Altered Barrier Function Research Articles

Diet restriction that improves longevity and health span also modifies gut microbiology

The importance of gut microbiota in gut health and health in general has received considerable attention. The elderly are known to have reduced diversity and decreased relative abundance of desirable microbiata. These age‐related changes in gut microflora could diminish health span. For example, reductions in the relative abundance of important sub‐populations such as Clostridium cluster IV and bifidobacteria might result in reduced formation of short chain fatty acids, altered epithelial cell maintenance, and altered barrier function of the gut epithelium in the elderly. Those changes in the GI microbiota have previously been linked to impaired immune function prevalent in older individuals and may result in a greater susceptibility to disease. It is well established that diet restriction leads to improved health and increased longevity in many species. In addition, resistant starch diets improve gut function and metabolic status. Does part of this increase in health involve an improvement in gut function and specifically in gut microbiota? The data from 2‐year old C57BL/6 mice fed either resistant starch or were diet restricted show similar trends for the bacteria levels measured by qRT‐PCR. Both treatments increase Bifidobacterium and total Clostridium IV. These data support our hypothesis that dietary fermentable fiber mimics many of the effects of diet restriction on health span and longevity. Supported by National Starch and LSU AgCenter.

Effects of age and allergen exposure on transepidermal water loss in a house dust mite-sensitized beagle model of atopic dermatitis

Transepidermal water loss (TEWL) was measured as assessment of barrier function in house dust mite-sensitized atopic (n = 24) and normal (n = 21) beagle dogs before and after allergen challenge. Dogs of the two groups were matched for age and housed in the same environment. Ten sites were evaluated including both areas of high and low predisposition to atopic lesion formation. On day 0 (before allergen exposure) and day 3 (3 days after allergen exposure), TEWL measurements were taken using an open-chamber evaporimeter. On day 0, significant differences (one-way ANOVA) were found between normal and atopic dogs in the chin, pinna, periocular region, axilla, antebrachium and thorax. Within the atopic group, a significant increase (one-way ANOVA) in TEWL was found in the axilla on day 3. Such a difference was particularly marked in young dogs (chin, pinna, axilla, antebrachium and groin). Within the normal group, allergen challenge did not change TEWL except for the chin. When atopic and nonatopic sites were compared within the atopic group, a significant difference in TEWL was found on day 0. A two-way ANOVA showed a significant effect of site, a significant effect of time but no site x time (i.e. allergen exposure) interaction. It is concluded that the house dust mite-sensitized atopic beagle dogs used in this study have altered barrier function in 'atopic' areas and that such differences are more evident in young individuals and are aggravated by allergen exposure.

Even low-grade inflammation impacts on small intestinal function

Independent of the cause and location, inflammation - even when minimal - has clear effects on gastrointestinal morphology and function. These result in altered digestion, absorption and barrier function. There is evidence of reduced villus height and crypt depth, increased permeability, as well as altered sugar and peptide absorption in the small intestine after induction of inflammation in experimental models, which is supported by some clinical data. Identification of inflammatory factors which may promote the process of gastrointestinal dysfunction as well as clinical research to verify experimental observations of inflammatory modulation of gastrointestinal function are required. Moreover, nutritional strategies to support functional restitution are needed.

Serotonin Transport and Metabolism in the Mammary Gland Modulates Secretory Activation and Involution

Serotonin [5-hydroxytryptamine (5-HT)] is an important local regulator of lactation homeostasis; however, the roles for the serotonin reuptake transporter and monoamine oxidase have not been known. The aim of the study was to determine whether drugs that impact 5-HT affect human lactation physiology. We conducted laboratory studies of human and animal models and an observational study of the onset of copious milk secretion in postpartum women at a university medical center. We studied women expecting their first live-born infant; exclusion criteria were: referred to the medical center for another medical condition, known contraindication to breastfeed, and less than 19 yr of age and unable to obtain parental consent. The mothers were interviewed. The cell and animal studies consisted of a variety of biochemical, pharmacological, and genetic interventions. The human subjects outcome was prevalence of delayed onset of copious milk secretion. The cell and animal outcomes were physiological and morphological. Inhibiting serotonin reuptake in mammary epithelial cells altered barrier function, and the effects were amplified by coadministering a monoamine oxidase inhibitor. Direct delivery of fluoxetine by slow-release pellets caused localized involution. TPH1 knockout mice displayed precocious secretory activation. Among a cohort of 431 women, those taking SSRI were more likely (P = 0.02) to experience delayed secretory activation. Medications that perturb serotonin balance dysregulate lactation, and the effects are consistent with those predicted by the physiological effects of intramammary 5-HT bioactivity. Mothers taking serotonergic drugs may need additional support to achieve their breastfeeding goals.

