Reduced expression of the lncRNA NRON is a potential hallmark of the CMV-amplified CD8+ T cell accumulations commonly seen in older humans.

The characteristic accumulation of late-stage differentiated CD8+ T cells is enhanced by lifelong latent cytomegalovirus (CMV) persistence, which makes it challenging to screen for subclinical biomarkers of immune aging in the elderly. We systematically identified predominantly preformed, long, noncoding RNAs (lncRNAs) as integrative biomarkers of CD8+ T cell aging in 14 elderly CMV carriers over 80 years of age. After sorting the CD28nullCD8+ T cell subset and its CD28bearingCD8+ counterpart in five nonagenarians, we profiled the differential expression of lncRNAs and genes in CD28nullCD8+ T cells via array detection. We focused on 11 differentially expressed antisense lncRNAs and cross-referenced them with previously identified age-accumulated lncRNAs to create a set of candidates in CD28nullCD8+T cells. We performed intracellular validation on the age-accumulated candidate lncRNAs paired with their antisense target genes using quantitative polymerase chain reaction (qPCR). Simultaneously, we sorted the CMVpp65-specific CD8+ T cell subset and its counterpart from participant cells with the HLA-A-*0201 genotype. The validated age-accumulated lncRNAs in CD28nullCD8+ T cells were intracellularly cross-validated in CMVpp65CD8+ T cells. Finally, we identified the immunity-related gene(s) that acted as potential target(s) to the cross-validated age-accumulated lncRNA(s), using bioinformatics techniques. The potential regulation of the final identified lncRNA-gene pair(s) was simultaneously predicted in two pathway-integrated networks. We concluded that expression of an age-accumulated lncRNA (NRON) was decreased, whereas that of its immunity-related target gene (NFAT) was increased, in both CD28nullCD8+ T cells and CMVpp65CD8+ T cells of elderly individuals with persistent CMV infection. The identification of NRON as a potential biomarker suggests that NRON contributes to CMV-enhanced CD28nullCD8+ T cell aging by modulating phosphorylation and/or IL-4-dependent NFAT signaling.

Correlation between T lymphocyte subsets in peripheral blood lymphocytes and 2-year all-cause mortality in an apparently healthy elderly Chinese cohort.

BACKGROUND: Few data have been acquired on the predictive value of age-related T-lymphocyte subsets among older individuals. The present study has determined the distribution of T-cell phenotypes and their correlation to 2-year mortality in a cohort of Chinese male seniors. METHODS: A total of 101 asymptomatic elderly individuals with laboratory homeostasis were enrolled at baseline. Three age subgroups were categorized as young (65 - 74 years old), middle (75 - 84 years old), and old (≥ 85 years) for age-related comparison. T-cell subsets in peripheral blood were measured by multi-colored flow cytometry. RESULTS: At baseline, there was a mild negative correlation by age for total lymphocytes and CD3(+) T-cells. The frequency of CD28 and CD95 demonstrated a "curved" rather than linear tendency by age. At 2-year follow-up, little change of T-cell distribution was found among those who remained alive (as survivors) comparing the data at baseline to the 2-year time point. Immune risk phenotypes were distinctly demonstrated between survivors and non-survivors. CONCLUSIONS: Since few studies have studied on the distribution of T-lymphocyte subsets in an elderly Chinese population, our results have not only provided reference values of T-subsets for aged Chinese men, but confirmed the immune risk phenotypes among elderly Chinese. The inappropriate age-dependent trajectory of CD28(-)/CD8(+) and CD95(-)/CD8(+) by age, which suggested 85 might be an inflexion point of age during T-cell ageing, warrants further exploration of the underlying mechanisms of T-cell ageing.

Multilevel model estimation of age-dependent individual-specific trajectories for left ventricular echocardiographic indexes in an asymptomatic elderly cohort.

