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Equine foamy virus (EFVeca) is a foamy virus of non-primate origin and among the least-studied members of this retroviral subfamily. By sequence comparison, EFVeca shows the highest similarity to bovine foamy virus. In contrast to simian, bovine or feline foamy viruses, knowledge about the epidemiology of EFVeca is still limited. Since preliminary studies suggested EFVeca infections among horses in Poland, we aimed to expand the diagnostics of EFVeca infections by developing specific diagnostic tools and apply them to investigate its prevalence. An ELISA test based on recombinant EFVeca Gag protein was developed for serological investigation, while semi-nested PCR for the detection of EFVeca DNA was established. 248 DNA and serum samples from purebred horses, livestock and saddle horses, Hucul horses and semi-feral Polish primitive horses were analyzed in this study. ELISA was standardized, and cut off value, sensitivity and specificity of the test were calculated using Receiver Operating Characteristic and Bayesian estimation. Based on the calculated cut off, 135 horses were seropositive to EFVeca Gag protein, while EFVeca proviral DNA was detected in 85 animals. The rate of infected individuals varied among the horse groups studied; this is the first report confirming the existence of EFVeca infections in horses from Poland using virus-specific tools.

Background: The 2016 European Tobacco Products Directive (TPD) required Member States (MS) to implement new regulations for electronic cigarettes (ECs). We conducted a longitudinal study to assess changes over 2 years in smokers' support for EC policies and identify predictors of support in seven European countries after TPD implementation., Methods: Prospective cohort surveys were conducted among adult smokers in Germany, Greece, Hungary, Poland, Romania, Spain and England in 2016 (n = 9547; just after TPD) and 2018 (n = 10 287; 2 years after TPD). Multivariable logistic regression models employing generalized estimating equations assessed changes in support for four EC policies, and tested for country differences and strength of key predictors of support., Results: Banning EC use in smoke-free places was supported by 53.1% in 2016 and 54.6% in 2018 with a significant increase in Greece (51.7-66.0%) and a decrease in Spain (60.1-48.6%). Restricting EC/e-liquid nicotine content was supported by 52.2 and 47.4% in 2016 and 2018, respectively, with a significant decrease in England (54.2-46.5%) and Romania (52.5-41.0%). An EC promotion ban was supported by 41.1 and 40.2%. A flavour ban was supported by 33.3% and 32.3% with a significant increase in Hungary (34.3-43.3%). Support was generally higher in Poland, Hungary and Greece vs. England. Support was lower among dual and EC-only users, and low-income smokers., Conclusions: Smokers in all countries strongly supported banning EC use in smoke-free places and restricting nicotine content after TPD implementation, with no clear trends for changes in policy support., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: Advertising, promotion and sponsorship of electronic cigarettes (ECAPS) have increased in recent years. Since May 2016, the Tobacco Products Directive 2014/40/EU (TPD2) prohibits ECAPS in various advertising channels, including media that have cross-border effects. The objective of this study was to investigate changes in exposure to ECAPS in a cohort of smokers from six European Union member states after implementation of TPD2., Methods: Self-reported exposure to ECAPS overall and in various media and localities was examined over two International Tobacco Control Policy Evaluation survey waves (2016 and 2018) in a cohort of 6011 adult smokers from Germany, Greece, Hungary, Poland, Romania and Spain (EUREST-PLUS Project) using longitudinal generalized estimating equations models., Results: Self-reported ECAPS exposure at both timepoints varied between countries and across examined advertising channels. Overall, there was a significant increase in ECAPS exposure [adjusted odds ratio (aOR): 1.25, 95% CI: 1.09-1.44]. Between waves, no consistent patterns of change in ECAPS exposure across countries and different media were observed. Generally, ECAPS exposure tended to decline in some channels regulated by TPD2, particularly on television and radio, while exposure tended to increase in some unregulated channels, such as at points of sale., Conclusions: The findings suggest that the TPD2 was generally effective in reducing ECAPS in regulated channels. Nonetheless, further research is warranted to evaluate its role in reducing ECAPS exposure, possibly by triangulation with additional sources of data., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: The European Tobacco Products Directive (TPD), which went into effect in May 2016, regulates packaging design and labelling of cigarettes and roll-your-own (RYO) tobacco. The aim of the current study was to examine whether smokers and recent quitters in six European Union (EU) countries (Germany, Greece, Hungary, Poland, Romania and Spain) reported noticing TPD-related changes to packaging, and correlates of noticing these changes., Methods: Cross-sectional data from the Wave 2 of the ITC 6 European Country Survey in 2018 after implementation of the TPD. Bivariate analyses included adult smokers (n = 5597) and recent quitters (n = 412). Adjusted logistic regression analyses were restricted to the subset of current smokers (n = 5597) and conducted using SAS-callable SUDAAN., Results: Over half of smokers (58.2%) and 30% of quitters noticed at least one of five types of TPD-related pack changes. Over one-quarter of all respondents noticed changes to health warnings (30.0%), standardized openings (27.7%), minimum pack unit size (27.9%), and the removal of tar, nicotine and carbon monoxide information (26.7%) on packaging. Cross-country differences