Brain aquaporin 1 (AQP1) and AQP4 are involved in cerebrospinal fluid (CSF) homeostasis and might participate in the origin of hydrocephalus. Studies have shown alterations of perivascular AQP4 expression in idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer’s disease (AD). Due to the overlapping of clinical signs between iNPH and certain neurological conditions, mainly AD, specific biomarkers might improve the diagnostic accuracy for iNPH. The goal of the present study was to analyze and quantify the presence of AQP1 and AQP4 in the CSF of patients with iNPH and AD to determine whether these proteins can be used as biomarkers of iNPH. We examined AQP1 and AQP4 protein levels in the CSF of 179 participants (88 women) classified into 5 groups: possible iNPH (81 participants), hydrocephalus associated with other neurological disorders (13 participants), AD (41 participants), non-AD dementia (32 participants) and healthy controls (12 participants). We recorded each participant’s demographic and clinical variables and indicated, when available in the clinical history, the record of cardiovascular and respiratory complications. An ELISA showed virtually no AQP content in the CSF. Information on the vascular risk factors (available for 61 patients) confirmed some type of vascular risk factor in 86% of the patients with possible iNPH and 58% of the patients with AD. In conclusion, the ELISA analysis showed insufficient sensitivity to detect the presence of AQP1 and AQP4 in CSF, ruling out the possible use of these proteins as biomarkers for diagnosing iNPH. Idiopathic normal pressure hydrocephalus (iNPH) is a disease commonly observed in older adults and is generally underdiagnosed because the obvious symptoms are frequently interpreted as indicative of the elderly condition. The prevalence in the general population is still unclear but figures ranging from 0.5% to 2.9% are indicated among those aged 65 and older .The typical clinical triad of this potentially reversible neurological condition consists of gait disturbance, dementia and urinary incontinence, combined with ventriculomegaly –4. AlthoBrain aquaporins (AQPs), particularly AQP4 and AQP1, are water channel proteins that facilitate the flow of water through the brain compartments and play an important role in CSF homeostasis ,9. RecenThe obvious participation of AQP4 in CSF transport in the brain and interstitial fluid clearance as a key element of the glymphatic system, as well as the lack of reliable biological markers in the differential diagnosis of iNPH and AD, lead to the conclusion that brain AQPs might be involved in the development of these pathological conditions and might help monitor and differentiate the two diseases. Thus, the aim of this study is to determine whether ventricular enlargement in elderly patients with iNPH and patients with AD might correlate with abnormal expression of either AQP1 or AQP4 in the brain, affecting the abundance of these proteins in CSF.This observational, cross-sectional, and retrospective study was designed to analyze the expression of AQPs on CSF samples of patients with “probable iNPH” against patients with a different diagnosis, and healthy controls. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of University Hospital Virgen Macarena and University Hospital Virgen del Rocío .CSF samples were donated, after signing informed consent, by 179 patients  aged over 50 years old. They were recruited from December 2018 until January 2020 from Virgen del Rocío University Hospital, Seville, Spain. For diagnosis, patients were classified into 5 groups :I- Groups 1–2 (possible iNPH and unlikely iNPH). 94 patients were recruited from the Department of Neurosurgery and Neurology. These patients presented with gait disorder (with or without cognitive impairment or urinary incontinence) and cranial magnetic resonance imaging (MRI) showing free communication between the ventricular system and subarachnoid space and an Evans’ index >0.30. They were evaluated by expert neurologists considering the diagnosis of iNPH. The workup included Hellström’s scale ; cranialII- Groups 3–4 (AD suspected). Both groups were recruited from the Neurology Department and included patients with cognitive complaints in whom AD was suspected after a comprehensive clinical, neuropsychological, and radiological evaluation. Group 3 was made up by 41 patients who met the International Working Group (IWG) 2 criteria  for typiIII. Group 5 . This group was recruited by Department of Anesthesia and made up by 12 not-cognitively-impaired patients for whom a CSF sample was obtained during spinal anesthesia for non-neurological surgical indications.For the analysis, demographic and clinical variables were recorded for each participant, including gender, age at study inclusion, time from symptoms onset, and clinical diagnosis . CSF samAfter extraction, CSF was aliquoted and kept at -80°C until use in the Biobank of the University Hospital Virgen del Rocío, Seville, Spain.HEK293T-plated cells at approximately 80% confluence were transfected with AQP4-EGFP construct using Lipofectamine 2000 (Invitrogen) as described by Sánchez-Gomar et al. . Thirty-For the AQP CSF determination, CSF was collected into polypropylene tubes and stored at -80°C until analysis by ELISA. Quantification of the AQP1 and AQP4 protein content in 100 μL of CSF from patients and healthy controls was performed using commercial ELISA kits for AQP1 and for AQP4 , following the manufacturer’s protocol. The optical density obtained in the ELISA plates was measured with a spectrophotometer at 450 nm (Thermo Scientific). For the standard curve, we used lyophilized protein of either AQP1 or AQP4 provided with the kit. Homogenates of the protein extracts were prepared for use as positive controls for the presence of AQP4. Bloody CSF was also included in the analysis as a positive control for AQP1 detection, given that AQP1 is highly abundant in human erythrocytes .The specific number of CSF analyzed in each experimental condition (n) are exposed in each figure. Data are presented as mean ± standard deviation (SD). For all the statistical analysis performed, data were tested for normality  and equal variance (Brown-Forsythe test). Because the data do not adjust to a normal distribution, the non-parametric Kruskall-Wallis test was carried out. All statistical analysis were conducted by GraphPad Prism8 software.A combination of various tests that includes a neurological examination, neuroimaging study and tap test are usually performed to diagnose iNPH ,27; howeWe used two specific ELISA kits to determine the presence of AQP1 and AQP4 in CSF –32. The 2 = 0.9932) that ensured the exact determination of AQP1 protein in the detection range covered by the standard curve. As shown in Two CSF samples from patients with hemorrhagic hydrocephalus who were initially excluded were subsequently included in a second round of assays. The inclusion of these two CSF samples, which were slightly contaminated by erythrocytes that abundantly express AQP1 in their plasma membrane, provide us with a perfect positive control for the ELISA test and demonstrate that, if AQP1 was present, we would be able to detect it in our analysis. As shown in Therefore, the estimated AQP1 concentration was above the lower detection limit (LDL) of 0.09 ng/mL in only a few CSF samples from the various patient groups . In mostRegarding AQP4 detection in CSF, given that AQP4 is not expressed in the erythrocyte membrane, we generated a specific positive control for this assay to further demonstrate the adequacy of the commercial ELISA test for detecting AQP4 proteins. HEK cells were transfected with a plasmid encoding for human AQP4, and a homogenate of proteins extracted from these cells was prepared at different concentrations and used in a trial test of the ELISA assay. In the case of AQP4, the standard curve covered a determination range from 0.156 ng/mL to 10 ng/mL ; and theIn 2011, Blocher et al.  were theTwo previous studies ,34 exploThe reason behind the almost complete lack of these proteins in the CSF of the patients with iNPH and not just a reduction in AQP levels as indicated by other authors remains unclear. Differences in the kits’ sensitivity could offer one explanation, but that was not the case for our study. Inadequate criteria for extrapolating the protein concentrations might have been the cause, as observed in the case of AQP4. Specifically, the detection range for the ELISA kit used in the study by Arighi et al. was 0.156 ng/mL to 10 ng/ mL , indicatAs in AD, the loss of perivascular AQP4 in iNPH has been observed by immunohistochemical analysis ,13,35, wAlthough iNPH is generally considered an idiopathic condition, its etiology appears to combine several factors. Abnormal CSF dynamics, resulting from alterations in CSF pulsation due to variations in cardiac rhythm, respiration rate, or brain compliance, can play a role . The assWe observed the presence of cardiovascular and respiratory risk factors that could contribute to iNPH in the clinical history of a subset of patients . InteresFinally, we would like also to say that this study is not free of limitations. For instances, none of the different criteria used for diagnosis show high enough sensitivity and specificity for diagnosing iNPH, so some overlap between groups 1–2 could be present. Also, the apparently clear separation between groups 1 and 3 has recently been challenged by a new line of evidence that supports a continuum between AD and iNPH . But in In summary, AQPs have been widely implicated in CSF homeostasis. AQP1 in the choroid plexus and AQP4 in brain perivascular blood vessels play important roles in CSF production and clearance. Alterations in the glymphatic system associated with the reduction or redistribution of AQP4 expression appear to participate in the etiology of iNPH and AD. However, the shedding of AQP1 and AQP4 into the CSF does not appear to occur in any of these diseases, at least not at levels detectable by ELISA. Thus, detecting AQPs in CSF as biomarkers for diagnosing iNPH and distinguishing these patients from those with AD appears unlikely, at least with the detection level offered by the currently available commercial ELISA kits.AQP1 and AQP4 proteins are almost undetectable in the CSF of patients with iNPH using commercial ELISA tests. These AQPs were also undetectable or rarely present in the CSF of patients with AD and healthy controls. AQP1 and AQP4 cannot be used as CSF biomarkers to distinguish iNPH from AD.


