- Email: [email protected]
Nitrite and nitrate contents in CSF of patients with multiple sclerosis-elevated levels in active disease
70
Poster Abstracts / Journal o f Neuroimmunology 90 (1998) 13-105
389
392
Retrograde Axonal Transport Reduction During CNS D e m y e l i n a t i o n in T h e i l e r ' s Virus-infected Mice D. Ure, M. Rodriguez, MayoClinic, USA
Association Between C. P n e u m o u l a e and MS SjY. Yao. S. Sriram, C. Stratton, W. Mitchell, MSResearchLab., Fanderbilt-
StallworthRehabilitationHosp., Tennessee,USA
Chronic infection of SJL mice with "l~eiler's virus produces extensive inflammation and demyelination of the spinal cord due to virus persistence in glial cells and macrophages in the white matter. The mice also show progressive neurologic dysfunction. We have hypothesized that the neurologic deficits in part reflect secondary injury to neurons resulting from effects of inflammation/demyelination on axons. To study neuronal function we have quantitated retrograde axonal transport of the fluorescent tracer. Fluorogold, following lower thoracic implantation of dye-saturated Gelfoam. In preliminary experiments analysis of the midbrain mbrospinal neurons, whose axons descend into the spinal cord, revealed that only half as many neurons were labeled in chronically infected mice as compared to uninfected mice, indicative of injury to axons or possibly retrograde death of the neurons. The reduction in retrograde labeling correlated with the reduction in motor function as measured by the Rotarod assay. Thus, axonal damage induced by inflammation and/or demyelination might contribute significantly to neurologic dysfunction.
_Q.biective: To test whether there is an association between C pneumoniae infection and Multiple Sclerosis. ~ : Although current opinion favors MS to be an autoimmtme disease directed against self neural antigens, epidemiologic studies have argued for an infectious agent. Design/Methods: We have tested for. a) pmsenca o f C. pneumoniae, using P C R to D N A products o f major outer membrane protein (MOMP) in C S F o f secondary progressive MS patients and other neurologic disease controls (OND); b) presence o f culture positivity o f 6". pneumoniae; and e) the presence of antibody to M O M P and whole EB lysates using Western blot techniques. Results and Conclusions: In 44% patients with MS, C. pneumoniae waS cultured from the CSF and all controls were culture negative. In 19/20 MS patients, PCR signal to M O M P gene products were identified. Only 2 o f 19 controls showed a signal to M O M P gene products. Antibody titers to EB antigens were elevated in 17/20 MS patients. These observation suggest a correlation between infection o f the CNS with C. pneumoniae and the development o f MS.
390
393
Differentiation o f B r a i n M a e r o p i t a g e s in S l Y Infection: T h e Role of P e r i v a s e u l a r Cells in E a r l y C N S Infection & S l Y Eneepitalitis K. Willim~_~sD. Panley, S. Westmoreland, H~ Knight, V. Sasseville, A. Lackner,
Nitrite a n d N i t r a t e Contents in C S F of Patients w i t h Multiple Sclerosis-elevated Levels in A c t i v e Disease L. Brtmdin, E. Morcos, T. Olsson, N.P. Wikhnd, M. Andersson, Karolinska
HarvardMedicaISchool,NewEnglandRegionalPrimateResearchCenter,MA, USA
Hospital Sweden
Identification of distinct monocyte/macrophage subpopulations in the CNS that are the target for infection by HIV/SIV is not resolved. In this study, we have used immunohistochemistry to define phenotypic differences between the resident parenchymal micmgtia, perivascular macrophages, and multinucleated giant cells (MNGC). All of these cells express antigens of myeloid lineage including complement receptor 3 (CD11b), CD4, and CD68. Perivascular macrophages and MNGC but not parenchymal microglia expressed LPS receptor (CD14) and LCA (CD45). Perivascular macrophages and parenchymul microglia upregulate or express de novo CD11b, CD40, and B7-2 (CD86) with higher level of expression of these antigens within the subcortical white matter. In several cases MNGC had downregulated CD4. Using this immunophenotypic pattern to differentiate between brain maerophages in conjunction with either simultaneous immunostaining for viral protein or in situ hybndization for viral nucleic acid, we demonstrate that a population of perlvascular brain macrophages is the major cell infected in the CNS of SlY infected macaques. These cells can be immune selected using flow cytometry and FACsorting or immune selection with antibodycoated magnetic beads. Immunohistochemistry for viral UP120, in situ hybridization, QC-PCR, and CEMX174 assays using immune selected cells demonstrate that CD14 + cells are the major cell infected by SlY during peak vimmia and in animals with SIV encephalitis.
