Reliability, stability during long-term storage, and intra-individual variation of circulating levels of osteopontin, osteoprotegerin, vascular endothelial growth factor-A, and interleukin-17A.

BACKGROUND: Studies in many populations have reported associations between circulating cytokine levels and various physiological or pathological conditions. However, the reliability of cytokine measurements in population studies, which measure cytokines in multiple assays over a prolonged period, has not been adequately examined; nor has stability during sample storage or intra-individual variation been assessed. METHODS: We assessed (1) analytical reliability in short- and long-term repeated measurements; (2) stability and analytical reliability during long-term sample storage, and (3) variability within individuals over seasons, of four cytokines-osteopontin (OPN), osteoprotegerin (OPG), vascular endothelial growth factor-A (VEGF-A), and interleukin-17A (IL-17A). Measurements in plasma or serum samples were made with commercial kits according to standard procedures. Estimation was performed by fitting a random or mixed effects linear model on the log scale. RESULTS: In repeated assays over a short period, OPN, OPG, and VEGF-A had acceptable reliability, with intra- and inter-assay coefficients of variation (CV) less than 0.11. Reliability of IL-17A was poor, with inter- and intra-assay CV 0.85 and 0.43, respectively. During long-term storage, OPG significantly decayed (- 33% per year; 95% confidence interval [- 54, - 3.7]), but not OPN or VEGF-A (- 0.3% or - 6.3% per year, respectively). Intra- and inter-assay CV over a long period were comparable to that in a short period except for a slight increase in inter-assay CV of VEGF-A. Within-individual variation was small for OPN and VEGF-A, with intra-class correlations (ICC) 0.68 and 0.83, respectively, but large for OPG (ICC 0.11). CONCLUSIONS: We conclude that OPN and VEGF-A can be reliably measured in a large population, that IL-17A is suitable only for small experiments, and that OPG should be assessed with caution due to degradation during storage and intra-individual variation. The overall results of our study illustrate the need for validation under relevant conditions when measuring circulating cytokines in population studies.

Naive CD4 T Cells Highly Expressing the Inflammatory Chemokine Receptor CXCR3 Increase with Age and Radiation Exposure in Atomic Bomb Survivors.

The numbers of naive T cells that react to novel pathogens not yet encountered by an immune system, decrease during aging, mainly due to age-associated involution of the thymus. CD45RA+ naive CD4 T cells consist of heterogeneous populations, including highly CXCR3-expressing cells that appear during the homeostatic proliferation of naive T cells and exhibit enhanced type-1 inflammatory phenotypes. Based on previous evidence of radiation-associated reductions in thymic function and peripheral blood naive CD4 T cells, we hypothesized that the homeostatic proliferation of naive CD4 T cells compensates for deficits in peripheral T-cell populations after radiation injury, which may increase the proportion of CXCR3high cells in naive CD4 T cells and enhance inflammation. The statistical models employed in this study revealed positive associations between the number of CXCR3high naive CD4 T cells and age as well as radiation dose among 580 Hiroshima atomic bomb survivors. In addition, the CXCR3high cells in these survivors increased not only with the levels of homeostatic cytokines, IL6 and IL7, but also with those of inflammatory indicators, CXCL10 and CRP. These results suggest that thymic T-cell production deficiency due to radiation and aging results in enhanced homeostatic proliferation that drives the appearance of CXCR3high naive CD4 T cells poised for an inflammatory response. Molecular mechanisms and clinical relevance of increasing CXCR3high cells in naive CD4 T populations should be further investigated in the context of inflammatory disease development long after radiation exposure.

Spectra and characteristics of somatic mutations induced by ionizing radiation in hematopoietic stem cells.

