Flow Cytofluorimetric Analysis of Anti-LRP4 (LDL Receptor-Related Protein 4) Autoantibodies in Italian Patients with Myasthenia Gravis.

BACKGROUND: 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. METHODS: The aim of this study was to test the presence and diagnostic significance of anti-LRP4 autoantibodies in an Italian population of 101 myasthenic patients (55 dSN, 23 AChR positive and 23 MuSK positive), 45 healthy blood donors and 40 patients with other neurological diseases as controls. All sera were analyzed by a cell-based antigen assay employing LRP4-transfected HEK293T cells, along with a flow cytofluorimetric detection system. RESULTS: We found a 14.5% (8/55) frequency of positivity in the dSN-MG group and a 13% frequency of co-occurrence (3/23) in both AChR and MuSK positive patients; moreover, we report a younger female prevalence with a mild form of disease in LRP4-positive dSN-MG individuals. CONCLUSION: Our data confirm LRP4 as a new autoimmune target, supporting the value of including anti-LRP4 antibodies in further studies on Myasthenia gravis.

A Study of Inflammatory/Necrosis Biomarkers in the Fracture of the Femur Treated with Proximal Femoral Nail Antirotation.

Pertrochanteric fractures are common injuries in adults and source of morbidity and mortality among the elderly. Different surgical techniques were recommended for their treatment but undoubtedly they add an additional inflammatory trauma along the fracture itself. Many attempts to quantify the degree of approach-related trauma are carried out through measurements of systemic inflammatory parameters. In this study we prospectively analyzed laboratory data of 20 patients over eighty with pertrochanteric fracture of the femur treated with proximal femoral nail antirotation (PFNA). This is an excellent device for osteosynthesis because it can be easily and quickly inserted by a mini-incision providing stable fixation and early full mobilization. Serum tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), and plasma creatin kinase (CK) were evaluated 1 hour preoperatively and 24 hours postoperatively. Our results show that PFNA did not induce significant increments in serum levels of inflammatory cytokines TNF-α and IL-6; CRP was elevated preoperatively in correlation with waiting time for surgery; CRP and CK showed a significant increment in the first postoperatory day; CK increment was correlated with surgical time length. We conclude that, for the markers we analyzed, PFNA shows a low biomechanical-inflammatory profile that represents an advantage over other techniques.

Poly-autoimmunity in patients with myasthenia gravis: A single-center experience.

We evaluated the co-occurrence of autoimmune diseases (ADs) in a large population of myasthenia gravis (MG) patients from a single center. 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.

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.

A genome-wide association study of myasthenia gravis.

IMPORTANCE: 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. OBJECTIVE: To identify genetic variants that alter susceptibility to myasthenia gravis, we performed a genome-wide association study. DESIGN, SETTING, AND PARTICIPANTS: DNA was obtained from 1032 white individuals from North America diagnosed as having acetylcholine receptor antibody-positive myasthenia gravis and 1998 race/ethnicity-matched control individuals from January 2010 to January 2011. These samples were genotyped on Illumina OmniExpress single-nucleotide polymorphism arrays. An independent cohort of 423 Italian cases and 467 Italian control individuals were used for replication. MAIN OUTCOMES AND MEASURES: We calculated P values for association between 8,114,394 genotyped and imputed variants across the genome and risk for developing myasthenia gravis using logistic regression modeling. A threshold P value of 5.0×10(-8) was set for genome-wide significance after Bonferroni correction for multiple testing. RESULTS: In the overall case-control cohort, we identified association signals at CTLA4 (rs231770; P=3.98×10(-8); odds ratio, 1.37; 95% CI, 1.25-1.49), HLA-DQA1 (rs9271871; P=1.08×10(-8); odds ratio, 2.31; 95% CI, 2.02-2.60), and TNFRSF11A (rs4263037; P=1.60×10(-9); odds ratio, 1.41; 95% CI, 1.29-1.53). These findings replicated for CTLA4 and HLA-DQA1 in an independent cohort of Italian cases and control individuals. Further analysis revealed distinct, but overlapping, disease-associated loci for early- and late-onset forms of myasthenia gravis. In the late-onset cases, we identified 2 association peaks: one was located in TNFRSF11A (rs4263037; P=1.32×10(-12); odds ratio, 1.56; 95% CI, 1.44-1.68) and the other was detected in the major histocompatibility complex on chromosome 6p21 (HLA-DQA1; rs9271871; P=7.02×10(-18); odds ratio, 4.27; 95% CI, 3.92-4.62). Association within the major histocompatibility complex region was also observed in early-onset cases (HLA-DQA1; rs601006; P=2.52×10(-11); odds ratio, 4.0; 95% CI, 3.57-4.43), although the set of single-nucleotide polymorphisms was different from that implicated among late-onset cases. CONCLUSIONS AND RELEVANCE: Our genetic data provide insights into aberrant cellular mechanisms responsible for this prototypical autoimmune disorder. They also suggest that clinical trials of immunomodulatory drugs related to CTLA4 and that are already Food and Drug Administration approved as therapies for other autoimmune diseases could be considered for patients with refractory disease.

