Role of the epithelial sodium channel (ENaC) and its positive regulator, the channel-activating protease 1 (CAP1) in skin

Summary: The mammalian epidermis is a pluristratified epithelium composed of 90% keratinocytes, and its main function is to serve as barrier for the body. The epithelial sodium channel (ENaC), formed by three homologous subunits α, β and γ is found in a variety of epithelia including epidermis. Previous studies showed that ENaC modulates different aspects of epidermal differentiation, such as synthesis of differentiation-specific proteins and lipid secretion. ENaC plays also a critical role in sodium homeostasis of renal and pulmonary epithelia, and its activity is thereby well controlled by hormones and non-hormonal factors, such as the serine protease CAP1 (channel-activating protease 1), also termed prostasin encoded by Prss8 gene. Serine proteases are proteolytic enzymes involved in numerous physiological and pathological processes in the epidermis. In order to evaluate the role of β and γENaC in epidermis, we analyzed the skin phenotype of β and γENaC null mutant (βENaC-/- and γENaC-/-) mice in comparison with the phenotype of αENaC-deficient mice. Furthermore, keratin14-specific CAP1-deficient mice (Prss8lox/Δ /K14-Cre) were generated in order to unveil the role of the serine protease CAP1 in epidermal development and function. This study reveals that the skin phenotype of βENaC and γENaC null mutant mice is less severe than the one of αENaC-deficient mice. However, all these mice present a common premature lipid secretion in the mid-granular layer of the epidermis. Further, the composition of the lipids of the stratum corneum in αENaC-deficient mice is strongly altered, suggesting that epidermal barrier function is compromised. K14-specific CAP1-deficient newborn mice are born at the expected Mendelian ratio, but die soon after birth, showing that CAP1 is required for postnatal survival. The epidermis of these mice exhibits striking malformations of the stratum corneum showing hyperkeratosis. These defects seriously affect both inward and outward epidermal barrier function, leading to rapid and fatal dehydration. As in αENaC-deficient mice, the lipid composition of the stratum corneum of K14-specific CAP1-deficient mice is disturbed. Furthermore, lack of CAP1 leads to the selective loss of filaggrin monomers, important for keratins aggregation and skin moisturization, and to an increased of aberrant profilaggrin precursors. In conclusion, both ENaC and CAP1 expression in the epidermis are crucial for keratinocyte differentiation processes and/or barrier function. Since the abnormalities in K14-specific CAP1-deficient mice resemble key features of human skin ichthyosis, in particular Harlequin ichthyosis, the study of ENaC and CAP1 mutant mice might allow new insights into mechanisms underlying skin diseases. Résumé: L'épiderme des mammifères est un épithélium pluristratifié, protégeant le corps contre les perturbations extérieures et la déshydratation. Le canal épithélial à sodium (ENaC), formé de trois sous-unités α, β et γ, est exprimé dans de nombreux épithélia, comme l'épiderme. Des études ont montré que l'absence de la sous-unité αENaC modulait différents aspects de la différenciation des kératinocytes de l'épiderme, comme la synthèse de protéines spécifiques ou la sécrétion de lipides dans la couche granulaire de l'épiderme. ENaC joue également un rôle crucial dans l'homéostasie du sodium dans les épithélia électriquement étanches, comme l'épithélium rénal ou pulmonaire. L'activité de ENaC est par conséquent finement régulée, en partie par des hormones, mais aussi par des facteurs non-hormonaux, telle que la sérine protéase CAP1 (« channel-activating protease 1 >>) (nommée également prostasine et codée par le gène Prss8). Le but de ce travail a donc été d'étudier le rôle des sous-unités β et γENaC dans l'épiderme en comparaison avec celui de la sous-unité α en utilisant des souris mutantes βENaC-/- et γENaC-/-. Dans un deuxième temps, le phénotype d'une souris chez qui CAP1 a été spécifiquement invalidé dans l'épiderme (Prsslox/Δ/K14-Cre) a été analysé, dans le but de mettre en évidence le rôle de cette protéase dans l'épiderme. Comme déjà montré pour les souris αENaC-/-, la sécrétion des lipides dans la couche granulaire de l'épiderme des souris