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Urinary tract infection frequency in MS exacerbations
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P l l .07 ANALYSIS OF T CELL EPITOPE SPECIFICITY DIFFERENTIATING PATHOGENIC AND NON-PATHOGENIC THEILER'S MURINE ENCEPHALOMYELITITS VIRUS B.S. Kim, R.L Yaoch, G. Rasmussen. K. Kerekes and C.A. Kappel Northwestern University Medical School, Chicago, EL 6061 l Introduction: lntracerebral inoculation of snsceptible strains of mice with Thcilcr's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination. Like other picornaviruses, TMEV consists of only 4 viral capsid proteins. Based on studies with other picornavirusos, VPI appears to be the most important for viral pathogenesis and protective immunity to the viruses. Materials and Methods: Standard methods were used for T cell cloning, selection of T cell hybridomas, T cell proliferation, and plaque isolation of virus clones. GST-fusion proteins and synthetic pcptidcs wcr¢ also used. Results: We have identified a major T cell epitop¢ and defined the region within the VP1 protein based on the rcactivities ofT cell clones derived from dcmyelinating spinal cords of virus-infected SJL mice. Interestingly, these VPIreactive clones recognizing the region of VP1233.2so did not recognize a spomancousiy mutated virus (M2) displaying extremely low-pathogenicity. Our preliminary results indicate that the mutant virus has a single base alteration leading to a lysine --~ arginine substitution within the T cell epitop¢. Conclusions: We believe that this VPI T cell epitopc may play an important role in the pathogenesis of viral demyelinating disease. (Supported by NIH grants, NS28752 and NS33008)
P06.08 B I N D I N G P R O P E R T I E S OF A MYELIN G L Y C O L I P I D - S P E C I FIC HUMAN M O N O C L O N A L A N T I B O D Y (MAB)DERIVED FROM A M U L T I P L E SCLEROSIS (MS) P A T I E N T E.Kirschning,
G.Rutter,
H.Uhlig
and R . D e r n i c k
H e i n r i c h - P e t t e - I n s t i t u t Exp. Virol. Immunol., M a r t i n i s t r a s s e 52, 20251 Hamburg, G e r m a n y Introduction: G a l a c t o c e r e b r o s i d e s u l f a t e ( S u l f a tide) reactive human m o n o c l o n a l a n t i b o d y (mAb) DSIF8 was tested for its a b i l i t y to b i n d to the surface of c u l t u r e d o l i g o d e n d r o c y t e s (OLs). M a t e r i a l and Methods: B i n d i n g was r e v e a l e d by immuno double labeling of rat brain cell cultures with glial c e l l - s p e c i f i c murine m a r k e r mAbs in light m i c r o s c o p y and by immunogold labeling in e l e c t r o n microscopy. Results: M A b DSIF8 selectively bound to the surface of a n t i - g a l a c t o c e r e b r o s i d e (GalC)-positive OLs but not to a s t r o c y t e s or OL precursors. Conclusion: Future studies will show, whether mAb DSIF8 inhibits the d i f f e r e n t i a t i o n of OL precursors, as has b e e n d e s c r i b e d for a murine mAb specific for GalC and sulfatide.
