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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

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.

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.

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.

Background The telomeric 3′-overhang (G-tail) length is essential for the biological effects of telomere dysfunction in vitro, but the association of length with aging and cardiovascular risk is unclear in humans. We investigated the association between the telomere G-tail length of leukocytes and cardiovascular risk, age-related white matter changes (ARWMCs), and endothelial function. Methods Patients with a history of cerebrovascular disease and comorbidity were enrolled (n = 102; 69 males and 33 females, 70.1 ± 9.2 years). Total telomere and telomere G-tail lengths were measured using a hybridization protection assay. Endothelial function was evaluated by ultrasound assessment of brachial flow-mediated dilation (FMD). Findings Shortened telomere G-tail length was associated with age and Framingham risk score (P = 0.018 and P = 0.012). In addition, telomere G-tail length was positively correlated with FMD values (P = 0.031) and negatively with the severity of ARWMCs (P = 0.002). On multivariate regression analysis, telomere G-tail length was independently associated with FMD values (P = 0.022) and the severity of ARWMCs (P = 0.033), whereas total telomere length was not associated with these indicators. Interpretation Telomere G-tail length is associated with age and vascular risk factors, and might be superior to total telomere length as a marker of endothelial dysfunction and ARWMC severity., Graphical abstract, Highlights • Telomere G-tail length was measured using a hybridization protection assay in patients with vascular risk factors. • Telomere G-tail length was independently related to endothelial function and age-related white matter changes. • Telomere G-tail length might be a promising biomarker of vascular damage.

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.

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 TCRβ 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

The relationships between commitments of dendritic cells (DCs) and T cells in human hematopoietic stem cells are not well understood. In this study, we enumerate and characterize conventional DC and plasmacytoid DC precursors in association with T cell and thymus-derived types of NK cell precursors among CD34+ hematopoietic progenitor cells (HPCs) circulating in human peripheral blood. By limiting-dilution analyses using coculture with stroma cells expressing Notch1 ligand, the precursor frequencies (PFs) of DCs in HPCs were found to significantly correlate with T cell PFs, but not with NK cell PFs, among healthy donors. Clonal analyses showed that the majority of T/NK dual- and T single-lineage precursors—but only a minority of NK single-lineage precursors—were associated with the generation of DC progenies. All clones producing both DC and T cell progenies were found with monocyte and/or granulocyte progenies, suggesting DC differentiation via myeloid DC pathways. Analyses of peripheral blood HPC subpopulations revealed that the lineage split between DC and T/NK cell progenitor occurs at the stage prior to bifurcation into T and NK cell lineages. The findings suggest a strong linkage between DC and T cell commitments, which may be imprinted in circulating lymphoid-primed multipotent progenitors or in more upstream HPCs.

Gastric cancer (GC) is one of the cancers that reveal increased risk of mortality and incidence in atomic bomb survivors. The incidence of gastric cancer in the Life Span Study cohort of the Radiation Effects Research Foundation (RERF) increased with radiation dose (gender-averaged excess relative risk per Gy = 0.28) and remains high more than 65 years after exposure. To assess a possible role of gene-environment interaction, we examined the dose response for gastric cancer incidence based on immunosuppression-related IL-10 genotype, in a cohort study with 200 cancer cases (93 intestinal, 96 diffuse and 11 other types) among 4,690 atomic bomb survivors participating in an immunological substudy. Using a single haplotype block composed of four haplotype-tagging SNPs (comprising the major haplotype allele IL-10-ATTA and the minor haplotype allele IL-10-GGCG, which are categorized by IL-10 polymorphisms at -819AG and -592TG, +1177TC and +1589AG), multiplicative and additive models for joint effects of radiation and this IL-10 haplotyping were examined. The IL-10 minor haplotype allele(s) was a risk factor for intestinal type gastric cancer but not for diffuse type gastric cancer. Radiation was not associated with intestinal type gastric cancer. In diffuse type gastric cancer, the haplotype-specific excess relative risk (ERR) for radiation was statistically significant only in the major homozygote category of IL-10 (ERR = 0.46/Gy, P = 0.037), whereas estimated ERR for radiation with the minor IL-10 homozygotes was close to 0 and nonsignificant. Thus, the minor IL-10 haplotype might act to reduce the radiation related risk of diffuse-type gastric cancer. The results suggest that this IL-10 haplotyping might be involved in development of radiation-associated gastric cancer of the diffuse type, and that IL-10 haplotypes may explain individual differences in the radiation-related risk of gastric cancer.

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.

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 naive 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.

