In vivo lung function evaluation showed airway hyperresponsiveness to methacholine in mutant mice. The expression of nuclear factor-kappa B (p65-NF-kB) in lung of VAChT-deficient mice were higher than in wild-type mice, whereas a decreased expression of janus-kinase 2 (JAK2) was observed in the lung of mutant animals. Our findings show the first evidence that cholinergic deficiency impaired lung function and produce local inflammation. Our data supports the notion that cholinergic system modulates airway inflammation by modulation of JAK2 and NF-kB pathway. We proposed that intact cholinergic pathway is necessary to maintain the lung homeostasis.Acetylcholine (ACh) plays a crucial role in physiological responses of both the central and the peripheral nervous system. Moreover, ACh was described as an anti-inflammatory mediator involved in the suppression of exacerbated innate response and cytokine release in various organs. However, the specific contributions of endogenous release ACh for inflammatory responses in the lung are not well understood. To address this question we have used mice with reduced levels of the vesicular acetylcholine transporter (VAChT), a protein required for ACh storage in secretory vesicles. VAChT deficiency induced airway inflammation with enhanced TNF-α and IL-4 content, but not IL-6, IL-13 and IL-10 quantified by ELISA. Mice with decreased levels of VAChT presented increased collagen and elastic fibers deposition in airway walls which was consistent with an increase in inflammatory cells positive to MMP-9 and TIMP-1 in the lung.  Pulmonary diseases such as asthma, acute lung inflammation and chronic obstructive pulmonary disease (COPD) represent major threats to human health. In common, they involve complex immune responses in which inflammatory and epithelial cells release elevated levels of pro-inflammatory Th1/Th2 cytokines such as IL-6, TNF-α, IL-4 and also the regulatory cytokine IL-10 .Total RNA from lung or spinal cord was extracted with Trizol , analyzed for quality on agarose gel and absorbance ratios of 260/280 and 260/230 nm, and reverse transcribed to cDNA using the SuperScript III cDNA kit (Invitrogen Life Technologies). Gene expression was evaluated by real-time qRT-PCR using a Rotor Gene  and SYBR Green as fluorescent dye (Qiagen) with GAPDH as a housekeeping gene. The reaction conditions were as follows: 95°C for 5 minutes, then 40 cycles of 95°C for 5 seconds and 60°C for 10 seconds. PCR products were run on agarose gel to confirm the size of the fragment and specificity of amplification. Primers used and annealing temperatures are presented: GAPDH  and VAChT . Data were obtained as ct values  and used to determine Δct values (Δct = (ct of the target gene)—(ct of the housekeeping gene). Data were expressed as arbitrary units using the following transformation . MMP-9 was less than twice greater in VAChT KDHOM, whereas the increase in TIMP-1 was approximately three times higher than observed in WT group, tipping in favor of decrease proteolysis supporting the turn-over of the extracellular matrix fibers and the remodeling [An imbalance in MMP-9 and TIMP-1 expression level is involved in ECM matrix deposition . Therefomodeling .HOM presented an increase in positive cells for MMP-9 and TIMP-1 (arrows) around airways compared to WT.Representative photomicrographs stained with Picro-Sirius to detect collagen fibers were shown in Given the increase in inflammation in VAChT deficient mice, we then evaluated both Respiratory system resistance (Rrs) and elastance (Ers) at baseline and post-challenge (after dose-response curve to methacholine) in wild-type and mutant mice. The absolute values of Rrs and Ers obtained in the methacholine dose-response curve are shown in milieu in the lung. These effects were associated with an increase in infiltration of inflammatory cells edema and increased in the number of cells expressing NF-kB and a reduction in JAK-2 levels in lung. These results suggest that long-term cholinergic deficiency affects pulmonary inflammation, pointing out the importance of acetylcholine in control pulmonary homeostasis.The major finding of the present study was that VAChT deficiency induces a pro-inflammatory Known sources of ACh for the lung are the parasympathetic neurons which are dependent on VAChT to release ACh , 30, airHOM mice have approximately 65% reduction in the levels of VAChT in the whole body. Here, we checked the VAChT mRNA expression in spinal cord and lung and confirmed this reduction, that was around 80 and 60%, respectively. These data were also confirmed by the reduction in VAChT protein content both in lung and in spinal cord. Furthermore, the absence of ACh induced a reduction in body weight and in the time of wire hang test in mutant mice. These data corroborate previously results that VAChT mice are myasthenic and had impairment in neuromuscular development and function [In order to evaluate the effects of cholinergic reduction in lung, we used genetically modified mice with cholinergic dysfunction. These mice were produced by targeting the VAChT gene. The release of ACh in these animals is proportional to the levels of VAChT expression , 30 and function , 16.Inflammatory responses are characterized by both endothelial permeability alteration and inflammatory cell recruitment. We noticed both phenomena in mutant mice in which we found increased mononuclear cells, peribronchial edema around airways and increase in the amount of total protein in BALF when compared to wild-type mice. Additionally, an increase in the number of macrophages, lymphocytes, eosinophils and neutrophils was recovered in BALF of mutant mice. Although the inflammatory response was mild, is important to note that these animals were not submitted to any stressors to induce lung inflammation. To our knowledge, these data show for the first time that VAChT reduction induces pulmonary inflammation.We evaluated pro-inflammatory cytokines  and the regulatory cytokine IL-10. Mutant mice presented higher levels of TNF-α and IL-4 in lung homogenates when compared to wild-type mice, while no difference was observed in the levels of IL-6, IL-13 and IL-10 between these two groups. IL-6 is an important cytokine involved in infection and in traumas, and is secreted primary by T cells and macrophages. IL-13 as IL-4 are more involved in allergic inflammation and are increase in experimental model of asthma  and IL-1Several studies have previously indicated that cholinergic activity has a fundamental role in anti-inflammatory responses in different experimental models. Vagotomized mice presented an increase in inflammatory cells in peritoneal fluid after septic peritonitis, enhancing early and late inflammatory responses . Howeverper se the ECM remodeling since macrophages and other cells release different types of profibrotic mediators [The persistence of chronic inflammation induces tissue repair , 34, 35.ediators . An imbaediators .HOM group related to baseline values (WT group), TIMP-1 increased approximately more than three fold, consistent with the dynamic turnover of ECM components that can occurs in inflammatory disease [We also found higher number of positive inflammatory cells to MMP-9 and TIMP-1 in airways in VAChT mutants compared to wild-type mice, with the increase in TIMP-1 more prominent than the increase in MMP-9. That is, while MMP-9, which degrades ECM, increased less that twice in VAChT KD disease . These rAlthough no difference in basal lung function was observed, VAChT KD mice presented an increased in maximal response of respiratory system resistance to a bronchoconstrictor agonist. Airway smooth muscle contraction is an important determinant of lung mechanical alterations in a presence of a bronchoconstrictor. However, it should be noted that the presence of inflammation, edema and airway remodeling can also increase Rrs responses by reducing airway lumen diameter. In this context, mutant animals presented increased airway inflammation, edema and remodeling which could explain the hyperresponsiveness observed. Another possibility is that changes in muscarinic receptors that could appear in mutant mice as a compensatory mechanism due to ACh deficiency. In this regard, we evaluated M2 protein in lung and we did not found any difference between the groups. Verbout et al.  showed tConsidering the mechanisms involved in VAChT deficiency-induced pulmonary inflammation, recently it has been suggested that stimulation of cholinergic receptors suppresses acute lung inflammation in mice, probably activating α7nAChR . Some auJAK-2 expression in lung of VAChT mutant mice was reduced. Janus kinases (JAKs) are regulators of signaling through cytokine receptors and the role of JAK2-STAT3 pathway in inflammation was not completely elucidated. Particularly in lung, this pathway was poorly investigated. The inhibition of JAK-2 prevents LPS-induced STAT-3 tyrosine phosphorylation . In turnin vivo is the difficult to distinguish the effect of each pathway. In lung, is not totally clear if the source of ACh by non-neuronal cells is dependent of VAChT. Other mechanism of ACh secretion in epithelial cells has been suggested mainly related to ACh release directly from the cytoplasm [Considering that both neuronal (parasympathetic nerves) and non-neuronal  sources of ACh co-exist in lung, the limitation of the present study performed ytoplasm , 54. Is ytoplasm  showed iytoplasm  pointed milieu observed was associated to increased p65-NF-kB and inhibition of JAK-2 expression. Importantly, these data suggest that intact cholinergic tone is an important mechanism to keep in check exacerbated inflammatory responses in order to maintain the lung homeostasis. Because the lung are constantly exposed to compounds from the environmental that can induce inflammatory responses, this regulatory mechanism may be relevant to avoid aberrant reactions in response to irrelevant stimuli.In conclusion, we showed for the first time that long-term VAChT deficiency induced airway hyperresponsiveness, inflammation and remodeling in a murine model of allergic airway inflammation. The pro-inflammatory


