Native Intestine Research Articles

Combinational Effect of Intestinal and Hepatic CYP3A5 Genotypes on Tacrolimus Pharmacokinetics in Recipients of Living Donor Liver Transplantation

For living donor liver transplantation, the genetic association of CYP3A5 genotype of recipient's native intestine and donor's liver allograft with tacrolimus pharmacokinetics has not been explained completely considering liver regeneration time. The goal of the study was to investigate the longitudinal effects of recipient-donor combinational CYP3A5 genotypes on tacrolimus dose-normalized concentration (C/D ratio) in blood. Tacrolimus blood concentrations were measured for 58 Korean adult living donor liver transplant recipients on tacrolimus-based immunosuppressants during 4 years of follow-up. CYP3A5 was genotyped for both recipient and donor, and the recipient-donor combinational genetic effect on tacrolimus C/D ratios were evaluated as a function of time after adjusting for covariates including demographics and clinical variables. CYP3A5 expresser recipients grafted from CYP3A5 expresser donors consistently had the least C/D ratio throughout the entire study period, whereas CYP3A5 expresser recipients grafted from CYP3A5 nonexpresser donors had an intermediate, and CYP3A5 nonexpresser recipients grafted from CYP3A5 nonexpresser donors had the largest C/D ratio (all P < 0.01). The CYP3A5 nonexpresser recipients grafted from CYP3A5 expresser donors showed a significant decrease from the largest to the intermediate in C/D ratio for the first month. CYP3A5 genotypes of both recipient and donor were important factors influencing pharmacokinetic variability of tacrolimus. The recipient-donor combinational genetic effect on C/D ratio changed over time after transplantation.

Conditional knockout of the Slc5a6 gene in mouse intestine impairs biotin absorption.

The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.

Tissue-Engineered Small Intestine Demonstrates Digestive and Absorptive Capability

Introduction: Short bowel syndrome (SBS) leads to severe morbidity and mortality. Current surgical treatments, including intestinal transplantation, are expensive and require intensive medical therapy. Previously, we demonstrated that tissue-engineered small intestine (TESI) rescues Lewis rats in a model of SBS. We recently transitioned our technique to a mouse model in order to study the cellular and molecular mechanisms of TESI formation. We have shown that TESI is morphologically similar to native small intestine with a fully differentiated epithelium and an innervated muscularis.

Differential expression of human riboflavin transporters -1, -2, and -3 in polarized epithelia: A key role for hRFT-2 in intestinal riboflavin uptake

Transport of riboflavin (RF) across both the brush border membrane (BBM) and basolateral membrane (BLM) of the polarized enterocyte occurs via specific carrier-mediated mechanisms. Although, three human riboflavin transporters (hRFTs), i.e., hRFT-1, hRFT-2 and hRFT-3 are expressed in the intestine, little is known about the cell surface domain(s) at which these specific hRFTs are expressed. Here, we used live cell confocal imaging of intestinal epithelial Caco-2 and renal MDCK cells to show that the hRFT-1 is mainly expressed at the BLM, hRFT-2 is exclusively expressed at the apical membrane, while hRFT-3 is mostly localized inside intracellular vesicular structures (with some expression at the BLM). Further the level of hRFT-2 mRNA expression in Caco-2 cells and in native human intestine is significantly higher than that of hRFT-1 and -3; hRFT-2 was also more efficient in transporting 3H-RF than hRFT-1 and -3. These findings implied an important role for hRFT-2 in intestinal RF uptake, a conclusion that was further supported by findings of hRFT-2 gene-specific siRNA knockdown investigation. These results show that members of the hRFT family are differentially expressed in polarized epithelia, and that the apically expressed hRFT-2 plays a key role in intestinal RF accumulation.

Loss of PDZ-adaptor protein NHERF2 affects membrane localization and cGMP- and [Ca2+]- but not cAMP-dependent regulation of Na+/H+exchanger 3 in murine intestine

Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore- or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal- and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport.