Sirolimus and cyclosporine A alter barrier function in renal proximal tubular cells through stimulation of ERK1/2 signaling and claudin-1 expression

Alteration of the tight junction complex in renal epithelial cells can affect renal barrier function and perturb normal kidney homeostasis. The immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) used in combination demonstrated beneficial effects in organ transplantation but this combination can also result in increased adverse effects. We previously showed that CsA treatment alone caused an alteration of the tight junction complex, resulting in changes in transepithelial permeability in Madin-Darby canine kidney distal tubular/collecting duct cells. The potential effect of SRL on transepithelial permeability in kidney cells is unknown. In this study, subcytotoxic doses of SRL or CsA were found to decrease the paracellular permeability of the porcine proximal tubular epithelial cells, LLC-PK1 cell monolayers, which was detected as an increase in transepithelial electrical resistance (TER). The cotreatment with SRL and CsA was found to increase TER in a synergistic manner. CsA treatment increased total cellular expression and membrane localization of the tight junction protein claudin-1 and this further increased with the combination of SRL/CsA. SRL and CsA treatment alone or in combination stimulated the phosphorylation of ERK1/2. The MEK-ERK1/2 pathway inhibitor, U0126, reduced the SRL, CsA, and CsA/SRL-induced increase in TER. U0126 also reduced the CsA and CsA/SRL-induced increase in the membrane localization of claudin-1. Alterations in claudin-2 and claudin-4 were also detected. However, the results suggest that the modulation in expression and localization of claudin-1 appears to be pivotal in the SRL- and CsA-induced modulation of the epithelial barrier function and that modulation is regulated by ERK1/2 signaling pathway.

Selective activation of signal transducer and activator of transcription (STAT) proteins STAT1 and STAT3 in human endothelial cells infected with Rickettsia rickettsii

A majority of typical pathologic sequelae of rickettsial diseases, such as altered barrier function of endothelial cells leading to compromised vascular permeability, manifest as non-cardiogenic pulmonary oedema ⁄ acute respiratory distress syndrome and parenchymal cerebral oedema, which can be attributed to disseminated infection and damage of the vascular endothelium [1]. Endothelial cells infected in vitro with R. rickettsii or R. conorii undergo profound phenotypic modifications to an ‘activated’ state due to induction of a number of immediate early responsive prothrombotic and proadhesive genes [2]. Ample published evidence also indicates an important role for interferons (IFNs) in host response and clearance of pathogenic Rickettsia species [3]. In general, both type I (a, b) and type II (c) IFNs modulate the innate immune response through Janus kinase (JAK)-STAT (Signal transducer and activator of transcription) mediated gene regulation. JAK-STAT pathway transmits signals originating from extracellular ligands and those from within the cell directly to the nucleus to control various gene promoters. There are four known Janus kinases (JAK1-3 and TYK2) and seven STAT proteins (STAT1-4, 5a, 5b, and 6). To better understand the immune response of vascular endothelium to infection with spotted fever rickettsiae, we have initiated efforts to investigate the contributions of the JAK-STAT signalling pathway to the pathophysiology of human spotted fever group (SFG) rickettsioses. Standard laboratory protocols established and described earlier were used for in vitro infection of cultured human umbilical vein-derived endothelial cells (HUVECs) [4]. Briefly, cells were incubated in antibiotic-free culture medium with approximately 6 · 10 pfu of R. rickettsii organisms for every cm of culture area. Indirect immunofluorescent staining and citrate synthase (gltA)-based quantitative PCR confirmed that this experimental protocol consistently resulted in infection of ‡80% of cells with three to five rickettsiae at 6 h [5]. Because phosphorylation of specific target residues triggers subsequent dimerisation and nuclear translocation, activation of different STAT proteins was determined by denaturing polyacrylamide gel electrophoresis of total protein lysates from R. rickettsii-infected HUVECs followed by Western blot analysis using phosphorylation state-specific antibodies. As expected, uninfected endothelial cells were found to have either very low or negligible basal phosphorylation levels of all investigated STAT proteins. In response to R. rickettsii infection, however, the phosphorylation of STAT3 and STAT1 was clearly induced (Fig. 1), while that of STAT2, STAT5 and STAT6 remained unaltered at all times (not shown). Interestingly, there were noticeable differences in the kinetics of activation of STAT1 and STAT3 in response to infection. The levels of p-STAT3 in infected cells were two to three times higher than corresponding controls at early times of 1.5 and 3 h and then declined to basal levels at 8 h post-infection (Fig. 1a). In contrast, the levels of p-STAT1 did not display significant changes early during the infection and started to increase at about 6 h post-infection with as much as four-fold and eight-fold induction at 12 h and 24 h post-infection, respectively (Fig. 1b). These results indicate that activation of only selective STAT proteins occurs in response to R. rickettsii infection and highlight the unique differences in kinetics of their activation. Corresponding author and reprint requests: Sanjeev K. Sahni, PhD, Department of Microbiology and Immunology, Room 211132, PO Box 672, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA E-mail: sanjeev_sahni@urmc.rochester.edu

