RBP4 plays an important role in mammalian embryonic development and that mutations in RBP4 can be used for the marker-assisted selection of animal reproductive traits. However, there are few studies on the regulation of reproduction and high-prolificacy traits by RBP4 in goats. In this study, the 5′ flanking sequence of RBP4 was amplified, and a G>C polymorphism in the promoter region -211 bp (g.36491960) was detected. An association analysis revealed that the respective first, second and third kidding number and mean kidding number of nanny goats with CC and GC genotypes  were significantly higher (p < 0.05) than those with the GG genotype . The luciferase assay showed that luciferase activity was increased in C allele individuals compared with that in G allele individuals. A competitive electrophoretic mobility shift assay (EMSA) showed that individuals with the CC genotype had a stronger promoter region binding capacity than those with the GG genotype. In addition, transcription factor prediction software showed that the RBP4 g.36491960G>C mutation added a novel binding site for transcription factor DP-1 (TFDP1). RT–qPCR results showed that the expression of TFDP1 was significantly higher in the high-prolificacy group than in the low-prolificacy group, and the expression of RBP4 was higher in both the CC and GC genotypes than that in the GG genotype. TFDP1 overexpression significantly increased the expression of RBP4 mRNA (p < 0.05) and the expression of the cell proliferation factors cyclin-D1, cyclin-D2 and CDK4 (p < 0.05). The opposite trend was observed after interference with TFDP1. Both the EdU and CCK-8 results showed that TFDP1 expression could regulate the proliferation of goat ovarian granulosa cells. In summary, our results showed that RBP4 g.36491960G>C was significantly associated with fecundity traits in goats. The g.36491960G>C mutation enhanced the transcriptional activity of RBP4 and increased the expression of RBP4, thus improving the fertility of Yunshang black goats.Retinol-binding protein 4 (RBP4), a member of the lipocalin family, is a specific carrier of retinol (vitamin A) in the blood. Numerous studies have shown that  PDGFRB, MARCH1, KDM6A, CSN11S, SIRT3, KITLG, GHR, ATBF1, INHA, GNRH1, and GDF9 have been identified as potential candidate genes for reproductive traits in goats [FER1L4 and SRD5A2 may be associated with high fecundity in goats [Goats are an economically important animal for daily human consumption, providing meat, milk, skins, and wool . Yunshanin goats ,13,14. Fin goats . The kidin goats .RBP4) belongs to the family of lipid transport proteins whose main function is to transfer retinol from the liver and adipose tissue to the blood and other tissues [RBP4 in adipose tissue is regulated by 17-β-oestradiol [RBP4 is expressed during the critical period of porcine gestation, plays an important role in embryonic development, and can significantly increase litter size in commercial pigs [Retinol, also known as vitamin A, functions through the binding and transport of its carrier proteins, the retinol binding proteins (RBPs). Retinol is involved in various reproductive processes in living organisms, including steroidogenesis, follicular development, oocyte maturation and early embryonic development, and is a key nutrient required for the normal development of many tissues ,18,19,20 tissues ,22. It f tissues . RBP4, w tissues ,25. The stradiol . RBP4 castradiol . In goatial pigs ,29,30,31RBP4, determined the relationship between the gene and kidding number in Yunshang black goats by association analysis, and further explored the potential molecular mechanism of RBP4. All these data are important for elucidating the molecular characteristics of RBP4 and understanding the molecular mechanisms of how RBP4 regulates high fecundity in Yunshang black goats.Moreover, the epigenome, comprising different mechanisms such as DNA methylation, remodeling, histone tail modifications, chromatin microRNAs and long noncoding RNAs, interacts with environmental factors such as nutrition, pathogens, and climate to influence the expression profile of genes and the emergence of specific phenotypes ,33. MultAll experimental procedures involved in this study were approved by the Animal Welfare Division of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (IAS-CAAS) . Ethical approval was provided by the animal ethics committee of IAS-CAAS (No. IAS2021-25).Blood samples were obtained from 400 female Yunshang black goats  aged two to five years old in Honghe Hani and Yi Autonomous Prefecture in the Yunnan Province of China; all experimental subjects were kept under the same conditions. Blood was collected from the jugular vein (10 mL/sample) using EDTA-K2 anticoagulation tubes and then stored at −20 °C. Ovarian tissues were obtained from slaughtered female goats of three different RBP4 genotypes  based on the results of genotype sequencing. All experimental goats used for molecular experiments were euthanized. Female goats were divided into two groups based on phenotypic data: low-prolificacy (an average kidding number of <2) and high-prolificacy groups (an average kidding number of ≥2); three healthy Yunshang black goats were randomly selected from each group for ovarian tissue collection. Immediately after collection, ovarian tissues were placed in liquid nitrogen and transferred to the laboratory, where they were stored at −80 °C.2O and 0.5 μL 10 nmol/L each of upstream and downstream primers . The steps are described in the instructions of the Tiangen ‘Blood/Cell/Tissue Genomic DNA Extraction Kit’ . To prevent degradation, agarose gel electrophoresis and UV spectrophotometry were used to assess the quality and concentration of DNA after storage at −20 °C. PCR amplification of the upstream promoter region of RBP4 was performed to screen for SNPs that may affect the binding site of the RBP4 transcription factor. Primers were designed based on GenBank number NC_030833.1 from the National Center for Biotechnology Information (NCBI) database. A PCR was performed using a 20 μL mix containing 1 μL DNA sample (diluted to 5–50 ng/μL), 10 μL 2× Taq Master Mix , 8 µL ddH primers . The ampRBP4 g.36491960G>C was genotyped in 400 female goats using the SNP-KASP genotyping technique. The primers and probes used are shown in t-tests. All results are reported as the mean ± SEM.The allele frequency, heterozygosity and polymorphism information content were calculated using Excel. The association analyses between different genotypes and kidding numbers of Yunshang black goats were performed according to Li et al. . The staRBP4 was analysed using DNAstar software . The core promoter of RBP4 was predicted by Promoter 2.0 , and the transcriptional binding site of the promoter was predicted by JASPAR .The SNP sequence of Total RNA was extracted from samples (tissue or cell) using TRIzol reagent according to the manufacturer’s instructions . The quality and concentration of RNA were assessed by agarose gel electrophoresis and UV spectrophotometry, respectively. A total of 1 μg of RNA was used for reverse transcription . The synthesized cDNA was stored at −20 °C.RBP4 sequence obtained from the NCBI database (GenBank NC_030833.1) was used to design primers for RT–qPCR (RPL19) (GenBank NC_030826.1) was used as a reference gene. According to the genotyping results, three genotypes existed for RBP4 g.36491960G>C: wild-type pure homozygote GG, mutant heterozygote GC and mutant pure homozygote CC. The difference in RBP4 expression between different genotypes of RBP4 promoter region SNPs was verified for the three groups with three biological replicates each. RT–qPCR was performed using a TaKaRa SYBR premixed Ex Taq II kit (TaKaRa Bio). The 20 µL reaction system included 2.0 µL cDNA template, 10 µL SYBR PremixExTaq, 0.8 µL each forward and reverse primers, and 6.4 µL ddH2O. Three replicates of each sample were performed. The RT–qPCR protocol was as follows: 40 cycles of 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 30 s.The goat  RT–qPCR , and the−CtΔΔ method [The relative expression of genes was calculated using the 2t method . First, 2, 1 µL 200 mM EDTA and 2 µL labelled probe (except the negative control) and then supplemented with DEPC water to make 20 µL. The mixture was allowed to incubate at 20 °C for 25–30 min. The samples were mixed with 4 μL of 6× loading buffer on a 5.5% nondenaturing polyacrylamide gel for electrophoresis (1 h pre-electrophoresis had been performed prior to electrophoresis); the electrophoresis conditions were 150 V for 60 min. After completion, the electrophoresis was then rotated at 300 mA for 30 min. Crosslinking was performed by exposure at 20 cm under a UV lamp for 20 min. At the end of the process, the antibody was placed in a closure solution for 20 min and reacted with a 300-fold dilution for 30 min. Finally, the elution was coupled with a chemiluminescent solution, and imaging showed the binding bands of DNA to transcription factors.EMSA was performed according to the instructions in the competitive EMSA kit . Briefly, 20 µL of the reaction system was mixed with 2 µg of nuclear extract in the presence of 2 µL binding buffer (10×), 1 µL poly (dI. dC), 1 µL 50% glycerol, 1 µL 1% NP-40, 1 µL 100 mM MgClRBP4 g.36491960G>C mutation site on its transcriptional efficiency, the promoter region of the GG genotype containing the TFDP1 binding site +/− 100 bp was amplified and cloned into pGL3-basic to obtain pGL3-G. The promoter region containing the CC genotype was amplified +/− 100 bp and cloned into pGL3-basic to obtain pGL3-C.To determine the effect of the RBP4 versus proliferation factors in the transcription factor overexpression group versus the interference group, four vectors  were constructed for cell transfection, and three biological replicates were used for each group. The TFDP1 CDS of Yunshang black goats was amplified and cloned into the pIRES2-EGFP  vector, resulting in the overexpression vector pIRES2-EGFP-TFDP1. TFDP1 was cloned into the PLKO.1-PURO (ampicillin) vector via AgeI and EcoRI to construct the TFDP1 interfering plasmid (PLKO.1-PURO-TFDP1) in Yunshang black goats . The plasmid was extracted with a QIAGEN® Plasmd Midi Kit .For subsequent validation experiments of 5 cells/mL and cultured at 37 °C under 5% CO2. Goat granulosa cells were extracted from >5 mm follicles, which represents the follicular phase. After the isolated granulosa cells had grown across the culture plates, they were passaged and used for subsequent experiments. The tests were conducted using GCs of up to five generations, which were still proliferating [2, and 95% humidity. Ovarian granulosa cells were cultured in DMEM/F-12 medium , and HEK293T cells were cultured in DMEM ; both were supplemented with 10% foetal bovine serum  and 100 U/mL penicillin/streptomycin . The HEK293T cell line was thawed and transfected at 1 × 105 cells/well in 24-well plates for dual luciferase reporter gene assays. Ovarian granulosa cells were transfected at 5 × 106 cells/well in six-well plates to measure gene expression. Goat TFDP1 and proliferation factor primers based on the NCBI database were used for RT–qPCR (Primary ovarian granulosa cells (GCs) were isolated from the ovaries of goats according to the method described by Du et al. . The goaferating . The FSH RT–qPCR .