were observed across all measures, with noticing all pack changes markedly lower in Spain than the other countries. Correlates of noticing specific pack changes included sociodemographic characteristics, smoking behaviours and related to packaging., Conclusions: This is one of the first cross-country studies to examine the extent to which TPD changes to cigarette and RYO tobacco packaging are being noticed by smokers and recent quitters. Findings indicate that the majority of smokers noticed at least one type of pack change, but this varied across countries and sub-populations., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: There is currently no comprehensive legislation protecting non-smokers and children from second-hand smoke (SHS) exposure in private cars at the European Union (EU) level. This study aims to assess smokers' support for smoke-free cars legislation in six EU countries., Methods: Data come from the EUREST-PLUS ITC Europe Surveys: Wave 1 (2016, n = 6011) and Wave 2 (2018, n = 6027) conducted in Germany, Greece, Hungary, Poland, Romania and Spain. Support for smoke-free cars carrying pre-school children and non-smokers and voluntary implementation of smoke-free cars were assessed among adult smokers. Generalized estimating equations models were used to assess changes in support between waves., Results: In 2018, 96.3% [95% confidence interval (CI) 95.4-97.0%] of the overall sample supported smoke-free legislation for cars carrying pre-school children, representing an increase of 2.4 percentage points in comparison to 2016. Smoke-free legislation for cars transporting non-smokers was supported by 85.2% (95% CI 83.1-87.1%) of smokers' in 2016 and 90.2% (95% CI 88.6-91.7%) in 2018. Among smokers who owned cars, there was a significant 7.2 percentage points increase in voluntary implementation of smoke-free cars carrying children from 2016 (60.7%, 95% CI 57.2-64.0%) to 2018 (67.9%, 95% CI 65.1-70.5%). All sociodemographic groups of smokers reported support higher than 80% in 2018., Conclusion: The vast majority of smokers in all six EU countries support smoke-free legislation for cars carrying pre-school children and non-smokers. This almost universal support across countries and sociodemographic groups is a clear indicator of a window of opportunity for the introduction of comprehensive legislation to protect non-smokers and children from SHS exposure in cars., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: Tobacco product packaging is a key part of marketing efforts to make tobacco use appealing. In contrast, large, prominent health warnings are intended to inform individuals about the risks of smoking. In the European Union, since May 2016, the Tobacco Products Directive 2014/40/EU (TPD2) requires tobacco product packages to carry combined health warnings consisting of a picture, a text warning and information on stop smoking services, covering 65% of the front and back of the packages., Methods: Key measures of warning label effectiveness (salience, cognitive reactions and behavioural reaction) before and after implementation of the TPD2, determinants of warning labels' effectiveness and country differences were examined in a longitudinal sample of 6011 adult smokers from Germany, Greece, Hungary, Poland, Romania and Spain (EUREST-PLUS Project) using longitudinal Generalized Estimating Equations (GEE) models., Results: In the pooled sample, the warning labels' effectiveness increased significantly over time in terms of salience (adjusted OR = 1.18; 95% CI: 1.03-1.35), while cognitive and behavioural reactions did not show clear increases. Generally, among women, more highly educated smokers and less addicted smokers, the effectiveness of warning labels tended to be higher., Conclusion: We found an increase in salience, but no clear increases for cognitive and behavioural reactions to the new warning labels as required by the TPD2. While it is likely that our study underestimated the impact of the new pictorial warning labels, it provides evidence that health messages on tobacco packaging are more salient when supported by large pictures., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: This study presents perceptions of the harmfulness of electronic cigarettes (e-cigarettes) relative to combustible cigarettes among smokers from six European Union (EU) countries, prior to the implementation of the EU Tobacco Products Directive (TPD), and 2 years post-TPD., Methods: Data were drawn from the EUREST-PLUS ITC Europe Surveys, a cohort study of adult smokers (≥18 years) from Germany, Greece, Hungary, Poland, Romania and Spain. Data were collected in 2016 (pre-TPD: N = 6011) and 2018 (post-TPD: N = 6027). Weighted generalized estimating equations were used to estimate perceptions of the harmfulness of e-cigarettes compared to combustible cigarettes (less harmful, equally harmful, more harmful or 'don't know')., Results: In 2016, among respondents who were aware of e-cigarettes (72.2%), 28.6% reported that they perceived e-cigarettes to be less harmful than cigarettes (range 22.0% in Spain to 34.1% in Hungary). In 2018, 72.2% of respondents were aware of e-cigarettes, of whom 28.4% reported perceiving that e-cigarettes are less harmful. The majority of respondents perceived e-cigarettes to be equally or more harmful than cigarettes in both 2016 (58.5%) and 2018 (61.8%, P > 0.05). Overall, there were no significant changes in the perceptions that e-cigarettes are less, equally or more harmful than cigarettes, but 'don't know' responses significantly decreased from 12.9% to 9.8% (P = 0.036). The only significant change within countries was a decrease in 'don't know' responses in Spain (19.3-9.4%, P = .001)., Conclusions: The majority of respondents in these six EU countries perceived e-cigarettes to be equally or more harmful than combustible cigarettes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association.)

