Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis?

Unicellular organisms, such as the protozoan parasite Leishmania, can be stimulated to show some morphological and biochemical features characteristic of mammalian apoptosis. This study demonstrates that under a variety of stress conditions such as serum deprivation, heat shock and nitric oxide, cell death can be induced leading to genomic DNA fragmentation into oligonucleosomes. DNA fragmentation was observed, without induction, in the infectious stages of the parasite, and correlated with the presence of internucleosomal nuclease activity, visualisation of 45 to 59 kDa nucleases and detection of TUNEL-positive nuclei. DNA fragmentation was not dependent on active effector downstream caspases nor on the lysosomal cathepsin L-like enzymes CPA and CPB. These data are consistent with the presence of a caspase-independent cell death mechanism in Leishmania, induced by stress and differentiation that differs significantly from metazoa.

Early production of IL-4 in susceptible mice infected with Leishmania major rapidly induces IL-12 unresponsiveness.

Previous results have documented a burst of IL-4 mRNA that peaks in draining lymph nodes of susceptible BALB/c mice 16 h after infection with Leishmania major. The importance of this early IL-4 response in subsequent Th2 cell maturation is supported by observations showing that 1) neutralization of IL-4 at the initiation of infection or 2) administration of IL-12, which results in an inhibition of the 16 h IL-4 mRNA burst, inhibits Th2 cell development. However, both treatments are effective in hampering Th2 cell development only if given at a time when IL-4 has been produced for <48 h. At this time after infection, lymph node CD4+ T cells from BALB/c mice no longer respond to IL-12. This IL-12 unresponsiveness is prevented in mice treated with anti-IL-4 Abs at the initiation of infection. Finally, the inhibition of Th2 development in BALB/c mice treated with anti-IL-4 Abs at the onset of infection results from maintenance of IL-12 responsiveness, since it requires IL-12. Together, these results reveal a narrow window of time, between 16 h and <48 h after infection, during which IL-4 produced rapidly in BALB/c mice renders T cells unresponsive to IL-12, allowing their differentiation toward the Th2 phenotype.

Leishmania metacaspase: an arginine-specific peptidase.

The purpose of this chapter is to give insights into metacaspase of Leishmania protozoan parasites as arginine-specific cysteine peptidase. The physiological role of metacaspase in Leishmania is still a matter of debate, whereas its peptidase enzymatic activity has been well characterized. Among the different possible expression systems, metacaspase-deficient yeast cells (Δyca1) have been instrumental in studying the activity of Leishmania major metacaspase (LmjMCA). Here, we describe techniques for purification of LmjMCA and its activity measurement, providing a platform for further identification of LmjMCA substrates.

MyD88 and TLR9 dependent immune responses mediate resistance to Leishmania guyanensis infections, irrespective of Leishmania RNA virus burden.

Infections with Leishmania parasites of the Leishmania Viannia subgenus give rise to both localized cutaneous (CL), and metastatic leishmaniasis. Metastasizing disease forms including disseminated (DCL) and mutocutaneous (MCL) leishmaniasis result from parasitic dissemination and lesion formation at sites distal to infection and have increased inflammatory responses. The presence of Leishmania RNA virus (LRV) in L. guyanensis parasites contributes to the exacerbation of disease and impacts inflammatory responses via activation of TLR3 by the viral dsRNA. In this study we investigated other innate immune response adaptor protein modulators and demonstrated that both MyD88 and TLR9 played a crucial role in the development of Th1-dependent healing responses against L. guyanensis parasites regardless of their LRV status. The absence of MyD88- or TLR9-dependent signaling pathways resulted in increased Th2 associated cytokines (IL-4 and IL-13), which was correlated with low transcript levels of IL-12p40. The reliance of IL-12 was further confirmed in IL12AB-/- mice, which were completely susceptible to infection. Protection to L. guyanensis infection driven by MyD88- and TLR9-dependent immune responses arises independently to those induced due to high LRV burden within the parasites.

