Concanavalin A-Dependent Cell-mediated Cytotoxicity Assay: The CDCMC assay was used to measure activated lymphocyte killing, as published elsewhere. In brief, the murine mastocytoma cell line P815 (1-2×10 cells/ml) was suspended in 0.3 ml of medium; 0.3 ml of Tris phosphate buffer, ph 7.4; and 100 to 200 μCi of sodium 5ICr chromate (Amersham Corp). The P815 target cells were then incubated for 30 min at 37°C, washed three times and resuspended in medium at 1.25 x 104 viable cells/ml. Con A, 80 n-g/ml, was added to the target cells to give a 2 x concentration relative to the final concentration in the assay. One hundred microliters of 5lCr-labeled P815 cells was then dispensed into triplicate wells of a 96 well V-bottom microtiter plate (Nunc).
One hundred microliters of viable lymphocytes was added to each wall of P815 cells and effector-to-target cells ratio were varied by twofold dilutions from 100:1 to 6.25:1, with the number of target cells kept constant. The effector cell concentration was 1.25×10 cells/ml. After 3 h at 37°C, 100 p,l of supernatant from triplicate samples was harvested and the 5ICr radioactivity determined in a gamma spectrophotometer (model 1185, Searle Analytic, Inc). The percent cytotoxicity was calculated by the formula: ([cpm experimental — cpm spontaneous] [cpm maximum—cpm spontaneous]) X 100. Spontaneous release was 10 percent of the maximum release. The cytotoxicity data obtained at the aboveindicated effector^to-target cell ratios was a linear ratio, and therefore, we report data only at 100:1. The SD of the mean cpm of experimental maximum and spontaneous groups was usually 10 percent and always <15 percent. The lowest level of activity considered to be detectable is 4 percent. add comment
Ten patients with IPF were studied; nine had a pathologic diagnosis of usual interstitial pneumonia (UIP) and one had a diagnosis of desquamative interstitial pneumonia (DIP). Chest roentgenograms showed bilateral diffuse interstitial infiltrates for all patients except the patient with DIP (who had a normal roentgenogram). Gallium scans were variably positive for UIP patients, but negative in the patient with DIP. Pulmonary function tests were consistent with moderate to severe restrictive lung disease (Fig 1, pretreatment pulmonary functions represented as closed symbols).
Figure 1. Longitudinal pulmonary function studies. Pulmonary function was measured by the percent predicted VC and by percent predicted diffusing capacity for carbon monoxide (Dsb). Nine patients were treated with prednisone (1 mg/kg/day) until their VC and Dsb was stable for one month. One patient (•) was begun on the same regimen but self-tapered the medication to one-half the prescribed dose. Four patients had improved pulmonary function with therapy (mean ± SEM increase in Dsb = 30 percent, +2 percent; p<0.001). Six patients failed to improve pulmonary function. Closed symbols = pretreatment; open symbols = posttreatment.