A retrospective multicenter study on clinical and serological parameters in patients with MuSK myasthenia gravis with and without general immunosuppression.

Introduction: Muscle-specific kinase (MuSK)- myasthenia gravis (MG) is caused by pathogenic autoantibodies against MuSK that correlate with disease severity and are predominantly of the IgG4 subclass. The first-line treatment for MuSK-MG is general immunosuppression with corticosteroids, but the effect of treatment on IgG4 and MuSK IgG4 levels has not been studied. Methods: We analyzed the clinical data and sera from 52 MuSK-MG patients (45 female, 7 male, median age 49 (range 17-79) years) from Italy, the Netherlands, Greece and Belgium, and 43 AChR-MG patients (22 female, 21 male, median age 63 (range 2-82) years) from Italy, receiving different types of immunosuppression, and sera from 46 age- and sex-matched non-disease controls (with no diagnosed diseases, 38 female, 8 male, median age 51.5 (range 20-68) years) from the Netherlands. We analyzed the disease severity (assessed by MGFA or QMG score), and measured concentrations of MuSK IgG4, MuSK IgG, total IgG4 and total IgG in the sera by ELISA, RIA and nephelometry. Results: We observed that MuSK-MG patients showed a robust clinical improvement and reduction of MuSK IgG after therapy, and that MuSK IgG4 concentrations, but not total IgG4 concentrations, correlated with clinical severity. MuSK IgG and MuSK IgG4 concentrations were reduced after immunosuppression in 4/5 individuals with before-after data, but data from non-linked patient samples showed no difference. Total serum IgG4 levels were within the normal range, with IgG4 levels above threshold (1.35g/L) in 1/52 MuSK-MG, 2/43 AChR-MG patients and 1/45 non-disease controls. MuSK-MG patients improved within the first four years after disease onset, but no further clinical improvement or reduction of MuSK IgG4 were observed four years later, and only 14/52 (26.92%) patients in total, of which 13 (93.3%) received general immunosuppression, reached clinical remission. Discussion: We conclude that MuSK-MG patients improve clinically with general immunosuppression but may require further treatment to reach remission. Longitudinal testing of individual patients may be clinically more useful than single measurements of MuSK IgG4. No significant differences in the serum IgG4 concentrations and IgG4/IgG ratio between AChR- and MuSK-MG patients were found during follow-up. Further studies with larger patient and control cohorts are necessary to validate the findings.

Plasma ceramides relate to mild cognitive impairment in middle-aged men: The Maastricht Study.

Introduction: There is an urgent need for biomarkers identifying individuals at risk of early-stage cognitive impairment. Using cross-sectional data from The Maastricht Study, this study included 197 individuals with mild cognitive impairment (MCI) and 200 cognitively unimpaired individuals aged 40 to 75, matched by age, sex, and educational level. Methods: We assessed the association of plasma sphingolipid and ceramide transfer protein (CERT) levels with MCI and adjusted for potentially confounding risk factors. Furthermore, the relationship of plasma sphingolipids and CERTs with magnetic resonance imaging brain volumes was assessed and age- and sex-stratified analyses were performed. Results: Associations of plasma ceramide species C18:0 and C24:1 and combined plasma ceramide chain lengths (ceramide risk score) with MCI were moderated by sex, but not by age, and higher levels were associated with MCI in men. No associations were found among women. In addition, higher levels of ceramide C20:0, C22:0, and C24:1, but not the ceramide risk score, were associated with larger volume of the hippocampus after controlling for covariates, independent of MCI. Although higher plasma ceramide C18:0 was related to higher plasma CERT levels, no association of CERT levels was found with MCI or brain volumes. Discussion: Our results warrant further analysis of plasma ceramides as potential markers for MCI in middle-aged men. In contrast to previous studies, no associations of plasma sphingolipids with MCI or brain volumes were found in women, independent of age. These results highlight the importance of accounting for sex- and age-related factors when examining sphingolipid and CERT metabolism related to cognitive function.

Anti-GAD65 autoantibody levels measured by ELISA and alternative types of immunoassays in relation to neuropsychiatric diseases versus diabetes mellitus type 1.

