Objective:To study the anti-inflammatory effect of immune milk. Method:Twenty four strains of pathogenic bacteria (including 12 strains of pathogenic E.coli, 8 strains of Salmonella, 3 strains of Shigella and 1 strain of Yersinia enterocolitica) originated from human intestinal tract were selected as antigens, and dairy cows were immunized. Results:Vaccination could not increase the concentration of IgG in the immune milk, but significantly highten the specificity of milk antibodies. The antibody titers against 24 pathogenic bacteria in immune milk was 128, 64 folds of control regular milk. Conclusion:Feeding of immune milk could inhibit edema of rat footpad induced by carrageenin and formaldehyde and the formation of acestoma of mice caused by cotton embedded hypophloedally. Meanwhile, the PGE2 level in inflammatory footpad of rat fed with immune milk was remarkably lower than that of rat fed with regular milk. These results indicate that immune milk has anti-inflammatory property.

Objective To study the regulatory T cells (Treg-cell) frequencies in patients with childhood acute leukemia and evaluate its clinical application value by investigating the relationship between the increasing numbers of Treg cells and minimal residual disease of leukemia (MRD), Methods Foxp3-FITC/CD25-PE/CD4-PerCP/CD3-APC four-color staining flow cytometry was established to identify Treg cells. Treg cells frequencies both in 10 healthy controls and in 33 patients with newly diagnosed childhood acute leukemia ( B-ALL 17 cases, T-ALL 9 cases, AML 7 cases) were detected. The possibility of the accumulation of Treg cells being the prognostic marker for acute leukemia was evaluated by comparing the results of Treg cells frequency with that of MRD. Results The percentage of Treg cells in CD+4 CD+3 T cells was M = 8. 09% in normal bone marrows, which was significantly different from the results in the bone marrows of newly diagnosed childhood acute leukemia ( M = 12.77% , U = 3.41, P < 0.01 ), but it showed no significantly differences among B-ALL, T-ALL and AML groups. No association was observed between the expression of Treg cells and clinical-biologic characteristics studied. In addition, Treg cells frequency in MRD positive group was significantly different from that in MRD continuously negative group ( M = 14. 74% vs M=11.3%, t =252.5,P<0.05). Conclusions Consistent with results from solid tumor, the study identifies a significantly increased numbers of Treg cells in patients with childhood acute leukemia. The situation of accumulation of Treg cells is closely associated with MRD results during chnical remission. High level of Treg cells may cause poor prognosis and increase the possibility of relapse.

Objective:Selected virulence factors more than and high virulent Aeromonas hydrophia strain made into inactivated vaccine,to study the immunization effect of inactivated vaccine.Methods:Crucian were vaccinated with formalin-killed vaccine via in-traperitoneal injection.Controls were injected with the same volumes of saline.Then the antibody titres, histopathology and relative percent survival were analyzed from samples of both groups.Results: The antibody in the indirect agglutination reaction could be detected in vaccinated fish once a weeks after immunization and reached highest level 6 weeks after immunization.The histopathology analysis indicated that the vaccine had a good protective effect on crucian target organs.Vaccinated fish showed 100%relative percent survival and the immune period would be 6 month.Conclusion:The vaccine in this study has a significant protective effect on crucian and may be used as effective fish vaccines against bacterial septicemia.

