An internal control to validate the extraction and amplification was also included. The limit of detection (LoD) of the assay was four copies/reaction for HTLV-1 and 10.9 copies/reaction for HTLV-2. The diagnostic sensitivity of the platform was 94.6% for HTLV-1, 78.6% for HTLV-2, and the specificity was 100% for both viruses. Cross-reactions of the test with human viruses including HAV, HBV, HCV, HIV-1/2, and parvovirus B19 were not observed. During the multicentric validation, the test was used to screen a total of 692 blood samples obtained from previously confirmed HTLV-positive individuals. From these, 91.1% tested positive being concordant with the previously obtained results. In conclusion, our duoplex-RT-PCR-HTLV1 /2 presented adequate efficiency for HTLV-1/2 differentiation showing high sensitivity and specificity. Therefore, it can be a suitable tool for confirmation of suspected and inconclusive HTLV cases, prenatal and pre-transplant diagnosis, in Brazil and in other countries HTLV-endemic countries.Human T cell lymphotropic virus (HTLV) is the caustive agent of two main conditions i. e., the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and the adult T-cell leukemia/lymphoma (ATLL). HTLV diagnosis is based on serological and molecular approaches; however, an accurate and validated method is still needed. The objective of this study was to establish a rapid and sensitive molecular test to confirm and discriminate HTLV 1/2 types. The test validation was performed as a multicentric study involving HTLV confirmation centers throughout Brazil. Proviral DNA was extracted from whole blood and the amplification was performed using in-house designed primer and probe sets targeting the  The HTLSince 1993, for transfusion purposes, screening of voluntary blood donors for HTLV-1/2 has been mandatory in Brazil . DespiteIn Brazil, the recommended algorithm for HTLV-1/2 diagnosis is based on conventional serological methods such as enzyme-linked immunosorbent assay (ELISA), particle agglutination, and chemiluminescence immunoassay. Western blot (WB) and/or line immunoassay (INNO-LIA) are usually applied as confirmatory and discriminatory diagnoses. However, indeterminate WB results can reach approximately 50% , 17, leain-house tests (The use of nucleic acid testing (NAT) platforms for HTLV viral gene detection instead of WB or line immune assay can be applied due to the higher sensitivity and specificity. Several centers have developed their own NAT assays generically called se tests –20. Howese tests . These ase tests –20. One se tests . AdditioThus, we have developed and validated a molecular platform with high sensitivity using a qualitative multiplex real-time PCR (duoplex-RT-PCR-HTLV-1/2) for both screening and discriminatory diagnosis of HTLV-1 and 2 infections. Additionally, this platform has been validated as a multicentric study in four main HTLV centers in Brazil, located in the states of São Paulo (two centers), Bahia, and Pará. The last two are where the majority of HTLV positive cases in Brazil are concentrated.n = 145), Institute of Infectious Diseases “Emilio Ribas” (IIER), São Paulo, (n = 137), HTLV Reference Center, Bahiana School of Medicine and Public Health, Salvador, Bahia (n = 179), and Tropical Medicine Center, Federal University of Pará, Belém, Pará (n = 226). Additionally, 198 blood samples were obtained from volunteer blood donors at the Regional Blood Center of Ribeirão Preto at the Faculty of Medicine of Ribeirão Preto and 90 blood samples from HIV, HCV, or HBV infected individuals from the Infectious Diseases Clinic at the Faculty of Medicine of Ribeirão Preto, USP. For the duoplex validation step, we calculated the number of tests that should be performed according to the incidence of HTLV in our region (0.01%)  . For the® , and HBV MONOLISA® (Bio-Rad), respectively. The HTLV-1/2 positive samples were confirmed by WB analyses  and by nested PCR through amplification of the tax and LTR regions for HTLV-1 and pxA, pxB, and LTR regions for HTLV-2 , Architect HIV Ag/Ab Combo ; HCV MONOLISAr HTLV-2 . Samplesouse PCR .The study was approved by the Institutional Ethics Committee at the Clinical Hospital of Ribeirão Preto, School of Medicine of Ribeirão Preto, University of São Paulo (Process No. 12.027/2009) and all participants signed an informed consent form.5 to 100 copies/reaction.The positive control used for HTLV-1 was DNA extracted from the MT-2 cell line (93121518/ECACC), with 2.1 copies of the virus per cell . The HTLThe genomic DNA extraction was performed from peripheral blood by ReliaPrep Blood gDNA Miniprep System DNA isolation kit  according to the manufacturer's instructions. The DNA concentration and purity was measured by using Nanodrop 2000c .® probes were designed for the pol gene for HTLV-1 and HTLV-2. The sets of primers and probes were designed to ensure the specific detection of the most important HTLV-1/2 subtypes. Beta globin gene was used as internal amplification control (IAC) which allows for the verification of the extraction efficiency. For the design of the oligonucleotides Primer Express® 3.0  and Oligo Analyzer v. 3.1  software were used. Primers and probe sequences are indicated in Complete reference genomes of HTLV-1 and−2  were aligned using BioEdit v. 7.2.3 software  in order to design primer and probe sets  at consesingleplex format, and then multiplex in order to compare linear correlation (r2), efficiency of qPCR reactions and shape of the amplification curves. The evaluation of the commercial PCR master mixes applied included intensity of fluorescence (ΔRn), the format of the amplification curve and the appearance of the Ct during the amplification. Distinct concentrations of primers (250–500 nM) and probes (100–300 nM) were used to adjust the best concentration of each. After these initial steps, duoplex-RT-PCR-HTLV1/2 reactions were standardized in a final volume of 25 μl with the reaction mixture consisting of 12.5 μl GoTaq® Probe qPCR Master Mix (Promega) and 5 μl of DNA , 500 nM for each forward and reverse primer for HTLV-1, and 200 nM of the probe. For HTLV-2 detection, two sets of reverse primers used simultaneously were designed to improve the detection. For instance, 500 nM of the forward primer, 250 nM of the reverse 1 primer, and 250 nM of the reverse 2 primer, and 200 nM of probe were used. The amplification of IAC was performed using 250 nM of each primer and 200 nM of probe. The following cycling parameters were applied: initial activation step at 50°C/2 min, denaturation at 95°C/10 min, followed by 45 cycles at 95°C/15 s and 60°C/1 min. Amplification data acquisition was carried out using ABI Prism 7500 Real-time PCR system . For all the runs, positive control (DNA from the MT2 and Gu cell line) and IAC were used. After the steps of standardization, a ready-to-use mix composed of primers, probes, and buffers was produced and sent to the participating centers for the multicentric study. Positive controls were produced using DNA from MT2 and Gu cell lines at 103 viral copies/reaction to obtain a reproducible and robust control. For clinical samples, HTLV-1 and HTLV-viral gene amplification limit Ct is <40 to be considered detectable. The homogeneity and stability of these lots of ready-to-use mix were evaluated before their use. Homogeneity was checked by randomly sampling five aliquots of the mix. Stability was evaluated by freezing and defrizzing the mixes (20 times) and checking at intervals of 120 days.The assays were optimized initially as 5, 104, and 103 proviral copies/reactions. The qPCR reactions were performed in duplicates of each dilution in three separate runs, using a different batch of reagents manipulated by three different operators in three separate days.The obtained variations for the Ct values were all within the postulated limit of within 5% , 25, 26.5 viral copies was prepared using MT-2 and Gu cell lines as standards. The stock dilution was submitted to serial dilutions until the 62.5, 31.25, 15.625, 7.81, 3.9, 1.95, and 0.975 proviral copies were obtained. Eight replicates of each dilution were pipetted in three independent assays. Probit analysis was used to calculate LoD  as supplied by the SPSS version 17.0 software .For the analytical sensitivity or limit of detection , a stock dilution of 10in vitro and in silico tests were performed. For in vitro validation, hepatitis A virus, HBV, HCV, HIV-1, HIV-2, and parvovirus B19 panels obtained from the National Institute for Biological Standards and Control (NIBSC) were tested with our platform. In addition to this, we tested 30 samples from HIV-infected patients undergoing HAART treatment, 30 samples from HBV-infected individuals, and 30 samples from HCV-infected individuals. For in-silico validation we performed comparisons of our primers and probes with public databases in order to verify possible similarities with other organisms.To evaluate the analytical specificity, We evaluated three hundred and twenty samples that showed positive results for HTLV-1/2-serology, WB and/or positive nested-PCR. Among them, 278 were for HTLV-1 positive individuals and 42 were for HTLV-2 positive patients. To calculate the diagnostic sensitivity we used the following formula: diagnostic sensitivity = true positive individuals/.In order to check this parameter, 288 samples from HTLV-1/2 serologically negative subjects were tested with our platform. The diagnostic sensitivity was calculated using the following equation: diagnostic specificity = true negative individuals .5, 104, and 103 viral copies/reactions with five replicates of each dilution. Three runs were performed on the same day, using the same batch of reagents and prepared by a single operator.For the precision tests, the parameters of repeatability (intra-assay) and reproducibility (inter-assay) were evaluated. The repeatability test was performed from three dilutions of the positive controls (MT-2 + Gu) containing 105, 104, and 103 viral copies/reaction with five replicates of each dilution. Three runs using the optimized test were performed on alternate days, using the same batch of reagents and prepared by three different operators.The reproducibility test was performed with three dilutions of the positive controls (MT-2 + Gu) containing 10The robustness was evaluated by the “chess table” method, in which a positive sample is pipetted alongside a negative sample in the qualitative duoplex-RT-PCR-HTLV1/2 plate.t-test was used to assess the stability of controls and reagents. One-way ANOVA test was applied to evaluate the homogeneity and stability of the batch of oligonucleotide mix and positive control. The Probit analysis (SPSS) was used to evaluate the analytical sensitivity of the platform.Precision analysis, reproducibility (intra-assay), repeatability (inter-assay), and comparison of singleplex and multiplex reaction formats using real-time PCR (qPCR) were evaluated using the mean, standard deviation and the coefficient of variation of the cycle threshold (Ct). The singleplex format, we generated a standard curve by serial dilutions from 105 to 100 viral copies/reaction of the MT-2 (HTLV-1) and Gu (HTLV-2) positive controls. The efficiency for the probe and primer sets for HTLV-1, HTLV-2, and internal control (IC) were >95% and the linear correlations (r2) was higher than 0.99.In order to evaluate singleplex and the multiplex qPCR reactions. We observed a high and similar efficiency between the singleplex and the multiplex format, 93%−100% and 94%, respectively. Correlation (r2) was also suitable in both formats  as shown in In addition, standard curve dilutions were also applied to compare the efficiency and accuracy between the in silico against public databases (BLAST) to avoid cross-reactivity with other critical human viruses. Primer/probe sequences demonstrated 100% similarity to HTLV-1, HTLV-2, and beta globin (IAC), while in silico they did not cross-react with other viral types, making this set of primers/probes suitable for the molecular assay. In vitro analytical specificity was evaluated by two strategies: initially, primers/probes were tested on a control panel containing viral genomes obtained from other human pathogens like HAV, HBV, HCV, HIV-1/2, and B19V . This parameter evaluation performed with a total of 90 DNA samples obtained from patients with HIV, HCV, and HBV infections showed 100% specificity, meaning that no cross-reaction with the established diagnostic platform was observed.The 95% LoD of the proposed platform was achieved using two fold serial dilutions (62 to 1 copies/reaction) in eight replicates. The LoD was 4 copies/reaction (range 3–6 copies/reaction) and 11 copies/reaction (range 7–17 copies/reaction) for HTLV-1 and HTLV-2, respectively. Analytical specificity was evaluated From 320 samples tested (278 HTLV-1 and 42 HTLV-2), the duoplex-RT-PCR-HTLV1/2 reached 94.6% of sensitivity for HTLV-1 and 78.6% for HTLV-2. All reactions were performed in duplicate. For calculating the specificity of the assay, 288 samples  were tested resulting in no PCR amplification or high Ct values (Ct>40 were considered as negative), therefore demonstrating 100% specificity.5, 104, and 103 copies/reaction) of MT-2 