Association of Torquetenovirus Viremia with Physical Frailty and Cognitive Impairment in Three Independent European Cohorts.

INTRODUCTION: Immunosenescence and inflammaging have been implicated in the pathophysiology of frailty. Torquetenovirus (TTV), a single-stranded DNA anellovirus, the major component of the human blood virome, shows an increased replication rate with advancing age. An elevated TTV viremia has been associated with an impaired immune function and an increased risk of mortality in the older population. The objective of this study was to analyze the relation between TTV viremia, physical frailty, and cognitive impairment. METHODS: TTV viremia was measured in 1,131 nonfrail, 45 physically frail, and 113 cognitively impaired older adults recruited in the MARK-AGE study (overall mean age 64.7 ± 5.9 years), and then the results were checked in two other independent cohorts from Spain and Portugal, including 126 frail, 252 prefrail, and 141 nonfrail individuals (overall mean age: 77.5 ± 8.3 years). RESULTS: TTV viremia ≥4log was associated with physical frailty (OR: 4.69; 95% CI: 2.06-10.67, p < 0.0001) and cognitive impairment (OR: 3.49, 95% CI: 2.14-5.69, p < 0.0001) in the MARK-AGE population. The association between TTV DNA load and frailty status was confirmed in the Spanish cohort, while a slight association with cognitive impairment was observed (OR: 1.33; 95% CI: 1.000-1.773), only in the unadjusted model. No association between TTV load and frailty or cognitive impairment was found in the Portuguese sample, although a negative association between TTV viremia and MMSE score was observed in Spanish and Portuguese females. CONCLUSIONS: These findings demonstrate an association between TTV viremia and physical frailty, while the association with cognitive impairment was observed only in the younger population from the MARK-AGE study. Further research is necessary to clarify TTV's clinical relevance in the onset and progression of frailty and cognitive decline in older individuals.

Association between fat-soluble vitamins and self-reported health status: a cross-sectional analysis of the MARK-AGE cohort.

Self-rated health (SRH) is associated with higher risk of death. Since low plasma levels of fat-soluble vitamins are related to mortality, we aimed to assess whether plasma concentrations of vitamins A, D and E were associated with SRH in the MARK-AGE study. We included 3158 participants (52 % female) aged between 35 and 75 years. Cross-sectional data were collected via questionnaires. An enzyme immunoassay quantified 25-hydroxyvitamin D and HPLC determined α-tocopherol and retinol plasma concentrations. The median 25-hydroxyvitamin D and retinol concentrations differed significantly (P < 0·001) between SRH categories and were lower in the combined fair/poor category v. the excellent, very good and good categories (25-hydroxvitamin D: 40·8 v. 51·9, 49·3, 46·7 nmol/l, respectively; retinol: 1·67 v. 1·75, 1·74, 1·70 µmol/l, respectively). Both vitamin D and retinol status were independently associated with fair/poor SRH in multiple regression analyses: adjusted OR (95 % CI) for the vitamin D insufficiency, deficiency and severe deficiency categories were 1·33 (1·06-1·68), 1·50 (1·17-1·93) and 1·83 (1·34-2·50), respectively; P = 0·015, P = 0·001 and P < 0·001, and for the second/third/fourth retinol quartiles: 1·44 (1·18-1·75), 1·57 (1·28-1·93) and 1·49 (1·20-1·84); all P < 0·001. No significant associations were reported for α-tocopherol quartiles. Lower vitamin A and D status emerged as independent markers for fair/poor SRH. Further insights into the long-term implications of these modifiable nutrients on health status are warranted.

Do low molecular weight antioxidants contribute to the Protection against oxidative damage? The interrelation between oxidative stress and low molecular weight antioxidants based on data from the MARK-AGE study.

