Host triacylglycerols shape the lipidome of intracellular trypanosomes and modulate their growth

Intracellular infection and multi-organ colonization by the protozoan parasite, Trypanosoma cruzi, underlie the complex etiology of human Chagas disease. While T. cruzi can establish cytosolic residence in a broad range of mammalian cell types, the molecular mechanisms governing this process remain poorly understood. Despite the anticipated capacity for fatty acid synthesis in this parasite, recent observations suggest that intracellular T. cruzi amastigotes may rely on host fatty acid metabolism to support infection. To investigate this prediction, it was necessary to establish baseline lipidome information for the mammalian-infective stages of T. cruzi and their mammalian host cells. An unbiased, quantitative mass spectrometric analysis of lipid fractions was performed with the identification of 1079 lipids within 30 classes. From these profiles we deduced that T. cruzi amastigotes maintain an overall lipid identity that is distinguishable from mammalian host cells. A deeper analysis of the fatty acid moiety distributions within each lipid subclass facilitated the high confidence assignment of host- and parasite-like lipid signatures. This analysis unexpectedly revealed a strong host lipid signature in the parasite lipidome, most notably within its glycerolipid fraction. The near complete overlap of fatty acid moiety distributions observed for host and parasite triacylglycerols suggested that T. cruzi amastigotes acquired a significant portion of their lipidome from host triacylglycerol pools. Metabolic tracer studies confirmed long-chain fatty acid scavenging by intracellular T. cruzi amastigotes, a capacity that was significantly diminished in host cells deficient for de novo triacylglycerol synthesis via the diacylglycerol acyltransferases (DGAT1/2). Reduced T. cruzi amastigote proliferation in DGAT1/2-deficient fibroblasts further underscored the importance of parasite coupling to host triacylglycerol pools during the intracellular infection cycle. Thus, our comprehensive lipidomic dataset provides a substantially enhanced view of T. cruzi infection biology highlighting the interplay between host and parasite lipid metabolism with potential bearing on future therapeutic intervention strategies

Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNF and IFN

In cutaneous leishmaniasis, Leishmania amazonensis activates macrophage double-stranded, RNA-activated protein kinase R (PKR) to promote parasite growth. In our study, Leishmania major grew normally in RAW cells, RAW-expressing dominant-negative PKR (PKR-DN) cells, and macrophages of PKR-knockout mice, revealing that PKR is dispensable for L. major growth in macrophages. PKR activation in infected macrophages with poly I:C resulted in parasite death. Fifty percent of L. major-knockout lines for the ecotin-like serine peptidase inhibitor (ISP2; Δisp2/isp3), an inhibitor of neutrophil elastase (NE), died in RAW cells or macrophages from 129Sv mice, as a result of PKR activation. Inhibition of PKR or NE or neutralization of Toll-like receptor 4 or 2(TLR4 or TLR2) prevented the death of Δisp2/isp3. Δisp2/isp3 grew normally in RAW-PKR-DN cells or macrophages from 129Sv pkr−/−, tlr2−/−, trif−/−, and myd88−/− mice, associating NE activity, PKR, and TLR responses with parasite death. Δisp2/isp3 increased the expression of mRNA for TNF-α by 2-fold and of interferon β (IFNβ) in a PKR-dependent manner. Antibodies to TNF-α reversed the 95% killing by Δisp2/isp3, whereas they grew normally in macrophages from IFN receptor–knockout mice. We propose that ISP2 prevents the activation of PKR via an NE-TLR4-TLR2 axis to control innate responses that contribute to the killing of L. major.—Faria, M. S., Calegari-Silva, T. C., de Carvalho Vivarini, A., Mottram, J. C., Lopes, U. G., Lima, A. P. C. A. Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNβ

Actividades de crianças do pré-escolar e educadores de infância com o computador em portugal

Muito se tem escrito hoje sobre a integração das tecnologias de informação e comunicação (TIC) na escola e diversas pesquisas têm vindo a demonstrar a importância da familiarização da criança desta idade com a tecnologia, quer porque esta faz parte inquestionável do mundo que a rodeia, quer pela relevância educativa das experiências que lhe pode proporcionar. No entanto existem muito poucos estudos que nos descrevam e analisem processos efectivos de integração da tecnologia na Escola em geral, e ao nível da educação Pré-Escolar em particular. Este foi o ponto de partida da nossa investigação, verificar as práticas de crianças e Educadores de Infância com as TIC, mais especificamente o computador, em Portugal. Elaborámos um questionário que foi enviado, via correio electrónico, para Educadores de Infância de todo o país e como forma de triangulação de dados foram realizadas observações em dois jardins-de-infância, mais concretamente nas aulas de informática das crianças do pré-escolar. Terminamos com conclusões baseadas nos dados recolhidos e algumas considerações finais sobre o tema

