Performance of two neutral oral contrast agents in CT enterography.

INTRODUCTION: This study compares the performance of two neutral oral contrast agents in CT enterography (CTE). Mannitol 2.5%, an oral osmotic agent, is compared with psyllium fibre (Metamucil). Both these agents are commonly used, but to our knowledge, they have not been compared in CTE. METHODS: CTE data were collected from 25 consecutive studies for both mannitol and psyllium fibre between 2011 and 2013. All images were reviewed by two radiologists and one registrar blinded to the oral contrast used. Each quadrant was assessed for maximum distension, proportion of bowel loops distended, presence of inhomogeneous content and bowel wall visibility. Overall subjective quality and whether the contrast agent reached the caecum were also assessed. Patients were invited to answer a questionnaire regarding tolerability of the preparations. RESULTS: Wall visibility was rated good in 100% of the mannitol studies, compared with 71% of the psyllium fibre studies, in the right lower quadrant (P = 0.01). No statistically significant difference between groups was observed in either maximal distension or proportion of loops distended in any quadrant. Inhomogeneous material was observed in 12% of the mannitol cases and 86% of the psyllium fibre cases (P < 0.0001). In all mannitol cases, the contrast reached the caecum, compared with 50% of psyllium fibre cases (P < 0.0001), and 36% of the mannitol studies were considered excellent, compared with 20% of the psyllium fibre studies (P = 0.03). CONCLUSION: Mannitol achieves studies of better quality and is now the preferred oral contrast for CTE studies at Auckland City Hospital.

Modeling of the in vivo kinetics of antioxidants delineates suitable parameters for selecting potential antioxidant adjuvants for cancer therapy.

To find in vivo behaviors of an antioxidant when used as an adjuvant cancer therapy, a more detailed integrated pharmacokinetic scheme is needed. Major reaction parameters associated with the sequential routes from ingestion to decay of an antioxidant were used in mathematical analysis, which included absorption rate coefficient k(a), quenching rate coefficient of the antioxidant k(q1) and tissue quenching rate coefficient k(r). The model was then treated with computer simulation using cited decay rate coefficients and some assumed parameters. When intestinal absorption rate coefficient k(a) becomes larger, retention time of antioxidant in plasma would be prolonged. moreover, k(a) had no effect on either quenching ability of antioxidants or tissue recovering capability. in quenching plasma ROS, the larger the quenching coefficient k(q1), the shorter peak- and the life-times would be for the secondary free radicals that are formed in primary quenching. Conclusively, it is suggestive to prescribe an antioxidant therapy with an appropriate values of k(a) and larger values of k(q1).

Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure.

We have analysed the incorporation of [(3)H]sucrose and [(3)H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [(3)H]sucrose or [(3)H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2-3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [(3)H]Sucrose uptake was twice that of [(3)H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [(3)H]sucrose and [(3)H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [(3)H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [(3)H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [(3)H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum.

Fluid absorption in endoscopic surgery.

Fluid absorption is an unpredictable complication of endoscopic surgery. Absorption of small amounts of fluid (1-2 litre) occurs in 5-10% of patients undergoing transurethral prostatic resection and results in an easily overlooked mild transurethral resection (TUR) syndrome. Large-scale fluid absorption is rare but leads to symptoms severe enough to require intensive care. Pathophysiological mechanisms consist of pharmacological effects of the irrigant solutes, the volume effect of the irrigant water, dilutional hyponatraemia and brain oedema. Other less widely known factors include absolute losses of sodium by urinary excretion and morphological changes in the heart muscle, both of which promote a hypokinetic circulation. Studies in animals, volunteers and patients show that irrigation with glycine solution should be avoided. Preventive measures, such as low-pressure irrigation, might reduce the extent of fluid absorption but does not eliminate this complication. Monitoring the extent of absorption during surgery allows control of the fluid balance in the individual patient, but such monitoring is not used widely. However, the anaesthetist must be aware of the symptoms and be able to diagnose this complication. Treatment should be based on administration of hypertonic saline rather than on diuretics. New techniques, such as bipolar resectoscopes and vaporizing instead of resecting tissue, result in a continuous change of the prerequisites for fluid absorption and its consequences.

