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PubMed1975

Differentiation of Entamoeba: a new medium and optimal conditions for axenic encystation of E. invadens.

Nutritional and culturing requirements for efficient axenic encystation of Entamoeba invadens have been studied. A simple and reliable axenic encystation medium has been developed. It contains 0.5% tryptic digest of casein, 0.5% yeast extract, and 5% dialyzed serum in 5 mM potassium phosphate, pH 7.0. Mass encystation (avg 70%) occurred within 30 hr when axenically growing trophozoites of E. invadens IP-l were transferred to this medium before they entered stationary growth phase. Mass encystation of E. invadens PZ occurred similarly, but less reproducibly. Two E. histolytica strains did not encyst. Experiments established that differentiation did not depend upon changes in the external environment after amebase were transferred to encystation medium and, therefore, was initiated by the shift from growth to encystation medium.

Ascorbic Acid↗
PubMedDate not supplied

[Growth inhibition of Entamoeba histolytica and E. invadens produced by pomegranate root (Punica granatum L.)].

The purpose of the present work has been to evaluate the biological activity of alkaloids and tannins extracted from roots of Punica granatum L. on axenic cultures from Entamoeba histolytica and E. invadens, strains. Initially, the total aqueous extract and later some of its components obtained by thin layer chromatography were tested. These compounds were identified by chromatography as alkaloids or tannins. The density of amebic cultures was determined with a hemocytometer after 48 and 96 hours of incubation, which was calculated by the difference between number of trophozoites obtained at the times chosen and the number of amoebae inoculated. Two milliliters of aqueous extract had higher activity on cultures from E. histolytica than E. invadens strains, producing growth inhibitions of about 100 and 40 per cent respectively. Alkaloid concentrations of 1 mg/ml had no amebicide activity, however tannins at concentrations of 10 micrograms/ml for E. histolytica, and 100 micrograms/ml for E. invadens were sufficient to produce an growth inhibition about 100 per cent. Tannic acid was also tested on the cultures of E. histolytica observing an high inhibitory activity on the growth, this effect was produced at 0.01 mg/ml was similar to that observed with the tannins mixture.

Alkaloids↗
PubMedDate not supplied

[Determination of cytoplasmic glycogen in 4 strains of amoeba of the genus Entamoeba].

The purpose of this work was made to determine the cytoplasmic glycogen concentration in trophozoites from four different strains of Entamoeba genus. We used IP-1 and PZ from E. invadens; HM-2 and HK-9 from E. histolytica strains, axenically cultured in TP-S-1 medium. The growth of different strains was determined each 24 hours during 10-days, with the aid of a hemocytometer. The quantification of cytoplasmic glycogen was made by two methods: 1) Montgomery and 2) Krisman. The glycogen concentrations were measured in the four strains during their growing. The results did not show any difference, remained as a constant concentration in each strain. These concentrations quantified by Montgomery method were similar in all strains (35 pg/cell), except in HM-2 strain which showed a less concentration of this polysaccharide (28 pg/cell) than the other strain. These results were different when Krisman method was used, both IP-1 and PZ strains had a similar glycogen concentrations about 19 pg/cell, however HM-2 and HK-9 strains had a higher glycogen concentration (21 pg/cell) than E. invadens strains.

Analysis of Variance↗
PubMed1990

[Microcultures of Entamoeba].

The purpose of this study was the development of a microculture system for Entamoeba using ultrathin culture chambers, characterized by constant culture conditions, where the growing amebas can constantly be observed under the microscope, and which allows rapid variations in the characteristics of the culture medium, so as to determine the optimal conditions for growth and encystment of the ameba. After a lag phase the number of amebas increased following exponential kinetics. The E. invadens distribution in the culture chamber continued up to a total surface of (444 mm2) with in 7 days. On the whole E. invadens showed a tendency of not forming aggregates and growing until a confluent monolayer was reached. On the other hand, E. histolytica grew faster, covering the internal surface of the chamber in 5 days, and its growth went beyond a monolayer, since it covered all the internal walls, forming multiple aggregates. Encystment was induced in E. invadens by changing the feeding medium in the chamber for encystment medium. The encystment we observed was lower than that previously reported.

Animals↗
PubMed1990

Entamoeba histolytica: naturally occurring infections in baboons.