and Gu cells. The coefficient of variation (CV) for Cts values was <5% . The multiplex PCR reactions were performed in two replicates of each dilution in three separated runs, using a distinct batch of reagents manipulated by three different operators on three alternate days. The CV for Cts values was within the 5% limit, as illustrated in We evaluated the intra-assay using three standard dilutions . A total of 692 HTLV samples with previously confirmed diagnosis were tested . In Ribemultiplex qPCR platform—duoplex-RT-PCR-HTLV1/2—for the simultaneous detection and discrimination of HTLV-1 and HTLV-2. The established assay is sensitive and reproducible, easy to perform in molecular biology laboratories with appropriate conditions for clinical diagnosis; it is essentially a low-cost operation compared to the available confirmatory methods such as WB and line immunoassay.In the current study, we optimized and validated a qualitative _pol, see ® Green, Molecular Beacons, and TaqMan® probes  and international patent applications (United States of America No. 15/517.449 and European Union patent application No. 158484220).Up to now, there is no molecular confirmatory platform for HTLV detection available commercially. The development and the detailed validation of our duoplex qPCR qualitative test for HTLV-1/2 diagnosis (confirmatory and discriminatory), which is ready-to-use, timesaving and presents low risk for sample cross-contamination. This technique was performed in a unique reaction tube to detect and discriminate between HTLV-1 and HTLV-2. Performance tests showed high sensitivity and specificity compared to the singleplex assays and to classical confirmatory methods for HTLV-1/2 diagnosis such as WB. Therefore, this platform can be implemented as a fast HTLV diagnosis and confirmation and to monitor HTLV-infected patients in the clinical centers for the correct management as well as counseling to avoid transmission. Despite the obtained suitable validation parameters of our assay, we highlight that it is important to include more HTLV-2-infected patients and perform the validation processes in other national and international centers engaged in HTLV diagnosis.In conclusion, we developed, optimized and validated a The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Clinical Hospital of School Medicine—Process number 20009119.0.1001.5440. The Ethics Committee waived the requirement of written informed consent for participation.MR-J idealized the proposal, performed major experiments and analysis, and wrote the original draft. ER, SS, TA, MP, and DL performed major experiments and analysis and reviewed the original draft. MS supervised sample collection and diagnosis of HTLV-1-infected and healthy individuals. VO performed major experiments and analysis and reviewed the original draft. BG-C, BF, AP, JS, OT, and MS supervised sample collection and diagnosis of HTLV-1-infected and healthy individuals, as well as ambulatory follow-up of HTLV-1 infected individuals. JC, DC, and SK idealized the proposal, supervised and reviewed the original draft. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported by Financiadora de Estudos e Projetos (FINEP/Process number 1387/10), Centro de Terapia Celular (CTC/FAPESP)/Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP—Process number 2013/08135-2), FAPESP n. 2018/07239-2; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq—Processo 465539/2014-9), CNPq n. 301275/2019-0, CNPq n. 301677/2018-2, Fundação Hemocentro de Ribeirão Preto (FUNDHERP); and scholarship from FM/FMUSP to JC.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.


In boys, we found a higher age, daily insulin dose, TG, TG/HDL ratio, TC/HDL ratio, systolic BP, N/L ratio and lower HDL, and eGFR across UA tertiles (p = 0.01–0.0001). Similar results were found in girls but not for TG and systolic BP. In boys, the odds ratio (OR) of high levels of TG/HDL ratio, TC/HDL ratio, BP and mildly reduced eGFR (MRGFR) increased for 0.5 mg/dL of UA. Instead, in girls an increased levels of 0.5 mg/dL of UA were associated with high OR of TC/HDL ratio, N/L ratio and MRGFR. Uric acid may represent a useful marker for identifying youth with T1D at high cardiometabolic risk, and this association appears to vary by sex.The aim of this study was to investigate the association between uric acid (UA) and cardiometabolic risk factors (CMRFs) by sex in youth with type 1 diabetes (T1D). Retrospective data collected from 1323 children and adolescents  with T1D recruited in 9 Italian Pediatric Diabetes Centers were analyzed. CMRFs included UA, HbA A robust association between UA and risk of incident cardiovascular events, mortality and an early onset of glomerular filtration rate (GFR) decline has also been reported in adults with type 1 diabetes (T1D)7. The mechanisms linking UA to cardio-renal disease risk are not fully elucidated yet. However, the effect of UA might be mediated by the activation of oxygen free radicals which in turn increase oxidative stress and inflammation8.Several cross-sectional and longitudinal studies have reported the independent role of uric acid (UA) as a predictor of cardiovascular morbidity, mortality and incident chronic kidney disease (CKD), in adult populations, especially with type 2 diabetes11.Several cross-sectional studies in pediatric populations, primarily performed in obese children, demonstrated that high UA levels are associated to metabolic syndrome and its individual components, nonalcoholic fatty liver disease, reduced estimated GFR (eGFR) and glucose dysmetabolism13. This is an important issue which deserves clarification since it is well known that cardiovascular prevention should start as early as possible, not only in children with obesity and type 2 diabetes but also in those with T1D16. Furthermore, a potential sex-related difference in the relationship between UA and CMRF has not been well explored so far13. Therefore, the aim of the present study was to evaluate the cardiometabolic risk (CMR) profile associated with UA levels in a large sample of children and adolescents with T1D. In addition, we assessed whether the UA-related CMR profile is different among the two sexes.In contrast, in adolescents with T1D, the relationship between UA levels and cardiometabolic risk factors (CMRFs) is controversial and little exploredp = 0.01–0.0001)  age of 13.5 ± 3.1 years. The clinical and biochemical characteristics of the study population are summarized in Table p = 0.04–0.0001)  Table .Table 3AThe age-adjusted proportions (mean and 95% CI) of youth with abnormal CMRFs across tertiles of UA both in boys and girls are shown in Fig. In boys, the odds ratio of high levels of TG/HDL ratio, TC/HDL ratio, BP and MRGFR increased for 0.5 mg/dL of UA; instead, in girls a 0.5 mg/dL increase in UA was associated with high odds ratio of TC/HDL ratio, N/L ratio and MRGFR Table .Table 4OOur study showed a potential association between UA and CMRFs in children and adolescents with T1D. This association suggests that UA could be a potential marker of cardiometabolic risk in this population. Furthermore, our study showed that the CMR risk profile associated with UA is worse in males than in females.1. Furthermore, prospective studies have shown the usefulness of UA in predicting incident cardio-renal events both in individuals with T1D and type 2 diabetes7.High levels of UA have been reported in adults with hypertension, obesity and metabolic syndrome11. These studies have been performed in obese youth, whereas few and conflicting observations have been reported in youth with T1D13. Indeed, Lytvyn et al. could not detect any relationship between UA and cardio-renal abnormalities in their study conducted in small sample of 180 adolescents with T1D13. In contrast, Słomiński et al. demonstrated a positive association between UA and both nephropathy and subclinical inflammatory marker concentrations in boys but not in girls with T1D12. Differences findings between studies might be related to sample size, age range and other specific characteristics of the study populations, such as ethnicity.In children, recent studies have shown a strong association between UA, nonalcoholic fatty liver disease mildly reduced eGFR and glucose dysmetabolismIn our study, we performed a comprehensive analysis including markers of low grade-inflammation and UA levels separately in the two sexes. To the best of our knowledge, our study demonstrates for the first time a robust association between CMRFs and UA in children and adolescents with T1D, which was more pronounced in boys than in girls.11. This difference is likely due to a distinct roles of sex hormones and a higher muscle mass in males17. In our study population, we did not observe any difference in UA levels between the two sexes, and this could be related most of participants being normal weight as well as to the specific age range.Uric acid levels are higher in adult males than females, as well in obese boys than girls19.In childhood, high values of UA are influenced by ethnicity, age and sex, so a universal definition of abnormal UA levels is lacking. In non-obese youth values above 6 or 7 mg/dL in boys and 5 or 6 mg/dL in girls were previously considered “elevated”20. This result could lead to undermining the usefulness of assessing UA in clinical practice in youth with T1D. Despite this, our study showed a robust negative association between 'high normal' levels of UA and eGFR, in line with our previous studies22 and extends a positive association between UA and different CMRFs in young people with T1D. Interestingly, the results differed by sex, with boys having an increased risk for high TG/HDL, TC/HDL ratio, high BP and MRGFR, whereas girls showed a high risk of abnormal TC/HDL ratio, N/L ratio and MRGFR.In children and adolescents with T1D levels of UA are lower than healthy individuals23. This suggests the link between UA, even in a high normal range, and elevated BP levels that may be useful to identify boys with T1D at high risk of hypertension.The different impact of UA in males vs females is still undefined. In our sample, we confirm in boys an association between high BP and high levels of UA as reported in a Caucasian sample of obese youthIn the present study, an association between the TG/HDL ratio and UA in boys was also detected. Insulin resistance may represent the link between the TG/HDL ratio and UA.24 and a more sensitive predictor of cardiovascular events in adults than total cholesterol25. Interestingly, the association between UA and high TC/HDL ratio is shared by the two sexes, independently of BMI. This further supports the usefulness of UA as marker of early atherosclerosis in both sexes.A further novel finding from our study is the potential association between high levels of UA and high TC/HDL ratio. This lipid ratio is considered a marker of atherogenic dyslipidemia10, whereas this association has been little explored in T1D, especially in young people13. The strong associations we found between UA and MRGFR in both boys and girls is in line with our previous reports22. The strength of this association sustains the evaluation of UA in clinical practice to identify youth with T1D at risk of eGFR decline in both sexes. Our results might have influenced by the specific formula used to calculate eGFR. At present, there is no consensus regarding the best method for estimating GFR in children and adolescents with T1D. We used a recently validated formula for the pediatric population, which relies on the use of creatinine and it is easily implemented into clinical practice26.In adults and obese children, the relationship between UA and MRGFR is well established27. In particular, recent studies demonstrated that a higher N/L ratio represents a useful marker to identify diabetic kidney disease28. In our sample we observed a robust association between N/L ratio and high levels of UA in girls, but not in boys. This association may be supported by higher percentage of girls with concomitant autoimmune diseases as compared to boys. This finding contrasts with those form Słomiński et al.12, who reported a positive association between UA and subclinical inflammatory marker concentrations only in boys but not in girls. The reasons for this discrepancy are unknown and warrant further study.Of particular interest is the link between a high N/L ratio, as surrogate of low-grade inflammation, and high levels of UA in girls. The N/L ratio has been recently studied in several conditions, such as cardiac, vascular, and kidney disease where the low-grade inflammation was potentially involvedSome limitations of the present study need to be acknowledged. Firstly, being a retrospective multicenter, clinic-based study, data were collected and analyzed across different centers. However, all laboratories were standardized and used similar methods that were aligned. In addition, it is important to acknowledge that the study methods reflect real-word data collection and therefore closely reflect what can be implemented in daily clinical practice. The study population lacked diversity in terms of ethnicity, and this may limit the generalization of the study findings. However, despite