P12.05 INTERFERON 8i:TA-1B INDUCED ULCERATIVE SKIN LESIONS IN MS. R. L. Knobior. C.L. Kelley, F. Trantas, G.F. Webster* and F. D. Lublin. Multiple Sclerosis Comprehensive Clinical Center, Division of Neuroimmunology, Department of Neurology, *Department of Dermatology, Thomas Jefferson University, Philadelphia, PA 19107, USA Ihtroduction. Interferon Beta -1B has been used as a treatment for multiple sclerosis, administered subcutaneously. Side effects have usually been of limited duration, and have included "flu-like" symptoms, rashqike skin reactions and elevated liver enzymes. Materials and Methods. Patients were openly enrolled onto a treatment regimen with 8 mlU of Interferon Beta-lB, administered subcutaneously every other day. Patients were carefully trained in the preparation and injection technique prior to initiation of therapy, and followed at 6 weeks and then at 3 month intervals after the initiation of therapy. Results. Within 3 to 6 months of the initiation of therapy, ulcerative skin lesions had developed at multiple injection sites in 4 el 200 patients. There was confinement of these lesions to a 1-2 cm region surrounding an injection site. These lesions were not do to scratching or infection, and there was no evidence of cellulitis. Biopsies of these skin lesions showed inflammatory cells, and the lesions rapidly disappeared upon withdrawal of therapy Conclusion. Interferon Beta-lB had previously been shown to produce similar ulcerative skin lesions only in AIDS patients treated subcutaneously. This adverse event had not previously cccurred in the 8 years of treatment of patients with MS. Although the precise cause of these ulcerative skin lesions has not yet been determined, their association to injection sites mJc',n~.~t~the nossibilitv of local cvtokine mediated mechanisms.
P06.09 URINARY TRACT INFECTION FREQUENCY IN M S - ~ . X A C E R B A T ~ . - Golclberg, C.L. Kelley, F. Trantas, F. D. Lublin and R. L. Knobler. Multiple Sclerosis Comprehensive Clinical Center, Division of Neuroimmunoiogy, Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107, USA Introduction. Infections may act as a "super-antigen" in the context of aufoimmune diseases such as MS. We sought to determine the incidence of urinary tract infections (UTIs) in patients at the time of admission for treatment of an exacerbation of multiple sclerosis (MS). Methods. All patients admitted to Thomas Jefferson University Hospital dudng 1992, for the treatment of exacerbations of MS were evaluated. 3-5 WBCs and 49,000-99,000 organisms were required for inclusion as a UTI. Results. 175 of 295 patients admitted for the treatmsnt of an acute exacerbation of MS had both urinalysis and cultures performed. 79 of the 175 had positive UTI findings (45%), regardless of urinary symptoms, while 96/175 had negative findings. Organisms most frequently encountered included E. colt (21%), mixed Gram positives (14%), group D Enferococci (13%), Sfaph. aureus (12%), Pseudomonas aeru. (6%), Proteus mirabilis (50) and others (29%). Discussion. Of 157 patients tested within the first five days of admission for treatment of an exacerbation of MS, 45% (70), had positive UTI findings, with different organisms predominant in different individuals. Therefore, no single bacterial organism is uniformly associated with exacerbations of MS. The duration of neurological symptoms prior to admission is not available from this retrospective analysis, so the issue of whether a neurogenic bladder led to UTI vs. UTI tnggering an exacerbation t~n n~t vat h~ an.~wnmd.
P14.09 INTRACEREBRAL URIC ACID AS A MARKER OF BRAIN INFECTION WITH MHV-4. R, L. Knololer. G. M. Alexander, J. Grothusen, J. Joseph and F. D. Lublin. Division of Neuroimmunoiogy, Thomas Jefferson University, Philadelphia, PA 19107, USA IFitroduction. Intracerebral infection with mouse hepatitis virus type-4 (MHV-4) provides a model system for the study of virus induced neurological illness, including fatal encephalomyelitis and demyelination. Materials and Methods. Brain specimens were assayed by high pressure liquid chromatography (HPLC) to search for molecules that might correlate with virus infection of the brain. Anesthetized mice were inoculated intracerebrally into the right cerebral hemisphere with 2 x 103 plaque forming units of MHV-4 in a 0.05 ml volume. Results. With HPLC, a peak was identified in the striatum of mice intracerebrally infected with MHV-4, that initially co-elutecl with epinephrine. Separation from epinephrine occurred after switching solvent systems. However, the new peak co-eluted with known samples of uric acid in both solvent systems, and disappeared after treatment el the sample with unease. This peak was not present following either intracerebral injection of an equal volume of normal saline, nor injection of even 1000 fold more vires intraperitoneally and damaging the blood brain barrier by needle puncture. Conclusion. Intracerebral uric acid production serves as a marker of neurological involvement due to MHV-4 induced disease.
