Micronutrients at Supplemental Levels, Tight Junctions and Epithelial Barrier Function: A Narrative Review.

Disease modifiers, whether from cancer, sepsis, systemic inflammation, or microbial pathogens, all appear to induce epithelial barrier leak, with induced changes of the Tight Junctional (TJ) complex being pivotal to the process. This leak-and the ensuant breakdown of compartmentation-plays a central role in disease morbidity on many levels. Accumulation of lung water in the luminal compartment of airways was a major driver of morbidity and mortality in COVID-19 and is an excellent example of the phenomenon. Increasing awareness of the ability of micronutrients to improve basal barrier function and reduce barrier compromise in pathophysiology may prove to be a low-cost, safe, and easily administered prophylactic and/or therapeutic option amenable to large populations. The growing appreciation of the clinical utility of supplemental doses of Vitamin D in COVID-19 is but one example. This narrative review is intended to propose a general theory on how and why micronutrients-at levels above normal dietary intake-successfully remodel TJs and improve barrier function. It discusses the key difference between dietary/Recommended Daily Allowance (RDA) levels of micronutrients versus supplemental levels, and why the latter are needed in disease situations. It advances a hypothesis for why signal transduction regulation of barrier function may require these higher supplemental doses to achieve the TJ remodeling and other barrier element changes that are clinically beneficial.

Calcitriol modifies tight junctions, improves barrier function, and reduces TNF-α-induced barrier leak in the human lung-derived epithelial cell culture model, 16HBE 14o.

Using the 16HBE 14o- human airway epithelial cell culture model, calcitriol (Vitamin D) was shown to improve barrier function by two independent metrics - increased transepithelial electrical resistance (TER) and reduced transepithelial diffusion of 14 C-D-mannitol (Jm ). Both effects were concentration dependent and active out to 168 h post-treatment. Barrier improvement associated with changes in the abundance of specific tight junctional (TJ) proteins in detergent-soluble fractions, most notably decreased claudin-2. TNF-α-induced compromise of barrier function could be attenuated by calcitriol with a concentration dependence similar to that observed for improvement of control barrier function. TNF-α-induced increases in claudin-2 were partially reversed by calcitriol. The ERK 1,2 inhibitor, U0126, itself improved 16HBE barrier function indicating MAPK pathway regulation of 16HBE barrier function. Calcitriol's action was additive to the effect of U0126 in reducing TNF- α -induced barrier compromise, suggesting that calcitriol may be acting through a non-ERK pathway in its blunting of TNF- α - induced barrier compromise. This was supported by calcitriol being without effect on pERK levels elevated by the action of TNF-α. Lack of effect of TNF- α on the death marker, caspase-3, and the inability of calcitriol to decrease the elevated LC3B II level caused by TNF-α, suggest that calcitriol's barrier improvement does not involve a cell death pathway. Calcitriol's improvement of control barrier function was not additive to barrier improvement induced by retinoic acid (Vitamin A). Calcitriol improvement and protection of airway barrier function could in part explain Vitamin D's reported clinical efficacy in COVID-19 and other airway diseases.

The multiphasic TNF-α-induced compromise of Calu-3 airway epithelial barrier function.

Purpose: Airway epithelial barrier leak and the involvement of proinflammatory cytokines play a key role in a variety of diseases. This study evaluates barrier compromise by the inflammatory mediator Tumor Necrosis Factor-α (TNF-α) in the human airway epithelial Calu-3 model. Methods: We examined the effects of TNF-α on barrier function in Calu-3 cell layers using Transepithelial Electrical Resistance (TER) and transepithelial diffusion of radiolabeled probe molecules. Western immunoblot analyses of tight junctional (TJ) proteins in detergent soluble fractions were performed. Results: TNF-α dramatically reduced TER and increased paracellular permeability of both 14C-D-mannitol and the larger 5 kDa probe, 14C-inulin. A time course of the effects shows two separate actions on barrier function. An initial compromise of barrier function occurs 2-4 hours after TNF-α exposure, followed by complete recovery of barrier function by 24 hrs. Beginning 48 hrs. post-exposure, a second more sustained barrier compromise ensues, in which leakiness persists through 144 hrs. There were no changes in TJ proteins observed at 3 hrs. post exposure, but significant increases in claudins-2, -3, -4, and -5, as well as a decrease in occludin were seen at 72 hrs. post TNF-α exposure. Both the 2-4 hr. and the 72 hr. TNF-α induced leaks are shown to be mediated by the ERK signaling pathway. Conclusion: TNF-α induced a multiphasic transepithelial leak in Calu-3 cell layers that was shown to be ERK mediated, as well as involve changes in the TJ complex. The micronutrients, retinoic acid and calcitriol, were effective at reducing this barrier compromise caused by TNF-α. The significance of these results for airway disease and for COVID-19 specifically are discussed.

