We investigated variations in sensitivity of an immunochemical (I-FOBT) and a guaiac (G-FOBT) faecal occult blood test according to type and location of lesions in an average-risk 50- to 74-year-old population. Screening for colorectal cancer by both non-rehydrated Haemoccult II G-FOBT and Magstream I-FOBT was proposed to a sample of 20 322 subjects. Of the 1615 subjects with at least one positive test, colonoscopy results were available for 1277. A total of 43 invasive cancers and 270 high-risk adenomas were detected. The gain in sensitivity associated with the I-FOBT was calculated using the ratio of sensitivities (RSN) according to type and location of lesions, and amount of bleeding. The gain in sensitivity by using I-FOBT increased from invasive cancers (RSN=1.48 (1.16–4.59)) to high-risk adenomas (RSN=3.32 (2.70–4.07)), and was inversely related to the amount of bleeding. Among cancers, the gain in sensitivity was confined to rectal cancer (RSN=2.09 (1.36–3.20)) and concerned good prognosis cancers, because they involve less bleeding. Among high-risk adenomas, the gain in sensitivity was similar whatever the location. This study suggests that the gain in sensitivity by using an I-FOBT instead of a G-FOBT greatly depends on the location of lesions and the amount of bleeding. Concerning cancer, the gain seems to be confined to rectal cancer. It is now established that screening by measuring faecal occult bleeding in average-risk populations can detect asymptomatic colorectal cancers and precancerous lesions (high-risk adenomas). A number of recently reviewed randomised trials have established the efficacy of average-risk population screening using the Haemoccult II guaiac faecal occult blood test (G-FOBT) to reduce specific mortality related to colorectal cancer . The first 30 000 attendants to screening were offered the possibility to join a study comparing an I-FOBT (Immudia/RPHA) and the conventional G-FOBT. This analysis focuses on the 20 322 subjects having performed both tests from 1 June 2004 to 31 December 2005.−1 of Hb in the buffer, which corresponds to 0.1–0.2 mg Hb per gram stool. The subject and practitioner were informed of the overall screening procedure result, but were blinded to each individual test result. In the case of a positive result (i.e. at least one of the FOBT result was positive), the subject was invited to undergo a colonoscopy. The colonoscopy was performed blinded to the I-FOBT and G-FOBT results. The study was approved by the local ethics committee  and all participants gave written informed consent.The design of the study has been detailed in a previous publication . However, if the colonoscopy was incomplete because of obstructing tumours or if the incomplete colonoscopy had been completed by another colonic examination  that failed to reveal any polypoid lesion, the results were included in the analysis. Hyperplastic polyps were not included as neoplasia. Advanced colonic neoplasia was defined as high-risk adenoma  or invasive cancer. Intramucosal carcinoma and carcinoma n=20 322). Among the 1615 subjects with a positive screening test (positive G-FOBT and/or I-FOBT), 1387 (85.9%) performed the indicated colonoscopy. The frequency of subjects undergoing endoscopic examination did not differ depending on which FOBT was positive . Fifty-nine subjects with incomplete colonoscopy, and 27 for whom information on the polypoid lesion was insufficient, were excluded from the analysis. Consequently, 1277 subjects with at least one positive test and having completed a satisfactory colonoscopy were included in the analysis.The population of eligible subjects comprised of all subjects with both I-FOBT and G-FOBT analysable results who had also given informed consent, from 1 June 2004 to 31 December 2005  was restricted to subjects classified as positive by at least one of the tests, the sensitivity and specificity of each test could not be directly estimated. We therefore compared the sensitivity of each test to detect high-risk adenomas or invasive cancers according to the location of lesions using the ratio of sensitivities (RSN) . Brieflyequation .The maximal faecal occult bleeding measured by the Magstream I-FOBT was compared according to location and type of lesions among subjects with a maximum of one lesion.Statistical analysis was performed using SAS software version 9.1 (SAS Institute).+I+), guaiac test positive and immunochemical test negative (G+I−), guaiac test negative and immunochemical test positive (G−I+). The positive predictive value (PPV) was lower for I-FOBT than for G-FOBT with regard to invasive cancers , and higher with regard to high-risk adenomas (24.3 vs 19.5%). Using the RSN, P<10−3). Concerning cancer, the increase in sensitivity of I-FOBT compared to G-FOBT was far higher for rectal cancers than for other localisations. In fact, the gain in sensitivity for cancer was restricted to invasive cancers located in the rectum , the gain for other localisations being non-significant. The extent of gain in sensitivity in screening for colorectal cancer when using I-FOBT instead of G-FOBT depends on the type and the location of lesions. It was higher for high-risk adenomas than for cancers. Among cancers, the gain in sensitivity was essentially confined to rectal cancers and concerned good prognosis cancers. This gain in sensitivity was related to the small amount of bleeding observed in such lesions.Our study has several drawbacks. First of all, because subjects for whom both tests were negative underwent no further evaluation by colonoscopy, we were unable to provide an estimation of sensitivity and specificity for each test. However, the use of RSN allowed us to quantify the potential gain obtained through the substitution of G-FOBT by I-FOBT for each lesion type and location. Moreover, despite our large sample size, the number of discordant pairs was low. Therefore, the size of confidence intervals was probably underestimated  and asymptomatic cancers is higher for rectal than for colonic cancers . Moreoveversus the colon , whereas The Magstream I-FOBT relies on an antibody directed against the globin moiety of human Hb. Deteriorated Hb is not detected. Immunochemical faecal occult blood test may underestimate the intensity of bleeding in proximal polypoid lesions and, therefore, be less sensitive to such lesions. This could explain why Our previous studies have demonstrated that I-FOBT is preferable to G-FOBT from a public health point of view because the availability of quantitative Hb measurement offers the possibility to choose a positivity rate offering both high sensitivity and high specificity (


Genetic interactions for common disorders are likely numerous and weak. Using the 200 replicates of the Genetic Analysis Workshop 16 (GAW16) Problem 3 simulated data, we compared the statistical power to detect weak gene-gene interactions using a haplotype-based test in the UNPHASED software with genotypic mixed model (GMM) and additive mixed model (AMM) mixed linear regression model in SAS. We assumed a candidate-gene approach where a single-nucleotide polymorphism (SNP) in one gene is fixed and multiple SNPs are at the second gene. We analyzed the quantitative low-density lipoprotein trait (heritability 0.7%), modulated by simulated interaction of rs4648068 from 4q24 and another gene on 8p22, where we analyzed seven SNPs. We generally observed low power calculated per SNP (≤ 37% at the 0.05 level), with the haplotype-based test being inferior. Over all tests, the haplotype-based test performed within chance, while GMM and AMM had low power (~10%). The haplotype-based and mixed models detected signals at different SNPs. The haplotype-based test detected a signal in 50 unique replicates; GMM and AMM featured both shared and distinct SNPs and replicates . Overall, the statistical signal for the weak gene-gene interaction appears sensitive to the sample structure of the replicates. We conclude that using more than one statistical approach may increase power to detect such signals in studies with limited number of loci such as replications. There were no results significant at the conservative 10 With efforts to uncover more genetic variation for common polygenic disorders, there is accrued interest in the analysis of genetic interactions . It is vThe subjects consist of 6,476 related and unrelated individuals from the Framingham Heart Study as made available for the GAW16 Problem 3 simulated data by dbGAP. We used two phenotype definitions in separate analyses. A first quantitative phenotype that was used for haplotype-based analyses only was the covariate-adjusted multivariate linear regression residual of the low-density lipoprotein (LDL) variable, averaged over the three visits and standardized. The covariates used for the adjustment were sex, age, diet, medication use, high-density lipoprotein (HDL), triglyceride level, and smoking. The main quantitative phenotype used for both haplotype-based and mixed model analyses was the LDL measurement at the first visit, regression-adjusted for age and sex only (excluding subjects on lipid-lowering medication). The simulated LDL measurement at the first visit did not show notable departure from the normal distribution in the first replica, suggesting that non-normality would not be an important factor in this study.NFKB1  gene at chromosome 4q24 was fixed in the analyses: it is involved in the simulated gene-gene interaction with the lipoprotein lipase precursor (LPL) gene with 0.7% heritability for the LDL trait. Seven SNPs from the 500 k chip, with minor allele frequencies ranging from 0.07-0.22, were selected in the LPL gene region at 8p22 as per HapMap gene position 19,841,058 to 19,869,049, encompassing an additional 10 kb on each side as available. These SNPs are: rs263, rs271, rs11570892, rs3200218, rs2410616, rs2898493, and rs17482753; respective chromosomal positions are 19856900, 19857092, 19857982, 19867897, 19868351, 19872959, 19873001, and 19876926. Figure D' measure of linkage disequilibrium obtained using 849 unrelated subjects in the computer program Haploview [The SNP rs4648068 in the 0) of equal contributions for all gene combinations (in haplotype form) sharing the same alleles at the conditioning marker, versus the alternative hypothesis (H1) of differential multiplicative contributions from the test marker. The test uses a likelihood-ratio chi-square statistics to compare models with and without the interaction terms. The frequency of gene combinations was limited to 0.01. Gene-gene interaction using SNP-SNP testing (in haplotype form) was evaluated between rs4648068 and each SNP in the LPL gene region one at a time. Gene-gene interaction was also evaluated using SNP-haplotype testing between rs4648068 and each consecutive pair of SNPs in the LPL gene region one pair at a time as haplotypes, e.g., rs263-rs271, rs271-rs11570892, etc. The SNP-haplotype testing was conducted only in the analysis of the first phenotype.The UNPHASED software implements a likelihood approach for primarily haplotype-based analysis of data, which can include both familial and unrelated subjects . The tesLPL gene region:We