Entamoeba chattoni frequently occurs as an intestinal infection in non-human primates. It has been isolated from both wild and captive animals. Morphologically this amoeba resembles E. histolytica. E. histolytica has also been isolated from non-human primates on a number of occasions but these isolations have been from captive animals. In recent years identification of E. histolytica has been enhanced by the introduction of iso-enzyme electrophoresis methods. This technology has been widely applied to amoebae isolated from humans from many parts of the world. Limited work using iso-enzyme electrophoresis on non-human primates in captivity has confirmed the earlier parasitological studies mentioned above as both E. histolytica and E. chattoni were isolated; furthermore zymodeme (strain or species based on the characteristic iso-enzyme electrophoretic pattern) analysis demonstrated that these two organisms could be readily distinguished by this method. The importance of establishing whether E. histolytica occurs in wild primates as a true zoonosis was emphasised by these studies, particularly from the point of view of potential disease transmission. A study of baboons (Papio ursinus) in South African game reserves has been initiated and three surveys have been conducted in the Kruger National Park; these baboons are considered to live in isolation from humans. Of the 210 isolations attempted 65-75% yielded E. chattoni while E. histolytica were cultured from the faeces of 1-3% of the baboons. Although the prevalence rates varied in each of the 3 study locations both species of amoeba were found in baboons from all of them. All the E. histolytica isolated thus far have been non-pathogenic zymodemes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1969

Entamoeba histolytica-like amoebae occurring in man.

Six Entamoeba histolytica-like amoebae of the Laredo-type are compared with classical E. histolytica strains with regard to morphology, temperature-tolerance, response to hypotonic solutions, antigenic make-up, pathogenicity, sensitivity to drugs, and biochemistry. Sharp discontinuities are shown to exist between the 2 groups in some parameters, sufficient, under certain conditions, to justify creation of a new species designation for the Laredo-type amoebae. Nevertheless, this action is not recommended at the present time because of the small number of "aberrant" strains so far isolated, and because of the limited comparative studies so far carried out.Entamoeba moshkovskii is compared with the Laredo-type E. histolytica. No consistent distinction can be made between the 2 groups except for the sources of the strains-the former is free-living and the latter parasitic. It is therefore suggested that E. moshkovskii may actually be a parasite of the Laredo type, only incidentally encountered in free-living environments because of its wide temperature and tonicity tolerances. Since, here too, only limited studies are so far available, it is suggested that the term "moshkovskii-like E. histolytica" be used until sufficient data are accumulated to assign these amoebae to a more precise location in the taxonomic scheme.

Amebicides↗
PubMed1994

Complement sensitivity of Entamoeba histolytica and various nonpathogenic amoeba species.

Culture forms of the potentially pathogenic Entamoeba histolytica were compared to those of the nonpathogenic species of E. dispar, E. hartmanni, E. coli, Endolimax nana, and E. moshkovskii regarding the sensitivity to lysis by human complement activated through the alternative pathway. E. dispar was found unique in its complement resistance; all other nonpathogenic isolates resembled E. histolytica in that they were complement sensitive. Thus, a state of complement sensitivity is not a particular property of potentially pathogenic amoebae.

Animals↗
PubMed1998

Differentiation of pathogenic and non-pathogenic Entamoeba: has the question been answered?

Pathogenic and non-pathogenic Entamoeba have been separated into two distinct species. Recently, the non-pathogenic E. dispar has been cultivated axenically. However, the genetic variability among different clones from the same strain, experimental production of hybrid clones which may differ from their parents, and the possibility of invasiveness of E. dispar, are some phenomena which may indicate that the last word on distinctiveness of the species has not yet been said.

Animals↗
PubMed1982

Seroconversion to Entamoeba histolytica among short-term travelers to Mexico.

Paired serum samples were evaluated for the presence of antibodies to Entamoeba histolytica in 326 healthy college students from the United States who lived in Guadalajara, Mexico, for an average of four weeks. One hundred eighty of these students had an enteric illness develop, but no stool test results were positive for ameba. An indirect hemagglutination assay was the serologic method used, and no seroconversion was demonstrates. This finding confirms the belief that amebiasis is a rare cause of diarrhea among short-term travelers to Mexico.

Antibodies, Bacterial↗
PubMed1998

Entamoeba gingivalis identified in a left upper neck nodule by fine-needle aspiration: a case report.

