A comprehensive analysis of the clinical characteristics and laboratory features in 179 patients with autoimmune autonomic ganglionopathy

Journal of Autoimmunity xxx (xxxx) xxxx

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A comprehensive analysis of the clinical characteristics and laboratory features in 179 patients with autoimmune autonomic ganglionopathy Shunya Nakanea,b,∗, Akihiro Mukainoa,b, Osamu Higuchic,d, Maeda Yasuhiroc,d,e, Koutaro Takamatsua, Makoto Yamakawaa, Mari Wataria, Nozomu Tawaraa, Kei-ichi Nakaharaa, Atsushi Kawakamif, Hidenori Matsuoc,e, Yukio Andoa a

Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Department of Molecular Neurology and Therapeutics, Kumamoto University Hospital, Kumamoto, Japan c Department of Clinical Research, National Hospital Organization Nagasaki Kawatana Medical Center, Nagasaki, Japan d Department of Neuroimmunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan e Department of Neurology, National Hospital Organization Nagasaki Kawatana Medical Center, Nagasaki, Japan f Department of Immunology and Rheumatology, Unit of Translational Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan b

A R T I C LE I N FO

A B S T R A C T

Keywords: Autoimmune autonomic ganglionopathy (AAG) Anti-ganglionic acetylcholine receptor (gAChR) antibodies Autonomic dysfunction Autoantibody: 123I- metaiodobenzylguanidine (MIBG) myocardial scintigraphy

The clinical importance of autoantibodies against the ganglionic acetylcholine receptor (gAChR) remains to be fully elucidated. We aimed to identify the clinical characteristics of autoimmune autonomic ganglionopathy (AAG) in patients with gAChR autoantibodies. For this cohort investigation, serum samples were obtained from patients with AAG between 2012 and 2018 in Japan. We measured the levels of autoantibodies against gAChRα3 and gAChRβ4 and evaluated clinical features, as well as assessing the laboratory investigation results among the included patients. A total of 179 patients tested positive for antibodies, including 116 gAChRα3-positive, 13 gAChRβ4-positive, and 50 double antibody-positive patients. Seropositive AAG patients exhibited widespread autonomic dysfunction. Extra-autonomic manifestations including sensory disturbance, central nervous system involvement, endocrine disorders, autoimmune diseases, and tumours were present in 118 patients (83%). We observed significant differences in the frequencies of several autonomic and extra-autonomic symptoms among the three groups. Our 123I-metaiodobenzylguanidine myocardial scintigraphy analysis of the entire cohort revealed that the heart-to-mediastinum ratio had decreased by 80%. The present study is the first to demonstrate that patients with AAG who are seropositive for anti-gAChRβ4 autoantibodies exhibit unique autonomic and extra-autonomic signs. Decreased cardiac uptake occurred in most cases, indicating that 123I- metaiodobenzylguanidine myocardial scintigraphy may be useful for monitoring AAG. Therefore, our findings indicate that gAChRα3 and gAChRβ4 autoantibodies cause functional changes in postganglionic fibres in the autonomic nervous system and extra-autonomic manifestations in seropositive patients with AAG.

1. Introduction Autoimmune autonomic ganglionopathy (AAG) is a rare disease that presents with various autonomic symptoms. The ganglionic neuronal

nicotinic acetylcholine receptor (gAChR) mediates fast synaptic transmission in all peripheral autonomic ganglia in the autonomic nervous system, comprising two α3 subunits and three β4 subunits [1,2]. However, the frequency of symptoms, extra-autonomic manifestations

Abbreviations: AAG, autoimmune autonomic ganglionopathy; Abs, autoantibodies; AChRs, acetylcholine receptors; AI, antibody index; CSF, cerebrospinal fluid; CVRR, coefficient of variation in R-R intervals; gAChR, ganglionic acetylcholine receptor; GI, gastrointestinal; GL, Gaussia luciferase; H/M, heart to mediastinum; IVIg, intravenous immunoglobulin; IVMP, intravenous methylprednisolone; LIPS, luciferase immunoprecipitation system; MIBG, metaiodobenzylguanidine; nAChR, nicotinic acetylcholine receptor; PSL, prednisolone; RLU, relative luminescence unit; SD, standard deviations ∗ Corresponding author. Department of Molecular Neurology and Therapeutics, Kumamoto University Hospital, 1-1-1, Honjo, Chuouku, Kumamoto-shi, Kumamoto, 860-8556, Japan. E-mail addresses: [email protected] (S. Nakane), [email protected] (A. Mukaino), [email protected] (O. Higuchi), [email protected] (M. Yasuhiro), [email protected] (K. Takamatsu), [email protected] (M. Yamakawa), [email protected] (M. Watari), [email protected] (N. Tawara), [email protected] (K.-i. Nakahara), [email protected] (A. Kawakami), [email protected] (H. Matsuo), [email protected] (Y. Ando). https://doi.org/10.1016/j.jaut.2020.102403 Received 9 October 2019; Received in revised form 29 December 2019; Accepted 1 January 2020 0896-8411/ © 2020 Published by Elsevier Ltd.

Please cite this article as: Shunya Nakane, et al., Journal of Autoimmunity, https://doi.org/10.1016/j.jaut.2020.102403

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guidelines described below. Patients with involvement of at least one autonomic domain and a positive Ab level were considered to have Abpositive AAG. Patients with alternative causes of autonomic dysfunction were eliminated from the study. Four patients were excluded due to insufficient data. Finally, we reviewed clinical survey data and summaries for 179 Japanese patients with seropositive AAG (mean age: 59 ± 20 years; 105 men and 74 women), which were collected along with pre-treatment serum samples. 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. The samples were later sent to Nagasaki Kawatana Medical Center or Kumamoto University Hospital. Comprehensive clinical, neurological, and serological assessments were performed for all patients with seropositive AAG [3,6]. At least two neurologists performed the neurological examinations. Our specific questionnaires and consent forms were sent to the referring neurologists, and the data were sorted and analysed in Kumamoto, Japan. Our questionnaire included items related to the following: (1) age, sex, clinical diagnosis, age at disease onset, antecedent infection, and mode of symptom onset; (2) autonomic manifestations; (3) extra-autonomic manifestations (e.g., sensory disturbance, motor symptoms, deep tendon reflexes, gait, and other neurological findings); (4) comorbid diseases (e.g., endocrine disorders, tumours, and autoimmune diseases); (5) autonomic test results including 123I-MIBG myocardial scintigraphy findings; and (6) other laboratory findings (e.g., cerebrospinal fluid (CSF) and other Ab tests) [3,6]. Acute and subacute symptom onset were defined as reaching peak autonomic symptoms within 3 months, while chronic onset was defined as reaching peak autonomic symptoms after 3 months. Moreover, we confirmed the present history of autonomic symptoms that appeared between several days and 2 weeks after antecedent infectious episodes. We reviewed the information of the seropositivity for anti-nuclear antibodies, anti-double stranded DNA antibodies, rheumatoid factor, and anti–SS–A/SS-B antibodies.

