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.     

In vitro killing of Entamoeba histolytica trophozoites by interferon-gamma-activated mouse macrophages

The effect of murine interferon gamma (IFN-gamma) on macrophage activation for amoebicidal activity was examined. Peritoneal macrophages were harvested from C57BL/6 mice and preincubated with IFN-gamma and/or lipopolysaccharide (LPS). In vitro amoebicidal activity of these macrophages was determined by trypan blue exclusion test against a virulent strain of E. histolytica (IP:0682:1). It was found that in vitro amoebicidal activity was evident in macrophage monolayers treated with both IFN-gamma and LPS. Macrophages treated with IFN-gamma alone did not develop cytotoxic activity unless they were exposed to LPS as a second triggering signal. The ability of IFN-gamma to prime macrophages to respond to trigger signals of LPS and develop cytotoxicity increased with time of incubation, the highest response being observed after 24 h. There was a dose-dependent relationship between the concentrations of both IFN-gamma and LPS used to activate macrophages and the number of dead trophozoites. These data suggest that macrophages are important in host defense against amoebiasis.     

Cytokine activation of murine macrophages for in vitro killing of Entamoeba histolytica trophozoites.

Macrophage-mediated effector mechanisms against the protozoan parasite Entamoeba histolytica were studied. Unstimulated macrophages were inefficient at killing E. histolytica trophozoites in vitro and were killed by the trophozoites. Conversely, immature cells of the mononuclear phagocyte lineage (promonocytes) were shown to display a strong spontaneous amebicidal activity. The acquisition of macrophage amebicidal activity following cytokine treatment was investigated. Gamma interferon, tumor necrosis factor alpha, and macrophage colony-stimulating factor 1, or combinations thereof, were shown to endow murine bone marrow-derived macrophages with significant amebicidal activity. Low doses of gamma interferon and tumor necrosis factor alpha and of gamma interferon and colony-stimulating factor 1 were shown to act synergistically in this phenomenon. This enhancement of amebicidal activity was shown to operate on bone marrow-derived macrophages, elicited peritoneal macrophages, and, to a much lesser extent, spleen macrophages. Although acquisition of amebicidal activity was associated with a strong respiratory burst, the addition of oxygen-free radical scavengers showed that the killing activity was approximately 45% H2O2 dependent. In addition, amebicidal activity by macrophages was shown to be contact dependent and was inhibited by 61% with the protease inhibitor tosyl lysyl chloromethyl ketone. Our results indicate that immunologic production of gamma interferon, tumor necrosis factor alpha, and colony-stimulating factor 1 could be important in the activation of macrophages for host defense against amebiasis and that promonocytes are strong effector cells against virulent amebae.     

Entamoeba histolytica alters arachidonic acid metabolism in macrophages in vitro and in vivo.

Entamoeba histolytica infections are associated with a state of transient suppression of cell-mediated immunity. Macrophages, the most important cells in host defence and control of invasive amoebiasis, in infected animals have been found to be deficient in effector functions and accessory cell potential. However, little is known of the cellular mechanisms responsible for the down-regulation. This study investigated whether macrophage dysfunction in amoebiasis is associated with altered macrophage arachidonic acid (AA) metabolism. Resident peritoneal macrophages (PMO) from naive gerbils produced enhanced levels of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) in response to live E. histolytica trophozoites, to diffusible excretory/secretory products released from live amoebae in millicells and to freeze-thawed soluble amoebic proteins that were inhibitable by indomethacin (INDO) and nordihydroguaiaretic acid (NDGA), respectively. In contrast to PMO from naive gerbils, PMO from animals with amoebic liver abscesses at 10, 20 and 30 days post-infection (p.i.) released high basal levels of PGE2 and LTC4. In response to zymosan stimulation, PMO from infected animals produced two- and fourfold less PGE2 and LTC4, respectively, as compared to uninfected controls. AMO showed high constitutive basal release of PGE2 and LTC4. In response to amoebic and zymosan stimulation, AMO at 10 days p.i. produced significantly higher levels of PGE2 than AMO at 20 days p.i., while AMO at 30 days p.i. were refractory to stimulation to produce higher than basal levels of PGE2. Early (10 days) and late (20-30 days) AMO were refractory to amoebic and zymosan stimulation for enhanced LTC4 release. Pretreatment of AMO with AA substrate restored optimal PGE2 and LTC4 biosynthesis, but the cells were generally unresponsive to zymosan stimulation to produce augmented levels of LTC4. These results strongly suggest that intrinsic or secreted products or both from E. histolytica can induce profound alteration of eicosanoid formation in cyclooxygenase and lipoxygenase pathways in macrophages from naive and infected animals and that AA metabolism by AMO is sequentially modified during the course of the disease.     