Retinoic acid improves baseline barrier function and attenuates TNF-α-induced barrier leak in human bronchial epithelial cell culture model, 16HBE 14o.

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-α-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in 14C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-α-induced 16HBE barrier leak, attenuating 60% of the TNF-α-induced leak to 14C-mannitol and 80% of the leak to 14C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-α resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward "band-shift" in occludin Western immunoblots. The presence of RA partially reversed TNF-α's effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-α-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a "cytokine storm" may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.

Epithelial barrier function properties of the 16HBE14o- human bronchial epithelial cell culture model.

The human bronchial epithelial cell line, 16HBE14o- (16HBE), is widely used as a model for respiratory epithelial diseases and barrier function. During differentiation, transepithelial electrical resistance (TER) increased to approximately 800 Ohms × cm2, while 14C-d-mannitol flux rates (Jm) simultaneously decreased. Tight junctions (TJs) were shown by diffusion potential studies to be anion-selective with PC1/PNa = 1.9. Transepithelial leakiness could be induced by the phorbol ester, protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the proinflammatory cytokine, tumor necrosis factor-α (TNF-α). Basal barrier function could not be improved by the micronutrients, zinc, or quercetin. Of methodological significance, TER was observed to be more variable and to spontaneously, significantly decrease after initial barrier formation, whereas Jm did not significantly fluctuate or increase. Unlike the strong inverse relationship between TER and Jm during differentiation, differentiated cell layers manifested no relationship between TER and Jm. There was also much greater variability for TER values compared with Jm. Investigating the dependence of 16HBE TER on transcellular ion conductance, inhibition of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel with GlyH-101 produced a large decrease in short-circuit current (Isc) and a slight increase in TER, but no significant change in Jm. A strong temperature dependence was observed not only for Isc, but also for TER. In summary, research utilizing 16HBE as a model in airway barrier function studies needs to be aware of the complexity of TER as a parameter of barrier function given the influence of CFTR-dependent transcellular conductance on TER.

Improving Transient Transfection Efficiency in a Differentiated, Polar Epithelial Cell Layer.

Polar, differentiated epithelial cell culture models (especially at confluence) are difficult to transfect compared with the higher transfection efficiencies that one obtains with relatively less differentiated, nonpolar cell culture models. Here, we sought to develop a strategy to enhance the efficiency of transfecting polar, differentiated epithelial cells. We found that chemically abrading the differentiated CACO-2 human intestinal epithelial cell layer by a trypsin and EDTA pretreatment (before the use of detergent-like transfection reagents) dramatically improved transfection efficiency in this polar, differentiated model. Although this treatment did improve the transfection efficiency, it also induced leakiness in the epithelial barrier by both opening tight junctional complexes and by creating holes in the cell layer because of low-level cell death and detachment. Thus, this approach to enhance the transfection efficiency of polar, differentiated cells will be useful for assessment of the effect of the transfected/expressed protein on (re)formation of an epithelial barrier rather than on a functional barrier itself.

Intestinal barrier tightening by a cell-penetrating antibody to Bin1, a candidate target for immunotherapy of ulcerative colitis.

Patients afflicted with ulcerative colitis (UC) are at increased risk of colorectal cancer. While its causes are not fully understood, UC is associated with defects in colonic epithelial barriers that sustain inflammation of the colon mucosa caused by recruitment of lymphocytes and neutrophils into the lamina propria. Based on genetic evidence that attenuation of the bridging integrator 1 (Bin1) gene can limit UC pathogenicity in animals, we have explored Bin1 targeting as a therapeutic option. Early feasibility studies in the dextran sodium sulfate mouse model of experimental colitis showed that administration of a cell-penetrating Bin1 monoclonal antibody (Bin1 mAb 99D) could prevent lesion formation in the colon mucosa in part by preventing rupture of lymphoid follicles. In vivo administration of Bin1 mAb altered tight junction protein expression and cecal barrier function. Strikingly, electrophysiology studies in organ cultures showed that Bin1 mAb could elevate resistance and lower 14 C-mannitol leakage across the cecal mucosa, consistent with a direct strengthening of colonic barrier function. Transcriptomic analyses of colitis tissues highlighted altered expression of genes involved in circadian rhythm, lipid metabolism, and inflammation, with a correction of the alterations by Bin1 mAb treatment to patterns characteristic of normal tissues. Overall, our results suggest that Bin1 mAb protects against UC by directly improving colonic epithelial barrier function to limit gene expression and cytokine programs associated with colonic inflammation.

Improvement of Human-Oral-Epithelial-Barrier Function and of Tight Junctions by Micronutrients.

