Journal of the Royal Society of Medicine Volume 85 January 1992
Department of Health for the introduction of a national screening programme. Many unanswered questions remain, including: (1) What information and follow-up needs to be provided for anti-HCV positive donors and their general practitioners? (2) After HCV, should other viruses of limited public
Immunological mechanisms of demyelination Keywords. demyelination; multiple sclerosis; Guillain-Barr6 syndrome
Although the primary causes of multiple sclerosis (MS) and Guillain-Barre syndrome (GBS) remain unknown, there is much circumstantial evidence to support the idea that autoimnaune mechanisms play an important part in the demyelination which is the hallmark of their pathology. For autoimmune responses to damage the myelin sheath, the blood-brain barrier must be breached. Either T-cells may reach and recognize relevant peptides or antibodies may react with neural antigens, the complement system may be activated, and macrophages provide a final common pathway for myelin damage and debris clearance. These subjects were all reviewed at a recent joint meeting of the Sections of Neurology and Clinical Immunology & Allergy. Dr D Male (Department of Neuropathology, Institute of Psychiatry, London) spoke on the bloodbrain barrier. The concept of a blood-brain barrier was first proposed to explain why the majority of large serum molecules and many small ones are excluded from the parenchyma of the brain. The physiological basis of the barrier lies in the highly specialized cerebral endothelium and underlying astrocyte foot processes'. Cerebral endothelium differs from all other endothelia (except retinal endothelium), in having continuous tight junctions around the cell, which severely limits the diffusion of molecules. In addition the number ofvesicles normally seen in these cells is very small, which limits the potential for active non-specific transport of serum molecules. On the other hand, the endothelium does have a number of receptors, such as the transferrin receptor, which are required to move specific essential nutrients across the cells. From an immunological viewpoint, endothelium is important in the development of immune reactions in the brain, since it limits the movement of antigens out of the brain and controls the movement of immunologically important molecules (antibodies, complement, etc) and leukocytes into the CNS. This cellular traffic is also usually very limited, and this has been ascribed to a combination of the high surface charge and lack of leukocyte adhesion molecules on brain endothelium. Nevertheless, when immune
53
health import such as HTLV-1 also be considered for routine screening? Chris Mattock Bill Brace Senior Registrars in Haematology on attachment at North London Blood Transfusion Centre
reactions do develop in brain there is a considerable increase in leukocyte traffic across the endothelium, particularly of lymphocytes, and this is accompanied by a partial breakdown of the barrier to serum molecules. There are several potential routes for antigen to leave the brain, to sensitize lymphocytes in the immune system. These include: (1) the cerebrospinal fluid, and then via the arachnoid granulations to the draining sinuses; (2) by reverse transcytosis across the brain endothelium; and, (3) lymphatics overlying the cribriform plate and draining to cervical lymph nodes. Although there is evidence that all three routes are available, the third, drainage to cervical lymphatics, appears to be most important immunologically, since antigen injection into CNS induces sensitized lymphocytes in these nodes. At least five explanations have been proposed for the partial breakdown of the barrier during immune reactions: (1) increased vesicular transport2 (2) formation of intracellular channels across the endothelium (3) opening of the intercellular tight junctions (4) cytotoxic damage to the cells (5) molecular movement associated with leucocyte traffic. The evidence shows there to be an increased number of vesicular profiles in the vascular endothelium during experimental allergic encephalomyelitis (EAE) and in other acute conditions. Also the electrical resistance of the endothelium drops as cells migrate across it in vitro, suggesting that alterations in molecular movement may also be important. Although it is possible to show CD4+ or CD8+ T cell mediated cytotoxicity to brain endothelium in vitro, it is thought that such damage could only occur in vivo during the most severe immune reactions3. Two principal factors appear to control lymphocyte movement across the endothelium. These are the state of activation ofthe endothelium as determined by cytokines, and the state of activation of the lymphocytes depending on their phase of the cell cycle4. Brain endothelium responds to TNF-a and JEN-y in vitro, showing enhanced expression of intercellular adhesion molecules (ICAM-1) and in vivo both ICAM-1 and vascular addressins have been shown during immune reactions. Equally important are the migrating cells themselves. In a dividing population of lymphocytes in vitro a particular group appear to be able to migrate across endothelium. In vivo it is notable that many migrating cells are in cell cycle5.
Report of joint meeting of Sections of Neurology and Clinical Immunology & Allergy, 13 May 1991
0141-0768/92/ 010053-05/$02.00/0 © 1992 The Royal Society of Medicine
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Journal of the Royal Society of Medicine Volume 85 January 1992
Dr D Wraith (Department of Pathology, University of Cambridge) then spoke on T cell recognition of myelin basic protein. T helper (Th) cell activation is a central feature of most normal immune and autoimmune responses6. Activation follows occupation of Th cell receptors by a complex ligand formed between antigenic peptides and class II molecules of the major histocompatibility complex (MHC). Activated Th cells produce lymphokines which have been shown to influence the level of activity of B lymphocytes, cytotoxic T (Tc) lymphocytes and cells involved in inflammatory responses. For this reason, Th cell activation plays an important role in autoimmune processes and consequently serves as a target for immune intervention. There are three main targets for intervention: (i) T cell receptors (TcR), if limited TcR usage can be demonstrated, (ii) CD4 molecules, more likely to cause non-specific immune suppression, and(iii) class II MHC molecules, implicated by the close association between specific class II alleles and susceptibility to autoimmune disease. Antibodies to any of these targets have been shown previously to inhibit the induction of and, in some cases, produce reversal of experimental autoimmune diseases7. Unfortunately, the use of xenogeneic antibodies is complicated by host antiglobulin responses and this has led a number of groups to seek alternative approaches. Structural studies have indicated that MHC molecules contain a single peptide binding site and functional studies have demonstrated the ability of peptides to compete for this site both in vitro and in vivo. These observations raise the possibility of designing allelespecific MHC blocking agents with potential for immune intervention. Recently, Dr Wraith's group have demonstrated the use of this strategy for the prevention of EAE in mice. EAE is induced by injection of myelin basic protein (MBP) or peptides derived from it. The resulting demyelinating disease is Th cell mediated and can be inhibited by antibodies to class I1 MHC molecules, disease associated TcRs and Th cell accessory molecules such as CD4. There is notable epitope dominance in murine models of EAE. Encephalitogenic T cells from H-2u mice respond predominantly to the N-terminal 1-9 peptide of MBP, whereas the majority of disease-inducing lymphocytes from SJL (H-2s) mice respond to the more C-terminal 87-98 peptide. This epitope dominance is a direct consequence of 'determinant selection' by predisposing MHC molecules. The role of individual amino acids of peptide 1-9 in T cell recognition by H-2u restricted Th cells has been defined8. Residue 4 is important for I-Au binding and single amino acid substitutions have indicated that the peptide associates with a 'hydrophobic pocket' in I-Au. Peptide binding and lymph node T cell activation studies have shown that residues 3 and 6 act as determinants for TcR interactions. Combined substitutions at position 3 and 4 generated a peptide with increased affinity for I-Au, which failed to activate encephalitogenic T cells and which competed for recognition both in vitro and in vivo. A peptide substituted at positions 3 and 4 has been used to inhibit competitively the induction of EAE in H-2u mice9. Another peptide, substituted with alanine at position 4 alone, displayed enhanced binding to I-Au and increased antigenicity for encephalitogenic T cells in vitro but surprisingly was not immunogenic in vivo.
