Mast cell tryptase controls paracellular permeability of the intestine: role of protease-activated receptor 2 and beta-arrestins

Tight junctions between intestinal epithelial cells prevent ingress of luminal macromolecules and bacteria and protect against inflammation and infection. During stress and inflammation, mast cells mediate increased mucosal permeability by unknown mechanisms. We hypothesized that mast cell tryptase cleaves protease-activated receptor 2 (PAR2) on colonocytes to increase paracellular permeability. Colonocytes expressed PAR2 mRNA and responded to PAR2 agonists with increased [Ca2+]i. Supernatant from degranulated mast cells increased [Ca2+]i in colonocytes, which was prevented by a tryptase inhibitor, and desensitized responses to PAR2 agonist, suggesting PAR2 cleavage. When applied to the basolateral surface of colonocytes, PAR2 agonists and mast cell supernatant decreased transepithelial resistance, increased transepithelial flux of macromolecules, and induced redistribution of tight junction ZO-1 and occludin and perijunctional F-actin. When mast cells were co-cultured with colonocytes, mast cell degranulation increased paracellular permeability of colonocytes. This was prevented by a tryptase inhibitor. We determined the role of ERK1/2 and of beta-arrestins, which recruit ERK1/2 to PAR2 in endosomes and retain ERK1/2 in the cytosol, on PAR2-mediated alterations in permeability. An ERK1/2 inhibitor abolished the effects of PAR2 agonist on permeability and redistribution of F-actin. Down-regulation of beta-arrestins with small interfering RNA inhibited PAR2-induced activation of ERK1/2 and suppressed PAR2-induced changes in permeability. Thus, mast cells signal to colonocytes in a paracrine manner by release of tryptase and activation of PAR2. PAR2 couples to beta-arrestin-dependent activation of ERK1/2, which regulates reorganization of perijunctional F-actin to increase epithelial permeability. These mechanisms may explain the increased epithelial permeability of the intestine during stress and inflammation.

Cyclodextrin-mediated enhancement of riboflavin solubility and corneal permeability

Cyclodextrins are water-soluble cyclic oligosaccharides consisting of six, seven, and eight α-(1,4)-linked glucopyranose subunits. This study reports the use of different cyclodextrins in eye drop formulations to improve the aqueous solubility and corneal permeability of riboflavin. Riboflavin is a poorly soluble drug with a solubility up to 0.08 mg mL–1 in deionized water. It is used as a drug topically administered to the eye to mediate UV-induced corneal cross-linking in the treatment of keratoconus. Aqueous solutions of β-cyclodextrin (10–30 mg mL–1) can enhance the solubility of riboflavin up to 0.12–0.19 mg mL–1, whereas the higher concentration of α-cyclodextrin (100 mg mL–1) achieved a lower level of enhancement of 0.11 mg mL–1. The other oligosaccharides were found to be inefficient for this purpose. In vitro diffusion experiments performed with fresh and cryopreserved bovine cornea have demonstrated that β-cyclodextrin enhances riboflavin permeability. The mechanism of this enhancement was examined through microscopic histological analysis of the cornea and is discussed in this paper.

(PDF, 406 KB) On L3 acquisition and phonological permeability: a new test case for debates on the mental representation of non-native phonological systems

The goal of this article is to make an epistemological and theoretical contribution to the nascent field of third language (L3) acquisition and show how examining L3 development can offer a unique view into longstanding debates within L2 acquisition theory. We offer the Phonological Permeability Hypothesis (PPH), which maintains that examining the development of an L3/Ln phonological system and its effects on a previously acquired L2 phonological system can inform contemporary debates regarding the mental constitution of postcritical period adult phonological acquisition. We discuss the predictions and functional significance of the PPH for adult SLA and multilingualism studies, detailing a methodology that examines the effects of acquiring Brazilian Portuguese on the Spanish phonological systems learned before and after the so-called critical period (i.e., comparing simultaneous versus successive adult English-Spanish bilinguals learning Brazilian Portuguese as an L3).

