Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome.

The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.     

In vivo T cell activation, in vitro defective IL-2 secretion, and response to influenza vaccination in elderly women.

IL-2 secretion in response to mitogenic stimulation, assayed in vitro, is significantly reduced in circulating T lymphocytes isolated from healthy old people, but the significance of this abnormality and how it relates to in vivo IL-2 secretion remain unclear. We found that IL-2 secretion in response to PHA plus PMA by peripheral blood T cells isolated from 10 out of 32 (31%) healthy old individuals (mean age 86 yr, range 74-97) was significantly decreased compared with results obtained in 23 younger individuals (mean age 34 yr, range 23-46). This IL-2 secretion defect in vitro was reversible after a 3-day incubation in the absence of activators. The 10 healthy old individuals who had defective IL-2 secretion in vitro also showed increased levels of serum IL-2. T cells from 22 healthy old and 22 young individuals, who had normal IL-2 secretion (geometric mean +/- log of 1 SD: 139 +/- 0.3 U/ml and 212 +/- 0.31 U/ml, respectively) in vitro, showed a remarkable transient T cell defect in IL-2 secretion (15 +/- 0.47 U/ml for the old, 54 +/- 0.28 U/ml for the young) 15 days after influenza vaccination. IL-2 secretion became normal again 30 days after vaccination. The T cell-IL-2 activity, expressed as a T cell-IL-2 activity score (calculated as the logarithm of the serum IL-2 U/ml divided by the logarithm of the IL-2 secretion U/ml, in vitro) was significantly increased in elderly non-responders after influenza vaccination (mean +/- 1 SD: 1.4 +/- 0.51) compared with elderly (0.44 +/- 0.13) and younger responders (0.3 +/- 0.2). Our data suggest that in vitro defective IL-2 secretion is a consequence of T cell activation which seems to occur in a significant proportion of healthy elderly individuals and may be clinically relevant inasmuch as it appears to prevent the normal vaccine-induced antibody response.     

In situ hybridization analysis of the age-associated decline in IL-2 mRNA expressing murine T cells

Only a small decrease in the number of L3T4- cells was observed in the Con A-stimulated splenocyte cultures of old mice as compared to young, which cannot account for the threefold decrease in IL-2 production. Northern and dot blot analysis of RNA from splenocytes containing equivalent numbers of L3T4+ cells from young and old mice showed that cells from old mice express less IL-2 mRNA after mitogenic stimulation than cells from young mice. Direct analysis by in situ hybridization of stimulated splenocytes from young and old mice then showed approximately a threefold decrease in the percentage of IL-2 mRNA expressing cells in the spleens of old mice as compared to young (8.7 +/- 4.1% old; 28.7 +/- 11.7% young). The average level of expression of IL-2 mRNA was not significantly different between cells from young and old mice; however, there were approximately 40% fewer cells expressing an intermediate to high amount of IL-2 mRNA in old mice as compared to young (26.3% vs 41.8%). These data suggest that the decrease in IL-2 production with age is associated primarily with a decrease in the frequency of IL-2 mRNA-expressing cells in old mice, especially in those cells expressing intermediate to high levels of IL-2 mRNA.