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In this paper, we demonstrate that circulating antibodies from chronic periodontitis patients reacting with atrial β(1)-adrenoceptors (β(1)-ARs) act as an inducer of soluble CD40 ligand (sCD40L) release and prostaglandin E(2) (PGE(2)) generation. By enzyme-linked immunosorbent assay using β(1) synthetic peptide (with an amino acid sequence identical to the second loop of human myocardial β(1)-ARs) as a coating antigen, we demonstrated reactivity against the second extracellular loop on human myocardial β(1)-ARs. This autoantibody present in the serum of chronic periodontitis patients was significantly correlated with the release of sCD40L and PGE(2). The release of sCD40L was blunted by atenolol, SP600125 and β(1) synthetic peptide, and PGE(2) generation was inhibited by DuP 697 and slightly by FR122049. The effects of the antibody incubated with isolated rat atria upregulated sCD40L release with an increase of PGE(2) production and c-Jun N-terminal kinase phosphorylation. These results indicate that in chronic periodontitis patients, there is a positive association between sCD40L release and PGE(2) generation via the action of β(1)-AR antibodies.

Background: The presence of serum autoantibodies against β(1) adrenoreceptors (β(1)-ARs) in human gingival fibroblast from patients with periodontitis inhibits primary cell-specific growth and induces over-expression of pro-inflammatory mediators. Serum β(1)-AR autoantibodies from patients with periodontitis react with myocardium and modify cardiac contractility. The relationship between the presence of serum β(1)-AR autoantibodies and alterations in heart rate variability (HRV) was also studied., Methods: An enzyme-linked immunosorbent assay (ELISA) using cardiac and gingival fibroblast membranes or synthetic peptides corresponding to the second extracellular loop of human β(1)-AR was used to detect serum autoantibodies. The HRV was assessed from RR interval files generated from 22:00 to 08:00 hours. The autoantibody effects on contractility were measured on spontaneous rat isolated atria., Results: Circulating autoantibodies from 36 patients with periodontitis and 20 healthy individuals (controls) interacted with fibroblasts, the cardiac surface, and β(1)-AR synthetic peptides. The distributions of serum antibodies against gingival and myocardium membranes and β(1)-AR synthetic peptide were 88.8%, 77.7%, and 92.8%, respectively. Moreover, 88.5% of patients with periodontitis whose sera were positive against β(1)-AR synthetic peptide had decreased HRV. The corresponding affinity-purified anti-β(1)-AR peptide IgG displayed partial agonist-like activity modifying the isolated atria contractility., Conclusion: This manuscript describes that patients with periodontitis showed increased levels of serum IgG with reactive activity against β(1)-AR. Those patients demonstrated decrease in heart rate, and IgG derived from their sera induced aberrant contractility of heart atrium. We propose that periodontitis increases the risk of cardiovascular diseases, although it increases anti-β(1)-AR autoantibody that alters myocardial contractility., (© 2011 John Wiley & Sons A/S.)

Background: We demonstrated that serum immunoglobulin G (IgG) from patients with primary Sjögren's syndrome (pSS), interacting with the second extracellular loop of human glandular M(3) muscarinic acetylcholine receptors (M(3) mAChR), trigger the production of matrix metalloproteinase-3 (MMP-3) and prostaglandin E(2) (PGE(2))., Methods: Enzyme-linked immunosorbent assays (ELISAs) were performed in the presence of M(3) mAChR synthetic peptide as antigen to detect in serum the autoantibodies. Further, MMP-3 and PGE(2) production were determined in the presence of anti-M(3) mAChR autoantibodies., Results: An association was observed between serum and anti-M(3) mAChR autoantibodies and serum levels of MMP-3 and PGE(2) in pSS patients. Thus, we established that serum anti-M(3) mAChR autoantibodies, MMP-3 and PGE(2) may be considered to be early markers of pSS associated with inflammation. Affinity-purified anti-M(3) mAChR peptide IgG from pSS patients, whilst stimulating salivary-gland M(3) mAChR, causes an increase in the level of MMP-3 and PGE(2) as a result of the activation of phospholipase A(2) (PLA(2)) and cyclooxygenase-2 (COX-2) (but not COX-1)., Conclusions: These results provide a novel insight into the role that cholinoceptor antibodies play in the development of glandular inflammation. This is the first report showing that an antibody interacting with glandular mAChR can induce the production of pro-inflammatory mediators (MMP-3/PGE(2))., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We demonstrate that patients with primary Sjögren's syndrome (pSS) produce functional IgG autoantibodies that interact with the glandular M(3) muscarinic acetylcholine receptors (mAChRs). These autoantibodies act as a partial muscarinic agonist, increasing prostaglandin E(2) (PGE(2)) and cyclic AMP production through modifying Na(+)/K(+)-ATPase activity, but also interfere with the secretory effect of the parasympathetic neurotransmitter. The IgG from patients with pSS has two effects on the submandibular gland. On the one hand, it may act as an inducer of the proinflammatory molecule (PGE(2)) that, in turn, inhibits Na(+)/K(+)-ATPase activity. On the other hand, it plays a role in the pathogenesis of dry mouth, abolishing the Na(+)/K(+)-ATPase inhibition and the net K(+) efflux stimulation of the salivary gland in response to the authentic agonist pilocarpine, decreasing salivary fluid production.

