Unhealthy alcohol use among university students is cause for concern, yet the level of help seeking behavior for alcohol use is low within the student population. Electronic brief interventions delivered via the Internet present an alternative to traditional treatments and could enable the delivery of interventions on a population basis. Further evidence is needed of the effectiveness of Internet-delivered interventions and of their generalizability across educational institutions.Our objective was to evaluate the effectiveness across 4 UK universities of a Web-based intervention for student alcohol use.In total, 1112 participants took part. Participants were stratified by educational institution, gender, age group, year of study, and self-reported weekly consumption of alcohol and randomly assigned to either the control arm or to the immediate or delayed intervention arms. Intervention participants gained access to the intervention between weeks 1 to 7 or weeks 8 to 15, respectively. The intervention provided electronic personalized feedback and social norms information on drinking behavior accessed by logging on to a website. Participants registered interest by completing a brief screening questionnaire and were then asked to complete 4 further assessments across the 24 weeks of the study. Assessments included a retrospective weekly drinking diary, the Alcohol Use Disorders Identification Test (AUDIT), and a readiness-to-change algorithm. The outcome variable was the number of units of alcohol consumed in the last week. The effect of treatment arm and time on units consumed last week and average units consumed per drinking occasion were investigated using repeated measures multivariate analysis of covariance (MANCOVA). In addition, the data were modeled using a longitudinal regression with time points clustered within students.MANCOVA revealed a main effect of time on units of alcohol consumed over the last week. A longitudinal regression model showed an effect of assessment across time predicting that participants who completed at least 2 assessments reduced their drinking. The model predicted an additional effect of being assigned to an intervention arm, an effect that increased across time. Regression analysis predicted that being male or being assigned to an intervention arm increased the odds of not completing all assessments. The number of units of alcohol consumed over the last week at registration, age, university educational institution, and readiness to change were not predictive of completion.Delivering an electronic personalized feedback intervention to students via the Internet can be effective in reducing weekly alcohol consumption. The effect does not appear to differ by educational institution. Our model suggested that monitoring alone is likely to reduce weekly consumption over 24 weeks but that consumption could be further reduced by providing access to a Web-based intervention. Further research is needed to understand the apparent therapeutic effect of monitoring and how this can be utilized to enhance the effectiveness of brief Web-based interventions. Unhealthy alcohol use among university students continues to be of concern given the immediate and long-term physical, psychological, and social consequences of this behavior -4. The lAlcohol interventions using the Internet are being developed that build on the established brief interventions evidence base. These interventions are viewed as having potential benefit to those who have not sought traditional modes of support or treatment . In addiRecent systematic reviews of electronic forms of alcohol intervention have indentified 17 randomized controlled trials involving young people ,12. A reOur recent randomized controlled trial suggested that Web-based interventions for students, incorporating brief personalized feedback and social norms information, can be effective in reducing per occasion alcohol consumption among UK students . The resThe mode of student recruitment varied across educational institution; however, all used some form of e-recruitment . In addition, some educational institutions posted paper-based posters around campus and provided verbal reminders to their students .Interested students were invited to register during October 2007 (Time 0). The trial was conducted over a 27-week period beginning in November 2007 (Time 1).Students who registered were asked to complete a retrospective drinking diary and the Alcohol Use Disorders Identification Test (AUDIT); these assessments are described in more detail below. At Time 0, students were also asked to provide details of their demographics including age, gender, ethnicity, graduate status, and area of study.Only students who were consumers of alcohol and provided details of their alcohol consumption at Time 0 were eligible to enter the current study. All students who consumed alcohol at least once every 6 months were included in the current study. This was deemed important because (1) if found to be effective, the intervention would be made available to all students, and, therefore, an evaluation of the effect on those consuming below hazardous or harmful levels is required as it is possible such information could have an adverse impact, and (2) previous research has shown that misperceptions of social norms are not only the domain of those engaging in risky behavior, and, therefore, to correct misperceptions across the population, feedback was necessary for all students.Eligible students were stratified by educational institution, gender, age group (above or below 21 at entry to undergraduate degree program), graduate student status, and self-reported weekly consumption of alcohol (within or above sensible drinking guidelines).The study was an RCT with 3 arms: 1 control arm (assessment only) and 2 intervention arms (immediate and delayed access). Participants were stratified by educational institution, gender, age group, year of study, and self-reported weekly consumption of alcohol and randomly assigned to either the control or to the immediate or delayed intervention arms. Participants were not blind to their allocation.After allocation to study arm, participants were emailed further information about the study along with an electronic link to the initial Time 1 (week 1) assessment. All contact with participants was via email. The Time 1 email contained an embedded link to either the control assessment electronic survey (completed by those in the control arm and in the delayed intervention arm) or to the intervention website (for those in the immediate intervention arm). Both websites contained identical project information and assessments (question presentation was the same between the two websites). Before completing their assessment participants were provided with a study briefing and provided informed consent online. Participants were advised that their having completed the earlier registration assessment did not mean they had to consent to taking part in the trial. Once participants in the intervention arm had completed their assessment, they received brief personalized feedback and social norms information via the website. Participants who did not complete the Time 1 assessment during week 1 were sent weekly reminders for up to 3 weeks (or until they had completed the assessment).Participants in the immediate and delayed intervention arms had access to the intervention during weeks 1 through 7 or weeks 8 through 15, respectively. Regardless of which arm they were allocated to, participants were assessed at 5 time points.Following the initial assessment at Time 1, additional assessments were completed at week 8 (Time 2), week 16 (Time 3), and week 24 (Time 4) see . The folThe study was approved by the Leeds East National Health Service (NHS) Research Ethics Committee and, where required by education institutional procedures, also by the relevant university ethics committee.2).From previous work we ascertained that the average natural logarithm of the number of units of alcohol consumed over the last week plus 1 for students is approximately 1.3 with a standard deviation of 0.58 and, hence, a variance of 0.34. A change in natural logarithm of the number of units consumed over the last week plus 1 over the intervention period will therefore have a variance of less than 0.68 . We have taken it to be equal to 0.49  and asked to indicate how many of each they had consumed each day for the last 7 days. The number of drinks was then converted to standard UK units of alcohol (1 unit = 8 grams of pure ethanol) using published UK government guidelines. This retrospective drinking diary method has been previously used within a student population , and theParticipants completed the AUDIT at Times 0 through 4. AUDIT is a 10-item measure investigating the quantity and frequency of alcohol consumption, problems related to use, and dependence symptoms. Items are scored on a scale of 0 to 4, and a cutoff score of 8 is recommended for the identification of possible hazardous or harmful drinking ,19. The Readiness to change drinking behavior was measured (at Times 1 through 4) using a 3-question algorithm developed by Epler et al . The queParticipants in the immediate and delayed intervention arms gained access to Unitcheck (www.unitcheck.co.uk). Unitcheck provides personalized feedback on alcohol consumption and social norms information. This feedback was available every time participants visited the website and completed the online survey. The online feedback, delivered after students completed the retrospective drinking diary alongside a number of other questions outlined below, consisted of 3 main sections: (1) feedback on level of alcohol consumption, (2) social norms information, and (3) generic information.Participants were presented with statements indicating the number of alcohol units they consumed per week and the associated level of health risk. Statements were standardized for each risk level and gave advice about whether personal alcohol consumption should be reduced or maintained within the current sensible levels . The numPersonalized statements were presented that summarized the percentage of university students who report drinking less alcohol than they consume. This was calculated relative to the risk level generated in section 1 of the feedback. The frequency of students within