Дисертації з теми "Lung epithelial barrier function"

Le syndrome de détresse respiratoire aiguë est une cause majeure d'insuffisance respiratoire avec un taux de mortalité élevé. Elle se caractérise par des lésions alvéolaires diffuses, un œdème alvéolaire et une défaillance respiratoire hypoxémique qui entraînent de lourds coûts de santé. Actuellement, les traitements disponibles pour le SDRA restent principalement de soutien, et aucune approche pharmacologique n'est traduite avec succès en application clinique. Il existe deux processus majeurs au cours du développement physiopathologique du SDRA qui conduisent à la formation d'un œdème pulmonaire :dysfonctionnement de la barrière alvéolaire et altération de la clairance du liquide alvéolaire suite à une lésion épithéliale alvéolaire et à une inflammation. Il a été indiqué que le récepteur des produits de glycation avancée (RAGE) était impliqué au cours de ces processus, avec le potentiel élevé de sa forme soluble en tant que biomarqueur pour le diagnostic et le pronostic du SDRA. Les agents halogénés volatils, tels que le sévoflurane ou l'isoflurane, sont de plus en plus utilisés dans les unités de soins intensifs comme agents sédatifs avec leurs caractéristiques intrinsèques idéales en tant que sédatifs. De plus, de nombreuses études précliniques et cliniques indiquent ses effets protecteurs pulmonaires chez les patients atteints de SDRA.Cependant, les mécanismes de ces effets bénéfiques restent à clarifier.Les principaux objectifs de ce travail de thèse sont multiples, à travers des approches expérimentales et modèles translationnels in vivo et in vitro du SDRA,1) Évaluer les effets protecteurs pulmonaires bénéfiques du sévoflurane dans le SDRA, y compris ses effets sur les caractéristiques physiologiques du SDRA, la clairance du liquide pulmonaire et la perméabilité alvéolaire.2) Étudier le mécanisme précis des effets observés du sévoflurane, y compris des études mécanistiques et la fonction et l'expression des protéines impliquées.3) Explorer le rôle de RAGE dans les lésions et la réparation de l'épithélium pulmonaire et son éventuel rôle de médiation des effets bénéfiques du sévoflurane.Au cours de ce travail de thèse, nous avons avancé sous plusieurs angles : Premièrement, nos travaux ont trouvé dans notre modèle de cicatrisation des cellules A549, le rôle important de RAGE dans la réparation des lésions pulmonaires processus, car son ligand, HMGB1, et les AGE ont favorisé la cicatrisation des plaies dépendante de RAGE des cellules épithéliales alvéolaires pulmonaires, ce qui est possiblement expliqué par une migration et une prolifération cellulaires améliorées. Deuxièmement, nos travaux sur des modèles murins de SDRA, trouve une diminution des indices de perméabilité et des structures épithéliales préservées dans les cellules et les souris, au moins en partie, augmentant l'expression de ZO-1 et l'inhibition de l'activité de RhoA et de pMLC ainsi que le réarrangement du cytosquelette d'actine suite à une lésion épithéliale pulmonaire . De plus, RAGE peut jouer un rôle médiateur dans les effets du sévoflurane sur les lésions pulmonaires aiguës. De plus, nos travaux sur des modèles de SDRA porcins in vivo ont confirmé les effets protecteurs pulmonaires du sévoflurane sur les caractéristiques du SDRA, avec une oxygénation améliorée, une perméabilité alvéolaire restaurée et une AFC améliorée. Notre étude suggère que l'effet protecteur du sévoflurane sur l'AFC peut s'expliquer par la restauration de l'expression pulmonaire altérée des canaux épithéliaux AQP-5, Na, K, ATPase et ENaC pendant le SDRA.Dans l'ensemble, ces travaux de thèse expliquent plus précisément les effets protecteurs des agents halogénés et la nouvelle révélation de son mécanisme potentiel, et conforte ainsi le grand intérêt pour l'utilisation de la sédation inhalée en soins intensifs pour les patients atteints de SDRA. Ce travail pourrait donner de nouvelles perspectives pour la recherche sur les effets du sévoflurane sur le SDRA et sa résolution
Acute respiratory distress syndrome (ARDS) is a major cause of respiratory failurewith a high mortality rate. It is characterized by diffuse alveolar damage, alveolar edema, and hypoxemic respiratory loss which cause heavy healthcare costs. Currently, available treatments for ARDS remain primarily supportive, and no pharmacological approach is successfully translated into clinical application. There are two major processes during the physiopathological development of ARDS that lead to the formation of lung edema:alveolar barrier dysfunction and the impairment of alveolar fluid clearance following alveolar epithelial injury and inflammation. The receptor for advanced glycation end products (RAGE) was indicated to be involved during those processes, with the high potential of its soluble form as a biomarker for ARDS diagnostic and prognostic. Volatile halogenated agents, such as sevoflurane or isoflurane, are increasingly used in intensive care units as sedative agents with their ideal intrinsic characteristics as a sedative. Furthermore, numerous pre-clinical and clinical studies indicate its lung protective effects for ARDS patients.However, its mechanisms of such beneficial effects remain to be clarified.The main objectives of this thesis work are multiple, through experimental andtranslational in vivo and in vitro models of ARDS, to1) Asses the beneficial lung protective effects of sevoflurane in ARDS, including its effects on ARDS physiological features, lung fluid clearance, and alveolar permeability.2) Investigate the precise mechanism of observed effects of sevoflurane, including mechanistic studies and involved proteins' function and expression.3) Explore the role of RAGE in lung epithelial injury and repair and its eventualmediation role of the beneficial effects of sevoflurane.During this thesis work, we advanced from many angles: First, our work found in ourA549 cells wound healing model, the important role of RAGE in the lung injury repairprocess, as its ligand, HMGB1, and AGEs promoted RAGE-dependent wound healing oflung alveolar epithelial cells, which is possible through enhanced cell migration and proliferation.Secondly, our work in murine in vitro and in vivo ARDS models, animprovement of experimental features, with decreased indices of permeability and preserved epithelial structures in cells and mice, by at least in a part, increasing expression of ZO-1 and the inhibition of RhoA activity and pMLC as well as actin cytoskeleton rearrangement following lung epithelial injury. Additionally, RAGE may play a mediating role in the effects of sevoflurane on acute lung injury. Furthermore, our work in porcine in vivo ARDS models confirmed the lung protective effects of sevoflurane on ARDS features, with improved oxygenation, restored alveolar permeability, and improved AFC. Our study suggests theprotective effect of sevoflurane on AFC may be explained by the restoration of impaired lung expression of epithelial channels AQP-5, Na, K, ATPase, and ENaC during ARDS.Taken together, this thesis work explained more precisely the protective effects ofhalogenated agents and the new revelation of its potential mechanism, and hence supports the high interest in the use of inhaled sedation in intensive care for ARDS patients. This work may give some new insights for research on the effects of sevoflurane on ARDS and its resolution.Keywords: Acute respiratory distress syndrome; Sevoflurane; Lung epithelial barrierfunction; Lung wound repair; Alveolar fluid clearance; Epithelial channels: Junction proteins;Intracellular pathways; Receptor for advanced glycation end-products

