The innate immune response to Entamoeba histolytica lipopeptidophosphoglycan is mediated by toll-like receptors 2 and 4.

Entamoeba histolytica is a human pathogen that may invade the intestinal mucosa, causing amoebic colitis or hepatic abscesses when the trophozoites travel through the portal circulation to the liver. Lipopeptidophosphoglycan (LPPG) is a molecular pattern of E. histolytica recognized by the human immune system. Here we report that LPPG is exposed on the cell surface of E. histolytica trophozoites, and is recognized by the host through toll-like receptor (TLR) 2 and TLR4. Correspondingly, human embryonic kidney (HEK)-293 cells were rendered LPPG responsive through overexpression of TLR2 or TLR4/MD2. Moreover, co-expression of CD14 enhanced LPPG signal transmission through TLR2 and TLR4. The interaction of LPPG with TLR2 and TLR4 resulted in activation of NF-kappaB and release of interleukin (IL)-10, IL-12p40, tumour necrosis factor (TNF)-alpha, and IL-8 from human monocytes. Consistent with these findings, responsiveness of mouse macrophages lacking TLR2 expression (TLR2-/-) or functional TLR4 (TLR4d/d) to E. histolytica LPPG challenge was impaired while double deficient macrophages were unresponsive. In contrast to wild-type control and TLR2-/- animals succumbing to lethal shock syndrome, TLR4d/d mice were resistant to systemic LPPG challenge-induced pathology.

Surface properties and in vitro cytopathic effect of various strains of Trichomonas vaginalis.

The in vitro cytopathic effect of four strains of Trichomonas vaginalis on cultured epithelial monolayers was analyzed through electrophysiology and electron microscopy. Interaction of trichomonads of two virulent strains (GT-10 and GT-13) with cultured MDCK cell monolayers mounted in Ussing chambers produced a rapid decrease in transepithelial resistance to less than 30% of control values after only 15 min. By 30 min the electrical resistance was practically abolished by the virulent parasites. In contrast, of two attenuated strains of trichomonads (GT-3 and GT-7) analyzed under similar conditions, GT-3 trophozoites required 180 min to reduce transepithelial resistance to 9% of control values, while monolayers in contact with GT-7 parasites still showed 28% of control values at this time of incubation. Sequential scanning electron microscopy confirmed the much faster and widespread cytopathic effect of virulent parasites. In contrast, the slow lytic process produced by attenuated trophozoites was reduced to focal areas of direct contact with epithelial cells. Another difference was found by measurement of the surface charge of the four strains of T. vaginalis by means of cell microelectrophoresis. While the two virulent strains showed a negative surface charge, the two attenuated strains had no detectable surface charge at neutral pH. When parasites were incubated with cationized ferritin and studied with transmission electron microscopy the surface of virulent trichomonads appeared heavily labeled, whereas the surface of attenuated parasites had only sparse and irregular ferritin binding.

Giardia lamblia: effect of infection with symptomatic and asymptomatic isolates on the growth of gerbils (Meriones unguiculatus).

Gerbils (Meriones unguiculatus) were intragastrically inoculated with axenic Giardia lamblia cultures from symptomatic and asymptomatic children. All isolates were able to colonize the duodenum. However, the colonization capacity of the symptomatic isolates was significantly higher compared to that of the asymptomatic ones. Despite the different colonization capacity of the isolates, the growth curves of infected animals were significantly lower than those of controls. The study demonstrates that acute giardia infections are capable of altering the corporal development of the host. These results may suggest that not only symptomatic, but also asymptomatic giardiasis in children, often unnoticed by parents and clinicians, could be causing a silent detriment in their nutritional status.

Pathogenesis of intestinal amebiasis: from molecules to disease.

In spite of a wealth of knowledge on the biochemistry and cellular and molecular biology of Entamoeba histolytica, little has been done to apply these advances to our understanding of the lesions observed in patients with intestinal amebiasis. In this review, the pathological and histological findings in acute amebic colitis are related to the molecular mechanisms of E. histolytica pathogenicity described to date. Infection of the human colon by E. histolytica produces focal ulceration of the intestinal mucosa, resulting in dysentery (diarrhea with blood and mucus). Although a complete picture has not yet been achieved, the basic mechanisms involved in the production of focal lytic lesions include complex multifactorial processes in which lectins facilitate adhesion, proteases degrade extracellular matrix components, porins help nourish the parasite and may also kill incoming polymorphonuclear leukocytes and macrophages, and motility is used by the parasite to invade deeper layers of the colon. In addition, E. histolytica has developed mechanisms to modulate the immune response during acute infection. Nevertheless, much still needs to be unraveled to understand how this microscopic parasite has earned its well-deserved histolytic name.