Abstract 4880: Novel Role of P90RSK-mediated Sentrin/Sumo-specific Proteases 2 (SENP2) Phosphorylation on Disturbed Flow and H2O2-induced Apoptosis in Endothelial Cells

The atheroprotective roles of steady laminar flow (s-flow) and its inhibition against pro-inflammatory responses and endothelial cells (EC) apoptosis has been extensively studied. Emerging evidence suggested that disturbed flow (d-flow) and reactive oxygen species (ROS) are critical factors to induce EC dysfunction, which includes changes in vasoregulation, inflammatory activation, and altered barrier function with EC apoptosis. However, it remains unclear how d-flow induces EC dysfunction. We performed a survey of which kinases can be activated by d-flow, but not s-flow. We found that two kinases, p90RSK and PKC ζ , were particularly activated under d-flow, but not s-flow in EC. D-flow and H2O2 (50 μ M) significantly increased apoptosis in EC (5.79±0.5 fold compared with control; p<0.01). Interestingly, we found that H2O2 increased nuclear export of p53 and p53-Bcl2 binding. The contribution of p53-SUMOylation on p53 nuclear export has been reported, and cytosolic p53-Bcl2 binding inhibits Bcl2 anti-apoptotic effect and induces apoptosis. Overexpression of p90RSK increased p53-SUMOylation, and adenovirus containing dominant negative form of p90RSK (Ad-DN-p90RSK) transduction significantly inhibited H2O2-mediated p53-SUMOylation, p53 nuclear export, p53-Bcl2 binding, and apoptosis, suggesting the critical role of p90RSK in p53-SUMOylation and subsequent apoptosis. Since four isoforms of p90RSK have similar function in EC, we used DN-p90RSK instead of siRNA which may downregulated all the isoforms. In addition, we determined SENP2 as a novel target of p90RSK based on the following results; depletion of SENP2 increased p53-SUMOylation and apoptosis in EC, overexpression of p90RSK significantly decreased SENP2 de-sumoylation activity, H2O2-mediated SENP2 nuclear export was significantly inhibited by Ad-DN-p90RSK transduction, and in vitro kinase assay showed that SENP2 was a good substrate of p90RSK. Finally, en face confocal microscopy analysis using apoE−/− mice also revealed the activation of p90RSK and the up-regulation of p53 expression at d-flow, but not at s-flow area, supporting the patho-physiological consequence of p90RSK-SENP2 and subsequent apoptosis on d-flow-mediated acceleration of atherosclerosis.

Remodeling of the tight junction during recovery from exposure to hydrogen peroxide in kidney epithelial cells

