Quercetin improves and protects Calu-3 airway epithelial barrier function.

Introduction: In light of the impact of airway barrier leaks in COVID-19 and the significance of vitamin D in COVID-19 outcomes, including airway barrier protection, we investigated whether the very common dietary flavonoid quercetin could also be efficacious in supporting airway barrier function. Methods: To address this question, we utilized the widely used airway epithelial cell culture model, Calu-3. Results: We observed that treating Calu-3 cell layers with quercetin increased transepithelial electrical resistance while simultaneously reducing transepithelial leaks of 14C-D-mannitol (Jm) and 14C-inulin. The effects of quercetin were concentration-dependent and exhibited a biphasic time course. These effects of quercetin occurred with changes in tight junctional protein composition as well as a partial inhibition of cell replication that resulted in decreased linear junctional density. Both of these effects potentially contribute to improved barrier function. Quercetin was equally effective in reducing the barrier compromise caused by the pro-inflammatory cytokine TNF-α, an action that seemed to derive, in part, from reducing the elevation of ERK 1/2 caused by TNF-α. Discussion: Quercetin improved Calu-3 barrier function and reduced TNF-α-induced barrier compromise, mediated in part by changes in the tight junctional complex.

Zinc Gluconate Induces Potentially Cancer Chemopreventive Activity in Barrett's Esophagus: A Phase 1 Pilot Study.

BACKGROUND: Chemopreventive effects of zinc for esophageal cancer have been well documented in animal models. This prospective study explores if a similar, potentially chemopreventive action can be seen in Barrett's esophagus (BE) in humans. AIMS: To determine if molecular evidence can be obtained potentially indicating zinc's chemopreventive action in Barrett's metaplasia. METHODS: Patients with a prior BE diagnosis were placed on oral zinc gluconate (14 days of 26.4 mg zinc BID) or a sodium gluconate placebo, prior to their surveillance endoscopy procedure. Biopsies of Barrett's mucosa were then obtained for miRNA and mRNA microarrays, or protein analyses. RESULTS: Zinc-induced mRNA changes were observed for a large number of transcripts. These included downregulation of transcripts encoding proinflammatory proteins (IL32, IL1ß, IL15, IL7R, IL2R, IL15R, IL3R), upregulation of anti-inflammatory mediators (IL1RA), downregulation of transcripts mediating epithelial-to-mesenchymal transition (EMT) (LIF, MYB, LYN, MTA1, SRC, SNAIL1, and TWIST1), and upregulation of transcripts that oppose EMT (BMP7, MTSS1, TRIB3, GRHL1). miRNA arrays showed significant upregulation of seven miRs with tumor suppressor activity (-125b-5P, -132-3P, -548z, -551a, -504, -518, and -34a-5P). Of proteins analyzed by Western blot, increased expression of the pro-apoptotic protein, BAX, and the tight junctional protein, CLAUDIN-7, along with decreased expression of BCL-2 and VEGF-R2 were noteworthy. CONCLUSIONS: When these mRNA, miRNA, and protein molecular data are considered collectively, a cancer chemopreventive action by zinc in Barrett's metaplasia may be possible for this precancerous esophageal tissue. These results and the extensive prior animal model studies argue for a future prospective clinical trial for this safe, easily-administered, and inexpensive micronutrient, that could determine if a chemopreventive action truly exists.

Quantitative histopathologic assessment of perfusion MRI as a marker of glioblastoma cell infiltration in and beyond the peritumoral edema region.

