Bacterial DNAemia in Older Participants and Nonagenarian Offspring and Association With Redox Biomarkers: Results From MARK-AGE Study.

Aging and age-related diseases have been linked to microbial dysbiosis with changes in blood bacterial DNA concentration. This condition may promote chronic low-grade inflammation, which can be further aggravated by antioxidant nutrient deficiency. Low plasma carotenoids are associated with an increased risk of inflammation and cellular damage and predict mortality. However, no evidence is yet available on the relationship between antioxidants and the blood bacterial DNA (BB-DNA). Therefore, this study aimed to compare BB-DNA from (a) GO (nonagenarian offspring), (b) age-matched controls (Randomly recruited Age-Stratified Individuals from the General population [RASIG]), and (c) spouses of GO (SGO) recruited in the MARK-AGE project, as well as to investigate the association between BB-DNA, behavior habits, Charlson Comorbidity Index (CCI), leucocyte subsets, and the circulating levels of some antioxidants and oxidative stress markers. BB-DNA was higher in RASIG than GO and SGO, whereas GO and SGO participants showed similar values. BB-DNA increased in smokers and males with CCI ≥ 2 compared with those with CCI ≤ 1 within RASIG. Moreover, BB-DNA was positively associated with lymphocyte, neutrophil, and monocyte counts, but not with self-reported dietary habits. Higher quartiles of BB-DNA were associated with low lutein and zeaxanthin and elevated malondialdehyde plasma concentrations in RASIG. BB-DNA was also positively correlated with nitric oxide levels. Herein, we provide evidence of a reduced BB-DNA in individuals from long-living families and their spouses, suggesting a decreased microbial dysbiosis and bacterial systemic translocation. BB-DNA was also associated with smoking, CCI, leukocyte subsets, and some redox biomarkers in older participants.

Zinc-Induced Metallothionein in Centenarian Offspring From a Large European Population: The MARK-AGE Project.

Metallothionein (MT) family are cysteine-rich proteins that regulate zinc (Zn) homeostasis and protect against oxidative damage. Studies in transgenic mice have shown that MT favorably influence longevity, although their role in human aging is not completely understood. Within the European multicenter study MARK-AGE, we analyzed MT induction after Zn treatment in peripheral blood mononuclear cells (PBMCs) and its relation with redox biomarkers in 2,936 age-stratified subjects (35-75 years) including the general population (RASIG), centenarian offspring (GO), and their spouses (SGO). We found that the lymphocyte capability to induce MT in response to Zn is not affected by aging. However, GO participants showed lower Zn-induced MT and increased basal expression of MT1A, MT1X, and ZnT-1 genes than RASIG subjects. Moreover, Zn-induced MT levels were found to be inversely related with oxidative stress markers (plasma protein carbonyls, 3-nitrotyrosine, and malondialdehyde) in the whole population, but not in GO subjects. In conclusion, our results support the hypothesis that the response to Zn is attenuated in PBMCs of centenarian offspring compared to the general population as a consequence of a tighter control of Zn homeostasis which is likely to provide them constant protection against stress stimuli over the whole lifespan.

Human endogenous retrovirus HERV-K(HML-2) env expression is not associated with markers of immunosenescence.

Ageing of the human immune system, or immunosenescence, is characterised by distinct changes in the proportion of the various cell types, e.g., increase of the CD14+ monocytic cells, decrease of CD19+ B lymphocytes, and changes in T cell subpopulations, namely increase of CD4+ and CD8+ cells which have lost the costimulatory CD28 antigen. Currently, it is believed that the lifelong antigenic burden may be one of the inducers of immunosenescence. Thus far, only one exogenous stimulus, cytomegalovirus infection, has shown to be a major factor in this respect. To find other possible candidates, we evaluated the role of the evolutionary youngest group of human endogenous retroviruses, namely HERV-K(HML-2), on immunosenescence. HERVs exist in the genome as proviruses, but their activation has been detected in several immunopathologic conditions. The expression of HERV-K(HML-2) env was observed to be lower in the peripheral blood mononuclear cells of nonagenarians (n=61) than in those of young controls (n=37). These mRNA levels did not correlate with the age-associated differences in the proportions of CD14+, CD4+CD28- and CD8+CD28- cells, but in the case of CD19+ B cells a strong positive correlation was observed in the nonagenarians. Thus, these data suggest that HERVs do not function as antigenic drivers of immunosenescence. On the contrary, expression of HERV-K(HML-2) env is associated with more youthful levels of B cells.

Associations between Specific Redox Biomarkers and Age in a Large European Cohort: The MARK-AGE Project.

