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Biomedical subjects

G T Keusch

Publications and source records attributed to G T Keusch.

At least 19 recordsLinked to original sources

Shiga toxin induces superoxide production in polymorphonuclear cells with subsequent impairment of phagocytosis and responsiveness to phorbol esters.

The role of inflammatory cells in the pathogenesis of hemolytic-uremic syndrome induced by Shiga toxin (Stx)-producing Escherichia coli remains unclear. The hypothesis that Stx has direct effects on polymorphonuclear cell (PMN) viability and function was examined by measuring apoptosis, necrosis, phagocytosis, and spontaneous and phorbol myristate acetate (PMA)-stimulated production of reactive oxygen intermediates. PMN from 6 healthy persons were exposed to medium, Stx1 (0.01-100 ng/mL), or heat-inactivated Stx1 or Stx1 B subunit (100 ng/mL). Stx1 induced a prominent dose-dependent respiratory burst from PMN at doses as low as 0.01 ng/mL; they were less responsive to PMA stimulation and had reduced ability for phagocytosis. This dysfunction was not due to cell death, as the magnitude of apoptosis and necrosis of PMN treated with Stx1 (100 ng/mL) for 20 h was identical to that of medium control. These results suggest that Stx has direct effects on PMN that could contribute to tissue injury early in the disease.

Apoptosis

Relative influences of sex, race, environment, and HIV infection on body composition in adults.

BACKGROUND: The factors that control body composition in disease are uncertain. OBJECTIVE: We planned to compare the relative influences of HIV infection, sex, race, and environment on body composition. METHODS: We analyzed results of body composition studies performed by bioelectrical impedance analysis in 1415 adults from 2 cohorts: white and African American men and women from the United States, and African men and women (279 HIV-infected and 1136 control). The effects of sex and HIV infection on weight, body cell mass, and fat-free mass were analyzed by using both unadjusted and age-, weight-, and height-adjusted data. RESULTS: Control men weighed more and had more body cell mass and fat-free mass than did control women, although control women had more fat. The strongest correlates with body composition were height and weight, followed by sex. HIV infection, age, environment, and race. Control men and women weighed more and had more body cell mass, fat-free mass, and fat than did HIV-infected men. However, differences in body composition between HIV-infected and control groups were strongly influenced by sex. Of the differences in weight between HIV-infected and uninfected subjects, fat-free mass accounted for 51% in men but only 18% in women, in whom the remainder was fat. Sex effects were similar in African and American groups. CONCLUSIONS: Sex has a marked effect on the changes in body composition during HIV infection, with women losing disproportionately more fat than men. Sex-related differences in body composition were narrower in the HIV-infected groups. Race and environment had smaller effects than sex and HIV infection.

Adolescent

Responses of human intestinal microvascular endothelial cells to Shiga toxins 1 and 2 and pathogenesis of hemorrhagic colitis.

Endothelial damage is characteristic of infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). Because Stx-mediated endothelial cell damage at the site of infection may lead to the characteristic hemorrhagic colitis of STEC infection, we compared the effects of Stx1 and Stx2 on primary and transformed human intestinal microvascular endothelial cells (HIMEC) to those on macrovascular endothelial cells from human saphenous vein (HSVEC). Adhesion molecule, interleukin-8 (IL-8), and Stx receptor expression, the effects of cytokine activation and Stx toxins on these responses, and Stx1 and Stx2 binding kinetics and bioactivity were measured. Adhesion molecule and IL-8 expression increased in activated HIMEC, but these responses were blunted in the presence of toxin, especially in the presence of Stx1. In contrast to HSVEC, unstimulated HIMEC constitutively expressed Stx receptor at high levels, bound large amounts of toxin, were highly sensitive to toxin, and were not further sensitized by cytokines. Although the binding capacities of HIMEC for Stx1 and Stx2 were comparable, the binding affinity of Stx1 to HIMEC was 50-fold greater than that of Stx2. Nonetheless, Stx2 was more toxic to HIMEC than an equivalent amount of Stx1. The decreased binding affinity and increased toxicity for HIMEC of Stx2 compared to those of Stx1 may be relevant to the preponderance of Stx2-producing STEC involved in the pathogenesis of hemorrhagic colitis and its systemic complications. The differences between primary and transformed HIMEC in these responses were negligible. We conclude that transformed HIMEC lines could represent a simple physiologically relevant model to study the role of Stx in the pathogenesis of hemorrhagic colitis.

