The 3-year probabilities of OS and DFS of this intermediate group were 72.6% and 66.5%, which were slightly improved than the lower and higher groups. Most importantly, these data suggest that the transfused nucleated cells from G-CSF-primed BM above than 5.20×108/kg could achieve remarkably lower TRM in haplo-HSCT receiving G-CSF-mobilized BM and PBSCs harvests. These encouraging results suggested that we could improve the efficacy of haplo-HSCT by adjusting the component and relative ratio of transfused graft cells. Nevertheless, the above findings should be confirmed in a randomized prospective comparative research with adequate follow-up.To determine the influence of graft composition in haplo-HSCT, we summarized the long-term consequences of 251 consecutive transplantations from haploidentical donors. For donor-recipient HLA3/6-matched setting, 125 cases used G-CSF-mobilized BM and PBSCs mixtures, while 126 cases only used G-CSF-mobilized PBSCs in HLA4/6-matched transplantation. On the one hand, we wanted to explore the effect of harvests (CD34+ cells and TNCs dosages) on transplantation outcome in the context of haplo-HSCT no matter HLA4/6 or HLA3/6-matched setting. On the other hand, for patients using G-CSF-mobilized BM and PBSCs combination in HLA3/6-matched setting, we attempted to analyze whether TNCs or CD34+ cells from G-CSF-mobilized BM or G-CSF-mobilized PBSCs play the most paramount role on transplantation prognosis. Collectively, patients with hematologic malignancies receiving G-CSF-primed BM and PBSCs harvests had comparable consequences with patients only receiving G-CSF-mobilized PBSCs. Moreover, when divided all patients averagely according to the total amount of transfused nucleated cells, 3-year TRM of the intermediate group (13.06-18.05×10 Nowadays, allogeneic hematopoietic stem cell transplantation  is the exclusively potential curative treatment for patients with hematological malignancies. In China, haploidentical HSCT (haplo-HSCT) has been widely accepted as an alternative approach for treating patients with high-risk hematological diseases who do not own a human leukocyte antigen (HLA) identical sibling donor and urgently need transplantation –3.More recently, we have successfully established an original haplo-HSCT protocol using idarubicin (IDA) intensified conditioning regimen and incorporation of antithymocyte globulin (ATG) and basiliximab as graft-versus-host disease (GVHD) prophylaxis. In our system, we adopted the mixture of granulocyte colony-stimulating factor (G-CSF)-mobilized bone marrow (BM) and peripheral blood stem cells (PBSCs) and an overall dose of 9mg/kg ATG in HLA3/6-matched setting. While in HLA4/6-matched transplantation, we only used G-CSF-primed PBSCs and a total dosage of 6mg/kg ATG. Remarkably, our encouraging haplo-HSCT protocol could remarkably enhance the survival in high-risk acute leukemia patients with considerably lower relapse incidence and acceptable transplantation-related mortality (TRM), and could be considered as a preferable therapeutic option for high-risk population  dosages) and the optimal thresholds of the transplanted harvests in haplo-HSCT. Thus, identification of the most appropriate threshold of the transplanted harvests is warranted as a potential strategy to improve prognosis in haplo-HSCT platform.A total of two hundreds and fifty-one consecutive patients who were diagnosed with hematological malignancies and underwent the first allo-HSCT from an HLA-haploidentical donor between August 2014 and April 2021 were enrolled in the present study. This time period was determined to allow a minimum follow-up of 12 months after allo-HSCT for surviving patients. All patients and donors provided written informed consent, in accordance with the Declaration of Helsinki. The studies involving human participants were reviewed and approved by the Institutional Review Board on Medical Ethics at Tongji Medical College of Huazhong University of Science and Technology. The characteristics and details of patients and transplantations were delineated as in HLA match was defined on the basis of 6/6 serological match at HLA-A, B and -DR loci. Among potential family members, HLA-A and HLA-B was typed by intermediate-resolution DNA techniques, whereas HLA-DRB1 was performed through high-resolution DNA techniques. The recipient of haplo-HSCT was transplanted from a family member who was matched at one HLA haplotype with the patient but differed to a variable extent among the HLA-A, B and DR antigens of the unshared haplotype. Donors were preferentially selected on the basis of HLA-matched loci, donor specific antibodies (DSA), younger age, male gender, and better performance conditions.Recombinant human G-CSF  with subcutaneous administration was utilized to prime BM and/or peripheral blood stem cells. In the G-CSF-mobilized PBSCs alone cohort, G-CSF-mobilized PBSCs were infused into the recipient after collection without manipulation on the fourth and fifth day (on the sixth day if needed). Meanwhile, in the G-CSF-primed PBSCs and BM combination group, G-CSF-mobilized BM was harvested on fourth day, and G-CSF-primed PBSCs were collected on the fifth day (and on the sixth day if needed). The unmanipulated BM and PBSCs were transfused into the recipients after collection. For exploring the influence of graft contents on transplant outcomes in the whole population, the number of infused TNCs and CD34+ cells were stratified averagely into low, intermediate and high categories. Then, the G-CSF-mobilized BM and PBSCs cohort was classified into dichotomized variables according to cutoff levels of infused nucleated cells or CD34+ cells harvested from G-CSF-primed BM or G-CSF-mobilized PBSCs due to the limited numbers.Generally, patients who were diagnosed with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) used IDA-BUCY2 regimen or traditional BUCY2 regimen, while patients with acute lymphoblastic leukemia (ALL) received regimens of IDA-intensified total body irradiation (TBI)-CY or traditional TBI-CY, which were consistent with our previous reports , 12–14. 2 on day +1 and 10 mg/m2 on days +3, +6, and +11. Mycophenolate mofetil  was administered orally from day -9, reduced to half-dose until day +30, finally discontinued on day +60. Basiliximab (20 mg by 40-minute i.v. infusion) was administered on day 0 and day +4. Decrement of immunosuppressants was based on the presence or absence of severe GVHD, infectious diseases, and relapse risks, starting at 2-4 months after allo-HSCT, aiming at cessation by approximately 6 months without manifestation of GVHD. aGVHD and cGVHD were diagnosed and graded according to the standard criteria (Prophylaxis against GVHD consisted of cyclosporine A (CsA), short-term methotrexate (MTX), mycophenolate mofetil and basiliximab. CsA at 2.5 mg/kg/day was initiated intravenously on day -1 with target levels of 150-250 ng/mL and switched to oral form when patients were able to tolerate. The administration of MTX was given to patients at 15 mg/mcriteria .9/L for three consecutive days, and platelet recovery was defined as platelets higher than 20×109/L without transfusions during the preceding seven days. Morphologic and cytogenetic analysis of BM using flow cytometry and qRT-PCR were scheduled for 1, 3, 6, 9, and 12 months and subsequently every 3 months during 12 to 24 months after allo-HSCT, as well as whenever clinically indicated. BM chimerism was evaluated at the same time through PCR amplification of short tandem repeats (STR).Neutrophil recovery was defined as achieving an absolute neutrophil count higher than 0.5×10Supportive care after allo-HSCT, including the usage of empirical antibiotics, prophylaxis and therapy for cytomegalovirus (CMV), and hepatic veno-occlusive disease (HVOD) prevention were executed as our previous reports , 12–14.P-value less than 0.10 with any endpoint in the univariate Cox analysis were eligible for inclusion in the multivariate Cox regression analysis. Statistical analyses were performed using SPSS 22.0 and GraphPad Prism 6. All statistical analysis was two-sided, and P-value less than 0.05 indicated statistical significance.Comparisons among different cohorts were analyzed by Chi-squared or Fisher’s exact tests for categorical variables and Mann-Whitney U test for continuous variables, respectively. The probabilities of hematopoietic reconstitution, acute and chronic GVHD, relapse and TRM were analyzed by the cumulative incidence estimator to accommodate competing risks. Incidences of time-dependent outcomes were calculated by the Kaplan-Meier method and compared by the log-rank test. For univariate analyses, continuous variables were categorized and the median used as a cutoff point. Cox proportional hazards model was applied to calculate the interaction between patient/graft characteristics and various transplant outcomes. Factors known to influence the outcome and factors associated with a vs. 23.0%, P<0.001), the two cohorts were well-balanced in terms of other parameters including patients’ age, gender, primary disease, risk classification, HCT-CI, donor-recipient relationship, donor-recipient CMV status, conditioning regimen, numbers of infused nucleated cells and CD34+ cells . The median follow-up times for surviving cases were 39 months, 41 months, respectively (P=0.127).A total of 251 patients were included in the present study, with 125 in G-CSF-primed BM and PBSCs group and 126 cases in G-CSF-mobilized PBSCs group, respectively. Characteristics of the patient and transplantation between two cohorts were summarized in vs. 2.4%, P=0.500), the others achieved hematopoietic reconstitution smoothly. Successful neutrophil recovery was achieved in G-CSF-mobilized BM and PBSCs cohort after a median of 11 days , which was resemble to that in the G-CSF-mobilized PBSCs cohort with a median of 11 days  (P=0.998). No remarkable difference was observed in the platelet engraftment between two cohorts ; G-CSF-mobilized PBSCs group, 12 days , P=0.504).Five patients in G-CSF-mobilized BM and PBSCs group and three in G-CSF-mobilized PBSCs group failed to acquire neutrophil reconstitution and subsequently died of lethal infection (4.0% P=0.105). 