Objective To investigate the effects of serotonin(5-HT) on the ionic channels currents and membrane potentials of single guinea pig proximate colon myocytes. Methods Cell images before and after contraction were captured and analyzed with an imag e analysis software. Channels currents, action potential (AP) and resting potent ial (RP) were recorded with an EPC-9 amplifier. Results The direct actions of 10 ?mol/L 5-HT to myocytes resulted in followin g effects: Firstly, the exogenous stimulated peak values of AP reduced to 60%?7 .3%(n=7,P

AIM To study the effects of progesterone on contractile activity of smooth muscle stripes and the large conduction Ca 2+ activated K + currents (BK Ca ) of single colonic myocytes in female guinea pig proximal colons METHODS Stripes and single cells were separated acutely from guinea pig proximal colon Contractions of stripes were measured by isonic transducer, and Ca 2+ activated K + currents were recorded with an Axopatch 1D amplifier under conventional whole cell patterns RESULTS 64 8 ?mol?L -1 progesterone produced longitudinal stripes significantly re laxation(0 1792 g?0 0873 g, n =6, P

Objective To investigate the role of Zn 2+and Zn2+-coordinating glutamic acid residues ( 71Glu and 80Glu ) in the superantigenic activities of staphylococcal enterotoxin C2 ( SEC2 ) .Methods Over-lap PCR was used to amply genes encoding recombinant mutant proteins rSEC 2 ( E71A), rSEC2 (E80A) and rSEC2 (E71A/E80A).The mutant proteins were expressed in E.coli BL21 (DE3) and puri-fied by affinity chromatography .The differences of biological activities between rSEC 2 and its mutants were compared in vitro.The effects of Zn 2+on the superantigenic activities of rSEC 2 were evaluated by analyzing the proliferation of splenic lymphocytes in the presence or absence of ethylenediaminetetraacetic acid ( EDTA) .Results The substitution of glutamic acid residue at position 71 and 80 by alanine residue had no significant effects on the superantigenic activities and the conformational stability of rSEC 2 mutants.How-ever, traces of Zn2+(10μmol/L) could significantly enhance rSEC2-induced proliferation of splenic lympho-cytes, and only certain amount of Zn 2+could completely restore rSEC2-induced proliferation in the presence of EDTA.Conclusion This study indicated that mutations at Zn 2+-coordinating glutamic acid residues had no significant effects on the conformational stability and the superantigenic activities of SEC 2.Zn2+might play a role in regulating the superantigenic activities of SEC 2 through some indirect ways .

Objective:To investigate the improved superantigen activity of SEC2(T20L/G22E) compared with recombinant staphylococcal enterotoxins C 2 ( rSEC2 ).Methods: The proliferation of spleen lymphocytes and T-cell subpopulations induced by rSEC2 and SEC2(T20L/G22E) were examined by WST-1 and flow cytometry separately,and the gene expression of cytokines and Vβspecificities were quantified by real-time PCR.Results: WST-1 and Flow cytometry assays showed that the superantigen activity of SEC2(T20L/G22E) was improved due to enhanced T-cell stimulating potency,resulting in massive activation of T-cells,particularly CD4+and CD8+T-cells.Quantitative real-time PCR assay showed that despite similar Vβspecificities induced by rSEC 2 and SEC2 (T20L/G22E),the quantities of activated T-cells bearing specific Vβwere different,and SEC2(T20L/G22E) could stimulate more gene expression of associated cytokines simultaneously.Conclusion: The results strongly suggested that the increased SEC 2 ( T20L/G22 E)-TCR-binding affinity contributed to more T-cells activation and cytokine release ,which elicit powerful immune activition.

A tumor-targeting recombinant fusion immunotoxin B-L-SEC2 was constructed by fusing staphylococcal enterotoxin C2 (SEC2) and an anti-HER-2 single-chain Fv B1 through a peptide linker, and expressed in E. coli strain BL21 (DE3) with an improved expression vector pASK75-EX as inclusion body. The denatured inclusion body was purified with Ni-NTA chelate agarose, and then re-natured by dialysis. FACS and MTT assays indicated that the re-natured fusion immunotoxin B-L-SEC2 could target the HER-2 over-expressing breast tumor cell SK-Br-3 in vitro, and inhibit the growth of SK-Br-3.

