Changes In Barrier Function Research Articles

Sebaceous Gland, Hair Shaft, and Epidermal Barrier Abnormalities in Keratosis Pilaris with and without Filaggrin Deficiency

Although keratosis pilaris (KP) is common, its etiopathogenesis remains unknown. KP is associated clinically with ichthyosis vulgaris and atopic dermatitis and molecular genetically with filaggrin-null mutations. In 20 KP patients and 20 matched controls, we assessed the filaggrin and claudin 1 genotypes, the phenotypes by dermatoscopy, and the morphology by light and transmission electron microscopy. Thirty-five percent of KP patients displayed filaggrin mutations, demonstrating that filaggrin mutations only partially account for the KP phenotype. Major histologic and dermatoscopic findings of KP were hyperkeratosis, hypergranulosis, mild T helper cell type 1-dominant lymphocytic inflammation, plugging of follicular orifices, striking absence of sebaceous glands, and hair shaft abnormalities in KP lesions but not in unaffected skin sites. Changes in barrier function and abnormal paracellular permeability were found in both interfollicular and follicular stratum corneum of lesional KP, which correlated ultrastructurally with impaired extracellular lamellar bilayer maturation and organization. All these features were independent of filaggrin genotype. Moreover, ultrastructure of corneodesmosomes and tight junctions appeared normal, immunohistochemistry for claudin 1 showed no reduction in protein amounts, and molecular analysis of claudin 1 was unremarkable. Our findings suggest that absence of sebaceous glands is an early step in KP pathogenesis, resulting in downstream hair shaft and epithelial barrier abnormalities.

Development of an in vitro model for studying the penetration of chemicals through compromised skin

The conventional safety approach that includes dermal absorption of pharmaceutical or consumer products uses models that are based on intact skin. However, when products are intended for application to skin with a less effective barrier, such as in new-born infants, or in cases where the skin is mildly damaged or diseased, there are instances where absorption through compromised skin is also important. A tape stripping procedure was investigated using dermatomed pig skin to assess if an in vitro model could replicate the typical changes in barrier function observed in humans with compromised skin. The relationship between Trans-Epidermal Water Loss (TEWL), Electrical Resistance (ER) and Tritiated Water Flux (TWF), markers of skin barrier function in OECD 428 studies was investigated. There was a step-wise reduction in ER from normal (control) skin following 5, 10, 15 or 20 tape strips. This was mirrored by increases in both TWF and TEWL. An in vitro experimental protocol using 5 tape strips, ER and dermatomed pig skin provided a rapid, robust and reproducible approach equivalent to the 3–4-fold increases in TEWL observed clinically in compromised skin.

A longitudinal study of skin barrier function in pregnancy and the postnatal period.

It is unknown whether skin's barrier function changes in pregnancy. Trans Epidermal Water Loss (TEWL) refers to the total amount of water loss through the skin and TEWL can be measured non-invasively as an index of skin barrier function. We measured TEWL during and after pregnancy to evaluate pregnancy-related skin barrier function. This was a prospective, longitudinal cohort study of 52 low-risk, first-time pregnant women nested within the Screening for Pregnancy Endpoints (SCOPE) Ireland study. TEWL (gwater/m2/h) was measured three times during pregnancy: 19-21 weeks, 27-32 weeks and 36 weeks; and three times postnatally: 2-4 days, 2 months and 6 months post-delivery. Data were analysed using SPSS 18.0 and P > 0.05 was considered statistically significant. A rise in TEWL was seen between each visit with the highest readings, exceeding the normal range of 0-20 gwater/m(2)/h, recorded at two months post-delivery. Forty women attended at two months post-delivery of whom 22 women had an average reading between 0 and 20 gwater/m(2)/h; 10 women had an average reading between 21 and 40 gwater/m(2)/h and 8 women had an average reading between 41 and 75 gwater/m(2)/h. Readings had returned to an average of 0-20 gwater/m(2)/h at six months postnatally. TEWL increases slightly in pregnancy and the postnatal period. The clinical significance of this is unclear and requires further investigation.

