Genotypic evaluation of antimicrobial resistance in Staphylococcus spp. isolated from bovine clinical mastitis / [Avaliação genotípica da resistência antimicrobiana em Staphylococcus spp. isolados de mastite clínica bovina]

Bovine clinical mastitis caused by Staphylococcus spp. is a serious and widespread disease in the world of dairy farming. Antimicrobial therapy is of fundamental importance in the prevention and treatment of infectious mastitis, but the indiscriminate use of antimicrobials acts as a determining factor for the spread of the disease. The present study evaluated the resistance profiles of 57 Staphylococcus spp. isolated from bovine clinical mastitis to beta-lactams and gentamicin, relating characteristics of phenotype (in vitro susceptibility tests) and genotype (detection and expression of genes encoding resistance - mecA, mecALGA251, blaZ, femA, femB, and aacA-aphD - using PCR and RT-PCR, respectively). One or more genes coding for resistance to different antimicrobials were detected in 50 Staphylococcus spp. isolates. The femA and femB genes were the most frequent (75.4% for both). The observed expression of the genes was as follows: blaZ (60%), femA (39.5%), aacA-aphD (50%), femB (32.6%), mecA (8.3%), and mecALGA251 (0%). Considering the relevance of the genus Staphylococcus to bovine mastitis, this study aimed to elucidate aspects regarding the genotypic and phenotypic profiles of these microorganisms so as to contribute to the development of effective strategies for mastitis control.(AU)

A mastite clínica bovina causada por Staphylococcus spp. é uma doença grave e generalizada no mundo da pecuária leiteira. A terapia antimicrobiana é de fundamental importância na prevenção e no tratamento da mastite infecciosa, mas o uso indiscriminado de antimicrobianos atua como fator determinante para a disseminação da doença. O presente estudo avaliou os perfis de resistência de 57 Staphylococcus spp. isolados de mastite clínica bovina em relação ao uso de betalactâmicos e gentamicina, relacionando características do fenótipo (testes de suscetibilidade in vitro) e genótipo (detecção e expressão de genes que codificam resistência - mecA, mecALGA251, blaZ, femA, femB, e aacA-aphD - usando PCR e RT-PCR, respectivamente). Um ou mais genes que codificam resistência a diferentes antimicrobianos foram detectados em 50 Staphylococcus spp. isolados. Os genes femA e femB foram os mais frequentes (75,4% para ambos). A expressão observada dos genes foi a seguinte: blaZ (60%), femA (39,5%), aacA-aphD (50%), femB (32,6%), mecA (8,3%) e mecALGA251 (0%). Considerando-se a relevância do gênero Staphylococcus para a mastite bovina, este estudo teve como objetivo elucidar aspectos referentes aos perfis genotípico e fenotípico desses microrganismos, a fim de contribuir para o desenvolvimento de estratégias eficazes para o controle da mastite.(AU)

Examination of preliminary behavioral and effective connectivity findings from treatment response to citalopram in cocaine use disorder: A dynamic causal modeling study.

We sought effective (directional) connectivity parameters associated with response to citalopram in cocaine use disorder (CUD) by conducting a functional magnetic resonance imaging (fMRI) experiment with participants diagnosed with CUD (n = 13) and matched healthy controls (HC; n = 17). CUD participants showed a positive correlation between bilateral DLPFC-to-putamen effective connectivity and treatment effectiveness score. These preliminary results support further investigation of prefrontal-striatal interactions in response to treatment in CUD.

Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses.

