Thomas Rechsteiner

A new heterotopic transplant animal model of intestinal fibrosis.

Background:Severe mucosal tissue damage requiring efficient wound healing is a main feature of inflammatory bowel disease but excessive tissue repair promotes fibrosis. The clinical investigation of fibrosis is confined to the limited amount of biological material available from patients. This makes the establishment of a new animal model, a highly desirable goal. We investigated whether intestinal fibrosis occurs after heterotopic transplantation of small bowel resections in rats. Methods:Donor (Brown Norway or Lewis rats) small bowel resections were transplanted subcutaneously into the neck of recipients (Lewis rats). Grafts were explanted 2, 7, 14, and 21 days after transplantation. Results:Heterotopic intestinal transplants remained viable for 21 days. Rapid loss of crypt structures at day 2 after intestinal transplantation was followed by lymphocyte infiltration and obliteration of the intestinal lumen by fibrous tissue at day 21. Loss of the intestinal epithelium was confirmed by the lack of cytokeratin staining in immunohistochemistry. Collagen expression was increased with time after transplantation as confirmed by real-time PCRs, Elastica van Gieson, and Sirius Red staining. Lumen obliteration was connected with increased expression of potent mediators of fibrosis such as &agr;5&bgr;6 integrin, interleukin (IL)-13, and transforming growth factor &bgr;. Myofibroblast phenotype was demonstrated by the presence of both &agr;-smooth muscle actin and vimentin in the obliterated lumen. Conclusions:We established a method for heterotopic transplantation of small bowel resections. A variety of histologic and molecular features of fibrosis were observed in the heterotopic intestinal grafts which suggests, that this new in vivo model will be instrumental in studying pathogenesis and treatment of intestinal fibrosis.

Effects of a short-term rehabilitation program on airway inflammation in children with cystic fibrosis.

Respiratory therapy in cystic fibrosis (CF) consists of airway clearance, infection control, and reduction of airway inflammation. It is well recognized that physical activity as well as daily chest physiotherapy, enhance airway clearance. We investigated the effects of pulmonary rehabilitation, including physical activity and chest physiotherapy, on airway inflammation in children with CF.

MicroRNA-223 controls the expression of histone deacetylase 2 : a novel axis in COPD

Reduced activity of histone deacetylase 2 (HDAC2) has been described in patients with chronic obstructive pulmonary disease (COPD), but the mechanisms resulting in decreased expression of this important epigenetic modifier remain unknown. Here, we employed several in vitro experiments to address the role of microRNAs (miRNAs) on the regulation of HDAC2 in endothelial cells. Manipulation of miRNA levels in human pulmonary artery endothelial cells (HPAEC) was achieved by using electroporation with anti-miRNAs and miRNA mimics. Target prediction software identified miR-223 as a potential repressor of HDAC2. In subsequent stimulation experiments using inflammatory cytokines known to be increased in patients with COPD, miR-223 was found to be significantly induced. Functional analysis demonstrated that overexpression of miR-223 decreased HDAC2 expression and activity in HPAEC. Conversely, HDAC2 expression and activity was preserved in anti-miR-223-treated cells. Direct miRNA-target interaction was confirmed by reporter gene assay. In a next step, reduced expression of HDAC2 was found to increase the levels of the chemokine fractalkine (CX3CL1). In vivo studies confirmed elevated expression levels of miR-223 in mice exposed to cigarette smoke and in emphysematous lung tissue from LPS-treated mice. Moreover, a significant inverse correlation of miR-223 and HDAC2 expression was found in two independent cohorts of COPD patients. These data emphasize that miR-223, the most prevalent miRNA in COPD, controls expression and activity of HDAC2 in pulmonary cells, which, in turn, might alter the expression profile of chemokines. This pathway provides a novel pathogenic link between dysregulated miRNA expression and epigenetic activity in COPD.Key messagesHistone deacetylase 2 is directly targeted by miR-223.Levels of miR-223 are induced by interleukin-1β and tumor necrosis factor-α.miR-223 controls the expression of fractalkine by targeting histone deacetylase 2.miR-223 levels are increased in COPD mouse models.miR-223 levels inversely correlate with HDAC2 expression in COPD patients.

THE MACROLIDE CLARITHROMYCIN INHIBITS EXPERIMENTAL POST-TRANSPLANT BRONCHIOLITIS OBLITERANS

Chronic allograft dysfunction in form of bronchiolitis obliterans is the most important hurdle to improved longterm survival after clinical lung transplantation to date. Recently, it was observed that the progression of bronchiolitis obliterans in lung transplant recipients might be inhibited by macrolide antibiotics. The authors therefore tested whether macrolide therapy can attenuate fibrous obliteration of airways in an animal model of bronchiolitis obliterans. Rats with heterotopic tracheal allografts were treated intraperitoneally with clarithromycin and compared to untreated transplanted animals with respect to allograft histology and expression of selected cytokines. At day 21 after transplantation, the tracheal allografts of treated animals were free of fibrous material or partially occluded dependent of clarithromycin dosage. Untreated animals had completely obliterated allografts. In treated animals, tumor necrosis factor alpha (TNF-α) was down-regulated early (5 days) and late (21 days) post transplant, whereas interferon gamma (IFN-γ) expression was decreased only early after transplantation. Transforming growth factor beta (TGF-β) expression was not affected. Therapy with low-dose macrolides in post-transplant obliterative bronchiolitis is based on their immunomodulatory rather than antimicrobial properties. In the setting of lung transplantation, macrolides primarily act as modulators of the early inflammatory response to stressed, damaged, or infected cells.