Nitric oxide (NO) is synthesized by the NO synthetase, an enzyme existing in three isotbrms. The macrophage isoform, (i-NOS) is induced by cytokines during inflammatory processes and tumor growth. This enzyme has been shown to be localized in multiple sclerosis (MS) lesions. Objective: The role of NO lbrmation in the pathogenesis of MS is unclear. Our objective were to analyze CSF levels of nitrite and nitrate in MS patients in different phases of the disease and thereby better understand the disease mechanism in MS. Methodology: Seventeen MS patients were recruited and scored according to the degree of progression/activity of the disease at the time of lumbar puncture. The CSF samples were analyzed for nitrite and nitrate content using capillary electrephoresis technology. Results: In patients with progressive/active MS trite levels were significantly increased as compared to patients with slow progression or no clinically detectable activity of the disease. Conclusions: First, this finding suggests that nitrite and nitrate analysis could provide a clinical means for determinations of disease activity in MS and potentially in other neuroinflammatory diseases. Second, our results may indicate that NO formation may constitute an important pathogenetic mechanism for demyelinisation and neuronal damage in MS.
391
394
Apoptotie Cell Death Following M o u s e H e p a t i t i s V i r u s - l n d u e e d D e m y e l i n a t i o n of the C N S G. Wu, S. Perlman, Universityoflowa, USA
P r o d u c t i o n of M o n o d o n a l A n t i - M B P l g M by E B V - t r a n s f o r m e d B Cell Lines f r o m Patients w i t h Stable Multiple S d e r o s i s P. Annunziata. C. Cioni, S. Pluchino, UniversityofStena, Italy
Infection nfthc murine central nervous system (CNS) with mouse hepatitis virus, strain JHM (MHV-JHM) can cause acute encephalitis or, in a percentage of mice protected with maternal antibody, chronic hind-limb paralysis associated with demyelination of the CNS. This chronic disease serves as a model for the human disease Multiple Sclerosis. Although it is clear that T-cells are necessary for viral clearance, it is unknown whether the immune system plays a necessary rote in the development of demyelmation. Recent evidence indicates that adoptive transfer of T-cells to an immtmodeficicnt mouse infected with MHV can result in demyelination. CTL "killing depends on two effector mechamsms: pcrforin mediated evtolysis and Fas/Fas ligand induced apoptosis. To determine whether an apoptoUc process is involved in CNS disease following infection ~ith MHV-JHM. two experiments have been done. First. the extent of apoptosis during a tunecoutse of acute disease was evaluated. Brains and spuml cords from mice infected with MHV-JHM at days 4, 5, 6 and 7 post-inoculation were evaluated using a TUNEL assay. Second, double labeling of TUNEL-positive cells with immtmohisax;hemical labels for various cell markets and viral antigen was done. Results demonstrate that apoptosis occurs to an extensive degree following acute infection with MHV-JHM. Furthermore. a majority nf cells undergoing apoptosis in a, immunodefieient mouse with subacute disease are not infected with virus. Overall, preliminary results suggest that apoptosis is invol~ed in MHV-JHM-indueed demyelination.
:~be rule af t,szral i m . m i ~ directed ao3inst ~ _ t i n ~ in maltiple sclerosis (NS) dsm,elimtion process iB uw/e~c, m~entlV, d,=,~tL-~t=d sign~icaut association of high levels of CSF I~4 binding tn ~ basic protein (~P) with e ~ l y benign cota~e in MS (A.nu.ziata et al., 1997). To assess ~ ¢ 6 - ~ hhis respoose aL.¢~ place in the p e r i ~ blood, ~ established ~3V-transformed Bi lines f l ' ~ 10 patients wih"l acd;ive, 5 T,,rii~* s'Im~ible I~3 and4 ncrmsl heslb'~ subjecbs. W~r,,:~/cr'~d. a n t i ~ I~W ,,,ms found i n rune 10 active, ,in 3/'5 stable p a t i ~ (P=O.(]~2), arc] i n 4/4 r,armal s u b j ~ (I*=O.OQ1). 'nzis respcr~se ~,~s k~pp~ ( r lambda chain i n stable patients and mostly k~ppa chain i n normal subjects. "n'w~se f i n dir~ d~rm~,~ U~at a ~ imbrue re~atmse n ~ n l r ~ ~ is
e t i e i t s d eitl'w~ i n s t a b l e 1~ p a t i e n t s o r i n nm'roa}_ healiah~ mtbject~.