Few studies have been performed on the individual-specific trajectory of left ventricular aging as assessed by echocardiography in an asymptomatic elderly cohort. In the present study, a representative cohort of elderly men, who were long-term asymptomatic for cardiovascular issues, were selected from an ongoing observational aging study. Annual echocardiographic data were used to establish an age-dependent hierarchical model. Based on two-level linear regression results, four echocardiographic indexes [left ventricular mass (LVmass; -1.872 g/yr), posterior ventricular wall thickness (-0.048 mm/yr), fraction shortening (0.097/yr), and transmitral peak A velocity (-0.006 m·s(-1)·yr(-1))] changed significantly with increasing age and were age- and subject-dependent. The most characterized results of the study were the significant, age-related, within-individual variances in echocardiographic results, which were observed using the likelihood ratio test at an occasion-dependent level. Of these, fluctuated amplitudes of two systolic variables [i.e., LVmass (con/age = -0.012 ± 0.004; P = 0.0007) and fraction shortening (con/age = -0.001 ± 0.004; P = 0.05)] were significantly attenuated with increasing age within individuals. On the other hand, the age-related variability of four diastolic Doppler variables [i.e., peak A velocity (con/age = 0.003 ± 0.002; P = 0.0009), peak E velocity (con/age = 0.004 ± 0.003; P = 0.01), E/A ratio (con/age = 0.007 ± 0.003; P = 0.0002), and deceleration time of E wave (con/age = 0.025 ± 0.007; P < 0.0001)] significantly increased with increasing age within individuals. The age-related individual variability of left ventricular indexes observed in this continuous asymptomatic cohort may reflect the mechanism of preclinical, individualized heart aging. In conclusion, successfully fitted multilevel models were applied as a valuable tool to determine the mechanism of individual cardiac aging in the elderly.

Optimization of conditions for transfection with the Sofast gene vector.

We previously reported the synthesis and characterization of a novel cationic polymer gene vector. The present article further explored and optimized the working conditions of the Sofast gene vector both in vitro and in vivo, and improved its performance. The transfection conditions of Sofast, such as cell type, cell density, transfection time, N/P values and analysis time after transfection, were further explored. Moreover, the effects of the fusion peptide diINF-7 on transfection efficiency were examined. Sofast was successfully applied for the transfection of exogenous genes into more than 40 types of cell lines derived from humans, mice, monkeys and other species. When the cells were 50-80% confluent, Sofast possessed a better transfection efficiency. In most cases, Sofast also had a higher transfection efficiency when it was used to transfect cells that were seeded for several hours and had adhered to the substrate. The results from in vitro experiments indicate that the recommended Sofast to DNA mass ratio is 16:1, and the optimum analysis time after transfection is 48 h. The salt concentration in the Sofast working solution markedly affected the transfection efficiency. When conducting in vivo transfection, the working solution should be salt-free, whereas for in vitro transfection, it is more appropriate for the working solution to include certain salt concentrations. Finally, the results confirm that diINF-7 significantly promotes the transfection efficiency of Sofast. In conclusion, the present research not only established the optimal conditions for Sofast in the transfection of commonly used cells, but also built the foundations for in vivo and in vitro applications of Sofast, as well as its use in clinical practice.

Further evaluation of a novel nano-scale gene vector for in vivo transfection of siRNA.

In this research, a lipid-cationic polymer (LCP) containing the side-chain branching of brassidic acid was synthesized using chemical methods. As a gene vector for small interfering ribonucleic acid (siRNA) transfection, the efficiency and biosafety of LCP were preliminarily evaluated to investigate its possible application on tumor gene therapy. The toxicity, side-effects, and biosafety of LCP were investigated in animals based on the results of in vitro experiments. The siRNA against cyclooxygenase-2 (COX-2) was transfected by LCP to interfere with the COX-2 expression in nude-transplanted tumors. Hematoxylin and eosin stains, immunohistochemistry, reverse transcription-polymerase chain reaction, and Western blot were performed to evaluate the efficiency of LCP for siRNA transfection. The animal toxicity experiment showed that a high concentration of LCP had a low toxic effect on animals and did not induce allergic or pyrogenic reactions. The results from the in vivo transfection indicated that LCP could efficiently transfect siRNA and silence the target gene expression. The LCP gene vector for siRNA transfection is highly efficient during in vivo transfection and had low toxicity. From all aspects of tumor gene therapy and basic research, LCP is valuable for scientific research and medical applications.