Objective　To construct a recombinant lentiviral expression vector for human lymphocyte activation gene 3 (LAG3) and generation of monoclonal cell lines that preferentially express LAG3 by transfection of the vector into mouse fibroblast cells 3T3. Methods　After extracting total RNA extracted from human peripheral blood mononuclear cells, the RNA is reversely transcribed into cDNA. The LAG3 extracellular and transmembrane region sequences are amplified by PCR using high-fidelity DNA polymerase. The PCR products are double-digested with the restriction endonucleases EcoRⅠ and NotⅠ, then ligated with the lentiviral vector pTSB-copGFP to construct the recombinant expression vector pTSB-LAG3-copGFP, which is subsequently transformed into Escherichia coli DH5α. Positive clonal bacteria are selected by PCR, and the plasmids are extracted and sequenced for verification. The recombinant vector pTSB-LAG3-copGFP, along with packaging plasmids psPAX2 and pMD2.0G, are co-transfected into human embryonic kidney 293T cells to assemble and release virus particles, the virus infected 3T3 cells were collected. During the puromycin selection of infected 3T3 cells, the limited dilution method is used to obtain 3T3 monoclonal cells that stably express LAG3. Real-time fluorescent quantitative PCR, immunofluorescence and flow cytometry were utilized to verify the transcription of LAG3 mRNA and the expression of LAG3 protein respectively. Results　Sequencing of the recombinant pTSB-LAG3-copGFP lentiviral vector plasmid reveals that the amplified LAG3 sequence contains a synonymous mutation in the His codon at nucleotide position 1 697 bp within the LAG3 transmembrane region, which aligns with the standard LAG3 sequence (accession number NM_002286.6) in GenBank. The 3T3 cells infected by pTSB-LAG3-copGFP packaging virus screened with puromycin. A total of 20 LAG3+copGFP+-3T3 monoclonal cell lines were obtained, all of which exhibited transcription of LAG3 mRNA. The monoclonal cell line MC-6 exhibits the highest transcriptional level of LAG3. Effective expression and distribution of LAG3 protein on the cell membrane and cytoplasmic organelle membranes in MC-6 indicated by immunofluorescence and flow cytometry. Conclusion　The pTSB-LAG3-copGFP lentiviral vector was successfully constructed. LAG3+copGFP+-3T3 monoclonal cell lines overexpressing lymphocyte activating 3 were efficiently established, laying the foundation for subsequent studies on the relationship between LAG3 and the development of chronic infectious diseases such as hepatitis B, as well as the interventional treatment of LAG3.

BACKGROUND: Disease-modifying therapies have been approved for the treatment of relapsing multiple sclerosis (RMS). The present study aims to examine the safety of teriflunomide in Chinese patients with RMS. METHODS: This non-randomized, multi-center, 24-week, prospective study enrolled RMS patients with variant (c.421C>A) or wild type ABCG2 who received once-daily oral teriflunomide 14 mg. The primary endpoint was the relationship between ABCG2 polymorphisms and teriflunomide exposure over 24 weeks. Safety was assessed over the 24-week treatment with teriflunomide. RESULTS: Eighty-two patients were assigned to variant ( n  = 42) and wild type groups ( n  = 40), respectively. Geometric mean and geometric standard deviation (SD) of pre-dose concentration (variant, 54.9 [38.0] μg/mL; wild type, 49.1 [32.0] μg/mL) and area under plasma concentration-time curve over a dosing interval (AUC tau ) (variant, 1731.3 [769.0] μg∙h/mL; wild type, 1564.5 [1053.0] μg∙h/mL) values at steady state were approximately similar between the two groups. Safety profile was similar and well tolerated across variant and wild type groups in terms of rates of treatment emergent adverse events (TEAE), treatment-related TEAE, grade ≥3 TEAE, and serious adverse events (AEs). No new specific safety concerns or deaths were reported in the study. CONCLUSION: ABCG2 polymorphisms did not affect the steady-state exposure of teriflunomide, suggesting a similar efficacy and safety profile between variant and wild type RMS patients. REGISTRATION NCT04410965, https://clinicaltrials.gov .
Humans ; Crotonates/adverse effects* ; Toluidines/adverse effects* ; Nitriles ; Hydroxybutyrates ; Female ; Male ; Adult ; ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics* ; Middle Aged ; Multiple Sclerosis, Relapsing-Remitting/genetics* ; Prospective Studies ; Young Adult ; Neoplasm Proteins/genetics* ; East Asian People

