[Objective] To explore the feasibility of using whole blood as a source of NK cells for allogeneic CAR NK cell therapy and activated NK cell reinfusion therapy, and initially construct a technical system for the separation and purification of NK cells from whole blood. [Methods] All peripheral blood mononuclear cells (PBMCs) were enriched from 400 mL of whole blood by manual separation and machine separation, respectively. The erythrocyte loss rate, PBMCs number, NK cell purity of the two methods were compared. NK cells were sorted from PBMCs by three separation and enrichment methods as immunomagnetic bead negative selection method, platelet lysate culture expansion and PERCOLL density gradient separation method, and the purity and yield of NK cells, the activity of NK cells and the tumor-killing ability of the three separation and enrichment methods were compared. [Results] The proportion of NK cells in the lymphocyte population was higher in the manual separation method than in the machine separation method[(13.16±5.16)% vs (8.56±3.92)%, P<0.05]; the number PBMCs was lower in the manual separation method than in the machine separation method[(4.09±1.80)×10⁸vs (6.49±2.16)×10⁸, P<0.05], and there was no difference in the red blood cell loss between the two methods (P>0.05). The purity of NK cells isolated and enriched from PBMCs by manual separation method using immunomagnetic was (96.77±2.31)%; the yield was (56.27±10.47)%; the inhibition of tumor proliferation was (38.67±14.05)%; and the tumor killing rate was (19.90±8.05)%. The purity of NK cells isolated and enriched from PBMCs by manual separation method using platelet lysis culture expansion method was the highest at day 7, which was (54.84±15.80)%; the cell expansion multiple could reach 16.92±6.28 at day 7; the in vitro tumor killing rate of NK cells was (15.83±5.5)%; the tumor inhibition rate was (44.33±13.5)%; and there was no difference in the toxicity and activity of NK cells between the two methods (P>0.05). The purity of NK cells isolated and enriched by PERCOLL density gradient separation method was (15.83±5.82)%, and the yield was (14±6.25)%, which was significantly lower than the other two methods. [Conclusion] PBMCs isolated from whole blood by manual separation and NK cells enriched by negative selection with immunomagnetic beads have the potential to provide NK cell materials for CAR-NK cell therapy, and NK cells enriched by platelet lysate-conditioned medium have the potential to provide NK cells for large-scale NK cell activation reinfusion therapy.