Poster Abstracts / Journal o f Neuroimmunology 90 (1998) 13-105
389
392
Retrograde Axonal Transport Reduction During CNS D e m y e l i n a t i o n in T h e i l e r ' s Virus-infected Mice D. Ure, M. Rodriguez, MayoClinic, USA
Association Between C. P n e u m o u l a e and MS SjY. Yao. S. Sriram, C. Stratton, W. Mitchell, MSResearchLab., Fanderbilt-
StallworthRehabilitationHosp., Tennessee,USA
Chronic infection of SJL mice with "l~eiler's virus produces extensive inflammation and demyelination of the spinal cord due to virus persistence in glial cells and macrophages in the white matter. The mice also show progressive neurologic dysfunction. We have hypothesized that the neurologic deficits in part reflect secondary injury to neurons resulting from effects of inflammation/demyelination on axons. To study neuronal function we have quantitated retrograde axonal transport of the fluorescent tracer. Fluorogold, following lower thoracic implantation of dye-saturated Gelfoam. In preliminary experiments analysis of the midbrain mbrospinal neurons, whose axons descend into the spinal cord, revealed that only half as many neurons were labeled in chronically infected mice as compared to uninfected mice, indicative of injury to axons or possibly retrograde death of the neurons. The reduction in retrograde labeling correlated with the reduction in motor function as measured by the Rotarod assay. Thus, axonal damage induced by inflammation and/or demyelination might contribute significantly to neurologic dysfunction.
_Q.biective: To test whether there is an association between C pneumoniae infection and Multiple Sclerosis. ~ : Although current opinion favors MS to be an autoimmtme disease directed against self neural antigens, epidemiologic studies have argued for an infectious agent. Design/Methods: We have tested for. a) pmsenca o f C. pneumoniae, using P C R to D N A products o f major outer membrane protein (MOMP) in C S F o f secondary progressive MS patients and other neurologic disease controls (OND); b) presence o f culture positivity o f 6". pneumoniae; and e) the presence of antibody to M O M P and whole EB lysates using Western blot techniques. Results and Conclusions: In 44% patients with MS, C. pneumoniae waS cultured from the CSF and all controls were culture negative. In 19/20 MS patients, PCR signal to M O M P gene products were identified. Only 2 o f 19 controls showed a signal to M O M P gene products. Antibody titers to EB antigens were elevated in 17/20 MS patients. These observation suggest a correlation between infection o f the CNS with C. pneumoniae and the development o f MS.
390
393
Differentiation o f B r a i n M a e r o p i t a g e s in S l Y Infection: T h e Role of P e r i v a s e u l a r Cells in E a r l y C N S Infection & S l Y Eneepitalitis K. Willim~_~sD. Panley, S. Westmoreland, H~ Knight, V. Sasseville, A. Lackner,
Nitrite a n d N i t r a t e Contents in C S F of Patients w i t h Multiple Sclerosis-elevated Levels in A c t i v e Disease L. Brtmdin, E. Morcos, T. Olsson, N.P. Wikhnd, M. Andersson, Karolinska
HarvardMedicaISchool,NewEnglandRegionalPrimateResearchCenter,MA, USA
Hospital Sweden
Identification of distinct monocyte/macrophage subpopulations in the CNS that are the target for infection by HIV/SIV is not resolved. In this study, we have used immunohistochemistry to define phenotypic differences between the resident parenchymal micmgtia, perivascular macrophages, and multinucleated giant cells (MNGC). All of these cells express antigens of myeloid lineage including complement receptor 3 (CD11b), CD4, and CD68. Perivascular macrophages and MNGC but not parenchymal microglia expressed LPS receptor (CD14) and LCA (CD45). Perivascular macrophages and parenchymul microglia upregulate or express de novo CD11b, CD40, and B7-2 (CD86) with higher level of expression of these antigens within the subcortical white matter. In several cases MNGC had downregulated CD4. Using this immunophenotypic pattern to differentiate between brain maerophages in conjunction with either simultaneous immunostaining for viral protein or in situ hybndization for viral nucleic acid, we demonstrate that a population of perlvascular brain macrophages is the major cell infected in the CNS of SlY infected macaques. These cells can be immune selected using flow cytometry and FACsorting or immune selection with antibodycoated magnetic beads. Immunohistochemistry for viral UP120, in situ hybridization, QC-PCR, and CEMX174 assays using immune selected cells demonstrate that CD14 + cells are the major cell infected by SlY during peak vimmia and in animals with SIV encephalitis.
Nitric oxide (NO) is synthesized by the NO synthetase, an enzyme existing in three isotbrms. The macrophage isoform, (i-NOS) is induced by cytokines during inflammatory processes and tumor growth. This enzyme has been shown to be localized in multiple sclerosis (MS) lesions. Objective: The role of NO lbrmation in the pathogenesis of MS is unclear. Our objective were to analyze CSF levels of nitrite and nitrate in MS patients in different phases of the disease and thereby better understand the disease mechanism in MS. Methodology: Seventeen MS patients were recruited and scored according to the degree of progression/activity of the disease at the time of lumbar puncture. The CSF samples were analyzed for nitrite and nitrate content using capillary electrephoresis technology. Results: In patients with progressive/active MS trite levels were significantly increased as compared to patients with slow progression or no clinically detectable activity of the disease. Conclusions: First, this finding suggests that nitrite and nitrate analysis could provide a clinical means for determinations of disease activity in MS and potentially in other neuroinflammatory diseases. Second, our results may indicate that NO formation may constitute an important pathogenetic mechanism for demyelinisation and neuronal damage in MS.
391
394
Apoptotie Cell Death Following M o u s e H e p a t i t i s V i r u s - l n d u e e d D e m y e l i n a t i o n of the C N S G. Wu, S. Perlman, Universityoflowa, USA
P r o d u c t i o n of M o n o d o n a l A n t i - M B P l g M by E B V - t r a n s f o r m e d B Cell Lines f r o m Patients w i t h Stable Multiple S d e r o s i s P. Annunziata. C. Cioni, S. Pluchino, UniversityofStena, Italy
Infection nfthc murine central nervous system (CNS) with mouse hepatitis virus, strain JHM (MHV-JHM) can cause acute encephalitis or, in a percentage of mice protected with maternal antibody, chronic hind-limb paralysis associated with demyelination of the CNS. This chronic disease serves as a model for the human disease Multiple Sclerosis. Although it is clear that T-cells are necessary for viral clearance, it is unknown whether the immune system plays a necessary rote in the development of demyelmation. Recent evidence indicates that adoptive transfer of T-cells to an immtmodeficicnt mouse infected with MHV can result in demyelination. CTL "killing depends on two effector mechamsms: pcrforin mediated evtolysis and Fas/Fas ligand induced apoptosis. To determine whether an apoptoUc process is involved in CNS disease following infection ~ith MHV-JHM. two experiments have been done. First. the extent of apoptosis during a tunecoutse of acute disease was evaluated. Brains and spuml cords from mice infected with MHV-JHM at days 4, 5, 6 and 7 post-inoculation were evaluated using a TUNEL assay. Second, double labeling of TUNEL-positive cells with immtmohisax;hemical labels for various cell markets and viral antigen was done. Results demonstrate that apoptosis occurs to an extensive degree following acute infection with MHV-JHM. Furthermore. a majority nf cells undergoing apoptosis in a, immunodefieient mouse with subacute disease are not infected with virus. Overall, preliminary results suggest that apoptosis is invol~ed in MHV-JHM-indueed demyelination.
:~be rule af t,szral i m . m i ~ directed ao3inst ~ _ t i n ~ in maltiple sclerosis (NS) dsm,elimtion process iB uw/e~c, m~entlV, d,=,~tL-~t=d sign~icaut association of high levels of CSF I~4 binding tn ~ basic protein (~P) with e ~ l y benign cota~e in MS (A.nu.ziata et al., 1997). To assess ~ ¢ 6 - ~ hhis respoose aL.¢~ place in the p e r i ~ blood, ~ established ~3V-transformed Bi lines f l ' ~ 10 patients wih"l acd;ive, 5 T,,rii~* s'Im~ible I~3 and4 ncrmsl heslb'~ subjecbs. W~r,,:~/cr'~d. a n t i ~ I~W ,,,ms found i n rune 10 active, ,in 3/'5 stable p a t i ~ (P=O.(]~2), arc] i n 4/4 r,armal s u b j ~ (I*=O.OQ1). 'nzis respcr~se ~,~s k~pp~ ( r lambda chain i n stable patients and mostly k~ppa chain i n normal subjects. "n'w~se f i n dir~ d~rm~,~ U~at a ~ imbrue re~atmse n ~ n l r ~ ~ is
e t i e i t s d eitl'w~ i n s t a b l e 1~ p a t i e n t s o r i n nm'roa}_ healiah~ mtbject~.