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La diffusione pandemica della malattia Covid-19 impone un enorme sforzo di identificazione degli individui già entrati in contatto con il virus o tuttora portatori dell’infezione anche tra gli individui pauci- o asintomatici. A fronte di questa esigenza le attuali capacità diagnostiche appaiono su scala mondiale fortemente sottodimensionate1. L’invenzione prevede la messa a punto di un test diagnostico rapido e semplice capace di rivelare la presenza del virus SARS-CoV-2 e delle immunoglobuline secretorie specifiche di classe IgA (sIgA) nella saliva/escreato del probando. Il test deve essere concepito come un test domiciliare (home testing) operato direttamente dal probando ed interpretato dal medico tramite semplice documentazione fotografica o App dedicata. L’evidenziazione del virus si basa su un test di agglutinazione nel liquido salivare di particelle di latex (Latex Agglutination Test) colorate, precedentemente sensibilizzate con un anticorpo (monoclonale) rivolto contro la Spike Protein (S1) virale, su una superfice di vetro o carta; la rivelazione potrà avvenire entro pochi minuti ad occhio nudo. La abbondanza e ripetitività dell’antigene bersaglio sulla superficie di SARS-CoV-2 rendono questo virus particolarmente efficace come agente agglutinante. Per la rilevazione delle sIgA anti SARS-CoV-2 nello stesso tipo di campione viene utilizzata una reazione immunocromatografica su striscetta (Lateral flow test) simile a quella comunemente utilizzata per la rivelazione qualitativa di IgG ed IgM specifiche in campioni di sangue; in questo caso immunoglobuline anti-IgA vengono immobilizzate sulla “linea di cattura”.

The use of rituximab (RTX), an anti-CD20 monoclonal antibody (Ab), in refractory myasthenia gravis (MG) is associated with a better response in patients with Abs to the muscle-specific tyrosine kinase (MuSK) than in other MG subgroups. Anti-MuSK Abs are mostly IgG4 with proven pathogenicity and positive correlation with clinical severity. The rapid and sustained response to RTX may be related to MuSK Ab production by short-lived Ab-secreting cells derived from specific CD20+ B cells. Here, we investigated the long-term effects of RTX in nine refractory MuSK-MG patients with a follow-up ranging from 17 months to 13 years. In patients’ sera, we titrated MuSK-specific IgG (MuSK-IgG) and MuSK-IgG4, along with total IgG and IgG4 levels. Optimal response to RTX was defined as the achievement and maintenance of the status of minimal manifestations (MM)-or-better together with a ≥ 50% steroid reduction, withdrawal of immunosuppressants, and no need for plasma-exchange or intravenous immunoglobulin. After a course of RTX, eight patients improved, with optimal response in six, while only one patient did not respond. At baseline, MuSK-IgG and MuSK-IgG4 serum titers were positive in all patients, ranging from 2.15 to 49.5 nmol/L and from 0.33 to 46.2 nmol/L, respectively. MuSK Abs mostly consisted of IgG4 (range 63.80–98.86%). RTX administration was followed by a marked reduction of MuSK Abs at 2–7 months and at 12–30 months (p < 0.02 for MuSK-IgG and p < 0.01 for MuSK-IgG4). In patients with a longer follow-up, MuSK Ab titers remained suppressed, paralleling clinical response. In the patient who achieved long-term complete remission, MuSK-IgG4 was no longer detectable within 2 years, while MuSK-IgG remained positive at very low titers up to 10 years after RTX. In the patient who did not respond, MuSK-IgG and MuSK-IgG4 remained unchanged. In this patient series, total IgG and IgG4 transiently decreased (p < 0.05) at 2–7 months after RTX. The different trends of reducti

Background: Multiple sclerosis (MS) is an autoimmune disease for which auto-antibodies fully validated as diagnostic and prognostic biomarkers are widely desired. Recently, an immunoreactivity against the inward rectifying potassium channel 4.1 (KIR4.1) has been reported in a large proportion of a group of MS patients, with amino acids 83–120 being the major epitope. Moreover, a strong correlation between anti-KIR4.183–120and anti-full-length-protein auto-antibodies titer was reported. However, this finding received limited confirmation. Objective: Validation of the diagnostic potential of anti-KIR4.183–120antibodies in 78 MS patients, 64 healthy blood donors, and 42 individuals with other neurological diseases. Methods: Analysis of anti-KIR4.183–120antibodies by enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum we produced as a new ELISA reliability control. Additionally, evaluation of reactivity against 293-T cells transiently transfected with full-length KIR4.1 by flow cytometry. Results: We found antibodies to KIR4.183–120only in 13 out of 78 (16.6%) MS patients; among these, only 2 were positive for anti-full-length KIR4.1 antibodies. Conclusion: Employing a new reliability control and a new cytofluorometric assay, we cannot support anti-KIR4.183–120auto-antibodies as a reliable biomarker in MS.