Objective To establish a method for determination of pyriclobenzuron (PBU) residues in rice and paddy environments, and to determine the residual amounts and observe the degradation dynamics of PBU. Methods In July 2022, the paddies of Zhejiang Academy of Agricultural Sciences were selected as experimental fields, and were divided into the blank control group (no pesticide application), the 1-fold-concentration pesticide group (1 kg/667 m²), and the 5-fold-concentration pesticide group (5 kg/667 m²), with a 100 m² area in each group. At the early tillering stage of rice, 20% suspension of PBU sulfate was sprayed once in the 1-fold-concentration and 5-fold-concentration pesticide groups, and rice plants, paddy water and soil samples were collected 2 h, and 1, 2, 3, 5, 7, 11, 14, 21, 28, 35, 49 d and 63 d following spraying PBU, while rice straw, field soil, brown rice and rice husk samples were collected 98 d following spraying. PBU was extracted and purified in samples using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) pretreatment technique, and the PBU contents were determined in samples using ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The solvent standard working solution and matrix standard working solution were prepared. A linear regression equation was fitted between PBU concentration (x-axis) and peak area (y-axis), and the ratio of the slope (k) of the matrix standard curve to the slope (K) of the solvent standard curve was calculated to evaluate the matrix effect of PBU in samples. According to the Guidelines for Pesticide Residue Testing in Crops (NY/T 788—2018), the addition levels of PBU were set at 0.005, 0.050, 5.000, 1 000.000 mg/kg in rice plants, 0.005, 0.050, 2.000, 10.000 mg/kg in paddy water, 0.005, 0.050, 2.000 mg/kg in soil, and 0.005, 0.050, 5.000 mg/kg in brown rice and rice husks. The recovery and relative standard deviation (RSD) of PBU addition were calculated to evaluate the effectiveness of UPLC-MS/MS for determination of PBU contents. The first-order kinetic equation of PBU concentration was fitted in samples at different sampling time points to analyze the trends in PBU degradation in rice plants, paddy water, and soil, and the half-life of PBU was calculated in different samples. Results There was a good linear relationship between the mass concentration and peak area of PBU at concentrations of 0.000 1 to 0.020 0 mg/kg under solvent and matrix conditions (R² = 0.985 8 to 0.999 7, t = -0.47 to 1.62, all P values < 0.01). The matrix effects of PBU were 70.26%, 65.42% and 65.12% in rice plants, brown rice and rice husks, indicating a matrix-inhibitory effect, and the matrix effect was 87.06% in soils, indicating a weak matrix effect. The recovery of PBU addition was 77.61% to 100.12% in different samples, with RSD of 1.43% to 6.74%, and a limit of quantification (LOQ) of 0.005 mg/kg, and the addition recovery and RSD met the requirements of the Guidelines for Pesticide Residue Testing in Crops (NY/T 788—2018), validating the effectiveness of UPLC-MS/MS assay. Following spraying PBU at a dose of 1 kg/667 m², the half-life of PBU was 6.24 d in rice plants and 3.43 d in paddy water samples, respectively. The final residues of PBU were lower than the LOQ of 0.005 mg/kg in brown rice and rice husk samples 98 d following spraying PBU. Following spraying PBU at a dose of 5 kg/667 m², the half-life of PBU was 15.75 d in rice plants and 7.62 d in paddy water samples, respectively. The final residue of PBU was lower than the LOQ of 0.005 mg/kg in brown rice 98 d following spraying PBU, and the final residue of PBU was 0.049 mg/kg in rice husks. Conclusions A simple, and highly accurate and precise UPLC-MS/MS assay has been developed for determination of PBU residues in rice plants and paddy environments through extraction and purification of PBU from matrix samples using QuEChERS pretreatment. After spraying PBU in paddies, the concentration of PBU gradually decreases in rice plants and paddy water over time, and the final residual concentration is low.

Acute respiratory distress syndrome (ARDS) is a common respiratory emergency, but current clinical treatment remains at the level of symptomatic support and there is a lack of effective targeted treatment measures. Our previous study confirmed that inhalation of hydrogen gas can reduce the acute lung injury of ARDS, but the application of hydrogen has flammable and explosive safety concerns. Drinking hydrogen-rich liquid or inhaling hydrogen gas has been shown to play an important role in scavenging reactive oxygen species and maintaining mitochondrial quality control balance, thus improving ARDS in patients and animal models. Coral calcium hydrogenation (CCH) is a new solid molecular hydrogen carrier prepared from coral calcium (CC). Whether and how CCH affects acute lung injury in ARDS remains unstudied. In this study, we observed the therapeutic effect of CCH on lipopolysaccharide (LPS) induced acute lung injury in ARDS mice. The survival rate of mice treated with CCH and hydrogen inhalation was found to be comparable, demonstrating a significant improvement compared to the untreated ARDS model group. CCH treatment significantly reduced pulmonary hemorrhage and edema, and improved pulmonary function and local microcirculation in ARDS mice. CCH promoted mitochondrial peripheral division in the early course of ARDS by activating mitochondrial thioredoxin 2 (Trx2), improved lung mitochondrial dysfunction induced by LPS, and reduced oxidative stress damage. The results indicate that CCH is a highly efficient hydrogen-rich agent that can attenuate acute lung injury of ARDS by improving the mitochondrial function through Trx2 activation.