We demonstrated that circulating antibodies from schizophrenia patients, which interact with cerebral M1 muscarinic acetylcholine receptors (M1 mAChRs), trigger production of nitric oxide (NO), prostaglandin E2 (PGE2) and matrix metalloproteinase-3 (MMP-3), and act as inducers of cyclooxygenase-1 (cox-1) and inducible nitric oxide synthase (iNOS) mRNA expression in the rat frontal cortex. The corresponding affinity-purified anti-M1 peptide IgG from schizophrenia patients, while stimulating cerebral M1 mAChRs, increases NOS activity, PGE2 and MMP-3 production associated with iNOS over-activity and mRNA expression. Moreover, PGE2 and MMP-3 production is the result of cox-1 expression and activity. All these effects were inhibited by pirenzepine or haloperidol and mimicked the action of the authentic mAChR agonist. Concurrent analysis of the effects of iNOS, phospholipase C, protein kinase C and calcium/calmodulin inhibition showed that antibody up-regulation of NOS activity, PGE2 and MMP-3 production is under the control of the endogenous NO signalling system. These results provide evidence of the role that cholinergic receptor antibodies play in the development of cerebral inflammation, which shows that an antibody that interacts with cerebral mAChRs can induce expression of pro-inflammatory mediators, and support the participation of an autoimmune process in a particular group of chronic schizophrenia patients.

Background and Objective: Autoimmune mechanisms may contribute to the pathogenesis of periodontal disease. Autoantibodies with the potential to bind and activate beta(1)-adrenoceptors (beta(1)-AR) of human gingival fibroblasts were studied to provide evidence of altered humoral immune response in chronic periodontal disease., Material and Methods: Flow cytometry and enzyme-linked immunosorbent assay using cell culture-adherent gingival fibroblasts and/or their purified membranes and/or a synthetic peptide corresponding to the second extracellular loop of human beta(1)-AR were used to detect serum antibodies. The effects of antibodies from chronic periodontal disease patients on PGE(2) generation and CD40 expression were also tested., Results: Circulating immunoglobulin G (IgG) from chronic periodontal disease patients (but not from normal individuals) interacted with the fibroblast surface, activating beta(1)-AR. Atenolol or CGP 20712 (beta 1-AR antagonists) and beta(1) synthetic peptide inhibited the interaction of IgG with beta(1)-AR. Immunoglobulin G from chronic periodontal disease patients also displayed agonist-like activity associated with specific beta(1)-AR activation, increasing PGE(2) generation and CD40 overexpression. The corresponding affinity-purified anti-beta(1)-AR peptide IgG mimicked these effects. Both effects were prevented by inhibition of cyclo-oxygenase., Conclusion: This article supports the participation of humoral immune alterations in chronic periodontal disease resulting in postsynaptic functional deregulation. Overproduction of proinflammatory mediators (PGE(2) and CD40 expression) is induced as a consequence of antibody-beta(1)-AR interaction. The PGE(2)-CD40-IgG axis may play a part in the pathophysiological mechanisms underlying the inflammatory process in chronic periodontal disease.

The presence of circulating antibodies from primary Sjögren Syndrome (pSS) patients enable to interact with rat cerebral frontal cortex by activating muscarinic acetylcholine receptors (mAChR). ELISA assay for PGE2 generation, nitric oxide synthase (NOS) activity was measured in cerebral frontal cortex slices by production of [U-14C]-citruline and mRNA isolation/quantitative PCR for COX-1 and COX-2 gene expression were carried out. By ELISA assay, it was shown that IgG from pSS patients reacted to cerebral frontal cortex cell surface and with human M1 and M3 mAChR. Beside pSS IgG displayed an agonistic-like activity stimulating NOS activity and PGE2 production associated with an increased COX-1 mRNA gene expression, without affecting COX-2 mRNA levels. Inhibition of phospholipase A2 (PLA2) and NOS prevented pSS IgG effects upon both PGE2 production and COX-1 mRNA levels. The results support the notion that serum IgG auto antibodies in pSS patients target cerebral mAChR may have pathogenic role in immune neuroinflammation and on cognitive dysfunction present in pSS patients.

Previous studies have demonstrated that antibodies against cholinoreceptors of exocrine glands correlate with dry mouth in persons with primary Sjögren syndrome (pSS). The aim of the present investigation was to establish if serum IgG antibodies (pSS IgG) were able to interact with cholinoreceptors in rat submandibular gland-dependent stimulation of cyclooxygenase 2 (COX-2) mRNA expression and PGE(2) production. Our findings indicated that pSS IgG-stimulating M(3), M(4), and M(1) cholinoreceptors exerted an increase in COX-2 mRNA without affecting COX-1 mRNA expression and increased PGE(2) production. Inhibitors of phospholipase A(2), COX- s, L-type calcium channel currents, and Ca(2+)-ATPase from sarcoplasmic reticulum prevented the pSS IgG effect on PGE(2) production. An ionophore of calcium mimicked pSS IgG action, suggesting a crucial role of calcium homeostasis in the cholinoreceptor-stimulated increase in PGE(2) production. Moreover, the amounts of PGE(2) in saliva and in sera from persons with pSS were significantly higher than in pre- or post-menopausal women. These findings illustrate the importance of autoantibodies to cholinoreceptors in the generation of chronic inflammation of target tissues in SS.