111 patients in G-CSF-primed BM and PBSCs cohort as well as 111 patients in G-CSF-mobilized PBSCs group were eligible to be evaluated for cGVHD. The cumulative incidence of total cGVHD at 2 years after allo-HSCT in G-CSF-mobilized BM and PBSCs cohort and G-CSF-primed PBSCs group were 30.3%, 26.5%, separately . The cumulative incidence cGVHD at 3 years of two cohorts were 32.9%, 28.1%, respectively .For the G-CSF-primed BM and PBSCs population, aGVHD occurred in 39 cases after a median period of 45 days post-transplantation. Among patients who developed aGVHD, 35 cases were I-II aGVHD, while four were III-IV aGVHD. It must be noteworthy that two I-II aGVHD and two III-IV aGVHD episodes occurred after utilizing donor lymphocyte infusion (DLI) because of primary recurrence. Thirty-one patients had only one organ involved , whereas six patients had two organs involved and only two had three organs involved. While in G-CSF-mobilized PBSCs group, 32 patients developed aGVHD including 24 cases with I-II aGVHD and eight with III-IV aGVHD. And one I-II aGVHD as well as six cases with III-IV aGVHD occurred after the application of DLI. Twenty-three patients had one organ involved , five cases had two organs involved and four had three organs involved. In patients receiving G-CSF-mobilized BM and PBSCs, I-IV aGVHD occurred at a median period of 43 days  with the +100 days cumulative incidence of 30.1%, whereas in G-CSF-primed PBSCs group, the cumulative incidence at day +100 was 20.3% and the median interval was 46 days  (hazard ratio (HR), 1.527; 95% confidence interval (CI): 0.915-2.547, vs. 34.7%, HR, 0.725; 95% CI: 0.451-1.166, P=0.185). In patients receiving G-CSF-primed BM and PBSCs grafts, 30 cases suffered relapse of primary disease at a median duration of 7 months  after allo-HSCT. Among all cases of relapse, 20 were hematological relapse, 6 were cytogenetic relapse, while 4 were extramedullary relapse. Meanwhile, in 126 patients receiving G-CSF-mobilized PBSCs, relapse occurred in 39 cases at a median period of 5 months , including 29 hematological relapse, 7 cytogenetic relapse as well as 3 extramedullary relapse.As exhibited in the vs. 14.6%, HR, 0.815; 95% CI: 0.402-1.652, P=0.570, vs. 39.7%, P=0.386). The incidence of CMV reactivation in G-CSF-mobilized BM and PBSCs cohort was 71.2%, which was not different from that in G-CSF-primed PBSCs group of 67.5% (P=0.521). Due to intensive surveillance and pre-emptive antiviral therapy, no onset of CMV associated diseases was observed in both cohorts.As for the cumulative incidences of 3-year TRM, there was no obvious difference between G-CSF-mobilized BM and PBSCs cohort and G-CSF-primed PBSCs group (11.5% P=0.127). As presented in vs. 61.4%, respectively . Furthermore, 3-year probabilities of DFS for patients in two cohorts were 65.4% vs. 57.4%, respectively .For the entire study cohort, 3-year probabilities of OS and DFS were 65.4%, 61.3%, separately. The median follow-up period after allo-HSCT among alive cases between G-CSF-mobilized BM and PBSCs cohort and G-CSF-primed PBSCs group were 39 months (22–91) and 41 months (22–92), respectively (vs. 31 cases). In G-CSF-mobilized BM and PBSCs cohort, nine cases died of pneumonia, two of graft failure, one of aGVHD, one of cGVHD, and one of unknown reasons. In contrast, pulmonary infection-related deaths (seven cases) and graft failure-related deaths (six cases) were common seen in the G-CSF-mobilized PBSCs group. One case died of aGVHD, two of cGVHD, and one of intracranial infection. Out of 17 patients who died due to infection, eight cases died before engraftment (5 in the G-CSF-primed BM and PBSCs cohort and 3 in the G-CSF-mobilized PBSCs cohort). A total of 38 patients in the G-CSF-mobilized BM and PBSCs cohort and 48 patients in the G-CSF-mobilized PBSCs group experienced death during the study process. Recurrence of primary disease was the most frequent reason of death in two groups . Patients having high-risk factors experienced significantly higher relapse, worse three-year OS and DFS . Patients receiving intensified regimen had reduced relapse, improved OS and DFS . Occurrence of III-IV aGVHD was prominently correlated with higher TRM, inferior OS and DFS . Patients developing cytomegaloviremia had higher TRM (P=0.036), while those with limited cGVHD had better OS and DFS .As shown in 8/kg, 13.06-18.05×108/kg, 18.05-48.35×108/kg. The three cohorts were comparable in terms of clinical characteristics which were shown in P=0.310). Three-year TRM of 13.06-18.05×108/kg group was only 4.9%, which was remarkably reduced when compared to lower and higher groups with corresponding value 18.3%, 19.6% (P=0.026). The 3-year OS probabilities were 57.8%, 72.6% and 65.7%, respectively (P=0.164). And 3-year DFS probabilities were 56.8%, 66.5% and 61.0%, separately (P=0.395). These results shed some light on the slightly improved real-life outcomes of haplo-HSCT through controlling the infused TNCs of harvests. Then, we divided all patients averagely into three groups on the basis of the amount of infused CD34+ cells: 1.65-5.21×108/kg, 5.21-7.60×108/kg, 7.60-25.10×108/kg. Nevertheless, no remarkable correlations between the amount of total CD34+ cells and transplant outcome indicators were found.We then attempted to explore the influence of graft contents on transplant outcomes in the whole population with hematologic malignancies undergoing haplo-HSCT. Firstly, we divided all cases into three subgroups averagely according to the number of infused TNCs: 3.83-13.06×108/kg, 5.20-16.30×108/kg. The two groups were comparable in terms of clinical characteristics which were shown in P=0.145). Encouragingly, 3-year TRM of higher G-CSF-primed BM nucleated cells group was significantly reduced when compared to contemporaneous lower group . The proportion of patients suffering from infectious events was significantly reduced in the higher G-CSF-primed BM nucleated cells cohort . The 3-year OS and DFS probabilities of two cohorts were 74.6% versus 65.8%, 69.5% versus 63.6%, respectively . However, it was interesting to note that there were no actually significant correlations between transplantation outcomes and factors including the amounts of G-CSF-primed BM CD34+ cells, nucleated and CD34+ cells in G-CSF-primed PBSCs harvests.We finally tried to answer which pivotal factor resulted in the substantially better survival benefit in the G-CSF-mobilized BM and PBSCs cohort under the circumstance of haplo-HSCT. Due to the limited number, the G-CSF-mobilized BM and PBSCs cohort was classified into dichotomized groups in terms of median infused nucleated cells or CD34+ cells harvested from G-CSF-primed BM or G-CSF-mobilized PBSCs, respectively. We firstly testified the relationship between transplant outcomes and the amount of nucleated cells in G-CSF-mobilized BM harvest: 0.51-5.20×108/kg and 18.05×108/kg might be the optimal dosage in the specific context of haplo-HSCT. Last but most important, our current study for the first time proved that the infused nucleated cells from G-CSF-primed BM above than 5.20×108/kg could achieve significantly improved NRM in haplo-HSCT being grafted with G-CSF-mobilized BM and PBSCs harvests.To our best knowledge, the current research represents the first formal comparison of G-CSF-mobilized BM and PBSCs harvests with G-CSF-primed PBSCs only in the context of haplo-HSCT. Our results encouragingly demonstrated that patients with hematologic malignancies receiving G-CSF-mobilized BM integrated with PBSCs harvests in HLA3/6-matched setting had resemble outcomes regardless of relapse, TRM and survival, when compared with patients only receiving G-CSF-primed PBSCs in HLA4/6-matched HSCT. Surprisingly, we proved that the total amount of infused TNCs between 13.06×10It is now generally acknowledged that G-CSF mobilization is a crucial link during allo-HSCT, because it accelerates cell cycling of pluripotent hematopoietic stem progenitor cells and enhances their amounts in the graft harvests . In addi8/kg showed faster hematopoietic reconstitution and lower TRM  progenitor cells, which might harbor immunoregulatory functions and allow the breakthrough of MHC barriers between donors and recipients , Key Scientific Research Project of Henan University (No. 20A320021), Joint Co-construction Project of Henan Medical Technology Research Plan (No. 2018020028), Key Science and Technology Project of Henan Province (No. SBGJ202103054), Natural Science Foundation of Hubei Province  and Collaborative Innovation Center of Hematology of China.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. 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Juvenile systemic lupus erythematosus (j-SLE) is a rare chronic autoimmune disease affecting multiple organs. Ranging from minor features, such as headache or mild cognitive impairment, to serious and life-threatening presentations, j-neuropsychiatric SLE (j-NPSLE) is a therapeutic challenge. Thus, the diagnosis of NPSLE remains difficult, especially in pediatrics, with no specific biomarker of the disease yet validated.To identify central nervous system (CNS) disease biomarkers of j-NPSLE.A 5-year retrospective tertiary reference monocentric j-SLE study. A combination of standardized diagnostic criteria and multidisciplinary pediatric clinical expertise was combined to attribute NP involvement in the context of j-SLE. Neopterin and interferon-alpha (IFN-α) protein levels in cerebrospinal fluid (CSF) were assessed, together with routine biological and radiological investigations.p = 0.0008). Neopterin and IFN-α protein levels in CSF were significantly higher at diagnosis of j-NPSLE with CNS involvement than after resolution of NP features (respectively p = 0.0015 and p = 0.0010) upon immunosuppressive treatment in all patients tested (n = 10). Both biomarkers correlated strongly with each other .Among 51 patients with j-SLE included, 39% presented with j-NPSLE. J-NPSLE was diagnosed at onset of j-SLE in 65% of patients. No specific routine biological or radiological marker of j-NPSLE was identified. However, CSF neopterin levels were significantly higher in active j-NPSLE with CNS involvement than in j-SLE alone (CSF IFN-α and neopterin constitute promising biomarkers useful in the diagnosis and monitoring of activity in j-NPSLE.The online version contains supplementary material available at 10.1007/s10875-022-01407-1. Systemic lupus erythematosus (SLE) is a chronic auto-immune disease affecting multiple organs, including the central and peripheral nervous system. SLE is a rare disease with an estimated global prevalence of 47/100,000 –4. NP inNotably, diagnostic biomarkers of NPSLE with central nervous system (CNS) involvement are limited, particularly in children, and none have been validated as demonstrating robust