Quantitative analysis of homo- and heterodimerization of muscarinic acetylcholine receptors in live cells

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 M1, M2, and M3 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 M1, M2, or M3 mAChR is predominantly composed of high affinity homodimers. Saturation curve analysis of cells expressing two receptor subtypes demonstrates the existence of high affinity M1/M2, M2/M3, and M1/M3 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 M2 receptor exhibits much higher susceptibility than the M3 receptor to agonist-induced down-regulation. Coexpression of M3 mAChR with increasing amounts of the M2 subtype in JEG-3 cells resulted in an increased agonist-induced down-regulation of M3, suggesting a novel role of heterodimerization in the mechanism of mAChR long term regulation.Fil: Goin, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Nathanson, Neil M.. University of Washington. School of Medicine; Estados Unido

Chagasic achalasia: Muscarinic antibodies and their actions

Fil: Bilder, Claudio Rubén. Fundación Favaloro; ArgentinaFil: Goin, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

Gastrointestinal Involvement in Chagas Disease

Achalasia and megacolon are the second most common manifestations of chronic Chagas disease (ChD) in endemic areas of Central and South America. Twenty or even more years after the initial infection, approximately one-third of infected people develop cardiac and/ or gastrointestinal abnormalities as typical chronic damages of ChD. The chronic phase of ChD is characterized by the damage of myenteric (intrinsic) and autonomic (extrinsic) neurons. A decreased density of enteric glial cells can be detected in patients with megaesophagus and megacolon and a loss of interstitial cells of Cajal in the latter patients. These lesions affect the complex mechanisms of neural, molecular, and cellular interactions that modulate the motor activity and other specific functions of the alimentary tract. Evidence for serum antibodies with the ability to recognize similar epitopes in both Trypanosoma cruzi and host antigens suggested that molecular mimicry could play a substantial role in the pathophysiology of chronic ChD. In fact, a high prevalence of circulating antibodies against M2 acetylcholine muscarinic receptor (M2R) in ChD patients with achalasia and megacolon has been found. These antibodies bind to and activate M2R, exhibiting agonist-like activity. Anti-M2R antibodies can both enhance tonic contraction in lower esophagus and distal colon by direct stimulation and also by counteracting the relaxant effect of drugs that increase cAMP accumulation (i.e. beta-adrenergic agonists). The biochemical and functional effects of these antibodies on esophageal and colon smooth muscle could play an important role in the pathophysiology of achalasia and megacolon secondary to ChD.Fil: Bilder, Claudio Rubén. Universidad Favaloro. Facultad de Medicina; ArgentinaFil: Goin, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

New Palaeogene metatherians from the Quebrada de Los Colorados Formation at Los Cardones National Park (Salta Province, Argentina)

The Quebrada de Los Colorados Formation (Los Cardones National Park, Salta Province, north-western Argentina), is an Eocene–Oligocene unit well represented in the Calchaquí Valley area. Here we describe a new metatherian association recorded from the base of this formation, inferred as middle Eocene. Represented taxa are: Sparassodonta, family indet.: Patene simpsoni; Polydolopimorphia, Bonapartheriiformes, Argyrolagoidea: family, genus, and species indet.; and Bonapartherioidea, Prepidolopidae: Punadolops alonsoi and Coloradolops cardonensis gen. et sp. nov. Patene simpsoni is also found in the Tonco Valley, near Los Cardones, and at São José de Itaboraí, Rio de Janeiro, Brazil. Punadolops alonsoi was originally described from the Geste Formation in Salta and Catamarca provinces. Hitherto, no other Argyrolagoidea have been found in north-western Argentina in the Palaeogene. Coloradolops cardonensis is unique to the locality and levels here described. The preservation of part of the skull constitutes the best prepidolopid remains known. North-western Argentina is a key region for the study of the evolution of South American mammals, since it has a singular geographical location between the Neotropics and the southernmost part of South America. Due to biogeographical constraints, it may lead to a better comprehension of the continental distribution of metatherian lineages during Cenozoic times.Fil: Chornogubsky Clerici, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas; ArgentinaFil: Zimicz, Ana Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Bio y Geociencias del NOA. Universidad Nacional de Salta. Facultad de Ciencias Naturales. Museo de Ciencias Naturales. Instituto de Bio y Geociencias del NOA; ArgentinaFil: Goin, Francisco Javier. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. División Paleontología Vertebrados; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernicola, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; ArgentinaFil: Payrola Bosio, Patricio Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Bio y Geociencias del NOA. Universidad Nacional de Salta. Facultad de Ciencias Naturales. Museo de Ciencias Naturales. Instituto de Bio y Geociencias del NOA; ArgentinaFil: Cardenas, Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigación en Paleobiología y Geología; Argentin

