Entamoeba histolytica and Entamoeba dispar infection in children in Bangladesh

The prevalence of infection by the invasive parasite Entamoeba histolytica and the noninvasive parasite Entamoeba dispar was determined in 2000 children in Bangladesh. Antigen detection identified more cases of E. histolytica-E. dispar infection than did culture or microscopy. Microscopic identification of E. histolytica-E. dispar complex infection in stool did not equate with the diagnosis of amebic dysentery because most amebic infections in this population were due to E. dispar: Urban children with diarrhea had a 4.2% prevalence of E. histolytica infection and a 6.5% prevalence of E. dispar infection; rural asymptomatic children had a 1.0% prevalence of E. histolytica infection and a 7.0% prevalence of E. dispar infection. Shigella dysenteriae and Shigella flexneri infections were more frequent in children who also had Entamoeba infection, a potentially important consideration for the empiric treatment of dysentery in this population.     

Sensitivity of Entamoeba histolytica and Entamoeba dispar patient isolates to human complement

Twenty-one Entamoeba histolytica and 56 Entamoeba dispar patient isolates were investigated for their sensitivity to the classical and alternative pathway of human complement, E. histolytica and E. dispar patient isolates were differentiated by polymerase chain reaction and hexokinase isoenzyme typing. It was found that 90.3% (+/- 12.0%) of the trophozoites of E. histolytica were lysed after 30 min by the alternative pathway of complement in the presence of 50% human serum (19 isolates showed lysis rates higher than 80%), whereas E. dispar cells were less susceptible to the alternative pathway as 68.8% (+/- 28.2%) of lysis occurred. However, 23 of the E. dispar isolates were lysed between 100 and 80% (90.9% +/- 9.1%), demonstrating that about half of the tested E. dispar isolates were highly sensitive to complement lysis. Only 11 of the E. dispar isolates were proven to be ?resistant' to the alternative pathway of complement and were lysed less than 40%. These results are in conflict to earlier publications, describing resistance of E. dispar to complement lysis (Hamelmann et al. 1992, 1993).     

Axenic cultivation of Entamoeba histolytica from liver abscess and its zymodeme

A local strain of Entamoeba histolytica, the HTH-56: MUTM from a human liver abscess was successfully axenized. The culture was initially established monoxenically in Diamond's TYI-S-33 medium in the presence of Crithidia luciliae and maintained at 34 +/- 0.5 degrees C. After 5 passages it was adapted to axenic cultivation by addition of 0.02% Bacto agar in Diamond's TYI-S-33 medium in place of Crithidia. Subcultures or replacement with fresh complete media were done twice or thrice for 7 days, after which the agar was omitted and a stable culture was obtained. Isoenzyme analysis showed that this strain of E. histolytica belonged to the zymodeme II pattern, which is one out of 10 pathogenic zymodemes of E. histolytica most commonly found among the virulent strains.     

Mechanisms of innate resistance to Toxoplasma gondii infection

The interaction of protozoan parasites with innate host defences is critical in determining the character of the subsequent infection. The initial steps in the encounter of Toxoplasma gondii with the vertebrate immune system provide a striking example of this important aspect of the host-parasite relationship. In immuno-competent individuals this intracellular protozoan produces an asymptomatic chronic infection as part of its strategy for transmission. Nevertheless, T. gondii is inherently a highly virulent pathogen. The rapid induction by the parasite of a potent cell-mediated immune response that both limits its growth and drives conversion to a dormant cyst stage explains this apparent paradox. Studies with gene-deficient mice have demonstrated the interleukin-12 (IL-12)-dependent production of interferon gamma (IFN-gamma) to be of paramount importance in controlling early parasite growth. However, this seems to be independent of nitric oxide production as mice deficient in inducible nitric oxide synthase (iNOS) and tumour necrosis factor receptor were able to control early growth of T. gondii, although, they later succumbed to infection. Nitric oxide does, however, seem to be important in controlling persistent infection; treating chronic infection with iNOS metabolic inhibitors results in disease reactivation. Preliminary evidence implicates neutrophils in effector pathways against this parasite distinct from that described for macrophages. Once initiated, IL-12-dependent IFN-gamma production in synergy with other proinflammatory cytokines can positively feed back on itself to induce 'cytokine shock'. Regulatory cytokines, particularly IL-10, are essential to down-regulate inflammation and limit host pathology.     