βENaC-/- et γENaC-/- est prématurée. Cependant, l'hyperplasie et l'expression anormale des protéines marqueurs de la différenciation présents chez les souris αENaC-/- n'ont pas été observés dans l'épiderme des souris βENaC-/- et γENaC-/-. La composition lipidique de la couche cornée des souris αENaC-/- est fortement altérée suggérant que la fonction de barrière de l'épiderme de ces souris est compromise. Les souris mutantes CAP1 ont quant à elles révélé des malformations sévères de leur couche cornée, affectant la fonction de barrière de leur épiderme et conduisant à la mort de ces souris par déshydratation quelques jours après leur naissance. De plus, la composition en lipides de la couche cornée ainsi que la taille des cellules cornées, les cornéocytes, de ces souris sont modifiées par rapport aux souris contrôles. L'invalidation de la protéine CAP1 dans l'épiderme conduit aussi à la perte de la filaggrine, une protéine cruciale pour l'agrégation des kératines dans la couche cornée et le maintien du niveau d'hydratation de la peau, et à l'accumulation de ses précurseurs. En conclusion, l'expression de ENaC et de CAP1 est cruciale pour la différenciation de l'épiderme et/ou sa fonction de barrière. De plus, le phénotype des souris mutantes CAP1 présente des caractéristiques qui ressemblent à celles observées dans certaines pathologies humaines cutanées, comme l'ichthyose d'Harlequin. L'étude des souris mutantes ENaC et CAP1 pourrait donc apporter de nouvelles connaissances dans les mécanismes impliqués dans l'ichthyose d'Harlequin ou d'autres maladies de la peau chez l'homme. Résumé tout public: La peau est le plus grand organe vital du corps humain. Sa fonction principale est de protéger le corps comme une barrière, contre les agressions extérieures et la déshydratation. De nombreuses maladies de la peau résultent d'une perte de fonction de cette barrière. Bien que les pathologies cutanées soient très bien décrites, leur cause génétique n'est en général pas encore connue. La souris est alors un modèle de choix pour la recherche fondamentale. En effet, grâce aux progrès récents de la science, le génome de la souris peut aujourd'hui être modifié dans le but d'étudier le rôle de nombreuses protéines. Dans différents organes, comme le rein et le poumon, le canal épithélial à sodium (ENaC), composé de trois sous-unités protéiques homologues (α, β, et γ), joue un rôle essentiel dans la réabsorption du sodium. L'activité de ENaC est régulée par de nombreux facteurs hormonaux et non-hormonaux, telle que la protéase CAP1 (« channel-activating protease 1 »). L'invalidation de la sous-unité αENaC chez la souris a permis de montrer que dans la peau, le canal ENaC est impliqué dans la différenciation des cellules de l'épiderme et la croissance des poils. Durant ce travail, le phénotype des souris chez qui la protéine βENaC, γENaC ou CAP1 a été invalidée (souris mutantes), a été étudié dans le but de mieux comprendre le rôle des sous-unités du canal ENaC et de son régulateur CAP1 dans la peau. Les résultats de ce projet ont montré que les souris mutantes βENaC et γENaC présentent un épiderme anormal avec une synthèse prématurée de lipides dans la couche granulaire, suggérant l'implication de ENaC dans la fonction de barrière de la peau. De plus, quand CAP1 est invalidé de manière totale chez les souris, le développement embryonnaire est perturbé et ces souris meurent avant la naissance. CAP1 a donc été invalidé spécifiquement dans l'épiderme des souris. Ces souris mutantes « épiderme-spécifique » naissent normalement, mais meurent peu après la naissance de déshydratation. La couche superficielle de l'épiderme, appelée couche cornée, de ces souris est malformée et ne confère plus à la peau sa fonction de barrière. De plus, les composants de la couche cornée, les cellules cornées entourées de lipides, sont sévèrement altérés. Le phénotype de ces souris ressemble aux caractéristiques présentes chez les patients atteints d'ichthyoses, en particulier l'ichthyose d'Harlequin. En conclusion, le canal ENaC ainsi que son régulateur CAP1 jouent un rôle clé dans les processus de différenciation de l'épiderme et/ou de sa fonction de barrière. De plus, les souris mutantes pour CAP1 et ENaC se révéleront peut-être comme des modèles appropriés dans l'étude de l'ichthyose d'Harlequin ou d'autres maladies cutanées.

IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus

Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

The roles of PKCδ and Src kinases in the glucocorticoid induction and the TGF-β superinduction of the mouse mammary tumor virus transcription in B lymphocytes

Résumé : Le virus tumoral de la glande mammaire de la souris (MMTV) est un rétrovirus provoquant le développement de tumeurs dans les glandes mammaires des souris susceptibles femelles. Au cours de son évolution, le virus s'est adapté et s'exprime dans des cellules spécialisées. Les lymphocytes B sont les premières cellules infectées et elles sont essentielles pour la propagation de l'infection aux glandes mammaires. Dans notre étude, le virus MMTV a été utilisé afin d'examiner les voies de signalisation induites par les glucocorticoïdes (dexaméthasone (dex), une hormone stéroïdienne) et le transforming growth factor-f3 (TGF-P, une cytokine), deux molécules impliquées dans l'activation de la transcription à partir du promoteur du MMTV dans les cellules B. Le TGF-P seul n'influence pas l'activité du promoteur du MMTV. Par contre, en synergie avec dex, le TGF-P provoque une super-induction de l'expression du promoteur par rapport à une stimulation par le glucocorticoïde seul. Cette super-induction est régulée par une famille de protéines, les Smads. Ainsi, dans les lymphocytes B, l'utilisation du MMTV a permis de mettre en évidence une nouvelle synergie entre les glueocortieoïdes et le TGF-p. pans ce travail, l'utilisation d'inhibiteurs pharmacologiques et de mutants « dominant-négatifs » nous a pet mis de démontrer qu'une Protéine Kinase C delta (PKC5) active est impliquée dans la transduction du signal lors de la réponse au dex ainsi que celle au TGF-P. Néanmoins, la PKC5 est régulée différemment dans chaque voie spécifique : la voie du TGF-p nécessitait l'activation du PKC5 par diacylglycerol (DAG) et la phosphorylation de tyrosines spécifiques, alors que la voie impliquant les glucocorticoïdes ne le nécessitait pas. Nous avons aussi démontré qu'une tyrosine kinase de la famille Src est responsable de la phosphorylation des tyrosines sur la PKC5. Les essais de kinase in vitro nous ont permis de découvrir que plusieurs Src kinases peuvent phosphoryler la PKC6 dans les cellules B et qu'elles étaient constitutivement actives. Enfin, nous avons montré qu'il existe une interaction protéine - protéine induite par dex, entre le récepteur aux glucocorticoïdes (GR) et la PKC5 dans les cellules B, une association qui n'a pas été démontrée auparavant. Par ailleurs, nous avons analysé les domaines d'interactions entre PKC5 et GR en utilisant les essais de «GST pull-down». Nos résultats montrent que le domaine régulateur de la PKC5 et celui qui interagit avec l'ADN du GR sont impliqués. En résumé, nous avons trouvé que dans une lignée lymphocytaire B, le virus MMTV utilise des mécanismes pour réguler à la fois la transcription et la voie de signalisation qui sont différents de ceux utilisés dans les cellules mammaires épithéliales et les fibroblastes. Nos découvertes pourraient être utilisées comme modèles pour l'étude de gènes cellulaires impliqués dans des processus tels qu'inflammation, immunité ou cancérogénèse. Summary: Mouse Mammary Tumor Virus (MMTV) is a retrovirus that causes tumors in the mammary glands of susceptible female mice and has adapted evolutionarily to be expressed in specialized cells. The B lymphocytes are the first cells to be infected by the MMTV and are essential for the spread of infection to the mammary glands. Here, we used the MMTV as a model system to investigate the signalling cascade induced by giucocorticoids (dexamethasone, "dex", a steroid hormone), and by Transforming Growth Factor-beta (TGF-P, a cytokine) leading to its transcriptional activation in B lymphocytes. By itself, TGF-I3 does not affect the basal activity of the MMTV promoter. However, TGF-13 significantly increases glucocorticoid-induced expression, through its effectors, the Smad factors. Thus, MMTV in B cells demonstrates a novel synergism between glucocorticoids and TGF-16. In this thesis project, we present evidence, based on the use of pharmacological inhibitors and of dominant-negative mutants, that an active Protein Kinase C delta (PKC6) is required as a signal transducer for the dex response and for the TGF-P superinduction as well. The PKC6 is differentially regulated in each specific pathway: whereas the TGF-13 superinduction required PKC6 to be activated by diacylglycerol (DAG) and to be phosphorylated at specific tyrosine residues, the glueocorticoid-induced pathway did not. We also showed that a protein tyrosine kinase of the Src family is responsible for the phosphorylation of tyrosines on PKC6. By performing in vitro kinase assays, we found that several Src kinases of B cells were able to phosphorylate PKC6 and that they were constitutively active. Finally, we demonstrate a dex-dependent functional protein-protein interaction between the glucocorticoid receptor (GR) and PKC6 in B cells, an association that has not been previously described. We further analysed the interacting domains of PKG6 and GR using in vitro GST pull-down assays, whereby the regulatory domain of PKC6 and the extended DNA-binding domain of the GR were involved. In summary, we found that in B-lymphoid cell lines, MMTV uses novel mechanisms of transcriptional control and signal transduction that are different from those at work in mammary epithelial or fibroblastic cells. These findings will be used as model for cellular genes involved in cellular processes such as immune functions, inflammation, or oncogenic transformation that may have a similar pattern of regulation.

