Lung cancer mortality in the European uranium miners cohorts analyzed with a biologically based model taking into account radon measurement error

The biologically based two-stage clonal expansion (TSCE) model is used to analyze lung cancer mortality of European miners from the Czech Republic, France, and Germany. All three cohorts indicate a highly significant action of exposure to radon and its progeny on promotion. The action on initiation is not significant in the French cohort. An action on transformation was tested but not found significant. In a pooled analysis, the results based on the French and German datasets do not differ significantly in any of the used parameters. For the Czech dataset, only lag time and two parameters that determine the clonal expansion without exposure and with low exposure rates (promotion) are consistent with the other studies. For low exposure rates, the resulting relative risks are quite similar. Exposure estimates for each calendar year are used. A model for random errors in each of these yearly exposures is presented. Depending on the used technique of exposure estimate, Berkson and classical errors are used. The consequences for the model parameters are calculated and found to be mostly of minor importance, except that the large difference in the exposure-induced initiation between the studies is decreased substantially.     

Radon-associated lung cancer risk among French uranium miners: modifying factors of the exposure–risk relationship

Radon is classified as a known pulmonary carcinogen in humans. A better understanding of the effects of low exposure and time-dependent factors, modifying the lung cancer risk is of continued interest. We present analyses of the exposure–risk relationship in the French cohort of uranium miners updated until 1999 and including five additional years of follow-up. These new analyses provide a better opportunity to look at low radon exposures with longer follow-up intervals, and allow consideration of new modifying factors, such as physical activity, mine location and job type. The cohort includes 5,086 miners, and 159 lung cancer deaths have been observed among these over a follow-up of more than 30 years. The exposure–risk relationship was estimated using excess relative risk models, which allow investigation of several modifying factors such as period of exposure, time since exposure, age at exposure, duration of exposure, exposure rate, job type, mine type and physical activity. The analysis confirms the association between radon exposure and lung cancer risk (ERR per 100 WLM = 0.58, P < 0.01). Period of exposure and physical activity appear as major modifying factors. Higher risks are observed for hard physical activity works. The effect of hard physical activity persists when the period of exposure is taken into account (ERR per 100 WLM = 2.95, P < 0.01).     

The influence of multiple types of occupational exposure to radon, gamma rays and long-lived radionuclides on mortality risk in the French "post-55" sub-cohort of uranium miners: 1956-1999

The adverse health effects of radon on uranium miners, especially on their lungs, are well documented, but few studies have considered the effects of other radiation exposures. This study examined the mortality risks associated with exposure to radon, external γ rays and long-lived radionuclides (LLR) in the French "post-55" sub-cohort, which includes uranium miners first employed between 1956 and 1990 for whom all three types of exposure were assessed individually. Exposure-risk relationships were estimated with linear excess relative risk models and a 5-year lag time. The post-55 sub-cohort includes 3377 miners, contributing 89,405 person-years, followed up through the end of 1999 with a mean follow-up of 26.5 years. Mean cumulative exposure was 17.8 WLM for radon, 54.7 mSv for γ rays, and 1,632 Bq.m(-3).h for LLR. Among the 611 deaths observed, 66 were due to lung cancer. Annual individual exposures were significantly correlated. Increased mortality was observed for lung cancer (SMR = 1.30; 95% CI: 1.01, 1.65) and for brain and central nervous system (CNS) cancer (SMR = 2.00; 95% CI: 1.09, 3.35). Cumulative exposure to radon, γ rays and LLR was associated only with a significant risk of lung cancer. These new results could suggest an association between lung cancer and exposure to γ rays and LLR. They must nonetheless be interpreted with caution because of the correlation between the types of exposure. The calculation of organ doses received by each of these exposures would reduce the collinearity.     

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.     