W06.02 ANAI.YSIS OF PLP-SPECIFIC T CELLS SUGGESTS T C I ~ L RECEPTOR CDR3 CONSERVATION AMONG HUMAN ENCEPHAL1TOGENIC T CELLS T.Kondo, T . Y a m a m u r a , J-i. lnobe, T. Ohashi, K. T a k a h a s h i a n d T.Tabira D e p a r t m e n t of D e m y e l i n a t i n g Disease a n d Aging, National I n s t i t u t e of Neuroscience, NCNP, Tokyo, J a p a n Introduction: We h a v e recently d e m o n s t r a t e d t h a t r o d e n t e n c e p h a l i t o g e n i c T cells t e n d to e x p r e s s p a r t i c u l a r s e q u e n c e motifs in t h e i r T cell r e c e p t o r (TCR). It n e e d s to be i n v e s t i g a t e d w h e t h e r this can b e e x t r a p o l a t e d to h u m a n T cells. M a t e r i a l s a n d M e t h o d s : T cell lines (TCL) specific for PLP d e t e r m i n a n t s w e r e e s t a b l i s h e d f r o m the blood of MS. T h e i r TCR I~-chains w e r e s e q u e n c e d a f t e r a m p l i f i c a t i o n w i t h i n v e r s e PCR. R e s u l t s : T h e TCR CDR3 w e r e e n r i c h e d in a l i p h a t i c r e s i d u e s (leu, Val) a n d Gly, a n d c o n t a i n e d c h a r a c t e r i s t i c s e q u e n c e m o t i f s c o m p o s e d of t h e s e r e s i d u e s . This f e a t u r e is s h a r e d b y t h o s e of MBP-specific "IEL a n d MS b r a i n - d e r i v e d T cells in t h e l i t e r a t u r e . C o n c l u s i o n : l l u m a n e n c e p h a l i t o g e n i c T cells m a ) be u n i q u e in t h e c o m p o s i t i o n of a m i n o acids in t h e i r TCR CDR3.
P l l .07 ANALYSIS OF T CELL EPITOPE SPECIFICITY DIFFERENTIATING PATHOGENIC AND NON-PATHOGENIC THEILER'S MURINE ENCEPHALOMYELITITS VIRUS B.S. Kim, R.L Yaoch, G. Rasmussen. K. Kerekes and C.A. Kappel Northwestern University Medical School, Chicago, EL 6061 l Introduction: lntracerebral inoculation of snsceptible strains of mice with Thcilcr's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination. Like other picornaviruses, TMEV consists of only 4 viral capsid proteins. Based on studies with other picornavirusos, VPI appears to be the most important for viral pathogenesis and protective immunity to the viruses. Materials and Methods: Standard methods were used for T cell cloning, selection of T cell hybridomas, T cell proliferation, and plaque isolation of virus clones. GST-fusion proteins and synthetic pcptidcs wcr¢ also used. Results: We have identified a major T cell epitop¢ and defined the region within the VP1 protein based on the rcactivities ofT cell clones derived from dcmyelinating spinal cords of virus-infected SJL mice. Interestingly, these VPIreactive clones recognizing the region of VP1233.2so did not recognize a spomancousiy mutated virus (M2) displaying extremely low-pathogenicity. Our preliminary results indicate that the mutant virus has a single base alteration leading to a lysine --~ arginine substitution within the T cell epitop¢. Conclusions: We believe that this VPI T cell epitopc may play an important role in the pathogenesis of viral demyelinating disease. (Supported by NIH grants, NS28752 and NS33008)
P06.08 B I N D I N G P R O P E R T I E S OF A MYELIN G L Y C O L I P I D - S P E C I FIC HUMAN M O N O C L O N A L A N T I B O D Y (MAB)DERIVED FROM A M U L T I P L E SCLEROSIS (MS) P A T I E N T E.Kirschning,
G.Rutter,
H.Uhlig
and R . D e r n i c k
H e i n r i c h - P e t t e - I n s t i t u t Exp. Virol. Immunol., M a r t i n i s t r a s s e 52, 20251 Hamburg, G e r m a n y Introduction: G a l a c t o c e r e b r o s i d e s u l f a t e ( S u l f a tide) reactive human m o n o c l o n a l a n t i b o d y (mAb) DSIF8 was tested for its a b i l i t y to b i n d to the surface of c u l t u r e d o l i g o d e n d r o c y t e s (OLs). M a t e r i a l and Methods: B i n d i n g was r e v e a l e d by immuno double labeling of rat brain cell cultures with glial c e l l - s p e c i f i c murine m a r k e r mAbs in light m i c r o s c o p y and by immunogold labeling in e l e c t r o n microscopy. Results: M A b DSIF8 selectively bound to the surface of a n t i - g a l a c t o c e r e b r o s i d e (GalC)-positive OLs but not to a s t r o c y t e s or OL precursors. Conclusion: Future studies will show, whether mAb DSIF8 inhibits the d i f f e r e n t i a t i o n of OL precursors, as has b e e n d e s c r i b e d for a murine mAb specific for GalC and sulfatide.
