A modified application of the luciferase immunoprecipitation systems for detecting antibodies to the G protein-coupled receptors

Background:　When multipass transmembrane molecules are located on the cell surface, there may be interaction with notonly bioactive molecules but also pathogenic molecules in areas protruding outside the cell. In antibody-mediated autoimmunedisorders, it has been found that the autoantibodies occasionally attack membrane molecules on the cell surface, thus causingthe disease such as myasthenia gravis. In such cases, highly sensitive autoantibody detection technology is required for earlydiagnosis. However, autoantibody analysis technology that is specialized for membrane molecules is still under development.Here we demonstrate a novel method for detecting of antibodies against the extracellular portions of multipass transmembranemolecules.Methods:　Antibodies for muscarinic acetylcholine receptor type3 (M3R) were detected with two kinds of luciferase immunoprecipitaionsystems (LIPS), conventional LIPS (cLIPS) and its modified application, termed modified LIPS (mLIPS). In mLIPS, antibodiesagainst extracellular portions of membrane molecules could be preferentially detected.Results:　An antibody to the amino-terminal portion of human M3R was detected with modified LIPS with a high sensitivity. Incontrast, an antibody to the carboxyl-terminal portion was not detected with mLIPS, because it did not interact with intracellularportions of M3R in living cells. We also found antibodies for M3R in a patient serum with Sjogren’s syndrome.Conclusion:　Our technology has a promising future, and we hope that it will be applied in the analysis of antibodies against adiverse range of multipass transmembrane molecules, including GPCRs

Detecting gastrointestinal manifestations in patients with systemic sclerosis using anti-gAChR antibodies

Background: Patients with systemic sclerosis (SSc) complicated by gastrointestinal dysmotility are difficult to treat and have high mortality. To clarify the pathogenesis of gastrointestinal manifestations, we aimed to demonstrate the association among the clinical features of SSc, the serological markers, the autoantibodies against nicotinic acetylcholine receptor at autonomic ganglia (gAChR). Methods: Fifty patients were enrolled and divided into two groups according to the presence or absence of gastrointestinal manifestations, and the characteristics were analyzed between these two groups. We measured biomarkers and the autoantibodies against two gAChRα3 and β4 subunits to test sera samples. Furthermore, patients were classified based on the presence or absence of anti-gAChR autoantibodies, and their clinical features were compared. Results: In patients with SSc and gastrointestinal manifestations, digital ulcers were more frequent (p = 0.050) and VEGF expression was significantly higher (p = 0.038). Seven subjects with SSc were seropositive for α3 subunit, whereas one patient was seropositive for β4 subunit. The mean level of anti-gAChRα3 autoantibodies in SSc patients with gastrointestinal manifestations was significantly higher than that in SSc patients without gastrointestinal manifestations (p = 0.001). The group of patients with SSc and gAChR autoantibodies had significantly higher endostatin levels (p = 0.046). Conclusions: This study is the first to demonstrate that clinical characteristics of SSc patients with seropositivity for gAChR autoantibodies. Patients with SSc have circulating autoantibodies against gAChR, which may contribute to gastrointestinal manifestations associated with this disease, suggesting that gAChR-mediated autonomic neurotransmission may provide a pathomechanism for gastrointestinal dysmotility in SSc

Intravenous cyclophosphamide treatment for systemic lupus erythematosus with severe autonomic disorders confirmed by head-up tilt table test: A case series

Autonomic disorders are common in patients with systemic lupus erythematosus (SLE), but the therapeutic strategy and methods for evaluating the effects of therapy have not been established. We describe the three cases of SLE patients who developed severe autonomic disorders as demonstrated by the head-up tilt table test (HUT). All three patients were treated by intensive immunosuppressive treatments including intravenous cyclophosphamide (IVCY); their HUT results all became negative. Our cases suggest that IVCY treatment can be a good therapeutic option for severe autonomic disorders in SLE patients. The HUT is a useful objective method for the diagnosis of and the evaluation of longitudinal therapeutic effects on autonomic disorders in SLE patients with orthostatic intolerance

