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.

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.

Hepatitis B Vaccine Non-Responders Show Higher Frequencies of CD24highCD38high Regulatory B Cells and Lower Levels of IL-10 Expression Compared to Responders.

Background: The cellular mechanisms involved in the lack of protective antibody response after hepatitis B vaccination are still rather unclear. Regulatory B cells (Breg) known as modulators of B-and T-cell responses may contribute to poor vaccine responsiveness. The current study aimed to investigate the role of regulatory B cells (Breg) in hepatitis B vaccine non-responsiveness after immunization with second- or third-generation hepatitis B vaccines. Method: We performed comparative phenotypic and frequency analysis of Breg subsets (CD24+CD27+ and CD24highCD38high Breg) in second-generation hepatitis B vaccine non-responders (2nd HBvac NR, n = 11) and responders (2nd HBvac R, n = 8) before (d0), on day 7 (d7), and 28 (d28) after booster vaccination. Cryopreserved peripheral blood mononuclear cells were stimulated ex vivo with a combination of CpG, PMA, and Ionomycin (CpG+P/I) and analyzed for numbers and IL-10 expression levels of Breg by flow cytometry-based analyses. Results: Flow cytometry-based analyses revealed elevated frequencies of CD24+CD27+ Breg at all time points and significantly higher frequencies of CD24highCD38high Breg on d0 (p = 0.004) and 28 (p = 0.012) in 2nd HBvac NR compared to 2nd HBvac R. In parallel, we observed significantly lower levels of CpG+P/I-induced IL-10 expression levels of CD24+CD27+ and CD24highCD38high Breg (d0: p < 0.0001; d7: p = 0.0004; d28: p = 0.0003 and d0: p = 0.016; d7: p = 0.016, respectively) in 2nd HBvac NR compared to 2nd HBvac R before and after booster immunization. Frequencies of CD24+CD27+ and CD24highCD38high Breg significantly decreased after third-generation hepatitis B booster vaccination (d7: p = 0.014; d28: p = 0.032 and d7: p = 0.045, respectively), whereas IL-10 expression levels of both Breg subsets remained stable. Conclusion: Here we report significantly higher frequencies of CD24highCD38high Breg in parallel with significantly lower IL-10 expression levels of CD24+CD27+ and CD24highCD38high Breg in 2nd HBvac NR compared to 2nd HBvac R. Anti-HBs seroconversion accompanied by a decrease of Breg numbers after booster immunization with a third-generation hepatitis B vaccine could indicate a positive effect of third-generation hepatitis B vaccines on Breg-mediated immunomodulation in hepatitis B vaccine non-responders.

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.

Antioxidants N-Acetylcysteine and Vitamin C Improve T Cell Commitment to Memory and Long-Term Maintenance of Immunological Memory in Old Mice.

Aging is characterized by reduced immune responses, a process known as immunosenescence. Shortly after their generation, antigen-experienced adaptive immune cells, such as CD8+ and CD4+ T cells, migrate into the bone marrow (BM), in which they can be maintained for long periods of time within survival niches. Interestingly, we recently observed how oxidative stress may negatively support the maintenance of immunological memory in the BM in old age. To assess whether the generation and maintenance of immunological memory could be improved by scavenging oxygen radicals, we vaccinated 18-months (old) and 3-weeks (young) mice with alum-OVA, in the presence/absence of antioxidants vitamin C (Vc) and/or N-acetylcysteine (NAC). To monitor the phenotype of the immune cell population, blood was withdrawn at several time-points, and BM and spleen were harvested 91 days after the first alum-OVA dose. Only in old mice, memory T cell commitment was boosted with some antioxidant treatments. In addition, oxidative stress and the expression of pro-inflammatory molecules decreased in old mice. Finally, changes in the phenotype of dendritic cells, important regulators of T cell activation, were additionally observed. Taken together, our data show that the generation and maintenance of memory T cells in old age may be improved by targeting oxidative stress.

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.

The impact of body mass index on adaptive immune cells in the human bone marrow.

