- Email: [email protected]
Comparison of CSF findings and MRI with GD-DTPA in patients with acute optic neuritis (AON)
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p05.03 ANTIBODIESTO MYCOBACTERIAL65 kDa and 70 kDa HEATSHOCKPROTEINS IN NEURODEGENERATIVEDISEASES" U. Fiszer1, S. Fredrikson2, J. Gajda~, A. CztonkowskaI [1] Departmentof CarsbrovaecularDiseases, Institute of Psychiatryand Neurology, Warsaw, Poland.[2] Departmentof Neurology,KarolinskaInstitute,Huddinge,Sweden. Introduction: In Parkinson'sdisease(PD) and Wilson'5 disease (WD) immune abnormalities havebeen reported, including the elevatedy,5÷ T cells population. Hsp could be targets for yc5+ T lymphocytes. Increassd levels of antibodies to hsp have been found in severaldiseases. Mstsrllls lind Methods:We haveexaminedserum of 22 patients with PD, 18 patients with WD who demonstrated neurological symptoms and 15 patients with other non-inflammatory neurological diseases (OND), Antibodies were assessed using an enzyme-linked irnmunosorbent assay 5ccording to Danieli and all. (J. Autoimmunity, 1992, 5, 443), The plates weracoatedwith recombinantmycopacterial hsp 65 (Mycobacterium bovls BCG, batch MA-12A) and hsp 70 (Mycobacterium tuberculosum batch M'r70.6A), a gift from Dr. J. D. A. van Embden. Fbsults: Studies haveshown that mean absorbanca ratio of ssra from patients with WD waa significantly greaterthan that of sarafrom patientswith OND (tested against IgG anti-hsp 65 and IgG anti-hsp 70). There were no differences betwasn mean absorbance ratio of sara from patients with PD and OND. Conclusion,, It is difficult to establish the role of immunity to hsp in WD characterized by the toxic accumulation of copper in a number of organs, particularly tha liver and brain. Increased hap expression, could induce hsp immunity. Alternatively,the antibodies may bs presentas a conssquancs of prior infections. * This investigation received flnencisl support from "the UNOP/WorldBank/WHOSpecial programme for Research and Trainig in Tropical Diseases (TDR)"and 5"tare Committee for Scientific Research in Poland
W04.01 THE Dw2 ASSOCIATION IN HULTIPLE SCLEROSIS EXTENDS TO THE DRB5 L O C U S A Foode]ll,2,J Hillertl,3, 0 Olerup1,2 ICenter for BioTechnology,NOVUI'I,end 2Departments of Clinical Immunology and 3Neurology, Karolinska Institute,Huddinge, Sweden I n t r o d u c t i o n : It is well established that multiple sclerosis (MS) is associated with the HLA haplotype defined as Dw2 with cellular techniques and as DR15,D06 with serological typing methods The Dw2 haplotype has been specified with genomic typing techniques to be D R B I • I 5 O I , D O A I * O I O 2 , D Q B I * 0 6 0 2 . The aim of this study was to define the DRB5 allele(s) on the MS associated DR-DO haplotype. Materials sad Methods: 55 MSpatients and 32 healthy controls carrying the DR2 haplotype were subtyped for ORB5alleles using PCRamplification with sequence-specific primers (PCR-SSP). Results: All the 5SDR2-positiveMS patients and 30 of the 32 DR2-positive controls carried the DRB5*OIO! allele. Thus, the DRB5*OIOI association in MS is as strong as the association with other parts of the Dw2 haplotype, i.e. DRBI*I501, OQAI*OI02DQB l * 0 6 0 2 .
