BACKGROUND: T cell dysfunction, which includes exhaustion, anergy, and senescence, is a distinct T cell differentiation state that occurs after antigen exposure. Although T cell dysfunction has been a cornerstone of cancer immunotherapy, its potential in transplant research, while not yet as extensively explored, is attracting growing interest. Interferon regulatory factor 4 (IRF4) has been shown to play a pivotal role in inducing T cell dysfunction. METHODS: A novel ultra-low-dose combination of Trametinib and Rapamycin, targeting IRF4 inhibition, was employed to investigate T cell proliferation, apoptosis, cytokine secretion, expression of T-cell dysfunction-associated molecules, effects of mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways, and allograft survival in both in vitro and BALB/c to C57BL/6 mouse cardiac transplantation models. RESULTS: In vitro , blockade of IRF4 in T cells effectively inhibited T cell proliferation, increased apoptosis, and significantly upregulated the expression of programmed cell death protein 1 (PD-1), Helios, CD160, and cytotoxic T lymphocyte-associated antigen (CTLA-4), markers of T cell dysfunction. Furthermore, it suppressed the secretion of pro-inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17. Combining ultra-low-dose Trametinib (0.1 mg·kg -1 ·day -1 ) and Rapamycin (0.1 mg·kg -1 ·day -1 ) demonstrably extended graft survival, with 4 out of 5 mice exceeding 100 days post-transplantation. Moreover, analysis of grafts at day 7 confirmed sustained IFN regulatory factor 4 (IRF4) inhibition, enhanced PD-1 expression, and suppressed IFN-γ secretion, reinforcing the in vivo efficacy of this IRF4-targeting approach. The combination of Trametinib and Rapamycin synergistically inhibited the MAPK and mTOR signaling network, leading to a more pronounced suppression of IRF4 expression. CONCLUSIONS Targeting IRF4, a key regulator of T cell dysfunction, presents a promising avenue for inducing transplant immune tolerance. In this study, we demonstrate that a novel ultra-low-dose combination of Trametinib and Rapamycin synergistically suppresses the MAPK and mTOR signaling network, leading to profound IRF4 inhibition, promoting allograft acceptance, and offering a potential new therapeutic strategy for improved transplant outcomes. However, further research is necessary to elucidate the underlying pharmacological mechanisms and facilitate translation to clinical practice.
Animals ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Interferon Regulatory Factors/metabolism* ; Heart Transplantation/methods* ; T-Lymphocytes/immunology* ; Sirolimus/therapeutic use* ; Pyridones/therapeutic use* ; Graft Survival/drug effects* ; Pyrimidinones/therapeutic use* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Male ; Signal Transduction/drug effects*

Objective:To establish the reference intervals of reticulocyte parameters for children in the Beijing area.Methods:1 766(856 males and 910 females) healthy children aged from 1 to 18 years old in the Beijing area were studied, the infant group (≥1-3 years old) included 146 participants; the preschool group (>3-6 years old) had 449 participants; the school age group (>6-13 years old) had 646 participants and the adolescent group (>13-18 years old) had 525 participants. Seven parameters were measured in venous blood by SySmex XN-20 (A1) automatic blood cell analyzer, which included reticulocyte percentage (RET%), absolute reticulocyte count (RET#), low fluorescence reticulocyte (LFR), medium fluorescence reticulocyte (MFR), high fluorescence reticulocyte (HFR), immature reticulocyte fraction (IRF) and reticulocyte hemoglobin equivalent (RET-He). After the test results were collected, the reference intervals were established according to the percentile ( P2.5, P97.5). As the reference intervals were established, venous blood samples from 109 healthy children in Beijing were collected to verify the reference intervals according to WS/T 402-2012 "Define and determine the reference intervals in clinical laboratory". Results:The reference interval of 7 Reticulocyte parameters for children aged 1-18 years without age and sex grouping,reference interval (RET%): 0.74%-2.06%; absolute reticulocyte count (RET#): (34.5-101.5)×10 9/L; low fluorescence reticulocyte (LFR): 86.6%-96.5%; medium fluorescence reticulocyte (MFR): 3.2%-11.5%; high fluorescence reticulocyte (HFR): 0%-2.1%; immature reticulocyte fraction (IRF): 3.5%-13.4%; reticulocyte hemoglobin equivalent (RET-He): 28.5-34.2 pg. Over 90% test results of samples from the 109 children ranged within the reference ranges. Conclusion:This study established convincible reference intervals of seven reticulocyte parameters for healthy children aged from 1 to 18 in the Beijing area was established, and reference interval verification passed.