Die Durchfallerkrankung Giardiasis wird durch den Protisten Giardia duodenalis ausgelöst. Die Infektion erfolgt fäkal-oral, meist über kontaminiertes Trinkwasser. Der Parasit kolonisiert den oberen Bereich des Dünndarms und heftt sich an das Epithel, wodurch es die Krankheitsbeschwerden auslöst. Allerdings sind Details über die Mechanismen der Pathogenese unbekannt. Dazu kommt, dass der Ausgang einer Infektion fallspezifisch starken Schwankungen unterworfen ist, von selbst-limitierend bis chronisch und asymptomatischer Kolonisierung bis hin zur schweren Enteritis. Ein möglicher Pathomechanismus ist der Wegfall der Barrierefunktion des Dünndarmepithels, z.B. durch Beeinträchtigung von tight junctions oder Zelltod. In dieser Arbeit wurden Effekte von G. duodenalis auf in vitro Modellsysteme des humanen Dünndarmepithels untersucht. Dazu wurden hauptsächlich Daten über die Barrierefunktion sowohl von der weit verbreiteten Caco-2 Zelllinie, als auch über ein neu etabliertes humanes Dünndarmorganoidsystem, erhoben. Es konnte gezeigt werden, dass mehrere - mitunter in der Literatur als hochvirulent beschriebene - G. duodenalis Isolate zu keinerlei Beeinträchtigung der Barrierefunktion oder irgendeiner anderen untersuchten potenziellen Schädigung an zwei unterschiedlichen Caco-2 Zelllinien unter diversen Infektions- und Kulturbedingungen führte. Jedoch andererseits das neu entwickelte Dünndarmorganoidsystem mit pseudo-luminalem Medium TYI S 33 reproduzierbar die Zerstörung des Epithelmodells mit Zellverlust, Zelltod (apoptotisch und nicht-apoptotisch), Störung der tight junctions (Abbau und Dislokation von Claudinen und ZO-1) und den Verlust von Mikrovilli innerhalb ein bis zwei Tage nach Parasiteninfektion zeigen konnte. Zudem wurde das Auftauchen von ClCa-1-Signalen unter andauerndem Infektionsstress beobachtet, was die Differenzierung bzw. Metaplasie zu Becherzellen nahelegt, jedoch keine Wirtsreaktion auf die Gewebszerstörung zu sein scheint.
The protozoan parasite Giardia duodenalis is the etiological agent for the intestinal diarrheal disease giardiasis. Infections are acquired via the fecal-oral route, mostly via uptake of cysts from contaminated drinking water. The colonization of the hosts’ duodenum and upper jejunum and the attachment of Giardia trophozoites onto the epithelium is the cause of a variety of gastrointestinal complaints but the exact pathomechanisms are unknown. Furthermore, the outcome of Giardia infections varies greatly between individuals, ranging from self-limiting to chronic, and asymptomatic to severe enteritis. One proposed mechanism for the pathogenesis is the breakdown of intestinal barrier function, e.g. by tight junction impairment or induction of cell death. In this work, effects of G. duodenalis on in vitro models of the human small intestinal epithelium were investigated by studying mainly barrier-related properties and changes of widely used Caco-2 cells as well as newly established human small intestinal organoid-derived monolayers (ODMs). It could be shown that several isolates of G. duodenalis, some described as highly virulent, fail to induce barrier dysfunction or any other investigated pathological effect on two Caco-2 cell lines under various infection and culturing conditions. On the other side, by developing a new organoid-based model system and the use of luminal mock medium TYI-S-33, considerable epithelial disruption (including loss of cells), cell death (apoptosis and non-apoptotic), tight junction impairment (degradation and dislocation of claudins and ZO-1), and microvilli depletion reproducibly induced by G. duodenalis trophozoites between one and two days after infection could be observed. Moreover, emergence of ClCa-1 positive cells with ongoing parasite infections suggest epithelial differentiation or metaplasia towards goblet cells, which is furthermore not associated to tissue damage.

