The relative performance of ART sealant and fluoride-releasing resin sealant in preventing fissure caries in permanent molars was compared in a randomized clinical trial conducted in southern China .After obtaining ethical approval, healthy schoolchildren who had permanent first molars with occlusal fissures which were sound but deep or presented with only incipient caries were recruited for the study. Included molars were randomly allocated into one of four parallel study groups in units of left/right teeth per mouth. Two of the four groups adopted the methods of ART or fluoride-releasing resin sealant placement while the other two groups adopted the topical fluoride application methods. Fissure status of the molars in each group was evaluated every 6 months. Development of dentine caries and sealant retention over 24 months in the molars in the two sealant-using groups was compared in this report. Outcome on cost-effectiveness of all four groups over 36 months will be reported elsewhere.At baseline, a total of 280 children (383 molars) with mean age 7.8 years were involved for the two sealant groups. After 24 months, 261 children (357 molars) were followed. Proportions of molars with dentine caries were 7.3% and 3.9% in the ART sealant and fluoride-releasing resin sealant groups, respectively . Life-table survival analysis showed that sealant retention  rate over 24 months for the resin sealant (73%) was significantly higher than that (50%) for the ART sealant (p < 0.001). Molar survival (no development of dentine caries) rates in the ART sealant (93%) and fluoride-releasing resin sealant (96%) groups were not significantly different (p = 0.169). Multilevel logistic regression (GEE modeling) accounting for the effects of data clustering and confounding factors confirmed this finding.Though the retention of fluoride-releasing resin sealant was better than that of the ART sealant, their effectiveness in preventing fissure caries in permanent molars did not differ significantly over 24 months. ART sealants could be a good alternative when and where resources for resin sealant placement are not readily available. Pits and fissures in the first molars are the most susceptible sites for dental caries in the permanent dentition ,2 and coSealing the pits and fissures of molars and premolars for prevention of dental caries was first introduced in the 1960s . It is nAlthough a number of different types of glass ionomers have been used previously as sealants, largely low-viscosity Type III sealants and ART sealants using high viscous type II restorative GIC, systematic reviews comparing GIC sealants with resin sealants have almost always, perhaps unfairly, pooled these different types of GIC sealants under a single category which makes true specific comparisons difficult. Consequently, inconsistent findings have been found regarding the comparative effectiveness of GIC to resin sealant in preventing fissure caries ,8 and thStrengthened highly viscous (type II restorative) GIC has the property of rapid setting, considerably reduced moisture sensitivity in the early setting stage and low solubility in oral fluids  which maA randomized controlled trial aimed at comparing the effectiveness of four different methods in preventing pit and fissure caries in permanent molars was conducted in southern China. The aim of this article was to report on the 24-month results of this trial, targeting specifically on the relative effectiveness of a self-cured ART sealant and that of a light-cured fluoride-releasing resin sealant in fissure caries prevention in permanent first molars of schoolchildren. Results on long-term cost-effectiveness of all four groups over a 36-month period will be reported elsewhere. The null hypothesis to test in this report was that there was no difference between the effectiveness of the two types of fissure sealants.This study was a randomized clinical trial with four parallel groups . Besides the two sealant-adopting groups compared here, the other two groups involved fluoride application. Ethical approval of the study was obtained from the Institutional Review Board of the University of Hong Kong. It was carried out in Shenzhen, China where the water supply was not fluoridated but fluoride toothpaste was common in the market. The concurrent dental caries prevalence of the 12-year-old children in Shenzhen when this study was started was 29.8% (mean DMFT score: 0.54) and 66.7% of the dental caries was nested in the permanent first molar [Children aged 7 to 9 years attending two of the largest primary schools in Shenzhen were invited to participate in the study. Children with written parental consent were clinically examined by dentists in their school. Children who did not have any major general health problems and had permanent first molars with occlusal fissures which were deep (base of fissure cannot be seen) or presented with signs of incipient caries (opacity and discoloration seen when viewed wet), similar to ICDAS code 2  were incIncluded molars were randomly allocated into one of four parallel study groups  in units of left/ right teeth per subject. If molars of both sides of one mouth were included, the molars on one side of the mouth were assigned to one group and those on the other side were assigned to another group. If only molars on the same side of one mouth were included, only one group was assigned to the mouth. At least one and at most two groups would be assigned in the same mouth. Two groups using topical fluorides would not be assigned in the same mouth. There were five possible combinations of two out of the four study groups  and these were assigned with numbers 1 to 5. Papers with the numbers written on were put into an envelope to be drawn by an assistant to decide the group combination of the included molars of a subject. A coin was then thrown to decide which side of the molars would be assigned the group with a smaller group number in the combination. If only molars of one side were included, the other group in the selected combination would be discarded.Molars allocated in the two sealant groups received single placement of the fluoride-releasing resin sealant  or ART sealant . Oral hygiene instruction was provided to all children in the study at baseline. The oral health