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Biomedical subjects

W F Stenson

Publications and source records attributed to W F Stenson.

At least 37 records · Page 2Linked to original sources
PubMed1997

Crypt stem cell survival in the mouse intestinal epithelium is regulated by prostaglandins synthesized through cyclooxygenase-1.

Prostaglandins (PGs) are important mediators of epithelial integrity and function in the gastrointestinal tract. Relatively little is known, however, about the mechanism by which PGs affect stem cells in the intestine during normal epithelial turnover, or during wound repair. PGs are synthesized from arachidonate by either of two cyclooxygenases, cyclooxygenase-1 (Cox-1) or cyclooxygenase-2 (Cox-2), which are present in a wide variety of mamalian cells. Cox-1 is thought to be a constitutively expressed enzyme, and the expression of Cox-2 is inducible by cytokines or other stimuli in a variety of cell types. We investigated the role of PGs in mouse intestinal stem cell survival and proliferation following radiation injury. The number of surviving crypt stem cells was determined 3.5 d after irradiation by the microcolony assay. Radiation injury induced a dose-dependent decrease in the number of surviving crypts. Indomethacin, an inhibitor of Cox-1 and Cox-2, further reduced the number of surviving crypts in irradiated mice. The indomethacin dose response for inhibition of PGE2 production and reduction of crypt survival were similar. DimethylPGE2 reversed the indomethacin-induced decrease in crypt survival. Selective Cox-2 inhibitors had no effect on crypt survival. PGE2, Cox-1 mRNA, and Cox-1 protein levels all increase in the 3 d after irradiation. Immunohistochemistry for Cox-1 demonstrated localization in epithelial cells of the crypt in the unirradiated mouse, and in the regenerating crypt epithelium in the irradiated mouse. We conclude that radiation injury results in increased Cox-1 levels in crypt stem cells and their progeny, and that PGE2 produced through Cox-1 promotes crypt stem cell survival and proliferation.

Animals↗
PubMed1997

Leukotrienes in ulcerative colitis: results of a multicenter trial of a leukotriene biosynthesis inhibitor, MK-591.

BACKGROUND & AIMS: Leukotrienes (LTs) are believed to be important in the pathogenesis of ulcerative colitis (UC). The aim of this study was to determine whether inhibition of LT biosynthesis with a 5-lipoxygenase inhibitor (MK-591) induces remission in patients with mild to moderate UC. METHODS: One hundred eighty-three patients with mild to moderately active UC enrolled in this randomized parallel group, double-blind study. Patients received placebo or MK-591 at a dose of 12.5, 50, or 100 mg twice daily for 8 weeks. A subset of patients underwent rectal dialysis to determine LTB4 concentration. RESULTS: MK-591 reduced LTB4 concentrations in rectal dialysate at the final determination. The median percent of baseline LTB4 concentration for 100 mg taken twice daily was 1.4% (n = 4); for 50 mg taken twice daily, 16.5% (n = 6); for 12.5 mg taken twice daily, 12% (n = 6); and for placebo, 78% (n = 6). There was no correlation between reduction of LTB4 and remission. Patients in remission at week 8 were as follows: placebo, 9 of 44 (20.5%); 100 mg taken twice daily, 11 of 43 (25.6%); 50 mg taken twice daily, 8 of 49 (16.3%); and 12.5 mg taken twice daily, 4 of 47 (8.5%) (P > 0.10). CONCLUSIONS: MK-591 markedly inhibited LT biosynthesis, but it did not differ significantly from placebo in clinical efficacy. Inhibition of LT biosynthesis was not effective as a single therapeutic modality in active UC.

Adolescent↗
PubMed1996

Quantification of cholesterol tracers by gas chromatography--negative ion chemical ionization mass spectrometry.

