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Biomedical subjects

D Mirelman

Publications and source records attributed to D Mirelman.

At least 91 records · Page 5Linked to original sources
PubMed1986

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.

Animals↗
PubMed1986

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.

Animals↗
PubMed1985

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.

Animals↗
PubMed1985

Antibacterial activity of the pancreatic fluid.

The antibacterial activity of canine pancreatic fluid was investigated in an attempt to understand the resistance of this organ, when intact, to ascending bacterial infections. The pancreatic fluid demonstrated bactericidal activity against Escherichia coli, Shigella species, Salmonella species, and Klebsiella pneumoniae; bacteriostatic activity against coagulase-positive and coagulase-negative staphylococci and Pseudomonas aeruginosa; and fungistatic activity against Candida albicans. There was no demonstrable antibacterial activity against Bacteroides fragilis and Streptococcus faecalis. The antibacterial activity was dialyzable and pH dependent, but independent of heat, the activity of several digestive pancreatic enzymes, and the bacterial inoculum. Electron micrographs of Escherichia coli exposed to pancreatic fluid did not demonstrate changes in the bacterial cell wall. Tracer studies of susceptible bacteria demonstrated decreased leucine uptake when briefly exposed to pancreatic fluid. The antibacterial activity was found by column chromatography to be a small molecular peptide. It is likely that pancreatic antibacterial factors protect the pancreas from ascending bacterial infections and operate along with other factors in the homeostasis of the upper small bowel flora.

Animals↗
PubMed1985

Isolation, purification, and partial characterization of an enterotoxin from extracts of Entamoeba histolytica trophozoites.

Soluble cell-free extracts of pathogenic Entamoeba histolytica, as well as serum-free minimal media in which trophozoites are incubated, contain substances that cause the rapid rounding up and detachment of tissue-cultured monolayers of mammalian cells (cytopathic activity) and induce fluid secretion in ligated intestinal loops of indomethacin-pretreated rats (enterotoxic activity). A semiquantitative assay for the determination of the cytopathic activity based on the rate of detachment of tissue-cultured baby hamster kidney cells was developed. Two peaks containing cytopathic activity were obtained upon gel filtration of the soluble extracts: peak I, with over 60% of the activity, emerged in the 30,000 to 50,000 molecular weight region, and peak II, containing the remaining activity, was in the 15,000 to 25,000 molecular weight region. The activity of peak I was found to be heat labile and inhibited by sialoglycoproteins such as fetuin and mucin (5 mg/ml), as well as by sialic acid. Protease inhibitors such as antitrypsin, pepstatin, phenylmethylsulfonyl fluoride, metaloprotease inhibitors, and bacitracin had no effect on the cytopathic activity. Marked inhibition of cytopathic activity was observed, however, with iodoacetamide and p-chloromercuribenzoate, which affect sulfhydryl groups. The toxic material in peak II was found to have ionophoric activity and was not inhibited by sialic acid-containing compounds. The materials from both peaks had enterotoxic activity in intestinal ligated loops. The active substance from peak I was further purified (200X) on an agarose-fetuin affinity column, yielding one major protein band with an apparent molecular weight of ca. 30,000 on sodium dodecyl sulfate. Amino acid analysis revealed that the protein was very poor in sulfur amino acids. The sialic acid-sensitive toxic activity was higher in known virulent strains such as HM-1:IMSS and could be markedly augmented after preincubation of the trophozoites with certain Escherichia coli strains.

Animals↗
PubMed1984

Virulence of Entamoeba histolytica trophozoites. Effects of bacteria, microaerobic conditions, and metronidazole.

The association of axenically grown trophozoites of Entamoeba histolytica strains HK-9 or HM-1:IMSS with various types of gram-negative bacteria for relatively short periods markedly increased their virulence, as evidenced by their ability to destroy monolayers of tissue-cultured cells. Interaction of trophozoites with bacteria that were heat inactivated, glutaraldehyde fixed, or disrupted by sonication, or bacteria treated with inhibitors of protein synthesis, did not augment amebic virulence. Lethally irradiated bacteria, however, retained their stimulative properties and trophozoites that ingested bacteria were protected from the toxic effects of added hydrogen peroxide. An increase in virulent properties of amebae was also found in experiments carried out under microaerobic conditions (5% O2, 10% CO2). The augmentation of amebic virulence due to association with bacteria was specifically blocked by metronidazole, but not by tetracycline or aminoglycosides, and the rate of metronidazole uptake in stimulated trophozoites was two to three times higher. The results obtained suggest that virulence of axenically grown E. histolytica trophozoites may depend to a considerable extent on the cell's reducing power. Both microaerobic conditions and the association with bacteria apparently stimulate the electron transport system of the ameba. Bacteria may function as broad range scavengers for oxidized molecules and metabolites through the contribution of enzymatic systems, components, or products.