Attenuation of airway obliteration by ciprofloxacin in experimental posttransplant bronchiolitis obliterans.

Background. Ciprofloxacin is widely used to treat respiratory tract infections. Like other fluoroquinolones, ciprofloxacin has immunomodulatory effects; however, it is unknown whether these effects are beneficial in the setting of lung transplantation. We investigated potential immunomodulatory effects of ciprofloxacin in a model of posttransplant bronchiolitis obliterans. Methods. The heterotopic tracheal transplantation model in rats was used. Three groups received ciprofloxacin and underwent different immunosuppressive regimens of cyclosporine A, that is, no immunosuppression, insufficient immunosuppression, or low-dose immunosuppression. Three groups underwent the same immunosuppressive regimen but had no ciprofloxacin treatment. Tracheas were harvested after 21 days and examined with respect to histology and expression of selected cytokines. Results. The allografts of animals treated with ciprofloxacin showed less airway obliteration compared with allografts of untreated animals. When combimed with low-dose immunosuppression ciprofloxacin showed beneficial effects in preventing airway obliteration and rejection of the respiratory epithelium. Cytokine gene expression of the allografts treated with ciprofloxacin was higher with respect to transforming growth factor-&bgr; and equal with respect to tumor necrosis factor-&agr; and interferon-&ggr; compared with controls. When applied in combination with cyclosporine A, ciprofloxacin lowered the expression of transforming growth factor-&bgr; and tumor necrosis factor-&agr; and increased interferon-&ggr; expression. Conclusion. Ciprofloxacin attenuates airway rejection after tracheal transplantation. Genetic expression of mediators that are known to play an important role in mediating rejection in this model supports an immunomodulatory and antifibrotic role of ciprofloxacin. These findings suggest that further clinical studies are needed to investigate whether ciprofloxacin in addition to its bactericidal effect might be beneficial in the treatment of human posttransplant bronchiolitis obliterans.

Extended measurements of exhaled nitric oxide in adult lung transplant recipients.

Natalia Ridao-Cano Ana I Sanchez-Fructuoso Antolina Rodriguez-Moreno Alberto Barrientos Servicio de Nefrología Hospital Clínico San Carlos Madrid, Spain The authors declare no conflict of interest. Address correspondence to: Natalia Ridao-Cano, M.D., Servicio de Nefrología. Hospital Clinico San Carlos, C/ Profesor Martin Lagos s/n, Madrid 28040, Spain. E-mail: nataliaridaocano@yahoo.es N.R.-C., A.S.-F., A.R.-M., and A.B. participated in research design; N.R.-C., A.S.-F., and A.B. participated in the writing of the article; N.R.-C. and A.R.-M. participated in the performance of the research; and N.R.-C. and A.B. participated in data analysis. Received 15 March 2010. Accepted 29 March 2010. Copyright

EXTRACORPOREAL PHOTOPHERESIS IN A RAT MODEL OF PULMONARY FIBROSIS

Extracorporeal photopheresis has anti-inflammatory properties. The development of pulmonary fibrosis includes inflammatory episodes. This study evaluates effects of extracorporeal photopheresis in experimental pulmonary fibrosis. The bleomycin model of pulmonary fibrosis was used. Two groups of 4 rats received intratracheal bleomycin to induce fibrosis. The treatment group received infusions of photochemically treated leukocytes harvested from syngeneic animals. All animals were sacrificed at day 21 after fibrosis induction and analyzed with respect to lung histology and hydroxyproline content, cellular composition of bronchoalveolar lavages, serum and lavage concentrations of transforming growth factor-beta, interferon-gamma, and interleukin-10, and expression of selected genes in the lung. Interleukin-10 and transforming growth factor-beta protein concentrations increased in the plasma of treated animals, whereas the interferon-gamma protein concentration was higher in bronchoalveolar lavages. Interferon-gamma gene expression was up-regulated in the lung tissue of treated animals. No significant differences between treated and untreated animals were found with respect to hydroxyproline, histology, and lavage cell count. To conclude, extracorporeal photopheresis has positive molecular effects but does not attenuate experimental lung fibrosis with respect to histology, hydroxyproline, and lavage cell count in the applied treatment regimen. Further investigations of extracorporeal photopheresis in experimental pulmonary fibrosis are justified.