Objectives To detemline whether apoptotic cell death is involved in rat cardiac allograft rejection and investigate the relevance of apoptosis with acute rejection and its implication.Methods Groups of Wistar rats underwent heterotopic heart transplantation from allogeneic SD or syngeneic Wistar rats.The cardiac grafts were harvested at 1,3,5,or 7 days after transplantation and underwent the detection of apoptotic cell death using in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling(TUNEL).Histopathological rejeclion grade and apoptotic index(AI)were analyzed.Results The incidence of apoptotic cells was increased steadily over time in allografts,in contrast to syngeneic grafts.The apoptotic cells in allografts were mainly cardiac myocytes and few infiltrating lymphocytes.The AI of rejection grade 1,2,3 and 4 was significantly higher than that of rejection grade 0(P<0.01).Conclusions TUNEL can display apoptosis of single cell in situ.Apoptosis is an important mechanism of tissue injury in acute cardiac allograft rejection in rats.Myocyte apoptosis can be used as a valuable index to estimate the injury of grafts and monitor acute rejection.

Objective To evaluate the effect of Toxoplasma gondii infection on immune function of the rat recipients and onset of Toxoplasmosis after heart transplantation and its correlation with the use of Cyclosporin A(CsA). Methods ELISA was used to detect recipient's specific circulating antigen (CAg) and antibodies (IgG, IgM) after the transplantation. T lymphocyte subsets in peripheral blood were examined by using immunofluorescence stain and flow cytometry (FCM) before and after heart allograft 5,10,15,20 days in rats. Results The use of CsA increased the risk of infection by T. gondii and accelerated the increase of CD8 + T lymphocyte after the transplantation. The incidence of donor acquired T.gondii infection was higher than that of reactivated silent infection in recipients before operation. The percentage of CD8 + T lymphocyte was evidently elevated due to the onset of toxoplasmosis and the ratio CD4 +/CD8 + was reduced or inverted in the meanwhile. Conclusion The immune suppression after use of CsA was the main reason leading to an activation of the silent infection of T.gondii . CD8 + was the main cytotoxic cell elevated during the infection.

Objective To evaluate the relationship between the use of CsA and onset of toxoplasma gondii after organ transplantation and study the effect of pathogen acquired path on toxoplasma infectious incidence.Methods ELISA was used to detect recipient's specific CAg and antibodies (IgG, IgM) after organ transplantation of rats. Results The incidence of donor acquired T.gondii infection was higher than that of reactivation of recipient latent infection before operation. The use of CsA could increase the risk of infection by T. gondii after organ transplantation. The use of CsA influenced the detection of anti IgG,IgM sometimes but had no effect in early diagnosis of T.gondii infection through detection of CAg.Conclusion The use of CsA was the main reason leading to infection of toxoplasma gondii after organ transplantion. Detection of specific anti IgG,IgM combined with clinical symptom could be used in early diagnosis.

ObjectiveTo investigate mechanisms for IL-27 induced proliferation and differentiation of peripheral blood CD4+ T cells in primary biliary cirrhosis (PBC).MethodsPeriperal blood CD4+ T cells were isolated from patients with PBC,chonic hepatitis B (CHB) and health controls (HCs).After IL-27 stimulation,proliferation ability of CD4+ T cells was evaluated by CCK-8 kit,and cytokines were analyzed by ELISA.Real-time PCR was employed to assay mRNA expression of T-bet and GATA3 in CD4+ T cells.p-STAT-1 and pSTAT-3 expression in CD4+ T cells were detected by Western blot.ResultsEnhanced proliferation of CD4+ T cells was found in all subjects after IL-27 stimulation.However,the proliferation ability in patients with PBC was greater than that in CHB and HCs ( P＜0.001 ).Levels of IL-2 and IFN-γ in supernatant from IL-27-incubated PBC blood CD4+ T cells were higher than that from CHB and HCs (P＜0.001 ).In normal situation,T-bet mRNA of CD4+ T cells in PBC group was higher than that in CHB group (P=0.007).Furthermore,after IL-27 stimulation,elevated T-bet mRNA expression and GATA3 inhibition were found in patients with PBC.High expression of p-STAT-1 and p-STAT-3 in blood CD4+ T cells were found in PBC,CHB and HCs after stimulation by IL-27.But their expression in patients with PBC were higher than those in patients with CHB and HCs.ConclusionProliferation of blood CD4+ T cells could be induced by IL-27 in patients with PBC.The signaling pathways of p-STAT-1,p-STAT-3 were involved to induce Th1 immune response and related cytokines expression.This study implicated that IL-27 may play important roles in early inflammation damage in PBC.