Downregulation of Expression of Xenobiotic Efflux Genes is Associated with Pelvic Pouch Inflammation in Ulcerative Colitis

As many as 50% of patients with ulcerative colitis who have undergone ileal pouch-anal anastomosis develop de novo inflammation in the ileal pouch after surgery. With the use of microarray technology, we investigated what gene expression changes occur in the pelvic pouch after surgery for ulcerative colitis and how these changes vary by pouch outcome. Patients who had undergone ileal pouch-anal anastomosis and closure of ileostomy had biopsy specimens from the pouch and pre-pouch ileum prospectively collected. The subjects were allocated into 4 outcome groups: no pouchitis, pouchitis, Crohn's disease-like phenotype, and familial adenomatous polyposis controls. RNA was extracted and transcriptomes were analyzed using a genome-wide approach. The statistical significance of each gene was assessed, and raw P values were corrected for multiple comparisons. The expression levels of 2733 transcripts in the pouch were significantly associated with outcome. These genes could be classified into 3 categories: regulation of the immune system, modification of the extracellular matrix, and xenobiotic activity. Contrary to the first 2 categories, genes involved in xenobiotic activity, such as ABCB1, had lower expression in the pouchitis and Crohn's disease-like groups compared with the no pouchitis and familial adenomatous polyposis groups. Transporters of compounds including xenobiotics are downregulated in recurrent disease after ileal pouch-anal anastomosis, whereas inflammatory pathways are upregulated. These findings corroborate the hypothesis that changes in barrier function could contribute to development of intestinal inflammation.

Variable roles of the H1‐H3 receptors and PKC in histamine‐induced barrier dysfunction in cultured endothelial cells from different sources (672.6)

We tested the hypothesis that histamine‐induced endothelial barrier dysfunction requires different receptors and signaling pathways depending upon endothelial cell (EC) source. Changes in barrier function elicited by 10 μM histamine were compared between human umbilical vein EC (HUVEC) and human dermal microvascular EC (HMVEC‐D) monolayers, using determination of transendothelial electrical resistance (TER). H1, H2, and H3 receptors were blocked with 10 μM mepyramine, 10 μM cimetidine, and 0.01‐1 μM ciproxifan, respectively. Inhibitors of PKC (GFX109203X), PI3K (PI828) and phospholipase C (U73122) were used to evaluate downstream signals. Western blots and immunofluorescence microscopy analysis demonstrated H1, H2, and H3 expression in both EC types. Blockade of H1, H2, or H3 significantly inhibited the histamine‐induced decrease in TER in HMVEC‐D. In contrast, only blockade of H1 significantly attenuated the histamine‐induced drop in TER in HUVEC. Inhibition of either PKC or PI3K significantly attenuated histamine‐induced barrier dysfunction in HUVEC, but combined inhibition of both was required to attain significant attenuation in HMVEC‐D. Inhibition of PLC also inhibited the histamine‐induced decrease in TER in HMVEC‐D, but interestingly, combined PLC and PKC inhibition did not. This study suggests a common role for H1, but differential roles for H2 and H3 in histamine‐induced endothelial barrier dysfunction among different endothelial cell types. In addition, PKC appears to have either a promoting or inhibitory role depending on the endothelial cell source. Supported by NIH R01HL098215.Grant Funding Source: NIH R01HL098215

The Expression of the Nectin Complex in Human Breast Cancer and the Role of Nectin-3 in the Control of Tight Junctions during Metastasis

