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The presence of autoantibodies directed against the muscle nicotinic acetylcholine receptor (AChR) is the most common cause of myasthenia gravis (MG). These antibodies damage the postsynaptic membrane of the neuromuscular junction and cause muscle weakness by depleting AChRs and thus impairing synaptic transmission. As one of the best-characterized antibody-mediated autoimmune diseases, AChR-MG has often served as a reference model for other autoimmune disorders. Classical pharmacological treatments, including broad-spectrum immunosuppressive drugs, are effective in many patients. However, complete remission cannot be achieved in all patients, and 10% of patients do not respond to currently used therapies. This may be attributed to production of autoantibodies by long-lived plasma cells which are resistant to conventional immunosuppressive drugs. Hence, novel therapies specifically targeting plasma cells might be a suitable therapeutic approach for selected patients. Additionally, in order to reduce side effects of broad-spectrum immunosuppression, targeted immunotherapies and symptomatic treatments will be required. This review presents established therapies as well as novel therapeutic approaches for MG and related conditions, with a focus on AChR-MG., (Copyright © 2022. Published by Elsevier B.V.)

Background: The etiology of thymic epithelial tumors is unknown. Murine polyomavirus strain PTA has been shown to induce thymomas in mice. Recently, using diverse molecular techniques, we reported the presence of human polyomavirus 7 (HPyV7) in thymic epithelial tumors. In the present study, we investigated the prevalence of Merkel cell polyomavirus (MCPyV) in thymic epithelial tumors., Methods: Thirty-six thymomas were screened for MCPyV by PCR and subsequently tested by DNA and RNA in situ hybridization and immunohistochemistry. Twenty-six thymomas were diagnosed with myasthenia gravis (MG)., Results: MCPyV DNA was detected by PCR in 7 (19.4%) of the 36 thymic epithelial tumors and in six of these, the presence of MCPyV was confirmed by fluorescence situ hybridization. Of these, 3 (28.6%) revealed weak MCPyV LT-antigen protein expression. In addition, one of the MCPyV positive thymomas tested positive for MCPyV LT RNA with RNAscope. Of interest, two out of the three thymomas that previously tested positive for MCPyV by immunohistochemistry also tested positive for HPyV7. One of the 11 MG-negative and 2 of the 25 MG-positive were positive for MCPyV., Conclusions: MCPyV DNA and MCPyV protein expression can be detected in human epithelial thymoma; however, to a far lesser extent than HPyV7. Our data strongly indicate that because of its infrequent detection and weak expression, MCPyV is unlikely to play an important role in the etiopathogenesis of human thymomas., (© 2019 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction. Most patients have pathogenic autoantibodies against the acetylcholine receptor (AChR). In the last years a novel subpopulation of MG patients has been described that harbors antibodies against low-density lipoprotein receptor-related protein 4 (Lrp4), another postsynaptic neuromuscular antigen. In early-onset AChR MG (EOMG), the thymus plays an important role in immunopathogenesis, and early thymectomy is beneficial. It is still unknown if the thymus plays any role in Lrp4-MG. In this pilot study, we compared thymus samples from four patients with Lrp4-MG (one pre-treated with immunosuppressive drugs), four non-MG controls and five EOMG patients (not pretreated with immunosuppressive drugs). Immunohistochemistry of the Lrp4-MG thymi revealed normal architecture, with normal numbers and distribution of B-cells, lymphoid follicles and Hassall's corpuscles. Primary CD23 + lymphoid follicles were similarly infrequent in Lrp4-MG and control thymic sections. In none of the control or Lrp4-MG thymi did we find secondary follicles with CD10 + germinal centers. These were evident in 2 of the 5 EOMG thymi, where primary lymphoid follicles were also more frequent on average, thus showing considerable heterogeneity between patients. Even if characteristic pathological thymic changes were not observed in the Lrp4 subgroup, we cannot exclude a role for the thymus in Lrp4-MG pathogenesis, since one Lrp4-MG patient went into clinical remission after thymectomy alone (at one year follow-up) and one more improved after thymectomy in combination with immunosuppressive therapy., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The α7 acetylcholine receptor (AChR) has been linked with the onset of psychotic symptoms and we hypothesized therefore that it might also be an autoimmune target. Here, we describe a new radioimmunoassay (RIA) using iodine 125-labelled α-bungarotoxin and membrane extract from transfected HEK293 cells expressing human α7 AChR. This RIA was used to analyze sera pertaining to a cohort of 711 subjects, comprising 368 patients diagnosed with schizophrenia spectrum disorders, 140 with bipolar disorder, 58 individuals diagnosed of other mental disorders, and 118 healthy comparison subjects. We identified one patient whose serum tested positive although with very low levels (0.2 nM) for α7 AChR-specific antibodies by RIA. Three out of 711 sera contained antibodies against iodine 125-labelled α-bungarotoxin, because they precipitated with it in the absence of α7 AChR. This first evidence suggests that autoantibodies against α7 AChR are absent or very rare in these clinical groups., Competing Interests: The authors have declared that no competing interests exist.

