Lack of enhanced oxygen consumption by polymorphonuclear leukocytes on phagocytosis of virulent Salmonella typhi.

Human polymorphonuclear leukocytes exhibit an enhanced rate of oxygen consumption during phagocytosis of relatively avirulent strains of Salmonella typhi or Staphylococcus aureus. However, phagocytosis of a virulent strain of Salmonella typhi is not associated with augmented oxygen consumption. The ability of a bacterial strain to alter the postphagocytic rate of oxygen consumption of polymorphonuclear leukocytes may be related to its in vivo virulence.

Pathogenesis of shigella diarrhea. Serum anticytotoxin antibody response produced by toxigenic and nontoxigenic Shigella dysenteriae 1.

The serum antitoxin response to the cytotoxin contained in preparations of Shigella dysenteriae 1 (Shiga's bacillus) exotoxin was studied in natural and experimental infections of man. Natural infection resulted in the rapid appearance of toxin-neutralizing antibody, which disappeared some time between 9 and 18 mo after infection. Experimental infection of human volunteers provided the opportunity to study immunoglobulin class of the antibody in sera obtained serially from 7 to 50 days after infection. Neutralizing antibody was present only in the IgM fraction isolated by sucrose density gradient ultracentrifugation. This was confirmed by the use of solid-phase immunoaffinity chromatography. Even though the time-course and immunoglobulin class of the antitoxin antibody response was similar to that previously observed for anti-O polysaccharide antibody, the biologically active cytotoxin was shown to be highly susceptible to destruction by proteolytic enzymes. Sera from subjects infected with a virulent invasive chlorate-resistant Shiga mutant thought to be "nontoxigenic" also contained antibody which was similarly restricted to the IgM fraction. Biologically active cytotoxin was recovered when this mutant organism was grown in liquid media with controlled ion concentration. The mutant cytotoxin was heat labile, neutralized by antiwild-type cytotoxin antibody, and was separable by isoelectric focusing into two fractions with pI 7.2 and 6.1 like the wild-type toxin. These studies show that cytotoxin antigen is produced during in vivo infection with Shiga bacilli, resulting in a serum antitoxin antibody response. Without explanation is the restriction of the antibody to the IgM class and lack of evidence for an IgG antibody to the protein cytotoxin. Finally, mutant strain 725, previously designated "nontoxigenic," was shown to produce biologically active cytotoxin in vitro and, in experimentally infected volunteers, to result in a serum IgM antibody similar to that observed during infection with the wild-type strain.

Mechanisms of endotoxin tolerance. The role of the spleen.

Splenectomy markedly impaired the production of circulating anti-endotoxin antibodies during the initial 10 days after .v. administration of a Boivin preparation of Escherichia coli endotoxin (ET) in both rabbit and man. Increase in antibodies with secondary (flocculating and bactericidal) activities were virtually abolished, whereas increases in antibodies with primary (binding) activity were significantly reduced. On the basis of these findings, splenectomized rabbit and man were employed to test the hypothesis that the early phase (less than 72 h) of pyrogenic tolerance to endotoxin is independent of anti-endotoxin antibody but that such antibody contributes significantly to the later phase (less than or equal to 72 h) of tolerance. In the splenectomized rabbit, the initial pyrogenic reponses to ET and the subsequent tolerant responses at 24 and 48 h were comparable to sham-operated controls...

The role of endotoxin during typhoid fever and tularemia in man. IV. The integrity of the endotoxin tolerance mechanisms during infection.

Volunteers infected with Salmonella typhosa develop a remarkable hyperreactivity to the pyrogenic and subjective toxic activities of homologous (S. typhos) and heterologous (Pseudomonas) endotoxins. The present studies quantitate this augmented reactivity and demonstrate by three differing approaches that significant tolerance to these endotoxins can be readily induced within the framework of the hyperreactive state. Thus, (a) tolerance induced before illness by repeated daily intravenous injections of the endotoxins remained demonstrable during overt illness, (b) daily intravenous injections of the endotoxins begun during overt illness evoked progressively increasing tolerance, and (c) continuous intravenous infusions of S. typhosa endotoxin during illness rapidly induced a pyrogenic refractory state. Despite unequivocal activation of the endotoxin tolerance mechanisms by any of the above methods, the febrile and toxic course of typhoid fever proceeded unabated. Similarly, in other volunteers with Pasteurella tularensis infection, continuous intravenous infusions of S. typhosa endotoxin evoked initial hyperreactive febrile and subjective toxic responses followed by rapid appearance of a pyrogenic refractory state without modification of the underlying clinical illness. These observations suggest that circulating endotoxin plays no major role in pathogenesis of the sustained fever and toxemia during typhoid fever and tularemia in man. The mechanisms responsible for the systemic hyperreactivity to endotoxin during typhoid fever and tularemia were further investigated. Low grade endotoxemia, nonspecific effects of tissue injury, impaired ability of the reticuloendothelial system to clear circulating endotoxin, and production of cytophilic antibodies capable of sensitizing leukocytes to endotoxin did not appear responsible. Inflammatory reactions to intradermal S. typhosa endotoxin increased significantly during typhoid fever. However, since no such dermal hyperreactivity developed to Pseudomonas endotoxin during typhoid fever nor to S. typhosa endotoxin during tularemia, the systemic hyperreactivity to bacterial endotoxins during typhoid fever and tularemia could not presently be ascribed to enhanced levels of acquired hypersensitivity.