Molecular changes in Entamoeba histolytica in response to bacteria

Entamoeba histolytica, the protozoan parasite, is the causative agent of amoebiasis. The degree of virulence, as inferred from invasiveness, of potentially pathogenic strains may be regulated by both host and parasite factors that determine the gut environment. One such factor that plays an important role is the bacterial flora in the gut. Previous studies have clearly shown that bacterial flora is an important determinant of virulence in E. histolytica. However, the exact nature of changes induced in E. histolytica in response to bacteria and their role in virulence is not clear. In this study the levels of a number of molecules potentially important in virulence mechanisms were determined in E. histolytica cells grown with and without normal human bacterial flora, using enzyme-linked immunosorbent assay. Significant changes were observed only after the E. histolytica cells had been adapted to grow with bacterial flora for a number of generations, and not in short term culture.     

Complement resistance of pathogenic Entamoeba histolytica mediated by trypsin-sensitive surface component(s)

Pathogenic forms of the protozoan parasite Entamoeba histolytica were reported previously to resist the cytolytic effect of the alternative complement pathway (AP) only temporarily during exposure to complement. In contrast, nonpathogenic forms of E. histolytica had been found to show AP resistance as a stable property. We studied the mechanisms of AP resistance of the two forms. Upon exposure to AP activity, resistant pathogenic or nonpathogenic forms bound significantly less C3 products than complement-sensitive pathogenic amebae, indicating that the two resistant forms both inhibited AP amplification. Various enzymatic treatments and inhibition of membrane mobility by cytochalasin B and glutaraldehyde fixation showed that the mechanisms of AP inhibition differed between pathogenic and nonpathogenic forms; in contrast to nonpathogenic forms, pathogenic amebae required intact membrane mobility and a trypsin-sensitive surface component(s) to inhibit AP activation.     

A beta 1 integrin-like molecule in Entamoeba histolytica

Human invasive amoebiasis is highly destructive, causing rapid necrosis and liquefaction of all tissues reached by the trophozoites. Degradation of extracellular matrix components (EMC) has been demonstrated during invasion of the basal lamina. Pursuing the idea that trophozoites might behave similarly to other invasive cells with respect to their interaction with EMC, plasma membrane proteins biochemically or functionally related to integrins were looked for. A 140 kDa molecular mass membrane protein from Entamoeba histolytica trophozoites with the characteristics of a beta 1 integrin-like fibronectin receptor was identified.     

Involvement of superoxide dismutase and pyruvate:ferredoxin oxidoreductase in mechanisms of metronidazole resistance in Entamoeba histolytica

Metronidazole resistance has been induced in an axenic strain of Entamoeba histolytica (HTH-56:MUTM) following continuous exposure to steadily increasing drug concentrations. The drug-resistant line is routinely maintained in normally lethal levels of metronidazole (10 microM). Resistance to this concentration of drug was developed over 177 days. Decreased pyruvate:ferredoxin oxidoreductase (PFOR) activity in anaerobic organisms is one mechanism of metronidazole resistance but in entamoeba, PFOR activity was not decreased in metronidazole-resistant parasites as determined by immunofluorescent assays and immunoblotting studies. 2-Oxoacid oxidoreductase activity, which appeared to be due to a single enzyme, PFOR, was evident with pyruvate as well as the alternative substrates, alpha-ketobutyrate, alpha-ketoglutarate and oxaloacetate. A marked increase in superoxide dismutase (SOD) activity was detected in metronidazole-resistant E. histolytica. Increased SOD activity has not previously been documented as a mechanism of drug resistance although SOD has been associated with a range of stress situations in other organisms.     

Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection

The Babesia bovis merozoite surface antigen-1 (MSA-1) is an immunodominant, neutralization-sensitive merozoite surface antigen encoded by a polymorphic gene family. MSA-1 antigenic polymorphism results in a complete lack of immunologic cross-reactivity among strains. It is unknown how rapidly this antigenic shift occurs, or whether it evolves in the mammalian host. To determine whether the dominant epitopes encoded by a single msa-1 gene copy vary during the course of a single infection, the antibody response to these epitopes was measured after infection of cattle with the Mo7 biologically cloned strain of B. bovis using an Mo7 gene copy-specific enzyme-linked immunosorbent assay. Antibodies against MSA-1 encoded by this gene copy were detected by postinoculation (PI) day 15 in each of 5 experimentally infected animals. Importantly, detectable antibody persisted in all carrier animals without a significant decrease in optical density through 12 mo PI, at which time the experiment was terminated. The results indicate that immunodominant epitopes expressed by a single gene copy of msa-1 do not undergo marked antigenic shift typical of the gene family during the course of a single infection in the mammalian host. The results are compatible with the limited MSA-1 polymorphism reported in some geographically defined endemic populations.     