We report here the sequence and functional characterization of a recombinantly expressed autoantibody (mAb 131) previously isolated from a myasthenia gravis patient by immortalization of thymic B cells using Epstein-Barr virus and TLR9 activation. The antibody is characterized by a high degree of somatic mutations as well as a 6 amino acid insertion within the VHCDR2. The recombinant mAb 131 is specific for the γ-subunit of the fetal AChR to which it bound with sub-nanomolar apparent affinity, and detected the presence of fetal AChR on a number of rhabdomyosarcoma cell lines. Mab 131 blocked one of the two α-bungarotoxin binding sites on the fetal AChR, and partially blocked the binding of an antibody (mAb 637) to the α-subunit of the AChR, suggesting that both antibodies bind at or near one ACh binding site at the α/γ subunit interface. However, mAb 131 did not reduce fetal AChR ion channel currents in electrophysiological experiments. These results indicate that mAb 131, although generated from an MG patient, is unlikely to be pathogenic and may make it a potentially useful reagent for studies of myasthenia gravis, rhabdomyosarcoma and arthrogryposis multiplex congenita which can be caused by fetal-specific AChR-blocking autoantibodies.

Autoantibodies against ion channels are the cause of numerous neurologic autoimmune disorders. Frequently, such pathogenic autoantibodies have a restricted epitope-specificity. In such cases, competing antibody formats devoid of pathogenic effector functions (blocker antibodies) have the potential to treat disease by displacing autoantibodies from their target. Here, we have used a model of the neuromuscular autoimmune disease myasthenia gravis in rhesus monkeys (Macaca mulatta) to test the therapeutic potential of a new blocker antibody: MG was induced by passive transfer of pathogenic acetylcholine receptor-specific monoclonal antibody IgG1-637. The effect of the blocker antibody (IgG4Δhinge-637, the hinge-deleted IgG4 version of IgG1-637) was assessed using decrement measurements and single-fiber electromyography. Three daily doses of 1.7 mg/kg IgG1-637 (cumulative dose 5 mg/kg) induced impairment of neuromuscular transmission, as demonstrated by significantly increased jitter, synaptic transmission failures (blockings) and a decrease in the amplitude of the compound muscle action potentials during repeated stimulations (decrement), without showing overt symptoms of muscle weakness. Treatment with three daily doses of 10 mg/kg IgG4Δhinge-637 significantly reduced the IgG1-637-induced increase in jitter, blockings and decrement. Together, these results represent proof-of principle data for therapy of acetylcholine receptor-myasthenia gravis with a monovalent antibody format that blocks binding of pathogenic autoantibodies.

Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies are pathogenic., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies that target proteins at the neuromuscular junction, primarily the acetylcholine receptor (AChR) and the muscle-specific kinase. Because downstream of kinase 7 (Dok-7) is essential for the full activation of muscle-specific kinase and consequently for dense clustering of AChRs, we hypothesized that reduced levels of Dok-7 increase the susceptibility to passive transfer MG. To test this hypothesis, Dok-7 expression was reduced by transfecting shRNA-coding plasmids into the tibialis anterior muscle of adult rats by in vivo electroporation. Subclinical MG was subsequently induced with a low dose of anti-AChR monoclonal antibody 35. Neuromuscular transmission was significantly impaired in Dok-7-siRNA-electroporated legs compared with the contralateral control legs, which correlated with a reduction of AChR protein levels at the neuromuscular junction (approximately 25%) in Dok-7-siRNA-electroporated muscles, compared with contralateral control muscles. These results suggest that a reduced expression of Dok-7 may play a role in the susceptibility to passive transfer MG, by rendering AChR clusters less resistant to the autoantibody attack., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Muscle-specific kinase (MuSK) myasthenia gravis (MG) is hallmarked by the predominant involvement of bulbar muscles and muscle atrophy. This might mimic amyotrophic lateral sclerosis (ALS) presenting with bulbar weakness. We encountered four cases of MuSK MG patients with an initial misdiagnosis of ALS. We analyzed the clinical data of the four misdiagnosed MuSK MG patients, and investigated the presence of MuSK autoantibodies in a group of 256 Dutch bulbar-onset ALS patients using a recombinant MuSK ELISA and a standard MuSK radioimmunorecipitation assay. Clues for changing the diagnosis were slow progression, clinical improvement, development of diplopia and absence of signs of upper motor neuron involvement. No cases of MuSK MG were identified among a group of 256 bulbar ALS patients diagnosed according to the revised El Escorial criteria. A misdiagnosis of ALS in patients with MuSK MG is rare. We recommend to carefully consider the diagnosis of MuSK MG in patients presenting with bulbar weakness without clear signs of upper motor neuron dysfunction., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: We have recently reported the presence of the Human polyomavirus 7 (HPyV7) in human thymic epithelial tumors as assessed by diverse molecular techniques. Here we report on the co-expression of p16, retinoblastoma protein (pRb) and phosphorylated retinoblastoma protein (phospho-Rb) in human thymic epithelial tumors in relation to HPyV7., Methods: PRB, phospho-RB and p16 expression was assessed by immuno-histochemistry in 37 thymomas and 2 thymic carcinomas. 17 thymomas (46 %) and 1 thymic carcinoma (50 %) were recently tested positive for HPyV7. In addition, 20 follicular hyperplasias were tested., Results: Expression of pRb was observed in 35 thymomas (94.6 %), in 16 thymomas (43.2 %) the expression was strong. Phospho-Rb was observed in 31 thymomas (83.8 %). 19 thymomas (51.4 %) showed immunoreactivity for p16 of which 8 thymomas revealed very strong p16 expression. No p16 expression was detected in thymic carcinomas. In addition, no significant correlation between the presence of HPyV7 and pRb-, phospho-Rb- and p16-expression could be established. No correlation between pRb, phospho-Rb, p16 and WHO staging, Masaoka-Koga staging or the presence of MG was found. All 20 follicular hyperplasias showed expression of pRb and less expression of phospho-Rb., Conclusions: Although polyomaviruses have been shown to interact with cell cycle proteins no correlation between the presence of HPyV7 and the expression of pRb, phospho-Rb and p16 in human thymic epithelial tumors was observed. In as much HPyV7 contributes to human thymomagenesis remains to be established. Our data indicate pRb, phospho-Rb and p16 expression are rather unlikely to be involved in HPyV7 related thymomagenesis.

Introduction: Although the molecular genetics possibly underlying the pathogenesis of human thymoma have been extensively studied, its etiology remains poorly understood. Because murine polyomavirus consistently induces thymomas in mice, we assessed the presence of the novel human polyomavirus 7 (HPyV7) in human thymic epithelial tumors., Methods: HPyV7-DNA Fluorescence in situ hybridization (FISH), DNA-polymerase chain reaction (PCR), and immunohistochemistry (IHC) were performed in 37 thymomas. Of these, 26 were previously diagnosed with myasthenia gravis (MG). In addition, 20 thymic hyperplasias and 20 fetal thymic tissues were tested., Results: HPyV7-FISH revealed specific nuclear hybridization signals within the neoplastic epithelial cells of 23 thymomas (62.2%). With some exceptions, the HPyV7-FISH data correlated with the HPyV7-DNA PCR. By IHC, large T antigen expression of HPyV7 was detected, and double staining confirmed its expression in the neoplastic epithelial cells. Eighteen of the 26 MG-positive and 7 of the 11 MG-negative thymomas were HPyV7-positive. Of the 20 hyperplastic thymi, 40% were HPyV7-positive by PCR as confirmed by FISH and IHC in the follicular lymphocytes. All 20 fetal thymi tested HPyV7-negative., Conclusions: The presence of HPyV7-DNA and large T antigen expression in the majority of thymomas possibly link HPyV7 to human thymomagenesis. Further investigations are needed to elucidate the possible associations of HPyV7 and MG.

Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from nine early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 h and were no longer detectable at 24 h. Bortezomib therefore appears promising for treating MG and possibly other Ab-mediated autoimmune or allergic disorders, especially when given in short courses at modest doses before the standard immunosuppressive drugs have taken effect., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR-MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR-MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR-MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein-Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR-MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR-MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR-MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

It is unclear to what extent neuropathological changes contribute to brain inflammation observed in temporal lobe epilepsy (TLE). Here, we compared cytokine levels between histopathologically-confirmed sclerotic hippocampi and histopathologically-confirmed normal hippocampi from TLE patients. We analyzed a similar cytokine panel in the hippocampi of amygdala-kindled rats and we evaluated neuropathological changes by immunohistochemistry. In TLE patients, cytokine levels were not significantly different between sclerotic and non-sclerotic hippocampi. Though kindling resulted in increased astrocyte activation, cytokine levels and microglia activation were unchanged. These results suggest that the chronic epileptic state in TLE can also occur in the absence of intracerebral inflammation. Highlights., (Copyright © 2014 Elsevier B.V. All rights reserved.)

