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PubMed1991

Down-regulation of murine lymphocyte responsiveness to mitogens after treatment with antigens of Entamoeba histolytica.

Injection of mice with pathogenic Entamoeba histolytica (strain HM1-IMSS) antigens resulted in a decreased capacity of splenocytes to respond to mitogen-induced blastogenesis following a challenge with concanavalin A (Con A), phytohemagglutinin (PHA), and lipopolysaccharide (LPS), whereas no inhibition was observed in mice that had previously been injected with equivalent amounts of non-pathogenic E. histolytica-like Laredo antigens. Depletion of adherent cells in the splenocyte preparation indicated that these cells were not a major contributor to the observed immunosuppression. Quantification of splenic T-lymphocyte subsets demonstrated a significant decrease in Thy-1+ and Lyt-1+ cells, but Lyt-2+ cells were not affected. Splenocytes treated with pathogenic amoebic antigens in vitro affected the capacity of these cells to respond optimally to Con A- and LPS-induced blastogenesis but not to that induced by PHA. These findings demonstrate that amoebic antigens affect lymphocyte function and may be important co-factors in the immunoregulation and pathogenesis of amoebiasis.

Animals↗

PubMed1989

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

We analyzed the influence of Panmede and horse serum concentrations on the growth of five Entamoeba 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 of E. histolytica trophozoites depended on the lot quality of Panmede, and their maximal values ranged from 8 x 10(3) to 8.9 x 10(4) 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 x 10(4) to 1.8 x 10(5) amoebae/ml, whereas yields obtained with HM1 ranged from 3 x 10(4) to 1.2 x 10(5) amoebae/ml. Thus, the optimal serum concentration in TPS-1 medium that caused maximal growth of E. histolytica cultures depended on the quality of the serum lot and proved to be specific for each of the five E. 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 of E. histolytica trophozoites in TPS-1 medium can be distinctly improved by selecting appropriate lots of Panmede and horse serum and using optimal serum concentrations.

Animals↗

PubMed1990

A new medium containing antibiotics for the xenic cultivation of Entamoeba gingivalis.

Diamond's trypticase-yeast extract-serum-gastric mucin (TYSGM-9) medium was studied for its suitability to support the xenic growth of the oral protozoan Entamoeba gingivalis. Amoebic growth was found to be best when the inoculum for transfer was 0.1 ml, the incubation temperature was 35 degrees C, and the interval between transfers was 48 h. These parameters were also useful for controlling the growth of accompanying bacteria. In addition, bacterial growth in xenic stock cultures, which had a direct effect on the activity of E. gingivalis trophozoites, was kept to a minimum by addition of the antibiotics piperacillin, erythromycin, neomycin, and penicillin. Varying substitutions and selected supplementations of TYSGM-9 medium led to the development of an improved medium for E. gingivalis. Supplements most beneficial for the growth of E. gingivalis trophozoites were ascorbic acid, ferric ammonium citrate, and special NCTC 107 vitamin mixture. As compared with TYSGM-9 medium (6-10 E. gingivalis trophozoites observed per field), the new E. gingivalis medium supported excellent growth (16-20 E. gingivalis trophozoites observed per field during optimal growth) of the oral protozoan. The medium is suitable for clinical isolation of E. gingivalis.

Animals↗

PubMed1989

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

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

Amebiasis↗

PubMed1995

Identification of Entamoeba histolytica intracellular phospholipase A and lysophospholipase L1 activities.

Entamoeba histolytica phospholipase A and lysophospholipase activities from a vesicular subcellular fraction (P30) were analyzed. The products, obtained using specific substrates labeled with 14C or 3H, indicated the presence of phospholipase A1 and A2 as well as lysophospholipase L1 activities. The enzymes detected could participate in phospholipid metabolism and the alkaline phospholipase A2 may contribute to E. histolytica cytopathogenicity.

Animals↗

PubMed1993

Dose- and time-dependent functional and structural damage to the colon mucosa by Entamoeba histolytica trophozoite lysates.

Analysis of the initial interaction between Entamoeba histolytica trophozoites and the large intestine is impossible in humans and very difficult in experimental animals. To circumvent this obstacle we treated the luminal side of full-thickness rabbit colon segments mounted in Ussing-type chambers with trophozoite lysates of the E. histolytica HM1 virulent strain. Exposure to lysates for up to 90 min produced dose- and time-dependent effects on the colon, consisting of (a) increased decay rates for potential difference, short-circuit current, and transmural resistance and (b) mucosal damage ranging from vacuolation at the bases and shortening of epithelial cells to the loss of intercellular junctions, destruction of microvilli, and necrosis of interglandular epithelial zones. This acute model of intestinal amebiasis is sensitive, fast, and reliable.