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

Clinical Features of Autoimmune Autonomic Ganglionopathy and the Detection of Subunit-Specific Autoantibodies to the Ganglionic Acetylcholine Receptor in Japanese Patients

Autoimmune autonomic ganglionopathy (AAG) is a rare acquired channelopathy that is characterized by pandysautonomia, in which autoantibodies to ganglionic nicotinic acetylcholine receptors (gAChR) may play a central role. Radioimmunoprecipitation (RIP) assays have been used for the sensitive detection of autoantibodies to gAChR in the serum of patients with AAG. Here, we developed luciferase immunoprecipitation systems (LIPS) to diagnose AAG based on IgGs to both the α3 and β4 gAChR subunits in patient serum. We reviewed the serological and clinical data of 50 Japanese patients who were diagnosed with AAG. With the LIPS testing, we detected anti-α3 and -β4 gAChR antibodies in 48% (24/50) of the patients. A gradual mode of onset was more common in the seropositive group than in the seronegative group. Patients with AAG frequently have orthostatic hypotension and upper and lower gastrointestinal tract symptoms, with or without anti-gAChR. The occurrence of autonomic symptoms was not significantly different between the seropositive and seronegative group, with the exception of achalasia in three patients from the seropositive group. In addition, we found a significant overrepresentation of autoimmune diseases in the seropositive group and endocrinological abnormalities as an occasional complication of AAG. Our results demonstrated that the LIPS assay was a useful novel tool for detecting autoantibodies against gAChR in patients with AAG

A modified application of the luciferase immunoprecipitation systems for detecting antibodies to the G protein-coupled receptors

Background:　When multipass transmembrane molecules are located on the cell surface, there may be interaction with not only bioactive molecules but also pathogenic molecules in areas protruding outside the cell. In antibody-mediated autoimmune disorders, it has been found that the autoantibodies occasionally attack membrane molecules on the cell surface, thus causing the disease such as myasthenia gravis. In such cases, highly sensitive autoantibody detection technology is required for early diagnosis. However, autoantibody analysis technology that is specialized for membrane molecules is still under development. Here we demonstrate a novel method for detecting of antibodies against the extracellular portions of multipass transmembrane molecules. Methods:　Antibodies for muscarinic acetylcholine receptor type3 (M3R) were detected with two kinds of luciferase immunoprecipitaion systems (LIPS), conventional LIPS (cLIPS) and its modified application, termed modified LIPS (mLIPS). In mLIPS, antibodies against extracellular portions of membrane molecules could be preferentially detected. Results:　An antibody to the amino-terminal portion of human M3R was detected with modified LIPS with a high sensitivity. In contrast, an antibody to the carboxyl-terminal portion was not detected with mLIPS, because it did not interact with intracellular portions of M3R in living cells. We also found antibodies for M3R in a patient serum with Sjögren’s syndrome. Conclusion:　Our technology has a promising future, and we hope that it will be applied in the analysis of antibodies against a diverse range of multipass transmembrane molecules, including GPCRs

Postural Orthostatic Tachycardia in a Patient with Type 2 Diabetes with Diabetic Neuropathy

24-year-old Japanese man with type 2 diabetes mellitus and diabetic neuropathy was admitted to our ward to evaluate the cause of orthostatic intolerance. During a head-up tilt test, his heart rate increased from 105 to 155 beats/minute within 3 minutes, and chest discomfort began. He was diagnosed with postural orthostatic tachycardia syndrome (POTS), and orthostatic intolerance disappeared after β-blocker treatment. Scintigraphy using123I-metaiodobenzylguanidine showed decreased cardiac uptake. Power spectral analysis of heart rate variability for 24 hours in Holter electrocardiography demonstrated decreases in both sympathetic and parasympathetic nervous system activities, with a greater decrease in parasympathetic activity than sympathetic activity. The relative sympathetic hyperactivity in the present patient with diabetic neuropathy seemed to be related to POTS