Because of its high sensitivity, gas chromatography negative ion chemical ionization mass spectrometry (GC-NCI-MS) is a potentially valuable analytical tool for the study of cholesterol metabolism. Of several derivatives prepared for potential use in tracer studies pentafluorobenzoyl cholesterol was selected because it formed rapidly at ambient temperature and was stable for long periods, could be detected at a level of 1 fmol, and yielded a mass spectrum in which the molecular ion was the principal component. Hexadeuterated cholesterol tracer ([26,26,26,27,27,27-2H6]cholesterol) could be detected in dilutions up to 2700 in unlabeled cholesterol by selected ion monitoring with a coefficient of variation averaging 3.2%. In seven normal subjects tracer cholesterol was infused intravenously and plasma cholesterol enrichment was determined after 4 h. The measured rapidly miscible cholesterol pool was 391.0 +/- 38.6 mg cholesterol/kg. Negative ion mass spectrometry of pentafluorobenzyol cholesterol will facilitate analysis of both small amounts of natural cholesterol and labeled cholesterol in applications where sensitivity is critical.

Benzoates↗
PubMed1996

Quantification of distinct molecular species of platelet activating factor in ulcerative colitis.

The aim of this study was to characterize the synthesis and metabolism of platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by colonic mucosa from patients with ulcerative colitis and healthy individuals. Tissue was obtained by endoscopic biopsy and by scraping the mucosa from surgical resections. Tissue was assayed for the various molecular species of PAF and its biologically inactive metabolite lyso-PAF using gas chromatography/mass spectrometry. Mucosa from surgical resections for ulcerative colitis contained C16:0 PAF (mean = 156 ng/g of mucosa), but not C18:0 PAF, PAF was not identified in mucosa from normal surgical resections or in endoscopic biopsies from either patients with ulcerative colitis or normal individuals. Both C16:0 lyso-PAF and C18:0 lyso-PAF were found in mucosa from normal and ulcerative colitis surgical resections and in endoscopic biopsies from ulcerative colitis and normal tissue. Levels of lyso-PAF were similar in ulcerative colitis and normal mucosa. Incubation of mucosa from areas of active inflammation in ulcerative colitis with the calcium ionophore A23187 increased the levels of C16:0 PAF by 2-3 fold and also increased the levels of C16:0 lyso-PAF. Addition of 3H-PAF to endoscopic biopsies from either normal individuals or patients with ulcerative colitis resulted in hydrolysis to 3H-lyso-PAF. The data on colonic mucosal levels of PAF are consistent with the results of earlier studies measuring PAF in patients with ulcerative colitis by bioassay. This study examines the synthesis and metabolism of specific molecular species of PAF in ulcerative colitis for the first time.

Adult↗
PubMed1996

Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease.

BACKGROUND & AIMS: Inducible nitric oxide synthase (iNOS) is generated in several cell types by treatment with lipopolysaccharides or cytokines. Earlier studies suggested that ulcerative colitis is associated with increased NO produced by iNOS; however, the cellular source of the NO synthesis was not identified. A possible mechanism of NO-induced cellular damage is through its interaction with superoxide to produce peroxynitrite, which reacts with tyrosine to form nitrotyrosine in cellular proteins. METHODS: Using immunoperoxidase microscopy with a new monospecific human iNOS antibody (NO-53), the cellular distribution of iNOS and nitrotyrosine was examined using human colonic mucosa from normal bowel, ulcerative colitis, Crohn's disease, and diverticulitis. RESULTS: Intense focal iNOS labeling was localized to the inflamed colonic epithelium in ulcerative colitis, Crohn's disease, and diverticulitis but was not detectable in the uninflamed epithelium. Nitrotyrosine labeling was also observed in the inflamed colonic epithelium and was associated with nearby iNOS staining; nitrotyrosine was undetectable in normal mucosal epithelium. iNOS and nitrotyrosine were also detected in lamina propria mononuclear cells and neutrophils. CONCLUSIONS: These findings suggest that iNOS is induced in the inflamed human colonic epithelium and is associated with the formation of peroxynitrite and the nitration of cellular proteins.

Aged↗
PubMed1995

Platelet-activating factor acetylhydrolases in Caco-2 cells and epithelium of normal and ulcerative colitis patients.