Enhancement in corneal permeability of riboflavin using calcium sequestering compounds

Ethylenediaminetetraacetic acid, ethylenediamine-N,N′-disuccinic acid and ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid are polyaminocarboxylic acids that are able to sequester metal ions. Calcium is implicated in maintenance of intercellular matrix, zonula occludens (tight junctions) and zonula adherens of epithelium and endothelium cells. Corneal epithelium is impervious to many aqueous formulations due to it being lipophilic, whereby transcellular drug transit is resisted, whilst tight junctions restrict access via the paracellular route. Research has shown that integrity of tight junctions breaks down through loss of Ca2+ for endothelial and epithelial cells. This study investigates different Ca2+ sequestering compounds and their effect on corneal permeability of riboflavin at physiological pH. Riboflavin is a topically administered ocular drug applied during UV-induced corneal cross-linking for the treatment of keratoconus.

Impact of the enhanced permeability and retention (EPR) effect and cathepsins levels on the activity of polymer-drug conjugates

Polymer-drug conjugates have demonstrated clinical potential in the context of anticancer therapy. However, such promising results have, to date, failed to translate into a marketed product. Polymer-drug conjugates rely on two factors for activity: (i) the presence of a defective vasculature, for passive accumulation of this technology into the tumour tissue (enhanced permeability and retention (EPR) effect) and (ii) the presence of a specific trigger at the tumour site, for selective drug release (e.g., the enzyme cathepsin B). Here, we retrospectively analyse literature data to investigate which tumour types have proved more responsive to polymer-drug conjugates and to determine correlations between the magnitude of the EPR effect and/or expression of cathepsin B. Lung, breast and ovarian cancers showed the highest response rate (30%, 47% and 41%, respectively for cathepsin-activated conjugates and 31%, 43%, 40%, across all conjugates). An analysis of literature data on cathepsin content in various tumour types showed that these tumour types had high cathepsin content (up to 3835 ng/mg for lung cancer), although marked heterogeneity was observed across different studies. In addition, these tumour types were also reported as having a high EPR effect. Our results suggest that a pre-screening of patient population could bring a more marked clinical benefit.

Drug permeability in 16HBE14o- airway cell layers correlates with absorption from the isolated perfused rat lung

The permeability of the lung is critical in determining the disposition of inhaled drugs and the respiratory epithelium provides the main physical barrier to drug absorption. The 16HBE14o- human bronchial epithelial cell line has been developed recently as a model of the airway epithelium. In this study, the transport of 10 low molecular weight compounds was measured in the 16HBE14o- cell layers, with apical to basolateral (absorptive) apparent permeability coefficients (P(app)) ranging from 0.4 x 10(-6)cms(-1) for Tyr-D-Arg-Phe-Phe-NH(2) to 25.2x10(-6)cms(-1) for metoprolol. Permeability in 16HBE14o- cells was found to correlate with previously reported P(app) in Caco-2 cells and absorption rates in the isolated perfused rat lung (k(a,lung)) and the rat lung in vivo (k(a,in vivo)). Log linear relationships were established between P(app) in 16HBE14o- cells and P(app) in Caco-2 cells (r(2)=0.82), k(a,lung) (r(2)=0.78) and k(a,in vivo) (r(2)=0.68). The findings suggest that permeability in 16HBE14o- cells may be useful to predict the permeability of compounds in the lung, although no advantage of using the organ-specific cell line 16HBE14o- compared to Caco-2 cells was found in this study.

The permeability parameter of the outer membrane of pseudomonas aeruginosa Varies with the concentration of a test substrate, cephalosporin C

The permeability parameter (C) for the movement of cephalosporin C across the outer membrane of Pseudomonas aeruginosa was measured using the widely accepted method of Zimmermann & Rosselet. In one experiment, the value of C varied continuously from 4·2 to 10·8 cm3 min-1 (mg dry wt cells)-1 over a range of concentrations of the test substrate, cephalosporin C, from 50 to 5 μm. Dependence of C on the concentration of test substrate was still observed when the effect of a possible electric potential difference across the outer membrane was corrected for. In quantitative studies of β-lactam permeation the dependence of C on the concentration of β-lactam should be taken into account.

