Masayuki Hakoda

Radiation dose-dependent increases in inflammatory response markers in A-bomb survivors

Purpose : 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. Materials and methods : 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. Results : 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. Conclusions : 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.

Increased somatic cell mutant frequency in atomic bomb survivors.

Frequencies of mutant T-cells in peripheral blood, which are deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT) activity, were determined for atomic bomb survivors by direct clonal assay using a previously reported method (Hakoda et al., 1987). Results from 30 exposed survivors (more than 1 rad exposed) and 17 age- and sex-matched controls (less than 1 rad exposed) were analyzed. The mean mutant frequency (Mf) in the exposed (5.2 X 10(-6); range 0.8-14.4 X 10(-6)) was significantly higher than in controls (3.4 X 10(-6); range 1.3-9.3 X 10(-6)), which was not attributable to a difference in non-mutant cell-cloning efficiencies between the 2 groups, which were virtually identical. An initial analysis of the data did not reveal a significant correlation between individual Mfs and individual radiation dose estimates when the latter were defined by the original, tentative estimates (T65D), even though there was a significant positive correlation of Mfs with individual frequency of lymphocytes bearing chromosome aberrations. However, reanalysis using the newer revised individual dose estimates (DS86) for 27 exposed survivors and 17 controls did reveal a significant but shallow positive correlation between T-cell Mf values and individual exposure doses. These results indicate that HPRT mutation in vivo in human T-cells could be detected in these survivors 40 years after the presumed mutational event.

Optimal Range of Serum Urate Concentrations to Minimize Risk of Gouty Attacks During Anti-Hyperuricemic Treatment

To find an optimal range of urate concentrations wherein the risk of attacks during the initial 6 months of treatment is minimized, data from 350 gouty patients treated with anti-hyperuricemic drugs were retrospectively analyzed. We determined the optimal range of urate concentrations to be 4.6-6.6 mg/dl. If urate concentrations were within this range, the risk ratio of an attack as opposed to outside of the range was 0.705 (95% confidence interval, 0.629-0.791). The increase (or decrease) in urate concentration in one month associated with minimal risk of gouty attacks was also determined. The lowest risk ratio of attack (0.451) occurred at a range of -0.1 to 0.6 mg/dl/month increase in urate concentrations (95% confidence interval, 0.310-0.655). In conclusion, we propose that urate concentrations during the initial 6 months of anti-hyperuricemic therapy should be maintained within a range of 4.6-6.6 mg/dl, and reduction in the urate concentrations during treatment should be as slow as possible.

Measurement of in vivo HGPRT-deficient mutant cell frequency using a modified method for cloning human peripheral blood T-lymphocytes.

Approximately 80% of human peripheral blood T-lymphocytes could be cloned in the presence of crude interleukin-2, phytohemagglutinin, and X-irradiated autologous lymphocytes and Raji B-cells. This modified cloning method was used to measure the in vivo frequency of HGPRT-deficient mutant T-lymphocytes. Repeated experiments using blood from the same individuals revealed that the frequency of mutant cells was almost constant for each individual even though the cloning efficiency of lymphocytes varied somewhat from experiment to experiment. Approximately 80% of both wild-type unselected and 6-thioguanine-resistant colonies had helper/inducer and about 20% had suppressor/cytotoxic T-lymphocyte markers. No difference was observed in the distribution of lymphocyte subsets in relation to colony type.

Mortality of rheumatoid arthritis in Japan: a longitudinal cohort study.

Objective: To determine the mortality risk of Japanese patients with rheumatoid arthritis, taking into account lifestyle and physical factors, including comorbidity. Methods: 91 individuals with rheumatoid arthritis were identified during screening a cohort of 16 119 Japanese atomic bomb survivors in the period 1958 to 1966. These individuals and the remainder of the cohort were followed for mortality until 1999. Mortality risk of the rheumatoid patients was estimated by the Cox proportional hazards model. In addition to age and sex, lifestyle and physical factors such as smoking status, alcohol consumption, blood pressure, and comorbidity were included as adjustment factors for the analysis of total mortality and for analysis of mortality from each cause of death. Results: 83 of the rheumatoid patients (91.2%) and 8527 of the non-rheumatoid controls (52.9%) died during mean follow up periods of 17.8 and 28.0 years, respectively. The age and sex adjusted hazard ratio for mortality in the rheumatoid patients was 1.60 (95% confidence interval, 1.29 to 1.99), p<0.001. Multiple adjustments, including for lifestyle and physical factors, resulted in a similar mortality hazard ratio of 1.57 (1.25 to 1.94), p<0.001. Although mortality risk tended to be higher in male than in female rheumatoid patients, the difference was not significant. Pneumonia, tuberculosis, and liver disease were significantly increased as causes of death in rheumatoid patients. Conclusions: Rheumatoid arthritis is an independent risk factor for mortality. Infectious events are associated with increased mortality in rheumatoid arthritis.