To discover the levels of NLR family pyrin domain-containing 3 (NLRP3) in the cerebrospinal fluid (CSF) from adult patients with community-acquired bacterial meningitis (CABM).We enrolled 34 patients with CABM, 20 patients with viral meningitis (VM), and 25 patients with non-inflammatory neurological disease. Data on standard clinical parameters, scores, and outcomes were obtained from clinical records, and inflammasome levels in the CSF were measured by an enzyme-linked immunosorbent assay. The area under the receiver operating characteristic curve (AUROC) was used to quantify the diagnostic and prognostic performance of CSF NLRP3 as a biomarker of CABM.The levels of NLRP3 were elevated in the CSF of patients with CABM, but levels for ASC, caspase-1, or other inflammasomes did not vary significantly. CSF NLRP3 was positively correlated with clinical severity and with the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte count, albumin quotient , and immunoglobulin G quotient (QIgG). Patients with unfavorable outcomes had higher levels of NLRP3 in the CSF, which were correlated with several blood indicators, including NLR, PLR, and lymphocyte and monocyte counts.Our results suggested that the level of CSF NLRP3 could represent the severity of CABM in adults. CSF NLRP3 may be a good biomarker for the diagnosis of CABM and for the discrimination between CABM and VM. It may also be a better biomarker for predicting the prognosis of adult patients with CABM when compared to the NLR or the lymphocyte and monocyte counts. Community-acquired bacterial meningitis (CABM) is a rare disease affecting all age groups, with an annual incidence of approximately three cases per 100,000 adults in developed countries . DespiteThe activation of toll-like receptors (TLRs) is a key event in meningeal inflammation and a pivotal factor for meningitis-associated tissue damage . In addiThe inflammasome is a complex composed of a variety of proteins that initiate the activation of caspase-1 leading to the cleavage of the pro-inflammatory cytokines IL-1β and IL-18 . Four maThe aims of this study were to measure NLRP3 levels in the CSF from patients with CABM, viral meningitis (VM), and non-inflammatory neurological disease; to characterize the correlation between clinical outcomes and NLRP3 levels; and to determine whether NLRP3 levels from the CSF could be used as a diagnostic and prognostic biomarker for bacterial meningitis in clinical practice.Streptococcus pneumoniae, six cases (17.65%) of Klebsiella pneumoniae, four cases (11.76%) of Escherichia coli, four cases (11.76%) of Listeria monocytogenes, two cases (5.88%) of Staphylococcus aureus, one case (2.94%) of Bacteroides fragilis, one case (2.94%) of human staphylococcal human subspecies, one case (2.94%) of acne propionic acid bacillus, one case (2.94%) of constellation streptococcus constellation subspecies, one case (2.94%) of Streptococcus suis, one case (2.94%) of Streptococcus pharyngitis, and one case (2.94%) of Enterobacter cloacae.This retrospective cohort study enrolled 34 patients with CABM, 20 patients with VM, and 25 patients with non-inflammatory neurological disease treated at the Department of Neurology of the First Affiliated Hospital of Zhengzhou University from June 2019 to June 2021. The study design was approved by the Zhengzhou University Ethics Committee. The inclusion criteria were as follows: (1) age ≥18 years; (2) disease first diagnosed at the Department, without previous antibacterial or anti-inflammatory treatment; and (3) confirmed bacterial meningitis by positive CSF microscopy/culture or high clinical suspicion of meningitis due to next-generation sequencing (NGS)-positive detection and increased cell count and/or decreased blood/glucose ratio in the CSF . We exclPatients from the VM group were diagnosed by NGS detection of viral DNA/RNA in the CSF. The etiologies of VM included eight cases of herpes simplex virus, five cases of Epstein–Barr virus, three cases of cytomegalovirus, two cases of varicella-zoster virus, and two cases of Coxsackie virus. The control group consisted of patients diagnosed with other non-inflammatory neurological diseases, including dizziness, headache, and hysteria.Data collected from all patients included medical history , Glasgow Coma Scale (GCS) on admission, clinical symptoms on admission, laboratory findings on admission , clinical course, outcome, neurological status at discharge [determined by the Glasgow Outcome Scale (GOS)] or 3 months after onset [determined by the modified Rankin Scale (mRS) score], and treatment. Two experienced neurologists independently evaluated the CABM severity according to the GCS scores at admission, the GOS scores at discharge, and the mRS scores at follow-up. Favorable outcomes were defined as those presenting an mRS score in the 0–2 range, and unfavorable outcomes were defined as those within the 3–5 range . All metin vitro culture, and electrophoresis tests. The remaining volume was immediately centrifuged at 4000 g for 10 min at 4°C. The supernatant was then transferred into polypropylene tubes and stored at −80°C. Sample treatment was completed within 60 min.CSF was collected from all patients on the date of venous blood collection. After a standard lumbar puncture, 15 ml of CSF was collected. From this sample, 10 ml was used for routine biochemical and cytological tests, NGS, via instrument analysis. In parallel, the centrifuge precipitation method was used for manual microscopic examination, classification, and reexamination. The albumin quotient  and immunoglobulin G quotient (QIgG) were detected via immunoelectrophoresis. CSF second-generation sequencing was performed via high-throughput analysis by the Beijing Institute of Genomics .All blood samples collected were tested for biomarkers at the Biochemistry Laboratory of the First Affiliated Hospital of Zhengzhou University. Fasting venous blood was drawn from all patients on the morning of the second day after admission. Blood samples were used for routine blood tests and for the measurement of inflammatory markers. For routine CSF analysis, humoral cells were counted and classified Commercial sandwich enzyme-linked immunosorbent assay (ELISA) kits were used to measure inflammasome levels in the CSF. Target proteins included NLRP1 (CSB-EL015864HU), NLRP3 (CSB-E15885h), NLRC4 (CSB-EL015862HU), AIM2 (CSB-EL001499HU), ASC (CSB-EL019114HU), and caspase-1 (CSB-E13025h). All kits were manufactured by Cusabio . Assays were performed according to the manufacturer’s instructions. All standards and samples were measured in duplicate, and CSF samples were not diluted. Optical densities were determined using a microplate reader .t-test was used to compare data between two groups. The median (interquartile range) was used to characterize the results for non-normally distributed data, and the Mann–Whitney U test was used in this case to perform comparisons between groups. Categorical variables were expressed as percentage of the total number of cases and were compared using the Chi-square test or Fisher’s exact test. Univariate and multivariate analyses were used to identify the factors that could distinguish between the groups. Correlations between the profiles were assessed using Spearman’s rank analysis or Pearson’s correlation analysis, as appropriate. The area under the receiver operating characteristic curve (AUROC) was used to quantify the performance of biomarkers for a given diagnosis. The cutoff values for outcome prediction were selected on the basis of the highest sum of sensitivity and specificity. Statistical significance was set at p < 0.05.Statistical analysis was performed using the SPSS software package . The data were tested for normality, and normally distributed data were expressed as mean ± standard deviation. The Kruskal–Wallis test was used for comparison between groups. An independent Student’s n = 34), VM patients (n = 20), and CTLs (n = 25) are shown in The demographic data, clinical features, and laboratory data of CABM patients , platelet-to-lymphocyte ratio (PLR), and levels of C-reactive protein (CRP) and procalcitonin (PCT), while at the same time exhibiting a significant decrease in the lymphocyte count and the lymphocyte-to-monocyte ratio (LMR) when compared with the other two groups Table 1.p < 0.001, p < 0.001, The results of the ELISA tests performed to measure the levels of CSF NLRP1, NLRP3, NLCR4, AIM2, ASC, and caspase-1 are shown in S. pneumoniae (29.41%), K. pneumoniae (17.65%), E. coli (11.76%), and L. monocytogenes (11.76%). Mean CSF NLRP3 (ng/ml) was 2,124 ± 1,396 for S. pneumoniae infection, 2,606 ± 1,497 for K. pneumoniae infection, 2,181 ± 2,386 for E. coli infection, and 1,213 ± 996.9 for L. monocytogenes infection. However, there was no significant difference in CSF NLRP3 among different bacteria  values for the CABM group in the receiver operating characteristic (ROC) curve analysis , suggesting that they may also be good predictors of CABM prognosis  and damage-associated molecular patterns (DAMP) are the critical initial stimuli that activate NLRP3 . The olipathways . NLRP3-dpathways .K. pneumoniae infection, but decreased in L. monocytogenes infection. However, due to the small sample size, there was no significant difference in CSF NLRP3 among different bacterial infections. We needed to expand the sample size for further analysis.We also compared NLRP3 levels in CSF from CABM patients caused by different bacteria. The expression of CSF NLRP3 seemed to be increased in To date, high levels of NLRP3 in the CSF have been reported in several neuroinflammatory diseases such as anti-N-methyl-D-aspartate receptor encephalitis , autoimmSome previous studies have proposed new biomarkers for bacterial meningitis, such as serum or CSF PCT , neutropWe further analyzed the correlation between CSF NLRP3 levels and some clinical parameters. The results showed that CSF NLRP3 had a good correlation with some immune system or immune response parameters such as the NLR, the PLR, and the lymphocyte count, suggesting that CSF NLRP3 may be closely associated to the innate and adaptive immune systems. A previous study on rats  had showOur study had some limitations. The sample size was somewhat small, and therefore the potential usefulness of CSF NLRP3 as a biomarker for CABM needs to be verified with a larger cohort. In addition, data from the blood and cerebrospinal fluid were obtained from standard hospital laboratory tests, which made us fail to further compare some newly discovered meaningful indicators in recent years. Finally, the study focused exclusively on adult patients, leaving the potential value of CSF NLRP3 as a biomarker in a pediatric context as a hypothesis to be tested in future studies.To summarize, we found substantially higher levels of NLRP3 in the CSF of adults with CABM. These levels were positively correlated with clinical severity, the NLR, the PLR, Qalb, and QIgG, and they were also negatively correlated with favorable outcomes and with the lymphocyte count, suggesting that the NLRP3 inflammasome might play an important role in the pathogenesis of CABM in adults. In addition, we found for the first time that CSF NLRP3 may be a good biomarker for the diagnosis of CABM and for the discrimination between CABM and VM cases in adults. It may also prove to be a useful potential biomarker for predicting the prognosis of individual CABM cases in adults.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of Zhengzhou University (2019-KY-018). The patients/participants provided their written informed consent to participate in this study.ZG carried out the experiments, performed the analysis, and wrote the manuscript. CZ contributed to the collection of the clinical data. YL and LJ contributed to data analysis. RD and YY edited the manuscript. JT collaborated with us and contributed to the design of the study. YJ designed the study and contributed to the writing and editing of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (NSFC 82001290 to ZG).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.


