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Differential gene expression in multiple sclerosis lesions identified using cDNA microarrays
P o s t e r A b s t r a c t s / J o u r n a l o f N e u r o i m m u n o l o g y 9 0 (1998) 1 3 - 1 0 5
71
395
398
Differential Gene Expression in Multiple Sclerosis Lesions Identified
Clinical Outcome for Patients with Traumatic Brain Injury Correlates with the Quota Between the Anti-inflammatory 1L-lra
Using eDNA Microarrays K.G. Becker, LCG, NHGR1,NIH, USA,L.W. Whitney, NIB, N1NDS,NIH, USA, M.L. Bittner, LCG, NHGRI,N1H, USA,H.F. McFarland, W.E. Biddison, NtB, NINDS, NIH, USA,J.M. Trent, LCG, NHGRI,NIH, USA
and t h e P r o - i n f l a m m a t o r y 1 L - 6 i n L i q v o r E. Danielsson. UniversityofUppsala,Sweden, K. Alheim, S. Tingsborg, Stockholm University,Sweden,P. Enblad, L. Persson, Universityof Uppsala, Sweden,T. Bart fai, Stockholm University,Sweden
The pathogenesis of the demyelinating process in multiple sclerosis (MS) is characterized by a complex pattern of gene expression. Thus far, few genes that potentially contribute to tbe pathogenesis of MS bave been identified. Using cDNA microarray technology, we have performed preliminary experiments in MS specimens monitoring the expression panem of over 5000 human genes. Total RNA for microarray hybridization was obtained from samples of human brains from patients with MS, comparing acute, chronic-active, and chronic plaques; to adjacent normal white matter. Differentially expressed genes included: TNF-c~ receptor, IL-15 receptor, fibronectin, IRF-2, c-Rel, high density lipoprotein binding protein, apolipoprotein-J, SOD-1, STAT-3, 7B-integrin, PLP, TGF-[32, and lkB, among others. In addition we are combining laser capture microdissection, in situ-hybridization, and immunohistochemistry to identify the various cell types within MS plaques in which gene expression changes occur. This highly parallel approach should be valuable in identifying genes contributing to the complex processes found in MS and other neurological disorders.
Inflammatory mechanisms have been proposed as mediators of secoodary, deleterious, events in traumatic brain injury (TBI). We were interested in determining whether an increased level of anti-inflammatory mediators in TIll has an advantageous effect on the clinical outcome in severe TBI. IL-6 was used as a pro-inflammatory marker and IL-Ira as an anti-inflammatory marker. Liqvor samples fi-om intraventricular drainage from eight TB[ patients were collected daily and analysed for IL-6 and IL-lra using EI,ISA. Intraventrieutar catheters were inserted one to three days after the injury and remained in place for up to five days. All patients were observed in a neurointanaive care unit The Glasgow outcome scale (GOS) at three months was used as final outcome. Of the six patients that survived, two became dependent and four made a good or excellent recovery. The two patients that died had a severe clinical status initially and the interleukin quota (1L-Ira/1L-6) were low or attenuated during the observation period. Of the patients that were initially conmtus¢ but survived, the cytokine index rose significantly during the first week after the injury. The eytokine quota determined in liquor seemed to be predictive of clinical outcome following severe TBI, where a low index predicts a deterioration of the patient's health and even deaft~ while a high index paints to a positive clinical outcome. From our studies on eight patients we hereby propose that an increased quota IL-Ira/IL-6 attenuates the inflammatory response in severe TBI in adult patients and hence improves the clinical outcome.
396
399
Anti-MOG Antibody Response in Multiple Sclerosis:
E n h a n c e d E x p r e s s i o n o f M C P - 1 , R a n t e s a n d MIP-IoL,[3 in Demyelinating Multiple Sclerosis Lesions and HumanAdnit
A First Step Towards an Immunopathological Subtype of MS ? tL Egg, M. Reindl, E. Dilitz, E Deisenhammer, T. Berg_er, Universityoflnnsbruck,
Austria
Astrocyte Cultures J.P. van der Voorn, AcademicHospital, VrijeUniversiteU,The Netherlands, J. Tekstra, I L H J . Beelen, Vroe Universiteit,TheNetherlands, C.P. Tensen, P. van der Valk, C.J.A. De Groot. AcademicHospital, Vrye Universiteit,TheNetherlands
Recent evidence given from experimental animal models and neuropathologieal studies suggests that in a subtype of MS-pathology myelin destruction may be caused by demyelinating antibodies directed against the myelin oligodeodrocyte glycoprotein (MOG). Therefore we decided to investigate whether there is a corresponding immunopatbological subtype of MS-patients characterized by a prominent anti-MOG autoantibody response. Using the method described in our abstract ..Anti-MOG antibody responses in multiple sclerosis (l): reliable testsystems provide reliable results" we analyzed serum- and cerebrospinal fluid (CSF) samples of 140 patients with clinically definite MS as well as 100 patients with other neurological diseases. The incidence of anti-MOG antibodies in our MS-patients was about 40% with antibody lives up to 1:50.0(}0.However, the incidence of these antibodies alone can not answer the question whether the presence of anti-MOG antibodies is due to a primary or secondary autoimmune response. Therefore we have characterized the duration of disease and the clinical course of these MS-patients (relapsthg-remiuing; secondary progressive; primary progressive; optic neuritis only). Furthermore we have analyzed serial serum probes in order to investigate the intraindividual behavior of anti-MOG antibodies os a marker of disease activity and treatment dependency. However, anti-MOG antibodies were also present in low frequencies in other neurological diseases, but only MS-patients bad a high spaeific and high-titre antiMOG autoimmune response. In conclusion our data suggest that the occurrence of anti-MOG antibodies in MS-patients may indicate a distinct immunpathological subtype of MS, urging further investigations about the biological activity and immunological relevance of these antibodies.