Adhesiveness↗
PubMed1984

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).

Animals↗
PubMed1984

Entamoeba histolytica: virulence enhancement of isoenzyme-stable parasites.

Pathogenic Entamoeba histolytica isolated from patients with clinical amoebiasis can be differentiated from nonpathogenic E. histolytica obtained from asymptomatic carriers on the basis of the electrophoretic pattern of their isoenzymes. Virulence of different strains of axenically grown trophozoites of Entamoeba histolytica, as determined by various laboratory tests, such as damage to tissue culture monolayers, or their ability to cause an hepatic abscess in a hamster, are known to vary considerably. Reassociation of trophozoites of strain HK-9 with certain Escherichia coli strains for short periods of time markedly augmented their virulence, as tested by the above-mentioned methods. The bacterial association, however, did not cause any change in the electrophoretic pattern of amoebic isoenzymes (zymodeme).

Amikacin↗
PubMed1984

Enhancement by retinoic acid of the sensitivity of different tumor cell lines to the sialic acid-specific toxin of Entamoeba histolytica.

Treatment of several tumor cell lines, including the murine melanomas B16 and S91 and the human sarcoma Hs791 and Hs705, with retinoic acid resulted in an increased sialylation of specific cell surface membrane sialoglycoproteins. This treatment also augmented the sensitivity of these cells to the cytopathic effects of a sialic acid-specific toxin from Entamoeba histolytica. In contrast, a similar treatment with retinoic acid of a retinoic acid-resistant mutant clone S91-C154, which does not increase sialylation of cell surface glycoproteins, failed to alter the susceptibility of the cells to the E. histolytica toxin. These results imply that cell surface sialoglycoproteins serve as receptors for the amoebic toxin.

Animals↗
PubMed1984

Adherence of bacteria to pediatric intravenous catheters and needles and its relation to phlebitis in animals.

The adherence of bacteria to pediatric IV catheters and needles was studied. Scanning electron micrographs showed that bacteria adhered well to the catheters and needles, mainly to non-smooth surface areas. In vitro quantitative determination, with the use of radiolabeled bacteria, revealed differences in the affinity of bacteria for the various IV cannula materials. The adherence per square area was greatest for plastic catheters, less for steel needles, and least for siliconized needles. Mean values for the adherence of Staphylococcus aureus to these cannulae were 37.9-40.3 X 10(5) bacteria/cm2 for the plastic catheters; 10.2 X 10(5) bacteria/cm2 for the steel needles, and 7.2-7.6 X 10(5)/cm2 for the siliconized needles. Removal of the glutaraldehyde-fixed bacteria adhered to the cannulae, after their placement in veins of rabbits, was lower for the plastic catheters than the IV needles. The appearance and severity of venous phlebitis produced by the various cannulae was determined in an animal model. The degree of the inflammatory response elicited correlated with the in vitro bacterial adherence, indicating that bacterial adherence plays a role in the appearance of cannula-associated phlebitis. In view of our results and other previous observations of lower rate of infections with the use of IV needles, it is suggested that needles should be preferred to plastic catheters whenever possible. The described in vitro assay for bacterial adherence can be used to determine the adherent properties of IV cannulae, which should be considered in any future cannula design.

Adhesiveness↗
PubMed1984

The effect of postnatal age on the adherence of Shigella flexneri, Escherichia coli 0124, and E. coli 0128 to guinea pig intestinal cells.