Entamoeba dispar: ultrastructure, surface properties and cytopathic effect.

The cytological features of Entamoeba dispar, recently recognized by biochemical and molecular biology criteria as a distinct species, were compared to those of Entamoeba histolytica When cultured under axenic conditions, living trophozoites of E. dispar strain SAW 76ORR clone A were more elongated in form, had a single frontal pseudopodium, and showed a noticeable uroid. In sections of E. dispar trophozoites stained with Toluidine blue, characteristic areas of cytoplasmic metachromasia were seen due to the presence of large deposits of glycogen, seldom found in E. histolytica strain HM1:IMSS. Under the light microscope the periphery of the nucleus in E. dispar was, lined by finer, more regularly distributed dense granules. With transmission electron microscopy the surface coat of E. dispar was noticeable thinner. In addition. E. dispar had a lower sensitivity to agglutinate with concanavalin A and a higher negative surface charge, measured by cellular microelectrophoresis. The cytopathic effect of E. dispar was much slower, analyzed by the gradual loss of transmural electrical resistance of MDCK epithelial cell monolayers mounted in Ussing chambers. Whereas in E. histolytica phagocytosis of epithelial cells plays an important role in its cytopathic effect. E. dispar trophozoites placed in contact with MDCK cells showed only rare evidence of phagocytosis. The results demonstrate that the morphology of E. dispar is different to that of E. histolytica, both at the light microscopical and the ultrastructural levels. In addition they show that E. dispar in axenic culture has a moderate cytopathic effect on epithelia] cell monoLayers. However, when compared to E. histolytica, the in vitro lytic capacity of E. dispar is much slower and less intense.

Trichomonas vaginalis: in vitro phagocytosis of lactobacilli, vaginal epithelial cells, leukocytes, and erythrocytes.

This paper explores the interaction of two strains of Trichomonas vaginalis, of high and low virulence, with the cell types present in the microenvironment of the parasite during human infections. With the use of transmission and scanning electron microscopy the sequence of internalization by T. vaginalis of Döderlein's lactobacilli, and of vaginal epithelial cells, leukocytes, and erythrocytes was documented. Furthermore, the degradation of ingested material by colocalization of acid phosphatase activity in phagocytic vacuoles was demonstrated. Phagocytosis of all cell types analyzed was found in both strains studied, although the highly virulent strain internalized target cells more rapidly than the less virulent one. Ultrastructural evidence indicated that phagocytosis takes place through two distinct mechanisms, only one involving the formation of a phagocytic stoma, characteristic of professional phagocytes. T. vaginalis phagocytosis may be both an efficient means of obtaining nutrients for the parasite and a significant factor in the pathogenesis of trichomonal infections of the human genitourinary tract.

Entamoeba histolytica: liver invasion and abscess production by intraperitoneal inoculation of trophozoites in hamsters, Mesocricetus auratus.

Intraperitoneal inoculation of axenically cultured Entamoeba histolytica trophozoites constitutes an easy to perform, highly reproducible procedure for inducing amebic liver abscesses in hamsters. Efficiency in abscess production (95% of infected animals after 1 week) was similar to data reported using direct intrahepatic or intraportal inoculation. The morphological sequence of infection shows that amebas in the peritoneal cavity initially produce a large exudate constituted mainly of acute inflammatory cells. These cells form a rim of polymorphonuclear leukocytes surrounding the amebas, which adhere to the trophozoite and can sometimes be observed polarized to one end of the parasite, suggesting capping of surface receptors. Early stages are also characterized by the production of distant inflammatory reactions in the hepatic portal spaces. At 6 h postintraperitoneal inoculation, larger foci of inflammatory reactions surrounding amebas are developed in the peritoneum, extending to and damaging the liver surface membranes as well as the serosa of other internal organs. Thereafter, tissue damage progresses deeper into the liver parenchyma, and a few days later, coalescing granulomas and large necrotic areas are observed in the liver tissue. Based on the present morphological time-sequence study, we suggest that inflammatory cells associated with E. histolytica trophozoites play an important role in commencing the damage of liver sheaths and producing the subsequent parenchymal lesions. The simplicity and reliability of this model are important factors to consider when large numbers of experimentally induced amebic liver abscesses are needed.

