Immunomodulation in progeny from thymectomized primiparous mice exposed to benzo(a)pyrene during mid-pregnancy.

Previous studies have shown that Benzo(a)pyrene (B(a)P3) given to non-thymectomized (NTX) female mice alters expression of T cell subsets and suppresses cell mediated immunity (CMI) and humoral immunity (HI) in the progeny. Thus, maternal exposure to B(a)P may influence changes in progeny immune status. To understand how maternal cellular and humoral factors influence embryonic development of progeny immunity, adult female mice were thymectomized (TX) at 6 weeks, mated and injected with 150 microg B(a)P)/g body weight at 12 days of pregnancy. After B(a)P exposure, the following studies were performed: (A) Maternal reproductive capacity and survival rate of progeny; (B) Detection of T cells in progeny thymus; (C) Functional characteristics of progeny thymus or spleen. Maternal thymectomy and B(a)P exposure reduced average litter size by 40%. Serological sensitivity of thymus cells with anti-Thyl + complement occurred at a higher dilution of mAb in progeny from TX mothers exposed to B(a)P, suggesting that B(a)P-thymectomy led to increased sensitivity of developing thymocytes to mAb plus complement. Progeny from TX mothers exposed to B(a)P showed enhanced thymic CMI, but suppressed splenic CMI and HI. Thus, thymectomy prevents CMI immunosuppression by B(a)P, while HI is still suppressed. These results indicate that the maternal thymus is necessary for incurring the effect of B(a)P on progeny CMI.

The primary structure of an Entamoeba histolytica beta-hexosaminidase A subunit.

An Entamoeba histolytica gene (hex-A1) that encodes subunit A of the lysosomal enzyme beta-hexosaminidase has been cloned and sequenced. The inferred 59 kDa hex-A1 protein has the same molecular weight and 32% amino acid residue identity with the human and mouse proteins and 28% residue identity with the Dictyostelium protein. Northern blot analysis identified a mRNA of approximately 1.6 kb, which is in agreement with the expected size of a mRNA encoding the 522 amino acid hex-A1 protein. Southern blot analysis indicated the presence of at least two beta-hexosaminidase A subunit genes.

Entamoeba motility: dynamics of cytoplasmic streaming, locomotion and translocation of surface-bound particles, and organization of the actin cytoskeleton in Entamoeba invadens.

The dynamics of cytoplasmic streaming, retrograde translocation of externally bound particles and locomotion by Entamoeba invadens were compared. Locomoting amoebae were monopodial, exhibited fountain flow cytoplasmic streaming and translocated externally bound erythrocytes to the rear of cells. The rates of rearward flow of peripheral cytoplasmic vacuoles and of the externally bound particles were equal to the rate of cell forward locomotion. Rhodamine-phalloidin staining revealed a distinct cortical polymerized actin cytoskelton. This was least evident about the periphery of the advancing pseudopod, increased in density toward the rear of the cell and was most concentrated in the uroid. A monoclonal anti-eucaryotic actin antibody, which recognized monomeric Entamoeba actin on immunoblots, stained trophozoites by indirect immunofluorescence throughout the cytoplasm, but not in the cortical regions stained by rhodamine-phalloidin. This and other evidence implied that the antibody recognized only unpolymerized actin in Entamoeba. We propose that locomotion, cytoplasmic streaming and translocation of externally bound particles are driven by a common actin-based mechanism in Entamoeba, possibly involving retrograde cortical actin flow and recycling.

High resolution two-dimensional protein gel evidence of differential gene expression during Entamoeba encystation.

As an initial step of investigation into the regulatory mechanisms of encystation in Entamoeba, we compared the protein profiles of newly formed cysts and trophozoites of E. invadens using high resolution two-dimensional PAGE and digitized video image analysis of silver stained gels. A total of 155 proteins unique to trophozoites and a total of 72 proteins unique to cysts were detected. Five of the most prominent cyst specific proteins were identified as candidates for further study. These results imply that extensive changes in gene expression accompany the progression of this parasite through its life cycle.

Actin associated proteins of Entamoeba histolytica.

Treatment of E. histolytica HM1-IMSS trophozoite extracts to conditions that produce gels of actin and associated cytoskeletal proteins in other ameboid cells caused formation of macroscopic actin rich complexes (ARCs). The one-dimensional PAGE protein profile of this ARC was similar to those of Dictyostelium and Acanthamoeba actin gels. Formation of the E. histolytica ARCs was enhanced by added lipids. In addition to actin, the ARC was enriched with proteins that showed cross-reactivity to antibodies to alpha-actinin and the 50K actin binding protein (elongation factor 1 alpha) from Dictyostelium. E. histolytica ARCs appear to be comprised of a number of actin cytoskeleton proteins and provide a source for their isolation and characterization.