Hes1 regulates corneal development and the function of corneal epithelial stem/progenitor cells.

Hes1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of Hes1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in Hes1-null mice. Hes1-null corneas manifested abnormal junctional specialization, cell differentiation, and less cell proliferation ability. Worthy of note, Hes1 is expressed mainly in the corneal epithelial stem/progenitor cells and is not detected in the differentiated corneal epithelial cells. Expression of Hes1 is closely linked with corneal epithelial stem/progenitor cell proliferation activity in vivo. Moreover, forced Hes1 expression inhibits the differentiation of corneal epithelial stem/progenitor cells and maintains these cells' undifferentiated state. Our data provide the first evidence that Hes1 regulates corneal development and the homeostatic function of corneal epithelial stem/progenitor cells.

Fibronectin modulates thymocyte-thymic epithelial cell interactions following Trypanosoma cruzi infection

Developing thymocytes interact with thymic epithelial cells (TECs) through cell-cell interactions, TEC-derived secretory moieties and extracellular matrix (ECM)-mediated interactions. These physiological interactions are crucial for normal thymocyte differentiation, but can be disrupted in pathological situations. Indeed, there is severe thymic atrophy in animals acutely infected with Trypanosoma cruzi due to CD4+CD8+ thymocyte depletion secondary to caspase-mediated apoptosis, together with changes in ECM deposition and thymocyte migration. We studied an in vitro model of TEC infection by T. cruzi and found that infected TEC cultures show a reduced number of cells, which was likely associated with decreased proliferative capacity, but not with increased cell death, as demonstrated by bromodeoxyuridine and annexin-V labelling. The infected TEC cultures exhibited increased expression of fibronectin (FN), laminin (LM) and type IV collagen. Importantly, treatment with FN increased the relative number of infected cells, whereas treatment with anti-FN or anti-LM antibodies resulted in lower infection rates. Consistent with these data, we observed increased thymocyte adhesion to infected TEC cultures. Overall, these results suggest that ECM molecules, particularly FN, facilitate infection of the thymic epithelium and that the consequent enhancement of ECM expression might be associated with changes in TEC-thymocyte interactions.

