A 5-aza-2'-deoxycytidine/valproate combination induces cytotoxic T-cell response against mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive tumour with a limited response to conventional therapy. The aim of this study was to evaluate the anticancer effect of a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-azaCdR), and two histone deacetylase inhibitors, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA). Human mesothelioma cells were treated with each epigenetic drug, either alone or in combinations. The cytotoxic effects on treated cells and the expression of specific tumour antigens were evaluated. The recognition of treated cells by a specific CD8+ T-cell clone was also measured. Additionally, the effect of combined treatments was tested in a murine model of mesothelioma. We showed that VPA and SAHA synergised with 5-azaCdR to kill MPM cells and induce tumour antigen expression in the remaining living tumour cells. As a consequence, tumour cells expressing these antigens were recognised and lysed by specific CD8+ cytotoxic T-cells. In vivo, treatment with 5-azaCdR/VPA inhibited tumour growth, and promoted lymphocyte infiltration and an immune response against tumour cells. Appropriate epigenetic drug combinations, in addition to inducing mesothelioma cell death, also affect the immunogenic status of these cells. This property could be exploited in clinical investigations to develop MPM treatments combining chemotherapeutic and immunotherapeutic approaches.

[Mortality in nuclear workers of the French electricity company: period 1968-2003]. / Cohorte des travailleurs du nucléaire à Electricité de France: mortalité des agents statutaires sur la période 1968-2003.

BACKGROUND: We conducted a mortality study on a cohort of French nuclear workers employed at Electricité de France (EDF). A first cancer mortality analysis had covered the period 1968-1994. This paper presents results from a mortality analysis including nine additional years of follow-up to cover workers employed from 1968 to 2003. METHODS: The cohort includes 22393 workers, 97% of whom are males. Employment data were updated using the EDF personnel file. Vital status was ascertained using the French National Registry of Population, and further completed using EDF personnel and pension files. Causes of death were obtained from the National registry of causes of death. Standardised Mortality Ratios (SMR) were computed using national rates as references. Variations of all causes and all cancers SMRs were studied according to demographic and occupational characteristics. RESULTS: At the study end point (31/12/2003), 74% of workers are still in active employment. Only 0.3% of workers are lost to follow-up. The median duration of follow-up is 20 years. Causes are ascertained for 96% of deaths. The total number of deaths is 874, 307 of which are cancer deaths. SMRs for all causes and cancers show a significant deficit compared to the French national mortality. No significant excess was observed for any of the cancer sites studied. Non-significant excesses are observed for pancreatic, pleural, kidney and brain cancer. Significant variations of all causes SMRs according to age at study entry and attained age are observed. Significant variations of all causes and all cancers SMRs according to diploma at employment are observed, with a reduced SMR for a higher level of diploma. CONCLUSION: There is a significant deficit of mortality compared to the general population, reflecting a strong Healthy Worker Effect. Although nine years of follow-up were added, this cohort is made up of young workers, most of whom are still in active service. Regular updating of the follow up of this cohort is planned, aiming for an occupational health surveillance of workers occupationally exposed to ionizing radiation in France.

Mortality risk in the French cohort of uranium miners: extended follow-up 1946-1999.

OBJECTIVES: This paper presents the risk of death from lung cancer and from other causes of death for the French cohort of uranium miners through 1999 and estimates associations with radon exposure. METHODS: The cohort includes men employed as uranium miners for at least 1 year between 1946 and 1990. For each miner, vital status and cause of death were obtained from the national registry, and radon exposure was reconstructed for each year. Standardised mortality ratios were computed with national mortality rates as references. Exposure-risk relationships were estimated by Poisson regression, with a linear excess relative risk (ERR) model and a 5-year lag. RESULTS: The cohort included 5086 miners and 153 063 person-years of exposure. The mean duration of follow-up was 30.1 years. In all 4140 miners exposed to radon, the average cumulative exposure was 36.6 working level months (WLM). There were 1411 deaths of miners <85 years of age. The miners did not differ significantly in overall mortality from the general male population. The analysis confirmed an excess risk of lung cancer death (n = 159; SMR = 1.43; 95% CI: 1.22 to 1.68), which increased significantly with cumulative radon exposure (ERR per 100 WLM = 0.71; 95% CI: 0.29 to 1.35). The ERR per unit exposure was much higher after 1955, when the accuracy of exposure measurement improved substantially (ERR per 100 WLM = 2.00; 95% CI: 0.91 to 3.65). A significant excess of kidney cancer deaths was observed (n = 20; SMR = 2.0; 95% CI: 1.22 to 3.09), which was not associated with cumulative radon exposure. No excess was observed for other causes of death, except silicosis (n = 23; SMR = 7.12; 95% CI: 4.51 to 10.69). CONCLUSIONS: The analysis confirmed the excess risk of death from lung cancer associated with low radon exposure. An excess risk of death from kidney cancer was also observed, apparently not associated with cumulative radon exposure.

