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Four year follow-up of multiple sclerosis (MS) patients treated with alpha-2A interferon
180
P06.13 y-INTERFERON
LYMPHOOYTES
AOTIVATES
A
FROM MULTIPLE
NEW
Ca 2+
SCLEROSIS
INFLUX IN T-
PATIENTS
G. Martins 1,2, E. Clemsnti 3, E. Brambilla 1, L. Moiola 1,2, G. Comi2, L.M.E. Gdmaldil, = 1Neuroimmunology Unit - DIBIT and =Dept. of Neurology, Univ. of Milano, San Raffaete Scientifi~ Institute, Milano, ttaty; 3Chair of Pharmacology, Faculty of Pharmacy, Univ. of Reggio Calabria, Catanzaro, Italy. INTRODUCTION: Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system of unknown etiology. Activated T-lymphocytes are considered essential in the pathogenesis of MS. They o~rate throug h a complex nstwork of cytoldnes among which 7" the relationship between activated T-cell, ?-[FN and intracellular Ca 2+ in lymphe~ from 8t MS patients, 35 control patients and 22 healthy subjects by r,onventional fluodmetry. RESULTS: Expomm= to 7-IFN of Tlymphocytes from MS patients activates, via a protein kinase C-mediatsd pathway, a new 7-1FN-actlvated Ca=+ influx, functionally coupled to the y-IFN receptor. The influx, mainly expressed by CD4* T-lymphocytes, was found in 24 of 32 (75%) patients with clinically active MS and in 26 of 59 (44%) patients with stable MS. The influx was found in only 6 of 35 (17%) control patients and in 3 of 22 (14%) healthy subjects studied: CONCLUSIONS: Our resulls document the appearance in MS lymphocytes of a 7-1FNactivated, protein kinase C-dependent, Ca2. influx which might be due to the expression of a new cation-specific plssmalemma channel. This finding suggests that at least part of 7-1FN contribution to the pathogsnesis of MS is exertsd through a CaS+-dependent regulation of T-lymphocyte activity.
P03.11 AN I M M U N O H I S T O C H E M I C A L S T U D Y OF THE PDGF IN THE M U L T I P L E S C L E R O S I S A C U T E PLAQUES. A.R.Massaro, G.Carbone, A.Carnevale "Sacro Cuore" University Medical School, Rome, Italy. PDGF is involved in the regulation of growth end differen tiation of glial cells. It was found that type-i astrocy~ tee produce a PDGF-Iike molecule to control the O-2A glial progenitor cell differentiation into oligodendrocytes end type-2 sstrocytes. PDGF also increases the expression of ~fl4CClass II antigens on antigen-presenting cells, and mo dulates ~-interferon production by helper T-cells. These-features prompted us to make a preliminary imm~nohistoche mical study in order to localize the PDGF in normal and MS brain white matter. Paraffin sections of acute MS plaques end apparently healthy white matter were treated with a polyclonal goat anti-human PDGF IgG (Collaborative Res.,Ine.). Specimens of white matter fr~n patients who died for non-neurologlcal diseases were taken as controls. A Veetastain-ABC kit for goat-IgG was then used. Mieroseo pie observation showed that anti-PDGF stained the normal-white matter of both control and MS patients, while the acute plaques did not stain. This observation could suggest that a PDGF-Iike antigen is present in normal white matter.0n the other hand,the possibility that formaline fixation and paraffine embedding could alterate the antige nic properties of the tissue must be taken into account.
P16.02
THREE Y E A R T R E A T M E N T W I T H A L P H A - 2 A I N T E R F E R O N OF A B E H ~ E T D I S E A S E P A T I E N T A.R.Massaro, A.Carnevale, C.Rumi "Sacro Cuore"Univarslty Medical School, Rome, Italy. A 28 year-old woman with recurrent aphtous ulcers, cutane our lesions,ocular manifestations, and neurological syrup~ toms associated with almormal brain ~ I , of 14 years dura tion, was d i a g n o s e d as having Behfe~ disease. The coursewas relapslng-remltting, with ocular and neurological sequelae. No treatment was efficient in stopping the recurrent relapse@. T r e a ~ t with human recombinant =(-2A inte rferon (3xlO'IU IM every three days) was then started inorder to demonstrate e possible Beneficial effect on the course of the disease and on s o m e laboratory parameters we monitored. General and neurological conditions, with brain MRI and lymphocytes subsets were followed-up, together with routine laboratory tests. During the three year we noticed: a sharp reduction of the m~cocutaneous end ocular manifestation, which were also much less pronounced end less painful; a significant improvement of the neurological examination; a clear reduction in the previously frequent epileptic seizures; no appearance of new lesions at the serial MRI controls, neither gadolinium enhae~ent ; and a no~mallzation of the E~G. The lymphocytes subsets showed a marked increase of CD8 and a decrease of CD4 cells. No significant side effects were shown, nor alterations of the routine laboratory tests.