clinical utility in the few studies undertaken of j-NPSLE –13. In aThe aim of our study was to identify CNS biomarkers associated with j-NPSLE in order to provide accurate diagnostic tools for clinicians and help with monitoring of disease activity.Patients identified through the French database of rare diseases who met the 2019 European League Against Rheumatism (EULAR) ACR classification criteria for SLE with onset < 16 years (j-SLE) followed at a tertiary reference center for autoimmune diseases in children, Robert-Debré hospital, Paris, France, from January 2017 to March 2022, were included. These patients were investigated according to the same routine-care procedure over the entire period: suspected cases of j-NPSLE were evaluated by a multidisciplinary pediatric team  and longitudinally assessed during their follow-up. Standardized criteria  and multidisciplinary consensus were combined to attribute NP features to j-SLE. Isolated headaches were excluded. CSF neopterin and IFN-α were not considered for classification and only analyzed after group attribution. The study protocol followed ethics guidelines . Data processing was approved by the AP-HP  data protection office (registration number N°20200227113056). All samples were collected with informed consent. The study was approved by the Comité de protection des personnes Ile de France II and the French advisory committee on data processing in medical research (ID-RCB: 2014-A01017-40).Demographic information, past medical history, clinical features and laboratory findings, treatments, outcome, brain computed tomography scans, magnetic resonance imaging (MRI), and electroencephalogram (EEG) were recorded. All patients with j-NPSLE underwent brain MRI with non-contrast–enhanced MR angiography  using either a 1.5 or 3 T MRI scanner. Global disease activity and cumulative organ damage were respectively quantified by the SLE Disease Activity Index (SLEDAI) and the Systemic Lupus International Collaborating Clinics (SLICC)/ACR damage index. Active NPSLE status was attributed with ACR and DSM V criteria and multidisciplinary expertise. Inactive NPSLE status was defined as the resolution of acute clinical signs which had led to the diagnosis of j-NPSLE.N-methyl-D-aspartate receptor) were assessed by immunohistochemistry on rat brains slices. Neopterin was determined by liquid chromatography coupled with tandem mass spectrometry. IFN-α was quantified by different methods in serum and CSF: biological titration of antiviral activity [Lumbar puncture analysis included cytology, protein, bacteriology, oligoclonal bands, CNS auto antibodies, neopterin, and IFN-α. CNS auto antibodies  and percentages (%). Fisher’s exact test was used for categorical data. Given the small sample size, continuous data were compared using Mann–Whitney’s test. A paired data test was not performed in the principal analysis for neopterin and pan-IFN-α in j-NPSLE because of the consequences in terms of data loss. A p-value below 0.05 was considered statistically significant. Analyses were performed with GraphPad PRISM (version 9).Medians and interquartile ranges were used for quantitative variable description. Qualitative variables were described as number of patients  at last follow-up evaluation . We collected 38 CSF samples from 28 individuals, comprising 20 j-NPSLE patients with CNS involvement (19 and 11 samples during active and inactive disease respectively) and 8 controls (j-SLE where NPSLE was discarded). The following assessments were available: CSF IFN activity in 18 active and 9 inactive j-NPSLE and 8 controls, blood IFN activity in 19 active and 9 inactive j-NPSLE and 7 controls, CSF pan-IFN-α measured by Simoa in 9 active and 5 inactive j-NPSLE patients and 3 controls, serum pan-IFN-α measured by Simoa in 8 active and 5 inactive j-NPSLE and 3 controls, CSF neopterin in 18 active and 11 inactive j-NPSLE and 7 controls, 17 ISG scores in peripheral blood in 5 active and 7 inactive j-NPSLE and 5 controls, taken concomitant to CSF sampling  and shown in a flow chart Figure . Median ng Figs. .Table 1Cn = 5), diffuse micro-voltage (n = 3), encephalopathy with disturbance in maintaining vigilance (n = 1), focal slow waves (n = 3), focal spikes (n = 2), and diffuse spikes (n = 1). CSF analysis was performed at first neuropsychiatric manifestation during the study period in all j-NPSLE patients , associated with an increased white blood cell count , and isolated pleocytosis  were the only abnormalities recorded. Oligoclonal bands were noted in 4 of 15 patients tested (27%). We observed positivity of CNS autoantibodies in none of the 12 patients tested. Brain MRI was performed in all j-NPSLE patients, and abnormal nonspecific morphological features were reported in 19 of 20 patients (95%): a mild enlargement of supratentorial anterior subarachnoid spaces associated with slight widening of sulci was seen in most patients , often after the use of corticosteroid treatment  and weight loss . White matter hyperintensities (WMH) on T2 or FLAIR were observed in 13 patients, without any correlation between their number or size and clinical features. Only one patient displayed ischemic brain lesions on MRI, after severe myocarditis and low brain blood-flow, without any feature of MRI inflammation.Electroencephalogram (EEG) nonspecific features were reported in 11/14 investigated j-NPSLE patients (79%): diffuse slow activity . ISG expression (IFN signature) was assessed in active j-NPSLE, inactive j-NPSLE, and j-SLE controls in 5, 7, and 5 patients, respectively, and median of IFN signature was not significantly different between active j-NPSLE patients and active j-SLE controls (median: 13 (n = 5) vs 19 (n = 5), p = 0.8968, data not shown). CSF IFN activity was assessed in active j-NPSLE, inactive j-NPSLE, and j-SLE controls in 18, 9, and 8 patients, respectively, as negative in 83% (n = 15/18) of active j-NPSLE  and not statistically different . In contrast, levels of pan-IFN-α in the CSF measured using the more sensitive Simoa digital ELISA were significantly higher in active versus inactive j-NPSLE . CSF neopterin and pan-IFN-α levels correlated strongly . On the contrary, levels of pan-IFN-α in the serum measured by Simoa were not significantly different in active versus inactive j-NPSLE, nor j-SLE controls . Interferon activity was higher in serum than in CSF for all j-NPSLE patients, except for one, who is currently being assessed for a monogenic cause of SLE by whole-genome sequencing. All serum IFN-α concentrations measured with the Simoa pan-IFN-α assay were higher than those in the CSF in j-NPSLE patients, except for one patient who had also a diagnosis of AGS by homozygous mutation c.529G > A, p.Ala177Thr in RNASEH2B . A decrease of CSF IFN-α concentrations, measured with the Simoa pan-IFN-α assay (n = 5), and CSF neopterin (n = 10) upon immunosuppressive treatment was observed in all patients tested, correlating with improvement of SLE and j-NPSLE features  after 24-month-median follow-up. Rapid diagnosis and subsequent appropriate treatments are therefore a major challenge. However, biological biomarkers are scarce in (j)-NPSLE. For instance, it has been suggested that anti-ribosomal P proteins or antiphospholipid antibodies may play a role in the pathogenesis of psychiatric complications of SLE, but this hypothesis remains controversial , 21. No We have first reported that CSF neopterin was able to distinguish active j-NPSLE from inactive j-NPSLE and j-SLE controls. Neopterin production is mainly mediated by IFN-gamma stimulation in response to Th1 cellular immune system activation, and CSF neopterin levels are already used to assess CNS inflammation , 23, andWe have also shown that CSF IFN-α levels assessed by Simoa, although mostly lower than concomitant blood concentration, are associated with active central j-NPSLE, decreasing with resolution of NP features upon immunosuppressive treatments. CSF IFN-α might therefore also constitute an interesting biomarker, as suggested in serum for SLE activity and relapse , 30. WhiA statistically significant difference was observed for neopterin between NPSLE and non-NPSLE groups, but not for CSF pan-IFN-α levels — probably due to a lack of statistical power, since so few non-NPSLE patients were assessed using this test. For ethical reasons, CSF of patients without j-NPSLE suspicion was not collected. Moreover, as this is a retrospective study, lumbar puncture was often performed after starting immunosuppressive drugs, which may decrease these biomarkers. In addition, CSF was not always tested after disease resolution.Among adult patients, other new NPSLE biomarkers have been identified in previous studies: CSF α-Klotho, lipocalin-2, macrophage colony-stimulating factor and IgM, and serum IL-6, miR-23a, and miR-155 . HoweverRegarding other biomarkers, non-specific brain MRI defects were noticed in almost all j-NPSLE patients. Cerebral atrophy and white matter FLAIR or T2 hyperintensities were the most common manifestations, as previously described in the literature , 35. HowOur study is limited by the retrospective design and its setting in a tertiary center, likely resulting in an over-representation of patients with severe SLE. Selection bias could also be linked to our nationally recognized expertise in the field of j-NPSLE, thereby influencing the prevalence of j-NPSLE that we observed.Further research is necessary to better understand j-NPSLE-pathophysiology and offer personalized treatments, adapted to systematic clinical evaluation and accurate biomarker measurement in homogeneous cohorts. If CSF IFN-α is confirmed as being associated to NPSLE activity, this cytokine might be an interesting therapeutic target in the future, with the use of JAK-inhibitors or IFN-receptor antagonists in severe and refractory j-NPSLE, the latter best at crossing the blood–brain barrier , 38. MorJ-NPSLE is a diagnostic challenge for clinicians in the absence of current validated specific j-NPSLE markers. We have reported, to our knowledge for the first time, that patients with active j-NPSLE patients with CNS involvement display a specific biological profile with increased CSF pan-IFN-α and CSF neopterin levels. Further prospective investigations are warranted to assess the specific link between active j-NPSLE and these biomarkers, which could enable earlier targeted treatment and improve clinical outcome.Supplementary file1 (DOCX 740 kb)Below is the link to the electronic supplementary material.