related behaviors of the children including the toothbrushing habits and the frequency of taking sweet snacks and drinks were asked and recorded by the examiners/assistants.Sealants were provided in the schools by four operators with help from chair-side assistants. The operators were two independent dentists from a local public hospital who carried out most of the sealant placement and the two aforementioned examiners who provided a small proportion of the treatments when the two independent dentists were not available. The chief examiner (YX) had good previous experience in providing the treatment while the other three dentists had little experience. Hands-on training on providing the two types of sealant was provided to the less experienced operators by YX in a compromised clinical environment before the start of this study. In sealant placement, the molar was isolated with cotton rolls. Occlusal surfaces of molars in resin sealant group were etched with 37% phosphoric acid for 15–20 seconds, washed with water which was removed by suction connected to a portable dental unit, and then dried with air blow from a 3-in-1 syringe attached to the dental unit. Resin sealant was then applied and light-cured for 20 seconds using a LED curing-light. Occlusal surfaces of molars in ART sealant group were conditioned with the liquid component of the glass ionomer for 10–15 seconds, cleaned by cotton pellets soaked with water, and then dried with cotton pellets. GIC material was hand mixed according to the manufacture’s instruction and placed using the finger-press technique . CompletDevelopment of dentine caries (ICDAS Code 4–6) and sealant retention  in the molars was assessed blindly every 6 months by the same two calibrated dentists involved in the baseline examination. Intra-oral LED light, disposable plane front-surface mouth mirrors were used in the examinations. CPI probes to aid the diagnosis of dental caries and sharp sickle-shaped dental explorers to aid the assessment of sealant retention were used when necessary. Fissures with fully retained sealants were regarded as sound. The primary outcome was development of dentine caries in the study molars.A 10% random sample, selected one out of every 10 subjects using systematic random sampling method by the dental assistant, was re-examined during every examination (baseline and follow-up). Inter-examiner reproducibility between the two examiners in this study during baseline examination and follow-up evaluation were monitored.In sample size calculation, it was anticipated that around 90% of molars receiving resin sealant would not have dentin caries after 24 months . To deteData was input into computer and analysis was performed using SPSS 20.0 . Chi-square test was used to compare the caries incidence rates. Life-table survival analysis was used to compare the cumulative molar survival (no dentine caries developed) rates and sealant retention  rates over 24 months of the two groups. Considering the relatively short observation period and the low caries progression rate, a multi-level logistic regression analysis using generalized estimating equation (GEE) modeling which used single-time-point outcome data was also performed. Outcome was reported at the tooth level, and a two-level structure  was adopted. The dependent variable was presence of dentine caries at the 24-month examination. Independent variables included those at the child level: gender (boy/girl), age, snacking (≥2 times daily or not), toothbrushing (≥2 times daily or not); as well as variables at the tooth level: treatment , status at baseline (sound or present with signs of incipient caries), molar location (upper/lower), baseline DIAGNOdent reading (≤15/16-39) of the molar, group combination during random allocation  and period of sealant retention (0–4 times 6 months). Interaction effects between and among the independent variables were considered. Exchangeable and independent correlation structure of the clustering of molars in each child was also assessed, and the model yielding the lowest adjusted quasi-likelihood under the independence model criterion (QIC) value was selected as the final model.Among the 1203 children screened, a total of 317 children (45.7% boys) with 744 molars were recruited for this study. At baseline, a total number of 280 children (44% boys) with 383 permanent first molars were included into the two sealant groups in the study  children with 357 (93.2%) molars were followed at 24 months for the two sealant groups. There was no statistically significant difference between the two groups in the distribution of children and molars lost to follow-up. No statistically significant differences were found between the children followed at 24 months and those lost to follow-up regarding their background and oral health behaviors as well as the distribution of the molars. No complaints from the children and no adverse effect of the treatments were found during this study.The proportions of study molars with dentine caries at the 24-month examination in the resin and ART sealant groups were 3.9% (7 out of 178) and 7.3% (13 out of 179) respectively (p = 0.171). Results of the Life-table survival analysis showed that the cumulative molar survival (no dentine caries developed) rates over 24 months in the resin sealant and ART sealant groups were 96% and 93% respectively (p = 0.169). The proportions of sealants retained at the 24-month examination in the resin and ART sealant groups were 78.7% (140 out of 178) and 55.3% (99 out of 179) respectively (p < 0.001). The cumulative sealant retention  rate over 24 months was 75% for the resin sealant and 52% for the ART sealant (p < 0.001). The mean number of survival periods (in units of 6 months) were 3.4 (SD = 1.2) and 2.7 (SD = 1.5) for the resin and ART sealants, respectively (p < 0.001). Molar survival (no dentine caries developed) and sealant retention in the two groups over the 24-month period of this study is shown in Figure Regarding the design of the study, cross-over effect of fluoride on the effectiveness of sealant placement in fissure caries prevention might exist, the amount of which cannot be estimated in this study. Despite this, similar proportions of molars in the two sealant groups were exposed to a topical fluoride