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Claudins (CLDN1-CLDN24) are a family of tight junction proteins whose dysregulation has been implicated in tumorigeneses of many cancer types. In colorectal cancer (CRC), CLDN1, CLDN2, CLDN4, and CLDN18 have been shown to either be upregulated or aberrantly expressed. In the normal colon, CLDN1 and CLDN3-7 are expressed. Although a few claudins, such as CLDN6 and CLDN7, are expressed in CRC their levels are reduced compared to the normal colon. The present review outlines the expression profiles of claudin proteins in CRC and those that are potential biomarkers for prognostication.

Die Entwicklung echter Epithelien markiert den Beginn der Evolution der Eumetazoa. Ein wichtiges Merkmal ist das Vorhandensein von zellulären Junctions, d. h. von Occludensjunctions (Septate oder Tight Junctions) und Adhärenzjunctions. Zwei wichtige Proteine, die an diesen Verbindungen beteiligt sind, sind Claudine in Septate/Tight Junctions, die parazelluläre Poren bilden und den Wasser- und Ioneneinstrom kontrollieren, und klassische Cadherine in Adherens Junctions, die die richtige Zell-Zell-Adhäsion ermöglichen. Hydra vulgaris, ein einfacher Hydrozoe, exprimiert alle Gene, die für die Bildung von Adherens und Septate Junctions erforderlich sind, und eignet sich ideal für die Untersuchung der Evolution von Epithelien, da sie zur Schwestergruppe der Bilateria gehören und an der Basis der Eumetazoa positioniert sind. Bislang wurden 14 Claudin-Orthologe in Hydra vulgaris beschrieben. In dieser Studie identifizierten und charakterisierten wir insgesamt 33 Claudin-Orthologe auf der Grundlage ihrer Sequenz, Domänenorganisation und des vorhergesagten Faltungsverhaltens. Darüber hinaus zeigen Expressionskarten, die auf Einzelzell-RNA-Sequenzierung basieren, eine zelltyp- und gewebespezifische Kombination von Claudinen und eine starke Überlappung der ubiquitär exprimierten Claudin-1, -7 und -12 und der fußscheibenspezifischen Claudin-19 und -31, was einen heterophilen Porenaufbau plausibel macht. Diese Studie ist sowohl der letzte Teil einer ersten detaillierten Charakterisierung eines Claudin-Orthologs in einem nicht-bilateralen Modellsystem als auch ein Ausgangspunkt für die weitere Untersuchung der Funktionalität von Claudinen und der molekularen Architektur von Septatejunctions. Hydra vulgaris exprimiert ein einziges klassisches Cadherin-Ortholog, das in späten Nematozyten und ektodermalen Tentakelbatteriezellen hochreguliert ist. Wir haben ein RNAi-mediiertes Knockdown-Protokoll auf der Basis von Elektroporation von Polypen etabliert und die Funktion von Cadherinen b, The development of true epithelia marks the start of the evolution of Eumetazoa. One important characteristic is the existence of sealing junctions, i.e. occluding junctions (Septate or Tight Junctions) and Adherens Junctions. Two major proteins involved in these junctions are Claudins in Septate/Tight Junctions, forming paracellular pores and control water and ion influx, and classic Cadherins in Adherens Junctions, facilitating proper cell-cell adhesion. Hydra vulgaris, a simple hydrozoan, expresses all genes required for establishing both Adherens and Septate Junctions, and is ideal for investigating the evolution of epithelia, as they are part of the sister group of Bilateria and are located at the base of Eumetazoa. So far, 14 Claudin-orthologs have been described in Hydra vulgaris. In this study, we identified and characterized a total of 33 Claudin-orthologs based on their sequence, domain organization and predicted folding behaviour. Furthermore, expression maps based on single cell RNA sequencing show a cell-type and tissue specific combination of Claudins and a high overlap of the ubiquitously expressed Claudin-1, -7 and -12 and the basal disc specific Claudin-19 and -31, making a heterophilic pore assembly plausible. This study is both, the final part of a first detailed characterization of a Claudin-ortholog in a non-Bilaterian model system and a starting point to further investigate the functionality of Claudins and the molecular architecture of septate junctions. Hydra vulgaris expresses a single classic Cadherin ortholog, that is upregulated in late nematocytes and ectodermal tentacle battery cells. We established a RNAi mediated knock-down protocol based on electroporation of polyps and investigated the function of Cadherins in nematocyte incorporation and head regeneration. Here, I am able to show that the incorporation of nematocytes into the battery cells is disturbed and that the early wound healing after decapitation is delayed. This study hints, Masterarbeit Universität Innsbruck 2023

Die Entwicklung echter Epithelien markiert den Beginn der Evolution der Eumetazoa. Ein wichtiges Merkmal ist das Vorhandensein von zellulären Junctions, d. h. von Occludensjunctions (Septate oder Tight Junctions) und Adhärenzjunctions. Zwei wichtige Proteine, die an diesen Verbindungen beteiligt sind, sind Claudine in Septate/Tight Junctions, die parazelluläre Poren bilden und den Wasser- und Ioneneinstrom kontrollieren, und klassische Cadherine in Adherens Junctions, die die richtige Zell-Zell-Adhäsion ermöglichen. Hydra vulgaris, ein einfacher Hydrozoe, exprimiert alle Gene, die für die Bildung von Adherens und Septate Junctions erforderlich sind, und eignet sich ideal für die Untersuchung der Evolution von Epithelien, da sie zur Schwestergruppe der Bilateria gehören und an der Basis der Eumetazoa positioniert sind. Bislang wurden 14 Claudin-Orthologe in Hydra vulgaris beschrieben. In dieser Studie identifizierten und charakterisierten wir insgesamt 33 Claudin-Orthologe auf der Grundlage ihrer Sequenz, Domänenorganisation und des vorhergesagten Faltungsverhaltens. Darüber hinaus zeigen Expressionskarten, die auf Einzelzell-RNA-Sequenzierung basieren, eine zelltyp- und gewebespezifische Kombination von Claudinen und eine starke Überlappung der ubiquitär exprimierten Claudin-1, -7 und -12 und der fußscheibenspezifischen Claudin-19 und -31, was einen heterophilen Porenaufbau plausibel macht. Diese Studie ist sowohl der letzte Teil einer ersten detaillierten Charakterisierung eines Claudin-Orthologs in einem nicht-bilateralen Modellsystem als auch ein Ausgangspunkt für die weitere Untersuchung der Funktionalität von Claudinen und der molekularen Architektur von Septatejunctions. Hydra vulgaris exprimiert ein einziges klassisches Cadherin-Ortholog, das in späten Nematozyten und ektodermalen Tentakelbatteriezellen hochreguliert ist. Wir haben ein RNAi-mediiertes Knockdown-Protokoll auf der Basis von Elektroporation von Polypen etabliert und die Funktion von Cadherinen b, The development of true epithelia marks the start of the evolution of Eumetazoa. One important characteristic is the existence of sealing junctions, i.e. occluding junctions (Septate or Tight Junctions) and Adherens Junctions. Two major proteins involved in these junctions are Claudins in Septate/Tight Junctions, forming paracellular pores and control water and ion influx, and classic Cadherins in Adherens Junctions, facilitating proper cell-cell adhesion. Hydra vulgaris, a simple hydrozoan, expresses all genes required for establishing both Adherens and Septate Junctions, and is ideal for investigating the evolution of epithelia, as they are part of the sister group of Bilateria and are located at the base of Eumetazoa. So far, 14 Claudin-orthologs have been described in Hydra vulgaris. In this study, we identified and characterized a total of 33 Claudin-orthologs based on their sequence, domain organization and predicted folding behaviour. Furthermore, expression maps based on single cell RNA sequencing show a cell-type and tissue specific combination of Claudins and a high overlap of the ubiquitously expressed Claudin-1, -7 and -12 and the basal disc specific Claudin-19 and -31, making a heterophilic pore assembly plausible. This study is both, the final part of a first detailed characterization of a Claudin-ortholog in a non-Bilaterian model system and a starting point to further investigate the functionality of Claudins and the molecular architecture of septate junctions. Hydra vulgaris expresses a single classic Cadherin ortholog, that is upregulated in late nematocytes and ectodermal tentacle battery cells. We established a RNAi mediated knock-down protocol based on electroporation of polyps and investigated the function of Cadherins in nematocyte incorporation and head regeneration. Here, I am able to show that the incorporation of nematocytes into the battery cells is disturbed and that the early wound healing after decapitation is delayed. This study hints, Masterarbeit Universität Innsbruck 2023

The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the third most common foodborne illness in the United States. CpE induces gut breakdown by disrupting barriers at cell-cell contacts called tight junctions (TJs), which are formed and maintained by claudins. Targeted binding of CpE to specific claudins, encoded by its C-terminal domain (cCpE), loosens TJ barriers to trigger molecular leaks between cells. Cytotoxicity results from claudin-bound CpE complexes forming pores in cell membranes. In mammalian tissues, ∼24 claudins govern TJ barriers-but the basis for CpE's selective targeting of claudins in the gut was undetermined. We report the structure of human claudin-4 in complex with cCpE, which reveals that enterotoxin targets a motif conserved in receptive claudins and how the motif imparts high-affinity CpE binding to these but not other subtypes. The structural basis of CpE targeting is supported by binding affinities, kinetics, and half-lives of claudin-enterotoxin complexes and by the cytotoxic effects of CpE on claudin-expressing cells. By correlating the binding residence times of claudin-CpE complexes we determined to claudin expression patterns in the gut, we uncover that the primary CpE receptors differ in mice and humans due to sequence changes in the target motif. These findings provide the molecular and structural element CpE employs for subtype-specific targeting of claudins during pathogenicity of C. perfringens in the gut and a framework for new strategies to treat CpE-based illnesses in domesticated mammals and humans.

The bacterium Clostridium perfringens causes severe, sometimes lethal gastrointestinal disorders in humans, including enteritis and enterotoxemia. Type F strains produce an enterotoxin (CpE) that causes the third most common foodborne illness in the United States. CpE induces gut breakdown by disrupting barriers at cell-cell contacts called tight junctions (TJs), which are formed and maintained by claudins. Targeted binding of CpE to specific claudins, encoded by its C-terminal domain (cCpE), loosens TJ barriers to trigger molecular leaks between cells. Cytotoxicity results from claudin-bound CpE complexes forming pores in cell membranes. In mammalian tissues, ∼24 claudins govern TJ barriers-but the basis for CpE's selective targeting of claudins in the gut was undetermined. We report the structure of human claudin-4 in complex with cCpE, which reveals that enterotoxin targets a motif conserved in receptive claudins and how the motif imparts high-affinity CpE binding to these but not other subtypes. The structural basis of CpE targeting is supported by binding affinities, kinetics, and half-lives of claudin-enterotoxin complexes and by the cytotoxic effects of CpE on claudin-expressing cells. By correlating the binding residence times of claudin-CpE complexes we determined to claudin expression patterns in the gut, we uncover that the primary CpE receptors differ in mice and humans due to sequence changes in the target motif. These findings provide the molecular and structural element CpE employs for subtype-specific targeting of claudins during pathogenicity of C. perfringens in the gut and a framework for new strategies to treat CpE-based illnesses in domesticated mammals and humans.