OBJECTIVES: The risk factors and role of mother‒child gut microbiota in pediatric inflammatory bowel disease (PIBD) remain unclear. We aimed to explore the clinical risk factors associated with PIBD, analyze the characteristics of gut microbiota of children and their mothers, and examine the correlation of the microbial composition in mother‒child pairs. METHODS: We conducted a case-control study including children with PIBD and their mothers as the case group, as well as healthy children and their mothers as the control group. Questionnaires were used to collect information such as family illness history and maternal and early-life events. Fecal samples were collected from the children and mothers for microbiota 16S ribosomal RNA (rRNA) sequencing to analyze the composition and its potential association with PIBD. RESULTS: A total of 54 pairs of cases and 122 pairs of controls were recruited. A family history of autoimmune disease and antibiotic use during pregnancy were associated with an increased risk of PIBD, and a higher education level of the father was associated with a decreased risk of PIBD. Children with PIBD and mothers exhibited different gut microbiota compared to healthy children and mothers. Similarities were observed in the gut microbiota of mothers and children in the same groups. Some bacterial biomarkers of mothers discovered in this study had the power to predict PIBD in their offspring. CONCLUSIONS PIBD is influenced by maternal risk factors and has unique gut microbiota characteristics. The mother‒child gut microbiota is closely related, suggesting the transmission and influence of the gut microbiota between mothers and children. This study highlights the potential pathogenesis of PIBD and provides a basis for developing targeted interventions.
Humans ; Gastrointestinal Microbiome ; Female ; Risk Factors ; Case-Control Studies ; Male ; Child ; Inflammatory Bowel Diseases/etiology* ; Adult ; RNA, Ribosomal, 16S/genetics* ; Feces/microbiology* ; Mothers ; Pregnancy ; Child, Preschool

Objective:To investigate the effects of maternal antibiotics during the pre-natal period on the develop-ment of microglia and anxiety-like behaviors in offspring.Methods:Female SD rats were randomly divided into a maternal control group(CON-M)and a maternal antibiotic group(ABX-M),with 5 rats in each group.The antibiotic group received oral administration of compound antibiotics for 7 days before conception,and the control group received oral administration of the same dose of 0.9％saline during the same period.Subsequently,the two groups of female rats completed the pregnancy and delivery process at the same time,and their male offspring were retained and marked as the offspring control group(CON-O)and the offspring antibiotic group(ABX-O),respectively.The anxiety-like behaviors of offspring rats at 4 weeks and 8 weeks of age were evaluated by open field test and elevated plus maze test,respectively.The number and morphology of microglia in the hippocampus(HIP),cingulate gyrus(CG)and basolater-al nucleus of amygdale(BLA)of the brain of each group of offspring rats were detected by laser confocal microscopy im-aging.Results:The body weight and number of offspring were reduced in the ABX-M group.The 4-week-old ABX-O group showed an increase in anxiety-like behaviors,accompanied by an increase in the number and immature phenotype of microglia in the hippocampus,cingulate gyrus,and amygdala.Compared with the 8-week-old CON-O group,the 8-week-old ABX-O group showed obvious anxiety-like behaviors,and the number and immature phenotype of microglia in the hippocampus,cingulate gyrus,and amygdala of the brain continued to increase.Conclusion:Maternal antibiotic exposure during the pre-pregnancy period significantly affects the number and maturation of microglia in offspring,lead-ing to anxiety-like behaviors,and its effects can persist into adulthood.

Objective:To investigate the pattern of Mesothelin(MSLN)-specific T-cell(CTLs)immune reconstitution after allogeneic hematopoietic stem cell transplantation.Methods:Two cases of Mesothelin-positive patients with acute myeloid leukemia(AML)were screened,and the number of MSLN-CTLs and its subpopulations,the expression of surface PD-1/CTLA-4/TIM-3 im-mune exhaustion molecules,and the functions of secreted cytokines TNF-α and IFN-γ were monitored by flow cytometry after trans-plantation.They were compared with WT1-CTLs and cytomegalovirus(CMV)CTLs.Meanwhile,the relationship between CTL recon-stitution and Minimal residual disease(MRD)and leukemia recurrence was analyzed.Results:After transplantation,MSLN-CTLs,WT1-CTLs and CMV-CTLs can be detected,and intracellular factors were secreted.The phenotypes of WT1 specific CD8+T cells,MSLN specific CD8+T cells and CMV specific CD8+T cells were mainly TEM subsets and TEMRA subsets,and the TEM subsets of CMV specific CD8+T cells were more obvious.Compared with CMV-CTLs,the proportions of T Naive,TCM and TEMRA subsets were relatively higher in MSLN-specific CD8+T cells and WT1-specific CD8+T cells,and the expression levels of PD-1,CTLA-4 and TIM-3 were higher in MSLN-CTLs and WT1-CTLs.The dynamic changes of MSLN-CTLs and WT1-CTLs after transplantation were related to leukemia load and the chimerism rate of donor and recipient.Conclusion:After allogeneic hematopoietic stem cell transplantation,im-mune recovery to MSLN is found,which is different from WT1-CTLs and CMV-CTLs.

