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Graduate

Investigation of the molecular alterations of macrophage function during intracellular infection by Leishmania donovani revealed both adverse and positive influences of this protozoan on host cell function. Chapter I delineates a negative effect which this parasite has on signal transduction pathways in its host cell. In macrophages put in contact, or infected with L. donovani, c-fos gene expression mediated through protein kinase A was unaffected under conditions where there was an impairment of protein kinase C-mediated c-fos gene expression. Selective impairment of protein kinase C-, or, calmodulin-dependent protein kinase-mediated signal transduction in the macrophage was found to influence the establishment of infection. Chapters two and three describe a positive enhancement of macrophage function by L. donovani. Intramacrophage infection with L. donovani was shown to enhance host cell viability in the absence of growth factor. This was attributable to the elaboration of a soluble factor(s) by infected macrophages into the cell culture supernatant, which enhanced macrophage viability in a manner independent of cell replication. Further characterization of the mechanism of this enhancement revealed that L. donovani infection, and lipophosphoglycan, inhibited macrophage death by apoptosis. Cell supernatants derived from L. donovani infected cells were also capable of inhibiting macrophage apoptosis. To identify the active factor in infected cell supernatants, the cytokine gene expression profile of L. donovani infected macrophages was delineated and possible candidate cytokines were further investigated. Levels of TNF-$ alpha$ capable of causing an abrogation of apoptosis were found to be produced by infected macrophages. However, antibody neutralization of TNF in infected cell cultures could not reverse the inhibition of apoptosis by L. donovani, implicating the involvement of multiple factors in the abrogation of apoptosis by L. donovani.

The cellular transformation of the Leishmania protozoan parasite from the promastigote to the amastigote stages takes place in the phagolysosomal compartment of vertebrate host macrophage cells. This cytodifferentiation is a prerequisite for parasite survival and for the establishment of the infection in the mammalian host. The differential screening of a L. donovani amastigote cDNA library using stage-specific cDNA probes allowed the identification of the A2 genes, which are only expressed by the parasite as an amastigote. A2 specific transcripts are developmentally expressed in Leishmania in response to a combination of pH and temperature shifts, conditions associated with the transfer from the insect vector to the phagolysosomal compartment in macrophages. The coding genes are clustered on a 850 kb chromosome and arranged in tandem arrays with copies of another gene, the A2rel gene. The developmental expression is mediated post-transcriptionally and involves elements located in the 3$ prime$ untranslated region; accurate processing by trans splicing is essential for the A2 mRNAs developmental accumulation in cells transferred into phagolysosomal conditions. The A2 protein product is composed mostly of highly repeated domains, carries a functional signal peptide at its amino-terminal end, and shares sequence homology with other developmentally expressed proteins in unrelated parasites of humans. Using A2 locus sequences, vectors and transfection systems were designed and developed to differentially express reporter or selectable markets specifically in the amastigote stage.

Graduate

Ulm, Univ., Diss., 2007.

Thesis (D. SC.)--University of Michigan, 1924. / "Reprinted from the Journal of infectious diseases, vol. 36, no. 3, March, 1925."