Since postnatal development of the gastrointestinal tract has an important effect on its microbial flora and may influence the types of intestinal infections, we examined the effect of age on bacterial adherence to intestinal epithelial cells. Radiolabeled bacteria were incubated with guinea pig enterocytes released by treating loops of the intestine with solutions containing EDTA, dithiothreitol, and citrate. Nonbound bacteria were separated from intestinal cells by sedimentation on a Percoll gradient. The colonic cells avidly bound Shigella flexneri (64 bacteria per cell), Escherichia coli 0124 (59), and E. coli 0128 (53). The adherence process was Ca2+ and temperature dependent, was inhibited by fucose, glucose, and mannose, and was shown to be mediated by a carbohydrate-binding protein (lectin) on the colonic cells. Adherence of these bacteria to intestinal cells of newborn animals was only 15-25% of the adherence to adult animal cells and increased gradually, reaching adult values at about 2 weeks of age. The lectin activity, which was determined by agglutination of bacteria, was secreted with the colonic mucus. It was undetectable in the newborn animal, appeared gradually with age, and its titer correlated with the adherent capability of the colonic cells. E. coli 0128 was the only one of the bacteria tested which significantly adhered to the ileum (19 bacteria per cell) in a process inhibited by mannose. This adherence was mediated by a mannose-sensitive lectin in the bacterial pili, and not on the intestinal cells. The postnatal age had no effect on the adherence to the ileum; the newborn animal had the same adherence capability as the adult one.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesiveness↗
PubMed1983

Interactions between Entamoeba histolytica, bacteria and intestinal cells.

Axenically grown pathogenic and non-pathogenic isolates of Entamoeba histolytica have been shown to adhere to mammalian epithelial cells and bacteria by virtue of carbohydrate-binding proteins present on their cell surfaces. The interaction of amoeba isolates of low pathogenicity with a variety of gram-negative bacteria, mainly Escherichia coli strains which are readily ingested by the amoebae after relatively short periods, significantly increased the ability of the trophozoites to: (a) destroy and ingest intestinal epithelial cells; (b) secrete a cytopathic substance which morphologically affects a variety of tissue-cultured cells; and (c) cause hepatic abscesses in hamsters. Addition of carbohydrates that inhibit the lectin-mediated attachment of bacteria to amoebae prevented the enhancement of virulence. Interaction of the amoebae with bacteria that were heat-inactivated, glutaraldehyde-fixed or disrupted by sonication, as well as with bacteria precoated with antibodies or concanavalin A, did not lead to an increase in virulence. Moreover, short prior treatments of the bacteria with inhibitors of protein synthesis, but not with cell-wall synthesis inhibitors, also prevented the stimulation. The results indicate that interactions of amoebae with certain bacteria may be responsible for the increase in amoebic virulence.

Adhesiveness↗
PubMed1983

Entamoeba invadens and E. histolytica: separation and purification of precysts and cysts by centrifugation on discontinuous density gradients of Percoll.

The different cell forms in the life cycle of Entamoeba invadens (trophozoites, precysts, and cysts) were rapidly and quantitatively separated on density step gradients of polyvinylpyrolidone-coated colloidal silica particles (Percoll). With this method, the gradual process of encystation by E. invadens trophozoites could be monitored. Percoll gradients were also efficient in separating trophozoites of Entamoeba histolytica and bacteria. After purification on Percoll, trophozoites display no evidence of damage when examined by light microscopy and no loss in viability as judged by their ability to multiply.

Centrifugation, Density Gradient↗
PubMed1983

Adherence and ingestion of Escherichia coli serotype 055 by trophozoites of Entamoeba histolytica.

Carbohydrate-binding activity present on the Entamoeba histolytica cell surfaces was found to mediate the adherence of two types of bacteria, Escherichia coli serotype 055 and Salmonella greenside 050. Adherence was inhibited by low-molecular-weight carbohydrates (10 mg/ml) such as galactose, lactose, and N-acetylgalactosamine, as well as by asialofetuin and the lipopolysaccharide extracted from E. coli 055. Mild periodate oxidation of the bacteria inhibited their adherence, whereas heat inactivation, glutaraldehyde fixation, or gamma-irradiation had no effect. On the other hand, pretreatment of trophozoites with glutaraldehyde, cytochalasin B, or cold (5 degrees C) abolished adherence. None of these treatments, however, affected the attachment of bacteria that contain on their cell surface type I pili with mannose-binding capacity. These findings lend further support to our earlier observations on how amoebae interact with bacteria.

Adhesiveness↗