamic macrophage activity and its modulation upon therapeutics. PP13-10

Morphometry of the corpus callosum: An early marker of brain atrophy in patients with probable multiple sclerosis Shmuel Miron, Or Reshef, Yael Nissan, Anat Achiron Multiple Sclerosis, Sheba Medical Center, Ramat-Gan, Israel

PP13-09

In vivo macrophage activity imaging in inflammatory brain lesions of multiple sclerosis using MRI Mitsunori KANAGAKI, Vincent DOUSSET, Bruno BROCHET, and Klaus G. PETRY University Victor Segalen Bordeaux 2, EA2966 Neurobiology of Myelin Disorders, Bordeaux, France Objective: In multiple sclerosis (MS), activated microglia and blood-borne macrophages play a pivotal role in the inflammatory process causing myelin and axonal loss. We present a new MR imaging modality to monitor in vivo macrophage brain infiltration in MS and its rat model of experimental autoimmune encephalomyelitis (EAE) with ultra-small superparamagnetic iron oxide (USPIO) nanoparticles. Methods: USPIO were intravenously given in both EAE rats and 10 MS patients during the acute attack. MRI-USPIO was performed 24 h later on MR scanners (1.5 T for humans, 4.7 T for rats) in comparison with gadolinium (Gd), MRI marker of blood–brain barrier permeability. In EAE rats, we evaluated histologically the underlying physiopathology with immunomarkers.

Background: Modern neuroimaging techniques provide information about brain structure. In multiple sclerosis (MS) it is possible to study axonal loss known to occur in the early stage of the disease. Irreversible axonal loss is associated with brain atrophy, and overtime lead to clinical disability. Objective: Investigate whether changes in the area of the corpus callosum (CC) are detected early in patients with probable MS. Methods: Brain MRI were performed in 28 subjects (20 females, 8 males) with a clinically isolated demyelinating event suggestive of MS (probable MS). 24 healthy subjects matched for age and gender served as controls. The mid-sagittal brain MR image was used for measurement of the CC (total area) and its sub-regions (anterior, middle, posterior). Results: The area of the CCn in patients with probable MS did not differ significantly from healthy subjects matched for age and sex. However, the total area of the CCn in the probable MS group significantly diminished from 539.6 ± 81.6 mm2 at baseline, to 516.9 ± 57.3 mm2 after one year (p < 0.05). The decrease was mainly attributed to the middle region of the CC that diminished from 227.0 ± 51.3 mm2 at baseline to 209.9 ± 25.4 mm2 after one year (p < 0.05). Conclusions: Measurement of CC area and its sub-regions is a reliable tool to detect early changes in the CC. Our findings demonstrate that structural changes can be detected even in patients with probable MS, and thus support the role of early treatment even in patients with probable MS.

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Abstracts

Poster Session 14: MS-therapy

Abstracts PP14-01

DNA microarray analysis identifies CXCR3 and CCR2 ligand chemokines as early IFN-responsive genes in peripheral blood lymphocytes Jun-ichi Satoha,b, Yusuke Nanria, Wakiro Satoa, Takashi Yamamuraa

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involved in IL1 activation. The induced pro-apoptotic pathways included up-regulation of apoptotic inductors like CD38 and BCL2, and caspase activators like CARD4, CASP4 and CASP10. The anti-apoptotic genes like TGFB1, SNCA, GPX1 and IGF1R were down-regulated. Conclusions: Short-term Rebif treatment resulted in immunomodulatory effects related to anti-adhesion effects involving integrin family genes, suppression of IL1-related inflammation and stimulation of apoptosis, all important to control RRMS disease activity.

a

Department of Immunology, National Institute of Neuroscience, NCNP; Department of Bioinformatics, Meiji Pharmaceutical University, Tokyo, Japan b

Objective: To identify systematically interferon-beta (IFNB)-responsive genes in peripheral blood mononuclear cells (PBMC). Background: IFNB reduces the frequency of relapses and the number of new MRI lesions in RRMS. However, a proportion of the patients discontinue the treatment due to various adverse effects, most of which emerge at the early phase of the treatment. No biomarkers predicting detrimental responses of IFNB are available. Methods: Total RNA of PBMC incubated with 50 ng/ml rhIFNB in vitro was processed for cDNA microarray and real-time RT-PCR analysis. Results: Among total 1258 genes, IFNB elevated the expression of 107 and 87 but reduced 22 and 23 genes at 3 and 24 h. Upregulated genes were categorized into conventional IFN-response markers, components of IFNsignaling pathways, chemokines, cytokines, regulators of apoptosis and DNA damage, and heat shock proteins. IFNB markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) active on effector Th1 cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) active on neutrophils. Eleven genes including ISG15, IFI616, IRF7, TAP1 and TNFAIP6 were elevated in 13 RRMS patients during IFNB treatment for 3 to 6 months. Conclusions: IFNB elevated immediately the expression of chemokines with relevance to early adverse effects in MS. PP14-02

Gene expression patterns identify short-term interferon-β-1a (Rebif) treatment effects in multiple sclerosis A. Achirona,b, Y. Snira, D. Magalashvilia, A. Feldmana, P. Sonisa and M. Gurevicha a

Multiple Sclerosis Center, Sheba Medical Center, Tel-Hashomer, RamatGan, Israel; bSackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel

Introduction: Interferon-β-1a (Rebif) is one of the leading immunomodulatory drugs for the treatment of relapsing–remitting multiple sclerosis (RRMS). However, despite the well documented efficacy, it remains unclear what mechanisms are involved and how Rebif treatment alters the clinical course of the disease. Methods: For all experiments we used cDNA Affymetrix microarrays (U133A). We analyzed peripheral blood samples from 12 RRMS patients, before and 3 months after initiation of Rebif treatment. Data analysis was performed using t-test, TNoM (Threshold Number of Misclassification) and Info-test. Most informative genes (MIGs) were defined as those with pvalue <0.05 in all 3 statistical tests. Results: Rebif treatment resulted in 890 MIGs. MS pathogenesis-related effects of Rebif include adhesion, inflammation and apoptosis. Rebifinduced adhesion effects were characterized by down-regulation of the integrin genes like ITGA2B, ITGB3, ITGAE and ITGB5. Additionally, LIMS1, CD9, CD44 and SELP gene-transcripts, mediating cell adhesion, and TNFRSF4 that directly affects adhesion of activated T-cells to vascular endothelium, were suppressed. The anti-inflammatory effects of Rebif were mainly related to suppression of IL1 inflammatory-pathways and involved over-expression of IL1RN, and inhibition of CXCL5 and TOLLIP that are

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Attaining RNA expression signatures in peripheral blood cells of interferon beta 1a (IM) treated multiple sclerosis (MS) patients over time R. Goertschesa, D. Koczana, P. Serrano-Fernándeza, S. Möllera, UK. Zettlb Institute of Immunology, University of Rostock, Rostock, Germany; bClinic of Neurology, University of Rostock, Rostock, Germany

a

This systematic time-course gene expression study focuses on the autoimmune disease MS and the effect of therapeutic intervention by Interferon-beta-1a (IFNβ1a). It is the aim to detect surrogate markers for drug-related molecular candidates in human PBMCs, to infer mechanisms of action affiliated to the effects/pathways of applied compound, and to discern RNA-signatures derived from (non-)responders to treatment. IFNβ therapy in patients with MS has proven efficacious, but appears suboptimal in terms of therapy responder rate. Large scale analysis of gene transcripts over time provide an unprecedented view of the complexity of studied disease and generates a huge data set that shall serve molecular group classifications. Employing the Affymetrix HGU133 set (33,000 genes) we perform an ongoing full genome study monitoring 20 patients receiving IFNβ1a (i.m., once weekly) over a period of 24 months. Samples were collected and directly processed before first treatment, after 1 week, 4 weeks, 1 year and will be drawn at corresponding dates. Systematic transcriptome analyses (supervised learning and literature-mining tools) are realized. Comparing time points, 115 genes showed increased or decreased expression levels in minimum 75% of the patients. 5 were IFN-modulated, 20 immune response-related, 5 identified in host defense and 3 in inflammatory responses. IFNβ1a may also alter the stability and coordination of cell components, as 30 genes were implicated in protein and lipid metabolism, cell adhesion and signaling, cytoskeletal (re-)arrangement and the ubiquitin pathway. The differential expression of the set of genes could be an important ancillary diagnostic tool. PP14-04

Longitudinal gene expression profiling in PBMCs of multiple sclerosis (MS) patients receiving Interferon-beta-1b therapy D. Koczana, R. Goertschesa, S. Möllera, P. Serrano-Fernándeza, HJ Thiesena, UK. Zettlb Institute of Immunology, University of Rostock, Rostock, Germany; bClinic of Neurology, University of Rostock, Rostock, Germany

a

A systematic time-dependent and comprehensive gene expression analysis from Interferon-beta-1b (IFNβ1b) treated MS patients will provide a rational basis for optimized or novel therapeutic strategies aimed at modulation of the disease course. Applying time-course microarray experiments to find new prognostic and diagnostic markers in peripheral blood cells after therapeutic intervention opens great perspectives regarding responsiveness or patient subclassification and has an ethical impact on the acceptance of costintensive treatment.

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Using the Affymetrix HGU133 set (approximately 33,000 genes) we performed a full genome study monitoring 18 patients over a period of 2 years. Samples were taken before first treatment and after 48 h, 4 weeks, 12 and 24 months. Only transcripts passing the detection threshold of the statistical algorithm MAS 5.0 were used for analysis. In order to distinguish molecular subgroups of treated patients based on generated RNA-profiles and clinical parameters, linear correlation models, supervised learning algorithms applying the bootstrap method and literature-mining tools were applied. Dynamic expression changes in individual RNA profiles could be ascertained; compared with pre-treatment expression level, 10 genes show differential expression at all time points. Introduction of profiling results into interaction exploring software generated valuable candidates (FCER1A; IFIT1, 3; HSXIAPAF1) for improved understanding of IFNb1b mechanism of action. The high proportion of IFN-regulated genes and the presence of MX1, a short-termed molecular indicator for IFNβ-activity, consistently induced in most of the patients support the validity of selected candidate genes. Being both immediately detectable and long lasting, the differential expression of these genes could be helpful for prospective purposes. PP14-05

Acute and steady state effects of interferon-beta in multiple sclerosis evaluated by gene expression profiling F. Sellebjerga, J. Larsenb, P. Dattaa, K. Rienecka, I. Alsingb, A. Oturaia, A. Svejgaardb, P.S. Sørensena, L.P. Ryderb a

Danish MS Research Center, Department of Neurology, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark; bDepartment of Clinical Immunology, Copenhagen Univeristy Hospital Rigshospitalet, Copenhagen, Denmark Treatment of multiple sclerosis (MS) with interferon (IFN)-beta modifies immune cell activation and cytokine secretion, adhesion molecules and chemokine receptor expression, and matrix metalloproteinases. We have recently found that the acute immunological effects of treatment with IFNbeta (i.e., within 24 h of drug injection) differ from the effects seen at steady state (i.e., more than 24 h after drug injection). We used the Affymetrix GeneChip Focus array with more than 8500 different genes displayed to study changes in gene expression in 10 patients treated with IFN-beta1a (30 μg intramuscularly once weekly). Blood samples for the study of mRNA expression in purified mononuclear cells (MNC) were drawn before the first injection of IFN-beta1a and 14–20 h after the first injection of IFN-beta1a (T1). After three months of treatment new blood samples were obtained before the injection of IFN-beta (T2) and after the injection of IFN-beta (T3). After the first injection of IFN-beta (T1) the expression of 283 genes differed significantly from baseline values (T0; p < 0.05 with Bonferroni correction). Among 135 up-regulated genes, 57 were induced at least two-fold. Among 148 down-regulated genes, 11 were at least 50% reduced. Gene expression at baseline and T2 did not differ significantly. After three months of treatment with IFN-beta, the injection of IFN-beta resulted in slightly less pronounced changes in gene expression, but there was no significant difference in the gene expression profile at T1 and T3. Treatment with IFNbeta results in acute changes but no major, persisting changes in blood MNC mRNA expression. PP14-06

In vitro inhibition of human CD4+ CD25+ regulatory T lymphocyte function by interferon-β through activation of autocrine/paracrine dopaminergic pathways Mauro Zaffaronia, Marco Cosentinob, Marco Ferrarib, Franca Marinob, Elena Carcanob, Federica Saporitib, Emanuela Rasinib, Raffaella Bombellib, Michela Perusinb, Angelo Ghezzia, Giancarlo Comia,c, Sergio Lecchinib

a

Multiple Sclerosis Study Centre, Hospital of Gallarate, Gallarate, Italy; Department of Clinical Medicine, Section of Experimental and Clinical Pharmacology, University of Insubria, Varese, Italy; cS. Raffaele Scientific Institute and University, Milan, Italy b

Impairment of CD4+CD25+ T lymphocyte (Treg) function might contribute to break down immune tolerance in patients with multiple sclerosis. We investigated in vitro the ability of Interferon β (IFN-β) to affect human Treg function as defined by production of IL-10 and TGF-β. Treg were isolated from healthy donors' blood by immunomagnetic sorting and cultured. IL-10 and TGF-β mRNAs and proteins were measured by RTPCR and ELISA respectively. Incubation with IFN-β 1000 IU/ml significantly reduced IL-10 and TGF-β in Treg at both mRNA and protein levels (IL-10: mRNA = − 51.8 ± 12.0%, protein = − 35.3 ± 10.3%; TGF-β: mRNA = − 42.7 ± 16.7%, protein = − 46.9 ± 17.6%; P < 0.001 vs. control in all cases). As we recently reported that IFN-β induces the release of endogenous catecholamines from human lymphocytes, we first measured catecholamines in unstimulated Treg by HPLC-ED: we found elevated intracellular levels comparable to those in activated lymphocytes. We therefore tested whether the effects of IFN-β could be antagonized by selective antagonists for dopaminergic or adrenergic receptors: the D1-like receptor antagonist SCH23390 completely reversed the effects of IFN-β. This is the first report showing a direct effect of IFN-β on human Treg function. The effect involves the activation of D1-like receptor pathways, likely through the autocrine/paracrine action of endogenous dopamine released from Treg by IFN-β itself. In view of the role or Treg in the regulation of the immune response in health and disease, further studies are urgently needed to assess the clinical significance of these findings. Supported by Italian Multiple Sclerosis Foundation (FISM), grant n. 2003/R/67. PP14-07

Interferon β-1a (Rebif) induced regulatory pathways in multiple sclerosis M. Gurevicha, Y. Snira and A. Achirona,b a

Multiple Sclerosis Center, Sheba Medical Center, Tel-Hashomer, RamatGan, Israel; bSackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel Introduction: Interferon β-1a (Rebif) is an effective treatment known to reduce disease activity and progression in patients with relapsing–remitting multiple sclerosis (RRMS). Using microarray gene-expression analysis we have recently identified 890 genes that characterize Rebif treatment effects in RRMS. Objective: Analysis of Rebif-induced regulatory pathways in order to further elucidate the hierarchy of biological functions associated with treatment effects in RRMS. Methods: Using an integrative model we classified the 890 genes within the Rebif signature into 23 groups, according to their chromosomal location. For each intra-chromosomal group of genes we reconstructed regulatory pathways, using Bayesian networks and identified their regulating genes. Next, we identified common motifs of transcription factors in the promoter region of these regulating genes. Results: The analysis resulted in 71 intra-chromosomal regulatory pathways, regulated by 110 regulating genes. Analysis of the motifs in the promoter region of the regulatory genes identified 48 transcription factors that added information for identification of interacting biological pathways induced by Rebif in RRMS. An interesting example for a previously unknown mechanism identified is the over-expression of zinc finger CCCH type domain containing 1 gene (ZC3HDC1). ZC3HDC1 was found directly regulate caspase recruitment domain family, member 4 gene (CARD4) that plays a significant role in apoptosis. This suggests that ZC3HDC1 is a regulating Rebif inducible gene that indirectly enhances apoptosis.

Abstracts Conclusions: Using an integrative approach for gene expression analysis we identified Rebif induced regulatory pathways in RRMS. PP14-08

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(3.3 ± 3.5 fold increases), it showed significantly different compared with before treatment (p < 0.05). Conclusion: Our results demonstrated that IFNβ1b might have played a role in controlling the auto-reactive T cells through an increased of Treg cells and also its relevance to the treatment of MS.

Interferon-β1a-induced expression of brain-derived neurotrophic factor in human T lymphocytes

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Z. Mangal, C. Pullankavumkal, V. S. Manda, H.-P. Hartung and O. Neuhaus

The effects of interferon-β1b on expression of CXCR3 on circulating T cells in multiple sclerosis patients

Department of Neurology, Heinrich Heine University, Düsseldorf, Germany Objective: To investigate the effects of interferon (IFN)-β1a (Rebif) on the expression of brain-derived neurotrophic factor (BDNF) and other neurotrophic factors in human T lymphocytes. Background: Neurodegeneration correlates with progression of disability in multiple sclerosis (MS). IFN-β1a reduces progression of sustained disability in MS, suggesting neuroprotective properties. BDNF and other neurotrophic factors capable of promoting neural cell survival may contribute to “neuroprotective immunity” in MS. Design/methods: For in vitro analyses, mitogen-activated peripheral blood lymphocytes (PBL) obtained from untreated MS patients or from healthy donors were treated with IFN-β1a. Their proliferative activity was compared to the secretion and transcription of BDNF as assessed by standard ELISA and by real-time PCR. Results: Proliferative activity of mitogen-activated PBL was inhibited by IFN-β1a in a dose-dependent manner. In contrast, IFN-β1a induced BDNF expression both in activated and in non-activated PBL. Highest BDNF levels were induced at IFN-β1a concentrations between 1000 and 5000U/ml, whereas higher or lower concentrations induced lower BDNF levels. BDNF mRNA levels, other neurotrophic factors and pro- and anti-inflammatory cytokines are currently being investigated. In addition, for ex vivo analyses, serum levels and mRNA levels of BDNF are being assessed in blood obtained from untreated or Rebif-treated MS patients. Conclusions: Our results support the hypothesis that IFN-β1a may promote neuroprotection by induction of BDNF expression. PP14-09

Modification of Interferon-β1b on CD4+CD25+high regulatory T cells and expression of FOXP3 mRNA in multiple sclerosis Shimizu, Y1, Ota, K2, Kawahata, K3, Ohara, K1, Ohashi, T1 and Iwata, M1 1

Department of Neurology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan; 2Tokyo University of Science. Tokyo, Japan; 3 University of Tokyo Graduate School of Medicine, Tokyo, Japan

Background: CD4+CD25+ regulatory T cells have suppressive auto-reactive and potent regulatory properties. Especially, CD4+CD25+high regulatory T cells (Treg) are anergic to antigenic stimulation and actively downregulate activation. Recently, the transcription factor Foxp3 gene has been implicated as a key element in balancing immune responses and is expressed in CD4+CD25+ Tr cells. The present study investigates whether Interferon (IFN)-β1b affects the inducing CD4+CD25high Treg cells and FOXP3mRNA expression. Method: We studied the frequency of CD4+CD25high Treg cells by flow cytometry analysis and the level of expression of FOXP3mRNA by quantitative real-time PCR in peripheral mononuclear cells (PBMC) from 18 healthy subjects and 18 multiple sclerosis (MS) patients before and after treating with IFN-β1b. Results: The frequency of CD4+CD25high Treg cells and the level of FOXP3 mRNA expression in PBMC showed no difference between healthy subjects and MS patients in the remission phase. The mean percentage of the frequencies of CD4+CD25high Treg cells in MS patients for 2 years after IFN-β1b treatment (2.7 ± 2.4%) were slightly higher than before treatment (2.0 ± 1.3%) (not statistically significant). Also an increase FOXP3mRNA expression was observed in MS patients at 3 months after IFN-β1b treatment