Objective:The mouse kidney transplantation model presents challenges in terms of surgical difficulty and low success rate,making it difficult to master.This study aims to provide a crucial model for transplantation immunology research by modifying and developing novel techniques for mouse kidney transplantation. Methods:A total of 57 pairs of mice were used to establish and compare the modified and innovative surgical techniques for mouse kidney transplantation.Three different surgical models were established,including the abdominal suture technique for orthotopic kidney transplantation,the abdominal cuff technique for orthotopic kidney transplantation,and the cervical cuff technique for ectopic kidney transplantation.BALB/c or C57BL/6 male mice,aged 8 to 12 weeks and weighed 20 to 25 g with specified pathogen free-grade were served as the donor mice or the recipient mice.The surgical technique characteristics,key surgical times,complications,and pathological examination in the early postoperative period were summarized and compared. Results:Three different surgical models of mouse kidney transplantation were successfully established.The comparison of warm ischemic time for the 3 groups of mice showed no statistical significance(P=0.510 4).The abdominal suture group had the shortest total operation time of the donor compared with the abdominal cuff group and the cervical cuff group[(18.3±3.6)min vs(26.2±4.7)min and(22.8±2.5)min;both P<0.000 1].There was a significant difference in cold ischemia time among the 3 groups(all P<0.000 1),with(60.8±4.1)min in the cervical cuff group,(43.3±5.0)min in the abdominal suture group,and(88.8±6.7)min in the abdominal cuff group.Due to different anastomosis methods,the cervical cuff group had the shortest time[(17.6±2.7)min],whereas the abdominal cuff group had the longest time[(38.8±5.4)min].The total operation time for the recipients showed significant differences(P<0.000 1),with the abdominal suture group having the shortest time[(44.0±6.9)min],followed by the cervical cuff group[(64.1±5.2)min],and the abdominal cuff group[(80.0±6.0)min]being the longest.In the 32 mice of the abdominal suture group,there were 6 with intraoperative bleeding,including 1 arterial intimal injury bleeding and 5 with bleeding after vessel opening.Six mice had ureteral complications,including ureteral bladder anastomotic stenosis,necrosis,and renal pelvis dilation.Two mice had postoperative abdominal infections.In the abdominal cuff group,there was no intraoperative bleeding,but 6 mice showed mild arterial stenosis and 5 showed venous stenosis,4 arterial injury,4 arterial thrombosis,and 2 ureteral complications.No postoperative infections occurred in the mice.In the cervical cuff group,no intraoperative bleeding,arterial intimal injury,arterial/venous stenosis,or thrombosis were found in 13 mice.Five mice had ureteral complications,including ureteral necrosis and infection,which were the main complications in the cervical cuff group.The renal function in mice of the 3 groups remained stable 7 days after surgery.Hematoxylin and eosin staining and periodic acid-Schiff staining showed no significant differences in terms of acute rejection among the 3 surgical methods(all P>0.05). Conclusion:All 3 surgical methods are able to successfully establish mouse kidney transplantation models,with no significant differences observed in the short-term graft survival and acute rejection.The modified abdominal suture technique and abdominal cuff technique have their respective advantages in research applications.The novel cervical cuff technique for ectopic kidney transplantation model is relatively simple to be prepared and causes less trauma to the mice,providing more options for studies involving xenotransplantation,secondary transplantation,and local lymphatic drainage.However,the difficulty in harvesting the donor kidney and the high incidence of ureteral infections need further validation in long-term survival.This study holds important reference value for choosing the type of mouse kidney transplantation model for different research needs.

