ABSTRACT
Previous studies showed that circulating autoantibodies against M2 muscarinic receptors (anti-M2R Ab) are associated with decreased cardiac parasympathetic modulation in patients with chronic Chagas disease (CD). Here we investigated whether the exposure of M2R to such antibodies could impair agonist-induced receptor activation, leading to the inhibition of associated signaling pathways. Preincubation of M2R-expressing HEK 293T cells with serum IgG fractions from chagasic patients with cardiovascular dysautonomia, followed by the addition of carbachol, resulted in the attenuation of agonist-induced Gi protein activation and arrestin-2 recruitment. These effects were not mimicked by the corresponding Fab fractions, suggesting that they occur through receptor crosslinking. IgG autoantibodies did not enhance M2R/arrestin interaction or promote M2R internalization, suggesting that their inhibitory effects are not likely a result of short-term receptor regulation. Rather, these immunoglobulins could function as negative allosteric modulators of acetylcholine-mediated responses, thereby contributing to the development of parasympathetic dysfunction in patients with CD.
Subject(s)
Autoantibodies/immunology , Autonomic Nervous System Diseases/immunology , Chagas Disease/immunology , Receptor, Muscarinic M2/immunology , Adult , Aged , Allosteric Regulation , Autoantibodies/metabolism , Autoantibodies/pharmacology , Autonomic Nervous System Diseases/etiology , Autonomic Nervous System Diseases/metabolism , Autonomic Nervous System Diseases/physiopathology , Carbachol/pharmacology , Chagas Disease/complications , Chagas Disease/metabolism , Chagas Disease/physiopathology , Cholinergic Agonists/pharmacology , Female , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , HEK293 Cells , Heart Rate , Humans , Male , Middle Aged , Receptor, Muscarinic M2/drug effects , Receptor, Muscarinic M2/metabolism , beta-Arrestin 1/metabolismABSTRACT
In this study, we assessed the effectiveness of a live, attenuated Salmonella enterica serovar Typhi (S. Typhi) vaccine strain as a cancer immunotherapy in a mouse model of metastatic T-cell lymphoma. EL4 tumor-bearing C57BL/6J mice immunized with S. Typhi strain CVD 915, by injection into the tumor and the draining lymph node areas, displayed a significant decrease in tumor growth, a reduction in the mitotic index (MI) of tumors, a delayed development of palpable lymph node metastases and most importantly improved survival, compared to untreated mice. Besides, complete tumor regression was achieved in a small number of bacteria-treated mice. A successful therapeutic response associated with a significant reduction of tumor mass was evident as early as 5 days after treatment. The administration of Salmonella to tumor-bearing mice promoted early cellular infiltration (mainly neutrophils) within the tumor, and was accompanied by a decreased intratumoral interleukin 10 production as well as by leukocyte expansion in tumor draining lymph nodes. A tumor-specific memory immune response was induced in most of cured animals, as evidenced by the lack of tumor growth after a rechallenge with the same tumor. EL4 cells cultured with live Salmonella failed to proliferate and underwent apoptosis in a dose-dependent, time-dependent, and contact-dependent manner. To our knowledge, these results demonstrate for the first time the efficacy of a S. Typhi vaccine strain as an oncolytic and immunotherapeutic agent against a highly malignant tumor and support the use of S. Typhi-based vaccine strains in cancer therapy.
