Susceptibility of Entamoeba histolytica to oxidants.

The effect of hydrogen peroxide and hypochlorite on culture forms of Entamoeba histolytica trophozoites was examined by using two strains of E. histolytica, virulent (IP:0682:1) and nonvirulent (DKB). The amoebae were incubated with various concentrations of hydrogen peroxide and hypochlorite, and their viability was determined at different times after incubation. When the viability of the virulent and nonvirulent strains was compared to different oxidant strengths, it became apparent that the virulent strain was less susceptible than the nonvirulent one to the cytotoxic effect of hydrogen peroxide and hypochlorite. Our studies further showed that the toxic effect was both time and dose dependent. To confirm that the killing of amoebae in this system was associated with the presence of hydrogen peroxide, amoebae were incubated with hydrogen peroxide and catalase. Catalase reduced the killing effect of hydrogen peroxide to the control level. These data confirmed previous observations of the susceptibility of amoebic trophozoites to hydrogen peroxide and also demonstrated susceptibility to hypochlorite.     

Development of inflammation and augmented chemotactic responsiveness of murine peritoneal macrophages following treatment with Entamoeba histolytica trophozoites

The accumulation of inflammatory cells in the peritoneal cavity of C57BL/6 mice was examined following intraperitoneal injection of Entamoeba histolytica trophozoites. Two different strains of E. histolytica were used: a virulent strain (IP:0682:1) and a non-virulent strain (DKB). Injection of 10(6) trophozoites of either strain resulted in significant increases in the numbers of total peritoneal cells, macrophages and polymorphonuclear cells as compared to either saline-injected control mice or mice injected with 10-fold lower doses of trophozoites. The in vitro chemotactic response of macrophages from amoebae-induced exudates was also examined. Macrophages from mice treated with strain IP:0682:1 or DKB strain trophozoites were more responsive to complement-derived chemotactic factors than macrophages from saline-injected mice. This increase was significant on day 2 and persisted at enhanced levels until day 20 when the experiment was terminated. In addition, it was found that trophozoites activated normal mouse serum resulting in the production of serum-derived chemotactic activity.     

Activation of macrophages by Entamoeba histolytica extracts in mice

The effect of Entamoeba histolytica extracts on the production of inflammatory macrophages and the release of hydrogen peroxide (H2O2) and superoxide (O2-) from these cells was examined in C57BL/6 mice. Two different strains of E. histolytica, either virulent (IP:0682:1) or nonvirulent (DKB), were used in this study. The number of macrophages recovered from the peritoneal cavity of mice treated 5 days previously with 150 micrograms of either strain of amoebic extracts was significantly higher than in the saline-treated groups. Macrophages from mice treated with 150 micrograms of the IP:0682:1 strain of amoebic extracts exhibited a powerful burst of oxidative metabolis when triggered with phorbol myristate acetate (PMA). Extract from the non-virulent strain was much less effective in activating macrophages for the production of oxidative metabolites. Thus, a crude extract preparation from E. histolytica, particularly the virulent strain, induces a strong macrophage inflammatory response in which the cells also produce high levels of H2O2 and O2-.     

Adhesion of Entamoeba histolytica trophozoites to human erythrocytes.

To understand the mechanism of Entamoeba histolytica adhesion, we characterized the binding of trophozoites to human erythrocytes (RBC) in suspension by measuring the kinetics of amoeba-RBC complex formation. Adhesion was very efficient, since most of the amoebae were complexed with RBC after only 5 min at 37 degrees C in mixtures containing 10(4) amoebae and 10(6) RBC per ml; the adhesion rate depended on amoeba and RBC concentrations, but not on the A, B, and O human blood groups, and was maximal at 37 degrees C and pH 7.3 in the presence of 4 mM Ca2+ and 1 mM Mg2+. Adhesion was prevented if trophozoites were fixed with glutaraldehyde, but only decreased slightly if RBC were previously fixed; it decreased in the absence of glucose and was inhibited as a function of the concentration of cytochalasin B and of the metabolic inhibitors bathophenanthroline and 8-hydroxyquinoline. From these results we conclude that E. histolytica adhesion is an active process that depends on the amoebal cytoskeleton and metabolic energy and on the mobility of both amoebal and RBC surface ligands.     

Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica.