application method in the same mouth. In addition, both types of sealant used in this study are fluoride-releasing materials. Thus, the above mentioned factor was considered to be balanced between the two sealant groups. Such factor was considered as a confounding factor and included in the logistic regression modeling during data analysis. The results show that it did not have a significant effect on the outcome of this study. Accumulation of the caries-preventive effect may be raised when combined application of different topical fluoride regimes are provided . The fluIn this study, attrition bias due to the subjects and molars lost to follow-up was considered not a major problem because the following two reasons. Firstly, no statistically significant differences were found between the children followed at 24 months and those lost to follow-up regarding their background and oral health behaviors as well as the distribution of the molars. This indicates that the subjects and the molars lost to follow-up in this study at the 24 months probably did not differ substantially from those followed. Secondly, there was no statistically significant difference between the two groups in the distribution of children and molars lost to follow-up. The risk of detection bias was not totally avoided in this study. First of all, it is a common problem for studies comparing ART sealant with resin sealant because of the distinctly different appearance of the two types of sealants. Secondly, the two examiners placed a small number of sealants in this study after they had finished all the baseline examinations and when the other two independent dentists were not available. The latter factor is probably not too significant as the first follow-up evaluation took place 6 months after the sealant placement.Resin sealant placement in this study is regarded as the positive control because its effectiveness in fissure caries prevention has been well established . Over thThere was no statistically significant difference regarding the 24-month dentine caries incidence rates as well as the molar survival rates between the two sealant groups in this study. Multivariate two-level logistic regression analysis (GEE modeling) which can account for the effects of confounding factors and data clustering was adopted . ResultsThere is currently no systematic review specially targeted on the comparison between ART sealant and light-cured resin sealant in fissure caries prevention. A literature search yielded four comparable original studies ,30-32. OThe 24-month retention rate of the resin sealant in this study is lower than those commonly reported in other studies which are around 80% ,33. The The 24-month retention rate of ART sealant in this study  is generally lower than those reported in previous comparable studies conducted under similar field settings which ranged 50-72% over a longer 3-year period ,34-36. IAlthough a significantly lower retention rate and shorter retention time than those of the fluoride-releasing resin sealant were found for the ART sealant in this study, the effectiveness of ART sealant in fissure caries prevention did not differ significantly from that of the resin sealant used in this study. This might be explained by the findings of Beiruti et al. that high-viscosity GIC sealants had a four times higher chance of preventing caries development in re-exposed pits and fissures of occlusal surfaces in first molars than resin sealant over a 1- to 3-year period . It is aIn the final GEE modeling in this study, it was also found that presence of early caries in the occlusal surfaces of the molars before sealant placement and shorter retention of sealant on the molars significantly increased the risk of developing dentine caries in the pits and fissures. These findings are consistent with those of other studies ,6,26,38.Comparing the placement of the two types of sealants, it is noted that the number and training of dental personal required as well as the time used are similar. However, the set-up and running of an ART sealant program for children in schools will be easier than those of a resin sealant program. It is because the equipment required for ART sealant placement is rather simple (only a few hand instruments) whereas that for resin sealant placement requires an electrically powered dental unit. Given that the two methods yield similar effectiveness in fissure caries prevention, their relative affordability, availability, and simplicity should be considered when making a choice between the two. ART sealant might be more appropriate than resin sealant for use in less developed areas or in outreach dental service programs. In addition, the use of ART sealant instead of resin sealant will prevent the harmful exposure of children to additional Bisphenol A (BPA) releasing materials since systematic review has shown that BPA can be released from resin-based pit and fissure sealant . BPA exhWithin the limitation of this study, it is concluded that though the retention of fluoride-releasing resin sealant was better than that of the ART sealant, their effectiveness in preventing fissure caries in permanent molars did not differ significantly over 24 months. ART sealants could be a good alternative when and where resources for resin sealant placement are not readily available.ART: Atraumatic restorative treatment; GIC: Glass ionomer cement; SDF: Silver diammine fluoride solution; NaF: Sodium fluoride varnish.The authors declare that they have no competing interests.BL helped the carrying out of the study and prepared the manuscript. YX participated in the study’s design, performed the study as well as assisted the preparation of the manuscript. EL and CC participated in the study’s design, supervised the carrying out of this study, and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6831/14/54/prepub


