Objective:To construct a vector that suits the construction of large phage antibody libraries.Methods:Hie phage antibody vector p3MH was modified by insertion of synthesized oligos and PCR mediated site-specific mutations. Hie resultant vector was checked by expression of phage antibody, ELBA and in vivo recombination. Results: Vector pAL was obtained by following modifications of p3MH: insertion of Ioxp511 and loxp sequences, substitution of Lac promoter by Ara promoter, and modification of the cloning site for antibody genes. pAL was proved capable of expressing functional Fab phage antibodies under tighter control. In ere+ bacteria, pAL exhibited loxp-cre mediated recombination. Conclusion: pAL is useful for construction of large phage antibody libraries.

Until now, the exactly effect of Kupffer cells (KCs) on inducing immune tolerance or aggravating acute rejection is still unknown. Activated by various way after liver transplantation, have the ability of phagucytosis the apoptositic T cells, up-regulated expression of FasL and many Th2/Th3 cytokines, such as interleukin-10 and transforming growth factor-lB. These up-regulated cytokines could induce the apoptosis of Th1 cells and enhance the proliferation and differentiation of the Th2 cells, finally induce the immune tolerance, However, the activated KCs also have the ability of expression many cytokine-dependent molecules,such as class Ⅱ major histocompatibility antigens, adhesion molecule and costimulatory molecules which could enhance the function of the antigen presentation, increase the expression of Thl cytokine and aggravate the acute rejection after liver transplantation. It maybe relate to the ratio of theTh1/Th2 cells determined by the complicated net of the cytokine produced by the activated Kupffer cells: the predominance of Th2 cells could induce the immune tolerance, on the contrary, the acute rejection proceed.

Objective This study has explored the role of antibody against annexin A2 in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). Methods Using purified recombinant annexin A2, IgG anti-annexin A2 antibody was measured by ELISA in 101 APS patients, 41 SLE patients with thrombosis, 124 SLE patients without thrombosis and 120 healthy controls. Results The positive rate of IgG anti-annexin A2 antibody in APS patients and SLE patients with thrombosis was 21.8%, 26.8%, respectively, they were all significantly higher than in SLE patients without thrombosis (6.5%). IgG anti-annexin A2 antibody was associated with thrombosis and/or pregnancy morbidity (P<0.01). Conclusion Anti-annexin A2 antibody is associated with thrombosis and/or pregnancy mnrbidity. It suggests that anti-annexin A2 antibody may be helpful in identifying in some potential AIRS.

NIT-1 cells were exposed to various concentrations of glucose for 24,48,and 72 hours.The content of triglyceride in NIT-1 cells increased in dose-and time-dependent manners (P ＜ 0.05).Long-term exposure of NIT-1 cells to high glucose concentrations caused an inverse "v"-like induction of HSL mRNA and protein expressions,which increased from beginning,and then decreased,along with similar changes of lipolysis.These results suggest that the adaptation of HSL may play an important role in regulating the intracellular triglyceride pool and the development of glucolipotoxicity in pancreatic β cells.

Objective To investigate the effects of sevoflurane anesthesia on the expression of c-Jun N-terminal kinase (JNK) and neuronal apoptosis in hippocampus in juvenile rats.Methods Forty healthy male SD rats, aged 30-35 days, weighing 100-110 g, were randomly divided into 2 groups (n = 20 each): control group (group C) and sevoflurane group (group S) . Group C inhaled a gas mixture of oxygen and air for 5 h and group S 3% sevoflurane for 5 h. The concentration of oxygen in both groups was maintained at 30% . Ten rats in each group were scarified at 1 h after regaining consciousness and the hippocampi removed for determination of phospho-JNK expression (by immuno-histochemistry and Western blot) and neuronal apoptosis (by TUNEL) . Another 10 rats were selected at 24 h after regaining consciousness to assess the cognitive function using Morris water maze. Results Compared with group C, phospho-JNK expression was significantly up-regulated, the number of apoptotic neurons increased, the latency prolonged and the duration of staying at the original platform quadrant shortened in group C ( P ＜ 0.05 or 0.01) . Conclusion Inhalation of 3.0% sevoflurane can induce neuronal apoptosis in hippocampus by activating JNK signaling pathway, thus leading to cognitive decline in juvenile rats.