A redox steady state is important in maintaining vital cellular functions and is therefore homeostatically controlled by a number of antioxidative agents, the most important of which are enzymes. Oxidative Stress (OS) is associated with (or/and caused by) excessive production of damaging reactive oxygen and/or nitrogen species (ROS, RNS), which play a role in many pathologies. Because OS is a risk factor for many diseases, much effort (and money) is devoted to early diagnosis and treatment of OS. The desired benefit of the "identify (OS) and treat (by low molecular weight antioxidants, LMWA)" approach is to enable selective treatment of patients under OS. The present work aims at gaining understanding of the benefit of the antioxidants based on interrelationship between the concentration of different OS biomarkers and LMWA. Both the concentrations of a variety of biomarkers and of LMWA were previously determined and some analyses have been published by the MARK-AGE team. For the sake of simplicity, we assume that the concentration of an OS biomarker is a linear function of the concentration of a LMWA (if the association is due to causal relationship). A negative slope of this dependence (and sign of the correlation coefficient) can be intuitively expected for an antioxidant, a positive slope indicates that the LMWA is pro-oxidative, whereas extrapolation of the OS biomarker to [LMWA] = 0 is an approximation of the concentration of the OS biomarker in the absence of the LMWA. Using this strategy, we studied the effects of 12 LMWA (including tocopherols, carotenoids and ascorbic acid) on the OS status, as observed with 8 biomarkers of oxidative damage (including malondialdehyde, protein carbonyls, 3-nitrotyrosine). The results of this communication show that in a cross-sectional study the LMWA contribute little to the redox state and that different "antioxidants" are very different, so that single LMWA treatment of OS is not scientifically justified assuming our simple model. In view of the difficulty of quantitating the OS and the very different effects of various LMWA, the use of the "identify and treat" approach is questionable.

Using mid infrared technology as new method for the determination of the dwell time of salivary substitutes on three dimensional gingiva models.

BACKGROUND: Many people suffer from dry mouth (xerostomia) due to radiotherapy treatment of head and neck cancer, diseases like Sjogren's syndrome or as adverse effects to prescribed medications. Salivary substitute products like gels or sprays are often used for treatment. Efficacy of those oral care products are regularly assessed by validated or even not validated questionnaires. To determine the adhesion effect over time more objectively a new and sensitive method was established. The following study was designed to assess the dwell time of different oral care products in vitro. METHOD: Two different types of surfaces were covered with oral care products and washed using a definite protocol with artificial saliva salt solution. First, oral care gels or oral care sprays were spread to a polystyrene surface of 2.25 cm(2), then onto cell based three-dimensional gingiva models. The surfaces were washed ten times with artificial saliva salt solution. The resulting washing solutions were examined using mid infrared spectroscopy in order to detect ingredients of the oral care products. RESULTS: All assessed oral care gels or oral care sprays and their components were detected very sensitive. Even traces of the products were detected in the eluent and thus enabled to differentiate the dwell times of the different products. In general, the dwell time of oral care gels on polystyrene or gingiva models was longer than that of oral care sprays. The use of gingiva models improved the differentiation between different products. CONCLUSIONS: MIR spectroscopy turned out to be a sensitive method to detect salivary substitutes. Differences between single components and different products can be detected. The described method is a simple, reliable and easy process to evaluate the dwell time of oral care products in vitro and thus a useful tool to design optimised salivary substitute products. ETHICS: This is an in vitro study. No ethics or consent was required for this study.

Development and validation of cardiometabolic risk predictive models based on LDL oxidation and candidate geromarkers from the MARK-AGE data.

