AIM: To establish the monoclonal antibody against human B lymphocyte stimulator (hBLyS) by DNA immunization and analyse its characterization. METHODS: The 858 bp DNA fragment of hBLyS was cloned into pcDNA3 plasmids. The cloned insert was identified by both sequence analysis and double digestion of the recombinant plasmid with restriction enzymes Xho I and EcoR I . After the splenocytes from BALB/c mice immunized with the recombinant plasmid of pcDNA3/hBLyS were fused with myeloma cells SP2/0,the hybridoma which can produce monoclonal antibodies against hBLyS were obtained. The specificity of anti-BLyS monoclonal antibody from hybridoma was verified by ELISA, Western blot and flow cytometry. RESULTS: The recombinant mammalian cell expression vector of pcDNA3/hBLyS was constructed, the sequence of the insert gene was identified to be the sequence encoding hBLy S antigen. The culture supernatants of hybridoma 9c10 were tested to be the monoclonal antibody with specificity against hBLyS on human peripheral blood CD3 + T cell activated by hIFN-? by ELISA, Western blot and flow cytometry. CONCLUSION: The monoclonal antibodies against hBLyS with high activity and specificity have been established successfully, and will be an useful tool in the studies of relationship between hBLyS and human autoimmunity diseases.

AIM: To investigate the effect of caveolin-1 on the endothelin-1 (ET-1)-induced vascular smooth muscle cells (VSMC) proliferation. METHODS: The -thymidine (TdR) incorporation, immunofluorescence assays and western blotting were used in this study. RESULTS: The ETA receptor specific antagonist BQ123 inhibited the increase in TdR incorporation in response to ET-1 on VSMC. Immunofluorescence assays showed that caveolin-1 was mostly distributed in plasmalemma of VSMC. After 24 h treatment of VSMC with ET-1, the expression of caveolin-1 in VSMC was significantly decreased. Western blotting showed that ET-1 inhibited the expression of caveolin-1 in VSMC, BQ123 reversed the effect of ET-1. CONCLUSION: Caveolin-1 was mostly distributed in plasmalemma of VSMC. ET-1 downregulated caveolin-1 expression in VSMC via ETA receptor.

AIM: To study the roles and mechanisms of ERKs and intracellular free calcium in cardiomyocyte hypertrophic response induced by endothelin-1(ET-1). METHODS: (1) Neonatal rat cardiomyocyte hypertrophic response was assayed by measuring cell surface area and protein content; (2) ERKs activity was determined by Whatman Paper Filter method; (3) Intracellular free calcium concentration (［Ca2+］i) was measured using Fura-2/AM as a fluorescent indicator. RESULTS: (1) ET-1 could increase total protein production, surface area, ERKs activity and ［Ca2+］i in cultured cardiomyocyte in dose-dependent manner at concentrations ranging from 10-9 to 10-7 mol/L. And this effect could be abolished by BQ123, an antagonist of ETA receptor, partly inhibited by PTX, but not by BQ788, an antagonist of ETB receptor.（2）The activation of ERKs and the increase of ［Ca2+］i induced by ET-1 were obviously inhibited by PD98059, a selective ERKs kinase inhibitor, and nifedipine, a calcium channel blocker, respectively. Both antagonists partially inhibited ET-1-stimulated cardiomyocyte hypertrophic response. (3) Staurosporine, a selective PKC inhibitor, could inhibit ET-1-stimulated cardiomyocyte hypertrophic response and increase of ［Ca2+］i, but not affect the activation of ERKs. CONCLUSION: Cardiomyocyte hypertrophic response induced by ET-1 is mediated by ETA receptor coupled to PTX-sensitive G-protein, which involves at least two signalling pathways: PKC-mediated increase of ［Ca2+］i , and PKC-independent activation of ERKs.