Ionizing radiation (IR) is a major source of cellular damage and the immediate cellular response to IR has been well characterized. But the long-term impact of IR on cell function and its relationship with aging are not known. Here, we examined the IR effects on telomere length and other biomarkers 50 to 68 years post-exposure (two time points per person) in survivors of the atomic bombing at Hiroshima during WWII. We found that telomere length of leukocytes was inversely correlated with the dose of IR (p=0.008), and this effect was primarily found in survivors who were exposed at younger ages; specifically those

Past exposure to atomic bomb (A-bomb) radiation has exerted various long-lasting deleterious effects on the health of survivors. Some of these effects are seen even after >60 yr. In this study, we evaluated the subclinical inflammatory status of 442 A-bomb survivors, in terms of 8 inflammation-related cytokines or markers, comprised of plasma levels of reactive oxygen species (ROS), interleukin (IL)-6, tumor necrosis factor α (TNF-α), C-reactive protein (CRP), IL-4, IL-10, and immunoglobulins, and erythrocyte sedimentation rate (ESR). The effects of past radiation exposure and natural aging on these markers were individually assessed and compared. Next, to assess the biologically significant relationship between inflammation and radiation exposure or aging, which was masked by the interrelationship of those cytokines/markers, we used multivariate statistical analyses and evaluated the systemic markers of inflammation as scores being calculated by linear combinations of selected cytokines and markers. Our results indicate that a linear combination of ROS, IL-6, CRP, and ESR generated a score that was the most indicative of inflammation and revealed clear dependences on radiation dose and aging that were found to be statistically significant. The results suggest that collectively, radiation exposure, in conjunction with natural aging, may enhance the persistent inflammatory status of A-bomb survivors.—Hayashi, T., Morishita, Y., Khattree, R., Misumi, M., Sasaki, K., Hayashi, I., Yoshida, K., Kajimura, J., Kyoizumi, S., Imai, K., Kusunoki, Y., Nakachi, K. Evaluation of systemic markers of inflammation in atomic-bomb survivors with special reference to radiation and age effects.

It is not yet known whether hematopoietic stem and progenitor cells (HSPCs) are compromised in the aging population of atomic bomb (A-bomb) survivors after their exposure nearly 70 years ago. To address this, we evaluated age- and radiation-related changes in different subtypes of circulating HSPCs among the CD34-positive/lineage marker-negative (CD34(+)Lin(-)) cell population in 231 Hiroshima A-bomb survivors. We enumerated functional HSPC subtypes, including: cobblestone area-forming cells; long-term culture-initiating cells; erythroid burst-forming units; granulocyte and macrophage colony-forming units; and T-cell and natural killer cell progenitors using cell culture. We obtained the count of each HSPC subtype per unit volume of blood and the proportion of each HSPC subtype in CD34(+)Lin(-) cells to represent the lineage commitment trend. Multivariate analyses, using sex, age and radiation dose as variables, showed significantly decreased counts with age in the total CD34(+)Lin(-) cell population and all HSPC subtypes. As for the proportion, only T-cell progenitors decreased significantly with age, suggesting that the commitment to the T-cell lineage in HSPCs continuously declines with age throughout the lifetime. However, neither the CD34(+)Lin(-) cell population, nor HSPC subtypes showed significant radiation-induced dose-dependent changes in counts or proportions. Moreover, the correlations of the proportions among HSPC subtypes in the survivors properly revealed the hierarchy of lineage commitments. Taken together, our findings suggest that many years after exposure to radiation and with advancing age, the number and function of HSPCs in living survivors as a whole may have recovered to normal levels.

This study aims to deepen understanding of lymphocyte phenotypes related to the course of hepatitis C virus (HCV) infection and progression of liver fibrosis, in a cohort of atomic-bomb survivors. The study subjects comprise three groups: 162 HCV persistently infected, 145 spontaneously cleared, and 3511 uninfected individuals. We found increased percentages of peripheral blood TH1 and total CD8 T cells and decreased percentages of NK cells in the HCV persistence group, compared with the other two groups, after adjustment for age, gender, and radiation exposure dose. Subsequently, we found that increased TH1 cell percentages in the HCV persistence group were significantly associated with an accelerated time-course reduction in platelet counts―accelerated progression of liver fibrosis―while TC1 and NK cell percentages were inversely associated with the progression. This study suggests that TH1 immunity is enhanced by persistent HCV infection, and that percentages of peripheral TH1, TC1, and NK cells may help predict progression of liver fibrosis., This research was based on RERF Research Protocols 3-09, 4-02, 2-00, 9-92, and was supported in part by the U.S. National Institute of Allergy and Infectious Diseases (NIAID Contract HHSN272200900059C).

Our previous study showed that radiation exposure reduced the diversity of repertoires of memory thymus-derived cells (T cells) with cluster of differentiation (CD)- 4 among atomic-bomb (A-bomb) survivors. To evaluate the maintenance of T-cell memory within A-bomb survivors 60 years after radiation exposure, we examined functionally distinct memory CD4 T-cell subsets in the peripheral blood lymphocytes of the survivors.Three functionally different subsets of memory CD4 T cells were identified by differential CD43 expression levels and measured using flow cytometry. These subsets consist of functionally mature memory cells, cells weakly responsive to antigenic stimulation, and those cells functionally anergic and prone to spontaneous apoptosis.The percentages of these subsets within the peripheral blood CD4 T-cell pool all significantly increased with age. Percentages of functionally weak and anergic subsets were also found to increase with radiation dose, fitting to a log linear model. Within the memory CD4 T-cell pool, however, there was an inverse association between radiation dose and the percentage of functionally mature memory cells.These results suggest that the steady state of T cell memory, which is regulated by cell activation and/or cell survival processes in subsets, may have been perturbed by prior radiation exposure among A-bomb survivors.