Entamoeba histolytica modulates the nitric oxide synthase gene and nitric oxide production by macrophages for cytotoxicity against amoebae and tumour cells.

Nitric oxide (NO) is the major cytotoxic molecule produced by activated macrophages for cytotoxicity against Entamoeba histolytica trophozoites. In the present study, we determined whether E. histolytica infection and soluble amoebic proteins affected macrophage cytotoxicity against amoebae and tumour cells by modulating the inducible NO synthase gene (iNOS) and NO (measured as nitrite, NO2-) and tumour necrosis factor-alpha (TNF-alpha) production. Amoebic liver abscess-derived macrophages [days 10, 20, 30 post-infection (p.i.)] stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) showed increased cytotoxicity against L929 cells (TNF-alpha-sensitive), but were refractory for killing amoebae and P815 cells (both NO-sensitive), concomitant with low NO2- production (< 4 microM/10(6) cells). In contrast, peritoneal and spleen macrophages at 10 and 20 days p.i. activated with IFN-gamma and LPS demonstrated increased killing of amoebae, and L929 and P815 cells concomitant with high NO2- production (> 12 microM/10(6) cells). Pretreatment of mouse bone marrow-derived macrophages with amoebic proteins suppressed IFN-gamma and LPS-induced amoebicidal (33%) and tumoricidal (44-49%) activities, with a corresponding decrease in TNF-alpha (56%) and NO (41%) production as well as TNF-alpha (41%) and iNOS (27%) mRNA by Northern blot analyses as compared to untreated activated controls. Inhibition of prostaglandin E2 (PGE2) biosynthesis in abscess and naive macrophages pretreated with amoebic proteins augmented IFN-gamma- and LPS-induced killing of L929 cells and TNF-alpha production, but failed to increase killing of P815 cells and amoebae as well as iNOS mRNA levels or NO production. These results suggest that E. histolytica selectively induces dysfunction of macrophage cytotoxicity by modulating iNOS mRNA expression and NO production independent from TNF-alpha and PGE2 allowing the parasites to survive within the host by impairing host immune responses.     

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.     

Transforming growth factor-beta 1 primes macrophages for enhanced expression of the nitric oxide synthase gene for nitric oxide-dependent cytotoxicity against Entamoeba histolytica.

Nitric oxide (NO) produced by activated macrophages is the major cytotoxic molecule for in vitro cytotoxicity against Entamoeba histolytica trophozoites. Transforming growth factor-beta 1 (TGF-beta 1) is a potent negative regulator of several macrophage functions, including NO production. In this study, we investigated the effect of TGF-beta 1 on macrophage nitric oxide synthase (mac-NOS) mRNA expression and NO production for macrophage cytotoxicity against E. histolytica trophozoites. TGF-beta 1 by itself was incapable of inducing mouse bone marrow-derived macrophage (BMM) amoebicidal activity and NO production (as measured by nitrite). In contrast, TGF-beta 1 pretreatment (4 hr) primed BMM for an enhanced amoebicidal activity of 15% and 23% in response to (interferon-gamma) IFN-gamma+tumour necrosis factor-alpha (TNF-alpha) or IFN-gamma+lipopolysaccharide LPS, concomitant with increased NO production of 85% and 27%, respectively. TGF-beta 1 pretreatment increased NO production in response to IFN-gamma+TNF-alpha/LPS stimulation in a time- and dose-dependent manner. By Northern blot analysis, the increased NO production of TGF-beta 1-pretreated BMM was preceded by markedly enhanced expression of mac-NOS mRNA. The priming effect of TGF-beta 1 on NO production was critically dependent on both a TNF-alpha (> or = 100 U) and a LPS (> or = 100 ng) triggering dose in the presence of IFN-gamma. TGF-beta 1 pretreatment enhanced TNF-alpha mRNA expression, but had no effect on TNF-alpha production in culture supernatants after 4 hr of stimulation with IFN-gamma+TNF-alpha/LPS; however, at a later time-point (16-48 hr), even though the levels of TNF-alpha mRNA expression were unaffected, TNF-alpha production was reduced. These data demonstrate that TGF-beta 1 priming for increased mac-NOS mRNA expression for NO-dependent cytotoxicity against E. histolytica in response to IFN-gamma+TNF-alpha/LPS stimulation may be involved in the modulation of a TNF-alpha triggering signal by TGF-beta 1.     