BACKGROUND: Conventional MRI fails to detect regions of glioblastoma cell infiltration beyond the contrast-enhanced T1 solid tumor region, with infiltrating tumor cells often migrating along host blood vessels. PURPOSE: To quantitatively and qualitatively analyze the correlation between perfusion MRI signal and tumor cell density in order to assess whether local perfusion perturbation could provide a useful biomarker of glioblastoma cell infiltration. STUDY TYPE: Animal model. SUBJECTS: Mice bearing orthotopic glioblastoma xenografts generated from a patient-derived glioblastoma cell line. FIELD STRENGTH/SEQUENCES: 7T perfusion images acquired using a high signal-to-noise ratio (SNR) multiple boli arterial spin labeling sequence were compared with conventional MRI (T1 /T2 weighted, contrast-enhanced T1 , diffusion-weighted, and apparent diffusion coefficient). ASSESSMENT: Immunohistochemistry sections were stained for human leukocyte antigen (probing human-derived tumor cells). To achieve quantitative MRI-tissue comparison, multiple histological slices cut in the MRI plane were stacked to produce tumor cell density maps acting as a "ground truth." STATISTICAL TESTS: Sensitivity, specificity, accuracy, and Dice similarity indices were calculated and a two-tailed, paired t-test used for statistical analysis. RESULTS: High comparison test results (Dice 0.62-0.72, Accuracy 0.86-0.88, Sensitivity 0.51-0.7, and Specificity 0.92-0.97) indicate a good segmentation for all imaging modalities and highlight the quality of the MRI tissue assessment protocol. Perfusion imaging exhibits higher sensitivity (0.7) than conventional MRI (0.51-0.61). MRI/histology voxel-to-voxel comparison revealed a negative correlation between tumor cell infiltration and perfusion at the tumor margins (P = 0.0004). DATA CONCLUSION: These results demonstrate the ability of perfusion imaging to probe regions of low tumor cell infiltration while confirming the sensitivity limitations of conventional imaging modalities. The quantitative relationship between tumor cell density and perfusion identified in and beyond the edematous T2 hyperintensity region surrounding macroscopic tumor could be used to detect marginal tumor cell infiltration with greater accuracy. LEVEL OF EVIDENCE: 1 Technical stage: 2 J. Magn. Reson. Imaging 2019;50:529-540.

Cobalt chloride compromises transepithelial barrier properties of CaCo-2 BBe human gastrointestinal epithelial cell layers.

BACKGROUND: Elevation of the transcription factor HIF-1 is a prominent mediator of not only processes that accompany hypoxia, but also the tumor microenvironment and tissue regeneration. This study uses mediators of "chemical hypoxia" to ask the question whether HIF-1α elevation in a healthy epithelial cell layer leads to leakiness in its tight junctional seals. METHODS: Transepithelial electrical resistance and transepithelial diffusion of 14C-D-mannitol and other radiolabeled probes are used as indicators of transepithelial barrier function of CaCo-2 BBe human gastrointestinal epithelial cell layers cultured on permeable supports. Western immunoblot analyses of integral tight junctional proteins (occludin and claudins) are used as further indicators of barrier function change. RESULTS: Cobalt, an inhibitor of the prolyl hydroxylase enzymes governing HIF-1α breakdown in the cell, induces transepithelial leakiness in CaCo-2 BBe cell layers in a time and concentration-dependent manner. This increased leakiness is accompanied by significant changes in certain specific integral tight junctional (TJ) proteins such as a decreased level of occludin and increased level of claudin-5. Similar results regarding barrier function compromise also occur with other chemical inhibitors of HIF-1α breakdown, namely ciclopiroxolamine (CPX) and dimethyloxalylglycine (DMOG). The increased leak is manifested by both decreased transepithelial electrical resistance (Rt) and increased paracellular diffusion of D-mannitol (Jm). The induced transepithelial leak shows significant size selectivity, consistent with induced effects on TJ permeability. Less-differentiated cell layers were significantly more affected than well-differentiated cell layers regarding induced transepithelial leak. A genetically modified CaCo-2 variant with reduced levels of HIF-1ß, showed reduced transepithelial leak in response to cobalt exposure, further indicating that elevation of HIF-1α levels induced by agents of "chemical hypoxia" is responsible for the compromised barrier function of the CaCo-2 BBe cell layers. CONCLUSIONS: Exposure to inducers of chemical hypoxia elevated HIF-1α levels and increased transepithelial leak. The degree of epithelial differentiation has significant effects on this action, possibly explaining the varying effects of HIF-1 modulation in epithelial and endothelial barrier function in different physiological and pathophysiological conditions.

Omeprazole induces gastric permeability to digoxin.

