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D W Acheson

Publications and source records attributed to D W Acheson.

At least 19 recordsLinked to original sources
PubMed1999

Effect of shiga toxin on NaCl transport in rat distal colon.

BACKGROUND: Shiga toxin causes net fluid secretion in rabbit jejunum by selectively targeting, and inhibiting protein synthesis in, absorptive villous cells. The effect of Shiga toxin on the colon, where it is presumably produced, is not known. This study was undertaken to investigate the effect of Shiga toxin on the rat distal colon. METHODS: Net absorption of water and Na was determined by in vivo perfusion of closed loops of rat colon pre-exposed to Shiga toxin or saline. Unidirectional and net fluxes of 22Na and 36Cl were measured in vitro under voltage-clamp conditions across rat distal colon mucosa pre-exposed to Shiga toxin. Shiga toxin binding to sections of rat distal colon was localized by immunohistochemistry. Protein synthesis was measured in surface and crypt colonocytes with 3H-leucine incorporation. RESULTS: In the in vivo perfusion studies net absorption of Na and water was increased in Shiga toxin-treated colon compared with controls (P < 0.01). In the studies carried out in vitro, J(net)Na and J9net)Cl across Shiga toxin-treated mucosa were found to be significantly higher than in control tissue (P < 0.001 and P < 0.01, respectively). Net absorption of Na or Cl did not increase further in the presence of 25 mM butyrate, indicating the absence of short-chain fatty acids (SCFA)-linked NaCl absorption in Shiga toxin-treated colon. Moreover, Shiga toxin-treated colon failed to respond to theophylline, which induced secretion in the normal colon. Immunohistochemistry showed Shiga toxin binding to crypt cells but not to surface cells in the distal colon. Shiga toxin inhibited protein synthesis (by 27.3%) in crypt cells but not in surface cells (P < 0.05). CONCLUSIONS: An unexpected increase in water and NaCl absorption was noted in Shiga toxin-treated rat distal colon, which appears to result from selective effects of the toxin on secretory crypt cells.

Animals↗
PubMed1999

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↗
PubMed1999

Shiga toxins stimulate secretion of interleukin-8 from intestinal epithelial cells.

In the 1980s, Shiga toxin (Stx)-producing Escherichia coli O157:H7 (STEC) was identified as a cause of hemorrhagic colitis in the United States and was found to be associated with hemolytic uremic syndrome (HUS), a microangiopathic hemolytic anemia characterized by thrombocytopenia and renal failure. The precise way that Stxs cause hemorrhagic colitis and HUS is unclear. Stxs have been thought to cause disease by killing or irreversibly harming sensitive cells through a nonspecific blockade of mRNA translation, eventually resulting in cytotoxicity by preventing synthesis of critical molecules needed to maintain cell integrity. Because STEC is noninvasive, we have been exploring the host-toxin response at the level of the gastrointestinal mucosa, where STEC infection begins. We have found that Stx is capable of interleukin-8 (IL-8) superinduction in a human colonic epithelial cell line. Despite a general blockade of mRNA translation, Stx treatment results in increased IL-8 mRNA as well as increased synthesis and secretion of IL-8 protein. Our data suggest that an active Stx A subunit is required for this activity. Ricin, which has the same enzymatic activity and trafficking pathway as Stx, has similar effects. Exploration of the effects of other protein synthesis inhibitors (cycloheximide, anisomycin) suggests a mechanism of gene regulation that is distinct from a general translational blockade. Use of the specific p38/RK inhibitor SB202190 showed that blocking of this pathway results in decreased Stx-mediated IL-8 secretion. Furthermore, Stxs induced mRNA of the primary response gene c-jun, which was subsequently partially blocked by SB202190. These data suggest a novel model of how Stxs contribute to disease, namely that Stxs may alter regulation of host cell processes in sensitive cells via activation of at least one member of the mitogen-activated protein kinase family in the p38/RK cascade and induction of c-jun mRNA. Stx-induced increases in chemokine synthesis from intestinal epithelial cells could be important in augmenting the host mucosal inflammatory response to STEC infection.

Anisomycin↗
PubMed1999

Shiga toxins 1 and 2 translocate differently across polarized intestinal epithelial cells.

Shiga toxin-producing Escherichia coli (STEC) is an important food-borne pathogen that causes hemolytic-uremic syndrome. Following ingestion, STEC cells colonize the intestine and produce Shiga toxins (Stx), which appear to translocate across the intestinal epithelium and subsequently reach sensitive endothelial cell beds. STEC cells produce one or both of two major toxins, Stx1 and Stx2. Stx2-producing STEC is more often associated with disease for reasons as yet undetermined. In this study, we used polarized intestinal epithelial cells grown on permeable filters as a model to compare Stx1 and Stx2 movement across the intestinal epithelium. We have previously shown that biologically active Stx1 is able to translocate across cell monolayers in an energy-dependent, saturable manner. This study demonstrates that biologically active Stx2 is also capable of movement across the epithelium without affecting barrier function, but significantly less Stx2 crossed monolayers than Stx1. Chilling the monolayers to 4 degrees C reduced the amount of Stx1 and Stx2 movement by 200-fold and 20-fold respectively. Stx1 movement was clearly directional, favoring an apical-to-basolateral translocation, whereas Stx2 movement was not. Colchicine reduced Stx1, but not Stx2, translocation. Monensin reduced the translocation of both toxins, but the effect was more pronounced with Stx1. Brefeldin A had no effect on either toxin. Excess unlabeled Stx1 blocks the movement of (125)I-Stx1. Excess Stx2 failed to have any effect on Stx1 movement. Our data suggests that, despite the many common physical and biochemical properties of the two toxins, they appear to be crossing the epithelial cell barrier by different pathways.