Entamoeba histolytica extract and interferon-gamma activation of macrophage-mediated amoebicidal function.

The effect of recombinant murine interferon-gamma (IFN-gamma) and E. histolytica extract (E.h.E.) on macrophage (M phi) activation for amoebicidal activity was examined. Peritoneal macrophages were harvested from C57BL/6 and A/J mice and preincubated with IFN-gamma and/or E.h.E. It was found that amoebicidal activity could be induced in both C57BL/6 and A/J-derived macrophages by pretreatment with IFN-gamma and E.h.E. Pretreatment of the M phi with E. histolytica extract or IFN-gamma alone did not result in the activation of significant cytotoxic activity against E. histolytica trophozoites. In the presence of IFN-gamma, E.h.E. had a dose-dependent effect on the activation of M phi amoebicidal function.     

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-.     

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).     

Use of indomethacin to demonstrate enterotoxic activity in extracts of Entamoeba histolytica trophozoites.

The present study was designed to develop and characterize animal models for the assay of enterotoxic activity in extracts of Entamoeba histolytica trophozoites. Marked water and electrolyte secretion occurred in both in vivo rabbit ileal loops and rat colon loops exposed to clarified sonic fluids of E. histolytica strain HM-1 trophozoites (10(6)/ml) when the animals were first administered indomethacin (0.1 mg/kg). No effect on intestinal absorption was observed in animals exposed to Entamoeba extracts alone or after administration of a lower (0.01 mg/kg). No effect on intestinal absorption was observed in animals exposed to Entamoeba extracts alone or after administration of a lower (0.01 mg/kg) dose of indomethacin. Higher doses (greater than or equal to 1 mg/kg) of indomethacin inhibited extract-induced secretion. No enterotoxic activity was detected with or without indomethacin, using extracts from the nonpathogenic E. histolytica-like Laredo strain, even at 10-fold-higher cell concentrations. The HM-1 enterotoxic activity was heat labile. Prior exposure of the loop lumen to fetuin (100 micrograms/ml) blocked the secretory response to subsequent HM-1 extract exposure, but postexposure of the loop to fetuin did not block secretion that had already been established by the amoeba extract. No histological changes were seen associated with the amoeba extract-induced secretion. The data suggest that E. histolytica HM-1 strain elaborates an enterotoxic activity capable of causing consistent secretion in the mammalian intestine that has had its mucosal cytoprotection impaired by indomethacin.     

Localization of myosin heavy chain A in the human pathogen Entamoeba histolytica.

To recognize myosin II in trophozoites of the human pathogen Entamoeba histolytica, a specific antimyosin polyclonal serum was raised against a fusion protein consisting of a 146-amino-acid fragment of the myosin II heavy chain A of E. histolytica (MhcA) fused with beta-galactosidase. The hybrid protein was encoded by a chimera gene formed by a DNA fragment, from the mhcA gene, amplified by polymerase chain reaction and fused with the lacZ gene of Escherichia coli. Polymerase chain reaction-amplified DNA is located within the region encoding the tail domain of myosin. This antibody recognized a 250-kDa protein in extracts of E. histolytica trophozoites. Confocal microscope analysis of antibody-labelled trophozoites indicated that MhcA localizes at the posterior pole of locomoting cells and concentrates within the uroid. These results might indicate that MhcA is involved in movement and in the uroid formation which help amoebas to escape the host immune response. These data are the first evidence indicating that myosin exists in E. histolytica. In addition, two other peptides were found in myosin-enriched extracts of amoebas, indicating that other myosins may be present in this parasite.     

Sexual dimorphism in the control of amebic liver abscess in a mouse model of disease