Many patients are referred to multiple sclerosis (MS) tertiary centers to manage brain white matter hyperintensities (WMH). Multiple diagnoses can match in such situations, and we lack proper tools to diagnose complex cases.This study aimed to prospectively analyze and correlate with the final diagnosis, cerebrospinal fluid (CSF) interleukin (IL)-1β, soluble IL-2 receptor (CD25), IL-6, IL-10, and kappa free light chains (KFLC) concentrations in patients presenting with brain WMH.All patients over 18 years addressed to our MS tertiary center for the diagnostic workup of brain WMH were included from June 1, 2020, to June 1, 2021. Patients were separated into three groups—MS and related disorder (MSARD), other inflammatory neurological disorder (OIND), and non-inflammatory neurological disorder (NIND) groups—according to clinical presentation, MRI characteristics, and biological workup.n = 88), OIND (n = 35), and NIND (n = 53). Median CSF KFLC index and KFLC intrathecal fraction (IF) were higher in MSARD than in the OIND and NIND groups; p < 0.001 for all comparisons. CSF CD25 and IL-6 concentrations were higher in the OIND group than in both the MSARD and NIND groups; p < 0.001 for all comparisons. KFLC index could rule in MSARD when compared to NIND  or OIND . These results were similar to those with oligoclonal bands . In contrast, elevated CSF CD25 and IL-6 could rule out MSARD when compared to OIND .A total of 176 patients  were included. The diagnosis was MSARD  vasculitis. These results need to be confirmed within more extensive and multicentric studies. Still, they sustain that KFLC, CSF CD25, and CSF IL-6 could be reliable biomarkers in brain WMH diagnostic workup for differentiating MSARD from other brain inflammatory MS mimickers. Multiple sclerosis (MS) is a chronic inflammatory and demyelinating central nervous system (CNS) disease. It presents as relapsing clinical demyelinating events or a progressive worsening neurological deficit disease with suggestive white matter hyperintensities on the brain or spinal cord MRI T2-weighted images. Clinical research has focused on diagnosing MS as early as possible to prevent relapse and disability by initiating disease-modifying treatments. In this condition, many patients may have an early demyelinating disease diagnosis: i) after a single demyelinating event , 2 or iiThe immunopathology of MS is complex and implicates a large number of cells. CD4+ Th1 and Th17 cells are thought to promote while CD4+ Th2 and Treg cells are thought to downregulate inflammation in MS –10. B ceDuring the last decade, many biomarkers have been explored. Kappa free light chains (KFLC), low-weighted immunoglobulin compounds, are a reliable biomarker in MS –16. ThisTherefore, in this study, we prospectively evaluated the expression of KFLC biomarkers and CSF concentration of IL-1β, sIL-2R (CD25), IL-6, and IL-10, and we correlated each biomarker measurement with diagnosis in patients referred to our MS center for suspected MS.All patients referred to our MS tertiary center in the University Hospital of Nice, France, were eligible for the study from June 1, 2020, to June 1, 2021. Patients were included if they i) were at least 18 years old and ii) had brain white matter hyperintensities on MRI T2-weighted images. According to routine care, all patients underwent the same diagnostic workup with a blood and CSF analysis and 3-T brain MRI.At the end of the diagnostic workup, patients were separated into three groups according to their diagnosis. First, patients were divided as having an inflammatory or a non-inflammatory CNS disorder according to clinical presentation, MRI , and biology (identification of blood or CSF red flags for MS). All non-inflammatory diagnoses were pooled together as a control group named non-inflammatory neurological disorder (NIND). Patients identified as having an inflammatory CNS disorder were separated into two groups. Patients who fulfilled the 2017 McDonald criteria for MS and clinically isolated syndrome (CIS) , 20, werA non-opposition to research was obtained for each patient according to French law. Our institutional review board approved the study design, and the study was registered on Clinical Trial (NCT05056740) as the CyBIRD (Cytokine and Brain Inflammatory Related Disorders) Study.Blood and CSF were collected the same day for all patients. Fluids were sent within 2 h after collection into the Immunology Laboratory of Nice’s University Hospital.®  with the serum-free light chain immunoassay Freelite® . Serum and CSF albumin were also measured with the same turbidimetric analyzer and permitted to calculate KFLC index and KFLC intrathecal fraction (KFLC IF) as follows:Detection of OCBs was performed by isoelectric focusing and subsequent immunoglobulin using IgG-specific antibody staining. OCB patterns were evaluated by experienced biologists and classified as negative or positive. A cutoff ≥2 CSF-restricted bands was used to define OCB positivity. CSF KFLC was measured on fresh samples using the turbidimetric analyzer Optilite(i) KFLC index = KFLC quotient/albumin quotient with:(ii) KLC IF was determined with Reiber’s formula :The turbidimetric analyzer’s lower detection limit (LDL) for KFLC was 0.33 mg/L. For patients with CSF KFLC concentration lower than the LDL, an empirical value of KFLC = LDL/2 = 0.16 mg/L was assigned.For cytokine measurement, CSF was directly centrifuged and kept frozen at −80°C until there were enough stored samples to perform analysis (16-well cartridges). CSF was thawed once, just before cytokine analysis. CSF IL-1β, sIL-2R (or CD25), IL-6, and IL-10 concentration were determined using a mixture of 25 μl of CSF and buffer, in a custom-designed cartridge Ella  for the detection of IL-1β, sIL-2R, IL-6, and IL-10, according to the manufacturer’s instructions. The LDL of ELISA kits for cytokine measurement was 0.32 pg/ml for IL-1β, 6.56 pg/ml for sIL-2R, 0.5 pg/ml for IL-6, and 1.16 pg/ml for IL-10. As for CSF KFLC measurement, when the CSF cytokine concentration was under the LDL, an empirical CSF cytokine value of LDL/2 was assigned.www.easymedstat.com).Statistical analysis was performed using the online application EasyMedStat  curves were used to assess the ability of each biomarker to predict MSARD diagnosis and to calculate the area under the curve (AUC). DeLong’s test was performed to make pairwise comparisons of the predictive biomarkers according to MSARD diagnosis. The test implementation follows “Fast Implementation of DeLong’s Algorithm for Comparing the Areas Under Correlated Receiver Operating Characteristic Curves, by Xu Sun and Weichao Xu.” An optimal threshold that best discriminates MSARD from control populations was then determined with Youden’s index. Based on the defined threshold values, patients were classified as positive or negative for each biomarker as a binary result. Sensitivity, specificity, and positive and negative predictive values were then calculated for each biomarker. All comparisons were two-tailed. To identify the impact of demographic and clinical features on each biomarker concentration, CSF KFLC, IL-6, and CD25 were included in a multivariate linear regression model. According to the three identified groups, the explanatory variables were age, gender, disease duration, immune-modifying drug use at sampling, and final diagnosis. Data were checked for multicollinearity with the Belsley–Kuh–Welsch test. Patients with missing data were excluded from the analysis. The differences were considered significant when the p-value was <0.05.Data were presented as means with their SD for continuous values and counts and percentages for categorical variables for descriptive statistics. The data’s normality and heteroscedasticity were assessed using the Shapiro–Wilk and Levene’s tests. Constant values were compared using the Mann–Whitney U test. When more than two groups needed to be compared, the Kruskal–Wallis test was performed with a p = 0.002) and NIND patients (p = 0.001). All MSARD patients, except for RIS, experienced a clinical demyelinating event, while 63% and 0% in the OIND and NIND groups, respectively, experienced the same (p < 0.001 for both comparisons). All groups were comparable for immune treatment exposure at sampling. MSARD patients had lower CSF protein level (p < 0.001), CSF white blood cell count (p < 0.001), and albumin quotient (p < 0.001) than had OIND patients but had a higher level of CSF immunoglobulin G (IgG) and positive OCB status . All characteristics are shown in In the study period, two hundred seventeen patients have been referred to our center for brain white matter T2 hyperintensities. Forty-one patients were excluded because of subnormal brain MRI or age <18 years. One hundred seventy-six patients were included in the study. After the diagnostic workup, patients were separated into the following groups: 88 patients (50%) in the MSARD group, 35 (20%) in the OIND group, and 53 (30%) in the NIND group , p < 0.001 for KFLC index; 10.11% (IQR 38.10%), p < 0.001 for KFLC IF) and the OIND group (0.43 (IQR 1.03) mg/L, p = 0.001 for CSF KFLC; 4.53 (IQR 7.35), p < 0.001 for KFLC index; 60.21% (IQR 66.98%), p < 0.001 for KFLC IF).Median values of CSF KFLC . There was no difference of KFLC biomarkers values between MS and RIS patients (3.4 (IQR 11.2) mg/L, 48.5 (IQR 213.6), and 94.2% (IQR 73.9%), for CSF KFLC (p = 0.403), KFLC index (p = 0.377), and KFLC IF (p = 0.320), respectively).In the MSARD group, median values of CSF KFLC (figure in the p < 0.001), and the NIND group (15.7 (IQR 