The predictive value of the susceptibility to oxidation of LDL particles (LDLox) in cardiometabolic risk assessment is incompletely understood. The main objective of the current study was to assess its relationship with other relevant biomarkers and cardiometabolic risk factors from MARK-AGE data. A cross-sectional observational study was carried out on 1089 subjects (528 men and 561 women), aged 40-75 years old, randomly recruited age- and sex-stratified individuals from the general population. A correlation analysis exploring the relationships between LDLox and relevant biomarkers was undertaken, as well as the development and validation of several machine learning algorithms, for estimating the risk of the combined status of high blood pressure and obesity for the MARK-AGE subjects. The machine learning models yielded Area Under the Receiver Operating Characteristic Curve Score ranging 0.783-0.839 for the internal validation, while the external validation resulted in an Under the Receiver Operating Characteristic Curve Score between 0.648 and 0.787, with the variables based on LDLox reaching significant importance within the obtained predictions. The current study offers novel insights regarding the combined effects of LDL oxidation and other ageing markers on cardiometabolic risk. Future studies might be extended on larger patient cohorts, in order to obtain reproducible clinical assessment models.

Association Between TTV Viremia, Chronic Inflammation, and Ischemic Heart Disease Risk: Insights From MARK-AGE and Report-Age Projects.

The implication of Torquetenovirus (TTV) in ischemic heart disease (IHD) has not been thoroughly explored. This study investigated the association between TTV viremia, pro-inflammatory cytokines, and IHD risk in an aging population. This cross-sectional study included 900 non-IHD subjects and 86 individuals with IHD (aged 55-75 years) selected from the MARK-AGE project. Results were verified in another independent Report-Age cohort, including 94 inpatients with chronic IHD and 111 inpatients with non-IHD (aged 65-96 years). Multivariable logistic regression in the MARK-AGE cohort revealed that male sex, TTV viremia ≥4log, Cu/Zn ratio, diabetes, hypertension, and smoking were significant IHD predictors. Notably, TTV viremia ≥4log independently increased the IHD risk (odds ratio [OR]: 2.51, 95% confidence interval [CI]: 1.42-4.43), confirmed in the Report-Age cohort (OR: 4.90, 95% CI: 2.32-10.39). In a RASIG subgroup, individuals with TTV viremia ≥4 log, both with and without IHD, exhibited increased plasma pro-inflammatory cytokine levels (IFN-γ, IL-1ß, IL-6, IL-10, IL-12p70, TNF-α) compared to those with TTV viremia <4 log. No significant difference in cytokine production was observed between IHD patients and non-IHD with TTV viremia ≥4 log. A positive correlation between TTV viremia and DNA methylation estimator of leukocyte telomere length was observed in Report-Age patients. Additionally, IHD Report-Age patients with TTV viremia ≥4 log displayed higher NLR and SIRI index than those with TTV viremia <4 log. In conclusion, a high TTV viremia is associated with an elevated IHD risk in the older population, potentially arising from an augmented pro-inflammatory response and immunosenescence.

High-resolution quantitative metabolome analysis of urine by automated flow injection NMR.

Metabolism is essential to understand human health. To characterize human metabolism, a high-resolution read-out of the metabolic status under various physiological conditions, either in health or disease, is needed. Metabolomics offers an unprecedented approach for generating system-specific biochemical definitions of a human phenotype through the capture of a variety of metabolites in a single measurement. The emergence of large cohorts in clinical studies increases the demand of technologies able to analyze a large number of measurements, in an automated fashion, in the most robust way. NMR is an established metabolomics tool for obtaining metabolic phenotypes. Here, we describe the analysis of NMR-based urinary profiles for metabolic studies, challenged to a large human study (3007 samples). This method includes the acquisition of nuclear Overhauser effect spectroscopy one-dimensional and J-resolved two-dimensional (J-Res-2D) (1)H NMR spectra obtained on a 600 MHz spectrometer, equipped with a 120 µL flow probe, coupled to a flow-injection analysis system, in full automation under the control of a sampler manager. Samples were acquired at a throughput of ~20 (or 40 when J-Res-2D is included) min/sample. The associated technical analysis error over the full series of analysis is 12%, which demonstrates the robustness of the method. With the aim to describe an overall metabolomics workflow, the quantification of 36 metabolites, mainly related to central carbon metabolism and gut microbial host cometabolism, was obtained, as well as multivariate data analysis of the full spectral profiles. The metabolic read-outs generated using our analytical workflow can therefore be considered for further pathway modeling and/or biological interpretation.