Amebic liver abscess (ALA) is the most common extraintestinal manifestation of human infection by the enteric protozoan parasite Entamoeba histolytica. In contrast to intestinal infection, ALA greatly predominates in males but is rare in females. Since humans are the only relevant host for E. histolytica, experimental studies concerning this sexual dimorphism have been hampered by the lack of a suitable animal model. By serial liver passage of cultured E. histolytica trophozoites in gerbils and mice, we generated amebae which reproducibly induce ALA in C57BL/6 mice. Interestingly, all animals developed ALA, but the time courses of abscess formation differed significantly between the genders. Female mice were able to clear the infection within 3 days, whereas in male mice the parasite could be recovered for at least 14 days. Accordingly, male mice showed a prolonged time of recovery from ALA. Immunohistology of abscesses revealed that polymorphonuclear leukocytes and macrophages were the dominant infiltrates, but in addition, gamma,delta-T cells, NK cells, and natural killer T (NKT) cells were also present at early times during abscess development, whereas conventional alpha,beta-T cells appeared later, when female mice had already cleared the parasite. Interestingly, male and female mice differed in early cytokine production in response to ameba infection. Enzyme-linked immunospot assays performed with spleen cells of infected animals revealed significantly higher numbers of interleukin-4-producing cells in male mice but significantly higher numbers of gamma interferon (IFN-gamma)-producing cells in female mice. Early IFN-gamma production and the presence of functional NKT cells were found to be important for the control of hepatic amebiasis as application of an IFN-gamma-neutralizing monoclonal antibody or the use of NKT knockout mice (Valpha14iNKT, Jalpha 18(-/-)) dramatically increased the size of ALA in female mice. In addition, E. histolytica trophozoites could be reisolated from liver abscesses of Jalpha18(-/-) mice on day 7 postinfection, when wild-type mice had already cleared the parasite. These data suggest that the sexual dimorphism in the control of ALA is due to gender-specific differences in early cytokine production mediated at least in part by NKT cells in response to E. histolytica infection of the liver.     

Amebic infection in the human colon induces cyclooxygenase-2

We sought to determine if infection of the colon with Entamoeba histolytica induces the expression of cyclooxygenase-2 and, if it does, to determine the contribution of prostaglandins produced through cyclooxygenase-2 to the host response to amebic infection. Human fetal intestinal xenografts were implanted subcutaneously in mice with severe combined immunodeficiency and allowed to grow; the xenografts were then infected with E. histolytica trophozoites. Infection with E. histolytica resulted in the expression of cyclooxygenase-2 in epithelial cells and lamina propria macrophages. Infection with E. histolytica increased prostaglandin E(2) (PGE2) levels 10-fold in the xenografts and resulted in neutrophil infiltration, as manifested by an 18-fold increase in myeloperoxidase activity. Amebic infection also induced an 18-fold increase in interleukin 8 (IL-8) production and a >100-fold increase in epithelial permeability. Treatment of the host mouse with indomethacin, an inhibitor of cyclooxygenase-1 and cyclooxygenase-2, or with NS-398, a selective inhibitor of cyclooxygenase-2, resulted in (i) decreased PGE(2) levels, (ii) a decrease in neutrophil infiltration, (iii) a decrease in IL-8 production, and (iv) a decrease in the enhanced epithelial permeability seen with amebic infection. These results indicate that amebic infection in the colon induces the expression of cyclooxygenase-2 in epithelial cells and macrophages. Moreover, prostaglandins produced through cyclooxygenase-2 participate in the mediation of the neutrophil response to infection and enhance epithelial permeability.     

The tumor necrosis factor alpha-stimulating region of galactose-inhibitable lectin of Entamoeba histolytica activates gamma interferon-primed macrophages for amebicidal activity mediated by nitric oxide.

Entamoeba histolytica adheres via galactose-lectin (Gal-lectin) to human colonic mucins and intestinal epithelial cells as a prerequisite to amebic invasion. Native Gal-lectin is a protective antigen in the gerbil model of amebiasis. Amino acids 596 to 1082 of Gal-lectin mediate E. histolytica adherence to target cells and stimulate tumor necrosis factor alpha (TNF-alpha) production by naive murine bone marrow macrophages (BMM). Resistance to amebiasis requires an effective cell-mediated immune response against E. histolytica trophozoites mediated by nitric oxide (NO) released from activated macrophages. Herein, we determine whether the TNF-alpha-stimulating region of Gal-lectin can activate gamma interferon (IFN-gamma)-primed BMM for NO production and amebicidal activity. Native Gal-lectin (100 to 500 ng/ml) stimulated TNF-alpha and inducible nitric oxide synthase (iNOS) mRNA expression in IFN-gamma-primed BMM as did lipopolysaccharide (100 ng/ml). Primed BMM produced TNF-alpha and NO in response to Gal-lectin in a dose-dependent manner. Antilectin monoclonal antibody IG7, which recognizes a domain (amino acids 596 to 818) of the TNF-alpha mRNA-stimulating region of Gal-lectin, specifically inhibited TNF-alpha and iNOS mRNA induction and TNF-alpha and NO production by primed BMM in response to Gal-lectin (100 ng/ml). Simultaneous treatment of BMM with IFN-gamma and Gal-lectin (100 ng/ml) activated the cells to kill E. histolytica trophozoites, whereas IFN-gamma treatment alone had no effect. In the presence of monoclonal antibody 1G7 or aminoguanidine (an iNOS inhibitor), NO production and amebicidal activity were inhibited >80%. These results suggest that the TNF-alpha-stimulating region of native Gal-lectin is a potent stimulus of IFN-gamma-primed BMM for NO production, which is essential for host defense against amebiasis.     