each risk level was established from data collected as part of an earlier university survey  and checGeneric information provided standard advice on calculating units and the general health risks of high levels of consumption and outlined sensible drinking guidelines publicized in the United Kingdom. Tips for sensible drinking and the contact details of both local and national support services were also presented.The effect of treatment arm and time on units consumed the previous week and average units consumed per drinking occasion were investigated using repeated measures multivariate analysis of covariance  (MANCOVA). Within this analysis, the dependent variable was units consumed, the independent variable was treatment arm, and the covariate was the natural logarithm of the number of units consumed the previous week at registration. All analyses were carried out on the basis of condition allocation with the last known value brought forward. Due to the data being positively skewed, the natural logarithm of 1 plus the number of units consumed was used for all analyses. The number of units presented within the text and tables is the number of original scale units. These analyses were undertaken using SPSS, version 15 .Previous research has suggested differential attrition according to treatment arm and some trials have observed relatively high rates of attrition. These trial characteristics render the traditional repeated measures MANCOVA problematic, specifically liable to dropout bias. Therefore, an additional analysis of the primary outcome data  was planned that could accommodate these characteristics , namely,A total of 2306 students registered interest in being involved in the project. Of these, 2005 eligible students were randomized to a study arm and invited to take part see .Of the 1112 students enrolled in the study, 816 (73%) were female. Participants’ age ranged from 18 to 67 years . The majority of participants (95%) were undergraduate students, and 92% were predominately white/white British, based on self-reported choice from among several categories of ethnicity. Participants came from a range of subject areas, the largest of which were: medicine and health (319/1112 or 29%), sciences (175/1112 or 16%), arts (155/1112 or 14%), and social sciences (140/1112 or 13%). In all, 4 universities (3 English and 1 Scottish) took part in the study. The 4 universities were heterogeneous in terms of student populations . The majority of participating students came from university A (591/1112 or 53%), followed by university B (344/1112 or 31%), university C (106/1112 or 10%), and university D (71/1112 or 6%).Of the 1112 students enrolled in the study, 57% (637) scored 8 or more on the AUDIT and, therefore, their drinking could be considered potentially hazardous or harmful. At registration, the mean number of units of alcohol consumed over the previous week was 23.2 (SD 25.5). The majority of participants (829/1112 or 75%) reported consuming alcohol at least once a week. A further 15% (169/1112) reported fortnightly consumption with 10% (114/1112) consuming either once a month (n=80) or once every 6 months (n=34). Regarding readiness to change, at Time 1, a quarter of participants (276/1112 or 25%) were in the precontemplation phase, almost one-third (319/1112 or 29%) were in the contemplation phase, and approximately one-fifth (246/1112 or 22%) were in the action phase. The remaining participants had either missing data (196/1112 or 18%) or had at that time identified themselves as nondrinkers (75/1112 or 7%). The demographics of participants by treatment arm are provided in 3,3324 = 6.42, P < .01). Pairwise comparisons showed a significant decrease between Time 1 and all other time points  but no significant differences between Time 2, Time 3, or Time 4. There was however no significant time by treatment arm interaction .Alcohol consumed over the last week and per average occasion is shown in 3,3324 = 0.53, P = .67). There was a significant time by treatment arm interaction . Further analysis revealed a significant time by consumption effect for the control arm  with significant reductions between Time 1 and Time 2 (P < .01) and between Time 1 and Time 3 and between Time 1 and Time 4 (P < .01). There was also a significant time effect for the delayed intervention arm  with significant reductions between Time 1 and Time 2, Time 1 and Time 3, and Time 1 and Time 4 (P < .01). No significant time effect was observed in the immediate intervention arm .Repeated measures MANCOVA revealed no main effect of time on average units consumed per drinking occasion over the previous week , sex, and treatment arm (dichotomized to treatment/no treatment), and time since treatment . Readiness to change, if access to the intervention was immediate or delayed, and level of consumption at Time 0 were all excluded from the final model as they did not add significantly to the prediction. The longitudinal regression model showed a significant effect of assessment (without intervention) on change across time, showing that participants who completed at least two assessments reduced their drinking . The modR                2 value of 0.06 and an interclass correlation coefficient of 0.14, indicating that there was significant variation between participants and over time. The model predicted that at week 24 without any assessment completion (ie. completing only Time 0 assessment), females drank 11.5 units per week while males drank 16.0 units. As can be seen in 2 = 67.4, P < .001; see Regarding the possible effects of differential assessment completion, 26% (293/1112) of participants completed all 5 assessments with the remaining 74% (819/1112) of participants being classified as nonassessment completers . Regression analysis showed that age, education institution, previous week unit consumption at Time 0, and readiness to change were unrelated to completion. The box plot summarizing units consumed over the previous week at Time 0 supported the regression analysis . The difThis study suggests that delivering an electronic brief intervention to students can be effective in reducing alcohol consumption. The results showed a significant effect of time and/or assessment completion. In addition, the intervention had an independent effect on reducing the number of units of alcohol consumed weekly. Neither educational institution nor time of academic year had an impact on intervention effectiveness. To the authors’ knowledge, these results are the first evaluation of an electronic Web-based feedback and social norms alcohol intervention to include students from multiple universities from outside of North America.The current results are in line with previous research on electronic Web-based interventions that have reported significant decreases in total consumption per week -28. In sAccording to our analysis, assessment alone had a significant effect on drinking. It is, therefore, possible that completing an assessment led participants to monitor and reflect on their own behavior leading to a decrease in consumption. Such a decrease in consumption by assessed participants is apparent in the literature ,30. Our Reactivity to assessment has been documented in the literature within randomized controlled trials -33 and wThe high proportion of students identified as drinking at potentially hazardous or harmful levels suggests that students engaging in unhealthy drinking behavior are interested in engaging with Web-based resources, and this is a strength of the current study. The ability to include such students is admirable given some studies have reported engaging a greater proportion of low-level consumers . This iThis study is the first outside of North America to engage students from multiple educational institutions and to include medium-term follow-up. A number of limitations need to be considered, however, when interpreting the results. First, although 75% of intervention participants accessed the intervention, the percentage that engaged with follow-up assessments was considerably lower, with only 26% (293/1112) completing all 5 assessments. Second, while participants from 4 educational institutions were involved, the relatively low number from some educational institutions highlights the difficulties in multisite interventions. In particular, it draws attention to the need to understand further how to engage students from universities that have not traditionally delivered health information to their students and who are perhaps not as receptive to receiving brief interventions via this medium. Third, while there was a 4-month follow-up period, these results say little about the longer term impact of the intervention. The long-term impact of electronic brief interventions is still uncertain. Nor is it understood how, if at all, repeated access to such interventions is likely to support behavior change. This is of particular importance to Web-based interventions that can be a repeated source of feedback to students at a time that they choose. It is a potential strength of e-interventions that students could be encouraged to repeatedly access feedback while incurring minimal additional costs. Fifth, the longitudinal regression analysis cannot determine if it is monitoring or willingness to be monitored that accounts for the reduction in consumption. The analysis attempted to account for this; however, we cannot rule out that willingness to be monitored (rather than engaging in the monitoring process itself) is an alternative explanation.This RCT confirms that Web-based interventions for alcohol can be effective in student samples. That the effect does not appear to vary across educational institutions is encouraging. It is hypothesized that differences in levels of recruitment across educational institutions are likely to be related to use of the Internet to deliver health information to students. Further work is needed, however, to understand how contextual factors can best be optimized in order to engage students. Within this study, much of the observed effect was apparently due to self-monitoring, but there was an additional effect of the intervention. Research is needed to understand how the individual elements of the personalized feedback interact with this self-monitoring effect in order to enhance the effectiveness of Web-based interventions.