An Oral Salmonella-Based Vaccine Inhibits Liver Metastases by Promoting Tumor-Specific T-Cell-Mediated Immunity in Celiac and Portal Lymph Nodes: A Preclinical Study

Primary tumor excision is one of the most widely used therapies of cancer. However, the risk of metastases development still exists following tumor resection. The liver is a common site of metastatic disease for numerous cancers. Breast cancer is one of the most frequent sources of metastases to the liver. The aim of this work was to evaluate the efficacy of the orally administered Salmonella Typhi vaccine strain CVD 915 on the development of liver metastases in a mouse model of breast cancer. To this end, one group of BALB/c mice was orogastrically immunized with CVD 915, while another received PBS as a control. After 24 h, mice were injected with LM3 mammary adenocarcinoma cells into the spleen and subjected to splenectomy. This oral Salmonella-based vaccine produced an antitumor effect, leading to a decrease in the number and volume of liver metastases. Immunization with Salmonella induced an early cellular immune response in mice. This innate stimulation rendered a large production of IFN-γ by intrahepatic immune cells (IHIC) detected within 24 h. An antitumor adaptive immunity was found in the liver and celiac and portal lymph nodes (LDLN) 21 days after oral bacterial inoculation. The antitumor immune response inside the liver was associated with increased CD4(+) and dendritic cell populations as well as with an inflammatory infiltrate located around liver metastatic nodules. Enlarged levels of inflammatory cytokines (IFN-γ and TNF) were also detected in IHIC. Furthermore, a tumor-specific production of IFN-γ and TNF as well as tumor-specific IFN-γ-producing CD8 T cells (CD8(+)IFN-γ(+)) were found in the celiac and portal lymph nodes of Salmonella-treated mice. This study provides first evidence for the involvement of LDLN in the development of an efficient cellular immune response against hepatic tumors, which resulted in the elimination of liver metastases after oral Salmonella-based vaccination.Fil: Vendrell, Alejandrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Mongini, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Gravisaco, Maria Jose Federica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Canellada, Andrea Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Tesone, Agustina Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Goin, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Waldner, Claudia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

Impairment of agonist-induced M2 muscarinic receptor activation by autoantibodies from chagasic patients with cardiovascular dysautonomia

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.Fil: Beltrame, Sabrina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Carrera Paez, Laura Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Auger, Sergio Ricardo. Gobierno de la Ciudad de Buenos Aires. Hospital de Agudos "D. F. Santojanni"; ArgentinaFil: Sabra, Ahmad H.. Gobierno de la Ciudad de Buenos Aires. Hospital de Agudos "D. F. Santojanni"; ArgentinaFil: Bilder, Claudio Rubén. Fundación Favaloro; ArgentinaFil: Waldner, Claudia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Goin, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

Therapeutic effects of Salmonella Typhi in a mouse model of T-cell lymphoma

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, timedependent, 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.Fil: Vendrell, Alejandrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Gravisaco, María J.. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Inv. Agropecuarias; ArgentinaFil: Goin, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Pasetti, Marcela F.. University of Maryland; Estados UnidosFil: Herschllik, Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: de Toro, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Rodriguez, Carla Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Comision Nacional de Energia Atomica; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Larotonda, Gerardo. Lavet Lab Argentina; ArgentinaFil: Mongini, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Waldner, Claudia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentin