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Exogenously administered (MET)-enkephalin alters the expression of CD4+ T lymphocytes in inbred strains of mice
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W06.06 TRANS-,SPECIES ACTIVATION OF A MYELIN SA~IC P R O ' J ~ ~ P ~ ' C l F I C HUMAN T CELL CLONE USING APC FROM MHC-COMPATIBLE RHESUS MONKEYS E. Melql*, B. A. 't Hart#, R. E. Bontrop#, R. M. Hoch*, A. Iglesias*, R. de Wanl Melefyt+, H. Fickenscher ~, ]. M011er-Fleckenateint, B. Fleekensteln ~, H. Wekerle*, R. HoNfeld*', and M. Jonker# "Dpt. of Nsuroimmunol., Max-Planek Institute of Psychiatry, Martinsried, Germany; #BPRC, Rijswijk, Netherlands; tlnstitut for Kliniscbe und Molekulore Virol., Univ. Erlangen-N0rnberg, Erlangen, Germany; *Human Immunol. Dpt, DNAX, USA; "Dpt. of Neurni., Univ. of Munich, Germany. For present address of E.M. see ~ Introduction: The pathogenic potential of human autoreactive T cell lines cen only be tested in vivo. There are two principal requirements for an animal model in which human T cells and host animal cells interact, First, sufficient numbers of human T cells must be availble for transfer. Second, the human T cells must recognize their antigen in the context of host MHC and adhesion molecules. The first requirement can be mat by transformation of human Ag-specifie T cells with Herpesvirus saimiri {HVS) {Weber at el. PNAS 90:11049-11053). Materiels, Methods and Results To meet the second requirement, we identified one /qLA-DR81*0301 restricted CD4 ÷ Thl T cell clone that can recognize MBP (peptide 29-48) in the context of Mamu-DRBI "0305 or -DR81"0306. The HVS-transformed T cells could be stimulated with MBP and rhesus PBMC to produce IFN-y. Conclusion: In certain MHC combinations, both HVS-transformed and nontransformed human T cells can recognize their Ag on rhesus APC.
Pl1.11 Detection of Human Heq:esvirus-6 sequences in the plaques of Multiple Sclerosis patients by PCR. E. Merelli 1, P. Barozzi 2. M. LcvanP. G L. Mancardi 3. I Clinic of Neurology, 2 Center for Experimental Haematology, University of Modena: 3 Clinic of Neurology, University of Geuova - Italy. Human H e r ~ i m s - 6 (HHV-6) is a ubiquitous lymphotropic virus also tropic for glioblastoma and neuroblastoma cell lines as well as for embryonic gila, and its possible involvement in neurologic disorders has been suggested. In a previous study, we have found significantly higher anti-HHV-6 antibody titers in multiple Sclerosis (MS) patients than in healthy contmts, but HHV-6 specific sequences were detected in the PBMCs of only one case (J Neurol Neurosurg P~chtatry 1993; 56: 917-919). To determine whether these resolls were due to the u n s ~ E c role of the PBMCs in the p ¢ t h o g ~ m e ~ l t m of MS. we decided to examine the demyelinated brain plaques of three MS patients in acute phase of inflammation and the medullary plaques of one patient affected by optic neuromyelitis. We used the polymerase chain reaction (PCR) assay on the DNA extracted from formalin-fixed and paralfin embedded tissues to look for the presence of HHV-6 sequences. The pair of primers used was derived from the ZVHI4 seqnence located to the left end of the HHV-6 (strain GS) gcnome. HHV-6 sequences were clearly identifiable only in the medulla specimen of the patient with optic neuromyelitis examined. At present we cannot draw any conclusion in favour of or against a possible role of HHV-6 in the development of stone MS cases. However. the presence of HI-IV-6 geuome in the demyelinated lesion is strongly suggestive and supports further sludies to clarify the eneephalitogeuic natere of this vires.