IntroductionNectins are a family of integral protein molecules involved in the formation of functioning Adherens and Tight Junctions (TJ). Aberrant expression is associated with cancer progression but little is known how this effects changes in cell behaviour. This study aimed to ascertain the distribution of Nectins-1 to -4 in human breast cancer and the effect on junctional integrity of Nectin-3 modulation in human endothelial and breast cancer cells.MethodsA human breast tissue cohort was processed for Q-PCR and immunohistochemistry for analysis of Nectin-1/-2/-3/-4. Nectin-3 over-expression was induced in the human breast cancer cell line MDA-MB-231 and the human endothelial cell line HECV. Functional testing was carried out to ascertain changes in cell behaviour.ResultsQ-PCR revealed a distinct reduction in node positive tumours and in patients with poor outcome. There was increased expression of Nectin-1/-2 in patients with metastatic disease, Nectin-3/-4 was reduced. IHC revealed that Nectin-3 expression showed clear changes in distribution between normal and cancerous cells. Nectin-3 over-expression in MDA-MB-231 cells showed reduced invasion and migration even when treated with HGF. Changes in barrier function resulted in MDAN3 cells showing less change in resistance after 2h treatment with HGF (p<0.001). Nectin-3 transformed endothelial cells were significantly more adhesive, irrespective of treatment with HGF (p<0.05) and had reduced growth. Barrier function revealed that transformed HECV cells had significantly tighter junctions that wildtype cells when treated with HGF (p<0.0001). HGF-induced changes in permeability were also reduced. Overexpression of Nectin-3 produced endothelial cells with significantly reduced ability to form tubules (p<0.0001). Immunoprecipitation studies discovered hitherto novel associations for Nectin-3. Moreover, HGF appeared to exert an effect on Nectin-3 via tyrosine and threonine phosphorylation.ConclusionsNectin-3 may be a key component in the formation of cell junctions and be a putative suppressor molecule to the invasion of breast cancer cells.

Role of Vascular and Lymphatic Endothelial Cells in Hantavirus Pulmonary Syndrome Suggests Targeted Therapeutic Approaches

Hantaviruses in the Americas cause a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). Hantaviruses nonlytically infect microvascular and lymphatic endothelial cells and cause dramatic changes in barrier functions without disrupting the endothelium. Hantaviruses cause changes in the function of infected endothelial cells that normally regulate fluid barrier functions. The endothelium of arteries, veins, and lymphatic vessels are unique and central to the function of vast pulmonary capillary beds that regulate pulmonary fluid accumulation. We have found that HPS-causing hantaviruses alter vascular barrier functions of microvascular and lymphatic endothelial cells by altering receptor and signaling pathway responses that serve to permit fluid tissue influx and clear tissue edema. Infection of the endothelium provides several mechanisms for hantaviruses to cause acute pulmonary edema, as well as potential therapeutic targets for reducing the severity of HPS disease. Here we discuss interactions of HPS-causing hantaviruses with the endothelium, roles for unique lymphatic endothelial responses in HPS, and therapeutic targeting of the endothelium as a means of reducing the severity of HPS disease.

Vitrigel-EIT (eye irritancy test) methods provide a brief assay system for estimating the changes of barrier function and architecture after exposing test chemicals to a human corneal epithelium model

Vitrigel-EIT (eye irritancy test) methods provide a brief assay system for estimating the changes of barrier function and architecture after exposing test chemicals to a human corneal epithelium model

Changes in villus‐like projections of corneocytes from the facial skin in normal infants with or without infantile eczema; useful parameter to assess barrier function

Skin barrier function changes rapidly during the first year of life in infants. Measuring skin barrier function in infants is difficult because of the burden on patients and their parents or guardians. A simple method for assessing barrier function in infants that can be performed quickly and would enable swift treatment and skin care management. We used laser microscopy to investigate villus-like projections (VPs) observed on the lower surface of corneocytes obtained on tape strips from 18 normal Japanese infants at the ages of 1, 3, and 6 months. We also performed transepidermal water loss and corneocyte size measurements. A dermatologist clinically assessed the cutaneous findings. We rapidly measured skin barrier function changes during the first 6 months of life in infants. During the study period, very slight erythema and pityriasis were the primary reasons for skin rash, which were generally most severe at 3 months. The results showed that barrier function declined at approximately 3 months of age in nearly all subjects. Therefore, we found that normal barrier function of infantile skin becomes temporarily impaired after birth. We conclude that even in patients with very mild skin rashes, multiple objective indices of skin barrier function deteriorate simultaneously. Observation of VPs on the surface of corneocytes using laser microscopy is a rapid, painless, and effective method of monitoring skin barrier function in infants, and VP score offers a useful index for assessing barrier function.

