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Schematic representation of the acetylcholine receptor (AChR) α3-Gaussia luciferase (GL) 8990. For the ganglionic AChR (gAChR)-LIPS assay, human embryonic kidney (HEK) 293 cells were transfected with an expression plasmid for the gAChRα3 or β4-GL reporter.
Source publication
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 pati...
Citations
... Very rarely, a7-nAChR antibodies have been reported in Rasmussen's encephalitis, schizophrenia (13) and in some myasthenia gravis patients (14)(15)(16)(17). Regarding a4b2and a7-nAChR autoimmunity, there is one study showing the presence of such antibodies in a patient with AAG and late-onset encephalopathy (18) and non-encephalopathic seropositive AAG patients have been identified to possess antibodies against the a4 subunit of nAChRs (19,20). ...
Background
Neuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the central nervous system (CNS), playing critical roles in brain function. Antigenicity of nAChRs has been well demonstrated with antibodies to ganglionic AChR subtypes (i.e., subunit α3 of α3β4-nAChR) and muscle AChR autoantibodies, thus making nAChRs candidate autoantigens in autoimmune CNS disorders. Antibodies to several membrane receptors, like NMDAR, have been identified in autoimmune encephalitis syndromes (AES), but many AES patients have yet to be unidentified for autoantibodies. This study aimed to develop of a cell-based assay (CBA) that selectively detects potentially pathogenic antibodies to subunits of the major nAChR subtypes (α4β2- and α7-nAChRs) and its use for the identification of such antibodies in “orphan” AES cases.
Methods
The study involved screening of sera derived from 1752 patients from Greece, Turkey and Italy, who requested testing for AES-associated antibodies, and from 1203 “control” patients with other neuropsychiatric diseases, from the same countries or from Germany. A sensitive live-CBA with α4β2-or α7-nAChR–transfected cells was developed to detect antibodies against extracellular domains of nAChR major subunits. Flow cytometry (FACS) was performed to confirm the CBA findings and indirect immunohistochemistry (IHC) to investigate serum autoantibodies’ binding to rat brain tissue.
Results
Three patients were found to be positive for serum antibodies against nAChR α4 subunit by CBA and the presence of the specific antibodies was quantitatively confirmed by FACS. We detected specific binding of patient‐derived serum anti‐nAChR α4 subunit antibodies to rat cerebellum and hippocampus tissue. No serum antibodies bound to the α7-nAChR-transfected or control-transfected cells, and no control serum antibodies bound to the transfected cells. All patients positive for serum anti‐nAChRs α4 subunit antibodies were negative for other AES-associated antibodies. All three of the anti‐nAChR α4 subunit serum antibody-positive patients fall into the AES spectrum, with one having Rasmussen encephalitis, another autoimmune meningoencephalomyelitis and another being diagnosed with possible autoimmune encephalitis.
Conclusion
This study lends credence to the hypothesis that the major nAChR subunits are autoimmune targets in some cases of AES and establishes a sensitive live-CBA for the identification of such patients.
... Our institution established the detection system of gnAChR α3 and β4 antibodies by a luciferase immunoprecipitation system (LIPS) assay in 2011, and since 2012, we have responded to requests for assays using serum samples from patients with any autonomic symptoms who visited a hospital in Japan [8,9]. We examined 1787 serum samples of 1381 patients with any autonomic symptoms who visited teaching and general hospitals throughout Japan between January 2012 and August 2018 [8,9]. ...
... Our institution established the detection system of gnAChR α3 and β4 antibodies by a luciferase immunoprecipitation system (LIPS) assay in 2011, and since 2012, we have responded to requests for assays using serum samples from patients with any autonomic symptoms who visited a hospital in Japan [8,9]. We examined 1787 serum samples of 1381 patients with any autonomic symptoms who visited teaching and general hospitals throughout Japan between January 2012 and August 2018 [8,9]. The serum samples were centrifuged at 3000 rpm for 10 min and were then stored in cryovial tubes at −80 °C within 2 h of collection. ...
... In the present study, we detected serum gnAChRα3 and gnAChRβ4 antibodies using the LIPS assay [9]. A National Institutes of Health group previously developed this efficient quantitative approach for the analysis of antibodies against human autoantigens in serum samples from patients [10,11]. ...