BACKGROUND: Obesity has been associated with chronic inflammation and oxidative stress. Both conditions play a determinant role in the pathogenesis of age-related diseases, such as immunosenescence. Adipose tissue can modulate the function of the immune system with the secretion of molecules influencing the phenotype of immune cells. The importance of the bone marrow (BM) in the maintenance of antigen-experienced adaptive immune cells has been documented in mice. Recently, some groups have investigated the survival of effector/memory T cells in the human BM. Despite this, whether high body mass index (BMI) may affect immune cells in the BM and the production of molecules supporting the maintenance of these cells it is unknown. METHODS: Using flow cytometry, the frequency and the phenotype of immune cell populations were measured in paired BM and PB samples obtained from persons with different BMI. Furthermore, the expression of BM cytokines was assessed. The influence of cytomegalovirus (CMV) on T cell subsets was additionally considered, dividing the donors into the CMV- and CMV+ groups. RESULTS: Our study suggests that increased BMI may affect both the maintenance and the phenotype of adaptive immune cells in the BM. While the BM levels of IL-15 and IL-6, supporting the survival of highly differentiated T cells, and oxygen radicals increased in overweight persons, the production of IFNγ and TNF by CD8+ T cells was reduced. In addition, the frequency of B cells and CD4+ T cells positively correlated with BMI in the BM of CMV- persons. Finally, the frequency of several T cell subsets, and the expression of senescence/exhaustion markers within these subpopulations, were affected by BMI. In particular, the levels of bona fide memory T cells may be reduced in overweight persons. CONCLUSION: Our work suggests that, in addition to aging and CMV, obesity may represent an additional risk factor for immunosenescence in adaptive immune cells. Metabolic interventions may help in improving the fitness of the immune system in the elderly.

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.

CD28 and CD57 define four populations with distinct phenotypic properties within human CD8+ T cells.

After repeated antigen exposure, both memory and terminally differentiated cells can be generated within CD8+ T cells. Although, during their differentiation, activated CD8+ T cells may first lose CD28, and CD28- cells may eventually express CD57 as a subsequent step, a population of CD28+ CD57+ (DP) CD8+ T cells can be identified in the peripheral blood. How this population is distinct from CD28- CD57- (DN) CD8+ T cells, and from the better characterized non-activated/early-activated CD28+ CD57- and senescent-like CD28- CD57+ CD8+ T cell subsets is currently unknown. Here, RNA expression of the four CD8+ T cell subsets isolated from human PBMCs was analyzed using microarrays. DN cells were more similar to "early" highly differentiated cells, with decreased TNF and IFN-γ production, impaired DNA damage response and apoptosis. Conversely, increased apoptosis and expression of cytokines, co-inhibitory, and chemokine receptors were found in DP cells. Higher levels of DP CD8+ T cells were observed 7 days after Hepatitis B vaccination, and decreased levels of DP cells were found in rheumatoid arthritis patients. More DP and DN CD8+ T cells were present in the bone marrow, in comparison with PBMCs. In summary, our results indicate that DP and DN cells are distinct CD8+ T cell subsets displaying defined properties.

Peripheral antibody concentrations are associated with highly differentiated T cells and inflammatory processes in the human bone marrow.

BACKGROUND: Antigen-experienced immune cells migrate back to the bone marrow (BM), where they are maintained in BM survival niches for an extended period. The composition of T cell subpopulations in the BM changes with age, leading to an accumulation of highly differentiated T cells and a loss of naïve T cells. While innate immune cells are also affected by age, little is known about interactions between different adaptive immune cell populations maintained in the BM. In this study, the phenotype and function of innate and adaptive immune cells isolated from human BM and peripheral blood (PB) was analysed in detail using flow cytometry, to determine if the accumulation of highly differentiated T and B cells, supported by the BM niches, limits the maintenance of other immune cells, or affects their functions such as providing protective antibody concentrations. RESULTS: Total T cells increase in the BM with age, as do highly differentiated CD8+ T cells which no longer express the co-stimulatory molecule CD28, while natural killer T (NKT) cells, monocytes, B cells, and naïve CD8+ T cells all decrease in the BM with age. A negative correlation of total T cells with B cells was observed in the BM. The percentage of B cells in the BM negatively correlated with highly differentiated CD8+CD28- T cells, replicative-senescent CD8+CD57+ T cells, as well as the CD8+CD28-CD57+ population. Similar correlations were seen between B cells and the frequency of highly differentiated T cells producing pro-inflammatory molecules in the BM. Interestingly, plasma concentrations of diphtheria-specific antibodies negatively correlated with highly differentiated CD8+CD57+ T cells as well as with exhausted central memory CD8+ and CD4+ T cells in the BM. A negative impact on diphtheria-specific antibodies was also observed for CD8+ T cells expressing senescence associated genes such as the cell cycle regulator p21 (CDKN1A), KLRG-1, and elevated levels of reactive oxygen species (ROS). CONCLUSION: Our data suggest that the accumulation and maintenance of highly differentiated, senescent, and exhausted T cells in the BM, particularly in old age, may interfere with the survival of other cell populations resident in the BM such as monocytes and B cells, leading to reduced peripheral diphtheria antibody concentrations as a result. These findings further highlight the importance of the BM in the long-term maintenance of immunological memory.