Background. Serological levels of free immunoglobulin light chains (FLCs), produced in excess of heavy chains during synthesis of immunoglobulins by plasma cells, can be considered a direct marker of B cell activity in different systemic inflammatory-autoimmune conditions and may represent a useful predictor of rituximab (RTX) therapeutic efficacy, as reported for rheumatoid arthritis and systemic lupus erythematosus. Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction with antibodies (abs) targeting the acetylcholine receptor (AChR) or the muscle-specific tyrosine kinase (MuSK), inducing muscle weakness and excessive fatigability. As MG course may be remarkably variable, we evaluated the possible use of FLCs as biomarkers of disease activity. Subjects and Methods. We assessed FLC levels in 34 sera from 17 AChR-MG and from 13 MuSK-MG patients, in comparison with 20 sera from patients with systemic autoimmune rheumatic diseases and 18 from healthy blood donors, along with titers of specific auto-abs and IgG subclass distribution. Results. We found a statistically significant increase in free. chains in both AChR-and MuSK-MG patients, while free lambda chain levels were increased only in AChR-MG. We also observed a significant reduction of both free kappa and lambda chains in 1/4 MuSK-MG patients along with specific abs titer, two months after RTX treatment. Conclusions. From our data, FLCs appear to be a sensitive marker of B cell activation in MG. Further investigations are necessary to exploit their potential as reliable biomarkers of disease activity.

Myasthenia graviswith antibodies to themuscle-specific tyrosine kinase(MuSK+ MG)is a rare diseasewith distinctive pathogenic mechanisms and clinical features. An acute onset and predominant bulbar muscle weakness are very commonand highly suggestive of the disease.On the other hand, amore indolent course, atypical ocular presentation, and signs of cholinergic hyperactivity may complicate the diagnosis. ThoughMuSK+ MGis still a severe disease, over the years we have observed a steady reduction in the rate of respiratory crisis and a significant improvement in the clinical outcome, both likely related to earlier diagnosis and timely treatment. Despite the improved management, MuSK+ MG patients tend to remain dependent on long-term immunosuppressive treatment and may develop permanent disabling weakness. In uncontrolled studies, B cell depletion with rituximab proved effective in most patientswith refractory disease, inducing prolonged clinical responses associatedwith a sustained reduction of serum antibody levels. Promising results fromexperimental studies and case reports suggest that both 3,4-diaminopyridine and albuterol may be effective as symptomatic agents.

Background Myasthenia gravis (MG) with antibodies to the muscle-specific kinase (MuSK) has a characteristic phenotype. Ocular manifestations have not been systematically evaluated. Objective To investigate the features of extrinsic ocular muscle involvement in patients with MuSK-MG. Methods We retrospectively evaluated the prevalence, time of onset, clinical pattern and outcome of ocular symptoms in 82 patients with a clinical follow-up ≥2 years. Results Ocular manifestations were observed in 79 patients (96.4%) and were the presenting symptoms in 48 (58.5%). Intermittent diplopia with subtle ophthalmoparesis was the most common complaint, ptosis was generally symmetrical and conjugated gaze paresis occurred in 35% of the patients. Ocular manifestations responded well to prednisone and partially to symptomatic treatment. A few patients developed chronic symmetrical ophthalmoparesis, associated with persistent weakness in other muscle groups. All patients with ocular presentation progressed to generalised disease, though weakness spread to other muscle groups was considerably delayed in a few cases. Conclusions In MG with antibodies to MuSK, ocular manifestations were as frequent as in other disease subtypes. Symmetrical ophthalmoparesis with conjugated gaze limitation was rather common and associated with low functional disability. A proportion of these patients developed chronic eye muscle paresis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease for which auto-antibodies fully validated as diagnostic and prognostic biomarkers are widely desired. Recently, an immunoreactivity against the inward rectifying potassium channel 4.1 (KIR4.1) has been reported in a large proportion of a group of MS patients, with amino acids 83-120 being the major epitope. Moreover, a strong correlation between anti-KIR4.183-120 and anti-full-length-protein auto-antibodies titer was reported. However, this finding received limited confirmation. OBJECTIVE: Validation of the diagnostic potential of anti-KIR4.183-120 antibodies in 78 MS patients, 64 healthy blood donors, and 42 individuals with other neurological diseases. METHODS: Analysis of anti-KIR4.183-120 antibodies by enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum we produced as a new ELISA reliability control. Additionally, evaluation of reactivity against 293-T cells transiently transfected with full-length KIR4.1 by flow cytometry. RESULTS: We found antibodies to KIR4.183-120 only in 13 out of 78 (16.6%) MS patients; among these, only 2 were positive for anti-full-length KIR4.1 antibodies. CONCLUSION: Employing a new reliability control and a new cytofluorometric assay, we cannot support anti-KIR4.183-120 auto-antibodies as a reliable biomarker in MS.