Background: The emergence of new types of tobacco and tobacco-related products on the European Union (EU) market has precipitated the possibility for both poly-tobacco use and transitions between products. In the EU, the regulatory environment has shifted with the implementation of the European Tobacco Products Directive (TPD) in May 2016, which may influence consumer transitions between products., Methods: The aim of this paper was to examine trends and transitions in tobacco products from 2016 to 2018 -before and after implementation of the TPD in the EU. Data come from Wave 1 (pre-TPD) and Wave 2 (post-TPD) of the EUREST-PLUS ITC Six European Country Survey, a cohort study of adults who at the time of recruitment were smokers from six EU countries- Germany, Greece, Hungary, Poland, Romania, and Spain. D (N = 3195). Bivariate and logistic regression analyses of weighted data was conducted using SAS-callable SUDAAN., Results: Overall, among those who smoked factory-made cigarettes (FM) only at Wave 1, 4.3% switched to roll-your-own tobacco (RYO) only. Among RYO only users at Wave 1, 17.0% switched to FM only, however compared to all other countries, respondents from Hungary had the highest percentage of FM only users at Wave 1 switch to RYO only at Wave 2 (18.0%)., Conclusions: The most prominent transition overall was from smoking RYO exclusively at Wave 1 to smoking FM tobacco exclusively at Wave 2, however this varied across countries. As the tobacco control regulatory environment of the EU develops, it is important to continue to monitor transitions between types of products, as well as trends in cessation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: The EUREST-PLUS ITC Europe surveys aim to evaluate the impact of the European Union's Tobacco Products Directive (EU TPD) implementation within the context of the WHO FCTC. This article describes the methodology of the 2016 (Wave 1) and 2018 (Wave 2) International Tobacco Control 6 European (6E) Country Survey in Germany, Greece, Hungary, Poland, Romania and Spain; the England arm of the 2016 (Wave 1) and 2018 (Wave 2) ITC 4 Country Smoking and Vaping (4CV) Survey; and the 2016 (Wave 10) and 2017 (Wave 11) ITC Netherlands (NL) Survey. All three ITC surveys covering a total of eight countries are prospective cohort studies with nationally representative samples of smokers., Methods: In the three surveys across the eight countries, the recruited respondents were cigarette smokers who smoked at least monthly, and were aged 18 and older. At each survey wave, eligible cohort members from the previous waves were retained, regardless of smoking status, and dropouts were replaced by a replenishment sample., Results: Retention rates between the two waves of the ITC 6E Survey by country were 70.5% for Germany, 41.3% for Greece, 35.7% for Hungary, 45.6% for Poland, 54.4% for Romania and 71.3% for Spain. The retention rate for England between ITC 4CV1 and ITC 4CV2 was 39.1%; the retention rates for the ITC Netherlands Survey were 76.6% at Wave 10 (2016) and 80.9% at Wave 11 (2017)., Conclusion: The ITC sampling design and data collection methods in these three ITC surveys allow analyses to examine prospectively the impact of policy environment changes on the use of cigarettes and other tobacco products in each country, to make comparisons across the eight countries., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: The study assessed awareness and use of heated tobacco products (HTPs) and factors that influenced these issues among cigarette smokers from six European countries in 2016 (Wave 1) and 2018 (Wave 2)., Methods: A survey was conducted among a nationally representative sample of cigarette smokers aged 18 years or older from Germany, Greece, Hungary, Poland, Romania and Spain in 2016 (N = 6011) and 2018 (N = 6027; 53% of smokers from the previous wave were retained, regardless of smoking status and dropouts were replaced by a replenishment sample of smokers). Data were collected through face-to-face interviews. Estimates were produced using weighted data. The study presents the cross-sectional results., Results: Awareness of HTPs increased from 8% to 17% between the two waves. At Wave 1, 1.1% of the smokers declared having used HTPs at least once during their lifetime; and at Wave 2, this increased to 1.9% (around 1% or less in four countries, except for Greece and Romania where it was around 4%). Factors associated with HTPs use among those who had ever heard about these products at Wave 1 were country of residence, being a daily cigarette smoker and ever use of electronic cigarettes. At Wave 2, ever use of HTPs was significantly higher among those who had tried to quit smoking combustible cigarettes in the last 12 months, had tried electronic cigarettes during lifetime and perceived HTPs as less dangerous than combustible cigarettes; the country of residence was also associated with HTPs use., Conclusion: This study offers insights into the behaviours and perceptions of European adult smokers regarding HTPs, an important emerging issue in the field of tobacco control., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: We examined quit attempts, use of cessation assistance, quitting beliefs and intentions among smokers who participated in the 2018 International Tobacco Control (ITC) Europe Surveys in eight European Union Member States (England, Germany, Greece, Hungary, the Netherlands, Poland, Romania and Spain)., Methods: Cross-sectional data from 11 543 smokers were collected from Wave 2 of the ITC Six European Country (6E) Survey (Germany, Greece, Hungary, Poland, Romania and Spain-2018), the ITC Netherlands Survey (the Netherlands-late 2017) and the Four Countries Smoking and Vaping (4CV1) Survey (England-2018). Logistic regression was used to examine associations between smokers' characteristics and recent quit attempts., Results: Quit attempts in the past 12 months were more frequently reported by respondents in the Netherlands (33.0%) and England (29.3%) and least frequently in Hungary (11.5%), Greece (14.7%), Poland (16.7%) and Germany (16.7%). With the exception of England (35.9%), the majority (56-84%) of recent quit attempts was unaided. Making a quit attempt was associated with younger age, higher education and income, having a smoking-related illness and living in England. In all countries, the majority of continuing smokers did not intend to quit in the next 6 months, had moderate to high levels of nicotine dependence and perceived quitting to be difficult., Conclusions: Apart from England and the Netherlands, smokers made few quit attempts in the past year and had low intentions to quit in the near future. The use of cessation assistance was sub-optimal. There is a need to examine approaches to supporting quitting among the significant proportion of tobacco users in Europe and increase the use of cessation support as part of quit attempts., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: Article 20 of the European Tobacco Product Directive (TPD), which went into effect in May 2016, regulates electronic cigarettes (e-cigarettes) in the European Union (EU). The aim of this study was to evaluate changes in e-cigarette use, design attributes of the products used and awareness of e-cigarette labelling and packaging among smokers from six EU Member States (MS) before and after TPD implementation., Methods: Data come from Wave 1 (2016, pre-TPD) and Wave 2 (2018, post-TPD) of the ITC Six European Country Survey among a sample of smokers and recent quitters who use e-cigarettes from six EU MS. Weighted logistic generalized estimating equations regression models were estimated to test the change in binary outcomes between Waves 1 and 2 using SAS-callable SUDAAN., Results: In 2018, current daily/weekly e-cigarette use among adult smokers was just over 2%, but this varied from the highest in Greece (4%) to lowest in Poland (1.2%). From Waves 1 to 2, there was a significant increase in respondents reporting noticing and reading health and product safety information on leaflets inside e-cigarette packaging (8.39-11.62%, P < 0.001). There were no significant changes between waves of respondents reporting noticing or reading warning labels on e-cigarette packages/vials., Conclusions: e-cigarette use among smokers in these six EU countries is low. Although reported noticing and reading leaflets included in the packaging of e-cigarettes increased significantly from before to after the TPD, there was no significant change in reported noticing and reading of warning labels. Findings indicate the importance of continued monitoring of TPD provisions around e-cigarettes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: The prevalence of roll-your-own tobacco (RYO) in Europe has been increasing. The aim of this study was to investigate transitions between factory-made (FM) cigarettes and RYO in a longitudinal sample of European smokers, and their perceptions of relative harmfulness and knowledge of health effects., Methods: We used data collected from the EUREST-PLUS ITC 6 European Country (6E) Surveys in 2016 (n = 6011 smokers) and in 2018 (n = 6027) in Germany, Greece, Hungary, Poland, Romania and Spain. A total of 3195 cohort respondents were interviewed in both years. Use of RYO and FM, knowledge of health effects of smoking as well as perceptions about RYO were assessed. We used logistic regression models to explore sociodemographic correlates of transitions from one product to the other, of perceptions and knowledge related to smoking health effects., Results: Approximately 7.4% of exclusive FM smokers transitioned to RYO and 29.5% of exclusive RYO smokers transitioned to FM cigarettes from 2016 to 2018. RYO use in 2018 was more frequent among smokers of low education and income, but none of these factors were associated with transitions. Most RYO smokers perceived RYO as cheaper than FM and 21.7% of them considered RYO to be less harmful than FM. Knowledge of the health effects of smoking was not associated with type of product smoked., Conclusions: RYO is popular among European smokers; its lower cost seems to be a major factor for RYO users; reasons for transitions to and from RYO are less clear and need to be further investigated., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association.)