Sertoli Cell Is a Potential Target for Perfluorooctane Sulfonate–Induced Reproductive Dysfunction in Male Mice

Perfluorooctane sulfonate (PFOS) is associated with male reproductive disorders, but its targets and mechanisms are poorly understood. We used in vitro and in vivo models to explore the roles of Sertoli cells and the blood-testis barrier (BTB) in PFOS-induced male reproductive dysfunction. First, we used primary Sertoli cell to estimate PFOS-induced cytotoxicity, junction proteins expression, and the changes of barrier function. ICR mice were then administered PFOS (0.25-50mg/kg/day) for 4 weeks. Sperm count, ultrastructure and permeability of the Sertoli cell-based BTB, and testicular PFOS were estimated. Furthermore, the expression and localization of proteins related to junctions between Sertoli cells and mitogen-activated protein kinase (MAPK) signaling pathway were evaluated. Apparent decreases in sperm count were found. PFOS significantly increased vacuolization in Sertoli cells in seminiferous tubules and BTB ultrastructural disassembly, which subsequently increased BTB permeability and testicular PFOS levels, which was confirmed by in vitro results that PFOS decreased transepithelial electrical resistance between Sertoli cells. Additionally, PFOS decreased the expression of junction proteins in Sertoli cells, which was further confirmed by in vivo results that PFOS decreased or dislocated junction proteins (i.e., ZO-1, occludin, claudin-11, and connexin-43) and increased proteins related to the MAPK signaling pathway (i.e., Erk and p38), whereas basal ectoplasmic specialization proteins did not change. The results were confirmed by SB203580, a p38 MAPK selective inhibitor. Sertoli cells appear to be a new cellular target for PFOS. Together with disruption of BTB integrity and function, these cells play an important role in PFOS-induced male reproductive toxicity.

Comparison of PLGA and lecithin/chitosan nanoparticles for dermal targeting of betamethasone valerate

Poly(lactide-co-glycolide) (PLGA) and lecithin/chitosan (LC) nanoparticles were prepared to evaluate the difference in the behavior upon administration on skin, for steroidal treatment. For this purpose, betamethasone-17-valerate (BMV)-loaded nanoparticles with a narrow size distribution and high entrapment efficiency were prepared. Permeation studies showed that both polymeric nanoparticles enhanced the amount of BMV in epidermis, which is the target site of topical steroidal treatment, when compared with commercial formulation. 1.58-Fold increase was determined in the epidermis concentration of BMV by LC nanoparticles with respect to PLGA nanoparticles. Nanoparticles were diluted in chitosan gel (10%, w/w) to prepare suitable formulation for topical application. Accumulation from both gel formulations were found significantly higher than commercial formulation in skin layers (p < 0.05). In addition, pharmacodynamic responses were also investigated as anti-inflammatory and skin-blanching parameters. Both formulations significantly improved these parameters although they contained 10 times less amount of BMV than commercial cream. Moreover, TEWL measurement exhibited no barrier function changes upon the application of nanoparticles on skin. Overall, both nanoparticles improved the localization of BMV within skin layers; but when compared with PLGA nanoparticles, the LC nanoparticles could be classified as a better candidate for topical delivery vehicle in the treatment of various dermatological inflammatory diseases.

Enhanced dermal delivery of diflucortolone valerate using lecithin/chitosan nanoparticles: in-vitro and in-vivo evaluations

The objective of this study was to prepare a suitable formulation for dermal delivery of diflucortolone valerate (DFV) that would maintain the localization in skin layers without any penetration and to optimize efficiency of DFV. Drug-loaded lecithin/chitosan nanoparticles with high entrapment efficiency (86.8%), were successfully prepared by ionic interaction technique. Sustained release of DFV was achieved without any initial burst release. Nanoparticles were also incorporated into chitosan gel at different ratios for preparing a more suitable formulation for topical drug delivery with adequate viscosity. In ex-vivo permeation studies, nanoparticles increased the accumulation of DFV especially in the stratum corneum + epidermis of rat skin without any significant permeation. Retention of DFV from nanoparticle in chitosan gel formulation (0.01%) was twofold higher than commercial cream, although it contained ten times less DFV. Nanoparticles in gel formulations produced significantly higher edema inhibition in rats compared with commercial cream in in-vivo studies. Skin blanching assay using a chromameter showed vasoconstriction similar to that of the commercial product. There were no barrier function changes upon application of nanoparticles. In-vitro and in-vivo results demonstrated that lecithin/chitosan nanoparticles in chitosan gel may be a promising carrier for dermal delivery of DFV in various skin disorders.

