Objective To clone the human soluble interleukin-6 receptor(hsIL-6R) gene and expression in insect cell line. Methods The hsIL-6R gene was cloned into plasmid pAcGP67B. After co-transfection of recombinant plasmid and wild type AcNPV DNA, the rAcNPV was confirmed by the end point dilution assay and dot blot. Then it was purified by plaque assay. Results SDS-PAGE showed molecular weight of the expressed product was about 47 000. The expressed recombinant protein was confirmed to be specific and capable of binding its ligand IL-6 by Western blot and ligand-receptor binding assays. Conclusions Secretory expression of hsIL-6R gene in baculovirus expression system was indicated. The expressed product had immunological and biological activities.

BACKGROUNDThe role of tumor-infiltrating lymphocytes (TILs) in the immunopathogenesis of individual cancer is not clear and is a challenge for anti-tumor immunotherapy. This study aimed to investigate the effects of interleukin (IL)-18 and -12 on cytotoxic functions of TILs.

METHODSTILs from postoperative gastric cancer patients were costimulated with IL-18 and IL-12. SGC-7901 tumor cells were pre-incubated with TILs and subcutaneously injected into BALB/C SCID mice. The function of TILs was evaluated by measuring tumor sizes in tumor-bearing mice, T helper (Th)1 (tumor necrosis factor (TNF)-α, interferon (IFN)-γ) and Th2 cytokine levels (IL-10 and IL-4) in serum and cytotoxicity of mouse natural killer (NK) and CD8(+) T cells.

RESULTSIL-18 and IL-12 synergistically inhibited the growth of SGC-7901 cells in vivo and significantly extended the survival rate of SGC-7901-bearing mice (66.7% vs. 13.7%, P < 0.01). Moreover, TILs could promote the secretion of TNF-α and IFN-γ ((130.34 ± 7.65) vs. (210.63 ± 12.31) pg/ml, P < 0.01; (14.23 ± 1.97) vs. (30.52 ± 2.12) pg/ml, P < 0.01), and downregulate IL-10 and IL-4 secretion ((103.72 ± 11.21) vs. (61.36 ± 5.41) pg/ml, P = 0.021; (49.36 ± 4.67) vs. (28.48 ± 3.86) pg/ml, P = 0.024).

CONCLUSIONIL-18 and IL-12 can synergistically enhance cytotoxic functions of TILs from human gastric cancer.

Adult ; Aged ; Animals ; CD8-Positive T-Lymphocytes ; immunology ; metabolism ; Cell Line, Tumor ; Female ; Humans ; In Vitro Techniques ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-12 ; pharmacology ; Interleukin-18 ; pharmacology ; Interleukin-4 ; metabolism ; Lymphocytes, Tumor-Infiltrating ; drug effects ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Middle Aged ; Stomach Neoplasms ; immunology ; Tumor Necrosis Factor-alpha ; metabolism ; Xenograft Model Antitumor Assays

OBJECTIVETo obtain the monoclonal antibody against hexon protein of human adenovirus.

METHODSBALB/c mice were immunized with purified recombinant hexon protein, and the spleen cells of the mice were isolated and fused with myloma cells. Four hybridoma cell strains were screened by indirect ELISA and cultured, and the sensitivity, specificity and virus neutralizing activity were analyzed with ELISA, Western blotting and neutralizing test.

RESULTSThe mouse ascites produced by these hybridoma cells contained specific monoclonal antibodies against hexon protein of human adenovirus as identified by ELISA and Western blot, and the antibody generated by 4C6 strain showed human adenovirus type 3-neutralizing activity.

CONCLUSIONThe monoclonal antibodies against hexon protein with high specificity have been successfully obtained, and these antibodies can be useful in developing assays for early diagnosis of HAdV3 infection and also in study of therapeutic drugs of the infection.

Adenoviruses, Human ; chemistry ; immunology ; Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Blotting, Western ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Humans ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; immunology

Objective To evaluate the clinical application and significance of lengthen-stem bipolar-femur prosthetic replacement for the treatment of old-age patients with intertrochanteric fracture osteoporosis.Methods 28 cases of patients aging from 75 to 99 years old of intertrochanteric fracture osteoporosis treated with lengthen- stem cemented bipolar prosthesis were studicd from March 2000 to December 2006.After taking the blank margin, the bones of different sizes were replaced and the steel wire was fixed.After determining the depth of the front an- gle,the artificial bone was placed.Results After 28 examples attaining the following-up examination for 7 months to 3 years,with an average of 1.5 years,its function according to Harris standard was evaluated 3 months after the operation,8 examples were excellent,13 examples good,5 examples pass,2 examples inferior.The excellent or good rate reached 75% ,with no abnormal cases,no joint dislocation during the followed-up period.1 example had the phantom phenomenon 1 year after the operation.2 examples among the inferior had got more serious internal medicine disease which affected the restoring function.1 example died of the internal medicine disease 1 year after the operation.Conclusion By using the lengthen-stem bipolar-femur prosthetic replacement for the treatment of old patients with inrertrochanteric fracture osteoporosis,the patients will restore quickly after the operation and can carry a heavy load at an early time.The illness complication and the mortality rate will be redaced.But its related disease must be strictly dealt with and the surgery operating skill must be grasped.