This peptide also inhibited the induction of disease in H-2u mice8. The mechanism by which this latter peptide prevented EAE is not clear. A trivial explanation for the ability of analogues of peptide 1-9 to inhibit disease induction in H-2u mice is their ability to compete effectively with the encephalitogenic peptide for binding to I-Au. However, it is possible that analogues of disease-inducing peptides could modulate the immune response by generating regulator cells or Th cells of a nonpathogenic type. Is MHC blockade a feasible approach? This question has been addressed directly in a recent study using unrelated peptides to inhibit induction of EAE by a peptide derived from murine proteolipid protein (PLP)10. Prevention of disease was achieved by administering an unrelated I-As binding peptide at a separate site from the autoantigen. Inhibition was due to binding of the inhibitory peptide to I-As. Further work is required to answer the following questions: (i) where in antigen presenting cells do peptides associate with MHC class II molecules and can inhibitors be targeted to this compartment? (ii) can inhibitors with higher affinity and increased stability be prepared? (iii) will it be possible to design MHC blocking agents which are non-immunogenic? (iv) can MHC 'blockers' reverse established disease? Dr H Willison (Institute of Neurological Sciences, Southern General Hospital, Glasgow) then spoke on antibodies to complex glycolipids and glycoproteins in peripheral nerve disease (paper prepared with Drs N Gregson, A Ilyas and R Quarles). It has become increasingly recognized that circulating antibodies to carbohydrate determinants found on complex glycolipids and glycoproteins may be important aetiological factors in the pathogenesis of a variety of peripheral nerve disorders including neuropathies associated with IgM paraproteinaemia, multifocal motor neuropathies and neuronopathies, chronic inflammatory demyelinating neuropathies and Guillain-Barr6 syndrome. Work by ourselves1",12 and many others in this field13 focused on the group of peripheral neuropathies associated with IgM paraproteinaemia. In our -studies, reactivity of the monoclonal IgM with-the myelin-associated glycoprotein (MAG) and antigenically related cross-reactive glycolipids accounted for over half of the cases of IgM paraproteinaemic neuropathy; epitope mapping studies showed that the fine specificity of these antibodies varied widely. The remaining monoclonal IgM antibodies react with a variety of different glycoconjugate antigens including GM1 and other gangliosides""4"6 An association between motor neuron disease (MND) and IgM paraproteinaemia has been recognized for many years but it is only recently that GM1 ganglioside has been identified as the antigen for the monoclonal paraprotein in some cases'6. Patients have also been described with multifocal motor neuropathies resembling MND in whom high titres of polyclonal IgM anti-GM1 antibody are detected and who have a good clinical response to immuno-
suppressive therapy'7.
Following the recognition ofthese atypical patients several screening studies on larger groups of patients with classical MND and amyotrophic lateral sclerosis (ALS) were performed. Anti-GM1 IgM antibodies were
Journal of the Royal Society of Medicine Volume 85 January 1992
found in over 50% of patients in some studies'8. In our studies'9, 90% of normal controls-had an anti-GM1 IgM titre below 1/180 and this value was therefore designated as the upper limit of normal. By this criterion 9.6% of the normal controls were positive (n=31, mean titre 1/79). Among the disease groups the following percentage of patients were positive: 16% with ALS (n=77, mean titre 1/106), 17% with chronic spinal muscular atrophy (n=24, mean titre 1/102), 32% with multifocal motor neuropathy (n-34, mean titre 1/440) and 28% with mixed motor and sensory neuropathies (n-42, mean titre 1/381). However, detectable titres of anti-GM1- antibodies have also been found in a wide variety of both normal and disease controls including SLE and multiple sclerosis. In view of the high incidence of these antibodies among control patients-and the lack of any appropriate experimental models, their clinical significance remains uncertain20. The search for antibodies to neural antigens in Guillain-Barre syndrome has been active for' many years. We reported2l -antibodies to a variety of gangliosides including LM1, GDlb, GDla and GTlb in about 20% of patients. Several subsequent studies have also demonstrated the presence of anti-acidic glycolipid antibodies (including GM1)in patients-with GBS. On average, approximately 20% of patients with GBS are positive by ELISA and/or TLC for IgM or IgG anti-GM1 antibodies. Evidence that antibodies to neutral glycolipids including Forsaman,antigen are elevated in GBS sera compared with controls22 has recently been refuted23; this discrepancy may be due to different methodological approaches. GM1 ganglioside shares the terminal structure of the ganglio series of glycosphingolipids, the Gal (31-3) GalNAc moiety, with. asialo-GMl and GDlh gangliosides; many anti-GM1 antibodies have been shown to bind to this Gal (11-3) GalNAc deter' minant 5'7. Other antibodies react with GM1 and GM2 suggesting that they react with the inner core structure common to the two gangliosides'4. Some antibodies are relatively specific for GM1, indicating that they react with a broader, portion of the oligosaccharide structure than the Gal (j31-3) GalNAc determinant'3. Studies are currently underway to investigate the molecular basis for the variations in fine specificity seen among anti-GM1 antibodies, to determine the pattern of antibody repertoires used in different diseases, and to describe the pathogenic effects, if any, of these antibodies in appropriate experimental models. Dr J Zajicek (Department of Neurology, Addenbrooke's Hospital, Cambridge) then spok-e on complement and oligodendrocyte-myelin injury (pgper prepared with Dr N Scolding). The complement system may be activated by the classical pathway, which conventionally requires specific antibody, or the alternative pathway, triggered by contact with, for example, certain bacterial surfaces. The subsequient cascade yields a variety of products which have three principal biological activities: soluble kinins (C3a and C5a) with pro-inflmmatoy properties; the surface bound opsonin C3b, which promotes macrophag adhesion and phagocytogis; and the membaneattachconwlex, which inserts into target cell membranes and causes cell damge. The combination of CNS inflamm tion, oligodendrocyte loss, and macrophage-mediated myelin removal24 raises the possibility that each group of complement