Subject(s)
Immunotherapy/methods , Lymphoma, T-Cell/therapy , Salmonella typhi/immunology , Typhoid-Paratyphoid Vaccines/therapeutic use , Animals , Apoptosis , Cell Line, Tumor , Cell Proliferation , Female , Interleukin-10/biosynthesis , Lymph Nodes/immunology , Lymphatic Metastasis/prevention & control , Lymphoma, T-Cell/immunology , Lymphoma, T-Cell/microbiology , Male , Mice , Mice, Inbred C57BL , Mice, Nude , Mitotic Index , Typhoid-Paratyphoid Vaccines/immunology , Vaccines, Attenuated/therapeutic useABSTRACT
Although previous pharmacological and biochemical data support the notion that muscarinic acetylcholine receptors (mAChR) form homo- and heterodimers, the existence of mAChR oligomers in live cells is still a matter of controversy. Here we used bioluminescence resonance energy transfer to demonstrate that M(1), M(2), and M(3) mAChR can form constitutive homo- and heterodimers in living HEK 293 cells. Quantitative bioluminescence resonance energy transfer analysis has revealed that the cell receptor population in cells expressing a single subtype of M(1), M(2), or M(3) mAChR is predominantly composed of high affinity homodimers. Saturation curve analysis of cells expressing two receptor subtypes demonstrates the existence of high affinity M(1)/M(2), M(2)/M(3), and M(1)/M(3) mAChR heterodimers, although the relative affinity values were slightly lower than those for mAChR homodimers. Short term agonist treatment did not modify the oligomeric status of homo- and heterodimers. When expressed in JEG-3 cells, the M(2) receptor exhibits much higher susceptibility than the M(3) receptor to agonist-induced down-regulation. Coexpression of M(3) mAChR with increasing amounts of the M(2) subtype in JEG-3 cells resulted in an increased agonist-induced down-regulation of M(3), suggesting a novel role of heterodimerization in the mechanism of mAChR long term regulation.
Subject(s)
Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Structure, Quaternary , Receptors, Muscarinic/chemistry , Receptors, Muscarinic/metabolism , Animals , Carbachol/metabolism , Cell Line , Cholinergic Agonists/metabolism , Dimerization , Fluorescence Resonance Energy Transfer , Humans , Protein Isoforms/genetics , Receptors, Muscarinic/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
It has been proposed that anti-myocardial antibodies (Ab) against neurotransmitter (NT) receptors are involved in the immunopathology of chronic Chagas' heart disease. We demonstrated that an anti-Trypanosoma cruzi monoclonal Ab (mAb), CAK20.12, binds to murine cardiac beta-adrenergic and muscarinic acetyl choline (mACh) receptors eliciting abnormal physiological responses on normal heart. No cross-linking requirement for mAb actions was demonstrated using Fab fragment derived from CAK20.12. mAb binding to synthetic peptides from the second extracellular loop of both beta1-adrenergic and mACh receptors, demonstrated by ELISA, identified the region of NT receptors involved. Cross-reactivity between these peptides and T. cruzi antigen was confirmed by binding inhibition assays. These results support the existence of cross-reactivity due to molecular mimicry between a parasite antigen and the major antigenic epitopes present on both beta1-adrenergic and M2-ACh receptors. Its possible relationship with cardiac dysfunction during chronic stage of Chagas' disease is also discussed.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Protozoan/pharmacology , Myocardial Contraction/drug effects , Pindolol/analogs & derivatives , Receptor, Muscarinic M2/immunology , Receptors, Adrenergic, beta-1/immunology , Trypanosoma cruzi/immunology , Adrenergic beta-Antagonists , Analysis of Variance , Animals , Binding, Competitive/drug effects , Binding, Competitive/physiology , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Enzyme-Linked Immunosorbent Assay/methods , Epitopes/metabolism , Epitopes/pharmacology , Immunoglobulin Fab Fragments/metabolism , In Vitro Techniques , Iodine Isotopes/pharmacokinetics , Mice , Mice, Inbred BALB C , Muscarinic Antagonists/pharmacokinetics , Myocardial Contraction/physiology , Pindolol/pharmacokinetics , Quinuclidinyl Benzilate/pharmacokinetics , Radioimmunoassay/methods , Radioligand Assay/methods , Receptor, Muscarinic M2/chemistry , Receptors, Adrenergic, beta-1/chemistry , Titrimetry/methods , Trypanosoma cruzi/chemistryABSTRACT
We examined the effect of long-term agonist exposure on muscarinic acetylcholine receptor expression and function in embryonic chicken retinal cells. Long-term carbachol exposure induced a time- and concentration-dependent decrease in M2, M3 and M4 muscarinic receptor numbers. Kinetic analyses revealed a first-order process with similar rate constants for all three subtypes. Both the maximal decrease and the agonist potency for regulation of M3 were significantly higher than those for M2 and M4. Upon agonist removal, M2 and M4 numbers returned to control values, but M3 recovery after 24 h was no higher than 40%. Agonist treatment did not alter the levels of receptor mRNAs. Receptor inactivation with a covalent alkylating antagonist demonstrated that the partial M3 protein recovery was not due to a decreased intrinsic basal rate of synthesis, suggesting that it is induced by agonist treatment. Prolonged carbachol exposure induced concomitant decreases in muscarinic-mediated inhibition of cyclic AMP accumulation which were completely reversed after agonist removal. Sustained receptor activation also promoted significant decreases in muscarinic receptor-stimulated phosphoinositide turnover, which were only partially reversed after agonist removal. These data demonstrate subtype-specific regulation of the expression and function of muscarinic receptors in the retina.