Conclusion: The class II haplotype associated with FIS extend to the ORB5 locus and can now be specified as DRB I * I501,DRBS~OIOI, DQA I *0102,DQB l *0602.
w03.04 C~mAcr~mzA~oN OF A COMP~ENT Can R E c ~ ' r o R (C3aR) ON ASTROCYr£s A. ]~CHENKOe, P. G ~ U E 1, S. ANDRF,~, E. SUMER~U 1, M. LAMACZ8, H. VAUDRY8 and M. FONTAINEI 1 INSERM U-78, Bois-Guilloume, France,
Bioweporofiom, St Pel~lrsbourg, R 1 / ~ ,
e Reseorch Institute of Highly Pure a INSERM U-413, Mont-Soint-
Aigno., P ~ n ~ S t u d i e s on c u l t i v a t e d r o d e n t a s t r o c y t e s a n d h u m a n glloma cell lines h a v e b r o u g h t evidence for a c o m p l e m e n t b i o s y n t h e s i s into t h e c e n t r a l n e r v o u s s y s t e m (CNS). A c t i v e f r a g m e n t o f C3, like C3a, m i g h t c o n t r i b u t e to an i n f l a m m a t o r y reaction i n t o t h e CNS. U s i n g b i n d i n g and cross-linking e x p e r i m e n t s w i t h 125I-C3a, we were able to characterize a C3aR o n five d i f f e r e n t glloma cell lines. Binding o f C3a was s h o w n t o be s a t u r a b l e and reversible. The b i n d i n g was i n h i b i t e d by e q u i m o l a r a m o u n t o f anti-C3a Mab (clone G10). The C3aR was e x p r e s s e d at a high level w i t h a n a v e r a g e o f 5.105 sites/cell. I n c u b a t i o n o f glioma cell lines or normal rat astrocYtes w i t h C3a induced a transient i n c r e a s e o f i n t r a c e l l u l a r Ca +++. Cross-linking e x p e r i m e n t s r e v e a l e d a 126Ilabeled complex o f 80kD, i n d i c a t i n g a m o l e c u l a r m a s s o f approx. 70kD for t h e C3aR.
W07.01 OTHER CNS MACROPHAGES BUT NOT FRESHLY ISOLATED ADULT MICROGLIA ACT AS ~ FOR MBP-REACTIVE T CELLS IN VITRO Andrew. L. Ford and Jonathon D. Sedgwick. Centenary Institute of Cancer Medicine and Cell Biology, Royal Prince Alfred Hospital, Sydney, Australia. Introduction. Cells of the macmphage/monocyte lineage in the CNS arc the cell types thought most likely to be the predominant CNS APC by virtue of their propensity to constitutive or inducible MHC class II expression in vivo. But what can the substantial population of CNS-residera microglia do in this context? Materials and Methods. We previously defined a flow cytometric phcnotype for microglia (CD45 l°w CDllb/c+) that distingpished these cells,frqm all blood-derived leukocytes, the latter being CIM5mgn. Within the CD45mg n population are other CNS-associatod macrophages, distinct from microglia, which also express the CD1 lb/c molecule. Cells have been recovered from the normal CNS as well as from rats with graft versus host disease in which the microglla arc highly activated (increased number, size and MHC class II expression), and the two CDllb/c + populations sorted to homogeneity. Results. When added to MBP-reactivf ?1"cells in vitro in the presence of whole MBP or peptide, it is the CD45mg n CNS macrophages, which exhibit the most potent APC capacity (T cell proliferation and 1L-2 secretion). There is also some evidence that this same cell population is presenting 'endogenous' MBP (that is, antigen picked up in the CNS), as T cells proliferate to some extent in the presqnce of these cells but in the absence of added MBP. Surprisingly, microglia (CD45l°w CDI lb/c +) whether activated or not, are consistently poor in terms of their ability to stimulate T cells to proliferation or cytokine secretion, even in the presence of added MBP or peptide. Conclusions. Unlike 'microglial' cells derived from fetal or newborn CNS by long term cell culture, those directly from the adult CNS show little evidence of antigen presenting cell function, even when MHC class II expression induced in vivo is substantial. Conversely, 'other' CNS macrophages, which could include perivascular cells or choroid macrophages, are potent APC. The role of microglia in the stimulation or perpetuation of CNS T cell responses needs to be reconsidered.