T cell repertoire in DQ5-positive MuSK-positive myasthenia gravis patients.

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.

PTPN22 and myasthenia gravis: replication in an Italian population and meta-analysis of literature data.

Polymorphisms in PTPN22 are associated with many autoimmune diseases; while rs2476601 is supposed to play a major role, other experimental data suggest that rs2488457 may be even more important. Results in myasthenia gravis are controversial. In 356 Italian myasthenic patients and 439 controls genotyped for both polymorphisms, we found that rs2476601 was not associated with myasthenia, presence of autoantibodies, thymus pathology, sex or onset age unlike previous studies on other European populations (confirmed by the present meta-analysis). On the other hand, while rs2488457 was not associated with myasthenia or thymus pathology, we found a correlation of rs2488457 with low autoantibody titers and a trend of association with a less severe disease. Both polymorphisms were in tight linkage disequilibrium in controls, not in patients. Our results suggest that SNPs in this gene different from rs2476601, and/or epigenetic interactions, could play a greater role.

Anti-acetylcholinesterase antibodies associate with ocular myasthenia gravis.

In MG, anti-AChR or anti-MuSK abs impair neuromuscular transmission. Partial inhibition of AChE can ameliorate symptoms, while a complete block causes a cholinergic blockade. We found anti-AChE abs in 115/240 MG patients, with no correlation with sex, age at onset, thymus pathology, presence of anti-AChR or anti-MuSK antibodies. We found a correlation with the ocular form of the disease, and with milder forms of MG not requiring immunosuppressants; moreover, when we considered only those patients who were off AChEI therapy, we found that ocular patients were positive for anti-AChE abs, while generalized patients were negative. According to an experimental model, we hypothesize that anti-AChE abs could contribute to ptosis through an inhibition of the sympathetic innervation of the tarsal muscle.

Response to therapy in myasthenia gravis with anti-MuSK antibodies.

Myasthenia gravis (MG) with antibodies against the muscle-specific tyrosine kinase (MuSK abs) is often a severe disease requiring aggressive treatment. Various immunosuppressive (IS) regimens have been employed; the efficacy of plasma exchange is unanimously recognized, while the indication for thymectomy is controversial. We evaluated the response to therapy in 57 MuSK-positive patients (12 M/45 F) comparing our experience with other authors' results. Disease severity and response to treatment were graded according to MG Foundation of America; follow-up ranged from 0.5-29 years. Owing to both MG severity and the unsatisfactory response to cholinesterase inhibitors, most patients (54/57) needed IS treatment, and 35 received one or more courses of plasma exchange and intravenous immunoglobulin. At the end of follow-up, the rate of complete remission was 8.8%, and IS treatment had been withdrawn in only 10/54 patients. The extent of therapeutic response varied considerably. With conventional IS therapy (prednisone alone or in combination with azathioprine or cyclosporine), most patients achieved good control of their disease, but 30% of them were left with permanent facial and bulbar weakness. In patients with refractory disease, the use of mycophenolate mofetil and rituximab proved very effective, as also reported by other authors. In our and others' experience, MuSK-positive MG markedly improves with IS therapy, although, in comparison with the AChR-positive disease, it is characterized by a lower remission rate, as a higher proportion of patients remain dependent on treatment. Thymectomy is mostly considered scarcely effective; however, at present, no firm conclusions can be drawn on its role in the treatment of this form of MG.

