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Biomedical subjects

G T Keusch

Publications and source records attributed to G T Keusch.

At least 37 records · Page 2Linked to original sources

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↗

Expression and purification of Shiga-like toxin II B subunits.

Shiga-like toxins (SLTs), which are produced by certain strains of Escherichia coli, are composed of enzymatically active A and B subunit multimers responsible for the toxin's binding. We have previously purified large amounts of the SLT-I B subunit by using a hyperexpression vector in Vibrio cholerae under the control of the trc promoter. In this study we examined various expression vectors to maximize yields of the SLT-II B subunit. The SLT-II B subunit has been expressed by using both the T7 promoter and the tac promoter in E. coli. When expressed from a plasmid containing the structural gene for SLT-II B deleted of the leader sequence, SLT-II B was able to form multimers when cross-linked, although SLT-II B production from this plasmid was unreproducible. SLT-II B expressed in all three systems appeared to form unstable multimers, which did not readily bind to a monoclonal antibody which preferentially recognizes B subunit multimers. SLT-II B expression was not increased by moving any of the plasmids into V. cholerae. Polyclonal antibodies raised to SLT-II B in rabbits recognized B subunit in SLT-II holotoxin yet were poorly neutralizing. SLT-II B was also expressed as a fusion protein with maltose-binding protein and could be cleaved from maltose-binding protein with factor Xa. Although the expression vectors were able to make large amounts of SLT-II B, as determined by Western blotting (immunoblotting), the levels of purified SLT-II B subunit were low compared with those obtained previously for SLT-I B subunit, probably because of instability of the multimeric SLT-II B subunit.

ATP-Binding Cassette Transporters↗

Globotriaosylceramide, Gb3, is an alternative functional receptor for Shiga-like toxin 2e.

We reexamined the binding specificity of the Shiga-like toxin variant associated with porcine edema disease, SLT2e, which is reported to be more cytotoxic for Vero cells than for HeLa cells, by using receptor-deficient cells and a liposomal insertion system for purified glycolipids. We found that SLT2e preferentially uses globotetraosylceramide as a receptor but can also cause cytotoxicity by using globotriaosylceramide, the SLT2 receptor. We conclude that the differential cytotoxicity of SLT2e on HeLa and Vero cells is a function of both the receptor preference of the toxin and the specific glycolipid content of the target cells being used.

Animals↗

Maturational regulation of globotriaosylceramide, the Shiga-like toxin 1 receptor, in cultured human gut epithelial cells.

Differentiated villus intestinal epithelial cells express globotriaosylceramide, the Shiga-like toxin 1 (SLT-1) receptor, and are sensitive to toxin-mediated cytotoxicity, whereas undifferentiated crypt cells neither express Gb3 nor respond to toxin. To investigate if SLT-1 receptors are maturationally regulated in human intestinal cells, we examined the effect of butyrate, a known transcriptional regulator of differentiation genes in many cell types, using cultured colonic cancer-derived epithelial cell lines. Exposure to butyrate increased villus cell marker enzymes such as alkaline phosphatase, sucrase, and lactase, expression of toxin receptors, and sensitivity to SLT-1 in villus-like CaCo-2A and HT-29 cells. These effects were reversibly inhibited by preincubation of CaCo-2A cells with actinomycin D or cycloheximide. Butyrate-treated CaCo-2A cells unable to bind fluoresceinated SLT-1 B subunit were undifferentiated as assessed by alkaline phosphatase activity. HT-29 cells induced to differentiate by another signal, glucose deprivation, upregulated receptor content and response to toxin. Crypt-like T-84 cells responded to butyrate with a modest increase in alkaline phosphatase and toxin binding, but no induction of sucrase or lactase, and no change in sensitivity to toxin. The results demonstrate that expression of SLT-1 toxin receptors and toxin sensitivity are coregulated with cellular differentiation in cultured intestinal cells.

Adenocarcinoma↗

Stimulation of gastrointestinal antibody to Shiga toxin by orogastric immunization in mice.

Shiga toxin (ST) is a protein toxin of Shigella dysenteriae type 1, a causative agent of severe diarrhoea and dysentery. In this report we describe the gastrointestinal secretory antibody response of mice following orogastric immunization with ST. Gastrointestinal secretions were sampled by a gastrointestinal lavage technique weekly for 5 weeks after initial immunization. Assay of lavage samples by ELISA showed that mice vaccinated orogastrically with various doses of ST developed gastrointestinal antibody to ST in a dose-dependent manner. Serum anti-ST activity developed by 5 weeks after initial immunization. The ability of ST to act as a mucosal immune adjuvant was investigated by coadministration of ST and keyhole limpet haemocyanin. In contrast to cholera toxin, a potent adjuvant, ST did not demonstrate adjuvant activity. The mouse gastrointestinal lavage model could be useful for further analysis of the cellular basis of ST immunogenicity.