Plasmacytoid dendritic cells (pDC) are type I interferon-producing cells with critical functions in a number of human illnesses; however, their molecular regulation is incompletely understood. Here we show the role of Src family kinases (SFK) in mouse and human pDCs. pDCs express Fyn and Lyn and their activating residues are phosphorylated both before and after Toll-like receptor (TLR) stimulation. Fyn or Lyn genetic ablation as well as treatment with SFK inhibitors ablate pDC (but not conventional DC) responses both in vitro and in vivo. Inhibition of SFK activity not only alters TLR-ligand localization and inhibits downstream signalling events, but, independent of ex-vivo TLR stimulation, also affects constitutive phosphorylation of BCAP, an adaptor protein bridging PI3K and TLR pathways. Our data identify Fyn and Lyn as important factors that promote pDC responses, describe the mechanisms involved and highlight a tonic SFK-mediated signalling that precedes pathogen encounter, raising the possibility that small molecules targeting SFKs could modulate pDC responses in human diseases.

Principales factores de riesgo cardiovasculares y adherencia al tratamiento en pacientes del centro de salud familiar Yanequén, Negrete, Chile 2015 / Main cardiovascular risk factors and treatment continuity of patients from the Yanequén family health center, Negrete, Chile 2015

Introducción: Los factores de Riesgo Cardiovasculares (FRCVs) son condiciones de cada individuo que pueden aumentar el riesgo de desarrollar una enfermedad cardiovascular (ECV). El objetivo del presente trabajo es identificar los principales Factores de Riesgo Cardiovasculares presentes en una población de pacientes ingresados al Programa de Salud Cardiovascular el Centro de Salud Familiar Yanequén en el año 2015 y su adherencia al tratamiento, con la finalidad de conocer la realidad local y poder trabajar en estrategias para la promoción de estilos de vida saludables. Materiales y métodos: Se realizó un estudio Descriptivo, retrospectivo y Transversal mediante la entrevista médico paciente y los datos del tarjetón del Programa de Salud Cardiovascular (PSCV). Se seleccionó una muestra de 109 pacientes adultos de ambos sexos ingresados al PSCV en el año 2015 hasta el mes de diciembre. Se seleccionaron personas de ambos sexos, mayores de 15 años que tengan las siguientes enfermedades: Hipertensión arterial, Diabetes Mellitus, Dislipidemia. Se excluyeron a aquellos Pacientes con datos incompletos. Resultados: EL 50% DE LOS FRCV correspondieron a: sedentarismo, Dieta no Saludable, Dislipidemia, Obesidad e hipertensión arterial. La adherencia al tratamiento es positiva con más del 50% de pacientes que cumplen con éste. Discusión: Los principales FRCV son el sedentarismo, Dieta no Saludable, Dislipidemia y, Obesidad, por lo tanto es necesario implementar medidas de promoción de la Salud con base en estilos de vida saludable para disminuir el riesgo de padecer ECV

Introduction: The Cardiovascular Risk Factors (CVRFs) are conditions from every individual that can increase the cardiovascular disease risk (CVD). The objective of this paper is to identify the main CVRFs present in a population of admitted patients in the Cardiovascular Health Program (CVHP) from the Family Health Center (FHC) Yanequén in the 2015 and their continuity in the treatment, with the purpose of knowing the local reality and to be able to work on strategies for promoting healthier life styles. Materials and methods: A Descriptive, retrospective and transversal study has been realized through the doctor-patient interview and the CVHP information. The Sample was about 109 adult patients from both sex admitted in the CVHP in the year 2015 until december. The Inclusion Criteria was people from both sex above 15 years old, with the following diseases: high blood pressure, Diabetes Mellitus, Dyslipidemia, and above 55 years old smokers. Patients with incomplete information were excluded. Results: 50% of the CVRFs were: sedentary lifestyle, unhealthy diet, dyslipidemia, obesity and high blood pressure. The treatment continuity was positive with more than 50% of patients that attended the CVHP. Discussion: The main CVRFs are the sedentary life style, unhealthy diet, dyslipidemia and obesity, so it is necessary to implement Health promotion based on healthy lifestyles to decrease the risk of CVDs

Chlamydia felis: Lack of association between clinical signs and the presence of the cryptic plasmid.