Periodontal bone defects, primarily caused by periodontitis, are highly prevalent in clinical settings and manifest as bone fenestration, dehiscence, or attachment loss, presenting a significant challenge to oral health. In regenerative medicine, harnessing developmental principles for tissue repair offers promising therapeutic potential. Of particular interest is the condensation of progenitor cells, an essential event in organogenesis that has inspired clinically effective cell aggregation approaches in dental regeneration. However, the precise cellular coordination mechanisms during condensation and regeneration remain elusive. Here, taking the tooth as a model organ, we employed single-cell RNA sequencing to dissect the cellular composition and heterogeneity of human dental follicle and dental papilla, revealing a distinct Platelet-derived growth factor receptor alpha (PDGFRA) mesenchymal stem/stromal cell (MSC) population with remarkable odontogenic potential. Interestingly, a reciprocal paracrine interaction between PDGFRA⁺ dental follicle stem cells (DFSCs) and CD31⁺ Endomucin⁺ endothelial cells (ECs) was mediated by Vascular endothelial growth factor A (VEGFA) and Platelet-derived growth factor subunit BB (PDGFBB). This crosstalk not only maintains the functionality of PDGFRA⁺ DFSCs but also drives specialized angiogenesis. In vivo periodontal bone regeneration experiments further reveal that communication between PDGFRA⁺ DFSC aggregates and recipient ECs is essential for effective angiogenic-osteogenic coupling and rapid tissue repair. Collectively, our results unravel the importance of MSC-EC crosstalk mediated by the VEGFA and PDGFBB-PDGFRA reciprocal signaling in orchestrating angiogenesis and osteogenesis. These findings not only establish a framework for deciphering and promoting periodontal bone regeneration in potential clinical applications but also offer insights for future therapeutic strategies in dental or broader regenerative medicine.
Receptor, Platelet-Derived Growth Factor alpha/metabolism* ; Humans ; Neovascularization, Physiologic/physiology* ; Dental Sac/cytology* ; Single-Cell Analysis ; Transcriptome ; Mesenchymal Stem Cells/metabolism* ; Bone Regeneration ; Animals ; Dental Papilla/cytology* ; Periodontium/physiology* ; Stem Cells/metabolism* ; Regeneration ; Angiogenesis