Background: The European (EU) Tobacco Product Directive (TPD) was implemented in May 2016 to regulate the design and labelling of cigarettes and roll-your-own tobacco. At the same time, the UK introduced standardized packaging measures, whereas Germany, Greece, Hungary, Poland, Romania and Spain did not. This study examines the impact of introducing standardized packaging in England using a quasi-experimental design., Methods: Data from adult smokers in Waves 1 (2016; N=9547) and 2 (2018; N=9724) from the International Tobacco Control Policy Evaluation surveys (England) and EUREST-PLUS surveys (Germany, Greece, Hungary, Poland, Romania and Spain) were used. Generalized estimating equations were used to estimate changes in pack/brand appeal, salience of health-warning labels (HWLs) and perceived relative harm of different brands in England (where larger HWLs and standardized packaging were implemented), vs. each EU country (where only larger HWLs were implemented)., Results: There was an increase in the percentage of respondents from Germany, Hungary and Poland reporting they did not like the look of the pack (4.7%, 9.6%, and 14.2%, respectively), but the largest increase was in England (41.0%). Moreover, there was a statistically significant increase in the salience of HWLs in Hungary, Poland and Romania (17.0%, 13.9%, and 15.3%, respectively), but the largest increase was in England (27.6%). Few differences were observed in cross-country comparisons of the perceived relative harm of different brands., Conclusions: Findings suggest that standardized packaging reduces pack appeal and enhances the salience of HWLs over and above the effects of larger HWLs. Findings provide additional evidence and support for incorporating standardized packaging into the EU TPD., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Foamy viruses (FVs) are widely distributed and infect many animal species including non-human primates, horses, cattle, and cats. Several reports also suggest that other species can be FV hosts. Since most of such studies involved livestock or companion animals, we aimed to test blood samples from wild ruminants for the presence of FV-specific antibodies and, subsequently, genetic material. Out of 269 serum samples tested by ELISA with the bovine foamy virus (BFV) Gag and Bet antigens, 23 sera showed increased reactivity to at least one of them. High reactive sera represented 30% of bison samples and 7.5% of deer specimens. Eleven of the ELISA-positives were also strongly positive in immunoblot analyses. The peripheral blood DNA of seroreactive animals was tested by semi-nested PCR. The specific 275 bp fragment of the pol gene was amplified only in one sample collected from a red deer and the analysis of its sequence showed the highest homology for European BFV isolates. Such results may suggest the existence of a new FV reservoir in bison as well as in deer populations. Whether the origin of such infections stems from a new FV or is the result of BFV inter-species transmission remains to be clarified.

