Modulation of M(2) muscarinic receptor-receptor interaction by immunoglobulin G antibodies from Chagas' disease patients.

Circulating immunoglobulin (Ig)G antibodies against M(2) muscarinic acetylcholine receptors (M(2) mAChR) have been implicated in Chagas' disease (ChD) pathophysiology. These antibodies bind to and activate their target receptor, displaying agonist-like activity through an unclear mechanism. This study tested the ability of serum anti-M(2) mAChR antibodies from chronic ChD patients to modulate M(2) muscarinic receptor-receptor interaction by bioluminescence resonance energy transfer (BRET). Human embryonic kidney (HEK) 293 cells co-expressing fusion proteins M(2) mAChR-Renilla luciferase (RLuc) and M(2) mAChR-yellow fluorescent protein (YFP) were exposed to the serum IgG fraction from ChD patients, and BRET between RLuc and YFP was assessed by luminometry. Unlike serum IgG from healthy subjects and conventional muscarinic ligands, ChD IgG promoted a time- and concentration-dependent increase in the BRET signal. This effect neither required cellular integrity nor occurred as a consequence of receptor activation. Enhancement of M(2) receptor-receptor interaction by ChD IgG was receptor subtype-specific and mediated by the recognition of the second extracellular loop of the M(2) mAChR. The monovalent Fab fragment derived from ChD IgG was unable to reproduce the effect of the native immunoglobulin. However, addition of ChD Fab in the presence of anti-human Fab IgG restored BRET-enhancing activity. These data suggest that the modulatory effect of ChD IgG on M(2) receptor-receptor interaction results from receptor cross-linking by bivalent antibodies.

Interaction of human chagasic IgG with human colon muscarinic acetylcholine receptor: molecular and functional evidence.

BACKGROUND AND AIMS: Gastrointestinal disorders is one of the clinical manifestations of chronic Chagas' disease. The pathogenesis seems to be associated with autonomic dysfunction. Here, we consider the muscarinic cholinoceptor mediated alteration in distal colon function in chagasic megacolon. PATIENTS: Patients were divided into four groups: group I, chronic chagasic patients with megacolon; group II, chronic chagasic patients without megacolon; group III, non-chagasic patients with megacolon; and group IV, normal healthy volunteers (control). METHODS: Binding assay and immunoblot of cholinoceptors from human and rat colon and enzyme immunoassay (ELISA) using a synthetic 24mer peptide corresponding to the second extracellular loop of human M2 muscarinic acetylcholine receptors (mAChR) were used to detect the presence of serum antibodies. The effect of antibodies on basal tone and 3',5'-cyclic monophosphate (cAMP) production of human and rat distal colon strips were also tested. RESULTS: Group I but not the other groups had circulating antibodies capable of interacting with human colon activating M2 mAChR, as they competed with binding of specific radioligand to mAChR and interacted with the second extracellular loop of human M2 mAChR. Moreover, affinity purified anti-M2 peptide IgG from group I, in common with monoclonal antihuman M2 mAChR, recognised bands with a molecular weight corresponding to colon mAChR. This antibody also displayed an agonist-like activity, increasing basal tone and decreasing cAMP accumulation. Both effects were blunted by AF-DX 116 and neutralised by the synthetic peptide. CONCLUSIONS: In chagasic patients with megacolon there are antibodies that can recognise and activate M2 mAChR. The implications of these autoantibodies in the pathogenesis of chagasic megacolon is discussed.

Functional implications of circulating muscarinic cholinergic receptor autoantibodies in chagasic patients with 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.

Cardiac M(2) muscarinic cholinoceptor activation by human chagasic autoantibodies: association with bradycardia.

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.

Role of CD8+ and WC-1+ gamma/delta T cells in resistance to Mycobacterium bovis infection in the SCID-bo mouse.

The role of various effector T cell populations in the bovine immune response to Mycobacterium bovis infection is poorly understood. This is largely due to the difficulties associated with performing in vivo challenge studies in the natural host species. In this report, we utilized a fetal bovine-severe combined immunodeficient (SCID-bo) xenochimeric mouse model to study the protective role of two putative effector cell types, CD8+ T cells and a subpopulation of gamma/delta T cells that express WC-1, a member of the cysteine-rich scavenger receptor superfamily (CRSR). We demonstrate that CD8+ T cells play a key role in protection and contribute substantially to bovine IFN-gamma mRNA levels at 30 days post-infection. The role of WC-1 bearing cells to protection was less definitive but our results suggest that this population may play a pivotal role early in infection. Granuloma architecture was altered in anti-WC-1 (ILA29) but not anti-CD8 (ILA51) -treated animals, suggesting that this population may be involved in recruitment of various cell types to sites of infection.

Circulating antibodies against nicotinic acetylcholine receptors in chagasic patients.

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.

The nitric oxide synthase of mouse spermatozoa.