Epithelial sodium channels (ENaC) are located throughout the epithelial lining of the respiratory tract and play a crucial role in ion and fluid homeostasis of the lungs. Increasing ENaC activity through stimulation of β₂-adrenergic receptors has been shown to increase sodium and fluid reabsorption from the airspace to the interstitial space. In cystic fibrosis lung disease there is a hyperabsorption of sodium through ENaC which results in dehydration of the airway surface liquid. Previous work has identified a common functional genetic variant of SCNN1A, the gene encoding the ENaC alpha-subunit. This variant manifests as an alanine to threonine substitution at amino acid 663 (T663), with the T663 variant resulting in a more active channel due to a greater number of channels in the membrane. We sought to determine the influence of the T663 variant on exhaled ions, pulmonary function, and the diffusing capacity of the lungs in healthy subjects as well as in patients with cystic fibrosis. We used exercise, which can increase endogenous epinephrine by up to 1000 fold at peak exercise, and albuterol, an exogenous β₂-adrenergic agonist, to stimulate ENaC activity. In healthy individuals we hypothesized that the T663 variant would be beneficial for lung function due to a greater fluid removal, which could improve gas transfer in a healthy lung. In the CF patients we predicted that the T663 variant would be detrimental to lung function due to an exaggerated absorption of sodium and drying/thickening of the mucus layer in the airways. Measurements of exhaled sodium were made in the healthy subjects at baseline, 30, 60, and 90 minutes post-albuterol administration. Subjects with the A663 variant had higher baseline exhaled sodium and a significant decrease in exhaled sodium by 90 minutes after β₂-adrenergic stimulation with albuterol, suggesting a removal of sodium from the airways. No changes in exhaled sodium were seen in the T663 variant in response to albuterol. In response to exercise the A663 variant had a greater increase in the diffusing capacity of the lung than the T663 variant, possibly due to differences in alveolar sodium and therefore fluid handling. Taken together, these results suggest that healthy humans with the A663 variant can increase ENaC activity in response to β₂-adrenergic stimulation, whereas individuals with the T663 variant have a diminished capacity for increasing ENaC activity in response to β₂-adrenergic stimulation. In CF patients, the T663 variant had significantly lower baseline pulmonary function, weight, and body mass index. In response to exercise, patients with the T663 variant had a greater increase in the diffusing capacity of the lungs, possibly due to purinergic inhibition of ENaC. Finally, we recruited additional CF patients to confirm our pulmonary function findings. Individuals with at least one allele resulting in the T663 variant had significantly lower body mass index, and tended to have lower exhaled chloride and pulmonary function. These results suggest greater dehydration of the lung in CF patients with the T663 variant. Overall, these results may suggest that the T663 variant modifies disease severity in CF, although more work is certainly warranted to confirm this result.

Inflammatory bowel disease (IBD) is associated with an inappropriate immune response to the gut microbiota and disruption of intestinal homeostasis. IBD patients and experimental animal models have consistently shown alterations in the gut microbiota composition. However, these studies have mainly focused on faecal microbiota samples taken after the onset of inflammation and IBD establishment. The colonic microbiota inhabits both the gut lumen and the mucus layer covering the intestinal epithelium. Thus, information about mucus-resident microbiota is not necessarily conveyed in the routine microbiota analyses of faecal samples. To address potential changes in microbial composition and function before the onset of IBD, we compared both mucus and faecal microbiota in the mdr1a-/- spontaneous model of colitis over times that we histologically defined as before onset of colitis, during and after colitis onset. We showed that alterations in microbiota composition preceded the onset of intestinal inflammation and that these changes were evident in the mucus, but not in faeces. This altered microbiota composition was coupled with a reduced inner mucus layer, indicating a compromised mucus barrier prior to colitis development. Upon emergence of inflammation, compositional differences were found in both mucus and faecal microbial communities. Spatial segregation of microbiota with intestinal mucosa was also disrupted on disease onset which we hypothesise contributes to a more severe intestinal pathology. Therefore, our data indicate that microbial changes start locally in the mucus and then proceed to the faecal matter concomitantly with colitis development. Next, we examined whether microbial gene functional potential and endogenous metabolite profiles followed alterations in gut microbiota taxonomic composition. Our findings showed that the microbial gene content was similar between mdr1a-/- mice and wild-type littermate controls, demonstrating stability of the gut microbiome at the face of ensuing gut inflammation. In further support of these findings, urinary metabolite analysis revealed that metabolite profiles were unaffected by intestinal inflammation. Metabolites previously reported to change in IBD were similar between mdr1a-/- and wild-type mice at stages preceding and during inflammation. We also found that changes in metabolite profiles did not correlate with colitis scores. However, metabolite changes could discriminate mdr1a-/- mice from wild-type controls, suggesting they could have value in predicting risk of IBD with a potential clinical use in at least a subset of individuals with MDR1A polymorphisms. To assess whether changes in antimicrobial proteins (AMPs) accounted for observed differences in mucus microbiota composition, we also investigated the expression of regenerating islet-derived protein 3 γ (Reg3γ), angiogenin 4 (Ang4), β-defensin 1 and resistin-like molecule beta (Relm-β) in the colon. We found similar levels of these AMPs as well as IgA-producing plasma cells between mdr1a-/- and wild-type mice, suggesting that other factors contribute to alterations in microbiota composition. Overall, our data indicate that the mdr1a-/- is a good model of colitis, as it enables us to look at pre-clinical changes in the gut microbiota. This work suggests the importance of mucus sampling for sensitive detection of microbiota changes. Furthermore, metabolite profiling may be a helpful way to discriminate genetic susceptibility to disease.

L’objectif de cette thèse est de mettre en évidence le rôle des canaux ioniques de l’épithélium alvéolaire dans la pathogenèse du syndrome de détresse respiratoire aiguë. Des études cliniques ont montré que l’absorption anormale de l’œdème pulmonaire par l’épithélium alvéolaire secondaire à la libération de médiateurs inflammatoires est une manifestation caractéristique du syndrome de détresse respiratoire aiguë. Dans une première étude, nous avons démontré que l’activité élevée du transforming growth factor -b1 (TGF-b1) présent dans les alvéoles lors de détresse respiratoire aiguë provoque un œdème pulmonaire en réduisant le transport vectoriel de sodium et de fluides à travers l’épithélium alvéolaire. Puis, nous avons montré que l’interleukine -1b (IL-1b) permet l’activation de TGF-b1 via un mécanisme cellulaire dépendant de l’intégrine avb6. Finalement, dans une dernière étude, nous avons montré que IL-1b affecte directement et indirectement le transport vectoriel de sodium et d’eau à travers l’épithélium alvéolaire. Cette réduction de transport de fluides s’est révélée être due en majeure partie à une diminution de la présence du canal sodium épithélial (ENaC) à la membrane apicale des cellules épithéliales alvéolaires, causée par l'inhibition de l’activité du promoteur d’ENaC par un mécanisme dépendant des MAP kinases. L’ensemble de ces études démontre que TGF-b1 et IL-1b affectent la biosynthèse d’ENaC, et suggère un rôle clé pour ces médiateurs dans la persistance de l'œdème pulmonaire chez les patients atteints de détresse respiratoire aiguë
The objective of this thesis is to investigate the role of abnormalities in alveolar epithelial ion channel function in the pathogenesis of acute lung injury. Clinical studies have demonstrated that impaired alveolar fluid clearance associated with the release of inflammatory mediators within the distal airspace of the lung is a characteristic feature of acute lung injury. Therefore, we examined the potential effect of these mediators on ion transport across the alveolar epithelium. In the first study, we demonstrated that increased transforming growth factor -b1 (TGF-b1) activity in distal airspaces during acute lung injury promoted pulmonary edema by reducing alveolar epithelial sodium and fluid transport. In the second study we showed that in alveolar epithelial cells, interleukin -1b (IL-1b) activated TGF-b1 via an integrin avb6-dependent mechanism. Finally in the last study, we demonstrated that IL-1b could also directly and independently reduce the alveolar epithelial sodium and fluid transport. The reduction in fluid transport was shown to be attributable in large part to a decrease in apical membrane expression of the epithelial sodium channel (ENaC) in lung epithelial cells. The decreased cell surface expression of ENaC was mediated through a MAP kinase-dependent inhibition of ENaC promoter activity. In summary, the studies presented here demonstrate that IL-1b and TGF-b1 down-regulate ENaC biosynthesis and indicate a critical role for these mediators in the impaired fluid clearance of patients with acute lung injury