Fecal steroids in diarrhea. I. Acute shigellosis.

Fecal bile acid and neutral sterol patterns of five healthy adult male volunteers, who were challenged by a virulent Shigella flexneri 2a (M42-43) strain and developed dysentery were studied. It was observed that cholic acid was increased from 1.9 +/- 0.4% of total bile acid in the feces before infection to 14.5 +/- 2.1% during diarrhea (P less than 0.001). Chenodeoxycholic acid also was increased from 3.2 +/- 0.7 to 8.7 +/- 3.2% in diarrhea but the difference was not significant statistically. Deoxycholic and lithocholic acids constituted 34.1 +/- 4.1 and 40.5 +/- 2.8%, respectively, of total bile acid in the normal controls as compared to 13.9 +/- 2.5 and 24.8 +/- 2.5% for the same subjects during diarrhea (P less than 0.005). Total excretion of bile acids, expressed as mg/kg of body weight per day, were higher in diarrhea (5.4 +/- 1.0) than that in controls (4,2 +/- 1.0) but the difference was not statistically significant. In the neutral sterol fraction, unmodified cholesterol was increased during diarrhea (86.2 +/- 8.7 versus 25.0 +/- 4.8% of total cholesterol metabolites in controls, P less than 0.001). Coprostanol was decreased in shigellosis (12.2 +/- 8.2 versus 65.8 +/- 4.7% in controls, P less than 0.001). Epicoprostanol, coprostanone, and unidentified cholesterol metabolites also were reduced in shigellosis. The effect of diarrhea on the plant sterols was not as consistent. However, unidentified plant sterols were reduced significantly in shigellosis stools. Total excretion of cholesterol metabolites and plant sterols, when expressed as mg/kg of body weight per day, were 6.8 +/- 1.7 and 0.6 +/- 0.2), respectively, in Shigellosis. These values were not significantly different from the corresponding values for controls (10.3 +/- 3.0 and 0.8 +/- 0.2). One subject's stool samples were studied during infection for the sequence of bile acid alteration. A progressive reduction of bacterial activity upon fecal steroids was evident following the initial diarrheal episode. The production of coprostanol was correlated with 7 alpha-dehydroxylation of cholic acid (r = 0.937, P less than 0.001) and chenodeoxycholic acid (r = 0.755, P less than 0.01).

A modified leukocyte nitroblue tetrazolium test in acute bacterial infection.

The increased ability to leukocytes to reduce nitroblue tetrazolium (NBT) has been used to detect the presence of systemic bacterial infection. This test has been utilized to evaluate infections and leukocyte dysfunction in children, but has not been extensively applied to traumatized patients or infected volunteers. Moreover, the technic as originally described presented methodologic difficulties. In this study of 889 such patients, a modified NBT test provided excellent differentiation of 63 systemic bacterial infections (NBT score greater than or equal to 10%) from non-infectious fevers, local enteric diseases, and certain viral and plasmodial infections (NBT score less than or equal to 9%). Splenectomy was associated with a transient false-positive score and clinical typhoid fever with a false-negative response

The problem of emesis during oral glucose-electrolytes therapy given from the onset of severe cholera.

In an attempt to obviate the need for intravenous fluids by preventing dehydration, 57 adult volunteers who experienced induced clinical cholera during a vaccine development programme were treated from the onset of diarrhoea with oral glucose-electrolytes therapy. 44 individuals with mild to moderately profuse diarrhoea (less than 8 L. total volume) were maintained in normal water and electrolyte balance with oral therapy alone. 13 individuals with severe diarrhoea (greater than 8 L. total volume) could not be maintained in balance with oral therapy alone, due chiefly to emesis during the first day of illness. Emesis occurred in the absence of significant dehydration or acidosis. Since emesis precludes effective early oral therapy in severe cases, domiciliary oral therapy is unlikely to eliminate cholera mortality. Rural diarrhoea treatment centres using oral therapy with limited amounts of intravenous fluids when needed, could reduce case fatality from cholera and related diarrhoeas virtually to zero with least expense.

