A Targeted Complement Inhibitor CRIg/FH Protects Against Experimental Autoimmune Myasthenia Gravis in Rats via Immune Modulation.

Antibody-induced complement activation may cause injury of the neuromuscular junction (NMJ) and is thus considered as a primary pathogenic factor in human myasthenia gravis (MG) and animal models of experimental autoimmune myasthenia gravis (EAMG). In this study, we tested whether CRIg/FH, a targeted complement inhibitor, could attenuate NMJ injury in rat MG models. We first demonstrated that CRIg/FH could inhibit complement-dependent cytotoxicity on human rhabdomyosarcoma TE671 cells induced by MG patient-derived IgG in vitro. Furthermore, we investigated the therapeutic effect of CRIg/FH in a passive and an active EAMG rodent model. In both models, administration of CRIg/FH could significantly reduce the complement-mediated end-plate damage and suppress the development of EAMG. In the active EAMG model, we also found that CRIg/FH treatment remarkably reduced the serum concentration of autoantibodies and of the cytokines including IFN-γ, IL-2, IL-6, and IL-17, and upregulated the percentage of Treg cells in the spleen, which was further verified in vitro. Therefore, our findings indicate that CRIg/FH may hold the potential for the treatment of MG via immune modulation.

Blocking of IL-6 suppresses experimental autoimmune myasthenia gravis.

Suppressive regulatory T cells (Treg) and pathogenic T helper 17 (Th17) cells are two lymphocyte subsets with opposing activities in autoimmune diseases. The proinflammatory cytokine IL-6 is a potent factor in switching immune responses in vivo from the induction of Treg to pathogenic Th17 cells. We studied the Treg and Th17 cell compartments in experimental autoimmune myasthenia gravis (EAMG) and healthy control rats in order to assess whether the equilibrium between Treg and Th17 cells is perturbed in the disease. We found that Th17 cell-related genes are upregulated and Treg-related genes are downregulated in EAMG. The shift in favor of Th17 cells in EAMG could be reversed by antibodies to IL-6. Administration of anti-IL-6 antibodies to myasthenic rats suppressed EAMG when treatment started at the acute or at the chronic phase of disease. Suppression of EAMG by anti-IL-6 antibodies was accompanied by a decrease in the overall rat anti-AChR antibody titer and by a reduced number of B cells as compared with control treatment. Administration of anti-IL-6 antibodies led to down-regulation of several Th17 related genes including IL-17, IL-17R, IL-23R and IL-21 but did not affect the number of Treg cells in the lymph nodes. These data identify IL-6 as an important target for modulation of autoimmune responses.

Novel complement inhibitor limits severity of experimentally myasthenia gravis.

OBJECTIVE: Complement mediated injury of the neuromuscular junction is considered a primary disease mechanism in human myasthenia gravis and animal models of experimentally acquired myasthenia gravis (EAMG). We utilized active and passive models of EAMG to investigate the efficacy of a novel C5 complement inhibitor rEV576, recombinantly produced protein derived from tick saliva, in moderating disease severity. METHODS: Standardized disease severity assessment, serum complement hemolytic activity, serum cytotoxicity, acetylcholine receptor (AChR) antibody concentration, IgG subclassification, and C9 deposition at the neuromuscular junction were used to assess the effect of complement inhibition on EAMG induced by administration of AChR antibody or immunization with purified AChR. RESULTS: Administration of rEV576 in passive transfer EAMG limited disease severity as evidenced by 100% survival rate and a low disease severity score. In active EAMG, rats with severe and mild EAMG were protected from worsening of disease and had limited weight loss. Serum complement activity (CH(50)) in severe and mild EAMG was reduced to undetectable levels during treatment, and C9 deposition at the neuromuscular junction was reduced. Treatment with rEV576 resulted in reduction of toxicity of serum from severe and mild EAMG rats. Levels of total AChR IgG, and IgG(2a) antibodies were similar, but unexpectedly, the concentration of complement fixing IgG(1) antibodies was lower in a group of rEV576-treated animals, suggesting an effect of rEV576 on cellular immunity. INTERPRETATION: Inhibition of complement significantly reduced weakness in two models of EAMG. C5 inhibition could prove to be of significant therapeutic value in human myasthenia gravis.

Prevention of experimental autoimmune myasthenia gravis by rat Crry-Ig: A model agent for long-term complement inhibition in vivo.