Objective To evaluate the relative efficacy of different dosages of Calf Pulmonary Surfactant (Calsurf) administration in premature infants with established respiratory distress syndrome (NRDS).Methods Four neonatal intensive care units in Jiangsu province were enrolled.Premature infants,birth weight ＜ 2 500 g,with NRDS,received 70 mg/kg (61 cases)or 100 mg/kg (69 cases)Calsurf.Clinical and respiratory parameters were recorded.The primary outcome measures were blood gas analysis of 1 h,12 h and 36 h after administration,the need for oxygenation and ventilatory requirements and the adverse events of NRDS.Results Arterial oxygen tension[pa (O2)] results in a significant improvement(80.27-±36.81) mmHg,(73.03 ±24.94) mmHg and (72.35 ± 24.72) mmHg at 1 h,12 h and 24 h in higher dose group(P ＜ 0.05),(67.95 ± 23.79) mmHg,(72.35 ± 24.72) mmHg in 24 h,as compared with the lower dose group at the same time [(67.07 ± 19.94) mmHg,(62.93 ± 21.71) mmHg,(67.95 ±-23.79) mmHg] (P ＞ 0.05).Inspired oxygen (FiO2) and pa (O2) decreased after administration in two groups and the duration of FiO2 decline lasted to 48 h (all P ＜ 0.05).The oxygen index (OI) was improved after Calsurf administration,especially in the infants who received 100 mg/kg of Calsurf with 6.1 ± 2.8,5.6 ± 3.3,5.5 ± 3.5,5.8 ± 4.5,5.3 ± 3.1 in 1 h,12 h,24 h,36 h,48 h,respectively(P ＜ 0.01).The arterial-to-alveolar oxygen tension ratio (a/APO2) of 100 mg/kg group was reduced significantly in 1 h,12 h,24 h,36 h,48 h with 0.39 ±0.22,0.42 ±0.20,0.45 ± 0.22,0.44 ± 0.22,0.46 ± 0.21 as compared with 0.27 ± 0.18 which was at the time point before administration (P ＜ 0.01).Although not statistically significant,the mean time of duration of mechanical ventilation and oxygen inhalation of higher dose group were decreased as compared to the lower dose group [(94.54 ± 113.44) h vs (109.27 ± 124.87) h (P＞0.05) and (259.10 ±280.45) vs (372.31 ±398.08) h(P ＞0.05)].There were no significant differences in the rates of other adverse events such as pneumonia,pneumothorax,intracranial hemorrhage,patent ductus arteriosus (PDA),pneumorrhagia,necrotizing enterocolitis,septicemia between two groups (P ＞ 0.05).Conclusions Calsurf given to preterm infants with NRDS at a dose of 100 mg/kg resulted in a higher Pa (O2),a/APO2,better OI and lower FiO2 as compared with those receiving 70 mg/kg.The need for mechanical ventilation and oxygen supplement were reduced with higher-dose administration.Large dose of Calsurf did not increase the risk of complications as mentioned above.

BACKGROUND:The enamel matrix derivative has been used in the clinical treatment of severe periodontitis; however, the mechanism(s) by which enamel matrix derivative promotes periodontal regeneration is stil obscure. OBJECTIVE:To explore the effects of enamel matrix derivatives on the differentiation and proliferation of periodontal ligament stem cels. METHODS:Periodontal ligament stem cels were isolated and identified from human teeth. Cloning forming efficiency, surface antigen expression and pluripotency were detected and identified. Enamel matrix derivatives with different concentrations (20, 50, 100 mg/L) were used to culture periodontal ligament stem cels for 2 and 4 weeks. Colagen synthesis and mineralized nodule formation were detected using Trichrom staining and Von Kosa’s staining, respectively; real-time RT-PCR was employed to detect expressions of colagen type I, osteocalcin, and RUX2; MTT and cel growth rate assay were used to detect the proliferation of periodontal ligament stem cels. RESULTS AND CONCLUSION:Periodontal ligament stem cels were spindle-shaped and showed a higher colony forming efficiency than periodontal ligament cels. The expressions of surface antigens of periodontal ligament stem cels-CD105, CD29, CD45, CD44 were respectively 99.8%, 99.7%, 1.26%, 98.8%, indicating periodontal ligament stem cels have the multilineage differentiation potential. Enamel matrix derivatives improve the colagen synthesis and mineralization nodule formation of periodontal ligament stem cels in a time-dose dependent manner. They also can improve the expression of osteogensis-related genes colagen type I, osteocalcin, RUX2 and proliferation of periodontal ligament stem cels.