ABCD1 gene. Approximately 20% of X-ALD female carriers may develop neurological symptoms. Skewed X chromosome inactivation (XCI) has been proposed to influence the manifestation of symptoms in X-ALD carriers, but data remain conflicting so far. We identified a three generation kindred, with five heterozygous females, including two manifesting carriers. XCI pattern and the ABCD1 allele expression were assessed in order to determine if symptoms in X-ALD carriers could be related to skewed XCI and whether skewing within this family is more consistent with genetically influenced or completely random XCI.X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder caused by mutations in the XIST promoter mutations, were not consistent with our results.We found a high frequency of skewing in this family. Four of five females had skewed XCI, including two manifesting carriers favoring the mutant allele, one asymptomatic carrier favoring the normal allele, and one female who was not an X-ALD carrier. Known causes of skewing, such as chromosomal abnormalities, selection against deleterious alleles, ABCD1 allele would be associated with the manifestation of heterozygous symptoms. Furthermore, XCI skewing in this family is genetically influenced. However, the underlying mechanism remains to be substantiated by further experiments.Our data support that skewed XCI in favor of the mutant  ABCD1 gene, which maps to chromosome Xq28. It is biochemically characterized by accumulation of pathognomonic saturated very long chain fatty acids (VLCFAs) in body fluids and tissues. It has been demonstrated that X-ALD not only affects males, but also female carriers. Approximately 20% of X-ALD carriers develop symptoms like adrenomyeloneuropathy (AMN) in middle age or later X-linked adrenoleukodystrophy , the most common peroxisomal disorder, is an inherited neurodegenerative disease caused by mutations in the XIST promoter mutations. However, in most cases, the cause of skewing is unknown.Skewing of X chromosome inactivation (XCI) is a process in mammals in which one X in every XX female somatic cell is transcriptionally silenced, and the determination of the X chromosome to be inactivated is usually random. Females are expected to have two cell populations: one with the maternal and one with the paternal X as the active chromosome. Skewed XCI is arbitrarily defined as more than 80% of cells showing a preferential inactivation of the same X chromosome Unfavorable skewing of XCI, where the X chromosome carrying a mutant allele is the predominantly active X, has been found in manifesting carriers of several X-linked disorders Here, we describe a family in which two females expressed the AMN-like phenotype. Our objective was to determine if symptoms in X-ALD carriers could be related to skewed XCI and whether skewing within this family is consistent with genetically influenced XCI.The protocols for this study were evaluated and approved by the Ethics Committee of Fuzhou General Hospital. Written informed consent was obtained from all participants in this study. The ethics committee approved this consent procedure.The proband , II2 wasVLCFAs were quantified in plasma by standard procedures.ABCD1 gene coding region, including flanking splicing sites, was amplified using appropriate primers. The purified PCR products were sequenced using an ABI 3730 Sequencer, and the mutation was characterized. Corresponding PCR products of normal controls  were also analyzed by restrictive digestion with Rse I to exclude the possibility of polymorphism. Pathogenicity of the mutation was assessed by in Silico analysis using three algorithms .Peripheral blood was collected from the female participants of this family and genomic DNA was extracted with a DNA isolation kit . The entire ABCD1 allele (data not shown), showing that the ABCD1 allele-specific expression pattern mirrored the XCI pattern, probably because one allele of ABCD1 was usually silenced as result of the XCI.In addition, two carriers from two unrelated X-ALD families were found to have skewed XCI in favor of the wild-type XCI pattern was determined by quantitation of the methylation status of the polymorphic human androgen receptor (AR) locus according to Allen et al Besides the presented family, we also analyzed blood samples from 25 asymptomatic carriers and 10 noncarriers from 19 unrelated X-ALD families and 50 age-matched healthy female controls. The females who were uninformative at the AR locus were not included in the analysis.ABCD1 allele was predominantly active. Total RNA was isolated from the peripheral blood of the female carriers with an RNA isolation kit , and 800 ng of total RNA was reverse transcribed with reverse transcriptase . A fragment of 700 bp spanning the mutation was amplified with the forward primer: 5′-ACTGGCCCTGTCGTTCCG-3′, and the reverse primer: 5′-GTTGCGGGCAATAGTGAAG-3′, and the PCR product was then sequenced. T cloning of the corresponding PCR product was also performed and a total of 25 colonies were picked, from which recombinant plasmids with inserted PCR product were isolated and sequenced.Because genes are transcribed only from the active X chromosome, RNA transcripts were analyzed to investigate whether the mutant or the wild-type XIST in female participants of the family as described PCR primers were used to amplify the minimal promoter of Cytogenetic analyses were performed by standard methods on PHA-stimulated peripheral blood lymphocytes. Bands were visualized at a resolution of 400–550.Plasma VLCFAs assay showed the amount of C26:0, the ratios of C24/C22 and C26/C22 were higher than normal in I 1, II3, II5 and III2 .ABCD1 gene, at codon 283 (CAC→CGC), which resulted in the replacement of histidine by arginine (p.His283Arg). The mutation was not detected in 200 alleles of normal controls by restriction analysis with Rse I (data not shown). There was no other mutation detected in the ABCD1 gene coding region of this family.Sequence analysis of the PCR products of the female participants revealed a heterozygous mutation in I 1, II3, II5 and III2, but not in II7 , A1–E1. Digestion with Dde I . These dXCI patterns of X-ALD carriers, related noncarriers and normal controls were summarized in ABCD1 allele in manifesting carriers II5 and II3, respectively, indicating that the mutant allele was predominantly expressed. In contrast, the wild-type allele was predominantly expressed in asymptomatic carrier III2.Sequencing chromograms , A2–E2 aXIST minimal promoter did not reveal any abnormalities of the amplified fragment (data not shown).The standard cytogenetic analyses showed the extremely skewed manifesting female (II5) had a normal karyotype. The sequence analysis of the ABCD1 gene. For three reasons, it is unlikely a polymorphism or rare variant. First, the mutation was not detected in 200 alleles of normal controls. Second, the histidine at codon 283 is conserved among multiple species  was identified, which has not been reported in X-ALD database or other published data, indicating that it is a novel mutation in the  species . Third, While it is often stated that in recessive X-linked disorders heterozygotes are asymptomatic, it is known that to some X-linked disorders the dichotomy between recessive and dominant does not apply and there are conditions where heterozygosity may represent a disease state. Besides X-ALD, these may include haemophilia A, myotubular myopathy, Duchenne muscular dystrophy and others ABCD1 mutation on the maternal X chromosome and the skewed pattern of XCI that prevents the expression of the wild-type ABCD1 allele. We propose that the convergence of these is related with the clinical manifestations in these two heterozygous females, which is in agreement with the previous data of Maier et al.Is skewed XCI responsible for ALD phenotype in female carriers? Conflicting results have been reported by different authors: Maier et al ABCD1 gene transcription and XCI pattern in blood leukocytes. Interestingly, four of five females in our study had skewed XCI, including two symptomatic sisters (II5 and II3) favoring the mutant allele, one asymptomatic carrier (III2) favoring the normal allele, and one female (II7) who was not an X-ALD carrier. This distribution suggested a genetic influence over XCI in this family.In this family, five females from three consecutive generations were assessed for the We have ruled out chromosomal abnormalities as a mechanism of XCI skewing in a symptomatic carrier (II5). We have not formally determined the karyotypes of the other family members with skewed XCI. However, it is unlikely that their skewed XCI is due to an X-autosome translocation, as none have the completely skewed XCI expected in these translocations. Further, it is most likely that all of the skewed XCI in this family is due to a common mechanism. Therefore, we are not pursuing X-autosome translocations as a mechanism in any of the family members.ABCD1 mutation, as transcript analysis showed that skewing can be associated both with predominance of the mutant and the wild-type ABCD1 allele. This observation does not go together with the reports of Salsano et al ABCD1 mutations confer a proliferative advantage leading to XCI skewing in favor of the X chromosome with the mutation, however, this phenomenon could not be explained. In addition, there was no other known disease mutation segregating in this family to predispose them to unbalanced XCI.Some X-linked gene mutations conveying a proliferative advantage or disadvantage to a cell might lead to XCI skewing. However, it is unlikely that XCI skewing is related to the inheritance of the XIST is essential for X chromosome inactivation. The XIST gene has been fully sequenced, but its functional domains have not yet been fully identified. Mutations in the promoter of the XIST gene have been reported to cause familiar non-random XCI Transcription of ABCD1 allele would be associated with the manifestation of heterozygous symptoms. Furthermore, XCI skewing in this family is genetically influenced. However, there is no obvious explanation for the XCI skewing in our case. The underlying mechanism triggering this phenomenon remains to be substantiated by further experiments. One could propose that there is an influence of a yet unidentified X-linked or autosomal locus and modifying factors affecting the mechanism.In conclusion, our data support that skewed XCI in favor of the mutant ABCD1 allele-specific expression pattern analysis is available in the laboratory.Our finding implies that the demonstration of skewed XCI in a carrier does not necessarily result in a clinically detrimental influence. However, the finding of unfavorable skewed XCI in a carrier would be a valuable parameter for early identification of the carriers with a high risk of becoming symptomatic and for whom a possible preventive therapy would be warranted. Moreover, an analysis of XCI over time can be useful in the assessment of females with atypical presentations of ALD. Therefore, the possible role that XCI pattern plays in clinical manifestations of ALD carriers should be told in ALD-related counseling, while mentioning that the techniques for XCI and the