Despite its vital role in innate immunity, complement is involved in a number of inflammatory pathologies and has therefore become a therapeutic target. Most agents generated for anti-complement therapy have short half-lives in plasma, or have been of mouse or human origin, thereby limiting their use either to murine models of disease or to short-term therapy. Here we describe the generation of a long-acting rat therapeutic agent based on the rat complement inhibitor, Crry. Characterisation of various soluble forms of Crry demonstrated that the amino-terminal four short-consensus repeat domains were required for full regulatory and C3b-binding activities. Fusion of these domains to rat IgG2a Fc generated an effective complement inhibitor (rCrry-Ig) with a circulating half-life prolonged from 7 min for Crry alone to 53 h for rCrry-Ig. Systemic administration of rCrry-Ig over 5 weeks generated a weak immune response to the recombinant agent, however this was predominantly IgM in nature and did not neutralise Crry function or cause clearance of the agent from plasma. Administration of rCrry-Ig completely abrogated clinical disease in a rat model of myasthenia gravis whereas soluble Crry lacking the immunoglobulin Fc domain caused a partial response. rCrry-Ig not only ablated clinical disease, but also prevented C3 and C9 deposition at the neuromuscular junction and inhibited cellular infiltration at this site. The long half-life and low immunogenicity of this agent will be useful for therapy in chronic models of inflammatory disease in the rat.

[The mechanism of prophylactic effects of nasal tolerance with a dual analogue on experimental autoimmune myasthenia gravis in young mice].

OBJECTIVE: To study the prophylactic effects of nasal tolerance with a dual analogue, Lys262-Ala207, on the mouse model of experimental autoimmune myasthenia gravis (EAMG) and the underlying mechanism. METHODS: Mouse model of EAMG was induced by intraperitoneal injection of mAb35. Lys262-Ala207 or PBS was given nasally before 10 days (study group A and control group A) or on the day (study group B and control group B) of immunization for 10 days. Clinical syndromes were evaluated after immunization. Serum level of acetylcholine receptor antibody (AChR-Ab) IgG was detected using ELISA. The number of monouclear cells expressing CD4+ and CD4+ CD25+T from spleen was measured using flow cytometry. RESULTS: Compared with the corresponding control groups, the clinical syndromes were improved (P<0.01) in mice from the study groups A and B. The positive rate of the repetitive nerve stimulation (RNS) test in the study groups A and B was significantly lower than that in the corresponding control groups (P<0.01). The study group A showed lower positive rate of RNS than the study group B (P<0.05). The serum levels of AChR-Ab IgG in the study groups A and B (15.01+/-1.09 and 19.23+/-1.31 microg/mL) decreased compared with that in the corresponding control groups (28.12+/-1.28 and 29.35+/-1.28 microg/mL) (P<0.01). The study group A mice had lower serum AChR-Ab IgG levels than the study group B (P<0.01). The number of CD4+ CD25+T cells in the study groups A (4.516+/-0.598%) and B (3.671+/-0.300%) increased significantly compared with that in the corresponding control groups (2.661+/-0.411% and 2.412+/-0.500%) (P<0.01) and more CD4+ CD25+T cells were found in the study group A than in the study group B (P<0.01). CONCLUSIONS: Nasal administration with dual analogues may ameliorate clinical syndromes in EAMG rats, which may be associated with decreased serum AChR-Ab IgG levels and increased number of CD4+ CD25+T cells from spleen.

Markedly enhanced susceptibility to experimental autoimmune myasthenia gravis in the absence of decay-accelerating factor protection.

Myasthenia gravis (MG) is an autoimmune neuromuscular transmission disorder characterized by loss of acetylcholine receptors (AChR's) due primarily to the production of anti-AChR autoantibodies. In this study we investigated whether the presence of decay-accelerating factor (DAF or CD55), an intrinsic complement regulator, protects against the development of disease. Experimental autoimmune MG was induced in Daf1(-/-) mice (devoid of neuromuscular DAF protein) and their Daf1(+/+) littermates by injection of rat anti-AChR mAb McAb-3. After twenty-four hours, grip strength assessment revealed that Daf1(-/-) mice exhibited hold times of less than 30 seconds, compared with more than 8 minutes for the Daf1(+/+) controls. The weakness was reversed by edrophonium, consistent with a myasthenic disorder. Immunohistochemistry revealed greatly augmented C3b deposition localized at postsynaptic junctions, and radioimmunoassays showed more profound reductions in AChR levels. Electron microscopy demonstrated markedly greater junctional damage in the Daf1(-/-) mice compared with the Daf1(+/+) littermates. Control studies showed equivalent levels of other cell surface regulators, i.e., Crry and CD59. The results demonstrate that mice that lack DAF are markedly more susceptible to anti-AChR-induced MG, which simulates the primary mechanism in the human disease, and strongly suggest that in disease flares complement inhibitors might have therapeutic value.