Background: Anti-GAD65 autoantibodies (GAD65-Abs) may occur in patients with epilepsy and other neurological disorders, but the clinical significance is not clear-cut. Whereas high levels of GAD65-Abs are considered pathogenic in neuropsychiatric disorders, low or moderate levels are only considered as mere bystanders in, e.g., diabetes mellitus type 1 (DM1). The value of cell-based assays (CBA) and immunohistochemistry (IHC) for GAD65-Abs detection has not been clearly evaluated in this context. Objective: To re-evaluate the assumption that high levels of GAD65-Abs are related to neuropsychiatric disorders and lower levels only to DM1 and to compare ELISA results with CBA and IHC to determine the additional value of these tests. Methods: 111 sera previously assessed for GAD65-Abs by ELISA in routine clinical practice were studied. Clinical indications for testing were, e.g., suspected autoimmune encephalitis or epilepsy (neuropsychiatric cohort; n = 71, 7 cases were initially tested positive for GAD65-Abs by ELISA), and DM1 or latent autoimmune diabetes in adults (DM1/LADA cohort (n = 40, all were initially tested positive)). Sera were re-tested for GAD65-Abs by ELISA, CBA, and IHC. Also, we examined the possible presence of GAD67-Abs by CBA and of other neuronal autoantibodies by IHC. Samples that showed IHC patterns different from GAD65 were further tested by selected CBAs. Results: ELISA retested GAD65-Abs level in patients with neuropsychiatric diseases was higher than in patients with DM1/LADA (only retested positive samples were compared; 6 vs. 38; median 47,092 U/mL vs. 581 U/mL; p = 0.02). GAD-Abs showed positive both by CBA and IHC only if antibody levels were above 10,000 U/mL, without a difference in prevalence between the studied cohorts. We found other neuronal antibodies in one patient with epilepsy (mGluR1-Abs, GAD-Abs negative), and in a patient with encephalitis, and two patients with LADA. Conclusion: GAD65-Abs levels are significantly higher in patients with neuropsychiatric disease than in patients with DM1/LADA, however, positivity in CBA and IHC only correlates with high levels of GAD65-Abs, and not with the underlying diseases.

Reemergence of pathogenic, autoantibody-producing B cell clones in myasthenia gravis following B cell depletion therapy.

Myasthenia gravis (MG) is an autoantibody-mediated autoimmune disorder of the neuromuscular junction. A small subset of patients (<10%) with MG, have autoantibodies targeting muscle-specific tyrosine kinase (MuSK). MuSK MG patients respond well to CD20-mediated B cell depletion therapy (BCDT); most achieve complete stable remission. However, relapse often occurs. To further understand the immunomechanisms underlying relapse, we studied autoantibody-producing B cells over the course of BCDT. We developed a fluorescently labeled antigen to enrich for MuSK-specific B cells, which was validated with a novel Nalm6 cell line engineered to express a human MuSK-specific B cell receptor. B cells (≅ 2.6 million) from 12 different samples collected from nine MuSK MG patients were screened for MuSK specificity. We successfully isolated two MuSK-specific IgG4 subclass-expressing plasmablasts from two of these patients, who were experiencing a relapse after a BCDT-induced remission. Human recombinant MuSK mAbs were then generated to validate binding specificity and characterize their molecular properties. Both mAbs were strong MuSK binders, they recognized the Ig1-like domain of MuSK, and showed pathogenic capacity when tested in an acetylcholine receptor (AChR) clustering assay. The presence of persistent clonal relatives of these MuSK-specific B cell clones was investigated through B cell receptor repertoire tracing of 63,977 unique clones derived from longitudinal samples collected from these two patients. Clonal variants were detected at multiple timepoints spanning more than five years and reemerged after BCDT-mediated remission, predating disease relapse by several months. These findings demonstrate that a reservoir of rare pathogenic MuSK autoantibody-expressing B cell clones survive BCDT and reemerge into circulation prior to manifestation of clinical relapse. Overall, this study provides both a mechanistic understanding of MuSK MG relapse and a valuable candidate biomarker for relapse prediction.

