We report the case of a cystic fibrosis patient colonized with a smooth-morphotype form of Mycobacterium abscessus who developed acute respiratory failure with the emergence of an isogenic rough (R) variant while he was recovering from peritonitis-induced shock. This report emphasizes the role of R forms in severe M. abscessus infections. (Fig. 1). CASE REPORTIn December 2003, the patient was admitted to the emergency unit of Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France, with a colic perforation and diffuse peritonitis secondary to a stercolith. Septic shock with severe hypoxemia occurred on day 1, requiring mechanical ventilation, inotropic adrenaline support, and treatment with combined antibiotics (ceftazidime, vancomycin, tobramycin, and ornidazole), hydrocortisone hemisuccinate, and drotrecogin alpha. Laboratory parameters showed lymphopenia (absolute lymphocyte count, 1,060/mm 3 ) and raised serum transaminase levels (alanine aminotransferase level, 80 IU/liter, three times the upper limit of the normal range). The patient's clinical status slowly improved. Bronchial aspiration performed on day 6 yielded A. fumigatus and a rough (R) variant of M. abscessus (M. abscessus CF01-R). Positive A. fumigatus antigenemia results led to the initiation of antifungal therapy on day 9.Unexpectedly, septic shock recurred on day 11. Severe respiratory failure was present (ratio of the partial pressure of oxygen in arterial blood to the fraction of inspired oxygen [PaO 2 /FiO 2 index], 190), associated with patchy alveolar consolidation (Fig. 2). A surgical lung biopsy (right thoracotomy) was performed on day 12: the biopsy specimen culture was positive for an R-morphotype isolate of M. abscessus as the sole pathogen, with concordant histology showing a granulomatous epithelioid reaction with giant cells in areas of peribronchovascular fibrosis and multiple microabscesses. Antibiotic therapy was shifted empirically to imipenem-cilastin, amikacin, and clarithromycin on day 13. Bronchial aspiration performed on day 13 yielded results similar to those from the lung biopsy, thus confirming the diagnosis of M. abscessus respiratory infection (7). Antibiotic susceptibility testing performed on several isolated strains (Institut Pasteur, Centre National de Références, Paris, France) showed the strains to be susceptible to the prescribed regimen. Bronchoalveolar lavage on day 32 still yielded an R-morphotype isolate of M. abscessus. Nebulized amikacin was added to the systemic anti-M. abscessus therapy on day 56. The patient became apyrexial on day 77, and mechanical ventilation was stopped on day 83. The patient was discharged on day 128 (April 2004) under a regimen of clarithromycin monotherapy, which was maintained until April 2005. Sputum samples remained repeatedly positive for M. abscessus (R form) throughout treatment (Fig. 1) (24) and multilocus sequence typing (data not shown) confirmed the clonal nature of the isolates of different morphotypes (Fig. 3). These results established the persistence ...

In cystic fibrosis (CF), abnormal control of cellular Ca(2+) homeostasis is observed. We hypothesized that transient receptor potential canonical (TRPC) channels could be a link between the abnormal Ca(2+) concentrations in CF cells and cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. We measured the TRPC and CFTR activities (using patch clamp and fluorescent probes) and interactions (using Western blotting and co-immunoprecipitation) in CF and non-CF human epithelial cells treated with specific and scrambled small interfering RNA (siRNA). The TRPC6-mediated Ca(2+) influx was abnormally increased in CF compared with non-CF cells. After correction of abnormal F508 deletion (del)-CFTR trafficking in CF cells, the level of TRPC6-dependent Ca(2+) influx was also normalized. In CF cells, siRNA-TRPC6 reduced this abnormal Ca(2+) influx. In non-CF cells, siRNA-TRPC6 reduced the Ca(2+) influx and activity wild-type (wt)-CFTR. Co-immunoprecipitation experiments revealed TRPC6/CFTR and TRPC6/F508 del-CFTR interactions in CF or non-CF epithelial cells. Although siRNA-CFTR reduced the activity of wt-CFTR in non-CF cells and of F508 del-CFTR in corrected CF cells, it also enhanced TRPC6-dependent Ca(2+) influx in non-CF cells, mimicking the results obtained in CF cells. Finally, this functional and reciprocal coupling between CFTR and TRPC6 was also detected in non-CF ciliated human epithelial cells freshly isolated from lung samples. These data indicate that TRPC6 and CFTR are functionally and reciprocally coupled within a molecular complex in airway epithelial human cells. Because this functional coupling is lost in CF cells, the TRPC6-dependent Ca(2+) influx is abnormal.

In CF small airways, defective Cl(-) secretion combined with unregulated (persistent) Na(+) absorption results in ASLdepletion.

Cystic fibrosis (CF) airway epithelia exhibit altered Cl(-) and Na(+) transport properties and increased IL-8 secretion. In the present study, we examined whether a small proportion of cells with a normal phenotype could normalize the ion transport and IL-8 secretion properties of a CF airway epithelial cell layer. We obtained three types of primary cultures of human bronchial epithelial cells: one composed of 100% non-CF cells, one of 100% CF cells, and one of 10% non-CF and 90% CF cells ("cocultures"). Measurement of the bioelectric properties in Ussing chambers revealed that the cocultures displayed Cl(-) and Na(+) transports similar to those observed in the 100% non-CF cultures and significantly different from CF cultures. IL-8 concentration in the coculture supernatant was not different from non-CF cultures, but was significantly lower than in CF cultures. This study provides evidence that 10% bronchial epithelial cells expressing a normal phenotype are sufficient to functionally correct a primary culture of CF bronchial epithelial cells in vitro. We postulate that 10% cells with a non-CF phenotype can be used as a goal for the design of gene therapy and cell therapy trials for CF lung disease.