Direct measurement of T cell subset kinetics in vivo in elderly men and women

The age-associated decline in immunocompetence is paralleled by changes in the proportions of PBL subpopulations. In turn, the size and composition of the peripheral lymphocyte pool is determined by input from the thymus and bone marrow and by the balance of proliferation and death in each lymphocyte subpopulation. We compared the kinetics of lymphocyte subtypes in young (seven of eight CMV seronegative) and healthy elderly human subjects (six of seven CMV seropositive), using deuterated glucose DNA labeling in vivo to measure rates of T cell proliferation and disappearance. For CD45RO(+) cells of both CD4(+) and CD8(+) subtypes and for CD4(+)CD45RA(+) cells the kinetics of proliferation and disappearance were remarkably similar between elderly and young subjects. In the young, the kinetics of CD8(+)CD45RA(+) cells with a naive phenotype resembled those of CD4(+)CD45RA(+) cells. However, CD8(+)CD45RA(+) T cells from the elderly exhibited a predominantly primed phenotype, and for this subset, although the proliferation rate was similar to that of other CD45RA(+) cells, the disappearance rate of labeled cells was greatly decreased compared with that of all other T cell subsets. Our data provide a direct demonstration that there are no substantial changes in in vivo kinetics for most T cell populations in healthy elderly compared with young subjects. However, primed CD8(+)CD45RA(+) cells show unusual kinetic properties, indicating the persistence of these cells in the blood and dissociation of proliferation from disappearance.     

Lymphocyte maintenance during healthy aging requires no substantial alterations in cellular turnover

In healthy humans, lymphocyte populations are maintained at a relatively constant size throughout life, reflecting a balance between lymphocyte production and loss. Given the profound immunological changes that occur during healthy aging, including a significant decline in T‐cell production by the thymus, lymphocyte maintenance in the elderly is generally thought to require homeostatic alterations in lymphocyte dynamics. Surprisingly, using in vivo ²H₂O labeling, we find similar dynamics of most lymphocyte subsets between young adult and elderly healthy individuals. As the contribution of thymic output to T‐cell production is only minor from young adulthood onward, compensatory increases in peripheral T‐cell division rates are not required to maintain the T‐cell pool, despite a tenfold decline in thymic output. These fundamental insights will aid the interpretation of further research into aging and clinical conditions related to disturbed lymphocyte dynamics.     

Alteration of circadian rhythmicity of CD3+CD4+ lymphocyte subpopulation in healthy aging

The CD4+ T helper/inducer and the CD8+ T suppressor/cytotoxic are major lymphocyte subsets that play a key role in cell-mediated immunity. Aging-related changes of immune function have been demonstrated. The purpose of this study is to analyze the dynamics of variation of these specific lymphocyte subsets in the elderly. In our study cortisol and melatonin serum levels were measured and lymphocyte subpopulation analyses were performed on blood samples collected every four hours for 24 hours from fifteen healthy young middle-aged subjects (age range 36-55 years) and fifteen healthy elderly male subjects (age range 67-79 years). A clear circadian rhythm was validated for the time-qualified changes of CD3+ and CD4+ cells with acrophase at night and for the time-qualified changes of CD8+ cells with acrophase at noon in young middle-aged subjects and for the time-qualified changes of CD3+ cells with acrophase at night and for the time-qualified changes of CD8+ cells with acrophase at noon in elderly subjects. No clear circadian rhythm was validated for the time-qualified changes of CD4+ cells in elderly subjects. No statistically significant correlation among lymphocyte subsets was found in elderly subjects. In elderly subjects CD3+ lymphocyte percentage was higher in the photoperiod and in the scotoperiod and cortisol serum level were higher in the scotoperiod in respect to young middle-aged subjects. In the elderly there is an alteration of circadian rhythmicity of T helper/inducer lymphocytes and this phenomenon might contribute to the aging-related changes of immune responses.     