H. Nakajimaa, T. Hosokawaa, M. Suginoa, F. Kimuraa, T. Hanafusaa, T. Ikemotob, and A. Shimizub a

Department of Internal Medicine I, Osaka Medical College, Takatsuki, Osaka, Japan; bDepartment of Central Laboratory, Osaka Medical College, Takatsuki, Osaka, Japan

Background: Multiple sclerosis (MS) is an autoimmune disease in whose pathogenesis Th1 cells play an important role. It has recently become clear that interferon-β1b (IFN-β1b) treatment is effective in ameliorating relapsing–remitting multiple sclerosis (RRMS). The treatment efficacy of IFN-β1b for multiple sclerosis is potentially attributable to the immune regulatory properties of the drugs. Method: We compared the expression of Th1-related CXCR3 chemokine receptors and Th2-related CCR4 chemokine receptors on T cells derived from MS patients and those derived from healthy controls. Also, we have investigated the chemokine receptor expressions in MS patients undergoing IFN-β1b therapy. The expression of these Th1/Th2-related chemokine receptors were assessed at the baseline and longitudinally over a period of 24 months after the start of treatment in 10 RRMS patients grouped as 5 responders and 5 non-responders according to their clinical response to IFNβ1b therapy. Results: The percentage of CXCR3-expressing CD4+ T cells in patients with MS was significantly elevated compared with those of healthy controls. Treatment with IFN-β1b reduced the percentage of CXCR3-expressing CD4+ T cells in both responders and non-responders during the first 12 month. At 24th months after the treatment, the percentage of CXCR3expressing CD4+ T cells for responders was still reduced. However, that for non-responders returned to a level at baseline. Conclusion: the percentage of CXCR3-expressing CD4+ T cells would be a marker of immunological activity in MS patients, and IFN-β1b can correct the Th1/Th2 imbalance. PP14-11

Beneficial effect of interferon-β treatment in patients with multiple sclerosis is associated with transient increase in serum IL-6 level in response to interferon-β injection Y. Nakatsuji, M. Nakano, M. Moriya, H. Kishigami, C. Tatsumi, S. Tada, S. Sadahiro, T. Naka, K. Mitani, M. Funauchi, T. Azuma, S. Watanabe, M. Kinoshita, K. Kajiyama, Y. Yuasa, M. Kaido, MP. Takahashi, I. Naba, T. Hazama, S. Sakoda Department of Neurology, Osaka University Graduate School of Medicine, and Osaka Neurological Research Consortium, Japan Objective: To elucidate whether untoward effects of IFN-β treatment such as headache and skin reaction assessed easily at bedside, and the background of each patient may have relationship with serum cytokine levels or not. In addition, we investigated if we could predict responder to treatment through these analyses. Methods: Twenty-five patients with multiple sclerosis (MS) before IFN-β 1b therapy were enrolled. Chronological blood sampling was performed 0, 10, and 24 h after injection of IFN-β. Body temperature, headache, arthralgia, and the size of skin reaction at the injected site were monitored for the first week. Serum levels of IFN-γ, TNF-α, IL-6, IL-10, and TGF-β were

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measured by enzyme-linked immunosorbent assay (ELISA), and the interactions between the change of cytokine levels and variables including headache, arthralgia, fever, size of skin reaction, age, EDSS scores at the initiation of the therapy and after one-year treatment, relapse rate before and after treatment, and type of MS were analyzed. Results: Transient increase in the serum IL-6 level in response to IFN-β administration correlates with headache, arthralgia, less relapse rate prior to the therapy, milder disability assessed by EDSS score at the initiation of the treatment, and less progression of disability during treatment. Conclusion: It may be helpful to assess serum IL-6 level at 2 points, before and 10 h after administration of IFN-β, for the prediction of the efficacy of IFN-β therapy. PP14-12

Analysis of neutralising antibodies in the Betaferon® in newly emerging multiple sclerosis for initial treatment (BENEFIT) study M. S. Freedmana, L. Kapposb, C. H. Polmanc, H.-P. Hartungd, G. Edane, D. H. Millerf, X. Montalbang, L. Bauerh, S. Dahmsh, C. Pohlh and R. Sandbrinkh The Ottawa Hospital, Ottawa, Ontario, Canada; bUniversity Hospital, Basel, Switzerland; cVrije Universiteit Medical Center, Amsterdam, The Netherlands; dHeinrich-Heine-Universität, Düsseldorf, Germany; eClinique Neurologique, Rennes, France; fThe National Hospital for Neurology and Neurosurgery, London, UK; gHospitals Vall d'Hebron, Barcelona, Spain; h Schering AG, Berlin, Germany

a

Neutralising antibodies (NAb) develop with all current immunomodulatory treatments for multiple sclerosis, but controversy exists about their clinical relevance. The BENEFIT study assessed the safety, tolerability and efficacy of interferon beta-1b (IFNB-1b; Betaferon®) 250 μg in patients with clinically isolated syndrome. Patients were randomised to IFNB-1b (n = 292) or placebo (n = 176) and treated for 24 months or until clinically definite MS (CDMS) was diagnosed. NAb for IFNB-1b were measured using the MxA assay on samples taken at baseline, every 6 months thereafter, and at end of study (EOS); NAb titres >=20 were considered positive. The impact of NAb on time to CDMS was evaluated by proportional hazards regression in all IFNB-1b patients with at least one post-baseline NAb sample. Additional analyses of the impact of NAb were performed focussing on patients with longer exposure to IFNB-1b, i.e. in patients with EOS after 180 days, 270 days and 360 days, respectively. The percentage of NAb-positive IFNB-1b-treated patients at each visit (month 6 to month 24) ranged from 16.5% to 26.1%. Reversion to NAbnegative status was observed in 22.7% of the NAb-positive patients. There was no significant effect of the presence of NAb on time to CDMS in any of the analyses performed: hazard ratios for NAb-positive versus NAb-negative patients were 0.63 (all IFNB-1b-treated patients), 1.01 (patients with EOS after 180 days), 1.14 (270 days) and 0.91 (360 days). In conclusion after 24 months on study, no effect of NAb on time to CDMS was detected. PP14-13

Qualitative differences of neutralizing antibodies against the different interferon beta preparations F. Deisenhammer, C. Gneiss, M. Reindl, T. Berger Department of Neurology, Innsbruck Medical University, Austria Interferon beta (IFNb) is a first line therapy for multiple sclerosis (MS). Three different types of IFNb are available, IFNb-1a for i.m. or s.c. application and IFNb-1b. These three preparations have different potentials to induce neutralizing antibodies (NAB) which is not only related to different dosing and routes of application. Recognition of epitopes is one

factor, where IFNβ-1a treated patients show significantly higher median binding titers against the N-terminal end of IFNβ than IFNb-1b treated patients (800 vs. 200). Another factor is affinity, showing significantly higher affinity values for IFNβ-1a treated patients as compared to patients on IFNb-1b (296 +/− 50.7 vs. 265 +/− 31.5; values obtained by titration of antigen–antibody binding using increasing concentrations of sodium isothiocyanate). The potency of binding antibodies (BAB) to neutralize IFNb is also different among the different preparations. Twelve% of BAB positive patients treated with IFNb-1a i.m. are also NAB positive compared to 51% of IFNb-1a s.c. and 37% of IFNb-1b treated patients (P < 0.0001). These biochemical characteristics contribute to the different abilities of the IFNb preparations to induce NAB and to their different dynamic of NAB development. Consequences for treatment decisions and therapy monitoring will be discussed as well as potential strategies to overcome NAB development or reversion to NAB negativity. PP14-14

Effects of IFN-β, leptin and simvastatin on LIF secretion by T lymphocytes of MS patients and healthy controls Joris Vanderlocht, Jerome Hendriks, Koen Venken, Piet Stinissen and Niels Hellings Hasselt University, Biomedical Research Institute and Transnationale Universiteit Limburg, School of Life Sciences, Diepenbeek, Belgium In Multiple Sclerosis (MS), oligodendrocyte injury is believed to be caused by an aberrant immune response initiated by autoreactive T cells. Increasing evidence indicates that inflammatory responses in the central nervous system are not exclusively detrimental, but may also exert protective effects. Such protective effects are potentially mediated by the local secretion of neurotrophic factors by immune cells. We previously reported that T cells and monocytes produce leukemia inhibitory factor (LIF), a member of the neuropoietic family of neurotrophic factors, both in vitro and in inflammatory MS lesions. In the present study, we report a reduced LIF production by CD4+ T cells of relapsing remitting MS patient as compared to secondary progressive MS patients and healthy controls. Furthermore, immunomodulatory agents such as leptin, IFN-β and simvastatin were studied for their potential to alter secretion of LIF and other cytokines by T cells and monocytes. Low doses of simvastatin, but not IFN-β or leptin enhanced LIF secretion by CD4+ T cells of RR-MS patients, but not in healthy controls. We further demonstrated that LIF did not influence viability, proliferation and cytokine secretion of T cells. Together these data provide new information on the regulation of LIF secretion by immune cells. Further insights into the complex regulation of neurotrophic factors such as LIF may prove useful for treatment of MS. PP14-15

Identification and selection of disease related autoreactive T cells from early multiple sclerosis patients for specific T-cell vaccination M. Mandela,b,c, A. Achirona,b,d, M. Gurevichb and G. Lavied Sackler School of Medicine Tel-Aviv University, bNeurogenomic Laboratory, cBlood Center and dMultiple Sclerosis Center, Sheba Medical Center, Tel-Hashomer, Israel

a

CD4+ T cells sensitized against myelin epitopes are believed to play a role in the pathogenesis of MS. One experimental approach to treat MS, termed T cell vaccination (TCV), involves patient immunization with attenuated autoreactive T-cell lines responsive to myelin antigens. Distinguishing disease-related autoreactive lineages from normal T cell lineages reactive to myelin autoantigens in culture is essential for vaccine preparation. T-cell lines can also be generated from healthy subjects. Using

Abstracts microarray analyses we identified a unique autoimmune gene expression fingerprint of MOG-responsive T cell lines with over-expression of IGFBP3, VEGF, BCL-2 and Lifeguard. This imprint is absent in myelinresponsive T-cell lines from healthy subjects. Over-expression of BCL2 suggested resistance of MS autoreactive lines to apoptosis. We thus protracted T cell exposure to antigen during line selection from the commonly used 16–18 days to ∼45 days, at which time normal human T cells undergo apoptosis. MS T cell lines generated were 93–99% CD4+ oligoclonal (4–9 TcR Vβ isoforms), IFNγ producing T cells bearing >90% CD45RO+ determinants. We stimulated MOG responsive healthy subject T cell lines with the proinflammatory SDF1α, TNFα or IFNγ. Treated cells expressed higher levels of IGF-BP3, VEGF and BCL-2 genes mimicking the autoimmune fingerprint. Autoimmune fingerprint-bearing lines were insensitive to additional cytokine stimulation. While most P-MS patient lines express the autoimmune fingerprint and cannot be further stimulated, several MBP-reactive lines derived from P-MS bear healthy T cell features. This fingerprint enable distinguishing disease-related from healthy lineages within an MS or P-MS individual and enable better line selection for TCV.

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study the effects of TCV on disease activity as measured by magnetic resonance imaging (MRI). Twenty-nine MS patients that demonstrated MRI activity in a pre-entry period of 6 months were randomized into active (TCV) (n = 20) and placebo (n = 9) groups. Three immunisations with irradiated CSF vaccines (5–10 million cells) or placebo were administered subcutaneously with an interval of 2 months. Patients were monitored for 12 months after the final immunisation for MRI activity (every 2 months), clinical scores and immune responses. The vaccinations were well tolerated and no toxicity or adverse effects were reported. Anti-myelin T cell responses were reduced after TCV in 16 out of 20 MS patients. The mean number of active MRI lesions and the volume of active MRI lesions and T2 lesions was reduced in the treated group, but not in the placebo group. These differences were however not statistically significant. The mean EDSS scores remained stable in both groups. Patients with a high immune response to the vaccine cells showed a trend for improved MRI and clinical responses to TCV. Despite the lack of statistically significant differences due to the low patient number, this study further demonstrates safety and feasibility of TCV in MS patients, and suggests that possible therapeutic effects of TCV may be more prominent in patients that show high immune responses to the vaccine.

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T-cell receptor Vbeta restriction in autoreactive T cell lines from probable MS patients T. Barliyaa, A. Achironb,c, G. Lavieb and M. Mandela,c Blood Center, bMultiple Sclerosis Center, Sheba Medical Center, TelHashomer; cSackler School of Medicine. Tel-Aviv University, Israel

Immunoadsorption plasma pheresis therapy for the treatment of refractory attacks of multiple sclerosis T. Ohashia, K. Otab, Y. Shimizua, K. Oharaa, C. Takeuchia, M. Iwataa

a

Tokyo Women's Medical University, Tokyo, Japan; bTokyo University of Science, Tokyo, Japan

a

T cell lines reactive to synthetic myelin peptides were generated from peripheral blood of probable multiple sclerosis patients (P-MS), within three months of their first demyelinating attack. The attenuated autoreactive cell lines served as T cell vaccines in these patients. We analyzed by Immunoscope the TcR Vβ profiles of these lines. These cell lines were >98% CD4+ and >90% CD45RO+. Oligoclonal compositions of TcR Vβ family isoforms of 21 tested were identified. Vβ15 was present at a very high frequency in 9 of the 12 T-cell lines (75%) from 5 of the 6 patients (83.3%) which were examined. Vβ9 occurred in 8 of the 12 T cell lines (66.6%) from 5 of the 6 patients (83.3%) examined. Vβ7, Vβ5.1, Vβ6 and Vβ21 also occurred in higher frequencies of 50%, 42%, 42% and 30% respectively. It is noteworthy that the combination of Vβ9,15 was found in 5 of the 12 tested lines and the combination of Vβ 6, 7, 15, 21 in 5 other lines. Despite the apparent degenerate nature of the autoreactive TcR there appears to be common denominators in myelin antigens because stimulation of PBMC from these P-MS patients with a tetanus toxin epitope did not stimulate these autoreactive populations. Our findings indicate existence of Vβ clusters in PMS patients which occur in repetitive combinations. These clusters are evident in T-cell lines generated in response to various myelin antigens within a given patient, irrespective of the nature of the specific antigen.

The effectiveness of immunoadsorption plasma pheresis (IAPP) therapy for treating acute exacerbations of multiple sclerosis (MS) has not been well evaluated; thus, an accumulation of therapeutic experiences for the disease is important. In addition, IAPP has recently been reported to have a preventive efficacy, which added to its establishment as a preventive therapy. We report 4 MS patients with acute or subacute exacerbations. These patients have been unresponsive to intravenous methyl prednisolone (IVMP) therapy and intravenous immunoglobulin therapy but showed improvement after IAPP therapy. IAPP therapy was well tolerated and effective for a patient with acute exacerbation during pregnancy. Three MS patients who showed frequent exacerbations even after the introduction of interferon β-1b therapy had no relapses while undergoing periodical IAPP therapy that aimed to prevent relapses. Adverse events of IAPP therapy included deep venous thrombosis, catheter infection, and anaphylactic symptom. We concluded that IAPP therapy is a therapeutic option, especially for the patients with fulminant attack or with opticospinal form. It is relatively safe and applicable for patients with acute exacerbation during pregnancy. IAPP therapy could be evaluated further as an option for the prevention of relapsing–remitting MS in the near future.

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T-cell vaccination in ms with CSF-derived activated CD4+ Tcells: Results of a placebo-controlled trial

IVIg decreases matrix metalloproteinase-9 production, not tissue inhibitor of metalloproteinase-1, by PBMC of multiple sclerosis patients

P. Stinissen1, R. Medaer1, N. Hellings1, F. Barkhof 2 and Jef Raus1 K. Okadaa, S. Tsujia 1

Hasselt University, Biomedisch Onderzoeksinstituut, and Transnational University Limburg, School of Life Sciences, Diepenbeek, Belgium; 2Image Analysis Center, VU Medical Center, Amsterdam, The Netherlands Activated anti-myelin T-cells accumulate in the cerebrospinal fluid (CSF) of MS patients, indicating that these T cells may represent a source of disease-related T cells. A previous pilot trial of T cell vaccination (TCV) with activated CD4+ T cells derived from CSF demonstrated safety, feasibility and immune effects in MS patients. A double-blind placebocontrolled trial was performed with early relapsing–remitting MS patients to

a

Department of Neurology, University of Occupational and Environmental Health., Kitakyushu, Japan Objective: To determine whether IVIg decreases matrix metalloproteinase (MMP)-9 production by peripheral blood mononuclear cells (PBMC) of multiple sclerosis (MS) patients. Methods: PBMC were prepared from relapsing and remitting MS patients (n = 10), who were not administered corticosteroid within 90 days or have never been introduced with interferon-β, and the healthy control (n = 10).

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PBMC were incubated with or without lipopolysaccharide (LPS, 1 μg/ml) for 1 h, and then IVIg (0.1–10 mg/ml) was added for 24 h. Moreover, to study the neutralizing effect of IVIg on MMP-9, PBMC were incubated with LPS (1 μg/ml) for 24 h, and then IVIg (0.1–10 mg/ml) was added for 1 h. MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 in the supernatant were measured using ELISA. Statistical analysis was performed using Mann–Whitney U test. Result: MMP-9 was significantly higher in the MS group than the control group both in the condition with and without LPS. Although TIMP-1 was up-regulated with LPS in both of the two groups, there was no significant difference between the two groups. The MMP-9/TIMP-1 ratio was significantly higher in MS group than the control group. IVIg decreased MMP-9 but not TIMP-1 dose dependently both in the two groups. IVIg decreased the MMP-9/TIMP-1 ratio dose dependently both in the two groups. Suppressive effects of IVIg on MMP-9 production were not by neutralizing effects. Conclusion: These results demonstrated that MMP-9 production is upregulated in MS and suppressed by IVIg. PP14-20

The changes of serum cytokines in the treatment of IAPP for Multiple Sclerosis Ohji S, Tomioka R, Mitsui T, Yoshida N, Iguchi T, Takahama M, Ohnuki M, Nomura K Saitama Medical Center, Department of Neurology, Saitama Medical University, Saitama, Japan [Purpose] The immuno adsorption plasma pheresis (IAPP) has recently become available for the treatment of MS. However, the mechanism of IAPP is still unclear. In this study, we measured serum cytokines to investigate the mechanism of immuno-modulation effects of IAPP. [Materials and methods] IAPP was carried out in 4 patients with MS in the active stage, that could not have enough clinical improvement with steroid therapy. The adsorption column was used TR350 (Asahi Kasei Medical. Co. Ltd). Serum cytokines (IL-1beta, 2, 4, 5, 6, 8, 10, 12, IFNgamm, TNF-alfa) were determined by using Beads Array Method (BD Bioscience Co.), in both outflow and inflow of IAPP circulation, and in the outflow of circulation just before and after the treatment. [Results] In the study of the changes of cytokines in both outflow and inflow while a treatment, IL-6, 8, 10 decreased in inflow, comparing to outflow. Also, in the outflow of circulation just before and after an IAPP, IL6, 8, 10 increased just after IAPP in all 4 cases. [Conclusion] The adsorption column removed various cytokines nonspecifically. IAPP for MS stimulated the production of mainly Th2 cytokine IL-10, and made immuno-state shift to Th2 balance. Consequently, the reactive immuno-modulation effects might have been occurred. PP14-21

Natalizumab effects on immune cell responses in multiple sclerosis M. Niinoa, C. Bodnera, M.L. Simarda, S. Alataba, D. Ganoa, H.J. Kima, M. Trigueiroa, D. Racicotb, C. Guéretteb, J.P. Antela, A. Fourniera, F. Grand'Maisonb, A. Bar-Ora a

Montreal Neurological Institute of McGill University, Montreal, Canada; Department of Neurology, Hopital Charles LeMoyne, Quebec, Canada

b

Objective: To study in vivo biological effects of natalizumab on immune cell phenotype and function in MS patients. Background: While natalizumab holds promise as an effective therapy for MS, the emergence of cases of PML creates the imperative to better define mechanisms underlying both beneficial as well as adverse drug effects. Development of simple assays that measure these effects could prove useful in immune monitoring of patients on this emerging therapy.