products might be relevant to the pathogenesis of inflammatory demyelination. In vitro studies of the cell biology of oligodendrocyte injury provide some insight into these possibilities25. These experiments indicate -that rat oligodendrocytes activate syngeneic complement in the absence of specific antibody, leading to cell lysis. Serum from decomplemented rats does not exhibit this cytolytic activity; neither does serum depleted in vitro of either C1 or C9. Oligodendrocytes exposed to serufia stain immuntochemically for surface C9, but not mun globulin. Astrcytes are not susceptible, while bipotential O-2A progenitor cells are lysed only by a high concentration ofserum. Differentiation and maturation of O-2AprogeniMstor cllsintoisassociated with ineasg susceptibiity to attackby complement. In addition to acquiing a cell-surface complementactivating component during maturation, oligodendrocytes also appear to be deficient in membraneanchored complement regulatory proteins In reactive lysis studies, when MAC formation in EDTA-treated human serum was triggered by the introduction of exogenously prepared activated C5b6, oligodendrocytes were again selectively lysed (Zajicek J, -Wing MG, Lachmannw PJ, Compston DAS, in preparation. Furthermore, the addition of human CD59 (which prevents C9 incorporation into the membrane attack complex) protects against both reactive lysis and the lytic effects of normal human serum, supporting the suggestion that rat oligodendrocytes lack one or more terminal complement regulatory proteins. During attack by sub-lethal concentrations of complement, numerous vesicles, brightly staining for C9, appear on the surface of.oligodendrocytes after 2 min exposure, which- have disappeared after a further 5 min. Concurrently, oligodendrocyte-derivedparticulate material bearing surface C9 appears in culture supernatants, suggesting that, following complement activation, oligodendrocytes resist lysis by the vesicular removal of complement m e attack complexesm. During reversible injury,, a transient rise in intracellular calcium and fall in ATP are seen, but oligodendrocytes recover and remain able later to express myelin antigens which are markers of oligodendrocyte maturation. The factors responsible for targeting macrophages against oligodendrocytes may also be investigated in vitro. The results indicate that anti-oligodendrocyte antibodies are able.- to direct macrophages onto oligodendrocytes and totrigger myelin phagocytosis?7. While complement alone does not exhibit this activity using resting macrophages, preliminary results suggest that activated macrophages and, more pertinently, activated microglia, can be targeted onto oligodendrocytes in vitro by. complement alone. A number of in vivo observation&also suggest that complement may be significant in the pathogenesis of demyelnation. Complement activation -products C3a and C4a are present in cerebrospinal fluid of patients with demyelinating disease, and levels of the terminal component 09 are reduced, implying that complement is activated within the CNS; complemelit components can also be: demonstrated immunohistochemically within lesions in a pen.vascular distribution. Vesicular material, identical .to that released by oligodendrocytes injured by complement in vitro, is also present in the cerebrospinal fluid of patients with,demyelination, but not controls with structural CNSlesions26. Finally,
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Journal of the Royal Society of Medicine Volume 85 January 1992
immunofluorescence staining of CNS tissue from patients with multiple sclerosis shows the co-location of complement and antibody on particles within macrophages in demyelinating lesions2. These observations are consistent with the hypothesis that blood-brain barrier breakdown, which is likely to be mediated by T lymphocytes, initiates CNS inflammation by exposing oligodendrocytes to serum complement. Reversible or lytic oligodendrocytemyelin damage might be followed by demyelination and/or macrophage recruitment, and the process may be amplified by consequent secondary cellular and humoral immune responses. In the final presentation Professor K Toyka spoke on the role of macrophages in immune-mediated demyelination. In most tissues macrophages are the principal cells expressing MHC class II and presenting antigenic peptides to the T-cell antigen receptor. They produce many cytotoxic factors including oxygen and nitric oxide radicals, ILl, IL6, TNFci, and prostaglandins and leukotrienes which damage blood tissue barriers and act as chemotactic signals for haematogenous inflammatory leukocytes. When activated, macrophages synthesize complement components. They can also bind to specific antibodies by means of antibody dependent cell-mediated cytotoxicity. What are the equivalent cells in the central and peripheral nervous systems? In the CNS the macrophage-equivalents are the microglia which are derived from bone-marrow monocytes during ontogeny. Microglia may be the cell type which initiates immune-mediated local inflammation once it has been activated by invading T-cells. It has been shown that interferon-'y is the main inducer of MHC class II upregulation on microglia in vivo. Astrocytes also have the potential to produce immune mediators and can function as antigen presenting cells in cell culture. At present, it seems unlikely that they are important in fulfilling this function in vivo. In the PNS resident macrophages are present which may initiate immune-mediated inflammation. At present, it seems unlikely that Schwann cells exert macrophage-like functions though they can function as antigen presenting cells in vitro. Microglial cells derived from neonatal rat brain and cultured to more than 95% purity can produce proinflammatory and cytotoxic mediators. Prostaglandin production can be stimulated with phorbolmyristate, Ca-ionophore, lipopolysaccharide, or IFN--y. The microglia also produces cytotoxic oxygen and, as very recently shown, nitric oxide radicals. Microglial cells produce these active metabolites in a dose dependent fashion upon stimulation with IFN--y. Astrocytes do produce very little nitric oxide but to a much lesser extent. The macrophage is the main effector cell at sites of inflammation. Macrophages are attracted during the induction and amplification phase by antigen-specific activated T cells and, possibly, by activated resident macrophages or microglia. In the early lesions of the peripheral nerve inflammatory disease, experimental allergic neuritis (EAN), abundant macrophages can be seen around blood vessels and associated with demyelinated axons29~31. The macrophages contain myelin debris which they have phagocytosed. Early axonal degeneration may be found and is considered to be caused by bystander damage. The crucial question is whether
the macrophages are important causative agents or merely involved in a clearing-up operation. In favour of a causative role is the ability of macrophages taken at disease onset to produce cytotoxic oxygen radicals. Furthermore when animals are treated with the natural oxygen radical scavengers, superoxide dismutase or catalase, before the onset of EAN, functional deficit can be prevented32. The histological evidence of inflammation is also reduced. The neurological deficit and inflammatory changes are also reduced by blocking eicosanoid production with indomethacin or even more selective cyclo-oxygenase blockersm3. Macrophages produce and activate complement, and complement activation may interfere with normal nerve conduction. The terminal complement component membrane attack complex can be detected on the surface of myelin sheaths in early but not late EAN. EAN in rats can be suppressed by depletion of complement with cobra venom factor3. There is transient expression of IFN-y by endoneurial cells in early EAN, and IFN--y is probably the most important activation signal for macrophages (and microglia)35. Treatment with antibodies to IFN-'y profoundly reduces disease activity. The speakers at the meeting made clear that the whole range of immunological instruments has a part to play in orchestrating immune-mediated demyelination. The process must be introduced by breaching the blood-nerve or blood-brain barrier in which activated T cells and inflammatory mediators play an important part. The mast cells resident in the perineurium, endoneurium and meninges may also be involved. Antibody-mediated or T cell-mediated reactions against specific myelin antigens have been demonstrated in experimental and human diseases. Complement undoubtedly plays its conventional role in amplifying the inflammatory process and may have a special role in damaging oligodendrocytes, even in the absence of specific antibody. Macrophages are active throughout immune-mediated demyelination, presenting antigen at an early stage, initiating the process of demyelination and marching triumphantly offthe stage bearing the myelin debris in a grand finale. R A C Hughes Department of Neurology Guy's Hospital, London References 1 Bradbury MW. The structure and function of the blood-brain barrier. Fedn Proc 1984;43:186 2 Broadwell RD. Transcytosis of molecules through the blood-brain barrier: a cell biological perspective and critical appraisal. Acta Neuropath (Berl) 1989;79:117 3 Sedgwick J, Hughes CCW, Male DK, MacPhee IAM, Ter Meulen V. Antigen-specific damage to brain vascular endothelial cells mediated by encephalitogenic and nonencephalitogenic CD4+ T cell lines in vitro. JImmunol
