Myasthenia Gravis Patient Sera Research Articles

Visualization and characterization of complement activation in acetylcholine receptor antibody seropositive myasthenia gravis.

There are no blood biomarkers to monitor treatment effects in myasthenia gravis (MG) or studies visualizing the acetylcholine receptor (AChR) antibody-induced membrane attack complex (MAC) at the human muscle membrane. This study aimed to compare levels of complement activation products and native complement components in MG patients and healthy controls (HCs) and to model the AChR antibody-mediated attacks in human muscle cells. We assessed the complement components and activation product levels with enzyme-linked immunosorbent assay and magnetic bead-based sandwich assays in plasma and sera of 23 MG patients and matched HCs. Receiver operator characteristic (ROC) curve analysis evaluated the diagnostic accuracy. Complement levels were correlated with the myasthenia gravis composite (MGC) scores. AChR+ MG modeling in human muscle cells used sera from nine MG patients and three HCs. MG patients had significantly higher plasma levels of C3a (p < .0001), C5 (p = .0003), and soluble C5b-9 (sC5b-9; p < .0001) than HCs. The ROC curve analysis showed a clear separation between MG patients and HCs for plasma C3a (AUC = 0.9720; p < .0001) and sC5b-9 (AUC = 0.8917, p < .0001). MG patients had higher levels of plasma complement Factor I (FI; p = .0002) and lower properdin levels (p < .0001). The MGC had moderate correlations with plasma Factor B (FB), FI, and Factor H. AChR+ MG patient sera triggered the deposition of MAC and reduced AChRs. We suggest validating plasma C3a and sC5b-9 as blood biomarkers for complement activation in MG. Further, the in vitro study allowed visualization of MAC deposition after applying AChR+ MG sera on human muscle cells.

Serum metabolomic profile of myasthenia gravis and potential values as biomarkers in disease monitoring

ObjectiveSerum metabolites from 19 myasthenia gravis (MG) patients and 15 normal controls were analyzed via untargeted metabolomics, including 6 pre/post-treatment paired MG patients, to assess the value of serum metabolites as biomarkers in monitoring MG. MethodDifferential metabolites between MG patients and normal controls were identified through liquid and gas chromatography-mass spectrometry simultaneously. Principal component analysis and orthogonal partial least squares-discriminant analysis were conducted to identify the differential metabolites. Candidate metabolites and pathways associated with MG were selected through a random forest machine learning model. ResultA total of 310 differential metabolites were identified with a threshold of variable projected importance > 1 and P value < 0.05. Among these, 158 metabolites were upregulated and 152 were downregulated. The random forest machine learning model selected 5 metabolites as potential biomarkers associated with MG: lignoceric acid (AUC=0.944), uridine diphosphate-N-acetylglucosamine (AUC=0.951), arachidonic acid (AUC=0.951), beta-glycerophosphoric acid (AUC=0.933), and L-Asparagine (AUC=0.877). Further analysis using 6 paired MG patients pre- and post-immunosuppression treatment revealed 25 upregulated and 6 downregulated metabolites in post-treatment serum, which might be relevant to disease intervention. The significance remains elusive due to the limited number of patients. ConclusionA subset of differential metabolites was identified in the serum of MG patients, some of which changed with immunosuppressive therapy. Small molecule metabolites may serve as valuable biomarkers for disease monitoring in MG.

Residual serum fibrinogen as a universal biomarker for all serotypes of Myasthenia gravis

Myasthenia Gravis (MG) is an autoimmune disease associated with severe neuromuscular weakness. Diagnostic confirmation of MG is typically delayed and secured in about 85% and 50% of patients with generalized and ocular MG, respectively with serum antibodies. We have identified a sensitive and specific diagnostic biomarker for various MG serotypes with quantitative proteomics. Serum proteomes of 18 individuals (MG patients, healthy controls (HC), Rheumatoid Arthritis (RA) were quantified in a pilot study and occurrence of high residual fibrinogen was validated by immunoblotting and further investigated by targeted mass spectrometry on the sera of 79 individuals (31 MG of various serotypes, 30 HC, 18 RA). Initial proteomic analysis identified high residual fibrinogen in MG patient sera which was then validated by antibody-based testing. Subsequently, a blinded study of independent samples showed 100% differentiation of MG patients from controls. A final serological quantification of 14 surrogate peptides derived from α-, β-, and γ-subunits of fibrinogen in 79 individuals revealed fibrinogen to be highly specific and 100% sensitive for MG (p < 0.00001), with a remarkable average higher abundance of > 1000-fold over control groups. Our unanticipated discovery of high levels of residual serum fibrinogen in all MG patients can secure rapid bedside diagnosis of MG.

