Attention-Deficit/ Hyperactivity Disorder (ADHD) is one of the most common childhood neurobehavioral conditions. Symptoms related to this disorder cause a significant impairment in school tasks and in the activities of children’s daily lives; an early diagnosis and appropriate treatment could almost certainly help improve their outcomes.The current study, part of the Models Of Child Health Appraised (MOCHA) project, aims to explore the age at which children experience the onset or diagnosis of ADHD in European countries.A systematic review was done examining the studies reporting the age of onset/diagnosis (AO/AD) of ADHD in European countries (28 European Member States plus 2 European Economic Area countries), published between January 1, 2010 and December 31, 2019. Of the 2276 identified studies, 44 met all the predefined criteria and were included in the review.The lowest mean AO in the children diagnosed with ADHD alone was 2.25 years and the highest was 7.5 years. It was 15.3 years in the children with ADHD and disruptive behaviour disorder. The mean AD ranges between 6.2 and 18.1 years.Our findings indicate that there is a wide variability in both the AO and AD of ADHD, and a too large distance between AO and AD. Since studies in the literature suggest that an early identification of ADHD symptoms may facilitate early referral and treatment, it would be important to understand the underlying reasons behind the wide variability found.CRD42017070631.PROSPERO registration: The online version contains supplementary material available at 10.1186/s12888-021-03547-x. Attention-Deficit/Hyperactivity Disorder (ADHD), which is one of the most common childhood neurobehavioral conditions, has been characterized by continually increasing global prevalence rates over the past few decades . A globaThese conflicting figures have triggered the hypothesis that ADHD is either over diagnosed , 6, undeChildren with unmanaged ADHD often experience unnecessary impairments and detrimental long-term consequences leading to high personal and societal costs , 9. EarlHealthcare professionals specialized in child psychology, including the American Academy of Paediatrics, advise screening for the disorder early as the preschool period  so that Multiple factors may affect the perception of the disorder by family members and healthcare providers and thus the timing of its diagnosis and treatment . MoreoveParents play a central role in recognizing behavioural problems early in their children, their perception, awareness and acceptance of the disease, as their decision to accompany the child to a specialist . Once paMany clinicians depend on and utilize the Diagnostic and Statistical Manual of Mental Disorders (DSM)  for guidAlthough the heterogeneity in the methodology of diagnosing of ADHD has resulted in a high variability in prevalence rates around the world , differeWithin the Models Of Child Health Appraised (MOCHA) project , which hThe current work set out to examine the studies involving children with ADHD in European countries that report their age at onset or diagnosis.Following a systematic review approach and a standardized method of Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) , we searDSM started in 2000 and finished in 2013 [We searched the Medline (PubMed) database for studies in the literature examining ADHD onset or diagnosis published between January 1, 2010 and December 31, 2019. It was decided to limit the review to the last decade because it is the one in which the clinical guideline’s recommendations previously produced by many parties had to be consolidated also with the fifth revision of the  in 2013 . The seaThe abstracts of all the articles were read and the full version of the papers for those seemingly fulfilling the selection criteria were retrieved.Studies were included in this review if they reported the AO or AD for ADHD and were conducted in or referred to data from a European country.We utilized a standardized form for data extraction that included the following items: the authors’ names, the year of publication, the country in which the study was performed, the journal in which the study was published, the type of study, the aim of the study, the year in which the study was performed, the types of persons composing the study sample (including age and sample size), the diagnostic criteria adopted for the diagnosis, and, of course, the AO/ADs.Two of the authors (IR and BC) screened all the articles; any differences in viewpoints that arose were resolved through discussion with the third author (NM).The initial PubMed search yielded 2276 studies Fig. . After tThe characteristics of the studies included are outlined in Table One article reported both the AD and the AO, 34 studies reported only the AD, and 9 reported only the AO. The majority of the studies included in this review were conducted in Sweden (7 articles) and in Germany (5 articles), followed by 3 countries publishing 4 articles each.Table In the majority of the articles, the diagnostic criteria used to define ADHD symptoms or to formulate a diagnosis of ADHD was the DSM. One study conducted in Sweden  reportedThe Composite International Diagnostic Interview (CIDI) version 3.0 was used to determine the presence of ADHD according to the DSM-IV criteria in the article by Tuithof and collaborators . DevelopWe also identified 15 papers using the ICD to define ADHD symptoms or to make an ADHD diagnosis. Among these papers, one article  used botIn three articles, multiple sources of information were taken into consideration for the diagnosis of ADHD. The diagnostic criteria of the DSM-IV and the ICD-9/ICD-10 were adopted and the results of Conner’s questionnaire were considered in the six countries involved in the study by Hodgkins and collaborators . In the The semistructured Schedule for Affective Disorders and Schizophrenia for School-Age Children, Present and Lifetime Version (K-SADS-PL) is a DSM-IV-based diagnostic interview procedure that was used in some of the articles to support ADHD diagnosis , 53, 59.Two studies , 66 usedRead codes have become the de facto standard for coding diagnoses, operations, and procedure for all national data sets and statistics on hospital and community health services in the UK.Lastly, the diagnostic criteria utilized were not specified in six articles. In three papers , 45, 58,Eight out of 10 of the studies presenting information on the AO reported the mean, median or age range at symptom onset in the sample of children being studied , 51, 53.The study by Muller and colleagues  reportedPolanczyk et al.  focused Thirty-two of the 35 studies presenting information on the AD of ADHD reported its mean, median, range or distribution, one study presented the peak of the ADHD incidence in males and females ; in two The age range in the 24 studies reporting the mean AD was 6.2 to 18.1 years. The lowest mean value, which was reported by Andreou and Trott , concernGranström et al.  reportedTwo studies reported the mean age for ADHD diagnosis for a specific group of countries , 43. TakADHD, one of the most common childhood neurodevelopmental disorders, is characterized by a pattern of inattention and/or impulsivity and hyperactivity, behaviours that can have a dramatic impact on children and on family life . Since eThe current study set out to investigate age at the time of onset and/or diagnosis of ADHD in children living in European countries by examining the studies published between January 1, 2010 and December 31, 2019 reporting on the AO and AD of ADHD. The study’s most important finding was that there is a wide variability in both.Much of the variability could be attributed to discrepancies in study methods. Differences in study designs, ranging from case-control, cohort, to cross-sectional, could have affected the AO/AD as a cohort study could more accurately identify the AO and thus the incidence of ADHD with respect to a cross-sectional retroactive study basing its figures on parent reports.The studies also show differences in sampling methods. Several studies, in fact, used convenience samples from clinic-based studies; others were based on registry data or medical records. It is reasonable to hypothesise that referred patient samples have lower AO/AD compared to community cohorts given the differences in the severity of the disorder in these populations.The source of information  can also significantly influence the figures on the AO/AD registered by the different studies. In a multi-country cross-section study by Caci et al.  which asThe articles by Genuneit et al.  and PohlThe presence of comorbidities is an exceedingly important consideration when ADHD diagnosis is being discussed. ADHD symptoms can overlap with those of other disorders, including autism spectrum disorder, disorders of mood and conduct, oppositional defiant disorder, learning difficulties, impaired motor control, poor executive functions , communication difficulties, sleep disorders, tics/Tourette syndrome, epilepsy, and anxiety disorders, that commonly coexist with ADHD.p-value = 0.07). This result suggests that children with comorbidities related to ADHD have a greater probability of being diagnosed with ADHD at a younger age, presumably because they already have access to some kind of healthcare services and are being monitored by medical specialists.Socanski et al. , who comThus, the characteristics of enrolled populations (exclusion/inclusion criteria) and the sample size enormously undermine the evaluation and comparison of studies as well as a formal summary of the results. The application of meta-analysis is also impossible due to the lack of publication by all the studies of the absolute values.The criteria used to diagnose the condition is another important factor that could affect the heterogeneity in the studies considered.The two main diagnostic systems currently being used are the ICD-10 and the DSM-V . Both sySeveral research teams have been concerned about the implications of increasing the age of onset to 12 years . Some inTo our knowledge, this study represents the first systematic review of the AO and AD of ADHD in European countries. Although we did adhere to PRISMA guidelines  to ensurThe first is that studies not published in English as well as those not available in PubMed were not taken into consideration.We would also like to point out that the 44 articles included in this systematic review refer to studies conducted in 13 European countries.Despite these limitations, and those methodological of analysed studies, the review offers new insights into the timing of the onset and diagnosis of ADHD.. Studies in the literature suggest that identifying ADHD symptoms early on can facilitate early referral and treatment, and thus limit its cost in personal and societal terms [One of the key functions of primary care is to recognize the symptoms of an illness at an early stage. As far as childhood illnesses are concerned, neurodevelopmental disorders are relatively common and increasing in Europe. Early diagnosis makes it possible to contemplate and implement opportune treatment strategies thus reducing, in this case, some of ADHD’s adverse current and future consequences in the child and family. This study provides a preliminary overview on the timing of the onset and the diagnosis of ADHD in children living in European countries. The long term validity and heterogeneity of the classification systems used to guide diagnoses and the factors behind the social, cultural and genetic differences affecting the timing of identification of the syndrome need further analysis. The fact that Germany has a much earlier AO and AD with respect to the UK and the Netherlands is just one example of differences that need to be clarifiedal terms , 82. To al terms . HoweverAdditional file 1. PRISMA checklist.


