- Email: [email protected]
Interferon-γ mRNA expression in immediately EX vivo CSF T cells
149
P17.01 DIFFERENTIAL IMMEDIATE EARLY GENE (lEG) RESPONSE OF THE CNS ENDOTHELIUM TO CALCIUM-MEDIATEDINJURY. Sawsen Abu-Shakra. W. Freij, R. Baiabenov, F Nachtmen, J.P. Muizalaar,end P. Dore-Duffy. Center for Neurotrauma, Wayne State University School of Medicine, Detroit, Michigan 48201. I n t r o d u c t i o n : Recent studies underscore the role of vascular endothelium in injury and repair mechanisms. Cerebrovascular microvessals composed of endothelial calls (EC) end pericyfas (PC) are highly sensitive to changes such as hypoxia, inflammatory stimuli, and trauma. These responses may make the difference between maintenance of homeostasis or injury extension. We hypothesize that lEG induction in PC end EC is an important regulatory mechanism which modulates responses to trauma. Methods: Primary cultures of CN'3 EC and PC were prepared from isolated rat cerebral microvessels. EC contained 1% or less PC, and PC had no EC contamination. Cultures received calcium ionophore, A23187 (luM) for varying periods, and analyzed for expression of zif268 mRNA by northern analysis. Results : CNS EC showed low constitutive levels of zif268, whereas PC basal levels were high. In response to A23187, CNS EC zlf268 is significantly upregulatod by 1 hr. In PC, however, zif268 levels were not significantly altered. This may mean that PC are maximally upregulated for zif268 or unresponsive to A2.3187. Conclusions: Differences in constitutive expression of zif268 may suggest differences in trophic factor influences between PC and EC. However, as zif268 encodes a transcription factor and has been associated with cell plasticity and learning, its constitutive expression in PC is intriguing, zif268 is an appealing candidate for a "plasticity gene" and is likely to be important in trauma.
P08.01 ~EURDIMMUNOLDGYOF~.Ai~pHINE~DD~CTIDN R. A~.: AZ~yev~ Z. N. " A l t y e v . Psycho-Nev~OIOgy'D~spR~eP'o~Baku city T h i r t y male morphine a d d i c t i o n pa%ients aged ( 2 9 . 0 + 7 . 0 years> c o n t i t u t e d t h e s t u d y samle. The l e v e l s o f dopaminl s o m a t o t r o p i ~ , cGMP, and a l l t h e n a t u r e T-lymphocytes p o p u l a t i o n ~ were significantly reduced Jr, nonabst inence c h r o n i c morphine dependencea a d d i c t i o n compared w i t h c o n t r o l s . Whereas t h e l e v e l o f s e r o t o n i n , GABA~ ACTH, pPolactin~ cAMP, and t h e number o f B lymphocytesaand~ pPothymocytes were found t o be h i g h e r t h a t t h e eontnol values. The c o n c e n t r a t i o n o f i ~ m u n o g l o b u l i n s a l s o were h i g h e r than the h e a l t h y i n d i v i d u a l s . The agents a d m i n i s t e r e d may be d i v i d e d i n t o t w o g r o u p s w i t h r e s p e c t t o t h e i r a c t i o n on t h e r a t i n g o f c r a v i n g For mo~'phine and t h e i ~ e f f e c t on t h e n e u r o e n d o c r i n e and immunological parameters. The f i r s t group i n c l u d e s p a r l o d e l and i n t e r f e r o n . In a d d i c t i o n t o an decreasing t h e rating of c r a v i n g for morphine thes~ agonts promoted a t~'end towards normaiizatior~; the second g~oup - d a l a r g i n led t o a d e t e r i o r a t i o n t h e r a t z n g o f c r a v i n g f o r morphine and i n e r e a sed d i s t u r b a n c e o f t h e neuroendoc;~ine and i ~ u n o l o g i c a l parameters.
