Pesticides exposure in pregnant Argentinian women: Potential relations with the residence areas and the anthropometric neonate parameters.

Intrauterine environment is the first chemical exposure scenario in life, through transplacental transfer. In this study, the aim was to determine concentrations of organochlorine pesticides (OCPs) and selected current use pesticides in the placentas of pregnant women from Argentina. Socio-demographic information, the mother's lifestyle and neonatal characteristics were also analysed and related to pesticides residue concentrations. Thus, 85 placentas were collected at birth, from an area of intensive fruit production for the international market, in Patagonia Argentina. Concentrations of 23 pesticides including, trifluralin (herbicide), the fungicides chlorothalonil and HCB, and the insecticides chlorpyrifos, HCHs, endosulfans, DDTs, chlordanes, heptachlors, drins and metoxichlor, were determined by GC-ECD and GC-MS. Results were first analysed all together and then grouped by their residential settings, in urban and rural groups. Total mean pesticide concentration was 582.6 ± 1034.4 ng/g lw, where DDTs (325.9 ± 950.3 ng/g lw) and chlorpyrifos (188.4 ± 365.4 ng/g lw) showed a high contribution. Pesticide levels found exceeded those reported in low, middle and high income countries from Europe, Asia and Africa. In general, pesticides concentrations were not associated with neonatal anthropometric parameters. When the results were analysed by residence place, significantly higher concentrations of total pesticides and chlorpyrifos (Mann Whitney test p = 0.0003 and p = 0.032, respectively) were observed in placentas collected from mothers living in rural settings compared to urban areas. Rural pregnant women presented the highest pesticide burden (5.9 µg), where DDTs and chlorpyrifos were the major constituents. These results suggested that all pregnant women are highly exposed to complex pesticide mixtures, including banned OCPs and the widely used chlorpyrifos. Based on the pesticide concentrations found, our results warn of possible health impacts from prenatal exposure through transplacental transfer. This is one of the first reports of both chlorpyrifos and chlorothalonil concentrations in placental tissue, and contributes to the knowledge of current pesticide exposure in Argentina.

The impact of pesticides on the macroinvertebrate community in the water channels of the Río Negro and Neuquén Valley, North Patagonia (Argentina).

Agriculture represents the second most important economic activity in the North Patagonian Region of Argentina and non-selective insecticides are still being used with significant implications to the quality of the environment. The range of concentrations (µg/L) determined for azinphosmethyl, chlorpyrifos, and carbaryl in drainage channels were from non-detected to 1.02, 1.45, and 11.21, respectively. Macroinvertebrate abundance and taxon richness in drainage channels were significantly lower in November compared to the other sampling months (October, February). The decrease in taxon richness observed in November was associated with chlorpyrifos and azinphosmethyl peak concentrations. The most remarkable changes were the decrease in sensitive taxa such as Baetidae and the increase in some tolerant taxa such as Chironomidae and Gastropoda.For all three pesticides, the acute hazard quotient exceeded the risk criteria for invertebrates. The effects of the three pesticides on aquatic organisms, characterized by joint probability curves, showed that the LC50 of 10% of the species were exceeded five and three times by the concentrations of azinphosmethyl and chlorpyrifos during the study period, respectively. However, the correlation between the pesticide concentrations and both taxon richness and abundance of macroinvertebrates at each site (irrigation and drainage channels) was indicative that only chlorpyrifos was negatively correlated with both parameters (Spearman r2 - 0.61, p = 0.0051 and Spearman r2 - 0.59, p = 0.0068 for taxon richness and abundance correlation, respectively). We conclude that macroinvertebrate assemblages in drainage channels were highly affected by chlorpyrifos levels.

Neonatal, placental, and umbilical cord blood parameters in pregnant women residing in areas with intensive pesticide application.