Subject(s)
Gene Expression Regulation , Receptors, Muscarinic/metabolism , Retina/metabolism , Adenylyl Cyclases/metabolism , Animals , Carbachol/pharmacology , Cells, Cultured , Chick Embryo , Colforsin/pharmacology , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Gene Expression Regulation/drug effects , Muscarinic Agonists/pharmacology , Muscarinic Antagonists/pharmacology , RNA, Messenger/metabolism , Receptor, Muscarinic M2 , Receptor, Muscarinic M3 , Receptor, Muscarinic M4 , Receptors, Muscarinic/drug effects , Receptors, Muscarinic/genetics , Retina/cytology , Retina/embryology , Time , Type C Phospholipases/metabolismABSTRACT
Este trabajo demuestra, en pacientes chagásicos, la presencia de auto-anticuerpos circulantes reactivos contra el tercer dominio extracelular del receptor muscarínico M2, humano utilizando un péptido sintético correspondiente a la secuencia 169-192 del receptor, mediante técnicas de ELISA e immunoblotting. Anticuerpos anti-péptido purificados por inmunoafinidad revelaron actividad muscarínica cardíaca, ya que deprimieron la contractilidad, inhibieron la producción de AMPc e incrementaron los niveles de GMPc; estos efectos fueron bloqueados específicamente por el péptido sintético y por atropina. Comprobamos una fuerte asociación entre presencia de autoanticuerpos anti-péptido M2 y manifestaciones de disautonomia en los pacientes, sugiriendo su posible utilidad como marcadores tempranos de disautonomia.
Subject(s)
Humans , Adult , Middle Aged , Autoantibodies/isolation & purification , Chagas Disease/immunology , Receptors, Muscarinic/immunology , Amino Acid Sequence , Autonomic Nervous System Diseases/immunology , Biomarkers , Chagas Disease/blood , Cyclic AMP , Cyclic GMP , Enzyme-Linked Immunosorbent Assay , Immunoblotting , Myocardial Contraction , PeptidesABSTRACT
Este trabajo demuestra, en pacientes chagásicos, la presencia de auto-anticuerpos circulantes reactivos contra el tercer dominio extracelular del receptor muscarínico M2, humano utilizando un péptido sintético correspondiente a la secuencia 169-192 del receptor, mediante técnicas de ELISA e immunoblotting. Anticuerpos anti-péptido purificados por inmunoafinidad revelaron actividad muscarínica cardíaca, ya que deprimieron la contractilidad, inhibieron la producción de AMPc e incrementaron los niveles de GMPc; estos efectos fueron bloqueados específicamente por el péptido sintético y por atropina. Comprobamos una fuerte asociación entre presencia de autoanticuerpos anti-péptido M2 y manifestaciones de disautonomia en los pacientes, sugiriendo su posible utilidad como marcadores tempranos de disautonomia. (AU)
