Eosinophilic "empyema" associated with crack cocaine use.

Smoking of crystalline cocaine, known as "crack" cocaine, has been associated with eosinophilic pneumonitis, but not with pleural effusions. We describe a patient with eosinophilic pneumonitis with an eosinophilic "empyema" after using "crack" cocaine. The illness resolved with corticosteroids. We hypothesised that his effusion would have increased levels of eosinophil cytokines that promote oedema, and found a marked increase in pleural vascular endothelial growth factor (VEGF) and smaller increases in interleukins IL-5, IL-6, and IL-8. In the setting of "crack" use, we suggest that a pleural effusion that appears grossly to be pus should be evaluated for eosinophilic inflammation. Such eosinophilic effusions may respond to corticosteroids alone, consistent with a non-infectious process driven by proinflammatory cytokines.

Epidermal and hair follicle transglutaminases. Partial characterization of soluble enzymes in newborn mouse skin.

Treatment of skins of newborn mice with the neutral protease Dispase in order to separate dermis and epidermis causes pronounced changes in the levels of transglutaminase activity in the epidermis. Two soluble transglutaminases, one anionic enzyme and one cationic enzyme, of Mr approximately 90,000 and approximately 50,000, respectively, are extracted from epidermis; and the activities of both enzymes increase as a function of the time of Dispase treatment of skin. When the anionic Mr approximately 90,000 enzyme is incubated with Dispase after its chromatographic isolation from epidermal extracts, it is converted to a lower molecular weight enzyme. Hair follicles isolated from dermis prepared by a 12-h Dispase treatment of the skin of newborn mice contain two soluble cationic transglutaminases, one of which is indistinguishable from that of epidermis and the other which is not seen in epidermis. Both of these hair follicle enzymes are of Mr approximately 50,000 and appear to exist in monomeric form. They have been partially purified. Based upon these findings, we suggest that transglutaminase processing and control occur during normal differentiation of keratinocytes in epidermis and of hair follicle epidermal cells in dermis and that production of the proper forms of the enzyme may be essential to the formation of mature cornified envelopes and hair shafts, respectively.

Sporulation and enterotoxin production by mutants of Clostridium perfringens.

The ability of Clostridium perfringens type A to produce an enterotoxin active in human food poisoning has been shown to be directly related to the ability of the organism to sporulate. Enterotoxin was produced only in a sporulation medium and not in a growth medium in which sporulation was repressed. Mutants with an altered ability to sporulate were isolated from an sp(+) ent(+) strain either as spontaneous mutants or after mutagenesis with acridine orange or nitrosoguanidine. All sp(0) (-) mutants were ent(-). Except for one isolate, these mutants were not disturbed in other toxic functions characteristic of the wild type and unrelated to sporulation. A total of four of seven osp(0) mutants retained the ability to produce detectable levels of enterotoxin. None of the ent(-) mutants produced gene products serologically homologous to enterotoxin. A total of three sp(-) mutants, blocked at intermediate stages of sporulation, produced enterotoxin. Of these mutants, one was blocked at stage III, one probably at late stage IV, and one probably at stage V. A total of three sp(+) revertants isolated from an sp(-) ent(-) mutant regained not only the ability to sporulate but also the ability to produce enterotoxin. The enterotoxin appears to be a sporulation-specific gene product; however, the function of the enterotoxin in sporulation is unknown.

Clostridium perfringens Type A Food Poisoning I. Response of the Rabbit Ileum as an Indication of Enteropathogenicity of Strains of Clostridium perfringens in Monkeys.

Diarrhea and vomiting have been experimentally produced in monkeys after oral challenge with viable cells or culture filtrates of certain strains of Clostridium perfringens that previously had been shown to produce either fluid accumulation in the ligated ileum or overt diarrhea in the nonligated ileum of the rabbit, or both. Strains (or their culture filtrates) which did not produce a response in the rabbit likewise produced no symptoms in monkeys.

Clostridium perfringens Type A Food Poisoning II. Response of the Rabbit Ileum as an Indication of Enteropathogenicity of Strains of Clostridium perfringens in Human Beings.

