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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.

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 β-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 β-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.

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. Recently, the importance of the bone marrow (BM) in the maintenance of antigen-experienced adaptive immune cells has been documented. 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 obesity may represent an independent risk factor supporting immunosenescence, in addition to aging and CMV. Metabolic interventions may help in improving the fitness of the immune system in the elderly.

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

Objectives Populations are aging worldwide. This paper summarizes some of the challenges and opportunities due to the increasing burden of infectious diseases in an aging population. Results Older adults typically suffer elevated morbidity from infectious disease, leading to increased demand for healthcare resources and higher healthcare costs. Preventive medicine, including vaccination can potentially play a major role in preserving the health and independence of older adults. However, this potential of widespread vaccination is rarely realized. Here, we give a brief overview of the problem, discuss concrete obstacles and the potential for expanded vaccination programs to promote healthy aging. Conclusion The increasing healthcare burden of infectious diseases expected in aging populations could, to a large extent, be reduced by achieving higher vaccination coverage among older adults. Vaccination can thus contribute to healthy aging, alongside healthy diet and physical exercise. The available evidence indicates that dedicated programs can achieve substantial improvements in vaccination coverage among older adults, but more research is required to assess the generalizability of the results achieved by specific interventions (see Additional file 1). Electronic supplementary material The online version of this article (10.1007/s41999-018-0040-8) contains supplementary material, which is available to authorized users.

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.

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.

A substantial proportion of CD8+ T cells in adults lack the expression of the CD28 molecule, and the aging of the immune system is associated with a steady expansion of this T cell subset. CD28−CD8+ T cells are characterized by potent effector functions but impaired responses to antigenic challenge. CD28 acts as the primary T cell costimulatory receptor, but there are numerous additional receptors that can costimulate the activation of T cells. In this study, we have examined such alternative costimulatory pathways regarding their functional role in CD28−CD8+ T cells. Our study showed that most costimulatory molecules have a low capacity to activate CD28-deficient T cells, whereas the engagement of the CD2 molecule by its ligand CD58 clearly costimulated proliferation, cytokine production, and effector function in this T cell subset. CD58 is broadly expressed on APCs including dendritic cells. Blocking CD58 mAb greatly reduced the response of human CD28−CD8+ T cells to allogeneic dendritic cells, as well as to viral Ags. Our results clearly identify the CD58/CD2 axis as the primary costimulatory pathway for CD8 T cells that lack CD28. Moreover, we show that engagement of CD2 amplifies TCR signals in CD28−CD8+ T cells, demonstrating that the CD2–CD58 interaction has a genuine costimulatory effect on this T cell subset. CD2 signals might promote the control of viral infection by CD28−CD8+ T cells, but they might also contribute to the continuous expansion of CD28−CD8+ T cells during chronic stimulation by persistent Ag.

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.

CD4+ regulatory T cells have been intensively studied during aging, but little is still known about age-related changes of other regulatory T cell subsets. It was, therefore, the goal of the present study to analyze CD8+human leukocyte antigen-antigen D related (HLADR)+ T cells in old age, a cell population reported to have suppressive activity and to be connected to specific genetic variants. We demonstrate a strong increase in the number of CD8+HLADR+ T cells with age in a cohort of female Sardinians as well as in elderly male and female persons from Austria. We also show that CD8+HLADR+ T cells lack classical activation molecules, such as CD69 and CD25, but contain increased numbers of checkpoint inhibitory molecules, such as cytotoxic T lymphocyte-associated antigen 4, T cell immunoglobulin and mucin protein-3, LAG-3, and PD-1, when compared with their HLADR- counterparts. They also have the capacity to inhibit the proliferation of autologous peripheral blood mononuclear cells. This suppressive activity is, however, decreased when CD8+HLADR+ T cells from elderly persons are analyzed. In accordance with this finding, CD8+HLADR+ T cells from persons of old age contain lower percentages of checkpoint inhibitory molecules than young controls. We conclude that in spite of high abundance of a CD8+ regulatory T cell subset in old age its expression of checkpoint inhibitory molecules and its suppressive function on a per cell basis are reduced. Reduction of suppressive capacity may support uncontrolled subclinical inflammatory processes referred to as "inflamm-aging."

