Helicobacter pylori (HP) infection appears protective against oesophageal adenocarcinoma (EA) risk. Matrix metalloproteinases (MMPs) are released in the presence of HP infection. In MMP2 wild-type individuals, HP was significantly protective of EA risk . Matrix metalloproteinases may modulate the EA–HP relationship. Helicobacter pylori (HP) infection protects against EA  . EpidemiA number of factors influence the chronic atrophic gastritis severity. Myeloperoxidase (MPO) released from inflammatory cells and manganese superoxide dismutase 2 (SOD2) enhance inflammation and tissue damage, thereby increasing the severity of chronic atrophic gastritis . A total of 101 age- and gender frequency-matched healthy controls were composed of lifetime cancer-free, GERD-free, non-blood-related family members  and friends of other cancer/surgical patients. For all participants, a standardised interviewer-administered questionnaire collected information on age, gender, race, body mass index, smoking status/history and HP infection status. Body mass index in the third decade of life was used as a surrogate of healthy adult weight decades prior to the development of EA. Over 90% of the participants were born in the United States.Local institutional review boards approved the study. All cases gave written consent, were >18 years old and were diagnosed within the last 6 months. All had histologically confirmed EA that was deemed endoscopically (or at the time of resection) to have a tumour centre located at or above the midpoint of the gastro-oesophageal junction, with at least two-thirds of the tumour bulk located in the oesophagus  using the ABI Prism 7900HT Sequence Detection System ; conditions and primers are available upon request. SOD2 Ala16Val and MPO −463 G/A genotyping was performed as described previously . The Helicobacter pylori infection status and CagA status were evaluated using Helicoblot 2.1 .Serum was processed within 1 h of collection and stored at −70°C, with no freeze–thaw cycles prior to analyses, and was run in mixed blinded batches. For quality control determinations of laboratory techniques, positive and negative controls and blinded duplicate samples were run. Alternative genotyping approaches  to verify technical reliability and accuracy were used as required. A second scientist checked all laboratory interpretation independently, and a blinded third scientist arbitrated discrepancies.t-tests, where appropriate. If homozygous variant genotype frequencies were very low, heterozygous and homozygous variant genotypes were combined. Hardy–Weinberg equilibrium was tested using the χ2 test. Using standardised kit criteria, we defined four different categories of HP infection status based on the antibodies detected with the Helicoblot 2.1 kit: ever infection, current infection, infection with CagA or infection with VacA strains . Where appropriate, crude and adjusted odds ratios (AOR) and 95% confidence intervals (CI) for the risk of EA were calculated. For adjusted analyses, we included adult body mass index, smoking status, age and gender. To test for a gene–HP interaction, an interaction term was added to the logistic regression model (likelihood ratio tests). The SAS macro HAPPY was used for haplotype determination and n=9), IIA (n=18), IIB (n=17), III (n=22), IVA (n=5) and IVB (n=29). The proportions of cases and controls treated for HP infection were 16 and 12%, respectively (P>0.30). There were no overall associations between each polymorphism and the EA risk. The overall AOR between HP infection and EA was 0.71 (95% CI=0.4–1.0). Duplicate genotyping was performed for at least 30% of subjects for each polymorphism with 100% concordance. Serologic analysis had 99% concordance of 100% duplicates.Baseline demographics are shown in MMP2 −1306 polymorphism and HP infection: in 58 cases/56 controls carrying the MMP2 −1306C/C wild-type genotype, having HP infection at any time in their life was strongly protective against EA . In contrast, in 35 cases/43 controls carrying MMP2 C/T or T/T (associated with lower promoter activity), this protective effect was lost . When we specifically analysed different definitions of HP status such as current, VacA+ or CagA+ infections for their HP–EA risk association, the protective effect of HP remained significant in the wild-type genotype of MMP2 −1306C/C (AOR ranged from 0.16 to 0.35) and abrogated in patients carrying any variant allele. The corresponding interaction model and interaction term were statistically significant (P<0.001). When using several other definitions of HP infection status, the MMP2–HP interaction terms were similarly significant: MMP2 and CagA+ infection , and MMP2 and current HP infection . Thus, both stratified analysis and interaction models point to an MMP2–HP relationship.The strongest and most consistent association was found between the MMP3 −1171 6A/6A  and MMP12 −82 A/A , but not for their corresponding variant genotypes. The MMP1 −1607, MMP3 −6A/5A and MMP12 −82A/G polymorphisms are in linkage disequilibrium , but none of the evaluated polymorphisms are linked with the MMP2 −1306 C/T polymorphism . Haplotype analyses of the MMP1-MMP3-MMP12 polymorphisms resulted in weaker associations when compared with the analyses of individual polymorphisms (data not shown).When evaluating other MMP polymorphisms, ever infection with HP was also associated with a significantly decreased EA risk in the subset of patients who carried the wild-type genotypes, MPO −463 G/A or with SOD2 Ala16Val.No significant protective effects of HP infection on the EA risk were found with any genotype subsets of To our knowledge, the question of gene–HP interaction in EA risk has not been addressed. The protective effects of HP infection on the risk of developing EA have been postulated, based on a number of epidemiologic studies (MMP2 −1306 C/C genotype, whereas no protective effect could be detected among the variant genotypes. MMP2 is part of the MMP family, a group of enzymes responsible for the degradation of extracellular matrix components. MMP2 levels were increased in the presence of HP infection and contributed to tissue damage in HP-associated gastritis (MMP2 expression (MMPs led to similar results.The most significant finding of our study was the increased protection against the risk of developing EA by HP infection in individuals carrying the wild-type MMP polymorphisms were associated independently with gene–HP interactions, this is a novel finding that warrants further exploration.Our results should be interpreted cautiously, given the modest sample size and multiple comparisons, although even a Bonferroni correction would still leave the primary gene–HP interaction and stratified wild-type models statistically significant. In addition, the serologic evaluation of prior HP infection over time is imperfect, and may be affected by antibiotic treatment. Nonetheless, because not just one but several In conclusion, we were able to demonstrate for the first time a modulation of the protective effect of HP on EA risk by several polymorphisms in the MMP pathway. These intriguing results will need confirmation in a larger prospective setting, particularly one that also explores the relationships between Barrett's oesophagus, GERD and EA prognosis.


Strongyloides ratti, comparing single and repeated dose infections, on the host immune response that was elicited. We considered and compared infections of the same size, but administered in different ways. We considered infection size in two ways: the maximum dose of worms administered and the cumulative worm exposure time. We found that both infection regimes resulted in Th2-type immune response, characterised by IL4 and IL13 produced by S. ratti stimulated mesenteric lymph node cells, anti-S. ratti IgG1 and intestinal rat mast cell protease II (RMCPII) production. We observed some small quantitative immunological differences between different infection regimes, in which the concentration of IL4, IL13, anti-S. ratti IgG1 and IgG2a and RMCPII were affected. However, these differences were quantitatively relatively modest compared with the temporal dynamics of the anti-S. ratti immune response as a whole.Nematode infections are a ubiquitous feature of vertebrate life. In nature, such nematode infections are acquired by continued exposure to infective stages over a prolonged period of time. By contrast, experimental laboratory infections are typically induced by the administration of a single (and often large) dose of infective stages. Previous work has shown that the size of an infection dose can have significant effects on anti-nematode immune responses. Here we investigated the effect of different infection regimes of  Trickle infections of N. brasiliensis in the rat can establish long lived (e.g. 12 weeks ) infections e.g. 3 weeks) infections N. brasiliensis infections, the worm burdens late in infection may not have been larger in size than the residual population that remains at the ‘end’ of a single dose infection N. brasiliensis trickle infections N. brasiliensis infections differ.Different infection regimes have been most thoroughly investigated for Trichuris muris infections, Th1-type immune responses occurred in animals given repeated low dose infections, i.e. a trickle-style infection; latterly, the immune response developed into a protective Th2-type response i.e. trickle or single dose) or the size of the dose given could be responsible for the different immunological effects. For Strongyloides ratti infections in rats, the host immune response changes both qualitatively from a Th1- to a Th2-type immune response and the Th2-type response increases quantitatively with higher dose S. ratti infections Immune responses elicited by, and to varying extents protective against, nematode infection are typically of a T helper 2 (Th2)-type, particularly characterised by production of the cytokines IL-4 and IL-13 Single, high-dose infections typical of experimental studies may be of limited relevance to understanding the biology of natural infections i.e. the product of the number of worms (or quantity of antigen) given to a host and the time for which the host is exposed to these worms (or antigen). Both these measures of ‘dose’ will differ between single or trickle dose infection regimes. Thus, consider three hosts  given to a host. Alternatively, dose could be the worm (or antigen) exposure time, ee hosts : host A Strongyloides ratti in rats to (i) investigate the effects of single dose and trickle dose infection regimes on the host immune response, and (ii) examine the temporal dynamics of host immune responses to single dose and trickle dose infections.Here we have used experimental laboratory infections of Strongyloides ratti is a natural parasite of brown rats, Rattus norvegicus. Other Strongyloides species infect a range of mammalian and other vertebrate hosts, including humans S. ratti occurs via the nasopharyngeal region S. ratti do not elicit genotype-specific immune responses i.e. trickle) infections (‘Trickle 5’ and ‘Trickle 10’) or as (ii) single dose infections . These three single dose infection regimes acted as control groups, because they received the same dose measured by (a) the maximum number of worms or (b) the worm exposure time as the two trickle infection regimes . The concentration of anti-S. ratti IgG1, IgG2a and IgG2b and total IgE in serum and of of anti-S. ratti IgA and of rat mast cell protease II (RMCPII) in intestinal tissue were also measured by ELISA S. ratti infections of rats