The effect of feeding human beings individual strains of Clostridium perfringens or culture filtrates thereof was examined. The strains selected for challenge included both those which had previously been shown to produce fluid accumulation in the ligated ileum or overt diarrhea when injected into the nonligated ileum of the rabbit, or had produced both, and those which did not regularly produce these responses. Challenge doses prepared by allowing each strain to grow in beef stew for 3 hr at 46 C resulted in a 61% incidence of diarrhea when rabbit-positive cells were used. No diarrhea occurred among the subjects fed rabbit-negative strains prepared in a similar manner. The procedures employed in preparing the challenge dose appeared to influence the results obtained. When cell-free filtrates were fed, 4 of 15 persons consuming filtrates from rabbit-positive strains developed diarrhea. All subjects fed filtrates from rabbit-negative strains remained free from diarrhea. Serological tests were carried out to compare the identity of the strains of C. perfringens consumed by the subjects and those excreted in the feces. Heat resistance measured as D(100) values varied greatly among the rabbit-positive strains.

Influence of water activity on the growth of Clostridium perfringens.

Each of four strains of Clostridium perfringens was grown in modified fluid thioglycolate medium which was adjusted to yield selected water activity (a(w)) levels. The adjustments to secure the desired a(w) levels were made with NaCl, KCl, or glucose. At each a(w) level, further modification was effected to produce four pH values. Cultures were incubated at either 37 or 46 C. The solute used to achieve the reduced a(w) levels appeared to have a definite effect on the magnitude of growth achieved, the rate of growth, and the limiting a(w) at which growth would occur. Use of glucose as the controlling solute permitted growth at the lowest a(w) level tested, 0.960, and yielded the greatest magnitude of growth as measured by turbidity values, at all of the a(w) levels investigated. Cultures grown in the medium with added KCl generally demonstrated the longest lag times and the least amount of growth. Regardless of specific solute used, as the a(w) level was lowered and the pH value decreased within each a(w) level, the rate and amount of growth were lessened. It appeared, however, that low pH values had less effect on inhibiting growth at low a(w) levels than at higher a(w) levels. Those cultures incubated at 46 C generally exhibited shorter lag periods than those at 37 C, although the maximal growth attained was somewhat less than that achieved at 37 C. The response to all of the investigated conditions was similar for each of the four strains tested.

Ileal loop fluid accumulation and production of diarrhea in rabbits by cell-free products of Clostridium perfringens.

The ability of cell extracts and culture filtrates of various strains of C. perfringens to produce ileal loop fluid accumulation and overt diarrhea in rabbits was tested. Good correlation was obtained in the ability of whole cells and a toxic factor (present in cell extracts and concentrated culture filtrates) to produce both fluid accumulation in ileal loops and diarrhea when injected into the normal ileum of the rabbit. The toxic factor was present in cell-free preparations when cells were grown in a sporulation medium, but not when they were grown in an asporogenic medium. The factor was shown to be heat labile, nondialyzable, and was inactivated by Pronase but not by trypsin, lipase, or amylase. Loss of activity occurred at pH 1.0, 3.0, 5.0, and 12.0.

Rabbit ileal loop response to strains of Clostridium perfringens.

The ligated loop of the rabbit intestine was investigated as a possible experimental model for the study of Clostridium perfringens food poisoning. The method of preparation of the challenge inoculum was important in determining whether a given strain would provoke a response. When cultures were grown for 4 hr at 37 C in Skim Milk (Difco), 14 of 29 type A strains isolated from food-poisoning outbreaks consistently produced exudation of fluid and consequent dilation of the ileal segments. In contrast, 15 of the 18 strains derived from other sources failed to elicit a response. By use of different inoculum preparations, nearly all strains could be made to give at least an occasional positive loop reaction. Diarrhea was not obtained in rabbits by intraluminal injection into the normal ileum or by per os administration of the cultures. Lecithinase, purified and in concentrated culture supernatant fractions, failed to produce a response in the isolated ileal loops.

Improved medium for sporulation of Clostridium perfringens.

