Mary Carmen Valenzano

Esomeprazole induces upper gastrointestinal tract transmucosal permeability increase

Background  Proton pump inhibitors (PPIs) are one of the most widely used drug classes in the US and are now frontline medications for gastro‐oesophageal reflux disease (GERD) and dyspepsia. In a previous work, we observed that a transmucosal, upper gastrointestinal (GI) leak exists in Barrett’s oesophagus (BO) patients. PPI medications are commonly used by Barrett’s patients.

Age- and diet-related increase in transepithelial colon permeability of Fischer 344 rats.

When transepithelial permeability of rat distal colon is evaluated on the basis of transepithelial electrical resistance, age does not have an effect. Age likewise did not affect the decrease in resistance brought about by phorbol ester exposure. However, age was shown to correlate with increased transepithelial permeability when diffusion of the nonelectrolyte, d-mannitol, was used as an indicator. A phorbol ester-induced increase in transepithelial permeability to d-mannitol was observed to increase with age. Basal permeability to d-mannitol was significantly higher in older rats when the animals were allowed to age on a high-fat diet. Distance from the rectum was shown to be a potential complicating factor in these studies, since distal colon closer to the rectum was observed to have lower transepithelial permeability. The potential effect of such increased leakiness on the increased frequency of colon cancer in older individuals is discussed.

Transepithelial Leak in Barrett’s Esophagus

Using orally administered sucrose as a probe of gastrointestinal permeability, this study focused on determining whether Barrett’s metaplasia exhibits a paracellular transepithelial leak to small nonelectrolytes. Subjects in five separate classes (nonendoscoped, asymptomatic controls; endoscoped, asymptomatic controls; gastroesophageal reflux disease without mucosal complications; grossly visible esophagitis; and Barrett’s esophagus) consumed a sucrose solution at bedtime and collected all overnight urine. Urine volume was measured and sucrose concentration was determined by high-performance liquid chromatography. Patients with Barrett’s were observed to exhibit a transepithelial leak to sucrose whose mean value was threefold greater than that seen in healthy control subjects or patients with reflux but without any mucosal defect. A parallel study of claudin tight junction proteins in endoscopy biopsy samples showed that whereas Barrett’s metaplasia contains dramatically more claudin-2 and claudin-3 than is found in normal esophageal mucosa, it is markedly lower in claudins 1 and 5, indicating very different tight junction barriers.

Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients.

The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.

Comparison of three integral tight junction barrier proteins in Barrett's epithelium versus normal esophageal epithelium.

rified DNA was used to determine the sex and to obtain a genetic fingerprint of the specimens. The cancer fragments showed a female phenotype, thereby excluding our patient as a possible source. The genetic fingerprint of the adenoma, however, was identical to the one of our patient (Table 1). Confounding of specimens is rare but cannot be completely avoided. As illustrated by our case, this can occur in the endoscopy room while tissue fragments are collected. In addition, specimens can be mislabeled, and during processing fragments from other specimens (so-called “floaters”) can be incorporated into the analysis (1). As soon as a mix-up is suspected by an unlikely finding, thorough investigation is critical, because misassignment of a diagnosis can delay appropriate treatment or lead to repeat procedures or unnecessary invasive treatment. The ideal test to determine the presence of a mix-up should be able to positively identify a person. Until recently, the identification of a person relied on the blood group and the human leukocyte antigens (2). Unfortunately, these tests lack the power to positively identify a person. For example, a discordance of the blood groups clearly indicates a different source, but the probability of different blood groups in two unrelated white persons is only approximately 60% (1). On the other hand, of course, the identification of the blood group does not prove the identity of the person(s). Almost unequivocal identification became possible only after the introduction of DNA fingerprinting (3). The probability of two unrelated persons having identical fingerprints is typically less than one in several millions. The only prerequisite to perform such a test is the availability of a tissue or blood sample to positively identify the person under question. It is almost always possible to extract enough DNA from tiny, even formalin-fixed and paraffin-embedded biopsy fragments, often even from individual pieces. There are few case reports in the literature in which this technology was used to correctly identify tissue, blood, and urine samples (2, 4, 5). We believe that this technology can be useful to all in the medical field who might be confronted with a possible tissue, blood, or urine mix-up. However, the analysis should only be performed in a laboratory with the necessary experience in forensic genetics.

Enhancement of Tight Junctional Barrier Function by Micronutrients: Compound-Specific Effects on Permeability and Claudin Composition

Amid an increasing number of reports in the literature concerning epithelial barrier enhancement by various nutrient compounds, there has never been a study performing side-by-side comparisons of these agents in a single epithelial model. We compare five nutrient compounds (previously reported in various epithelial models to enhance barrier function) regarding their ability to increase transepithelial electrical resistance (Rt) and decrease transepithelial mannitol permeability (Jm) across LLC-PK1 renal epithelial cell layers. The effects of these nutrients on the abundance of various tight junctional proteins are also compared. In the overall group of nutrients tested - zinc, indole, quercetin, butyrate and nicotine - only nicotine failed to improve barrier function by either parameter. Nicotine also was without effect on tight junctional proteins. Quercetin simultaneously increased Rt and decreased Jm. Zinc, butyrate and indole only exhibited statistically significant enhancement of Rt. Each of these four effective nutrient compounds had unique patterns of effects on the panel of tight junctional proteins studied. No two compounds produced the same pattern of effects. This unique pattern of effects on tight junctional complex composition by each compound establishes the chance for additive or even synergistic improvement of barrier function by combinations of compounds. A synergistic effect of the combination of quercetin and zinc on Rt is shown.