Gut permeability in autism spectrum disorders

Objective To test whether gut permeability is increased in autism spectrum disorders (ASD) by evaluating gut permeability in a population-derived cohort of children with ASD compared with age- and intelligence quotient-matched controls without ASD but with special educational needs (SEN). Patients and Methods One hundred thirty-three children aged 10–14 years, 103 with ASD and 30 with SEN, were given an oral test dose of mannitol and lactulose and urine collected for 6 hr. Gut permeability was assessed by measuring the urine lactulose/mannitol (L/M) recovery ratio by electrospray mass spectrometry-mass spectrometry. The ASD group was subcategorized for comparison into those without (n = 83) and with (n = 20) regression. Results There was no significant difference in L/M recovery ratio (mean (95% confidence interval)) between the groups with ASD: 0.015 (0.013–0.018), and SEN: 0.014 (0.009–0.019), nor in lactulose, mannitol, or creatinine recovery. No significant differences were observed in any parameter for the regressed versus non-regressed ASD groups. Results were consistent with previously published normal ranges. Eleven children (9/103 = 8.7% ASD and 2/30 = 6.7% SEN) had L/M recovery ratio > 0.03 (the accepted normal range cut-off), of whom two (one ASD and one SEN) had more definitely pathological L/M recovery ratios > 0.04. Conclusion There is no statistically significant group difference in small intestine permeability in a population cohort-derived group of children with ASD compared with a control group with SEN. Of the two children (one ASD and one SEN) with an L/M recovery ratio of > 0.04, one had undiagnosed asymptomatic celiac disease (ASD) and the other (SEN) past extensive surgery for gastroschisis.

Effect of blood contamination on the shear bond strength at resin/dentin interface in primary teeth

Purpose: To assess in vitro the shear bond strength at the resin/dentin interface in primary teeth after contamination with fresh human blood. Methods: 75 crowns of primary molars were embedded in acrylic resin and mechanically ground to expose a flat dentin surface. The specimens were randomly assigned to five groups (n=15), according to the surface treatment. Group I (control) had no blood contamination. The other groups were blood-contaminated and subjected to different post-contamination protocols: in Group 2, the surfaces were rinsed with water; in Group 3, the surfaces were air-dried; in Group 4, the surfaces were rinsed and air-dried; and in Group 5, no post-contamination treatment was done. In all groups, a 3-mm dentin bonding site was demarcated, Single Bond adhesive system was applied and resin composite cylinders were bonded. After 24 hours in distilled water, shear bond strength was tested at a crosshead speed of 0.5 mm/minute. Results: Means (in MPa) were: Group 1: 7.1 (+/- 4.2); Group 2: 4.0 (+/- 1.8); Group 3: 0.9 (+/- 0.7); Group 4: 3.9 (+/- 2.2) and Group 5: 1.3 (+/- 1.5). Data were analyzed statistically by the Kruskal-Wallis test at 5% significance level. Groups 2 and 4 were similar to each other (P > 0.05) and both ware similar to Group 1 (P > 0.05). These groups (2, 3 and 4) had statistically significantly higher bond strengths than Groups 3 and 5 (P < 0.05). Blood contamination negatively affected the shear bond strength to primary tooth dentin. Among the blood-contaminated groups, water-rinsed specimens had higher bond strengths than those that were exclusively air-dried or not submitted to any post-contamination protocol before adhesive application.