BACKGROUND & AIMS: Platelet-activating factor (PAF) is a potent inflammatory mediator implicated in the pathogenesis of inflammatory bowel disease and necrotizing enterocolitis. Metabolism by platelet-activating factor acetylhydrolase (PAF-AH) is the major pathway for platelet-activating factor degradation. The aim of this study was to investigate the possible role of intestinal epithelium as a source of PAF-AH. METHODS: Intracellular and secreted PAF-AHs were characterized in human colonic epithelial cells isolated from histologically normal mucosa and inflamed mucosa from patients with ulcerative colitis and in the human intestinal epithelial cell line Caco-2 by measuring the metabolism of [3H]-PAF to [3H]lysoPAF. RESULTS: Human colonic epithelial cells and Caco-2 cells synthesize and secrete PAF-AH as shown by in vitro hydrolysis of [3H]PAF to [3H]-lysoPAF in cell lysates and conditioned media. Both intracellular and secreted PAF-AHs are calcium-independent and substrate-specific for phospholipids similar to PAF. Epithelial cells from involved areas of resections for ulcerative colitis had increased levels of secreted PAF-AH and decreased levels of intracellular PAF-AH compared with epithelial cells from histologically normal areas. CONCLUSIONS: Human colonic epithelial cells and Caco-2 cells produce intracellular and secreted PAF-AHs, which are distinct proteins. This is the first demonstration of PAF-AH production by epithelial cells.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1994

Entamoeba histolytica interactions with polarized human intestinal Caco-2 epithelial cells.

To model the initial pathogenic effects of Entamoeba histolytica trophozoites on intestinal epithelial cells, the interactions of E. histolytica HM1-IMSS trophozoites with polarized human intestinal Caco-2 cell monolayers grown on permeabilized filters were examined. Trophozoites, when incubated with the apical surface of the monolayers at 37 degrees C, induced a rapid decrease in transepithelial resistance over 15 to 60 min. The transmonolayer resistance response was not associated with changes in short-circuit current but was associated with an increase in mannitol flux, suggesting that the drop in resistance reflected a nonselective increase in epithelial permeability rather than stimulation of electrogenic ion transport. This response preceded the earliest detection of morphologic disruption of monolayer integrity by light or electron microscopy. Apical injury to the monolayer was detected by ultrastructural studies which revealed a loss of brush border in regions of contact between epithelial cells and amebas and by chromium release assays where a small increase in the apical release of 51Cr from the monolayer (6% over background) was observed. The transmonolayer resistance response was inhibited when the temperature was reduced to 4 degrees C and by addition of cytochalasin D (1 microgram/ml) to the medium at concentrations that did not directly affect transmonolayer resistance. Application of amebic lysates or medium conditioned by coincubation of amebas with Caco-2 monolayers failed to lower transmonolayer resistance, suggesting that this effect was not mediated by soluble amebic cytotoxins. Polarized Caco-2 monolayers grown on permeable filters provide a useful model for studying the initial interactions of E. histolytica trophozoites with intestinal epithelial cells.

Animals↗
PubMed1994

Mechanisms of transit of lipid mediators of inflammation and bacterial peptides across intestinal epithelia.

The transit of two lipid mediators of inflammation, leukotriene B4 (LTB4) and prostaglandin E2 (PGE2), and a formylated peptide produced by intestinal bacteria, N-formylmethionylleucylphenylalanine (FMLP), across Caco-2 cell monolayers was characterized and compared with the transit of mannitol, a hexose known to cross epithelial monolayers by paracellular pathways. The permeability of less mature low-resistance ( < 200 ohm.cm2) monolayers to all four test compounds was similar, but as monolayers matured and the transmonolayer resistance increased, the transit of LTB4, PGE2, FMLP, and mannitol decreased to different degrees, resulting in a selectivity of permeability to the four test compounds in the order LTB4 > PGE2 > mannitol > FMLP. The transit of all four test compounds across Caco-2 cell monolayers was bidirectional, nonsaturable, and energy independent. A small portion of the added LTB4 was incorporated into the cells, whereas the other three compounds were not. Thus the transit of PGE2, mannitol, and FMLP across Caco-2 monolayers appears to be solely by the paracellular pathway, whereas the transit of LTB4 also involves the paracellular pathway but may also involve diffusion through the cell membrane and around tight junctions.

Bacterial Proteins↗
PubMed1993

Induction of epithelial arachidonate 12-lipoxygenase at active sites of inflammatory bowel disease.