Oxidative stress and antioxidants play a role in age-related diseases and in the aging process. We here present data on protein carbonyls, 3-nitrotyrosine, malondialdehyde, and cellular and plasma antioxidants (glutathione, cysteine, ascorbic acid, uric acid, α-tocopherol, and lycopene) and their relation with age in the European multicenter study MARK-AGE. To avoid confounding, only data from countries which recruited subjects from all three study groups (five of eight centers) and only participants aged ≥55 years were selected resulting in data from 1559 participants. These included subjects from (1) the general population, (2) members from long-living families, and (3) their spouses. In addition, 683 middle-aged reference participants (35-54 years) served as a control. After adjustment for age, BMI, smoking status, gender, and country, there were differences in protein carbonyls, malondialdehyde, 3-nitrotyrosine, α-tocopherol, cysteine, and glutathione between the 3 study groups. Protein carbonyls and 3-nitrotyrosine as well as cysteine, uric acid, and lycopene were identified as independent biomarkers with the highest correlation with age. Interestingly, from all antioxidants measured, only lycopene was lower in all aged groups and from the oxidative stress biomarkers, only 3-nitrotyrosine was increased in the descendants from long-living families compared to the middle-aged control group. We conclude that both lifestyle and genetics may be important contributors to redox biomarkers in an aging population.

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study.

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project 'MARK-AGE'. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly.

Cardiometabolic and Inflammatory Biomarkers as Mediators Between Educational Attainment and Functioning at the Age of 90 Years.

BACKGROUND: Low socioeconomic status is associated both with poorer functioning and elevated levels of inflammatory and cardiometabolic biomarkers; however, knowledge of such relations for the oldest old is limited. Our aim was to study whether education is associated with cardiometabolic (cholesterol levels, body mass index, and leptin) and inflammatory (C-reactive protein, interleukin-6, interleukin-1Ra) biomarkers for the 90-year-olds who participated in the Vitality 90+ study. In addition, we investigated whether these biomarkers explain educational inequalities in functioning. METHODS: All persons in Tampere, Finland, who were born in 1909 or 1910, were invited to participate, irrespective of their health status or dwelling place. The sample consisted of 262 participants who went through the home interview and blood tests. The socioeconomic status indicator used was the highest education, and physical functioning was assessed using the Barthel index. The association of education with individual and combined biomarker scores, and with functioning, was analyzed cross-sectionally applying generalized linear models. RESULTS: The low- and mid-level-educated participants had greater odds of belonging to the high risk group in cardiometabolic biomarkers than did the high-educated. Differences were statistically significant in three individual biomarkers (high-density lipoprotein-cholesterol, leptin, and body mass index) and in a cardiometabolic score. There were no educational differences in inflammatory biomarkers. When all biomarkers were combined, they mediated educational differences in functioning on an average of 23%. After controlling for smoking, alcohol use and diseases, biomarkers mediated part of the differences between the mid-level- and high-educated. CONCLUSIONS: High education was associated with better cardiometabolic biomarkers and functioning among the 90-year-olds. In part, educational inequalities in functioning were explained by cardiometabolic biomarkers.

Length of paternal lifespan is manifested in the DNA methylome of their nonagenarian progeny.

The heritability of lifespan is 20-30%, but only a few genes associated with longevity have been identified. To explain this discrepancy, the inheritance of epigenetic features, such as DNA methylation, have been proposed to contribute to the heritability of lifespan.We investigated whether parental lifespan is associated with DNA methylation profile in nonagenarians. A regression model, adjusted for differences in blood cell proportions, identified 659 CpG sites where the level of methylation was associated with paternal lifespan. However, no association was observed between maternal lifespan and DNA methylation. The 659 CpG sites associated with paternal lifespan were enriched outside of CpG islands and were located in genes associated with development and morphogenesis, as well as cell signaling. The largest difference in the level of methylation between the progeny of the shortest-lived and longest-lived fathers was identified for CpG sites mapping to CXXC5. In addition, the level of methylation in three Notch-genes (NOTCH1, NOTCH3 and NOTCH4) was also associated with paternal lifespan.There are implications for the inheritance of acquired traits via epigenetic mechanisms in mammals. Here we describe DNA methylation features that are associated with paternal lifespan, and we speculate that the identified CpG sites may represent intergenerational epigenetic inheritance.

Association of Body Mass Index and Waist Circumference With Physical Functioning: The Vitality 90+ Study.