used the mixed linear regression model implemented in PROC MIXED in SAS version 9.1.3 . To evaluate significance of the genotypic SNP-SNP interaction in the presence of potential marginal genotypic effects, we used the following full model for the main LDL phenotype and each of seven SNPs from the 0 is the fixed rs4648068, SNPi is from the LPL gene region (i = 1-7), and pedno is the pedigree ID included as a random effect to control for familial dependence. For the GMM test, SNPs were represented by three genotype classes (two homozygotes and a heterozygote). For the AMM test, the number of minor alleles in a genotype was coded as 0, 1, or 2. We used Type 3 tests of fixed effects to evaluate statistical significance.where LDL is measured at the first visit excluding subjects on lipid medication, SNPIn our first haplotype-based analyses of 200 replicates with the first LDL definition, there was low power (≤ 15%) at the 0.05 significance level with markers rs3200218 and rs2898493 in either SNP-SNP tests or SNP-haplotype tests involving these markers (not shown). The SNP-SNP testing was more powerful than SNP-haplotype testing .LPL gene region, while UNPHASED produced an interaction signal only with rs3200218 located between these SNPs, which is the only SNP to have a detectable marginal effect in either GMM or AMM without the interaction signal. The GMM and AMM produced an opposite pattern of interaction signals at rs263 and rs271, and both produced a signal at rs17482753. The AMM detected marginal SNP effects more frequently than GMM. When calculated over all SNP tests, the haplotype-based test had no power to detect the interaction, while both GMM and AMM had low power.Table Figure We generally observed low statistical power to detect a weak simulated candidate gene-gene interaction for the LDL trait with heritability of 0.7%, where one SNP was fixed and multiple SNPs are at the second gene. There was a small increase in power per SNP with the haplotype-based tests when LDL was not adjusted for HDL and triglyceride level, potentially retaining useful variation (the LPL candidate gene is simulated to contribute to HDL with a heritability of 0.003). In further analyses the haplotype-based test was inferior to GMM or AMM, particularly when power was calculated over all SNP tests.The haplotype-based test detected gene-gene interaction with a single SNP in the region, while GMM and AMM had shared and specific SNPs. We also examined the relationship between the specific methods used and the replicates that produced any gene-gene interaction signals. While there was some overlap in successful replicates with the GMM and AMM models, each of the three models identified distinct replicates, for a total of 183/200 replicates producing a detectable gene-gene interaction signal. Notably, the haplotype and mixed model tests did not share a single successful replica. The sole SNP identified with low power in the haplotype-based interaction test was also the only one with a robust marginal effect in both the GMM and AMM . Accordingly, the interaction signal from this particular SNP could be obscured in the mixed models by the strong marginal effect. The inability of the haplotype-based test to detect the weak interaction at other SNPs compared with mixed models suggests overall inferiority of the former approach for this simulated genetic interaction. In this data set, the weak gene-gene interaction followed different genetic patterns in different replicates, and its detection was sensitive to the particular structure of the replicates.-7 genome-wide level for a 500 k chip, indicating that these or similarly powered approaches would not detect such a weak gene-gene interaction from a genome-wide scan.The systematic use of more than one analytical approach may help to increase power to identify a weak gene-gene interaction in a limited number of test loci such as in a replication study. The replication sample may not necessarily present an identical genetic pattern to that of the original study. It might be expected that sensitivity to sample structure would be less of an issue for stronger gene-gene interactions. Factors beyond statistical interaction patterns, such as the presence of marginal genetic effects, may decrease the power to detect genetic interaction in mixed models. Finally, we note that not a single result from any analysis was significant at the conservative 10AMM: Additive mixed model; GAW16: Genetic Analysis Workshop 16; GMM: Genotypic mixed model; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; LPL: Lipoprotein lipase precursor; SNP: Single-nucleotide polymorphismThe authors declare that they have no competing interests.IPK performed statistical analysis, drafted the manuscript, and participated in the study design and interpretation. MPD participated in the study design, interpretation, and finalization of the manuscript.


A growing number of observational and epidemiological studies have suggested that mental illness, in particular mood disorders, is associated with reduced dietary intake and/or cellular abundance of omega-3 polyunsaturated fatty acids (PUFA). This has prompted researchers to test the efficacy of omega-3 PUFA in a range of different psychiatric disorders. We have critically reviewed the double blind placebo controlled clinical trials published prior to April 2007 to determine whether omega-3 PUFA are likely to be efficacious in these disorders.Most trials involved a small number of participants but were largely well designed.  Omega-3 PUFA were well tolerated by both children and adults with mild gastrointestinal effects being the only consistently reported adverse event. For schizophrenia and borderline personality disorder we found little evidence of a robust clinically relevant effect. In the case of attention deficit hyperactivity disorder and related disorders, most trials showed at most small benefits over placebo. A limited meta-analysis of these trials suggested that benefits of omega-3 PUFA supplementation may be greater in a classroom setting than at home. Some evidence indicates that omega-3 PUFA may reduce symptoms of anxiety although the data is preliminary and inconclusive. The most convincing evidence for beneficial effects of omega-3 PUFA is to be found in mood disorders. A meta-analysis of trials involving patients with major depressive disorder and bipolar disorder provided evidence that omega-3 PUFA supplementation reduces symptoms of depression. Furthermore, meta-regression analysis suggests that supplementation with eicosapentaenoic acid may be more beneficial in mood disorders than with docosahexaenoic acid, although several confounding factors prevented a definitive conclusion being made regarding which species of omega-3 PUFA is most beneficial. The mechanisms underlying the apparent efficacy of omega-3 PUFA in mood disorders compared to schizophrenia are discussed as is a rational for the possibly greater efficacy of EPA compared to DHA. While it is not currently possible to recommend omega-3 PUFA as either a mono- or adjunctive-therapy in any mental illness, the available evidence is strong enough to justify continued study, especially with regard to attentional, anxiety and mood disorders. PUFA are so-called because they are not 'saturated' with hydrogen atoms at multiple (poly) locations within the molecule and, as a result, contain two or more carbon-carbon double bonds. They form one of the three main classes of fatty acids, the others being saturated, in which all available hydrogen atom positions are filled, and monounsaturated, in which a single carbon-carbon double bond exists. PUFA are subdivided into the omega-3 (n-3) series (the first double bond is 3 carbons from the end (omega) carbon atom of the molecule) that are synthetically derived from linoleic acid (LA), and the omega-6 (n-6) series which are derived from alpha-linolenic acid (ALA), both 18 carbon atom containing fatty acids. LA and ALA are termed essential fatty acids because mammalian cells are unable to synthesize these fatty acids from simpler precursors. LA can be converted sequentially via a biosynthetic pathway into other omega-6 fatty acids, the 18 carbon gamma linolenic acid (GLA), and the 20 carbon arachidonic (AA) and dihomogammalinolenic acids (DGLA). Similarly, ALA is converted into longer chain omega-3 fatty acids such as 20 carbon eicosapentaeoic acid (EPA) and 22 carbon docosahexaenoic acid (DHA). Increasing evidence indicates, however, that although LA and ALA Omega-3 PUFA are of particular interest from a nutritional standpoint since the intake of these fatty acid is considered to be low in Western diets . They haThis review considers the findings of the clinical trials that form the basis of these developments and whether or not they offer convincing support for the use of omega-3 PUFA as a treatment for mental illness. To accomplish this we utilized PubMed and PsycINFO to identify placebo-controlled clinical trials of omega-3 fatty acids in psychiatric disorders. The strategy employed was to search for articles which contained search terms from list 1  and from list 2 . We also searched using Pubmed for omega-3, n-3, alpha linoleic, eicosapentaenoic, docosahexaenoic, EPA, DHA, or ALA limited by clinical trial. We reviewed these trials with respect to their findings, statistical analysis and overall trial quality.For all trials we assessed the evidence that trial participants who had received omega-3 PUFA had improved psychopathology compared to those who received placebo treatment. This was accomplished by extracting or calculating the change-from-baseline scores for the appropriate clinical rating scale for each disorder, and then calculating the mean difference in change-from-baseline scores between the placebo and omega-3 PUFA groups. A standardized or unstandardized difference value of zero indicates that there was no difference in the effect upon psychopathology between the two groups. For the attention deficit hyperactivity disorder (ADHD) and depression trials we constructed Forest plots of the standardized mean differences in change-from-baseline scores for the comparable trials. For the depression trials, we calculated an inverse-variance-weighted overall estimate of effect using the random effects method  as well The quality of the trial design was assessed using the Jadad scoring system  which taThe effect of omega-3 PUFA upon psychopathology was investigated in a range of psychiatric disorders that we will consider by general disease class.Attention deficit/hyperactivity disorder (ADHD) is a common disorder of school age children . Estimates vary but the prevalence of ADHD is thought to be in the range of 2 – 7% of children . As the Over 80% of children with ADHD in the US are treated with stimulants such as methylphenidate . While efficacious in some patients, these medications have significant side effects including decreased appetite, insomnia, impaired growth and irritability . AlthougInterest in the interaction between diet and attentional disorders is currently high with Joshi and colleagues  reportinThe three other trials differed from the others in that they did not utilize children with formally diagnosed ADHD but rather ADHD-related symptoms, as determined using one or more rating scales. As such the subject groups are likely to be more heterogeneous than found in the trials conducted by Voigt and colleagues  and by Hp < 0.00001. Information provided in the report suggests two possible explanations. Firstly, the authors state [A further trial  used a srs state  that thers state  that "grThe most recent trial  assessina priori hypothesis. Furthermore, there was not sufficient comparable data to attempt to calculate an overall estimate of effect. From these limited data it appears that teachers may tend to report a greater beneficial effect than do parents, and that there may be a greater effect on inattention than on hyperactivity.The variety of disorders and outcome measures used in the ADHD-related trials made direct comparison amongst them difficult. Three trials ,30,32 utIn summary, omega-3 containing PUFA supplements did not perform better than placebo in children with clinically diagnosed ADHD, but may have had a modest effect on attention and hyperactivity symptoms in children with DCD. The data are, however, inconclusive at this time. A trial of a similarly formulated supplement to that used in the DCD trial  in cliniThe investigation of the efficacy of omega-3 PUFA in anxiety disorders is a field in its infancy. Anxiety disorders comprise a variety of disorders characterised by abnormal fears and phobias such as panic disorder, generalised anxiety disorder, obsessive compulsive disorder and social phobia . To dateFatty acids have also been the focus of intense study in the psychotic disorder schizophrenia . Schizopa priori outcome measure, however, and, in any case, the clinical significance of a 25% improvement in symptoms is unclear [Despite the inconsistent nature of the fatty acid abundance data, a number of clinical trials utilising fatty acid supplementation in schizophrenia defined according to DSM criteria have been carried out. The earliest trials utilized n-6 fatty acids, mainly gamma linoleic acid. The outcome of these small studies was largely negative . More re unclear . The aut unclear . The triPeet and Horrobin  followeda priori outcome measure was intended to be scores at 12 weeks, the difference between the two groups had in fact diminished by this time point. Unfortunately, the statistical analysis and trials outcomes were only reported in detail for the 9 week scores, although the authors do not highlight this point in their analysis. The failure to use the a priori outcome measure, along with deficits in other reporting aspects of the trial, diminishes our confidence in the robustness of this trial's findings.Besides the researchers lead by Peet, two other groups have investigated the use of EPA in schizophrenia using double-blind placebo controlled trials. Fenton and colleagues  administIn general, clinical trials of omega-3 fatty acids in schizophrenia, despite being mostly of high quality, have produced inconsistent results and small effects sizes of doubtful clinical significance. Although the trial by Peet and Horrobin  does repPersonality disorders  are a type of mental illness in which patients form inflexible patterns of actions and thoughts . They arMajor depressive disorder (MDD) is a serious affective illness  with a lSuch studies have lead several investigators to conduct clinical trials of omega-3 fatty acids in patients with depressive disorders Table . The triAn apparent antidepressant effect of ethyl-EPA was also reported by Nemets and colleagues . These iMost recently, omega-3 PUFA supplementation is reported to be beneficial in depression occurring in children. Childhood depression is less common than that in adults but is nevertheless estimated to affect 2 – 4% of children . Nemets not efficacious [Like MDD, bipolar disorder (BD) is a disease of mood . It is licacious  . Keck and colleagues  administThe results of the bipolar disorder trials are somewhat supportive of an anti-depressant effect of omega-3 fatty acids similar to that observed in MDD, although one trial did reported omega-3 PUFA to be ineffective. One concern of treating depression in patients with bipolar disorder is that the therapy will exacerbate manic episodes. However none of the trials indicated that omega-3 PUFA has any effect upon mania, either negative or positive. Since all trials utilized supplements containing predominantly EPA, no conclusions can be drawn with respect to the relative efficacy in BD, if any, of the various species of omega-3 PUFA.There is evidence of omega-3 PUFA deficiency in women who develop postpartum depression (PPD) . PPD is 2) is markedly reduced. The predicted overall standardised mean difference  of EPA or a predominantly EPA mixture were used would be 1.18, whereas for a DHA or predominantly DHA mixture, the predicted overall SMD would be 0.06 . This suggests that EPA, rather than DHA, is efficacious in the alleviating the symptoms of depression, with an SMD value suggesting that EPA has a major effect upon the symptoms of depression. Two points should be note however. Firstly, as we have previously discussed, the negative DHA trial conducted by Silvers and colleagues [The use of the comparable ratings of depression by most investigators allowed us to compare the trials to determine if there was evidence that omega-3 PUFA had a significant mood-elevating effect in either MDD or bipolar disorder. Combining all of the MDD and bipolar trials using meta-analytical techniques shows that omega-3 fatty acid supplementation is significantly more effective than placebo in the treatment of depression Figure . The maglleagues  may havelleagues  utilisedand mood disorders. Moreover, if omega-3 PUFA were found to enhance dopaminergic and/or serotonergic neurotransmission, then the existing pharmacological data would predict that they should be effective in the treatment of mood, attentional and anxiety disorders. The literature is, however, ambivalent. An extensive series of experiments conducted by Chalon and colleagues [The clinical trials data is therefore supportive of n-3 PUFA playing a significant role in mood disorders, while providing weaker support for their ability to enhance attention and reduce anxiety, and little or no support for the amelioration of the symptoms of schizophrenia. Furthermore, for mood disorders, there is some evidence that EPA is more efficacious then DHA in reducing the symptoms of depression. We will now briefly consider whether the psychoactive characteristics of omega-3 PUFA are biologically plausible from a neuroscientific perspective, and how the findings emanating from the clinical trials might be explained . A usefulleagues  has indilleagues ,93. Morelleagues . Such filleagues  results lleagues ,65. Morelleagues . Using alleagues , but whilleagues , both oflleagues . Dopaminlleagues  would prlleagues  used is lleagues .inhibits this signaling pathway. Furthermore, although EPA is a minor brain fatty acid compared to the abundant DHA [At the biochemical level how might omega-3 PUFA deficiency and supplementation affect the nervous system? The conventional view  of omegadant DHA , both omdant DHA , a findiin vitro observation obtained using the U937 monocyte derived cell line which indicates that long term supplementation with EPA can increase the stimulated release of AA from membrane phospholipids, whereas DHA has no effect [2 [Differential effects of EPA and DHA have, however, been reported (for example see  and 105105). Of o effect . This roffect [2 . Althougffect [2 . Indeed,ffect [2 . Such a and AA, whereas a 1% EPA diet increased DHA and EPA but decreased AA levels in erythrocytes [and omega-6 PUFA into the membrane, but at higher doses they decrease omega-6 incorporation. It is of interest to note that of the two clinical trials examining the ability of omega-3 PUFA to ameliorate attentional deficits which reported a significant improvement in symptoms, in both cases a combination of omega-3 and omega-6 PUFA were administered [Stimulation of AA-dependent signaling by EPA has also been suggested by Horrobin and colleagues, but via a different mechanism: the ability of omega-3 PUFA to increase the abundance of AA in membrane phospholipids . This aghrocytes . A similhrocytes . It is lhrocytes . The mechrocytes . As suchnistered ,32. It wnistered , would hnistered ,81. On tnistered , althougnistered ,112. Altnistered . Thus, aBesides their role as modulators of AA-dependent signaling, it is emerging that both EPA and DHA are precursors of bioactive metabolites in their own right. For example, EPA can be metabolised by cyclooxygenase to form the 3-series prostaglandins . Most reThe available data are not supportive of omega-3 PUFA being efficacious in the treatment of schizophrenia or borderline personality disorders, while the investigation of their use in anxiety disorders is at an early stage. Of all the illnesses considered, the strongest evidence supports the use of omega-3 PUFA in the treatment of mood disorders with approximately 1 – 2 g/day of omega-3 PUFA appearing to be effective. An ability of omega-3 PUFA to treat attentional deficits is suggested by some trials although strong evidence is lacking. Larger trials will hopefully be conducted which allow more definitive statements to be made regarding the efficacy of omega-3 PUFA in these disorders over longer time periods, as well as determining the most beneficial omega-3 PUFA formulation to use.All of the clinical trials described in this review had one conclusion in common – that omega-3 fatty acids had no serious side effects . These compounds, therefore, represent an attractive and novel treatment modality. Although the clinical trials varied in quality of design and tended to be small in terms of participants, further investigation of the use of omega-3 PUFA to treat at least some mental health problems is warranted. Many recent trials have utilised omega-3 formulations containing a high proportion of EPA compared to other omega-3 types such as DHA. We did find some evidence to support the use of such a high-EPA formulation for treatment of depressive symptoms although more study of the possibly differential efficacies of omega-3 PUFA species is required. Moreover, it should be noted that some evidence suggests that the use of formulations containing only EPA and lacking any DHA may actually be less effective than an EPA/DHA mixture, and that combinatorial therapies which include mixtures of both omega-3 and omega-6 PUFA should be considered . This maOverall, based on the available evidence, it is not possible to firmly recommend omega-3 PUFA as either sole or adjunctive treatment for any of the mental illnesses considered. On the other hand, the research work done to date is certainly exciting and offers a strong foundation on which to base future clinical investigations.The author(s) declare that they have no competing interests.BR, JS and LS all contributed to the collation of trials data and their critical review. LS performed the meta-analysis. All authors have read and approved the final manuscript.