Objective To investigate the correlation between immune inflammation and overactivity of T helper cells in childhood asthma by cell proliferation assay and activation induced cell death in vitro.Methods Th1/Th2/Th17 cytokines were determined by cytometric bead array.Cell proliferation and activation induced cell death were detected when CD4+ T cells were purified by magnetic beads and stimulated by PHA and antiCD3.At last,mRNA of Fas,FasL and Bcl-2 were mesured by real-time PCR.Results Cytokines of IL-4(2.451± 1.052ng/L vs 1.796 ±0.615 ng/L,P =0.018),IL-10( 1.920 ±0.813ng/L vs 1.390 ±0.162ng/L,P =0.006)and TNF(5.112 ±5.842 ng/L vs 1.506 ±0.551 ng/L,P =0.009) in sera of asthma group were higher than those in control group.Compared to control group,proliferation ability of CD4 + T cells in asthma group was greater ( OD450:0.498 ± 0.052 vs 0.274 ± 0.032,P ＜ 0.001 ) and apoptosis rate was lower( 35.62 ± 0.05 ％ vs 65.28±3.85％,P ＜0.001 ).mRNA expression of Fas in asthma group was lower but Bcl-2 was higher than those in control group.Conclusion It is implicated that defective expression of Fas and over expression of Bcl-2 in CD4+ T cells may contribute to apoptosis inhibition and cell proliferation,which could explain overeactivity of CD4 + T cells and lvmphocvte infiltration in childhood asthma.

Objective To investigate the relationship between cardiac vagus nerves and changes of connexins(Cx)and intracellular gap junction(GJ)distribution pattern in superior vena cava(SVC)myosleeve in dog with atrial fibrillation(AF).Methods Twenty four hybrid dogs were divided into sham operation group(Sham group,n=8),SVC-AO fat pad removed group(RM group,n=8)and SVC-AO fat pad reserved group(RS group,n=8).In RM group and RS group,right atrial pacing was performed at a frequency of 500～650/min for 6 weeks to establish AF model.AF was induced by programmed stimulation or burst stimulation of atrial pacing.The expression and distribution of Cx40 and Cx43 in the SVC myosleeve tissue in three groups were analyzed by immunofluorescence staining.Transmission electron microscopy was used to observe the uhrastructural organization of gap junction(GJ).Results The rate of inducing sustained AF(＞ 15 min)in RS group was significantly higher than that in RM group (P ＜ 0.01).The expression of Cx40 and Cx43 in the SVC myosleeve in sham group and RS group were significantly higher than that in RM group(P ＜ 0.05).Furthermore,the expression of Cx40 and Cx43 in RS group were obviously higher than that in sham group(P ＜0.05).The ratio of end-to-end to side-to-side in RS group was lower than that in Sham group and RM group.Comparing with RM group,the channel of GJ became shorter and wider in RS group(P ＜0.05).Sarcomere was dissolved and mitochondrion showed vacuole degeneration in RS group.Conclusion The remodeling of Cx40 and Gx43 in SVC myosleeve tissue may be mediated by vagus nerves.It is conducive to the maintenance and stability of AF.However,this effect can be weakened by removing SVC-AO fat pad of canine.

Objective To investigate the population and role of IL-10+ CD19+ regulatory B cell (Breg) in patients with chronic hepatitis B.Methods Patients with acute hepatitis B (AHB) (n =28),chronic hepatitis B (CHB) (n =31) and normal subjects (n =25) were collected from Changshu No.2 People's Hospital between 2011 June and 2012 October.Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with CpG ODN 2006 and PMA.Flow cytometry was used to analyze the population of IL-10-CD19 + Breg,CD4 + CD25high Treg,and ELISA was used to analyze the concentration of IL-10 in culture supernatant.Results The population of Breg in Peripheral blood of the CHB group [1.28％ (1.05％-2.20％)] was higher than that in the AHB group [0.87％(0.55％-1.22％)] and the HCs group [0.89％ (0.51％-1.37％)] (P =0.001,0.006),and the difference between the AHB group and the HCs group was not statistically significant (P=0.669).Breg in the CHB group [14.30％ (10.70％-16.70％)] was higher than that in the AHB group [10.30％ (7.05％-13.30％)] and the HCs group [10.40％(6.85％-12.60％)] (P =0.003,0.001),treg in the CHB group [5.80％ (4.20％-9.10％)] was also higher than that in the AHB group [4.05％ (2.53％-5.40％)] and the HCs group [4.50％ (2.55％-5.50％)] (P ＜0.001,P =0.005),and there was no significantly difference between the AHB group and the HCs group (Breg:P =0.796 ; Treg:P =0.227).Spearman correlation analysis showed that Breg was positively correlated with Treg in the CHB group (r =0.50,P =0.004),however there was no significantly correlation in the AHB group and the HCs group (r =-0.15,P =0.462; r =0.09,P =0.669).The concentration of IL-10 in the CHB group was higher than that in the AHB group and the HCs group (P ＜ 0.001),and the difference between the AHB group and the HCs group was not statistically significant (P=0.341).Spearman correlation analysis showed that IL-10 were positively correlated with the population of Breg in the CHB group (r =0.409,P =0.022).Conclusion The poluations of regulatory B cell and regulatory T cell increased in patients with chronic hepatitis B,and Breg cell might play the immune regulation role through secreting IL-10 in chronic HBV infection.