Dose conversion of radon exposure according to new epidemiological findings.

In 1993, ICRP-65 recommended that dose conversion of radon exposure should be based on the comparison of detriments between radon exposure and effective dose. The lifetime detriment from the radon exposure was projected according to the epidemiological studies of uranium miners then available. The projection model (GSF) was multiplicative with temporal and age-at-exposure modification. Since 1993, new studies of uranium miners have appeared and many original studies were updated. In addition, projections of the risk have been improved by including further modifying factors as for instance in BEIR VI. New analyses were completed in the Czech and French studies of uranium miners with accurate estimates of exposures based on extensive radon measurements. The resulting estimates of excess absolute lifetime risk per unit exposure in working level months (WLM) from these models lead to dose conversion of 10 mSv WLM(-1) for the BEIR VI model and 8 mSv WLM(-1) for the joint Czech-French model in contrast to the conversion of 5 mSv WLM(-1) for the GSF model.

Mortality from diseases other than cancer following low doses of ionizing radiation: results from the 15-Country Study of nuclear industry workers.

BACKGROUND: Ionizing radiation at very high (radio-therapeutic) dose levels can cause diseases other than cancer, particularly heart diseases. There is increasing evidence that doses of the order of a few sievert (Sv) may also increase the risk of non-cancer diseases. It is not known, however, whether such effects also occur following the lower doses and dose rates of public health concern. METHODS: We used data from an international (15-country) nuclear workers cohort study to evaluate whether mortality from diseases other than cancer is related to low doses of external ionizing radiation. Analyses included 275 312 workers with adequate information on socioeconomic status, over 4 million person-years of follow-up and an average cumulative radiation dose of 20.7 mSv; 11 255 workers had died of non-cancer diseases. RESULTS: The excess relative risk (ERR) per Sv was 0.24 [95% CI (confidence intervals) -0.23, 0.78] for mortality from all non-cancer diseases and 0.09 (95% CI -0.43, 0.70) for circulatory diseases. Higher risk estimates were observed for mortality from respiratory and digestive diseases, but confidence intervals included zero. Increased risks were observed among the younger workers (attained age <50 years, identified post hoc) for all groupings of non-cancer causes of death, including external causes. It is unclear therefore whether these findings reflect real effects of radiation, random variation or residual confounding. CONCLUSIONS: The most informative low-dose radiation study to date provides little evidence for a relationship between mortality from non-malignant diseases and radiation dose. However, we cannot rule out risks per unit dose of the same order of magnitude as found in studies at higher doses.

The 15-Country Collaborative Study of Cancer Risk Among Radiation Workers in the Nuclear Industry: design, epidemiological methods and descriptive results.

Radiation protection standards are based mainly on risk estimates from studies of atomic bomb survivors in Japan. The validity of extrapolations from the relatively high-dose acute exposures in this population to the low-dose, protracted or fractionated environmental and occupational exposures of primary public health concern has long been the subject of controversy. A collaborative retrospective cohort study was conducted to provide direct estimates of cancer risk after low-dose protracted exposures. The study included nearly 600,000 workers employed in 154 facilities in 15 countries. This paper describes the design, methods and results of descriptive analyses of the study. The main analyses included 407,391 nuclear industry workers employed for at least 1 year in a participating facility who were monitored individually for external radiation exposure and whose doses resulted predominantly from exposure to higher-energy photon radiation. The total duration of follow-up was 5,192,710 person-years. There were 24,158 deaths from all causes, including 6,734 deaths from cancer. The total collective dose was 7,892 Sv. The overall average cumulative recorded dose was 19.4 mSv. A strong healthy worker effect was observed in most countries. This study provides the largest body of direct evidence to date on the effects of low-dose protracted exposures to external photon radiation.