Troponin I (TnI) and Myosin Binding Protein C (MyBP-C) are the prime targets in the myofilaments. TnI phosphorylation lowers myofibrillar Ca2+-sensitivity and increases the speed of Ca2+-dissociation and relaxation (lusitropic effect). Recent studies have shown that this relationship between Ca2+-sensitivity and TnI phosphorylation may be unstable. In familial cardiomyopathies, both dilated and hypertrophic (DCM and HCM), a mutation in one of the proteins of the thin filament often results in the loss of the relationship (uncoupling) and blunting of the lusitropic response. For familial dilated cardiomyopathy in thin filament proteins it has been proposed that this uncoupling is causative of the phenotype. Uncoupling has also been found in human heart tissue from patients with hypertrophic obstructive cardiomyopathy as a secondary effect. Recently, it has been found that Ca2+-sensitizing drugs can promote uncoupling, whilst one Ca2+-desensitizing drug Epigallocatechin 3-Gallate (EGCG) can reverse uncoupling. We will discuss recent findings about the role of uncoupling in the development of cardiomyopathies and the molecular mechanism of the process.Contraction in the mammalian heart is controlled by the intracellular Ca Contraction is initiated by Ca2+ release from the sarcoplasmic reticulum via the Ryanodine receptor and is terminated by Ca2+-uptake by the ATP-powered sarcoplasmic Ca2+ pump (SERCA). Ca2+ binds to troponin C (TnC), the Ca2+ receptor of the contractile apparatus to switch on contractile interactions between actin and the myosin motor protein in the thick filaments   and troponin I (TnI) within the cardiac myofibril. TnI is the inhibitory component of the trimeric troponin molecule that makes up the CaEngland, , that TnEngland,  and that2+-sensitivity by TnI phosphorylation is quite a labile system and that mutations associated with cardiomyopathies in particular, can lead to disruption of the system. This was first noted with mutations in TnI that caused hypertrophic cardiomyopathy (HCM)  it was found that in all of these DCM-causing mutations the myofilament Ca2+-sensitivity is independent of the level of TnI phosphorylation. Therefore, by analogy with the S22/23A transgenic mice, it was proposed that this uncoupling was necessary and sufficient to cause the DCM phenotype  phosphorylation levels in situ is very important. Quantitative methods such as Top-down mass spectrometry and phosphate affinity SDS-PAGE has clearly established that serines 22 and 23 are the main amino acids phosphorylated in native heart tissue in rats, mice or humans  phosphorylation and Ca2+-dependent specific chelators of phosphoproteins, when added to standard SDS-PAGE, phosphoproteins are retarded in proportion to the number of phosphates per mole of protein  generally have a low level of phosphorylation (0.1–0.4 mols Pi/mol TnI)  or increased by PKA treatment  with isolated human cardiac Tn in reconstituted thin filaments. By manipulating the level of Tn phosphorylation and then measuring the level using phosphate affinity SDS-PAGE (Phos-Tags)  phosphorylation, that phosphorylation of troponin (Tn) modulates Cas Figure   was not affected by PKA phosphorylation of cTnI  tropomyosin  and troponin T (TnT) (4 mutations recorded to date) in addition to 5 mutations in cTnC and 5 mutations in TnI. The currently known mutations causing uncoupling are summarized in Table These seminal studies on uncoupling investigated mutations in the regions of TnI and TnC that could be directly involved in the phosphorylation-dependent interaction that modulates Ca2+-sensitivity. It was found that when TnI was fully phosphorylated, the mutations had different effects on Ca2+-sensitivity of thin filaments compared to non-failing; some increased Ca2+-sensitivity , some decreased it  whereas for α-actin E361G and α-Tropomyosin E54K there was no change in Ca2+-sensitivity. This confirmed that the simple hypothesis that Ca2+-sensitivity is always reduced by DCM-causing mutations, that we and others had proposed, is no longer tenable . The HCM phenotype has been generally thought to be due to mutations increasing myofilament CaMarston, . In addiMarston, , cTnT K2Marston, , α-TropoMarston, , cardiacMarston,  and cTnIMarston,   the hypertrophied interventricular septum causes LVOTO (left ventricular outflow tract obstruction) and pressure overload. Several abnormalities in the contractile proteins in septal tissue from HOCM patients have been observed including; low phosphorylation levels of TnI and MyBP-C . Of particular interest are the Ca2+-sensitizing drugs EMD57033 and Bepridil -Epigallocatechin 3-Gallate], is a Cag Figure . Most stg Figure .2+-sensitivity of isolated cardiac myofibrils. It also blunted the PKA-dependent calcium desensitization induced by phosphorylation at Ser 22/23 and uncoupled the effects of phosphorylation from β-adrenergic stimulation  increased Ca2+ regulation is due to the interaction of a 30 amino acid N-terminal extension of TnI, containing the PKA-specific phosphorylation sites at Ser 22 and 23, with cTnC   than surrounding residues. When the Ser 22 and 23 were phosphorylated in silico, the two serines become more mobile relative to arginines 20 and 21 suggesting a weakening of their interaction with TnC. In addition, Ca2+ becomes more exposed to solvent and the interaction of the “switch peptide” with TnC is altered. Thus, coupling can be accounted for by the formation of a weak ionic complex between TnC and TnI Ser 22 and 23 that is destabilized by phosphorylation.The molecular dynamics simulations indicate a possible structure of TnI N-terminus interacting with TnC   can be found that increase Ca2+-sensitivity but do not uncouple.Whilst it is recognized that the uncoupling phenomenon provides a satisfactory molecular mechanism for thin-filament based mutations that cause DCM, the role of uncoupling in HCM is not as clear. Since CaMarston, , it is lUncoupling inevitably leads to blunting of the response to β-adrenergic agonists but the lack of response to dobutamine is of course not only due to uncoupling. Heart failure is associated also with desensitization of β-receptors, such that the activation of PKA is attenuated, or the activity of phosphatase increased whilst coupling is intact (Houser and Margulies, Patient studies from the 80 and 90 s using echocardiography showed that IDCM and HCM patients could be classified into dobutamine responders and non-responders and that the non-responders have a poor prognosis whilst the responders can respond to treatment (Borow et al., 2+-sensitivity coupling mechanism. Our recent finding that EGCG is capable of recoupling in vitro, although it has different effects in vivo (Feng et al., This dichotomy would suggest that different treatments would be optimum for the two cases. Drugs are available that impact on β-receptors but so far no drugs act positively on the TnI-phosphorylation-CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Studies of the relationship between sedentary behaviors and overweight among children and adolescents show mixed results. The fourth Thai National Health Examination Survey data collected between 2008 and 2009 were used to explore this association in 5,999 children aged 6 to 14 years. The prevalence of overweight defined by the age- and gender-specific body mass index cut-points of the International Obesity Task Force was 16%. Using multiple logistic regression, computer game use for more than 1 hour a day was found to be associated with an increased risk of overweight (adjusted odds ratio (AOR) = 1.4; 95% confidence interval: 1.02–1.93). The effect of computer game use and TV viewing on the risk for overweight was significantly pronounced among girls who spent ≤3 days/week in 60 minutes of moderate-intensity physical activity . On the contrary, these sedentary behaviors did not exert significant risk for overweight among boys. The moderating effect on risk of overweight by physical inactivity and media use should be taken into consideration in designing the interventions for overweight control in children and adolescents. Tracking societal changes is essential for identification of potential areas for targeted interventions. Childhood obesity has emerged as a significant health problem in a transitional society. Rapidly changing dietary practices and a sedentary lifestyle have led to a high prevalence of childhood overweight and obesity among school-aged children  in developing countries between 2004 and 2010: 41.8% in Mexico, 22.1% in Brazil, 13.3–22.3% in South Africa, 27.9% in Argentina, and 2.8–28% in India [Increased energy content in diet, decreased levels of physical activity, and increased sedentary lifestyles as well as a number of cultural and environmental factors have been identified as the causes of obesity in children –6. In thChanges in several social and environmental factors have been suggested as causes of the “obesity epidemic” among children, for example, reduced physical education at school, increased homework loads, school vending machines, TV, larger food portion sizes, fast-food restaurants, video games, and many others . Time spIn order to gain an understanding of the current rising prevalence of childhood overweight, we used recent data from the fourth National Health Examination Survey (NHES IV) to investigate the associations of SB  with overweight among children aged 6 to 14 years in Thailand. In addition, we examined whether the effect of screen time on overweight varied by physical activity status and gender.The National Health Examination Surveys have been conducted every 5 years since 1991. The fourth National Health Examination Survey (NHES IV) 2008-2009 conducted by the National Health Examination Survey Office was designed to represent the noninstitutionalized Thai population using a multistage stratified sampling based on 2008 Thai population registers [The NHES IV enrolled 29,485 subjects aged 1 to 60+ years. The final sample size of the 1- to 14-year-old subjects was 9,035 individuals (response rate of 92.8%). The response rate for boys and girls was 92.4% and 93.1%, respectively. This report presents an analysis of the data from the school-aged population: 5,999 children aged 6 to 14 years.Subjects were weighed in light clothes using a Tanita scale and recorded to the nearest 0.1 kg. Standing heights were measured by trained research assistants using a locally made stadiometer and recorded to the nearest 0.1 cm.Demographic and socioeconomic data were obtained by interviewing the parents. Information regarding dietary intake and physical and sedentary activities was taken by interviewing the parents of children under 10 years of age and the subjects themselves for those aged 10 years and older. The questions for media use were “How many hours a day during the past month did you watch television?” and “How many hours a day during the past month did you play computer games?” For physical activity, the question was “How many days during the past week did you have physical activity that increased your breathing rate and heart rate for at least 60 minutes?”Overweight was defined using the age- and gender-specific BMI cut-points of the International Obesity Task Force . For medThe National Ethical Review Committee for Research in Human Subjects, Ministry of Public Health, approved the study. The sampled families were informed of the data collection process and verbal permission was obtained.P < 0.001). Male subjects also engaged in moderate-intensity physical activity significantly more than the females (P < 0.001). No gender difference was noted in the frequency of having high energy snacks and family income levels.The associations of media use and physical activity with overweight are shown in The relationship between media use and overweight was further explored by a subgroup analysis according to the PA level . The effBy using the data from the recent National Health Examination Survey, we found that computer game use for more than 1 hour a day was associated with an increased risk for overweight while engagement in moderate-intensity PA significantly decreased the likelihood of overweight among the 6- to 14-year-old female subjects. Further analysis showed that the effect of computer game use and TV viewing on the risk for overweight was significant among girls, but not boys, who spent ≤3 days/week in 60 minutes of moderate-intensity PA.The association between the level of PA and weight status has been shown to be nonuniform in the recent systematic review of cross-sectional studies . Like ouThe positive association between sedentary behaviors, namely, TV viewing or playing video/computer games, and child weight status found in this study is consistent with the majority of previous studies in the recent systematic review . ResearcWe found physical activity to be a moderator of the relation between sedentary activities  and overweight. Girls who had 60 minutes of moderate-intensity PA ≤ 3 days/week were