Animals↗

PubMed1995

Cyclic nucleotide changes induced in human leukocytes by a product of axenically grown Entamoeba histolytica that inhibits human monocyte locomotion.

Pulse exposure of human mononuclear phagocytes to the monocyte locomotion-inhibitory factor produced by Entamoeba histolytica (i.e., the 369- to 765-Da chromatographic fraction obtained from the supernatant fluid of axenically grown E. histolytica) led to a swift increase in the intracellular concentration of adenosine 3':5' cyclic monophosphate (cAMP). A weaker response was observed in human polymorphonuclear leukocytes, the locomotion of which, however, is not inhibited by this amebic factor. The same chromatographic fraction obtained from the axenic medium control lacked this effect, at least upon mononuclear phagocytes. On the other hand, both the monocyte locomotion-inhibitory factor and the axenic medium control, possibly through shared cultured medium components, induced comparable increases in guanosine 3':5' cyclic monophosphate (cGMP) in human mononuclear phagocytes and in polymorphonuclear leukocytes, thus suggesting that the latter-nucleotide is not critical for the leukotactic inhibitory phenomenon. Our results suggest that like other leukotactic inhibitors, the monocyte locomotion-inhibitory factor produced by E. histolytica operates through modulations of intracellular cAMP.

Animals↗

PubMed1991

The role of mannose in the receptor of the monocyte locomotion inhibitory factor produced by Entamoeba histolytica.

The effect of the monocyte locomotion inhibitory factor (MLIF) produced by Entamoeba histolytica is diminished, if not cancelled, when human monocytes are pre-exposed to concanavalin A or sodium periodate, respectively, but not when MLIF is pretreated with sodium periodate. When the MLIF-inhibited monocyte locomotion assays were performed in the presence of 12 different carbohydrates, only the runs containing D-mannose, 4-O-beta-galactosyl-mannoside or mannan revealed a significant reduction in the inhibitory effect. Finally, the MLIF activity was virtually absorbed out with mannan-coupled Sepharose 4B beads. This suggests that D-mannose constitutes an essential part of the receptor for MLIF on the human monocyte membrane.

Animals↗

PubMed1992

Adherence of pathogenic and non-pathogenic Entamoeba histolytica strains to neutrophils.

The adherence of polymorphonuclear leukocytes (PMNs) to eight pathogenic and nine nonpathogenic strains of Entamoeba histolytica was examined. No difference between pathogenic and nonpathogenic strains was found. The addition of different carbohydrates confirmed the importance of the 170-kDa lectin of E. histolytica in binding to PMNs, corroborated by the finding that treatment of PMNs with galactosidase inhibited adherence. Inhibition of the microfilament system of E. histolytica using cytochalasin B resulted in a loss of adherence to PMNs. Inhibition of the microtubule system using nocodazole did not affect adherence. Preincubation of the trophozoites with serum resulted in enhanced adherence, but the serum factor responsible for this effect could not be identified. Fibronectin, vitronectin, integrins (CD11/CD18 molecules), complement, and mannose-binding protein did not seem to mediate adherence between E. histolytica and PMNs. In summary, these results indicate that defective adherence mechanisms are not a common feature of nonpathogenic E. histolytica strains.

Acetylgalactosamine↗

PubMed1992

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

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

Animals↗

PubMed1993

Entamoeba histolytica: changes in the zymodeme of cloned nonpathogenic trophozoites cultured under different conditions.

In this paper we report the occurrence of changes in the migration of certain isoenzymes of a cloned strain (MAV-CINVESTAV) of Entamoeba histolytica. This strain was isolated from an asymptomatic carrier and showed an initial nonpathogenic zymodeme I. The transfer of polyxenic trophozoites from Robinson's medium (7% serum) to Jones' medium (30% serum) provoked changes in isoenzyme expression, resulting in the conversion of zymodeme I to a zymodeme that was similar to the XVII zymodeme except for two slow-running bands and a single fast-running band that were detected for hexokinase (HK). This XVII-like zymodeme reverted to zymodeme I when trophozoites were cultured under monoxenic conditions in TY1-S-33 medium (10% serum). When we cultured cloned strain MAV-CINVESTAV under axenic conditions in TY1-S-33 medium, trophozoites expressed a pathogenic zymodeme with two fast-running HK bands and a beta-phosphoglucomutase band. In addition, phagocytosis and the ability of the trophozoites to destroy cell-culture monolayers were expressed only in trophozoites cultured under axenic conditions. The axenization procedure required the presence of live Fusobacterium symbiosum and is also described herein.