Renal ischemia–reperfusion injury results in oxidative stress-induced alterations in barrier function. Activation of the mitogen-activated protein (MAP) kinase pathway during recovery from oxidative stress may be an effector of oxidant-induced tight junction reorganization. We hypothesized that tight junction composition and barrier function would be perturbed during recovery from oxidative stress. We developed a model of short-term H 2O 2 exposure followed by recovery using Madin Darby canine kidney (MDCK II) cells. H 2O 2 perturbs barrier function without a significant cytotoxic effect except in significant doses. ERK-1/2 and p38, both enzymes of the MAP kinase pathway, were activated within minutes of exposure to H 2O 2. Transient exposure to H 2O 2 produced a biphasic response in the transepithelial electrical resistance (TER). An initial drop in TER at 6 h was followed by a significant increase at 24 h. Inhibition of ERK-1/2 activation attenuated the increase in TER observed at 24 h. Expression of occludin initially decreased, followed by partial recovery at 24 h. In contrast, claudin-1 levels decreased and failed to recover at 24 h. Claudin-2 levels were markedly decreased at 24 h; however, inhibition of ERK-1/2 activation was protective. Occludin and claudin-1 localization at the apical membrane on immunofluorescence images was fragmented at 6 h after H 2O 2 exposure with subsequent recovery of appropriate localization by 24 h. MDCK II cell recovery after H 2O 2 exposure is associated with functional and structural modifications of the tight junction that are mediated in part by activation of the MAP kinase enzymes ERK-1/2 and p38.

Maintenance of an Acidic Stratum Corneum Prevents Emergence of Murine Atopic Dermatitis

Neutralization of stratum corneum (SC) adversely impacts key epidermal functions, including permeability barrier homeostasis and SC integrity. Conversely, acidification of SC improves these functions in developmentally impaired (neonatal or aged) skin, and enhances function in normal skin. Hence, we hypothesized that acidification could alter the course of inflammatory dermatoses, which invariably exhibit an increased SC pH. Maintenance of a low pH by topical applications of the polyhydroxyl acid, lactobionic acid, during the repeated-challenge phase inhibited the development of oxazolone-induced atopic dermatitis (AD). Neither gross/histological dermatitis nor altered barrier function developed, and emergence of epidermal hyperplasia was prevented; however, cytokine generation decreased. Acidification also largely normalized the development of hapten-induced changes in eosinophil/mast cell densities, density of chemoattractant receptor-homologous molecule expressed on TH2-positive lymphocytes, and serum IgE levels. The pH-induced improvement in barrier function most likely accounts for the anti-inflammatory activity, which could be further attributed to normalization of both lamellar body secretion and lamellar bilayer formation. Acidification of SC alone substantially prevents development of barrier abnormalities and downstream immune abnormalities during the elicitation phase of murine AD. These results provide direct evidence for the "outside-inside" pathogenesis of AD and further suggest that maintenance of an acidic SC pH could prevent the emergence of AD in humans.

Aquaporin5 (AQP5) modulation as a novel mechanism to regulate airway barrier function

Airflow creates shear forces on the apical surface of airway epithelial cells, the effects of which are largely unknown. In isolated mouse trachea, airflow induced shear stress enhanced epithelial barrier function (permeability of evans blue dye‐4% albumin). Primary human airway epithelial cells differentiated in an air‐interface were exposed to shear stress (3 dynes/cm2) using Krebs solution, leading to increased barrier function (FITC‐dextran permeability) and reduced the abundance of AQP5, an apical water channel, by sequential activation of TRPV4 and VGCC. 16HBE cells, without endogenous AQP5, were infected with either an adeno‐AQP5 expressing or control GFP virus. The presence of AQP5 reduced barrier function and allowed further modulation of barrier function in response to shear stress. Altering barrier function by expressing AQP5 altered receptor‐ligand interactions. Apical ligands (EGF,IFNƒ×) interact with basolateral receptors only when paracellular permeability is increased (induced by AQP5 overexpression). Similarly, Substance P (basolateral ligand) only interacts with apical receptors when AQP5 was overexpressed. We hypothesize that regulation of AQP5 modulates the airway epithelia barrier, which is critical in regulating signaling. This important homeostatic mechanism to regulate epithelial responses may be disrupted in pathologic conditions. Supported by HL085763 and FAMRI.

The food contaminant deoxynivalenol, decreases intestinal barrier permeability and reduces claudin expression

'The gastrointestinal tract represents the first barrier against food contaminants as well as the first target for these toxicants. Deoxynivalenol (DON) is a mycotoxin that commonly contaminates cereals and causes various toxicological effects. Through consumption of contaminated cereals and cereal products, human and pigs are exposed to this mycotoxin. Using in vitro, ex vivo and in vivo approaches, we investigated the effects of DON on the intestinal epithelium. We demonstrated that, in intestinal epithelial cell lines from porcine (IPEC-1) or human (Caco-2) origin, DON decreases trans-epithelial electrical resistance (TEER) and increases in a time and dose-dependent manner the paracellular permeability to 4 kDa dextran and to pathogenic Escherichia coli across intestinal cell monolayers. In pig explants treated with DON, we also observed an increased permeability of intestinal tissue. These alterations of barrier function were associated with a specific reduction in the expression of claudins, which was also seen in vivo in the jejunum of piglets exposed to DON-contaminated feed. In conclusion, DON alters claudin expression and decreases the barrier function of the intestinal epithelium. Considering that high levels of DON may be present in food or feed, consumption of DON-contaminated food/feed may induce intestinal damage and has consequences for human and animal health.

