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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.

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.

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.

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. Supplementary Information The online version contains supplementary material available at 10.1007/s11357-021-00347-9.

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.

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

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&ndash, 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 (&minus, 3.31% difference per increase in medication group), &beta, carotene (&minus, 11.44%), &alpha, 8.50%) and positive associations with retinol (+2.26%), &alpha, tocopherol/cholesterol (+2.89%) and &gamma, 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, &alpha, and &gamma, 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.

Many currently used vaccines are less immunogenic in the elderly compared to young adults. The impact of latent infection with Cytomegalovirus (CMV) on vaccine-induced antibody responses has been discussed controversially. We have demonstrated that recall responses to diphtheria vaccination are frequently insufficient in elderly persons and that antibody concentrations decline substantially within 5 years. In the current study we show that within a cohort of healthy elderly (n = 87; median age 71 years, range 66–92) antibody responses to a booster vaccination against diphtheria do not differ between CMV-negative and CMV-positive individuals 4 weeks after vaccination.. However, the goal of diphtheria-vaccination is long-term protection and this is achieved by circulating anti-toxin antibodies. Diphtheria-specific antibody concentrations decline faster in CMV-positive compared to CMV-negative older adults leading to an increased proportion of persons without protective antibody concentrations 5 years after booster vaccination and endangering long-term protection. This finding could be relevant for vaccination schedules. Electronic supplementary material The online version of this article (doi:10.1186/s12979-017-0099-y) contains supplementary material, which is available to authorized users.

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. published

Many current vaccines are less immunogenic and less effective in elderly compared to younger adults due to age-related changes of the immune system. Most vaccines utilized in the elderly contain antigens, which the target population has had previous contact with due to previous vaccination or infection. Therefore, most studies investigating vaccine-induced immune responses in the elderly do not analyze responses to neo-antigens but rather booster responses. However, age-related differences in the immune response could differentially affect primary versus recall responses. We therefore investigated the impact of age on primary and recall antibody responses following hepatitis B vaccination in young and older adults. Focused gene expression profiling was performed before and 1 day after the vaccination in order to identify gene signatures predicting antibody responses. Young (20-40 years;  = 24) and elderly (>60 years;  = 17) healthy volunteers received either a primary series (no prior vaccination) or a single booster shot (documented primary vaccination more than 10 years ago). Antibody titers were determined at days 0, 7, and 28, as well as 6 months after the vaccination. After primary vaccination, antibody responses were lower and delayed in the elderly compared to young adults. Non-responders after the three-dose primary series were only observed in the elderly group. Maximum antibody concentrations after booster vaccination were similar in both age groups. Focused gene expression profiling identified 29 transcripts that correlated with age at baseline and clustered in a network centered around type I interferons and pro-inflammatory cytokines. In addition, smaller 8- and 6-gene signatures were identified at baseline that associated with vaccine responsiveness during primary and booster vaccination, respectively. When evaluating the kinetic changes in gene expression profiles before and after primary vaccination, a 33-gene signature, dominated by IFN-signaling, pro-inflammatory cytokines, inflammasome components, and immune cell subset markers, was uncovered that was associated with vaccine responsiveness. By contrast, no such transcripts were identified during booster vaccination. Our results document that primary differs from booster vaccination in old age, in regard to antibody responses as well as at the level of gene signatures. Clinical Trial Registration: www.clinicaltrialsregister.eu, this trial was registered at the EU Clinical Trial Register (EU-CTR) with the EUDRACT-Nr. 2013-002589-38.

Do men die young and sick, or do women live long and healthy? By trying to explain the sexual dimorphism in life expectancy, both biological and environmental aspects are presently being addressed. Besides age-related changes, both the immune and the endocrine system exhibit significant sex-specific differences. This review deals with the aging immune system and its interplay with sex steroid hormones. Together, they impact on the etiopathology of many infectious diseases, which are still the major causes of morbidity and mortality in people at old age. Among men, susceptibilities toward many infectious diseases and the corresponding mortality rates are higher. Responses to various types of vaccination are often higher among women thereby also mounting stronger humoral responses. Women appear immune-privileged. The major sex steroid hormones exhibit opposing effects on cells of both the adaptive and the innate immune system: estradiol being mainly enhancing, testosterone by and large suppressive. However, levels of sex hormones change with age. At menopause transition, dropping estradiol potentially enhances immunosenescence effects posing postmenopausal women at additional, yet specific risks. Conclusively during aging, interventions, which distinctively consider the changing level of individual hormones, shall provide potent options in maintaining optimal immune functions.