The pathology of multiple sclerosis (MS) is characterized by breakdown of the blood-brain-barrier, accompanied by infiltration of macrophages and T cells into the central nervous system (CNS). The migration o f these cells into the CNS parenchyma may be partly regulated by chemokines. In MS lesions o f different cellular activity the distribution and cellular localization of the fichemokines MCP-I, RANTES, M I P - l a / ~ have been studied. In addition, we determined the m R N A expression and protein secretion of MCP-1 and RANTES by cultured haman adult astrocytes using northern blot analysis and ELISA. MCP-1 and RANTES inununoreactivity was exclusively found in reactive astrocytes in active and chronic active MS lesions, whereas M I P - l a and [3 was strongly expressed in phagocytic macrophages and less abundant in reactive astrocytes. Furthermore, M C P - I and to a lesser extent RANTES were expressed at m R N A level and produced by human adult astrocytes hi vitro after stimulation with proinflammatory mediatorso These results suggest a role for fS-chemokines in the recruitment and activation of inflammatory cells, and thereby contributing to the development of MS lesions.
397
400 RANTESand MCP-1 Secretion by Mononudear Cells of Patients
Localization of NFkBin
Multiple Sclerosis Lesions: Implications
for Oligodendroeyte Damage B. Bonetti, C. Stegagno, G. Moretto, N. Rizzuto, B. Cannella, C.S. Paine,
with Multiple Sclerosis G. De Luea~ M. Reale, C. Iarlori, A. Lugaresi, P. Conti, D. Gambi, Universityof
Universityof Verona,Italy
Chieti,ltaly
Oligodendrocytes are a major target of autnimmune response in multiple sclerosis (MS) lesions. However, little is known about the mechanisms underlying their damage: in fact. despite their expression of pm-apoptotic molecules (e.g. Fas and tumor necrosis factor receptors), these cells do not undergo programmed cell death in MS plaques. O n the other hand, in addition to its apoptutic effect, it is known that TNFc~ can also generate sunrival signals through the activation and nuclear translecation of NFKB. Aim of this study was to investigate the expression and cellular localization o f NFr,.B proteins by immunohistochemistry on chrome active and silent MS lesions. We found no reactivity for NFr,.B in the absence of inflammation; in active lesions, either infiltrating lymphocytes and glial cells showed nuclear and\or cytoplasmic staining for NFr,B proteins. While positive fymphocytes and astrocvt,es were seen throughout the entire plaque, cells morphologically resembling oligodendrocytes with NFr,.B nuclear reactivity were mainly localized at the edge of active lesions and in the surrounding white matter. We hypothesize that activation of NFr, B. possibly TNFc~-mediated. may be a mechanism accounting for the absence of apoptusis on oligedandrocytes in MS lesions.
Chemokines axe a family of proinflamnmtory cytokines which induce chemotaxis and mediate monocyte-maerophage activation. Several experimental findings suggest their involvement in the modulation o f immune responses and their role in mediating leucocyte entry into CNS tissues during immune-mediated inflammation. MCP-I and R A N T E S production by unstimulated and PHA stimulated mononuclear cell cultures after 24 h was measured with commercially available ELISA kits (Endogen, Inc. Wobum, MA, USA). Twenty patients with definite multiple sclerosis (MS) (13 w o m e n , 7 men: range 28 - 41 yrs) and twenty health subjects served as control (CL) were studied. Ten patients were treated with 1FNbetalb since twelve months. Our preliminary results showed significantly enhanced amounts of RANTES and MCP-I in unstimulated and PHA stimulated monocytes cultures in both groups of patients compared to controls. Lower levels of spontaneous R A N T E S production were found in patients treated with IFNbetalb. These data suggest that chemokines play a role in MS pathogenesis and might be modulated by 1FNbeta.
