Appearance of sialoglycoproteins in encysting cells of Entamoeba histolytica.

Amoeba-bacterium cultures of Entamoeba histolytica transferred to a hypoosmotic medium depleted of nutrients changed morphologically and biochemically. The cells ejected grains of rice starch, rounded up, and formed a distinct cell wall that was resistant to detergent, bound the sialic acid-specific lectin from Limulus polyphemus, and became fluorescent with Calcofluor M2R. A subpopulation of these cells displayed more than one nucleus. All these signs are characteristic of encysting cells and were also observed in cysts obtained from a human patient. The morphological changes were accompanied by the appearance of two new glycoproteins with apparent molecular sizes of 100 and 150 kilodaltons which contained sialic acid. Sialic acid has been reported to be absent from trophozoites of Entamoeba species. The presence of this sugar residue on cyst-specific proteins parallels recently reported findings during the encystation of the related reptilian parasite Entamoeba invadens. This may indicate a basic role for sialic acid in the encystation of Entamoeba parasites.

A stage-specific sialoglycoprotein in encysting cells of Entamoeba invadens.

A novel sialoglycoprotein with an apparent molecular mass of approximately 250 kDa was detected on the surface of cysts of Entamoeba invadens. Sialic acid was identified in this glycoprotein by gas chromatography after methanolysis; N-acetyl- and N-glycolyl neuraminic acid were identified by thin layer chromatography in hydrolysates of partially purified preparations of the 250 kDa glycoprotein as well as in whole cysts. The sialoglycoprotein is stage-specific and could be detected by binding of wheat germ agglutinin and a specific monoclonal antibody (JAM3) only to precysts and mature cysts but not to trophozoites. A 250 kDa protein could be metabolically labeled with [35S]methionine. This, together with the absence of such a glycoprotein in the encystation medium, suggests that the 250 kDa sialoglycoprotein is not an adsorbed serum glycoprotein. Indirect evidence suggests that the parasite may utilize serum components as a source for sialic acid.

Isolation and partial characterization of the hexokinase isoenzymes from pathogenic and non-pathogenic strains of Entamoeba histolytica.

Isoenzyme electrophoretic patterns (zymodemes) are increasingly used to distinguish between pathogenic and non-pathogenic strains of Entamoeba histolytica. Isolates of E. histolytica from asymptomatic and symptomatic cases have been shown to differ in the electrophoretic mobility of their hexokinase and phosphoglucomutase isoenzymes. The hexokinase isoenzymes from a non-pathogenic strain and from a pathogenic strain of E. histolytica were purified by fast protein liquid chromatography in several steps, which included a separation by size, chromatofocusing, and anion exchange chromatography. The isoenzymes differed in their isoelectric points, which ranged from pH 4.8-5.4, but had very similar kinetic properties and almost identical apparent molecular weights (48,000) in sodium dodecyl sulfate polyacrylamide gels, as well as on gel filtration columns. Comparison of tryptic peptide analysis of each of the isoenzymes indicated considerable homology between the non-pathogenic and pathogenic forms. Antibodies produced against each of the two pathogenic hexokinase isoenzymes inhibited their enzymatic activity. The antibodies also inhibited the activity of the isoenzymes of the non-pathogenic strain. Our findings suggest that the isoenzymes have structural similarities, and that the pathogenic ones differ from the non-pathogenic ones in their electromobility due to post-translational modifications.

Changes in isoenzyme patterns of a cloned culture of nonpathogenic Entamoeba histolytica during axenization.

The axenization of an Entamoeba histolytica isolate with a nonpathogenic isoenzyme electrophoretic pattern (zymodeme) was recently achieved for the first time (15). Forty days after the cells were transferred to the medium used for axenic cultivation, the amebae developed virulence properties, and the zymodeme converted to a pathogenic pattern. To exclude the possibility that the original isolate consisted of two zymodeme populations and that conditions of growth selected for a particular population, the experiment was repeated with a cloned culture of a nonpathogenic (zymodeme III) strain, E. histolytica SAW 1734R clAR, isolated by and obtained from P. G. Sargeaunt. Axenization was accomplished, as before, by transferring trophozoites to TYI-S-33 medium containing a mixture of antibiotics to suppress the growth of the associated bacterial flora and a nutritional supplement consisting of gamma-irradiated bacteria. A change in the hexokinase and phosphoglucomutase isoenzyme pattern was observed 21 days after the amebae had been transferred to the axenic medium but before complete axenization of the amebae had occurred. The change in zymodeme was accompanied by an increase in virulence, as evidenced by the ability of fewer amebae to induce hepatic abscesses in hamsters. A reverse conversion to a nonpathogenic zymodeme was also accomplished by reassociating and subculturing the newly converted pathogenic trophozoites of strain SAW 1734R clAR with the bacterial flora that accompanied this ameba in the original xenic culture. The electromobilities of the hexokinase isoenzymes changed back to their original pattern 7 days after the amebae were returned to xenic growth conditions. Our in vitro results demonstrate that culture conditions and bacterial flora can cause changes in the zymodeme and virulence of a cloned ameba isolate and raise the concern that this could happen also in vivo. Thus, the finding of a particular zymodeme in a culture of E. histolytica isolated from a carrier should not be used to predict a clinical condition or serve as a basis for the recommendation of therapy.