Adjuvants, Immunologic↗

Pathogenesis of Shigella diarrhea: XVII. A mammalian cell membrane glycolipid, Gb3, is required but not sufficient to confer sensitivity to Shiga toxin.

Shiga toxin recognizes a galactose-alpha 1-->4-galactose terminal glycolipid, globotriaosylceramide (Gb3), in sensitive mammalian cells and is translocated by endocytosis to the cytoplasm, where it blocks protein synthesis. To determine if Gb3 is both required and sufficient for toxicity, Gb3 content in cells was altered by blocking key biosynthetic or degradative path enzymes with specific inhibitors. The resulting decrease or increase in cellular Gb3 was associated with a decrease or increase in binding of and response to Shiga toxin. Toxin-resistant Gb3-deficient variants of sensitive cells fused with liposomes containing Gb3 but not globotetraosylceramide (Gb4) became susceptible, whereas fusion of Gb3 liposomes to naturally resistant Gb3-deficient CHO cells increased toxin binding but not cytotoxicity. These data demonstrate that Gb3 is required, but not sufficient, for the action of Shiga toxin and suggest the existence of a toxin translocation mechanism linked to surface glycolipids that is not expressed in CHO cells.

Animals↗

Cryptosporidium parvum infection of Caco-2 cell monolayers induces an apical monolayer defect, selectively increases transmonolayer permeability, and causes epithelial cell death.

Caco-2 cells were grown on permeable filters and infected with Cryptosporidium parvum. Infection rates exceeded 50% of target cells with a sufficient inoculum dose of parasites. Infection induced a dose- and time-dependent fall in transmonolayer resistance, which was closely related to both the inoculum dose and the number of parasites detected by immunofluorescence. Caco-2a, MDBK, and MDBK subclone F5D2 evidenced similar declines in resistance when grown and infected under similar circumstances. Caco-2 monolayers became permeable to molecules of < or = 1,000 Da but continued to remain impermeable to exogenously added, or endogenously produced, proteins of > or = 1,881 Da. We found that infected monolayers released up to 50% of the total cellular lactase dehydrogenase into apical media, but not basal media, and that the vital dye propidium iodide avidly stained infected cells, and often parasites, when added to the apical reservoir. Cryptosporidium infection of Caco-2 monolayers increases transmonolayer permeability, induces an apical cellular and monolayer defect, and causes cell death.

Animals↗

Attachment of Cryptosporidium parvum sporozoites to MDCK cells in vitro.

The initial attachment of Cryptosporidium parvum sporozoites to host cells in vivo may be a critical event in the pathogenesis of this infection. The molecular basis of attachment and the conditions influencing this host-parasite interaction have not been studied systematically. Therefore, we have developed a sporozoite attachment model by using paraformaldehyde-fixed Madin-Darby canine kidney (MDCK) cells. Attachment of sporozoites to fixed MDCK cells was quantitated by indirect immunofluorescence and confirmed by transmission electron microscopy. Attachment in this system was time, temperature (37 degrees C), and pH (7.2 to 7.6) dependent. Dose-response studies demonstrated that the attachment of sporozoites to fixed MDCK cells was a saturable process. Attachment was enhanced in the presence of 10 mM manganese, 1 mM calcium, and 1 to 10 mM zinc. Attachment of sporozoites to MDCK cells was inhibited in a dose-dependent manner by polyclonal anti-Cryptosporidium antisera and by purified immunoglobulin G (IgG). This model will be useful for the study of parasite and host cell molecules involved in the initial interaction of C. parvum sporozoites with their target cell.

Adhesiveness↗

Developmentally regulated Gb3 galactosyltransferase and alpha-galactosidase determine Shiga toxin receptors in intestine.

The receptor for Shiga toxin on rabbit intestinal microvillus membranes (MVMs) has been identified as a developmentally regulated glycolipid, globotriaosylceramide [galactose alpha 1-4 galactose beta 1-4 glucose beta 1-1 ceramide (Gb3)]. MVM Gb3 levels increase markedly in the third week of life, concomitant with fluid secretory responses to the toxin. To study mechanisms controlling developmental regulation of MVM Gb3, we measured the specific synthetic Gb3 galactosyltransferase and degradative alpha-galactosidase activities and subcellular distribution of Gb3 at various ages. Quantitative high-performance liquid chromatography demonstrated a similar developmental pattern in both microsomal and MVM Gb3, indicating that late expression of MVM Gb3 is not due to delayed migration of Gb3 from the microsomal to the MVM. The specific Gb3 galactosyltransferase activity increased with age, with a sharp increase seen at 18 days of age, whereas alpha-galactosidase activity followed an inverse pattern. Thus, regulation of both synthetic and degradative pathways for Gb3 appears to explain the observed changes in Gb3 levels with age. The nature of the signals for developmental regulation of Gb3 levels is unknown, as are the physiological consequences of altered MVM glycolipid composition other than mediating response to Shiga toxin.

Aging↗