71
395
398
Differential Gene Expression in Multiple Sclerosis Lesions Identified
Clinical Outcome for Patients with Traumatic Brain Injury Correlates with the Quota Between the Anti-inflammatory 1L-lra
Using eDNA Microarrays K.G. Becker, LCG, NHGR1,NIH, USA,L.W. Whitney, NIB, N1NDS,NIH, USA, M.L. Bittner, LCG, NHGRI,N1H, USA,H.F. McFarland, W.E. Biddison, NtB, NINDS, NIH, USA,J.M. Trent, LCG, NHGRI,NIH, USA
and t h e P r o - i n f l a m m a t o r y 1 L - 6 i n L i q v o r E. Danielsson. UniversityofUppsala,Sweden, K. Alheim, S. Tingsborg, Stockholm University,Sweden,P. Enblad, L. Persson, Universityof Uppsala, Sweden,T. Bart fai, Stockholm University,Sweden
The pathogenesis of the demyelinating process in multiple sclerosis (MS) is characterized by a complex pattern of gene expression. Thus far, few genes that potentially contribute to tbe pathogenesis of MS bave been identified. Using cDNA microarray technology, we have performed preliminary experiments in MS specimens monitoring the expression panem of over 5000 human genes. Total RNA for microarray hybridization was obtained from samples of human brains from patients with MS, comparing acute, chronic-active, and chronic plaques; to adjacent normal white matter. Differentially expressed genes included: TNF-c~ receptor, IL-15 receptor, fibronectin, IRF-2, c-Rel, high density lipoprotein binding protein, apolipoprotein-J, SOD-1, STAT-3, 7B-integrin, PLP, TGF-[32, and lkB, among others. In addition we are combining laser capture microdissection, in situ-hybridization, and immunohistochemistry to identify the various cell types within MS plaques in which gene expression changes occur. This highly parallel approach should be valuable in identifying genes contributing to the complex processes found in MS and other neurological disorders.
Inflammatory mechanisms have been proposed as mediators of secoodary, deleterious, events in traumatic brain injury (TBI). We were interested in determining whether an increased level of anti-inflammatory mediators in TIll has an advantageous effect on the clinical outcome in severe TBI. IL-6 was used as a pro-inflammatory marker and IL-Ira as an anti-inflammatory marker. Liqvor samples fi-om intraventricular drainage from eight TB[ patients were collected daily and analysed for IL-6 and IL-lra using EI,ISA. Intraventrieutar catheters were inserted one to three days after the injury and remained in place for up to five days. All patients were observed in a neurointanaive care unit The Glasgow outcome scale (GOS) at three months was used as final outcome. Of the six patients that survived, two became dependent and four made a good or excellent recovery. The two patients that died had a severe clinical status initially and the interleukin quota (1L-Ira/1L-6) were low or attenuated during the observation period. Of the patients that were initially conmtus¢ but survived, the cytokine index rose significantly during the first week after the injury. The eytokine quota determined in liquor seemed to be predictive of clinical outcome following severe TBI, where a low index predicts a deterioration of the patient's health and even deaft~ while a high index paints to a positive clinical outcome. From our studies on eight patients we hereby propose that an increased quota IL-Ira/IL-6 attenuates the inflammatory response in severe TBI in adult patients and hence improves the clinical outcome.
396
399
Anti-MOG Antibody Response in Multiple Sclerosis:
E n h a n c e d E x p r e s s i o n o f M C P - 1 , R a n t e s a n d MIP-IoL,[3 in Demyelinating Multiple Sclerosis Lesions and HumanAdnit
A First Step Towards an Immunopathological Subtype of MS ? tL Egg, M. Reindl, E. Dilitz, E Deisenhammer, T. Berg_er, Universityoflnnsbruck,
Austria
Astrocyte Cultures J.P. van der Voorn, AcademicHospital, VrijeUniversiteU,The Netherlands, J. Tekstra, I L H J . Beelen, Vroe Universiteit,TheNetherlands, C.P. Tensen, P. van der Valk, C.J.A. De Groot. AcademicHospital, Vrye Universiteit,TheNetherlands
Recent evidence given from experimental animal models and neuropathologieal studies suggests that in a subtype of MS-pathology myelin destruction may be caused by demyelinating antibodies directed against the myelin oligodeodrocyte glycoprotein (MOG). Therefore we decided to investigate whether there is a corresponding immunopatbological subtype of MS-patients characterized by a prominent anti-MOG autoantibody response. Using the method described in our abstract ..Anti-MOG antibody responses in multiple sclerosis (l): reliable testsystems provide reliable results" we analyzed serum- and cerebrospinal fluid (CSF) samples of 140 patients with clinically definite MS as well as 100 patients with other neurological diseases. The incidence of anti-MOG antibodies in our MS-patients was about 40% with antibody lives up to 1:50.0(}0.However, the incidence of these antibodies alone can not answer the question whether the presence of anti-MOG antibodies is due to a primary or secondary autoimmune response. Therefore we have characterized the duration of disease and the clinical course of these MS-patients (relapsthg-remiuing; secondary progressive; primary progressive; optic neuritis only). Furthermore we have analyzed serial serum probes in order to investigate the intraindividual behavior of anti-MOG antibodies os a marker of disease activity and treatment dependency. However, anti-MOG antibodies were also present in low frequencies in other neurological diseases, but only MS-patients bad a high spaeific and high-titre antiMOG autoimmune response. In conclusion our data suggest that the occurrence of anti-MOG antibodies in MS-patients may indicate a distinct immunpathological subtype of MS, urging further investigations about the biological activity and immunological relevance of these antibodies.