Encystation of Entamoeba invadens IP-1 is induced by lowering the osmotic pressure and depletion of nutrients from the medium.

Trophozoites of Entamoeba invadens IP-1 can be induced to encyst in simple solutions composed of semipermeable constituents (buffer, salts, or sugars) provided that their osmotic pressure is in the range of 60-160 mosmol/kg. Optimal yield of mature cysts was obtained when the osmotic pressure of the medium was 110 mosmol/kg. Encystation could be obtained in the absence of serum although higher yields were obtained in its presence. No difference in the yield of mature cysts was found when either dialyzed or full serum was used. High yields of encystation were obtained (greater than 70%) in the presence of 5% serum in solutions of NaCl, KCl, or MgSO4, suggesting that the mechanism of encystation is not induced via sodium or potassium channels. Cysts were obtained in the presence of 72 mM glucose, indicating that depletion of a carbon source is not the only requirement for encystation. A rapid change in the density of the Entamoeba cells was observed upon transfer of trophozoites (density 1.061-1.073 g/ml) from growth medium to the low osmotic pressure encystation solutions. Within the first 2 min their density decreased (to 1.050 g/ml), but it soon increased, reaching within 30 min a density higher than 1.120 g/ml. As the encystation process continued to completion, the density of the cells gradually decreased, the mature cysts reaching a density of 1.049-1.061 g/ml.

Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence.

In xenic culture, isolates of Entamoeba histolytica from asymptomatic carriers are characterized, with rare exception, by possession of a nonpathogenic zymodeme. During the process of axenizing such an isolate, strain CDC:0784:4, a change in the pattern of the isoenzymes from nonpathogenic zymodeme I to pathogenic zymodeme II was observed 40 days after the amebae had been transferred from a medium for xenic cultivation to one used for axenic cultivation, but before axenization of the amebae had actually occurred. Axenization was accomplished by feeding the amebae lethally irradiated bacteria while suppressing and finally eradicating with antibiotics the bacterial flora accompanying the amebae in the original xenic culture. The change in zymodeme was accompanied by a change in virulence as evidenced by the ability of the amebae to produce hepatic abscesses in hamsters and to destroy monolayers of tissue culture cells. Two explanations are offered for the observed changes in zymodeme and virulence: a zymodeme is not a stable inherent property of the ameba. Alternatively, the original isolate consisted of two zymodeme populations and the conditions of growth selected for one or the other of the populations. In either case, our results suggest that the finding of a particular zymodeme in a culture of E. histolytica isolated from an asymptomatic carrier of the parasite cannot be used to predict a clinical condition or serve as a basis for the recommendation of therapy.

Changes in cell surface proteins and glycoproteins during the encystation of Entamoeba invadens.

Changes in cell surface components of axenically grown trophozoites of Entamoeba invadens which occur during encystation were followed. Protein patterns of trophozoites, immature and mature cyst forms, were analyzed by sodium dodecyl sulphate gel electrophoresis. Total protein profiles of trophozoites and cyst forms stained by Coomassie blue gave similar patterns. In contrast, a number of different bands were observed in gels stained with the carbohydrate-specific Schiff's reagent as well as when nitrocellulose blottings were treated with 125I-radiolabelled wheat germ or soybean agglutinins. The most notable differences were bands at 250 and 95-105 kDa present in the cyst forms and absent in the trophozoites, and two bands at 70 and 75 kDa present in the latter and missing in the cysts. Labelling of trophozoites and cyst cell surfaces by iodination with lactoperoxidase revealed a number of protein bands which were exposed on the trophozoite surface and missing in the cysts. Moreover, gel electrophoresis patterns of non-reduced or reduced samples also differed considerably, indicating that a number of proteins are linked by disulphide bonds. This study shows that specific glycoproteins are produced during cyst formation.

The occurrence of antibodies to hidden and exposed determinants of surface antigens of Trichinella spiralis.

Mice were infected per os with Trichinella spiralis and their lymphocytes were removed and fused with mouse myeloma cell line P3 x 63Ag8653P3 for the selection of monoclonal antibodies to biochemically defined, stage-specific surface antigens of 3 parasite developmental stages: muscle larvae, adults and newborn larvae. Two separate antibodies against a defined single surface antigen of each stage were isolated. In each separate case the pair of monoclonal antibodies precipitated the same component from detergent-solubilized surface antigen preparations, but only one was able to bind to the surface of the living worm. The other must therefore be directed against an antigenic epitope which is obscured in the intact worm surface. The latter type of antibody is unlikely to be involved in the initial phase of parasite rejection and hence is another example of a non-protective host antibody response. The stimulus for its synthesis may be release of surface antigen, which does occur in vitro. One surface antigen of the newborn larvae is only detected by antibody in the first 6 h after birth; thereafter its presence is obscured as other antigens appear. The major surface antigen of the infective larvae contains carbohydrate determinants which are not available at the parasite surface. In addition, it displays great molecular heterogeneity but all variants appear to be derived from a common polypeptide structure.