The 15-Country Collaborative Study of Cancer Risk among Radiation Workers in the Nuclear Industry: estimates of radiation-related cancer risks.

A 15-Country collaborative cohort study was conducted to provide direct estimates of cancer risk following protracted low doses of ionizing radiation. Analyses included 407,391 nuclear industry workers monitored individually for external radiation and 5.2 million person-years of follow-up. A significant association was seen between radiation dose and all-cause mortality [excess relative risk (ERR) 0.42 per Sv, 90% CI 0.07, 0.79; 18,993 deaths]. This was mainly attributable to a dose-related increase in all cancer mortality (ERR/Sv 0.97, 90% CI 0.28, 1.77; 5233 deaths). Among 31 specific types of malignancies studied, a significant association was found for lung cancer (ERR/Sv 1.86, 90% CI 0.49, 3.63; 1457 deaths) and a borderline significant (P = 0.06) association for multiple myeloma (ERR/Sv 6.15, 90% CI <0, 20.6; 83 deaths) and ill-defined and secondary cancers (ERR/Sv 1.96, 90% CI -0.26, 5.90; 328 deaths). Stratification on duration of employment had a large effect on the ERR/Sv, reflecting a strong healthy worker survivor effect in these cohorts. This is the largest analytical epidemiological study of the effects of low-dose protracted exposures to ionizing radiation to date. Further studies will be important to better assess the role of tobacco and other occupational exposures in our risk estimates.

p53 cellular tumor antigen: analysis of mRNA levels in normal adult tissues, embryos, and tumors.

The relative levels of mRNA specific for the mouse p53 cellular tumor antigen were determined in various normal adult tissues, embryos, and tumors. All tumors studied contained concentrations of p53 mRNA well above those present in most normal tissues. Normal spleen, however, had p53 mRNA levels comparable to those found in some tumors, despite the fact that they contained barely detectable p53 protein. This apparent discrepancy was found to be due to the extremely rapid turnover rate of p53 in the spleen (half-life, approximately equal to 6 min). In developing fetuses, a marked reduction of p53 mRNA levels was manifest from day 11 onwards, whereas the levels during organogenesis (days 9 to 11) were comparable to those found in undifferentiated embryonic stem cells and in some tumors.

The neuroprotective adenosine-activated signal transduction pathway involves activation of phospholipase C.

We have demonstrated before that exposure of neuronal cultures to poisoning by iodoacetic acid (IAA) followed by "reperfusion" (IAA-R insult), results in severe cytotoxicity, which could be markedly attenuated by prior activation of the adenosine A1 receptors. We also have demonstrated that adenosine activates a signal transduction pathway (STP), which involves activation of PKC epsilon and opening of KATP channels. Here, we provide proof for the involvement also of phospholipase C (PLC) in the neuronal protective adenosine-activated STP. R-PIA, a specific A1 adenosine receptor agonist, was found to enhance neuronal PLC activity and protect against the IAA-R insult. The PLC inhibitor U73122, abrogated both R-PIA-induced effects. These results demonstrate that activation of PLC is a vital step in the neuronal protective adenosine-induced STP.

Liver subcellular fatty acid profiles of chicks fed diets containing hydrogenated fats and varying linoleate levels.