The pathology of multiple sclerosis (MS) is characterized by breakdown of the blood-brain-barrier, accompanied by infiltration of macrophages and T cells into the central nervous system (CNS). The migration o f these cells into the CNS parenchyma may be partly regulated by chemokines. In MS lesions o f different cellular activity the distribution and cellular localization of the fichemokines MCP-I, RANTES, M I P - l a / ~ have been studied. In addition, we determined the m R N A expression and protein secretion of MCP-1 and RANTES by cultured haman adult astrocytes using northern blot analysis and ELISA. MCP-1 and RANTES inununoreactivity was exclusively found in reactive astrocytes in active and chronic active MS lesions, whereas M I P - l a and [3 was strongly expressed in phagocytic macrophages and less abundant in reactive astrocytes. Furthermore, M C P - I and to a lesser extent RANTES were expressed at m R N A level and produced by human adult astrocytes hi vitro after stimulation with proinflammatory mediatorso These results suggest a role for fS-chemokines in the recruitment and activation of inflammatory cells, and thereby contributing to the development of MS lesions.
397
400 RANTESand MCP-1 Secretion by Mononudear Cells of Patients
Localization of NFkBin
Multiple Sclerosis Lesions: Implications
for Oligodendroeyte Damage B. Bonetti, C. Stegagno, G. Moretto, N. Rizzuto, B. Cannella, C.S. Paine,
with Multiple Sclerosis G. De Luea~ M. Reale, C. Iarlori, A. Lugaresi, P. Conti, D. Gambi, Universityof
Universityof Verona,Italy
Chieti,ltaly
Oligodendrocytes are a major target of autnimmune response in multiple sclerosis (MS) lesions. However, little is known about the mechanisms underlying their damage: in fact. despite their expression of pm-apoptotic molecules (e.g. Fas and tumor necrosis factor receptors), these cells do not undergo programmed cell death in MS plaques. O n the other hand, in addition to its apoptutic effect, it is known that TNFc~ can also generate sunrival signals through the activation and nuclear translecation of NFKB. Aim of this study was to investigate the expression and cellular localization o f NFr,.B proteins by immunohistochemistry on chrome active and silent MS lesions. We found no reactivity for NFr,.B in the absence of inflammation; in active lesions, either infiltrating lymphocytes and glial cells showed nuclear and\or cytoplasmic staining for NFr,B proteins. While positive fymphocytes and astrocvt,es were seen throughout the entire plaque, cells morphologically resembling oligodendrocytes with NFr,.B nuclear reactivity were mainly localized at the edge of active lesions and in the surrounding white matter. We hypothesize that activation of NFr, B. possibly TNFc~-mediated. may be a mechanism accounting for the absence of apoptusis on oligedandrocytes in MS lesions.
Chemokines axe a family of proinflamnmtory cytokines which induce chemotaxis and mediate monocyte-maerophage activation. Several experimental findings suggest their involvement in the modulation o f immune responses and their role in mediating leucocyte entry into CNS tissues during immune-mediated inflammation. MCP-I and R A N T E S production by unstimulated and PHA stimulated mononuclear cell cultures after 24 h was measured with commercially available ELISA kits (Endogen, Inc. Wobum, MA, USA). Twenty patients with definite multiple sclerosis (MS) (13 w o m e n , 7 men: range 28 - 41 yrs) and twenty health subjects served as control (CL) were studied. Ten patients were treated with 1FNbetalb since twelve months. Our preliminary results showed significantly enhanced amounts of RANTES and MCP-I in unstimulated and PHA stimulated monocytes cultures in both groups of patients compared to controls. Lower levels of spontaneous R A N T E S production were found in patients treated with IFNbetalb. These data suggest that chemokines play a role in MS pathogenesis and might be modulated by 1FNbeta.