Nitric oxide synthase (NOS) was evidenced in mature mouse spermatozoa by means of biochemical techniques and Western blot. During 120 min of incubation, 10(7) spermatozoa synthesized 7 +/- 2 pmol of L-[14C]citrulline. Besides, L-citrulline formation depended on the incubation time and on the concentration of L-arginine present in the incubation medium. Different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) but not aminoguanidine, inhibited L-[14C]citrulline formation. Western-blot analysis of solubilized sperm proteins revealed a unique band of M(r)=140 kDa with the neural, endothelial and inducible NOS antisera tested. These results provide evidence that mature mouse sperm contains a NOS isoform and that spermatozoa have the potential ability to synthesize NO, suggesting a role for endogenous NO on mammalian sperm function.

Participation of nitric oxide signaling system in the cardiac muscarinic cholinergic effect of human chagasic IgG.

The possible role of altered humoral immune response in the pathogenesis of the chronic chagasic cardioneuromyopathy was examined by analyzing the interaction of IgG from T. cruzi infected patients with cardiac muscarinic acetylcholine receptors (mAChR). Human chagasic IgG by activating cardiac M2 mAChR, simulated the agonist actions triggering negative inotropic effect, inositol phosphate accumulation, nitric oxide synthase stimulation and increased production of cyclic GMP. Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase activities; prevented chagasic IgG effects on signaling pathways involved in M2 mAChR activation. In addition, sodium nitroprusside or 8-bromo cyclic GMP, mimicked the chagasic IgG effect associated with cholinergic-mediated cellular transmembrane signals. Moreover, these chagasic IgG immunoprecipitated the mAChRs solubilized from cardiac membranes. By means of SDS-PAGE and immunoblotting analysis, chagasic sera recognized a band of 70-75 kDa. The major protein recognized by chagasic IgG had an Rf coincident with the peak of [3H] propylbenzilylcholine mustard with an apparent molecular weight similar to that of mAChRs, which disappeared in the presence of atropine. The specificity of this interaction was checked by immunoprecipitation of rat cardiac mAChR and immunoblotting of pure human M2 mAChRs. Chronic interaction of chagasic IgG with myocardial mAChRs, behaving as a muscarinic agonist, might lead to cell dysfunction or tissue damage. Also, these antibodies could produce desensitization, internalization or degradation of mAChRs; explaining the progressive blockade of mAChRs in myocardium with parasympathetic denervation, a phenomenon that has been described in the course of Chagas' cardioneuromyopathy.

Desensitization and sequestration of human m2 muscarinic acetylcholine receptors by autoantibodies from patients with 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.

Interaction of human chagasic IgG with the second extracellular loop of the human heart muscarinic acetylcholine receptor: functional and pathological implications.

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.

Involvement of nitric oxide synthase and protein kinase C activation on chagasic antibodies action upon cardiac contractility.

We have already demonstrated the presence of antibodies in the sera of chagasic patients with the ability to interact with neurotransmitter receptors triggering several intracellular pathways of transduction signals. Here we show that, chagasic IgG induced protein kinase C (PKC) translocation to rat cardiac membranes and this effect was inhibited by muscarinic cholinergic blockers atropine and AF-DX 116 pointing to the participation of M2 receptors in this effect. It was also able to stimulate nitric oxide synthase (NOS) activity and this action was blunted by phospholipase C (PLC) and PKC inhibitors indicating that the production of nitric oxide (NO) would be the consequence of the cascade of enzymatic pathways triggered by mAChR activation. PKC and NOS activities were involved in chagasic IgG negative inotropic actions on rat isolated myocardium as its effects were blunted by staurosporine and L-N-monomethyl arginine. Furthermore, low concentrations of chagasic IgG inhibited the cardiac mechanical action of carbachol in a non-competitive manner. These data suggested that PKC activation in myocardium by chagasic IgG would be involved in its physiological actions by modulating NOS activity. The participation of PKC-mediated phosphorylation of mAChR leading to receptor desensitization as one of the causes of dysautonomia is also discussed.

[Interaction of chagasic autoantibodies with the third extracellular domain of the human heart muscarinic receptor. Functional and pathological implications]. / Interacción de autoanticuerpos chagásicos con el tercer dominio extracelular del receptor muscarínico cardíaco humano.

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.

Modification of cholinergic-mediated cellular transmembrane signals by the interaction of human chagasic IgG with cardiac muscarinic receptors.