Animals were sampled for parasitological and immunological analyses. To do this, for each animal, faeces were collected on the days p.i. indicated (above). The faeces were cultured and the number of larvae that developed was determined, as previously described 2 conforming as closely as possible to a normal distribution 1, and -0.101 for RMCPII. Transformed data for each parameter were then scaled to have a mean of zero and variance of 1. For spleen IL4, spleen IL13, IgA, IgG2a and IgG2b, maximum likelihood estimates of λ failed to converge and/or quantile-quantile plots indicated that no value of λ provided a suitable transformation. For these measures, a binary response corresponding to presence/absence was used, where presence was defined as a detectable level of immunoglobulin isotype or cytokine (relative to corresponding negative control reactions). For analyses of immune parameters as a response variable, linear models were used for immune parameters that could be normalised, and generalised linear models (GLMs) with a binomial error structure were used for parameters as presence/absence data. For analyses of intestinal worm burden and per capita fecundity as response variables, GLMs with a negative binomial error structure were used. Principal component analysis 1 and RMCPII. All analyses were performed in R v2.5.0 (www.r-project.org).We sought to normalise the data for each of the immune parameters using a Box-Cox transformation i.e. the number of parasitic females recovered from a host) of all 5 infection regimes through time is shown in 2, p<0.001, S. ratti infections 2, p<0.001, The intestinal worm burden  number of worms or (b) worm exposure time (1 (p<0.01) and RMCPII (p<0.01). For dose measured as worm exposure time, significant and positive associations were found with MLN IL4 (p<0.001), MLN IL13 (p<0.01), MLN IFNγ (p<0.05), IgA (p<0.01), IgG1 (p<0.001) and RMCPII (p<0.001). We then tested formally which, if either, of these two measures of dose better explained the observed variation in the measured immune parameters. This showed that the number of worms was a better explanatory variable for the concentration of IL4 produced by MLNs stimulated with parasite antigen and the concentration of RMCPII; in contrast, worm exposure time was a better explanatory variable for the concentration of IL13 produced by MLN stimulated with parasite antigen and the concentration of anti-S. ratti IgG1 and IgG2a  in this way, will remain relatively constant through time.Intestinal worm burden differed markedly between treatment groups, with maximum worm burden appearing to occur earlier in higher dose infections , consistper capita fecundity (Trichuris trichiura and Ascaris lumbricoides infection 1 and IgG2a changed during an infection; the concentration of IgA increased to a peak and then declined, whereas the concentration of IgG1 and IgG2a both increased steadily to a plateau; and the concentration of IgE remained relatively unchanged (T. trichiura infections where parasite-specific IgG1–4 increased to a peak in 8–11 year old cohorts and declined by age 12–15, whereas IgE increased steadily Our immunological results are consistent with previous findings from experimental and field infections. Here we found that the concentration of IL4 late in an infection and of IL-13 throughout an infection, both produced by MLN, were associated with reduced worm burdens  and 4; aecundity  and 7. Tnchanged . These rS. ratti-IgG1 and of RMCPII were also positively correlated. Principal component analysis indicated that PCA1 correlated positively to all the immune measures analysed (1 and RMCPII. PCA1 may therefore reflect general immune activation following infection (The individual immune parameters measured showed correlated and concerted responses within this experiment . The conanalysed , with thnfection , whereasThe experimental and statistical approach that we describe here goes some way towards interpreting the correlated suite of immune responses elicited by experimental nematode infection in the context of age-intensity profiles found in natural field infections


GLUT9 gene in two genome-wide association analyses of Italian and British populations. Elevated serum UA levels are often found in conjunction with the metabolic syndrome. Hyperuricemia is the major risk factor for gout and has been associated with increased cardiovascular morbidity and mortality. The aim of the present study was to further elucidate the association of polymorphisms in GLUT9 with gout and coronary artery disease (CAD) or myocardial infarction (MI). To test our hypotheses, we performed two large case-control association analyses of individuals from the German MI Family Study.Serum uric acid (UA) levels have recently been shown to be genetically influenced by common polymorphisms in the GLUT9 gene. All four SNPs demonstrated highly significant association with gout. SNP rs6855911, located within intron 7 of GLUT9, showed the strongest signal with a protective effect of the minor allele with an allelic odds ratio of 0.62 . Importantly, this finding was not influenced by adjustment for components of the metabolic syndrome or intake of diuretics. Secondly, 1,473 cases with severe CAD or MI and 1,241 healthy controls were tested for the same four GLUT9 SNPs. The analyses revealed, however, no significant association with CAD or with MI. Additional screening of genome-wide association data sets showed no signal for CAD or MI within the GLUT9 gene region.First, 665 patients with gout and 665 healthy controls, which were carefully matched for age and gender, were genotyped for four single nucleotide polymorphisms (SNPs) within or near the GLUT9 gene strongly influence the risk for gout but are unlikely to have a major effect on CAD or MI in a German population.Thus, our results provide compelling evidence that common genetic variations within the  Gout is the most frequent inflammatory joint disease in men above 40 years of age GLUT9 gene, also known as SLC2A9, to significantly influence serum UA levels The role of hyperuricemia as an independent risk factor for coronary artery disease (CAD) and myocardial infarction (MI) has been discussed controversially for many years since several but not all epidemiologic studies found an independent association of elevated serum UA with cardiovascular disease GLUT9 polymorphisms in the phenotypic expression of gout and their possible role in susceptibility to CAD, assuming an independent influence of hyperuricemia on the development of cardiovascular disease. For this purpose, we examined two large, separate case-control samples of individuals from the German MI Family Study and included also the results from two CAD/MI GWA studies at the GLUT9 gene region to analyse potential additive effects.The aims of this study were thus to further assess the association of n  =  7,575). Recruitment process, selection criteria and study details have been reported previously All individuals of this study participated in the German MI Family Study . Severe CAD was defined as treatment with percutaneous coronary intervention or coronary artery bypass graft. Resting blood pressure was taken according to MONICA guidelines after participants had been resting in a sitting position The phenotype gout was carefully established using two levels of evidence: 1. medical history readings at time of inclusion, two-year and five-year follow-up interview; and 2. self-reported history of gout. The diagnosis of MI was established according to the MONICA (Monitoring Trends and Determinants in Cardiovascular Disease) diagnostic criteria  with the diagnosis of gout were selected from the German MI Family Study. All gout cases fulfilled the above mentioned criteria for “gout”. Controls were unrelated individuals from our German MI Family Study who did neither have any indication for gout nor were they medicated with uricostatics or uricosuric agents at any time during follow-up.A total of n  =  665) according to age and gender to the gout cases. Further phenotypic details are shown in We carefully matched the gout-free controls  and 1,241 unrelated CAD/MI-free control individuals . Cardiovascular risk factors and phenotypic details are summarized in GLUT9 SNPs were selected from a public reference database  based on the following criteria: (1) evidence for significant association with serum UA levels from two recent publications (rs6855911 and rs7442295) Four GLUT9 gene according to NM_001001290 .Genomic DNA was isolated from whole blood samples using the PureGene DNA Blood Kit . DNA samples were genotyped using 5′ exonuclease TaqMan® technology . For each genotyping experiment 10 ng DNA was used in a total volume of 5 µl containing 1× TaqMan® Genotyping Master Mix (Applied Biosystems). PCR reaction and post-PCR endpoint plate read was carried out according to the manufacturer's instructions using the Applied Biosystems 7900HT Real-Time PCR System. Sequence Detection System software version 2.3 (Applied Biosystems) was used to assign genotypes applying the allelic discrimination test. Case and control DNA was genotyped together on the same plates with duplicates of samples (15%) to assess intraplate and interplate genotype quality. No genotyping discrepancies were detected. Assignment of genotypes was performed by a person without knowledge of the proband's affection status. Additionally, comparison between genotype calls from TaqMan assays and Affymetrix GeneChip® Human Mapping 500K Array Set were carried out in 288 DNA samples on all four SNPs . Again, no discrepancies were found. SNPs were mapped to the GLUT9 gene region GLUT9 region with 500 kb on either side of the gene  were included (Genome-wide data from two recently published screens for polymorphisms associated with CAD or MI were employed to survey the complete included .GLUT9 gene region Data of the HapMap phase II release 22 data were used to assess linkage disequilibrium (LD) patterns of the GLUT9 SNPs deviated from Hardy-Weinberg equilibrium, actual and predicted genotype counts of both groups were compared by χ2-test. Differences in allele frequencies between dichotomous traits were calculated employing the same method. Genotypes were coded for both dominant and recessive effects . The additive genetic model was calculated using Armitage's trend test. Multiple logistic regression analysis was used to examine the association of GLUT9 SNPs with either phenotype (gout or CAD/MI) allowing adjustment for relevant covariates  with their 95% confidence intervals (CI) were reported. A two-sided p-value ≤0.05 was considered statistically significant.To determine whether the genotypes of cases and controls of all variates , 2. DiffAssociation analyses were performed using JMP IN 7.0.1  and PLINK v1.00 n  =  665) were matched for age and gender to control individuals (n  =  665). Prevalence of cardiovascular risk factors and cardiovascular disease was high in both, gout cases and gout-free controls. Gout cases were more often treated with diuretics as compared to controls. In addition, in gout cases, the prevalence of type 2 diabetes and increased BMI was higher, whereas in gout-free controls HDL-C levels were elevated. However, we found no significant difference in number of reported MI or CAD events between gout cases (67.5% CAD/MI) and gout-free controls (65.9% CAD/MI). Prevalence of hypercholesterolemia, hypertension and smoking were equally distributed between the two groups  as compared to controls   . Interes  1,241) .GLUT9 intron 7, 6, and 4, respectively. Additionally, rs12510549, 