Impaired Innate Immunity in Tlr4−/− Mice but Preserved CD8+ T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8+ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-γ secreting CD8+ T cells specific for H-2Kb-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2−/−, Tlr4−/−, Tlr9−/− or Myd88−/− mice generated both specific cytotoxic responses and IFN-γ secreting CD8+ T cells at levels comparable to WT mice, although the frequency of IFN-γ+CD4+ cells was diminished in infected Myd88−/− mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-γ, TNF-α and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4−/− mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi

Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia.

ABSTARCT: Hepatitis B virus (HBV) occult infection (OBI) is a risk factor to be taken into account in transfusion, hemodialysis and organ transplantation. The aim of this study was to identify and characterize at the molecular level OBI cases in patients with end-stage liver disease. METHODS: Sixty-six liver samples were obtained from patients with diagnosis of end-stage liver disease submitted to liver transplantation in Medellin (North West, Colombia). Samples obtained from patients who were negative for the surface antigen of HBV (n = 50) were tested for viral DNA detection by nested PCR for ORFs S, C, and X and confirmed by Southern-Blot. OBI cases were analyzed by sequencing the viral genome to determine the genotype and mutations; additionally, viral genome integration events were examined by the Alu-PCR technique. RESULTS: In five cases out of 50 patients (10%) the criteria for OBI was confirmed. HBV genotype F (subgenotypes F1 and F3), genotype A and genotype D were characterized in liver samples. Three integration events in chromosomes 5q14.1, 16p13 and 20q12 affecting Receptor-type tyrosine-protein phosphatase T, Ras Protein Specific Guanine Nucleotide Releasing Factor 2, and the zinc finger 263 genes were identified in two OBI cases. Sequence analysis of the viral genome of the 5 OBI cases showed several punctual missense and nonsense mutations affecting ORFs S, P, Core and X. CONCLUSIONS: This is the first characterization of OBI in patients with end-stage liver disease in Colombia. The OBI cases were identified in patients with HCV infection or cryptogenic cirrhosis. The integration events (5q14.1, 16p13 and 20q12) described in this study have not been previously reported. Further studies are required to validate the role of mutations and integration events in OBI pathogenesis

Mechanisms of Resistance to Noncovalent Bruton's Tyrosine Kinase Inhibitors

Background Covalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood. Methods We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors. Results Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors.Conclusions Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.

Avaliação do nível de conhecimento e de atitudes preventivas da população sobre a leishmaniose visceral em Belo Horizonte, Minas Gerais, Brasil Assessment of knowledge and preventive attitudes concerning visceral leishmaniasis in Belo Horizonte, Minas Gerais State, Brazil

Objetivou-se avaliar o nível de conhecimento e algumas atitudes preventivas em relação à leishmaniose visceral em Belo Horizonte, Minas Gerais, Brasil, em 2006. Foi feito um estudo de caso-controle, com visitas domiciliares e questionário semi-estrurado. Comparou-se dois grupos: (1) 82 casos humanos de leishmaniose visceral ocorridos em 2004 e (2) 164 controles, constituídos por vizinhos dos casos. A leishmaniose visceral acometeu mais em crianças, com aumento do risco de contrair leishmaniose visceral de 109,77 vezes para menores de dez anos. O homem demonstrou ter 2,57 vezes mais chances de adoecer que a mulher. A escolaridade da população mostrou-se baixa (68,3% não completaram o ensino médio). Cinqüenta por cento dos casos desconheciam-na quando foram infectados e apenas 1,2% conhecia o vetor. Conhecer algo sobre a leishmaniose visceral minimizou o risco de adoecer em 2,24 vezes. Quanto às atitudes de proteção, o risco de se contrair leishmaniose visceral diminui em 1,94 vez para pessoas que mantêm limpos os domicílios ou que levam o cão ao veterinário. Em Belo Horizonte, o conhecimento da população perante a leishmaniose visceral é superficial e as atitudes preventivas inespecíficas.<br>The main objective of this study was to evaluate knowledge concerning visceral leishmaniasis and attitudes used to prevent the disease in Belo Horizonte, Minas Gerais State, Brazil, in 2006. A case-control study was conducted, with home visits and a questionnaire. The odds ratio was calculated, comparing 82 cases of human visceral leishmaniasis in 2004 and 164 controls (neighbors of cases). The disease was more frequent in children (OR = 109.77). Visceral leishmaniasis was 2.57 times more likely in males than in females. Overall schooling level was low (68.3% of subjects had not completed secondary school). Half of the cases did not know what visceral leishmaniasis was, and only 1.2% could identify the vector. Having basic knowledge of visceral leishmaniasis reduced the odds of acquiring the disease by 2.24 times. Keeping the house clean and taking pet dogs to the veterinarian reduced the risk by 1.94 times. In Belo Horizonte, the population's level of knowledge on visceral leishmaniasis is superficial, and preventive attitudes are inaccurate

Tuberculose no Brasil: construção de um sistema de vigilância de base territorial Tuberculosis in Brazil: construction of a territorially based surveillance system

