Telomerase-based pharmacologic enhancement of antiviral function of human CD8+ T lymphocytes

Telomerase reverse transcribes telomere DNA onto the ends of linear chromosomes and retards cellular aging. In contrast to most normal somatic cells, which show little or no telomerase activity, immune cells up-regulate telomerase in concert with activation. Nevertheless, during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8(+) CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8(+) T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity. The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation. Our study is the first to use a pharmacological telomerase-based approach to enhance immune function, thus directly addressing the telomere loss immunopathologic facet of chronic viral infection.     

Simultaneous flow cytometric analysis of two cell surface markers,telomere length,and DNA content

BACKGROUND: Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate the harsh conditions needed for DNA denaturation during hybridization of the telomere-specific PNA probe. We overcame these problems and developed a method for measuring telomere length in cell subsets characterized by the expression of two surface antigens. METHODS: Alexa Fluor 488 and Alexa Fluor 546 were used for cell surface staining. Antigen-antibody complexes were covalently cross-linked onto the cell membrane before Flow-FISH. Cells were hybridized with a PNA probe conjugated to cyanine 5 (Cy5). Hoechst 33342 (HO342) was added for determination of cellular DNA content. For assay standardization, we added an aliquot of a single batch of 1,301 cells to each sample as an internal control before hybridization with the PNA probe. Samples were prepared in duplicate and analyzed on a standard three-laser BD LSR flow cytometer. For assay validation, the same samples were analyzed in parallel to correlate the percentage of telomere length of the sample versus 1,301 control cells to the mean size of terminal restriction fragments (TRFs) of DNA as determined by Southern gel analysis. RESULTS: The method permitted clear identification of lymphocyte subsets in samples hybridized for Flow-FISH, with subset frequencies comparable to those of untreated samples. At a concentration of 10 nM, the Cy5-labeled telomere-specific PNA probe produced a bright fluorescence signal well separated from background. Addition of HO342 in low concentration did not interfere with Cy5 telomere fluorescence, produced adequate DNA histograms, and permitted clear identification of cell phenotype. The probe concentration of 10 nM also proved optimal for inclusion of 1,301 control cells for assay standardization. Telomere length estimations by the current method correlated highly with TRF calculations by Southern gel hybridization (r(2)= 0.9, P = 0.0003). Application of our protocol to the analysis of human CD8CD28 lymphocyte subsets showed that CD8(+bright)CD28(-) lymphocytes generally exhibit shorter telomeres than CD8(+bright)CD28(+) cells. These data concurred with previous results of telomere shortening in CD8(+)CD28(-) T cells that were obtained by using different techniques. CONCLUSIONS: The multiparameter Flow-FISH protocol permitted rapid determination of differences in telomere length in subpopulations characterized by two surface markers without prior cell separation.     

Role of perfusion and diffusion in 14CO2 exchange in the rabbit lung

The rate of transfer of H14CO-3 and 14CO2 from the alveoli to the capillaries was studied in rabbit lungs perfused without erythrocytes. Aliquots of 0.5 ml of buffered solutions containing these 14C indicators and 3H2) were injected into the distal airways, and the recoveries of 14C and 3H were compared in the left atrial outflow. It was assumed that 3H2O had equilibrated between the alveoli and fluid leaving the pulmonary capillaries, and a decline in the initial 14C recovery relative to that of 3H was attributed to incomplete equilibration of 14C between these compartments. No disequilibrium of 14C could be detected at pH 7.4 when excess carbonic anhydrase was present. When the pH was increased to 8.4, 14C equilibration was only 69% complete at 36 ml/min and 41% complete at 160 ml/min. Confirmatory evidence was obtained that carbonic anhydrase is associated with the endothelial side of the alveolar-capillary barrier but is absent on the epithelial surface. The data suggest that the barrier is at least 600 times more permeable to 14CO2 than to H14CO-3, and diffusion of 14CO2 would not limit exchange at normal pH unless pulmonary flow reached extremely high values.     

New evidence for active sodium transport from fluid-filled rat lungs

The hypothesis that fluid reabsorption from the air spaces is mediated at least in part by active transport of Na+ was investigated in six sets of experiments conducted in isolated fluid-filled rat lungs. Fluid reabsorption was monitored by following the changes in the air space concentration of labeled albumin. We found that incorporation of bicarbonate rather than a nonvolatile buffer (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) in the air space solution more than doubled the rate of fluid reabsorption. Addition of 10(-4) M amiloride to the air space solution reduced the rate of fluid reabsorption over a 2-h experiment from 1.2 +/- 0.1 to 0.7 +/- 0.1 ml and decreased reabsorption of both labeled and unlabeled Na+ from the air spaces. To show that Na+ could be reabsorbed from the air spaces even if the concentrations of Na+ in the perfusate increased above those in the air space, mannitol (150 mM) was added to the perfusate and air space solutions and the concentrations of Na+ and Cl- were reduced to 90 and 60 mM, respectively. Mannitol diffuses across the pulmonary epithelium very slowly, and it osmotically restrained the movement of water out of the air spaces. Na+ concentrations in the perfusate increased by 10 +/- 2 mM, but concentrations in the air space remained unchanged. Despite an increasingly unfavorable concentration gradient for Na+, 0.2 mmol Na+ and 0.6 ml water were reabsorbed from the air spaces in 2 h. Ouabain (10(-4) M) did not appear to slow fluid reabsorption in the presence of mannitol, but it reduced K+ secretion into the air spaces and increased K+ appearance in the perfusate in a manner consistent with inhibition of Na+-K+-adenosinetriphosphatase at the basolateral surface of the epithelial cells. Fluid reabsorption was not altered when the lungs were exposed to a hypotonic solution (185 mM), but secretion of K+ into the air spaces was accelerated and K+ was lost from the perfusate. These experiments are consistent with active Na+ transport from the air spaces.     

Abrupt changes in mixed venous blood gas composition after the onset of exercise

It has been assumed that increases in both O2 uptake and ventilation occurring within the first few seconds after the onset of exercise cannot be the result of changes in blood gas composition reaching the central circulation because of the circulatory delay from the exercising limbs (A. Krogh and J. Lindhard, J. Physiol. Lond. 42: 112-136, 1913). We sought to validate this assumption by measuring the time course of pulmonary arterial blood gases during the transition from rest to exercise. Six healthy men underwent pulmonary arterial catheterization and then performed transitions from rest to moderate cycle ergometer exercise. An anaerobic sampling manifold withdrew 19 samples of blood during the rest-to-exercise transition; sampling interval was usually 4 s. Blood gas analysis showed that, on average, from rest-to-steady-state exercise, O2 saturation (Svo2) fell from 71 to 41% and mixed venous PCO2 (PvCO2) rose from 42 to 59 Torr. Contrary to our expectations, Svo2 decreased and PvCO2 increased with no discernible latency after exercise onset (by 10% and 2 Torr, respectively, within 6 s). The half time for the Svo2 decrease was 32 s, whereas for the PvCO2 increase it was 80 s. The time course of superior vena cava blood gas composition was determined in several experiments; no rapid changes after exercise onset were found. We conclude that at exercise onset there is a rapid fall in Svo2 and rise in PvCO2 well in advance of arrival of blood produced by exercising legs.(ABSTRACT TRUNCATED AT 250 WORDS)