Shear strength of the bond to primary dentin: influence of Er:YAG laser irradiation distance

The aim of this study was to assess in vitro the influence of Er:YAG laser irradiation distance on the shear strength of the bond between an adhesive restorative system and primary dentin. A total of 60 crowns of primary molars were embedded in acrylic resin and mechanically ground to expose a flat dentin surface and were randomly assigned to six groups (n = 10). The control group was etched with 37% phosphoric acid. The remaining five groups were irradiated (80 mJ, 2 Hz) at different irradiation distances (11, 12, 16, 17 and 20 mm), followed by acid etching. An adhesive agent (Single Bond) was applied to the bonding sites, and resin cylinders (Filtek Z250) were prepared. The shear bond strength tests were performed in a universal testing machine (0.5 mm/min). Data were submitted to statistical analysis using one-way ANOVA and the Kruskal-Wallis test (p < 0.05). The mean shear bond strengths were: 7.32 +/- 3.83, 5.07 +/- 2.62, 6.49 +/- 1.64, 7.71 +/- 0.66, 7.33 +/- 0.02, and 9.65 +/- 2.41 MPa in the control group and the groups irradiated at 11, 12, 16, 17, and 20 mm, respectively. The differences between the bond strengths in groups II and IV and between the bond strengths in groups II and VI were statistically significant (p < 0.05). Increasing the laser irradiation distance resulted in increasing shear strength of the bond to primary dentin.

Effect of different bleaching systems on the ultrastructure of bovine dentin

The aim of this study was to evaluate in vitro the effect of different in-office bleaching systems on the surface morphology of bovine dentin. Thirty tooth fragments measuring 4 x 4mm, containing enamel and dentin, were obtained from the crowns of extracted bovine incisors. Samples were subjected to simulated intracoronal bleaching techniques using conventional (Opalescence Endo (R) and Whiteness Super Endo (R)) and light-activated systems (Opalescence Xtra (R) and Whiteness HP Maxx (R)). Controls were treated with either sodium perborate mixed with 10% hydrogen peroxide or no bleaching agent. The samples were observed under SEM and the recorded images were evaluated for topographic alterations. The ultrastructural alterations of dentin observed in this study varied greatly between groups according to the products used. Higher pH products (Whiteness HP Maxx (R) and Opalescence Xtra (R)) associated with in-office techniques yielded better maintenance of dentin ultrastructure. Apparently, both low pH and hydrogen peroxide oxidation play a role in altering the ultrastructure of dentin during internal dental bleaching. The use of alkaline products with reduced time of application (in-office techniques) may decrease such morphological alterations.

Adhesion to Er : YAG laser-prepared dentin after long-term water storage and thermocycling

This in vitro study evaluated the microtensile bond strength of a resin composite to Er:YAG-prepared dentin after long-term storage and thermocycling. Eighty bovine incisors were selected and their roots removed. The crowns were ground to expose superficial dentin. The samples were randomly divided according to cavity preparation method (I-Er:YAG laser and II-carbide bur). Subsequently, an etch & rinse adhesive system was applied and the samples were restored with a resin composite. The samples were subdivided according to time of water storage (WS)/number of thermocycles (TC) performed: A) 24 hours WS/no TC; B) 7 days WS/500 TC; C) 1 month WS/2,000 TC; D) 6 months WS/12,000 TC. The teeth were sectioned in sticks with a cross-sectional area of 1.0-mm(2), which were loaded in tension in a universal testing machine. The data were subjected to two-way ANOVA, Scheffe and Fisher`s tests at a 5% level. In general, the bur-prepared group displayed higher microtensile bond strength values than the laser-treated group. Based on one-month water storage and 2,000 thermocycles, the performance of the tested adhesive system to Er:YAG-laser irradiated dentin was negatively affected (Group IC), while adhesion of the bur-prepared group decreased only within six months of water storage combined with 12,000 thermocycles (Group IID). It may be concluded that adhesion to the Er:YAG laser cavity preparation was more affected by the methods used for simulating degradation of the adhesive interface.

