Specific humoral and cellular immunity induced by Trypanosoma cruzi DNA immunization in a canine model.

Chagas disease has a high incidence in Mexico and other Latin American countries. Because one of the most important known methods of prevention is vector control, which has been effective only in certain areas of South America, the development of a vaccine to protect people at risk has been proposed. In this study, we assessed the cellular and humoral immune response generated following immunization with pBCSP and pBCSSP4 plasmids containing the genes encoding a trans-sialidase protein (present in all three forms of T. cruzi) and an amastigote specific glycoprotein, respectively, in a canine model. Thirty-five beagle dogs were divided randomly into 5 groups (n=7) and were immunized twice intramuscularly with 500 µg of pBCSSP4, pBCSP, pBk-CMV (empty plasmid) or saline solution. Fifteen days after the last immunization the 4 groups were infected intraperitoneally with 500,000 metacyclic trypomastigotes. The fifth group was unimmunized/infected. The parasitaemia in the immunized/infected dogs was for a shorter period (14 vs. 29 days) and the parasite load was lower. The concentration of IgG1 (0.612±0.019 O.D.) and IgG2 (1.167±0.097 O.D.) subclasses was measured (absorbance) 15 days after the last immunization with both recombinant plasmids, the majority of which were IgG2. The treatment of parasites using the serum from dogs immunized with pBCSP and pBCSSP4 plasmids produced 54% (±11.8) and 68% (±21.4) complement-mediated lysis, respectively. At 12 h post immunization, an increase in cytokines was not observed; however, vaccination with pBCSSP4 significantly increased the levels of IFN-γ and IL-10 at 9 months post-infection. The recombinant plasmid immunization stimulated the spleen cell proliferation showing a positive stimulatory index above 2.0. In conclusion, immunization using both genes effectively induces a humoral and cellular immune response.

Plasmid DNA immunization with Trypanosoma cruzi genes induces cardiac and clinical protection against Chagas disease in the canine model.

The only existing preventive measure against American trypanosomosis, or Chagas disease, is the control of the transmitting insect, which has only been effective in a few South American regions. Currently, there is no vaccine available to prevent this disease. Here, we present the clinical and cardiac levels of protection induced by expression to Trypanosoma cruzi genes encoding the TcSP and TcSSP4 proteins in the canine model. Physical examination, diagnostic chagasic serology, and serial electrocardiograms were performed before and after immunization, as well as after experimental infection. We found that immunization with recombinant plasmids prevented hyperthermia in the acute phase of experimental infection and produced lymphadenomegaly as an immunological response against the parasite and additionally prevented heart rate elevation (tachycardia) in the acute and/or chronic stages of infection. Immunization with T. cruzi genes encoding the TcSP and TcSSP4 antigens diminished the quality and quantity of the electrocardiographic abnormalities, thereby avoiding progression to more severe developments such as right bundle branch block or ventricular premature complexes in a greater number of dogs.

The MHC2TA gene polymorphisms are not associated with restenosis after coronary stenting in Mexican patients.

OBJECTIVE: The aim of this study was to test for association between MHC2TA gene polymorphisms and risk for restenosis after coronary stent placement in a group of Mexican patients. METHODS: The MHC2TA-168A>G (rs3087456), 1614C>G (rs4774), and 2536G>A (rs2229320) single nucleotide polymorphisms were genotyped using 5' exonuclease TaqMan genotyping assays in a group of 202 patients, who underwent coronary artery stenting. Basal and procedure coronary angiography were analyzed, looking for angiographic predictors of restenosis and follow-up angiography was performed to screen for binary restenosis. RESULTS: The results obtained in this study showed that the frequency of the three polymorphisms studied was similar in patients with and without restenosis. Univariate analysis showed that the use of drug-eluting stent (DES) reduces the risk of developing restenosis (p<0.001, OR=0.26). In contrast, the diameter<2.5mm of the stent and bifurcations increased the risk of developing restenosis (p=0.049, OR=1.74 and p=0.041, OR=1.8). CONCLUSION: The present study suggests that the MHC2TA polymorphisms are not involved in the risk of developing restenosis after coronary stent placement.

DNA Vaccine Treatment in Dogs Experimentally Infected with Trypanosoma cruzi.

