Beneficial effect of tocilizumab in myasthenia gravis refractory to rituximab.

Muscle fatigue associated with myasthenia gravis is caused by autoantibodies interfering with neuromuscular transmission. Immunomodulating treatment is widely used in moderate to severe myasthenia, although the use of newer biological drugs except rituximab is rare. We describe the effect of tocilizumab, a blocker of interleukin-6 signalling, in two female myasthenia patients with high titres of serum acetylcholine receptor antibodies and insufficient response to rituximab. The first patient had been treated with high dose immunoglobulins regularly for several years and the second patient had been treated both with different oral immune suppressants and immunoglobulins before testing a low dose of rituximab without significant clinical effect. Subsequent treatment with tocilizumab resulted in clinical improvement within a few months. The first patient was switched back to rituximab, which resulted in worsening until tocilizumab was restarted. Tocilizumab can be a therapeutic option in cases not responding to rituximab.

Successful autologous haematopoietic stem cell transplantation for refractory myasthenia gravis - a case report.

Myasthenia gravis (MG) is an autoimmune disease, with immune reactivity against the post-synaptic endplate of the neuromuscular junction. Apart from symptomatic treatment with choline esterase blockers, many patients also require immunomodulatory treatment. Despite existing treatment options, some patients are treatment refractory. We describe a patient with severe MG refractory to corticosteroids, four oral immunosuppressants, cyclophosphamide, rituximab and bortezomib who was treated with autologous haematopoietic stem cell transplantation. Two years after this, the patient has significantly improved in objective tests and in quality of life and leads an active life. Diplopia is her only remaining symptom and she is completely free of medication for MG. We believe that autologous haematopoietic stem cell transplantation can be an effective therapeutic option for carefully selected cases of severe, treatment refractory MG.

Concomitant autoimmunity in myasthenia gravis--lack of association with IgA deficiency.

A marked increase in concomitant autoimmune diseases has previously been noted in patients with myasthenia gravis (MG). We show that these diseases occur both before and after the onset of MG and that the process is not influenced by thymectomy. IgA deficiency (IgAD), which is strongly associated with the same HLA haplotype as early onset MG, has recently been suggested to be an autoimmune disease. However, there was no increase in the prevalence of IgAD in a large cohort of Swedish MG patients.

Utilizing twins concordance rates to infer the predisposition to myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disorder in which patients experience muscular fatigability due to the presence of anti-acetylcholine receptor (AChR) antibodies which inhibit signal transduction across the neuro-muscular junction. Like all complex disorders, disease is caused by an interaction between genetic and environmental factors. Although several genes have been identified which appear to be associated with MG, both classic twin studies and current multi-gene models are insufficient to explain either disease pathogenesis or inheritance. We examined the literature on MG to determine both mono- and dizygotic twin concordance rates, and used this data to (1) estimate the proportion of the population with underlying genetic predisposition to MG and the frequency of the environmental component and (2) derive the number of inherited genetic regions that are required to confer predisposition to MG. Using a MZ twin concordance rate of 35.5%, and a dizygotic rate of approximately 4-5% (based on family data), the probability of encountering environmental components necessary to develop MG in an individual with genetic predisposition is approximately 52.4%, making the frequency of predisposition (1:5240) roughly twice the rate of incidence. Furthermore, the number of genetic regions co-inherited between affected individuals is between two and four, which may be large haplotypes with interacting activity. Determining these haplotypes, by fully sequencing associated regions in cases and controls to identify mutations present, may therefore be a practically step toward the understanding of complex disease.

The CD45 77C/G allele is not associated with myasthenia gravis - a reassessment of the potential role of CD45 in autoimmunity.

BACKGROUND: The G allele of the CD45 77C/G SNP (rs17612648), which has previously been suggested to be associated with autoimmune disorders, was genotyped in 446 Swedish myasthenia gravis (MG) patients and 2303 matched controls. RESULTS: There was no association between the polymorphism and patient group as a whole (p = 0.199), nor with clinical subgroups. Our results add to a growing number of studies unable to find association between the 77C/G polymorphism and autoimmune disorders. One control sample, from an adult blood donor, was homozygous for the G allele, yet negative for a panel of auto-antibodies, representing the first homozygous individual studied in this respect. CONCLUSIONS: The 77C/G mutation does not predispose to MG, and its role in autoimmunity may have to be re-evaluated.