these limitations, the present study, based on a large sample size and data collection, highlighted an important role of UA as potential early marker of cardiometabolic risk.In conclusion, our study suggests that UA levels in youth with T1D could be useful to identify those at higher cardio-metabolic risk, although sex-related differences need to be taken into account. If confirmed by future prospective studies, the present findings could lead towards the implementation of UA as part of the set of investigations required in all youth with T1D from early stages of the disease to support prediction and prevention of cardio-metabolic complications.This multicenter retrospective cross-sectional study including Caucasian children and adolescents with T1D consecutively recruited within 16 months from 1 January 2019 to 30 April 2020 in 9 Italian Pediatric Diabetes Centers that are part of the Diabetes Study Group of the Italian Society for Pediatric Endocrinology and Diabetology.Medical history, biochemical and clinical data were retrieved from clinical records. Inclusion criteria were: availability of data regarding UA, diagnosis of T1D (at least 1 positive autoantibody), diabetes duration > 1 year, age 5–18 years, availability of anthropometric and biochemical data. Finally were analyzed records of 1323 youth. The study protocol was approved by the Ethics Committees of the participating Centers  and was conducted in accordance with the declaration of Helsinki and good clinical practice guidelines. Informed written consent was obtained from all the parents and/or patients before their inclusion in the study. The clinical and biochemical parameters were anonymously entered in a database using an alphanumeric and progressive code.1c, total cholesterol (TC), triglycerides (TG), HDL, Neutrophil (N) and Lymphocyte (L) counts, albuminuria and insulin regimen (multiple daily injections vs continuous subcutaneous insulin infusion) and doses (IU/kg/day). Weight and height were collected by a single trained operator in each center. BMI-SDS was calculated based on the Italian BMI normative data29.For all study participants, the following data were recorded by each of the nine participating centers: age, sex, height, weight, body mass index (BMI), blood pressure (BP), serum creatinine (Scr), UA, HbA1c was assessed by High-Performance Liquid Chromatography; TC, TG and HDL were determined by enzymatic methods; N and L counts were measured using an automated analyzer.In all centers HbA30. The relationship between Scr, age, sex, BMI and enzymatic method was explored by a linear regression analysis using Scr as dependent variable and age, sex, BMI and enzymatic method as covariate. We obtained a B coefficient for enzymatic method that was subtracted from the value of Scr assessed by Jaffé method. The value of Scr assessed by Jaffé method resulted identical to that obtained with enzymatic method as elsewhere described21.Scr was analyzed in 4 centers by enzymatic method and in 5 centers using Jaffé IDMS traceable method, while UA by the uricase method in all centers31.The following parameters: TG to HDL (TG/HDL), TC to HDL (TC/HDL) and Neutrophil to Lymphocyte ratio (N/L) were calculated31:32. Albuminuria was defined as normal to mildly increased ; moderately increased ; severely increased 32. The presence of albuminuria was defined by positivity in at least two different measurements.Estimated glomerular filtration rate (eGFR) was calculated using Schwartz-Lyon equation33. BP was measured, after 5 min of resting in a quiet room, using an appropriate sized arm cuff on the right arm and an aneroid sphygmomanometer. Three measurements were obtained every 2 min and the mean of the last two values were used in the analyses.BP was measured following the recommendations by the European Society of Hypertensionwww.iso9000.it/)34.All blood samples for biochemical analyses were collected in each center after 12 h of fasting. Although laboratory analyses were performed in different laboratories, these are all part of the Italian National Health System and are certified according to International Standards ISO 9000  was defined as moderately increased  or severely increased  32. The presence of albuminuria was defined by positivity in at least two different measurements36.Presence of puberty in all patients was identified as breast development in girls and testicular growth to at least 4 mL in volume in boys; pre-puberty in case of absence of any sign of pubertyth percentile for age, sex and height in children aged less than 13 years or BP ≥ 130/80 mmHg in adolescents (age ≥ 13 years)37.High BP was defined using criteria proposed by the American Academy of Pediatrics: BP ≥ 95th percentile of the study population. Mildly reduced eGFR was defined by a value between 60–89 mL/min/1.73 m232.High TG/HDL ratio, TC/HDL ratio and N/L ratio were defined by the 751c, TG, TG/HDL ratio, TC/HDL ratio, N/L ratio and insulin dose, the statistical analysis of these variables was applied after log transformation and back transformation to natural units to allow presentation in the text and tables. Between groups differences in continuous variables were assessed with Student’s t-test. Analysis of variance (ANOVA) was applied to evaluate differences across tertiles of UA. The χ2 and Fisher’s exact tests, as appropriate, were used to compare categorical variables. Exact tests were performed using the Monte Carlo method. To evaluate the association  between the CMRFs and UA, a logistic regression analysis was performed using the high levels of each CMRF as dependent variable and centers, age, insulin dose (IU/kg/day), BMI-SDS and 0.5 mg/dL of UA as covariates. Statistical analysis was performed using the IBM SPSS Statistics for Windows, Version 27. A p-value < 0.05 was considered statistically significant.Data are expressed as mean ± standard deviation, median and interquartile range, or absolute and relative frequencies, unless otherwise stated. Given the skewed distribution of HbAThe study protocol was approved by the ethics committees of all participating centers and was conducted in accordance with the declaration of Helsinki and good clinical practice guidelines.


Human T-cell leukemia virus type 1 (HTLV-1) is the causal agent of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. Its tropism is known to be broad in cultured cell lines, while in vivo data support a more selective transmission toward CD4+ T cells and the limited targeting of other hematopoietic cell types. An essential condition for HTLV-1 infection is cell-to-cell contact, to which both virological synapse and viral biofilm have been suggested to strongly contribute. As cell lines and animal models each present their own limitations in studying HTLV-1 replication, we have explored the use of an ex vivo model based on the secondary lymphoid tonsillar tissue. HIV-1 luciferase-expressing pseudotyped viruses bearing the HTLV-1 envelope protein at their surface were first shown to recapitulate the wide spectrum of infectivity of HTLV-1 toward various cell lines. Tonsil fragments were next exposed to pseudotyped viruses and shown to be reproducibly infected. Infection by HTLV-1 Env-pseudotyped viruses was blocked by different anti-gp46 antibodies, unlike infection by HIV-1 virions. The dose-dependent infection revealed a gradual increase in luciferase activity, which was again sensitive to anti-gp46 antibodies. Overall, these results suggest that the ex vivo tonsil model represents a reliable alternative for studying HTLV-1 replication and potentially viral latency, as well as early clonal formation. The human T-cell leukemia virus type 1 (HTLV-1) was the first retrovirus to be isolated in humans ,3,4 and HTLV-1 has a large tropism in vitro but has been shown to be more restricted to CD4+ T cells in vivo, although previous studies had provided evidence of the presence of proviral DNA in other hematopoietic  as well as non-hematopoietic cells ,10,11,12Alternative cell culture models have been proposed for studying viral infection. These include organoid tissues and cultured tissue fragments, often referred to as tissue explants ,22,23. TIn this study, we have thereby examined the susceptibility of tonsil tissue toward HTLV-1 infection. Using HTLV-1 Envelope-pseudotyped viruses, we demonstrate the reproducible and specific permissiveness of this ex vivo model to infection. These results provide evidence that the ex vivo tonsil model could represent an alternative model to study HTLV-1 replication in lymphoid tissue and clonal evolution.Cell lines. HEK293T (human embryonic kidney) and HOS-CXCR4 (human osteosarcoma) cells were grown in DMEM medium supplemented with 10% FBS and antibiotics (1% penicillin-streptomycin). The human Jurkat E6.1 T cell line was maintained in RPMI medium containing 10% FBS and antibiotics.env gene under the SV40 and CMV promoter, respectively [Plasmids and antibodies. The plasmid pNL4-3.Luc.E-R+ expresses the luciferase gene and is defective for Envelope protein synthesis  . pHXB-Luectively ,34. Antiectively  and monoectively ,37, both5) were transfected with a reporter viral DNA construct (7 μg) in the presence or absence of an env expression vector (3 μg) using the calcium phosphate protocol . At 48 h post-transfection, supernatants were harvested, passed through a 0.22 μm filter and stored at −80 °C until further use. The quantification of the viral production was conducted using a home-made p24 ELISA assay, as previously described [Production of pseudotyped particles. HEK293T cells (4 × 10escribed .2 blocks. Tissues (two fragments) were then overlaid on each collagen sponge gel , presoaked in supplemented RPMI medium in 6-well plates. A volume of 300 μL of additional supplemented medium was then added to allow tonsil sections to be at the air-liquid interface. Plates were incubated at 37 °C for 24 h before infection.Ex vivo tonsil model. Human tonsils were provided by the department of otorhinolaryngology of the Centre Hospitalier de l’Université Laval  from routine tonsillectomy within 4 h of excision. The tonsils were first soaked and rinsed in PBS containing antibiotics for 45 min and then cut into thin 2 to 3 mmp value lower than 0.05.Infection of cell lines and the ex vivo tonsil model. HTLV-1 Envelope-pseudotyped viral particles and HXB-Luc viruses  were added to HEK293T, HOS-CXCR4, or Jurkat E6.1 cells in 96-well plates, which were then incubated for 24 h (or up to 96 h) at 37 °C. After the removal of half of the volume, cells were lysed in lysis buffer  and incubated for 20 min. For tonsil fragments, various amounts of viruses were added on top of each fragment and then incubated for 72 h. Next, tissues were retrieved from the sponge gels and individually added to 50 μL of medium to which 5X lysis buffer was added, followed by the mincing of tissues. In certain infection experiments, viral samples were preincubated with anti-gp46 antibodies for 5 min at 37 °C. Luciferase activity was measured in a MLX Microtiter plate reader , using luciferin as the substrate, as previously described . All staThe tonsil tissue represents a second lymphoid organ, which can be used as a model for lymph nodes. We were thus interested in assessing if HTLV-1 could be studied in this environment through a previously published protocol .env expression vectors (pCAG- and pSV-based) in HEK293T cells. The produced virions were tested in different cell types, such as HEK293T, Jurkat E6.1, and HOS-CXCR4, and were shown to lead to significant luciferase activity, which was sensitive to blocking LAT-27 antibodies, but not to non-specific IgG antibodies  . We thusThe infection of tonsil tissue by pseudotyped viruses was performed next. Our results clearly showed infection, which was dependent on gp46, as shown by the blocking capacity of different anti-gp46 antibodies. Furthermore, HIV-1 HXB-Luc infection was not impacted by these antibodies, again demonstrating specificity. We were however limited in the amount of pseudotyped particles to be added, due to the maximal volume that can be reliably added on top of each tonsil block. Concentrated viruses would have improved the signal. As another limitation, we were not successful in initiating infection with cell-free infectious, replication-competent viruses (data not shown), although this might be expected, given the strict reliance on cell-to-cell contact for infection. Although mouse and simian models have obvious advantages for studying HTLV-1 in terms of replication and pathogenesis, they remain challenging. The ex vivo tonsil model offers several advantages and has actually been used for HIV-1 and other viruses ,41,42,43In conclusion, our infection experiments of tonsil tissue with HTLV-1 Envelope-pseudotyped viruses suggest that this ex vivo model provides an adequate environment to study HTLV-1 infection, although we are aware that infection experiments with fully replicating HTLV-1 viruses will be required to confirm its use. We are nonetheless confident that the future optimization of this model will allow us to study HTLV-1 infection in lymphoid tissues and address important questions in terms of the early generation of clones and their characteristics.