With the deepening of the public hospital reform and the implementation of a series of policies,the external environment of the hospital has undergone profound changes,and the operation and management of the hospital has faced unprecedented pressure.Against this background,the case hospital has set up a special operation management team,established an operation evaluation index system that integrates business and finance,written an analysis report on"one policy for one department",set up an"operation management MDT clinic",and issued a list of operation problems to functional departments.A series of measures,such as the establishment of a set of operation management system with multi-line synergy,big data support,lean operation and evidence-based decision-making,have strongly guaranteed the scientific decision-making and efficient implementation of the hospital's operation management,and played a good role in the hospital's medical business,operation efficiency,cost structure,and management of the medical insurance fund,etc.The practical exploration of the operation management model has further improved the medical service level of the hospital and accelerated the pace of improving quality and increasing efficiency of the hospital.It is necessary to pay more attention to the refined management,continuously optimize the operation management system,and continuously promote the high-quality development of the hospital.

Objective:To establish a modified one-stage clotting assay (mOSA) based on the STA-R Evolution coagulation analyzer for quantifying emicizumab (EMI) concentration and to preliminarily evaluate its analytical performance; meanwhile to explore the clinical utility of the standard one-stage clotting assay (sOSA) in indirectly predicting EMI levels through surrogate factor Ⅷ (FⅧ) activity.Methods:A total of 30 pediatric patients with hemophilia A (HA) treated with EMI in the Hemophilia Treatment Center of Shenzhen Children′s Hospital from January 2023 to March 2025 were enrolled, and 48 post-treatment plasma samples were collected. EMI standards (2.5~100 μg/ml) were prepared using FⅧ-deficient plasma to establish the mOSA detection system. The linearity, accuracy, and precision of the method were evaluated. Surrogate FⅧ activity was measured by sOSA to estimate EMI concentrations, and its correlation with mOSA-derived EMI concentrations was analyzed using Spearman correlation analysis. The equivalent FⅧ activity in patient plasma samples was measured using a human chromogenic substrate assay-based FⅧ activity detection reagent, and Spearman correlation analysis was employed to evaluate its correlations with both the EMI concentrations measured by the mOSA method and estimated by the sOSA method respectively.Results:The established mOSA method for EMI detection showed excellent linearity in the range of 2.5?100 μg/ml ( Y=1.047 X?1.033, R 2=0.995, P<0.001). Average spike recovery rates at 25, 50, and 75 μg/ml were 101.55%(25.39/25.00), 105.31%(52.66/50.00), and 98.20%(73.65/75.00), respectively. Coefficients of variations of within-and inter-batch were 3.47%?4.80% and 6.30%?8.96%, respectively. A prediction model for EMI concentration was established as follows: estimated EMI concentration (μg/ml)=0.095×[alternative FⅧ activity (%) measured by sOSA]+2.652 ( R2=0.999, P<0.001). Validation demonstrated a strong correlation between the EMI concentration measured by the mOSA method and the EMI concentration estimated by the sOSA method ( r=0.989, P<0.001), with good consistency ( Y=1.014 X+0.684, R2=0.972, P<0.001). Both the EMI concentration measured by the mOSA method and the EMI concentration estimated by the sOSA method showed extremely strong correlations with the equivalent FⅧ activity ( r=0.986 and 0.987, respectively; P<0.001 for both). Conclusions:The mOSA system established on the STA-R Evolution analyzer demonstrates robust linearity, accuracy, and reproducibility, fulfilling clinical requirements for therapeutic drug monitoring of EMI. The sOSA method provides reliable indirect estimation of EMI concentrations through surrogate FⅧ activity, offering critical support for emergency decision-making.