Lack of association of the CIITA -168A→G promoter SNP with myasthenia gravis and its role in autoimmunity.

BACKGROUND: The major histocompatibility complex class II transactivator (CIITA) regulates MHC class II gene expression. A promoter SNP -168A→G (rs3087456) has previously been shown to be associated with susceptibility to several immune mediated disorders, including rheumatoid arthritis (RA), multiple sclerosis (MS) and myocardial infarction (MI). Myasthenia gravis (MG) is an autoimmune disorder which has previously been shown to be associated with polymorphisms of several autoimmune predisposing genes, including IL-1, PTPN22, TNF-α and the MHC. In order to determine if allelic variants of rs3087456 increase predisposition to MG, we analyzed this SNP in our Swedish cohort of 446 MG patients and 1866 controls. RESULTS: No significant association of the SNP with MG was detected, neither in the patient group as a whole, nor in any clinical subgroup. The vast majority of previous replication studies have also not found an association of the SNP with autoimmune disorders. CONCLUSIONS: We thus conclude that previous findings with regard to the role of the CIITA -168A→G SNP in autoimmunity may have to be reconsidered.

Identification of CTLA-4 isoforms produced by alternative splicing and their association with myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness induced by autoantibodies against the acetylcholine receptor. CTLA-4 (CD152) plays an inhibitory role in the immune response and has been suggested to be involved in the pathophysiology of MG. In this study, we focused on alternative CTLA-4 mRNA expression in PBMCs from MG patients. We defined two new isoforms of CTLA-4 mRNA that arise due to alternative splicing. By semi-quantitative RT-PCR analysis, we observed a lower expression of sCTLA-4 mRNA relative to the membrane form in MG patients. In addition, the MG patients had lower levels of sCTLA-4 mRNA in PBMCs compared to healthy controls, as assessed by real-time PCR. One of the spliced isoforms (LCTLA-4) was more prevalent in MG patients compared to healthy controls. The alternative splicing was not associated with sex, thymectomy, serum levels of anti-AChR, immunosuppressive treatment or the four CTLA-4 gene polymorphisms analyzed. This study reveals an abnormal spectrum of mRNA expression of CTLA-4 in MG patients, which marks the importance of studying gene expression of alternative splicing.

PTPN22 R620W promotes production of anti-AChR autoantibodies and IL-2 in myasthenia gravis.

In order to investigate the potential involvement of PTPN22 R620W in the pathogenesis of myasthenia gravis (MG), we performed a case-control study including 409 Swedish MG patients and 1557 normal controls. The W620 variant was significantly overrepresented in patients (odds ratio, 1.52; 95% confidence interval, 1.21-1.90; p=0.00027). Incubation of patient (n=100) derived PBMC cells with the autoantigen, the acetylcholine receptor, resulted in a significantly higher number of cells producing anti-AChR antibodies and IL-2 in W620 carriers, suggesting that PTPN22 W620 may be a loss-of-function variant in MG.

Programmed Death-1: from gene to protein in autoimmune human myasthenia gravis.

The key role of an inhibitory receptor, Programmed Death-1, has been evaluated in 273 patients with autoimmune myasthenia gravis. At the genetic level, SNP's genotyping showed no significant association to the disease. Gene expressions in patients were not different from that in controls. Interestingly, at the cell-surface protein level, there were significant elevated levels of PD-1 on T cells and its ligand PD-L1 on monocytes in the patients compared to controls. However, we could not demonstrate any secreted soluble forms of PD-1 among the patients and controls. Thus, our study shows PD-1 might have a natural regulatory property behind MG.

Soluble costimulatory factors sCD28, sCD80, sCD86 and sCD152 in relation to other markers of immune activation in patients with myasthenia gravis.