Objective: To explore the correlation between characteristics of myocardial energy expenditure (MEE) and the degree of diastolic dysfunction in patients of heart failure with preserved ejection fraction (HFpEF) and its clinical significance.Methods: 125 consecutive patients diagnosed with HFpEF in the Department of Cardiology, Affiliated Zhongshan Hospital of Dalian University from January 2018 to October 2018 were enrolled. According to the degree of diastolic dysfunction, patients were divided into group A (8 ≤ E/e' ≤15) and group B (E/e'> 15), and MEE was calculated, patients finished 1-year clinical follow-up.Results: The level of MEE in group A was significantly lower than that in group B (p < 0.05). During 1-year follow up, MEE over 3145.69 kcal/systole was associated with increased risk of death as compared to patients with MEE less 3145.69 kcal/systole, and in patients with MEE over 101.68 kcal/min than in patients with MEE less than 101.68 kcal/min.Conclusion: There is a significant correlation between MEE and diastolic dysfunction and MEE over 3145.69 kcal/systole as well as MEE over 101.68 kcal/min are linked with increased risk of 1-year mortality in HFpEF. Heart failure (HF) is a severe manifestation of various heart diseases, associated with high rate of mortality and re-hospitalization. The worldwide prevalence of HF is 1.0–2.0%, ≥10% among people with age over 70ys is . Heart fn = 69, aged 70.90 ± 10.97 years, 46.4% males) and group B .Patients who were admitted to the Department of Cardiology of Affiliated Zhongshan Hospital of Dalian University and diagnosed as HFpEF from January 2018 to October 2018 were selected to participate in the study. The study protocol was approved and carried out in accordance with recommendations of the ethic committee of Affiliated Zhongshan Hospital of Dalian University. The total of 125 of enrolled participates have signed the informed consents in accordance with the Declaration of Helsinki . They weThe peak blood flow (E) at the early stage of mitral valve diastole was measured according to the color Doppler echocardiogram. Left ventricular ejection fraction (LVEF) was measured at 1 year follow-up. The primary endpoint is the all-cause mortality at the 1-year follow-up.2, septal E/e’ ratio>15, and TRVmax>2.8 m/s (HFpEF was defined according to 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic HF : 1) HF s>2.8 m/s .1) Unable to express medical history and co-examination due to unconsciousness, mental retardation or vague speech; 2) Combined with malignant hypertension, acute myocardial infarction, aortic dissection, severe arrhythmia  or pacemaker implant; 3) Various congenital heart diseases; 4) Heart valve disease, pericardial disease, hypertrophic cardiomyopathy; 5) Take medicine that improves myocardial energy metabolism in the past 3 months ; 6) Those who have serious diseases of other systems, such as malignant tumors, liver cirrhosis, renal failure, and bleeding diseases.Patient’s morning height (H): The patient stood upright on the floor of the height meter on bare foot, with the heel, metatarsal bone, and the midpoint of shoulder blades abutting on the height meter post. The level plate of the altimeter was moved to the top of the patient’s head. The recorder’s line of sight was flushed with the lowest point of the upper edge of the patient’s tragus and the lower edge of the orbit. Measurement was recorded the value in meters.Morning weight (W): The patient took off clothes and socks, stood on the weight scale, and the value was recorded in kilograms.Laboratory parameters, including blood lipid, fast blood glucose, glycated hemoglobin and N-terminal pro-brain natriuretic peptide (NT-proBNP) were obtained in all participants. Tandem mass spectrometry was used to detect valine, leucine, and free carnitine with peripheral blood collected by dried blood spot method. Blood pressure, heart rate, electrocardiogram and 6-min walking test (6MWT) were measured after hospital admission.–4All the echocardiographic parameters including left ventricular ejection fraction (LVEF), peak blood flow (E) in early mitral valve diastole, early mitral annulus velocity (e'), stroke volume (SV), tricuspid regurgitation velocity (TR velocity), ventricular septal thickness (IVSD), left ventricular posterior wall systolic thickness (PWTs) and diastolic thickness (PWTd), left ventricular end-systolic diameter (LVIDs), end-diastolic diameter (LVIDd) were measured with PHILIPS EPIQ7 ultrasound system, E/e' as well as left ventricular short axis shortening rate (FS%) was calculated. Aortic valve ejection time (ET) was measured by Doppler blood flow spectrum in three continuous cardiac cycles, and the average value was obtained. The mean arterial pressure (MAP), cardiac output (CO), cardiac index (CI), left atrial volume index (LAVI), left ventricular mass index (LVMI), left ventricular end-systolic peripheral wall stress (cESS) were calculated, and MEE was calculated by related formula . A skillt test (for continuous variables), Mann-Whitney U test (for non-parametric variables). Spearman’s correlation analysis. Receiver operating characteristics (ROC) curve analysis was carried out to investigate the efficacy of MEE and Metabolomics in predicting end point and the best cut-off values were determined on the basis of Youden index. Based on these cut-off values, patients were divided into two groups for each parameter. Kaplan–Meier curve analysis was used for event-free survival analysis in between these groups. Statistical significance was defined as p < 0.05. Data was analyzed with SPSS 25.0 software.The continuous variables were reported as the mean ± standard deviation (SD) and the categorical variables were expressed as the number of patients and percentages. Kolmogorov-Smirnov tests were used to assess the normality of the data distribution. The comparisons between groups were performed with p > 0.05).The clinical data of the whole participates was shown in p = 0.013) and MEE per minute  were lower in group A than in group B; MEE was positively correlated with NT-proBNP  and negatively correlated with the 6-min walking distance  (p > 0.05). MEE per minute in overweight (BMI≥24) group was higher than the other group , MEE per minute was lower in mild degree HF group (6MWT>450) than in moderate degree HF group (150<6MWT≤450)  (p = 0.033). Additionally, LVEF were lower than baseline in both two groups  and  (p = 0.013) (p = 0.019) (p = 0.040) (p = 0.008) (The data of echocardiography parameters and MEE was presented in = 0.001) . However < 0.05) ; During  < 0.05) ; In ROC = 0.013) . All the= 0.019) ; MEE (kc= 0.040) . The stu= 0.008) .Data released in 2012 shows that the prevalence of HF in developed countries will increase by 46% by 2030, and the incidence of HF will double for every 10-year increased in age . SeveralSome experts pointed out that MEE increased significantly with the decrease of LVEF and the increase of cardiac function grade, and was closely related to the NT-proBNP level . It is n2), MEE , MEE  in E/e' >15 group is significantly higher than that in 8 ≤ E/e'≤15 group. LAVI values and TR velocity in E/e' >15 group had a tendency of higher than that in 8 ≤ E/e'≤15 group. For HFpEF patients, MEE is significantly correlation with the degree of diastolic dysfunction. The results suggested that the worse the diastolic function of HFpEF, the more energy consumption per minute and even per stroke is, In other words, the augmented myocardial oxygen and energy consumption, together with aggravated neurohumoral activity might coexist in HFpEF patients with severely diastolic dysfunction, which may further lead to the depletion of cardiac energy reserves in HFpEF patients and establish the vicious circle of above unfavorable features in HFpEF patients.Our preliminary study findings demonstrated that E/e' ratio, cESS .In the study of Chen , brancheHowever, We found that compared with metabolomics, the method of MEE measured by echocardiogram could reflect the degree of diastolic dysfunction of patients with HFpEF better, and also has predictive value. What’s more, this study reflect that patients with HFpEF who are elderly, overweight, female sex population spend more MEE, suggesting that elderly, obese and female patients should undergo more detailed screening, to improve the detection rate of HFpEF. This conclusion same as found that women, African American, obese, and hypertensive have more HFpEF patients, and upregulated genes in oxidative phosphorylation pathways in HFpEF were associated with obesity .Although some drugs known to have remodeling effect in the myocardial tissue  which coThe worse the diastolic function, the greater the myocardial energy consumption to maintain the heart function to some extent. HFpEF trials like VICTORIA , EMPERORThe latest study defined Diastolic dysfunction (DD) severity with three predominant parameters  is for and predicting the outcome in HFmrEF and HFrEF patients with various cardiac rhythms. Severe DD serves as an independent determinant for all-cause mortality for both HFmrEF and HFrEF patients after adjustment of clinical and other echocardiographic covariates. This finding might be interpreted as a hint for DD being the ‘straw that breaks the camel’s back’ for CV death in HFrEF, but not in HFmrEF patients. MEE might be a part of mechanism . As a siThe deficiency of this study is that the 1 year follow up data is limited to LVEF and mortality, without detailed diastolic function MEE calculations, and It is to note that the number of enrolled patients is relatively small and from a single medical center in our study, and future studies with large patient cohort are needed to validate present findings. Patients were followed up for a long time to observe the clinical symptoms, cardiac function classification, objective biochemical, echocardiogram indicators and quality of life. In this study all-cause mortality was analyzed during follow-up and cardiovascular mortality, MACE and HF rehospitalization data were not collected during follow-up, future studies is required to overcome this study limitation.It is of importance to observe the serial changes of MEE parameters and explore the impact dynamic MEE changes on outcome of HFpEF patients and observe the impact of medication on these parameters and the dynamic prognostic implication of theses parameters, the upcoming study in our center will address this issue.