AIMS--To assess the reliability of the detection of erythrophagocytic amoebic trophozoites in stool samples in the diagnosis of dysentery associated with invasive Entamoeba histolytica. METHODS--Amoebic culture was carried out on single stool samples collected from patients from Mexico, Colombia, and Bangladesh. The stools had been examined by light microscopy. Amoebic dysentery was diagnosed when erythrophagocytic E histolytica trophozoites were observed in a case of bloody diarrhoea. E histolytica isolates were characterised by isoenzyme electrophoresis and results correlated with microscopical findings in stools. Statistical analysis was performed using the chi 2 test. RESULTS--Where erythrophagocytic amoebae had been observed in dysenteric stool specimens the E histolytica phenotype was invariably invasive (p < 0.0001). Observation of erythrophagocytic amoebae in dysentery is 100% specific and predictive of infection with invasive E histolytica. When amoebic culture-positive cases only are considered it is 96% sensitive. In this study E histolytica of zymodeme XIV was more commonly associated with amoebic dysentery than zymodeme II. There was no significant difference between the carriage rate of invasive and non-invasive E histolytica in non-dysenteric diarrhoea. Asymptomatic subjects carried non-invasive E histolytica more frequently than invasive E histolytica. Patients with non-amoebic dysentery, when shown to be infected with E histolytica, carried non-invasive strains (12%). CONCLUSIONS--Sensitivity and specificity of microscopical examination of a single stool specimen for diagnosing amoebic dysentery is very high; intestinal carriage of invasive E histolytica detected by culture is not necessarily an indication of active disease as patients with diarrhoea and asymptomatic subjects shed invasive and non-invasive E histolytica. There are possibly two subpopulations of invasive E histolytica with different pathogenic potential which can be differentiated by zymodeme analysis.     

Phorbol esters specifically enhance the cytolytic activity of Entamoeba histolytica.

Entamoeba histolytica causes invasive amebiasis by lysis of host tissue and inflammatory cells. The in vitro cytolysis of target Chinese hamster ovary (CHO) cells by axenic E. histolytica trophozoites (strain HM1:IMSS) is a calcium- and phospholipase A-dependent event initiated by the binding to the target cell of the galactose-inhibitable surface lectin of the parasite. We utilized phorbol esters as a probe to determine whether an amebic protein kinase C has a role in the cytolytic event. The addition of phorbol 12-myristate 13-acetate (PMA) at 10(-6) or 10(-7) M resulted in a greater than twofold enhancement of amebic killing of target CHO cells over 30 min (P less than 0.01). Prior exposure of only the amebae, but not the CHO cells, to PMA produced a similar effect (P less than 0.01). The inactive analog 4-alpha-phorbol had no effect on amebic killing of CHO cells. The PMA-mediated enhancement of amebic cytolysis persisted for up to 60 min after a 5-min exposure; however, after a 30-min exposure to PMA (10(-6) M) there was no augmentation of amebic killing of CHO cells. PMA (10(-6) M) did not promote adherence of parasites to CHO cells but did enhance amebic cytolysis of previously adherent target cells (P less than 0.01). Sphingosine, a specific inhibitor of protein kinase C, abolished both the PMA-stimulated and the basal cytolytic activity of E. histolytica. PMA enhanced CHO cell cytolysis by the less virulent wild-type strain H-303:NIH (P less than or equal to 0.02) but did not augment the activity of the less virulent strain H-200:NIH or two avirulent clones of HM1 (L6 and C919). In summary, these experiments with the phorbol esters and sphingosine as probes to modulate the activity of protein kinase C indicate participation of a parasite protein kinase C in the cytolytic activity of virulent, axenic E. histolytica trophozoites and thus in the pathogenesis of amebiasis.     

Catalase and superoxide dismutase activities in virulent and nonvirulent Staphylococcus aureus isolates.

Catalase and superoxide dismutase (SOD) activities of virulent and nonvirulent isolates of Staphylococcus aureus were compared. The mean value of catalase activity for intact cell suspensions was 2,773 +/- 1,049 Kat f units (Kat f is defined as the ratio of the velocity constant of catalase at 0 min to the protein content in grams per milliliter); that of nonvirulent isolates was 154 +/- 92 Kat f units. The mean value of the catalase activities for lysates of virulent isolates was 260 +/- 120 Kat f units, and that of nonvirulent isolates was 31 +/- 19 Kat f units. Catalase levels in intact cells as well as in cell lysates were significantly different for virulent than for nonvirulent S. aureus isolates (P less than 0.001). The mean value of SOD activities was 20.85 +/- 11.48 U (1 U is defined as the amount of SOD required to inhibit the rate of reduction of cytochrome c by 50%) for virulent cell lysates, compared with a mean of 5.39 +/- 2.89 U for nonvirulent cell lysates. The SOD levels in virulent and nonvirulent isolates were significantly different (P less than 0.001). The virulence of the S. aureus isolates was determined by comparing weight gains of neonatal mice injected with virulent or nonvirulent strains. The percent weight gain of neonatal mice injected with virulent isolates was significantly lower than that of those injected with nonvirulent isolates.     