Objective:To discuss the effect of Boschnikia rossica polysaccharides rapa polysaccharides(BRPS)on lipopolysaccharide(LPS)-induced inflammatory responses in the THP-1 macrophages,and to clarify its mechanism.Methods:The THP-1 monocytes were differentiated into the macrophages,and the inflammation model was established using LPS to induce the THP-1 macrophages.CCK-8 method was used to detect the survial rates of the THP-1 macrophages after treated with different concentrations(0,100,200,500,1 000,and 2 000 μg·L-1)of LPS and different concentrations(0,12.5,25.0,50.0,100.0,and 200.0 mg·L-1)of BRPS to select the concentrations for the subsequent experiments.The THP-1 macrophages were divided into blank group,model group,low dose of BRPS group(25.0 mg·L-1 BRPS),medium dose of BRPS group(50.0 mg·L-1 BRPS),and high dose of BRPS group(100.0 mg·L-1 BRPS).P38 inhibitor SB203580,ERK inhibitor U0126,c-Jun N-terminal kinase(JNK)inhibitor SP600125,and nuclear factor of kappa B(NF-κB)inhibitor BAY11-7082 were used to verify the effects on THP-1 cells.The THP-1 cells were divided into control group,LPS group,inhibitor group,100.0 mg·L-1 BRPS group,and inhibitor+100.0 mg·L-1 BRPS group.ELISA method was used to detect the levels of tumor necrosis factor α(TNF-α),interleukin(IL)-6,and IL-1β in culture fluid of the THP-1 macrophages in various groups;DCFH-DA fluorescence probe method was used to detect the reactive oxygen species(ROS)levels in the THP-1 macrophages in various groups;Hoechst33342/PI fluorescence staining method was used to detect the membrane damage in the THP-1 macrophages in various groups;JC-1 fluorescence staining was used to observe mitochondrial membrane potential in the THP-1 macrophages in various groups;Western blotting method was used to detect the expression levels of cyclooxygenase-2(COX-2),high mobility group protein B1(HMGB1),NOD-like receptor thermal protein domain assciated protein 3(NLRP3),cysteinyl aspartate specific protease(Caspase)-1,gasdermin D(GSDMD)-N,IL-1β,mitogen-activated protein kinase(MAPK),and nuclear factor-kappa B(NF-κB)related proteins in the THP-1 macrophages in various groups.Results:The CCK-8 method results showed that when the LPS concentration was 100-2 000 μg·L-1,the survival rates of the THP-1 macrophages were over 90%.Compared with 0 μg·L-1 LPS group,the IL-6 levels in culture fluid of the THP-1 macrophages in 100,200,500,1 000,and 2 000 μg·L-1 LPS group were increased(P<0.05),indicating a significant enhancement of the inflammatory response in the macrophages,so 100 μg·L-1 LPS was used to construct the inflammation model.After treated with 12.5,25.0,50.0,100.0,and 200.0 mg·L-1 BRPS,the survival rates of the THP-1 macrophage were 91.2%,93.8%,91.4%,90.6%,and 91.8%,respectively,so 25.0,50.0,and 100.0 mg·L-1 BRPS were selected as the drug concentrations for low,medium,and high doses of BRPS groups in the subsequent experiments.The ELISA results showed that compared with blank group,the levels of IL-6,TNF-α,and IL-1β in culture fluid of the THP-1 macrophages in model group were increased(P<0.05);compared with model group,the levels of IL-6,TNF-α,and IL-1β in low,medium,and high doses of BRPS groups were decreased(P<0.05).The DCFH-DA fluorescence probe method results showed that compared with blank group,the ROS level in the THP-1 macrophages in model group was increased(P<0.05);compared with model group,the ROS levels in low,medium,and high doses of BRPS groups were decreased(P<0.05).The Hoechst33342/PI fluorescence staining results showed that compared with blank group,the degree of membrane damage in the THP-1 macrophages in model group was increased;compared with model group,the degrees of membrane damage in low,medium,and high doses of BRPS groups were decreased.The JC-1 fluorescence staining results showed that compared with blank group,the mitochondrial membrane potential in the THP-1 macrophages in model group was decreased significantly;compared with model group,the mitochondrial membrane potential in low,medium,and high doses of BRPS groups were increased gradually.The Western blotting results showed that compared with blank group,the expression levels of COX-2,HMGB1,NLRP3,Caspase 1,GSDMD-N,and IL-1β proteins and the ratios of p-P38/P38,p-ERK/ERK,p-JNK/JNK,and p-NF-κB/NF-κB in the THP-1 macrophages in model group were increased(P<0.05);compared with model group,the expression levels of HMGB1,NLRP3,Caspase-1,GSDMD-N,and IL-1β proteins and the ratios of p-P38/P38,p-ERK/ERK,p-JNK/JNK,and p-NF-κB/NF-κB in the THP-1 macrophages in medium and high doses of BRPS groups were decreased(P<0.05),the expression levels of NLRP3,Caspase-1,and IL-1β proteins in the cells in low dose of BRPS group were decreased(P<0.05),the expression level of COX-2 protein in the cells in high dose of BRPS group was decreased(P<0.05).Compared with control group,the ratios of p-P38/P38,p-ERK/ERK,p-JNK/JNK,and p-NF-κB/NF-κB,and the expression level of IL-1β protein in the THP-1 macrophages in LPS group were increased(P<0.05);compared with LPS group,the ratios of p-P38/P38,p-ERK/ERK,p-JNK/JNK,and p-NF-κB/NF-κB,and the expression level of IL-1β protein in the THP-1 macrophages in inhibitor group,100 mg·L-1 BRPS group,and inhibitor+100 mg·L-1 BRPS group were decreased(P<0.05);compared with inhibitor group,the ratios of p-P38/P38,p-ERK/ERK,p-JNK/JNK,and p-NF-κB/NF-κB in the THP-1 macrophages in inhibitor+100 mg·L-1 BRPS group were decreased(P<0.05).Conclusion:BRPS inhibits the inflammatory response of the THP-1 macrophages,which may be related to the MAPK and NF-κB signaling pathways regulated by BRPS.

Pancreatic cystic neoplasm(PCN)is a category of pancreatic tumors with significant heterogeneity.In recent years,the detection rate of PCN has been increasing,and it has gradually become a concern of clinicians.Endoscopic ultrasound(EUS)can be close to the pancreas for scanning and biopsy,and it has certain advantages in the diagnosis and treatment of PCN.This review mainly summarizes the latest progress of EUS in the diagnosis and treatment of PCN.Cyst fluid molecular markers,such as Kirsten rat sarcoma viral oncogene homolog,GNAS complex locus,Von Hippel-Lindau tumor suppressor gene,as well as emerging endoscopic technologies such as EUS-guided needle based confocal laser endomicroscopy and through-the-needle biopsy,have all showed the potential to significantly improve the diagnostic accuracy of PCN.EUS-guided ablation is an emerging minimally invasive treatment technique for PCN,with the efficacy and safety of chemical ablation being supported by a substantial amount of research.