Intravenous immunoglobulin suppresses experimental myasthenia gravis: immunological mechanisms.

Intravenous immunoglobulin (IVIG) administration has been beneficially used in the treatment of several autoimmune disorders including myasthenia gravis (MG), although its mechanism of action is still not clear. To study the optimal conditions of IVIG treatment and delineate its mechanism of action we established a suitable model in rat experimental autoimmune MG (EAMG). We show that IVIG has a suppressive effect on the clinical symptoms of ongoing EAMG that is associated with decreased AChR-specific cellular and humoral immune reactivity. Costimulatory factors and cytokine profile analyses suggest that IVIG immunomodulation in EAMG involves suppression of B and Th1-type T cell responses with no generation of T-regulatory cells. Our data contribute to the understanding of the immunological mechanisms underlying IVIG treatment in MG and in other autoimmune disorders.

Vaccination with a MHC class II peptide in Alum and inactive pertussis strongly ameliorates clinical MG in C57BL/6 mice.

We have investigated the efficacy of immunization against peptides from predisposing MHC class II molecules in human-compatible adjuvants for ameliorating experimental autoimmune myasthenia gravis (EAMG). C57BL/6 mice were immunized three times with the peptide I-Abetab62-76 in Alum+killed pertussis organisms (PT) prior to two injections with tAChR. The treatment greatly reduced the occurrence and severity of clinical MG relative to controls that received saline/Alum+PT or none. It also reduced antibody and T-cell responses against tAChR. The results have important implications for the possible immunotherapy of MG by targeting disease-associated MHC.

Curcumin ameliorates experimental autoimmune myasthenia gravis by diverse immune cells.

Curcumin is a traditional Asian medicine with diverse immunomodulatory properties used therapeutically in the treatment of many autoimmune diseases. However, the effects of curcumin on myasthenia gravis (MG) remain undefined. Here we investigated the effects and potential mechanisms of curcumin in experimental autoimmune myasthenia gravis (EAMG). Our results demonstrated that curcumin ameliorated the clinical scores of EAMG, suppressed the expression of T cell co-stimulatory molecules (CD80 and CD86) and MHC class II, down-regulated the levels of pro-inflammatory cytokines (IL-17, IFN-γ and TNF-α) and up-regulated the levels of the anti-inflammatory cytokine IL-10, shifted the balance from Th1/Th17 toward Th2/Treg, and increased the numbers of NKR-P1(+) cells (natural killer cell receptor protein 1 positive cells, including NK and NKT cells). Moreover, the administration of curcumin promoted the differentiation of B cells into a subset of B10 cells, increased the anti-R97-166 peptide IgG1 levels and decreased the relative affinity indexes of anti-R97-116 peptide IgG. In summary, curcumin effectively ameliorate EAMG, indicating that curcumin may be a potential candidate therapeutic agent for MG.

The effect of CD3-specific monoclonal antibody on treating experimental autoimmune myasthenia gravis.

CD3-specific monoclonal antibody was the first one used for clinical practice in field of transplantation. Recently, renewed interests have elicited in its capacity to prevent autoimmune diabetes by inducing immune tolerance. In this study, we tested whether this antibody can also be used to treat another kind of autoimmune disease myasthenia gravis (MG) and explored the possible mechanisms. MG is caused by an autoimmune damage mediated by antibody- and complement-mediated destruction of AChR at the neuromuscular junction. We found that administration of CD3-specific antibody (Fab)2 to an animal model with experimental autoimmune myasthenia gravis (EAMG) (B6 mice received 3 times of AChR/CFA immunization) could not significantly improve the clinical signs and clinical score. When the possible mechanisms were tested, we found that CD3 antibody treatment slightly down-regulated the T-cell response to AChR, modestly up-regulating the muscle strength. And no significant difference in the titers of IgG2b was found between CD3 antibody treated and control groups. These data indicated that CD3-specific antibody was not suitable for treating MG, an antibody- and complement- mediated autoimmune disease, after this disease has been established. The role of CD3-specific antibody in treating this kind of disease remains to be determined.