Objective:To investigate and summarize pediatric patients with severe Mycoplasma pneumoniae pneumonia (MPP) presenting with varied clinical and chest imaging features in order to guide the individualized treatment. Methods:This was a retrospective cohort study. Medical records of clinical, imaging and laboratory data of 505 patients with MPP who were admitted to the Department Ⅱ of Respirology Center, Beijing Children′s Hospital, Capital Medical University from January 2016 to October 2023 and met the enrollment criteria were included. They were divided into severe group and non-severe group according to whether lower airway obliterans was developed. The clinical and chest imaging features of the two groups were analyzed. Those severe cases with single lobe ≥2/3 consolidation (lobar consolidation) were further divided into subtype lung-necrosis and subtype non-lung-necrosis based on whether lung necrosis was developed. Comparison on the clinical manifestations, bronchoscopic findings, whole blood C-reactive protein (CRP) and other inflammatory indicators between the two subtypes was performed. Comparisons between two groups were achieved using independent-sample t-test, nonparametric test or chi-square test. Univariate receiver operating characteristic (ROC) curve analyses were performed on the indicators such as CRP of the two subtypes. Results:Of the 505 cases, 254 were male and 251 were female. The age of the onset was (8.2±2.9) years. There were 233 severe cases, among whom 206 were with lobar consolidation and 27 with diffuse bronchiolitis. The other 272 belonged to non-severe cases, with patchy, cloudy infiltrations or single lobe <2/3 uneven consolidation or localized bronchiolitis. Of the 206 cases (88.4%) severe cases with lobar consolidation, 88 harbored subtype lung-necrosis and 118 harbored subtype non-lung-necrosis. All 206 cases (100.0%) presented with persistent high fever, among whom 203 cases (98.5%) presented with inflammatory secretion obstruction and plastic bronchitis under bronchoscopy. Of those 88 cases with subtype lung-necrosis, there were 42 cases (47.7%) with dyspnea and 39 cases (44.3%) with moderate to massive amount of pleural effusion. There were 35 cases (39.8%) diagnosed with lung embolism during the disease course, of which other 34 cases (38.6%) were highly suspected. Extensive airway mucosal necrosis was observed in 46 cases (52.3%), and the level of their whole blood CRP was significantly higher than that of subtype non-lung-necrosis (131.5 (91.0, 180.0) vs. 25.5 (12.0, 43.1) mg/L, U=334.00, P<0.001). They were regarded as subtype "lung consolidation-atelectasis-necrosis". Of those 118 cases with subtype non-lung-necrosis, 27 cases (22.9%) presented with dyspnea and none were with moderate to massive amount of pleural effusion. Sixty-five cases (55.1%) presented with plastic bronchitis and localized airway mucosal necrosis was observed in 32 cases (27.1%). They were deemed as subtype "lung consolidation-atelectasis". ROC curve analyses revealed that whole blood CRP of 67.5 mg/L on the 6-10 th day of disease course exhibited a sensitivity of 0.96, a specificity of 0.89, and an area under the curve of 0.97 for distinguishing between these two subtypes among those with lobar consolidation. Conclusions:Pediatric patients with severe MPP present with lobar consolidation or diffuse bronchiolitis on chest imaging. Those with lobar consolidation harbor 2 subtypes as "lung consolidation-atelectasis-necrosis" and "lung consolidation-atelectasis". Whole blood CRP of 67.5 mg/L can be applied as an early discriminating indicator to discriminate between these two subtypes.

Infant, Newborn ; Humans ; Retrospective Studies ; Case-Control Studies ; Tracheotomy ; Respiration, Artificial

Objective:To study the outcomes and post-discharge follow-up of neonatal tracheotomy in neonatal intensive care unit(NICU).Methods:This study included patients who were admitted to NICU in Beijing Children′s Hospital from January, 2016 to August, 2021, and less than 28 days or 44 weeks(corrected age)on admission, and required tracheotomy.The patients were divided into tracheotomy group and the non-tracheotomy group (the parents signed to refuse the tracheotomy) according to whether perform tracheotomy.Demographic data, general hospitalization information, diagnosis, indications for tracheotomy, follow-up outcomes at 3/6/12 months of age after discharge of patients were collected and analyzed.Results:Totally 26 patients were included in this study, 14 cases in tracheotomy group and 12 cases in non-tracheotomy group.The average gestational age was(37.7±3.80)weeks and(38.99±1.83)weeks, and birth weight was(2 823.57±948.89)g and (3 320.83±378.76)g, respectively.There were no significant differences in sex, gestational age, birth weight, age on admission, weight on admission, age at diagnosis, ratio of endotracheal intubation for respiratory support on admission between two groups( P>0.05). The commonest indications of tracheotomy group were bilateral vocal cord paralysis(50.0%) and congenital anomaly/defect of throat/larynx(21.4%), and the commonest indications of non-tracheotomy group were bilateral vocal cord paralysis(50.0%) and vocal cord/subglottic mass(25.0%), and there was no significant difference between two groups( P>0.05). The rate of discharge-against-medical order of tracheotomy and non-tracheotomy group was 7.14% and 66.67%( P=0.003), respectively.The total follow-up rate of tracheotomy and non-tracheotomy group was 88.9% and 38.9%, while the follow-up rates at 3 months, 6 months, and 12 months were 100.0% vs. 50.0%, 83.3% vs. 41.7%, and 81.8% vs. 25.0%, respectively, whose differences were statistically significant(all P<0.05). In the 14 cases of tracheotomy group, 3 cases died, 4 cases successfully removed the tracheal cannula, 5 cases did not remove the tracheal cannula, and 2 cases were lost. Conclusion:Bilateral vocal cord paralysis is the commonest indication of neonatal tracheotomy.Parents′ compliance in the tracheotomy group is significantly higher than that in non-tracheotomy group.To give caring skill training for parents of neonates with tracheotomy before discharge is beneficial for improving the overall prognosis of children.