Diagnosis and Management of Seronegative Myasthenia Gravis: Lights and Shadows.

Myasthenia gravis (MG) is an antibody-mediated neuromuscular disease affecting the neuromuscular junction. In most cases, autoantibodies can be detected in the sera of MG patients, thus aiding in diagnosis and allowing for early screening. However, there is a small proportion of patients who have no detectable auto-antibodies, a condition termed "seronegative MG" (SnMG). Several factors contribute to this, including laboratory test inaccuracies, decreased antibody production, immunosuppressive therapy, immunodeficiencies, antigen depletion, and immune-senescence. The diagnosis of SnMG is more challenging and is based on clinical features and neurophysiological tests. The early identification of these patients is needed in order to ensure early treatment and prevent complications. This narrative review aims to examine the latest updates on SnMG, defining the clinical characteristics of affected patients, diagnostic methods, management, and therapeutic scenarios.

The relationship between serum iron levels and AChR-Ab and IL-6 in patients with myasthenia gravis.

To investigate the correlation between serum iron (SI) levels and acetylcholine receptor antibodies (AChR-Ab) and interleukin 6 (IL-6) in patients with myasthenia gravis (MG). A total of 76 patients with myasthenia gravis (MG group) between July 2015 to March 2018, and another 50 healthy subjects during the same period were selected for this study. SI levels in the peripheral blood serum of all participants were measured using the colorimetric method (a SI level of < 8.95 umol/L was considered the standard for SI deficiency). Enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA) were used to detect the expression and levels of IL-6 and AChR-Ab in the peripheral blood of all participants. The presence and levels of IL-6 and AChR-Ab in the serum of MG patients and normal healthy subjects were compared. The levels of IL-6 and AChR-Ab in MG patients with normal SI levels and those with SI deficiency were analyzed. The SI deficiency rate, AChR-Ab positivity rate, AChR-Ab levels, and IL-6 levels in the MG group were significantly higher than those in the control group (73.68% vs. 26.00%, 81.58% vs. 0.00%, 1.05 ± 0.40 nmol/L vs. 0.21 ± 0.09 nmol/L, and 183.54 ± 35.26 ng/mL vs. 121.43 ± 28.45 ng/mL, respectively; all p-values were < 0.01). In MG patients, the levels of AChR-AB and IL-6 in the SI deficiency group were significantly higher than those in the normal SI group (1.15 ± 0.34 nmol/L vs. 0.81 ± 0.45 nmol/L and 193.12 ± 31.70 ng/mL vs. 156.74 ± 31.19 ng/mL, respectively; all p-values were < 0.01). The correlation analysis showed that SI levels were negatively correlated with AChR-AB and IL-6 levels in MG patients (r = -0.776, r = -0.663, both p-values were < 0.01). Iron deficiency in MG patients and SI levels are negatively correlated with AChR-Ab and IL-6 levels in MG patients.

Effects of Follicular Helper T Cells and Inflammatory Cytokines on Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disorder mediated by antibodies against the acetylcholine receptors (AChR) of the skeletal muscles. An imbalance in various T helper (Th) cells, including Th1, Th2, Th17, Th22 and follicular helper T (TFH) cells, has been found associated with immunological disturbances. In this study, we aim to investigate the role of the Th cells in peripheral blood of MG patients. A total of 33 MG patients and 34 age matched controls were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradient centrifugation assay. The proportion of TFH cells in PBMC were analyzed using flow-cytometry assay by determining the levels of cellular markers CD4, CXCR5, CD45RO, CD45RA and ICOS and PD-1. The levels of IFN-γ, IL-4, IL-17 and IL-21 in serum were analyzed by Cytometric Bead Array. The serum IL-22 level was analyzed by ELISA. The frequency of TFH cells in PBMCs was higher than those in healthy subjects and correlated to the severity of MG patients. The levels of pro-inflammatory cytokines IFN-γ, IL-17 and IL-21 were elevated in the serum of MG patients, while there were no significant differences regarding the levels of IL-4 and IL-22 between MG patients and control subjects. Our findings suggest that Th cells and their cytokines balance of MG patients are involved in the clinical condition or severity of MG disease.