Spectra and frequencies of spontaneous and X-ray-induced somatic mutations were revealed with mouse long-term hematopoietic stem cells (LT-HSCs) by whole-genome sequencing of clonal cell populations propagated in vitro from single isolated LT-HSCs. SNVs and small indels were the most common types of somatic mutations, and increased up to twofold to threefold by whole-body X-irradiation. Base substitution patterns in the SNVs suggested a role of reactive oxygen species in radiation mutagenesis, and signature analysis of single base substitutions (SBS) revealed a dose-dependent increase of SBS40. Most of spontaneous small deletions were shrinkage of tandem repeats, and X-irradiation specifically induced small deletions out of tandem repeats (non-repeat deletions). Presence of microhomology sequences in non-repeat deletions suggested involvement of microhomology mediated end-joining repair mechanisms as well as nonhomologous end-joining in radiation-induced DNA damages. We also identified multisite mutations and structural variants (SV), i.e., large indels, inversions, reciprocal translocations, and complex variants. The radiation-specificity of each mutation type was evaluated from the spontaneous mutation rate and the per-Gy mutation rate estimated by linear regression, and was highest with non-repeat deletions without microhomology, followed by those with microhomology, SV except retroelement insertions, and multisite mutations; these types were thus revealed as mutational signatures of ionizing radiation. Further analysis of somatic mutations in multiple LT-HSCs indicated that large fractions of postirradiation LT-HSCs originated from single LT-HSCs that survived the irradiation and then expanded in vivo to confer marked clonality to the entire hematopoietic system, with varying clonal expansion and dynamics depending on radiation dose and fractionation.

Lifelong cytomegalovirus and early-LIFE irradiation synergistically potentiate age-related defects in response to vaccination and infection.

While whole-body irradiation (WBI) can induce some hallmarks of immune aging, (re)activation of persistent microbial infection also occurs following WBI and may contribute to immune effects of WBI over the lifespan. To test this hypothesis in a model relevant to human immune aging, we examined separate and joint effects of lifelong latent murine cytomegalovirus (MCMV) and of early-life WBI over the course of the lifespan. In late life, we then measured the response to a West Nile virus (WNV) live attenuated vaccine, and lethal WNV challenge subsequent to vaccination. We recently published that a single dose of non-lethal WBI in youth, on its own, was not sufficient to accelerate aging of the murine immune system, despite widespread DNA damage and repopulation stress in hematopoietic cells. However, 4Gy sub-lethal WBI caused manifest reactivation of MCMV. Following vaccination and challenge with WNV in the old age, MCMV-infected animals experiencing 4Gy, but not lower, dose of sub-lethal WBI in youth had reduced survival. By contrast, old irradiated mice lacking MCMV and MCMV-infected, but not irradiated, mice were both protected to the same high level as the old non-irradiated, uninfected controls. Analysis of the quality and quantity of anti-WNV immunity showed that higher mortality in MCMV-positive WBI mice correlated with increased levels of MCMV-specific immune activation during WNV challenge. Moreover, we demonstrate that infection, including that by WNV, led to MCMV reactivation. Our data suggest that MCMV reactivation may be an important determinant of increased late-life mortality following early-life irradiation and late-life acute infection.

Development of diabetes mellitus following hormone therapy in prostate cancer patients is associated with early progression to castration resistance.

To identify risk factors for the prognosis of prostate cancer (PC), we retrospectively analyzed the impact of lifestyle-related disorders as well as PC characteristics at initial diagnosis on the progression to castration-resistant PC (CRPC) in PC patients undergoing hormone therapy. Of 648 PC patients, 230 who underwent hormone therapy and met inclusion criteria were enrolled in this study. CRPC developed in 48 patients (20.9%). Univariate analysis using Cox proportional hazard model indicated that newly developed diabetes mellitus (DM) following hormone therapy (postDM), but not preexisting DM, as well as PC characteristics at initial diagnosis including prostate-specific antigen (PSA) ≥ 18 were significantly associated with the progression to CRPC. A similar tendency was also observed in the relationship between newly developed hypertension following hormone therapy and CRPC progression. On the other hand, neither dyslipidemia nor hyperuricemia, regardless the onset timing, exhibited any association with CRPC progression. In multivariate analysis, postDM and PSA ≥ 18 were extracted as independent risk factors for CRPC progression (adjusted hazard ratios, 3.38 and 2.34; p values, 0.016 and 0.019, respectively). Kaplan-Meier analysis and log-rank test clearly indicated earlier progression to CRPC in PC patients who developed postDM or had relatively advanced initial PC characteristics including PSA ≥ 18. Together, the development of lifestyle-related disorders, particularly DM, following hormone therapy, as well as advanced PC characteristics at initial diagnosis is considered to predict earlier progression to CRPC and poor prognosis in PC patients undergoing hormone therapy.

