Antibodies to the α3 subunit of the ganglionic-type nicotinic acetylcholine receptors in patients with autoimmune encephalitis.

Since autonomic dysfunction is closely associated with autoimmune encephalitis (AE), the objective of this study was to determine the autonomic symptoms and the prevalence of anti-α3 subunit of the ganglionic-type nicotinic acetylcholine receptor (gAChRα3) antibodies in the patients with AE. We reviewed the clinical features of 19 AE patients, and specifically analyzed sera for anti-gAChRα3 antibodies using the luciferase immunoprecipitation system (LIPS) assay. Cardiovascular autonomic symptoms were found to be common in patients with AE, and hypersalivation was seen only in patients with NMDAR encephalitis. LIPS detected anti-gAChRα3 antibodies in the sera from patients with AE (5/29, 26%). This study is the first to demonstrate that clinical characteristics including autonomic symptoms of AE patients with seropositivity for gAChR autoantibodies. It will be important to verify the role of gAChR antibodies in autonomic dysfunction and brain symptoms to clarify the pathogenesis of AE.

Increased serum acetylcholine receptor α1 subunit protein in anti-acetylcholine receptor antibody-positive myasthenia gravis.

Complement-dependent disruption of motor endplate is detected in anti-acetylcholine receptor (AChR) antibody-positive myasthenia gravis (MG). We measured serum AChR α1 subunit protein levels, which may be associated with neuromuscular damage, in 55 patients with MG (47 were seropositive and 8 were negative) and in 20 controls. Serum AChR α1 subunit protein concentrations were higher in patients with anti-AChR antibody-positive MG than those in controls (P = .04), were negatively correlated with MG activities of daily living score (P = .01), and tended to be higher in ocular MG than in generalized MG. AChR α1 subunit protein elevation may be related to seropositive MG pathogenesis, especially in the ocular type.

Fatigue scores correlate with other self-assessment data, but not with clinical and biomarker parameters, in CIS and RRMS.

BACKGROUND: Fatigue is common in multiple sclerosis and is associated with reduced quality of life. This study aimed to assess the correlation between fatigue scores and data from other self-assessment questionnaires, neuropsychological tests and neuroimaging, as well as data on neuroimmunological markers in cerebrospinal fluid (CSF) and serum/plasma, in clinically isolated syndrome (CIS) and relapsing remitting MS (RRMS). METHODS: Modified fatigue impact scale (MFIS) scores were determined in 38 patients with newly diagnosed CIS or RRMS at baseline and after one year in a prospective longitudinal cohort study. Non-parametric correlation analyses were used to assess associations between MFIS scores and other self-assessment questionnaire data (Hospital Anxiety and Depression scale (HAD), Multiple Sclerosis Impact Scale 29 (MSIS-29) and Short Form 36 (SF-36)), as well as with neuropsychological test performances (e.g. Auditory Consonant Trigram Test (ACTT)), clinical parameters (e.g. disease duration and expanded disability status scale (EDSS)), magnetic resonance imaging (MRI) data (number of T2 lesions in brain MRI and total brain volume) and several neurodegenerative/neuroinflammatory markers in CSF and serum/plasma (IL-1ß, IL-6, CXCL1, CXCL10, CXCL13, CCL-22 in plasma; neurofilament light chain (NFL) in serum; IL-6, CXCL1, CXCL10, CXCL13, CCL22, NFL and chitinase-3-like-1 (CHI3L1) in CSF. CSF and serum/plasma from 21 age- and sex-matched healthy controls were available for comparison. RESULTS: At both baseline and one-year follow-up, fatigue scores correlated significantly with HAD, MSIS-29 and SF-36 scores and ACTT performance (Spearman´s rho 0.45-0.78, all p ≤ 0.01) but not with the other neuropsychological test results, disease duration, EDSS ratings, number of T2 lesions, total brain volume or neurodegenerative/neuroinflammatory markers, including neurofilament light chain levels in CSF and serum. In group comparisons, MFIS scores were similar in patients fulfilling no evidence of disease activity-3 (NEDA-3) (n = 18) and patients not fulfilling NEDA-3 (n = 20) during one year of follow-up (p > 0.01). CONCLUSIONS: In this cohort of patients with newly diagnosed CIS and RRMS, fatigue scores were associated with mood, disease impact on daily life and quality of life as well as with alterations of attentive functions. Study results indicate that subjective fatigue scores are not well reflected by some commonly used and objectively measurable disease parameters like EDSS, T2 lesions and NFL levels.