The Physical Activity and Sedentary Behaviour Assessment Questionnaire (PASBAQ), used within the Health Survey for England (HSE) at 5-yearly intervals, is not included annually due to funding and interview-length constraints. Policy-makers and data-users are keen to consider shorter instruments such as the Short-form International Physical Activity Questionnaire (IPAQ) for the annual survey. Both questionnaires were administered in HSE 2012, enabling comparative assessment in a random sample of 1252 adults.Relative agreement using prevalence-adjusted bias-adjusted Kappa (PABAK) statistics was estimated for: sufficient aerobic activity ; inactivity (MVPA<30minutes/week); and excessive sitting (≥540minutes/weekday). Cross-sectional associations with health outcomes were compared across tertiles of MVPA and tertiles of sitting time using logistic regression with tests for linear trend.P for trend = 0.003 and 0.004, respectively) and obesity .Compared with PASBAQ data, IPAQ-assessed estimates of sufficient aerobic activity and inactivity were higher and lower, respectively; estimates of excessive sitting were higher. Demographic patterns in prevalence were similar. Agreement using PABAK statistics was fair-to-moderate for sufficient aerobic activity (0.32–0.49), moderate-to-substantial for inactivity (0.42–0.74), and moderate-to-substantial for excessive sitting (0.49–0.75). As with the PASBAQ, IPAQ-assessed MVPA and sitting each showed graded associations with mental well-being (women: Capturing habitual physical activity and sedentary behaviour through brief questionnaires is complex. Differences in prevalence estimates can reflect differences in questionnaire structure and content rather than differences in reported behaviour. Treating all IPAQ-assessed walking as moderate-intensity contributed to the differences in prevalence estimates. PASBAQ data will be used for population surveillance every 4 to 5 years. The current version of the Short-form IPAQ was included in HSE 2013–14 to enable more frequent assessment of physical activity and sedentary behaviour; a modified version with different item-ordering and additional questions on walking-pace and effort was included in HSE 2015. Both physical inactivity and sedentary behaviour (characterised by activities involving prolonged sitting) are independent risk factors for chronic diseases such as cardiovascular disease (CVD) and obesity ,2. ClustFeasibility and costs are important considerations for choosing self-report or device-based methods to assess physical activity or sedentary behaviour. Despite decreasing costs for device-based measures, reported methods remain less expensive than device-based methods, especially for large studies . Data coDue to funding and interview-length constraints, the PASBAQ cannot be included each year. This prevents the inclusion of physical activity and sedentary behaviour data in analyses in the survey years when it is not collected . Both poQuestionnaire design is an iterative process. It is well recognised that efforts should continue in developing high-quality self-report measures of physical activity and sedentary behaviour, including a need for shorter instruments for use in large national studies where questionnaire space is limited . In EnglBoth the PASBAQ and Short-form IPAQ were administered to the same set of participants in HSE 2012 allowing us an opportunity to undertake a comparative assessment to examine the usefulness of including a shorter instrument in future annual rounds of the HSE to complement occasional use of the PASBAQ. Given this intended application, the most important correlation is that between the two self-report instruments, and not their level of agreement with device-based methods.The aim of this study was to compare the physical activity and sedentary behaviour data obtained from the two instruments. More specifically, our objectives were to: (1) compare PASBAQ- and IPAQ-assessed prevalence estimates of sufficient aerobic activity, inactivity, and excessive sitting, and examine the similarities in patterning across population subgroups; (2) quantify their level of agreement in identifying the same set of participants as being above or below commonly used duration thresholds; and (3) compare their cross-sectional associations with a range of physical health and mental health variables. These objectives are consistent with the use of physical activity questionnaires in large-scale surveys to produce estimates for population subgroups rather than to detect individual behaviour change as in the clinical setting .HSE data is used to monitor progress on many national health objectives, including physical activity in 1998, 2003, 2004, 2006, 2008, and 2012 . DetailsTrained interviewers measured participants’ height and weight and assessed their demographic characteristics, self-reported health, and health behaviours using computer-assisted personal interviewing. Following this, participants had a visit from a trained nurse. The response rate for both the main interview and nurse-visit (within co-operating households) was 56%. The PASBAQ was administered during the main interview; additionally, for participants in the fourth quarter of fieldwork (October 2012-February 2013), the Short-form IPAQ was administered during the nurse-visit, which occurs a few days to a few weeks later. Our analytical sample consisted of 1252 participants aged ≥16years who completed both questionnaires. The distribution of the analytical sample for the number of days between the main interview and nurse-visit was as follows: 1–13 days (28%), 14–27 days (28%), 28–41 days (20%), and 42 days or more (24%).breathe faster, feel warmer or sweat” [out of breath or sweaty”. Sedentary behaviour during leisure-time, on weekdays and at weekend days, was assessed using a set of questions on the usual amount of time spent in: (1) television viewing ) and (2) any other (non-television-viewing) sitting, including reading and computer use.Detailed information on the PASBAQ is available elsewhere . Brieflyr sweat” . Intensir sweat” ,39 and aAs part of the main interview, participants aged 16–74 engaged in employment were asked on how many workdays, in the last four weeks, their work included: (1) climbing of stairs/ladders, or (2) lifting, carrying or moving heavy loads, followed by a question about the average time spent on that activity on a typical workday. Including these activities for participants in specific occupations allows an assessment of overall volumes of moderate-to-vigorous physical activity (MVPA), and as such is taken into account in the estimation of adherence to current UK physical activity recommendations .Participants reported the frequency and duration of: (1) vigorous , (2) moderate (carrying light loads and bicycling at a regular pace), and (3) walking activities, as well as the average time spent sitting on a weekday, including sitting at work, during the last seven days.Self-report instruments such as the PASBAQ and IPAQ prompt participants to report activities lasting at least 10 minutes, reflecting global recommendations on physical activity for health which state that aerobic activity should be performed in bouts of at least 10 minutes duration . FurtherSummary measures of physical activity and sedentary behaviour derived from the PASBAQ and Short-form IPAQ are outlined in Time spent sitting was calculated as the sum of television and non-television-viewing: sedentary behaviour  was defined as spending 540 minutes or more sitting on weekdays. Time spent in moderate-intensity physical activity (MPA) was calculated as minutes per week (frequency × duration) spent in: (1) “heavy” domestic activity; (2) “heavy” manual/gardening activity; (3) moderate-intensity walking; (4) occupational activity (as described above); and (5) a subset of sports/exercise . WalDuring the last 7 days, how much time did you usually spend sitting on a weekday”. The frequency and duration of walking was assessed but not its intensity. In our primary analysis all IPAQ-assessed walking was assumed to be of at least moderate-intensity. This assumption was made for two reasons. First, our assumption was in line with a number of previous studies which assumed all walking to be of at least moderate-intensity by assigning MET values of 3.3 or 4.0 in accordance with the IPAQ scoring protocol [Time spent sitting was derived from the single-item “protocol  and the protocol ,39. Seco2), and was grouped into three categories: normal weight (18.5–24.9kg/m2), overweight (25.0–29.9kg/m2), and obese (≥30.0kg/m2). Participants with BMI <18.5kg/m2 were excluded from BMI-specific analyses due to small numbers. Annual household income was established using a card showing 30 bands . Equivalised household income was calculated  and grouped into tertiles. Three blood pressure readings were taken . Resting heart rate (RHR), a marker of physical fitness [Single measurements of height and weight were taken using standard protocols. Body mass index (BMI) was computed as weight in kilogrammes (kg) divided by height in metres squared . Hypertension was defined as systolic blood pressure of ≥140mmHg and/or diastolic blood pressure of ≥90mmHg and/or current use of medication to lower blood pressure [Positive mental well-being was measured by the Warwick-Edinburgh Mental Well-Being Scale (WEMWBS) . Responspressure . Total cpressure .c). The concordance correlation coefficient evaluates the degree to which paired data fall on the line of equality  [Relationships between PASBAQ- and IPAQ-assessed MVPA and sitting time (expressed as continuous measures) were summarised using the Pearson correlation (r) and Lin’s concordance correlation coefficient (P origin) ,47. Our  origin) . Three sAnalyses were run separately using each instrument to compare prevalence estimates of: (1) sufficient aerobic activity (MVPA ≥150minutes/week); (2) inactivity (MVPA <30minutes/week); and (3) excessive sedentary behaviour (sitting ≥540minutes/weekday). The threshold duration for aerobic activity was chosen to compare levels of adherence to current UK physical activity recommendations ; the samThe Kappa statistic was used as a measure of relative agreement between instruments that was not attributable to chance . HoweverP-value used as a test for linear trend. These analyses were conducted on age-standardised data, using the 2012 English household population as standard, to take into account the differences in age profile across the categories of MVPA and of sitting. We assessed difference in linear trend across the instruments using the significance level of the coefficient for an interaction term , which was added to the model including both instruments as a main effect.Analyses were run separately using each instrument to compare cross-sectional associations with other health variables. Sex-specific prevalence estimates of obesity, having a low WEMWBS score, CVD, current cigarette smoking, drinking more than twice in excess of recommended daily alcohol limits on the heaviest drinking day in the last 7 days, hypertension, and raised cholesterol were computed according to the PASBAQ- and IPAQ-based tertiles of time spent in MVPA and the tertiles of time spent sitting. Logistic regression was performed with the health outcome as the dependent variable and the three categories of MVPA and of sitting as the independent variable. First, the tertiles were entered in the models using two indicator variables with the group representing the lowest tertile as reference. An adjusted Wald test was used for testing overall significance. Secondly, the tertiles were entered as one continuous variable with the Selection bias potentially results in estimates for subgroups not being representative of the true levels in the entire study population . The HSEPASBAQ questions on usual walking-pace and effort (aged ≥65years) are used to distinguish between light- and moderate-intensity walking: with only the latter counting towards adherence to UK physical activity recommendations. Earlier versions of the Short-form IPAQ included questions about walking-pace. These have since been removed given their minimal contribution to estimates of reliability and validity with accelerometer data used as the criterion . Domain-Consistent with HSE reporting, a subset of occupational physical activity for participants in a specific set of occupations  was accoP<0.05).Data management was performed using SPSS version 20.0 , and analysis was conducted using Stata version 13.1  accounting for the complex sample design. Tests of statistical significance were based on two-sided probability . It is made clear in the advance letters and information leaflets that participation in the survey is entirely voluntary, and that participants may decline to answer individual questions, withdraw or stop at any time, or refuse any particular measurement if they wish to do so. Interviewers and nurses will often repeat this information in their introductions and when they are setting up appointments, and throughout the interview as necessary. Indeed, many individuals do refuse to participate in the survey; others may refuse individual questions, decline to continue part way through an interview or refuse physical measurements. It is also standard practice to conduct interviews and nurse visits some time after an appointment has been made so that individuals have a chance to reflect on their agreement before the appointment takes place. The procedures used in the HSE to obtain informed consent are very closely scrutinised by a National Health Service (NHS) ethics committee each year. Information leaflets and both the content and wording of questionnaires are also carefully reviewed by the ethics committees. Ethical approval for HSE 2012 was obtained from the Oxfordshire A Research Ethics Committee (reference 10/H0604/56). This study is a secondary analysis of previously collected data and so additional ethical approval was not required.P<0.001) and contained more married than single people (P = 0.024), but did not differ with regard to other socio-demographic characteristics such as gender, socioeconomic status, and self-reported health conditions. 