Grainyhead-like 2 (Grhl2) is a transcription factor regulating cell adhesion genes. Grhl2 acts as an epithelial-mesenchymal transition suppressor, and it is a proto-oncogene involved in estrogen-stimulated breast cancer proliferation. However, its expression during ovarian hormone-dependent mammary ductal development remains obscure. We here examined Grhl2 expression in the mammary gland of normal and steroid-replaced ovariectomized mice. Grhl2 protein signals were detected in both the mammary luminal epithelial and myoepithelial nuclei. The ratio and density of Grhl2-positive nuclei increased after the onset of puberty and progressed with age, whereas Grhl2-negative epithelial cells were detected in mature ducts. Claudin 3, claudin 4, claudin 7, and E-cadherin gene expression in the mammary gland was upregulated, and their expression was highly correlated with Grhl2 gene expression. Furthermore, Grhl2 mRNA expression and ductal lumen width were significantly increased by the combined treatment of estrogen and progesterone compared with estrogen alone. These results suggest that Grhl2 expressed in the luminal epithelial and myoepithelial cells from the early phase of ductal development, controlling the expression of cell adhesion molecules to establish functional ducts

Grainyhead-like 2 (Grhl2) is a transcription factor regulating cell adhesion genes. Grhl2 acts as an epithelial-mesenchymal transition suppressor, and it is a proto-oncogene involved in estrogen-stimulated breast cancer proliferation. However, its expression during ovarian hormone-dependent mammary ductal development remains obscure. We here examined Grhl2 expression in the mammary gland of normal and steroid-replaced ovariectomized mice. Grhl2 protein signals were detected in both the mammary luminal epithelial and myoepithelial nuclei. The ratio and density of Grhl2-positive nuclei increased after the onset of puberty and progressed with age, whereas Grhl2-negative epithelial cells were detected in mature ducts. Claudin 3, claudin 4, claudin 7, and E-cadherin gene expression in the mammary gland was upregulated, and their expression was highly correlated with Grhl2 gene expression. Furthermore, Grhl2 mRNA expression and ductal lumen width were significantly increased by the combined treatment of estrogen and progesterone compared with estrogen alone. These results suggest that Grhl2 expressed in the luminal epithelial and myoepithelial cells from the early phase of ductal development, controlling the expression of cell adhesion molecules to establish functional ducts

The blood–testis barrier (BTB) is thought to be indispensable for spermatogenesis because it creates a special environment for meiosis and protects haploid cells from the immune system. The BTB divides the seminiferous tubules into the adluminal and basal compartments. Spermatogonial stem cells (SSCs) have a unique ability to transmigrate from the adluminal compartment to the basal compartment through the BTB upon transplantation into the seminiferous tubule. Here, we analyzed the role of Cldn11, a major component of the BTB, in spermatogenesis using spermatogonial transplantation. Cldn11-deficient mice are infertile due to the cessation of spermatogenesis at the spermatocyte stage. Cldn11-deficient SSCs failed to colonize wild-type testes efficiently, and Cldn11-deficient SSCs that underwent double depletion of Cldn3 and Cldn5 showed minimal colonization, suggesting that claudins on SSCs are necessary for transmigration. However, Cldn11-deficient Sertoli cells increased SSC homing efficiency by >3-fold, suggesting that CLDN11 in Sertoli cells inhibits transmigration of SSCs through the BTB. In contrast to endogenous SSCs in intact Cldn11-deficient testes, those from WT or Cldn11-deficient testes regenerated sperm in Cldn11-deficient testes. The success of this autologous transplantation appears to depend on removal of endogenous germ cells for recipient preparation, which reprogrammed claudin expression patterns in Sertoli cells. Consistent with this idea, in vivo depletion of Cldn3/5 regenerated endogenous spermatogenesis in Cldn11-deficient mice. Thus, coordinated claudin expression in both SSCs and Sertoli cells expression is necessary for SSC homing and regeneration of spermatogenesis, and autologous stem cell transplantation can rescue congenital defects of a self-renewing tissue.

This work probes the binding kinetics of COOH-terminus of Clostridium perfringens enterotoxin (c-CPE) and claudin expressing MCF-7 cells using force spectroscopy with optical tweezers. c-CPE is of high biomedical interest due to its ability to specifically bind to claudin with high affinity as well as reversibly disrupt tight junctions whilst maintaining cell viability. We observed single-step rupture events between silica particles functionalized with c-CPE and MCF-7 cells. Extensive calibration of the optical tweezers’ trap stiffness and displacement of the particle from trap center extracted a probable bond rupture force of ˜ 18 pN. The probability of rupture events with c-CPE functionalized silica particles increased by 50% compared to unfunctionalized particles. Additionally, rupture events were not observed when probing cells not expressing claudin with c-CPE coated particles. Overall, this work demonstrates that optical tweezers are invaluable tools to probe ligand-receptor interactions and their potential to study dynamic molecular events in drug-binding scenarios.

von MMag. Dr. Marie-Kristin Eder, Summary in englischer Sprache, Universität Innsbruck, Diplomarbeit, 2019, (VLID)4606464

The human pathogenic bacterium Clostridium perfringens secretes an enterotoxin (CpE) that targets claudins through its C-terminal receptor-binding domain (cCpE). Isoform-specific binding by CpE causes dissociation of claudins and tight junctions (TJs), resulting in cytotoxicity and breakdown of the gut epithelial barrier. Here, we present crystal structures of human claudin-9 (hCLDN-9) in complex with cCpE at 3.2 and 3.3 Å. We show that hCLDN-9 is a high-affinity CpE receptor and that hCLDN-9-expressing cells undergo cell death when treated with CpE but not cCpE, which lacks its cytotoxic domain. Structures reveal cCpE-induced alterations to 2 epitopes known to enable claudin self-assembly and expose high-affinity interactions between hCLDN-9 and cCpE that explain isoform-specific recognition. These findings elucidate the molecular bases for hCLDN-9 selective ion permeability and binding by CpE, and provide mechanisms for how CpE disrupts gut homeostasis by dissociating claudins and TJs to affect epithelial adhesion and intercellular transport.

STUDY QUESTION: How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to samples from a large cohort of pre/peripubertal boys? SUMMARY ANSWER: The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients. WHAT IS KNOWN ALREADY: Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved. STUDY DESIGN SIZE, AND DURATION: Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in-vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical fi

STUDY QUESTION: How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to samples from a large cohort of pre/peripubertal boys? SUMMARY ANSWER: The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients. WHAT IS KNOWN ALREADY: Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved. STUDY DESIGN SIZE, AND DURATION: Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in-vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical fi

Bakterielle Toxine stellen eine wirkungsvolle und effektive Alternative zur Therapie von Tumorerkrankungen dar. Das vom Clostridium perfringens Typ A produzierte Clostridium perfringens enterotoxin (CPE) gehört zu der Gruppe der porenbildenden Toxine und weist eine rezeptorspezifische zytotoxische Wirkung auf, welche über die Membranrezeptoren Cldn3 und Cldn4 entfaltet wird. Diese liegen vor allem in Epithelialkarzinomen wie dem Brust-, Prostata-, oder Kolon-, sowie dem Pankreaskarzinom (PK) stark hochreguliert vor. Ziel dieser Arbeit war die Anwendung des neuen selektiven und effizienten „Onkoleaking“ Suizid-Gentherapie Konzepts für die Behandlung von Cldn3 / 4 überexprimierender PK unter Verwendung eines nicht-viralen translations-optimierten CPE exprimierenden Vektors (optCPE). Weiterhin sollte in dieser Arbeit der genaue molekulare Mechanismus der CPE-vermittelten Zytotoxizität in vitro und auch in vivo analysiert werden. Für die in vitro Analysen wurden verschiedene humane PK Zelllinien, Patienten abgeleitete Xenotransplantate (PDX) und deren abgeleiteten Zellen bezüglich ihrer Cldn3 / 4 Expression und Sensitivität sowohl gegenüber rekombinantem CPE (rekCPE) als auch nach optCPE Gentransfer untersucht. Es konnte eine positive Korrelation zwischen der Effizienz CPE vermittelter Zytotoxizität und der Höhe der Cldn3 / 4 Überexpression gezeigt werden. Des Weiteren wurde die Verfügbarkeit und Zugänglichkeit der CPE Rezeptoren für die Toxinbindung als kritischer Faktor für die durch Porenbildung induzierte Zytotoxizität beschrieben. Auch eine detaillierte Analyse verschiedener apoptotischer und nekrotischer Signalwege und deren Schlüsselmoleküle waren vom besonderen Interesse. Von noch größerer Wichtigkeit war jedoch die Anwendbarkeit und der Nachweis der antitumoralen Wirksamkeit der optCPE-basierten Suizid-Gentherapie mit Hilfe des intratumoralen Jet-Injektion Gentransfers in verschiedenen Luziferase-exprimierenden CDX und PDX Modellen des PK. Alle in vivo Studien, Bacterial toxins have evolved to an effective therapeutic option for cancer therapy and numerous studies demonstrated their antitumoral potential. The Clostridium perfringens enterotoxin (CPE), produced by the anaerobic Clostridium perfringes bacteria, is a pore-forming (oncoleaking) toxin, which binds to its receptors claudin-3 and -4 (Cldn3 / 4) and exerts a selective, receptor-dependent cytotoxicity. The transmembrane tight junction proteins Cldn3 and Cldn4 are known CPE receptors and are highly upregulated in several human epithelial cancers such as breast, colon, ovarian and pancreatic cancer. This study aimed at the evaluation of the potential of oncoleaking gene therapy using a non-viral translation optimized CPE vector (optCPE) as a new suicide approach for the treatment of Cldn3 / 4 overexpressing pancreatic cancer (PC) in vitro and in vivo. We demonstrated the successful in vitro use of optCPE gene transfer in a panel of human PC cells and more importantly patient derived PC xenograft (PDX) derived cells. We showed significant reduction of cell viability in all Cldn3 / 4 overexpressing PC cells after optCPE transfection. Furthermore a positive correlation between CPE cytotoxicity and level of claudin expression was shown. We revealed accessibility of CPE receptors for toxin binding as determining for optCPE mediated cytotoxicity. Since investigation of optCPE induced cell death mechanism was of particular interest, detailed analyses of apoptotic and necrotic key players were performed. By this, caspase dependent- and independent apoptosis and necrosis activation after gene transfer was demonstrated, which was dependent on amount of expressed optCPE and accessibility of Cldn. More importantly, this study demonstrated the applicability and antitumoral efficacy of optCPE gene therapy by the non-viral intratumoral jet-injection gene transfer in vivo in different luciferase-expressing CDX and PDX pancreatic cancer models. The animal experiments demonstrated t