A child suffering from attention deficit hyperactivity disorder (ADHD) faces many difficulties in social as well as academic performances. School teachers’ knowledge and attitude towards ADHD play a vital role in early detection and referral of the child to treatment centers. Few existing reports, however, indicate the alarming rate at which the problem is highly neglected in sub-Saharan Africa. The present study is designed to determine the knowledge, attitude, and factors that affecting elementary school teachers about ADHD.An institutional-based cross-sectional study design was conducted in Gondar town and other towns nearby Gondar from February 24 to March 24, 2020. Data were collected through structured self -administered questionnaires using the Knowledge of Attention Deficit Disorders Scale and ADHD-specific attitudes measurement tools. Then, it was entered into Epi-info version 7 and exported to SPSS version 20 for analysis. Bivariable and multivariate logistic regressions were fitted to identify factors associated with the knowledge and attitude of elementary school teachers. Variables having a p-value < 0.05 at 95% CI were considered statistically significant.Of 636 respondents, about 44.8%  and 84.1%  of elementary school teachers had good knowledge and a favorable attitude towards ADHD, respectively. Having a diploma and above , reading ADHD leaflets  and search ADHD on the internet  were significantly associated with teachers knowledge to ADHD; whereas, working experience in teaching a child with ADHD  and watching ADHD on mass media  were positively predicts teachers attitude towards ADHD.the proportion of teachers’ knowledge towards ADHD was low; in contrast, their attitude was relatively satisfactory. Strengthening teachers’ educational upgrading system, frequent and fair distribution of leaflets written to address ADHD, installation of an internet system to the schools, and continuous ADHD awareness creation programs through mass media are highly recommended. Attention deficit with or without hyperactivity disorder (ADHD) is one of the most frequently reported neuropsychiatric disorders, especially in school-age children . AccordiThe global prevalence of ADHD was estimated at 5–7% in children and 2.5% in adults , 6. EvenADHD can have an impact on a student’s academic and social performance due to difficulties in maintaining attention, failure to complete tasks, forgetfulness, and excessive non-goal directed physical activities . MoreoveTo give timely treatment following early detection, elementary school teachers have been taking the highest place, since most children spend most of their time in schools and interact often with teachers on a daily basis even more than their parents or physicians, suggests that schools play a very important role in the early detection and management of ADHD , 24.Thus, teachers play a central role in early detection and advising parents on managing their children, and implementing classroom and behavioral management strategies , 26. TheSeveral factors that can affect teachers knowledge and attitude towards ADHD; some of them are, age, sex, marital status, and educational level of the teachers, and searching on the internet about ADHD were factors that affecting the knowledge and attitude of teachers towards ADHD –34.In view of the problem and importance of early detection for effective treatment, different studies should be undertaken to show the prevalence and possible factors that affecting teachers’ knowledge and attitude towards ADHD with its possible recommendation. However, there is a paucity of studies in the nation, Ethiopia, therefore, this study aimed to assess the knowledge and attitude of elementary school teachers about ADHD and its associated factors in Gondar town, Northwest Ethiopia. This could be helpful to design new policies to tackle the problem and make a healthy generation.An institution-based cross-sectional study design was conducted from February 24 to March 24, 2020, and data were collected from 636 Gondar and nearby town’s public and private elementary school teachers. Gondar town is located in Amhara National Regional State, Ethiopia which is about 727 km Northwest of Addis Ababa . Administratively, Gondar town has a total of 24 kebeles with 13 urban and 11 rural kebeles. Based on reports published by the Central Statistical Agency in 2011, Gondar town has an estimated total population of 254,420 . The towAll elementary school teachers working at public and private elementary schools in Gondar and nearby towns were considered as a source population. Elementary school teachers who are working in the selected Gondar and nearby town’s elementary schools were enrolled as a study population.All elementary school teachers working at the selected public and private elementary schools in Gondar and nearby towns who were present at the time of data collection were included in the study. Those participants who had a family member with diagnosed ADHD were excluded from the study, since incorporating those teachers having a family member with ADHD might have affected the true magnitude.2 = 1.96, the margin of error = 5%.2 critical value of 95% CI = 1.96, p-proportion (0.564) of knowledge taken from similar previous study’s , which indicated that there is a knowledge gap in this population about the problem, therefore, a comprehensive and well-designed strategy will be mandatory for making a timely solution to minimize the possibility of delayed diagnosis of ADHD and classroom mistreatment –42.This study finding was in line with a study conducted in Cape Town (South Africa) . NonetheOn the other hand, the current finding was lower than studies conducted in Canada 68%, Colombia 48.52%, Saudi Arabia 58.9%, another Saudi Arabia study 72%, India 49%, and South Africa 78%, , 34, 46.In the current study, having good knowledge about ADHD was significantly associated with participants’ educational level of diploma and degree than those having a certificate. The same has been reported by others , 32. It This study came up with having good knowledge was statistically significant with teachers reading ADHD leaflets. This might be due to the content of leaflets is minimal with a brief description of the information and easy to handle , 49. HenThe study evidenced that the higher odds of having Good knowledge were observed among participants of the internet user. The same has been reported . This miAn important finding observed in this study is participant’s attitudes about ADHD, the study revealed that 84.1% of elementary school teachers were detected to have a favorable attitude, which indicated that the existing attitude towards ADHD is good. Even though, to address the issues at large for covering the rest of the participants having an unfavorable attitude, a well-designed strategy and policy still needed. The current finding was higher than a study done in Iran, South Africa, Egypt, and Nigeria , 46, 55.Regarding factors associated with the participant's attitude towards ADHD; The odds of having a favorable attitude was higher among elementary school teachers who had a teaching experience of a child with ADHD. This supported finding is in line with a study conducted in Saudi Arabia . This coFinally, this study found that the likelihood of having a favorable attitude was higher among elementary school teachers watching any mass media programs. This might be due to the mass media may play an important role in sharing health information and increasing awareness about health since media not only spread awareness but also report new knowledge, scientific information, and research findings to the public which has ultimately helped in the change of attitude of the audience for achieving better health , 58.Doing the study in this overlooked area of mental health (ADHD) especially in the country with a paucity of researches was the good looks of the study and the findings could alarm the responsible body’s pleasing to the eye on Childhood mental health. Nonetheless, the fact that the study was a cross-sectional survey, it is difficult to derive causal relation. Likewise, it would have been more novel if it was conducting a training and re-administer the survey to teachers to see if attitudes/knowledge of ADHD has changed. Moreover, it would have been more soundable if it incorporates the socio-economic status of the teachers as this may possibly also have an impact on the knowledge and attitudes of ADHD.The proportion of elementary school teachers who had good knowledge of ADHD was low, whereas, the proportion of teachers who had a favorable attitude towards ADHD was relatively satisfactory. Educational status, reading the leaflet, and searching on the internet were variables significantly associated with the knowledge of teachers about ADHD. On the other hand, the experience of teaching children with ADHD and watching mass media were significantly associated with the attitude of elementary school teachers regarding ADHD. Therefore, to enhance teachers’ understanding of ADHD and implementation of behavioral strategies in the classroom, teachers with lower educational status like certificates could be encouraged to seek training regarding ADHD. Moreover, policymakers should consider the incorporation of ADHD and other childhood mental health issues in primary school teachers’ curriculum. Likewise, Frequent and fair distribution of leaflets that are talking about ADHD, installation of the internet to the schools considering easy accessibility, and continuous ADHD awareness creation through mass media are highly recommended.


Attention-deficit/hyperactivity disorder (ADHD) and co-existing psychiatric/psychological impairments as well as adverse childhood experiences (ACEs) are common among young offenders. Research on their associations is of major importance for early intervention and crime prevention. Intermittent explosive disorder (IED) warrants specific consideration in this regard. To gain sophisticated insights into the occurrence and associations of ADHD, IED, ACEs, and further psychiatric/psychological impairments in young  offenders, we used latent profile analysis (LPA) to empirically derive subtypes among 156 young offenders who were at an early stage of crime development based on their self-reported ADHD symptoms, and combined those with the presence of IED. We found four distinct ADHD subtypes that differed rather quantitatively than qualitatively . Additional IED, ACEs, and further internalizing and externalizing problems were found most frequently in the severe ADHD subtype. Furthermore, females were over-represented in the severe ADHD subtype. Finally, ACEs predicted high ADHD symptomatology with co-existing IED, but not without IED. Because ACEs were positively associated with the occurrence of ADHD/IED and ADHD is one important risk factor for on-going criminal behaviors, our findings highlight the need for early identification of ACEs and ADHD/IED in young offenders to identify those adolescents who are at increased risk for long-lasting criminal careers. Furthermore, they contribute to the debate about how to best conceptualize ADHD regarding further emotional and behavioral disturbances. Delinquency committed by adolescents and young adults is a common phenomenon; however, whereas most juveniles overcome offending when entering adulthood, some of them continue to develop long-lasting criminal careers . With rePsychiatric impairments have been related to elevated risk of delinquency in young people: High rates of various psychiatric disorders were found among young detainees –5. AlthoAttention-deficit/hyperactivity disorder (ADHD) is one externalizing