235 kb upstream of GLUT9, was included in the analysis . In contrast, there was a deviation from the HWE in gout cases .Genotype distributions and allele frequencies in gout case-control cohort are shown in p  =  2.9*10−7 was detected for genotype distribution of marker rs6855911. We found a protective effect of the minor allele with an allelic OR of 0.62 (95% CI 0.52–0.75) (p  =  4.6*10−8). Hence, effect of marker rs6855911 on susceptibility to gout is independent of common cardiovascular risk factors and several components of the metabolic syndrome.Strong association with gout was observed for all four markers . The hig52–0.75) . MultivaIn addition to allele frequency and genotype distribution, we analyzed additive, dominant and recessive genetic models with no substantial increase in significance .2 possible haplotypes could be observed with only two (AATT and GGCC) having frequencies >0.05 in cases and controls of the gout sample. The most common haplotype AATT  was less frequently present in healthy individuals as compared to patients with gout , i. e. representing a risk haplotype, resulting in a p-value of 2.2*10−7. The second common haplotype (GGCC) codes for a protective haplotype with frequency in controls of 0.183 and frequency in cases of 0.126, respectively (p  =  4.5*10−5). After adjustment for BMI, type 2 diabetes, and medication with diuretics in a multivariate logistic regression analysis, the association results remained significant (p  =  1.3*10−7 for AATT and p  =  2.9*10−5 for GGCC).According to the high LD between the SNPs, four-marker haplotypes were constructed spanning a 340-kb genomic region. Nine of the 42-value exceeded highest observed χ2-value.Both, single marker and haplotype association analysis remained significant after 100,000 permutations. No permutation χSeparate analyses in females and males revealed similar odds ratios in allele frequency comparisons but slightly higher significance levels in males  than in n  =  1,473 cases and n  =  1,241 CAD-free controls (p>0.4).Our power calculation indicated that at a significance level of 0.05 and with a two-sided alternative hypothesis, our CAD/MI sample with controls  had moren  =  1,114) as a sub-sample of CAD patients in a separate analysis. Again, no significant difference in allele or genotype frequencies of the four genotyped markers between MI cases and disease-free controls  was observed . Multivariate logistic regression analysis adjusting for cardiovascular risk factors  both on single SNPs and haplotypes in CAD/MI case-control sample did not reveal positive association results (data not shown). Separate analysis for male and female individuals did not point to gender-specific effects of the four SNPs and CAD/MI in trend test (p>0.18).No association with CAD was observed for any of the analyzed SNPs . To evaln  =  916 CAD/MI cases compared to n  =  916 controls neither SNPs nor haplotypes achieved statistical significance, even after adjustment for age of first manifestation of CAD/MI, male gender, type 2 diabetes, hypercholesterolemia, hypertension, smoking, and BMI (data not shown).To gain maximum power for detection of even small effects, we included all appropriate individuals from our German MI Family Study in the CAD/MI case-control sample. This included an overlap of 220 individuals from gout case sample in CAD/MI case sample, 337 individuals from gout control sample in CAD/MI case sample, 108 individuals from gout case sample in CAD/MI control sample, and 217 individuals from gout control sample in CAD/MI control sample, respectively. After an initial round of association testing with CAD/MI, we excluded all overlapping individuals from the analysis. Again, in remaining GLUT9 gene could exert influence on susceptibility to CAD or MI, we analyzed the data from two recently conducted GWA studies p-values were obtained for association with CAD/MI (WTCCC) and MI (Cardiogenics). A total of 292 SNP markers located in GLUT9 gene region (214 kb) and a more extended region of 1,000 kb around the GLUT9 gene were analyzed  to detect a true effect.For all genotyped markers, we found moderate deviation from HWE in gout cases. Generally, deviations from HWE can point to either a sampling bias or population stratification. Whereas HWE deviations in a control sample suggest the possibility of genotyping errors, HWE deviations in cases can also be the result of true association GLUT9 SNPs and gout came from analysis of the Framingham SNP Health Association Resource (SHARe) posted on the NCBI dbGaP website at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgistudy_idphs000007.v2.p1. Here, n  =  1,345 adult participants of the largest 310 pedigrees in the Framingham Heart Study were genotyped with the Affymetrix 100K GeneChip p-values for the phenotype gout (defined as history of gout at any examination cycle) were available (accession: pha001406.1). However, the four SNPs analyzed in our study  were not included on the Affymetrix 100K GeneChip. For GLUT9, marker rs10516195 located in intron 3 near to and in LD with our SNPs (7.6 kb upstream from rs6449213) showed high significance with gout (p  =  3.7*10−8).Additional evidence for a reliable positive association between n  =  186 cases and n  =  186 controls, respectively) limits the conclusion of gender-specific effects mediated by polymorphisms in GLUT9 gene on gout.In our study, we found stronger significance between genotyped markers and gout in males than in females. However, odds ratios have the same order of magnitude with a risk reduction of about 40%. It is known that serum UA levels and incidence of gout are higher in males, but tend to equalize with increasing age GLUT9 showed significant association with gout in our study. HapMap data describe a large LD block covering two-thirds of GLUT9 and a large proportion of upstream intergenic sequence, including WDR1 gene GLUT9 gene, could be identified by Li et al. GLUT9 gene region analysed in our study were located in intronic and intergenic regions. A possible effect of a functional variant beyond changes in amino-acid composition of translated protein could lead to altered gene expression, which can be modulated by the concerted action of multiple transcription factors over long distances and, thus, be influenced by multiple SNPs GLUT9 gene is not fully understood yet. It is known, however, that GLUT9 plays an important role as transmembrane sugar transporter and is highly expressed in liver and kidney tubules GLUT9 gene polymorphisms with the phenotype “gout”. However, without the knowledge of serum UA levels in our study, we cannot exclude more direct effects of GLUT9 gene variants on gout.Also, SNP rs12510549, located 235 kb upstream to GLUT9 with CAD/MI. In addition, GWA studies on CAD/MI revealed no significant association signal of SNP markers in the extended GLUT9 region. Hence, it is unlikely for polymorphisms in GLUT9 to be major contributors to the risk for CAD or MI. Moreover, a pathophysiologic link between gout and CAD via genetic variations within the GLUT9 gene appears doubtful. No difference in the prevalence of CAD/MI in the gout case-control sample was observed: 67.5% and 65.9% of the gout cases and gout-free controls, respectively, were affected by CAD/MI . Therefore, we adjusted for male gender and other cardiovascular risk factors with no increase in significance. Additionally, the two analyzed GWA studies on CAD/MI included a more balanced gender distribution between cases and controls . Here, separate analysis of the four SNPs  revealed no association with MI, even after adjustment for male gender.GLUT9 gene are associated with increased susceptibility to gout. Furthermore, these SNPs are unlikely to play a causal role in the pathogenesis of CAD or MI. Additional functional analyses are warranted to further elucidate the role of GLUT9 in the pathophysiology of hyperuricemia and gout.In conclusion, our study demonstrates strong evidence that polymorphisms in http://www.cardiogenics.imbs-luebeck.de/Cardiogenics data: http://www.wtccc.org.uk/info/summary_stats.shtmlWTCCC data: http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgistudy_id phs000007.v2.p1Framingham SHARe: http://pngu.mgh.harvard.edu/∼purcell/plink/PLINK: http://www.broad.mit.edu/mpg/haploview/HaploView: http://www.psycho.uni-duesseldorf.de/abteilungen/aap/gpower3/G*Power: http://www.hapmap.org/HapMap:


The cost-effectiveness of LCNB and NLBB was estimated using a microsimulation model. The sensitivity of LCNB (0.97) and resource use and costs of LCNB and NLBB were derived from a multicentre consecutive cohort study among 973 women who consented in getting LCNB and NLBB, if LCNB was negative. Sensitivity analyses were performed. Replacing NLBB with LCNB would result in approximately six more breast cancer deaths per year (in a target population of 2.1 million women), or in 1000 extra life-years lost from breast cancer (effect over 100 years). The total costs of management of breast cancer (3% discounted) are estimated at £4676 million with NLBB; introducing LCNB would save £13 million. The incremental cost-effectiveness ratio of continued NLBB  In Western societies, breast cancer is the most common malignancy in women. This disease and the ensuing mortality are considered the major public health problems. Mass screening for breast cancer is deemed effective in reducing breast cancer mortality. Studies have shown that early detection of breast cancer may reduce breast cancer mortality  were obtained from a prospective multicentre consecutive cohort study (the COBRA study) . This stTo study the impact of LCNB and NLBB on health effects and costs, we used the simulation model MISCAN . Health effects were considered for the remaining life expectancy of the cohort simulated (maximum age 100 years).We simulated three scenarios: (A) the situation without mass screening; (B) mass screening with NLBB as diagnostic work-up for palpable and nonpalpable breast lesions; and (C) mass screening with LCNB replacing NLBB as diagnostic work-up for nonpalpable lesions, followed by NLBB if the core biopsy indicates normal breast tissue or a high-risk lesion . We usedFigure 1NLBB was considered to be the gold standard diagnostic procedure with 100% sensitivity. The test characteristics of LCNB were obtained from the COBRA study.The costs of the diagnostic phase were calculated as the resource use in the diagnostic stage multiplied with cost-per-unit estimates. The relevant resource items were LCNB, NLBB, histopathological examination, hospitalisation, day care, general practitioner (GP), diagnostic mammography, other diagnostic procedures (e.g. ultrasound and MR imaging), FNA and outpatient visit. The actual economic costs per unit of LCNB, NLBB and histopathological examination were obtained from five participating Dutch hospitals. Due to practice variation, the costs of hospitalisation for diagnostic excision biopsy were averaged for treatment setting  . The cosThe costs of breast cancer treatment were obtained from previous studies . To take account of time preference, the costs estimates over time were adjusted with 3% (baseline) and 5%  annual discount rate. To compute the cost-effectiveness, equal discount rates were applied to the effects as well , respectively .Fourth, we simulated LCNB scenarios where 100 or 85% of referred women, respectively, would have further histopathologic assessment after clinical mammography and physical examination.Finally, we varied the distribution of nonpalpable lesions at screening. In the basic