Adolescent ; Child ; Child, Preschool ; Endoscopy ; Esophageal and Gastric Varices ; surgery ; Female ; Humans ; Infant ; Ligation ; Male

Materials Testing ; instrumentation ; Plastics ; chemistry ; Stress, Mechanical ; Temperature ; Water

Objective To observe the expression of tumor necrosis factor-α induced protein 8 like-2 (TIPE2) in CD4 + CD25 + regulatory T cells ( CD4 + CD25 + Tregs) and analyze its potential effect on immunosuppressive activity of CD4 + CD25 + Tregs. Methods CD4 + CD25 + Tregs were purified from spleen of the BALB/c mice by using magnetic cell sorting system.The expressions of TIPE2 mRNA and protein in CD4 + CD25 + Tregs were detected by using RT-PCR and Western blot.CD4 + CD25 +Tregs were further infected with the recombinant lentiviruses that carried small interference RNA(siRNA)to knock down the TIPE2 expression.Based on the expressions of cell surface molecules including cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and forkhead/winged helix transcription factor p3 (Foxp3) detected by flow cytometry and the levels of cytokines including interleukin (IL)-10 and transforming growth factor (TGF)-3 examined by ELISA in CD4 + CD25 + Tregs,the functional role of TIPE2 in controlling suppressive activity of CD4 + CD25 + Tregs was analyzed.In the meantime,the proliferation activities of T effector cells were assayed by MTT test. Results A 147 bp TTPE2 gene band and a clear TIPE2 band with a molecular mass of approximately 21 000 in CD4 + CD25 + Tregs were detected by using Westem blot.Cell surface molecule as well as cytokine expressions were significantly down-regulated when the CD4 + CD25 + Treg stimulated and activated by anti-CD3/CD28 was cultured for 24 hours after the siRNA silenced CD4 + CD25 + Treg cells ( P ＜ 0.01 ).Meanwhile,the suppression role of CD4 +CD25 + Tregs on the proliferation activity of T effector cells was weakened obviously ( P ＜ 0.05 ). Conclusions As an important immunoregulatory molecule,TIPE2 not only expresses in the CD4 + CD25 +Tregs,but also affects the immunosuppressive function of CD4 + CD25 + Tregs.

AIM: The aim of this study was to reveal the regulatory role of glutathione (GSH) in the transcriptional activity of activating transcription factor-2 (c-Jun/ATF-2) and nuclear factor-?B (NF-?B) of macrophages induced by angiotensin Ⅱ(AngⅡ). METHODS: Macrophage intracellular GSH was determined by fluorophotometry, and buthionine-[S,R]-sulfoximine(BSO)was used for depletion of intracellular GSH. The phosphorylation of c-Jun/ATF-2 and expression of NF-?B p65 were determined by immunoblot, and the activity of NF-?B was determined by electrophoresis mobility shift assay (EMSA). The c-Jun/ATF-2 was also determined by Immunohistochemical staining. RESULTS: The GSH content in the macrophage was decreased in cells that were lipid-peroxidized with AngⅡ (1.0 (?mol/L)) for 30 min and 60 min, respectively, followed by an adaptive GSH increase in the presence of AngⅡ (1.0 (?mol/L)) for longer time. In parallel, exposure to AngⅡfor 60 min also decreased macrophage GSH content in a dose-dependent manner. The GSH of RAW 264.7 cells were depleted by BSO, a specific inhibitor of GSH synthesis, and incubation for 18 h with 0.5 mmol/L BSO was sufficient for complete depletion of intracellular GSH. The phosphorylation of c-Jun/ATF-2 could be induced by the AngⅡ (1.0 (?mol/L)), whereas it did not occur in glutathione-depleted RAW 264.7 macrophages. The activation of NF-?B could also be induced by the AngⅡ (1.0 (?mol/L)), but it did not occur in glutathione-depleted RAW 264.7 macrophages. CONCLUSION: These data provide evidences that the intracellular glutathione redox may participate in the regulation of transcription activity of c-Jun/ATF-2 and NF-?B in macrophages. [