In rural populations, the proximity to areas with intensive pesticide application represents a risk factor of xenobiotic exposure. Here, we investigated whether newborns born to mothers residing in an area with intensive pesticide application show alterations in placental and neonatal morphometric standards, umbilical cord blood (UCB) biochemical parameters, and/or biomarkers related to oxidative stress and oxidative damage. Samples were collected from 151 healthy pregnant women residing in a rural area (rural group; RG) during the pesticide spraying (SS) and nonspraying (NSS) seasons, as well as from women from an urban population (control group; CG), and grouped according to the delivery type (vaginal or cesarean). In the vaginal delivery group, the placental weight and placental index were higher in the RG groups than in the CG (p = 0.01), whereas in the cesarean delivery group, newborn weight was lower in the RG-SS group than in the CG. In the RG-SS group, UCB erythrocyte osmotic fragility and the DNA damage index (DI) were higher, and superoxide dismutase (SOD) activity was lower than in the RG-NSS group. Acetylcholinesterase and SOD activities were found to be inversely correlated with the DI.

Residuos de plaguicidas organofosforados y carbamatos en aguas subterráneas de bebida en las zonas rurales de Plottier y Senillosa, Patagonia Norte, Argentina / Organophosphate and carbamate pesticide residues in drinking groundwater in the rural areas of Plottier and Senillosa, North Patagonia, Argentina

Plottier y Senillosa son dos localidades contiguas que forman parte del área de producción agrícola del Alto Valle de Río Negro y Neuquén, Patagonia Norte. Entre los plaguicidas más utilizados predominan las familias de neonicotinoides, carbamatos y organofosforados. El conocimiento de los niveles de estos compuestos en aguas superficiales y subterráneas se ha convertido en un tema de interés social debido a su posible impacto en el ambiente y la salud. El objetivo del presente trabajo fue determinar niveles de residuos de plaguicidas en agua subterránea utilizada para bebida en zonas rurales de Plottier y Senillosa. Se analizaron carbamatos: propoxur, carbofuran, pirimicarb, carbaril y organofosforados: clorpirifos, dimetoato, metidation, fenaminfos, triazofos, fosmet y metil azinfos, por cromatografía gaseosa (GC), en seis sitios de muestreo, durante la época de aplicación y no aplicación de insecticidas. No se detectaron residuos de los plaguicidas estudiados por el método analítico utilizado. El límite de detección fue 0,010 µg/L para organofosforados y 0,030 µg/L para carbamatos. Estos resultados indican que las buenas prácticas agrícolas aplicadas en la zona así como las características fsicoquímicas del suelo, su espesor y el contenido de materia orgánica y las propiedades fsicoquímicas de los plaguicidas estudiados son factores que contribuyen favorablemente a la calidad del agua de bebida de la población rural.

Plottier and Senillosa are two adjoining towns that belong to the agricultural production area of Upper Valley, Río Negro and Neuquén, North Patagonia. Among the most widely used pesticides, neonicotinoids, carbamates and organophosphates are predominant. The levels of these compounds in surface and groundwater have become a topic of public concern, since the possible impact on health and environment. The aim of this study was the determination of pesticide residue levels in drinking ground-water in the rural areas of Plottier and Senillosa. Carbamates: propoxur, carbofuran, pirimicarb, carbaryl and organophosphate: chlorpyrifos, dimethoate, methidathion, fenaminfos, triazophos, phosmet, azinphos methyl, were analyzed by GC in six sampling sites during insecticide application and non-application season. No pesticide residues were detected by the analytical method used. The detection limit for organophosphates was 0.010 µg/L and 0.030 µg/L for carbamates. These results indicate that good agricultural practices, the physicochemical characteristics of the soil, its thickness, the content of organic matter and the physicochemical properties of pesticides studied are factors that positively contribute to the drinking water quality in the rural population.

Alteration of syncytiotrophoblast mitochondria function and endothelial nitric oxide synthase expression in the placenta of rural residents.

The impact of environmental organophosphate (OP) pesticide exposure on respiratory complexes, enzymatic antioxidant defense activities, and oxidative damage markers in the syncytiotrophoblast and cytotrophoblast mitochondria was evaluated. Placental progesterone (PG) levels and endothelial nitric oxide synthase (eNOS) expression were studied. Samples from women non-exposed (control group-CG) and women living in a rural area (rural group-RG) were collected during pesticide spraying season (RG-SS) and non-spraying season (RG-NSS). In RG-SS, the exposure biomarker placental carboxylesterase decreased and syncytiotrophoblast cytochrome c oxidase activity increased, while 4-hydroxynonenal levels decreased. PG levels decreased in RG-SS and in the RG. Nitric oxide synthase expression decreased in RG, RG-SS and RG-NSS. No significant changes in mitochondrial antioxidant enzyme activities were found. These results suggest that the alteration of syncytiotrophoblast mitochondrial complex IV activity and steroidogenic function may be associated to pesticide exposure. Reduction in placental PG and eNOS expression may account for low newborn weight in RG.