(coexistence with autoimmune rheumatic diseases and tumours, brain involvement, sensory disturbance, and endocrine disorders), laboratory findings, and prognosis of AAG remain largely unknown [3,4]. Approximately 50% of patients with AAG possess autoantibodies (Abs) against the gAChR [5,6]. Originally, gAChR Abs found in patients with AAG were thought to be specific for acetylcholine receptors (AChRs) containing the α3 subunit and were not assumed to bind non-specifically to nicotinic AChRs (nAChRs). Several previous reports have described the autonomic symptoms of gAChR Ab-positive AAG as severe, widespread, and predominantly post-ganglionic in distribution. Moreover, such studies have revealed that Ab levels are correlated with disease severity and pathogenesis [7,8]. Our previous study was the first to identify a subunit-specific Ab for gAChR (i.e., anti-gAChRβ4) [6]. Another subunit-specific Ab for gAChR, anti-gAChRα3, has been associated with several autonomic dysfunctions in an experimental model of AAG [9–12]. Despite our previous findings, the clinical characteristics of patients with AAG who are seropositive for the gAChRβ4 Ab remain unknown. Therefore, we evaluated the frequency of individual autonomic symptoms and extra-autonomic manifestations such as sensory disturbance, central nervous system (CNS) involvement, endocrine disorders, and comorbid diseases (i.e., autoimmune diseases, tumours) in patients with AAG testing positive for gAChRα3 Abs, gAChRβ4 Abs, or both [3,13]. Differentiating AAG from other neurological disorders presenting with autonomic dysfunction via clinical and laboratory tests is often difficult [14]. In addition to anti-gAChR Ab levels, effective clinical tools are required to improve AAG diagnosis and monitoring. Neuroimaging data may aid in identifying clinically relevant post-ganglionic denervation. Metaiodobenzylguanidine (MIBG) is a physiological analogue of noradrenaline, which is taken up by the myocardium and actively transported into the noradrenaline granules of sympathetic nerve terminals by the noradrenaline transporter. Indeed, 123I-MIBG myocardial scintigraphy may assist in evaluating damage to postganglionic fibres of the cardiac sympathetic nerve based on decreases in the heartto-mediastinum (H/M) ratio. We previously mentioned the usefulness of 123I-MIBG myocardial scintigraphy for AAG diagnosis [3]. Myocardial scintigraphy is generally used to evaluate autonomic function in patients with suspected Lewy body disease or autonomic neuropathies such as diabetic neuropathy or familial amyloid polyneuropathy [15–18]. However, no similar studies have been performed among patients with AAG. Therefore, the secondary aim of the present study was to determine the value of 123I-MIBG myocardial scintigraphy for AAG diagnosis, relative to other laboratory evaluations. The present study aimed to determine the clinical characteristics of patients with AAG testing positive for gAChRβ4 Ab. We examined the frequency of autonomic and extra-autonomic manifestations in 179 patients with AAG testing positive for gAChRα3 Abs, gAChRβ4 Abs, or both Abs. Furthermore, our study highlights the value of 123I-MIBG myocardial scintigraphy for AAG diagnosis.

2.3. Luciferase immunoprecipitation system (LIPS) assay for the detection of anti-gAChR Abs In the present study, we detected serum gAChRα3 and gAChRβ4 Abs using the LIPS assay [6]. A National Institutes of Health research group previously developed this efficient quantitative approach for analysing antibodies against human autoantigens in serum samples [19,20]. We previously established that LIPS assays can be used to diagnose AAG based on the presence of immunoglobulin Gs (IgGs) to both the α3 and β4 subunits of the gAChR in serum samples [4,6]. Levels of these gAChR Abs were measured at Nagasaki Kawatana Medical Center and Kumamoto University Hospital, as previously described. To evaluate the diagnostic accuracy of this LIPS assay, we verified the cut-off points for all data collected in the previous study [4,6,21,22]. Based on the anti-gAChRα3 and β4 Abs data from healthy controls, cut-off values were calculated as the mean plus three standard deviations (SDs) from the mean [4,6,21,22]. In the present study, Ab levels were expressed as an Ab index (AI), which was calculated as follows: AI = (measured value in the serum sample [relative luminescence units (RLU)])/(cut-off value [RLU]). The normal AI value established based on data from healthy individuals was < 1·0.

2. Materials and methods 2.1. Standard protocol approvals and patient consent All patients provided written, informed consent before participating in the present study. The Human Ethics Committees at the Nagasaki Kawatana Medical Center and Kumamoto University Hospital (Japan) approved this study (approval number 2011-21 and 1281, respectively).