P04.04 C O M P A R I S O N OF CSF FINDINGS A N D MRI WITH GD-DTP~ IN P A T I E N T S W I T H A C U T E OPTIC NEURITIS (AON). J.Frederiksenl,H.Larsson2,P.Christiansen2,F.Se] lebjergl. Dept. of Neurology, Gentofte Hospital and D e p t . o f MRI, H v i d o v r e Hospital, Denmark. INTRODUCTION: The interrelationships of MRI and the b e l o w m e n t i o n e d CSF analyses were examined p r o s p e c t i v e l y in c o n s e c u t i v e l y referred patients w i t h idiopathic m o n o s y m p t o m a t i c AON. M A T E R I A L S AND METHODS: 44 p a t i e n t s ( 3 0 F ; a g e d 1857 years) u n d e r w e n t MRI 1-43 days and a lumbar tap 8-49 days from onset of ON. RESULTS: Of the 6 p a t i e n t s with enhancing lesions 5 showed OB,1 abnormal IgG-index and 2 CFS leucocytosis. Of the remaining 38 patients these a b n o r m a l i t i e s were found in 39%,24% and 29%, respectively. Of the 19 patients with abnormal MRI p r i o r to Gd-DTPA, 68% showed OB, 26% i n c r e a s e d IgG-index and 32% CSF leococytosis as c o m p a r e d to 28%, 26% and 28% of the 25 p a t i e n t s w i t h n o r m a l MRI. CONCLUSION: OB, but not increased IgG-index or CSF l e u c o c y t o s i s was m o r e frequent in patients w i t h a b n o r m a l MRI than with normal MRI.
W05.07 y8 T CELL-INDUCED LYSIS OF HUMAN OLIGODENDROCYTES: A HEAT SHOCK PROTEIN MEDIATED MECHANISM? M.S. Freedman I andR. Bitar2. IDepartment ofMedicine (Neuroingy), University of Ottawa, Ottawa, Ontario and 2McGiIIUniversity, Montrdal, Quebec, CANADA. Introduction: Human oligodendrocytes (OGC), RPMI 8226 and Daudi cells all express heat shock proteins (HSP) and induce the expansion of y8 T cells, whereas cell lines lacking in HSP expression (e.g. U937) do not. In contrast, OGC, RPMI 8226, Dandi and U937 are all effectively and about equally lysed by y6 T cells. Since the ligand for y6 T cell induced cytotoxicity is not known, we compared the relative susceptibility among these cell types in order to determine whether the OGC ligand more closely resembled that of other HSP expressing cells. Materials & Methods: y8 T cells were expanded from PBMC using plates coated with anti-¥aTCR mAb (TCR 8-1) and rhlL-2. Relative lysis was determined using a cold target competition assay, wherein unlabeled ("cold") cells were titred into a fixed E:T ratio of20:1 y8 T cell:SICr-lnbeledtargets (cold:hot ratio from 5-40:1). The fewer cold targets needed to effectively compete for y8 T cell-induced lysis, the higher the affinity for the 78 T cell relative to the hot target cell. Results: 8226>Dandi cells inhibited the lysis of either 8226 or Dandi cells (41% vs. 20% and 39% vs. 14%; cold:hot ratio of 20:1). The U937 cells, ineffective at cold inhibition of either 8226 (4%) or Daudi (1%) cells, were however most capable of self-inhibition (66%) compared to either the 8226 (25%) or Daudi's (40%). 8226>Dandi>U937 inhibited the lysis of OGC (61% vs. 22% vs. 9%). Conclusion: These data suggest distinctively different ligands on 8226 and Daudi vs. U937 for y8 T cell-induced lysis, but the ligand on the HSP-expressing 8226 and Daudi cells is most similar to that on OGC. Whether this ligand represents HSP or an associated molecule has yet to be discerned.