W12.04 FOUR Y E A R F O L L O W - U P OF M U L T I P L E S C L E R O S I S (MS) PATIENTS TREATED WITH ALPHA-2A INTERFERON. A . R . M a s s a r o , A . C a r n e v a l e , C.Rumi, A . L a u d i s i o "Sacro Cuore"University Medical Sehool, Rome, Italy. We previously reported promising results with the use of htmmn recombinant c~-ZA interferon in 14 MS patients, all over 40 years of age, for a period of 2 years (ISNI Congress'91). Here we report about an additional two year period of observation on the same patients, 7 of whom decided to continue the treatment. The treatment schedule was slightly modified: 3xlO~IU IM every three days, without any interruption. The patients were evaluated by using eli doliniom, iymphoeytes subsets,and
routine
laboratory
tests.
None of the treated patients presented any major side effects, nor alterstions in the routine tests. Of the 7 treated patients, only one relapsed during the observation pe riod,with no increase on the Kurtzke's scales at the final biennial evaluation. Of the 7 untreated patients, only 2 remained stable; the others experienced relapses, some of them with a permanent residual w o r s e n . ~ F r o m this dr~rn we can suggest that: Long-term treatment with ~-interferon appears to be safe even after 4 years; the supposed benefi cial effect experienced during the first biennium is lost when the treatment is stopped; continuing the treatment probably mantains a positive biological activity on the course of MS. W11.02 RELEVANCE OF H U M O R A L A N D cr~ J.ULAR IMMUNEFY IN THE CENTRAL NERVOUS SYSTEM IN HAbl/TSP. M. Mat~ai and Y. Kuroda Div. Neurol., Dept. Internal Medicine, Saga Medical School, Saga, JAPAN
l n . . ~ t i o n : HAM/TSP is a disease rarity of chrodic m ~ y a~ociated wirehuman T l y n ~ virustypeI.A l t h ~ m a w / i a ~ have revealedthe~ ofimmone ~ m thisdls~fdWr,~ pathological pmceses keding m the Msion fornmfion in h'le central nefvons system (CNS) have not been clarif~d.
Material~a~l Metho~: Twea~-foor patleats with HAM/FSP were compared with 39 patients with other nonintlmnnmtt~ neamiogkal diseaaus in temls of IgG nod ~,2-imcroginbelin leveb ia the us~tm3qtim111~1 (CSF) nod imra. blood.brain ~ ' ~ lgG sy.:.~,=, in ,d4~t~n to ~ ~ of o~el~ infiltrates in the CSF and mmonucle~ ~ 04NC3 in the pe~0heml blood. MNC isolated were singl~stained with monoclonal anfil~dy (MoAb) CD3, CD20, or CD56, a~l a/so double-stained with combinalions of MoAb: CIM or CDg plus CDila, CD25, CD28, or CD45RO. Results: There were sigaifr.not increases in CSF IgG and [~2-microglobulin levels and a ~ of CD8+ T cells of a CDI l a + C D 4 5 R ~ . phenotype in the CSF of HAM/FSP. By conwast, a ~igaificmR incteue in activated CD4+ help~ T cells with CD25 rod/or CD45RO tattgam was observed in the blood o~ HAM/TSp. The CSF lgG leve.~ ssd de sovo IgG synthesis rate w~e si~dft~m~y dem.ussed with the dimaan ~l~immion,
one,=~on: Potentially ¢~olox~ ~ l l ~ ~ T ceils may play hn l m [ ~ U n t~'[~ I~ the L'V~3-1i~tth~-bTl~KlPO'rb-'~.