FTY720 decreases ceramides levels in the brain and prevents memory impairments in a mouse model of familial Alzheimer's disease expressing APOE4.

The protection mediated by the bioactive sphingolipid sphingosine-1-phosphate (S1P) declines during Alzheimer's disease (AD) progression, especially in patients carrying the apolipoprotein E ε4 (APOE4) isoform. The drug FTY720 mimics S1P bioactivity, but its efficacy in treating AD is unclear. Two doses of FTY720 (0.1 mg / kg and 0.5 mg / kg daily) were given by oral gavage for 15 weeks to transgenic mouse models of familial AD carrying human apolipoprotein E (APOE) APOE3 (E3FAD) or APOE4 (E4FAD). After 12 weeks of treatment, animals were subjected to behavioral tests for memory, locomotion, and anxiety. Blood was withdrawn at different time points and brains were collected for sphingolipids analysis by mass spectrometry, gene expression by RT-PCR and Aß quantification by ELISA. We discovered that low levels of S1P in the plasma is associated with a higher probability of failing the memory test and that FTY720 prevents memory impairments in E4FAD. The beneficial effect of FTY720 was induced by a shift of the sphingolipid metabolism in the brain towards a lower production of toxic metabolites, like ceramide d18:1/16:0 and d18:1/22:0, and reduction of amyloid-ß burden and inflammation. In conclusion, we provide further evidence of the druggability of the sphingolipid system in AD.

Novel treatment strategies for acetylcholine receptor antibody-positive myasthenia gravis and related disorders.

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.

Autoimmune Encephalitis With mGluR1 Antibodies Presenting With Epilepsy, but Without Cerebellar Signs: A Case Report.

OBJECTIVE: To describe the unique case history of a patient with mGluR1 antibodies, with mainly limbic and without cerebellar symptoms. METHODS: A 50-year-old woman initially presented with focal seizures with epigastric rising and déjà-vu sensations, next to cognitive complaints, and musical auditory hallucinations. MRI, EEG, and neuronal autoantibody tests were performed. RESULTS: EEG findings showed slow and sharp activity (sharp waves and sharp-wave-slow-wave complex) in the left temporal lobe. A test for autoantibodies was negative initially. Because of persistent symptoms, serum and CSF were tested 4 years later and found positive for mGluR1 antibodies. Treatment started with monthly IV immunoglobulins and azathioprine that was replaced by mycophenolate mofetil later. Especially cognitive symptoms and hallucinations did not respond well to the treatment. During treatment, mGluR1 antibodies remained present in CSF. DISCUSSION: Whereas cerebellar symptoms are present in 97% of mGluR1-positive cases, our patient presented without ataxia. Therefore, we suggest that the clinical presentation of patients with mGluR1 antibodies is probably more diverse than previously described. Testing for mGluR1 antibodies should be considered in patients with limbic encephalitis and epilepsy, especially when negative for more common antibodies.

Effects of Sex, Age, and Apolipoprotein E Genotype on Brain Ceramides and Sphingosine-1-Phosphate in Alzheimer's Disease and Control Mice.

Apolipoprotein ε4 (APOE)4 is a strong risk factor for the development of Alzheimer's disease (AD) and aberrant sphingolipid levels have been implicated in AD. We tested the hypothesis that the APOE4 genotype affects brain sphingolipid levels in AD. Seven ceramides and sphingosine-1-phosphate (S1P) were quantified by LC-MSMS in hippocampus, cortex, cerebellum, and plasma of <3 months and >5 months old human APOE3 and APOE4-targeted replacement mice with or without the familial AD (FAD) background of both sexes (145 animals). APOE4 mice had higher Cer(d18:1/24:0) levels in the cortex (1.7-fold, p = 0.002) than APOE3 mice. Mice with AD background showed higher levels of Cer(d18:1/24:1) in the cortex than mice without (1.4-fold, p = 0.003). S1P levels were higher in all three brain regions of older mice than of young mice (1.7-1.8-fold, all p ≤ 0.001). In female mice, S1P levels in hippocampus (r = -0.54 [-0.70, -0.35], p < 0.001) and in cortex correlated with those in plasma (r = -0.53 [-0.71, -0.32], p < 0.001). Ceramide levels were lower in the hippocampus (3.7-10.7-fold, all p < 0.001), but higher in the cortex (2.3-12.8-fold, p < 0.001) of female than male mice. In cerebellum and plasma, sex effects on individual ceramides depended on acyl chain length (9.5-fold lower to 11.5-fold higher, p ≤ 0.001). In conclusion, sex is a stronger determinant of brain ceramide levels in mice than APOE genotype, AD background, or age. Whether these differences impact AD neuropathology in men and women remains to be investigated.