OBJECTIVE:To study the effect of polysaccharides of Radix scrophulariae on immune function in mice under nor-mal physiological and hypoimmunical state. METHODS:80 mice were randomly divided into normal group,model group,treat-ment high-dose,medium-dose,low-dose groups and physiological high-dose,medium-dose,low-dose groups,10 in each group. Mice in normal group and model group were received distilled water (10 mL/kg) intragastrically,treatment high-dose,medi-um-dose,low-dose groups and physiological high-dose,medium-dose,low-dose groups were administrated 0.16,0.08,0.04 g/kg drug,ig,once a day,for 7 d. From the third day,mice in model group and treatment high-dose,medium-dose,low-dose groups were respectively received cyclophosphamide(100 mg/kg),ip,for 4 d to induce hypoimmunical model. Carbon clearance test was adopted to determine the carbon clearance indexes and organs(chest,spleen)indexes of mice. Another 80 mice were grouped with the same administration,serum half hemolytic value (HC50) was determined after compressed Mianyang red blood cell sensitiza-tion. Then another 80 mice were grouped with the same administration,1% dinitrofluorobenzene method was used to induce de-layed hypersensitivity in mice;its ear swelling was determined,as well as IL-2,IL-4,immunoglobulin G(IgG),immunoglobulin M(IgM),γ-interferon(IFN-γ)contents in serum,and the proliferation in vitro of splenic lymphocytes were detected. RESULTS:High-dose polysaccharides can promote the increasing of immune organ and carbon clearance indexes(P<0.05);enhance the inten-sity of delayed type hypersensitivity (HC50 increasing)(P<0.01) and improve the decreasing of serum hemolysin in model mice (P<0.01);promote the splenic lymphocytes proliferation and increase IL-2,IL-4,IgG,IgM,IFN-γ contents in serum(P<0.05 or P<0.01). CONCLUSIONS:Polysaccharides can enhance the immune function in mice under normal physiological and hypoim-munical state induced by cyclophosphamide.

Objective To investigate the protective effects of ischemic preconditioning on myocardial ischemia/reperfusion injury in diabetic rats.Methods Twenty-eight healthy male rats were injected streptozotocin at dose of 45 mg/kg by tail and be fed with normal diet for 4 weeks,then rats were randomly divided into iscbemia/reperfusion (I/R) group and ischemic preconditioning (IP) group.ST segment of electrocardiograph changes and arrhythmias of all rats were recorded before ischemia and 0,15,30 minuets after ischemia and 0.5,2 h after reperfusion.TTC staining was performed to determine myocardial infarct size.TUNEL assay was used to assesse cardiomyocyte apoptosis.The expression of antiapoptotic gene (Bcl-2) and proapoptotic (Bax) was determined by immunohistochemistry.Results Compared with I/R group,ST segment elevation of patients in IP group decrease from (0.675 ±0.150) mV to (0.489 ±0.161) mV at 30 min after ischemia(P ＜0.05).Meanwhile the onset of ventricular premature contraction(VPC) in IP group was (18.21 ± 5.36) min,later than that of control group ((6.47 ± 4.28) min,t =5.241,P =0.000).The duration of VPC was (6.07 ± 4.33) min,shorter than that of I/R group ((16.71 ± 5.48) min,t =4.924,P ＜ 0.01)).The incidence of ventricular tachycardia (VT) and ventricular fibrillation (VF) of lP group remarkably decreased compared with I/R group (VT:57.14％ (8/14) vs.14.29％ (2/14),x2 =5.600,P =0.018 ; VF:50.00％ (7/ 14) vs.14.29％ (2/14),x2 =4.094,P=0.043).The myocardial infarct size in IP group was (12.50 ± 9.45) ％,smaller than that of I/R group ((37.50 ± 11.40)％,t =3.211,P =0.006).Cardiomyocyte apoptotic index (AI) was attenuated in IP group than that of I/R group((24.31 ± 3.12)％ vs.(19.01 ± 4.32)％,t =3.227,P =0.006),which was correlate with increased the ratio of Bcl-2/Bax((0.103 ±0.045) vs.(0.221 ±0.101),t =2.670,P =0.015).Conclusion IP treatment for diabetic rats shows a protect effect on myocardial I/R injury through attenuating myocardial apoptosis,and increasing the ratio of Bcl-2/Bax.