Immature dendritic cell exosomes suppress experimental autoimmune myasthenia gravis.

Immature dendritic cell-derived exosomes (iMDEX) display a certain degree of immunosuppressive activity in autoimmune diseases. However, the role of iMDEX in experimental autoimmune myasthenia gravis (EAMG) is still unclear. Therefore, we tested the effects of mouse bone marrow (BM)-derived iMDEX on tolerance induction in a mouse model of EAMG. In this study, we found that the CELLine culture system produced more exosomes, the morphology and phenotype of these exosomes were found to be identical when compared with traditional cell culture. And, iMDEX(1000) ameliorated the progression of EAMG by reducing AChR-reactive lymphocyte proliferation, AChR antibody levels and pro-inflammatory cytokine levels.

Prevention of passively transferred experimental autoimmune myasthenia gravis by an in vitro selected RNA aptamer.

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are mainly caused by autoantibodies directed against acetylcholine receptors (AChR) located in the postsynaptic muscle membrane. Previously, we isolated an RNA aptamer with 2'-fluoropyrimidines using in vitro selection techniques that acted as an effective decoy against both a rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the AChR, and a significant fraction of patient autoantibodies with MG. To investigate the therapeutic potential of the RNA, we tested the ability of the RNA aptamer to protect the receptors in vivo from mAb198. Clinical symptoms of EAMG in rats engendered by passive transfer of mAb198 were efficiently inhibited by a truncated RNA aptamer that was modified with polyethylene glycol, but not by control scrambled RNA. Moreover, the loss of AChR in the animals induced by the antibody was also significantly blocked with the modified RNA aptamer. These results suggested that RNA aptamers could be applied for antigen-specific treatment for autoimmune diseases including MG.

Adoptive protection from experimental myasthenia gravis with T cells from mice treated nasally with acetylcholine receptor epitopes.

Nasal administration of synthetic CD4(+) epitopes of the acetylcholine receptor (AChR) prevents experimental myasthenia gravis (EMG) in C57Bl/6 mice, but not in IL4-deficient C57Bl/6 (IL4(-/-)) mice. Here we verify that nasal tolerance requires IL4, by showing that CD4(+) cells from C57Bl/6 mice treated nasally with a pool of AChR CD4(+) epitopes protected IL4(-/-) mice from EMG and caused a reduced production of anti-AChR antibody. CD4(+) cells from C57Bl/6 mice treated with unrelated peptides or sham-treated did not induce protection. CD4(+) cells from C57Bl/6 mice treated with just one AChR peptide protected IL4(-/-) mice from EMG without affecting antibody synthesis.

Prevention of passively transferred experimental autoimmune myasthenia gravis by Fab fragments of monoclonal antibodies directed against the main immunogenic region of the acetylcholine receptor.

The muscle acetylcholine receptor loss, responsible for the clinical symptoms of myasthenia gravis, is due mainly to mechanisms dependent on the bivalent character of the anti-receptor antibodies. In cell culture, univalent Fab fragments of monoclonal antibodies (mAbs) directed against the main immunogenic region (MIR) of the acetylcholine receptor are able to protect the receptor against the action of the intact antibodies. To investigate the potential therapeutic use of this approach, we examined the ability of the Fab fragment of anti-MIR mAb195 (Fab195) to protect the receptor in vivo against two anti-MIR mAbs. Because of the rapid clearance of Fab fragments from the circulation, Lewis rats were treated repeatedly with Fab195. The Fab fragment significantly protected muscle receptors against antibody-mediated loss and was very efficient in providing protection against clinical symptoms when its administration was commenced before, simultaneously with, or 2 h after, mAb injection. Twenty-four hours after mAb injection, the protected rats only showed mild myasthenic symptoms, whereas those which only received intact antibodies were moribund or dead. These results suggest that, once modified to ensure their low immunogenicity and a long half-life, anti-MIR Fab fragments might be useful in the specific immunotherapy of myasthenia gravis.

Vaccination with a MHC class II peptide attenuates cellular and humoral responses against tAChR and suppresses clinical EAMG.