Modulation of tumor necrosis factor production by macrophages in Entamoeba histolytica infection.

The macrophage-derived mediator tumor necrosis factor alpha (TNF) is a cytokine with pleiotropic effects. TNF exhibits potent immunologic and inflammatory properties in parasitic diseases. The present study examined the production of TNF by macrophages isolated from gerbils infected with Entamoeba histolytica and by naive macrophages in response to amoebae in vitro. Amoebic liver abscess-derived macrophages produced low constitutive basal levels of TNF; in response to lipopolysaccharide (LPS) stimulation, TNF production was enhanced by 14-, 11-, and 6-fold at 10, 20, and 30 days postinfection, respectively. Amoebic liver abscess-derived macrophages pretreated with either recombinant gamma interferon (IFN-gamma) or the cyclooxygenase inhibitor indomethacin augmented TNF production in response to soluble amoebic proteins and LPS. Kupffer cells and peritoneal and spleen macrophages from infected animals did not release TNF constitutively in vitro. However, TNF production in response to LPS stimulation was significantly higher at 10 and 20 days postinfection. Macrophages from infected and naive animals pretreated with recombinant IFN-gamma or indomethacin produced increased amounts of TNF in response to LPS but not in response to soluble amoebic protein stimulation. Pretreatment of naive macrophages with amoebic proteins inhibited LPS-induced TNF production by 69 to 79%; the effect of the amoebic proteins was partially reversed by indomethacin pretreatment. In contrast, IFN-gamma- and LPS-activated naive macrophages produced enhanced levels of TNF in response to live amoebae and soluble amoebic proteins. Our results demonstrate that TNF production by macrophages is altered during E. histolytica infection and in response to amoebae and suggest a role for IFN-gamma and prostaglandin E2 in regulating TNF production during the infection.     

Effect of silica on resistance of mice to Entamoeba histolytica infection.

The role of macrophages in intestinal amoebiasis in mice has been investigated. The effect of injuring host macrophages on the course of infection was examined by using strains selected as being either genetically susceptible (C3H/HeJ, C57BL/6) or genetically resistant (A/J) to amoebiasis. Mice were treated with an intravenous injection of silica particles 1 day before infection with 2.5 X 10(5) or 5 X 10(5) polyxenic trophozoites of Entamoeba histolytica. The animals were killed at various times after infection, and the parasite burden in the cecum was measured. Silica treatment dramatically increased the growth of parasites in the susceptible mice. The same trend was evident, although less marked, in the resistant mice. The effect of silica treatment in experimental amoebiasis was much more pronounced in animals inoculated with 5 X 10(5) amoebae than in those with 2.5 X 10(5) amoebae. These data suggest that macrophages play an important role in host defense against amoebiasis in mice.     

Effects of Panmede and various horse serum concentrations on the axenic cultivation ofEntamoeba histolytica strains in TPS-1 medium

We analyzed the influence of Panmede and horse serum concentrations on the growth of fiveEntamoeba histolytica strains (HK9, HM1, HM2, HM3 and HM38) axenically cultivated in TPS-1 medium. Panmede was evaluated by comparing the growth of strain HM1 in medium prepared with each of 15 Panmede lots; the yields ofE. histolytica trophozoites depended on the lot quality of Panmede, and their maximal values ranged from 8×10³ to 8.9×10⁴ amoebae/ml. The growth-promoting effect of eight lots of horse serum on strains HK9 and HM1 were studied using a single Panmede lot of good quality. Yields obtained with strain HK9 ranged from 8×10⁴ to 1.8×10⁵ amoebae/ml, whereas yields obtained with HM1 ranged from 3×10⁴ to 1.2×10⁵ amoebae/ml. Thus, the optimal serum concentration in TPS-1 medium that caused maximal growth ofE. histolytica cultures depended on the quality of the serum lot and proved to be specific for each of the fiveE. histolytica strains investigated. It ranged from 18% (v/v) for strain HM2 to 28% (v/v) for strain HM1. Our results reveal that the growth ofE. histolytica trophozoites in TPS-1 medium can be distinctly improved by selecting appropriate lots of Panmede and horse serum and using optimal serum concentrations.     