Churra sheep was performed. In the protocol design phase, 125 ewes were exocervically inseminated, and their fertility was assessed based on the number of inseminations, physiological state, body condition, estrus detection–insemination interval and vaginal fluids in cervix. That protocol was repeated in six consecutive years. In all ewes, fertilities based on the timing of insemination after estrus detection were very high. Lactating ewes produced better results than did dry ewes, which was probably because of the better feeding of the former. In addition, double insemination increased the fertility of ewes, whose estrus was detected within 16 h of onset. Body condition and amount of vaginal fluid were correlated with fertility. In conclusion, exocervical inseminations at natural estrus can produce acceptable fertility and prolificity in Churra ewes, avoiding physiological problems derived from the use of hormonal treatments.Hormonal treatments used to synchronize estrus in sheep artificial insemination procedures can cause several physiological problems that can affect negatively fertility and animal health; however, alternative protocols based on the detection of natural estrus offer a more sustainable option while reaching successful fertility results. Therefore, in this study, an artificial insemination protocol at natural estrus in organic Churra sheep was performed. In the first phase (AI protocol development), 125 ewes were exocervically inseminated, and their fertility was assessed based on the following factors: number of AI, physiological state, body condition, estrus detection–AI interval, and vaginal fluids in cervix. That protocol was repeated for six consecutive years. In all individuals, fertilities based on the timing of insemination after estrus detection were very high. Lactating ewes produced better results than did dry ewes, which was probably because of the better feeding of the former. In addition, double insemination increased the fertility of ewes whose estrus was detected within 16 h of onset. Body condition and amount of vaginal fluid were correlated with fertility. Exocervical inseminations at natural estrus can produce acceptable fertility and prolificity in Churra ewes.Hormonal treatments used in the artificial insemination (AI) of sheep can cause several physiological problems that can affect negatively fertility and animal health; however, AI protocols based on the detection of natural estrus offer a more sustainable option and can achieve high fertility. In this study, an AI protocol at natural estrus in organic  In Spain, artificial insemination (AI) in sheep is used as an improvement genetic method within purebred selection programs. Livestock farms that are in the herd book of a breed can benefit from the selection scheme of that breed. To do so, they use AI to test males of high genetic value , rebreedThe current regulations on organic farming  prohibitChurra sheep under the European Organic certification. We hypothesized that AI procedures based on sheep natural estrus can produce high fertility without increasing economic costs, which would eliminate the need for other procedures that require the administration of hormones for estrus synchronization.The main objective of this study was to quantify the efficiency of AI performed at natural estrus in Churra sheep farm in northwestern Spain that was part of the Churra breed selection scheme. The farm was surrounded by the Los Arribes del Duero Natural Park, near Portugal (41°24′23″ N–6°15′38″ W). The dehesa ecosystem predominated the area, with its typical semi-arid Mediterranean climate and poor-quality siliceous soils, which make the area ideal for extensive livestock farming. Specifically, the sheep flock was reared under a semi-extensive management system because the ewes spent nights outdoors, although weaned animals were milked twice a day for at least three months after lambing.The research was performed in an organic The first two years of the 8-year experiment were used to develop the best AI protocol, and in the remaining years, the established AI protocol was repeated. The Ethic Committee for Animal Experiments of the University of Salamanca approved the procedures performed in this study. Furthermore, the care and use of animals were in accordance with the Spanish Policy for Animal Protection (RD 53/2013), which meets the European union Directive 2010/63 on the protection of animal used for experimental and other scientific purposes.9 spermatozoa/mL were accepted for insemination. After that test, following the method of Evans and Maxwell [v/v) diluent at a concentration of 8 × 108 spermatozoa/mL, cooled until 15 °C, and loaded into mini-plastic straws .An AI program must be established well in advance of estrus detection because of the availability and time needed to collect semen as well as its limited shelf life. In this study, the process occurred at the “Ovigen” Genetic Improvement and Selection Center , 60 km from the organic farm. Semen from adult Churra rams with proven fertility was collected daily by means of an artificial vagina, and selection of the rams was coordinated by the National Association of Sheep Breeders of the Churra Breed (ANCHE). The testing procedures of the extracted semen included pH, color, mass activity , and concentration (spermatozoa per ml of semen). Following Moss et al. , only ej Maxwell , fresh sIn general, an AI protocol for natural estrus involves several phases; specifically, selection of dates for insemination, method of detection of natural estrus in ewes, optimal time from the detection of estrus to perform insemination, and expected fertility. In Spain, the optimal reproductive season for sheep is in September and October, and the entire experiment was conducted in these months.To detect estrus in the ewes in a flock of 700 sheep, three vasectomized rams with harness markers with chalk for marking mounted ewes were used. Following Gibbons and Cueto , rams weFollowing Glowatzki-Mullis , the AniGiven the complexity and operational limitations of having to perform inseminations for each of the three groups of ewes based on when they were marked by the vasectomized males , the AI process of groups G2 and G3 were combined into a single group (G23), and therefore, they contained ewes that were inseminated between 16 and 29 h after the detection of estrus. Once the AI protocol was accepted, it was implemented in the farm in six consecutive years. In that period, 581 ewes , physiological state (lactating or dry), body condition (1–5), number of hours post estrus detection until insemination , and vaginal fluid in the cervix —had on fertility. To that end, and given that all of the previous factors were categorical variables, a SAS Proc CATMOD  , originaChurra populations and other Spanish breeds that have the same type of semen but received induced estrus, e.g., 0.31 in the Churra breed [Castellana breed [Rasa Aragonesa breed [Fertility (lambings/inseminated ewes) and prolificity (lambs/ewe) of the first two years in which the AI protocol was developed are shown in ra breed  and 0.39ra breed  but lessna breed  and the sa breed . Mean fesa breed . In addisa breed ,25.Daily variability in fertility was high in the first year (from 0 to 0.38); however, except for the first day of the year of the study, daily fertilities were similar, although the daily sample size was small. The null result on the first day might indicate a failure with the marking method, because the semen and the application technique were the same on all days. Mean prolificacy rates were 1.23 and 1.25 in the first and second year, respectively, which were acceptable for ewes that did not receive hormonal stimulation.Multinomial associations between the factors and fertility indicated by the CATMOD suggest that none of the factors included in the analysis had a significant association with fertility over the rest of the factors ; therefop > 0.05) (The mean fertility of ewes that were inseminated either once (0.38) or twice (0.42) did not differ significantly ( > 0.05) , which iThe effect of the second insemination was most evident in ewes in the G1 group, which showed a 0.08 increase in fertility. In the ewes in the G3 group (inseminated later after estrus detection), fertility in the first and second insemination did not differ significantly. It would be interesting to perform double inseminations on ewes that have been detected in estrus no later than 16 h after onset. Furthermore, our results suggest that an early first insemination (<16 h) is less effective than a late insemination (16–24 h) .Lactating ewes had a higher fertility than did dry ewes, but the difference was not statistically significant . Others The reproductive performance of ewes subjected to AI is regulated largely by nutritional state, which can be evaluated visually based on body condition. In our study, all of the ewes had body scores between 2 and 4 on the 5-point scale . The resFertility in each of the three groups defined based on the number of hours elapsed since estrus detection until insemination did not differ significantly , althougAnalysis of vaginal fluid in the cervix area is essential for a successful AI procedure because the quantity and physicochemical characteristics dictate whether spermatozoa can pass through the cervix and fertilize the ovum .Although fertility and vaginal fluid volume were not correlated significantly, the highest fertility occurred among the ewes that had an average volume of vaginal fluid, and the lowest fertility was among the ewes that had very high fluid volumes . Other sChurra breed for induced estrus AI protocols [The average fertility in the last six years of the study was 0.39 , which irotocols . In addirotocols .p < 0.01) among rams. About a quarter (24.19%) of the rams had fertilities < 0.20, 25.80% had fertilities between 0.20 and 0.40, 45.16% had fertilities between 0.40 and 0.80, and 4.8% of the rams had fertilities > 80%. Male fertility is influenced by a multitude of intrinsic factors such as animal age and semen factors characteristics [The number of ewes inseminated by the AI protocol developed in our study per ram was very small because it depended on the number of ewes marked on that day and on the availability of semen from the individual ram . The numeristics ,32; howeeristics , the proeristics , and eveeristics  are othep = 0.451). Average fertilities among the last six years of the study differed significantly (p < 0.01) (p < 0.05) between ewes that differed in the duration of the estrus detection-AI interval  (0.38) and those marked in the day (G23) (0.39) did not differ significantly ( < 0.01) , and theinterval .Differences in fertility among the six years were similar to the results of Kukovics et al.  in AwassRegarding the economic costs associated with AI at natural estrus, they are generally reduced to the daily travel of the inseminator from the semen production and collection center to the farm for at least one week. However, this cost is offset by savings in the cost of hormonal treatments. Palacios 2010  estimateThe results of this study suggest that natural estrus insemination can substitute for induced estrus insemination without a reduction in fertility or an increase in economic cost. The only difference is an increase in the labor needed on the days of insemination. Fertilities were very high in all groups and did not differ significantly based on the timing of insemination after estrus detection. Lactating ewes produced better results than did dry ewes, which was probably because of the better feeding of the former. Double insemination increased the fertility of ewes whose estrus was detected within 16 h of onset. The other factors evaluated such as body condition and abundance of vaginal fluid in the cervix were correlated with fertility in ways that were similar to other studies, although differences were not statistically significant in our study.The implementation of the AI protocol used in this study for six years confirmed that exocervical inseminations at natural estrus can provide acceptable fertility and prolificity.


