Caspase-9 as a target for Pneumocystis pneumonia therapy.
Caspase-9 as a target for Pneumocystis pneumonia therapy.
复制标题
Caspase-9 作为肺孢子菌肺炎治疗的靶标。
DOI：
10.1111/j.1550-7408.2006.00205.x
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Lee,Chao-Hung
中科院分区：
文献类型：
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作者：
Lasbury,MarkE;Wang,Shao-Hung;Zhang,Chen;Liao,Chung-Ping;Durant,PamelaJ;Tschang,Dennis;Lee,Chao-Hung
MATERIALS AND METHODSFemale Sprague–Dawley rats (120–140 g) were obtained from Harlan (Indianapolis, IN) and were divided into four groups. The first group of rats, referred to as Normal rats, were uninfected immunocompetent rats. The second group of rats consisted of Normal rats challenged with 7.8 x 106 P. carinii organisms by transtracheal inoculation as described previously (Lasbury, Durant, and Lee 2003). The third group of rats included dexamethasone-treated rats (Dex); they were immunosuppressed by providing drinking water containing 1.8 mg/ml dexamethasone. The fourth group of rats consisted of dexamethasone-treated rats transtracheally inoculated with 7.8 x 106 P. carinii organisms (Bartlett et al. 1988) and these were called Dex-Pc rats. Alveolar macrophages were obtained by bronchoalveolar lavage (BAL) and identified based on their morphology and reaction with the anti-RMA antibody that is specific for alveolar macrophages (Lasbury et al. 2003). To test the effects of BAL fluids or caspase inhibitors on apoptosis of alveolar macrophages, the cells were cultured in RPMI medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum, 1 mM pyruvate, 1% non-essential amino acids, 14mM glucose, 17.9 mM NaCO3, 10 mM HEPES buffer, 100 U/ml penicillin and 0.1 mg/ml streptomycin. The cells were incubated in tissue culture plates for 1 h at 37 1C in 5% CO2 to allow attachment of the macrophages and then the appropriate BAL fluid with or without 10 μg/ml of each caspase inhibitor was added for 48h as described previously (Lasbury et al. 2006).The TUNEL assay to detect DNA fragmentation, a characteristic feature of apoptosis, was performed as described previously (Lasbury et al. 2006). Immunoblotting was performed to detect activated caspases using antibodies against activated caspase-3,-8,-9, or-10 as described previously (Lasbury et al. 2006).