OBJECTIVEThe study was to investigate the impact of cord blood CD(3)AK cell culture supernatant (CS) on proliferation, differentiation and apoptosis of HL-60 cells.

METHODSHL-60 cells were treated with different concentrations of CS (10%, 15%, 20%) for 3 days, 6 days and 9 days, and the same cells of control group were not treated with CS. The growth of induced cells was assessed with Trypan blue staining and cell counting with cytometer. The differentiation marker CD(11b) on the cell surface and cell-cycle was analyzed by flow cytometry (FCM), cell morphology (Wright-Giemsa staining) and NBT test to determine the extent of differentiation. Meanwhile, the changes of the apoptosis of the cells induced by 20% CS at different time points (3, 6 and 9 days) were analyzed by TUNNEL-POD, and the apoptotic characteristics of cells were observed.

RESULTSThe growth of HL-60 cell was inhibited as CS-inducing time and the dose of CS increased. At the same time, but HL-60 cell number in G(0)/G(1) phase of cell-cycle increased, but HL-60 cell number in S phase decreased compared with untreated group. The HL-60 cells induced by 20% CS for 9 days showed that (52.7 +/- 1.8)% of cells were at G(0)+G(1) phase and (43.8 +/- 1.1)% were at S phase (P < 0.05), which demonstrated that HL-60 cells induced by 20% CS underwent G(0)/G(1) phase cell-cycle arrest. The volume of the differentiated cells was enlarged gradually as CS-inducing time prolonged. After 3 days the differentiating cells began to express differentiating marker CD(11b) on the cell surface and the nuclei morphology of the differentiated cells was also changed and NBT-stained cells increased in number with the increased dose of CS increased. Three days after induction by 20% CS, the induced cells began to show signs of apoptosis and the apoptotic percentage of induced cells gradually increased with CS-induction time. The rate of apoptosis of cells was (33.3 +/- 2.3)% at 9 days (P < 0.01).

CONCLUSIONCS could not only inhibit the growth of HL-60 cells but also induce the differentiation and apoptosis in HL-60 cells.

Apoptosis ; Cell Culture Techniques ; Cell Cycle ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Culture Media ; chemistry ; Fetal Blood ; chemistry ; HL-60 Cells ; Humans

OBJECTIVETo construct and identify a adenovirus vector of the expression of connective tissue growth factor (CTGF) and to explore the role of CTGF in the metabolism of glucose and lipid.

METHODSThe over-expressed plasmid of CTGF was cloned, and then the CTGF sequences were cloned into pAdTrack-CMW vector. The reformed E. coli BJ5183-sensitive bacteria that contain pAdEasy-1 were transformed with lined vector cut by Pme I enzyme. The recombinant adenovirus vector was cut with Pac I enzyme and obtained, then transfected 293A cells to produce virus. Through three times of amplification, the adenovirus infected the primary hepatocytes to determine the infection efficiency and CTGF expression. The mice were starved for several time periods, and then the liver RNA was extracted for real-time PCR to detect the expressions of CTGF under different nutritional conditions.

RESULTSThe adenovirus of CTGF was successfully produced with an infection efficiency of 90%. The expressions of the CTGF were different under different nutritional conditions and showed a coincidence with the expression of peroxisome proliferators-activated receptor gamma coactivator 1 alpha. After the mice were starved for 24h, the expression of CTGF increased by (2.38 +/- 0.51) folds; after the mice were starved for 48 h, the expression of CTGF increased by (2.95 +/- 0.57) folds (P < 0.05).

CONCLUSIONCTGF is speculated to be involved in the metabolism of glucose and lipids.

Adenoviridae ; genetics ; Animals ; Cell Line ; Connective Tissue Growth Factor ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; Mice ; Mice, Inbred C57BL ; Plasmids ; Transfection

OBJECTIVETo construct a replication-defective recombinant adenovirus expressing the ORF2 (112-660aa) antigen of hepatitis E virus (HEV) and evaluate its immunization effect in BALB/c mice by mucosal inoculation.

METHODSThe HEV ORF2 gene encoding for 112-660aa was amplified from plasmid pUC-HEV and inserted into the transfer vector pTrack-CMV. The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E. coli strain BJ5183. Taking the advantage of the high efficient homologous recombination machinery presented in bacteria, the recombinant adenovirus backbone plasmid was generated in BJ5183, and then was transfected into 293 cells. Recombinant Adenoviruses were propagated in 293 cells with high titers. 8-week-old BALB/c mice were inoculated intraperitoneally and intranasally with 10(7) pfu recombinant adenovirus each on weeks 0, 3, 5, 7, 10.

RESULTSBoth groups of mice induced humoral IgG immune response with the highest titers 1:1,000 and 1:10,000 each. Only the group inoculated intranasally could induce mucosal IgA immune response.

CONCLUSIONSThe adenoviral recombinant can stimulate specific humoral and mucosal immune response in mice and is potentially to be used as a candidate vaccine for the treatment of HEV infection.

