Common denominators in the immunobiology of IgG4 autoimmune diseases: What do glomerulonephritis, pemphigus vulgaris, myasthenia gravis, thrombotic thrombocytopenic purpura and autoimmune encephalitis have in common?

IgG4 autoimmune diseases (IgG4-AID) are an emerging group of autoimmune diseases that are caused by pathogenic autoantibodies of the IgG4 subclass. It has only recently been appreciated, that members of this group share relevant immunobiological and therapeutic aspects even though different antigens, tissues and organs are affected: glomerulonephritis (kidney), pemphigus vulgaris (skin), thrombotic thrombocytopenic purpura (hematologic system) muscle-specific kinase (MuSK) in myasthenia gravis (peripheral nervous system) and autoimmune encephalitis (central nervous system) to give some examples. In all these diseases, patients’ IgG4 subclass autoantibodies block protein-protein interactions instead of causing complement mediated tissue injury, patients respond favorably to rituximab and share a genetic predisposition: at least five HLA class II genes have been reported in individual studies to be associated with several different IgG4-AID. This suggests a role for the HLA class II region and specifically the DRβ1 chain for aberrant priming of autoreactive T-cells toward a chronic immune response skewed toward the production of IgG4 subclass autoantibodies. The aim of this review is to provide an update on findings arguing for a common pathogenic mechanism in IgG4-AID in general and to provide hypotheses about the role of distinct HLA haplotypes, T-cells and cytokines in IgG4-AID

Guidelines for pre-clinical assessment of the acetylcholine receptor-specific passive transfer myasthenia gravis model - recommendations for methods and experimental designs.

Antibodies against the muscle acetylcholine receptor (AChR) are the most common cause of myasthenia gravis (MG). Passive transfer of AChR antibodies from MG patients into animals reproduces key features of human disease, including antigenic modulation of the AChR, complement-mediated damage of the neuromuscular junction, and muscle weakness. Similarly, AChR antibodies generated by active immunization in experimental autoimmune MG models can subsequently be passively transferred to other animals and induce weakness. The passive transfer model is useful to test therapeutic strategies aimed at the effector mechanism of the autoantibodies. Here we summarize published and unpublished experience using the AChR passive transfer MG model in mice, rats and rhesus monkeys, and give recommendations for the design of preclinical studies in order to facilitate translation of positive and negative results to improve MG therapies

A Striational Muscle Antigen and Myasthenia Gravis-Associated Thymomas Share an Acetylcholine-Receptor Epitope

The coincidence of autoantibodies against the acetylcholine receptor (AChR) and muscle striational antigens (SA) is a characteristic finding in thymoma-associated myasthenia gravis (MG), but their origins are still unresolved. Some common muscle antigens that were shown to be targets of anti-SA autoantibodies in thymoma-associated MG have also been detected in normal or neoplastic thymic epithelial cells, suggesting that the release of (eventually altered) antigens from the thymic tumors could elicit SA autoimmunity. In contrast to this model, we report here that titin, which is a recently reported target of SA autoimmunity, is not expressed in thymomas. In addition, we show that skeletal muscle type-II fibers exhibit a striational immunoreactivity with monoclonal antibody mAb155, which was previously identified to label a very immunogenic cytoplasmic epitope of the AChR and neoplastic epithelial cells of MGassociated thymomas. We conclude from these findings that titin autoimmunity in thymoma-associated MG is either due to a molecular mimicry mechanism involving tumor antigens (other than titin) or is a secondary phenomenon following release of titin from muscle. Based on the common immunoreactivity of the AChR, a striational antigen and thymoma, we suggest as the pathogenetic mechanism of thymoma-associated MGa "circulus vitiosus" in which SA autoimmunity could help maintain the AChR autoimmunity that is primarily elicited by the thymomas

IL-21 and IL-21 Receptor Expression in Lymphocytes and Neurons in Multiple Sclerosis Brain

IL-17–producing CD4+ T cells (Th-17) contribute to the pathogenesis of experimental autoimmune encephalomyelitis and are associated with active disease in multiple sclerosis (MS). In addition to IL-17, Th-17 cells can also express IL-21, IL-22, and IL-6 under Th-17–polarizing conditions (IL-6 and transforming growth factor-β). In this study we investigated IL-21 and IL-21 receptor (IL-21R) expression in MS lesions by in situ hybridization and immunohistochemistry. We detected strongly IL-21+ infiltrating cells predominantly in acute but also in chronic active white matter MS lesions in which IL-21 expression was restricted to CD4+ cells. In contrast, IL-21R was much more broadly distributed on CD4+, CD19+, and CD8+ lymphocytes but not major histocompatibility complex class-II+ macrophages/microglia. Interestingly, in cortical areas we detected both IL-21 and IL-21R expression by neurons. These findings suggest role(s) for IL-21 in both the acute and chronic stages of MS via direct effects on T and B lymphocytes and, demonstrated for the first time, also on neurons

Crystal Structure of the Monomeric Extracellular Domain of α9 Nicotinic Receptor Subunit in Complex With α-Conotoxin RgIA: Molecular Dynamics Insights Into RgIA Binding to α9α10 Nicotinic Receptors