C1q is the initiator of the classical complement pathway and, as such, is essential for efficient opsonization and clearance of pathogens, altered self-structures, and apoptotic cells. The ceramide transporter protein (CERT) and its longer splicing isoform CERTL are known to interact with extracellular matrix components, such as type IV collagen, and with the innate immune protein serum amyloid P. In this article, we report a novel function of CERT in the innate immune response. Both CERT isoforms, when immobilized, were found to bind the globular head region of C1q and to initiate the classical complement pathway, leading to activation of C4 and C3, as well as generation of the membrane attack complex C5b-9. In addition, C1q was shown to bind to endogenous CERTL on the surface of apoptotic cells. These results demonstrate the role of CERTs in innate immunity, especially in the clearance of apoptotic cells.

Myasthenia gravis (MG) is an autoimmune disease in which autoantibodies, most commonly directed against the acetylcholine receptor (AChR), impair neuromuscular transmission and cause muscle weakness. In this study, we utilized two-dimensional difference in-gel electrophoresis (2D-DIGE) to analyze the muscle's proteomic profile at different stages of experimental autoimmune myasthenia gravis (EAMG). We identified twenty-two differentially expressed proteins, mainly related to metabolic and stress-response pathways. Interestingly, these identified proteins have also been associated with other contraction-impairing muscle pathologies (e.g. inclusion body myositis), suggesting a similar response of the muscle to such conditions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We studied Ig heavy chain (VDJ) sequences and antigen reactivity of 412 immortalized B cell lines from the peripheral blood of 10 multiple sclerosis (MS) patients, 4 clinically isolated syndrome (CIS) patients and 6 healthy controls (HCs). 78/238 (32.8%) MS and CIS B cell lines were part of 9 clonally expanded B cell populations, of which 5 were present in multiple patients. Increased VH1 gene family usage was evidenced for MS B cells, with 29.2% expressing VH1-69. Affinity maturation in MS and CIS was indicated by increased Ig VDJ mutations. Autoantibody producing B cells reactive to intracellular antigens were significantly higher in MS (25%) and CIS (28%) patients than in HCs (5%), including 3/9 expanded B cell clones. Specificity for phosphatidylcholine was observed for 1/9 B cell clones. These findings indicate clonally expanded autoreactive B cells with affinity maturation in the peripheral blood in MS and CIS., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Myasthenia gravis is caused by antibodies to the acetylcholine receptor, muscle-specific kinase, low-density lipoprotein receptor-related protein 4, or possibly yet unidentified antibodies. The mechanisms by which these antibodies interfere with the function of postsynaptic proteins include complement activation, antigenic modulation by crosslinking of the target proteins, competition with ligand binding sites, or steric hindrance which inhibits conformational changes or binding to associated proteins. Screening for auto-antibodies to different postsynaptic targets, and also for low-affinity antibodies, is contributing to a more accurate diagnosis of MG patients. Further studies into the specific pathophysiological pathways of the several MG subforms might help to develop new, more antigen specific, therapies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Fetal asphyctic preconditioning, induced by a brief episode of experimental hypoxia-ischemia, offers neuroprotection to a subsequent more severe asphyctic insult at birth. Extensive cell stress and apoptosis are important contributing factors of damage in the asphyctic neonatal brain. Because ceramide acts as a second messenger for multiple apoptotic stimuli, including hypoxia/ischemia, we sought to investigate the possible involvement of the ceramide pathway in endogenous neuroprotection induced by fetal asphyctic preconditioning. Global fetal asphyxia was induced in rats by clamping both uterine and ovarian vasculature for 30 min. Fetal asphyxia resulted in acute changes in brain ceramide/sphingomyelin metabolic enzymes, ceramide synthase 1, 2, and 5, acid sphingomyelinase, sphingosine-1-phosphate phosphatase, and the ceramide transporter. This observation correlated with an increase in neuronal apoptosis and in astrocyte number. After birth, ceramide and sphingomyelin levels remained high in fetal asphyxia brains, suggesting that a long-term regulation of the ceramide pathway may be involved in the mechanism of tolerance to a subsequent, otherwise lethal, asphyctic event.