® Luciferase Assay System .The plasmids were transfected into HEK293T cell lines using Lipofectamine 2000  according to the manufacturer’s instructions. First, HEK293T cells were seeded on 24-well plates, and transfection was started after reaching 60–80% confluence. Diluted Lipofectamine 2000 reagent was then added to Opti-MEM medium and mixed. Master mixtures of different construct vectors were prepared by diluting the construct vectors in Opti-MEM medium and mixing well. The diluted construct vector was added to each tube of diluted Lipofectamine 2000 reagent (1:1) and incubated for 25 min. Finally, the construct vector-lipid complexes were added to the cells. Forty-eight hours after transfection, the cells were harvested and processed to measure luciferase activity. The remaining steps were performed according to the protocol of the Dual-Glo4 cells/well and cultured in DMEM/F12 medium containing 1% penicillin–streptomycin and 10% FBS for 24 h. The Opti-MEM medium was replaced, and the plasmids were transfected into the granulosa cells using Lipofectamine 2000. The liquid was removed and replaced with the original medium again, and subsequent experiments were performed. Proliferation experiments were performed with three replicates per group. A vector with GFP labelling was used to detect the efficiency of transfection, and the results showed that the cell transfection efficiency reached 70–80% . After transfection treatment of the cells, the culture medium was changed after 6 h, and 10 μL of CCK-8 reagent was added to each well. The cells were incubated for 2 h, and the optical density values were detected at 450 nm with a microplate reader  at 0, 6, 12, 24 and 48 h.The CBeyoClick™ EdU-488 Cell Proliferation Assay Kit  was used to determine the proliferation status of goat ovarian granulosa cells according to the manufacturer’s guidelines. Briefly, 6-well plates were used to add EdU at a final concentration of 10 μM per well after transfecting pIRES2-TFDP1 with PLKO.1-PURO-TFDP1 during 6 h of incubation. The cells were washed three times with PBS , fixative was added for 15 min at room temperature and then removed, and the cells were rinsed three times with a washing solution. The cells were incubated with 1 mL of 0.3% Triton X-100 PBS for 15 min at room temperature to increase the permeability of the cell membrane. In addition, 500 μL of the prepared Click reaction solution was added to each well. The cells were incubated for 30 min at room temperature (protected from light) to observe and quantify the number of cells stained with EdU. Three regions of view were randomly selected for statistical analysis.RBP4 g.36491960G>C  (p > 0.05).The results of enotypes . The pop < 0.50) . The chiRBP4 g.36491960G>C on reproductive traits in Yunshang black goats, an independent samples t-test was used to analyse the association between g.36491960G>C and the first, second and third kidding and the mean number of kids. RBP4 g.36491960G>C was highly significantly associated with the number of first kids in Yunshang black goats (p < 0.01), and the number of kids in individuals with the CC genotype was significantly higher than those with the GC and GG genotypes (p < 0.05) (p < 0.05). The number of kids born in the third litter and the mean number of kids born to individuals with the CC and GC genotypes were both highly significantly higher those in the GG genotype (p < 0.01); there were no significant differences between the GC and CC genotypes. These results suggested that the g.36491960G>C polymorphism in the RBP4 promoter region was involved in the regulation of the kidding number in Yunshang black goats.To further investigate the effect of  < 0.05) . Both thRBP4 promoter region, pGL3-basic, pGL3-C and pGL3-G were transfected into HEK293T cells. The dual luciferase reporter assay showed that the luciferase activity of plasmid pGL3-C was significantly higher than those of pGL3-G and pGL3-basic (p < 0.05)  A. The RBgenotype B. The EMgenotype C. These RBP4 g.36491960G>C polymorphic site combined with transcription factors using the online tool JASPAR revealed the highest score for the newly added transcription factor TFDP1 at this locus (p < 0.05) (Prediction of the is locus A. To fur < 0.05) B.RBP4 expression, the plasmids pIRES2-TFDP1 and PLKO.1-PURO-TFDP1 were transfected into goat granulosa cells. The RT–qPCR results showed that TFDP1 expression levels were significantly increased or decreased after overexpression and interference with TFDP1, respectively, in goat granulosa cells (p < 0.05) (RBP4 was significantly increased after overexpression of TFDP1 (p < 0.05) and significantly decreased after interference with TFDP1 (p < 0.01) (To verify the role of TFDP1 in regulating  < 0.05) A,B. The  < 0.01) C,D.p < 0.05 or p < 0.01)  A,B. Both < 0.01) C,E. Simi < 0.01) D,E. All RBP4 directs embryonic development by binding to retinol [RBP4 can significantly affect backfat thickness [RBP4 on goat fertility, a mutation in the promoter region of RBP4 g.36491960G>C in Yunshang black goats was identified by PCR amplification. An association analysis with kidding number revealed that the mutation significantly increased the number of Yunshang black goats born in the first, second and third litters. These results suggest that RBP4 might be a candidate molecular marker for breeding high fertility breeds such as Yunshang black goats. Furthermore, a comparison of expression in the ovaries of RBP4 homozygous individuals with different genotypes showed that expression was significantly higher in individuals with the CC genotype than those with the GG and GC genotypes. This suggests that RBP4 is an important candidate gene associated with high fertility traits in goats. Previously, no association between reproduction and RBP4 has been reported in studies of reproductive traits in goats or sheep. However, in studies of candidate genes associated with high fertility in pigs, RBP4 SNPs were found to significantly increase total litter size [RBP4 polymorphism had a significant effect on the total number of offspring per litter. There are many other studies showing a relationship between RBP4 polymorphisms and litter size [RBP4 can enhance the proliferation and invasion of HTR8 cells by inhibiting PI3K/AKT signalling [RBP4 may also play a role in promoting cell growth and development to some extent.Granulosa cells (GCs) are known to proliferate in the follicles of female ovarian tissue and are important somatic cells surrounding the oocyte . The mai retinol . Availab retinol , human o retinol ,50,51,52 retinol . It has hickness  and foethickness . In thister size . For exater size ,59,60,61gnalling . This reRBP4 on goat reproductive traits, transcription factor prediction revealed that the RBP4 g.36491960G>C mutation added a new binding site for the transcription factor TFDP1. TFDP1 is a member of the TFDP family [TFDP1 deletion leads to extraembryonic developmental defects and lethality [TFDP1. Therefore, we constructed overexpression and interference vectors for the transcription factor TFDP1 and then transfected them into goat GCs separately to assay RBP4 expression and GC proliferation. The results showed that the expression of TFDP1 was positively correlated with the expression of RBP4. Our results indicate that the transcription factor TFDP1 can influence the proliferation of goat GCs by affecting the expression of RBP4. This is consistent with the finding of Elis et al. that RBP4 is able to induce the proliferation of bovine GCs [RBP4 has been shown to promote mitosis in GCs [TFDP1 was able to influence the proliferation of goat GCs through the regulation of RBP4.Gene expression is a complex process influenced by several biological regulatory elements, among which transcription initiation is essential for gene expression . TranscrP family . The E2FP family ,67,68,69P family . It has ethality . Thus, wvine GCs . Althougs in GCs ,74. ThesRBP4 was significantly associated with the kidding number of Yunshang black goats and resulted in increased RBP4 promoter activity. This mutation creates a new binding site for the transcription factor TFDP1, which increases RBP4 promoter activity and influences the proliferation of GCs in goats (In summary, the process of gene transcriptional expression is very complex and remains largely unknown. Our study provided a new perspective: a g.36491960G>C mutation in the 5′ flanking region of in goats . This st


The prognosis of patients with lung cancer who demonstrate pleural plaques intraoperatively, which may be associated with exposure to asbestos, is unclear. Here, we compared the clinicopathological characteristics and prognosis of these patients to those of patients without pleural plaques.We included patients who underwent curative-intent resection for non-small cell lung cancer. We retrospectively investigated the relationship of intrathoracic findings of pleural plaques with clinicopathological features and prognosis.P < 0.001) and the pathological stage (P = 0.021) were higher in patients with pleural plaques. Overall survival was significantly worse in patients with pleural plaques , and the same finding was noted in clinical stage I patients . In multivariable analysis, the presence of pleural plaques was a significant predictor of overall survival in patients with clinical stage I . In the analysis among patients with emphysema more severe than Goddard score 5 points or interstitial pneumonia, overall survival was significantly worse in those with pleural plaques than in those without pleural plaques .Pleural plaques were found in 121/701 patients (17.3%) during surgery. The incidence of squamous cell carcinoma (Patients with non-small cell lung cancer who underwent resection and demonstrated pleural plaques intraoperatively had a significantly worse prognosis. It is important to recognize the presence of pleural plaques intraoperatively, and our findings will be useful in determining the treatment and follow-up strategy for such patients with lung cancer and pleural plaques on intrathoracic examination.The online version contains supplementary material available at 10.1186/s12885-022-09600-6. Pleural plaques are one of the most common manifestations associated with asbestos exposure . The preAsbestos can easily be released into the atmosphere and inhaled, leading to various diseases such as asbestosis  and mesoRegarding the pathogenesis of pleural plaques as noted by Hillerdal , inhaledKure in Hiroshima prefecture, Japan, where our facility is located, flourished as a naval port , and curThis retrospective study was approved by the Institutional Review Board of our institute . Because this is a retrospective study, it was not practical to obtain informed consent from all patients; therefore, patient consent was obtained using informed consent documents with an opt-out process. The study was carried out in accordance with institutional guidelines which were established based on the Declaration of Helsinki. The present study included patients who underwent curative-intent resection for NSCLC between April 2009 and March 2020 at the National Hospital Organization, Kure Medical Center, and Chugoku Cancer Center. We excluded patients who underwent induction therapy and for whom complete resection was not achieved. We also excluded patients with clinical stage 0 NSCLC because follow-up time from detection to resection varied among patients with pure ground-glass nodule, and their prognosis is known to be extremely good . Figure 18F]-fluoro-2-deoxy-D-glucose (FDG-PET/CT), brain magnetic resonance imaging (MRI), and pulmonary function tests were performed preoperatively to determine the clinical stage and treatment strategies. Clinical stage was determined based on the 8th edition of the TNM classification of malignant tumors ; P < 0.001)  or death from lung cancer (P = 0.164), the incidence of death from respiratory diseases other than lung cancer was higher in patients with pleural plaques (P = 0.002). RSS  and RFS . In the multivariable analysis, the presence of pleural plaques was also a significant predictor of OS  (Table In the univariable analysis of patients with clinical stage I, the presence of pleural plaques was a significant predictor of OS (hazard ratio [HR], 2.984; 95% CI, 1.916–4.648; P = 0.001; Fig. P < 0.001; Fig. P < 0.001; Supplemental Figure P = 0.287; Supplemental Figure In the analysis of clinical stage I patients with radiological emphysematous changes with GS ≥ 5 points or IP (characteristics are shown in Supplemental Table In our study, the prognosis of patients with NSCLC who underwent resection and demonstrated pleural plaques intraoperatively was worse than that of patients without pleural plaques. This finding was also noted in the analysis limited to patients with clinical stage I NSCLC. In the overall population, lung cancer in patients with pleural plaques had more invasive features than that in patients without pleural plaques; the pathological stage was more advanced and squamous cell carcinomas were more common. Moreover, there were more deaths due to respiratory diseases among patients with pleural plaques. These facts might have led to a worse prognosis in patients with NSCLC patients who had pleural plaques. To our knowledge, the present study is the first to investigate the prognosis of patients with NSCLC who underwent resection and had pleural plaques