B-esterase determination and organophosphate insecticide inhibitory effects in JEG-3 trophoblasts.

The placenta and trophoblasts express several B-esterases. This family includes acethylcholinesterase (AChE), carboxylesterase (CES) and butyrylcholinesterase (BChE), which are important targets of organophosphate insecticide (OP) toxicity. To better understand OP effects on trophoblasts, B-esterase basal activity and kinetic behavior were studied in JEG-3 choriocarcinoma cell cultures. Effects of the OP azinphos-methyl (Am) and chlorpyrifos (Cp) on cellular enzyme activity were also evaluated. JEG-3 cells showed measurable activity levels of AChE and CES, while BChE was undetected. Recorded Km for AChE and CES were 0.33 and 0.26 mM respectively. Native gel electrophoresis and RT-PCR analysis demonstrated CES1 and CES2 isoform expression. Cells exposed for 4 and 24 h to the OP Am or Cp, showed a differential CES and AChE inhibition profiles. Am inhibited CES and AChE at 4 h treatment while Cp showed the highest inhibition profile at 24 h. Interestingly, both insecticides differentially affected CES1 and CES2 activities. Results demonstrated that JEG-3 trophoblasts express AChE, CES1 and CES2. B-esterase enzymes were inhibited by in vitro OP exposure, indicating that JEG-3 cells metabolization capabilities include phase I enzymes, able to bioactivate OP. In addition, since CES enzymes are important for medicinal drug activation/deactivation, OP exposure may interfere with trophoblast CES metabolization, probably being relevant in a co-exposure scenario during pregnancy.

Environmental pesticide exposure modulates cytokines, arginase and ornithine decarboxylase expression in human placenta.

To evaluate the cytokine balance and enzymatic alterations induced by environmental pesticide exposure during pregnancy, this transversal study explored placentas derived from non-exposed women (control group-CG), and from women living in a rural area (rural group-RG), collected during intensive organophosphate (OP) pesticide spraying season (RG-SS) and during non-spraying season (RG-NSS). The exposure biomarkers blood cholinesterase and placental carboxylesterase (CaE) were significantly decreased in RG-SS. Among the cytokines studied IL-8, IL-6, TNFα, IL-10, TGFß and IL-13, the expression frequency of IL-13 increased in RG-SS. Arginase and ornithine decarboxylase (ODC) enzymes were induced in syncytiotrophoblast and endothelial cells. Interestingly, the decrease in CaE activity was associated with arginase and ODC activity induction. These findings suggest that environmental pesticide exposure impacts the placenta by increasing the expression frequency of the anti-inflammatory cytokine IL-13, which may be related to the up-regulation of enzymes implicated in tissue repair.

Effects of the organophosphate insecticides phosmet and chlorpyrifos on trophoblast JEG-3 cell death, proliferation and inflammatory molecule production.

Epidemiological data have associated environmental organophosphate insecticide (OP) exposure during pregnancy with fetal growth deficits. To better understand OP injury that may adversely affect pregnancy, we used the JEG-3 choriocarcinoma cell line, which provide a recognized in vitro model to study placental function. The effects of the OP phosmet (Pm) and chlorpyrifos (Cp) on JEG-3 cells viability, proliferation, cell cycle and inflammatory molecule production were evaluated. Both insecticides affected cellular viability in a concentration- and time-dependent manner, inducing apoptosis and decreasing [(3)H]-thymidine incorporation. However, only Pm reduced DNA synthesis independently of cellular death and decreased the cell percentage at the S-phase. Unlike apoptosis, TNFα production varied with the concentration tested, suggesting that other TNFα independent mechanisms might trigger cell death. No induction of the inflammatory molecule nitric oxide was detected. The mRNA levels of pro-inflammatory IL-6, IL-17 and the anti-inflammatory IL-13 cytokines were differentially modulated. These findings show that Pm and Cp generate a specific toxicity signature, altering cell viability and inducing an inflammatory cytokine profile, suggesting that trophoblasts may represent a possible target for OP adverse effects.