To determine if epithelial lipoxygenases are regulated by mucosal inflammation, we examined the distribution of arachidonate 12-lipoxygenase in healthy colonic tissue and in involved and uninvolved sections of colon with inflammatory bowel disease. Immunohistochemistry of formaldehyde-fixed, paraffin-embedded intestinal tissue using two anti-12-lipoxygenase antibodies and indirect biotin-avidin-peroxidase detection demonstrated that, in contrast to tissue from normal colon (n = 8), in which 12-lipoxygenase antigen was undetectable in mucosal epithelial cells, the mucosal and glandular epithelium of inflamed colon in ulcerative colitis (n = 4) or Crohn's disease (n = 4) exhibited markedly positive immunostaining with anti-12-lipoxygenase antibodies. Immunoblotting of whole cell extracts with anti-12-lipoxygenase antibodies showed immunoperoxidase staining of a single protein band that comigrated with purified 12-lipoxygenase (in relative molecular weight: M(r) = 72,000) in mucosal samples from inflamed colon from subjects with ulcerative colitis or Crohn's disease but no comparable band in samples from uninvolved sections of the same colons or from colons of subjects without inflammatory bowel disease. Assays of 12-lipoxygenase activity indicated a corresponding increase in enzymatic activity in the same mucosal samples. The increased levels of 12-lipoxygenase antigen in the mucosal and glandular epithelial cells in regions of colon affected by inflammatory bowel disease, the corresponding increases in 12-lipoxygenase activity, and the absence of detectable 12-lipoxygenase antigen or enzymatic activity in the same cell types in noninflamed colonic tissue all suggest that epithelial cell 12-lipoxygenase is induced by local mediators of colonic inflammation.

Adult↗
PubMed1993

Regulation of paracellular permeability in Caco-2 cell monolayers by protein kinase C.

Caco-2 cells are an enterocyte-like cell line derived from a human colonic adenocarcinoma. Paracellular permeability was assessed in monolayers of these cells by transmonolayer resistance and by the permeation of [3H]mannitol across the monolayer. Paracellular permeability was increased by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (50 nM), carbachol (500 microM), and the combination of carbachol (50 microM) and monolein (100 microM), an inhibitor of diacylglycerol kinase, as manifested by a decrease in transmonolayer resistance and an increase in mannitol permeation. The effects of all of these stimuli on transmonolayer resistance were inhibited by staurosporine (3 nM), an inhibitor of PKC. The effects of carbachol plus monolein were also inhibited by atropine (0.1 microM), a muscarinic antagonist. Treatment of the monolayers with each of the stimuli was associated with translocation of PKC activity from cytosol to a membrane-associated state. Stimulation of Caco-2 cell monolayers with phorbol myristate acetate or with the combination of carbachol and monolein was also associated with phosphorylation of the MARCKS protein, an endogenous substrate of PKC. These data support the hypothesis that intestinal paracellular permeability is regulated by the activity of enterocyte PKC and demonstrate that the increase in paracellular permeability induced by binding of carbachol to the muscarinic receptor is mediated by activation of PKC.

Adenocarcinoma↗
PubMed1992

Dietary supplementation with fish oil in ulcerative colitis.

OBJECTIVE: To determine the efficacy of fish oil supplementation in patients with active ulcerative colitis. DESIGN: Multicenter, randomized, double-blind, placebo-controlled, crossover trail with 4-month treatment periods (fish oil and placebo) separated by a 1-month washout. SETTING: Four gastroenterology divisions. PATIENTS: Twenty-four patients with active ulcerative colitis entered the study. Five dropped out, and one was noncompliant. Eighteen patients completed the study. All patients had active disease as manifested by diarrhea and rectal inflammation. INTERVENTIONS: Treatment with prednisone and sulfasalazine was continued. Fish oil supplementation consisted of 18 Max-EPA (eicosapentaenoic acid) capsules daily (eicosapentaenoic acid, 3.24 g; and docosahexaenoic acid, 2.16 g). Placebo supplementation consisted of 18 identical capsules containing isocaloric amounts of vegetable oil. MEASUREMENTS: Patients were evaluated at study entry and after each diet period. Evaluations included a review of symptoms, flexible sigmoidoscopy, rectal biopsy, and rectal dialysis to measure prostaglandin E2 and leukotriene B4 levels. RESULTS: Fish oil supplementation resulted in a significant decrease in rectal dialysate levels of leukotriene B4 from 71.0 to 27.7 pg/mL (average change, -43.3 pg/mL; 95% CI, -83 to -3.6). Significant improvements were seen in acute histology index (average change, -8.5 units from a baseline of 10.5 units; CI, -12.9 to -4.2) and total histology index (average change, -8.5 units from a baseline of 14.80; CI, -13.2 to -3.8) as well as significant weight gain (average weight gain, 1.74 kg, CI, 0.94 to 2.54). No significant changes occurred in any variable during the placebo period. Seven patients received concurrent treatment with prednisone. During the fish oil supplementation period, the mean prednisone dose decreased from 12.9 mg/d to 6.1 mg/d and rose from 10.4 mg/d to 12.9 mg/d during the placebo diet period (P greater than 0.20). CONCLUSIONS: Four months of diet supplementation with fish oil in patients with inflammatory bowel disease resulted in reductions in rectal dialysate leukotriene B4 levels, improvements in histologic findings, and weight gain.

Adult↗
PubMed1992

Caco-2 cell transfection by rat intestinal alkaline phosphatase cDNA increases surfactant-like particles.

The rat enterocyte produces a particle with surfactant-like properties (including a whorled appearance, enrichment for dipalmitoyl phosphatidylcholine, and ability to lower surface tension) that also is enriched for intestinal alkaline phosphatase. Human Caco-2 cells grown on polycarbonate filters were utilized to study the secretion of these particles and exhibited whorls and strands of unilamellar membranes, particularly concentrated at the apical pole or near junctional complexes. Concentrated culture medium from these cells separated on continuous NaBr gradients revealed a fraction at density = 1.07 g/l enriched for phosphatidylcholine and intestinal alkaline phosphatase. This fraction contained membranous sheets containing alkaline phosphatase, detected by immunolocalization. Phosphatidylcholine comprised 54% of phospholipid in this fraction, compared with 20% in brush borders. When Caco-2 cells were transfected with cDNA encoding rat intestinal alkaline phosphatase, cellular phosphatase activity increased twofold, but activity in the medium increased 14-fold to > 200 (average 32)-fold. Ultrastructurally, compared with mock-transfected cells or cells transfected with human placental alkaline phosphatase, transfection with rat intestinal alkaline phosphatase cDNA led to intracellular and extracellular accumulation of surfactant-like particles. We conclude that surfactant-like particles are produced by Caco-2 cells, and their production can be enhanced by transfection with a cDNA encoding a protein known to be associated with such particles.

Alkaline Phosphatase↗
PubMed1992

Metabolism of oxygenated derivatives of arachidonic acid by Caco-2 cells.

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated separately with 3H8-labeled preparations of three different lipid mediators of inflammation: 5-hydroxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, and leukotriene B4. Both [3H8]5-hydroxyeicosatetraenoic and [3H8]12-hydroxyeicosatetraenoic acids were taken up and metabolized by Caco-2 cells, but [3H]leukotriene B4 remained unmetabolized in the incubation medium. [3H]5-hydroxyeicosatetraenoic acid was esterified into cellular phospholipids (15%) and triglycerides (4%) but did not undergo beta-oxidation. When [3H]12-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, 14% underwent two cycles of beta-oxidation to form [3H]8-hydroxyhexadecatrienoic acid, and 3% underwent three cycles of beta-oxidation to form [3H]6-hydroxytetradecadienoic acid, both of which were released into the media. [3H]12-Hydroxyeicosatetraenoic acid was also esterified into cellular phospholipids (13%), but none was esterified into cellular triglycerides.

Adenocarcinoma↗
PubMed1992

The role of the mucosal immune system in inflammatory bowel disease.

Continued delineation of the major factors that lead to intestinal inflammation will provide critical insights into many of the pathophysiologic events leading to tissue destruction in IBD. The exploration of exciting and important new areas, such as the role of adhesion molecules, proinflammatory cytokines, and the activation of lymphocytes and phagocytes, will contribute significantly to a better understanding of the mechanisms that sustain the intestinal inflammatory process. Determining the mechanisms of amplification and perpetuation of intestinal inflammation as well as learning more about the natural suppression of intestinal inflammation by the normal cellular and cytokine networks of the mucosal immune system will open exciting new therapeutic approaches. It is encouraging to see realistic and testable working models emerge from the combined efforts of many committed investigators who have been engaged in studying the role of the mucosal immune system in the pathophysiology of IBD. A great deal more remains to be learned in this rapidly advancing area, and we can look forward with confidence to continued advances in the study of IBD.

Antibody Formation↗
PubMed1992

Trafficking of exogenous fatty acids within Caco-2 cells.

Dietary fatty acids (FAs) crossing the apical plasma membrane of small intestinal enterocytes are targeted to different metabolic pathways than serum FAs crossing the basolateral membrane. This apparent compartmentalization of FA metabolism in enterocytes was further investigated using a model human enterocyte-like intestinal cell line. [3H]Oleic acid bound to bovine serum albumin (BSA) was added to the apical or basolateral surfaces of confluent monolayers of Caco-2 cells growing on uncoated polycarbonate filters. In other experiments, [3H]oleic acid incorporated into micelles with taurocholate (+/- 2-monoacylglycerol) was added apically. Caco-2 cells absorbed oleic acid bound to BSA from both the apical and basolateral surfaces at the same rate. Oleic acid in micellar solution was absorbed more efficiently than oleic acid bound to BSA. Regardless of its site or mode of presentation, the majority of the incorporated oleic acid was found in triglycerides. Only a small fraction was subjected to beta-oxidation or esterification into phospholipids. Most of the incorporated oleic acid was still retained intracellularly at 24 h. The polarity of triglyceride secretion was influenced by the experimental conditions. Triglyceride secretion was not significantly polarized when oleic acid-BSA was presented apically. However, the ratio of basolateral to apical secretion at 24 h was 9:1 for oleic acid-BSA presented basolaterally. For oleic acid in taurocholate micelles there was a trend toward polarity of secretion to the apical media (apical to basolateral ratio = 2:1). The inclusion of 2-monoacylglycerol in oleic acid-taurocholate micelles did not augment triglyceride synthesis or secretion. These differences indicate that compartmentation of FA metabolism in Caco-2 cells is influenced by the site of FA presentation. Northern and Western blot hybridization studies indicated that the liver fatty acid-binding protein but not the intestinal fatty acid-binding protein gene is expressed in these cells. The absence of this latter 15 kDa protein indicates that it is not required by Caco-2 cells for the synthesis of triglycerides or for the polarized export of triglyceride. These studies indicate that the Caco-2 cell line will be a useful model system for studying the polarization of FA trafficking/metabolism in enterocytes and defining the role of intracellular fatty acid binding proteins in these processes.

Adenocarcinoma↗
PubMed1991

Aggregated bovine IgG inhibits mannose receptor expression of murine bone marrow-derived macrophages via activation.

We previously described the presence of an inhibitory protein contained in the 20 to 40% (NH4)2SO4 precipitable fraction of FCS that down-regulates expression of mannose receptors on bone marrow-derived macrophages. We now identify aggregated bovine IgG as the main inhibitory component. Heat-aggregated bovine IgG was capable of down-regulating expression of the macrophage mannose receptor in a dose-dependent manner without inducing changes in ligand affinity whereas neither F(ab')2 fragments nor nonaggregated IgG displayed any inhibitory effect. Depleting of IgG from heat inactivated FCS by protein G affinity chromatography completely removes the inhibitory activity. Moreover, readdition of the Ig eluate from the protein G chromatography column restored inhibition in a dose-dependent manner. Macrophages were able to clear exogenously added aggregated bovine IgG, thus leading to loss of inhibitory activity in macrophage-conditioned media as compared to sham-conditioned media containing aggregated IgG. These results indicate that aggregated IgG down-regulates mannose receptor expression by macrophage activation via interaction with Fc-gamma R.

Animals↗