2.4. Clinical assessment of autonomic and extra-autonomic function We examined the presence or absence of the following functions controlled by the autonomic nervous system, as reported in our previous study: syncope or orthostatic hypotension and orthostatic intolerance; arrhythmia; pupillary dysfunction; sicca complex; coughing episodes; skin dryness or hypohidrosis/anhidrosis indicating heat intolerance; upper gastrointestinal (GI) system problems; diarrhoea or constipation indicating dysfunction of the lower GI system; dysuria or urinary retention needing catheterisation for bladder dysfunction; and sexual dysfunction [3,4,6]. The presence or absence of clinically

2.2. Study design and participants We obtained 1787 serum samples (from 1381 patients) from teaching and general hospitals throughout Japan between January 2012 and August 2018 (Supplementary data 1). Clinical diagnoses were made in each hospital. All hospitals follow the same diagnosis 2

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2.6. Laboratory evaluations for autonomic function

obvious landmarks such as vomiting, diarrhoea or constipation for dysfunction of the digestive system, dysuria or urinary retention needing catheterisation for urinary dysfunction, syncope or orthostatic hypotension for orthostatic intolerance, mydriasis for pupillary dysfunction, and dryness of the skin on palpation for hypohidrosis/anhidrosis was assessed by bedside examinations of the patients, reviewing the patients' records, and interviewing the patients’ families. Urinary symptoms were estimated by nocturnal or diurnal urinary frequency, a sensation of urgency, urinary incontinence, voiding difficulty, and retention. Constipation was considered to be present if there were no stools for > 3 days. Orthostatic hypotension was defined as a fall of at least 30 mmHg in the systolic blood pressure within 10 min of rising from a supine position. It was assessed using the head-up tilt test unless the general condition of the patient was unsuitable. We also evaluated the presence or absence of the following extraautonomic manifestations: sensory disturbance and other neurological findings, as well as comorbid diseases (e.g., endocrine disorders, tumours, and autoimmune diseases). We included four symptoms in the sensory disturbance category: paraesthesia, dysesthesia, nerve conduction study abnormalities, and no symptoms. Sensory examinations included pinprick, temperature, light touch, vibratory sensation, and joint position tests. After excluding neurodegenerative diseases, patients with any CNS symptoms (cerebral, cerebellar, brainstem, and/or spinal cord) were considered to exhibit CNS involvement. We distinguished CNS involvement from other neurological disorders such as multiple system atrophy based on the results of clinical assessments, laboratory tests, and imaging studies. With regard to the diagnosis of comorbid autoimmune diseases, the diagnosis of Sjögren's syndrome was confirmed using the diagnostic criteria proposed by the American European Consensus Group and/or the Japanese Ministry of Health criteria for SS diagnostics [23,24]. The diagnosis of Hashimoto thyroiditis was confirmed using the Guidelines for the diagnosis of chronic thyroiditis proposed by the Japan Thyroid Association [25]. The diagnosis of AAG was confirmed using clinical criteria including frank orthostatic hypotension, gastrointestinal dysmotility, and seropositive for anti-gAChR autoantibodies [26,27]. Subsequently, we divided patients into three groups (gAChRα3 Ab-positive, gAChRβ4 Abpositive, and double Ab-positive). We then compared autonomic symptoms and extra-autonomic manifestations among these three groups.

Each patient underwent various tests for autonomic function, including the Schellong test, head-up tilt test, measurement of the coefficient of variation in R-R intervals (CVRR), noradrenaline infusion test, pupillary response to local instillation test, assessment of plasma levels of catecholamines, sweat testing, quantitative sudomotor axon reflex test, 123I-MIBG myocardial scintigraphy, and cystometry. We extracted and utilised the data for resting CVRR, 123I-MIBG myocardial scintigraphy, and CSF analyses for patients with seropositive AAG. Only these examinations were performed in approximately half of the included patients. RR intervals were continuously recorded for 3 min with an ECG machine. One-hundred-and-fourteen participants (114/179, 64%) maintained a supine position with normal breathing with at least 10 min of rest before recording began. Resting CVRR was calculated as a percentage of the SD of the last 100 RR intervals divided by their mean (M). Thus, CVRR (%) = (SD/M)*100. Ninety-three CSF samples (93/179, 52%) that had been obtained at the time of the diagnostic spinal tap, within 1 month of symptom onset, were frozen and stored at −80 °C for subsequent analyses. We collected routine CSF data (CSF proteins and cell count). Elevated CSF protein levels were defined according to criteria used in laboratories in the hospitals involved in this study. Pleocytosis was defined as a CSF white cell count of > 5 cells/μl. Albuminocytologic dissociation was defined as elevated CSF protein levels with a CSF cell count of < 50 cells/μl per the Brighton criteria [30]. In this study, we evaluated 85 (85/179, 48%) seropositive AAG cases that had had 123I-MIBG myocardial scintigraphy performed, and we studied and compared the H/M ratios in scintigraphy in the three groups. A dose of 111 MBq of MyoMIBG was injected intravenously for clinical studies utilising 123I-MIBG (Fujifilm RI Pharma, Tokyo, Japan). Patients in Japan use no specific preparations before this test, other than avoiding medications (e.g. tricyclic antidepressants, Ca2+ blockers) that affect myocardial 123I-MIBG uptake. Early images were usually acquired between 15 and 30 min after administration, and late images were acquired at 3–4 h after administration. Regions of interest included the whole heart (H) and mediastinum (M) of the front image. The H/M ratio of myocardial 123I-MIBG uptake in regions of interest was calculated. This study evaluated H/M ratios from early and delayed images (normal range ≥ 2.20, respectively) [31,32].

2.5. Compass

2.7. Statistical analysis

The patients with AAG enrolled since April 2014 completed a selfadministered questionnaire. COMPASS is a shortened version of the Composite Autonomic Symptom Score and was designed to quantitatively assess autonomic symptoms [28]. It has six subscale weighted scores in the following domains: orthostatic intolerance (four items; range, 0–40), vasomotor (three items; range, 0–5), secremotor (four items; range, 0–15), gastrointestinal (12 items; range, 0–25), bladder (three items; range, 0–10), and pupillomotor (five items; range, 0–5). COMPASS is weighted according to published scoring methods to yield a total score of 0–100, with a score of 100 representing the highest, most severe degree of the autonomic symptom burden. The mean ± standard deviation score in healthy control subjects for this questionnaire is reported to be 9.67 ± 8.1 [29]. In the present study, 136 subjects completed the questionnaire in Japanese within 15 min. However, we excluded the questions related to the vasomotor and pupillomotor domains, because it is occasionally difficult for the Japanese to judge the colour changes in the skin on an individual basis, and it is not the custom for middle-aged and older persons to wear sunglasses or tinted glasses in Japan. The total scores were calculated by summation of the individual item scores, with a possible maximum score of 90 [13].

