Biomedical subjects
Publications and source records attributed to R A Finkelstein.
Vibrio cholerae produces a phenolate-type siderophore that stimulates growth of the organism in low-iron medium. This compound is similar, but not identical, to enterochelin, the siderophore produced by Salmonella and Escherichia coli.
Strains of Neisseria gonorrhoeae isolated from disseminated gonococcal infections have enhanced ability to acquire iron in an experimental host when compared with urogenital gonococcal isolates from patients with uncomplicated infections.
Heat-labile enterotoxin from Escherichia coli is immunologically related to both subunits, A and B, of cholera enterotoxin as demonstrated by neutralization and immunodiffusion.
Immunodiffusion and biological neutralization studies demonstrated that the heat-labile enterotoxin (LT) from Escherichia coli has antigenic determinants in common with each of the isolated subunits (A and B) of the enterotoxin (choleragen) from Vibrio cholerae. Each of the enterotoxins also possesses unique antigenic specificities. Monospecific antiserum to LT was prepared by immunization with antigens derived by immune precipitation of E. coli cell-free supernatant with isolated specific anticholeragenoid antibodies. This antiserum neutralized the biological acitivity of both LT and cholera enterotoxin and recognized antigens of both in immunodiffusion. This antiserum was adsorbed with choleragenoid to remove antibodies directed against the shared "B" immunological determinants. The neutralizing effect of the antiserum on cholera toxin was completely removed, but the neutralizing activity against the E. coli preparations was retained, although somewhat reduced. Antisera to the isolated subunits (A and B) of cholera enterotoxin neutralized the biological activity of cholera enterotoxin and LT. These antisera also recognized the homologous and heterologous antigens in immunodiffusion. Multiple forms or conformations of LT and its components may explain the diversity of the properties which have been reported for it.
The ability of potential pathogens to acquire iron in a host is an important determinant of both their virulence and the nature of the infection produced. Virulent gram-negative bacteria are capable of acquiring sufficient iron from the host because their virulence (for chick embryos) is unaffected by exogenous iron. Avirulent mutants which are apparently limited in their ability to acquire iron could be isolated from the virulent strains. The lethality of these mutants was significantly enhanced by exogenous iron. Reduction of the relatively high serum iron saturation of chick embryos (to levels more closely approximating those in man) by pretreatment with iron-binding proteins or endotoxin inhibits the lethality of some virulent bacteria. Those bacteria whose virulence was reduced include the Shigella, Vibrio cholerae and strains of Neisseria gonorrhoeae, all of which are nondisseminating pathogens in the normal human host. Pathogens which produce septicemic and disseminating infections such as Neisseria meningitidis, Haemophilus influenzae type B, Escherichia coli possessing K-1 antigen, Pseudomonas aeruginosa and Salmonella typhimurium and disseminating strains of N. gonorrhoeae were, in general, unaffected by reduced serum iron saturation. These disseminating bacteria appeared to produce greater quantities of compounds (siderophores) which stimulated microbial growth in low-iron media than did the nondisseminating pathogens. Thus, the gram-negative bacteria tested can be divided into four major classes according to their responses to modifications in iron levels in the chick embryo model and these results correlate with the nature of the infections which they typically produce in man.
Large crystals of the cholera toxin were grown; their dimensions, symmetry (P21), order, and resistance to radiation make them ideally suited for a high-resolution x-ray structure determination. There is one molecule (approximately 84,000 daltons) per asymmetric unit, and therefore the lattice reveals no molecular symmetry. Two distinct bioassays indicate that the protein from dissolved crystals retains full biological activity.
Agar medium containing Congo red dye differentiates virulent and avirulent colonies of Shigella, Vibrio cholerae, Escherichia coli, and Neisseria meningitidis. Like virulent plague bacilli, wild-type cells of these species absorb the dye and produce red colonies. Mutants or colonial variants have been isolated that fail to absorb the dye and produce colorless colonies. These mutants exhibit reduced virulence in the chicken embryo model, but their virulence is enhanced by supplementation with iron. Of those species tested, only Neisseria gonorrhoeae isolates failed to grow in the presence of this dye. Inhibition of growth by Congo red may thus provide a simple means for differentiating gonococci from other Neisseria.
Imferon, an iron-dextran complex, enhances the growth of Neisseria gonorrhoeae and N. meningitidis. The use of Imferon as a replacement for ferric nitrate, in a defined supplement for GC agar significantly increased the average colony sizes of both gonococci and meningococci. In comparison with Thayer-Martin medium, Imferon agar increased the speed and rate of isolation of gonococci from clinical specimens.
Exceptional strains of gonococci that grow on Congo red agar were found to be penicillin-resistant non-penicillinase producers.
Murine splenocytes and tumor cells bind cholera enterotoxoid (choleragenoid). Four hours after sensitization, choleragenoid-coated cells were lysed in the presence of anti-cholergenoid serum and complement, indicating that the binding was stable. Choleragenoid-coated cells were unable to sensitize spleen cells from normal or choleragenoid primed syngeneic mice into displaying a cytotoxic effect against choleragenoid-coated target cells in the T cell-mediated cytotoxicity assay. Cells coated with both choleragenoid and trinitrophenyl (TNP) groups did sensitize syngeneic spleen cells to display a cytotoxic effect against target cells bearing choleragenoid and TNP or TNP alone, but not choleragenoid alone. These data demonstrate that the mere binding of a foreign component to lymphoid cells is not sufficient to allow sensitization of cytotoxic T cells.