We present a unique case of a 37-yr-old patient who underwent a left partial glossectomy, modified neck dissection, and radiation therapy in December 1990. In June 1991 the patient developed a 0.6 cm erythematous nodule in the left upper portion of her neck adjacent to the surgical scar. A fine-needle aspiration (FNA) of the nodule was performed. Cytologic examination revealed the presence of numerous organisms morphologically consistent with Entamoeba gingivalis. Although E. gingivalis can be found in specimens from the oral cavity, especially following radiation, it is rare to find these organisms within lesions of the head and neck region. In this case, it is believed that the patient had an increased number of organisms within her oral cavity secondary to radiation therapy. Radiation therapy also may have contributed to the formation of a fistula tract between the oral cavity and the surgical incision site which resulted in the formation of a small inflammatory nodule in the upper portion of the neck. The organisms could then travel from the oral cavity to the nodule in the neck thus explaining their presence in the FNA material.

Adult↗
PubMed2000

A subunit vaccine candidate region of the Entamoeba histolytica galactose-adherence lectin promotes interleukin-12 gene transcription and protein production in human macrophages.

The cysteine-rich region of the 170-kDa subunit galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica is a subunit vaccine candidate and a protective antigen in the gerbil model of amebiasis. Macrophage-mediated immunity is important for protection against E. histolytica and is activated by Th1 cytokines. As Th1 differentiation is promoted by IL-12, we investigated what portion of the Gal-lectin could stimulate IL-12 in human THP-1 macrophages. Native Gal-lactin stimulated IL-12 p40 / p35 mRNA expression in a dose- and time-dependent manner as measured by reverse transcriptase-PCR. Human immune serum and Gal-lectin mAb inhibition studies identified amino acids (aa) 596 - 998 as immunogenic and containing the IL-12 inducing domain. IFN-gamma priming augmented Gal-lectin-induced IL-12 mRNA expression independent of TNF-alpha and IL-1beta, and was required for IL-12 p70 protein production from macrophages and human peripheral blood mononuclear cells. Gal-lectin plus IFN-gamma stimulated IL-12 p40 and p35 gene transcription with stable mRNA transcripts and a differential requirement for protein synthesis. These results suggest that aa 596 - 998 of the Gal-lectin can confer Th1-mediated protection against amebiasis through IL-12 induction.

Animals↗
PubMed1981

Adhesion properties of Entamoeba histolytica.

Trophozoites of Entamoeba histolytica adhere to and phagocytize red blood cells and bacteria. Furthermore, in the initial step of the amoebic infectious process the parasite attaches to intestinal epithelial cells. A lectin (carbohydrate-binding protein) which apparently has a role in the attachment of the parasite to host cells was found in trophozoites of E. histolytica. When amoeba cells were disrupted by freeze-thawing, the lectin activity, as determined by haemagglutination of human erythrocytes, remained associated with the sedimented membrane fraction. This activity was pH dependent and heat and oxidation-sensitive, and was destroyed by proteolysis and on autoincubation. Moreover, the lectin activity was inhibited by a variety of N-acetylglucosamine-containing compounds such as chitin and chitin oligosaccharides, bacterial peptidoglycan, rabbit colonic mucus, bovine and human serum, an IgA fraction isolated from human colostrum, and IgG from sera of amoebiasis patients. These glycoconjugates also interfered with the adherence of intact radiolabelled amoeba trophozoites to human intestinal epithelial cells as well as their attachment to red blood cells. Although the lectin activity and the toxin-like activity previously found in E. histolytica seem to be two separate substances, they share a number of properties which suggest that they are related and may have a function in pathogenicity.

Acetylglucosamine↗
PubMed1983

Interactions between Entamoeba histolytica, bacteria and intestinal cells.

