Objective To observe the effect of combination of Tju103 and CTLA4-Ig on engraftment, graft versus host disease (GVHD), graft versus leukemia (GVL) and anti-infection post major histocompatibility complex (MHC) haploidentical bone marrow transplantation in mice in order to seek an effective access to transplantation with less GVHD and more potential GVL and anti-infection.Methods In the presence of the recipient's antigen (normal CB6F1, H-2 bd) as a stimulus for induction of specific immune tolerance, T cells from the MHC haploidentical donors (C57BL/6, H-2 b) were first in vitro cultured with Tju103 and CTLA4-Ig, then were transfused with the donors' bone marrow cells into the preconditioned recipients. At last, the effect of combination of Tju103 and CTLA4-Ig on hematopoietic rebuilding, GVHD, GVL and anti-infection was observed in compared with CsA, Tju103 and CTLA4-Ig as controls.Results The only irradiated group (group A): All the mice (10 mice) died of failure of hematopoiesis within 11 days post irradiation, of which most (8 mice) died within 4～7 days post transplantation. The CTX-treated leukemia group (group B): All the mice (10 mice) died of leukemia within 16～23 days post leukemia cells infusion (11～18 days post BMT). CTX treatment prolonged the survival time. The only transplanted group (group C): The mice began to die from day 16 post transplantation, and all (10 mice) died of GVHD within 3 weeks. The CsA prophylaxis group (group D): 4 mice died within 8～22 days after transplantation, of which one died of leukemia, two died of infection and one died of GVHD, and the remaining 6 survived over 30 days post transplantation. The Tju103 treated group (group E): 4 mice died within 9～26 days post transplantation, of which one died of leukemia, one died of infection and two died of GVHD, and the remaining 6 mice survived over 30 days post transplantation. The CTLA4-Ig treated group (group F): 3 mice died within 14～23 days after transplantation, of which one died of infection and two died of GVHD, and the remaining 7 survived over 30 days post transplantation. The Tju103/CTLA4-Ig treated group (group G): one died of GVHD on day 19 after transplantation, and the remaining 9 mice survived over 30 days post transplantation. Conclusions CsA, Tju103 or CTLA4-Ig alone could prolong survival time and reduce incidence and degree of GVHD severity. But CTLA4-Ig could spare much ability of GVL and anti-infection while Tju103, just like CsA, couldn't. Combination of both was the most favorable way for transplantation with the most remarkable efficiency on prolongation of survival time and reduction of GVHD.

Objective To observe the effect of Ly49A transfected lymphocytes on graft versus host disease (GVHD) and graft versus leukemia (GVL) effect post allogeneic bone marrow transplantation. Methods Ly49A gene was transfected into lymphocytes of C57BL/6 mice by retrovirus and the expression rate of Ly49A receptor was detected by flow cytometer. The murine model of allogeneic acute GVHD was established in this way: the donor was C57BL/6(H 2 b). The recipient was BALB/c(H 2 d), which was injected EL9611 cells. After the irradiation (TBI, 60 Co 9.0 Gy) the recipient accepted the transplantation of mixed spleen cells and bone marrow cells to establish the GVHD model. The effects of Ly49A transfected spleen cells on GVHD and GVL post allogeneic bone marrow transplantation were detected. Results The expression rate of Ly49A receptor was ( 42.20 ? 4.87 )?% for lymphocytes transfected with pLXSN Ly49A; ( 18.67 ? 2.48 )?% for lymphocytes transfected with pLXSN and ( 18.73 ? 3.82 )?% for untransfected control respectively. In the case of allo BMT (C57BL/6 H 2 b→BALB/c H 2 d), survival time was ( 6.50 ? 2.41 ) days for the irradiation group; ( 20.90 ? 2.88 ) days for cyclophosphomide therapy group; ( 17.10 ? 4.65 ) days for bone marrow cells mixed with lymphocytes transplantation group; ( 17.40 ? 5.32 ) days for bone marrow cells mixed with pLXSN transfected lymphocytes transplantation group; ( 35.20 ? 12.52 ) days for bone marrow cells mixed with Ly49A transfected lymphocytes transplantation group, which was much longer than the survival time of any of the above group ( P = 0.000 ). Conclusion The Ly49A transfected lymphocytes transplantation could alleviate GVHD and retain GVL effect on acute GVHD model post allo BMT.