model, all DCIS and lesions <1 cm in diameter (i.e. T1a and T1b) were considered nonpalpable, whereas T1c and T2+ lesions were regarded palpable. As a high estimate, we considered 75% of all lesions to be nonpalpable, with the proportion of nonpalpable tumours gradually decreasing with tumour size .in situ (DCIS) and 38% (322 of 858) invasive carcinoma. With the LCNB procedure, 481 of 494 malignancies were detected: malignancy being the initial diagnosis at core biopsy (470 cases) or the diagnosis at surgery following a nonconclusive diagnosis  at core biopsy (11 cases). Thus, the overall sensitivity of the LCNB procedure was 0.97 (95% CI: 0.96–0.99). The sensitivity for DCIS was 0.94 (161 of 172) (95% CI: 0.90–0.97) and for invasive breast cancer 0.99 (320 of 322) (95% CI: 0.99–1.00).Of the nonpalpable breast lesions, 58% (494 of 858) were malignant according to the gold standard: 20% (172 of 858) involved ductal carcinoma in situ cancers (DCIS), 42 700 (36.6%) T1a or T1b tumours, and 57 400 (49.3%) T1c or T2+ tumours. In the LCNB scenario, 115 800 cancers are detected at screening (4.7 per 1000 women screened). The tumour size distribution is somewhat less favourable, with 15 700 (13.5%) in situ tumours, 42 500 (36.7%) T1a or T1b tumours, and 56 700 (49.8%) T1c or T2+ tumours.In the NLBB scenario, 116 500 cancers are detected at screening (4.8 per 1000 women screened): 16 400 (14.1%) Without screening, the total cost of the management of breast cancer would be £4369 million (3% discounted) for the next 100 years, of which £910 million (21%) would be spent on diagnostics. If screening is introduced, the total costs would increase by £307 million (7.0%) with NLBB as diagnostic work-up and by £294 million (6.7%) if LCNB is applied. Thus, the net (discounted) cost reduction of LCNB compared with NLBB would be £13 million, which can be mainly attributed to a reduction in diagnostic costs.vs LCNB is about £12 482 per life-year gained, that is extra costs of £12.70 million for NLBB compared to LCNB for 1017 additional life-years gained by NLBB.The costs per life-year gained resulting from screening would be £1515 with NLBB and £1459 for screening with LCNB (both costs and effects 3% discounted). The incremental cost-effectiveness ratio of NLBB vs LCNB was the highest . The low cost of LCNB resulted in an incremental cost-effectiveness ratio of £21 430. The high cost of LCNB and lower thresholds for breast biopsy resulted in negative incremental cost-effectiveness ratios .vs LCNB, that is, about £12 500 per life-year saved, may be ‘borderline’ in political decision-making about health-care services in the Netherlands, and will be well within the bounds of acceptable cost-effectiveness in many other countries. Further evaluation of quality of life in patients who underwent LCNB vs NLBB might be undertaken for a more elaborate comparison of particularly the long-term effects of LCNB vs NLBB in terms of quality-adjusted life-years.The current study shows that LCNB may be considered a cost-effective alternative to NLBB, that is, considerable cost savings are achieved at probably acceptable losses in terms of life-years. We estimated that, with the introduction of LCNB, costs of diagnosis and (primary) treatment of breast cancer would decrease by about £500 000 per year. Continued use of NLBB, on the other hand, would save more life-years. The pertaining incremental cost-effectiveness ratio of continued NLBB vs LCNB is most sensitive to relatively small variations in the sensitivity of LCNB. We considered NLBB as the gold standard diagnostic procedure, assuming 100% sensitivity. A recent study into the diagnostic accuracy of open-breast biopsy, however, showed that the sensitivity of open-breast biopsy dropped to 0.99 after 2 years of follow-up and to 0.96 after 5 years of follow-up, when taking into account the so-called interval lesions . This is comparable to the incidence in the United Kingdom (about 136 per 100 000 women). There is no ground to assume that comparable test characteristics of LCNB cannot be achieved in other Western medical settings too. Finally, introduction of LCNB replacing NLBB for the diagnosis of nonpalpable lesions on a large scale, particularly in countries where breast screening results in an increased number of biopsies, will have a marked impact on health care costs.


In this study, we extended the in vitro observation using an ex vivo model of prostate cancer tissues and assessed the prognostic significance of the PI3K/Ak/NF-κB signalling determinants.The PI3K/Akt signalling pathway, induced by epidermal growth factor receptor (EGFR) and Her-2, is involved in the constitutive activation of NF-κB subunit p65. Data were analysed using Spearman's rho test, Kaplan–Meier curves and multivariate Cox regression analysis. In addition, a non-supervised hierarchical clustering analysis was applied to stratify patients according to prognostic groups in terms of risk of recurrence.We analysed a prostate cancer tissue microarray of 63 patients for the expression of total and activated EGFR, Her-2 receptors and the signalling molecules PTEN, phospho-PTEN, Akt, phospho-Akt and the NF-κB. EGFR, phospho-EGFR, phospho-Her-2, ErbB3 and nuclear NF-κB were associated with the overall biochemical recurrence (BCR) of patients. The non-supervised hierarchical clustering analysis resulted in the separation of patients into five groups according to BCR.The concomitant overexpression of activated EGFR and Her-2 was correlated with the nuclear expression of NF-in vitro data on ErbB involvement in NF-κB activation and shows evidence for a significant role of ErbB/PI3K/Akt/NF-κB signalling in the progression of prostate cancer.These results validate the previous  The net result of this process is translation to the nucleus of NF-κB  to evaluate the profile of expression of EGFR, Her-2, ErbB3, PI3K, PTEN, Akt, p65 and their phosphorylated forms. The staining with phospho-ErbB3 antibody was too weak to be analysed and was thus not included in this study. Statistical correlations of the protein expression in tissues allowed us to associate signalling events according to the activation of EGFR, Her-2 and PTEN, as well as that of p65. Indeed, this also allowed us to conduct hierarchical analysis to provide a holistic view of pathway interactions and their association with prognosis. This is the first systematic analysis of ErbB receptors and the downstream signalling pathway PI3K/Akt in prostate cancer tissues.In this study, we sought to extend these Paraffin-embedded human primary PCa specimens (collected blocks from 1993 to 2000) from patients who provided informed consent were reviewed. A total of 64 specimens from patients who had a radical prostatectomy were included in our study to create a tissue array. Criteria for the retrospective cohort study to create tissue arrays were the following: (a) no pre- or postoperative treatment was used before biochemical recurrence (BCR) and (b) all cases had a clinical follow-up of at least 3 years or until death. Patients were followed for an average range of 72 months. No age difference was observed between the group of patients who relapsed and the group who did not. Preoperative prostate-specific antigen (PSA) levels were available for 62 patients. Postoperative PSA was available for all patients. Non-failure of PSA was defined as PSA remaining below 0.3 ng/ml after radical prostatectomy. Recurrence-free interval was defined as the time between the date of surgery and the date of first PSA increase above 0.3 ng/ml. The final staging, grading and histological diagnosis were based on the Hôpital Notre-Dame  pathology department report in agreement with the review by an independent pathologist. Ethics approval was obtained from the local IRB committee.Tissue arrays containing a total of 384 cores of prostate tissues  were bui2O2/methanol. To unmask antigen, the slides were submerged in 95 °C citrate buffer  or in EDTA buffer  for 15 min. The tissues were blocked for 15 min with a protein-blocking, serum-free reagent  and incubated with different antibodies for 60 min at room temperature in a humid chamber. The optimal concentration for each primary antibody was determined by serial dilutions. The arrays were then incubated with a secondary biotinylated antibody (Dako Cytomation) for 15 min, followed by incubation with a streptavidin–peroxidase complex . Reaction products were developed using diaminobenzidine (brown stain) containing 0.3% H2O2 as a substrate for peroxidase. Nuclei were counterstained with diluted haematoxylin (blue stain). Tumour sections were inspected at × 20 and × 40 magnification. Epithelial zones were scored according to the staining intensity of the cytoplasm, nucleus or membrane . In cores in which staining was of variable intensity, the average intensity was reported. Each array was independently analysed in a blind study by two independent observers. Interrating correlation was >70%. When strong differences in scoring between the two observers (more than 1 unit per core) occurred, the core was re-evaluated to reach a concordant scoring between the two observers. The average of all cores with cancer from the same patient was used for analysis. No decrease in staining intensity on older paraffin blocks was observed.In brief, tissue arrays were deparaffinised in toluene and rehydrated in a gradient of ethanol. Subsequently, endogenous peroxidase activity was quenched by treatment with 0.3% HM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT/1 mM NaF/0.5% NP-40/ 0.5 mM PMSF/0.2 mM sodium orthovanadate/2 μg ml–1 of aprotinin, leupeptin and pepstatin), boiled in loading buffer, separated by SDS–PAGE and transferred on a nitrocellulose membrane under refrigerated conditions (60 V for 2 h). The membrane was saturated with 5% milk/PBS/0.1% Tween-20. Immunodetection was carried out as described in the protocol of the ECL kit . Membranes were incubated for 2 h at room temperature with the specific antibody (0.5–1 μg ml–1), washed twice with PBS/0.05% Tween-20 and incubated for another 30 min at room temperature with peroxidase-conjugated antibodies .The antibodies and conditions used in IHC are summarised in Statistical analysis was carried out with SPSS software 11.0 . To avoid bias due to a different efficiency of antibody hybridisation on different array slides, we calibrated each slide for interslide comparison. Spearman's rho correlation test (two tailed) was used to estimate the correlation with clinicopathological variables. Survival curves were plotted using the Kaplan–Meier analysis and the log-rank test was used to test for significant differences. Receiver operative characteristic (ROC) curves were used to determine the threshold value for each marker. Cox univariate and multivariate proportional hazard models were used to estimate the hazard ratio for each marker. Multivariate analysis was carried out using a forward stepwise hazard model on univariate analysis required for entry into the model. Only three categorical variables were included in the multivariate Cox regression model to avoid an overfitting situation. These variables were chosen on the basis of their significance in the univariate model and were as follows: surgical margins, Gleason score and pathological stage.We carried out hierarchical clustering analysis using Genespring software  on the filtered data set. We used the distance branch of 0.8 with a Pearson correlation as a similarity metric.r=0.63 and r=0.45, respectively, P<0.001, Spearman's rho test; Table 3). Similarly, EGFR