CD4

The progressive increase of the aged population worldwide mandates new strategies to ensure sustained health and well-being with age. The development of better and/or new vaccines against pathogens that affect older adults is one pivotal intervention in approaching this goal. However, the functional decline of various physiological systems, including the immune system, requires novel approaches to counteract immunosenescence. Although important progress has been made in understanding the mechanisms underlying the age-related decline of the immune response to infections and vaccinations, knowledge gaps remain, both in the areas of basic and translational research. In particular, it will be important to better understand how environmental factors, such as diet, physical activity, co-morbidities, and pharmacological treatments, delay or contribute to the decline of the capability of the aging immune system to appropriately respond to infectious diseases and vaccination. Recent findings suggest that successful approaches specifically targeted to the older population can be developed, such as the high-dose and adjuvanted vaccines against seasonal influenza, the adjuvanted subunit vaccine against herpes zoster, as well as experimental interventions with immune-potentiators or immunostimulants. Learning from these first successes may pave the way to developing novel and improved vaccines for the older adults and immunocompromised. With an integrated, holistic vaccination strategy, society will offer the opportunity for an improved quality of life to the segment of the population that is going to increase most significantly in numbers and proportion over future decades.

There is an increased susceptibility and mortality in the elderly due to pneumonia caused by Streptococcus pneumoniae. We aimed to assess the inflammatory cell composition with respect to age in pneumococcal pneumonia patients. Neutrophilic granulocytes and various lymphocyte and macrophage subpopulations were immunohistochemically quantified on lung tissue specimens of young (n = 5; mean age 8.4 years), middle-aged (n = 8; mean age 55.9 years) and elderly (n = 9; mean age 86.6 years) pneumonia patients with microbiologically proven S. pneumoniae pneumonia. We discovered a higher percentage of neutrophilic granulocytes in elderly as opposed to young patients (95 vs. 75%, p = 0.012). Conversely, young patients versus elderly patients had more alveolar macrophages (CD11c+: 20 vs. 9%, p = 0.029) and M1 macrophages (CD14+: 30 vs. 10%, p = 0.012 and HLA-DR+: 52 vs. 11%, p = 0.019). There was no significant difference concerning M2 macrophages and lymphocytes. Comparison of young patients with middle-aged patients showed similar significant results for alveolar macrophages (p = 0.019) and subsignificant results for M1 macrophages and neutrophilic granulocytes (p < 0.08). This is the first study characterizing the inflammatory infiltrate of pneumococcal pneumonia in situ. Our observations improve the understanding of the innate immune mechanisms of pneumococcal lung infection and point at the potential of therapies for restoring macrophage function and decreasing neutrophilic influx in order to help prevent or cure pneumonia.

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.

The life-long homeostasis of memory CD8(+) T cells as well as persistent viral infections have been shown to facilitate the accumulation of highly differentiated CD8(+) CD28(-) T cells, a phenomenon that has been associated with an impaired immune function in humans. However, the molecular mechanisms regulating homeostasis of CD8(+) CD28(-) T cells have not yet been elucidated. In this study, we demonstrate that the miR-23∼24∼27 cluster is up-regulated during post-thymic CD8(+) T-cell differentiation in humans. The increased expression of miR-24 in CD8(+) CD28(-) T cells is associated with decreased expression of the histone variant H2AX, a protein that plays a key role in the DNA damage response (DDR). Following treatment with the classic chemotherapeutic agent etoposide, a topoisomerase II inhibitor, apoptosis was increased in CD8(+) CD28(-) when compared to CD8(+) CD28(+) T cells and correlated with an impaired DDR in this cell type. The reduced capacity of CD8(+) CD28(-) T cell to repair DNA was characterized by the automated fluorimetric analysis of DNA unwinding (FADU) assay as well as by decreased phosphorylation of H2AX at Ser139, of ATM at Ser1981, and of p53 at Ser15. Interleukin (IL)-15 could prevent etoposide-mediated apoptosis of CD8(+) CD28(-) T cells, suggesting a role for IL-15 in the survival and the age-dependent accumulation of CD8(+) CD28(-) T cells in humans.

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.

Immunologic memory is a critical feature of the adaptive immune system to fight recurrent infections. However, the mechanisms that shape the composition and function of the human memory T-cell pool remain incompletely understood. We here demonstrate that post-thymic human T-cell differentiation was associated with the downregulation, but not loss, of the inhibitory molecule CD5. The sensitivity of human CD8+ and CD4+ memory T cells to interleukin (IL)–15 was inversely associated with the level of CD5 expression. CD5 expression was downregulated by IL-15–mediated signaling in vitro and CD5lo memory T cells accumulated in the bone marrow. Persistent antigenic stimulation, as in the case of cytomegalovirus infection and rheumatoid arthritis (RA), was also associated with an increased number of CD5lo memory T cells. In conclusion, CD5 may be a useful marker to identify memory T-cell subsets with distinct responsiveness to the homeostatic cytokine IL-15.

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.

To identify new genetic regulators of cellular aging and senescence, we performed genome-wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress-induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.

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.

Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.