Correlation of HLA-DQ and TNF-α gene polymorphisms with ocular myasthenia gravis combined with thyroid-associated ophthalmopathy.

The present study aims to explore the correlation of human leucocyte antigen (HLA)-DQ and tumour necrosis factor (TNF)-α gene polymorphisms with ocular myasthenia gravis (OMG) combined with thyroid-associated ophthalmopathy (TAO). From March 2009 to March 2015, 56 OMG patients complicated with TAO (OMG + TAO group), 134 patients diagnosed with OMG only (OMG group) and 236 healthy individuals (control group) were enrolled in the present study. PCR-sequence specific primer (PCR-SSP) was used for HLA-DQ genotyping and PCR-restriction fragment length polymorphism (PCR-RFLP) for TNF-α genotyping. ELISA kit was applied to detect acetylcholine receptor antibody (AchRAb) level and chemiluminescence immunoassay (CLIA) to measure thyroid-associated antibody (T-Ab) level. Logistic regression analysis was carried out to analyse the risk factors for OMG combined with TAO. DQA1*0103 showed lower frequency in the OMG group than in the control group. DQA1*0301 showed increased and DQB1*0601 showed decreased frequency in the OMG + TAO group. DQB1*0501 showed higher frequency in the OMG and OMG + TAO groups than in the control group. Patients carrying TNF-α -863C > A (CA + AA) might confront with greater risks of OMG combined with TAO. Frequency of DQA1*0103/*0301 and DQB1*0501/*0601, and TNF-α -863C > A, -238G > A and -308G > A were associated with the levels of AchRAb and T-Ab. TNF-α -863C > A (CA + AA) and high level of T-Ab were risk factors for OMG combined with TAO. Our results demonstrate that TNF-α -863 polymorphism is possibly correlated with the risk of OMG combined with TAO.

Megakaryocytes and platelets express nicotinic acetylcholine receptors but nicotine does not affect megakaryopoiesis or platelet function.

In our previous investigations we have shown that platelets and their precursors express nicotinic α7 acetylcholine receptors (nAChRα7) that are involved in platelet function and in vitro differentiation of the megakaryoblastic cell line MEG-01. In this study, we were interested in the expression analysis of additional nAChR and the effects of nicotine in an ex vivo model using megakaryocytic cells differentiated from cord blood derived CD34(+) cells (CBMK) and an in vivo model using blood samples from smokers. CBMK were differentiated with thrombopoietin (TPO) for up to 17 days. Quantitative real-time PCR (QRT-PCR), Western blot analysis and flow cytometry were used to investigate nAChR expression (nAChRα7, nAChRα4, nAChRß2) and nicotine effects. In blood samples of 15 nonsmokers and 16 smokers platelet parameters (count, mean platelet volume--MPV and platelet distribution width--PDW) were determined as indicators for changes of in vivo megakaryopoiesis. Platelet function was determined by the use of whole blood aggregometry and flow cytometry. The functional role of nAChR was evaluated using specific antagonists in aggregometry. CHRNA7, CHRNA4 and CHRNB2 gene transcripts and the corresponding proteins could be identified in CBMK during all stages of differentiation. Platelets contain nAChRα7 and nAChRß2 but not nAChRα4. Nicotine had no effect on TPO-induced differentiation of CBMK. There was no significant difference in all platelet parameters of the smokers compared to the nonsmokers. In line with this, cholinergic gene transcripts as well as the encoded proteins were equally expressed in both the study groups. Despite our observation of nAChR expression in megakaryopoiesis and platelets, we were not able to detect effects of nicotine in our ex vivo and in vivo models. Thus, the functional role of the nAChR in these cells remains open.