Objective:To explore the relationship between endotracheal tube-bacterial biofilm (ETT-BF) in mechanically ventilated neonates and ventilator-associated pneumonia (VAP).Methods:A total of 30 mechanically ventilated neonates whose mechanical ventilation time were ≥48 h in the Department of Neonatology in The Second Affiliated Hospital of Wenzhou Medical University from January 2019 to January 2020 were included.According to the indwelling time of endotracheal tube, all cases were divided into three groups including group A(two to six days), group B(seven to 14 days) and group C (over 14 days). The morphological results of ETT-BF were scanned by scanning electron microscope (SEM). The incidence of VAP, the positive rates of strains isolated from endotracheal tube surface and lower respiratory tract secretion, the detection of strains and drug resistance were analyzed. Chi-squared test were used for statistical analysis.Results:The results of SEM showed that sheet matrix could be observed on the surface of the inner cavity of endotracheal tube in three days of tracheal catheter retention, and cocci adhered to it in four days. With prolonged indwelling time of endotracheal tube, the structure of bacterial biofilm (BF) had improved.The positive rate of strains isolated from the secretion of lower respiratory tract in 30 neonates was 23.3%(7/30) and all of them were Gram-negative bacteria. There was no patient developed VAP in group A, while there were two patients with VAP in group B, and five patients with VAP in group C. The incidences of VAP in the three groups were statistically significant ( χ2=10.82, P=0.004). There was no significant difference in the positive rate of strains isolated from the surface of endotracheal tube under different indwelling time in 30 cases ( χ2=1.03, P=0.598). Among of 13 neonates in group A, there were seven strains isolated from ETT-BF, mainly Gram-positive bacteria which turned out to be mainly Gram-negative bacteria with the prolongation of endotracheal tube indwelling time. Of the seven VAP cases, strains isolated from the lower respiratory tract secretion were consistent with the strains isolated from the surface of the corresponding endotracheal tube in five cases, which were Serratia liquefaciens, Klebsiella acidogenes, Serratia marcescens, Flavobacterium meningosepticum and Stenotrophomonas maltophilia, and the drug resistance was consistent. Conclusions:The colonization bacteria of early ETT-BF may come from the upper respiratory tract, with less migration which rarely causes VAP. With the prolongation of endotracheal tube indwelling time, the incidence of VAP in neonates increases. The same pathogen can be found in the ETT-BF and lower respiratory tract secretion. The source of pathogen needs further study.

TAM(tumor-associated macrophage)is a kind of immune cell in tumor microenvironment,which mainly exists in tumor matrix to mediate inflammatory reaction.Generally,TAM can be stimulated by different cytokines to polarize into M1 macrophage or M2 macrophage with different phenotypes.In the early stage of tumor,M1 macrophages in TAM exhibit pro-inflammatory and anti-tumor activities,while as tumor progresses,TAMs tend to polarize into M2 macrophages to play anti-inflammatory and tumor-promoting roles.A large number of studies have shown that TAM is closely related to tumor growth,invasion,metastasis and poor prognosis.Therefore,targeting TAM has become the focus of anti-tumor immunotherapy.This paper has summarized the origin of TAM,and introduced the specific roles of TAM in promoting tumor proliferation,angiogenesis,migration and invasion and the formation of immunosuppressive environment.At the same time,the research progress of TAM-targeted anti-tumor immu-notherapy in recent years was discussed from 3 aspects:inhibiting monocyte recruitment,promoting TAM apoptosis and reshaping TAM phenotype.

Digestive tract diseases， especially digestive tract tumors， including liver cancer， pancreatic cancer， and colorectal cancer， have high incidence in China. Digestive tract tumor is one of the top 10 cancers in terms of the number of new cases and deaths in the world， and the incidence and mortality of tumor diseases have been increasing year by year. Therefore， the prevention and treatment of tumors is particularly important. With the application and promotion of traditional Chinese medicine in the medical field and the rapid development of molecular biology and pharmacology， more and more potential active components of Chinese medicinal materials have been extracted and studied. These active components can inhibit tumor cells in a multi-target and multi-pathway manner. Cinobufotalin is an effective component extracted from the skin of Bufo bufo gargarizans. It has been prepared into a variety of agents with anti-tumor， immunomodulatory， cardiac boosting， pain-easing， anti-inflammatory， and swelling-relieving activities. In clinical practice， cinobufotalin is mainly used to assist the treatment of liver cancer， lung cancer， colorectal cancer， gastric cancer and other malignant tumors， which can reduce the adverse reactions of patients in the middle and late stages and improve the quality of life and five-year survival rate of patients. The available studies of molecular mechanism have demonstrated that cinobufotalin can play a therapeutic role by inducing cell apoptosis， regulating cell cycle， inhibiting cell proliferation and angiogenesis， modulating immune response， reversing multidrug resistance， enhancing radiochemotherapy sensitivity， inhibiting tumor inflammation， invasion， and metastasis， etc. This review focuses on the clinical application and mechanism of cinobufotalin against digestive tract tumors in recent years， aiming to provide a theoretical basis for the anti-tumor research of cinobufotalin， promote the application of cinobufotalin in tumor treatment， and facilitate the further research and development of this compound.

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; metabolism ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Enterovirus A, Human ; drug effects ; genetics ; growth & development ; immunology ; Fibroblasts ; drug effects ; virology ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; metabolism ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; immunology ; Mice ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Receptors, Scavenger ; chemistry ; genetics ; immunology ; Receptors, Virus ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sf9 Cells ; Spodoptera ; Thermodynamics