Animals↗

PubMed1993

Biochemical and immunological studies on soluble antigens of Entamoeba histolytica.

The soluble antigens of Entamoeba histolytica trophozoites were analysed in detail by biochemical and immunochemical methods. The antigen was highly complex and heterogeneous as revealed by Sephacryl S-300 column chromatography, which showed four distinct fractions. The molecular mass of fractions FI, FII, FIII and FIV was 660, 170, 65 and 13 kDa, respectively. Protein was the major constituent in crude soluble antigen (CSA) and fractions FI and FII (67%, 80% and 90%, respectively). Polysaccharide was predominant in the FIII fraction (59%). Antigenic activity observed after different physico-chemical treatments revealed that CSA and FI antigens were predominantly glycoprotein in nature. However, the antigenicity of FIII antigen was greatly reduced after sodium meta-periodate treatment, whereas no alteration in reactivity was discerned after trypsin treatment. Sodium dodecyl sulphate-polyacryl-amide gel electrophoresis (SDS-PAGE) analysis demonstrated nearly 28 Coomassie blue bands for CSA and 20, 16, 15 and 3 polypeptide bands for the FI, FII, FIII and FIV fractions, respectively. The molecular mass of the polypeptides of these bands ranged from 210 to 20 kDa. Antigenic activity was observed in CSA and in the first three fractions, both in counter immunoelectrophoresis (CIEP) and in enzyme-linked immunosorbent assay (ELISA). However, the highest antigenic activity was noted in fraction FI. Major immunoreactive polypeptides of CSA and FI antigens against whole trophozoite antibody were observed in the 10- to 170-kDa regions. However, major differences in the immunoreactivity of the two antigens were noted at 116 and 14 kDa for FI antigen and at 84, 30 and 20 kDa for CSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Entamoeba histolytica cysts with a defective wall formed under axenic conditions.

Axenic HK9 Entamoeba histolytica strain amoebae, maintained in PEHS medium, displayed several cystic characteristics that involve an active process of cystic wall formation, cellular volume and density diminution, and one or two nuclear divisions. The differentiation process was asynchronic, beginning after the logarithmic growth phase. The axenic cysts, which were maintained in a 50 mOsm/kg medium at 4 degrees C for 72 h, produced growing trophozoites within 1-7 days of incubation at 36 degrees C in fresh medium. Negative results were obtained with trophozoites submitted to the above treatment, and with axenic cysts maintained in double-distilled water at 4 degrees C for 24 h, or in 0.1% sarkosyl, for 10 min at room temperature instead of 55 mosmol/kg medium. Thus, the HK9 E. histolytica strain, cultured in PEHPS, produced under axenic conditions a small proportion of mature, metabolically active cysts, but with an immature or abnormal wall.

Animals↗

PubMed1993

Interaction between trophozoites of Entamoeba histolytica and the human intestinal cell line HT-29 in the presence or absence of leukocytes.

Studies on the interaction between trophozoites of Entamoeba histolytica of pathogenic or non-pathogenic origin and epithelial cells of the human intestine can contribute to the understanding of the pathogenesis of invasive amoebiasis. We have examined the interaction of virulent E. histolytica with the human colonic carcinoma cell line HT-29. Differentiated HT-29 cells are comparable to the mucosa cells to which E. histolytica attaches physiologically. Adherence between E. histolytica trophozoites and HT-29 cells was effectively inhibited by glycoconjugates containing galactose, indicating the importance of the 170-kDa lectin of E. histolytica in binding to intestinal cells. Adherence was not significantly inhibited by glycoconjugates containing N-acetyl-glucosamine, indicating that the 220-kDa lectin of E. histolytica is not involved in binding to HT-29 cells. The destruction of HT-29 cells by pathogenic E. histolytica was dependent on adherence. The destruction was enhanced when polymorphonuclear granulocytes were added to the E. histolytica trophozoites.

Adenocarcinoma↗

PubMed1994

Inhibitory activity of saccharomyces yeasts on the adhesion of Entamoeba histolytica trophozoites to human erythrocytes in vitro.