disorder, which has received increased attention in research on juvenile and adult delinquency. Previous findings indicate that children with ADHD (with and without psychiatric comorbidity) show an elevated risk of early, persistent, and versatile crime involvement –13. MetaHowever, research on the association between ADHD and delinquency has faced several complications. There has been an ample debate about the conceptualization of ADHD. Sole reliance on an ADHD diagnosis risks to undermine specific differences among the three presentations  described in the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) ; yet, thWith regard to further examining emotional and behavioral problems accompanying ADHD, one psychiatric disorder appears of specific relevance: intermittent explosive disorder (IED). According to DSM-5 , IED refAdditionally, neglecting shared etiological factors that contribute to ADHD, co-existing emotional/behavioral problems, and delinquency holds the risk to draw inadequate conclusions and implications. Adverse childhood experiences (ACEs) display such influencing factors. ACEs are common among young offenders and were proven to contribute to ADHD and the occurrence and maintenance of delinquency , 36–44. The present study intended to overcome some of the abovementioned shortcomings of previous research . First, according to the dimensional character and heterogeneity of ADHD , 23, 51,The present study was conducted as part of a pilot study that aimed to examine the feasibility of scientific data assessment in the below-mentioned juvenile detention center. The study protocol was approved by the ethical review board of the Medical Council in Rhineland-Palatine, Germany (reference number: 837.290.17 (11124); approval date: 21st September 2017). Study procedures were performed in accordance with the ethical standards of the Declaration of Helsinki. Data assessment took place at the juvenile detention center in Worms, Germany. The German juvenile law is usually applied for young people aged 14–21 years (at the time of offending); however, the maximum age may be expanded under specific circumstances  are summarized for one verdict). Juvenile detention is defined as an educational intervention for adolescents and young adults. In contrast, youth prison reflects a measure of punishment. Thus, juvenile detention is usually arranged for adolescents or young adults with rather minor offenses who have not yet been involved in chronic crime. The maximum duration of juvenile detention is 30 days. All adolescents and young adults who had to stay for at least 7 days in juvenile detention between May 2018 and May 2019 were provided study information via mail about three weeks before the beginning of sentence. There were no further inclusion or exclusion criteria. The document contained information about (1) study procedures, (2) the voluntariness of participation including the possibility to withdraw consent at any time, (3) the scientific purpose of data collection, (4) the anonymization of data, and (5) the fact that consent or denial of study participation would not have any consequences concerning the given sentence.In case of study participation, written informed consent was given by the detainee  at the beginning of sentence. Self-report questionnaires were handed over on the first day of detention to be filled out at the detainees’ private detention rooms. Completing the questionnaires took about one hour. In case a participant did not understand the content of single items, she/he could ask a responsible staff member at the juvenile detention center for assistance. Completed questionnaires were collected in a closed box.M = 18.53 years, SD = 2.13 years). The mean length of detention was 2.11 weeks . Detainees showed an average school education of 9.29 years . Males and females did not differ concerning age, length of detention, or years of education (p > 0.05).Since the present study represents (parts of) a pilot/feasibility study, we did not apply any sample size calculations in advance. Data were collected from a consecutive sample of a total of 161 adolescents or young adults  with a mean age of 18.48 years . For the present study, data were considered only from those detainees who had given full information on the questionnaires concerning ADHD and IED symptoms, leaving a total of 156 participants  between 14 and 25 years , property offenses , breach of narcotics law , breach of school law/excessive school skipping , driving without driver`s license , and others . No sex differences were found, χ2(5) = 4.46, p = 0.485.Index offenses (most severe per participant) included (grievous) bodily harm (ADHD symptoms based on the Utah Criteria were assessed by the German version of the SR-WRAADDS (WR-SB) , 60, 61,α for all subscales were between 0.77 and 0.90.Satisfactory psychometric properties were proven for the English and German versions of the WR-SB , 60. In IED was assessed using the IED-SQ . The IEDк = 0.80), test–retest reliability (к = 0.71), 82% sensitivity, 97% specificity, and overall accuracy = 0.90  are naturally sorted” [Some qualifications must be considered for the interpretation of the present findings. Due to the primary purpose as pilot/feasibility study, data was solely assessed by self-reports and not expanded to external evaluation  by clinical experts. The validity of self-reports on ADHD symptoms, further psychiatric impairments, and ACEs has been debated, especially in forensic samples , 6, 83.  sorted” . Further sorted” .In conclusion, the present study showed that not only chronic and severe criminals but also young offenders at the beginning of their delinquent development are afflicted with ADHD, IED, ACEs, and further internalizing and externalizing problems. Our findings highlight the need for early identification of crime-promoting factors in young offenders, which requires standardized but individualized assessment of the young offenders’ (or high-risk samples’) risks and needs by psychiatrically/psychologically educated experts. Practitioners in psychiatry, psychology, and law enforcement as well as politicians and other stakeholders need to work together towards the realization and implementation of tailored interventions, not only to protect society from future crime and reduce respective economic costs, but also to allow young offenders and high-risk youths to develop functional and crime-free future prospects.


The hUC-MSCs caused significant decreases in emphysema severity by measuring the mean linear intercept (MLI) and destructive index (DI). A decrease in neutrophils (%) and an increase in lymphocytes (%) in bronchoalveolar lavage fluid (BALF) were observed in emphysematous mice after hUC-MSC treatment. Lung levels of interleukin (IL)-1β, C-X-C motif chemokine ligand 1 (CXCL1)/keratinocyte chemoattractant (KC), and matrix metalloproteinase (MMP)-12 significantly decreased after hUC-MSC administration. Significant reductions in tumor necrosis factor (TNF)-α, IL-1β, and IL-17A in serum occurred after hUC-MSC administration. Notably, the cell viability of lung fibroblasts improved with hUC-MSCs after being treated with CS extract (CSE). Furthermore, the hUC-MSCs-conditioned medium (hUC-MSCs-CM) restored the contractile force, and increased messenger RNA expressions of elastin and fibronectin by lung fibroblasts. In conclusion, hUC-MSCs reduced inflammatory responses and emphysema severity in CS-induced emphysematous mice.Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were shown to have potential for immunoregulation and tissue repair. The objective of this study was to investigate the effects of hUC-MSCs on emphysema in chronic obstructive pulmonary disease (COPD). The C57BL/6JNarl mice were exposed to cigarette smoke (CS) for 4 months followed by administration of hUC-MSCs at 3 × 10 Chronic obstructive pulmonary disease (COPD) is currently one of the world's highest causes of mortality and ranks fifth worldwide in terms of disease burden –3. AboutMesenchymal stem cells , multipotent SCs, have high self-renewal and differentiation capacities . Recent in vivo -MSCs have a higher differential capacity, lower immunogenicity, and less age-related dysfunction compared to adult SCs . Other ain vivo .Lung fibroblasts were shown to have an important role in repairing damaged lung tissues after CS exposure . HoweverDespite the efficacy of MSCs in ameliorating acute lung damage, few studies have investigated the effects of hUC-MSCs on chronic CS-induced emphysema. The objective of this study was to investigate the therapeutic efficacy of hUC-MSCs in emphysema.n = 8–10 per group) were obtained from the National Laboratory Animal Center . Mice were housed in plastic cages and supplied with Lab Diet 5001  and water ad libitum. A light/dark cycle of 12 h/12 h was maintained. The room temperature was set to 22 ± 2°C, and relative humidity to 55 ± 10%.The animal study was approved by the Animal and Ethics Review Committee of the Laboratory Animal Center at Taipei Medical University, Taipei, Taiwan (IACUC: LAC-2017-0231). Male C57BL/6JNarl mice  was monitored using a DustTrak monitor . 2.5 mass concentration during CS exposure. The average PM2.5 mass concentration was 90.5 ± 40.6 mg/m3 during the first 15 min. It reached a maximum level of about 154.3 ± 58.2 mg/m3 after 4 min of cigarette combustion, and then the mass concentration declined to the baseline level after 16 min. Simultaneously, mice exposed to CS-free high-efficiency particulate air (HEPA)-filtered room air (RA) served as the control group.An emphysema mouse model was established by whole-body exposure to CS for 4 months. Details of the CS exposure system were previously reported . Briefly2 at 37°C for 3 days. hUC-MSCs were characterized using flow cytometry  to detect expressions of cluster of differentiation (CD) markers  and human leukocyte antigen–antigen D related (HLA-DR). As presented in Details of hUC-MSC preparation were previously reported . Briefly6 cells/kg body weight (BW) for CS + MSC-L, 1 × 107 cells/kg BW for CS + MSC-M, and 3 × 107 cells/kg BW for CS + MSC-H. The administered dose of hUC-MSCs was referenced to our previous reports . The mean linear intercept (MLI) and destructive index (DI) were used to evaluate the presence of emphysema. The MLI was assessed by counting the number of the alveolar walls intercepted in the grid lines, according to previously described methods , 28. TheBALF was centrifuged at 1,500 rpm for 10 min at 4°C. Cell pellets were resuspended in PBS. Numbers of neutrophils, lymphocytes, monocytes, and eosinophils were quantified by a hematology analyzer . Data are expressed as percentages (%) of total cell counts.Lysis buffer was prepared from 490 μL of lysis reagent  containing 5 μL of a protease inhibitor  and 5 μL of ethylenediaminetetraacetic acid. Lung tissues were homogenized in lysis buffer using a homogenizer .A CBA  was used to quantify levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, chemokine (C-X-C motif) ligand 1/keratinocyte chemoattractant (CXCL1/KC), and IL-17A in BALF, lung, and serum samples. Matrix metalloproteinase (MMP)-12 was determined in lung samples by an ELISA . Quantification of these markers in lung samples was normalized to the total protein. All measurements were undertaken in accordance with the manufacturers' instructions.Human lung fibroblasts (MRC-5 cells) were obtained from the Food Industry Research and Development Institute  and cultured in T75 flasks with Eagle's minimum essential medium  supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate.6 cells) were cultured in T75 flasks with 15 mL of hUC-MSC culture medium for 24 h. After