Natural Killer Group 2 Member D Cell Recruitment Driven by Major Histocompatibility Complex Class I Chain–Related Antigens A and B: A Possible Mechanism During Acute Intestinal Allograft Rejection in the Mouse

Intestinal allograft rejection occurs frequently despite potent T-cell depletion protocols. We investigated the interaction of major histocompatibility complex class I chain–related antigens A and B (MICA/B; a ligand for natural killer [NK] cells) and NK group 2 member D (NKG2D) cells as an alternative mechanism for acute rejection (AR) of the intestinal graft. Heterotopic intestinal allotransplantation was performed from BalbC to C57Bl mice. Samples of grafted and native intestine were obtained at days 1, 3, 6, and 8 after transplantation (n = 4–6). We performed immunostaining for MICA/B and NKG2D. Moderate AR with increased crypt apoptosis was observed at day 6 and advanced AR with crypt destruction and mucosal sloughing was present by day 8. Low MICA/B levels were observed in grafted and native intestines on day 1. MICA/B expression gradually increased in the grafts during AR but not in the native intestines. The up-regulation was found mostly in the crypts. NKG2D+ cell counts that increased in the graft colocalized with MICA/B. The increase was most prominent in the crypt and villus. Together, these results suggest that MICA/B up-regulation and its subsequent interaction with the NK cells may represent an important link between innate and adaptive immune responses early after intestinal transplantation.

Characterization of Expression in Mice of a Transgene Containing 3.3 kb of the Human Lactase-Phlorizin Hydrolase (LPH) 5′ Flanking Sequence

The regulation of human intestinal lactase-phlorizin hydrolase remains incompletely understood. One kb of pig and 2kb of rat 5'-flanking sequence controls correct tissue, cell, topographic, and villus LCT expression. To gain insight into human LCT expression, transgenic mouse lines were generated from 3.3kb of human LPH 5' flanking sequence from a lactase persistent individual fused to a human growth hormone (hGH) reporter bounded by an insulator. Four lines were identified in which reporter expression was specifically detectable in the intestine and no other organ, two of which demonstrated hGH expression specific to small and large intestine. Quantitative RT-PCR was carried out on proximal to distal segments of small intestine at fetal days 16.5 and 18.5 and at birth, postnatal days 7 and 28 in line 22. In fetal intestine, hGH expression demonstrated a proximal to distal gradient similar to that in native intestine. There was no significant difference between hGH expression levels at 7 and 28days in segment 3, the midpoint of the small intestine, where expression of endogenous lactase is maximal at 7days and declines significantly by 28days. Distal small intestine displayed high levels of hGH expression in enteroendocrine cells, which were shown to be a subset of the PYY cells. Thus, a 3.3-kb LPH 5' flanking sequence construct from a lactase persistent individual is able to maintain postnatal expression in transgenic mice post weaning.

Gene ablation for PEPT1 in mice abolishes the effects of dipeptides on small intestinal fluid absorption, short-circuit current, and intracellular pH

PEPT1 function in mouse intestine has not been assessed by means of electrophysiology and methods to assess its role in intracellular pH and fluid homeostasis. Therefore, the effects of the dipeptide glycilsarcosin (Gly-Sar) on jejunal fluid absorption and villous enterocyte intracellular pH (pH(i)) in vivo, as well as on enterocyte[(14)C]Gly-Sar uptake, short-circuit current (I(sc)) response, and enterocyte pH(i) in vitro were determined in wild-type and PEPT1-deficient mice and in mice lacking PEPT1. Immunohistochemistry for PEPT1 failed to detect any protein in enterocyte apical membranes in Slc15a1(-/-) animals. Saturable Gly-Sar uptake in Slc15a1(-/-) everted sac preparations was no longer detectable. Similarly, Gly-Sar-induced jejunal I(sc) response in vitro was abolished. The dipeptide-induced increase in fluid absorption in vivo was also abolished in animals lacking PEPT1. Since PEPT1 acts as an acid loader in enterocytes, enterocyte pH(i) was measured in vivo by two-photon microscopy in SNARF-4-loaded villous enterocytes of exteriorized jejuni in anesthetized mice, as well as in BCECF-loaded enterocytes of microdissected jejunal villi. Gly-Sar-induced pH(i) decrease was no longer observed in the absence of PEPT1. A reversal of the proton gradient across the luminal membrane did not significantly diminish Gly-Sar-induced I(sc) response, whereas a depolarization of the apical membrane potential by high K(+) or via Na(+)-K(+)-ATPase inhibition strongly diminished Gly-Sar-induced I(sc) responses. This study demonstrates for the first time that proton-coupled electrogenic dipeptide uptake in the native small intestine, mediated by PEPT1, relies on the negative apical membrane potential as the major driving force and contributes significantly to intestinal fluid transport.