Objective To investigate if the administration of CORM-2 can provide protection against renal ischemia-reperfusion injury (IRI).Method Murine renal ischemia was induced by clamping left renal pedicles for 40 min with vascular micro damps at 32 C,then the contralateral kidney was removed.CORM-2 or vehicle was administered via intravenous infusion 1 h before the onset of ischemia.The blood plasma and renal samples were obtained at 24 h after reperfusion to assess renal function and cellular injury.Plasma Cr and BUN levels,HE and TUNEL were performed to estimate the magnitude of renal damage.Kidneys were retrieved from indicated animals at various time points after renal IRI,and the sections were prepared for histological evaluation.MPO staining procedures were performed to assess the neutrophils infiltration in the renal IRI.Besides,Immunofluorescent stain of TNF-α was performed on the kidneys which were retrieved from indicated animals to determine the production of inflammatory mediators in renal I/R.Results The plasma Cr and BUN were significantly increased at 24 h after reperfusion in IRI control mice,and CORM-2 treatment could markedly diminish the increase of plasma Cr and BUN in mice subjected to I/R.In parallel,histological analysis demonstrated that CORM2 treatment markedly reduced apoptosis of the renal tubular epithelium cells and hemorrhage.IRI caused marked infiltration and accumulation of the MPO-positive neutrophils in renal interstitium.Administration of CORM-2 before ischemia dramatically inhibited neutrophils infiltration as compared with IRI or iCORM-2 group.Furthermore,we confirmed that CORM-2 markedly decreased production of TNF-α.Conclusion Carbon monoxidereleasing molecule CORM-2 could ameliorate inflammation to protect against the renal IRI in mice.

Objective To investigate whether rat adipose-derived stem cells (rASCs) could resist human xenoantibody-dependent complement-mediated lysis and to explore its possible mechanisms.Method SD rat ASCs were isolated,rASCs at passage 2 to 8 were used for the following studies and rat lymphocytes were harvested as control cells.α-Gal expression was detected by flow cytometry.After incubation of rASCs with 20％ normal human serum (NHS) or heat inactivated normal human serum (HINHS),flow cytometry was used to detect cytotoxicity,IgG or IgM binding,and C3c,C4c and C5b-9 deposition.Result We successfully established the method to isolate and culture rASCs.The morphology of rASCs remained unchanged after passages.rASCs were positive for tell surface markers of CD44 and CD90,while negative for CD45 and MHC-Ⅱ.As compared with rLCs,rASCs significantly resisted human natural antibody and complement-mediated lysis when incubated with 20％ NHS in vitro (20.42％ ± 2.80％ vs 51.84％ ± 6.70％,P ＜ 0.01).Mechanistically,rASCs expressed lower level of α-Gal (13.97 ± 0.33 vs.24.47 ± 3.03,P＜0.05),which was correlated with decreased binding of human xenoreactive IgG and IgM (IgM:9.4 ± 2.0 vs.107.2± 4.8,P＜0.01; IgG:5.73 ± 1.0 vs.27.49 ± 3.9,P＜0.01) and reduced deposition of complements C3c,C4c and C5b-9 (C3c:294.6 ± 38.02 vs.1924 ± 509.4,P＜0.05; C4c:35.23 ± 3.1vs.177.3 ± 37.17,P＜0.05; C5b-9:5.63 ± 1.74 vs.37.05 ± 7.4,P＜0.01).Conclusion These data demonstrated that the resistance of rASCs to human xenoantibody and complement-mediated lysis is associated with low expression of xenoantigen a-Gal and inhibition of MAC (membrane attack complex) formation.