P06.14
P08.11
CHANGES IN INTERI.~UKIN-I BETA (IL-I BETA) AND AMYLOID PREC~ P R O T E ~ (APP) E ~ N ]FOLLOWING T I ~ U M A T I C BRAIN INJURY 1N THE RAT Multes~ L ~ . 1, Hillhocae, E.W.2, Cann~ AJ. 3, Cmu:ly,J.M. 1, Sharpies, p.M. 4 1MRC Neurochemicel Pathology Unit, Newcasde General Hospitsl, 3pae~alric Neurcecieace Research Unit, University of Newcastle Upon Tyne, 2Depm'tment of Medicine and 4institute of Child Health. University of Bmtol, United Kingdom
EXOGENOUSLY ADMINISTERED (MET)-ENKEPHALIN ALTERS THE EXPRESSION OF CD4 + T LYMPHOCYTES IN INBRED STRAINS OF MICE
Head injm'y is a major cause of death and acquired handicap in children and young adults.Head trauma is also implicated in the p s ~ h o ~ s of dementia pugifistica (DP) and has been identified as n risk factor for Ab,heimec's Disease (AD). Rapid deposition of the AD prot~a, APP, has been observed following a single severe heeds. T h e h i F e a : a m p ~ i ~ a m i c a l a ~ ~ a : e p t i t ~ to,he ~,-~,,d~__,y pathology of head in~u~ ~ isalsoprimaail" y invalv¢(JIn the~ 0 g Y of AD~ D P also shares many featuresin common with A D including delx~itionof APP/A4. Thus it has beea hypothcsisedthathead trauma may play an important rolein the pathogenesis of A D and conversely A P P may also play an important role in the secondary pathology of head ~ IL- l beta has been shown to uprcgulateextxession of the APP gene in vitro and there is evidence to suggest IL-1 beta expression is upregulated in b ~ - - a t ~ , cells in the Ix'ain after head injury. In this study we have used immtmocytochemistry to daoraeterian the time course and disiribution of IL-I beta and APP expression and microgfial,cell activation in a fluid percussion model of concussive brain injury in the rat. Preliminary data indicates early (424hrs) activalion of mimo~ial cells in the ipsilateml on~ex and hippoeampos. This is spatially and temporally associnted with increased APP and IL-1 beta immunoreactivity. These results support a r d e for inflammatory mica~inl cells and IL-I beta in th~ aoeumulation of AFP and the secondary pathology in the hippoeampus following head trauma.
P l l .10 CHARACTERIZATION OF PASSIVELY TRANSFERRED CELLS CAPABLE OF PROVIDING PROTECTION AGAINST TMEVINDUCED DEMYELINATING DISEASE IN BALB/c MICE. Ro~er W. Melvold, S/morn N. N/chobo~ Mauro C. Dal Canto and S t e v i ~ D. M/ller. Dinma~nsn~ of ~ v d n m u m o l o z v and Pa-tlmloav, Northwcetimn ~ Mud/cal Schc~L Clntca _~o. ll]inolL US& 60611i Introdtsetinn. Infection with Tbeihr's Murizw Emx~halmnyelRis Virus (TMEV) leads to u ~ ~ t o r y d ~ y e l t n a U n g disease in some strulns/suhe~eins of mice. Among BALB/c subs~ains, mmceptibility can be either genetically determined (BALB/cAnNCr) or Induced in resistant subet/~-:--~ (BALB/cByJ) by low d o ~ irradiation prior to /_n~t~on. Both B A L E / ~ C r and irradiated EALB/cSyJ mice can be protected from development of demyelimling dMeane by the adopt/ve t~ansfer of splann cells from u ~ I r r ~ / n l ~ c t e d BALB/cByJ donor. a n d Ma4h~Is. Ado1~ve tzemf~r ~ l ~ n / m ~ s were done using donc~ S l ~ poohs ~ or depleted of various lymphocyte subpopuMtions, Results. Protection s g a i m t demyelinating disease is best ass
M. Metier. C.P. Ho, J. Acosta State University of New York, College at Old Westbury Old Westhury, New York, 11568 USA Introduction: The focus of this study was to elucidate the interaction between an exogeaously administered opiold on both developing and mature T lymphocytes in mice of diffenmt ages and differant genetic backgrounds. Mala'tals and Methods: Mice of several distinct inbred lines were administered 0.02mg/kg of animal weight (me0-enkopludin over a period of 7 or 14 days (via a subcuameons ommtic pump). Thymus m d lymph nedm were ~amvedat specified times (3, 7, 10 or 10, !4, 17 days ~ deug__t~toneat~. ~.~tl~e extracted cells separated by a magnetic column and analyzed by flow cytometry. Results: In nnce of the RF/J strain there is an increase in CD4 + T cells, but notinCD8 + ceils, that is detectable by day 7 and persists for up to6 days after ~lmini~ratiou ofdrttg. An increase in CD4 + Tcellsin mice of the Balb/C strain is only evidenced during drug treatment. In addition, there are differences that are delmmdant on the age and disease state of the animals. We have also demonstsated that we can suppr~msthis effect on the immtme system by altering the doee of the opinld. Condmkm: Previously we had shown that the levels of endogenous (met)enkephalinvsxieddeponding on the age, genetic~ and ~ state of the animal. In thesestndieswe have demonstrated thatthe effectof exoge~mmly edtomistared drug is dependant on the mmaeparameters.