Spatiotemporal variations of diterpene production in the brown macroalga <Emphasis Type="Italic">Bifurcaria bifurcata</Emphasis> from the western coasts of Brittany (France)

Bifurcaria bifurcata, a temperate brown macroalga, is known to show spatial fluctuations in its diterpene content along the northwestern coasts of France (Brittany). In the aim to identify environmental factors which could influence the occurrence of a particular chemical type, several populations of B. bifurcata were collected in summer 2009 and winter 2010. Their chemical composition was studied through thin layer chromatography (TLC) and nuclear magnetic resonance (NMR) analyses. Results showed that specific diterpenes are biosynthesized depending on seasons and abiotic factors, such as hydrodynamism or substratum. Exposed sites were characterized by thalli of B. bifurcata producing two main diterpenoids, bifurcane, and eleganediol, whereas thalli from sheltered sites showed crude extracts containing a major diterpene, eleganolone. On these last sites, another diterpene (bifurcanone) is only expressed in winter and was thus considered as a seasonal chemomarker. The term “chemotype” applied to a population is proposed and discussed.     

Impact of measurement error in radon exposure on the estimated excess relative risk of lung cancer death in a simulated study based on the French Uranium Miners’ Cohort

Measurement error (ME) can lead to bias in the analysis of epidemiologic studies. Here a simulation study is described that is based on data from the French Uranium Miners’ Cohort and that was conducted to assess the effect of ME on the estimated excess relative risk (ERR) of lung cancer death associated with radon exposure. Starting from a scenario without any ME, data were generated containing successively Berkson or classical ME depending on time periods, to reflect changes in the measurement of exposure to radon (²²²Rn) and its decay products over time in this cohort. Results indicate that ME attenuated the level of association with radon exposure, with a negative bias percentage on the order of 60% on the ERR estimate. Sensitivity analyses showed the consequences of specific ME characteristics (type, size, structure, and distribution) on the ERR estimates. In the future, it appears important to correct for ME upon analyzing cohorts such as this one to decrease bias in estimates of the ERR of adverse events associated with exposure to ionizing radiation.     

Masked Selection: A Straightforward and Flexible Approach for the Selection of Binders Against Specific Epitopes and Differentially Expressed Proteins by Phage Display

Phage display is a well-established procedure to isolate binders against a wide variety of antigens that can be performed on purified antigens, but also on intact cells. As selection steps are performed in vitro, it is possible to focus the outcome of the selection on relevant epitopes by performing some additional steps, such as depletion or competitive elutions. However in practice, the efficiency of these steps is often limited and can lead to inconsistent results. We have designed a new selection method named masked selection, based on the blockade of unwanted epitopes to favor the targeting of relevant ones. We demonstrate the efficiency and flexibility of this method by selecting single-domain antibodies against a specific portion of a fusion protein, by selecting binders against several members of the seven transmembrane receptor family using transfected HEK cells, or by selecting binders against unknown breast cancer markers not expressed on normal samples. The relevance of this approach for antibody-based therapies was further validated by the identification of four of these markers, Epithelial cell adhesion molecule, Transferrin receptor 1, Metastasis cell adhesion molecule, and Sushi containing domain 2, using immunoprecipitation and mass spectrometry. This new phage display strategy can be applied to any type of antibody fragments or alternative scaffolds, and is especially suited for the rapid discovery and identification of cell surface markers.Hybridoma (1) and phage-display recombinant antibody systems (2) are currently the predominant methods for isolating monoclonal antibodies (Abs).¹ Display of recombinant Abs on the surface of bacteriophage M13 has numerous advantages compared with conventional hybridoma technology. When combined with the use of large non-immune libraries, phage Ab selection represents a rich source of binders that can be isolated in a fraction of the time needed for hybridoma-based approaches. Moreover, this in vitro selection method permits the selection of binders against toxic, non-immunogenic or highly conserved antigens, which is not easily performed using the conventional hybridoma techniques. Importantly, it can be used to isolate fully human antibody fragments (3). Consequently, phage display rapidly became an established procedure for the isolation of binders against a wide variety of antigens.Phage display-based antibody isolation typically relies on the use of recombinant proteins for several steps, including immunizations (if needed), library enrichment by selection on immobilized antigen, screening, and characterization of antibodies in terms of specificity and affinity (4). This procedure is efficient but depends on the availability of purified recombinant proteins. Unfortunately, some surface molecules, such as G-protein coupled receptors, cannot be easily expressed and purified in their native conformation. Some molecules with large extracellular domains may adopt a specific conformation upon interaction with other cell surface proteins, thereby forming complexes that are cumbersome to produce by recombinant expression. Moreover, many standard screening practices, such as the adsorption of recombinant proteins on plastic, may significantly alter protein conformations (5). For these reasons, Abs selected on the basis of binding to a recombinant protein may not bind the native conformation of this protein. It is thus of high interest to develop procedures entirely based on the use of intact cells expressing the receptor of choice. However, in this case, an extra step is necessary to enrich for phage-Abs binding to the receptor of interest rather than to other cell surface proteins. Because selection steps are performed in vitro, it is possible to influence the outcome of a selection by performing some additional steps such as deletion steps (also named negative selection) prior to positive selections to remove unwanted specificities or cross-reactions (6), by alternating the source of the antigen (7), or by using a competitive elution with a ligand or an existing monoclonal antibody to favor the selection of binders against a precise epitope (8).Along this line, it would be of very high interest to establish a procedure able to reliably guide the selection toward an unknown but relevant antigen within a complex mixture, such as a tumor maker overexpressed at the surface of intact cells, or in a cell lysate. Indeed, during the past two decades, there has been a growing interest in approaches aiming at discovering new diagnosis biomarkers and identifying new potential surface markers for targeted therapy. Several studies have described the use of phage display and libraries of recombinant antibodies for the isolation of tumor specific binders (9–15), leading in some cases to the identification of new tumor markers (16, 17). Most of these strategies are relying on the use of depletion steps on normal samples followed by a selection step on tumor samples. Unfortunately, this procedure often leads to inconsistent results and its efficiency can be a limiting factor in complex situations such as the selection of antibodies against unknown overexpressed tumor antigens.We have designed a new selection method, named masked selection, which is relying on the blockade of unwanted epitopes to favor the targeting of relevant ones. We demonstrate the efficiency of this method by selecting binders against a specific portion of a fusion protein, by selecting binders against two members of the seven transmembrane receptor family and a tyrosine kinase receptor using intact transfected HEK cells, or by selecting binders against unknown breast cancer markers not expressed on normal samples, as shown by flow cytometry and immunohistochemistry. The universality and efficiency of this approach should ultimately lead to the rapid selection of specific binders and the development of diagnostic and targeted therapies in various settings.     

