Tilivalline- and Tilimycin-Independent Effects of Klebsiella oxytoca on Tight Junction-Mediated Intestinal Barrier Impairment

Klebsiella oxytoca causes antibiotic-associated hemorrhagic colitis and diarrhea. This was attributed largely to its secreted cytotoxins tilivalline and tilimycin, inductors of epithelial apoptosis. To study whether Klebsiella oxytoca exerts further barrier effects, T84 monolayers were challenged with bacterial supernatants derived from tilivalline/tilimycin-producing AHC6 or its isogeneic tilivalline/tilimycin-deficient strain Mut-89. Both preparations decreased transepithelial resistance, enhanced fluorescein and FITC-dextran-4kDa permeabilities, and reduced expression of barrier-forming tight junction proteins claudin-5 and -8. Laser scanning microscopy indicated redistribution of both claudins off the tight junction region in T84 monolayers as well as in colon crypts of mice infected with AHC6 or Mut-89, indicating that these effects are tilivalline/tilimycin-independent. Furthermore, claudin-1 was affected, but only in a tilivalline/tilimycin-dependent manner. In conclusion, Klebsiella oxytoca induced intestinal barrier impairment by two mechanisms: the tilivalline/tilimycin-dependent one, acting by increasing cellular apoptosis and a tilivalline/tilimycin-independent one, acting by weakening the paracellular pathway through the tight junction proteins claudin-5 and -8

Campylobacter concisus Impairs Sodium Absorption in Colonic Epithelium via ENaC Dysfunction and Claudin-8 Disruption

The epithelial sodium channel (ENaC) can increase the colonic absorptive capacity for salt and water. Campylobacter concisus is a common pathogenic epsilonproteobacterium, causing enteritis and diarrhea. It can induce barrier dysfunction in the intestine, but its influence on intestinal transport function is still unknown. Therefore, our study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR (epithelial cell line HT-29/B6 transfected with glucocorticoid and mineralocorticoid receptors) cell model and mouse colon. In Ussing chambers, C. concisus infection inhibited ENaC-dependent Na+ transport as indicated by a reduction in amiloride-sensitive short circuit current (-55%, n = 15, p < 0.001). This occurred via down-regulation of β- and γ-ENaC mRNA expression and ENaC ubiquitination due to extracellular signal-regulated kinase (ERK)1/2 activation, predicted by Ingenuity Pathway Analysis (IPA). In parallel, C. concisus reduced the expression of the sealing tight junction (TJ) protein claudin-8 and induced claudin-8 redistribution off the TJ domain of the enterocytes, which facilitates the back leakage of Na+ ions into the intestinal lumen. In conclusion, C. concisus caused ENaC dysfunction via interleukin-32-regulated ERK1/2, as well as claudin-8-dependent barrier dysfunction-both of which contribute to Na+ malabsorption and diarrhea

Effect of ACTH on cortisol metabolism and characterization of 11ß- hydroxysteroid dehydrogenase isoforms in different tissues of the guinea pig