Thesis (M.S.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed July 22, 2009). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 57-63).

The passage of fluid, ions and macromolecules across the epithelium is controlled primarily by epithelial tight junctions. Altered epithelial permeability is associated with lung disease, and barrier function is impaired by the Th2 cytokine IL-13. This thesis investigates the signalling pathways involved in the modulation of the epithelial barrier by IL-13 stimulation. Initial experiments demonstrated that the human sub-bronchial epithelial cell line Calu-3 could be easily manipulated when grown using an air-liquid culture system. Expression of various key tight junction proteins was demonstrated, as well as a high trans-epithelial resistance (TER) for up to 7 days. Stimulation with IL-13 resulted in a decrease in TER compared with controls and this decrease was shown to be prevented with the PI3K inhibitor ZSTK474. IL-13 did not increase paracellular permeability of the epithelial monolayer to FITC-dextran from the apical to the basolateral chamber and ZSTK474 did not influence FITC-dextran flux. Immunocytochemistry showed that the expression of the tight junction protein claudin 2 was increased by IL-13 stimulation and this change in expression was shown to be PI3K dependent with the PI3K inhibitor ZSKT474 preventing the increase. Further studies were carried out in an attempt to uncover the PI3K isoform responsible for the effects seen on both the TER and the TJ expression. It was shown that inhibition of the p110α isoform with PIK75 mimicked the result observed with the pan-PI3K inhibitor ZSTK474 and prevented the IL-13-induced claudin 2 upregulation. However none of the PI3K isoform inhibitiors showed the prevention of TER, as shown by the pan PI3K inhibitor ZSTK474. The role of STAT6 in TJ modulation was shown to be similar to that of PI3K, in that inhibition of STAT6 had a positive effect on the epithelial barrier by preventing the IL-13-induced TER decrease and the increase in the expression of claudin 2. In addition, both PI3K inhibition and STAT6 inhibition demonstrated effects on basal TER and claudin 2 expression, indicating that both pathways are involved in maintenance of epithelial barrier integrity.

Ergot alkaloids present in endophyte-infected (E+) tall fescue are thought to be the causative agent of fescue toxicosis, a syndrome affecting cattle in the eastern United States. Many of the observed signs of fescue toxicosis are thought to be attributed to peripheral vasoconstriction; however, there are data indicating that ergot alkaloids can alter blood flow to the gut. An experiment was conducted using right ruminal artery and vein collected from heifers shortly after slaughter. Vessels were mounted in a multi-myograph to determine the vasoconstrictive potentials of ergot alkaloids present in E+ tall fescue. Results indicated ergot alkaloids have the potential to induce vasoconstriction of foregut vasculature. A second experiment was conducted to determine if the additional ergot alkaloids present in E+ tall fescue increase the vasoconstrictive response above that of ergovaline. Results indicated that ergovaline is the main alkaloid responsible for vasoconstriction in bovine vessels. A third study was performed to determine the effect of ergot alkaloids on ruminal epithelial blood flow in the washed rumen of steers exposed to E+ or endophyte-free (E-) tall fescue seed. Steers were dosed with seed followed by a washed rumen experiment with differing levels of ergovaline incubated in the rumen. Results indicated that E+ tall fescue seed treatment reduced ruminal epithelial blood flow. Additionally, incubating ergovaline in the rumen during the washed rumen further decreased epithelial blood flow and volatile fatty acid (VFA) absorption. A final study was conducted to determine the acute effects of ergot alkaloids on isolated rumen epithelial absorptive and barrier functions and the potential for ruminal ergovaline absorption. Results indicate that acute exposure to ergot alkaloids does not alter the absorptive or barrier function of rumen epithelium and ergovaline is absorbed from the rumen. Data from this series of experiments have shown that ergot alkaloids from E+ tall fescue can induce vasoconstriction of blood vessels in the foregut of cattle, reduce blood flow to the rumen epithelium, and decrease VFA absorption. The decrease in nutrient absorption could contribute to the observed symptoms of fescue toxicosis, including depressed growth rates and general unthriftiness.

Previous findings revealed that engagement of SGLT-1 by orally administered D-glucose protects the intestinal mucosa from lipopolysaccharides (LPS)-induced injury. Here, we tested whether engagement of SGLT-1 protected also from doxorubicin (DXR)/5-fluorouracil (5-FU)-induced and DSS-induced intestinal injuries using a new synthetic compound named BLF501 as SGLT-1 agonist. A large set of experiments was performed in order to assess damages induction and recovery after treatment with DXR/5-FU and DSS with or without co-treatment with BLF501 with particular attention for intestinal epithelial integrity. We evaluated the preservation of correct epithelial structure, correct formation and functionality of junctional systems, electrophisiological properties and physiological functionality of small and large intestine. In conclusion, oral administration of BLF501 greatly accelerated recovery from mucosal injury induced by DXR (alone or in combination with 5-FU) and DSS. These data suggest a novel therapeutic approach to reduce the severity of chemotherapy-induced mucositis and a new approach for IBDs treatment.