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) 1H 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.

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. published

Many genes have been shown to be involved in the decline in immune function of the elderly. However, normal numbers of myeloid and lymphoid colonies can be grown from elderly bone marrow under optimal conditions and some thymic function is preserved well into adult life. It may also be possible to reverse partially declining thymic function by IL-7 treatment. Peripheral B and T cells show evidence of dysregulation with production of large clones, changes in subset distribution and altered signalling and cytokine production, particularly decreased IL-2 production in the mouse. The identification of these defects may lead to relatively simple procedures to improve vaccination for the elderly.

The immune system undergoes profound age-related changes, including a gradual increase in the production and circulation of proinflammatory cytokines. Despite the known capacity of fibroblasts to produce cytokines, little is known so far about the inflammatory response of fibroblasts to cellular stress such as viral and/or bacterial infection in the context of aging. Therefore the aim of this study was to analyze the levels of IL6 and IL8 secretion in supernatants of human skin fibroblasts from young and elderly persons. Cytokine and chemokine secretion was analyzed before and after in vitro infection of the cells with Cytomegalovirus (CMV) and/or stimulation with Lipopolysaccharide (LPS). The exposure of fibroblasts to these agents caused inflammatory changes, reflected by the secretion of both the cytokine IL6 and the chemokine IL8 by fibroblasts from young as well as elderly persons. The cytokine/chemokine production induced by either agent alone could be further increased by co-stimulation of the cells with both stimuli. The level of protein secretion was dependent on the chronological age of the fibroblasts. Stimulated human skin fibroblasts from elderly donors produced higher amounts of IL6 as well as IL8 than fibroblasts from young donors. These differences were more pronounced for IL6 than for IL8. The inflammatory response of fibroblasts to stimulation differed among donors and did not correspond to the responsiveness of whole blood derived from the same person. In summary lifelong CMV-infection may act as an in vivo trigger for inflammatory changes by increasing the inflammatory response to bacterial products such as LPS. It may thus contribute to age-related inflammatory processes, referred to as ‘inflamm-aging’., Highlights ► CMV and LPS induced cytokine production of skin fibroblasts depends on donor age. ► Stimulated fibroblasts from elderly donors produce higher amounts of IL6 and IL8. ► CMV-infection can increase the inflammatory response to bacterial products (LPS). ► The inflammatory response to LPS stimulation is different in individual cell types.

Recently, the BM has been shown to play a key role in regulating the survival and function of memory T cells. However, the impact of aging on these processes has not yet been studied. We demonstrate that the number of CD4+ and CD8+ T cells in the BM is maintained during aging. However, the composition of the T cell pool in the aged BM is altered with a decline of naïve and an increase in TEM cells. In contrast to the PB, a highly activated CD8+CD28– T cell population, which lacks the late differentiation marker CD57, accumulates in the BM of elderly persons. IL-6 and IL-15, which are both increased in the aged BM, efficiently induce the activation, proliferation, and differentiation of CD8+ T cells in vitro, highlighting a role of these cytokines in the age-dependent accumulation of highly activated CD8+CD28– T cells in the BM. Yet, these age-related changes do not impair the maintenance of a high number of polyfunctional memory CD4+ and CD8+ T cells in the BM of elderly persons. In summary, aging leads to the accumulation of a highly activated CD8+CD28– T cell population in the BM, which is driven by the age-related increase of IL-6 and IL-15. Despite these changes, the aged BM is a rich source of polyfunctional memory T cells and may thus represent an important line of defense to fight recurrent infections in old age.