Day-old male broiler chickens were fed semipurified diets containing 5% lipid from one of four different lipid sources: corn oil (CO), partially hydrogenated soybean oil (HSBO), a spent restaurant grease (SRG) and a purified mixture of triolein, tripalmitin and tristearin (OPS). Diets CO and HSBO contained adequate amounts of linoleic acid, but diets SRG and OPS were deficient in linoleate. In addition, SRG and HSBO contained trans isomers of 16:1 and 18:1. The diets were fed for 3 wk to determine the effects of low linoleate levels and trans isomers on fatty acid profiles in liver microsomes, mitochondria and cytosol. Chicks fed HSBO had the highest body weights, while those fed SRG and OPS had the lowest. The incidence and severity of dermatitis were similar for all treatments. The proportions of linoleate and arachidonate in lipids from liver subcellular fractions were reduced significantly in chicks fed OPS and SRG; however, levels of 20:3 omega 9 were not increased. Feeding HSBO, which is high in both linoleate and linolenate, resulted in higher levels of 18:3 omega 3 and 20:5 omega 3 in liver subcellular fractions and lower levels of 20:4 omega 6 than those seen in chicks fed CO. The isomeric forms of 18:1 present in the partially hydrogenated fats (HSBO and SRG) appeared to be incorporated into the lipids of liver fractions. The results of this study show that dietary lipids influence fatty acid profiles of chick liver microsomes, mitochondria and cytosol. Decreases in linoleate and arachidonate in these organelles occur before overt essential fatty acid (EFA), deficiency signs in chicks fed EFA-deficient diets.

A novel spectrophotometric assay for lipase activity utilizing cis-parinaric acid.

A new spectrophotometric assay for determining the activity of acylglycerol hydrolases (lipases, E.C. 3.1.1.3) was developed and optimized for yeast lipase (Candida cylindracea). Studies with porcine pancreatic lipase were also conducted and the influence of various detergents and divalent cations on the assay was evaluated. The assay uses cis-parinaric acid (PnA), a naturally occurring fatty acid that has unique spectroscopic properties, and takes advantage of the reversible binding of fatty acids to bovine serum albumin (BSA). Free PnA has an ultraviolet absorption peak at 321.2 nm. When PnA is bound to BSA, however, the peak shifts to 324.2 nm. The assay mixture contains 6 microM PnA, 1 microM BSA, 75 microM triolein, and 0.3 mM taurocholate in a 50 mM tris-HCl buffer with 1 microM EDTA. The release of oleic acid from triolein is monitored over time by measuring the ratio of optical densities (OD) at 319.0 and 329.0 nm. Initially, there is maximum binding of PnA to BSA, and the OD ratio is approximately 1.0. Upon addition of lipase, PnA is displaced from the BSA by oleic acid released from triolein, and the OD ratio increases to a maximum of about 1.8. However, when calcium is present in the reaction mixture an insoluble calcium-PnA complex forms, resulting in a progressive decrease in OD at both 319.0 and 329.0 nm. The kinetic assay described here is simple, rapid, sensitive, reproducible, inexpensive, and it can be adapted to measure the activity of a variety of calcium-independent lipases. Under similar assay conditions, activities for Candida cylindracea lipase obtained with this assay are similar to those obtained with 14C-labelled triolein.

Alteration of lipid metabolism in Japanese quail by feeding oat hulls and brans.

Five varieties of oat brans fed for 4 weeks at 2.27% and oat hulls fed at 11.36% in a semi-purified diet containing .5% cholesterol were ineffective in lowering cholesterol levels in the serum of 5-week-old Japanese quail. Oat brans and hulls reduced the level of fat in livers of both males and females. No differences in cholesterol levels were observed between sexes, but hypertriglyceridemia and high liver fat contents were more evident in females. This may be the result of sexual maturation stimulated by a 14L:10D photoperiod.

Seasonal correction factors for estimating radon exposure in dwellings in France.

Indoor radon concentrations are subject to seasonal variation with a maximum in winter and a minimum in summer. Procedures to correct for seasonal variation are necessary in order to get an unbiased estimate of the annual average radon concentration from data based on short-period radon measurements. To obtain correction factors, we apply the model developed by Pinel et al to the French database of indoor radon measurements (measurements performed as part of the indoor radon case-control study and of the national radon measurement campaign). For 6-month measurements, the correction factors vary from 0.87 to 1.17 and agree with those previously published. These results might be applicable when assessing indoor radon concentrations with regard to recommended action levels.

