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PubMed2007

Activation of dendritic cells by the Gal-lectin of Entamoeba histolytica drives Th1 responses in vitro and in vivo.

Amebiasis is a human disease caused by the protozoan intestinal parasite Entamoeba histolytica. Vaccine development has focused on the parasite's surface galactose-N-acetyl-D-galactosamine inhibitable lectin (Gal-lectin) as a protective antigen. The Gal-lectin is immunogenic and has been shown to induce Th1 cytokines in vitro and in vivo. The immunological basis of the protective immune response elicited by the Gal-lectin is unknown. In this study, we investigated the response of BALB/c bone marrow-derived DC to E. histolytica Gal-lectin. Incubation of immature DC with Gal-lectin resulted in activation and maturation after 24 h. FACS analysis demonstrated an up-regulation of DC maturation markers CD80, CD86, CD40 and MHC class II upon exposure to Gal-lectin. The Gal-lectin also induced DC production of IL-12, indicating a Th1 response. Gal-lectin-activated DC were able to stimulate T cell proliferation in an allogeneic mixed leukocyte reaction and adoptive transfer of Gal-lectin-treated DC into naïve mice resulted in IFN-gamma-producing Gal-lectin-sensitized T cells. The activation of DC by Gal-lectin was mediated by MAPK and NF-kappaB. These findings indicate that E. histolytica Gal-lectin is a potent vaccine antigen capable of directly initiating DC maturation and activation characterized by Th1 cytokine production.

Adoptive Transfer↗
PubMed2006

Differentiation of Entamoeba histolytica from Entamoeba dispar by PCR-coupled nonisotopic SSCP analysis.

Entamoeba histolytica is a pathogenic protozoan parasite, which causes amoebic colitis, dysentery and liver abscesses in humans. Since the cyst and small trophozoite stages of this parasite are indistinguishable by light microscopy from Entamoeba dispar (which is nonpathogenic), specific diagnosis is compromised. To overcome this limitation, a PCR-coupled SSCP approach, utilising a sequence difference of 4.6% in a short region ( approximately 173-174 bp) of the small subunit of nuclear ribosomal DNA, was evaluated for the differentiation of the two species of Entamoeba. Including a range of well-defined control DNA samples (n = 67) to evaluate the specificity of the PCR, 45 DNA samples representing E. histolytica and E. dispar from human faecal samples were tested by SSCP, and unequivocal delineation between the species was achieved. This SSCP approach should provide a practical tool for the specific diagnosis of E. histolytica in humans and for investigating its epidemiology.

Animals↗
PubMed1990

Evaluation of the ELISA test for detection of Entamoeba histolytica in feces.

The clinical utility of an ELISA test with monoclonal antibodies to detect antigen of Entamoeba histolytica in feces was evaluated in 150 patients with gastrointestinal symptoms. Each subject was examined by rectosigmoidoscopy with rectal smear and/or a triple stool search for ova-bacteria-parasite (OBP); in addition, one stool sample was collected for the ELISA test. All the tests were independent and double blind. E. histolytica was detected by OBP and/or rectosigmoidoscopy in 66 patients; 61 patients had other parasites; and in 23, no parasites were identified. Of all patients, 116 were positive for the ELISA test. Of these, E. histolytica was identified in 52. In 47, other parasites were identified and in 17, no parasites were found. The ELISA test with a monoclonal antibody against E. histolytica antigen showed higher sensitivity than the standard diagnostic methods: the ability to detect the presence of E. histolytica antigen regardless of the destruction of the parasite or of the error due to misidentification of the parasite resulting from faulty preparation of the samples.

Adolescent↗
PubMed2003

Antiprotozoal activity of the constituents of Teloxys graveolens.

Antiprotozoal activity-guided fractionation of a methanol extract of the aerial parts of Teloxys graveolens led to the isolation of one coumarinic acid derivative, melilotoside, and five flavonoids, pinocembrine, pinostrobin, chrysin, narcissin and rutin. Among them, melilotoside exhibited the most potent activity toward Entamoeba histolytica and Giardia lamblia (IC(50) 12.5 and 16.8 micro g/mL, respectively). Narcissin showed selectivity against E. histolytlica (IC(50) 17.2 micro g/mL). The results support data for the traditional use of T. graveolens in some gastrointestinal diseases.

Amaranthaceae↗
PubMed2001

Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene.