Tumour-associated monoclonal antibodies  radiolabelled with iodine-131 were given intracavitary  to patients with malignant effusions. Ten out of 13 effusions  responded completely with no fluid reaccumulation between 3 and 18 months. No clinical or other toxicity was observed. This new method of treatment for recurrent malignant effusions is non-toxic and effective resulting in improved quality of life, and, in some cases, prolongation of survival.


Oryza sativa (rice) and Zizania latifolia manifested substantial methylation re-patterning and rampant mobilization of two TEs, a copia retrotransposon Tos17 and a MITE mPing. It was not known however whether other types of TEs had also been transpositionally reactivated in these introgressants, their relevance to alteration in cytosine methylation, and their impact on expression of adjacent cellular genes.It is widely recognized that interspecific hybridization may induce "genome shock", and lead to genetic and epigenetic instabilities in the resultant hybrids and/or backcrossed introgressants. A prominent component involved in the genome shock is reactivation of cryptic transposable elements (TEs) in the hybrid genome, which is often associated with alteration in the elements' epigenetic modifications like cytosine DNA methylation. We have previously reported that introgressants derived from hybridization between Dart TE family was transpositionally reactivated followed by stabilization in all three studied introgressants  derived from introgressive hybridization between rice (cv. Matsumae) and Z. latifolia, while the TEs remained quiescent in the recipient rice genome. Transposon-display (TD) and sequencing verified the element's mobility and mapped the excisions and re-insertions to the rice chromosomes. Methylation-sensitive Southern blotting showed that the Dart TEs were heavily methylated along their entire length, and moderate alteration in cytosine methylation patterns occurred in the introgressants relative to their rice parental line. Real-time qRT-PCR quantification on the relative transcript abundance of six single-copy genes flanking the newly excised or inserted Dart-related TE copies indicated that whereas marked difference in the expression of all four genes in both tissues (leaf and root) were detected between the introgressants and their rice parental line under both normal and various stress conditions, the difference showed little association with the presence or absence of the newly mobilized Dart-related TEs.We document in this study that the Dart-related TEs in the parental rice line (cv. Matsumae), which was accompanied with a moderate alteration in the element's cytosine methylation. Significant difference in expression of the Dart-adjacent genes occurred between the introgressants and their rice parental line under both normal and various abiotic stress conditions, but the alteration in gene expression was not coupled with the TEs.Introgressive hybridization has induced transpositional reactivation of the otherwise immobile  It is widely recognized that hybridization between genetically differentiated natural plant populations may cause structural genomic changes  as well as perturbation of epigenetic state of the recipient genome , and both may result in heritable phenotypic novelties -7. TheseZizania latifolia (a distantly related species to Oryza) into rice has caused an array of genetic and epigenetic instabilities in the recipient rice genome [copia retrotransposon Tos17 and a MITE (mPing) [Zizania introgressants was confined to these two elements or other TEs also experienced reactivation.At least circumstantial evidence has indicated that for the hybridization- associated genomic shock to occur, a symmetric hybrid genome is not a prerequisite; instead, introgression or integration of "foreign" chromatin or DNA segments via introgressive hybridization or other means  might as well produce the "shocking" effects on the recipient genome . For exae genome ,20, and  (mPing) . Given t (mPing) , it is iDart transposon family belongs to the hAT superfamily of class II TEs, and which was first characterized by Fujino and colleagues [Dart was found as transcriptionally active in several rice tissues [Dart and its deletion-derivative called nDart can be transpositionally active in certain rice genotypes that harbour active Dart, even under normal growing conditions [Dart copies can be reactivated by 5-azacytidine treatment [Tos17 and mPing, the Dart/nDart represents another family of highly active TEs endogenous to the rice genome.The rice lleagues . Dart wa tissues . Moreovenditions . In addireatment ,26. AppaDart TE family was transpositionally reactivated in the same set of rice-Zizania introgressants that showed rampant mobilization of Tos17 and mPing [The aim of this study was to investigate (1) whether the nd mPing ; (2) wheDart1 [Dart-related elements to amplify a 296 bp fragment within the ORF region, designed by Fujino et al. [HindIII-digested genomic DNAs of the three representative introgressants of rice-Zizania  together with their recipient rice parental line (Matsumae) and the donor species Z. latifolia. Since there are only two restriction site of HindIII within the full-length Dart , the Southern blotting results suggest that most of the Dart copies were likely transpositionally reactivated in the rice-Zizania introgressants, and produced an array of excisions and reinsertions in the introgressants  developed by Tsugan et al. (2006) [iDart1-51/nDart1-3 and iDart1s/nDart1-101s subgroups with the primer pairs used, indicating they were mainly targeting to overlapping genomic regions , we suspect that these bands were not resulted from TD, but from the homo-amplification by the MseI-adaptor primers in the silver-stained profiles, in which all amplicons were visible . To test this, we sequenced 17 bands isolated from the Z. latifolia lanes; mainly those bands the positions of which were either identical or proximal to bands existing in the introgressant(s) but not in the rice parent were selected to maximize the likelihood of detecting possible Zizania introgression. We found that all the sequenced bands contained the same MseI-adaptor primer at both ends, and none contained the terminus of Dart-related TE that should be expected for a bona fide TD band. This suggested that (1) the novel bands in the introgressant(s) were not likely due to direct introgression from the donor species, Z. latifolia; (2) there was probably no homologue of Dart-related TEs in Z. latifolia, consistent with the Southern blotting and Dart-specific PCR amplification results   to isolas Figure  and on ts Figure . The tabde novo in one or more of the introgressants). We found that for each of the clones sequenced, the expected terminus encompassing the 19-bp terminal inverted repeat (TIR) of Dart was identified, indicating that these isolated bands were resulting from real TD rather than from homo-amplification by the adaptor primers. Further sequence analysis identified 15 and 21 of the variant TD bands as most likely resulted from excision and insertion events, respectively, because these bands contained the typical 8-bp target-site duplication (TSD) of Dart  in the TD profiles. These variant bands included 30 putative excisions (present in Matsumae but absent from at least one of the introgressants) and 30 putative insertions  at positions 678 nt and 2910 nt, respectively, and hence, in theory digestion with this enzyme would produce three fragments for each of the conserved Dart copies: the delineated internal body-region (2232 bp in length) and the 5'- and 3'-terminus together with their contiguous flanks , or there had been insertions by related or unrelated sequences within the two restriction sites, and hence producing larger-sized fragments after the enzyme restriction. Nonetheless, those Dart-related elements that gave rise to the 2232 bp band were amenable to methylation analysis of the body-region, because there had been no apparent internal truncations, as detailed below.Given our previous results that cytosine methylation of these rice-Matsumae , and theMatsumae ,24,27-30s Figure . We noteHpaII and MspI, to the BamHI-digests, and using probes specific to each of the three regions to assess their cytosine methylation state in the introgressants relative to Matsumae. We obtained the following results: (1) there had been no detectable internal truncations between the two BamHI sites, as no clear, smaller-sized bands than the expected 2232 bp were detected in this enzyme digest    to a moderate extent, methylation alteration including both hypo- and hypermethylation occurred in the introgressants relative to their rice parental line Matsumae.We next performed the second round restriction by adding each of the pair of isoschizomers, d Figure ; (3) the) Figure ; (5) com) Figure ; (7) coms Figure . Taken tDart-related TEs in the introgressants  from the whole genome sequence of Nipponbare and used them as queries for similarity search against the full-length cDNA database of rice http://cdna01.dna.affrc.go.jp/cDNA. We identified six genes meeting the criteria of unique-copy, encoding either known-function or hypothetical proteins, and identical amplification among all lines on genomic DNA templates , showed significant down- and up-regulation, respectively, in the root tissue of RZ1 and RZ2; (2) gene 2, to which a Dart copy being inserted 3469 bp upstream in one introgressant (RZ1), showed significant up-regulation in both the leaf and root tissues (but more markedly in leaf) of all three introgressants though at variable degrees; (3) gene 3, to which a Dart copy being inserted 1467 bp downstream in one introgressant (RZ1), showed significant up-regulation in the leaf tissue of introgressants RZ2 and RZ35, and in the root tissue of all three introgressants at variable degrees; (4) gene 4, for which a Dart copy residing 2062 bp upstream being excised from all three introgressants , showed significant up-regulation in the leaf tissue of all three introgressants, and in the root tissue of introgressants RZ1 and RZ35 at variable degrees; (5) gene 5, to which a Dart copy being inserted into the 9th exon in all three introgressants , showed significant up-regulation only in the root tissue of introgressant 35; (6) gene 6, to which a Dart copy being inserted into the 2nd intron in two introgressants (RZ1 and RZ2), showed significant up-regulation only in the root tissue of introgressant 35. Albeit the dramatic changes (predominantly up-regulation) in transcription of all six studied genes in the introgressants relative to in the rice parental line, we noted that the changes were not associated with the presence or absence of the newly transposed Dart-related TEs under normal growing conditions.The seemingly random distribution of the excisions and insertions of the s Figure  raised t Figures  and 5b.  