Background: Pelvic inflammatory disease (PID) has been associated with ovarian cancer risk. To clarify the role of Chlamydia trachomatis and other infectious agents in the development of ovarian cancer, we evaluated the association of serologic markers with incident ovarian cancer using a staged approach in two independent populations., Methods: Studies included: 1) a case-control study in Poland (244 ovarian cancers/556 control subjects) and 2) a prospective nested case-control study in the PLCO Cancer Screening Trial (160 ovarian cancers/159 control subjects). Associations of serologic marker levels with ovarian cancer risk at diagnostic as well as higher thresholds, identified in Poland and independently evaluated in PLCO, were estimated using multivariable adjusted logistic regression., Results: In the Polish study, antibodies (based on laboratory cut-point) against the chlamydia plasmid-encoded Pgp3 protein (serological gold standard) were associated with increased ovarian cancer risk (adjusted odds ratio [OR] = 1.63, 95% confidence interval [CI] = 1.20 to 2.22); when a positive result was redefined at higher levels, ovarian cancer risk was increased (cut-point 2: OR = 2.00, 95% CI = 1.38 to 2.89; cut-point 3 [max OR]: OR = 2.19, 95% CI = 1.29 to 3.73). In the prospective PLCO study, Pgp3 antibodies were associated with elevated risk at the laboratory cut-point (OR = 1.43, 95% CI = 0.78 to 2.63) and more stringent cut-points (cut-point 2: OR = 2.25, 95% CI = 1.07 to 4.71); cut-point 3: OR = 2.53, 95% CI = 0.63 to 10.08). In both studies, antibodies against other infectious agents measured were not associated with risk., Conclusions: In two independent populations, antibodies against prior/current C. trachomatis (Pgp3) were associated with a doubling in ovarian cancer risk, whereas markers of other infectious agents were unrelated. These findings lend support for an association between PID and ovarian cancer., (Published by Oxford University Press 2018.)