Serum and lymphocyte levels of heat shock protein 70 in aging: a study in the normal Chinese population

Heat shock proteins (Hsps) have been reported to play an important role in both physiological and pathological processes. Hsps also may serve as biomarkers for evaluating disease states and exposure to environmental stresses. Whether Hsp levels in serum and lymphocytes are correlated with age and sex is largely unknown. In this study, we analyzed serum Hsp70 (the most abundant mammalian Hsp) levels by using Western dot blot in 327 healthy male donors aged between 15 and 50 years. We also investigated the association between Hsp70 levels and age in lymphocytes of 80 normal individuals aged between 40 and 77 years because various chronic diseases increase after the age of 40 years. Our data showed that serum Hsp70 levels were positively correlated with age in subjects aged between 15 and 30 years (P < 0.05) but negatively correlated with age in subjects aged between 30 and 50 years (P < 0.05). Serum Hsp70 levels were the highest in individuals aged between 25 and 30 years among all age groups. In the lymphocyte study there also was a significant age-related decrease in Hsp70 levels in lymphocytes of individuals older than 40 years. The Hsp70 levels were negatively correlated with age (r = -3.708, P < 0.0001) but not with sex (r = -10.536, P = 0.452). This suggests that both serum and lymphocyte Hsp70 levels are age-related and that these may be linked to age-related stress. Thus, age is an important factor in using serum and lymphocyte Hsp70 as biomarkers to evaluate the disease states or exposure to environmental stresses (or both).     

Genetic heterogeneity of heat shock protein synthesis as a factor determining the resistance to stressors in mammalia

The relationship between the levels of 70 kDa family heat shock protein (Hsp) synthesis and lymphocyte sensitivity to stressors was investigated. Lymphocyte cultivation in mitogen deprived culture medium and/or the cell treatment with alkylating agents have been used as a stress challenge. Model experiments with two inbred murine strains genetically contrasting by the sensitivity to alkylating agents demonstrated that the basic level of Hsp synthesis depends on genotype. The quantity Hsp70 mRNA, as well as intracellular level of the proteins, in BALB/c was significantly higher than those in C57BL/6 mice. The mice, which were characterized by higher Hsp levels, demonstrated higher resistance to alkylating agent action. The induction of surplus amount of Hsp by heat shock increased the cell resistance to an alkylating agent melphalan. Lymphocyte isolated from high Hsp producers BALB/c mice were more resistant to apoptotic signals induced by mitogen deprivation.     

Influence of age,castration, and testosterone on T cell subsets in healthy and leukemia grafted mice

The distribution of T cell subsets in pubertal (2 months) and post-pubertal (10 months) mice showed a significant decrease in the percentage of CD4+ splenocytes and peripheral blood lymphocytes (PBL) with age, unlike the percentage of CD8+ cells in PBL, which remained unchanged. The change in the distribution of T cell subsets in the spleen and blood occurred in 2 months old castrated mice, as in 10 months old animals. P388 tumor grew better in post-pubertal and in castrated mice than in young mice. The intact mice survived longer than the castrated ones. The relative number of CD4+, CD8+ and CD2+ splenocytes was lower in transplanted intact mice than that in controls. The CD8+ and CD2+ subsets in the blood of 2 months transplanted mice were higher than those in controls, whereas in PBL, in 10 months old and castrated mice, the T lymphocyte subsets remain unchanged. Depo-testosterone (DT) injection strongly reduced weight and tumor growth in all the intact and castrated animals. A significant correlation is observed between the tumor weight and testosterone level in the plasma of the 2 months old DT treated mice. Moreover, DT injection induced a significant increase in the percentage of blood CD8+ cells in all the batches. These data indicate that physiologically, androgens affect the age-related distribution of lymphocyte T subsets and suggest that they slow down tumor growth, besides causing a direct effect, through an immunological process.     

Constitutive expression of heat shock proteins Hsp90, Hsc70, Hsp70 and Hsp60 in neural and non-neural tissues of the rat during postnatal development