Axenically grown pathogenic and non-pathogenic isolates of Entamoeba histolytica have been shown to adhere to mammalian epithelial cells and bacteria by virtue of carbohydrate-binding proteins present on their cell surfaces. The interaction of amoeba isolates of low pathogenicity with a variety of gram-negative bacteria, mainly Escherichia coli strains which are readily ingested by the amoebae after relatively short periods, significantly increased the ability of the trophozoites to: (a) destroy and ingest intestinal epithelial cells; (b) secrete a cytopathic substance which morphologically affects a variety of tissue-cultured cells; and (c) cause hepatic abscesses in hamsters. Addition of carbohydrates that inhibit the lectin-mediated attachment of bacteria to amoebae prevented the enhancement of virulence. Interaction of the amoebae with bacteria that were heat-inactivated, glutaraldehyde-fixed or disrupted by sonication, as well as with bacteria precoated with antibodies or concanavalin A, did not lead to an increase in virulence. Moreover, short prior treatments of the bacteria with inhibitors of protein synthesis, but not with cell-wall synthesis inhibitors, also prevented the stimulation. The results indicate that interactions of amoebae with certain bacteria may be responsible for the increase in amoebic virulence.

Adhesiveness↗
PubMed1985

Secretory hormones of Entamoeba histolytica.

Watery diarrhoea as distinct from dysentery is occasionally seen in intestinal amoebiasis, suggesting a component of intestinal secretion. To study the pathogenesis of this watery diarrhoea, we evaluated the effect of lysates of Entamoeba histolytica on active intestinal electrolyte transport using rabbit ileum and rat colon studied by the Ussing chamber-voltage clamp technique. Amoebic lysates added to the ileal and colonic mucosal surfaces did not alter electrolyte transport; in contrast, addition to the ideal and colonic serosal surfaces caused an increase in short-circuit current which was transient in the ileum but more prolonged in the colon. This increase in current corresponded to inhibition of active Na+ and Cl- absorption and apparent stimulation of Cl- secretion in rat colon. In rabbit ileum, the short-circuit current response was: (1) dependent on serosal Ca2+, (2) inhibited by serosally applied verapamil, (3) associated with reversible desensitization, and (4) only partially inhibited by heating. These characteristics were similar to those of a series of neurohumoral substances present in mammalian intestinal mucosa which affect active electrolyte transport by increasing the permeability of the basolateral membrane to Ca2+. Substances with these properties identified include serotonin, substance P and neurotensin. All three substances were shown to be present in amoebic lysates by radioimmunoassay. Serotonin was also present by high performance liquid chromatography (HPLC) and thin layer chromatography, and neurotensin by HPLC. Prostaglandins were not present by radioimmunoassay. Attempts were made to determine which of these neurohumoral substances contributed to the changes in intestinal transport caused by amoebic lysates. Serotonin was thought to be involved, from the inhibition of the transport effect of amoebic lysate on rat colon by anti-serotonin antibody and by bufotenine, which inhibits the effect of serotonin on ion transport. Prostaglandins also appeared to be involved, since pretreatment with PGE2 or indomethacin inhibited the effects of amoebic lysate on transport. We conclude that amoebae contain neurohumoral substances, including serotonin, neurotensin and substance P, which may be important in the intestinal secretion caused by amoebae. Serotonin appears partially responsible for the intestinal secretion. In addition, amoebae may induce prostaglandin synthesis by the intestinal mucosa which could also contribute to the secretory response. The relation between the neurohumoral substances which act by Ca2+ and the prostaglandins presumably caused to be synthesized in the intestinal mucosa is not known.

Animals↗
PubMed2001

Entamoeba histolytica trophozoites activated by collagen type I and Ca(2+) have a structured cytoskeleton during collagenase secretion.

A peculiar characteristic of Entamoeba histolytica trophozoites is their capacity to invade human tissues. One of the cellular determinants of invasion may include adhesion to extracellular matrix components such as collagen, induction, and secretion of electron-dense granules (EDG) and tissue digestion. The mechanism and receptors involved in this process are not well understood. Previous results suggested that cytoskeleton plays a very important role during EDG secretion. We present evidence suggesting that adhesion to collagen and activation of EDG secretion are integrin-dependent events, since beta1 subunits detected by antibodies are concentrated at membrane sites where collagen and actin were colocalized. Furthermore, the involvement of actin, vimentin, and tubulin in restructuring cytoskeleton during EDG secretion was evident, since cytoskeleton isolation was possible exclusively in activated cells. Studies of immunolocalization of tubulin, actin, and vimentin by immunofluorescence and transmission electron microscopy suggest a role for cytoskeleton in EDG secretion.

Actins↗
PubMed2004

Dynamics of endocytic traffic of Entamoeba histolytica revealed by confocal microscopy and flow cytometry.