Elevated expression of interleukin-33 in myasthenia gravis patients

Myasthenia gravis (MG) is an archetypal autoimmune disorder of the neuromuscular junction. The imbalance of inflammatory cytokines are involved in the pathogenesis of MG. IL-33, a member of the IL-1 family, plays a key immune-modulation role in several autoimmune disease. However, its regulatory role in MG remains unclear. Here, we demonstrated that IL-33 expression in the serum of MG patients was significantly increased. We further proved that the serum levels of IL-33 were significantly negative correlated with the expression levels of TSLP. Increased serum IL-33 levels positively correlated with the upregulation of IL-17A levels and the quantitative myasthenia gravis (QMG) score in MG patients. Our findings indicate that IL-33 plays a potent immuno- enhancing role in the pathogenesis of MG by downregulating TSLP, consequently affecting the development of Th17 cells in MG.

Altered Gut Microbiota in Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune-mediated disorder, the etiology of which involves both environmental factors and genetics. While the exact factors responsible for predisposition to MG remain elusive, it is hypothesized that gut microbiota play a critical role in the pathogenesis of MG. This study investigated whether gut microbiota are altered in MG patients by comparing the fecal microbiota profiles of MG patients to those of age- and sex-matched healthy controls. Phylotype profiles of gut microbial populations were generated using hypervariable tag sequencing of the V4 region of the 16S ribosomal RNA gene. Fecal short-chain fatty acids (SCFAs) were assessed by gas chromatographic analyses. The results demonstrated that, compared to the healthy cohort, the gut microbiota of the MG group was changed in terms of the relative abundances of bacterial taxa, with sharply reduced microbial richness, particularly in the genus Clostridium. The fecal SCFA content was significantly lower in the MG group. Furthermore, microbial dysbiosis was closely related to the levels of inflammatory biomarkers in the sera of MG patients.

Profile of upregulated inflammatory proteins in sera of Myasthenia Gravis patients

This study describes specific patterns of elevated inflammatory proteins in clinical subtypes of myasthenia gravis (MG) patients. MG is a chronic, autoimmune neuromuscular disease with antibodies most commonly targeting the acetylcholine receptors (AChRab), which causes fluctuating skeletal muscle fatigue. MG pathophysiology includes a strong component of inflammation, and a large proportion of patients with early onset MG additionally present thymus hyperplasia. Due to the fluctuating nature and heterogeneity of the disease, there is a great need for objective biomarkers as well as novel potential inflammatory targets. We examined the sera of 45 MG patients (40 AChRab seropositive and 5 AChRab seronegative), investigating 92 proteins associated with inflammation. Eleven of the analysed proteins were significantly elevated compared to healthy controls, out of which the three most significant were: matrix metalloproteinase 10 (MMP-10; p = 0.0004), transforming growth factor alpha (TGF-α; p = 0.0017) and extracellular newly identified receptor for advanced glycation end-products binding protein (EN-RAGE) (also known as protein S100-A12; p = 0.0054). Further, levels of MMP-10, C-X-C motif ligand 1 (CXCL1) and brain derived neurotrophic factor (BDNF) differed between early and late onset MG. These novel targets provide valuable additional insight into the systemic inflammatory response in MG.

Expression of nicotinic acetylcholine receptor subunits in HEp-2 cells for immunodetection of autoantibody specificities in sera from Myasthenia gravis patients.

Myasthenia gravis (MG) is an autoimmune disease characterized by the formation of pathogenic autoantibodies mostly targeting the nicotinic acetylcholine receptor (AChR). The AChR is composed of two alpha subunits and one subunit of each beta, delta and gamma (fetal AChR), or epsilon (adult AChR), respectively. Serological diagnostics is commonly done by radioimmunoassay (RIA). Here we used an indirect immunofluorescence assay with MG patient sera on transiently transfected HEp-2 cells expressing selected components of the AChR. Our data show that already 12 out of 13 MG patient sera showed autoantibody binding to HEp-2 cells transfected to express the alpha subunit solely. Interestingly, 11 out of 13 patient sera reacted positive with cells transfected to reconstitute the complete fetal AChR, but only 6 out of 13 sera showed positive signals with cells expressing the components of adult AChR. Moreover, there was no strict correlation of the serum concentration required to obtain clear-cut fluorescence signals to the antibody titer as measured by RIA. It will be an interesting topic to further investigate if the optimal serum dilution for indirect immunofluorescence as well as the autoantibody binding preferences to defined AChR subunits and to the adult versus the fetal receptor variant could provide additional predictive value in MG diagnostics.