Uncovering the Relationship between Selenium Status, Age, Health, and Dietary Habits: Insights from a Large Population Study including Nonagenarian Offspring from the MARK-AGE Project.

An inadequate selenium (Se) status can accelerate the aging process, increasing the vulnerability to age-related diseases. The study aimed to investigate plasma Se and Se species in a large population, including 2200 older adults from the general population (RASIG), 514 nonagenarian offspring (GO), and 293 GO Spouses (SGO). Plasma Se levels in women exhibit an inverted U-shaped pattern, increasing with age until the post-menopausal period and then declining. Conversely, men exhibit a linear decline in plasma Se levels with age. Subjects from Finland had the highest plasma Se values, while those from Poland had the lowest ones. Plasma Se was influenced by fish and vitamin consumption, but there were no significant differences between RASIG, GO, and SGO. Plasma Se was positively associated with albumin, HDL, total cholesterol, fibrinogen, and triglycerides and negatively associated with homocysteine. Fractionation analysis showed that Se distribution among plasma selenoproteins is affected by age, glucometabolic and inflammatory factors, and being GO or SGO. These findings show that sex-specific, nutritional, and inflammatory factors play a crucial role in the regulation of Se plasma levels throughout the aging process and that the shared environment of GO and SGO plays a role in their distinctive Se fractionation.

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.

Circulating cell-free DNA in health and disease - the relationship to health behaviours, ageing phenotypes and metabolomics.

Circulating cell-free DNA (cf-DNA) has emerged as a promising biomarker of ageing, tissue damage and cellular stress. However, less is known about health behaviours, ageing phenotypes and metabolic processes that lead to elevated cf-DNA levels. We sought to analyse the relationship of circulating cf-DNA level to age, sex, smoking, physical activity, vegetable consumption, ageing phenotypes (physical functioning, the number of diseases, frailty) and an extensive panel of biomarkers including blood and urine metabolites and inflammatory markers in three human cohorts (N = 5385; 17-82 years). The relationships were assessed using correlation statistics, and linear and penalised regressions (the Lasso), also stratified by sex.cf-DNA levels were significantly higher in men than in women, and especially in middle-aged men and women who smoke, and in older more frail individuals. Correlation statistics of biomarker data showed that cf-DNA level was higher with elevated inflammation (C-reactive protein, interleukin-6), and higher levels of homocysteine, and proportion of red blood cells and lower levels of ascorbic acid. Inflammation (C-reactive protein, glycoprotein acetylation), amino acids (isoleucine, leucine, tyrosine), and ketogenesis (3-hydroxybutyrate) were included in the cf-DNA level-related biomarker profiles in at least two of the cohorts.In conclusion, circulating cf-DNA level is different by sex, and related to health behaviour, health decline and metabolic processes common in health and disease. These results can inform future studies where epidemiological and biological pathways of cf-DNA are to be analysed in details, and for studies evaluating cf-DNA as a potential clinical marker.

Microbiome in Blood Samples From the General Population Recruited in the MARK-AGE Project: A Pilot Study.

The presence of circulating microbiome in blood has been reported in both physiological and pathological conditions, although its origins, identities and function remain to be elucidated. This study aimed to investigate the presence of blood microbiome by quantitative real-time PCRs targeting the 16S rRNA gene. To our knowledge, this is the first study in which the circulating microbiome has been analyzed in such a large sample of individuals since the study was carried out on 1285 Randomly recruited Age-Stratified Individuals from the General population (RASIG). The samples came from several different European countries recruited within the EU Project MARK-AGE in which a series of clinical biochemical parameters were determined. The results obtained reveal an association between microbial DNA copy number and geographic origin. By contrast, no gender and age-related difference emerged, thus demonstrating the role of the environment in influencing the above levels independent of age and gender at least until the age of 75. In addition, a significant positive association was found with Free Fatty Acids (FFA) levels, leukocyte count, insulin, and glucose levels. Since these factors play an essential role in both health and disease conditions, their association with the extent of the blood microbiome leads us to consider the blood microbiome as a potential biomarker of human health.