In Germany, annual vaccination against seasonal influenza is recommended for certain target groups ). In season 2009/10, vaccination against pandemic influenza A(H1N1)pdm09, which was controversially discussed in the public, was recommended for the whole population. The objectives of this study were to assess vaccination coverage for seasonal (seasons 2008/09-2010/11) and pandemic influenza (season 2009/10), to identify predictors of and barriers to pandemic vaccine uptake and whether the controversial discussions on pandemic vaccination has had a negative impact on seasonal influenza vaccine uptake in Germany.We analysed data from the ‘German Health Update’ (GEDA10) telephone survey  and a smaller GEDA10-follow-up survey , which were both representative of the general population aged ≥18 years living in Germany.Overall only 8.8% of the adult population in Germany received a vaccination against pandemic influenza. High socioeconomic status, having received a seasonal influenza shot in the previous season, and belonging to a target group for seasonal influenza vaccination were independently associated with the uptake of pandemic vaccines. The main reasons for not receiving a pandemic vaccination were ‘fear of side effects’ and the opinion that ‘vaccination was not necessary’. Seasonal influenza vaccine uptake in the pre-pandemic season 2008/09 was 52.8% among persons aged ≥60 years; 30.5% among HCW, and 43.3% among chronically ill persons. A decrease in vaccination coverage was observed across all target groups in the first post-pandemic season 2010/11 .Seasonal influenza vaccination coverage in Germany remains in all target groups below 75%, which is a declared goal of the European Union. Our results suggest that controversial public discussions about safety and the benefits of pandemic influenza vaccination may have contributed to both a very low uptake of pandemic vaccines and a decreased uptake of seasonal influenza vaccines in the first post-pandemic season. In the upcoming years, the uptake of seasonal influenza vaccines should be carefully monitored in all target groups to identify if this trend continues and to guide public health authorities in developing more effective vaccination and communication strategies for seasonal influenza vaccination. In Germany, annual influenza epidemics usually occur during the winter months December to March. In the last decade, an estimated zero to 19,000 excess deaths per year were attributable to influenza virus infections . Severe Annual vaccination has proven to be an effective method to reduce the burden of influenza disease . In GermDuring the pandemic, vaccination against A(H1N1)pdm09 was subject to controversial discussions, not only in Germany but also in many other countries worldwide -9. Main To develop target group specific communication strategies and to enhance compliance with the official recommendations it is important to monitor vaccine uptake in each of the target groups and to understand factors that influence uptake. This applies not only for annual influenza vaccination campaigns but also for the planning of future vaccination campaigns during a pandemic. The influence of the 2009/2010 pandemic situation on seasonal influenza vaccine uptake in Germany in the post-pandemic seasons has so far not been investigated. For this purpose, we utilized data from the large  ‘German Health Update 2010’ (GEDA10) telephone survey and a smaller GEDA10 follow-up survey . The objectives of our study were (1) to assess seasonal influenza vaccination coverage for seasons 2008/09 to 2010/11, (2) to assess pandemic influenza vaccination coverage for season 2009/10, (3) to identify predictors of and barriers to pandemic vaccine uptake, and (4) to detect a potential influence of the pandemic situation on seasonal influenza vaccine uptake in the first post-pandemic season (2010/11).The GEDA survey design has been described previously ,13,16. IIn this study we present data from GEDA10 which was conducted between 22 September 2009 and 10 July 2010. Since the annual GEDA survey was not conducted in 2010/2011, we conducted a follow-up interview among a subsample of 2.493 GEDA10 respondents (from now on referred to as the GEDA follow-up survey) from 1 April to 2 July 2011 to assess seasonal influenza vaccine uptake for the post pandemic season 2010/11. Based on a sample size calculation, 385 subjects were needed to estimate a prevalence of 50% (“worst case scenario”) vaccine uptake with a confidence interval of +/− 5%. Our sample size of ~2,400 subjects for the follow-up survey was based on the premise to estimate a prevalence (i.e. vaccination coverage) of 50% for up to six subgroups .Since both surveys were conducted by RKI, the ownership of the data lies with RKI and we did not have to obtain permission to use the data for this study. Similar to the previous GEDA survey (GEDA09), a public use file of the GEDA10 dataset will be provided soon. Data from the GEDA follow-up survey will not be openly available.To control for possible selection biases, weighting factors for the GEDA10 sample were constructed by taking age, sex, educational status, geographical region, and household size into consideration. Potential participants of the follow-up survey were sampled disproportionally to their weighting factors in GEDA10. We applied this method to avoid that groups which were already underrepresented in GEDA10 become underrepresented again at the sampling stage in the follow-up sample and thus to prevent further bias. Weighting factors for the follow-up survey were constructed in a first step on the basis of the values calculated for GEDA10 , and, in a second step, on population data gathered in the Microcensus 2008 , taking Information on seasonal influenza vaccination status for season 2008/2009 was collected from all participants of GEDA10. Between 1 January 2010 and 10 July 2010 respondents were additionally asked to provide information on seasonal and pandemic influenza vaccination status for season 2009/10. Unvaccinated survey participants of GEDA10 were additionally asked to state their reasons for not receiving a pandemic influenza vaccination. Information on seasonal vaccination status for season 2010/11 was collected from all respondents of the GEDA follow-up survey. In the follow-up survey, respondents were additionally asked by whom they were vaccinated  and in which month they received the vaccination against seasonal influenza in season 2010/11. One should note that GEDA10 does not cover the paediatric population (aged 0–17 years) for which pandemic vaccination was also recommended in Germany. Information on vaccination coverage in this particular age-group was therefore not available from this data source.We calculated the response for GEDA10 by using Response Rate 3 as defined by the American Association for Public Opinion Research (AAPOR) . ResponsSocio-economic status levels were created as described by Lampert and Kroll on the basis of self-reported educational, income, and professional status of survey respondents . In accoData analysis was performed using PASW 18.0 for Windows . Proportions were calculated by using procedures for the analysis of complex samples. Univariate analyses were conducted to determine associations between pandemic influenza vaccine uptake and socio-demographic, health-related and professional factors. A p-value ≤0.05 was considered to indicate a statistically significant difference. Odds ratios (OR) and 95% confidence intervals (CI) were calculated as appropriate. Multivariable analysis was performed by entering variables potentially associated with vaccine uptake  into a multivariable logistic regression model in a first step, followed by step-wise backward removal of variables with a p-value >0.05 to produce a final model. Interaction terms were included to account for effect modification between independent variables.In total, 22,050 telephone interviews were conducted during the study period of GEDA10 and 2,493 participants were re-interviewed for the follow-up survey. An overview of the survey populations is given in Table Information on seasonal influenza vaccination status was available for over 99.8% in each of the study samples for the three seasons under investigation (seasons 2008/09-2010/11). Vaccination coverage for the three seasons by sex, age group, place of residence, and target group is presented in Table Figure Information on pandemic influenza vaccine uptake was available for 99.9% of the respective study population in GEDA 10 n=13,048). Vaccination coverage by sex, age group, place of residence, socio-economic status and different target groups for seasonal influenza vaccination is presented in Table ,048. VacThe most frequently reported reasons for not receiving the vaccination were (1) ‘fear of side effects of pandemic vaccines’ , (2) ‘pandemic vaccination is not necessary’ , (3) ‘pandemic vaccination not officially recommended for me’ , and (4) ‘reject vaccinations in general’ .Results of univariate and multivariable analysis of factors potentially associated with pandemic influenza vaccine uptake are shown in Table The aim of this study was to assess the uptake of seasonal influenza vaccines in specific target groups for seasons 2008/09 and 2009/10, as well as for pandemic influenza vaccines during the pandemic season 2009/10 in Germany in the total adult population by using data from a large population-representative telephone survey. By using data from a smaller follow-up survey, our study moreover provides the only so far available data on seasonal influenza vaccination coverage in Germany for the post-pandemic season 2010/11. Overall, only 8.8% of the adult population in Germany followed the official recommendation and received a vaccination against pandemic influenza in season 2009/10. The follow-up survey revealed a decrease in seasonal influenza vaccine uptake in the first post-pandemic season across all target groups when compared to the pre-pandemic season 2008/09, most prominent among HCW. With an average coverage of 50% in the elderly, 41% in the chronically ill, and 28% in HCW in seasons 2008/09 to 2010/11, the EU goal of reaching a seasonal influenza vaccination coverage of at least 75% in the target groups [Having received a seasonal influenza shot in the pre-pandemic season was the strongest predictor for receiving pandemic influenza vaccination in our study. The high correlation between seasonal and pandemic influenza vaccine uptake highlights the significance of habitual behaviour with regard to influenza vaccination decisions. In addition and independent from this factor, persons belonging to at least one of the recommended target groups for seasonal influenza vaccination were significantly more likely to receive a pandemic influenza vaccination than persons not belonging to a target group. Our results are broadly in line with the findings of a prospective monitoring survey on pandemic influenza vaccination in Germany  and two A higher uptake of seasonal influenza vaccines in season 2009/10 was observed in the follow-up survey population when compared to the total GEDA10 study population in 2009/10. It is therefore very likely that the point estimates for seasonal influenza vaccination coverage for the 2010/11 season, which were based on data from the same follow-up survey, were also overestimated. Taking into consideration that acceptance of seasonal influenza vaccination was higher in the follow-up survey population, our study results suggest that seasonal influenza vaccine uptake in the recommended target groups in Germany has decreased in the post-pandemic season 2010/11, not only in comparison to season 2008/09 but also to the pandemic season 2009/10  among 3,002 parents of children aged 0–13 years, 35% stated that they reject particular vaccinations for their children, but only 1% of parents reject vaccinations in general . Since oOur study has some limitations that need to be acknowledged. Calculation of influenza vaccination coverage was based on self-reported vaccination status and may therefore be prone to recall problems. However, it was shown in several studies that self-report of influenza vaccination status has an adequate degree of validity ,36. FurtIn conclusion, poor compliance with official vaccination recommendation resulting in low uptake of pandemic influenza vaccines during the pandemic season 2009/10 suggests that public communication strategies and vaccination campaigns during the influenza A(H1N1)pdm09 pandemic in Germany were not successful. In addition, our results raise concerns that controversial discussions about the safety and necessity of pandemic influenza vaccines may have contributed to decreased seasonal influenza vaccine uptake in the first post-pandemic season. It is therefore crucial to develop concerted communication strategies based on the lessons learned from the 2009/10 influenza pandemic and to include them in the national pandemic preparedness plan. This should be done, not only with respect to a competent handling of pandemic situations but also to avoid a decrease in the acceptance of vaccinations in general. In this respect, communication strategies and different modes of communication to specific target groups should be evaluated and implemented already in non-crisis situations, to be enhanced during a pandemic influenza situation or other public health crisis. This is of particular importance, since seasonal influenza vaccine uptake in the recommended target groups in Germany stagnated at a low level since 2005  and doesThe authors have declared no conflict of interest.All authors made substantial contributions to the study. SM was involved in the development of the GEDA10 study design and contributed to the Methods section. MMB, DW, and OW developed the study design of the GEDA10 follow-up survey. MMB analysed the data in consultation with DW, GF, OW and GK. MMB wrote the draft version of the manuscript. All authors have read, carefully reviewed and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/938/prepub