Exposición ambiental a plaguicidas: impacto en parámetros moleculares, bioquímicos y morfométricos de la tríada madre-placenta-feto / Environmental exposure to pesticides: impact on molecular, biochemical and morphometric parameters of the mother-placenta-fetus triad

INTRODUCCION:La salud prenatal representa un problema relevante de salud públic, y uno de los temas prioritarios es la exposición ambiental a los plaguicidas.OBJETIVO:Identificar modificaciones en biomarcadores de efecto en la tríada madre-placenta-feto, mecanismos involucrados y asociaciones con el desarrollo "in utero".METODOS:Se realizó un estudio prospectivo con 776 embarazadas del Alto Valle del Río Negro, donde se aplican organofosforados (OF) intensivamente. Se utilizaron criterios de inclusión/exclusión, determinándose parámetros bioquímicos, moleculares y morfométricos en población de comunidades rurales (PoR) en período de pulverizaciones (PP) y de receso (PR) y en grupo control (PoC). Se registraron las alteraciones del embarazo.RESULTADOS:En PP, la Por presentó exposición a OF, y se vieron afectados los niveles de progesterona, cortisol y sulfato de dehidroepiandrosterona séricos durante el embarazo. En sangre de cordón aumentó la actividad de catalasa eritrocitaria. En la placenta se modificaron biomarcadores de estrés oxidativo, de funcionalidad mitocondrial, la expresión de TNFò e IL-13 y el contenido de la proteína Bcl-2. En concordancia, en células JEG-3 expuestas a OF se observó estrés oxidativo, apoptosis y aumento en la expresión de dichas citoquinas. La talla y el perímetro cefálico ajustados por sexo y edad gestacional del neonato fueron menores en PoR que en PoC (p<0,010 y p<0,05, respectivamente), mientras que el porcentaje de neonatos con perímetro cefálico menor al percentilo 5 fue mayor. En PoR se registró un mayor porcentaje de amenaza de aborto. Los niveles maternos de cortisol de primer y segundo trimestre de embarazo de PoR se asociaron negativamente con el peso y el perímetro cefálico.CONCLUSIONES:En PoR se afectaron parámetros de la tríada y del desarrollo intrauterino. El aumento en el nivel de cortisol materno sobreexpondría al feto a esta hormona, afectando la programación del eje hipotalámico-adrenal y la capacidad cognitiva.

INTRODUCTION: Prenatal health represents a relevant problem in public health. Special attention should be given to pesticide environmental exposure.OBJECTIVE: To identify changes in biomarkers of effect in the triad mother-placenta-fetus, mechanisms involved and their association with the in utero development.METHODS: Prospective study of 776 pregnante woman living in the High Valley of Río Negro, where organophosphate pesticides (OP) are intensively applied. The study used inclusion/exclusion criteria. Biochemical, molecular and morphometric parameters were determined in rural residents (RR) in pulverization (PP) and recess periods (RP) and in a control group (CG). Pregnancy alterations were registered.RESULTS: In PP, RR were exposed to OP, while serum progesterone, cortisol and dehydroepiandrosterone sulfate levels were affected during pregnancy. In cord blood, erythrocyte catalase activity was increased. In the placenta, there was a change in stress oxidative biomarkers, mitochondrial function biomarkers, the expression of TNFò and IL-13 and the protein Bcl-2 content. In line with the above, JEG-3 cells exposed to OP showed oxidative stress, apoptosis and increase in these cytokines expression. In RR, the newborn length and heah circumference adjusted by sex and gestational age were lower (p<0.010 and p<0.05, respectively) than in CG. Meanwhile, the percentage of newborns with head circumference lower than percentile 5 was higher. In RR, a higher percentage of threatened abortion was registered. Cortisol blood levels at first and second trimester of pregnancy were negatively associated with newborn weight and head circumference.CONCLUSIONS: Parameters of the triad and intrauterine development were affected in RR. The increase in the level of maternal cortisol would overexpose the fetus to this hormone, affecting the programming of hypothalamic-adrenal axis and the cognitive ability.

