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Zur Pathogenese der Antibiotikakolitis: Wirkung von Clostridium difficile Toxin A und B auf die humane Kolonschleimhaut in vitro

Antibiotic-associated colitis: Effect of clostridium difficile toxins A and B on human colonic mucosa in vitro

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Zusammenfassung

Grundlagen: Clostridium difficile ist der Erreger der Antibiotika-assoziierten Enterokolitis. In Tierstudien schädigt Clostridium difficile Toxin A, aber nicht Toxin B die Schleimhaut des Gastrointestinaltraktes. Die Wirkung der Toxine auf die humane Kolonschleimhaut ist unbekannt. Deshalb untersuchten wir in dieser Studie die Wirkung von Clostridium difficile Toxin A (TxA) und Toxin B (TxB) auf die humane Kolonschleimhaut in vitro.

Methodik: Schleimhautpräparationen humanen Kolons wurden in Ussing-Kammern eingebracht und 5 h mit Puffer (Kontrollen) oder Puffer, der an der luminalen Seite 5 bis 20 µg/ml TxA bzw. 0,06 bis 5,0 µg/ml TxB enthielt, inkubiert. Potentialdifferenz (PD), Kurzschlußstrom (Isc) wurden gemessen, der Widerstand (R) errechnet. Das Ausmaß der Mukosaschädigung wurde an mit Hämatoxylin und Eosin (HE) gefärbten Schnitten mittels computergesteuerter Morphometrie gemessen. Das filamentöse (F-) Aktin des Epithelzellskelettes wurde mit Rhodamin beladenem Phalloidin auf Gefrierschnitten gefärbt und im Fluoreszenzmikroskop untersucht.

Ergebnisse: Während die Kontrollen eine normale Morphologie aufwiesen, zeigte die Histologie eine konzentrationsabhängige Schädigung des Oberflächenepithels der Kolonschleimhaut durch TxA und TxB. Das Epithel der Krypten blieb auch bei höchsten Konzentrationen intakt. Entsprechend waren in der Morphometrie nach 5 h luminaler Inkubation mit 5, 10 und 20 µg/ml TxA jeweils 18,6 ± 3%, 33,3 ± 2% bzw. 54 ± 2% der Mukosa geschädigt. Nach Inkubation mit 0,25, 0,5 und 1,0 µg/ ml TxB waren jeweils 9,8 ± 0,6%, 15,6 ± 0,2% bzw. 28,6 ± 0,3% geschädigt (p <0,05). Elektrophysiologie: Zu Versuchsbeginn betrug R in der Kontrollgruppe 174 ± 13 Ohm x cm2, nach 5 h 177 ± 11 Ohm x cm2. 5 h Inkubation mit 5, 10 bzw. 20 µg/ml TxA führte zu einem R-Abfall von jeweils etwa 58, 82 bzw. 136 Ohm x cm2. 5 h Inkubation mit 0,5, 1,0 bzw. 5 µg/ml TxB verursachte einen R-Abfall von jeweils etwa 85, 94 bzw. 169 Ohm x cm2 (p <0.05). Fluoreszenzmikroskopie: Beide Toxine zerstörten das F-Aktin des Zellskelettes der Epithelzellen.

Schlußfolgerungen: Histologie, Morphometrie und Elektrophysiologie zeigen, daß Clostridium difficile TxA und B das Epithel der humanen Kolonschleimhaut in vitro konzentrationsabhängig schädigen und Toxin B, was die Konzentration betrifft, etwa 10mal wirksamer ist als Toxin A.

Summary

Background: In animal models of Clostridium difficile enteritis, toxin A, but not toxin B, appears to mediate intestinal damage. In this study we investigated the morphologic and electrophysiologic effects of purified C. difficile toxins A and B on human colonic mucosa in vitro.

Methods: Human colonic mucosal sheets were mounted in Ussing chambers and incubated with buffer or luminal buffer containing 5 to 20 µg/ml of toxin A and 0.06 to 10 µg/ml of toxin B for 5 h. Potential difference (PD) and short-circuit current (Isc) were measured, resistance (R) was calculated. Histology and Morphometry were performed on hematoxyline and eosine (HE) stained paraffin embedded sections. Flourescent staining of Factin was performed using rhodamin labelled phalloidin.

Results: Histology: Both toxins caused a dose dependent damage of the surface epithelium, while the epithelium lining the crypts remained intact. Morphometry showed that luminal incubation with 5, 10 and 20 µg/ml of TxA caused mucosal damage of 18.6 ± 3%, 33.3 ± 2% and 54 ± 2% respectively. Inkubation with 0.25, 0.5 and 1.0 µg/ml of TxB caused damage of 9.8 ± 0.6%, 15.6 ± 0.2% and 28.6 ± 0.3% respectively (p <0.05). Electrophysiology: At the beginning of the experiments R was 174 ± 13 Ohm x cm2 in the controlgroup, after 5 h R was 177 ± 11 Ohm x cm2. 5-h incubation of tissues with 5, 10 and 20 µg/ml of TxA caused R to drop by 58, 82 and 136 Ohm x cm2, respectively. 5-h incubation with 0.5, 1.0 bzw. 5 µg/ml of TxB caused R to drop by 85, 94, and 169 Ohm x cm2, respectively (p <0.05). Flourescent microscopy of phalloidin stained sections showed that both toxins caused disruption and condensation of cellular filamentous (F-) actin.

Conclusions: Our results demonstrate that the human colon is approximately 10 times more sensitive to the damaging effects of toxin B than toxin A, suggesting that toxin B may be more important that toxin A in the pathogenesis of C. difficile colitis in man.

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Authors and Affiliations

  1. Klinische Abteilung für Allgemeinchirurgie, Universitätsklinik für Chirurgie, Währinger Gürtel 18–20, A-1090, Wien, Austria

    M. Riegler, B. Teleky, G. H. Hamilton, J. Zacherl, G. Bischof, E. Cosentini &  E. Wenzl

  2. Institut für klinische Pathologie der Universität Wien, Austria

    R. Sedivy

  3. Abteilung für Gastroenterologie des Universitätsspitals Boston, Massachusetts, USA

    Ch. Pothoulakis & J. Th. LaMont

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  1. M. Riegler
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  9. J. Th. LaMont
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Riegler, M., Sedivy, R., Pothoulakis, C. et al. Zur Pathogenese der Antibiotikakolitis: Wirkung von Clostridium difficile Toxin A und B auf die humane Kolonschleimhaut in vitro. Acta Chir Austriaca 29, 95–101 (1997). https://doi.org/10.1007/BF02619754

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