Disease-specific signature of serum miR-20b and its targets IL-8 and IL-25, in myasthenia gravis patients.

Myasthenia gravis (MG) is an autoimmune disorder characterized by antibodies directed against components of the neuromuscular junction. Currently, the diagnosis and therapeutic evaluation rely on the serum acetylcholine receptor (AchR) antibody titer, which is not reliable for monitoring. The disruption of the menus had been implicated in many immunological disorders, including MG. A quantitative PCR was used to evaluate the miR-20b level. ELISA was used to determine the levels of IL-8 and IL-25 in serum. Quantitative MG scores (QMGS) were used to examine the clinical manifestations. Here, we report that miR-20b, an immune- and cancer-related miRNA, is decreased in the serum of MG patients and correlates negatively with QMGSs in the pretreatment stage. Furthermore, after treatment with prednisone acetate, levels of miR-20b recover but remain negatively correlated with the QMGS. We also identified that IL-8 and IL-25 are targets of miR-20b via the luciferase reporter system. Both of these are increased in MG and correlate negatively with miR-20b. Furthermore, IL-8 and IL-25 levels are decreased following treatment with prednisone acetate. Our data suggest that miR-20b might be a potential biomarker for MG.

Serum proteomic, peptidomic and metabolomic profiles in myasthenia gravis patients during treatment with Qiangji Jianli Fang

BackgroundQiangji Jianli Fang (QJF) has been used for treatment of myasthenia gravis (MG) in China. However, our understanding of the effects of QJF against MG at the molecular level is limited. This study aims to investigate the effects of QJF treatment of MG patients on the protein, peptide and metabolite levels in serum.MethodsHigh-throughput proteomic, peptidomic and metabolomic techniques were applied to investigate serum samples from 21 healthy individuals and 47 MG patients before and after QJF treatment via two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time of flight mass spectrometry and liquid chromatography Fourier transform mass spectrometry, respectively.ResultsAfter QJF treatment, the expression levels of peptides m/z 1865.019, 2021.128 and 1211.668 of complement C3f increased (P = 0.004, P = 0.001 and P = 0.043, respectively), while that of peptide m/z 1739.931 of component C4b decreased (P = 0.043), in the serum of MG patients. The levels of γ-aminobutyric acid (P = 0.000) and coenzyme Q4 (P = 0.000) resumed their normal states.ConclusionQJF could inhibit the activity of the complement system and restore the normal levels of metabolites.

Characterization of peripheral blood acetylcholine receptor-binding B cells in experimental myasthenia gravis

In myasthenia gravis (MG), the neuromuscular transmission is impaired by antibodies (Abs) specific for muscle acetylcholine receptor (AChR). Anti-AChR Abs can be detected in the serum of MG patients, although their levels do not correlate with disease severity. In this study, we developed a flow cytometric assay for the detection of peripheral blood AChR-specific B cells to characterize B cell phenotypes associated with experimental autoimmune myasthenia gravis (EAMG). Alexa-conjugated AChR was used as a probe for AChR-specific B cells (B220+Ig+). Mice with EAMG had significantly elevated frequencies of AChR-specific IgG2+ and IgM+ B cells. While the frequencies of IgG2+ B cells and plasma anti-AChR IgG2 levels significantly correlated with the clinical grades of EAMG, the frequencies of IgM+ B cells and plasma anti-AChR IgM levels did not. These results indicate that the frequency of AChR-specific and IgG1+ (mouse IgG2 equivalent) peripheral blood B cells and anti-AChR IgG1 levels could be potential biomarkers for MG disease severity.