Bakterielle Toxine stellen eine wirkungsvolle und effektive Alternative zur Therapie von Tumorerkrankungen dar. Das vom Clostridium perfringens Typ A produzierte Clostridium perfringens enterotoxin (CPE) gehört zu der Gruppe der porenbildenden Toxine und weist eine rezeptorspezifische zytotoxische Wirkung auf, welche über die Membranrezeptoren Cldn3 und Cldn4 entfaltet wird. Diese liegen vor allem in Epithelialkarzinomen wie dem Brust-, Prostata-, oder Kolon-, sowie dem Pankreaskarzinom (PK) stark hochreguliert vor. Ziel dieser Arbeit war die Anwendung des neuen selektiven und effizienten „Onkoleaking“ Suizid-Gentherapie Konzepts für die Behandlung von Cldn3 / 4 überexprimierender PK unter Verwendung eines nicht-viralen translations-optimierten CPE exprimierenden Vektors (optCPE). Weiterhin sollte in dieser Arbeit der genaue molekulare Mechanismus der CPE-vermittelten Zytotoxizität in vitro und auch in vivo analysiert werden. Für die in vitro Analysen wurden verschiedene humane PK Zelllinien, Patienten abgeleitete Xenotransplantate (PDX) und deren abgeleiteten Zellen bezüglich ihrer Cldn3 / 4 Expression und Sensitivität sowohl gegenüber rekombinantem CPE (rekCPE) als auch nach optCPE Gentransfer untersucht. Es konnte eine positive Korrelation zwischen der Effizienz CPE vermittelter Zytotoxizität und der Höhe der Cldn3 / 4 Überexpression gezeigt werden. Des Weiteren wurde die Verfügbarkeit und Zugänglichkeit der CPE Rezeptoren für die Toxinbindung als kritischer Faktor für die durch Porenbildung induzierte Zytotoxizität beschrieben. Auch eine detaillierte Analyse verschiedener apoptotischer und nekrotischer Signalwege und deren Schlüsselmoleküle waren vom besonderen Interesse. Von noch größerer Wichtigkeit war jedoch die Anwendbarkeit und der Nachweis der antitumoralen Wirksamkeit der optCPE-basierten Suizid-Gentherapie mit Hilfe des intratumoralen Jet-Injektion Gentransfers in verschiedenen Luziferase-exprimierenden CDX und PDX Modellen des PK. Alle in vivo Studien, Bacterial toxins have evolved to an effective therapeutic option for cancer therapy and numerous studies demonstrated their antitumoral potential. The Clostridium perfringens enterotoxin (CPE), produced by the anaerobic Clostridium perfringes bacteria, is a pore-forming (oncoleaking) toxin, which binds to its receptors claudin-3 and -4 (Cldn3 / 4) and exerts a selective, receptor-dependent cytotoxicity. The transmembrane tight junction proteins Cldn3 and Cldn4 are known CPE receptors and are highly upregulated in several human epithelial cancers such as breast, colon, ovarian and pancreatic cancer. This study aimed at the evaluation of the potential of oncoleaking gene therapy using a non-viral translation optimized CPE vector (optCPE) as a new suicide approach for the treatment of Cldn3 / 4 overexpressing pancreatic cancer (PC) in vitro and in vivo. We demonstrated the successful in vitro use of optCPE gene transfer in a panel of human PC cells and more importantly patient derived PC xenograft (PDX) derived cells. We showed significant reduction of cell viability in all Cldn3 / 4 overexpressing PC cells after optCPE transfection. Furthermore a positive correlation between CPE cytotoxicity and level of claudin expression was shown. We revealed accessibility of CPE receptors for toxin binding as determining for optCPE mediated cytotoxicity. Since investigation of optCPE induced cell death mechanism was of particular interest, detailed analyses of apoptotic and necrotic key players were performed. By this, caspase dependent- and independent apoptosis and necrosis activation after gene transfer was demonstrated, which was dependent on amount of expressed optCPE and accessibility of Cldn. More importantly, this study demonstrated the applicability and antitumoral efficacy of optCPE gene therapy by the non-viral intratumoral jet-injection gene transfer in vivo in different luciferase-expressing CDX and PDX pancreatic cancer models. The animal experiments demonstrated t

Background. A n increase of leaky vasculature is vital for the growth and metastasis of hepatocellular carcinoma (HCC). The paracellular permeability-regulating proteins in tumor vessels and adjacent sinusoids have not been studied in HCC patients. Methods. Expression of an endothelial tight junction protein claudin-5 (CL-5) and a standard endothelial marker CD34 were immunohistochemically examined in resected specimens from 51 HCC cases. The relationship between hepatitic or fibrotic grade and CL-5 expression pattern in sinusoidal endothelial cells (SECs) was evaluated in the tumor-adjacent tissues. Microvessel density (MVD) highlighted by CD34 or CL-5 was examined in tumor tissues. Results. In the normal liver, a ubiquitous CL-5 expression was seen in SECs, the arteries, and portal veins but not in the central veins. Sinusoidal CL-5 expression was down-regulated according to the increase of hepatitic or fibrotic grade. Poor differentiation and vasculobiliary invasion were significantly associated with a lower CL-5-MVD but not CD34-MVD. By multivariate analysis, vasculobiliary invasion and lower CL-5-MVD were independent factors associated with a lower postoperative overall survival rate. Conclusions. Attenuated CL-5 expression in SECs may be related to SEC dysfunction in injured liver. Down-regulated CL-5 expression in tumor vessels may serve as a potential marker for poor prognosis in HCC.

Background: The subtype of claudin-low breast cancer can be reliably determined only by gene-expression profiling. Attempts have been made to develop immunohistochemical surrogates, which nearly always focus on membranous claudin expression. Methods: We assessed the immunohistochemical expression of both membranous and cytoplasmic claudins 3, 4 and 7 in a series of 197 non-metastatic breast cancers, enriched with triple-negative breast cancers (TNBCs; 60%). The expression of epithelial-to-mesenchymal transition-regulating transcription factors Sip1, Zeb1 and vimentin had previously been determined in the same material. Results: In multivariate analysis, strong cytoplasmic claudin 3 expression was associated with poor relapse-free survival (RFS), disease-free survival, distant disease-free survival, breast cancer-specific survival and overall survival among TNBC patients (for RFS, RR 5.202, 95% CI 1.210–22.369, p = 0.027, vs. T-class, RR 0.663, 95% CI 0.168–2.623, p = 0.558, and N-class, RR 3.940, 95% CI 0.933–16.631, p = 0.062). Cytoplasmic claudin 3 expression was also associated with strong nuclear Sip1 expression (p = 0.000053), TNBC phenotype (p = 0.012) and within them, non-basal-like phenotype (p = 0.026). Cytoplasmic claudin 7 was associated with dismal RFS (RR 6.328, 95% CI 1.401–28.593, p = 0.016, vs. T-class, RR 0.692, 95% CI 0.242–1.982, p = 0.493, and N-class, RR 2.981, 95% CI 1.1016–8.749, p = 0.047). Low cytoplasmic expression of claudins 3, 4 and 7 together also predicted poor RFS (RR 6.070, 95% CI 1.347–27.363, p = 0.019, vs. T-class, RR 0.677, 95% CI 0.237–1.934, p = 0.467, and N-class, RR 3.167, 95% CI 1.079–9.290, p = 0.036). Conclusions: Immunohistochemical expression levels of cytoplasmic claudins 3 and 7 appear to be novel prognostic factors in TNBC.

Bakterielle Toxine stellen eine wirkungsvolle und effektive Alternative zur Therapie von Tumorerkrankungen dar. Das vom Clostridium perfringens Typ A produzierte Clostridium perfringens enterotoxin (CPE) gehört zu der Gruppe der porenbildenden Toxine und weist eine rezeptorspezifische zytotoxische Wirkung auf, welche über die Membranrezeptoren Cldn3 und Cldn4 entfaltet wird. Diese liegen vor allem in Epithelialkarzinomen wie dem Brust-, Prostata-, oder Kolon-, sowie dem Pankreaskarzinom (PK) stark hochreguliert vor. Ziel dieser Arbeit war die Anwendung des neuen selektiven und effizienten „Onkoleaking“ Suizid-Gentherapie Konzepts für die Behandlung von Cldn3 / 4 überexprimierender PK unter Verwendung eines nicht-viralen translations-optimierten CPE exprimierenden Vektors (optCPE). Weiterhin sollte in dieser Arbeit der genaue molekulare Mechanismus der CPE-vermittelten Zytotoxizität in vitro und auch in vivo analysiert werden. Für die in vitro Analysen wurden verschiedene humane PK Zelllinien, Patienten abgeleitete Xenotransplantate (PDX) und deren abgeleiteten Zellen bezüglich ihrer Cldn3 / 4 Expression und Sensitivität sowohl gegenüber rekombinantem CPE (rekCPE) als auch nach optCPE Gentransfer untersucht. Es konnte eine positive Korrelation zwischen der Effizienz CPE vermittelter Zytotoxizität und der Höhe der Cldn3 / 4 Überexpression gezeigt werden. Des Weiteren wurde die Verfügbarkeit und Zugänglichkeit der CPE Rezeptoren für die Toxinbindung als kritischer Faktor für die durch Porenbildung induzierte Zytotoxizität beschrieben. Auch eine detaillierte Analyse verschiedener apoptotischer und nekrotischer Signalwege und deren Schlüsselmoleküle waren vom besonderen Interesse. Von noch größerer Wichtigkeit war jedoch die Anwendbarkeit und der Nachweis der antitumoralen Wirksamkeit der optCPE-basierten Suizid-Gentherapie mit Hilfe des intratumoralen Jet-Injektion Gentransfers in verschiedenen Luziferase-exprimierenden CDX und PDX Modellen des PK. Alle in vivo Studien, Bacterial toxins have evolved to an effective therapeutic option for cancer therapy and numerous studies demonstrated their antitumoral potential. The Clostridium perfringens enterotoxin (CPE), produced by the anaerobic Clostridium perfringes bacteria, is a pore-forming (oncoleaking) toxin, which binds to its receptors claudin-3 and -4 (Cldn3 / 4) and exerts a selective, receptor-dependent cytotoxicity. The transmembrane tight junction proteins Cldn3 and Cldn4 are known CPE receptors and are highly upregulated in several human epithelial cancers such as breast, colon, ovarian and pancreatic cancer. This study aimed at the evaluation of the potential of oncoleaking gene therapy using a non-viral translation optimized CPE vector (optCPE) as a new suicide approach for the treatment of Cldn3 / 4 overexpressing pancreatic cancer (PC) in vitro and in vivo. We demonstrated the successful in vitro use of optCPE gene transfer in a panel of human PC cells and more importantly patient derived PC xenograft (PDX) derived cells. We showed significant reduction of cell viability in all Cldn3 / 4 overexpressing PC cells after optCPE transfection. Furthermore a positive correlation between CPE cytotoxicity and level of claudin expression was shown. We revealed accessibility of CPE receptors for toxin binding as determining for optCPE mediated cytotoxicity. Since investigation of optCPE induced cell death mechanism was of particular interest, detailed analyses of apoptotic and necrotic key players were performed. By this, caspase dependent- and independent apoptosis and necrosis activation after gene transfer was demonstrated, which was dependent on amount of expressed optCPE and accessibility of Cldn. More importantly, this study demonstrated the applicability and antitumoral efficacy of optCPE gene therapy by the non-viral intratumoral jet-injection gene transfer in vivo in different luciferase-expressing CDX and PDX pancreatic cancer models. The animal experiments demonstrated t