An��lise das cita����es das disserta����es de mestrado e teses de doutorado em sa��de p��blica (1990-1994): estudo explorat��rio

Parte de an��lise de disserta����es de mestrado e teses de doutorado defendidas, de 1990 a 1994, nos cursos de p��s-gradua����o em sa��de p��blica da Escola Nacional de Sa��de P��blica da Funda����o Oswaldo Cruz (ENSP/Fiocruz), Faculdade de Sa��de P��blica da Universidade de S��o Paulo (FSP/USP) e Instituto de Medicina Social da Universidade Estadual do Rio de Janeiro (IMS/UERJ), visando a detectar aspectos referentes ��s caracter��sticas dos documentos citados, como subs��dios para estabelecimento de indicadores necess��rios �� avalia����o da produ����o cient��fica brasileira no campo da sa��de p��blica. O conjunto das cita����es amostradas (6 019) de 266 disserta����es de mestrado e 84 teses de doutorado revelou que os artigos de peri��dicos contribu��ram com maior n��mero de cita����es (46,7%); o percentual de livros foi mais representativo para as teses de doutorado; a maior concentra����o dos documentos citados encontra-se no conjunto publicado de 6 a 10 anos da defesa da disserta����o/tese; o idioma portugu��s predominou nas cita����es (49,3%) e o ingl��s para os artigos de peri��dicos; os alunos valeram-se mais de disserta����es/teses de outras institui����es do que das escolas que freq��entaram.

A fluorescent peptide substrate for the surface metalloprotease of Leishmania.

A fluorescent oligopeptide substrate for the promastigote surface protease (PSP) of Leishmania was designed using the data reported for the substrate specificity of the enzyme (Bouvier, J., Schneider, P., Etges, R. J., and Bordier, C. 1990. Biochemistry 29, 10113-10119). The indole fluorescence of the tryptophan residue was efficiently quenched through resonance energy transfer by an N-terminal dansyl group located five amino acid residues away. The heptapeptide, dansyl-A-Y-L-K-K-W-V-NH2, was cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 8.8 x 10(6) M-1sec-1. Hydrolysis by the enzyme results in a time-dependent increase of fluorescence intensity of 3.7-fold. Assays can be designed based on the tryptophan fluorescence at 360 nm or by individual product analyses using thin-layer chromatography. The synthetic substrate is readily cleaved by the metalloprotease at the surface of fixed promastigotes. The specificity and sensitivity of such internally quenched fluorescent peptide substrate will facilitate the identification of novel inhibitors for the enzyme and aid in detailed studies on its enzymology.

Intelig��ncia obtida pela aplica����o de data mining em base de teses francesas sobre o Brasil

O assunto Brasil foi analisado na base de teses francesas DocTh��ses, compreendendo os anos de 1969 a 1999. Utilizou-se a t��cnica de Data Mining como ferramenta para obter intelig��ncia e conhecimento. O software utilizado para a limpeza da base DocTh��ses foi o Infotrans, e, para a prepara����o dos dados, empregou-se o Dataview. Os resultados da an��lise foram ilustrados com a aplica����o dos pressupostos da Lei de Zipf, classificando-se as informa����es em trivial, interessante e ru��do, conforme a distribui����o de freq����ncia. Conclui-se que a t��cnica do Data Mining associada a softwares especialistas �� uma poderosa aliada no emprego de intelig��ncia no processo decis��rio em todos os n��veis, inclusive o n��vel macro, pois oferece subs��dios para a consolida����o, investimento e desenvolvimento de a����es e pol��ticas.

A Biblioteca Digital de Teses e Disserta����es da Universidade de S��o Paulo

Apresenta����o dos aspectos principais da Biblioteca Digital de Teses e Disserta����es da Universidade de S��o Paulo (USP): o processo de desenvolvimento adotado para a implementa����o do site, a tecnologia utilizada, a arquitetura e a funcionalidade. Discuss��o sobre a Biblioteca Digital no processo de p��s-gradua����o da USP. Relato das v��rias decis��es n��o-t��cnicas adotadas ao longo do projeto, que tiveram grande impacto no resultado final.

