Histolytica Cells Research Articles

Growth stimulation by serum in Entamoeba histolytica is associated with protein tyrosine dephosphorylation

Very little protein tyrosine phosphorylation was observed in growing (exponential-phase) Entamoeba histolytica cells by immunoblotting and quantitative immunofluorescence. After 1 h of serum deprivation, two proteins (42 and 38 kDa in SDS-PAGE) were tyrosine phosphorylated and two more proteins (96 and 63 kDa) also showed tyrosine phosphorylation when examined after 4 h of serum deprivation. Intense enhancements of anti-phosphotyrosine immunofluorescence levels were observed during this period of serum withdrawal. Membrane-associated tyrosine kinase activity reached a peak (3.5-fold increase) 1 h after serum deprivation and decreased thereafter reaching a basal level by 2 h of serum deprivation. Interestingly, tyrosine kinase activities remained unaffected by serum stimulation (2–60 min) of serum-deprived cells. Also, during this period of serum stimulation tyrosine phosphorylated proteins of serum-deprived cells were dephosphorylated. Tyrosine phosphatase activities were suppressed during serum deprivation and on serum addition to serum-deprived cells tyrosine phosphatase activities increased significantly. Our data attest that protein tyrosine phosphorylation was associated with growth inhibition of E. histolytica and serum stimulation of E. histolytica produced tyrosine phosphatase activation and protein tyrosine dephosphorylation.

Growth stimulation by serum in Entamoeba histolytica is associated with protein tyrosine dephosphorylation.

Very little protein tyrosine phosphorylation was observed in growing (exponential-phase) Entamoeba histolytica cells by immunoblotting and quantitative immunofluorescence. After 1 h of serum deprivation, two proteins (42 and 38 kDa in SDS-PAGE) were tyrosine phosphorylated and two more proteins (96 and 63 kDa) also showed tyrosine phosphorylation when examined after 4 h of serum deprivation. Intense enhancements of anti-phosphotyrosine immunofluorescence levels were observed during this period of serum withdrawal. Membrane-associated tyrosine kinase activity reached a peak (3.5-fold increase) 1 h after serum deprivation and decreased thereafter reaching a basal level by 2 h of serum deprivation. Interestingly, tyrosine kinase activities remained unaffected by serum stimulation (2-60 min) of serum-deprived cells. Also, during this period of serum stimulation tyrosine phosphorylated proteins of serum-deprived cells were dephosphorylated. Tyrosine phosphatase activities were suppressed during serum deprivation and on serum addition to serum-deprived cells tyrosine phosphatase activities increased significantly. Our data attest that protein tyrosine phosphorylation was associated with growth inhibition of E. histolytica and serum stimulation of E. histolytica produced tyrosine phosphatase activation and protein tyrosine dephosphorylation.

Mucoid presentation of acute enterocolitis in children: a hospital‐based case‐control study

A hospital‐based case‐control study was carried out to clarify the characteristics of mucoid presentation of acute enterocolitis in children. One hundred sixty‐eight cases of acute mucoid enterocolitis (study population) were compared with 200 cases of watery diarrhoea and 118 cases of blood dysentery (control groups) on the basis of clinical characteristics and findings on stool examination. Study and control groups were comparable with respect to age, body weight and nutritional status. There was no significant difference in clinical characteristics (duration of diarrhoea, stool frequency, presence of vomiting, fever and dehydration) between patients suffering from mucoid enterocolitis and blood dysentery. However, watery diarrhoea patients had significantly high frequencies of vomiting (p = 0.00001) and dehydration (p = 0.00001). High numbers of microscopic red blood cells (mean±SD: 40.8 ± 16.8) and white blood cells (40.6 ± 18.0) were present in faecal samples of the patients with mucoid enterocolitis, which is indicative of infection caused by enteroinvasive enteropathogens. Shigella was a commonly identified enteropathogen in patients with mucoid enterocolitis (40.5%) and in patients with dysentery (46.6%), with no statistically significant difference (p= 0.30). Isolation of Salmonella was statistically similar in study and control groups. However, Entamoeba histolytica was detected in significantly high frequency in patients with mucoid enterocolitis as compared to the patients with dysentery (p = 0.0004) and watery diarrhoea (p = 0.00004). Our results indicate that mucoid enterocolitis patients are infected with enteroinvasive enteropathogens, and that stool examination is useful in establishing the aetiological diagnosis. □Acute enterocolitis, diarrhoea, dysentery, Entamoeba histolytica, faecal red and white blood cells, mucoid presentation, Shigella, Salmonella

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.