Chagas disease is a chronic and potentially lethal disorder caused by the parasite Trypanosoma cruzi, and an effective treatment has not been developed for chronic Chagas disease. The objective of this study was to determine the effectiveness of a therapeutic DNA vaccine containing T. cruzi genes in dogs with experimentally induced Chagas disease through clinical, pathological, and immunological analyses. Infection of Beagle dogs with the H8 T. cruzi strain was performed intraperitoneally with 3500 metacyclic trypomastigotes/kg body weight. Two weeks after infection, plasmid DNA immunotherapy was administered thrice at 15-day intervals. The clinical (physical and cabinet studies), immunological (antibody and cytokine profiles and lymphoproliferation), and macro- and microscopic pathological findings were described. A significant increase in IgG and cell proliferation was recorded after immunotherapy, and the highest stimulation index (3.02) was observed in dogs treated with the pBCSSP4 plasmid. The second treatment with both plasmids induced an increase in IL-1, and the third treatment with the pBCSSP4 plasmid induced an increase in IL-6. The pBCSP plasmid had a good Th1 response regulated by high levels of IFN-gamma and TNF-alpha, whereas the combination of the two plasmids did not have a synergistic effect. Electrocardiographic studies registered lower abnormalities and the lowest number of individuals with abnormalities in each group treated with the therapeutic vaccine. Echocardiograms showed that the pBCSSP4 plasmid immunotherapy preserved cardiac structure and function to a greater extent and prevented cardiomegaly. The two plasmids alone controlled the infection moderately by a reduction in the inflammatory infiltrates in heart tissue. The immunotherapy was able to reduce the magnitude of cardiac lesions and modulate the cellular immune response; the pBCSP treatment showed a clear Th1 response; and pBCSSP4 induced a balanced Th1/Th2 immune response that prevented severe cardiac involvement. The pBCSSP4 plasmid had a better effect on most of the parameters evaluated in this study; therefore, this plasmid can be considered an optional treatment against Chagas disease in naturally infected dogs.

Electrolyzed Oxidizing Water Modulates the Immune Response in BALB/c Mice Experimentally Infected with Trypanosoma cruzi.

Chagas disease is a major public health problem in Latin America. The mixed Th1/Th2 immune response is required against Trypanosoma cruzi. Electrolyzed oxidizing water (EOW) has been shown to have germicidal efficacy. The objective of this study was to evaluate the EOW effectiveness in T. cruzi-infected BALB/c mice clinically, immunologically, and histologically. The severity of the infection was assessed by parasitaemia, general health condition, mortality, mega syndromes, and histological lesions. IgG, TNF-alpha, IFN-gamma, and IL-1 beta levels were quantified. The EOW administration showed a beneficial effect on parasitaemia, general physical condition, and mortality. High levels of IgG1 at 50 days postinfection were observed. Prophylactic EOW treatment was able to induce a predominantly TH1 immune response based on an IgG2a levels increase at the late acute phase, and a 10-fold increase of INF-gamma in whole acute phase. EOW was able to control the acute phase infection as effectively as benznidazole. Splenomegaly was caused by EOW treatment and lymphadenopathy was stimulated by T. cruzi infection in all groups. Severe tissue damage was not prevented by EOW treatments. Moderate efficacy may be due to immunomodulatory properties and not to a direct toxic effect on the parasite.

Seropositivity for Trypanosoma cruzi in domestic dogs from Sonora, Mexico.

BACKGROUND: Chagas disease is an important health problem in Latin America due to its incapacitating effects and associated mortality. Studies on seropositivity for Trypanosoma cruzi in Mexican dogs have demonstrated a direct correlation between seropositivity in humans and dogs, which can act as sentinels for the disease in this region. The objective of this study was to determine the seropositivity for T.cruzi infection in dogs from Sonora, a northern borderstate of Mexico. METHODS: Responsible pet owners were selected at random from an urban area of Empalme municipality, Sonora, Mexico, and from there, 180 dog samples were collected. Anti-T. cruzi antibodies were determined using the enzyme-linked immunosorbent assay (ELISA) method. Reactive ELISA sera were processed by indirect immunofluorescence to confirm the presence of anti-T. cruzi antibodies. For the statistical analysis, chi-square tests were conducted. RESULTS: Dogs' sera showed a seropositivity rate of 4.44%. The rate of seropositivity was not associated with the dogs' age, sex, or socioeconomics pertaining to the geographical area. One sample (1/180, 0.55%) showed the acute state of the disease. CONCLUSIONS: The study found a presence of anti-T. cruzi antibodies in dogs in this area, which suggests vector transmission. There is a need for active surveillance programs throughout the state of Sonora and vector control strategies should also be implemented in endemic regions.