OBJETIVO: Analisar a ocorrência da tuberculose, identificando variáveis definidoras de situações coletivas de risco que determinam sua distribuição espacial, como subsídio à implantação de um sistema de vigilância de base territorial para controle da tuberculose. MÉTODOS: Estudo ecológico realizado no período 1996-2000, em Olinda, município da região metropolitana do Recife, PE. A mediana do número de casos de tuberculose, notificados por setor censitário, serviu como ponto de corte para caracterização das áreas de alta e baixa transmissão. Um modelo de regressão logística, utilizando essa variável resposta, permitiu estimar as "odds-ratio" de algumas variáveis socioeconômicas do Censo Demográfico de 2000 e de outras co-variáveis relacionadas com a transmissão da doença. RESULTADOS: A tuberculose em Olinda apresentou altas taxas de incidência no período (média de 111 casos por 100.000 habitantes). Verificou-se que são significativamente associadas à ocorrência da tuberculose, as variáveis: média de moradores por domicílio (OR=2,2; IC 95%: 1,3; 3,6); existência de famílias com mais de um caso no período (OR=5,1; IC 95%: 2,3; 11,3); e presença de casos de retratamento (OR=6,8; IC 95%: 2,7; 17,1). Setores censitários com a ocorrência desses dois últimos eventos concentraram 45% do total de casos do período, representando apenas 28% da população do município. CONCLUSÕES: Duas das três variáveis explicativas associadas a maiores taxas de incidência da doença são informações que devem ser monitorizadas, em nível local, pelo sistema de vigilância da tuberculose. O simples mapeamento de casos de retratamento e de domicílios com ocorrência de repetidos casos, permitiria refinar o foco de atenção em micro-áreas prioritárias para intervenções intensivas, como forma de enfrentar o problema da tuberculose.<br>OBJECTIVE: To analyze the occurrence of tuberculosis and to identify variables that define situations of collective risk that determine the spatial distribution of the disease, as backing for implementing a territory-based surveillance system for tuberculosis control. METHODS: This was an ecological study performed in Olinda, a municipality in the metropolitan region of Recife, State of Pernambuco, between 1996 and 2000. The median number of notified tuberculosis cases in each census tract served as the cutoff point for characterizing areas of high and low transmission. A logistic regression model using this response variable allowed odds ratios for some socioeconomic variables from the 2000 demographic census and other covariates related to the transmission of the disease to be estimated. RESULTS: Tuberculosis in Olinda presented high incidence rates during the study period (average of 111 cases per 100,000 inhabitants). Significant associations with the occurrence of tuberculosis were found for the variables of average number of inhabitants per household (OR=2.2; 95% CI: 1.3; 3.6); existence of families with more than one case during the study period (OR=5.1; 95% CI: 2.3; 11.3); and presence of cases of retreatment (OR=6.8; 95% CI: 2.7; 17.1). The census tract where the latter two events occurred accounted for 45% of the total number of cases during the study period, while representing only 28% of the population of Olinda. CONCLUSIONS: The two explanatory covariates that were strongly associated with higher incidence rates of the disease are events that need to be carefully monitored at a local level by the tuberculosis surveillance system. Simply by mapping out retreatment cases and households with more than one case, attention could be focused on small areas with high priority for intensive intervention, thus facing up to the tuberculosis problem

Effect of nitrogen doses on disease severity and watermelon yield Influência de doses de nitrogênio na severidade de doenças e na produtividade da melancia

Nitrogen fertilization is an important step for watermelon (Citrullus lanatus) production due to its influence over yield, fruit quality, and disease severity. Currently, the gummy stem blight (Didymella bryoniae) and the downy mildew (Pseudoperonospora cubensis) can be taken as the most important watermelon diseases, since they impose severe impairment to the crop. Furthermore, studies focusing on plant responses to nitrogen fertilization regarding fruit yield and quality, and disease resistance are rare. Hence, the present study aimed at evaluating the effect of nitrogen doses on fruit yield and quality, and on disease prevalence in watermelon. Two experiments were carried out at the Universidade Federal de Tocantins, employing sprinkler irrigation, in an area previously used to grow watermelon. The experimental design was blocks at random, with five treatments (N doses, applied twice as side-dressing, in kg ha-1, as follows: T1= control treatment without N, T2= 20, T3= 40, T4= 80, and T5= 120), and four replications. Urea (45% N) was used as the N source. In the first assay, the highest fruit yield and average weight were observed when 40 kg ha-1 of N were applied. The highest severity of the gummy stem blight was observed when the highest nitrogen doses were applied (80 and 120 kg ha-1). In the second assay, the highest severity levels of the gummy stem blight, as well as of mildew, were observed again when N doses were the highest (80 and 120 kg ha-1). The lowest severity levels for both diseases were observed in the control treatment.<br>A adubação nitrogenada é importante para a cultura da melancia (Citrullus lanatus) por afetar diretamente a produtividade, a qualidade dos frutos e a severidade de doenças. Atualmente, o crestamento gomoso (Didymella bryoniae) e o míldio (Pseudoperonospora cubensis) podem ser consideradas as doenças mais importantes da melancia devido aos prejuízos que podem causar na lavoura. Estudos envolvendo a aplicação de doses de nitrogênio e seus efeitos na produtividade, qualidade de frutos e manifestação de doenças são escassos. Desta forma, o presente trabalho teve como objetivo avaliar diferentes doses de nitrogênio e seus efeitos na produtividade, características dos frutos e na prevalência de doenças da melancieira. Foram conduzidos dois ensaios na Universidade Federal do Tocantins, sob irrigação por aspersão, em área anteriormente cultivada com melancia. Foi utilizado o delineamento experimental de blocos ao acaso, com cinco tratamentos (doses de nitrogênio, aplicados em duas vezes em cobertura, em kg ha-1, sendo T1= testemunha sem nitrogênio em cobertura, T2= 20, T3= 40, T4= 80 e T5= 120) e quatro repetições. A uréia foi utilizada como fonte de nitrogênio (45% de N). No ensaio I, verificou-se maior produtividade e maior peso médio de frutos no tratamento que recebeu 40 kg ha-1. Níveis mais altos de severidade do crestamento gomoso foram verificados nos tratamentos que receberam maiores dosagens de nitrogênio (80 e 120 kg). No ensaio II, nos tratamentos com 80 e 120 kg ha de N foram observados os níveis mais altos de míldio e crestamento gomoso. Menores níveis dessas doenças foram verificados no tratamento testemunha