8.60) pg/ml, p < 0.001). Similar to CSF CD25, median values of CSF IL-6 (p < 0.001), and the NIND group (2.68 (IQR 2.07) pg/ml, p < 0.001). CSF IL-10 concentration was under the LDL in most of the MSARD patients (67%) and the NIND patients (91%). Median values of CSF IL-10 (p < 0.001) and the NIND group (0.58 (IQR 0.1) pg/ml, p = 0.002).CSF concentration of IL-1β was often under the LDL of the analyzer . Therefore, median values of CSF IL-1β  pg/ml), p = 0.836. Median values of CSF IL-6  against a non-inflammatory-mimicking disease by the comparison of the MSARD and NIND groups and ii) against another inflammatory-mimicking disease by the comparison of the MSARD and OIND groups. CSF IL-1β was not analyzed because of its low CSF concentration in most patients.p < 0.001 for all comparisons). The thresholds that best separated MSARD from NIND were 8.4 for KFLC index, 73.1% for KFLC IF, 21.5 pg/ml for CSF CD25, 2.0 pg/ml for CSF IL-6, and 1.2 pg/ml for CSF IL-10. All data are shown in KFLC index and KFLC IF had similar, and good, overall performances  to diagnose MSARD compared to NIND. However, CSF CD25, CSF IL-6, and CSF IL-10 had lower performances  than both KFLC biomarkers . In contrast with the comparison with the NIND group, in this situation, CSF CD25 and CSF IL-6 showed good diagnostic performances , statistically similar to the KFLC index . However, diagnostic performances of CSF IL-10  were lower than those of both KFLC index (p = 0.02) and IL-6 (p < 0.001). The thresholds that best separated MSARD from OIND were 13.1 for KFLC index, 82.8% for KFLC IF, 41.5 pg/ml for CSF CD25, 4.1 pg/ml for CSF IL-6, and 2.4 pg/ml for CSF IL-10. All data are shown in When comparing MSARD to OIND, KFLC index showed better diagnostic performances than KFLC IF . The comparison between MS and CNS infection did not seem valid, while only two patients presented with a CNS infection in our cohort. The CSF CD25 concentration (p = 0.755). Nevertheless, CSF CD25 could separate MS from CNS vasculitis  or other types of OIND . Finally, CSF IL-6 (p < 0.001) and from CNS vasculitis . However, median CSF IL-6 concentrations were not different between MS and the other OIND (p = 0.392).As shown in p = 0.423, 0.508, and 0.891, respectively), gender , immune-modifying drug use at sampling , or disease duration . The only factor associated with elevated CSF KFLC was MSARD diagnosis , and the only one associated with elevated CSF CD25 and IL-6 was OIND diagnosis . All data are shown in Based on the linear regression multivariate analysis model, CSF KFLC, CSF CD25, and CSF IL-6 concentrations were not influenced by age  may strongly recommend MSARD diagnosis regardless of the chosen control population. KFLC index has the advantage of being more sensitive than OCBs but suffered from less specificity. These results are consistent with previous retrospective , 16 and We found that CSF CD25 and CSF IL-6 concentrations were lower in MSARD than in OIND. However, these biomarkers cannot rule in MSARD, while NIND patients also express low CSF CD25 and IL-6 concentrations. Nevertheless, high CSF CD25 and IL-6 could be helpful in rolling out MSARD diagnosis, while it would favor another MS-mimicking inflammatory CNS disease. Of note, elevated CSF CD25 presents the highest positive predictive value for OIND diagnosis, more than low KFLC index or negative OCBs. However, CSF CD25 lacks diagnostic performance in separating MSARD from NMOSD and MOGAD, whereas IL-6 seems to be an effective tool in such situations. This is why we think that CSF CD25 and CSF IL-6 should both be used in practice. Moreover, CSF KFLC, CD25, and IL6 concentrations were not influenced by age, gender, disease duration, or immune treatment used during sampling. This point is important, while diagnostic biomarkers need to be efficient at any time of the diagnostic workup.Our results are consistent with previously published data, showing that a high KFLC index or KFLC IF is associated with MS diagnosis , 22, 24.Our findings agree with other retrospective studies that found an increased concentration of CSF IL-6 in NMOSD , 28, 29 Nonetheless, in clinical practice, a spinal tap is not performed in all suspected MS patients, while MS diagnostic criteria are based on the presence of a typical demyelinating event and MRI presentation . HoweverOur study suffers from several limits. First, being a monocentric study, our results need to be confirmed by others, even if these results are consistent with multiple retrospective data. Second, our cohort’s small size and heterogeneity, particularly in the OIND group, do not permit us to conclude on the effectiveness of these biomarkers for the different MS-mimicking diseases. However, it allows figuring out which biomarker may help in rolling in or rolling out MSARD. Third, it would have been interesting to measure serum IL-1β, CD25, IL-6, and IL-10 to calculate cytokine indexes, but our routine diagnostic workup of white matter hyperintensities does not include these analyses. Nevertheless, this study is pragmatic, evaluating these biomarkers prospectively in daily practice for the diagnostic workup of suspected MS. We think that these results will increase the etiological diagnostic accuracy of such patients.In patients presenting for a diagnostic workup of MRI white matter hyperintensities, elevated CSF activated B-cell biomarkers such as KFLC index or KFLC IF strongly suggest MSARD. In contrast, elevated CSF IL-6 and CD25 suggest another inflammatory-mimicking disease. These findings need to be confirmed in other prospective cohort studies within larger samples.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.ML and CL-F designed the study, collected the data, performed the data analysis, and drafted the manuscript. CL, LM, MC, and SB helped in designing the study, data collection, preparation of the manuscript, and review for intellectual content. VB and BS-P performed the biomarker analysis and helped in reviewing the manuscript for intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: TNF-α has a major role in the pathogenesis of Crohn's disease (CD). In contrast, GM-CSF may be beneficial for its anti-inflammatory role in a subset of patients with CD with antibodies against GM-CSF as seen in prior trials of GM-CSF which resulted in clinical improvement in CD. We developed butanol purified Food Allergy Herbal Formula-2 (B-FAHF-2) by refining FAHF-2. FAHF-2 suppressed TNF-α production by human peripheral blood mononuclear cells (PBMCs) and colonic mucosa, and abrogated colitis in a murine model. We sought to examine the effect of B-FAHF-2 and the herbs that comprise it on TNF-α and GM-CSF production as a potential herbal therapy for the treatment of CD.Methods: B-FAHF-2 was examined using high pressure liquid chromatography (HPLC) and compared to the original formulation, FAHF-2. PBMCs from pediatric patients with CD were cultured with lipopolysaccharide and B-FAHF-2, individual herbs or medium alone. Colonic biopsy specimens were cultured with or without B-FAHF-2. TNF-α and GM-CSF were measured by enzyme-linked immunosorbent assay (ELISA). B-FAHF-2 efficacy was tested in vivo in the CD45Rbhi transfer model.Results: B-FAHF-2 had a similar HPLC fingerprint as FAHF-2 but decreased TNF-α production by PBMCs and colonic mucosa from pediatric CD subjects at 20% of the FAHF-2 dose. B-FAHF-2 increased GM-CSF production by PBMCs and colonic mucosa from pediatric CD subjects including those with antibodies to GM-CSF. Of B-FAHF-2's herbal constituents, only Huang Bai suppressed TNF-α and increased GM-CSF production. In the murine model, B-FAHF-2 treatment alleviated colitis.Conclusions: B-FAHF-2 decreased TNF-α production by PBMCs and colonic mucosa from pediatric subjects at a lower dose than FAHF-2. B-FAHF-2 also increased GM-CSF production by PBMCs independent of antibodies. B-FAHF-2 may have a benefit in CD patients. Crohn's disease (CD), a form of inflammatory bowel disease (IBD), is a life-long disease characterized by chronic and relapsing inflammation of the gastrointestinal tract. The prevalence of CD varies based on region, race, age, and environment. The annual incidence of CD ranges from 16.7 to 318.5 per 100,000 in North American, 0.6 to 322 per 100,000 in Europe, and 0.88 to 67.9 per 100,000 in Asia and the Middle East . Panax ginseng was extracted twice with 80% aqueous ethanol, then filtered, combined, and evaporated until there was no residual ethanol. The other 7 herbs were combined and boiled twice in water (1.5 h each time). The decoction was collected, combined, and purified by ethanol precipitation. Panax ginseng was mixed with the 7 herbs extract, dried into a powder, and then combined with the Ganoderma lucidum powder to form FAHF-2  certified facility. Herbal components of FAHF-2 are listed in m FAHF-2 , 18.Extensive quality control, analytical chemistry data and batch consistency pertaining to the formula have been published previously . We idenin vivo models of peanut allergy . B-FAHF-2 tablets were ground, and a 22.5 mg/mL solution was prepared using 1:1 ratio of mobile phase mixture. The B-FAHF-2 solution was centrifuged at 10,000 rpm for 10 minutes. 10μL of the supernatant was injected into the HPLC system and separated on a ZORBAX SB-C18  column . The mobile phase A was made of 0.1% of formic acid aqueous solution. Mobile phase B was acetonitrile. The separation was performed at 1 min/mL flow rate following a linear gradient elution of 2–25% mobile phase B in 45 min, 25–35% B in the following 25 min, 35–55% in the next 15 min, 55–75% in another 10 min. This mobile phase composition was maintained for 5 min and then rapidly switched to 2% mobile phase B. An equivalent amount of FAHF-2 formula, 99 mg/mL, was also prepared following the same procedure as for B-FAHF-2. 