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Various techniques have been applied to the detection of skin reactivity associated with heat-labile Escherichia coli enterotoxin in fermenter-grown cultures of enterotoxigenic strains in syncase medium and in trypticase soy broth. Isolated products that were homogeneous, as determined by disc electrophoresis and sodium dodecyl sulfate gel electrophoresis, differed immunologically and in physicochemical characteristics depending on the strain and medium used, even though the products had similar specific activities in skin tests and in Chinese hamster ovary cells. In support of observations on porcine E.coli enterotoxin, and in contrast to the enterotoxin of Vibrio cholerae, the products were single polypeptide chains with molecular weights ranging from approximately 35,000 ot over 100,000 daltons. Proteolytic cleavage during culture and purification might account for some of the variations observed. The isolated products were almost 10(6)-fold less active than purified choleragen in causing morphologic alterations of Chinese hamster ovary cells, approximately 1,000-fold less active in skin tests, and at least 100-fold less active in rabbit ileal loops. In addition, only 1/100 as much active protein was produced by the strains employed as by V. cholerae. It is possible that accessory or host-derived factors are required. The most effective large-scale procedure for isolation from trypticase soy broth culture supernatants was a sequence of concentration by ultrafiltration, judicious (NH4)2SO4 precipitation, gel filtration on Sephadex G-150, and gel filtration on Agarose A5m, from which the E. coli products (like choleragen) are retarded in their elution. Toxin was isolated from an enterotoxigenic strain that caused diarrhea (total volume, 60-liters) for four days in a patient who had visited Mexico.
Colonization of the intestinal epithelium by Vibrio cholerae was examined in two model systems, in ligated ileal loops of adult rabbits and in the patent gut of infant rabbits, using both scanning and transmission electron microscopy. Time studies in the adult model showed a lag period of up to 1 h before the attachment of significant numbers of the vibrios. The bacteria appeared initially in small patches on the sides of the villi, predominantly along the transverse furrows. The number of adherent bacteria steadily increased, reaching a maximum between 4 and 7 h, when a dense mat of bacteria several layers thick covered much of the villi. After this time there was a rapid decline in the number of V. cholerae bound. By 12 to 16 h only a few bacteria could be seen on the surface of the villi, which had a rough, patchy appearance at these later times. Globular protrusions, with vibrios attached, may play a role in the clearance of bacteria. Colonization and clearance in the patent intestine of the infant rabbit occurred much as in the adult model. However, the bacteria adhered more uniformly and there was no lag in attachment. In both models the majority of bacteria were aligned horizontally with the epithelial surface, but some were attached in an end-on manner, with their flagella extending into the lumen. The bacteria adhered via their surface coats directly to the tips of the microvilli, except for a few vibrios that were partly embedded into the brush border. Some changes in the microvilli occurred as a consequence of the bacterial attachment.
A woman returning from Mexico was hospitalized as an emergency patient with hypovolemic shock due to dirrheal disease of less than 1-day duration. Her clinical course was similar to that of severe cholera--she excreted greater than 60 liters of stool and urine in a 4-day period. The etiological agent was a non-enteropathogenic serotype but enterotoxigenic strain of Escherichia coli (063:NM). The patient responded both agglutinating and antitoxic antibodies against this strain and its enterotoxin. An "enteropathogenic serotype," 0111:B4, was also isolated but this finding had no etiological significance.
Scientifically controlled field studies have established that parenterally administered killed vibrio vaccines or somatic antigen preparations offer only limited degrees of protection in certain population groups and have made it obvious that new approaches to the immunoprophylaxis of cholera are needed. It has now also been established that the symptoms of cholera result from the action of the cholera enterotoxin (choleragen) on the epithelial cells of the small intestine. Immulogically related enterotoxins have been incriminated in other newly recognized diarrheal diseases (e.g., those caused by Escherichia coli and "non-agglutinable" (NAG)vibrios). Additionally, volunteer studies have shown that induced cholera results in rather solid and lasting immunity against homologous re-challenge thus proving that immunity against cholera is feasible. Although numerous studies have demonstrated the efficacy of purely antitoxic immunity in experimental animal models, the protective effect of parenterally administered glutaraldehyde treated toxoid in man has shown to be limited, at best. The protection attained following an attack of cholera suggests that local immune mechanism may be of predominant importance. Immunity has been stimulated, experimentally in mice, by toxin antigen administered per os on a single occasion. Choleragenoid, which is non-toxic but binds to the same receptors as cholera toxin, has been shown to provide immediate resistance as well as later immunity to toxin challenge. More ideal, however, would be a colonizing mutant of V. cholerae which elaborates a non-toxic cross-reactive material (CRM) like choleragenoid and which could stimulate local antitoxic as well as anti-vibrio immune mechanisms. A tox-mutant of V. cholerae which is avirulent for man has been shown to elicit substantial immunity in man but the ideal CRM-mutant has yet to be found.
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Matings between strains of Vibrio cholerae differing in toxinogenicity, nutritional requirements, and antibiotic susceptibilities were performed in order to determine the location of the gene tox that controls production of cholera enterotoxin. Segregation analysis shows that tox is linked to a gene required for histidine biosynthesis. Our data indicate that the tox gene is located on the bacterial chromosome and not on a plasmid in the strains of V. cholerae studied.