Traditional Chinese medicines (TCMs) possess a rich historical background, unique theoretical framework, remarkable therapeutic efficacy, and abundant resources. However, the modernization and internationalization of TCMs have faced significant obstacles due to their diverse ingredients and unknown mechanisms. To gain deeper insights into the phytochemicals and ensure the quality control of TCMs, there is an urgent need to enhance analytical techniques. Currently, two-dimensional (2D) chromatography, which incorporates two independent separation mechanisms, demonstrates superior separation capabilities compared to the traditional one-dimensional (1D) separation system when analyzing TCMs samples. Over the past decade, new techniques have been continuously developed to gain actionable insights from complex samples. This review presents the recent advancements in the application of multidimensional chromatography for the quality evaluation of TCMs, encompassing 2D-gas chromatography (GC), 2D-liquid chromatography (LC), as well as emerging three-dimensional (3D)-GC, 3D-LC, and their associated data-processing approaches. These studies highlight the promising potential of multidimensional chromatographic separation for future phytochemical analysis. Nevertheless, the increased separation capability has resulted in higher-order data sets and greater demands for data-processing tools. Considering that multidimensional chromatography is still a relatively nascent research field, further hardware enhancements and the implementation of chemometric methods are necessary to foster its robust development.

Objective To analyze the influencing factors of survival of patients with airway stenosis requiring clinical interventions after lung transplantation. Methods Clinical data of 66 patients with airway stenosis requiring clinical interventions after lung transplantation were retrospectively analyzed. Univariate and multivariate Cox’s regression models were adopted to analyze the influencing factors of survival of all patients with airway stenosis and those with early airway stenosis. Kaplan-Meier method was used to calculate the overall survival and delineate the survival curve. Results For 66 patients with airway stenosis, the median airway stenosis-free time was 72 (52,102) d, 27% (18/66) for central airway stenosis and 73% (48/66) for distal airway stenosis. Postoperative mechanical ventilation time [hazard ratio (HR) 1.037, 95% confidence interval (CI) 1.005-1.070, P=0.024] and type of surgery (HR 0.400, 95%CI 0.177-0.903, P=0.027) were correlated with the survival of patients with airway stenosis after lung transplantation. The longer the postoperative mechanical ventilation time, the higher the risk of mortality of the recipients. The overall survival of airway stenosis recipients undergoing bilateral lung transplantation was better than that of their counterparts after single lung transplantation. Subgroup analysis showed that grade 3 primary graft dysfunction (PGD) (HR 4.577, 95%CI 1.439-14.555, P=0.010) and immunosuppressive drugs (HR 0.079, 95%CI 0.022-0.287, P<0.001) were associated with the survival of patients with early airway stenosis after lung transplantation. The overall survival of patients with early airway stenosis after lung transplantation without grade 3 PGD was better compared with that of those with grade 3 PGD. The overall survival of patients with early airway stenosis after lung transplantation treated with tacrolimus was superior to that of their counterparts treated with cyclosporine. Conclusions Long postoperative mechanical ventilation time, single lung transplantation, grade 3 PGD and use of cyclosporine may affect the survival of patients with airway stenosis after lung transplantation.