Commercially available statistical software (SigmaPlot®; SPSS, Inc., Chicago, IL, USA) was used to analyse the data. When comparing clinical findings among the three groups (gAChRα3 Ab-positive group, gAChRβ4 Ab-positive group, and double-positive group), normally distributed data were analysed via one-way analyses of variance (ANOVA). To compare non-normally distributed data among the three groups, we utilised a one-way ANOVA on ranks. To quantify the relationship between the COMPASS score and the levels of autoantibodies against gAChRα3 and gAChRβ4, the correlation coefficient was used. For all analyses, the level of statistical significance was set at P < 0.05. 3. Results 3.1. Clinical features of patients with seropositive AAG Although patients were divided into subgroups based on the time course of their illness, chronic AAG was the predominant subtype in our study (133/179, 74%) (Table 1). Among those with chronic AAG, 12 patients (12/133, 9%) experienced an antecedent event, whereas 13 of 46 (28%) patients in the acute AAG group experienced an antecedent event. Twenty-five patients (14%) reported antecedent events shortly before the start of autonomic symptoms: flu-like symptoms (n = 11), 3

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Table 1 Clinical characteristics of seropositive AAG patients. Feature

TOTAL

Patients positive for gAChRα3 Abs

Patients positive for gAChRβ4 Abs

Patients positive for gAChRα3 and gAChRβ4 Abs

P-value

Number of patients (%) gAChRα3 Abs (AI) gAChRβ4 Abs (AI) Gender, number of male patients (%) Age (years) Onset age, (years) Number of patients with acute or subacute onset (%) Number of patients with antecedent events (%)

179 1.864 ± 1.446 0.903 ± 0.545 106 (59%) 59 ± 20 55 ± 21 46 (26%)

116 1.529 ± 0.634 0.551 ± 0.185 60 (52%) 58 ± 20 54 ± 20 28 (24%)

13 0.640 ± 0.303 1.330 ± 0.323 10 (77%) 55 ± 20 49 ± 24 4 (31%)

50 2.423 ± 1.147 1.522 ± 0.582 35 (70%) 64 ± 18 60 ± 20 14 (28%)

0.035* 0.127 0.150 0.795

26 (15%)

13 (11%)

0 (0%)

12 (24%)

0.030*

Values are given as means ± SD. *p < 0.05 was considered statistically significant among the three groups (Patients positive for gAChRα3 Abs, gAChRβ4 Abs, and gAChRα3 and gAChRβ4 Abs).

thymoma (n = 1), and seminoma (n = 1). One-hundred-and-sixteen patients (65%) demonstrated single seropositivity for anti-gAChRα3 Abs, while 13 patients (7%) demonstrated single seropositivity for anti-gAChRβ4 Abs. Fifty patients (28%) were positive for both Abs. Table 1 presents the clinical features of these three AAG groups. We observed significant differences in gender and antecedent events among the three groups. The frequencies of antecedent events were 11%, 0%, and 24% in the gAChRα3 Ab-positive, gAChRβ4 Ab-positive, and double-positive groups, respectively (Table 1). We also observed significant differences in the frequency of several autonomic (sicca complex, coughing episodes, anhidrosis, upper GI dysfunction, and bladder dysfunction) and extra-autonomic symptoms (sensory disturbance) among the three groups (Fig. 3). OH, sicca, upper GI dysfunction, bladder dysfunction, and sensory disturbance were more frequent in the gAChRβ4 Ab-positive and double-positive groups than in the gAChRα3 Ab-positive group, except for coughing episodes (Supplementary data 3).

enterocolitis (n = 9), herpesvirus infection (n = 2), epididymitis (n = 1), chlamydia infection (n = 1), or surgery (n = 1) (Table 1). In 114 patients (66%), the initial symptoms of seropositive AAG were orthostatic hypotension and orthostatic intolerance (e.g., light headedness, palpitations, or syncope) (Table 1). 3.2. Frequencies of autonomic symptoms and extra-autonomic manifestations Patients with AAG exhibited widespread autonomic dysfunction including orthostatic hypotension, orthostatic intolerance, and lower GI tract dysfunction (Fig. 1). Extra-autonomic manifestations were observed in 148 patients (83%) (Fig. 2 and Supplementary data 2). Eighty patients (45%) experienced subjective sensory disturbance including numbness and other symptoms. Fifty-nine patients (33%) exhibited CNS involvement including psychiatric symptoms. Frequent signs of CNS involvement included changes in personality (e.g., abnormal behaviour, emotional instability, restlessness), cognitive impairment, parkinsonism, and ataxia (n = 18, n = 13, n = 12, and n = 8, respectively). Endocrine disorders were identified in 26 patients (15%): hyponatraemia (n = 9), amenorrhoea (n = 6), syndrome of inappropriate antidiuretic hormone secretion (SIADH) (n = 5), panhypopituitarism (n = 1), hyperprolactinemia (n = 1), and others. Fiftythree patients (30%) presented with other autoimmune diseases. Sjögren's syndrome (SS) (including five secondary SS cases) and Hashimoto's disease were confirmed in 20 and 13 patients, respectively. Tumours were diagnosed in 19 patients (11%); ovarian tumours (n = 5), lung cancer (n = 5), gastric cancer (n = 3), prostate cancer (n = 2), maxillary sinus cancer (n = 1), mediastinal tumour (n = 1),

3.3. COMPASS We confirmed the COMPASS data in 136/179 (76%) patients with AAG. The COMPASS data were as follows: median total score, 36.3 ± 17.1; median orthostatic intolerance score, 21.8 ± 12.9; median secretomotor score, 4.7 ± 3.8; median gastrointestinal score, 7.1 ± 3.9; and median bladder score, 2.6 ± 2.8. There was a nonsignificant correlation between the COMPASS score, including the total and each subdomain score, and the levels of autoantibodies against gAChRα3 and gAChRβ4 (Table 2). Fig. 1. Autonomic symptoms in patients with seropositive AAG. Patients with seropositive AAG exhibited widespread autonomic dysfunction. OH, OI, and lower gastrointestinal (GI) tract dysfunction were frequently observed. Five patients (2·8%) exhibited Adie's tonic pupil. Upper GI dysmotility syndromes, including achalasia, diffuse oesophageal spasm, and gastroparesis, were confirmed in 13 patients (7·3%). Paralytic ileus was confirmed in 11 patients (6·1%).

4

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Fig. 2. Extra-autonomic symptoms in patients with seropositive AAG. Most patients with seropositive AAG exhibited extra-autonomic manifestations (148/179, 83%). AAG = autoimmune autonomic ganglionopathy; OH = orthostatic hypotension; OI = orthostatic intolerance; CNS = central nervous system.

