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Human lung and segments of pulmonary artery were obtained at lung transplantation from 6 patients with IPAH 1 man and 5 women; mean age, 24.5 4.7 years, SEM ; .8 For control experiments, pulmonary artery samples were taken from 5 patients with bronchogenic carcinoma 4 men and 1 woman; mean age, 59.0 4.1 years ; , who had no evidence of pulmonary hypertension at the time of lung lobectomy. Samples of the pulmonary arteries were obtained from the most distal area from the carcinoma in the resected lobe. Patients with bronchogenic carcinoma received no systemic chemotherapy or radiation therapy before lung lobectomy. All cell culture experiments were carried out after the approval of the Human Ethics Committee of Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, and written informed consent was obtained from all patients before the procedure. We isolated and cultured SMCs by using the methods described previously, with minor modifications.9, 10 Pulmonary artery SMCs were isolated from the peripheral segments of the artery smaller than 1 mm in outer diameter. For the enzymatic disaggregation of SMCs, a mixture of 10 mL phosphate-buffered saline PBS ; and 100 g collagenase was used in a shaking water bath for 15 minutes at 37C. The adventitia and endothelial cell layers were removed. The vessels were cut into 2-mm-long sections and plated in 6-well culture plates containing Dulbecco modified Eagle's medium DMEM; Nissui ; supplemented with 10% fetal bovine serum FBS ; and 0.1 mg mL kanamycin. They were incubated in a humidified 5% CO2 atmosphere at 37C. The medium was changed every 3 days. After reaching confluence, the cells were subcultured by a trypsin 0.05% ; ethylenediaminetetraacetic acid EDTA ; 0.02% ; treatment. Are usually recommended to be part of second-line regimens in most resourcelimited settings. EFV is the NNRTI of choice in children who are on rifampicin, if treatment needs to start before anti-tuberculous therapy is completed. For drug dosages and regimens see Appendix 2, page 348 and etoposide.
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