Attenuation of Collagen-Induced Arthritis in rat by nicotinic alpha7 receptor partial agonist GTS-21.

This research was performed to observe the effect of GTS-21 on Collagen Induced Arthritis (CIA). CIA model was used and after the onset of arthritis, the rats were divided into three groups based on their clinical symptoms score. Two groups were intraperitoneally (IP) injected daily with GTS-21 (1 mg/kg, 2.5 mg/kg) for a week, whereas phosphate buffered saline (PBS) was used for the control group. Cytokine titers, radiological, and histological examinations were performed at different time points after treatment with GTS-21. Compared with those of the control, the levels of TNF-α, IL-1, and IL-6 in the serum were significantly reduced after GTS-21 management. In addition, radiological results show that bone degradation was inhibited as well. Moreover, the hematoxylin and eosin (H&E) staining indicated that the histological score was significantly alleviated in the therapeutic group. Tartrate-resistant acid phosphatase (TRAP) stain-positive cells were also detected in the destruction of the articular cartilage, which was significantly reduced compared with the control group. This study provides the first evidence on the effect of GTS-21 as a potential treatment for RA.

Niacin and biosynthesis of PGD2by platelet COX-1 in mice and humans.

The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1-dependent formation of PGD2 and PGE2 followed by COX-2-dependent production of PGE2. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD2 receptor DP1. NSAID-mediated suppression of COX-2-derived PGI2 has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD2. Here, we show that PGD2 biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD2 biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD2 was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD2, like PGI2, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy.

Role of α7 nicotinic acetylcholine receptors in regulating tumor necrosis factor-α (TNF-α) as revealed by subtype selective agonists.

Immunological responses to protect against excessive inflammation can be regulated by the central nervous system through the cholinergic anti-inflammatory pathway wherein acetylcholine released from vagus nerves can inhibit inflammatory cytokines. Although a role for the α7 nicotinic acetylcholine receptor (α7 nAChR) in mediating this pathway has been suggested, pharmacological modulation of the pathway by selective agonists remains to be further elucidated. In this study, the role of α7 nAChRs in the regulation of TNF-α release was investigated using high affinity and selective α7 nAChR agonists in mouse peritoneal macrophage and human whole blood in vitro, and in mouse serum in vivo. In mouse peritoneal macrophages, LPS-induced TNF-α release in vitro was inhibited by a selective α7 nAChR agonist, A-833834 (5-[6-(5-Methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyridazin-3-yl]-1H-indole), and that effect was attenuated by α7 nAChR antagonist methyllycaconitine. The inhibitory effect of A-833834 on LPS-induced TNF-α release was also observed in human whole blood in vitro. I.v. LPS-induced TNF-α release in mouse serum was attenuated following i.p. administration of A-833834. Similarly, i.v. LPS-induced TNF-α release in mouse serum was also attenuated following i.p. administration of A-585539, another α7 nAChR agonist with limited brain penetration, suggesting that these effects are mediated by peripheral α7 nAChRs. A-833834 was also efficacious in suppressing TNF-α release in mouse serum following oral administration in zymosan-induced peritonitis. These studies collectively demonstrate that selectively targeting α7 nAChRs could offer a novel therapeutic modality to treat acute and chronic inflammatory disease states.

Human platelets express functional alpha7-nicotinic acetylcholine receptors.