Objective: Myasthenia gravis (MG) is an autoimmune disease mediated mainly by anti-acetylcholine receptor (AChR) antibodies. The thymus plays a primary role in MG pathogenesis. As we recently showed an inflammatory and antiviral signature in MG thymuses, we investigated whether pathogen-sensing molecules could contribute to an anti-AChR response., Methods: We studied the effects of toll-like receptor agonists on the expression of α-AChR and various tissue-specific antigens (TSAs) in human thymic epithelial cell (TEC) cultures. As polyinosinic-polycytidylic acid (poly[I:C]), which mimics double-stranded RNA (dsRNA), stimulated specifically α-AChR expression, the signaling pathways involved were investigated. In parallel, we analyzed the expression of dsRNA-signaling components in the thymus of MG patients, and the relevance of our data was investigated in vivo in poly(I:C)-injected mice., Results: We demonstrate that dsRNA signaling induced by poly(I:C) specifically triggers the overexpression of α-AChR in TECs and not of other TSAs. A poly(I:C) effect was also observed on MG TECs. This induction is mediated through toll-like receptor 3 (TLR3) and protein kinase R (PKR), and by the release of interferon (IFN)-β. In parallel, human MG thymuses also display an overexpression of TLR3, PKR, and IFN-β. In addition, poly(I:C) injections specifically increase thymic expression of α-AChR in wild-type mice, but not in IFN-I receptor knockout mice. These injections also lead to an anti-AChR autoimmune response characterized by a significant production of serum anti-AChR antibodies and a specific proliferation of B cells., Interpretation: Because anti-AChR antibodies are highly specific for MG and are pathogenic, dsRNA-signaling activation could contribute to the etiology of MG., (Copyright © 2012 American Neurological Association.)

Myasthenia gravis (MG) is treated primarily with broad-spectrum immuno-suppressants such as prednisone or azathioprine, which normally require several months to reduce autoantibody titers significantly. This delay may be caused by the resistance of the main antibody-producing cells, the plasma cells, to these drugs. In particular, long-lived plasma cells are resistant to immunosuppressive treatments and can produce (auto-) antibodies for months. Bortezomib is a proteasome inhibitor approved for treating patients with multiple myeloma, a plasma cell malignancy. Recent preclinical studies in cell cultures and animal models, and clinical studies in organ-transplant recipients, have demonstrated that bortezomib can kill non-neoplastic plasma cells within hours. This suggests that proteasome inhibitors could also be used for rapidly reducing autoantibody production in autoimmune diseases. We have begun to assess their potential in MG., (© 2012 New York Academy of Sciences.)

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)-coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation-related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet-induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance.

Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to human SAP. GPBP is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Also GPBP is found in plasma and in the extracellular matrix. In the present study, we demonstrate that GPBP specifically binds SAP in its physiological conformations, pentamers and decamers. The START domain in GPBP is important for this interaction. SAP and GPBP form complexes in blood and partly colocalize in amyloid plaques from Alzheimer disease patients. These data suggest the existence of complexes of SAP and GPBP under physiological and pathological conditions. These complexes are important for understanding basement membrane, blood physiology, and plaque formation in Alzheimer disease.

Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, (14)C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (P = 0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation.

Ceramides are lipids that are abundant in brain tissue where they have an important structural role in cellular membranes. Ceramides are also powerful intracellular signalling molecules controlling cell death, growth and differentiation. So far, the ceramide transfer protein (CERT), a shorter splice variant of the Goodpasture antigen-binding protein (GPBP), is the only known protein with the ability to shuttle ceramide from the endoplasmic reticulum to the Golgi apparatus. GPBP/CERT are widely distributed in the central nervous system where they act as key factors for normal brain development and homeostasis. Ceramide accumulates in neurons during acute neurodegeneration. The objective of this study was to define whether levels of the ceramide transfer protein GPBP/CERT are altered in the acute neurodegenerative process. We used design-based stereology to quantify the number of GPBP/CERT immunoreactive cells in the striatum of 6-hydroxydopamine (6-OHDA) lesioned rats as an animal model of Parkinson's disease (PD). In addition, gray value measurement was performed to quantify GPBP/CERT immunoreactivity-levels within individual cells. No difference in the striatal expression levels of GPBP/CERT proteins was found between diseased and control animals, suggesting that the expression pattern of GPBP/CERT in the striatum is not affected in the 6-OHDA rat model of PD., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

In myasthenia gravis (MG), the neuromuscular transmission is impaired mainly by auto-antibodies against the acetylcholine receptor (AChR) or MuSK. In about 5% of the MG patients, however, the auto-antigen is still unknown. We investigated whether these idiopathic MG patients (iMG) have auto-antibodies against ErbB proteins, which influence the AChR density at the NMJ. Our results show reduced mRNA expression levels of ErbB4 in thymus tissue of iMG patients compared to AChR-MG and non-MG patients, but we could not detect anti-ErbB antibodies in sera of iMG patients. Therefore, our results do not support a role for ErbB receptors as auto-antigens in iMG patients., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Myotonic dystrophy 1 (DM1) is characterized by a wide range of clinical features. We aimed to verify the presence of peripheral nerve involvement in a large cohort of DM1 patients and to determine clinical consequences. A total of 93 patients underwent detailed neurological examination and nerve conduction studies. Additionally, balance impairment was assessed with the Berg Balance Scale and health status was evaluated with the SF-36 health survey. Sensory symptoms were not reported and mild sensory signs were found in six patients. Electrophysiological abnormalities consistent with a diagnosis of neuropathy were found in 16 patients (17%). Peripheral nerve involvement was significantly associated with decreased muscle strength (p = 0.001) and absence of Achilles-tendon reflexes (p = 0.003), but not with age or duration of neuromuscular symptoms. It had no significant effect on balance, mental or physical health. In conclusion, peripheral nerve involvement may be one of the multisystemic manifestations of DM1, but is usually subclinical. Other causes should be excluded when sensory symptoms or signs are severe., (© 2011 Peripheral Nerve Society.)

Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.

Background: The involvement of muscle triacylglycerol (TAG) storage in the onset of insulin resistance is questioned and the attention has shifted towards inhibition of insulin signalling by the lipid intermediate diacylglycerol (DAG). The enzyme 1,2-acylCoA:diacylglyceroltransferase-1 (DGAT1) esterifies a fatty acyl-CoA on DAG to form TAG. Therefore, the aim of the present study was to investigate if unilateral overexpression of DGAT1 in adult rat Tibialis anterior (TA) muscle will increase conversion of the lipid intermediate DAG into TAG, thereby improving muscle insulin sensitivity., Methodology/principal Findings: The DGAT1 gene construct was injected in the left TA muscle of male rats on chow or high-fat (45% kcal) diet for three weeks, followed by application of one 800 V/cm and four 80 V/cm pulses, using the contralateral leg as sham-electroporated control. Seven days after electroporation, muscle specific insulin sensitivity was assessed with a hyperinsulinemic euglycemic clamp using 2-deoxy-[3H]glucose. Here, we provide evidence that unilateral overexpression of DGAT1 in TA muscle of male rats is associated with an increased rather than decreased DAG content. Strikingly, this increase in DAG content was accompanied by improved muscle insulin sensitivity. Interestingly, markers of muscle lipolysis and mitochondrial function were also increased in DGAT1 overexpressing muscle., Conclusions/significance: We conclude that unilateral DGAT1 overexpression can rescue insulin sensitivity, possibly by increasing DAG and TAG turnover in skeletal muscle. In case of a proper balance between the supply and oxidation of fatty acids in skeletal muscle, the lipid intermediate DAG may not exert harmful effects on insulin signalling.

Electroporation is a widely used technique for enhancing the efficiency of DNA delivery into cells. Application of electric pulses after local injection of DNA temporarily opens cell membranes and facilitates DNA uptake. Delivery of plasmid DNA by electroporation to alter gene expression in tissue has also been explored in vivo. This approach may constitute an alternative to viral gene transfer, or to transgenic or knock-out animals. Among the most frequently electroporated target tissues are skin, muscle, eye, and tumors. Moreover, different regions in the central nervous system (CNS), including the developing neural tube and the spinal cord, as well as prenatal and postnatal brain have been successfully electroporated. Here, we present a comprehensive review of the literature describing electroporation of the CNS with a focus on the adult brain. In addition, the mechanism of electroporation, different ways of delivering the electric pulses, and the risk of damaging the target tissue are highlighted. Electroporation has been successfully used in humans to enhance gene transfer in vaccination or cancer therapy with several clinical trials currently ongoing. Improving the knowledge about in vivo electroporation will pave the way for electroporation-enhanced gene therapy to treat brain carcinomas, as well as CNS disorders such as Alzheimer's disease, Parkinson's disease, and depression., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The etiology of the sporadic form of Alzheimer's disease (AD) remains largely unknown. Recent evidence has suggested that gene-environment interactions (GxE) may play a crucial role in its development and progression. Whereas various susceptibility loci have been identified, like the apolipoprotein E4 allele, these cannot fully explain the increasing prevalence of AD observed with aging. In addition to such genetic risk factors, various environmental factors have been proposed to alter the risk of developing AD as well as to affect the rate of cognitive decline in AD patients. Nevertheless, aside from the independent effects of genetic and environmental risk factors, their synergistic participation in increasing the risk of developing AD has been sparsely investigated, even though evidence points towards such a direction. Advances in the genetic manipulation of mice, modeling various aspects of the AD pathology, have provided an excellent tool to dissect the effects of genes, environment, and their interactions. In this paper we present several environmental factors implicated in the etiology of AD that have been tested in transgenic animal models of the disease. The focus lies on the concept of GxE and its importance in a multifactorial disease like AD. Additionally, possible mediating mechanisms and future challenges are discussed.

