Recent studies suggest de novo mutations may involve the pathogenesis of autism and attention-deficit/hyperactivity disorder (ADHD). Based on the evidence that excessive alcohol consumption may be associated with an increased rate of de novo mutations in germ cells (sperms or eggs), we examine here whether the risks of autism and ADHD are increased among individuals with a family history of alcohol use disorders (AUDs). The standardized incidence ratios (SIRs) of autism and ADHD among individuals with a biological parental history of AUDs were 1.39 (95% CI 1.34–1.44) and 2.19 (95% CI 2.15–2.23), respectively, compared to individuals without an affected parent. Among offspring whose parents were diagnosed with AUDs before their birth, the corresponding risks were 1.46 (95% CI 1.36–1.58) and 2.70 (95% CI 2.59–2.81), respectively. Our study calls for extra surveillance for children with a family history of AUDs, and further studies examining the underlying mechanisms are needed.DOI:http://dx.doi.org/10.7554/eLife.02917.001 Children learn to talk, manage their emotions, and control their behavior in a period when the brain is developing rapidly. The first signs of several developmental disorders, such as autism and attention-deficit/hyperactivity disorder (ADHD), may also emerge during this period. Children with autism may have difficulties with social interactions and communication, while those with attention-deficit/hyperactivity disorder may struggle to pay attention to a task and may be more active than other children.Autism or ADHD are diagnosed based on the child's behavior because the underlying causes of the disorders are not well understood. Both genes and the environment have been linked to the conditions; and it was recently suggested that certain common genetic mutations are more common in children with ADHD or autism. However, as some of the mutations linked to autism are not found in the genes of the affected children's parents, it is likely that they occurred in either of the sperm or the egg cell from the parents.Exposure to harmful substances in the environment can cause mutations in egg or sperm cells, or alter the expression of genes without changing the gene sequence. Excessive alcohol consumption is one environmental factor that can mutate genes or alter gene expression. Here, Sundquist et al. have looked to see if there is a relationship between a child having a parent with an alcohol use problem and the child's risk of developing autism or ADHD.Examining national medical registries identified 24,157 people with autism and 49,348 with ADHD in Sweden between 1987 and 2010. Sundquist et al. discovered that autism and ADHD were more common in individuals who had a parent with a history of an alcohol use disorder than in those whose parents had no history of an alcohol use disorder. There was also an even greater risk of either condition if the parent had been diagnosed with an alcohol use problem before the birth of the child.Adopted children who had a biological parent with an alcohol use disorder were at a greater risk of autism and ADHD than those whose adoptive parent had an alcohol use disorder. However, as very few adopted parents were diagnosed with an alcohol use problem, it is important to be cautious about drawing firm conclusions from this observation.Sundquist et al. estimate that around 4% of autism cases and 11% of ADHD cases could be avoided if parents abstained from heavy alcohol consumption. Though these findings are consistent with parents with an alcohol use disorder being more likely to pass on mutations to their children, there are also other possible explanations. As such, further research examining the underlying cause is still needed.DOI:http://dx.doi.org/10.7554/eLife.02917.002 Autism is a neural development disorder characterized by impaired social interaction and verbal and non-verbal communication . AttentiIn The risks of autism and ADHD among individuals with a family history of AUDs are presented in To examine whether the observed association in In In this population-based cohort study from Sweden of 24,157 and 49,348 individuals with autism and ADHD, respectively, we found that the risks were significantly increased among individuals with a family history of AUDs, after adjusting for potential confounding factors. The incidence rates of autism and ADHD were somewhat more pronounced when the diagnosis of AUDs in parents occurred before the birth of their offspring. The time-relationship supports that the observed association between parental AUDs and autism and ADHD in offspring may be causal. In addition, we studied the incidence among adoptees to disentangle the contribution by environmental factors. However, we could not draw definite conclusions from these results, as the cases were very few among adoptees with an affected adoptive parent. The PAF was 3.6 and 11.0% for autism and ADHD, respectively, if excess alcohol consumption could be avoided in their parents.There are a few possible biological explanations for the high observed risks of autism and ADHD among individuals with a family history of AUDs. Firstly, they could be explained by an increased rate of de novo mutations among individuals with AUDs, confirming our hypothesis. It should be noted that although a few de novo mutations have been found to be related to autism , associaOne possible reason for the high risks of autism and ADHD among offspring with mothers with AUDs is that mothers with AUDs may continue drinking alcohol during pregnancy, causing long-term structural, behavioral, and cognitive damage to their children. It is known that alcohol can be carried to the placenta and that the concentration of alcohol in the unborn baby's bloodstream is the same as that in the mother. Mothers who consume alcohol during pregnancy may give birth to a baby with fetal alcohol syndrome (FAS). In general, the more severe the mother's drinking problem is during pregnancy, the more severe are the symptoms of FAS in infants . ChildreAround 4% of autism and 11% of ADHD in offspring can be avoided if parents avoid excessive alcohol consumption. It should be noted that even moderate alcohol consumption during pregnancy may affect the child's cognitive score , suggestAn important strength of this study is that all the data were retrieved from Swedish registers with high quality and coverage. In addition, the number of patients included is large enough to guarantee reliable risk estimates. The prospective study design and the completeness of the follow-up of patients are other major advantages of the present study. Moreover, we adjusted for a number of confounding factors and thereby minimized confounding.One limitation of this study is that only individuals with autism and ADHD who visited either primary health care or a hospital were included. Another limitation is the lack of information on some individual-level risk factors, such as parental drinking habits of moderate nature, psychosocial factors, and sociocultural behaviors. In addition, it is likely that the identification of AUD in parents could be underreported.In summary, individuals with a family history of AUDs had increased risks of autism and ADHD, which calls for further study to examine the underlying mechanisms and for clinical attention and extra surveillance for children with a family history of AUDs.This study was approved by the Ethics Committee at Lund University, Sweden. Individuals with autism and ADHD were identified from four Swedish Registers: the primary health care registers covering the counties of Skåne (1987–2010) and Stockholm (2001–2007) and the National Swedish Hospital Discharge Register (1987–2010) and Outpatient Register (2001–2010). The primary health care register in Skåne (the southernmost province of Sweden), PASIS, contains data on individuals (around 1.2 million) who visited primary health care in Skåne between 1987 and 2010. The primary health care register in Stockholm covers 75 primary health care centers for the period 2001–2007. The Swedish Hospital Discharge Register was founded in 1964–65 by the National Board of Health and Welfare, and has had complete nationwide coverage since 1987. The Outpatient Register contains data on all visits to outpatient clinics in Sweden since 2001. A recent external review suggested that the overall accuracy of the Swedish Hospital Registry is approximately 85–95% . IndividWe further linked individuals with autism and ADHD to the Swedish Multi-Generation Register to identify their parents . The SweAdditional linkages were made to the Statistics Sweden's Total Population Register (≥1990) and the Population a Housing Census (<1990) to obtain information on individual-level characteristics, such as year of birth, sex, and country of birth; to the Cause of Death Register to identify date of death; and to the Emigration Registry to identify date of emigration. All linkages were performed using individual national identification numbers, which were replaced with serial numbers in order to preserve anonymity.We calculated person-years at risk of autism and ADHD among individuals with a family history of AUDs  from the date of birth, immigration, or 1 January 1987, whichever came last, until the date of diagnosis of autism or ADHD, death, emigration, or the end of the study period (31 December 2010), whichever came first. Standardized incidence ratios (SIRs) were calculated as the ratio of the observed and expected numbers of cases . The exp review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and 2 reviewers.Thank you for sending your work entitled “Autism and Attention-Deficit/Hyperactivity Disorder among Individuals with a Family History of Alcohol Use Disorders” for consideration at The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.Your paper presents intriguing data on the association between a diagnosis of alcoholism in at least one of the parents and autism or ADHD in the offspring. The following points must be addressed in your revision. Although all six items are important we consider concern #1 below as absolutely critical. You must convince us that the preponderance of the evidence is in line with your hypothesis, i.e., that the putative germ line mutations induced by alcohol occurred before the birth of the child. Our decision to publish will primarily hinge on this demonstration.1) The most important concern we have is that you may have misinterpreted the directionality of the causal relation. How do you know that the putative germ cell mutations that you attribute to heavy and chronic alcohol consumption in the parents occurred before the conception of the affected offspring? You must carry out analyses and add evidence to demonstrate that the diagnosis of alcoholism  occurred before the birth of the child. This is the only analytical framework that can bolster your underlying hypothesis. Having a child affected by autism or ADHD can lead parents to seek emotional relief in alcohol drinking, which would also create a statistical association. Therefore, it is paramount that you document the temporality of the association.2) Although the attempt to distinguish between risks in biological and adoptive parents is commendable much of your reasoning hinges on the lack of significance among the latter to advance the notion of a causal mechanism. With at most nine parents for ADHD and one father for autism the analysis for cases among adoptive parents is imprecise, and thus meaningless. You must refrain from making sweeping statements about the differences between findings in biological and adoptive parents as being in support of your hypothesis.3) The argument that the effect is greater for maternal than paternal alcoholism calls for a specific test for effect modification for the data summarized in 4) 5)  1) The most important concern we have is that you may have misinterpreted the directionality of the causal relation. How do you know that the putative germ cell mutations that you attribute to heavy and chronic alcohol consumption in the parents occurred before the conception of the affected offspring? You must carry out analyses and add evidence to demonstrate that the diagnosis of alcoholism  occurred before the birth of the child. This is the only analytical framework that can bolster your underlying hypothesis. Having a child affected by autism or ADHD can lead parents to seek emotional relief in alcohol drinking, which would also create a statistical association. Therefore, it is paramount that you document the temporality of the association.This is a good point. We have now also calculated the risk of autism and ADHD in offspring when their parents were diagnosed with alcohol use disorder (AUDs) before the birth of the offspring, and the results are shown in 2) Although the attempt to distinguish between risks in biological and adoptive parents is commendable much of your reasoning hinges on the lack of significance among the latter to advance the notion of a causal mechanism. With at most nine parents for ADHD and one father for autism the analysis for cases among adoptive parents is imprecise, and thus meaningless. You must refrain from making sweeping statements about the differences between findings in biological and adoptive parents as being in support of your hypothesis.Thanks for this comment. We have modified the Discussion section to avoid any imprecise conclusions.3) The argument that the effect is greater for maternal than paternal alcoholism calls for a specific test for effect modification for the data summarized inWe agree with this comment. However, the differences in offspring risk between maternal and paternal alcoholism are rather small and probably not clinically meaningful. We have therefore omitted the argument that the effect is greater for maternal as opposed to paternal alcoholism.4)should be more explicit and indicate in additional columns the number of persons at risk and hopefully the person-time accumulated in each category.This is a good point. We have included the number of individuals at risk and the person–years of follow-up in these tables.5)needs to be better explained. It seems that your study did not confirm the expected trend of increased autism risk with advanced paternal age.This is correct. We could not confirm an increased risk of autism with advanced paternal age, and we have deleted this table because our study focused on the risk of autisms and ADHD among offspring with a family history of AUDs rather than the potential effect of advanced parental age on autism risk.