The switch of intestinal Slc26 exchangers from anion absorptive to HCO3− secretory mode is dependent on CFTR anion channel function

CFTR has been recognized to function as both an anion channel and a key regulator of Slc26 anion transporters in heterologous expression systems. Whether this regulatory relationship between CFTR and Slc26 transporters is seen in native intestine, and whether this effect is coupled to CFTR transport function or other features of this protein, has not been studied. The duodena of anesthetized CFTR-, NHE3-, Slc26a6-, and Scl26a3-deficient mice and wild-type (WT) littermates were perfused, and duodenal bicarbonate (HCO(3)(-)) secretion (DBS) and fluid absorptive or secretory rates were measured. The selective NHE3 inhibitor S1611 or genetic ablation of NHE3 significantly reduced fluid absorptive rates and increased DBS. Slc26a6 (PAT1) or Slc26a3 (DRA) ablation reduced the S1611-induced DBS increase and reduced fluid absorptive rates, suggesting that the effect of S1611 or NHE3 ablation on HCO(3)(-) secretion may be an unmasking of Slc26a6- and Slc26a3-mediated Cl(-)/HCO(3)(-) exchange activity. In the absence of CFTR expression or after application of the CFTR(inh)-172, fluid absorptive rates were similar to those of WT, but S1611 induced virtually no increase in DBS, demonstrating that CFTR transport activity, and not just its presence, is required for Slc26-mediated duodenal HCO(3)(-) secretion. A functionally active CFTR is an absolute requirement for Slc26-mediated duodenal HCO(3)(-) secretion, but not for Slc26-mediated fluid absorption, in which these transporters operate in conjunction with the Na(+)/H(+) exchanger NHE3. This suggests that Slc26a6 and Slc26a3 need proton recycling via NHE3 to operate in the Cl(-) absorptive mode and Cl(-) exit via CFTR to operate in the HCO(3)(-) secretory mode.

TEXTILE COMPOSITE MATERIALS FOR SMALL INTESTINE REPLACEMENT

Abstract Down to the present day there are no sufficient techniques for a small intestine replacement, mostly because of the high standards for such implants. An indication for the need of novel operation techniques is the small patient survival rate of just 80 % for isolated small intestine transplantation and 62 % for combined liver-small intestine transplantation. The five year survival rates of the patients are merely 42 %. In order to overcome these limitations the authors are developing a partly resorbable textile-foam-composite for small intestine replacement The novel implant consists of a non resorbable textile PVDF mesh which is foamed with a micro porous, resorbable, and drug loaded polymer. The resorbable polymer serves on the one hand as initial sealing, therefore no intestine substance and bacteria can leak out into surrounding tissue, on the other hand it needs to be micro porous in order to ensure cell ingrowth. For the macro porous textile mesh warp knitting technology is used. The warp knitted tubular structure remains inside the body as a long term implant and provides mechanical support to ingrowing cells. In order to evaluate biomechanical properties of the warp knitted tubular PVDF meshes to compare them to the mechanical characteristics of small intestine tissue, tensile tests were conducted. Results of tensile tests on warp knitted structures with three different loop densities of 8, 12, and 16 loops per cm were compared to tensile tests on native small intestine tissue probes. The recorded curves of small intestine and warp knitted structures showed similar characteristics. The two characteristic Young Modules as well as the curve progression of the warp knitted structure with 12 loops per cm showed good accordance to the values of the native small intestine. Morphological analysis of the textile structures by digital image processing showed adequate pore size and porosity of the textile mesh.