Roles of target cell membrane carbohydrate and lipid in Entamoeba histolytica interaction with mammalian cells.

Latex beads and liposomes carrying glycoproteins with carbohydrate sequences recognized by an Entamoeba histolytica galactose-specific binding protein were assessed for their ability to adhere to trophozoites and to stimulate amoeba actin polymerization. Glycoprotein-conjugated beads bound significantly to amoebae but did not stimulate actin polymerization. Glycoprotein-bearing liposomes bound to amoebae and did enhance actin polymerization, as do recognized glycosphingolipid-bearing liposomes (G. B. Bailey, E. D. Nudelman, D. B. Day, C. F. Harper, and J. R. Gilmour, Infect. Immun. 58:43-47, 1990). Liposome-stimulated actin polymerization occurred only if the vesicle contained negatively charged phospholipid. It was concluded that both glycoprotein and glycosphingolipid glycans on the target cell surface are involved in attachment to E. histolytica but do not themselves induce the transmembrane signals that lead to cytoskeleton activation and target destruction. This requires interaction with lipids of the target membrane bilayer.

Specificity of glycosphingolipid recognition by Entamoeba histolytica trophozoites.

The ability of purified glycosphingolipids to enhance liposome-stimulated Entamoeba histolytica actin polymerization was assessed as a means of defining the specificity of mammalian cell membrane lipid glycan recognition by this parasite. Synthetic liposomes containing a variety of individual glycosphingolipids bearing neutral, straight-chain oligomeric glycans with galactose or N-acetylgalactosamine termini stimulated rapid (90-s) polymerization of amoeba actin. Glycans with terminal N-acetylglucosamine residues were not stimulatory at all or were only weakly stimulatory. Glycans with glucose, N-acetylglucosamine, galactose, and N-acetylgalactosamine as the penultimate residue were recognized. Attachment of N-acetylneuraminate to the terminal residue of a stimulatory glycosphingolipid eliminated activity; attachment of fucose to the penultimate sugar reduced activity. Glycans with a terminal beta 1-4 or 1-3 glycosidic bond were most effective; glycans with terminal alpha 1-4 or 1-3 glycosides were less effective. The activity of glycans with both beta- and alpha-linked terminal glycosides was inhibited by lactose, suggesting recognition of both configurations by a single amoeba protein. The ability of liposomes to stimulate actin polymerization reflected the extent of liposome phagocytosis.

Use of non-cellular models to study the interaction of E. histolytica with mammalian cells.

We have utilized liposomes constructed with individual mammalian cell membrane glycosphingolipids and latex beads conjugated with glycoproteins as models to investigate the molecular specificity and mechanism of interaction of E. histolytica with target cells. Synthetic liposomes constructed with a variety of glycosphingolipids bearing neutral, straight chain glycans with galactose or N-acetylgalactosamine termini stimulated rapid (90 sec), contact dependent polymerization of E. histolytica actin. Glycans with terminal N-acetylglucosamine residues were not, or only weakly, stimulatory. Attachment of N-acetylneuraminate to the terminal residue of stimulatory glycosphingolipids eliminated activity. Attachment of fucose to the penultimate sugar reduced glycan recognition. Latex beads conjugated with asialofetuin (galactose beta 1-4 glycan termini) adhered to amoebae more effectively than fetuin conjugated beads (N-acetylneuraminate termini) or agalactosyl asialofetuin conjugated beads (N-acetylglucosamine termini). However, none of the glycoprotein conjugated beads stimulated contact mediated polymerization of E. histolytica actin. The carbohydrate specificity of E. histolytica interaction with non-cellular particles resembles that observed with whole target cells our results demonstrate that carbohydrate recognition specificity extends to lipid as well as protein bound glycoconjugates. Further, these studies suggest that the biochemical consequences of binding to glycosphingolipids differ from those resulting from interaction with glycoprotein. This may be relevant to the mechanism of mammalian cell attack by this pathogen.

Stimulation by target cell membrane lipid of actin polymerization and phagocytosis by Entamoeba histolytica.

The identity of molecules of mammalian target cells that stimulate contact-dependent attack by Entamoeba histolytica was sought using human erythrocytes (RBC) as a model. Protein-free liposomes prepared from RBC membrane lipids stimulated the same rapid E. histolytica actin polymerization and phagocytosis as did whole target cells. Liposomes constructed from the major phospholipids of RBC stimulated these responses but only if a negatively charged phospholipid was included. The addition to these liposomes of digalactosyl diglyceride significantly enhanced their stimulatory activity. The results demonstrate that ligands that trigger attack-related responses by E. histolytica reside in the target cell membrane lipid fraction and suggest roles for both glycolipid and phospholipid components.