P12.05 INTERFERON 8i:TA-1B INDUCED ULCERATIVE SKIN LESIONS IN MS. R. L. Knobior. C.L. Kelley, F. Trantas, G.F. Webster* and F. D. Lublin. Multiple Sclerosis Comprehensive Clinical Center, Division of Neuroimmunology, Department of Neurology, *Department of Dermatology, Thomas Jefferson University, Philadelphia, PA 19107, USA Ihtroduction. Interferon Beta -1B has been used as a treatment for multiple sclerosis, administered subcutaneously. Side effects have usually been of limited duration, and have included "flu-like" symptoms, rashqike skin reactions and elevated liver enzymes. Materials and Methods. Patients were openly enrolled onto a treatment regimen with 8 mlU of Interferon Beta-lB, administered subcutaneously every other day. Patients were carefully trained in the preparation and injection technique prior to initiation of therapy, and followed at 6 weeks and then at 3 month intervals after the initiation of therapy. Results. Within 3 to 6 months of the initiation of therapy, ulcerative skin lesions had developed at multiple injection sites in 4 el 200 patients. There was confinement of these lesions to a 1-2 cm region surrounding an injection site. These lesions were not do to scratching or infection, and there was no evidence of cellulitis. Biopsies of these skin lesions showed inflammatory cells, and the lesions rapidly disappeared upon withdrawal of therapy Conclusion. Interferon Beta-lB had previously been shown to produce similar ulcerative skin lesions only in AIDS patients treated subcutaneously. This adverse event had not previously cccurred in the 8 years of treatment of patients with MS. Although the precise cause of these ulcerative skin lesions has not yet been determined, their association to injection sites mJc',n~.~t~the nossibilitv of local cvtokine mediated mechanisms.
P06.09 URINARY TRACT INFECTION FREQUENCY IN M S - ~ . X A C E R B A T ~ . - Golclberg, C.L. Kelley, F. Trantas, F. D. Lublin and R. L. Knobler. Multiple Sclerosis Comprehensive Clinical Center, Division of Neuroimmunoiogy, Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107, USA Introduction. Infections may act as a "super-antigen" in the context of aufoimmune diseases such as MS. We sought to determine the incidence of urinary tract infections (UTIs) in patients at the time of admission for treatment of an exacerbation of multiple sclerosis (MS). Methods. All patients admitted to Thomas Jefferson University Hospital dudng 1992, for the treatment of exacerbations of MS were evaluated. 3-5 WBCs and 49,000-99,000 organisms were required for inclusion as a UTI. Results. 175 of 295 patients admitted for the treatmsnt of an acute exacerbation of MS had both urinalysis and cultures performed. 79 of the 175 had positive UTI findings (45%), regardless of urinary symptoms, while 96/175 had negative findings. Organisms most frequently encountered included E. colt (21%), mixed Gram positives (14%), group D Enferococci (13%), Sfaph. aureus (12%), Pseudomonas aeru. (6%), Proteus mirabilis (50) and others (29%). Discussion. Of 157 patients tested within the first five days of admission for treatment of an exacerbation of MS, 45% (70), had positive UTI findings, with different organisms predominant in different individuals. Therefore, no single bacterial organism is uniformly associated with exacerbations of MS. The duration of neurological symptoms prior to admission is not available from this retrospective analysis, so the issue of whether a neurogenic bladder led to UTI vs. UTI tnggering an exacerbation t~n n~t vat h~ an.~wnmd.