Remission of myasthenia gravis with MuSK antibodies during ruxolitinib treatment.

In recent years, the discovery of new autoantigens and the use of sensitive assays have expanded the clinical spectrum of myasthenia gravis (MG). In particular, antibodies binding to clustered acetylcholine receptors and to the low-density lipoprotein receptor-related protein 4 have not only bridged a significant gap in diagnosis but also have relevant clinical implications. MG management includes different therapeutic options, from symptomatic agents as the only therapy in mildly affected cases to combined long-term immunosuppression and thymectomy in patients with severe disabling disease. MG biological diversity can influence the response to therapies and should be taken into account when planning treatment. Biologic agents are promising, though their use is currently limited to patients with refractory disease.

Myasthenia gravis (MG) patients with antibodies against the muscle specific tyrosine kinase (MuSK +) have predominantly involvement of cranio-bulbar muscles and do not display thymus pathology, as do acetylcholine receptor antibody seropositive (AChR +) MG patients. In search of novel biomarkers for MuSK + MG, we evaluated circulating serum microRNAs. Four analyzed microRNAs were specifically elevated in MuSK + MG patient serum samples: let-7a-5p, let-7f-5p, miR-151a-3p and miR-423-5p. The circulating microRNA profile in MuSK. + MG differs from the profile previously observed in the serum of AChR + MG, thus indicating the etiological difference between these two entities. We propose that the identified microRNAs could serve as potential serum biomarkers for MuSK + MG.

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Characterization of B cells in muscle-specific kinase antibody myasthenia gravis. OBJECTIVE: To characterize B-cell subsets in patients with muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). METHODS: In accordance with Human Immunology Project Consortium guidelines, we performed polychromatic flow cytometry and ELISA assays in peripheral blood samples from 18 patients with MuSK MG and 9 healthy controls. To complement a B-cell phenotype assay that evaluated maturational subsets, we measured B10 cell percentages, plasma B cell-activating factor (BAFF) levels, and MuSK antibody titers. Immunologic variables were compared with healthy controls and clinical outcome measures. RESULTS: As expected, patients treated with rituximab had high percentages of transitional B cells and plasmablasts and thus were excluded from subsequent analysis. The remaining patients with MuSK MG and controls had similar percentages of total B cells and naïve, memory, isotype-switched, plasmablast, and transitional B-cell subsets. However, patients with MuSK MG had higher BAFF levels and lower percentages of B10 cells. In addition, we observed an increase in MuSK antibody levels with more severe disease. CONCLUSIONS: We found prominent B-cell pathology in the distinct form of MG with MuSK autoantibodies. Increased BAFF levels have been described in other autoimmune diseases, including acetylcholine receptor antibody-positive MG. This finding suggests a role for BAFF in the survival of B cells in MuSK MG, which has important therapeutic implications. B10 cells, a recently described rare regulatory B-cell subset that potently blocks Th1 and Th17 responses, were reduced, which suggests a potential mechanism for the breakdown in immune tolerance in patients with MuSK MG.

Myasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoantibodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4 (LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG.

In recent years, the discovery of new autoantigens and the use of sensitive assays have expanded the clinical spectrum of myasthenia gravis (MG). In particular, antibodies binding to clustered acetylcholine receptors and to the low-density lipoprotein receptor-related protein 4 have not only bridged a significant gap in diagnosis but also have relevant clinical implications. MG management includes different therapeutic options, from symptomatic agents as the only therapy in mildly affected cases to combined long-term immunosuppression and thymectomy in patients with severe disabling disease. MG biological diversity can influence the response to therapies and should be taken into account when planning treatment. Biologic agents are promising, though their use is currently limited to patients with refractory disease.