Trichomonas vaginalis: ultrastructural bases of the cytopathic effect.

The in vitro cytopathic effect of Trichomonas vaginalis on epithelial cells was explored through the interaction of trophozoites of the virulent strain GT-10 with MDCK monolayers. The interaction was analyzed through electrophysiology, video microscopy, and transmission and scanning electron microscopy. Electrical measurements revealed that living parasites produced severe damage to the cell monolayers within 30 min, manifested as a rapid decrease in transepithelial resistance. Microscopic observations demonstrated that when placed in contact with epithelial cells, trichomonas formed clumps through interdigitations and transient plasma membrane junctions between adjacent parasites. Also, attached trophozoites adopted an ameboid shape. The in vitro cytopathic action of T. vaginalis on MDCK cells was initially evident by modifications of the plasma membrane, resulting in opening of tight junctions, membrane blebbing, and monolayer disruption. After 15 min of interaction the damage was focal, concentrating at sites where parasite clumps adhered to the monolayer. At 30 min practically all MDCK cells were dead, whether or not trichomonas were attached to them. These events were followed by detachment of lysed cells and complete disruption of the monolayer at 60 min. Electron microscopy demonstrated a peculiar form of adhesion that appears to be specific for trichomonas, in which the basal surface of T. vaginalis formed slender channels through which microvilli and cytoplasmic fragments of epithelial cells were internalized. The same sequence of lytic events was found with the less virulent GT-3 strain. However, the time course of cytolysis with GT-3 parasites was much slower, and lysis was limited to areas of attachment of T. vaginalis.

Entamoeba histolytica: mechanism of surface receptor capping.

Binding of antibodies to the surface of the parasitic protozoan Entamoeba histolytica induces capping and release of antigen-antibody complexes, in a process that may be an effective means of evading the host immune response. To analyze the mechanism of capping in E. histolytica trophozoites, optimal conditions were determined for cap induction by sera from patients with amebic liver abscess, by polyclonal anti-amebic antibodies, and by concanavalin A. Cocapping of actin and myosin was documented by immunofluorescent staining of capped amebas using phalloidin, monoclonal anti-actin antibodies, and polyclonal anti-myosin antibodies. Immunoblots confirmed the presence of actin and myosin in isolated caps. These observations demonstrate that amebic cytoskeletal components participate in E. histolytica capping of surface receptors.

Effects of albendazole on Entamoeba histolytica and Giardia lamblia trophozoites.

Albendazole, a benzimidazole carbamate commonly used for the treatment and control of intestinal helminthic infections, is also useful for the treatment of giardiasis. Therefore, it is of interest to determine whether the drug has activity against other intestinal protozoa, such as E. histolytica. The present results demonstrate that albendazole inhibits the growth of E. histolytica trophozoites in axenic cultures and induces fine structural changes such as polyribosome aggregation and loss of cytoplasmic vacuoles at concentrations up to 10 micrograms/ml. The viability of E. histolytica trophozoites was not affected by the drug. In contrast, lower concentrations of albendazole showed dramatic effects on G. lamblia trophozoites. These included loss of adhesiveness, striking modifications of the overall morphology of giardias, disassembly of the ventral disk, and loss of viability after prolonged treatment. The results provide further evidence on the potent antigiardial activity of albendazole and indicate that, at the concentrations used, the drug has no antiamebic activity.

Entamoeba histolytica: electrophoretic analysis of isolated caps induced by several ligands.

Caps induced by several ligands including sera from patients with amebic liver abscess (ALA) were isolated by differential centrifugation. Purification was verified by scanning and transmission electron microscopy. Electrophoretic profiles obtained by SDS-PAGE and Western blots were compared when probed with the ligand used to induce capping. It was confirmed that sera from different patients recognize different proteins. Indeed, electrophoretic profiles reveal that capped membrane proteins vary according to the ligand used to induce capping. This would agree with the supposition that E. histolytica might use this process as a mechanism to evade the host's immune response, by clearing the cell surface from recognized antigens.