Thalidomide modulates Mycobacterium leprae-induced NF-κB pathway and lower cytokine response

AbstractIt is widely accepted that tumor necrosis factor alpha (TNF-α) plays a critical role in the development of tissue and nerve damage in leprosy and during the reactional episodes of acute inflammation. Thalidomide (N-α-phthalimidoglutarimide), a drug used to treat leprosy reaction, modulates immune response, inhibits inflammation and NF-κB activity. Here we investigated whether thalidomide inhibits NF-κB activation induced by Mycobacterium leprae, p38 and ERK1/2 MAPK activation. EMSA and supershift assays were performed to investigate NF-κB activation in response to M. leprae and its modulation following in vitro treatment with thalidomide. Luciferase assay was assayed in transfected THP-1 cells to determine NF-κB transcriptional activity. Flow cytometry and immunofluorescence were used to investigate p65 accumulation in the nucleus. Immunoblotting was used to investigate p38 and ERK1/2 phosphorylation. Following activation of PBMC and monocytes with M. leprae, the formation and nuclear localization of NF-κB complexes composed mainly of p65/p50 and p50/p50 dimers was observed. Induction of NF-κB activation and DNA binding activity was inhibited by thalidomide. The drug also reduced M. leprae-induced TNF-α production and inhibited p38 and ERK1/2 activation. Definition of the activation mechanisms in cells stimulated with M. leprae can lead to the development of new therapy applications to modulate NF-κB activation and to control the inflammatory manifestations due to enhanced TNF-α response as observed in leprosy and in leprosy reactions

Identification of di-substituted ureas that prevent growth of trypanosomes through inhibition of translation initiation

Some 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) activate heme-regulated kinase causing protein synthesis inhibition via phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) in mammalian cancer cells. To evaluate if these agents have potential to inhibit trypanosome multiplication by also affecting the phosphorylation of eIF2 alpha subunit (eIF2 alpha), we tested 25 analogs of 1,3-diarylureas and cHAUs against Trypanosoma cruzi, the agent of Chagas disease. One of them (I-17) presented selectivity close to 10-fold against the insect replicative forms and also inhibited the multiplication of T. cruzi inside mammalian cells with an EC50 of 1-3 mu M and a selectivity of 17-fold. I-17 also prevented replication of African trypanosomes (Trypanosoma brucei bloodstream and procyclic forms) at similar doses. It caused changes in the T. cruzi morphology, arrested parasite cell cycle in G1 phase, and promoted phosphorylation of eIF2 alpha with a robust decrease in ribosome association with mRNA. The activity against T. brucei also implicates eIF2 alpha phosphorylation, as replacement of WT-eIF2 alpha with a non-phosphorylatable eIF2 alpha, or knocking down eIF2 protein kinase-3 by RNAi increased resistance to I-17. Therefore, we demonstrate that eIF2 alpha phosphorylation can be engaged to develop trypanosome-static agents in general, and particularly by interfering with activity of eIF2 kinases.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)CNPqFundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ)NIHUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04039032 Sao Paulo, SP, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Parasitol Mol, Rio De Janeiro, RJ, BrazilInst Butantan, Sao Paulo, SP, BrazilUniv Sao Paulo, Inst Ciencias Biomed, Dept Microbiol, Sao Paulo, SP, BrazilBrigham & Womens Hosp, Dept Med, Hematol Lab Translat Res, 75 Francis St, Boston, MA 02115 USAHarvard Med Sch, 75 Francis St, Boston, MA 02115 USAUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04039032 Sao Paulo, SP, BrazilFAPESP: 2015/20031-0FAPESP: 2014/01577-2CNPq: 445655/2014-3NIH: R01 CA152312Web of Scienc

The GTPase TcRjl of the human pathogen Trypanosoma cruzi is involved in the cell growth and differentiation