Background: Functional gastrointestinal disorders (FGIDs), including functional dyspepsia (FD) and irritable bowel syndrome (IBS), are characterized by chronic and recurrent gastrointestinal symptoms. Clinically, FD and IBS often resemble gastrointestinal dysmotility caused by autoimmune autonomic neuropathy. We examined the seropositive frequency of autoantibodies against ganglionic nicotinic acetylcholine receptors (gnAChRs) in patients presenting with FGIDs. Objective: To elucidate the seropositivity of gnAChR antibodies and the clinical features of seropositive FD and IBS. Materials and Methods: We measured autoantibodies against the gnAChR α3 and β4subunits using luciferase immunoprecipitation systems. Serum samples from patients with any autonomic symptoms were obtained from hospitals in Japan between January 2012 and August 2018 (1787 serum samples of 1381 patients). We selected FD and IBS patients and compared the clinical characteristics and prevalence of autonomic symptoms between those with seropositive and seronegative IBS and FD. Results: Nine IBS and two FD cases (one comorbid case with IBS) were found. We found four patients (36.4%) in whom gnAChR antibodies were positive in these eleven patients. Sicca symptoms were observed in three of four cases (75%) of seropositive FGID compared with zero of seven cases (0%) of seronegative FGID. Conclusions: We found patients with gnAChR antibodies in FD and IBS patients. These data will be valuable for elucidating the pathophysiology of these FGIDs and developing new treatment strategies.
... The Mayo Clinic, in particular, has been at the forefront of research at both the clinical and basic levels [3][4][5][6][7][8][9]. Based on their studies, we established a detection system for autoantibodies to gAChR in Japan and have reported on the clinical features of patients with AAG in the country [10,11]. Most recently, we established a murine model of human AAG by active immunization with a recombinant nAChR α3 subunit fusion protein based on cDNA encoding residues 1-205 of the human nAChR α3 [12]. ...
... Future studies should strive to improve the accuracy of diagnosis not only by testing for autonomic nervous system function, but also by combining pathology and imaging tests to confirm the presence or absence of Lewy bodies. We have noted the usefulness of MIBG myocardial scintigraphy as a tool for diagnosing and understanding the pathogenesis of AAG [10,11]. Among Japanese patients with AAG who underwent MIBG myocardial scintigraphy, 80% had a decreased cardiac uptake. ...
Autoimmune autonomic ganglionopathy (AAG) is a disease of autonomic failure caused by ganglionic acetylcholine receptor (gAChR) autoantibodies. Although the detection of autoantibodies is important for distinguishing the disease from other neuropathies that present with autonomic dysfunction, other factors are important for accurate diagnosis. Here, we provide a comprehensive review of the clinical features of AAG, highlighting differences in clinical course, clinical presentation, and laboratory findings from other neuropathies presenting with autonomic symptoms. The first step in diagnosing AAG is careful history taking, which should reveal whether the mode of onset is acute or chronic, followed by an examination of the time course of disease progression, including the presentation of autonomic and extra-autonomic symptoms. AAG is a neuropathy that should be differentiated from other neuropathies when the patient presents with autonomic dysfunction. Immune-mediated neuropathies, such as acute autonomic sensory neuropathy, are sometimes difficult to differentiate, and therefore, differences in clinical and laboratory findings should be well understood. Other non-neuropathic conditions, such as postural orthostatic tachycardia syndrome, chronic fatigue syndrome, and long COVID, also present with symptoms similar to those of AAG. Although often challenging, efforts should be made to differentiate among the disease candidates.
... in the ulnar nerve). Serum autoantibodies to gAChR were not detected in a luciferase immunoprecipitation system (LIPS) assay (Nakane et al., 2015). ...
... Luciferase immunoprecipitation system (LIPS). The predominant laboratory method for the detection of antibodies against ganglionic nAChRs, is radioimmunoprecipitation assay (RIPA) using radio-iodinated epibatidinelabeled α3β4 nAChRs [62]. Furthermore, Luciferase immunoprecipitation system (LIPS) is another method which detects antibodies to individually expressed α3 or β4 nAChR single subunit [62]. ...