Prophylactic and therapeutic DNA vaccines against Chagas disease.

Chagas disease is a zoonosis caused by Trypanosoma cruzi in which the most affected organ is the heart. Conventional chemotherapy has a very low effectiveness; despite recent efforts, there is currently no better or more effective treatment available. DNA vaccines provide a new alternative for both prevention and treatment of a variety of infectious disorders, including Chagas disease. Recombinant DNA technology has allowed some vaccines to be developed using recombinant proteins or virus-like particles capable of inducing both a humoral and cellular specific immune response. This type of immunization has been successfully used in preclinical studies and there are diverse models for viral, bacterial and/or parasitic diseases, allergies, tumors and other diseases. Therefore, several research groups have been given the task of designing a DNA vaccine against experimental infection with T. cruzi. In this review we explain what DNA vaccines are and the most recent studies that have been done to develop them with prophylactic or therapeutic purposes against Chagas disease.

Experimental Vaccines against Chagas Disease: A Journey through History.

Chagas disease, or American trypanosomiasis, which is caused by the protozoan parasite Trypanosoma cruzi, is primarily a vector disease endemic in 21 Latin American countries, including Mexico. Although many vector control programs have been implemented, T. cruzi has not been eradicated. The development of an anti-T. cruzi vaccine for prophylactic and therapeutic purposes may significantly contribute to the transmission control of Chagas disease. Immune protection against experimental infection with T. cruzi has been studied since the second decade of the last century, and many types of immunogens have been used subsequently, such as killed or attenuated parasites and new DNA vaccines. This primary prevention strategy appears feasible, effective, safe, and inexpensive, although problems remain. The objective of this review is to summarize the research efforts about the development of vaccines against Chagas disease worldwide. A thorough literature review was conducted by searching PubMed with the terms "Chagas disease" and "American trypanosomiasis" together with "vaccines" or "immunization". In addition, reports and journals not cited in PubMed were identified. Publications in English, Spanish, and Portuguese were reviewed.

The -974C>A (rs3087459) gene polymorphism in the endothelin gene (EDN1) is associated with risk of developing acute coronary syndrome in Mexican patients.

Endothelial dysfunction plays an essential role in the development and progression of atherosclerotic lesions. Endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) are considered important molecules in the endothelial dysfunction process. The aim of the present study was to evaluate the role of eNOS and ET-1 (EDN1) gene polymorphisms as susceptibility markers for acute coronary syndrome (ACS). Six polymorphisms (rs1799983, rs2070744, rs1800783, rs3087459, rs1800541, and rs5369) of eNOS and EDN1 genes were analyzed by 5' exonuclease TaqMan genotyping assays in a group of 452 patients with ACS and 283 healthy controls. The results showed increased frequencies of the A allele of the END1-914 C>A (rs3087459) polymorphism in ACS patients when compared to controls (OR=1.56, Pc=0.01). Under an additive model, the "AA" genotype was associated with an increased risk of developing ACS, adjusted for gender, hypertension, dyslipidemia, alcohol consumption, smoking, and diabetes (OR=1.56, p=0.045). Linkage disequilibrium analysis showed one EDN1 haplotype (AT) with increased frequency in ACS patients when compared to healthy controls (OR=1.65, Pc=0.0015). The "AT" haplotype was associated with the risk of developing ACS after adjusting for cardiovascular risk factors using multiple logistic analysis. In this case, the adjusted OR was 1.73 for the AT haplotype (Pc=0.0018). In summary, resulting data suggest that the END1-914 C>A gene polymorphism could be involved in the risk of developing ACS in Mexican individuals.

Effect of the plasmid-DNA vaccination on macroscopic and microscopic damage caused by the experimental chronic Trypanosoma cruzi infection in the canine model.

The dog is considered the main domestic reservoir for Trypanosoma cruzi infection and a suitable experimental animal model to study the pathological changes during the course of Chagas disease (CD). Vaccine development is one of CD prevention methods to protect people at risk. Two plasmids containing genes encoding a trans-sialidase protein (TcSP) and an amastigote-specific glycoprotein (TcSSP4) were used as DNA vaccines in a canine model. Splenomegaly was not found in either of the recombinant plasmid-immunized groups; however, cardiomegaly was absent in animals immunized only with the plasmid containing the TcSSP4 gene. The inflammation of subendocardial and myocardial tissues was prevented only with the immunization with TcSSP4 gene. In conclusion, the vaccination with these genes has a partial protective effect on the enlargement of splenic and cardiac tissues during the chronic CD and on microscopic hearth damage, since both plasmids prevented splenomegaly but only one avoided cardiomegaly, and the lesions in heart tissue of dog immunized with plasmid containing the TcSSP4 gene covered only subepicardial tissue.