Activated mouse macrophages kill Entamoeba histolytica trophozoites by releasing reactive nitrogen intermediates.

Mouse macrophages activated by gamma interferon (IFN-gamma) and bacterial lipopolysaccharide (LPS) are highly cytotoxic for the enteric protozoan parasite Entamoeba histolytica. Herein, we show that this killing by activated macrophages is L-arginine dependent, inasmuch as it was blocked by exogenous arginase or NG-monomethyl-L-arginine. These two inhibitors had no effect on E. histolytica cytolytic activity against L929 fibroblasts. Also, macrophage killing of E. histolytica always correlated with nitrite presence in the supernatant fluids. Finally, it was shown that addition of excess iron or the reductant sodium dithionite to activated macrophages blocked their ability to kill E. histolytica. Overall, this suggests that killing of E. histolytica by activated macrophages depends on the production of reactive nitrogen intermediates which leads to critical iron loss and protozoan parasite death.     

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.     

Differences in cap formation between invasive Entamoeba histolytica and non-invasive Entamoeba dispar

The rapid redistribution of surface antigen-antibody complexes in trophozoites of the human protozoan parasite Entamoeba histolytica, in a process known as capping, has been considered as a means of the parasite to evade the host immune response. So far, capping has been documented in the invasive E. histolytica, whereas the mobility of surface components in the non-invasive Entamoeba dispar is not known. E. dispar does not induce liver lesions in rodent experimental models, in contrast to the liver abscesses produced by E. histolytica in the same animal model. We have therefore analyzed the mobility of surface receptors to the lectin concanavalin A and of Rab11, a membrane-associated protein, in both species of Entamoebae by confocal fluorescence microscopy and transmission and scanning electron microscopy. The great majority of E. histolytica trophozoites became morphologically polarized through the formation of well-defined caps at the posterior pole of the parasite. Actin colocalized with the lectin caps. Antibodies against the membrane protein Rab 11 also produced capping. In striking contrast, in E. dispar, the mobility of concanavalin A surface receptors was restricted to the formation of irregular surface patches that did no progress to constitute well-defined caps. Also, anti-Rab 11 antibodies did not result in capping in E. dispar. Whether the failure of E. dispar to efficiently mobilize surface molecules in response to lectin or antibodies as shown in the present results is related to its non-invasive character represents an interesting hypothesis requiring further analysis.     

Cytokine Genes are Down-Regulated When Attachment of Entamoeba histolytica to HT-29 Colon Epithelial Cells is Prevented

Entamoeba histolytica can cause invasive disease by disruption of the intestinal barriers and subsequent lysis of the intestinal cells. Adherence to and contact dependent killing of host cells requires the galactose inhibitable lectin. To elucidate the mechanism whereby E. histolytica influences host defence, the authors assessed the change of proinflammatory cytokine genes expressed by colon epithelial cells in response to co-culture with E. histolytica trophozoites and carbohydrates, including galactose, N -acetyl-galactosamine or N -acetyl-lactosamine, which prevented E. histolytica from attaching to epithelial cells. After HT-29 human colon epithelial cells were co-cultured with E. histolytica trophozoites in the presence or absence of carbohydrates (0.1–100 m m ), RNA was extracted from the epithelial cells by an acid guanidinium thiocyanate–phenol–chloroform method. Cytokine gene expression was assessed by quantitative RT-PCR using a synthetic internal standard, and proteins were determined by ELISA. IL-8 mRNA expressed by HT-29 cells in response to E. histolytica trophozoites was downregulated in the presence of galactose, N -acetyl-galactosamine or N -acetyl-lactosamine (0.1–100 m m ), and this was paralleled by decreased IL-8 protein secretion. GM-CSF and IL-1α/β mRNAs were also downregulated in those cells in the presence of these agents. These results suggest that the expression of proinflammatory cytokine genes could be inhibited by preventing E. histolytica from attaching to the host's colon epithelial cells.