Figures . The res Figures . Specifivs. their rice parental line under the various stress conditions and 5-AC treatment, though were substantially different, were again not coupled with the presence or absence of the newly mobilized Dart-related TEs. However, the fact that at least in one of the studied rice lines, expression of each of the four genes (1 to 4) was up-regulated by 5-AC treatment  and cold, which have been widely studied in rice . We alsot Figure  suggestsde novo genetic and epigenetic instabilities can be induced by the process per se, including transcriptional activation and mobilization of cryptic transposable elements (TEs) [Oryza sativa) and Z. latifolia had induced rampant mobilization of two TEs, a copia-like LTR retrotransposon Tos17 and a MITE mPing [Dart-related TEs were also transpositionally reactivated in the introgressants, although the elements were totally quiescent in the parental rice cultivar Matsumae. We validated the excisions and insertions by transposon-display (TEs) and sequencing, which ruled out genomic rearrangements as the major cause for the dramatically altered hybridization patterns detected by Southern blotting in the introgressants.Hybridization is prevalent in plants, which plays important roles in genome evolution, and may lead to speciation at both the homoploid level and followed by genome doubling  ,7,33-37.ts (TEs) ,29,38-42ts (TEs) ,17. We hTE mPing ,43. In tDart-related TEs were similarly hypermethylated along their entire length in the introgressants and their rice parental line Matsumae. Nonetheless, moderate alteration in the methylation patterns was discernible in the introgressants. Furthermore, given that the introgressants were at the 9th-selfed generation and stabilized in both phenotype and DNA fingerprinting patterns [Dart-related TEs, which however were either largely reverted to the original pattern and/or those individuals with more drastically altered patterns had been purged out during the sexual reproduction, probably due to reduced fitness. Therefore, it remains a formal possibility that alteration in cytosine methylation had been associated with mobility of the Dart-related TEs in the introgressants, a scenario gaining increased empirical support in a vast range of TEs and organisms [Although numerous studies have established correlative or causal links between TE activation and alteration in the element's cytosine methylation state ,30,44,45patterns , we coulrganisms ,28,30,46Dart-related TEs exhibited significantly altered expression in the introgressants relative to their rice parental line under both normal and several abiotic-stress conditions. However, the altered gene expression in the introgressants was not coupled with the TE excisions or insertions, suggesting that other regulatory mechanisms were responsible for the altered gene expression in the introgressants. Because unbiased amplifications between the introgressants and their rice parental line were observed when their genomic DNA was used as templates, it is likely that epigenetic regulation was involved. The observation that for most of the genes, these rice lines exhibited sharply differential response to 5-AC treatment corroborated this possibility, which also accords with our previous results showing that substantial re-patterning of cytosine methylation occurred in the introgressants for amny genomic loci [An array of studies in both plants and animals has established that activity of TEs, particularly LTR retrotransposons, may significantly impact expression and function of their adjacent genes ,41. Nonemic loci . FurtherZ. latifolia has induced transpositional reactivation of another distinct family of cryptic TEs in the parental rice genome, namely, the Dart-related TEs, suggesting that introgression of chromatin from a realted alien species might have caused a general breakdown of the host cellular machinery responsible for repressive control of TE activity. Transposition of the Dart-related TEs was accompanied with a moderate alteration in the element's cytosine methylation in the introgressants. In addition, results of this study showed that extensive alteration in expression of a set of Dart-adjacent, protein-coding genes occurred in the introgressants relative to their rice parental line, under both normal and various abiotic stress conditions. Nonetheless, the alteration in gene expression was not coupled with excision or insertion of the Dart-related TEs, implicating other regulatory mechanism(s) was underpinning the changes in gene expression in these novel rice introgressants.Results of this study have extended our previous findings by documenting that introgressive hybridization between rice and Zizania latifolia Griseb, were used in this study [th selfed generation) were homogeneous in phenotype and DNA fingerprinting patterns, and exhibited heritable, novel morphological characteristics in multiple traits compared with their rice parental cultivar Matsumae [Three introgression lines  derived from a cross between rice (cv. Matsumae) and is study . The thrMatsumae ,21. The Zizania introgressants  and their rice parental line cv. Matsumae were disinfected and thoroughly rinsed, and placed on petri-dishes covered with half-strength Murashige and Skoog (MS) medium in darkness at 25°C. For the three kinds of abiotic stress treatments, seedlings were grown to the 3-leaf-stage, and then aqueous solutions respectively containing 5 mM CuSO4 , 5 mM HgCl2 , 10 mM NaCl  and 10 mM NaHCO3  containing the half-strength MS medium were added, and grown for one more week. For cold stress, the 3-leaf-stage seedlings were grown in the medium at 12°C for one week. The 5-azacytidine (5-AC) treatment was conducted by treating the germinating seeds in the medium containing 50 mM 5-azacytidine (Sigma) for one week and then thoroughly rinsed with ddH2O and allowed the seedlings to grow in the medium up to the same stage as the other treatments. In all cases mock-control seedlings grown in the half-strength MS medium alone was included.Healthy and uniform seeds of three rice-Zizania introgressants  and their rice parental line Matsumae by a modified CTAB method. To assess possible genetic changes in the patterns of the Dart-related TEs, the genomic DNA  of each line (the mock-control) was digested by HindIII. To test for possible alteration in cytosine methylation of the Dart-related TEs, the genomic DNA of each line (the mock-control) was first digested with BamHI , followed by a second round of digestion with a pair of isoschizomers, HpaII and MspI, that recognize the same sequence 5'-CCGG but with differential sensitivity to methylation of the two cytosine residues. Digested DNA were run on 1% agarose gel and transferred onto Hybond N+ nylon membrane  by the alkaline transfer method recommended by the manufacturer.Genomic DNA was isolated from leaf tissue of young seedlings at the same developmental stage from the various treatments and mock of the three rice-Dart-related TEs were designed to amplify the fragments to be used as hybridization probes. The primers are: (1) for the 5'-region of Dart-related TEs: Dart5'-forward: 5'- aaatagggcatgaaccccagc, Dart5'-reverse: 5'-ggtcgaaatcacccaaggtg; (2) for the 3'-region of Dart-related TEs: Dart3'-forward: 5'-tccagaccaaccccagtagaa, Dart3'-reverse: 5'-aaaaaaagcaaaggaaatgtataagg; (3) for the body-region of Dart-related TEs: Dart-body-forward: 5'-ctagagaggattatcttagcgtagttgtt, Dart-body-reverse: 5'-cttcttcttacctgtagtggggatag. Authenticity of the amplified fragments was verified by sequencing. The fragments were then agarose gel-purified and labelled with fluorescein-11-dUTP by the Gene Images random prime-labelling module (Amersham-Pharmacia Biotech). Hybridizations were done with the Gene Images CDP-Star detection module (Amersham-Pharmacia Biotech). After washing twice with 0.2×SSC, 0.1% SDS for 50 min. The filters were exposed to X-ray films for chemiluminescence signal detection.In total, three pairs of primers specific respectively to the 5'-, 3'- and the body-region of the MseI and subject to transposons display (TD) following the protocol essentially as reported [Dart-related TEs were