To help explain a role of the Shiga toxin family in hemorrhagic colitis and hemolytic-uremic syndrome in humans, it has been hypothesized that these toxins cause direct damage to the vascular endothelium. We now report that Shiga toxin purified from Shigella dysenteriae 1 does indeed have a direct cytotoxic effec on vascular endothelial cells in cultures. Human umbilical vein endothelial cells (HUVEC) in confluent monolayers were reduced 50% by 10-8 M Shiga toxin after a lag period of 48 to 96 h. In comparison, nonconfluent HUVEC were reduced 50% by 10-10 M Shiga toxin within a 24-h period. These data suggest that dividing endothelial cells are more sensitive to Shiga toxin than are quiescent cells in confluent monolayers. Both confluent and nonconfluent HUVEC specifically bound '25I-Shiga toxin. However, in response to the toxin, rates of incorporation of [3H]leucine into protein were more severely reduced in nonconfluent cells than in confluent cells. Toxin inhibition of protein synthesis preceded detachment of cells from the substratum. The specific binding of '251-Shiga toxin to human endothelial cells and the cytotoxic response were both toxin dose dependent and neutralized by anti-Shiga toxin antibody. Heat-denatured Shiga toxin was without the cytotoxic effect. In addition, the complete culture system contained less than 0.1 ng of bacterial endotoxin per ml, as measured by the Limulus amoebocyte lysate test.

The viscosit ies of solut ions of t hree polysty rene fractions in t hree solve nts of v ar y ing solvent power were m easured at t wo temperat ures. The relat ive vi scosit ies of the syst em s investigated ranged from 1.03 to 43.The appli cability of two empirical expressions for the concen tration depende nce, nam ely t he Martin equation and t he Baker relation, is examined . In addit ion, t he results are represented by m eans of poly nomials of suitable degr ee. The numerical procedures for t he e valuatio n of t he coeffi cien ts are discussed in detail. In t he concent ration r an ge investigated , t he in troduction of a reduced co ncen t rat ion scale S = ['1] c, places t he viscosity-con cen t ration curves for different molec ular weights in t he same solven t on a more nearly common scale. This scale, S, is simply rela ted t o another reduced scale c/co. H ere Co represen ts t he concentration a t which t he equivalent spher es of t he coiling m olec ul es, a s d efin ed at infinite dilu tion, would just begin to o verlap . At c/co « 1, t he con cent ration dependence can be described in terms of hydrody namic in teract ion. This intera ction involves single molecul es and can also involve t he int rinsic v iscosity and interactions of aggregates of low order . An a ttempt is made t o d educe from t he viscosi ty data and on t he basis of cer tain hydrody namic results, t he equilibrium constant s and relative p opulations of s uch aggregates. R easonable values are obt ained . On approaching Co, t he a verage volume a vailable t o a chain molec ule in a good solve nt is redu ced because of t he cage form ed by its nearest neighbors. The effective press ure is just t he internal osmo tic press ure. This leads to an expression for t he concent rat ion depe ndence of t he viscosi ty, in terms of t he vi rial coeffi cie nts of osmotic press ure, luolec ular weight, and size. These equat ions are s hown to be in satisfactory agreemen t wi t h t he experimen tal data. In part icul ar , in t he neighborhood of Co one obtains r easo nabl e values for t he molec ular exte nsio n f actors of t he chain .

Shiga toxin has been purified in milligram quantities to near homogeneity from cell lysates of Shigella dysenteriae 1 strain 3818-0. Purification involved an initial ultracentrifugation, ammonium sulfate fractionation, chromatography on DEAEcellulose and carboxymethyl cellulose, gel filtration, and preparative isoelectric focusing in sucrose gradients. The purified toxin was resolved by discontinuous polyacrylamide gel electrophoresis into a major cytotoxic protein band and a closely migrating, cytotoxic protease-nicked minor band. Antiserum generated by immunization with glutaraldehyde-inactivated toxin was shown to be monospecific against S. dysenteriae cell lysates. This highly purified toxin was cytotoxic to HeLa cells, enterotoxic in rabbit ileal loops, and lethal to mice. Monospecific antiserum to the toxin neutralized completely these toxin activities in both purified toxin preparations and crude shigella cell lysates.

Shiga toxin purified to near homogeneity from cell lysates of Shigella dysenteriae 1 inhibited protein and deoxyribonucle acid syntheses in intact HeLa cells. Inhibition was dependent on toxin concentration and time of incubation. A minimal latent period of 30 min was observed with saturating doses of toxin. Ribonucleic acid synthesis, uptake of alpha-aminoisobutyric acid, and maintenance of intracellular K+ concentrations were not affected until well after maximal inhibition of protein and deoxyribonucleic acid syntheses. The inhibitory effect of toxin was sensitive to heat inactivation and was prevented by antibody neutralization. Several cytotoxic components were separated by polyacrylamide gel electrophoresis of the purified toxin preparations; all inhibited protein and deoxyribonucleic acid syntheses equally.

A 1994 outbreak of acute hemorrhagic conjunctivitis in Israel was caused by an enterovirus 70 strain that was distinct from previously reported strains. Characterization was by electron microscopy (eye washes), reverse transcription-PCR (RT-PCR; eyewash, specimens, eye swabs, and tears), and sequence analysis of RT-PCR-amplified fragments from the 5 noncoding region and VP1.

Toxin A and toxin B preparations of Clostridium difficile have been shown to affect metabolic functions of intact HeLa cells with different kinetics. The cytotoxins were purified from dialyzed filtrates of C. difficile strain VPI 10463 by hydrophobic interaction chromatography and ion-exchange chromatography and were concentrated by dialysis or by ultrafiltration. The toxins, which are immunologically unrelated, were analyzed by polyacrylamide gel electrophoresis and by immunochemistry with the Western blot technique. Toxin A was resolved into one major cytotoxic protein and a minor, rapidly migrating species that did not comigrate with toxin B. Toxin B was resolved into one major and three minor cytotoxic proteins. One protein comigrating with toxin A had no cytotoxic activity. The highly purified toxin A at 1.0 mg/ml caused loss of intracellular K+ and inhibition of protein synthesis in HeLa cells within 1 h. These effects correlated with morphological changes indicating cytotoxicity. At lower protein concentrations of toxin A (10to 100-fold less), however, cytotoxic effects were seen at 120 min, whereas no changes in K+ levels or protein synthesis were yet evident. The toxin B preparation, 1,000-fold more toxic than toxin A, was diluted to equivalent cytotoxicity as measured in the overnight assay. Toxin B caused loss of K+ and inhibition of protein synthesis well after cytotoxic morphological changes were complete. In contrast, at higher protein concentrations (2to 2,000-fold more), intracellular K+ was lost completely by 120 min. The effects on cell rounding and protein synthesis were incomplete at 120 min, but increased with the toxin B concentration.

Shiga toxin purified from Shigella dysenteriae 1 was cytotoxic to cultured epithelial cells from human colon and ileum. The cytotoxicity, which affected only about 50% of treated cells, was neutralized by rabbit antiserum monospecific for Shiga toxin and mediated by protein synthesis inhibition.

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