Heterogeneity of DNA content and expression of cell cycle genes in axenically growing Entamoeba histolytica HM1:IMSS clone A

The cell division cycle of Entamoeba histolytica was studied using multi-parametric flow cytometry in asynchronous and partially synchronised cells. Dynamic changes in the DNA synthesis and DNA content of axenically growing trophozoites were observed by using 5-bromo-2′-deoxyuridine (BrdU) uptake and DNA specific fluorochromes. It was observed that DNA synthesis in these cells continues beyond the typical S-phase stop point when DNA duplication is complete. Asynchronously growing E. histolytica cells could be synchronised by serum starvation followed by serum re-addition. BrdU incorporation in synchronised cells showed that cell synchrony is maintained for at least one generation time, in which the G1 phase lasts for 2–3 h and the S-phase lasts for 5–6 h. Analysis of our results revealed that E. histolytica trophozoites, growing in axenic medium, are made up of a heterogenous population of euploid and polyploid cells. The number of polyploid cells increases with age of the cells in culture. Expression of putative cell cycle and signal transduction markers was studied using specific antibodies and changes in their expression levels have been correlated with changes in the DNA content. Based upon our results we could identify G1, S and G2 phases of the cell cycle of E. histolytica and also predict the mechanism underlying the generation of polyploidy in these cells, which may have significant effects on its biology and pathogenesis.

Interaction between Entamoeba histolytica and intestinal epithelial cells involves a CD44 cross-reactive protein expressed on the parasite surface.

This study shows that Entamoeba histolytica binds hyaluronic acid. The binding molecule was identified as an 80-kDa membrane protein and was recognized by anti-CD44 monoclonal antibodies. These data indicate that a CD44 cross-reacting adherence molecule is expressed on E. histolytica.

Invasion and metastasis mechanisms in Entamoeba histolytica and cancer cells. Some common cellular and molecular features

Cell invasion in multicellular organisms is a phenomenon usually attributed to malignant cells. The most threatening behavior of malignant neoplasms is their capacity to spread to neighboring structures and to reach distant organs, where they multiply in a disorderly manner (Fidler, 1990). Invasion, however, is not an exclusive phenomenon of malignant transformed cells. Phagocytes have the capacity to invade as part of their normal biological activity, and fibroblasts and endothelial cells display invasive ability during tissue repair (Armstrong, 1985). Some pathogens also colonize and invade host tissues. Entamoeba histolytica, the protozoon responsible for human amebiasis, becomes pathogenic only when it is

Serum-independent and serum-dependent cytoadherence in the interaction of Entamoebahistolytica with mammalian target cells

Entamoeba histolytica kills target cells'only on direct contact, suggesting that trophozoite-mediated cytolysis is initiated by the contact between trophozoites and target cells. We have shown that adherence between E. histolytica and target cells (polymorphonuclear granulocytes, erythrocytes, Chinese hamster ovary cells, human colon carcinoma cells) was inhibited by specific carbohydrates, and adherence between E. histolytica and polymorphonuclear granulocytes (PMN) was enhanced by preincubation of the trophozoites with serum. Inhibition of adherence clearly paralleled inhibition of cytolysis and phagocytosis of target cells. Cytolysis of PMN, however, was not increased by preincubation of the trophozoites with serum. These results suggest that the effector functions of trophozoites are only dependent on carbohydrate-specific adherence mechanisms mediated by the amoebic Gal/GalNAc-binding lectin. E. histolytica trophozoites themselves can be killed by PMN, depending on the virulence of the trophozoites. PMN could not kill E. histolytica trophozoites more effectively when the adherence was enhanced by preincubation of the trophozoites with serum or when adherence was only mediated by serumdependent mechanisms.

Possible role of calmodulin in the secretion of Entamoeba histolytica electron-dense granules containing collagenase.

Entamoeba histolytica cells secrete electron-dense granules (EDGs) that have collagenase activity. To study the possible involvement of calmodulin (CaM) on EDG secretion, the effect of several CaM antagonists (TFP, R24571, W-7, W-5, dibucaine and DL-propranolol) was tested on this cellular function. Except for W-5 and dibucaine, the rest of these compounds inhibited EDG secretion. Transmission electron microscopy of collagen-activated trophozoites showed numerous EDGs located in or near the surface membrane. In contrast, trophozoites incubated with TFP showed no EDGs. Protein kinase C inhibitors (H-7, ML-9) had no effect on EDG secretion, suggesting that CaM antagonists acted by selectively inhibiting CaM. These results suggest that a CaM-dependent process is involved in EDG secretion.