Ulcerative colitis is a relapsing and remitting inflammatory bowel disease involving the large bowel. The current hypothesis on the pathogenesis of UC is that an abnormal innate immune response in genetically susceptible individuals, combined with environmental factors, result in excessive activation of the adaptive immune system within lamina propria. The role of abnormal barrier function is widely accepted. Using transcriptomic analysis of punch biopsies of patients with quiescent UC, CLDN8 was identified as grossly downregulated in the intestine. In this thesis, loss of Cldn8, a tight junction (TJ) molecule, was shown to result in reduced susceptibility of mice to DSS-induced colitis. Cldn8 knock out (Cldn98-KO) mice, had smaller increase in intestinal permeability to 3H-mannitol, reduced neutrophils and macrophages in inflammatory cell infiltrate in lamina propria during the early phase of inflammation. The inner layer of mucous is sterile in naïve Cldn8-KO and WT mice, and remains sterile after the animals have been exposed to DSS-water for 12 hours. Transcriptomic analysis between Cldn8-KO and WT mice did not reveal any significant differences between the two groups at different time points. After correction for multiple-testing, no differentially-expressed genes remained. These results suggest that downregulation of CLDN8 in patients with UC is a physiologic response by the intestine to increase local defences against luminal pathogens.

Thesis (Ph.D.)--Boston University
HIV-infected macrophages are frequently present in semen of untreated HIV-infected men. After intercourse, there is evidence that these cells infiltrate from the vaginal epithelial surface into the mucosa (apical to basal infiltration) and transmit HIV to a new host. Since women with cervicovaginal inflammation are at an increased risk of acquiring HIV, we hypothesized that epithelial inflammation enhances cell-associated HIV transmission via increased seminal leukocyte infiltration into the vaginal mucosa. In order to investigate this mechanism, we utilized an in vitro reconstructed human vaginal epithelium that is structurally similar to normal human mucosa. We have used confocal microscopy to characterize the kinetics of apical to basal infiltration of Phorbol-12- myristate-13-acetate activated U937 macrophages as well as non-infected and HIV-infected primary monocyte-derived macrophages (MDMs) through this vaginal mucosal model. Within two hours of placement on the apical side of the epithelium, both non-infected and HIV-infected macrophages were abundant within the uppermost cell layers (stratum corneum) and a subpopulation of these cells were observed to have infiltrated through intact epithelial junctions to reach depths ranging from 50-200 µm. Infiltration of both non-infected and infected cells was significantly enhanced when the Epivaginal tissues were pre-treated with TNF-alpha, an important pro-inflammatory cytokine. Through microarray analysis and Enzyme-linked immunosorbent assay (ELISA), we demonstrated that exposure to TNF-alpha upregulated transcription and/or expression of several chemotactic factors and matrix metalloproteases, the presence of which may have contributed to increased leukocyte infiltration into inflamed epithelium. Additionally, TNF-alpha treatment resulted in a dramatic increase in epithelial expression of intercellular adhesion molecule 1 (ICAM-1). These data provide evidence that HIV-infected macrophages can infiltrate into the normal human vaginal epithelium from the vaginal lumen after intercourse, and that cervicovaginal inflammation enhances infiltration. These studies provide further insight into potential mechanisms of cell-associated HIV transmission and suggest that strategies to inhibit vaginal inflammation, and/or block leukocyte attachment and infiltration may be a relevant approach to prevent HIV transmission.