An essential role for gamma interferon in innate resistance to Shigella flexneri infection

The purpose of this experiment was to study endurance performance and substrate storage and utilization in fat- or carbohydrate-fed rats. Ninety-nine rats were randomly divided into three groups and over 4 wk were fed either a carbohydrate-rich [CHO; 10% total energy content in the diet (E%) fat, 20 E% protein, 70 E% carbohydrate] diet or one of two fat-rich diets (65 E% fat, 20 E% protein, 15 E% carbohydrate) containing either saturated (Sat) or monounsaturated fatty acids (Mono). Each dietary group was randomly assigned to a trained (6 days/wk, progressive to 60 min, 28 m/min at a 10% incline) or a sedentary group. Rats were killed either before or after a treadmill endurance run to exhaustion. Training increased endurance (206%), but diet composition did not affect endurance in either trained or sedentary rats. beta-Hydroxyacyl-CoA dehydrogenase activity was increased in fat-fed but not carbohydrate-fed rats (P < 0.05). Respiratory exchange ratio during the initial phase of exercise was lower after the Mono compared with the Sat diet (P < 0. 05) and higher after the CHO than the Sat diet (P < 0.05). Thus adaptation to a high-fat diet containing a moderate amount of carbohydrates did not induce enhanced endurance in either trained or untrained rats; however, substrate utilization was modulated by both amount and type of dietary fat during the initial stage of exercise in trained and sedentary rats.     

Detoxicating enzymes of Entamoeba histolytica and their detoxifying roles

OBJECTIVE: To investigate the Entamoeba histolytica living in the low oxygen concentration colon of the host and how does it survive in the circumstance after invading the tissues with high oxygen concentration while obtaining oxygen without being damaged by the toxins. MATERIAL AND METHODS: E. histolytica cultured for 48 hours was collected, centrifuged, rinsed, ultrasonically shattered and again centrifuged, and the activities of superoxide dismutase and the catalase in the supernatant were determined. The catalase and peroxidase were identified by the electron microscopic enzyme cytochemical reaction technique. RESULTS: E. histolytica contained 122.42 +/- 15.47 U/mgpr of superoxide dismutase, 126.05 +/- 17.04 K/mgpr of catalase and peroxidase, and all of them are detoxifying enzymes. Catalase and peroxidase were located within microsomes and lysosome-like organelles respectively. CONCLUSIONS: E. histolytica contains the detoxifying enzymes as superoxide dismutase, catalase and peroxidase, that may prevent the aerobic metabolism from being poisoned by the activated oxygen free radical (superoxide anion radical and hydrogen peroxide) produced in this process, suggesting that the detoxifying function of these enzymes play an important defensive role in the survival of E. histolytica.     

Monoclonal antibody-based immunohistochemical demonstration of Entamoeba histolytica in liver tissues of experimentally infected hamster (Mesocricetus auratus)

Histopathological changes and the presence of Entamoeba histolytica trophozoites was sequentially followed after intrahepatic inoculation of the parasites in 42 hamsters, 35 of which received no treatment whereas the remaining seven were treated with metronidazole. The liver tissues were examined for amoebic trophozoites by a monoclonal antibody (mAb)-based immunofluorescence assay (IFA), a mAb-based immunoperoxidase (IPx) and H & E staining. The number of hamsters developing abscesses was increased with time and was highest on day 30. Cellular infiltration with inflammatory cells and glycogen depletion were observed as early as day 5, followed thereafter by more intense inflammation of portal canals, periportal fibrosis, bile duct proliferation and hepatocyte degeneration. In 7 metronidazole-treated hamsters, no obvious pathological damage was seen. In a group of seven hamsters each, both IPx and IFA were positive in 3, 3, 4, 5 and 4 hamsters and in 3, 4, 3, 3 and 5 hamsters on days 5, 10, 15, 20 and 30, respectively. In 18 control hamsters, IPx, IFA and H & E were all negative. If the result from H & E was used as a gold standard, agreement between H & E and IFA and H & E and IPx were 91.4%, and 88.6%, respectively. Sensitivity and specificity were 93.8% and 89.5%, respectively for IFA, and 93.8% and 84.2%, respectively for IPx.     

Amphotericin B-induced resistance to Pseudomonas aeruginosa infection in mice

We evaluated the effects of amphotericin B (AmB) against Pseudomonas aeruginosa (P. aeruginosa) infection in mice. Pretreatment with 2 mg/kg of AmB 24 hours before infection significantly increased the survival rates of mice intraperitoneally infected with either P. aeruginosa or Escherichia coli. To evaluate the mechanism of this AmB-induced resistance to infection, we conducted a number of experiments. Peritoneal macrophages exposed in vitro to AmB showed superior bactericidal activity compared to that of control macrophages. Interleukin-1 production by peritoneal macrophages from mice pretreated with 2 mg/kg of AmB was significantly higher than that in control mice. Serum tumor necrosis factor level after intravenous injection of P. aeruginosa was also higher in mice pretreated with 2 mg/kg of AmB than in control mice. These data indicate that AmB induces resistance to P. aeruginosa in mice. Furthermore AmB-induced activation of peritoneal macrophages and their production of interleukin-1 and tumor necrosis factor appeared to play important roles in this phenomenon.     