10μL of FAHF-2 supernatant was analyzed on the HPLC system. Data was collected and processed with Waters' Empower software.n = 29) and inflamed colonic biopsy specimens (n = 20) were collected from newly diagnosed pediatric CD patients (8–19 years old) naïve to medications that could alter immune responses including: immune-modulators, biologics or steroids. CD patients were diagnosed based on standard clinical, radiographic and endoscopic criteria. Blood and colonic biopsy specimens were also collected from non-IBD control subjects (n = 12). Patient characteristics are outlined in Human studies were approved by the Institutional Review Board of the Icahn School of Medicine at Mount Sinai (No. 11-00808). All participants provided written informed consent for participation in the study. Blood samples  for 24 h followed by LPS (2 μg/ml) stimulation or no stimulation for an additional 24 h  with density gradient centrifugation. Purified PBMCs were cultured in medium containing 5% FBS with or without FAHF-2 (250 μg/ml), B-FAHF-2 (60 μg/ml), individual herbs in B-FAHF-2 (doses noted above) or dexamethasone  in complete RPMI (10% FBS and GPS) with phosphatase and protease inhibitors overnight. The volume of culture medium was based upon the weight of each sample so that the size of the biopsies could be standardized. Supernatants were filtered and TNF-α and GM-CSF were assessed by ELISA (BD Biosciences) per the manufacturer's instructions.Serum was tested by ELISA. Briefly, samples were diluted serially in 4-fold increments from 1/100 to 1/25,000 and assayed for the presence of IgG and IgA against GM-CSF , as well as irrelevant control recombinant proteins. Each plate included positive control sera from patients with pulmonary alveolar proteinosis as well as a negative control healthy donor serum pool, expected, respectively, to react or not with GM-CSF.+, CD11b+, CD11c+, CD19+, and B220+ cells by negative selection. The resulting CD4+ enriched population was labeled with FITC-conjugated anti-CD4 Ab, APC-conjugated anti-CD62L Ab and PE-conjugated anti-CD45RB Ab (eBiosciences). Subpopulations of CD4+ cells were sorted by flow cytometry. CD4+CD45RBhi T cells (3.5 x 105) were adoptively transferred by intraperitoneal injection into recipient RAG1−/− mice. Transfer was confirmed by flow cytometry of peripheral blood in all mice after 2–4 weeks. To prevent the progression to colitis, the recipient mice were fed B-FAHF-2 (50 mg/day) daily by gavage 24 h after CD4+CD45RBhi T cells transfer. Control mice received water by gavage. Weights were recorded semi-weekly. All mice were sacrificed once any mouse lost 20% of their initial weight. Colonic histology was scored for inflammatory infiltrates and epithelial damage by a pathologist blinded to the treatment group , the slides were blocked with 20% goat serum in PBS for 1 h at room temperature, and then incubated with CD4-FITC and F4/80-APC  overnight at 4°C. DAPI was used to stain cell DNA.Animal studies were approved by the Institutional Animal Care and Use Committee at Mount Sinai. Cells were obtained from C57BL/6 wild type mice and enriched using an EasySep Cell Isolation Kit  that depletes CD8nt group . A totalT-tests with Wilcoxon signed-rank test were used when comparing the same samples without and with treatment. Comparisons between multiple groups were done using one-way ANOVA. This was followed by either non-parametric Mann-Whitney U test or Bonferroni analysis when appropriate. Statistical differences between groups for the colitis model were determined by Mann-Whitney T-test assuming non-normally distributed data. Data analysis was done using Prism software . A value of p < 0.05 was considered statistically significant. P-values are indicated by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Paired After butanol extraction of FAHF-2 was completed, B-FAHF-2 and FAHF-2 were examined by HPLC to ensure that the compounds were similar. HPLC fingerprints  demonstrp < 0.01) (p < 0.001)  . TNF-α l< 0.001) . No cytop < 0.05)  .Thus, like FAHF-2, B-FAHF-2 significantly suppressed TNF-α production by PBMCs and inflamed colonic mucosa from pediatric subjects with CD.FAHF-2 had an immunomodulatory effect that is best demonstrated by its stimulation of increased production of GM-CSF while at the same time suppressing production of multiple inflammatory chemokines  and HGF from PBMCs from pediatric subjects with CD .p < 0.05). B-FAHF-2 also stimulated significantly increased production of GM-CSF from biopsies from pediatric subjects with CD (p < 0.05).Therefore, using GM-CSF as representative of the immunomodulatory capacity FAHF-2, we tested the effect of B-FAHF-2 on GM-CSF secretion by unstimulated PBMCs from all 29 pediatric subjects with CD. PBMCs cultured with B-FAHF-2 produced more GM-CSF than when cultured in medium alone  (p < 0.0 with CD  (p < 0.0p < 0.05)   despite Prunus mume (Wu Mei), Zanthoxylum bungeanum (Chuan Jiao), Angelica sinensis (Dang Gui), Zingiber officinalis (Gan Jiang), Cinnamomum cassia (Gui Zhi), Phellodendron chinense (Huang Bai), Coptis chinensis (Huang Lian), Panax ginseng (Hong Shen) and Ganoderma lucidum (Ling Zhi)] was tested for the ability to suppress production of TNF-α from LPS stimulated PBMCs from pediatric subjects with CD. LPS caused a marked increase in production of TNF-α. B-FAHF-2, Dexamethasone (Dexa), Huang Bai (HB), Huang Lian (HL) and Dang Gui (DG) significantly reduced production of TNF-α   .p < 0.05, p < 0.001, and p < 0.05, respectively)  and Huang Bai (HB) induced significantly increased production of GM-CSF by unstimulated PBMCs from pediatric subjects with CD (ctively) .Overall, only B-FAHF-2 and Huang Bai (HB) could both decrease TNF-α and increase GM-CSF production by PBMCs unlike any of the other individual herbs.hi T cell transfer model of colitis, treatment with FAHF-2 decreased colitis progression as evidenced by decreased weight loss, histological inflammation and production of TNF-α, IFN-γ, IL-6, and IL-17 from the colon    (In the CD45RBght loss  and had  < 0.05) . The B-Fctively) . The scoed group . Inflammctively) . Levels Thus, B-FAHF-2 alleviates colitis and inhibits the inflammatory milieu associated with colitis.In this continuation of our studies on FAHF-2 as a potential treatment for CD, we first sought to lessen the pill burden of FAHF-2 required for human trials by purifying the active compounds and then by examining the individual herbs. Our data demonstrates that the purified product, B-FAHF-2, was effective at suppression of TNF-α production by both PBMCs and mucosal biopsies from pediatric subjects with CD at 20% of the dose of FAHF-2. In a murine model, B-FAHF-2 was effective at alleviating colitis at this same dose. B-FAHF-2 also caused an increase in GM-CSF production even in those subjects with antibodies against it. Of the individual herbs within FAHF-2 and B-FAHF-2, only Huang Bai was effective at both suppressing TNF-α and stimulating GM-CSF production.ex vivo from human PBMCs and colonic mucosa nor its ability to abrogate colitis in a murine model. The components removed by butanol purification were non-medicinal components including fiber, wax, proteins and starch. Given that B-FAHF-2 has a similar HPLC fingerprint as FAHF-2, and similar effects on TNF-α suppression , U01 DK1241654 (to SG), and partially supported by NIH 2R01 AT001495-01A1 (to X-ML).X-ML formerly had patents with Herbal Springs for FAHF-2 for treating food allergy. SG is a named inventor of a patent application related to GM-CSF autoantibodies in immune-related colitis. This is filed through the Icahn School of Medicine at Mount Sinai and is currently unlicensed. SG reports consultancy and/or advisory roles for Merck and OncoMed, and research funding from Bristol-Myers Squibb, Celgene, Genentech, Immune Design, Janssen R&D, Pfizer, Regeneron, and Takeda. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Classical swine fever (CSF), caused by CSF virus (CSFV), is a highly contagious swine disease with high morbidity and mortality, which has caused significant economic losses to the pig industry worldwide. Biosecurity measures and vaccination are the main methods for prevention and control of CSF since no specific drug is available for the effective treatment of CSF. Although a series of biosecurity and vaccination strategies have been developed to curb the outbreak events, it is still difficult to eliminate CSF in CSF-endemic and re-emerging areas. Thus, in addition to implementing enhanced biosecurity measures and exploring more effective CSF vaccines, other strategies are also needed for effectively controlling CSF. Currently, more and more research about anti-CSFV strategies was carried out by scientists, because of the great prospects and value of anti-CSFV strategies in the prevention and control of CSF. Additionally, studies on anti-CSFV strategies could be used as a reference for other viruses in the Flaviviridae family, such as hepatitis C virus, dengue virus, and Zika virus. In this review, we aim to summarize the research on anti-CSFV strategies. In detail, host proteins affecting CSFV replication, drug candidates with anti-CSFV effects, and RNA interference (RNAi) targeting CSFV viral genes were mentioned and the possible mechanisms related to anti-CSFV effects were also summarized. Pestivirus genus