... The predominant laboratory method for the detection of antibodies against ganglionic nAChRs, is radioimmunoprecipitation assay (RIPA) using radio-iodinated epibatidinelabeled α3β4 nAChRs [62]. Furthermore, Luciferase immunoprecipitation system (LIPS) is another method which detects antibodies to individually expressed α3 or β4 nAChR single subunit [62]. LIPS represents a nonradioactive alternative assay and offers the ability to screen many different antigens in a complex mixture in parallel, however, it detects single peptides/subunits from autoantigens which would not be expected to assemble conformationally [63]. ...
Nicotinic acetylcholine receptors (nAChRs) are widely expressed in many and diverse cell types, participating in various functions of cells, tissues and systems. In this review, we focus on the autoimmunity against neuronal nAChRs, the specific autoantibodies and their mechanisms of pathological action in selected autoimmune diseases. We summarize the current relevant knowledge from human diseases as well as from experimental models of autoimmune neurological disorders related to antibodies against neuronal nAChR subunits. Despite the well-studied high immunogenicity of the muscle nAChRs where autoantibodies are the main pathogen of myasthenia gravis, autoimmunity to neuronal nAChRs seems infrequent, except for the autoantibodies to the ganglionic receptor, the α3 subunit containing nAChR (α3-nAChR), which are detected and are likely pathogenic in Autoimmune Autonomic Ganglionopathy (AAG). We describe the detection, presence and function of these antibodies and especially the recent development of a cell-based assay (CBA) which, contrary to until recently available assays, is highly specific for AAG. Rare reports of autoantibodies to the other neuronal nAChR subtypes include a few cases of antibodies to α7 and/or α4β2 nAChRs in Rasmussen encephalitis, schizophrenia, autoimmune meningoencephalomyelitis, and in some myasthenia gravis patients with concurrent CNS symptoms. Neuronal-type nAChRs are also present in several non-excitable tissues, however the presence and possible role of antibodies against them needs further verification. It is likely that the future development of more sensitive and disease-specific assays would reveal that neuronal nAChR autoantibodies are much more frequent and may explain the mechanisms of some seronegative autoimmune diseases.
... Dysautonomia was diagnosed based on autonomic medical history, physical examination including postural variations in blood pressure, and/or neurological evaluation [10,24]. We examined anti-gAChR (gAChRα3 and gAChRβ4) antibodies using the luciferase immunoprecipitation system (LIPS) assay [25], anti-P/Q type voltage-gated calcium channel (VGCC) antibodies by an immunoprecipitation method [26], and autoantibody panels of paraneoplastic neurological syndrome (Mayo Medical Laboratories, Paraneoplastic Autoantibody Evaluation) using patient serum. Disease severity was graded according to the Common Terminology Criteria for Adverse Events version 5 (CTCAEv5). ...
Objective
The purpose of this study is to report the clinical characteristics of dysautonomia associated with immune checkpoint inhibitors (ICIs).
Methods
We reported two patients with autoimmune autonomic ganglionopathy (AAG) occurring as immune-related adverse events (irAEs). We also performed a review of previous case reports presenting dysautonomia during ICI therapy. Moreover, we conducted pharmacovigilance analyses using the US Food and Drug Administration Adverse Events Reporting System (FAERS) to investigate dysautonomia associated with ICI.
Results
Two patients in our care developed both AAG and autoimmune encephalitis following ICI therapy for lung cancers. We comprehensively reviewed 13 published cases (M:F = 11:2, mean onset age of 53 years) with ICI-associated dysautonomia including AAG (n = 3) and autonomic neuropathy (n = 10). Of these, ICI monotherapy was performed in seven and combination ICI use in six. In 6 of 13 patients, dysautonomia appeared within one month after the start of ICIs. Orthostatic hypotension was observed in 7 and urinary incontinence or retention in five. All patients except three showed gastrointestinal symptoms. Anti-ganglionic acetylcholine receptor antibodies were undetectable. All but two patients received immune-modulating therapy. Immuno-modulating therapy was effective in three patients with AAG and two patients with autonomic neuropathy, but ineffective in the others. Five patients died, of either the neurological irAE (n = 3) or cancer (n = 2). The pharmacovigilance analyses using FAERS showed that ipilimumab monotherapy and the combination of nivolumab and ipilimumab constituted significant risks for developing dysautonomia, consistent with the review of literature.
