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Interferon regulatory factor-1 in peripheral blood mononuclear cells from patients with multiple sclerosis
72
P o s t e r A b s t r a c t s / J o u r n a l o f Neuroimmuru~logy 9 0 (1998) 1 3 - 1 0 5
401
404
Interferon Regulatory Factor-1 in Peripheral Blood Manonudear Cells from Patients with Multiple Sclerosis J. Drulovic, D. Popadic, M.M. Stojkovic, Z. Levic, S. Mesaros, N. Stojsavljevic,
Autoantibodies from the Sera and Cerebrospinal Fluid of Patients
UniversityofBelgrade, l~ugoslavia
Interl~ron mgulmory factor-I (1RF-I) is a member of a family of la'aascription factors that regulate an array of genes involved in cell growth, differentiation and death. Studies on mice with a targeted dissruption of the tRF-I germ revealed the involvement of IRF-I in the regulation of an immane ~ and pathogenesis of autdimmtme disease. The aim of this study was to analyse the level of constitutive exFa:ssion of IRF-I in peripheral blood mononuclear cells (PBMC) from l~ieras with multiple sclerosis (MS) and ~ 1 subjects. [RF-I mRNA was determined in 47 patients with definite MS (26 p~ents with active and 21 with stable disease) and 23 patientswith othernetrologiesl diseases. To analyse IRF-I messenger RNA expression pattern of PBMC, we applied a c o m p l ~ DNA polHnemse chain reason technique. Values are given as IRF-1/[3 actin n~o. ~ median IRF-I/13 actin ratio was lower in patients with active MS (0.28) in comparison to beth stable (0A7) and control pa6ents (0.72) although the difference did ~ reach the level of statistical significance. Having in mind that IRF-I gene expression is regulated by several cytokines besides interl~rons,the data showing lower level of expression of IRF-I in the patients with active MS might be explained by the well known imbalance in cytokine production in MS. It is also telrtpung to speculate t l ~ the olr,eawed differences in IRF-I expression could be involved in the dysfunction of the Fas system recently dascnbed in MS patients,
with Multiple Sderosis Effectively Hydrolyze DNA and RNA G.A. Nevinsky, Novosibirsklnstitute of Bworganic Chemistry,Russia, 0.0. Favorova. Russian StateMedical University,Moscow, A.G. Baranovskii, Army Hospital333, Novosibirsk,Russia, T.G. Kanyshkova, lnstimte ofBioorganicChemistry,Pushchino, A.S. Mogilnitskii, Army Hospital333, Novosibirsk,Russia, A.N. Boiko, V.A. Natunov, V.N. Buneva, T.A. Zargarova, E.I. Gusev, Russian StateMedical Universi~ Moscow Multiple sclerosis (MS) is an inflammatory putative autoimmune demyelinatmg disorder of the central nervous system. Catalytic DNA- and RNA-hydrolyzing autoantibodies (Abs) were detected in some autoimmane pathologies. We measured DNA- and RNA-hydrolyzing activities of highly purified DNAbinding Abs from MS patients. The l g G fractions of Abs from sera of 69 out of 72 and from cerebrospinal fluid of 5 oat of 5 MS patients displayed DNA- and RNA-hydrolyzing activities stable to acid and alkaline shock. Homogeneous Fab fragments isolated from IgG fractions effectively catalyzed hydrolysis o f DNA and RNA and separated hght chains (both K and )~ types) catalyzed hydrolysis of DNA. DNA- and RNA-hydsolyzing Abs were not detected in scra from 50 healthy, individuals. Abs from MS patients showed pmnoanced differences in the specificities toward various model RNAs when compared with the known ribonucleases from human blood and tissues and the described RNA-hydrolyzing Abs. They had as high the specific enzymatic activity in hydrolysis o f RNA as the pancreatic ribonuclease A being the fwst example of Abs with high catalytic activity. These and other data indicate that the capabifity of nucleic acids hydrolysis is an intrinsic property of Abs from MS patients. Catalytic Abs are expected to be involved in MS aetiopathogeoesis.