BACKGROUND: Both obesity and underweight are associated with impaired physical functioning, but related information on the oldest old population is scarce. Our purpose was to examine whether body mass index, waist circumference (WC), and their combination are associated with physical performance and activities of daily living (ADL) disability in 90-year-old women and men. METHODS: Data are from the Vitality 90+ Study, which is a population-based study of persons with age ≥90 years living in the area of Tampere, Finland. Altogether 416 women and 153 men, aged 90-91 years, provided data on body mass index, WC, chair stand, and Barthel Index. Comorbidity, physical exercise, smoking history, living residence, and sample year were used as covariates in multinomial logistic and logistic regression models. RESULTS: Women in the highest WC tertile had lower physical performance and were more likely unable to perform the chair stand than women in the lowest WC tertile. Women in the highest WC tertile were also more likely to have ADL disability, compared to the lowest WC tertile. In women, overweight and obesity were associated with ADL disability, but not when WC was included in the model. Men with body mass index ≥25 kg/m(2) and WC < sex-specific median were less likely to have ADL disability. Similarly classified women were less likely to have low performance or unable to perform chair stand (marginally significant). CONCLUSIONS: High WC in the oldest old women, but not in men, is associated with both poor physical performance and ADL disability.

Vascular cell adhesion molecule 1, soluble Fas and hepatocyte growth factor as predictors of mortality in nonagenarians: the Vitality 90+ study.

BACKGROUND: Ageing is characteristically accompanied by changes in vascular endothelial markers and growth factor as well as increased cellular death. We analysed the associations of the plasma levels of vascular cell adhesion molecule-1 (VCAM-1), hepatocyte growth factor (HGF) and soluble Fas (sFAS), and their combinations, with 4-year mortality to identify new biomarkers. METHODS: A total of 238 individuals, both community-dwelling and institutionalised, aged 89 91 years and participating in the Vitality 90+ study were included. Biomarkers of endothelial function (VCAM-1), growth factor (HGF) and a marker of apoptosis (sFAS) were determined from plasma using Luminex® technology. This newly-determined data was combined with earlier data, e.g., 4-year mortality and medical history. RESULTS: Subjects who died during the follow-up had higher baseline plasma levels of VCAM-1, sFas, and HGF. When other known risk factors were adjusted for, subjects in the highest concentration tertile for VCAM-1 (HR 1.85; 95% CI, 1.12-3.05) and HGF (HR 2.22; 95% CI, 1.33-3.71) had higher mortality compared to those in the lowest tertile. In the adjusted analyses, when compared to subjects with none of the biomarkers in the highest concentration tertile, mortality was also higher when sFas and VCAM-1 were simultaneously (HR 2.03; 95% CI, 1.13-3.64) or all three were simultaneously (HR 3.63; 95% CI, 1.65-7.97) in the highest concentration tertile. CONCLUSIONS: Our results suggest that increased concentrations of these biomarkers, separately and in combination, associate with mortality among the aged and are prognostic markers of death.

Transcriptional analysis reveals gender-specific changes in the aging of the human immune system.

Aging and gender have a strong influence on the functional capacity of the immune system. In general, the immune response in females is stronger than that in males, but there is scant information about the effect of aging on the gender difference in the immune response. To address this question, we performed a transcriptomic analysis of peripheral blood mononuclear cells derived from elderly individuals (nonagenarians, n = 146) and young controls (aged 19-30 years, n = 30). When compared to young controls, we found 339 and 248 genes that were differentially expressed (p<0.05, fold change >1.5 or <-1.5) in nonagenarian females and males, respectively, 180 of these genes were changed in both genders. An analysis of the affected signaling pathways revealed a clear gender bias: there were 48 pathways that were significantly changed in females, while only 29 were changed in males. There were 24 pathways that were shared between both genders. Our results indicate that female nonagenarians have weaker T cell defenses and a more prominent pro-inflammatory response as compared to males. In males significantly fewer pathways were affected, two of which are known to be regulated by estrogen. These data show that the effects of aging on the human immune system are significantly different in males and females.

Inflammation, adiposity, and mortality in the oldest old.