p05.03 ANTIBODIESTO MYCOBACTERIAL65 kDa and 70 kDa HEATSHOCKPROTEINS IN NEURODEGENERATIVEDISEASES" U. Fiszer1, S. Fredrikson2, J. Gajda~, A. CztonkowskaI [1] Departmentof CarsbrovaecularDiseases, Institute of Psychiatryand Neurology, Warsaw, Poland.[2] Departmentof Neurology,KarolinskaInstitute,Huddinge,Sweden. Introduction: In Parkinson'sdisease(PD) and Wilson'5 disease (WD) immune abnormalities havebeen reported, including the elevatedy,5÷ T cells population. Hsp could be targets for yc5+ T lymphocytes. Increassd levels of antibodies to hsp have been found in severaldiseases. Mstsrllls lind Methods:We haveexaminedserum of 22 patients with PD, 18 patients with WD who demonstrated neurological symptoms and 15 patients with other non-inflammatory neurological diseases (OND), Antibodies were assessed using an enzyme-linked irnmunosorbent assay 5ccording to Danieli and all. (J. Autoimmunity, 1992, 5, 443), The plates weracoatedwith recombinantmycopacterial hsp 65 (Mycobacterium bovls BCG, batch MA-12A) and hsp 70 (Mycobacterium tuberculosum batch M'r70.6A), a gift from Dr. J. D. A. van Embden. Fbsults: Studies haveshown that mean absorbanca ratio of ssra from patients with WD waa significantly greaterthan that of sarafrom patientswith OND (tested against IgG anti-hsp 65 and IgG anti-hsp 70). There were no differences betwasn mean absorbance ratio of sara from patients with PD and OND. Conclusion,, It is difficult to establish the role of immunity to hsp in WD characterized by the toxic accumulation of copper in a number of organs, particularly tha liver and brain. Increased hap expression, could induce hsp immunity. Alternatively,the antibodies may bs presentas a conssquancs of prior infections. * This investigation received flnencisl support from "the UNOP/WorldBank/WHOSpecial programme for Research and Trainig in Tropical Diseases (TDR)"and 5"tare Committee for Scientific Research in Poland
W04.01 THE Dw2 ASSOCIATION IN HULTIPLE SCLEROSIS EXTENDS TO THE DRB5 L O C U S A Foode]ll,2,J Hillertl,3, 0 Olerup1,2 ICenter for BioTechnology,NOVUI'I,end 2Departments of Clinical Immunology and 3Neurology, Karolinska Institute,Huddinge, Sweden I n t r o d u c t i o n : It is well established that multiple sclerosis (MS) is associated with the HLA haplotype defined as Dw2 with cellular techniques and as DR15,D06 with serological typing methods The Dw2 haplotype has been specified with genomic typing techniques to be D R B I • I 5 O I , D O A I * O I O 2 , D Q B I * 0 6 0 2 . The aim of this study was to define the DRB5 allele(s) on the MS associated DR-DO haplotype. Materials sad Methods: 55 MSpatients and 32 healthy controls carrying the DR2 haplotype were subtyped for ORB5alleles using PCRamplification with sequence-specific primers (PCR-SSP). Results: All the 5SDR2-positiveMS patients and 30 of the 32 DR2-positive controls carried the DRB5*OIO! allele. Thus, the DRB5*OIOI association in MS is as strong as the association with other parts of the Dw2 haplotype, i.e. DRBI*I501, OQAI*OI02DQB l * 0 6 0 2 .