Titelblatt und Inhaltsverzeichnis 1\. Einleitung 5 2\. Materialien und Methoden 12 3\. Ergebnisse 20 4\. Diskussion 44 5\. Zusammenfassung 61 6\. Abkürzungsverzeichnis 63 7\. Anhang 64 8\. Lebenslauf 65 9\. Danksagung 66 10\. Literaturverzeichnis 67Die 11ß-Hydroxysteroid-Dehydrogenase (11ßHSD) des Meerschweinchens ist für die reversible Umwandlung von Kortisol (F) zu Kortison (E) zuständig. Nur F, die 11-Hydroxy-Form, ist am Gluko- und Mineralokortikoidrezeptor aktiv, so dass die 11ßHSD eine entscheidende Rolle bei der Wirkung und Selektivität der Steroidhormone spielt. Bislang sind zwei Isoenzyme bekannt: Die 11ßHSD Typ 1 (11ßHSD1) ist in fast allen Geweben nachweisbar und funktioniert in vivo fast ausschließlich als reduzierendes und damit aktivierendes Enzym. Sie ist vermutlich für die Feinregulation des ?Glukokortikoid-Tonus? in den verschiedenen Zielgeweben verantwortlich. Die 11ßHSD Typ 2 (11ßHSD2) ist ein ausschließlich oxidierendes und damit inaktivierendes Enzym, das die Protektion des unselektiven Mineralokortikoidrezeptors vor hohen Kortisolkonzentrationen gewährleistet und dementsprechend exklusiv in den mineralokortikoiden Zielgeweben exprimiert wird. Nach Vorarbeiten der Arbeitsgruppe, in denen die Isoenzyme des Meerschweinchens in Leber (11ßHSD1) und Niere (11ßHSD1/2) charakterisiert wurden, war das Ziel dieser Arbeit den Einfluss von Stress (=ACTH-Injektionen) auf die Aktivität dieser Isoformen zu untersuchen. Außerdem sollte eine Charakterisierung der Isoenzyme in Homogenaten weiterer Organe durchgeführt werden. Zunächst wurden Leber- und Nierengewebeschnitte von sechs Meerschweinchen hinsichtlich ihres Kortisolmetabolismus nach dreitägiger subkutaner in vivo ACTH-Behandlung untersucht. Als Kontrollgruppe dienten dieselbe Anzahl von Tieren, die mit Kochsalzlösung (NaCl) behandelt wurden. Nach der Tötung wurden die Gewebeschnitte mit radioaktiv markiertem F oder E inkubiert, die Steroide mittels Dünnschichtchromatographie (TLC) getrennt und der Umsatz (in Prozent) errechnet. Es zeigte sich nach ACTH-Behandlung eine hochsignifikante Zunahme der Reduktionsreaktion in Leber (70,3% ACTH vs. 50,2% NaCl) und Niere (56,8% ACTH vs. 39,6% NaCl). Gleichzeitig kam es zu einer signifikanten Abnahme der Oxidationsreaktion (Leber: 23,9% ACTH vs. 33,7% NaCl; Niere: 28,6% ACTH vs. 33,9% NaCl), so dass das Reaktionsgleichgewicht nach dreitägiger in vivo ACTH- Behandlung eindeutig zu Gunsten des F verschoben wurde. Im Einklang mit diesen Ergebnissen zeigte sich eine signifikante Zunahme des Verhältnisses von Tetrahydrokortisol/Tetrahydrokortison im 24h Urin, was Ausdruck einer Steigerung der hepatischen 11ßHSD1 Aktivität ist. Die Plasmakonzentration von F, E, Progesteron, 17-OH-Progesteron und Androstendion waren bei den ACTH- Tieren nach Behandlung hochsignifikant erhöht. Zusammenfassend konnten wir in den Untersuchungen an Gewebeschnitten zeigen, dass die Erhöhung des Plasmakortisolspiegels durch ACTH nicht ausschließlich durch adrenale Sekretion verursacht wird, sondern auch durch verstärkte hepatische und renale Aktivierung von inaktivem E zu F. Dies ist durch eine Aktivitätssteigerung der 11ßHSD1 bedingt. Daher scheint vor allem die Leber als relativ großes Organ mit hoher 11ßHSD1-Aktivität neben den Nebennieren zur Stressadaptation durch eine gesteigerte Kortisolproduktion beizutragen, wohingegen andere Organe, die 11ßHSD1 exprimieren, vermutlich ihren Bedarf an aktivem Glukokortikoid in einer auto- oder parakrinen Weise über dieses Enzym regulieren. Des weiteren wurde Homogenate mit radioaktiv markiertem F oder E, sowie gegebenenfalls mit Kosubstrat inkubiert, die Steroide wiederum mittels TLC getrennt und der Umsatz errechnet. Eine gesicherte Reduktaseaktivität mit NADPH Präferenz als Ausdruck einer 11ßHSD1 Aktivität zeigte sich in absteigender Reihenfolge in Leber, Niere, Nebenniere und Lunge der Meerschweinchen. Andererseits ließ sich eine Oxidaseaktivität mit NAD+ Präferenz als Ausdruck einer 11ßHSD2 Aktivität in Niere, Nebenniere, Kolon, Lunge und Herz nachweisen (ebenfalls in absteigender Reihenfolge). In der Leber zeigte sich zudem eine NAD+-abhängige Oxidation, die bei fehlender Expression der 11ßHSD2 in diesem Organ Ausdruck der Aktivität eines dritten, von unserer Arbeitsgruppe bereits zuvor charakterisierten Isoenzyms in der Meerschweinchenleber ist.In man and in rodents the hepatic 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is a functional oxidoreductase preferring NADP+/NADPH as cosubstrates. In contrast, the renal isoenzyme (11ß-HSD2) mediates in vivo only oxidation of natural glucocorticoids (GK) with NAD+ as cosubstrate. It is widely accepted that the function of the renal 11ß-HSD2 is the protection of mineralocorticoid (MC) receptors from high cortisol concentrations by inactivating cortisol to cortisone, thus prohibiting cortisol access to the MC receptor, which binds aldosterone and cortisol with similar affinity. The function of the hepatic 11ß-HSD1 is not yet well understood but it is thought that it plays a pivotal role in regulating the intracellular level of active GK. We have recently characterized the 11ß-HSD isoenzymes in the guinea pig liver and kidney (Quinkler et al. 1997 J Endocrinol). In this work we investigated the in vivo effects of corticotropin (ACTH) on 11ß-HSDs in these organs and furthermore characterized the 11ß-HSD isoenzymes in homogenates of other tissues. Guinea pigs were treated with ACTH (twice daily injections of 10 IU depot ACTH1-24 for three days) and saline respectively. Tissue slices of liver and kidney were incubated with 3H-labeled cortisol or cortisone, and the conversion of substrate to product was measured by thin layer chromatography. Furthermore different plasma steroid concentrations as well as urinary cortisol metabolites were measured by high pressure liquid chromatography or radioimmunoassay respectively. Then homogenates of different guinea pig tissues (adrenal gland, kidney, lung, heart, liver, colon) were incubated with 3H-labeled cortisol or cortisone and cosubstrate (NAD/H, NADP/H) and the conversion of substrate to product measured as described above. Three days of in vivo ACTH treatment significantly increased the reductase activity in liver (50,2% vs. 70,3% conversion) and kidney slices (39,6% vs. 56,8% conversion). At the same time oxidase activity was significantly decreased in both liver (33,7% vs. 23,9%) and kidney slices (33,9% vs. 28,6%). Furthermore 11ß-HSD1 activity assessed by measurement of the urinary ratio of tetrahydrocortisol [THF] + 5?-THF /tetrahydrocortisone was significantly increased after ACTH treatment compared to the control group. Plasma levels of cortisol, cortisone, progesterone, 17-OH-progesterone and androstendione increased significantly following in vivo ACTH treatment. In homogenates we could show clear-cut activity of 11ßHSD1 in liver > kidney > adrenal > lung and activity of 11ßHSD2 in kidney > adrenal > colon > lung > heart. In conclusion we have shown that the stress adaptation of the guinea pig is not solely caused by increased adrenal secretion of cortisol but also through an increased 11ßHSD1 activity in liver and kidney. Therefore, liver and kidney might contribute to stress adaptation in an endocrine fashion, whereas other organs expressing 11ßHSD1 might regulate their needs for cortisol in an autocrine way

Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum

BACKGROUND: Giardia lamblia causes infection of the small intestine, which leads to malabsorption and chronic diarrhoea. AIM: To characterise the inherent pathomechanisms of G lamblia infection. METHODS: Duodenal biopsy specimens from 13 patients with chronic giardiasis and from controls were obtained endoscopically. Short‐circuit current (I(SC)) and mannitol fluxes were measured in miniaturised Ussing chambers. Epithelial and subepithelial resistances were determined by impedance spectroscopy. Mucosal morphometry was performed and tight junction proteins were characterised by immunoblotting. Apoptotic ratio was determined by terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labelling staining. RESULTS: In giardiasis, mucosal surface area per unit serosa area was decreased to 75% (3%) of control, as a result of which epithelial resistance should increase. Instead, epithelial resistance of giardiasis biopsy specimens was decreased (19 (2) vs 25 (2) Ω cm(2); p<0.05) whereas mannitol flux was not significantly altered (140 (27) vs 105 (16) nmol/h/cm(2)). As structural correlate, reduced claudin 1 expression and increased epithelial apoptosis were detected. Furthermore, basal I(SC) increased from 191 (20) in control to 261 (12) µA/h/cm(2) in giardiasis. The bumetanide‐sensitive portion of I(SC) in giardiasis was also increased (51 (5) vs 20 (9) µA/h/cm(2) in control; p<0.05). Finally, phlorizin‐sensitive Na(+)–glucose symport was reduced in patients with giardiasis (121 (9) vs 83 (14) µA/h/cm(2)). CONCLUSIONS: G lamblia infection causes epithelial barrier dysfunction owing to down regulation of the tight junction protein claudin 1 and increased epithelial apoptoses. Na(+)‐dependent d‐glucose absorption is impaired and active electrogenic anion secretion is activated. Thus, the mechanisms of diarrhoea in human chronic giardiasis comprise leak flux, malabsorptive and secretory components