Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice.

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness associated with acetylcholine receptor (AChR), muscle-specific receptor kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4)-antibodies. MuSK-antibodies are predominantly of the non-complement fixing IgG4 isotype. The MuSK associated experimental autoimmune myasthenia gravis (EAMG) model was established in mice to investigate immunoglobulin (Ig) and cytokine responses related with MuSK immunity. C57BL/6 (B6) mice immunized with 30µg of recombinant human MuSK in incomplete or complete Freund's adjuvant (CFA) showed significant EAMG susceptibility (>80% incidence). Although mice immunized with 10µg of MuSK had lower EAMG incidence (14.3%), serum MuSK-antibody levels were comparable to mice immunized with 30µg MuSK. While MuSK immunization stimulated production of all antibody isotypes, non-complement fixing IgG1 was the dominant anti-MuSK Ig isotype in both sera and neuromuscular junctions. Moreover, MuSK immunized IgG1 knockout mice showed very low serum MuSK-antibody levels. Sera and MuSK-stimulated lymph node cell supernatants of MuSK immunized mice showed significantly higher levels of IL-4 and IL-10 (but not IFN-γ and IL-12), than those of CFA immunized mice. Our results suggest that through activation of Th2-type cells, anti-MuSK immunity promotes production of IL-4, which in turn activates anti-MuSK IgG1, the mouse analog of human IgG4. These findings might provide clues for the pathogenesis of other IgG4-related diseases as well as development of disease specific treatment methods (e.g. specific IgG4 inhibitors) for MuSK-related MG.

Expression of γ-tubulin in non-small cell lung cancer and effect on patient survival.

INTRODUCTION: It has been reported that overexpression and altered compartmentalization of γ-tubulin may contribute to tumorigenesis and tumor aggressiveness in a variety of human malignancies. We have shown that γ-tubulin expression and cellular distribution pattern is also altered in non-small cell lung cancer (NSCLC) (Histol. Histopathol. 2012; 27: 1183-1194). In the present study we examined the relationship between γ-tubulin expression and patient overall survival (OS). MATERIAL AND METHODS: Immunohistochemistry was performed, with well-characterized anti-γ-tubulin antibodies, on 109 formalin-fixed, paraffin-embedded NSCLC specimens (p-TNM stage I-III). γ-Tubulin labeling indexes (LIs) were determined, and the association of γ-tubulin expression with clinicopathological parameters was evaluated. To analyze OS rates according to γ-tubulin LIs, patients were categorized into three groups: those with low (0-30%), intermediate (31-69%) or high (70-100%) γ-tubulin LI. Association of clinicopathological parameters and γ-tubulin with survival were examined using univariate and multivariate Cox regression analysis. RESULTS: No statistically significant association was seen between γ-tubulin overexpression and histological type, tumor differentiation, p-TNM stage and adenocarcinoma subtyping. Longer survival was observed in the high γ-tubulin LI group of patients with p-TNM stages II+III when compared to intermediate or low γ-tubulin LI groups, but the difference was not statistically significant (p=0.066). On the other hand, when combined low and intermediate γ-tubulin LI groups (p-TNM stages II+III) where compared to high γ-tubulin LI group, statistically significant longer survival was observed in high γ-tubulin group (p=0.021). CONCLUSION: Our findings suggest that level of γ-tubulin expression may have an impact on patient survival at more advanced NSCLC stages.

Antigen-specific apheresis of pathogenic autoantibodies from myasthenia gravis sera.

