The Effect of Natalizumab Therapy On Immune Surveillance of the Central Nervous System in Multiple Sclerosis Patients

Abstracts each TCRm included ELISA and flow cytometric analysis, demonstrating specific binding to recombinant HLA-A2 protein and T2 cells loaded with relevant peptide. The TCR mimics were then used to confirm and directly quantify specific peptide-MHC complexes on mDCs. These mAbs recognize peptide presented in the context of MHC class I molecules, thus mimicking the binding specificity of TCRs. TCRm staining of vaccine-treated DCs showed detection and quantification of each peptide-HLA epitope. We next compared antibody staining intensity to CTL activation threshold and showed that the level of specific peptideHLA complexes correlates directly to CTL activation. These findings suggest that TCRms may be important tools for determining the potency of DC-based vaccines. doi:10.1016/j.clim.2006.04.418

OR.114. Protective Anti-Helicobacter Immunity Is Induced with CTA1-DD Adjuvant By Intranasal Immunization and Is Mediated By Th1 Cells. Ali Akhiani,1 Anneli Stensson,2 Karin Schon,2 Nils Lycke.2 1 Clinical Bacteriology, Goteborg University, Goteborg, Sweden; 2Clinical Immunology, Goteborg University, Goteborg, Sweden. Safe and efficacious adjuvants are much needed to facilitate the development of mucosal vaccines. Here, we have asked whether our non-toxic vaccine adjuvant, CTA1DD, can enhance protective immunity against Helicobacter pylori-infection. Intranasal immunizations with H. pylori lysate together with CTA1-DD-adjuvant induced significant protection in C57Bl/6 mice, almost as strong as similar immunizations using cholera toxin (CT)-adjuvant. Protection remained strong even at 8 weeks post-challenge and the bacterial colonization was reduced by 20-fold compared to lysate-immunized controls. Although CTA1-DD was designed to bind to B-cells, AMT-mice developed significant, but lower, level of protection following immunization. Intranasal immunizations with CT-adjuvant in C57Bl/6 mice resulted in the development of severe post-immunization gastritis at 2 and 8 weeks post-challenge, whereas the degree of gastritis was substantially lower in the CTA1DD-immunized mice. Protection induced by both CTA1-DDand CT-adjuvant was associated with a strong local infiltration of CD4+ T-cells in the gastric mucosa and recall responses to specific Ag elicited substantial IFN-gamma production, indicating Th1-dominance. These findings clearly demonstrate that CTA1-DD-adjuvant is a promising candidate to be further exploited in the development of a mucosal vaccine against H. pylori-infection. doi:10.1016/j.clim.2006.04.419

OR.115. Functional Mimic of CD40L Homotrimers By C3-Symetric Peptide Scaffolds. Sylvie Fournel,1 Sebastien Wieckowski,1 Weimin Sun,1 Nathalie Trouche,1 Helene Dumortier,1 Alberto Bianco,1 Olivier Chaloin,1 Mohammed Habib,2 Jean-Christophe

S47 Peter,1 Pascal Schneider,3 Bernard Vray,2 Johan Hoebeke,1 Gilles Guichard.1 1UPR9021, IBMC, Strasbourg, France; 2laboratoire d’immunologie experimentale, Universite Libre de Bruxelles, Brussels, Belgium; 3Department of Biochemsitry, University of Lausanne, Epalinges, Switzerland. Interaction between CD40, a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily, and its ligand CD40L, is essential for the development of humoral and cellular immune responses. Selective blockade or activation of this pathway provide the ground for the development of new treatments against immunologically baseddiseases and malignancies. Like other members of the TNF superfamily, CD40L monomers self-assemble around a three-fold symmetry axis to form non-covalent homotrimers that can each bind three receptor molecules. We have developed mimetics of CD40L by integrating 3-fold symmetry as a design principle(1). We reasoned that such trimeric architectures besides providing the correct geometry for receptor trimerization, could also achieve tight binding to CD40 with receptor-binding elements of modest surface areas. Molecules built by anchoring a short, linear CD40binding motif onto rigid macrocyclic cores were found by surface plasmon resonance to interact with CD40, and to compete with the binding of CD40L homotrimers. Like soluble CD40L, these small molecules (3-kDa) induced apoptosis of murine and human lymphoma cell lines and stimulated the in vitro maturation of a murine dendritic cell line. CD40 mimetics alone did not induce proliferation of murine B-cells, but like soluble CD40L synergized with an agonist anti-CD40 antibody or with IL-4. Moreover, we have shown that CD40L mimetics promote control of parasitemia by enhancing CD8+ cells producing interferon-g during experimental Trypanosoma cruzi infection. Such architectures based on rigid C3-symmetrical cores may thus represent a general approach to mimic homotrimers of the TNF superfamily. (1) S. Fournel, S. Wieckowski, W. Sun, N. Trouche, H. Dumortier, A. Bianco, O. Chaloin, M. Habib, J.-C. Peter, P. Schneider, B. Vray, R. E. Toes, R. Offringa, C. J. M. Melief, J. Hoebeke, G. Guichard. Nature Chem. Biol. 2005 1, 377-382. doi:10.1016/j.clim.2006.04.420

OR.116. The Effect of Natalizumab Therapy On Immune Surveillance of the Central Nervous System in Multiple Sclerosis Patients. Olaf Stuve,1,6 Christina Marra,2,6 Keith Jerome,3,6 Linda Cook,3,6 Petra Cravens,1,6 Sabine Cepok,4,6 Elliot Frohman,1,6 Theodore Phillips,5,6 Gabriele Arendt,4,6 Bernhard Hemmer,4,6 Nancy Monson,1,6 Michael Racke.1,6 1 Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX; 2Neurology, University of Washington, Seattle, WA; 3Laboratory Medicine, University of Washington, Seattle, WA; 4Neurology, Heinrich Heine University, Dusseldorf, Germany; 5Multiple Sclerosis Center at Texas Neurology, Baylor University Medical Center, Dallas, TX; 6Center for Immunology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX.