AbstractThe protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas Disease, undergoes through a complex life cycle where rounds of cell division and differentiation occur initially in the gut of triatominae vectors and, after transmission, inside of infected cells in vertebrate hosts. Members of the Ras superfamily of GTPases are molecular switches which play pivotal regulatory functions in cell growth and differentiation. We have previously described a novel GTPase in T. cruzi, TcRjl, which belongs to the RJL family of Ras-related GTP binding proteins. Here we show that most of TcRjl protein is found bound to GTP nucleotides and may be locked in this stage. In addition, we show that TcRjl is located close to the kinetoplast, in a region corresponding possibly to flagellar pocket of the parasite and the expression of a dominant-negative TcRjl construct (TcRjlS37N) displays a significative growth phenotype in reduced serum medium. Remarkably, overexpression of TcRjl inhibits differentiation of epimastigotes to trypomastigote forms and promotes the accumulation of intermediate differentiation stages. Our data suggest that TcRjl might play a role in the control of the parasite growth and differentiation

Intranasal immunization with chitosan microparticles enhances LACK-DNA vaccine protection and induces specific long-lasting immunity against visceral leishmaniasis

Development of a protective vaccine against Leishmania depends on antigen formulation and adjuvants that induce specific immunity and long-lasting immune responses. We previously demonstrated that BALB/c mice intranasally vaccinated with a plasmid DNA encoding the p36/LACK leishmanial antigen (LACK-DNA) develop a protective immunity for up to 3 months after vaccination, which was linked with the systemic expression of vaccine mRNA in peripheral organs. In this study, LACK-DNA vaccine was associated with biocompatible chitosan microparticles cross-linked with glyceraldehyde (CMC) to boost the long-lasting immunity against the late Leishmania infantum challenge. Infection at 7 days, 3 or 6 months after vaccination resulted in significantly lower parasite loads when compared with non-vaccinated controls. Besides, LACK-DNA-chitosan vaccinated mice showed long-time protection observed after the late time point challenge. The achieved protection was correlated with an enhanced spleen cell responsiveness to parasite antigens, marked by increased proliferation and IFN-γ as well as decreased IL-10 production. Moreover, we found diminished systemic levels of TNF-α that was compatible with the better health condition observed in LACK-DNA/CMC vaccinated-infected mice. Together, our data indicate the feasibility of chitosan microparticles as a delivery system tool to extend the protective immunity conferred by LACK-DNA vaccine, which may be explored in vaccine formulations against Leishmania parasite infections

Evaluating the intervening factors in patient safety: focusing on hospital nursing staff

OBJECTIVE To evaluate intervening factors in patient safety, focusing on hospital nursing staff. METHOD The study is descriptive, with qualitative approach, excerpt from a larger study with analytical nature. It was undertaken in a public hospital in Fortaleza, CE, Brazil, between January and June 2013, with semi-structured interviews to 70 nurses, using Thematic Content Analysis. RESULTS The principal intervening factors in patient safety related to hospital nursing staff were staff dimensioning and workload, professional qualification and training, team work, being contracted to the institution, turnover and lack of job security, and bad practice/disruptive behaviors. These aspects severely interfere with the establishment of a safety culture in the hospital analyzed. CONCLUSION It is necessary for managers to invest in nursing staff, so that these workers may be valued as fundamental in the promotion of patient safety, making it possible to develop competences for taking decisions with focus on the improvement of quality care.OBJETIVO Avaliar fatores intervenientes na segurança do paciente, com enfoque na equipe de Enfermagem hospitalar. MÉTODO Estudo descritivo, com abordagem qualitativa, recorte de pesquisa de maior abrangência de caráter analítico. Desenvolveu-se em um hospital público de Fortaleza, CE, Brasil, entre janeiro a junho de 2013, por meio entrevistas semiestruturadas com 70 enfermeiros assistenciais, empregando-se Análise de Conteúdo Temática. RESULTADOS Os principais fatores intervenientes na segurança do paciente relacionados à equipe de Enfermagem encontrados foram: dimensionamento de pessoal e carga de trabalho; formação e capacitação profissional; trabalho em equipe; vínculo empregatício, rotatividade e falta de estabilidade; má prática/comportamentos destrutivos. Tais aspectos interferem sobremaneira no estabelecimento da cultura de segurança na instituição analisada. CONCLUSÃO São necessários investimentos, por parte dos gestores, nos recursos humanos de Enfermagem, para que estes trabalhadores sejam valorizados como fundamentais na promoção da segurança do paciente, possibilitando o desenvolvimento de competências para a tomada de decisão com foco na melhoria da qualidade assistencial.OBJETIVO Evaluar los factores intervinientes en la seguridad del paciente, con énfasis en el equipo de Enfermería hospitalaria. MÉTODO Estudio descriptivo, con abordaje cualitativo, recorte de investigación de mayor alcance de carácter analítico. Se desarrolló en un hospital público de Fortaleza, CE, Brasil, entre enero y junio de 2013, por medio de entrevistas semiestructuradas con 70 enfermeros asistenciales, empleándose el Análisis de Contenido Temático. RESULTADOS Los principales factores intervinientes en la seguridad del paciente relacionados con el equipo de Enfermería encontrados fueron: dimensionamiento de personal y carga laboral; formación y capacitación profesional; trabajo en equipo; vínculo de empleo, rotatividad y falta de estabilidad; mala práctica/comportamientos destructivos. Dichos aspectos interfieren demasiado en el planteamiento de la cultura de seguridad de la institución analizada. CONCLUSIÓN Son necesarias inversiones, por parte de los gestores, en los recursos humanos de Enfermería, para que se valoren esos trabajadores como fundamentales en la promoción de la seguridad del paciente, posibilitando el desarrollo de competencias para la toma de decisiones con énfasis en la mejoría de la calidad asistencial