The varied organ tropisms and clinical presentations of infection by Entamoeba histolytica have stimulated interest in the role of parasite genetic diversity in virulence. We investigated genetic diversity among 54 E. histolytica isolates from Bangladesh by analyzing polymorphism in the serine-rich gene by nested PCR on DNA extracted from stool and liver aspirate pus. We detected both size and restriction site polymorphisms among the isolates within this endemic area. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded 25 distinct DNA banding patterns among the 42 stool isolates and an additional 9 distinct patterns among the 12 liver abscess isolates. Approximately half of the isolates had unique polymorphisms. Interestingly, the majority of E. histolytica from the liver had polymorphisms which were not present in intestinal isolates from the same geographic area. These data are consistent with the existence of genetic differences between E. histolytica which cause intestinal and those which cause hepatic disease. We conclude that there is genetic diversity within E. histolytica isolates from an endemic population as reflected in serine-rich E. histolytica protein gene polymorphism. The correlation of genetic differences with the pathogenic potential of E. histolytica strains and the implications of genetic diversity for the immunoprophylaxis of amebiasis require further study.

Amino Acid Sequence↗
PubMed1993

Molecular mechanisms of invasion by Entamoeba histolytica.

Amebiasis is the third leading cause of death due to parasitic disease. Adherence to and contact-dependent killing of host cells requires the galactose-inhibitable lectin, a heterodimeric glycoprotein composed of heavy and light subunits. The cysteine-rich extracellular domain of the heavy subunit has identity with beta 1 integrins, complement receptor CD59, and complement components C8 and C9; the light subunit sequence is unlike any other sequenced protein. Monoclonal antibodies to the cysteine-rich domain identify pathogenic-specific domains, have adherence-inhibitory and -enhancing properties, block contact-dependent cytotoxicity, and abrogate complement C5b-9 resistance. The purified lectin has galactose-binding activity and confers C5b-9 resistance to susceptible amebae. The accumulated evidence points to the same cell surface galactose-inhibitable lectin as a mediator of two activities required for invasion: adherence and complement resistance.

Animals↗
PubMed1986

[A rare cause of 2 unclear fatalities: undiagnosed acute and chronic amebiasis].

Two cases are reported of unexpected and unclarified death caused by an infection with Entamoeba histolytica. The first case concerned a German whose disease was not diagnosed during the asymptomatic phase (2 years' duration) or in the acute phase during the last 3 weeks before death. This is typical of the disease. In the final phase, he developed a hepatic abscess. Death occurred as a consequence of bilateral apoplexia in the adrenal glands. In the second case a Turk fell ill with gastralgia and diarrhea. Developing hepatic insufficiency with comsumptive coagulopathy, he died after 6 days. Intoxication and/or intolerance of fructose were assumed as tentative diagnoses. These two cases show clearly that even outside an endemic area, one has to reckon with the possibility of amebiasis.

Adrenal Glands↗
PubMed1990

Comparative study of aminosidine, etophamide and nimorazole, alone or in combination, in the treatment of intestinal amoebiasis in Kenya.

417 patients suffering from intestinal amoebiasis were randomly allocated to 6 different treatment groups in a controlled study in 3 District Hospitals in Kenya. The patients received either aminosidine (A), etophamide (E), nimorazole (N), or the combinations NA, NE, EA. Treatment in all cases was given twice daily for 5 days. Before and after treatment, rectosigmoidoscopy was done in each patient, and stool examination with characterization of invasive (IF) and non invasive (NIF) forms of amoeba was done daily throughout treatment, and on Days 15, 30 and 60 of follow-up. Clinical cure was good after all the treatments, varying from 90 to 100%; parasitological cure at the end of treatment was 100% in the NA and EA treatments groups, and 98% in A group. The incidence of relapses was nil in the EA group, followed by 3% in NA and 6% in A groups. Anatomical cure (healing of ulcers) was 97.8% in the NA group, 95.5% in the N group and 88.5% in the A group. Drug tolerance was excellent or good after all the treatments, except that the EA combination produced diarrhoea in 76.5% of patients. Overall analysis of the findings, including tolerance of the various treatments, showed that aminosidine either alone or in combination with nimorazole gave the best results. Ulcers seen on rectosigmoidoscopy were more common in patients excreting invasive forms of amoebae in their stools.

Acetamides↗
PubMed1975

Virulence of bacteria-associated, Crithidia-associated, and axenic Entamoeba histolytica: experimental hamster liver infections with strains from patients and carriers.