Influence of ABCB1 polymorphisms and haplotypes on tacrolimus nephrotoxicity and dosage requirements in children with liver transplant

The aim of this study was to investigate the influence of genetic polymorphisms in ABCB1 on the incidence of nephrotoxicity and tacrolimus dosage-requirements in paediatric patients following liver transplantation. Fifty-one paediatric liver transplant recipients receiving tacrolimus were genotyped for ABCB1 C1236>T, G2677>T and C3435>T polymorphisms. Dose-adjusted tacrolimus trough concentrations and estimated glomerular filtration rates (EGFR) indicative of renal toxicity were determined and correlated with the corresponding genotypes. The present study revealed a higher incidence of the ABCB1 variant-alleles examined among patients with renal dysfunction (> or =30% reduction in EGFR) at 6 months post-transplantation (1236T allele: 63.3% vs 37.5% in controls, P= 0.019; 2677T allele: 63.3% vs. 35.9%, p = 0.012; 3435T allele: 60% vs. 39.1%, P= 0.057). Carriers of the G2677->T variant allele also had a significant reduction (%) in EGFR at 12 months post-transplant (mean difference = 22.6%; P= 0.031). Haplotype analysis showed a significant association between T-T-T haplotypes and an increased incidence of nephrotoxicity at 6 months post-transplantation (haplotype-frequency = 52.9% in nephrotoxic patients vs 29.4% in controls; P= 0.029). Furthermore, G2677->T and C3435->T polymorphisms and T-T-T haplotypes were significantly correlated with higher tacrolimus dose-adjusted pre-dose concentrations at various time points examined long after drug initiation. These findings suggest that ABCB1 polymorphisms in the native intestine significantly influence tacrolimus dosage-requirement in the stable phase after transplantation. In addition, ABCB1 polymorphisms in paediatric liver transplant recipients may predispose them to nephrotoxicity over the first year post-transplantation. Genotyping future transplant recipients for ABCB1 polymorphisms, therefore, could have the potential to individualize better tacrolimus immunosuppressive therapy and enhance drug safety.

Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium

The intestinal Menkes copper Atpase (Atp7a) gene is strongly induced by iron deficiency in the rat intestine. We sought to develop an in vitro model to understand the mechanism of this induction by performing molecular studies in native rat intestine and in intestinal epithelial (IEC-6) cells. IEC-6 cells express Atp7a, and induction was noted with iron deprivation. 5' Rapid amplification of cDNA ends and PCR experiments revealed three splice variants in rat intestine and IEC-6 cells; all variants were strongly induced during iron deprivation (five- to sevenfold). The splice variants presumably encode proteins that would either contain the extreme NH(2) terminus of the protein (containing copper binding domain 1) or not. We thus hypothesized that more than one version of Atp7a protein exists. Antibodies against this NH(2)-terminal region of the protein were developed (named N-term) and used along with previously reported antibodies (against more COOH-terminal regions, termed 54-10) to perform immunoblotting and immunolocalization studies. Results with the 54-10 antiserum revealed an Atp7a protein variant of approximately 190 kDa that localized to the trans-Golgi network of IEC-6 cells and trafficked to the plasma membrane with copper loading. Using the N-term antiserum, however, we noted protein of approximately 97 and 64 kDa. The 97-kDa protein was cytosolic and nuclear, whereas the 64-kDa protein was nuclear specific. Immunolocalization analyses with the N-term antiserum showed strong staining of nuclei in IEC-6 and Caco-2 cells and in rat intestine. We conclude that novel Atp7a protein variants may exist in rat and human intestinal epithelial cells, with different intracellular locations and potentially distinct physiological functions.

Tissue-Engineered Small Intestine and Stomach Form from Autologous Tissue in a Preclinical Large Animal Model