Intracellular reactive oxygen species level in blood cells of atomic bomb survivors is increased due to aging and radiation exposure.

Although reactive oxygen species (ROS) play important roles in immune responses, excessive ROS production and accumulation might enhance the risk of inflammation-related diseases. Moreover, impaired immune function and the acceleration of pre-clinically persistent inflammation due to aging and radiation exposure have been observed in atomic bomb (A-bomb) survivors more than 60 years post-exposure. Meanwhile, the effects of aging and radiation exposure on ROS production in immune cells have not been characterized. This study investigated the relationship between intracellular ROS (H2O2 and O2•-) levels in blood cells or T cell subsets and serum iron, ferritin, and C-reactive protein (CRP) levels, as well as how these variables are affected by age and radiation exposure in A-bomb survivors. We examined 2495 Hiroshima A-bomb survivors. Multiple linear regression models adjusted for confounding factors indicated that intracellular O2•- levels in monocytes, granulocytes, and lymphocytes, and particularly in memory CD8+ T cells, including effector memory and terminally differentiated effector memory CD8+ T cells, increased with radiation dose. Additionally, serum iron, ferritin, and CRP levels affected intracellular ROS levels in specific blood cell types and T cell subsets. Serum CRP levels increased significantly with increasing age and radiation dose. Finally, when divided into three groups according to serum CRP levels, dose-dependent increases in the intracellular O2•- levels in blood cells and central memory and effector memory CD8+ T cells were most prominently observed in the high-CRP group. These results suggest that an increase in the levels of certain intracellular ROS, particularly after radiation exposure, might be linked to enhanced inflammatory status, including elevated serum CRP levels and reduced serum iron levels. This study reveals that aging and radiation exposure increase oxidative stress in blood cells, which is involved in impaired immune function and accelerated pre-clinically persistent inflammation in radiation-exposed individuals.

Modifying Effect of Chronic Atrophic Gastritis on Radiation Risk for Noncardia Gastric Cancer According to Histological Type.

The findings from previously published studies have suggested that radiation exposure is associated with increased mortality and incidence of gastric cancer. However, few cohort studies have incorporated risk factors such as Helicobacter pylori (H. pylori) infection or chronic atrophic gastritis (CAG). The current study is aimed at evaluating the modifying effect of CAG on radiation risk of noncardia gastric cancer by histological type, by reanalyzing data from a nested case-control study conducted within the longitudinal clinical cohort of atomic bomb survivors. The analysis was restricted to 297 intestinal- or diffuse-type noncardia cases and 873 controls rematched to the cases on gender, age, city, and time and type of serum storage, and countermatched on radiation dose. Multivariable-adjusted relative risks [95% confidence interval (CI)] of noncardia gastric cancer were 3.9 (2.1-7.2) for H. pylori IgG seropositivity with cytotoxin-associated gene A (CagA) IgG low titer, 2.6 (1.9-3.6) for CAG, 1.9 (1.3-2.8) for current smoking, and 1.4 (1.1-1.9) for 1 Gy irradiation. Among subjects without CAG, the relative risk (95% CI) of noncardia gastric cancer at 1 Gy was 2.3 (1.4-3.7), whereas relative risk (95% CI) at 1 Gy was 1.1 (0.8-1.5) among subjects with CAG (for the overall interaction, P = 0.012). By histological type, the risk at 1 Gy was high for diffuse type without CAG, with adjusted relative risk (95% CI) of 3.8 (2.0-7.6), but was not high for diffuse type with CAG or for intestinal-type irrespective of CAG status. The results indicate that radiation exposure is associated with increased risk of diffuse-type noncardia gastric cancer without CAG, and this association exists despite adjustment for H. pylori infection and smoking habit.