402
405
Expression of Blood and CSFAdhesion Molecules in Multiple Sclerosis: Corrdations to Neurological Disability and Volumes of Plaques and Atrophy I. Elovaara, M. Lalla, M Ukkonen, J. Peltola, T. Lehtimaki, P. Dastidar, Tampers
CytokineAlteratian of Co-stimulatory Molecules in MS L.G. Filion. J. Chabot, A. Kumar, M. Frcedm an, Universityof Ottawa, Canada
UniversityHospital, Finland
We analyzed the expression of VLA-4, LFA-I, VCAM-I and 1CAM-I immunocytochemically in 23 patients with relapsing MS, 20 patients with secondary progressive MS and iI controls. The results were related to EDSS and RFSS and to volumes of T1 and T2 plaques and brain atrophy analyzed by MRI. Highest expressions of VLA-4 and LFA-I on blood leukocytes were found in patients with relapsing MS in exacerbation. In patients with secondary progressive MS these adhesion proteins were also increased compared to controls, but lower than in relapsing MS. The correlation to MRI volumetric measurements revealed weak correlation only between TI plaques and the blood VLA-4 or LFA-I. No correlations were found between the adhesion molecule expressions in blood and EDSS. The upregulated expression of adhesion molecules on leukocytes from MS patients facilitates their transmigration into the brain. In stable MS increased adhesion molecule expression may mean sustained potential for inflammation and demyelinafish.
The exact mechanism by which ~-'~okitm the~l~" positively affects the course of lois is m~lear, We hypothesize that modifying the expression o f eo-stimulatoryi molecules (CDS0, CD86, CD28, CD40, CD40L) on antigen presenting cells (APC) (B cells, monocyt¢~ (/vie)) or the T cells nugbt account for this action. PBMC from MS patients or normal controls were cultured in vitro with IFNI] or rIL-10 for 24 Ill'. The expression ofceU ~uface markers w e r e e _ ~ r d n e d by flow c'~tometry. IFNI3 down regulated CD80 but not CD86 on M e from normal controls (n~5) hut IL-10 down regulated CD86 only. In MS cell, two distinct: populations o f M e in terms of CD80 and CD86 were seen. IFNI3 down reg~tlated M e exptessmn of CD80 and CD86 in 3/4 patients; bat IL-10 down regulated i CD86 0nly (3/4 patient~ CD40 and CD40L expression were increas~ in response ~to IFN[5((3/4 patients), but a larger response was observed in rrL-10 t~ated ~-nltures_ The co.-sUmulatory mol~'ul~s on MS APC respond differently tO IFN'~ and rIL-10, when eompared with normal controls. The altered response o f APC! of MS patients suggest that these cells may play a role in the pathogenesis of the disease. Supported hy: Multiple Sclerosig Society of Canada
403
406
C o r t i s o l a n d I n t e r l e u k i n (IL)-6 i n P o s t - M o r t e m C e r e b r o s p i n a l F l u i d of M u l t i p l e S c l e r o s i s ( M S ) P a t i e n t s a n d C o n t r o l s Z.A. Erkut NetherlandslnstituteforBrain Research, E. Endert, AMC, Netherlands, I. Huitinga, D,E Swaab, NetherlandslnstituteforBrainResearch
Decreases in IL-4 Secretion by Invariant V~24Jc~QT edls in P e r i p h e r a l B l o o d o f P a t i e n t s w i t h M u l t i p l e S c l e r o s i s (MS)
The hypothalamo-pituitary-adrenal(HPA)-axisactivity is increased in muhiplesclerosis(MS) as appears fromthe incre~ed cotlisoland corticotxopth(ACTH)levelsand hypothalamiccorticotropinreleasing hormone (CRH) neuronal activityI. This increase in the HPA-axisactivity and thus the elevatedcoaisol levelsare consideredto be crucial for the defense mechanismof the patient against the immune-mediatedpatho-guneUcmechanismof the disease. Cytokines play an important role in the patho-physiologyof multiple sclerosis (MS). Some cytokines, like interleukin(IL)-6. act as prthnflammatoryagents and are presumed to mediate the exacerbations of the disease. In additionto its direct immune-regulatoryeffect. IL-6also plays a role in the communicationbetween the neuro..endocaneJ~adil~'nune systems. It activatesthe HPA-axis. pre.sumahlythroughstimulationof the hypothalanucCRH cells.~mdin turn, it is underthe feed-back control of adrenalcorticostemids.To investigatewhether 1[,-6levelis associatedwith the activation of HPA-axlsduring MS, we analyzed the post-mortemventricularCSFzof 22 MS patientsand 61 controls for conisol and IL-6.The effects of age, sex. sepsis, corticosteroidtherapy and morphine on the CSF conisol and IL-6levelshave also been investigated. The results showed a 2-fold increase in CSF cortisol levels in MS patients in comparison to controls (p--0.005),but no differencein IL-6levels.There w~ an age related increase of cortisolin controls (R=0.37, p=0.013), but not in MS patients. Cortisoland I1.-6levelswere correlated in MS patients (R--0.67, p=0.017), but not in controls. Our results indicatedthat in the CSF of chronic MS patients, IL-6levelis under the influenceof cortisol. I: Erkut et al. (1995) J Neuroimmunok62: 27-33; 2: Post-monem CSF w~ obtained from the Netherlands BrainBank (coordinatorDr. R. Ravid).Thisstudywas supportedby FoundationFriends of MS Research (The Netherlands) and by TUBITAK(Turkey).