Bacterial Toxins

Attachment of Cryptosporidium parvum sporozoites to human intestinal epithelial cells.

An enzyme-linked immunosorbent assay-based attachment model using the human intestinal cell line Caco-2A was developed to study attachment of Cryptosporidium parvum sporozoites in vitro and to assess potential inhibitors of sporozoite binding. In this system, attachment was related to sporozoite dose, incubation time, and host cell differentiation status. Polyclonal antibodies to C. parvum as well as glycoprotein inhibitors of a sporozoite lectin reduced attachment. This model will be a valuable tool in elucidating specific molecules and mechanisms involved in sporozoite-host cell attachment.

Adhesiveness

In vivo transduction with shiga toxin 1-encoding phage.

To facilitate the study of intestinal transmission of the Shiga toxin 1 (Stx1)-converting phage H-19B, Tn10d-bla mutagenesis of an Escherichia coli H-19B lysogen was undertaken. Two mutants containing insertions in the gene encoding the A subunit of Stx1 were isolated. The resultant ampicillin-resistant E. coli strains lysogenic for these phages produced infectious H-19B particles but not active toxin. These lysogens were capable of transducing an E. coli recipient strain in the murine gastrointestinal tract, thereby demonstrating that lysogens of Shiga toxin-converting phages give rise to infectious virions within the host gastrointestinal tract.

Ampicillin Resistance

An in vitro model of infection of human biliary epithelial cells by Cryptosporidium parvum.

Cryptosporidium parvum infection in the immunosuppressed host is frequently complicated by biliary tract involvement. The recent production of human biliary epithelial cell lines was exploited to develop an in vitro model of biliary cryptosporidiosis. Infection with C. parvum oocysts was detected by IFA and ELISA and confirmed by transmission electron microscopy. Inoculation of monolayers with 10(4) to 5 X 10(5) oocysts/well resulted in a dose-dependent increase in infection. Time-course experiments showed that the number of parasitic stages was maximal at 18-24 h after inoculation. Infection was significantly enhanced by bile at concentrations of 50 and 100 microg/mL and inhibited by 400 microg/mL paromomycin. Infection of human biliary cells with C. parvum can be consistently achieved and monitored by use of IFA or ELISA. This system will be of use in evaluating mechanisms of C. parvum infection and response to therapeutic agents in biliary cryptosporidiosis.

Amebicides

Plasma cytokines, cytokine antagonists, and disease progression in African women infected with HIV-1.

OBJECTIVES: To examine the relation of circulating cytokines and cytokine antagonists to the progression of human immunodeficiency virus type 1 (HIV-1) disease. DESIGN: Cross-sectional analysis. SETTING: An ambulatory acquired immunodeficiency syndrome (AIDS) research clinic in Kinshasa, Zaire. PATIENTS: 48 women with AIDS, 51 women with HIV infection who were clinically asymptomatic, and 11 female controls who did not have HIV infection, all from Zaire. MEASUREMENTS: Plasma levels of interleukin-1beta, tumor necrosis factor-alpha (TNF-alpha), interleukin-6, interleukin-8, interferon-gamma, interleukin-1beta receptor antagonist (interleukin-1Ra), and TNF soluble receptor p55 (TNFsRp55) were assayed by specific radioimmunoassays. Plasma levels of interferon-gamma were assayed by commercial enzyme-linked immunosorbent assay. The Wilcoxon rank-sum test was used to assess the significance of mean and median differences between groups. RESULTS: Of the 48 patients with AIDS, circulating interleukin-1beta was detected in 2, TNF-alpha in 4, interleukin-6 in 3, and interleukin-8 in 12. None of these factors were seen in any of the 11 controls. Median values of interleukin-1beta (320 pg/mL), TNF-alpha (210 pg/mL), and interleukin-8 (750 pg/mL) were elevated in HIV-infected asymptomatic patients compared with patients with AIDS (2-, 2.6-, and 18.7-fold higher, respectively; P < 0.001). Interleukin-1Ra and TNFsRp55 levels were substantially higher than interleukin-1beta and TNF-alpha levels in HIV-infected asymptomatic patients (73- and 14-fold, respectively) and were higher than those in patients with AIDS (17.8- and 1.74-fold, respectively). CONCLUSION: High circulating levels of the proinflammatory cytokines interleukin-1beta and TNF-alpha, combined with an excess of their natural inhibitors interleukin-1Ra and TNF-sRp55, were seen in clinically asymptomatic HIV-1-positive African women but not in African women with AIDS or in HIV-negative controls. Circulating cytokine antagonists may play a clinical role in modulating cytokine-associated symptoms in the early phases of HIV infection.