The association of integration patterns of human papilloma virus and single nucleotide polymorphisms on immune- or DNA repair-related genes in cervical cancer patients.

The present study investigated the association between single nucleotide polymorphisms (SNPs) in immune- or DNA repair-related genes and the integration pattern of human papillomavirus (HPV), a promising prognostic marker in cervical cancer. The HPV integration patterns of cervical cancer patients were determined by polymerase chain reaction and in situ hybridization, and categorized as episomal (group A), single-copy or multi-copy tandem repetition integrated (group B), and undetectable HPV types (group C). After sample and SNP quality control, 166,505 SNPs in 161 samples (38, 111, and 12 patients in groups A, B, and C, respectively) were examined. None of the SNPs reached genome-wide significance, and several candidate SNPs for future study were selected, including rs10999435 on chromosome 10q22, rs1322054 on chromosome 9q32-33, and rs10902171 on chromosome 11p15. Luciferase assay identified rs1322054 as the primary functional variant to regulate gene expression in immune cell. Further studies are needed to determine the genetic background of different integration patterns of HPV in cervical cancer patients.

Protective association of HLA-DRB1*13:02, HLA-DRB1*04:06, and HLA-DQB1*06:04 alleles with cervical cancer in a Korean population.

Human leukocyte antigen (HLA) class II alleles have been previously associated with cervical cancer. However, these associations vary widely across racial and ethnic groups. Therefore, we evaluated the effect of HLA class II alleles on cervical cancer in a Korean population. HLA-DRB1, HLA-DQB1, and HLA-DQA1 alleles were analyzed in 457 cervical cancer patients and compared to those of 926 control subjects. The odds ratio (OR) of each allele between the patients and controls was calculated using the logistic regression model. Patients, had significantly lower frequencies of HLA-DRB1 and HLA-DQB1 alleles than control subjects: HLA-DRB1*13:02:01 (4.4% vs 8.8%; OR 0.48, 95% confidence interval (CI) 0.27-0.84; p = 0.001), HLA-DRB1*04:06 (2.1% vs 4.7%; OR 0.44, 95% CI 0.20-0.97; p = 0.033), and HLA-DQB1*06:04:01 (2.3% vs 5.0%; OR 0.46, 95% CI 0.22-0.94; p = 0.021). No significant association was observed for HLA-DQA1. Protective associations between HLA-DRB1*13:02, HLA-DRB1*04:06, and HLA-DQB1*06:04 alleles and cervical cancer were found in the Korean population.

Impact of early life exposure to ionizing radiation on influenza vaccine response in an elderly Japanese cohort.

The objective of this study was to evaluate effects of whole body radiation exposure early in life on influenza vaccination immune responses much later in life. A total of 292 volunteers recruited from the cohort members of ongoing Adult Health Study (AHS) of Japanese atomic bomb (A-bomb) survivors completed this observational study spanning two influenza seasons (2011-2012 and 2012-2013). Peripheral blood samples were collected prior to and three weeks after vaccination. Serum hemagglutination inhibition (HAI) antibody titers were measured as well as concentrations of 25 cytokines and chemokines in culture supernatant from peripheral blood mononuclear cells, with and without in vitro stimulation with influenza vaccine. We found that influenza vaccination modestly enhanced serum HAI titers in this unique cohort of elderly subjects, with seroprotection ranging from 18 to 48% for specific antigen/season combinations. Twelve percent of subjects were seroprotected against all three vaccine antigens post-vaccination. Males were generally more likely to be seroprotected for one or more antigens post-vaccination, with no differences in vaccine responses based on age at vaccination or radiation exposure in early life. These results show that early life exposure to ionizing radiation does not prevent responses of elderly A-bomb survivors to seasonal influenza vaccine.

Radiation- and Age-Associated Changes in Peripheral Blood Dendritic Cell Populations among Aging Atomic Bomb Survivors in Japan.

