OBJECTIVETo construct eukaryotic expression vectors containing three different haplotype cDNAs of human CIITA gene.

METHODcDNA fragments of three different CIITA haplotypes were obtained by inducing one or two single nucleotide mutations of wild type recombinant plasmid EBS-NPL-CIITA cDNA, which correspond to two non-homonymy single nucleotide polymorphism (SNP) sites in the coding region of human CIITA gene, using overlap extension PCR site-directed mutagenesis technology. The above-mentioned three haplotype cDNAs were respectively cloned to EBS-NPL-CIITA linearized vectors. Positive clones were identified by colonial PCR and restriction endonuclease digestion and were sent to be sequenced. Then eukaryotic expression vectors containing four different haplotypes and an empty vector EBS-NPL were transfected into HepG2 cells respectively. HLA-DR was detected by indirect cell immunofluorescence technique.

RESULTSThe cDNA fragments of three different human CIITA haplotypes were successfully constructed, and the eukaryotic expression vectors containing three different haplotype cDNAs of human CIITA gene were obtained. No expression of HLA-DR was observed in the original HepG2 cells and empty vector transfected HepG2 cells and the expression of HLA-DR emerged in the HepG2 cells transfected with four eukaryotic expression vectors.

CONCLUSIONThe eukaryotic expression vectors containing three different haplotype cDNAs of human CIITA gene were successfully constructed, and they are essential for our further study of the functional differences of them.

Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Genetic Vectors ; Haplotypes ; Hep G2 Cells ; Humans ; Molecular Sequence Data ; Nuclear Proteins ; genetics ; Trans-Activators ; genetics

OBJECTIVETo investigate the relationship between the susceptibility to cirrhosis and the single nucleotide polymorphisms (SNPs) at C-1350T and G-944C loci of class II transactivator (CIITA) gene promoter IV in chronic HBV carriers.

METHODSC-1350T and G-944C loci of CIITA gene promoter IV were analyzed by sequence-specific primer PCR (PCR-SSP) in 544 chronic HBV carriers and 125 non-HBV infected healthy blood donors.

RESULTSAmong the chronic viral hepatitis B patients, there were significantly decreased frequencies of CC and TG haplotypes, and significantly increased frequency of CG haplotype among patients with liver cirrhosis (CG vs. CC: chi2=8.274, df=1, P < 0.01; CG vs. TG: chi2 = 15.027, df =1, P <0.01). There were no significant differences in the frequencies of CC and TG haplotypes between chronic hepatitis B and liver cirrhosis patients (chi2 = 1.231, df =1, P < 0.05). There were significantly increased frequencies of CC/CC (Group 1) genotype and genotypes contained CG haplotype (Group 3), and significantly decreased frequencies chi2= 7.176, df = 1, P < 0.01; Group1 vs Group 4, chi2 = 19.818, df = 1, P < 0.01; Group 3 vs Group 2, chi2 = 11.423, df = 1, P < 0.01; Group 3 vs Group 4, chi2 = 34.226, df = 1, P < 0.01; Group 1 vs Group 3, chi2 = 0.009, df = 1; Group 2 vs Group 4, chi2 = 2.176, df = 1).

CONCLUSIONPolymorphisms at -1350 and -944 loci of CIITA gene promoter IV are associated with susceptibility to liver cirrhosis in chronic HBV carriers. The chronic HBV carriers bearing CC/CC genotype or genotypes containing CG haplotype progress into liver cirrhosis with more probability.

Adult ; Female ; Genetic Predisposition to Disease ; Haplotypes ; Hepatitis B, Chronic ; genetics ; Humans ; Liver Cirrhosis ; genetics ; Male ; Middle Aged ; Nuclear Proteins ; genetics ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Trans-Activators ; genetics

OBJECTIVETo study the possible association between interferon regulatory factor 5 (IRF5) gene polymorphism and allergic rhinitis (AR).

METHODSSix independent single nucleotide polymorphism (SNP, rs729302, rs4728142, rs3807306, rs2070197, rs11770589, rs2280714) were analyzed. The genotype and allele frequencies were detected in 110 AR patients and 101 healthy controls in Singapore Chinese population by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP).

RESULTSRs729302 was rejected as it was not polymorphic. For others SNP, no statistically significant difference was detected in genotype between AR and healthy control group (χ(2) value were 0.21, 5.02, 0.01, 2.91, 0.37, all P > 0.05). No statistically significant difference was detected in allele frequencies between AR and healthy control group (χ(2) value were 0.00, 2.78, 0.01, 2.31, 0.00, all P > 0.05).