Methods: Blood was obtained before and after serial monthly natalizumab infusions to track functional expression of VLA-4 and migratory capacity of immune cells. The impact of infusion on activation thresholds of immune cells was evaluated. Results: Pre-infusion VLA-4 expression differed across immune cell subsets. Natalizumab significantly, albeit partially, diminished VLA-4 expression on circulating immune cells. Treatment significantly decreased migratory capacity of immune cells, correlating well with changes in VLA-4 expression. Effects of a single dose were not saturating and did not persist through the monthly dose-interval. Infusion effects varied across patients but were remarkably stable within individual patients, over multiple infusions. Treatment significantly modulated proliferative responses of immune cells. Interpretation: We provide first proof-of-concept that in vivo natalizumab diminishes migratory capacity of human immune cells. Our prospective study shows that effects of therapy likely: (i) differ for distinct immune cell subsets, (ii) are not sustained over current dose interval, (iii) have unique profiles in individual patients, and (iv) include modulation of activation thresholds of immune cells. Monitoring these parameters could be relevant to ongoing safety and efficacy considerations. PP14-22

Phase I/II trial of a MBP encoding DNA plasmid (BHT-3009) alone or combined with atorvastatin for treatment of multiple sclerosis Hideki Garren1, Jack Antel5, Amit Bar-Or5, Caroline Anita Bodner5, Denise Campagnolo3, Jill Gianettoni1, Farzaneh Jalili5, Norman Kachuck4, Yves Lapierre5, Masaaki Niino5, Joel Oger6, Mary Price3, Susan Rhodes3, Fu-Dong Shi3, Frank Valone1, Timothy Vollmer3, Leslie Weiner4, Lawrence Steinman2 Bayhill Therapeutics, Palo Alto, USA; 2Stanford University, Palo Alto, USA; 3Barrow Neurological Institute, Phoenix, USA; 4University of Southern California, Los Angeles, USA; 5Montreal Neurological Institute, Montreal, Canada; 6University of British Columbia, Vancouver, Canada

1

Objective: To assess safety and immune modulation by BHT-3009 (a DNA plasmid that expresses full-length human MBP) in MS patients. Methods: We are conducting a 30 patient, randomized, double-blind, placebo-controlled trial in relapsing MS patients. Within this trial we are measuring several immune parameters including CFSE based antigenspecific T cell proliferation and intracellular cytokine production by PBMCs. Results: All thirty patients have been randomized. To date, the twenty patients in dose cohorts 1 (0.5 mg) and 2 (1.5 mg) have all completed treatment. There were 22 treatment-related adverse events (AEs), all of which were mild/moderate: 12 on placebo and 10 on BHT-3009 arms. Data from the CFSE assay on four patients from two centers who ran this assay in the first two cohorts demonstrate antigen-specific reduction in MBP reactive T cells by week 9 of treatment in three of them. In the first patient, the proliferation of MBP83–99 reactive IFN-γ positive cells decreased from 25.9% to 1.2%. In a second patient, the proliferation of MBP83–99 reactive IFN-γ positive cells decreased from 13.3% to 5.4%. In the third patient, the proliferation of whole MBP reactive IFN-γ positive cells decreased from 2.27% to 0.79%. In all three of these patients the proliferation to tetanus toxoid did not decrease, pointing to antigen specificity following BHT-3009 treatment. In a fourth patient, there was no change in either the MBP83–99, whole MBP or tetanus proliferative responses. Conclusion: The data indicate that BHT-3009 is safe and may suppress immune responses in an antigen-specific manner. PP14-23

No signs of immunoactivation in the cerebrospinal fluid during infliximab treatment M. Anderssona, E. Svenungssonb, M. Khademia, J. Lampab, L. Brundina and E. Wallströma

Abstracts a

Neuroimmunology Unit, Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden; b Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden

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neuroprotective properties in neuroinflammatory disorders such as multiple sclerosis. PP14-25

Background: Anti-tumor necrosis factor α (anti-TNFα) therapy, including infliximab treatment, for rheumatic diseases has been associated with rare cases of new or exacerbated neuroinflammatory disorders such as multiple sclerosis and optic neuritis. In multiple sclerosis, anti-TNFα therapy may increase disease activity. There is evidence for an increased peripheral T cell reactivity, measured as enhanced IFNγ production, during anti-TNFα therapy of rheumatoid arthritis. This may potentially provoke neuroinflammation, since systemic IFNγ administration has been demonstrated to exacerbate multiple sclerosis. We therefore hypothesize that the few cases of clinical neuroinflammatory disorders observed after anti-TNFα therapy of rheumatic diseases represents the extreme end of a commonly occurring minor intrathecal immune activation, which in most cases does not give any overt neurological dysfunction. Objective and methods: To test this hypothesis, we determined the expression of IFNγ, TNFα and IL-10 mRNA in cerebrospinal fluid cells and peripheral blood mononuclear cells, the levels of nitric oxide oxidation products in cerebrospinal fluid and serum, the cerebrospinal fluid cell counts and IgG indices, in ten patients with polyarthritis before and during infliximab treatment. Results: No significant signs of intrathecal immune activation were recorded. In the systemic compartment, induction of IFNγ expression during infliximab treatment was demonstrated. Conclusions: Intrathecal immune activation, as measured by the employed assays, during infliximab therapy is not a common phenomenon and we thereby refute our initial hypothesis. Our finding of increased systemic IFNγ expression demonstrates that a systemic pro-inflammatory component is part of the diverse effects of anti-TNFα therapy. PP14-24

Statins inhibit neuronal cell injury promoted by activated T lymphocytes V. S. Manda, J. V. Opatz, H.-W. Müller, H.-P. Hartung and O. Neuhaus

Lymphocyte homeostasis and regulatory cells in multiple sclerosis V. H. Robertsona, A. L. Coxa, S. A. J. Thompsona, J. L. Jonesa, D. A. S. Compstona and A. J. Colesa a

Department of Neurosciences, The University of Cambridge, Cambridge, United Kingdom

The hallmark of autoimmune diseases such as multiple sclerosis (MS) is the breakdown of self-tolerance. Naturally occurring regulatory T cells (Tregs), which preferentially express high levels of CD25 and the transcription factor forkhead box P3 (FoxP3), are critical to the maintenance of peripheral tolerance and prevention of autoimmune disease. In MS patients Treg numbers are normal but they are functionally impaired compared to healthy controls. We have treated a cohort of patients with MS using Campath-1H, a monoclonal antibody that induces a profound and prolonged T cell lymphopaenia. In the first few months after treatment, there is a five-fold increase in serum interleukin-7 (IL-7), a cytokine that promotes homeostatic proliferation, and an over-representation of cells expressing high levels of CD25 in the depleted T cell pool. We sought to determine whether these were truly Tregs using a functional assay involving the fluorescent dye carboxy fluoroscein diacetate succinimidyl ester (CFSE). IL-7 is reported to induce the transient expression of high levels of CD25 on CD4+ cells; however, these cells do not have suppressor cell function. We investigated the role of IL-7 in the genesis of these CD25hi cells; spiking healthy control human peripheral blood mononuclear cells (PBMCs) with increasing concentrations of recombinant human IL-7 resulted in an increase in the percentage of CD4+cells that express high levels of CD25, as determined by flow cytometry. In the post-Campath-1H lymphopaenic patient, IL-7 appears to link homeostatic mechanisms driving lymphocyte reconstitution with the enrichment of putative regulatory T-cells.

Department of Neurology, Heinrich Heine University, Düsseldorf, Germany PP14-26 Background: Cell–cell contact interactions play a major role in promoting neuronal cell injury in neuroinflammatory diseases. Activated T lymphocytes cause injury in a cell contact-dependent non antigen-restricted manner. We hypothesize that statins could offer neuroprotection by down regulating T-cell activation, cell–cell contact-dependent interactions and matrix metalloproteinase (MMP) expression. Methods: Statin- and control-treated peripheral blood lymphocytes were co-cultured with two neuronal (SHSY-5Y and NB-1) and one astrocytic cell line (LN18). Functional antibodies directed against CD147, CD98, HLA-DR and HLA-ABC attenuating T-cell activation were pulsed on T cells and then presented to target cells. Neurons were also incubated with different proinflammatory cytokines and chemokines. Cell functions were assessed by MTT assay, microscopy, ELISA, FACS and zymography. Results: Neurons were highly susceptible to cytotoxicity caused by activated T cells. Neuronal cell viability was restored when T cells were pretreated with statins (which again was reversed by mevalonate) and also when prepulsed with T-cell activation antibodies (CD147 and CD98 but not HLA-DR and HLA-ABC). Proinflammatory cytokines and chemokines did not promote neuronal apoptosis. Further we observed a decrease in MMP-9 in statin-treated cultures. Cell apoptosis was specific to neuronal cell lines as the astrocytic cell line did not show a similar behaviour. Conclusion: Treatment with statins down regulates cell activation markers which mediate the neuronal insult. Cell death on neurons is to be further reconfirmed by MAP-2 immunoreactivity on neurons and detection of nucleosomes in culture medium. Thus, statins may promote

Neurotrophin secretion after lymphodepletion with Campath-1H in multiple sclerosis J. L. Jonesa, S. Thompsona, B. S. Simpsona, A. L. Coxa, D. A. S. Compstona and A. J. Colesa a

Department of Neurology, Cambridge University, UK

Multiple sclerosis (MS) is a chronic neurological disorder thought to be driven by auto-reactive T lymphocytes, resulting in acute inflammatory axonal transaction, focal demyelination and oligodendrocyte destruction, with subsequent chronic axonal loss. The therapeutic monoclonal anti-CD52 antibody, Campath-1H, induces a prolonged T lymphopaenia. Treatment of patients with early, aggressive, relapsing–remitting MS results in a 94% reduction in relapse rate and a sustained improvement in fixed disability (as assessed by the Expanded Disability Severity Score). Such reported efficacy is unparalleled in trials of other MS therapies. Early improvement may represent abrogation of inflammation-associated conduction block, but this fails to explain sustained improvement seen at 12 to 24months. We hypothesise that growth factors are secreted in the context of immune reconstitution after Campath-1H treatment, and that these may promote neuronal survival and repair. ELISA (Enzyme-Linked Immunosorbant Assay) was used to determine concentrations of neurotrophins in peripheral blood mononuclear cell

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(PBMC) culture supernatants pre- and post-Campath treatment. Our results show that patients' PBMCs secrete neurotrophic factors after Campath-1H treatment: we found an increase in Ciliary Neurotrophic Factor (CNTF), a substance recently shown to enhance myelin formation; an even greater elevation in Brain Derived Neurotrophic Factor (BDNF); but a dramatic reduction in insulin-like growth factor-1 (IGF-1) secretion. We explore the functional significance of these observations using a well established in vitro model, in which neurons derived from rat embryonic cortices are cultured and exposed to PBMC conditioned media. PP14-27

B cell homeostasis and the role of B cell activating factor in patients with Multiple Sclerosis treated with Campath-1H S.A.J. Thompsona, J.L. Jonesa, A.L. Coxa, B.L.C. Wrighta, D.A.S. Compstona and A.J. Colesa

induced TNFRSF 18 (GITR) is upregulated in TR cells and its triggering leads either to apoptosis or proliferation of TR cells. In this study we measured the mRNA and protein levels of FOXP3, CTLA-4 and GITR in TR cells in interferon treated relapsing–remitting multiple sclerosis patients before and after corticosteroid treatment. All patients were examined according to expanded disability status scores (EDSS) to be in relapse. Fresh whole blood was obtained from MS patients. CD25+CD4+T cells were isolated from PBMCs and total RNA was extracted. The quantification of FOXP3, CTLA-4 and GITR mRNA level was performed by Real-time PCR. The quantification of protein level was performed by Western blot followed by densitometry. All patients have been tested for antibody mediated neutralizing activity against interferon-beta molecule. Our results indicate a clear trend in which corticosteroid treatment results in upregulation of GITR molecule. Opposite FOXP3 and CTLA-4 expression profile is shown to be downregulated. PP14-29

a

Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK

We examined the dynamics of the reconstituting B cell repertoire in patients with relapsing–remitting Multiple Sclerosis (MS) who had been treated with Campath-1H, an anti-CD52 monoclonal antibody. Untreated MS patients had a greater proportion of mature naïve (CD27−23+) and fewer memory (CD27+) B cells compared with healthy controls. One month after Campath-1H, there was a profound lymphopenia. By three months, the total number of B cells had recovered, while T cell numbers remained depleted even at twelve months post treatment. Within the B cell population, mature naïve cells remained the dominant cell type, but memory cells were further decreased, in contrast to the relative dominance of memory cells in the depleted T cell pool. One month after treatment the proportion of transitional type 1 (CD27−/ CD23−) B cells, the recent bone marrow emigrants, increased dramatically, suggesting they are principally responsible for the reconstitution. Also at one month, serum B cell activating factor (BAFF) increased significantly; but by month three levels had dropped and we observed a switch from T1 to mature naïve B cell phenotype. This is in agreement with published work showing the importance of BAFF in naïve B cell maturation and survival. However, ex vivo neutralisation of BAFF did not alter B cell proliferation in our patients. PP14-28

Differential expression of FOXP3, CTLA-4 and GITR gene in CD25+CD4+ T regulatory cells before and after corticosteroid treatment during relapses in interferon treated multiple sclerosis patients N. Grigoriadisa, I. Paspaltsisb, D.Tsiantoulasa, G. Deretzic V. Giantzia, T. Sklaviadisb,d, I. Milonasa.

Immunomodulatory therapies and low dose Azathioprine reverse partially and significantly visual and neurological deficits in progressive multiple sclerosis Dan Milder Prince of Wales Hospital, Sydney, Australia Objective: To determine the effect of the addition of low dose Azathioprine to immunomodulatory therapies in progressive demyelinating disease. Methods: Azathioprine 50 mg daily was added to an immunomodulatory therapy in patients with progressive multiple sclerosis and progressive demyelinating optic neuropathy. Patients were observed for twenty four to forty eight months. Results: Partial and significant reversals of deficits in cognition, visual acuity, colour vision, balance, strength, sensation, and micturition followed the addition of low dose Azathioprine to immunomodulatory therapies in all patients. Reversals in visual acuity were significant (p = 0.016) as were reversals in colour vision (p = 0.009). Vision in the patient with progressive demyelinating optic neuropathy of five years improved after the commencement of “combination therapy”, deteriorated when either the immunomodulator or Azathioprine was withdrawn and improved when the “combination” was reinstituted. The effect of “combination therapy” upon visual acuity was significant (p < 0.001) as was the effect on colour vision (p < 0.005) and has been maintained for two and a half years. Conclusion: The addition of low dose Azathioprine to immunomodulatory therapies may ameliorate visual and neurological deficits in progressive multiple sclerosis. Therapy instituted in the early phase may diminish progression and lessen disability. Visual acuity and Ishihara scores may be robust in vivo “therapeutic markers”. The long term adverse consequences of therapy remain to be determined. A large scale randomised controlled study is needed.

a

B' Department of Neurology, Laboratory of Experimental Neurology and Neuroimmunology, AHEPA University Hospital, Thessaloniki, Greece; b Prion Disease Research Group, Laboratory of Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece; c Department of Neurology, Ippokration Hospital, Thessaloniki, Greece; d Centre for Research and Technology-Hellas, Institute of Agrobiotechnology, Thessaloniki, Greece CD25+CD4+ T regulatory (TR) cells seem to play a crucial role in the maintenance of immunological self-tolerance in autoimmune diseases such as multiple sclerosis (MS). Recent experimental data have shown that Forkhead box P3 (FOXP3) is highly expressed in TR cells and is responsible for their function. Cytotoxic T lymphocyte antigen 4 (CTLA-4) is constitutively expressed in TR cells; it is responsible for the costimulation of TR cells and the direct suppression of effector T cells. Glucocorticoid

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The double edged sword of treating autoimmunity: Insights from therapeutic lymphocyte depletion in the treatment of multiple sclerosis A. L. Coxa, B. Wrighta, S. A. J. Thompsona, J. L. Jonesa, V. H. Robertsona, D. A. S. Compstona and A. J. Colesa a

Department of Clinical Neurosciences, University of Cambridge, United Kingdom Two consequences of the treatment of multiple sclerosis with the lymphocyte depleting monoclonal antibody Campath-1H have been

Abstracts observed; prolonged T cell lymphopaenia and the emergence of novel autoimmune diseases. It has been shown that this coincides with a period of lymphopaenia induced proliferation (LIP). Animal models of autoimmune disease employ LIP to induce susceptibility to disease, suggesting that homeostatic proliferation inherently results in the loss of self tolerance. The treatment of multiple sclerosis offers a unique opportunity to study this phenomenon in humans. We have conducted a survey of all autoimmune diseases arising during the treatment of multiple sclerosis with Campath-1H at our institution. The autoreactivity of lymphocytes prior to and following this treatment was measured by challenging carboxy fluoroscein diacetate succinimidyl ester labelled PBMCs with the putative autoantigens myelin basic protein and thyroid stimulating hormone receptor extracellular domain. We report an incidence of autoimmunity after treatment with Campath-1H of 39%. The incidence was greatest in those patients receiving a single pulse of Campath-1H, a group who were also older and had more advanced multiple sclerosis. A variety of autoimmune conditions have been identified, including organ specific, haematological and systemic autoimmunity; the most prevalent was thyroid autoimmune disease. An eightfold increase in the autoreactivity of lymphocytes emerging during LIP, which resides within the CD4+ T cell compartment, is demonstrated. We hypothesise that this may, at least in part, be responsible for the emergence of autoimmunity in patients undergoing lymphocyte depletion for the treatment of multiple sclerosis. PP14-31

Simvastatin affects microglia cell motility H.F. Kuipersa,b, A.A.C. Rappertc, A.M. Mommaasa, E.S. van Haastertb, P. van der Valkb, H.W.G.M. Boddekec, K.P.H. Biberc and P.J. van den Elsena,b

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a

Leiden University Medical Center, Leiden, The Netherlands; b VU University Medical Center, Amsterdam, The Netherlands; cUniversity Medical Center Groningen, Groningen, The Netherlands Statin treatment is proposed to be a new potential therapy for multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. Although their anti-inflammatory properties have been studied extensively, the effects of statins on brain cells, however, are until now poorly investigated. We therefore evaluated the effects of simvastatin treatment on the migratory capacity of brain microglial cells, key elements in the pathogenesis of MS. Using FACS analysis, we show that exposure of human and murine primary microglial cells to simvastatin reduces cell surface expression of the chemokine receptors CCR5 and CXCR3. In addition, simvastatin treatment specifically abolishes chemokine-induced microglial cell motility, as determined by chemotaxis assays. Furthermore, using cytoskeleton staining and structural electron microscopy we show that simvastatin treatment of microglia alters actin cytoskeleton distribution and leads to changes in intracellular vesicles. These data clearly show that simvastatin inhibits several immunologically relevant features of microglia, which may provide a rationale for statin treatment in MS. Supported by the Dutch MS Research Foundation (grant 00-407 MS).

