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Delivery of antigens by oral route is considered as the mainly strategy of immunization against enteric pathogens. However, it could result in the induction of oral tolerance, a state of unresponsiveness that could exacerbate the infection. Entamoeba histolytica is the enteric protozoan parasite responsible for the highest levels of morbidity and mortality worldwide. With the aim of induce a secretory immune response against amebic antigens, we immunized a group of C3H/HeJ mice by oral route with a 29 kDa cysteine-rich protein recognized by human salivary IgA fused to the cholera toxin subunit B (CREHP-CTxB). Mice were immunized once a week during a month with 100 ug of CREHP/CTxB in presence of bicarbonate. Two control groups of mice received CTxB and PBS, respectively. Serum and fecal samples were collected before immunization and the day of sacrifice, 7 days after the last dose. Analysis of the local and systemic humoral responses against the fused protein by ELISA showed the raising of both secretory IgA antibodies in feces and high levels of IgM antibodies in sera of immunized mice but not in the controls groups. However, the analysis of the cellular response by spleen cell proliferation and incorporation of 3H-thymidine, showed that T cell from the mice immunized with CREHP/CTxB were unable to respond to Con A and anti-CD3 antibodies. By contrast, T cells from control groups showed high levels of proliferation under those stimulus. The results suggest the generation of an oral tolerance of the suppressive type with an E. histolytica antigen fused to CTxB. [ABSTRACT FROM AUTHOR]

More than one quarter of human world's population is exposed to intestinal helminth parasites. The Taenia solium tapeworm carrier is the main risk factor in the transmission of both human neurocysticercosis and porcine cysticercosis. Sex steroids play an important role during T. solium infection, particularly progesterone has been proposed as a key immunomodulatory hormone involved in susceptibility to human taeniosis in woman and cysticercosis in pregnant pigs. Thus, we evaluated the effect of progesterone administration upon the experimental taeniosis in golden hamsters (Mesocricetus auratus). Intact female adult hamsters were randomly divided into 3 groups: progesterone-subcutaneously treated; olive oil-treated as the vehicle group; and untreated controls. Animals were treated every other day during 4 weeks. After 2 weeks of treatment, all hamsters were orally infected with 4 viable T. solium cysticerci. After 2 weeks post infection, progesterone-treated hamsters showed reduction in adult worm recovery by 80%, compared to both vehicle-treated and non-manipulated infected animals. In contrast to control and vehicle groups, progesterone treatment diminished tapeworm length by 75% and increased proliferation rate of leukocytes from spleen and mesenteric lymph nodes of infected hamsters by 5-fold. The latter exhibited high expression levels of IL-4, IL-6 and TNF-α at the duodenal mucosa, accompanied with polymorphonuclear leukocytes infiltration. These results support that progesterone protects hamsters from the T. solium adult tapeworm establishment by improving the intestinal mucosal immunity, suggesting a potential use of analogues of this hormone as novel inductors of the gut immune response against intestinal helminth infections and probably other bowel-related disorders.

Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.

Entamoeba histolytica antigens recognized by salivary IgA from infected patients include the 29 kDa antigen (Eh29), an alkyl hydroperoxide reductase. Here, we investigate the potential of recombinant Eh29 and an Eh29-cholera toxin subunit B (CTxB) fusion protein to confer protection against intestinal amoebiasis after oral immunization. The purified Eh29-CTxB fusion retained the critical ability to bind ganglioside GM(1), as determined by ELISA. Oral immunization of C3H/HeJ mice with Eh29 administered in combination with a subclinical dose of whole cholera toxin, but not as an Eh29-CTxB fusion, induced elevated levels of intestinal IgA and serum IgG anti-Eh29 antibodies that inhibited trophozoites adherence to MDCK cell monolayers. The 80% of immunized mice seen to develop IgA and IgG immune responses showed no evidence of infection in tissue sections harvested following intracecal challenge with virulent E. histolytica trophozoites. These results suggest that Eh29 is capable of inducing protective anti-amoebic immune responses in mice following oral immunization and could be used in the development of oral vaccines against amoebiasis., ((c) 2010 Elsevier Inc. All rights reserved.)