TLR2, TLR4 and Protein kinase R (PKR) induced Type I Interferon sustains infection of Leishmania donovani in macrophages

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, provoking liver and spleen tissue destruction that is lethal unless treated. The parasite replicates in macrophages and modulates host microbicidal responses. We have previously reported that neutrophil elastase (NE) is required to sustain L. donovani intracellular growth in macrophages through the induction of interferon beta (IFN-β). Here, we show that the gene expression of IFN-β by infected macrophages was reduced by half when TLR4 was blocked by pre-treatment with neutralizing antibodies or in macrophages from tlr2 (-/-) mice, while the levels in macrophages from myd88(-/-) mice were comparable to those from wild-type C57BL/6 mice. The neutralization of TLR4 in tlr2 (-/-) macrophages completely abolished induction of IFN-β gene expression upon parasite infection, indicating an additive role for both TLRs. Induction of type I interferon (IFN-I), OASL2, SOD1, and IL10 gene expression by L. donovani was completely abolished in macrophages from NE knock-out mice (ela2 (-/-)) or from protein kinase R (PKR) knock-out mice (pkr (-/-)), and in C57BL/6 macrophages infected with transgenic L. donovani expressing the inhibitor of serine peptidase 2 (ISP2). Parasite intracellular growth was impaired in pkr (-/-) macrophages but was fully restored by the addition of exogenous IFN-β, and parasite burdens were reduced in the spleen of pkr (-/-) mice at 7 days, as compared to the 129Sv/Ev background mice. Furthermore, parasites were unable to grow in macrophages lacking TLR3, which correlated with lack of IFN-I gene expression. Thus, L. donovani engages innate responses in infected macrophages via TLR2, TLR4, and TLR3, via downstream PKR, to induce the expression of pro-survival genes in the host cell, and guarantee parasite intracellular development

Neutrophil elastase promotes Leishmania donovani infection via interferon-β

Visceral leishmaniasis is a deadly illness caused by Leishmania donovani that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of L. major named inhibitor of serine peptidases (ISP2) inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF and IFN production and parasite survival. We report poor intracellular growth of L. donovani in macrophages from knock-out mice for NE (ela-/-), TLR4 or TLR2. NE and TLR4 co-localized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of ela-/- mice were reduced and accompanied by increased nitric oxide and decreased TGF production. Expression of ISP2 was not detected in L. donovani and a transgenic line constitutively expressing ISP2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected ela-/- macrophages displayed significantly lower IFN mRNA than background mice macrophages and the intracellular growth of was fully restored by exogenous IFN. We propose that L. donovani utilizes the host NE-TLR machinery to induce IFN necessary for parasite survival/growth during early infection. Low or absent expression of parasite ISP2 in L. donovani is necessary to preserve the activation of the NE-TLR pathway