We have developed a B cell immortalization method for low B cell numbers per well using simultaneous B cell stimulation by CpG2006 and B cell infection by Epstein-Barr virus (EBV), followed by an additional CpG2006 and interleukin-2 (IL-2) stimulus. Using this method, immunoglobulin G (IgG)-producing immortalized B cell lines were generated from peripheral blood IgG(+)CD22(+) B cells with an efficiency of up to 83%. Antibody can already be obtained from the culture supernatant after 3-4 weeks. Moreover, clonality analysis demonstrated monoclonality in 87% of the resulting immortalized B cell lines. Given the high immortalization efficiency and monoclonality rate, evidence is provided that no further subcloning is necessary. An important application of this B cell immortalization method is the characterization of (autoreactive) antibodies from patients with autoimmune disease. This could eventually lead to the identification of new autoantigens, disease markers or targets for therapy., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Myasthenia Gravis (MG) is an antibody-mediated autoimmune disorder affecting the postsynaptic membrane of the neuromuscular junction (NMJ). MG is characterized by an impaired signal transmission between the motor neuron and the skeletal muscle cell, caused by auto-antibodies directed against NMJ proteins. The auto-antibodies target the nicotinic acetylcholine receptor (nAChR) in about 90% of MG patients. In approximately 5% of MG patients, the muscle specific kinase (MuSK) is the auto-antigen. In the remaining 5% of MG patients, however, antibodies against the nAChR or MuSK are not detectable (idiopathic MG, iMG). Although only the anti-nAChR and anti-MuSK auto-antibodies have been demonstrated to be pathogenic, several other antibodies recognizing self-antigens can also be found in MG patients. Various auto-antibodies associated with thymic abnormalities have been reported, as well as many non-MG-specific auto-antibodies. However, their contribution to the cause, pathology and severity of the disease is still poorly understood. Here, we comprehensively review the reported auto-antibodies in MG patients and discuss their role in the pathology of this autoimmune disease.

Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies that are either directed to the muscle nicotinic acetylcholine receptor (AChR) or to the muscle-specific tyrosine kinase (MuSK). These autoantibodies define two distinct subforms of the disease-AChR-MG and MuSK-MG. Both AChR and MuSK are expressed on the postsynaptic membrane of the neuromuscular junction (NMJ), which is a highly specialized region of the muscle dedicated to receive and process signals from the motor nerve. Autoantibody binding to proteins of the postsynaptic membrane leads to impaired neuromuscular transmission and muscle weakness. Pro-inflammatory antibodies of the human IgG1 and IgG3 subclass modulate the AChR, cause complement activation, and attract lymphocytes; together acting to decrease levels of the AChR and AChR-associated proteins and to reduce postsynaptic folding. In patients with anti-MuSK antibodies, there is no evidence of loss of junctional folds and no apparent loss of AChR density. Anti-MuSK antibodies are predominantly of the IgG4 isotype, which functionally differs from other IgG subclasses in its anti-inflammatory activity. Moreover, IgG4 undergoes a posttranslational modification termed Fab arm exchange that prevents cross-linking of antigens. These findings suggest that MuSK-MG may be different in etiological and pathological mechanisms from AChR-MG. The effector functions of IgG subclasses on synapse structure and function are discussed in this review.

Myasthenia gravis is a prototype anti-receptor autoimmune disease. Antibodies against proteins at the neuromuscular junction cause a defect in the signal transmission from nerve terminal to the damaged postsynaptic membrane. This issue of Autoimmunity reviews the mechanisms that lead to the destruction of the neuromuscular junction and the role of the thymus in myasthenia gravis and its animal models. In addition, this issue explores recent advances in diagnosis and treatment of this disease.

The Goodpasture antigen-binding protein (GPBP) and its splice variant the ceramide transporter (CERT) are multifunctional proteins that have been found to play important roles in brain development and biology. However, the function of GPBP and CERT is controversial because of their involvement in two apparently unrelated research fields: GPBP was initially isolated as a protein associated with collagen IV in patients with the autoimmune disease Goodpasture syndrome. Subsequently, a splice variant lacking a serine-rich domain of 26 amino acids (GPBPDelta26) was found to mediate the cytosolic transport of ceramide and was therefore (re)named CERT. The two splice forms likely carry out different functions in specific sub-cellular localizations. Selective GPBP knockdown induces extensive apoptosis and tissue loss in the brain of zebrafish. GPBP/GPBPDelta26 knock-out mice die as a result of structural and functional defects in endoplasmic reticulum and mitochondria. Because both mitochondria and ceramide play an important role in many biological events that regulate neuronal differentiation, cellular senescence, proliferation and cell death, we propose that GPBP and CERT are pivotal in neurodegenerative processes. In this review, we discuss the current state of knowledge on GPBP and CERT, including the molecular and biochemical characterization of GPBP in the field of autoimmunity as well as the fundamental research on CERT in ceramide transport, biosynthesis, localization, metabolism and cell homeostasis.