intraoperatively.In the present study, we focused on the presence or absence of intrathoracic findings of pleural plaques, rather than the often-reported pleural plaques on CT scans , 2, 18. Pleural plaques are irregularly distributed on the parietal and diaphragmatic pleura and may occasionally be identified on the visceral pleura. Pleural plaques also tend to follow rib lines, often extending across one or more intercostal spaces . We consAsbestos exposure is an acknowledged risk factor for primary lung cancer . AlthougIn our study, the incidence of death from respiratory diseases other than lung cancer was higher in patients with pleural plaques. Asbestos induces several respiratory diseases, including asbestosis and pleuritis . MoreoveThere are several limitations to this study. First, the inherent limitations of a single-institution, retrospective study are recognized. Secondly, there was no information on individual exposure to asbestos, and thus, an analysis based on the history of exposure to asbestos could not be performed. Information on the presence of asbestos bodies in the lungs, fibrosis, and other pathological findings associated with asbestos would have been ideal. Third, there was also no information about the preoperative CT findings of pleural plaques. We investigated the outcomes in patients with pleural plaques at the time of lung cancer surgery in the present study, and we would like to examine the discrepancy between CT findings and intraoperative findings in a future study. Fourth, not only the incidence of respiratory disease, but also the incidence of death from causes other than respiratory disease and lung cancer was higher in patients with pleural plaques. The incidence of death from other cancers appeared to be higher in patients with pleural plaques, however, the reason for this is unknown. Finally, the presence of pleural plaques was confounded by other factors, such as sex and smoking. In addition, because oncological factors such as stage and histological type were among the outcomes, it was not possible to adjust for these factors with methods such as propensity score matching. However, the presence of pleural plaques on intrathoracic examination was a significant predictor of OS in the multivariable analysis containing other factors that could affect prognosis. This indicates that the presence of pleural plaque is independently significant from other factors.Despite the above-mentioned limitations, our study seems important, as there have been no other studies on resected NSCLC with asbestos exposure or intrathoracic findings of pleural plaques. Most past studies –10 on asPatients with NSCLC who underwent resection and were identified with pleural plaques intraoperatively had a significantly worse prognosis. Careful follow-up after resection is needed because these patients tend to have lung cancer with invasive characteristics and die from respiratory disease. It is important to recognize the presence of pleural plaques intraoperatively, and our findings will be useful in determining the treatment and follow-up strategy for patients with lung cancer who have pleural plaques or a history of exposure to asbestos.Additional file 1: Figure S1. Prognosis of all included patients.Additional file 2: Figure S2. Prognosis of patients with clinical stage I cancer.Additional file 3: Figure S3. Prognosis of clinical stage I patients with emphysematous change with Goddard score ≥5 points or interstitial pneumonia.Additional file 4: Table S1. Detail of death from respiratory disease other than lung cancer.Additional file 5: Table S2. Detail of death from other than respiratory disease and lung cancer.Additional file 6: Table S3. Characteristics of patients with stage I.Additional file 7: Table S4. Characteristics of clinical stage I patients with emphysema with Goddard score ≥5 points or interstitial pneumonia.


ObjectivesThe use of autologous grafts is a key aspect of contemporary septorhinoplasty. When septal cartilage is deficient, auricular cartilage serves as a biocompatible, readily accessible alternative. Our study aimed to assess donor site patient-reported outcome measures (PROMs) where auricular cartilage has been harvested for use in septorhinoplasty, adding to the limited existing literature on this topic.DesignA dual-centre, single-surgeon retrospective analysis of patients undergoing septorhinoplasty surgery with augmentation using auricular cartilage grafts was conducted. Grafts were harvested using an anterior anti-helical approach. Patients were followed up at one week, three months and 12 months post-operatively. Donor site outcomes were assessed across several physical and psychological domains by adapting the EAR-Q questionnaire, which was administered via telephone consultation. Responses were quantified using a Likert scale.ResultsA total of 22 patients met our inclusion criteria. Four were lost to follow-up, five were non-responders and one case was excluded due to documentation of body dysmorphic disorder. A significant proportion of patients reported no reduction in quality of life (QOL) or confidence attributed to donor site cosmesis. High satisfaction was noted with anti-helical donor site scars. Although noticeable differences in shape and symmetry were reported, these had negligible effects across psychological domains.ConclusionsPreliminary results suggest high levels of patient satisfaction, with minimal physical and psychological donor site sequelae following auricular cartilage harvest in septorhinoplasty. Subsequent studies should involve the use of validated questionnaires, coupled with larger patient cohorts in order to provide further data for statistical analysis. The use of autologous cartilage grafts to achieve satisfactory long-term functional and aesthetic results is a key aspect of contemporary septorhinoplasty -3. SucceImportant surrogate markers of success in septorhinoplasty surgery are patient perception and satisfaction. To date, there are limited studies that formally assess both physical and psychological patient-reported outcome measures (PROMs) at the donor site where auricular cartilage has been harvested for septorhinoplasty surgery.A single-surgeon, dual-centre retrospective case-note analysis of patients undergoing septorhinoplasty surgery with auricular cartilage grafts between January 2016 and December 2019 was conducted. An anterior approach for graft harvest via incision along the anti-helix is our preferred technique, relating to ease of access, minimal requirement for retraction, and optimal visualisation of the anterior limits of dissection. Patients were followed up at one-week, three-month and 12-month intervals. Exclusion criteria included non-anterior/anti-helical approaches to graft harvesting, revision/multiple harvesting attempts at the same site, and a follow-up period of fewer than 12 months. Donor site outcomes were assessed across several physical and psychological domains by identifying and adapting key items defined within the EAR-Q questionnaire developed by Klassen et al. . Semi-stPhysical domains included donor site pain, numbness, sensitivity, hearing impairment, scarring, as well as perceived alteration in size, shape and symmetry. Psychological domains included the effect of donor site appearance on level of confidence, self-consciousness and QOL. A four-point Likert scale  was utilised to quantify responses within each domain. Data were collated over telephone by two independent investigators located at each hospital site using a standardised questionnaire.A total of 22 patients were identified. Of these, four patients were excluded due to insufficient follow-up, five patients were non-respondents and one case was excluded due to documented evidence of body dysmorphic disorder, which we felt would have an adverse skew on the outcome and overall data. Of the 12 patients meeting our inclusion criteria, seven were male and 5 were female, with a mean age of 42.1 years SD = 13.3). The majority of cases  requiring use of autologous conchal cartilage were documented as revision cases. The percentage mode of responses per domain is summarised in Tables .3. The mThe most common response skewed towards Likert scores of 0, suggesting overall high patient satisfaction. Eighty-three percent of patients n = 10) reported no reduction in QOL from donor site cosmesis. Ninety-two percent (n = 11) were unaffected by the appearance of anti-helical donor site scarring. Variable responses were received when asked about long-term post-operative pain; 66% of patients (n = 8) stated they never had pain, while sixteen percent (n =2) reported constant donor site pain, with the remaining patients falling in between. Eighty-three percent of patients reported no numbness (n = 10) and 75% stated they never suffered donor site sensitivity (n = 9). Eighty-three percent of patients noted no post-operative deficit in hearing (n = 10). Although noticeable differences in shape and symmetry when compared to the contralateral ear were reported, with 58% of patients in each domain (n = 7) responding with scores of one to three, a corresponding drop in satisfaction across psychological domains was not seen in the majority of respondents. Ninety-two percent of patients (n = 11) stated they had no reduction in post-operative confidence due to the appearance of the donor's ear. Pre- and post-operative clinical photographs of one patient are included for reference in Figures 0 reporteAutologous cartilage is a readily available and biocompatible graft material in nasal reconstruction. A review of the literature suggests that the auricle and rib are common donor sites in septorhinoplasty surgery ,2,9. AurThe results of our study suggest overall high patient satisfaction at the donor site when conchal cartilage is harvested in septorhinoplasty surgery. This is in spite of outcomes affirming previously published objective data about post-operative changes in shape and symmetry of the donor ear . Indeed,PROMs investigating donor site outcomes traditionally focus on autologous costal cartilage . The majNevertheless, our results suggest high patient satisfaction in the other physical domains. Notably, the majority of patients (n=10) reported no post-operative issues with hearing, despite potential alteration in external ear anatomy. Amin et al. published an objective assessment of the relationship between pinna morphology and hearing, using rubber-composite models coupled with a sound processor, generating decreased sound intensity detection with wedge resection . HoweverAlthough the PROMs included herein provide useful preliminary data, there were several limitations of this study. The overall cohort of patients incorporated was low, with a lack of homogeneity in follow-up duration. Inclusion of data from a greater number of surgeons may alter the overall conclusions through a greater cohort of patients, although this may have an impact on uniformity due to variations in surgical technique. In addition, outcomes were dependent on inherently subjective data, and using adjunctive tools to obtain objective measurements would refine our study. Data collection was performed retrospectively, and additional information about the temporal changes in patient perception at each stage of follow-up could be obtained using a prospective approach. Furthermore, a non-validated PROM tool was utilised for data collection, and results are thus subject to measurement and analysis errors. An optimal design for future research would be to implement the use of a validated data collection tool, such that conclusions can be made with a greater degree of confidence. Including data on patients who have undergone cartilage harvest from the ribs would allow for comparative analysis of techniques. Analysis of the relationship between septorhinoplasty outcomes and patient perception of the auricular donor site would also be an area for further research.The results of this pilot study suggest high levels of patient satisfaction, with minimal physical and psychological donor site sequelae following auricular cartilage harvest in septorhinoplasty. Subsequent studies should involve the use of validated questionnaires to provide further data for analysis. The inclusion of a PROM database within the literature would assist surgeons during the pre-operative/planning process.