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Poster Session 15: MS-clinical studies

Abstracts PP15-01

215

Department of Immunology, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan

Late onset multiple sclerosis in Japan

Recently, Japan changed drastically to a high speed of aging society, it is very likely that the number of late-onset (clinical onset after 50 years of age) multiple sclerosis (MS) increase. The purpose of this study was to investigate the late-onset Japanese MS patients in our hospital and compare the reported case from different ethnic background for correct diagnosis and therapeutics. We enrolled 20 late-onset MS patients from our hospital. For each patients, age, gender, clinical presentation, disease course, neurological involvement, disease duration, disability score (EDSS) were assessed and evaluated with the change of brain, spinal MRI as well as CSF and/or serum. More than 70% of the patients were female and good response to steroid pulse therapy at relapse. Conclusion: There seemed often encountered the difficult case for making differential diagnosis of late-onset MS. In such cases serial MRI scan (brain and spinal cord) as well as long term clinical follow up might required for correct diagnosis. Poser's criteria fits better than McDonald criteria, because of the aging effect of the MRI lesions. Although the early relapse is very common, the prognosis and the response to steroidal therapy of late-onset MS is better. In Japan, neurodegenerative processes are not so prominent compare to other countries. Taking into account the above reasons, the use of immunomodulant therapy is the first recommendation.

Objective and methods: The accurate diagnosis of multiple sclerosis (MS) is often hampered by a difficulty in differential diagnosis of neurological diseases mimicking MS, such as Sjögren syndrome (SjS) and neuroBehçet's disease. By gene expression profiling of peripheral blood T cells on a DNA microarray, we recently found that MS is classified into four distinct subgroups with different therapeutic responses to interferon-beta (IFNB), separated from healthy control subjects (Satoh et al. Neurobiol Dis 18: 537– 550, 2005; J Neuroimmunol, 2006 in press). To determine the expediency of DNA microarray analysis for a support in differential diagnosis of MS and prediction of IFNB response in clinical practice, an 18-item questionnaire was faxed to certified neurologists of 1709 hospitals in Japan. Results: The answer rate was 19% (319/1709). Great numbers (54% and 56%) of neurologists experienced difficulties in discriminating MS from tumors and cerebrovascular accidents, where 70 and 4 cases required pathological diagnosis, respectively. Nine neurologists suggested difficulties in differential diagnosis of MS and SjS. Approximately a half (46%) of IFNB-treated MS patients showed satisfaction with the treatment, while 25% of the patients were grouped into poor responders, composed of dropouts due to the induction of severe relapses or adverse effects. The great majority (86%) of neurologists showed a favorable answer to the expediency of DNA microarray analysis for prediction of IFNB response. Conclusions: Even by certified neurologists, the accurate diagnosis of MS is often difficult. The potential power of gene expression profiling of peripheral blood T cells makes most neurologists to have the expediency of DNA microarray technology for differential diagnosis of MS and prediction of therapeutic response to IFNB.

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PP15-04

Characteristics of late-onset multiple sclerosis

Coincidence of multiple sclerosis and autoimmune diseases

K. Yokoyama, M. Fukuda, Y. Mizuno Department of Neurology Juntendo University School of Medicine, Japan

Kondo M, Nagakane Y, Kuriyama N, Tokuda T, Mizuno T, Nakagawa M

1

J. Szilasiová, 1Z. Gdovinová, 1E. Kahancová, 2. Macejová

Department of Neurology, Faculty Hospital, Šafarik University, Košice, Slovakia, 2Department of Internal Medicine, Faculty Hospital, Šafarik University, Košice, Slovakia

1

Department of Neurology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan Background and objective: Few patients with multiple sclerosis (MS) were more than 65 years old. According to McDonald's diagnostic criteria for MS, the criteria would best apply to patients between 10 and 59 years of age and special care must be taken in making a diagnosis of MS in those who were younger or older. Although some cases of late-onset MS were reported, clinical characters of late-onset MS were still unknown. Therefore we evaluated characteristics of late-onset MS in our hospital. Methods: Thirty patients with MS admitted to our hospital from April, 1991 to March, 2006. Late onset is defined as onset after 65 years old and we found out three patients (68–77 years old, one woman and two men). Clinical course and MRI findings of these patients were evaluated. Results: All three patients with late-onset MS underwent relapse and remission. The sites of the lesion were optic nerve and spinal cord in one patient, cerebrum and spinal cord in two patients. All their symptoms were temporarily improved after steroid therapy. Two patients with late-onset MS had poor prognosis, but in one patient, subcutaneous administration of Interferon β1b was effective for preventing relapse for 2.5 years. Conclusion: Characters of late-onset MS were similar to Japanese ordinary type of opticospinal form. Interferon β1b therapy may provide good prognosis in late-onset MS. PP15-03

Questionnaire on the expediency of DNA microarray analysis for differential diagnosis of multiple sclerosis and prediction of therapeutic response to interferon-beta Yusuke Nanri, Jun-ichi Satoh, Wakiro Sato, Takashi Yamamura

Background: Multiple sclerosis (MS) could be associated with other autoimmune diseases (ADs). The aim of our study was to quantify the coincidence MS with an ADs and to characterize the MS with other AD. Patients and methods: Data of 194 patients with definite diagnosis of MS (134 women and 60 men, aged 36.5 ± 9.1 years, range 18–62 years, disease duration 8.6 ± 6.4 years), defined by the Poser criteria, were retrospective analysed. 28 (15%) of them had benign, 111 (57%) relapsing–remitting, 43 (22%) secondary progressive and 12 (6%) primary progressive MS. The two groups — MS with and without ADs were compared. Disability status was assessed by the Expanded Disability Status Scale (EDSS). Results: The median EDSS-value was 3.1 ± 1.6 (range 1–8). 14 (7.2%) of the patients with MS had other AD. The most frequent ADs were: autoimmune thyroiditis 7, uveitis 2, rheumatoid arthritis 1, ulcerative colitis 1, coeliac disease 1, asthma bronchiale 1, psoriasis vulgaris 1, spondyloarthritis 1 (one patient had autoimmune thyroiditis and rheumatoid arthritis). These ADs preceded MS diagnosis and interferon beta therapy in all cases. There were found no significant differences in the age and EDSS between MS group with and without ADs. Conclusion: Our results showed similar findings as other reports. Neurologic disability is not significantly influenced by coincidence MS and other AD. Autoimmune thyroiditis showed the strongest coexistence with MS, but this may reflect the high prevalence in common population. PP15-05

Two cases of biopsy-proven tumefactive multiple sclerosis Yoshii S, Iizuka T, Masuda R, Ujiie S, Hamada J, Sakai F

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Department of Neurology, School of Medicine, Kitasato University, Sagamihara, Japan Objectives: to report two cases of steroid-resistant tumefactive multiple sclerosis (MS), which improved on combination therapy including repeated steroid pulse, intravenous immunoglobulin (IVIG) or plasma exchanges (PE). Methods: Case reports. Results: the first case is a 21-year-old woman who presented with convulsive seizure following the onset of headache and fever. Brain MRI showed multiple enhancing brain tumors. She underwent brain biopsy, which showed abundant accumulation of lipid-laden macrophages with perivascular lymphocytes compatible with acute MS. She did not improve following steroid pulse therapy, but clinical symptoms and brain MRI findings gradually became stabilized after repeated combination therapy consisting of steroid pulse and IVIG, followed by interferon beta. The second case is a 61-year-old man who presented with gradual progressive memory loss. He was admitted to another hospital where under a diagnosis of brain tumor he underwent open brain biopsy, which showed no malignancy. After given a diagnosis of MS, he received steroid pulse therapy without beneficial effect. He also received steroid pulse therapy followed by IVIG at the second hospital, but he progressed relentlessly and became bed-ridden. He was transferred to this hospital. Brain MRI showed enhancing massive tumors involving the corpus callosum. He initially received IVIG with steroid pulse therapy because he was in a septic state. He underwent PE followed by interferon beta. His neurological deficits remained unchanged but the activity of MS on MRI markedly decreased. Conclusion: patients with tumefactive MS should be treated intensively. Repeated combination therapy may be effective if began during earlystage of MS.

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Three cases of relapsing multiple sclerosis presenting with only nausea and vomiting H. Fukauraa, S. Takahashia and Y. Terayamaa a

Neurology, Iwate Medical University, Morioka, Japan

Two male and one female patients with multiple sclerosis (MS) were admitted to our hospital because of nausea and vomiting. Upper gastroenterological examination showed no abnormalities. Magnetic resonance imaging showed high signal intensity area (dorsal portion of medulla) in Gd-DTPA enhanced T1 weighted image. After the treatment of intravenous methylprednisolone sodium, nausea and vomiting improved. Lesions involving the dorsal motor nucleus of the vagus, the nucleus ambiguous or the autonomic nuclei in the medullary reticular formation are usually associated with vomiting and upper gut motility disturbances. We speculate that their nausea and vomiting may have been of autonomic origin. Immunological features are also reported. We believe our cases represent an unusual manifestation of MS. PP15-08

Depression score in multiple sclerosis patients Ohara, K1, Ota, K2, Shimizu, Y1, Ohashi, T1 and Iwata, M1 1

Department of Neurology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan; 2Tokyo University of Science, Tokyo, Japan

PP15-06

A case report of the patient with tumefactive demyelinating lesion masqueraded brain tumor M. Apiwattanakul Prasat Neurological Institute, Bangkok, Thailand Objective: Tumefactive demyelinating lesion is quite rare in clinical practice. Most of these patients have clinical manifestation and imaging simulate brain tumors. Surgical biopsy is usually performed to differentiate between two conditions. We would like to present a case of this disease. Method: A case report of Thai young man who presented with progressive left hemiparesis and right side dullness headache in the past two months. Magnetic resonance imaging and surgical biopsy were done to demonstrate his pathology. Result: MRI revealed hyposignal T1 weighted/hypersignal T2 weighted and Fluid-Attenuated Inversion Recovery (FLAIR) at right frontoparietal white matter area with gadolinium enhancement in the lesion which some of these were not completed rings. There is also another lesion at left parietooccipital area but lesser in size and enhancement. Magnetic resonance spectroscopy (MRS) shown dropping of N-acetyl aspartate (NAA) peak with modest increased in choline (Cho)/phosphocreatine (Cr) ratio which was compatible with demyelinating disease. Histopathology demonstrated foamy macrophage, perivascular lymphocytic infiltration, prominent reactive astrocyte and special stains revealed markedly loss of myelin with myelin debris. The patient was diagnosed with tumefactive demyelinating lesion and his clinical status was improved after high dose steroid administration. Conclusion: In patient who presents with progressive neurological deficit and mass liked lesion in neuroimaging, in addition to tumor such as glioma, a variant form of demyelinating disease such as tumefactive demyelinating lesion should be in differential diagnosis. But biopsy should be always done to discriminate between two conditions because of difference in treatment and prognosis.

Background: Some patients of multiple sclerosis (MS) have psychic symptoms. Depression is observed as one of the frequent psychic symptoms of MS. However, many papers, researched psychological interventions for MS, were published, but they did not form any definite conclusions. Methods: We used Beck Depression Inventory (Second Edition). To investigate MS depression, 27 MS patients (7 males, 22 females; 24 relapse and remitting type, 2 secondary progressive type, 3 with clinical isolated syndrome; average age: 43) were compared and categorized or examined with respect to age, depressive score, EDSS, morbidity period, those receiving interferon beta 1b therapy and those who did not, total frequency of relapse, and frequency of relapse the last year. Results: There were no significant differences. Between depressive score and EDSS, numbers of cerebral lesions, total frequency of relapse, frequency of relapse in the last year, among patients with high EDSS. Conclusion: Depressive scores in MS patients not associated with any clinical examination in this study may suggest that depression is not constitutive of the disease. PP15-09

Characterization of the T cell receptor repertoire in neuromyelitis optica: T cell activity is up-regulated compared to multiple sclerosis Y. Warabia,b, K. Yagia, H. Hayashia and Y. Matsumotob Tokyo Metropolitan Neurological Hospital, Tokyo, Japan; bTokyo Metropolitan Institute for Neuroscience, Tokyo, Japan

a

To characterize T cell immunity in neuromyelitis optica (NMO), we examined the T cell receptor (TCR) repertoire in NMO patients with complementarity-determining region 3 (CDR3) spectratyping and compared the results with those from multiple sclerosis (MS) patients and healthy subjects. From 11 patients with NMO, 21 MS patients and 25 healthy subjects, peripheral blood lymphocytes (PBL) were isolated.

Abstracts Written consent was obtained from all the subjects and the study was approved by the Institute Review Board. RNA was extracted from PBL and cDNA was synthesized, then, amplified using primer pairs for TCR to determine CDR3 spectratypes. Both NMO and MS patients had a larger number of clonally expanded Vβ genes than healthy subjects. Moreover, NMO patients had a significantly larger number of expanded Vβs than MS patients. The detailed analysis revealed that Vβ1 and Vβ13 were significantly activated in NMO. These results reflected unique pathophysiology of NMO, which is distinguishable from that of MS. Furthermore, longitudinal examinations of the TCR repertoire demonstrated that the number of clonally expanded Vβs in NMO correlates with the Kurtzke Expanded disability status scale (EDSS). Although the activation pattern of the TCR repertoire in relapsing–remitting MS (RRMS) was similar to that in NMO, secondary progressive MS (SPMS) patients with longer disease durations and higher EDSS scores consistently had a smaller number of clonally expanded Vβs than RRMS patients. Detailed TCR investigations will provide useful information to evaluate the clinical and immunological status of NMO and MS and to develop effective immunotherapies.

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cerebral lesions after IFNβ-1b was discontinued. Case 1: A 30-year-old woman, who was diagnosed MS and started IFNβ-1b in 2004, discontinued IFNβ-1b due to transient liver dysfunction in February, 2005. IFNβ-1b was resumed from March, 2005. Five months later, August, 2005, brain MRI was taken when she had mild headache. The MRI demonstrated a large cystic lesion with ring enhancement in the right frontal white matter. Since the lesion expanded in size in September, 2005, high-dose intravenous methylprednisolone therapy was done. In October, 2005, the lesion shrank and there was no ring enhancement. Case 2: A 26-year-old woman, who was diagnosed MS and started IFNβ-1b in 2003, discontinued IFNβ-1b due to depressive state in June, 2005. Four months later, October, 2005, brain MRI revealed a large cystic lesion with ring enhancement in the left parietal white matter. Without any treatments, the lesion became smaller in December, 2005. Discussion: Although pathogenetic mechanism of developing cystic cerebral lesions remains unknown, our cases suggest that discontinuation of IFNβ might be linked to the development of the cystic lesions. In order not to overslip such lesions after discontinuation of IFNβ-1b, we should check brain MRI regularly or when any cerebral symptoms are noticed regardless of severity. PP15-13

PP15-10

Brain MRI findings in Japanese MS patients with NMO-IgG

Analysis of the opticospinal, conventional forms of multiple sclerosis, and neuromyelitis optica in Taiwan

I. Nakashima, K. Fujihara, I. Miyazawa, T. Misu, and Y. Itoyama

Jen Jen, Su; Chih Chao, Yang

Department of Neurology, Tohoku University School of Medicine, Sendai, Japan

Department of Neurology, National Taiwan University Hospital, Taiwan

Magnetic resonance imaging (MRI) was investigated in 15 Japanese multiple sclerosis (MS) patients with NMO-IgG, a specific serum marker for neuromyelitis optica (NMO). Among the 15 MS patients with NMO-IgG, 7 patients (47%) had brain MRI lesions and 14 patients (93%) had longitudinally extensive lesions on spinal cord MRI. The only patient without the longitudinally extensive spinal cord lesion, however, had extensive spinal cord atrophy indicating a history of an extensive cord lesion. Among the 7 patients with brain MRI lesions, 5 had only optic neuritis and transverse myelitis as clinical symptoms. The other 2 patients had no episode of optic neuritis but had histories of cognitive dysfunction, hemiparesis, or cerebellar ataxia. On brain MRI, diffuse white matter lesions, juxtacortical lesions, callosal lesions, periventricular lesions, and longitudinally extensive lesions along pyramidal tracts were commonly observed. Cerebellar hemisphere lesions were seen in one patient who developed cerebellar ataxia, and hypothalamic lesions were seen in another patient who showed hyperprolactinemia at the relapse of visual disturbance. Cavity formation was observed in none of the patients. Although some brain MRI lesions seen in the patients with NMO-IgG resembled those of the classic form of MS in shape, typical ovoid lesions with open-ring enhancement were not observed in any patient with NMO-IgG. Longitudinally extensive lesions along the pyramidal tract seemed specific for the patients with NMO-IgG. Brain lesions were not rare in the patients with NMO-IgG, but the radiological features of the lesions were rather atypical for MS. PP15-11

Large “cystic” cerebral lesions in multiple sclerosis patients after discontinuation of IFNβ-1b: A report of two cases

Multiple sclerosis (MS) is an inflammatory demyelinating disease of unknown etiology, and increasing evidence suggests that it is heterogeneous. According to this clinical course, MS can be described as relapsing– remitting type (RR-MS), secondary progressive type (SP-MS), or primary progressive type (PP-MS). On the other hand, concerning the sites of involvement, two further MS subtypes in Japan were separated into opticospinal MS (OS-MS), in which the clinically estimated main lesions are confined to the optic nerves and spinal cord, the conventional MS (C-MS), which shows disseminated lesions in the central nervous system (CNS), including the cerebrum, cerebellum or brainstem. Neuromyelitis optica (NMO) is an inflammatory demyelinating disease that selectively affects optic nerves and spinal cord. Typically, NMO has a worse outcome than MS, and frequent and early relapse. A specific IgG for NMO was described in NMO and Asian optic-spinal form of MS, but not detected at classical (western) form of MS. But whether OS-MS is NMO? There were still many debates and discussions. In Taiwan there were increasing numbers of multiple sclerosis and there were many patients presenting optic nerve and spinal cord involvement but less brain involvements. We try to evaluate the patients of multiple sclerosis admitted at one primary medical center in Taiwan, to classify them into OSMS, conventional MS (C-MS) and NMO then to analyze whether there were differences between their clinical course, prognosis and neuroimage studies and tried to correlate the relationship of OS-MS and NMO in Taiwan. PP15-14