Recombinant expression of the AChR-alpha1 subunit for the detection of conformation-dependent epitopes in Myasthenia Gravis

Detection of autoantibodies associated with neurological disease typically involves immunoprecipitation of radioactively labeled native proteins. We explored whether single receptor subunits, fused to Renilla luciferase (Ruc), could detect patient autoantibodies in Luciferase Immunoprecipitation Systems. Myasthenia Gravis patient sera were tested for conformational autoantibodies to only the α1-subunit of the nicotinic acetylcholine receptor (AChR). Using a panel of 10 AChR-α1 fragments, AChR-α1-Δ5-Ruc demonstrated the highest immunoreactivity with a conformational-specific antibody and the highest sensitivity in a pilot cohort. Testing a larger cohort with AChR-α1-Δ5-Ruc demonstrated 21% sensitivity and 97% specificity. A point mutation within Ruc increased the diagnostic performance of AChR-α1-Δ5 (32% sensitivity, 97% specificity). The 125I-α-bungarotoxin multi-subunit AChR assay demonstrated 63% sensitivity and 97% specificity. These findings highlight the difficulty in detecting Myasthenia Gravis conformational epitopes across assay formats and lay the foundation for detecting autoantibodies to defined recombinant chains of the AChR and potentially other neurotransmitter receptors.

A rapid, fluorescence‐based assay for detecting antigenic modulation of the acetylcholine receptor on human cell lines

Myasthenia gravis (MG) is an autoimmune disease affecting approximately 40,000 patients in the United States. One of the major mechanisms of disease pathology in MG is the binding, internalization, and eventual destruction of acetylcholine receptors (AChR) at the neuromuscular junction by cross-linking AChR-specific autoantibodies. This process, known as antigenic modulation, ultimately attenuates the ability of muscle cells to contract in response to signals from neurons, leading to muscle weakness and fatigue. For this reason, antigenic modulation of the AChR on cultured cells has become an important diagnostic tool for assessing the pathogenicity of AChR-specific autoantibodies. Traditionally, these assays have been done using radiolabeled AChR ligands such as (125)I alpha-bungarotoxin to determine relative AChR number. Here, we present a high-throughput immunofluorescent flow cytometry-based assay that can be used to quantify AChR levels on the cell surface and assess the efficacy of molecules designed to rescue antigenic modulation. AChR levels were quantified on human muscle cells before and after treatment with AChR antibodies via immunofluorescent labeling with the AChR monoclonal antibodies, mAb210 and mAb B3, followed by flow cytometry of EDTA-treated cells. Using a novel, flow cytometry-based assay, antigenic modulation of the AChR was demonstrated on human cells using both AChR-specific monoclonal antibody and MG patient serum. The degree of antigenic modulation was dose responsive to antibody levels and could be reversed by preincubating antibodies with soluble AChR alpha subunit extracellular domain. A rapid, nonradioactive assay was developed to determine the potential of AChR-specific antibodies in the serum of MG patients to bind and down-regulate the AChR. This assay can be used to assess the ability of putative therapeutics that rescue antigenic modulation and could be developed for the treatment of MG.

Effect of Acetylcholine Receptor Source on Detection and Quantification of Anti–Acetylcholine Receptor–Binding Antibody

Myasthenia gravis (MG),1 an illness characterized by sporadic muscle weakness that increases with activity, is a common autoimmune disease with an estimated population prevalence of 10 to 70 per 100 000 (1). Diagnosis of MG is primarily based on clinical findings but may be confirmed by laboratory test results demonstrating the presence of antibodies that recognize the neuromuscular nicotinic acetylcholine receptor (AChR). Use of an RIA precipitation method enables detection of anti-AChR antibodies in approximately 85% of patients with generalized MG(2). The AChR source, however, has been shown to affect the detection and quantification of antibodies present in the serum of MG patients(3). The AChR expressed by the rhabdomyosarcoma (RD) cell line TE671/RD has been used in the detection and quantification of anti-AChR antibodies, but results obtained with this method have not been identical to results obtained with assays based on alternative receptor sources(3)(4)(5). To investigate this phenomenon, we used our routine, in-house validated method(4)(5) to assay patient serum samples submitted to ARUP Laboratories, the national esoteric reference laboratory owned by …

The clinical significance of anti-heat shock cognate protein 71 antibody in myasthenia gravis