Patients with human immunodeficiency virus-associated cryptococcal meningitis (HIV-CM) have persistent intracranial inflammation despite negative cerebrospinal fluid (CSF) fungal cultures after optimal treatment for CM, which could be devastating for the central nervous system. However, a definitive treatment strategy for persistent intracranial inflammation despite optimal antifungal therapies is undefined.We identified 14 HIV-CM patients with persistent intracranial inflammation and conducted a 24-week, prospective, interventional study. All participants received lenalidomide  on days 1 to 21 of a 28-day cycle. Follow-up lasted for 24 weeks with visits at baseline and weeks 4, 8, 12, and 24. The primary endpoint was the change in clinical manifestations, routine CSF parameters, and MRI findings after lenalidomide treatment. An exploratory analysis was made on changes in cytokine levels in CSF. Safety and efficacy analyses were undertaken in patients who received at least one dose of lenalidomide.P = 0.009). The median protein concentration in CSF decreased from 1.4 (0.7–3.2) g/L at baseline to 0.9 (0.6–1.4) at week-4 (P = 0.004). The median albumin concentration in CSF decreased from 79.2 (48.4–149.8) mg/L at baseline to 55.3 (38.3–89.0) mg/L at week-4 (P = 0.011). The WBC count, protein level, and albumin level in CSF remained stable and approached a normal range through week-24. There was no significant change in immunoglobulin-G, intracranial pressure (ICP), or chloride-ion concentration at each visit. Brain MRI demonstrated multiple lesions to be absorbed post-therapy. Levels of tumor necrosis factor-α granulocyte colony stimulating factor, interleukin (IL)-6, and IL-17A decreased significantly during 24-week follow-up. Two (14.3%) patients had mild skin rash, which resolved spontaneously. Lenalidomide-related serious adverse events were not observed.Of 14 participants, 11 patients completed the 24 weeks of follow-up. Rapid clinical remission following lenalidomide therapy was observed. Clinical manifestations  were reversed fully by week-4 and remained stable during follow-up. A significant reduction in white blood cell (WBC) count in CSF was noted occurred at week-4 (Lenalidomide could improve persistent intracranial inflammation in HIV-CM patients significantly and was well tolerated without serious adverse events observed. And the additional randomized controlled study is required to further validate the finding.The online version contains supplementary material available at 10.1186/s12974-023-02717-w. Cryptococcus neoformans. CM occurs primarily in immunocompromised hosts, such as people living with the human immunodeficiency virus (PLHIV) [Cryptococcal meningitis (CM) is a type of invasive infectious disease caused by the fungus  (PLHIV) . CM is a (PLHIV) . It has  (PLHIV) . Fatalit (PLHIV) .Recently, the mortality of patients with HIV-CM has decreased significantly thanks to active antifungal therapy, aggressive management of increased intracranial pressure (ICP), and timely antiretroviral therapy (ART) initiation . After lStudies have shown that the phenomena described above occur because of brain injury mediated by persistent intracranial inflammation . DespiteMechanistic studies have suggested that the persistent intracranial inflammation in CM is characterized by high levels of T helper 1 (Th1) cell-associated proinflammatory cytokines such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, granulocyte colony-stimulating factor (G-CSF), and interleukin (IL)-6 in CSF , 17. ThuThe TNF-α antagonist lenalidomide has been shown to exert immunomodulatory and anti-inflammatory effects, and is a widely used and efficacious anti-tumor agent , 21. As We conducted a prospective interventional clinical trial to determine if lenalidomide improved the clinical outcomes of HIV-CM patients suffering from persistent intracranial inflammation. We assessed the clinical features of enrolled individuals in terms of clinical presentations, laboratory features, and neuroimaging characteristics, and we evaluated clinical outcomes after 6 months of follow-up. In addition, the cytokine concentrations in the CSF of participants were measured and compared before and after lenalidomide therapy.www.chictr.org/cn/; ChiCTR1900023184).The trial protocol (and other documents) was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University  in accordance with the Helsinki Declaration 1964 and its later amendments. All patients received detailed information about the proposed treatment option. Written informed consent was obtained from all patients. This study is registered with the China Clinical Trial Registry . The primary inclusion criterion was HIV-CM patients with a positive diagnosis of CNS inflammation despite accepting ART and successful induction therapy for CM . HIV-RNA was measured by TaqMan 2.0 , which had a lower limit of quantification of 20 copies/mL. Furthermore, at each visit, CSF samples were collected by lumbar puncture to measure CSF parameters and immunological indices. Information regarding neuroimaging characteristics was collected at screening as well as at weeks 8 and 24  on days 1 to 21 of a 28-day cycle to avoid severe myelotoxicity. Treatment and observation lasted 6 months. Before enrollment, eligible individuals were screened in addition to undergoing laboratory tests, and a comprehensive medical history was taken. Subjects who met the inclusion criteria but not the exclusion criteria were enrolled in the study. The time point at which lenalidomide treatment was planned to be initiated was considered to be the baseline time , medical history, laboratory parameters, and MRI findings were obtained from medical records or hospital databases.The primary endpoint of this pilot study was the change in clinical manifestations of participants, routine CSF parameters, and imaging findings after lenalidomide treatment. Routine CSF parameters were cell counts as well as levels of protein and glucose. The response for lenalidomide therapy based on imaging was assessed by magnetic resonance imaging (MRI) of the brain. Clinical data, routine CSF parameters, and neuroimaging information were compared between values at baseline and 4, 8, 12, and 24 weeks post-treatment. Drug safety was evaluated mainly through adverse events, vital signs, and laboratory parameters. At each visit, vital signs were assessed and height and weight measured; laboratory tests for FBC, biochemistry, coagulation function, and CD4CSF samples were collected from participants. They were centrifuged immediately and the supernatant was stored at − 80 °C until testing. The concentration of cytokines in blood was determined by a magnetic bead-based multiplex panel . In 14 patients, levels of the following cytokines in CSF were evaluated: TNF-α, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, IL-17A, interferon-gamma induced protein 10, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, G-CSF, granulocyte–macrophage colony-stimulating factor, soluble CD40 ligand, complement C3, complement factor H (CFH), fibroblast growth factor-2, platelet-derived growth factor-AA, growth-related oncogene, alpha 2-macroglobulin (α2M), apolipoprotein AI (Apo AI), apolipoprotein E (Apo E), amyloid beta 1–42, total tau, and phosphorylated tau. Cytokine concentrations were expressed in pg/mL and determined before and after lenalidomide therapy  was considered significant, and P-values were adjusted for multiple comparisons via a segmentation method. Statistical analyses were undertaken with SPSS 23 . Each parameter was tested for a normal distribution with SPSS 23. Analyses of enrichment of genes of cytokines were undertaken using the Gene Ontology (GO) database (www.geneontology.org/). Charts were generated using Prism 8 .Demographic and laboratory data are expressed as median (interquartile range (IQR)) for continuous variables. Categorical variables are described as numbers and percentages. Efficacy and safety endpoints are reported based on the full-analysis set, which consisted of the data of all patients who received at least one dose of lenalidomide. Clinical and imaging data were recorded in Excel™ 2019 . The Wilcoxon matched-pair signed-rank test and McNemar test were applied to compare baseline and post-treatment values. Between January 2017 and October 2020, we screened 99 patients who had successful induction therapy for HIV-CM, 72 of whom had developed CNS inflammation post-ART. Finally, 14 patients were found to be eligible for our study on the basis of our inclusion and exclusion criteria /µL. For 11 participants, the HIV-RNA load in plasma was below the limit of detection of the assay. The predominant clinical manifestations were fever , headache,  altered mentation , and seizures   years. At baseline, the median time from the CM diagnosis to ART initiation was 25.5 18.5–36.5) days. The median time from ART initiation to lenalidomide treatment was 558 (314.0–882.3) days. The median CD48.5–36.5 All participants received at least one cycle of lenalidomide. When all participants had completed the first cycle of lenalidomide therapy (week-4), clinical presentations such as fever, headache, and convulsions had reversed fully. Only one (7.1%) patient continued to show altered mentation but had recovered by week-12 . A significant reduction in the WBC count (× 106/L) occurred at week-4 from baseline  vs. week-0: 35 , P = 0.009). Simultaneously, levels of protein and albumin in CSF decreased significantly in a short time. The median protein concentration in CSF decreased from 1.4  g/L at baseline to 0.9  g/L at week-4 (P = 0.004). The median albumin concentration in CSF decreased from 79.2 (48.4–149.8) mg/L at baseline to 55.3  mg/L at week-4 (P = 0.011). In CSF, the WBC count as well as levels of protein and albumin remained stable and approached the normal range through week-24 , P = 0.009; protein = 0.7  g/L, P = 0.003; albumin = 42.4  mg/L). After lenalidomide therapy for 24 weeks, the immunoglobulin-G level (mg/dL) in CSF was also decreased, though not significantly so (week-24: 11.2 (6.4–36.0) vs. week-0: 13.1 , P = 0.263). However, there was a slight increase in ICP after 4 weeks of lenalidomide treatment, though the difference was not significant  vs. 150.0 (135.0–210.0), P = 0.248). There was no significant difference in ICP between week-24 and baseline. There was no significant change in CSF chloride-ion concentration at each visit, but the glucose level in CSF increased slightly  at week-24 from that at baseline . Three  participants had an ischemic injury that improved and one (1/1 100%) patient had a hemorrhagic lesion that was absorbed.The neuroimaging characteristics of all participants at baseline are