The oral epithelium represents a major interface between an organism and its external environment. Improving this barrier at the molecular level can provide an organism added protection from microbial-based diseases. Barrier function of the Gie-3B11-human-gingival-epithelial-cell-culture model is enhanced by the micronutrients zinc, quercetin, retinoic acid, and acetyl-11-keto-ß-boswellic acid, as observed by a concentration-dependent increase in transepithelial electrical resistance and a decrease in transepithelial 14C-d-mannitol permeability. With this improvement of tight-junction (TJ)-barrier function (reduced leak) comes a pattern of micronutrient-induced changes in TJ claudin abundance that is specific to each individual micronutrient, along with changes in claudin subcellular localization. These micronutrients were effective not only when administered to both cell surfaces simultaneously but also when administered to the apical surface alone, the surface to which the micronutrients would be presented in routine clinical use. The biomedical implications of micronutrient enhancement of the oral-epithelial barrier are discussed.

The Host Microbiome Regulates and Maintains Human Health: A Primer and Perspective for Non-Microbiologists.

Humans consider themselves discrete autonomous organisms, but recent research is rapidly strengthening the appreciation that associated microorganisms make essential contributions to human health and well being. Each person is inhabited and also surrounded by his/her own signature microbial cloud. A low diversity of microorganisms is associated with a plethora of diseases, including allergy, diabetes, obesity, arthritis, inflammatory bowel diseases, and even neuropsychiatric disorders. Thus, an interaction of microorganisms with the host immune system is required for a healthy body. Exposure to microorganisms from the moment we are born and appropriate microbiome assembly during childhood are essential for establishing an active immune system necessary to prevent disease later in life. Exposure to microorganisms educates the immune system, induces adaptive immunity, and initiates memory B and T cells that are essential to combat various pathogens. The correct microbial-based education of immune cells may be critical in preventing the development of autoimmune diseases and cancer. This review provides a broad overview of the importance of the host microbiome and accumulating knowledge of how it regulates and maintains a healthy human system. Cancer Res; 77(8); 1783-812. ©2017 AACR.

Zinc reduces epithelial barrier compromise induced by human seminal plasma.

Human semen has the potential to modulate the epithelial mucosal tissues it contacts, as seminal plasma (SP) is recognized to contain both pro- and anti-barrier components, yet its effects on epithelial barrier function are largely unknown. We addressed the role of human SP when exposed to the basal-lateral epithelial surface, a situation that would occur clinically with prior mechanical or disease-related injury of the human epithelial mucosal cell layers in contact with semen. The action of SP on claudins-2, -4, -5, and -7 expression, as well as on a target epithelium whose basolateral surface has been made accessible to SP, showed upregulation of claudins-4 and -5 in CACO-2 human epithelial cell layers, despite broad variance in SP-induced modulation of transepithelial electrical resistance and mannitol permeability. Upregulation of claudin-2 by SP also exhibited such variance by SP sample. We characterize individual effects on CACO-2 barrier function of nine factors known to be present abundantly in seminal plasma (zinc, EGF, citrate, spermine, fructose, urea, TGF, histone, inflammatory cytokines) to establish that zinc, spermine and fructose had significant potential to raise CACO-2 transepithelial resistance, whereas inflammatory cytokines and EGF decreased this measure of barrier function. The role of zinc as a dominant factor in determining higher levels of transepithelial resistance and lower levels of paracellular leak were confirmed by zinc chelation and exogenous zinc addition. As expected, SP presentation to the basolateral cell surface also caused a very dramatic yet transient elevation of pErk levels. Results suggest that increased zinc content in SP can compete against the barrier-compromising effect of negative modulators in SP when SP gains access to that epithelium's basolateral surface. Prophylactic elevation of zinc in an epithelial cell layer prior to contact by SP may help to protect an epithelial barrier from invasion by SP-containing STD microbial pathogens such as HPV or HIV.

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-ß (IL1ß) 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) + IL1ß (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 + IL1ß 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.

Novel Colitis Immunotherapy Targets Bin1 and Improves Colon Cell Barrier Function.

BACKGROUND: Ulcerative colitis (UC) is associated with defects in colonic epithelial barriers as well as inflammation of the colon mucosa resulting from the recruitment of lymphocytes and neutrophils in the lamina propria. Patients afflicted with UC are at increased risk of colorectal cancer. Currently, UC management employs general anti-inflammatory strategies associated with a variety of side effects, including heightened risks of infection, in patients where the therapy is variably effective. Thus, second generation drugs that can more effectively and selectively limit UC are desired. AIM: Building on genetic evidence that attenuation of the Bin1 (Bridging integrator 1) gene can limit UC pathogenicity in the mouse, we pursued Bin1 targeting as a therapeutic option. METHODS: Mice were injected with a single dose of Bin1 mAb followed by oral administration of 3 % DSS in water for 7 days. RESULTS: In this study, we offer preclinical proof of concept for a monoclonal antibody (mAb) targeting the Bin1 protein that blunts UC pathogenicity in a mouse model of experimental colitis. Administration of Bin1 mAb reduced colitis morbidity in mice; whereas unprotected mice is characterized by severe lesions throughout the mucosa, rupture of the lymphoid follicle, high-level neutrophil and lymphocyte infiltration into the mucosal and submucosal areas, and loss of surface crypts. In vitro studies in human Caco-2 cells showed that Bin1 antibody altered the expression of tight junction proteins and improved barrier function. CONCLUSIONS: Our results suggest that a therapy based on Bin1 monoclonal antibody supporting mucosal barrier function and protecting integrity of the lymphoid follicle could offer a novel strategy to treat UC and possibly limit risks of colorectal cancer.