Chronic chagasic cardiomyopathy (CCC) is characterized mainly by a dilated cardiomyopathy complicated by frequent and complex ventricular arrhythmias and/or conduction defects. The aim of the present study was to evaluate functional implications of neurotransmitter receptor autoantibodies in vivo. Sera from chagasic patients were used to measure the level of autoantibodies to peptide fragments from the M2 cholinergic and beta1 adrenergic receptors. Optical density values and the frequency of anti-M2 and anti-beta1 antibodies were significantly higher in the indeterminate form and in CCC patients than in normal individuals. There was no correlation between levels of autoantibodies and clinical parameters of ventricular dysfunction, as assessed by echocardiography. Patients presenting with chronotropic insufficiency in exercise test had higher levels of anti-M2 but not anti-beta1 autoantibodies. Although anti-M2 and anti-beta1 antibodies do not appear to play a role in the pathophysiology of the heart failure that accompanies severe CCC, anti-M2 cholinergic autoantibodies may contribute to the pathogenesis of Chagas' disease dysautonomia.

In this paper we demonstrate that, circulating antibodies from schizophrenic patients interacting with cerebral M1 muscarinic acetylcholine receptors (M1 mAChRs), can act as an inducer of m1 mAChR-mRNA, and neuronal nitric oxide synthase (nNOS) mRNA gene expression of rat frontal cortex. The different signaling pathways involved in the autoantibody's actions, were characterized. As previously reported serum autoantibodies from schizophrenic patients reacted against neural cells surface inhibiting the binding of the specific mAChR radioligand to rat cerebral frontal cortex membrane. Moreover, by ELISA using M1 synthetic peptide (with identical aminoacid sequence to human M1 mAChR) as coating antigen we demonstrated the reactivity against the second extracellular loop of human cerebral M1 mAChR. The corresponding affinity-purified anti M1 peptide IgG (anti M1 peptide IgG) from schizophrenic patients by stimulation of M1 mAChR exerted an increase in m1 mAChR-mRNA and nNOS-mRNA levels, that significantly correlated with the accumulation of phosphoinositides (IPs) and activation of NOS (alpha = 0.05). All these effects were blunted by pirenzepine and mimicked the action of the authentic agonist. Concurrent analysis of the effects of nNOS, phospholipase C (PLC) and calcium/calmodulin (CaM) inhibition on both, m1 mAChR-mRNA and nNOS-mRNA levels, showing that antibody up-regulation mRNA level is under the control of endogenous nitric oxide (NO) signaling system. On the basis of our results, the activation of M1 mAChR by schizophrenic autoantibody appears to induce nNOS-mRNA expression and reciprocally, the activation of NOS up-regulates m1 mAChR gene expression. These results gave support to the participation of an autoimmune process in a particular group of chronic schizophrenic patients.

We demonstrated the presence of circulating antibodies from Sjögren syndrome (SS) patients enable to interact with rat cerebral frontal cortex by activating muscarinic acetylcholine receptors (mAChRs). IgG from SS and IgG from normal subjects were studied by flow cytometry, enzyme immunoassay (ELISA), and radioligand binding assays. By flow cytometric and ELISA procedures, it was shown that IgG from SS patients reacted to cerebral frontal cortex cell surface. SS IgG was able to interact with mAChR by inhibiting 3H-QNB binding to its specific receptor. Besides, SS IgG displayed an agonistic-like activity associated to specific M1 and M3 mAChR activation, increasing nitric oxide synthase (NOS) isoform activities. Neuronal (n) and endothelial (e) NOS-mRNA gene expression of rat frontal cortex is induced by SS IgG. This article supports the participation of humoral immune alterations in SS, resulting in central parasympathetic functional deregulation. These antibodies alter mAChR activation, NOS activity, and eNOS and nNOS gene expression.

Previous studies have demonstrated that antibodies against muscarinic acetylcholine receptors (mAChRs) from exocrine glands, correlates with Sjögren syndrome (SS) in the majority of patients. The aim of the present investigation was to establish if serum IgG antibodies present in SS interacts with cerebral mAChRs. Results show that anti-cerebral IgG are present in the sera of 40% SS patients studied. Autoantibodies were able to interact with mAChRs of cerebral frontal cortex membranes inhibiting the [(3)H]QNB binding to its specific receptor. Moreover, tested by ELISA and dot blot they recognized the synthetic peptides corresponding to the second extracellular loop of human M(1) and M(3) mAChR. In addition, the corresponding affinity-purified anti-M(1) and anti-M(3) peptide IgGs displayed an agonistic activity, stimulating phosphoinositide hydrolysis. The results support the notion that serum IgG autoantibodies in SS patients target cerebral mAChRs may have some role in the pathogenesis of higher cognitive dysfunction present in SS patients.