Rapid polymerization of Entamoeba histolytica actin induced by interaction with target cells.

Within 5 s of challenge of Entamoeba histolytica trophozoites with red blood cells (RBC), attachment and deformation of target cells occurred at multiple sites on the amoeba surface. Many trophozoite-target interfaces were outlined with a ring of polymerized amoeba actin, revealed by rhodamine-phalloidin staining of glutaraldehyde-fixed and Triton-X 100-extracted cells. The beginnings of phagocytic pseudopods rimmed many targets. The phagocytic membrane and underlying actin network grew uniformly about a target cell, which became dramatically elongated and constricted, sometimes severed, as it entered the amoeba. Total engulfment of RBC targets occurred within 10 s. By methanol extraction and spectrofluorimetric measurement of bound rhodamine-phalloidin we were able to quantitate polymerized actin in amoebae. Interaction with target cells was accompanied by a net increase of up to twofold in the average polymerized actin content of trophozoites. This reached a maximum during the period of most active phagocytosis (4 min after challenge at 25 degrees C), and declined as phagocytic activity diminished (8-16 min). Challenge with latex beads of similar size and number, which E. histolytica phagocytized more slowly than RBC, induced neither a detectable increase in polymerized actin content nor appearance of polymerized actin at the contact interface. RBC inhibited phagocytosis of latex beads, but the reverse did not occur. The results demonstrate a rapid, recognition-specific stimulation of reorganization of the actin cytoskeleton of E. histolytica induced by binding to target cells. Vigorous phagocytic activity is frequently an immediate consequence of cell-cell contact, which emphasizes the importance of this process in the contact-mediated attack mechanism of this pathogen. The quantitative assay of polymerized actin may be useful in further studies of this mechanism.

Chemotaxis by Entamoeba histolytica.

A micropore membrane procedure to assay taxis by Entamoeba histolytica is described and the results of studies of responses to a variety of soluble substances, bacteria, an rat colon washings using this procedure are reported. Trophozoites migrated in blind well chambers through 8-micron pore size polycarbonate membranes but not nitrocellulose membranes up to 12 micron pore size. Amoebae were attracted toward fresh axenic culture medium (TYI-S), an enzymatic hydrolysate of casein (Trypticase), and a partially purified preparation of N-acetylneuraminic acid from egg mucin, but not purified N-acetylneuraminate or a variety of other low molecular weight metabolites. The response was verified as chemotaxis by checkerboard analysis. Amoebae migrated most dramatically toward suspensions of all of seven bacterial species tested, including motile and non-motile, gram-negative and gram-positive rods and cocci. This response was diminished when the bacteria concentration gradient was eliminated. The response to bacteria culture filtrates was less than 10% of that to bacterial suspensions. A response to clarified washings from the rat colon was detected; this was diminished but not eliminated by filter sterilization of the washings. We concluded that some soluble molecules, possibly of intermediate molecular size, whole bacteria, and both soluble and particulate components of the rat colon provide tactic stimuli for E. histolytica. Scanning electron micrographs of trophozoites migrating towards attractants through membranes showed narrow, extended pseudopodia entering the membrane pores, and enlarging spheres exiting as the cells proceeded through.

Entamoeba histolytica trophozoites in the lumen and mucus blanket of rat colons studied in vivo.

Trophozoites of Entamoeba histolytica HM-1 were cultivated axenically in TYI-S medium. The amoebae were then transferred into this medium lacking serum (TYI) and inoculated into in vivo colon loops of adult Sprague-Dawley rats. The trophozoites were rapidly absorbed by the mucus, and few were found free in the luminal fluid by 1 h. By 4 h, the amoebae began to reappear in the lumen, aggregated in sloughed mucus blanket fragments. The colon was examined histologically and by scanning electron microscopy. There was no evidence of invasion or even brush-border attachment by the trophozoites within 4 h. In TYI, trophozoite motility was low. Exposure to the colonic lumen environment for 5 min in this medium significantly increased motility. However, as the trophozoites became absorbed to mucus fragments, their observed motility virtually ceased despite some morphological evidence of pseudopod extension. Erythrophagocytosis was not significantly affected by either exposing trophozoites to TYI washings of the colonic lumen, or by the more complete medium, TYI-S, in which the amoebae were significantly more motile. Two major mucus glycoprotein oligosaccharide end-group sugars, L-fucose and N-acetyl-neuraminic acid, were tested for their effects on trophozoite motility in both TYI and TYI-S. L-Fucose reduced motility; the sialic acid increased motility. It is concluded that the intestinal lumen contains several compartments, including the luminal fluid and the mucus blanket, and that Entamoeba trophozoites exist in a highly motile state in the former and a low motility state in the latter. The mucus blanket provided a significant barrier to trophozoite access to intestinal epithelium target tissue.