The 22q11.2DS group showed a higher rate of ADHD inattentive subtype , and fewer hyperactive‐impulsive symptoms compared to the clinical group . The 22q11.2DS ADHD group parents reported fewer oppositional defiant disorder/conduct disorder symptoms  and a higher rate of generalized anxiety disorder  in relation to the clinical group. Two percent of the 22q11.2 DS ADHD sample had received ADHD treatment. The results were similar when the 22q11.2 ADHD group was compared to the population cohort ADHD group. Conclusions: The clinical presentation of ADHD and patterns of co‐morbidity in 22q11.2DS is different from that in idiopathic ADHD. This could lead to clinical under‐recognition of ADHD in this group. Examining psychopathology in 22q11.2DS can provide insights into the genetic origins of psychiatric problems with implications beyond the 22q11.2DS population. © 2015 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics Published by Wiley Periodicals, Inc.Background: Although attention deficit‐hyperactivity disorder (ADHD) is the most prevalent psychiatric disorder in children with 22q11.2DS, it remains unclear whether its clinical presentation is similar to that in children with idiopathic ADHD. The aim of this study is to compare the ADHD phenotype in children with and without 22q11.2DS by examining ADHD symptom scores, patterns of psychiatric comorbidity, IQ and gender distribution. Methods: Forty‐four children with 22q11.2DS and ADHD (mean age = 9.6), 600 clinic children (mean age = 10.8) and 77 children with ADHD from a population cohort (mean age = 10.8) participated in the study. Psychopathology was assessed using parent‐report research diagnostic instruments. Results: There was a higher proportion of females in the 22q11.2DS ADHD sample in relation to the clinical sample (χ As a supplementary analysis, we further compared the 22q11.2DS and ADHD group with a sample of children with ADHD identified from a large population cohort (population‐based ADHD).http://medicine.cardiff.ac.uk/psychological-medicine-neuroscience/areas-research/copy-number-variant-research/research-projects/). ECHO participants are referred from 11 genetics clinics within the UK, as well as a number of charities and also through word of mouth. To date, 113 children with 22q11.2DS have taken part in the ECHO study. For the purpose of the present study, children with 22q11.2DS meeting a DSM‐IV research diagnosis of ADHD , after conducting a research diagnostic interview (see below), were included. The mean age of this sample was 9.61(SD = 2.1), range: 6–14 years. Only one child was receiving ADHD treatment in form of stimulant medication. Presence of the deletion was confirmed for all children with 22q11.2DS by the relevant Medical Genetics laboratories. The study was approved by the appropriate research National Health System (NHS) ethics and R&D committees. The carers of the children provided informed written consent to participate prior to recruitment and assent/consent was also obtained from the children.Participants were drawn from three different studies. Children with 22q11.2DS and ADHD were selected from an ongoing study of children with copy number variations   were recruited from child psychiatry and pediatric outpatient clinics for a genome‐wide association and CNV study of ADHD [see Williams et al., http://www.bris.ac.uk/alspac/researchers/data-access/data-dictionary/).Seventy‐seven received a research diagnosis of DSM‐IV ADHD (mean age: 10.8 (0.2), range: 10–12) (population‐based ADHD). These were recruited for the Avon Longitudinal Study of Parents and Children (ALSPAC). ALSPAC started in 1991–1992, when all pregnant women residing in the southwest of England were recruited. The initial ALSPAC cohort consisted of 14,062 live births and 13,988 infants still alive at 12 months [Boyd et al., The parent version of the Child and Adolescent Psychiatric Assessment (CAPA) [Angold et al., The parent Development and Well‐Being Assessment (DAWBA) [Goodman et al., Taking into account evidence showing that questionnaires may over‐estimate prevalence of psychopathology  [Wechsler, Data analysis was conducted using Stata (version 13) for Windows 7. To compare the mean differences in age, IQ and symptom counts between the two groups, t‐tests were used or in the case of non‐normally distributed variables, Mann–Whitney U tests were used. Chi‐square tests were used to compare the rates of psychopathology between the groups. The Wilcoxon matched‐pairs signed‐rank test was used to examine whether the ADHD phenotype between the 22q11.2DS and the clinical ADHD sample was similar after matching for IQ, sex, and age.Table P = 0.01) and there was a higher proportion of males in the clinical than in the 22q11.2DS sample (P < 0.001). There were no IQ differences between the 22q11.2DS ADHD and the clinical ADHD samples (P = 0.06).There were age and gender differences between the 22q11.2DS ADHD and the clinical ADHD samples (see Table P < 0.001). Furthermore, the 22q11.2DS ADHD group had a lower rate of the ADHD combined subtype (30%) in relation to the clinical ADHD group (82%) (P < 0.001). With regard to ADHD symptoms, the two groups were similar in terms of inattention symptoms (P = 0.07). However, the clinical ADHD sample had more hyperactive‐impulsive (P < 0.001) and total symptoms (P < 0.001) than the 22q11.2DS ADHD group  compared to the clinical ADHD (8%) (P = 0.03). Rates of ODD, separation anxiety disorder (SAD), MDD diagnoses, and tic disorder were similar between the groups. However, the clinical ADHD sample had a higher rate of CD diagnosis compared to the 22q11.2DS ADHD sample (P < 0.001).The 22q11.2DS ADHD sample had higher GAD rates than the clinical ADHD sample (P < 0.001), while there were no differences in the rate of tic disorder or in symptom counts of depression, worries, and sleep problems.With regard to ODD/ CD symptoms, the clinical ADHD sample showed higher levels compared to the 22q11.2DS ADHD sample   to evaluate whether intellectual disability IQ explained these findings. The results were found to be broadly similar to those based on the full samples Table .Analyses based on males only  also yielded comparable results Table .P = 0.04).We matched the 22q11.2DS ADHD sample and the clinical ADHD sample on age, IQ, and gender and repeated the analyses. The results remained similar Table , with thFinally, we repeated the analyses by comparing the 22q11.2DS ADHD group with a population‐based ADHD group and the results were also similar Table  with theThe results of this study suggest that the ADHD phenotype is different in children with 22q11.2DS compared to children with ADHD recruited through child psychiatry and pediatric outpatient clinics or identified from a population cohort. To our knowledge, our study is the first to report that children with 22q11.2DS and ADHD show fewer ODD/CD symptoms when compared to children with idiopathic ADHD and children from a population‐based study. Moreover, our study indicated that children with 22q11.2DS and ADHD show a lower rate of CD diagnoses and a higher rate of GAD diagnoses when compared to children with idiopathic ADHD. Another noticeable difference in ADHD subtypes was also present. The majority of children with 22q11.2DS met criteria for the DSM‐IV inattentive ADHD subtype, whereas the majority of children in the other two samples had the DSM‐IV combined subtype. This appears to be because while they endorsed a similar number of inattention symptoms, they endorsed fewer hyperactive and impulsive symptoms than the clinical and population cohort of children with ADHD. This is in agreement with a previous study [Antshel et al., Studies comparing psychiatric comorbidities in populations with ADHD have indicated that children with the inattentive ADHD subtype show more anxiety/depression symptoms in comparison to children with the hyperactive ADHD subtype, who tend to show more conduct problems . Psychiatric comorbidity has been associated with more adverse outcomes which may be due to the combined effects of the disorders [DuPaul et al., This is the largest study to date to compare children with 22q11.2DS and a large sample of children with ADHD from clinical settings. Nevertheless, differences in methodologies and ascertainment strategies need to be kept in mind when interpreting the findings. The children in the longitudinal population cohort had already been participants for 10 years at the time the ADHD and other assessments used in this study were conducted and attrition bias may have influenced the findings. Moreover, referral bias could also apply to both the 22q11.2DS sample (given that people from a higher socio‐economic background are more likely to be tested in genetics clinics) and the clinical ADHD group . This could also explain the low rates of inattentive subtype among children in the clinical cohort. Ideally, our findings should have been based on reports from multiple sources; however, obtaining teacher reports was outside the remit of the 22q11.2 DS study. Another limitation is that we did not examine autism‐spectrum disorder, although this is reported to frequently co‐occur with ADHD in 22q11.2DS [Niklasson et al., The ADHD phenotype in 22q11.2DS is different in comparison to the ADHD phenotype of clinical and population cohort samples. Children with 22q11.2DS and ADHD showed fewer ODD/CD symptoms, lower rates of CD diagnoses, and higher rates of GAD diagnoses. The study of psychopathology in 22q11.2DS and in other CNVs that have been recently implicated in risk of ADHD [Williams et al.,


