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Immunology. Apr 2002; 105(4): 458–465.
PMCID: PMC1782682

Age-related telomere length dynamics in peripheral blood mononuclear cells of healthy cynomolgus monkeys measured by Flow FISH


Telomere length is a good biomarker to study the cellular senescence as well as aging of an organism, because it regulates the replicative capacity of vertebrate somatic cells. To demonstrate age-related telomere length dynamics in the peripheral blood mononuclear cells (PBMC) of the cynomolgus monkey, we introduced a novel method of measuring telomere length by fluorescence in situ hybridization with a Peptide Nucleic Acid (PNA) labelled probe and flow cytometry (Flow FISH). A highly significant correlation was observed between the intensity of telomere-specific fluorescence by Flow FISH and telomere length by Southern blot analysis (R = 0·923, n = 22). The intensity of telomere fluorescence in PBMC significantly decreased with age in 55 monkeys aged from 0 to 34 years and this decrease corresponded to the loss of 62·7 base pairs per year (R = − 0·52, P < 0·00004). We also analysed the expression of naive cell-associated markers, CD28, CD62L and CD45RA/CD62L in T lymphocytes of 47 cynomolgus monkeys. An age-related increase in the CD28 subset was observed in CD8+ T lymphocytes in monkeys less than 11 years old and in CD4+ T lymphocytes in monkeys over 23 years old, respectively. The percentage of CD62L+ subsets was significantly decreased with age in both CD4+ (R = − 0·55) and CD8+ T lymphocytes (R = − 0·73). From the comparison of telomere length among PBMC, CD62L+ and CD62L T lymphocytes, it was clearly evident that loss of naive subsets results in the shortening of telomere length in vivo. These results show that this method can be applicable to studying the turnover and precursor-progeny of PBMC in cynomolgus monkeys as an animal model of aging.


Telomeres are unique protein–DNA complexes that consists of G-rich hexanucleotide repeats (TTAGGG)n in vertebrate cells.1 At the termini of eukaryotic linear chromosomes, these complexes play an important role in maintaining the integrity of chromosomes by protecting against inappropriate recombination and random end-to-end fusions of chromosomes and in preventing the incomplete DNA replication of chromosomes in cell division. For complete DNA replication, conventional DNA polymerase requires template primer at the ends of chromosomes and it results in a loss of terminal telomere DNA in the absence of compensatory mechanisms.2,3 Telomeres have therefore been identified as a candidate for ‘mitotic clocks’, reflecting residual replicative capacity as well as replicative history, and have attracted particular attention in studies of cellular senescence and organism aging.13

It has often been reported that normal somatic cells enter a state of replicative senescence and become incapable of further cell divisions after a limited number of cell divisions.4 Because the capacity for extensive cell division and clonal expansion of lymphocytes is essential to maintain the effective function of the immune system, it is of great interest to understand whether shortening the telomere length results in a decline in the replicative capacity of lymphocytes and finally induces replicative senescence of lymphocytes in vivo.

On the other hand, telomere length has been used as a powerful tool for the analysis of cell division under physiological circumstances in which the clonal expansion was hard to monitor. Therefore, measurement of telomere length has been widely used to analyse lineage or precursor–product relationships and rates of cell division.58

Concerning this, age-related telomere shortening in various subpopulations of blood cells in human subjects was recently reported by a new technique, fluorescence in situ hybridization with a Peptide Nucleic Acid (PNA) labelled probe and flow cytometry (Flow FISH).911 This method made it possible to carry out single cell analyses, and it is relatively easy to apply to the analysis of multiple cell populations.2

The importance of the macaque monkey as an experimental model has been increased because of its close phylogenetic relationship to humans.1214 To understand the differences and similarities of the immune systems of humans and the macaque monkey is an essential step in interpreting experimental research data, but there are few reports on the immune system of the healthy macaque monkey. We recently reported the age-dependent remodelling of peropheral blood mononuclear cells (PBMC) in the cynomolgus monkey (Macaca fascicularis) and the usefulness of non-human primates as a model for studying the mechanism of aging in the immune system.15,16 Considering age-related change in telomere length, fundamental data on the relationship between telomere length changes and age-related phenotypic remodelling of PBMC in the monkey are necessary in order to use monkeys in aging studies.