designed. Two specific primers (TDPrm1/TDPrm2 and MseI+C/G) were respectively combined with the selective-amplification primers targeting the adapters, while the most external primer (TIR+N/MseI+3) was used for further validation of the isolated TD bands. Detailed information concerning adapters and primers are listed in Additional files Dart-related TEs, and recovered for sequencing.The genomic DNA (approximately 300 ng per sample) was cut with reported . Three cDart-related TEs, the contiguous flanking regions were extracted and used to query the Nipponbare genome sequence http://rgp.dna.affrc.go.jp by BlastN, and a set of locus-specific primers each being downstream of the Dart but compatible with the Dart-specific TD primers were designed with Primer 3 http://biocore.unl.edu/cgi-bin/primer3/primer3_www.cgi and those produced the expected results were listed in Tables Dart-specific TD primers to reproduce the putative excisions and insertions identified by TD in the introgressant(s), and the amplicons were then sequenced for validation.Based on the sequencing results by identifying the expected 3'-terminus of the Total RNA was isolated from the same young leaf tissue as used for DNA isolation and also from the root tissue of the same seedlings of the various treated and mock lines, with the Trizol Reagent (Invitrogen) according to the manufacturer's protocol. The RNA was then treated with DNaseI (Invitrogen) to eliminate possible genomic DNA contamination before being reverse transcribed with the SuperScript RNase H- Reverse Transcriptase (Invitrogen).Dart-related TEs in the introgressant(s) was studied by quantitative real-time-RT-PCR using gene-specific primers  was used as internal control with the primer pairs 5'-atgccattctccgtctt and 5'-gctcctgctcgtagtc. The choice of the β-actin gene as the internal control was based on previous investigation showing that expression of this house-keeping gene between the tissues and under the various stress conditions was constant [The expression of genes adjacent to the newly excised or inserted constant . Conditiconstant .NW carried out major parts of the experiments, analyzed the data and drafted the manuscript. HW, HW, DZ, CL, YW, XO and ZD participated in all the experiments. BL designed the work and finalized the manuscript. All authors read and approved the final manuscript.Primers used in the transposon-display (TD) assay.Click here for fileGene-specific primers used in expression analysis by real-time qRT-PCR.Click here for file


The individual is exposed to a complex mixture of POPs being life-long beginning during critical developmental windows. Exposure to POPs elicits a number of species- and tissue-specific toxic responses, many of which involve the aryl hydrocarbon receptor (AhR). The aim of this study was to compare the actual level of integrated AhR transcriptional activity in the lipophilic serum fraction containing the actual POP mixture among Inuits from different districts in Greenland, and to evaluate whether the AhR transactivity is correlated to the bio-accumulated POPs and/or lifestyle factors.Human exposure to lipophilic persistent organic pollutants (POPs) including polychlorinated dibenzo-The study included 357 serum samples from the Greenlandic districts: Nuuk and Sisimiut (South West Coast), Qaanaaq (North Coast) and Tasiilaq (East Coast). The bio-accumulated serum POPs were extracted by ethanol: hexane and clean-up on Florisil columns. Effects of the serum extract on the AhR transactivity was determined using the Hepa 1.12cR mouse hepatoma cell line carrying an AhR-luciferase reporter gene, and the data was evaluated for possible association to the serum levels of 14 PCB congeners, 10 organochlorine pesticide residues and/or lifestyle factors.In total 85% of the Inuit samples elicited agonistic AhR transactivity in a district dependent pattern. The median level of the AhR-TCDD equivalent (AhR-TEQ) of the separate genders was similar in the different districts. For the combined data the order of the median AhR-TEQ was Tasiilaq > Nuuk ≥ Sisimiut > Qaanaaq possibly being related to the different composition of POPs. In overall, the AhR transactivity was inversely correlated to the levels of sum POPs, age and/or intake of marine food.i) We observed that the proportion of dioxin like (DL) compounds in the POP mixture was the dominating factor affecting the level of serum AhR transcriptional activity even at very high level of non DL-PCBs; ii) The inverse association between the integrated serum AhR transactivity and sum of POPs might be explained by the higher level of compounds antagonizing the AhR function probably due to selective POP bioaccumulation in the food chain. Some POPs e.g. PCBs and persistent organochlorine pesticides, that still are released into the environment due to ongoing use in developing countries and improper storage or disposal in developed countries, can be transported to the Arctic regions by atmospheric and oceanic currents and have been found to bioaccumulate and biomagnify in the Arctic marine food web  were evaluated using the Student t-test with statistical significant level of p ≤ 0.05 (Microsoft Excel).The AhR-TEQs values of serum extract were obtained by interpolation of the AhRag values onto the parallel TCDD dose-response sigmoid Hill curve and only the AhRag values being significantly higher than the solvent control and in the linear range of the TCDD dose-response curve were used to calculate AhR-TEQ .ad hoc tests was performed. Test for equal variances was performed using Levene's test. The least-significant difference (LSD) test was used if the variables showed equal variance; otherwise Dunett T3 test was used. The comparison of variables between the genders was performed by independent Student t-test.Natural logarithmic transformation of AhR-TEQ/AhRcomp and POP data improved the normality (checked by Q-Q plots) and homogeneity of variance, and the statistical analysis was performed on the ln-transformed data. The comparisons of different variables  among the districts were performed with One-way ANOVA test. When ANOVA showed statistical significant differences complementary multiple comparison Bivariate correlations were evaluated by Pearson correlation analyses.The overall associations between the AhR-mediated activities and POPs across the study groups and/or across genders were assessed by comparing the regression lines for each study group/gender by using multiple regression analysis.Up to date few studies on AhR transcriptional activity in human serum have been reported ,36-39, aThe inter-district variations in serum levels of POPs, AhR-TEQ and AhRcomp were also assessed by linear regression models and age was in these models considered as a potential confounder of these variables.The statistical analysis was performed on SPSS 13.0 . The statistical significant level was set to p = 0.05.As reported , the ageFor the men, similar median levels of Σ PCB_14, Σ pesticide and Σ POP were observed among Nuuk, Qaanaaq and Tasiilaq and all were significantly higher than that of Sisimiut with the order of Nuuk ≥ Qaanaaq ≥ Tasiilaq > Sisimiut. Tasiilaq and Qaanaaq women had significantly higher POPs median levels than that of Nuuk and Sisimiut women Table  as reporThe dioxin-like PCBs (DL-PCBs) determined in this study were CB105, CB118 and CB156. The Nuuk men had higher median percentage of ΣDL-PCB of the Σ PCB_14 than men from other districts (Nuuk > Tasiiaq ≥ Qaanaaq ≥ Sisimiut). No significant difference of ΣDL-PCB percentage between women from different districts was observed but a lower trend was observed for Qaanaaq women Table . Women h91% of all male and 79% of all female serum samples showed significant agonistic AhR transcriptional activity. The levels of AhR-TEQ differed significantly among the districts for the separate gender Table  &1B. TheTCDD)50. The median AhRcomp activity of men was in the order of Qaanaaq ≥ Sisimiut ≥ Tasiilaq > Nuuk, reflecting the higher percentage of samples elicited inhibition of the TCDD induced AhR activity in the serum samples of Nuuk and Tasiilaq .Pearson correlation analyses showed that most PCB congeners were significantly and mutually intercorrelated , whereas CB 28, CB52, CB128 and CB101 had a relatively lower correlation coefficients to other PCB congeners . Also the determined pesticides were intercorrelated (r > 0.75 – 0.99). The grouping variables (ΣPCB_14 and Σ pesticide) intercorrelated with r = 0.97 and the sum of determined POPs as Σ POP was therefore acceptable.Multiple regression analysis of the combined districts and genders data was performed to analyze for homogeneity/heterogeneity of the associations between POPs and AhR-TEQ/AhRcomp .Scattered and few significant correlations of AhR-TEQ/AhRcomp and the single PCB and pesticides and/or Σ DL-PCBs were observed (data not shown). Since the congeners of PCBs were highly intercorrelated and the AhR-TEQ represents the integrated activity of the POP mixture, it is hard to assess which of the compounds having the highest impact on the AhR-TEQ/AhRcomp level.As found earlier , for theAs shown in Table The AhRcomp data was also in overall inversely related to age and/or the n3/n6 ratio, but also the seabird intake seemed to be a determinant. A negative correlation for AhRcomp to seabird intake was observed for the Qaanaaq men and all men data . Also the association of the AhR transcriptional activity to the determined POPs differed between the districts and genders. We observed for the combined data an inverse