Las patologías funcionales (síndrome del intestino irritable, IBS) e inflamatorias gastrointestinales (enfermedad inflamatoria intestinal, IBD) se caracterizan por alteraciones de la función barrera epitelial, con un aumento de la permeabilidad, y cambios en la microbiota intestinal. Los receptores de tipo Toll (TLRs) participan en el reconocimiento bacteriano en el intestino y en el control neuroinmune local, estando, por tanto, implicados en la regulación de la función barrera del epitelio intestinal. El objetivo de este trabajo ha sido caracterizar la implicación de los receptores TLR5 y TLR7 en la regulación de la función barrera epitelial del colon. Para ello se ha caracterizado la función barrera epitelial, tanto en condiciones in vitro (electrofisiología y permeabilidad a macromoléculas en un sistema de cámaras de Ussing), como in vivo (permeabilidad a macromoléculas), tras la sobre-estimulación local de los receptores TLR5 y TLR7 con agonistas selectivos, flagelina e imiquimod, respectivamente, en rata y ratón. Los efectos en la función barrera se han caracterizado en condiciones normales, en estados de permeabilización del epitelio con DMSO, y en condiciones de inflamación (colitis inducida por dextrano sulfato de sodio -DSS-). Con la finalidad de definir el mecanismo de acción, se ha valorado la dinámica de las uniones estrechas epiteliales (expresión génica de proteínas -RT-qPCR- y distribución celular -inmunohistoquímica-) y la activación inmune local (expresión de citoquinas pro-inflamatorias). Los resultados obtenidos muestran que la sobre-estimulación del TLR7 del colon in vivo mejora la función barrera epitelial en la rata en condiciones fisiológicas, observando una reducción dosis-dependiente de la permeabilidad epitelial a macromoléculas evaluada en las cámaras de Ussing. No obstante, en condiciones de permeabilización del epitelio con DMSO, la sobre-estimulación del TLR7 causa un empeoramiento de la función barrera valorada in vivo. En ratones, la sobre-estimulación del TLR7 cólico in vitro no tiene efecto. Sin embargo, en un modelo de colitis inducida por DSS, reduce el aumento de la permeabilidad epitelial causado por la inflamación. Por tanto, parecen existir diferencias especie-específicas en los efectos de la sobre-estimulación del TLR7 cólico, pudiéndose observar tanto acciones promotoras como lesivas de la función barrera epitelial. La sobre-estimulación del TLR5 cólico agrava la disfunción de la barrera asociada a la inflamación (colitis inducida por DSS) en el ratón, incrementando la permeabilidad a macromoléculas. Sin embargo, la adición del agonista del TLR5, flagelina, en las cámaras de Ussing no afecta a la función barrera epitelial, ni en condiciones fisiológicas, ni durante la inflamación. En ningún caso, estos efectos moduladores de la función barrera se asociaron a cambios en la expresión génica de las principales proteínas de las uniones estrechas (claudina-2, claudina-3, ocludina, tricelulina, molécula de adhesión de la unión de tipo 1 y Zonula Occludens 1) ni a su distribución celular (claudina-2, claudina-3 y ZO-1). De la misma forma, los factores moduladores de la barrera, quinasa de la cadena ligera de la miosina y pro-glucagón (precursor del péptido similar al glucagón de tipo 2), tampoco presentaron cambios en su expresión asociados a la sobre-estimulación del TLR5 o del TLR7. Finalmente, se observó un efecto inmunomodulador receptor-específico. La sobre-estimulación del TLR7 reveló efectos potencialmente protectores al reducir la expresión de la citoquina pro-inflamatoria IL12-p40. Por el contrario, la sobre-estimulación del TLR5 tendió a aumentar la expresión de marcadores pro-inflamatorios, sugiriendo, por tanto, efectos pro-lesivos. En conclusión, estos resultados muestran la importancia de las interacciones microbiota-hospedador mediadas por TLRs en el control de la función barrera epitelial intestinal. Tanto el TLR7 como el TLR5 cólicos pueden considerarse potenciales dianas terapéuticas para el control de la función barrera y las respuestas inmunes locales en desórdenes funcionales e inflamatorios gastrointestinales como el IBD y el IBS.
Functional (irritable bowel syndrome, IBS) and inflammatory (inflammatory bowel disease, IBD) gastrointestinal disorders are characterized by an altered epithelial barrier function, with an increased permeability, and changes in the intestinal microbiota. Toll-Like Receptors (TLRs) participates in bacterial recognition within the intestine and in local neuro-immune control, thus participating in the regulation of intestinal epithelial barrier function. The objective of this work has been to characterize the implication of TLR5 and TLR7 in the regulation of colonic epithelial barrier function. For this, colonic epithelial barrier function has been studied in vitro (electrophysiology and permeability to macromolecules in a Ussing chamber system), as well as in in vivo conditions (permeability to macromolecules), after the local over-stimulation of TLR5 and TLR7 with selective agonists, flagellin and imiquimod, respectively, in rats and mice. The effects on barrier function have been studied in normal conditions, under states epithelial permeabilization with DMSO, and in conditions of inflammation -dextran sulfate sodium (DSS)-induced colitis-. In order to characterize the mechanisms of action, dynamics of tight junction (gene expression -RT-qPCR- and cellular distribution -immunohistochemistry- of tight junction proteins) and the presence of a local immune activation (gene expression of pro-inflammatory cytokines) were assessed. The results obtained indicate that the in vivo over-stimulation of colonic TLR7 improves epithelial barrier function in rats in physiological conditions, with a dose-dependent reduction in epithelial permeability to macromolecules, as assessed in Ussing chambers. However, under conditions of epithelial permeabilization with DMSO, the over-stimulation of TLR7 deteriorates barrier function, as assessed in vivo. In mice, the in vitro over-stimulation of colonic TLR7 was without effects. However, in a model of DSS-induced colitis, imiquimod reduces inflammation-induced increased epithelial permeability. Therefore, specie-specific differences seemed to exist for the barrier effects associated to the over-stimulation of colonic TLR7, leading to either protective or damaging actions on epithelial barrier function, depending upon the experimental conditions. The over-stimulation of colonic TLR5 aggravates the barrier dysfunction associated to inflammation (DSS-induced colitis) in mice, increasing the permeability to macromolecules. However, the direct addition of flagellin to the Ussing chambers did not affect epithelial barrier function, neither in physiologic conditions nor during inflammation. Regardless the conditions considered, TLR5/7-mediated modulatory actions on barrier function were not associated to changes in gene expression of the main tight junction-related proteins (claudin-2, claudin-3, occludin, tricellulin, junctional adhesion molecule 1 and Zonula Occludens 1). Moreover, no changes in the cellular distribution of tight junction proteins (claudin-2, claudin-3 y ZO-1) was observed. Likewise, TLR5/7 over-stimulation was not associated to changes in the expression of the barrier-modulating factors myosin light chain kinase and proglucagon (precursor of glucagon-like peptide 2). Finally, TLR-specific immunomodulatory effects were also observed. Over-stimulation of TLR7 revealed potential protective effects, reducing the expression of the pro-inflammatory cytokine IL12-p40. In contrast, over-stimulation of TLR5 tended to increase the expression of pro-inflammatory markers, thus suggesting pro-damaging effects. In conclusion, these results provide evidence of the importance of TLRs-dependent host-microbial interactions in the control of intestinal epithelial barrier function. Colonic TLR5 and TLR7 should be considered potential therapeutic targets for the control of barrier function and local immune responses in functional and gastrointestinal disorders, such as IBD and IBS.

Doctor of Philosophy
Department of Diagnostic Medicine/Pathobiology
Ronette Gehring
Milk composition has a dynamic nature, and the composition varies with stage of lactation, age, breed, nutrition and health status of the udder. The changes in milk composition seem to match the changes in the expression of membrane proteins in secretory mammary epithelial cells that are needed for the movement of molecules from blood to milk and vice versa (Nouws and Ziv, 1982). Thus, an understanding of transporter expression, function and regulation in mammary epithelial cells can provide insight into mammary gland function and regulation. The goal of this project was to elucidate (molecularly and functionally) the role of drug transporters in the barrier function of an epithelial monolayer cultured from an immortalized bovine mammary epithelial cell line (BME-UV). To characterize the regulation (expression and function) of these drug transporters in BME-UV cells after exposure to cytokine TNF-α for selected periods of time. Representative members of drug transporters of the SLC (OCT and OAT) and ABC (P-glycoprotein) superfamilies were chosen for this project. In the first study, the involvement of a carrier-mediated transport system in the passage of organic cation (TEA) and anion (EsS) compounds was elucidated across the BME-UV monolayer. In the second study, molecular and functional expression of bOAT isoforms in BME-UV cells were studied. The final study characterized the effects of cytokine TNF-α on the expression and function of P-glycoprotein, an efflux pump, in BME-UV cells. Cytokine TNF-α exposure induced the expression of ABCB1 mRNA and increased P-glycoprotein production in BME-UV cells, resulting in a greater efflux of digoxin, a known P-glycoprotein substrate, back into the apical fluid. The expression, function, and regulation of these transporters in the mammary gland has important implications for understanding the barrier function of the mammary epithelium and, in more specific, for characterizing the role of these transporters in the accumulation and/or removal of specific substrates from milk and/or plasma. Moreover, this study provides an in vitro cell culture model of mammary epithelium to characterize mammary epithelial cell function during inflammation.