We demonstrated the presence of circulating antibodies from schizophrenic patients able to interact with cultured astrocytes activating muscarinic acetylcholine receptors (mAChRs). Sera and purified IgG from 15 paranoid schizophrenic and 15 age-matched normal subjects were studied by indirect immunofluorescence (IFI), flow cytometry, dot blot, enzyme immunoassay (ELISA), and radioligand competition assays. Astrocyte membranes and/or a synthetic peptide, with identical amino acid sequence of human M(1) and M(2) mAChR, were used as antigens. By IFI and flow cytometry procedures, we proved that serum purified IgG fraction from schizophrenic patients, reacted to astrocyte cell surface. The same antibodies were able to inhibit the binding of the specific mAChR radioligand (3)H-QNB. Using synthetic peptide for dot blot and ELISA, we demonstrated that these antibodies reacted against the second extracellular loop of human cerebral M(1) and M(2) mAChR. Also, the corresponding affinity-purified antipeptide antibody displayed an agonistic-like activity associated to specific M(1) and M(2) mAChR activation, increasing inositol phosphates accumulation and decreasing cyclic AMP production, respectively. This article gives support to the participation of an autoimmune process in schizophrenia disease., (Copyright 2004 Wiley-Liss, Inc.)

The goal of this study was to investigate whether cruzipain, a Trypanosoma cruzi immunodominant antigen, was able to induce antibodies reactive to the cardiac M(2) muscarinic acetylcholine receptor (M(2) mAChR). Immunization with cruzipain that was devoid of enzyme activity triggered IgG antibodies against cardiac M(2) mAChR. By radioligand competition assay we proved that the anti-cruzipain IgG fraction, purified from serum, inhibited binding of the specific M(2) mAChR radioligand [(3)H]quinuclidinyl benzilate. We also demonstrated that anti-cruzipain IgG reacted against the second extracellular loop of the M(2) mAChR. The corresponding affinity-purified serum anti-M(2)e2 IgG (reacting against a synthetic peptide corresponding to this loop in humans) displayed agonist-like activity associated with specific M(2) mAChR activation - increase of cGMP, inositol phosphate accumulation and nitric oxide synthase activity - triggering a decrease in myocardial contractility. Moreover, the same IgG fraction decreased heart frequency, related to inhibition of adenylate cyclase activity. These results imply that cruzipain plays a role in the production of antibodies against M(2) mAChR, which have been related to the pathogenesis of dysautonomic syndrome described in Chagas' disease.

Saliva IgA autoantibodies against M(3) muscarinic acetylcholine receptors (mAChRs) could be a new marker for the diagnosis for Sjögren syndrome (SS) dry mouth. Saliva IgA from dry mouth primary SS (pSS) or secondary SS patients tested by ELISA recognized membrane parotid gland acinar cell antigens and the synthetic 25-mer peptide corresponding to the second extracellular loop of human M(3) mAChRs. Moreover, the IgA fraction was able to inhibit the [(3)H]QNB binding to parotid acinar membrane mAChRs. In addition, the IgA prevented carbachol stimulation of protein secretion by the parotid gland. As controls, IgA and saliva from women without dry mouth and from normal control subjects gave negative results on ELISA, binding, and biological assays, thus demonstrating the specificity of the reaction. IgA autoantibodies against mAChR may be considered among the immunoglobulin factors implicated in the pathophysiology of the development of pSS dry mouth and could be a new marker for differentiating SS dry mouth from non-SS dry mouth.

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 M(1) 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 M(1) mAChR radioligand [(3)H]pirenzepine. Immunoblotting experiments showed that IgG from schizophrenic patients revealed a band with a molecular mass coincident to that labeled by an anti-M(1) mAChR Ab. Using synthetic peptide for dot blot and ELISA, we demonstrated that these Abs reacted against the second extracellular loop of human cerebral M(1) 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.

Isolated congenital heart block may be associated with autoimmune disorder such as Sjögren Syndrome and systemic lupus erythematosus. In this work we demonstrate circulating autoantibodies against neonatal heart M1 muscarinic acetylcholine receptor (mAChR) in the sera of children with congenital heart block. This antibody were able to react with the second extracellular loop of the human M1 mAChR as demonstrated using a synthetic peptide in enzyme immune assay and binding assay. Affinity purified anti-peptide IgG as well as total IgG from children with congenital heart block, interfered with the specific radioligand occupancy from neonatal heart M1 mAChR, interacting irreversibly. The antipeptide antibodies also displayed an 'agonist-like' activity, i.e. decreased contractility, activated nitric oxide synthase activity and increased production of cyclic GMP. All of these effects were selectively blunted by pirenzepine and neutralized by the synthetic M1 peptide. Both binding and biological effects were obtained using neonatal rat heart instead adult heart and were independent of Ro/SS-A and La/SS-B antibodies and were also absent in the sera of normal children. A clinical relevance of these findings is demonstrated by a strong association between the existence of circulating M1 mAChR antipeptide antibodies and the presence of isolated congenital heart block, making these antibodies a proper marker of this disease., (Copyright 2001 Academic Press.)