OBJECTIVE: Nicotinic acetylcholine receptors, especially α7 (nAChRα7), form Ca(2+) channels and are expressed on a variety of neuronal and nonneuronal cells. Also, megakaryocytic cells have been shown to contain components of a nonneuronal cholinergic system, including acetylcholine and acetylcholine esterase. However, the corresponding nAChRs and their role in platelet function have not been demonstrated until now. Our previous platelet transcriptome data indicated the presence of nAChR gene transcripts. METHODS AND RESULTS: Here, we present evidence that human platelets and megakaryocytic precursor cells express nAChRα7 subunits, as revealed by mRNA and protein expression. The subunits form functional Ca(2+) channels, as demonstrated by Ca(2+) entry in platelets induced by the nAChRα7-selective agonist PNU-282987. PNU-282987 also enhanced fibrinogen receptor activation induced by classical platelet agonists (the thromboxane A(2) analog U46619 and ADP). Furthermore, agonist-induced platelet aggregation was significantly inhibited by the nAChRα7-selective antagonists α-bungarotoxin and methyllycaconitine. CONCLUSIONS: Ca(2+) influx via nAChRα7 channels represents a novel pathway for human platelets with significant impact on platelet function. Because platelets were suggested to contain acetylcholine, we conclude that on activation, stored acetylcholine is released, which activates nAChRα7 channels and thereby contributes to maintaining intracellular Ca(2+) levels and supporting platelet activation.

Cholinesterase activity and mRNA level of nicotinic acetylcholine receptors (alpha4 and beta2 Subunits) in blood of elderly Chinese diagnosed as Alzheimer's disease.

The aim of the study is to investigate the cholinergic deficit in Alzheimer's disease (AD) and identify candidate blood biomarkers for the diagnosis of the disease. Twenty-nine elderly Chinese diagnosed with AD and 33 age-matched controls were selected. The activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in plasma were detected by a spectrophotometric method, and the mRNA levels of alpha4 and beta2 nicotinic acetylcholine receptor (nAChR) subunits in blood leukocytes were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that AChE activity in plasma was significantly lower in the AD group than in normal controls, while BuChE activity did not show any differences between AD and controls; mRNA levels of both alpha4 and beta2 nAChR subunits in blood leukocytes were significantly lower in the AD group than in controls. The AChE activity and the mRNA levels of alpha4 and beta2 nAChR subunits in the AD patients were also significantly correlated with cognitive test scores. No differences of AChE in plasma or alpha4 and beta2 nAChR subunits in blood leukocytes were detected between smoking and non-smoking subjects. The results indicated that the decreases in the activity of AChE and in the mRNA levels of nAChR alpha4 and beta2 subunits from the peripheral blood of patients with AD might serve as supplementary indicators for the clinical diagnosis of AD.

Increased antibodies for the alpha7 subunit of the nicotinic receptor in schizophrenia.

One of the etiological theories of schizophrenia is dysregulation of the immune system. Autoantibodies specific for the alpha7 subunit of the nicotinic receptor could potentially contribute to the pathophysiology of the disease. In this study, positive antibodies specific for the receptor were found to exist in 23% of the patients diagnosed with schizophrenia (n=21). On the average, levels for the antibody were elevated in the schizophrenia patient population than in controls. The data also suggests that there is a significant correlation between antibody titer and age, lending support to the neurodegenerative nature of the disease.

Differentiating nicotine- versus schizophrenia-associated decreases of the alpha7 nicotinic acetylcholine receptor transcript, CHRFAM7A, in peripheral blood lymphocytes.

Nicotine addiction is prevalent in individuals with schizophrenia. Nicotine activation of nicotinic receptors (nAChRs) is time- and dose-dependent, but gene expression analyses often rely on qualitative self- or family-reported measures of smoking. We sought lymphocyte surrogates for cerebral alpha7-nAChR activity and tested if receptor transcription correlated with concurrently measured serum biomarkers for smoking [cotinine, C-reactive protein (CRP)]. PCR surveys to detect lymphocytic alpha7-related isoforms identified CHRFAM7A as the only consistently amplifiable transcript. In 20 smoking-matched people (n = 10 schizophrenia, n = 10 controls), we found significantly lower CHRFAM7A in cotinine and self-reported smokers versus nonsmokers (p

Association of idiopathic generalized epilepsy with polymorphisms in the neuronal nicotinic acetylcholine receptor subunits.