being washed with PBS, the culture medium was replaced with 10 mL of α-MEM basal medium  and incubated for 48 h. The subsequent serum-free culture medium was collected and served as hUC-MSCs-CM.To collect hUC-MSCs-CM, hUC-MSCs  by impinging onto 30 mL of α-MEM (Invitrogen) with a firm filter. The cigarette contained 10 mg of tar and 0.8 mg of nicotine. Fresh CSE was collected to serve as 100% CSE and immediately used for cell experiments.MRC-5 cells were treated with 8% CSE for 24 h and then indirectly cocultured with hUC-MSCs for another 48 h. Cell viability of MRC-5 cells was determined by a cell counting kit-8 .5/cells) seeded in six-well plates were treated with 8% CSE for 24 h. After CSE exposure, cells were cultured in hUC-MSCs-CM for 24 h. The cell contractile force was measured using a collagen-based cell contraction assay kit . Messenger (m)RNA expressions of elastin and fibronectin were analyzed by a quantitative polymerase chain reaction (qPCR), according to the manufacturer's instructions.MRC-5 cells . p < 0.05 was considered statistically significant.Data are presented as the mean ± standard deviation (SD). Multiple groups were compared by an analysis of variance (ANOVA) with Tukey's p < 0.001). Furthermore, the DI (%) significantly decreased after hUC-MSC administration .Results of the histological analysis are shown in p < 0.001). In contrast, lymphocytes (%) significantly increased after hUC-MSC administration compared to the CS group . There was no statistical difference in monocytes (%) or eosinophils (%) among the groups. Also, we observed no statistical difference in TNF-α, IL-1β, CXCL1/KC, or IL-17A in BALF after hUC-MSC administration (As shown in stration .p < 0.05) and CXCL1/KC  in lung lysates significantly decreased after hUC-MSC administration compared to the CS group (p < 0.001). There was no significant change in TNF-α or IL-17A levels among all groups.Levels of IL-1β , IL-1β , and IL-17A  compared to the CS group. No significant reduction in CXCL1/KC was found when compared among all groups.TNF-α, IL-1β, CXCL1/KC, and IL-17A levels in serum of mice were examined . hUC-MSCp < 0.05). The contractile force of MRC-5 cells as determined by the collagen gel surface area was significantly reduced by hUC-MSCs-CM treatment compared to the CSE group  and proteases (MMP-9 and−12) in the lungs of rats decreased by MSC administration after CS exposure for 11 weeks . In addiThe emphysema severity was significantly decreased by hUC-MSCs in emphysematous mice based on the MLI and DI results. Previous studies showed a decrease in emphysematous lesions in the lungs of mice due to bone marrow (BM)-MSCs , 61. OthFibroblasts play an important role in regulating COPD severity. We observed that the cell viability of lung fibroblasts increased by hUC-MSC administration after CSE exposure. In addition, hUC-MSCs-CM restored collagen's contractile force in lung fibroblasts after treatment with the CSE. MSCs-conditioned medium (MSCs-CM) was compatible with MSCs in attenuating inflammation in bronchopulmonary dysplasia . A previin vivo are still unclear, which should be evaluated in future work.There are a few limitations in this study. We observed an increase in lymphocytes in BALF of mice due to hUC-MSC administration. The different subgroups of lymphocytes, including regulatory T cells, were not determined in our study. In addition, interactions of hUC-MSCs with lymphocytes are not fully understood. The pulmonary function and the underlying mechanism of the hUC-MSCs in COPD will be determined in the future. Moreover, the adverse effects of fibroblasts by hUC-MSCs In conclusion, hUC-MSCs reduced the emphysema severity and inflammatory responses in mice with CS-induced emphysema. hUC-MSCs increased the proliferation of fibroblasts after CSE exposure. hUC-MSCs may mitigate COPD in mice after CS exposure.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of the National Cheng Kung University Hospital Institutional Review Board . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Animal and Ethics Review Committee of the Laboratory Animal Center, Taipei Medical University, Taipei, Taiwan (IACUC: LAC-2017-0231).H-CC and X-YC contributed to interpretation of the data and completion of the manuscript. H-CC, WL, and KC contributed substantially to the concept, design, interpretation of the data, and completion of the study and manuscript. Y-YC and C-HC contributed substantially to the completion of the study. T-CH contributed to the establishment of the cigarette smoke generation system and particle measurement. All authors contributed to critically revising the manuscript for important intellectual content and read and approved the final manuscript.This study was supported by a Grant from Meridigen Biotech Co., Ltd., Taipei, Taiwan (2017-TR-VIV-001). The fund was used for designing the study, collecting, analyzing, interpreting the data, and writing the manuscript.WL, C-HC, and Y-CW are employed by Meridigen Biotech Co., Ltd. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this review, we undertake a critical appraisal of eight published studies providing first evidence that a history of attention-deficit/hyperactivity disorder (ADHD) may increase risk for the later-life development of a neurodegenerative disease, in particular Lewy body diseases (LBD), by up to five-fold. Most of these studies have used data linked to health records in large population registers and include impressive sample sizes and adequate follow-up periods. We identify a number of methodological limitations as well, including potential diagnostic inaccuracies arising from the use of electronic health records, biases in the measurement of ADHD status and symptoms, and concerns surrounding the representativeness of ADHD and LBD cohorts. Consequently, previously reported risk associations may have been underestimated due to the high likelihood of potentially missed ADHD cases in groups used as “controls”, or alternatively previous estimates may be inflated due to the inclusion of confounding comorbidities or non-ADHD cases within “exposed” groups that may have better accounted for dementia risk. Prospective longitudinal studies involving well-characterized cases and controls are recommended to provide some reassurance about the validity of neurodegenerative risk estimates in ADHD. Attention-deficit hyperactivity disorder (ADHD) is a psychiatric disorder beginning in childhood that is characterized by core symptoms of inattention, impulsivity, and hyperactivity , and the authors evaluated the risk of subsequent basal ganglia diseases including LBD  and representativeness of the exposed cohort—were flawed across almost all studies: including non-ADHD hyperkinetic syndromes or restricting the sample to those with numerous outpatient visits or hospitalizations results in samples that do not likely represent most ADHD cases, and non-exposed cases were also not systematically assessed to rule out ADHD. Likely, these methodological issues result in a conservative estimate of true effects; that is, without the “noise” generated by the inclusion of non-ADHD cases in the exposed group and potentially missed ADHD in the non-exposed group, associations between exposure and outcome may be even stronger. A second possibility is that the inclusion of non-ADHD cases in the exposed group is driving observed effects. As previously noted, many non-ADHD hyperkinetic disorders—which were included in the exposed cohort in four of the five cohort studies  in most studies. However, six (Curtin et al., These studies provide tentative support for ADHD as a potential risk factor for later development of a neurodegenerative disease. Due to the methodological limitations and biases we have identified, we argue that the true association between ADHD and neurodegeneration is not yet identifiable. The sources of biases identified here should be considered in future studies to ascertain the true relative risk of neurodegeneration in patients with ADHD. Accurate and systematic diagnoses of ADHD and neurodegeneration are needed.BC conceptualized the work. SB, MS, and BC made substantial contributions to the critical analysis and interpretation of data. SB, MS, and BC wrote initial drafts of the manuscript. SB and BC wrote the final draft. All authors revised it critically for important intellectual content, approved the version to be published, and agree to be accountable for all aspects of the work.The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Effective treatments for acute-on-chronic liver failure (ACLF) are lacking. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have been applied in tissue regeneration and repair, acting through paracrine effects, cell fusion, and actual transdifferentiation. The present study was designed to investigate the therapeutic potential of hUC-MSCs in acute-on-chronic liver injury (ACLI) and ACLF rat models.6 hUC-MSCs, 4×106 hUC-MSCs, or 0.9% sodium chloride as a control. The rats were sacrificed at 1, 2, 4, and 6 weeks (ACLI rats) or 4, 12, and 24 h (ACLF rats). The blood and liver tissues were collected for biochemical and histological investigation.Wistar rats aged 6 weeks were intraperitoneally administered porcine serum (PS) at a dose of 0.5 mL twice per week for 11 weeks to generate an immune liver fibrosis model. After 11 weeks, rats with immune liver fibrosis were injected intravenously with lipopolysaccharide (LPS) to induce an ACLI model or combined LPS and D-galactosamine  to induce an ACLF model. The rats with ACLI or ACLF were injected intravenously with 2×10The application of hUC-MSCs in rats with ACLI and ACLF led to a significant decrease in the serum levels of ALT, AST, TBil, DBil, ALP, ammonia, and PT, with ALB gradually returned to normal levels. Inflammatory cell infiltration and collagen fiber deposition in liver tissues were significantly attenuated in ACLI rats that received hUC-MSCs. Inflammatory cell infiltration and apoptosis in liver tissues of ACLF rats that received hUC-MSCs were significantly attenuated. Compared with those in the rats that received 0.9% sodium chloride, a significant reduction in proinflammatory cytokine levels and elevated serum levels of hepatocyte growth factor (HGF) were found in ACLF rats that received hUC-MSCs. Furthermore, Notch, IFN-γ/Stat1, and IL-6/Stat3 signaling were inhibited in ACLI/ACLF rats that received hUC-MSCs.hUC-MSC transplantation can improve liver function, the degree of fibrosis, and liver damage and promote liver repair in rats with ACLI or ACLF, mediated most likely by inhibiting Notch signaling and reversing the imbalance of the Stat1/Stat3 pathway.The online version contains supplementary material available at 10.1186/s13287-021-02468-6. Chronic liver disease (CLD) refers to a type of disease with a history of cirrhosis or noncirrhosis of the liver for more than 6 months. According to the latest global disease burden research data, 1.5 billion people worldwide suffered from CLD in 2017, the most common types of which are nonalcoholic fatty liver disease (NAFLD), viral hepatitis, and alcoholic liver disease . The curBacterial /virus infection or acute alcoholic hepatitis that occurs on the basis of CLD can induce innate immunity or an acquired immune response and induce systemic inflammation (SI). In severe cases, it progresses to systemic inflammatory response syndrome (SIRS), and the more severe the SIRS is, the worse the prognosis . Serum l+ cells to promote hepatocyte regeneration, but its ability to repair liver tissue is limited [Therefore, immune regulation and promotion of liver cell regeneration are two important aspects of ACLF therapy. According to