Bakterielle Toxine stellen eine wirkungsvolle und effektive Alternative zur Therapie von Tumorerkrankungen dar. Das vom Clostridium perfringens Typ A produzierte Clostridium perfringens enterotoxin (CPE) gehört zu der Gruppe der porenbildenden Toxine und weist eine rezeptorspezifische zytotoxische Wirkung auf, welche über die Membranrezeptoren Cldn3 und Cldn4 entfaltet wird. Diese liegen vor allem in Epithelialkarzinomen wie dem Brust-, Prostata-, oder Kolon-, sowie dem Pankreaskarzinom (PK) stark hochreguliert vor. Ziel dieser Arbeit war die Anwendung des neuen selektiven und effizienten „Onkoleaking“ Suizid-Gentherapie Konzepts für die Behandlung von Cldn3 / 4 überexprimierender PK unter Verwendung eines nicht-viralen translations-optimierten CPE exprimierenden Vektors (optCPE). Weiterhin sollte in dieser Arbeit der genaue molekulare Mechanismus der CPE-vermittelten Zytotoxizität in vitro und auch in vivo analysiert werden. Für die in vitro Analysen wurden verschiedene humane PK Zelllinien, Patienten abgeleitete Xenotransplantate (PDX) und deren abgeleiteten Zellen bezüglich ihrer Cldn3 / 4 Expression und Sensitivität sowohl gegenüber rekombinantem CPE (rekCPE) als auch nach optCPE Gentransfer untersucht. Es konnte eine positive Korrelation zwischen der Effizienz CPE vermittelter Zytotoxizität und der Höhe der Cldn3 / 4 Überexpression gezeigt werden. Des Weiteren wurde die Verfügbarkeit und Zugänglichkeit der CPE Rezeptoren für die Toxinbindung als kritischer Faktor für die durch Porenbildung induzierte Zytotoxizität beschrieben. Auch eine detaillierte Analyse verschiedener apoptotischer und nekrotischer Signalwege und deren Schlüsselmoleküle waren vom besonderen Interesse. Von noch größerer Wichtigkeit war jedoch die Anwendbarkeit und der Nachweis der antitumoralen Wirksamkeit der optCPE-basierten Suizid-Gentherapie mit Hilfe des intratumoralen Jet-Injektion Gentransfers in verschiedenen Luziferase-exprimierenden CDX und PDX Modellen des PK. Alle in vivo Studien, Bacterial toxins have evolved to an effective therapeutic option for cancer therapy and numerous studies demonstrated their antitumoral potential. The Clostridium perfringens enterotoxin (CPE), produced by the anaerobic Clostridium perfringes bacteria, is a pore-forming (oncoleaking) toxin, which binds to its receptors claudin-3 and -4 (Cldn3 / 4) and exerts a selective, receptor-dependent cytotoxicity. The transmembrane tight junction proteins Cldn3 and Cldn4 are known CPE receptors and are highly upregulated in several human epithelial cancers such as breast, colon, ovarian and pancreatic cancer. This study aimed at the evaluation of the potential of oncoleaking gene therapy using a non-viral translation optimized CPE vector (optCPE) as a new suicide approach for the treatment of Cldn3 / 4 overexpressing pancreatic cancer (PC) in vitro and in vivo. We demonstrated the successful in vitro use of optCPE gene transfer in a panel of human PC cells and more importantly patient derived PC xenograft (PDX) derived cells. We showed significant reduction of cell viability in all Cldn3 / 4 overexpressing PC cells after optCPE transfection. Furthermore a positive correlation between CPE cytotoxicity and level of claudin expression was shown. We revealed accessibility of CPE receptors for toxin binding as determining for optCPE mediated cytotoxicity. Since investigation of optCPE induced cell death mechanism was of particular interest, detailed analyses of apoptotic and necrotic key players were performed. By this, caspase dependent- and independent apoptosis and necrosis activation after gene transfer was demonstrated, which was dependent on amount of expressed optCPE and accessibility of Cldn. More importantly, this study demonstrated the applicability and antitumoral efficacy of optCPE gene therapy by the non-viral intratumoral jet-injection gene transfer in vivo in different luciferase-expressing CDX and PDX pancreatic cancer models. The animal experiments demonstrated t

Bakterielle Toxine stellen eine wirkungsvolle und effektive Alternative zur Therapie von Tumorerkrankungen dar. Das vom Clostridium perfringens Typ A produzierte Clostridium perfringens enterotoxin (CPE) gehört zu der Gruppe der porenbildenden Toxine und weist eine rezeptorspezifische zytotoxische Wirkung auf, welche über die Membranrezeptoren Cldn3 und Cldn4 entfaltet wird. Diese liegen vor allem in Epithelialkarzinomen wie dem Brust-, Prostata-, oder Kolon-, sowie dem Pankreaskarzinom (PK) stark hochreguliert vor. Ziel dieser Arbeit war die Anwendung des neuen selektiven und effizienten „Onkoleaking“ Suizid-Gentherapie Konzepts für die Behandlung von Cldn3 / 4 überexprimierender PK unter Verwendung eines nicht-viralen translations-optimierten CPE exprimierenden Vektors (optCPE). Weiterhin sollte in dieser Arbeit der genaue molekulare Mechanismus der CPE-vermittelten Zytotoxizität in vitro und auch in vivo analysiert werden. Für die in vitro Analysen wurden verschiedene humane PK Zelllinien, Patienten abgeleitete Xenotransplantate (PDX) und deren abgeleiteten Zellen bezüglich ihrer Cldn3 / 4 Expression und Sensitivität sowohl gegenüber rekombinantem CPE (rekCPE) als auch nach optCPE Gentransfer untersucht. Es konnte eine positive Korrelation zwischen der Effizienz CPE vermittelter Zytotoxizität und der Höhe der Cldn3 / 4 Überexpression gezeigt werden. Des Weiteren wurde die Verfügbarkeit und Zugänglichkeit der CPE Rezeptoren für die Toxinbindung als kritischer Faktor für die durch Porenbildung induzierte Zytotoxizität beschrieben. Auch eine detaillierte Analyse verschiedener apoptotischer und nekrotischer Signalwege und deren Schlüsselmoleküle waren vom besonderen Interesse. Von noch größerer Wichtigkeit war jedoch die Anwendbarkeit und der Nachweis der antitumoralen Wirksamkeit der optCPE-basierten Suizid-Gentherapie mit Hilfe des intratumoralen Jet-Injektion Gentransfers in verschiedenen Luziferase-exprimierenden CDX und PDX Modellen des PK. Alle in vivo Studien, Bacterial toxins have evolved to an effective therapeutic option for cancer therapy and numerous studies demonstrated their antitumoral potential. The Clostridium perfringens enterotoxin (CPE), produced by the anaerobic Clostridium perfringes bacteria, is a pore-forming (oncoleaking) toxin, which binds to its receptors claudin-3 and -4 (Cldn3 / 4) and exerts a selective, receptor-dependent cytotoxicity. The transmembrane tight junction proteins Cldn3 and Cldn4 are known CPE receptors and are highly upregulated in several human epithelial cancers such as breast, colon, ovarian and pancreatic cancer. This study aimed at the evaluation of the potential of oncoleaking gene therapy using a non-viral translation optimized CPE vector (optCPE) as a new suicide approach for the treatment of Cldn3 / 4 overexpressing pancreatic cancer (PC) in vitro and in vivo. We demonstrated the successful in vitro use of optCPE gene transfer in a panel of human PC cells and more importantly patient derived PC xenograft (PDX) derived cells. We showed significant reduction of cell viability in all Cldn3 / 4 overexpressing PC cells after optCPE transfection. Furthermore a positive correlation between CPE cytotoxicity and level of claudin expression was shown. We revealed accessibility of CPE receptors for toxin binding as determining for optCPE mediated cytotoxicity. Since investigation of optCPE induced cell death mechanism was of particular interest, detailed analyses of apoptotic and necrotic key players were performed. By this, caspase dependent- and independent apoptosis and necrosis activation after gene transfer was demonstrated, which was dependent on amount of expressed optCPE and accessibility of Cldn. More importantly, this study demonstrated the applicability and antitumoral efficacy of optCPE gene therapy by the non-viral intratumoral jet-injection gene transfer in vivo in different luciferase-expressing CDX and PDX pancreatic cancer models. The animal experiments demonstrated t

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p = 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p = 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p = 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p = 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p = 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p = 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p = 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p = 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p = 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Altered claudin expression has been described in colon, prostatic, ovarian, and breast carcinoma. However, the role of epigenetic modifications in these genes and their role in colorectal cancer is unknown. We aimed our study to investigate whether claudin protein expression and methylation of CLDN can influence the tumorigenesis of colorectal cancer. A total of 31 patients diagnosed with colorectal carcinoma was used in this study. Immunohistochemical staining was used to study protein expression in both tumor and the adjacent nonneoplastic mucosa of claudin 1, 4, and 7. To detect the DNA methylation pattern of CLDN1, 4, and 7, genomic DNA was extracted from both the tumor and the adjacent nonneoplastic mucosa. Methylation analysis was carried out using bisulfite pyrosequencing. Cell membrane staining intensity of all claudins was found significantly lower in colorectal cancer tissues when compared to paired normal mucosa (p = 0.001). For claudin 4, the percentage of cells staining positively was also significantly reduced (p = 0.04). In normal mucosa, cytoplasm showed no staining for claudins in any patient, whereas in paired colorectal cancer tissues, significant cytoplasmic staining appeared both for claudin 1 (p = 0.04) and claudin 4 (p = 0.01). Tumor samples were significantly hypomethylated in CLDN1 (p < 0.05). In conclusion, our results show that CLDN1 is significantly hypomethylated in tumor samples and that the membrane staining intensity for claudin 1, 4, and 7 is significantly lower in colorectal cancer tissues than in adjacent nonneoplastic tissue. Colorectal cancer cells showed dystopic cytoplasmic location of claudins.