Objective To explore the differentiation of allogeneic naive T cells to regulatory T cells (Tregs) and T helper (TH) 1/2/17 cells by coculture with bone marrow-derivedimmature dendritic cells (irnDC).Method Bone marrow-derived imDC were cultivated from Balb/c mice.Lipopolysaccharide-stimulated DC were harvested as mature dendritic cells (mDC) and unstimulated cells were collected as imDC.Then irnDC or mDC were cocultured with allogeniec naive T cells,respectively.TH1 cytokines [interferon-γ (IFN-γ) and interleukin (IL)-2],TH2 cytokines (IL-4 and IL-10),and TH17 cytokine (IL-17) of co-cultured cells were detected by enzyme linked immunospot assay.CD4+ Forkhead box p3 (FoxP3) + Treg proportion in CD4+ cells in the co-cultured system with IL-2 and transforming growth factor-β1 (TGF-β1) was analyzed by flow cytometry.Result As compared with mDC,na(i)ve T cells cocultured with imDC secreted much less IFN-γ (11.67 ± 2.18 vs.182.00±23.71,P＜0.01),IL-2 (26.67±2.96 vs.318.30± 18.62,P＜0.01),IL-4 (17.00±3.78 vs.45.33±3.48,P＜0.01),IL-10(7.00±1.00vs.158.70±10.90,P＜0.001) and IL-17 (0.66 ± 0.33 vs.238.30 ± 24.39,P＜0.001).Furthermore,imDC induced more CD4+ FoxP3+ Tregs than mDC after adding IL-2 and TGF-β1 in the coculture system for 7 days (22.70 ± 1.53 ％ vs.5.42 ± 1.27％,P＜0.01).Conclusion imDC are more effective to induce na ve T cells to Tregs,but not differentiate to TH 1/TH 2/TH 17 cells.These findings provide in vitro experimental evidence for induction of transplant tolerance by adoptive transfer of imDC.

Objective To establish a rapid and high sensitive assay of real-time fluorescence loop-mediated isothermal amplification assay for clinical detection of HIV-1 DNA.Methods Four loop primers were constructed for loop-mediated isothermal amplification ( LAMP) assay based on six conserved regions selected from HIV gene sequence .SYBR Green Ⅰdye was added into the established LAMP assay and its specificity and sensitivity were evaluated .Results The real-time fluorescence LAMP assay for the detection of HIV-1 DNA was successfully established .It was used for the detection of HIV-1 DNA among 200 patients with HIV infection, of which 195 cases were positive.Moreover, 50 healthy subjects were found HIV-1 DNA negative tested by the real-time fluorescence LAMP assay .Quantitative testing for HIV-1 DNA showed that the lowest and the highest detectable amount were 51 copies/ml and 8.21×106 copies/ml respectively, and the average amount was 5.78×105 copies/ml.HIV viral loads ranging from 1×105 to 10×105 were detected in 162 of 200 patients (83.08%).The ten times dilution method showed that the lowest detection limit of the assay was 50 copies/ml.The crossover experiment indicated that the specificity of the assay was 100%as none of HBV-DNA, HSV-DNA and HCV-RNA was determined by the assay .Conclusion The present study shows that 97.5%of the patients with HIV infection are confirmed HIV-1 DNA positive by the real-time fluorescence LAMP assay , suggesting that the real-time fluorescence LAMP assay is a rapid and sensi-tive assay with high specificity and could be applied for clinical detection of HIV-1 DNA.

Objective To explore the impact of different hemoglobin on postoperative cognitive functions in female patients underwent laparoscopic surgery. Methods 215 female patients who were underwent laparoscopic total hysterectomy or laparoscopic myomectomy from January 2016 to December 2016 in our hospital were selected. The patients were divided into 3 groups according to their hemoglobin:Group N with normal hemoglobin ,Group A with hemoglobin concentration of 9 ~ 11 g/L ,and Group B with hemoglobin concentration of 6 ~ 9 g/L. Same anesthesia and recovery plan were performed in all patients. Basic and operative information were recorded. The score of MoCA on the day before surgry ,the day after surgry ,3 days after surgry and 30 days after surgry were recorded. Blood samples were collected on the day before surgry and the day after surgry for the determina-tion of serum S-100β protein and IL-6 concentrations. Results No significant difference was found in basic and operative information among three groups. The recovery time was much longer in Group B than that in Group N. No difference were found among three group in the score of MoCA on the day before surgry and 30 days after surgry ,while the score in the day after surgry and 3 days after surgry were lower in Group B com-pared with Group N. The incidence of POCD were higher in Group B compared with Group N. Compared with the day before surgry ,the concentration of serum S-100β protein and IL-6 were much higher. And compared with Group N ,the concentration of serum S-100β protein was higher in Group A and B in the day after surgry , while IL-6 was higher in Group B. The concentration of serum S-100βprotein and IL-6 were much higher in Group B compared with Group A in the day after surgry. Conclusion Female patients with hemoglobin concen-tration of 6 ~ 9 g/L are susceptible to POCD ,which might be contributed by operative central nerve system damage induced by excessive inflammation.