63.3% of participants in the analytical sample met current physical activity recommendations (MVPA ≥150minutes/week) according to the PASBAQ, compared with 61.2% in the full sample (data not shown).Of the 8291 adults interviewed in HSE 2012, 8173 completed the PASBAQ. 2325 individuals were interviewed in the fourth quarter, and, of these, 1252 co-operated with the nurse-visit and completed the Short-form IPAQ. Statistically significant differences in demographic characteristics were examined by comparing the 1252 participants in the analytical sample (PASBAQ with IPAQ) with the subset of the main HSE 2012 sample that comprised 6921 participants with PASBAQ but without IPAQ data . The anac<0.30), reflecting the large divergence of the data points from the fitted regression line, and the divergence of the fitted line from the 45-degree line of equality through the origin.Scatterplots of the continuous measures showed the familiar pattern of increased scatter as time spent in MVPA and time spent sitting on weekdays increased Figs  and 2. Pvs. 8.1%), 45–64 (18.3% vs. 5.0%), and in the highest income category (25.8% vs. 2.5%).Unadjusted Kappa statistics for sufficient aerobic activity and for inactivity varied across population subgroups, being highest for groups with the lowest volumes of MVPA: participants aged ≥65years, in the lowest income group, and in the group with the highest values of resting heart rate. Agreement across BMI categories failed to show any consistent pattern. PABAK statistics for sufficient aerobic activity and for inactivity showed a different pattern in some instances, with values being highest for men, participants aged 16–44, and in the group with normal weight. Relative agreement for excessive sitting also varied across subgroups, with values being highest for participants aged ≥65years and in the group with the highest values of resting heart rate.Quadratic weighted Kappa statistics across the PASBAQ- and IPAQ-based tertiles were higher for MVPA (κ = 0.31 to 0.42) than for time spent sitting (κ = 0.12 to 0.35) . DiffereP-values for group differences  and for linear trend. P-values for the PASBAQ × IPAQ term examining the difference in linear trend across the instruments are also shown. PASBAQ data showed a number of apparent dose-response associations, some of which were seen only among men or women. Higher MVPA was associated with lower odds of having a low WEMWBS score , lower odds of being classed as obese (women: P for trend = 0.001), and marginally significant lower odds of reporting CVD (women: P for trend = 0.069). IPAQ-assessed MVPA showed similar graded associations with having a low WEMWBS score (women: P for trend = 0.003), obesity (women: P for trend = 0.007), and was marginally significant for CVD (men: P for trend = 0.069). Null associations across both questionnaires were found for excessive alcohol consumption, hypertension, and raised cholesterol , having a low WEMWBS score (men: P for trend = 0.053), and reporting CVD . IPAQ-assessed sitting showed graded associations with health outcomes for women but not for men. For women, more time spent sitting was associated with increased odds of being classed as obese (P for trend = 0.014) and increased odds of having a low WEMWBS score (P for trend = 0.004) (= 0.004) . Null asExcluding all walking from MVPA narrowed the gap in prevalence estimates, but the IPAQ-based estimates of sufficient aerobic activity remained higher than the PASBAQ . PrevaleValidation studies have compared self-reported data on physical activity and sedentary behaviour with device-based methods such as accelerometry ,56 and pIPAQ-assessed prevalence estimates of sufficient aerobic activity (MVPA ≥150minutes/week) and inactivity (MVPA<30minutes/week) were higher and lower respectively than the PASBAQ. IPAQ-assessed estimates of excessive sitting (sitting ≥540minutes/weekday) were also higher. Demographic patterns in prevalence estimates were similar. PABAK statistics showed fair-to-moderate agreement for sufficient aerobic activity (ranging from 0.32 to 0.49), moderate-to-substantial agreement for inactivity (0.42 to 0.74), and moderate-to-substantial agreement for excessive sitting (0.49 to 0.75). Agreement based on the PABAK varied across population subgroups for activity and for inactivity, being highest among groups with the highest volumes of MVPA. As with PASBAQ data, IPAQ-assessed MVPA showed graded associations with having a low score on a positive mental well-being scale (WEMWBS), obesity, and reported CVD. IPAQ-assessed sitting showed graded associations with positive mental well-being and obesity for women but not for men. Higher estimates of adherence to physical activity recommendations using the Short-form IPAQ compared with longer, more detailed instruments was also found in similar studies conducted in the United States  and Austvs. 7 days respectively). This is exacerbated when the index date of completing the questionnaire differs, as occurred in this study. Secondly, the exclusion / inclusion of work-based activities in the PASBAQ and IPAQ respectively partly explains the higher IPAQ-assessed estimates of sufficient aerobic activity and excessive sitting, and lower estimates of inactivity. Thirdly, the different approaches to capturing intensity . We would anticipate that excluding light-intensity walking from MVPA would result in lower prevalence estimates of sufficient aerobic activity, and higher estimates of inactivity, than those shown in this study; it would also improve the classification of participants across broad categories of MVPA. A recent analysis of UK Biobank data showed self-reported walking-pace to be a strong predictor of all-cause mortality [“During the last 7 days, how much time did you spend sitting on an average weekday?”). An example was also added to illustrate how participants should report their answer.Following our comparison study, four main modifications were made to the current version of the Short-form IPAQ (for inclusion in HSE 2015). First, the order of questions was reversed so that questions on walking appeared first, followed by moderate- and then vigorous-activities in order to minimise both spill-over effects and the potential double-counting of activities. Secondly, two questions about walking-pace and effort (taken from the PASBAQ) were added to collect data on intensity. Including them will enable data-users to distinguish between light- and moderate-intensity walking in the same way as with PASBAQ data , suggesting that behaviour change between the PASBAQ and IPAQ did not materially influence our findings. As the IPAQ was administered only in the fourth quarter of fieldwork, spanning the winter months, the findings of our study cannot be assumed to be generalizable to the full HSE year. Although the analytical sample was older on average than the rest of the HSE sample, it did not differ with regard to other socio-demographic characteristics such as gender, socioeconomic status, and self-reported health conditions. The older age of the analytical sample slightly reduced the representativeness of our data, but it did not influence our findings as we compared the two instruments using data collected from the same sample. Finally, the cross-sectional nature of the study precludes us from making any inferences about direction or causality.Our study had a number of limitations. In the HSE 2012, the PASBAQ and IPAQ were positioned in the main interview and nurse-visit respectively, and so were administered on average one month apart. Participant responses to the IPAQ may have been influenced by their earlier responses to the PASBAQ, e.g. participants may have modified their behaviour in the period of time between instruments, or have been influenced by the context of other questions. A crossover design—in which participants are randomised to a balanced ordering of the instruments –would have enabled us to control for an order effect when comparing the instruments. However, a crossover design was not possible in our study as it is essential to use a standardised protocol to administer the PASBAQ for the purposes of using HSE data to monitor changes over time in adherence to UK physical activity recommendations. We examined the sensitivity of our results by conducting a multiple linear regression analysis of the difference in time spent in MVPA (PASBAQ—IPAQ) using the number of days between data collection points as an independent variable adjusted for age and sex. The number of days between data collection points was not a significant predictor of the difference in MVPA . This complements the detailed information collected by the PASBAQ at approximately 5-yearly intervals. This enables descriptive analysis of broad discrete categories of physical activity and sedentary behaviour, and its cross-sectional associations with health. For example, its inclusion in HSE 2013 enabled analysis of the association between shift-working and physical activity  .Feasibility and costs are important considerations for choosing self-report or device-based methods to assess physical activity or sedentary behaviour. Despite decreasing costs for device-based measures, reported methods remain less expensive than device-based methods, especially for large studies. Obtaining high quality data from reported methods requires choosing the right instruments and using them correctly. Capturing the multi-dimensional nature of habitual physical activity and sedentary behaviour through brief questionnaires is complex. Differences in prevalence estimates can reflect differences in questionnaire structure and content—and the analytical assumptions they impose on the data—rather than differences in reported behaviour. Treating all IPAQ-assessed walking as moderate-intensity contributed to the differences in prevalence estimates based on thresholds of MVPA, and the fair-to-moderate strength of agreement. PASBAQ data will continue to be used for population surveillance at 4- to 5-yearly intervals. The Short-form IPAQ was included in HSE 2013–14 to enable more frequent assessment of physical activity and sedentary behaviour; a modified version with different item-ordering and additional questions on walking-pace and effort was included in HSE 2015.S1 FigThis figure compares the proportion of participants categorised with physical health and mental health outcomes according to the PASBAQ and Short-form IPAQ assessed tertiles of time MVPA excluding all walking, by gender, age-group, equivalised household income, resting pulse rate (RHR), and BMI status.(TIF)Click here for additional data file.S2 FigThis figure compares the proportion of participants categorised with physical health and mental health outcomes according to the PASBAQ and Short-form IPAQ assessed tertiles of time spent in MVPA excluding occupational physical activity, by gender, age-group, income, resting pulse rate (RHR), and BMI category.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableEstimates of MVPA excluded walking.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 TableEstimates of MVPA excluded occupational physical activity.(DOCX)Click here for additional data file.