An animal model of myasthenia gravis (MG), termed experimental autoimmune MG (EAMG), can be induced in C57BL/6 (B6, H-2b) mice by immunization with Torpedo californica acetylcholine receptor (tAChR). We have investigated the effect of vaccination with MHC class II peptide I-A beta(b)62-76 on clinical EAMG and on T cell and antibody (Ab) responses against tAChR. B6 mice were vaccinated with the peptide (25 microg/mouse) four times prior to two injections with tAChR. The incidence of clinical EAMG in vaccinated mice was 14% (3 out of 22 mice) compared to 48% (17 out of 35 mice) in control non-vaccinated or PBS-immunized mice. The T cells of the vaccinated group showed lower proliferative responses to tAChR and to T-cell epitope-containing tAChR alpha-chain peptides than the T cells of controls. In addition, the Ab responses in the vaccinated group was also lower against tAChR and some of the B-cell epitope-containing tAChR alpha-chain peptides.

Suppression of myasthenia gravis by antigen-specific mucosal tolerance and modulation of cytokines and costimulatory factors.

We have shown that mucosal administration of recombinant fragments corresponding to the human acetylcholine receptor (AChR) alpha subunit suppresses chronic ongoing experimental autoimmune myasthenia gravis (EAMG) in rats. Treated animals exhibit a Th1 to Th2/Th3 shift in their cytokine profile and downregulation of costimulatory factors. However, application of a xenogeneic recombinant fragment may have limitations when considered as a possible approach for the treatment of MG in humans. We therefore tested the potential of a syngeneic fragment and of long synthetic peptides to suppress EAMG. We found that a syngeneic fragment corresponding to the extracellular region of the rat AChR alpha subunit was as effective as the formerly described human xenogeneic fragment in suppressing ongoing EAMG. This is encouraging in view of the potential use of mucosally administered recombinant AChR fragments for the treatment of MG in humans. However, in severely affected individuals, this antigen-specific approach may need to be supported by direct modulation of cytokines and costimulatory factors known to be involved in the pathogenesis of EAMG. To test the potential of this approach, myasthenic rats were injected by antibodies either to the proinflammatory cytokine IL-18 or to the costimulatory factor CD40L. These treatments act via different mechanisms, but both lead to the alleviation of clinical symptoms even when given at the chronic phase of EAMG. We suggest that antagonists to key cytokines and/or costimulatory factors be used to augment antigen-specific treatments of myasthenia such as mucosal administration of AChR recombinant fragments.

The role of adjuvants in the efficacy of a peptide vaccine for myasthenia gravis.

Myasthenia gravis (MG) and its animal model, experimental autoimmune (EA) MG, are caused by interference with neuromuscular transmission by autoantibodies against the nicotinic acetylcholine receptor (AChR) on muscle. Previously, we have shown that two peptides, denoted RhCA 67-16 and RhCA 611-001, designed to be complementary in structure to the main immunogenic region and the dominant Lewis rat T cell epitope (alpha-chain residues 100-116) of the AChR, respectively, are effective vaccines that prevent EAMG in rats by inducing antiidiotypic/clonotypic antibodies (Ab) and lowering levels of AChR Ab. These studies employed keyhole limpet hemocyanin (KLH) as a carrier and complete Freunds adjuvant (CFA). In advance of a clinical trial the present study tested the efficacy of RhCA 611-001 when combined with different adjuvants that are approved for use in humans. Adjuvants chosen for comparison were incomplete Freunds adjuvant (IFA) and aluminum hydroxide (Alum). As a second goal we evaluated diphtheria toxin (DT) as an alternative carrier protein to KLH. Alum was found to be an effective adjuvant, particularly when used with the peptide conjugated to DT. This combination of carrier and adjuvant provided protection against EAMG comparable with that observed with CFA and KLH. Using enzyme-linked immunosorbent assays for Ab against RhCA 611-001, it was found that disease protection is qualitatively, but not quantitatively, related to the anti-peptide Ab response. Our results demonstrate a vaccine formulation that should be useful in the first soon-to-be-conducted clinical trials of peptide vaccines to specifically correct aberrant T and B cell responses in an autoimmune disease.

Prophylactic effect of probiotics on the development of experimental autoimmune myasthenia gravis.