In aging-related diseases, miR-140-5p and miR-140-3p expressions are modulated. The seric levels of these two miRNAs are used as circulating biomarkers and may represent predictive tools. They are also considered key actors in the pathophysiology of aging-related diseases. miR-140-5p/-3p repress targets regulating cell proliferation, apoptosis, senescence, and inflammation. This work focuses on the roles of miR-140-3p and miR-140-5p in aging-related diseases, details their regulation , and reviews the molecular targets of theses miRNAs involved in aging pathophysiology.microRNAs (miRNAs) are small single strand non-coding RNAs and powerful gene expression regulators. They mainly bind to the 3′UTR sequence of targeted mRNA, leading to their degradation or translation inhibition.  Caenorhabditis elegans, by the Ambros and Ruvkun groups [The first microRNA (miRNA), lin-4, was described in 1993 in the nematode n groups . Since tmiRNAs are small non-coding RNAs involved in gene expression regulation. The average length of miRNAs is 22 nucleotides (nt), of which seven correspond to the seed region (nucleotides 2 to 8). Genes encoding microRNAs are intergenic or intragenic. In the first case, a specific promoter regulates transcription; in the second case, miRNA genes are mainly located in introns, whereas few of them are located in exons ,6,7. Conventionally, miRNAs genes are transcribed to primary miRNAs  using RNA polymerase II. Then, pri-miRNA is processed by a complex including DiGeorge Syndrome Critical Region 8 protein (DGCR8) and a RNase III enzyme Drosha, which cleaves the pri-miRNA into a 70 nt stem-loop precursor miRNA (pre-miRNA) ,10,11,12Several non-classical miRNA biogenesis pathways have also been described, independent of Drosha, Dicer, or AGO proteins . MirtronIntronic miRNAs genes are embedded into a host gene and are mostly processed according to the protein-encoding gene in which they are hosted. In a few cases, intronic miRNA expression differs from the expression of the host gene: miRNA transcription can be independent from the host gene promoter, as the miRNA genes can be regulated by their own independent promoter ,24,25,26MiRNAs mainly regulate gene expression due to messenger RNA (mRNA) interactions, inside the RISC complex: the seed region located to the 5′ end of microRNAs usually targets the 3′ untranslated transcript region (3′UTR) of mRNA, leading to mRNA degradation or translation inhibition, according to the base complementarity between these two sequences. In a few cases, a miRNA targets the 5′UTR of mRNA or coding sequence, and this interaction has been reported to induce translation under specific conditions  ,28,29,30Considering all these mechanisms, miRNAs are considered major actors in gene expression regulation. In addition, one microRNA is rarely specific to one mRNA, and one mRNA can be targeted by different microRNAs sharing the same seed region. Many studies and reviews are available reporting miRNA involvement in physiological pathways and developmental processes, as well as their deregulation under pathological conditions leading to diseases, such as cancers or genetic diseases. They also have been proposed as potential biomarkers or therapeutic targets in various pathological contexts ,35,36,37WW domain containing E3 Ubiquitin protein ligase 2 (WWP2) human gene. In mice, miR-140 gene is localized in the 16th intron of WWP2 gene [miR-140 gene transcription is independent of the transcription of its host gene, and is regulated by transcription factors, such as Nuclear factor of activated T-cells 3 (NFAT3) and Mothers against decapentaplegic homolog 3 (SMAD3) [Gene encoding microRNA-140-5p/-3p is located on chromosome 16q22.1, and is hosted in the 15th intron of the WP2 gene ,38. Its  (SMAD3) . A large (SMAD3)  is a tar (SMAD3) , and miR (SMAD3) .This review focuses on the roles of miR-140-3p and miR-140-5p in neurodegenerative diseases, atherosclerosis, visual acuity loss, osteoporosis, and osteoarthritis, which are all age-related diseases, and details the molecular targets of these miRNAs involved in aging pathophysiology.Neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease, or post-stroke infarction, lead to cognitive impairment with aging. Several miRNAs have recently been considered as neuroregulators, as well as neuroprotectors, leading to their consideration as biomarkers or therapeutic targets for neuronal cell death .phosphatase and tensin homolog-induced putative kinase 1 (PINK1), involved in mitochondrial function preservation [A Disintegrin and Metalloproteinase 10 (ADAM10) and SRY-box transcription factor 2 (SOX2), a transcription factor that positively regulates ADAM10 transcription. ADAM10 expression is thus reduced in AD, leading to Aβ plaque accumulation [To our knowledge, no study has evidenced miR-140-5p/-3p involvement in Parkinson’s disease. In contrast, two recent publications have highlighted the neurotoxic role of miR-140-5p in AD, based on the study of different AD models. AD is caused by accumulation of β-amyloid (Aβ) in the brain, forming neurotoxic amyloid plaques. miR-140-5p overexpression was observed in hippocampal tissues from a rat model of AD, and associated with neurological function impairment. In hippocampal mouse neurons from an in vitro model of AD, miR-140-5p overexpression (using mimics transfection) led to reactive oxygen species (ROS) production and mitochondrial dysfunction, by directly targeting ervation . miR-140mulation .Plasmatic miR-140-5p/-3p has also been proposed as a predictive tool for cognitive function evaluation during aging: miR-140-5p levels are positively associated with cognitive performance measures . miR-140Vascular Endothelial Growth Factor A (VEGFA), a pro-angiogenic factor and inhibits endothelial cell properties in vitro  under normoxic and hypoxic conditions [Similarly, the miR-140-5p plasmatic level is enhanced during acute ischemic stroke in humans . Interesnditions .These studies suggest that miR-140-5p is downregulated after ischemic stroke. Preventing the downregulation of miR-140-5p and, to a lesser extent, miR-140-3p could be neuroprotective and represent a new therapeutic approach, by inhibiting neuro-inflammation and abrogating angiogenesis following ischemic brain injury.Atherosclerosis (AS) is one of the main factors leading to cardiovascular diseases, which are the leading cause of death worldwide . AS is cOpposite results were published concerning the role of miR-140-5p in lipid accumulation into macrophages-derived foam cells: in a cellular model of AS macrophages (ox-LDL treated THP1 cells), miR-140-5p expression was inversely correlated with ox-LDL macrophages content, suggesting that in AS, miR-140-5p is downregulated. miR-140-5p could participate as a brake in AS, by targeting TLR4. Indeed, miR-140-5p mimic transfection in AS macrophages prevented lipids accumulation, ROS production, and cell apoptosis .ABCA1 gene to induce its expression [−/− mice, an in vivo model of AS, overexpression of miR-140-5p accelerated plaque formation in aorta, in association with reduced levels of RFX7 and ABCA1 in this tissue [In contrast, Zhao and colleagues demonstrated, in similar in vitro model, that miR-140-5p expression was enhanced in ox-LDL-treated macrophages. They showed that miR-140-5p silences Regulatory Factor X7 (RFX7), a transcription factor that binds to the promoter of pression . This gepression . miR-140s tissue .−/− mice, miR-140-5p was enhanced compared to wild type mice, thus increasing oxidative stress, resulting from the increase of ROS production and the decrease of antioxidant molecules (superoxide dismutase (SOD) and gluthathione (GSH)) [miR-140-5p is also involved in VSMC alterations during AS. In AS aorta from ApoEe (GSH)) . Sirtuine (GSH)) ,62. In re (GSH)) ,63. miR-e (GSH)) .LDLR. This prediction was experimentally confirmed in human hepatic cells, in which miR-140-5p inhibits the cell surface LDLR expression and reduces LDL-cholesterol absorption, while simvastatine reverts this phenomenon, as this drug is able to reduce miR-140-5p expression [LDL-cholesterol clearance from blood circulation also contributes to reducing atherosclerotic plaque formation. Plasmatic LDL-cholesterol endocytosis, via LDL-receptors (LDLR) on hepatocytes, is a major event controlling plasmatic LDL-cholesterol levels. In humans, the seed region of miR-140-5p was predicted to hybridize with 3′UTR of pression . miR-140pression . Moreovepression .−/− mice, leading to MKK6 and TP53RK proteins overexpression, compared to control aorta. These two kinases have been reported to be involved in signaling cascades playing a pivotal role in endothelial injury [mitogen-activated protein kinase 1 (MAPK1), encoding a serine/threonine protein kinase that belongs to the MAPK family. Activation of MAP kinase signaling results in the production of inflammatory cytokines and chemokines via NF-kB nuclear importation [Concerning miR-140-3p, this miRNA was significantly downregulated in AS aorta from ApoEl injury ,68. Simiortation ,70.C-Myb and B-cell lymphoma 2 (BCL-2) are no longer targeted by miR-140-3p, causing their upregulation. C-Myb is a transcription factor known to induce ASMC proliferation, while BCL-2 is an outer membrane mitochondrial anti-apoptotic protein [In a vascular injury model , miR-140-3p was downregulated in arterial smooth muscle cells (ASMC) isolated from restenosis artery wall compared to normal arteries, resulting in abnormal ASMC proliferation enhancement and apoptosis inhibition, which contributed to arterial damage. In this model,  protein .