Purpose: The authors investigated whether circulating autoantibodies against M(3) muscarinic acetylcholine receptors (mAChRs) could be a new marker for diagnosis for primary and secondary Sjögren syndrome (SS) dry eye., Methods: Enzyme-linked immunosorbent assay (ELISA) using both rat exorbital lacrimal gland acinar cell membranes and synthetic 25-mer peptide as antigens was used to determine autoantibodies against acinar cells and M(3) mAChRs. Also, nitric oxide synthase (NOS) activity was assessed to determine the biological effect of these autoantibodies in relation to the M(3) mAChR., Results: Sera from dry eye primary SS (pSS) or secondary SS (sSS) patients tested by ELISA recognized membrane lacrimal gland acinar cells antigens and the synthetic 25-mer peptide, corresponding to the second extracellular loop of human M(3) mAChRs. Moreover, the IgG fraction and the corresponding affinity-purified anti-M(3) peptide autoantibodies from the same patients were able to activate NOS coupled to lacrimal gland M(3) mAChRs. As controls, IgG and sera from women without dry eye with or without rheumatoid arthritis and from normal control subjects gave negative results on ELISA and biological assay; thus demonstrating the specificity of the reaction., Conclusions: Autoantibodies against mAChR may be considered among the serum factors implicated in the pathophysiology of the development of pSS dry eyes and could be a new marker to differentiate SS dry eyes from non-SS dry eyes.

Chagas' disease is caused by a parasite, Trypanosoma cruzi, which is widely distributed in South and Central America. Dysautonomias, derangements of sympathetic and parasympathetic nervous system function, are seen fairly often during the chronic course of Chagas' disease. Many infected subjects developed, in the course of the disease, neurogenic cardiomyopathy or digestive damage. Our investigations show the existence of circulating antibodies in Chagas' disease that bind to beta-adrenergic and muscarinic cholinergic receptor (mAChR). The neurotransmitter receptor-autoantibody interaction triggers in the cells intracellular signal transductions that alter the physiological behavior of the target organs, leading to tissue damage. Moreover, the deposit of autoantibodies behaving as agonists induces desensitization and/or down regulation of the receptors. This in turn can lead to a progressive blockade of them with sympathetic and parasympathetic denervation. Using synthetic peptides for immunoblotting and enzyme immunoassay, we demonstrated that these autoantibodies reacted against the second extracellular loop of the human heart beta 1 adrenoceptor and M2 cholinoceptor. Also, the corresponding affinity-purified antipeptide antibodies displayed an agonist-like activity associated with specific receptor activation. A strong association between circulating antipeptide M2 mAChR autoantibodies and the presence of patients' low heart rate variability index, bradycardia and cardiac or esophageal autonomic dysfunction in chronic chagasic patients was verified. This fact make these antipeptide antibodies a proper marker of cardiac neuromyopathy and achalasia.

Background & Aims: Autoantibodies against M(2)-muscarinic acetylcholine receptors (M(2) mAChR) have been reported in patients with chronic Chagas' disease who have cardiac dysautonomia. The aim of this study was to investigate the presence of such antibodies in chronic chagasic and non-chagasic patients with esophageal achalasia and their ability to activate M(2) mAChR in the isolated esophagus., Methods: Enzyme-linked immunosorbent assay was used to detect serum immunoglobulin (Ig) G antibodies against a synthetic 24-mer peptide corresponding to the second extracellular loop of human M(2) mAChR. The effects of both total serum IgG and affinity-purified antipeptide antibodies on the contractile activity and adenosine 3', 5'-cyclic monophosphate (cAMP) production in rat esophageal strips were also tested., Results: Circulating IgG antibodies from chagasic achalasia patients recognized the M(2)-peptide more often than those from non-chagasic achalasia patients (P < 0.0005) and normal subjects (P < 0.0001). A strong association between the existence of circulating anti-M(2) mAChR antibodies and the presence of achalasia in chagasic patients was found (P < 0.01). Both the total IgG fraction and anti-M(2)-peptide antibodies increased the basal tone, reduced the relaxant effect of isoproterenol, and decreased cAMP accumulation in esophageal strips, displaying a muscarinic agonist-like activity on M(2) mAChR., Conclusions: Patients with chronic Chagas' disease have circulating autoantibodies against M(2) mAChR. These antibodies could be involved in the pathophysiological mechanism of chagasic achalasia.

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 effect of affinity 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: chi(2) = 37.5, p < 0. 00001; Valsalva manoeuvre: chi(2) = 70.0, p < 0.00001). The antipeptide autoantibodies also showed agonist activity, decreasing the rate of contraction and cAMP production. The effects on rat atria resembled the effects 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.

Isolated congenital heart block may be associated with primary Sjögren syndrome. In this work we describe circulating antibodies in the sera of primary Sjögren syndrome patients that are able to interact with neonatal myocardium by activating muscarinic acetylcholine receptors of M1 subtype. We report on the presence of autoantibodies against the second extracellular loop of human M1 muscarinic acetylcholine receptors in primary Sjögren syndrome mothers whose children have congenital heart block using a synthetic peptide in indirect immunofluorescence technique. Autoantibodies from primary Sjögren syndrome patients gave positive image on neonatal atria but not on adult atria slices. The synthetic M1 peptide selectively abrogated indirect immunofluorescence recognition. The primary Sjögren syndrome-immunoglobulin G also displayed an 'agonist like' activity modifying the intracellular events associated with muscarinic acetylcholine receptor activation. 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 leads to activation of nitric oxide synthase and soluble guanylate cyclase. All of these effects were selectively blunted by pirenzepine and neutralized by M1 synthetic peptide. These biological effects were not obtained using adult instead of neonatal rat atria and neither occurred with the sera of normal healthy women of childbearing age. It could be concluded that antibodies against neonatal M1 muscarinic acetylcholine receptor may be another serum factor to be considered in the pathophysiology of the development of congenital heart block associated with primary Sjögren syndrome mothers.

