MSc (Microbiology) / Department of Microbiology

Made available in DSpace on 2014-06-12T15:52:35Z (GMT). No. of bitstreams: 2 arquivo5024_1.pdf: 448901 bytes, checksum: 4d971b7e87c950e13777aa7be6ce24c9 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2003 / Vários relatos da literatura revelam ser a prevalência de Entamoeba dispar maior do que Entamoeba histolytica nos indivíduos que vivem no Nordeste brasileiro, a partir de estudos utilizando Enzyme Linked Immnosorbent Assay (ELISA), imunodifusão em gel e zimodemos. Este trabalho consistiu em determinar a prevalência dessas formas de amebas mediante o uso de detecção imunocoprológica de antígeno específico para E. histolytica e Reaction Chain Polimerase (PCR) do DNA genômico extraído de trofozoitos cultivados de amostras de fezes. A presença de amebas tetranucleadas foi investigada em 1437 amostras de fezes de indivíduos vivendo em Macaparana, cidade da zona da mata norte de Pernambuco; em 346 amostras de escolares com idades de 3 a 14 anos morando em uma favela do Recife e em 109 amostras de imunodeprimidos (104 HIV positivos e 05 transplantados) atendidos no Hospital das Clínicas da UFPE (2002 e 2003). Dessas amostras, 59 (4.1%) e 45 (13%) foram positivas para aquelas coletadas das populações de Macaparana e das crianças do Recife, respectivamente, enquanto que nenhuma foi positiva para as obtidas dos imunodeprimidos. Todas as amostras foram negativas para a presença de adesina galactose específica de E. histolytica, inclusive as amostras dos pacientes imunosuprimidos. As amostras com amebas tetranucleadas cultivadas em meio de Robinson foram positivas para trofozoítos em 31 daquelas coletadas das populações de Macaparana e 21 das de Recife. A partir da amplificação por PCR de seqüências espécie-específicas de DNA genômico, extraído desses trofozoítos, foi possível identificar E. dispar em 23 e 19 amostras dos habitantes de Macaparana e das crianças do Recife, respectivamente, enquanto que nenhuma amplificação foi observada para E. histolytica. As demais amostras (08 e 02 paraMacaparana e Recife, respectivamente) foram negativas para ambas as espécies. Estes resultados corroboram aqueles previamente descritos que mostravam a prevalência de E. dispar (ameba não patogênica) nestas populações. Ademais, validam o emprego do kit imunocoprológico como alternativa a PCR na identificação de E. dispar e E. histolytica. Finalmente, o polimorfismo genético das cepas de E. dispar das amostras coletadas da população de Macaparana e de crianças do Recife foi investigado com o uso de marcadores moleculares específicos para E. dispar, Dsp1/Dsp2 e Dsp5/Dsp6. Das 42 amostras analisadas, 39 amplificaram os loci 1-2 e 5-6. O dendrograma resultante desta análise revelou uma alta variabilidade entre os isolados para esta região. Entretanto, uma comparação entre as freqüências dos produtos de amplificação para as duas localidades, através de teste de Qui-quadrado, mostrou que a incidência de uma banda obtida do locus 5-6, foi significativamente diferente entre Recife e Macaparana, evidenciando a potencialidade desta técnica para abordar questões relativas à distribuição geográfica

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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.

No description available.

No description available.

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 (M.Sc. (Biochemistry)) -- Mahidol University, 1973. / Supported by the Rockefeller Foundation.