Ample data exists about the high precision of three-dimensional (3D) scanning devices and their data acquisition of the facial surface. However, a question remains regarding which facial landmarks are reliable if identified in 3D images taken under clinical circumstances. Sources of error to be addressed could be technical, user dependent, or patient respectively anatomy related. Based on clinical 3D photos taken with the 3dMDface system, the intra observer repeatability of 27 facial landmarks in six cleft lip (CL) infants and one non-CL infant was evaluated based on a total of over 1,100 measurements. Data acquisition was sometimes challenging but successful in all patients. The mean error was 0.86 mm, with a range of 0.39 mm (Exocanthion) to 2.21 mm (soft gonion). Typically, landmarks provided a small mean error but still showed quite a high variance in measurements, for example, exocanthion from 0.04 mm to 0.93 mm. Vice versa, relatively imprecise landmarks still provide accurate data regarding specific spatial planes. One must be aware of the fact that the degree of precision is dependent on landmarks and spatial planes in question. In clinical investigations, the degree of reliability for landmarks evaluated should be taken into account. Additional reliability can be achieved via multiple measuring. Objective evaluation of the face is challenging. Meaningful assessment by basic measurements is hindered by the complex three-dimensional (3D) anatomy of the face because of its specific but not perfect symmetry. Anthropometry, the science of measuring the characteristics of the body , has deaRegarding the underlying bony structures, 3D evaluations based on computed tomography data have become more and more routine , 3. HoweThe state-of-the-art method for facial soft tissue evaluation and documentation is direct measurement and two-dimensional (2D) photography –6. Both Modern computer technology has opened the door to the development of computed tomography , 14 and  In the facial region, these scanning devices are applied in anthropometry –28 to coObviously, it is important to evaluate the precision and reliability of new technology before applying it in clinical routines , 40. ThiOur hypothesis was that, despite the technically sufficient precision of 3D face scanning techniques with errors below 1 mm , 22–24, The aim of the study was to investigate the reliability of landmark assessment utilizing virtual 3D face models of children with cleft lips (CLs) acquired under clinical circumstances with the 3dMDface imaging system. The main goal was to identify landmarks that are reliable in marking for study purposes. Later on, strategies should be developed to achieve the highest possible repeatability and precision.Data about the technical precision of all kinds of 3D imaging systems is available in the literature –24. Our Six data sets of infants between six and 18 months old-all with uni- or bilateral CL-were acquired. One additional infant without any craniofacial deformity served as a control in order to identify difficulties specifically related to CL. All images were taken under clinical conditions, meaning for example, no special skin preparation-except drying off saliva if necessary-was performed. Also no special lightning concept away from the flash system integrated into the imaging system was utilized. Finally all pictures were taken by medical personal in the presence of at least one parent.The study design satisfied the criteria of the local ethics committee for being exempted from individual Institutional Review Board approval. The study design thereby fulfilled the guidelines of the Declaration of Helsinki about Ethical Principles for Medical Research Involving Human Subjects.All data were acquired with artificial lighting using the 3dMDface System  . RegistrEach subject data acquisition was performed repeatedly until no better 3D model was practically achievable; images with obvious facial expressions were discarded. The acquisition process was considered complete if one image covered all 27 landmarks chosen for evaluation. In cases in which infants became increasingly noncompliant, the acquisition process was stopped and the image covering most of the 27 landmarks was chosen for further evaluation. An example used for a resulting virtual 3D model is shown in The dataset of the chosen image was transfered to a laptop computer via USB-Stick. Further data processing was performed utilizing the 3dMD-Patient-Software  that comes with a capture device. A total of 27 landmarks were labeled on the surface of each virtual face. Landmarks were chosen due to clinical relevance and spread over the face with emphasis on aesthetically relevant regions . Softwarx-, y-, and z-coordinates of these markings were directly saved into xls-files. These files then were imported into Excel for Mac 2011  for further analysis.To match a typical retrospective study situation, one observer labeled all landmarks on five consecutive days and again after a break of one week. From the 3dMD-Patient-Software the Since all measurements were performed within one virtual face model, the coordinates were identical for identical points without the necessity of superimposition or registration. Since no real truth coordinates serving as references can be derived by any means, a “mean coordinate” was calculated out of the six individual measurements. The target registration error lculated , 44, 45.A mean TRE for each landmark in all 3D models was calculated in order to identify landmark-specific precision. Overall, the concept of analysis was analogous to that in previously published studies concentrating on the technical precision of the system and the influence of involuntary facial movements , 43. AllA sufficient 3D image was acquired for all infants. However, it did require multiple attempts in all cases. Two to 14  captures were performed per child. Based on this data an image acquisition session with a child takes between 5 and 15 minutes time if measured from entering the room until leaving again. The necessary system start up and registration process (about 5 minutes) is not included and we strongly recommend to have the system ready once the child comes into the room since in most cases compliance is best at the beginning of the session and on should not waste that time spot for procedures that can be done ahead.For the most part, incorrect head positioning was the reason for the insufficient imaging. The system's low capture time of 1.5 milliseconds guaranteed sharp images under most circumstances, but a long shutter lag sometimes allowed the children to move their faces partially out of the capture region before images were acquired. This could be resolved, for example, via a technical improvement involving a short shutter lag time. Any attempts of gently holding the head position in place mostly resulted in agitation of the infant including facial expressions leading to 3D representations that were inappropriate for craniofacial anthropometry. This issue of little children tending to move and thereby interfering with imaging is also a problem in conventional 2D photography  and evenAnother common reason for necessary reacquisition was that prominent areas compromised the view of less prominent areas, resulting in poor or even missing 3D representations. Basically, this also is a problem of positioning: to achieve optimal imaging, an approximate 15-degree angled camera view from below is necessary .Five craniofacial landmarks  could not be labeled on all subjects since the relevant regions were not sufficiently represented in the 3D model (compare the second column in Insufficiently represented landmarks appeared in five (71%) of the seven models. Only two (29%) were labeled completely. However, all other landmarks  other than the above mentioned  were accurately represented in all of the 3D models and, therefore, labeled on all subjects.The target registration errors for each landmark are given in descriptive statistics in Even though that is not statistically significant, it is interesting to note that the control patient without CL provides the best overall accuracy. We believe this is due to the CL anatomy, which is less likely to be completely captured by the camera system. In addition the cleft region tends to be covered with saliva.On the other hand, the differences in TRE between the best and the worst landmarks  are much more distinct.Of the 27 landmarks, 21 (78%) show reliability better than onemm in mean and another four (15%) are within the range of 1–1.5 mm. Only two (7%) landmarks were revealed to be worse than 1.5 mm . These ry- and z-axes. The error in the x-axis, which represents the width of the mandible, is only 0.84 mm (the mean for the soft gonion right and left) compared to 1.20 mm (y-axis) and 1.67 mm (z-axis). Therefore, this landmark can very well be used to, for example, evaluate the width of the mandible. This leads to the claim that prior to any clinical study involving 3D imaging for the sake of anthropometric evaluation, the landmarks to be studied must not only be evaluated by general means but, depending on the clinical demands, precision and reliability in terms of the three special dimensions need to be analyzed in detail.For the soft gonion, as the least reliable landmark, it can be shown that the TRE is mainly a result of bad definition in An obvious downside of this study is the small number of subjects, combined with the focus on intraobserver reliability. However, we believe this to have been overcome by the conclusive results obtained which are in line with previous studies involving model heads , childreThe fact that only one 3D imaging system was evaluated is not a downside to the study results since the systems on the market provide more or less the same technical precision –24 and tThe technique of 3D photography seems to be valid for facial soft tissue analysis. Data can be acquired under clinical conditions and, without loss of precision, retrospectively analyzed under study conditions. This is a new option for craniofacial measurements that cannot be achieved by direct anthropometric measurements or 2D photography. However, one must be aware and take into account the fact that every landmark has its own level of precision and reliability that also depends on the spatial plane in question. In addition, landmarks are differently influenced by involuntary facial movements .Researchers conducting any study utilizing 3D imaging techniques should investigate their evaluation concept in advance. The revealed information  needs to be discussed and taken into account in order to come to conclusions. The fact that in our study CL infants showed a lower level of accuracy compared to the non-CL infant underlines this fact.More investigations are necessary regarding inter-observer reliability, which might be an issue for bigger studies or meta-analysis of multiple scientific studies. Patient-specific short- and long-term factors, such as involuntary mimicking or weight changes, must be defined regarding their level of influence .The upcoming technology of 4D (video data) acquisition , 36 willBesides all this facts that focus on exact landmark identification and clinical study issues 3D photography in our eyes is a great tool for objective documentation of a person's facial appearance. This objective documentation is not only an important issue in the evaluation of surgical results by the means of pre-postcomparison. It also plays an important rule when medico-legal questions are raised. We believe the 3D imaging technique to be the “natural” further development of conventional photography.The technical precision of 3D-soft-tissue imaging techniques, such as those provided by the 3dMDface System, are of great help in acquiring soft tissue surface data of the human face in an objective way. However, all anthropological evaluation concepts based on this data must meet high standards regarding precision and reliability. Due to the complex situation with the degree of reliability that is strictly dependent on the landmark and axis in question, we conclude that prestudies addressing the baseline accuracy of any evaluation strategy are mandatory.Utilizing the mean of multiple measurements instead of a single measurement could clearly reduce the risk of corrupting the data during the evaluation process.


