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.

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 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.

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 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. )

Objective To investigate the role of C-Jun N-terminal kinase (JNK) in epithelial mesenchymal transition (EMT) induced by transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells(RPMCs). Methods RPMCs were harvested from the peritoneum of male Sprague-Dawley rats, then cultured in DMEM/F12 medium with 15% (V/V) FBS. After stimulation with TGF-β1, the expression of a-smooth muscle actin (α-SMA), E-cadherin and collagen I were detected in RPMCs. In some groups, the ceils were pretreated with SP600125, a specific inhibitor of JNK, for 4 hours before incubation with TGF-β1. The protein expression of phosphorylated JNK was detected by Western blotting. The mRNA and protein expression ofα-SMA, E-cadherin and collagen I were examined with RT-PCR and Western blotting, respectively.The intracellular distribution and expression of α-SMA was determined by indirect immunofluorescence. Results TGF-β1 could significantly increase the expression of α-SMA and collagen I, and decrease the expression of E-cadherin in RPMCs. TGF-α1 could stimulate the expression of phosphorylated JNK at 5 minutes with the peak at 10 minutes (P<0.01). The addition of SP600125 effectively inhibited TGF-β1-induced high expression of α-SMA and collagen I (P<0.05), and prevented TGF-β1-induced down-regulation of E-cadherin expression in RPMCs (P<0.05). The indirect immunofluorescence showed that the expression of intracellular α-SMA in RPMCs stimulated by TGF-β1 for 48 h increased significantly, which could be inhibited by SP600125. Conclusions JNK regulates epithelial mesenchymal transition induced by TGF-β1 in rat peritoneal mesothelial cells. JNK inhibitor may be used as a novel therapeutic agent for peritoneal fibrosis.

Objective To investigate the effect of high mobility group box-1 protein (HMGB1) on cytokine expression in splenic dendritic cell (DCs). Method DCs isolated from the spleens of male Wistar rats were seed-ed on 96-well (1×10s cells/well) cell culture plates, and the cells were stimulated with HMGB1 for various length of time or in different concentrations. (1) The time-dependent response between HMGB1 and tumor necrosis factor-α(TNF-α) as well as interleukin-12 (IL-12) gene/protein expressions: 24 wells of DCs were dividedly into six groups including 24 h-normal controls (n=4), 48 h-normal controls (n=4), 72 h-normal controls (n=4), and 24 h-HMGB1 treated group (n=4), 48 h-HMGB1 treated group (n=4) as well as 72 h-HMGB1 treated group (n=4), respectively. Among three HMGB1-treated groups, DCs were stiraulated by 1 μg/mL HMGB1. DCs were denatured in cell culture plates to determine gene expression of IL-12 as well as TNF-α, and supernatants were harvested to determine Il-12 as well as TNF-α protein levels. (2) The dose-dependent response between HMGB1 and TNF-α as well as IL-12 gene/protein expressions: 16 wells of DCs were dividedly into four groups in-cludingnormal controls (n=4), 0.1 μg/mL HMGB1 treated group (n=4), 1 μg/mL HMGB1 treated group (n =4), and 10 μg/mL HMGB1 treated group (n=4), respectively. After stimulated for 48 h, DCs were dena-tured in cell culture plates to determine gene expression of IL-12 as well as TNF-α, and supernatants were harvest-ed to determine IL-12 as well as TNF-α protein levels. Total RNA was extracted from cells using the single-step technique of acid guanidinium thiocyanate-chloroform extraction according to the manufacturer' s instruction (Promega, Madison, WI). mRNA for TNF-α and IL-12 were quantified by SYBR Green two-step, real-time re-verse transeription-polymerase chain reaction taking glyceraldehyde-3-phesphate dehydrogenase (GAPDH) as an internal standard. Levels of IL-12 and TNF-α in cell culture supernatants were determined with ELISA, strictly fol-lowing the protocols provided by the manufacturer. Data were analyzed with a one-way ANOVA. A P-values <0.05 were considered statistically significant. Results After stimulation with 1 μg/mL HMGB1, IL-12 and TNF-α protein and gene expressions in rat splenic DCs were markedly up-regulated at 24 h to 72 h (P<0.05 or P<0.01), and the expression levels of IL-12 and TNF-α peaked at 48 h (P<0.01). When DCs were cultured in the presence of 0.1 μg/mL, 1 μg/mL, and 10 μg/ml HMGBI for 48 h, expressions of IL-12 and TNF-α were also significantly up-regulated (P<0.01), and values of these cytokines were highest in 1 μg/mL HMGB1-treated group (P<0.01). Conclusions These data suggest that HMGB1 appears to be a potential immunostimulatory signal that induced DC maturation, and HMGB1 stimulation can result in marked up-regulation of IL-12 as well as TNF-α synthesis and release in splenic DCs.

Objective To investigate the immunological activity change of regulatory T cells (Treg) and discuss its significance in the outcomes of patients with multiple organ dysfunction syndrome (MODS) and severe burn. Methods A total of 106 patients with total burn surface area (TBSA) larger than 30% were included in the study and randomly divided into three groups according to the burn area: Group Ⅰ (TBSA of 30%-49%, n = 41), Group Ⅱ (TBSA of 50% -69%, n = 34) and Group Ⅲ (TBSA of 70%-99%, n = 31). According to the development of MODS, patients were divided into MODS group (n =21) and non-MODS group (n =85). The patients with MODS were further divided into non-survival group (n = 16) and survival group (n = 5) based on their outcomes. Healthy volunteers were served as normal control (n = 25). Peripheral blood samples were collected at days 1,3,7, 14 and 21 postburn. The immunomagnetic separation technique was applied to separate and purify CD4+ CD25+Tregs in peripheral blood, and phenotypes (CTLA-4) were analyzed by flow cytometry and the contents of interleukin-10 released in the supernatants were determined by ELISA. Results Expression of CTLA-4 and level of IL-10 were significantly increased in burn patients compared with normal control group, with statistical differences. The expression of CTLA-4 and level of IL-10 were significantly increased in patients with severe burns at all time points. The expression of CTLA-4 and level of IL-10 in MODS group were much higher than those in non-MODS group at days 3-21 postburn (P ＜ 0.01). Among the MODS patients, the expression of CTLA-4 and level of IL-10 in the survival group were obviously lower than those in the non-survival group at days 3-21 postburn (P ＜ 0.05 or P ＜ 0.01). Conclusions After severe burn injury, expressions of the markers on CD4 + CD25 + Treg surface and secretion of cytokines produced by CD4 + CD25 + Tregs show significant difference in patients with different born areas, MODS development and survival state. CD4 + CD25 + Treg may play an important role in the pathogenesis of immunoregulation, MODS and mortality of burn patients through secretion of inhibitory cytokines.