−/− mice, miR-140-3p was negatively regulated by circRNA_36781, also called circRNA_ABCA1 [As described for miR-140-5p (see above), several non-coding RNAs are predicted or proven to sponge miR-140-3p in AS. In plasma from coronary atherosclerotic heart disease patients, miR-140-3p was inversely correlated with the lncRNA nuclear enriched abundant transcript 1 (NEAT1) ; and in During aging, visual acuity loss occurs. Age-related macular degeneration (AMD) is the leading cause of visual impairment in Europe . AMD is In age-related cataract, another cause of visual acuity loss occurring during aging, no studies have shown a role for miR-140-5p/-3p, unlike other miRNAs .Osteoporosis is a systemic bone metabolic disorder, characterized by dysregulation of bone turnover, associated with low bone density and mass and accelerated bone loss, leading to bone fragility and fractures . OsteopoOsteogenic differentiation was induced in a model of bone marrow mesenchymal stem cells (BMSCs) submitted to tensile strain, a mechanical stimulation mimicking physical exercise at the cellular level, together with lncRNA-MEG3 expression enhancement; the latter represses miR-140-5p expression through a “sponging” mechanism . NeverthConversely, an elevated seric level of miR-140-3p could be a biomarker of osteoporosis, osteopenia, and fractures in post-menopausal women, as miR-140-3p was overexpressed in the serum of osteoporotic post-menopausal women, compared to healthy post-menopausal women . These rThe role of miR-140-3p in osteoblastic differentiation has still not been totally elucidated, as opposite results have been reported : FushimiOsteoarthritis (OA) is a chronic degenerative joint disease that affects quality of life in elderly populations. OA is associated with articular pain and stiffness, leading to functional disabilities. Several predisposing factors have been identified such as age, gender, obesity, and traumatic injury. Extracellular matrix (ECM) remodeling is impaired, in response to chondrocytes senescence and lesser proteoglycan secretion by chondrocytes. High levels of proinflammatory cytokines ) and matrix-degrading enzymes  and a disintegrin and metalloproteinase metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5)) are secreted in OA cartilage and synovial fluid. The elevated levels of reactive oxygen species (ROS) associated with oxidative stress cause oxidative damage, leading to OA . During miR-140 gene sequence in the intronic region of Wwp2 gene led to the abolishment of both miR140-3p and miR-140-5p expression in these miR-140−/− mice, whereas transgenic mice overexpressing both miR140-3p and miR-140-5p in cartilage were generated, using pri-miR-140 insertion near the cartilage-specific Col2a1 promoter [−/− mice exhibited a mild skeletal phenotype with a short stature, and during aging, OA damage was associated with ECM degradation and cartilage fibrosis. Moreover, transgenic mice were protected from OA development in a model of antigen-induced arthritis [miR-140-3p and miR-140-5p are physiologically expressed in cartilage and chondrocytes, and have been highlighted as key actors in OA ,88. In npromoter . Mouse drthritis . miR-140rthritis  and manyrthritis ,89,92. Trthritis ,94.miR-140 gene, and Interleukin-1β (IL-1β) stimulation [Regulatory mechanisms for miR-140-5p are opposed, independently of those regulating WWP2: NFAT3 increases miR-140-5p levels in OA-chondrocytes, and SOX9 physiologically stimulates miR-140-5p expression during chondrogenesis ,95. Convmulation ,88,96. Mmulation .In an in vitro model of OA chondrocytes (IL-1β treatment), miR-140-5p was decreased compared to non-stimulated chondrocytes. In this model, miR-140-5p transfection prevented chondrocytes senescence, and ECM degradation, as the expression of two major ECM components (COL2 (collagen type II) and ACAN (Aggrecan)), was restored, in association with a reduction of ECM degradation enzymes . ComparamiR-140-3p is also expressed in cartilage to a greater extent than miR-140-5p . SimilarWWP2 expression modulation. This recent development has only been used at this stage to identify miR-140-5p/-3p targets, but opens up great possibilities as a tool to further evaluate the role of miRNAs in AO and to assess potential new therapies [Considering all these data, miR-140-5p/-3p represent serious targets for OA treatment. As described previously, intra-articular injection of miR-140-5p in animal models of OA slows down OA progression ,94,98. Therapies .miR-140-5p/-3p have been widely studied in the context of cancer, but also in diseases related to aging, particularly in osteoarthritis. Both miR-140-5p and miR-140-3p are derived from the same gene, but have different targets. Despite this, these two miRNAs are often involved in the same pathologies with similar or opposite actions. They are key actors in the pathophysiology of many diseases and/or can be used as predictive circulating biomarkers.A list of miRNAs regulating aging-related pathways, such as senescence, DNA damage response, has been established; the miRNAs involved in physiological aging are thus, grouped under the term “geromiRs” . Even if


Strongyloides hyperinfection syndrome, tuberculosis, liver cancer, lymphoma other than adult T-cell leukemia-lymphoma and cervical cancer  is an important human pathogen. A fair estimate indicates that at least 5–10 million people harbor the virus worldwide  1, 2, and 3 . Subsequently, a second HTLV-1 isolate, HTLV-1MB was described , PTLV-2 (with type HTLV-2) and PTLV-3 (with type HTLV-3). Since then, there were no other suggested changes in the nomenclature nor in HTLV classification.In 1991, the 5th Report of ICTV  with numbers either in Roman (I and II) or in Arabic  according to the preference of the authors. All of these different denominations are the cause of confusion and do not help to increase awareness both among healthcare workers or the lay community. Nowadays when search sites are essential to collate an understanding of infection and disease, important gaps appear unless all the permutations are entered as illustrated in Although there were several names used before and after the definition of the genus A change of HTLV-1 denomination back to “human T-cell leukemia virus 1” has been proposed  and the second during the 18th International Conference on Human Retrovirology . Although this might be taken into consideration in the process to define a virus name when there is more than one candidate for the same taxon, the policy of the ICTV is that, as far as possible, decisions on questions of taxonomy and nomenclature should reflect the majority view of the appropriate virologic constituency . A fact that undeniably links HTLV-1 and HTLV-2 is that they are both lymphotropic viruses and this tropism characteristic should be the main aspect to be kept in their present denomination.https://talk.ictvonline.org/information/w/ictv-information/383/ictv-code), essential principles of virus nomenclature are: (i) to aim for stability; (ii) to avoid or reject the use of names which might cause error or confusion; (iii) to avoid the unnecessary creation of names.According to the International Code of Virus Classification and Nomenclature of the ICTV (Considering that: (i) the names “Human T-lymphotropic virus 1” and “Human T-lymphotropic virus 2” are comprehensive in their meaning and have been widely used, (ii) the fact that the clinical presentation goes beyond leukemia, (iii) the possibility of misleading healthcare professionals once ATL is a rare clinical manifestation, and most importantly (iv) the term “leukemia virus” is detrimental to patients as it would add stress to those diagnosed with this infection, we recommend that the name should not only be maintained but followed strictly as defined by the ICTV, which is the only institution capable of analyzing and defining viral taxonomy, since 1966. It would be desirable if editors of scientific journals could also stick to the appropriate denominations in viral taxonomy (including HTLV) to ensure a consistency in the published literature. Since the 7th Report of the ICTV in 2000, we are perpetuating an equivocal denomination. It is time to reach a consensus for the sake of consistency to refer properly to such a burden among the viruses of human medical importance.All authors contributed to the writing and approved the final version of the manuscript.AV (#302935/2021-5), LM (#314209/2021-2), and RI (#312979/2018-5) are supported and acknowledge the Research Grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq. GT was supported by the Imperial College Biomedical Research Center.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.