Tissue-engineered small intestine, stomach, large intestine, esophagus, and gastroesophageal (GE) junction have been successfully formed from syngeneic cells, and employed as a rescue therapy in a small animal model. The purpose of this study is to determine if engineered intestine and stomach could be generated in an autologous, preclinical large animal model, and to identify if the tissue-engineered intestine retained features of an intact stem cell niche. A short segment of jejunum or stomach was resected from 6-wk-old Yorkshire swine. Organoid units, multicellular clusters with predominantly epithelial content, were generated and loaded onto biodegradable scaffold tubes. The constructs were then implanted intraperitoneally in the autologous host. Seven wk later, all implants were harvested and analyzed using histology and immunohistochemistry techniques. Autologous engineered small intestine and stomach formed. Tissue-engineered intestinal architecture replicated that of native intestine. Histology revealed tissue-engineered small intestinal mucosa composed of a columnar epithelium with all differentiated intestinal cell types adjacent to an innervated muscularis mucosae. Intestinal subepithelial myofibroblasts, specialized cells that participate in the stem cell niche formation, were identified. Moreover, cells positive for the putative intestinal stem cell marker, doublecortin and CaM kinase-like-1 (DCAMKL-1) expression were identified at the base of the crypts. Finally, tissue-engineered stomach also formed with antral-type mucosa (mucus cells and surface foveolar cells) and a muscularis. We successfully generated tissue-engineered intestine with correct architecture, including features of an intact stem cell niche, in the pig model. To our knowledge, this is the first demonstration in which tissue-engineered intestine was successfully generated in an autologous manner in an animal model, which may better emulate a human host and the intended therapeutic pathway for humans.

The Role of the NHERF Family of PDZ Scaffolding Proteins in the Regulation of Salt and Water Transport

The four members of the NHERF (Na(+)/H(+) exchanger regulatory factor) family of PDZ adapter proteins bind to a variety of membrane transporters and receptors and modulate membrane expression, mobility, interaction with other proteins, and the formation of signaling complexes. All four family members are expressed in the intestine. The CFTR (cystic fibrosis transmembrane regulator) anion channel and the Na(+)/H(+) exchanger NHE3 (Na/H exchanger- isoform 3) are two prominent binding partners to this PDZ-adapter family, which are also known key players in the regulation of intestinal electrolyte and fluid transport. Experiments in heterologous expression systems have provided a number of mechanistic models how NHERF protein interactions can affect the function of their targets at the molecular level. Recently, NHERF1, 2, and 3 knockout mice have become available, and this review summarizes the reports on electrolyte and fluid transport regulation in the native intestine of these mice.

Dual role of the Na+/H+ exchanger isoform 3 for PEPT1‐mediated H+/dipeptide cotransport in native murine intestine

This study investigates the molecular function of NHE3 in H+‐dipeptide transport regulation in the native intestine. Luminal application of the dipeptide Gly‐Sar resulted in a strong increase in murine small intestinal fluid absorption in vivo, Isc response in chambered jejunal mucosa, and a decrease in pHi in BCECF‐loaded enterocytes in isolated jejunal villi. Genetic ablation of PEPT1 abolished Gly‐Sar‐induced fluid absorption, Isc response, and enterocyte acidification. Genetic ablation, pharmacological or second‐messenger‐mediated inhibition of NHE3 also reduced Gly‐Sar‐induced fluid absorption in vivo and Isc response in vitro, but enhanced enterocyte acidification. Genetic ablation of Slc26a6 also enhanced enterocyte acidification, but did not alter either Gly‐Sar induced fluid absorption or Isc response. The transmembrane proton gradient was irrelevant for sustained H+/dipeptide transport rates, but the luminal Na+ concentration, enterocyte Na+/K+ ATPase activity, and luminal membrane potential were essential determinants. PEPT1 is the only H+/dipeptide uptake pathway, and NHE3 function is essential for sustained H+/dipeptide uptake rates in the native intestine. We speculate that NHE3 is the major Na+‐uptake pathway into enterocytes during dipeptide‐induced volume increase, and therefore essential to the maintenance of membrane potential negativity.