3.4. Laboratory findings

addition, there were no significant differences in pleocytosis, CSF protein levels, or albuminocytologic dissociation among the groups.

Table 3 summarises the results of our laboratory examinations. The H/M ratio in the early and/or delayed phase of 123I-MIBG myocardial scintigraphy decreased in 79% of patients tested. Specifically, 78% (40/ 51) of patients were positive for gAChRα3 Abs, while 80% (68/85) of patients were positive for gAChRβ4 Abs. Twenty-five of the 50 doublepositive patients (50%) underwent myocardial scintigraphy, 84% (21/ 25) of whom tested positive. The CVRR value decreased in 77% (54/ 70), 75% (9/12), and 63% (20/32) of patients who were gAChRα3 Abpositive, gAChRβ4 Ab-positive, and double-positive, respectively. In

4. Discussion In the present study, we investigated the clinical symptoms involving autonomic and extra-autonomic manifestations, and laboratory findings among a diagnosed cohort of 179 patients with AAG who tested positive for gAChRα3 Abs, gAChRβ4 Abs, or both. The gAChR Abs have the potential to physiologically block the ganglionic synaptic transmission in the both of sympathetic and parasympathetic nervous

Fig. 3. Evaluation of autonomic and extra-autonomic symptoms in patients with AAG. We observed significant differences in the frequency of several autonomic and extra-autonomic symptoms among the three groups (i.e., gAChRα3 Ab-positive group, gAChRβ4 Ab-positive group, and the gAChRα3 and gAChRβ4 Abdouble-positive group), including sicca complex, coughing episodes, anhidrosis, upper GI dysfunction, bladder dysfunction, and sensory disturbances. gAChR = ganglionic acetylcholine receptor; AAG = autoimmune autonomic ganglionopathy; Abs = autoantibodies; GI = gastrointestinal. 5

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Table 2 Correlation analysis between the COMPASS score in 139 AAG patients and the levels of autoantibodies against gAChRα3 and gAChRβ4.

gAChRα3 Abs

gAChRβ4 Abs

Level Level Level Level Level Level Level Level Level Level

vs vs vs vs vs vs vs vs vs vs

COMPASS COMPASS COMPASS COMPASS COMPASS COMPASS COMPASS COMPASS COMPASS COMPASS

total score orthostatic intolerance score secremotor score gastrointestinal score bladder score total score orthostatic intolerance score secremotor score gastrointestinal score bladder score

Correlation coefficient

P-value

−0.081 −0.097 0.147 −0.088 −0.110 0.020 0.001 0.110 −0.031 0.016

0.348 0.261 0.089 0.306 0.206 0.814 0.911 0.204 0.715 0.852

Values are given as means ± SD.

system [1,12]. Our findings indicated that autonomic and extra-autonomic dysfunction (i.e., sicca complex, anhidrosis, upper GI dysfunction, bladder dysfunction, and sensory disturbance) occurred more frequently in patients seropositive for gAChRβ4 Abs than in those seropositive for gAChRα3 Abs. Transgenic mice lacking the gAChRβ4 subunit exhibit profound autonomic failure such as bladder dysfunction and GI dysmotility, suggesting that the β4 subunit is required for ganglionic neurotransmission [33–36]. Given previous findings, future studies should investigate the localisation of gAChRβ4 and the effects of gAChRβ4 Abs on cell-surface nAChRs [9–11,37,38]. Nonetheless, the present study is the first to demonstrate that patients with AAG who test positive for anti-gAChRβ4 Abs exhibit autonomic and extra-autonomic signs that differ from those of patients testing positive for anti-gAChRα3 Abs. Interestingly, extra-autonomic manifestations occur in over 80% of patients with seropositive AAG [3,4]. The nAChRs comprise a family of abundantly expressed ligand-gated cation channels found throughout the peripheral and central nervous systems, especially in the hypothalamus. They are involved in various neurobiological systems that have been implicated in the pathophysiology of psychiatric symptoms. In addition to their roles in cholinergic neurotransmission, nAChRs modulate dopamine function and hypothalamic-pituitary-adrenal axis activity [39–42]. Therefore, we speculated that nAChRα3 and nAChRβ4 Abs may play a role in extra-autonomic manifestations. Anti-gAChRα3 and anti-gAChRβ4 Abs, in addition to new Abs to other subunits of nAChRs in the dorsal root ganglion and brain, may also be associated with extra-autonomic manifestations, especially sensory disturbance, CNS involvement, and endocrine disorders [3,4,39]. The patients with seropositive AAG had the coexistence of autoimmune diseases. Autonomic dysfunctions have been reported in association with Sjögren's syndrome, systemic sclerosis, systemic lupus erythematosus, rheumatoid and arthritis [43,44]. Although AAG and the other autoimmune

diseases can coexist due to the same background of autoimmunity, previous reports have referred to anti-gAChR antibodies in these autoimmune diseases [6,22,42–44]. Previous studies reported that high levels of the gAChR Abs are not required for severe dysfunction [7,45]. In the present study, we could not observe a significant correlation between the COMPASS score and the levels of the gAChR Abs. There is a possibility that changes in the gAChR Ab levels in AAG correlate intra-individually with the severity of the clinical symptoms, just like the relationship between the changes in AChR Ab titre and clinical severity in myasthenia gravis [46]. In the future, we should examine the relationship between the chronological change in the gAChR Ab levels and the clinical severity including the COMPASS score over the entire course (i.e. pre- and post-immunotherapy) in each case with AAG. H/M ratios of 123I-MIBG myocardial scintigraphy abnormalities among patients with AAG were greater than those observed for other tests of autonomic function in the present study. Although previous authors have described methods for evaluating sudomotor dysfunction in patients with AAG, no other studies have reported 123I-MIBG myocardial scintigraphy findings for patients with AAG [47]. The frequency of abnormalities in 123I-MIBG myocardial scintigraphy indicates that dysfunction of postganglionic fibres is common among patients with AAG. However, we observed no significant correlation between decreases in the H/M ratio and gAChR Ab levels in our study. In our previous report, we described three seropositive patients with AAG who exhibited improvements in H/M ratio and autonomic symptoms following immunotherapy, suggesting that damage due to AAG is reversible, and that neuronal losses are not permanent [3]. The H/M ratio in the early phase reflects the distribution and density of cardiac sympathetic nerves, while that in the delayed phase reflects the function of the cardiac sympathetic nerves [48–50]. In our illustrative case, we did not observe sufficient improvements in the delayed phase H/M ratio.