Exposición ambiental a plaguicidas: impacto en parámetros moleculares, bioquímicos y morfométricos de la tríada madre-placenta-feto / Environmental exposure to pesticides: impact on molecular, biochemical and morphometric parameters of the mother-placenta-fetus triad

INTRODUCCION:La salud prenatal representa un problema relevante de salud públic, y uno de los temas prioritarios es la exposición ambiental a los plaguicidas.OBJETIVO:Identificar modificaciones en biomarcadores de efecto en la tríada madre-placenta-feto, mecanismos involucrados y asociaciones con el desarrollo "in utero".METODOS:Se realizó un estudio prospectivo con 776 embarazadas del Alto Valle del Río Negro, donde se aplican organofosforados (OF) intensivamente. Se utilizaron criterios de inclusión/exclusión, determinándose parámetros bioquímicos, moleculares y morfométricos en población de comunidades rurales (PoR) en período de pulverizaciones (PP) y de receso (PR) y en grupo control (PoC). Se registraron las alteraciones del embarazo.RESULTADOS:En PP, la Por presentó exposición a OF, y se vieron afectados los niveles de progesterona, cortisol y sulfato de dehidroepiandrosterona séricos durante el embarazo. En sangre de cordón aumentó la actividad de catalasa eritrocitaria. En la placenta se modificaron biomarcadores de estrés oxidativo, de funcionalidad mitocondrial, la expresión de TNFò e IL-13 y el contenido de la proteína Bcl-2. En concordancia, en células JEG-3 expuestas a OF se observó estrés oxidativo, apoptosis y aumento en la expresión de dichas citoquinas. La talla y el perímetro cefálico ajustados por sexo y edad gestacional del neonato fueron menores en PoR que en PoC (p<0,010 y p<0,05, respectivamente), mientras que el porcentaje de neonatos con perímetro cefálico menor al percentilo 5 fue mayor. En PoR se registró un mayor porcentaje de amenaza de aborto. Los niveles maternos de cortisol de primer y segundo trimestre de embarazo de PoR se asociaron negativamente con el peso y el perímetro cefálico.CONCLUSIONES:En PoR se afectaron parámetros de la tríada y del desarrollo intrauterino. El aumento en el nivel de cortisol materno sobreexpondría al feto a esta hormona, afectando la programación del eje hipotalámico-adrenal y la capacidad cognitiva.

INTRODUCTION: Prenatal health represents a relevant problem in public health. Special attention should be given to pesticide environmental exposure.OBJECTIVE: To identify changes in biomarkers of effect in the triad mother-placenta-fetus, mechanisms involved and their association with the in utero development.METHODS: Prospective study of 776 pregnante woman living in the High Valley of Río Negro, where organophosphate pesticides (OP) are intensively applied. The study used inclusion/exclusion criteria. Biochemical, molecular and morphometric parameters were determined in rural residents (RR) in pulverization (PP) and recess periods (RP) and in a control group (CG). Pregnancy alterations were registered.RESULTS: In PP, RR were exposed to OP, while serum progesterone, cortisol and dehydroepiandrosterone sulfate levels were affected during pregnancy. In cord blood, erythrocyte catalase activity was increased. In the placenta, there was a change in stress oxidative biomarkers, mitochondrial function biomarkers, the expression of TNFò and IL-13 and the protein Bcl-2 content. In line with the above, JEG-3 cells exposed to OP showed oxidative stress, apoptosis and increase in these cytokines expression. In RR, the newborn length and heah circumference adjusted by sex and gestational age were lower (p<0.010 and p<0.05, respectively) than in CG. Meanwhile, the percentage of newborns with head circumference lower than percentile 5 was higher. In RR, a higher percentage of threatened abortion was registered. Cortisol blood levels at first and second trimester of pregnancy were negatively associated with newborn weight and head circumference.CONCLUSIONS: Parameters of the triad and intrauterine development were affected in RR. The increase in the level of maternal cortisol would overexpose the fetus to this hormone, affecting the programming of hypothalamic-adrenal axis and the cognitive ability.