Neutrophil Interactions With ICAM-1 at the Luminal Surface of the Intestinal Epithelium Enhance Neutrophil Survival and Alter Epithelial Barrier Function

Neutrophil transepithelial migration (TEM) is a hallmark of inflammatory bowel disease (IBD), and is associated with epithelial injury and impaired barrier function. While TEM has been extensively modeled, little is known about neutrophil interactions with ligands expressed on the luminal surface of the intestinal epithelium. ICAM-1 is a well-recognized signaling receptor and endothelial ligand for migrating neutrophils. Its expression is also induced on the luminal (apical) membrane of intestinal epithelial cells (IEC) exposed to interferon γ (IFNγ), and in the colonic mucosa of individuals with active IBD. However, the role of ICAM-1 in mediating functional effects of neutrophil interactions with intestinal epithelial cells is undefined. ICAM-1 expression in cultured IECs and mouse intestinal epithelium was induced by exposure to IFNγ (100U/mL, 24h) and IFNγ+TNFα (500ng each, 24h, ip) respectively, and analyzed by flow cytometry and immunofluorescence. The effects of neutrophil-IEC interactions, and specifically of ICAM-1 engagement by antibody crosslinking on the regulation of epithelial barrier function was examined in T84 IECs by measuring changes in transepithelial resistance (TER) and permeability to 4kDa FITC-Dextran, and by using an intestinal loop model from anesthetized mice, measuring absorption of FITC-dextran from the intestinal lumen into the circulation. We confirmed upregulation of ICAM-1 expression in IFNγ treated T84 cells, in the intestinal epithelium of patients with IBD, and further showed induction in ICAM-1 expression in-vivo in murine small intestine pre-treated with IFNγ and TNFa. In cultured IECs, expression of ICAM-1 enhanced neutrophil adhesion to the apical epithelial membrane. The majority (˜60%) of apically adhered neutrophil that completed TEM were found to exhibit luminal crawling behavior, which was significantly enhanced (>33%) when T84 cells were pre-exposed to IFNγ. Neutrophil interactions with apical epithelial membrane of IFNγ pretreated IECs also resulted in enhanced neutrophil survival as evident by decreased apoptosis. The effects on neutrophil crawling and apoptosis were ICAM-1-dependent and were reversed by treatment with an anti-ICAM-1 function-blocking antibody. Furthermore, neutrophil interactions with the apical epithelial membrane resulted in time-dependent decrease in TER. This decrease was prevented by inhibition of neutrophil adhesion and crawling using an anti-CD11b/CD18 function-blocking antibody, suggesting that neutrophil induced effects on barrier function were due to direct engagement of ligands on epithelial surface. Experiments modeling neutrophil apical engagement using antibody-mediated crosslinking of cell surface ICAM-1 in cultured IECs and in-vivo, using murine intestinal loop model confirmed a direct role for ICAM-1 engagement in regulating epithelial barrier function. Antibody crosslinking of ICAM-1 resulted in time-dependent decrease in TER (up to 30%) and increased flux of FITC-dextran (˜2.5-fold at 4 hours) in-vitro and increased flux of FITC-dextran (˜1.6-fold) in-vivo. ICAM-1 mediated changes in barrier function were accompanied by actin rearrangement and dependent on MLCK. These studies suggest that neutrophil engagement of apically expressed ICAM-1during IBD results in signaling events that can inhibit neutrophil clearance and alter barrier. Such interactions may contribute to further recruitment of inflammatory cells and impede resolution of inflammation. Thus, targeting ICAM-1 in the intestinal epithelium may provide new/improved therapeutic approaches for treating IBD.