Idiopathic generalized epilepsy (IGE) refers to a common group of epilepsies, and genetic factors play an important role in the pathogenesis of these disorders. Mutations in CHRNA4 and CHRNB2 are associated with some cases of familial epilepsies classified as autosomal-dominant nocturnal frontal lobe epilepsies. We aimed to evaluate whether polymorphisms of CHRNA4 and CHRNB2 are associated with IGE. A total of 75 children with IGE and 80 normal control subjects were included in the study. Each genetic polymorphism was typed by polymerase chain reaction (PCR)-based restriction analysis. The genotypes and allelic frequencies of each polymorphism were compared between the IGE patients and controls. The results showed that genotype and allelic frequency for CHRNB2 did not differ significantly between the groups. However, the genotype proportion of the CHRNA4 (Ser543Ser) gene in both groups was significantly different (P<0.0001). The T allele frequency was significantly higher (P=0.0126) in patients with IGE compared to healthy controls. The odds ratio (OR) for developing IGE in individuals with the CHRNA4 (Ser543Ser)-T homozygote was 4.9 (95% confidence interval (CI), 1.71-14.04) compared to individuals with two copies of the CHRNA4 (Ser543Ser)-C allele. This study demonstrates that the CHRNA4 gene may be one of the susceptibility factors for IGE.

Nicotine inhibits the production of proinflammatory mediators in human monocytes by suppression of I-kappaB phosphorylation and nuclear factor-kappaB transcriptional activity through nicotinic acetylcholine receptor alpha7.

Macrophages/monocytes and the proinflammatory mediators, such as tumour necrosis factor (TNF)-alpha, prostaglandin E(2) (PGE(2)), macrophage inflammatory protein (MIP)-1alpha and MIP-1alpha, play a critical role in the progression of immunological disorders including rheumatoid arthritis, Behçet's disease and Crohn's disease. In addition, the nicotinic acetylcholine receptor-alpha7 (alpha7nAChR) subunit is an essential regulator of inflammation. In this study, we evaluated the expression of the alpha7nAChR subunit on human peripheral monocytes and the effect of nicotine on the production of these proinflammatory mediators by activated monocytes. Fluorescein isothiocyanate (FITC)-labelled alpha-bungarotoxin demonstrated the cell surface expression of the alpha7nAchR subunit. Pretreatment with low-dose nicotine caused inhibition of TNF-alpha, PGE(2), MIP-1alpha and MIP-1alpha production, and mRNA expression of TNF-alpha, MIP-1alpha and MIP-1alpha and COX-2 in lipopolysaccharide (LPS)-activated monocytes. These suppressive effects of nicotine were caused at the transcriptional level and were mediated through alpha7nAChR. Nicotine suppressed the phosphorylation of I-kappaB, and then inhibited the transcriptional activity of nuclear factor-kappaB. These immunosuppressive effects of nicotine may contribute to the regulation of some immune diseases.

Alpha7-acetylcholine receptor antibodies in two patients with Rasmussen encephalitis.

Rasmussen encephalitis (RE) sera were screened for antibodies to human alpha7 nicotinic acetylcholine receptors (nAChRs) using electrophysiology, calcium imaging, and ligand binding assays. Sera from two of nine patients with RE blocked ACh-induced currents through alpha7 nAChRs and the ACh-induced rise in intracellular free calcium ([Ca2+]i) and inhibited (125)I-alpha-bungarotoxin binding in cells expressing alpha7 nAChRs. Thus, the alpha7 nAChR is a potential target for pathogenic antibodies in patients with RE.

Increased alpha 7 nicotinic acetylcholine receptor protein levels in Alzheimer's disease patients.