Background: There are limited numbers of reports on the association of lymphotoxin-alpha (LTA) genotypes with gastric cancer. Methods: A nested case–control study was carried out in the longitudinal cohort of atomic bomb survivors using stored sera before diagnosis (mean, 2.3 years) and blood cells. Enrolled were 287 cases with noncardia gastric cancer of diffuse and intestinal types and three controls per case selected from cohort members matched on age, gender, city, and time and type of serum storage and counter-matched on radiation dose. Results: LTA 252GG and GA genotypes were associated with the prevalence of Helicobacter pylori IgG seropositivity and higher antibody titer against H. pylori cytotoxin-associated gene A (CagA) protein in controls and they were an independent risk factor for noncardia gastric cancer of diffuse type (RR = 2.8 (95% CI: 1.3–6.3), p = .01, and RR = 2.7 (95% CI: 1.5–4.8), p

Lung cancer is a leading cause of cancer death worldwide. Prevention could be improved by identifying susceptible individuals as well as improving understanding of interactions between genes and etiological environmental agents, including radiation exposure. The epidermal growth factor receptor (EGFR)-signaling pathway, regulating cellular radiation sensitivity, is an oncogenic cascade involved in lung cancer, especially adenocarcinoma. The cytosine adenine (CA) repeat number polymorphism in the first intron of EGFR has been shown to be inversely correlated with EGFR production. It is hypothesized that CA repeat number may modulate individual susceptibility to lung cancer. Thus, we carried out a case-cohort study within the Japanese atomic bomb (A-bomb) survivor cohort to evaluate a possible association of CA repeat polymorphism with lung cancer risk in radiation-exposed or negligibly exposed (5 mGy) A-bomb survivors. First, by dividing study subjects into Short and Long genotypes, defined as the summed CA repeat number of two allelesor = 37 andor = 38, respectively, we found that the Short genotype was significantly associated with an increased risk of lung cancer, specifically adenocarcinoma, among negligibly exposed subjects. Next, we found that prior radiation exposure significantly enhanced lung cancer risk of survivors with the Long genotype, whereas the risk for the Short genotype did not show any significant increase with radiation dose, resulting in indistinguishable risks between these genotypes at a high radiation dose. Our findings imply that the EGFR pathway plays a crucial role in assessing individual susceptibility to lung adenocarcinoma in relation to radiation exposure.

Using flow cytometry, we quantified the number of micronucleated reticulocytes in peripheral blood of whole-body X-irradiated mice in order to evaluate the radiation sensitivity and the induced genomic instability of the hematopoietic system. An acute effect of radiation dose as small as 0.1 Gy was detectable 2 days after irradiation, and the radiation dose effect was significantly greater in BALB/c mice than in C57BL/6 mice, that is, 3.0- and 2.3-fold increases in frequencies of micronuclei were noted in the two groups of mice, respectively. Even 1 year after irradiation, mice irradiated with 2.5 Gy of X-rays showed significantly increased frequencies of micronucleated reticulocytes, that is, 1.6- and 1.3-fold increases in BALB/c and C57BL/6 mice, respectively. However, this delayed effect was not apparent when the same mice were analyzed for T-cell receptor mutant frequencies in splenocytes. A significant mouse strain difference in the delayed radiation effect on micronucleated reticulocyte frequencies was noted as well. The results indicate that delayed genomic effects of irradiation on the murine hematopoietic system can persist in vivo for prolonged periods, and that there are mouse strain differences in sensitivity to radiation-induced genomic instability.

We have previously shown that natural cytotoxic activity of peripheral blood lymphocytes was inversely related to cancer development based on a prospective cohort study. The genetic fraction of cytotoxic activity needs to be clarified, identifying individuals immunogenetically susceptible to cancer. A case-control study within the cohort members was designed: 102 cancer cases with peripheral lymphocyte DNA available and three control groups, each of which consisted of 204 subjects with each tertile level of cytotoxic activity. We first compared two control groups with high and low cytotoxic activity in terms of the single nucleotide polymorphisms in the natural killer complex gene region on chromosome 12p, identifying the haplotype alleles that were associated with the activity. Next, cancer risks were assessed for these haplotypes. We found two haplotype blocks, each of which generated two major haplotype alleles: low-activity-related LNK1 (frequency 0.478 and 0.615 in groups with high and low activity, respectively; P < 0.00008) and high-activity-related HNK1 (0.480 and 0.348; P < 0.0001), LNK2 (0.711 and 0.821; P < 0.0002), and HNK2 (0.272 and 0.174; P < 0.0008). These NKG2D haplotype alleles showed a significant difference between cases (0.632 for LNK1 and 0.333 for HNK1) and controls (0.554 for LNK1 and 0.406 for HNK1). The haplotype HNK1/HNK1 revealed a decreased risk of cancer (odds ratio, 0.471; 95% confidence interval, 0.233-0.952) compared with LNK1/LNK1. Individuals who are genetically predisposed to have low or high natural cytotoxic activity can in part be determined by NKG2D haplotyping, which in turn reveals an increased or decreased risk of cancer development. (Cancer Res 2006; 66(1): 563-70)