Transpar��ncia e gest��o do conhecimento por meio de um banco de teses e disserta����es: a experi��ncia do PPGEP/UFSC

Em 1995, o Programa de P��s-Gradua����o em Engenharia de Produ����o da Universidade Federal de Santa Catarina (PPGEP/UFSC) digitalizou e disponibilizou, de forma institucional, teses e disserta����es defendidas no programa. Inicialmente um reposit��rio de documentos digitais, o BTD (Banco de Teses e Disserta����es) do PPGEP transformou-se em sistema ��nico de conjuga����o de recursos bibliom��tricos e inform��tricos, tendo como base as teses e disserta����es do programa e a medi����o de acessos. Al��m dos produtores de conhecimento (alunos e professores), o BTD atende a tomadores de decis��o e a gestores do sistema de p��s-gradua����o, subsidiando-os com informa����es sobre n��vel de interesse e interc��mbio de conhecimento entre cada uma das ��reas de concentra����o do programa. O presente artigo apresenta a origem, principais caracter��sticas, fundamentos te��ricos, funcionais e as perspectivas de desenvolvimento do projeto BTD.

Tem��tica das disserta����es e teses em ci��ncia da informa����o no Programa de P��s-Gradua����o em Ci��ncias da Comunica����o da USP

Estudo de natureza descritiva cujo objetivo foi tra��ar um panorama tem��tico das disserta����es de mestrado e teses de doutorado apresentadas ao Programa de P��s-gradua����o em Ci��ncias da Comunica����o ��rea de concentra����o: Ci��ncia da Informa����o e Documenta����o da Escola de Comunica����es e Artes da Universidade de S��o Paulo, no per��odo de 1979 a 2002. O universo de estudo constituiu-se de 114 documentos (75 disserta����es de mestrado e 39 teses de doutorado). As vari��veis da produ����o analisada referiram-se �� distribui����o temporal, tem��ticas abordadas e sua rela����o com as linhas de pesquisa departamentais. As disserta����es/teses foram agrupadas nas categorias da Lista de Cabe��alho de Assunto adotada pelo Library and Information Science Abstracts (LISA). Observou-se que a produ����o discente da p��s-gradua����o manteve-se est��vel no per��odo, com tend��ncia a aumentar. Na an��lise tem��tica, as categorias 19.0 Other Fringe Subjects [Outros Assuntos Correlatos] e 12.0 Bibliographc Records [Registro Bibliogr��fico] foram as mais abordadas, detendo respectivamente, 22,81% e 15,54% da produ����o. Foram identificados como temas de maior interesse para a elabora����o dos trabalhos: a����o cultural, sistemas e linguagens de indexa����o, materiais em C&T e medicina, bibliotecas p��blicas e meios de comunica����o de massa. Contatou-se que as tem��ticas abordadas na produ����o refletem as caracter��sticas da ��rea de concentra����o e das linhas de pesquisa.

In BALB/c mice, IL-4 production during the initial phase of infection with Leishmania major is necessary and sufficient to instruct Th2 cell development resulting in progressive disease.

In contrast to intact BALB/c mice, BALB/c mice rendered deficient in Vbeta4+ CD4+ T cells develop a Th1 response to infection with Leishmania major and are resistant. Vbeta4-deficient BALB/c mice are unable to generate the early IL-4 transcription occurring in Vbeta4 Valpha8 CD4+ T cells of BALB/c mice within 1 day of infection. Here we demonstrate that treatment of Vbeta4-deficient BALB/c mice with IL-4 during the first 64 h after infection instructs Th2 cell development and susceptibility to infection. The demonstrated inability of IL-4 to reverse the resistant phenotype of BALB/c mice treated with anti-CD4 mAb the day before infection suggest that these effects of IL-4 require its interaction with CD4+ T cells. In contrast to draining lymph node cells from BALB/c mice, cells from Vbeta4-deficient BALB/c mice remain responsive to IL-12 following infection. Strikingly, administration of IL-4 to Vbeta4-deficient BALB/c mice renders their lymph node cells unresponsive to IL-12 by down-regulating IL-12R beta2-chain expression. This study directly demonstrates that in BALB/c mice IL-4 is necessary and sufficient to initiate the molecular events steering Th2 cell maturation and susceptibility to L. major.