In the present study, we wished to demonstrate the age-related change in telomere length and the relationships between telomere length and the PBMC phenotype in healthy monkeys. We reported here that the rate of shortening of telomere length in the PBMC of the cynomolgus monkey was 62·7 bp per year determined by the Flow FISH method and it was evident that loss of naive T-lymphocyte subsets resulted in shortening of telomere length in vivo.

Materials and methods

Animal subjects

Heparinized peripheral blood samples were collected from 55 apparently healthy cynomolgus monkeys aged from 0 to 34 years. All the monkeys were bred and reared in the Tsukuba Primate Centre, National Institute of Infectious Diseases.17 This study was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals in the National Institute of Infectious Diseases.


The PBMC were isolated by density centrifugation with Ficoll–Paque (Ficoll-Hypaque; Amersham Pharmacia Biotech, Uppsala, Sweden), and washed twice with phosphate-buffered saline (PBS). They were resuspended with RPMI-1640 medium containing 10% fetal calf serum (FCS). Typically, 2 × 105 PBMC were stained with the following antibodies: fluorescein isothiocyanate (FITC)-labelled anti-CD4 (NU-TH/1; Nichirei, Tokyo, Japan), anti-CD45RA (2H4; Coulter, Hialeah, FL), phycoerythrin (PE)-labelled anti-CD28 (L293; Becton Dickinson), anti-CD62L (Leu8; BD) and R-PE-Cy5-labelled anti-CD8 (DK25; Dako, Glostrup, Denmark). After staining, the PBMC were analysed by means of fluorescence-activated cell sorting (FACSCalibur; Becton Dickinson).


The average telomere length was measured by Flow FISH,11 In brief, 5×105–10 × 105 PBMC were washed in 1 ml 0·1% bovine serum albumin (BSA)-PBS, and were divided equally into two 1·5-ml tubes. After centrifugation for 15 seconds at 15 000 g, the supernant was removed and the cell pellets were resuspended in the hybridization mixture (105 cells/100 µl), which consisted of 70% deionized formamide (Gibco BRL, Rockville, MD), 20 mm Tris, 1% BSA, with or without 0·3 µg/ml telomere-specific FITC-conjugated (CCCTAA)3 PNA probe (PE Biosystems, Framingham, MA).11 After heat denaturation of DNA for 10 min at 80°, followed by hybridization for 2 hr at room temperature in the dark, the cells were washed three times with 1 ml washing buffer and resuspended with 7-aminoactinomycin D (7-AAD; Molecular Probes, Eugene, OR) solution (0·06 µg/ml) at 105 cells/100 µl followed by incubation for 2 hr at room temperature. Samples were analyzed immediately or stored at 4° until analysis. Calibration beads (Immuno-Brite Standards Kit, Coulter, Hialeah, FL) were used before and after each experiment to correct daily shifts in the linearity of the flow cytometry and fluctuations in the intensity and alignment of the laser. Net fluorescence intensity was calculated by subtracting the mean background intensity from the mean telomere-specific fluorescence intensity.

Determination of the actual telomere length by Southern blot analysis

To estimate actual telomere length from the telomere-specific fluorescence intensity by Flow FISH, we performed Southern blot analysis with the same samples (n = 22). Briefly, equal amounts of genomic DNA (5 µg) were digested with AluI (Gibco BRL) and then loaded onto a 0·5% 20 × 25 cm agarose gel. The gels were run by pulse-field electrophoresis for 17·5 hr at 6 V/cm using a programmable power inverter (PPI-200, MJ Research, Watertown, MA).18 The gel was then subjected to depurination, denaturation and neutralization before Southern blotting onto a nylon membrane (Biodyne A membrane, PALL, Washington DC). The blotted membranes were hybridized with a biotin-labelled telomere probe (CCCTAA)5 overnight at 62°. The DNA was visualized with a chemiluminescent detection system (Phototope Detection kit, New England BioLabs, Beverly, MA) following the protocol provided, and then exposed to film (X-Omat Film, Kodak, Rochester, NY). The signal density was analysed using Scion Image software (Scion Corporation, Frederick, MD).