Bovine foamy virus (BFV), or bovine spumaretrovirus, is an infectious agent of cattle with no obvious disease association but high prevalence in its host. Here, we report two complete BFV sequences, BFV-Riems, isolated in 1978 in East Germany, and BFV100, isolated in 2005 in Poland. Both new BFV isolates share the overall genetic makeup of other foamy viruses (FV). Although isolated almost 25 years apart and propagated in either bovine (BFV-Riems) or nonbovine (BFV100) cells, both viruses are highly related, forming the European BFV clade. Despite clear differences, BFV-Riems and BFV100 are still very similar to BFV isolates from China and the United States, comprising the non-European BFV clade. The genomic sequences presented here confirm the concept of high sequence conservation across most of the FV genome. Analyses of cell culture-derived genomes reveal that proviral DNA may specifically lack introns in the env-bel coding region. The spacing of the splice sites in this region suggests that BFV has developed a novel mode to express a secretory but nonfunctional Env protein.

Chromosomal instability is a known hallmark of many cancers. DNA polymerases represent a group of enzymes that are involved in the mechanism of chromosomal instability as they have a central function in DNA metabolism. We hypothesized that genetic variation in the polymerase genes may affect gene expression or protein configuration and by that cancer risk and clinical outcome. We selected four genes encoding for the catalytic subunits of the polymerases β, δ, θ and ζ (POLB, POLD1, POLQ and REV3L, respectively) and two associated proteins (MAD2L2 and REV1) because of their previously reported association with chromosomal instability and/or tumorigenesis. We selected potentially functional and most informative tagging single nucleotide polymorphisms (SNPs) for genotyping in a population-based series of 783 Swedish breast cancer (BC) cases and 1562 controls. SNPs that showed a significant association in the Swedish population were additionally genotyped in a Polish population consisting of 506 familial/early onset BC cases and 568 controls. SNPs in all three polymerase ζ subunit genes associated either with BC risk or prognosis. Two SNPs in REV3L and one SNP in MAD2L2 associated with BC risk: rs462779 (multiplicative model: OR 0.79, 95% CI 0.68-0.92), rs3204953 (dominant model: OR 1.28, 95% CI 1.05-1.56) and rs2233004 (recessive model: OR 0.49, 95% CI 0.28-0.86). Homozygous carriers of the minor allele C of the third SNP in REV3L, rs11153292, had significantly worse survival compared to the TT genotype carriers (HR 2.93, 95% CI 1.34-6.44). Minor allele carriers of two REV1 SNPs (rs6761391 and rs3792142) had significantly more often large tumours and tumours with high histological grade and stage. No association was observed for SNPs in POLB, POLQ and POLD1. Altogether, our data suggest a significant role of genetic variation in the polymerase ζ subunit genes regarding the development and progression of BC.