Previous immunological studies in atomic bomb survivors have suggested that radiation exposure leads to long-lasting changes, similar to immunological aging observed in T-cell-adaptive immunity. However, to our knowledge, late effects of radiation on dendritic cells (DCs), the key coordinators for activation and differentiation of T cells, have not yet been investigated in humans. In the current study, we hypothesized that numerical and functional decreases would be observed in relationship to radiation dose in circulating conventional DCs (cDCs) and plasmacytoid DCs (pDCs) among 229 Japanese A-bomb survivors. Overall, the evidence did not support this hypothesis, with no overall changes in DCs or functional changes observed with radiation dose. Multivariable regression analysis for radiation dose, age and gender effects revealed that total DC counts as well as subpopulation counts decreased in relationship to increasing age. Further analyses revealed that in women, absolute numbers of pDCs showed significant decreases with radiation dose. A hierarchical clustering analysis of gene expression profiles in DCs after Toll-like receptor stimulation in vitro identified two clusters of participants that differed in age-associated expression levels of genes involved in antigen presentation and cytokine/chemokine production in cDCs. These results suggest that DC counts decrease and expression levels of gene clusters change with age. More than 60 years after radiation exposure, we also observed changes in pDC counts associated with radiation, but only among women.

Radiation- and Age-Associated Changes in Peripheral Blood Dendritic Cell Populations among Aging Atomic Bomb Survivors in Japan.

Previous immunological studies in atomic bomb survivors have suggested that radiation exposure leads to long-lasting changes, similar to immunological aging observed in T-cell-adaptive immunity. However, to our knowledge, late effects of radiation on dendritic cells (DCs), the key coordinators for activation and differentiation of T cells, have not yet been investigated in humans. In the current study, we hypothesized that numerical and functional decreases would be observed in relationship to radiation dose in circulating conventional DCs (cDCs) and plasmacytoid DCs (pDCs) among 229 Japanese A-bomb survivors. Overall, the evidence did not support this hypothesis, with no overall changes in DCs or functional changes observed with radiation dose. Multivariable regression analysis for radiation dose, age and gender effects revealed that total DC counts as well as subpopulation counts decreased in relationship to increasing age. Further analyses revealed that in women, absolute numbers of pDCs showed significant decreases with radiation dose. A hierarchical clustering analysis of gene expression profiles in DCs after Toll-like receptor stimulation in vitro identified two clusters of participants that differed in age-associated expression levels of genes involved in antigen presentation and cytokine/chemokine production in cDCs. These results suggest that DC counts decrease and expression levels of gene clusters change with age. More than 60 years after radiation exposure, we also observed changes in pDC counts associated with radiation, but only among women.

Fate Decision Between Group 3 Innate Lymphoid and Conventional NK Cell Lineages by Notch Signaling in Human Circulating Hematopoietic Progenitors.

The role of Notch signaling in human innate lymphoid cell (ILC) differentiation is unclear, although IL-7 and IL-15 promote differentiation of natural cytotoxicity receptor (NCR) NKp44+ group 3 ILCs (NCR+ILC3s) and conventional NK (cNK) cells from CD34+ hematopoietic progenitor cells (HPCs) ex vivo. In this study, we analyzed the functions of Notch in the differentiation of NCR+ILC3s and cNK cells from human HPC subpopulations circulating in peripheral blood by limiting dilution and clonal assays using high-throughput flow cytometry. We demonstrated that Notch signaling in combination with IL-7 induced NCR+ILC3 differentiation, but conversely suppressed IL-15-dependent cNK cell generation in CD45RA+Flt-3-c-Kitlow, a novel innate lymphocyte-committed HPC subpopulation. In contrast, Notch signaling induced CD45RA-Flt-3+c-Kithigh multipotent HPCs to generate CD34+CD7+CD62Lhigh, the earliest thymic progenitor-like cells, which preserved high cNK/T cell potential, but lost NCR+ILC3 potential. These findings implicate the countervailing functions of Notch signaling in the fate decision between NCR+ILC3 and cNK cell lineages at different maturational stages of human HPCs. Inhibition of Notch functions by Abs specific for either the Notch1 or Notch2 negative regulatory region suggested that both Notch1 and Notch2 signals were involved in the fate decision of innate lymphocyte-committed HPCs and in the generation of earliest thymic progenitor-like cells from multipotent HPCs. Furthermore, the synergistic interaction between Notch and IL-7 in NCR+ILC3 commitment was primarily explicable by the induction of IL-7 receptor expression in the innate lymphocyte-committed HPCs by Notch stimulation, suggesting the pivotal role of Notch in the transcriptional control required for human NCR+ILC3 commitment.