An improved sporulation medium has been developed in which all five strains of Clostridium perfringens tested exhibited a 100- to 10,000-fold increase in numbers of spores when compared with spore yields in SEC medium under comparable conditions. In addition, three of five strains produced a 100- to 1,000-fold increase, with the remaining two strains yielding approximately the same numbers of spores, when compared with strains cultured in Ellner medium. At the 40-hr sampling time, 18 of 27 strains produced a 10- to 100-fold increase in numbers of spores in our medium, when compared to spore production obtained in a medium recently reported by Kim et al. The new medium contained yeast extract, 0.4%; proteose peptone, 1.5%; soluble starch, 0.4%; sodium thioglycolate, 0.1%; and Na(2)HPO(4). 7H(2)O, 1.0%. In some cases, the spore yield could be increased by the addition of activated carbon to the new medium. The inclusion of activated carbon in the medium resulted in spores with slightly greater heat resistance than spores produced in the new medium without added carbon or in SEC or in Ellner medium. The major differences in heat resistance of the various strains appeared to be genetically determined rather than reflections of a particular sporulation medium. A definite heat-shock requirement was shown for four of four strains, with the optimal temperature ranging from 60 C for a heat-sensitive strain to 80 C for a heat-resistant strain. Heating for 20 min at the optimal temperature resulted in a 100-fold increase over the viable count obtained after heating for 20 min at 50 C.

Effect of cookery and holding on hams and turkey rolls contaminated with Clostridium perfringens.

Canned hams, turkey rolls, and ground-beef casseroles were inoculated with a mixture of vegetative cells and spores of selected strains of Clostridium perfringens, in approximately known numbers. After cooking and holding at different temperatures for various times, samples of the food were plated directly on sulfadiazine-polymixin-sulfite-agar. In all cases, small but measurable percentages of the organisms survived cookery. The number of cells viable after cookery of the ham or turkey was influenced by the position of the slice of meat in the roast as well as by the final temperature to which the product was heated. Plate counts for turkey or beef casserole held at temperatures in the range of 5 to 10 C for 48 hr indicated stabilization of the population or a tendency to decrease. At 24 C, the multiplication of cells was apparent in 4 hr and rapid in 6 hr. When the food was maintained at 68 C, populations remained viable for 6 hr and the counts did not change markedly. In turkey maintained at 37 C, the number of cells increased sharply within 4 hr.

Some properties of heat-resistant and heat-sensitive strains of Clostridium perfringens. I. Heat resistance and toxigenicity.

Heat resistance at 100 C (D-values), sporulating ratios, toxigenicity for mice, and lecithinase activity (as micrograms per milliliter of enzyme, ascertained by the lecithovitellin reaction) were determined for four strains of Clostridium perfringens. A definite inverse relationship between thermal resistance and toxigenicity was found. The D-values ranged from 17.6 for the most heat-resistant strain to 0.3 for the strain possessing the least heat resistance, with corresponding lecithinase activities from 25 to 133 mug/ml of enzyme. The sporulating ratios did not differ greatly between the strains. The heat stability of the toxin was greater at 100 C than at 75 C. There was a noticeable difference between the heat stabilities of the toxin in the culture fluids of the heat-sensitive and heat-resistant strains at pH 7.0 when the toxic filtrates were held at 100 C. At a holding temperature of 75 C, a similar but lesser difference was observed at pH 5.5. Heat resistance and lecithinase activity did not change when a substrain of the least heat-resistant parent strain was obtained through heat selection by a single transfer, or when the most heat-resistant strain was transferred serially 12 times.

Mice and monkeys as assay animals for Clostridium perfringens food poisoning.

Spores and vegetative cells of Clostridium perfringens, in combination with meat or starch paste, sterile culture filtrates, lecithinase, and phosphorylcholine, were administered to mice and rhesus monkeys in an attempt both to evaluate the animals as test agents and, if possible, to elucidate the active factors producing food-poisoning symptoms caused by this organsim. Some of the preparations were administered to the monkeys by stomach tube; others, in gelatin capsules which were treated with formaldehyde so that the release of their contents was delayed and presumably reached the intestines of the animals. Any changes in intestinal passage times and in consistency of stools of the animals were observed, and the counts of C. perfringens in the feces of the monkeys previous and subsequent to treatment were recorded. The results obtained were inconclusive. Diarrhea occurred only relatively infrequently in both species, regardless of the substance fed or the mode of administration. The changes in intestinal passage times were not great, although in the monkeys there appeared to be a slight trend toward reduction as the magnitude of the bacterial load increased. Phosphorylcholine appeared to have little, if any, effect in reducing intestinal passage time of mice or monkeys. No procedures explored in these experiments could be said to be satisfactory as a means of animal assay for food poisoning strains of C. perfringens since typical symptoms did not appear with regularity.