Age, Sex, and BMI Influence on Copper, Zinc, and Their Major Serum Carrier Proteins in a Large European Population Including Nonagenarian Offspring From MARK-AGE Study.

The analysis of copper (Cu) and zinc (Zn) along with their major serum carriers, albumin (Alb) and ceruloplasmin (Cp), could provide information on the capacity of humans to maintain homeostasis of metals (metallostasis). However, their relationship with aging, sex, body mass index, as well as with nutritional and inflammatory markers was never investigated in a large-scale study. Here, we report results from the European large-scale cross-sectional study MARK-AGE in which Cu, Zn, Alb, Cp, as well as nutritional and inflammatory parameters were determined in 2424 age-stratified participants (35-75 years), including the general population (RASIG), nonagenarian offspring (GO), a well-studied genetic model of longevity, and spouses of GO (SGO). In RASIG, Cu to Zn ratio and Cp to Alb ratio were higher in women than in men. Both ratios increased with aging because Cu and Cp increased and Alb and Zn decreased. Cu, Zn, Alb, and Cp were found associated with several inflammatory as well as nutritional biomarkers. GO showed higher Zn levels and higher Zn to Alb ratio compared to RASIG, but we did not observe significant differences with SGO, likely as a consequence of the low sample size of SGO and the shared environment. Our results show that aging, sex, body mass index, and GO status are characterized by different levels of Cu, Zn, and their serum carrier proteins. These data and their relationship with inflammatory biomarkers support the concept that loss of metallostasis is a characteristic of inflammaging.

Ageing affects subtelomeric DNA methylation in blood cells from a large European population enrolled in the MARK-AGE study.

Ageing leaves characteristic traces in the DNA methylation make-up of the genome. However, the importance of DNA methylation in ageing remains unclear. The study of subtelomeric regions could give promising insights into this issue. Previously reported associations between susceptibility to age-related diseases and epigenetic instability at subtelomeres suggest that the DNA methylation profile of subtelomeres undergoes remodelling during ageing. In the present work, this hypothesis has been tested in the context of the European large-scale project MARK-AGE. In this cross-sectional study, we profiled the DNA methylation of chromosomes 5 and 21 subtelomeres, in more than 2000 age-stratified women and men recruited in eight European countries. The study included individuals from the general population as well as the offspring of nonagenarians and Down syndrome subjects, who served as putative models of delayed and accelerated ageing, respectively. Significant linear changes of subtelomeric DNA methylation with increasing age were detected in the general population, indicating that subtelomeric DNA methylation changes are typical signs of ageing. Data also show that, compared to the general population, the dynamics of age-related DNA methylation changes are attenuated in the offspring of centenarian, while they accelerate in Down syndrome individuals. This result suggests that subtelomeric DNA methylation changes reflect the rate of ageing progression. We next attempted to trace the age-related changes of subtelomeric methylation back to the influence of diverse variables associated with methylation variations in the population, including demographics, dietary/health habits and clinical parameters. Results indicate that the effects of age on subtelomeric DNA methylation are mostly independent of all other variables evaluated.

Medication Intake Is Associated with Lower Plasma Carotenoids and Higher Fat-Soluble Vitamins in the Cross-Sectional MARK-AGE Study in Older Individuals.

