Isoenzyme characterisation of Entamoeba histolytica into pathogenic and non-pathogenic zymodemes substantiated previously held views that this parasite con5titutes two distinct strains or even sub-species that are morphologically identical but vary in their pathogenicity. A reappraisal of the epidemiology of amoebiasis and investigation of the patho-physiological relationships between these pathogenic and non-pathogenic zymodemes and their host was therefore indicated. Only pathogenic zymodemes were isolated from hospitalised patients with amoebic liver abscess (ALA) and amoebic dysentery (AD). In the amoebiasis endemic peri-urban population of Durban, I. histolytica occurred at an overall prevalence of 10%. Carriers of non-pathogenic zymodemes constituted 9% of the population. A key observation was that asymptomatic infections with pathogenic zymodemes occurred at a prevalence of 1%. Higher prevalence of E. histolytica occurred in association with poor sanitary conditions. Furthermore., both pathogenic and non-pathogenic zymodemes tended to cluster into family units suggesting person-to-person transmission of the parasite by the faecal-oral route. Although invasive amoebiasis occurs far more frequently in males than females (8:1) both pathogenic and non-pathogenic zymodemes are equally distributed in male and female E. histolytica cyst passers. Ninety percent of carriers of pathogenic zymodemes spontaneously cleared their infections and remained asymptomatic throughout the study period of 2 years while 10% developed AD which required treatment with metronidazole. No spontaneous changes in zymodemes from the non-pathogenicto the pathogenic type was observed in a longitudinal study. The serological response of asymptomatic carriers of pathogenic zymodemes (100% seropositive) was identical to that of patients with ALA or AD with a high proportion (94-100%) of them being strongly seropositive. The prevalence of seropositivity amongst subjects who were not infected by E. histolytica (13% seropositive) was not statistically different (p>0,5) from that of the random population of this endemic area (19% seropositive) and carriers of non-pathogenic zymodemes (21% positive); the prevalence of strongly seropositive reactions among this group was only between 2-4%. It is concluded that a positive serological response is directly due to past or present contact with pathogenic zymodemes. This is further substantiated by the observation that the proportion of seropositive subjects was found to increase dramatically in a population near Cape Town where an outbreak of invasive amoebiasis (ALA and AD) occurred indicating a high prevalence of pathogenic zymodemes in this community. Another community in northern Transvaal (Gazankulu) where ALA and AD does not occur was, as expected, uniformly seronegative. Axenic growth of pathogenic zymodemes was possible but could not be accomplished with the non-pathogenic zymodemes. Even though monaxenic growth together with Trypanosoma cruzi was possible with both strains, the pathogenic zymodemes tended to grow more prolificly. No zymodeme changes from non-pathogenic to pathogenic and vice versa were observed with such changes in culture conditions. Cyst production by the pathogenic zymodemes in vivo was confirmed experimentally, thereby demonstrating the ability of pathogenic E. histolytica to independently complete their life-cycle thus giving it the ability to propagate itself successfully as a species. / Thesis (M.D.)-University of Natal, Durban, 1989.

Entamoeba histolytica, etiological agent of amebiasis, continues to be a significant threat to human health worldwide. The disease affects 10% of the world's population and leads to an estimated 100, 000 deaths a year. The parasite's surface Gal-lectin is an immunodominant protein that also mediates colonization and pathogenicity. The Gal-lectin is the most promising vaccine candidate against amebiasis. However, the immune mechanisms involved in protection against disease remain unclear. The objective of this study was to characterize the immunological basis of the host defense mechanisms using a Gal-lectin based vaccine. Exposure of the Gal-lectin with immature dendritic cells increased cell maturation and activation and upregulated co-stimulatory molecules and pro-inflammatory cytokines production. Dendritic cell activation was dependent on NF-kappaB and MAPK activation. In vaccination studies, the adjuvant effect of CpG-ODN, a synthetic oligodeoxynucleotide capable of stimulating Th1 immune responses enhanced the immune response to the Gal-lectin when administered systemically or mucosally. Protected animals had elevated anti-Gal-lectin serum and stool IgA antibodies capable of blocking parasite adherence in vitro. Analysis of cytokine responses in vaccinated and protected animals revealed increased IFN-gamma production compared to controls. Finally, E. histolytica DNA was shown to activate macrophages in a TLR9 and MYD88-dependent manner. Immunized gerbils with Gal-lectin and E. histolytica DNA induced protective immunity against a challenge infection. Taken together, these findings underscore the importance of multivalent subunit vaccines in Th1 mediated immune responses in host defense against amebiasis.