Conclusion: The class II haplotype associated with FIS extend to the ORB5 locus and can now be specified as DRB I * I501,DRBS~OIOI, DQA I *0102,DQB l *0602.
w03.04 C~mAcr~mzA~oN OF A COMP~ENT Can R E c ~ ' r o R (C3aR) ON ASTROCYr£s A. ]~CHENKOe, P. G ~ U E 1, S. ANDRF,~, E. SUMER~U 1, M. LAMACZ8, H. VAUDRY8 and M. FONTAINEI 1 INSERM U-78, Bois-Guilloume, France,
Bioweporofiom, St Pel~lrsbourg, R 1 / ~ ,
e Reseorch Institute of Highly Pure a INSERM U-413, Mont-Soint-
Aigno., P ~ n ~ S t u d i e s on c u l t i v a t e d r o d e n t a s t r o c y t e s a n d h u m a n glloma cell lines h a v e b r o u g h t evidence for a c o m p l e m e n t b i o s y n t h e s i s into t h e c e n t r a l n e r v o u s s y s t e m (CNS). A c t i v e f r a g m e n t o f C3, like C3a, m i g h t c o n t r i b u t e to an i n f l a m m a t o r y reaction i n t o t h e CNS. U s i n g b i n d i n g and cross-linking e x p e r i m e n t s w i t h 125I-C3a, we were able to characterize a C3aR o n five d i f f e r e n t glloma cell lines. Binding o f C3a was s h o w n t o be s a t u r a b l e and reversible. The b i n d i n g was i n h i b i t e d by e q u i m o l a r a m o u n t o f anti-C3a Mab (clone G10). The C3aR was e x p r e s s e d at a high level w i t h a n a v e r a g e o f 5.105 sites/cell. I n c u b a t i o n o f glioma cell lines or normal rat astrocYtes w i t h C3a induced a transient i n c r e a s e o f i n t r a c e l l u l a r Ca +++. Cross-linking e x p e r i m e n t s r e v e a l e d a 126Ilabeled complex o f 80kD, i n d i c a t i n g a m o l e c u l a r m a s s o f approx. 70kD for t h e C3aR.
W07.01 OTHER CNS MACROPHAGES BUT NOT FRESHLY ISOLATED ADULT MICROGLIA ACT AS ~ FOR MBP-REACTIVE T CELLS IN VITRO Andrew. L. Ford and Jonathon D. Sedgwick. Centenary Institute of Cancer Medicine and Cell Biology, Royal Prince Alfred Hospital, Sydney, Australia. Introduction. Cells of the macmphage/monocyte lineage in the CNS arc the cell types thought most likely to be the predominant CNS APC by virtue of their propensity to constitutive or inducible MHC class II expression in vivo. But what can the substantial population of CNS-residera microglia do in this context? Materials and Methods. We previously defined a flow cytometric phcnotype for microglia (CD45 l°w CDllb/c+) that distingpished these cells,frqm all blood-derived leukocytes, the latter being CIM5mgn. Within the CD45mg n population are other CNS-associatod macrophages, distinct from microglia, which also express the CD1 lb/c molecule. Cells have been recovered from the normal CNS as well as from rats with graft versus host disease in which the microglla arc highly activated (increased number, size and MHC class II expression), and the two CDllb/c + populations sorted to homogeneity. Results. When added to MBP-reactivf ?1"cells in vitro in the presence of whole MBP or peptide, it is the CD45mg n CNS macrophages, which exhibit the most potent APC capacity (T cell proliferation and 1L-2 secretion). There is also some evidence that this same cell population is presenting 'endogenous' MBP (that is, antigen picked up in the CNS), as T cells proliferate to some extent in the presqnce of these cells but in the absence of added MBP. Surprisingly, microglia (CD45l°w CDI lb/c +) whether activated or not, are consistently poor in terms of their ability to stimulate T cells to proliferation or cytokine secretion, even in the presence of added MBP or peptide. Conclusions. Unlike 'microglial' cells derived from fetal or newborn CNS by long term cell culture, those directly from the adult CNS show little evidence of antigen presenting cell function, even when MHC class II expression induced in vivo is substantial. Conversely, 'other' CNS macrophages, which could include perivascular cells or choroid macrophages, are potent APC. The role of microglia in the stimulation or perpetuation of CNS T cell responses needs to be reconsidered.