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-10Bitstream added on 2014-06-13T19:22:42Z : No. of bitstreams: 1 santiago_meb_dr_jabo.pdf: 510527 bytes, checksum: 23f153f852b4ef52ff4f3278f8a62f07 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente estudo investiga in vitro a ação leishmanicida do imunomodulador fosfolinoleato-palmitoleato de magnésio e amônio proteico (PMAPA) e sua ação citotóxica em células mononucleares, e in vivo seu efeito imunomodulador em cães naturalmente infectados por Leishmania spp. Para avaliar a viabilidade celular após a ação do P-MAPA utilizou-se o ensaio colorimétrico com (3 - (4,5-dimetil-2-il) -2,5-difenil tetrazolium brometo (MTT), onde células mononucleares derivadas de sangue periférico foram cultivadas com diferentes concentrações de P- MAPA; 0, 50, 100 e 200 μg/mL em ambiente úmido a 37°C, com 5% de CO2 por 72 horas. A ação leishmanicida do imunomodulador foi avaliada pelo mesmo método utilizando-se a forma promastigosta de Leishmania (L.) chagasi. Para avaliar a ação terapêutica do PMAPA, vinte cães sem raça definida, com sintomas clínicos compatíveis com leishmaniose e positivos na detecção de anticorpos anti-leishmania pelo método ELISA, foram tratados. Dez cães receberam 15 doses do imunomodulador (2,0 mg/kg), por via intramuscular, e dez receberam solução fisiológica como placebo. Os grupos foram acompanhados para observação da remissão de sinais clínicos, no grupo tratado e controle foram colhidas amostras de pele antes e após a administração do imunomodulador para quantificação da carga parasitária, ao final do tratamento amostras de sangue periférico foram utilizadas para o isolamento de mononucleares e investigação do perfil linfocitário do tipo T e produção das citocinas IL-10, IFN-gama e IL-2 em sobrenadante de cultura celular. A quantificação da carga parasitária nas amostras de pele foi realizada pelo método da PCR em tempo real. O perfil linfocitário do tipo T foi realizado por citometria de fluxo, e a quantificação das citocinas em sobrenadante de cultura... / This study investigates the in vitro leishmanicidal action of the immunomodulator-phospholinoleate palmitoleato magnesium and ammonium protein (P-MAPA) together with its cytotoxic action in mononuclear cells. It also investigates, in vivo, its immunomodulatory effect in dogs naturally infected by Leishmania spp. To assess cell viability after the action of P-MAPA, the colorimetric assay with (3 - (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) was used. There mononuclear cells derived from peripheral blood and were cultured with different concentrations of P-MAPA, 0, 50, 100 and 200 μg/mL in a humid environment at 37 ° C with 5% CO2 for 72 hours. The leishmanicidal action of immunomodulators was evaluated using the same method to form promastigosta of Leishmania (L.) chagasi. To assess the therapeutic action of P-MAPA, twenty mongrel dogs, with clinical symptoms compatible with leishmaniasis and positive detection of anti-leishmania antibodies by ELISA assay were treated. Ten dogs received 15 doses of the immunomodulator (2.0 mg / kg) intramuscularly, and ten received saline solution as placebo. The groups were followed for observation of clinical signs of remission; in the both groups, skin samples were collected before and after administration of the immunomodulator to quantify the parasite load. At the end of treatment, peripheral blood samples were used for the isolation of mononuclears, the profile investigation of type T lymphocytes, together with the production of IL-10, IFN-gamma and IL-2 cytokines in cell culture supernatant. The quantification of parasite load in the samples was performed by PCR in real time. The profile of type T lymphocytes was performed by flow cytometry, and quantification of cytokines in culture supernatants by ELISA capture... (Complete abstract click electronic access below)

Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, 2006. / Submitted by Thaíza da Silva Santos (thaiza28@hotmail.com) on 2009-09-25T19:44:43Z No. of bitstreams: 1 2006_Maria Elizabeth M C Dorval.pdf: 5823254 bytes, checksum: d0288003f2151f98537c46e000f4a4c1 (MD5) / Approved for entry into archive by Tania Milca Carvalho Malheiros(tania@bce.unb.br) on 2009-09-28T13:15:03Z (GMT) No. of bitstreams: 1 2006_Maria Elizabeth M C Dorval.pdf: 5823254 bytes, checksum: d0288003f2151f98537c46e000f4a4c1 (MD5) / Made available in DSpace on 2009-09-28T13:15:03Z (GMT). No. of bitstreams: 1 2006_Maria Elizabeth M C Dorval.pdf: 5823254 bytes, checksum: d0288003f2151f98537c46e000f4a4c1 (MD5) Previous issue date: 2006 / Os estudos epidemiológicos realizados permitiram o isolamento e a identificação de Leishmania (Leishmania) amazonensis em pacientes procedentes do município de Bela Vista, Mato Grosso do Sul, ampliando-se, portanto, o conhecimento da distribuição geográfica desta espécie de parasito. As capturas de flebotomineos foram realizadas no período de fevereiro de 2004 a janeiro de 2006, na mata, utilizando-se armadilhas automáticas luminosas (AAL), de Shannon e de Disney, e em abrigo de animais domésticos, com AAL. Com o emprego dos três tipos de armadilhas capturou-se 1.999 espécimes pertencentes a três subtribos, oito gêneros e 19 espécies de Phlebotominae. Brumptomyiina: Brumptomyia avellari, Br. brumpti e Brumptomyia sp; Lutzomyiina: Evandromyia aldafalcaoae, Ev. bourrouli, Ev. cortelezzii, Ev. evandroi, Ev. lenti, Ev. teratodes, Ev. termitophila, Lutzomyia longipalpis, Pintomyia christenseni e Sciopemyia sordellii; Psychodopygina: Bichromomyia flaviscutellata, Nyssomyia whitmani, Psathyromyia aragaoi, Ps. campograndensis, Ps. punctigeniculata e Ps. shannoni. Do total de espécimes capturados, 22,7% foram nas armadilhas de Shannon (33,9% das fêmeas de Ps. punctigeniculata; 7,7% de Bi. flaviscutellata e 4,4% de Lu. longipalpis), 17,7% nas AALs (na mata, 70,6% dos espécimes de Brumptomyia e em abrigos de animais domésticos, 80,2% de Lu. longipalpis) e 59,6% nas armadilhas de Disney (57,6% de Ev. bourrouli e 41,4% de Bi. flaviscutellata, sendo 81,7% das fêmeas, desta espécie). Pela primeira vez, se registra a presença de Ev. evandroi no Estado. Bi. flaviscutellata esteve distribuída em todos os ambientes de mata amostrados, com freqüência mais regular nos meses secos, porém, mostrou pico em março. Não foi observada a infecção natural por Le .(Le.) amazonensis em Bi. flaviscutellata, porém, seu pico de captura coincidente com o período da infecção natural em hamsters (Mesocricetus auratus) sentinelas, permitem supor a existência de um ciclo enzoótico na área, com a infecção acidental e periódica do homem que adentra a área, uma vez que fêmeas deste flebotomíneo compareceram, embora com freqüência baixa, em armadilhas de Shannon, sugerindo a sua antropofilia. Além da presença de vetores de Le. (Le.) amazonensis e Le. (Le.) chagasi, constatou-se ainda a presença de Ny. whitmani, reconhecido vetor de Le. braziliensis, no entanto, em baixa freqüência. ______________________________________________________________________________________ ABSTRACT / The epidemiological studies undertaken permitted the isolation and identification of Leishmania (Leishmania) amazonensis in patients from the municipality of Bela Vista, Mato Grosso do Sul State, Brazil, thus expanding our knowledge of this parasite’s distribution. The captures of phlebotomines were undertaken from February 2004 to January 2006, using automatic light traps (ALTs), Shannon traps and Disney traps in forested environments and ALTs in domestic animal shelters. With the three types of traps, a total of 1,999 specimens belonging to three subtribes, eight genera and 19 species of Phlebotominae were captured: Brumptomyiina: Brumptomyia avellari, Br. brumpti and Brumptomyia sp; Lutzomyiina: Evandromyia aldafalcaoae, Ev. bourrouli, Ev. cortelezzii, Ev. evandroi, Ev. lenti, Ev. teratodes, Ev. termitophila, Lutzomyia longipalpis, Pintomyia christenseni and Sciopemyia sordellii; Psychodopygina: Bichromomyia flaviscutellata, Nyssomyia whitmani, Psathyromyia aragaoi, Ps. campograndensis, Ps. punctigeniculata and Ps. shannoni. Of this total, 22.7% were captured on the Shannon traps (33,9% of the females being of Ps. punctigeniculata; 7.7% of Bi. flaviscutellata and 4.4% of Lu. longipalpis); 17.7% in the ALTs (in the forest 70.6% of the specimens captured belonged to Brumptomyia and in the domestic animal shelters, 80.2% belonged to Lu. longipalpis) and 59.6% in the Disney traps (57.6% of Ev. bourrouli and 41.4% of Bi. faviscutellata; 81.7% of the females being of this species). The occurrence of Ev. evandroi in the State is here registered for the first time. Bi. Flaviscutellata occurred in all the forested environments sampled, with more regular frequencies during the dry period of the year, though showing a peak in March. No natural infection by Le. amazonensis was observed in Bi. flaviscutellata, however its peak of capture coincident with the period of natural infection in hamsters (Mesocricetus auratus) used as bait in Disney traps, suggests the existence of an enzootic cycle in the area, with periodic and accidental infection of people who enter it, since females of this sandfly were captured (although in low frequency) on the Shannon traps, indicating their anthropophily. Besides the presence of the vectors of Le. amazonensis and Le. chagasi, Ny. whitmani, a known vector of Le. braziliensis, was found, albeit in very low frequency.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Biotecnologia. / Made available in DSpace on 2012-10-23T19:44:54Z (GMT). No. of bitstreams: 1 248379.pdf: 6044909 bytes, checksum: 3ff74fbefc084ae3b89c236795505d7c (MD5)