Heat shock proteins (Hsps) are a group of highly conserved proteins, that are constitutively expressed in most cells under normal physiological conditions. Previous work from our laboratory has shown that neurons in the adult brain exhibit high levels of Hsp90 and Hsc70 mRNA and protein, as well as basal levels of Hsp70 mRNA. We have now investigated the expression of Hsp90, Hsc70, Hsp60 and Hsp70 in neural and non-neural tissues of the rat during postnatal development, a time of extensive cell differentiation. Western blot analysis revealed constitutive expression of these Hsps early in postnatal development. Developmental profiles of these Hsps suggest that they are differentially regulated during postnatal development of the rat. For example, while levels of Hsp90 decrease somewhat in certain developing brain regions, levels of Hsp60 show a developmental increase, and Hsc70 protein is abundant throughout postnatal neural development. Low basal levels of Hsp70 are also observed in the developing and adult brain. A pronounced decrease in Hsp90 and Hsc70 was observed during postnatal development of the kidney while levels of Hsp60 increased. In addition, tissue-specific differences in the relative levels of these Hsps between brain and non-brain regions were found. Immunocytochemical studies demonstrated a neuronal localization of Hsp90, Hsc70 and Hsp60 at all stages of postnatal development examined as well as in the adult, suggesting a role for Hsps in both the developing and fully differentiated neuron. The developmental expression of subunit IV of cytochrome oxidase was similar to that of Hsp60, a protein localized predominantly to mitochondria.     

Aging,caffeine, and information processing: an event-related potential analysis

Structural and energetic processes in information processing were studied in young and elderly subjects. A visually focussed selective search task was used, in which subjects had to select relevant information, followed by controlled memory search processes to locate a target item. Caffeine was used to manipulate the energetic state of the subjects. During task performance event-related potentials (ERPs) and reaction time (RT) were recorded.Subjects were 15 young and 15 elderly healthy, non-smoking, moderate caffeine consumers (250–600 mg/day). Before the experimental sessions they abstained from caffeine for ≥12 h. In the experiment subjects received 250 mg caffeine or placebo dissolved in decaffeinated coffee.RT data seem to indicate that aging effects are at least partly due to a shift in the speed-accuracy trade-off. ERP results provide evidence for decreased levels of energy resources in the elderly. The identification of relevant information and stimulus evaluation processes were delayed in the elderly, suggesting an additional effect of aging on structural processes. Caffeine improved performance and increased the amplitude of the N1, N2b, and P3b, in both young and old subjects. These results suggest that caffeine makes more energy resources available for task performance. The effects of aging on P3b latency were counteracted by caffeine. Other caffeine effects did not differ significantly between young and elderly subjects.     

Age-Related Circadian Rhythm of DHEAS Plasma Levels in Male Subjects

The aging influences the endocrine temporal structure, including DHEAS which can be considered as a biomarker of aging, since its levels gradually decrease in older subjects. The aim of this work was to observe the circadian rhythms of DHEAS, prolactin, cortisol and body temperature, in healthy elderly male subjects (73.7 ± 2.5 years) compared with healthy young subjects (27.2 ± 6.6 years). The results documented that in our subjects no significant age-related differences in prolactin levels existed. In elderly subjects cortisol levels were weakly enhanced in comparison with young subjects. DHEAS showed a preserved circadian rhythm, but markedly lower rhythm adjusted mean (74.38 ± 10.29 versus 273.63 ± 26.39) (p &lt; 0.001) and decreased amplitude of oscillation (p &lt; 0.001), when expressed as absolute value, in elderly subjects when compared with young subjects. In elderly subjects the DHEAS circadian rhythm modifications could represent an impairment of of the endocrine temporal structure.     

Age-related lung tissue remodeling due to the local distribution of MMP-2, TIMP-2, TGF-β and Hsp70

Lung tissue remodeling requires complex interactions of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), transforming growth factor (TGF) family and heat shock protein 70 (Hsp70). We evaluated the appearance and distribution of MMP-2, TIMP-2, TGF-β1 and Hsp70 in lung tissue using immunohistochemistry. Stained structures were graded semiquantitatively. Overall, more MMP-2, TIMP-2, TGF-β1 and Hsp70 were observed in bronchial cartilage, bronchial and alveola repithelium, and among alveolar macrophages. We evaluated mostly alveolar macrophages, bronchial epithelial cells and mucosal fibroblasts stained for TGF-β1, MMP-2 and TIMP-2. We also assessed strong or moderate correlations between numbers of cells containing TGF-β1, MMP-2, TIMP-2 in patients ≥ 60 years old. The presence of less TGF-β1 and more MMP-2, TIMP-2 and Hsp70 containing cells in all tissue groups indicated that local regulation was more dependent on MMP-2, TIMP-2 and Hsp70 distribution. Fewer TIMP-2, Hsp70 and TGF-β1 immunoreactive cells in younger individuals and increased expression of Hsp70 in elderly individuals demonstrated the influence of aging in lung remodeling. Findings of MMP-2, TIMP-2 and TGF-β1 immunoreactive cells in elderly individuals indicate lung remodeling due to aging.     