IL-6 regulates MCP-1, ICAM-1 and IL-6 expression in human myoblasts.

The inflammatory reaction in autoimmune polymyositis and rejection of transplanted myoblasts is characterized by mononuclear cell infiltration. In other settings monocytes are locally recruited by an IL-6-induced IL-8-to-MCP-1 switch. IL-6, upon binding to soluble gp80 (sIL-6R), can interact with membrane-bound ubiquitously expressed gp130 and activate virtually all cells (transsignaling). We found that human myoblasts could use transsignaling to produce IL-6, MCP-1 and ICAM-1; the addition of sIL-6R, binding to IL-1beta-induced IL-6, greatly increases IL-6 production. These in vitro data support the hypothesis that locally secreted IL-6 can target monocyte chemotaxis and leukocytes trafficking through an IL-6, MCP-1 and ICAM-1 modulation.

IL-6-deficient mice show impaired inflammatory response in a model of myosin-induced experimental myositis.

Inflammatory/immune reactions against muscle cells are responsible for the damage in idiopathic inflammatory myopathies. We investigated the role of IL-6, a cytokine known to contribute to local leukocyte accumulation, in a model of myosin-induced experimental myositis. After injection of rabbit myosin in CFA/pertussis toxin, normal mice develop clinically evident muscle deficit and damage, as demonstrated by myofiber necrosis and leukocyte infiltration, while IL-6-deficient mice have no clinical or histological signs of muscle damage. This study evidences that selective deficiency of IL-6 directly or indirectly hinders the local inflammatory response and its harmful effects in this model of muscle damage.

Clinical correlates with anti-MuSK antibodies in generalized seronegative myasthenia gravis.

The term seronegative myasthenia gravis (SNMG) refers to the generalized disease without detectable anti-acetylcholine receptor (anti-AChR) antibodies. In these patients, IgG antibodies against the muscle-specific kinase (MuSK) have been described, which reduced agrin-induced AChR clustering in vitro. We have assayed anti-MuSK antibodies in 78 patients with SNMG, who have been followed for many years in our Institution. Here we describe the clinical phenotype of the 37 patients whose results were positive on this assay. MG with anti-MuSK antibodies was characterized by a striking prevalence of female patients (eight men and 29 women). Age of onset ranged from 6 to 68 years, with 56.8% of patients presenting under 40 years of age. All these patients shared a similar pattern of muscle weakness, with prevalent involvement of cranial and bulbar muscles and a high frequency of respiratory crises; the involvement of limb muscles was comparatively less severe and inconsistent. Single-fibre-EMG confirmed the most sensitive examination in the EMG diagnosis of MuSK-positive disease, while, owing to weakness topography, repetitive nerve stimulation in limb muscles was diagnostic in 56.8% of cases. The effect of edrophonium (or neostigmine) injection was equivocal or negative in 11 of 37 patients (29.7%), and the response to oral pyridostigmine was even more unsatisfactory, ranging from mild benefit to overt intolerance. In thymectomized patients, thymus was normal for age or atrophied, and no benefit from surgery was noticed. Thirty-five of 37 patients were given immunosuppressive therapy and 22 received plasma-exchange. The course of the disease was often characterized by periodic exacerbation phases requiring hospitalization and even assisted ventilation; plasma-exchange produced marked improvement in these cases. At the end of the observation period, most patients, although improved, were still symptomatic, having developed permanent facial and pharyngeal weakness together with some atrophy of facial muscles. MuSK-negative disease was comparatively more heterogeneous. Most patients were affected with mild to moderate symptoms and responded well to pharmacological treatment; however, a few subjects in this group had severe refractory disease, poorly responsive to both acetylcholinesterase inhibitors and immunosuppressants.