The co-occurrence of autoimmune diseases (ADs) in a large population of myasthenia gravis (MG) patients from a single center was evaluated. Our survey included 984 patients, 904 with anti-acetylcholine receptor antibodies and 80 with anti-muscle specific kinase antibodies. The anti-acetylcholine receptor positive population included patients with early-onset (age at onset ≤50 years), late-onset and thymoma-associated disease. Follow-up ranged 2-40 years. Two-hundred and fourteen ADs were diagnosed in 185 patients; 26 of them had two or more ADs in association with MG. Thyroid disorders were the most common and, together with vitiligo and thrombocytopenia, occurred in all disease subsets. Otherwise, there was a broad variability with partial overlap among patient groups. The highest rate of ADs was observed in early-onset patients, while clusters, i.e. 2 or more ADs other than MG in the same individual, were more common among thymoma cases. Thirty-four diseases were diagnosed at the same time, 88 occurred before and 92 after the onset of MG. On multivariate analysis, immunosuppressive treatment was the only independent variable which negatively influenced the risk of developing other ADs in our cohort.

Myasthenia gravis is a chronic, autoimmune, neuromuscular disease characterized by fluctuating weakness of voluntary muscle groups. Although genetic factors are known to play a role in this neuroimmunological condition, the genetic etiology underlying myasthenia gravis is not well understood.

Myasthenia gravis (MG) is a prototypical antibody-mediated disease characterized by muscle weakness and fatigability. Serum antibodies to the acetylcholine receptor and muscle-specific tyrosine kinase receptor (MuSK) are found in about 85% and 8% of patients respectively. We have previously shown that more than 70% of MG patients with MuSK antibodies share the HLA DQ5 allele. The aim of the present study was to analyze the T cell receptor (TCR) repertoire specific for recombinant human MuSK protein. We used the CDR3 TRBV-TRBJ spectratyping (immunoscope) to analyze the T cell response to MuSK from 13 DQ5+ MuSK-MG patients and from 7 controls (six DQ5+ MuSK negative subjects and one DQ5- DQ3+ MuSK positive patient). DQ5+ MuSK-MG patients but not controls used a restricted set of TCR VJ rearrangements in response to MuSK stimulation. One semiprivate (TRBV29-TRBJ2.5) rearrangement was found in 5/13 patients, while 4 other semiprivate (one in TRBV28-TRBJ2.1 and in TRBV3-TRBJ1.2, and two in TRBV28-TRBJ1.2) rearrangements were differently shared by 4/13 patients each and were absent in controls. When we sequenced the TRBV29-TRBJ2.5 rearrangement, we obtained 26 different sequences of the expected 130 bp length from 117 samples of the 5 positive patients: two common motifs GXGQET/TEHQET were shared in 4 patients as semiprivate motifs. Thus, the MuSK-specific T-cell response appears to be restricted in DQ5+ MuSK-MG patients, with a semiprivate repertoire including a common motif of TRBV29. This oligoclonal restriction of T cells will allow the identification of immunodominant epitopes in the antigen, providing therefore new tools for diagnosis and targeted therapy.

The skeletal muscle has been long viewed as a site of election for immunization due to the sustained stay of the antigens in this tissue. Besides a passive function during immune priming, however, recent studies have shed light on an active role of muscle cells (fibers, satellite cells, and stromal cells) in inflammatory and immune response. In this context, the chemical nature of the antigen (into tissues and cells?) and the choice of adjuvants contribute to determine the fate of the antigen itself and the resulting immune response. In particular, this applies to modern vaccine approaches that involve the use of nucleic acid to locally synthesize the protein antigen. Based on these novel concepts, the possibility of manipulating therapeutically the mechanisms of peripheral tolerance at the level of muscle tissue will be discussed in the light of the recent literature.

Increasing evidence suggests a contribution of epigenetic processes in promoting cancer and autoimmunity. Myasthenia gravis (MG) is an autoimmune disease mediated, in approximately 80% of the patients, by antibodies against the nicotinic acetylcholine receptor (AChR+). Moreover, epithelial tumours (thymomas) are present in about 10-20% of the patients, and there is indication that changes in DNA methylation might contribute to the risk and progression of thymomas. However, the role of epigenetics in MG is still not completely clarified. In the present study we investigated if a common polymorphism (-579G>T: rs1569686) in the promoter of the DNMT3B gene coding for the DNA methyltransferase 3B, an enzyme that mediates DNA methylation, increases the risk to develop MG or MG-associated thymomas. The study polymorphism was selected based on recent reports and a literature meta-analysis suggesting association with increased risk of various types of cancer. We screened 324 AChR+ MG patients (140 males and 184 females, mean age 56.0 ± 16.5 years) and 735 healthy matched controls (294 males and 441 females, mean age 57.3 ± 15.6 years). 94 of the total MG patients had a thymoma. While there was no association with the whole cohort of MG patients, we found a statistically significant association of the DNMT3B-579T allele (OR = 1.51; 95% CI=1.1-2.1, P = 0.01) and the TT homozygous genotype (OR = 2.59; 95% CI=1.4-4.9, P = 0.006) with the risk of thymoma. No association was observed in MG patients without thymoma, even after stratification into clinical subtypes. Present results suggest that the DNMT3B-579T allele might contribute to the risk of developing thymoma in MG patients, particularly in homozygous TT subjects.