Chlamydia felis is an obligate intracellular bacterial pathogen that infects cats, causing severe conjunctivitis associated with upper respiratory tract disease (URTD). In the present study, 186 cats from three non-commercial catteries in São Paulo, SP, Brazil were evaluated. The detection of Chlamydia felis was performed by PCR. The clinical severity was scored from 1 to 4, with a score of 4 as the most severe manifestation. The total occurrence of C. felis was of 18.82% (35/186) of cats overall, but notably, 58.06% (18/31) of infected cats originated from a single cattery. All animals harboring C. felis had URTD clinical signs and higher scores (3 and 4). In addition, C. felis occurrence was associated with the presence of cryptic plasmid. However, the virulence and clinical severity were not correlated.

Microbiological analysis and sensitivity profile of Staphylococcus spp. in subclinical mastitis of dairy goats / Análise microbiológica e perfil de sensibilidade do Staphylococcus spp. em mastite subclínica de caprinos leiteiros

Subclinical mastitis in goats causes economic losses and risks to public health. Given the need for research that shows the most isolated staphylococci species and sensibility tests comparing the resistance between coagulase-negative (CNS) and positive Staphylococcus (CPS) goats with subclinical mastitis, the aim of this study was to identify the microorganisms isolated from milk samples of goats with subclinical mastitis, as well as define the staphylococci species and determine the sensitivity profile of Staphylococcus spp. to antimicrobials. To collect samples, tests were performed for mug of black background and California mastitis test (CMT), collecting milk from CMT positive animals. A total of 226 samples from seven herds of dairy goats was collected and forwarded to the laboratory, where they were seeded for the isolation of the microorganism and implementing the antibiotic sensibility test. Of these, 122 samples had bacterial growth and the most isolated staphylococci species were: S. epidermidis (24.55%), S. lugdunensis (15.40%) and S. intermedius (13.64%). Samples showed increased resistance to antimicrobials: penicillin (81.8%), oxacillin (60.0%) and ampicillin (55.5%). Greater sensitivity to: enrofloxacin (99.1%), erythromycin (98.2%), gentamicin (98.2%) and vancomycin (98.2%) were observed. The S. epidermidis showed higher antimicrobial resistance to amoxicillin and penicillin than S. lugdunensis and S. intermedius. Similar resistance in vitro between CNS and CPS was observed to most antimicrobials. It is important to control the overuse of antibiotics to prevent the emergence of resistant strains.

A mastite subclínica em caprinos acarreta prejuízos econômicos e riscos à saúde pública. Tendo em vista a necessidade de pesquisas que demonstrem as espécies de estafilococos mais isoladas e os testes de sensibilidade que comparem a resistência entre Staphylococcus coagulase negativa (SCN) e positiva (SCP) de cabras com mastite subclínica, os objetivos do presente estudo foram identificar os microrganismos isolados de amostras de leite de cabras com mastite subclínica, bem como definir as espécies de estafilococos e determinar o perfil de sensibilidade de Staphylococcus spp. aos antimicrobianos. Para realizar a coleta das amostras, foram executados os testes da caneca de fundo preto e California mastitis test (CMT) com o leite dos animais reagentes ao CMT. Coletaram-se 226 amostras provenientes de sete rebanhos de caprinos leiteiros, as quais foram encaminhadas para o laboratório, onde foram semeadas para o isolamento do microrganismo e a realização do teste de antibiograma. Dessas amostras, 122 tiveram crescimento bacteriano e as espécies mais isoladas de estafilococos foram: S. epidermidis (24,55%), S. lugdunensis (15,40%) e S. intermedius (13,64%). As amostras apresentaram maior resistência aos antimicrobianos penicilina (81,8%), oxacilina (60,0%) e ampicilina (55,5%). Observou-se maior sensibilidade para enrofloxacina (99,1%), eritromicina (98,2%), gentamicina (98,2%) e vancomicina (98,2%). O S. epidermidis apresentou maior resistência antimicrobiana para a amoxicilina e a penicilina do que o S. lugdunensis e o S. intermedius. Foi verificada uma resistência in vitro semelhante entre os estafilococos coagulase negativa e positiva para a maioria dos antimicrobianos testados. É importante o controle do uso abusivo de antimicrobianos para evitar o surgimento de cepas resistentes.

