Dora Barchan

Blockade of CD40 ligand suppresses chronic experimental myasthenia gravis by down-regulation of Th1 differentiation and up-regulation of CTLA-4.

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-dependent Ab-mediated autoimmune disorders, in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Th1-type cells and costimulatory factors such as CD40 ligand (CD40L) contribute to disease pathogenesis by producing proinflammatory cytokines and by activating autoreactive B cells. In this study we demonstrate the capacity of CD40L blockade to modulate EAMG, and analyze the mechanism underlying this disease suppression. Anti-CD40L Abs given to rats at the chronic stage of EAMG suppress the clinical progression of the autoimmune process and lead to a decrease in the AChR-specific humoral response and delayed-type hypersensitivity. The cytokine profile of treated rats suggests that the underlying mechanism involves down-regulation of AChR-specific Th1-regulated responses with no significant effect on Th2- and Th3-regulated AChR-specific responses. EAMG suppression is also accompanied by a significant up-regulation of CTLA-4, whereas a series of costimulatory factors remain unchanged. Adoptive transfer of splenocytes from anti-CD40L-treated rats does not protect recipient rats against subsequently induced EAMG. Thus it seems that the suppressed progression of chronic EAMG by anti-CD40L treatment does not induce a switch from Th1 to Th2/Th3 regulation of the AChR-specific immune response and does not induce generation of regulatory cells. The ability of anti-CD40L treatment to suppress ongoing chronic EAMG suggests that blockade of CD40L may serve as a potential approach for the immunotherapy of MG and other Ab-mediated autoimmune diseases.

Suppression of ongoing experimental myasthenia by oral treatment with an acetylcholine receptor recombinant fragment

Myasthenia gravis (MG) is an autoimmune disorder in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. In an attempt to develop an antigen-specific therapy for MG, we administered a nonmyasthenogenic recombinant fragment of AChR orally to rats. This fragment, corresponding to the extracellular domain of the human AChR alpha-subunit (Halpha1-205), protected rats from subsequently induced experimental autoimmune myasthenia gravis (EAMG) and suppressed ongoing EAMG when treatment was initiated during either the acute or chronic phases of disease. Prevention and suppression of EAMG were accompanied by a significant decrease in AChR-specific humoral and cellular responses. The underlying mechanism for the Halpha1-205-induced oral tolerance seems to be active suppression, mediated by a shift from a T-helper 1 (Th1) to a Th2/Th3 response. This shift was assessed by changes in the cytokine profile, a deviation of anti-AChR IgG isotypes from IgG2 to IgG1, and a suppressed AChR-specific delayed-type hypersensitivity response. Our results in experimental myasthenia suggest that oral administration of AChR-specific recombinant fragments may be considered for antigen-specific immunotherapy of myasthenia gravis.

Suppression of experimental myasthenia gravis, a B cell-mediated autoimmune disease, by blockade of IL-18

Interleukin‐18 (IL‐18) is a pleiotropic proinflammatory cytokine that plays an important role in interferon gamma (IFN‐γ) production and IL‐12‐driven Th1 phenotype polarization. Increased expression of IL‐18 has been observed in several autoimmune diseases. In this study we have analyzed the role of IL‐18 in an antibody‐mediated autoimmune disease and elucidated the mechanisms involved in disease suppression mediated by blockade of IL‐18, using experimental autoimmune myasthenia gravis (EAMG) as a model. EAMG is a T cell‐regulated, antibody‐mediated autoimmune disease in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Th1‐ and Th2‐type responses are both implicated in EAMG development. We show that treatment by anti‐IL‐18 during ongoing EAMG suppresses disease progression. The protective effect can be adoptively transferred to naive recipients and is mediated by increased levels of the immunosuppressive Th3‐type cytokine TGF‐β and decreased AChR‐specific Th1‐type cellular responses. Suppression of EAMG is accompanied by down‐regulation of the costimulatory factor CD40L and up‐regula‐tion of CTLA‐4, a key negative immunomodulator. Our results suggest that IL‐18 blockade may potentially be applied for immunointervention in myasthenia gravis.—Im, S.‐H., Barchan, D., Maiti, P. K., Raveh, L., Souroujon, M. C., Fuchs, S. Suppression of experimental myasthenia gravis, a B cell‐mediated autoimmune disease, by blockade of IL‐18. FASEB J. 15, 2140–2148 (2001)

Modulation of the anti-acetylcholine receptor response and experimental autoimmune myasthenia gravis by recombinant fragments of the acetylcholine receptor.

Myasthenia gravis (MG) is a neuromuscular disorder of man caused by a humoral response to the acetylcholine receptor (AChR). Most of the antibodies in MG and in experimental autoimmune myasthenia gravis (EAMG) are directed to the extracellular portion of the AChR α subunit, and within it, primarily to the main immunogenic region (MIR). We have cloned and expressed recombinant fragments, corresponding to the entire extracellular domain of the AChR α subunit (Hα1 – 210), and to portions of it that encompass either the MIR (Hα1 – 121) or the ligand binding site of AChR (Hα122 – 210), and studied their ability to interfere with the immunopathological anti‐AChR response in vitro and in vivo. All fragments were expressed as fusion proteins with glutathione S‐transferase. Fragments Hα1 – 121 and Hα1 – 210 protected AChR in TE671 cells against accelerated degradation induced by the anti‐MIR monoclonal antibody (mAb)198 in a dose‐dependent manner. Moreover, these fragments had a similar effect on the antigenic modulation of AChR by other anti‐MIR mAb and by polyclonal rat anti‐AChR antibodies. Fragments Hα1 – 121 and Hα1 – 210 were also able to modulate in vivo muscle AChR loss and development of clinical symptoms of EAMG, passively transferred to rats by mAb 198. Fragment Hα122 – 210 did not have such a protective activity. Our results suggest that the appropriate recombinant fragments of the human AChR may be employed in the future for antigen‐specific therapy of myasthenia.