summarized in Table P = 0.005), and its concentration tended to decline within 24 weeks. Levels of G-CSF and IL-6 in CSF were high. At baseline, the median level (pg/mL) of G-CSF and IL-6 was 83.7  and 191.4 , respectively. Compared with that at baseline, the median G-CSF level decreased by 90.1% at week-4  vs. week-0: 83.7 (IQR 3.9–562.2), P = 0.005) and decreased by 96.5% at week-24 , P = 0.008). The IL-6 level (pg/mL) decreased at week-4 from that at baseline, and remained diminished at week-24  vs. week-0: 191.4 , P = 0.008))  decreased significantly from baseline to week-4  to 8.1 , 9/L) dropped to its lowest value at week-12  vs. week-0: 6.8 , P = 0.002) and the platelet count (109/L) at week-8  vs. week-0: 260.0 , P = 0.001), both of which remained within the normal IQR. Furthermore, a significant decrease in the neutrophil count (109/L) was noted, which reached a minimum at week-12  vs. week-0 3.8 , P = 0.002)  and 4 low absolute neutrophil count (1 grade I and 3 grade II) was reported. The median WBC count  before enrollment. Among these patients (who had achieved successful induction therapy for CM), cryptococcal culture of CSF became negative and viral loads in plasma tended to be stable. The median interval between starting antifungal treatment and lenalidomide treatment was 581.0 (IQR 340.5–906.5) days. Long-term antifungal therapy did not improve persistent intracranial inflammation. Levels of CSF indicators continued to be abnormal, and clinical manifestations did not improve significantly (or even worsened). These phenomena were likely because of paradoxical immune reconstitution inflammatory syndrome (IRIS) . ParticiRecently, it has been postulated that anti-inflammatory actions and protection by neurons are important against chronic CNS inflammation. In the past, corticosteroids have been used to treat persistent intracranial inflammation . Guo andLenalidomide is thought to have more powerful immunomodulatory functions, but fewer side-effects, than thalidomide . DiffereThere are several limitations to our study. The principal limitation is the small sample single-arm design, it is possible confounding of time may have occurred, which prevents us from drawing definitive conclusions. We did this because identifying a suitable control for a randomized study is challenging with no other approved treatment options for this patient population in China in 2017. It’s difficult to ethically randomize them to a control group. In the past, corticosteroids have been tried to treat these patients, but serious adverse effects including severe infection, osteoporosis, and obesity have given us pause . PatientThe findings of this study demonstrated that lenalidomide was safe to use, well-tolerated, and there may be clinical benefit of lenalidomide in HIV-CM patients who develop persistent intracranial inflammation. Furthermore, lenalidomide exerts an immunomodulatory effect upon proinflammatory cytokines in CSF. Given the close association between cytokines and neuroinflammation , lenalidAdditional file 1. Definition of primary HIV infection. Diagnostic criteria for HIV-CM. Inclusion criteria. Exclusion criteria. Table S1. Follow-up procedures and items for the clinical trial. Table S2. Abbreviations for cytokines in CSF. Table S3. Baseline clinical characteristics of enrolled participants. Table S4. Change in routine CSF parameters from baseline to after 24 weeks of treatment. Table S5. Safety analysis of lenalidomide. Figure S1. Study flow. Figure S2. Measurement of levels of proinflammatory cytokines in CSF. Figure S3. Analysis of enrichment of the genes for proinflammatory cytokines in CSF. Figure S4. Change in complete blood count after lenalidomide treatment.


Patients with AE associated with intracellular antigens had increased CSF-NFL (p = 0.003) but not T-tau levels compared to controls. When adjusted for age, CSF-NFL but not CSF-T-tau was higher in patients with encephalitis associated with intracellular antigens as compared to those with encephalitis associated with extracellular antigens (p = 0.032). Total tau and NFL levels were not significantly altered in patients with encephalitis associated with extracellular antigens compared to controls. NFL in the total cohort correlated with neurological signs of cerebellar dysfunction, peripheral neuropathy, presence of CV2 positivity, presence of an underlying tumor and a more detrimental clinical outcome. AE patients with abnormal MRI findings displayed higher NFL levels compared to those without, albeit with no statistical significance (p = 0.07). Using receiver operating characteristic curve analysis, CSF-NFL levels with a cut-off value of 969 pg/mL had a sensitivity and specificity of 100% and 76.19%, respectively, regarding the detection of underlying malignancies. Our findings suggest that neuronal integrity is preserved in autoimmune encephalitis associated with extracellular antigens and without the presence of tumor. However, highly increased NFL is observed in AE associated with intracellular antigens and presence of an underlying tumor. CSF-NFL could potentially be used as a diagnostic biomarker of underlying malignancies in the clinical setting of AE.The aim of this study was to evaluate the association of neuronal damage biomarkers (neurofilament light chain (NFL) and total tau protein (T-tau)) in the CSF of patients with autoimmune encephalitis (AE) with the presence of an underlying malignancy and to determine correlations with patient characteristics. The study comprised 21 patients with encephalitis associated with antibodies against intracellular ( Encephalitis is an inflammatory brain condition with many possible causes, and in 40–50% of cases the etiology cannot be established . There aA definite diagnosis of paraneoplastic or autoimmune encephalitis can be difficult because of the broad spectrum of disease manifestations. Clinical presentation is regularly not limited to a well-defined syndrome and not specific enough to identify those patients with underlying malignancies . ConventIncreased levels of neuroaxonal damage markers (neurofilament light chain (NFL) and total tau protein (T-tau)) have been found in patients with both paraneoplastic and non-paraneoplastic autoimmune syndromes ,6, as weTo determine whether processes of neuronal and neuroaxonal injury associate with clinical course, disease severity and the presence of an underlying tumor in antibody-mediated encephalitis, we measured biomarkers of these processes in CSF from patients presenting with antibody-mediated encephalitis and from cognitively normal individuals.n = 11, mean age: 69 ± 12) and those with detectable antibodies to an extracellular antigen . Ten healthy controls (mean age: 71.7 ± 4) undergoing minor surgery  were included for comparisons. The control group presented no cognitive complaints and no neurological, psychiatric or other major diseases. They all had normal cognitive function as assessed by a semi-structured interview based on the Mini-Mental State Examination (MMSE) and within-normal-limit scores for neuropsychological testing based on the Hamilton Depression Rating Scale (HAM-D) prior to operation. Prior history of underlying malignancy was negative.A total of 31 adults was included in the study. All participants were recruited during the years 2012–2020 and all fulfilled the criteria for autoimmune encephalitis as defined by Graus et al. . Of thesDemographic and clinical data at initial presentation and at follow-up were collected in all cases retrospectively. The presence of underlying malignancies was also reported. All patients underwent a complete physical and neurological examination, evaluating focal and peripheral symptoms/signs, autonomic dysfunction, psychiatric symptoms, cognitive impairment and movement disorders. A battery of neuropsychological tests was performed in all patients which included: (a) the Mini-Mental State Examination (MMSE); (b) the Frontal Assessment Battery (FAB); (c) the 5-word immediate and delayed recall (5WT); and (d) the 15-point spontaneous and copy CLOX drawing  tests. Neuropsychiatric tests included the 15-item Geriatric Depression Scale (GDS).Laboratory investigations included electroencephalogram (EEG); electromyography (EMG); 3 or 1.5 Tesla MRI brain (some earlier MRIs were not available due to the retrospective nature of the study); abdomen, chest and pelvis CT; a standard commercially available panel of paraneoplastic and autoimmune antibodies  in the serum and/or CSF; CSF analysis ; and Positron Emission Tomography and Computed Tomography (PET-CT) if necessary.Lumbar puncture was performed at 10–11 am, after overnight fasting, at the L3–L5 interspace, according to recently proposed recommendations on standardized operating procedures for CSF biomarkers [® Elisa, UmanDiagnostics AB, Sweden) according to the manufacturers’ instructions. The concentration of total tau and NFL (picograms per milliliter) were calculated using standard curves. The detection limit of NF-light® Elisa is 33 pg/mL. All CSF analyses were performed in samples collected during disease presentation (upon disease exacerbation).The CSF levels of total tau protein and NFL were measured in duplicate by enzyme-linked immunosorbent assays  curves were applied. Biomarker levels were compared between groups of interest using a univariate analysis of covariance (ANCOVA), with age added as a variable covariate. Correlations were assessed by Spearman’s correlation coefficient. Statistical analysis was performed using GraphPad Prism-9.Data were first tested for normal distribution. Due to non-normal distribution, the Mann–Whitney rank-sum test was used to compare between groups. A two tailed n = 2; Ma2, n = 1; CV2/CRMP5, n = 1; CV2/CRMP5 and Yo, n = 1; CV2/CRMP5 and Hu, n = 2; Zic4, n = 1; GAD, n = 2; and amphiphysin, n = 1) and 10 patients had antibodies against extracellular antigens . Malignancy was detected in 10 patients. Of these patients, eight had antibodies against intracellular antigens and two had antigens against neuronal cell surface antigens. Demographic/clinical data and paraclinical data of the patients are reported in Of the 21 patients with encephalitis included in the study, 11 had antibodies against intracellular antigens , both CV2 and Yo  and both CV2 and Hu , while the highest CSF-tau levels were found in patients with antibodies against Ma2/Ta , CV2 and Hu  and Zic4  (p = 0.0043) and the controls  .Of the total study cohort, the highest CSF-NFL levels were found in patients with antibodies against CV2 alone  . When th 0.0033) A. NFL lep = 0.0001) . Moreover, CSF-tau was lower in the patients with antibodies to cell surface antigens than in the controls   compared to those with antibodies against cell