Glutamine-enriched enteral nutrition in very low birth weight infants. Design of a double-blind randomised controlled trial [ISRCTN73254583].

BACKGROUND: Enteral feeding of very low birth weight (VLBW) infants is a challenge, since metabolic demands are high and administration of enteral nutrition is limited by immaturity of the gastrointestinal tract. The amino acid glutamine plays an important role in maintaining functional integrity of the gut. In addition, glutamine is utilised at a high rate by cells of the immune system. In critically ill patients, glutamine is considered a conditionally essential amino acid. VLBW infants may be especially susceptible to glutamine depletion as nutritional supply of glutamine is limited in the first weeks after birth. Glutamine depletion has negative effects on functional integrity of the gut and leads to immunosuppression. This double-blind randomised controlled trial is designed to investigate the effect of glutamine-enriched enteral nutrition on feeding tolerance, infectious morbidity and short-term outcome in VLBW infants. Furthermore, an attempt is made to elucidate the role of glutamine in postnatal adaptation of the gut and modulation of the immune response. METHODS: VLBW infants (gestational age <32 weeks and/or birth weight <1500 g) are randomly allocated to receive enteral glutamine supplementation (0.3 g/kg/day) or isonitrogenous placebo supplementation between day 3 and 30 of life. Primary outcome is time to full enteral feeding (defined as a feeding volume >/= 120 mL/kg/day). Furthermore, incidence of serious infections and short-term outcome are evaluated. The effect of glutamine on postnatal adaptation of the gut is investigated by measuring intestinal permeability and determining faecal microflora. The role of glutamine in modulation of the immune response is investigated by determining plasma Th1/Th2 cytokine concentrations following in vitro whole blood stimulation.

Experimental assessment of chemotherapy-induced early intestinal damage in colon cancer the lactulose-mannitol permeability test.

AIMS AND BACKGROUND: Although chemotherapy plays an important role in the management and cure of cancer, it has undesiderable side effects mostly affecting the bone marrow and gastrointestinal tract, which greatly limit patient compliance and treatment efficacy. METHODS: The lactulose-mannitol test was used to assess intestinal mucosa damage 48 hours after the end of the first adjuvant chemotherapy cycle with 5-fluorouracil (5-FU) and levamisole in 12 patients with colon cancer. Fifteen age- and sex-matched subjects were studied as controls. The excreted amount of lactulose and mannitol was expressed as the percentage of the administered doses recovered in the urine as well as their ratio. RESULTS: The percent urinary recovery of lactulose was significantly (P < 0.001) higher in colon cancer patients (1.1 +/- 0.5%) than in the control group (0.3 +/- 0.03%), whereas the mannitol recovery was only slightly reduced in the former. As a result, the lactulose/mannitol excretion ratio was significantly (P < 0.001) higher in colon cancer patients (0.07 +/- 0.03) than in the control group (0.01 +/- 0.01). CONCLUSIONS: As assessed by the lactulose-mannitol test, the combined chemotherapy regimen with 5-FU and levamisole affects mainly the barrier function of the intestinal mucosa rather than its absorption capacity. The toxic effect seems to be attributable to the 5-FU molecule rather than to levamisole. The lactulose-mannitol test is a simple, safe and reliable tool to evaluate chemotherapy-induced early damage to the intestinal epithelium, in particular when new kinds of substances are being administered. Its use in clinical practice seems appropriate to establish the correct timing of drug administration, thereby enhancing treatment efficacy and improving patient compliance.

Permeability characteristics of parental and clonal human intestinal Caco-2 cell lines differentiated in serum-supplemented and serum-free media.