Effect of allozyme heterozygosity on basal and induced levels of heat shock protein (Hsp70), in juvenile Concholepas concholepas (Mollusca)

Organisms cope physiologically with extreme temperature by producing heat shock proteins (HSPs). Expression of Hsp70 enhances thermal tolerance and represents a key strategy for ectotherms to tolerate elevated temperature in nature. Synthesis of these proteins, together with other physiological responses to elevated temperatures, increases energy demands. A positive association between multiple and single locus heterozygosity (MLH and SLH, respectively) and individual fitness has been widely demonstrated. In molluscs, MLH can decrease routine metabolic rates and improve energetic status. Juvenile Concholepas concholepas live in the intertidal zone and are constantly exposed to temperature fluctuations. Thus, these young individuals are exposed both to thermal risks and the large metabolic costs required to cope with thermal stress. We evaluated the effects of allozyme MLH and SLH on basal (control animals) and induced (stressed animals) levels of the Hsp70 in juveniles C. concholepas. Juveniles (n = 400) were acclimated at 16 °C for 2 weeks; then 100 animals were exposed to 24 °C (stress) and 100 were kept at 16 °C (control) for 2 and 7 days. The variability of 20 loci was analyzed by starch gel electrophoresis. For SLH effects we used 7 polymorphic loci. We quantified expression of Hsp70 by Western blot analyses. Hsp70 expression increased markedly (~ 90%) with temperature. We found a positive association between MLH and basal and induced levels of Hsp70 in the 2-day exposure experiment. Regardless of temperature, Hsp70 levels increased with MLH (r² = 0.7 and 0.9, for basal and induced levels, respectively) reaching maximal levels in juveniles with intermediate and high MLH levels (2 and 3 loci), and decreasing slightly (but not significantly) in juveniles with highest MLH (≥ 4 heterozygous loci). However, after 7 days of exposure to thermal stress, less heterozygous juveniles attained the same levels of Hsp70 than more heterozygous juveniles. Given the faster increment of Hsp70 in C. concholepas juveniles with intermediate-high levels of MLH, these individuals could be less affected by thermal stress in the intertidal zone. We found an association between specific loci genotype and higher Hsp70 levels (basal or induced). In comparison to homozygous juveniles, heterozygous juveniles for several loci showed higher Hsp70. However, these associations were not for the same loci in juveniles exposed to high temperature for 2 and 7 days. This suggests genotypic variation at some allozyme loci could be more important in the period of initial response to high temperature and others can be more important in the response to the chronic temperature stress.     

Genetic heterogeneity of heat shock protein synthesis as a factor determining the resistance of mammalia to stressors

The relationship between the levels of 70 kDa family heat shock protein (Hsp) synthesis and lymphocyte sensitivity to stressors was investigated. Lymphocyte cultivation in mitogen deprived culture medium and (or) the cell treatment with alkylating agents have been used as a stress challenge. On the model of two inbred murine strains genetically contrasting by the sensitivity to alkylating agents we succeeded in demonstration that the basic level of Hsp synthesis depends on genotype. The quantity Hsp mRNA, as well as the intracellular level of the proteins were significantly higher in BALB/c than in C57BL/6 mice. The mice characterized by higher Hsp levels demonstrated higher resistance to alkylating agent action. The induction of surplus amount of Hsp by heat shock increased the cell resistance to the alkylating agent melphalan. Lymphocyte isolated from high Hsp producers, BALB/c mice, were more resistant to apoptotic signals induced by mitogen deprivation.     

Subpopulations of human T lymphocytes. X. Alterations in T, B, third population cells, and T cells with receptors for immunoglobulin M (Tmu) or G (Tgamma) in aging humans.

Peripheral blood from 120 healthy subjects, of whom 59 were young (35.5 +/- 9.6 years) and 61 aging (69.2 +/- 4.2 years), was examined for the proportions and numbers of lymphocyte populations, with a battery of surface markers. Absolute lymphocyte count and T,B and third population cells were comparable in both groups. Tgamma cell proportions were significantly (p less than 0.001) increased in aging subjects when compared with the young subjects. However, this difference was more significant (p less than 0.001) when aged females were compared with the young females as compared to aged males vs young males (p less than 0.05). When data were anlayzed for absolute numbers of Tmu and Tgamma cells, a similar significant decrease in Tmu, and increase in Tgamma cells were observed. Interestingly, when these data were anlayzed according to gender, significant differences in Tmu and Tgamma cell number were observed between young and old females but not between young and old men. Implications of these results are discussed.     