Table 3 Results of laboratory tests in seropositive AAG patients. Variable

Total

Patients positive for gAChRα3 Abs

Patients positive for gAChRβ4 Abs

Patients positive for both gAChRα3 and gAChRβ4 Abs

Number of patients Number of patients who underwent CVRR (%) CVRR Decrease in CVRR value (%) CVRR value Number of patients who underwent scintigraphy (%) H/M ratio Decrease in H/M ratio in early and/or delayed phase (%) H/M ratio in early phase H/M ratio in delayed phase Number of patients who underwent CSF analysis (%) CSF Lymphocytic pleocytosis (%) Elevation of CSF protein (%) Albuminocytologic dissociation (%) CSF cell count,/μl CSF protein, mg/dl

179 114 (64%) 83 (72%) 2.37 ± 2.21 85 (48%) 68 (80%)

116 70 (60%) 54 (77%) 2.38 ± 2.35 51 (44%) 40 (78%)

13 12 (92%) 9 (75%) 2.13 ± 1.40 9 (69%) 7 (78%)

50 32 (64%) 20 (63%) 2.43 ± 2.18 25 (50%) 21 (84%)

0.077 0.303 0.942 0.207 0.839

1.96 ± 0.67 1.83 ± 0.90 93 (52%) 13 (14%) 45 (48%) 34 (37%) 3.8 ± 8.1 64.0 ± 57.1

1.98 ± 0.73 1.87 ± 1.00 55 (47%) 7 (13%) 24 (44%) 17 (31%) 4.0 ± 9.8 71.2 ± 69.2

1.94 ± 0.54 1.87 ± 0.74 9 (69%) 2 (22%) 5 (56%) 3 (33%) 4.3 ± 5.5 51.9 ± 32.9

1.94 ± 0.80 1.76 ± 0.75 29 (58%) 4 (14%) 16 (55%) 14 (48%) 3.4 ± 4.9 54.4 ± 31.2

0.881 0.667 0.199 0.750 0.548 0.288 0.101 0.947

6

P-value

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S. Nakane, et al.

References

Thus, in some cases of AAG, postganglionic dysfunction may be due to functional impairments in the postganglionic fibres caused by Abs against gAChRs. Furthermore, our findings suggest that 123I-MIBG myocardial scintigraphy can aid in monitoring the effects of immunotherapy among patients with AAG. The present study possesses several limitations of note. First, we could not treat all laboratory examinations equally because the autonomic testing equipment varied among the hospitals involved in this study. Second, we could not obtain information regarding drugs that may have interfered with MIBG uptake in sympathetic nerve terminals. Third, further studies are required to determine whether 123I-MIBG myocardial scintigraphy has equal diagnostic value for AAG in patients with parkinsonism or diabetes mellitus. Despite these limitations, our neuroimaging findings are in accordance with current hypotheses regarding the pathogenesis of AAG. In conclusion, our findings may help to elucidate the pathophysiological mechanisms underlying AAG mediated by anti-gAChR Abs, and to explain how disruption of gAChR function in autonomic and dorsal root ganglia and the brain induces distinct extra-autonomic manifestations. Therefore, detection of gAChR Abs can aid in the diagnosis of patients with AAG who exhibit autonomic signs and extra-autonomic manifestations. As we observed decreases in cardiac MIBG uptake in most seropositive patients with AAG, our study further indicates that 123 I-MIBG myocardial scintigraphy may be useful for monitoring patients with AAG. Finally, as AAG provides a unique human model of selective nicotinic AChR dysfunction, our findings have implications for understanding other neurological diseases, including neuropsychiatric disorders associated with nicotinic AChR dysfunction.