W10.01 CHARACTERIZATION OF C D 4 " C D S " c t ~ T C E L L S IN AUTOIMMUNE ENCEPHALOMYELITIS ~ h i l l t U / ~ 2 t , S. Ahe2; T. Shin 1, T. Kojima t and Y. I~ihara 1 l Depm'anent of Neuropethogy, Toky~ Metropolitan Institute for N ~ 2 DepurUnent of Neuropethology, Niigata University Brain Research Institute
Experimental autoimmuue encephalomyelitis (EAE) is a T cell-mediated disease characterized by the i~sonce of T cells and ~ e s mainly in the spirml cord. Recent studies have shown that eacephalitogenic T cells he~ a ~ number of V~ chains of T cell receptor (TCR) and play an important role not only in recraitment of inflammatory ceils, but also in lesion formation in the central nervoussysmm (UNSy.q:I6~,e~, theklneficsof ehcepffalll~efiii: T ~P.~reffial n p(x, rly underslood so far. In the present study, we addregsed this question by
characterizinguaiqnoT cellswhicha l ~ inthes,,~hnC,h~_space(SAS) of fats wilh the early stage of EAE. Row cytomctric analysis revealed timt more than 70% of T ceils in SAS we~ CD4"CD8- (double negative, DN) aftd bore a ~ TCR. Furthermore, the predominant V[~ in this lglgelation was shown to he V~t8.2 by ~ co~ s~. W e thench)n~ RT-PCR FroduasamplR-~d~ n g primexs for V[$8.2 and seqaenced nacleOti-l-l-l-l-l-l-l-~Of the j ~ _ _ ~ region of each clone. It was revealed that some clones isolated floe DN SAS T cells had a common motif sequence which was reported to be characteristic for enccphalitogenic T cells. Furthermore, the same sequence was recognized in clones from spinal cord and ! y ~ l i node CD4+ T cells of the same rat. DN SAS T cells were also detected in rats with passive EAE induced by ltmsfer of CD4 + e,cephalitogenic T cells. These findings suggest that DN ~ T cells in the SAS are a direct precursor population of encephalitogenic T cells in the CNS parenchyma.
P06.13 y-INTERFERON
LYMPHOOYTES
AOTIVATES
A
FROM MULTIPLE
NEW
Ca 2+
SCLEROSIS
INFLUX IN T-
PATIENTS
G. Martins 1,2, E. Clemsnti 3, E. Brambilla 1, L. Moiola 1,2, G. Comi2, L.M.E. Gdmaldil, = 1Neuroimmunology Unit - DIBIT and =Dept. of Neurology, Univ. of Milano, San Raffaete Scientifi~ Institute, Milano, ttaty; 3Chair of Pharmacology, Faculty of Pharmacy, Univ. of Reggio Calabria, Catanzaro, Italy. INTRODUCTION: Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system of unknown etiology. Activated T-lymphocytes are considered essential in the pathogenesis of MS. They o~rate throug h a complex nstwork of cytoldnes among which 7" the relationship between activated T-cell, ?-[FN and intracellular Ca 2+ in lymphe~ from 8t MS patients, 35 control patients and 22 healthy subjects by r,onventional fluodmetry. RESULTS: Expomm= to 7-IFN of Tlymphocytes from MS patients activates, via a protein kinase C-mediatsd pathway, a new 7-1FN-actlvated Ca=+ influx, functionally coupled to the y-IFN receptor. The influx, mainly expressed by CD4* T-lymphocytes, was found in 24 of 32 (75%) patients with clinically active MS and in 26 of 59 (44%) patients with stable MS. The influx was found in only 6 of 35 (17%) control patients and in 3 of 22 (14%) healthy subjects studied: CONCLUSIONS: Our resulls document the appearance in MS lymphocytes of a 7-1FNactivated, protein kinase C-dependent, Ca2. influx which might be due to the expression of a new cation-specific plssmalemma channel. This finding suggests that at least part of 7-1FN contribution to the pathogsnesis of MS is exertsd through a CaS+-dependent regulation of T-lymphocyte activity.