Mechanism of nasal tolerance induced by a recombinant fragment of acetylcholine receptor for treatment of experimental myasthenia gravis

Acetylcholine receptor (AChR) is the major autoantigen in myasthenia gravis (MG) and experimental autoimmune MG (EAMG). Here we analyze the mechanisms involved in suppression of ongoing EAMG in rats by nasal administration of a recombinant fragment from the human AChR alpha-subunit. We demonstrate that such a fragment, expressed without a fusion partner, confers nasal tolerance that can be adoptively transferred. Our observations suggest that the underlying mechanism of this nasal tolerance is active suppression involving a shift from a Th1 to a Th2/Th3-regulated AChR-specific response which may be mediated by down regulation of costimulatory factors.

Analysis and modulation of the immune response of mice to acetylcholine receptor by anti-idiotypes

Anti-idiotypes were raised in mice against three well-characterized anti-acetylcholine receptor (AChR) monoclonal antibodies (mcAbs), as well as against polyclonal mouse anti-AChR antibodies. In binding experiments, the anti-idiotypic antibodies inhibited the binding of AChR only to the immunizing idiotype. However, a less restricted specificity was found in in vivo experiments. Mice producing anti-idiotypes were challenged with AChR and the idiotypic composition of their anti-AChR response was analysed using specific rabbit anti-idiotypic antibodies. It was found that preimmunization with a certain idiotype leads to the preferential suppression of this particular idiotype in the polyclonal response to AChR. However, preimmunization with either polyclonal or monoclonal anti-AChR antibodies resulted in a reduction of the overall anti-Torpedo AChR and anti-muscle AChR titers. This reduction was greater than would be expected from the representation of each of the respective idiotypes in the polyclonal anti-AChR serum, and may imply that in addition to the immunizing idiotype other anti-AChR idiotypes are also suppressed. Our results suggest that anti-idiotypes may have a potential for the modulation of the autoimmune response directed against AChR in myasthenia.

Protective molecular mimicry in experimental myasthenia gravis

Protein databases were searched for microbial sequences that bear amino acid similarities with identified T- or B-cell epitopes within the human alpha-subunit of acetylcholine receptor (AChR). One peptide, derived from Haemophilus influenzae, exhibits 50% homology to an identified T-cell epitope of AChR alpha-subunit. This peptide was shown to have a protective effect in experimental autoimmune myasthenia gravis (EAMG). Pretreatment of rats with the mimicry peptide attenuated the induction and progression of EAMG. These effects were accompanied by a reduced T-cell response to AChR, diminished IL-2, IL-12, IFN-gamma and IL-4 levels, as well as decreased humoral response to self-AChR.

Synthetic peptides and their antibodies in the analysis of the acetylcholine receptor.

The amino acid sequence of the entire acetylcholine receptor (AChR) of electric fish and of several other species has been established with the advent of gene cloning and recombinant DNA technologies employed by several groups (for review see Patrick et al., this volume). The sequence data have been the basis of predictions concerning the transmembrane orientation of the AChR subunits, as well as the possible location of the acetylcholine ( ACh) binding site, the glycosylation and phosphorylation sites, and the main immunogenic determinants. In our laboratory we are employing synthetic peptides from various regions in the AChR molecule, and their specific antibodies, for structure and function analysis of the AChR. In the following we would like to report on our recent studies on the application of synthetic peptides and their antibodies for (a) mapping the cholinergic binding site of AChR (b) preparation of species-specific anti-AChR antibodies; (c) identification of highly immunogenic regions in the receptor; and (d) analysis and localization of phosphorylation sites in AChR. Synthetic peptides corresponding to sequences from the AChR a-subunit were employed for studying the first three topics (a-c), and synthetic peptides corresponding to sequences from the AChR &subunit were employed in the study of the fourth one (d). The peptides that were synthesized and analyzed for these studies are listed in FIGURE 1.

The ligand binding domain of the nicotinic acetylcholine receptor: Immunological analysis

The interaction of the acetylcholine receptor (AChR) binding site domain with specific antibodies and with α‐bungarotoxin (α‐BTX) has been compared. The cloned and expressed ligand binding domain of the mouse AChR α‐subunit binds α‐BTX, whereas the mongoose‐expressed domain is not recognized by α‐BTX. On the other hand, both the mouse and mongoose domains bind to the site‐specific monoclonal antibody 5.5. These results demonstrate that the structural requirements for binding of α‐BTX and mcAb 5.5, both of which interact with the AChR binding site, are distinct from each other.

Antigen-Specific Therapy of Experimental Autoimmune Myasthenia Gravis: Mucosal tolerance with recombinant fragments of the human acetylcholine receptor

Two important observations in 1973 resulted in a breakthrough in Myasthenia Gravis (MG) research. The first by Fambrough et al [1] showed reduced levels of acetylcholine receptor (AChR) at the neuromuscular junction of patients with MG and the second, by Patrick and Lindstrom [2], demonstrated that rabbits immunized with AChR develop an autoimmune response to AChR, resulting in muscle weakness, and a decrementai electromyogram response to repeated nerve stimulation, which are characteristic to MG. These two pivotal observations were seminal for elucidating and understanding the autoimmune nature of MG, and prompted a vast amount of research into both MG and its experimental animal model, experimental autoimmune myasthenia gravis (EAMG).