Adenoviruses, Human ; genetics ; Animals ; Hepatitis Antigens ; genetics ; immunology ; Immunoglobulin A ; immunology ; Immunoglobulin G ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa ; immunology ; Peritoneum ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Hepatitis Vaccines ; Viral Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo observe anti-HEV IgG response to vaccination of recombinant antigen fragments and evaluate its protection from Hepatitis E Virus infection in rhesus monkeys (Macaca mulatta).

METHODSTwelve monkeys were divided into three groups and immunized respectively with three different recombinant antigens: namely Ag1 (carboxyl terminal 431 amino acids of ORF2), Ag2 (128aa fragment at the carboxyl terminal of ORF2), and Ag3 (full length ORF3 ligated with two ORF2 fragments encoded by 6743-7126nt and 6287-6404nt). The monkeys were challenged intravenously with fecal suspension from experimentally infected rhesus monkeys, and the other three monkeys served as the placebo group for challenge with HEV. The dynamic changes of the levels of ALT and anti-HEV IgG were examined. Pathological changes of liver tissue were observed by light microscope. Excretion of virus was detected by RT-nPCR.

RESULTSHepatic histopathology of two monkeys in the placebo group was consistent with acute viral hepatitis, and ALT was elevated 3-4 weeks after inoculated with virus, up to 10-20 times higher than normal level. The liver tissue of monkeys immunized with antigen kept normal, ALT in several monkeys elevated mildly, and anti-HEV IgG conversation occurred at 1-2 weeks after vaccination, with the titer reaching 1:12,800. The virus RNA could be detected by RT-nPCR from days 7 to 50 in monkeys of control group, and from days 7 to 21 in vaccinated monkeys after challenged with virus.

CONCLUSIONSThe recombinant antigens could induce the production of anti-HEV IgG, which protected rhesus monkeys from acute Hepatitis symptoms related to HEV infection.

Animals ; Antigens, Viral ; immunology ; Hepatitis E ; prevention & control ; Hepatitis E virus ; immunology ; Immunoglobulin G ; immunology ; Macaca mulatta ; RNA, Viral ; blood ; Recombinant Proteins ; immunology ; Vaccination ; Viral Hepatitis Vaccines ; immunology

OBJECTIVETo report the outcome of long bone nonunion of humerus, femur and tibia treated with locking internal fixation and bone graft.

METHODSFrom February 2003 to October 2006, locking internal fixation and bone grafting were employed to treat 5 cases at humerus, 33 cases at femur, 23 cases at tibia. Forty-four of the patients were men, and 17 were women. The mean age was 38 years (range 7-70 years). The nonunion had resulted from failure of internal fixation in 47 cases, failure of external fixation in 5 cases, infection in 9 cases. The history of nonunion lasted from 10 to 156 months (mean 19 months). There were 42 patients treated with locking compression plate (LCP), and 19 patients with less invasive stabilization system (LISS). For bone grafting, autogenous ilium was used in 55 patients, autogenous ilium and allograft bone was used in 3 patients, allograft bone and Wright DBM artificial bone was used in 3 patients.

RESULTSAll the 61 patients were followed up for an average 12 months (range 6-24 months) only to reveal solid bone union in all the fracture, with a mean healing time of 4.8 months (ranged from 4 to 6 months). No loosening or breakage of the implants occurred in this series. The Knee Society Scores (KSS) was used to evaluate knee function in 47 patients with peri-knee joint nonunion, excellent result were seen in 35 patients, good in 7 patients, fare in 1 patients, poor in 4 patients.

CONCLUSIONLocking internal fixation can be used to treat effectively bone nonunion at the humerus, femur and tibia.

Adolescent ; Adult ; Aged ; Bone Plates ; Child ; Female ; Femoral Fractures ; surgery ; Follow-Up Studies ; Fracture Fixation, Internal ; methods ; Fractures, Ununited ; surgery ; Humans ; Humeral Fractures ; surgery ; Male ; Middle Aged ; Retrospective Studies ; Tibial Fractures ; surgery ; Treatment Outcome

OBJECTIVETo evaluate the clinical result of the treatment of old femoral neck fracture and nonunion with free vascularized fibular grafting.

METHODSFrom November 2000 to December 2005, 29 cases with old femoral neck fracture and nonunion had been treated by free vascularized fibular grafting with an average follow-up of 28.5 months.

RESULTSAll the fracture were healed without any severe complications. And the healing time was 4-6 months (5.6 months on average). During the follow-up, the hips of 28 cases got well-function, and the average Harris hip score was 88.2. One case came about with osteonecrosis of femoral head after one year and finally accepted THA after 2 years.

CONCLUSIONSThe free vascularized fibular grafting is a valuable procedure to treat old femoral neck fracture and nonunion.

Adult ; Bone Transplantation ; methods ; Female ; Femoral Neck Fractures ; surgery ; Fibula ; blood supply ; transplantation ; Follow-Up Studies ; Fractures, Ununited ; surgery ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome ; Young Adult