The regular use of medication may interfere with micronutrient metabolism on several levels, such as absorption, turnover rate, and tissue distribution, and this might be amplified during aging. This study evaluates the impact of self-reported medication intake on plasma micronutrients in the MARK-AGE Project, a cross-sectional observational study in 2217 subjects (age- and sex-stratified) aged 35-75 years from six European countries that were grouped according to age. Polypharmacy as possible determinant of micronutrient concentrations was assessed using multiple linear regression models adjusted for age-group, dietary fruit, vegetables, and juice intake, and other confounders. Younger participants reported taking fewer drugs than older participants. Inverse associations between medication intake and lutein (-3.31% difference per increase in medication group), ß-carotene (-11.44%), α-carotene (-8.50%) and positive associations with retinol (+2.26%), α-tocopherol/cholesterol (+2.89%) and γ-tocopherol/cholesterol (+1.36%) occurred in multiple adjusted regression models. Combined usage of a higher number of medical drugs was associated with poorer status of carotenoids on the one hand and higher plasma concentrations of retinol, α- and γ-tocopherol on the other hand. Our results raise concerns regarding the safety of drug combinations via the significant and surprisingly multifaceted disturbance of the concentrations of relevant micronutrients.

Prevalence and Loads of Torquetenovirus in the European MARK-AGE Study Population.

Torquetenovirus (TTV) viremia has been associated with increased mortality risk in the elderly population. This work aims to investigate TTV viremia as a potential biomarker of immunosenescence. We compared levels of circulating TTV in 1813 participants of the MARK-AGE project, including human models of delayed (offspring of centenarians [GO]) and premature (Down syndrome [DS]) immunosenescence. The TTV load was positively associated with age, cytomegalovirus (CMV) antibody levels, and the Cu/Zn ratio and negatively associated with platelets, total cholesterol, and total IgM. TTV viremia was highest in DS and lowest in GO, with intermediate levels in the SGO (spouses of GO) and RASIG (Randomly Recruited Age-Stratified Individuals From The General Population) populations. In the RASIG population, TTV DNA loads showed a slight negative association with CD3+T-cells and CD4+T-cells. Finally, males with ≥4log TTV copies/mL had a higher risk of having a CD4/CD8 ratio<1 than those with lower viremia (odds ratio [OR] = 2.85, 95% confidence interval [CI]: 1.06-7.62), as well as reduced CD3+ and CD4+T-cells compared to males with lower replication rates (<4log), even after adjusting for CMV infection. In summary, differences in immune system preservation are reflected in the models of delayed and premature immunosenescence, displaying the best and worst control over TTV replication, respectively. In the general population, TTV loads were negatively associated with CD4+ cell counts, with an increased predisposition for an inverted CD4/CD8 ratio for individuals with TTV loads ≥4log copies/mL, thus promoting an immune risk phenotype.

Antioxidants linked with physical, cognitive and psychological frailty: Analysis of candidate biomarkers and markers derived from the MARK-AGE study.

Frailty among elderly people leads to an increased risk for negative health outcomes. To prevent frailty, we need a better understanding of the underlying mechanisms and early detection of individuals at risk. Both may be served by identifying candidate (bio)markers, i.e. biomarkers and markers, for the physical, cognitive, and psychological frailty domains. We used univariate (Rank-ANOVA) and multivariate (elastic net) approaches on the RASIG study population (age range: 35-74 years, n = 2220) of the MARK-AGE study to study up to 331 (bio)markers between individuals with and without frailty for each domain. Biomarkers and markers identified by both approaches were studied further regarding their association with frailty using logistic regression. Univariately, we found lower levels of antioxidants, including ß-cryptoxanthin and zeaxanthin, in those who were physically, cognitively or psychologically frail. Additionally, self-reported health was worse in these three frail groups. Multivariately, we observed lower levels of ß-cryptoxanthin and zeaxanthin in the cognitively frail. Levels of these carotenoids were inversely associated with the risk of being cognitively frail after adjusting for confounders. Antioxidants and self-reported health are potential (bio)markers to detect persons at risk of becoming frail. The biomarkers identified may indicate the involvement of inflammation in frailty, especially for physical and cognitive frailty.

Protection against Tetanus and Diphtheria in Europe: The impact of age, gender and country of origin based on data from the MARK-AGE Study.