P03.11 AN I M M U N O H I S T O C H E M I C A L S T U D Y OF THE PDGF IN THE M U L T I P L E S C L E R O S I S A C U T E PLAQUES. A.R.Massaro, G.Carbone, A.Carnevale "Sacro Cuore" University Medical School, Rome, Italy. PDGF is involved in the regulation of growth end differen tiation of glial cells. It was found that type-i astrocy~ tee produce a PDGF-Iike molecule to control the O-2A glial progenitor cell differentiation into oligodendrocytes end type-2 sstrocytes. PDGF also increases the expression of ~fl4CClass II antigens on antigen-presenting cells, and mo dulates ~-interferon production by helper T-cells. These-features prompted us to make a preliminary imm~nohistoche mical study in order to localize the PDGF in normal and MS brain white matter. Paraffin sections of acute MS plaques end apparently healthy white matter were treated with a polyclonal goat anti-human PDGF IgG (Collaborative Res.,Ine.). Specimens of white matter fr~n patients who died for non-neurologlcal diseases were taken as controls. A Veetastain-ABC kit for goat-IgG was then used. Mieroseo pie observation showed that anti-PDGF stained the normal-white matter of both control and MS patients, while the acute plaques did not stain. This observation could suggest that a PDGF-Iike antigen is present in normal white matter.0n the other hand,the possibility that formaline fixation and paraffine embedding could alterate the antige nic properties of the tissue must be taken into account.
P16.02
THREE Y E A R T R E A T M E N T W I T H A L P H A - 2 A I N T E R F E R O N OF A B E H ~ E T D I S E A S E P A T I E N T A.R.Massaro, A.Carnevale, C.Rumi "Sacro Cuore"Univarslty Medical School, Rome, Italy. A 28 year-old woman with recurrent aphtous ulcers, cutane our lesions,ocular manifestations, and neurological syrup~ toms associated with almormal brain ~ I , of 14 years dura tion, was d i a g n o s e d as having Behfe~ disease. The coursewas relapslng-remltting, with ocular and neurological sequelae. No treatment was efficient in stopping the recurrent relapse@. T r e a ~ t with human recombinant =(-2A inte rferon (3xlO'IU IM every three days) was then started inorder to demonstrate e possible Beneficial effect on the course of the disease and on s o m e laboratory parameters we monitored. General and neurological conditions, with brain MRI and lymphocytes subsets were followed-up, together with routine laboratory tests. During the three year we noticed: a sharp reduction of the m~cocutaneous end ocular manifestation, which were also much less pronounced end less painful; a significant improvement of the neurological examination; a clear reduction in the previously frequent epileptic seizures; no appearance of new lesions at the serial MRI controls, neither gadolinium enhae~ent ; and a no~mallzation of the E~G. The lymphocytes subsets showed a marked increase of CD8 and a decrease of CD4 cells. No significant side effects were shown, nor alterations of the routine laboratory tests.
W12.04 FOUR Y E A R F O L L O W - U P OF M U L T I P L E S C L E R O S I S (MS) PATIENTS TREATED WITH ALPHA-2A INTERFERON. A . R . M a s s a r o , A . C a r n e v a l e , C.Rumi, A . L a u d i s i o "Sacro Cuore"University Medical Sehool, Rome, Italy. We previously reported promising results with the use of htmmn recombinant c~-ZA interferon in 14 MS patients, all over 40 years of age, for a period of 2 years (ISNI Congress'91). Here we report about an additional two year period of observation on the same patients, 7 of whom decided to continue the treatment. The treatment schedule was slightly modified: 3xlO~IU IM every three days, without any interruption. The patients were evaluated by using eli doliniom, iymphoeytes subsets,and
routine
laboratory
tests.
None of the treated patients presented any major side effects, nor alterstions in the routine tests. Of the 7 treated patients, only one relapsed during the observation pe riod,with no increase on the Kurtzke's scales at the final biennial evaluation. Of the 7 untreated patients, only 2 remained stable; the others experienced relapses, some of them with a permanent residual w o r s e n . ~ F r o m this dr~rn we can suggest that: Long-term treatment with ~-interferon appears to be safe even after 4 years; the supposed benefi cial effect experienced during the first biennium is lost when the treatment is stopped; continuing the treatment probably mantains a positive biological activity on the course of MS. W11.02 RELEVANCE OF H U M O R A L A N D cr~ J.ULAR IMMUNEFY IN THE CENTRAL NERVOUS SYSTEM IN HAbl/TSP. M. Mat~ai and Y. Kuroda Div. Neurol., Dept. Internal Medicine, Saga Medical School, Saga, JAPAN
l n . . ~ t i o n : HAM/TSP is a disease rarity of chrodic m ~ y a~ociated wirehuman T l y n ~ virustypeI.A l t h ~ m a w / i a ~ have revealedthe~ ofimmone ~ m thisdls~fdWr,~ pathological pmceses keding m the Msion fornmfion in h'le central nefvons system (CNS) have not been clarif~d.