Primary Sjögren Syndrome is a chronic autoimmune disease characterized by exocrine gland dysfunction. Here we present evidence of the activation of nitric oxide signaling cascade by circulating antibodies of patients with Sjögren Syndrome in rat submandibular glands. Constitutive nitric oxide synthase and cyclic GMP levels are modulated by Sjögren IgGs through the activation of muscarinic acetylcholine receptors on the glands. The effects are similar to those produced by the agonist carbachol and blocked by the antagonist atropine. The involvement of M1 subtype of muscarinic receptors is proposed since both a synthetic peptide homologous to an extracellular domain of M1 receptor and pirenzepine, a selective M1 antagonist, partially blocked the effects. We conclude that Sjögren Syndrome antibodies can activate nitric oxide signaling in submandibular glands by interacting with muscarinic acetylcholine receptors., (Copyright 1999 Academic Press.)

Evidence accumulated by our investigations over the years give adequate proof for the existence of circulating antibodies in Chagas disease which bind to beta adrenergic and muscarinic cholinergic receptor of myocardium. The interaction of agonist-like antibodies with neurotransmitter receptors, triggers in the cells intracellular signal transductions that alter the physiological behaviour of the target organs. These events convert the normal cells into pathologically active cells. The interaction of antibodies with heart beta adrenergic and cholinergic receptors triggers physiologic, morphologic, enzymatic and molecular alterations, leading to tissue damage. The analysis of the prevalence and distribution of these antibodies reveals a strong association with cardiac and esophageal autonomic dysfunction in seropositive patients in comparison with those without alteration of the heart and esophagus autonomic disorders: therefore, the presence of these antibodies may partially explain the cardiomyoneurophathy and achalasia of Chagas disease, in which the sympathetic and parasympathetic systems are affected. The deposit of autoantibodies behaving like an agonist on neurotransmitter receptors, induceds desensitization and/or down regulation of the receptors. This in turn, could lead to a progressive blockade of neurotransmitter receptors, with sympathetic and parasympathetic dennervation, a phenomenon that has been described during the course of Chagas cardioneuropathy and achalasia. The clinical relevance of these findings is the demonstration, using biomolecules, of a strong association between the existence of circulating autoantibodies against peptides corresponding to the second extracellular loop of the human heart beta, adrenoceptor and M2 cholinoceptor in chagasic patients, and the presence of dysautonomic symptoms, making these autoantibodies a proper early marker of heart and digestive autonomic dysfunction.

Objective: Cardiac tissue from chagasic mice was studied to evaluate the expression and biological activity of beta-adrenoceptors in association with circulating beta-adrenoceptor-related autoantibodies., Methods: BALB/c inbred mice that were either treated or not treated with atenolol (2.5 mg/kg) and infected or not infected with 1 x 10(4) trypomastigotes (CA-1 strain) were sacrificed weekly up to week nine. Morphological, binding and contractility studies were performed on the four different groups of animals. The effect of their serum antibodies was also assayed in binding and contractility studies on normal heart preparations., Results: Hearts from chagasic myocarditis mice showed a beta-adrenoceptor-related dysfunction, with a decrease in heart contractility, impaired response to exogenous beta-adrenoceptor agonist and a significant reduction in beta-adrenergic binding sites. Those effects were maximum at eight-nine weeks post-infection and were improved by treating infected mice with atenolol. In addition, serum or IgG from chagasic myocarditis mice was capable of interacting with cardiac beta-adrenoceptors, reducing the number of binding sites and inhibiting the contractile response to exogenous norepinephrine. IgG effects that were observed in normal myocardium, were highest in sera from mice eight-nine weeks post-infection and correlate with the degree of myocarditis. Moreover, chagasic autoantibodies from infected mice recognized a peptide corresponding to the sequence of the second extracellular loop of the human beta 1-adrenoceptor., Conclusions: (1) The development of alterations in beta-adrenergic receptors, related to cardiac dysfunction, may be associated with the presence of circulating antibodies against these receptors and (2) it is possible that the chronic deposits of these autoantibodies in cardiac beta-adrenoceptors could lead to a progressive blockade with sympathetic denervation, a phenomenon that has been described in the course of chagasic myocarditis.

IgG obtained from sera of primary Sjögren's syndrome (pSS-IgG) patients and its interaction with M3 muscarinic cholinoceptors of rat exorbital lacrimal glands were studied by indirect immunofluorescence (IFI) and binding assay. Primary Sjögren's syndrome IgG stained epithelial cells with a continuous fluorescence pattern. The IFI imagen was attenuated by incubating the pSS-IgG with a synthetic peptide corresponding to the second extracellular loop of M3 muscarinic cholinoceptor. Primary SS-IgG was also able to bound irreversibly to muscarinic acetylcholine receptors (mAChRs) displacing the specific cholinergic antagonist QNB. Moreover, these antibodies triggered intracellular signals coupled to M3 muscaric cholinoceptors such as nitric oxide synthase (NOS) activation and cGMP production. Both primary Sjögren's syndrome IgG effects mimicked carbachol action and were abrogated by specific muscarinic antagonist 4-DAMP. The nitric oxide pathway through muscarinic cholinoceptors activation by pSS-IgG on rat exorbital lacrimal gland is also described. We proposed that chronic interaction of these autoantibodies on lacrimal gland muscarinic acetylcholine receptors could lead to tissue damage through nitric oxide release after immunological stimulation.

