Pore-forming protein from Entamoeba histolytica forms voltage- and pH-controlled multi-state channels with properties similar to those of the barrel-stave aggregates.

Pore-forming protein from Entamoeba histolytica forms cation-selective channels in planar bilayers. With increasing potentials, the open-state probability of these channels decreases, and channel aggregates collapse (Young, J.D.-E. and Cohn, Z.A. (1985) J. Cell. Biochem. 29, 299-308). In this communication we report the following observations: (i) incorporation of the pore in black-lipid membranes was stimulated by membrane potential, (ii) pores were rectifying, (iii) breakdown of pores resulted in a continuous spectrum of subconductance states, (iv) the open-state probability increased strongly with pH. This pattern of behaviour is similar to that of the barrel-stave aggregates (alamethicin and related toxins). We therefore conclude that the amebal pores, like those of the barrel-stave class, may consist of complexes involving variable numbers of membrane-spanning subunits.

Pathogenic and non-pathogenic Entamoeba: pore formation and hemolytic activity.

Pore-forming activity in planar lipid bilayers and liposomes of extracts from differentially pathogenic Entamoeba and the capacity of trophozoites and subcellular fractions to lyse human red blood cells (hrbc) were investigated. In all amebas studied, the two activities paralleled each other. They were high in E. histolytica irrespective of the virulence of the particular strain, but low in non-pathogenic E. histolytica-like amebas of human origin as well as in E. invadens, which is pathogenic for reptiles, and in E. moshkovskii isolated from sewage. We conclude that the capacities to insert pores and to lyse are not sufficient for virulence although they may be necessary. The subcellular distribution of the hemolytic activity of E. histolytica and its sensitivity to a variety of inhibitors and activators differ from those of other known amebic cytotoxic activities including pore formation. Therefore, there may be an additional constituent of E. histolytica involved in the cytotoxicity of the parasite.

Effects of amiloride on Na+ content and pinocytosis in Entamoeba histolytica.

Amiloride, a blocker of Na+ leak and Na+-H+ exchange in animal cells, caused cells of Entamoeba histolytica to release Na+ (up to 40% of their original Na+ content within 90 min, at an amiloride concentration of 3 mM); K+ content was not affected. By comparing the unidirectional uptake of 22Na+ with that of the fluid-phase marker 125I-labeled poly(vinylpyrrolidone) we established that the amiloride-induced Na+ loss was due to inhibition of pinocytic Na+ uptake rather than to blockage of an amiloride-sensitive transport system in the plasma membrane. Amiloride penetrated the cells, and both its intracellular concentration and its effect on pinocytosis increased with pH. The permeant weak base quinacrine similarly inhibited pinocytosis in a pH-dependent manner. We conclude that the effect of amiloride on pinocytosis and, consequently, on Na+ content was due to its properties as a permeant weak base.

Effects of cytochalasin B on Na+ content and cell volume of Entamoeba histolytica.

Cells of Entamoeba histolytica accumulated K+ and extruded Na+ compared to the concentrations of those ions present in the growth medium. Pinocytic activity, measured by the uptake of horseradish peroxidase of 125I-polyvinylpyrrolidone, was high (up to 0.3 ml/ml cells per h). Upon addition of cytochalasin B, at a concentration (20 microM) that completely blocked pinocytosis, cells lost up to 40% of their Na+ content within 90 min; K+ content was not affected or increased slightly compared to control cells without the inhibitor. Cation loss was associated with cell shrinkage. The dose-response curves for the effects of cytochalasin B on pinocytosis and Na+ content were identical. These data provide direct evidence that pinocytosis is an important component of the homeostatic system for Na+.

Immunology of Entamoeba histolytica in human and animal hosts.

Although Entamoeba histolytica induces humoral and cellular immune responses in both human and animal hosts, there is no indication of postinfection immunity in humans; in contrast, several other mammals are protected by prior infection or immunization. The exacerbation of the disease by immunosuppression suggests a protective function of still-unknown defense mechanisms. Specific local and circulating antibodies are produced regularly during invasive amebiasis. Although serum antibodies, together with complement, are lytic to the trophozoites in vitro, the poor correlation of these antibodies with resistance contradicts a protective capacity in vivo. The parasite may evade harm by shedding antigen-antibody complexes from its surface. Demonstration of immediate-type skin reactions, elevated IgE titers, and specific antiamebic IgE suggests that anaphylaxis occurs. The function of the anaphylactic reaction in pathology and resistance remains to be studied. Delayed hypersensitivity parallels healing or resistance and is retarded in human hepatic amebiasis. This observation is consistent with a protective role of cell-mediated immunity.

Mitogenicity of Entamoeba histolytica extracts for murine lymphocytes.

Aqueous extracts or aqueous extracts of delipidated Entamoeba histolytica (E.h.e.) contain a mitogenic principle for murine lymphocytes. As detected by [3H]-thymidine incorporation and blast transformation, E.h.e. acted predominantly on T cells of splenic origin, but not on thymocytes or bone marrow cells. Furthermore, E.h.e. induced proliferation of a subset of non-T-cells which is present in the spleen of athymic nude mice, adhered to nylon wool, but could not be activated to produce antibody. It seems possible that polyclonal activation of lymphocytes by E. histolytica might play a role in the disturbance of the immune system as manifested in the impaired cell mediated immune response of E. histolytica infected hosts.

Surface properties of Entamoeba: increased rates of human erythrocyte phagocytosis in pathogenic strains.