within the Flaviviridae family. CSFV is an enveloped, single-stranded, positive-sense RNA virus containing a 12.3 kb RNA genome, which consists of a 5′-untranslated region (5′-UTR), a single large open reading frame (ORF), and a 3′-UTR. A polypeptide precursor could be encoded by ORF of CSFV and then cleaved into four structural proteins  and eight non-structural proteins  through the processing of the polypeptide precursor by viral and cellular proteases  is a highly contagious swine disease characterized by high fever, multiple hemorrhages, and gastrointestinal symptoms with high morbidity and mortality, which has caused significant economic losses to the pig industry worldwide . CSF in roteases  1,3,4].,3,4.PestBased on partial sequences of CSFV 5′-UTR, E2, and NS5B, phylogenic typing of CSFV isolates worldwide was carried out. In general, CSFV strains could be classified into three genotypes  and eleven subgenotypes  ; subgenoCSFV strains’ virulence, ranging from high, moderate, to low virulence, is the crucial determinant of CSFV pathogenicity in pigs. CSFV infection can lead to an acute, subacute, chronic, or asymptomatic disease in pigs depending on CSFV strains’ virulence and other factors, such as the host’s age and immune status . AlthougCurrently, the World Organization for Animal Health (OIE) lists 38 CSF-free members, which mainly locate in North America, part of South America, Oceania, and a large part of the European Union. CSF is still endemic in Asia, South and Central America, Eastern Europe, and parts of Africa. Additionally, specific zones in Brazil, Colombia, and Ecuador are declared CSF-free by OIE, while the other zones in these countries remain CSF-endemic . AdditioBiosecurity measures are crucial for preventing and controlling CSF. According to the guidance of OIE, strict sanitary prophylaxis is the first barrier to prevent CSF outbreaks . In brie® , containing a baculovirus-expressed E2 glycoprotein of vaccine C-strain of genotype 1.1, was reported to provide complete protection to pigs against lethal challenge with virulent Shimen strain of genotype 1.1 and heterologous strains of genotype 2 [Currently, prophylactic vaccination is still the primary strategy for preventing and controlling CSF in CSF-endemic regions. Traditional CSF live attenuated vaccines (LAVs) strains, such as C-strain, LPC strain, LK-VNIVViM strain, GPE-strain, Thiverval strain, and PAV-250 strain, could provide an effective, rapid, and solid immune protection against CSFV infection and have been widely used to fight CSF . Althougnotype 2 ,29.Despite decades of efforts, it is still difficult to eliminate CSF in endemically affected regions and re-emerging areas ,31. The The interaction between virus and host exists in the process of virus infection. After viral infection, the host will quickly initiate the innate immune response to achieve the goal of eliminating the virus. Previous studies have demonstrated that many host proteins had anti-CSFV activity . In geneSince host proteins mentioned above can inhibit CSFV replication, it is valuable to develop drugs that can activate the functions of these anti-CSFV proteins. Theoretically, these drug candidates can also exert antiviral effects. Additionally, these antiviral proteins can be used to prepare transgenic pigs. Pigs that over-express antiviral genes can theoretically resist or reduce CSFV infection.Different host proteins might play the opposite role in the process of viral infection. In addition to the host proteins with antiviral effects mentioned above, there is another kind of host protein, which is utilized or hijacked by the virus and might promote virus replication. Previous studies have revealed that some host proteins played an important role in the process of CSFV infection or replication and are required for the life cycle of CSFV . They haSince these proteins are potential anti-CSFV targets, scientists can consider developing some molecules that inhibit the functions of these proteins. Theoretically, these drug candidates can inhibit CSFV replication and have potential value in CSF treatment. Importantly, while verifying the antiviral effects of these drug candidates, it is necessary to test whether these drug candidates affect cell viability and/or have an adverse effect on pigs. Since the host proteins, required for CSFV infection or replication, might also play an important role in the cell life. While destroying the functions of these proteins exerts antiviral effects, the normal functions of cells or the body might also be disrupted.pro [S-transferase pulldown, laser confocal microscopy or other methods to further verify the potential interaction obtained by high-throughput screening and then to evaluate the impact of interactions on CSFV replication. We believe that studies on the interaction between host protein and CSFV viral protein will be conducive to the discovery of novel antiviral proteins or antiviral targets.Additionally, according to possible mechanism by which host proteins affect CSFV replication , we founpro , and intpro , which mpro ,91,92,93Staphylococcus aureus nuclease fused with CSFV capsid protein (Cap-SNase) [rns, E1, and E2) are located on the external part of viral particles. These glycoproteins form heterodimeric and/or homodimeric complexes, which are important for the stability of viral glycoproteins and the ability of viral infection. Some molecules targeting the glycosylation process, such as GP6 [Many efforts have been made for developing anti-CSFV drugs. CSFV life cycle mainly includes virus attachment, receptor binding and virus entry, virus uncoating, viral RNA replication, translation and processing of viral proteins, virion morphogenesis, and virus release . It was p-SNase)  could suh as GP6 , glycosyh as GP6 ,105, andh as GP6 ,107, couMoreover, when studying the mechanism of CSFV replication, scientists found that many intracellular signaling pathways or biological processes were involved in the process of CSFV replication and many molecules had anti-CSFV activity by targeting these intracellular signaling pathways or biological processes . (1) Botpro, p7, NS3, NS4A, NS5A, NS5B, and C [RNA interference (RNAi) is an intracellular mechanism for post-transcriptional gene silence induced by small interfering RNAs (siRNAs) of about 21–23 nt, which are homologous to the mRNA of target genes . RNAi haB, and C ,129,130,B, and C  revealedB, and C ,132. TheB, and C .CSF is an ancient zoonotic disease, which has caused substantial economic losses to the pig industry worldwide. The control of this disease is still a major problem for the pig industry. Currently, it is difficult to completely eradicate CSF in CSF-endemic and re-emerging areas through biosecurity and vaccination strategies. Therefore, it is meaningful to develop anti-CSFV drugs or other strategies. In this review, we summarized the anti-CSFV strategies into several aspects including host proteins with anti-CSFV effects, host proteins whose function defect exert anti-CSFV effects, drug candidates with anti-CSFV effects, and RNAi targeting CSFV viral genes. Many host proteins and chemical molecules exert anti-CSFV effects by targeting the process of virus replication including viral attachment and entry, viral genome replication, translation and post-translational modification of viral proteins, and the assembly of the viral particle . FurtherAfter summarizing and analyzing the results of previous anti-CSFV research, we make some suggestions, which might benefit further anti-CSFV research. (1) Animal experiments are needed for the evaluation of the effect of anti-CSFV strategies. We found that cells were selected as the main object for the anti-CSFV research in previous reports, and animal experiments were rarely involved. Thus, we recommend that researchers could use animals to evaluate the effects of antiviral strategies after antiviral strategies have achieved good antiviral effects at the cellular level. (2) It is necessary to evaluate the safety of antiviral strategies to animals, because some antiviral strategies may be toxic to animals. For example, Dai et al.  reportedIf effective anti-CSFV drugs, targeting the CSFV life cycle or CSFV-host interaction, are developed, approved, and commercialized, they will be beneficial to the prevention and control of CSF, especially during the outbreak of CSF. The use of anti-CSFV drugs in combination with vaccination will be an effective emergency preventive measure against CSF for pigs in infected zone during CSF outbreak. Notably, the selected anti-CSFV drugs need to be proven not to affect the immune effect of vaccines. The use of anti-CSFV drugs might establish a robust antiviral state in pig herd and inhibit CSFV replication and spread , which w