surface antigens  C. The ad= 0.092) C. In our= 0.092) .p = 0.04), CRMP5/CV2 positivity , dysmetria , cerebellar ataxia , balance disorder , respiratory failure , peripheral neuropathy , tumor presence  and death . Moreover, CSF-NFL levels were found to correlate with specific neuropsychological variables; significant correlations were observed among CSF-NFL with decreasing MMSE values , FAB  and Clox2 —a pattern of cognitive disturbances indicative of loss of visuomotor functionalities. Moreover, CSF-NFL showed a negative correlation with levels of total IgG in the serum . Finally, a moderate correlation was found between CSF-NFL and CSF-tau levels (Spearman’s R = 0.4793 p = 0.0279).In the total patient cohort, CSF-NFL levels were found to correlate with the following parameters (Spearman’s R): age at disease onset , GAD positivity , NMDAR positivity , bradykinesia , dysdiadochokinesia , weight loss , peripheral neuropathy , tumor presence , partial recovery (regarding disease evolution)  and death  .CSF-T-tau levels in the total patient cohort correlated with age at disease onset , hippocampal asymmetry . CSF-T-tau levels were not statistically different among patients with normal and abnormal brain MRI findings . Patients with abnormal MRI findings displayed high NFL levels  compared to those without . However, this comparison did not reach statistical significance (p = 0.07).Only one patient displayed typical findings suggestive of limbic encephalitis with a FLAIR-intense signal in the limbic system A,B and ap = 0.0007) and the controls  . CSF-tau levels were also found to be increased in patients with encephalitis associated with tumors  compared to those without   compared to those without  B. The ad = 0.04) D. Nevertr ataxia .ROC curve analysis was performed to investigate the possible diagnostic utility of CSF-NFL and CSF-T-tau levels for the detection of underlying tumors. Only CSF-NFL showed an acceptable diagnostic performance; we found that CSF-NFL levels at a cut-off of 969 pg/mL could differentiate the patients with encephalitis associated with tumors from those without tumors and the controls, with an area under the ROC curve of 0.93, excellent sensitivity (100%) and moderate specificity (76%) E,F.In the present study, we measured NFL and T-tau levels in the CSF of patients with neurological syndromes associated with autoantibodies to cell surface and intracellular/synaptic antigens. We found that patients with encephalitis associated with intracellular antigens had increased CSF-NFL but not T-tau levels as compared to controls. Furthermore, CSF-NFL and CSF-T-tau were higher in cases of encephalitis associated with intracellular antigens compared to those associated with cell surface/synaptic antigens. After controlling for age, only NFL levels differed between cell surface/synaptic and intracellular antigens. Finally, we found that CSF-NFL levels at a cut-off value of 969 pg/mL have a potential diagnostic utility in detecting underlying malignancies. Longitudinal analyses and validation of NFL as a candidate biomarker for paraneoplastic encephalitis in larger multicenter cohorts have to be carried out for its establishment in clinical practice.Antibodies in AE are mostly directed against neuronal surface antigens. By binding to excitatory transmitter receptors , inhibitory transmitter receptors , ion channel subunits and cell adhesion molecules  or soluble synaptic proteins (LGI1), they cause initially reversible neuronal dysfunction . AutoantIntracellular neuronal antigens, i.e., Hu, Ri, Yo and Ma2, are known to be targeted by autoantibodies in paraneoplastic neurological syndromes (PNSs) and these autoantibodies serve as valuable biomarkers indicating the presence of underlying tumors . PrimariNeuropathological studies of patients with PNSs have mainly evaluated the immunological aspects of disease pathogenesis, with an emphasis on characterizations of inflammatory infiltrates , gliosis, microglia activation, neuronal loss and neuronophagia. Nevertheless, a detailed investigation of tissue markers of neurodegeneration has not been performed. NFL is mainly expressed in high-caliber myelinated axons within the central nervous systems, especially in subcortical regions, and is considered to indicate neuroaxonal damage and degeneration, irrespective of cause . Herein,This study has several limitations, including those associated with the retrospective sampling of CSF from a relatively small cohort of patients assessed at one academic medical center and the limited numbers of sequential MRIs for some patients . Nevertheless, the extended presentation of clinical information is one of the major powers of the present study. Prospective evaluation of greater numbers of patients with autoimmune encephalitis is required to validate the findings. The pathomechanisms of autoimmune encephalitis are relevantly different for each antibody and thus larger groups with the same antibody with/without tumors are needed to finally answer our research questions. Of note, we should keep in mind that the only way to diagnose autoimmune encephalitis with certainty is by pathology. Based on clinical symptoms alone, it is extremely challenging to identify the differences in CSF markers and the specificity of lesions, if existent, on imaging.The patterns of CSF biomarkers of neuronal and neuroaxonal damage in autoimmune neurological syndromes associated with antibodies against extracellular antigens showed distinct features compared to those associated with antibodies against intracellular antigens—a finding that implicates a difference in pathogenetic mechanisms between them. Our findings suggest that CSF-NFL could potentially be used as a diagnostic biomarker of encephalitis with underlying malignancies. In the CSF of antibody-mediated encephalitis, the relative maintenance of biomarkers of synaptic and/or neuroaxonal integrity (CSF-tau and NFL), possibly reflects distinct antibody-mediated effects. Future studies with larger cohorts are warranted to validate the benefit of NFL and total tau measures in clinical practice.


Spodoptera frugiperda. In this study, we screened two PO genes specifically expressed in the hemocyte transcriptome data of the fall armyworm. qRT-PCR results showed that the mRNA expression level of two PO genes gradually increased at different developmental stages. In different tissues, we found that the SfPO2 gene mainly existed in the hemolymph. As an important enzyme for activating the PO gene, the SfPAE gene existed in the midgut and hemolymph. When these two genes were silenced by RNA interference, the pathogenicity of Vip3Aa19 toxin increased significantly. A PO activity assay also confirmed the contribution of two PO genes to insect immune defenses against the Vip3Aa protein.Phenoloxidase (PO) is an important part of the innate immune system of the fall armyworm, Bacillus thuringiensis. Vip3Aa19 is secreted and expressed by Bacillus thuringiensis (Bt) at the middle exponential growth phase and is a kind of protein with efficient insecticidal activity against Spodoptera frugiperda. However, immune responses of the target insects have been regarded as a hindrance to Bt pathogenicity. This paper reports two phenoloxidase (PO) genes (SfPAE and SfPO2) identified from the hemocyte transcriptome data of the fall armyworm, Spodoptera frugiperda. qRT-PCR validation results showed that the expression levels of two PO genes were significantly upregulated after Vip3Aa19 (LC50 = 4.98 µg/g) toxin treatment compared with those of S. frugiperda fed an insecticide-free artificial diet. Meanwhile, two PO genes were expressed from the egg to adult stages even without an immune challenge. We noticed that at all developmental stages investigated in the S. frugiperda, SfPAE was generally expressed at a higher level than SfPO2. However, after Vip3Aa19 treatment, the SfPO2 gene mRNA expression level was significantly elevated in response to the toxin challenge. An injection of a specific double-stranded RNA (dsRNA) against POs could suppress its expression. The third instar larvae of S. frugiperda treated with dsRNA were much more susceptible to Vip3Aa19 toxin than the control larvae were. Notably, the mortality rate was nearly 90% after a dsPO2 injection. These results proved that SfPO2 was more important for the survival of S. frugiperda. Finally, RNA interference and then PO activity detection revealed that PO genes mainly existed in the hemolymph and played an important role in immune defense against Bt toxin.Phenoloxidase (PO) is an important enzyme in the cellular immune system and is involved in defense against a wide range of pathogens, including  Insects have extremely high species diversity in nature and have formed their innate immune system in their long-term evolution. When exogenous pathogenic microorganisms or the external environment threaten their survival and reproduction, the innate immune system will play a protective role in insects . Insect Spodoptera frugiperda , has seriously threatened the safety of maize production in China since its invasion in 2019 [S. frugiperda and results in increasing the use of insecticides [S. frugiperda to several pesticides [Bacillus thuringiensis (Bt) is a Gram-positive bacterium with spore formation and exhibits a significant entomopathogenic effect [Metarhizium rileyi suppresses the number of S. frugiperda hemocytes and activates humoral immunity of the host S. frugiperda [S. frugiperda play a significant role in immune regulation in Micrococcus luteus and Escherichia coli. Meanwhile, they have been made to identify and characterize these genes in S. frugiperda [Ostrinia furnacalis, beet armyworm, Spodoptera exigua, and silkworm, Bombyx mori, have developed resistance to insecticides, and PO activity in the hemolymph was significantly increased [Plutella xylostella, and the higher PO activity may be attributed to the stress of the Cry1Ac toxin [B. thuringiensis in the logarithmic growth phase [S. frugiperda remains highly sensitive only to Vip3A among many commercially grown Bt maize in the Americas [S. frugiperda to explore the relationship between PO genes and Bt protein.The fall armyworm (FAW),  in 2019 . Chemicasticides . Transgesticides ,12,13. Bc effect . Howeverc effect . Suppresc effect ,17. For ugiperda . Studiesncreased ,22. StudAc toxin . Bt vegeth phase . So far,Americas . In thisS.frugiperda increased significantly after feeding on an artificial diet containing Vip3Aa19 toxin, and the higher PO activity may be attributed to the stress of Vip3Aa toxin. Thus, we want to know what the responses of phenoloxidase and its related genes are, and whether that response is related to the Bt insecticides resistance. This study identified