Biomedical subjects

Russell L Delude

Publications and source records attributed to Russell L Delude.

28 records · Page 2Linked to original sources

PubMed2003

Proinflammatory cytokines increase the rate of glycolysis and adenosine-5'-triphosphate turnover in cultured rat enterocytes.

OBJECTIVE: Measurements of steady-state adenosine-5'-triphosphate (ATP) levels in tissue samples from patients or experimental animals with sepsis or endotoxemia provide little information about the rate of ATP production and consumption in these conditions. Accordingly, we sought to use an in vitro "reductionist" model of sepsis to test the hypothesis that proinflammatory cytokines modulate ATP turnover rate. DESIGN: In vitro "reductionist" model of sepsis. SETTING: University laboratory. SUBJECTS: Cultured rat enterocyte-like cells. INTERVENTIONS: IEC-6 nontransformed rat enterocytes were studied under control conditions or following incubation for 24 or 48 hrs with cytomix, a mixture of tumor necrosis factor-alpha (10 ng/mL), interleukin-1beta (1 ng/mL), and interferon-gamma (1000 units/mL). To measure ATP turnover rate, ATP synthesis was acutely blocked by adding to the cells a mixture of 2-deoxyglucose (10 mM), potassium cyanide (8 mM), and antimycin A (1 microM). ATP content was measured at baseline (before metabolic inhibition) and 0.5, 1, 2, 5, and 10 mins later. Log-linear ATP decay curves were generated and the kinetics of ATP utilization thereby calculated. MEASUREMENTS AND MAIN RESULTS: ATP consumption rate was higher in cytomix-stimulated compared with control cells (3.11 +/- 1.39 vs. 1.25 +/- 0.66 nmol/min, respectively; p <.01). Similarly, the half-time for ATP disappearance was shorter in cytomix-stimulated compared with control cells (2.63 +/- 1.00 vs. 6.21 +/- 3.49; p <.05). In contrast to these findings, the rate of ATP disappearance was similar in cytokine-naïve and immunostimulated IEC-6 cells when protein and nucleic acid synthesis were inhibited by adding 50 microg/mL cycloheximide and 5 microg/mL actinomycin D to cultures for 4 hrs. The rates of glucose consumption and lactate production were significantly greater in cytomix-stimulated compared with controls cells. CONCLUSIONS: Incubation of IEC-6 cells with cytomix significantly increased ATP turnover. Increased ATP turnover rate was supported by increases in the rate of anaerobic glycolysis. These findings support the view that proinflammatory mediators impose a metabolic demand on visceral cells. In sepsis, cells may be more susceptible to dysfunction on the basis of diminished oxygen delivery and/or mitochondrial dysfunction.

Adenosine Triphosphate↗

PubMed2003

Ethyl pyruvate provides durable protection against inflammation-induced intestinal epithelial barrier dysfunction.

Ethyl pyruvate (EP) has been shown to be an effective anti-inflammatory agent. Herein, we sought to test the following hypotheses: 1) the pharmacological effects of EP persist after cells have been exposed to the compound in vitro, even if the cultures are washed to minimize the amount of EP that is retained in the media; 2) the pharmacological effects of EP persist in vivo, even after waiting a prolonged period (i.e., 6 h) after the last dose of the compound; and 3) the in vivo pharmacological effects of EP are distinct from those of the closely related compound, sodium pyruvate. Incubation of Caco-2 human enterocyte-like monolayers with cytomix, a mixture of interleukin-1beta, interferon-gamma, and tumor necrosis factor, increased permeability to the fluorescent macromolecule, FITC-labeled Dextran (mol wt 4,000 Da). Co-incubation of the cells with 5 mM EP ameliorated cytomix-induced hyperpermeability and induction of iNOS mRNA expression. EP was associated with similar pharmacological effects when cells were pre-incubated with the compound for 24 h prior and then washed extensively prior to adding the cytokine cocktail. Injecting C57Bl/6 mice with lipopolysaccharide (LPS) resulted in gut barrier dysfunction and hepatocellular injury. Although equivalent doses of both EP and sodium pyruvate ameliorated these phenomena, EP was more efficacious than pyruvate. Pretreatment with EP ameliorated the deleterious effects of LPS, even when the duration between the last dose of EP and the endotoxic challenge was 6 h. We conclude that EP provides durable protection against some of the deleterious effects of LPS or pro-inflammatory cytokines.

Animals↗

PubMed2003

Ethyl pyruvate ameliorates intestinal epithelial barrier dysfunction in endotoxemic mice and immunostimulated caco-2 enterocytic monolayers.