The aim of this study was to define the permeability characteristics of the parental Caco-2/ATCC cell line and of three clonal lines (Caco-2/TC7, Caco-2/AQ, Caco-2/15) differentiated in serum-supplemented or in serum-free defined medium. The Caco-2 cells were grown in DMEM supplemented with either 10% foetal calf serum or insulin-transferrin-selenium and lipids (cholesterol, palmitic acid, oleic acid) for up to 24 days after seeding on polyethylene terephthalate filter inserts (1 microm pore diameter). The permeability of the cell monolayer was assessed by measuring trans-epithelial electrical resistance (TEER) and the apparent permeability (Papp) of the extracellular marker mannitol during differentiation from day 6 until day 24. In all lines TEER values increased during differentiation reaching a plateau value around day 15 after seeding, while the Papp for mannitol decreased sharply around day 8 and levelled off thereafter. Substantial differences were observed in the maximal TEER values achieved during differentiation in the four lines examined (Caco-2/TC7

Amelioration of dextran sulfate colitis by butyrate: role of heat shock protein 70 and NF-kappaB.

Butyrate enemas have been demonstrated to ameliorate inflammation in ulcerative colitis. The mechanism of this protective effect of butyrate is not known, and this study examines the effect of butyrate on epithelial function, inducible heat shock protein 70 (HSP70) expression, and NF-kappaB activation in experimental colitis. Colitis was induced in rats by oral dextran sulfate sodium (DSS) and by butyrate or saline enemas. Mucosal barrier function was assessed by electrical resistance and [14C]mannitol permeability. HSP70 production was determined by [35S]methionine labeling, Western blot analysis, and immunohistochemistry. Activation of heat shock factors (HSFs) and NF-kappaB was evaluated by EMSA. Butyrate showed a significant protection against the decrease in cell viability, increase in mucosal permeability, and polymorphonuclear neutrophil infiltration seen in DSS colitis. Butyrate inhibited HSP70 expression in DSS colitis and also inhibited the activation of HSF and NF-kappaB. Thus butyrate enema was found to be cytoprotective in DSS colitis, an effect partly mediated by suppressing activation of HSP70 and NF-kappaB.

Improvement of the experimental setup to assess cutaneous bioavailability on human skin models: dynamic protocol.

Human skin models, such as EpiDerm and Episkin, are not easily mounted into static or dynamic diffusion cells that are commonly used to perform bioavailability studies with human skin ex vivo. For various reasons, such as fragility, small sample size, and other morphological constraints, skin absorption studies with human skin models are often carried out on the delimited skin surface obtained by gluing a ring onto the reconstituted epidermis and manually exchanging the receptor solution. However, such an experimental setup is prone to artifacts. Discontinuous removal of the receptor fluid leads to alternating sink conditions, and an area of application smaller than the area in contact with the receptor fluid, as well as imperfect seal of the glued ring, may result in inaccurate penetration rates. Human skin models were shown to be relatively easily mounted into In-Line cells (PermeGear Inc.), vertical diffusion cells which appear to be appropriately designed for such a purpose. In-Line cells allowed accurate determination of solute penetration as well as automated sampling of receptor fluid. Excised human skin can be mounted into these cells as well, making it possible to compare penetration rates through different types of skin samples under identical conditions. Using mannitol as a reference compound, penetration profiles and epidermal distribution similar to those obtained with human skin ex vivo were obtained both with EpiDerm and Episkin. Under the present conditions, human skin models were more permeable to mannitol than excised human skin, which was only slightly permeable to mannitol. Due to these experimental innovations and to the good agreement with the absorption characteristics through human skin ex vivo, EpiDerm and Episkin seem to be promising human skin models for testing the cutaneous bioavailability of topical products in vitro.

Effect of dietary fatty acids on the intestinal permeability of marker drug compounds in excised rat jejunum.

The aim of this study was to explore the effects of diets containing saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and omega-3 and omega-6 polyunsaturated fatty acids (omega-3 and omega-6 PUFA, respectively) on the passive and active transport properties of rat jejunum using marker compounds. Rats were fed diets supplemented with 18.4% (w/w) lipid (4 groups) or standard rat chow (1 group) for a period of 30 days. At the end of the dietary period, mucosal scrapings were taken for the determination of membrane phospholipids, and the apparent jejunal permeability of radiolabelled marker compounds was determined using modified Ussing chambers. Changes in the phospholipid content of the brush border membrane reflected the different lipid content of the diets. The passive paracellular permeability of mannitol was not significantly affected by the fatty acid composition of the diet, although there was a trend toward decreased mannitol permeability in the rats fed both the omega-3 and omega-6 PUFA diets. In comparison, the transcellular diffusion of diazepam was reduced by 20% (P < 0.05) in rats fed diets supplemented with omega-3 and omega-6 PUFA. In the lipid-fed rats, the serosal to mucosal flux of digoxin, an intestinal P-glycoprotein substrate, was reduced by 20% (P < 0.05) relative to the chow-fed group, however there were no significant differences between the different lipid groups. The active absorption of D-glucose via the Na+-dependent transport pathway was highest in the SFA, MUFA and PUFA omega-3 dietary groups, intermediate in the low-fat chow group and lowest in the PUFA omega-6 group, and was positively correlated with short-circuit current. These studies indicate that dietary fatty acid changes can result in moderate changes to the active and passive transport properties of excised rat jejunum.