P16.03 I M M U N O R E G U L A T O R ¥ EFFECTS O F R E C O M M N A N T MICROVESSEL ENDOTHELIAL CELLS. INTERACTIONS W I T H IFN-y. Ariel Miller I , Neural Laeir2, Michel Revel3, Silvia Honigman I , Amelia ~ - - - - ~ ' ] ' - ~ Nitza l.ad~ 1; 1Carmel and 2Ramham ~ , biafra & 3The W ~ Inetimteof Science,Rehovot. Israel. lntreduetion: The mechanism(s) of action responsible for the effects of IFN-p are still elusive although suggestions include anti-viral effect, Ihe enhancement of NK or mplmmsm T 0all activity and oRooaition of the effects of inflammatory cytokines. Matllelrlals mud Metlt~la: As vascular endothelial ceils are active partinilmam in iullanmmaney end demyelianting ixocesses,wede~led toexamine the e f f ~ of IPN,~oa the e x ~ of major histoec~imtibilitym m ~ x (MHC) l~*nepmdee~ aad the adhesion molecules ICAM-I on human miecoveslal ~ad01hl~ial cLqls (HUVEC). R e s u l t s : Intact human umbelical EC did not eXln¢~ MHC clmm-lI (Ia) molecules, lncuhetio~ of F.C with IFN-y (0.1-5{}0 IU/ml) induced Ia expression in a dose dependent mamaar. Treatment of EC mmolayers with recombinant hanura IFN-I~(10-500 IUlml) foe 96 hours failed to induce la antigens, howevec, c o - i ~ of EC with IFN-,f (10 IU/m]) and IFN-~ (10-500 IU/ml) resulted in signific4mt downml~lation of la molecules expressionwbiea was ~ r , ~ l y ~ on IPN-I~ceeesnmdion. M H C class-I and 1CAM-I mofect~a were ~ eXlmmaed on HC. S p a a ~ expmsmen of ICAM-I molecule w ~ uralffected by II~I.,.¢ but e~ahamsedby IFN-~ in a dine dqmadant maanec. IvK-ICcinm-i expremion on EC were euhanced by IFN-.y u web u by I R ~ . CUlllehraiuu: our study 8 u ~ t s that both IFN-y and I F I ~ exestlt . ' :~ endothelial cells with ¢fifferential modulatory effects on various cell surface markers.
W06.06 TRANS-,SPECIES ACTIVATION OF A MYELIN SA~IC P R O ' J ~ ~ P ~ ' C l F I C HUMAN T CELL CLONE USING APC FROM MHC-COMPATIBLE RHESUS MONKEYS E. Melql*, B. A. 't Hart#, R. E. Bontrop#, R. M. Hoch*, A. Iglesias*, R. de Wanl Melefyt+, H. Fickenscher ~, ]. M011er-Fleckenateint, B. Fleekensteln ~, H. Wekerle*, R. HoNfeld*', and M. Jonker# "Dpt. of Nsuroimmunol., Max-Planek Institute of Psychiatry, Martinsried, Germany; #BPRC, Rijswijk, Netherlands; tlnstitut for Kliniscbe und Molekulore Virol., Univ. Erlangen-N0rnberg, Erlangen, Germany; *Human Immunol. Dpt, DNAX, USA; "Dpt. of Neurni., Univ. of Munich, Germany. For present address of E.M. see ~ Introduction: The pathogenic potential of human autoreactive T cell lines cen only be tested in vivo. There are two principal requirements for an animal model in which human T cells and host animal cells interact, First, sufficient numbers of human T cells must be availble for transfer. Second, the human T cells must recognize their antigen in the context of host MHC and adhesion molecules. The first requirement can be mat by transformation of human Ag-specifie T cells with Herpesvirus saimiri {HVS) {Weber at el. PNAS 90:11049-11053). Materiels, Methods and Results To meet the second requirement, we identified one /qLA-DR81*0301 restricted CD4 ÷ Thl T cell clone that can recognize MBP (peptide 29-48) in the context of Mamu-DRBI "0305 or -DR81"0306. The HVS-transformed T cells could be stimulated with MBP and rhesus PBMC to produce IFN-y. Conclusion: In certain MHC combinations, both HVS-transformed and nontransformed human T cells can recognize their Ag on rhesus APC.