association between AhR-TEQ and POPs where the Qaanaaq women had main influence. The negative association of AhRcomp activity to POPs was mainly contributed by Nuuk and Tasiilaq subjects.In the present study we compared the As reported , the POPThe pattern of regional differences of serum AhR-TEQ levels did not follow the district-distribution of POPs levels. The AhR-TEQ level of Nuuk men having the highest POP level was lower than that of Sisimiut men with the lowest POPs level, whereas Qaanaaq women with relatively higher POPs level elicited the lowest AhR-TEQ level. However, the regional differences of AhR-TEQ seemed to mimic the district variation of the ratio of the ΣDL-PCB to the ΣPCB_14. For both sexes, the DL-PCB proportion of Qaanaaq subjects was in general lower than that of Nuuk, Tasiilaq and Sisimiut  and consistently lower AhR-TEQ level was observed for Qaanaaq men and women . Similarly, male participants in all districts had higher POP burden but lower DL-PCB proportion and accordingly a tendency of lower AhR-TEQ level compared to female was observed in the present study. Moreover, we also found that individuals with higher ΣDL-PCB/Σ PCB_14 ratio tend to have higher AhR-TEQ levels while those with higher Σ POP have significantly lower AhR-TEQ than individuals with lower Σ POP (data not shown). Our study further support the previous report that DL-PCBs were major contributors of the total WHO-TEQ concentration in the plasma of Arctic Inuits . Therefos = 0.20, p = 0.001), combined with their higher AhR-TEQ level, suggesting European Caucasians were more close to DL-compounds sources.Ayotte and coworkers reported that the average plasma WHO-TEQ level of Inuit adults living in Arctic Quebec was 184 pg/g lipid , being cKnowing that the measured net integrated AhR induced activity of the lipophilic serum extract are in the linear range , and running a TCDD dose-response measurement in parallel, we suggest that the chemical mixture in serum extract can further increase or inhibit the TCDD induced AhR mediated activity (termed as AhRcomp). In the present study, the AhRcomp differed between districts and between sexes. Compared to Qaanaaq and Sisimiut men, Nuuk and Tasiilaq men had the lowest AhRcomp median level being in accordance with the higher frequency of samples eliciting inhibition of the TCDD induced AhR activity, suggesting the presence of compounds having the potency to compete with TCDD for the AhR activation. The higher incidence of further increase of the TCDD induced effect observed for Sisimiut and Qaanaaq men as well as women from all districts suggest the presence of compounds interacting with TCDD for AhR mediated effects. The differences in the incidence of further increase and inhibition of TCDD induced activity might be caused by the different POP composition of Inuits from the different districts and for genders also differences in diet intake might have an impact on the AhR function ,10.In vivo and in vitro studies have reported that PCB mixtures and some individual PCB congeners antagonized TCDD effects [Humans are exposed to a combination of several xenobiotics. It is likely that the interaction between these contaminants, additivity and/or synergy and/or antagonism, will be reflected in the final toxic effect.  effects ,46, and  effects . A mixtu effects . For the effects ,17,23, w effects ,48 and H effects . Chlorda effects . Further effects -54 or an effects ,56. Prev effects ,57. More effects ,58. Also effects . As humaThe biomagnification of PCBs in the aquatic food-chain is congener-specific. For most marine mammals, non-DL-PCBs are observed more resistant to biodegradation than DL-PCBs  and a prIn overall, the serum AhR-TEQ/AhRcomp negatively correlated to age and n-3/n-6 ratio, being in accordance to the frequency of marine food intake and POPs bioaccumulation over time. For the separate districts and genders a scattered correlation of AhR-TEQ/AhRcomp and lifestyle factors was observed. This non-consistent pattern might be explained by relative low statistical power for the separate gender and/or districts and needs further confirmation.The inverse correlation of AhR-TEQ to seafood intake given by the n-3/n-6 ratio, suggests that the AhR transcriptional activity might follow the intake of n-6 fatty acids possibly with the origin from non-marine diet being supported by our previous report . Moreover, it was reported that the important source of DL-compounds for the population of the western world is dairy food intake -66. In tAlthough the correlations found in the present study were weak, the chance finding may be excluded because the significant correlations were higher than 5%.In the present study, the AhR transcriptional activation of serum extract was measured as an integrated net AhR-TEQ value without providing information on the levels of individual congeners. This bioassay responds to environmental contaminants acting through the AhR-pathway and allows the detection of chemicals that are not included in the chemical analytical method based on the standard TEF scheme. However, as discussed previously , upon seThe POPs and serum AhR transcriptional activity differed among Greenlandic districts, suggesting that the difference of serum AhR transcriptional activity depends on the composition of bioaccumulated POPs. The observed inverse correlation of POPs and AhR-TEQ or AhRcomp data suggests the presence of compounds with antagonistic impact on the AhR signalling pathway, probably due to the selective PCB bioaccumulation in the food chain. Further study is needed to elucidate this preliminary conclusion.AhR aryl hydrocarbon receptorAhRag agonistic AhR activityAhRcomp competitive AhR activityCALUX Chemical activated luciferase gene expressionDDT 2, 2-bis (p-chlorophenyl)-1, 1, 1-trichloroethaneDL dioxin-likeGC-MS gas chromatography – mass spectrometryHCB hexachlorobenzeneβ-HCH HexachlorocyclohexanePAHs polycyclic aromatic hydrocarbonsPCDDs/PCDFs polychlorinated dibenzo-p-dioxins/furansPCBs polychlorinated biphenylsPOPs persistent organic pollutantsTCDD 2,3,7,8-tetrachlorodibenzo-p-dioxinTEFs Toxic Equivalency FactorsTEQs TCDD toxic equivalentsThe author(s) declare that they have no competing interests.ML and EBJ were responsible for design of the AhR transcriptional activity study and preparation of the manuscript; ML performed the mechanistic work and data evaluation. BD has provided the epidemiological data concerning POPs, questionnaires and fatty acids. All authors have read and approved the final manuscript.Multiple regressions of the combined study groups. The data provided represents the statistical analysis of the homogeneity/heterogeneity of the associations between POPs and AhR-TEQ/AhRcomp among the districts and genders.Click here for file


Febrile neutropenia carries a high risk of sepsis complications, and the identification of biomarkers capable to identify high risk patients is a great challenge. Angiopoietins (Ang -) are cytokines involved in the control microvascular permeability. It is accepted that Ang-1 expression maintains endothelial barrier integrity, and that Ang-2 acts as an antagonizing cytokine with barrier-disrupting functions in inflammatory situations. Ang-2 levels have been recently correlated with sepsis mortality in intensive care units.We prospectively evaluated concentrations of Ang-1 and Ang-2 at different time-points during febrile neutropenia, and explored the diagnostic accuracy of these mediators as potential predictors of poor outcome in this clinical setting before the development of sepsis complications.Patients that evolved with septic shock (n = 10) presented higher levels of Ang-2 measured 48 hours after fever onset, and of the Ang-2/Ang-1 ratio at the time of fever onset compared to patients with non-complicated sepsis (n = 31). These levels correlated with sepsis severity scores.Our data suggest that imbalances in the concentrations of Ang-1 and Ang-2 are independent and early markers of the risk of developing septic shock and of sepsis mortality in febrile neutropenia, and larger studies are warranted to validate their clinical usefulness. Therapeutic strategies that manipulate this Ang-2/Ang-1 imbalance can potentially offer new and promising treatments for sepsis in febrile neutropenia. Despite improvements in supportive care, sepsis remains the most common cause of death in intensive care units, with mortality rates of 30% to 50 ,2. RecenFebrile neutropenia (FN) in patients with hematologic malignancies is characterized by increased susceptibility to sepsis complications, and a higher risk of septic shock, with mortality ranging from 2-21% . Most paRecruitment of patients took place at the Bone Marrow Transplantation Unit of University of Campinas between March 2008 and March 2009. Patients were included if they fulfilled the following criteria: (1) diagnosis of hematological malignancies, and (2) admission as inpatients for intensive chemotherapy  or hematopoietic stem-cell transplantation (HSCT). Patients were invited to participate before the initiation of any chemotherapy regimen. Fever (T ≥ 38.0°C) at admission was the only exclusion criteria. The study was performed in accordance with the Declaration of Helsinki and approved by the local Ethics Committee. Informed written consent was obtained from all patients prior to collection of samples. Only patients that presented fever during neutropenia (defined as a neutrophil count <500 μl) were included in the second phase of the study. Descriptive data consisting of