P04.04 C O M P A R I S O N OF CSF FINDINGS A N D MRI WITH GD-DTP~ IN P A T I E N T S W I T H A C U T E OPTIC NEURITIS (AON). J.Frederiksenl,H.Larsson2,P.Christiansen2,F.Se] lebjergl. Dept. of Neurology, Gentofte Hospital and D e p t . o f MRI, H v i d o v r e Hospital, Denmark. INTRODUCTION: The interrelationships of MRI and the b e l o w m e n t i o n e d CSF analyses were examined p r o s p e c t i v e l y in c o n s e c u t i v e l y referred patients w i t h idiopathic m o n o s y m p t o m a t i c AON. M A T E R I A L S AND METHODS: 44 p a t i e n t s ( 3 0 F ; a g e d 1857 years) u n d e r w e n t MRI 1-43 days and a lumbar tap 8-49 days from onset of ON. RESULTS: Of the 6 p a t i e n t s with enhancing lesions 5 showed OB,1 abnormal IgG-index and 2 CFS leucocytosis. Of the remaining 38 patients these a b n o r m a l i t i e s were found in 39%,24% and 29%, respectively. Of the 19 patients with abnormal MRI p r i o r to Gd-DTPA, 68% showed OB, 26% i n c r e a s e d IgG-index and 32% CSF leococytosis as c o m p a r e d to 28%, 26% and 28% of the 25 p a t i e n t s w i t h n o r m a l MRI. CONCLUSION: OB, but not increased IgG-index or CSF l e u c o c y t o s i s was m o r e frequent in patients w i t h a b n o r m a l MRI than with normal MRI.
W05.07 y8 T CELL-INDUCED LYSIS OF HUMAN OLIGODENDROCYTES: A HEAT SHOCK PROTEIN MEDIATED MECHANISM? M.S. Freedman I andR. Bitar2. IDepartment ofMedicine (Neuroingy), University of Ottawa, Ottawa, Ontario and 2McGiIIUniversity, Montrdal, Quebec, CANADA. Introduction: Human oligodendrocytes (OGC), RPMI 8226 and Daudi cells all express heat shock proteins (HSP) and induce the expansion of y8 T cells, whereas cell lines lacking in HSP expression (e.g. U937) do not. In contrast, OGC, RPMI 8226, Dandi and U937 are all effectively and about equally lysed by y6 T cells. Since the ligand for y6 T cell induced cytotoxicity is not known, we compared the relative susceptibility among these cell types in order to determine whether the OGC ligand more closely resembled that of other HSP expressing cells. Materials & Methods: y8 T cells were expanded from PBMC using plates coated with anti-¥aTCR mAb (TCR 8-1) and rhlL-2. Relative lysis was determined using a cold target competition assay, wherein unlabeled ("cold") cells were titred into a fixed E:T ratio of20:1 y8 T cell:SICr-lnbeledtargets (cold:hot ratio from 5-40:1). The fewer cold targets needed to effectively compete for y8 T cell-induced lysis, the higher the affinity for the 78 T cell relative to the hot target cell. Results: 8226>Dandi cells inhibited the lysis of either 8226 or Dandi cells (41% vs. 20% and 39% vs. 14%; cold:hot ratio of 20:1). The U937 cells, ineffective at cold inhibition of either 8226 (4%) or Daudi (1%) cells, were however most capable of self-inhibition (66%) compared to either the 8226 (25%) or Daudi's (40%). 8226>Dandi>U937 inhibited the lysis of OGC (61% vs. 22% vs. 9%). Conclusion: These data suggest distinctively different ligands on 8226 and Daudi vs. U937 for y8 T cell-induced lysis, but the ligand on the HSP-expressing 8226 and Daudi cells is most similar to that on OGC. Whether this ligand represents HSP or an associated molecule has yet to be discerned.