Outcome predictors for patients with stage II/III gastric cancer who undergo gastrectomy and S-1 adjuvant chemotherapy.

Predictors of survival in patients with stage II/III gastric cancer (GC) who received tegafur/gimeracil/oteracil (S-1) adjuvant chemotherapy (ACT) subsequent to gastrectomy were examined. Additionally, the association between dose intensity of S-1 and survival rate was investigated. A total of 62 patients with stage II/III gastric cancer were retrospectively evaluated, each of whom had received a curative D2 gastrectomy and S-1 ACT. The relative performance (RP; administered/planned S-1 doses ×100%), body mass index (BMI), prognostic nutritional index (PNI) and body weight (BW) were calculated, and the association of survival with these factors and other clinicopathological parameters was examined. The 1-year treatment continuation rate for S-1 was 94.2%, excluding patients who experienced cancer recurrences during their ACT year. The initial S-1 reduction rate was 38.7%. Patients with stage II/IIIA disease exhibited significantly improved 5-year overall survival rates compared with patients with stage IIIB GC, 81.6/73.7 vs. 33.8% (P<0.01). No association between RP and survival was observed. BMI, BW and PNI were significantly decreased following surgery compared with preoperative states. In the univariate analysis, postoperative BW loss (BMI loss), pathological stage and >7 lymph node metastases were significantly associated with outcome (P<0.05); in the multivariate analysis, postoperative BW loss >10.6% and pathological stages were independent prognostic factors for survival. Continuing S-1 ACT for the full year exhibited a greater effect on survival compared with dosage. Early postoperative nutritional deterioration may decrease the survival rates in these patients.

Aging-related changes in human T-cell repertoire over 20years delineated by deep sequencing of peripheral T-cell receptors.

Recent deep sequencing studies on T-cell receptor (TCR) repertoire have provided robust data to characterize diversity of T-cell immune responsiveness to a wide variety of peptide antigens, including viral and tumor antigens. The human TCR repertoire declines with age, but this decline has not been fully investigated longitudinally in individuals. Using a deep sequencing approach, we analyzed TCRß repertoires longitudinally over approximately 20years, with ages ranging from 23 to 50years at the start (23 to 65years overall), in peripheral-blood CD4 and CD8 T-cell populations that were collected and cryopreserved 3 times at intervals of approximately 10years from each of 6 healthy adults (3 men and 3 women). Sequence data at the hypervariable complementarity determining region 3 (CDR3) in the TCRB gene locus were evaluated by applying a random-coefficient statistical regression model. Two outcomes were analyzed: total number of distinct TCRB CDR3 sequences as a TCR diversity metric, and clonality of the T-cell populations. TCR repertoire diversity decreased (p<0.001) and frequencies of clonal populations increased (p=0.003) with age in CD8 T cells, whereas CD4 T cells retained fairly diverse TCR repertoires along with relatively low clonality. We also found that approximately 10-30% and 30-80% of read sequences in CD4 and CD8 T cells, respectively, overlapped at different ages within each individual, indicating long-term stable maintenance of T-cell clonal composition. Moreover, many of the most frequent TCRB CDR3 sequences (i.e., top T-cell clones) persisted over 20years, and some of them expanded and exerted a dominating influence on clonality of peripheral T-cell populations. It is thus possible that persistence or expansion of top T-cell clones is a driver of T-cell immunity aging, and therefore represents a potential interventional target.