In vitro Entamoeba histolytica adhesion to human endothelium: a comparison using two strains of different virulence

Extraintestinal dissemination of Entamoeba histolytica is frequently manifested by the life-threatening amebic liver abscess (ALA). The hepatic establishment of amebas implies invasion of blood vessels and contact with the endothelium. By means of a fluorescence-based quantitative adhesion assay, we assessed the binding to human endothelial cells of two E.?histolytica strains of different virulence. The highly virulent strain (L-A) adhered substantially more strongly to unstimulated endothelium than the nonvirulent one (BG3). Attachment of L-A was increased by treatment of endothelial cells with interleukin-1β (IL1β). Other proinflammatory cytokines such as interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) did not modify the spontaneous adhesion capacity of amebas. For purposes of comparison we also performed adhesion of the parasites to skin fibroblasts. Adhesion to this cell type was quite low (相似文献   

Comparison of repeated DNA from strains of Entamoeba histolytica and other Entamoeba

Restriction enzyme digestion patterns of total genomic DNA revealed the presence of highly repeated DNA in Entamoeba histolytica and E. histolytica-like (Laredo type) amoebae of humans, E. invadens and E. invadens and E. terrapinae of reptiles, and E. moshkovskii isolated from sewage polluted waters. Homology with Plasmodium berghei ribosomal DNA was striking. Northern blot analysis of E. histolytica RNA provided further evidence that the highly repeated DNA codes for ribosomal RNA. The distribution of restriction enzyme sites on repeated DNA of the Entamoeba was highly polymorphic. Thus it was possible, based on EcoRI digestion patterns, to distinguish between strains of E. histolytica and the other species of Entamoeba. Additionally, these investigations at the molecular level corroborate previously reported physiological and biochemical evidence indicating the E. histolytica-like amoebae and E. histolytica are not conspecific. EcoRI fragments from repeated DNA of E. histolytica (strain HM-1: IMSS) were cloned in the plasmid vector pTZ18R. A subfragment from one of these clones proved to be a useful hybridization probe in distinguishing E. histolytica from the other Entamoeba.     

Specific labeling of cysteine proteinases in pathogenic and nonpathogenic Entamoeba histolytica.

Growth of Entamoeba histolytica trophozoites was inhibited by 50% at low concentrations (2.0 micrograms/ml) of the diazopeptidyl inhibitor benzyloxycarbonyl-leucyl-L-tyrosyldiazomethane (Z-L-Leu-L-Tyr-CHN2). Iodination of the tyrosine residue lowered the growth inhibitory efficacy of the diazopeptidyl inhibitor (50% inhibition, approximately 10 micrograms/ml). However, even at this concentration, practically all of the cysteine proteinase activity of the cells was irreversibly inactivated as shown by fluorescence microscopy with the dipeptide substrate L-Arg-L-Arg-4-methoxy-beta-napthylamide or colorimetrically with azocasein as the substrate. Growth of trophozoites of E. histolytica from various strains, including both pathogenic and nonpathogenic zymodemes, was similarly inhibited. The concentration of inhibitor required to inactivate the proteinase activity of nonpathogenic cells was lower. Lysates from trophozoites grown in the presence of sublethal concentrations of 125I-labeled protease inhibitor (10 micrograms/ml) showed as many as eight radioactive bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular sizes, 73, 68, 56, 40, 39, 35, 29, and 27 kilodaltons). Two of these bands (molecular sizes, 29 and 27 kilodaltons) could be seen in gels of the cytoplasmic fraction, whereas the high-molecular-size bands were mostly associated with the membrane fraction. The radioactive bands in pathogenic and nonpathogenic strains were very similar with only minor differences. The results obtained show that E. histolytica cells, irrespective of their pathogenicity, possess a number of cysteine proteinases of similar molecular sizes which are vital for cell growth.     