Cell sorting

To isolate CD4+ CD62L+, CD4+ CD62L, CD8+ CD62L+ and CD8+ CD62L T lymphocytes from individual monkeys (n = 5, 7–22 years), the PBMC were reacted with the following monoclonal antibodies (mAbs), FITC-labelled anti-CD14 (Leu-M3; Becton Dickinson), anti-CD16 (LeuTM-11a; BD), anti-CD20 (LeuTM-16; Becton Dickinson) and PE-labelled anti-CD62L at 4° for 1 hr and were then washed with 10% FCS-RPMI-1640 medium. The desired cells, CD14 CD16 CD20 CD62L+ and CD14 CD16 CD20 CD62L T lymphocytes, were sorted out using Epics Elites (Coulter, Hialeah, FL). Two sorted T-lymphocyte subsets were stained once more with mAbs, PE-labelled anti-CD4 (NU-TH/1; Nichirei) and R-PE-Cy-5-labelled anti-CD8, and then sorted into CD4+ and CD8+ subsets, respectively. The purity of the sorted cells was always > 95%. The isolated CD4+ CD62L+, CD4+ CD62L, CD8+ CD62L+ and CD8+ CD62L T lymphocytes were kept in 10% FCS-RPMI-1640 medium at 4° until Flow FISH.


The relationships between variables were tested by simple regression analysis and extended Tukey's multiple comparison tests. Statistical analysis was conducted with statistica (Statsoft Inc, Tulsa, OK), and differences were accepted as significant at P < 0·05.


Age-related change in telomere length of PBMC in the cynomolgus monkey

To determine the rate of telomere shortening with age in the PBMC of the cynomolgus monkey, we isolated PBMC from the blood of 55 normal cynomolgus monkeys aged 0–34 years and analysed relative telomere by Flow FISH with telomere-specific PNA probe as previously described11 (Fig. 1). Hybridized PBMC were gated on forward scatter versus 7-AAD fluorescence dot plot histogram (Fig. 1b) and then the telomere-specific fluorescence of PBMC was calculated by subtracting the mean background fluorescence from the mean telomere-specific fluorescence obtained with the PNA probe (Fig. 1c).

Figure 1
Flow FISH in PBMC of healthy cynomolgus monkeys. Isolated PBMC was hybridized with or without a telomere-specific PNA probe. Region 1 (R1) in (a) shows the distribution of hybridized PBMC. To obtain the fluorescence histogram (c), the cells were gated ...

To estimate actual telomere length from telomere-specific fluorescence obtained from Flow FISH, we measured telomere length directly by Southern blot analysis as described in the Materials and methods. Because there was a highly significant correlation between the two methods (Fig. 2a, R = 0·923), we used the slope shown in Fig. 2(a) as a conversion factor to calculate the actual telomere length from the intensity of telomere-specific fluorescence. The calculated conversion factor was 1 fluorescence arbitrary unit (AU) = 15·6 base pairs (bp) and the average telomere length of PBMC of 55 cynomolgus monkeys was 14·2 ± 1·2 kilobase pairs (kbp) calculated from this conversion factor.

Figure 2
Age-related shortening of telomere length in PBMC. The telomere length in PBMC from 55 healthy cynomolgus monkey (aged 0–34 years old) was determined by Flow FISH. To determine actual telomere length, the intensity of telomere fluorescence in ...

As shown in Fig. 2(b), a highly significant relationship (R = − 0·52, P < 0·00004) was observed between telomere length and age in the cynomolgus monkey, as observed in humans. The annual rate of telomere shortening was found to be 4·02 AU, which was equivalent to 62·7 bp per year.

Age-related changes in a naive T-lymphocyte subset in PBMC of the cynomolgus monkey

The accurate marker of naive T lymphocytes is still controversial but several markers, CD28, CD62L, CD45RA and CD95 have been used for the identification of naive and effector/memory T lymphocytes.19,20 We analysed age-related changes in the expression of these markers in isolated PBMC of 47 healthy cynomolgus monkeys aged from 0·5 to 34 years.