We performed a cross-sectional examination of the cytokine secretion of invariant Va24JaQ T cells in patients with MS as compared to healthy controls. Vc~24+ T cells were directly sorted from the blood of 11 patients with relapsing remitting MS (RR-MS), 5 patients with progressive MS (CP-MS) and 9 healthy controls into single wells and expanded in vitro for analysis of IL-4 and IFN-7 secretion. A total of 322 independent T cell clones were generated that were sequenced and stimulated with TCR cross-linking for cytokine secretion. There was a tendency for lower numbers of IL-4 secreting Va24JaQ invariant T cells in RR-MS patients as compared to patients with CP-MS or controls. No differences in IFN-~, secretion were observed. While further subjects need to be examined, these data are consistent with the hypothesis that alterations in the function of V~t24J~xQ invariant T cells may contribute to the induction of human autoimmune disease.
R. Gauslhle, T. Trollmo, D. Hafler, HarvardMedicalSchool, USA
P o s t e r A b s t r a c t s / J o u r n a l o f Neuroimmuru~logy 9 0 (1998) 1 3 - 1 0 5
401
404
Interferon Regulatory Factor-1 in Peripheral Blood Manonudear Cells from Patients with Multiple Sclerosis J. Drulovic, D. Popadic, M.M. Stojkovic, Z. Levic, S. Mesaros, N. Stojsavljevic,
Autoantibodies from the Sera and Cerebrospinal Fluid of Patients
UniversityofBelgrade, l~ugoslavia
Interl~ron mgulmory factor-I (1RF-I) is a member of a family of la'aascription factors that regulate an array of genes involved in cell growth, differentiation and death. Studies on mice with a targeted dissruption of the tRF-I germ revealed the involvement of IRF-I in the regulation of an immane ~ and pathogenesis of autdimmtme disease. The aim of this study was to analyse the level of constitutive exFa:ssion of IRF-I in peripheral blood mononuclear cells (PBMC) from l~ieras with multiple sclerosis (MS) and ~ 1 subjects. [RF-I mRNA was determined in 47 patients with definite MS (26 p~ents with active and 21 with stable disease) and 23 patientswith othernetrologiesl diseases. To analyse IRF-I messenger RNA expression pattern of PBMC, we applied a c o m p l ~ DNA polHnemse chain reason technique. Values are given as IRF-1/[3 actin n~o. ~ median IRF-I/13 actin ratio was lower in patients with active MS (0.28) in comparison to beth stable (0A7) and control pa6ents (0.72) although the difference did ~ reach the level of statistical significance. Having in mind that IRF-I gene expression is regulated by several cytokines besides interl~rons,the data showing lower level of expression of IRF-I in the patients with active MS might be explained by the well known imbalance in cytokine production in MS. It is also telrtpung to speculate t l ~ the olr,eawed differences in IRF-I expression could be involved in the dysfunction of the Fas system recently dascnbed in MS patients,