demographics, diagnosis, clinical data, and disease severity scores were obtained from the medical records. Twenty healthy individuals volunteered to determine a normal reference range for Ang-1 and Ang-2 levels .2 < 32 mmHg, and a microbiologically proven or clinically evident source of infection. Septic shock was present in patients in which sepsis was complicated with hypoperfusion or hypotension , despite adequate volume resuscitation. Patients were subdivided into two outcome groups: sepsis (non-complicated) and septic shock. Severity of illness was assessed by calculating the Sequential Organ Failure Assessment (SOFA) score [An infectious etiology was assumed for all patients with post chemotherapy neutropenia with new onset fever, in accordance with FN management protocols. Blood and urine cultures were immediately obtained and broad-spectrum antibiotics were initiated . Sepsis,A) score  daily afA) score ,22.Venous blood was drawn at enrollment (baseline), within 12 hours after first episode of neutropenic fever, and 48 hours thereafter. Samples were immediately centrifuged at 3000 rpm  and plasma and serum was stored at -80°C until analysis. Samples were processed by the same investigator. Serum levels of Ang-1 and Ang-2 were measured in duplicate using a commercial enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer's instructions. Interassay coefficient of variations were 2.95% for Ang-1 and 6.87% for Ang-2 samples with concentrations within the range observed in our study. Von Willebrand Factor (VWF) levels were measured in duplicate in citrate plasma samples using a rabbit anti-human VWF peroxidase conjugate .p value less than or equal to 0.05 was considered statistically significant. All statistical analysis were performed with the SPSS package  and the GraphPad Prism Software .Patients were divided in two outcome subgroups according to the presence of absence of septic shock at any time point before the resolution of FN. Differences in continuous variables between patients from each subgroup, and between patients and healthy controls were analyzed using the Mann-Whitney rank sum test. Categorical variables were compared using the Fisher's exact test. Data are expressed as median and range unless otherwise stated. Correlation (Spearman's rank correlation) and linear regression analysis were performed between sepsis severity scores and angiopoietin concentrations. Receiver operator characteristics (ROC) procedures were used to identify optimal cut-off values of angiopoietin concentrations to differentiate patients with non-complicated sepsis and patients with septic shock. A second analysis was performed to explore the effect of angiopoietin concentrations on 30-day mortality, calculated from the day of fever onset. Survival curves were estimated using the Kaplan-Meier method. Parameters independently associated with survival were identified by univariate and multivariate Cox proportional hazards models. Variables found to be statistically significant at a 10% level in the univariate analyses were included in the multivariate model. Different models were established, and variable selection was performed by different forward and backward procedures, with comparable results. A A total of 60 patients fulfilled the primary criteria for study entry, of which 41 patients experienced neutropenic fever and completed the study Figure .At baseline, no difference in Ang-1 levels could be observed between patients with non-complicated sepsis and with septic shock Table . No statAt baseline, no statistically significant difference could be observed in levels of Ang-2 between patients from the two groups  compared to patients with non-complicated sepsis . After 48 hours, the Ang-2/Ang-1 ratio was higher in patients that developed septic shock  compared to patients with non-complicated sepsis , but statistical significance was not reached (Mann-Whitney test: P = 0.06)  and Ang-2/Ang-1 ratio (both at fever onset and 48 hours thereafter). We then performed multivariate Cox regression analysis incorporating these variables, and the only variable that remained statistically significant in the multivariate setting was the Ang-2/Ang-1 ratio, measured at fever onset . Figure Post chemotherapy FN in patients with hematological malignancies is a condition that carries a high risk of sepsis complications with mortality rates as high as 21% , usuallyAngiopoietins are a family of vascular growth factors with critical roles in embryonic and postnatal angiogenesis. Ang-1 and Ang-2 both act on the Tie2 tyrosine kinase receptor found primarily on endothelial cells, but appear to play antagonist roles . ApparenIn our prospective study we explored the time-course of Ang-1 and Ang-2 in a population of patients with high risk of sepsis complications before the development of severe sepsis. As far as we are aware, the significance of Ang-1 and Ang-2 levels has not been studied in patients with FN. An additional contribution of our study is that we prospectively evaluated the significance of Ang-1 and Ang-2 levels at an earlier time-point in the development of sepsis (which developed in a median time of 4.1 days after fever onset), as opposed to studies that evaluated patients at the ICU. This difference is evidenced by the median SOFA score of our patients (4 range 1-8) compared to higher median SOFA scores (16 range 1-22) from other key studies . Last, AFor this study we only included patients with hematological malignancies and FN after intensive chemotherapy that were treated as inpatients from day one of chemotherapy until the resolution of neutropenia. By doing so, we intended to obtain a representative sample of patients with high risk of sepsis complications. Furthermore, the fact that all patients were treated as inpatients allowed us to standardize important variables that could affect the validity of our results such as the time between fever onset and sample collection. So as to limit as much as possible the influence of different diagnosis and chemotherapy regimens on our results, Ang-1 and Ang-2 levels were also collected immediately before the initiation of chemotherapy, allowing differences between outcome groups to be compared not only as absolute values, but also as fold-increase from baseline levels, which did not alter our results (data not shown). In fact, neither Ang-1 nor Ang-2 levels were significantly different before the initiation of chemotherapy between patients with non-complicated sepsis and septic shock, supporting the uniformity of our group of patients as far as the two study variables were concerned.The main finding of our study is that the relative concentration of Ang-1 and Ang-2 are different in subgroups of patients with FN that evolve to non-complicated sepsis compared to patients that develop septic shock, and that evaluation of these two proteins within the first 48 hours after neutropenic fever, before the development of any signs and symptoms of septic shock, is a promising tool to discriminate high risk patients with FN. In accordance with previous studies, Ang-2 levels were significantly higher in patients with septic shock compared to patients with non-complicated sepsis. This difference was not present at the time of fever onset, rose sharply and became evident after 48 hours, when levels in the poorer outcome group were 8 times higher than in the good outcome group. Importantly, Ang-2 baseline levels were similar. As in previous studies, we were not able to detect any significant difference of Ang-1 levels between study groups. However, when the relative concentrations of these two antagonistic cytokines were evaluated, we did observe a relative deficiency of Ang-1 compared to Ang-2 levels in patients that developed septic shock, that was evident early at the time of fever onset. It has been known for more than a decade that Ang-1 can protect adult vasculature against VEGF-induced plasma leakage . Ang-1 hRecently, the time course of Ang-1 and Ang-2 release was evaluated in a model of human endotoxemia , which sThere are certain limitations of our study that need to be acknowledged to avoid overinterpretation of its conclusions. Research about new diagnostic tools follow a sequence of phases along which the questions answered by each phase progress from the demonstration that a biomarker behaves differently in diseased and normal individuals (phase I) to the ultimate demonstration of improved clinical outcomes after its incorporation (phase IV) . Ours isIn summary, our data suggest that imbalances in the concentrations of Ang-1 and Ang-2 present already at the time of the first fever peak is an independent marker of the risk of developing septic shock and of 30-day mortality in FN. Further studies are warranted to validate this new biomarker as clinically relevant tool in the daily care of these patients. In addition, therapeutic strategies designed to manipulate the Ang-2/Ang-1 imbalance can offer a new and promising paradigm for the treatment of sepsis and septic shock in patients with FN.The authors declare that they have no competing interests.BEA enrolled patients, recorded clinical data, performed laboratory analysis and contributed to manuscript production; SALM and TMGS performed laboratory analysis; FJPA performed statistical analysis and reviewed the manuscript; CAS, IL and JMA contributed to the study design and reviewed the manuscript; EVDP designed the study, analyzed data and contributed to manuscript production. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/143/prepub