Evaluation of the BBMEC model for screening the CNS permeability of drugs.

Combinatorial synthesis and high-throughput pharmacology screening have greatly increased compound throughput in modern drug-discovery programs. For CNS drugs, it is also important to determine permeability to the blood--brain barrier. Yet, given the increased pace of discovery, it difficult to conduct this screen in a timely fashion. In this presentation, we describe several improvements to an existing CNS permeability screen, the bovine brain microvessel endothelial cell (BBMEC) model. By implementation of these incremental process improvements, we have achieved a robust, facile screen for determination of CNS permeability of multiple compounds.

Zinc supplementation tightens "leaky gut" in Crohn's disease.

OBJECTIVES: Small intestinal permeability is often increased in patients with Crohn's disease and may be pathogenic for clinical relapses. No effective prophylactic treatment is available for these patients. The aim of this study was to ascertain whether zinc supplementation may improve intestinal permeability. METHODS: We studied 12 patients with quiescent Crohn's disease who had been in remission for at least 3 months and had increased intestinal permeability on two separate occasions within the last 2 months. Patients received oral zinc sulfate supplements (110 mg three times a day) for 8 weeks and were followed-up for 12 months thereafter to monitor relapses. RESULTS: We found that the lactulose/mannitol ratio was significantly higher before supplementation than after (0.041 +/- 0.003 versus 0.026 +/- 0.005). During follow-up, 10 patients had normal intestinal permeability and did not relapse; of the remaining two who had increased intestinal permeability, one relapsed. CONCLUSIONS: Our findings show that zinc supplementation can resolve permeability alterations in patients with Crohn's disease in remission. Improving intestinal barrier function may contribute to reduce the risk of relapse in Crohn's disease.

Transport measurements across Caco-2 monolayers of different organic and inorganic selenium: influence of sulfur compounds.

The transport and uptake of the most common Se compounds, selenate (SeO42-), selenite (SeO3(2-)), selenomethionine, and selenocystine, were investigated using confluent monolayers of Caco-2 cells, a human carcinoma cell line. Comparative measurements were performed in the absorptive (apical to basolateral side) and exsorptive (basolateral to apical side) directions. Apparent permeability coefficients (Papp), calculated from transport experiments in the absorptive direction, showed increasing values in the following rank order: about 1 x 10(6) cm/s < mannitol < SeO3(2-) < or = selenocystine < selenomethionine < SeO4(2-) < or = about 16 x 10(4) cm/s. The ratios of the Papp measured in the absorptive versus exsorptive directions indicated that only the organic forms presented a net polarized transport (Papp ratio >> 1), suggesting the presence of a transcellular pathway. No significant excretion was observed. The transport of selenomethionine was inhibited by its sulfur analog, methionine, suggesting a common transport mechanism. In contrast, an inhibition of the transport of selenocystine by cysteine was not observed. From the two substrates tested, sulfate and thiosulfate, only thiosulfate inhibited the transport of SeO4(2-) . This effect was also observed for SeO32- (i.e., was unspecific), which questioned the assertion of a common transport for sulfate and SeO4(2-) and may confirm the paracellular pathway of SeO42- suggested by the Papp ratio of about 1. The addition of glutathione (GSH) in large excess had no consequence on the passage of SeO3(2-) but strongly increased the uptake (about fourfold). The liquid chromatography - mass spectrometry (LC-MS) data showed that, in the ionic condition of incubation medium, GSH promptly reduced SeO3(2-) (< or = 2 min) in its elemental form Se0, which cannot ascribe to selenodiglutathione a direct role in the effect of GSH.