Adhesion to target cells represents the first step in infection by Entamoeba histolytica. Binding of axenic amoeba (HMI strain) to human red cells in vitro was employed as a model of the adhesion process. The influence of precontact of trophozoites with suspensions of live Saccharomyces boulardii yeasts, their fractions (membranes and yeast-content supernatant before and after filtration to eliminate the membrane) or yeast culture medium before and after fermentation was investigated. N-Acetylgalactosamine (GalNAC) was employed as the reference inhibitory sugar. The percentage of amoebae bearing red cells after pretreatment of amoebae with the various suspensions and derivates was determined. Adhesion was also evaluated by scanning electron microscopy (SEM). Pretreatment of amoebae with the live yeast suspension led to a significant reduction in the percentage of adhesion [32% vs 70% in the phosphate-buffered saline (PBS) control]. Reduced adhesion was also observed with the filtered and unfiltered supernatant of the yeast suspension homogenate [32% and 34%, respectively, vs 69% in the PBS control], yeast culture medium at the end of fermentation [49% vs 76% in the PBS control] and GalNAC [32% vs 72% in the PBS control]. SEM showed a decrease in the number of amoebae bearing red cells and a reduction in the number of red cells adhering to amoebae. We conclude that substances produced by the yeasts compete with red cells for adhesion sites on amoebae.

Acetylgalactosamine↗

PubMed1994

Purification and immunologic characterization of a 30-kDa cysteine proteinase of Entamoeba histolytica.

A 30-kDa cysteine proteinase was purified from extracts of axenically grown trophozoites of Entamoeba histolytica strain HM1:IMSS. The purification procedure involved two consecutive chromatographic steps. Sequence analysis revealed high similarity with histolysin and with other 27-kDa cysteine proteinase. Western-blot analysis using F(ab')2 fragments of a polyclonal antibody raised against the purified enzyme revealed that when the amebic extract was prepared in the absence of proteinase inhibitors there were many positive bands ranging in relative molecular weight from 115 to 12.5 kDa, but when the extract was prepared in the presence of proteinase inhibitors there was only a single 30-kDa positive band. Similar results were obtained with immunoprecipitates. This phenomenon would suggest the formation of multimer aggregates of the 30-kDa cysteine proteinase after partial proteolysis.

Amino Acid Sequence↗

PubMed1992

Adherence between Entamoeba histolytica trophozoites and undifferentiated or DMSO-induced HL-60 cells.

Differentiation of promyelocytic HL-60 cells along the pathway toward granulocytes using dimethyl-sulfoxide (DMSO) led to increased expression of the adhesion molecules CD11b (Mac-1), CD11c (p150,95), and CD35 (CR1). Undifferentiated and differentiated HL-60 cells adhered similarly to Entamoeba histolytica trophozoites, and monoclonal antibodies against adhesion molecules CD11b and CD11c did not inhibit adherence. We therefore suggest that CD11b and CD11c are not involved in the adherence between polymorphonuclear granulocytes and E. histolytica trophozoites.

Animals↗

PubMed1993

Entamoeba histolytica cyst passers. Clinical profile and spontaneous eradication of infection.

The present study was carried out to examine whether Entamoeba histolytica cyst passers suffer from any parasite-related bowel symptoms and to assess the frequency of spontaneous eradication of this infection. The study was carried out in two parts. In part I, stool samples were collected at random from 3536 individuals living in rural communities around Delhi. E. histolytica was isolated by the culture technique in 345 (9.7%) subjects. There was no increase in the prevalence rate of bowel symptoms in the culture-positive compared to the culture-negative subjects. One hundred twenty-four (36%) of the culture-positive subjects agreed to take part in a longitudinal study; the subjects were left untreated and clinical assessment and stool examinations were carried out at three-month intervals. One hundred eighteen (95.2%) subjects had eradicated their parasite spontaneously at the end of one year; none developed any features of invasive amebiasis. Part II of the study was carried out on 625 patients attending our Gastroenterology Clinic. Positive cultures of E. histolytica were obtained from 99 (15.2%) patients. Again, there was no increase in the prevalence rate of bowel complaints in the culture-positive compared to the culture-negative subjects. Moreover, histological appearances of the rectal biopsy specimens were not significantly different between the two groups. Twenty-eight (28.2%) patients agreed to the longitudinal study and all eradicated the parasite spontaneously within five months; none developed any evidence of invasive amebiasis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