[1] S. Vernino, J. Lindstrom, S. Hopkins, Z. Wang, P.A. Low, Muscle Study Group, Characterization of ganglionic acetylcholine receptor autoantibodies, J. Neuroimmunol. 197 (2008) 63–69. [2] Z. Wang, P.A. Low, S. Vernino, Antibody-mediated impairment and homeostatic plasticity of autonomic ganglionic synaptic transmission, Exp. Neurol. 222 (2010) 114–119. [3] S. Nakane, A. Mukaino, Y. Maeda, O. Higuchi, H. Matsuo, Y. Ando, Extra-autonomic manifestations in autoimmune autonomic ganglionopathy: a Japanese survey, J. Neurol. Neurosurg. Psychiatry 88 (2017) 367–368. [4] S. Nakane, A. Mukaino, O. Higuchi, M. Watari, Y. Maeda, M. Yamakawa, K. Nakahara, K. Takamatsu, H. Matsuo, Y. Ando, Autoimmune autonomic ganglionopathy: an update on diagnosis and treatment, Expert Rev. Neurother. 18 (2018) 953–965. [5] S. Vernino, P.A. Low, R.D. Fealey, J.D. Stewart, G. Farrugia, V.A. Lennon, Autoantibodies to ganglionic acetylcholine receptors in autoimmune autonomic neuropathies, N. Engl. J. Med. 343 (2000) 847–855. [6] S. Nakane, O. Higuchi, M. Koga, T. Kanda, K. Murata, T. Suzuki, H. Kurono, M. Kunimoto, K.I. Kaida, A. Mukaino, W. Sakai, Clinical features of autoimmune autonomic ganglionopathy and the detection of subunit-specific autoantibodies to the ganglionic acetylcholine receptor in Japanese patients, PLoS One 10 (2015) e0118312. [7] J.K. Cutsforth-Gregory, A. McKeon, E.A. Coon, D.M. Sletten, M. Suarez, P. Sandroni, W. Singer, E.E. Benarroch, R.D. Fealey, P.A. Low, Ganglionic antibody level as a predictor of severity of autonomic failure, Mayo Clin. Proc. 93 (2018) 1440–1447. [8] P. Sandroni, P.A. Low, Other autonomic neuropathies associated with ganglionic antibody, Auton. Neurosci. 146 (2009) 13–17. [9] V.A. Lennon, L.G. Ermilov, J.H. Szurszewski, S. Vernino, Immunization with neuronal nicotinic acetylcholine receptor induces neurological autoimmune disease, J. Clin. Investig. 111 (2003) 907–913. [10] S. Vernino, P.A. Low, V.A. Lennon, Experimental autoimmune autonomic neuropathy, J. Neurophysiol. 90 (2003) 2053–2059. [11] S. Vernino, L.G. Ermilov, L. Sha, J.H. Szurszewski, P.A. Low, V.A. Lennon, Passive transfer of autoimmune autonomic neuropathy to mice, J. Neurosci. 24 (2004) 7037–7042. [12] S. Vernino, P. Sandroni, W. Singer, P.A. Low, Invited article: autonomic ganglia: target and novel therapeutic tool, Neurology 70 (2008) 1926–1932. [13] A. Mukaino, H. Minami, H. Isomoto, H. Hamamoto, E. Ihara, Y. Maeda, O. Higuchi, T. Okanishi, Y. Kokudo, K. Deguchi, F. Sasaki, Anti-ganglionic AChR antibodies in Japanese patients with motility disorders, J. Gastroenterol. 53 (2018) 1227–1240. [14] D.S. Goldstein, C. Holmes, R. Imrich, Clinical laboratory evaluation of autoimmune autonomic ganglionopathy: preliminary observations, Auton. Neurosci. 146 (2009) 18–21. [15] M. Mäntysaari, J. Kuikka, J. Mustonen, K. Tahvanainen, E. Vanninen, E. Länsimies, M. Uusitupa, Noninvasive detection of cardiac sympathetic nervous dysfunction in diabetic patients using 123I-metaiodobenzylguanidine, Diabetes 41 (1992) 1069–1075. [16] Y. Ando, K. Obayashi, Y. Tanaka, T. Tsuji, M. Uchino, M. Takahashi, M. Ando, Radiolabelled meta-iodobenzylguanidine in assessment of autonomic dysfunction, Lancet 343 (1994) 984–985. [17] S. Orimo, E. Ozawa, S. Nakade, T. Sugimoto, H. Mizusawa, 123I-metaiodobenzylguanidine myocardial scintigraphy in Parkinson's disease, J. Neurol. Neurosurg. Psychiatry 67 (1999) 189–194. [18] K. Nakajima, M. Yamada, 123I-Meta-iodobenzylguanidine sympathetic imaging: standardization and application to neurological diseases, Chonnam. Med. J. 52 (2016) 145–150. [19] P.D. Burbelo, K.H. Ching, C.M. Klimavicz, M.J. Iadarola, Antibody profiling by luciferase immunoprecipitation systems (LIPS), J. Vis. Exp. 1549 (2009). [20] P.D. Burbelo, E.E. Lebovitz, A.L. Notkins, Luciferase immunoprecipitation systems for measuring antibodies in autoimmune and infectious diseases, Transl. Res. 165 (2015) 325–335. [21] Y. Maeda, K. Migita, O. Higuchi, A. Mukaino, H. Furukawa, A. Komori, M. Nakamura, S. Hashimoto, S. Nagaoka, S. Abiru, H. Yatsuhashi, Association between anti-ganglionic nicotinic acetylcholine receptor (gAChR) antibodies and HLA-DRB1 alleles in the Japanese population, PLoS One 11 (2016) e0146048. [22] A. Mukaino, S. Nakane, O. Higuchi, H. Nakamura, T. Miyagi, K. Shiroma, T. Tokashiki, Y. Fuseya, K. Ochi, M. Umeda, T. Nakazato, Insights from the ganglionic acetylcholine receptor autoantibodies in patients with Sjögren's syndrome, Mod. Rheumatol. 26 (2016) 708–715. [23] C. Vitali, S. Bombardieri, R. Jonsson, H.M. Moutsopoulos, E.L. Alexander, S.E. Carsons, T.E. Daniels, P.C. Fox, R.I. Fox, S. Kassan, S.R. Pillemer, Classification criteria for Sjögren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group, Ann. Rheum. Dis. 61 (2002) 554–558. [24] H. Tsuboi, S. Hagiwara, H. Asashima, H. Umehara, A. Kawakami, H. Nakamura, H. Sano, K. Tsubota, Y. Ogawa, E. Takamura, I. Saito, Validation of different sets of criteria for the diagnosis of Sjögren's syndrome in Japanese patients, Mod. Rheumatol. 23 (2013) 219–225. [25] Guidelines for the Diagnosis of Chronic Thyroiditis. Japan Thyroid Association. http://www.japanthyroid.jp/en/guidelines.html#Chr. [26] K. Kimpinski, V. Iodice, S. Vernino, P. Sandroni, P.A. Low, Association of N-type calcium channel autoimmunity in patients with autoimmune autonomic ganglionopathy, Auton. Neurosci. 150 (2009) 136–139. [27] A. Mckeon, E.E. Benarroch, Autoimmune autonomic disorders, Handb. Clin. Neurol. 133 (2016) 405–416.

Declaration of competing interest None of the authors have any conflicts of interest to disclose. Acknowledgements The authors would like to thank Dr. S. Orimo (Department of Neurology, Kanto Central Hospital, Tokyo, Japan) for helpful discussions. The authors are grateful to Drs. Masataka Umeda, Kunihiro Ichinose, Hideki Nakamura, Hitomi Minami, Hajime Isomoto, Akio Ido, Kiyoshi Migita, and Kazuhiko Nakao for useful discussions. The authors are indebted to members of Kumamoto University Hospital Department of Neurology and Nagasaki Kawatana Medical center Department of Neurology for discussing some issues for this study and to Nana Kusumoto, Yuka Okumura, Keiko Hida, Haruna Akaishi, and Haruka Ikezaki for providing excellent secretarial support. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jaut.2020.102403. Funding This study was supported by the Ministry of Health, Labor, and Welfare, Japan, and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (JSPS KAKENHI Grant Numbers 16K09695 and 19H03549). Author contributions Conceived and designed the experiments: SN, AM, OH, HM, and YA. Performed the experiments: SN, AM, OH, and YM. Collected the samples and summarised the cases: SM, AM, KT, MY, MW, NT, KN and AK. Analysed the data: SN, AM, OH, AK, HM, and YA. Wrote the paper: SN, AM, OH, AK, HM, and YA. 7

