Correlation of DNA content, p53, T antigen, and V antigen in simian virus 40-infected human diploid cells.

Human diploid fibroblasts (HDF) have a finite life span in cell culture which can be extended when transformed with simian virus 40 (SV40). Flow cytometric analysis of SV40-HDF transformation allowed DNA content changes to be correlated with the appearance, quantity, and distribution of T antigen, p53, and V antigen, three proteins associated with this process. These studies demonstrated a shift in the DNA content to tetraploidy, which was correlated with the age of the SV40-HDF but not the time of infection. A significant increase of the epitope recognized by PAb122 to host p53 and the epitope PAb101 to SV40 T antigen occurred at the same time the tetraploid population appeared. However, an antigen reactive with SV40 V antibody was present at high levels in most of the population early after infection, but the levels declined with time. The percentage of PAb101-T antigen-positive cells increased more rapidly in cells infected at a late passage, and this was concomitant with the shift in DNA content to tetraploid. Analysis of the mean fluorescence of total, gated populations (G1, G2, and greater than G2) demonstrated that a threshold level of p53 and T antigen was reached in each compartment of the cell cycle. As the transformed phenotype appeared, a population of cells was continually released into the supernatant, and although these cells had a DNA pattern similar to the monolayer cells, the T antigen and p53 levels were 3-5 times higher in the tetraploid G2 cells. These studies correlated the expression of proteins associated with viral transformation in HDF which vary with time and shift in DNA content.

Detection of an early cytomegalovirus antigen with two-color quantitative flow cytometry.

An early cytomegalovirus (CMV) antigen was detected with a monoclonal antibody by two-color fluorescent flow cytometry. With the aid of a human diploid fibroblast cell strain, FLOW 2000, infected with the AD169 strain of CMV, the viral antigen and the DNA content of infected or uninfected cells were measured. There was no evidence of change in the cell-cycle distribution of the infected cells. The viral antigen was detected within 30 minutes following virus adsorption at 0.1 and 1.0 plaque-forming units/cells; and the percentage of positive cells increased with time and viral dosage. All stages of the cell cycle were susceptible to viral infection and the average fluorescence was greater than the background fluorescence. Flow cytometry detected the viral antigen earlier than conventional immunofluorescent microscopy and cell culture for CMV cytopathological effect (CPE). Ten bronchoalveolar lavages assayed by flow cytometry and conventional diagnostic procedures demonstrated that flow cytometry might be useful in early diagnosis for CMV infection.

Analysis of simian virus 40 infection of CV-1 cells by quantitative two-color fluorescence with flow cytometry.

Quantitative two-color fluorescent analysis of Simian virus (SV40) infection of permissive CV-1 cells was investigated. Analysis included by quantitation of cellular DNA, the early viral tumor (T) antigen with a monoclonal antibody, and late viral (V) antigens with a polyclonal antibody. T antigen was detected in all phases of the cell cycle at 6 and 12 h, after SV40 infection of growth arrested cells. At later time intervals, the percentage of T-antigen-positive cells increased with the induction of the cells into successive rounds of DNA synthesis and an increase in tetraploid-polyploid cells. The amount of T antigen per cell increased as the cells entered the successive stages of the cell cycle (G0/G1----G2 + M----tetraploid S and G2 + M). The V antigen from adsorbed virus was detected immediately after infection. Synthesis of V antigen began in late S and G2 + M phases of the cell cycle. This quantitative analysis allows a definitive determination of antigen per cell in a population correlated with the cell cycle and may be useful in correlating viral and cellular events with transformation.

Analysis of intracellular antigens by flow cytometry.

Quantitative immunofluorescence is routinely used in flow cytometric assay of cell surface antigens. Intracellular antigens have not been as tractable. Recent publications (Proc Natl Acad Sci 80:5573-5577, 1983; Cytometry 6:208-214, 1985) and the results presented here demonstrate that highly specific staining and subsequent quantitative analysis are not only possible but rather easily achieved. High purity antibodies and optimized fixing and staining technique are required. Under conditions presented in this paper, 97% of fluorescein specific signal is specific to the T antigen of SV40 when monoclonal antibody to this antigen is used with a transformed cell line. Three levels of quantitative analysis are discussed: estimation of the fraction of positive cells in a mixed +/- population, estimation of the average content of antigen in a population of cells, and measurement of the distribution of antigen content within a population of cells. Results are presented that demonstrate that relatively low specific signal (measured as percentage of total signal) can be tolerated to achieve the first level and that the current methods available that produce a high specific signal are sufficient to achieve the second level. The third level will require further research aimed at lowering the variation introduced by the method of measurement.

Identifying a susceptible subgroup: effects of the Pittsburgh air pollution episode upon schoolchildren.

Pulmonary function test results on 224 parochial schoolchildren collected during and after the Pittsburgh air pollution episode of November 1975 were reanalyzed to determine whether a small subgroup of susceptible children could be defined. Individual regressions of three-quarter second forced expiratory volumes (FEV.75) and forced vital capacities (FVC) on time over the six-day study period were calculated, and the distributions of individual slopes for the four exposed and two control schools were compared. Excesses of strong upward trends in the exposed areas would suggest effects of suspended particulate air pollution by indicating significant improvement following the episode. A highly statistically significant excess of strong upward trends in the FVC among exposed students was observed, and was consistent by sex and by school within sex. Approximately 10--15% of the students appear susceptible to an average impairment of about 20% of the FVC. The findings are limited by the small number of subjects with strong post-episode upward trends in the FVC, and by lack of validation by replication of the study design, but do suggest that episode levels of suspended particulates induce lung damage, and that this may occur only in a small susceptible subgroup. Children with low baseline pulmonary function values, histories of asthma, or with acute respiratory symptoms immediately following the episode were not found to be especially susceptible to these effects of suspended particulates.