Despite improvements in therapy, the prognosis for non-small cell lung cancer (NSCLC) patients remains poor, and cure is only possible in localized tumors after surgical resection. A new generation of targeted cancer drugs has led to the expectation that lung cancer therapy can be significantly improved, but these drugs are today only an option in a small subset of NSCLC patients, and their effect is temporary. Therefore, the aim of this thesis was to characterize NSCLC in order to find new treatment targets and to evaluate biomarkers that further optimize therapy selection. In Paper I, the expression of the potential treatment targets claudin 6 and claudin 18.2 were evaluated based on immunohistochemical- and gene expression analysis. High ectopic protein and gene expression were demonstrated for both claudins in small subgroups of NSCLC. Clinical trials using humanized monoclonal antibodies against both proteins are ongoing in other cancer forms and may be extended to NSCLC. In Paper II, the prognostic impact of the inflammatory mediator cyclooxygenase 2 (COX-2) was evaluated. No prognostic significance was found in a meta-analysis incorporating gene expression data of 1337 NSCLC patients. Likewise, COX-2 protein expression in tumor cells was not associated with survival in two independent NSCLC cohorts. However, in one of the analyzed cohorts, higher COX-2 expression in the tumor stroma was associated with longer survival and may therefore be a subject for further investigation. In Paper III, tumor and stromal COX-2 protein expression was examined in patients treated with the COX-2 inhibitor celecoxib in order to evaluate if COX-2 expression is a predictive biomarker for benefit of celecoxib therapy. Celecoxib did not prolong overall survival neither in the whole cohort nor in patients stratified according to COX-2 expression in tumor or stromal cells. Noteworthy, a tendency towards longer survival was again demonstrated in patients with high COX-2 stromal expres

Despite improvements in therapy, the prognosis for non-small cell lung cancer (NSCLC) patients remains poor, and cure is only possible in localized tumors after surgical resection. A new generation of targeted cancer drugs has led to the expectation that lung cancer therapy can be significantly improved, but these drugs are today only an option in a small subset of NSCLC patients, and their effect is temporary. Therefore, the aim of this thesis was to characterize NSCLC in order to find new treatment targets and to evaluate biomarkers that further optimize therapy selection. In Paper I, the expression of the potential treatment targets claudin 6 and claudin 18.2 were evaluated based on immunohistochemical- and gene expression analysis. High ectopic protein and gene expression were demonstrated for both claudins in small subgroups of NSCLC. Clinical trials using humanized monoclonal antibodies against both proteins are ongoing in other cancer forms and may be extended to NSCLC. In Paper II, the prognostic impact of the inflammatory mediator cyclooxygenase 2 (COX-2) was evaluated. No prognostic significance was found in a meta-analysis incorporating gene expression data of 1337 NSCLC patients. Likewise, COX-2 protein expression in tumor cells was not associated with survival in two independent NSCLC cohorts. However, in one of the analyzed cohorts, higher COX-2 expression in the tumor stroma was associated with longer survival and may therefore be a subject for further investigation. In Paper III, tumor and stromal COX-2 protein expression was examined in patients treated with the COX-2 inhibitor celecoxib in order to evaluate if COX-2 expression is a predictive biomarker for benefit of celecoxib therapy. Celecoxib did not prolong overall survival neither in the whole cohort nor in patients stratified according to COX-2 expression in tumor or stromal cells. Noteworthy, a tendency towards longer survival was again demonstrated in patients with high COX-2 stromal expres

Bladder cancer displays an aggressive phenotype in the muscle-invasive phase, and is associated with a high mortality rate. Therefore, novel molecular therapeutic targets are needed to improve patient survival. A monoclonal antibody against the extracellular domain of the claudin-4 (CLDN4) tight junction protein was established by immunizing rats with a plasmid vector encoding human CLDN4. A hybridoma clone, producing a rat monoclonal antibody recognizing CLDN4 (clone 4D3), was obtained. Immunohistochemistry by using the 4D3 antibody showed that CLDN4 expression was associated with local invasion, nodal metastasis, distant metastasis, and advanced stage in 86 cases of bladder cancer. The 4D3 antibody inhibited growth, invasion, and survival, associated with abrogation of the intratumoral microenvironment; lowered concentrations of epidermal growth factor and vascular endothelial growth factor were found in three-dimensional cultures of T24 and RT4 cells. In combination with cisplatin therapy, 4D3 enhanced cisplatin cytotoxicity by increasing cellular permeability, leading to increased intracellular cisplatin concentrations. In mouse models of subcutaneous tumors and lung metastasis, 4D3 enhanced tumor growth inhibition, alone and with concurrent cisplatin treatment. The anti-tumor activity of the newly established 4D3 antibody suggests that it may be a powerful tool in CLDN4-targeting therapy, and in combination with chemotherapy., 博士（医学）・甲第649号・平成28年3月15日, Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Despite improvements in therapy, the prognosis for non-small cell lung cancer (NSCLC) patients remains poor, and cure is only possible in localized tumors after surgical resection. A new generation of targeted cancer drugs has led to the expectation that lung cancer therapy can be significantly improved, but these drugs are today only an option in a small subset of NSCLC patients, and their effect is temporary. Therefore, the aim of this thesis was to characterize NSCLC in order to find new treatment targets and to evaluate biomarkers that further optimize therapy selection. In Paper I, the expression of the potential treatment targets claudin 6 and claudin 18.2 were evaluated based on immunohistochemical- and gene expression analysis. High ectopic protein and gene expression were demonstrated for both claudins in small subgroups of NSCLC. Clinical trials using humanized monoclonal antibodies against both proteins are ongoing in other cancer forms and may be extended to NSCLC. In Paper II, the prognostic impact of the inflammatory mediator cyclooxygenase 2 (COX-2) was evaluated. No prognostic significance was found in a meta-analysis incorporating gene expression data of 1337 NSCLC patients. Likewise, COX-2 protein expression in tumor cells was not associated with survival in two independent NSCLC cohorts. However, in one of the analyzed cohorts, higher COX-2 expression in the tumor stroma was associated with longer survival and may therefore be a subject for further investigation. In Paper III, tumor and stromal COX-2 protein expression was examined in patients treated with the COX-2 inhibitor celecoxib in order to evaluate if COX-2 expression is a predictive biomarker for benefit of celecoxib therapy. Celecoxib did not prolong overall survival neither in the whole cohort nor in patients stratified according to COX-2 expression in tumor or stromal cells. Noteworthy, a tendency towards longer survival was again demonstrated in patients with high COX-2 stromal expres

Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies.

Despite improvements in therapy, the prognosis for non-small cell lung cancer (NSCLC) patients remains poor, and cure is only possible in localized tumors after surgical resection. A new generation of targeted cancer drugs has led to the expectation that lung cancer therapy can be significantly improved, but these drugs are today only an option in a small subset of NSCLC patients, and their effect is temporary. Therefore, the aim of this thesis was to characterize NSCLC in order to find new treatment targets and to evaluate biomarkers that further optimize therapy selection. In Paper I, the expression of the potential treatment targets claudin 6 and claudin 18.2 were evaluated based on immunohistochemical- and gene expression analysis. High ectopic protein and gene expression were demonstrated for both claudins in small subgroups of NSCLC. Clinical trials using humanized monoclonal antibodies against both proteins are ongoing in other cancer forms and may be extended to NSCLC. In Paper II, the prognostic impact of the inflammatory mediator cyclooxygenase 2 (COX-2) was evaluated. No prognostic significance was found in a meta-analysis incorporating gene expression data of 1337 NSCLC patients. Likewise, COX-2 protein expression in tumor cells was not associated with survival in two independent NSCLC cohorts. However, in one of the analyzed cohorts, higher COX-2 expression in the tumor stroma was associated with longer survival and may therefore be a subject for further investigation. In Paper III, tumor and stromal COX-2 protein expression was examined in patients treated with the COX-2 inhibitor celecoxib in order to evaluate if COX-2 expression is a predictive biomarker for benefit of celecoxib therapy. Celecoxib did not prolong overall survival neither in the whole cohort nor in patients stratified according to COX-2 expression in tumor or stromal cells. Noteworthy, a tendency towards longer survival was again demonstrated in patients with high COX-2 stromal expres

Despite improvements in therapy, the prognosis for non-small cell lung cancer (NSCLC) patients remains poor, and cure is only possible in localized tumors after surgical resection. A new generation of targeted cancer drugs has led to the expectation that lung cancer therapy can be significantly improved, but these drugs are today only an option in a small subset of NSCLC patients, and their effect is temporary. Therefore, the aim of this thesis was to characterize NSCLC in order to find new treatment targets and to evaluate biomarkers that further optimize therapy selection. In Paper I, the expression of the potential treatment targets claudin 6 and claudin 18.2 were evaluated based on immunohistochemical- and gene expression analysis. High ectopic protein and gene expression were demonstrated for both claudins in small subgroups of NSCLC. Clinical trials using humanized monoclonal antibodies against both proteins are ongoing in other cancer forms and may be extended to NSCLC. In Paper II, the prognostic impact of the inflammatory mediator cyclooxygenase 2 (COX-2) was evaluated. No prognostic significance was found in a meta-analysis incorporating gene expression data of 1337 NSCLC patients. Likewise, COX-2 protein expression in tumor cells was not associated with survival in two independent NSCLC cohorts. However, in one of the analyzed cohorts, higher COX-2 expression in the tumor stroma was associated with longer survival and may therefore be a subject for further investigation. In Paper III, tumor and stromal COX-2 protein expression was examined in patients treated with the COX-2 inhibitor celecoxib in order to evaluate if COX-2 expression is a predictive biomarker for benefit of celecoxib therapy. Celecoxib did not prolong overall survival neither in the whole cohort nor in patients stratified according to COX-2 expression in tumor or stromal cells. Noteworthy, a tendency towards longer survival was again demonstrated in patients with high COX-2 stromal expres