ACLF pathogenesis, early immunosuppressive therapy can block SIRS and reduce hepatocyte necrosis and extrahepatic organ dysfunction caused by immune damage. A meta-analysis indicated that early treatment of HBV-ACLF with glucocorticoids can significantly improve the survival rate without increasing the incidence of secondary infection and bleeding . However limited .Mesenchymal stem cells (MSCs) have the potential for self-renewal and multipotent differentiation. In theory, they can repair diseased cells or reconstruct normal liver cells and tissues through transplantation, providing new ideas for the treatment of liver injury diseases , 21. HumIn the present study, ACLI and ACLF were induced in rats, and different doses of hUC-MSCs were transplanted to investigate the effectiveness and mechanism of hUC-MSC transplantation.Wistar rats weighing 120–150 g were obtained from the Experimental Animal Research Center of Hubei Province . Animal care and all experimental procedures were approved by ethical review by the Laboratory Animal Management and Use Committee of the Center for Disease Control of Hubei Province.hUC-MSCs were provided by Shenzhen Beike Cell Engineering Research Institute. hUC-MSCs from Wharton’s jelly were isolated by a non-enzymatic method and culture-expanded as described in previous report. The inf6 cells/mL per rat (n = 12) or hUC-MSCs at a concentration of 4×106 cells/mL per rat (n = 12). In the control group, the rats received 1 mL of 0.9% sodium chloride (n = 12). Three rats in each group were sacrificed at 1, 2, 4, and 6 weeks after hUC-MSCs or 0.9% sodium chloride injection, and blood samples and liver tissues were collected for biochemical and histological investigation   at a dose of 0.5 mL twice per week for 11 weeks to generate an immune liver fibrosis model. After 11 weeks, rats with immune liver fibrosis were intravenously injected with lipopolysaccharide (LPS)  at a dose of 50 μg/kg to generate an ACLI model. After 1 h, the rats were divided into three groups. In the hUC-MSC groups, the rats underwent intravenous tail vein transplantation of hUC-MSCs at a concentration of 2×106 cells/mL per rat (n = 20) or 4×106 cells/mL per rat (n = 20). In the control group, control rats received 1 mL of 0.9% sodium chloride (n = 20). Three to four rats in each group were sacrificed at 4, 12, and 24 h after hUC-MSC injection, and blood samples and liver tissues were collected for biochemical and histological investigation  twice per week for 11 weeks to generate an immune liver fibrosis model. After 11 weeks, rats with immune liver fibrosis were intravenously injected with LPS at a dose of 50 μg/kg. Thirty minutes later, D-galactosamine   was intraperitoneally injected at a dose of 600 mg/kg to induce an ACLF model . After 1The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBiL), direct bilirubin (DBiL), alkaline phosphatase (ALP), and albumin (ALB), and plasma level of ammonia were detected with an automatic analyzer .The plasma prothrombin time (PT) and international normalized ratio (INR) were detected with an automatic analyzer .Liver tissues were fixed in 4% paraformaldehyde solution. The liver tissue samples were processed using the paraffin block technique in wax, deparaffinization in xylene, and dehydration in alcohol. The samples were then sectioned (5 μm) and stained with hematoxylin-eosin (HE) to evaluate the pathological changes and Masson staining to demonstrate the collagen deposition that had occurred in the liver. The sections were then examined and photographed by light microscopy .Immunohistochemistry staining was conducted using a previously described method . The folThe serum levels of hyaluronic acid (HA) and N-procollagen type III peptide (PIIINP) were detected by ELISA kits . The serum levels of TNF-α, IFN-γ, IL-6, interleukin-1β (IL-1β), transforming growth factor-β1 (TGF-β1), interleukin-4 (IL-4), IL-10, and HGF were detected by ELISA kits .-△△Ct method was used to analyze the real-time PCR data. The primers were provided in Table SThe total RNA was isolated from liver tissues with TRIzol regent  according to the manufacturer’s instructions. Approximately 1 μg of total RNA from each sample was used to synthesize cDNA using the HiScript II Q RT SuperMix for qPCR  according to the manufacturer’s specification. Then, quantitative real-time PCR was performed by using SYBR Green Master Mix , with 15 s at 95°C and 60 s at 60°C for 40 cycles. GAPDH was used as the reference gene for calculations. The 2Protein was extracted from liver tissues with RIPA lysis buffer  containing protease inhibitor  and phosphatase inhibitor . Equal protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. The membranes were incubated sequentially with appropriate primary antibodies and secondary antibodies. Immune complexes were visualized by ECL detection reagent , and band intensities were determined using the BandScan software. Primary antibodies used in this study were as follows: rabbit anti-GAPDH mAb , mouse anti-Stat3 mAb , rabbit anti-Phospho-Stat3 mAb , rabbit anti-Stat1 mAb , rabbit anti-Phospho-Stat1 mAb , rabbit anti-CyclinD1 mAb , rabbit anti-c-Myc mAb , and mouse anti-Bcl2 mAb . Secondary antibodies used in this study were horseradish peroxidase-conjugated anti-mouse IgG  and anti-rabbit IgG .P <0.05 was considered statistically significant.All data are expressed as the mean ± standard deviation (SD). Comparisons between two groups were assessed by the Mann-Whitney U test. Kruskal-Wallis test with Dunn’s multiple comparisons post-test analysis was used to compare among three groups. At 11 weeks post-PS injection, the HA (232.1±90.39) and PIIINP (85.26±36.23) levels in the treated group were significantly increased compared to those in the normal group Fig. a. These 6 hUC-MSCs returned to normal levels, but serum ALT, AST, and TBil levels and other indicators did not return to normal levels after hUC-MSC treatment for 6 weeks , a large amount of CD90-positive cells were presented in the liver tissues from both ACLI and ACLF rats received hUC-MSC therapy, while not in the liver tissues from rats received 0.9% sodium chloride Figure S. This re6 hUC-MSC treatment group rats, while collagen deposition in the 4×106 hUC-MSC treatment group rats was not completely relieved . There was no significant difference between 2×106 hUC-MSCs and 4×106 hUC-MSCs in inhibiting α-SMA and desmin expression in rat liver. However, the expression levels of MMP9 and TIMP1 in rat livers were not significantly different between the control group and the hUC-MSC treatment group . The expression of HGF and PCNA in rats receiving 4×106 hUC-MSCs or 2×106 hUC-MSCs showed no significant difference, but strangely, there was no significant increase in the expression of AFP in the 4×106 hUC-MSC treatment group at 24 h. At 24 h after hUC-MSC transplantation, the expression of AFP and PCNA in liver tissues of the control group and in those of the 4×106 hUC-MSC treatment group was not significantly different, while the expression of CK18 and HGF in liver tissues of the rats treated with 4×106 hUC-MSCs was significantly higher than that in in liver tissues of the rats in the control group (P < 0.05)  was significantly higher than that in the control rats from 4 to 24 h (P < 0.001)  Fig. a–c. In a01) Fig. d.Fig. 7After injection with hUC-MSCs in ACLI rats, serum levels of TNF-α, IFN-γ, IL-6, IL-1β, TGF-β1, and IL-4 gradually decreased, while IL-10 levels gradually increased Fig. .Fig. 8T6 hUC-MSCs were lower than those in rats treated with 2×106 hUC-MSCs although there was no statistically different      secrete a large amount of ECM, which eventually leads to collagen formation and fibrosis/cirrhosis . HSCs arSublarge hepatic necrosis is the main pathological feature of ACLF and the pathological basis of liver failure . The livIn this study, there were no differences in the expression of AFP and PCNA in the liver tissues of ACLF rats in the control group and in the liver tissues of rats in the high-dose hUC-MSC group after 24 h. On the one hand, the liver itself may have certain reactive hepatocyte regeneration after liver injury; on the other hand, this result may be related to the small sample size and the control of the hUC-MSC infusion process. As mentioned above, the PT and INR of ACLF rats treated with hUC-MSCs or 0.9% sodium chloride showed a large variation after 24 h, and there was no significant difference between the hUC-MSC treatment group and the control group.SIRS is the initiating factor of ACLF and the driving factor of disease progression . When afIn addition, this study showed that high-dose hUC-MSC treatment was superior to low-dose hUC-MSC treatment in improving the liver function and inhibiting the production of proinflammatory cytokines in ACLI and ACLF rats. These results indicated that the high dose may have better efficacy, especially in the treatment of ACLF, which may exert comprehensive effects, such as immune regulation, liver function improvement, antifibrosis, and hepatocyte regeneration promotion. The specific infusion dose needs to be further optimized.In summary, the present study proved for the first time that hUC-MSC transplantation has a good effect on both ACLI and ACLF rats. hUC-MSC transplantation can improve liver function, reduce liver injury/hepatocyte necrosis, and promote hepatocyte regeneration. hUC-MSCs may regulate the degree and progress of the immune response through paracrine mechanisms by upregulating HGF production to inhibit HSC activation, thereby interrupting the pathophysiological process of ACLI/ACLF, promoting the repair of liver tissue, and finally achieving the effect of treating ACLI/ACLF. And the downregulation on Notch, IFN-γ/Stat1, and IL-6/Stat3 signaling may contribute to the effect of hUC-MSCs in fibrosis inhibiting and hepatocyte regeneration. This study provided a theoretical basis and preclinical research evidence for the clinical application of hUC-MSC transplantation to treat ACLI and ACLF. However, due to the limited number of rats in this study, the long-term efficacy and the improvement in mortality due to hUC-MSC treatment for ACLF were not studied. Further study is required to evaluate the role of hUC-MSCs in decreasing the mortality of ACLF, and optimization of hUC-MSC treatment, especially in combination with agents targeting Notch and Stat1/Stat3 pathways.Additional file 1: Figure S1. The Phenotypes of hUC-MSCs. Flow cytometry analysis of hUC-MSCs for MSCs related markers. hUC-MSCs used for infusion were stained with CD105 (a), CD90 (b), CD73 (c), CD45 (d) and CD34 (e).Additional file 2: Table S1. Primers for quantitative real-time PCR analysis.Additional file 3: Figure S2. The integration of transplanted hUC-MSCs in the injured liver. Liver sections from ACLF and ACLI rats 24 hours post-hUC-MSC transplantation or 0.9% sodium chloride injection as a control were used for immunohistochemically staining of human-specific CD90.Additional file 4: Figure S3. The expression of Notch signaling pathway related genes and Stat1/Stat3 signaling molecules in ACLI and ACLF rats. Total RNA and protein were extracted from the liver tissues from rats with ACLI or ACLF transplanted with hUC-MSCs or 0.9% sodium chloride as a control (n = 3). The mRNA expression levels of Notch1, Hes1, and P21 in ACLI rats were detected by quantitative PCR (a). Total protein was subjected to western blotting analysis. Relative protein expression of Stat1, pStat1, Stat3 and pStat3 in ACLF rats were quantitated (b). Relative protein expression of Stat1, pStat1, Stat3, pStat3, c-Myc, Bcl2 and CyclinD1 in ACLI rats were quantitated (c-f). Data were presented as the mean ± SD. *P < 0.05.