two differentially expressed PO genes, SfPAE (S. frugiperda phenoloxidase-activating enzyme-like) and SfPO2 (S. frugiperda phenoloxidase subunit 2-like) expressed in hemocyte transcriptome data of S. frugiperda treated with Vip3Aa19 toxin. Using RNA interference (RNAi), the significance of POs in defending the insect from a Vip3Aa19 toxin infection was assessed. Meanwhile, a study about the activity of PO enzyme after an infection with Vip3Aa19 toxin was conducted. The present study aims to analyze the alteration of PO gene expression and PO activity in S. frugiperda in response to Vip3Aa19 toxin on different tissues and larval carcass tissues. This study offers insights into the physiological roles of PO genes in the immune system of S. frugiperda.In insects, PO is an important immune system protein. When insects are invaded by foreign objects, humoral and cellular immunity are the main means of defense, and PO plays an important role in this process. Our study found that the phenoloxidase activity of S. frugiperda larvae were obtained from a laboratory colony at the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences . The original larvae were collected from cornfields in Mangshi, Dehong Prefecture, Yunnan Province, China, and cultured continuously on an insecticide-free artificial diet with 65 ± 5% RH, 26.5 ± 0.5 °C and 14 h L:10 h D.S. frugiperda neonates and third instar larvae. Initially, the Vip3Aa19 toxin stock solution (1 mg/mL) was prepared by dissolving the toxin in a sodium carbonate buffer . The Vip3Aa19 toxin solution was diluted with distilled water to eight concentration gradients  before mixing into an artificial diet. The calculation method of protein addition is a gradient concentration of protein (µg/g) multiplied by 10 g then divide by 1 mg/mL. Each 24-well plate contained 9 g of the artificial diet and Vip3Aa19 toxin diluted to a total of 1 mL. When the agar artificial diet was cooled below 42 °C, 1 mL of the Vip3Aa19 toxin diluent was added and stirred well. The cut and mixed artificial diet was placed in a 24-well cell culture plate, ensuring that each well contained the same amount of the artificial diet. One neonate or third instar larva was placed on the surface of the diet in each well using a fine brush. These plates were sealed with sealing film, a small hole was punctured on each well, and then placed in the rearing room . Each concentration was replicated in three plates and contained a total of 72 larvae. Neonatal mortality was checked after seven days. Third instar larvae mortality was estimated after 10 days. Because the third instar larvae are well-developed, they already have fine immune ability, needing more time for observation. If larvae died or could not move coordinately, they were considered dead. The number of larval deaths and survivals in three 24-well plates at each concentration gradient was recorded statistically. The data were processed with Polo Plus.The Vip3Aa19 toxin was provided by Beijing DaBeiNong Biotechnology Co., Ltd. . Vip3Aa19 toxin was used to check the toxicity against S. frugiperda were treated with an LC50 (4.98 µg/g) dose of Vip3Aa19 for 48 h with the same method as described in Third instar larvae of S. frugiperda. Meanwhile, 24 third instar larvae were used to extract the midgut, hemocyte and fat body.Total RNA samples were extracted using each developmental stage of Total RNA was extracted from each sample with the TRIzol method. The quality of the total RNA from individual tissue samples was evaluated with electrophoresis in 1% agarose gels, and RNA was quantified spectrophotometrically with a NanoDrop 2000 spectrophotometer  and an Agilent Bioanalyzer 2100 . All RNA samples were stored at −80 °C. First-strand cDNA was synthesized using an RT™ All-in-One Master Mix Kit . cDNA synthesis was carried out using 1 μg of total RNA. Anchored oligo (dT) was used to synthesize cDNA. The final cDNA samples were stored at −20 °C until further analysis.SfPAE and SfPO2 genes in each developmental stage and in different tissues of S. frugiperda were analyzed using qRT-PCR. The SYBR green  method was used to evaluate the expression level of two genes in different tissues of S. frugiperda . qRT-PCR primer pairs were designed using Primer 3 (http://bioinfo.ut.ee/primer3/ (accessed on 13 October 2022)) ) . A two-sne GADPH ,27.S. frugiperda were utilized to examine the role of two PO genes against Bt toxicity by RNAi. The SfPAE and SfPO2 primers were designed to synthesize dsRNA. Meanwhile, specific primers dsGFP-F and dsGFP-R were designed according to a GFP sequence as control . To restrict insect movement, they were cooled for two minutes before the injection. Control groups were either injected with equivalent volumes of ddH2O. Meanwhile, a similar dsRNA construct of the green fluorescence protein gene, dsGFP, was generated as a control. RNA was extracted from whole third instar larvae at different time points  after feeding an artificial diet, and qRT-PCR was used to detect the gene interference efficiency. The procedures were performed as above.The third instar larvae of  control . Double-S. frugiperda were treated with dsRNA specific to POs as described above. The results of qRT-PCR showed that the expression of PO genes decreased significantly after 48 h of injection of dsRNA. Therefore, 48 h was selected to feed the artificial diet of the 3rd instar larvae of S.frugiperda and mixed with Vip3Aa19 toxin. The procedures were performed the same as in 2O; injection dsGFP; injection dsPAE; injection dsPO2) were replicated four times, and each replication used 24 larvae and contained a total of 96 larvae. After 48 h, the 72 fine surviving larvae were selected and fed an artificial diet supplemented with Vip3Aa19 toxin. Larvae mortality and the weights of surviving larvae were estimated after 10 days.Third instar larvae of S. frugiperda was dissected and washed using an ice-cold Mannitol buffer . The midgut tissue was transferred to a glass homogenizer and homogenized using an ice-cold lysis buffer . The homogenate was transferred to a microfuge tube, placed on ice for 10 min and centrifuged . The supernatant was recovered, and the total protein extracted from the midgut was used to assess PO activity. The hemolymph was extracted by cutting the proleg of the larvae, and oozing hemolymph was collected in a sterile microfuge tube held on ice and centrifuged . The hemocyte-free supernatant was used to assess PO activity. The HLS was prepared by cutting the proleg of the larva, and oozing hemolymph was collected in a microfuge tube containing 500 μL of ice-cold iso-osmotic buffer  held on ice. This hemocyte suspension was centrifuged , and the supernatant was discarded. The loosely packed hemocyte pellet was washed twice by centrifuge  using the iso-osmotic buffer. Then, the iso-osmotic buffer was discarded, and the pellet was homogenized using 100 μL of ice-cold lysis buffer. The homogenate was centrifuged , and the supernatant (HLS) was used to assess PO activity. The protein concentrations in the samples were estimated using an Easy Protein quantitative kit , and the sample concentrations were set to 1 mg/mL using Tris-buffered saline  before subjecting to PO activity.PO activity was analyzed in the midgut, hemolymph and hemocytes lysate supernatant (HLS). The midgut of the third instar larva of −1 mg protein−1 [In vitro PO activity in the midgut and hemolymph fractions (serum and HLS) was assessed in the non-treated control, Vip3Aa19 toxin treatment, only RNAi treatment and both RNAi- and Vip3Aa19-toxin-treated samples. PO activity was determined by incubating the samples prepared from the midgut and hemolymph with 5 mM L-3,4-dihydroxyphenylalanine  in Tris-HCl buffer . This was done by incubating 20 μL of the midgut or serum or HLS samples with 180 μL of L-DOPA in a 96-well plate for 10 min at RT. The formation of dopachrome was measured at 480 nm in a spectrophotometer  against a blank, and six replicates were maintained for all prepared samples. PO activity was expressed as units minrotein−1 . One uni50 value for Vip3Aa19 toxin was calculated using Polo Plus software . Data related to all experiments and the relative mRNA expression levels of the PO genes were analyzed using the SPSS 20.0 Software Package, and Tukey’s test was used to determine the variance of the significance level (p < 0.05) . All figures were plotted by GraphPad Prism 5.0 software .The LCS. frugiperda treated with Vip3Aa19 toxin, which were SfPAE (S. frugiperda phenoloxidase-activating enzyme-like) and SfPO2 (S. frugiperda phenoloxidase subunit 2-like). S. frugiperda is sensitive to Vip3Aa19 toxin. The LC50 of Vip3Aa19 toxin to third instar larvae was 4.98 µg/g , the most widely used and successful microbial insecticide, has specific high insecticidal activity against field pests [S. frugiperda has developed different degrees of resistance to insect-resistant maize expressing Cry1Ab and Cry1F proteins [S. frugiperda. Meanwhile, the sensitivity of S. frugiperda to Bt toxin is closely related to the expression of immune gene POs and PO enzyme activity.PO plays an important role in the growth and development of insects and is a key enzyme for melanin synthesis and insect immune processes, and PO activity is regarded as an important marker of host immunity ,29. B. tld pests . In Amerproteins . Recent proteins . Our stuS. frugiperda was used as the research material, and Vip3Aa19 was used to infect S. frugiperda to explore its innate immune system, especially the response of PO genes to Bt when infected with Vip3Aa19 toxin. Specific upregulated SfPAE (S. frugiperda phenoloxidase-activating enzyme-like) and SfPO2 (S. frugiperda phenoloxidase subunit 2-like) were screened by hemocyte transcriptome data of S. frugiperda treated with Vip3Aa19 toxin. qRT-PCR results revealed that the mRNA expression levels of the two PO genes in the hemocyte were significantly upregulated after treatment with Vip3Aa19 toxin compared to those in the untreated group. Recent research demonstrates that PO activity and mRNA transcriptional expression and protein levels of the PPO gene in resistant strains are all significantly higher than in the susceptible strain of P. xylostella [SfPO2 was higher than that of SfPAE. Both PO genes are specifically upregulated upon Vip3Aa19 toxin invasion. The innate immune system response is central for insects to sequester invading pathogens. POs were already identified as indispensable for the S. frugiperda innate immunity in both cellular and humoral aspects [SfPAE and SfPO2 are expressed in all developmental stages and tissues of S. frugiperda. SfPAE gene expression levels upregulated with increasing age, and the mRNA expression level reached the maximum