Previous animal and patient-based studies have shown that omeprazole induces a transepithelial paracellular gastric leak. This study reports on the potential for an omeprazole-induced leak of drugs with narrow therapeutic windows. Ussing chamber experiments investigated the effects of omeprazole on rat gastric corpus permeability to the drugs, digoxin and phenytoin. Digoxin (780 MW) permeated the gastric mucosa at an accelerated rate in the presence of omeprazole. This leak could contribute to dangerous elevations of blood digoxin levels in certain situations. Omeprazole was found to have no effect on the flux rate of phenytoin (252 MW). The tight-junctional leak generated by omeprazole thus exhibits specificity to the types of molecules it allows to permeate through the gastric mucosa. This leak may pose a clinical danger by increasing drug uptake into the bloodstream, a phenomenon which would act synergistically with the effect of omeprazole on inhibiting liver cytochrome P450s that remove drugs from the bloodstream, thereby elevating drug blood levels.

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. AIM: To examine if the PPI, esomeprazole, affects the barrier function of the upper GI tract. METHODS: The sucrose permeability test (SPT) was used to assess the possible effect of the PPI, esomeprazole, on upper GI leak in 37 first-time-presenting GERD patients and 25 healthy controls. RESULTS: Esomeprazole induced a significant transmucosal leak in the upper GI tract of patients taking the drug for the first time. The leak occurred quickly, within days of first taking the drug. The leak was also reversed within days of stopping the medication. CONCLUSIONS: This is the first patient-based study showing that a PPI compromises upper GI barrier function. There are potential implications for transmucosal leak of other medications that a patient on a PPI may be taking, as well as possible leak of endogenous peptides/proteins. The clinical consequences of this phenomenon are currently unknown, but are potentially important.

Restriction of sulfur-containing amino acids alters claudin composition and improves tight junction barrier function.

Restriction of sulfur-containing amino acids (SCAA) has been shown to elicit a similar increase in life span and decrease in age-related morbidity as caloric restriction. The singular importance of epithelial barrier function in both physiological homeostasis and prevention of inflammation raised the issue of examining the effect of SCAA restriction on epithelial tight junction structure and permeability. Using a well-described in vitro, epithelial model, the LLC-PK(1) renal epithelial cell line, we studied the effects of SCAA restriction in culture medium. Reduction of methionine by 90%, cysteine by 50%, and total elimination of cystine resulted in dramatically lower intracellular pools of these amino acids and their metabolite, taurine, but the intracellular pools of the non-SCAA were all elevated. Cell growth and differentiation were maintained, and both confluent cell density and transepithelial short circuit current were unaffected. Certain tight junctional proteins, such as occludin and claudins-1 and -2 were not altered. However, claudins-3 and -7 were significantly decreased in abundance, whereas claudins-4 and -5 were markedly increased in abundance. The functional result of these structural changes was improved barrier function, as evidenced by increased transepithelial electrical resistance and decreased transepithelial (paracellular) diffusion of D-mannitol.

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.

Epithelial barriers, compartmentation, and cancer.

Epithelial cells, and the tight junctions between them, form a polarized barrier between luminal and serosal fluid compartments and segregate luminal growth factors from their basal-lateral receptors. Breakdown of this barrier should allow access of growth factors in the luminal fluid to their receptors on the basal-lateral cell membranes, as recently demonstrated for heregulin and erbB receptors in airway epithelia. It should also allow luminal growth factors to access the stroma. This property may have adaptive value for epithelial tissues in general, as an elegant response to injury, but may also promote cancer formation in premalignant epithelial tissues in which the tight junctions have become chronically leaky to growth factors.

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.

Phorbol ester treatment increases paracellular permeability across IEC-18 gastrointestinal epithelium in vitro.