~07.05 THE C08"nMULATORY MOLECULE B'/ 18 E]Cw~Ei~ED IN CULllJRED HUMAN MICROGLIA AND IN MULlrlPLE SCLEROGB AculrE LESIONS. R. De Slmone t, A. Glsmpeolo 2, B. Giometto 3, P. Gallo3, G. Levi1, C. Peschle2 and F..~ohll 1. IL8~. of ~ ~ System ~ o l l j f s [ o J ~ f
P04.01 DETERGENT SOLUBILITYOF CLASS I ANTIGENS PRESENT IN CSF. JC A L V A R E Z - C E R M E N O , L M VILLAR, M NOCITO, JG SEGOVIA, A SERRANO, C PALACIOS, P G O N Z A L E Z PORQUE. The source of soluble class I antigens in cerebrospinal fluid (CSF) has not been s t u d i e d in detail, however, an active s e c r e t i o n from activ a t e d lymphocytes w i t h i n b l o o d - b r a i n b a r r i e r (BBB) has b e e n suspected. A n o t h e r p o s i b i l i t y is s h e d d i n g from classic m e m b r a n e - b o u n d H L A (mHLA) Both proteins differ in t h e i r s o l u b i l i t y properties; sHLA s e c r e t e d b y l y m p h o c y t e s is t o t a l l y found in aqueous phases w h i l e m H L A has higher hydrophobicity. W e have p e r f o r m e d a s o l u b i l i t y study of CSF sHLA in T r i t o n TX 114 in CSF from patients w i t h MS, or m e n i n g i t i s w i t h BBB damage. We found that in MS cases w i t h normal BBB function, over 99 percent of CSF H L A appears in the aqueous phase. In cases w i t h b r e a k d o w n of BBB, 10 to 30% of HLA is h y d r o p h o b i c (similar to values found in serum) (p<0.05). We c o n c l u d e that the increased levels of s H L A in MS are due to intrathecal l y m p h o c y t e synthesis, w h i l e in the cases of BBB damage, the h y d r o p h o b i c H L A originates from serum.
P05.01 MYELIN OLIGODENDROCYTE GLYCOPROTEIN RELAPSING DEMYELINATING EAE IN MICE
(MOG)
INDUCES
S. Amorl. J.K. O'Neill2, N. Oroome3, M,M. Morris t and D. Baker 2 1Department of Immunology.Ray.e Inslltute, UMDS, at.Thomas' Ho~pitni, Londoa, UK; 2Department of Clinical Ophthalmology,Imzituteof Ophthnimniogy,London, UK; 3Multiple Scl¢ro~isSocietyPeptld¢Laboratory,OxfordBrookesUniversity.Oxford,UK. Introduction: MOG is a specific, myelin antigen which may provide a target for antibody.mediated demyelination, following T cell mediated blood-brain barrier dysfunction. MOG is also a minor myelin protein, which has complicated purification of sufficient m a ~ a l to determine encephaiitogenic potential in vivo. Materials and Methods: Overlapping (8 amino acids) synthetic 15mar peptidus corresponding to the whole MOG molecule were used to actively sensitise EAEsusceptible Biozzi AB/H (H-2dqt) and SJL (H-2s) mice. Results: In contrast to the identification of single encephaiitogenic epitopes within the PLP (residues 54-76) and MBP (residues 12-35) molecules io AB/H mice, multiple epitopes within MOG residues 1-22, 43-57 and 134-148 induced clinical and/or histological disease in Biozzi AB/H mice. However in the SJL mouse a single epitope wifltin residues 92-106 was capable of inducing clinical and histological EAE of a comparable magnitude as that observed following immunisation with either PLP or MBP peptides. Furthermore MOG-immtmised SJL mice exhibited a relapsing disease progression and demyelinated CNS lesions. Conclusions: Immune responses to MOG induce clinical EAE, clinically silent histological lesions and a relapsing-remitting, demyelinating disease. Therefore MOG must be considered as a potential autoantigen in multiple sclerosis, where different MHC haplotypes may respond to distinct regions of the molecule.