Low-Positive Antibody Titer against Helicobacter pylori Cytotoxin-Associated Gene A (CagA) May Predict Future Gastric Cancer Better Than Simple Seropositivity against H. pylori CagA or against H. pylori

Background: To investigate the IgG antibody titer against Helicobacter pylori CagA as a risk factor for future noncardia gastric cancer. Methods: A nested case-control study was done in the longitudinal cohort of atomic bomb survivors using stored sera before diagnosis (mean, 2.3 years). Enrolled were 299 cancer cases and 3 controls per case selected from cohort members matched on age, gender, city, and time and type of serum storage and countermatched on radiation dose. Results: H. pylori IgG seropositive with CagA IgG low titer was the strongest risk factor for noncardia gastric cancer [relative risk (RR), 3.9; 95% confidence interval (95% CI), 2.1-7.0; P < 0.001], especially for intestinal-type tumor (RR, 9.9, 95% CI, 3.5-27.4; P < 0.001), compared with other risk factors, H. pylori IgG seropositive with CagA IgG negative (RR, 2.2; 95% CI, 1.3-3.9; P = 0.0052), H. pylori IgG seropositive with CagA IgG high titer (RR, 2.0; 95% CI, 1.3-3.2; P = 0.0022), chronic atrophic gastritis (RR, 2.4; 95% CI, 1.8-3.3; P < 0.001), current smoking (RR, 2.3; 95% CI, 1.4-3.5; P < 0.001), or radiation dose (RR, 2.1; 95% CI, 1.2-3.1; P = 0.00193). Current smoking showed significantly higher risk for diffuse-type than intestinal-type tumors (P = 0.0372). Radiation risk was significant only for nonsmokers, all noncardia, and diffuse-type gastric cancers. Conclusions: A low CagA IgG titer is a useful biomarker to identify a high-risk group and it also provides a clue to understanding host-pathogen interaction. (Cancer Epidemiol Biomarkers Prev 2007;16(6):1224–8)

T-Cell Responses to Mitogens in Atomic Bomb Survivors: A Decreased Capacity to Produce Interleukin 2 Characterizes the T Cells of Heavily Irradiated Individuals

Abstract Kusunoki, Y., Hayashi, T., Morishita, Y., Yamaoka, M., Maki, M., Hakoda, M., Kodama, K., Bean, M. A. and Kyoizumi, S. T-Cell Responses to Mitogens in Atomic Bomb Survivors: A Decreased Capacity to Produce Interleukin 2 Characterizes the T Cells of Heavily Irradiated Individuals. 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.

Somatic mutation in peripheral lymphocytes of former workers at the Okunojima poison gas factory.

The former workers at the Okunojima poison gas factory comprise a high risk group for malignant tumors such as respiratory tract cancer. Demonstration of injury to somatic cell genes in this group may provide important data for evaluating the association between mustard gas and malignant tumors. So we measured the frequency of T lymphocytes lacking the hypoxanthine guanine phosphoribosyl transferase (HGPRT) activity, by cloning with interleukin 2 (IL2). In this study, we performed cloning of T lymphocytes lacking the HGPRT activity using recombinant IL2 (rIL2) and observed an increased frequency of somatic mutation in poison gas workers who had had more chances to be exposed to mustard gas and those who had worked for a longer period. This result suggested that inhalation of small amounts of mustard gas damaged somatic cell genes, resulting in carcinogenesis.

Urine alkalization facilitates uric acid excretion

BackgroundIncrease in the incidence of hyperuricemia associated with gout as well as hypertension, renal diseases and cardiovascular diseases has been a public health concern. We examined the possibility of facilitated excretion of uric acid by change in urine pH by managing food materials.MethodsWithin the framework of the Japanese governments health promotion program, we made recipes which consist of protein-rich and less vegetable-fruit food materials for H+-load (acid diet) and others composed of less protein but vegetable-fruit rich food materials (alkali diet). Healthy female students were enrolled in this consecutive 5-day study for each test. From whole-day collected urine, total volume, pH, organic acid, creatinine, uric acid and all cations (Na+,K+,Ca2+,Mg2+,NH4+) and anions (Cl-,SO42-,PO4-) necessary for the estimation of acid-base balance were measured.ResultsUrine pH reached a steady state 3 days after switching from ordinary daily diets to specified regimens. The amount of acid generated ([SO42-] +organic acid-gut alkai) were linearly related with those of the excretion of acid (titratable acidity+ [NH4+] - [HCO3-]), indicating that H+ in urine is generated by the metabolic degradation of food materials. Uric acid and excreted urine pH retained a linear relationship, where uric acid excretion increased from 302 mg/day at pH 5.9 to 413 mg/day at pH 6.5, despite the fact that the alkali diet contained a smaller purine load than the acid diet.ConclusionWe conclude that alkalization of urine by eating nutritionally well-designed food is effective for removing uric acid from the body.

Selection against blood cells deficient in hypoxanthine phosphoribosyltransferase (HPRT) in Lesch-Nyhan heterozygotes occurs at the level of multipotent stem cells

Lesch-Nyhan syndrome is caused by a severe genetic deficiency of hypoxanthine phosphoribosyltransferase (HPRT) and is characterized by central nervous system disorders, gout, and in some cases, macrocytic anemia. Women heterozygous for HPRT deficiency are healthy but their somatic cells are mosaic for enzyme deficiency owing to random inactivation of the X chromosome. Frequencies of red blood cells and T cells deficient in HPRT are significantly lower than the expected 50% in heterozygotes, suggesting that HPRT-negative blood cells are selected against in heterozygotes. To determine at which stage of hematopoiesis such selection occurs, we determined the frequencies of HPRT-negative T, B and erythroid precursor cells in three heterozygotes. Since the cloning efficiencies of T and B cells and colony forming efficiency of burst-forming unit erythroid (BFU-E) for sample from Lesch-Nyhan patients were similar to those of normal cells, HPRT deficiency does not seem to render the differentiated cells less efficient for proliferation. However, the frequencies of HPRT-negative T and B cells, and BFU-E were all less than 10% in each of the three heterozygotes. Although the frequencies of HPRT-negative cells showed tenfold variations between the heterozygotes, each heterozygote had similar frequencies of HPRT-negative cells in the three cell types. These results suggest that HPRT is important at early stages of hematopoiesis, but less so after the cells have differentiated into T cells, B cells and erythroid precursor cells.