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.

Acute Inhibition of MEK Suppresses Congenital Melanocytic Nevus Syndrome in a Murine Model Driven by Activated NRAS and Wnt Signaling.

Congenital melanocytic nevus (CMN) syndrome is the association of pigmented melanocytic nevi with extra-cutaneous features, classically melanotic cells within the central nervous system, most frequently caused by a mutation of NRAS codon 61. This condition is currently untreatable and carries a significant risk of melanoma within the skin, brain, or leptomeninges. We have previously proposed a key role for Wnt signaling in the formation of melanocytic nevi, suggesting that activated Wnt signaling may be synergistic with activated NRAS in the pathogenesis of CMN syndrome. Some familial pre-disposition suggests a germ-line contribution to CMN syndrome, as does variability of neurological phenotypes in individuals with similar cutaneous phenotypes. Accordingly, we performed exome sequencing of germ-line DNA from patients with CMN to reveal rare or undescribed Wnt-signaling alterations. A murine model harboring activated NRAS(Q61K) and Wnt signaling in melanocytes exhibited striking features of CMN syndrome, in particular neurological involvement. In the first model of treatment for this condition, these congenital, and previously assumed permanent, features were profoundly suppressed by acute post-natal treatment with a MEK inhibitor. These data suggest that activated NRAS and aberrant Wnt signaling conspire to drive CMN syndrome. Post-natal MEK inhibition is a potential candidate therapy for patients with this debilitating condition.

Drug delivery of zinc to Barrett's metaplasia by oral administration to Barrett's esophagus patients.

BACKGROUND: Delivery of a pharmacologically effective drug dosage to a target tissue is critical. Barrett's epithelia are a unique challenge for drug delivery of orally administered zinc due to rapid transit down the esophageal lumen, incomplete absorptive differentiation of these epithelia, and the use of proton-pump inhibitor drugs abrogating intestinal uptake of supplemental zinc. METHODS: Barrett's esophagus patients were administered oral zinc gluconate (26 mg zinc twice daily) for 14 days prior to biopsy procurement. Barrett's biopsies were analyzed for total zinc content by atomic absorption spectroscopy and by western immunoblot for cellular proteins known to be regulated by zinc. RESULTS: Cellular levels of both the Znt-1 transport protein and the alpha isoform of PKC were over 50% lower in the zinc treatment group. CONCLUSION: Oral zinc administration can result in effective delivery of zinc to Barrett's epithelia with resulting effects on intracellular signal transduction.

Zinc enhancement of LLC-PK(1) 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 zinc's 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. Zinc's 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.

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 (R(t)) and decrease transepithelial mannitol permeability (J(m)) 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 R(t) and decreased J(m). Zinc, butyrate and indole only exhibited statistically significant enhancement of R(t). 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 R(t) is shown.

Transepithelial leak in Barrett's esophagus patients: the role of proton pump inhibitors.

AIM: To determine if the observed paracellular sucrose leak in Barrett's esophagus patients is due to their proton pump inhibitor (PPI) use. METHODS: The in vivo sucrose permeability test was administered to healthy controls, to Barrett's patients and to non-Barrett's patients on continuous PPI therapy. Degree of leak was tested for correlation with presence of Barrett's, use of PPIs, and length of Barrett's segment and duration of PPI use. RESULTS: Barrett's patients manifested a near 3-fold greater, upper gastrointestinal sucrose leak than healthy controls. A decrease of sucrose leak was observed in Barrett's patients who ceased PPI use for 7 d. Although initial introduction of PPI use (in a PPI-naïve population) results in dramatic increase in sucrose leak, long-term, continuous PPI use manifested a slow spontaneous decline in leak. The sucrose leak observed in Barrett's patients showed no correlation to the amount of Barrett's tissue present in the esophagus. CONCLUSION: Although future research is needed to determine the degree of paracellular leak in actual Barrett's mucosa, the relatively high degree of leak observed with in vivo sucrose permeability measurement of Barrett's patients reflects their PPI use and not their Barrett's tissue per se.