P02.01 I~r~;RFERON-~ m R N A EXPRESSION IN' ~ L t , VIVO CS]F T CRI.I.~
T E L Y EX
Manon Blain, Jnsuphine Nalbantoglu, Jack P. Antel
2L~. of HenlMolo~-Oncelogy, Istltufo Superiors di Sant~, Rome; 311
Neu,,ofoglcC~c, Univer~ of Padova, Padova, #sly. Introduction: To alucldeta tha mechsnlsms of T cell activation in the CNS, expression of B7, a major cosltmulatow molecule, was examined: 1) in vttro, in two APC popelatlons of tha brain, mlcloglla and aatrocytes, and 2) in situ, in post-mortam brain tissue from multiple solercets (MS) petlants. Methods and Results: Using Immunocytocheminal and PCR techniques, we found that in cultures from the human amb~onl¢ brain mlcroglla, but not astrocytes, constitutively express B7. Mlcrogllal expression of B7 wa~ Increased by IFN-7, but not TNF-a, IL-11~,M-CSF, GM-CSF or LPS. Human mlcrnglis also expressed the accessory molecules LFA-1, LFA-3 and ICAM-1. In sciive MS lesions, B7 Immunoreactlvlty was detected both on infiltrating macrophngex and activated mlcrnglia, while in chronic lesions only pedvaecular cells were faintly mined. No B7 immunoreacUvlty was found in brain tumor, Alzhelmer dlsance and normal control brain tissue specimens. Conclusions: Taken together the shove findings suggest that B7 may play s mla in T cell activation and lesion development In MS and that the regulated expression of B7 on micro~ia may contribute in the local stimulation of T cell proliferation and effector functions. (Supported by CNR grant n.93.00472.PF40, Project on Aging).
Montreal Nanrologteal Institute Introduction - We compared interferon (IFN)-,y mRNA expression between immediately ex-vivo CSF- and peripheral blood (PB)-derived T cells using a PCR amplification technique to overcome the limitations imposed by cell numbers present in CSF samples. Materials and Matheds - Total RNA was extracted from 3-5 cc of CSF and from T cells isolated from PB by gradient centrifugation and E + rosetting, c D N A was synthesized by reverso transcribing the R N A using random primers. The eDNA was amplified using specific primers for CD3 and IFN-~; the product was analyzed on agarose gels. Results - Using CSF samples which contained 2-5xl0' cells, a PCR product was obtained using both the CD3 and IFN-,y primers. Using PB cDNA derived from up to 10x greetar T-cell number than in the CSF samples, only a CD3 primer product was obtained. Conclusion - The expression of IFN-,y mRNA in immediately ex-vivo T cells derived from CSF, but not PB, indicates furthar distinction in properties of cells in the systemic and intrathecal compartments, with activation being a characteristic of the formar.
P17.01 DIFFERENTIAL IMMEDIATE EARLY GENE (lEG) RESPONSE OF THE CNS ENDOTHELIUM TO CALCIUM-MEDIATEDINJURY. Sawsen Abu-Shakra. W. Freij, R. Baiabenov, F Nachtmen, J.P. Muizalaar,end P. Dore-Duffy. Center for Neurotrauma, Wayne State University School of Medicine, Detroit, Michigan 48201. I n t r o d u c t i o n : Recent studies underscore the role of vascular endothelium in injury and repair mechanisms. Cerebrovascular microvessals composed of endothelial calls (EC) end pericyfas (PC) are highly sensitive to changes such as hypoxia, inflammatory stimuli, and trauma. These responses may make the difference between maintenance of homeostasis or injury extension. We hypothesize that lEG induction in PC end EC is an important regulatory mechanism which modulates responses to trauma. Methods: Primary cultures of CN'3 EC and PC were prepared from isolated rat cerebral microvessels. EC contained 1% or less PC, and PC had no EC contamination. Cultures received calcium ionophore, A23187 (luM) for varying periods, and analyzed for expression of zif268 mRNA by northern analysis. Results : CNS EC showed low constitutive levels of zif268, whereas PC basal levels were high. In response to A23187, CNS EC zlf268 is significantly upregulatod by 1 hr. In PC, however, zif268 levels were not significantly altered. This may mean that PC are maximally upregulated for zif268 or unresponsive to A2.3187. Conclusions: Differences in constitutive expression of zif268 may suggest differences in trophic factor influences between PC and EC. However, as zif268 encodes a transcription factor and has been associated with cell plasticity and learning, its constitutive expression in PC is intriguing, zif268 is an appealing candidate for a "plasticity gene" and is likely to be important in trauma.