National cancer incidence is estimated using the incidence/mortality ratio in countries with local incidence data: is this estimation correct?

BACKGROUND: In countries with local cancer registration, the national cancer incidence is usually estimated by multiplying the national mortality by the incidence/mortality (I/M) ratio from pooled registries. This study aims at validating this I/M estimation in France, by a comparison with estimation obtained using the ratio of incidence over hospital discharge (I/HD) or the ratio of incidence over health insurance data (long-duration diseases, I/LDD). METHODS: This comparison was performed for 22 cancer sites over the period 2004-2006. In France, a longitudinal I/M approach was developed relying on incidence and mortality trend analyses; here, the corresponding estimations of national incidence were extracted for 2004-2006. The I/HD and I/LDD estimations were performed using a common cross-sectional methodology. RESULTS: The three estimations were found similar for most cancers. The relative differences in incidence rates (vs. I/M) were below 5% for numerous cancers and below 10% for all cancers but three. The highest differences were observed for thyroid cancer (up to +21% in women and +8% in men), skin melanoma (up to +13% in women and +8% in men), and Hodgkin disease in men (up to +15%). Differences were also observed in women aged over 60 for cervical cancer. Except for thyroid cancer, differences were mainly due to the smoothing performed in the I/M approach. CONCLUSION: Our results support the validity of I/M approaches for national estimations, except for thyroid cancer. The longitudinal version of this approach has, furthermore, the advantage of providing smoothed estimations and trend analyses, including useful birth-cohort indicators, and should thus be preferred.

Modelling the effect of breast cancer screening on related mortality using French data.

INTRODUCTION: This study aimed at modelling the effect of organized breast cancer screening on mortality in France. It combined results from a Markov model for breast cancer progression, to predict number of cases by node status, and from relative survival analyses, to predict deaths. The method estimated the relative risk of mortality at 8 years, in women aged 50-69, between a population screened every two years and a reference population. METHODS: Analyses concerned cases diagnosed between 1990 and 1996, with a follow-up up to 2004 for the vital status. Markov models analysed data from 3 screening programs (300,000 mammographies) and took into account opportunistic screening among participants to avoid bias in parameter's estimates. We used survival data from cancers in the general population (n=918, 7 cancer registries) and from screened cancers (n=565, 3 cancer registries), after excluding a subgroup of screened cases with a particularly high survival. Sensitivity analyses were performed. RESULTS: Markov model main analysis lacked of fit in two out of three districts. Fit was improved in stratified analyses by age or district, though some lack of fit persisted in two districts. Assuming 10% or 20% overdiagnosed screened cancers, mortality reduction was estimated as 23% (95% CI: 4, 38%) and 19% (CI: -3, 35%) respectively. Results were highly sensitive to the exclusion in the screened cancers survival analysis. Conversely, RR estimates varied moderately according to the Markov model parameters used (stratified by age or district). CONCLUSION: The study aimed at estimating the effect of screening in a screened population compared to an unscreened control group. Such a control group does not exist in France, and we used a general population contaminated by opportunistic screening to provide a conservative estimate. Conservative choices were systematically adopted to avoid favourable estimates. A selection bias might however affect the estimates, though it should be moderate because extreme social classes are under-represented among participants. This modelling provided broad estimates for the effect of organized biennial screening in France in the early nineteen-nineties. Results will be strengthened with longer follow-up.

Multi-state Markov models in cancer screening evaluation: a brief review and case study.

This work presents a brief overview of Markov models in cancer screening evaluation and focuses on two specific models. A three-state model was first proposed to estimate jointly the sensitivity of the screening procedure and the average duration in the preclinical phase, i.e. the period when the cancer is asymptomatic but detectable by screening. A five-state model, incorporating lymph node involvement as a prognostic factor, was later proposed combined with a survival analysis to predict the mortality reduction associated with screening. The strengths and limitations of these two models are illustrated using data from French breast cancer service screening programmes. The three-state model is a useful frame but parameter estimates should be interpreted with caution. They are highly correlated and depend heavily on the parametric assumptions of the model. Our results pointed out a serious limitation to the five-state model, due to implicit assumptions which are not always verified. Although it may still be useful, there is a need for more flexible models. Over-diagnosis is an important issue for both models and induces bias in parameter estimates. It can be addressed by adding a non-progressive state, but this may provide an uncertain estimation of over-diagnosis. When the primary goal is to avoid bias, rather than to estimate over-diagnosis, it may be more appropriate to correct for over-diagnosis assuming different levels in a sensitivity analysis. This would be particularly relevant in a perspective of mortality reduction estimation.