Limited data are available regarding the long-term clinical outcomes of second-generation drug-eluting stents (DES) versus first-generation DES in patients with coronary chronic total occlusion (CTO) who undergo percutaneous coronary intervention (PCI). The aim of this study was to compare the clinical outcomes of second-generation DES with those of first-generation DES for the treatment of CTO.p = 0.72) or major adverse cardiac events . After propensity score matching, the incidences of cardiac death  and MACE  were still similar in both groups. Furthermore, no significant differences were observed between sirolimus-eluting, paclitaxel-eluting, zotarolimus-eluting, and everolimus-eluting stents regarding the incidence of cardiac death or MACE.Between March 2003 and February 2012, 1,006 consecutive patients with CTO who underwent successful PCI using either first-generation DES (n = 557) or second-generation DES (n = 449) were enrolled in a multicenter, observational registry. Propensity-score matching was also performed. The primary outcome was cardiac death over a 2-year follow-up period. No significant differences were observed between the two groups regarding the incidence of cardiac death (first-generation DES versus second-generation DES; 2.5% vs 2.0%; hazard ratio [HR]: 0.86; 95% confidence interval [CI]: 0.37 to 1.98; This study shows that the efficacy of second-generation DES is comparable to that of first-generation DES for treatment of CTO over 2 years of follow-up. Percutaneous coronary intervention (PCI) of chronic total occlusion (CTO) lesions is a challenging procedure due to the difficulty in crossing the CTO and the high restenosis rates after PCI –4. HowevDrug-eluting stents (DES) were developed for enhanced stent durability compared with BMS by inhibiting in-stent neointimal hyperplasia. Sirolimus-eluting and paclitaxel-eluting stents (SES and PES), hereafter referred to as first-generation DES, are superior to BMS with respect to the in-stent restenosis rate and target lesion revascularization after CTO PCI –10. HoweThis study was conducted from prospective registries at two tertiary medical centers, Samsung Medical Center and Bucheon Sejong Hospital, in South Korea. Between March 2003 and February 2012, 2,659 consecutive patients were enrolled. The inclusion criteria for the registries were: 1) at least 1 CTO detected on a diagnostic coronary angiograph; and 2) symptomatic angina and/or a positive functional ischemia study. Exclusion criteria included: 1) previous coronary bypass grafting; 2) history of cardiogenic shock or cardiopulmonary resuscitation; and 3) ST-segment elevation acute myocardial infarction (MI) during the preceding 48 hours. A CTO lesion was defined as the obstruction of a native coronary artery with a Thrombolysis In Myocardial Infarction (TIMI) flow grade 0 and an estimated duration longer than 3 months (4). Duration was estimated based on the interval from the last episode of acute coronary syndrome (ACS). For patients with no history of ACS, duration was estimated from the first episode of exertional angina consistent with the location of the occlusion or previous coronary angiogram , 20, 21.Of the 2,659 patients included in the registry, 477 patients who underwent CABG and 787 patient who treated with medical therapy only were excluded. Of the patients who performed PCI, 1,196 patients (80.2%) underwent successful revascularization. Among them, 1,006 patients who underwent PCI with DES implantation and achieved angiographic success were finally included in this analysis .Experienced clinical research physicians and coordinators from an individual clinical research organization collected baseline clinical, angiographic and procedural characteristics from hospital charts or hospital databases according to prespecified definitions. Clinical follow-up of the registry after index coronary angiography was performed at 1, 3, 6, and 12 months, and every year thereafter. Collection of follow-up information was mainly conducted through review of inpatient and outpatient hospital charts by the clinical research coordinators, and additional follow-up information was collected through a telephone interview with patients and was confirmed with the Korean national database using a citizen registration number unique to each individual.All baseline and procedural cine coronary angiograms were reviewed and quantitatively analyzed at the angiographic core laboratory  with an automated edge-detection system  using standard definitions . The extCoronary interventions were performed according to a standard technique. All procedures and treatments, including periprocedural and postprocedural medication regimens, were performed according to current practice guidelines. All patients, regardless of stent type, received a 300 mg loading dose of aspirin and a 300 to 600 mg loading dose of clopidogrel before the coronary intervention, unless they had previously received these antiplatelet medications. DES were used without restriction; the duration of dual antiplatelet therapy and the use of glycoprotein IIb/IIIa antagonists was left to the discretion of the operator.The primary outcome was cardiac death over a 2-year follow-up period. The secondary outcomes were all-cause death, MI, repeat revascularization, and major adverse cardiac events (MACEs). Repeat revascularization included target vessel revascularization (TVR) and non-TVR treated with PCI or CABG. MACEs included cardiac death, MI, and repeat revascularization. All deaths were considered to be of cardiac origin unless a definite noncardiac cause could be established. MI was defined as an elevation of the creatine kinase-MB fraction or the troponin-T/troponin-I level greater than the upper normal limit with concomitant ischemic symptoms or electrocardiographic findings indicative of ischemia . Periprot-test for continuous variables and by the chi-square test or Fisher’s exact test when the Cochran rule was not met for categorical variables. The Cox proportional hazards model was used to estimate the hazard ratio (HR) and 95% confidence interval (CI) for clinical outcomes between two groups. Cumulative event rates were estimated by the Kaplan-Meier method, and treatment effects were assessed using stratified log-rank statistics. We also adjusted for differences in clinical and angiographic characteristics by performing propensity score matching. The “psmatching” custom dialogue was used in conjunction with SPSS version 21 . The psmatching program performs all analyses in R  though the SPSS R-Plugin (version 2.14.2). Using the propensity score matching method, we created 557 matched pairs of patients. The adequacy of propensity matching was calculated by the overall balance test . All tests were 2-tailed, and p values < 0.05 were considered to indicate statistical significance.Continuous variables are expressed as means ± SDs, and categorical variables are presented as absolute numbers and proportions (%). Overall comparisons between groups were performed by Student’s The design of the study was approved by Institutional Review Board at Samsung Medical Center and Bucheon Sejong Hospital. The institutional review boards approved this study and waived the requirement for informed consent. All patients’ records/information were anonymized and de-identified prior to analysis.Of the 2,659 patients included in the registry, 1,006 patients received only first-generation  or second-generation DES . In the first-generation group, 59.4% of the recipients received SES , whereas 40.6% of the recipients received PES . In the second-generation group, 62.4% of the recipients received EES  and 36.6% of the recipients received ZES .The patients in the first-generation group were more likely to be male and less likely to have had a history of dyslipidemia or ACS compared with the patients in the second-generation group . As showThe median follow-up duration for all surviving patients was 1,265 days . As expected, the median follow-up duration was shorter for patients treated with second-generation DES [median 881 days ] compared with patients treated with first-generation DES . The disparity in follow-up duration between the two groups, which was unavoidable because of the later development of second-generation DES, was accounted for in the analysis by considering only the first 24 months after PCI.p = 0.72). The rates of all-cause death, MI, repeat revascularization, and MACE were also not different between the two groups  observed in the overall population were consistent in these subgroups . No signIn this study, we demonstrated that first-generation and second-generation DES are similarly effective in the context of CTO lesions. No differences were found between the two groups regarding the overall incidence of cardiac death or the overall incidence of MACE, despite the mid-term (two years) period of observation. After propensity score matching of all the patients, the incidences of cardiac death and MACE in the two groups were still comparable. Subgroup analyses according to DES type  also revealed similar incidences of cardiac death and MACE. In fact, previous meta-analyses substantially differ from the current one. In the meta-analysis by Lanka et al.  only 5 sEES have been found to be superior to PES , 27–29 ap < 0.01) compared with first-generation DES (SES and PES). Although this study had a relatively large number of patients, the registry was from a single center and was not randomized; moreover, the follow-up duration was only 12 months. In contrast to this study, Moreno et al [p = 0.52) and MACE . Park et al also performed a prospective randomized study of 160 patients with CTO and found that ZES was comparable to SES with respect to cardiac death  and repeat revascularization . Although these two studies were prospective randomized studies, their sample sizes were relatively small and the follow-up duration was only 12 months. Therefore, we compared the impacts of second-generation and first-generation DES on clinical outcomes in patients with CTO from a large-scale, multicenter registry with a relatively long-term follow-up duration.Several randomized and nonrandomized studies have compared outcomes between first-generation and second-generation DES after PCI in CTO lesions. Valenti et al  examinedno et al , Park etno et al , and Almno et al  observedSecond-generation DES have several advantages for PCI for CTO lesions. First, stent platforms of cobalt or platinum-chromium alloys are thinner and more deliverable than the platforms used in first-generation DES. This improved flexibility and deliverability might increase the success rate in treating CTO lesions. Moreover, second-generation DES produce a weaker inflammatory response and stimulate more rapid vessel re-endothelialization. Despite these improvements of second-generation DES, similar incidences of cardiac death, MI, and MACE have been observed in patients with bifurcated lesions  and ACS Our study did have several limitations. First, the study design was nonrandomized due to the nature of the registry data, which means that confounding factors may have affected the results. Second, we lacked comprehensive data regarding possible alterations of medical therapies over the follow-up period. Third, we did not routinely perform angiographic follow-up examination on our patients; thus, angiographic adverse events may have been underestimated. Fourth, although the vital status of all patients, including those lost to follow-up, was confirmed with the Korean national database using a citizen registration number that is unique to each individual, we cannot exclude the possibility of under-reporting of clinical outcomes other than death such as nonfatal MI and stent thrombosis. Fifth, our attempt to mitigate the unavoidable disparity in follow-up duration due to the time lag between the development of first-generation and second-generation DES limited our outcome analysis to 2 years. Furthermore, newer specialized revascularization devices for the treatment of CTO lesions may have been used more often in the second-generation DES group, which may have led to overestimation or underestimation of MACEs. Finally, the present analysis included patients who were treated over a long period of time. During this time, changes in PCI strategies may have impacted the clinical outcomes, irrespective of the stent type used.Our results suggest that the efficacy of second-generation DES is similar to that of first-generation DES for patients with CTO who undergo PCI, at least over a 2 year follow-up period.S1 DatasetIt includes data of CTO patients with file format of Microsoft Excel.(XLSX)Click here for additional data file.