Probable NMO IgG in Turkish patients with Devic's disease and MS G. Akman-Demira, G. Demira, M. Mutlua, S. Icoz a, M. Kurtuncua, N. Yesilota, M. Eraksoya

T. Takahashi, I. Nakashima, T. Misu, Y. Shiga, K. Fujihara, Y. Itoyama a

Department of Neurology, Tohoku University School of Medicine, Sendai, Japan Interferon (IFN) β is an established disease-modifying therapy for multiple sclerosis (MS). However, the following course after discontinuation of IFN-β is not fully known. Here we report two patients who developed large “cystic”

Istanbul University, Istanbul Faculty of Medicine, Department of Neurology, Turkey Neuromyelitis optica (NMO), or Devic's disease is a disorder of the demyelinating disease spectrum which involves mainly the optic nerves and the spinal cord within the central nervous system (CNS). Recently, abnormalities of the humoral immune system have been shown in cases

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with NMO, with an antibody against aquaporin-4. Here we aimed to look for NMO IgG in Turkish cases with Devic's disease in comparison to cases with classical multiple sclerosis (MS) and healthy controls (HC). Serum samples from 14 patients with Devic's disease, 14 cases with MS and 15 HC were assessed using an indirect immunofluorescence kit containing monkey cerebrum, cerebellum, intestinum and HEP2 cells. The sections were assessed blinded to the clinical diagnosis. 8 patients with Devic's disease showed staining around the vessels and pial surfaces in cerebrum and cerebellum sections. On the other hand none of the MS cases or HC showed such a staining. 5 out of 8 positive Devic's showed strong binding, and among these 4 had severe clinical involvement. On the other hand, among the cases with Devic's disease that were negative, 2 had severe disease while 4 had milder forms of the disease. However this difference did not reach statistical significance. This relatively small cohort of patients suggest that the presence of NMO IgG might have an association with the disease course and severity. Such a relationship should be further assessed in larger series. PP15-15

Acute disseminated encephalomyelitis in Japan: A population-based study of pediatric patients H. Torisua, R. Kiraa, S. Yasumotob, Y. Ishizakic, Y. Murakamid, M. Shimonoe, S. Nagamitsuf, M. Masuzakib, M. Amamotog, R. Kondoh, T. Uozumii, M. Aibej, K. Gondoa, T. Hanaic, T. Matsuishif, A. Shirahatae, A. Mitsudomeb, and T. Haraa Kyushu University, Fukuoka, Japan; bFukuoka University, Fukuoka, Japan; cFukuoka Children's Hospital, Fukuoka, Japan; dOmuta City

a

General Hospital, Omuta, Japan; eUniversity of Occupational and Environmental Health, Kitakyushu, Japan; fKurume University, Kurume, Japan; gKitakyushu City Yahata Hospital, Kitakyushu, Japan; hAso Iizuka Hospital, Iizuka, Japan; Nippon Steel Yawata Memorial Hospital, Kitakyushu, Japan; jKyushu Rosai Hospital, Kitakyushu, Japan Acute disseminated encephalomyelitis (ADEM) has recently been studied in several countries due to the development and wide spread of imaging technology, but few epidemiologic studies of ADEM have been undertaken, especially in Asian countries. We conducted a multicentric study on childhood ADEM, acute transverse myelitis (ATM) and multiple sclerosis (MS) by epidemiologic approach from 1998 to 2003 at Fukuoka Prefecture in southern Japan, where almost 5 million people lived. We identified 38 patients with disseminated demyelination of the central nervous system; 26 with ADEM, 4 with TM and 8 with MS. An estimated incidence of ADEM was 0.64/100,000, median age at onset was 5.7 years, and male–female ratio was 2.3:1. The median age at onset (9.3 years) of MS was significantly higher than that of ADEM. Nineteen patients with ADEM (73%) experienced febrile illnesses within one month before the onset. As a prodromal symptom of ADEM, fever was observed in 73% of patients. Initial symptoms included disturbance of consciousness (50%), motor symptoms (50%), and seizures (31%). Laboratory studies revealed pleocytosis (81%) and protein elevation (34%) in cerebrospinal fluid. Administration of high-dose methylprednisolone was effective (84%). Neurological sequelae such as mild motor disturbance occurred in 30%. Compared with previous reports of ADEM in other countries, the onset age and sex ratio in Japan were similar, but the proportions of fever and seizures were higher. There might be distinct clinical features of ADEM in Asian countries.

Abstracts

Poster Session 16: PNS diseases

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PP16-01

Intraneural γδ T cells during experimental autoimmune neuritis T. Fujiokaa, T. Kudekena, T. Kiyozukaa, Y. Iwasaki, T. Kurihara and A. M. Rostamib a

b

Toho University, Tokyo, Japan; Thomas Jefferson University, Philadelphia, USA

simultaneously before reaching peripheral nervous microenvironment. Supported by CAPES, FAPESP. PP16-03

Expression of caveolin-1 in experimental autoimmune neuritis Meejung Ahna, Changjong Moona, Heechul Kima, Jeeyoung Leea, Changsung Kohb, You Matsumotoc, Taekyun Shina⁎, a

To investigate the infiltration of γδ T cells in peripheral nervous system during autoimmune neuritis, FACS analysis of the infiltrating lymphocytes and quantitative PCR for the intraneural expression of γδ TCR mRNA in the cauda equina (CE) of female Lewis rats with SP26induced experimental autoimmune neuritis (EAN) were sequentially studied. Total RNA was extracted from CE sampled at four, seven, 10, 14, 18, 21, 26 and 32 days post-immunization (DPI; n = 4). Complementary DNA was then analyzed by quantitative PCR for γδ TCR and house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions. The value of γδ TCR mRNA products was expressed by the ratio to the value of GAPDH PCR products of the same rat. For FACS analysis, mononuclear cells recovered from the homogenated CE sampled at 13 and 17 DPI (n = 6) were stained by anti-rat CD3-FITC or γδ TCRPE monoclonal antibody. The rats developed EAN at 10 DPI, peaked at 14 to 18 DPI then recovered. γδ TCR mRNA was negligible at 0 DPI, increased at 4 DPI only transiently then started to increase at 10 DPI again. It reached the peak at 14–18 DPI then recovered to the basal level by 32 DPI. FACS analysis revealed that γδ T cell populations among total T cells in the CE were always higher than those in the other organs (peripheral blood, lymph nodes and spleens) of the same rats. Our data suggest two waves of γδ T cell infiltration one of which might be proinflammatory while the other might be anti-inflammatory.

Department of Veterinary Medicine, Cheju National University, Jeju 690756, South Korea; bDepartment of Medical Technology, School of Allied Medical Science, Shinshu University, Matsumoto, Japan; cDepartment of Molecular Neuropathology, Tokyo Metropolian Institute for Neuroscience, Fuchu, Tokyo, Japan The expression of caveolin-1 and the related molecule endothelial nitric oxide synthase (eNOS) was analyzed in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that caveolin-1 was significantly increased in the sciatic nerves with EAN upon initiation of cell infiltration during the early and peak stage (days 10 and 14 post-immunization, p.i.), and declined thereafter. The pattern of eNOS expression over the course of EAN largely matched that of caveolin1. Immunohistochemistry showed that, in EAN lesions, intense caveolin-1 immunostaining was seen in ED1-positive macrophages, as well as intensely in vascular endothelial cells and Schwann cells, which constitutively expressed caveolin-1 in normal sciatic nerves. Consequently, we postulated that caveolin-1 expression increased in the sciatic nerves with EAN, possibly mediating either molecular trafficking and nitric oxide generation partly through the activation of eNOS in vascular endothelial cells and Schwann cells, as well as in inflammatory macrophages in EAN and /or cellular apoptosis of inflammatory cells. The functional role of caveolin-1 in EAN will be discussed.

PP16-02

PP16-04

TNF-alpha and iNOS in peripheral nervous system during experimental autoimmune neuritis

Regulation of genes for immune system-associated molecules in Schwann cells (SC) by cyclic adenosine monophosphate (cAMP) in vitro

C. L. R. de la Hoza, F. R. Castrob, L. M. B. Santosb and F. Langonec RP Lisak, B Bealmear, JA Benjamins, B Yao and S Land Department of aCellular Biology, bMicrobiology and Immunology, and c Physiology and Biophysics, Institute of Biology, Unicamp, Campinas, SP, Brazil

Department of Neurology, Wayne State University, Detroit, USA; Center for Molecular Medicine and Genetics, Wayne State University, Detroit, USA

Tumor necrosis factor-alpha (TNF-α) and inducible nitric oxide synthase (iNOS) are involved in inflammatory processes such as Guillain Barré Syndrome and its animal model, Experimental Autoimmune Neuritis (EAN). We have investigated the presence of TNF-α and iNOS in peripheral nervous system during EAN. At different stages of the disease, spinal roots adjacent to lumbar intumescence, cauda equina, sensitive ganglia and sciatic nerves were obtained from induced-EAN Lewis rats and processed for double iNOS and TNF-α fluorescence immunohistochemistry. Large oval TNF-α+ cells and smaller rounded TNF-α+/iNOS+ cells were evidenced. All experimental groups, including the control group showed TNF-α+ cells around ganglia and in perineurium, with slight differences among groups. At the onset of clinical signs, few TNFα+/iNOS+ cells were found in blood vessels of cauda equina and sciatic nerve. During the acute phase there has been an increase in the amount of these cells around vessels and inflammatory infiltrates in ganglia, cauda equina and the other spinal roots. Just before the peak stage, TNF-α+/iNOS+ cells in these regions and in sciatic nerve were widely distributed around numerous infiltrates. In the recovery stage, some TNF-α+/iNOS+ cells have been still found in cauda equina and sciatic nerve vessels, while in the resolution stage they were not evidenced. Evolution of EAN clinical signs may be related to TNF-α and iNOS-immunoreactive cells in different nervous regions. Probably these cells are stimulated to produce TNF-α and iNOS

As part of our ongoing studies of interactions between cytokines and SC, we studied the effects of cAMP on gene regulation at early (6 h) and intermediate (24 h) time points. Incubation of SC in vitro with 8-Br-cAMP at high concentration simulates many aspects of axolemmal effects on SC. Neonatal rat SC were incubated with l mM 8-Br-cAMP for 6 and 24 h. RNA was extracted from SC and analyzed with Affymetrix microarray gene chips. These chips contain >28,000 genes. Up or down regulation of expression compared to unstimulated SC was set at very high stringency (p < 0.001). We found changes in expression of many genes including several originally associated with the immune system. At 6 h we observed upregulation for genes for tumor necrosis factor (TNF)-induced protein 6, CD200, cytokineinduced neutrophil chemoattractant-2, CD24, CXCL2 and transforming growth factor (TGF)B-induced transcript 4. Down regulation was noted in the gene for TGFB-induced transcript 1. At 24 h there was upregulation of the genes for CXCR4, integrinB4, hypoxia induced factor (HIF)-la responsive RTP801, HIF-la and TGFB-induced transcript 4. Down regulation was noted for genes for IgG Fc binding protein 1, TNFRSF member 12a (receptor for TWEAK), integrinB, integrinB-binding protein 1 and endothelial monocyte-activating polypeptide. Thus SC differentiation is associated with changes in expression of genes for immune system associated molecules, which may be important in SC differentiation, and in interaction of SC with the immune system.

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PP16-05

PP16-07

Effects of acidic phospholipids on antiganglioside antibodies in GBS: Role of the disialosyl residue

Campylobacter coli enteritis and Guillain–Barré syndrome: No evidence of molecular mimicry and serological relationship

Minako Hirakawa1,2, Masami Sada1, Shoji Tsuji2, Susumu Kusunoki1

Kei Funakoshia, Michiaki Kogaa, Masaki Takahashib, Koichi Hirataa, Nobuhiro Yukia

Department Neurology, Kinki University, Osaka, Japan; 2Department Neurology, University of Tokyo, Tokyo, Japan

1

a

Department of Neurology, Dokkyo Medical University, Tochigi, Japan; Department of Microbiology, Tokyo Metropolitan Institute of Public Health, Tokyo, Japan

b

[Introduction] We have reported the increase of the activity of the antiGM1 IgG antibodies in Guillain–Barré syndrome when a mixture of such acidic phospholipids as phosphatidic acid (PA) and GM1 was an antigen in ELISA. In contrast, phospholipids provided no obvious enhancing effect on the anti-GQ1b IgG in Miller Fisher syndrome. One possible reason of such different effects is the difference in the preceding infections, GM1 epitope of pathogens being presented with a phospholipid to the patients. Another possibility is the difference of the physiochemical properties of two antigens; GQ1b has two disialosyl residues having strongly negative charge, while GM1 does not. [Methods] To investigate the former possibility, we sensitized rabbits with a mixture of GM1 and PA (GM1/PA) to see whether the antibodies thus produced show higher titer against GM1/PA or not. To clarify the role of charge influences, we examined the effect of the phospholipids on IgG against GD1b, which shares a disialosyl residue with GQ1b and the terminal Gal-GalNAc structure with GM1. [Results and Discussion] There was no significant difference in the antibody specificities in rabbits sensitized either with GM1 or with GM1/PA. The anti-GD1b IgG activities with anti-Gal-GalNAc specificity were increased when GD1b/PA was an antigen, whereas those monospecific to GD1b were not. Thus, the enhancing effect of PA may not be due to the presence of a phospholipid within the bacteria of the antecedent infection. It may depend on whether the antibody recognizes disialosyl residue or not. PP16-06

Identification of new antigenic candidates in autoimmune neuropathies by serological proteome analysis Masumi Endoh Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan In order to identify new antigenic candidate proteins present in peripheral nervous tissue, we performed proteome mapping and immunoproteomics analysis using patients sera of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Guillain–Barre syndrome (GBS), anti-myelin-associated glycoprotein neuropathy (anti-MAG), and leprosy patients. The homogenate of peripheral nervous tissue from healthy subject was applied to two-dimensional electrophoresis on two or more gels run in parallel. One was stained with SyproRuby used for spot cutting and protein sequencing, and the other gel was electroblotted onto PVDF membrane. The transferred-membrane was immunostained with patients sera, and then immunoreactive spots were visualized by chemiluminescence substrate. Immunoreactive signals of membrane and protein spots on gel were matched by using Decyder software (Amersham Biosciences). The matched protein spots were picked from gel, and analyzed by MALDI-TOF MS sequencing of tryptic peptides. Over 30 of antigenic proteins reacted on immunoblots membrane with CIDP, GBS and anti-MAG sera. A few immunoreactive spots were detected with leprosy sera and patterns of spot maps varied greatly among individual patients of leprosy. More than 20 spots were successfully matched with SyproRuby stained gels, which were further selected for MS analysis for protein sequencing. In conclusion, serological proteome analysis may provide a useful tool for the identification of novel autoimmune antigens, whose relevance to pathogenically events in autoimmune neuropathies remains to be defined.

Campylobacter coli was isolated from two Guillain–Barré syndrome (GBS) patients who had anti-GM1 and GD1a IgG antibodies. To determine whether C. coli is a causative agent, we examined the hypothesis that production of anti-ganglioside antibodies is induced by ganglioside-mimics on that bacterial lipo-oligosaccharide (LOS), as in C. jejuni-associated GBS. LOSs of both C. coli isolates had very weak reactivities with anti-GM1 and anti-GD1a IgG monoclonal antibodies, whereas those of some GBS-related C. jejuni isolates had strong reactivities. Anti-GM1 and anti-GD1a IgG antibodies from the two patients were not absorbed as much by the LOSs of their isolates as were those of GBS-related C. jejuni strains. These findings do not support the hypothesis of ganglioside mimicry on C. coli isolates' LOSs. We next made a serological assay of recent C. coli infection in 74 patients with GBS, 26 with Fisher syndrome (FS), 49 with other neurological diseases (OND), and 37 normal controls (NC) using the bacterial outer membrane protein as antigen. Eight (11%) GBS and 2 (8%) FS patients had two or three classes of IgG, IgM, and IgA anti-C. coli antibodies. Anti-C. jejuni IgG and IgA antibody titers were significantly higher than those of anti-C. coli (respectively, p = 0.03 and 0.01). This suggests that anti-C. coli antibodies cross-react with C. jejuni protein. We concluded that a C. coli infection was not the cause of GBS in our patients. Both isolation of a microorganism from, and the positive infectious serology of, GBS patients do not always indicate the causal agent. PP16-08

Campylobacter jejuni-isolate neuropathies: Clinical spectrum and bacterial features K. Kimotoa, M. Kogaa, M. Odakaa, K. Hirataa, M. Takahashib, J. Lic, M. Gilbertc, N. Yukia a

Department of Neurology, Dokkyo Medical University School of Medicine, Kitakobayashi 880, Mibu, Shimotsuga, Tochigi 321-0293, Japan; b Department of Microbiology, Tokyo Metropolitan Institute of Public Health, Tokyo, Japan; cInstitute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada Background: Clinical and serological studies have suggested that Guillain–Barré syndrome (GBS) and atypical GBS with preserved deep tendon reflexes (DTR) form a continuous spectrum with Fisher syndrome (FS), FS/GBS overlap, Bickerstaff's brainstem encephalitis (BBE), BBE/ GBS overlap, acute ophthalmoparesis (AO), ataxic GBS, and acute oropharyngeal palsy. Objective: To clarify the spectrum of neurological disorders that occur subsequent to Campylobacter jejuni enteritis. Methods: C. jejuni-isolated patients with various neurological conditions were recruited. Bacterial features of each isolate were investigated. Results: Diagnoses for the C. jejuni-isolate patient were GBS (n = 90), FS (n = 22), DTR-preserved GBS (n = 10), FS/GBS (n = 6), BBE (n = 1), BBE/ GBS (n = 2), AO (n = 3), ataxic GBS (n = 1), and acute oropharyngeal palsy (n = 3). Isolates from DTR-preserved GBS were similar to those of GBS in serotype (HS:19), genotype (lipo-oligosaccharide [LOS] locus class A or B, cst-II genotype [Thr51]), and GM1 or GD1a epitope expression on LOS. FS/GBS overlap, BBE, BBE/GBS overlap, AO, ataxic GBS, and acute oropharyngeal palsy isolates were similar to those

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of FS in serotype (HS:2 or HS:4-complex), genotype (LOS locus class A or B, cst-II genotype [Asn51]), and GQ1b epitope expression on LOS. Conclusions: The bacterial findings support the proposal that GBS and DTR-preserved GBS form a continuous spectrum together with FS, FS/ GBS overlap, BBE, BBE/GBS overlap, AO, ataxic GBS, and acute oropharyngeal palsy. PP16-09

Clinical features of child Guillain–Barré syndrome associated with anti-ganglioside antibodies

GBS, and Haemophilus influenzae was rare (1%) unlike in FS. Antiganglioside IgG antibodies were detected in 68% of the PCB patients and anti-GT1a antibody was the most common (51%), in most cases it had crossreactivity with GQ1b. Anti-ganglioside antibody profiles were similar in pure PCB and GBS overlap populations. Conclusions: Although PCB regularly is accompanied by other overlapping conditions, considerable number of patients was classified as pure PCB, suggesting that PCB is a distinct variant of GBS. GBS overlap was potentially defined as severe condition of PCB. PCB appeared to be closer to GBS than to FS, based on the antecedent infectious agents and autoantibody profile. PP16-11

Y. Nishimotoa, K. Susukib and N. Yukib

Long-term prognosis of Guillain–Barre syndrome a

Department of Pediatrics, Nanki Fukushi Center, Wakayama, Japan; b Department of Neurology, Dokkyo University School of Medicine, Tochigi, Japan

Mieko Ogino, Yutaka Ogino, Sachiko Irie, Naomi Kanazawa, Toyokazu Saito, Fumihiko Sakai

Objective: To clarify the association between anti-ganglioside IgG antibodies and clinical features of child GBS. Methods: We retrospectively studied 32 GBS patients less than 16-yearold. The IgG antibodies to GM1, GM1b, GD1a, or GalNAc-GD1a were examined by ELISA. Information on preceding infectious symptoms, neurological findings, electrodiagnoses, treatments, and outcomes was obtained. Results: The anti-ganglioside IgG antibodies were positive in 14 of 32 patients. Patients with preceding gastro-intestinal tract infection were 8 (57%) in positive group (n = 14), but 2 (11%) in negative group (n = 18) (p = 0.01). Neurological findings did not differ between positive and negative groups. Nerve conduction studies showed that 9 patients (64%) in positive group were diagnosed as having axonal pattern, but 2 (11%) in negative group (p = 0.006). All the patients had favorable outcome. At the final follow-up, however, Hughes grade 1 was found in 9 patients (64%) in positive group and 2 (11%) in negative group (p < 0.001). Furthermore, periods from neurological onset to the final follow-up were significantly longer in positive group (median days, 384) than those in negative group (median days, 144). Conclusions: Child GBS with anti-ganglioside IgG antibodies often have preceding gastro-intestinal tract infection, the axonal pattern, and slight residual symptoms. Anti-ganglioside antibody testing may be helpful for the subtype classification of child GBS as well as for that of adult GBS.