Temporal processing (TP) is associated with functions such as perception, verbal skills, temporal perspective, and future planning, and is intercorrelated with working memory, attention, and inhibitory control, which are highly impaired in individuals with attention deficit hyperactivity disorder (ADHD). Here we evaluate TP measures as potential endophenotypes in Caribbean families ascertained from probands affected by ADHD. A total of 232 individuals were recruited and clinically evaluated using an extensive battery of neuropsychological tasks and reaction time (RT)-based task paradigms. Further, the heritability (genetic variance underpinning phenotype) was estimated as a measure of the genetics apportionment. A predictive framework for ADHD diagnosis was derived using these tasks. We found that individuals with ADHD differed from controls in neuropsychological tasks assessing mental control, visual-verbal memory, verbal fluency, verbal, and semantic fluency. In addition, TP measures such as RT, errors, and variability were also affected in individuals with ADHD. Moreover, we determined that only omission and commission errors had significant heritability. In conclusion, we have disentangled omission and commission errors as possible TP endophenotypes in ADHD, which can be suitable to assess the neurobiological and genetic basis of ADHD. A predictive model using these endophenotypes led to remarkable sensitivity, specificity, precision and classification rate for ADHD diagnosis, and may be a useful tool for patients’ diagnosis, follow-up, and longitudinal assessment in the clinical setting. Attention deficit hyperactivity disorder (ADHD) is a complex neurodevelopmental disorder that affects up to 5–16% of children and adolescents worldwide ,4,5,6,7.Given the highly variable clinical manifestations of ADHD, it has been suggested that endophenotypes might be a better approach to dissect genetic and neurophysiological basis of ADHD. The endophenotype concept refers to markers that are more directly connected to neuropsychological, behavioral, or neuroanatomical components associated with the disease ,12,13. ESeveral ADHD studies have identified potential cognitive endophenotypes in neuropsychological tasks ,20,21,22TP is associated with functions such as perception, verbal skills, temporal perspective, and future planning. These time-dependent functions are intercorrelated with other impaired functions in ADHD such as working memory, attention, and to a lesser degree inhibitory control. These key timing deficits appear to survive when these functions are controlled, suggesting independent cognitive deficits in the temporal domain . Strong RTs are defined as the elapsed time  between the presentation of a stimulus until the reaction or response and constitute a measure of information processing. Errors of commission provide an index of faster information activation  , while oIn addition to the aforementioned deficits, deficiencies in the perception and subjective appreciation of time are equally relevant ,27. ADHDTo our knowledge, there are no reports of family-based studies in Caribbean communities, especially in those of significant African admixture, that investigate different measures of TP as possible cognitive endophenotype in patients diagnosed with ADHD. Here we explored the definition of TP endophenotypes in a sample of 232 individuals belonging to 67 nuclear families from Barranquilla, Colombia, the biggest city of the Northern Colombian Caribbean coast, who were clinically characterized using neuropsychology batteries as well as TP measures using RT-based tasks. Our overarching hypothesis was that there were specific patterns of TP and the existence of different endophenotypes in this community that represent a significant variance of the ADHD symptomatology, which could play an important role in neuropsychological models of TP ,40,41,42We recruited 232 individuals from 67 nuclear families segregating ADHD with at least a single ADHD-affected individual, from patients attending our research program in ADHD advertised in the Grupo de Neurociencias del Caribe’s website . All indWe performed an extensive clinical, neurological, and neuropsychological evaluation to define ADHD status and the presence of other comorbidities as described elsewhere ,57,58,59This test assesses individual’s ability of selective attention  and requires individuals to suppress automatic responses in favor of a specific response requested by the evaluator . The tesSimilar to Toulouse-Pierrón test , the croThis is a paper-and-sheet test with two parts. In Part A, numbers 1 to 25 are randomly presented in a paper sheet. Individuals should connect, as fast as possible, the numbers in consecutive order using a straight line . In Part B numbers 1 to 13 and letters A to L are randomly in a paper sheet. The task consists of connecting, alternating in consecutive order, numbers and letters  ,75,76,77This is a computerized test in which stimuli consists of letters presented every 250 ms ,79,80. AWe administered a two-part Go/No-Go (GNG) task using visual signals ,83. PartU test for two independent samples. Uncorrected Cohen’s d was calculated to measure the effect size for all variables . I. I100]. We found that ADHD affected individuals differed from unaffected individuals in omissions and perseverance of the CPT . OmissioIt is noteworthy that the COST showed a differential pattern between ADHD affected and unaffected individuals in our sample . Indeed,Our findings also indicate that ADHD affected individuals committed more lecture errors in the Stroop task compared to unaffected individuals and this measure was also heritable and there is a tendency for ADHD affected to spend more time to complete the task . This diIn addition to genetic aspects, the aforementioned neuropsychological alterations have been proposed as working models in ADHD for the development of more precise diagnostic tools, and to obtain better treatment responses ,132,133.The idea of a TP deficit in ADHD is coherent with different models, where TP is a complex cognitive process that involves multiple components to modulate time-associated tasks. In ADHD, TP highlights the importance of time perception and task-specific demands and coincides with current ADHD models. The ARPA-based predictive model of ADHD diagnosis includes, in addition to demographic variables such as age and sex, three TP endophenotypes derived from neuropsychological instruments used to assess TP . This prADGRL3, SNAP-25, FGF1 and DRD4 genes, which were previously reported to be associated with ADHD [Future research directions could potentially include the study of TP measures, RT variability and the trial-by-trial performance variability using entropy-based measures and transitions between states  that might be useful to better understand, from a different perspective, the differential evolution of pattern of individuals with ADHD when compared to controls. Using our family-based cohort, these findings could be subsequently integrated to complex segregation, and genetic linkage and association studies of TP towards the development of more precise and differential ADHD diagnosis ,145,146.ith ADHD ,151,152.ith ADHD ,154,155.


Pediatric obesity and Attention Deficit Hyperactivity Disorder (ADHD) are rising health concerns in the United States, especially among Hispanic children and adolescents. Research on Hispanic children and adolescents indicates disproportionately higher prevalence rates of obesity in this community but scant data on ADHD prevalence rates. In contrast, a plethora of research studies across the general population examines the relationship between childhood obesity and ADHD. In addition, there is a lack of research that examines the role of ethnicity and sub-ethnic group correlations in ADHD, particularly in the Hispanic population. Existing studies in the general population indicate ADHD may be a risk factor for being overweight compared to normal controls. The objective of the present study is to examine the prevalence of obesity in children with ADHD compared to children in the general population in a predominately Hispanic sample on the US-Mexico border. A total of 7,270 pediatric medical records were evaluated. The retrospective analysis included Body Mass Index (BMI) and related health variables, and ethnicity and showed that children with ADHD are more likely to be underweight. In conclusion, no significant relationship existed between obesity and ADHD among Hispanic children on the US-Mexico Border, and instead we found the opposite correlation. Pediatric obesity in the United States is increasing rapidly, with a prevalence rate of about 32% of children and adolescents classified as overweight . ObesityHispanic children are more likely to be impacted by obesity-related chronic diseases such as Type 2 Diabetes due to higher insulin resistance and appearance of pre-diabetes than non-Hispanic White children . PediatrExtensive research has documented the impact of health inequality factors, such as lower socioeconomic status (SES) and lack of health insurance, on childhood obesity for Hispanic children. When including migration status, Hispanic children with a low SES are at a greater risk for developing negative health behavior patterns and higher BMI scores . In addiIncreased obesity rates are also prevalent in children with psychiatric disorders such as ADHD, the most common childhood neurodevelopmental disorder . StudiesThe Texas Tech University Health Sciences Center at El Paso operates as a medical teaching healthcare system on the US-Mexico Border. El Paso is a US-Mexico border community with a predominantly Hispanic population and is within close proximity to Ciudad Juarez a major metropolitan city of Mexico. The community is a particularly unique population in that both Mexican-born and US-born, individuals travel back and forth. While we do not have data on the patients’ migration circulatory patterns, we understand that the population is cross-border mobile.We performed a retrospective analysis of electronic health records of pediatric patients receiving services from one of two clinics – our Child and Adolescent Psychiatry clinic and our Geneneral Pediatrics clinic. Patient records were included if the patient was between 6–17 years of age on the date of the visit. Patients records were considered in the analysis if the patient was diagnosed with ADHD meeting Diagnostic and Statistical Manual of Mental Disorders V (DSM-5) criteria. Only the most recent patient visit was considered for analysis in the study.Body mass index calculations were determined using the weight and height at the time of the visit and included age and sex-specific differences. The calculation for BMI was determined using the Center for Disease Control and Prevention (CDC) guidelines for pediatric patients. As a part of routine care, height, weight, age, and sex-specific information is obtained at the time of the visit. Pediatric BMI categories were separated into the following categories: (1) Underweight (<5th percentile); (2) Normal or Healthy Weight (5–85th percentile); Overweight (85–95th percentile); or Obese (>95th percentile).Pediatric patient records selected for data analysis included controls (without a diagnosis of ADHD at any time point) and pediatric patients diagnosed with ADHD (at any time point). Patients were diagnosed with ADHD through (a) a comprehensive developmental history, (b) clinician interview, and (c) direct observation. Patient records were sorted into one of the four subtypes of ADHD. The four subsets included: (1) ADHD-combined type, (2) ADHD-unspecified type, (3) ADHD-predominantly inattentive type, and (4) ADHD-predominantly hyperactive/other types. In addition, we considered any patients diagnosed with ADHD at any point for analysis in the ADHD group.Furthermore, we analyzed patients by ethnicity and language. The definitions for ethnicity and language were pre-determined using Medicaid/Medicare measures, and direct data collection methods were used for this information. For analysis purposes, patients were categorized into traditional general categories by ethnicity – Hispanic or Latino and Not Hispanic or Latino, and language – English or Spanish or Other/missing. The Institutional Review Board (IRB) approved the study. Personal protected information in data collection was de-identified.t-test and Fisher’s Exact test depending on the data distribution. A univariate and multivariable relative risk regression reviewed the risk of ADHD as compared to non-ADHD patients. Relative risk (rr), 95% confidence interval, and p-values describe the models. p-values were considered statistically significant at 5%. Analysis of the data was performed by using Stata software, SE edition.Key descriptive variables  were summarized. Association and relationships between the variables were analyzed using an unpaired We extracted 7,270 electronic health records of pediatric patients receiving clinic services between 2017–2018 . The meaT-Test and Fisher’s exact test, a marginal statistical significance was detected in BMI categories as shown in p < 0.001). Differences were detected among Hispanic or Latino or Not Hispanic or Latino ethnicity diagnosed with ADHD and among those diagnosed with ADHD (p < 0.001). Findings for language detected significant mean differences by language by ADHD status (p < 0.001) (Based on the unpaired < 0.001) .p-value of 0.047. However, ADHD status showed moderate association with the normal weight category . Further, the ADHD group had a decreased risk of being in the overweight category .Logistical regression analyses revealed that ADHD status leads to increased risk of being underweight (<5th percentile) and is statistically significant with a The analysis indicated a 60% decreased risk of being female and being diagnosed with ADHD, indicating the risk of ADHD might be more prevalent in males. There is also a 44% lower risk of being Hispanic and being diagnosed with ADHD. Related to insurance type, a 50% increased risk of being diagnosed if a patient has private insurance versus Medicare/Medicaid was detected .p < 0.006). No significant associations were detected when accounting for ethnicity, but the language showed a significant association to ADHD subset status (p < 0.002). No significant association was found between ADHD subset status and insurance status (p > 0.68)  .When the variables were compared individually with ADHD status, an increased risk of being underweight was identified. Females, Hs, and primary Spanish speakers had a decreased risk of having ADHD. Overall, after considering the quantitative version of BMI and adjusting for gender, ethnicity, and language as the children’s weight increased, there was a lower risk of being diagnosed with ADHD (children diagnosed with ADHD were less likely to be overweight) .This study shows that Hispanic children with ADHD are more likely to be underweight than overweight. While these results add to the growing body of research on the association between ADHD and obesity that indicates lower effects in children, it raiseWhile the focus of the study was to examine the relationship between ADHD diagnosis and BMI, the decreased risk of an ADHD diagnosis associated with being of Hispanic ethnicity. The present findings are consistent with other population-based studies that indicate lower rates of ADHD diagnosis among Hispanic children compared to non-Hispanic children . HoweverRelated to the present study, the methods for diagnosing patients with ADHD are through clinical interview with the patients and parents or guardians and the reporting of symptoms. Perhaps, during those provider-patient interactions, parents or guardians attitudes and beliefs influenced the reporting of symptoms related to mental illness. Prior studies conducted by An interesting result from this retrospective analysis is the finding of increased risk of being diagnosed with ADHD if using private insurance compared to those with Medicaid-related insurance. This is also found in other studies . The reaThere are important limitations to the present retrospective analysis. Primarily, this study relies on less rigorous research methods for diagnosing ADHD than a prospective study. The means for calculating BMI are standardized, yet the methods for arriving at ADHD diagnosis are more variable. Considering our findings on Hispanic ethnicity and ADHD diagnosis and BMI status, interpreting the factors behind these findings would require greater exploration using prospective techniques.Finally, there are limited studies that have examined ADHD and obesity in children in predominantly Hispanic populations, even fewer that explore unique geographic locations where two countries converge, such as this region on the US-Mexico border. Patients with ADHD are less likely to be overweight and obese than those without ADHD. As obesity continues to be a prevalent health problem in American children, it is important to continue finding possible interventions to decrease the rate of obesity in the pediatric population. Further investigations into understanding what factors contribute to these increased rates of ADHD based on ethnicity and insurance status must be conducted to improve diagnosis, treatment and the overall health of communities.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The research protocol has been reviewed by the Institutional Review Board (IRB) at TTUHSC-El Paso. The IRB Committee, Texas Tech University Health Sciences Center, El Paso have reviewed and approved the research protocol for the collection of the participants data for the study. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.AS and SM contributed to this study by initiating the project idea and design, writing the IRB proposal that was submitted and approved, and interpreting the results as they apply to clinical medicine. KC provided the clinical consultation. LA completed the statistical analysis, Texas Tech University Health Science Center El Paso, division of biostatistics and epidemiology. BG conceptualized, analyzed, writing, and editing of the manuscript. ER, JVM, VDP, and SI revised and edited the work critically for important intellectual content and final approval of the version to be published. All authors contributed to the article and approved the submitted version.The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Escherichia coli and purified by glutathione affinity column respectively. The optimal antigen (VP56-3) was screened by enzyme-linked immunosorbent assay (ELISA). Adjuvant FlaB was also expressed in E. coli and purified by Ni2+ affinity column. Subsequently, we prepared the oral vaccines using sodium alginate as a carrier. The vaccine (SA-VP56-3/FlaB) forms microsphere (1.24 ± 0.22 μm), examined by transmission electron microscopy, scanning electron microscopy, and dynamic light scattering assay. SA-VP56-3/FlaB vaccine has excellent stability, slow-release, and low toxicity by dynamic light scattering assay, release dynamic assay, in vivo fluorescence imaging system, hemolytic activity and cytotoxicity. Then we vaccinated grass carp orally with SA-VP56-3/FlaB and measured immune-related parameters , immune-related genes , IgZ in hindgut)). The results showed that SA-VP56-3/FlaB significantly induced strong immune responses, compared to other groups. The highest survival rate achieved in SA-VP56-3/FlaB microencapsulated vaccine (56%) in 2 weeks post GCRV challenge, while 10% for the control group. Meanwhile, the tissue virus load in survival grass carp is lowest in SA-VP56-3/FlaB group. These results indicated that SA-VP56-3/FlaB could be a candidate oral vaccine against GCRV-II infection in aquaculture.Grass carp reovirus (GCRV) is highly infectious and lethal to grass carp, causing huge economic losses to the aquaculture industry annually. Currently, vaccination is the most effective method against viral infections. Among the various vaccination methods, the oral vaccination is an ideal way in aquaculture. However, low protective efficiency is the major problem for oral vaccination owing to some reasons, such as antigen degradation and low immunogenicity. In our study, we screened the antigenic epitopes of GCRV-II and prepared an oral microencapsulated vaccine using sodium alginate (SA) as a carrier and flagellin B (FlaB) as an adjuvant, and evaluated its protective effects against GCRV-II infection in grass carp. The full length and three potential antigenic epitope regions of GCRV-II VP56 gene were expressed in  VP56-3 had the strongest anti-serum binding capacity.Oral vaccine increased protection by 46%.Oral vaccine can slow-release and resistant to intestinal protease action.Ctenopharyngodon idella) is one of the most economically important cultured fish in China  VP56 protein and adjuvant FlaB delivered by sodium alginate against GCRV-II infection to develop a handling stress-free oral vaccine that is easily applicable for mass vaccination of fish with GCRV-II at farm level.C. idella kidney (CIK) cells at 28°C with 5% CO2, in DMEM  medium supplemented with 10% FBS , 100 U/mL penicillin , and 100 U/mL streptomycin .Grass carp reovirus (GCRV-II) used in this experiment is GCRV 097, stored in our laboratory. GCRV 097 was propagated in Escherichia coli (DH5a or DE3plys)  used to express the recombinant protein is stored in our lab in glycerol at -80°C.The Healthy grass carp  obtained from the Xingfu Farm  were maintained in fiberglass tanks (300 L of water) at 28 ± 1°C. The grass carp were fed with commercial feed  once a day (12:00 am), and after two weeks of temporary rearing, the grass carp were divided into 15 tanks with 50 fish per tank. The experiment was divided into five groups, including the control group, SA group, SA-FlaB group, SA-VP56-3 group, SA-VP56-3/FlaB group, each group had three parallel, total of 150 fish (50 × 3), each group of fish was fed once a day for 28 days, feeding rate is 3% of fish weight. Feeding normal feed after the viral challenge. All animal experiments described comply with the National Guidelines for the Care and Use of Laboratory Animals (CNAS-CL06:2018) and were approved by the Committee on the Ethics of Animal Experiments of Huazhong Agricultural University, China (HZAUFI-2021-0028).The antigenic epitopes and hydrophilicity of VP56 (GenBank: MK675081.1) were analyzed by the DNA Star program, and the full length of VP56 was divided into three fragments, VP56-1 (0-420 bp), VP56-2 (421-939 bp), and VP56-3 (940-1461 bp).BamH I and Xho I . The recombinant plasmid  was transformed into E. coli (DH5a) and positive clones were selected by PCR and sequenced by QingKe Bioscience and Technology Company .Total RNA of GCRV 097 (GCRV-II) was extracted with RNAiso Plus kit (TaKaRa), the purification and concentration were measured by a NanoDrop 2000 spectrophotometer , and the quality was evaluated using 1.5% agarose gel electrophoresis. mRNAs were reverse-transcribed into cDNAs respectively with MMLV reverse transcriptase, RNase inhibitor , hexamer random primer. The primers for VP56-1, VP56-2, VP56-3, VP56 were listed in E. coli (DE3plys) and incubated in LB medium at 37°C for 4 h. Then IPTG (1 mmol/L) was added