Skin integrity testing and monitoring of in vitro tape stripping by capacitance‐based sensor imaging

Despite the frequent use of porcine ear skin for tape-stripping experiments, the peculiarities of this skin type have not been characterised in detail yet. Thus, different techniques were employed to investigate the skin surface structure of porcine ear skin and the changes in barrier function during in vitro tape stripping. To this end, the potential of capacitance-based skin hydration imaging as a means of skin quality control was investigated for the first time. The porcine ear model was characterised before and during tape stripping using transepidermal water loss (TEWL) measurements, capacitance-based sensor imaging, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and optical light microscopy. The capacitance-based sensor was found to deliver precise information about the quality of the employed skin sites before and during tape stripping. The removal of stratum corneum proteins was highly reproducible even for different porcine ear types. The mean greyscale values showed an excellent linear correlation to the corresponding TEWL values and the respective penetration depth. Optical light microscopy confirmed the presence of canyons on the surface of porcine ear skin. The results suggest that the capacitance-based sensor is a suitable tool for skin integrity testing of porcine ear skin in vitro and for monitoring changes in skin barrier function.

Mo1103 Blast-Type Traumatic Brain Injury Alters Intestinal Contractility and Barrier Integrity in Rats

Pathophysiologic and clinicalmanifestations of stress-induced intestinal disorders are initiated by disturbances in intestinal barrier function. Corticotropin releasing factor (CRF) receptor signaling pathways have been shown to be a central pathway by which stress mediates its deleterious effects on the intestinal barrier; however, the precise mechanisms remain poorly understood. In addition, few studies exist that utilize translational large animal models to study CRF pathways in intestinal dysfunction. Here we utilized In Vivo and ex vivo porcine models to investigate the mechanism of intestinal barrier dysfunction mediated by CRF. To determine the role of CRF signaling in stress-induced barrier dysfunction, 16-d old piglets were administered the dual CRF receptor 1 (CRF1)/CRF receptor 2(CRF2) antagonist Astressin B (30 μg/kg), 30 min prior to subjecting pigs to early weaning stress. At 24 h post-weaning stress, ileum was harvested and mounted on Ussing chambers for measurement of intestinal barrier function as determined by transepithelial electrical resistance (TER) and mucosalto-serosal flux of 3H-mannitol. Weaning stress caused significant reductions (p<0.05) in TER and elevations (p<0.01) in 3H-mannitol flux in control pigs; however, pre-treatment with Astressin B ameliorated deleterious changes in barrier function in weaned pigs. In an ex vivo Ussing chamber model, application of CRF to porcine ileum caused elevations in FITC dextran (4 kDa) flux that were blocked by pre-treating tissues with the CRF1 antagonist antalarmin and the CRF1/CRF2 antagonist Astressin B. Application of the selective CRF1 agonist stressin1 to porcine ileum increased FD4 flux and reduced TER compared with CRF and vehicle treatments (p<0.05). CRF and stressin1 were shown to induce mast cell activation and release of mast cell-derived TNF-α and tryptase. CRF and stressin1 mediated increases in FD4 flux were inhibited by pre-treating tissues with protease inhibitors and anti-TNF-α antibodies. These studies demonstrate that in the porcine ileum, stress induces increases in paracellular permeability that is mediated by CRF1. This pathway involves the release of mast cell-derived proteases and TNF-α that contribute to disturbances in intestinal barrier function.

Mo1102 Mechanisms of Stress-Induced Intestinal Barrier Dysfunction in the Porcine Intestine