To study cerebrospinal fluid neurofilament light chain (CSF-NfL) levels as a prognostic biomarker in pediatric Guillain-Barré syndrome (GBS).Prospective study enrolling 26 pediatric GBS patients and 48 healthy controls (HCs) from neurology units between 2017 to 2021. The CSF-NfL levels were measured by enzyme-linked immunosorbent assay. The children's disability levels were evaluated using Hughes Functional Score (HFS) at nadir, 1 month, and 6 months after onset. The receiver operating characteristic (ROC) curve derived from logistic regression (with age as a covariate) was used to assess the prognostic value of CSF-NfL on the possibility of walking aided at 1 month after symptom onset.t = 6.754, p < 0.001). At follow- up, the mean CSF-NfL levels after treatment (65.69 pg/mL) declined significantly . CSF-NfL levels upon admission were significantly associated with the HFS at nadir . Moreover, the mean CSF-NfL levels in GBS patients with poor prognosis (130.47pg/mL) were significantly higher than that in patients with good prognosis (104.87pg/mL) . ROC curve analysis of the predictive value of CSF-NfL levels with respect to the inability to walk unaided within 1 month showed a significant difference .The mean CSF-NfL levels were significantly increased in GBS patients (111.76 pg/mL) as compared to that in HCs (76.82 pg/mL) (CSF-NfL levels were increased in pediatric GBS patients. High CSF-NfL level predicted worse motor function, and was strongly associated with poor short-term prognosis of pediatric GBS. We propose a biomarker for early prediction of outcome in pediatric GBS, which would be applicable for clinical practice and efficacy of treatment in the future. Campylobacter Jejuni, Mycoplasma Pneumoniae and other factors such as vaccinations, surgery, and malignancy (Guillain–Barré syndrome (GBS) is currently the most common cause of acute flaccid paralysis in children and is thought to be provoked by an aberrant immune response. Potential triggering factors include viruses such as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), Zika virus, cytomegalovirus (CMV), Epstein–Barr virus (EBV), and influenza virus, bacteria such as lignancy . Since Glignancy . HoweverMany studies have shown that patients with GBS differ considerably in their clinical manifestation and prognosis , 5. ChilAs the most important axonal damage biomarker, neurofilament levels have emerged as important in neurological disorders, such as multiple sclerosis , AlzheimWe enrolled 26 pediatric GBS patients (under 14 years of age) who were admitted to our hospital between September 2017 and January 2021. Patients fulfilling levels 1 or 2 of the Brighton criteria of GBS  were inc®). Sample processing was carried out according to the manufacturer's instructions and protocol. All samples were measured in duplicates. The mean intra- and inter-assay coefficients of variation for mean CSF-NfL level (111.76 pg/mL) were 1.9and 4.5%, respectively.CSF samples of GBS patients were collected upon admission and 2 weeks after the treatment. All samples were collected into a polypropylene tube and stored at −80°C until the analysis. Samples were analyzed by two investigators blinded to clinical data using the Nf-light kit (ELISA kits: Uman Diagnostics NF-light(1) Limb disability was assessed by Hughes Functional Scale (HFS) at nadir, 1 month, and 6 months after onset. Patients with HFS ≥ 3 were categorized as poor outcomes, and patients with HFS <3 were categorized as good outcomes. Other clinical manifestations, such as facial paralysis, paraesthesias, autonomic dysfunction, and mechanical ventilation were also analyzed. (2) The pleocytosis and protein levels of CSF were measured within 4 weeks of weakness onset. (3) Electrophysiologic examinations were performed at least twice and patients were classified into acute inflammatory demyelinating polyneuropathy (AIDP) and acute motor axonal neuropathy (AMAN) according to the Hughes electrodiagnostic criteria .2 tests. The continuous variables were tested by the t-test or Mann–Whitney U test. The associations between basal characteristics and CSF-NfL levels were analyzed using Pearson correlation or Spearman's correlation coefficient. The receiver operating characteristic (ROC) curve derived from logistic regression (with age as a covariate) was used to evaluate the prognostic value of CSF-NfL on the probability of walking dependently. Statistical significance was set at 0.05.Statistical analyses were performed using the IBM SPSS Statistics 24. Categorical data were shown as proportions, and continuous data were shown as mean ± SD or medians with IQR. Differences in proportions were tested by χt = 6.754, p < 0.001)  were recruited. The mean CSF-NfL level for the GBS patients upon admission was 111.76 ± 26.33 pg/mL. Forty-eight healthy age-matched controls  were included, and their mean CSF-NfL level was 76.82 ± 17.96 pg/mL. CSF-NfL levels were significantly higher in the GBS patients than in healthy controls (< 0.001) .t = 6.693, p < 0.001)   . The CSF= 0.053) .rs = 0.461, p = 0.018) . Other clinical features and CSF-NfL levels are summarized in CSF-NfL levels upon admission were significantly associated with the Hughes Functional Score (HFS) calculated at nadir (= 0.018) . Meanwhirs = 0.002, p = 0.991), and receiver operating characteristic (ROC) curve analysis of the predictive value of CSF protein levels showed no statistical difference .There was no association between CSF protein levels and prognosis in GBS patients  , which was not the case for the GBS patients . Analysis of covariance suggested no influence of age on CSF-NfL concentrations , whereas the age-adjusted group difference was preserved (p = 0.009). ROC curves derived from logistic regression (with age as a covariate) were used to analyze the potential predictive value of CSF-NfL levels with respect to unable to walk unaided (Hughes grade of 3 and more) within 1 month, which showed a statistically significant difference   . A corre= 0.006) .Guillain-Barré syndrome (GBS) is one of the most common causes of acute flaccid paralysis in children, and is characterized by progressive, relatively symmetrical limb weakness with or without paresthesia . The proNeurofilament light protein (NfL) is one of the most important intracellular neuronal skeletal proteins and is secreted into CSF during axonal damage . In the CSF albuminocytological dissociation phenomenon could further assist the diagnosis of GBS . CSF proHigher CSF-NfL levels in adult GBS had also been associated with a poorer clinical prognosis . In the Our study was also subject to some limitations. First, the association of CSF-NfL levels and electrophysiological subtypes was not evaluated because of a smaller number of cases. Secondly, this was a small prospective study that recruited 26 pediatric GBS patients. We will further conduct some prospective clinical studies on the prognosis of children with GBS based on the present study.In conclusion, CSF-NfL levels are increased in pediatric GBS patients, higher CSF-NfL predicted worse motor function and were strongly associated with poor short-term prognosis of pediatric GBS. We proposed a biomarker for early prediction of outcome in the pediatric GBS, applicable for clinical practice and future treatment efficacy.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.This study was approved by the Ethics Committee of the Children's Hospital of Hebei Province. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.LZ and YZ collected serum and CSF samples. JL acquired the electrophysiological data. ZZ completed the statistical analysis. MJ designed the experiments, interpreted the results, and drafted the initial manuscript. KL reviewed the data and revised the manuscript. SS revised the initial draft and wrote the final manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Medical Science Research Key Project Plan of Hebei Province in 2020(20200223).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In recent years, there has been growing interest in the use of thyme and oregano essential oils in feed formulations to promote growth in chicken broilers. Thyme and oregano essential oils are considered promising ingredients to replace antibiotics as growth promotors. The aim of this study was to evaluate the impact of thyme and oregano essential oils on growth performance, broiler health, and the incidence of muscle abnormalities at different slaughter ages. This study showed that the addition of thyme and oregano essential oils, individually or in combination, significantly increased body weight compared to the control group. Thyme and oregano essential oils improved the feed conversion factor, which indicates lower feed intake (feed intake did not change according to our results) with higher meat production. Muscle abnormalities increased with the addition of thyme and oregano essential oils to broiler diets, which could be due to the increase in the growth rate. In conclusion, the inclusion of thyme and oregano oils in broiler chicken feed resulted in an improvement in the growth performance of broiler chickens.The objective of this study was to investigate the effects of thyme and oregano essential oils (as growth promotors), individually and in combination, on the health, growth performance, and prevalence of muscle abnormalities in broiler chickens. Six hundred day-old Cobb 500 hybrid chickens were randomized into four dietary treatment groups with three replicates each. Chicks in the control group (C) received a basal diet, while the experimental treatment groups received basal diets containing 350 mg/kg of thyme oil (T1), 350 mg/kg of oregano oil (T2), and 350 mg/kg of thyme and oregano oil (T3). Growth performance parameters were evaluated at 14, 28, and 42 days. The broilers in treatments T1 and T2 had significantly higher body weights than the control group. The feed conversion ratio was the lowest in chicks who received oregano oil, followed by those fed thyme oil. The overall prevalence of growth-related breast muscle abnormalities (including white striping and white striping combined with wooden breast) in groups receiving essential oils  was significantly higher than in the control group (C). The thyme and oregano oil diets showed no significant differences in antibody titers against Newcastle disease or interferon-γ (INF-γ) serum levels. In conclusion, thyme and oregano oils had a positive impact on the growth performance of broiler chickens but increased the incidence of growth-related breast muscle abnormalities. Health concerns and regulatory restrictions on the use of antibiotics motivated the researchers to evaluate several alternatives to antibiotics. It was found that the use of different combinations of additives  exhibited positive effects on the growth performance of broilers . ExtractThymus vulgaris) and oregano (Origanum vulgare L.) are rich in several functional compounds such as carvacrol, thymol, lutein, and zeaxanthin, which play an important role in broiler health and growth performance [Clostridium perfringens [Extracts of thyme  and wooden breast (WB) . In thisMudalal et al.  examinedMost previous research results agreed that essential oils have antimicrobial ,7,30, anIn particular, Newcastle disease (ND) is considered one of the most serious diseases affecting broiler flocks worldwide, causing severe losses in the poultry sector . BiosecuTo our knowledge, there are few studies that investigated the effects of thyme and oregano oils as a mixture on the health, growth performance, and prevalence of muscle abnormalities of broilers reared under commercial conditions. Therefore, the objective of this study was to examine the possible effects of thyme and oregano oils and a combination of both oils on the performance parameters, health status, and meat characteristics of broiler chicks as well as on the prevalence of muscle abnormalities from 1 day to 42 days of age.In this study, 600 one-day-old Cobb 500 hybrid broiler chicks were randomly divided into four groups of 150 chicks each, and each group was replicated three times. The chicks from the first treatment group received a basal ration (starter and grower) as a control group (C) . The ratThe feed conversion ratio was calculated as feed intake (g) per mean body weight (g) for each replicate of the treatment groups. The feed intake was calculated on a weekly basis, taking into account differences in feed weight. In addition, the weight of each broiler was recorded weekly.The feed conversion ratio (FCR), body weight (BW), and feed intake (FI) were determined at different ages .n = 21/group). Breasts were weighed using a balance with a sensitivity of 0.01 g.Seven broilers from each replicate were slaughtered at 42 days of age using a manual operation technique  based on previously described criteria ,36. BreaThe color trait  of raw breast meat was measured in triplicate using a Chroma Meter CR-410 , and the skin-side surface of each fillet was considered a measuring point.