Altered sphingolipid function in Alzheimer's disease; a gene regulatory network approach.

Sphingolipids (SLs) are bioactive lipids involved in various important physiological functions. The SL pathway has been shown to be affected in several brain-related disorders, including Alzheimer's disease (AD). Recent evidence suggests that epigenetic dysregulation plays an important role in the pathogenesis of AD as well. Here, we use an integrative approach to better understand the relationship between epigenetic and transcriptomic processes in regulating SL function in the middle temporal gyrus of AD patients. Transcriptomic analysis of 252 SL-related genes, selected based on GO term annotations, from 46 AD patients and 32 healthy age-matched controls, revealed 103 differentially expressed SL-related genes in AD patients. Additionally, methylomic analysis of the same subjects revealed parallel hydroxymethylation changes in PTGIS, GBA, and ITGB2 in AD. Subsequent gene regulatory network-based analysis identified 3 candidate genes, that is, SELPLG, SPHK1 and CAV1 whose alteration holds the potential to revert the gene expression program from a diseased towards a healthy state. Together, this epigenomic and transcriptomic approach highlights the importance of SL-related genes in AD, and may provide novel biomarkers and therapeutic alternatives to traditionally investigated biological pathways in AD.

The search for an autoimmune origin of psychotic disorders: Prevalence of autoantibodies against hippocampus antigens, glutamic acid decarboxylase and nuclear antigens.

The etiology of psychotic disorders is still unknown, but in a subgroup of patients symptoms might be caused by an autoimmune reaction. In this study, we tested patterns of autoimmune reactivity against potentially novel hippocampal antigens. Serum of a cohort of 621 individuals with psychotic disorders and 257 controls were first tested for reactivity on neuropil of rat brain sections. Brain reactive sera (67 diseased, 27 healthy) were further tested for antibody binding to glutamic acid decarboxylase (GAD) isotype 65 and 67 by cell-based assay (CBA). A sub-cohort of 199 individuals with psychotic disorders and 152 controls was tested for the prevalence of anti-nuclear antibodies (ANA) on HEp2-substrate as well as for reactivity to double-stranded DNA, ribosomal P (RPP), and cardiolipin (CL). Incubation of rat brain with serum resulted in unidentified hippocampal binding patterns in both diseased and control groups. Upon screening with GAD CBA, one of these patterns was identified as GAD65 in one individual with schizophrenia and also in one healthy individual. Two diseased and two healthy individuals had low antibody levels targeting GAD67 by CBA. Antibody reactivity on HEp-2-substrate was increased in patients with schizoaffective disorder, but only in 3 patients did antibody testing hint at a possible diagnosis of systemic lupus erythematosus. Although reactivity of serum to intracellular antigens might be increased in patients with psychotic disorder, no specific targets could be identified. GAD antibodies are very rare and do not seem increased in serum of patients with psychotic disorders.

CERTL reduces C16 ceramide, amyloid-ß levels, and inflammation in a model of Alzheimer's disease.

BACKGROUND: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer's disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-ß (Aß) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. METHODS: A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-ß (Aß), Aß aggregation process in presence of CERTL, and the resulting changes in Aß toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. RESULTS: Here, we report that CERTL binds to APP, modifies Aß aggregation, and reduces Aß neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aß formation, and modulates microglia by decreasing their pro-inflammatory phenotype. CONCLUSION: Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.