Recently, a key role in memory T cell homing and survival has been attributed to the bone marrow (BM) in mice. In the human BM, the repertoire, function, and survival niches of CD4+ and CD8+ T cells have not yet been elucidated. In this study, we demonstrate that CD4+ and CD8+ effector memory T cells accumulate in the human BM and are in a heightened activation state as revealed by CD69 expression. BM-resident memory T cells produce more IFN-γ and are frequently polyfunctional. Immunofluorescence analysis revealed that CD4+ and CD8+ T cells are in the immediate vicinity of IL-15–producing BM cells, suggesting a close interaction between these two cell types and a regulatory role of IL-15 on T cells. Accordingly, IL-15 induced an identical pattern of CD69 expression in peripheral blood CD4+ and CD8+ T cell subsets. Moreover, the IL-15–inducible molecules Bcl-xL, MIP-1α, MIP-1β, and CCR5 were upregulated in the human BM. In summary, our results indicate that the human BM microenvironment, in particular IL-15–producing cells, is important for the maintenance of a polyfunctional memory CD4+ and CD8+ T cell pool.

Since Looney at al. published their seminal paper a decade ago [1] it has become clear that many of the differences in T cell immunological parameters observed between young and old people are related to the age-associated increasing prevalence of infection with the persistent β-herpesvirus HHV-5 (Cytomegalovirus). Ten years later, studies suggest that hallmark age-associated changes in peripheral blood T cell subset distribution may not occur at all in people who are not infected with this virus [[2]; Derhovanessian et al., in press]. Whether the observed changes are actually caused by CMV is an open question, but very similar, rapid changes observed in uninfected patients receiving CMV-infected kidney grafts are consistent with a causative role [3]. This meeting intensively discussed these and other questions related to the impact of CMV on human immune status and its relevance for immune function in later life.

Tumor necrosis factor-like cytokine 1A (TL1A) is expressed in endothelial cells and contributes to T-cell activation, via an extracellular fragment TL1A(L72-L251), generated by ectodomain shedding. Fragments of TL1A, referred to as vascular endothelial growth inhibitor, were found to induce growth arrest and apoptosis in endothelial cells; however, the underlying mechanisms remained obscure. Here, we show that full-length TL1A is the major detectable gene product in both human umbilical vein endothelial cells and circulating endothelial progenitor cells. TL1A expression was significantly enhanced in senescent circulating endothelial progenitor cells, and knockdown of TL1A partially reverted senescence. TL1A overexpression induced premature senescence in both circulating endothelial progenitor cells and human umbilical vein endothelial cells. We also identified a novel extracellular fragment of TL1A, TL1A(V84-L251), resulting from differential ectodomain shedding, which induced growth arrest and apoptosis in human umbilical vein endothelial cells. These findings suggest that TL1A is involved in the regulation of endothelial cell senescence, via a novel fragment produced by differential ectodomain shedding.

The human model within MARK-AGE project contains innovative concepts.

Many candidate biomarkers of human ageing have been proposed in the scientific literature but in all cases their variability in cross-sectional studies is considerable, and therefore no single measurement has proven to serve a useful marker to determine, on its own, biological age. A plausible reason for this is the intrinsic multi-causal and multi-system nature of the ageing process. The recently completed MARK-AGE study was a large-scale integrated project supported by the European Commission. The major aim of this project was to conduct a population study comprising about 3200 subjects in order to identify a set of biomarkers of ageing which, as a combination of parameters with appropriate weighting, would measure biological age better than any marker in isolation. (C) 2015 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Neopterin is produced from human monocyte-derived macrophages and dendritic cells upon Stimulation with interferon-y, and increased neopterin concentrations thus indicate cell-mediated immune activation. In healthy individuals increasing neopterin concentrations are found with increasing age, which is also evident from the agedependency of reference values of serum neopterin concentrations. Increase of neopterin concentrations in the elderly may relate to, e.g., altered T lymphocyte differentiation with age. In this study, serum neopterin concentrations of 138 persons (median age: 34 years; interquartile range 29.0 - 67.8 years) were investigated and compared to age and to the percentage of CD28+CD45RA+ or CD28+CD45RO+ subsets among CD8+ T cells. With increasing age, the percentage of CD28+CD45RA+ in CD8+ T-cells decreased (Spearman's rank correlation coefficient: rs = -0.561; p < 0.0001) accompanied by an increase of the percentage of CD28+CD45RO+ in CD8+ T-cells (rs = +0.221; p < 0.01). Serum neopterin concentrations increased with age (rs = -+0.541; p < 0.0001). This increase of neopterin concentration was accompanied by a decreased percentage of CD28+CD45RA+ in CD8+ T-cells (r+ = -0.287; p < 0.001). Multivariate analyses revealed that the inverse relationship between the percentage of CD28+CD45RA+ in CD8+ T-cells and neopterin concentrations was at least partly independent from age. Thus, investigation allows to conclude that an increase of neopterin concentrations with older age is accompanied by a loss of naive CD28+CD45RA+ CD8+ T-cells. Data suggest that the subset of CD28+CD8+ T-cells, which is developing in states of sustained immune activation, is important for a chronic production of interferon-γ which in turn gives rise to increased neopterin concentrations.