Pesquisa disciplinar do corpus documental das teses de doutorado do PPGCI-IBICT/UFRJ: aplicando princ��pios e categorias para estudo interdisciplinar da ci��ncia da informa����o no Brasil

A partir do hist��rico da ci��ncia da informa����o no Brasil, realizou-se estudo visando a investigar, mediante princ��pios e categorias, a rela����o das disciplinas com a ci��ncia da informa����o. O modelo aplicado dividiu o corpus de 58 teses em tr��s categorias: disciplinas como conjuntos distintos, j�� que os conceitos e m��todos dessa categoria n��o s��o suficientes para formalizar um quadro interdisciplinar. A segunda pela intera����o dos conceitos abordados, com encadeamento das quest��es colocadas pelas disciplinas na busca de solu����o para o problema proposto. A terceira remete �� integra����o entre a disciplina e a ci��ncia da informa����o, apresentando uma linguagem comum. O resultado do estudo visa a contribuir para a cria����o de um m��todo de pesquisa interdisciplinar para a p��s-gradua����o brasileira.

Some of the early events underlying Th2 cell maturation and susceptibility to Leishmania major infection in BALB/c mice.

The first experimental evidence for the development of polarized CD4+ Th1 and Th2 responses in vivo has been obtained using the murine model of infection with Leishmania major, an intracellular parasite of macrophages in their vertebrate host. Genetically determined resistance and susceptibility to infection with this parasite have been clearly demonstrated to result from the development of polarized Th1 and Th2 responses, respectively. Using this model system, the dominant role of cytokines in the induction of polarized CD4+ responses has been validated in vivo. The requisite role of IL-4 in mediating both Th2 differentiation and susceptibility to infection in BALB/c mice has directed interest towards the search for evidence of IL-4 production early after infection and identification of its cellular source. We have been able to demonstrate a burst of IL-4 production in susceptible BALB/c mice within the first day of infection with L. major and could establish that this rapidly produced IL-4 instructed Th2 lineage commitment of subsequently activated CD4+ T cells and stabilized this commitment by downregulating IL-12 Rbeta2 chain expression, resulting in susceptibility to infection. Strikingly, this early IL-4 response to infection resulted from the cognate recognition of a single epitope in a distinctive antigen, LACK, from this complex microorganism by a restricted population of CD4+ T cells that express Vbeta4-Valpha8 T cell receptors.

Detection of serum antibodies against Leishmania 94 kDa antigen in visceral and cutaneous leishmaniosis due to Leishmania infantum.

Leishmania promastigotes polypeptides are analyzed by immunoblotting with sera from patients infected with different Leishmania species and presenting visceral or cutaneous infections. These sera recognize Leishmania polypeptides in several molecular masses. The major findings of this study are as follow. 1) The Leishmania 94 kDa antigen, which is specifically recognized by all sera from L. infantum-infected patients with visceral infection, is recognized by some sera from L. infantum-infected patients presenting cutaneous infection. 2) All patients with cutaneous infections due to L. tropica, L. amazonensis, or L. guyanensis do not develop anti-94 kDa antibodies, whatever the Leishmania species used as antigens. 3) Difference in electrophoretic mobilities is seen between the 94 kDa antigen identified by sera from Leishmania infantum-infected patients, and the antigen both recognized by the Concavalin A lectin and a rabbit antiserum raised against deglycosylated Promastigote Surface Protease.

Rapid IL-4 production by Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells in resistant mice treated once with anti-IL-12 or -IFN-gamma antibodies at the onset of infection with Leishmania major instructs Th2 cell development, resulting in nonhealing lesions.

Rapid production of IL-4 by Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) T cells expressing the V beta 4-V alpha 8 TCR chains has been shown to drive aberrant Th2 cell development and susceptibility to Leishmania major in BALB/c mice. In contrast, mice from resistant strains fail to express this early IL-4 response. However, administration of either anti-IL-12 or -IFN-gamma at the initiation of infection allows the expression of this early IL-4 response in resistant mice. In this work we show that Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells also expressing the V beta 4-V alpha 8 TCR chains are the source of the early IL-4 response to L. major in resistant mice given anti-IL-12 or -IFN-gamma Abs only at the onset of infection. Strikingly, these cells were found to be required for the reversal of the natural resistance of C57BL/6 mice following a single administration of anti-IL-12 or -IFN-gamma Abs. Together these results suggest that a deficiency in mechanisms capable of down-regulating the early IL-4 response to L. major contributes to the exquisite susceptibility of BALB/c mice to L. major.