The costimulatory factors CD28, CD80, CD86 and CD152 needed to start and turn off an immune response are present as membrane receptors and soluble proteins. There was no difference in the serum levels of soluble costimulatory molecules in 153 healthy controls and 118 patients with myasthenia gravis. However, we could confirm that the soluble forms of ICAM-1 and CD25 were increased in patients. The concentrations of the soluble costimulatory proteins seemed to be rather constant in individual patients, despite changes in clinical presentation. Thus, the soluble costimulatory factors do not seem to constitute reliable markers for disease activity in myasthenia gravis.

The thymus is a source of B-cell-survival factors-APRIL and BAFF-in myasthenia gravis.

The accumulation of B cells in the thymus is a common feature of myasthenia gravis (MG). To understand whether factors enhancing B-cell survival are increased in MG, we studied the expression of APRIL, BAFF and three of their receptors in the thymus. In hyperplastic thymi, macrophages expressed APRIL and BAFF, and germinal-center B cells, BAFF-R. CD138-positive plasma cells were abundant in MG thymi. By contrast, BCMA-positive plasma cells were scarce. The expression of APRIL and BAFF in MG thymi may reflect the establishment of an environment favorable to B-cell survival.

Dendritic cells activate autologous T cells and induce IL-4 and IL-10 production in myasthenia gravis.

Dendritic cells (DC), as initiators and orchestrators of immune responses, control both naive and primed T cell responses. Depending on their maturation stage, DC promote immunity or tolerance. Here we investigated (1) the phenotype and cytokine secretion patterns of IL-10-modulated immature DC (IL-10-DC) and lipopolysaccharide (LPS)-driven mature DC (LPS-DC) in comparison with unmodulated immature DC (imDC) and (2) the effects of IL-10-DC, and of LPS-DC, vs. imDC on autologous T cell responses in patients with myasthenia gravis (MG) compared with healthy controls (HC). All three types of DC derived from MG significantly increased the levels of CD4+CD25+ T cells and of their subfraction expressing CD69, when compared to DC derived from HC. IL-10-DC induced production of IL-10 and IL-4 by T cells from MG patients, but only IL-10 production from HC. LPS-DC activated autologous T cells as reflected by augmented CD25, CD69 and CTLA-4 expression on CD4+ T cells, without differences between MG and HC. This was associated with increased production of both Th1 (IFN-gamma) and Th2 (IL-10 and IL-4) cytokines by T cells. These results indicate that DC-induced activation of autologous T cells is more pronounced in MG than in HC. In addition, DC-induced T cell responses in MG vs. HC are more Th2-prone.

Genotypes of CCR2 and CCR5 chemokine receptors in human myasthenia gravis.

The aim of this study was to examine the association of human autoimmune myasthenia gravis (MG) with two DNA polymorphisms of the chemokine receptors CCR5-Delta 32 and CCR2-64I. CCR2 and CCR5 interact primarily with the human CC family ligands CCL2 (formerly called monocyte chemoattractant protein; MCP-1), CCL3 and CCL4 (macrophage inflammatory protein-1 alpha and -1 beta; MIP-1 alpha/beta), and their main function is to recruit leukocytes from circulation into the tissues, thus playing an important role in human inflammatory disorders. A PCR-based genotyping method was used to determine the genetic variation at the CCR5 gene and an automated real-time Pyrosequencing technology was employed for the analysis of G right curved arrow A point mutation at the CCR2 gene. Results obtained from 158 patients and 272 healthy controls demonstrate no evidence of association between genetic variants of CCR2 and CCR5 with MG and its clinical manifestations. CCR2-64I and CCR5-Delta 32 genotypes are thus unlikely to be involved in protection or predisposition to MG.

Abnormal expression of CTLA-4 by T cells from patients with myasthenia gravis: effect of an AT-rich gene sequence.

Cytolytic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in the down-regulation of antigen-activated immune responses. The aberrant CTLA-4 expression is characterized by low surface and intracellular levels of CTLA-4 protein, impaired up-regulation of CTLA-4 in T cells in response to ConA stimulation and high levels of soluble CTLA-4 (sCTLA-4) in serum. The serum levels of sCTLA-4 are positively correlated with the serum concentration of antibodies against the acetylcholine receptor. The (AT)(n) polymorphism in the 3'-untranslated region contributes to decreased mRNA stability and, hence, to reduced expression of CTLA-4.