Mature three-spined stickleback males use spiggin threads secreted from their kidney to glue together nest material. This requires strongly hypertrophied renal proximal tubular cells, which compromises renal osmoregulatory function during the breeding period. Experimental evidence suggests that the intestine takes over hypotonic fluid secretion at that stage but the mechanism is unexplored. To unravel the molecular mechanism we analyzed and compared transcript levels of several membrane proteins involved in water and salt transport in intestinal and renal tissues, in non-mature males (NM), mature males (MM), and mature females (MF). Aquaporin paralogs aqp1a, -30, -8aa, -8ab, -10a, and -10b, two Na+,K+-ATPase alpha-1 subunit isoforms (nka547, nka976), Na+,K+,2C1(-)-, and Na+,CI--cotransporters (nkcc1a, nkcc2,ncc), the cystic fibrosis transmembrane conductance regulator (cftr) and two claudin isoforms (cldn2, cldn15a) were expressed in the intestine and kidney in all groups. There were no differences in aqp and cldn expression between intestines of NM and MM; nkcc2 was lower and nka levels tended to be higher in intestines of MM than in NM. In the kidney, aqp1 and aqp8ab levels were lower in MM than in NM, whereas aqp3a, nkcc1a, cldn15a, and spiggin were markedly elevated. This was accompanied by marked hypertrophy of kidney tubules in MM. The data support an altered kidney function in terms of water handling in mature males, whereas there was no support for modified trans-epithelial water permeability or salt-secretory activity in the intestine of mature males. Salt-absorptive activity in the intestine may, however, be down-regulated during male maturation.

Claudins (CLDNs) are central components of tight junctions that regulate epithelial-cell barrier function and polarity. Altered CLDN expression patterns have been demonstrated in numerous cancer types and lineage-specific CLDNs have been proposed as therapy targets. The objective of this study was to assess which fraction of patients with non-small-cell lung cancer (NSCLC) express CLDN6 and CLDN18 isoform 2 (CLDN18.2). Protein expression of CLDN6 and CLDN18.2 was examined by immunohistochemistry on a tissue microarray (n=355) and transcript levels were supportively determined based on gene expression microarray data from fresh-frozen NSCLC tissues (n=196). Both were analyzed with regard to frequency, distribution and association with clinical parameters. Immunohistochemical analysis of tissue sections revealed distinct membranous positivity of CLDN6 (6.5%) and CLDN18.2 (3.7%) proteins in virtually non-overlapping subgroups of adenocarcinomas and large-cell carcinomas. Pneumocytes and bronchial epithelial cells were consistently negative. Corresponding to the protein expression, in subsets of non-squamous lung carcinoma high mRNA levels of CLDN6 (7-16%) and total CLDN18 (5-12%) were observed. Protein expression correlated well with total mRNA expression of the corresponding gene (rho=0.4-0.8). CLDN18.2 positive tumors were enriched among slowly proliferating, thyroid transcription factor 1 (TTF-1)-negative adenocarcinomas, suggesting that isoform-specific CLDN expression may delineate a specific subtype. Noteworthy, high CLDN6 protein expression was associated with worse prognosis in lung adenocarcinoma in the univariate [hazard ratio (HR): 1.8; p=0.03] and multivariate COX regression model (HR: 1.9; p=0.02). These findings encourage further clinical exploration of targeting ectopically activated CLDN expression as a valuable treatment concept in NSCLC., De 2 första författarna delar förstförfattarskapet. (The 2 first authors contributed equally to the study)

Tight junction is composed of transmembrane proteins important for maintaining cell polarity and regulating ion flow. Among these proteins are the tissue-specific claudins, proteins that have recently been suggested as tumor markers for several different types of cancer. An altered claudin expression has been observed in colon, prostatic, ovarian, and breast carcinoma. The aim of this study was to analyze the allele frequencies of three common single nucleotide polymorphisms (SNPs) in the genes for claudin 1 and claudin 7 in colon cancer (CC) patients and in a control population of healthy blood donors. Pyrosequencing was used to genotype the CLDN1 SNP rs9869263 (c.369C>T), and the CLDN7 SNPs rs4562 (c.590C>T) and rs374400 (c.606T>G) in DNA from 102 formalin fixed paraffin embedded (FFPE) colon cancer tissue, and 111 blood leukocyte DNA from blood/plasma donors. These results were correlated with clinical parameters such as TNM stage, tumor localization, tumor differentiation, complexity index, sex, and age. We found that there was a significant association between the CLDN1 genotype CC in tumor samples and a higher risk of colon cancer development (OR 3.0, p < 0.001). We also found that the CLDN7 rs4562 (c.590C>T) genotype CT had a higher risk of lymph node involvement (p = 0.031) and a lower degree of tumor differentiation (p = 0.028). In the control population, the allele frequencies were very similar to those in the HapMap cohort for CLDN7. The CLDN1 rs9869263 genotype (c.369C>T) was related to increased risk of colon cancer, and the CLDN7 rs4562 genotype (c.590C>T) was related to tumor differentiation and lymph node involvement in colon carcinoma. Further studies are warranted to ascertain their potential uses as biomarkers predicting tumor development, proliferation, and outcome in this disease.

Tight junction is composed of transmembrane proteins important for maintaining cell polarity and regulating ion flow. Among these proteins are the tissue-specific claudins, proteins that have recently been suggested as tumor markers for several different types of cancer. An altered claudin expression has been observed in colon, prostatic, ovarian, and breast carcinoma. The aim of this study was to analyze the allele frequencies of three common single nucleotide polymorphisms (SNPs) in the genes for claudin 1 and claudin 7 in colon cancer (CC) patients and in a control population of healthy blood donors. Pyrosequencing was used to genotype the CLDN1 SNP rs9869263 (c.369C>T), and the CLDN7 SNPs rs4562 (c.590C>T) and rs374400 (c.606T>G) in DNA from 102 formalin fixed paraffin embedded (FFPE) colon cancer tissue, and 111 blood leukocyte DNA from blood/plasma donors. These results were correlated with clinical parameters such as TNM stage, tumor localization, tumor differentiation, complexity index, sex, and age. We found that there was a significant association between the CLDN1 genotype CC in tumor samples and a higher risk of colon cancer development (OR 3.0, p < 0.001). We also found that the CLDN7 rs4562 (c.590C>T) genotype CT had a higher risk of lymph node involvement (p = 0.031) and a lower degree of tumor differentiation (p = 0.028). In the control population, the allele frequencies were very similar to those in the HapMap cohort for CLDN7. The CLDN1 rs9869263 genotype (c.369C>T) was related to increased risk of colon cancer, and the CLDN7 rs4562 genotype (c.590C>T) was related to tumor differentiation and lymph node involvement in colon carcinoma. Further studies are warranted to ascertain their potential uses as biomarkers predicting tumor development, proliferation, and outcome in this disease., Funding Agencies:Nyckelfonden, Örebro University Hospital, Örebro, SwedenLions cancer research foundation, Uppsala, Sweden

Acute pancreatitis is a common disease, Finland being among the countries with the highest incidence. The majority of patients have a mild, self-limiting disease. However, 20% of these patients develop severe necrotizing pancreatitis with a mortality rate of 7 to 25%. The mechanisms for developing the severe disease are not known, it is not possible to accurately forecast the severity of the disease and there is no curative treatment yet. This study was aimed at analyzing the early phase of acute experimental porcine oedematous and necrotizing pancreatitis. In Study I, the pancreatic microcirculatory changes were measured and the expression of tight junction proteins (claudins-2, -3, -4, -5 and -7) and the rate of apoptosis in the pancreas were all measured. In Study II, bacterial translocation to the blood in the portal vein blood or to the mesenteric lymph nodes was analyzed and the intestinal expression of tight junction proteins (claudins-2, -3, -4, -5 and -7) and the intestinal apoptosis/ proliferation rates were measured. The basic histology of the jejunum and colon were analyzed. Study III analyzed which cytokines are released from the pancreas to the portal venous blood. In Study IV, the ultrastructure of the epithelium of the jejunum and colon was analyzed and the expression of adherens junction proteins, E-cadherin and β-catenin, were measured from both jejunum and colon. The first study (I) showed that membranous immunoreactivity of claudin-2 in acinar cells appeared in the pancreas during acute oedematous and necrotizing pancreatitis. The expressions of claudins -3, - 4, - 5 and 7 were unaffected. The second study (II) showed that bacterial translocation from the gut was not present at the beginning of acute porcine pancreatitis. The expressions of claudins-2 and -5 do not become altered; however, there might be some decrease in claudin-3 expression in the colon and decrease in the expression of claudins-4 and -7 in the jejunum in necrotizi, Tiivistelmä Akuutti haimatulehdus on yleinen sairaus, jonka ilmaantuvuus Suomessa on verrattain suuri. Suurimmalla osalla potilaista tauti on lievä ja itsestään paraneva. Kuitenkin 20 %:lle potilaista kehittyy vaikea haimatulehdus, johon liittyy 7–25 %:n kuolleisuus. On epäselvää, miksi toisinaan kehittyy vaikea tautimuoto. Taudin vaikeusastetta ei voida etukäteen tarkasti ennustaa, eikä tautiin ole parantavaa hoitoa. Väitöskirjatyön tarkoituksena oli tutkia lievän ja vaikean haimatulehduksen varhaisvaihetta kokeellisessa sikamallissa. Työssä I mitattiin haiman mikroverenkierron muutoksia, tutkittiin tiivisliitosproteiinien klaudiini-2:n, -3:n, -4:n, -5:n ja -7:n ilmenemistä sekä apoptoosin määrää haimassa. Toisessa työssä tutkittiin mahdollista bakteeritranslokaatiota porttilaskimovereen ja vatsaontelon imusolmukkeisiin, mitattiin suoliston tiivis liitos-proteiinien klaudiinien-2, -3, -4, -5 ja -7 ilmenemistä ja suoliston apoptoosin ja soluproliferaation määrää. Mahdollisia muutoksia ohut- ja paksusuolen perushistologiassa analysoitiin. Kolmannessa työssä mitattiin sytokiinipitoisuuksia porttilaskimoverestä. Neljännessä työssä analysoitiin ohut- ja paksusuolen mikrorakennetta elektronimikroskopian avulla ja mitattiin vyöliitosproteiinien E-cadherin ja β-catenin määrää. I työssä todettiin klaudiini-2:n ilmaantuvan haiman asinaarisolujen solukalvoille lievässä ja vaikeassa kokeellisessa haimatulehduksessa. Klaudiinien 3,- 4,- 5 ja 7 esiintyminen haimassa ei muuttunut. II työssä todettiin, että bakteeritranslokaatiota ei tapahtunut seuranta-aikana. Suolistossa klaudiinien-2 ja -5 ilmenemisessä ei tapahtunut muutoksia. Klaudiini-3:n ilmenemisessä paksusuolessa ja klaudiinien -4 ja -7 ilmenemisessä ohutsuolessa saattaa tapahtua vähenemistä vaikeassa haimatulehduksessa. Tutkimustoimenpide itsessään aiheutti ohut- ja paksusuolen apoptoosin lisääntymistä. III työn mukaan tulehdusvaste oli erilainen akuutissa lievässä ja vaikeassa kokeellisessa haimatulehduksessa.