The human retroviruses HTLV-1 (human T cell leukemia virus type 1) and HIV-1 persist in vivo as a reservoir of latently infected T cell clones. It is poorly understood what determines which clones survive in the reservoir. We compared >160,000 HTLV-1 integration sites  from T cells isolated ex vivo from naturally infected individuals with >230,000 HTLV-1 integration sites  from in vitro infection to identify genomic features that determine selective clonal survival. Three statistically independent factors together explained >40% of the observed variance in HTLV-1 clonal survival in vivo: the radial intranuclear position of the provirus, its genomic distance from the centromere, and the intensity of local host genome transcription. The radial intranuclear position of the provirus and its distance from the centromere also explained ~7% of clonal persistence of HIV-1 in vivo. Selection for the intranuclear and intrachromosomal location of the provirus and host transcription intensity favors clonal persistence of human retroviruses in vivo. Clonal persistence of HTLV-1 and HIV-1 favors specific intranuclear sites and, independently, low local host transcription. S2C). The rank order of chromosome preference for in vivo survival was also correlated between the two viruses, albeit less strongly .While initial integration (in vitro) of HTLV-1 occurs in proportion to chromosome size (fig. S1)  , where F denotes the respective frequency of integration sites in that window.To identify the within-chromosome features that favor or disfavor survival of integrated HTLV-1 proviruses in vivo, we divided the human genome  into discrete 1-Mb windows. We define the HTLV-1 survival index in each window as Ln: Survival is favored when the provirus is integrated closer to the centromere and progressively disfavored toward the telomeres (fig. S4). Survival of the integrated provirus correlated significantly less strongly with the distance from the telomere than with the distance to the centromere (fig. S5).We define the distance from each window to the centromere as the absolute distance between the midpoint of each window and the midpoint of the centromere. The results show a strong negative correlation between the HTLV-1 survival index and the distance from the centromere  on both et al. . To corroborate the observation that proviral survival is associated with proximity to the nuclear lamina by an independent approach, we used a DamID dataset produced in T cells . FurtherLast, we used the recently reported spatial position inference of the nuclear genome (SPIN) method, which combines data from TSA-seq, DamID, and Hi-C to build a model that defines a set of spatial localization states of chromatin relative to nuclear bodies, reflecting a gradient of radial position from the nuclear lamina to the nuclear speckles .The highest gene density is often located in “T” bands of the human genome, many of which are telomeric . Similarly, at the level of 1-Mb windows, there was a strong negative correlation between expression intensity and the survival index for both HTLV-1 and HIV-1 , the expression intensity, and the distance to the centromere—remain significant independent predictors, together explaining ~40% of the observed variation in the HTLV-1 survival index  in rare, self-limiting bursts , We previously showed that the HTLV-1 provirus binds the chromatin architectural protein CTCF  clones. However, the reservoir of HIV-1–infected cells that can persist indefinitely during highly active ART is maintained partly by clonal proliferation  and the distance from the centromere. This observation contrasts with the fact that the nuclear speckle-associated domains, which are enriched in transcriptionally active genes, are strongly favored for the initial integration of HIV-1. However, in contrast with HTLV-1, only approximately 7% of the variation in proviral survival of HIV-1 can be explained by these factors: We postulate that this difference is due to the difference between the two retroviruses in the relative importance of infectious spread and mitotic spread  are detailed in table S1. Raw FASTQ data were used and processed in parallel to ensure consistency and comparability of data.www.bioinformatics.babraham.ac.uk/projects/trim_galore) to remove low-quality (q = 20) and adapter bases (using minimum overlap of three bases) and subsequently aligned against a combined reference of hg38 human genome and HTLV-1 upstream sequence using the Burrow-Wheeler Aligner (BWA)  integration sites were sequenced in parallel. Any integration sites found in the donor cell line or in >1 infection assay were excluded from analysis.https://doi.org/10.5281/zenodo.6353523 and https://github.com/ImperialCollegeLondon/intsite-2022.Random sites were selected from the hg38 genome reference using the R package intSiteRetriever , and repeatedly observed sites were removed from each dataset to ensure nonredundancy. See For the analysis of 1-Mb windows across the human genome, discrete windows along each chromosome were defined from position 1 to the chromosome terminus. One-megabase windows that overlap the end of the chromosome were excluded. To improve mapping confidence, 1-Mb windows with at least 1-kb overlap with an ENCODE exclusion list region (F) is calculated in each defined genomic region  as the proportion of all integration sites of a given dataset present in that region. We define a clone survival index (CSI) for each specified genomic region  asFin vivo or Fin vitro = 0), and the corresponding 1-Mb window is excluded from statistical analysis.The proviral integration site frequency  (https://bioconductor.org/packages/release/bioc/html/hiAnnotator.html).DNase-seq data were retrieved from the ENCODE project using the following criteria: organ, blood; cell, leukocyte; biosample, GM12878 or CD4-positive, alpha-beta memory T cell; genome assembly, GRCh38; genome assembly, GRCh38; and file type, “bed narrowpeak.” For GM12878, two replicate experiments are reported; a site is recorded as within N bases of a DHS if this condition is satisfied in both experimental replicates. Genomic distances are cumulative; e.g., the integration sites within 10 kb of a DHS also include the integration sites within 1 kb. Annotation of the nearest DHS to each integration site was done using the hiAnnotator R package , 156 datasets were available from 135 targets. Where more than one dataset was available for the same target, the larger dataset was used. Annotation of the nearest TFBS to each integration site from each type was made using the hiAnnotator R package.Histone modification ChIP-seq datasets were retrieved from the ENCODE project using the following criteria: organ, blood; cell, leukocyte; biosample, GM12878 or CD4-positive, alpha-beta memory T cell; genome assembly, GRCh38; file type, “bigwig”; and output type, “fold change over control.” Only those based on two replicates are used. Where more than one dataset was available for the same target, the larger dataset was used. Histone modification signal was averaged over fixed windows in the regions flanking each integration (or random) site using the UCSC bigWigAverageOverBed tool . A Y-excluded reference genome (hg38F) was used ; Bowtie2 (For consistency of reference (hg38), raw FASTQ data reported by Chen et al. (LAD data (table S2) were mapped by Robson Chromosome positions, chromosome arms, gaps, and centromere data were retrieved from the UCSC table browser  using the biomaRt R package (http://dcc.blueprint-epigenome.eu/#/files) based on the following criteria: cell type, central memory CD4-positive, alpha-beta T cell; and tissue, venous blood. Using discrete 1-Mb windows across the human genome, the mean expression signal from uniquely mapped reads per window was calculated using the UCSC bigWigAverageOverBed tool  under grant agreement no 282510 - BLUEPRINT.This study makes use of data generated by the BLUEPRINT Consortium. A full list of the investigators who contributed to the generation of the data is available at


Background: The emergence of acute-onset functional neurological symptoms, the focus of this study, is one of three stress responses related to immunisation. This case–control study documents the experience of 61 young people with past or current functional neurological disorder (FND) in relation to the COVID-19 vaccination program in Australia. Methods: Information about the young person’s/parent’s choice and response pertaining to COVID-19 vaccination was collected as part of routine clinical care or FND research program follow-up. Results: 61 young people treated for FND  and 46 healthy controls  were included in the study. Vaccination rates were high: 58/61 (95.1%) in the FND group and 45/46 (97.8%) in the control group. In the FND group, 2 young people  presented with new-onset FND following COVID-19 vaccination; two young people with resolved FND reported an FND relapse ; and two young people with unresolved FND  reported an FND exacerbation. In the control group no FND symptoms were reported. Conclusions: Acute-onset FND symptoms following COVID-19 vaccination are uncommon in the general population. In young people prone to FND, COVID-19 vaccination can sometimes trigger new-onset FND, FND relapse, or FND exacerbation. Diagnostic and Statistical Manual of Mental Disorders  no longer requires identification of a precipitating stressor, young people with FND—and their families—commonly report a triggering stressor or event against the background of cumulative stress or adverse childhood experiences (ACEs) .,35.2 areBy contrast, the mechanisms underpinning the second and third clusters—functional neurological symptoms and post-immunisation illness—are less well understood, poten-tially much more complex, and the focus of current research efforts .In this article we document the experience of 61 young people with past or current treatment for FND in the Mind–Body Program (2018–2022) in relation to the New South Wales COVID-19 vaccination program and the occurrence—new onset, exacerbation, or recurrence—of acute-onset functional neurological symptoms (cluster 2 above). We also report the experience of healthy controls who volunteered to take part in the FND research program.Participants were 61 young people who had been referred for treatment for FND in the Mind–Body Program at The Children’s Hospital at Westmead (October 2018 to September 2022) and who had agreed to participate in the FND research program. All young people with FND had undergone a comprehensive neurology assessment and had been given a positive diagnosis of FND (DSM-5 criteria) by a paediatric neurologist  see  and the The Sydney Children’s Hospital Network Ethics Committee approved the study (HREC/18/SCHN/232). Participants and their legal guardians provided written informed consent.In August/September 2022, information about the young person’s/parent choice per-taining to COVID-19 vaccination was collected as part of routine clinical care or as part of follow-up—looking at outcomes over time—for the FND research program. For young people treated in the past, questions were asked over the phone  and age (t(84.40) = −0.303; p = 0.763).The final study group interviewed about their vaccination experience (August/September 2022) comprised 61 young people (47 girls and 14 boys) aged 12.08 to 21.83 years  who had been or were being treated for FND and 46 healthy controls (34 girls and 12 boys) aged 10.83 to 21.5 years . The groups were matched for sex  and depression  were the most common). In addition, 21 (34.4%) had reported suicidal ideation, and 16 (26.2%) had suffered from one or more comorbid functional syndromes: functional gut disorder , postural orthostatic tachycardia syndrome (POTS) , and frequent vasovagal events .In the FND group, the clinical presentations of the 61 young people—at the time of their admission for treatment into the Mind–Body Program—had been diverse see . They prp < 0.001)) (see p < 0.001)).On admission to the research program, young people in the FND group reported a greater number of ACEs  compared to healthy controls  (t(70.57) = 8.22; 01)) see . On admin = 59), their medical status at the time of vaccination—or when pre-sented with the option of vaccination—was as follows: fully resolved FND ; resolved FND with a pattern of short-lived relapses in the face of stress ; and unresolved FND   were referred for treatment to the Mind–Body Program after their FND illness was triggered by COVID-19 vaccination. For the rest of the cohort (.8%) see .n = 34, 55.7%), depression , and PTSD  being the most common (see Forty-seven (77.0%) young people in the FND group were experiencing mental health symptoms at the time of vaccination (or option of vaccination), with anxiety .The vaccination rate in the FND group was 58/61 (95.1%). The vaccination rate in healthy control group was 45/46 (97.8%). There was no statistical difference between groups on vaccination rate —and her family—reported that she had not had the vaccine because her mother had experienced neurological symptoms  and cardiac symptoms  fol-lowing COVID-19 vaccination. In this context the adolescent (and her parents) were con-cerned that the vaccine could potentially trigger cardiac and neurological symptoms in the adolescent.The rates of FND relapse, FND exacerbation, and first-presentation FND following COVID-19 vaccination in our FND cohort are reported in Case 1. Relapse of FND symptoms post COVID-19 vaccination in a child whose FND had resolvedAn 18-year-old female with a past history of FND , coupled with comor-bid back pain, nausea, fatigue, dizziness, and functional gut symptoms, presented with FND relapse. Three years earlier, the initial episode of FND was triggered by a sporting injury . Other stressors at the time had included bullying and the death of her grandfather from a long illness. The young woman had recovered from the FND illness more than two years earlier. At the time of vaccination, she had current diag-noses of POTS and anxiety. Two weeks