N = 18,232 youths 5–15 years old) included semi-structured interview DSM-IV diagnoses and parent-rated SDQ scores. Areas under the curve for SDQ subscales were good (0.81) to excellent (0.96) across sex and age groups. Hyperactivity/inattention scale scores of 10+ increased odds of ADHD by 21.3×. For discriminating ADHD from other diagnoses, accuracy was fair (<0.70) to good (0.88); Hyperactivity/inattention scale scores of 10+ increased odds of ADHD by 4.47×. The SDQ is free, easy to score, and provides clinically meaningful changes in odds of ADHD that can guide clinical decision-making in an evidence-based medicine framework.Early, accurate identification of ADHD would improve outcomes while avoiding unnecessary medication exposure for non-ADHD youths, but is challenging, especially in primary care. The aim of this paper is to test the Strengths and Difficulties Questionnaire (SDQ) using a nationally representative sample to develop scoring weights for clinical use. The British Child and Adolescent Mental Health Survey ( Attention-deficit/hyperactivity disorder (ADHD) affects 3 % of boys and 1 % of girls in the United Kingdom –4. It isThe overarching goal of this study is to evaluate how the SDQ for Parents could help in the clinical identification of ADHD conceptualized as a discrete category, differentiating it from other sources of externalizing behavior. To do this, we evaluated how the SDQ  performsThe current study used the data from The British Child and Adolescent Mental Health Survey 1999 , which wTrained child and adolescent psychiatrists reviewed both the verbatim accounts and the answers to the Development and Well-Being Assessment   before aParents, teachers, and eligible 11–15-year-old children were invited to complete the SDQ , a 25 itThe DAWBA is a widely used semi-structured interview that involves child and parent interviews alongside a teacher questionnaire. The child/parent interviews and teacher questionnaires assess current and recent past psychiatric symptoms and their impact on functioning in children. The DAWBA is based on diagnostic criteria (ICD-10 and DSM-IV) and focuses on anxiety disorders, depressive disorders, ADHD and conduct disorders. A clinical diagnostic rating is informed by triangulation of these three sources.The validity of the clinical diagnoses derived from the DAWBA have been demonstrated by concordance with case note screening in a clinical sample of children aged 11–15 years , and witThe 25-item SDQ generates scores for five subscales confirmed through factor analysis . The parNonparametric estimates of the area under the curve (AUC) from receiver operating characteristic (ROC) analyses quantified the diagnostic efficiency of the SDQ H/I, CD and TD subscale scores. A rough guideline for evaluating AUC values is: <0.70 poor, 0.70–0.79 fair; 0.80–0.89 good; and 0.90–1.00 excellent , althougAUCs were calculated for the target condition of any ADHD using SDQ subscale scores, to evaluate whether the TD or subscales scores (H/I and CD) were better able to discriminate youth with any ADHD disorder from other youth in the sample. Venkatraman’s permutation test compared ROC curves . ModeratFinally, we calculated diagnostic likelihood ratios (DLRs) for optimal cut-points yielding the best balance between sensitivity and specificity from the ROC curves . DLRs baAll analyses were done using SPSS-Version 22.0 and pROC package in R .n = 264) as well subgroups characterized by inattention (n = 110) and by hyperactivity (n = 35). Mean age and family size did not differ significantly across the groups. Half of the non-ADHD group was male, whereas this was significantly higher in each of the ADHD groups, comprising over 2/3 of the sample for each. Relative to the non-ADHD group, all three ADHD groups  had a significantly higher percentage of white children, single parent family background, parental unemployment and mothers with no educational qualifications. The ADHD groups had also experienced three or more life events in the past year. For clinical variables, the ADHD groups reported poorer child and parent health and family functioning, as well as higher rates of neurodevelopmental problems.Table The AUCs for hyperactivity/inattention, conduct problems and total difficulties scales from the SDQ ranged from good (0.81) to excellent (0.96) in male and female subsamples and at different age ranges Table a. Based p > 0.05) for the H/I subscale, supporting the use of a single set of cutoff scores for the entire sample.No significant AUC differences were found between gender and age groups (A score of 5+ (from a possible range of 0–10) on the H/I subscale had a DLR of 2.3, and a score of 10 yielded a DLR of 21.3, reflecting a large increase in the post-test probability of any ADHD in this national community sample Table .Table 4Dn = 685 with any emotional diagnosis, n = 608 with any anxiety diagnosis, n = 136 with less common psychiatric diagnosis, etc.). These evaluated the ability of the SDQ to discriminate “which diagnosis” instead of a general “sick versus well” comparison. The AUCs for H/I, CP and TD scales ranged from poor (<0.70) to good (0.88)  to combine the information yields a precise estimate of 57 %, and using the probability nomogram recommended in EBM provides a close, quick approximation  in the H/I subscale produced a DLR of 1.8, and a score of 10 with a DLR of 4.47  to apply Bayes’ Theorem.As an example, using McGee’s mnemonic , a likeln = 35) limited our ability to test SDQ performance between ADHD subtypes. Although SDQ Teacher data were gathered, teacher ratings were used as a piece of evidence in establishing the formal diagnosis using the DAWBA; thus there would be criterion contamination that would exaggerate the apparent accuracy of teacher report because it contributed to both the predictor and the criterion [Though the SDQ has clear clinical utility, the subsamples of youth with ADHD subtypes diagnosis (particularly hyperactivity, riterion . Future riterion , 43. In riterion .Finally, ADHD can be conceptualized either as a source of group differences or as a constructivist variable. We note that even a constructivist definition of ADHD also has issues of reliability and measurement error. For example, patients could misread a checklist, or misconstrue the nature of the item. Clinicians frequently interpret the same responses differently—multiple studies have shown that even when presented with videotaped interviews  or vigneThe SDQ is a free, easy-to-use measure that has demonstrated utility as an ADHD disorder screening measure in community, and between youth experiencing mental health problems in the community in the UK. Current results suggest that elevated scores on the subscales of the SDQ increase the likelihood that an individual meets criteria for any ADHD, by a factor of more than 20 compared to healthy peers, and by a factor of 4.5 compared to other youths with commonly diagnosed mental health issues (as reflected by DLR in Table


We theorise that in some cases Attention Deficit Hyperactivity Disorder (ADHD) predisposes to narcolepsy and hypersomnia, and that there may be a shared pathophysiology with various addictions [Reward Deficiency Syndrome (RDS)]. Reticence to acknowledge such connections may be due to a narrow nosological framework. Additionally, we theorise that the development of narcolepsy on a baseline of ADHD/RDS leads to an additional assault on the dopaminergic reward system in such individuals. In this study, we propose to test these hypotheses by using a combination of broad genetic screening, and neuroimaging with and without pharmacological intervention, in those with pure ADHD, pure narcolepsy, and the combined ADHD-narcolepsy phenotype. Results of this proposed study may reveal a common pathophysiology of ADHD, narcolepsy and RDS, and perhaps an additional compromise to the reward system in those with combined ADHD-narcolepsy. If the evidence supports the hypothesis that indeed there is a shared pathophysiology for narcolepsy with RDS and thus its subtype ADHD, early intervention/preventative treatment amongst those with ADHD may be beneficial with the putative dopaminergic compound KB220Z™. Primary Disorder of Vigilance. Sultan and associates  that may share a common pathophysiology and perhaps genes suggestive of pleiotropy. This cluster of disorders was titled Affective Spectrum Disorder (ASD), and interestingly includes both ADHD and narcolepsy , 11. Folvia receptors on dopaminergic neurons, and indirectly by augmenting glutamatergic excitability of dopaminergic neurons via increasing NMDA receptor number) to this cascade. This 1b. pathway is associated with narcolepsy.In this same vein of nosological integration, there is Reward Deficiency Syndrome (RDS). RDS is a cluster of syndromes across addictive, compulsive, and impulsive spectrums  where there is a dysregulation of dopamine that makes an impact on the reward system. Remarkably, through innervation and various neural cascades, RDS encompasses various pathologies across multiple neurotransmitter systems, which ultimately lead to a dysregulation of this same dopaminergic reward system -17. For Taq A1 allele variant of the D2 dopamine receptor, in addition to genes for COMT, GABA, serotonin, etc. [Despite the tight linkage with HLA-DQB1*0602, narcolepsy appears to be polygenic, and thus associated with the interaction of various genes and environmental factors , 20. Onein, etc. -17. Intein, etc. , and RDoin, etc. , 14, we in, etc. , to furtin, etc. , 1, 6, min, etc.  in thoseKB220Z™ is a complex that has been extensively studied in pre-clinical and human trials . Note: tPrior to commencing the study, all research will be approved by the local ethics committee/institutional review boards in accordance with the ethical standards of the 1964 Declaration of Helsinki. Furthermore, informed consent will be obtained from all participants. Diagnoses of ADHD, narcolepsy, and the combined phenotype will be confirmed by a clinical interview with a licensed clinician, in addition to medical records.