Mortality among workers monitored for radiation exposure at the French nuclear fuel company.

A cohort of 9,285 nuclear workers employed at the French company AREVA NC specializing in the nuclear fuel cycle was established. Vital status, causes of death, employment characteristics and annual exposure to ionizing radiation were reconstructed for each individual over the time period 1977-2004. Standardized mortality ratios (SMRs) were computed using national mortality rates as an external reference. Tests for trends in mortality with duration of employment and cumulative external dose were performed. The all-cause and all-cancer mortality was significantly lower than expected from the French population. No significant excess among cancer sites studied was observed. Significant positive trends with cumulative dose were observed for colon and liver cancer and for respiratory diseases. Isolated significant trends should be carefully interpreted and considered in line with the large number of trend tests performed.

Risk of cancer after low doses of ionising radiation: retrospective cohort study in 15 countries.

OBJECTIVES: To provide direct estimates of risk of cancer after protracted low doses of ionising radiation and to strengthen the scientific basis of radiation protection standards for environmental, occupational, and medical diagnostic exposures. DESIGN: Multinational retrospective cohort study of cancer mortality. SETTING: Cohorts of workers in the nuclear industry in 15 countries. PARTICIPANTS: 407 391 workers individually monitored for external radiation with a total follow-up of 5.2 million person years. MAIN OUTCOME MEASUREMENTS: Estimates of excess relative risks per sievert (Sv) of radiation dose for mortality from cancers other than leukaemia and from leukaemia excluding chronic lymphocytic leukaemia, the main causes of death considered by radiation protection authorities. RESULTS: The excess relative risk for cancers other than leukaemia was 0.97 per Sv, 95% confidence interval 0.14 to 1.97. Analyses of causes of death related or unrelated to smoking indicate that, although confounding by smoking may be present, it is unlikely to explain all of this increased risk. The excess relative risk for leukaemia excluding chronic lymphocytic leukaemia was 1.93 per Sv (< 0 to 8.47). On the basis of these estimates, 1-2% of deaths from cancer among workers in this cohort may be attributable to radiation. CONCLUSIONS: These estimates, from the largest study of nuclear workers ever conducted, are higher than, but statistically compatible with, the risk estimates used for current radiation protection standards. The results suggest that there is a small excess risk of cancer, even at the low doses and dose rates typically received by nuclear workers in this study.

Distinct steps in the penetration of adenylate cyclase toxin of Bordetella pertussis into sheep erythrocytes. Translocation of the toxin across the membrane.

Adenylate cyclase (AC) toxin from Bordetella pertussis penetrates eukaryotic cells and upon activation by calmodulin generates unregulated levels of intracellular cAMP. The process of toxin penetration into sheep erythrocytes was resolved into three consecutive steps including insertion, translocation, and intracellular cleavage. Insertion of the toxin into the cell membrane occurred over a wide temperature range (4-36 degrees C). In contrast, translocation of the toxin, i.e. transfer of the NH2-terminal catalytically active fragment across the membrane, occurred only above 20 degrees C and was highly temperature-dependent. While a single exposure of the toxin to Ca2+ was sufficient for its insertion into the plasma membrane, toxin translocation required exogenous Ca2+ at mM concentrations. Translocation was not affected by pretreatment of cells with trypsin, N-ethylmaleimide, and sodium carbonate at alkaline pH. The NH2-terminal fragment of the toxin was cleaved in the cell releasing the 45-kDa active AC into the cytosol. The cleavage was blocked by treatment of cells with N-ethylmaleimide. It is hypothesized that the COOH-terminal portion of the toxin creates in the membrane a channel through which the NH2-terminal fragment is translocated.