Interleukin (IL)-6 is a multifunctional cytokine with a critical role in inflammatory, immunoregulatory and haemopoietic responses. Its receptor consists of an ubiquitously expressed membrane transducing element (gp130) and of the specific element IL-6R-alpha (gp80), present only on hepatocytes and some leukocyte subsets. IL-6R-alpha also exists as soluble protein (sIL-6R) that, in the presence of IL-6, forms a complex able to bind gp130 and, thanks to the mechanism called trans-signaling, transduces IL-6 effect through tyrosine phosphorylation and activation of the signal transducer and transcription activator (STAT)-3. The aim of this study was to analyze the bidirectional relationships between platelet aggregation and IL-6-dependent effects. While platelets do not produce IL-6, we found that resting platelets express gp130, but not gp80, on their membranes. Upon activation by thrombin or calcium ionophore A23187, but not by ADP, the IL-6R-alpha is released in soluble form, while cangrelor, the specific inhibitor of P2Y12 receptor, can partially inhibit sIL-6R release. This sIL-6R is biologically active and, in the presence of IL-6, can trigger IL-6 trans-signaling, inducing an autocrine activation loop (as measured by an increase in gp80 and gp130 content) and STAT3 phosphorylation. On the other hand, IL-6 trans-signaling has no effect on platelet degranulation or aggregation by itself, nor on thrombin-induced platelet aggregation. Our data add an important piece to the puzzle of thrombosis and inflammation: in the presence of IL-6, which can be produced by stressed endothelial cells, the platelet-derived IL-6 trans-signaling could be crucial for the evolution of inflammation within a damaged vessel.

Myasthenia gravis with antibodies to muscle-specific tyrosine kinase (MuSK-MG) is generally considered a severe disease because of the associated weakness distribution with prevalent involvement of bulbar muscles and a rapidly progressive course and early respiratory crises. Its treatment can be unrewarding, owing to poor response to acetylcholinesterase inhibitors in most patients, disease relapses in spite of high-dose immunosuppression, and development of permanent bulbar weakness. High-dose prednisone plus plasma exchange is the recommended approach for treating rapidly progressive bulbar weakness. In the disease management, oral steroids proved effective, plasma exchange produced marked, albeit short-term, improvement, while conventional immunosuppressants were comparatively less effective. Rituximab is a promising treatment for refractory MuSK-MG; in uncontrolled studies, nearly all treated patients achieved significant improvement with substantial decrease of medication. It is yet to be clarified whether the early use of rituximab could prevent the permanent bulbar weakness, which constitutes a relevant disability in these patients.

The skeletal muscles are the major living component of the human body. They are constituted by stable cells, the myofibres, and by adult multipotent stem cells, the satellite cells, which can multiply to regenerate and repair the damaged tissues. Injections of DNA in muscle cells have been used to produce recombinant proteins with opposite goals: somatic reparation of genetic defects, which needs to elicit no inflammatory or immune response, and DNA vaccination, which needs a robust immune response. Because of possible therapeutical interventions, a growing body of information is being produced dealing with every aspect of the myofibres during inflammatory and autoimmune responses: skeletal muscle-antigen presenting cell (APC) interaction and intrinsic APC capabilities of myoblasts and myocytes, the response to released cytokines and their endogenous production, the regulation of Toll-like receptors and major histocompatibility complex expression. According to these data, the muscle tissue is now emerging no longer as a passive bystander, but more as an active player that, when correctly manipulated, can drive tolerance or immunization to these de novo produced proteins. In the present review, we summarize the recent developments on the control of muscle immune function.

We evaluated HLA-DRB1*, DQA1*, and DQB1* allele profile in MuSK-MG in comparison with a control population and non-thymoma early onset AChR-MG (AChR-EOMG). We confirm the strong positive association of AChR-EOMG with DRB1*03 DQA1*0501 and DQB1*0201 alleles as previously reported. MuSK-MG appears to be strongly associated with DRB1*16 and DQB1*0502 alleles.