It has previously been reported that hemizygous mutant fraction (Mf) at the glycophorin A (GPA) locus in erythrocytes increased with radiation dose in heterozygotes among Hiroshima and Nagasaki atomic bomb survivors. In the present study, we analyzed the relationship between GPA Mf and cancer risk using newly developed cancers among previously cancer-free subjects whose GPA Mf had been measured between 1988 and 1996. Among 1,723 survivors (1,117 in Hiroshima and 606 in Nagasaki), we identified 186 subjects who developed a first cancer by the end of 2000. We compared the radiation dose responses of GPA Mf between cancer and cancer-free groups using a linear-quadratic model fit by multiple regression analysis in combination with age, sex, and city. The slope of the GPA Mf dose-response curve was significantly higher in the cancer group than in the cancer-free group among Hiroshima subjects. Moreover, no significant difference of GPA Mf between cancer and cancer-free groups was found in unexposed controls in the two cities. The same conclusions were obtained using a linear dose-response model and by further analysis using Cox regression of cancer incidence. These findings suggest that there might be interindividual variation in mutability of somatic genes and that Hiroshima survivors who have higher mutability in response to radiation exposure would be expected to have a higher probability of suffering radiation-related cancer.

Suzuki, G., Shimada, Y., Hayashi, T., Akashi, M., Hirama, T. and Kusunoki, Y. An Association between Oxidative Stress and Radiation-Induced Lymphomagenesis. Radiat. Res. 161, 642–647 (2004). It is generally thought that reactive oxygen species (ROS) play an important role in carcinogenesis. However, direct evidence supporting this idea is still lacking. In the present study, we measured ROS in thymocytes at the thymic prelymphoma stage in C57BL/6 mice. Mice (n = 20) were irradiated at 1.6 Gy/week for 4 consecutive weeks and the levels of ROS were measured 8 to 11 weeks later by dehydrorhodamine 123, which accumulated in mitochondria and became fluorescent dye upon oxidation. Unirradiated littermates (n = 17) served as controls. Thymic prelymphoma cells were diagnosed by the aberrant CD4/CD8 staining profile and monoclonal or oligoclonal T-cell receptor gene rearrangement. A significant fraction of mice (11/13) bearing thymic prelymphoma cells exhibited elevated levels of ROS in thymocytes (P < 0....

Our previous studies have revealed a clear dose-dependent decrease in the percentage of naïve CD4 T cells that are phenotypically CD45RA+ in PBL among A-bomb survivors. However, whether there is a similar radiation effect on CD8 T cells has remained undetermined because of the unreliability of CD45 isoforms as markers of naïve and memory subsets among the CD8 T-cell population. In the present study, we used double labeling with CD45RO and CD62L for reliable identification of naïve and memory cell subsets in both CD4 and CD8 T-cell populations among 533 Hiroshima A-bomb survivors. Statistically significant dose-dependent decreases in the percentages of CD45RO-/CD62L+ naïve cells were found in the CD8 T-cell population as well as in the CD4 T-cell population. Furthermore, the percentages of CD45RO+/CD62L+ and CD45RO+/CD62L- memory T cells were found to increase significantly with increasing radiation dose in the CD8 T-cell population but not in the CD4 T-cell population. These results suggest that the prior A-bomb exposure has induced long-lasting deficits in both naïve CD4 and CD8 T- cell populations along with increased proportions of these particular subsets of the memory CD8 T-cell population.

The well-documented increases in malignant tumours in the A-bomb survivors have recently been supplemented by reports that non-cancer diseases, including cardiovascular disease, may also have increased in incidence with increasing radiation dose. Given that low-level inflammatory responses are widely accepted as a significant risk factor for such diseases, we undertook a detailed investigation of the long-term effects of ionizing radiation on the levels of the inflammatory markers C-reactive protein (CRP) and interleukin 6 (IL-6) in A-bomb survivors.Blood samples were taken from 453 participants in a long-term epidemiological cohort of A-bomb survivors. Plasma levels of CRP and IL-6 were measured using standard antibody-mediated procedures. Relationships between CRP or IL-6 levels and radiation dose were then investigated by multivariate regression analysis. Blood lymphocytes from each individual were used for immunophenotyping by flow cytometry with murine monoclonal antibodies to CD3, CD4 and CD8.CRP levels were significantly increased by about 31% Gy(-1) of estimated A-bomb radiation (p=0.0001). Higher CRP levels also correlated with age, male gender, body mass index and a history of myocardial infarction. After adjustments for these factors, CRP levels still appeared to have increased significantly with increasing radiation dose (about 28% increase at 1Gy, p=0.0002). IL-6 levels also appeared to have increased with radiation dose by 9.3% at 1Gy (p=0.0003) and after multiple adjustments by 9.8% at 1Gy (p=0.0007). The elevated CRP and IL-6 levels were associated with decreases in the percentages of CD4(+) helper T-cells in peripheral blood lymphocyte populations.Our results appear to indicate that exposure to A-bomb radiation has caused significant increases in inflammatory activity that are still demonstrable in the blood of A-bomb survivors and which may lead to increased risks of cardiovascular disease and other non-cancer diseases.