KIR cluster in a large and well-characterized sample of worldwide human populations in the Human Genome Diversity Project—Centre d'Etude du Polymorphisme Humain (HGDP-CEPH) panel in order to better understand the patterns of diversity in the region. Comparison of KIR data with that from other genomic regions allows control for strictly demographic factors; over 500,000 additional genomic markers have been typed in this panel by other investigators and the data made publicly available. Presence/absence frequencies and haplotypic associations for the KIR region are analyzed in the 52 populations comprising the panel and in accordance with major world regions . These data represent the first overview of KIR population genetics in the well-documented HGDP-CEPH panel and suggest different evolutionary histories and recent selection in the KIR gene cluster.In the present study, we investigate patterns of variation in the The online version of this article (doi:10.1007/s00251-012-0629-x) contains supplementary material, which is available to authorized users. KIR)  class I molecules as their ligand, the ligands for most of the stimulatory KIR have not been identified definitively. The balance between inhibitory and activating KIR and their specific ligands results in a finely tuned innate-adaptive immune response. The inhibitory KIR, partnered with alternative NK receptor complexes (CD94:NKG2A), ensure that NK cells are tolerant of healthy autologous cells and responsive to cells with compromised HLA class I expression, as what occurs frequently in virus-infected and tumor cells. The stimulatory KIR contribute to the activation of NK cells in response to infection and malignancy. Our recent work and that of others has shown that KIR and their HLA ligands are associated in a variety of autoimmune diseases , variability in gene content haplotypes is responsible for significant diversity both within and between populations. While many KIR haplotypes have been identified in human populations, they are generally categorized into two groups, A and B  exists between the centromeric and telomeric sections of the o et al.  consistsKIR complex via repeated recombination and duplication events . However, direct comparisons between populations are hindered by a lack of equivalence across data sets due to variations in typing methodology and the number of loci typed, as well as clarity regarding population collection and limited data regarding other genomic markers. In the present study, we investigate patterns of variation in the KIR cluster in a large and well-characterized sample of worldwide human populations in the Human Genome Diversity Project—Centre d'Etude du Polymorphisme Humain (HGDP-CEPH) panel .The 52 populations comprising the HGDP-CEPH represent seven major world regions. KIR loci in the HGDP-CEPH, we utilized our high throughput single nucleotide polymorphism (SNP)-based KIR genotyping assay developed using the SEQUENOM™ MassARRAY  on the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer platform . Briefly, the two-tiered analysis includes 38 “capture” primer pairs to amplify specific KIR genes in the region surrounding the SNPs to be queried; this is followed by 39 homogenous mass extend primer reactions to differentiate individual SNP patterns for the 16 KIR genes on the MALDI-TOF platform. These assays were run using the KIR sequence alignment in the Immuno Polymorphism Database  using version 1.4.0. Our typing system is regularly assessed against all known alleles in order to insure that all loci will be detected.To genotype the A treemap of HGDP-CEPH sample size and number of unique KIR genotypes was constructed using the “tmPlot” function in the “Treemap” package Tennekes  for the KIR loci were obtained by direct counting. Gene frequencies were estimated according to Lynch and Milligan (KIR2DL2L3 (KIR2DL2/KIR2DL3), gene frequencies were obtained by direct counting. A direct counting approach was also utilized to obtain an estimate for gene frequencies for the KIR3DL1S1 locus. There is evidence that in some populations there are haplotypes that lack this locus .Carrier frequencies for the Milligan  using thMilligan . For theKIR carrier frequencies was constructed using the “heatmap” function in the base “stats” package for the R language for statistical computing  algorithm for haplotype frequency estimation that accommodates KIR-specific constraints. Our constrained EM algorithm uses an a priori list of known/possible haplotypes to restrict the space of possible haplotype patterns due to known constraints on gene content variation and allelic variation. The constrained approach differs from traditional EM implementations, in which the set of all possible haplotypic combinations is generated from the observed genotypic data with no restrictions on possible haplotype patterns. The list of user-designated a priori haplotypes is said to “span” the set of observed genotypes in a study if all observed genotypes can be generated from at least one pair of haplotypes in the a priori list. If pairs of haplotypes from the a priori list do not account for all of the observed genotypes in the sample , care must be taken in the interpretation of the resulting estimates. Our algorithm uses sharp constraints, meaning that if the a priori haplotypes do not span the observed genotypes a warning/error message is produced and the a priori set must be increased. We began with an a priori list of haplotypes taken from published data based on sequence analysis of seven KIR haplotype frequency data were plotted using an adaptation of a shell script written by Owen Solberg for analysis of HLA allele data (www.pypop.org/popdata).Global heatmaps for haplotype frequency data were generated using Generic Mapping tools  across all study populations. Only individuals for whom KIR genotyping was performed were included in the analysis of the SNP data. Empirical p values (pemp) represent the proportion of the distribution of the correlations for the additional markers that were greater in absolute value than the true correlation.Distance from Africa was calculated using Addis Abbaba, Ethiopia as the central point  and the latitude and longitude for each population sample Table , compute Wickham . Data wepemp represents the proportion of the (two-tailed) distribution above or below the FST values obtained for the KIR loci.A similar approach was employed to obtain an empirical distribution for FST values from the same markers. Here, FST was calculated according to Nei  using anKIR gene content profile (genotype) detected and the number of observations in the HGDP-CEPH population are shown in Fig. n = 25) is seen in only one individual, and five genotypes have not been previously reported in 108 populations listed on AFND. The six most frequent KIR genotypes in the HGDP-CEPH each have a frequency of ≥5 % worldwide, and together account for over two thirds of all genotypes observed in the panel  in the diversity panel are observed in the NAN Melanesian sample with one of these found in three individuals. In addition, two of the NAN Melanesian private genotypes have not been previously reported in any population tested to date. Three other previously unreported genotypes were detected in the HGDP-CEPH: one each in the Druze (Middle East), Orcadian (Europe), and Mbuti Pygmies (Africa) populations.Each KIR, with only two or three different genotypes; however, the sample sizes for these populations are among the smallest in the panel. In contrast, the Palestinian population is the most diverse, with 18 unique genotypes, but the sample size is one of the largest in the panel. In order to visualize the relationship between sample size and KIR genotypic diversity in the HGDP populations, we constructed a treemap, which displays a graphical representation of hierarchical data via variation in color and area  are observed in each population; however, there is a wide range among populations. Several Asian populations in the sample are extremely homogenous for the rea Fig. . While tKIR loci typed in the HGDP-CEPH populations are given in Table x-axis, KIR loci) of the strong linkage disequilibrium between the loci associated with the A and B haplotypes that define the two major clades. The heatmap clearly illustrates the much higher frequencies for all A haplotype loci, as well as subnodes within both major clusters along affiliation with the centromeric and telomeric intervals of the KIR region. Clustering in the second dimension  reveals two major clades according to the relative carrier frequency of the B haplotype loci. Within the larger cluster defined by higher overall carrier frequencies of B-haplotype-associated loci, specific patterns of genotypic variation are clearly seen in the independent clustering of the three African hunter-gather populations (the San and Biaka and Mbuti Pygmies); these three populations are characterized by high overall B haplotype frequencies, with the notable exception of KIR2DS1, KIR2DS3, and KIR3DS1. A separate cluster with the two Oceanic populations is defined by high frequencies for most B haplotype loci. A third cluster is composed of three Amerindian populations , where KIR2DS3 is virtually absent. The large cluster associated with generally lower B haplotype frequencies is populated primarily by the remaining African and East Asian populations. The African populations are further distinguished by exceptionally low frequencies of the telomeric B haplotype loci.Gene and carrier frequencies for all of the KIR genotypes is relatively robust , a wide range in the A haplotype frequency is observed between populations, from 8 to 80 %  with which it is generally associated are extremely low. Additionally, the less common Cen-B3 motif, which appears to be a fusion between Cen-A and Cen-B1, and is characterized by the presence of KIR2DL3, KIR2DL5, KIR2DS3S5, and KIR2DP1, is observed at very high frequencies in the two pygmy populations  between KIR3DS1 carrier frequencies with distance from Africa .In contrast to the centromeric l-B Fig. , correspica Fig. , compariica Fig.  which reKIR2DL2L3 locus, and the telomeric haplotypes are marked by the KIR3DL1S1 locus. As gene frequencies give a better view of genetic differentiation than carrier frequencies, we focused on these loci, assuming that the gene frequencies we obtain via direct counting will in general have less missing data regarding gene content on the second chromosome. While there are clear limitations to this assumption (due to genotypes in some populations with deletion of the KIR3DL1S1 locus) as noted in the “Materials and methods” section, we determined that genotypes with this deletion are not common in most populations in our sample. Analysis of KIR3DL1S1 carrier status in our sample set