Animals↗
PubMed1999

Isogenic lysogens of diverse shiga toxin 2-encoding bacteriophages produce markedly different amounts of shiga toxin.

We produced isogenic Escherichia coli K-12 lysogens of seven different Shiga toxin 2 (Stx2)-encoding bacteriophages derived from clinical Shiga toxin-producing E. coli (STEC) isolates of serotypes O157:H7, O145, O111, and O83 to assess the variability among these phages and determine if there were phage-related differences in toxin production. Phage genomic restriction fragment length polymorphisms (RFLP) and superinfection resistance studies revealed significant differences among these phages and allowed the seven phages to be placed into five distinct groups. Experiments revealed striking differences in spontaneous phage and toxin production that were correlated with the groupings derived from the RFLP and resistance studies. These results suggest that the genotype of the Stx2 prophage can influence the level of phage release and toxin expression by host strains and thus may be relevant to STEC pathogenesis.

Bacterial Toxins↗
PubMed1999

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↗
PubMed1998

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↗
PubMed1997

Coexpression of the B subunit of Shiga toxin 1 and EaeA from enterohemorrhagic Escherichia coli in Vibrio cholerae vaccine strains.

A promoterless gene for the Shiga toxin 1 B subunit (stxB1) has been placed under transcriptional control of the Vibrio cholerae heat shock gene htpG. A chromosomal enterohemorrhagic Escherichia coli fragment containing eaeA and 400 bp of upstream DNA was added to the construct, downstream of stxB1; no transcription terminators were located between the two genes. The plasmid construct was confirmed by DNA sequencing; in vitro transcription-translation studies demonstrated expression of EaeA from the plasmid. The htpGp-->stxB1, eaeA construct was inserted into lacZ on the chromosome of Peru2, an El Tor V. cholerae strain with both attRS1 sequences and the entire cholera toxin genetic element deleted, and into lacZ in JRB10, a Peru2 derivative that has a second copy of htpGp-->stxB1 also inserted in the V. cholerae virulence gene irgA. Two plasmid constructs, one containing stxB1 under the control of the tac promoter and another containing htpGp-->stxB1,eaeA, were transformed into Peru2. Expression of StxB1 by these constructs was quantified by enzyme-linked immunosorbent assay and was highest in the plasmid construct with stxB1 under the control of the tac promoter. Localization of EaeA to the outer membrane of the vector strains was demonstrated both by Western blotting and by immunofluorescence with an anti-EaeA antibody. A rabbit model for colonization by V. cholerae was used to compare the immune responses to the two heterologous antigens, StxB1 and EaeA, expressed by these strains. Rabbits immunized with Peru2 transformed with a plasmid carrying tac-->stxB1 developed neutralizing serum anti-StxB1 immunoglobulin G antibody responses. One of two rabbits immunized with a strain carrying a chromosomal copy of eaeA developed a marked immune response against EaeA. The plasmid construct containing htpGp-->stxB1,eaeA was unstable, producing low levels of StxB1 in vitro and not evoking anti-EaeA antibody responses in vivo following oral immunization. Chromosomal insertion of eaeA may be preferred for future expression of this antigen in V. cholerae vaccine constructs.

Adhesins, Bacterial↗
PubMed1997

Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli.

An enzyme-linked immunosorbent assay for the detection of Shiga toxins (Premier EHEC assay; Meridian Diagnostics, Inc.) was compared to conventional sorbitol-MacConkey culture for the recovery of enterohemorrhagic Escherichia coli. A total of 74 enteric pathogens, including 8 E. coli O157:H7 isolates, were recovered from 974 stool specimens. Two of these specimens were not tested by Premier assaying due to insufficient sample and are not considered in the data analysis. The Premier EHEC assay detected the 6 evaluable specimens which were culture positive for E. coli O157:H7 and identified an additional 10 specimens as containing Shiga toxin. Seven isolates were recovered from these 10 specimens by an immunoblot assay and were confirmed as toxin producers by a cytotoxin assay. Of these seven, four isolates were serotype O157:H7, one was O26:NM, one was O6:H-, and one was O untypeable:H untypeable. Three specimens contained Shiga toxin by both EHEC immunoassaying and cytotoxin testing; however, no cytotoxin-producing E. coli could be recovered. The sorbitol-MacConkey method had a sensitivity and a specificity of 60 and 100%, respectively, while the Premier EHEC assay had a sensitivity and a specificity of 100 and 99.7%, respectively, for E. coli O157:H7 only. The Premier EHEC assay also detected an additional 20% Shiga toxin-producing E. coli (STEC) that were non-O157:H7. Thus, the Premier EHEC assay is a sensitive and specific method for the detection of all STEC isolates. Routine use would improve the detection of E. coli O157:H7 and allow for determination of the true incidence of STEC other than O157:H7. The presence of blood in the stool and/or the ages of the patients were poor predictors of the presence of STEC. Criteria need to be determined which would allow for the cost-effective incorporation of this assay into the routine screen for enteric pathogens in high-risk individuals, especially children.

Bacterial Toxins↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1995

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↗