The biology of foamy viruses, their mode of transmission and disease potential in their natural host and after interspecies transmission are largely unknown. To gain insights into the prevalence of bovine foamy virus (BFV) and its zoonotic potential, enzyme-linked immunosorbent assays (ELISAs) were established to determine antibody responses against Gag, Env, and the non-structural protein Bet in bovine serum and milk. In Polish cattle, strong Gag reactivity was most frequent (41.5%) and strongly associated with Bet antibodies, Env antibodies were less frequent. German cattle showed a low overall BFV antibody prevalence of 6.8%. Besides clearly BFV-positive animals, a substantial number of weakly reacting cattle were identified. BFV-specific antibodies were also detectable in milk. BFV was isolated from PBLs and milk cells of BFV-positive cattle but not from antibody-negative or weakly reacting animals. The implications of these findings for the potential interspecies transmission of BFV to humans will be discussed.

The analysis of short tandem repeats is a widely used method to estimate relatedness between closely related populations or individuals. The AmpFlSTR PCR Amplification Kit has 15 highly variable autosomal markers of tetranucleotide repeats and is principally made to identify individuals and first- or second-degree relatives. However, in many studies one is searching for individuals who are related through more than one generation. We wanted to test whether the amplification kit can also be used to identify more distantly related individuals. Therefore we compared 16 different methods that calculate genetic distance with regard to each method's ability to cluster more distantly related individuals from two test families. Among all the tested methods Nei et al.'s (1983) DA distance performed well in clustering family members within a group of unrelated individuals for a broad range of scenarios. However, second-degree relatives were difficult to cluster with any of the examined methods when other family members were absent. With a simulation we further estimated how many markers would actually be needed to detect a certain degree of relatedness. According to this simulation, one would need at least 123 independent microsatellite markers to detect third-degree relatives with 90% probability. In conclusion, the 15 STR markers in the amplification kit are suitable for detecting only very closely related individuals or entire families.

Important aspects of the inverse relation between physical activity and colon cancer risk are still under discussion. In 2000-2003, 239 incident cases of colorectal cancer confirmed by histopathology and 239 hospital-based controls, matched by age and gender, were enrolled. In standardized interviews, data on occupational and recreational physical activity for ages 20, 30, 40, 50 and 60 years were collected from 98 colon cancer cases, 141 rectal cancer cases, and from 193 controls. Besides lifestyle and sociodemographic characteristics, a detailed food frequency questionnaire was assessed. In multivariate logistic regression for colon cancer, significant risk reductions for the highest quartile of total physical activity were found for almost all ages. For lifetime mean physical activity, the multivariate odds ratio for the highest quartile was 0.37 [95% confidence interval (CI) 0.17, 0.83]. For lifelong constantly high-exercisers compared with lifelong non-exercisers an odds ratio of 0.26 (95% CI 0.08, 0.84) was estimated. For rectal cancer, no consistent association with physical activity was found. No confounding effects were observed but the authors found effect modification with total energy intake. These data support an inverse association of colon cancer risk and physical activity which is most expressed if activity is kept up throughout life.

Binding of IGF-1 to the type I IGF receptor starts a signalling cascade that plays an important role in regulating cell proliferation, differentiation and apoptosis. The interaction between the IGF-1 and its receptor is mainly regulated by a binding protein, IGFBP 3. We studied a CA repeat polymorphism 969 bp upstream of the transcription start site in the IGF-1 gene and an A-202 C polymorphism in the IGFBP 3 gene and tested their association with breast cancer risk using four case-control series with a total of 787 cases and 900 controls. We did not find any association between the breast cancer risk and the IGF-1 repeat length (19 versus non-19) or the IGFBP 3 A-202 C polymorphism in the postmenopausal breast cancer series or in women diagnosed for breast cancer under the age of 50. In the familial breast cancer series we observed a non-significantly increased odds-ratio (OR) in homozygotes for the non-19 alleles of the IGF-1 gene (OR 1.51, 95% CI 0.96-2.39, p=0.07). Similarly, in the familial breast cancer series we detected an increased frequency of the IGFBP 3 -202 C allele carriers (OR 1.50, 95% CI 1.05--2.14, p=0.03). The association was stronger in individuals homozygous for these alleles (OR 3.76, 95% CI 1.44-v-9.81, p=0.006). Thus, the polymorphisms in the IGF-1 and IGFBP 3 genes associated with an increased risk of breast cancer in familial cases carrying the variant alleles.