The enteric protozoan parasite Entamoeba histolytica is the etiologic agent of the disease amebiasis which is characterized by colitis or hepatic lesions. Amebae colonize the colon by binding to mucous glycoproteins (mucins). Secretory mucins provide the gel nature to mucus and are a vital component of epithelial barrier function. Mucins prevent contact-dependent cytolysis of colonic cells by E. histolytica. To possibly circumvent this barrier, the parasite secretes a potent yet unidentified mucin secretagogue, which could deplete the stored mucin pool and render the mucous layer less protective. The objective of this study was to investigate the molecular mechanisms by which E. histolytica modulates colonic mucin exocytosis. We showed that E. histolytica converts exogenous arachidonic acid to prostaglandin E2 (PGE2), a known mucin secretagogue and potential mechanism by which the parasite evokes mucin secretion. Conversion was via a novel cyclooxygenase-like activity and was inhibitable with the known cyclooxygenase inhibitor aspirin. To study E. histolytica-mucin interactions, we developed an in vitro model of LS174T human colonic epithelial cells that secrete mucin constitutively and in response to mucin agonists. Highly purified mucins isolated from LS174T cells markedly inhibited amebic adherence to target cells and the mucous barrier protected the LS174T monolayers from amebic cytolysis. We have identified that Gal and GalNAc residues (O-linked sugars) of mucins are the protective moiety as O- but not N-linked glycosylation inhibitors decreased their protective effect. To understand how mucins are regulated during intestinal amebiasis and in the inflamed gut, we determined that PGE2 binds the EP4 receptor on LS174T cells and in rat colon to stimulate cyclic adenosine monophosphate-dependent mucin exocytosis. Taken together, these studies delineate how E. histolytica modulates host responses during infection to allow the parasite to survive and persist in th

The pathogenesis of the enteric protozoan parasite Entamoeba histolytica remains poorly understood. Moreover, the host responses during the early periods of interaction in the gut remain to be clarified. In this study I investigated the cell specific responses to the parasite and the importance of cross talk between epithelial-immune cells that could potentially influence the outcome of infection, with a central focus on Nuclear factor (NF)-kappaB. NF-kappaB is a ubiquitous transcription factor that plays a critical role in mucosal inflammation and its regulation by E. histolytica has not been studied so far. Gal-lectin is a well characterized parasite virulence factor and vaccine candidate. I first characterized the interactions between Gal-lectin and macrophages and found that several proinflammatory genes are upregulated as early as 2h. The Gal-lectin activated NF-kappaB and up-regulated Toll like receptor-2 expression in an NF-kappaB- and p38 Mitogen Activated Protein (MAP) kinase-dependent manner. As intestinal epithelial cells (IEC) form the first line of active host defense against mucosal pathogens, I determined the interaction between ameba soluble proteins and naive IEC. I observed that the parasite could elicit a chemokine response via activation of PI3 kinase and phosphorylation of p65 subunit to induce monocyte chemoattractant protein-1. The consequent recruitment of immune cells could be responsible for colonic inflammation. Finally, I made the novel observation that in macrophage-primed IEC, ameba proteins elicited a cytoprotective stress response. Using a combination of siRNA and over expression studies, I demonstrated that amebic proteins increased the expression and phosphorylation of Heat shock protein (Hsp) 27 thereby enhancing its association with and subsequent inhibition of Inhibitory kappaB kinase (IKK). The resulting inhibition of NF-kappaB could be a potential mechanism that explains the absence of inflammation in the majority of infected individuals. Taken together, the findings of this study open up a new facet in the pathogenesis of amebiasis and unravel a novel paradigm to study host-parasite interactions in the gut.

The heat shock response was studied in the intestinal parasitic protozoan Entamoeba histolytica. Temperature shifts from 37$ sp circ$C to 44$ sp circ$C enhanced the synthesis of five major heat shock (or stress) proteins (HSP) of 100, 50, 42, 37, and 28 kDa. Similarly, exposure of amebae to lymphokine activated macrophages and hydrogen peroxide caused HSP expression. Heat shock caused the reversible inhibition of amebic adherence to Chinese hamster ovary cells and human colonic mucin binding to trophozoites by ${>80 %}$. This was due to a decrease in the surface expression of the Gal/GalNAc adherence lectin and a marked reduction in the lectin mRNA expression. However, the presence of target Chinese hamster ovary cells during recovery at 37$ sp circ$C augmented amebic adherence. These results suggest that E. histolytica trophozoites produce a variety of HSP in response to different stimuli and can modulate the expression of the surface adherence lectin which maybe important in pathogenesis.