Kitasato University School of Medicine, Department of Neurology, Sagamihara, Japan

PP16-10

PP16-12

Pharyngeal–cervical–brachial variant of Guillain–Barré syndrome: Analysis of 100 cases

Continuous spectrum of Fisher syndrome and Bickerstaff's brainstem encephalitis

T. Nagashimaa,b, M Kogaa, M. Odakaa, K. Hirataa, N. Yukia

M. Itoa, M. Odakaa, M. Kogaa, K. Hirataa, and N. Yukia

a

a

Background: Pharyngeal–cervical–brachial weakness (PCB) is considered as a variant of Guillain–Barré syndrome (GBS). There have been no studies of a large number of PCB patients because of its rarity. Objective: To elucidate the clinical profiles of PCB patients. Methods: Patients presenting progressive weakness predominant in the pharynx, neck, and upper limbs within 4 weeks were recruited. Clinical features, antecedent infections, and anti-ganglioside antibodies were investigated. Results: Diagnoses of 100 patients were pure PCB (n = 32), deep tendon reflexes-preserved PCB (n = 8), GBS overlap (n = 29), Fisher syndrome (FS) overlap (n = 26), and Bickerstaff's brainstem encephalitis overlap (n = 5). Intratracheal intubation was significantly frequent in GBS overlap than in pure PCB (48% vs. 19%, p = 0.01). Serological test showed that frequent agents of antecedent infections in PCB were (31%) and cytomegalovirus (6%), as in typical

Background: Whether Bickerstaff's brainstem encephalitis (BBE) is a distinct disease entity or a variant of Fisher syndrome (FS) remains unclear because there have been no clinical studies of a large number of patients with FS and BBE. Objective: To elucidate the nosological relationship between FS and BBE. Methods: Patients presenting progressive, relatively symmetric external ophthalmoplegia and ataxia within 4 weeks were recruited. Clinical features were analyzed. Antecedent infections and anti-ganglioside antibodies were investigated. Results: Recruited patients were classified into typical FS (n = 267) and typical BBE associated with consciousness disturbance (n = 34). Age distribution, sex ratio, and frequency of antecedent illness did not differ significantly between FS and BBE. The BBE patients frequently required endotracheal intubation, and showed CSF pleocytosis. In FS and BBE, serologic evidence of recent Campylobacter jejuni (21 and 23%) was common as well as that of recent Haemophilus influenzae infection (8

Department of Neurology, Dokkyo Medical University School of Medicine, Kitakobayashi 880, Mibu, Shimotsuga, Tochigi 321-0293, Japan; bInstitute of Brain and Blood Vessels, Mihara Memorial Hospital, Otamachi 366, Isesaki, Gunma 372-0006, Japan

[Objective] There are few reports to relate to long-term prognosis of Guillain–Barre syndrome (GBS) and Fischer syndrome (FS) because of good convalescent of patients with these diseases. However, some patients have severe sequences and sometimes they have a recurrent GBS. To understand the condition of a patient with long-term rehabilitation and of a recurrence cases, we evaluated long-term prognosis. [Method] We investigate a clinical symptom, neurological examination, nerve conduction study and anti-ganglioside antibody titers of the cases with GBS or FS after the onset from 2 to 17 years. [Results] Only one case out of 26 cases that we were able to evaluate this time, showed serious sequences who had the axonal type of GBS which developed in China. Most cases recovered for everyday life even in the most severe patients group who needed mechanical ventilation. However, the neurological examination showed slight abnormality, and there were many cases to have abnormality for H wave in particular by nerve conduction study. Some cases have a positive of an anti-ganglioside antibody for a long term without the exacerbation and the recurrence of a symptom. [Conclusions] Generally it is thought that an axonal type is poor prognosis, but it is important that there were many good cases as for the long-term convalescence.

Department of Neurology, Dokkyo Medical University School of Medicine, Tochigi, Japan

Abstracts and 6%): Anti-GQ1b IgG antibodies were highly positive (81 and 62%): MRI showed brain abnormality (3 and 23%): Abnormal EEG findings were noted (25 and 57%). Conclusions: Common antecedent infections as well as common autoantibodies in this large study gave conclusive evidence that both conditions are closely related and form a continuous spectrum. Brain abnormality was detected in FS, although the frequency was lower than in BBE. BBE could be positioned as a CNS subtype of FS associated with consciousness disturbance. PP16-13

A bedside prognostic scoring system for Guillain–Barré syndrome R. van Koningsvelda, E. W. Steyerbergb, P. A. van Doornc, B. C. Jacobsa,c Departments of aNeurology, bPublic Health, cImmunology; Erasmus Medical Center, Rotterdam, The Netherlands Objectives. Guillain–Barré syndrome (GBS) is an immune-mediated polyneuropathy with a highly variable clinical course and outcome. Predicting outcome is important for counselling and identifying high-risk groups to guide future trials. In this study a bedside scoring system was developed and validated to predict in an early disease phase the long term prognosis, i.e. the inability to walk independently at six months. Methods. The derivation set included 388 GBS patients from two Dutch trials and one Dutch pilot study. Sixteen potential prognostic factors were tested univariately. A scoring system was developed based on the regression coefficients of these variables in a multivariable logistic regression model. Model performance was quantified regarding discrimination (area under receiver operating curve (ROC)) and calibration (graphically). Internal validation was performed by bootstrap resampling. Results. Several factors had a significant prognostic value in univariate analysis, but the 3 key predictive variables in multivariate analysis were age, preceding diarrhoea and GBS disability grade at two weeks after admission. The scoring system ranged from 0 to 7, including three categories for age (≤40, 40–60, >60), two for diarrhoea (absent/present) and five for GBS disability grade at two weeks. The predictions showed a high association with observed outcomes and very good discriminative ability (area under ROC 0.85). The scoring system is currently validated in another large set of GBS patients. Conclusion. The scoring system based on three simple bedside clinical characteristics present at two weeks after admission accurately predicts the inability to walk independently at six months in patients with GBS. PP16-14

Spectrum of neurological diseases associated with antibodies to minor gangliosides GM1b and GalNAc–GD1a M. Tatsumotoa, M. Kogaa, M. Gilbertb, M. Odakaa, K. Hirataa, S. Kuwabarac and N. Yukia a

Department of Neurology, Dokkyo University School of Medicine, Tochigi, Japan; bInstitute for Biological Sciences, National Research Council of Canada, Canada; and cDepartment of Neurology, Chiba University School of Medicine, Chiba, Japan

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chromatography. Results: The clinical diagnoses for 456 anti-GM1bpositive patients were Guillain–Barré syndrome (GBS; 71%), atypical GBS with preserved deep tendon reflexes (12%), Fisher syndrome (10%), Bickerstaff's brainstem encephalitis (2%), ataxic GBS (2%), acute ophthalmoparesis (1%). For 193 anti-GalNAc–GD1a-positive patients, the diagnoses were GBS (70%), atypical GBS (16%), Fisher syndrome (10%), and Bickerstaff's brainstem encephalitis (3%). Of the patients with GBS or atypical GBS, 28% of the 381 anti-GM1b-positive and 31% of the 166 antiGalNAc–GD1a-positive patients had neither anti-GM1 nor anti-GD1a antibodies. Of those patients with Fisher syndrome, Bickerstaff's brainstem encephalitis, ataxic GBS, or acute ophthalmoparesis, 33% of the 67 antiGM1b-positive and 52% of the 25 anti-GalNAc–GD1a-positive patients had no anti-GQ1b antibodies. Purity of the isolated GM1b was confirmed by thin-layer chromatography. Conclusion: Autoantibodies against GM1b and GalNAc–GD1a are associated with GBS, Fisher syndrome, and related conditions. These antibodies should provide useful serological markers for identifying patients who have atypical GBS with preserved deep tendon reflexes, ataxic GBS, Bickerstaff's brainstem encephalitis, or acute ophthalmoparesis, especially for those who have no antibodies to GM1, GD1a, or GQ1b. PP16-15

In vitro acute conduction block of rat sciatic nerve induced by anti-GM1 antibody positive sera Y. Kuzumoto, M. Shioyama, H. Hara and S. Kusunoki Department of Neurology Kinki University School of Medicine, OsakaSayama, Japan Antiganglioside antibodies which are frequently present in the sera from patients with autoimmune neuropathies, may be involved in the pathogenetic mechanism. But the precise effects of the antibodies on the peripheral nerves are still unknown. We studied the effects of the anti-GM1 antibodies on the peripheral nerve electrophysiologically. The rat sciatic nerve was put on the recording chamber. Human and rabbit sera with high titer of anti GM1 antibody were applied to a pool located between the proximal and distal stimulation electrodes in the chamber. We stimulated the nerve at the proximal and distal stimulation sites and recorded the compound nerve action potentials (CNAP) at the more distally-situated recording site. Proximally-stimulated CNAP (P) was compared with distally-stimulated one (D). Human and rabbit anti-GM1 antibody-positive sera caused significant reduction of P/D ratio, which suggested conduction block, within a few hours. Human and rabbit control sera did not cause such reduction. Human and rabbit anti GM1 anti-sera that was preheated at 56 °C for 30 min did not produce the conduction block. It was suggested that anti GM1-positive human and rabbit sera produce conduction block in rat sciatic nerve in vitro. The complement system may play an important role for the development of the conduction block. PP16-16

Anti-GM1 IgG antibodies mediate complement-dependent destruction of sodium channel clusters in peripheral motor nerve fibers K. Susukia,b, M. N. Rasbandb, K. Tohyamac, K. Hirataa, H. Babad, N. Yukia

Objectives: To clarify the neurological disease spectrum associated with autoantibodies against minor gangliosides GM1b and GalNAc–GD1a and to develop a new method for GM1b preparation. Methods: IgG and IgM antibody reactivity against gangliosides GM1, GM1b, GD1a, GalNAc– GD1a, and GQ1b in sera from 6800 consecutive patients who had various neurological conditions. Clinical features and their correlation with those autoantibodies were analyzed. GM1b was produced from asialo-GM1 by means of Campylobacter jejuni sialyltransferase and isolated by column

a

Department of Neurology, Dokkyo Medical University School of Medicine, Tochigi, Japan; bDepartment of Neuroscience, University of Connecticut Health Center, Farmington, USA; cThe Center for Electron Microscopy and Bio-Imaging Research, Central Research Laboratories, Iwate Medical University, Morioka, Japan; dDepartment of Molecular Neurobiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan

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Abstracts

In axonal Guillain–Barré syndrome, anti-ganglioside antibodies could cause peripheral motor nerve conduction failure. The earliest pathological change is lengthening of the nodes of Ranvier in the ventral roots. Saltatory conduction along the myelinated axons depends on the accumulation of voltage-gated sodium (Nav) channels at nodes. Paranodal axo-glial junctions flanking nodes are important for the appropriate ion channel clustering. Nav channel dysfunction may cause the initial motor nerve conduction block in axonal Guillain–Barré syndrome. To prove this, we examined nodal lesions in an animal model. By immunizing with gangliosides, rabbits produced anti-GM1 IgG antibodies and developed acute limb weakness with a monophasic course. Electron micrographs showed abnormally lengthened nodes in ventral roots from the paralyzed rabbits. Immunohistochemical studies revealed deposit of IgG, C3 component of complement, and membrane attack complex on the nodes in ventral roots from the rabbits during the acute phase of the illness. GM1 epitope was revealed at the IgGdeposited lesions. The number of affected nodes with complement deposit was reduced during the recovery phase. Clusters of Nav channels, βIV spectrin (a cytoskeletal protein at nodes), and contactin-associated protein (Caspr; axonal component of the paranodal junction) were altered or disappeared on the abnormally lengthened nodes with complement deposit. Anti-GM1 antibodies mediate complement-dependent destruction of nodes of Ranvier and disrupt Nav channel clusters. These findings confirm the hypothesis that Nav channel dysfunction is responsible for the initial nerve conduction block and consequent motor weakness in axonal Guillain–Barré syndrome. PP16-17

Higher sensitivity in detecting anti-myelin associated glycoprotein (MAG) IgM autoantibody when using antigen purified from human brain T. D. Jaskowskia, H. E. Princed, R. W. Greera, C. M. Litwina,b and H. R. Hilla,b,c a

Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology and the bDepartment of Pathology, c Pediatrics and Medicine, University of Utah School of Medicine, Salt Lake City, Utah, USA; dFocus Diagnostics, Cypress, California, USA

Objective: To compare MAG IgM results in patients suspected of having autoimmune neuropathy using different methods and different sources of antigen. Materials and methods: Seventy-five sera from patients suspected of having autoimmune neuropathy, and sent to Focus Diagnostics for MAG IgM testing (Western blot; human MAG), were split, de-identified and sent blind to ARUP Laboratories. These 75 sera were assessed for the following IgM autoantibodies at ARUP Laboratories: anti-myelin by indirect fluorescent antibody (IFA) using primate peripheral nerve; anti-MAG by Western blot using antigen purified from primate; anti-MAG by enzyme immunoassay (EIA) using antigen purified from human brain; anti-sulfate-3glucuronyl paragloboside (SGPG) by EIA using antigen purified from bovine. Results: When compared to the MAG IgM results obtained by Focus Diagnostics (Western blot using human MAG), the following percent agreement, sensitivity and specificity were calculated for the MAG IgM Western blot (primate MAG) and MAG IgM EIA (human MAG): 85.3, 72.5, 100.0 and 89.3, 97.5, 80.0, respectively. There were 11 of 40 MAG Western blot (human MAG) positive sera that were negative by MAG Western blot using antigen purified from primate. Nine of these 11 (81.8%) sera were also positive by MAG EIA and myelin IFA. There were 7 of 35 MAG Western blot (human MAG) negative sera that were positive by MAG EIA. Six of these 7 (85.7%) sera also gave positive results for SGPG by EIA. Conclusion: When assessing sera from patients suspected of having autoimmune neuropathy for MAG IgM, these data suggest a higher sensitivity in methods using human MAG. Moreover, these data suggest that

EIA may be more sensitive than Western blot in detecting IgM autoantibody against human MAG. PP16-18

Epidemiology of chronic inflammatory demyelinating polyneuropathy in Japan Masahiro Iijima1,4, Yumiko Oki1,4, Keiko Mori1,4, Haruki Koike1,4, Naoki Hattori1,4, Akiko Tamakoshi2, Kimiyoshi Arimura3,4, Gen Sobue1,4 1

Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Department of Preventive Medicine/Biostatistics and Medical Decision Making, Nagoya University Graduate School of Medicine, Nagoya, Japan; 3Department of Neurology and Geriatrics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; 4The Refractory Neuropathy Research Committee in Japan., Tokyo, Japan Chronic inflammatory demyelinating polyneuropathy (CIDP) is characterized as an acquired and symmetrical motor and sensory disturbance brought about by immune-mediated segmental demyelination in peripheral nerves. Although this epidemiologic information has already been reported in Europe and Oceania, it is little known in Asia. We aimed to clarify the crucial prevalence and incidence rate in Japan and performed a large epidemiologic study throughout Japan. This study covered the whole Japanese population and investigated the number of CIDP patients in combination with each sex, age group (adult and juvenile) and geographic distribution. We investigated the prevalence and incidence number of patients in general and university hospitals with either a department of neurology, internal medicine or pediatrics during November 2004 to August 2005. The number of patients was reported by all hospitals with a department of neurology and by randomly extracted hospitals with internal medicine and pediatrics corresponding to its number of beds. The number of Japanese patients used for the calculation of the prevalence and incidence rate follows the national census of 2005. As a result, we clarify that the annual prevalence rate is 1.91/100,000 and the incidence rate is 0.57/100,000 in Japan, which is very similar to the results in other counties. Additionally, both the prevalence and incidence rate are almost the same in each prefecture throughout Japan. In conclusion, the epidemiology of CIDP shows universality not only from the aspect of ethnic distribution but also geographical distribution worldwide as well as in Japan. PP16-19

CD1E polymorphism in chronic inflammatory demyelinating polyneuropathy (CIDP) G. Lee, H. Bang, D. Latov, T. H. Brannagan, R. L. Chin, H.W. Sander and N. Latov Cornell University Weill Medical College, New York, USA Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) is an autoimmune disease that targets the myelin sheaths in peripheral nerve. The target antigen is unknown and there is no linkage to the major histocompatibility (MHC) locus that is involved in protein antigen presentation. As glycolipid antigens are implicated in other autoimmune neuropathies, we investigated CIDP patients for DNA polymorphisms in CD1A and CD1E, members of the CD1 gene family involved in the processing and presentation of lipid antigens to T-cells. Patients with IgM monoclonal gammopathy (MG) associated neuropathy were also investigated. We found that patients with CIDP (n = 23) were more than twice as likely to have the CD1E 01/01 genotype than normal controls (n = 28) (p < 0.01). A negative association between MG neuropathy (n = 15) and

Abstracts CD1E 01/01 was also observed, but this finding did not reach statistical significance (p = 0.018). The association with CD1E 01/01 implicates immune reactivity to a lipid antigen in the pathogenesis of CIDP. Furthermore, the gene product of CD1E 01 may have a higher affinity for a putative CIDP antigen than other forms of CD1E, or homozygosity may result in higher expression of 01, facilitating antigen presentation, and enhancing susceptibility to disease. This points to the CD1E pathway as a potential target for therapeutics.

