Entamoeba histolytica is the protozoan parasite that causes amoebic dysentery and amoebic liver abscesses in humans. For many years it was believed to be a primitive organism because it lacks many typical eukaryotic features including mitochondria. Recently, two genes that in other organisms encode proteins normally found in the mitochondrion have been isolated, giving evidence for the secondary loss of mitochondrial function in E. histolytica. These are the pyridine nucleotide transhydrogenase (PNT) and the mitochondrial chaperonin cpn60 genes. In this study we isolated and characterised a gene encoding a mitochondrial-type heat shock protein 70 from E. histolytica. cDNA and genomic library clones have been isolated and sequenced. Comparison with previously published sequences confirmed the assumption that E. histolytica comes from mitochondrion - bearing ancestors. Southern blot hybridisation revealed there are two copies of the gene in the genome. Northern blot analysis revealed two transcripts hybridising to the mt-hsp70 probe that differ in length and which are induced by heat shock. In addition, an apparently noncoding, polyadenylated RNA that is also induced by heat shock is encoded immediately upstream of the mitochondrial-type hsp70 gene. Expression analysis was also performed in four other Entamoeba species. Partial cpn60, PNT, and mt-hsp70 genes were isolated and the size of the mRNAs and their heat shock induction levels were investigated by hybridisation to these probes. The similarity of the mt-hsp70 amino terminus to those of hydrogenosomal proteins in conjunction with the phylogenetic analyses suggests it is also likely to be targeted to the mitochondrion-derived organelle of E. histolytica known as the mitosome.

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Entamoeba histolytica is a protozoan parasite and the causative agent of amoebiasis. While infection is associated with suppression of cell-mediated, immunity, drug-cured patients are resistant to reinvasion by amoebae. Macrophages are the principal effector cells in host defence against E. histolytica via production of nitric oxide which is cytotoxic for the parasite. The objective of this study was to determine the T cell cytokine responses associated with host defence against E. histolytica . A mixed Th1/Th2 (Th0) response predominated at days 5--10 of amoebic liver abscess development in gerbils, as indicated by spleen and hepatic lymph node cell IL-2 (Th1 marker) and IL-4 (Th2 marker) production. However, T cell responses were profoundly suppressed at day 20 of infection. Serum collected at day 20, but not at other times, markedly suppressed T cell proliferative responses by inhibiting IL-2 production. A switch to a Th1 response occurred after day 20 of infection. Following drug-abbreviation of infection at day 20, animals were completely resistant to challenge infection in the liver and demonstrated a Th1 response. The Gal-lectin 170-kDa heavy subunit of E. histolytica is a protective antigen in gerbils and a potential subunit vaccine candidate. We determined which region of the Gal-lectin stimulates IL-12 production, as IL-12 is key to inducing Th1 cytokine responses. Native Gal-lectin plus interferon-gamma stimulated IL-12 p40 and p35 gene transcription and IL-12 p70 protein production in human macrophages. Using a panel of anti-170-kDa subunit monoclonal antibodies in inhibition studies, aa 596--998 was identified as the IL-12-inducing domain. These results suggest that this portion of the Gal-lectin has potential for use as a subunit vaccine to induce Th1-mediated immunity against E. histolytica .

Infections with Entamoeba histolytica are associated with impaired cell mediated immunity by an unknown mechanism. Macrophages are the most important cells in host defense against invasive amebiasis. The present study investigated the effect of E. histolytica on macrophage functions. Macrophages isolated from gerbils with amebic liver abscess and naive macrophages exposed to soluble amebic proteins induced profound alteration of eicosanoid formation both in cyclooxygenase and lipoxygenase pathways by enhanced PGE$ sb2$ and LTC$ sb4$ production. TNF-$ alpha$ production by macrophages was altered locally in amebic granulomas and at systemic sites during the infection and in response to amebic proteins stimulation in vitro. PGE$ sb2$ produced by macrophages in response to amebic proteins was involved in the down-regulation of TNF-$ alpha$ production. E. histolytica-induced dysfunction of macrophage cytotoxicity against amebae and tumor cells occurred by suppressing NO and by enhancing PGE$ sb2$ production. Amebic proteins suppressed the induction. of IFN-$ gamma$-induced bone marrow macrophage class II MHC Ia molecule synthesis and I-A$ beta$ mRNA expression by stimulating PGE$ sb2$ production. Taken together, these results demonstrate that E. histolytica induced PGE$ sb2$ production plays a central role in the suppression of macrophage effector and accessory cell functions.

Entamoeba histolytica is a protozoan parasite and the causative agent of amebiasis. Activated macrophages are the main host effector cells in host defence against E. histolytica, through the production of nitric oxide (NO) which is cytotoxic for the parasite. NO is upregulated by tumor necrosis factor-alpha (TNF-$ alpha$) produced by macrophages. The objective of this study was to determine the effect of amebic components on TNF-$ alpha$ and NO production by macrophages. Soluble E. histolytica proteins stimulated naive macrophages for enhanced TNF-$ alpha$ mRNA expression through PKC signal transduction. E. histolytica-induced TNF-$ alpha$ mRNA expression was unstable, and macrophages pretreated with E. histolytica proteins expressed reduced levels of TNF-$ alpha$ mRNA in response to LPS or IFN-$ gamma$ + LPS. In contrast, the purified galactose-adherence lectin (Gal-lectin) of E. histolytica stimulated naive macrophages for stable TNF-$ alpha$ mRNA expression and protein production. Furthermore, IFN-$ gamma$ primed macrophages produced TNF-$ alpha$ and NO in response to the Gal-lectin. Naive macrophages exposed to Gal-lectin + IFN-$ gamma$ were activated to kill E. histolytica trophozoites in vitro by NO. Anti-lectin monoclonal antibodies that recognize non-overlapping epitopes of the kDa heavy subunit of the Gal-lectin identified amino acids 596-1082 as important in mediating amebic adherence to target cells and TNF-$ alpha$ mRNA induction in macrophages. Likewise, a region between amino acids 596-818 of the 170 kDa Gal-lectin, in conjunction with IFN-$ gamma$, activated macrophages for TNF-$ alpha$ and NO production and amebicidal activity. This research demonstrates the immunogenic potential of the E. histolytica Gal-lectin and the critical regions that could be used as a subunit vaccine candidate against amebiasis.