more likely to be at risk for overweight if they watched TV > 2 hours/day or played computer games > 1 hour/day than those who had such level of PA > 3 days/week. This moderating effect was not observed in boys. In a study of 9,278 Taiwanese adolescents, Yen et al. demonstrated that the relationship between increased BMI and a high level of TV viewing was found only in adolescents who exercised less than 1 hour/day, but not in those who exercised 1 hour/day or more . These fOur study has some limitations. As is the nature of surveys, one could not interpret the findings as causal. The PA and SB were collected by questionnaires. Given the publicized untoward effect of TV viewing and computer game use, the bias may be likely towards underreporting. Nevertheless, this study has its strength as a national representative study with a relatively large sample size. The findings could reflect the behavioral pattern of Thai children and a meaningful relationship of these behaviors to child overweight problem.The lives of children nowadays have been considerably affected by societal changes and media technologies. We found that computer game use for more than 1 hour a day and TV viewing for more than 2 hours a day increased the risk for overweight among girls who had a low PA level. These findings are helpful for developing effective interventions to promote healthy behaviors for the control of obesity. As computers are being used in schools and at home for educating children at a younger age, the risk of this electronic media should be communicated to the families and the public as well. Parents should be informed to discipline the use of a computer in order to control sedentary time. Tracking societal changes on lifestyles should be carried out regularly to identify potential areas for targeted interventions.


In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars  showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.An  Immunogenicity to protein based biotherapeutics is a complex process that involves numerous patient and product specific factors ,2. Monoc+-depleted PBMC, immortalized cell lines, dendritic cells/monocytes/macrophages co-cultured with autologous CD4+ T-cells, and artificial lymph node systems, to name a few -thymidine  in AIM-V culture media, and incubated for 18 h. Cultures were harvested onto filter mats  using a TomTec Mach III cell harvester (Hamden). Counts per minute (cpm) for each well were determined by scintillation counting using a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter  in paralux, low background counting. For data analysis, the stimulation index (SI) was calculated by dividing the proliferative response of the test well (cpm) by the proliferative response of the medium-only treated well (cpm) for each donor. A response was considered positive if the SI was greater than 2.0 (SI≥2.0) and statistically significant (p<0.05) as compared to medium-only treated wells using an unpaired two sample student’s t-test. The percentage of donors that responded (% donors responding) was calculated by taking the number of donors that had a positive response  over the entire time course (5–8 days) as a percentage of the total number of donors that were tested. Positive thresholds in all assays were previously established based on statistically derived cut points. Extensive assay development and previous studies have shown that this is the minimum signal to noise threshold allowing maximum sensitivity without detecting large numbers of false positive responses or omitting subtle immunogenic events.Culture plates for T-cell proliferation assays were incubated in a 5% CO6 cells/mL, and then challenged with 40 μg/mL of each sample. IL-2 Elispot Plates were incubated 8 days before developing according to the manufacturer’s instructions (R&D Systems). Dried plates were scanned on an Immunoscan Analyzer  and the number of spot forming cells (SFC) per well were determined using Immunoscan Version 4 software. The SI was calculated by dividing the number of IL-2 secreting cells in the test well (SFC) by the number of IL-2 secreting cells in the medium-only treated well (SFC) for each donor. A response was considered positive if the SI≥2.0 and used to calculate the percentage of donors that responded similar to the T-cell proliferation section.For the IL-2 Elispot readout, anti-human IL-2 Elispot plates  that were pre-wetted and coated overnight with IL-2 capture antibody  were used. PBMC were added to the IL-2 Elispot plates at a final concentration of 2–4 X 106 cells/ml to a 96-well Elispot plate and then challenged with 40 μg/mL of the original or glycated samples. The IFN-γ Elispot plates were incubated for 7 days and then developed according to the manufacturer’s instructions. Negative controls testing medium only or no cells as well as a PHA positive control were also included. The dried plates were scanned on an AID Elispot Reader version 7.0 , and SFC were determined using AID EliSpot Software v5.0. For data analysis, to highlight the potential differential response of glycated mAbs, the SI was calculated as the SFC for donors treated with glycated mAbs divided by the SFC for donors treated with the original mAbs before glycation treatment. An SI ≥ 2.0 was considered positive.For the IFN-γ Elispot readout, Elispot was performed using a kit according to the manufacturer’s instructions . Briefly, PBMC from 6 donors were added at a concentration of 1.5 X 10Multiplex cytokine analysis was performed on culture supernatants by Luminex technology using Milliplex human panel kits  and a Luminex FlexMAP 3D instrument with the XPonent version 4.0 software, as described by Joubert et al. . Cell cup values less than 0.05 were considered as statistically significant.Statistical analysis of the proliferative responses and concentration of cytokines determined by multiplex cytokine analysis was performed using a mixed effect analysis of variance model as previously described . BrieflyAggregated solutions were characterized for particle count, size distribution and morphology as described in our previous report . Aggregain vitro tool for assessing the relative risk of immunogenicity in the clinic, ten IgG1 and IgG2 biotherapeutic monoclonal antibodies, with known rates of clinical immunogenicity (in silico methods. It is important to note that the immunogenicity rates shown are based on the anti-drug antibody (ADA) responses associated with diverse disease indications and assay testing platforms with variable sensitivity. The responses of PBMC from 50 healthy naïve human donors were monitored 5 to 8 days for T-cell proliferation, the number of IL-2 secreting cells (Elispot), and the concentration of secreted cytokines (multiplex cytokine secretion).In order to appraise the IVCIA assay as an effective genicity  26,27],,27,in viblack circles) shows that the T-cell proliferative results were highly reproducible. For each mAb, the standard deviation between the percentages of donors that responded in each duplicate experiment was less than 4%. The correlation of the assay with the rate of clinical immunogenicity and the reproducibility of the results suggests that this assay is a reasonable approach for immunogenicity risk evaluation.To judge the reproducibility of the results, three representative mAbs , were tested in a duplicate experiment, on different days, and with different sets of 50 donors. in silico immunogenicity risk assessment analysis  class II alleles) showed that the non-tolerant aggretope content of FP1 and the three mutants is absent or very low (data not shown). This suggests that the risk of immunogenicity  for FP1 and the three mutants is low and relatively similar. To evaluate the risk of immunogenicity of the FP1 variants in the IVCIA assay, the responses of PBMC from 50 healthy human drug naïve donors were monitored up to 8 days for T-cell proliferation, and the number of IL-2 secreting cells. in silico analysis alone.To determine whether the IVCIA assay can be utilized to differentiate and rank order different biotherapeutic candidates during early drug product development, the response of the assay to different sequence variants of the same biotherapeutic was tested. FP1 is a biotherapeutic fusion protein of an enzyme homodimer fused to the Fc domain of a monoclonal antibody. Several sequence variants, including proteins containing either one (mutant-1 and mutant-2) or two (mutant-3) mutations in amino acid residues, or a construct lacking the Fc domain (FP1 (no Fc)) were evaluated. All of the FP1 mutants had similar secondary and tertiary structures, and similar profiles of chemically modified amino acids. However, under accelerated stress conditions, some differences in the stability and aggregation profiles were observed (data not shown), indicating that the change in amino acid sequence may have caused differences in other attributes. FP1 has not been tested in the clinic so the rate of clinical immunogenicity for this molecule is not known. However, in vitro . Fig. Fig8]. white and grey bars), although donor to donor differences could be observed (colored circles). Some donors showed slight differences in response to different lots of the same mAb , were tested. The response of the IVCIA assay to biosimilar mAbs was also tested and found to give similar responses across the population. in vitro. For example, mAb therapeutics such as Xolair and Herceptin, with low T-cell responses (≤10% of donors responded), would be ranked low on the scale for potential immunogenicity risk. Similarly, Remicade and Humira, that showed higher T-cell responses (≥10% of donors responded), will be ranked toward the right side of the scale with a higher risk for potential immunogenicity (>10% of donors responded)  . This thin vivo experiments, retrospectively assess manufacturing changes, and potentially operationalize in a manufacturing setting. Additional advantages include that these assays can: incorporate the HLA diversity of the human population, be evaluated in human donors with a known medical history, simulate potential immune cell activation, and contain a relevant mixture of immune cells without the confounding influence of other components of tissues and blood [There are multiple operational benefits to these types of cell-based assays including the ability to: screen the same donor with a large number of samples, test a variety of different sample types (i.e. highly stressed) that may not be safe to test in the clinic, achieve a production level that is not possible with nd blood ,23.in vivo, including: the route of administration and disposition of the biologic, processing of the biologic by the relevant professional antigen presenting cells, buffering of the response by the presence of other relevant cell types in physiologically relevant ratios, and the interaction with surrounding tissues and lymph nodes [in vitro may not be consistent with immunogenicity observed in the clinic [Although the IVCIA assay is useful for relative risk ranking and candidate selection, the utility of this assay is limited by several factors. It is notable that these assays lack many of the components that may contribute to an immune response ph nodes ,23. Evenph nodes ). For the clinic .in silico evaluation could not identify and rank order the variants as they are all predicted to have a similar risk of immunogenicity. A rank ordering of the FP1 candidates from highest to lowest risk (with FP1 mutant 1 > FP1 mutant 2 > FP1 mutant 3) was achieved by the IVCIA assay, confirming that minor mutations in the sequence, that were not differentiated by in silico analysis alone, could be detected by the T-cell functional assay. Although the different FP1 mutants did show differences in a few additional attributes (including the aggregation profile) which might impact the response in the IVCIA assay, the assay is capable of detecting both differences in sequence and differences in attributes that result from changes in the sequence. The sensitivity of T-cells in vitro to minor changes in sequence in viral antigens has been shown by others [The application of the IVCIA assay to the development of biopharmaceuticals can range from candidate selection at the early development phase to the late stage evaluation of specific attributes that may impact the risk of immunogenicity. y others . Howeverin vitro and induce weak and transient responses in vivo [During later stage development, the potential risk of immunogenicity due to attributes that can vary due to the manufacturing process  ,33–37 be in vivo ,21,22,42Herceptin, Xolair, Erbitux, and Avastin samples containing high levels of aggregates induced the highest response in the assay, although the mAbs before aggregation were not strong elicitors of a T-cell functional response. Furthermore, mAbs that caused a higher T-cell functional response, such as Remicade, mAb3 and Humira, were only slightly more immunogenic when stress treated. The lower response could be attributed to the more immunogenic nature of these molecules as observed by their higher T-cell functional responses before stress treatment (>10% donors responded). The response to aggregates could be detected as early as 20 hours post-stimulation as shown by increased levels of monocyte-specific cytokines , indicatOverall, this indicates that the relative response to aggregates in the assay does not correlate with the rate of clinical immunogenicity of the unstressed molecule. Aggregates may possess unique structural features or 3-dimensional epitopes that can lead to B-cell or T-cell activation (perhaps through enhanced uptake and subsequent activation of antigen presenting cells) in a manner that is not dependent on the T-cell epitope content of the linear amino acid sequence. In addition, two of the aggregated samples that induced the highest response (Herceptin and Avastin) did not have the highest number of particles present (Avastin was one of the lowest) suggesting that additional aggregate attributes (besides aggregate number) are contributing to the immune response observed. The assay also demonstrated the target mediated effect of Campath on T-cells. Campath, both before and after aggregation, showed no response in the assay, suggesting that the monomeric (or monomer fraction of the stirred sample that was not aggregated upon stress treatment) and/or aggregated Campath retained its ability to bind to the target in the assay. It is important to note that all of these aggregated samples are heavily aggregated and have particle numbers that are orders of magnitude above what would be in a marketed product. Future experiments that evaluate the threshold of aggregate activation in the IVCIA assay will be key to establishing the sensitivity of the assay. Further correlation between the threshold of activation in the IVCIA assay to the threshold of response in humans will be necessary for setting biologically relevant specifications during product manufacturing.in vivo [in vitro.Glycation is the non-enzymatic addition of sugars to proteins and occurs on lysine side chains and the N-terminal amine. Glucose is traditionally used as a carbon source during mammalian cell culture expression, although other sugars could be used. Glycation levels of antibody biotherapeutics are product quality attributes that can vary during the expression and manufacturing of the drug product, or after administration to patients in vivo ,43. Althin vivo –46, the The suitability of the IVCIA assay for comparing various lots of the same mAb, from both the same manufacturer and from different manufacturers (biosimilars), was demonstrated. The different lots that were compared had the same sequence but contained varying levels of certain product quality attributes. For example, Humira lots varied in several characteristics including: some N-linked glycan species  and number of sub-visible particles in the 2–10 μm and greater than 10 μm size ranges (data not shown). It is important to note that Humira is administered in a pre-filled syringe so particle numbers showed high variability between lots and are mostly silicone oil droplets (>98%). As another example, Erbitux lots also showed variability in some N-linked glycan species  and % high molecular weight by size exclusion chromatography (data not shown). The IVCIA assay showed a similar average profile across the population to different lots of the same mAb, although individual donor to donor differences could be observed. The IVCIA assay may therefore be an important comparability technique for showing both high-level similarity and detecting minor attribute-related differences.In conclusion, the IVCIA assay is an informative cell-based system for relative immunogenicity risk assessment, and has multiple applications from early development through the lifespan of the drug product.S1 FigThe HLA-DR allotypes of one representative set of 50 donors that was used in the assay is shown. Donors were selected to best represent the number and frequency of HLA-DR allotypes expressed in the world population.(PDF)Click here for additional data file.S2 FigPBMC from 50 human donors were challenged with the original and aggregated forms of several biotherapeutic mAbs. Multiplex cytokine analysis was performed to evaluate the level of secretion of signature cytokines after A) the early phase (20 h) and B) the late phase (7 days) post challenge. The average SI of positive donors at the early phase  or late phase  and percentage of donors that responded  to the aggregated mAb above the original mAb is shown. Representative cytokines that displayed the strongest responses are shown. Asterisks (*) highlight statistically significant differences (p<0.05). Black circles depict responding individuals and highlight the distribution of responses across the population tested.(PDF)Click here for additional data file.S3 FigDonors that were positive for T-cell proliferation in the IVCIA assay over the entire study (5–8 days) in response to A) the original mAbs or B) aggregated mAbs at the late phase were evaluated by multiplex cytokine analysis for the secretion of IL-10 on Day 7 (n = 50 donors). Not all donors were tested for IL-10 for some samples (grey circles). The percentage of donors that responded positively in the proliferation assay (purple bars) and the percentage of donors that responded positively for both proliferation and the secretion of IL-10 (green bars) are shown. A response was considered positive if the SI ≥ 2.0 (p<0.05) for proliferation or the SI ≥ 1.9 for IL-10 concentration (above the background response). The asterisk indicates that borderline T-cell responses were included (SI ≥ 1.9) in some cases. The scale bars at the top of each graph show the relative rate of clinical immunogenicity taken from the product label see . All rat(PDF)Click here for additional data file.