Purpose: The authors demonstrated that immunoglobulin G, present in the sera of patients with primary Sjögren syndrome (pSS), could recognize and activate muscarinic acetylcholine receptors (mAChRs) of rat exorbital acrimal gland., Methods: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and radioligand binding and biologic assays were used to demonstrate autoantibodies against mAChRs., Results: These autoantibodies recognized by means of SDS-PAGE and immunoblotting assay a band of approximately 70 kDa expressed on lacrimal gland membranes that comigrated with the peak of labeled mAChRs. Moreover, pSS IgG were able to inhibit, in an irreversible manner, the binding of [3H]quinuclidinyl benzilate to mAChRs of rat exorbital lacrimal glands and to simulate the biologic effect of mAChR agonists, because they trigger the activation of phosphoinositide turnover. Atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the effect and carbachol mimicked it, confirming that the M3 subtype mAChRs mediated pSS IgG action. As control, IgG from sera of women without pSS gave negative results on immunoblotting, binding, and biologic assays, thus demonstrating the specificity of the reaction., Conclusions: Autoantibodies against mAChRs may be considered among the serum factors implicated in the pathophysiology of the development of pSS dry eyes.

Human and experimental Chagas' disease causes peripheral nervous system damage involving neuromuscular transmission alterations at the neuromuscular junction. Additionally, autoantibodies directed to peripheral nerves and sarcolemmal proteins of skeletal muscle have been described. In this work, we analyse the ability of serum immunoglobulin factors associated with human chagasic infection to bind the affinity-purified nicotinic acetylcholine receptor (nAChR) from electric organs of Discopyge tschudii and to identify the receptor subunits involved in the interaction. The frequency of serum anti-nAChR reactivity assayed by dot-blot was higher in seropositive chagasic patients than in uninfected subjects. Purified IgG obtained from chagasic patients immunoprecipitated a significantly higher fraction of the solubilized nAChR than normal IgG. Furthermore, immunoblotting assays indicated that alpha and beta are the main subunits involved in the interaction. Chagasic IgG was able to inhibit the binding of alpha-bungarotoxin to the receptor in a concentration-dependent manner, confirming the contribution of the alpha-subunit in the autoantibody-receptor interaction. The presence of anti-nAChR antibodies was detected in 73% of chagasic patients with impairment of neuromuscular transmission in conventional electromyographical studies, indicating a strong association between seropositive reactivity against nAChR and electromyographical abnormalities in chagasic patients. The chronic binding of these autoantibodies to the nAChR could induce a decrease in the population of functional nAChRs at the neuromuscular junction and consequently contribute to the electrophysiological neuromuscular alterations described in the course of chronic Chagas' disease.

Chronic Chagas' disease is associated with pathologic changes of the cardiovascular, digestive, and autonomic nervous system, culminating in autonomic denervation and congestive heart failure. Previously, circulating autoantibodies that activate signaling by cardiac muscarinic acetylcholine receptors (mAChRs) have been described. However, it remains unclear whether the chagasic IgGs directly interact with the m2 mAChRs (predominant cardiac subtype), and, if so, whether chronic exposure of the mAChRs to such activating IgGs would result in receptor desensitization. Here we performed studies with purified and reconstituted hm2 mAChRs and demonstrate that IgGs from chagasic serum immunoprecipitated the mAChRs in a manner similar to an anti-m2 mAChR monoclonal antibody tested in parallel. The chagasic antibodies did not directly interact with the ligand binding site, because the binding of radiolabeled antagonist was unchanged by the addition of the chagasic IgG. In intact cells stably expressing the hm2 mAChR, the chagasic IgGs, but not normal IgGs, mimicked the ability of the agonist acetylcholine to induce two effects associated with agonist-induced receptor desensitization: a decrease in affinity for agonist binding to m2 mAChR and sequestration of the hm2 mAChRs from the cell surface. The results demonstrate that the chagasic IgGs can directly interact with and desensitize m2 mAChRs and provide support for the hypothesis of autoimmune mechanisms having a role in the pathogenesis of Chagas' cardioneuromyopathy.

Herein we demonstrate by ELISA and immunoblotting the presence in the sera of chagasic patients of circulating autoantibodies against the third extracellular domain of human muscarinic acetylcholine receptors by using a synthetic peptide corresponding to the sequence 169-192 of the receptor. Immunoaffinity purified antipeptide antibodies displayed cardiac muscarinic activity as decreased contractility and cAMP production and increased cGMP levels. These effects were specifically blocked by the synthetic peptide and by atropine. A strong association between the existence of circulating autoantibodies and the presence of dysautonomia was shown, making these autoantibodies an appropriate marker of heart autonomic dysfunction.