®, Cape Town, South Africa) was administrated via drinking water when the chicks were 12 days old, and this was repeated when the chicks were 22 days old. Blood samples were collected during the 1st, 3rd, and 5th weeks from the wing vein (n = 24). Each blood sample was left to coagulate at room temperature and was then centrifuged at 3000 rpm for 5 min.The freeze-dried live Newcastle Disease (ND) vaccine , following the method of Allan and Gough [The collected sera were subjected to the hemagglutination inhibition (HI) test, and the level of the anti-NDV antibody titer was determined. The HI tests were performed in microplates using two-fold dilutions of serum, 1% PBS-washed chicken red blood cells, and four hemagglutinating units of vaccinal LaSota NDV (Biovacnd Gough . Titers The interferon concentration was determined by an immunoenzymatic assay (ELISA). At three time points  the serum level of interferon-γ (INF-γ) was determined using ELISA kits, following the instructions enclosed in the manufactured kits . Eight standards of 0, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 pg/mL were added to the wells of the ELISA plate. Absorbance was measured at a wavelength of 450 nm. The interferon concentration was calculated using the standard curve.p < 0.05). Pearson’s correlation was used to test the relationships between pairs of continuous variables .The effects of the thyme and oregano oils on the growth performance, feed conversion ratio, and the incidence of muscle abnormalities were assessed using an ANOVA . Duncan’s test was employed to separate means in the case of the presence of statistical differences  in all treatments are shown in p < 0.05) in comparison to normal meat, while white-striped meat exhibited intermediate values.The effects of thyme and oregano extracts on color traits , pH, and breast weight are shown in p < 0.05) positive effects on the broilers’ humoral or cellular immune reactions to NDV treatments  was higher in the treated groups  than in the control group , respectively. These results may be attributed to an increase in the growth rate and the live weight of broilers at slaughter . PreviouThe addition of thyme and oregano extracts exhibited no effects on the color traits , pH, and breast weight. The incidence of muscle abnormalities  had no effect on the color traits  and pH but affected breast weight. Zambonelli et al.  found thp < 0.05) compared to normal meat, while white-striped meat exhibited intermediate values. Similar results were obtained by Tasoniero et al. [Meat affected by the WB abnormality exhibited a higher breast weight  positive effects on the humoral or cellular immune reactions of broilers to NDV in the treated groups (Dietary supplementation with thyme or oregano essential oils alone or in a mixture had no significant (d groups . No signIn conclusion, the addition of oregano oil was the most effective in improving the growth performance of broiler chickens and was better than thyme oils. The inclusion of thyme and oregano essential oils together had no positive impact on broiler health. While the essential oils of oregano and thyme improved the feed conversion factor, the incidence of muscle abnormalities increased, and this may be attributed to the increase in the growth rate. Therefore, it is important to consider the impact of these muscle abnormalities on meat quality when developing any growth promotion program.


Background: Given the significant role of neurodegeneration in the progression of multiple sclerosis (MS) and insufficient therapies, there is an urgent need to better understand this pathology and to find new biomarkers that could provide important insight into the biological mechanisms of the disease. Thus, the present study aimed to compare different neurodegeneration and axonal dysfunction biomarkers in MS and verify their potential clinical usefulness. Methods: A total of 59 patients, who underwent CSF analysis during their diagnostics, were enrolled in the study. Quantitative analysis of neurodegeneration biomarkers was performed through immunological tests. Oligoclonal bands were detected by isoelectric focusing on agarose gel, whereas the concentrations of immunoglobulins and albumin were measured using nephelometry. Results: Our studies showed that NfL, RTN4, and tau protein enabled the differentiation of MS patients from the control group. Additionally, the baseline CSF NfL levels positively correlated with the tau and MRI results, whereas the RTN4 concentrations were associated with the immunoglobulin quotients. The AUC for NfL was the highest among the tested proteins, although the DeLong test of the ROC curves showed no significant difference between the AUCs for NfL and RTN4. Conclusion: The CSF NfL, RTN-4, and tau levels at the time of diagnosis could be potential diagnostic markers of multiple sclerosis, although NfL seems to have the best clinical value. Multiple sclerosis (MS) is a chronic neurological disease characterized by focal inflammatory lesions in the gray and white matter, as well as progressive, diffuse neurodegeneration in the entire brain. The evidence from preclinical studies has shown that neurodegeneration is already apparent in MRI examination at the time of diagnosis . MoreoveOne neurodegeneration hallmark in MS patients is axonal damage and loss, which leads to brain and cervical atrophy, cortical thickening, disability, and cognitive dysfunction ,7,8,9. An = 37) and subjects with other noninflammatory neurological diseases as a control group (n = 22) , and all the patients signed an informed consent form before any procedure. The study population consisted of 59 subjects with neurological disorders , including patients with MS ((n = 22) . Study pIn this investigation, the diagnosis of MS was based on the MacDonald criteria (2017) . The subAll MS patients included in the study were diagnosed with relapsing–remitting MS. They had a history of at least one clinical attack, and there was no evidence of dissemination in time or atrophy according to magnetic resonance imaging (MRI). A radiologist examined all the MRI scan data of the enrolled patients. Patients were followed up clinically and radiologically according to routine clinical practice. The presence of oligoclonal bands (OCBs) in the cerebrospinal fluid and serum was also assessed. The disease severity in patients with MS was assessed using the Expanded Disability Status Scale (EDSS). All evaluations were rated between 1 and 2 points, indicating an early stage of the disease. Since the CSF was collected during the diagnostic process, none of the patients were on disease-modifying therapy during lumbar puncture. Matched CSF and serum samples from the patients were collected only once during disease diagnosis. Moreover, 23 out of 37 patients had OCBs in the CSF but not in serum (pattern type 2), whereas five had OCBs in the CSF and serum, with additional OCBs in the CSF (pattern type 3).The control group consisting of 22 neurological patients  was carefully selected on the basis of neurological, neuropsychological, and laboratory (blood and CSF) examinations, which allowed excluding the organic background of symptoms. Additionally, the concentrations of albumin and immunoglobulins in the CSF and serum were simultaneously assessed to calculate their quotients. In the control group, 16 patients had no bands in the CSF and serum (pattern type 1), whereas five had the same pattern of OCBs in the CSF and serum (pattern type 4).g, divided into 0.5 mL aliquots, and immediately frozen at −80 °C until analysis. After lumbar puncture, patient demographic and clinical data were stored. The tested biomarkers measured in the current study were assessed using the same batch of reagents in the Department of Neurodegeneration Diagnostics, Medical University of Białystok, Poland. CSF samples were run in duplicate. The quantitative analysis of tested proteins was prepared following each protein manufacturer’s instructions. CSF RTN-4 was measured using a human RTN4 (Reticulon 4) ELISA kit , CSF NfL was analyzed using a sandwich ELISA NF-light UmanDiagnostic kit , and CSF tau was determined using an Innotest Fujirebio kit  following the manufacturer’s instructions.CSF samples were drawn by lumbar puncture (LP), performed in the L3/L4 or L4/L5 interspace at the time of diagnosis. CSF was collected in sterile polypropylene tubes, centrifuged for 10 min at 2000× Alb, QIgG, QIgA, and QIgM) to assess the integrity of the blood–CSF barrier and intrathecal immunoglobulin production. Furthermore, the oligoclonal bands were assessed using isoelectric focusing on agarose gel  by immunoblotting, following the manufacturer’s protocol. Patients were classified as OCB-positive if they had more than one CSF OCB, but considered OCB-negative if they had 0–1 CSF OCBs.CSF and serum concentrations of albumin and immunoglobulins were measured using a nephelometer . Albumin and immunoglobulin quotients were calculated  and neurodegeneration biomarkers were different in patients with MS and controls. Additionally, receiver operating characteristic (ROC) curve analysis was performed to determine the diagnostic usefulness of the tested proteins as potential biomarkers in MS. DeLong’s test was used to determine whether the AUCs of tested biomarkers were statistically significantly different.Statistical analysis was conducted using the PMCMRplus package in the statistical software R . A p-valThe characteristics of participants included in our investigation are reported in p < 0.001) and IgM (p = 0.002), as well as QIgG (p < 0.001), QIgM (p < 0.001), and QIgA (p = 0.05), were significantly higher in relapsing–remitting MS as compared with controls. A