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The intestinal protozoan parasite Entamoeba histolytica affects 50 million people worldwide, causing 100,000 deaths per year (1). Intestinal amebiasis is characterized by colitis and severe dysentery. Despite our knowledge of the role of several pathogen and host factors in causing colonic inflammation, the cell-specific response to amebic infection is poorly understood. Moreover, the majority of research done to unravel the mechanism of amebic colitis has been focused on proinflammatory responses (2, 3) by epithelial cells and a role of NF-B (4) and chemokines such as IL-8 3 (5) as triggering events for inflammation. However, it is noteworthy that only 10% of E. histolytica-infected individuals show symptoms of intestinal inflammation (6), and none of the studies addressed the question of why only a minority of infected individuals develops amebic colitis.

The Gal/GalNAc lectin (Gal-lectin) of Entamoeba histolytica is a surface molecule involved in parasite adherence to host cells and is the most promising subunit vaccine candidate against amoebiasis. As macrophages are the major effector cells in host defense against amoebas, we studied the molecular mechanisms by which Gal-lectin activates macrophage. Microarray analysis showed that Gal-lectin up-regulated mRNAs of several cytokines and receptor genes involved in proinflammatory responses. The mechanism whereby the Gal-lectin regulates Toll-like receptor 2 (TLR-2) expression in macrophages was studied. Native Gal-lectin increased TLR-2 mRNA expression in a dose- and time-dependent fashion; peak response occurred with 1 microg/ml after 2 h stimulation. By immunoflourescence, enhanced surface expression of TLR-2 was observed after 12 h. With the use of nonoverlapping anti-Gal-lectin monoclonal antibodies that map to the carbohydrate recognition domain, amino acid 596-1082 was identified as the TLR-2 stimulating region. The Gal-lectin increased TLR-2 gene transcription, and the half-life of the mRNA transcripts was 1.4 h. Inhibition of nuclear factor (NF)-kappaB suppressed TLR-2 mRNA induction by the Gal-lectin. Moreover, cells pretreated with an inhibitor of p38 kinase (SB 208530) inhibited Gal-lectin induced TLR-2 mRNA expression by 40%. We conclude that the Gal-lectin activates NF-kappaB and MAP kinase-signaling pathways in macrophages culminating in the induction of several genes including TLR-2 and hypothesize that this could have a significant impact on macrophage activation and contribute to amoebic pathogenesis.

BackgroundWe have evaluated the therapeutic efficacy of AIGIV when given in combination with levofloxacin and the effective window of treatment to assess the added benefit provided by AIGIV over standard antibiotic treatment alone in a New Zealand white rabbit model of inhalational anthrax.MethodsRabbits were exposed to lethal dose of aerosolized spores of Bacillus anthracis (Ames strain) and treated intravenously with either placebo, (normal immune globulin intravenous, IGIV) or 15 U/kg of AIGIV, along with oral levofloxacin treatment at various time points (30–96 hours) after anthrax exposure.ResultsThe majority of treated animals (>88%) survived in both treatment groups when treatment was initiated within 60 hours of post-exposure. However, reduced survival of 55%, 33% and 25% was observed for placebo + levofloxacin group when the treatment was initiated at 72, 84 and 96 hours post-exposure, respectively. Conversely, a survival rate of 65%, 40% and 71% was observed in the AIGIV + levofloxacin treated groups at these time points.ConclusionsThe combination of AIGIV with antibiotics provided an improvement in survival compared to levofloxacin treatment alone when treatment was delayed up to 96 hours post-anthrax exposure. Additionally, AIGIV treatment when given as an adjunct therapy at any of the time points tested did not interfere with the efficacy of levofloxacin.

The role intestinal epithelial cells play in the pathogenesis of amebic colitis is poorly understood. Herein, we demonstrate that secreted and soluble ameba (Entamoeba histolytica) proteins (SAP) induce expression of the chemoattractant monocyte chemotactic protein (MCP) in the colonic epithelial cell lines Caco-2, T84, and LS174T. MCP-1 mRNA induction was both dose and time dependent, with peak induction occurring at 8 h and with 100 g/ml of SAP. Significant increase in MCP-1 protein expression was observed after 12 h. SAP failed to activate any of the mitogen-activated protein kinase pathways or IB kinase activity. Moreover, inhibiting the classical pathway of NF-B activation did not affect SAP-induced MCP-1 expression. Instead, we find that SAP-induced MCP-1 expression is dependent on posttranslational modification of the NFB p65 subunit. SAP induced phosphorylation of p65 and enhanced NF-B transcriptional activity, which are phosphatidylinositol 3-kinase (PI3 kinase) dependent. Treatment with PI3 kinase inhibitor LY290004 significantly abrogated the activation of Akt, p65, and MCP-1 mRNA induction. We conclude that colonic epithelial cells play a role in the initiation of inflammation by secreting chemokines in response to soluble ameba components.Entamoeba histolytica is an enteric protozoan parasite that is responsible for the disease amebiasis in humans. The disease affects 50 million people globally and is the fourth leading parasitic cause of death (14). Host inflammatory responses are thought to play an important role in the pathogenesis of intestinal amebiasis. However, the roles of intestinal epithelial cells (IEC) and mediators of colonic inflammation have not been fully elucidated.Invasive amebiasis is characterized by infiltration of immune cells such as leukocytes and lymphocytes (5). It has not been well established if this cellular infiltration is a cause or consequence of inflammation. The parasite has previously been shown to elicit interleukin-8 (IL-8) production from colonic epithelial cells (4, 15). IL-8 is a potent chemoattractant primarily for neutrophils, and its secretion alone does not explain the homing of other immune cells such as monocytes and lymphocytes seen in the amebic lesions (5). Monocyte chemotactic protein 1 (MCP-1) belongs to a group of C-C or ␤-chemokines, is produced by a variety of cells, including IEC, and is potently chemotactic for monocytes, lymphocytes, and basophils (10). Recently, amebic infection in a human intestinal xenograft model has been shown to increase a number of genes in the epithelial cells, including the MCP-1 gene (16). However, the mechanism of induction is not known and also it is not clear if ameba components themselves can directly induce this chemokine.The transcription factor NF-B regulates a number of genes involved in immune response and inflammation. It is composed of two subunits, most commonly of p65 and P50. Only p65 has the transactivating domains and hence is critical for NF-B activity. NF-B is activated by different pathways, includ...