in the adult stage. It indicated that SfPAE possibly not only functioned as one of the key factors to compete with defense stress in larvae, but also was endowed with the physical function in adults’ development and may be involved in the melanization and hardening of adult body wall and its extremity [SfPOs in the growth and development of S. frugiperda [SfPO2 was lower than that of SfPAE. This may be because SfPO2 mainly exists in the hemolymph of insects and is less expressed in other tissues [SfPAE is an important activating enzyme in proPO-AS. We speculate that SfPAE can activate a variety of POs genes to play an immune function of the host, so SfPAE may exist in both the hemocyte and hemolymph. With detection of PO gene expression in different tissues, we also found that SfPAE was mainly present in hemocytes in sensitive S. frugiperda populations, but it was also significantly expressed in the midgut compared to the fat body. SfPO2 mainly exists in the hemocyte with a small amount in the midgut. In Drosophila, PO mutants show increased susceptibility to pathogens [In this study, the sensitive lostella . PO usua aspects . This stxtremity . This reugiperda . The ove tissues . SfPAE iathogens . In addiathogens ,39, suggathogens . Additioathogens .SfPAE and SfPO2 genes in S. frugiperda. dsPAE and dsPO2 genes were injected and fed LC50 Vip3Aa19 toxin. It is noteworthy that the mortality of larvae was close to 90% after the dsPO2 injection. Meanwhile, silencing both PO genes made it difficult for S. frugiperda to gain weight over time. We boldly speculate that insects have difficulties successfully developing into adults and laying eggs. Four PPO genes have been reported in S. frugiperda [SfPO2 is a more important immunity gene in S. frugiperda with a specific immune function to Bt toxin. One finding also implies that suppressing immune responses may increase the gut region’s Bt toxicity and the possibility of the gut microbiome adapting to commensal pathogens in the hemocoel [S. frugiperda was significantly inhibited and they failed to molt, leading to sepsis and death. It could be speculated that the immune responses of PO may be the natural response of resistance.This study used RNA interference to silence hemocoel . In our S. frugiperda in the midgut, hemolymph and hemocytes after feeding Vip3Aa19 toxin and silencing the PO genes, we determined PO content in S. frugiperda fed on insecticidal protein Vip3Aa19 and silenced SfPAE and SfPO2 genes by an enzyme activity assay. The activity of PO in the midgut, hemolymph and hemocyte lysate supernatant increased significantly after feeding the Vip3Aa19 toxin compared with those in the control. Consistently with qRT-PCR results, the POs mRNA expression level was upregulated. As a result, we conclude that the increased PO activity in the tested tissues could be a response to Vip3Aa19 toxicity. It is known that insect PPO not only plays an important role in shielding hosts from invasion (immediate activation in vivo) but is also responsible for wound healing [SfPAE activity decreased after silencing, but after Vip3Aa19 toxin, SfPAE exercised a significant activation, so that more PPO was activated to PO to play an immunity function. Therefore, it is further confirmed that SfPAE also exists in the hemolymph and is positively correlated with PO. As a specific enzyme catalyzing PPO, PAE also plays an important role in the immune system [Zeugodacus tau induces abnormal puparia and low pupation rates, which is important for the normal transition from larvae to pupae [P. xylostella [In order to verify the POs activity of  healing ,43,44. Te system . In the to pupae . Similarlostella .In summary, innate immunity is an important part of insect defense against external damage. The PO gene is an important group in humoral and cellular immunity, but there are few studies on the pathogenicity of Bt to the fall armyworm. This study showed that PO genes are immune genes against Gram-positive bacteria and play dual roles in development and immune responses. The activity of PO increased significantly after feeding with Vip3Aa toxin. Similarly, the suppression of POs expression enhanced the pathogenicity of Vip3Aa19 toxin. POs, as a resistance factor against Bt, need to be tested for directly exhibiting the adverse effect on the bacteria or indirectly giving an antibacterial effect on indigenous gut bacteria to suppress septicemia due to the effect of Bt toxins.S. frugiperda, especially proPO-AS, to Vip3Aa19 toxin, was preliminarily analyzed by transcriptome sequencing and gene interference. Our results revealed that two PO genes were expressed in all development stages of S. frugiperda and increased with age. After silencing SfPAE and SfPO2 genes, the bioassay and POs activity was measured to verify the important role of two PO genes in innate immunity. In general, the results provide insights into the physiological roles of PO genes in the immune system of S. frugiperda.In this study, the response of the innate immune system of


Myelin oligodendrocyte glycoprotein antibody disease (MOG-AD) is characterized by its monophasic or relapsing course and inflammatory demyelinating condition which is unable to be classified in typical multiple sclerosis (MS) or other known neuroinflammatory conditions. In the condition of neuroinflammatory, activated microglia are essential for demyelination. The secreted ectodomain of soluble triggering receptor expressed on myeloid cells 2 (sTREM2), expressed by microglial cells, is associated with abnormal biological pathways. It is known that the cerebrospinal fluid (CSF) sTREM2 concentration is much higher in neuroinflammatory and neurodegeneration diseases. However, the role of activated microglia has not been reported in MOG-AD pediatric patients. For the first time, the increased CSF and serum sTREM2 concentration in pediatric patients with MOG-AD is investigated in this work, showing evidence of microglia activation in MOG-AD. CSF sTREM2 levels significantly correlated with clinical inflammatory indexes and adapted modified Rankin Scale score, indicating the potential value of sTREM2 as a severity biomarker. The frequency of anti-MOG antibody seropositivity is high in children with acute demyelinating syndrome presentations . In MOG-phalitis . This mophalitis . Even thphalitis .On the other hand, in neuroinflammation, demyelination is associated with activated microglia . TriggerThe objective of this work is to explore the relationship between the pediatric MOG-AD and the sTREM2 levels in serum and CSF. In the meantime, the correlation between the CSF sTREM2 levels and the clinical inflammatory indexes is investigated by analyzing the sTREM2 in CSF samples from the pediatric MOG-AD group and control group.The study included 19 control patients and 19 pediatric MOG-AD patients. Nineteen patients were selected from non-neuroinflammatory diseases as control groups, including two cases of mitochondrial disorders, one case of dizziness, one case of febrile convulsion, one case of migraine, three cases of somatization disorder, one case of acute concomitant strabismus, one case of intracranial hypertension, and nine cases of functional headache. Clinical data were retrospectively collected from hospitalized patients diagnosed with MOG-AD at the neurology department of Beijing Children’s Hospital between June 2018 and August 2021. The data was acquired from The FUTang Updating medical REcords (FUTURE) database. More details and the process of data cleaning have been presented in a previous publication . We recoAll patients with MOG-AD were in the acute phase, and the samples were collected prior to treatment. CSF and serum samples were immediately centrifuged and the supernatant was collected and stored at −80°C until the time of the ELISA assays.This study was approved by the Ethics Committee of Beijing Children’s Hospital Affiliated to Capital Medical University, Beijing, People’s Republic of China, and written informed consent was obtained from all selected participants and their parents.The CSF and serum analyses on sTREM2 were performed using a commercially available ELISA assay from Abcam  according to the manufacturer. Neat CSF samples and serum samples were analyzed on the same day using assays from the same lot to avoid inter-lot variations. CSF samples and serum samples from the control group were evenly distributed on the plates.p > 0.05), both in pediatric MOG-AD and control groups. The distribution of CSF WBC count and CSF protein concentration were not normal  in the pediatric MOG-AD group, but were normal  in the control groups. Differences between pediatric MOG-AD and control groups were assessed with an independent two-sample t-test when the data were parametric. Correlation coefficients were calculated using Pearson’s two-tailed correlation test when all variables satisfy normal distribution. Spearman’s two-tailed correlation test was employed for correlation coefficients when any variables did not satisfy normal distribution. Receiver operating characteristic (ROC) curves were performed to assess the diagnostic value.Data were analyzed by SPSS software (IBM SPSS 22.0 version) and Prism (GraphPad software 8.0 version). As a small sample quantity, the distribution of CSF and serum level of sTREM2 obey normal  to assess the disease severity . The CSFr = −0.25, p = −0.303, r = −0.219, p = 0.367, r = −0.009, p = 0.970, However, the serum sTREM2 level showed no correlation with CSF WBC count (N = 19) well from the control group (N = 19), with the area under the curve (AUC) of CSF sTREM2 levels of 0.861  the CSF and serum sTREM2 levels are higher in MOG-AD pediatric patients; (2) the CSF sTREM2 levels are correlated with CSF WBC count and CSF protein concentration; (3) the CSF sTREM2 levels are correlated with mRS score; and (4) the CSG sTREM2 levels could be a biomarker for MOG-AD diagnosis.Besides, we also noted that there are no relationships between the serum sTREM2 levels with both CSF WBC count and CSF protein concentration. These negative results demonstrated that, compared with serum sTREM2 level, the CSF sTREM2 level has more reference value in measuring the degree of inflammation and may indicate the time phase of the inflammatory process.As a soluble variant of TREM-2 detected in human biofluids , the difvia PI3K/Akt pathway and activates the microglial depending on NF-κB . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.AZ prepared the reagents, performed the experiments, and analyzed the data. CR and HC selected and clinically characterized the patients. JZ carried out statistical analyses. XR obtained the financial support. WZ designed the study. AZ wrote the manuscript with revisions from all authors.