Acute respiratory distress syndrome(ARDS)is a common respiratory emergency,but current clinical treatment remains at the level of symptomatic support and there is a lack of effective targeted treatment measures.Our previous study confirmed that inhalation of hydrogen gas can reduce the acute lung injury of ARDS,but the application of hydrogen has flammable and explosive safety concerns.Drinking hydrogen-rich liquid or inhaling hydrogen gas has been shown to play an important role in scavenging reactive oxygen species and maintaining mitochondrial quality control balance,thus improving ARDS in patients and animal models.Coral calcium hydrogenation(CCH)is a new solid molecular hydrogen carrier prepared from coral calcium(CC).Whether and how CCH affects acute lung injury in ARDS re-mains unstudied.In this study,we observed the therapeutic effect of CCH on lipopolysaccharide(LPS)induced acute lung injury in ARDS mice.The survival rate of mice treated with CCH and hydrogen inhalation was found to be comparable,demonstrating a significant improvement compared to the untreated ARDS model group.CCH treatment significantly reduced pulmonary hemorrhage and edema,and improved pulmonary function and local microcirculation in ARDS mice.CCH promoted mitochon-drial peripheral division in the early course of ARDS by activating mitochondrial thioredoxin 2(Trx2),improved lung mitochondrial dysfunction induced by LPS,and reduced oxidative stress damage.The results indicate that CCH is a highly efficient hydrogen-rich agent that can attenuate acute lung injury of ARDS by improving the mitochondrial function through Trx2 activation.

BACKGROUND: Several randomized controlled studies have suggested that the prophylactic use of proton pump inhibitors (PPIs) in intensive care unit (ICU) patients could not reduce the incidence of gastrointestinal bleeding (GIB) and may increase adverse events such as intestinal infection and pneumonia. Gut microbiota may play a critical role in the process. PPIs have been widely prescribed for GIB prophylaxis in patients with acute coronary syndrome (ACS). This study aimed to determine the short-term effects of PPI and histamine-2 receptor antagonist (H2RA) treatment on gut microbiota of ACS patients. METHODS: The study was designed as a single-blind, multicenter, three-parallel-arm, randomized controlled trial conducted at three centers in Beijing, China. We enrolled ACS patients at low-to-medium risk of GIB and randomized (2:2:1) them to either PPI ( n = 40), H2RA ( n = 31), or control group ( n = 21). The primary outcomes were the alterations in gut microbiota after 7 days of acid suppressant therapy. Stool samples were collected at baseline and 7 days and analyzed by 16S ribosomal RNA (rRNA) gene sequencing. RESULTS: There were no significant changes in the diversity of gut microbiota after the short-term use of acid suppressants, but the abundance of Fusobacterium significantly increased and that of Bifidobacterium significantly decreased, especially in PPI users. In addition, the abundance of some pathogenic bacteria, including Enterococcus and Desulfovibrio, was significantly elevated in the PPI users. The fecal microbiota of the PPI users included more arachidonic acid metabolism than that of control group. CONCLUSIONS: PPIs may increase the risk of infection by adversely altering gut microbiota and elevating arachidonic acid metabolism, which may produce multiple proinflammatory mediators. For ACS patients at low-to-medium risk of GIB, sufficient caution should be paid when acid-suppressant drugs are prescribed, especially PPIs. REGISTRATION www.chictr.org.cn (ChiCTR2000029552).
Humans ; Proton Pump Inhibitors/therapeutic use* ; Acute Coronary Syndrome/microbiology* ; Female ; Gastrointestinal Microbiome/drug effects* ; Male ; Middle Aged ; Histamine H2 Antagonists/therapeutic use* ; Aged ; Single-Blind Method