Development of a more rapid, reduced serum culture system for Caco-2 monolayers and application to the biopharmaceutics classification system.

The objectives were: (1) to develop a more rapid, reduced serum culture system for Caco-2 monolayers, relative to the traditional 21-day, 10% fetal bovine serum (FBS) system; and (2) to determine the biopharmaceutical drug classification of an oral therapeutic agent using this new system. Caco-2 cells were grown in the six well format on polycarbonate filters, in medium containing 2% iron supplemented calf serum (sCS) and a combination of growth factors and hormones. After 4 days in culture, permeabilities of three marker compounds (metoprolol, mannitol, and taurocholate) across monolayers were determined, and compared to permeabilities from the traditional 21-day, 10% FBS system, using cells at similar passage number. Cell morphology, degree of cell differentiation, and the presence of two efflux pumps were assessed. The 2% sCS model was also used to classify the permeability of an oral therapeutic agent as high or low. No difference in permeability was observed for metoprolol transport (P = 0.38) between the two culture methods, and the values obtained were independent of passage number of the cells. Mannitol permeability was about 2-fold higher from the 2% sCS system, as compared to the 10% FBS system. Taurocholate permeability was low indicating the 2% sCS culture at 4 days lacked this particular active transporter capability. Electron micrographs of cells grown in the 2% sCS system at 4 days revealed the presence of microvilli and tight junctions. P-glycoprotein and an efflux pump for furosemide were functionally present. The 2% sCS system indicated the oral therapeutic agent as highly permeable, which agreed with the 10% FBS system. This new system provides a rapid, accurate, and economical option for passive permeability determination, and appears to be applicable to the proposed Biopharmaceutics Classification System (BCS).

Nasal epithelial permeation of thymotrinan (TP3) versus thymocartin (TP4): competitive metabolism and self-enhancement.

PURPOSE: To investigate concentration dependent permeabilities and metabolism kinetics of thymotrinan (TP3) versus thymocartin (TP4) in nasal epithelium in vitro. METHODS: Excised bovine nasal mucosa was used as an in vitro model. Permeabilities were studied in a diffusion chamber, metabolism kinetics in a reflection kinetics set-up. Studies were performed at various TP3 and TP4 concentrations. The 3H-mannitol flux was measured to monitor junctional permeability. Potential Ca(2+)-complexation was investigated using a Ca(2+)-selective electrode. RESULTS: Permeability of TP3 was negligible at 0.1 and 0.2 mM and increased drastically above 0.4 mM up to -2 X 10(-5) cm s(-1). In the presence of 2 mM TP4 the TP3 permeabilites were significantly above (approximately 4 x 10(-5) cm s(-1)) the level of TP3 without TP4, and TP3 metabolism was totally inhibited. TP3 and TP4 showed a significant concentration dependent effect on the permeability of 3H-mannitol. A hyperosmolarity effect of the peptide solutions was excluded. Transepithelial electrical resistance (TEER; approximately 30 ohms cm2) was unchanged by either TP3 or TP4. At 1 mM TP3 the mucosal-to-serosal permeability was four times higher than serosal-to-mucosal, indicating enzyme polarization. In reflection kinetics studies, TP3 degradation was slightly higher on the mucosal than on the serosal side. TP3 and TP4 followed the same non-linear metabolism kinetics. CONCLUSIONS: Increase in permeability at high TP concentrations involves competitive enzyme saturation combined with self-enhanced paracellular permeation.

Transdermal permeation of neutral molecules by skin electroporation.

Electroporation of skin has recently been shown to enhance transport of charged molecules across skin by up to four orders of magnitude. This study demonstrates that high-voltage pulses can also increase transdermal permeation of two neutral model solutes, i.e. mannitol and water, up to 100-fold. The elevated flux results from the persistent increase in skin permeability following electroporation, rather than from electro-osmosis during pulsing. Control on transport was achieved by controlling the electrical parameters of the pulse, i.e. the pulse voltage, time constant and number.