Objective To prepare mouse anti-human lymphocyte activation gene 3 (LAG3) monoclonal antibody (mAb) and perform immunological identification of the antibody. Methods BALB/c mice were immunized with LAG3-mLumin-3T3 cells, which stably express the extracellular and transmembrane regions of human LAG3 in mouse 3T3 cells. The secretion of anti-human LAG3 antibodies in mouse serum was assessed using flow cytometry and immunofluorescence. SP2/0 cells were injected subcutaneously into the mice to elicit solid myelomas, and mouse myeloma cells were subsequently isolated. Spleen cells from the immunized mice were fused with the myeloma cells to establish hybridomas, which were then separated using the limiting dilution method. Flow cytometry was used to detect LAG3 mAbs in the hybridoma culture medium. To map the epitopes recognized by these mAbs, 3T3 cells expressing individual extracellular domains of LAG3(LAG3 domains 1/-2/-3/-4-3T3) were used. Flow cytometry was also applied to analyze LAG3 expression on activated human peripheral blood mononuclear cells (PBMC) before and after co-culture with the LAG3 mAbs. Results Mice immunized with the recombinant eukaryotic cell antigen produced anti-LAG3 antibodies. The generated hybridomas secreted mouse anti-human LAG3 mAbs, with each hybridoma line recognizing different LAG3 antigenic domains. Conclusion Mouse anti-human LAG3 mAbs were successfully generated, with different hybridoma clones secreting antibodies that recognize distinct LAG3 epitopes. These findings lay the groundwork for further studies into the biological properties of LAG3 and the development of diagnostic reagents and therapeutic blocking antibodies for cancer treatment.
Animals ; Humans ; Mice ; Lymphocyte Activation Gene 3 Protein ; Antibodies, Monoclonal/immunology* ; Mice, Inbred BALB C ; Hybridomas/immunology* ; Antigens, CD/genetics* ; Immunization ; Recombinant Proteins/immunology* ; Female ; Eukaryotic Cells/immunology* ; Flow Cytometry ; Epitopes/immunology*

OBJECTIVES: During the coronavirus disease 2019 (COVID-19) pandemic, a growing prevalence of racial and ethnic discrimination occurred when many Americans struggled to maintain healthy lifestyles. This study investigated the associations of racial and ethnic discrimination with changes in exercise and screen time during the pandemic in the United States. METHODS: We included 2,613 adults who self-identified as non-Hispanic White, non-Hispanic Black, non-Hispanic Asian, or Hispanic from the Health, Ethnicity, and Pandemic study, a cross-sectional survey conducted among a nationally representative sample of United States adults between October and November 2020. We assessed self-reported racial and ethnic discrimination by measuring COVID-19-related racial and ethnic bias and examined its associations with changes in exercise and screen time using multivariable logistic regression models. We analyzed data between September 2021 and March 2022. RESULTS: COVID-19-related racial and ethnic bias was associated with decreased exercise time among non-Hispanic Asian (odds ratio [OR], 1.46; 95% confidence interval [CI], 1.13 to 1.89) and Hispanic people (OR, 1.91; 95% CI, 1.32 to 2.77), and with increased screen time among non-Hispanic Black people (OR, 1.94; 95% CI, 1.33 to 2.85), adjusting for age, sex, education, marital status, annual household income, insurance, and employment status. CONCLUSIONS Racial and ethnic discrimination may have adversely influenced exercise and screen time changes among racial and ethnic minorities during the COVID-19 pandemic in the United States. Further studies are needed to investigate the mechanisms through which racial and ethnic discrimination can impact lifestyles and to develop potential strategies to address racial and ethnic discrimination as a barrier to healthy lifestyles.

Objective:To improve the prognosis stratification, especially early mortality(EM), of elderly patients with newly diagnosed multiple myeloma(NDMM).Methods:In this retrospective study, univariate and multivariate Cox regression analysis were conducted to identify the independent prognostic factors associated with overall survival(OS)and the chi-square test and multivariate Logistic analysis were used to identify the prognostic factors associated with EM in 223 elderly patients(age≥65 years)with NDMM from three centers in the country.Results:Increased NT-pro-BNP(≥300 pg/ml), ECOG-PS≥2 and stage Ⅲ R-ISS were identified as three independent adverse prognostic factors of OS.The rates of EM3, EM6, EM12 and EM24 were 12.1%, 20.1%, 32.2% and 60%, respectively.The most common cause for EM6(particularly EM3)was disease-related complications resulting from ineligibility for treatment due to poor physical performance, severe organ dysfunction or treatment discontinuation due to treatment intolerance, while the most common cause for EM12(particularly EM24)was disease progression or relapse mainly as a result of inadequate treatment.R-ISS staging failed to predict EM, while decreased eGFR, ECOG-PS≥2, and increased NT-pro-BNP were able to estimate the risk of EM, with increased NT-pro-BNP as a common independent factor for EM12( P=0.03)and EM24( P=0.015). Conclusions:R-ISS staging, which primarily reflects MM biology, cannot predict EM.However, factors such as NT-pro-BNP, eGFR and ECOG-PS associated with frailty and impairment of organ functions can be used to estimate the risk of EM, among which NT-pro-BNP may be the most important independent factor for EM.Therefore, incorporation of these frailty-related biomarkers into R-ISS staging may be able to more precisely estimate the prognosis and particularly early death of elderly patients with NDMM.