Objective To investigate the roles of TLR3 pathway activiated by polyI:C in proliferation and apoptosis of multiple myeloma (MM) RPMI8226 cell line.Methods RPMI8226 cells were cultured in RPMI 1640 with different dose of polyl:C.Cells were collected in different time.Proliferation and apoptosis were detected by CCK-8 kit and flow cytometry,separately.Results The proliferation of RPM18226 was inhibited by polyI:C,and it was dose and time dependent,24 h:12.30％ ±2.04％,22.50％±2.20％,37.90％ ±1.30％ ; 48 h:17.80％ ±1.52％,29.60％ ±0.85％,45.80％ ±1.68％ ;72 h:25.10％±1.01％,34.60％±1.27％,60.50％±2.08％,P＜0.05.RPMI8226 cells were incubated with 50 μg/ml,100 μg/ml and 200 μg/ml polyI:C for 48 h.Apoptotic rate were 5.60％ ±1.06％,8.71％ ±1.06％ and 13.93％ ±1.17％,P＜0.05.TLR3 and TRIF mRNA expression increased obviously and dose dependent,TLR3:1.41±0.10,2.24±0.16,4.08±0.13; TRIF:1.07±0.16,1.97±0.13,3.56±0.19,P＜0.05.Conclusion The proliferation of MM cells were inhibited by TLR3 pathway obviously,and apoptosis was induced by polyI:C.

Objective To investigate a new therapeutic pathway for primary biliary cirrhosis (PBC) by immune tolerance reestablishment in a PBC mouse model. Methods Spleenic cells from naive mice were incubated with M2 in the presence of ECDI and the ceils were injected into caudal vein of the mice which would be used for development of PBC model. Spleenic cells incubated with bovine serum albumin (BSA) were injected as controls. 16 weeks later, anti-mitoehondrial antibody (AMA) , alkaline phosphatase(AKP) and portal inflammation were assayed for evaluating the prevention effect. Results AMA positive rate in tolerance group was lower than that in BSA and PBC groups ( P = 0. 007, P = 0. 003 ). The difference between BSA and PBC was not significantly. Serum AKP levels in tolerance, BSA and PBC group were (80.5 ±9.8) U/L, (93.8 ±15.7) U/L and (92.5 ±17.7) U/L, separately. The level in tolerance group was lower than that in BSA and PBC groups (P =0.0095, P =0.029). The rates of portal areas with cell infiltration were 42. 67% ± 12. 30% , 57. 07% ± 11. 35% and 51. 53% ± 9. 96% , separately. The number of infiltrated portal tracts in tolerance group was less than that in PBC group (P = 0.039) and BSA group (P = 0. 0024). Conclusion PBC was prevented to some extent by reestablishing immune tolerance to M2 autoantigen. This provides clues for finding a better treatment proposal.

Objective To investigate the safety and efficacy of nephron-sparing surgery (NSS)for selective T2 stage renal tumor.Methods The surgical database of 26 patients treated with NSS for clinical T2 stage renal cell carcinomas between March 2010 and May 2013 were collected and analyzed retrospectively.There were 17 males and 9 females,with a mean age of 52 years (39-74 years),mean tumor size of 10.3 cm(7.2-16.5 cm),and mean R.E.N.A.L score of 7.5 (6-10).Patients'demographics,clinical characteristics,oncologic outcomes,renal function were reviewed.Results The renal masses were removed successfully and the surgical margins were negative.There were 21 (80.8％) cases of clear cell carcinoma,4 (15.4％) papillary carcinoma and 1 (3.8％) chromophobe carcinoma.The mean ischemia time was (28.3 ± 12.5) minutes (7 patients were clamp-free).Three patients needed transfusion,one experienced urine fistula and cured by conservative treatment,and one patient's renal function got progressive worsening and required long-term hemodialysis.The average serum creatinine was 121 μ mol/L before and 136 μmol/L after surgery (P =0.06).After a period of 22-47 months' follow-up,no patient had local recurrence or metastasis.Conclusions NSS can be safely performed and provide effective oncologic outcomes for selective patients with clinical T2 stage renal cell carcinomas.R.E.N.A.L nephrometry is an important factor and should be used to evaluate the feasibility of NSS.