To investigate the risk of epilepsy in stroke patients receiving and not receiving acupuncture treatment.Retrospective cohort study.This study was based on Taiwan's National Health Insurance Research Database that included information on stroke patients hospitalised between 1 January 2000 and 31 December 2004.We identified 42 040 patients hospitalised with newly diagnosed stroke who were aged 20 years and above.We compared incident epilepsy during the follow-up period until the end of 2009 in stroke patients who were and were not receiving acupuncture. The adjusted HRs and 95% CIs of epilepsy associated with acupuncture were calculated using multivariate Cox proportional hazard regression.Stroke patients who received acupuncture treatment (9.8 per 1000 person-years) experienced a reduced incidence of epilepsy compared to those who did not receive acupuncture treatment (11.5 per 1000 person-years), with an HR of 0.74 (95% CI 0.68 to 0.80) after adjustment for sociodemographic factors and coexisting medical conditions. Acupuncture treatment was associated with a decreased risk of epilepsy, particularly among stroke patients aged 20–69 years. The log-rank test probability curve indicated that stroke patients receiving acupuncture treatment had a reduced probability of epilepsy compared with individuals who did not receive acupuncture treatment during the follow-up period (p<0.0001).Stroke patients who received acupuncture treatment had a reduced risk of epilepsy compared with those not receiving acupuncture treatment. However, the protective effects associated with acupuncture treatment require further validation in prospective cohort studies. We used propensity score matching to reduce confounding effects in this study, and multivariate Cox proportional hazard models were used to control the residual confounding bias.Our study had no selection bias because all stroke patients hospitalised from 2000 to 2004 in Taiwan were analysed.Our data lack information on clinical risk scores, lesion characteristics, biochemical measures and the lifestyles of stroke patients,  and preventive administration of antiepileptic medications or engagement with rehabilitation programmes during the follow-up period were not considered.The lack of validity of diagnostic disease codes is also a limitation of the study; in addition our study could not validate the actual acupuncture points used in treatment.The mode of acupuncture treatment for stroke patients varied among traditional Chinese medicine (TCM) physicians.Stroke remains the leading cause of adult disability and death worldwide.6–8Acupuncture is a major part of traditional Chinese medicine (TCM) and is widely used in many countries.Using the Taiwan National Health Insurance Research Database (NHIRD), we conducted a nationwide, propensity score-matched, population-based, retrospective cohort study to investigate the risk of epilepsy in stroke patients who did and did not receive acupuncture treatment.Research data were obtained from the reimbursement claims of the Taiwan National Health Insurance Program, which was implemented in March 1995 and insures over 99% of the 23 million residents in Taiwan. NHIRD was established by the National Health Research Institutes in Taiwan and records all beneficiaries’ medical services, inpatient and outpatient demographics, primary and secondary diagnoses, procedures, prescriptions and medical expenditures, in the interest of public research. The validity of this database has been favourably evaluated, and research articles that have used this database have been published in prominent scientific journals worldwide.20–25The insurance reimbursement claims used in this study were from Taiwan's NHIRD. To ensure privacy, the patients were de-identified in the electronic database. This study was evaluated and approved by the Institutional Review Board of Taiwan's National Health Research Institutes NHIRD-100-122) and E-DA Hospital, Kaohsiung, Taiwan (2014012). Informed consent was not required because the patients’ identities were decoded and scrambled. This study was conducted in accordance with the Declaration of Helsinki.0-122 and20–25In this study, we used a similar approach using the same database in previous works.We defined stroke according to the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM 430–438). Eligible study participants included the 226 699 patients in the stroke cohort. Coexisting medical conditions included hypertension (ICD-9-CM 401–405), mental disorder (ICD-9-CM 290–319), diabetes (ICD-9-CM 250), hyperlipidaemia (ICD-9-CM 272.0–272.4), head injury , Parkinson's disease (ICD-9-CM 332), Alzheimer's disease (ICD-9-CM 331), brain cancer (ICD-9-CM 191) and regular renal dialysis (administration codes D8 and D9). Use of rehabilitation, anti-epilepsy drugs, anticoagulant, anti-platelet agents and lipid-lowering agents during the follow-up period was also considered in this study. During the index stroke admission, we identified stay in ICU, neurosurgery and length of hospital stay as potential confounding factors for the association between acupuncture treatment and post-stroke epilepsy. Under the condition of not experiencing previous epilepsy prior to stroke admission before 1 January 1996, cases of newly diagnosed epilepsy (ICD-9-CM 345) were further defined as at least one medical visit (including inpatient and outpatient care) with a physician's diagnosis after stroke admission.2 test was used to measure covariate balance and a p value <0.05 was suggested to represent meaningful covariate imbalance.We used a propensity score-matched pair method combined with frequency matching to analyse patients with and without acupuncture treatment. We developed a non-parsimonious multivariable logistic regression model to estimate a propensity score for patients with and without acupuncture treatment, irrespective of outcome. Clinical significance guided the initial choice of covariates in this model: age, sex, stroke subtype, low income, urbanisation, hypertension, mental disorder, diabetes, hyperlipidaemia, head injury, Parkinson's disease, Alzheimer's disease, brain cancer, renal dialysis, rehabilitation, anti-epilepsy drugs, anticoagulant, anti-platelet agents, lipid-lowering agents, stay in ICU, neurosurgery and length of hospital stay. We matched patients with acupuncture to patients without acupuncture using a greedy-matching algorithm with best to next-best matches until no more matches could be made. In the 1:1 matching procedure, all cases are initially matched to their ‘best’ control in the first iteration of the 8 to 1 Digit Match. The set of matched cases is then matched to the set of unmatched controls in N–1 additional iterations of the 8 to 1 Digit Match. If a case does not have one matched control, it is removed from the set of matches at the time it fails to receive a matched control. The corresponding control is also removed from the set of matches. The control is added back to the pool of unmatched controls, and allowed to match another case. This method can remove 98% of the bias from measured covariates. The χThe follow-up time, in person-years, was calculated for each subject until the diagnosis of epilepsy or until censoring because of death, withdrawal from the insurance system, or loss to follow-up. The outcome of this study was the person-years of new-onset epilepsy in stroke patients. This study's objective was to determine whether using acupuncture was associated with a reduced incidence of epilepsy in stroke patients.2 tests and t tests. We calculated HRs with 95% CIs for the risk of epilepsy after stroke associated with acupuncture treatment, adjusting for age, sex, low income and urbanisation in multivariate Cox proportional hazard regression models. The duration of observation for each person was calculated as the time until the individual was diagnosed with epilepsy or censored for death, migration, or discontinued enrolment in the insurance system. Adjusted HRs with 95% CIs for epilepsy associated with acupuncture treatment were calculated using multivariate Cox proportional hazard analyses with the variables categorised. We also performed age- and sex-stratified analyses to investigate the association between epilepsy and the use of acupuncture. SAS software V.9.1  was used for data analyses with a two-tailed probability, and p<0.05 was considered statistically significant.We first compared the distribution of sociodemographic factors and coexisting medical conditions between the stroke cohorts with and without acupuncture treatment using χAfter the propensity score matching procedure, no significant differences in sex, age, stroke subtype, low income, urbanisation, coexisting medical condition, ICU stay, neurosurgery or length of hospital stay were noted between stroke patients with and without acupuncture treatment .Compared to stroke patients who did not receive acupuncture treatment , stroke Among stroke patients, the risk of epilepsy exhibited a dose-dependent decrease with increasing use of acupuncture treatment . Figure In this nationwide, propensity score-matched, retrospective cohort study, we observed that stroke patients receiving acupuncture treatment exhibited a significantly lower risk of epilepsy during the follow-up period compared to those not receiving acupuncture treatment. A decreased risk of epilepsy after receiving acupuncture treatment was noted only among stroke patients aged 20–69 years. To our knowledge, this study is the first to report an association between using acupuncture and a reduced risk of post-stroke epilepsy.Older age, male sex, urbanisation, hypertension, diabetes mellitus, mental disorder, Parkinson's disease and Alzheimer's disease are known risk factors associated with stroke.Our study demonstrated that acupuncture treatment is associated with a decreased risk of epilepsy among male and female stroke patients aged 20–69 years. Age and sex have been previously shown to determine the medical conditions and complications of individuals with stroke.The incidence of post-stroke epilepsy varied depending on stroke type.Acupuncture treatment is one option for patients undertaking rehabilitation programmes,Functional MRI used in previous studies indicated that laser acupuncture activated the precuneus which is relevant to mood in the posterior default mode network, while needle acupuncture activated the parietal cortical region associated with the primary motor cortex.One of the strengths of this study was its large sample size and reduced selection bias. Second, our study consisted of a retrospective cohort design which used the NHIRD; this design provides stronger causal inference than case–control or cross-sectional designs. Third, to eliminate the influence of sociodemographic factors and coexisting medical conditions between stroke patients receiving and not receiving acupuncture treatment, we used a propensity score matched-pair procedure to select acupuncture and non-acupuncture treatment controls. To control for residual confounding effects in the association between a decreased risk of post-stroke epilepsy and acupuncture treatment, we applied multivariable Cox proportional hazard models to calculate the adjusted HRs and 95% CIs of epilepsy associated with acupuncture treatment. Finally, immortal time in observational studies may bias the results in favour of the treatment group, thereby overestimating the beneficial effects. To reduce such bias, we calculated person-years by correcting for immortal time in the acupuncture treatment group.Nevertheless, this study had certain limitations. First, we used insurance claims data, which lack information on clinical risk scores , lesion characteristics (location or size), biochemical measures and patients’ lifestyles, which have been reported as predictors of post-stroke epilepsy. Thus, this study does not provide information regarding the severity-dependent relationship between epilepsy and stroke. Second, preventive administration of antiepileptic medications or other rehabilitation programmes were not considered during the follow-up period. Thus, we were unable to evaluate the influence of these factors on the association between acupuncture and the decreased risk of post-stroke epilepsy. Third, although the accuracy of diagnosis codes in the database has been validated in previous studies,In conclusion, stroke patients receiving acupuncture treatment exhibited a reduced risk of epilepsy compared to patients not receiving acupuncture treatment. The association between decreased epilepsy risk and acupuncture use was observed in men, women, younger adults, and patients with any stroke subtype. However, this associated protective effect must be further assessed in randomised clinical trials to provide direct evidence regarding the effectiveness and mechanisms of acupuncture treatment for post-stroke epilepsy.


