Entamoeba histolytica trophozoites were inoculated into the liver of hamsters and serum nitrate/nitrite levels [expressed as nitric oxide (NO) production] were determined at different times during amebic liver abscess (ALA) development. We also tested the effects of NO synthase (NOS) inhibitors such as N(G)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine, and dexamethasone during ALA production. Since NOS activity has been correlated with expression of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) in tissues, we performed histochemistry studies to determine the activity of the latter in livers infected with E. histolytica trophozoites. Production of NO in serum was directly proportional to the size of ALAs, and NOS inhibitors caused low levels of NO and smaller ALAs. Our data suggest that NO does not have any lytic effect on E. histolytica trophozoites and is therefore incapable of providing protection against the amebic liver infection. In addition, NADPHd activity was detected histochemically in hepatocytes and inflammatory cells associated with focal necrosis containing trophozoites. The positive reactivity observed in these parasites may be attributable to a close biochemical similarity of NADPHd to the NADPH:flavin oxidoreductase described in E. histolytica by other investigators.

We studied the early in situ interactions of live and fixed Entamoeba histolytica trophozoites with hamster hepatic parenchymal and inflammatory cells using immunoperoxidase and immunoelectronmicroscopy. Close contact between trophozoites and endothelial cells and the diffusion of amoebic molecules from trophozoites towards nearby endothelial cells and distant hepatocytes were observed. The inflammatory cells around the amoebae and the remnants of parenchymal cells and hepatocytes located close to the lesion had a positive stain for amoebic molecules. In the amoebae, at the ultrastructural level, molecules were attached to the membranes and inside the vesicles. These molecules were apparently released into the space formed between the parasite and the endothelial cells. The endothelial cells and the nearby and distant hepatocytes captured amoebic molecules, and later they became necrotic. Contrarily, when fixed amoebae were inoculated, amoebic molecules were captured by endothelial cells and polymorphonuclear (PMN) leukocytes, but neither suffered any damage. In this work, we are presenting evidence clearly showing that some molecules of the amoeba can diffuse away long distances causing cytotoxic effects and even necrosis on hepatic cells of hamster liver without the need of the trophozoite being in close contact with the target cells. They also may promote lytic or proinflammatory effects by inducing the secretion of enzymes or cytokines in other nonparenchymal cells, like PMN leukocytes and endothelial cells. Our results suggest that the accepted mechanisms of cytotoxicity by amoebae are not exclusively restricted to the following sequence: adhesion, phagocytosis, and necrosis.

Amoebic liver abscesses (ALA) are the most frequent and severe extraintestinal clinical presentations of amoebiasis. During the early establishment of amoebae in the liver parenchyma, as well as during the extension of the tissue necrosis, parasites interact with the parenchymal liver cells and, as a consequence of these interactions, hepatocytes can be destroyed and host immune cells can become activated. However, little is known about the nature of these interactions in the liver or about the factors involved in the local immune response. In this investigation we studied the localization of Entamoeba histolytica trophozoites, TCD4+, TCD8+ cells, CD68+ macrophages and CD15+ neutrophils in human ALA using immunohistochemical techniques. Trophozoites were found close to undamaged hepatocytes in both lysed and non-lysed areas with either sparse or abundant inflammatory infiltrate. CD8+ cells were more abundant than CD4+ T cells. CD 68+ macrophages and CD15+ neutrophils were also detected, suggesting that neutrophils, macrophages and T cells might be related to the local host immune mechanisms in ALA. We also found that E. histolytica possesses proteins recognized by antibodies raised against inducible nitric oxide synthase.

Using immunocytochemical techniques, we studied the interaction of antibodies with Entamoeba histolytica trophozoites present during the development of amebic liver abscess. Hamsters were intrahepatically inoculated with HM1-IMSS axenic amebas and sacrificed at different days post-inoculation. IgG of rabbit anti-E. histolytica and IgG of rabbit anti-IgG of hamster were used, both labeled with peroxidase. With the rabbit anti-E. histolytica, all trophozoites present in hepatic lesions from 1-7 days post-inoculation were highly labeled. The IgG of rabbit anti-IgG of hamster intensively stained only those trophozoites present in lesions from 1-2 days post-inoculation. From day 3, the intensity and number of labeled trophozoites decreased progressively. The results suggest that the interaction between the amebas and the IgG of hamster is non-specific during the first 2 days. The absence of labeling in the chronic stages could be due to changes in the membrane antigens of the parasite or to alterations in the bloodstream around necrosis. Also, the anti-E. histolytica antibodies produced in the serum during the development of the hepatic disease are apparently incapable of reaching and interacting with the trophozoites present on the liver abscess. This can explain in part why antibodies do not have an important role in the defense of the host.