Distribution of the IL-1RN, IL-6, IL-10, INF-γ, and TNF-α Gene Polymorphisms in the Mexican Population.

BACKGROUND: Cytokines are a group of polypeptides with an important role in the inflammatory response. It has been suggested that certain polymorphisms located in several cytokine genes are associated with different diseases. The aim of the present study was to establish the gene frequency of 13 polymorphisms of the IL-1RN, IL-6, IL-10, INF-γ, and TNF-α genes in a Mexican population. These polymorphisms have been reported in several populations, with important variation in frequency according to the studied population. METHODS: Thirteen polymorphisms (rs419598, rs315951, rs2234663, rs3811058, rs1800796, rs2069827, rs1800896, rs1800871, rs1800872, rs1800629, rs2069709, rs2069710, and rs361525) were analyzed by 5' exonuclease TaqMan genotyping assays in a group of 248 healthy unrelated Mexican individuals. RESULTS: The results obtained showed that the studied Mexican population presents significant differences (p<0.05) in the distribution of the IL1RN (rs419598, rs315951, and and rs2234663), IL1F10 (rs3811058), IL6 (rs1800796, rs2069827), IL10 (rs1800896, rs1800871, and rs1800872), and TNF-α (rs1800629) polymorphisms when compared to Caucasian, Asian, and African populations. CONCLUSIONS: In summary, the distribution of the IL-1RN, IL-6, IL-10, and TNF-α cytokine gene polymorphisms distinguishes the studied Mexican population from other groups. Since the alleles of these cytokines are associated with the development of several inflammatory diseases, knowledge of the distribution of these alleles in the studied Mexican population could be helpful to understand their true role as a genetic susceptibility marker in this population.

[Genetic and genomic studies in the hypertension: an actualization of the genomic studies]. / Genética y genómica de la hipertensión arterial: una actualización.

The arterial hypertension is considered to be the main risk factor for myocardial infarction, heart failure, ventricular arrhythmias, kidney failure, blindness and other diseases. Arterial hypertension is a multifactorial disease derived from environmental, genetic, gender and ethnic factors. In recent years, the World Health Organization estimated that approximately 17.5 million of deaths were due to cardiovascular diseases worldwide and that this pathology will become the leading cause of death in the next decade. Data from the National Survey of Mexican Ministry of Health (2006), reported approximately 17 million hypertensive adults, equivalent to a prevalence of 30.8% among Mexican population. As a consequence, hypertension represents the leading cause of morbidity from 2000 to 2005 and is increasing in recent years. However, studies have failed to clearly identify the molecular and genetic mechanisms of this pathology so far. Nevertheless, using the new technologies for analysis of variants in the genome, several genes in different loci that confer susceptibility to develop hypertension have been identified. In this review we compared the different studies in genetics and genomics of the hypertension that have been made worldwide and in Mexico, with the aim of identifying important genes involved in susceptibility to the development of this pathology.

The MHC2TA gene polymorphisms are not associated with restenosis after coronary stenting in Mexican patients / Los polimorfismos del gen MHC2TA no se asocian con la reestenosis después del implante de stent coronario en pacientes mexicanos

Objective: The aim of this study was to test for association between MHC2TA gene polymorphisms and risk for restenosis after coronary stent placement in a group of Mexican patients. Methods: The MHC2TA-168A>G (rs3087456), 1614C>G (rs4774), and 2536G>A (rs2229320) single nucleotide polymorphisms were genotyped using 5' exonuclease TaqMan genotyping assays in a group of 202 patients, who underwent coronary artery stenting. Basal and procedure coronary angiography were analyzed, looking for angiographic predictors of restenosis and follow-up angiography was performed to screen for binary restenosis. Results: The results obtained in this study showed that the frequency of the three polymorphisms studied was similar in patients with and without restenosis. Univariate analysis showed that the use of drug-eluting stent (DES) reduces the risk of developing restenosis (p < 0.001, OR = 0.26). In contrast, the diameter< 2.5 mm of the stent and bifurcations increased the risk of developing restenosis (p = 0.049, OR = 1.74 and p = 0.041, OR = 1.8). Conclusion: The present study suggests that the MHC2TA polymorphisms are not involved in the risk of developing restenosis after coronary stent placement.