Significant decreases in the fraction of lymphocytes that are CD4(+) and increases in serum levels of some classes of immunoglobulin have been reported to occur in atomic bomb (A-bomb) survivors and in victims of the Chernobyl nuclear plant accident. To investigate the long-term effects of nuclear radiation on cellular immunity in more detail, we used limiting dilution assays with peripheral blood mononuclear cell preparations to analyze the T-cell responses of 251 A-bomb survivors exposed to less than 0.005 Gy and 159 survivors exposed to more than 1.5 Gy. The percentages of CD2-positive cells that were capable of proliferating in response to phytohemagglutinin (PHA) in the presence of exogenous interleukin 2 (IL2) did not differ substantially between distally exposed and more heavily exposed survivors. The heavily exposed survivors appeared to possess fewer T cells that were capable of proliferating in response to concanavalin A (Con A) or of producing interleukin 2. Assuming that CD4 T cells were the ones primarily responsible for producing IL2 in response to Con A, we were able to estimate how many cells in any given CD4 T-cell population were actually producing IL2. The results indicated that peripheral blood samples from heavily exposed survivors contained significantly fewer IL2-producing CD4 T cells than did similar samples from distally exposed survivors, indicating that significant exposure to A-bomb radiation may have a long-lasting negative effect on the capacity of CD4 T-cell populations to produce IL2.

Reduction of the naive T-cell population represents a deteriorating state in the immune system that occurs with advancing age. In animal model studies, obesity compromises the T-cell immune system as a result of enhanced adipogenesis in primary lymphoid organs and systemic inflammation. In this study, to test the hypothesis that obesity may contribute to the aging of human T-cell immunity, a thousand atomic-bomb survivors were examined for obesity status and ability to produce naive T cells, i.e., T-cell receptor excision circle (TREC) numbers in CD4 and CD8 T cells. The number of TRECs showed a strong positive correlation with naive T cell numbers, and lower TREC numbers were associated with higher age. We found that the TREC number was inversely associated with levels of obesity indicators (BMI, hemoglobin A1c) and serum CRP levels. Development of type-2 diabetes and fatty liver was also associated with lower TREC numbers. This population study suggests that obesity with enhanced inflammation is involved in aging of the human T-cell immune system. Given the fact that obesity increases the risk of numerous age-related diseases, attenuated immune competence is a possible mechanistic link between obesity and disease development among the elderly.

Inflammatory markers have been associated with increased risk of several cancers, including colon, lung, breast and liver, but the evidence is inconsistent. We conducted a nested case-control study in the longitudinal cohort of atomic-bomb survivors. The study included 224 hepatocellular carcinoma (HCC) cases and 644 controls individually matched to cases on gender, age, city and time and method of serum storage, and countermatched on radiation dose. We measured C-reactive protein (CRP) and interleukin (IL)-6 using stored sera obtained within 6 years before HCC diagnosis from 188 HCC cases and 605 controls with adequate volumes of donated blood. Analyses with adjustment for hepatitis virus infection, alcohol consumption, smoking habit, body mass index (BMI) and radiation dose showed that relative risk (RR) of HCC [95% confidence interval (CI)] in the highest tertile of CRP levels was 1.94 (0.72-5.51) compared to the lowest tertile (p = 0.20). RR of HCC (95% CI) in the highest tertile of IL-6 levels was 5.12 (1.54-20.1) compared to the lowest tertile (p = 0.007). Among subjects with BMI25.0 kg/m(2) , a stronger association was found between a 1-standard deviation (SD) increase in log IL-6 and HCC risk compared to subjects in the middle quintile of BMI (21.3-22.9 kg/m(2) ), resulting in adjusted RR (95% CI) of 3.09 (1.78-5.81; p = 0.015). The results indicate that higher serum levels of IL-6 are associated with increased HCC risk, independently of hepatitis virus infection, lifestyle-related factors and radiation exposure. The association is especially pronounced among subjects with obesity.

Chemical pollutants in the Great Lakes have found their way through the food chain into humans because of their environmental persistence and lipophilicity. Some epidemiological studies have claimed an association between metabolites of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT), polychlorinated biphenyls (PCBs), and polybrominated biphenyls (PBBs) and breast cancer, but others have reported no such association. We examined various halogenated hydrocarbons for their capacity to inhibit gap junctional intercellular communication (GJIC) in normal human breast epithelial cells (HBEC) when given as single compounds or as mixtures. The scrape-loading/dye transfer and fluorescent redistribution after photobleaching techniques were used to measure GJIC; immunostaining and Western and Northern analyses were performed on connexin 43 (Cx43) gap junction protein and message to determine how halogenated hydrocarbons might affect GJIC. DDT, dieldrin, and toxaphene inhibited GJIC in a dose-responsive manner after 90 min treatments. Dieldrin suppressed GJIC within 30 min with no recovery after 24 hr. Inhibition of GJIC by DDT and toxaphene was partially restored after 12 hr and fully restored after 24 hr. Several PCB and PBB congeners inhibited GJIC in a dose-responsive and time-dependent manner, but GJIC was almost restored to control values 24 hr after exposure. The highest concentrations of the individual chemicals that did not inhibit GJIC was determined, and mixtures containing two of these chemicals were tested for their ability to inhibit GJIC. Significant inhibition of GJIC was observed when cells were treated with a mixture of DDT and 2,4,5-hexachlorobiphenyl (2,4,5-HCB), dieldrin and 2,4,5-HCB, or dieldrin and 2,4,5-hexabromobiphenyl (2,4,5-HBB). These results indicate that halogenated hydrocarbons, alone or in specific combinations, can alter GJIC at the post-translational level. These results are consistent with the hypothesis that DDT, dieldrin, toxaphene, 2,3,4-HCB, 2,4,5-HCB, and 2,4,5-HBB could have tumor-promoting potential in human breast tissue. Images Figure 1. A Figure 1. B Figure 1. C Figure 1. D Figure 1. E Figure 1. F Figure 2. A Figure 2. B Figure 2. C Figure 2. D Figure 2. E Figure 2. F Figure 3. Figure 4. A Figure 4. B Figure 4. C Figure 4. D Figure 4. E Figure 4. F Figure 4. G Figure 4. H Figure 5. A Figure 5. B Figure 5. C Figure 6. Figure 6.