with Multiple Sderosis Effectively Hydrolyze DNA and RNA G.A. Nevinsky, Novosibirsklnstitute of Bworganic Chemistry,Russia, 0.0. Favorova. Russian StateMedical University,Moscow, A.G. Baranovskii, Army Hospital333, Novosibirsk,Russia, T.G. Kanyshkova, lnstimte ofBioorganicChemistry,Pushchino, A.S. Mogilnitskii, Army Hospital333, Novosibirsk,Russia, A.N. Boiko, V.A. Natunov, V.N. Buneva, T.A. Zargarova, E.I. Gusev, Russian StateMedical Universi~ Moscow Multiple sclerosis (MS) is an inflammatory putative autoimmune demyelinatmg disorder of the central nervous system. Catalytic DNA- and RNA-hydrolyzing autoantibodies (Abs) were detected in some autoimmane pathologies. We measured DNA- and RNA-hydrolyzing activities of highly purified DNAbinding Abs from MS patients. The l g G fractions of Abs from sera of 69 out of 72 and from cerebrospinal fluid of 5 oat of 5 MS patients displayed DNA- and RNA-hydrolyzing activities stable to acid and alkaline shock. Homogeneous Fab fragments isolated from IgG fractions effectively catalyzed hydrolysis o f DNA and RNA and separated hght chains (both K and )~ types) catalyzed hydrolysis of DNA. DNA- and RNA-hydsolyzing Abs were not detected in scra from 50 healthy, individuals. Abs from MS patients showed pmnoanced differences in the specificities toward various model RNAs when compared with the known ribonucleases from human blood and tissues and the described RNA-hydrolyzing Abs. They had as high the specific enzymatic activity in hydrolysis o f RNA as the pancreatic ribonuclease A being the fwst example of Abs with high catalytic activity. These and other data indicate that the capabifity of nucleic acids hydrolysis is an intrinsic property of Abs from MS patients. Catalytic Abs are expected to be involved in MS aetiopathogeoesis.
402
405
Expression of Blood and CSFAdhesion Molecules in Multiple Sclerosis: Corrdations to Neurological Disability and Volumes of Plaques and Atrophy I. Elovaara, M. Lalla, M Ukkonen, J. Peltola, T. Lehtimaki, P. Dastidar, Tampers
CytokineAlteratian of Co-stimulatory Molecules in MS L.G. Filion. J. Chabot, A. Kumar, M. Frcedm an, Universityof Ottawa, Canada
UniversityHospital, Finland
We analyzed the expression of VLA-4, LFA-I, VCAM-I and 1CAM-I immunocytochemically in 23 patients with relapsing MS, 20 patients with secondary progressive MS and iI controls. The results were related to EDSS and RFSS and to volumes of T1 and T2 plaques and brain atrophy analyzed by MRI. Highest expressions of VLA-4 and LFA-I on blood leukocytes were found in patients with relapsing MS in exacerbation. In patients with secondary progressive MS these adhesion proteins were also increased compared to controls, but lower than in relapsing MS. The correlation to MRI volumetric measurements revealed weak correlation only between TI plaques and the blood VLA-4 or LFA-I. No correlations were found between the adhesion molecule expressions in blood and EDSS. The upregulated expression of adhesion molecules on leukocytes from MS patients facilitates their transmigration into the brain. In stable MS increased adhesion molecule expression may mean sustained potential for inflammation and demyelinafish.