Children with early symptomatic psychiatric disorders such as Attention-Deficit/Hyperactivity Disorder (ADHD) and Autism Spectrum Disorder (ASD) have been found to have high rates of motor and/or perception difficulties. However, there have been few large-scale studies reporting on the association between Conduct Disorder (CD) and motor/perception functions. The aim of the present study was to investigate how motor function and perception relate to measures of ADHD, ASD, and CD.categorical variables of scoring above previously validated cut-off values for diagnosing ADHD, ASD, and CD on the one hand and motor and/or perception problems on the other hand were analysed using cross-tabulations, and the Fisher exact test. Associations between the continuous scores for ADHD, ASD, CD, and the subdomains Concentration/Attention, Impulsiveness/Activity, Flexibility, Social Interaction and Language, and the categorical factors age and gender, on the one hand, and the dependent dichotomic variables Motor control and Perception problems, on the other hand, were analysed using binary logistic regression in general estimated equation models.Parents of 16,994 Swedish twins (ages nine and twelve years) were interviewed using the Autism-Tics, ADHD and other Comorbidities inventory (A-TAC), which has been validated as a screening instrument for early onset child psychiatric disorders and symptoms. Associations between Male gender was associated with increased risk of Motor control and/or Perception problems. Children scoring above the cut-off for ADHD, ASD, and/or CD, but not those who were ‘CD positive’ but ‘ADHD/ASD negative’, had more Motor control and/or Perception problems, compared with children who were screen-negative for all three diagnoses. In the multivariable model, CD and Impulsiveness/Activity had no positive associations with Motor control and/or Perception problems.CD symptoms or problems with Impulsiveness/Activity were associated with Motor control or Perception problems only in the presence of ASD symptoms and/or symptoms of inattention. Our results indicate that children with CD but without ASD or inattention do not show a deviant development of motor and perceptual functions. Therefore, all children with CD should be examined concerning motor control and perception. If problems are present, a suspicion of ADHD and/or ASD should be raised. Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV)[Children with Autism Spectrum Disorders (ASD) and Attention-Deficit/Hyperactivity Disorder (ADHD) have been shown to have high rates of motor and perception difficulties in controlled studies-4. Befor(DSM-IV). DSM-IV et al.[Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5)[et al.[In the 1980s, Gillberg and Gillberg. The diaet al. have shoet al.. In the  (DSM-5), sensory[et al. describeet al.[Conduct Disorder (CD) is very common among children with ADHD and not et al. have shoet al.,24.et al.[et al. showed that individuals with persistent antisocial behaviour had a higher frequency of problems with spatial ability assessed at three-years of age compared to controls and individuals with non-persistent antisocial behaviour, and they made a hypothesis that early visuospatial (right hemisphere) impairments could interfere with early attachment, emotion recognition and emotional regulation leading to antisocial behaviour. In a study of criminal offenders, Raine et al.[et al.[Given the relationship between ADHD and CD, and the motor and/or perceptual dysfunction in ADHD, it seems likely that some of these symptoms would be present in CD. However, some questions remain as to whether only those children with co-occurring ADHD and CD would demonstrate these motor and/or perceptual problems, as well as the clinical significance of these symptoms in CD. We have only found a few studies describing the association between CD on one hand and motor and/or perceptual dysfunction on the other. In a population based follow-up study, Moffitt and Silva assessed children of 13 years of age and compared those with and without self-reported delinquency and with and without attention deficit disorder. Childreet al. studied et al., Raine ee et al. showed t[et al. found thClearly, more research is needed concerning the association between CD and motor problems/DCD and perception difficulties. Large-scale general population studies provide an excellent vehicle for studying such possible associations. In an earlier published article, we reported results including the heritability of motor problems, perception, ADHD, ASD, and CD from a large Swedish twin study.The objectives of the present study were to examine the distribution of problems regarding motor function and perception and their relation to childhood neuropsychiatric and behavioural problems in a large population-based twin study, taking gender and age into consideration. We wanted to test the hypothesis that there is an association between CD and motor and/or perceptual problems even among children with CD without ADHD or ASD. We also wanted to test the hypothesis that ADHD (with its subtypes attention deficit and hyperactivity/impulsivity) and ASD  all are associated with motor and/or perceptual problems.The Child and Adolescent Twin Study in Sweden (CATSS) is an on-going longitudinal twin study targeting all twins born in Sweden since 1 July 1992. Parents of twins are interviewed regarding their children’s somatic and mental health and social environment in connection with the children’s ninth or twelfth birthdays (CATSS-9/12). By January 2010, 8,610 parental interviews concerning 17,220 twins had been completed, with an overall response rate of 80% of all contacted families. If one The Autism-Tics, ADHD and other Comorbidities (A-TAC) inventory used in the interviews was developed  with a view to screening for all types of child and adolescent psychiatric disorders. The items in the A-TAC are organised in theoretically defined modules based on DSM-IV categories and criteria. For each question the answer ‘no’ is coded as ‘0’ , ‘yes, to some extent’ is coded as ‘0.5’ and ‘yes’ is coded as ‘1’. The A-TAC has been validated in several studies and has been shown to be a sensitive screening tool for ASD, ADHD, tics, learning disorders, and DCD,39. It hThe ‘Motor control problem’ scale of the A-TAC initially comprised five items. In one of the validation studies, the item: ‘does he/she have problems with smooth coordination of movement?’ was found to have the same level of predictive power for the clinical diagnosis of DCD as did all the five items taken as a module score. This siThe Perception scale of the A-TAC comprises five items, organised into one module. These five items are: ‘Does he/she seem disturbed by height differences such as in connection with climbing stairs, and so on?’ , ‘Does he/she have difficulty judging distance or size?’ , ‘Is he/she oversensitive to the touch of tight clothing?’ , ‘Is he/she particularly sensitive to certain sounds/noise?’ , and ‘Is he/she particularly sensitive to certain flavours, smells, or consistencies?’ Thus, the item ‘perception’ in the A-TAC contains questions concerning visual, auditory, sensory, olfactory and gustatory perception and, to some degree, proprio-sensibility (climbing stairs). To define ‘Perception disorder caseness’ , a cut-off of ≥ 2.5 points was used . The inThe ADHD scale of the A-TAC consists of 19 items organised into two modules/subdomains: Concentration/Attention and Impulsiveness/Activity. The scale has strong internal consistency and exceThe ASD scale of the A-TAC consists of 17 items organised into three modules/subdomains: Language, Social Interaction and Flexibility. The scale has both excellent internal consistency and predictive ability,38. ‘ASDThe CD scale of the A-TAC consists of five items, with good internal consistency and exceThe main modules/subdomains of the A-TAC scales were also analysed as continuous scale variables.The associations of ADHD, ASD, or CD caseness on the one hand, and DCD and Perception disorder caseness on the other hand, were analysed using the Fisher exact test . The associations of defined continuous covariates and categorical cofactors with DCD and Perception disorder were studied using binary logistic regression analyses in General Estimated Equation (GEE) models, and the relationship between the twins in each pair was taken into consideration. DCD and Perception disorder constituted the dichotomous dependent variables. Gender as a co-factor had significant odds ratios (ORs) for DCD and Perception disorder; hence GEE models were analysed by gender. The Fisher r-to-z transformation was used to test the significance of the difference between the Spearman correlations for prematurely born and full term children.The CATSS-9/12 study has ethical approval from the Karolinska Institute Ethical Review Board: Dnr 03–672 and 2010/507-31/1.P < 0.001). Perception disorder caseness was also somewhat more common in ADHD caseness plus CD caseness than in ADHD caseness ‘only’ .ADHD, ASD and, CD caseness was associated with much higher rates of DCD and Perception disorder caseness Table . CD caseP < 0.001), DCD caseness and CD caseness (P = 0.003) and Perceptual disorder caseness and CD caseness (P < 0.001). For perceptual disorder caseness we found that the correlation was stronger for full term children than for those born prematurely. Thus omitting prematurely born children from the analysis would strengthen the association between perceptual problems and ASD caseness. This shows that the strong correlations between caseness for ADHD and ASD and having motor and/or perceptual problems are not explicable only by the fact that many of the children were born prematurely.Children born prematurely have been shown to have a higher frequency of motor delays and motor difficulties than children born full term. Since mDimensional analyses with continuous A-TAC scores were used in the binary logistic regression analyses in GEE models and the dependence of each twin in a pair was taken into consideration. Male versus female gender was associated with an odds ratio (OR) of 1.86 (95% CI = 1.64 