Sentinel node mapping has been used for laryngeal carcinoma in several studies, with excellent results thus far. In the current study, we report our preliminary results on sentinel node mapping in laryngeal carcinoma using intra-operative peri-tumoral injection of a radiotracer.Patients with biopsy-proven squamous cell carcinoma of the larynx were included in the study. Two mCi/0.4 cc Tc-99m-phytate in four aliquots was injected on the day of surgery, after induction of anesthesia, in the sub-mucosal peri-tumoral location using a suspension laryngoscopy. After waiting for 10 minutes, a portable gamma probe was used to search for sentinel nodes. All patients underwent laryngectomy and modified radical bilateral neck dissection. All sentinel nodes and removed non-sentinel nodes were examined by hematoxylin and eosin (H&E) staining. Ten patients with laryngeal carcinoma were included. At least one sentinel node could be detected in five patients . One patient had pathologically involved sentinel and non-sentinel nodes .Sentinel node mapping in laryngeal carcinoma is technically feasible using an intra-operative radiotracer injection. In order to evaluate the relationship of T-stage and the laterality of the tumor with accuracy, larger studies are needed.  Sentinel node mapping is the standard method of regional lymph node staging in breast cancer and melanoma ,2. In thThe sentinel node mapping concept has also been used with various degrees of success in gynecological, urological, and gastrointestinal tumors -7. For cSentinel node mapping has been used for laryngeal carcinoma in several studies, with excellent results thus far -15. In tTen patients with biopsy-proven SCC of the larynx were included in the study. Patients had no history of neck treatment, including radiotherapy or previous surgery. All patients were cN0 on neck examination and/or para-clinical imaging. Any of the following signs on computed tomography (CT) imaging were considered exclusion criteria: nodes larger than 10 mm, central necrosis, non-oval shape of nodes, perinodal tissue enhancement.On the day of surgery, after induction of anesthesia, 2 mCi/0.4 cc Tc-99m-phytate in four aliquots was injected in the sub-mucosal peri-tumoral location using a suspension laryngoscopy. After waiting for 10 minutes, sentinel nodes were searched for using a portable gamma probe . Any lymph node with more than a two-fold count rate as compared with the background was considered as sentinel node and harvested .After excision of the sentinel nodes, the background of the neck area was checked for any residual activity. The location of the sentinel nodes was recorded according to their anatomical levels in the neck. All patients underwent laryngectomy and modified radical bilateral neck dissection. All sentinel nodes and removed non-sentinel nodes were examined by H&E staining. For sentinel nodes, step sectioning was also performed in order not to miss any small metastasis.Detection rate was defined as the ratio of patients with at least one detected sentinel node to all recruited patients. The false-negative rate was defined as the ratio of patients with involved neck lymph nodes without involved sentinel node to all patients with involved neck nodes and at least a harvested sentinel node. This study was approved by the local ethical committee of Mashhad University of Medical Sciences under the approval number 920275. All patients gave written informed consent before inclusion into the trial.Between May 2012 and Jan 2014, 10 patients with laryngeal SCC were included in the study. The mean age of the patients was 59.3 years (range: 48–69 years); nine patients were male and one was female (F/M: 1/9). All patients had T3 laryngeal tumors except for patient No. 5 who presented with T2 supra-glottic tumor. At least one sentinel node could be detected in five patients, which was bilateral in four patients (detection rate: 5/10=50%). One–to-four sentinel nodes could be identified in these patients. The median number of detected sentinel nodes in the patients was 2. Neck dissection yielded 6–51 lymph nodes. The median number of dissected lymph nodes was 24. One patient had pathologically involved sentinel and non-sentinel nodes . No other patient had lymph node involvement on pathological examination. Characteris- tics of the patients are shown in .Sentinel node mapping is an important aspect of lymph node staging in several solid tumors of the head and neck, including melanoma, high risk SCC, and Merkel cell carcinoma ,16,17. Several authors have previously reported their experience of sentinel node mapping in laryngeal carcinoma ,15,18-31Our preliminary results showed that sentinel node mapping using an intra-operative radiotracer injection is feasible in laryngeal carcinoma patients. We achieved a 50% detection rate and no false-negative cases. Tc-99m-phytate was used as the mapping material in this study as we have previously shown this to be a reliable tracer for sentinel node mapping . Intra-oThe sentinel node detection rate in our study was sub-optimal 50%) as compared with the previous studies shown in 0% as comThe larynx is a midline organ and can exhibit bilateral lymphatic drainage . This caAnother important aspect of sentinel node mapping is false-negative cases. No false-negative cases were revealed in this study, which is compatible with the results of the previous studies mentioned in Sentinel node mapping in laryngeal carcinoma is technically feasible using an intra-operative radiotracer injection. Larger studies are needed in order to evaluate the relationship of T-stage and the laterality of the tumor with accuracy of the technique.


Background and aims. Nano-ionomer (NI) interacts with tooth structures superficially, and there is a concern about the enamel bonding ability of mild self-etch Ketac primer. This study compared the effect of different adhesive procedures (self-etching and etch-and-rinse approach) on long-term marginal microleakage of nano-filled resin-modified glass-ionomer (NI) cervical restorations.Materials and methods. Class V cavities were prepared on 72 maxillary premolars. The teeth were divided into six groups: G1: No treatment (NC); G2: Ketac primer (K primer); G3: Etchant + Ketac primer (E+K primer); G4: Self-etch adhesive (Bond Force); G5: Etchant + Bond Force (E+Bond Force); G6: Etchant + Adper Single Bond (Etch and rinse adhesive). All the cavities were restored with Ketac N100. The samples were stored in water for 6 months and thermocycled for 2000 cycles. Marginal sealing was assessed using dye penetration technique. Data were analyzed with non-parametric tests (α=0.05).Results. All the adhesive pretreatments resulted in a lower marginal leakage than that of NC (P≤0.01), except for E+Bond Force at the dentin margin. There was no significant difference between K primer and Bond Force. Microleakage differed significantly between K primer pretreatment and E+K primer (P=0.003), E+Bond Force (P=0.002) and etch-and-rinse adhesive (P=0.001) at the enamel margin, but it did not differ at the dentin margin. E+ Bond Force group showed insignificantly lower leakage at the enamel margin and significantly higher leakage at the dentin margin (P=0.02).Conclusion. Etch-and-rinse adhesive and selective enamel etching along with self-etch adhesive/Ketac primer might improve marginal sealing of aged nano-ionomer restoration. However, NI seems to be more resistant to water degradation.9A novel, highly packed, nano-filled RMGI, Nano-ionomer  has been introduced to the dental market to restore small cavities. Incorporation of approximately 69% of filler by weight or 56% by volume, of which two-thirds are nano-fillers, has improved mechanical properties, wear resistance, color and polishability characteristics and resistance to biomechanical degradation.10 In addition, this high-filled RMGI exhibited lower polymerization shrinkage and lower coefficient of thermal expansion and fluoride release compared to RMGI.12 The two-paste mixing system of NI might facilitate its handling characteristics.8These advantageous properties might result in the widespread use of NI instead of RMGI in clinical situations.When comparing abrasion resistance, NI behaved as an intermediate material between RMGI and nanocomposite because it had an intermediate composition.11 NI interacts very superficially with the dentin and enamel without demineralization or hybrid layer formation. This interaction provided micromechanical interlocking into the substrate roughness that is most likely supported by chemical bonding of polyalkenoic acid copolymer with surface hydroxyapatite.11 However, in addition to chemical bonding, RMGI could establish micromechanical bonding of the monomeric component into partially demineralized smear layer-free tooth surfaces.12However, NI, in contrast to RMGI, has no self-adhesive capacity and the use of Ketac Nano primer before NI is an essential step to its bond to tooth structures. This self-etch primer (pH=3) contains the acrylic/itaconic acid copolymer dissolved in HEMA and water; its low acidity may not allow the primer to totally dissolve/remove the smear layer.11According to TEM observations, NI revealed a tight interface with the tooth structure. Nevertheless, despite its effective bonding, it bonded less effectively than RMGI after 24 hours.13 The chemical bonding is achieved through acid-base reaction at the adhesive interface of RMGI.14The chemical bonding might have played an important role in the stability of marginal integrity and excellent performance of RMGI in a clinical trial conducted by Peumans et al.15 The reduced enamel marginal adaptation of NI cervical restorations was reported after one year of clinical service.15On the other hand, the higher filler and resin content of NI compared to RMGI was reported to possibly result in a reduced acid-base reaction.18 On the other hand, some authors indicated that acid etching of the enamel could increase the bond strength of RMGI.19A number of studies have reported that the use of different self-etch adhesives could improve dentin bonding/sealing of RMGI.20A recent study has indicated that using self-conditioner and especially an etch-and-rinse adhesive instead of cavity conditioner for RMGI (Fuji II LC) and Ketac primer for NI (Ketac N100) could improve the bond strength to dentin.Therefore, this study was designed to evaluate the effects of different adhesive cavity pretreatments  on the long-term sealing ability of NI cervical restoration. The null hypothesis was that prior different adhesive procedures have no effect on the marginal sealing of Class V NI restorations after water aging.The research protocol was approved by the local ethics committee. Seventy-two sound human maxillary premolars recently extracted for orthodontic treatments were selected, cleaned and disinfected in 0.5% chloramine solution for two weeks. The teeth were then stored in distilled water at 4°C, until use in the experiment. Standard Class V cavities  were prepared