SUMMARYIn this study we demonstrate that IgG present in the sera of patients with primary Sjögren's syndrome (PSS) could bind and activate muscarinic acetylcholine receptors (mAChRs) of rat parotid gland. These antibodies were able to inhibit in a non-competitive manner the binding of 3 H-quinuclidinyl benzilate (QNB) to mAChRs of purified rat parotid gland membranes. Moreover, IgG from PSS could modify biological effects mediated by mAChR activation; i.e. decrease cAMP, increase phosphoinositide turnover without affecting cGMP. Atropine and 4-DAMP blocked all of these effects, and carbachol mimicked them, confirming the M 3 subtype mAChRs mediated PSS IgG action. Neither binding nor biological effect were obtained with IgG from sera of normal women. The prevalence of cholinergic antibody was 100% in PSS, and was independent of Ro/SS-A and La/SS-B antibodies. It could be concluded that antibody against mAChRs may be another serum factor to be considered in the pathophysiology of the development of PSS.

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Circulating antibodies from human and murine chagasic sera are able to interact with myocardium-activating neurotransmitter receptors. Here we reported the presence of autoantibodies against the second extracellular loop of the human heart muscarinic acetylcholine receptors (mAChR) in patients with Chagas' disease by using a synthetic 24-mer peptide in immunoblotting and enzyme immunoassay. Affinity-purified antipeptide IgG from chagasic patients, similar to monoclonal antihuman M2 mAChR, recognized bands with a molecular weight corresponding to the cardiac mAChR. The binding was inhibited by the peptide, assessing the specificity of the interaction. The antipeptide autoantibody also displayed an "agonist-like" activity modifying the intracellular events associated with mAChR activation, i.e., decreased contractility, increased cGMP, and decreased cAMP production. All of these effects on rat atria by chagasic antipeptide autoantibodies resemble the effects of the authentic agonist and those of the total polyclonal chagasic IgG, being selectively blunted by atropine and neutralized by the synthetic peptide corresponding in aminoacid sequence to the second extracellular loop of the human M2 mAChR. A clinical relevance of these findings is demonstrated by a strong association between the existence of circulating antipeptide autoantibodies in chagasic patients and the presence of dysautonomic symptoms, making these autoantibodies a proper early marker of heart autonomic dysfunction.

Orthodontic appliances serve as different impact zones and modify microbial adherence and colonization, acting as foreign reserves and possible sources of infection.

1 In this paper we have determined the different signalling pathways involved in muscarinic acetylcholine receptor (AChR)-dependent inhibition of contractility in rat isolated atria. 2 Carbachol stimulation of M2 muscarinic AChRs exerts a negative inotropic response, activation of phosphoinositide turnover, stimulation of nitric oxide synthase and increased production of cyclic GMP. 3 Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase, shifted the dose-response curve of carbachol on contractility to the right. These inhibitors also attenuated the muscarinic receptor-dependent increase in cyclic GMP and activation of nitric oxide synthase. In addition, sodium nitroprusside, isosorbide, or 8-bromo cyclic GMP, induced a negative inotropic effect, increased cyclic GMP and activated nitric oxide synthase. 4 These results suggest that carbachol activation of M2 AChRs, exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositides turnover via phospholipase C activation. This in turn, triggers cascade reactions involving calcium/calmodulin and protein kinase C, leading to activation of nitric oxide synthase and soluble guanylate cyclase.

We demonstrated the presence of circulating Abs from schizophrenic patients able to interact with cerebral frontal cortex-activating muscarinic acetylcholine receptors (mAChR). Sera and purified IgG from 21 paranoid schizophrenic and 25 age-matched normal subjects were studied by indirect immunofluorescence, flow cytometry, immunoblotting, dot blot, ELISA, and radioligand competition assays. Rat cerebral frontal cortex membranes and/or a synthetic peptide, with an amino acid sequence identical with that of human M1 mAChR, were used as Ags. By indirect immunofluorescence and flow cytometry procedures, we proved that serum-purified IgG fraction from schizophrenic patients reacted to neural cell surfaces from rat cerebral frontal cortex. The same Abs were able to inhibit the binding of the specific M1 mAChR radioligand [3H]pirenzepine. Immunoblotting experiments showed that IgG from schizophrenic patients revealed a band with a molecular mass coincident to that labeled by an anti-M1 mAChR Ab. Using synthetic peptide for dot blot and ELISA, we demonstrated that these Abs reacted against the second extracellular loop of human cerebral M1 mAChR. Also, the corresponding affinity-purified antipeptide Ab displayed an agonistic-like activity associated to specific receptor activation, increasing cyclic GMP production and inositol phosphate accumulation, and protein kinase C translocation. This paper gave support to the participation of an autoimmune process in schizophrenia.

Objective-To assess whether exposure of cardiac muscarinic acetylcholine receptors (mAChR) to activating chagasic antimyocardial immunoglobulins results in bradycardia and other dysautonomic symptoms associated with the regulation of heart rate. Methods-Trypanosoma cruzi infected patients with bradycardia and other abnormalities in tests of the autonomic nervous system were studied and compared with normal subjects. Antipeptide antibodies in serum were demonstrated by an enzyme linked immunosorbent assay using a synthetic 24-mer-peptide corresponding antigenically to the second extracellular loop of the human heart M 2 mAChR. The functional eVect of aYnity purified antipeptide IgG from chagasic patients on spontaneous beating frequency and cAMP production of isolated normal rat atria was studied.Results-There was a strong association between the finding of antipeptide antibodies in chagasic patients and the presence of basal bradycardia and an altered Valsalva manoeuvre (basal bradycardia: 2 = 37.5, p < 0.00001; Valsalva manoeuvre: 2 = 70.0, p < 0.00001). The antipeptide autoantibodies also showed agonist activity, decreasing the rate of contraction and cAMP production. The eVects on rat atria resembled the eVects of the authentic agonist and those of the total polyclonal chagasic IgG, being selectively blunted by atropine and AF-DX 116, and neutralised by the synthetic peptide corresponding in amino acid sequence to the second extracellular loop of the human M 2 mAChR. Conclusions-There is an association between circulating antipeptide autoantibodies in chagasic patients and the presence of bradycardia and other dysautonomic symptoms. Thus these autoantibodies are a marker of autoimmune cardiac autonomic dysfunction. The results support the hypothesis that autoimmune mechanisms play a role in the pathogenesis of chagasic cardioneuromyopathy. (Heart 1999;82:273-278)

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