Influence of dentin cavity surface finishing on micro-tensile bond strength of adhesives

The current trend toward minimal-invasive dentistry has introduced innovative techniques for cavity preparation. Chemical vapor deposition (CVD) and laser-irradiation technology have been employed as an alternative to the common use of regular burs in high-speed turbines. Objectives. The purpose of this study was to assess the influence of alternative techniques for cavity preparation on the bonding effectiveness of different adhesives to dentin, and to evaluate the morphological characteristics of dentin prepared with those techniques. Methods. One etch&rinse adhesive (OptiBond FL, Kerr) and three self-etch systems (Adper Prompt L-Pop, 3M ESPE; Clearfil SE Bond, Kuraray; Clearfil S3 Bond, Kuraray) were applied on dentin prepared with a regular bur in a turbine, with a CVD bur in a turbine, with a CVD tip in ultrasound and with an ErCr:YSGG laser. The micro-tensile bond strength (mu TBS) was determined after storage in water for 24 h at 37 degrees C, and morphological evaluation was performed by means of field -emission -gun scanning electron microscopy (Feg-SEM). Results. Feg-SEM evaluation revealed different morphological features on the dentin surface after the usage of both the conventional and alternative techniques for cavity preparation, more specifically regarding smear-layer thickness and surface roughness. CVD bur-cut, CVD ultra-sonoabraded and laser-irradiated dentin resulted in lower mu TBSs than conventionally bur-cut dentin, irrespective of the adhesive employed. Significance. The techniques, such as CVD diamond-bur cutting, CVD diamond ultra-sonoabrasion and laser-irradiation, used for cavity preparation may affect the bonding effectiveness of adhesives to dentin, irrespective of their acidity or approach. (C) 2007 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Effect of chemical treatment on the mechanical properties, water vapour permeability and sorption isotherms of gelatin-based films

Proteins contain hydrophilic groups, which can bind to water molecules through hydrogen bridges, resulting in water vapour adsorption. An increase in the degree of cross-linking can be a method to improve the cohesiveness force and functional properties of protein-based films. Thus, the objective of this work was to evaluate the effect of chemical treatment of gelatin with formaldehyde and glyoxal on the mechanical properties, water vapour permeability (WVP) and water vapour sorption characteristics of gelatin-based films. Films were produced using gelatin, with and without chemical treatment. The formaldehyde treatments caused a significant increase in the tensile strength and a reduction in the WVP of films. The Guggenheim-Anderson-De Boer and Halsey models could be used to model the sorption isotherms of films. It was observed that an increase in temperature produced a decrease in water sorption, and the chemical modifications did not affect the monolayer moisture content. Copyright (c) 2007 John Wiley & Sons, Ltd.

Influence of Er,Cr : YSGG laser treatment on the microtensile bond strength of adhesives to dentin

Purpose: In light of the concept of minimally invasive dentistry, erbium lasers have been considered as an alternative technique to the use of diamond burs for cavity preparation. The purpose of this study was to assess the bonding effectiveness of adhesives to Er,Cr:YSGG laser-irradiated dentin using irradiation settings specific for cavity preparation. Materials and Methods: Fifty-four midcoronal dentin surfaces, obtained from sound human molars, were irradiated with an Er,Cr:YSGG laser or prepared with a diamond bur using a high-speed turbine. One etch-and-rinse (Optibond FL/Kerr) and three self-etching adhesives (Adper Prompt L-Pop/3M ESPE, Clearfil SE Bond/Kuraray, and Clearfil S-3 Bond/Kuraray) were used to bond the composite to dentin. The microtensile bond strength (mu TBS) was determined after 24 h of storage in water at 37 degrees C. The Kruskal-Wallis test was used to determine pairwise statistical differences (p < 0.05). Prepared dentin surfaces, adhesive interfaces, and failure patterns were analyzed using a stereo microscope and Field-emission gun Scanning Electron Microscopy (Feg-SEM). Results: Significantly lower mu TBS was observed to laser-irradiated than to bur-cut dentin (p < 0.05), irrespective of the adhesive employed. Feg-SEM photomicrographs of lased dentin revealed an imbricate patterned substrate and the presence of microcracks at the dentin surface. Conclusion: Morphological alterations produced by Er,Cr:YSGG laser-irradiation adversely influence the bonding effectiveness of adhesives to dentin. Keywords: dentin, adhesion, adhesives, laser, ErCr:YSGG.