Corrigendum

CORRIGENDUMCorrigendumPublished Online:01 Feb 2009https://doi.org/10.1152/ajpcell.zh0-5839-corr.2009MoreSectionsPDF (178 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 295, September 2008 Volume 64, September 2008 Subramanian VS, Reidling JC, Said HM. Differentiation-dependent regulation of the intestinal folate uptake process: studies with Caco-2 cells and native mouse intestine. Am J Physiol Cell Physiol 295: C828–C835, 2008. First published July 23, 2008; doi:10.1152/ajpcell.00249.2008 ( http://ajpcell.physiology.org/cgi/content/full/295/3/C828).—In the version of this article initially published, in Fig. 2, it was not indicated that Western blot lane images in A and D, although run simultaneously on the same gels, were rearranged for clarity of presentation. As per recent American Physiological Society requirements on figure presentation, dividing lines have been added to indicate these changes, and disclosure of the arrangement has been added to the figure legend. The modified image and legend are presented here. Fig. 2.Level of expression of human reduced folate carrier (hRFC) and human proton-coupled folate transporter (hPCFT) protein and mRNA as well as activity of the SLC19A1 and SLC46A1 promoters in preconfluent, confluent, and postconfluent Caco-2 cells. A and D: Western blot analysis was performed using membranous fraction (150 μg) isolated from preconfluent, confluent, and postconfluent Caco-2 cells and specific anti-hRFC and anti-hPCFT polyclonal antibodies. Using an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL), immunoreactive bands were detected as described in materials and methods. Note that the lane images show proteins detected on a representative single blot; however, they were rearranged for clarity of presentation. B and E: quantitative real-time PCR was performed using hRFC (B) and hPCFT (E) gene-specific primers and total RNA (5 μg) from preconfluent, confluent, and postconfluent Caco-2 cells. Real-time PCR was performed as described in materials and methods. Data represent means ± SE of at least 3 independent sets of experiments and were normalized relative to β-actin and calculated using a relative relationship method supplied by the iCycler manufacturer (Bio-Rad, Hercules, CA). Note that the level of expression of hRFC and hPCFT in preconfluent cells was set at 1 for each figure and the expression during differentiation is in relation to that level; therefore the results are not representative of the levels of hRFC compared with hPCFT (hPCFT mRNA expression is ∼3-fold higher than hRFC at all 3 stages of differentiation). C and F: activity of full-length hRFC promoter B (C) and hPCFT promoters (F) in pGL3-Basic was determined following transient expression in preconfluent, confluent, and postconfluent Caco-2 cells. Luciferase activity was determined and normalized relative to the activity of simultaneously expressed Renilla luciferase as described in materials and methods. The results are expressed relative to the PGL3-Basic vector set at 1. Data represent means ± SE of at least 3 independent sets of determinations.Download figureDownload PowerPointThis article has no references to display. Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 296Issue 2February 2009Pages C385-C386 Copyright & PermissionsCopyright © 2009 the American Physiological Societyhttps://doi.org/10.1152/ajpcell.zh0-5839-corr.2009History Published online 1 February 2009 Published in print 1 February 2009 Metrics

PL12. Tissue-Engineered Small Intestine and Stomach Demonstrate Features of an Intact Stem Cell Niche in a Large Animal Model

Tissue-engineered small intestine has been successfully employed as rescue therapy after massive small bowel resection in the Lewis Rat, with tissue architecture that successfully recapitulates native small intestine including epithelium, muscularis, ganglion cells, and S100 positive tissue in the location of Meissner and Auerbach's plexi. Tissue engineered stomach has also been created with gastric glands, parietal cells. The purpose of this study was to generate and characterize engineered intestine in a preclinical, large animal model.

Impact of IntestinalCYP2C19Genotypes on the Interaction between Tacrolimus and Omeprazole, but Not Lansoprazole, in Adult Living-Donor Liver Transplant Patients

To assess the effects of intestinal cytochrome P450 2C19 on the interaction between tacrolimus and proton pump inhibitors, we examined the concentration/dose ratio [(ng/ml)/(mg/day)] of tacrolimus coadministered with omeprazole (20 mg) or lansoprazole (30 mg) to 89 adult living-donor liver transplant patients on postoperative days 22 to 28, considering the CYP2C19 genotypes of the native intestine and the graft liver, separately. The concentration/dose ratio of tacrolimus coadministered with omeprazole was significantly higher in patients with two variants (*2 or *3) for intestinal CYP2C19 (median, 6.38; range, 1.55-22.9) than intestinal wild-type homozygotes (median, 2.11; range, 1.04-2.54) and heterozygotes (median, 2.11; range, 0.52-4.33) (P = 0.010), but the extent of the increase was attenuated by carrying the wild-type allele in the graft liver even when patients were CYP3A5*1 noncarriers. Conversely, the CYP2C19 polymorphisms both in the native intestine and in the graft liver little influenced the interaction between tacrolimus and lansoprazole, but CYP3A5*1 noncarriers showed higher tacrolimus concentration/dose ratio than CYP3A5*1 carriers. Furthermore, our experiments in vitro revealed that lansoprazole had a stronger inhibitory effect on the CYP3A5-mediated metabolism of tacrolimus than omeprazole, although not significantly (IC(50) = 19.9 +/- 13.8 microM for lansoprazole, 53.7 +/- 6.1 microM for omeprazole). Our findings suggest that intestinal and graft liver CYP2C19 plays a relatively greater role in the metabolism of omeprazole than it does for lansoprazole, so that the effects of CYP3A5 on the metabolism of tacrolimus might be masked by the interaction with omeprazole associated with the CYP2C19 genotype.