BACKGROUND: Increased proinflammatory status is associated with both increased adiposity and higher mortality risk. Thus, it is paradoxical that mild obesity does not predict increased mortality in older adults. We investigated the association of inflammatory markers with body mass index (BMI), waist circumference (WC), and waist-to-hip ratio (WHR) in nonagenarians, and the combined effects of BMI, WC, WHR, and inflammatory status on mortality. METHODS: This study was based on a prospective population-based study, Vitality 90+, carried out in Tampere, Finland. Altogether, 157 women and 53 men aged 90 years were subjected to anthropometric measurements, blood samples, and a 4-year mortality follow-up. Inflammatory status was based on sex-specific median levels of interleukin-1 receptor antagonist (IL-1RA), interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor-α (TNF-α). RESULTS: In the unadjusted linear regression analyses, IL-1RA, CRP, and TNF-α were positively associated with BMI and WC in women, whereas in men IL-1RA was positively associated with BMI and IL-6 positively with WC. In the models adjusted for diseases, functional status, and smoking, IL-1RA and CRP were positively associated with BMI and WC in women. Low WC and WHR combined with low inflammation protected from mortality in women and high BMI and WC regardless of inflammation protected from mortality in men in the adjusted Cox regression analysis. CONCLUSIONS: In the oldest old, the effect of adiposity in combination with inflammatory status on mortality differs between men and women. More research is needed to disentangle the role of adiposity among the oldest old.

Aging-associated increase in indoleamine 2,3-dioxygenase (IDO) activity appears to be unrelated to the transcription of the IDO1 or IDO2 genes in peripheral blood mononuclear cells.

BACKGROUND: Old age is associated with increased levels of circulating pro-inflammatory cytokines, a phenomenon termed inflamm-aging. Elevated levels of pro-inflammatory cytokines have been associated with several age-associated diseases and with a shortened lifespan. Indoleamine 2,3-dioxygenase (IDO) has immunomodulatory properties and its activity is elevated in inflammation, autoimmune disorders and malignancies. We have previously shown that IDO activity is increased in nonagenarians compared to young individuals and that high IDO activity is associated with mortality at old age. FINDINGS: In this study our aim was to assess whether this difference in IDO activity in the plasma was due to the differential expression of either the IDO1 or IDO2 gene in peripheral blood mononuclear cells. Our results show that IDO1 and IDO2 are not differently expressed in nonagenarians compared to controls and that the expression of IDO genes is not associated with the level of IDO activity in the plasma. CONCLUSION: The level of IDO activity in the plasma is not regulated through the expression of IDO1 or IDO2 in the peripheral blood mononuclear cells.

Inflammatory markers and physical performance among nonagenarians.

BACKGROUND: Recent studies have suggested that inflammation may play an important role in aging and the development of disabilities, but knowledge about its importance in the development of muscle weakness and functional disabilities in very old people is limited. This study examined associations between inflammatory markers and physical performance among nonagenarians. METHODS: The population-based sample consisted of 197 women and 65 men aged 90 years. Physical performance was assessed according to the Barthel Index, the chair stand, and handgrip strength. Plasma levels of interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1Ra), and C-reactive protein (CRP) were determined. RESULTS: A gender-adjusted linear regression model showed that high levels of CRP, IL-6, and IL-1Ra were significantly associated with poor handgrip strength (p = .041, p = .023, p < .001, respectively). After adjustment for diseases, smoking and physical exercise high levels of IL-6 and IL-1Ra were still significantly associated with poor hand grip strength (p = .048, p = .004, respectively). In the gender-adjusted model, high levels of CRP, IL-6, and IL-1Ra were significantly associated with a worse Barthel Index (p = .009, p = .004, p = .004, respectively). High levels of CRP and IL-6 were still significantly associated with a worse Barthel Index after adjusted for diseases, smoking and physical exercise (p = .034, p = .041, respectively). In the chair stand, no significant association with inflammatory markers was found. CONCLUSIONS: Associations between high levels of inflammatory markers and worse handgrip strength as well as a worse Barthel Index result were evident among nonagenarians. However, the association with the chair stand was not significant.

Interleukin-1 receptor antagonist, interleukin-6, and C-reactive protein as predictors of mortality in nonagenarians: the vitality 90+ study.

BACKGROUND: Inflammation plays a major role in both aging and chronic disease. Longitudinal studies in very old people can improve our understanding of these processes. We investigated blood levels of C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1ra), and their combinations as predictors of mortality in nonagenarians. METHODS: This is a prospective population-based study including both community-dwelling and institutionalized nonagenarians enrolled in the Vitality 90+ Study. Altogether 285 persons participated in the baseline interview and gave blood. Information on chronic disease was drawn from health center registers. Data on mortality over 4 years were obtained from the Population Register Center. In Cox proportional hazards models, chronic disease and major risk factors were adjusted for. RESULTS: Plasma levels of IL-1ra, IL-6, and CRP were higher in persons who died during the follow-up than in those who survived. When sex, education, cardiovascular disease, diabetes, cancer, history of infections, high density lipoprotein cholesterol, Mini-Mental State Examination, body mass index, smoking status, and exercise were adjusted for, only IL-1ra was a significant predictor of mortality (hazard ratio [HR] 2.12; 95% confidence interval [CI], 1.24-3.62). Persons in the upper tertiles of both CRP and IL-1ra (HR 2.72; 95% CI, 1.25-6.00), or in the upper tertile of all three markers (HR 2.34; 95% CI, 1.23-4.61), had higher mortality than those who were not in the upper tertile in any of the markers. CONCLUSIONS: IL-1ra is a powerful prognostic marker in very old people. Our results implicate its role in the complex interaction between inflammatory markers in aging and disease.