Accurate and applicable reference intervals can provide important information for disease diagnosis, efficacy evaluation, and health monitoring. Age-dependent trends exist for many clinical laboratory indicators, and the interpretation of such indicators should consider the effect of age carefully. However, age-specific reference intervals have certain limitations in clinical application. Continuous reference intervals can not only help accurate interpretation of laboratory test results, but also provide a baseline value for dynamic monitoring of their changing trends. Based on the researches on pediatric reference intervals from our team, this paper introduces different algorithms for establishing continuous reference intervals, and their applicable conditions and implementation steps. The aim of this paper is to provide methodological guidance for the establishment of continuous reference intervals, so as to improve the quality of laboratory reports and the accuracy of clinical diagnosis in China.

Objective:To analyze the public demands for information about congenital birth defects in “Baidu zhidao” based on word frequency retrieval.Methods:Based on discussion between obstetrics and gynecology experts and epidemiological experts, the key words related to congenital birth defects were determined and the search strategy was formulated. Python 2.7 was used for web crawler search. Questions related to congenital birth defects were obtained on the “Baidu zhidao” platform, and then the R 4.0.2 software was used to process the data, complete the semantic analysis of keywords and statistical analysis of word frequency, and draw word cloud graph and polar chart to describe the key results.Results:A total of 16668 non-repetitive questions were retrieved from “Baidu zhidao” platform, and the frequency of semantic words was 15 371. Among them, 35.02% were the names and symptoms of congenital birth defects. In addition, the frequency of congenital heart disease was the highest (26.09%). The results of subject analysis of key words of birth defects showed that the average word frequency of diagnosis and treatment semantic words (49.55) was significantly higher than that of etiology and prevention semantic words (12.47). In addition, the key words of examination, cause, treatment, development and heredity were more frequently used in the semantic words related to the seven types of systemic malformations.Conclusion:The public in China has a high demand for information on congenital birth defect related diseases, and their causes, prevention and treatment, especially congenital heart disease.

Real world data (RWD) refers to the data generated in routine clinical practices, daily life, and real work environment and has been widely used in clinical or public health research. Still, issues related to the quality of RWD, such as incompleteness, inconsistency, and inaccuracy, would affect the validity of real-world research. To overcome the challenges due to the lack of standardization of real world source data, case report form based on clinical data interchange standards consortium (CDISC-CRF) on certain diseases was developed to promote the ecology construction of RWD based on the data standards set by the CDISC which has been widely used. Firstly, we described how to apply data standards to make up the gap between RWD and real world evidence. Then, the process was designed to build RWD ecology based on CDISC-CRF, in which the development technology of CDISC-CRF form is mainly introduced. Finally, the application prospect and significance of building real-world data based on disease-specific CDISC-CRF are described. It is believed that the present paper can provide a new idea for promoting the ecology construction of RWD in China.

Objective:To evaluate the heterogeneity in pediatric ETV6-RUNX1 acute lymphoblastic leukemia (ALL) by gene expression profile and to study clinical characteristics in different clusters.Methods:An improved advanced fragment analysis (iAFA) technique was developed to detect 57 marker genes in 264 pediatric ALL patients treated in Beijing Children’s Hospital from August 2016 to June 2019. The 56 ALL patients with ETV6-RUNX1 positive were evaluated by clinical characteristics in gene expression profile, immunophenotype and early response of chemotherapy in different clusters.Results:The 56 ETV6-RUNX1-positive patients were clustered into 2 groups of E/R-1 (45, 80.4%) and E/R-2 (11, 19.6%) . Spearman coefficient was 0.788 and 0.901 in E/R-2 and E/R-1, respectively. The median of initial platelet counts was 104 (27-644) and 50 (8-390) ( P<0.01) in E/R-2 and E/R-1, respectively. The median of proportion of initial bone marrow immature cells was 0.830 (0.270-0.975) and 0.935 (0.445-0.990) ( P<0.05) in E/R-2 and E/R-1, respectively. The most specific immunophenotype at initial diagnosis, CD22 +CD34 +CD20 -CD117 -CD56 -, mainly gathered in E/R-2 ( P<0.001) . Patients negative of minimal residual disease detected by flow cytometry (MRD-FCM) at day 33 were 5 (55.6%) and 32 (88.9%) in E/R-2 and E/R-1, respectively. There was no significant difference in the original analysis ( P=0.064) but difference in sensitivity analysis ( P=0.035) . Nevertheless, patients negative of MRD detected by polymerase chain reaction (MRD-PCR) at day 33 were 7 (77.8%) and 36 (100%) in E/R-2 and E/R-1, respectively, with significant difference ( P=0.047) . Conclusion:Gene expression profile shows heterogeneous in ETV6-RUNX1 ALL, and the E/R-2 profile indicates that these patients may have a less tendency to thrombocytopenia at the initial diagnosis but have poorer response to induction chemotherapy and may influence further outcome.