The phorbol ester, TPA, transiently increases the transepithelial permeability across the gastrointestinal epithelium formed by IEC-18. There was a significant decrease in transepithelial resistance (R(T)) between 0 and 1.5 hr, accompanied by increased flux of polyethylene glycol (4000 MW), suggesting that the increase was across the tight junction. By 2 hr, the decrease in R(T) reversed and maintained control level. The transepithelial permeability increase was prevented by coincubation with the protein kinase C (PKC) inhibitor bisindolylmaleimide. There was a rapid (within 15 min) translocation of PKC-alpha from the cytosolic to the "membrane-associated" compartment, followed by a down-regulation that was detectable within 60 min of TPA treatment. The down-regulation of PKC-alpha from the membrane was prevented by either calpain inhibitor I or MG-132 and resulted in a sustained permeability increase. The permeability changes were not accompanied by significant effects on the amount or localization of the tight junctional proteins, occludin and ZO-1. However, occludin did show a reversible increase in phosphorylation with TPA treatment. Together these data support a role for PKC-alpha-mediated regulation of barrier permeability in an in vitro model of small intestinal epithelium, perhaps through modulation of the phosphorylation state of the tight junctional protein, occludin.

The transient increase of tight junction permeability induced by bryostatin 1 correlates with rapid downregulation of protein kinase C-alpha.

The role of PKC-alpha in altered epithelial barrier permeability following the activation of PKC by TPA (12-O-tetradecanoyl phorbol 13-acetate) and bryostatin 1 in LLC-PK1 cells was investigated in this study. Like TPA, bryostatin 1 binds to and activates PKC but unlike TPA, it is not a tumor promoter. TPA at 10(-7) M induced a sustained 95% decrease in transepithelial electrical resistance (R(t)) across LLC-PK1 epithelial cell sheets, while 10(-7) M bryostatin 1 caused only a 30% decrease in R(t), which spontaneously reversed after 5 h. Simultaneous exposure of cell sheets to 10(-7) M TPA and 10(-7) M bryostatin 1 blunted the increase in epithelial permeability observed with 10(-7) M TPA alone. Co-incubation of cell sheets with bryostatin 1 and MG-132, a proteasomal inhibitor, caused a further decrease in R(t) at the 6-h time point and inhibited the recovery in R(t) seen with bryostatin 1 alone at this time point. TPA caused a rapid translocation of PKC-alpha from the cytosol to the membrane of the cell where it remained elevated. Bryostatin 1 treatment resulted in a slower translocation of PKC-alpha from the cytosol to the membrane and a much more rapid downregulation of PKC-alpha, with disappearance from this compartment after only 6 h. The classical PKC inhibitor Go6976 prevented the decrease in R(t) seen with TPA. Treatment of cells with TPA and bryostatin 1 resulted in a PKC-alpha translocation and downregulation profile which more closely resembled that seen with bryostatin 1 alone. Co-incubation of cells with MG-132 and bryostatin 1 caused a slower downregulation of PKC-alpha from the membrane fraction. Bryostatin 1 treatment of cells expressing a dominant/negative form of PKC-alpha resulted in a slower and less extensive decrease in R(t) compared to the corresponding control cells. For both TPA and bryostatin 1, the level of PKC-alpha in the membrane-associated fraction of the treated cells correlated closely with increased transepithelial permeability. Due to its transient effect on tight junction permeability, bryostatin 1 offers a novel pharmacological tool to investigate junctional physiology.

Plasma concentrations of soluble tumor necrosis factor receptor I and tumor necrosis factor during cardiopulmonary bypass.

BACKGROUND: Tumor necrosis factor-alpha (TNF) has been implicated in the development of postoperative morbidity after cardiopulmonary bypass for myocardial revascularization. Despite their postulated roles as modulators of TNF bioavailability, soluble TNF receptors have not been characterized in patients undergoing this procedure and is the focus of this study. METHODS: Soluble tumor necrosis factor receptor I (sTNFRI) and TNF were measured by immunoassay in plasma samples collected from 36 patients at events before, during, and after cardiopulmonary bypass. RESULTS: Plasma concentrations of sTNFRI averaged 1.39 ng/mL at the start of the operation. Preoperative sTNFRI concentrations were found to significantly correlate with a preoperative morbidity assessment score, age, duration of bypass, duration of supplemental oxygen, and length of hospital stay. Plasma sTNFRI increased in all of the patients during the procedure. Plasma concentrations of sTNFRI and TNF did not correlate at any time. CONCLUSIONS: Preoperative measurement of sTNFRI could potentially serve as a reliable indicator for prophylactic treatment with an anti-TNF therapy. Such a therapeutic approach might help attenuate inflammatory processes thought to underlie postoperative morbidity associated with cardiopulmonary bypass.