Bloom's syndrome (BS) is an autosomal recessive disorder conferring high propensity for cancer and displaying a high degree of genetic instability; the frequency of sister chromatid exchange is characteristically 10 times above background. The symmetrical four‐armed chromatid interchanges are much more readily detected in peripheral blood lymphocytes of BS patients, suggesting that the frequency of somatic recombination is also increased. In the present study, the rate of spontaneous loss of HLA‐A allele expression was estimated following fluctuation analysis in cultured T lymphocytes using a flow‐cytometric assay. It was found to be 10 times or more higher than normal in lymphocytes from a BS patient. Molecular and chromosome analyses showed that all 13 independent variants from the patient were most likely derived from somatic recombinations. Further tests for loss of heterozygosity at a closely linked proximal locus, HLA‐DQA1, showed that as many as half of the recombinants retained heterozygosity irrespective of the donor. The results suggest that the HLA region is hyperrecombinogenic in somatic cells and that the elevated recombination rate in BS cells results from the general increase at ordinary sites and not from random creation of unusual sites for recombination.

NKG2D is a primary activating receptor that triggers cell-mediated cytotoxicity in NK cells against tumor and virus-infected cells. We previously identified the NKG2D haplotypes in the natural killer gene complex region on chromosome 12p. Two major haplotype alleles, LNK1 and HNK1, were closely related to low and high natural cytotoxic activity phenotypes, respectively. Furthermore, the haplotype of HNK1/HNK1 has revealed a decreased risk of cancer compared with LNK1/LNK1. In the present study, using flow cytometry, we evaluated the functional effects of NKG2D haplotypes and five htSNPs in terms of the cell-surface expression of NKG2D protein on NK and CD8 T cells of peripheral blood among 732 atomic-bomb survivors. NKG2D expression on NK cells showed significant increases, in the order of LNK1/LNK1, LNK1/HNK1 and HNK1/HNK1 haplotypes (p for trend = 0.003), or with major homozygous, heterozygous, and minor homozygous genotypes for individual htSNPs (p for trend = 0.02–0.003). The same trend was observed for NKG2D expression on CD8 T cells. Our findings indicate that the NKG2D haplotypes are associated with the expression levels of NKG2D protein on NK and CD8 T cells, resulting in inter-individual variations in human cytotoxic response.

Levels of exposure to ionizing radiation are increasing for women worldwide due to the widespread use of CT and other radiologic diagnostic modalities. Exposure to ionizing radiation as well as increased levels of estradiol and other sex hormones are acknowledged breast cancer risk factors, but the effects of whole-body radiation on serum hormone levels in cancer-free women are unknown. This study examined whether ionizing radiation exposure is associated with levels of serum hormones and other markers that may mediate radiation-associated breast cancer risk. Serum samples were measured from cancer-free women who attended biennial health examinations with a wide range of past radiation exposure levels (N = 412, ages 26-79). The women were selected as controls for separate case-control studies from a cohort of A-bomb survivors. Outcome measures included serum levels of total estradiol, bioavailable estradiol, testosterone, progesterone, prolactin, insulin-like growth factor-1 (IGF1), insulin-like growth factor-binding protein 3 (IGFBP-3), and ferritin. Relationships were assessed using repeated-measures regression models fitted with generalized estimating equations. Geometric mean serum levels of total estradiol and bioavailable estradiol increased with 1 Gy of radiation dose among samples collected from postmenopausal women (17%(1Gy), 95% CI: 1%-36% and 21%(1Gy), 95% CI: 4%-40%, respectively), while they decreased in samples collected from premenopausal women (-11%(1Gy), 95% CI: -20%-1% and -12%(1Gy), 95% CI: -20%- -2%, respectively). Interactions by menopausal status were significant (P = 0.003 and P0.001, respectively). Testosterone levels increased with radiation dose in postmenopausal samples (30.0%(1Gy), 95% CI: 13%-49%) while they marginally decreased in premenopausal samples (-10%(1Gy), 95% CI: -19%-0%) and the interaction by menopausal status was significant (P0.001). Serum levels of IGF1 increased linearly with radiation dose (11%(1Gy), 95% CI: 2%-18%) and there was a significant interaction by menopausal status (P = 0.014). Radiation-associated changes in serum levels of estradiol, bioavailable estradiol, testosterone and IGF1 were modified by menopausal status at the time of collection. No associations with radiation were observed in serum levels of progesterone, prolactin, IGFBP-3 or ferritin.