Conclusion
ICIs can cause dysautonomia including AAG, and autonomic neuropathy is a neurological irAE.
... . . Detection of anti-gAChR antibodies using a luciferase immunoprecipitation system Serum antibodies against gAChR α3 and β4 subunits were quantified using a LIPS assay (13, 19), in which specific antibodies are bound to a Gaussia luciferase-tagged antigen and the bound antibody is quantified by measuring luciferase activity with a luminometer. The results are expressed as relative luminescence units (RLU). ...
... More recently, 0% of 2,628 patients with non-AAG neurological diseases were positive for antibodies to α3-nAChR (22). These reports suggest that anti-gAChR antibodies are usually undetectable or present at very low levels in the serum (16,19,23). Although these reports used radioimmunoprecipitation assay (RIA) as the measurement method, it has been proven that RIA and LIPS show the similar performance to detect anti-gAChR antibody (19). ...
... These reports suggest that anti-gAChR antibodies are usually undetectable or present at very low levels in the serum (16,19,23). Although these reports used radioimmunoprecipitation assay (RIA) as the measurement method, it has been proven that RIA and LIPS show the similar performance to detect anti-gAChR antibody (19). The high prevalence of anti-gAChR antibodies among our FNSD/CD patient cohort (27.1%) prompted us to hypothesize that anti-gAChR antibodies may be directly associated with autonomic symptoms in these patients. ...
Objective
Autoimmune autonomic ganglionopathy (AAG) is a rare disorder characterized by autonomic failure associated with the presence of anti-ganglionic acetylcholine receptor (gAChR) antibodies; however, several studies have reported that individuals with anti-gAChR antibodies present with central nervous system (CNS) symptoms such as impaired consciousness and seizures. In the present study, we investigated whether the presence of serum anti-gAChR antibodies correlated with autonomic symptoms in patients with functional neurological symptom disorder/conversion disorder (FNSD/CD).
Methods
Clinical data were collected for 59 patients presenting with neurologically unexplained motor and sensory symptoms at the Department of Neurology and Geriatrics between January 2013 and October 2017 and who were ultimately diagnosed with FNSD/CD according to the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition. Correlations between serum anti-gAChR antibodies and clinical symptoms and laboratory data were analyzed. Data analysis was conducted in 2021.
Results
Of the 59 patients with FNSD/CD, 52 (88.1%) exhibited autonomic disturbances and 16 (27.1%) were positive for serum anti-gAChR antibodies. Cardiovascular autonomic dysfunction, including orthostatic hypotension, was significantly more prevalent (75.0 vs. 34.9%, P = 0.008), whereas involuntary movements were significantly less prevalent (31.3 vs. 69.8%, P = 0.007), among anti-gAChR antibody-positive compared with -negative patients. Anti-gAChR antibody serostatus did not correlate significantly with the frequency of other autonomic, sensory, or motor symptoms analyzed.
Conclusions
An autoimmune mechanism mediated by anti-gAChR antibodies may be involved in disease etiology in a subgroup of FNSD/CD patients.
... . . Detection of anti-gAChR antibodies using a luciferase immunoprecipitation system Serum antibodies against gAChR α3 and β4 subunits were quantified using a LIPS assay (13, 19), in which specific antibodies are bound to a Gaussia luciferase-tagged antigen and the bound antibody is quantified by measuring luciferase activity with a luminometer. The results are expressed as relative luminescence units (RLU). ...
... More recently, 0% of 2,628 patients with non-AAG neurological diseases were positive for antibodies to α3-nAChR (22). These reports suggest that anti-gAChR antibodies are usually undetectable or present at very low levels in the serum (16,19,23). Although these reports used radioimmunoprecipitation assay (RIA) as the measurement method, it has been proven that RIA and LIPS show the similar performance to detect anti-gAChR antibody (19). ...
