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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Introduction: An amebic liver abscess is the most common presentation of extraintestinal amebiasis. This condition is the result of a parasite infection caused by Entamoeba histolytica. Materials and Methods: We report a case of a 53-year-old male who presented with abdominal pain in the right upper quadrant, jaundice, and a 10-kg weight loss within a 1-month span. Results and Conclusion: A wide range of symptoms and findings in the imaging tests suggestive of neoplasia, elevated levels of CA 19-9 and CA 125, and the presentation of biliary peritonitis as a complication makes this case a challenge for its approach and management. / Revisión por pares

Thesis (M.S.)--Marshall University, 2008. / Title from document title page. Includes abstract. Document formatted into pages: contains xiii, 126 p. Includes bibliographical references p. 113-118.

Invasive amebiasis, caused by the protozoan parasite Entamoeba histolytica, is one of the leading parasitic causes of mortality worldwide, and there are no vaccines available to control the disease. The heavy subunit of the E. histolytica Gal-lectin is regarded as a potential subunit vaccine candidate. A Th1 (cell-mediated) immune response is protective against invasive amebiasis, and DNA vaccination is a strategy to induce such a response against specific antigens. The objective of this study was to construct and test a Gal-lectin-based DNA vaccine against E. histolytica. DNA encoding as 894--1081 of the Gal-lectin heavy subunit was resynthesized using a gerbil codon frequency bias and inserted in a mammalian expression vector to generate the DNA vaccine pCISToGL6. Balb/c mice vaccinated intradermally developed a Gal-lectin-specific cellular immune response, as well as an anti-Gal-lectin humoral immune response. Serum antibodies recognized a recombinant portion of the Gal-lectin heavy subunit by immunoblot and ELISA, and bound to native Gal-lectin on the surface of live trophozoites, inhibiting adherence to target cells. The Gal-lectin-specific serum antibodies were of the IgG2a isotype, indicating that a Th1 response was stimulated by the vaccine. We were also interested in using DNA encoding IL-12, IL-18 or GM-CSF as genetic adjuvants co-injected with pCISToGL6 to potentiate the immune response. Since the DNA vaccine was destined to confer protection in the gerbil model of invasive amebiasis, we cloned gerbil IL-12 (p35 and p40), IL-18 and its convertase caspase-1, and GM-CSF. The proteins were expressed in mammalian cells and showed bioactivity in vitro. Taken together, these results have laid the foundation to optimize and test a working Gal-lectin with co-stimulatory molecules to elicit a Th1 immune response for protective immunity against invasive amebiasis.

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.