In this work we demonstrated that IgG present in the sera of patients with congenital heart block (CHB) and their mothers could bind and activate beta adrenoceptors and muscarinic cholinergic receptors of neonatal heart. These antibodies were able to inhibit in a noncompetitive manner the binding of [3H]QNB and [3H]CGP to muscarinic cholinergic and beta adrenoceptors of purified neonatal rat myocardial membranes, respectively. Moreover, IgG from children with CHB and their mothers could modify biological effects mediated by these neurotransmitter receptors, i.e., heart contractility and cAMP production. Neither binding nor biological effects were obtained using adult instead of neonatal rat atria. Both reactivities (adrenergic and cholinergic) were independent of Ro/SS-A and La/SS-B antibodies and were absent in the sera of normal women of childbearing age and of normal children. It could be concluded that antibodies against cardiac neurotransmitter receptors may be another serum factor (or factors) to be considered in the pathophysiology of the development of CHB.

To determine whether antibodies against beta adrenergic activity interact with neonatal cardiac cell receptors and alter their physiological behaviour. An "in vitro" experimental model measuring the frequency of contraction, the production of cAMP and binding assay on neonatal and adult rat atria was employed. Sera and IgG fraction from mothers of infants with congenital heart block (CHB) interact with neonatal rat atria increasing the frequency of contraction and cAMP production. These effects were abolished by propranolol, pointing to a beta adrenergic participation. IgG also competed with 3H-CGP to beta adrenergic receptors on neonatal cardiac membranes. Neither the contractile nor the cAMP effects or binding assay were obtained using adult instead of neonatal rat atria. Reactivity against cardiac neurotransmitter receptors may be another serum factor(s) to be considered in the pathophysiology of the development of CHB.

In human immune deficiency virus (HIV) disease, direct infection of heart tissue with HIV and repeated intestinal infections with opportunistic pathogens are thought to be the main cause of cardiac disease and diarrhoea respectively. A role for autoimmune phenomena may also be involved in the pathogeny of HIV disease. In this study, we demonstrate that immunoglobulins from the A and G classes from HIV positive patients are able to interfere with the function of the muscarinic cholinergic receptors from heart and gut. Both IgA and IgG HIV+ preparations decreased the tension of isolated atria and increased the tension of isolated ileum. The mechanical effect of carbachol was inhibited in both atria and ileum preparations, when they were preincubated with either IgA or IgG HIV+ fractions. An inhibitor of muscarinic cholinergic receptors (atropine) impaired the negative inotropic action of HIV+ immunoglobulins (Ig) on the heart and prevented the positive inotropic effect of HIV+ Igs on ileum. HIV+ IgA fraction was approximately ten fold more potent to interfere with the cholinergic function as compared to the IgG fraction. These results suggest that antibodies present in HIV+ serum may also modulate muscle's cholinergic activity in the heart and ileum from HIV+patients.

Evidence is presented showing that in experimental autoimmune myocarditis, there are certain components in IgG fraction of the sera that bind to myocardium muscarinic cholinergic receptors. The autoimmune IgG simulated the biologic effect of cholinergic agonists because (i) it increased cGMP levels, (ii) it decreased cAMP stimulated levels, and (iii) it reduced heart contractility and diminished reactivity to exogenous acetylcholine. Autoimmune IgG inhibited the binding of specific muscarinic cholinergic radioligand to purified myocardial membranes behaving as noncompetitive inhibitors. The recognition appears to be organ specific because the autoimmune IgG did not bind to muscarinic cholinergic receptors of urinary bladder. The presence of antibodies against antigens expressed in an accessible form to antibody in living myocardial cells might be related to some of the immunopathologic mechanisms participating in the pathogenesis of the experimental autoimmune myocarditis.

Background and Objective: Autoimmune mechanisms may contribute to the pathogenesis of periodontal disease. Autoantibodies with the potential to bind and activate β1-adrenoceptors (β1-AR) of human gingival fibroblasts were studied to provide evidence of altered humoral immune response in chronic periodontal disease. Material and Methods: Flow cytometry and enzyme-linked immunosorbent assay using cell culture-adherent gingival fibroblasts and/or their purified membranes and/or a synthetic peptide corresponding to the second extracellular loop of human β1-AR were used to detect serum antibodies. The effects of antibodies from chronic periodontal disease patients on PGE2 generation and CD40 expression were also tested. Results: Circulating immunoglobulin G (IgG) from chronic periodontal disease patients (but not from normal individuals) interacted with the fibroblast surface, activating β1-AR. Atenolol or CGP 20712 (beta 1-AR antagonists) and β1 synthetic peptide inhibited the interaction of IgG with β1-AR. Immunoglobulin G from chronic periodontal disease patients also displayed agonist-like activity associated with specific β1-AR activation, increasing PGE2 generation and CD40 overexpression. The corresponding affinity-purified anti-β1-AR peptide IgG mimicked these effects. Both effects were prevented by inhibition of cyclo-oxygenase. Conclusion: This article supports the participation of humoral immune alterations in chronic periodontal disease resulting in postsynaptic functional deregulation. Overproduction of proinflammatory mediators (PGE2 and CD40 expression) is induced as a consequence of antibody–β1-AR interaction. The PGE2–CD40–IgG axis may play a part in the pathophysiological mechanisms underlying the inflammatory process in chronic periodontal disease. [ABSTRACT FROM AUTHOR]