Ultrastructural and molecular analysis of Bowman's layer corneal dystrophies: an epithelial origin?

PURPOSE: Two mutations (R555Q and R124L) in the BIGH3 gene have been described in anterior or Bowman's layer dystrophies (CDB). The clinical, molecular, and ultrastructural findings of five families with CDB was reviewed to determine whether there is a consistent genotype:phenotype correlation. METHODS: Keratoplasty tissue from each patient was examined by light and electron microscopy (LM and EM). DNA was obtained, and exons 4 and 12 of BIGH3 were analyzed by polymerase chain reaction and single-stranded conformation polymorphism/heteroduplex analysis. Abnormally migrating products were analyzed by direct sequencing. RESULTS: In two families with type I CDB (CDBI), the R124L mutation was defined. There were light and ultrastructural features of superficial granular dystrophy and atypical banding of the "rod-shaped bodies" ultrastructurally. Patients from three families with "honeycomb" dystrophy were found to carry the R555Q mutation and had characteristic features of Bowman's dystrophy type II (CDBII). CONCLUSIONS: There is a strong genotype:phenotype correlation among CBDI (R124L) and CDBII (R555Q). LM and EM findings suggest that epithelial abnormalities may underlie the pathology of both conditions. The findings clarify the confusion over classification of the Bowman's layer dystrophies.

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures.

The epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the βENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 μm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 μm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.

Epithelial Na+ channel mutants causing Liddle's syndrome retain ability to respond to aldosterone and vasopressin.

Liddle's syndrome is a monogenic form of hypertension caused by mutations in the PY motif of the COOH terminus of beta- and gamma-epithelial Na+ channel (ENaC) subunits. These mutations lead to retention of active channels at the cell surface. Because of the critical role of this PY motif in the stability of ENaCs at the cell surface, we have investigated its contribution to the ENaC response to aldosterone and vasopressin. Mutants of the PY motif in beta- and gamma-ENaC subunits (beta-Y618A, beta-P616L, beta-R564stop, and gamma-K570stop) were stably expressed by retroviral gene transfer in a renal cortical collecting duct cell line (mpkCCDcl4), and transepithelial Na+ transport was assessed by measurements of the benzamil-sensitive short-circuit current (Isc). Cells that express ENaC mutants of the PY motif showed a five- to sixfold higher basal Isc compared with control cells and responded to stimulation by aldosterone (10(-6) M) or vasopressin (10(-9) M) with a further increase in Isc. The rates of the initial increases in Isc after aldosterone or vasopressin stimulation were comparable in cells transduced with wild-type and mutant ENaCs, but reversal of the effects of aldosterone and vasopressin was slower in cells that expressed the ENaC mutants. The conserved sensitivity of ENaC mutants to stimulation by aldosterone and vasopressin together with the prolonged activity at the cell surface likely contribute to the increased Na+ absorption in the distal nephron of patients with Liddle's syndrome.

Insulin-like growth factor-I and C-reactive protein during pregnancy and maternal risk of non-epithelial ovarian cancer: a nested case-control study.

BACKGROUND: Insulin-like growth factor-I (IGF-I) and C-reactive protein (CRP) may be positively associated with the risk of epithelial ovarian cancer (EOC) but no previous studies have investigated their associations with non-epithelial ovarian cancers (NEOC). METHODS: A case-control study was nested within the Finnish Maternity Cohort. Case subjects were 58 women diagnosed with sex cord-stromal tumors (SCST) and 30 with germ cell tumors (GCT) after recruitment. Control subjects (144 for SCST and 74 for GCT) were matched for age, parity, and date of blood donation of the index case. RESULTS: Doubling of IGF-I concentration was not related to maternal risk of either SCST (OR 0.97, 95% CI 0.58-1.62) or GCT (OR 1.13, 95% CI 0.51-2.51). Similarly, doubling of CRP concentrations was not related to maternal risk of either SCST (OR 1.10, 95% CI 0.85-1.43) or GCT (OR 0.93, 95% CI 0.68-1.28). CONCLUSIONS: Pre-diagnostic IGF-I and CRP concentrations during the first trimester of pregnancy were not associated with increased risk of NEOC in the mother. Risk factors for NEOC may differ from those of EOC.