Entamoeba histolytica, the protozoan parasite of humans, manifests constitutive endocytosis to obtain nutrients and, when induced to express invasive behavior, as a means of ingesting and processing host cells and tissue debris. E. histolytica trophozoites were grown in liquid axenic medium that contained fluorescently labeled fluid-phase markers, so that the kinetics of uptake, the transit of loaded endosomes through the cytoplasm, and the time of release of the markers could be monitored by flow cytometry. Confocal microscopy of live trophozoites revealed uptake of fluid by avid macropinocytosis and the occurrence of fusion between young and older endosomes, as well as between pinosomes and phagosomes containing bacteria. Endosomes were rapidly acidified, then gradually neutralized; finally, indigestible material was released. Transit of endosomes containing fluid-phase markers required about 2 h. Uptake and release of fluid-phase markers were impaired by drugs that inhibited actin dynamics and actin-myosin interaction; uptake was also impaired by inhibition of PI 3-kinase. A striking feature of the trophozoites was the great heterogeneity of their endocytic behavior.

Actin Cytoskeleton↗
PubMed2006

Rho signaling in Entamoeba histolytica modulates actomyosin-dependent activities stimulated during invasive behavior.

Interaction of Entamoeba histolytica trophozoites with target cells and substrates activates signaling pathways in the parasite. Phosphorylation cascades triggered by phospho-inositide and adenyl-cyclase-dependent pathways modulate reorganization of the actin cytoskeleton to form structures that facilitate adhesion. In contrast, little is known about participation of Rho proteins and Rho signaling in actin rearrangements. We report here the in vivo expression of at least one Rho protein in trophozoites, whose activation induced actin reorganization and actin-myosin interaction. Antibodies to EhRhoA1 recombinant protein mainly localized Rho in the cytosol of nonactivated amoebae, but it was translocated to vesicular membranes and to some extent to the plasma membrane after treatment with lysophosphatidic acid (LPA), a specific agonist of Rho activation. Activated Rho was identified in LPA-treated trophozoites. LPA induced striking polymerization of actin into distinct dynamic structures. Disorganization of these structures by inhibition of Rho effector, Rho-kinase (ROCK), and by ML-7, an inhibitor of myosin light chain kinase dependent phosphorylation of myosin light chain, suggested that the actin structures also contained myosin. LPA stimulated concanavalin-A-mediated formation of caps, chemotaxis, invasion of extracellular matrix substrates, and erythrophagocytosis, but not binding to fibronectin. ROCK inhibition impaired LPA-stimulated functions and to some extent adhesion to fibronectin. Similar results were obtained with ML-7. These data suggest the presence and operation of Rho-signaling pathways in E. histolytica, that together with other, already described, signaling routes modulate actomyosin-dependent motile processes, particularly stimulated during invasive behavior.

Actomyosin↗
PubMed1995

Characterization of adhesion plates induced by the interaction of Entamoeba histolytica trophozoites with fibronectin.

Entamoeba histolytica trophozoites are pleiomorphic and highly motile cells. Although scarce fibrous material can be identified in the cytoplasm as elements of an organized cytoskeleton, clearly defined actin-containing structures are formed at the site of cell-matrix contact upon the interaction of trophozoites with fibronectin (FN) and other cellular matrix substrates. The structures are reminiscent of the adhesion plaques or focal contacts found in higher eukaryotic cells, where actin filament bundles insert into specialized regions of the plasma membrane and function as signal transduction organelles. Thus, the formation of adhesion plates in this parasitic ameba could be related to the specific signaling responses involved in its invasive behavior. Here, we report the isolation of amebic adhesion plates and the results of their structural and molecular analyses. Filaments, with the characteristic diameter of F-actin, radiating from an electron-dense matrix, are the main feature. Actin is one of the main protein components of the plate; other proteins identified are a FN-binding protein--previously reported as a "putative" FN receptor--the actin-binding proteins myosin II, myosin I, alpha-actinin, vinculin, and tropomyosin. The presence of the isolated plates of several proteases and protein kinases, in particular pp125FAK, is also demonstrated. our results suggest that adhesion plates in amebas are dynamic membrane-cytoskeletal complexes participating not only in the attachment to FN substrates but also providing the structural basis for their involvement in parasite locomotion and invasiveness.

Actins↗