We first examined the mean percentages of CD28 subsets within both CD4+ and CD8+ T lymphocytes from monkeys in the four age groups, less than 5 years, 6–11 years, 12–22 years, over 23 years. Figure 3 shows that the proportion of CD28 subsets within the two different T lymphocyte subsets increased with age, but the increase in CD4+ CD28 T lymphocytes was less pronounced than that observed in the CD8+ T lymphocytes (data not shown), and a difference in the pattern of increase in the CD28 subpopulation was observed between the two T-lymphocyte subsets. In CD8+ T lymphocytes, a dramatic increase in the CD28 subset occurred mainly in the first 11 years and thereafter these cells increased gently throughout the remaining life (Fig. 3a). On the other hand, CD4+ CD28 T lymphocytes were rarely found among monkeys younger than 11 years (Fig. 3b), although there were a few monkeys (3/19;16%) who had a high proportion (> 5% of total CD4+ T lymphocytes) of these cells. In contrast, a marked increase in the proportion of these cells was observed in many of the elderly monkeys. In particular, a high proportion (> 5%) of CD4+ CD28 T lymphocytes were observed in about half the monkeys over 23 years. But there were also monkeys over 23 years who had only a very small proportion of these lymphocytes (< 1% of total CD4+ T lymphocytes).

Figure 3
Age-related change in the proportion of CD28 subsets within the CD4+ and CD8+ T lymphocytes. Bars indicate the means of proportions of CD28 T lymphocytes in difference age groups [<5 years (n = 11), 6–11 years (n = 8), ...

It has been reported that CD62L is closely associated with naive T lymphocytes as a major homing receptor, and this receptor was poorly expressed on CD8+ CD28 T lymphocytes which were considered to be primed memory cells,21 although all CD8+ CD28+ T lymphocytes did not express the CD62L molecule20 (our unpublished data).

As shown in Fig. 4(a), the proportion of CD62L+ subsets within both the CD4+ and CD8+ T lymphocytes significantly decreased with age, being more evident in the CD8+ (R = − 0·73, P < 0·000001) than in the CD4+ T lymphocytes (R = − 0·55, P < 0·0001). Individual variation was observed particularly in the proportion of CD4+ CD62+ T lymphocytes in same age monkeys.

Figure 4
Age-related change in the proportion of CD62+ T cells within the CD4+ and CD8+ T lymphocytes. R and P-values were analysed by simple linear regression test. The proportion of CD62+ subsets decreased significantly with age in both CD4 ([filled triangle] and broken ...

There are several recent reports stating that the co-expression of CD45RA and CD62L may be considered as the marker of the naive of T lymphocyte marker in human and monkey.19,22 We analysed age-related change in CD45RA+ CD62L+ naive subset in CD8+ T lymphocytes. Figure 4(b) shows that CD45RA+ CD62L+ naive subset within CD8+ T lymphocytes decreased significantly with age and the proportion of naive subset within CD8+ T lymphocytes became less than 5% in most monkeys (15/19, > 79%) over 20 years old.

Telomere length in CD62L+ and CD62L T lymphocytes

Our present results showed that both telomere length and percentage of naive cell markers expressing T lymphocytes decreased with age in the cynomolgus monkeys. Although telomere length is influenced by genetic difference at a given age,2 it is expected that the decrease in naive T lymphocytes directly affects the shortening of telomere length due to a 50–100-bp loss of telomere DNA per cell division.58 We therefore attempted by Flow FISH to determine how much telomere length changes in PBMC during the phenotypic change from CD62L+ to CD62L. For this purpose, CD4+ CD62L+, CD4+ CD62L, CD8+ CD62L+ and CD8+ CD62L T lymphocytes were sorted out from the PBMC of five monkeys (age range 7–22) as described in the Materials and methods. Figure 5(a) (10-year-old) shows a representative result of the flow cytometry data, in which the CD8+ CD62L T lymphocyte has a shorter telomere length than the CD8+ CD62L+ T lymphocyte. Figure 5(b) summarizes the results for five monkeys. Although there was individual difference in telomere length, in CD62L+ subsets it was generally longer than in CD62L subsets. The mean differences between CD62L+ and CD62L subsets in telomere length were 450 bp and 920 bp in the CD4 and CD8 T-lymphocyte subsets, respectively. These results suggested that the telomere length of PBMC was mainly affected by that of CD62L subsets in the same monkey.

Figure 5
Telomere length of CD62L+ and CD62L subsets in CD4 and CD8 T lymphocytes. (a) A representative histogram of sorted cells from a normal cynomolgus monkey (10-year-old). The telomere-specific fluorescence intensity of CD8+ CD62L was lower ...