Material~a~l Metho~: Twea~-foor patleats with HAM/FSP were compared with 39 patients with other nonintlmnnmtt~ neamiogkal diseaaus in temls of IgG nod ~,2-imcroginbelin leveb ia the us~tm3qtim111~1 (CSF) nod imra. blood.brain ~ ' ~ lgG sy.:.~,=, in ,d4~t~n to ~ ~ of o~el~ infiltrates in the CSF and mmonucle~ ~ 04NC3 in the pe~0heml blood. MNC isolated were singl~stained with monoclonal anfil~dy (MoAb) CD3, CD20, or CD56, a~l a/so double-stained with combinalions of MoAb: CIM or CDg plus CDila, CD25, CD28, or CD45RO. Results: There were sigaifr.not increases in CSF IgG and [~2-microglobulin levels and a ~ of CD8+ T cells of a CDI l a + C D 4 5 R ~ . phenotype in the CSF of HAM/FSP. By conwast, a ~igaificmR incteue in activated CD4+ help~ T cells with CD25 rod/or CD45RO tattgam was observed in the blood o~ HAM/TSp. The CSF lgG leve.~ ssd de sovo IgG synthesis rate w~e si~dft~m~y dem.ussed with the dimaan ~l~immion,
one,=~on: Potentially ¢~olox~ ~ l l ~ ~ T ceils may play hn l m [ ~ U n t~'[~ I~ the L'V~3-1i~tth~-bTl~KlPO'rb-'~.
W10.01 CHARACTERIZATION OF C D 4 " C D S " c t ~ T C E L L S IN AUTOIMMUNE ENCEPHALOMYELITIS ~ h i l l t U / ~ 2 t , S. Ahe2; T. Shin 1, T. Kojima t and Y. I~ihara 1 l Depm'anent of Neuropethogy, Toky~ Metropolitan Institute for N ~ 2 DepurUnent of Neuropethology, Niigata University Brain Research Institute
Experimental autoimmuue encephalomyelitis (EAE) is a T cell-mediated disease characterized by the i~sonce of T cells and ~ e s mainly in the spirml cord. Recent studies have shown that eacephalitogenic T cells he~ a ~ number of V~ chains of T cell receptor (TCR) and play an important role not only in recraitment of inflammatory ceils, but also in lesion formation in the central nervoussysmm (UNSy.q:I6~,e~, theklneficsof ehcepffalll~efiii: T ~P.~reffial n p(x, rly underslood so far. In the present study, we addregsed this question by
characterizinguaiqnoT cellswhicha l ~ inthes,,~hnC,h~_space(SAS) of fats wilh the early stage of EAE. Row cytomctric analysis revealed timt more than 70% of T ceils in SAS we~ CD4"CD8- (double negative, DN) aftd bore a ~ TCR. Furthermore, the predominant V[~ in this lglgelation was shown to he V~t8.2 by ~ co~ s~. W e thench)n~ RT-PCR FroduasamplR-~d~ n g primexs for V[$8.2 and seqaenced nacleOti-l-l-l-l-l-l-l-~Of the j ~ _ _ ~ region of each clone. It was revealed that some clones isolated floe DN SAS T cells had a common motif sequence which was reported to be characteristic for enccphalitogenic T cells. Furthermore, the same sequence was recognized in clones from spinal cord and ! y ~ l i node CD4+ T cells of the same rat. DN SAS T cells were also detected in rats with passive EAE induced by ltmsfer of CD4 + e,cephalitogenic T cells. These findings suggest that DN ~ T cells in the SAS are a direct precursor population of encephalitogenic T cells in the CNS parenchyma.