Experimental infections of the hamster liver were carried out with five strains from patients with clinical amoebiasis and ten strains from asymptomatic carriers. Inocula of comparatively small size (12000-36000 amoebae) were injected under the liver capsule. 1. The virulence of the patient strains varied from 21-96% (see article) and declined sharply within 7-15 weeks after elimination of the associated bacterial flora. The virulence of the carrier strains varied from 0-100%, probably fluctuating with changes in the concomitant bacterial flora (Table 1). 2. The interrelation between size of inoculum, period of bacteria-free growth, and virulence was demonstrated with a Crithidia-associated patient strains (Table 2). 3. A patient strain showed a faster decrease of virulence during axenic than in Crithidia-associated cultivation (Table 3). 4. Two successive passages through hamster liver resulted in a marked increase of virulence of two bacteria-free strains, lasting for several months (Table 4). 5. A significant enhancement of virulence of Crithidia-associated and axenic amoebae by reassociation with a mixed bacterial flora during two weeks, followed by elimination of the bacteria, was demonstrated with two strains. The restored virulence was lost again within a few weeks (Table 5). 6. The virulence of an attenuated patient strain did not become manifest by adding large numbers of dead amoebae to the inoculum (Table 6). 7. The pathology of the different lesions caused in the hamster liver by the amoebae is described, including one of a granulomatous type, frequently found after inoculation with bacteria-free amoebae. 8. In an attempt to explain the occurrence of strains differing in pathogenicity an hypothesis is put forward based on the idea of selection of virulence and avirulent amoebae.

Amebiasis↗
PubMed1975

Monoxenic and axenic cultivation of carrier and patient strains of Entamoeba histolytica.

All of five strains of Entamoeba histolytica, isolated from symptomatic cases of amoebiasis, could be adapted to axenic growth on the TP-S-1 medium of Diamond (1968). Four axenic strains were started from amoeba-Crithidia cultures; one could be axenized directly after isolation from a case of cutaneous amoebiasis. Attempts to monoxenize, resp. axenize strains, isolated from Dutch, asymptomatic carriers, were less successful. Only three out of ten strains could be submitted to bacteria-free growth. These three strains, however, originated probably from a recent case of intestinal amoebiasis. The results, suggesting that highly virulent strains can be easier cultivated bacteria-free than those with low or no virulence, are further discussed. The yield of axenic amoebae per tube fluctuates largely depending on many factors such as the strain, the number of transfers (i.e. degree of establishment), the quality of Panmede liver digest and serum in the TP-S-1 medium, and the care of manipulating the cultures. For optimal growth, a more acid medium was required in an amoeba-Crithidia culture than in an axenic culture. Multinucleated, giant amoebae were frequently observed in axenic cultures.

Amebiasis↗
PubMed1984

Caerulomycin, an antifungal antibiotic with marked in vitro and in vivo activity against Entamoeba histolytica.

The anti-amoebic action of the bipyridyl antibiotic caerulomycin was assessed in vitro and in vivo using various strains of Entamoeba histolytica from polyxenic, axenic and monoxenic cultures. Minimum inhibition concentrations of caerulomycin (metronidazole) were 7.5 (5), 15.6(1.95) and 60 (2.5) micrograms/ml against polyxenic, axenic and monoxenic cultures of E. histolytica, respectively. The ED50 values ascertained in golden hamsters (extraintestinal amoebiasis) and rats (intestinal amoebiasis) after the oral route were 136 and 199 mg/kg (X4), respectively. Metronidazole proved to be approximately four times more active against tissue forms of E. histolytica than caerulomycin [ED50 of metronidazole: less than 40 mg/kg (X4)]. The antibiotic was slightly superior to metronidazole in its action on lumen forms of E. histolytica [ED50 of metronidazole: 233 mg/kg (X4)]. The antibiotic was in some cases toxic to hamsters and rats within the therapeutic range.

2,2'-Dipyridyl↗
PubMed1978

Experimental muscular amebiasis in hamsters as a biological model.

Trophozoites of Entamoeba histolytica maintained in vitro in Pavlova's medium were inoculated by deep intramuscular injection into the proximal left hindleg of hamsters. Thioglycollate medium was utilized as a successful vehicle to induce the infection. The invasion of the muscular tissue by the vegetative forms caused the formation of abscesses with great destruction of muscular fibers. The lesions were limited to the muscular tissue of the femoral area. The number of trophozoites, the medium of thioglycollate as a vehicle, the volume of the inoculum and the trauma caused by the needle were important elements in the evolution of the muscular amebic abscesses. A limited trial of the amebicidal activity of metronidazole utilizing the amebic intramuscular infection was also performed.