COPD and levels of Hsp70 (HSPA1A) and Hsp27 (HSPB1) in plasma and lymphocytes among coal workers: a case-control study

This case-control study aimed to investigate whether the levels of Hsp70 (HSPA1A) and Hsp27 (HSPB1) in plasma and lymphocytes were associated with the risk of chronic obstructive pulmonary disease (COPD) among coal workers. A total of 76 COPD cases and 48 age-matched healthy controls from a group of coal workers were included. The case group consisted of 35 COPD patients whose condition was complicated with coal workers’ pneumoconiosis (CWP) and 41 COPD patients without CWP. Heat shock proteins (Hsps) in plasma and lymphocytes were detected by ELISA and flow cytometry, respectively. Multiple logistic regression models were applied to estimate the association between Hsp levels and COPD risk. Our results showed that plasma Hsp70 and lymphocyte Hsp27 levels were significantly higher and plasma Hsp27 levels were significantly lower in COPD cases than in controls (p < 0.01). No significant differences in lymphocyte Hsp70 levels were found between COPD cases and the matched subjects. Higher plasma Hsp70 levels (odds ratio (OR) = 13.8, 95 % confidence interval (CI) = 5.7–33.5) and lower plasma Hsp27 levels (OR = 4.6, 95 % CI = 2.0–10.5) were significantly associated with an increased risk of COPD after adjusting for confounders. Higher lymphocyte Hsp27 levels were only associated with an increased risk of COPD with CWP (OR = 6.6, 95 % CI = 2.0–22.1) but not with an increased risk of COPD without CWP (OR = 3.0, 95 % CI = 0.9–8.9). Additionally, there were strong joint effects of different Hsps on COPD risk. These results showed that higher levels of plasma Hsp70 and lower levels of plasma Hsp27 might be associated with an increased risk of COPD among coal workers. They may have the potential to serve as monitoring markers for COPD in coal workers.     

Hsp70 Induction and hsp70 Gene polymorphisms as Indicators of acclimatization under hyperthermic conditions

The possibility of using Hsp70 and hsp70 gene polymorphisms as markers of acclimatization was investigated. Volunteers (22) were subjected to an acclimatization regimen and blood analysed for Hsp70 (Hsp72) and hsp70 polymorphisms before and after a heat tolerance test. Physiological parameters denoting acclimatization, or not, were correlated to levels of Hsp70 and combination of hsp70 genes. Only individuals that acclimatized had decreased basal Hsp70 levels and increased ability to induce Hsp70 together with a specific hsp70 genotype combination. We propose that Hsp70 levels (basal vs. induced) with the genotype combination have the potential to be used as markers of acclimatization.     

Relationship between immune system and gram-negative bacteria. II. Natural killer cytotoxicity of Salmonella minnesota Rb 345-unbound human peripheral blood lymphocytes

Spontaneous binding of human peripheral blood lymphocytes (PBL) to bacteria represents a promising approach for the characterization of lymphocyte subsets mediating different functions. In the light of previous findings on the high degree of spontaneous adherence of S. minnesota Rb cells to PBL, we have evaluated the natural killer (NK) cytotoxicity of PBL subpopulations that fail to bind to Rb bacteria. The S. minnesota Rb-unbound cell fraction exhibits higher levels of cytotoxic capacity, which is related to a more elevated frequency of active NK cells, as determined in an agarose-single cell cytotoxic assay. Moreover, the cytotoxic activity of the unbound fraction is additionally boosted by interferon-alpha pretreatment. The effector cells bear Fc gamma receptors that are involved in NK cell lysis, because a decrease of NK activity is observed after immune complex modulation of the receptors. Finally, these cells, which display a high percentage (approximately 70%) of typical large granular lymphocyte morphology, express HNK-1, T10, T8, and M1 antigens, and to a lesser extent T3 and T4 antigens. These data indicate a selective enrichment of NK cells in the S. minnesota Rb-unbound fraction.