The ubiquitin-proteasome system and signal transduction pathways regulating Epithelial Mesenchymal transition of cancer.

Epithelial to Mesenchymal transition (EMT) in cancer, a process permitting cancer cells to become mobile and metastatic, has a signaling hardwire forged from development. Multiple signaling pathways that regulate carcinogenesis enabling characteristics in neoplastic cells such as proliferation, resistance to apoptosis and angiogenesis are also the main players in EMT. These pathways, as almost all cellular processes, are in their turn regulated by ubiquitination and the Ubiquitin-Proteasome System (UPS). Ubiquitination is the covalent link of target proteins with the small protein ubiquitin and serves as a signal to target protein degradation by the proteasome or to other outcomes such as endocytosis, degradation by the lysosome or specification of cellular localization. This paper reviews signal transduction pathways regulating EMT and being regulated by ubiquitination.

Influence of the Paracoccidioides brasiliensis14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

The fungal strain Paracoccidioides brasiliensisremains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensismolecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensisuses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

Rôle des cellules souches épithéliales de la cornée et de leur micro-environnement dans le traitement des destructions de la surface oculaire [Darwin or Lamarck? Understanding the ocular surface and its normal or abnormal differentiation in order to cure ocular surface destruction with corneal opacification].

According to the World Health Organization, 5.1% of blindnesses or visual impairments are related to corneal opacification. Cornea is a transparent tissue placed in front of the color of the eye. Its transparency is mandatory for vision. The ocular surface is a functional unit including the cornea and all the elements involved in maintaining its transparency i.e., the eyelids, the conjunctiva, the lymphoid tissue of the conjunctiva, the limbus, the lacrymal glands and the tear film. The destruction of the ocular surface is a disease caused by : traumatisms, infections, chronic inflammations, cancers, toxics, unknown causes or congenital abnormalities. The treatment of the ocular surface destruction requires a global strategy including all the elements that are involved in its physiology. The microenvironnement of the ocular surface must first be restored, i.e., the lids, the conjunctiva, the limbus and the structures that secrete the different layers of the tear film. In a second step, the transparency of the cornea can be reconstructed. A corneal graft performed in a healthy ocular surface microenvironnement will have a better survival rate. To achieve these goals, a thorough understanding of the renewal of the epitheliums and the role of the epithelial stem cells are mandatory.

The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells.

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-gamma, but not TNF-alpha or IL-1 beta, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-alpha and IL-1 beta synergized with IFN-gamma induction in all three cell types. High levels of IP-10 protein (> 800 ng/ml) were detected in supernatants of IFN-gamma/TNF-alpha-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-gamma is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-gamma is highly expressed) play an important role in the recruitment of activated T cells.

Multifocal epithelial tumors and field cancerization: stroma as a primary determinant.

It is increasingly evident that cancer results from altered organ homeostasis rather than from deregulated control of single cells or groups of cells. This applies especially to epithelial cancer, the most common form of human solid tumors and a major cause of cancer lethality. In the vast majority of cases, in situ epithelial cancer lesions do not progress into malignancy, even if they harbor many of the genetic changes found in invasive and metastatic tumors. While changes in tumor stroma are frequently viewed as secondary to changes in the epithelium, recent evidence indicates that they can play a primary role in both cancer progression and initiation. These processes may explain the phenomenon of field cancerization, i.e., the occurrence of multifocal and recurrent epithelial tumors that are preceded by and associated with widespread changes of surrounding tissue or organ "fields."