The assertion that ingestion of human erythrocytes is restricted to invasive strains of Entamoeba histolytica has not been evaluated previously by comparative studies. In this report we describe the in vitro ingestion of human erythrocytes by pathogenic and nonpathogenic Entamoeba. Microscopic evaluation of erythrophagocytosis by eight different Entamoeba grown in culture revealed that strains of E. histolytica isolated from cases of human dysentery show a much higher rate of erythrocyte ingestion than nonpathogenic strains. However, all strains are able to phagocytize erythrocytes. The extremely high rate of phagocytic activity shown by pathogenic E. histolytica could be one of the properties related to the pathogenicity of this parasitic protozoan.

[Phagocytosis of human erythrocytes by Entamoeba histolytica. Quantitative study]. / Fagocitosis de eritrocitos humanos por Entamoeba histolytica. Estudio cuantitativo.

The degree of eythrophagocytosis of two recently isolated strains of E. histolytica was measured by microscopic examination. Amebas isolated from a patient with amebic rectocolitis (strain HM22:IMSS, monoxenic) ingested human red blood cells faster and in larger numbers than trophozoites isolated from an asymptomatic carrier (HM27:IMSS, monoxenic). The results suggest that an increased rate of phagocytosis could be one of the surface properties characteristic of the invasive strains of E. histolytica.

Surface charge of trypanosoma cruzi. Binding of cationized ferritin and measurement of cellular electrophoretic mobility.

The surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi was evaluated by means of binding of cationized ferritin to the cell surface as visualized by electron microscopy, and by direct measurements of the cellular microelectrophoretic mobility (EPM). Epimastigote forms had a mean EPM of -0.52 micrometer-s-1-V-1-cm and were lightly labeled with cationized ferritin. In contrast, bloodstream trypomastigotes had a much higher EPM (-1.14), and the surface was heavily labeled with cationized ferritin. When trypomastigotes from staionary phase cultures were isolated on DEAE cellulose columns, the mean EPM was found to be significantly lower (-0.63), and labeling with cationized ferritin decreased. With a mixed population containing epimastigote, trypomastigote, and intermediate forms, EPM values ranging between -0.70 to -1.14 were found. From these observations we conclude that there is a definite increase in negative surface charge during development from epi- to trypomastigote forms of T. cruzi.

Surface properties related to concanavalin A-induced agglutination. A comparative study of several Entamoeba strains.

Pathogenic strains of Entamoeba histolytica are more easily agglutinated with concanavalin A (Con A) than strains isolated from human asymptomatic carriers. All three pathogenic strains studied here were found to agglutinate with low concentrations of Con A in contrast to various nonpathogenic axenic strains of amebas, characterized by their ability to grow at room temperature. Our present observations suggest that the extreme susceptibility of pathogenic strains of E. histolytica to agglutinate with Con A is related to their higher capacity for lectin binding and to their lack of detectable repulsive charges at the cell surface. The amount of fluorescein-tagged Con A bound to the surface was much higher in pathogenic strains. Only nonpathogenic strains showed a detectable negative surface charge as studied both by means of cell microelectrophoresis and by labeling cells with cationized ferritin at 0 degrees C. The mobility of surface Con A receptors estimated as the percentage of caps was comparable in all strains. Results of one strain cultured in axenic and monoxenic conditions suggested that bacteria can modify the behaviour of E. histolytica trophozoites by altering surface properties of the amebas.

Ir-gene control of antibody class production.

The antibody response in mice to DNP-insulin is under Ir-gene control. The Ir gene defects in two strains have been analyzed. In both cases the IgG immune response was impaired whereas the IgM response was not affected. One H-2 gene haplotype was characterized by lack of IgG response, independent of the immunization protocol. A second H-2 haplotype manifested a low response of IgG after immunization with Bordetella pertussis as an adjuvant but a high response after complete Freund's adjuvant. It is proposed that a low level of T cell help induces the production of IgM antibodies, intermediate levels allow few IgG clones to develop, and high levels induce a heterogeneous IgG response.

Similar combining sites of IgG1 and IgG2a antibodies in individual mice as judged from affinity and rate constant measurements.

Within individual sera, mouse IgG1 and IgG2a subclass antibodies against the antigenic determinant oligo-D-alanine are likely to have similar combining sites. The antibodies are characterized by two methods, by their rate of binding to antigen equipped sheep red blood cells and by the hapten inhibition of antigen binding. The two characteristics are shown to be independent. The subclass antibodies of thirteen different sera differ by a factor of about 10 in their rate constant and by a factor of about 50 in affinity for hapten. In contrast, IgG1 and IgG2a within the same serum have strikingly similar rate constants (factor 1.02-1.33) as well as very similar affinities (factor 1.1-2.7). Since it is highly improbable that correlation in two independent criteria occurs by chance, the IgG1 and IgG2a antibodies are assumed to have similar combining sites.

Similar specificity of IgG1 and IgG2a antibodies in individual mice.

In this study the previous finding of similar variable regions in individual IgG1 and IgG2a antibody populations is extended by the demonstration of similar fine specificity of IgG1 and IgG2a combining sites. Antibody populations from individual mice directed against oligo-D-alanine determinants were analyzed in their cross-reactivity towards 5 heterologous dipeptides. This was done by mixing antibody and hapten followed by determination of free antibodies in a kinetic red cells sensitization assay. The comparison of hyperimmune sera from 10 mice showed that genetically identical mice can differ significantly in their cross-reaction pattern. Within each serum the cross-reaction pattern was determined for IgG1 and IgG2a. With a few exceptions the same individual pattern was found in both IgG1 and IgG2a antibody populations. This was taken as evidence that the combining sites of IgG1 and IgG2a anti-oligo-D-alanine antibody populations in an individual mouse are similar.