Immunosenescence, defined as the age-associated dysregulation and dysfunction of the immune system, is characterized by impaired protective immunity and decreased efficacy of vaccines. An increasing number of immunological, clinical and epidemiological studies suggest that persistent Cytomegalovirus (CMV) infection is associated with accelerated aging of the immune system and with several age-related diseases. However, current evidence on whether and how human CMV (HCMV) infection is implicated in immunosenescence and in age-related diseases remains incomplete and many aspects of CMV involvement in immune aging remain controversial. The attendees of the 4th International Workshop on "CMV & Immunosenescence", held in Parma, Italy, 25-27th March, 2013, presented and discussed data related to these open questions, which are reported in this commentary., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

UnlabelledDemographic changes and a more active life-style in older age have contributed to an increasing public awareness of the need for lifelong vaccination. Currently many older persons have been vaccinated against selected pathogens during childhood but lack regular booster immunizations. The impact of regular vaccinations when started late in life was analyzed in an open, explorative trial by evaluating the immune response against tetanus and diphtheria in healthy older individuals. 252 persons aged above 60 years received a booster vaccination against tetanus, diphtheria, pertussis and polio and a subcohort (n=87) was recruited to receive a second booster vaccination against tetanus, diphtheria and pertussis 5 years later. The percentage of unprotected individuals at the time of enrollment differed substantially for tetanus (12%) and diphtheria (65%). Despite protective antibody concentrations 4 weeks after the first vaccination in almost all vaccinees, antibodies had again dropped below protective levels in 10% (tetanus) and 45% (diphtheria) of the cohort after 5 years. Protection was restored in almost all vaccinees after the second vaccination. No correlation between tetanus- and diphtheria-specific responses was observed, and antibody concentrations were not associated with age-related changes in the T cell repertoire, inflammatory parameters, or CMV-seropositivity suggesting that there was no general biological "non-responder type." Post-vaccination antibody concentrations depended on pre-existing plasma cells and B cell memory as indicated by a strong positive relationship between post-vaccination antibodies and pre-vaccination antibodies as well as antibody-secreting cells. In contrast, antigen-specific T cell responses were not or only weakly associated with antibody concentrations. In conclusion, our findings demonstrate that single shot vaccinations against tetanus and/or diphtheria do not lead to long-lasting immunity in many elderly persons despite administration at relatively short intervals. Sufficient antigen-specific B cell memory B generated by adequate priming and consecutive booster vaccinations and/or exposure is a prerequisite for long-term protection.Trial registrationEU Clinical Trials Register (EU-CTR); EudraCT number 2009-011742-26; www.clinicaltrialsregister.eu/ctr-search/trial/2009-011742-26/AT.

Alone among herpesviruses, persistent Cytomegalovirus (CMV) markedly alters the numbers and proportions of peripheral immune cells in infected-vs-uninfected people. Because the rate of CMV infection increases with age in most countries, it has been suggested that it drives or at least exacerbates “immunosenescence”. This contention remains controversial and was the primary subject of the Third International Workshop on CMV & Immunosenescence which was held in Cordoba, Spain, 15-16th March, 2012. Discussions focused on several main themes including the effects of CMV on adaptive immunity and immunosenescence, characterization of CMV-specific T cells, impact of CMV infection and ageing on innate immunity, and finally, most important, the clinical implications of immunosenescence and CMV infection. Here we summarize the major findings of this workshop. The Workshop was partially supported by the University of Cordoba and the Spanish Ministry of Science (SAF2011-16169-E)