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[39] R.C. Hogg, M. Raggenbass, D. Bertrand, Nicotinic acetylcholine receptors: from structure to brain function, Rev. Physiol. Biochem. Pharmacol. 147 (2003) 1–46. [40] N.S. Philip, L.L. Carpenter, A.R. Tyrka, L.H. Price, Nicotinic acetylcholine receptors and depression: a review of the preclinical and clinical literature, Psychopharmacology 212 (2010) 1–12. [41] M. Reuter, S. Markett, M. Melchers, C. Montag, Interaction of the cholinergic system and the hypothalamic-pituitary-adrenal axis as a risk factor for depression: evidence from a genetic association study, Neuroreport 23 (2012) 717–720. [42] J.Y. Streifler, Y. Molad, Connective tissue disorders: systemic lupus erythematosus, Sjögren's syndrome, and scleroderma, Handb. Clin. Neurol. 119 (2014) 463–473. [43] C. Ramos-Remus, S. Duran-Barragan, J.D. Castillo-Ortiz, Beyond the joints: neurological involvement in rheumatoid arthritis, Clin. Rheumatol. 31 (2012) 1–12. [44] T. Kondo, H. Inoue, T. Usui, T. Mimori, H. Tomimoto, S. Vernino, R. Takahashi, Autoimmune autonomic ganglionopathy with Sjögren's syndrome: significance of ganglionic acetylcholine receptor antibody and therapeutic approach, Auton. Neurosci. 146 (2009) 33–35. [45] Y. Li, A. Jammoul, K. Mente, J. Li, R.W. Shields, S. Vernino, A. Rae-Grant, Clinical experience of seropositive ganglionic acetylcholine receptor antibody in a tertiary neurology referral center, Muscle Nerve 52 (2015) 386–391. [46] S. Tzartos, M. Seybold, J. Lindstrom, Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies, Proc. Natl. Acad. Sci. U.S.A. 79 (1982) 188–192. [47] K. Kimpinski, V. Iodice, P. Sandroni, R.D. Fealey, S. Vernino, P.A. Low, Sudomotor dysfunction in autoimmune autonomic ganglionopathy, Neurology 73 (2009) 1501–1506. [48] M.C. Tobes, S. Jr Jaques, D.M. Wieland, J.C. Sisson, Effect of uptake-one inhibitors on the uptake of norepinephrine and metaiodobenzylguanidine, J. Nucl. Med. 26 (1985) 897–907. [49] M. Nakajo, K. Shimabukuro, H. Yoshimura, R. Yonekura, Y. Nakabeppu, P. Tanoue, S. Shinohara, Iodine-131 metaiodobenzylguanidine intra- and extravesicular accumulation in the rat heart, J. Nucl. Med. 27 (1986) 84–89. [50] J.C. Sisson, D.M. Wieland, P. Sherman, T.J. Mangner, M.C. Tobes, S. Jr Jacques, Metaiodobenzylguanidine as an index of the adrenergic nervous system integrity and function, J. Nucl. Med. 28 (1987) 1620–1624.

[28] D.M. Sletten, G.A. Suarez, P.A. Low, J. Mandrekar, W. Singer, Compass 31: a refined and abbreviated composite autonomic symptom score, Mayo Clin. Proc. 87 (2012) 1196–1201. [29] M.M. Cortez, S.K. Nagi Reddy, B. Goodman, J.L. Carter, D.M. Wingerchuk, Autonomic symptom burden is associated with MS-related fatigue and quality of life, Mult. Scler. Relat. Disord. 4 (2015) 258–263. [30] C. Fokke, B. van den Berg, J. Drenthen, C. Walgaard, P.A. van Doorn, B.C. Jacobs, Diagnosis of Guillain-Barré syndrome and validation of Brighton criteria, Brain 137 (2014) 33–43. [31] S. Orimo, E. Ozawa, S. Nakade, T. Sugimoto, H. Mizusawa, 123I-metaiodobenzylguanidine myocardial scintigraphy in Parkinson's disease, J. Neurol. Neurosurg. Psychiatry 67 (1999) 189–194. [32] K. Nakajima, K. Okuda, M. Yoshimura, S. Matsuo, H. Wakabayashi, Y. Imanishi, S. Kinuya, Multicenter cross-calibration of 123I metaiodobenzylguanidine heart-tomediastinum ratios to overcome camera-collimator variations, J. Nucl. Cardiol. 21 (2014) 970–978. [33] W. Xu, A. Orr-Urtreger, F. Nigro, S. Gelber, C.B. Sutcliffe, D. Armstrong, J.W. Patrick, L.W. Role, A.L. Beaudet, M. De Biasi, Multiorgan autonomic dysfunction in mice lacking the β2 and the β4 subunits of neuronal nicotinic acetylcholine receptors, J. Neurosci. 19 (1999) 9035–9298. [34] N. Wang, A. Orr-Urtreger, A.D. Korczyn, The role of neuronal nicotinic acetylcholine receptor subunits in autonomic ganglia: lessons from knockout mice, Prog. Neurobiol. 68 (2002) 341–360. [35] V.I. Skok, Nicotinic acetylcholine receptors in autonomic ganglia, Auton. Neurosci. 97 (2002) 1–11. [36] X. Simeone, R. Karch, A. Ciuraszkiewicz, A. Orr‐Urtreger, R. Lemmens‐Gruber, P. Scholze, S. Huck, The role of the nAChR subunits α5, β2, and β4 on synaptic transmission in the mouse superior cervical ganglion, Phys. Rep. 7 (2019) e14023. [37] Z. Wang, P.A. Low, J. Jordan, R. Freeman, C.H. Gibbons, C. Schroeder, P. Sandroni, S. Vernino, Autoimmune autonomic ganglionopathy: IgG effects on ganglionic acetylcholine receptor current, Neurology 68 (2007) 1917–1921. [38] Z. Wang, P.A. Low, S. Vernino, Antibody-mediated impairment and homeostatic plasticity of autonomic ganglionic synaptic transmission, Exp. Neurol. 222 (2010) 114–119.

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