The role of Toll-like receptors and adaptive immunity in the development of protective or pathological immune response triggered by the Trypanosoma cruzi protozoan.

Trypanosoma cruzi, the causal agent of Chagas disease, is an intracellular protozoan parasite that predominantly invades macrophages and cardiomyocytes, leading to persistent infection. Several members of the Toll-like receptor family are crucial for innate immunity to infection and are involved in maintaining tissue homeostasis. This review focuses on recent experimental findings of the innate and adaptive immune response in controlling the parasite and/or in generating heart and liver tissue injury. We also describe the importance of the host's genetic background in the outcome of the disease and emphasize the importance of studying the response to specific parasite antigens. Understanding the dual participation of the immune response may contribute to the design of new therapies for Chagas disease.

Importance of TLR2 on hepatic immune and non-immune cells to attenuate the strong inflammatory liver response during Trypanosoma cruzi acute infection.

BACKGROUND: Toll-like receptors (TLR) and cytokines play a central role in the pathogen clearance as well as in pathological processes. Recently, we reported that TLR2, TLR4 and TLR9 are differentially modulated in injured livers from BALB/c and C57BL/6 (B6) mice during Trypanosoma cruzi infection. However, the molecular and cellular mechanisms involved in local immune response remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we demonstrate that hepatic leukocytes from infected B6 mice produced higher amounts of pro-inflammatory cytokines than BALB/c mice, whereas IL10 and TGFß were only released by hepatic leukocytes from BALB/c. Strikingly, a higher expression of TLR2 and TLR4 was observed in hepatocytes of infected BALB/c mice. However, in infected B6 mice, the strong pro-inflammatory response was associated with a high and sustained expression of TLR9 and iNOS in leukocytes and hepatic tissue respectively. Additionally, co-expression of gp91- and p47-phox NADPH oxidase subunits were detected in liver tissue of infected B6 mice. Notably, the pre-treatment previous to infection with Pam3CSK4, TLR2-agonist, induced a significant reduction of transaminase activity levels and inflammatory foci number in livers of infected B6 mice. Moreover, lower pro-inflammatory cytokines and increased TGFß levels were detected in purified hepatic leukocytes from TLR2-agonist pre-treated B6 mice. CONCLUSIONS/SIGNIFICANCE: Our results describe some of the main injurious signals involved in liver immune response during the T. cruzi acute infection. Additionally we show that the administration of Pam3CSk4, previous to infection, can attenuate the exacerbated inflammatory response of livers in B6 mice. These results could be useful to understand and design novel immune strategies in controlling liver pathologies.

Induction of NADPH oxidase activity and reactive oxygen species production by a single Trypanosoma cruzi antigen.

Trypanosoma cruzi is an intracellular protozoan parasite that predominantly invades mononuclear phagocytes and is able to establish a persistent infection. The production of reactive oxygen species (ROS) by phagocytes is an innate defence mechanism against microorganisms. It has been postulated that ROS such as superoxide anion (O(2)), hydrogen peroxide and peroxynitrite, may play a crucial role in the control of pathogen growth. However, information on parasite molecules able to trigger ROS production is scarce. In this work, we investigated whether cruzipain, an immunogenic glycoprotein from T. cruzi, was able to trigger the oxidative burst by murine cells. By employing chemiluminiscense and flow-cytometric analysis, we demonstrated that cruzipain induced ROS production in splenocytes from non-immune and cruzipain immune C57BL/6 mice and in a Raw 264.7 macrophage cell line. We also identified an O(2)(-) molecule as one of the ROS produced after antigen stimulation. Cruzipain stimulation induced NOX2 (gp91(phox)) and p47(phox) expression, as well as the co-localisation of both NADPH oxidase enzyme subunits. In the current study, we provide evidence that cruzipain not only increased ROS production but also promoted IL-6 and IL-1ß cytokine production. Taken together, we believe these results demonstrate for the first time that cruzipain, a single parasite molecule, in the absence of infection, favors oxidative burst in murine cells. This represents an important advance in the knowledge of parasite molecules that interact with the phagocyte defence mechanism.