1990;145:2474 4 Male DK, Pryce G, Hughes CCW, Lantos PL. Lymphocyte migration into brain modelled in vitro: control by
lymphocyrte activation cytokines and antigen. Cell Immunol 1990;127:1 5 Cross AH, Cannella B, Brosnan OF, Raine CS. Homing to central nervous system vasculature by antigen-specific lymphocytes. I. Localisation of 14C-labelled cells during acute, chronic and relapsing experimental allergic encephalomyelitis. Lab Inuest 1990;63:162 6 Schwartz RHI. Immune responses (Ir) genes of the murine major histocompatibility complex. Adv Immunol 1986; 38:31
Journal of the Royal Society of Medicine Volume 85 January 1992 7 Wraith DC, McDevitt HO, Steinman L, Acha-Orbea H. T cell recognition as the target for immune intervention in autoimmune disease. Cell 1989;57:709 8 Wraith DC, Smilek DE, Mitchell DJ, Steinman L, McDevitt HO. Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunotherapy. Cell 1989;59:247-55 9 Urban JL, Horvath SJ, Hoad L. Autoimmune T cells: immune recognition of normal and variant epitopes and peptide-based therapy. Cell 1989;59: 257-71 10 Lamont AG, Sette A, Fujinami R, Colon SM, Miles C, Grey HM. Inhibition of experimental autoimmune encephalomyelitis induction in SJL/J mice by using a peptide with high affinity for I-As molecules. JImmunol 1990;145:1687 11 Quarles RH, Ilyas AA, Willison HJ. Antibodies to glycolipids in demyelinating diseases of the human peripheral nervous system. Chem Phys Lipids 1986; 42:235-48 12 Gregson NA, Leibowitz S. IgM paraproteinaemia, polyneuropathy and myelin-associated glycoprotein (MAG). Neuropathol Appl Neurobiol 1985;11:329347 13 Latov N. Antibodies to glycoconjugates in neurological disease. Clin Aspects Autoimmun 1990;4:18-29 14 Ilyas AA, Willison HJ, Dalakas MC, Whittaker JN, Quarles RH. Identification and characterization of gangliosides reacting with IgM paraproteins in three patients with neuropathy associated with biclonal gammopathy. J Neurochem 1988;51:851-8 15 Baba H, Daune GC, Ilyas AA, et al. Anti-GM1 ganglioside antibodies with differing fine specificities in patients with multifocal motor neuropathy. J Neuroimmunol 1989;25:143-50 16 Latov N, Hays AP, Donofrio PD, et al. Monoclonal IgM with unique specificity to gangliosides GM1 and GD1B and to lacto-N-tetraose associated with human motor neuron disease. Neurology 1988;38:763-8 17 Pestronk A, Cornblath DR, Ilyas AA, et al. A treatable multifocal neuropathy with antibodies to GM1 ganglioside. Ann Neurol 1988;24:73-8 18 Pestronk A, Adams RN, Clawson L, et al. Serum antibodies to GM1 ganglioside in amyotrophic lateral sclerosis. Neurology 1988;38:1457-61 19 Gregson NA, Jones D, Willison HJ. Antibodies against GM1 and other ganglioside in patients with motor neuron syndromes. In: Clifford Rose F, ed. New evidence in MND/ALS research. London: Smith-Gordon & Co. Ltd, 1991 20 Marcus DM, Latov N, Hsi BP, Gillard BK. Measurement and significance of antibodies against GM1 ganglioside. Report of a Workshop, 18 April 1989, Chicago, IL, USA. J Neuroimmunol 1989;25:255-9
21 Ilyas AA, Willison HJ, Quarles RH, et al. Serum antibodies to gangliosides in Guillain-Barre syndrome. Ann Neurol 1988;23:440-7 22 Koski CL, Chou DKH, Jungalwala FB. Anti-peripheral nerve myelin antibodies in GBS bind a neutral glycolipid of peripheral myelin and cross-react with Forssman antigen. J Clin Invest 1989;84:280-7 23 Ilyas AA, Mithen FA, Chen Z-W, Cook SD. Search for antibodies to neutral glycolipids in sera of patients with Guillain-Barre syndrome. JNeurol Sci 1991;102:67-75 24 Prineas JW. The neuropathology of multiple sclerosis. In: Handbook of clinical neurology. Amsterdam: Elsevier, 1985:213-57 25 Compston A, Scolding N, Wren D, Noble M. The pathogenesis of demyelinating disease: Insights from cell biology. Trends Neurosci 1991;14:175-182 26 Scolding NJ, Morgan BP, Houston A, Linington C, Campbell AK, Compston DAS. Vesicular removal by oligodendrocytes of membrane attach complexes formed by complement. Nature 1989;339:620-2 27 Scolding NJ, Compston DAS. Oligodendrocyte-macrophage interactions in vitro triggered by specific antibodies. Immunology 1990;72:127-32 28 Gay D, Esiri M. Blood-brain barrier damage in acute multiple sclerosis plaques. An immunocytochemical study. Brain 1991;114:557-72 29 Craggs RI, King RHM, Thomas PK. The effect of suppression of macrophage activity on the development of experimental allergic neuritis. Acta Neuropathol (Berl) 1988;23:326-31 30 Sobue G, Yamato S, Hirayama M, et aL. The role of macrophages in demyelination in experimental allergic neuritis. J Neurol Sci 1982;56:75-87 31 Trotter J, Smith ME. The role of phospholipases from inflamatoy macropha in demyelination. Neurochem Res 1986;11:349-61 32 Hartung H-P, Schafer B, Heininger K, Toyka KV. Suppression of experimental autoimmune neuritis by the oxygen radical scavengers superoxide dismutase and catalase. Ann Neurol 1988;23:453-60 33 Hartung H-P, Schafer B, Heininger K, Stoll G, Toyka KV. The role of macrophages and eicosanoids in the pathogenesis of experimental allergic neuritis. Brain 1988;111:1039-59 34 Stoll G, Schmidt B, Jander S, Toyka KV, Hartung HP. Presence of the terminal complement complex (C5b-9) precedes myelin degradation in acute demyelination in the peripheral nervous system of the rat. Ann Neurol 1991;30:147-55 35 Hartung H-P, Schifer B, Van der Meide PH, Fierz W, Heininger K, Toyka KV. The role ofinterferon-gamma in the pathogenesis of experimental autoimmune disease of the peripheral nervous system. Ann Neurol 1990; 27:247-57
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Department of Health for the introduction of a national screening programme. Many unanswered questions remain, including: (1) What information and follow-up needs to be provided for anti-HCV positive donors and their general practitioners? (2) After HCV, should other viruses of limited public
Immunological mechanisms of demyelination Keywords. demyelination; multiple sclerosis; Guillain-Barr6 syndrome