We describe the development of an outcome measure of activity and participation for patients with myotonic dystrophy type 1 using the Rasch measurement model. A 49-item questionnaire was completed by 163 DM1 patients. Data were subsequently analyzed with Rasch software to design the item set to fit model expectations. Through systematic investigation of response category ordering, model fit, item bias, and local response dependency, we succeeded in constructing a 20-item unidimensional scale of activity and participation (DM1-Activ). High internal consistency (PSI=0.95) and good test-retest reliability values of item difficulty hierarchy and patient location were demonstrated. Patient measures had acceptable correlations with MRC sum scores and MIRS grades (ICC=0.69 and 0.71, respectively), indicating good external construct validity. DM1-Activ is a practical, reliable and valid outcome measure that fulfils all clinimetric requirements. Further evaluation of this scale is needed to provide a nomogram for clinical use., (2010 Elsevier B.V. All rights reserved.)

The main immunogenic region (MIR) is a conformation-dependent region at the extracellular apex of alpha1 subunits of muscle nicotinic acetylcholine receptor (AChR) that is the target of half or more of the autoantibodies to muscle AChRs in human myasthenia gravis and rat experimental autoimmune myasthenia gravis. By making chimeras of human alpha1 subunits with alpha7 subunits, both MIR epitopes recognized by rat mAbs and by the patient-derived human mAb 637 to the MIR were determined to consist of two discontiguous sequences, which are adjacent only in the native conformation. The MIR, including loop alpha1 67-76 in combination with the N-terminal alpha helix alpha1 1-14, conferred high-affinity binding for most rat mAbs to the MIR. However, an additional sequence corresponding to alpha1 15-32 was required for high-affinity binding of human mAb 637. A water soluble chimera of Aplysia acetylcholine binding protein with the same alpha1 MIR sequences substituted was recognized by a majority of human, feline, and canine myasthenia gravis sera. The presence of the alpha1 MIR sequences in alpha1/alpha7 chimeras greatly promoted AChR expression and significantly altered the sensitivity to activation. This reveals a structural and functional, as well as antigenic, significance of the MIR.

Muscle-specific tyrosine kinase (MuSK) is essential for clustering of acetylcholine receptors (AChRs) at embryogenesis and likely also important for maintaining synaptic structure in adult muscle. In 5 to 7% of myasthenia gravis (MG) cases, the patients' blood contains antibodies to MuSK. To investigate the effect of MuSK-MG antibody on synapse regeneration, notexin was used to induce damage to the flexor digitorum brevis muscle. We administered aliquots of MuSK-MG patients' plasma to the flexor digitorum brevis twice daily for a period up to 21 days, and muscles were investigated ex vivo in contraction experiments. AChR levels were measured with (125)I-alpha-bungarotoxin, and endplates were studied with quantitative immunohistochemistry. In normal muscles and in 14-day regenerated muscles, MuSK plasma caused impairment of nerve stimulus-induced contraction in the presence of 0.35 and 0.5 mmol/L Ca(2+) with or without 100 to 400 nmol/L tubocurarine. Endplate size was decreased in regenerated muscles relative to controls; however, we did not observe such differences in muscle not treated with notexin. MuSK plasma had no effect on the amount and turnover rate of AChRs. Our results suggest that anti-MuSK antibodies influence the activity of MuSK molecules without reducing their number, thereby diminishing the size of the endplate and affecting the functioning of AChRs.

The Goodpasture antigen-binding protein (GPBP) plays a critical role in brain development. Knockdown of GPBP leads to loss of myelinated tracts in the central nervous system and to extensive apoptosis in the brain during early embryogenesis. GPBP was initially identified as a protein associated with the autoantigen in Goodpasture autoimmune syndrome, where it was shown to be a kinase that regulates type IV collagen organization. GPBP isoforms bind and transport ceramide from the endoplasmic reticulum to the Golgi apparatus and are therefore also known as ceramide transporters (CERT). Ceramide dysregulation is involved in autoimmunity and neurodegenerative disorders. In order to analyze the possible role of GPBP in neuroinflammation and neurodegeneration we studied the basal GPBP expression in normal rat brain. High levels of immunoreactivity were detected in neurons of the cerebral cortex, hippocampal formation, the basal ganglia, the olfactory bulb and nuclei of the thalamus, the hypothalamus and the septal area. Lower expression levels of GPBP were observed widely throughout the brain, suggesting that GPBP plays an important role in central nervous system neuron function.