Due to the successful implementation of vaccination strategies early-life morbidity and mortality due to infectious disease has been reduced dramatically. Vaccines against tetanus and diphtheria are among the most frequently used vaccines worldwide, but various studies in different European countries have shown that protection against tetanus and particularly against diphtheria is unsatisfactory in adults and older persons. In this study we analyzed tetanus- and diphtheria-specific antibody concentrations in 2100 adults of different age from 6 selected European countries (Austria, Belgium, Germany, Greece, Italy, Poland) in order to investigate differences in the level of protection against tetanus and diphtheria across Europe. Our data reveal that tetanus- and diphtheria-specific antibody concentrations vary greatly between countries, which is also reflected in the percentage of persons with antibody concentrations below the protective level (0.1IU/ml), which ranged from 2 to 31% percent for tetanus and 28-63% for diphtheria. In most countries, tetanus- and diphtheria-specific antibody concentrations decrease with age. This phenomenon is more pronounced in countries with generally low antibody levels, such as Italy, Poland and Greece. Interestingly, tetanus-specific antibody concentrations are generally higher in males than in females, which is probably due to vaccination during their military service or more frequent booster vaccinations after injuries, whereas no gender-related differences were found for diphtheria-specific antibodies. In conclusion, our study demonstrates that the European population is not fully protected against tetanus and diphtheria. Measures to improve protection should include a life-long perspective on vaccination, more education to increase awareness of and compliance with vaccination guidelines, and a harmonization of recommendations and incentives across Europe.

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.

Age-dependent expression of DNMT1 and DNMT3B in PBMCs from a large European population enrolled in the MARK-AGE study.

Aging is associated with alterations in the content and patterns of DNA methylation virtually throughout the entire human lifespan. Reasons for these variations are not well understood. However, several lines of evidence suggest that the epigenetic instability in aging may be traced back to the alteration of the expression of DNA methyltransferases. Here, the association of the expression of DNA methyltransferases DNMT1 and DNMT3B with age has been analysed in the context of the MARK-AGE study, a large-scale cross-sectional study of the European general population. Using peripheral blood mononuclear cells, we assessed the variation of DNMT1 and DNMT3B gene expression in more than two thousand age-stratified women and men (35-75 years) recruited across eight European countries. Significant age-related changes were detected for both transcripts. The level of DNMT1 gradually dropped with aging but this was only observed up to the age of 64 years. By contrast, the expression of DNMT3B decreased linearly with increasing age and this association was particularly evident in females. We next attempted to trace the age-related changes of both transcripts to the influence of different variables that have an impact on changes of their expression in the population, including demographics, dietary and health habits, and clinical parameters. Our results indicate that age affects the expression of DNMT1 and DNMT3B as an almost independent variable in respect of all other variables evaluated.

3,4-Dihydroxymandelic acid, a noradrenalin metabolite with powerful antioxidative potential.

The decarboxylated noradrenaline metabolite 3,4-dihydroxymandelic acid [DHMA, 2-(3,4-dihydroxyphenyl)-2-hydroxyacetic acid] occurs in different mammalian tissues, especially in the heart. To elucidate the physiological function of DHMA, the antioxidative and radical scavenging activity was determined by physicochemical and cell-based test systems. In the 2,2-diphenyl-1-picrylhydrazyl assay it shows a 4-fold higher radical scavenging activity compared to the standard antioxidants ascorbic acid, tocopherol, and butylated hydroxytoluene. DHMA is also a very potent superoxide radical scavenger and shows a 5-fold smaller IC(50) value compared to standard ascorbic acid. Again, in most cases the antioxidative power of DHMA against bulk lipid oxidation determined by accelerated autoxidation of oils is much higher than for the standard antioxidants. In soybean oil and squalene a DHMA/alpha-tocopherol mixture (1:1 w/w) shows a synergistic effect. Last but not least, 0.001 and 0.0005% levels of DHMA protect human primary fibroblasts against H(2)O(2)-induced oxidative stress as determined by the 2',7'-dichlorofluorescein assay.