Myasthenia gravis (MG) is an autoimmune disease in which autoantibodies attack the neuromuscular junction, the site at which nerve cells communicate with muscle fibres, impairing signal transmission and causing fluctuating muscle weakness that worsens with sustained activity. In most patients this dysfunction is reversible and improves with treatments that suppress the immune response. About 10 to 15 percent of patients do not respond adequately to standard therapy, and the mechanisms of this refractory course remain unclear.
The study is based on the hypothesis that in refractory patients the autoantibody attack causes irreversible damage to the neuromuscular junction, and that this damage sustains symptoms despite appropriate treatment. A further aim is to identify circulating biomarkers reflecting such damage that may help predict response to therapy.
The study includes adults with generalised MG positive for antibodies against the acetylcholine receptor, stratified by disease duration and treatment response into treatment-naive, treatment-sensitive and treatment-refractory MG. Subjects without neuromuscular disease and negative for these antibodies serve as controls. Blood samples (serum, plasma and mononuclear cells) are obtained from material left over from blood draws performed as part of routine care, together with clinical data including disease duration, symptom severity measured with validated scales (MG-ADL and QMG), antibody titre and treatment history. No study-specific visit or blood draw is required.
Antibodies purified from participants are applied to human neuromuscular organoids, three-dimensional models grown from stem cells of healthy donors that reproduce key features of the neuromuscular junction. Exposing these organoids to antibodies from patients at different disease stages reproduces the antibody-mediated attack under controlled laboratory conditions and allows the resulting structural and electrical changes to be measured. Molecules released by damaged organoids, including microRNAs and proteins, are identified and then measured in participants' blood. The immune profile of participants, including complement factors, lymphocyte subsets and cytokines, is characterised in parallel.
The study will determine whether irreversible neuromuscular junction damage distinguishes treatment-refractory MG from treatment-responsive disease, and whether specific circulating biomarkers can identify a refractory course.
Age >= 18 years
Established diagnosis of myasthenia gravis according to international criteria (fluctuating muscle weakness plus at least one of: positive anticholinesterase test; >10% decrement on repetitive nerve stimulation [SR-ENG]; or increased jitter on single-fiber EMG [SFEMG])
Anti-AChR antibody positivity confirmed by serological testing
Generalized MG stratified by disease duration and response to conventional immunosuppressive therapy into: treatment-naive MG (nMG); treatment-sensitive MG with stable disease and at least 12 months of follow-up (sMG); or treatment-refractory MG with at least 12 months of follow-up (rMG)
Seronegative (AChR-negative) generalized MG, or MG with anti-MuSK or other non-AChR antibodies
Purely ocular myasthenia gravis
Other concomitant neuromuscular disease (e.g., Lambert-Eaton myasthenic syndrome, polymyositis, muscular dystrophies)
Pregnancy or breastfeeding
Adults with acetylcholine receptor antibody-positive (AChR-positive) generalized myasthenia gravis, followed at two Italian tertiary referral centers for neuromuscular diseases, stratified by disease duration and response to conventional immunosuppressive therapy into treatment-naive, treatment-sensitive, and treatment-refractory subgroups. Both patients already followed at the participating centers (retrospective component) and newly evaluated patients (prospective component) are included. A control group of adults evaluated for neurological symptoms whose diagnostic work-up excluded neuromuscular disease is enrolled at the coordinating center only. Clinical data and residual (leftover) blood samples collected during routine clinical care are analyzed.
Cohort
Cross-sectional
Not listed
Antibody-induced change in neuromuscular junction electrophysiological activity in a patient serum-derived organoid assay
Change in spontaneous electrophysiological activity, measured as firing rate (Hz) by high-density multielectrode array (HD-MEA), in human neuromuscular organoids exposed to purified IgG from participants' serum. The primary comparison is between organoids exposed to IgG from treatment-refractory MG participants versus organoids exposed to IgG from treatment-sensitive MG, treatment-naive MG, and AChR-negative control participants, at the day 14 (chronic) exposure timepoint. The measure is assessed on a specimen-derived in vitro assay using participant-purified IgG, not on the participants directly. Lower firing rate indicates greater antibody-mediated neuromuscular junction dysfunction.
Antibody-induced change in neuromuscular junction morphology in a patient serum-derived organoid assay
Change in the alpha-Bungarotoxin/NF200 co-localization area, measured by confocal immunofluorescence and quantified with FIJI/ImageJ, in human neuromuscular organoids exposed to purified IgG from participants' serum. The comparison is between organoids exposed to IgG from treatment-refractory MG participants versus organoids exposed to IgG from treatment-sensitive MG, treatment-naive MG, and AChR-negative control participants, at the day 14 exposure timepoint. This measure is assessed on a specimen-derived in vitro assay using participant-purified IgG and supports the primary electrophysiological outcome. A reduced co-localization area indicates greater antibody-mediated neuromuscular junction damage.
Validation of candidate refractoriness biomarkers in participants' serum
Levels of candidate biomarkers, first identified in the serum-derived organoid models, measured in participants' serum to assess their association with a treatment-refractory disease course. Neuromuscular-enriched and inflammation-related microRNAs (such as miR-206, miR-133b, miR-1 and miR-499) are quantified by quantitative RT-PCR, and candidate proteins are quantified by immunoassay (Simple Plex/Ella or ELISA). Levels are compared across treatment-refractory, treatment-sensitive, and treatment-naive AChR-positive generalized MG participants and AChR-negative controls.
Immunological profile across MG treatment-response subgroups
Characterization of the immunological profile in participants' blood, compared across treatment-refractory, treatment-sensitive, and treatment-naive AChR-positive generalized MG participants and AChR-negative controls. Assessments include anti-AChR antibody titer, complement factors, lymphocyte subpopulations by flow cytometry (T subsets including CD4+, CD8+ and regulatory T cells; B subsets including naive, memory and plasma cells), and circulating cytokines and chemokines measured by immunoassay (Simple Plex/Ella or ELISA). The aim is to identify immunological features that distinguish treatment-refractory disease.
Correlation between clinical severity, immunological profile, and organoid alterations
Correlation between clinical severity of myasthenia gravis (MGFA classification, QMG score, MG-ADL score) and the immunological and molecular parameters measured in the study, including the immunological profile and the morphological and molecular alterations observed in the serum-derived organoid models. Associations are assessed using Spearman or Pearson correlation as appropriate, with multivariable regression to identify independent predictors of clinical severity and of a treatment-refractory course.
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Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico
Lead sponsor
IRCCS Ospedale San Raffaele
Collaborator