The exact mechanism by which ~-'~okitm the~l~" positively affects the course of lois is m~lear, We hypothesize that modifying the expression o f eo-stimulatoryi molecules (CDS0, CD86, CD28, CD40, CD40L) on antigen presenting cells (APC) (B cells, monocyt¢~ (/vie)) or the T cells nugbt account for this action. PBMC from MS patients or normal controls were cultured in vitro with IFNI] or rIL-10 for 24 Ill'. The expression ofceU ~uface markers w e r e e _ ~ r d n e d by flow c'~tometry. IFNI3 down regulated CD80 but not CD86 on M e from normal controls (n~5) hut IL-10 down regulated CD86 only. In MS cell, two distinct: populations o f M e in terms of CD80 and CD86 were seen. IFNI3 down reg~tlated M e exptessmn of CD80 and CD86 in 3/4 patients; bat IL-10 down regulated i CD86 0nly (3/4 patient~ CD40 and CD40L expression were increas~ in response ~to IFN[5((3/4 patients), but a larger response was observed in rrL-10 t~ated ~-nltures_ The co.-sUmulatory mol~'ul~s on MS APC respond differently tO IFN'~ and rIL-10, when eompared with normal controls. The altered response o f APC! of MS patients suggest that these cells may play a role in the pathogenesis of the disease. Supported hy: Multiple Sclerosig Society of Canada
403
406
C o r t i s o l a n d I n t e r l e u k i n (IL)-6 i n P o s t - M o r t e m C e r e b r o s p i n a l F l u i d of M u l t i p l e S c l e r o s i s ( M S ) P a t i e n t s a n d C o n t r o l s Z.A. Erkut NetherlandslnstituteforBrain Research, E. Endert, AMC, Netherlands, I. Huitinga, D,E Swaab, NetherlandslnstituteforBrainResearch
Decreases in IL-4 Secretion by Invariant V~24Jc~QT edls in P e r i p h e r a l B l o o d o f P a t i e n t s w i t h M u l t i p l e S c l e r o s i s (MS)
The hypothalamo-pituitary-adrenal(HPA)-axisactivity is increased in muhiplesclerosis(MS) as appears fromthe incre~ed cotlisoland corticotxopth(ACTH)levelsand hypothalamiccorticotropinreleasing hormone (CRH) neuronal activityI. This increase in the HPA-axisactivity and thus the elevatedcoaisol levelsare consideredto be crucial for the defense mechanismof the patient against the immune-mediatedpatho-guneUcmechanismof the disease. Cytokines play an important role in the patho-physiologyof multiple sclerosis (MS). Some cytokines, like interleukin(IL)-6. act as prthnflammatoryagents and are presumed to mediate the exacerbations of the disease. In additionto its direct immune-regulatoryeffect. IL-6also plays a role in the communicationbetween the neuro..endocaneJ~adil~'nune systems. It activatesthe HPA-axis. pre.sumahlythroughstimulationof the hypothalanucCRH cells.~mdin turn, it is underthe feed-back control of adrenalcorticostemids.To investigatewhether 1[,-6levelis associatedwith the activation of HPA-axlsduring MS, we analyzed the post-mortemventricularCSFzof 22 MS patientsand 61 controls for conisol and IL-6.The effects of age, sex. sepsis, corticosteroidtherapy and morphine on the CSF conisol and IL-6levelshave also been investigated. The results showed a 2-fold increase in CSF cortisol levels in MS patients in comparison to controls (p--0.005),but no differencein IL-6levels.There w~ an age related increase of cortisolin controls (R=0.37, p=0.013), but not in MS patients. Cortisoland I1.-6levelswere correlated in MS patients (R--0.67, p=0.017), but not in controls. Our results indicatedthat in the CSF of chronic MS patients, IL-6levelis under the influenceof cortisol. I: Erkut et al. (1995) J Neuroimmunok62: 27-33; 2: Post-monem CSF w~ obtained from the Netherlands BrainBank (coordinatorDr. R. Ravid).Thisstudywas supportedby FoundationFriends of MS Research (The Netherlands) and by TUBITAK(Turkey).
We performed a cross-sectional examination of the cytokine secretion of invariant Va24JaQ T cells in patients with MS as compared to healthy controls. Vc~24+ T cells were directly sorted from the blood of 11 patients with relapsing remitting MS (RR-MS), 5 patients with progressive MS (CP-MS) and 9 healthy controls into single wells and expanded in vitro for analysis of IL-4 and IFN-7 secretion. A total of 322 independent T cell clones were generated that were sequenced and stimulated with TCR cross-linking for cytokine secretion. There was a tendency for lower numbers of IL-4 secreting Va24JaQ invariant T cells in RR-MS patients as compared to patients with CP-MS or controls. No differences in IFN-~, secretion were observed. While further subjects need to be examined, these data are consistent with the hypothesis that alterations in the function of V~t24J~xQ invariant T cells may contribute to the induction of human autoimmune disease.
R. Gauslhle, T. Trollmo, D. Hafler, HarvardMedicalSchool, USA