Absence of Influence of Concomitant Administration of Rabeprazole on the Pharmacokinetics of Tacrolimus in Adult Living-donor Liver Transplant Patients: A Case–control Study

This study assesses the effects of rabeprazole on the pharmacokinetics of tacrolimus, considering the cytochrome P450 (CYP) 2C19 and CYP3A5 genotypes of living-donor liver transplant patients (native intestine) and their corresponding donors (graft liver). We examined the concentration/dose ratio of tacrolimus in transplant patients treated with (n=17) or without (n=38) rabeprazole at 10 mg/day on postoperative days 22-28. A stratified analysis revealed no significant differences between the control and rabeprazole groups in the median (range) concentration/dose ratio of tacrolimus [(ng/mL)/(mg/day)] for CYP2C19 extensive/intermediate metabolizers [2.71 (1.00-6.15) versus 2.55 (0.96-9.25); P=0.85] and for poor metabolizers [4.92 (2.44-7.00) versus 3.82 (2.00-7.31); P=0.68], respectively. Even based on the classification of CYP2C19 genotypes of donors, no significant difference in the concentration/dose ratio of tacrolimus was found for the two groups (CYP2C19 extensive/intermediate metabolizers, P=0.52; poor metabolizers, P=0.51). The same was observed for CYP3A5(*)1 carriers (P=0.97 for native intestine; P=0.87 for graft liver) and CYP3A5(*)3/(*)3 carriers (P=0.89 for native intestine; P=0.56 for graft liver). These findings suggest a safer dosing and monitoring of tacrolimus coadministered with rabeprazole early on after liver transplantation regardless of the CYP2C19 and CYP3A5 genotypes of transplant patients and their donors.

Regulation of mucosal structure and barrier function in rat colon exposed to tumor necrosis factor alpha and interferon gamma in vitro: A novel model for studying the pathomechanisms of inflammatory bowel disease cytokines

Objective. In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal transport and barrier function. The mechanisms of cytokine action have usually been studied in cell culture models only; therefore the aim of this study was to establish an in vitro model based on native intestine to analyze distinct cytokine effects on barrier function, mucosal structure, and inherent regulatory mechanisms. Material and methods. Rat colon was exposed to tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) in Ussing chambers. Transepithelial resistance (Rt) and 3H-mannitol fluxes were measured for characterization of the paracellular pathway. Transcellular transport was analyzed by horseradish peroxidase (HRP) flux measurements. Expression and distribution of tight junction proteins were characterized in immunoblots and by means of confocal laser-scanning microscopy (LSM). Results. Colonic viability could be preserved for 20 h in a specialized in vitro set-up. This was sufficient to alter mucosal architecture with crypt surface reduction. Rt was decreased (101±10 versus 189±10 Ω·cm2) with a parallel increase in mannitol permeability after cytokine exposure. Tight junction proteins claudin-1, -5, -7, and occludin decreased (45±10%, 16±7%, 42±8%, and 42±13% of controls, respectively), while claudin-2 increased to 208±32%. Occludin and claudin-1 translocated from the plasma membrane to the cytoplasm. HRP flux increased from 0.73±0.09 to 8.55±2.92 pmol·h−1·cm−2. Conclusions. A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFα and IFNγ alters mucosal morphology and impairs epithelial barrier function by up-regulation of the paracellular pore-former claudin-2 and down-regulation of the barrier-builders claudin-1, -5, and -7. These alterations resemble changes seen in IBD and thus underline their prominent role in IBD pathogenicity.