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.

In vitro and in vivo interaction between trophozoites of Entamoeba histolytica and gerbil lymphoid cells.

The in vitro and in vivo antiamoebic cytotoxic effects of peritoneal exudate cells and mesenteric lymph node lymphocytes of gerbils with cecal amoebiasis or those immunized with amoebic extract were investigated. A differential effect of the lymphoid cells against trophozoites of nonpathogenic and pathogenic strains of Entamoeba histolytica was observed. Nonpathogenic amoebae were more susceptible to killing by lymphoid cells than pathogenic amoebae in vitro and in vivo in infected or immunized animals. These data suggest that during the course of cecal amoebiasis in gerbils, a differential stimulation or depletion of cytotoxic cells in the lymphoreticular tissues occurs, resulting in an impaired cell-mediated immune response.

Interactions between trophozoites of Entamoeba histolytica and cells of the immune system.

The interaction between Entamoeba histolytica trophozoites and cells of the immune system was studied in vitro. Both immune as well as non-immune mononuclear cells (MNC) to some extent were capable of exerting a cytotoxic effect on E. histolytica trophozoites. Incorporation of immune serum directed against the trophozoites in the reaction mixture resulted in a marked increase in the killing capacity of MNC. Highest killing was observed (96.7 +/- 1.04%) with MNC obtained from animals immunized with fraction-I(F-I) of the crude amoebic extract in association with anti-F-I serum. This cytotoxic event was found to be independent of complement involvement and required very low antibody concentrations. This antibody-dependent cellular cytotoxicity against E. histolytica in vitro was found to correlate well with the results of experiments reported earlier by us and others where a high degree of protection has been shown after vaccination of animals with F-I followed by challenge with highly virulent strains of E. histolytica.

Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate.

Treatment of living entamoeba histolytica cells with low concentrations of concanavalin A (con A) and peroxidase results in redistribution of the plasma membrane con A receptors to one pole of the cell where a morphologically distinct region--the uroid--is formed. Capping of con A receptors is not accompanied by parallel accumulation of ruthenium red-stainable components. In capped cells, the pattern of distribution of acidic sites ionized at pH 1.8 (labeled by colloidal iron) at the outer surface and of membrane particles (integral membrane components revealed by freeze-fracture) is not altered over the uroid region. Cytochemistry of substrate-attached microexudate located in regions adjacent to E. histolytica cells demonstrates the presence of con A binding sites and ruthenium red- and alcian blue-stainable components and the absent of colloidal iron binding sites. In a previous report we demonstrated that glycerol-induced aggregation of the plasma membrane particles is accompanied by a discontinuous distribution of colloidal iron binding sites, while con A receptors and acidic sites ionized at pH 4.0 remain uniformly distributed over the cell surface. Taken together, our experiments show that, in E. histolytica cells, peripheral membrane components may move independently of integral components and, also, that certain surface determinants may redistribute independently of others. These results point to the complexity of the membrane structure-cell surface relationship in E. histolytica plasma membranes relative to the membrane of the erythrocyte ghost where integral components (the membrane-intercalated particles) contain all antigens, receptors, and anionic sites labeled so far. We conclude that fluidity of integral membrane components (integral membrane fluidity) cannot be inferred from the demonstration of the mobility of surface components nor, conversely, can the fluidity of peripheral membrane components (peripheral membrane fluidity) be assumed from demonstration of the mobility of integral membrane components.

Induced redistribution of membrane particles, anionic sites and con A receptors in Entamoeba histolytica.

THE extent to which the distribution of antigens and other receptors at the cell surface is determined by structural components within the plasma membrane is largely unknown. In erythrocyte ghost membranes, freeze-fracture and freezeetch studies have demonstrated that structural components, ‘membrane intercalated particles’, are the exclusive sites at the surface which bear A and B antigens1,2, wheat germ agglutinin3, influenza virus3, con A receptors (Pinto da Silva and Nicolson, unpublished), and anionic sites4. It is clear, however, that conclusions derived from the study of this membrane system cannot be freely extrapolated to living cells where the relationship cell surface-membrane structure may be more complex. We report a study of this relationship on the plasma membrane of living Entamoeba histolytica cells in conditions which induce a redistribution of membrane intercalated particles, anionic sites or con A receptors.