The mucosal surfaces of the gastrointestinal and respiratory tracts are the main interfaces between the environment and the host. All two are protected by continuous epithelia that prevents the entry of microbes and integrity loss of these epithelia commonly predispose to infection. On the other hand the epithelial surfaces provide essential absorptive functions for the intake of food and air. SGLT-1 is a co-transporter able to absorb D-glucose, against a concentration gradient, together with Na+. The expression of SGLT-1 on the apical membrane of enterocytes, the cells that line the gut and overlook to the intestinal lumen, is fundamental in order to obtain the maximum D-glucose absorption from the digested alimentary bolo that transits through the gut. Accumulating data support the notion that SGLT1 orchestrates a number of fundamental cellular processes besides its canonical absorptive function. Presented results show a novel role of SGLT-1: this protein if appropriately activated modulates the immune response and protect barrier function. Our recent findings indicate that the activation of SGLT-1, present at apical membrane of enterocytes inhibits bacteria-induced inflammatory processes and lifesaving treatments, assuming a role as an immunological player. The main drawback of this activation is the high level of glucose that must be administrated (2.5 g/kg) in vivo so to achieve protection. In this contest we have developed a new glucoderivatives, named BLF501, able to “activate” SGLT-1 in order to achieve the protection against damages induced by LPSs while avoiding the disadvantages caused by high glucose concentration.. Thus our new synthetic molecule BLF501, acting as a potent activator of SGLT-1 at very low dosages, might represent a new pharmacological drug for the treatment of IBD, given the cytoprotective and anti-inflammatory effects linked to SGLT-1 activation. Infact BLF501 stabilizes TJ-protein localization preventing INF-γ/TNF-α. or DSS-mediated degradation. BLF501 is very effective at preventing functional (FD-3 flux) and morphological (TJ protein) permeability defects induced by inflammatory or chemically stimuli. In vivo experiments utilizing a chemically-induced mouse model of intestinal inflammation, we found that mice with acute or chronic colitis BLF-501-treated not presents typical mucosal injury, shows a weight recovery and not develops severe clinical symptoms, including bleeding and dehydratation. The TJ protein has been reported to be deregulated in IBD. We have analyzed occludin and ZO-1 localization in colon tissue of different treatments and we have evaluated that intestinal permeability recovery observed with Ussing Chamber analysis is mediated by TJ protein protection. Results suggest that the BLF-501-mediated action involves stabilization of epithelial junction complex also in vivo. Moreover, we observed that activation of pro-inflammatory cytokines, such as TNF-α, and IL-12 in acute and chronic colitis was suppressed by oral administration of BLF-501. We have observed a marked increase of IL-10 levels in mice treated with BLF-501 and acute and chronic cycles of DSS in comparison with DSS alone or with untreated mice. The continuous IL-10 production from immune system components, guaranteeing an endogenous source of this anti-inflammatory cytokine, is able, to down- The sodium-dependent glucose transporter-1 (SGLT-1) molecule is expressed by intestinal epithelial cells and by pneumocytes. We show here that BLF501, induces protective anti-inflammatory effects in lung of mice exposed to aerosolized lipopolysaccharide (LPS) or to ovalbumin (OVA), as assessed by analysis of serum, brochoalveolar lavage and lung morphology of the mice in both experimental models. Findings in the OVA-induced asthma murine model that aerosol and oral, administration of BLF501 led to a marked decrease of bronchoalveolar cellular infiltrate and of IL-4, IL-5, NO and IgE levels, and increased levels of the anti-inflammatory cytokine IL-10, suggest the promise of SGLT-1 activation by BLF501 as a new approach to improving asthma pathology. We can conclude thatBLF-501 as ligand of SGLT-1 may be suggested as a new pharmacological approach for the treatment of different inflammatory diseases as IBD and asthma.

Indiana University-Purdue University Indianapolis (IUPUI)
As mass production of carbon nanoparticles (CNPs) continues to rise, the likelihood of occupational and environmental exposure raises the potential for exposure‐related health hazards. Although many groups have studied the effects of CNPs on biological systems, very few studies have examined the effects of exposure of cells, tissues or organisms to low, physiologically relevant concentrations of CNPs. Three of the most common types of CNPs are single wall nanotubes (SWNT), multi wall nanotubes (MWNT) and fullerenes (C60). We used electrophysiological techniques to test the effects of CNP exposure (40 μg/cm2 – 4 ng/cm2) on barrier function and hormonal responses of well characterized cell lines representing barrier epithelia from the kidney (mpkCCDcl4) and airways (Calu‐3). mpkCCDcl4 is a cell line representing principal cell type that lines the distal nephron in an electrically tight epithelia that aids in salt and water homeostasis and Calu‐3 is one of the few cell lines that produces features of a differentiated, functional human airway epithelium in vivo. These cell lines respond to hormones that regulate salt/water reabsorption (mpkCCDcl4) and chloride secretion (Calu‐3). In mpkCCDcl4 cells, after 48 hour exposure, the transepithelial electrical resistance (TEER) was unaffected by high concentrations (40 – 0.4 μg/cm2) of C60 or SWNT while lower, more relevant levels (< 0.04 μg/cm2) caused a decrease in TEER. MWNT decreased TEER at both high and low concentrations. CNT exposure for 48 hour did not change the transepithelial ion transport in response to anti‐diuretic hormone (ADH). In Calu‐3 cells, after 48 h of exposure to CNPs, fullerenes did not show any effect on TEER whereas the nanotubes significantly decreased TEER over a range of concentrations (4 μg/cm2‐0.004 ng/cm2). The ion transport response to epinephrine was also significantly decreased by the nanotubes but not by fullerenes. To look at the effect of exposure times, airway cells were exposed to same concentrations of CNPs for 24 and 1h. While the 48 h and 24 h exposures exhibited similar effects, there was no effect seen after 1h in terms of TEER or hormonal responses. In both the cell lines the magnitude of the transepithelial resistance change does not indicate a decrease in cellular viability but would be most consistent with more subtle changes (e.g., modifications of the cytoskeleton or changes in the composition of the cellular membrane). These changes in both the cell lines manifested as an inverse relationship with CNP concentration, were further corroborated by an inverse correlation between dose and changes in protein expression as indicated by proteomic analysis. These results indicate a functional impact of CNPs on epithelial cells at concentrations lower than have been previously studied and suggest caution with regard to increasing CNP levels due to increasing environmental pollution.