TGF-beta 1 and IL-10 modulate IL-1 beta-induced membrane and soluble ICAM-1 in human myoblasts.

We have previously shown that interleukin (IL)-1 beta and other inflammatory cytokines are able to induce the expression of membrane and soluble intercellular adhesion molecule (ICAM)-1 on human myoblasts. In this paper we found that IL-10 and transforming growth factor (TGF)-beta 1 are able to prevent IL-1 beta-induced membrane and soluble ICAM-1 protein expression on human myoblasts, with different time courses. The effect of both cytokines is associated to a reduction in ICAM-1 mRNA. Our findings suggest that IL-10 and TGF-beta 1 are able to influence the inflammatory process in muscle tissue at least in part by means of control of membrane and soluble ICAM-1.

Anti-p110 autoantibodies identify a subtype of "seronegative" myasthenia gravis with prominent oculobulbar involvement.

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness and pathogenetic autoantibodies directed against the nicotinic acetylcholine receptor (seropositive myasthenia gravis; SPMG). Nearly 15% to 20% of MG patients do not have these antibodies (seronegative myasthenia gravis; SNMG), but several evidence indicate that these patients have circulating pathogenic autoantibodies directed against other muscle antigens. Using the TE671 rhabdomyosarcoma cell line as an antigen source, we analyzed sera from 63 SNMG and 26 SPMG patients and 26 healthy blood donors by FACS analysis. We found that 40 of 63 SNMG patients and only 1 of 26 SPMG patients had IgG binding to the TE671 cell line. None of the sera bound to the unrelated MRC5 cell line. To identify the antigen, we analyzed sera immunoreactivity in more detail by immunoprecipitation of biotinylated membrane proteins from TE671 cells. When the immunoprecipitated proteins were separated by SDS-PAGE electrophoresis and then transferred to nitrocellulose membranes, we found that SNMG IgG identify a band corresponding to a protein with a molecular weight of 110 kDa (P110), which is not recognized by seropositive MG sera. This anti-P110 immunoreactivity is significantly associated with a distinct clinical picture characterized by a prominent involvement of ocular and bulbar muscles, with frequent respiratory problems (p < 0.005), and is recognized by a specific antimuscle specific kinase (MuSK) antiserum. In a recent article, the presence of anti-MuSK antibodies was described in SNMG. Our results confirm the presence of these antibodies in SNMG and suggest that anti-P110/MuSK autoantibodies identify a subtype of SNMG in which the different pathogenesis induces the distinct clinical picture.

Prolactin/cortisol ratio in rheumatoid arthritis.

Prolactin (PRL) and glucocorticoids are hormones involved in the regulation of the immune system. Rheumatoid arthritis (RA) is an inflammatory condition that presents a diurnal rhythm of disease activity. PRL/cortisol ratio, and IL-1beta and TNF-alpha levels were determined in patients with RA and in control subjects at 0600, 1000, 1400, 1800, 2200, and 0200 hours. In patients with RA we observed higher PRL/cortisol ratio at 0200 hours, whereas IL-1beta and TNF-alpha reached their highest serum levels at 0200 and 0600 hours. In patients with RA we observed an imbalance in favor of proinflammatory hormones as opposed to levels of antiinflammatory hormones during nocturnal hours together with increased levels of IL-1beta and TNF-alpha of the diurnal rhythm of disease activity.