Thesis (Ph. D.)--University of Virginia, 1998. / Spine title: Control of E. histolytica lectin. Includes bibliographical references (p. 93-105). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Virginia, 2000. / Includes bibliographical references (leaves 102-114). Also available online through Digital Dissertations.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-11T12:22:59Z No. of bitstreams: 1 Karla Graziela. Avaliação enzimática...pdf: 7967639 bytes, checksum: aadcd19e00e6ff487dba2f6a89822b03 (MD5) / Made available in DSpace on 2013-10-11T12:22:59Z (GMT). No. of bitstreams: 1 Karla Graziela. Avaliação enzimática...pdf: 7967639 bytes, checksum: aadcd19e00e6ff487dba2f6a89822b03 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Entamoeba histolytica, o agente etiológico da amebíase, constitui a segunda maior causa de morte por protozooses, sendo considerado um grave problema de saúde pública, particularmente em países em desenvolvimento. A morte celular induzida por este parasito entérico é dependente de contato e mediada por proteínas específicas, as quais modulam diversos eventos fisiológicos no hospedeiro. Dentre os mecanismos reguladores de tais respostas está a desfosforilação de grupos fosfotirosil de proteínas, através da atuação de proteína tirosina fosfatases (PTPases), tanto expressas na superfície celular como secretados pelo trofozoíto. Alguns estudos demonstram uma rápida diminuição nos níveis de fosforilação de resíduos tirosina em proteínas de células-alvo após o contato com E. histolytica. Estas PTPases têm sido descritas como tendo importante papel na patogênese da amebíase. O objetivo deste estudo foi analisar o perfil de atividade fosfatásica em diferentes cepas de E. histolytica, assim como caracterizar a sua sensibilidade a conhecidos inibidores de proteína tirosina fosfatases, e os efeitos destes moduladores em mecanismos envolvidos na patogênese, a exemplo de fagocitose e efeito citopático. Inicialmente, a sensibilidade aos moduladores da via de sinalização celular em trofozoítos de E. histolytica (cepas ICB – 452, ICB – CSP e HM1:IMSS) foi caracterizada através de avaliação da proliferação celular, onde inóculos de parasitos foram incubados em presença ou ausência de inibidores de PTPases. Dentre os moduladores testados, o derivado de vanádio bisperoxo (1, 10 - fenantrolina) oxovanadato de potássio (bpVphen) e o óxido de fenilarsina (PAO) demonstraram efetiva capacidade antiproliferativa. Estas células apresentaram uma maior sensibilidade ao PAO em comparação ao bpV(phen), com valores de IC50 para a cepa HM1:IMSS de 0,9 μM e 38,4 μM, respectivamente. A análise bioquímica revelou que há uma maior atividade secretória e ectofosfatásica na linhagem HM1:IMSS (24,48 nM p-NP/40 min./3 x 106 células e 297 nM p-NP/40 min./2 x 105 células, respectivamente), e uma redução desta atividade foi detectada na presença dos derivados de vanádio na superfície do parasito (31,3 nM p-NP/40 min./2 x 105 células). O mesmo não foi observado após a adição de PAO. A análise da eritrofagocitose e da destruição da monocamada de células epiteliais da linhagem MDCK, importantes marcadores de virulência neste patógeno, demonstrou significativa redução destes processos em trofozoítos tratados com os inibidores ortovanadato de sódio (OVS) e o bpV(phen), indicando a participação de PTPases nos mesmos. Estas mesmas alterações foram observadas após o tratamento do parasito com PAO, porém dados ultraestruturais de citoquímica enzimática não indicam a redução da atividade fosfatásica por este composto. Estes dados foram confirmados após a avaliação da atividade das frações purificadas de homogenatos solubilizados de trofozoítos de E. histolytica, onde detectou-se a redução dos níveis de atividade enzimática após a adição de vanadato e seus derivados à reação, o que não pôde ser observado após o acréscimo de PAO. Os resultados obtidos indicam que PTPases estão diretamente envolvidas em importantes funções celulares exercidas por trofozoítos de Entamoeba histolytica. Ademais, novos estudos que visem à elucidação dos possíveis modos de ação do composto PAO neste patógeno poderiam contribuir, consideravelmente, com o entendimento da biologia do parasito e, consequentemente, dos mecanismos patogênicos da amebíase. / The microaerophilic protozoan Giardia lamblia inhabits the upper small intestine mucosa of vertebrate hosts, where it is exposed to different concentrations of oxygen. Despite the fermentative metabolism, Giardia trophozoites consume O2 and produce oxygen free radicals and therefore mechanism for detoxification are required. Devoid of glutathione, Giardia express high concentrations of cystein-rich proteins (CRP, also known as variable surface protein or VSP), as an antioxidant defense. This mechanism involves redox cycling for maintenance of a reduced intracellular environment and protection from oxidative stress. In this regard, substances that interfere in the antioxidant response of this protozoan could comprise a powerful chemotherapeutic strategy for Giardia lamblia infection. Here, we analyzed the effects of DETC, a superoxide dismutase (SOD) inhibitor, on parasite proliferation, thiol expression, lipid peroxidation, free radicals detection and cell architecture. DETC inhibited parasite proliferation at levels similar to metronidazole and induced peroxidation of membrane, possibly by the increase of reactive species.Ultrastructural alteration were also observed. Since this protozoan is devoid of SOD, here present data indicate SOD-independ DETC effects. Thiol groups detection with the fluorescent probe o-phthaldialdehyde (OPA). Cells treated with DETC displayed washed out cytoplasm and structures indicative of autophagy. The peripheral vesicles also had an increased volume, presumably caused by homophilic fusion. Taken together these data indicate that DETC enhance the oxidative stress in Giardia trophozoites by reacting with thiol groups.

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