International audience; While the thymus is known to be essential for the initial production of T cells during early life, its contribution to immune development remains a matter of debate. In fact, during cardiac surgery in newborns, the thymus is completely resected to enable better access to the heart to correct congenital heart defects, suggesting that it may be dispensable during childhood and adulthood. Here, we show that young adults thymectomized during early childhood exhibit an altered T cell compartment. Specifically, absolute CD4+ and CD8+ T cell counts were decreased, and these T cell populations showed substantial loss of naive cells and accumulation of oligoclonal memory cells. A subgroup of these young patients (22 years old) exhibited a particularly altered T cell profile that is usually seen in elderly individuals (more than 75 years old). This condition was directly related to CMV infection and the induction of strong CMV-specific T cell responses, which may exhaust the naive T cell pool in the absence of adequate T cell renewal from the thymus. Together, these marked immunological alterations are reminiscent of the immune risk phenotype, which is defined by a cluster of immune markers predictive of increased mortality in the elderly. Overall, our data highlight the importance of the thymus in maintaining the integrity of T cell immunity during adult life.

Vaccines are an underused public health strategy for healthy aging. Considering the risks of vaccine-preventable diseases and the current low vaccine coverage rates in older European citizens, the two European geriatric and gerontological societies (European Union Geriatric Medicine Society [EUGMS] and International Association of Gerontology and Geriatrics-European Region [IAGG-ER]) convened a Joint Vaccine Working Group to develop a consensus document advocating routine vaccination of aging populations. The mandate of this Working Group was to improve the uptake of routine vaccinations in adults aged 60 years and over. The consensus statement underlines the need to establish, strengthen, and harmonize European policies that continue routine vaccinations to adulthood and that will include older populations. Improved vaccination rates will promote healthy aging by reducing the burden of vaccine-preventable infectious diseases in older populations, a population that is rapidly increasing in Europe.

In a recent article by Macchia et al.,1 the frequency, phenotype and function of peripheral CD4+ CD8+ T cells in rhesus macaques were described. The authors demonstrate that the double-positive population is mainly CD4hi CD8lo and that 78% of this population exhibits an effector memory phenotype. They also state that the frequency of double-positive T cells does not change with age. No data are available about the characteristics of peripheral CD4hi CD8lo or CD4lo CD8hi T cells in young and elderly persons, so it is difficult to assess the significance of the data by Maccia et al.1 for humans. We will therefore illustrate similarities and differences of peripheral CD4+ CD8+ T-cell subsets between rhesus macaques and humans and address the effect of age. As demonstrated by Laux et al.,2 we found a significant increase in the frequency of total peripheral CD4+ CD8+ T cells in the elderly compared to young persons (Table 1). Interestingly, CD4+ CD8+ T cells were enriched within the large cells of the lymphocyte population. Within the CD4+ CD8+ T-cell population we distinguished between CD4hi CD8lo, which expressed the CD8αα receptor and CD4lo CD8hi T cells which expressed the CD8αβ receptor (Fig. 1a). Immunofluorescence surface staining was performed as described elsewhere.3 Briefly, the antibodies used were CD3-peridinin chlorophyll protein, CD4-fluorescein isothiocyanate, CD8α-allophycocyanin and CD8β-phycoerythrin. Our data demonstrate that the number of CD4lo CD8hi T cells was increased in elderly compared to young persons, while the number of CD4hi CD8lo T cells was not affected by age (Table 1). In contrast to rhesus macaques, no dominance of the CD4hi CD8lo T cells was observed in either young or elderly persons, but a high variability in the frequency of CD4hi CD8lo T cells in both the young (0·06–1·15%) and the elderly (0·05–1·85%, Fig. 1a) was recorded. Table 1 Frequency of peripheral CD4+ CD8+, CD4lo CD8hi and CD4hi CD8lo T cells in young and elderly persons Figure 1 Frequency, phenotype and cytokine profile of peripheral human CD4lo CD8hi and CD4hi CD8lo T cells. (a) Representative fluorescence-activated cell sorter pictures showing the frequency of peripheral CD4lo CD8hi and CD4hi CD8lo T cells in four young (YD) ... Phenotypic analysis of CD4lo CD8hi and CD4hi CD8lo T cells from young persons (mean age ± SD: 27·6 ± 5·9 years, n = 9) did not reveal any differences,4 except for expression of natural killer group 2D (NKG2D), which was higher in the CD4lo CD8hi T-cell population (P = 0·004; Fig. 1b). In the elderly, however, CD4hi CD8lo T cells had a more differentiated phenotype compared to CD4lo CD8hi T cells, as demonstrated by a decreased expression of CD27 and an increased expression of CD56 and CD57 (Fig. 1b). The number of interleukin-2 (IL-2)-producing cells, as assessed by intracellular cytokine staining after stimulation with phorbol 12-myristate 13-acetate and ionomycin in the presence of brefeldin A,5 was also lower in CD4hi CD8lo than in CD4lo CD8hi T cells in elderly persons (Fig. 1c). No difference was observed in the number of cells producing IL-4, IL-10, interferon γ, tumour necrosis factor α and perforin between CD4lo CD8hi and CD4hi CD8lo T cells in both age groups. IL-2-producing CD25-expressing memory CD8+ T cells, which frequently coexpress CD4, have been shown to characteristically accumulate in a subgroup of healthy elderly persons who still have a good humoral response after influenza vaccination.3,6 As a result of the coexpression of CD4, double-positive cells can enhance interaction with antigen-presenting cells by serving as both an adhesion and a costimulatory molecule and they may interact with major histocompatibility complex class II.7 In conclusion, our data highlight several differences regarding the frequency and the composition of the CD4+ CD8+ T-cell population between rhesus macaques and humans. They also demonstrate that the size of the CD4lo CD8hi subset increases with age. It may therefore be of relevance to distinguish between CD4+ CD8+ T-cell subsets before assessing their functional significance in elderly humans.