Immunity of cholera in man: relative role of antibacterial versus antitoxic immunity.

Purified cholera toxoid is antigenic when given enterally and orally. Purified toxoid fails to provide protection against experimental challenge. Clinical cholera confers formidable protection against homologous or heterologous rechallenge. Failure to culture vibrios from intestinal fluid or stool of re-challenge volunteers suggests that the predominant immune mechanism is antibacterial rather than antitoxic.

The nature of endotoxin tolerance.

Certain of the mechanisms by which man develops pyrogenic tolerance to bacterial endotoxins have been considered. After an initial intravenous injection of toxin, two temporally distinct phases of tolerance can be discerned, early and late, each with very different characteristics. Early tolerance appears to be mediated by a non-antibody mechanism entailing a transiently occurring refractory state, apparently involving to a major degree decreased production of endogenous pyrogen by the macrophage system, particularly the hepatic macrophages. Late tolerance appears to be mediated by anti-endotoxin antibodies directed against both "O" and common core antigens which blunt the release of endogenous pyrogen from macrophages. The common core antigens are masked in the presence of the "O" antigenic side chains and become effective immunogens only when these "O" side chains are lacking. Accelerated reticuloendothelial system clearance of circulating endotoxin provides an ancillary protective mechanism in that it brings the toxin more efficiently into the macrophages that are refractory or protected by antibody. When endotoxin is administered repeatedly at closely spaced intervals, both the early phase (non-immune) and late phase (immune) mechanisms may become superimposed. In addition, a third mechanisms, enhanced detoxification capabilities of macrophages, also now appears to come into play. At any given time, it is the relative contribution of each mechanism, which in turn is dependent upon the immunization schedule, antigenicity of the endotoxin, dosage, and immunological competency of the host, that determines the expression of the endotoxin tolerant state.

Biohydrogenation of cholesterol as an index of bacterial 7 alpha-dehydroxylase activity.

Fecal steroid compositions of 82 human subjects of various ages and diets and gastrointestinal status were examined by gas liquid chromatography. Progressive increases in bacterial activities on both bile acids and neutral sterols were observed with the advance of age in infants and children. The patterns in the 4-year-olds approached those observed in adults. Bacterial activities on fecal steroids were found to be decreased in adult subjects with acute shigellosis and in those challenged by castor oil. In contrast, no significant changes in fecal steroid profiles were observed in the subjects with traveller's diarrhea associated with toxigenic Escherichia coli. The effects of diarrhea on fecal steroids of infants under 1 1/2 years were less consistent than those of adults. However, a close relationship was observed between the degree of 7 alpha-dehydroxylation of cholic acid (expressed as the ratio of deoxycholic to the sum of deoxycholic and cholic acids) and the percentage of cholesterol in the feces (r = -0.921, p less than 0.001). The correlation between the production of lithocholic acid and the percentage cholesterol was also good (r = -0.739, p less than 0.001). Analysis of neutral steroids may be a good index of intraluminal bile acid metabolism.

Biohydrogenation of cholesterol as an index of bacterial 7α-dehydroxylase activity.

Fecal steroid compositions of 82 human subjects of various ages and diets and gastrointestinal status were examined by gas liquid chromatography. Progressive increases in bacterial activities on both bile acids and neutral sterols were observed with the advance of age in infants and children. The patterns in the 4-year-olds approached those observed in adults. Bacterial activites on fecal steroids were found to be decreased in adult subjects with acute shigellosis and in those challenged by castor oil. In contrast, no significant changes in fecal steroid profiles were observed in the subjects with traveller's diarrhea assoicated with toxigenicEscherichia coli. The effects of diarrhea on fecal steroids of infants under 1(1/2) years were less consistent than those of adults. However, a close relationship was observed between the degree of 7α-dehydroxylation of cholic acid (expressed as the ratio of deoxycholic to the sum of deoxycholic and cholic acids) and the percentage of cholesterol in the feces (r= 0.921, p<0.001). The correlation between the production of lithocholic acid and the percentage cholesterol was also good (r=-0.739, p<0.001). Analysis of neutral steroids may be a good index of intraluminal bile acid metabolism.