A number of recent studies have documented changes in the immune system in the aged.19,23 Among these changes at the cellular level, the most pronounced one was a shift in cell population profiles, mainly represented by accumulation of memory T-lymphocyte subsets. Phenotypic transition from naive to memory T lymphocytes induced by antigen stimulation was characterized by extensive clonal expansion caused by cell division, which induced oligoclonal diversity and a rapid loss of telomere length of these lymphocytes.58,24 Eventually they enter a state of replicative senescence if they undergo a finite number of cell divisions. Since the effective function of the immune system is dependent on the capacity for extensive cellular proliferation and clonal expansion of lymphocytes, it is important to elucidate the relationships among the dynamics of telomere length, phenotypic change and the replicative capacity of lymphocytes.

There have been many studies on age-related changes in telomere length in the PBMC of humans.9,10 In particular, the rate of age-related telomere shortening in various subpopulations of blood cells in human detected by a new technique, Flow FISH, was recently reported. This method is characterized by in situ hybridization with a FITC-conjugated PNA probe specific for the telomeric sequence and quantification of fluorescence intensity of hybridized probe by flow cytometry.11 The advantages of this method were to be able to measure the telomere length of each single cell and to carry out analysis of heterogeneous cell populations with relative ease. Moreover, the ability to process a large number of samples quickly and easily is useful to compensate for variation of telomere length at any given age.911

Several studies suggested that the relationship between telomere length and cell proliferation capacity was more complex in vivo.3 Although rodents are used as experimental animals, there are many biochemical and physiological differences between rodents and human in telomeres and telomerase,1,25 a suitable animal model is therefore needed to clarify this relationship. We recently reported the age-dependent remodelling of PBMC in cynomolgus monkeys and the usefulness of non-human primates as models for studying the mechanism of aging in the immune system15,16 but there have been few reports on telomere dynamics in macaque monkeys,18,26 particularly in healthy ones.

In the present study we introduced Flow FISH to analyse the telomere length of PBMC in cynomolgus monkeys and showed the rate of telomere shortening with age in healthy monkeys. The average telomere length (14·2 ± 1·2 kbp) on Flow FISH was a very close to that in previously published data (14·1 ± 1·8 kbp) which was quantified by an improved method based on two-dimensional calibration of DNA sizes in cynomolgus monkeys.18 Although the telomere length in PBMC of cynomolgus monkeys (about 12–16 kbp) was greater than that of human (6–11 kbp), the rate of telomere shortening per year was 62·7 bp, which was almost equal to the 59 bp in human.9 It was interesting that although there are differences in the life span as well as in the average telomere length, the age-related shortening of the telomere length of the PBMC of cynomolgus monkeys was comparable to that of humans. In human studies, the rate of shortening (1000 bp/year) of telomere length of PBMC from newborn to 4 years was much greater than that (52 bp/year) of individuals later in life,9 but the small sample size of young monkeys in this study makes it hard to confirm that difference. Nevertheless, the rapid decrease in CD8+ CD28+ T lymphocytes during the first 5 years of life in cynomolgus monkeys suggests that telomere length may decrease rapidly in this period.15

Although appropriated markers of naive T lymphocytes have hitherto been unclear, several markers have been reported to be naive associated markers, for example, CD28, CD45RA and CD62L.19 It is believed that once a naive cell is primed with antigen, these naive cell-associated markers disappear from the cell surface during the course of cell activation21 so that an increase in CD28, CD45RA and CD62L T-lymphocyte subsets means a decrease in naive T-lymphocyte subsets in peripheral compartments. A decrease in naive cells is one of the major age-related changes and it results in inability to evoke a primary immune response to novel antigens.

Age-related augmentation of CD8+ CD28 T lymphocytes which were considered to be replicative senescence lymphocytes has been well documented in human studies.20,21 Our results showed that CD8+ CD28 T lymphocytes in cynomolgus monkeys increase with age, but the pattern of increase is different from that in humans. In the case of humans, an increase in CD8+ CD28 T lymphocytes occurs suddenly late in life (over 60 years of age),21 but in the case of monkeys, the increase in these subsets generally occurs relatively early in life, up to 11 years old. Previous single-strand conformational polymorphism results suggested that these CD8+ CD28 T lymphocytes in cynomolgus monkeys were a clonally expanded population similar to those in humans.27 Therefore, oligoclonal expansion of CD8+ CD28 T lymphocytes early in life may be considered as a feature of the immune system in cynomolgus monkeys. However, the cause remains to be elucidated.