Immunofluorescence Labeling of Lipid-Binding Proteins CERTs to Monitor Lipid Raft Dynamics.

Fluorescence microscopy is a powerful and widely used tool in molecular biology. Over the years, the discovery and development of lipid-binding fluorescent probes has established new research possibilities to investigate lipid composition and dynamics in the cell. For instance, fluorescence microscopy has allowed the investigation of lipid localization and density in specific cell compartments such as membranes or organelles. Often, the characteristics and the composition of lipid-enriched structures are determined by analyzing the distribution of a fluorescently labeled lipid probe, which intercalates in lipid-enriched platforms, or specifically binds to parts of the lipid molecule. However, in many cases antibodies targeting proteins have higher specificity and are easier to generate. Therefore, we propose to use both antibodies targeting lipid transporters and lipid binding probes to better monitor lipid membrane changes. As an example, we visualize lipid rafts using the fluorescently labeled-B-subunit of the cholera toxin in combination with antibodies targeting ceramide-binding proteins CERTs, central molecules in the metabolism of sphingolipids.

Novel neuronal surface autoantibodies in plasma of patients with depression and anxiety.

Neuronal surface autoantibodies (NSAbs) against various antigens cause autoimmune encephalitis. Some of these antigens are also involved in the pathology of depression and anxiety. To study whether NSAbs are more common in plasma of individuals with depression and anxiety than in controls, and to investigate if NSAbs correlate with disease status, plasma samples of 819 individuals with a current diagnosis of depression and/or anxiety, 920 in remission and 492 individuals without these disorders were included in this study. Samples were tested by a combination of immunohistochemistry (IHC), staining on live rat hippocampus neurons and cell-based assay (CBA). By IHC, 50 (2.2%) samples showed immunoreactivity to rat brain tissue, with no significant differences between the aforementioned groups (22/819 vs 18/920 vs 11/492, P > 0.99). In addition, eight IHC positive samples were positive for NSAbs on live neurons (7/819 vs 0/920 vs 1/492, P = 0.006). The IHC-staining patterns of these eight samples were atypical for autoimmune encephalitis and accordingly, they tested negative for known NSAbs by CBA. No obvious difference in the clinical characteristics between individuals with or without NSAbs was observed. In conclusion, novel NSAbs were rare but predominately found in patients with current anxiety or depression indicating they might affect mental health in a small group of patients.

Ceramide analog [18F]F-HPA-12 detects sphingolipid disbalance in the brain of Alzheimer's disease transgenic mice by functioning as a metabolic probe.

The metabolism of ceramides is deregulated in the brain of Alzheimer's disease (AD) patients and is associated with apolipoprotein (APO) APOE4 and amyloid-ß pathology. However, how the ceramide metabolism changes over time in AD, in vivo, remains unknown. Distribution and metabolism of [18F]F-HPA-12, a radio-fluorinated version of the ceramide analog N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide, was investigated in the brain of AD transgenic mouse models (FAD) on an APOE4 or APOE3 genetic background, by positron emission tomography and by gamma counter. We found that FAD mice displayed a higher uptake of [18F]F-HPA-12 in the brain, independently from the APOE4 or APOE3 genetic background. FAD mice could be distinguished from littermate control animals with a sensitivity of 85.7% and a specificity of 87.5%, by gamma counter measurements. Metabolic analysis of [18F]F-HPA-12 in the brain suggested that the tracer is degraded less efficiently in the FAD mice. Furthermore, the radioactive signal registered in the hippocampus correlated with an increase of Cer d18:1/20:2 levels measured in the same brain region by mass spectrometry. Our data gives additional proof that ceramide metabolism is different in FAD mice compared to controls. Ceramide analogs like HPA-12 may function as metabolic probes to study ceramide disbalance in the brain.

Unidentified Neuronal Surface IgG Autoantibodies in a Case of Hashimoto's Encephalopathy.