Objective To investigate the feasibility and clinical efficacy of laparoscopic disc hand assisted laparoscopic splenectomy(LDHALS) for patients with ruptured spleen.Methods LDHALS was performed in 14 patients with ruptured spleen,and among them,8 cases were combined with other visceral trauma.Results All cases were performed successfully by using LDHALS.The median operation time was 70 min(45～100 min).The average hospital stay was 7 days in 6 cases with only spleen trauma and 13.5 days in 8 cases combined with other injuries.No major operative complications and mortality occurred.Conclusion The LDHALS is safe,effective,simple and flexible performed for patients with ruptured spleen.

176.2,P0.05).Conclusions The tight adherence around catheter is formed in 5 days after PTCD.

Objective To investigate the impact of myocardial contrast echocardiography (MCE) on evaluating the myocardial perfusion in beagles with ischemic cardiomyopathy and cardiac resynchronization therapy (CRT),and to estimate the myocardial perfusion by myocardial blood flow (MBF) value.Methods Twelve adult beagles were randomly divided into two groups (CRT group and non-CRT group).Each beagle underwent a ligature in the first diagonal branch and a pacemaker was implanted.The pacing was started in CRT group,but was not started in non-CRT group.MCE and speckle tracking imaging were performed to evaluate the MBF value and circumferential strain (Cir1 2SD),radial strain (R12SD) and longitudinal strain (L12SD) of 12 segments of left ventricle at baseline,before CRT,and 4 weeks after CRT.Results There was no significant difference of LVEF,LVEDV,LVESV,Cir12SD,and R12SD between two groups at baseline or before CRT( P ＞0.05).After 4 weeks of CRT,LVEF,LVEDV,LVESV,Cir1 2SD,and R 12SD in two groups were ( 58.8 ± 8.2 ) ％ vs (39.5 ± 8.7 ) ％,(28.2 ± 2.9) ml vs (34.2 ± 2.5 ) ml,( 13.9± 2.6 ) ml vs (21.5± 4.7)ml,(29.1 ± 6.6)ms vs (46.5 ± 10.1)ms and (36.1 ± 10.7)ms vs (67.6± 11.2)ms( P ＜0.05 ).A,β,and MBF value between two groups were ( 13.6 ± 2.2 )dB vs ( 14.9 ± 3.0)dB,(5.1±1.1 )s- 1 vs (4.8 ± 2.1 )s- 1,(67.6 ± 12.1)dB/s vs (72.8 ± 8.6)dB/s( P ＞0.05) at baseline and were (7.4 ± 1.2)dB vs (7.3±2.7)dB,(3.9±0.9)s-1 vs (2.9±0.9)s-1,(23.4±4.2)dB/s vs (22.2±4.1)dB/s (P＞0.05)before CRT.After 4 weeks of CRT,A,β,and MBF value in CRT group were higher than those in non-CRT group (12.1 ± 1.8)dB vs (9.5 ± 1.7)dB,(4.7 ± 0.3)s-1 vs (3.1 ± 0.8)s-1,(47.2 ± 8.6)dB/s vs (29.5 ±4.2)dB/s,all P ＜0.05).Conclusions In beagles with ischemic cardiomyopathy,CRT can not only improve cardiac synchrony,but also increase myocardial perfusion.