Aim Toinvestigatetheeffectofrecombi-nant snake venom metalloproteinase inhibitor (rSVM-PI ) on neovascularization and its molecular mecha-nism.Methods Chickenchorioallantoicmembrane (CAM)assay was used to examine the antiangiogenic effect of rSVMPI.Alamar blue analysis was used to de-tect cell proliferation.Annexin V-FITC double labeling flow cytometry was used to assay cell apoptosis. Scratch marker was used to assay cell migration.Boy-den chamber analysis method was used to detect cells chemotaxis in vitro.Tube like structure(TLS)of HU-VECs was used to detect the ability of neovasculariza-tion in vitro.Real-time PCR and Western blot were used to assay the expressions of KDR and FGFR-1 inHUVECs. Results Thevasculardensityindex (VDI)of CAM was drastically decreased after rSVMPI treatment, chemotaxis of HUVECs in response of VEGF was inhibited in the presence of rSVMPI,TLS of HUVECs was less than control group.The expres-sions of KDR and FGFR-1 were down-regulated by re-al-timePCRandWesternblotassay.Conclusion rS-VMPI may inhibit neovascularization by blocking the VEGF-KDR or bFGF-FGFR signal transduction path-way.

Objective:To investigate the optimal emergency endoscopy timing in patients with esophagogastric variceal bleeding (EGVB).Methods:The clinical data of patients with EGVB emergency endoscopy in Renmin Hospital of Wuhan University from December 2018 to November 2020 were collected and analyzed. According to the time interval from admission to the start of emergency endoscopy, they were divided into emergency endoscopy group (<6 h, n=115) and early endoscopy group (6-24 h, n=57). The baseline data, clinical efficacy and postoperative situation of the two groups were compared, and the risk factors of 6-week mortality of EGVB emergency endoscopy were analyzed by univariate and multivariate analysis. Results:In terms of baseline characteristics, there were no significant differences in age, gender, causes, shock index, model for end-stage liver disease (MELD) score, charlson complication index (CCI) score, portal hypertension related complications between the two groups ( P<0.05). However, the albumin (ALB) in emergency endoscopy group was significantly lower than that in early endoscopy group ( P<0.001). There were significant differences in Child Pugh grading and Child Pugh score between the two groups ( P=0.002, P=0.001). In terms of endoscopic efficacy, the detection rate of bleeding site in emergency endoscopy group was significantly higher than that in early endoscopy group (90.4% and 73.7%, P<0.05). There was no significant difference in operation duration, immediate hemostasis success rate, 5-day rebleeding rate, rescue treatment demand and 6-week mortality between the two groups ( P>0.05). There was no significant difference in bleeding related death between the two groups ( P>0.05). In addition, there was no significant difference in blood product consumption, intensive care unit (ICU) stay and total hospital stay between the two groups ( P>0.05). Multivariate analysis showed that Child Pugh grade C ( P=0.018), MELD score ( P=0.005) and CCI score ( P=0.001) were independent risk factors for 6-week death outcome of EGVB patients, while emergency endoscopic intervention time was not related to 6-week death outcome ( P=0.5). Conclusions:The efficacy of early endoscopic intervention is no worse than that of emergency endoscopic intervention, except for the identification of bleeding site. Child-Pugh grade C, MELD score, and CCI score are the independent risk factors for 6-week mortality, while the timing of emergency endoscopy is not associate with 6-week mortality in EGVB patients.