Prolonged life expectancy in the 20th century has been one of humankind's greatest triumphs. However, the substantial increase in the human life span has ushered in a new concern: healthy aging. Because infectious diseases prominently contribute to morbidity in the particularly vulnerable elderly population, strategies for preventing these diseases would have a clear impact on improving healthy aging. Thus, vaccines and immunization strategies tailored for the elderly population are needed, and vaccines should be developed to take into consideration the peculiar age-induced variations of immune responsiveness. The conference "Ageing and Immunity" recently held in Siena, Italy, has reviewed and discussed several possible causes of immune senescence, as well as strategies for counteracting this waning of immune responsiveness and for restoring immunocompetence. In addition, examples of diseases that should be targeted by vaccination in the senior population were considered.

Background As a chronic antigenic stressor human Cytomegalovirus (CMV) contributes substantially to age-related alterations of the immune system. Even though monocytes have the greatest propensity for CMV-infection and seem to be an important host for the virus during latency, fibroblasts are also discussed to be target cells of CMV in vivo. However, little is known so far about general immunoregulatory properties of CMV in fibroblasts. We therefore investigated the immunoregulatory effects of CMV-infection in human lung fibroblasts and the impact on replicative senescence. Findings We observed that CMV-infection led to the induction of several immunoregulatory host cell genes associated with the innate and adaptive immune system. These were genes of different function such as genes regulating apoptosis, cytokines/chemokines and genes that are responsible for the detection of pathogens. Some of the genes upregulated following CMV-infection are also upregulated during cellular senescence, indicating that CMV causes an immunological phenotype in fibroblasts, which is partially reminiscent of replicative senescent cells. Conclusion In summary our results demonstrate that CMV not only affects the T cell pool but also induces inflammatory processes in human fibroblasts.

Der Alterungsprozess ist der Nahrboden, auf dem altersassoziierte Erkrankungen wachsen. Die Wissenschaftsdisziplin Gerontologie beschaftigt sich mit der Erforschung dieses normalen Prozesses der „biologischen Alterung“. Dieser Begriff bezeichnet den Verlust von Korperfunktionen, der zu einer progressiven Erhohung des Sterberisikos fuhrt. Studien in Modellorganismen zeigen, dass verschiedene pharmakologische Substanzen, genetische Interventionen sowie die Verminderung der Nahrungsaufnahme den Alterungsprozess verlangsamen konnen. Das Zellkulturmodell der zellularen Seneszenz ermoglicht es, diese Vorgange nah am menschlichen Organismus zu erforschen. Dazu werden isolierte Zellen aus verschiedenen Korpergeweben in vitro kultiviert, bis sie sich nicht mehr teilen konnen. Dieser Zustand wird „zellulare Seneszenz“ genannt. Mittlerweile konnte nachgewiesen werden, dass seneszente Zellen in vivo in zahlreichen Geweben akkumulieren und zur Pathogenese von altersassoziierten Erkrankungen beitragen.