Intracerebral microdialysis study of glutamate reuptake in awake, behaving rats.

The central nervous system has high-affinity uptake systems for the clearance of amino acid transmitters. These systems are found in both neurons and astrocytes. Previous studies have shown that the uptake of amino acid transmitters by astrocytes in culture can be modulated by adrenergic agents. The objectives of this study were to develop a methodology that evaluates the brain's reuptake capacity for glutamate in awake, behaving animals and to determine whether glutamate reuptake is under alpha-adrenergic regulation in the intact central nervous system. Male Sprague-Dawley rats weighing 250-450 g were used in this study. The extraction fraction of L-[3H]glutamate with [14C]mannitol as a reference was measured. The cortical extraction fraction of L-[3H]glutamate corrected for [14C]mannitol (EL-glu) reaches steady state rapidly and is both stable and repeatable. EL-glu is a measure of L-glutamate reuptake and not metabolism. EL-glu is decreased in a dose-dependent manner by the addition of the glutamate reuptake blocker D,L-threo-beta-hydroxyaspartic acid or unlabeled L- glutamate. In addition, EL-glu is increased in a dose-dependent manner by the alpha1-adrenergic agonist phenylephrine, and this increase is blocked by the alpha-adrenergic antagonist phentolamine.

Paracellular phosphate absorption in rat colon: a mechanism for enema-induced hyperphosphatemia.

The mechanism of colonic phosphate absorption is not well defined. We measured unidirectional phosphate fluxes across rat distal colon epithelium in the absence of transepithelial electrochemical gradients. Steady-state mucosal-to-serosal flux (Jms) was not different from the serosal-to-mucosal flux (Jsm), generating no net flux (Jnet = Jms - Jsm, was not different from "0'). Simultaneous fluxes of mannitol, a paracellular probe, exhibited an identical flux pattern, suggesting that phosphate flux across the colonic epithelium may be mediated through the paracellular pathway. Tight junction permeability was increased with mucosal addition of taurodeoxycholate (TDC, 2 mM) which caused a prompt increase in transepithelial conductance from 7.03 +/- 0.35 to 13.88 +/- 0.35 mS/cm2 (p < 0.001). This was associated with an increase in Jsm, but no change in Jms, for mannitol, resulting in a net flux in the secretary direction. Identical TDC-induced changes were observed in phosphate fluxes, again suggesting phosphate permeation through the intercellular, mannitol pathway. A significant correlation was observed between the permeability of phosphate and the permeability of mannitol, measured both in the mucosal-to-serosal and the serosal-to-mucosal directions and under both control and experimental (mucosal TDC) conditions. Thus, colonic phosphate transport is mediated through the paracellular pathway and enema with high phosphate concentrations (1,760 times blood concentration), can trigger rapid and massive phosphate absorption through this diffusive pathway.

Occludin is a functional component of the tight junction.

Occludin's role in mammalian tight junction activity was examined by 'labeling' the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cell with the Lac repressor gene. HygR clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting HygR/NeoR clones were plated on porous inserts and allowed to form tight junctions. Once steady state transepithelial electrical resistance was achieved, isopropyl- beta-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also co-localized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl- beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isopropyl- beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca2+ medium were treated with isopropyl- beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175 +/- 11 nm to 248 +/- 16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl- beta-D-thiogalactoside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.

The pro-drug sulindac may reduce the risk of intestinal damage associated with the use of conventional non-steroidal anti-inflammatory drugs.

To test the hypothesis that administration of a non-steroidal anti-inflammatory drug formulated as a pro-drug, inactive as a cyclooxygenase inhibitor until after absorption, might cause less intestinal damage than conventional non-steroidal anti-inflammatory drugs, intestinal permeation to 51Cr-EDTA and mannitol was assessed in healthy volunteers before and after oral treatment for 1 week with either the pro-drug sulindac or the conventional non-steroidal anti-inflammatory drug indomethacin. Indomethacin, but not sulindac, significantly increased intestinal permeation to 51Cr-EDTA and reduced haemoglobin and haematocrit; neither affect mannitol permeation.