after her first COVID-19 Pfizer vaccination, she developed recurrence of functional seizures, which continued every 2–3 days for six months before resolving with further treatment. She subsequently had two further vac-cinations (Pfizer and Moderna) with no issues see .Case 2. Relapse of FND symptoms post COVID-19 vaccination in a young woman whose FND had resolved but who had experienced a number of short relapses with stressA 17-year-old female with developmental disability and severe anxiety developed her first episode of FND at age 15. That episode had emerged in the following context: influenza vaccination; chest infection (that same week); allergic reaction (after eating an egg sandwich) with urticarial rash and difficulty breathing (week 3); extreme fatigue and a collapse event at school, with hypokalaemia on blood screen treated with potassium (week 4); and emergence of FND in the form of jerky movements (week 5). Over time, the patient’s FND symptoms came to include back arching (dystonic movements in her back), functional seizures, loss of coordination in the legs (astasia abasia), and shaking and tremoring, coupled with episodes of sudden stabbing pain and panic attacks (with long periods of hyperventilation). The death of a grandfather was an additional, concurrent stressor. Following full recovery, the patient had had short-lived FND relapses in the con-text of stress at school . She presented two days after her first COVID-19 Pfizer vaccination with dysphagia, sialorrhea, leg weakness, and exacerbation of functional seizures. She was admitted for nine days of rehabilitation as her dysphagia resulted in restriction of oral intake to the degree of becoming medically unstable. Her FND symptoms resolved with treatment. She subsequently had two further COVID-19 Pfizer vaccinations with no issues see .Case 3. Worsening of FND symptoms in unresolved FNDA 19-year-old birth-assigned female who identifies as male suffered with chronic FND from age 15: functional seizures, leg weakness , and time-limited episodes of whole-body stiffening and functional blindness that occurred on a weekly basis. The patient’s FND had occurred in the setting of cumulative stressors over many years. These included: bullying, exposure to violence, and exposure to a severe mental illness in an older sibling. Comorbid diagnoses at the time of vaccination included dissociative identity disorder, depression, and gender dysphoria. Three days after the second COVID-19 Pfizer vaccination, the patient experienced a recurrence of bilateral leg weakness and required a wheelchair to mobilise. The leg weakness continued for six months while the patient underwent outpatient rehabilitation. The time-limited FND episodes of whole-body stiffening and blindness continued to occur. She subsequently had a further COVID-19 Moderna vaccination with no issues see .Case 4. Worsening of FND symptoms in unresolved FNDA 15-year-old adolescent female aspiring to be a doctor first experienced FND symp-toms at age 13 while under academic stress. Her FND included weakness and dragging of the left leg, loss of coordination in the legs, tremor, loss of hearing and tinnitus, blotchy vision, light-headedness, nausea, and cognitive symptoms of being unable to think clearly. In the patient’s own words, “The root cause of my symptoms was my tendency to put pressure on myself. I just challenged myself so much that I eventually burnt out.” At the time of her first COVID-19 Pfizer vaccination, the patient was making excellent progress toward a full recovery. Her remaining symptoms included functional seizures (of decreasing frequency), tinnitus, and visual loss, as well as comorbid POTS and functional gut symptoms. Immediately following the first vaccination, the patient experienced a functional seizure. Following the functional seizure, her legs were weak and unsteady, she experienced ticcing a new symptom , and she needed to use a wheelchair. The leg weakness recurred following the second vaccination. On both occasions the symptoms resolved in a few days.Case 5. New-onset FND following COVID-19 vaccinationA 17-year-old female experienced new-onset FND following COVID-19 vaccination that led to admission into the Mind–Body Program for treatment. Immediately after her first Pfizer vaccination, she experienced pain and itchiness at the injection site. She woke the next day with a circular, raised, erythematous rash with central blistering that ex-tended along the entire right arm. The rash remained for a week. The day following the vaccination she also experienced nausea and vomiting as well as an unresponsive epi-sode that lasted for approximately 40 min.Three weeks following the vaccination, she had increased frequent collapses. She was admitted for further investigation: an electroencephalogram did not show any evidence of epileptiform activity. Her collapses continued for six months before the diagnosis of FND was made by a neurologist after further investigations (lumber puncture and magnetic resonance imaging). By this time the patient was experiencing new symptoms of limb weakness, muscle spasms (causing a shoulder subluxation), and functional tics, which were both verbal and motor. She was also experiencing irritable bladder, functional gut symptoms, and dizziness on standing secondary to POTS.Predisposing risk factors included the following: the death of four grandparents, death of an aunt, death of a close friend, death of three dogs, and sexual assault, all within the period of two years. Prior to the development of FND, this patient had been given a diagnosis of depression with posttraumatic stress disorder—related her traumatic expe-riences—and she was being managed by a community mental health team.The patient was admitted for a three-week rehabilitation admission. On discharge she was mobilising on a walking frame, and her tics had largely subsided. She had en-gaged in the tasks of learning physiological regulation strategies  and learCase 6. New-onset FND following COVID-19 vaccinationA 12-year-old girl experienced new onset of three different medical conditions—atypical narcolepsy  ,51,52,53The child presented to the emergency department with an 11-day history of hyper-somnolence that started two hours after her second vaccination. She was treated with dexamphetamine. Four weeks following the vaccination, she developed acute episodes of nausea, vomiting, and abdominal pain, coupled with an inability to tolerate oral intake. The patient’s pain was managed with simple analgesics; her distress, with olanzapine (as needed); and her nutritional needs, with feeds via a nasogastric tube. Two weeks follow-ing the vaccination, she developed bilateral lower limb weakness (necessitating a wheel-chair to mobilise). She also developed functional seizures that showed semiology changes across time: zoning out; episodes of collapse; generalised shaking; generalised shaking coupled with the patient hitting herself; episodes of aggression while the patient was in an altered state; and episodes where the patient ran around and around her hospital room while in an altered state . The child’s FND symptoms made the clinical picture very complex and excluded the possibility of clearly diagnosing cataplexy.The patient was discharged from hospital on day 97 with ongoing outpatient man-agement of narcolepsy (by the sleep team), functional gut disorder (by the family doctor), and FND, depression, and anxiety .Predisposing risk factors for FND and a functional gut disorder included the follow-ing: the stress of home schooling during COVID-19 lockdowns; increased stress (conflict) in the parental relationship, which had previously been amicable; conflict with peers in Year 6; loss of the opportunity to go to a performing arts high school; stress around transi-tion to high school; and a medical history of functional cough, functional abdominal pain, and untreated depression.In the FND group, 24 (39.3%%) reported that they had contracted COVID-19. Of these, 23 (37.7%) reported that they had contracted COVID following one or more vaccinations. No exacerbations of FND were reported in association with COVID-19 infections, and no prolonged illness (long COVID) was reported.In the control group, 31 (67.4%) reported that they had contracted COVID-19. Of these, 30 (96.8%) reported that they had been vaccinated, and they had contracted COVID-19 after one or more vaccinations. In this vaccinated subgroup, no neurological symptoms and no prolonged illness (long COVID) were reported.Additionally, in the control group, the adolescent girl who had not been vaccinated—because her mother had experienced neurological symptoms after being vaccinated—reported a prolonged illness (long COVID). After falling ill with COVID-19, she developed POTS—reflecting activation and dysregulation of the autonomic nervous system—alongside high temperatures and cold-like symptoms. The POTS symptoms slowly resolved after more than two months and recurred three times with subsequent viral infections (not COVID-19). In each case the autonomic symptoms preceded the adolescent’s cold-like symptoms. The symptoms were severe enough to require management by a cardiologist.The current study documents the experience of a cohort of 61 young people admitted to the Mind–Body Program for the treatment of FND (October 2018 to September 2022)—and 46 healthy controls—with respect to the COVID-19 vaccination program in New South Wales, Australia. Vaccination rates across the two groups were high. In the FND group see , two youThree previous follow-up studies of young people with FND at 4 years, 12 months, and 18 months have shown that 17.5%, 11.5%, and 4%, respectively, experienced relapse in response to subsequent stress. In the current study, of the 51 young people with past or current FND who were vaccinated for COVID-19, 4/51 (7.84%) experienced a relapse or a worsening of their symptoms with vaccination see . As abovStress-related responses to vaccination are well documented in the literature (see the three clusters described in the introduction) ,31,32,33hypothesis based on emerging research.In the clinical setting, the understanding of underlying biological mechanisms that underpin stress-related responses to vaccination is limited. For example, the 2019 updated World Health Organization user manual—Causality Assessment of an Adverse Event Following Immunization —FND presA large body of research highlights that stressors—whether they be physical or psy-chological—activate the stress response in a coordinated manner see  59]. By. By59]. Vaccination as a physical (bottom-up) stressor involves activation of the immune-inflammatory system via vaccine constituents and the mounting of an antibody response. The antibody response also involves activation of the sympathetic nervous system (a component of the autonomic nervous system) and the HPA axis . BecauseVaccination as a psychological (top-down) stressor involves activation of the stress system via mental processes (thoughts and feelings) ,62,63,64Previous literature has noted that young people are susceptible to communications of threat  from family members, peers, and social media. A group-contagion effect pertaining to mass outbreaks of FND and other functional somatic symptoms (see clusters 2 and 3 in the Introduction) is well documented. Such outbreaks appear to occur during times of threat or stress, including war and pandemics ,66. The The hypothesised mechanisms—working bottom-up and working top down—are not mutually exclusive. The mechanisms can occur in tandem and may reflect a set of neuroimmune–endocrine–psychological cascade of threat-related responses—the body–brain–mind response to signals of danger —that interact with each other in a mutually reinforcing way.Importantly, in most healthy young people, stress-system activation is short-lived. The stressor is dealt with, and body and brain systems return to normal function . In young people who de-velop FND, however, various components of the stress system—the HPA axis , the au-In this final section of the discussion we consider three potential biological mecha-nisms via which vaccination may trigger a cascade of stress-related responses and con-tribute to the process by which FND symptoms are triggered or exacerbated.First, the nervous system and the immune system are interdependent systems that communicate in a bidirectional manner—from the brain to the immune system and from the immune system to the brain . Any altSecond, neural networks are now conceptualised as neuron–glia networks—where microglia engage in reciprocal signalling with neurons to underpin brain function in health and disease ,83,84,85Third, young people with FND ,89 reporOnce vaccination or another stressor  has triggered the FND symptoms, a number of factors may contribute to symptom maintenance see  3,6,7,8,94,95,96A prospective design using a whole-population approach is needed to provide inci-dence statistics of acute-onset FND following COVID-19 vaccination in the general popu-lation. Such data will also tell us what FND symptoms are most common post COVID-19 vaccination. Such a design would also provide incidence data about the two other stress responses related to immunisation: acute anxiety-related responses (cluster 1) and a post-immunisation illness characterised by nonspecific functional somatic symptoms (cluster 3) (see introduction for the description of the three clusters). The lack of longitudinal outcome data for case 5 and case 6—who were still experiencing FND symptoms at the time this paper was written—is another limitation.Acute-onset FND symptoms following COVID-19 vaccination are uncommon in the general population. Notwithstanding, in young people prone to FND, COVID-19 vaccina-tion can trigger new-onset FND, FND relapse, or FND exacerbation. The biological mech-anisms involved are thought to be complex: a cascade of stress-related responses (includ-ing epigenetic reprograming) that lead to a sustained activation of neural  networks ,74,75 co