staining was also often associated with Her-2 and ErbB3 staining  and a very strong correlation was obtained between phospho-EGFR and phospho-Her-2 . A slightly weaker correlation was observed between Her-2 and ErbB3 .Membrane and cytoplasmic reactivity rates were evaluated in samples of 63 prostate cancer patients (cohort described in In 65% of patients (41 of 63), cancer tissues showed an overexpression of EGFR compared with normal tissues, whereas 79 and 81%  of patients showed an overexpression of cytoplasmic Her-2 and ErbB3, respectively . These rκB signalling pathway. For this purpose, we immunostained the tissue arrays with antibodies against phospho-Akt, PTEN, phospho-PTEN  and both were overexpressed in >60% of patient tumours.We also evaluated the activation status of the Akt/PI3K and NF-pho-PTEN  and 3. Pr=0.40, P=0.001, Spearman's test; Table 3). However, 4 out of 10 patients with a weak PTEN staining showed a strong phospho-PTEN staining  had a strong PTEN staining (⩾2.5) and only two patients showed a very weak (<0.5) or an absence of PTEN staining . Similarstaining . PTEN war=0.59, r=0.70, r=0.59, and P<0.001, respectively; In 45 out of 63 cases (71%), PI3K was highly overexpressed in tumour tissues compared with adjacent normal tissues. Only two patients presented a weak PI3K staining (below average intensity 1 on 2 cores). PI3K staining was exclusively cytoplasmic. The intensity of PI3K staining was strongly associated with the expression of PTEN, phospho-PTEN and phospho-Akt  also showed a nuclear staining of p65, suggesting a constitutive activation of NF-κB in these tissues. Phospho-p65 was present in the cytoplasm and a weak staining could be observed in a few cells.The p65 subunit of NF- tissues . Some tir=0.28, P=0.026 Spearman's test, r=0.11, P=0.37), as well as between phosphorylated p65 and nuclear p65 , even when the staining of phospho-p65 and cytoplasmic p65 strongly correlated .The presence of p65 in the nucleus of prostate cancer tissues was evaluated by the percentage of cells with a nuclear p65 staining. A mean of 2% of cells per tissue showed a p65 nuclear staining. In eight patients, cancer tissues had >10% positive nuclei. Interestingly, the presence of nuclear p65 was increased in patients with Gleason scores of >6, and globally correlated with Gleason scores  when continuous values were considered. However dichotomised values of nuclear p65 (high and low) did correlate with combined EGFR and Her-2 .We also evaluated the correlation between nuclear p65 and EGFR, Her-2 and ErbB3 expression. Surprisingly, the presence of nuclear p65 did not correlate with EGFR, Her-2 or ErbB3, either alone or with any other proteins tested . A trend towards significance was observed for EGFR and ErbB3 . Both phospho-PTEN and nuclear p65 correlated with the Gleason score . Only phospho-PTEN was significantly and inversely correlated with death due to prostate cancer  and a positive trend was observed with nuclear p65 staining  and phospho-Akt  in this cohort of radical prostatectomy patients.We evaluated the correlation between a strong staining of each antibody and the clinical parameters available for the patient cohort. No correlation was observed between any staining and metastatic spread, perineural infiltration or age . In contκB were associated with the overall BCR. Phospho-Akt showed a strong trend to associate with BCR (P=0.058). In addition, phospho-Akt and PI3K were significantly associated with early BCR at 2 years but not with overall BCR . EGFR staining was the most significant event associated with BCR . No combination of proteins, either phosphorylated receptors or signalling proteins, was more significant than EGFR alone (data not shown). In addition, only EGFR was statistically significant in multivariate Cox regression analysis when Gleason score, margin status and pathological stage were considered in the model .To evaluate the association between the expression of each protein and the BCR of prostate cancer patients in our cohort, we performed Kaplan–Meier curve analysis coupled with log-rank test and univariate Cox regression analysis. P=0.003, log rank). Interestingly, this combination of multiple markers does not correlate with any clinicopathological parameters and suggests that the combination may provide a new, clinically useful, independent prognostic parameter. Indeed, in Cox multivariate analysis, when clinical variables  were taken in the model, only EGFR and the multiple marker combination remained an independent predictive variable of BCR . No significant association of p-PTEN and survival was obtained in multivariate analysis. However, the significance of this result should be taken into consideration with the relatively low mortality rate (n=9) in this cohort of radical prostatectomy patients.No significant association of the proteins tested was obtained, with the exception of p-PTEN . A similar observation has already been reported in prostate cancer tissues, although the reason for this remains unclear (in vitro cell line observations in which PTEN expression is inversely correlated with activation of PI3K and Akt. Here, we also observed a positive correlation between p-Akt and p-PTEN. A possible explanation is that, although PTEN would be normally expected to downregulate p-Akt through PI3K, this is counterbalanced by a concomitant inhibition of PTEN by higher levels of pPTEN, which is itself functioning as a negative regulator, thus allowing the observed higher levels of PI3K and pAkt seen in tissues. This hypothesis would require further experimentation for validation.We observed that proteins associated with cell signalling, such as PTEN, p-PTEN, p-Akt and PI3K, correlated with the expression level of EGFR, Her-2 and ErbB3 receptors . The expin vitro analysis in cell lines (We also found that EGFR, Her-2 and ErbB3 are strongly correlated and associated with the activation of phospho-Akt, which suggests that these receptors share similar signalling pathways as reported by ll lines . Our stuThe present study also analysed and compared the prognostic value of the total expression of EGFR, Her-2 and ErbB3 receptors, as well as their activated forms in a series of primary prostate cancer specimens. The downstream signalling molecules PI3K, PTEN, phospho-PTEN and activated Akt were also analysed. A number of previous studies have already reported the association of EGFR, Her-2 and ErbB3 with PSA biochemical recurrence in prostate cancer patients (When individually compared with each other, the marker that most significantly correlated with PSA recurrence was EGFR. It showed a hazard ratio similar to nuclear p65. Multivariate analysis suggests that EGFR and p65 are two independent prognostic indicators in prostate cancer tissue, which also supports previous reports analysing these two markers separately. Therefore, EGFR and p65 may be interesting markers to help stratify patients for personalised therapy. However, only EGFR was an independent marker in multivariate analysis when Gleason, margin status and pathological stage were included in the model. As EGFR and p65 seem to be strong markers of recurrence in prostate cancer cells, further studies in larger cohorts would be of major interest to evaluate the clinical relevance of these two markers in prostate cancer tissues.Analysis of the expression level of PTEN and its inactivated phosphorylated form showed that neither correlated with BCR in patients. This result supports other reports showing no significant association between PTEN expression and biochemical recurrence (Interestingly, in this study, we applied a non-supervised hierarchical analysis of our set of markers, which identified five prognostically significant groups of patients. The same approach has already been successfully used for stratification of breast cancer tissues in TMAs, allowing the classification of breast cancers into clinically relevant prognostic groups. Cells from the patient group with the highest risk of recurrence group 5,  stained κB, which validates, for the first time in prostate cancer samples, previous in vitro data obtained in cell lines. The importance of PTEN in the constitutive activation of Akt was not validated in our cohort of patients. In addition, we show evidence that the ErbB/PI3K/Akt/ NF-κB signalling pathway is involved in prostate cancer progression and that a multiple marker approach identifying active signalling pathways may be prognostically more relevant than single markers. Further studies including larger patient cohorts should be investigated to confirm these initial results.In summary, our multimarker analysis provides evidence that EGFR, Her-2 and ErbB3 are involved in the constitutive activation of Akt and NF-


Increasing evidence suggests that high selenium levels are associated with diabetes and other cardiometabolic risk factors.We evaluated the association of serum selenium concentrations with fasting plasma glucose, glycosylated hemoglobin levels, and diabetes in the most recently available representative sample of the U.S. population.We used a cross-sectional analysis of 917 adults ≥ 40 years of age who had a fasting morning blood sample in the Mean serum selenium was 137.1 μg/L. The multivariable adjusted odds ratio [95% confidence interval (CI)] for diabetes comparing the highest quartile of serum selenium (≥ 147 μg/L) with the lowest (< 124 μg/L) was 7.64 (3.34–17.46). The corresponding average differences (95% CI) in fasting plasma glucose and glycosylated hemoglobin were 9.5 mg/dL (3.4–15.6 mg/dL) and 0.30% (0.14–0.46%), respectively. In spline regression models, the prevalence of diabetes as well as glucose and glycosylated hemoglobin levels increased with increasing selenium concentrations up to 160 μg/L.In U.S. adults, high serum selenium concentrations were associated with higher prevalence of diabetes and higher fasting plasma glucose and glycosylated hemoglobin levels. Given high selenium intake in the U.S. population, further research is needed to determine the role of excess selenium levels in the development or the progression of diabetes. Most Americans have selenium intake ranging from 60 to 220 μg/day , well abBecause there is a narrow range between selenium intake levels required for selenoprotein synthesis and toxic levels , these fn = 3,299), of whom 1,302 participated in the morning examination and had a fasting blood sample. Among these, 1,273 participants had serum selenium measurements. To minimize the possibility of reverse causation in the associations examined, we excluded participants with self-reported coronary heart disease (n = 156), stroke (n = 80), or cancer (n = 173), leaving 934 participants. We further excluded 16 participants with missing body mass index (BMI) and 1 participant missing cotinine concentration. The final sample size was 917.NHANES is conducted by the National Center for Health Statistics (NCHS) using a complex multistage sampling design to obtain a probability sample of the civilian non-institutionalized U.S. population. We used data from NHANES 2003–2004 , the mosCollection materials were screened for potential selenium contamination. Serum selenium was measured at the Trace Elements Laboratory at the Wadsworth Center of the New York State Department of Health  using inductively coupled plasma–dynamic reaction cell–mass spectrometry. The between-assay coefficients of variation for quality control pooled samples analyzed throughout the duration of the survey ranged from 2.5% to 2.9% .Plasma glucose was measured from a morning fasting sample