to 2.10) and 2.34 (95% CI = 1.77 to 3.09), in the prediction of Motor control and Perception problems respectively. Age was not associated with the risk of Motor control or Perception problems.P < 0.05) positively associated with both Motor control and Perception problems in both genders  association with Motor control problems only in boys. The strongest positive significant association in both genders was that between Flexibility and Motor control or Perception problems  and the subdomains of ASD  were analysed as continuous scale variables in a multivariable model together with the continuous CD-score Table . In thiss Table .This is probably the largest study ever performed on the relationship between symptoms relating to commonly diagnosed problems in child and adolescent psychiatry - ADHD, ASD, and, CD - and motor control and perception problems - commonly encountered but often not separately diagnosed, either as DCD or under any other label. ADHD and ASD, as ascertained on the basis of parent telephone interviews, showed strong and independent associations with such DCD-related problems, but CD without ‘comorbid’ ADHD or ASD did not. Associations were established both when phenomena were studied on a categorical basis and when they were considered from the point of view of continuous distributions. Parents were interviewed using the A-TAC, an instrument which is used for easy and quick detection of childhood psychiatric problems. Data frThe hypothesis that there is an association between CD and motor and/or perceptual problems was not supported by our study. The results seem to indicate that there are different forms of CD. One type of CD is associated with attention deficit and/or autistic symptoms and these children have a high frequency of motor and/or perceptual problems indicating a more ‘organic’ kind of disturbance. Another type of CD is associated with hyperactive and impulsive behaviour and children in this group have a low frequency of motor and/or perceptual problems and neither attention deficit nor autistic symptoms. Children with CD without either ADHD-symptoms or autistic symptoms also have a low frequency of motor and/or perceptual problems. Thus motor and/or perceptual problems among children with CD should lead a clinician to suspect comorbid attention deficit and/or autistic symptoms which might be of importance in efforts to find the right treatment and support.et al.[et al.[ADHD is often argued to be a heterogeneous condition with different subtypes; for example, the DAMP subtype of ADHD with motor and perception problems. Lambek et al. have deset al.. Robinso[et al. have peret al.[Cerebellar function is of importance for motor function and seems to be of importance in ASD as well as in ADHD,44. The et al., while tThe assessment of Motor control and Perception problems and of screen positivity for ADHD, ASD and, CD was based on information from parent interviews. In this study, we could not examine the children or obtain information from the teacher as in a thorough clinical assessment. Depending on only one subjective source of information is a weakness. The parents’ description of the child’s motor and perception functions and the result of a neurological examination of the child have been shown to have a low to moderate degree of correlation. On the Meeting criteria for CD caseness was positively associated with Motor control and/or Perception problems only in individuals who simultaneously met criteria for ADHD caseness and/or ASD caseness. High scores of the continuous variables Impulsiveness/Activity or CD in the absence of high scores of any of the variables Concentration/Attention, Flexibility, Social Interaction, or Language were not associated with Motor control and/or Perception problems. Thus, CD symptoms or problems with Impulsiveness/Activity seem to be associated with Motor control or Perception problems only in the presence of ASD symptoms and/or symptoms of inattention. This seems to indicate that children with CD without ASD or inattention do not show a deviant development of motor and perceptual functions. Children with CD should be examined concerning motor function and perception, and a suspicion of attention deficit disorder and/or ASD should be raised if the child is found to have motor and/or perceptual problems.Written informed consent was obtained from the patient’s guardian/parent/next of kin for the publication of this report.Diagnostic and Statistical Manual of Mental Disorders 4th edition/5th edition; GEE: General Estimated Equation models; MBD: Minimal Brain Dysfunction/Damage; OR: odds ratio; SPECT: Single Photon Emission Computed Tomography.ADHD: Attention-Deficit/Hyperactivity Disorder; ASD: Autism Spectrum Disorder; A-TAC: The Autism-Tics ADHD and other Comorbidities inventory; CATSS: Child and Adolescent Twin Study in Sweden; CD: Conduct Disorder; CI: confidence interval; DAMP: Deficits in Attention Motor control and Perception; DCD: Developmental Coordination Disorder; DSM-IV/5: All authors declare that we have no competing interests.PG was responsible for the design of the study, performed statistical analyses and drafted the manuscript. NK participated in the design of the study, performed statistical analyses and helped to draft the manuscript. HA, PL, CG and MR came with advice when interpreting and describing the results and helped to draft the manuscript. All authors read and approved the final manuscript.


The inflammatory nature of atherosclerosis provides a broad range of potential molecular targets for atherosclerosis imaging. Growing interest is focused on targets related to plaque vulnerability such as the co-stimulatory molecules CD80 and CD86. We investigated in this preclinical proof-of-concept study the applicability of the CD80/CD86-binding fusion protein belatacept as a probe for atherosclerosis imaging.Belatacept was labeled with indium-111, and the binding affinity was determined with CD80/CD86-positive Raji cells. In vivo distribution was investigated in Raji xenograft-bearing mice in single-photon emission computed tomography (SPECT)/CT scans, biodistribution, and ex vivo autoradiography studies. Ex vivo SPECT/CT experiments were performed with aortas and carotids of ApoE KO mice. Accumulation in human carotid atherosclerotic plaques was investigated by in vitro autoradiography.111In-DOTA-belatacept was obtained in >70 % yield, >99 % radiochemical purity, and ~40 GBq/μmol specific activity. The labeled belatacept bound with high affinity to Raji cells. In vivo, 111In-DOTA-belatacept accumulated specifically in Raji xenografts, lymph nodes, and salivary glands. Ex vivo SPECT experiments revealed displaceable accumulation in atherosclerotic plaques of ApoE KO mice fed an atherosclerosis-promoting diet. In human plaques, binding correlated with the infiltration by immune cells and the presence of a large lipid and necrotic core.111In-DOTA-belatacept accumulates in CD80/CD86-positive tissues in vivo and in vitro rendering it a research tool for the assessment of inflammatory activity in atherosclerosis and possibly other diseases. The tracer is suitable for preclinical imaging of co-stimulatory molecules of both human and murine origin. Radiolabeled belatacept could serve as a benchmark for future CD80/CD86-specific imaging agents.The online version of this article (doi:10.1186/s13550-015-0157-4) contains supplementary material, which is available to authorized users. The innate and adaptive immune systems interact in a well-orchestrated manner upon recognition of an antigen. After antigen exposure, professional antigen-presenting cells (APC), such as dendritic cells (DC), lose phagocytic action simultaneously gaining antigen-presenting capability by the upregulation of major histocompatibility complex (MHC) receptors and the co-stimulatory molecules CD80 B7.1) and CD86 B7.2) [ and CD86  and CD, 4.In atherosclerosis research, Buono et al. demonstrated that LDL receptor (Ldlr) knockout (KO) mice lacking CD80 and CD86 displayed a delayed atherosclerosis progression compared to control Ldlr KO mice . The invSince activated APCs that express CD80 and CD86 are a primary component of atherosclerotic lesions and the amount of APCs correlates with plaque vulnerability –11, CD80®, Bristol Myers Squibb) and belatacept  are 90-kDa fusion proteins of the extracellular domain of human CTLA-4 and a modified Fc fragment of a human IgG1, used in the therapy against rheumatoid arthritis and transplant rejection, respectively, inhibiting CD80/CD86-mediated T cell activation. Belatacept differs by two amino acids from abatacept and the CTLA-4 extracellular domain, resulting in a 10-fold increased inhibition of T cell co-stimulation in vitro [Abatacept  with mice regarding its accumulation in CD80/CD86-positive murine tissues and human CD80/CD86-positive xenografts. Moreover, ex vivo SPECT scans were acquired in a mouse model of atherosclerosis. Finally, the in vitro binding of 111In-DOTA-belatacept to human carotid plaques was compared with histological markers of plaque inflammation.In the present proof-of-concept study, we evaluated the potential of CD80/CD86-specific belatacept for atherosclerosis imaging. We labeled belatacept with the gamma-emitting nuclide indium-111  and characterized its binding affinity to human CD80/CD86 and its stability in plasma in vitro. The All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committees and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual patients included in the study prior to surgery.All applicable international, national, and institutional guidelines for the care and use of animals were followed, in particular the Swiss legislation and FELASA guidelines. All experiments with animals were approved by the veterinary office of the Kanton Zurich.