P14.09 INTRACEREBRAL URIC ACID AS A MARKER OF BRAIN INFECTION WITH MHV-4. R, L. Knololer. G. M. Alexander, J. Grothusen, J. Joseph and F. D. Lublin. Division of Neuroimmunoiogy, Thomas Jefferson University, Philadelphia, PA 19107, USA IFitroduction. Intracerebral infection with mouse hepatitis virus type-4 (MHV-4) provides a model system for the study of virus induced neurological illness, including fatal encephalomyelitis and demyelination. Materials and Methods. Brain specimens were assayed by high pressure liquid chromatography (HPLC) to search for molecules that might correlate with virus infection of the brain. Anesthetized mice were inoculated intracerebrally into the right cerebral hemisphere with 2 x 103 plaque forming units of MHV-4 in a 0.05 ml volume. Results. With HPLC, a peak was identified in the striatum of mice intracerebrally infected with MHV-4, that initially co-elutecl with epinephrine. Separation from epinephrine occurred after switching solvent systems. However, the new peak co-eluted with known samples of uric acid in both solvent systems, and disappeared after treatment el the sample with unease. This peak was not present following either intracerebral injection of an equal volume of normal saline, nor injection of even 1000 fold more vires intraperitoneally and damaging the blood brain barrier by needle puncture. Conclusion. Intracerebral uric acid production serves as a marker of neurological involvement due to MHV-4 induced disease.
W06.02 ANAI.YSIS OF PLP-SPECIFIC T CELLS SUGGESTS T C I ~ L RECEPTOR CDR3 CONSERVATION AMONG HUMAN ENCEPHAL1TOGENIC T CELLS T.Kondo, T . Y a m a m u r a , J-i. lnobe, T. Ohashi, K. T a k a h a s h i a n d T.Tabira D e p a r t m e n t of D e m y e l i n a t i n g Disease a n d Aging, National I n s t i t u t e of Neuroscience, NCNP, Tokyo, J a p a n Introduction: We h a v e recently d e m o n s t r a t e d t h a t r o d e n t e n c e p h a l i t o g e n i c T cells t e n d to e x p r e s s p a r t i c u l a r s e q u e n c e motifs in t h e i r T cell r e c e p t o r (TCR). It n e e d s to be i n v e s t i g a t e d w h e t h e r this can b e e x t r a p o l a t e d to h u m a n T cells. M a t e r i a l s a n d M e t h o d s : T cell lines (TCL) specific for PLP d e t e r m i n a n t s w e r e e s t a b l i s h e d f r o m the blood of MS. T h e i r TCR I~-chains w e r e s e q u e n c e d a f t e r a m p l i f i c a t i o n w i t h i n v e r s e PCR. R e s u l t s : T h e TCR CDR3 w e r e e n r i c h e d in a l i p h a t i c r e s i d u e s (leu, Val) a n d Gly, a n d c o n t a i n e d c h a r a c t e r i s t i c s e q u e n c e m o t i f s c o m p o s e d of t h e s e r e s i d u e s . This f e a t u r e is s h a r e d b y t h o s e of MBP-specific "IEL a n d MS b r a i n - d e r i v e d T cells in t h e l i t e r a t u r e . C o n c l u s i o n : l l u m a n e n c e p h a l i t o g e n i c T cells m a ) be u n i q u e in t h e c o m p o s i t i o n of a m i n o acids in t h e i r TCR CDR3.