The effects of Entamoeba histolytica lysates on human colonic mucins

Detergent lysates of Entamoeba histolytica trophozoites contained high levels of beta-N acetyl-D-glucosaminidase, beta-N acetyl-D-galactosaminidase and alpha-D-galactosidase activity, and lower but significant levels of five other glycosidases. Although these activities should have been capable of largely degrading the oligosaccharide side-chains of human colonic mucin, in fact only about one third of high MW mucin was degraded in 72 h and trypsin alone produced a similar effect. There was no evidence that these glycosidases were excreted and we conclude that they are unlikely to represent significant virulence factors for E. histolytica.     

Effects of aphidicolin on Entamoeba histolytica growth and DNA synthesis

We have previously demonstrated that DNA polymerase activity of Entamoeba histolytica is inhibited by aphidicolin, which is a specific inhibitor of eukaryotic nuclear replicative DNA polymerases. The present study was aimed to evaluate the effect of aphidicolin on growth and DNA synthesis by this parasite. Aphidicolin blocked the growth of axenic E. histolytica strain HM-1:IMSS. DNA synthesis was also inhibited by aphidicolin when assayed by incorporation of [3H]thymidine into the DNA. The inhibitory effect of aphidicolin on the growth of E. histolytica was abrogated by removal of the drug, and exposure to 3 microg/ml of the drug for at least 48 hr had little effect on the viability. Synchronous growth was observed in the recovery phase after removal of aphidicolin.     

Analysis of the 170-kDa lectin gene promoter of Entamoeba histolytica

The ultrastructure of the vegetative cell in the pollen of Ledebouria socialis Roth (Hyacinthaceae) was investigated from microspore mitosis to anthesis. As a result of the good preservation quality achieved with high-pressure freeze fixation and freeze substitution, novel structural features were observed. Extensive endomembrane compartments emerging at the onset of lipid and starch mobilization, were identified as protein bodies by using video-enhanced contrast light microscopy. Thus, proteins, apart from starch and lipids, represent a third class of important intermediary storage substances in developing pollen. The close spatial relationship between protein bodies, endoplasmic reticulum (ER), and storage lipids suggest that protein bodies and ER contribute to lipid digestion. Immediately prior to anthesis the protein bodies become transformed into unspecialized vacuoles as a result of the gradual dissolution of their contents; the formation of the protein bodies remains still to be elucidated. The ER proliferates extensively during pollen ontogenesis, thereby changing its ultrastructure and spatial organization. Microfilaments were detected during all developmental stages, in particular microtubule-associated single microfilaments. The microfilaments are likely to be composed of actin as shown by immunogold labeling.     

Molecular analysis of the Gal/GalNAc adhesin of Entamoeba histolytica

Attachment of Entamoeba histolytica to colonic epithelium and a variety of other target cells is mediated by a galactose/N-acetyl D-galactosamine (Gal/GalNAc) inhibitable adhesin. Seven monoclonal antibodies specific for nonoverlapping epitopes of the 170 kDa subunit have been shown to have distinct effects on adherence. Four of these monoclonal antibodies inhibit or have no effect on amebic adherence while two others enhance amebic adherence. The epitopes recognized by these seven monoclonal antibodies have been mapped to the extracellular cysteine rich region of the 170 kDa subunit. The conformational nature of the epitopes was examined by testing monoclonal antibody reactivity with isolated regions of the 170 kDa subunit expressed as fusion proteins in E. coli and also with denatured native adhesin. These analyses suggested that three of monoclonal antibodies recognized conformational epitopes while the remaining four recognized linear epitopes. The mapping of these monoclonal antibodies have identified functionally important regions of the Gal/GalNAc adhesin and have also shown that recombinant Gal/GalNAc adhesin, when expressed in E.coli, retained at least some of its native conformation.     

Effect of bacterial association on virulence of Entamoeba histolytica to baby hamster kidney cell monolayers

Axenic E. histolytica trophozoite strain NIH:200 and HMI:IMSS when co-associated with aerobic bacteria Escherichia coli strain K12 and serotype 056 showed marked increase in virulence as observed by destruction of baby hamster kidney (BHK) monolayers. However, when incubated with anaerobic bacterial strains Clostridium perfringens and Bacteroides fragilis virulence remained unaltered. Further, adherence of E. histolytica to BHK monolayer was found to be mediated by N-acetyl-D-galactosamine.