®, Bristol Myers Squibb) was conjugated to p-SCN-Bn-DOTA (S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid, B205, Macrocyclics, Dallas, TX) and labeled with indium-111 according to a published procedure [Commercially available belatacept  as optimized by incubation of a fixed amount of belatacept-DOTA with increasing amounts of 111In. The reaction mixture for the radiolabeling contained 300–350 μg belatacept-DOTA (175 μL), 0.1 M ammonium acetate buffer , and 111In . After 1 h incubation at room temperature (rt), the reaction was quenched by the addition of 50 mM EDTA solution , mixed and allowed to incubate for 5 min at rt. The product was purified, and its stability in phosphate-buffered saline (PBS) and plasma was investigated as described in Additional file For radiolabeling, each 10 μg protein conjugate was reacted with 4 MBq Cell culture, immunocytochemistry, binding studies with Raji and NCI-H69 cells and tissue staining are described in detail in Additional file For immunocytochemistry, the following primary antibodies were used: anti-CD80 1:100 , anti-CD86 1:200 , and anti-CTLA-4 1:100 . Additionally, the following secondary antibodies were used: goat anti-rabbit IgG H&L 1:1000  and goat anti-mouse IgG H&L 1:1000 . Cells were fixed in paraformaldehyde and permeabilized with methanol. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Binding affinity was determined in saturation binding and Lindmo assays with Raji and NCI-H69 cells as described in Additional file Foxn1nu) from Charles River Laboratories  were fed a normal chow diet ad libitum. At the age of 6 weeks, these immune-deficient mice were subcutaneously inoculated in their shoulder region with 1 × 107 Raji cells in 100 μL Matrigel  . NCI-H69 cells (1 × 107 in 100 μL Matrigel) were subcutaneously inoculated in the shoulder region of CD1 nu/nu mice 2 weeks afterwards . In vivo SPECT/CT scans, ex vivo autoradiography, and biodistribution experiments were performed 2 weeks after the second inoculation according to Additional file 111In-DOTA-belatacept  or 10 MBq 111In-DOTA-belatacept and 500 μg unlabeled belatacept (blockade). In vivo and ex vivo experiments were performed 48 h after tracer injection or as indicated.Five-week-old female CD1 nu/nu mice (Crl: CD1-tm1Unc/J) were obtained from Charles River Laboratories. Two C57BL/6 mice and two ApoE KO mice were fed a normal chow diet ad libitum. All other ApoE KO mice (n = 4) received a modified Western-type diet containing 21 % fat, 0.25 % cholesterol, and 19.5 % casein  ad libitum. In two ApoE KO animals, fed a modified Western-type diet for 4 weeks, flow-modifying implants (cuff) were placed around the right and left common carotid arteries according to previous publications, inducing atherosclerotic plaque formation up- and downstream [111In-DOTA-belatacept  or 10 MBq 111In-DOTA-belatacept and 500 μg unlabeled belatacept (blockade) was injected into the tail vein of the animals. Mice were euthanized 48 h after tracer injection for ex vivo SPECT/CT imaging and oil red o staining of the excised aorta and carotids. The two mice with implants were in addition scanned before euthanasia.Eleven-week-old male C57BL/6 mice (control) and four-week-old male ApoE KO mice  were drawn manually to include aorta and carotids. A VOI of equal size was drawn outside the tissue samples to define background radioactivity in the absence of tissue. Tissue-to-background and tissue baseline-to-blocked ratios were calculated from the averaged 5 voxels with the highest radioactivity per VOI.In vivo and ex vivo SPECT/CT scans were conducted with a four-head multiplexing multipinhole camera . The acquisition time per view depended on the amount of radioactivity at scan start in the field of view, and the resulting scan times ranged from 20 min to 1 h (in vivo scans) and 14.5 to 15.5 h (ex vivo scans). In vivo scans were performed 48 h after tracer injection or as indicated. For ex vivo studies, animals were euthanized ∼48 h after tracer injection and SPECT/CT images were acquired after sample preparation. For in vivo SPECT/CT scans, animals were anesthetized with isoflurane (1.5–4 %) in oxygen. SPECT data were reconstructed with HiSPECT software , and images were generated with the Nucline Software (Bioscan). The fused SPECT and CT data were analyzed using VivoQuant image post-processing software  and PMOD biomedical image quantification software . For the ex vivo scans, volumes of interest . Accumulated tissue radioactivity was expressed as % injected dose (Bq) per g tissue (% ID/g).Post-mortem biodistribution studies with inoculated CD1 nu/nu mice (Raji xenograft right and NCI-H69 xenograft left shoulder) were performed with three baseline animals and three blockade animals. Animals were euthanized with CO® Plus Storage Phosphor System (PerkinElmer). Radioactivity signals were quantified with the software OptiQuant .For ex vivo autoradiographic studies, CD1 nu/nu mice bearing either Raji or NCI-H69 xenografts on both shoulders were injected with ~10 MBq tracer and euthanized after 48 h as described above. Tumors were dissected, embedded in Tissue-Tek O.C.T. medium, and frozen at −80 °C, and sections of 5 μm were prepared using a cryostat. After drying, frozen sections were exposed to super-resolution imager plates  for 5 min and analyzed by a Cyclone® solution  at −80 °C. After thawing, plaques were embedded in Tissue-Tek O.C.T. medium and cryosections (20 μm) were prepared with a cryostat. Frozen sections were stored at −20 °C until further use.Atherosclerotic carotid plaques were obtained from patients undergoing carotid endarterectomy (CEA) at the University Hospital of Zurich, Switzerland. Indications for CEA were stroke, transient ischemic attack, on-pump surgery to prevent perioperative stroke, ultrasound diagnosis revealing a soft and unstable plaque, or other surgical interventions . Twenty-three patients were included in this study with an average age of 75.4 ± 6.8 years, 16 were of male gender, 4 females, and 3 were unknown. Plaque material was collected from all patients from the common, external, and internal carotid artery. Available details of the patients and the samples used in this study are shown in Additional file 2, 125 mM NaCl, 1 mM CaCl2, pH 7.4) supplemented with 0.5 % milk powder and 40 μM gammanorm . Gammanorm contains human immunoglobulins that block unspecific binding of belatacept to ubiquitous Fc receptors. Tissues were incubated with 17 nM 111In-DOTA-belatacept (specific activity 36.6 GBq/μM) diluted in the above-specified buffer (~100 μL) for 1 h on ice. For blockade conditions, additionally 10 μM unlabeled belatacept was added to the tracer solution. The sections were washed with HEPES buffer containing milk powder and gammanorm , 3× HEPES buffer , and finally 2× distilled water  and were dried at rt. Standards of 1 μL tracer solution dried on 0.87 cm2 filter papers were prepared as positive controls. The sections and the standards were exposed to a super-resolution imager plate (PerkinElmer) for 30 min and 14 h. The plate was scanned by a Cyclone® Plus Storage Phosphor System (PerkinElmer), and data was analyzed with the software OptiQuant . Specific binding was calculated from the difference between the integrated radioactivity signals per area (DLU/mm2) under baseline and blocking conditions, divided by the value under baseline conditions. Hematoxylin and eosin (HE) staining was performed with all sections to investigate tissue morphology and score each individual plaque specimen used in autoradiography according to Additional file 111In-DOTA-belatacept autoradiography with paraffin-embedded tissues and immunohistochemistry with cryosections were not successful. In addition, the poor resolution of the 111In autoradiography did not allow the microscopic localization of tracer accumulation in the sections. In total, 37 carotid specimens were used for autoradiography experiments. The distribution of the plaques per classification category and the individual details on patients and tissues are shown in Additional file Radiotracer binding was evaluated by in vitro autoradiography with sections of human carotid plaques (20 μm). Cryosections were thawed at rt for 30 min and preincubated for 10 min on ice in HEPES buffer  or Microsoft Excel. To assess the intergroup difference of two groups, a two-tailed unpaired Student’s p-SCN-Bn-DOTA was successfully conjugated to belatacept under aqueous conditions by reaction with lysine amino groups of the protein according to a published procedure [111In-DOTA-belatacept was obtained in a radiochemical yield of 73–78 %, a radiochemical purity of >99 %, and a specific activity of 36–41 GBq/μmol (n = 3).The bifunctional chelating agent rocedure . A 20-fo111In-DOTA-belatacept at 37 °C in PBS and human and murine plasma, respectively, was investigated by FPLC up to 72 h  of 17.6 nM for 111In-DOTA-belatacept was determined in a saturation binding assay with CD80/CD86-positive Raji cells  or 111In-DOTA-belatacept together with unlabeled belatacept (blockade). Under baseline conditions, the highest percentage of injected dose per g tissue (% ID/g) was found in the spleen, the axial and inguinal lymph nodes, the liver, and the salivary glands, followed by the Raji xenografts and the blood  and salivary glands (p = 0.0019).Quantitative radiotracer distribution in xenograft-bearing CD1 nu/nu mice was evaluated in an ex vivo biodistribution experiment with three animals each, 48 h after injection of 111In-DOTA-belatacept.ApoE KO mice on regular diet develop atherosclerotic plaques in the aortic arch and the descending aorta. To enhance and accelerate plaque formation, ApoE KO mice were fed a high-fat diet. In two of these mice, plaques were further provoked in the carotid arteries by shear stress-modifying implants . Radiotracer accumulation in the atherosclerotic plaques was evaluated by ex vivo SPECT/CT imaging to allow an accurate localization of the SPECT signal. Tissues were dissected 48 h after injection of 111In-DOTA-belatacept accumulated in defined regions of the carotids besides the aortic arch and the descending aorta  size of the lipid/necrotic core, (ii) number of immune cells (foam cells and macrophages), (iii) cap thickness, and (iv) cap rupture .In the in vitro autoradiography experiment, ept Fig. . Averageept Fig. . Howeverore Fig. . Radioacp-SCN-Bn-DOTA as bifunctional chelating agent. After purification, the product was obtained in high radiochemical yield and purity in