Objective To investigate the effects of different tidal volume ventilation on acute lung injury in rats. Methods Thlrty-two normal Wistar rats were randomly divided into four groups:control group,low tidal volume group(L- V_T),conventional tidal volume group(C-V_T)and high tidal volume group(H-V_T).The pathologic changes of the lungs were observed under macrography,light and electron microscope.The blood gas analysis(PaO_2),the counts of neutrophils (PMN),the levels of protein and the myloperoxidase(MPO)activities in bronchoalveolar lavage fluid(BALF)were measured by biochemical methods respectively.Results There were no distinct pathological differences between L-V_T group and control group under macrography,light and electron microscope.In the C-V_T and H-V_T groups,there were different degree of lung injuries under light and electron microscope,their PMN,MPO activity and protein level in BALF were significantly higher than those of control and L-V_T groups and their PaO_2 were significantly lower than those of control and L- V_T groups(P＜0.01,P＜0.05).The MPO activity and the protein level in BALF were also significantly higher than those of C-VT group(P＜0.01)Of the above indexes,there were no statistical differences between L-V_T group and control group(P＞0.05).Conclusion Conventional tidal volume ventilation alone,without any lung-protective strategy, could produce injuries to the normal lung tissues,while low tidal volume ventilation hadn't effects on them.The injury effects produced by mechanical ventilation was closely related to the recruitment and activation of neutrephils in the lung.

As a superantigen protein, Staphylococcal enterotoxin C2 (SEC2) activates the immune system effectively even in extremely low concentrations, and this property could be applied in adjuvant therapy against tumors and infectious diseases. In order to enhance the superantigen activity of SEC2, the residues at position 102-106 of native SEC2 were substituted for WWH, WWT and WWP by over-lap PCR, and three mutants named ST-1, ST-2 and ST-3 were obtained. Stimulating activity to murine lymphocytes proliferation and inhibiting activity to tumor cell growth of the three mutants were significantly improved compared with the native SEC2. Febrile activities of ST-1 and ST-3 were comparable with the native SEC2, but ST-2 showed markedly increased febrile activity than native SEC2. Moreover, the levels of IL-2, IFN-gamma and TNF-alpha secreted by T cells stimulated with the three mutants were significantly improved, which might be the possible reason for enhanced tumor cell growth inhibition activities. Furthermore, mVbeta8.2 gene transcription levels of murine splenocytes stimulated by the three mutants were dramatically increased compared with native SEC2, suggesting their increased affinities to TCR mVbeta8.2 molecular, which might be the main reason for their enhanced superantigen activities.
Animals ; Enterotoxins ; genetics ; immunology ; Female ; Interferon-gamma ; secretion ; Interleukin-2 ; secretion ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Mutant Proteins ; genetics ; immunology ; Receptors, Antigen, T-Cell ; immunology ; Superantigens ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; secretion

CAR-T cell therapy that targets surface antigens to kill tumor cells specifically has recently become another cornerstone in tumor immunotherapy. In this study, a lentiviral expression plasmid of CAR targeting human epidermal growth factor receptor 2 (HER2) was constructed by genetic engineering. The recombinant plasmid was co-transfected with other packaging plasmids into HEK293T cells by calcium phosphate precipitation to generate lenti-car, which are CAR lentiviral particles. HER2-specific CAR-T cells were obtained by transducing human peripheral blood mononuclear cells with lenti-car. Their specific inhibitory effects on HER2-positive and HER2-negative tumor cells were analyzed in vitro. The constructed CAR-T cells were specifically activated by HER2-expressing tumor cells as indicated by secretion of IFN-γ and IL-2. The inhibitory rate on HER2-positive SK-OV-3 cell line was (58.47±1.72)%, significantly higher than that on the mock-treated control group (P<0.05). The inhibitory rate on HER2-negative K562 cell lines was (11.74±2.37)%, which was not significantly different from that on the control group (P>0.05). Furthermore, when we transfected a HER2-expressing vector into K562, the inhibitory rate increased to (30.41±7.59)%, which was higher than that on HER2-negative K562 (P<0.05). Thus, the constructed second-generation HER2-specific CAR-T cells specifically suppressed growth of tumor cells overexpressing HER2 protein, suggesting that HER2-specific CAR-T cells might prove useful for immunotherapy of HER2-positive cancer.