The nuclear receptor coactivator 3 (NCOA3, also known as AIB1) is a coactivator of nuclear receptors like the estrogen receptor. NCOA3 is overexpressed in approximately 60% of primary human breast tumors, and high levels of NCOA3 expression are associated with tamoxifen resistance and worse survival rate. In contrast, NCOA3 deficiency suppresses v-Ha-ras-induced breast cancer initiation and progression in mice. Here, we analyzed the influence of NCOA3 coding single nucleotide polymorphisms on breast cancer risk by performing a case-control study using a German and a Polish study population and identified an association between NCOA3 polymorphisms and breast cancer. A joint analysis of the German and the Polish study population revealed a significant protective effect for the 1758G>C (Q586H) and 2880A>G (T960T) variants. In addition, haplotype analysis showed a protective effect of the 1758C-2880A and 1758G-2880G haplotypes (odds ratio 0.79; 95% confidence interval, 0.67-0.93; P = 0.004). Because of the impact of NCOA3 in antiestrogen therapy resistance, these polymorphisms might also influence therapy outcome in breast cancer.

A limited number of genes have been identified that explain heritable risks of breast cancer (BC). We searched for low-penetrant genes in an association study using two populations: 223 Finnish unselected patients and 172 Polish familial cases, both with locally collected healthy controls. Candidate genes included DNA repair genes, methylenetetrahydrofolate reductase (MTHFR) and cyclin D1 genes. The frequencies for single nucleotide polymorphisms (SNPs) were measured in the following genes: NBS1, XPC, XPD, XRCC1, XRCC3, MTHFR, and cyclin D1. Odds ratios (ORs) were calculated to the wild-type genotype. The positive findings in the Finnish series were repeated in the Polish series. Significant findings among Finns were associations to XPC exon 15, XPD exon 10 and XRCC3 exon 7, the latter of borderline significance. None of these results could be repeated in the Polish series. The XPC result among Finns was probably an artifact of the control group deviating from the Hardy-Weinberg Equilibrium (HWE). The attempt to repeat the result for the XPD polymorphism among Poles was probably not valid because the control group deviated from the HWE. We conclude that within statistical power of the present study, none of the tested polymorphisms associated with BC, with the probable exception of XPD.

Nonsmoking females age 35-65 years from Bremen, Germany (91 women), and Opole, Poland (98 women), were interviewed about their recent passive smoking exposure. We obtained urine samples at the time of interview and determined the concentration of cotinine as an indicator of tobacco smoke exposure. In Poland and in Germany, the vast majority of nonsmoking women are involuntarily exposed to environmental tobacco smoke (ETS). Polish women had slightly higher exposure levels than German women, with overall means of 9.93 and 8.65 ng cotinine/mg creatinine, respectively. Smoking by the husband was the major source of exposure in both study groups. In the Polish group, the work place was also an important source of ETS exposure. The validity of self-reported passive smoking exposure was found to be generally good; it was somewhat better in the German study group. A negative attitude toward tobacco smoke was slightly stronger among the German women. The percentage of women misreporting their active smoking status was low.

A hospital-based, multicenter, case-control study has been performed in Poland covering 741 incident stomach-cancer cases (520 males and 221 females) and the same number of controls. All stomach-cancer diagnoses were evaluated for histologic type according to the Lauren criteria. Fifty-one percent were of the intestinal type, 35 percent of the diffuse type, and 8.5 percent of the mixed type. The frequency of consumption of individual food items and several food groups was analyzed and the association of various foods with stomach cancer risk was evaluated after controlling for sex, age, occupation, education, and residency. Increased consumption of sausages was related significantly to gastric cancer risk, whereas increased consumption of cheese products, nonwhite bread, vegetables, and fruit was associated with decreased risk. A particularly strong decrease in risk was associated with consumption of radishes and onions. When consumption of fruits and vegetables, sausages, nonwhite bread, and cheese were introduced simultaneously in a multivariate model, independent effects were found only for fruit and vegetables, sausages, and nonwhite bread. The use of table salt, the frequency of eating hot meals, and an irregular eating pattern were also associated with increased risk, while additional consumption of fruit between meals showed reduced risk. If a reduction in vegetable and fruit consumption took place after marriage, an increased risk for stomach cancer was found, whereas augmented consumption of these food items after marriage decreased the risk. Separate risk models were calculated for stomach cancer of the intestinal and diffuse types, but both histologic varieties showed the same pattern of associations with dietary risk factors.

A method is proposed for transforming a class of models having an outcome variable with more than two levels into an equivalent binary model. The polychotomous logistic model is used to demonstrate the method. The equivalency to a simple logistic regression model after some data transformation (augmentation) is shown. The method is applied to the data from two case-control studies each with two control groups, and further applications are indicated.