The epithelial cell layer that lines the intestine creates a barrier, largely mediated by the tight junction (TJ) apparatus, which serves as a first line of protection from the contents in the lumen containing an enormous number of microbes. Cellular microbiology, the study of microbial-host interactions, is used to describe mechanisms that play a role in the way epithelial cells regulate barrier properties in the context of bacterial colonization. The research in this thesis had three aims: (1) to characterize the effects of candidate pathogenic bacteria on the epithelial barrier, (2) to determine if a beneficial microbe (a probiotic bacterium) could ameliorate the deleterious effects of a pathogenic infection on this barrier, and (3) to extend the investigation of probiotic mechanisms in the context of pro-inflammatory cytokine-mediated barrier dysfunction. In the first part of this thesis, two undercharacterized bacterial species purported to cause diarrheal illness, Escherichia albertii and Hafnia alvei, were employed in a polarized epithelial infection model with outcome measures including transepithelial electrical resistance (TER), macromolecular permeability, TJ protein immunofluorescence staining, and immunoblotting. A well characterized pathogen, enterohemorrhagic Escherichia coli (EHEC), serotype O157:H7, was used as a positive control to demonstrate deleterious effects on TJs. All of the bacteria tested decreased TER, but the effects on TJs and TJ protein expression were specific to the bacterial strain tested and the epithelial model cell line used. The second component of this thesis investigated how probiotics confer beneficial effects on epithelial barrier function. A probiotic bacterium, Lactobacillus rhamnosus GG (LGG), was employed to effectively block EHEC O157:H7 adherence to epithelial cells and prevent the ability of the pathogen to induce characteristic attaching-effacing lesions on epithelial cell surfaces. LGG ameliorated the pathogenic effects on barrier function normally induced by EHEC O157:H7, including prevention of decreased TER, increased permeability to a dextran probe, and rearrangement of tight junction architecture. The third section elucidated the role of LGG in the prevention of barrier disruption due to pro-inflammatory cytokine stimuli (IFN-γ and TNF-α). Using a polarized epithelial (Caco-2bbe) cell model, LGG treatment largely prevented cytokine-induced decreases in TER and TJ disruption. Furthermore, LGG suppressed the secretion of the chemokines interleukin-8 (CXCL-8) and eotaxin-1 (CCL-11), and the activation of NF-κB. Preliminary experimentation demonstrated a role for mitogen-activated protein kinases, with pharmacologic inhibition of extracellular signal related kinase (ERK-1/2) abolishing the protective effects of LGG. Taken together, the findings presented in this thesis demonstrate how cellular microbiology models can be used to study host-microbial interactions, giving insight as to how the intestinal epithelium regulates barrier function; characterizing enteropathogenic candidates, and the diversity in responses to these bacteria that is dependent on both the bacterial strain and the epithelial cell line tested; and elucidating the mechanisms of probiotic action to reduce the deleterious effects of infection and inflammation.

Mechanical ventilation inevitably exposes the delicate tissues of the airways and alveoli to a wide range of abnormal stresses that can induce pulmonary edema and initiate or exacerbate life-threatening conditions such as acute lung injury and acute respiratory distress syndrome. The goal of our research is to characterize the cellular trauma caused by the transient, abnormal mechanical stresses that arise when air is forced into a liquidoccluded airway. Using a fluid-filled parallel-plate flow chamber to model this 'airway reopening' process, our in vitro study specifically examined consequent increases in pulmonary epithelial plasma membrane rupture, paracellular permeability, and tight junction (TJ) protein disruption. Through this analysis, we observed a distinct, unexpected drop in pulmonary epithelial injury during particularly traumatic reopening events if DeltaPcell -- the reopening-induced fore-aft pressure difference exerted across the cells -- was greater than a critical value, DeltaPcrit. Namely, when DeltaP cell < DeltaPcrit, plasma membrane rupture, paracellular permeability and TJ disruption were magnified by increases in DeltaP cell. On the other hand, further enhancing DeltaPcell beyond DeltaP crit consistently and dramatically diminished cell wounding. From these results, we propose that a passive DeltaPcell-induced shift in pulmonary epithelial rheology when DeltaPcell > DeltaP crit prevents excessive cellular trauma during airway reopening which may be exploited to improve patient prognosis during mechanical ventilation
acase@tulane.edu

博士
國立臺北科技大學
工程科技研究所
101
Lactobacillus shows beneficial anti-inflammatory effects to Salmonella infection. The maintenance of the tight junction (TJ) integrity plays an importance role in avoiding bacterial invasion. Whether Lactobacillus could be used to regulate the TJ protein expression and distribution in inflamed intestinal epithelial cells was determined. Using the transwell co-culture model, Salmonella lipopolysaccharide (LPS) was apically added to polarized Caco-2 cells co-cultured with peripheral blood mononuclear cells in the basolateral compartment. LPS-stimulated Caco-2 cells were incubated with various Lactobacillus strains. TJ integrity was determined by measuring trans-epithelial electrical resistance across Caco-2 monolayer. Expression and localization of TJ proteins (zonula occludens (ZO)-1) were determined by western blot and immunofluorescence microscopy. Various strains of Lactobacillus were responsible for the different modulations of cell layer integrity. LPS was specifically able to disrupt epithelial barrier and change the location of ZO-1. Our data demonstrate that Lactobacillus could attenuate the barrier disruption of intestinal epithelial cells caused by Salmonella LPS administration. We showed that Lactobacillus strains are associated with the maintenance of the tight junction integrity and appearance. In this study we provide insight that live probiotics could improve epithelial barrier properties and this may explain the potential mechanism behind to their beneficial effect in-vivo.

The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.