Acquired Immunodeficiency Syndrome

Comparison of the effects of Shiga-like toxin 1 on cytokine- and butyrate-treated human umbilical and saphenous vein endothelial cells.

To examine the reported heterogeneity of endothelial cells to Shiga-like toxin 1 (Stx1), the responses of human umbilical (HUVEC) and saphenous (HSVEC) vein endothelial cells to cytokines, butyrate, and toxin were compared. Untreated HSVEC were generally more susceptible than were HUVEC to Stx1; pretreatment of either cell with lipopolysaccharide, interleukin-1 beta, or tumor necrosis factor-alpha enhanced Stx1 toxicity. Dexamethasone alone increased total globotriaosylceramide (Gb3) content and toxin binding but inhibited cytokine-enhanced cytotoxicity, whereas the differentiation agent, sodium butyrate, increased both Gb3 content and cytotoxicity responses to Stx1, most prominently in HSVEC. Stx1 toxicity directly correlated with the release of von Willebrand factor from HSVEC but not from HUVEC. Thus, HUVEC and HSVEC exhibit distinctive responses to Stx1, cytokines, and butyrate. This suggests the need for caution in extrapolating from in vitro studies utilizing one endothelial cell type to in vivo events during pathogenesis of Stx-mediated thrombotic microangiopathies.

Bacterial Toxins

Protective immunity to Shiga-like toxin I following oral immunization with Shiga-like toxin I B-subunit-producing Vibrio cholerae CVD 103-HgR.

This study addresses a mechanism for inducing systemic immunity to Shiga-like toxins by oral administration of a Shiga-like toxin I B-subunit-expressing Vibrio cholerae vaccine strain [CVD 103-HgR(pDA60)]. Two sets of three rabbits were given either CVD 103-HgR or CVD 103-HgR(pDA60) orally. All rabbits immunized with CVD 103-HgR(pDA60) developed neutralizing serum antibodies to Shiga-like toxin I. None of the controls developed such antibodies.

Administration, Oral

Regulation of the Shiga-like toxin II operon in Escherichia coli.

Investigations of the regulation of the bacteriophage-encoded Shiga-like toxin II (SLT-II) in Escherichia coli demonstrated that bacteriophages exhibit a regulatory impact on toxin production by two mechanisms. Firstly, replication of the toxin-converting bacteriophages brings about an increase in toxin production due to concomitant multiplication of toxin gene copies. Secondly, an influence of a phage-encoded regulatory molecule was demonstrated by using low-copy-number plasmid pADR-28, carrying a translational gene fusion between the promoter and proximal portion of slt-IIA and the structural gene for bacterial alkaline phosphatase (phoA). PhoA activity, reflecting the slt-II promoter activity, was significantly enhanced in E. coli strains which and been lysogenized with an SLT-I or SLT-II-converting bacteriophage (H-19B or 933W, respectively) or bacteriophage lambda. Both mechanisms are dependent on bacteriophage induction and hence are recA dependent. Moreover, the study revealed that the DNA-binding protein H-NS has a regulatory impact on both bacteriophage-mediated SLT-II synthesis and the activity of the slt-II promoter of plasmid pADR-28. While a slight impact of growth temperature on SLT-II expression was observed, no impact of either osmolarity, pH, oxygen tension, acetates, iron level, or utilized carbon source could be demonstrated.

Amino Acid Sequence

Translocation of Shiga toxin across polarized intestinal cells in tissue culture.