S48 Objective: To test whether treatment of multiple sclerosis (MS) with natalizumab, an antibody against VLA-4, interferes with central nervous system immune surveillance, leukocyte cell numbers and cellular phenotypes in cerebrospinal fluid (CSF) and peripheral blood (PB). Background: Natalizumab 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 blood were analyzed 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 significantly lower in natalizumabtreated 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: These data suggest that a low CSF CD4:CD8 ratio in natalizumab-treated patients may confer an increased risk of developing PML in these patients.

Abstracts were not sustained, but were recreated at each subsequent infusion. Notably, the magnitude of the infusion effect varied significantly across patients yet was remarkably stable in individual patients, over multiple infusions. Conclusions: We provide proof-of-concept that in vivo therapy diminishes VLA-4 functional expression and migratory capacity of immune cells. Natalizumab likely has different effects on trafficking of distinct cell subsets and can modulate their in vivo activation thresholds. The standard regimen does not fully block VLA-4 function, nor are biological effects sustained through the dosing interval. Individualized response profiles may prove to be of predictive value if future treatment and monitoring become possible. doi:10.1016/j.clim.2006.04.422

OR.118. Anti-Rheumatic and HMGB1 Blocking Effects of Oxaliplatin. Therese Ostberg,1 Karin Palmblad,1 Heidi Wahamaa,2 Maria Shoshan,3 Michael Lotze,4 Ulf Andersson,1 Helena Erlandsson Harris.2 1Department of Woman and Child health, Karolinska Institutet, Stockholm, Sweden; 2Department of Medicine, Karolinska Institutet, Stockholm, Sweden; 3 Department of Oncology Pathology, Karolinska Institutet, Stockholm, Sweden; 4Molecular Medicine Institute, University of Pittsburgh, Pittsburgh, PA.

doi:10.1016/j.clim.2006.04.421

OR.117. Prospective Monitoring of in Vivo Biological Effects of Natalizumab (AVLA-4, TysabriR R) in Patients with MS. Amit Bar-Or, Masaaki Niino, Marie-Lune Simard, Caroline Bodner, Sudabeh Alatab, Alyson Fournier, Dawn Gano, Ho Jin Kim. Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada. Background: Following approval for MS therapy, natalizumab was put on hold when several patients developed JC virus CNS reactivation. Any future use is predicated on gaining insights into the drug’s mechanisms of efficacy and toxicity in patients. Methods: blood was drawn prior to and after monthly infusions, over a 14-month period. VLA-4 expression was tracked on immune cell subsets by FACS, and migratory capacity, activation thresholds and cytokine profiles of immune cells were evaluated. Results: Pre-infusion VLA-4 expression was significantly higher in CD8 T-cells compared to CD4 T-cells (p b 0.0002), and higher yet on monocytes and B-cells. Natalizumab infusions diminished available VLA-4 on circulating T-cells (p = 0.004), more so than B-cells (p = 0.026) or monocytes (p = 0.032). Infusions significantly decreased migratory capacities of immune cells (p = 0.026), which correlated well with change in VLA-4 expression (r = 0.78; p b 0.04) in the same samples. In vivo natalizumab also modulated proliferative and cytokine responses of immune cells (p = 0.003). Effects of individual infusions

High mobility group box chromosomal protein 1 (HMGB1) is a nuclear protein present in all eukaryotic cells. In activated cells of myeloid origin HMGB1 can be translocated to the cytosol, followed by extracellulary release. Once released, HMGB1 can act as a cytokine, stimulating TNF, IL-1, and IL-6 production. We have previously demonstrated that HMGB1 is an important mediator in the pathogenesis of arthritis and that blockade of HMGB1 has beneficial effects. Oxaliplatin is a novel platinum based cytostatic drug with beneficial effects in cancer therapy. When administrated, oxaliplatin enters the cell and forms Pt-DNA adducts. These adducts can be recognized by HMGB1 which firmly binds to these structures of distorted DNA. We hypothesised that treatment with oxaliplatin could block the release of HMGB1 and thus be a potential anti-inflammatory and anti-rheumatic drug. Collagen-induced arthritis was provoked in DBA/1 mice by immunization with collagen type II. Mice were treated with single or double injections of oxaliplatin and the development of clinical arthritis was monitored. Further, in an HMGB1-ELISpot assay we investigated whether the addition of oxaliplatin to macrophage-like RAW cell cultures could inhibit the release of HMGB1. Mice treated with oxaliplatin once at the onset of clinical arthritis had significantly suppressed arthritis development for a period of 1 week. An additional injection of oxaliplatin after 1 week could further suppress disease development. By the ELISpot assay we could demonstrate that non-toxic concentrations of oxaliplatin suppressed the release of HMGB1 from activated RAW cells by half compared to control cultures. Our results indicate that oxaliplatin block HMGB1 release and can be used for downregulation of the