Myasthenia gravis (MG) is usually caused by autoantibodies against muscle nicotinic acetylcholine receptor (AChR), which is composed of five subunits (alpha(2)betagammadelta or alpha(2)betaepsilondelta). Current treatments, including plasmapheresis, are nonspecific, causing several side effects. We aim to develop an antigen-specific alternative to plasmapheresis, since the latter removes indispensable plasma components in addition to anti-AChR antibodies. We are developing a method for the selective depletion of the anti-AChR autoantibodies from patients' plasma through the construction of "immunoadsorbent" columns carrying AChR domains. We have expressed the extracellular domains (ECDs, amino acids approximately 1-210/220) of all human muscle AChR subunits in Pichia pastoris and, in preliminary experiments, in E. coli. The ECDs were immobilized (individually or mixed) on Sepharose beads, producing Sepharose-ECD columns, which were tested for their immunoadsorbing capacity on MG sera and shown to specifically eliminate major autoantibody fractions from several MG sera. The immobilized ECDs remained stable and did not dissociate from their matrix after incubation with serum, whereas the procedure was neither toxic nor immunogenic in two experimental rabbits. Testing the intact or antibody-depleted MG sera and the affinity purified autoantibodies showed that both the intact sera and the purified autoantibodies, but not the antibody-depleted sera, could induce AChR loss in cell cultures and experimental MG in rats. This preliminary study suggests that the myasthenic potency of MG sera is entirely due to their anti-AChR antibodies and therefore their depletion should be of therapeutic value. We conclude that ECD-mediated immunoadsorption can be used as an efficient, antigen-specific therapy for MG.

MuSK induced experimental autoimmune myasthenia gravis does not require IgG1 antibody to MuSK.

Sera of myasthenia gravis (MG) patients with muscle-specific receptor kinase-antibody (MuSK-Ab) predominantly display the non-complement fixing IgG4 isotype. Similarly, mouse IgG1, which is the analog of human IgG4, is the predominant isotype in mice with experimental autoimmune myasthenia gravis (EAMG) induced by MuSK immunization. The present study was performed to determine whether IgG1 anti-MuSK antibody is required for immunized mice to develop EAMG. Results demonstrated a significant correlation between clinical severity of EAMG and levels of MuSK-binding IgG1+, IgG2+ and IgG3+ peripheral blood B cells in MuSK-immunized wild-type (WT) mice. Moreover, MuSK-immunized IgG1 knockout (KO) and WT mice showed similar EAMG severity, serum MuSK-Ab levels, muscle acetylcholine receptor concentrations, neuromuscular junction immunoglobulin and complement deposit ratios. IgG1 and IgG3 were the predominant anti-MuSK isotypes in WT and IgG1 KO mice, respectively. These observations demonstrate that non-IgG1 isotypes can mediate MuSK-EAMG pathogenesis.

Specific immunoadsorption of the autoantibodies from myasthenic patients using the extracellular domain of the human muscle acetylcholine receptor alpha-subunit. Development of an antigen-specific therapeutic strategy.

Antibodies against the acetylcholine receptor (AChR) are the main pathogenic factor in myasthenia gravis (MG). Clinical improvement correlates well with a reduction in levels of circulating anti-AChR antibodies, and plasmapheresis is an efficient short-term MG treatment. The Sepharose-immobilized N-terminal extracellular domain of human muscle AChR alpha-subunit was used to immunoadsorb anti-AChR autoantibodies from 50 MG patients sera. The immunoadsorbents removed 60-94% of the anti-AChR antibodies in 10 sera and a mean of 35% from all samples combined. Immunoadsorption was fast, efficient, and the columns could be used repeatedly without any release or proteolysis of the polypeptide, suggesting the feasibility of antigen-specific MG immunoadsorption therapy.

Antigen-specific apheresis of autoantibodies in myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disorder affecting the neuromuscular junction, usually caused by autoantibodies against the acetylcholine receptor (AChR) or the muscle-specific kinase (MuSK). Our aim is the development of a therapy based on the selective extracorporeal elimination of anti-AChR or anti-MuSK antibodies. To this end, the extracellular domains of the AChR subunits and MuSK have been expressed in yeast to be used as adsorbents, after optimization, and to obtain large quantities of proteins with near-native structure. We have characterized these proteins with respect to their use as specific immunoadsorbents for MG autoantibodies, and have begun large-scale experiments in order to verify the feasibility of application of the method for therapy. Furthermore, we have initiated animal studies to test possible toxicity and safety issues of the adsorbents or the procedure itself. The successful completion of the scale-up and safety tests will allow the initiation of clinical trials.