... These reports suggest that anti-gAChR antibodies are usually undetectable or present at very low levels in the serum (16,19,23). Although these reports used radioimmunoprecipitation assay (RIA) as the measurement method, it has been proven that RIA and LIPS show the similar performance to detect anti-gAChR antibody (19). The high prevalence of anti-gAChR antibodies among our FNSD/CD patient cohort (27.1%) prompted us to hypothesize that anti-gAChR antibodies may be directly associated with autonomic symptoms in these patients. ...
Background
Autoimmune autonomic ganglionopathy (AAG) is a rare disorder characterized by autonomic failure associated with the presence of anti-ganglionic acetylcholine receptor (gAChR) antibodies; however, several studies have reported that individuals with anti-gAChR antibodies present with central nervous system (CNS) symptoms such as impaired consciousness and seizures. In the present study, we investigated whether the presence of serum anti-gAChR antibodies correlated with autonomic symptoms in patients with functional neurological symptom disorder/conversion disorder (FNSD/CD).
Methods
Clinical data were collected for 59 patients presenting with neurologically unexplained motor and sensory symptoms at the Department of Neurology and Geriatrics between January 2013 and October 2017 and who were ultimately diagnosed with FNSD/CD according to the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition. Correlations between serum anti-gAChR antibodies and clinical symptoms and laboratory data were analyzed. Data analysis was conducted in 2021.
Results
Of the 59 patients with FNSD/CD, 52 (88.1%) exhibited autonomic disturbances and 16 (27.1%) were positive for serum anti-gAChR antibodies. Cardiovascular autonomic dysfunction, including orthostatic hypotension, was significantly more prevalent (75.0% vs 34.9%, p = 0.008), whereas involuntary movements were significantly less prevalent (31.3% vs 69.8%, p = 0.007), among anti-gAChR antibody-positive compared with - negative patients. Anti-gAChR antibody serostatus did not correlate significantly with the frequency of other autonomic, sensory, or motor symptoms analyzed.
Conclusions
An autoimmune mechanism mediated by anti-gAChR antibodies may be involved in the etiology of FNSD/CD in a subgroup of patients.
... Autoimmune autonomic neuropathy (AAN) is a rare autonomic neuropathy that mainly affects middle-aged patients [1,2]. Several autoantibodies are suspected to mediate AAN, and some authors classify the disease into antibodypositive and antibody-negative cases [1,3], with elevated ganglionic acetylcholine receptor antibodies (gACHR-Ab) in 50% of patients [1]. ...
... Autoimmune autonomic neuropathy (AAN) is a rare autonomic neuropathy that mainly affects middle-aged patients [1,2]. Several autoantibodies are suspected to mediate AAN, and some authors classify the disease into antibodypositive and antibody-negative cases [1,3], with elevated ganglionic acetylcholine receptor antibodies (gACHR-Ab) in 50% of patients [1]. N-type calcium channel antibodies [4] have also been described. ...
... Autoimmune autonomic neuropathy (AAN) is a rare autonomic neuropathy that mainly affects middle-aged patients [1,2]. Several autoantibodies are suspected to mediate AAN, and some authors classify the disease into antibodypositive and antibody-negative cases [1,3], with elevated ganglionic acetylcholine receptor antibodies (gACHR-Ab) in 50% of patients [1]. N-type calcium channel antibodies [4] have also been described. ...
Objective
Autonomic small fibre neuropathy is described in patients with autoimmune autonomic neuropathy (AAN). Few data are available on somatosensory function and skin biopsies in AAN.
Methods
Retrospective analysis of 17 patients (51.2 ± 6.8 years, n = 7 males) with AAN, including autoantibodies, quantitative sensory testing (QST, n = 13) and intraepithelial nerve fibre density (IENFD) in skin biopsy ( n = 16). QST was performed according to the DFNS protocol over hands and feet dorsum. QST data were compared to healthy controls. Comparison of antibody-positive and antibody-negative cases.