Although the primary causes of multiple sclerosis (MS) and Guillain-Barre syndrome (GBS) remain unknown, there is much circumstantial evidence to support the idea that autoimnaune mechanisms play an important part in the demyelination which is the hallmark of their pathology. For autoimmune responses to damage the myelin sheath, the blood-brain barrier must be breached. Either T-cells may reach and recognize relevant peptides or antibodies may react with neural antigens, the complement system may be activated, and macrophages provide a final common pathway for myelin damage and debris clearance. These subjects were all reviewed at a recent joint meeting of the Sections of Neurology and Clinical Immunology & Allergy. Dr D Male (Department of Neuropathology, Institute of Psychiatry, London) spoke on the bloodbrain barrier. The concept of a blood-brain barrier was first proposed to explain why the majority of large serum molecules and many small ones are excluded from the parenchyma of the brain. The physiological basis of the barrier lies in the highly specialized cerebral endothelium and underlying astrocyte foot processes'. Cerebral endothelium differs from all other endothelia (except retinal endothelium), in having continuous tight junctions around the cell, which severely limits the diffusion of molecules. In addition the number ofvesicles normally seen in these cells is very small, which limits the potential for active non-specific transport of serum molecules. On the other hand, the endothelium does have a number of receptors, such as the transferrin receptor, which are required to move specific essential nutrients across the cells. From an immunological viewpoint, endothelium is important in the development of immune reactions in the brain, since it limits the movement of antigens out of the brain and controls the movement of immunologically important molecules (antibodies, complement, etc) and leukocytes into the CNS. This cellular traffic is also usually very limited, and this has been ascribed to a combination of the high surface charge and lack of leukocyte adhesion molecules on brain endothelium. Nevertheless, when immune
53
health import such as HTLV-1 also be considered for routine screening? Chris Mattock Bill Brace Senior Registrars in Haematology on attachment at North London Blood Transfusion Centre
reactions do develop in brain there is a considerable increase in leukocyte traffic across the endothelium, particularly of lymphocytes, and this is accompanied by a partial breakdown of the barrier to serum molecules. There are several potential routes for antigen to leave the brain, to sensitize lymphocytes in the immune system. These include: (1) the cerebrospinal fluid, and then via the arachnoid granulations to the draining sinuses; (2) by reverse transcytosis across the brain endothelium; and, (3) lymphatics overlying the cribriform plate and draining to cervical lymph nodes. Although there is evidence that all three routes are available, the third, drainage to cervical lymphatics, appears to be most important immunologically, since antigen injection into CNS induces sensitized lymphocytes in these nodes. At least five explanations have been proposed for the partial breakdown of the barrier during immune reactions: (1) increased vesicular transport2 (2) formation of intracellular channels across the endothelium (3) opening of the intercellular tight junctions (4) cytotoxic damage to the cells (5) molecular movement associated with leucocyte traffic. The evidence shows there to be an increased number of vesicular profiles in the vascular endothelium during experimental allergic encephalomyelitis (EAE) and in other acute conditions. Also the electrical resistance of the endothelium drops as cells migrate across it in vitro, suggesting that alterations in molecular movement may also be important. Although it is possible to show CD4+ or CD8+ T cell mediated cytotoxicity to brain endothelium in vitro, it is thought that such damage could only occur in vivo during the most severe immune reactions3. Two principal factors appear to control lymphocyte movement across the endothelium. These are the state of activation ofthe endothelium as determined by cytokines, and the state of activation of the lymphocytes depending on their phase of the cell cycle4. Brain endothelium responds to TNF-a and JEN-y in vitro, showing enhanced expression of intercellular adhesion molecules (ICAM-1) and in vivo both ICAM-1 and vascular addressins have been shown during immune reactions. Equally important are the migrating cells themselves. In a dividing population of lymphocytes in vitro a particular group appear to be able to migrate across endothelium. In vivo it is notable that many migrating cells are in cell cycle5.
Report of joint meeting of Sections of Neurology and Clinical Immunology & Allergy, 13 May 1991
0141-0768/92/ 010053-05/$02.00/0 © 1992 The Royal Society of Medicine
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Dr D Wraith (Department of Pathology, University of Cambridge) then spoke on T cell recognition of myelin basic protein. T helper (Th) cell activation is a central feature of most normal immune and autoimmune responses6. Activation follows occupation of Th cell receptors by a complex ligand formed between antigenic peptides and class II molecules of the major histocompatibility complex (MHC). Activated Th cells produce lymphokines which have been shown to influence the level of activity of B lymphocytes, cytotoxic T (Tc) lymphocytes and cells involved in inflammatory responses. For this reason, Th cell activation plays an important role in autoimmune processes and consequently serves as a target for immune intervention. There are three main targets for intervention: (i) T cell receptors (TcR), if limited TcR usage can be demonstrated, (ii) CD4 molecules, more likely to cause non-specific immune suppression, and(iii) class II MHC molecules, implicated by the close association between specific class II alleles and susceptibility to autoimmune disease. Antibodies to any of these targets have been shown previously to inhibit the induction of and, in some cases, produce reversal of experimental autoimmune diseases7. Unfortunately, the use of xenogeneic antibodies is complicated by host antiglobulin responses and this has led a number of groups to seek alternative approaches. Structural studies have indicated that MHC molecules contain a single peptide binding site and functional studies have demonstrated the ability of peptides to compete for this site both in vitro and in vivo. These observations raise the possibility of designing allelespecific MHC blocking agents with potential for immune intervention. Recently, Dr Wraith's group have demonstrated the use of this strategy for the prevention of EAE in mice. EAE is induced by injection of myelin basic protein (MBP) or peptides derived from it. The resulting demyelinating disease is Th cell mediated and can be inhibited by antibodies to class I1 MHC molecules, disease associated TcRs and Th cell accessory molecules such as CD4. There is notable epitope dominance in murine models of EAE. Encephalitogenic T cells from H-2u mice respond predominantly to the N-terminal 1-9 peptide of MBP, whereas the majority of disease-inducing lymphocytes from SJL (H-2s) mice respond to the more C-terminal 87-98 peptide. This epitope dominance is a direct consequence of 'determinant selection' by predisposing MHC molecules. The role of individual amino acids of peptide 1-9 in T cell recognition by H-2u restricted Th cells has been defined8. Residue 4 is important for I-Au binding and single amino acid substitutions have indicated that the peptide associates with a 'hydrophobic pocket' in I-Au. Peptide binding and lymph node T cell activation studies have shown that residues 3 and 6 act as determinants for TcR interactions. Combined substitutions at position 3 and 4 generated a peptide with increased affinity for I-Au, which failed to activate encephalitogenic T cells and which competed for recognition both in vitro and in vivo. A peptide substituted at positions 3 and 4 has been used to inhibit competitively the induction of EAE in H-2u mice9. Another peptide, substituted with alanine at position 4 alone, displayed enhanced binding to I-Au and increased antigenicity for encephalitogenic T cells in vitro but surprisingly was not immunogenic in vivo.