a fast and robust labeling reaction. Under the applied conditions, approximately two DOTA chelators were coupled to belatacept which is the intended range to minimize undesired chelator-mediated effects on pharmacokinetics [In this proof-of-concept study, we successfully established a radiolabeling procedure for the CD80/CD86-targeting fusion protein belatacept with indium-111 using kinetics . A high Kd value. Efforts to assess the binding affinity to mouse recombinant CD80 by differential scanning fluorimetry were not successful due to similar melting temperatures Tm of the recombinant CD80 and belatacept. Immune-reactive affinity assays were excluded due to interferences with the Fc portion of belatacept. However, the results of this study indicate that the radiolabeling modifications did not hamper high-affinity binding to human and murine CD80/CD86 in vitro and in vivo. The contribution of Fc receptor-mediated binding to the total binding of belatacept was negligible as concluded from in vitro autoradiography with human tissue and as expected from published data [The radiolabeled product bound to human Raji cells with a nanomolar hed data .111In-DOTA-belatacept specifically accumulated in CD80/CD86-positive tissues. Besides Raji xenografts, strong and specific accumulation was observed in the lymph nodes. The lymph nodes exhibit a resident population of APCs capable of expressing CD80 and CD86 in particular during the process of antigen presentation. Moreover, biodistribution studies revealed the highest radiotracer accumulation in the spleen, a peripheral lymphoid organ, and a high specific accumulation in the salivary glands of xenograft-bearing mice. This is consistent with the fact that APCs are constituents of human and murine salivary glands being involved in immune surveillance [In vivo SPECT/CT scans and ex vivo autoradiography experiments indicated that eillance , 21. Theeillance .111In-DOTA-belatacept is cleared from the blood.The accumulation of radioactivity in the Raji xenografts increased from 2 to 18 to 48 h p.i. This is in agreement with the relatively long half-life in the blood of IgG1 Fc fusion proteins. The half-life of the closely related abatacept in mice was ~90 h after i.v. injection . Indium-111In-DOTA-belatacept in the lymph nodes of the xenograft-bearing animals seen in SPECT and confirmed in the biodistribution experiments looks promising towards the imaging of small CD80/CD86-rich structures. We, therefore, proceeded to an animal model of atherosclerosis where lesions are of similar or smaller dimension. However, owing to the proximity of atherosclerosis-prone areas in the aorta and the carotids to other tissues with radiotracer accumulation, e.g. the liver, the salivary glands, and the thymus, SPECT/CT scans of aorta and carotids were performed ex vivo. Furthermore, perfusion of the excised tissue allowed to exclude any blood-related tracer radioactivity, considering the long residence time of 111In-DOTA-belatacept in the blood (still 7.6 % ID/g blood 48 h after injection). 111In-DOTA-belatacept accumulated in lipid-rich atherosclerotic plaques, and tracer accumulation was higher under baseline than blockade condition. Moreover, 111In-DOTA-belatacept accumulation in the aortas of ApoE KO animals was dependent on an atherosclerosis-promoting diet. However, several lipid-rich regions were not positive in SPECT. We can only speculate on the reasons. SPECT imaging with 111In-DOTA-belatacept may not have been sensitive enough to visualize all lesions, depending on their size and tracer uptake. The signal was close to the detection limit in the ex vivo scans. Alternatively and in addition, not all lipid-rich regions may be invaded by high numbers of CD80/CD86-positive cells. As discussed under limitations, we did not further investigate these vessel walls by immunohistochemistry. However, we found that the descending aortas of ApoE KO mice fed a high-fat diet contained significantly higher mRNA levels of CD80 than the descending aortas of wild type mice under normal diet . In agreement with our findings, the correlation between characteristics of plaque vulnerability and the lipid content of the lesions may be weak as derived from intravascular ultrasound measurements [The high and specific accumulation of urements .111In-DOTA-belatacept binding was associated with the infiltration of immune cells. It can be assumed that the majority of inflammatory cells within a plaque are APCs [111In-DOTA-belatacept uptake in lesions with a large lipid/necrotic core. Our results suggest that 111In-DOTA-belatacept accumulates specifically in atherosclerotic plaques, depending on their degree of inflammation and vulnerability.In human carotid tissue samples, are APCs . The incare APCs , 27. Thi111In-DOTA-belatacept is unfavorable in this respect, as it contributes to significant unspecific tissue radioactivity as observed in the in vivo scans. Imaging vulnerable plaques in coronary arteries would be even more challenging. According to the biodistribution results, radioactivity in myocardium was still relatively high 48 h p.i., although unspecific and lower than in the blood. This relatively high background level in the heart together with the respiratory and myocardium motion would hamper detection of atherosclerotic lesions in coronary arteries under these experimental conditions. A tracer with a faster clearance from the blood and CD80/CD86-negative tissues in combination with motion correction from simultaneously acquired magnetic resonance imaging (MRI) may improve the contrast between atherosclerotic lesions and myocardium in the future.Limiting factors are related to the small size of atherosclerotic plaques both in preclinical models and in humans. High specific accumulation of radioactivity and low spill-over from other tissues are required for the detection of lesions in the millimeter or submillimeter range . The lon111In-DOTA-belatacept in the absence of xenografts or atherosclerosis. In addition, belatacept binds with similar affinity to CD80 and CD86, which may reduce selectivity for a particular type of cell activation as compared to a CD80- or CD86-selective tracer. Due to technical limitations with available antibodies, we were not able to counterstain the atherosclerotic tissues from SPECT and autoradiography for CD80 and CD86, the proteins that we found upregulated in human vulnerable atherosclerotic plaques [Further limitations of our study are the low number of animals used for in vivo and ex vivo imaging and the lack of control animals to study lymph node accumulation of  plaques . Finally plaques .111In-DOTA-belatacept could serve as a benchmarking tracer in the preclinical development of novel CD80/CD86 imaging agents.Future studies have to focus on CD80- or CD86-selective small molecules and truncated versions of belatacept as well as abatacept, which has a higher affinity and selectivity for CD80 than CD86 in mouse . In this111In-DOTA-belatacept could serve as a research tool to study the approach of targeting CD80/CD86-rich APCs in atherosclerosis and possibly other inflammation-related diseases such as cancer, autoimmune disease, and rejection after organ transplantation. A highly expedient characteristic of belatacept is its immunoreactivity with both murine and human CD80/CD86 allowing the translation between species.CD80 and CD86 expressed by activated APCs are promising imaging targets in atherosclerosis. In this context, Additional file 1:Supplementary information. This file contains supplementary Tables 1–4. and Figures 1–4. and supplementary material and methods. (PDF 1198 kb)


Adults with attention deficit hyperactivity disorder (ADHD) often present with a lifelong pattern of core symptoms that is associated with impairments of functioning in daily life. This has a substantial personal and economic impact. In clinical practice there is a high need for additional or alternative interventions for existing treatments, usually consisting of pharmacotherapy and/or psycho-education. Although previous studies show preliminary evidence for the effectiveness of mindfulness-based interventions in reducing ADHD symptoms and improving executive functioning, these studies have methodological limitations. This study will take account of these limitations and will examine the effectiveness of Mindfulness Based Cognitive Therapy (MBCT) in further detail.N = 120 adults with ADHD. Patients will be randomised to MBCT in addition to treatment as usual (TAU) or TAU alone. Assessments will take place at baseline and at three, six and nine months after baseline. Primary outcome measure will be severity of ADHD symptoms rated by a blinded clinician. Secondary outcome measures will be self-reported ADHD symptoms, executive functioning, mindfulness skills, self-compassion, positive mental health and general functioning. In addition, a cost-effectiveness analysis will be conducted.A multi-centre, parallel-group, randomised controlled trial will be conducted in This trial will offer valuable information about the clinical and cost-effectiveness of MBCT in addition to TAU compared to TAU alone in adults swith ADHD.NCT02463396. Registered 8 June 2015.ClinicalTrials.gov  Attention Deficit Hyperactivity Disorder (ADHD) is a common neurodevelopmental disorder with a high persistence into adulthood. Results of a meta-analysis of six studies indicated the pooled prevalence of adult ADHD according to DSM-IV criteria to be 2.5 % (95 % CI 2.1-3.1) . In an iThe vast majority of ADHD patients in the Netherlands is offered stimulant medication after or parallel to psycho-education on ADHD. In accordance with the NICE guideline on ADHD  stimulanThe NICE guideline recommends group Cognitive Behavioural Therapy (CBT) for adults with ADHD whose response to medication is insufficient, who have difficulty adhering to drug treatment or who are not willing to use medication. Some studies have shown evidence for the efficacy of treatment with CBT in adults with ADHD, particularly when combined with medication . For exaA few studies on the effectiveness of mindfulness training for adults with ADHD have so