High-throughput transformation pipeline for a Brazilian japonica rice with bar gene selection.

The goal of this work was to establish a transformation pipeline for upland Curinga rice (Oryza sativa L. ssp. japonica) with bar gene selection employing bialaphos and phosphinothricin as selection agents. The following genes of interest: AtNCED3, Lsi1, GLU2, LEW2, PLD-alpha, DA1, TOR, AVP1, and Rubisco were cloned into the binary vector p7i2x-Ubi and were transferred into Agrobacterium strain EHA 105. Embryogenic calli derived from the mature embryos were transformed, and transgenic cells and shoots were selected on the medium supplemented with bialaphos or phosphinothricin (PPT) using a stepwise selection scheme. Molecular analyses were established using polymerase chain reaction and Southern blot for the bar gene and the NOS terminator. Overall, 273 putative transgenic plants were analyzed by Southern blot with 134 events identified. In total, 77 events had a single copy of the transgene integrated in the plant genome while 29 events had two copies. We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome. The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants. The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house.

[Efficacy of procainamide in the treatment of refractory ventricular fibrillation: report of 4 cases and a review of the literature]. / Eficacia de procainamida en el tratamiento de la fibrilación ventricular refractaria: descripción de 4 casos clínicos y revisión de la literatura.

Ventricular fibrillation is the most common malignant arrhythmia, found in up to 55% of patients who go on to experience cardiac arrest. Only monophasic or biphasic defibrillation has been shown to be effective. The efficacy of antiarrhythmic drugs is much lower and depends on how much time has elapsed since the onset of symptoms. In patients with persistent ventricular fibrillation refractory to shocks, treatment options are limited. We report 4 cases in which procainamide was administered at a dosage of 17 mg/kg in 1 minute. Heart rhythm was restored and pulse rate recovered in less than 3 minutes in all cases.

Characterization of a neutral polysaccharide with antioxidant capacity from red wine.

A neutral fraction (PS-SI) (0.3g/L) with MW of 74kDa, which contained galactose, arabinose, mannose, and glucose in the molar ratio of 1.0:0.6:0.4:0.2 was obtained by treatment of the whole polysaccharide extracted from red wine with cetrimide, followed by gel permeation chromatography. Spectroscopic and methylation analyses indicated that PS-SI is a mixture of neutral polysaccharides, consisting mainly of beta (1-->3)-linked galactopyranosyl residues, with side chains of galactopyranosyl residues at positions O-6. Arabinofuranosyl residues linked alpha (1-->5), alpha-mannopyranosyl and glucosyl residues appear to be components of different polysaccharides. The in vitro antioxidant capacity of fractions of wine polysaccharide was studied by hydroxyl radical scavenging and ORAC assays. Fraction PS-SI presented the strongest effect on hydroxyl radicals (IC(50)=0.21).

Type I interferon during viral infections: multiple triggers for a multifunctional mediator.

Type I interferons (IFN-I) orchestrate numerous biological and cellular processes and are essential elements during host antiviral defense. After recognition of highly conserved virus signatures, a complex network of signaling events is rapidly initiated and leads to IFN-I synthesis. These cytokines directly induce a strong antiviral state and exert several immune-regulatory actions aimed at preventing virus spread. On the other hand, viruses evolved to evade or subvert the IFN-I system for their own benefit. In the present article, we review selective aspects of IFN-I induction and functions during several viral infections and discuss the beneficial and detrimental roles of IFN-I illustrated during lymphocytic choriomeningitis virus (LCMV) infection in its natural host, the mouse.