Hashimoto's encephalopathy is an encephalitis of presumed autoimmune origin characterized by the presence of autoantibodies against thyroid proteins. We present a case of a young patient with pre-existing Hashimoto's thyroiditis and progressive cognitive complaints, absence-like episodes, and sporadic bilateral epileptiform frontal and frontotemporal activity. No abnormalities were observed during the neurological examination and on MRI. Antibodies to thyroid peroxidase (TPO) were elevated and remained positive while the symptoms were present. Levothyroxine and methylprednisolone did not ameliorate the complaints. Subsequent treatment with high-dose intravenous immunoglobulins (IVIG) led to improved cognitive functions and to the disappearance of the absence-like-episodes. Patient's serum, but not CSF, gave a characteristic IgG-specific hippocampal pattern in rat brain immunohistochemistry; this immunoreactivity was maintained after specific and complete depletion of TPO antibodies. Serum IgG bound to primary neurons in cell culture, likely targeting a yet unidentified neuronal surface antigen. The clinical response to IVIG suggests but does not prove, that the circulating novel autoantibodies may induce the encephalopathy. It would be of interest to investigate more patients with Hashimoto's encephalopathy for the presence of neuronal surface autoantibodies, to define their role in the disease and their target antigen(s).

Sphingolipids in Alzheimer's disease, how can we target them?

Altered levels of sphingolipids and their metabolites in the brain, and the related downstream effects on neuronal homeostasis and the immune system, provide a framework for understanding mechanisms in neurodegenerative disorders and for developing new intervention strategies. In this review we will discuss: the metabolites of sphingolipids that function as second messengers; and functional aberrations of the pathway resulting in Alzheimer's disease (AD) pathophysiology. Focusing on the central product of the sphingolipid pathway ceramide, we describ approaches to pharmacologically decrease ceramide levels in the brain and we argue on how the sphingolipid pathway may represent a new framework for developing novel intervention strategies in AD. We also highlight the possible use of clinical and non-clinical drugs to modulate the sphingolipid pathway and sphingolipid-related biological cascades.

Autoimmunity in psychotic disorders. Where we stand, challenges and opportunities.

Psychotic disorders are debilitating mental illnesses associated with abnormalities in various neurotransmitter systems. The development of disease-modifing therapies has been hampered by the mostly unknown etiologies and pathophysiologies. Autoantibodies against several neuronal antigens are responsible for autoimmune encephalitis. These autoantibodies disrupt neurotransmission within the brain, resulting in a wide range of psychiatric and neurologic manifestations, including psychosis. The overlap of symptoms of autoimmune encephalitis with psychotic disorders raised the question as to whether autoantibodies against a number of receptors, ion channel and associated proteins could ultimately be responsible for some forms of psychosis. Here we review our current knowledge, on antibody mediated autoimmunity in psychotic disorders, the different diagnostic methods and their limitations, as well as on varying therapeutic approaches targeting the immune system.

Characterization of pathogenic monoclonal autoantibodies derived from muscle-specific kinase myasthenia gravis patients.

Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by pathogenic autoantibodies that bind to membrane proteins at the neuromuscular junction. Most patients have autoantibodies against the acetylcholine receptor (AChR), but a subset of patients have autoantibodies against muscle-specific tyrosine kinase (MuSK) instead. MuSK is an essential component of the pathway responsible for synaptic differentiation, which is activated by nerve-released agrin. Through binding MuSK, serum-derived autoantibodies inhibit agrin-induced MuSK autophosphorylation, impair clustering of AChRs, and block neuromuscular transmission. We sought to establish individual MuSK autoantibody clones so that the autoimmune mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from 6 MuSK MG patients using a fluorescently tagged MuSK antigen multimer, then generated a panel of human monoclonal autoantibodies (mAbs) from these cells. Here we focused on 3 highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells, and at mouse neuromuscular junctions, where they colocalized with AChRs. These 3 IgG isotype mAbs (2 IgG4 and 1 IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The mAbs inhibited AChR clustering, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation, which suggests an alternative mechanism for inhibiting AChR clustering.

Low prevalence of Merkel cell polyomavirus in human epithelial thymic tumors.

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.