TLR2, TLR4 and TLR9 are differentially modulated in liver lethally injured from BALB/c and C57BL/6 mice during Trypanosoma cruzi acute infection.

Toll-like receptor (TLR) family is crucial for microbial elimination and homeostasis, and has an important immunoregulatory role. In this study, we comparatively analyze innate immune response and tissular injury elicited in BALB/c and C57BL/6 (B6) mice during acute Trypanosoma cruzi infection. The liver was the most affected tissue with numerous cellular infiltrates, apoptotic cells and necrotic areas. The apoptotic rate, evaluated by Hoescht stain, was highest in liver of B6. Infection increased transaminase activities in both mouse strains, although they were highest in B6. BALB/c showed sixfold higher parasitemias than B6 but the latter presented higher mortality (80%) than BALB/c (40%). To gain insight into the molecular basis, we investigated the TLRs commitment in liver. We found that, TLR2 and TLR4 were up-regulated in BALB/c while they were down-regulated in B6. However, TLR9 showed a diminution in BALB/c and an increase in B6 at the end of infection. Moreover, an intensified pro-inflammatory cytokine profile was observed in B6 and F4/80+ and Gr1+ leukocytes were the predominant cells in liver from both mouse strains. Thus, altered TLR2, TLR4 and TLR9 signalling and exacerbate inflammatory cytokine profile could be responsible of the fatal hepatic damage observed in infected B6.

Spleen B cells from BALB/c are more prone to activation than spleen B cells from C57BL/6 mice during a secondary immune response to cruzipain.

There is an increasing interest in the study of roles that B cells may play in regulating immune responses both in protection and in pathogenesis. However, little is known about additional immune functions of B cells independently of antibody production. In this study, we have assessed how the immunization with T-dependent antigens in different host genetic backgrounds affects several parameters of B cells during secondary immune responses. We have previously reported that BALB/c immunized with cruzipain, induced heart autoimmunity, whereas C57BL/6 mice were resistant. In a comparative study employing the same experimental model, we demonstrated that BALB/c-enriched spleen B cells presented higher ability to proliferate releasing elevated levels of IL-4. Moreover, spleen of immune BALB/c mice presented an increased number of germinal center and plasma cells as well as higher expression of B-cell activation markers (MHC class II, CD40, CD86). These findings demonstrate the influence of genetic background on B-cell activation and emphasize the importance of examining B-cell behavior in the context of the specific immunogens.

Immunisation with a major Trypanosoma cruzi antigen promotes pro-inflammatory cytokines, nitric oxide production and increases TLR2 expression.

Innate and adaptive immunity collaborate in the protection of intracellular pathogens including Trypanosoma cruzi infection. However, the parasite molecules that regulate the host immune response have not been fully identified. We previously demonstrated that the immunisation of C57BL/6 mice with cruzipain, an immunogenic T. cruzi glycoprotein, induced a strong specific T-cell response. In this study, we demonstrated that active immunisation with cruzipain was able to stimulate nitric oxide (NO) production by splenocytes. Immune cells also showed increased inducible nitric oxide synthase protein and mRNA expression. Spleen adherent cells secreted high levels of IFN-gamma and IL-12. Microbicidal activity in vitro was mainly mediated by reactive nitrogen intermediaries and IFN-gamma, as demonstrated by the inhibitory effects of NO synthase inhibitor or by IFN-gamma neutralisation. Specific T-cells were essential for NO, IFN-gamma and TNF-alpha production. Furthermore, we reported that cruzipain enhanced CD80 and major histocompatibility complex-II molecule surface expression on F4/80+ spleen cells. Interestingly, we also showed that cruzipain up-regulated toll like receptor-2 expression, not only in F4/80+ but also in total spleen cells which may be involved in the effector immune response. Our findings suggest that a single parasite antigen such as cruzipain, through adaptive immune cells and cytokines, can modulate the macrophage response not only as antigen presenting cells, but also as effector cells displaying enhanced microbicidal activity with reactive nitrogen intermediary participation. This may represent a mechanism that contributes to the immunoregulatory process during Chagas disease.

Cytokines and cell adhesion receptors in the regulation of immunity to Trypanosoma cruzi.

Pathophysiology of Chagas' disease is not completely defined, although innate and adaptative immune responses are crucial. In acute infection some parasite antigens can activate macrophages, and this may result in pro-inflammatory cytokine production, nitric oxide synthesis, and consequent control of parasitemia and mortality. Cell-mediated immunity in Trypanosoma cruzi infection is also modulated by cytokines, but in addition to parasite-specific responses, autoimmunity can be also triggered. Importantly, cytokines may also play a role in the cell-mediated immunity of infected subjects. Finally, leukocyte influx towards target tissues is regulated by cytokines, chemokines, and extracellular matrix components which may represent potential therapeutic targets in infected patients. Here we will discuss recent findings on the role of cytokines, chemokines and extracellular matrix components in the regulation of innate and adaptive immunity during T. cruzi infection.

Different signaling pathways are involved in cardiomyocyte survival induced by a Trypanosoma cruzi glycoprotein.

We have recently reported that Trypanosoma cruzi infection protects cardiomyocytes against apoptosis induced by growth factor deprivation. Cruzipain, a major parasite antigen, reproduced this survival effect by a Bcl-2-dependent mechanism. In this study, we have investigated the molecular mechanisms of cruzipain-induced cardiomyocyte protection. Neonatal BALB/c mouse cardiac myocytes were cultured under minimum serum conditions in the presence of cruzipain or T. cruzi (Tulahuen strain). Some cultures were pretreated with the phosphatidylinositol 3-kinase (PI3K) inhibitor Ly294002 or specific inhibitors of the mitogen-activated protein kinase (MAPK) family members such as the mitogen-activated protein kinase kinase (MEK1) inhibitor PD098059, Jun N-terminal kinase (JNK) inhibitor SP600125, p38 MAPK inhibitor SB203580. Inhibition of PI3K and MEK1 but not JNK or p38 MAPK increased the apoptotic rate of cardiomyocytes treated with cruzipain. Phosphorylation of Akt, a major target of PI3K, and ERK1/2, MEK1-targets, was achieved at 15 min and 5 min, respectively. In parallel, these kinases were strongly phosphorylated by T. cruzi infection. In cultures treated with cruzipain, cleavage of caspase 3 was considerably diminished after serum starvation; Bcl-2 overexpression was inhibited by PD098059 but not by Ly294002, whereas Bad phosphorylation and Bcl-xL expression were increased and differentially modulated by both inhibitors. The results suggest that cruzipain exerts its anti-apoptotic property in cardiac myocytes at least by PI3K/Akt and MEK1/ERK1/2 signaling pathways. We further identified a differential modulation of Bcl-2 family members by these two signaling pathways.

Immune response to a major Trypanosoma cruzi antigen, cruzipain, is differentially modulated in C57BL/6 and BALB/c mice.

BALB/c mice immunized with cruzipain, a major Trypanosoma cruzi antigen, produce specific and autoreactive immune responses against heart myosin, associated with cardiac functional and structural abnormalities. Preferential activation of the Th2 phenotype and an increase in cell populations expressing CD19+, Mac-1+ and Gr-1+ markers were found in the spleens of these mice. The aim of the present study was to investigate whether cardiac autoimmunity could be induced by cruzipain immunization of C57BL/6 mice and to compare the immune response elicited with that of BALB/c mice. We demonstrate that immune C57BL/6 splenocytes, re-stimulated in vitro with cruzipain, produced high levels of IFNgamma and low levels of IL-4 compatible with a Th1 profile. In contrast to BALB/c mice, spleens from cruzipain immune C57BL/6 mice revealed no significant changes in the number of cells presenting CD19+, Mac-1+ and Gr-1+ markers. An increased secretion of TGFbeta and a greater number of CD4+ TGFbeta+ cells were found in immune C57BL/6 but not in BALB/c mice. These findings were associated with the lack of autoreactive response against heart myosin and a myosin- or cruzipain-derived peptide. Thus, the differential immune response elicited in C57BL/6 and BALB/c mice upon cruzipain immunization is implicated in the resistance or pathogenesis of experimental Chagas' disease.