of participants who fasted 8–24 hr by the enzyme hexokinase method . DiabeteGlycosylated hemoglobin measurements were performed using the Primus CLC330 and Primus CLC385 boronate affinity high-performance liquid chromatography systems . The systems were standardized to the reference method used for the Information on sex, age, race/ethnicity, education, menopausal status, smoking, and use of vitamin/mineral supplements  was based on self-report. BMI was calculated by dividing measured weight in kilograms by measured height in meters squared. Serum cotinine was measured by isotope-dilution high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry .Participants were divided into quartiles of serum selenium concentration based on the weighted population distribution. We used multivariable linear regression to calculate adjusted means for plasma glucose and glycosylated hemoglobin differences and logistic regression to calculate odds ratios (ORs) for diabetes comparing each quartile of serum selenium with the lowest quartile. We used three models with progressive degrees of adjustment. Model 1 was adjusted for sex, age, race/ethnicity, and education. Model 2 was further adjusted for BMI, smoking, cotinine, and menopausal status. Model 3 was further adjusted for use of vitamin/mineral supplements. Because ignoring diabetes treatment may result in biased estimates of the association between selenium and glucose or glycosylated hemoglobin, we conducted an additional analysis using censored linear regression (model 4) to correct for the effect of medication for diabetes using NHANES survey weights . Tests fThe mean (± SD) serum selenium concentration in the study population was 137.1 ± 19.9 μg/L. The overall prevalence of diabetes was 10.0%. Participants with diabetes were more likely to be older, male, and non-Hispanic black or Mexican American and to have a higher BMI compared with participants without diabetes . Serum sp = 0.001). The multivariable adjusted OR [95% confidence interval (CI)] for diabetes comparing the highest selenium quartile (≥ 147 μg/L) with the lowest (< 124 μg/L) was 7.64   , model 34–17.46) , left. A4–17.46) .In multivariable adjusted models, the average differences (95% CI) comparing the highest with the lowest selenium quartile were 9.5 mg/dL (3.4–15.6 mg/dL) for fasting plasma glucose, and 0.30% (0.14–0.46%) for glycosylated hemoglobin , model 3n = 9,085) to 136.6 μg/L (134.4–138.7 μg/L) in NHANES 2003–2004 . This increasing trend in serum selenium levels highlights the importance of understanding the association between serum selenium levels and diabetes. Moreover, the NPC trial—a randomized, double-blind clinical trial to evaluate the efficacy of selenium supplementation (200 μg/day) for the prevention of cancer—showed an increased risk of diabetes after 7.7 years of follow-up  comparing selenium supplementation with placebo (p = 0.16) but not in the selenium plus vitamin E group   .Our findings are consistent with recent reports from several U.S. studies of positive associations of selenium concentrations with lipid levels  and withThe high soil content of selenium in several areas of the United States and the use of supplements  explain Many other countries, including most of Europe, have substantially lower serum selenium levels  , mainly An excess of reactive oxygen species may increase insulin resistance and affect pancreatic β-cell function , and somHigher selenium concentrations were associated with some diabetes risk factors such as increasing age. In contrast, selenium concentrations decreased with higher BMI, which is one of the stronger diabetes risk factors. The associations of selenium and diabetes reported were adjusted for these risk factors, reducing the possibility that the association between selenium and diabetes is driven by differences in established diabetes risk factors across selenium levels.The strengths of our study come from the rigorous sampling design, the quality of the study measurements, and the representativeness of the NHANES sample. Among its limitations, the cross-sectional design restricts our ability to evaluate the temporality of the observed association. It is possible that high serum selenium levels are related to body weight. Nevertheless, the observed association persisted after adjusting for BMI as well as for use of multivitamin/mineral supplements. The use of a single measurement of serum selenium may not reflect completely the known high within-person variability  and doesIn summary, high serum selenium concentrations were associated with higher prevalence of diabetes in a representative survey of U.S. adults conducted in 2003–2004. These findings are consistent with an earlier NHANES survey conducted in 1988–1994. Furthermore, these findings are supported by randomized evidence from the NPC trial and SELECT indicating that selenium supplementation may increase the risk of diabetes in selenium-replete populations such as that of the United States. High selenium concentrations have also been linked to increased lipid levels  and hype


To the Editor: Leptospirosis is a zoonotic disease that has now been identified as an emerging infectious disease  (panel A). Cases occurred in all age groups but were more common (90%) in those 25–74 years of age. Age-specific incidence was highest for persons 55–64 years of age; mean annual incidence was 0.57/100,000 population.During 2001–2006, of 7,733 suspected human cases of leptospirosis, 291 cases were confirmed. The major serotype identified was  (83.5%) , panel Apanel B); June–October accounted for 60% of cases, with a higher incidence of 0.022–0.028 cases/100,000 population. In October–December, monthly rainfall was below average (201.1 mm/month), but leptospirosis incidence was above average . Specifically, 25 (74%) of leptospirosis cases in October, 10 (42%) in November, and 4 (19%) in December were likely associated with several days of heavy rainfall from typhoons. Therefore, the typhoons may be the reason for high incidence in October–December.Rainfall data from the Central Weather Bureau of the Republic of China showed typically high rainfall (252–433 mm/month) in Taiwan during May–September. Heavy rains were followed by an increase in laboratory-confirmed cases of leptospirosis , panel B The annual incidence of leptospirosis in Taiwan is relatively lower than that in other countries with tropical or subtropical climates. Our study does not conclusively document the reason for lower incidence, although it does suggest an association between amount of rainfall and incidence. An understanding of the relationship between leptospirosis incidence and rainfall is indispensable for implementing appropriate preventive measures.


P < 0.001), and total protein, albumin, protein thiols, and copper levels were found to be significantly decreased in CRF patients compared to normal controls (P < 0.001). Ceruloplasmin levels were decreased significantly (P < 0.05), and there was no significant change in serum GST levels in CRF patients compared to normal controls. In conclusion, the significant increase in levels of MDA, and the decrease in levels of protein thiols, CP, and copper in uremia patients when compared to controls, reconfirms the presence of stress in this patient population. In view of the changes in other markers of oxidative stress, this absence of any significant change in the activity of GST in uremia patients compared to controls, warrants further study.The existence of oxidative stress and the higher incidence of cardiovascular diseases in association with uremia is well proved. The uremic status of serum copper, ceruloplasmin (CP), protein thiols, malonyldialdehyde (MDA), and glutathione S-transferase (GST) levels was studied. The study was carried out on 51 chronic renal failure (CRF) patients who were not on hemodialysis therapy and on 42 healthy controls. Serum urea, creatinine, and MDA levels were found to be significantly increased ( Enhanced oxidative stress has been well established in uremia.2 Oxidati47The production of ROS is a natural process; the phagocyte oxidant generation system which includes both polymorphonuclear neutrophils (PMNs) and monocyte-macrophage cells is based on the inducible production of ROS.9 The pha1in vivo measurement to assess oxidative stress is generally not feasible. Instead, lipids, proteins, carbohydrate, and nucleic acid have lifetimes ranging from hours to weeks after being modified by ROS, which makes them ideal markers of oxidative stress.[in vivo and most of the SH-groups are present over albumin and are major reducing groups present in our body fluids.[ROS are highly reactive compounds with a half-life of only seconds; therefore, their e stress. The totay fluids.15 Ceruloy fluids.17Copper is a transition metal ion and the great bulk of copper in the body is bound to storage and transport proteins in forms that are unable to act as pro-oxidants. Surprisingly, adequate copper is required to maintain antioxidant defenses within the body, and it has been demonstrated that copper deficiency in animals can have pro-oxidant effects. During lIn the present study, we aim to assess oxidative stress by determining relevant antioxidant levels and oxidative stress markers in CRF patients compared to normal controls.g for 15min for clear separation of serum.The study was carried out on 51 CRF patients who were not on hemodialysis therapy and on 42 healthy controls. The causes of CRF were: chronic glomerulonephritis (30 cases), diabetic nephropathy (16 cases), and unknown (five cases), and all of them were on conservative management. None of the patient groups received any form of antioxidant medication; all of them were on renal diet . The healthy controls were not on any kind of prescribed medication or dietary restrictions. Informed consent was taken from all subjects involved in the study and the study was approved by the Institutional Review Board. Blood samples (5mL) were drawn into plain vacutainers from the antecubital veins of healthy controls and CRF patients. The blood was allowed to clot for 30min and centrifuged at 2000 bis (2-nitrobenzoic acid) (DTNB), 1-chloro-2, 4-dinitrobenzene (CDNB), reduced glutathione (GSH), bathocuprine disulphonate disodium salt (BCDS), and p-phenyl diammonium dichloride (PPD) were obtained from Sigma Chemicals, St Louis, MO, USA. All other reagents were of chemical grade.Special chemicals such as 5', 5'-dithio-2EDTA in 0.2 M Na2HPO4, 20 μL 10 mM DTNB in 0.2 M Na2HPO4, and 100 μL serum was incubated at room temperature for five min; the absorbance was read at 412 nm. Appropriate sample and reagent blanks were prepared simultaneously and the respective absorbance was noted. Corrected absorbance values were used to calculate serum protein thiol content using a molar extinction coefficient of 1600/M/cm and values were expressed as μM. The calibration curve was produced using GSH dissolved in phosphate-buffered saline (PBS).[The reaction mixture containing 900 μL 2 mM Nane (PBS).One mL of reaction mixture containing 850 μL of 0.1M phosphate buffer, pH 6.5; 50 μL 20 mM CDNB , and 50 μL 20 mM GSH was preincubated at 37° C for 10 min. Reaction was started by adding 50 μL serum and GST activity was assayed kinetically. Reaction was followed at one minute intervals for five minutes by measuring the absorbance at 340 nm. GST was determined by using a molar extinction coefficient of 9.6/mM/cm and GST activity was expressed as IU/L.255/M/cm and the values were expressed as μM.