CD24 regulated gene expression and distribution of tight junction proteins is associated with altered barrier function in oral epithelial monolayers

BackgroundControl of intercellular penetration of microbial products is critical for the barrier function of oral epithelia. We demonstrated that CD24 is selectively and strongly expressed in the cells of the epithelial attachment to the tooth and the epithelial lining of the diseased periodontal pocket and studies in vitro showed that CD24 regulated expression of the epithelial intercellular adhesion protein E-cadherin.ResultsIn the present study, the barrier function of oral epithelial cell monolayers to low molecular weight dextran was assayed as a model for the normal physiological function of the epithelial attachment to limit ingress of microbial products from oral microbial biofilms. Paracellular transfer of low molecular weight dextran across monolayers of oral epithelial cells was specifically decreased following incubation with anti-CD24 peptide antibody whereas passage of dextran across the monolayer was increased following silencing of mRNA for CD24. Changes in barrier function were related to the selective regulation of the genes encoding zonula occludens-1, zonula occludens-2 and occludin, proteins implicated in tight junctions. More particularly, enhanced barrier function was related to relocation of these proteins to the cell periphery, compatible with tight junctions.ConclusionCD24 has the constitutive function of maintaining expression of selected genes encoding tight junction components associated with a marginal barrier function of epithelial monolayers. Activation by binding of an external ligand to CD24 enhances this expression but is also effective in re-deployment of tight junction proteins that is aligned with enhanced intercellular barrier function. These results establish the potential of CD24 to act as a potent regulator of the intercellular barrier function of epithelia in response to local microbial ecology.

Nitric oxide and airway epithelial barrier function: Regulation of tight junction proteins and epithelial permeability

Acute airway inflammation is associated with enhanced production of nitric oxide (NO ) and altered airway epithelial barrier function, suggesting a role of NO or its metabolites in epithelial permeability. While high concentrations of S-nitrosothiols disrupted transepithelial resistance (TER) and increased permeability in 16HBE14o− cells, no significant barrier disruption was observed by NONOates, in spite of altered distribution and expression of some TJ proteins. Barrier disruption of mouse tracheal epithelial (MTE) cell monolayers in response to inflammatory cytokines was independent of NOS2, based on similar effects in MTE cells from NOS2−/− mice and a lack of effect of the NOS2-inhibitor 1400W. Cell pre-incubation with LPS protected MTE cells from TER loss and increased permeability by H 2O 2, which was independent of NOS2. However, NOS2 was found to contribute to epithelial wound repair and TER recovery after mechanical injury. Overall, our results demonstrate that epithelial NOS2 is not responsible for epithelial barrier dysfunction during inflammation, but may contribute to restoration of epithelial integrity.

A Common Barrier Defect for Celiac Disease and Ulcerative Colitis

A Common Barrier Defect for Celiac Disease and Ulcerative Colitis

Molecular Pathogenesis of Inflammatory Bowel Disease: Relevance for Novel Therapies

The immense load of microorganisms within the gastrointestinal tract is a great challenge for the mucosal immune system. Whereas the vast majority of commensal bacteria should be tolerated, pathogenic organisms have to be attacked. During inflammatory bowel disease, the balanced interaction between the mucosal flora and the intestinal immune system is disturbed. Various defective components of this complex interaction have been described, such as different susceptibility genes, impaired innate immune responses and environmental factors, suggesting that inflammatory bowel diseases are multifactorial diseases. Based on new insights into the pathogenesis of inflammatory bowel disease, various targets for future drugs have been identified and new substances are emerging. The following article will review the current understanding of inflammatory bowel disease pathogenesis in context with genetic risk factors, imbalanced innate and acquired immune responses, and altered barrier function. Clinical treatment of the diseases will be summarized and emerging therapies as well as individual management based on recent insights into pathogenesis will be discussed.