Trypanosoma cruzi infection beats the B-cell compartment favouring parasite establishment: can we strike first?

Trypanosoma cruzi, the causative agent of Chagas' disease, may sabotage humoral response by affecting B cells at the different stages of its development. The present review highlights the contributions of our laboratory in understanding how T. cruzi hinders B-cell generation and B-cell expansion limiting host defence and favouring its chronic establishment. We discuss how homoeostatic mechanisms can be triggered to control exacerbated B-cell proliferation that favour T. cruzi infection by eliminating parasite-specific B cells. Specific targeting of evasion mechanisms displayed in T. cruzi infection, as in vivo Fas/FasL blockade or Gal-3 expression inhibition, allowed us to modulate B-cell responses enhancing the anti-parasite humoral immune response. A comprehensive understanding of the biology of the B cell in health and disease is strictly required to devise immunointervention strategies aimed at enhancing protective immune responses during infections.

1H,13C 2D NMR and X-ray studies of the products of the reaction between dibenzylidencyclohexanone and 6-amino-1,3-dimethyl uracil.

The reaction of the 6-amino-1,3-dimethyl uracil with the dibenzylidencyclohexanone (1), provided three adducts whose structures result from nucleophilic attack yielding the monoadduct 3 and two isomeric bisadducts (4 and 5) in moderate yields (50-60%). The structures obtained in this study were elucidated with 2D high resolution NMR experiments, variable temperature NMR and X-ray crystallographic studies. In compound 3, the tricyclic skeleton is essentially planar and the cyclohexane ring addopts an envelope conformation. The structures 4 and 5 correspond to two isomeric spiro compounds.

Interleukin-4 biases differentiation of B cells from Trypanosoma cruzi-infected mice and restrains their fratricide: role of Fas ligand down-regulation and MHC class II-transactivator up-regulation.

In the present work, we demonstrate that interleukin (IL)-4 is able to rescue B cells from Trypanosoma cruzi-infected mice, counteracting the strong apoptotic signals that these cells received in vivo. We have observed that IL-4 restrains the apoptosis of immunoglobulin (Ig)M(+) and IgG(+) B cells from infected and normal mice without inducing them to proliferate. In addition, IL-4 does not modify the quantity or quality of the antibodies secreted by B cells from infected mice, as it blocks their terminal differentiation to plasma cells and favors memory pathway. It is interesting that the protective effect of IL-4 over B cells from infected mice is mediated, at least partly, by the down-regulation of Fas ligand (FasL) expression, which leads to interference in the apoptosis executed by these B cells through the Fas/FasL death pathway. Accordingly, a marked up-regulation of the "FasL gene repressor" class II transactivator was observed, suggesting that this would be one mechanism underlying the IL-4-mediated FasL down-regulation.

Trypanosoma cruzi mitochondrial malate dehydrogenase triggers polyclonal B-cell activation.

It has been proposed that Trypanosoma cruzi, the aetiologic agent of Chagas' disease, produces mitogenic substances responsible for the polyclonal B-cell activation observed during the acute phase of the infection. Isolation and characterization of the molecules involved in the induction of polyclonal activation observed during infectious diseases have posed a great challenge for the immunologist over the last decade. In this work we report that a 33 kD protein obtained from an alkaline fraction of T. cruzi epimastigotes (FI) stimulates proliferation and promotes differentiation into antibody-secreting cells of normal murine B cells in a T-cell independent manner. By flow cytometry we also found that the 33 kDa protein induces an increase in the expression of MHC class II and B7.2 but not B7.1 molecules on the B-cell surface. Sequencing by mass spectrometry identified the T. cruzi 33 kD protein as hypothetical oxidoreductase, a member of the aldo/ketoreductase family. In this report we demonstrate that this protein is also present in the infective bloodstream trypomastigote form of the parasite and was identified as T. cruzi mitochondrial malate dehydrogenase (mMDH) by enzyme activity and by Western blotting using a specific mMDH polyclonal antiserum. The biologic relevance of mMDH-induced polyclonal activation concerning T. cruzi infection is discussed.