Human bone marrow adipocytes display distinct immune regulatory properties.

BACKGROUND: The bone marrow (BM) is a major reservoir of resting memory T cells and long-lived plasma cells, capable of providing protection against recurrent infections. Whether the age-related accumulation of adipose tissue in the BM affects the functionality and maintenance of memory cells is not well understood. METHODS: For the first time, we compare human femur marrow adipose tissue (fMAT) and subcutaneous white adipose tissue of the thigh (tsWAT) obtained from the same donors. Therefore, we used microarrays for comparative global gene expression analysis, and employed assays to analyse parameters of adipocyte biology, inflammation and oxidative stress. FINDINGS: We show that fMAT adipocytes differ significantly from tsWAT adipocytes regarding specific gene expression profiles including inflammatory responses and adipogenesis/adipocyte phenotype. Concomitant with considerably lower levels of CD36, a membrane-associated protein important for long-chain fatty acid uptake that is used as maturation marker, fMAT adipocytes are smaller and contain less triglycerides. fMAT adipocytes secrete similar levels of adiponectin and leptin as tsWAT adipocytes, and express increased levels of pro-inflammatory molecules concomitant with an elevated generation of reactive oxygen species (ROS) and impaired function of plasma cells in the BM. INTERPRETATION: Our findings suggest that fMAT is a unique type of adipose tissue containing small adipocytes with lower CD36 protein and triglyceride levels than tsWAT but high adipokine secretion. Moreover, fMAT adipocytes secrete high levels of pro-inflammatory cytokines, contributing to inflammation and impairment of plasma cell function in the BM, suggesting that fMAT has more immune regulatory functions than tsWAT.

Concentric and Eccentric Endurance Exercise Reverse Hallmarks of T-Cell Senescence in Pre-diabetic Subjects.

The peripheral T-cell pool undergoes a striking age associated remodeling which is accelerated by progressive insulin resistance. Exercise training is known to delay several aspects of T-cell senescence. The purpose of the current study was to investigate the effect of 3 weeks regular concentric or eccentric endurance exercise training on the composition of the T-cell compartment in pre-diabetic subjects. Sixteen male older adults with impaired glucose tolerance were recruited and performed either concentric exercise (CE) or eccentric exercise (EE) walking 3 times a week for 3 weeks. Fasting venous blood sampling was performed before training and after the training intervention. Various T-cell subpopulations were analyzed by flow cytometry. We did not find significant time × group effects (interaction) but found several significant time effects for cell type ratios and cell subsets proportions. There was an increase of the CD4+/CD8+ (0.55 ± 0.85%; p = 0.033) and CD4+/CD3+ ratio (5.63 ± 8.44%; p = 0.018) and a decrease of the CD8+/CD3+ ratio (-0.95 ± 1.64%; p = 0.049) after training. We found proportional increases of CD4+/CCR7+/CD45RO+ central memory cells (5.02 ± 7.68%; p = 0.030), naïve CD8+/CCR7+/CD45RO- (3.00 ± 6.68%; p = 0.047) and CD8+/CCR7+/CD45RO+ central memory cells (3.01 ± 3.70%; p = 0.009), while proportions of CD4+/CCR7-/CD45RO- TEMRA cells (-2.17 ± 4.66%; p = 0.012), CD8+/CCR7-/CD45RO- TEMRA cells (-5.11 ± 7.02%; p = 0.018) and CD16+ cells (-4.67 ± 6.45%; p = 0.016) decreased after training. 3 weeks of either CE or EE were effective in reversing hallmarks of T-cell senescence in pre-diabetic subjects. It is suggested that exercise stimulates production and mobilization of naïve T-cells, while differentiated TEMRA cells might disappear by apoptosis.