The α9 subunit of nicotinic acetylcholine receptors (nAChRs) exists mainly in heteropentameric assemblies with α10. Accumulating data indicate the presence of three different binding sites in α9α10 nAChRs: the α9(+)/α9(−), the α9(+)/α10(−), and the α10(+)/α9(−). The major role of the principal (+) side of the extracellular domain (ECD) of α9 subunit in binding of the antagonists methyllylcaconitine and α-bungarotoxin was shown previously by the crystal structures of the monomeric α9-ECD with these molecules. Here we present the 2.26-Å resolution crystal structure of α9-ECD in complex with α-conotoxin (α-Ctx) RgIA, a potential drug for chronic pain, the first structure reported for a complex between an nAChR domain and an α-Ctx. Superposition of this structure with those of other α-Ctxs bound to the homologous pentameric acetylcholine binding proteins revealed significant similarities in the orientation of bound conotoxins, despite the monomeric state of the α9-ECD. In addition, ligand-binding studies calculated a binding affinity of RgIA to the α9-ECD at the low micromolar range. Given the high identity between α9 and α10 ECDs, particularly at their (+) sides, the presented structure was used as template for molecular dynamics simulations of the ECDs of the human α9α10 nAChR in pentameric assemblies. Our results support a favorable binding of RgIA at α9(+)/α9(−) or α10(+)/α9(−) rather than the α9(+)/α10(−) interface, in accordance with previous mutational and functional data

Transmembrane orientation of an early biosynthetic form of acetylcholine receptor delta subunit determined by proteolytic dissection in conjunction with monoclonal antibodies

The transmembrane topology of acetylcholine receptor (AChR) delta subunit, synthesized in vitro and co-translationally integrated into dog pancreas rough microsomal membranes, was studied using limited proteolysis and domain-specific immunoprecipitation. Forty-four kilodaltons (kd) of the 65-kd delta subunit comprise a single fragment that is inaccessible to exhaustive proteolytic digestion from the cytoplasmic surface of the membrane by trypsin, chymotrypsin, thermolysin, and pronase. Previously, we have shown that this 44-kd “protected” fragment contains the amino terminus of the intact molecule and all of the core oligosaccharides (Anderson, D.J., P. Walter, and G. Blobel (1982) J. Cell Biol. 93: 501–506). Here we demonstrate that this domain can be further dissected into a 26-kd fragment, together with low molecular weight material, when the membranes are rendered permeable to trypsin by low concentrations of deoxycholate (Kreibich, G., P. Debey, and D. D. Sabatini (1973) J. Cell Biol. 58: 436–462). This 26-kd fragment contains all of the core oligosaccharides present on the intact subunit and therefore constitutes at least part, if not all, of the extracellular domain. The remaining low molecular weight material may derive from the membrane-embedded domain; our data imply that as much as 18 kd may be internal to the lipid bilayer. On the other hand, part of the cytoplasmic pole of AChR-delta can be recovered as a discrete, 12-kd fragment upon mild trypsinization of intact vesicles. We have used this 12-kd fragment to identify anti-AChR-delta monoclonal antibodies (mAbs) that react with the cytoplasmic domain of this subunit. Partial proteolytic fragmentation of the AChR in vitro translation products, in topologically well defined rough microsomes, may be used as a general assay to characterize the domain specificity of anti-AChR mAbs. For example, in the case of AChR-beta, we were able to identify two mAbs that recognize extracellular and cytoplasmic fragments, respectively

A Human Recombinant Autoantibody-Based Immunotoxin Specific for the Fetal Acetylcholine Receptor Inhibits Rhabdomyosarcoma Growth In Vitro and in a Murine Transplantation Model

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) γ-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms

Genetics of Myasthenia Gravis: A Case-Control Association Study in the Hellenic Population

Myasthenia gravis (MG) is an heterogeneous autoimmune disease characterized by the production of autoantibodies against proteins of the postsynaptic membrane, in the neuromuscular junction. The contribution of genetic factors to MG susceptibility has been evaluated through family and twin studies however, the precise genetic background of the disease remains elusive. We conducted a case-control association study in 101 unrelated MG patients of Hellenic origin and 101 healthy volunteers in order to assess the involvement of common genetic variants in susceptibility to MG. We focused on three candidate genes which have been clearly associated with several autoimmune diseases, aiming to investigate their potential implication in MG pathogenesis. These are interferon regulatory factor 5 (IRF-5), TNFα-induced protein 3 (TNFAIP3), also known as A20, and interleukin-10 (IL-10), key molecules in the regulation of immune function. A statistical trend of association (P=0.068) between IL-10 promoter single nucleotide polymorphisms (SNPs) and the subgroups of early and late-onset MG patients was revealed. No statistically significant differences were observed in the rest of the variants examined. As far as we are aware, this is the first worldwide attempt to address the possible association between IRF-5 and TNFAIP3 common genetic variants and the genetic basis of MG

A comprehensive analysis of the epidemiology and clinical characteristics of anti-LRP4 in myasthenia gravis

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