Objetivo: El propósito de este estudio fue evaluar la asociación de los polimorfismos del gen MHC2TA y el riesgo de desarrollar reestenosis, después del implante de stent coronario en un grupo de pacientes mexicanos. Métodos: Los polimorfismos de un solo nucleótido MHC2TA-168A>G (rs3087456), 1614C>G (rs4774) y 2536G>A (rs2229320), se determinaron en un grupo de 202 pacientes tratados con stent coronario. Los polimorfismos fueron evaluados utilizando ensayos de genotipificacion Taq-Man 5' exonucleasa. El procedimiento basal y la búsqueda de predictores de reestenosis fueron analizados por medio de angiografía coronaria, y seguimiento angiográfico con el fin de detectar reestenosis binaria. Resultados: Los resultados obtenidos en este estudio mostraron que la distribución génica de los tres polimorfismos estudiados fue muy similar, en pacientes con o sin reestenosis. Sin embargo, el análisis univariado mostró que el uso de los stent medicados reducen el riesgo de desarrollar reestenosis (p < 0.001, OR = 0.26). En contraste, con las bifurcaciones y el diámetro < 2.5 mm del stent que se incrementa el riesgo de desarrollar reestenosis (p = 0.049, OR = 1.74 y p = 0.041, OR = 1.8). Conclusión: El presente estudio sugiere que los polimorfismos del gen MHC2TA no están asociados con el riesgo de desarrollar reestenosis, después del implante de stent coronario.

Genética y genómica de la hipertensión arterial: una actualización / Genetic and genomic studies in the hypertension: an actualization

La hipertensión arterial es considerada la principal causa de riesgo para el desarrollo de infarto agudo al miocardio, falla cardiaca, arritmia ventricular, nefropatía, ceguera, entre otras. La hipertensión arterial es una enfermedad multifactorial en la que participan factores ambientales, genéticos e intrínsecos como raza y género. La Organización Mundial de la Salud ha estimado que la prevalencia de la hipertensión se incrementará drásticamente, de modo que en la próxima década será la primera causa de muerte a nivel mundial, debido al elevado número de muertes (17.5 millones) por enfermedades cardiovasculares registradas a nivel mundial. No obstante, los datos publicados en la Encuesta Nacional de la Secretaría de Salud en 2006, señalan aproximadamente 17 millones de hipertensos en población adulta y una prevalencia de 30.8%, cifra que indica que la hipertensión arterial aumentó su morbilidad del año 2000 al 2005. Sin embargo, poco se ha logrado en la comprensión de los mecanismos moleculares y genéticos involucrados en el desarrollo de esta patología y en consecuencia su prevalencia sigue aumentando drásticamente. Con el uso de las nuevas tecnologías para el análisis de las variantes en el genoma, se han identificado algunos genes en diferentes loci que confieren susceptibilidad al desarrollo de hipertensión arterial. La finalidad de esta revisión es hacer una comparación entre los diferentes estudios en genética y genómica relacionados con esta patología a nivel mundial y en nuestro país, con la finalidad de identificar genes clave que participen en la susceptibilidad al desarrollo de la hipertensión arterial.

The arterial hypertension is considered to be the main risk factor for myocardial infarction, heart failure, ventricular arrhythmias, kidney failure, blindness and other diseases. Arterial hypertension is a multifactorial disease derived from environmental, genetic, gender and ethnic factors. In recent years, the World Health Organization estimated that approximately 17.5 million of deaths were due to cardiovascular diseases worldwide and that this pathology will become the leading cause of death in the next decade. Data from the National Survey of Mexican Ministry of Health (2006), reported approximately 17 million hypertensive adults, equivalent to a prevalence of 30.8% among Mexican population. As a consequence, hypertension represents the leading cause of morbidity from 2000 to 2005 and is increasing in recent years. However, studies have failed to clearly identify the molecular and genetic mechanisms of this pathology so far. Nevertheless, using the new technologies for analysis of variants in the genome, several genes in different loci that confer susceptibility to develop hypertension have been identified. In this review we compared the different studies in genetics and genomics of the hypertension that have been made worldwide and in Mexico, with the aim of identifying important genes involved in susceptibility to the development of this pathology.