Regulated expression and effect of galectin-1 on Trypanosoma cruzi-infected macrophages: modulation of microbicidal activity and survival.

Galectin-1 is a beta-galactoside-binding protein with potent anti-inflammatory and immunoregulatory effects. However, its expression and function have not been assessed in the context of an infectious disease. The present study documents, for the first time, the regulated expression of galectin-1 in the context of an infectious process and its influence in the modulation of macrophage microbicidal activity and survival. A biphasic modulation in parasite replication and cell viability was observed when macrophages isolated from Trypanosoma cruzi-infected mice were exposed to increasing concentrations of galectin-1. While low concentrations of this protein increased parasite replication and did not affect macrophage survival, higher inflammatory doses of galectin-1 were able to commit cells to apoptosis and inhibited parasite replication. Furthermore, galectin-1 at its lowest concentration was able to down-regulate critical mediators for parasite killing, such as interleukin 12 (IL-12) and nitric oxide, while it did not affect IL-10 secretion. Finally, endogenous galectin-1 was found to be up-regulated and secreted by the J774 macrophage cell line cultured in the presence of trypomastigotes. This result was extended in vivo by Western blot analysis, flow cytometry, and reverse transcription-PCR using macrophages isolated from T. cruzi-infected mice. This study documents the first association between galectin-1's immunoregulatory properties and its role in infection and provides new clues to the understanding of the mechanisms implicated in host-parasite interactions during Chagas' disease and other parasite infections.

Regulated expression of galectin-1 during B-cell activation and implications for T-cell apoptosis.

Galectin-1 (GAL-1), a highly conserved beta-galactoside-binding protein, has shown immunomodulatory properties. In this study, we investigated the regulation of GAL-1 expression within the B-cell compartment using Trypanosoma cruzi infection as a natural model of in vivo B-cell activation. GAL-1 was found to be expressed on activated B cells from T. cruzi-infected mice, mainly localized at the cytosolic compartment. Expression of this protein was found to be modulated according to the activation state of the cells, revealing a significant increase in stimulated B cells that received signals via cross-linking of the B-cell receptor and CD40. It was found that GAL-1 was secreted by B cells to the extracellular milieu upon activation. Finally, purified GAL-1 produced by activated B cells induced apoptosis of T cells but not B cells and also influenced interferon-gamma cytokine production. Hence, the present study describes a potential mechanism by which B cells can regulate T-cell function and survival.

Carrageenans from chilean samples of Stenogramme interrupta (Phyllophoraceae): structural analysis and biological activity.

Carrageenans extracted from cystocarpic and tetrasporic Stenogramme interrupta were analysed by chemical and spectroscopic methods. The carrageenan from cystocarpic plants is composed predominantly of 0.5 M KCl-insoluble and 1 M KCl-soluble fractions. The insoluble fraction contained iota-carrageenan as the major component with alpha-carrageenan and pyruvated carrageenan as minor components. The soluble fraction is highly heterogeneous and did not contain the precursors mu- and nu-carrageenans. The polysaccharide from tetrasporic plants is composed of zeta- and lambda-carrageenans, and low sulfated galactans. It is soluble in KCl and partly cyclized by alkaline treatment. The antiviral and anticoagulant properties of the insoluble polysaccharide fraction from cystocarpic S. interrupta and the polysaccharide from tetrasporic S. interrupta are reported the results of which suggest promising antiherpetic activity.

Trypanosoma cruzi-induced immunosuppression: B cells undergo spontaneous apoptosis and lipopolysaccharide (LPS) arrests their proliferation during acute infection.