Objective To construct, express and purify human scFv antibody (S1) against the recombinant SAG1 of Toxoplasma fused to magainin, and observe its protective effect against Toxoplasma in infected mice. Methods The S1 scFv antibody gene amplified from phagmid S1/pIT-2 fused to magainin was cloned into procaryotic expression vector pET-32c. The recombinant plasmid S1M/pET-32c proved by DNA sequencing was transformed into E.coli BL21, and induced for fusion expression of S1M with IPTG. The expressed S1M was purified with Ni 2+ chelating HiTrap HP column and detected with SDS-PAGE. The effect of reduction of infection of Toxoplasma was observed through in vivo and in vitro experiments in mice. Results The fused gene of S1 and magainin was successfully cloned into procaryotic expression vector pET-32c proved by DNA sequencing. The recombinant S1M protein about 43 kDa was expressed in E.coli as inclusion body, and prepared with Ni 2+ column purification. Tachyzoite of Toxoplasma preincubated with S1M showed decreased infectivity in mice, the result of in vivo experiments showed that mice treated with S1M hadlonger survival time than the mice untreated. Conclusion The purified targeting antimicrobial peptide S1M could reduce the infectivity of tachyzoites of Toxoplasma in a certain extent and has a potential value for biological therapy of toxoplasmosis; otherwise, the constructed targeting antimic robial peptide S1M also provides a new model for biological therapy of toxoplasmosis.

BACKGROUND:Femoral neck fracture is mainly fixed by three inverted triangle cannulated screws. Scholars have proposed to add a cannulated screw to enhance the fixation strength of femoral neck fracture of Pauwels III type based on three cannulated screw fixation, but the stability is not verified. OBJECTIVE:To analyze the biomechanical stability and stress of the three and four cannulated screws for the treatment of the Pauwels III femoral neck fractures. METHODS:The CT imaging results of the fourth generation of artificial bone sawbones were imported into the Mimics software wherein a three-dimensional finite element model of the proximal femur was prepared and introduced in the 3-matic software. Models of middlesegment of femoral neck with Pauwels III fractures were established. Cannulated screw models were established with UG 8.0 software and introduced in the fractures models. Finally, finite element models of Pauwels III femoral neck fractures fixed with three and four screws were established. In the same condition, an axial load of 411 N was applied on the femoral head with Abaqus software. The displacement of two markers of the broken ends and internal fixation system Von Mises stress distribution were compared between the two models. RESULTS AND CONCLUSION:(1) The displacement was 0.42 mm in three screws model, and 0.17 mm in the four screws model. (2) Von Mises stress peak was 547 MPa and 27.8 MPa in both models. The peak value was lower in models of fourscrews than that of three screws. Stress concentration position was at the fracture site in both models. The stress range of models of four screws was more extensive and scattered. (3) Finite element analysis results demonstrated that four-screw implantation for Pauwels III femoral neck fractures had strong anti-shearing force and biomechanical stability. Clinical advantages need further clinical comparative study.

Objective To assess the efficacy and safety of monoclonal antibody rituximab combined with cyclophosphamide (CTX) in the treatment of refractory and recurrent autoimmune hemolytic anemia.Methods Seven cases with refractory and recurrent autoimmune hemolytic anemia ( including 1 case of Evans syndrome) were recruited during January,2007 to December,2010.Treatment regimens were as follows:rituximab:375 mg/m2,1 time/week,2-6 courses; CTX:1 g,1/10 d,2-7 courses; combined with intravenous immunoglobulin (IVIG) 5 g,1 time/week,given 1 day after rituximab administration.The efficacy and safety of this regimen were assessed during follow-up.Results All the patients showed good responses (7/7).Six patients achieved complete remission (6/7) and one achieved partial remission ( 1/7 ).Average follow-up time for the patients was 27 months.All patients remained in remission during the 12-month follow-up visits.Two patients showed elevated indirect bilirubin and increased reticulocyte counts within 24 months.One patient achieved complete remission after additional rituximab therapy,and another patient remained partial remission after cyclosporine therapy.At the time of 36-month follow-up visit,the patient relapsed and was retreated with 3 courses of rituximab combined with CTX and eventually achieved partial remission.All patients tolerated the treatment well with few mild side effects.Conclusions Rituximab combined with CTX is effective and relatively safe in patients with refractory and recurrent autoimmune hemolytic anemia.Additional treatment to relapse patients about 12-24 months after drug withdrawal continues to be effective.