This peptide also inhibited the induction of disease in H-2u mice8. The mechanism by which this latter peptide prevented EAE is not clear. A trivial explanation for the ability of analogues of peptide 1-9 to inhibit disease induction in H-2u mice is their ability to compete effectively with the encephalitogenic peptide for binding to I-Au. However, it is possible that analogues of disease-inducing peptides could modulate the immune response by generating regulator cells or Th cells of a nonpathogenic type. Is MHC blockade a feasible approach? This question has been addressed directly in a recent study using unrelated peptides to inhibit induction of EAE by a peptide derived from murine proteolipid protein (PLP)10. Prevention of disease was achieved by administering an unrelated I-As binding peptide at a separate site from the autoantigen. Inhibition was due to binding of the inhibitory peptide to I-As. Further work is required to answer the following questions: (i) where in antigen presenting cells do peptides associate with MHC class II molecules and can inhibitors be targeted to this compartment? (ii) can inhibitors with higher affinity and increased stability be prepared? (iii) will it be possible to design MHC blocking agents which are non-immunogenic? (iv) can MHC 'blockers' reverse established disease? Dr H Willison (Institute of Neurological Sciences, Southern General Hospital, Glasgow) then spoke on antibodies to complex glycolipids and glycoproteins in peripheral nerve disease (paper prepared with Drs N Gregson, A Ilyas and R Quarles). It has become increasingly recognized that circulating antibodies to carbohydrate determinants found on complex glycolipids and glycoproteins may be important aetiological factors in the pathogenesis of a variety of peripheral nerve disorders including neuropathies associated with IgM paraproteinaemia, multifocal motor neuropathies and neuronopathies, chronic inflammatory demyelinating neuropathies and Guillain-Barr6 syndrome. Work by ourselves1",12 and many others in this field13 focused on the group of peripheral neuropathies associated with IgM paraproteinaemia. In our -studies, reactivity of the monoclonal IgM with-the myelin-associated glycoprotein (MAG) and antigenically related cross-reactive glycolipids accounted for over half of the cases of IgM paraproteinaemic neuropathy; epitope mapping studies showed that the fine specificity of these antibodies varied widely. The remaining monoclonal IgM antibodies react with a variety of different glycoconjugate antigens including GM1 and other gangliosides""4"6 An association between motor neuron disease (MND) and IgM paraproteinaemia has been recognized for many years but it is only recently that GM1 ganglioside has been identified as the antigen for the monoclonal paraprotein in some cases'6. Patients have also been described with multifocal motor neuropathies resembling MND in whom high titres of polyclonal IgM anti-GM1 antibody are detected and who have a good clinical response to immuno-
suppressive therapy'7.
Following the recognition ofthese atypical patients several screening studies on larger groups of patients with classical MND and amyotrophic lateral sclerosis (ALS) were performed. Anti-GM1 IgM antibodies were
Journal of the Royal Society of Medicine Volume 85 January 1992
found in over 50% of patients in some studies'8. In our studies'9, 90% of normal controls-had an anti-GM1 IgM titre below 1/180 and this value was therefore designated as the upper limit of normal. By this criterion 9.6% of the normal controls were positive (n=31, mean titre 1/79). Among the disease groups the following percentage of patients were positive: 16% with ALS (n=77, mean titre 1/106), 17% with chronic spinal muscular atrophy (n=24, mean titre 1/102), 32% with multifocal motor neuropathy (n-34, mean titre 1/440) and 28% with mixed motor and sensory neuropathies (n-42, mean titre 1/381). However, detectable titres of anti-GM1- antibodies have also been found in a wide variety of both normal and disease controls including SLE and multiple sclerosis. In view of the high incidence of these antibodies among control patients-and the lack of any appropriate experimental models, their clinical significance remains uncertain20. The search for antibodies to neural antigens in Guillain-Barre syndrome has been active for' many years. We reported2l -antibodies to a variety of gangliosides including LM1, GDlb, GDla and GTlb in about 20% of patients. Several subsequent studies have also demonstrated the presence of anti-acidic glycolipid antibodies (including GM1)in patients-with GBS. On average, approximately 20% of patients with GBS are positive by ELISA and/or TLC for IgM or IgG anti-GM1 antibodies. Evidence that antibodies to neutral glycolipids including Forsaman,antigen are elevated in GBS sera compared with controls22 has recently been refuted23; this discrepancy may be due to different methodological approaches. GM1 ganglioside shares the terminal structure of the ganglio series of glycosphingolipids, the Gal (31-3) GalNAc moiety, with. asialo-GMl and GDlh gangliosides; many anti-GM1 antibodies have been shown to bind to this Gal (11-3) GalNAc deter' minant 5'7. Other antibodies react with GM1 and GM2 suggesting that they react with the inner core structure common to the two gangliosides'4. Some antibodies are relatively specific for GM1, indicating that they react with a broader, portion of the oligosaccharide structure than the Gal (j31-3) GalNAc determinant'3. Studies are currently underway to investigate the molecular basis for the variations in fine specificity seen among anti-GM1 antibodies, to determine the pattern of antibody repertoires used in different diseases, and to describe the pathogenic effects, if any, of these antibodies in appropriate experimental models. Dr J Zajicek (Department of Neurology, Addenbrooke's Hospital, Cambridge) then spok-e on complement and oligodendrocyte-myelin injury (pgper prepared with Dr N Scolding). The complement system may be activated by the classical pathway, which conventionally requires specific antibody, or the alternative pathway, triggered by contact with, for example, certain bacterial surfaces. The subsequient cascade yields a variety of products which have three principal biological activities: soluble kinins (C3a and C5a) with pro-inflmmatoy properties; the surface bound opsonin C3b, which promotes macrophag adhesion and phagocytogis; and the membaneattachconwlex, which inserts into target cell membranes and causes cell damge. The combination of CNS inflamm tion, oligodendrocyte loss, and macrophage-mediated myelin removal24 raises the possibility that each group of complement