Pl1.11 Detection of Human Heq:esvirus-6 sequences in the plaques of Multiple Sclerosis patients by PCR. E. Merelli 1, P. Barozzi 2. M. LcvanP. G L. Mancardi 3. I Clinic of Neurology, 2 Center for Experimental Haematology, University of Modena: 3 Clinic of Neurology, University of Geuova - Italy. Human H e r ~ i m s - 6 (HHV-6) is a ubiquitous lymphotropic virus also tropic for glioblastoma and neuroblastoma cell lines as well as for embryonic gila, and its possible involvement in neurologic disorders has been suggested. In a previous study, we have found significantly higher anti-HHV-6 antibody titers in multiple Sclerosis (MS) patients than in healthy contmts, but HHV-6 specific sequences were detected in the PBMCs of only one case (J Neurol Neurosurg P~chtatry 1993; 56: 917-919). To determine whether these resolls were due to the u n s ~ E c role of the PBMCs in the p ¢ t h o g ~ m e ~ l t m of MS. we decided to examine the demyelinated brain plaques of three MS patients in acute phase of inflammation and the medullary plaques of one patient affected by optic neuromyelitis. We used the polymerase chain reaction (PCR) assay on the DNA extracted from formalin-fixed and paralfin embedded tissues to look for the presence of HHV-6 sequences. The pair of primers used was derived from the ZVHI4 seqnence located to the left end of the HHV-6 (strain GS) gcnome. HHV-6 sequences were clearly identifiable only in the medulla specimen of the patient with optic neuromyelitis examined. At present we cannot draw any conclusion in favour of or against a possible role of HHV-6 in the development of stone MS cases. However. the presence of HI-IV-6 geuome in the demyelinated lesion is strongly suggestive and supports further sludies to clarify the eneephalitogeuic natere of this vires.
P06.14
P08.11
CHANGES IN INTERI.~UKIN-I BETA (IL-I BETA) AND AMYLOID PREC~ P R O T E ~ (APP) E ~ N ]FOLLOWING T I ~ U M A T I C BRAIN INJURY 1N THE RAT Multes~ L ~ . 1, Hillhocae, E.W.2, Cann~ AJ. 3, Cmu:ly,J.M. 1, Sharpies, p.M. 4 1MRC Neurochemicel Pathology Unit, Newcasde General Hospitsl, 3pae~alric Neurcecieace Research Unit, University of Newcastle Upon Tyne, 2Depm'tment of Medicine and 4institute of Child Health. University of Bmtol, United Kingdom
EXOGENOUSLY ADMINISTERED (MET)-ENKEPHALIN ALTERS THE EXPRESSION OF CD4 + T LYMPHOCYTES IN INBRED STRAINS OF MICE
Head injm'y is a major cause of death and acquired handicap in children and young adults.Head trauma is also implicated in the p s ~ h o ~ s of dementia pugifistica (DP) and has been identified as n risk factor for Ab,heimec's Disease (AD). Rapid deposition of the AD prot~a, APP, has been observed following a single severe heeds. T h e h i F e a : a m p ~ i ~ a m i c a l a ~ ~ a : e p t i t ~ to,he ~,-~,,d~__,y pathology of head in~u~ ~ isalsoprimaail" y invalv¢(JIn the~ 0 g Y of AD~ D P also shares many featuresin common with A D including delx~itionof APP/A4. Thus it has beea hypothcsisedthathead trauma may play an important rolein the pathogenesis of A D and conversely A P P may also play an important role in the secondary pathology of head ~ IL- l beta has been shown to uprcgulateextxession of the APP gene in vitro and there is evidence to suggest IL-1 beta expression is upregulated in b ~ - - a t ~ , cells in the Ix'ain after head injury. In this study we have used immtmocytochemistry to daoraeterian the time course and disiribution of IL-I beta and APP expression and microgfial,cell activation in a fluid percussion model of concussive brain injury in the rat. Preliminary data indicates early (424hrs) activalion of mimo~ial cells in the ipsilateml on~ex and hippoeampos. This is spatially and temporally associnted with increased APP and IL-1 beta immunoreactivity. These results support a r d e for inflammatory mica~inl cells and IL-I beta in th~ aoeumulation of AFP and the secondary pathology in the hippoeampus following head trauma.