Abscess↗
PubMed1991

Role of leukocytes and amebic proteinases in experimental rat testicular necrosis produced by Entamoeba histolytica.

To investigate the role of amebic proteinases and host leukocytes, we studied amebiasis experimentally in the rat testis. The degree of inflammation and necrosis produced by different strains was correlated with proteinase activity and with zymograms. Intratesticular injection of axenically grown trophozoites of a pathogenic strain (HM-1 of Entamoeba histolytica) produced indistinguishable lesions in normal animals and leukopenic rats (less than 1000 leukocytes/mm3), suggesting that granulocytes do not contribute to the formation of lesions in this model. Testicular lesions produced by five different strains of E. histolytica ranging from highly virulent to almost nonpathogenic were proportional to the proteinase activity of each amebic strain. Inhibition of amebic proteinases in vitro and subsequent injection into the rat testis markedly reduced the inflammatory lesions resulting from highly virulent E. histolytica. The pathogenicity of three other amebae (E. laredo, E. moshkovskii, and E. invadens) was generally proportional to their proteinase activity; however, E. laredo showed high proteinase activity and caused minimal tissue damage. These results suggest that the pathogenic potential of Entamoeba spp. in the rat testis may be related to the type as well as the level of their proteinase activity.

Animals↗
PubMed1989

Delayed-type hypersensitivity to Entamoeba histolytica in mice and hamsters: a comparison.

Delayed-type hypersensitivity (DTH) to live or fixed Entamoeba histolytica was induced and compared in mice and hamsters. Peak reactions were obtained 24 h post-challenge. In mice, challenge with 10(5) amoebae produced maximal, specific footpad swelling; in hamsters, 5 x 10(4) were required. Elicitation of DTH in mice was strongest 1 week after induction and remained comparatively high for 8 weeks. In hamsters, elicitability declined after 1-2 weeks. Cyclophosphamide increased footpad reactions in mice and hamsters when given 1 day prior to induction but not prior to challenge. Reactions were usually somewhat (but not significantly) stronger in mice than in hamsters, which was also evident from adoptive transfer experiments. Thus, differences in cell-mediated immunity as expressed by DTH in mice and hamsters do not explain the differential susceptibility of these animals to infection with this parasite. In hamsters, multiple footpad injections of live or fixed amoebae lowered the percentage of subsequent liver infections after i.p. injection of virulent amoebae.

Amebiasis↗
PubMed1992

Analysis of pathogenicity by restriction-endonuclease digestion of amplified genomic DNA of Entamoeba histolytica isolated in Pernambuco, Brazil.

The pathogenicity of 47 strains of Entamoeba histolytica isolated in Pernambuco, Brazil, was examined using the polymerase chain reaction (PCR) followed by restriction-endonuclease digestion. Electrophoretic patterns of PCR products digested with HinfI revealed that all strains were nonpathogenic. The results were entirely in accord with phenotypic properties such as isoenzyme patterns and the failure to bind a pathogenic-isolate-specific monoclonal antibody. When the sensitivity of PCR was examined, amplified products could be detected from template DNA equivalent to five trophozoites. These observations indicate that PCR amplification of genomic DNA and subsequent restriction-enzyme digestion is a useful strategy for obtaining a sensitive and accurate diagnosis. The present study also demonstrates that nonpathogenic strains of E. histolytica predominate in northeastern Brazil.

Animals↗
PubMed1995

Intestinal amebiasis: cyclic suppression of the immune response.

The cellular immune response was evaluated in a C3H/HeJ mouse model of intestinal amebiasis at 5-60 days postinoculation with Entamoeba histolytica. At various intervals, spleen lymphocytes were obtained from infected and noninfected control mice and cultured with concanavalin A (Con A), pokeweed mitogen (PWM), or ameba antigen. E. histolytica infection induced a cyclic depression of DNA synthesis when spleen lymphocytes were stimulated with a T-cell mitogen (Con A), a T- and B-cell (PWM) mitogen, or an ameba antigen. A similar response was observed in the determinations of interleukin-2 in the supernatants of Con A-stimulated spleen cells from infected mice. When spleen cells from E. histolytica-infected mice were stimulated with phorbol myristate acetate plus ionomycin, results indicated a signal-transduction defect. These alterations, observed at the cellular level, might facilitate invasion of the host by the parasite.

Animals↗