Results
70.6% of patients were antibody positive. 82.4% described at least one episode with sensory symptoms. Skin biopsies revealed reduced IENFD in 58.8% of patients, whereas neuropathic pain was only present in 41.2%. QST showed a nonregional increase for nonpainful thermal and mechanical detection rather than for mechanical pain thresholds. Compared to healthy controls, sensory loss for cold and warm detection thresholds and for the thermal sensory limen—the temperature difference between alternating warm and cold stimuli—was found on hands and feet (all p < 0.05). For nonpainful mechanical stimuli, the vibration detection threshold on the hand was increased ( p < 0.05). Of all pain thresholds, only the mechanical pain threshold was elevated for pinprick stimuli to the feet ( p < 0.05).
Interpretation
Findings are consistent with a sensory small fibre more than large fibre neuropathy in AAN. Sensory loss was comparably distributed across hands and feet, indicating that nerve fibre dysfunction was rather generalized. Serostatus was not a significant predictor of the small fibre deficit present in AAN.
... We have previously established and reported on the use of the LIPS assay for AAG diagnosis on the basis of IgGs to both the α3 and β4 gAChR subunits in human serum samples. 4 We measured the gAChR antibody levels at the Nagasaki Kawatana Medical Center and Kumamoto University Hospital, as described elsewhere. 4,14 Autoantibody levels were expressed as an antibody index which was calculated as the ratio of the relative luminescence units of the sample to the relative luminescence units of the cut-off value. ...
... 4 We measured the gAChR antibody levels at the Nagasaki Kawatana Medical Center and Kumamoto University Hospital, as described elsewhere. 4,14 Autoantibody levels were expressed as an antibody index which was calculated as the ratio of the relative luminescence units of the sample to the relative luminescence units of the cut-off value. The normal antibody index value was established to be < 1.0 based on data from healthy individuals. ...
... The questionnaire included six categories: (1) age, sex, clinical diagnosis, age at onset of disease, antecedent infection, and mode of symptom onset; (2) autonomic manifestations; (3) extraautonomic manifestations; (4) comorbid diseases (endocrine disorders, tumors, and autoimmune diseases); (5) autonomic testing; and (6) other laboratory findings. 4,14,15 With regard to the mode of symptom onset, acute onset and subacute onset were defined as autonomic symptoms reaching a peak within 3 months. Chronic onset was defined as autonomic symptoms reaching a peak after 3 months. ...
Background
Autoimmune autonomic ganglionopathy (AAG) is characterized by serum autoantibodies against the ganglionic acetylcholine receptor (gAChR). Immunomodulatory treatments may alleviate AAG symptoms, but the most appropriate treatment strategy is unclear.
Objective
This study aimed to confirm the effectiveness of treatments, particularly immunotherapy, in patients with seropositive AAG in Japan, as well as to determine the most effective treatment and the best assessment method for clinical response to treatment.
Methods
We collected data from a previous cohort study of patients with seropositive AAG. The clinical autonomic and extra-autonomic symptoms were objectively counted and subjectively assessed using the modified Composite Autonomic Symptom Score. Post-treatment changes in the gAChR antibody level were evaluated.
Results
Thirty-one patients received immunotherapy. Among them, 19 patients received intravenous methylprednisolone; 27, intravenous immunoglobulin; 3, plasma exchange; 18, oral steroids; 2, tacrolimus; 1, cyclosporine; and 1, mycophenolate mofetil. Patients who received immunotherapy showed improvements in the total number of symptoms (from 6.2 ± 2.0 to 5.1 ± 2.0) and modified Composite Autonomic Symptom Score (from 37.4 ± 15.3 to 26.6 ± 12.8). Orthostatic intolerance, sicca, and gastrointestinal symptoms were ameliorated by immunotherapy. Immunotherapy decreased the antibody levels (gAChRα3 antibodies, from 2.2 ± 0.4 to 1.9 ± 0.4, p = 0.08; gAChRβ4 antibodies, from 1.6 ± 0.1 to 1.0 ± 0.2, p = 0.002), but antibody levels increased in 10 patients despite immunotherapy. The rate of improvement in the total number of symptoms was higher in patients with combined therapy than in patients with non-combined therapy (70.7% vs 28.6%).
Conclusions
The scores in many items on the rating scale decreased after immunotherapy in patients with seropositive AAG, particularly in the combined immunotherapy group. However, more accurate assessment scales for clinical symptoms and multicenter randomized, placebo-controlled prospective studies are warranted to establish future treatment strategies.