Anti-heat shock cognate protein 71 antibody (HSC71 Ab) formation in the sera of myasthenia gravis (MG) patients was measured, and the correlation between HSC71 Ab titers, clinical features and therapy efficacies for MG patients were examined. Clinical evaluations were performed according to the MG Foundation of America (MGFA) clinical classification. Therapy efficacies were measured using the MG activities of daily living (MG-ADL) score. Before treatment, 38 out of 48 serum samples (79%) from MG patients showed positive HSC71 Ab titers. In the “therapy-responsive group”, HSC71 Ab titers significantly reduced, along with patient clinical improvements. Conversely, in the “therapy-resistant group”, HSC71 Ab titers did not decline. The use of tacrolimus resulted in improvement in clinical manifestations together with a reduction in HSC71 Ab titers in the “therapy-resistant group”. Thus, measurement of HSC71 Ab in the sera of MG patients seemed useful, as it appeared to reflect the effectiveness of treatment and allowed prediction of prognosis.

Establishment and analysis of serum two-dimensional gel electrophoresis profiles of myasthenia gravis patients with spleen and kidney deficiency syndrome

The purpose of this study was to establish two-dimensional gel electrophoresis (2-DE) profiles of serum of myasthenia gravis patients, and to identify the differential proteomic expressions between normal persons and myasthenia gravis patients with spleen and kidney deficiency syndrome. Samples of serum protein were extracted by repeated freeze-thaw method and separated by two-dimensional electrophoresis. Differential proteomic expressions between the myasthenia gravis patients and the normal control persons were identified by two-dimensional polyacrylamide gel electrophoresis, silver staining, image-master 2-DE software analysis, peptide mass fingerprinting based on matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), BioWorks and NCBI software database searching. The two-dimensional polyacrylamide gel electrophoresis profiles of serum proteins were successfully established by 2-DE. Twenty-one of the significant differential proteins were selected and identified by MALDI-TOF-MS. Eight of them were finally identified. The 2-DE profiles of serum proteins were established and the differential proteomic expressions were identified by proteome technique in our study. This can be an experimental basis for further research of the pathogenesis and treatment of myasthenia gravis.

Overexpression of Rapsyn in Rat Muscle Increases Acetylcholine Receptor Levels in Chronic Experimental Autoimmune Myasthenia Gravis

The primary autoantigen in myasthenia gravis, the acetylcholine receptor (AChR), is clustered and anchored in the postsynaptic membrane of the neuromuscular junction by rapsyn. Previously, we found that overexpression of rapsyn by cDNA transfection protects AChRs in rat muscles from antibody-mediated loss in passive transfer experimental autoimmune myasthenia gravis (EAMG). Here, we determined whether rapsyn overexpression can reduce or even reverse AChR loss in muscles that are already damaged by chronic EAMG, which mimics the human disease. Active immunization against purified AChR was performed in female Lewis rats. Rapsyn overexpression resulted in an increase in total muscle membrane AChR levels, with some AChR at neuromuscular junctions but much of it in extrasynaptic membrane regions. At the ultrastructural level, most endplates in rapsyn-treated chronic EAMG muscles showed increased damage to the postsynaptic membrane. Although rapsyn overexpression stabilized AChRs in intact or mildly damaged endplates, the rapsyn-induced increase of membrane AChR enhanced autoantibody binding and membrane damage in severe ongoing disease. Thus, these results show the complexity of synaptic stabilization of AChR during the autoantibody attack. They also indicate that the expression of receptor-associated proteins may determine the severity of autoimmune diseases caused by anti-receptor antibodies.

Effects of myasthenia gravis patient sera on human myoblast cultures

To investigate the role of acetylcholine receptor (AChR) and other anti-muscle autoantibodies in myasthenia gravis (MG). Since many of these autoantibodies target proteins with structural or signalling functions, we examined the effect of MG sera on muscle cell morphology. Primary human myoblast cultures were exposed to MG sera and morphological changes observed by light and fluorescence microscopy. MG patient sera caused changes in cell shape (cell retraction) and led to the formation of inclusion bodies and intracellular vesicles. A disordered arrangement of actin microfilaments was also observed. The effects were not complement-mediated, were both dose- and time-dependent, and appeared to correlate with disease severity of the MG donor. The factors responsible for these effects in vitro may also play a role in the pathogenesis of MG in vivo. Further study of these factors may improve our understanding of MG pathogenesis.