Lung cancers and chronic obstructive pulmonary disease (COPD) are the most common smoking-related lung diseases and both have high mortality rate. Tight junctions (TJ) are apical junctions between epithelial cells that regulate the permeability of epithelium and form the tight junction along with occludin. Dysfunction of the TJ and dysregulation of TJ proteins leads to a loss of cell-cell adhesion and a loss of cohesion as well as epitheliomesenchymal transition. These increase invasion of lung carcinomas and possibly predispose to the exacerbations in COPD. Therefore, the aim of this thesis was to study expression and regulation of claudins in different lung carcinomas and COPD. Carcinomas expressed claudins 1, 2, 3, 4, 5 and 7 in different variations. Claudin 5 expression was weak in all carcinoma types. Strong claudin 1, 4 and 7 expression was associated with better survival in squamous cell carcinoma and adenocarcinoma. Claudin 3 expression was associated with COPD in large airways. Claudins 3 and 4 was found to be stronger in small airways of smokers and COPD patients than in non-smokers. Transcription factor snail had prognostic value in lung carcinomas. Negative snail expression was associated with longer life expectancy in lung carcinoma patients. Negative snail expression was associated with up-regulated claudin 5 and 7 expression, while strong expression was associated with low claudin 1 and 3 expression. Transcription factors slug and twist were inversely associated with claudins 3 and 4 in small and large airways. Slug expression was higher in non-smokers than in COPD patients and smokers. Transcription factor knockdown increased claudin expression in normal bronchial cell line. Except for claudin 2 and 7, which were decreased. Adenocarcinoma-like cell line was not affected by snail knockdown and in squamous cell carcinoma-like cell line claudin 3, 4 and 7 expression was increased. Transcription factor snail knockdown inhibited invasion of cell, Tiivistelmä Keuhkosyöpä ja keuhkoahtaumatauti ovat yleisiä tupakoinnin aiheuttamia keuhkosairauksia, joissa on korkea kuolleisuus. Tupakointi aiheuttaa muutoksia keuhkojen epiteelisoluissa ja solujen välisissä liitoksissa. Tiivisliitokset solujen välillä säätelevät epiteelin rakennetta ja läpäisevyyttä. Klaudiinit ovat proteiineja, jotka muodostavat tiivisliitoksen yhdessä okkludiinin kanssa. Tiivisliitos proteiinien toimintahäiriöt voivat johtaa solujen välisten liitosten katoamiseen ja epiteelin hajoamiseen sekä epiteelisolujen muuntumiseen mesenkymaalisten solujan kaltaisiksi. Nämä seikat lisäävät invaasiota keuhkosyövissä ja saattavat altistaa pahenemisvaiheisiin keuhkoahtaumataudissa. Väitöskirjassa tutkittiin klaudiinien ilmentymistä ja säätelyä keuhkosyövässä ja keuhkoahtaumataudissa. Klaudiinien1, 2, 3, 4, 5 ja 7 esiintyminen keuhkosyövän histologisissa alatyypeissä vaihteli. Klaudiinien 1, 4 ja 7 voimakas ilmentyminen voitiin yhdistää pidempään elinikään potilailla, joilla oli levyepiteeli- tai adenokarsinooma. Klaudiini 3:n ilmentyminen liittyi keuhkoahtaumatautiin suurissa hengitysteissä. Klaudiinien 3 ja 4 voimakas ilmeneminen pienissä ilmateissä oli yleisempää keuhkoahtaumatautipotilailla ja tupakoitsijoilla kuin tupakoimattomilla henkilöillä. Transkriptiotekijä snailin puuttuminen keuhkosyövässä liittyi potilaiden pidempään elinaikaan. Klaudiinien 5 ja 7 ilmeneminen oli voimakkaampaa, kun snailin määrä oli vähäinen. Klaudiinien 1 ja 3 ilmeneminen väheni snail:in ollessa voimakas keuhkosyövässä. Traskriptiotekijöiden (slug ja twist) ilmeneminen liittyi käänteisesti klaudiinien ilmentymiseen pienissä ja suurissa ilmateissä. Slugin ilmeneminen oli voimakkaampaa tupakoimattomilla henkilöillä kuin tupakoivilla tai keuhkoahtaumatautia sairastavilla. Transkriptiotekijöiden snail, slug ja twist toiminnan estäminen lisäsi klaudiinien määrää normaaleissa keuhkon epiteelisoluissa. Poikkeuksen muodostivat klaudiinit 2 ja 7, joiden määrä väheni kun snail:in toimint

Mediators of epithelial permeability, angiogenesis and invasion may serve as prognostic indicators and targets for therapies in esophageal cancer (EC). The expressions of claudins, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor-A (VEGF), nitric oxide synthases (iNOS, eNOS, nNOS) and toll-like receptor 9 (TLR9) were evaluated by immunohistochemistry in EC. The results were compared with clinicopathological variables, tumor proliferation and apoptosis. All of the claudins were expressed in most of the cancers. Esophageal adenocarcinomas (EAC) displayed more often increased claudin 3 and 5 expression than esophageal squamous cell carcinomas (ESCC). Loss of claudin 3 expression associated with distant metastases in EC and a tendency in this direction was also observed for claudin 4. Cancers with stronger claudin 4 expression showed increased apoptosis in both EAC and ESCC. HIF-1α was present in most of the ECs and like iNOS more often in ESCC than in EAC. Strong HIF-1α expression tended to associate with positive VEGF immunostaining. In ESCC, both strong HIF-1α expression and VEGF positivity tended to associate with increased microvessel density (MVD). In EAC, tumors showing VEGF positivity associated with increased MVD outside the tumor. Patients with strong HIF-1α expression had more often distant metastases than other patients in EC. There was no VEGF expression in normal esophageal mucosa and T3-4 tumors tended to be more often VEGF positive than T1-2 tumors. The expression of TLR9 was more intensive in dysplasia than in normal epithelium and ESCC and abundant TLR9 expression could serve as a marker of squamous cell high grade dysplasia. Intensive TLR9 expression was associated with higher grade tumors and the presence of nodal and distant metastases in ESCC. EC and its progression may be related to increased angiogenesis regulated by VEGF and HIF-1α. In EC, claudin expression varies along with the histology of the tumor. Claudin e, Tiivistelmä Solukerrosten läpäisevyyttä, verisuonten uudismuodostusta ja kasvainsolujen liikkuvuutta säätelevät tekijät voivat toimia ruokatorvisyövän ennustetekijöinä ja hoidon kohteina. Tässä tutkimuksessa selvitettiin klaudiinien, hypoksia-indusoituvan tekijän 1α (HIF-1α), verisuonen endoteelin kasvutekijän A (VEGF), kolmen typpioksidisyntaasin (iNOS, eNOS ja nNOS) sekä tollin kaltaisen reseptorin 9 (TLR9) ilmentymistä ja merkitystä ruokatorvisyövässä immunohistokemiallisin menetelmin. Tuloksia arvioitiin suhteessa proliferaatioon ja apoptoosiin. Useimmat syöpäkasvaimet ilmensivät jokaista tutkittua klaudiinia. Ruokatorven rauhassyövässä ilmeni levyepiteelisyöpää useammin klaudiineja 3 ja 5. Klaudiinin 3 vähäinen ilmentyminen oli yhteydessä etäpesäkkeiseen tautiin. Sama suuntaus näkyi klaudiinin 4 kohdalla. Apoptoosia todettiin enemmän kasvaimissa, jotka ilmensivät muita enemmän klaudiini 4:ä. Useimmat syöpäkasvaimet ilmensivät HIF-1α:a. Sekä iNOS että HIF-1α ilmentyivät runsaammin levyepiteeli- kuin rauhassyövässä. Tutkimus viittasi yhteyteen voimakkaan HIF-1α:n ilmentymisen ja VEGF:n ilmentymisen välillä. Voimakas HIF-1α:n ilmentyminen ja VEGF:n ilmentyminen vaikuttivat liittyvän ruokatorven levyepiteelisyövän uudissuonituksen lisääntymiseen. Rauhassyövän lisääntynyt uudissuonitus kasvaimen ulkopuolella saattaa liittyä VEGF:n ilmentymiseen. Potilailla, joiden kasvaimissa HIF-1α:n ilmentyminen oli voimakasta, todettiin etäpesäkkeitä muita useammin. VEGF:a ei todettu normaalissa limakalvossa, ja sen ilmentyminen vaikutti olevan yleisempää syvälle kasvavissa kuin pinnallisissa syövissä. TLR9 ilmentyi voimakkaammin levyepiteelin dysplasiassa kuin normaalissa tai kasvainepiteelissä. Huonosti erilaistuvissa ja levinneissä kasvaimissa TLR9 ilmentyi voimakkaammin kuin muissa kasvaimissa. Ruokatorvisyövän synty ja eteneminen voivat liittyä HIF:n ja VEGF:n säätelemään verisuonten uudismuodostukseen. Klaudiinit saattavat vaikuttaa syövän käyttäytymiseen myös apoptoosin ja

The alveolar epithelium of the lung constitutes a unique interface with the outside environment. This thin barrier must maintain a surface for gas transfer while being continuously exposed to potentially hazardous environmental stimuli. Small differences in alveolar epithelial barrier properties could therefore have a large impact on disease susceptibility or outcome. Moreover, recent work has focused attention on the alveolar epithelium as central to several lung diseases, including acute lung injury and idiopathic pulmonary fibrosis. Although relatively little is known about the function and regulation of claudin tight junction proteins in the lung, new evidence suggests that environmental stimuli can influence claudin expression and alveolar barrier function in human disease. This review considers recent advances in the understanding of the role of claudins in the breakdown of the alveolar epithelial barrier in disease and in epithelial repair.

