Biomedical subjects
Publications and source records attributed to R Bracha.
A pseudophakic pilot of the Israeli air force flying an F-15 (Eagle) aircraft was followed up for three years. He experienced about 100 flying hours, 5% of the time under high g stress. The intraocular lens did not dislocate and no complications were observed. It seems that flying high performance fighter aircraft is not contraindicated in pseudophakic pilots.
Strains of Entamoeba histolytica which were isolated from symptomatic patients and which possess a characteristic pathogenic isoenzyme pattern (zymodeme) have extrachromosomal circular DNA molecules containing RNA genes and clusters of tandemly reiterated PvuI elements. The nucleotide sequence of comparable reiterated BamHI elements present in amebae with nonpathogenic zymodemes differs from that found in pathogenic ones. By using the polymerase chain reaction, it was demonstrated that the cloned, nonpathogenic E. histolytica strain SAW 1734R clAR also contains one or few of the tandemly repeated DNA PvuI elements characteristic of the pathogenic amebae. Sequences were detected by hybridization with the P-145 probe after in vitro amplification. Because of technical difficulties, it was impossible to resolve whether single copies of the nonpathogenic BamHI repetitive elements are present in pathogenic amebae. Our findings suggest that in the nonpathogenic amebae, the signal to start amplifying the PvuI-type elements may be induced during the process of elimination of bacterial associates from their growth environment.
Most individuals infected with Entamoeba histolytica are reported to be clinically asymptomatic. On the basis of the electrophoretic migration of hexokinase and phosphoglucomutase isoenzymes, two groups of E. histolytica isolates have been classified. Those derived from symptomatic cases were found to have fast-migrating hexokinase bands and were labeled pathogenic. The others, isolated from cyst passers, had (in most cases) slow-migrating bands and were called nonpathogenic. Differences between these two groups of E. histolytica were found recently at the DNA level. Two sets of different DNA probes derived from tandemly repeated sequences present in extrachromosomal circular DNA elements in each group of E. histolytica were characterized. Using these probes with procedures for direct hybridization of trophozoites on nylon membranes, we could correctly correlate hexokinase electromobility with the DNA hybridization signal of 81 different isolates of E. histolytica. The advantages of using DNA probes lie in their sensitivity (fewer than 200 trophozoites can be detected) and specificity. The probes hybridized only with amebae from the E. histolytica species and not with other enteric protozoa and can be useful as a diagnostic tool.
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.
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.
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.
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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.
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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.
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).
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.
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.
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.
Entamoeba histolytica trophozoites were found to be very selective in their interactions with bacteria. Two principal mechanisms were shown to be responsible for these interactions. Certain bacteria, such as a number of Escherichia coli and Serratia marcescens strains which are known to contain mannose-binding components on their cell surface, bound to mannose receptors on the amoeba membrane. This attachment was markedly inhibited by alpha-methylmannoside (0.5%), especially when the incubations were done at low temperature (5 degrees C). Other bacterial species, such as Shigella flexneri and Staphylococcus aureus, which do not possess a mannose-binding capacity, attached to the amoebae, but only with the aid of concanavalin A or after opsonization of the bacteria with immune serum. In both types of attachment, between 40 and 100 bacteria bound per amoeba, and considerable ingestion of bacteria into amoeba vacuoles was observed at 37 degrees C. The attachment of opsonized bacteria to the amoebae does not appear to be mediated by Fc receptors since Fab' dimers obtained after pepsin digestion of immunoglobulin were capable of mediating adherence. Furthermore, preincubation of the amoebae with aggregated human immunoglobulin G or with heat-inactivated immune serum and EDTA did not inhibit the attachment of opsonized bacteria. The attachment of opsonized bacteria was markedly inhibited by N-acetylglucosamine-containing glycoconjugates, such as peptidoglycan and chitin oligosaccharides, as well as by N-acetylgalactosamine. These results indicate that amoebae can attach and ingest bacteria either by using their membrane-associated carbohydrate-binding protein or by having their mannose-containing cell surface components serve as receptors.
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Two homologous strains of Escherichia coli, one of which completely lacked the cell envelope Braun's lipoprotein, were compared with respect to their peptidoglycan synthesis and assembly. Both strains were auxotrophic for diaminopimelic acid and their uptake of radiolabeled diaminopimelic acid was comparable. Analysis of subcellular fractions obtained after mechanical disruption of the cells in a French pressure cell and sedimentation of the cell envelopes showed the existence of a soluble, chromatographically immobile macromolecular peptidoglycan. This labeled peptidoglycan contained a reduced degree of peptide side chain cross-linkages (19 mol % of labeled residues as compared to that present in the insoluble cell sacculus, 27 mol %). In addition, approximately 20% of its peptide side chains terminated in pentapeptide structures versus 1 to 4% in the sacculus. Furthermore, the soluble peptidoglycan of the parent strain also contained covalently bound lipoprotein (4.6%). Extraction of the cell envelope fraction with detergents afforded an additional amount of soluble peptidoglycan. This material was quite similar, in its degree of cross-linkage and amount of covalently bound lipoprotein, to the peptidoglycan present in the detergent-insoluble sacculus. These results indicate that peptidoglycan strands which are, in part, covalently linked to lipoprotein are late stage synthesis intermediates which subsequently become covalently attached to the preexisting sacculus.
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