Objective To investigate the expression of TET2 and DLK1 mRNA in bone marrow CD3+ T cells of patients with myelodysplastic syndrome (MDS) and their clinical significance and to explore the potential mechanism of abnormal cell-mediated immunity.Methods CD3+ T cells were sorted by magnetic activated cell-sorting system.The expressions of TET2 and DLK1 mRNA in bone marrow CD3+ T cells from 26 MDS patients and 16 healthy controls were detected by fluorescence quantitative PCR.Results The expression of TET2 mRNA in CD3+ T cells was down-regulated in the MDS patients by (0.16 ±0.15) fold compared with the controls ( P ＜ 0.05 ).The expression of TET2 mRNA in CD3+ T cells of MDS patients was positively correlated with serum complement C3 ( r =0.404,P ＜ 0.05 ).The expression of DLK1 mRNA in CD3+ T cells was up-regulated in the MDS patients by (1.61 ±0.88) folds compared with the controls (P＜0.05).Grouped by the chromosomes,the patients with chromosome abnormalities presented significantly higher DLK1 mRNA level than those with normal chromosomes [ ( 1.45 ± 0.44 ) folds,P ＜0.05 ].The expression of DLK1 mRNA in CD3+ T cells of MDS patients was positively correlated with the proportion of bone marrow blasts ( r =0.343,P ＜ 0.05 ).Conclusions The mRNA expression of TET2 in CD3+ T cells of MDS patients was decreased while the mRNA expression of DLK1 was increased,which might decline the immune surveillance function.The findings would be useful for exploring the mechanism of immune tolerance.

To investigate the role of CD4+, CD25+, and CD127low regulatory T cells (Tregs) in multiple myeloma (MM). Methods:Levels of CD4+T cells and Tregs, as well as expression of CTLA-4 and apoptosis-related proteins, such as CD95, bcl-2, and Caspase3 of Tregs in peripheral blood of 30 patients with newly diagnosed cases, 27 patients under of complete remission (CR) from multiple myeloma patients, and 25 healthy adults were analyzed by flow cytometry. Results:The percentage of CD4+T cells in the untreated group was significantly lower than that of the control group (P<0.05). The percentage of Tregs in CD4+T cells in the untreated group was significantly higher than that of the CR group and control group (P<0.05), which in ISSⅢpatients of the untreated group was significantly higher than that in I/II(P<0.05). No significant difference of CD95 expression in Tregs was observed among the three groups. The expression of CTLA-4 in Tregs from the untreated group was significantly higher than that of the CR group (P<0.05) and control group (P<0.01), and so was in CR group than this in controls (P<0.05). The expression of bcl-2 in Tregs in the untreated group was significantly higher than that of the CR group (P<0.05) and control group (P<0.01), and so was in CR group than this in controls(P<0.05). The expression of Caspase3 in Tregs from the untreated group and CR group were all significantly lower than that of the control group (P<0.05). The percentage of Tregs in CD4+T cells in the untreated group was positively correlated with the proportion of bone marrow plasma cells (P<0.05). The percentage of Tregs in CD4+T cells from 15 MM patients who received bortezamib and dexamethasone (VD) chemotherapy was negatively correlated to the ratio of plasma cell reduction after the first VD chemotherapy (r=0.735, P<0.01). Conclusion:The level of Tregs in the peripheral blood of MM patients was positively correlated with tumor burden and progression of disease, but was negatively correlated with curative effect. The increased level of Tregs was associated with their strengthened anti-apoptosis function.