products might be relevant to the pathogenesis of inflammatory demyelination. In vitro studies of the cell biology of oligodendrocyte injury provide some insight into these possibilities25. These experiments indicate -that rat oligodendrocytes activate syngeneic complement in the absence of specific antibody, leading to cell lysis. Serum from decomplemented rats does not exhibit this cytolytic activity; neither does serum depleted in vitro of either C1 or C9. Oligodendrocytes exposed to serufia stain immuntochemically for surface C9, but not mun globulin. Astrcytes are not susceptible, while bipotential O-2A progenitor cells are lysed only by a high concentration ofserum. Differentiation and maturation of O-2AprogeniMstor cllsintoisassociated with ineasg susceptibiity to attackby complement. In addition to acquiing a cell-surface complementactivating component during maturation, oligodendrocytes also appear to be deficient in membraneanchored complement regulatory proteins In reactive lysis studies, when MAC formation in EDTA-treated human serum was triggered by the introduction of exogenously prepared activated C5b6, oligodendrocytes were again selectively lysed (Zajicek J, -Wing MG, Lachmannw PJ, Compston DAS, in preparation. Furthermore, the addition of human CD59 (which prevents C9 incorporation into the membrane attack complex) protects against both reactive lysis and the lytic effects of normal human serum, supporting the suggestion that rat oligodendrocytes lack one or more terminal complement regulatory proteins. During attack by sub-lethal concentrations of complement, numerous vesicles, brightly staining for C9, appear on the surface of.oligodendrocytes after 2 min exposure, which- have disappeared after a further 5 min. Concurrently, oligodendrocyte-derivedparticulate material bearing surface C9 appears in culture supernatants, suggesting that, following complement activation, oligodendrocytes resist lysis by the vesicular removal of complement m e attack complexesm. During reversible injury,, a transient rise in intracellular calcium and fall in ATP are seen, but oligodendrocytes recover and remain able later to express myelin antigens which are markers of oligodendrocyte maturation. The factors responsible for targeting macrophages against oligodendrocytes may also be investigated in vitro. The results indicate that anti-oligodendrocyte antibodies are able.- to direct macrophages onto oligodendrocytes and totrigger myelin phagocytosis?7. While complement alone does not exhibit this activity using resting macrophages, preliminary results suggest that activated macrophages and, more pertinently, activated microglia, can be targeted onto oligodendrocytes in vitro by. complement alone. A number of in vivo observation&also suggest that complement may be significant in the pathogenesis of demyelnation. Complement activation -products C3a and C4a are present in cerebrospinal fluid of patients with demyelinating disease, and levels of the terminal component 09 are reduced, implying that complement is activated within the CNS; complemelit components can also be: demonstrated immunohistochemically within lesions in a pen.vascular distribution. Vesicular material, identical .to that released by oligodendrocytes injured by complement in vitro, is also present in the cerebrospinal fluid of patients with,demyelination, but not controls with structural CNSlesions26. Finally,
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immunofluorescence staining of CNS tissue from patients with multiple sclerosis shows the co-location of complement and antibody on particles within macrophages in demyelinating lesions2. These observations are consistent with the hypothesis that blood-brain barrier breakdown, which is likely to be mediated by T lymphocytes, initiates CNS inflammation by exposing oligodendrocytes to serum complement. Reversible or lytic oligodendrocytemyelin damage might be followed by demyelination and/or macrophage recruitment, and the process may be amplified by consequent secondary cellular and humoral immune responses. In the final presentation Professor K Toyka spoke on the role of macrophages in immune-mediated demyelination. In most tissues macrophages are the principal cells expressing MHC class II and presenting antigenic peptides to the T-cell antigen receptor. They produce many cytotoxic factors including oxygen and nitric oxide radicals, ILl, IL6, TNFci, and prostaglandins and leukotrienes which damage blood tissue barriers and act as chemotactic signals for haematogenous inflammatory leukocytes. When activated, macrophages synthesize complement components. They can also bind to specific antibodies by means of antibody dependent cell-mediated cytotoxicity. What are the equivalent cells in the central and peripheral nervous systems? In the CNS the macrophage-equivalents are the microglia which are derived from bone-marrow monocytes during ontogeny. Microglia may be the cell type which initiates immune-mediated local inflammation once it has been activated by invading T-cells. It has been shown that interferon-'y is the main inducer of MHC class II upregulation on microglia in vivo. Astrocytes also have the potential to produce immune mediators and can function as antigen presenting cells in cell culture. At present, it seems unlikely that they are important in fulfilling this function in vivo. In the PNS resident macrophages are present which may initiate immune-mediated inflammation. At present, it seems unlikely that Schwann cells exert macrophage-like functions though they can function as antigen presenting cells in vitro. Microglial cells derived from neonatal rat brain and cultured to more than 95% purity can produce proinflammatory and cytotoxic mediators. Prostaglandin production can be stimulated with phorbolmyristate, Ca-ionophore, lipopolysaccharide, or IFN--y. The microglia also produces cytotoxic oxygen and, as very recently shown, nitric oxide radicals. Microglial cells produce these active metabolites in a dose dependent fashion upon stimulation with IFN--y. Astrocytes do produce very little nitric oxide but to a much lesser extent. The macrophage is the main effector cell at sites of inflammation. Macrophages are attracted during the induction and amplification phase by antigen-specific activated T cells and, possibly, by activated resident macrophages or microglia. In the early lesions of the peripheral nerve inflammatory disease, experimental allergic neuritis (EAN), abundant macrophages can be seen around blood vessels and associated with demyelinated axons29~31. The macrophages contain myelin debris which they have phagocytosed. Early axonal degeneration may be found and is considered to be caused by bystander damage. The crucial question is whether