Endothelial dysfunction and diabetes: roles of hyperglycemia, impaired insulin signaling and obesity

Endothelial dysfunction comprises a number of functional alterations in the vascular endothelium that are associated with diabetes and cardiovascular disease, including changes in vasoregulation, enhanced generation of reactive oxygen intermediates, inflammatory activation, and altered barrier function. Hyperglycemia is a characteristic feature of type 1 and type 2 diabetes and plays a pivotal role in diabetes-associated microvascular complications. Although hyperglycemia also contributes to the occurrence and progression of macrovascular disease (the major cause of death in type 2 diabetes), other factors such as dyslipidemia, hyperinsulinemia, and adipose-tissue-derived factors play a more dominant role. A mutual interaction between these factors and endothelial dysfunction occurs during the progression of the disease. We pay special attention to the possible involvement of endoplasmic reticulum stress (ER stress) and the role of obesity and adipose-derived adipokines as contributors to endothelial dysfunction in type 2 diabetes. The close interaction of adipocytes of perivascular adipose tissue with arteries and arterioles facilitates the exposure of their endothelial cells to adipokines, particularly if inflammation activates the adipose tissue and thus affects vasoregulation and capillary recruitment in skeletal muscle. Hence, an initial dysfunction of endothelial cells underlies metabolic and vascular alterations that contribute to the development of type 2 diabetes.

Allergy and the skin.

Allergic skin disorders include urticaria, angioedema, contact dermatitis and atopic dermatitis, but the model fitting most closely the systemic concept of allergy is atopic dermatitis (AD), the pathogenesis of which is linked to a complex interaction between skin barrier dysfunction and environmental factors such as allergens and microbes. In particular, an important advance was the demonstration that the mutation of the skin barrier protein filaggrin is related strictly to allergen sensitization and to the development of asthma in subjects with AD. The altered skin barrier function, caused by several factors, results in the passage of allergens through the skin and to systemic responses. A pivotal role in such a response is exerted by Langerhans cells which, via their immunoglobulin E (IgE) receptor, capture the allergens and present them to T cells. When T helper type 2 (Th2) cells are activated, the production of a proinflammatory cytokines and chemokines pattern sustains the persistence of inflammation. Known AD-related cytokines are interleukin (IL)-5, IL-13 and tumour necrosis factor (TNF)-alpha, with emerging importance for IL-17, which seems to drive airway inflammation following cutaneous exposure to antigens, and IL-31, which is expressed primarily in skin-homing Th2 cells. Skin-homing is another crucial event in AD, mediated by the cutaneous lymphocyte-associated antigens (CLA) receptor, which characterizes T cell subpopulations with different roles in AD and asthma.

T1269 Effects of Synthetic Zot Peptide Analog AT-1002 and Inhibitor AT-1001 On Gluten Induced Alterations in Barrier Function and Macrophage Recruitment in HLA-HCD4/DQ8 Mice

T1269 Effects of Synthetic Zot Peptide Analog AT-1002 and Inhibitor AT-1001 On Gluten Induced Alterations in Barrier Function and Macrophage Recruitment in HLA-HCD4/DQ8 Mice

M1174 Enteropathogenic E. coli-Induced Barrier Function Alteration Is Not a Consequence of Host Cell Death

M1174 Enteropathogenic E. coli-Induced Barrier Function Alteration Is Not a Consequence of Host Cell Death

EnteropathogenicE. coli-induced barrier function alteration is not a consequence of host cell apoptosis

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen that perturbs intestinal epithelial function. Many of the alterations in the host cells are mediated by effector molecules that are secreted directly into epithelial cells by the EPEC type III secretion system. The secreted effector molecule EspF plays a key role in redistributing tight junction proteins and altering epithelial barrier function. EspF has also been shown to localize to mitochondria and trigger membrane depolarization and eventual host cell death. The relationship, if any, between EspF-induced host cell death and epithelial barrier disruption is presently not known. Site-directed mutation of leucine 16 (L16E) of EspF impairs both mitochondrial localization and consequent host cell death. Although the mutation lies within a region critical for type III secretion, EspF(L16E) is secreted efficiently from EPEC. Despite its inability to promote cell death, EspF(L16E) was not impaired for tight junction alteration or barrier disruption. Consistent with this, the pan-caspase inhibitor Q-VD-OPH, despite reducing EPEC-induced host cell death, had no effect on infection-mediated barrier function alteration. Thus EPEC alters the epithelial barrier independent of its ability to induce host cell death.