reveals that only one individual out of 976 tested in the panel has a negative carrier status for both KIR3DL1 and KIR3DS1, i.e., is homozygous for the deletion in a previously unreported genotype  is an order of magnitude lower than that which is observed for autosomal markers genome-wide; comparison with FST value SNPs from the HGDP-CEPH database suggests that this FST value is lower than expectations due to chance alone (pemp < 0.05), suggesting a history of balancing selection on the locus.Genetic distances calculated from these gene frequencies for on Table  and compon Table  reveal tKIR diversity within populations is maintained by variation in gene content, and KIR differentiation between populations can be attributed in large part to frequency variation in common, shared haplotypes; this is evidenced by the fact that in most world regions, six genotypes account for the vast majority of observed variation. Nevertheless, numerous other genotypes are observed to varying degrees, highlighting the great degree of plasticity and potential for variation in gene content within the KIR cluster. Moreover, the distribution of genotypes in several populations is characterized by the virtual absence of the more common genotypes, suggesting that they are not requisite at the individual or population level.KIR genotype worldwide, which corresponds to homozygosity for the canonical A haplotype. The extreme examples of the Oceanic populations and the San are representative of a general trend towards region-specific gene content diversity, coupled with higher overall carrier frequencies of some or all stimulatory KIR; these populations occupying the more extreme ends of the spectrum of genetic divergence, specifically the (earliest diverged) African populations and the (most recently diverged) Amerindian and Oceanic populations, generally mirror patterns observed genome-wide , we did not find any individuals bearing the most common KIR genotypic variation in the HGDP-CEPH closely aligns with that reported in other population studies, analysis of KIR frequencies within the panel reveals novel observations and permits a more detailed understanding of particular region-specific and worldwide patterns. As previously noted, the stimulatory KIR3DS1 and KIR2DS1 are present at much lower frequencies in African populations , suggests negative selection on KIR2DS3 in Amerindians.Outside of Africa, this pattern of linkage disequilibrium placing KIR reveals that in most populations there is a very even balance between the inhibitory A haplotypic structure and the more stimulatory B haplotype, except within East Asia, where Cen-A predominates. These data are suggestive of a selective sweep within the centromeric KIR in East Asian populations, resulting in near fixation of the Cen-A motif. Previous work in the HGDP-CEPH and HapMap populations has suggested that recent sweeps of this nature have been relatively common in East Asia, compared to other world populations  events, contributing substantially to gene content variation. In the HGDP-CEPH populations outside of Africa, most notably the Oceanic and Amerindian populations, we observed extensive variation in gene content in the telomeric KIR, further supporting the notion of balancing, or diversifying selection on this section of the KIR.While present only at very low frequencies in African populations, the telomeric B haplotype loci are seen at increasing frequencies in populations outside of Africa, with a positive correlation of KIR in the HGDP-CEPH reveals significant evidence for balancing, diversifying selection within the telomeric region, and purifying selection and/or a history of population bottlenecks within the centromeric region, in general accordance with recent work showing different evolutionary histories for the centromeric and telomeric KIR Below is the link to the electronic supplementary material.


The relationship between leukocyte gene expression and recovery of respiratory function after injury may provide information on the etiology of multiple organ dysfunction.To find a list of genes for which expression after injury predicts respiratory recovery, and to identify which networks and pathways characterize these genes.Blood was sampled at 12 hours and at 1, 4, 7, 21 and 28 days from 147 patients who had been admitted to the hospital after blunt trauma. Leukocyte gene expression was measured using Affymetrix oligonucleotide arrays. A linear model, fit to each probe-set expression value, was used to impute the gene expression trajectory over the entire follow-up period. The proportional hazards model score test was used to calculate the statistical significance of each probe-set trajectory in predicting respiratory recovery. A list of genes was determined such that the expected proportion of false positive results was less than 10%. These genes were compared to the Gene Ontology for ‘response to stimulus’ and, using Ingenuity software, were mapped into networks and pathways.The median time to respiratory recovery was 6 days. There were 170 probe-sets representing 135 genes that were found to be related to respiratory recovery. These genes could be mapped to nine networks. Two known pathways that were activated were antigen processing and presentation and JAK- signaling.The examination of the relationship of gene expression over time with a patient's clinical course can provide information which may be useful in determining the mechanism of recovery or lack of recovery after severe injury. There has been improvement in the outcome for acute lung injury (ALI) Glue), a multi-centered study supported by the National Institute of General Medical Sciences (NIGMS), that aims to better describe genomic response in patients following severe injury or burns . This primes the innate immune system such that a secondary insult during this vulnerable window provokes an unbridled inflammatory response culminating in early MODS We have investigated the mechanisms critical for early priming of the innate immune system and have employed the circulating PMN as a surrogate for this response Although the MODS is the primary mechanism of morbidity in the patients in this study there was often no clear period between the end of resuscitation and the onset of MODS. For this reason we choose respiratory recovery as a surrogate for MODS. In addition respiratory recovery represents a positive clinical outcome for a patient and can be viewed as a marker of an improving overall health.In this paper, we apply novel statistical methods Glue study entry criteria included patients who had suffered a blunt trauma without isolated head injury, who had arrived at a hospital within 6 hours of the injury, and had either hypotension or an elevated base deficit. Subjects with anticipated survival of less than 24 hours, significant pre-existing organ dysfunction, or severe traumatic brain injury were excluded. Written informed consent was obtained from all patients or their legally authorized representative. Blood was sampled at 12 hours and at 1, 4, 7, 21 and 28 days after the blunt trauma and was hybridized to an Affymetrix HU133 plus 2.0 gene chip. The details of the clinical protocol and sample processing are described in The analysis presented in this report includes data on a subset of 147 patients admitted to one of seven participating institutions, between November 2003 and July 2006. Our paper focuses on a subset of 147 patients who had complete genomic profiles at the time of our analysis. The perfect-match model using dChip software (www.dChip.org) and gene expression values were log-transformed prior to any calculations. There were several steps taken to reduce the overwhelming dimensionality of the microarray. We first excluded probe-sets labeled ‘Absent’ over all arrays by the Affymetrix software. ‘Present/Absent’ labels in this technology indicate whether a probe-set was reliably detected or not. This step reduced the number to 48,992 probe-sets. Applying the assumption that the genes exhibiting temporal changes are potentially related to the outcome, we next excluded those probe-sets with a ‘low’ sample coefficient of variation, which we defined as having a coefficient of variation in the bottom half of the sample . This reduced the number of probe-sets to 11,461. Lastly, we performed the analysis of time-course microarrays using the EDGE Gene expressions were extracted from oligonucleotide probes by a The method used to obtain a list of statistically significant probe sets has been described previously Once we obtain a set of probe-set specific test statistics, we cannot use classical p-values to infer which probe-sets are important because the large number of hypothesis tests we have just done will result in an inflated type I error rate . Instead, we can assess the statistical significance of each test by sorting them in descending order by their absolute value and decide how many genes in the list were statistically significant using a false positive ratio of 10% As described above, 4,010 probe-sets were evaluated for an association with the time to respiratory recovery. Of these, 170 were identified as statistically significant when the proportion of false positive findings is set at the 10% level. Using the WebGestalt Response branch of the biological process. Each node contains the number of genes among the identified genes in our set that are represented in that particular ontology term . For example, a group of 30 genes were represented among the genes comprising response to stimulus GO term. The six genes that were represented in the inflammatory response GO term were ANXA1 (annexin A1), S100A8 , ALOX5AP (arachidonate 5-lipoxygenase-activating protein), S100A12 , CCR5 (chemokine (C-C motif) receptor 5), NR3C1 (glucocorticoid receptor).We next examined the Gene Ontology (GO) terms corresponding to our group of genes found significant by statistical hypothesis testing. These are listed in Focus genes. Connections for each focus gene were calculated by the percentage of its connections to other significant genes. A total of nine networks were found, six of which are shown in The 170 probe-sets with a false discovery rate (FDR) less than 0.1 were used in the network and pathway analysis. Affymetrix probe-set IDs were imported to the Ingenuity Pathway Analysis (IPA) software We next examined whether genes found in these networks can also be placed within known pathways. There exist large publicly available libraries of known networks and web application that allow for organized search and examination of these repositories. These web-based tools allowed us to examine further our list of selected genes and compare them to the configurations and content of known gene pathways. Pathways of highly interconnected genes were