the macrophages are important causative agents or merely involved in a clearing-up operation. In favour of a causative role is the ability of macrophages taken at disease onset to produce cytotoxic oxygen radicals. Furthermore when animals are treated with the natural oxygen radical scavengers, superoxide dismutase or catalase, before the onset of EAN, functional deficit can be prevented32. The histological evidence of inflammation is also reduced. The neurological deficit and inflammatory changes are also reduced by blocking eicosanoid production with indomethacin or even more selective cyclo-oxygenase blockersm3. Macrophages produce and activate complement, and complement activation may interfere with normal nerve conduction. The terminal complement component membrane attack complex can be detected on the surface of myelin sheaths in early but not late EAN. EAN in rats can be suppressed by depletion of complement with cobra venom factor3. There is transient expression of IFN-y by endoneurial cells in early EAN, and IFN--y is probably the most important activation signal for macrophages (and microglia)35. Treatment with antibodies to IFN-'y profoundly reduces disease activity. The speakers at the meeting made clear that the whole range of immunological instruments has a part to play in orchestrating immune-mediated demyelination. The process must be introduced by breaching the blood-nerve or blood-brain barrier in which activated T cells and inflammatory mediators play an important part. The mast cells resident in the perineurium, endoneurium and meninges may also be involved. Antibody-mediated or T cell-mediated reactions against specific myelin antigens have been demonstrated in experimental and human diseases. Complement undoubtedly plays its conventional role in amplifying the inflammatory process and may have a special role in damaging oligodendrocytes, even in the absence of specific antibody. Macrophages are active throughout immune-mediated demyelination, presenting antigen at an early stage, initiating the process of demyelination and marching triumphantly offthe stage bearing the myelin debris in a grand finale. R A C Hughes Department of Neurology Guy's Hospital, London References 1 Bradbury MW. The structure and function of the blood-brain barrier. Fedn Proc 1984;43:186 2 Broadwell RD. Transcytosis of molecules through the blood-brain barrier: a cell biological perspective and critical appraisal. Acta Neuropath (Berl) 1989;79:117 3 Sedgwick J, Hughes CCW, Male DK, MacPhee IAM, Ter Meulen V. Antigen-specific damage to brain vascular endothelial cells mediated by encephalitogenic and nonencephalitogenic CD4+ T cell lines in vitro. JImmunol
1990;145:2474 4 Male DK, Pryce G, Hughes CCW, Lantos PL. Lymphocyte migration into brain modelled in vitro: control by
lymphocyrte activation cytokines and antigen. Cell Immunol 1990;127:1 5 Cross AH, Cannella B, Brosnan OF, Raine CS. Homing to central nervous system vasculature by antigen-specific lymphocytes. I. Localisation of 14C-labelled cells during acute, chronic and relapsing experimental allergic encephalomyelitis. Lab Inuest 1990;63:162 6 Schwartz RHI. Immune responses (Ir) genes of the murine major histocompatibility complex. Adv Immunol 1986; 38:31
Journal of the Royal Society of Medicine Volume 85 January 1992 7 Wraith DC, McDevitt HO, Steinman L, Acha-Orbea H. T cell recognition as the target for immune intervention in autoimmune disease. Cell 1989;57:709 8 Wraith DC, Smilek DE, Mitchell DJ, Steinman L, McDevitt HO. Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunotherapy. Cell 1989;59:247-55 9 Urban JL, Horvath SJ, Hoad L. Autoimmune T cells: immune recognition of normal and variant epitopes and peptide-based therapy. Cell 1989;59: 257-71 10 Lamont AG, Sette A, Fujinami R, Colon SM, Miles C, Grey HM. Inhibition of experimental autoimmune encephalomyelitis induction in SJL/J mice by using a peptide with high affinity for I-As molecules. JImmunol 1990;145:1687 11 Quarles RH, Ilyas AA, Willison HJ. Antibodies to glycolipids in demyelinating diseases of the human peripheral nervous system. Chem Phys Lipids 1986; 42:235-48 12 Gregson NA, Leibowitz S. IgM paraproteinaemia, polyneuropathy and myelin-associated glycoprotein (MAG). Neuropathol Appl Neurobiol 1985;11:329347 13 Latov N. Antibodies to glycoconjugates in neurological disease. Clin Aspects Autoimmun 1990;4:18-29 14 Ilyas AA, Willison HJ, Dalakas MC, Whittaker JN, Quarles RH. Identification and characterization of gangliosides reacting with IgM paraproteins in three patients with neuropathy associated with biclonal gammopathy. J Neurochem 1988;51:851-8 15 Baba H, Daune GC, Ilyas AA, et al. Anti-GM1 ganglioside antibodies with differing fine specificities in patients with multifocal motor neuropathy. J Neuroimmunol 1989;25:143-50 16 Latov N, Hays AP, Donofrio PD, et al. Monoclonal IgM with unique specificity to gangliosides GM1 and GD1B and to lacto-N-tetraose associated with human motor neuron disease. Neurology 1988;38:763-8 17 Pestronk A, Cornblath DR, Ilyas AA, et al. A treatable multifocal neuropathy with antibodies to GM1 ganglioside. Ann Neurol 1988;24:73-8 18 Pestronk A, Adams RN, Clawson L, et al. Serum antibodies to GM1 ganglioside in amyotrophic lateral sclerosis. Neurology 1988;38:1457-61 19 Gregson NA, Jones D, Willison HJ. Antibodies against GM1 and other ganglioside in patients with motor neuron syndromes. In: Clifford Rose F, ed. New evidence in MND/ALS research. London: Smith-Gordon & Co. Ltd, 1991 20 Marcus DM, Latov N, Hsi BP, Gillard BK. Measurement and significance of antibodies against GM1 ganglioside. Report of a Workshop, 18 April 1989, Chicago, IL, USA. J Neuroimmunol 1989;25:255-9
21 Ilyas AA, Willison HJ, Quarles RH, et al. Serum antibodies to gangliosides in Guillain-Barre syndrome. Ann Neurol 1988;23:440-7 22 Koski CL, Chou DKH, Jungalwala FB. Anti-peripheral nerve myelin antibodies in GBS bind a neutral glycolipid of peripheral myelin and cross-react with Forssman antigen. J Clin Invest 1989;84:280-7 23 Ilyas AA, Mithen FA, Chen Z-W, Cook SD. Search for antibodies to neutral glycolipids in sera of patients with Guillain-Barre syndrome. JNeurol Sci 1991;102:67-75 24 Prineas JW. The neuropathology of multiple sclerosis. In: Handbook of clinical neurology. Amsterdam: Elsevier, 1985:213-57 25 Compston A, Scolding N, Wren D, Noble M. The pathogenesis of demyelinating disease: Insights from cell biology. Trends Neurosci 1991;14:175-182 26 Scolding NJ, Morgan BP, Houston A, Linington C, Campbell AK, Compston DAS. Vesicular removal by oligodendrocytes of membrane attach complexes formed by complement. Nature 1989;339:620-2 27 Scolding NJ, Compston DAS. Oligodendrocyte-macrophage interactions in vitro triggered by specific antibodies. Immunology 1990;72:127-32 28 Gay D, Esiri M. Blood-brain barrier damage in acute multiple sclerosis plaques. An immunocytochemical study. Brain 1991;114:557-72 29 Craggs RI, King RHM, Thomas PK. The effect of suppression of macrophage activity on the development of experimental allergic neuritis. Acta Neuropathol (Berl) 1988;23:326-31 30 Sobue G, Yamato S, Hirayama M, et aL. The role of macrophages in demyelination in experimental allergic neuritis. J Neurol Sci 1982;56:75-87 31 Trotter J, Smith ME. The role of phospholipases from inflamatoy macropha in demyelination. Neurochem Res 1986;11:349-61 32 Hartung H-P, Schafer B, Heininger K, Toyka KV. Suppression of experimental autoimmune neuritis by the oxygen radical scavengers superoxide dismutase and catalase. Ann Neurol 1988;23:453-60 33 Hartung H-P, Schafer B, Heininger K, Stoll G, Toyka KV. The role of macrophages and eicosanoids in the pathogenesis of experimental allergic neuritis. Brain 1988;111:1039-59 34 Stoll G, Schmidt B, Jander S, Toyka KV, Hartung HP. Presence of the terminal complement complex (C5b-9) precedes myelin degradation in acute demyelination in the peripheral nervous system of the rat. Ann Neurol 1991;30:147-55 35 Hartung H-P, Schifer B, Van der Meide PH, Fierz W, Heininger K, Toyka KV. The role ofinterferon-gamma in the pathogenesis of experimental autoimmune disease of the peripheral nervous system. Ann Neurol 1990; 27:247-57
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