Interestingly, an age-related increase in CD4+ CD28 T lymphocytes was observed in the present studies. Vallejo et al. reported that CD4+ CD28 T lymphocytes in human were caused by dysregulation of the apoptotic pathway and characterized by oligoclonal and autoreactive properties.28,29 Although several exceptions suggested the probability of another age-independent factor affecting the presence of these cells in peripheral blood, age related increase in CD4+ CD28 T lymphocytes itself might be considered as another hallmark of the aging immune system.

It has been well documented that expression of the CD62L+ molecule is associated with naive T lymphocytes as a homing receptor and it is expressed at a low level in primed memory T cells.21,30 In the present study it was observed that the percentage of CD62L+ T lymphocytes decreased with age in both CD4+ and CD8+ subsets. Although there was large individual variation in the proportion of CD4+ CD62+ T lymphocytes in same-age monkeys, this individual variation in the naive subset markers in CD4+ T lymphocytes was also observed in human CD4+ CD95 T lymphocytes in which the absence of CD95 expression was used as another naive-associated marker.19

In view of these results, it was expected that accumulation of memory T lymphocytes in the peripheral blood compartment of aged monkeys results in shortening of the telomere length in vivo. We demonstrated with Flow FISH that CD62L T lymphocytes have shorter telomere length than the CD62L+ in both CD4+ and CD8+ T-lymphocyte subsets. The difference between CD62L+ and CD62L subsets in average telomere length was 450 bp and 920 bp in the CD4+ and CD8+ T-lymphocyte subsets, respectively. This result demonstrated that a decrease in naive T-lymphocyte subsets (Fig. 4a) directly affects telomere shortening (Fig. 2a) in vivo. Assuming the rate of telomere shortening to be 50–100 bp per cell division,8 phenotypic transition from CD62L+ to CD62L was caused by 4–9 and 9–18 cell divisions in the CD4 and CD8 T-lymphocyte subsets, respectively.

In previous reports on humans, it was evident that loss of CD28 expression in the CD8+ T lymphocyte resulted in a 1·1 kb telomere shortening.6 This was slightly greater than that caused by the loss of CD62L expression in our study. One possible explanation for this difference is that the shortening rate per cell division in humans and monkeys may be different. Another is that loss of the CD62L molecule occurs prior to loss of the CD28 molecule in phenotypic transition. It can be expected from the results that CD62L was poorly expressed on CD8+ CD28 T lymphocytes, but all CD8+ CD28+ T lymphocytes did not express the CD62L receptor20,21 (our unpublished data). The individual telomere length of CD62L subsets was observed in order of age of monkeys, but that of CD62L+ subsets has no relationship to age, particularly in CD8+ T lymphocytes. This may indicate that genetic influence on telomere length still remains in naive T lymphocytes. We also observed that telomere length in PBMC was shorter than that of CD4+ or CD8+ T lymphocytes in old monkeys (18 and 22 years). This might be influenced by the telomere length in other subsets, for example, B lymphocytes, natural killer cells, and CD4+ CD8+ T lymphocytes (double-positive T lymphocytes), suggesting that the rate of shortening of telomere length is more rapid in these subsets than in T lymphocytes. It can be explained by our reports that CD4+ CD8+ T lymphocytes, which were characterized as resting memory T lymphocytes, increased with age15,16,31 and by our preliminary data that the telomere length of CD4+ CD8+ T lymphocytes was shorter than that of CD4+ or CD8+ single-positive T lymphocytes. But the telomere length of other subsets remains to be elucidated.

Taken together, the present results clearly show that Flow FISH can be applied to the study of telomere length dynamics and the precursor–progeny relationship of PBMC in cynomolgus monkeys. Considering the more complex mechanisms among telomere length, aging of an organism and cellular senescence in vivo, aging studies in monkeys are the most suitable animal models to elucidate these relationships in humans.


We thank Dr F. Ono, Mr K. Hanari, Mr T. Ono, Mr H. Narita, Mr H. Ogawa and Mr H. Ageyama for animal care and blood collection. This study was supported by funds for Comprehensive Research on Aging and Health from the Japan Foundation for Aging and Health.


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