Objective To investigate the relationship between polymorphisms of interleukin-4 receptor (IL-4R) gene (E375A, C406R, Q576R) and childhood asthma in Harbin. Methods Ninety-one (91) children with asthma were included, and 42 healthy children were enrolled as control. The genotype polymorphisms of IL-4R were determined by polymerase chain reaction-restriction fragment length polymorphism analysis. Results There was no significant difference in the distributions of the genotypes at both 1 124 A/C polymorphic sites (i.e. E375A polymorphism) and 1 902 A/G polymorphic site (i.e. Q576R polymorphism) in IL-4R between asthmatic group and control group (P > 0.05). There was significant difference in the distributions of the genotypes (CC, CR, RR) on 1 216 T/C polymorphic site (i.e. C406R polymorphism) in IL-4R between the two groups (P < 0.05). The R allele frequency was 22.94% in asthmatic group and 6.58% in control group, with significant difference between the two groups (P < 0.05), but the allele frequencies at this site did not satisfy Hardy-Weinberg equilibrium (P < 0.05). Conclusions Our data suggest that the E375A and Q576R polymorphisms in IL-4R is not associated with the development of asthma in children in Harbin. Further study is needed on the relationship between C406R polymorphism and asthma.

OBJECTIVETo establish a rapid specific method to identify the single-nucleotide polymorphisms (SNPs) of HBV polymerase gene region which are the methionine residue of the conserved YMDD motif.

METHODSTwo specific primers were designed to amplify interested gene region involved in SNPs which were also used as HBV DNA identification. Specific primers of SNaPshot were designed to detect 741A-G (YVDD), 743G-T (YIDD). The different fluorescent dye labeled ddNTP was used to further extend the strand of PCR product and was detected by ABI PRISM 310 Genetic Analyzer. Sera from 13 patients with chronic hepatitis B after lamivudine treatment were analyzed.

RESULTSAside from mutation of YMDD, there were mutations of 514C-A, 523C-A, 562T-A, 667C-A. The 13 samples were simultaneously tested with SNaPshot and DNA sequencing, the same results were obtained. The method of SNaPshot showed high specificity.

CONCLUSIONMutation of YMDD results in the changes of ATG codon, and there are new ATG codon in the upper strand of YMDD. SNaPshot technique is rapid, specific and accurate for the SNPs monitoring of HBV DNA mutation during lamivudine therapy. Two samples were determined by SnaPshot technique, identifying the co-existence of the mixed wild type and mutant type HBV infection.

Antiviral Agents ; therapeutic use ; Base Sequence ; DNA, Viral ; blood ; genetics ; Drug Resistance, Viral ; Gene Products, pol ; genetics ; Hepatitis B ; blood ; drug therapy ; virology ; Humans ; Lamivudine ; therapeutic use ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

Objective To determine the risk factors related to tuberculosis infection among people living with HIV/AIDS and to develop strategies for preventing the co-infection.Methods A 1:2matched nested case-control study was carried out to analyze the influencing factors of tuberculosis among people living with HIV/AIDS.Results 1018 people living with HIV/AIDS were followed up for one year with a total number of 736.75 person-years,among them 62 tuberculosis cases were diagnosed.The incidence density of tuberculosis among people living with HIV/AIDS was 8.42 persons per 100 person-years.Factors as education level(OR=0.483),vaccination history of Bacille Calmette Guerin(OR=0.561),CD_4~+ count T-lymphocyte(OR=0.356),unemployment(OR=1.976),living alone(OR=2.646),and smoking(OR=2.215)were significantly related to the prevalence of tuberculosis among people living with HIV/AIDS.Conclusion High education level,with vaccination history of Bacille Calmette Guerin and high level of CD_4~+ T-lymphocyte count were protective factors while being unemployed,living alone,and smoking habit were risk factors related to the prevalence of tuberculosis among people living with HIV/AIDS.