Thesis (Ph. D.)--University of Virginia, 1998. / Spine title: CRD of the E. histolytica Gall lectin. Includes bibliographical references (p. 71-87). Also available online through Digital Dissertations.

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Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-15T17:14:57Z No. of bitstreams: 1 Gustavo Santos. Padronização de condições experimentais.2011.pdf: 3612850 bytes, checksum: 6681f5cbe674b715c7de996edc26d29d (MD5) / Made available in DSpace on 2013-10-15T17:14:57Z (GMT). No. of bitstreams: 1 Gustavo Santos. Padronização de condições experimentais.2011.pdf: 3612850 bytes, checksum: 6681f5cbe674b715c7de996edc26d29d (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / O protozoário, Entamoeba histolytica, constitui a etiologia de milhares de óbitos anuais e, em muitos casos, a falta de saneamento, o grau de instrução e a falta de higiene da população podem favorecer a transmissão e a manutenção desses patógenos em uma comunidade. Por causar tantas mortes e problemas na saúde pública trabalhos que facilitem o estudo deste parasito fazem-se importantes. Uma vez que a padronização de cultivo de E. histolytica em placas de poços vai Existem indicações que mostram que este parasito pode se tornar resistente ao medicamento utilizado no tratamento desta protozoose, por isso, a busca por novas substâncias que possam atuar como tratamento alternativo é de suma importância. Portanto, o objetivo do presente trabalho foi otimizar e padronizar o cultivo e a contagem deste parasito in vitro, além de identificar substâncias com potencial amebicida, que possam ser utilizadas no futuro como fármacos no tratamento da amebíase, sugerindo também uma via possível de ação das substâncias que apresentaram os melhores efeitos. Para tanto, os trofozoítos foram cultivados em placas de 24 poços sobre diferentes condições, quatro métodos de contagem de células foram comparados e 74 (setenta e quatro) substâncias foram testadas. Destas 13 (treze) apresentaram uma inibição na proliferação axênica dos trofozoítos de cerca de 70%. Destas, três compostos foram estudados em mais detalhes, os mesoiônicos derivados da piperina (as MII, MVI e MIX). Estas substâncias pertencem ao grupo dos compostos mesoiônicos, substâncias formadas por um anel heteroatômico composto por nitrogênio, carbono e enxofre, capazes de atravessar membranas e interagir com biomoléculas. Além disso, alguns mesoiônicos são doadores de radicais NO e tais grupamentos são capazes de induzir uma morte celular semelhante à apoptose em E. histolytica, como sugerido pela expressão de fosfatidil-serina revelada por anexina-V. Confirmando os resultados descritos na literatura, estas substâncias foram capazes de induzir uma morte programada, porém observações da ultra-estrutura, tais como figuras de mielina, das células tratadas apontaram para autofagia que também foi evidenciada por testes com MDC gerando apoptose tipo II, que pode ser iniciada pela presença de ROS, que neste caso foram por DCFDA. / The protozoan parasite, Entamoeba histolytica, is the etiology of thousands of deaths annually and in many cases lack of sanitation, education level and poor hygiene of the population may facilitate the transmission and maintenance of these pathogens in a community. There are indications showing that this parasite may become resistant to the drug used in treatment of protozoal disease, so the search for new substances that can act as an alternative treatment is of paramount importance. Therefore, the aim of this study was to optimize and standardize the cultivation and enumeration of this parasite in vitro, and identify substances with potential amebicidal, which can be used in future as drugs for the treatment of amoebiasis, suggesting a possible route of action of the compounds that showed the greatest effects. For this purpose, trophozoites were cultured in 24-well plates under different conditions, four methods of cell count were compared and 74 (seventy four) substances were tested. Of these thirteen (13) showed an inhibition in the proliferation of axenic trophozoites of about 70%. Of these, three compounds were studied in more detail, the mesoionic derivatives of piperine (the MII, MVI and MIX). These substances belong to the group of mesoionic compounds, formed by a heteroatomic ring composed of nitrogen, carbon and sulfur, able to traverse membranes and interact with biomolecules. Moreover, some donors are mesoionic radicals NO and such groups are able to induce a cell death similar to apoptosis in E. histolytica, as suggested by the expression of phosphatidyl-serine revealed by annexin-V. Confirming the results described in the literature, these substances were capable to inducing a programmed death, but observations of the ultra-structure, such as myelin figures, treated cells pointed out that autophagy was also evidenced by tests with MDC generating apoptosis type II can be initiated by the presence of ROS, which in this case were by DCFDA.

MSc (Microbiology) / Department of Microbiology