Lipidomics provides an overview of lipid profiles in biological systems. Although blood is commonly used for lipid profiling, cerebrospinal fluid (CSF) is more suitable for exploring lipid homeostasis in brain diseases. However, whether an individual’s background affects the CSF lipid profile remains unclear, and the association between CSF and plasma lipid profiles in heathy individuals has not yet been defined. Herein, lipidomics approaches were employed to analyze CSF and plasma samples obtained from 114 healthy Japanese subjects. Results showed that the global lipid profiles differed significantly between CSF and plasma, with only 13 of 114 lipids found to be significantly correlated between the two matrices. Additionally, the CSF total protein content was the primary factor associated with CSF lipids. In the CSF, the levels of major lipids, namely, phosphatidylcholines, sphingomyelins, and cholesterolesters, correlated with CSF total protein levels. These findings indicate that CSF lipidomics can be applied to explore changes in lipid homeostasis in patients with brain diseases. Lipidomics provide an overview of lipid profiles in biofluids and tissues ,2,3. TheCSF lipid profiling can provide insights into the pathophysiology of brain diseases, such as multiple sclerosis, Alzheimer’s disease, and amyotrophic lateral sclerosis, as well as to screen for biomarkers for such diseases. High levels of CSF ceramides are associated with multiple sclerosis , elevateThe current study collected demographic information, as well as CSF and plasma samples, from 114 healthy subjects, and a lipidomics platform was used to analyze their lipid profiles. First, the CSF and plasma lipid profiles were compared and potential correlations were examined. Subsequently, the effects of CSF properties and background of the subjects on CSF lipids were evaluated. This study provides fundamental information regarding CSF lipid profiles, and on changes in CSF lipids due to the CSF condition and the individual background of each person. Cumulatively, these findings may represent a guideline for future CSF application for biomarker screening via lipidomics.n = 114 and 99, respectively). The CSF properties assessed included: neutrophil, lymphocyte, and red blood cell counts, as well as protein, glucose, and chloride concentrations. The information regarding subjects was not limited to age and sex, but also included allergies, smoking and alcohol drinking habits, postprandial time, height, and BMI, although some of these data were missing for some of the participants. Demographic and lifestyle-related information of the participants is shown in n = 93) was 0.5 to 18 h (median 2.33 h), the height range (n = 63) was from 146.6 to 180.1 cm (median 165 cm), and the BMI range (n = 63) was 17.72 to 32.46 (median 21.65).The present study enrolled 114 healthy subjects, from whom CSF and plasma lipid profiles were obtained  and statistical values ) are summarized in Next, the effect of CSF properties  on CSF lipid levels were examined. None of the combinations of these CSF properties were correlated (|r| < 0.4) . TherefoNext, the effects of age and sex, which are major factors affecting lipid levels in plasma, on CSF lipids were evaluated. No correlation was found (|r| < 0.4) between CSF total protein levels, age, and sex . CSF totAfter adjustment for CSF total protein levels, age, and sex, the effects of subject background on CSF lipid levels were determined. Since the information regarding the other subject backgrounds was random in some subjects, these factors were examined individually. Each categorical parameter was evaluated using effect size, whereas each numerical parameter was evaluated using correlation coefficients. Only three lipids, SM, SM(d42:1) for allergy, and PE for alcohol habit, were defined as being associated with the other subject backgrounds a. The inThe current study comprehensively demonstrated the profiles of CSF lipids and their association with plasma lipids in healthy subjects. A key finding of the present study was revealed in the comparison of CSF and plasma lipid profiles of the same subjects. The levels of CSF lipids were found to be far less than those of plasma lipids, and the composition of CSF lipids was clearly different from that of plasma lipids. For example, the FA side chains of PCs differed between CSF and plasma, wherein PCps were found in much higher abundance in the CSF than in plasma. In addition, only a limited number of lipids of the CSF and plasma were correlated; some of these were specific for age and sex. FA(22:6), corresponding to docosahexaenoic acid (DHA), and FA(20:5), corresponding to eicosapentaenoic acid (EPA), containing lipids were generally correlated, whereas correlations of several plasmalogens were specific for younger group subjects and males. Furthermore, the factors associated with CSF lipid levels were characterized. CSF total protein level was found to be the dominant factor regulating CSF lipid levels, except for several PEs and PEes, with over 90% of CSF lipid levels being associated with CSF total protein levels. Age was also a major factor regulating CSF lipid levels. DHA and EPA containing lipids were positively associated with age, whereas ether lipids, including plasmalogens, were negatively associated with age. Other factors, such as sex and allergy, were not associated with most CSF lipids, although several SMs were enriched in female and allergy-negative subjects.DHA is highly enriched in the brain and is essential for brain development and function ,24. EPA,Plasmalogens represent another class of important lipids required for brain function ,28. DecrThe characterization of CSF total protein levels as the dominant factor of CSF lipid levels is another feature of the present study. Levels of global classes of lipids, except for several PEs and PEes, are positively associated with CSF protein levels. The major CSF protein reported in healthy subjects was serum albumin, which accounts for over 50% of all proteins ,33. RepoThe present study has several limitations. First, although self-reported healthy subjects who had not taken any medication prior to at least one week before study start were recruited, asymptomatic disease was inevitable. In addition, the diet of the participant was not controlled, which may have affected the plasma and CSF lipid levels, although the limited postprandial effect on CSF lipids was in agreement with a previous study . FurtherSubjects were recruited at the National Center of Neurology and Psychiatry, Tokyo, Japan, through a local free magazine or our website announcement. All participants were biologically-unrelated Japanese individuals and underwent a structured interview using the Mini-International Neuropsychiatric Interview, Japanese version ,48, admig, 10 min, 4 °C) and the supernatant was dispensed into 0.5 mL aliquots in low-protein adsorption tubes  and stored in a deep freezer (−80 °C). For lipidomics, the samples were thawed once and re-dispensed into 120 μL aliquots in smaller low-protein adsorption tubes  and stored in a deep freezer (−80 °C) before use.CSF samples were obtained by lumbar puncture as described previously . Brieflyg, 10 min, 4 °C), dispensed in screw-capped polypropylene tubes , and stored in a deep freezer (−80 °C). For lipidomics, the samples were thawed once and re-dispensed into 120 μL aliquots in polypropylene tubes  and stored at −80 °C before use.Simultaneously, blood samples were collected via venipuncture into 7 mL EDTA-2Na-containing vacuum blood collection tubes . The samples were immediately centrifuged  and the National Institute of Health Sciences (no. 278). Written informed consent was obtained from all participants.Lipid extraction was performed using a Microlab NIMBUS workstation . CSF samples were mixed with 1 volume of methanol containing 2 μM of an internal standard (PC[1 2:0/12:0] and PE[12:0/12:0]), followed by 3 volumes of 1% formic acid in water. The mixed samples were then subjected to solid phase extraction with FastRemover C18  and MPE2 unit (Hamilton). The preparations were washed with water and 50% methanol, and then subjected to elution with methanol and isopropanol. The resulting lipid-containing solution was evaporated and reconstituted with 30 μL of methanol for lipidomics. Plasma samples were mixed with 9 volumes of methanol:isopropanol (1:1) containing 2 μM of internal standard (PC[12:0/12:0]). Mixed samples were filtered with FastRemover Protein (GL Science) using an MPE2 unit. The resulting lipid-containing solution was directly subjected to lipidomics.v:v:v]) with 0.1% formic acid and 10 mM ammonium formate, and the mobile phase B was composed of water:acetonitrile:isopropanol (1:10:90 [v:v:v]) with 0.1% formic acid and 10 mM ammonium formate. A multistep gradient was used as follows: the gradient was initiated at 10% solvent B and subsequently increased to 40% solvent B over 5 min. The mobile phase was further increased to 50% solvent B between 5 and 10 min, and finally changed to 100% between 10 and 18 min, prior to maintaining it at 100% mobile phase B for further 4 min. The column was equilibrated with 10% mobile phase B for 5 min before the next sample was injected.To obtain lipidomic data, we performed reversed-phase liquid chromatography  and MS , following a recently established lipidomics platform . An InerCompound Discoverer 2.0  was used with the raw data for peak extraction, annotation, identification, and lipid quantification, as described previously . Full MSp-values of unpaired Student’s t-test. FDR was calculated using p-values of Student’s t-tests for comparisons between groups and p-values of multiple regression model. The multiple regression analysis was conducted using R 3.6.0 software . In this study, a lipid level was considered significantly different if its FDR was <0.1.Significant differences in individual lipid levels between groups were assessed using FDR, except for the effect of allergy, alcohol consumption, smoking, postprandial effect, height, or BMI, which were assessed by The current study revealed and characterized detailed fundamental lipid profile of CSF in humans. As CSF is the biofluid with the closest association with the brain, this information is useful for exploring changes in lipid homeostasis in patients with neuropsychiatric disorders.