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Peripheral neuropathy in patients with multiple sclerosis Megumi Takeuchi1, Kohei Ota2, Takashi Ohashi1, Yuko Shimizu1, Atsuko Mochizuki1, Yumi Kimura1, Mutsumi Iijima1, Yoko Masuda1, Megumi Ubano 1, Makoto Iwata1 1

PP16-20

Diagnostic criteria and nationwide survey of Crow–Fukase syndrome in Japan K. Arimuraa, T. Hashiguchib, M. Osamea, and J. Kirac Neurology and Geriatrics and bLaboratory and Vascular Medicine, Kagoshima University, cNeurology, Kyushu University, Japan

a

We previously reported that VEGF was extremely elevated in Crow– Fukase syndrome (CFS) and was a key molecule of the pathogenesis of CFS. According to our clinical and experimental studies, we proposed the diagnostic criteria of CFS. To confirm the usefulness of the diagnostic criteria, we conducted a Japanese nationwide survey of CFS in 2003. Furthermore, we investigated the efficacy of various treatments for CFS. The estimated number of patients was about 340. Detailed medical questionnaires of 84 CFS patients were obtained (males: 49, females: 35, mean age: 57 years old). Mean duration of illness was 6.7 years, which suggested an improvement in the prognosis of this syndrome. However, mean annual frequency of hospitalization (2.7 times) and absence of any patient showing complete recovery indicated that CFS is still an intractable disease. Polyneuropathy (100%), edema (97%), and skin lesions (95%) were the most frequent manifestations, followed by organomegaly (79%) and endocrine abnormalities (78%). On laboratory examination, M-protein (89%) and high serum VEGF (98%) were detected in these patients. These findings surprisingly resembled the previous Japanese nationwide survey conducted by Nakanishi in 1982, but differed from the report of Dispensieri et al. (2003) done in the USA. According to Dispenzieri's diagnostic criteria, 11% of patients were not correctly diagnosed. Our proposed diagnostic criteria, which includes serum VEGF, correctly diagnosed CFS in all patients. Treatment by peripheral blood stem cell transplantation with high dose chemotherapy was increased and most responded well compared to conventional therapy. PP16-21

Highly concentrated vascular endothelial growth factor (VEGF) in platelets in Crow–Fukase (POEMS) syndrome T. Hashiguchia, K. Arimurab, M. Osameb and I. Maruyamaa a

Laboratory and Vascular Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan; bDepartment of Neurology and Geriatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan We report a marked difference in concentration of vascular endothelial growth factor (VEGF) between serum and plasma in patients with Crow– Fukase syndrome (CFS). The serum/plasma VEGF levels in four CFS patients were 8634/152, 5203/176, 3724/127 and 868/13 pg/ml, respectively. It is predicted that in CFS, local VEGF concentration is markedly elevated by aggregation of platelets containing excessive VEGF and their adhesion to vascular walls, resulting in excessive physiological activities of VEGF, which are considered deeply involved in the characteristic clinical manifestations observed in CFS. Our findings provide important information for developing more effective therapeutic trials.

Department of Neurology, Tokyo Women's Medical University, Tokyo, Japan; 2Tokyo University of Science, Tokyo, Japan

Background: Multiple sclerosis (MS) is characterized by the demyelination in the central nervous system, but a few studies have demonstrated the involvement of peripheral nerves. The prevalence of peripheral neuropathy in MS remains controversial. The aim of this study is to perform an electrophysiological evaluation of the peripheral nervous system in patients with MS. Methods: Subjects were 28 patients with MS, 7 males and 21 females and aged between 21 and 51 (mean 34). Motor conduction study in median, ulnar, peroneal and tibial nerves as well as sensory conduction in median, ulnar, and sural nerves were evaluated. Results: There was electrophysiological evidence of peripheral nervous system lesions in at least one nerve in 9 patients. Low compound muscle action potential was noted in 5 patients. Low sensory nerve action potential was noted in 1 patient. Slowed motor and/or sensory conduction velocity or prolonged distal latency was noted in 2 patients. Two patients had a history of Guillain– Barré syndrome. One patient was diagnosed as having chronic inflammatory demyelinating polyradiculoneuropathy as well as MS. Conclusions: This study demonstrated the peripheral nerve involvement in patients with MS. Further investigations are needed to clarify the immunological background affecting both central nervous system and peripheral nerves. PP16-23

Association of multiple sclerosis and chronic inflammatory demyelinating polyneuropathy: Possible common pathogenesis of central and peripheral demyelination S. Misawa, S. Kuwabara, M. Mori, S. Hayakawa and T. Hattori Department of Neurology, Chiba University School of Medicine, Chiba, Japan Objectives: To investigate the frequency of MS-CIDP association, and its correlation with clinical profiles. Methods: Consecutive 59 patients with MS and 61 patients with CIDP were studied. Results: Clinical evident association of the two disorders was found in the two patients (3% of the MS patients and 3% of the CIDP patients). In the two patients, MS preceded CIDP by 2 years and activity of the two diseases appeared to be parallel. Nerve conduction studies showed demyelination in the intermediate nerve segments without involvement of the distal nerve terminals. Another MS patient presented subclinical nerve conduction abnormalities suggestive of intermediate demyelination. Conclusions: The frequency of MS and CIDP association is approximately 3%. CIDP associated with MS is characterized by multifocal demyelination in the nerve trunk and no involvement of the distal nerve terminals. Myelin components common to the central and peripheral nervous systems may be target molecules in such cases. PP16-24

Clinical features of the neuropathy with high titer IgM antibody against gangliosides with disialosyl residue M. Sada, MD1, M. Hirakawa, MD, PhD1,2, Y. Mitsui, MD, PhD1 and S. Kusunoki, MD, PhD1 1

Department of Neurology, Kinki University School of Medicine, 377-2 Ohno-Higashi Osakasayama, Osaka 589-8511, Japan; 2Department of

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Abstracts

Neurology, School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-8655, Japan IgM paraproteins that bind to gangliosides with disialosyl residue have been reported to be specifically associated with sensory ataxic neuropathy. Among the sera in which we have examined antiganglioside antibody titer, there are 5 sera with high IgM antibody titer against GD3, GD1b, GT1b and GQ1b. We investigated the clinical feature, clinical course and response to the treatment of each case. Four of five patients had IgM antibody against all 4 gangliosides and had sensory ataxic neuropathy. The remaining one patient, who had IgM with high titier against GD3, GT1b and GQ1b but with very low titer against GD1b, had sensori-motor neuropathy with no ataxia. Patients were treated either by intravenous immunoglobulin (IVIg), corticosteroids, plasma exchange, immunoadsorption or immunosuppressants. All patients responded temporarily to those treatments. Two patients had IgM M-protein, and the other 2 patients had malignant hematological diseases. The treatment to the hematological diseases in the latter 2 patients also improved neuropathy. The importance of the anti-GD1b reactivity in the pathogenesis of sensory ataxic neuropathy was indicated. Several treatments were effective but the effects were temporary. PP16-25

Therapeutic effects of single administration of Cyclophosphamide in rat experimental autoimmune neuritis, a preclinical model of autoimmune peripheral neuropathies Gabriele Dati1, Ferdinando Nicoletti2, Roberto Di Marco2, Katia Mangano2, Beatrice Greco1, Paola Zaratin1 RBM Serono, Ivrea, Italy; Via Ribes, 1, 10010, Turin, Italy; 2Department of Biomedical Sciences, University of Catania, Italy; Via Androne, 83, 95124, Catania, Italy

1

High dose immunosuppressive therapies have been used to treat autoimmune diseases, including autoimmune peripheral neuropathies. These agents have been used to treat some cases of Guillain–Barré syndrome (GBS), Chronic Inflammatory Demyelinating Polyradiculoneuropathy (CIDP) and multifocal motor neuropathy (MMN), resistant to usual treatments, such as plasma exchange (PE) and intravenous immunoglobulin (IVIg). The long term use of high doses of these immunosuppressants is however limited due to their severe side effects: bone marrow suppression, hair loss, upset stomach and development of hemorrhagic cystitis. In order to assess the beneficial effect of short term treatment with lower doses of the alkylating immunosuppressive drug Cyclophosphamide (CY) on the development of a preclinical model of GBS and CIDP, the experimental autoimmune neuritis (EAN) in Lewis rats, a single dose of CY was administered to the animals 14 days post-immunization. The data obtained in our experiment shows that a single low dose administration of CY was highly effective in preventing clinical signs of EAN. These data suggest that CY deserves further consideration for its use in the treatment of both acute and chronic autoimmune peripheral neuropathies. In particular, since the acute phase of human GBS is usually ranging between 2 and 4 weeks, it is possible that one single administration of the drug would be sufficient to influence the course of the disease, thus avoiding the well known side and toxic effects provoked by prolonged exposure to CY. PP16-26

Intravenous immunoglobulin therapy for Miller Fisher syndrome Mori M, Kuwabara S, Hayakawa S, Misawa S, Tamura N and Hattori T Department of Neurology, Chiba University School of Medicine, Chiba, Japan

Background: There is no evidence whether intravenous immunoglobulin (IVIg) has beneficial effects for Miller Fisher syndrome (MFS) patients. Methods: Clinical recovery was reviewed in a consecutive series of 92 patients with MFS. The time-course of recovery was compared in patients treated with IVIg (n = 28), those treated with plasmapheresis (PP; n = 23), and those who did not receive these immune modulating treatments (n = 41). Results: The times required to start amelioration of external ophthalmoplegia and ataxia were slightly shorter for the IVIg group (ophthalmoplegia; median, 12.0 days; p = 0.04, ataxia; median 8.0 days, p = 0.027) than the control (ophthalmoplegia; 13.5 days, ataxia 10.0 days) groups, but the times required to disappear external ophthalmoplegia and ataxia were similar between the IVIg and the control and between the PP group and the control. One year after onset, 89 (96%) of the 92 patients had no ophthalmoparesis and ataxia irrespective of receiving immunomodulating treatment. Conclusions: In MFS, IVIg slightly hastens the amelioration of ophthalmoplegia, but does not affect the outcome, presumably because of a good natural recovery. PP16-27

Efficacy of immunoglobulin and steroid to variant type Guillain–Barré syndrome: In vitro study of the MEPP frequency increase M. Kishia, K. Kumamotob, K. Saitaa, K. Shioyaa, M. Nakazatoc, T. Kuriharad Department of Neurology and b Department of Internal Medicine, Miyazakihigashi National Hospital, Miyazaki, Japan; cIIIrd Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan; dDepartment of Neurology, Toho University Ohashi Medical Center, Tokyo, Japan

a

Variant type Guillain–Barré syndrome (V-GBS) patient's serum has miniature endplate potential (MEPP) frequency increase (MFI; so-called α-latrotoxin like effect) with anti-ganglioside antibodies, and these correlate to clinical symptoms. Mechanism of MFI is speculated as complement dependent nerve terminal destruction by autoantibody. The autoantibody and complement make membrane attack complex and create a lot of pores on the nerve terminal membrane. At first, the Ca+ inflow and concentrate gain increase MEPP frequency. Thereafter, too much Ca+ concentration gain injures the nerve terminal, resulting in disappearance of MEPP at the end. We were able to confirm this process on the mouse diaphragm, and we tried immunoglobulin (Ig) and prednisolone (PSL) treatment to this in vitro model. We chose a V-GBS patient whose serum had high MFI and anti-ganglioside antibodies. We prepared a C57BL/6J mouse diaphragm preparation bathed in the patient's serum (0.5 cc) and recorded MEPP using conventional grass microelectrode technique. We added immunoglobulin (Ig), prednisolone (PSL) or both of them to the patient's serum. There was complete suppression of MFI when we used 10 mg/ml Ig or 1 mM PSL. 2 or 5 mg/ml Ig did not completely suppress MFI. MEPP frequency increase was not suppressed by 0.1 mM PSL, but the appearance of the MEPP lost fibers was suppressed. One mg/ml Ig and 0.01 mM PSL did not influence the patient's serum action. Although the Ig was effective when the standard clinical dosage was used, 10 times the standard clinical dosage was necessary for make PSL effective. To confirm the synergistic effect of Ig and PSL, we used patient's serum containing the 0.1 mM PSL and 2 or 5 mg/ml Ig. However, there was no complete MFI suppression. We could not confirm usefulness of the Ig and PSL combination therapy by this in vitro model.

Abstracts

Poster Session 17: MG and inflammatory myopathies

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PP17-01

Studies on myoid cells in hyperplastic myasthenia gravis Kamo1, H. Tomoyasu4, M. Kobayashi1, N. Sakuragawa1, S. Inoue5, T. Hagiwara5, T. Kaneda5 and A. Kikuchi1,2,3

those of the control rats. The morphological changes, AChR-declustering muscle fibers have appeared in the MuSK-immunized rat (EDL, white muscle > soleus, red muscle). Conclusions: Our results suggest that anti-MuSK antibodies in MuSKimmunized rat may down-regulate MuSK and AChR, lead to the morphological changes in motor endplates. Further investigations will draw the conclusion whether MuSK Ab may be pathogenic or not.

1

Kitasato University, Sagamihara, Japan; 2The University of Tokyo, Tokyo, Japan; 3Institute of Animal Reproduction, Kasumigaura, Japan; 4Japan Red Cross Hospial, Tokyo, Japan; 5Nationall Hospital Organization, Nagoya Med Center, Nagoya, Japan We have previously reported that thymic myoid cells are possibly stemmed from plural sources and that some myoid cell clones produce growth factors, including an 80-kDa haemopoietic factor (80-K) and 100kDa haemopoietic biglycan (100-K), besides AChR. Those factor-producing cells were also abundantly detected in the hyperplastic myasthenic thymi (HMG-thymi). AChR (+) cells and myogenin (+) cells were in a subset of them, suggesting that 100-K (+)/AChR (+) double positive cells are matured myoid cells, whereas 100-K (+)/AChR (−) single positive cells their precursors. Numeral increases of myoid cells occurred at precursor stages, followed by concurrent production of large amounts of AChR and growth factors that may affect the AChR specific breakage of the anergic state. 80K/100-K appear to be a useful hallmark for understanding the immunological state of H-MG-thymi. However, little is known about the mechanisms of development of myoid cells that produce those factors, although recent studies on skeletal muscle development suggested that biglycan, decorin and other factors such as Pax3/7 are differentially expressed in embryonic and fetal myoblasts. In the present report, we use different types of myoid cells cloned from mice and rats, and hyperplastic thymic tissues to correlate between the regulatory factors and the growth factor production. We found Pax3 and 100-K in H-MG-thymi and marked immunostimulatory roles of 80-K/100-K purified from H-MG-thymi. We are currently testing the role of regulatory factors of myoid cells and their immunostimulatory products for better understanding of MG. PP17-02

Experimental autoimmune MuSK antibody-induced myasthenia gravis Masakatsu Motomura, M.D.a, Hirokazu Shiraishi, M.D.a, Katsumi Eguchi, M.D.a, Jiro Nakano, Ph.D.b, Toshiro Yoshimura, M.D.b, Takayasu Fukudome, M.D.c, Hidenori Matsuo, M.D.c, Mitsuhiro Tsujihata, M.D.d a

First Department of Internal Medicine, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan; b Health Sciences, Nagasaki University, Nagasaki, Japan; cDepartment.of Neurology Nagasaki Medical Center of Neurology, dNagasaki Kita Hospital, Nagasaki, Japan Heading: The aim of this study is to investigate the pathogenesis and characteristics of muscle-specific tyrosine kinase (MuSK) antibody in experimental autoimmune myasthenia gravis (EAMG) rat. Background: Some of generalized seronegative MG patients have antibodies (Ab) against MuSK. But it is not clear how the Ab cause myasthenic symptoms. We investigated the pathogenesis of MuSK Ab using rat. Methods: Female Lewis rats, aged 8 weeks were inoculated twice in multiple intradermal sites, either MuSK protein (10–100 μg) in complete Freund's adjuvant (CFA) and Bordetella pertussis as co-adjuvant or the controls. Soluble mouse MuSK-6xHis protein was purified from HEK293. Results: The weights of MuSK-immunized rats were lower than those of the control rats. The titers of antibodies to MuSK raised markedly compared with those of the controls. The electrophysiological examination using diaphragm was negative. The quantity of AChR and MuSK at the endplates of limb muscles were reduced in MuSK-immunized rats, compared with

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The thymic theme of acetylcholinesterase splice variants in myasthenia gravis Adi Gilboa-Geffen1, Paul Lacoste2, Lilach Soreq1, Geraldine Clairac2, Frederique Truffault2, Iftach Shaked1, Hermona Soreq1,3 and Sonia BerrihAknin2 The Hebrew University of Jerusalem, Israel; 2CNRS UMR8078-CCML, LePlessis Robinson, Paris, France

1

Myasthenia gravis (MG) is an autoimmune disease mediated by antibodies towards nicotinic acetylcholine receptors. Thymic abnormalities such as hyperplasia and thymoma are found in most MG patients. Here, we report that modified acetylcholinesterase (AChE) gene expression and properties are involved in these thymic pathologies. Microarray analysis demonstrated attenuation of the general decrease in gene expression, which is characteristic of adult human thymus, in patients with MG-induced thymic hyperplasia. This was accompanied by reduced expression of DNA replication-associated transcripts and by reduced levels of the “synaptic” AChE-S mRNA. Thymic extracts from MG patients further presented elevated amounts but reduced hydrolytic activities of the MG-induced AChE-R variant as compared with adult controls, accompanied by translocation of both AChE-R and the signaling protein kinase PKC-βII from the cytoplasmic to the membrane-associated fraction. To explore possible causal association of the modified AChE splicing pattern with thymic composition and function, we employed up-regulation of specific thymic AChE variants TgR mice overexpressing human (h)AChE-R and TgS mice overexpressing the membrane-associated (h)AChE-S variant. Both showed smaller thymic medulla than strain-matched FVB/N controls, suggesting differentiation imbalances. Intriguingly, TgS mice displayed smaller thymus weight, CD3+-high mature cell fractions with higher rate of spontaneous apoptosis as compared with controls. In contrast, TgR mice showed heavier thymuses, selectively enlarged CD4+CD8+ fractions and protection of cultured thymocytes from apoptosis. Together, the overexpression of AChE-R variant could explain the higher number of thymocytes in both the transgenic model and the human thymus associated with myasthenia gravis. PP17-04

The thymus is a source of B-cell survival factors – APRIL and BAFF – in myasthenia gravis Mathula Thangarajh1,2, Thomas Masterman1, Lars Helgeland3, Uroš Rot4, Malin V. Jonsson5, Geir Egil Eide6, Ritva Pirskanen7, Roland Jonsson2, Jan Hillert1 1

Neurology Division, Department of Clinical Neuroscience, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; 2Broegelmann Research Laboratory, The Gade Institute, University of Bergen, Bergen, Norway; 3Department of Pathology, Haukeland University Hospital, University of Bergen, Bergen, Norway; 4Department of Neurology, Medical Centre, Ljubljana, Slovenia; 5Department of Oral Sciences–Oral Pathology and Forensic Odontology, University of Bergen, Bergen, Norway; 6Center for Clinical Research, Haukeland University Hospital and Section for Epidemiology and Medical Statistics, Department of Public Health and Primary Health Care, University of Bergen, Bergen, Norway; 7Neurology