Probiotics are live bacteria that confer health benefits to the host physiology. Although protective role of probiotics have been reported in diverse diseases, no information is available whether probiotics can modulate neuromuscular immune disorders. We have recently demonstrated that IRT5 probiotics, a mixture of 5 probiotics, could suppress diverse experimental disorders in mice model. In this study we further investigated whether IRT5 probiotics could modulate the progression of experimental autoimmune myasthenia gravis (EAMG). Myasthenia gravis (MG) is a T cell dependent antibody mediated autoimmune disorder in which acetylcholine receptor (AChR) at the neuromuscular junction is the major auto-antigen. Oral administration of IRT5 probiotics significantly reduced clinical symptoms of EAMG such as weight loss, body trembling and grip strength. Prophylactic effect of IRT5 probiotics on EMAG is mediated by down-regulation of effector function of AChR-reactive T cells and B cells. Administration of IRT5 probiotics decreased AChR-reactive lymphocyte proliferation, anti-AChR reactive IgG levels and inflammatory cytokine levels such as IFN-γ, TNF-α, IL-6 and IL-17. Down-regulation of inflammatory mediators in AChR-reactive lymphocytes by IRT5 probiotics is mediated by the generation of regulatory dendritic cells (rDCs) that express increased levels of IL-10, TGF-ß, arginase 1 and aldh1a2. Furthermore, DCs isolated from IRT5 probiotics-fed group effectively converted CD4(+) T cells into CD4(+)Foxp3(+) regulatory T cells compared with control DCs. Our data suggest that IRT5 probiotics could be applicable to modulate antibody mediated autoimmune diseases including myasthenia gravis.

Ex vivo generated regulatory T cells modulate experimental autoimmune myasthenia gravis.

Naturally occurring CD4(+)CD25(+) regulatory T (Treg) cells are key players in immune tolerance and have therefore been suggested as potential therapeutic tools for autoimmune diseases. In myasthenia gravis (MG), reduced numbers or functionally impaired Treg cells have been reported. We have observed that PBL from myasthenic rats contain decreased numbers of CD4(+)CD25(high)Foxp3(+) cells as compared with PBL from healthy controls, and we have tested whether Treg cells from healthy donors can suppress experimental autoimmune MG in rats. Because the number of naturally occurring Treg cells is low, we used an approach for a large-scale ex vivo generation of functional Treg cells from CD4(+) splenocytes of healthy donor rats. Treg cells were generated ex vivo from CD4(+) cells by stimulation with anti-CD3 and anti-CD28 Abs in the presence of TGF-beta and IL-2. The obtained cells expressed high levels of CD25, CTLA-4, and Foxp3, and they were capable of suppressing in vitro proliferation of T cells from myasthenic rats in response to acetylcholine receptor, the major autoantigen in myasthenia. Administration of ex vivo-generated Treg cells to myasthenic rats inhibited the progression of experimental autoimmune MG and led to down-regulation of humoral acetylcholine receptor-specific responses, and to decreased IL-18 and IL-10 expression. The number of CD4(+)CD25(+) cells in the spleen of treated rats remained unchanged, but the subpopulation of CD4(+)CD25(+) cells expressing Foxp3 was significantly elevated. Our findings imply that Treg cells play a critical role in the control of myasthenia and could thus be considered as potential agents for the treatment of MG patients.

Pixantrone (BBR2778) reduces the severity of experimental autoimmune myasthenia gravis in Lewis rats.

Pixantrone (BBR2778) (PIX) and mitoxantrone share the same mechanism of action because both drugs act as DNA intercalants and inhibitors of topoisomerase II. PIX is an interesting candidate immunosuppressant for the treatment of autoimmune diseases because of its reduced cardiotoxicity compared with mitoxantrone. The clinical response to conventional immunosuppressive treatments is poor in some patients affected by myasthenia gravis (MG), and new but well-tolerated drugs are needed for treatment-resistant MG. PIX was tested in vitro on rat T cell lines specific for the immunodominant peptide 97-116 derived from rat acetylcholine receptor (AChR), and showed strong antiproliferative activity in the nanomolar range. We demonstrate in this study that PIX administration reduced the severity of experimental autoimmune MG in Lewis rats. Biological and immunological analysis confirmed the effect of PIX, compared with vehicle-treated as well as mitoxantrone-treated experimental autoimmune MG rats. Anti-rat AChR Abs were significantly reduced in PIX-treated rats, and AChR content in muscles were found increased. Torpedo AChR-induced T cell proliferation tests were found reduced in both in vitro and ex vivo experiments. The effectiveness and the reduced cardiotoxicity make PIX a promising immunosuppressant agent suitable for clinical investigation in MG, although additional experiments are needed to confirm its safety profile in prolonged treatments.