at the CEJ on the buccal surface with the gingival margin in dentin and the occlusal margin in enamel. The teeth were randomly divided into six groups of 12 each, according to different adhesive cavity pretreatments as follows:Group 1 (NC): Negative control with no treatmentGroup 2 (K Primer): Pretreatment with Ketac primerGroup 3 (E+K primer): Pretreatment with phosphoric acid etching + Ketac primerGroup 4 (Force Bond): Pretreatment with Force Bond self-etch adhesiveGroup 5 (E+Force Bond): Pretreatment with phosphoric acid etching + Force BondGroup 6 (Etch-and-rinse adhesive): Pretreatment with acid etching + Adper Single Bond.The materials used and their application procedures are presented in Score 0: No leakage visible at the tooth‒restoration interfaceScore 1: Penetration of dye along the cavity wall, but less than one-half of the lengthScore 2: Penetration of dye along the cavity wall, but short of the axial wallScore 3: Penetration of dye to and along the axial wallThe worst score from the two sections of each specimen was recorded. The microleakage data were analyzed using Kruskal-Wallis and Mann-Whitney statistical tests at a significance level of 5%.Dye penetration scores for the enamel and dentin margins in the six groups are presented in Tables No significant differences were found between groups 2 and 4, indicating approximately similar microleakage for K primer and Bond Force at both margins. At the enamel margin, there were significant differences between group 2 and groups 3, 5 and 6 , revealing a positive effect of acid etching on reducing microleakage; however, there were no significant differences at dentin margins. Comparison of groups 4 and 5 showed an insignificant lower leakage for group 5 than group 4 at the enamel margin, but a significantly higher leakage for group 5 (E+Bond Force) than group 4 (Bond Force) at the dentin margin (P=0.02). Group 6 (etch-and rinse adhesive) exhibited the lowest microleakage at both margins that was not different from group 4 at the dentin margin, while it was significantly different from group 4 at the enamel margin (P=0.03).21 In the current study, Ketac N100 restorations were subjected to thermocycling and water storage to challenge marginal integrity over time. The thermal fatigue stress and hydrolytic effect of water on the adhesive interface simulate conditions in the oral cavity, thereby predicting the behavior of the aged adhesive interface.22According to the results of this study, all the adhesive pretreatments significantly improved marginal sealing of the aged NI restorations compared to that of negative controls (with no treatment), except for E+Bond Force in which this improvement was not significant at the dentin margin. Ketac primer might act as a mild self-etch adhesive due to acidic monomers and photoinitiator content; it may create a resin covering on the primed surfaces after light-curing.15Maintaining the enamel and dentin margins of cervical restorations sealed against microleakage remains a major factor in clinical longevity.23 our results indicated relatively high microleakage at the enamel margin of aged NI restorations. Microleakage was significantly higher than at the dentin margin. Because of its low acidity, Ketac primer may not be able to interact sufficiently with the highly mineralized enamel structure. Recently, unstable bonding and increased microleakage with time were reported at the enamel margin of NI restorations.11 In addition, a one-year clinical trial of NI restorations disclosed enamel marginal deficiencies. These authors suggested that this decreased marginal adaptation was similar to the results obtained with self-etch adhesives in clinical evaluations.15Consistent with concerns about the long-term marginal sealing effectiveness of self-etch adhesives to the enamel,24 Imbery et al20 reported a higher bond strength of self-conditioner/NI compared to Ketac primer/NI to dentin. These authors suggested that the lower pH of self-conditioner (1.8) might have more completely removed the smear layer compared to Ketac primer.The self-etch adhesive, Bond Force used in this study, did not perform significantly better than Ketac primer in terms of marginal sealing. This self-etch adhesive (pH=2.3) contains a phosphoric acid monomer. It seems that despite the lower pH of Bond Force than Ketac primer, this adhesive was not capable of significantly lowering microleakage at both margins. In a recent study, the use of four self-etch adhesives prior to NI was reported to provide a higher or similar bond strength compared to that of Ketac primer, depending on their chemical compositions.25 using Silorane Adhesive System (pH=2.7) and Clearfil SE Bond (pH=2) instead of Ketac primer prior to NI could significantly improve early cervical sealing in open modified sandwich restorations.In a new study conducted by Shafiei and Akbarian,17 The water content in both NI and self-etch adhesive compositions might lead to compatibility between them.On the other hand, resin content of NI can create direct covalent bonds with the resin layer formed on the surface of self-etch adhesive, similar to that of RMGI.19 reported an early increase in bond strength of an RMGI to the enamel after using phosphoric acid.The results of the present study indicated that an additional etching prior to Ketac primer improved sealing at the enamel margin but had no effect on dentinal marginal sealing in aged restorations. Consistent with our results, Glasspoole et al9 It is well documented that this bonding plays a decisive role in marginal sealing and bonding durability.26 The thicker hybrid layer on acid-etched dentin observed in a TEM analysis by Coutinho et al27 in combination with a low-molecular-weight self-etching polymer resulted in greater early bond strength with an RMGI. Nevertheless, the lack of intimate interaction with hydroxyapatite-coated collagen led to instability of the interface in the long term.9 The same point may account for the higher microleakage obtained at the dentin margin of cavities in which acid etching was used before Bond Force/NI. However, this procedure led to an insignificant decrease in microleakage at the enamel margin (P=0.06). So far, no study has been performed on the effect of prior acid etching on bonding or sealing ability of Ketac primer/NI or self-etch adhesive/NI.Although acid etching prior to RMGI use was reported to provide better resin penetration into the underlying dentin and consequently better hybridization, this micromechanical interlocking might not benefit from additional chemical bonding.30 and even to the dentin32 was previously reported. However, some authors have proposed that acid etching of the dentin may reduce the adhesive potential of self-etch adhesives.30 It, therefore, appears that the bonding ability of self-etch adhesives to the etched dentin may be adhesive-specific.32 This assumption was relevant in the present study. The acid etching of the dentin prior to Bond Force increased the marginal leakage, whereas it displayed no effect on marginal leakage for Ketac primer.The beneficial effect of additional acid etching on the bonding efficacy of some self-etch adhesives/composite resin to the enamel30 Our results supported this recommendation, in particular for self-etch Ketac primer whose insufficient acidity is not capable of achieving reliable enamel bonding. The positive effect of selective enamel etching on the adhesive properties of Bond Force was indicated by a two-year clinical study.33In fact, enamel etching capability of self-etch adhesives is controversial. So, some authors have advised selective enamel etching to improve enamel bonding.According to our findings, pretreatment with an etch-and-rinse adhesive compared to Ketac primer significantly reduced microleakage at the enamel margin but this reduction was not significant at the dentin margin. Etch-and-rinse adhesive/NI yielded the best performance in terms of preventing marginal leakage after aging.34 demonstrating positive effect of etch-and-rinse adhesive on early marginal sealing of an RMGI (Vitremer). In a recent study, the highest bond strength of two RMGIs and NI to the dentin was attained by using an etch-and-rinse adhesive in short term.20 These results possibly supported the important role of the hybrid layer and resin tags in providing bonding durability, especially at the enamel margin. In addition, establishment of chemical bonding via using self-etch adhesive with partly demineralized dentin may be beneficial in terms of marginal sealing stability.9 However, etch-and-rinse adhesives were reported to have no effect on bonding of two RMGIs; the bonding may be more material-dependent than surface treatment-dependent.35This finding is in line with a study by Bayrak et al,36It was reported that dentin pretreatment using a HEMA-rich primer may compromise the formation of the absorption layer, and the acid-base reaction continuation in RMGI at the adhesive interface.Applying an etch-and-rinse adhesive prior to NI could lead to the formation of a hybrid layer into demineralized dentin and an adhesive layer on the dentin surface. Consequently, any chemical bonding of NI was not expected. However, a good union could be established between polyacrylic acid copolymer and resin content of both Adper Single Bond and NI. In this situation, bonding mechanism of NI to dentin is more like a composite resin than a glass-ionomer and the sealing ability of NI depends on the hybrid layer formation.Clinically, one advantage of using an etch-and-rinse or self-etch adhesive as surface pretreatment might be facilitation of the bonding procedures with simultaneous adhesive application for both NI and composite resin in a sandwich restoration. However, a decrease in fluoride release from NI following formation of an adhesive layer on the treated surface might be considered as a disadvantage. Nevertheless, clinicians would take advantage of the released fluoride from NI in the proximal surfaces of the adjacent teeth in an open sandwich restoration.Further studies of long-term bond strength and scanning electron microscopy analysis are required to confirm the results of the present study. Furthermore, clinical trials should be conducted on the long-term efficacy of different adhesive pretreatments for NI restorations.Under the limitations of this in vitro study, using etch-and-rinse adhesive and acid etching along with Ketac primer might be suggested in terms of marginal sealing of aged NI cervical restorations. However, when using a self-etch adhesive, selective enamel etching is recommended.The authors wish to thank the Vice-Chacellery of Shiraz University of Medical Sciences, Shiraz, Iran, for supporting this research (grant no. 92-5666); Dr Mehrdad Vossoughi, Assistant Professor, Center for Research Improvement, School of Dentistry, Shiraz University of Medical Sciences, for statistical analysis, and Dr Nasrin Shokrpour for help with the English language. This article is based on the thesis by M K. Etminan.