Objective To investigate the mutations of pedocyte molecules in patients with late onset familial focal segmental glomerular sclerosis (FSGS). Methods Thirty-one pedigrees of late onset familial FSGS in Department of Nephrology, Shanghai Ruijin Hospital from Sep 1997 to Oct 2007 were enrolled in this study. The diagnosis standard of familial FSGS was as follows:(1) the age of presentation was more than 12 years old. (2) in one pedigree, two or more individuals were proven as FSGS by renal biopsy, or at least one was proven to be FSGS by renal biopsy, the others presented renal insufficiency or pmteinuria without precise causes. One hundred unrelated healthy people were screened as control group. Genomic DNA extracted from peripheral blood cells were amplified by PCR and then sequenced for mutations of NPHS2, ACTN4 and TRPC6. Results A novel missense heterozygotic mutation L316P of ACTN4 was identified inone pedigree. The mean onset age of the affected members of this pedigree was (38.7±7.4) years old and their kidney injury progress was slow. Proteinuria of the proband's brother was not improved by immunosuppressor. All 3 affected members of this family had such heterozygotic mutation. A novel missense heterozygotic mutation Q889K of TRPC6 was found in another pedigree. The mean onset age of the affected members in this pedigree was (38.0±4.2) years old. Three members presenting renal disease in this family all had such heterozygotic mutation but with different clinical manifestations. A quiescent mutation G467G of TRPC6 was also identified. Above variants were not found in healthy controls. No NPHS2 mutation was found to cause familial FSGS in these pedigrees. Conclusions A novel mutation L316P of ACTN4 and a new mutation Q889K of TRPC6 are identified in Chinese patients of late onset familial FSGS. No NPHS2 mutation is found to induce FSGS in these pedigrees.

objective To analyze the clinicopathological features and prognosis of antiglomerular basement membrane(GBM)disease,and evaluate the efficacy and safety of double filtration plasmapheresis(DFPP). Methods A total of 35 hospitalized patients diagnosed as anti-GBM disease in our department were enrolled in the study.All the patients were divided into 3 groups according to the manifestations at admission.Group Ⅰ∶24 patients with severe pulmonary hemorrhage or rapidly progressive glomerulonephritis(RPGN)received pulse methylprednisolone with or without DFPP,and then followed by prednisone and CTX.Group Ⅱ∶5 patients without severe pulmonary hemorrhage and RPGN received prednisone and CTX.Group Ⅲ∶5 ESRD patients and 1 normal renal function patient did not receive immunosuppression therapy.Anti-GBM antibody titer of pre-and post-DFPP in 4 patients was measured consecutively,and removal rate was calculated.Results The mean age of all the patients was(41.1±16.6)years.Sixteen patients(45.7%)presented Goodpasture's syndrome.Eighteen patients(51.4%)had anti-GBM glomerulonephritis alone,whereas one suffered solely from pulmonary hemorrhage.20%patients had positive P-ANCA serology.54.2%crescentic glomerulonephritis and 7 with other glomerulonephritis were revealed by kidney biopsy in 24 patients.Patients in Group Ⅰ showed more severe manifestation at admission:higher Scr level,higher titer of anit-GBM antibody,greater percentage of crescents.Within the follow-up period,7 patients died and kidneys of 50%patients survived.No patient died in Group Ⅱ and Ⅲ.The elder age,anemia,higher Scr(＞300 μmol/L),oliguria or anuria,emergency hemodialysis at admission,and more glomerular sclerosis were predictors of poor prognosis.The anti-GBM antibody was negative after 4 to 6 sessions of DFPP.and the mean removal rate was 55%.During total 94 DFPP sessions,there was no unacceptable morbidity. Conclusions Different therapy strategy is necessary for anti-GBM disease with different clinical manifestations.DFPP is an effective and safe clearance way of anti-GBM antibody.