ASPM-associated stem cell proliferation is involved in malignant progression of gliomas and constitutes an attractive therapeutic target

Background

ASPM (Abnormal Spindle-like Microcephaly associated) over-expression was recently implicated in the development of malignant gliomas.

Results

To better characterize the involvement of ASPM in gliomas, we investigated the mRNA expression in 175 samples, including 8 WHO Grade II, 75 WHO Grade III and 92 WHO Grade IV tumors. Aspm expression was strongly correlated with tumor grade and increased at recurrence when compared to the initial lesion, whatever the initial grade of the primary tumor. ASPM expression also increased over serial passages in gliomaspheres in vitro and in mouse xenografts in vivo. Lentivirus-mediated shRNA silencing of ASPM resulted in dramatic proliferation arrest and cell death in two different gliomasphere models.

Conclusion

These data suggest that ASPM is involved in the malignant progression of gliomas, possibly through expansion of a cancer stem cell compartment, and is an attractive therapeutic target in glioblastoma multiforme.     

Effect of different conditioning treatments on total phenolic content and antioxidant activities in two Sargassacean species: Comparison of the frondose Sargassum muticum (Yendo) Fensholt and the cylindrical Bifurcaria bifurcata R. Ross

The effects of different conditioning treatments (fresh, freezing, freeze‐drying, oven‐drying and greenhouse‐drying) on the total phenolic content (TPC) and antioxidant activities of two brown algae, Sargassum muticum and Bifurcaria bifurcata, were investigated and compared. Phenolic compounds were extracted in a methanol/water (50:50) solution, and TPC was measured by the colorimetric Folin‐Ciocalteu assay. Antioxidant activity was assessed by the DPPH (2, 2‐diphenyl‐1‐picrylhydrazyl) radical scavenging assay and the β‐carotene bleaching method. The dried seaweeds showed lower phenolic contents and lower antioxidant capacities than the fresh and frozen ones, which suggests that the phenolic content and antioxidant activities are decreased by the drying treatments, especially, oven‐ and greenhouse‐drying. Relationships between TPC, antioxidant properties and conditioning treatments are discussed.