far been conducted, two uncontrolled pilot studies , 28 and The results of the above mentioned studies support the feasibility and efficacy of mindfulness training as a treatment for adults with ADHD and encourage further research. This approach offers a novel and potentially useful tool in the multimodal treatment of ADHD. Previous studies are conducted in one centre and lack a follow-up period. Some of them are small –29, uncoThe primary aim of this study is to investigate effectiveness of MBCT in addition to Treatment As Usual (TAU) versus TAU alone in adults with ADHD. We expect that MBCT can improve ADHD symptoms and overall functioning in two ways: a) by strengthening their attention regulation and executive functioning, resulting in a greater reduction of ADHD symptoms rated by a blinded clinician (primary outcome) and self-reported ADHD symptoms and a greater improvement of executive functioning (secondary outcomes); and b) by extending their repertoire of coping strategies to deal with core symptoms and functional impairments, resulting in a greater improvement of general functioning, positive mental health and self-compassion (secondary outcomes).Other aims of the study are to assess whether MBCT is a cost-effective intervention compared to TAU in patients with ADHD from a societal perspective and to assess the financial consequences of implementing mindfulness training in patients with ADHD from different perspectives . We expect that the implementation of this intervention will result in a reduction of medical and societal costs in the MBCT group compared to the TAU group.Furthermore, in order to gain insight in the working mechanisms of mindfulness, we will investigate whether possible clinical effects of MBCT can be attributed to changes in mindfulness skills, self-compassion and adherence to MBCT.The design of the study is multi-centre, parallel-group, randomised controlled superiority trial. Patients will be randomised to MBCT in addition to TAU or TAU alone. The study protocol has been ethically approved by CMO Arnhem-Nijmegen for all participating centres and registered under number 2014/206.The study population will consist of adults with ADHD. To enhance generalisability, the study is conducted in three different mental health centres for patients with ADHD in the south-east of the Netherlands: the Department of Psychiatry of the Radboudumc in Nijmegen, the Reinier van Arkel Group in the Jeroen Bosch hospital in ‘s Hertogenbosch and Dimence in Deventer. Each centre has a specialized outpatient clinic for adults with developmental disorders, like ADHD.th edition (DSM-IV-TR)  tpulation .Resource use will be collected and calculated with the latest version of the Trimbos/iMTA questionnaire for costs associated with psychiatric illness (TiC-P) . This seWe will use the 12-item Short Form (SF-12) Health Survey , a shortDuring the nine weeks of participation in the MBCT group, we will assess adherence to mindfulness exercises with a calendar on which patients fill out total minutes of formal (e.g. sitting meditation) and informal practice (e.g. mindful eating) on a daily basis. For patients allocated to the MBCT group, we will assess adherence during 6 and 9 months after baseline by questions in which patients are asked to estimate total minutes of formal and informal practice during an average week in the last three months. To check for compliance with the instruction to refrain from mindfulness training during the nine months of the study, patients in the TAU group will be asked whether they participated in an MBSR or MBCT course during the study at the different time points of assessment.We developed a protocol primarily based on the Mindfulness-Based Cognitive Therapy (MBCT) for recurrent depression . We madeThe protocol has been tested in three consecutive pilot groups of ADHD patients and has been adapted according to their feedback. The 8-week programme will consist of weekly sessions lasting 2.5 hours, and a silent day between session six and seven. In addition to the group sessions, participants will be instructed to practice six days a week for approximately 30 min a day and complete home practice assignments. The participants will receive a set of CDs containing guided mindfulness exercises for home practice ranging in length from 10 min to 45 min.http://mindfulnessteachersuk.org.uk/pdf/teacher-guidelines-2015.pdf) which include: a) a minimum of 150 h of education in MBSR/MBCT background and theory including a reflection report; b) relevant professional training; c) minimum of three years of practicing meditation regularly and attending retreats; d) having attended MBSR/MBCT as a participant; e) continued training; and f) giving a minimum of two courses per two year. The teachers will receive additional training in the study protocol at the start of the project. We will organise peer supervision meetings every three weeks during the intervention phase of the trial. Intervention integrity consisting of adherence to the original programme, the degree to which the intervention is delivered as intended and teacher competency, will be assessed by the Mindfulness-based Interventions-Teaching Assessment Criteria (MBI: TAC) [The MBCT will be taught by mindfulness teachers meeting the advanced criteria of the Association of Mindfulness Based Teachers in the Netherlands and Flanders and the internationally agreed good practice guidelines of the UK Network for Mindfulness-Based Teachers BI: TAC) . VideotaThe power calculation has been based on our previous randomised controlled study consisting of 103 adults with ADHD  in which we demonstrated a significant difference in post-assessment clinician-rated ADHD symptoms (CAARS-INV)  between Baseline socio-demographic and clinical characteristics of the MBCT group and the TAU group will be compared to check whether these characteristics will have been evenly distributed by randomisation. The primary analyses will be aimed at comparing results on the outcome measures at three months after baseline between the MBCT group and the TAU group, controlling for baseline levels and possible other baseline differences between intervention and control groups (despite randomisation).T-score greater than 65) and standard deviations. In addition, a Cohen’s d-type effect size will be calculated.The outcome data will be analysed and reported according to the CONSORT guidelines and will include both intention-to-treat analyses and per-protocol analyses with the treatment-adherent sample (patients in the MBCT group have to attend at least four of MBCT sessions and patients in the TAU group do not attend a mindfulness-based programme). Percentages of improved, recovered, unchanged and worsened patients in the intervention and control groups will be provided on the basis of predefined cut-off scores  costs; b) treatment response; and c) QALYs will be collected over the study period of nine months. Total costs for each patient will be obtained by multiplying the resource use as measured with the TiC-P with standard costs, based on the Dutch guideline for costing research . OverallIf MBCT is more costly and more effective than TAU, an incremental cost-effectiveness ratio (ICER) will be calculated by dividing the extra costs by the extra effects. ICERs are computed to obtain the costs per responder and the costs per QALY gained. This will give an estimation of the extra costs that are needed to gain one responder or QALY. If this is below the societal willingness to pay, mindfulness training is deemed cost-effective. If one comparator is less costly and more effective, it dominates the other and no ICER is necessary to determine cost-effectiveness. If MBCT is less effective and less costly, ICERs will be calculated representing the savings per QALY lost. Uncertainty surrounding the costs, effects and ICER will be addressed by means of bootstrapping.To gain insight in underlying working mechanism of mindfulness, we will test with a mediation analysis whether possible clinical effects of MBCT can be attributed to changes in mindfulness skills, self-compassion and adherence to MBCT. We will follow the approach suggested by Preacher, Hayes .Adult ADHD has a substantial personal and economic impact and consequently there is a high need for evidence-based treatments for this patient group. Although pharmacotherapy has shown to be effective in reducing ADHD symptoms and its economic impact, there is a call for psychosocial interventions to offer in addition to pharmacotherapy or as an alternative treatment. At this moment research examining the effectiveness of psychosocial interventions for adult ADHD is still in its infancy. Mindfulness training, such as MBCT, is a promising psychosocial intervention, which can be offered in a group format. Considered from an economic perspective, this is an advantage over treatment on individual basis. Previous studies support the feasibility of mindfulness training for this patient group and provide preliminary, but consistent evidence that this intervention reduces ADHD symptoms and improves executive functioning. However, the methodology of these studies is limited by single-centre enrolment –29, and To our knowledge, this is the first properly powered randomised controlled study in this field, that contains blinded outcome assessments and an intention-to-treat analysis. Besides, a follow-up period with a length of six months after the end of treatment is included to consider potential long-term effects of MBCT. To enhance generalisability, this study is conducted in three different mental health centres with expertise in treating adults with ADHD. Additionally, cost-effectiveness will be assessed to evaluate whether MBCT is a cost-effective intervention compared to the treatment as usual and to examine the financial consequences of implementing this intervention. This trial will offer valuable information on whether MBCT has an effect on clinician-rated and self-reported ADHD symptoms, executive functioning, general functioning, positive mental health, self-compassion and medical and societal costs in comparison to treatment as usual.