Use of indomethacin to demonstrate enterotoxic activity in extracts of Entamoeba histolytica trophozoites.

The present study was designed to develop and characterize animal models for the assay of enterotoxic activity in extracts of Entamoeba histolytica trophozoites. Marked water and electrolyte secretion occurred in both in vivo rabbit ileal loops and rat colon loops exposed to clarified sonic fluids of E. histolytica strain HM-1 trophozoites (10(6)/ml) when the animals were first administered indomethacin (0.1 mg/kg). No effect on intestinal absorption was observed in animals exposed to Entamoeba extracts alone or after administration of a lower (0.01 mg/kg). No effect on intestinal absorption was observed in animals exposed to Entamoeba extracts alone or after administration of a lower (0.01 mg/kg) dose of indomethacin. Higher doses (greater than or equal to 1 mg/kg) of indomethacin inhibited extract-induced secretion. No enterotoxic activity was detected with or without indomethacin, using extracts from the nonpathogenic E. histolytica-like Laredo strain, even at 10-fold-higher cell concentrations. The HM-1 enterotoxic activity was heat labile. Prior exposure of the loop lumen to fetuin (100 micrograms/ml) blocked the secretory response to subsequent HM-1 extract exposure, but postexposure of the loop to fetuin did not block secretion that had already been established by the amoeba extract. No histological changes were seen associated with the amoeba extract-induced secretion. The data suggest that E. histolytica HM-1 strain elaborates an enterotoxic activity capable of causing consistent secretion in the mammalian intestine that has had its mucosal cytoprotection impaired by indomethacin.

The mechanism of formation of inhibitor-induced ribosome helices in Entamoeba invadens.

Helices andaggregates of helices (chromatoid bodies) composed of ribosomelike particles appear in cysts and slow-growing trophozoites of Entamoeba invadens. We found that similar helix aggregates were formed abundantly in actively growing E. invadens trophozoites treated with a variety of direct or indirect inhibitors of protein synthesis. The inhibitor-induced helices appeared cytochemically and ultrastructurally identical to those seen in cysts. Numerous single helices and small arrays occurred randomly distributed throughout the trophozoite cytoplasm within 15 min after treatment with NaF, which rapidly and completely stopped all nucleic acid and protein synthesis. Cycloheximide (CH), which inhibited protein synthesis as effectively a NaF, stimulated aggregate formation more slowly, and only after a delay of 30-60 min. CH temporarily blocked NaF-stimulated aggregated formation. Aggregation was slowest with actinomycin-D, which strongly inhibited RNA synthesis but depressed protein synthesis only slowly. These results suggested that release of ribosomes from mRNA was required for aggregation. Inhibition by CH was reversible, and aggregates disappeared from CH-treated amebas shortly after they were transferred to inhibitor-free frowth medium. There was no evidence that helices assembled about a structural organizer within the cell or that the process involved metabloc activity. It was concluded that the inhibitor-induced helices were composed of mature, normally functional ribosomes and that helix formation was a spontaneous and reversible consequence of the accumulation withing the cell of free monosomes (or subunits) which were prevented from binding to mRNA.

Comparision of the structure and function of polysomal and helical ribosomes from Entamoeba invadens.

Some structural and functional properties of ribosomes from polysomes and from helix aggregates of Entamoeba invadens have been compared by sucrose gradient analysis and assays of in vitro protein synthesis. Actively growing trophozoites, lacking helices, presented normal polysome profiles in sucrose gradients. The single large ribosomal helix aggregate (chromoatoid body) of cysts diappeared as the cells were disrupted. Gradient profiles of cyst extracts contained predominantly large and small ribosome subunit peaks and no evidence of remaining helix fragments of mRNA-bound polysomes. Sequential profiles of trophozoites incubated with NaF or cycloheximide (which both stimulate ribosome aggregation, but at different rates) showed that polysome breakdown occurred before aggregates appeared and, again, that helices broke down to subunits in vitro. Radioactive ribosomes synthesized during vegetative growth were collected into helices during encystation. Subunits of these ribosomes cosedimented with comparable particles isolated from trophozoites. Ribosomes from both trophozoites and cysts were active in cell-free protein synthesis, although activity in cyst extracts required the addition of trophozoite-soluble fraction. It was concluded that ribosomes from polysomes and helices in E. invadens were probably identical and that the ability to form helices was an intrinsic property of mature mRNA-free ribosomes of this organism.