[The reaction mixture containing 1 mL 0.67% thiobarbituric acid (TBA), 500 μL 20% tricarboxylic acid (TCA), and 100 μL serum was incubated at 100° C for 20 min and centrifuged at 12,000 rpm for 5 min. The absorbance of the supernatant was read at 532 nm and MDA concentration was determined by using a molar extinction coefficient of 1.56 × 10ed as μM.The reaction mixture containing 3 mL PPD and 60 μL plasma was incubated at 37°C for 15 minutes. After incubation, 600 μL of 3% sodium azide was added and the absorbance was read at 546 nm. An appropriate sample blank was prepared simultaneously and its absorbance was noted. Plasma ceruloplasmin levels were calculated as (absorbance of test – absorbance of blank) × 237, and values were expressed as mg/dL.3/M/cm. Plasma copper values were expressed as μg/dL.[Reaction mixture was composed of 200 μL 6M guanidine hydrochloride containing 0.1M ascorbic acid in 0.1M acetate buffer, pH 4.8, and 100 μL plasma. After incubation for 10 minutes at room temperature, 200 μL 0.89 mM BCDS in 2M Tris was added and mixed by vortexing. The absorbance was read at 480 nm, as was that of appropriate sample and reagent blanks that were prepared simultaneously. Corrected absorbance values were used to calculate plasma copper levels using a molar extinction coefficient of 2.388 × 10as μg/dL.In CRF patients and healthy controls, serum total protein levels were determined using Biuret method, albumin level was measured by the bromocresol green dye-binding method, urea by the urease-glutamate dehydrogenase method, and creatinine by Jaffe's method using a Clinical Chemistry Autoanalyzer (Hitachi 912).P < 0.05 was considered to be statistically significant. Statistical analysis was performed using the Statistical Package for Social Sciences . Independent sample t-test was used to compare mean values and Pearson's correlation was applied to correlate the parameters.The results were expressed as mean ± standard deviation (SD). P < 0.001), whereas total protein, albumin, protein thiols, and copper levels were found to be significantly decreased in CRF patients compared to normal controls (P < 0.001). Ceruloplasmin levels had decreased significantly (P < 0.05) and there was no significant change in serum GST levels in CRF patients compared to normal controls. On applying Pearson's correlation, serum copper correlated negatively with urea , and creatinine ; but positively with total protein , albumin , protein thiols , ceruloplasmin  [P < 0.01) [As shown in Tables  < 0.01) , and MDA < 0.01) .Proteinuria is a well established finding in CRF patients. Total prCRF is an inflammatory condition and we hypothesized that the levels of CP, an acute phase protein, should increase in these patients. In contrast to our hypothesis, we observed that levels of CP decreased significantly in CRF patients as compared to the controls. Possible factors contributing to this observation were proteinuria and damage to this excreted protein due to the prevalent oxidative stress. Advanced glycation end products (AGEs) are formed during nonenzymatic, glyco-oxidation reactions involving plasma proteins. These reactions are enhanced in uremia where there is increased concentration of many α-oxaldehydes such as glyoxal, methylglyoxal etc. Like AOPPs, AGEs are also a class of uremic toxins.–35 ROS mBefore being excreted in urine, free copper may participate in metal ion-catalyzed Fenton and/or Haber-Wiess reactions to generate ROS. Some of the events associated with copper toxicity in uremia may be due to the generation of ROS by metal ions such as copper. Copper ions display high affinity for thiols and amino groups occurring in proteins. This indiscriminate binding may damage the structure of proteins, thus modifying their biological functions. Copper-dOther authors have suggested that serum copper, determined by TBARS levels, is the major determinant of serum lipid peroxidation status. Our studAs GST is involved in the detoxification of electrophiles and hydroperoxides, it is generally expected that its plasma levels should have increased in CRF patients due to enhanced oxidative stress. In contrast, we found no significant difference in the serum levels of GST in CRF patients when compared to healthy controls. A previous study described inhibition of RBC–GST activity with uremic plasma. Hence, tIn conclusion, our study reconfirms the observations of earlier studies, but with a different patient population in a different geographical location. We have observed increased uremic stress in CRF patients, which may be induced by metal ion. In agreement with previous studies, we found a deficiency of antioxidant levels in CRF patients, a finding that emphasizes the importance of antioxidant supplementation in these patients. In contrast to previous studies, we did not observe any change in activity of GST in CRF patients, a finding that warrants further study in different patient populations and with other experimental designs.


Kangaroo care (KC) has been widely using to improve the care of low birth weight infants. However, very little is known about cerebral hemodynamics responses in low birth weight infants during KC intervention. The objective of this study was to elucidate the response of cerebral hemodynamics during KC in low birth weight infants.2), heart rate (HR), respiration rate (RR) measured by electrocardiogram, and percentages of oxygen saturation (SpO2) measured by pulse oxymetry was monitored in 16 preterm infants (< 1600 g) in three sessions: before, during, and after KC. Using power spectral analysis, total power (TP), low-frequency  and high-frequency  bands, the ratio of LF/HF were calculated and normalized as %LF or %HF = LF or HF/TP × 100 (%).Near infrared spectroscopy measured regional cerebral oxygenation . The %LF of LrSO2 and RrSO2 was decreased during KC (p < 0.05) with decreased %HF in RrSO2 (p < 0.05). The %LF of HR was significantly increased during KC while %HF was decreased (p < 0.05). Mean and TP of RR was increased during KC (p < 0.01 respectively) with the increase of quiet sleep state (p < 0.05) and decreased after KC (p < 0.01). The %LF of RR was increased after KC (p < 0.05) with decreased %HF (p < 0.05); however, significant changes were not observed during KC.Significant differences were not observed in the mean rSO2 and HR might be associated with quiet sleep states. The results of this study may indicate the contribution of KC intervention to the activation of central nervous system and brain function. Further study is needed to determine the underlying physiology responsible for these differences.KC intervention appears to have influence on cerebral hemodynamics as well as cardiorespiratory parameters. The results of rSO Recently, skin – to – skin care, called kangaroo care (KC), has been widely practiced for preterm and low birth weight (LBW) infants in the neonatal intensive care unit (NICU). During kangaroo care, the mother holds a naked infant in a vertical position against the breasts so that the infant can achieve skin – to – skin contact. KC was first introduced in Bogotá, Columbia by Dr. Edgar Rey and Hector Martinez in 1978 as a way of compensation for the overcrowding of incubators in hospitals caring for preterm infants [Many studies have been performed to evaluate the psychological and physiological responses during KC in preterm infants -4. PositSpectral analysis of time series data using Fast Fourier transformation (FFT) has been widely utilized to study the autonomic nervous system -26. In p2) as a parameter of cerebral hemodynamics, heart rate (HR), respiratory rate (RR), and SpO2 in stable low birth weight infants during KC using power spectral analysis.In this study, we investigated cerebral hemodynamics in addition to cardiorespiratory parameters in preterm infants during KC intervention using power spectral analysis. Therefore, we investigated regional cerebral oxygenation  were enrolled in this study. All of the infants were stable and breathed spontaneously without supplemental oxygenation with postconceptional age ≥ 32 wks. Infants who had severe congenital malformations, severe asphyxia, and a potential cause of apnea other than immaturity, such as sepsis or intracranial hemorrhage, were excluded from the study. Three infants were excluded because of interrupted observation. Finally, 16 infants were selected for further analysis (Table 2). Regional cerebral oxygenation was measured with a near infrared spectroscopy, NIRS , with the two probes positioned on the bilateral frontoparietal areas. Physiological data were recorded at 10 second intervals (averaged over 10 second period) through a Wave Archiving System  and further analyzed. Data from the first 30 minutes during KC were excluded from the analysis to minimize the effects of changes due to the adaptation process. The data for rSO2 were recorded from both the left and right hemispheres independently .KC intervention was performed for one hour. The temperature of the room ranged from 27 to 28°C and the humidity ranged from 60 to 70% during KC. Infants were observed carefully and monitored in three conditions: 30 minutes in the incubators (before KC), 1 hour of KC intervention (during KC), and 30 minutes in the incubator again after KC intervention. Mothers were seated on a reclining chair at a 60° angle, wearing a front opening blouse. The infants were placed naked except for a diaper directly onto the skin between the breasts and covered with a light blanket. Infants were fed 1 hour before KC. All infants were continuously monitored with electrocardiogram to determine HR, RR and with pulse oxymetry for percentages of oxygen saturation . A repeated measures ANOVA was performed to analyze the differences of parameters among three sessions. Conventional statistics: mean, standard deviation, coefficient of variance (CV), range, normal distribution, and median were performed for all parameters. Data are expressed as median (range) or mean ± SD.2, LrSO2 and RrSO2 are shown in Table 2, LrSO2, and RrSO2, there were no significant differences among the three sessions; however, RR was significantly increased during KC and decreased after KC . Body temperature was increased by 0.3°C during KC (p < 0.01). The CV of HR was decreased by 2.2% during KC (p < 0.05), while it was increased again after KC by 2.9% (p < 0.05). There were no significant differences in the CVs of RR, SpO2, LrSO2, and RrSO2.Descriptive statistics of HR, RR, SpO2/Hz; before vs during and during vs after, p < 0.01). The TP of SpO2 had a similar tendency to that of HR. On the other hand, the TP of RR was significantly increased during KC and decreased after KC . In regional cerebral oxygenation, the TP of either LrSO2 or RrSO2 was significantly decreased during KC and increased again after KC .Changes in TP during the sessions were shown in Figure 2 was significantly decreased during KC for both LrSO2  and RrSO2 . The %HF was decreased during KC for RrSO2 , although there was no significant change in this parameter for LrSO2 during the sessions. For RR and SpO2, significant changes were not observed in %LF or HF during KC, although the %LF of RR after KC was significantly increased while the %HF was decreased  with an increased LF/HF ratio (p < 0.01). There were no significant differences in the ratio of HR, SpO2, LrSO2, and RrSO2 during the sessions , low – frequencies (0.02 – 0.20 Hz) reflect sympathetic activities such as the baroreceptor reflex, high-frequencies (0.2 – 2.0 Hz) reflect parasympathetic activities such as vagal activity, and the ratio of LF/HF power reflects the balance between