Fcµ receptor as a Costimulatory Molecule for T Cells.

Fc receptor for IgM (FcµR)-deficient mice display dysregulated function of neutrophils, dendritic cells, and B cells. The relevance of FcµR to human T cells is still unknown. We show that FcµR is mostly stored inside the cell and that surface expression is tightly regulated. Decreased surface expression on T cells from elderly individuals is associated with alterations in the methylation pattern of the FCMR gene. Binding and internalization of IgM stimulate transport of FcµR to the cell surface to ensure sustained IgM uptake. Concurrently, IgM accumulates within the cell, and the surface expression of other receptors increases, among them the T cell receptor (TCR) and costimulatory molecules. This leads to enhanced TCR signaling, proliferation, and cytokine release, in response to low, but not high, doses of antigen. Our findings indicate that FcµR is an important regulator of T cell function and reveal an additional mode of interaction between B and T cells.

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.

Immunological and morphological analysis of heterotopic ossification differs to healthy controls.

BACKGROUND: Formation of lamellar bone in non-osseus tissue is a pathological process called heterotopic ossification. It is the aim of this study to analyse the morphology and immunological status of patients with heterotopic ossification compared to individual healthy persons. METHODS: Human bone marrow and blood samples were obtained from 6 systemically healthy individuals and 4 patients during resection of heterotopic ossification from bone at hip arthroplasty. Bone was fragmented and treated with purified collagenase. Immunofluorescence surface staining was performed and analyzed with flow cytometry. Microcomputed tomography scanning was done performed at a resolution of 11 and 35 µm isometric voxel size respectively using a two different cone beam X-computer tomography systems and a microfocus X-ray tube. Subsequently the volume data was morphometrically analysed. RESULTS: The monocytes, stem cells, stroma cells and granulocytes progenitor cells were strongly reduced in the heterotopic ossification patient. Additionally a significant reduction of stromal stem cells cells and CD34 positive stem cells was observed. The frequency of NK-cells, B cells and T cells were not altered in the patients with heterotopic ossification compared to a healthy person. Micromorphometric parameters showed a lower content of mineralized bone tissue compared to normal bone. Mean trabecular thickness showed a high standard deviation, indicating a high variation in trabecular thickness, anisotropy and reducing bone strength. CONCLUSIONS: This work shows altered immunological distribution that is accompanied by a low decrease in bone volume fraction and tissue mineral density in the heterotopic ossification sample compared to normal bone. Compared to healthy subjects, this might reflect an immunological participation in the development of this entity.

GM-CSF improves the immune response to the diphtheria-component in a multivalent vaccine.

Multivalent tetanus and diphtheria toxoid containing vaccines belong to the most frequently applied vaccines. However, there is an imbalance in the degree of protection against the two antigens with insufficient long-term protection against diphtheria, particularly in the elderly population. We have previously reported a positive correlation between granulocyte macrophage-colony stimulating factor (GM-CSF) and the production of diphtheria-specific antibodies. Therefore, in the present study we analyzed the effects of in vivo applied recombinant GM-CSF on immunization with multivalent tetanus/diphtheria vaccine in mice of different age. In vivo application of GM-CSF lead to enhanced production of diphtheria-specific antibodies as well as more diphtheria-specific CD4+ T cells following vaccination with multivalent tetanus/diphtheria vaccine. In contrast, the humoral and cellular immune response to the tetanus component was unaltered. Furthermore, application of GM-CSF resulted in more splenic CD11b+ dendritic cells (DCs) with a higher MHC-II expression. GM-CSF also induced a stronger recruitment of CD11b+ DCs to the injected muscle. Most remarkably, GM-CSF was able to boost the diphtheria-specific immune response to the multivalent vaccine in aged mice. This study demonstrates that local administration of GM-CSF is able to improve immune responsiveness to the diphtheria component of multivalent tetanus/diphtheria vaccine in young and old mice. This information could be useful for the future design of vaccines for the elderly.