P l l .10 CHARACTERIZATION OF PASSIVELY TRANSFERRED CELLS CAPABLE OF PROVIDING PROTECTION AGAINST TMEVINDUCED DEMYELINATING DISEASE IN BALB/c MICE. Ro~er W. Melvold, S/morn N. N/chobo~ Mauro C. Dal Canto and S t e v i ~ D. M/ller. Dinma~nsn~ of ~ v d n m u m o l o z v and Pa-tlmloav, Northwcetimn ~ Mud/cal Schc~L Clntca _~o. ll]inolL US& 60611i Introdtsetinn. Infection with Tbeihr's Murizw Emx~halmnyelRis Virus (TMEV) leads to u ~ ~ t o r y d ~ y e l t n a U n g disease in some strulns/suhe~eins of mice. Among BALB/c subs~ains, mmceptibility can be either genetically determined (BALB/cAnNCr) or Induced in resistant subet/~-:--~ (BALB/cByJ) by low d o ~ irradiation prior to /_n~t~on. Both B A L E / ~ C r and irradiated EALB/cSyJ mice can be protected from development of demyelimling dMeane by the adopt/ve t~ansfer of splann cells from u ~ I r r ~ / n l ~ c t e d BALB/cByJ donor. a n d Ma4h~Is. Ado1~ve tzemf~r ~ l ~ n / m ~ s were done using donc~ S l ~ poohs ~ or depleted of various lymphocyte subpopuMtions, Results. Protection s g a i m t demyelinating disease is best ass
M. Metier. C.P. Ho, J. Acosta State University of New York, College at Old Westbury Old Westhury, New York, 11568 USA Introduction: The focus of this study was to elucidate the interaction between an exogeaously administered opiold on both developing and mature T lymphocytes in mice of diffenmt ages and differant genetic backgrounds. Mala'tals and Methods: Mice of several distinct inbred lines were administered 0.02mg/kg of animal weight (me0-enkopludin over a period of 7 or 14 days (via a subcuameons ommtic pump). Thymus m d lymph nedm were ~amvedat specified times (3, 7, 10 or 10, !4, 17 days ~ deug__t~toneat~. ~.~tl~e extracted cells separated by a magnetic column and analyzed by flow cytometry. Results: In nnce of the RF/J strain there is an increase in CD4 + T cells, but notinCD8 + ceils, that is detectable by day 7 and persists for up to6 days after ~lmini~ratiou ofdrttg. An increase in CD4 + Tcellsin mice of the Balb/C strain is only evidenced during drug treatment. In addition, there are differences that are delmmdant on the age and disease state of the animals. We have also demonstsated that we can suppr~msthis effect on the immtme system by altering the doee of the opinld. Condmkm: Previously we had shown that the levels of endogenous (met)enkephalinvsxieddeponding on the age, genetic~ and ~ state of the animal. In thesestndieswe have demonstrated thatthe effectof exoge~mmly edtomistared drug is dependant on the mmaeparameters.
P16.03 I M M U N O R E G U L A T O R ¥ EFFECTS O F R E C O M M N A N T MICROVESSEL ENDOTHELIAL CELLS. INTERACTIONS W I T H IFN-y. Ariel Miller I , Neural Laeir2, Michel Revel3, Silvia Honigman I , Amelia ~ - - - - ~ ' ] ' - ~ Nitza l.ad~ 1; 1Carmel and 2Ramham ~ , biafra & 3The W ~ Inetimteof Science,Rehovot. Israel. lntreduetion: The mechanism(s) of action responsible for the effects of IFN-p are still elusive although suggestions include anti-viral effect, Ihe enhancement of NK or mplmmsm T 0all activity and oRooaition of the effects of inflammatory cytokines. Matllelrlals mud Metlt~la: As vascular endothelial ceils are active partinilmam in iullanmmaney end demyelianting ixocesses,wede~led toexamine the e f f ~ of IPN,~oa the e x ~ of major histoec~imtibilitym m ~ x (MHC) l~*nepmdee~ aad the adhesion molecules ICAM-I on human miecoveslal ~ad01hl~ial cLqls (HUVEC). R e s u l t s : Intact human umbelical EC did not eXln¢~ MHC clmm-lI (Ia) molecules, lncuhetio~ of F.C with IFN-y (0.1-5{}0 IU/ml) induced Ia expression in a dose dependent mamaar. Treatment of EC mmolayers with recombinant hanura IFN-I~(10-500 IUlml) foe 96 hours failed to induce la antigens, howevec, c o - i ~ of EC with IFN-,f (10 IU/m]) and IFN-~ (10-500 IU/ml) resulted in signific4mt downml~lation of la molecules expressionwbiea was ~ r , ~ l y ~ on IPN-I~ceeesnmdion. M H C class-I and 1CAM-I mofect~a were ~ eXlmmaed on HC. S p a a ~ expmsmen of ICAM-I molecule w ~ uralffected by II~I.,.¢ but e~ahamsedby IFN-~ in a dine dqmadant maanec. IvK-ICcinm-i expremion on EC were euhanced by IFN-.y u web u by I R ~ . CUlllehraiuu: our study 8 u ~ t s that both IFN-y and I F I ~ exestlt . ' :~ endothelial cells with ¢fifferential modulatory effects on various cell surface markers.