P08.01 ~EURDIMMUNOLDGYOF~.Ai~pHINE~DD~CTIDN R. A~.: AZ~yev~ Z. N. " A l t y e v . Psycho-Nev~OIOgy'D~spR~eP'o~Baku city T h i r t y male morphine a d d i c t i o n pa%ients aged ( 2 9 . 0 + 7 . 0 years> c o n t i t u t e d t h e s t u d y samle. The l e v e l s o f dopaminl s o m a t o t r o p i ~ , cGMP, and a l l t h e n a t u r e T-lymphocytes p o p u l a t i o n ~ were significantly reduced Jr, nonabst inence c h r o n i c morphine dependencea a d d i c t i o n compared w i t h c o n t r o l s . Whereas t h e l e v e l o f s e r o t o n i n , GABA~ ACTH, pPolactin~ cAMP, and t h e number o f B lymphocytesaand~ pPothymocytes were found t o be h i g h e r t h a t t h e eontnol values. The c o n c e n t r a t i o n o f i ~ m u n o g l o b u l i n s a l s o were h i g h e r than the h e a l t h y i n d i v i d u a l s . The agents a d m i n i s t e r e d may be d i v i d e d i n t o t w o g r o u p s w i t h r e s p e c t t o t h e i r a c t i o n on t h e r a t i n g o f c r a v i n g For mo~'phine and t h e i ~ e f f e c t on t h e n e u r o e n d o c r i n e and immunological parameters. The f i r s t group i n c l u d e s p a r l o d e l and i n t e r f e r o n . In a d d i c t i o n t o an decreasing t h e rating of c r a v i n g for morphine thes~ agonts promoted a t~'end towards normaiizatior~; the second g~oup - d a l a r g i n led t o a d e t e r i o r a t i o n t h e r a t z n g o f c r a v i n g f o r morphine and i n e r e a sed d i s t u r b a n c e o f t h e neuroendoc;~ine and i ~ u n o l o g i c a l parameters.
~07.05 THE C08"nMULATORY MOLECULE B'/ 18 E]Cw~Ei~ED IN CULllJRED HUMAN MICROGLIA AND IN MULlrlPLE SCLEROGB AculrE LESIONS. R. De Slmone t, A. Glsmpeolo 2, B. Giometto 3, P. Gallo3, G. Levi1, C. Peschle2 and F..~ohll 1. IL8~. of ~ ~ System ~ o l l j f s [ o J ~ f
P04.01 DETERGENT SOLUBILITYOF CLASS I ANTIGENS PRESENT IN CSF. JC A L V A R E Z - C E R M E N O , L M VILLAR, M NOCITO, JG SEGOVIA, A SERRANO, C PALACIOS, P G O N Z A L E Z PORQUE. The source of soluble class I antigens in cerebrospinal fluid (CSF) has not been s t u d i e d in detail, however, an active s e c r e t i o n from activ a t e d lymphocytes w i t h i n b l o o d - b r a i n b a r r i e r (BBB) has b e e n suspected. A n o t h e r p o s i b i l i t y is s h e d d i n g from classic m e m b r a n e - b o u n d H L A (mHLA) Both proteins differ in t h e i r s o l u b i l i t y properties; sHLA s e c r e t e d b y l y m p h o c y t e s is t o t a l l y found in aqueous phases w h i l e m H L A has higher hydrophobicity. W e have p e r f o r m e d a s o l u b i l i t y study of CSF sHLA in T r i t o n TX 114 in CSF from patients w i t h MS, or m e n i n g i t i s w i t h BBB damage. We found that in MS cases w i t h normal BBB function, over 99 percent of CSF H L A appears in the aqueous phase. In cases w i t h b r e a k d o w n of BBB, 10 to 30% of HLA is h y d r o p h o b i c (similar to values found in serum) (p<0.05). We c o n c l u d e that the increased levels of s H L A in MS are due to intrathecal l y m p h o c y t e synthesis, w h i l e in the cases of BBB damage, the h y d r o p h o b i c H L A originates from serum.