to induce expression at 37°C for 4 h.The recombinant expression plasmids  were transformed into The bacteria were collected by centrifugation . The bacteria was resuspended with PBS (pH = 7.4) and pulverized with a high-pressure pulverizer (880 MPa). The supernatant and precipitate were collected by centrifugation . Purification of recombinant protein  according to the previous method . BrieflyThe grass carp were randomly divided into 5 groups, including the VP56-1 group, VP56-2 group, VP56-3 group, VP56 group, and control group (10 fish in each group). Each group of grass carp was injected intraperitoneally with an equal amount of protein , while the control group was injected with PBS . After three weeks, five fish from each group were anesthetized in MS-222, and blood was collected. The blood was placed at 4°C overnight, then serum was collected by centrifugation  and stored at -80°C.2SO4 and read at 450 nm of wavelength.Antigen binding capacity according to the previous method . Briefly50/mL and incubated at 37°C for 1 h. We added 100 µL of the diluted serum mixture to each well containing CIK cells and incubated the plate at 28°C for 1 h. Then, the mixtures were gently aspirated, and 0.2 mL of fresh DMEM supplemented with 2% FBS was added back to each well. The 96-well microplates were incubated at 28°C for 5-7 days. Negative serum was used as the control group. Survival rate determination of CIK cells by the 3--2,5-diphenyltetrazole (MTT) assay. Briefly, the cell culture medium was replaced with 10% MTT solution, and the cells were incubated at 37°C and 5% CO2 for 4 h. The absorbance at 595 nm was measured using a microplate reader .Neutralizing antibodies were determined according to the previous method . BrieflyRecombinant protein VP56-3 and adjuvant protein FlaB (preserved in our lab) were verified by 10% SDS-PAGE and WB. The protein samples (10 μL) were separated by 10% SDS-PAGE and the gels were stained using Coomassie Brilliant Blue G-250 . In addition, the gel was transferred to the NC membrane at 9 V and the membrane was closed using 5% skim milk powder diluted in TBST for 2 h. The membrane was washed three times with TBST, incubated with GST-tag mAb (VP56-3) or His-tag mAb (FlaB) diluted 1:2500 for 2 h, washed four times with TBST, diluted 1:2500 with HRP-labeled sheep anti-mouse IgG mAb was incubated with the membrane for 45 min, TBST washed three times, subsequently stained with Clarity TM Western ECL Substrate (Bio-Rad), and finally imaged by the Amersham Imager 600 .2 was added to the emulsified sodium alginate protein mixture to calcify and form microspheres, the precipitate was collected by centrifugation  and washed three times with 0.01 mol/L CH₃COONa (pH = 4.0). Then stirred with 0.8% chitosan (pH = 5.0) at 1500 r/min for 15 min, the precipitate was collected by centrifugation , washed three times, and stored in lyophilization.We prepared the oral vaccine SA-VP56-3/FlaB using the encapsulation method. Briefly, 1.6 mg/mL of VP56-3 and 0.4 mg/mL of FlaB were mixed well and then mixed thoroughly with 1.5% sodium alginate (Control was prepared by replacing the protein with PBS pH = 7.4). The mixed solution was slowly added dropwise at 4:6 to paraffin (containing emulsifier Span-80), stirred at 1500 rpm/min for 30 min. After full emulsification, the 8% (M/V) CaClCentrifuge 5 mL of SA-VP56-3/FlaB suspension, add SA-VP56-3/FlaB precipitate into the appropriate amount of PBS (pH = 7.4), ultrasonically break it up, and dissolve it fully. The supernatant was collected by centrifugation . The protein concentration was quantified by BCA kit, and the encapsulation efficiency and loading rate were calculated according to the formula:Where A denotes the amount of protein in the actual SA-VP56-3/FlaB, B denotes the amount of protein in the theoretical SA-VP56-3/FlaB, and C denotes the total mass of SA-VP56-3/FlaB (n = 3).The formed SA-VP56-3/FlaB were resuspended in distilled water. The particle size distribution and zeta potential of SA-VP56-3/FlaB were determined by dynamic light scattering using a Malvern Nano-ZS 90 laser particle size analyzer  at a detector angle of 90°, 670 nm, and temperature of 25°C. The morphology of SA-VP56-3/FlaB was observed by transmission electron microscopy  and scanning electron microscope .A hemolysis assay was performed with grass carp erythrocytes. Briefly, 3 mL of fresh peripheral blood was collected using sodium heparin tubes, and erythrocytes were collected by centrifugation . Flushing with PBS (pH = 7.4) and resuspending. Added 100 μL SA-VP56-3/FlaB and erythrocytes (100 μL) to a 96-well plate and incubate at 37°C for 2 h. After centrifugation , the absorbance of each well at 450 nm was measured using a microplate reader.4 per well), and then different concentrations of SA-VP56-3/FlaB, SA, and VP56-3/FlaB were added. After co-culture at 37°C for 1 h, the cell culture medium was replaced with 10% MTT solution. The cells were incubated at 37°C and 5% CO2 for 4 h. The absorbance at 595 nm was measured using a microplate reader.The cytotoxicity of SA-VP56-3/FlaB to CIK cells was measured by the MTT assay. Cells were inoculated into 96-well plates (1.0 × 10in vitro release of SA-VP56-3/FlaB was detected at 25°C for 36 h. 1 mL SA-VP56-3/FlaB (1 mg/mL) were placed into EP tubes with 1 mL PBS or intestinal fluid in a shaking incubator  at 100 rpm for 36 h at 25°C. The mixture was tested by a BCA Protein Assay Kit  at specified time intervals . The amount of protein in the supernatant at each time point was counted to produce an in vitro release curve.The release of VP56-3/FlaB from SA-VP56-3/FlaB was performed according to the previous description . In relein vivo optical imaging system.SA-VP56-3/FlaB were prepared by FITC-VP56-3 and FITC-FlaB under light-proof conditions. 5 mg SA-VP56-3/FlaB was resuspended to 5 mL PBS (pH = 7.4), and stored under light-proof conditions. 27 grass carp were randomly divided into 3 groups, including the control group, FITC-VP56-3/FlaB group, SA- FITC-VP56-3/FlaB group, and gavaged with 200 μL samples at 0 h, 6 h and 12 h (three grass carp each time). The control group was gavaged with 200 μL PBS (pH = 7.4). Grass carp were dissected to expose the abdominal cavity at 12 h, and the fluorescence in the intestine was analyzed using a small animal 7 TCID50/mL of 100 μL GCRV 097 on D29, and the mortality of each group was recorded and monitored daily. Blood, head kidney, spleen, and hindgut were taken from each group of grass carp on D0, D28, and D40.To evaluate the protective effect of SA-VP56-3/FlaB, we conducted a viral challenge experiment on grass carp. Briefly, grass carp were divided into five groups (n = 150). The feeding situations before challenge were as follows: common fodder (Control group), SA , SA-FlaB , SA-VP56-3 , SA-VP56-3/FlaB . Each group of fish was fed once a day for 28 days. Stopped feeding for one day before the challenge, and gave common fodder after challenge. Each group of grass carp was injected intraperitoneally with 10On D42, the viral load was measured by taking the head kidney, spleen, and hindgut of each group of grass carp (n = 5). Total RNAs of head kidney, spleen, and hindgut were extracted with RNAiso Plus kit, the purification and concentration were measured by a NanoDrop 2000 spectrophotometer , and the quality was evaluated using 1.5% agarose gel electrophoresis. mRNAs were reverse-transcribed into cDNAs respectively with MMLV reverse transcriptase, RNase inhibitor , hexamer random primer. The viral protein VP56 was measured by qRT-PCR using the primers VP56-Q . Fresh blood samples were placed overnight at 4°C. After centrifugation , the serum was collected for enzyme activity detection, and the remaining serum was stored at -80°C. Serum enzyme activities including total superoxide dismutase (TSOD), lysozyme (LZM), complement 3 (C3) were measured as a previous report . These t-△△CT).We collected the head kidney, spleen, and hindgut of each group of grass carp according to time points . Total RNAs of head kidney, spleen, and hindgut were extracted with RNAiso Plus kit and reverse transcribed into cDNAs according to the method in 2.5. The primers for qRT-PCR analyses were listed in Serum was collected from each group of grass carp on D28, and the dilution ratio was 1:500, 1:1000, 1:2000, 1:4000, 1:8000. Antigen binding capacity was determined for each group of grass carp serum as described in method 2.3.2.Serum was collected from each group of grass carp on D28, and the dilution ratio was 1:1, 1:10, 1:20, 1:40. Neutralizing antibody level was determined for each group of grass carp serum as described in method 2.3.2.The results were presented as means ± standard deviation (SD) and all the statistical analyses were done using SPSS 26.0 package. The experimental data were subjected to the Kruskal-Wallis test, followed by Dunn’s multiple comparison (with Bonferroni adjustment) to identify the significance (p < 0.05). Different superscript letters in each group  denote significant variations. The protection rates were analyzed by Mantel-Cox test, * denotes significant variation.E. coli was induced by IPTG to express proteins of suitable size , and the size of the recombinant proteins was verified by SDS-PAGE (The VP56 (1539 bp) gene, VP56-1 (420 bp) fragment, VP56-2 (522 bp) fragment, and VP56-3 (525 bp) fragment were cloned into the prokaryotic expression plasmid pGEX-4T-1, and The VP56-3 protein (44 kDa) and the adjuvant protein FlaB (34 kDa) were verified by SDS-PAGE and WB Table 1.7 TCID50/mL) injected intraperitoneally and survival was recorded daily for two weeks  and control group after oral immunization and viral challenge. On D28, the highest TSOD and LZM levels were found in the SA-VP56-3/FlaB group, which was significantly different from the other groups  and hindgut IgZ genes by qRT-PCR after oral immunization and viral challenge. The results showed that the expression levels of IFN1, CD8, MHC-II, and IgZ mRNA in the SA-VP56-3/FlaB group were significantly upregulated on D28, which was significantly different compared with the other groups  and the control group. We measured the expression levels of IgM mRNA in the head kidney and spleen after oral immunization and viral challenge by qRT-PCR. The results showed that the SA-VP56-3/FlaB group had the highest expression levels of IgM mRNA, which was significantly different from the other groups, and SA-VP56-3/FlaB induces higher levels of IgM mRNA than D40 at D28  with excellent stability, low toxicity, and slow-release, which can effective against hydrolysis by proteases of the intestine. Oral immunization of SA-VP56-3/FlaB can induce an effective immune response, significantly alleviate tissue viral load and reduce mortality. This research has developed a promising oral vaccine that can be easily applied in aquaculture, providing a strategy for against GCRV-II infection in aquaculture.The original contributions presented in the study are included in the article/All procedures of animal experiments were approved by the Ethical Committee on Animal Research at Huazhong Agricultural University (ID Number: HZAUFI-2021-0028).JS and CX conceived and designed the experiments, and wrote the manuscript. CX carried out most experiments and data analyses. MQ and XH performed qRT-PCR assay. ZL expressed and purified proteins. JS revised the manuscript critically. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Fundamental Research Funds for the Central Universities (2662021SCPY006) and National Key Research and Development Program of China (2018YFD0900504).The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.