Acute infection with Trypanosoma cruzi is characterized by multiple manifestations of immunosuppression of both cellular and humoral responses. B cells isolated at the acute stage of infection have shown marked impairment in their response to polyclonal activators in vitro. The present work aims at studying the B cell compartment in the context of acute T. cruzi infection to provide evidence for B cell activation, spontaneous apoptosis and arrest of the cell cycle upon mitogenic stimulation as a mechanism underlying B cell hyporesponse. We found that B cells from acutely infected mice, which fail to respond to the mitogen LPS, showed spontaneous proliferation and production of IgM, indicating a high level of B cell activation. Furthermore, these activated B cells also exhibited an increase in Fas expression and apoptosis in cultures without an exogenous stimulus. On the other hand, B cells from early acute and chronic infected mice did not present activation or apoptosis, and were able to respond properly to the mitogen. Upon in vitro stimulation with LPS, B cells from hyporesponder mice failed to progress through the cell cycle (G0/G1 arrest), nor did they increase the levels of apoptosis. These results indicate that B cell apoptosis and cell cycle arrest could be the mechanisms that control intense B cell expansion, but at the same time could be delaying the emergence of a specific immune response against the parasite.

Antibodies against Trypanosoma cruzi alkaline antigens are elicited in sera from acute but not chronic human chagasic patients.

The aim of this work was to study the antibody response of acute and chronic chagasic patients against a Trypanosoma cruzi alkaline fraction (FI) in comparison with the reactivity against a T. cruzi acidic antigen, the main cystein proteinase of the parasite named cruzipain, and "natural" antigens. FI-specific antibodies were detected only during the acute phase of the infection and IgM was the main isotype produced, whereas cruzipain-specific antibodies were detected during all phases of the infection. By means of immunoblot and sequencing analysis we identified a 47-kDa FI proteic band recognized by IgM from acute chagasic patients as the T. cruzi glutamate dehydrogenase (GluDH). Furthermore, the antibody response against isolated GluDH showed similar characteristics as the one against FI. We also observed a strict association between the reactivity of IgM against FI and GluDH and IgM natural antibodies. However, reactivity against these alkaline antigens was not modified after absorption of natural antibodies in sera from acute chagasic patients, indicating that these parasite antigens are not recognized by the polyspecific natural antibodies. The most important goal of this report is that for the first time the T. cruzi antigen isoelectric point has been associated with its ability to trigger immunological memory, raising a novel antigen property that should be considered in the selection of antigens used in Chagas' disease diagnostic test and in the design of a vaccine against T. cruzi infection.

A Trypanosoma cruzi alkaline antigen induces polyclonal B-cell activation of normal murine spleen cells by T-cell-independent, BCR-directed stimulation.

We have previously reported that a cytosolic alkaline fraction (FI) obtained from epimastigotes of Trypanosoma cruzi promotes the activation, proliferation and differentiation of normal murine B cells into antibody-secreting plasmocytes. Neither the mechanism nor the cells involved in the FI-induced polyclonal B-cell activation were established. In this work we report that accessory cells are required for FI-induced polyclonal B-cell activation as no proliferative responses were obtained following treatment of normal spleen mononuclear cells (NSMC) with L-leucine methyl ester. Furthermore, FI did not induce the expression of CD25 on T cells and it promoted the proliferation of a T-cell-depleted population, indicating that it acts in a T-independent manner. We observed that NSMC were stimulated in vitro by FI-released cytokines, such as interleukin (IL)-4, IL-6 and IL-10, which are involved in B-cell proliferation and differentiation. Interestingly, while significant amounts of interferon-gamma (IFN-gamma) were found in culture supernatants we did not observe detectable levels of IL-2. Additionally, we found that B-cell receptor (BCR) and major histocompatibility complex (MHC) class II antigens were involved in the proliferative response induced by FI because antibodies directed against cell-surface immunoglobulin M (IgM), CD45 and MHC class II molecules inhibited the FI-induced B-cell proliferation. CD40 ligand (CD40L) did not participate in such a phenomenon.