Ethyl pyruvate (EP) solution ameliorates ileal mucosal hyperpermeability and decreases the expression of several proinflammatory genes in ileal and/or colonic mucosa when it is used instead of Ringer's lactate solution (RLS) to resuscitate mice from hemorrhagic shock. To test the hypothesis that EP can ameliorate gut barrier dysfunction induced by other forms of inflammation, we incubated Caco-2 monolayers for 24 to 48 h with cytomix (a mixture of interferon-gamma, tumor necrosis factor-alpha, and interleukin-1beta) in the presence or absence of graded concentrations of EP or sodium pyruvate. Cytomix increased the permeability of Caco-2 monolayers to fluorescein isothiocyanate-labeled dextran (FD4; average molecular mass 4 kDa), but this effect was inhibited by adding 0.1 to 10 mM EP (but not similar concentrations of sodium pyruvate) to the culture medium. EP inhibited several other cytomix-induced phenomena, including nuclear factor-kappaB activation, inducible nitric oxide synthase mRNA expression, and nitric oxide production. Cytomix altered the expression and localization of the tight junctional proteins, ZO-1 and occludin, but this effect was prevented by EP. Delayed treatment with EP solution instead of RLS ameliorated ileal mucosal hyperpermeability to FD4 and bacterial translocation to mesenteric lymph nodes in mice challenged with lipopolysaccharide (LPS). These data support the view that EP ameliorates cytokine- and/or LPS-induced derangements in intestinal epithelial barrier function.

Adjuvants, Immunologic↗

PubMed2002

Cytokines induce HIF-1 DNA binding and the expression of HIF-1-dependent genes in cultured rat enterocytes.

Cellular adaptation to hypoxia depends, in part, on the transcription factor hypoxia-inducible factor-1 (HIF-1). Normoxic cells exposed to an inflammatory milieu often manifest phenotypic changes, such as increased glycolysis, that are reminiscent of those observed in hypoxic cells. Accordingly, we investigated the effects of cytomix, a mixture containing IFN-gamma, TNF, and IL-1beta on the expression of HIF-1-dependent proteins under normoxic and hypoxic conditions. Incubation of intestine-derived epithelial cells (IEC-6) under 1% O(2) increased HIF-1 DNA binding and expression of aldolase A, enolase-1, and VEGF mRNA. Incubation of normoxic cells with cytomix for 48 h also markedly increased HIF-1 DNA binding and expression of mRNAs for these proteins. Incubation of hypoxic cells with cytomix did not inhibit HIF-1 DNA binding or upregulation of HIF-1-dependent genes in response to hypoxia. Neither cytomix nor hypoxia increased steady-state levels of HIF-1alpha mRNA. Incubation of IEC-6 cells with cytomix induced nitric oxide (NO.) biosynthesis, which was blocked if the cultures contained l-N(G)-(1-iminoethyl)lysine hydrochloride (l-NIL). Treatment with l-NIL, however, failed to significantly alter aldolase A, enolase-1, and VEGF mRNA levels in normoxic cytomix-treated cells. Proinflammatory cytokines activate the HIF-1 pathway and increase expression of glycolytic genes in nontransformed rat intestinal epithelial cells, largely through an NO.-independent mechanism.

Animals↗

PubMed2002

Regulation of hypoxia-inducible factor 1 in enterocytic cells.

BACKGROUND: Mucosal hypoxia due to intestinal hypoperfusion is characteristic of a number of clinical disorders. An early event in the adaptive response to cellular hypoxia is the binding of hypoxia-inducible factor 1 (HIF-1) to cis-acting regulatory sites in target genes. METHODS: We characterized the expression of HIF-1 in transformed (Caco-2(BBe) and T84) and nontransformed human (FHs 74 Int) and rat (IEC-6) intestinal epithelial cell lines. RESULTS: The electrophoretic mobility shift assay detected increased HIF-1 DNA-binding activity in each cell line within 2 h of hypoxia (1% O2). HIF-1 binding was maximal within 4 h and remained stable for 24 h. HIF-1 DNA-binding activity was maximal in the established IEC-6 cell line below 2% oxygen, but HIF-1 DNA-binding activity was not detectable above 0.5% O2 in the primary human FHs 74 Int cell line. The nonspecific protein kinase inhibitor genistein (200 microM) inhibited HIF-1 binding at 4 h. Transfection of Caco-2 cells with a wild-type, but not a mutant, HIF-1-dependent luciferase expression vector resulted in a fourfold induction of reporter gene expression during hypoxia. CONCLUSIONS: In conclusion, HIF-1 regulates gene expression in enterocytes and an undefined phosphorylation event is important for O2 sensing.