Pathophysiologic and clinicalmanifestations of stress-induced intestinal disorders are initiated by disturbances in intestinal barrier function. Corticotropin releasing factor (CRF) receptor signaling pathways have been shown to be a central pathway by which stress mediates its deleterious effects on the intestinal barrier; however, the precise mechanisms remain poorly understood. In addition, few studies exist that utilize translational large animal models to study CRF pathways in intestinal dysfunction. Here we utilized In Vivo and ex vivo porcine models to investigate the mechanism of intestinal barrier dysfunction mediated by CRF. To determine the role of CRF signaling in stress-induced barrier dysfunction, 16-d old piglets were administered the dual CRF receptor 1 (CRF1)/CRF receptor 2(CRF2) antagonist Astressin B (30 μg/kg), 30 min prior to subjecting pigs to early weaning stress. At 24 h post-weaning stress, ileum was harvested and mounted on Ussing chambers for measurement of intestinal barrier function as determined by transepithelial electrical resistance (TER) and mucosalto-serosal flux of 3H-mannitol. Weaning stress caused significant reductions (p<0.05) in TER and elevations (p<0.01) in 3H-mannitol flux in control pigs; however, pre-treatment with Astressin B ameliorated deleterious changes in barrier function in weaned pigs. In an ex vivo Ussing chamber model, application of CRF to porcine ileum caused elevations in FITC dextran (4 kDa) flux that were blocked by pre-treating tissues with the CRF1 antagonist antalarmin and the CRF1/CRF2 antagonist Astressin B. Application of the selective CRF1 agonist stressin1 to porcine ileum increased FD4 flux and reduced TER compared with CRF and vehicle treatments (p<0.05). CRF and stressin1 were shown to induce mast cell activation and release of mast cell-derived TNF-α and tryptase. CRF and stressin1 mediated increases in FD4 flux were inhibited by pre-treating tissues with protease inhibitors and anti-TNF-α antibodies. These studies demonstrate that in the porcine ileum, stress induces increases in paracellular permeability that is mediated by CRF1. This pathway involves the release of mast cell-derived proteases and TNF-α that contribute to disturbances in intestinal barrier function.

Epidermal Transglutaminase (TGase 3) Is Required for Proper Hair Development, but Not the Formation of the Epidermal Barrier

Transglutaminases (TGase), a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members.

The Role of the Endothelium in HPS Pathogenesis and Potential Therapeutic Approaches

American hantaviruses cause a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). Hantaviruses nonlytically infect endothelial cells and cause dramatic changes in barrier functions of the endothelium without disrupting the endothelium. Instead hantaviruses cause changes in the function of infected endothelial cells that normally regulate fluid barrier functions of capillaries. The endothelium of arteries, veins, and lymphatic vessels is unique and central to the function of vast pulmonary capillary beds, which regulate pulmonary fluid accumulation. The endothelium maintains vascular barrier functions through a complex series of redundant receptors and signaling pathways that serve to both permit fluid and immune cell efflux into tissues and restrict tissue edema. Infection of the endothelium provides several mechanisms for hantaviruses to alter capillary permeability but also defines potential therapeutic targets for regulating acute pulmonary edema and HPS disease. Here we discuss interactions of HPS causing hantaviruses with the endothelium, potential endothelial cell-directed permeability mechanisms, and therapeutic targeting of the endothelium as a means of reducing the severity of HPS disease.

Expression of Epidermal CAMP Changes in Parallel with Permeability Barrier Status

Two critical defensive functions of the outer epidermis, the permeability barrier and antimicrobial defense, share certain structural and biochemical features. Moreover, 3antimicrobial peptides (AMP); i.e., mouse beta-defensin 3 (mBD3), mouse cathelicidin protein (mCAMP), and the neuroendocrine peptide, catestatin, all localize to the outer epidermis, and both mBD3 and mCAMP are secreted from epidermal lamellar bodies with other organelle contents that subserve the permeability barrier. These 3 AMP are up-regulated in response to acute permeability barrier disruption, while conversely, mCAMP−/− mice (unable to combatgram-positive pathogens) also display abnormal barrier homeostasis. To determine further whether these two functions are co-regulated, we investigated changes in immunostaining for these 3 AMP in skin samples in which permeability barrier function in mice had been either compromised or enhanced. Compromised or enhanced barrier function correlated with reduced or enhanced immunohistochemical expression of mCAMP, respectively, but conversely with Cst expression likely due to the role of this AMP as an endogenous inhibitor of cathelicidin expression. mBD3 expression correlated with experimental barrier perturbations, but poorly with developmental changes in barrier function. These studies show that changes in cathelicidin and Cst expression parallel changes in permeability barrier status, with a less clear relationship with mBD3 expression.

Role of the Gut in Visceral Fat Inflammation and Metabolic Disorders

Role of the Gut in Visceral Fat Inflammation and Metabolic Disorders