The possible role of altered humoral immunity in cardiac Chagas' disease was examined by analyzing the interaction of IgG and the corresponding F(ab')2 from Trypanosoma cruzi-infected patients with cardiac muscarinic cholinergic receptors (mAchR). Human chagasic IgG and its F(ab')2 simulated the agonist actions triggering the biological effects associated with cholinergic-mediated cellular transmembrane signals, i.e. stimulation of cGMP, inhibition of cAMP and a decrease in atrial contractility. Atropine blunted all of these effects while pertussis toxin prevented the inhibition of cAMP and contractility confirming the G-regulatory-protein-mediated response in the interaction of chagasic antibodies with cardiac mAchR. In addition, chagasic IgG and its F(ab')2 behaved as partial agonists activating the specific receptor and inhibiting in a noncompetitive manner the activity of an exogenous agonist (pilocarpine). Moreover, chagasic IgG immunoprecipitated the mAchR solubilized from cardiac membrane in a concentration-dependent fashion. By means of SDS-PAGE and immunoblotting analysis, chagasic serum was shown to recognize a band of approximately 75 kD. The electrophoretic studies of prelabeled immunoprecipitated proteins revealed a peak of [3H] propylbenzilylcholine mustard with an apparent molecular weight similar to that of mAchR, which disappeared in the presence of atropine. The presence of these antibodies in the serum of chagasic patients could explain the progressive receptor blockade in the parasympathetic branch of the cardiac autonomic nervous system associated with the cardioneuromyopathy described in the course of Chagas' disease.

Identification of antibodies with muscarinic cholinergic activity in human Chagas' disease: pathological implications.

We examined the possible role of altered humoral immunity in dysautonomic syndrome in Chagas' disease by analyzing the effect of sera and IgG on the binding of radioligand to heart muscarinic cholinergic receptors and on the contractility of myocardium. Human Chagasic IgG inhibited in a non-competitive manner the binding of [3H]quinuclidinyl benzilate to the cardiac cell membrane. Moreover, human Chagasic IgG behaved as a partial muscarinic cholinergic agonist, reducing heartcontractility and inhibiting the action of pilocarpine. The prevalence of the cholinergic antibody activity was higher in sera from T. cruzi-infected asymptomatic individuals with dysautonomic syndrome than in those without autonomic nervous system alterations. The presence of these antibodies could explain the progressive receptor blockade in the parasympathetic branch of the autonomic nervous system, leading to dysautonomia.

Antibodies against beta adrenoceptors in mothers of children with congenital heart block.

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.

Antibodies against beta adrenoceptors in mothers of children with congenital heart block.

To determine whether antibodies against beta adrenergic activity interact with neonatal cardiac cell receptors and alter their physiological behaviour. An [quot ]in vitro[quot ] 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.

Antibodies against beta adrenoceptors in mothers of children with congenital heart block

En este trabajo se empleó un modelo experimental "in vitro" en el cual se midió la frecuencia de las contracciones, la producción de AMPc y la la unión de ligandos específicos al receptor ß adrenérgicos en las aurículas de ratas neonatales y adultas; a fin de, determinar si anticuerpos con actividad ß adrenérgica interactuaban con receptores ß adrenérgicos cardíacos y alteraban su compartimiento fisiológico. Los sueros y la fracción IgG de madres de infantes con bloqueo cardíaco neonatal congénito incrementaron la frecuencia de las contracciones y la producciín de AMPc en el miocardio auricular neonato. Este efecto fue inhibido por propranolol, puntalizando una participación ß adrenérgica. La IgG también compitió con el radiologando específico por los receptores ß adrenérgicos (3H-CGP) en las membranas purificadas de miocardio neonatal. Ni los efectos biológicos ni los ensayos de unión específica fueron modificados cuando se usó miocardio de rata adulta. La reactividad contra adrenoreceptores cardíacos por parte de anticuerpos provenientes de madres de niños con bloqueo neonatal congénito, podría ser otro factor sérico que debería considerarse en la patofisiología del desarrollo del bloqueo neonatal congénito (AU)

Antibodies against beta adrenoceptors in mothers of children with congenital heart block

En este trabajo se empleó un modelo experimental "in vitro" en el cual se midió la frecuencia de las contracciones, la producción de AMPc y la la unión de ligandos específicos al receptor ß adrenérgicos en las aurículas de ratas neonatales y adultas; a fin de, determinar si anticuerpos con actividad ß adrenérgica interactuaban con receptores ß adrenérgicos cardíacos y alteraban su compartimiento fisiológico. Los sueros y la fracción IgG de madres de infantes con bloqueo cardíaco neonatal congénito incrementaron la frecuencia de las contracciones y la producciín de AMPc en el miocardio auricular neonato. Este efecto fue inhibido por propranolol, puntalizando una participación ß adrenérgica. La IgG también compitió con el radiologando específico por los receptores ß adrenérgicos (3H-CGP) en las membranas purificadas de miocardio neonatal. Ni los efectos biológicos ni los ensayos de unión específica fueron modificados cuando se usó miocardio de rata adulta. La reactividad contra adrenoreceptores cardíacos por parte de anticuerpos provenientes de madres de niños con bloqueo neonatal congénito, podría ser otro factor sérico que debería considerarse en la patofisiología del desarrollo del bloqueo neonatal congénito