Interleukin-6 modulates plasma cholesterol and C-reactive protein concentrations in nonagenarians.

OBJECTIVES: To establish whether the relationship between interleukin-6 (IL-6) and plasma lipid and C-reactive protein (CRP) concentrations is different in Finnish nonagenarians than in middle-aged subjects with lower inflammatory status. DESIGN: Cross-sectional. SETTING: Observational cohort study concentrating on the oldest old. PARTICIPANTS: Nonagenarians (n=291, mean age+/-standard deviation 90+/-1; 68 men, 223 women) who lived in the Tampere municipality in southern Finland and a middle-aged control population from the same area (n=227, aged 44+/-8). MEASUREMENTS: Plasma high sensitive CRP and lipid concentrations were analyzed using an automatic analyzer and IL-6 levels using enzyme-linked immunosorbent assay. RESULTS: Plasma concentrations of IL-6 (4.39+/-5.25 vs 1.88+/-1.98 pg/mL) and CRP (3.54+/-4.98 vs 1.53+/-1.91 mg/L) were significantly higher in nonagenarians than in middle-aged subjects (P<.001). In nonagenarians, plasma CRP levels increased (P<.001) and plasma total cholesterol (P=.006), low-density lipoprotein cholesterol (P=.02), and high-density lipoprotein cholesterol (P=.002) levels decreased according to IL-6 quartiles. In middle-aged subjects, similar associations were not found. CONCLUSION: The relationship between IL-6 and plasma CRP and cholesterol levels in nonagenarians with enhanced systemic inflammation differs from that of middle-aged subjects.

IgA levels are predictors of mortality in Finnish nonagenarians.

It has been demonstrated that in obviously healthy, very old people increased levels of inflammatory markers as well as some defects in T lymphocyte populations are strong predictors of mortality. Very little is known about the role of possible functional defects in antibody formation. To examine this, we now measured IgM, IgG and IgA concentrations in a cohort of 285 nonagenarians (67 males, 218 females). IgG and IgA levels were significantly higher than those of healthy middle-aged controls. The analyzed serum samples were taken at the age of 90-91 years. After 4 years, 20 males and 94 females had survived. To analyze the role in predicting mortality, the immunoglobulin data (as well as the measured CRP and IL-6 concentrations) were stratified according to this survival data. IgA levels (and CRP and IL-6 levels) were clearly higher in the nonsurvivors than in the survivors. These data imply that elevated serum IgA level, i.e. indicator of intestinal inflammation and/or defect in mucosal defence, is a strong mortality predicting factor.

Survival of AA and ANA rats during lifelong ethanol exposure.

BACKGROUND: Study of the long-term effects of chronic alcohol consumption in human populations is confounded by genetic and environmental factors. METHODS: The study was intended to investigate the effects on morbidity and survival of lifetime forced ethanol consumption in male and female AA (Alko, Alcohol) and ANA (Alko, Non-Alcohol) rats. The ethanol-exposed rats had 12% ethanol as the only available fluid from 3 to 24 months of age. The control groups had water. Rats that died during the experiment and those that were killed at 24 months of age were all autopsied, and the pathologic findings were recorded. RESULTS: Lifelong ethanol consumption did not change the survival rate of the rats, and had no significant effect on the rates of any of the pathologic measures in either the AA or ANA line of rats, suggesting that this may not be a good animal model for studying the detrimental effects of chronic alcohol. An unexpected, highly significant finding was observed: the AA rats, bred for high voluntary ethanol drinking, lived much longer than the ANA rats, bred for ethanol avoidance. The death rate by 24 months in the AA line was less than one-third of that in the ANA line. This difference was found regardless of whether the animals were maintained on alcohol or water, and in both genders. The AA rats had significantly lower rates of kidney disease, benign tumors, and cardiovascular disease than the ANA animals. CONCLUSIONS: Lifelong ethanol consumption increased neither the mortality nor the morbidity of AA and ANA line of rats. Genes selected in the development of the high drinking AA line have additional effects producing rats that are healthier and living longer than the ANA rats possessing genes resulting in alcohol avoidance.