We have previously reported 2 cases of healthy men showing in vivo monoclonal expansion of mature CD4- CD8- alpha beta T cells. In the present study, an additional 3 adults were found to exhibit such an expansion, among a total 464 adult donors studied. These 5 individuals were otherwise physiologically normal, with no history of severe illness and autoimmune disease at the time of examination. To investigate the mechanisms of the clonal expansion, further characterization of the clonal cells was attempted. No apparent preference for usage of the T cell receptor beta chain variable region was observed in the clonal T cells. These clonal T cells showed lectin-dependent or redirected antibody-dependent cell-mediated cytotoxicities, whereas they could not lyse autologous lymphoblastoid cell lines. Failure of Fas antigen expression was not observed for any of these clones. These results suggest that clonal expansion of CD4- CD8- alpha beta T cells frequently occurs in the periphery without any T cell abnormalities.

In this paper we summarize the long-term effects of A-bomb radiation on the T-cell system and discuss the possible involvement of attenuated T-cell immunity in the disease development observed in A-bomb survivors. Our previous observations on such effects include impaired mitogen-dependent proliferation and IL-2 production, decreases in naive T-cell populations, and increased proportions of anergic and functionally weak memory CD4 T-cell subsets. In addition, we recently found a radiation dose-dependent increase in the percentages of CD25(+)/CD127(-) regulatory T cells in the CD4 T-cell population of the survivors. All these effects of radiation on T-cell immunity resemble effects of aging on the immune system, suggesting that ionizing radiation might direct the T-cell system toward a compromised phenotype and thereby might contribute to an enhanced immunosenescence. Furthermore, there are inverse, significant associations between plasma levels of inflammatory cytokines and the relative number of naïve CD4 T cells, also suggesting that the elevated levels of inflammatory markers found in A-bomb survivors can be ascribed in part to T-cell immunosenescence. We suggest that radiation-induced T-cell immunosenescence may result in activation of inflammatory responses and may be partly involved in the development of aging-associated and inflammation-related diseases frequently observed in A-bomb survivors.

Patients who received hematopoietic cell transplants have an increased risk for a new malignancy. In addition to genotoxic regimens such as radiotherapy and chemotherapy, graft-versus-host disease (GVHD) is a risk factor for development of new malignancies in long-term survivors. To understand mechanisms underlying this malignant transformation, we evaluated genomic damage in several murine models of GVHD by enumerating reticulocytes containing micronuclei (MN) in the blood after semi-allogeneic (parent-into-F1) hematopoietic cell transplantation. On day 40 after transplantation, MN frequencies were significantly increased in unirradiated (C57BL6 x DBA/2) F1 (BDF1) and (BALB/c x C57BL6) F1 (CBF1) mice that received cells from C57BL6 (B6) donors. MN frequencies were not significantly increased in F1 mice that received cells from DBA/2 or BALB/c donors. Serum levels of tumor necrosis factor-alpha (TNF-alpha) were higher after transplantation with B6 donors than with DBA/2 or BALB/c donors. The results indicate that GVHD, without irradiation, can induce genomic damage associated with inflammatory reactions manifested by increased TNF-alpha levels.

Background: Although a number of reports regarding the role of reactive oxygen species (ROS) as the first step in cancer induction exist, few studies have investigated how vitamin C influences ROS in human plasma. Aim of the study: Using the ROS assay system, a method recently established by one of the authors, we aimed to evaluate the effect of vitamin C supplementation on serum ROS among subjects diagnosed with chronic gastritis. Methods: A total of 244 Japanese subjects with atrophic gastritis were randomized to take 5-year supplementation of either 50 mg or 500 mg of vitamin C. Results: The adjusted difference in the changes of total ROS between baseline and after 5-year supplementation was statistically significant between the intervention groups: 2.70 decrease (corresponds to 1.26% decrease) in the high-dose group and 4.16 increase (corresponds to 3.79% increase) in the low-dose group, p for difference = 0.01. Conclusion: Vitamin C was suggested to reduce oxidative stress among subjects with atrophic gastritis.