Murine T-cell clones against Entamoeba histolytica: in vivo and in vitro characterization

Eleven T-cell clones were raised from the spleens of BALB/c mice hyperimmunized against a crude soluble extract of Entamoeba histolytica trophozoites. Seven clones were of the Lyt-1+, and four of the Lyt-23+ phenotype. All clones proliferated in the presence of E. histolytica antigens but not to a purified protein derivative; five clones proliferated to a crude extract of the E. histolytica-like Laredo amoebae. Ten clones secreted T-cell growth factors in response to E. histolytica antigens. Two clones (Lyt-23+) mediated direct lymphocytotoxicity (73% and 86%) against amoebic trophozoites that was inhibited with rabbit anti-mouse TNF-alpha. Supernatants of five of the clones (all Lyt-1+) activated mouse peritoneal macrophages (Mphi) to kill E. histolytica trophozoites in vitro, seemingly independent of secreted reactive oxygen intermediates (O2- and H2O2) in the case of three clones supernatants. All of the clones that were activating Mphi to kill amoeba in vitro also mediated a local DTH reaction in mouse footpad. Our results demonstrate direct lymphocyte cytotoxicity via a cytolytic molecule antigenically related to TNF-alpha and lymphokines activating Mphi for amoebic killing by oxidative and non-oxidative mechanisms, the latter process mediated by a macrophage-activating factor (MAF) distinct from interferon-gamma (IFN-gamma).     

Effect of splenectomy on the size of amoebic liver abscesses and metastatic foci in hamsters.

The role of the spleen in hepatic amoebiasis in hamsters was studied. In hamsters receiving an intrahepatic inoculation of 10(5) trophozoites of axenic Entamoeba histolytica at 7 or 14 days postsplenectomy, the mean weight of metastatic foci increased significantly when compared with sham-splenectomized or intact controls. In contrast, when both splenectomy and intrahepatic inoculation with amoebae were carried out at the same time, there was not only a significant increase in the mean weight of metastatic foci but also in the liver abscess. It is suggested that the spleen is important for host defense against E. histolytica infection, especially in the reduction in the degree of metastatic spread from the primary site.     

Effect of recombinant tumor necrosis factor α on in vitro amoebicidal activity of murine Kupffer cells

The present study was undertaken to determine whether recombinant tumor necrosis factor α (TNF) could induce Kupffer cells to kill Entamoeba histolytica parasite in vitro. C57BL/6 mice were used in this study. The liver was perfused and Kupffer cells harvested and treated with TNF for 6 h. It was found that Kupffer cells treated with TNF are able to kill amoebic trophozoites in vitro. These results further show that amoebicidal activity of TNF-activated Kupffer cells is dependent on the ratio of Kupffer cells to amoebic trophozoites. The maximum amoebicidal activity of Kupffer cells was observed with the ratio of one Kupffer cell to five amoebae. This study also shows that the optimal concentration of TNF is required in the induction of amoebicidal activity in Kupffer cells (10⁵ units). It seems that both oxidative-dependent and -independent mechanisms are important for the killing of amoebae by the TNF-treated Kupffer cells. It is likely that TNF-treated Kupffer cells produce endogenous TNF or other cytotoxic molecules which are capable of mediating the parasite killing. Our results indicate that the immunologic production of TNF is important in the activation of Kupffer cells to kill amoebic trophozoites.     

An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces.

The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region.     

Entamoeba histolytica calreticulin: an endoplasmic reticulum protein expressed by trophozoites into experimentally induced amoebic liver abscesses

Entamoeba histolytica calreticulin (EhCRT) is remarkably immunogenic in humans (90-100% of invasive amoebiasis patients). Nevertheless, the study of calreticulin in this protozoan is still in its early stages. The exact location, biological functions, and its role in pathogenesis are yet to be fully understood. The aim of the present work is to determine the location of EhCRT in virulent trophozoites in vivo and the expression of the Ehcrt gene during the development of experimentally induced amoebic liver abscesses (ALA) in hamsters. Antibodies against recombinant EhCRT were used for the immunolocalization of EhCRT in trophozoites through confocal microscopy; immunohistochemical assays were also performed on tissue sections of ALAs at different times after intrahepatic inoculation. The expression of the Ehcrt gene during the development of ALA was estimated through both in situ RT-PCR and real-time RT-PCR. Confocal assays of virulent trophozoites showed a distribution of EhCRT in the cytoplasmic vesicles of different sizes. Apparently, EhCRT is not exported into the hepatic tissue. Real-time RT-PCR demonstrated an over-expression of the Ehcrt gene at 30 min after trophozoite inoculation, reaching a peak at 1-2 h; thereafter, the expression fell sharply to its original levels. These results demonstrate for the first time in an in vivo model of ALA, the expression of Ehcrt gene in E. histolytica trophozoites and add evidence that support CRT as a resident protein of the ER in E. histolytica species. The in vivo experiments suggest that CRT may play an important role during the early stages of the host-parasite relationship, when the parasite is adapting to a new environment, although the protein seems to be constitutively synthesized. Moreover, trophozoites apparently do not export EhCRT into the hepatic tissue in ALA.