Proton Pump Inhibitors Interfere With Zinc Absorption and Zinc Body Stores

Background Proton pump inhibitors (PPIs) cause a sharp elevation of gastro-duodenal luminal pH which in turn has resulted in reports of reduced absorption of magnesium and certain other nutrients. Methods Gastroesophageal reflux disease (GERD) patients on long-term PPI therapy (> 6 months) or healthy test subjects (not on any acid preventive or neutralizing medication) were administered oral doses of zinc gluconate (26.2 mg zinc, twice daily) for 14 days followed by 5 cc venous blood samples. Plasma was analyzed for total zinc content by atomic absorption spectrophotometry. Baseline plasma and red blood cell zinc levels were also measured in these two groups when not taking any zinc supplementation. Results Plasma zinc levels of healthy controls increased by 126% during the period of zinc supplementation compared to only a 37% increase for individuals on long-term PPI therapy. On their normal diet (with no zinc supplementation), PPI-users had a 28% lower plasma zinc level than healthy controls (P < 0.005). Conclusions PPI use dramatically reduces supplemental zinc uptake and can result in decreased zinc body stores. Certain individuals on long-term PPI therapy, such as infants being treated for colic, may be at risk for decreased systemic levels of trace metals needed for developmental, regenerative and immunological requirements.

Modification of Tight Junction Structure and Permeability by Nutritional Means

The relative abundance of various claudin proteins of LLC‐PK1 renal epithelial tight junctions (TJs) is modulated by culturing the cells in a medium that is sharply reduced in the sulfur‐containing amino acids, cysteine, cystine, and methionine. The functional result is an epithelial barrier that has a higher transepithelial electrical resistance and a decreased paracellular leak to D‐mannitol (i.e., improved barrier function). This is accomplished without affecting the cultures confluent cell density, its short circuit current, or its hallmark differentiated property, Na+‐dependent sugar transport. The implications of being able to enhance epithelial TJ barrier function by nutritional means are discussed, particularly in light of the ability of methionine‐restrictive diets to enhance life span and forestall age‐related morbidity.

Zinc enhancement of LLC-PK1 renal epithelial barrier function

BACKGROUND AND AIMS Earlier work by our group and others has documented improvement of epithelial barrier function in human gastrointestinal models. Here we tested zincs ability to improve a renal epithelial model. Our aim was to compare the functional and structural effects of zinc on the tight junctional (TJ) complexes of these two very distinct epithelial cell types. Zincs ability to achieve barrier enhancement in very different epithelial cell types by action upon distinct molecular targets in each epithelial model may suggest a fundamental general role for supplemental zinc in epithelial barrier improvement throughout the body. METHODS Cell layers were exposed to 50 or 100 μM zinc on both cell surfaces for 48 h followed by measurement of transepithelial electrical resistance (Rt) and transepithelial (14)C-mannitol flux (Jm). TJ proteins in cell layers were analyzed by Western immunoblot. RESULTS AND CONCLUSIONS Zinc supplementation improved the basal TJ barrier function of LLC-PK1 renal cell layers, exemplified by increased Rt and decreased Jm. These zinc-induced changes were also accompanied by decreased NaCl dilution potentials. Of the tight junctional proteins that were tested (occludin, claudins 1, 2, 3, 4, and 5, and tricellulin), we did not observe a zinc-induced change in abundance of any of them, in detergent-soluble fractions of lysates of confluent differentiated cell layers. However, examination of cytosolic fractions showed concentration-dependent increases in the levels of claudins -2 and -4 in this compartment as a result of supplemental zinc. The effects of supplemental zinc on the tight junctional complexes and barrier properties of this renal epithelial model are contrasted with zinc effects on the CACO-2 gastrointestinal model.

Sieving characteristics of cytokine- and peroxide-induced epithelial barrier leak: Inhibition by berberine

AIM To study whether the inflammatory bowel disease (IBD) colon which exhibits varying severity and cytokine levels across its mucosa create varying types of transepithelial leak. METHODS We examined the effects of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1-β (IL1β) and hydrogen peroxide (H2O2) - singly and in combinations - on barrier function of CACO-2 cell layers. Our focus was on the type (not simply the magnitude) of transepithelial leak generated by these agents as measured by transepithelial electrical resistance (TER) and transepithelial flux of (14)C-D-mannitol, (3)H-Lactulose and (14)C-Polyethylene glycol as radiolabeled probe molecules. The isoquinoline alkaloid, berberine, was then examined for its ability to reduce specific types of transepithelial leak. RESULTS Exposure to TNF-α alone (200 ng/mL; 48 h) induced a 50% decrease in TER, i.e., increased leak of Na(+) and Cl(-) - with only a marginal but statistically significant increase in transepithelial leak of (14)C-mannitol (Jm). Exposure to TNF-α + IFN-γ (200 ng/mL; 48 h) + IL1β (50 ng/mL; 48 h) did not increase the TER change (from TNF-α alone), but there was now a 100% increase in Jm. There however was no increase in transepithelial leak of two larger probe molecules, (3)H-lactulose and (14)C-polyethylene glycol (PEG). However, exposure to TNF-α + IFN-γ + IL1β followed by a 5 h exposure to 2 mmol/L H2O2 resulted in a 500% increase in (14)C-PEG leak as well as leak to the luminal mitogen, epidermal growth factor. CONCLUSION This model of graded transepithelial leak is useful in evaluating therapeutic agents reducing IBD morbidity by reducing barrier leak to various luminal substances.