Histone H2A T120 Phosphorylation Promotes Oncogenic Transformation via Upregulation of Cyclin D1.

How deregulation of chromatin modifiers causes malignancies is of general interest. Here, we show that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrate that this phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation.

Long-Term Effects of Radiation Exposure and Metabolic Status on Telomere Length of Peripheral Blood T Cells in Atomic Bomb Survivors.

In a series of studies of atomic bomb survivors, radiation-dose-dependent alterations in peripheral T-cell populations have been reported. For example, reduced size in naïve T-cell pools and impaired proliferation ability of T cells were observed. Because these alterations are also generally observed with human aging, we hypothesized that radiation exposure may accelerate the aging process of the T-cell immune system. To further test this hypothesis, we conducted cross-sectional analyses of telomere length, a hallmark of cellular aging, of naïve and memory CD4 T cells and total CD8 T cells in the peripheral blood of 620 atomic bomb survivors as it relates to age and radiation dose, using fluorescence in situ hybridization with flow cytometry. Since telomere shortening has been recently demonstrated in obesity-related metabolic abnormalities and diseases, the modifying effects of metabolic status were also examined. Our results indicated nonlinear relationships between T-cell telomere length and prior radiation exposure, i.e., longer telomeres with lower dose exposure and a decreasing trend of telomere length with individuals exposed to doses higher than 0.5 Gy. There were associations between shorter T-cell telomeres and higher hemoglobin Alc levels or fatty liver development. In naïve and memory CD4 T cells, radiation dose and high-density lipoprotein (HDL) cholesterol were found to positively interact with telomere length, suggesting that the decreasing trend of telomere length from a higher radiation dose was less conspicuous in individuals with a higher HDL cholesterol. It is therefore likely that radiation exposure perturbs T-cell homeostasis involving telomere length maintenance by multiple biological mechanisms, depending on dose, and that long-term-radiation-induced effects on the maintenance of T-cell telomeres may be modified by the subsequent metabolic conditions of individuals.

Metabolic Profile as a Potential Modifier of Long-Term Radiation Effects on Peripheral Lymphocyte Subsets in Atomic Bomb Survivors.

Immune system impairments reflected by the composition and function of circulating lymphocytes are still observed in atomic bomb survivors, and metabolic abnormalities including altered blood triglyceride and cholesterol levels have also been detected in such survivors. Based on closely related features of immune and metabolic profiles of individuals, we investigated the hypothesis that long-term effects of radiation exposure on lymphocyte subsets might be modified by metabolic profiles in 3,113 atomic bomb survivors who participated in health examinations at the Radiation Effect Research Foundation, Hiroshima and Nagasaki, in 2000-2002. The lymphocyte subsets analyzed involved T-, B- and NK-cell subsets, and their percentages in the lymphocyte fraction were assessed using flow cytometry. Health examinations included metabolic indicators, body mass index, serum levels of total cholesterol, high-density lipoprotein cholesterol, C-reactive protein and hemoglobin A1c, as well as diabetes and fatty liver diagnoses. Standard regression analyses indicated that several metabolic indicators of obesity/related disease, particularly high-density lipoprotein cholesterol levels, were positively associated with type-1 helper T- and B-cell percentages but were inversely associated with naïve CD4 T and NK cells. A regression analysis adjusted for high-density lipoprotein cholesterol revealed a radiation dose relationship with increasing NK-cell percentage. Additionally, an interaction effect was suggested between radiation dose and C-reactive protein on B-cell percentage with a negative coefficient of the interaction term. Collectively, these findings suggest that radiation exposure and subsequent metabolic profile changes, potentially in relationship to obesity-related inflammation, lead to such long-term alterations in lymphocyte subset composition. Because this study is based on cross-sectional and exploratory analyses, the implications regarding radiation exposure, metabolic profiles and circulating lymphocytes warrant future longitudinal and molecular mechanistic studies.

Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors.

Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AX (γH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34+Lin-) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure.