Restituting intestinal epithelial cells exhibit increased transducibility by adenoviral vectors

Background and aims While mature enterocytes are resistant to transduction by adenovirus type 5 (Ad5) vectors, undifferentiated cells are transduced much more efficiently. Our purpose was to study enterocyte transduction in models of intestinal wound healing. Methods Transduction was studied ex vivo using cultures of endoscopic biopsies and in vitro utilizing Caco-2 cells in models of mucosal wound healing. Vectors carried either the LacZ or the luciferase gene. CAR (coxsackievirus and adenovirus receptor) and integrins were studied with transduction inhibition and immunofluorescent staining. Results Increased transduction efficiency was observed for a subset of enterocytes with a flattened de-differentiated phenotype present at the edge of cultured biopsies. In the in vitro systems, expanding Caco-2 cell monolayers exhibited increased transducibility that was time- and dose-dependent, reaching virtually 100% in cells along the leading edge at high viral load. Bioluminescence activity of transduced expanding monolayers was up to 3-fold greater than that of non-expanding monolayers (‘fence’ system, 48 h, MOI 1000, p < 0.05). Mitomycin C pre-treatment did not affect levels of transduction in expanding monolayers. At the highest viral load tested, CAR or integrin blocking prior to virus application resulted in 39.4% and 45.4% reduction in transduction levels ( p < 0.05). Immunofluorescence revealed altered expression of CAR on the migrating edge of the Caco-2 cultures and the expression of CAR on the apical membrane of biopsy enterocytes. Conclusions Increased CAR and integrin accessibility in migrating enterocytes mediates increased transduction by Ad5 vectors. This subset of enterocytes provides a target for the delivery of genes of interest for both research and gene therapy applications. Copyright © 2006 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55907/1/981_ftp.pd

Resveratrol has antiinflammatory and antifibrotic effects in the peptidoglycan‐polysaccharide rat model of Crohn's disease

Background: Resveratrol has antiinflammatory and antifibrotic effects. Resveratrol decreases proliferation and collagen synthesis by intestinal smooth muscle cells. We hypothesized that resveratrol would decrease inflammation and fibrosis in an animal model of Crohn's disease. Methods: Peptidoglycan‐polysaccharide (PG‐PS) or human serum albumin (HSA) was injected into the bowel wall of Lewis rats at laparotomy. Resveratrol or vehicle was administered daily by gavage 1–27 days postinjection. On day 28, gross abdominal and histologic findings were scored. Cecal collagen content was measured by colorimetric analysis of digital images of trichrome‐stained sections. Cecal levels of procollagen, cytokine, and growth factor mRNAs were determined. Results: PG‐PS‐injected rats (vehicle‐treated) developed more fibrosis than HSA‐injected rats by all measurements: gross abdominal score ( P < 0.001), cecal collagen content ( P = 0.04), and procollagen I and III mRNAs ( P ≤ 0.0007). PG‐PS‐injected rats treated with 40 mg/kg resveratrol showed a trend toward decreased gross abdominal score, inflammatory cytokine mRNAs, and procollagen mRNAs. PG‐PS‐injected rats treated with 100 mg/kg resveratrol had lower inflammatory cytokine mRNAs (IL‐1β [3.50 ± 1.08 vs. 10.79 ± 1.88, P = 0.005], IL‐6 [17.11 ± 9.22 vs. 45.64 ± 8.83, P = 0.03], tumor necrosis factor alpha (TNF‐α) [0.80 ± 0.14 vs. 1.89 ± 0.22, P = 0.002]), transforming growth factor beta 1 (TGF‐β1) mRNA (2.24 ± 0.37 vs. 4.06 ± 0.58, P = 0.01), and histologic fibrosis score (6.4 ± 1.1 vs. 9.8 ± 1.0; P = 0.035) than those treated with vehicle. There were trends toward decreased gross abdominal score and decreased cecal collagen content. Procollagen I, procollagen III, and IGF‐I mRNAs also trended downward. Conclusions: Resveratrol decreases inflammatory cytokines and TGF‐β1 in the PG‐PS model of Crohn's disease and demonstrates a promising trend in decreasing tissue fibrosis. These findings may have therapeutic applications in inflammatory bowel disease. (Inflamm Bowel Dis 2011;)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90530/1/21843_ftp.pd

Role of intestinal P‐glycoprotein ( mdr1 ) in interpatient variation in the oral bioavailability of cyclosporine

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110064/1/cptclpt1997116.pd

Cyp3A gene expression in human gut epithelium

CYP3A4, a major Phase I xenobiotic metabolizing enzyme present in liver, is also present in human small bowel epithelium where it appears to catalyse significant 'first pass' metabolism of some drugs. To determine whether CYP3A4 or the related enzymes CYP3A3, CYP3A5, and CYP3A7 are present in other regions of the digestive tract, we used CYP3A-specific antibodies to examine histological sections and epithelial microsomes obtained from a human organ donor. CYP3A-related proteins were detected in epithelia throughout the digestive tract and in gastric parietal cells, in pericentral hepatocytes, and in ductular cells of the pancreas. Immunoblot analysis suggested that the major CYP3A protein present in liver, jejunum, colon, and pancreas was CYP3A4 or CYP3A3, whereas CYP3A5 was the major protein present in stomach. Both CYP3A4 and CYP3A5 mRNA were detectable in all regions of the digestive tract using the polymerase chain reaction (PCR); however, only CYP3A4 could be detected by Northern blot analysis. CYP3A7 mRNA was consistently detected only in the liver by PCR and CYP3A3 mRNA was not detected in any of the tissues. We conclude that CYP3A4 and CYP3A5 are present throughout the human digestive tract and that differences in the expression of these enzymes may account for inter-organ differences in the metabolism of CYP3 A substrates

Mechanisms of enhanced oral availability of CYP3A4 substrates by grapefruit constituents: Decreased enterocyte CYP3A4 concentration and mechanism-based inactivation by furanocoumarins

Grapefruit juice increases the oral availability of a variety of CYP3A4 substrates. It has been shown that recurrent grapefruit juice ingestion results in a loss of CYP3A4 from the small bowel epithelium. We now show that the reduction in intestinal CYP3A4 concentration is rapid; a 47% decrease occurred in a healthy volunteer within 4 hr after consuming grapefruit juice. To identify the specific components of the juice responsible for this effect, we used a recently developed Caco-2 cell culture model of human intestinal epithelium that expresses catalytically active CYP3A4. We found that grapefruit oil and two furanocoumarin constituents (6*,7*-dihydroxybergamottin and a closely related dimer) caused a dose-dependent fall in CYP3A4 catalytic activity and immunoreactive CYP3A4 concentration. The effect was selective in that concentrations of CYP1A1 and CYP2D6 did not fall, consistent with previous results obtained in vivo. Assays of various juices confirmed that 6*,7*-dihydroxybergamottin is the major furanocoumarin present and, although its concentration varies significantly among types and brands of grapefruit juice, it is consistently present in concentrations exceeding the IC50 (1 mM) for loss of midazolam 1*-hydroxylase activity determined in the Caco-2 cells. Studies with recombinant CYP3A4 revealed that 6*,7*-dihydroxybergamottin is a mechanism-based inactivator, which supports the idea that loss of CYP3A4 results from accelerated degradation of the enzyme. We conclude that the effect of grapefruit juice on oral availability of CYP3A4 substrates can be largely accounted for by the presence of 6*,7*-dihydroxybergamottin although other furanocoumarins probably also contribute

Effective in vivo and ex vivo gene transfer to intestinal mucosa by VSV-G-pseudotyped lentiviral vectors

<p>Abstract</p> <p>Background</p> <p>Gene transfer to the gastrointestinal (GI) mucosa is a therapeutic strategy which could prove particularly advantageous for treatment of various hereditary and acquired intestinal disorders, including inflammatory bowel disease (IBD), GI infections, and cancer.</p> <p>Methods</p> <p>We evaluated vesicular stomatitis virus glycoprotein envelope (VSV-G)-pseudotyped lentiviral vectors (LV) for efficacy of gene transfer to both murine rectosigmoid colon <it>in vivo </it>and human colon explants <it>ex vivo</it>. LV encoding beta-galactosidase (LV-β-Gal) or firefly-luciferase (LV-fLuc) reporter genes were administered by intrarectal instillation in mice, or applied topically for <it>ex vivo </it>transduction of human colorectal explant tissues from normal individuals. Macroscopic and histological evaluations were performed to assess any tissue damage or inflammation. Transduction efficiency and systemic biodistribution were evaluated by real-time quantitative PCR. LV-fLuc expression was evaluated by <it>ex vivo </it>bioluminescence imaging. LV-β-Gal expression and identity of transduced cell types were examined by histochemical and immunofluorescence staining.</p> <p>Results</p> <p>Imaging studies showed positive fLuc signals in murine distal colon; β-Gal-positive cells were found in both murine and human intestinal tissue. In the murine model, β-Gal-positive epithelial and lamina propria cells were found to express cytokeratin, CD45, and CD4. LV-transduced β-Gal-positive cells were also seen in human colorectal explants, consisting mainly of CD45, CD4, and CD11c-positive cells confined to the LP.</p> <p>Conclusions</p> <p>We have demonstrated the feasibility of LV-mediated gene transfer into colonic mucosa. We also identified differential patterns of mucosal gene transfer dependent on whether murine or human tissue was used. Within the limitations of the study, the LV did not appear to induce mucosal damage and were not distributed beyond the distal colon.</p

Marginal increase of sunitinib exposure by grapefruit juice

Clinical Oncolog

Modulation of Cytochrome P450 Metabolism and Transport across Intestinal Epithelial Barrier by Ginger Biophenolics

Natural and complementary therapies in conjunction with mainstream cancer care are steadily gaining popularity. Ginger extract (GE) confers significant health-promoting benefits owing to complex additive and/or synergistic interactions between its bioactive constituents. Recently, we showed that preservation of natural ‘‘milieu’’ confers superior anticancer activity on GE over its constituent phytochemicals, 6-gingerol (6G), 8-gingerol (8G), 10-gingerol (10G) and 6-shogaol (6S), through enterohepatic recirculation. Here we further evaluate and compare the effects of GE and its major bioactive constituents on cytochrome P450 (CYP) enzyme activity in human liver microsomes by monitoring metabolites of CYPspecific substrates using LC/MS/MS detection methods. Our data demonstrate that individual gingerols are potent inhibitors of CYP isozymes, whereas GE exhibits a much higher half-maximal inhibition value, indicating no possible herb-drug interactions. However, GE’s inhibition of CYP1A2 and CYP2C8 reflects additive interactions among the constituents. In addition, studies performed to evaluate transporter-mediated intestinal efflux using Caco-2 cells revealed that GE and its phenolics are not substrates of P-glycoprotein (Pgp). Intriguingly, however, 10G and 6S were not detected in the receiver compartment, indicating possible biotransformation across the Caco-2 monolayer. These data strengthen the notion that an interplay of complex interactions among ginger phytochemicals when fed as whole extract dictates its bioactivity highlighting the importance of consuming whole foods over single agents. Our study substantiates the need for an indepth analysis of hepatic biotransformation events and distribution profiles of GE and its active phenolics for the design of safe regimens