Regulation of protein kinase C-delta and -epsilon isoforms by phorbol ester treatment of LLC-PK1 renal epithelia.

BACKGROUND: LLC-PK1 renal epithelia are a widely used model for proximal tubular physiology and differentiation. Protein kinase C (PKC) has been observed to play a role in both processes. This study examines the subcellular distribution and down-regulation of PKC-delta and PKC-epsilon isoforms in phorbol ester-treated LLC-PK1 epithelia. METHODS: Cells were treated with 10-7 mol/L 12-O-tetradecanoyl phorbol 13-acetate (TPA) for up to seven days and were extracted as total cell lysates as well as cytosolic, membrane-associated (Triton-X soluble) and a third (Triton-X insoluble) fraction. The expression and cellular localization of PKC-delta and PKC-epsilon isoforms were then detected using Western immunoblot and immunofluorescence. RESULTS: Based on the use of an anti-PKC-delta monoclonal antibody, TPA was observed to cause a rapid decrease in total PKC-delta content, which then returned to near control levels by seven days of treatment. Immunofluorescence indicated that PKC-delta had a cytoskeletal localization within the cells, and a subtle cytoskeletal rearrangement occurred upon exposure to TPA. Western immunoblots showed that PKC-delta did not undergo the expected membrane translocation upon activation by TPA, but simply disappeared immediately from the cytosolic compartment. Conventional cell fractionation procedures such as homogenization and Triton extraction prior to Western immunoblot will, however, fail to evaluate completely PKC-delta in LLC-PK1 epithelia because of the highly stringent measures necessary to extract PKC-delta from the cytoskeletal compartment of these cells. Furthermore, we observed that a second (polyclonal) PKC-delta antibody may recognize phosphorylated forms of PKC-delta, which went unrecognized by the other antibody. PKC-epsilon was present in the cytosol, membrane, and Triton-X-insoluble fractions of the cells. TPA treatment resulted in a partial translocation of PKC-epsilon to both the membrane and Triton-X-insoluble fractions of the cell, but total PKC-epsilon remained essentially unchanged. CONCLUSIONS: The present data indicate that the localization of PKC-delta and subsequent redistribution within the LLC-PK1 cells in response to TPA treatment is highly unique and distinct from that of PKC-epsilon and PKC-alpha. An important methodological finding is that one given antibody may not recognize all phosphoproteins of a given PKC isoform.

Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets.

Activation of protein kinase C by exposure of LLC-PK1 renal epithelial cells to 10(-7) M TPA, a tumor promoting phorbol ester, results in a rapid and sustained increase in paracellular permeability as evidenced by a decrease in transepithelial electrical resistance. Occludin, the first identified transmembrane protein to be localized to the tight junction of both epithelial and endothelial cells is thought play an important role in tight junction barriers. Although transepithelial electrical resistance fell to less than 20% of initial values within 1 hour of TPA exposure, transmission electron microscopy showed no change in the gross morphology of the tight junction of cells treated with 10(-7) M TPA for up to 2 hours. Immunofluorescence microscopy revealed a more rapid change in the membrane distribution of ZO-1 compared to occludin in the TPA-treated cells. Immunoblot analysis indicated that occludin levels in total cell lysates as well as cytosolic, membrane (Triton-X soluble) and cytoskeletal (Triton-X insoluble) fractions remained unchanged for at least 2 hours in cells treated with 10(-7) M TPA compared to their corresponding control cells. As the phosphorylation state of occludin is thought to be important in both tight junction assembly and regulation, the effect of phorbol ester treatment on the phosphorylation of occludin was investigated. Surprisingly, activation of protein kinase C with 10(-7) M TPA resulted in a time-dependent decrease in threonine phosphorylation of occludin which correlated closely with the rapid decrease in transepithelial electrical resistance. This dephosphorylation of occludin, occurring after activation of a serine/threonine kinase by TPA, suggested that protein kinase C was not acting directly on this tight junction target protein. If occludin dephosphorylation is involved in increasing tight junction permeability, then protein kinase C is apparently further upstream in the signaling pathway regulating epithelial barrier function, with a downstream serine/threonine phosphatase acting upon occludin.

