Postural Orthostatic Tachycardia Syndrome Is Associated With Elevated G-Protein Coupled Receptor Autoantibodies.

Background The etiology of postural orthostatic tachycardia syndrome (POTS) is yet to be established. The disorder is often misdiagnosed as chronic anxiety or a panic disorder because the autonomic failure in these patients is not severe. A growing body of evidence suggests that POTS may be an autoimmune disorder. Antinuclear antibodies and elevations of ganglionic, adrenergic, and muscarinic acetylcholine receptor antibodies have all been reported. Methods and Results We collected detailed clinical symptoms of 55 patients diagnosed with POTS. We also evaluated serum levels of autoantibodies against 4 subtypes of G-protein coupled adrenergic receptors and 5 subtypes of G-protein coupled muscarinic acetylcholine receptors by ELISA. Our patients had a multitude of comorbidities, were predominantly young females, and reported viral-like symptoms preceding episodes of syncope. We detected a significant number of patients with elevated levels of autoantibodies against the adrenergic alpha 1 receptor (89%) and against the muscarinic acetylcholine M4 receptor (53%). Surprisingly, elevations of muscarinic receptor autoantibodies appeared to be dependent upon elevation of autoantibodies against the A1 adrenergic receptor! Four patients had elevations of G-protein coupled autoantibodies against all 9 receptor subtypes measured in our study. Five POTS patients had no elevation of any autoantibody; similarly, controls were also negative for autoantibody elevations. There was a weak correlation of clinical symptom severity with G-protein coupled autoantibodies. Conclusions Our observations provide further evidence that, in most cases, POTS patients have at least 1 elevated G-protein coupled adrenergic autoantibody and, in some instances, both adrenergic and muscarinic autoantibodies, supporting the hypothesis that POTS may be an autoimmune disorder.

VU6005806/AZN-00016130, an advanced M4 positive allosteric modulator (PAM) profiled as a potential preclinical development candidate.

This letter describes progress towards an M4 PAM preclinical candidate that resulted in the discovery of VU6005806/AZN-00016130. While the thieno[2,3-c]pyridazine core has been a consistent feature of key M4 PAMs, no work had previously been reported with respect to alternate functionality at the C3 position of the pyridazine ring. Here, we detail new chemistry and analogs that explored this region, and quickly led to VU6005806/AZN-00016130, which was profiled as a putative candidate. While, the ß-amino carboxamide moiety engendered solubility limited absorption in higher species precluding advancement (or requiring extensive pharmaceutical sciences formulation), VU6005806/AZN-00016130 represents a new, high quality preclinical in vivo probe.

Immunoadsorption to remove ß2 adrenergic receptor antibodies in Chronic Fatigue Syndrome CFS/ME.

INTRODUCTION: Infection-triggered disease onset, chronic immune activation and autonomic dysregulation in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) point to an autoimmune disease directed against neurotransmitter receptors. We had observed elevated autoantibodies against ß2 adrenergic receptors, and muscarinic 3 and 4 acetylcholine receptors in a subset of patients. Immunoadsorption (IA) was shown to be effective in removing autoantibodies and improve outcome in various autoimmune diseases. METHODS: 10 patients with post-infectious CFS/ME and elevated ß2 autoantibodies were treated with IA with an IgG-binding column for 5 days. We assessed severity of symptoms as outcome parameter by disease specific scores. Antibodies were determined by ELISA and B cell phenotype by flow cytometry. RESULTS: IgG levels dropped to median 0.73 g/l (normal 7-16 g/l) after the 4th cycle of IA, while IgA and IgM levels remained unchanged. Similarly, elevated ß2 IgG antibodies rapidly decreased during IA in 9 of 10 patients. Also 6 months later ß2 autoantibodies were significantly lower compared to pretreatment. Frequency of memory B cells significantly decreased and frequency of plasma cells increased after the 4th IA cycle. A rapid improvement of symptoms was reported by 7 patients during the IA. 3 of these patients had long lasting moderate to marked improvement for 6-12+ months, 2 patients had short improvement only and 2 patients improved for several months following initial worsening. CONCLUSIONS: IA can remove autoantibodies against ß2 adrenergic receptor and lead to clinical improvement. B cell phenotyping provides evidence for an effect of IA on memory B cell development. Data from our pilot trial warrants further studies in CFS/ME.

Immunoglobulin G from breast cancer patients in stage I stimulates muscarinic acetylcholine receptors in MCF7 cells and induces proliferation. Participation of nitric oxide synthase-derived nitric oxide.

INTRODUCTION: Muscarinic acetylcholine receptors (mAChR) belong to the G-protein-coupled receptor family and are extensively expressed in most cells in mammals. We had reported the expression of mAChR in murine and human breast tumors. METHODS: The presence of antibodies in the sera of patients with different tumors directed against self-proteins has been recently described. In this work, we investigated the presence of autoantibodies against mAChR in the sera of breast cancer patients in stage I (T1N0Mx-IgG). IgG purification was performed by affinity chromatography in protein G-agarose. We also studied the ability of these antibodies to modulate the proliferation of MCF-7 breast tumor cells by the MTS colorimetric assay. The ability of T1N0Mx-IgG to stimulate muscarinic signaling pathway via nitric oxide synthase was tested by Griess reaction. RESULTS: We demonstrated M(3) and M(4) receptors expression in MCF-7 cells. T1N0Mx-IgG promotes cell proliferation, mimicking the action of the muscarinic agonist carbachol. This effect was preferentially due to M(3) receptor activation in tumor cells via phospholipase C-induced nitric oxide liberation by calcium-dependent nitric oxide synthases. IgG from control patients was unable to produce this effect. DISCUSSION: IgG from patients with breast cancer in early stages could be promoting tumor progression by muscarinic activation, and its presence could be determining the prognosis of this illness.

Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice.

Acetylcholine (ACh) is a major regulator of visceral function exerting pharmacologically relevant effects upon smooth muscle tone and epithelial function via five types of muscarinic receptors (M1R-M5R). In this paper, we assessed the specificity of muscarinic receptor (MR) antibodies in immunohistochemical labelling on tissue sections by analysing specimens from wild-type and respective gene-deficient mice. Of 24 antibodies evaluated in this study, 16 were tested at 18 different conditions each, and eight of them in 21 different protocols, resulting in a total number of 456 antibody/protocol combinations. Each of them was tested at four antibody dilutions at minimum, so that finally, at least 1,824 conditions were evaluated. For each of them, dorsal root ganglia, urinary bladder and cross-sections through all thoracic viscera were investigated. In all cases where the antigen was available, at least one incubation condition was identified in which only select cell types were immunolabelled in the positive control but remained unlabelled in the pre-absorption control. With two exceptions (M2R antibodies), however, all antibodies produced identical immunohistochemical labelling patterns in tissues taken from corresponding gene-deficient mice even when the pre-absorption control in wild-type mice suggested specificity. Hence, the present data demonstrate the unpleasant fact that reliable immunohistochemical localisation of MR subtypes with antibodies is the exception rather than the rule. Immunohistochemical detection of MR subtype localisation in tissue sections of peripheral organs is limited to the M2R subtype utilising the most commonly used methodological approaches.