Background: Donor-derived cell-free DNA (ddcfDNA) has been suggested as an indicator of allograft injury in adult and pediatric kidney transplantation (KTx). However, the dynamics of ddcfDNA in pediatric KTx have not been investigated. In addition, it has not been demonstrated whether donor-recipient (D/R) size mismatch affect ddcfDNA level.Methods: Pediatric KTx recipients with a single donor kidney were enrolled and followed up for 1 year. ddcfDNA, calculated as a fraction (%) in the recipient plasma, was examined longitudinally within 3 months post-transplant. D/R size mismatch degree was described as D/R height ratio. The 33rd percentile of D/R height ratio (0.70) was used as the cut-off to divide the patients into low donor-recipient height ratio group (<0.70) and high donor-recipient height ratio group (≥0.70). The dynamics of ddcfDNA were analyzed and the impact factors were explored. Stable ddcfDNA was defined as the first lowest ddcfDNA. ddcfDNA flare-up was defined as a remarkable elevation by a proportion of >30% from stable value with a peak value >1% during elevation.Results: Twenty-one clinically stable recipients were enrolled. The median D/R height ratio was 0.83 (0.62–0.88). It took a median of 8 days for ddcfDNA to drop from day 1 and reach a stable value of 0.67% (0.46–0.73%). Nevertheless, 61.5% patients presented ddcfDNA>1% at day 30. Besides, 81.0% (17/21) of patients experienced elevated ddcfDNA and 47.6% (10/21) met the standard of ddcfDNA flare-up. Donor-recipient height ratio was an independent risk factor for ddcfDNA flare-up  and low donor-recipient height ratio (<0.70) was found to increase the risk of flare-up occurrence .Conclusions: ddcfDNA rebounds in many stable pediatric KTx recipients without rejection. This may be induced by significant D/R size mismatch and may affect its diagnostic performance at the early phase after pediatric KTx in children. Kidney transplantation (KTx) is the most effective treatment for children with end-stage renal disease (ESRD) , 2. Howede novo DSA (dnDSA) formation and future rejection (Donor-derived cell-free DNA (ddcfDNA) has emerged as a potential and promising biomarker for early detection of graft injury and rejection. The ddcfDNA level above 1% strongly correlates with acute rejection . Accompaejection .A high amount of ddcfDNA is released into peripheral circulation immediately after renal allograft reperfusion mainly due to ischemia-reperfusion injury (IRI), followed by a swift decrease to a stable low level . It's imThis is a single-center prospective cohort study conducted in the First Affiliated Hospital of Sun Yat-sen University. Children who received primary pediatric deceased-donor-kidney transplantation between December 2019 and June 2020 were consecutively investigated. All enrolled pediatric recipients were scheduled to receive sequential ddcfDNA detection within 3 months and were followed up for 1 year. Patients with post-transplant complications within 3 months including graft loss, rejection, and polyomavirus-associated nephropathy were excluded. Patients with substandard specimen quality and those who were unable to finish 1-year follow-up were also excluded.All patients and their legal representatives provided written informed consents for participation in the study. This study was approved by the institutional review board of the First Affiliated Hospital of Sun Yat-sen University and was registered at the official national Chinese Clinical Trial Registry (No. ChiCTR2000032333). This study was conducted in accordance with the principles of the World Medical Association Declaration of Helsinki and the declaration of Istanbul. No organs were from executed prisoners.Blood (8 mL) samples were drawn from enrolled patients using customized cfDNA blood collection tubes . Plasma was separated by centrifugation at 1,600×g for 10 min followed by a second centrifugation at 16,000×g for another 10 min. Plasma cfDNA was extracted with Circulating Nucleic Acid kit  and whole blood genomic DNA was extracted with DNA Blood mini kit . Purified DNA was quantified by Qubit 3.0 using the dsDNA HS Assay Kit (Life Technologies). Sequencing libraries were prepared by applying KAPA Hyper Prep kit (KAPA Biosystems) with 30 ng of input DNA. A total of 6,200 human single nucleotide polymorphism (SNP) loci were enriched by liquid hybridization based on SNP loci selection criteria . All seqThe ddcfDNA level was examined longitudinally and ddcfDNA fraction (%) was determined at day 1, day 4, day 7, day 14, day 30, day 60, and day 90, within an acceptable time window for each time point. Dynamic change of ddcfDNA was explored and depicted.Stable ddcfDNA was defined as the first lowest ddcfDNA value after transplantation. Elevation of ddcfDNA was observed in a proportion of study subjects after reaching the stable level. Therefore, the dynamic characteristics of ddcfDNA elevation was especially described as the followings. The occurrence time of ddcfDNA elevation was presumed as the previous detection time point before its elevation, and therefore it was the same as the time point when ddcfDNA reached a stable level. The duration time of ddcfDNA elevation was counted as the period from occurrence of ddcfDNA elevation to its peak level. Peak ddcfDNA was the highest level of ddcfDNA during the elevation. The proportion of ddcfDNA change from stable level to peak level was calculated as (peak ddcfDNA – stable ddcfDNA)/stable ddcfDNA. Slight fluctuation in ddcfDNA was justified and considered as random intra-patient variations , whereasBSA(m2) = 0.0061 × height(cm) + 0.0128 × weight (kg) −0.1529. In pediatric patients with kidney disease, height is preferred because ascites, edema or other complications can significantly affect weight. In this study, D/R size mismatch degree was described by the ratio of donor height to recipient height , human leukocyte antigen (HLA) mismatch, donor type, cold ischemia time (CIT), donor-recipient size mismatch, and immunosuppressive therapy were recorded. The size of child is often assessed by weight, height or BSA. BSA was calculated using Stevenson's BSA formula : BSA(m2)t height , 24. TheDelayed graft function (DGF) was defined as the need for dialysis within the first week post-transplant. All recipients received tacrolimus as a part of the maintenance immunosuppression regimen. Tacrolimus trough levels were measured at least once a week in the first month, every 2–3 weeks in 2–3 months, every 4–6 weeks in 4–6 months, and every 1–2 months after 6 months. In addition, the frequency of detection was adjusted according to the clinical condition. The tacrolimus target trough level was 6.0–10.0 ng/mL in the first month, 5.0–9.0 ng/mL within 3 months, and 5.0–8.0 ng/mL thereafter. The estimated glomerular filtration rate (eGFR) calculated with Schwartz formula was used for assessment of kidney graft function , 26. WitdnDSA, rejection and BK polyomavirus-associated nephropathy (BKVN) were prospectively recorded. After transplantation, DSA detection was conducted monthly within the first 3 months and every 3 months after that. Diagnosis of rejection and BKVN was confirmed by renal allograft biopsy following the 2018 Banff criteria  and GraphPad Prism 7 . Comparisons between groups were performed using Mann-Whitney U-test for continuous variables and Chi-square test or Fisher's exact test for categorical variables. Spearman correlation test was used to test correlation between variables. Logistic regression was used for the univariable and multivariable analysis of risk factors for ddcfDNA flare-up and post-transplant outcomes. Linear regression was used for univariable and multivariable analysis of stable ddcfDNA, ddcfDNA at day 90, occurrence time and duration time of ddcfDNA elevation. The variables with P < 0.10 in the univariable analysis were enrolled in the multivariable model. Demographic and clinical characteristics of recipients and donors, including donor-recipient size mismatch degree, were included as the independent variables in the regression models. Dynamics parameters of ddcfDNA were included in the regression models for post-transplant outcomes. Adjusted R square and Somer's Delta were used for model performance evaluation. For all analyses, a P < 0.05 was considered statistically significant.Continuous data were presented as median (lower quartile-upper quartile) if not especially mentioned. Categorical data were reported as counts and percentages. Statistical analysis was performed using IBM SPSS Statistics version 22.0 , R version 4.0.3  participants showed an elevation of ddcfDNA after stable time points . As for P = 0.002). The potential impact factors of ddcfDNA at day 90, the occurrence time and duration time of ddcfDNA elevation were summarized in R square, 0.358 vs. 0.311). Besides, DCD donor was found significantly correlated with the duration time of elevation .Linear analysis in p = 0.017). Factors including D/R height ratio, donor height, donor type, MPA type, and D/R BSA ratio were potential risk factors (P < 0.10). D/R height ratio, donor type and MPA type were included in the multivariable analysis of flare-up while donor height and D/R BSA ratio were excluded due to the correlations with D/R height ratio. The potential risk factors on flare-up were investigated by univariable and multivariable analysis in p = 0.029). Recipients were divided into low D/R height ratio (<0.70) and high D/R height ratio group (≥0.70) according to the 33rd percentile of D/R height ratio (0.70). Model 1 indicated that low D/R height ratio increased the risk of flare-up occurrence  compared with the high ratio group. The multivariable model 3 and 4 in r = −0.506, p = 0.034, The comparisons of flare-up and non-flare-up group were shown in dnDSA. There was one patient diagnosed with dnDSA but with a stable renal function. The eGFR of the 21 patients were 80.0 (65.2–91.3) ml/min/1.73 m2 and 86.8 (61.4–102.9) ml/min/1.73 m2 at 3 months and 1 year, respectively .This is the first study to investigate the dynamic characteristics of ddcfDNA in pediatric kidney transplantation and to explore the impact factors of ddcfDNA dynamics. We observed a rapid decline of ddcfDNA level within 1 week after its initial increase post-transplant and reached a stable level <1%. Nevertheless, ddcfDNA fluctuated and presented an abnormal elevation defined as ddcfDNA flare-up after the first week post-transplant. Further analysis revealed that ddcfDNA flare-up was correlated with donor-recipient size mismatch. This study depicted the early change of ddcfDNA in pediatric KTx recipients and facilitates interpretation in its clinical application.In this cohort of pediatric recipients, high-level ddcfDNA at day 1 rapidly declined and reached a stable level <1%. This dynamic characteristic was similar as the change pattern of ddcfDNA in adult KTx recipients , while tPuliyanda et al. suggested the ddcfDNA level of 1% as the cut-off value for ABMR diagnosis in pediatric KTx recipients, which was the same in adult KTx studies , 14. UnednDSA. Such abnormal elevation pattern was denoted as flare-up. We found that pediatric recipients with donor-recipient size mismatch were at high risk of ddcfDNA flare-up  of ddcfDNA was observed, and this may reflect the hyperfiltration injury of grafts caused by donor-recipient size mismatch. The study results suggest one should carefully interpret the clinical implication of ddcfDNA level at the early phase after pediatric kidney transplantation.The data presented in the study are deposited in the NCBI Sequence Read Archive, accession number PRJNA789391.The studies involving human participants were reviewed and approved by the Institutional Review Board of The First Affiliated Hospital of Sun Yat-sen University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.WN, XS, LL, HZ, and CWa: study design, data interpretation, and manuscript preparation. LL, JL, QF, CWu, RD, and SL: patient enrollment and informed consent. WN and XL: laboratory examination. WN and HZ: data analysis. WN, XS, JW, and EC: clinical data collection. SY: interpretation of pathological biopsies. XS, LL, HZ, and CWa: funding support, study supervision, and critical review of manuscript. HZ and CWa were responsible for the decision to submit for publication. All authors contributed to the article and approved the submitted version.This study was supported by Science and Technology Planning Project of Guangdong Province, China (2015B020226002 and 2017A020215012), National Natural Science Foundation of China (81870511), Key Scientific and Technological Program of Guangzhou City (201803040011), Guangdong Basic and Applied Basic Research Foundation (2020A1515010884), Guangdong Natural Science Foundation (2018A030313016), Guangdong Provincial Key Laboratory on Organ Donation and Transplant Immunology (2017B030314018 and 2020B1212060026), and Guangdong Provincial International Cooperation Base of Science and Technology .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. The reviewer BN declared a past co-authorship with several of the authors XS, LL, JL, QF, CWu, RD, HZ, and CWa to the handling editor.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.