Polyamines in Entamoeba invadens.

The polyamine content of Entamoeba was measured by a procedure that involved benzoylation followed by high performance liquid chromatography (h.p.l.c.). A high concentration of putrescine and significant amounts of spermidine and spermine were found in actively growing trophozoites and in the cyst forms of the organism. In contrast, trophozoites in stationary phase had greatly reduced amounts of putrescine and exhibited peaks in h.p.l.c., possibly indicative of acetylated polyamines. alpha-D,L-difluoromethylornithine (DFMO) lowered the concentration of polyamines in growing trophozoites, but did not inhibit the degree of proliferation. There is evidence for pathways of polyamine biosynthesis in Entamoeba other than through ornithine decarboxylase (ODC).

Heterogeneity of B-cells.

Two rat monoclonal antibodies, NIM-R2 and NIM-R3, were prepared that only recognize B-cells in murine secondary lymphoid tissues. Both showed differential reactivity in that they reacted most strongly with nonoverlapping populations of B-cells. They also reacted differentially with some cells of the primary lymphoid tissue, recognizing 90-95% of bone marrow cells, again in a nonoverlapping manner and with NIM-R2 accounting for twice as many cells as NIM-R3. The antibody NIM-R2, but not NIM-R3, also recognized thymocytes and red blood cells. The determinant recognized by NIM-R2 is of interest because it fluctuates at four defined stages of B-cell development: pre-B to immature B, activation of mature B, differentiation of memory cells, and differentiation of antibody-secreting cells. Particularly noteworthy was the differential reactivity of NIM-R2 with virgin and memory B-cells. Those cells that stained strongly with the antibody gave excellent primary responses, but were unable to transfer secondary responses, whereas weakly stained B-cells transferred memory, but could not generate primary antibody responses in vitro. Thus NIM-R2 recognized a cell-surface determinant that presumably decreases in density as primary B-cells differentiate into memory B-cells. A survey of sIg, I-A, Fc receptors, and major glycoproteins of B-cells from spleen, Peyer's patches, mesenteric lymph nodes, and peripheral lymph nodes failed to reveal any major differences between the cells from these different lymphoid organs.

B cell subpopulations in the mouse: analysis with monoclonal antibodies NIM-R2 and NIM-R3.

Two rat monoclonal antibodies, NIM-R2 and NIM-R3, have been produced using the rat myeloma line 210RCY3-Ag1.2.3 and spleen cells from Lou rats immunized with mouse spleen cell plasma membrane or cells. The antibodies identify nonoverlapping populations of surface Ig-positive cells in the spleen and a large (95%) proportion of bone marrow cells. Both recognize differentiation antigens in that the surface representation of the markers changes during the development of the cell. The NIM-R3 specificity does not appear until three weeks of age in both the spleen and bone marrow and may be on a more mature set of cells. In contrast, the NIM-R2 antibody, which stains the pre-B cell line 70Z/3 and binds to neonatal cells, may recognize pre-B cells in the bone marrow. There was no clear-cut correlation between the presence or absence of surface IgM, surface IgD or complement receptors on B cells positive or negative for either NIM-R2 or NIM-R3. Most interesting was the finding of identical total surface Ig densities on cells which stained weakly or strongly with NIM-R2, since these two B cell subpopulations are shown to be enriched for memory and virgin B cells, respectively. To bias the production of monoclonal antibodies to distinct populations of cells, the immunogen for the NIM-R3 fusion was depleted of cells strongly reactive with NIM-R2. This method is of general applicability in the production of monoclonal antibodies to complementary populations of cells.

Preparation and properties of a cytotoxic monoclonal rat anti-mouse Thy-1 antibody.

A hybrid cell line secreting a cytotoxic rat IgM antibody with specificity for both the 1 and 2 alleles of mouse Thy-1 was selected. The fusion was between rat spleen cells immunised to SBA spleen cell plasma membranes and the rat myeloma cell line 210RCY3-Ag123. The cell line can be propagated in Lou-strain rats to yield large volumes of antibody-containing ascites with a cytotoxic titre of 10(6)-10(7) for peripheral T cells. The antibody is relatively stable and can be used for indirect fluorescence assays in conjunction with a fluorochrome (e.g. fluorescein)-coupled goat anti-rat immunoglobulin (pre-absorbed on mouse immunoglobulin). More simply, it can be directly conjugated to a fluorochrome and used in association with an appropriately absorbed goat anti-mouse Ig coupled with a contrasting and different fluorochrome (e.g. rhodamine) to allow simultaneous enumeration of both T and B lymphocytes.