P05.01 MYELIN OLIGODENDROCYTE GLYCOPROTEIN RELAPSING DEMYELINATING EAE IN MICE
(MOG)
INDUCES
S. Amorl. J.K. O'Neill2, N. Oroome3, M,M. Morris t and D. Baker 2 1Department of Immunology.Ray.e Inslltute, UMDS, at.Thomas' Ho~pitni, Londoa, UK; 2Department of Clinical Ophthalmology,Imzituteof Ophthnimniogy,London, UK; 3Multiple Scl¢ro~isSocietyPeptld¢Laboratory,OxfordBrookesUniversity.Oxford,UK. Introduction: MOG is a specific, myelin antigen which may provide a target for antibody.mediated demyelination, following T cell mediated blood-brain barrier dysfunction. MOG is also a minor myelin protein, which has complicated purification of sufficient m a ~ a l to determine encephaiitogenic potential in vivo. Materials and Methods: Overlapping (8 amino acids) synthetic 15mar peptidus corresponding to the whole MOG molecule were used to actively sensitise EAEsusceptible Biozzi AB/H (H-2dqt) and SJL (H-2s) mice. Results: In contrast to the identification of single encephaiitogenic epitopes within the PLP (residues 54-76) and MBP (residues 12-35) molecules io AB/H mice, multiple epitopes within MOG residues 1-22, 43-57 and 134-148 induced clinical and/or histological disease in Biozzi AB/H mice. However in the SJL mouse a single epitope wifltin residues 92-106 was capable of inducing clinical and histological EAE of a comparable magnitude as that observed following immunisation with either PLP or MBP peptides. Furthermore MOG-immtmised SJL mice exhibited a relapsing disease progression and demyelinated CNS lesions. Conclusions: Immune responses to MOG induce clinical EAE, clinically silent histological lesions and a relapsing-remitting, demyelinating disease. Therefore MOG must be considered as a potential autoantigen in multiple sclerosis, where different MHC haplotypes may respond to distinct regions of the molecule.
P02.01 I~r~;RFERON-~ m R N A EXPRESSION IN' ~ L t , VIVO CS]F T CRI.I.~
T E L Y EX
Manon Blain, Jnsuphine Nalbantoglu, Jack P. Antel
2L~. of HenlMolo~-Oncelogy, Istltufo Superiors di Sant~, Rome; 311
Neu,,ofoglcC~c, Univer~ of Padova, Padova, #sly. Introduction: To alucldeta tha mechsnlsms of T cell activation in the CNS, expression of B7, a major cosltmulatow molecule, was examined: 1) in vttro, in two APC popelatlons of tha brain, mlcloglla and aatrocytes, and 2) in situ, in post-mortam brain tissue from multiple solercets (MS) petlants. Methods and Results: Using Immunocytocheminal and PCR techniques, we found that in cultures from the human amb~onl¢ brain mlcroglla, but not astrocytes, constitutively express B7. Mlcrogllal expression of B7 wa~ Increased by IFN-7, but not TNF-a, IL-11~,M-CSF, GM-CSF or LPS. Human mlcrnglis also expressed the accessory molecules LFA-1, LFA-3 and ICAM-1. In sciive MS lesions, B7 Immunoreactlvlty was detected both on infiltrating macrophngex and activated mlcrnglia, while in chronic lesions only pedvaecular cells were faintly mined. No B7 immunoreacUvlty was found in brain tumor, Alzhelmer dlsance and normal control brain tissue specimens. Conclusions: Taken together the shove findings suggest that B7 may play s mla in T cell activation and lesion development In MS and that the regulated expression of B7 on micro~ia may contribute in the local stimulation of T cell proliferation and effector functions. (Supported by CNR grant n.93.00472.PF40, Project on Aging).
Montreal Nanrologteal Institute Introduction - We compared interferon (IFN)-,y mRNA expression between immediately ex-vivo CSF- and peripheral blood (PB)-derived T cells using a PCR amplification technique to overcome the limitations imposed by cell numbers present in CSF samples. Materials and Matheds - Total RNA was extracted from 3-5 cc of CSF and from T cells isolated from PB by gradient centrifugation and E + rosetting, c D N A was synthesized by reverso transcribing the R N A using random primers. The eDNA was amplified using specific primers for CD3 and IFN-~; the product was analyzed on agarose gels. Results - Using CSF samples which contained 2-5xl0' cells, a PCR product was obtained using both the CD3 and IFN-,y primers. Using PB cDNA derived from up to 10x greetar T-cell number than in the CSF samples, only a CD3 primer product was obtained. Conclusion - The expression of IFN-,y mRNA in immediately ex-vivo T cells derived from CSF, but not PB, indicates furthar distinction in properties of cells in the systemic and intrathecal compartments, with activation being a characteristic of the formar.