sympathetic and parasympathetic activities -26,32. Sreported ,33, and d on HRV ,34. Furtreported ,36. Thesreported ,38, thisreported  or in th2 due to KC in LBW preterm infants through spectral characteristics. Therefore, it was difficult to compare the results of this study with other similar studies and apply to the clinical settings. Despite the sampling limitation, this reference data provide new understanding into the response of cerebral hemodynamics in preterm infants and should have significant implications to generalize the rSO2 responses with different modalities in LBW infants. Further studies are necessary to determine the clinical relevance of the present findings.To our knowledge, the present study was the first study to analyze the response of rSOThe results of this study revealed that changes of cerebral hemodynamics associated with KC position in preterm infants as well as cardiorespiratory parameters. These changes were especially apparent by power spectral analysis. Furthermore, the results of this study indicate that KC may contribute to the activation of central nervous system and brain function. Further study is needed to determine the underlying physiology responsible for these differences.2: left side regional cerebral oxygenation; RrSO2: right side regional cerebral oxygenation; HR: heart rate, RR: respiration rate; TP: total power; HF: high frequency; LF: low frequency; weeks, wks; NIRS: near infrared spectroscopy, ECG: electrocardiogram, HRV, heart rate variability.KC: Kangaroo care; LBW: low birth weight; NICU: neonatal intensive care unit; FFT: fast Fourier transformation; LrSOThe authors declare that they have no competing interests.EAB: Participated in all data collection, in the analysis and discussion of the results, and in the writing of the manuscript; MB: Mainly worked with first author and responsible for this study.; NO: Participated in all clinical and physiological data collection; SY: Participated in organized the study group with nursing staff.; ST: Participated in interpretation of clinical findings and theoretical support; HY: Introduced and established the NICU local area network system (LAN system) for physiological data recording and Participated in clinical suggestion about physiological rhythmicity; MK: Participated in theoretical support of physiological instability in neonates and physiological rhythmicity; YK: Organized the study group and checked the manuscript finally.The pre-publication history for this paper can be accessed here:


The world age-standardised prevalence of high-risk HPV (hrHPV) infection among 5038 UK women aged 20–59 years, with a low-grade smear during 1999–2002, assessed for eligibility for TOMBOLA  was 34.2%. High-risk HPV prevalence decreased with increasing age, from 61% at ages 20–24 years to 14–15% in those over 50 years. The age-standardised prevalence was 15.1, 30.7 and 52.7%, respectively, in women with a current normal, borderline nuclear abnormalities (BNA) and mild smear. In overall multivariate analyses, tertiary education, previous pregnancy and childbirth were associated with reduced hrHPV infection risk. Risk of infection was increased in non-white women, women not married/cohabiting, hormonal contraceptives users and current smokers. In stratified analyses, current smear status and age remained associated with hrHPV infection. Data of this type are relevant to the debate on human papillomavirus (HPV) testing in screening and development of HPV vaccination programmes. Infection with human papillomavirus (HPV) is necessary for the development of cervical cancer  and are Human papillomavirus population prevalence mainly depends on patterns of sexual exchange , which vSubjects were women assessed for eligibility for TOMBOLA , a randomised controlled trial (RCT) of alternative management policies and HPV triage .Women aged 20–59 years, resident in Grampian, Tayside or Nottingham, with a low-grade smear ) taken routinely in the UK national cervical screening programmes (CSPs) during 01/10/1999–31/10/2003, with no previous treatment for cervical lesions, were eligible for TOMBOLA. Recruitment was in two phases: 01/10/1999–12/03/2001 and 13/03/2001–31/10/2003. During phase one, eligible women had no abnormal smears in the previous 3 years; during phase two, they had up to one BNA smear in the previous 3 years. In phase one, women with a BNA smear were invited to a recruitment clinic approximately 6 months later for a follow-up smear and a swab for HPV testing. Women with a mild smear during both phases, or a BNA smear during phase two, were invited to a recruitment clinic approximately 2 months later and a swab for HPV testing was taken. The swab, an endocervical sample, was taken with a cytobrush; this was immersed in 2 ml of sterile phosphate-buffered saline containing 0.05% thiomersal.n=5514) consented to participate; 27 were subsequently excluded because the smear was inadequate. Five thousand and seventy-four (92%) of the remainder provided an HPV sample.A total of 52% of eligible women attended a recruitment clinic, of whom 95% . Adequate samples underwent HPV PCR using GP5+/6+ consensus primers, followed by enzyme immunoassay for detection of 14 high-risk types  . Human and viral DNA were extracted using the Qiagen UK kit  (QIAamp® DNA Mini Kit) following optimisation of the manufacturer's protocol. Each batch included negative controls containing only elution buffer AE. Extracted DNA was amplified and quantitated by type-specific real-time polymerase chain reaction (PCR) for the housekeeping gene, human betaglobin. Samples without recordable DNA levels were considered inadequate . For other women, the ‘current’ smear was the smear that triggered the invitation to participate in TOMBOLA. Six smear status categories were defined: normal and one previous BNA; BNA and no previous abnormal; BNA and one previous BNA; mild and no previous abnormal; mild and one previous BNA; and moderate or severe dyskaryosis and one previous BNA.χ2-tests. High-risk HPV prevalence was age-standardised to the truncated (20–59 years) world standard population. Multivariate unconditional logistic regression models were constructed to assess factors associated with hrHPV+ve risk. Since age modified the relationship between smear status and hrHPV status, these factors were fitted by a single variable, which combined the six smear status categories with two age groups (20–29 and ⩾30 years). The global likelihood ratio test (LRT) was used to assess the impact of lifestyle and socio-demographic factors on age-and-smear status-adjusted risk estimates; factors with P<0.1 were retained in the final model. To explore sub-group heterogeneity, the modelling was repeated stratifying by age  and current smear . All multivariate models had adequate fit , non-white ethnic groups  or those with a previous abnormal smear . Since two of three centres did not take swabs from menstruating women, the proportion providing a sample varied by centre . It did not vary by tertiary education level .Younger women, white women and those with a single BNA or mild smear were less likely to provide an HPV sample than older women (P(interaction) 0.0004). The overall age-standardised prevalence was 34.2% (95% CI 32.6–35.8). For women with a current normal smear, it was 15.1% (95% CI 12.6–17.6), for those with a current BNA smear 30.7% (95% CI 8.6–32.8) and with a current mild smear 52.7% (95% CI 48.6–56.8).The crude hrHPV prevalence was 39.2%  37.8–40.5). Positivity declined with increasing age , and increased with increasing smear grade. For all grades, prevalence and risk were higher in younger (20–29 years) than older (⩾30 years) women, but not by a constant amount  and physical activity .In multivariate age-stratified analyses of women aged 20–29 years, age and smear status were significantly associated with infection. Tertiary education and having had children were also significant risk factors, with risk estimates similar to those in unstratified analyses. In women aged 30–59 years, age and smear status were significantly associated with infection, as were tertiary education, having had children, ethnicity and smoking; effect sizes were similar to those in In multivariate smear-stratified analyses of all smear groups, age was significantly associated with infection. Having a college/university degree reduced infection risk in women with a current normal  or BNA smear  but not in those with a mild smear. In all smear strata, divorced/separated/widowed women had higher risk than married/co-habiting women . In the BNA strata only, being single also increased risk . Having been pregnant was inversely associated with infection in those with a current normal  or BNA smear , but not in those with a mild smear. Having had children was associated with reduced risk in all smear strata, only reaching statistical significance in the current normal group . Hormonal contraceptive use was associated with increased risk in the current normal  and BNA  strata, but not among the mild group. Barrier contraceptive use, caesarean delivery, smoking, physical activity and ethnicity were unrelated to infection in all strata.Our study was large, population-based and nested in a pragmatic RCT within the UK national CSPs. Among study participants, the current BNA : mild smear ratio (1.8 : 1) was close to that reported for the CSP screening age group in 2004–2005 (1.9 : 1) , suggestWhile TOMBOLA participation was 52% overall, it was lower among younger women and those resident in more deprived areas , groups The treatment of lesions, and possibly also the act of taking a smear, can potentially clear cervical HPV infection  was similar to that among women with a BNA smear from the UK ARTISTIC trial (31%) . In the Our crude (16.0%) and age-standardised (15.1%) prevalences among women with a current normal smear were similar to the pooled estimate from 27 PCR-based studies of cytologically normal women mainly from North America and Europe (16.2%) . HoweverNotable geographical variations in age-specific HPV curves have recently been described (n=223), precluding multivariate analysis of individual groups. Crude infection frequencies  are consistent with sexual behaviour data and suggest that the raised risk may be limited to black women.In the United Kingdom, white, black-African and black-Caribbean women have higher numbers of lifetime sexual partners, lower median age at first heterosexual intercourse and higher incidence of (non-HPV) sexually transmitted infections than women from Indian or Pakistani ethnic groups (vs never users 1.23 (95% CI 0.99–1.51); OR current users vs never users 1.46 (95% CI 1.24–1.73)). This appears compatible with the postulated mechanisms by which current OC use influences infection, which include the enhancing effect of oestrogens on cervical ectopy, thereby permitting potential carcinogens (including HPV) easier access to the transformation zone (High-risk HPV infection risk was >50% higher in women who had used OCs or other hormonal contraceptives. The latter have been studied little previously. Among ALTS participants, no association was found with injectable contraceptives or Norplant , in seveion zone .Our observation that having been pregnant was associated with reduced hrHPV infection risk is consistent with the IARC HPV Prevalence Surveys Study Group analysis of >15 000 women . HoweverIdentifying differences in the relative importance of risk factors in sub-groups can be informative – as is evident from the few previous studies using this analytical approach (