A number of species of euryhaline teleosts have the remarkable ability to adapt and survive in environments of extreme salinity, up to two or even three times the osmolality of seawater. This review looks at some of the literature describing the adaptive changes that occur, primarily with intestinal water absorption and with the properties of the gill epithelium. While there is much that is still not completely understood, recent work has begun to look at these adaptations at the cellular and molecular level. As with seawater osmoregulation, fish adapting to hypersaline conditions generally increase drinking rates and water absorption across the intestine by solute-linked transport. This process requires increased activity, expression levels and possibly changes in subunit isoforms of Na(+)/K(+)-ATPase pumps, along with increases in other components of the NaCl absorptive pathway. Additionally, recent studies have demonstrated the importance of luminal anion exchange activity and of luminal alkalinization, which can support absorption against increasing osmotic gradients by promoting divalent ion precipitation (CaCO(3)) and by conversion of HCO(3)(-) ions to CO(2). The steepness of the lumen to blood osmotic gradient vis-a-vis the NaCl absorptive capacity, along with the accumulation of non-absorbed Mg(++) and SO(4)(--), likely become the limiting factors for survival in hypersaline conditions. Of interest is the observation of predicted hyperosmotic fluid absorption by the intestine in several species. Adaptive changes in the gill epithelium are also critical in this process, allowing for secretion of absorbed NaCl from the extracellular fluids. Most notably there are important changes in the numbers and size of mitochondrion-rich (MR) cells, the sites of active secretion of Cl(-), which ultimately drives the overall process of NaCl secretion. Balance studies of intake and output clearly indicate that a decrease in the osmotic permeability of the gill epithelium mu

A number of species of euryhaline teleosts have the remarkable ability to adapt and survive in environments of extreme salinity, up to two or even three times the osmolality of seawater. This review looks at some of the literature describing the adaptive changes that occur, primarily with intestinal water absorption and with the properties of the gill epithelium. While there is much that is still not completely understood, recent work has begun to look at these adaptations at the cellular and molecular level. As with seawater osmoregulation, fish adapting to hypersaline conditions generally increase drinking rates and water absorption across the intestine by solute-linked transport. This process requires increased activity, expression levels and possibly changes in subunit isoforms of Na(+)/K(+)-ATPase pumps, along with increases in other components of the NaCl absorptive pathway. Additionally, recent studies have demonstrated the importance of luminal anion exchange activity and of luminal alkalinization, which can support absorption against increasing osmotic gradients by promoting divalent ion precipitation (CaCO(3)) and by conversion of HCO(3)(-) ions to CO(2). The steepness of the lumen to blood osmotic gradient vis-a-vis the NaCl absorptive capacity, along with the accumulation of non-absorbed Mg(++) and SO(4)(--), likely become the limiting factors for survival in hypersaline conditions. Of interest is the observation of predicted hyperosmotic fluid absorption by the intestine in several species. Adaptive changes in the gill epithelium are also critical in this process, allowing for secretion of absorbed NaCl from the extracellular fluids. Most notably there are important changes in the numbers and size of mitochondrion-rich (MR) cells, the sites of active secretion of Cl(-), which ultimately drives the overall process of NaCl secretion. Balance studies of intake and output clearly indicate that a decrease in the osmotic permeability of the gill epithelium mu

Die Tight Junction (TJ) reguliert den parazellulären Transport von Ionen, Wasser und Soluten an Epithelien und Endothelien und ist von entscheidender Bedeutung für die Aufrechterhaltung der Funktion von Organen und Geweben. Obwohl Claudin-3 zu den zuerst identifizierten und ubiquitär exprimierten Komponenten der TJ gehört, konnte seine spezifische Funktion bislang nicht geklärt werden. Für die funktionelle Charakterisierung des humanen Claudin-3-Proteins wurden stabile Überexpressionsklone der lecken Nierenepithel-Zelllinie MDCK II generiert. Die Überexpression von Claudin-3 führte zu einer deutlichen Änderung des TJ-Strangmusters sowie zu einer starken Zunahme des transepithelialen Widerstandes und einer verminderten Permeabilität für Ionen und Moleküle der Größe 332 Da und 4000 Da. Der parazelluläre Durchtritt von Wasser war unverändert. Claudin-3 konnte eindeutig als abdichtende Komponente der TJ identifiziert werden. Anhand des endothelialen Zellkulturmodells HUVEC wurden Expression und Regulation von Claudin-3 und anderen TJ-Proteinen unter dem Einfluss mechanischer Strömungsverhältnisse und des Sauerstoffpartialdrucks analysiert. Die Behandlung mit fehlender Wandschubspannung führte zur Hochregulation der abdichtenden TJ-Proteine Occludin, Claudin-3, Claudin-5 und Claudin-11, nicht aber Claudin-23. Die Regulation der einzelnen TJ-Komponenten wurde durch unterschiedliche Signalwege vermittelt, wobei der verstärkten Proteinexpression jeweils eine Hochregulation auf mRNA-Ebene zugrunde lag. Die kombinierte Behandlung mit fehlender Wandschubspannung und Hypoxie resultierte in einer sehr stark erhöhten Expression von Claudin-3. Durch die Hochregulation abdichtender TJ-Komponenten unter Bedingungen fehlender Wandschubspannung und Hypoxie, wie sie in verschiedenen physiologischen und pathologischen Situationen auftreten, könnte einem unerwünschten Durchtritt von Substanzen aus dem Blut in das umliegende Gewebe vorgebeugt werden., The tight junction (TJ) regulates the paracellular transport of ions, water and solutes in epithelia and endothelia and is of particular importance for a correct function of organs and tissues. Although claudin-3 is one of the first identified and ubiquitously expressed TJ components, its specific function was unsolved as yet. For functional characterization, human claudin-3 was stably overexpressed in the leaky epithelial cell line MDCK II. Overexpression of claudin-3 led to a marked alteration of TJ meshwork pattern, a strong increase in transepithelial resistance and a decrease in permeability for ions and paracellular tracers (332 or 4000 Da). Paracellular water transport was not affected. It was proved that claudin-3 acts as a „tightening“ TJ component. The endothelial cell culture model HUVEC was used for analysis of expression and regulation of claudin-3 and several other TJ proteins under different conditions of wall shear stress and oxygen saturation. Treatment with lacking wall shear stress led to an upregulation of the “tightening” TJ proteins occludin, claudin-3, claudin-5, and claudin-11, but not claudin-23. Upregulation of all proteins was due to increased mRNA levels. Apparently, different signaling pathways were involved in regulation of particular TJ components. Combined treatment with lacking shear stress and hypoxia resulted in drastically increased claudin-3 expression. Upregulation of tightening TJ components under lacking shear stress and hypoxic conditions as occuring in different physiological or pathological situations would limit the passage of solutes from the blood into the surrounding tissue.

Die Tight Junction (TJ) reguliert den parazellulären Transport von Ionen, Wasser und Soluten an Epithelien und Endothelien und ist von entscheidender Bedeutung für die Aufrechterhaltung der Funktion von Organen und Geweben. Obwohl Claudin-3 zu den zuerst identifizierten und ubiquitär exprimierten Komponenten der TJ gehört, konnte seine spezifische Funktion bislang nicht geklärt werden. Für die funktionelle Charakterisierung des humanen Claudin-3-Proteins wurden stabile Überexpressionsklone der lecken Nierenepithel-Zelllinie MDCK II generiert. Die Überexpression von Claudin-3 führte zu einer deutlichen Änderung des TJ-Strangmusters sowie zu einer starken Zunahme des transepithelialen Widerstandes und einer verminderten Permeabilität für Ionen und Moleküle der Größe 332 Da und 4000 Da. Der parazelluläre Durchtritt von Wasser war unverändert. Claudin-3 konnte eindeutig als abdichtende Komponente der TJ identifiziert werden. Anhand des endothelialen Zellkulturmodells HUVEC wurden Expression und Regulation von Claudin-3 und anderen TJ-Proteinen unter dem Einfluss mechanischer Strömungsverhältnisse und des Sauerstoffpartialdrucks analysiert. Die Behandlung mit fehlender Wandschubspannung führte zur Hochregulation der abdichtenden TJ-Proteine Occludin, Claudin-3, Claudin-5 und Claudin-11, nicht aber Claudin-23. Die Regulation der einzelnen TJ-Komponenten wurde durch unterschiedliche Signalwege vermittelt, wobei der verstärkten Proteinexpression jeweils eine Hochregulation auf mRNA-Ebene zugrunde lag. Die kombinierte Behandlung mit fehlender Wandschubspannung und Hypoxie resultierte in einer sehr stark erhöhten Expression von Claudin-3. Durch die Hochregulation abdichtender TJ-Komponenten unter Bedingungen fehlender Wandschubspannung und Hypoxie, wie sie in verschiedenen physiologischen und pathologischen Situationen auftreten, könnte einem unerwünschten Durchtritt von Substanzen aus dem Blut in das umliegende Gewebe vorgebeugt werden., The tight junction (TJ) regulates the paracellular transport of ions, water and solutes in epithelia and endothelia and is of particular importance for a correct function of organs and tissues. Although claudin-3 is one of the first identified and ubiquitously expressed TJ components, its specific function was unsolved as yet. For functional characterization, human claudin-3 was stably overexpressed in the leaky epithelial cell line MDCK II. Overexpression of claudin-3 led to a marked alteration of TJ meshwork pattern, a strong increase in transepithelial resistance and a decrease in permeability for ions and paracellular tracers (332 or 4000 Da). Paracellular water transport was not affected. It was proved that claudin-3 acts as a „tightening“ TJ component. The endothelial cell culture model HUVEC was used for analysis of expression and regulation of claudin-3 and several other TJ proteins under different conditions of wall shear stress and oxygen saturation. Treatment with lacking wall shear stress led to an upregulation of the “tightening” TJ proteins occludin, claudin-3, claudin-5, and claudin-11, but not claudin-23. Upregulation of all proteins was due to increased mRNA levels. Apparently, different signaling pathways were involved in regulation of particular TJ components. Combined treatment with lacking shear stress and hypoxia resulted in drastically increased claudin-3 expression. Upregulation of tightening TJ components under lacking shear stress and hypoxic conditions as occuring in different physiological or pathological situations would limit the passage of solutes from the blood into the surrounding tissue.