We compared the intact alpha7 nicotinic acetylcholine receptor (alpha7nAChR) protein levels in the peripheral blood leukocytes in 15 Alzheimer's disease (AD) patients and 13 normal elderly control subjects. Demographic data and Mini-Mental State Examination (MMSE) scores were obtained. Western blot analysis for alpha7nAChR protein levels in peripheral blood leukocytes was performed. There were no significant differences in sex and age between the AD and control groups. The mean MMSE score of the AD subjects was significantly lower than that of the normal control subjects (15.4 +/- 5.5 vs. 28.5 +/- 1.9 respectively; p < 0.001). The median value of normalized alpha7nAChR protein levels (optical density, arbitrary unit) of the AD group was significantly higher than that of the normal control group (0.6923 vs. 0.4803 respectively; p = 0.045, Mann-Whitney U test). The normalized alpha7nAChR protein levels showed a significant inverse correlation with the MMSE scores (Spearman rho = -0.45; p = 0.016; n = 28). Receiver Operating Characteristic curve analyses showed that the area under curve was 0.72 (95% CI 0.52- 0.87). If the cut-off of the alpha7nAChR protein level was >0.312, the sensitivity, specificity, positive predictive value and negative predictive value would be 80, 39, 60 and 63%, respectively. These findings showed that the alpha7nAChR protein levels would be a potentially useful diagnostic marker for AD.

[(3)H]Nicotine binding in peripheral blood cells of smokers is correlated with the number of cigarettes smoked per day.

The principal sites for biological action of tobacco products are thought to be the nicotinic acetylcholine receptors (nAChR). Nicotinic receptor subunit genes, therefore, represent an important gene family for study in nicotine addiction. They are localized in both brain and in the periphery. In brain these receptors appear to function as modulators of synaptic transmission; the function of peripheral receptors is not known. Nicotinic receptor levels in human brain are regulated by smoking in a dose-dependent manner. In peripheral blood, nicotinic receptors are present on both lymphocytes and polymorphonuclear cells (PMN). We have compared [(3)H]nicotine binding in PMN isolated from smokers and non-smokers. [(3)H]nicotine binding was increased in smokers and was correlated, as in brain, with tobacco use. Expression of both mRNA and protein in lymphocytes and PMN, for a subset of nicotinic receptor subunits, suggests that these cell types contain both alpha4beta2 and alpha3beta4 receptors.

Absence of (-) [3H]desmethoxyverapamil binding sites on human platelets and lack of evidence for voltage-dependent calcium channels.

The two major pathways for Ca2+ entry into cells are potential-sensitive channels and receptor-operated channels. The main object of this investigation was to identify which mechanism regulates Ca2+ entry into human platelets. Platelet stimulation with thrombin, adenosine diphosphate, platelet activating factor and arachidonic acid resulted in a concentration-dependent 2.5-3-fold increase in cytoplasmic free calcium concentration over the basal levels (140 +/- 32 nM or 104 +/- 21 respectively) as measured with the fluorescent dyes Quin-2 and Fura-2. Adrenaline and collagen had no effect in promoting intracellular Ca2+ increase as measured with Quin-2 and little effect when measured with Fura-2. Incubation of Quin-2-loaded platelets with the calcium antagonists verapamil and diltiazem, which are known to inhibit Ca2+ entry from voltage-gated channels in many types of cells, over the concentration range 10(-8) - 10(-4) M did not alter significantly either the resting or the cytoplasmic free Ca2+ after stimulation of platelets by several agonists. Moreover, the calcium antagonists exhibited little or no effect on aggregation and 5-hydroxytryptamine secretion induced by platelet activating factor, adenosine diphosphate, collagen or arachidonic acid in whole blood, platelet-rich plasma or washed platelets when employed at concentration ranges as above. Similar results were obtained in washed thrombin-stimulated platelets. High doses of verapamil (but not diltiazem) inhibited platelet aggregation and secretion in response to adrenaline. Direct radioligand binding studies with (-)[3H]desmethoxyverapamil showed that platelet membranes have no receptors for this drug, suggesting that Ca2+ entry occurs in human platelets via a pathway different from potential-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)