To identify patterns of stability and change in co-occurrence in children between 5–11 years, and to assess if they vary by socio-demographic factors.Data from 9548 singleton children from the UK Millennium Cohort Study (MCS) were assessed for co-occurrence of five common adverse conditions: wheeze; longstanding illness; unfavorable weight; injury; and socio-emotional difficulties. We summed adverse conditions (0–5) for each child at ages 5, 7, and 11 and identified co-occurrence (≥2 conditions). Using multinomial regression, we explored associations between co-occurrence trajectories and child’s sex and ethnicity, maternal education, and income quintile.45.6% of children experienced co-occurrence between 5–11 years (7% experienced constant co-occurrence). More children moved into co-occurrence than moved out (16.9 vs. 11.9%). Mutually-adjusted relative risk ratios (aRRR) showed a gradient by maternal education: compared to children with no co-occurrence whose mothers had a higher/degree, children whose mothers had no qualifications were more likely to move into ), out of ), have fluctuating ) or constant co-occurrence ). The same gradient (high vs. low) was evident for income quintiles. Girls were less likely to experience co-occurrence.Co-occurrence of adverse conditions is common during childhood, and trajectories are socially patterned. Child-focused care for lower-income children and boys early in life may prevent and reduce co-occurrence in later childhood. Poor health in childhood may be harmful to children’s social and academic development,,2 and prStudies exploring cross-sectional relationships between two or three health conditions report that obesity and emotional wellbeing, and variMultiple adverse health conditions in childhood may therefore present a particular burden for disadvantaged families and boys. Importantly, co-occurrence appears to be common in early childhooThis ‘equalization’ is suggested to arise when the influences of the family and home environment diminish, with (secondary) school, peers and youth culture playing a larger role in children’s lives. MoreoverUsing a population-based sample of UK children born between 2000–02, we therefore sought to explore whether the burden of co-occurrence in children changed between the ages of 5 and 11, and if changes differed by socio-demographic factors.We used data from the Millennium Cohort Study (MCS), a longitudinal study of children born in the UK between September 2000 and January 2002 which has been described elsewhere. Briefly,The Child and Youth Health conditions framework (CYPHOF)  and ChilFour socio-demographic variables were included in analyses. Child’s sex and ethnic group, highest level of maternal education and quintiles of household income  were obtained when the children were 9 months old. These latter two socio-economic variables were chosen to represent children’s early life influences, though we also tested whether results were similar using socio-economic indicators recording at later sweeps (see below).We first derived a co-occurrence index by summing the total number of adverse health conditions a child had at each of the three time points  . As we a3).Using the co-occurrence index, we created a binary variable at each age to categorize health burden. Preliminary analyses indicated that children with 0 or 1 adverse health condition did not differ significantly by sex, ethnicity or socio-economic variables. Children with no or one outcome were therefore coded 0, whilst children experiencing two or more adverse health conditions were coded 1 (‘co-occurrence’). Combining those with ‘co-occurrence’ (i.e. 2 or more conditions), alloweno co-occurrence, 2) into co-occurrence, 3) out of co-occurrence, 4) fluctuating co-occurrence and 5) constant co-occurrence. Generating these trajectories allowed us to explore the equalization hypothesis in more details, comparing prevalence of children moving into and out of co-occurrence between 5–11 years. Specifically, we compared how the risk of children moving into, out of, having fluctuating and constant co-occurrence between 5 and 11 years differed from those with no co-occurrence. We then assessed whether these trajectories were influenced by children’s socio-demographic factors and tested for possible effect modification.The cross-sectional binary markers of co-occurrence were used to generate five co-occurrence trajectories : 1) no cWe used multinomial regression analyses to estimate relative risk ratios (RRR) for these trajectories, using the no co-occurrence group as the baseline comparator. A RRR of less than one therefore indicates that risk is higher in reference trajectory, and when greater than one, the risk is higher in the comparison trajectory. We first conducted univariable analyses adjusting for sex, ethnic group, highest level of maternal education and quintile of household income when the cohort child was 9 months. We then conducted multivariable analyses, adjusting for all four socio-demographic variables simultaneously. As no effect modification by sex, maternal educational attainment, household income or ethnicity was apparent, we analyzed the sample as a whole.th sweep (at age 11).[Analyses were conducted using STATA 13/SE; sample w age 11).We conducted a number of sensitivity analyses. We tested whether the results obtained using measures of socioeconomic circumstances  at nine months differed when the equivalent measures recorded at ages 5 and 11 years were used. We also assessed whether results differed according to the age at which a child moved into or out of co-occurrence (i.e. at 7 vs. 11 years old). Finally, we analyzed the effect of data missingness, repeating the analyses using multiply imputed data, to examiPrevalences of the five adverse health conditions varied between ages 5, 7 and 11 , with noIn unadjusted analyses, boys (vs. girls), children of mothers with lower educational attainment (vs. those with a higher/degree) and children from lower income homes (vs. those in the highest income quintile) were at a greatest risk of all forms of co-occurrence (i.e. experiencing any trajectory). These children were more likely to move into, move out of, have fluctuating, and constant co-occurrence. Although there was a general trend for non-white children to move out of co-occurrence, due to small numbers of children in several ethnic groups, not all of these results reached statistical significance.The elevated risks for boys and children from more disadvantaged homes remained in models mutually adjusted for socio-demographic variables . Multiple imputation analyses showed the same pattern of results as those conducted using complete cases . ChildreAlmost half of children in this sample experienced co-occurrence between the ages of 5–11: more children moved into co-occurrence than out, and 7% of children had constant co-occurrence . Both cross-sectionally and longitudinally, co-occurrence was socially patterned; compared to children with no co-occurrence between ages 5 and 11, those who experienced co-occurrence  were more likely to be boys, have mothers with lower educational attainment, and be from lower income homes. Compared to white children, certain groups of non-white children appeared to be more likely to move out of co-occurrence over time. These inequalities in co-occurrence exited regardless of the age at which socio-economic indicators were measured , and when children moved into and out of co-occurrence (i.e. at age 7 or 11).1 We were however constrained by the available data, which meant we were only able to assess common health conditions reported in the study, and periods of time between data collection sweeps varied. Weight status was assessed using objective measures of height and weight at each time point, though the remaining physical and mental health conditions were assessed by main respondent report, generally the child’s mother. Parental report of child’s long-standing illness has been shown to accurately reflect recorded illness and child’s self-reported illness,[To our knowledge, no other study to date has assessed how co-occurrence of multiple adverse health conditions in children changes over time, with studies tending to explore only one or two health conditions at a time,–10 and u illness, and mate illness,,32 As as illness, we used  illness,The results presented here are based on complete case analysis. As tends to be the case in longitudinal cohort studies, remaining children included here were more likely to have mothers with higher educational attainment, come from higher income homes and be White. However, we used weighting to account for attrition in our analyses, and, using multiply imputed data to account for missingness, we showed results were very similar, indicating the robustness of our reported findings. We used a relatively simple measure of co-occurrence (an index) though there were many different combinations of adverse health conditions cross-sectionally and longitudinally. We also combined children with no or one health outcome, and those with two or more conditions, at each sweep as we sought to assess the difference between those with and without co-occurrence. In doing so, we may have masked the complexity between these different groups of children. Moreover, the adverse health conditions resulting in a classification of co-occurrence may have changed in some children over the measurement period while remaining the same in others. The trajectories are therefore a simplification of children’s patterns of co-occurring conditions over time. However, they provide an indication of the prevalence of multiple adverse health conditions and how these change during childhood, and whether this varies by a child’s socio-demographic characteristics. We considered other methods of analysis, which may have prevented the need for data reduction . However, these methods would not have allowed us to explore individual-level trajectories, children with and without co-occurrence and the complexity of movement between ages 5 and 11, as they necessitate aggregation of data at the sample level.Previous studies have explored co-occurrence cross-sectionally.,12 Our linto and constant co-occurrence, does not support this hypothesis. Although equalization has been shown to occur for individual health conditions, including two in our co-occurrence index ,[The equalization hypothesis suggests that such inequalities in health are reduced as children age, due to the greater influence of school and peers rather than the family and home environment. Our work health), we did n health), or that  health), ChildrenSingle conditions are generally used to measure the success of healthcare provision,,20 but tA number of conditions have been shown to occur together previously, and may share common etiologies . Yet deS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.