identified using the equation in antigen processing and presentation pathway, while genes CREBBP, IL12RB1, JAK2, LEPR, MIZ1, ARCN2, CCR5, were involved in the JAK-STAT signaling pathway, and genes CREBBP, PLP2, and RPS6KA3 were in the Signaling Pathway from G-Protein Families.For example, genes HLA-DMA, KIR2DL2, PSME1, TAP1, TAP2 were involved in the Among the genes associated with a shorter time to recovery are IL-12RB and CREB that indicate IL-2 activation and CCR5 increase, the indicators of increased immune lymphocyte function. The Janus family (JAK-STAT) signaling pathway plays a critical role in signal transduction mediated by cytokine and hormone receptors. The JAK-STAT pathway is used by interferons and type I cytokines (cell products that that may stimulate immunity). These cytokines and interferons activate Janus family tyrosine kinases (JAK kinases), which in turn activate STAT proteins. In addition, JAKs are involved in the signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. It has been well documented in the literature that the loss of JAK kinase function has been found to result in disease states such as severe-combined immunodeficiency and that the optimal JAK kinase activity is crucial for normal cellular responses Among the genes associated with a longer time to recovery is arachodoniate acid (ALOX5AP) that metabolizes into mediators of inflammation such as leukotrienes and prostaglandins and other ecocinoids which are associated with immune deactivation. Leukotrienes and prostaglandins act to increase vascular permeability and serves as chemoattractant for neutrophils. In addition, polymerase (DNA directed) iota (POLI) on the other hand has been reported to have enzymatic properties consistent with that of a somatic hypermutase and suggest that poliota may be one of the DNA polymerases hypothesized to participate in the hypermutation of immunoglobulin variable genes in vivo The major shortcoming of the current study is that it is based on a mixed cell population of leukocytes and genomic changes may reflect changes in this population. At the time of this study the technology was not available to obtain enough RNA from sorted cells to reliably run chips on pure cell populations. This technological hurdle has been overcome and we will soon be able to replicate this paper with pure cell populations.Describing all the findings of a study such as this is difficult because the essential output of our analysis is a list of 170 probe sets included in the supplement. These probe sets were then mapped into 135 genes, and the genes where identified with networks and pathways that are also described in the supplement. We described these networks and pathways that are known to be associated with inflammation in order to validate the process that was used to find the 170 probe sets. This does not contribute new knowledge. New knowledge comes from the further study of some of the 135 genes that are not generally known to be related to inflammation or associations of our findings with other work that we are not aware of. We invite the reader to do this. A much larger data set with extensive genomic and clinical data is available by application on our website


Lumbar disc degeneration (LDD) is a serious social and medical problem which has been shown to be highly heritable. It has similarities with peripheral joint osteoarthritis (OA) in terms of both epidemiology and pathologic processes. A few known genetic variants have been identified using a candidate gene approach, but many more are thought to exist. GDF5 is a gene whose variants have been shown to play a role in skeletal height as well as predisposing to peripheral joint OA. In vitro, the gene product growth differentiation factor 5 has been shown to promote growth and repair of animal disc. This study was undertaken to investigate whether the GDF5 gene plays a role in LDD.We investigated whether the 5′ upstream single-nucleotide polymorphism (SNP) variant rs143383 was associated with LDD, using plain radiography and magnetic resonance imaging to identify disc space narrowing and osteophytes, in 5 population cohorts from Northern Europe.P = 0.008).An association between LDD and the SNP rs143383 was identified in women, with the same risk allele as in knee and hip OA (odds ratio 1.72 [95% confidence interval 1.15–2.57], Our findings in 5 population cohorts from Northern Europe indicate that a variant in the GDF5 gene is a risk factor for LDD in women. Many more such variants are predicted to exist, but this result highlights the growth and differentiation cellular pathway as a possible route to a better understanding of the process behind lumbar disc degeneration. Lumbar disc degeneration (LDD) is a common cause of back pain, which is a major public health problem in Western countries. It is a common cause of work absenteeism, and its prevalence is increasing. LDD is known to be influenced by genetic factors, with estimated heritability of >70%. Little is known, however, of the genetic processes underlying the development of disc degeneration. A number of genetic loci have been investigated through a candidate gene approach. Many early reports of candidate gene associations in LDD, however, have not been replicated . LDD hasThe gene GDF5 is known to be involved in digit growth and certain monogenic disorders of skeletal development, and it was recently identified as playing a role in height . There iWe investigated the association between rs143383 and LDD in 5 independent population cohorts from Northern Europe  underwent radiography and coding of degenerative change as previously described  underwent plain radiography of the lateral lumbar spine, with LDD scored using a 4-point scale for disc space narrowing and anterior osteophytes over the 4 uppermost lumbar vertebrae.  Subjects were classified as having disc space narrowing (narrowing present at ≥2 levels), osteophytes (osteophytes present at ≥2 levels), or a combination (cases of narrowing plus osteophyte formation). Subjects in the Hertfordshire study (n = 336)  from the TwinsUK cohort who had previously undergone T2-weighted sagittal MRI of the lumbar spine were selected. The scans had been coded for degenerative change using a 4-point scale (0–3) for the following features: disc height, disc signal intensity, disc bulge, and anterior osteophyte. The individual traits were investigated, and subjects were considered to be cases if they had a score for a particular trait of ≥2 at 2 or more levels (L1/2 to L5–S1).P > 0.05).For the Rotterdam cohort, data on genotypes at rs143383 were obtained from the results of a genome-wide association scan using the version 3 Illumina Infinium II HumanHap550 SNP chip array. Genotyping procedures were followed according to the recommendations of the manufacturer (Illumina), as previously described . For the2. If heterogeneity existed (I2 >25%), a random-effects model was used for the analysis; otherwise, a fixed-effects model (inverse variance method) was applied. Since many reported associations of genetic variants are not yet replicated, we wished to determine the likelihood of our finding being a true positive, so the false-positive report probability was calculated  in each study separately. Meta-analysis of the results in the different cohorts was carried out in the statistical environment R, and forest plots were produced. Heterogeneity was evaluated with the Q statistic and Ilculated .Characteristics of the 5 population samples are shown in Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.) A significant association was found with the combination of disc space narrowing plus osteophytes in women, with an odds ratio (OR) of 1.72  (P = 0.008). Heterogeneity was not detected, so a fixed-effects model was used.Minor allele frequencies of rs143383 were broadly similar across the study groups and consistent with that reported in HapMap . Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131. Using a relatively high prior probability of 20%, the false-positive report probability was calculated, assuming expected effect sizes of OR 1.2 and OR 1.5, for the predisposing T allele. The false-positive report probability is the probability of no true association between a genetic variant and disease given a statistically significant finding, which depends not only on the observed P value but also on both the prior probability that the association is real and the statistical power of the test. The false-positive report probability for adjusted values was 36% and 12%, respectively, suggesting that this is not likely to be a false-positive finding.Distributions of the rs143383 genotypes and ORs unadjusted and adjusted for age are shown for women in each of the cohorts in Supplementary Table 1, available on the An association between the GDF5 SNP rs143383 and LDD in women is an important finding and is consistent with the known association of the T allele with OA . LDD is GDF5 is a proven variant in peripheral joint OA; this has been demonstrated in a number of populations. Furthermore, it is associated with skeletal height, suggesting mediation by either disc or vertebral body height or long bone length . GDF5 waP < 0.05 . This is further supported by the results of the false-positive report probability, suggesting that the results are more likely to be true positive than false positive.This study has a number of strengths and weaknesses. First, it combines a number of well-characterized cohorts that have been subjected to LDD phenotyping, albeit using different methods. The study of degenerative disease of the disc lags behind the study of hip, knee, and hand OA. In addition, LDD suffers from a lack of standardized epidemiologic definitions, which hampers attempts to study it in a systematic way. The use of twins and any relatedness within the other cohorts was taken into account. This study had 80% power to detect an OR for the T allele of 1.19 or higher for narrowing and 1.15 for osteophytes in the total sample size with We investigated whether the 5′-untranslated region SNP rs143383 GDF5 polymorphism was associated with LDD using 5 cohorts of European descent and found evidence of association in women. This SNP is thought to result in reduced transcription of GDF5, a prochondrogenic growth factor which has been shown to increase fiber size in the ligament complex model of knee instability in rats. One might postulate a number of mechanisms by which the SNP exerts its influence on LDD risk, including direct effects on the disc itself or by influence on periarticular structures such as the longitudinal spinal ligaments. Further work is needed to establish its mechanism of action, and this may reveal cellular pathways leading to a greater understanding of the pathophysiology of lumbar disc degeneration.