Escherichia coli strains producing Shiga toxins (Stx) 1 and 2 colonize the lower gastrointestinal tract in humans and are associated with gastrointestinal and systemic diseases. Stx are detectable in the feces of infected patients, and it is likely that toxin passes from the intestinal tract lumen to underlying tissues. The objective of this study was to develop an in vitro model to study the passage of Stx across intact, polarized cell monolayers. Translocation of biologically active Stx was examined in four cell lines grown on polycarbonate filters. Stx1 translocated across intestinal cell monolayers (CaCo2A and T84 cells) in an energy-requiring and saturable manner, while the monolayers maintained a high level of electrical resistance. Stx1 had no effect on electrical resistance or inulin movement across these cell lines for at least 24 h. Induction of specific Stx receptors with sodium butyrate reduced the proportion of toxin translocated across CaCo2A monolayers but had no major effect on the movement of horseradish peroxidase or [3H]inulin. We have shown that biologically active Stx1 is capable of moving across intact polarized intestinal epithelial cells without apparent cellular disruption, probably via a transcellular pathway. The data also suggest that the presence of Stx receptors on the apical surface of intestinal epithelial cells may offer some protection against the absorption of luminal Stx1.

Animals

Cryptosporidia--who is at risk?

Cryptosporidium parvum is a coccidian parasite originally described a century ago and, until recently, not considered to be a human pathogen. It has a complex life cycle, including both sexual and asexual reproduction, an auto-infectious cycle, and the ability to complete its development within a single host. The transmission form is a robust, environmentally resistant oocyst, excreted in the stool, which can exist for long periods of time in the environment. Because animals, in particular domesticated livestock, are its primary host, human infection is usually zoonotic. Oocysts often find their way into water supplies, and it resists chlorination and is incompletely filtered from processed drinking water supplies, even when filtration is working optimally. Transmission via ingestion of fecally contaminated swimming pool water, food, fomites, and sexual activities facilitating fecal-oral inoculation have been demonstrated. The major target of C. parvum in the host is the intestinal epithelial cell, resulting in diarrhea, sometimes profuse and persistent, although it may also infect other organs such as the gall bladder and lungs. Pathogenesis involves attachment, probably via a sporozoite lectin, invasion, probably involving apical organelles, replication within a parasitophorous vacuole with the host cell membrane, causing cellular dysfunction. Diagnosis is generally made by visualization of the oocyst form in stool by staining methods, the best of which appears to be auramine and fluorescence microscopy. Those at greatest risk are immunocompromised adults and children, especially those with AIDS, children in day care, travelers to endemic regions, dairy or cattle farm workers of their families or contacts, household contacts of cases or carriers, and possibly owners of infected dogs or cats or their neighbors. There is no specific therapy available, however in the immunocompetent host the illness is self-limited, lasting from a few days to 3 weeks, and long term carriage is uncommon. In the immunocompromised host, infection is prolonged, sometimes asymptomatic, but may result in chronic debilitating diarrhea with dehydration, malabsorption and wasting. Public health measures to reduce contamination of water supplies and vigilant surveillance will reduce the risk to populations. Reducing behaviors favoring fecal-oral transmission, such as certain sexual activities, and scrupulous hygiene in the day care setting would also reduce the likelihood of transmission but not eliminate it. Given our lack of knowledge about Cryptosporidium biology and pathogenesis, high priority should be given to research designed to increase our understanding of the organism and improve the chance of developing useful therapeutic or preventative drugs or strategies.

Adult

Fecal excretion of leukotriene C4 during human disease due to Shigella dysenteriae.

Fecal excretion of leukotriene C4 was determined in 26 individuals with dysentery and in 19 healthy controls. Of the patients, five were infected with Shigella dysenteriae type 1, 15 were infected with Shigella flexneri, two were infected with Shigella boydii, and four were infected with Shigella sonnei. Three of the healthy controls were infected with non-dysenteriae Shigellae. All isolates of Shigella dysenteriae type 1 produced Shiga toxin; the other strains were not toxigenic. Patients with dysentery due to Shigella dysenteriae type 1 excreted higher concentrations of leukotriene C4 (median, 3,234 pg/0.05 g of feces) than either ill individuals infected with non-dysenteriae Shigellae (median, 202 pg/0.05 g) or healthy carriers (median, 145 pg/0.05 g) and uninfected controls (median, 129 pg/0.05 g). We propose that Shiga toxin stimulates intestinal mast cells, which release leukotriene C4, contributing to the inflammatory response in Shigella dysenteriae type 1-associated dysentery.

Adult