comparison of the CSF levels of tested biomarkers is presented in p < 0.001). Moreover, the ratios of NfL to RTN4 and tau were calculated. Only the NFL/RTN4 ratio was significantly different between the MS and control groups (p = 0.03).r = 0.45; p = 0.01)  were observed. Additionally, the CSF RTN4 level was positively associated with log QIgA  and log QIgM . There were no correlations with other variables such as age, sex, and oligoclonal bands.In the group of all the MS patients, significant correlations between log NfL and tau ( = 0.01)  and demyp < 0.001), sensitivity of 0.78, and specificity and 0.77, compared to CSF RTN4 and tau. AUC values for the CSF levels of RTN4 and tau were comparable .p = 0.008). Furthermore, the DeLong test of the ROC curves showed no significant difference between the AUCs for RTN4 and tau  or the AUCs for NfL and RTN4  despite the highest value of the area under the ROC curve for NfL  was not found.Other studies have also demonstrated a correlation between NfL levels and radiological findings, in agreement with our findings. We observed a significant association between CSF NfL levels and MRI demyelinating lesions in treatment-naïve RRMS patients, which may indicate a worse prognosis in this group of patients. A similar correlation was not observed for tau and RTN4 levels. A relationship of elevated levels of NfL with brain atrophy and spinal cord volume loss has been described in the literature ,22,32,33Unexpectedly, even though RTN4 plays a role in two major pathomechanisms underlying MS , it seemingly has less diagnostic significance in the disease than NfL, particularly at disease onset. Considering the in vivo studies of Nikic et al. , which d+ [We found a positive correlation between immunoglobulins quotients and RTN4A concentrations, supporting the theory that RTN4A is more strongly involved in the central nervous system (CNS) inflammatory state and increases due to the potentiated immunological response after demyelination. Abundant overexpression of receptor NgR1 was demonstrated in the microglia, which plays a crucial role in CNS homeostasis, the transmission of axon potentials, and the mediation of immune responses . In vitr+ ,43. Addi+ ,45. In lIn the present study, we also evaluated the diagnostic performance of CSF NfL, RTN4, and tau levels as clinical biomarkers enabling the discrimination of MS using ROC curves. The AUC values discriminating between controls and MS patients were higher for NfL than other neurodegenerative biomarkers (RTN4 and tau). Although further analysis revealed that this difference between NfL and RTN4 was insignificant, both biomarkers could be valuable in multiple sclerosis. Further studies comparing different neurodegenerative biomarkers are necessary, particularly in combination with imaging methods.Our study has some limitations within which the findings need to be interpreted carefully. Firstly, in this study, we investigated a small population; however, we chose samples of patients with MS diagnosed in the same center and restored in the same facilities over similar periods. Additionally, collection procedures were standardized to avoid the influence of different conditions on the tested biomarker levels in our study cohort. Moreover, considering the promising results for NfL, our study will be continued, and other neurodegeneration biomarkers in the clinical practice of neurological disease will be explored and compared. Lastly, despite RTN4 reflecting major pathologies underlying MS , the combined analysis of NfL and RTN4 did not improve the accuracy of early diagnosis of MS patients. Therefore, additional empirical research is required to provide adequate answers regarding the early diagnosis of MS.Neurodegeneration is a crucial problem underlying MS; thus, more studies are needed in this area. The best-known marker of neurodegeneration is NfL, although its specificity is limited. Therefore, further research should be carried out to find a panel of biomarkers enabling stratification of patients and individualized selection of effective therapy. Even though selected neurodegeneration indicators in physiological conditions share the same functions, their role in pathological conditions appears to differ. Our research suggests that CSF levels of NfL, RTN4, and tau alone could potentially be applied in the early diagnosis of MS. However, combined analysis of the tested proteins did not improve the accuracy of early diagnosis. Given the importance of this medical problem in MS, further research is still needed.


Preclinical evidence suggests a cross-regulation between adenosine A2A and dopamine D2 receptors in this region and that it is linked to the sensitization of the dopamine system. However, the relationship between A2A receptor availability and schizophrenia has not been directly examined in vivo in patients with this disorder. To investigate, using positron emission tomography (PET), the availability of A2A receptors in patients diagnosed with schizophrenia in comparison to matched healthy controls. A2A receptor availability was measured using the PET tracer [11C]SCH442416. Twelve male patients with chronic schizophrenia were compared to 13 matched healthy subjects. All patients were medicated with antipsychotics and none presented with any motor or extrapyramidal symptoms. Binding potential (BPND), a ratio measure between specific and non-specific tracer uptake, were compared between the groups for the caudate, putamen, accumbens and globus pallidum. There was no differences between A2A receptor binding potential (BPND) of schizophrenia patients in the caudate (p = 0.16), putamen (p = 0.86), accumbens (p = 0.44) and globus pallidum (p = 0.09) to that of matched healthy subjects. There was also no significant correlation between [11C]SCH442416 binding and severity of psychotic symptoms (p = 0.2 to 0.82) or antipsychotic dosage (p = 0.13 to 0.34). By showing that A2A receptor availability in medicated patients with chronic male schizophrenia is not different than in healthy controls, this study does not support the primary role of this receptor in the pathogenesis of schizophrenia.Adenosine A Schizophrenia is a chronic mental health disorder that affects approximately 1% of the population. Although the precise neurobiological mechanisms underlying schizophrenia are still unknown, several lines of evidence show that striatal abnormalities are a core component of the pathophysiology of this disorder  in the A2A receptor (A2AR) in the basal ganglia of patients with this disorder when compared to healthy controls (Kurumaji and Toru n = 12) were recruited from the South London and Maudsley Foundation NHS Trust, and were compared to matched healthy controls (n = 13). All participants gave written informed consent to participate after receiving a full description of the study. The study protocol was approved by the National Research Ethics Service (NRES) and permission to administer radioactive substances was granted by the Administration of Radioactive Substances Advisory Committee (ARSAC), UK. All subjects had a physical, psychiatric and neurological examination. Diagnosis was performed by reviewing patients medical notes and the use of the Structured Clinical Interview for DSM-5 (SCID)  = 2300 ms, time echo (TE) = 2.98 ms, flip angle of 9°, time to inversion (TI) = 900 ms, matrix = 240 × 256) for co-registration with the PET images; fast GM T1 inversion recovery  (REF) and fluid and WM suppression  sequences for improving delineation of subcortical brain regions. All sequences used a 1-mm11C]SCH442416. Subjects were positioned supine with their transaxial planes parallel to the line intersecting the anterior–posterior commissure line. Head position was maintained with the help of individualised foam holders, monitored by video and subjects were repositioned if movement was detected. Subjects were in a resting state with low light. Intrascan notes for participant’s movement were acquired during scanning. Dynamic emission data were acquired continuously for 90 min following the injection of [11C]SCH442416. The dynamic images were reconstructed into 26 frames , using a filtered back projection algorithm (direct inversion Fourier transform) with a 128 × 128 matrix, zoom of 2.6, producing images with isotropic voxel size of 2 × 2 × 2 mm3 and a 5-mm isotropic Gaussian filter. Corrections were applied for attenuation, randoms and scatter. Arterial plasma samples were collected during the scan to enable metabolite corrected input functions to be determined.PET scans were acquired on a Siemens Biograph Hi-Rez 6 PET-CT scanner  following the injection of an intravenous bolus of SCH442416 BPND in the primary regions of interest ) and putamen ) or in secondary regions of interest ) and nucleus accumbens ) when compared to healthy controls , putamen , globus pallidus (r = 0.21 p = 0.28), nucleus accumbens ) or with severity of total psychotic symptoms , putamen , globus pallidus , nucleus accumbens ). There was also no significant correlation between [11C]SCH442416 BPND and severity of positive , putamen , globus pallidus , nucleus accumbens ), negative , putamen , globus pallidus , nucleus accumbens ) or general psychotic symptoms , putamen , globus pallidus , nucleus accumbens ). Finally, there was also no significant correlation between [11C]SCH442416 BPND and duration of illness , putamen , globus pallidus , nucleus accumbens ).There were no differences in the injected dose, injected mass and specific activity between patients with schizophrenia and healthy controls Table . PatientTo our knowledge, this is the first study to assess the A2A receptor in vivo in patients with schizophrenia. We found no difference in striatal A2A binding potential between patients diagnosed with schizophrenia and healthy controls in any of the basal ganglia regions examined.11C]-TMSX, but these showed several problems such as a variable affinity at striatal and non-striatal A2AR, and hence non-xanthine tracers are preferred . Another approach has been to use spectral analysis to identify the different components of the signal (Subrata et al. ND data from the study using the spectral analysis approach are lower than we obtained using our approach. However, it should be recognised that both our approach and spectral analysis approach for this tracer require further validation. Alternatively, future studies could make use of more recent radiotracers for imaging A2A with better kinetics properties, such as [18F]-MNI-444 (Barret et al. Several potential limitations affecting the interpretation of the results need to be considered. First, the challenges for quantifying A2AR with current in vivo ligands. The first measures of A2A receptors in healthy controls were made using xanthine tracers such as [H]-CGS21680 (Kurumaji and Toru Our results do not support the existing post-mortem imaging findings in schizophrenia. While Villar-Menéndez showed a reduction in A2AR binding, Kurmanji and Toru found an increase in approximately 35 to 55% in A2A receptor specific binding in the caudate and putamen of post-mortem brains, as measured by [3In conclusion, we showed for the first time in vivo that there is no significant differences in A2A receptor expression between patients and healthy controls, not supporting the adenosine hypofunction hypothesis of schizophrenia.