During early experimental amebic liver abscess in hamsters (EALAH), acute inflammation is primarily responsible for tissue damage. However, during the late stages of this process, the relative contribution to tissue destruction of both parasite factors and host response is unknown. In the present work, the role of the cellular immune response in tissue damage during EALAH is explored by using the immunosuppressor drug cyclosporine A (CsA). CsA treatment inhibits tissue damage after 72 h (but not at 24 h). Also, many well-preserved parasite clusters with minimal or no leukocyte influx and with minimal or no tissue destruction characterize the late stage of the process (7 days). The same results are observed with the immunosuppressor tacrolimus, but not with sirolimus; the latter drug does not cause immunosuppression in hamsters. On the other hand, similar results are observed with the antioxidant and anti-inflammatory N-acetylcysteine, with minimal immunosuppression in hamsters. These results suggest that, as in the early EALAH (24 h), during the late stages of the process (7 days), inflammation is also primarily responsible for tissue damage. However, lysosomal and cationic proteins are responsible for the early lesions, whereas reactive oxygen and nitrogen species are primarily involved in late stages.

To identify the Entamoeba histolytica antigens capable of inducing secretory IgA (sIgA) responses in humans, a cDNA library from the strain HM1:IMSS was immuno-screened with saliva from patients with intestinal amebiasis or amebic liver abscess. Clones isolated with sIgA antibodies from patients with intestinal amebiasis corresponded to the known serine-rich protein isoform, a 29 kDa cysteine-rich protein and 1-alpha elongation factor. Clones corresponding to enolase, cyclophilin, ribosomal protein L23a, and an Hsp70 family protein were isolated with sIgA from a patient with amebic liver abscess. A glutamic acid-rich peptide (EhGARP) positive with sIgA from a patient with amebic liver abscess was also isolated; for EhGARP, no homologs were found in the protein databases. The antigens isolated are potentially useful in the development of an oral vaccine or new diagnostic tools for amebiasis.

The present study analyzes the effects of zinc on Entamoeba histolytica activity and on its pathogenicity. Metal activity was evaluated in vitro with regard to the parasite's viability, replication, and adhesion to epithelial cells and in vivo with regard to its pathogenicity. The results obtained in vitro show that zinc at 1.0 mM concentration does not affect amebic viability; however, it does decrease amebic replication and adhesion (P < 0.001). In vivo studies performed on a model of experimental liver abscess in the hamster indicate that the intraperitoneal administration of a single dose of zinc at 48 h after the intrahepatic inoculation of amebic trophozoites significantly inhibits (P < 0.001) abscess development. The results indicate that zinc alters the functionality of the ameba in vitro as reflected by a decrease in replication and adhesion and in vivo as manifested by inhibition of amebic pathogenicity.

The secretory immune response in saliva from intestinal amoebiasis patients against antigens obtained from Entamoeba histolytica membranes was studied. Western blot analysis indicated that patient saliva contains secretory IgA antibodies against antigens with molecular masses ranging from 170 to 24 kDa, some of which were also recognized by saliva from healthy subjects. However, antigens of 170, 125, 46 and 37 kDa are recognized more frequently (> 90%) by the secretory IgA from patients with intestinal amoebiasis than by that from healthy subjects (< 10%).

Amebiasis is one of the main causes worldwide of morbidity and mortality by parasites. Application of recombinant DNA technology to the study of Entamoeba histolytica is bringing new light into our understanding of this remarkable protozoan parasite and of the disease it causes. New achievements affect the way we approach many essential questions about E. histolytica, from the mechanism of its pathogenicity to the definition of E. histolytica as a separate species from the nonpathogenic E. dispar. To give a single example, transfection of trophozoites is now possible and a new generation of studies taking advantage of this capability of manipulation is expected in the short term. Our goal with this review is to provide an updated and simple guide to the growing information on the molecular biology of E. histolytica.