Modification of tight junction function by protein kinase C isoforms.

The regulation of tight junction permeability by a variety of signal transduction pathways is summarized. An emphasis is placed on regulation of paracellular permeability by the protein kinase C family of isoforms, which involves the reporting of a large number of studies using the phorbol ester family of protein kinase C activators. The ability of protein kinase C activation to open epithelial barriers to a very wide range of solutes is emphasized, but then countered with discussion of the role of phorbol esters and protein kinase C activation in epithelial carcinogenesis. The ability of protein kinase C activation to enable growth factors to leak from luminal fluid compartments of epithelial tissues into lateral intercellular and interstitial fluid spaces may play a role in this carcinogenic action. An examination of protein kinase C effects on the phosphorylation states of tight junctional proteins suggests that downstream kinases and/or phosphatases mediate protein kinase C's effect on tight junction permeability. A role for protein kinase C in transepithelial drug delivery is questioned herein. The tight junctional leakiness associated with protein kinase C activation and apparently intrinsic to transformed epithelia suggests a potentially useful role for tight junction leakiness as a marker for early cancer diagnosis.

Increased tight junction permeability can result from protein kinase C activation/translocation and act as a tumor promotional event in epithelial cancers.

Exposure of LLC-PK1 epithelial cell cultures to phorbol ester tumor promoters causes immediate translocation of protein kinase C-alpha (PKC-alpha) from cytosolic to membrane-associated compartments. With a very similar time course, a dramatic and sustained increase in tight junctional (paracellular) permeability occurs. This increased permeability extends not only to salts and sugars but macromolecules as well. Fortyfold increases of transepithelial fluxes of biologically active EGF and insulin occur. Recovery of tight junction barrier function coincides with proteasomal downregulation of PKC-alpha. The failure to downregulate activated membrane-associated PKC-alpha has correlated with the appearance of multilayered cell growth and persistent leakiness of tight junctions. Accelerated downregulation of PKC-alpha results in only a partial and transient increase in tight junction permeability. Transfection of a dominant/negative PKC-alpha results in a slower increase in tight junction permeability in response to phorbol esters. In a separate study using rat colon, dimethylhydrazine (DMH)-induced colon carcinogenesis has been preceded by linear increases in both the number of aberrant crypts and transepithelial permeability, as a function of weeks of DMH treatment. Adenocarcinomas of both rat and human colon have been found to have uniformly leaky tight junctions. Whereas most human colon hyperplastic and adenomatous polyps contain nonleaky tight junctions, adenomatous polyps with dysplastic changes did possess leaky tight junctions. Our overall hypothesis is that tight junctional leakiness is a late event in epithelial carcinogenesis but will allow for growth factors in luminal fluid compartments to enter the intercellular and interstitial fluid spaces for the first time, binding to receptors that are located on only the basal-lateral cell surface, and causing changes in epithelial cell kinetics. Tight junctional leakiness is therefore a promotional event that would be unique to epithelial cancers.

The impact of antidepressants on sleep and anxiety: a comparative study of fluoxetine and dothiepin using the Leeds Sleep Evaluation Questionnaire.

Fluoxetine (20 mg) was compared to dothiepin (150 mg) in a multicentre, prospective, double blind, randomised clinical trial involving 125 patients with major depression treated for an initial phase of 6 weeks and then followed up for a further 6 months. There was no difference in the efficacy of the two drugs based on the results of established rating scales (MADRS, HAM-D, BPRS). The impact of both drugs on sleep measured using the Leeds Sleep Evaluation Questionnaire showed no significant differences between treatments, however drowsiness and disturbed sleep were reported more frequently as side effects with dothiepin. Symptoms of anxiety responded equally well to both treatments. The short term and long term tolerability of dothiepin was inferior to that of fluoxetine. The place of dothiepin in treatment should be reassessed in the light of its anticholinergic adverse event profile, particularly in the elderly. Copyright 2000 John Wiley & Sons, Ltd.