To investigate the impact and predictors of tympanostomy tube (TT) extrusion.A retrospective study on 258 ears underwent TT insertion during 2016-2018 at King Abdullah Specialized Children’s Hospital, Riyadh, Saudi Arabia. Patients were followed for 36-48 months postoperatively to detect the recurrence rate. The sample was divided into 2 groups based on extrusion time and were compared to determine the predictors of TT extrusion.Otitis media with effusion (OME) recurrence after TT insertion was detected in 28.7%. A shorter TT extrusion time was associated with a higher recurrence (p=0.002). Small TTs increased the probability of early TT extrusion .More than one-fourth of the patients who underwent TT insertion for OME developed recurrence. Tympanostomy tube extrusion earlier than 12 months was associated with a higher recurrence rate. Small TTs increased the probability of early TT extrusion. Otitis media with effusion (OME) is the most common cause of pediatric hearing loss. It is defined as the accumulation of fluids behind an intact tympanic membrane, with the absence of the signs and symptoms of acute infection. Most OME episodes resolve spontaneously without intervention. However, around 10% of cases require myringotomy with tympanostomy tube (TT) insertion due to failure of conservative treatment. Tympanostomy tube insertion is one of the most commonly carried out procedures in children with OME, aiming to release the middle ear effusion (MEE) and ventilate the middle ear. The complications of this procedure include otorrhea, myringosclerosis, atrophy of the tympanic membrane, formation of granulation tissue, and early extrusion of TT. Tympanostomy tube extrusion time is an essential factor that influences the recurrence rate of OME, where a longer extrusion time is associated with a lower recurrence rate. Variable factors were reported in the literature to affect the TT extrusion. These include patient-related factors (such as age and gender), surgery-related factors , and disease-related factors . This study investigates the TT extrusion time with its impact and predictors to provide more input to the current literature.Otitis media with effusion has a considerable recurrence rate post myringotomy with TT insertion, ranging between 19.9-53.2%.A retrospective cohort study was carried out at King Abdullah Specialized Children’s Hospital in Riyadh, Saudi Arabia. All pediatric patients (aged 14-year-old or younger) who were diagnosed with OME and underwent myringotomy with TT insertion between 2016-2018 were included in the study. The patients who have head and neck anomalies were excluded from the study.The diagnosis of OME existed based on clinical and tympanometric findings. The patients with persistent OME for more than 3 months with a documented hearing loss underwent myringotomy with TT insertion. Shah-type fluoroplastics TTs of 2 sizes were used; regular TTs (1.14 cm inner diameter with 1.58 cm interflange distance) and small TTs (0.76 cm inner diameter with 1 cm interflange distance). Small tubes were used in patients with narrow or tortuous external auditory canals, while all the other patients received regular TTs. Patients were followed for 36-48 months postoperatively in 2-3 months intervals to detect the recurrence rate.The data collection sheet included baseline clinical and demographic characteristics , history of otolaryngology-related conditions (such as recurrent acute otitis media and adenoid hypertrophy), OME-related details , surgery-related details , concurrent surgery (such as adenoidectomy or tonsillectomy), and recurrence status. The data were extracted from medical charts and Hospital Health Information System.The data were analyzed using Statistical Package for the Social Sciences, version 25.0 . The categorical data were summarized and reported as frequencies and proportions, while the continuous variables were summarized and reported as means and standard deviations (SD). The recurrence rate was estimated for all study samples, and based on extrusion time in 3 subgroups . Then, the study sample was divided into 2 groups based on TT extrusion time (≤12 and >12 months). The 2 groups were compared using univariable and multivariable regression analysis to determine the predictors of TT extrusion time.The study included 258 ears from 144 patients. The mean age of the patients was 5.19±4.0 years old, with males dominating the females (62.5% versus 37.5%). Bilateral OME constituted 89.6% of the cases, and more than two-thirds of the patients had adenoid hypertrophy (70.1%). The proportions of patients who underwent adenoidectomy were 63.2% and tonsillectomy were 33.3%.p=0.002). Otitis media with effusion recurrence after myringotomy with TT insertion was detected in 74 (28.7%) ears. Tympanostomy tube lasted more than 12 months in more than half (53.1%) of the cases, with a mean extrusion time of 13.96±6.5 months . A higheThe tube size was the only significant predictor of early TT extrusion . Small T The insertion of TTs helps improve hearing function and patients’ overall quality of life. However, the recurrence rate following TT insertion is still high. The recurrence rate in our study is 29%, which falls in the range of reported rates in the literature. The reported predictors of OME recurrence following TT insertion included patient-related factors , surgery-related factors , and disease-related factors .The majority of OME cases resolve spontaneously within 3 months. Myringotomy with TT insertion is carried out for patients with persistent MEE despite watchful waiting, which is seen in around 10% of cases. The present study showed a higher recurrence rate in ears with a shorter TT extrusion time  are associated with a shorter extrusion time and higher recurrence rate but fewer overall complications. On the other hand, long-term TTs  have a longer extrusion time and lower recurrence rate but a higher rate of complications. These complications include otorrhea, persistent perforation, myringosclerosis, atrophy of the tympanic membrane, formation of granulation tissue, and cholesteatoma. Moreover, Kim et al showed that thermoplastic elastomer TTs have a longer extrusion time than silicone TTs.Multiple studies investigated the factors that influence the timing of TT extrusion, but the results are variable. The present study found that the size of TTs was the only predictor of TT extrusion time. Small TTs extruded prematurely more frequently compared to regular tubes. The finding correlated with Lin et al This relationship could be explained by the decreasing incidence of OME and the growth of Eustachian tube (ET) as patients get older. However, our study and others showed no significant association between age and extrusion time. Ahn et al demonstrated that concurrent adenoidectomy decreases the need for revision TT insertion as it can relieve ET’s mechanical obstruction and reduce the pathogens load in the nasopharynx. However, our study showed no significant association between adenoid size and adenoidectomy with TT extrusion time, supporting the findings of Song et al.Adenoid hypertrophy and patient’s age were reported to influence the extrusion time in the literature. Younger children were more likely to have early extrusion than older patients in multiple studies. The MEE was divided into thick, serous, and negative intraoperative effusion in the present study. Our study showed no significant association between MEE characteristics and TT extrusion time, supporting the conclusion of Gibb et al and Maw et al. Moreover, there was no significant association between preoperative tympanometry findings and TT extrusion in the present study. Song et al and Leopold et al found that revision TT insertion was associated with a longer extrusion time. This association could be explained by the postoperative changes in the tympanic membrane resulting from the surgery. Previous surgery could result in scarring and atrophy of the tympanic membrane due to losing the middle fibrous layer during the healing process, affecting the tympanic membrane’s elasticity. Our study did not examine this relationship as it was confined to primary surgeries.The viscosity of MEE, which varies from thick mucus fluid to serous secretion, was reported to affect the recurrence rate of OME.Our sample was collected from one tertiary healthcare center and was confined to one shape and material of TTs. This limitation can affect the generalizability of the study. However, the present study investigated the predictors of TT extrusion time in a large sample with a long-term follow-up period. Additional prospective randomized clinical trials with a larger sample are needed to validate predictors of TT extrusion time.In conclusion, more than one-fourth of the patients who underwent TT insertion for OME developed recurrence within 3-4 years. Most TTs lasted in the tympanic membrane for more than 12 months; however, tube extrusion earlier than 12 months was associated with a higher recurrence rate. Small TTs increased the probability of early TT extrusion.