Objective To study the potential role of tumor necrosis factor-α (TNF-α) induction in the development of mucosal barrier dysfunction in rats caused by acute intestinal ischemia-reperfusion injury, and to examine whether pretreatment with monoclonal antibody against TNF-α (TNF-α MoAb) would affect the release of D(-)-lactate after local gut ischemia followed by reperfusion. Methods Anesthetized Sprague-Dawley rats underwent superior mesenteric artery occlusion for 75 min followed by reperfusion for 6 hr. The rats were treated intravenously with either TNF-α MoAb (20 mg/kg) or albumin (20 mg/kg) 30 min prior to the onset of ischemia. Plasma D(-)-lactate levels were measured in both the portal and systemic blood by an enzymatic spectrophotometric assay. Intestinal TNF-αmRNA expression as well as protein levels were also measured at various intervals. In addition, a postmortem examination was performed together with a macropathological evaluation based on a four-grade scoring system.Results Intestinal ischemia resulted in a significant elevation in D(-)-lactate levels in the portal vein blood in both the control and treatment groups ( P ＜0.05). However, animals pretreated with TNF-α MoAb at 6 hr after reperfusion showed significant attenuation of an increase in both portal and systemic D(-)-lactate levels when compared with those only receiving albumin (P ＜ 0.05). In the control animals, a remarkable rise in intestinal TNF-α level was measured at 0.5 hr after clamp release ( P ＜ 0.01); however, prophylactic treatment with TNF-α MoAb completely annulled the increase of local TNF-α levels seen in the control animals. Similarly, after anti-TNF-α MoAb administration, intestinal TNF-α mRNA expression was markedly inhibited, which showed significant differences when compared with the control group at 0.5 hr, 2 hr and 6 hr after the release of occlusion ( P ＜ 0.05-0.01 ). In addition, the pathological examination showed marked intestinal lesions that formed during ischemia, which were much worse upon reperfusion,particularly at the 6 hr time point. These acute injuries were obviously attenuated in animals receiving TNF-α MoAb.Conclusions It appeared that acute intestinal ischemia was associated with failure of the mucosal barrier, resulting in increased plasma D(-)-lactate levels in both portal and systemic blood. These results suggest that TNF-α appears to be involved in the development of local damage associated with intestinal ischemic injury. Moreover, prophylactic treatment with TNF-α MoAb exerts preventive effects on ischemia/ reperfusion-induced circulating D (-)-lactate elevation and gut injury. ( J Geriatr Cardiol 2004;1(2):119-124. )

Cell Membrane ; metabolism ; Gastrointestinal Stromal Tumors ; diagnosis ; metabolism ; Humans ; Immunohistochemistry ; Proto-Oncogene Proteins c-kit ; analysis

Objective:To clone and express the Staphylococcus aureus Efb(extracellular fibrinogen-binding protein) protein in Escherichia coli, to purify the expression product and prepare its functional antibody and to detect the functions of Efb protein for further studies on S.aureus infection.Methods: Efb gene was amplified by PCR using S.aureus NCTC-8325 genome DNA as template and cloned into the recombinant expression vectors pET28a. E.coli BL21(DE3) with the plasmid was induced with IPTG for protein production. The protein was purified by Ni~(2+) affinity chromatography. The function of Efb protein was determined by complement activity assay and inhibition ELISA.The polyclonal antibodies were prepared by immunizing the animals. Results: The purified recombinant Efb was obtained, which could inhibit the CH50 and AH50 effectively. The functional poly-antibodies of Efb were prepared.Conclusion:Efb could inhibit the classical pathway and alternative pathway of complement activation, and the antibodies against to Efb could block the inhibition of the classical pathway of complement activation induced by Efb.

Objective To explore the mechanism and inhibition of botulinum toxin type A (BTXA) on hypertrohic scar fibroblasts.Methods The cells were treated by 0 (control),0.2,0.4,0.8 U/ml BTXA for 48 h.Cell viability was detected by MTT assay.Cell apoptosis was detected by Hoechst staining.Cell cycle was detected by flow cytometry.The level of cell cycle related protein D1 (Cyclin D1),proliferation nuclear antigen (PCNA) and activation of phosphatidylinositol 3 kinase (PI3K) / protein kinase B (AKT) signaling pathway were assayed by western blot.Results Compared with control group(0.75±0.07),0.2,0.4,0.8 U/mL BTXA(0.59 ± 0.06,0.43 ± 0.04,0.34± 0.03) inhibited hypertrohic scar fibroblasts cell viability,increased cell apoptotic rate[control group(2.38±0.24)％;BTXA(15.79±1.54)％,(27.32±2.69)％,(38.46±3.90)％],down-regulated the expression of Cyclin D1(control group 1.57±0.18;BTXA 0.93±0.07,0.42±0.04,0.35±0.03) and PCNA(control group 1.46±0.16;BTXA 0.50±0.05,0.59±0.05,0.37±0.03),inhibited the expression of PI3K(control group 0.98±0.06;BTXA 0.49±0.04,0.50±0.04,0.39±0.03) and the phosphorylation of AKT(control group 1.38±0.08;BTXA 0.97±0.06,0.60±0.04,0.29± 0.02),made cell cycle arrested in G1 phase,The difference was statistically significant (P＜0.05).Conclusion These results suggested BTXA inhibit proliferation via blocking the activation of PI3K/AKT signal pathway and down-stream related cell cycle related protein.