Although it has been suggested that cardiovascular disease incidence is increased among atomic bomb survivors, the existence of a causal relationship between radiation exposure and atherosclerosis is unclear. Microbial infections, including those caused by Chlamydia pneumoniae, Helicobacter pylori and cytomegalovirus, have recently been implicated in atherosclerosis. Since immune function is somewhat impaired among atomic bomb survivors, their immune defense against such infections might be diminished. To investigate this possibility, we measured antibody levels to the above microorganisms in the sera of survivors. We found that the levels of IgG and IgA antibodies to Chlamydia pneumoniae decreased significantly with radiation dose, whereas the levels of IgG antibodies to Helicobacter pylori or cytomegalovirus remained unchanged. The inflammation marker C-reactive protein was significantly and positively associated with level of antibodies to Chlamydia pneumoniae only in heavily exposed (or=1000 mGy) survivors. These results may suggest that among atomic bomb survivors, immune response to Chlamydia pneumoniae is diminished and chronic inflammatory reactions related to Chlamydia pneumoniae infection are present.

i b 2 P 1 f c h w i o i d h b w a Atomic bomb survivors have a persistently increased risk f cancer, hepatitis, and cardiovascular and autoimmune iseases. There is no clear explanation for these late ffects of radiation exposure. One hypothesis is that radiaion causes chronic low-grade inflammation, with elevated irculating levels of cytokines. Proinflammatory cytokines, uch as interleukin 6, tumor necrosis factor (TNF) , and nterferon , and anti-inflammatory cytokines, such as inerleukin 10, are synthesized predominantly by macrohages and lymphocytes, and regulate the inflammatory esponse. Interleukin 6 in turn induces the synthesis of cute-phase plasma proteins, such as C-reactive protein. ncreased levels of inflammatory cytokines, even within the ormal range, have been associated with an increased risk of ardiovascular disease. Chronic low-grade inflammation ay also influence the production of immunoglobulins by B ells.

To study the long-term effects of radiation-induced T-cell depletion on the T-cell receptor (TCR) Vbeta repertoires of human peripheral CD4 T-cell populations, we measured the percentages of CD4 T cells representing each of the full range of possible TCR Vbeta families in a cohort of atomic bomb survivors. We then estimated the extent to which the expression levels for individual TCR Vbeta families differed from the average expression level for that particular TCR Vbeta family across the entire cohort. We found no evidence of a systematic change in the TCR Vbeta repertoires of the naïve CD4 T-cell populations, but memory CD4 T-cell TCR Vbeta family expression levels diverged significantly from the population average for counterpart families, especially in individuals who had been exposed to higher doses and were at least 20 years of age at the time of the bombing. Comparisons of the TCR Vbeta family expression profiles in the naïve and memory CD4 T-cell pools of the same group of adult survivors revealed that differences in the TCR Vbeta repertoires of these two types of CD4 T-cell pool were larger in more heavily exposed survivors than in unexposed controls. These findings suggest that the memory CD4 T-cell pools of individuals who received significant radiation doses in adulthood may well have become (and could still be) dependent upon a much less diverse complement of TCR Vbeta families than would otherwise have been the case.

We examined radiation effects on the relationship between diabetes development and genetic background in atomic-bomb (A-bomb) survivors. Our main aim in this study was to shed light on the role of genetic background in diabetes onset among A-bomb survivors by studying possible relationships between human leukocyte antigen (HLA) genotypes and the diabetes in patients and controls. We examined the effects of different HLA haplotypes on type 2 diabetes development by determining the DQA1 and DRB1 alleles of Hiroshima A-bomb survivors (111 diabetic patients and 774 controls) using the polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) method. We noted an increased risk of diabetes in the higher dose group among these patients (trend p = 0.001). The risk of the most heavily exposed group was significantly higher than that of the unexposed group or the low-dose group especially in survivors with the DQA1*03-DRB1*09 or DQA1*0401-DRB1*08 haplotypes (trend p = 0.002 or p = 0.05, respectively). By contrast, in people with other haplotypes, the risk did not increase significantly with increasing dose. These results suggest that individuals with specific HLA haplotypes may have an increased risk of diabetes with increased-dose categories.

We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer naïve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (naïve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (naïve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their naïve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.

Many studies have suggested a relationship between certain alleles of the human leukocyte antigen (HLA) and the prevalence of some diseases or the immunological responsiveness to certain antigens. Furthermore, our studies in the past have demonstrated decreased immune function among atomic-bomb survivors who were exposed to high doses of radiation. However, no studies have addressed the possibility of various degrees of radiation-induced immune suppression being dependent on HLA type. To investigate the possibility of differing frequency distributions of HLA type in the Hiroshima atomic bomb survivors, HLA-DQA1 alleles and HLA-DR antigens were typed for 291 survivors in a high-dose group (>1.5 Gy), 339 survivors in an intermediate-dose group (0.005-1.5 Gy), and 388 in a distally exposed control group (

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 microg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 microg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 microg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 microg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 microg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing 'initiated' cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.

A sensitive enzyme immunoassay for human epidermal growth factor (hEGF) is described. The anti-hEGF antibody was prepared by immunizing rabbits with hEGF, which was synthesized by Escherichia coli using the genetic engineering technique. The present assay system was based on the sandwiching of an antigen between anti-hEGF F(ab')2 precoated on a 96-well polystyrene plate and beta-D-galactosidase-labeled anti-hEGF Fab'. The range of measurable hEGF by this assay was 0.1-100 pg/well. Recoveries of hEGF added to serum and urine ranged between 94 and 108%. The intra- and inter-assay coefficients of variation were less than 6 and 8%, respectively. The results obtained by this assay method correlated well with those obtained by the radioimmunoassay method. By using this assay, the time course of serum hEGF levels in mice after the various administrations were also examined.