Animals↗

PubMed2002

HMGB1 B box increases the permeability of Caco-2 enterocytic monolayers and impairs intestinal barrier function in mice.

BACKGROUND & AIMS: High mobility group (HMG) B1 is a nonhistone nuclear protein that was recently identified as a late-acting mediator of lipopolysaccharide-induced lethality in mice. The proinflammatory actions of HMGB1 have been localized to a region of the molecule called the B box. METHODS: To determine whether HMGB1 or B box are capable of causing derangements in intestinal barrier function, we incubated cultured Caco-2 human enterocytic monolayers with recombinant human HMGB1 or a 74-residue truncated form of the protein consisting of the B box domain. RESULTS: Both HMGB1 and B box increased the permeability of Caco-2 monolayers to fluorescein isothiocyanate-labeled dextran (FD4) in a time- and dose-dependent fashion. The increase in permeability was reversible following removal of the recombinant protein. Exposure of Caco-2 cells to B box resulted in increased expression of inducible nitric oxide synthase messenger RNA and increased production of NO. When we used various pharmacologic strategies to inhibit NO production or scavenge NO or peroxynitrite (ONOO(-)), we abrogated B box-induced hyperpermeability. Administration of B box to wild-type mice increased both ileal mucosal permeability to FD4 and bacterial translocation to mesenteric lymph nodes. These effects were not observed in inducible nitric oxide synthase knockout mice. CONCLUSIONS: These data support the view that HMGB1 and B box are capable of causing alterations in gut barrier function via a mechanism that depends on the formation of NO and ONOO(-).

Animals↗

PubMed2002

Resuscitation from hemorrhagic shock with Ringer's ethyl pyruvate solution improves survival and ameliorates intestinal mucosal hyperpermeability in rats.

We previously showed that pretreatment with a solution of ethyl pyruvate in a calcium-containing balanced salt solution, Ringer's ethyl pyruvate solution (REPS), ameliorates gut mucosal damage in rats subjected to mesenteric ischemia/reperfusion. Herein, we sought to test the hypothesis that REPS would be beneficial as a post-treatment (i.e., resuscitation fluid) for hemorrhagic shock. Anesthetized Sprague-Dawley rats were bled to a mean arterial pressure (MAP) of 40 mmHg until 40% of shed blood was returned. The animals then were resuscitated over 60 min with the remaining shed blood plus twice the shed blood volume as either Ringer's lactate solution (RLS) or REPS. In Experiment 1, RLS or REPS was then infused for 3 h more (or until death) at 3 mL/kg/h. Read-outs were post-resuscitation ileal mucosal permeability to fluorescein-labeled Dextran with an average molecular mass of 4000 Da (FD4) and survival. Permeability, determined just before death (MAP < 40 mmHg) or after 4 h of resuscitation, was assessed using an ex vivo everted gut sac technique and is expressed as a clearance (nL/cm/min). In Experiment 2, the read-outs were ileal FD4 permeability measured at 60 min after starting resuscitation and gut and liver malondialdehyde (MDA) formation. FD4 clearance data were logarithmically transformed prior to performing statistical analyses. In Experiment 1, 4/8 (50%) of RLS-treated rats survived 4 h after resuscitation whereas 7/7 (100%) of REPS-treated rats survived (P< 0.05). Ileal FD4 clearances were 105 +/- 30*, 85 +/- 34*, and 38 +/- 7 for all rats treated with RLS, surviving rats treated with RLS, and rats treated with REPS, respectively (the asterisk indicates P < 0.05 vs. REPS). In Experiment 2, ileal FD4 clearances were 71 +/- 13* and 34 +/- 8 for rats treated with RLS and REPS (n = 5 each), respectively. Post-resuscitation levels of MDA in the ileum and liver were significantly lower in rats treated with REPS as compared with RLS. Resuscitation with REPS, a stable and nontoxic antioxidant solution, improves survival and ameliorates ileal mucosal permeability in a rat model of severe hemorrhagic shock.

Animals↗

PubMed2002

Ethyl pyruvate modulates inflammatory gene expression in mice subjected to hemorrhagic shock.

Administration of pyruvate, an effective scavenger of reactive oxygen species, has been shown to be salutary in numerous models of redox-mediated tissue or organ injury. Pyruvate, however, is unstable in solution and, hence, is not attractive for development as a therapeutic agent. Herein, ethyl pyruvate, which is thought to be more stable than the parent compound, was formulated in a calcium-containing balanced salt solution [Ringer ethyl pyruvate solution (REPS)] and evaluated in a murine model of hemorrhagic shock and resuscitation (HS/R). Resuscitation with REPS instead of Ringer lactate solution (RLS) significantly improved survival at 24 h and abrogated bacterial translocation to mesenteric lymph nodes and the development of increased ileal mucosal permeability to FITC-labeled dextran (4,000 Da) at 4 h. Mice treated with REPS instead of RLS also had lower circulating levels of alanine aminotransferase at 4 h. Treatment with REPS instead of RLS decreased activation of nuclear factor-kappaB in liver and colonic mucosa after HS/R and also decreased the expression of inducible nitric oxide synthase, tumor necrosis factor, cyclooxygenase-2, and interleukin-6 mRNA in liver, ileal mucosa, and/or colonic mucosa. These data support the view that resuscitation with REPS modulates the inflammatory response and decreases hepatocellular and gut mucosal injury in mice subjected to HS/R.

Animals↗

PubMed2002

Liposomal NAD(+) prevents diminished O(2) consumption by immunostimulated Caco-2 cells.

Accumulating data support the view that sepsis is associated with an acquired intrinsic derangement in the ability of cells to consume O(2), a phenomenon that has been termed "cytopathic hypoxia." We sought to use an in vitro "reductionist" model system using cultured cells stimulated with proinflammatory cytokines to test the hypothesis that cytopathic hypoxia is mediated, at least in part, by depletion of intracellular levels of NAD(+)/NADH secondary to activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We measured O(2) consumption by Caco-2 enterocytes growing on microcarrier beads after cells were incubated for 24 h under control conditions or with cytomix, a mixture of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma. Immunostimulated cells consumed O(2) at about one-half the rate of control cells, but this effect was largely prevented if any one of the following pharmacological agents was present during the period of incubation with cytomix: 4,5-dihydroxy-1,3-benzene disulfonic acid, a superoxide radical anion scavenger; 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger; 5,10,15,20- tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III], a peroxynitrite (ONOO(-)) decomposition catalyst; urate, an ONOO(-) scavenger; 3-aminobenzamide, a PARP inhibitor; or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide HCl, a chemically dissimilar and more potent PARP inhibitor. The decrease in O(2) uptake induced by cytomix was associated with decreased cellular levels of NAD(+)/NADH. The decrease in cellular NAD(+)/NADH content and the decrease in O(2) uptake induced by cytomix were completely abrogated if liposome-encapsulated NAD(+) was added to the cultures during immunostimulation. Empty liposomes also increased O(2) uptake by immunostimulated Caco-2 cells, but much less effectively than liposomes containing NAD(+). These data are consistent with the view that enterocytes exposed to proinflammatory cytokines consume less O(2) due to NAD(+)/NADH depletion secondary to activation of PARP by ONOO(-) or other oxidants.

Antineoplastic Agents↗

PubMed2002

Effect of hemorrhagic shock on gut barrier function and expression of stress-related genes in normal and gnotobiotic mice.

We sought to determine whether gut-derived microbial factors influence the hepatic or intestinal inflammatory response to hemorrhagic shock and resuscitation (HS/R). Conventional and gnotobiotic mice contaminated with a defined microbiota without gram-negative bacteria were subjected to either a sham procedure or HS/R. Tissue samples were obtained 4 h later for assessing ileal mucosal permeability to FITC dextran and hepatic and ileal mucosal steady-state IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and TNF mRNA levels. Whereas HS/R significantly increased ileal mucosal permeability in conventional mice, this effect was not apparent in gnotobiotic animals. HS/R markedly increased hepatic mRNA levels for several proinflammatory genes in both conventional and gnotobiotic mice. HS/R increased ileal mucosal IL-6 and COX-2 mRNA expression in conventional but not gnotobiotic mice. If gnotobiotic mice were contaminated with Escherichia coli C25, HS/R increased ileal mucosal permeability and upregulated expression of IL-6 and COX-2. These data support the view that the hepatic inflammatory response to HS/R is largely independent of the presence of potentially pathogenic gram-negative bacteria colonizing the gut, whereas the local mucosal response to HS/R is profoundly influenced by the microbial ecology within the lumen during and shortly after the period of hemorrhage.

Animals↗