Oligo-2,5-Adenylate Synthetase in Pulmonary Sarcoidosis and Idiopathic Pulmonary Fibrosis: Methods

Bronchoalveolar Lavage
Three 50-ml aliquots of 0.9 percent sterile saline solution were instilled into a bronchus in the left lingular division or the right middle lobe through a fiberoptic bronchoscope. The BALF was recovered by gentle suction immediately after the infusion of each aliquot. After centrifugation, the recovered cells were washed three times with phosphate-buffered saline solution (PBS). A small volume was removed for cell numeration and differential cell count. Hematoxylin-eosin staining on polymer (millipore) filter preparations was used to analyze the cell differentials, and a percentage was established from 500 cells made up of PAM, lymphocytes, and polymorphonuclear leukocytes (PMN).
Isolation of Monocytes and Lymphocytes from PBML, and Isolation of PAM and Lymphocytes From BALF Cells
From PBML of some cases (two SAR, two IPF, and three NC), monocytes and lymphocytes were isolated according to discontinuous Percoll gradients as previously described. The purity of monocytes and lymphocytes averaged 84 percent and 89 percent, respectively. The BALF cells of the seven cases were suspended in RPM1-1640 medium with 10 percent fetal calf serum at a density of 5 to 10 x 106/ml in 100-mm tissue culture dishes for 30 min at 37°C. Nonadherent cells were removed from the dishes by three sequential gentle washes. From the collected nonadherent cells, lymphocytes were isolated by discontinuous Percoll gradients. The purity of macrophages (the adherent cells) and lymphocytes was 95 percent and 94 percent, respectively. canadian neightbor pharmacy

Measurement of the Activity of 2,5 AS
We measured the levels of 2,5AS activity according to a previous method. Briefly, PBML and BALF cells were lysed in 60 |xl of solution buffer 1 (20 mmol/L HEPES-KOH [pH 7.5] containing 5 mmol/L MgCl2, 120 mmol/L KCl, 1 mmol/L DTT, 10 percent glycerol, and 0.5 percent Noidet P-40) at room temperature for 10 min, after which the lysate was centrifuged at 10,000 rpm for 15 min, and then the supernatant was collected and used in the assay for measurement of the levels of 2,5AS activity. The protein concentration was determined by the methods of the protein kit (Bio-Rad, Richmond, Calif). Ten micrograms of cell extract was added in solution 2 (10 mmol/L HEPES-KOH [pH 7.4] containing 90 mmol/L CH3COOK, 25 mmol/L [CH3COO]2Mg, 4 mmol/L ATP, 4 mmol/L fructose-1,6-biphosphate, 1 mmol/L DTT, 200 Jlg/ml polyinosinate-sytidylate and [H]ATP (1.25 ЦС0), and then each total volume was adjusted in 50 \i\ using the same buffer. Incubation was carried out at 33°C for 4 h, and then terminated by heating the mixture to 95°C for 2 min. The samples were diluted to 1 ml with 90 mmol/L KCl-20 mmol/L Tris-HCl buffer, at a pH of 7.2, and centrifuged for 5 min at 3,000 rpm. The supernatant was passed through a column (4 by 20 mm) of DEAE-cellulose (DE52, Whatman Inc, Clifton, NJ) equilibrated with this buffer. In order to remove nonreactive ATP, the columns were washed with 15 ml of the same buffer and then eluted with 2 ml of 350 mmol/L KCl-20 mmol/L Tris-HCl buffer, at a pH of 7.5. The radioactivity of each eluted sample was measured directly in 10 ml of solubilizer (PCS, Amersham Corp, Arlington Heights, III). The enzyme activity was represented as nanomoles of polymerized ATP per milligram of protein per hour (nmol/mg/h).

Enzyme Assay
Angiotensin-converting enzyme (ACE) activity was measured using a method based on colorimetry of the quinoneimine dye produced from the substrate hippuryl-L-histidyl-L-leucine.
Statistical Analysis
Data were expressed as the mean ± SEM. Data were analyzed by Student’s t test or by Mann-Whitney U test. The critical significance level was chosen at the point of 5 percent.

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