Abstracts Department, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden Background: The thymus is important for the selection and maturation of T cells. In disease, the thymus is capable of establishing a microenvironment conducive to the survival of auto-reactive T and B cells. A classical example is myasthenia gravis (MG) – an antibody-mediated autoimmune disease – in which anti-acetylcholine-receptor (AchR) antibodies are present. In MG, the thymus shows ectopic germinal centers (GC) that are also a source of antibodies. To understand the molecular mechanisms involved in the accumulation of B cells within myasthenic thymi, we evaluated the thymic expression of recently characterized B-cell survival factors, APRIL and BAFF. Methods: We used immunohistochemistry and two-color immunofluorescence to study protein expression of APRIL, BAFF and their receptorsBAFF-R, BCMA and CD138. Using ELISA, we quantified plasma APRIL and BAFF levels. Additionally, BAFF-R, BCMA and TACI mRNA levels in peripheral-blood B cells were quantified in patients and controls by real-time PCR. Results: APRIL and BAFF were abundantly expressed by thymic macrophages. BAFF-R expression was limited to B cells within the GC and was undetectable in thymoma. BCMA-positive plasma cells were abundant in patients with hyperplastic thymi. CD138-postive plasma cells were restricted to the perivascular space and thymic parenchyma. Soluble levels of APRIL and BAFF were not increased in patients compared to controls and did not correlate to anti-AchR levels. Levels of mRNA of BAFF-R, BCMA and TACI were comparable between patients and controls. Conclusions: Intrathymic expression of APRIL, BAFF and their receptors may support survival of autoreactive cells and contribute to MG pathogenesis. PP17-05

CD21 and Ki-67 expression in thymus of patients with myasthenia gravis and their correlation with clinical parameters J. Ikedaad, T. Nishisakab, T. Kohriyamac, H. Tokinobua, E. Imamuraa, M. Matsumotoc a

Department of Neurology, Hiroshima Prefectural Hospital, Hiroshima, Japan; bDepartment of Pathology and Laboratory Medicine, Hiroshima Prefectural Hospital, Hiroshima, Japan; cDepartment of Clinical Neuroscience and Therapeutics, Hiroshima University, Graduate School of Biomedical science, Hiroshima, Japan; d Department of Neurology, Suiseikai Kajikawa Hospital, Hiroshima, Japan Objectives: The aim of this study was to evaluate the area of thymic tissue per section (% of the total tissue), Ki-67 (cell proliferation marker) and CD21 (mature B cells, follicular dendritic cells) expression in thymus of patients with myasthenia gravis (MG) and to determine the potential correlation with clinical parameters. Methods: We analyzed thymic compartment in 17 patients (5 males and 12 females; mean age 51.9 years) with MG who underwent extended thymectomy. Two of the patients were in Myasthenia Gravis Foundation of America (MGFA) class I, 6 were in class II, and 9 were in class III. Results: Microscopic examination of thymus showed thymic hyperplasia in 8 (mean age 38.6 years), thymoma with hyperplasia in 3 (52.7 years), thymoma with involution in 3 (69.0 years) and involution in 3 (69.3 years). The area of thymic tissue and numbers of the germinal center (GC) for section expressed Ki-67 and CD21 had inverted correlation with age of onset. GC frequencies (GC/ mm2 of thymic tissue) with CD21 expression were significantly higher in hyperplasia (with or without thymoma) group than in involution (with or without thymoma) group. The area of the Ki-67 expression had positive correlation with MGFA class. Conclusion: Age of onset is the most strongest inverse factor of the area of thymic tissue and the numbers of the GC expressed Ki-67 and CD21. Increased proliferation in thymus accompany advanced disease stage.

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Autoimmunization in thymus in myasthenia gravis patients without detectable acetylcholine receptor or muscle specific kinase autoantibodies M I Leitea, M Jonesb, K Micklemb, A Vincenta, N Willcoxa a

Neurosciences Group, Department of Clinical Neurology, Weatherall Institute of Molecular Medicine and bNuffield Department of Clinical and Laboratory Sciences, John Radcliffe Hospital, University of Oxford, UK

The thymus is implicated in the pathogenesis of early onset-myasthenia gravis (EOMG) associated with AChR autoantibodies (AChRAb+). It shows hyperplasia of medullary thymic epithelial cells (mTEC) and lymph nodelike infiltrates with germinal centres (GC). AChR is expressed by the rare muscle-like thymic myoid cells, and isolated AChR subunits by TEC. In generalised MG without detectable AChR antibodies, some have antibodies to muscle specific kinase (MuSKAb+). The thymus is less well studied but is reportedly ‘normal-for-age’ or atrophic, and clinical benefits of thymectomy are unproven. We have re-assessed thymic samples from 30 AChRAb−/ MuSKAb− and 14 AChRAb−/MuSKAb+ patients, comparing them with 23 EOMG (AChRAb+) and 11 non-myasthenic adults by immunohistochemistry and double immunofluorescence staining of paraffin-embedded sections. Overall, we found few abnormalities in the MuSKAb+ thymi, only 4/14 showing small infiltrates. In ∼ 70% of the AChRAb−/MuSKAb− MG samples, however, we found substantial infiltrates (cytokeratin− areas in thymic medulla), though GC (CD20+/CD35+CD21+) numbers and sizes were modest (Leite et al., 2005). We now find that in AChRAb+ and nearly 50% of the AChRAb−/MuSKAb− MG thymi, there are myoid cells exposed to the infiltrates. In both groups, some mTECs and many ‘exposed’ myoid cells, particularly near GCs, show deposits of activated C3c (and sometimes C9) complement components. These results further implicate both mTECS in priming specific helper T cells and myoid cells in GC formation with autoantibody diversification/perpetuation. The similarities between AChRAb+ and AChRAb−/MuSKAb− MG thymi strongly suggest that they belong to the same spectrum. PP17-07

In vitro immune response in myasthenia gravis patients with anti-MuSK or anti-AChR antibodies V. Yılmaza,c, Ö. Güngör-Tunçerb, Y. G. Parmanb, P. Serdaroğlub, F. Deymeerb and G. Saruhan-Direskenelic I.U. a Institute of Experimental Medicine Research, Department of Immunology; Istanbul Medical Faculty Departments of bNeurology and c Physiology, Istanbul, Turkey Myasthenia gravis (MG) is an autoimmune disease caused by antibodymediated effector mechanisms. In 85–90% of MG patients, anti-acetylcholine receptor (AChR) antibodies (Ab) are present, whereas a subgroup of the remaining 10–15% of the patients have Ab against muscle-specific kinase (MuSK). The production of the Ab is mainly regulated by cytokines derived from Th1 or Th2 type cells. In this study, 60 MG patients and 23 healthy controls (HC) are included. Among the patients, 17 had anti-MuSK Ab and 18 had anti-AChR Ab only whereas 25 did not have any of these Ab. IFN-γ, IL-13 and IL-10 levels are measured in supernatants of Torpedo californica AChR or anti-CD3 stimulated peripheral blood mononuclear cells (PBMC) of patients and controls by ELISA. Spontaneous IL-10 secretion levels were higher (40.7 vs. 9.6 pg/ml, p < 0.001) and IL-13 levels were lower (1.1 vs. 3.8 pg/ml, p < 0.001) in MG than in HC. Cytokine levels upon anti-CD3 or AChR stimulations did not differ between groups. Anti-CD3 stimulated PBMC secreted higher levels of IFN-γ in anti-MuSK (−) (908.4 pg/ml, p = 0.001) and in anti-AChR (+) (2464 pg/ml, p < 0.001) subgroups than in anti-MuSK (+) patients (11.4 pg/

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Abstracts

ml). Anti-MuSK (−) subgroup had also higher IFN-γ levels compared to anti-MuSK (+) subgroup when stimulated with AChR (2.4 vs. 0.0 pg/ml, p = 0.03). IL-13 and IL-10 secretion levels and proliferative responses were not different between disease subgroups. Although regulatory cytokine activity is detected in MG spontaneously, differences between disease subgroups according to Ab are implicated by these results. This study was supported by the Brain Research Society (BAD) and Istanbul University Research Fund. PP17-08

Autoreactive T cells mediate NK cell degeneration in autoimmune disease Ruolan Liu1, Luc Van Kaer2, Antonio La Cava3, Mary Price1, Denise I Campagnolo1, Mary Collins4, Deborah A Young4, Timothy L Vollmer1 and Fu-Dong Shi1 1

Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ85013, USA; 2Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN37232, USA; 3 David Geffen School of Medicine, University of California, Los Angeles, CA90095, USA; 4Wyeth Research, Cambridge, MA02140, USA Emerging evidence indicates that NK cells play an important and complex role in autoimmune disease. Humans with autoimmune diseases often have reduced NK cell numbers and compromised NK cell functions. Mechanisms underlying this NK cell degeneration are not known. Here we show that, in an experimental model of human autoimmune myasthenia gravis induced by a self-antigen, the acetylcholine receptor, NK cells undergo expansion during the initiation of autoimmunity, followed by significant degeneration concomitant with the establishment of the autoreactive T cell response. NK cell degeneration was mediated by interleukin-21 derived from autoreactive CD4+ T cells. NK cell degeneration may signify the transition from innate immune triggering to emergence of autoreactive T cells, serving as a means evolved by the immune system to control excessive autoimmunity. PP17-09

Comparative analysis of two different assays in the detection of anti-MuSK antibodies

the support of radioisotopic facilities. In our opinion, IBBA is useful for research purpose to identify new antigens, while IPA provides a simple procedure to detect anti-MuSK abs in clinical routine. PP17-10

A new sandwich ELISA method to detect antibodies against native form of ryanodine receptor Takahiro Maruta1,2, Hiroaki Yoshikawa2,3, Yutaka Furukawa2, Kazuo Iwasa2, Masahito Yamada2 Department of Neurology, Keiju Medical Center, Nanao, Japan; 2Department of Neurology and Neurobiology of Aging, Graduate School of Medicine, Kanazawa University, Kanazawa, Japan; 3Health Service Center, Kanazawa University, Kanazawa, Japan

1

The purpose of this study is to design a new measurement method to detect serum autoantibodies against the ryanodine receptor (RyR) of striate muscle. With this new method of sandwich ELISA using the complex of anti-RyR monoclonal antibody and native form of RyR from rabbit striate muscle as the target antigen, we measured titers of the autoantibody to RyR from serum samples of 80 patients with myasthenia gravis (MG), 34 disease controls and 23 normal subjects. When titers that exceeded the average + 3 × standard deviation of normal samples was defined as the positive, 31 patients with MG (39%) turned to be positive. On the other hand, all samples from the disease and normal controls were negative. The positive rate was significantly high in aged and in thymoma-associated patients. Furthermore, it tended to be high in female and in patients with the generalized MG. In spite of no detection of antibodies to RyR from the samples without antibodies to acetylcholine receptor, there was no correlation with these two autoantibodies. These results suggest that our new method is useful to detect anti-RyR antibodies and is expected as the method to recognize the pathophysiological condition of each myasthenic patient with this antibody. PP17-11

Elevation of IL-12 p40 and its antibody is a specific serum marker for myasthenia gravis with thymoma

M. Marinoa, F. Scuderia, C. Provenzanoa, A. Evolib and E. Bartoccionia.

Hiroaki Yoshikawaa,c, Katsuaki Satob, Shigeki Edahiroc, Yutaka Furukawac, Takahiro Marutac, Kazuo Iwasac, Masahito Yamadac, Hideto Watanabed, Masaharu Takamorie

a

a

Anti-MuSK autoantibodies have been associated to “seronegative Myasthenia Gravis” (SNMG). Until now these antibodies have been detected by means of two different techniques: a home-made immunoblotting of biotinylated proteins assay (IBBA) and a radioimmunoprecipitation assay (IPA). The IBBA uses biotin-labeled membrane proteins from TE671 cells while the IPA uses 125-I-labeled purified recombinant antigen, the results being given as nmoles 125-I-MuSK precipitated per liter of serum. We compared home made IBBA with commercial IPA (RSR Limited). testing 130 sera: 20 from healthy blood donors, 65 from SNMG patients, and 45.from other neurological pathologies. No anti-MuSK abs were detected in control sera by both assays, while they were detected in 38 out of 65 (58.5%) SNMG patients tested by IPA, and in 42 out of 65 (64.6%) tested by IBBA. Five out of 65 sera tested were IBBA(+) and IPA(−), while only one serum was IBBA(−) and IPA(+); the concordance between the two tests was 95.4%, with comparable values of sensitivity and specificity. IBBA is a qualitative test, which detects several immunoreactivities against native muscle antigens; it is complex, not easy to standardize for routine and requires the support of cell culture facilities. IPA is a quantitative test, which employs a recombinant antigen (extracellular fragment, 1–490 aa) and allows to titre the anti-MuSK antibodies in patient sera, however, it requires

We measured the serum levels of various cytokines, and their antibodies in patients with myasthenia gravis (MG). The levels of IL-12 p40 were significantly elevated in 53.3% of MG patients with thymoma (n = 15). Titers of anti-IL-12 p40 antibodies were significantly elevated in 40.0% of MG with thymoma. We also tested antibodies against IL-12 p70 and found that it was significantly elevated in both MG with thymoma and thymoma without MG. There was a significant correlation between IL-12 p40 and its autoantibodies in thymoma with MG; however, there was no correlation in MG with LFH, MG with normal thymus or thymoma without MG. The titers of IL-12 p40 did not have a significant correlation with the histopathology of thymoma according to the WHO classification. Furthermore, the levels of IL-12 p40, anti-IL-12 p40 antibodies and anti-IL-12 p70 antibodies did not correlate with serum titers of anti-acetylcholine receptor (AChR) antibodies, clinical severity, or ratio of 201Tl accumulation in the thymus. Thymectomy followed by prednisolone administration for up to four years significantly

Institute of General Pathology and bDepartment of Neurosciences, Catholic University, Rome, Italy

Health Service Center Pathophysiology Laboratory Neurology and Neuroimmunology, Graduate School of Natural Science and Technology Kanazawa University, bDepartment of Pathology II Kanazawa Medical University, cDepartment of Neurology and Neurobiology of Aging, Graduate School of Medicine Kanazawa University, dInstitute for Molecular Science of Medicine, Aichi Medical University and eNeurological Center, Kanazawa Nishi Hospital, Kanazawa, Japan

Abstracts decreased the anti-AChR antibody levels and the IL-12 p40 levels, but not the anti-IL-12 p40 antibodies nor anti-IL-12 p70 antibodies. These observations suggest that both IL-12 p40 and its autoantibodies are associated with the immunopathology of MG with thymoma, which has distinct immunopathology compared to other MG. PP17-12

Long term effect of infrasternal mediastinoscopic thymectomy in myasthenia gravis H. Muraia, A. Uchiyamab, F. Meia, N. Shia, M. Kojimab and J. Kiraa Department of Neurology; bDepartment of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

a

Background: Endoscopic thymectomy is now widely performed because of relatively low invasion and less stress for patients. However its long term effect has not been sufficiently evaluated. Objective: To assess the long term effect of infrasternal mediastinoscopic thymectomy (IMT) and compare with transsternal thymectomy (TT) in myasthenia gravis (MG) patients. Methods: Among 41 MG patients who underwent thymectomy in our institute between January 1997 and December 2000, 16 patients who received IMT and 17 who received TT were enrolled in this study. QMG score and antiacetylcholine receptor antibody (anti-AChR) titer were evaluated before and 5 years after the surgery. Intracellular cytokine of peripheral blood CD4− positive T cells were analyzed by flowcytometry in 9 patients who underwent IMT, and IFNγ/IL-4 ratio was calculated. Results: After 5 years, QMG score reduced from 6.9 to 1.9 (p < 0.01) in IMT group and 7.5 to 2.5 (p < 0.01) in TT group. Anti-AChR titer reduced from 68.6 to 40.0 (p = 0.048) in IMT group and 201.1 to 106.2 (p = 0.017) in TT group. IFNγ/ IL-4 ratio increased from 9.40 to 22.28 (p = 0.039). Conclusion: IMT is as effective as TT, and it is considered to be suitable for MG. IMT seemed to alleviate MG symptoms by correcting the Th2 shift. PP17-13

Effect of thymectomy for late onset non-thymomatous myasthenia gravis N. Kawaguchia, S. Kuwabaraa, Y. Nemotoa, H. Takahashia, T. Fukutakea, K. Arimurab, M. Osameb, and T. Hattoria a

Department of Neurology, Chiba University, Graduate School of Medicine, Chiba, Japan; bThe Third Department of Internal Medicine, Kagoshima University, Kagoshima, Japan

Objectives: To investigate whether thymectomy is beneficial for nonthymomatous myasthenia gravis patients with mild generalized weakness and onset age >50 years. Patients and methods: Clinical course and outcomes were compared between 10 patients receiving thymectomy and 12 without it. Results: At the end of follow-up (mean 9.6 years after onset), the thymectomized group showed the greater percentage of clinical remission (no symptoms with or without medication; 50% versus 17%; p = 0.11), and the less frequency of the presence of generalized symptoms (30% versus 75%; p < 0.05). Conclusions: Thymectomy appears to be effective for late onset, nonthymomatous patients with generalized myasthenia gravis. PP17-14

Is excitation–contraction (E–C) coupling is impaired in myasthenia gravis? S. Kuwabara, M. Nakata, N. Kawaguchi, S. Misawa, M. Mori, and T. Hattori Department of Neurology, Chiba University School of Medicine, Chiba, Japan

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Objectives: To investigate whether E–C coupling of muscle is impaired in patients with generalized myasthenia gravis (MG). Methods: A total of 51 patients with MG were studied. Compound muscle action potentials (CMAPs) of the abductor pollicis brevis, and movement-related potentials using an accelerometer placed at the thumb tip were recorded after median nerve stimulation at the wrist. The E–C coupling time (ECCT) was estimated by a latency difference between CMAP and movement-related response. Antibodies against ryanodine receptor (RyR) and muscle specific receptor tyrosine kinase (MuSK), as well as acetylcholine receptor, were measured by immunoassays. Results: Compared with age-matched normal controls (n = 31), the mean ECCT was longer in patients with MG (p = 0.01). ECCT had no significant correlation with clinical severity, disease duration, and anti-RyR or MuSK antibodies, whereas the mean ECCT was shorter for patients treated with FK506 than for those not receiving this treatment (p = 0.03). Conclusions: In MG, E–C coupling appears to be impaired, possibly contributing to additional muscle weakness. FK506 may facilitate E–C coupling in MG patients. PP17-15

Tacrolimus treatment in elderly patients with myasthenia gravis H. Takahashi, N. Kawaguchi, Y. Nemoto, T. Hattori Department of Neurology, Graduate School of Medicine, Chiba University, Chiba, Japan Tacrolimus is an immunomodulating agent being widely used for patients with myasthenia gravis (MG), rheumatoid arthritis, and organ transplantation in Japan. Tacrolimus have effect to inhibit antiacetylcholine receptor (AChR) antibody production through suppressing helper B cell, and help uptake corticosteroid into nucleus. We evaluate the effect and adverse effect of tacrolimus for elderly patients with MG.