CONCLUSIONNo association is observed between IRF5 and AR in Singapore Chinese population.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Female ; Gene Frequency ; Genotype ; Humans ; Interferon Regulatory Factors ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Rhinitis, Allergic ; Rhinitis, Allergic, Perennial ; genetics ; Singapore ; Young Adult

AIM:To investigate the effects of vinpocetine on inflammation of brain in intracerebral hemorrhage ( ICH) rats and to explore the underlying mechanisms .METHODS:All rats were randomly divided into sham group , ICH group, ICH with low dose of vinpocetine treatment group , ICH with medium dose of vinpocetine treatment group , and ICH with high dose of vinpocetine treatment group .Except sham group , the rats in other groups were injected with type VII col-lagenase to establish ICH model , and then the rats in vinpocetine treatment groups were received vinpocetine at 0.5, 1.0 or 1.5 mg/kg by intraperitoneal injection once a day for 7 days.After corresponding treatment , the impairment of neurological function in the rats was scored and the water content of the brain tissues was measured .Moreover, the activity of myeloper-oxidase (MPO) was determined by ELISA, and the protein expression of Toll-like receptors 4 (TLR4), nuclear factor-κB (NF-κB), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molcule-1 (VCAM-1) in the brain tissues was determined by Western blot .RESULTS: Compared with ICH group , vinpocetine treatment significantly de-creased the scores of the impairment of neurological function and the water content of the brain tissues .Moreover, the activ-ity of MPO and the protein expression of TLR4, NF-κB, ICAM-1 and VCAM-1 were also reduced after vinpocetine treat-ment (P<0.05).CONCLUSION:Vinpocetine improves neurological function in ICH rats via suppression of inflamma -tion by inhibiting NF-κB signaling and expression of ICAM-1 and VCAM-1.

OBJECTIVETo study aberrant expression of cell cycle control genes in patients with myelodysplastic syndromes (MDS).

METHODSReverse transcription polymerase chain reaction (RT-PCR) was used to investigate the expression of cell cycle control genes (cyclin D2, cyclin D3, cyclin A1, cyclin E, CDK2, CDK4, CDK6, p21, p27, p57, Rb and E2F1) in bone marrow mononuclear cells (BMMNCs) from 29 normal control, 27 MDS and 19 de novo acute myeloid leukemia (AML).

RESULTSThe expression levels of cyclin D3 (0.65 +/- 0.17, P < 0.05) and cyclin A1 (0.48 +/- 0.04, P < 0.05) in MDS were higher than those in normal control and significantly lower than those in AML. The expression rates and levels of cyclin D2 (40.7% and 0.78 +/- 0.21) and cyclin E (51.9% and 0.52 +/- 0.10) in MDS were statistically higher than those in normal control and AML. The expression level of CDK2 in MDS (0.66 +/- 0.19, P < 0.01) was higher than that in normal control (0.42 +/- 0.04) and the expression rate of CDK6 in MDS (25.9%) higher than in normal control (3.4%, P < 0.05). There was no significant difference of the expression rates and levels of CDK4 in MDS, AML and normal control. The expression rates and levels of p21 (77.8% and 1.18 +/- 0.21) and p27 (48.1% and 1.14 +/- 0.40) in MDS were statistically higher than those in normal control and AML. The expression level of p57 in MDS (0.69 +/- 0.06) was higher than that (0.53 +/- 0.05, P < 0.01) in normal control but lower than in AML (0.96 +/- 0.16, P < 0.01). The expression rate (55.6%) and level (0.85 +/- 0.17) of Rb in MDS were significantly higher than those in normal control and AML. The expression rate (7.4%) and level (0.39 +/- 0.04) of E2F1 in MDS were comparable to those in normal control but lower than those in AML.

CONCLUSIONMDS clones have aberrant mechanism of cell cycle control: high expressions of cyclin family members, CDK2 and CDK6 may lead to high proliferation; high expression of p21 and p27 may cause the G1 phase arrest.

Adolescent ; Adult ; Cell Cycle Proteins ; genetics ; Child ; Cyclin-Dependent Kinase Inhibitor Proteins ; genetics ; Cyclin-Dependent Kinases ; genetics ; E2F1 Transcription Factor ; genetics ; Female ; Gene Expression Profiling ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; pathology ; Retinoblastoma Protein ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

To evaluate the efficacy and safety of interferon-alpha-2b (IFN-α-2b) in polycythemia vera patients(PV patient) with or without post-polycythemic myelofibrosis (post-PV MF), 30 patients with mutated JAK2V617F were enrolled in this study, from which 29 patients were evaluable. The percentage of mutated JAK2V617F allele (V617F%) was evaluated by real-time polymerase chain reaction (RT-PCR) before and after treatment with IFN-α-2b. The correlation of V617F allele burden with the major clinical outcomes was studied. Adverse effects appeared in patients was observed. The results showed that the median follow-up was 24 (12 - 42) months for 29 evaluable patients. Complete hematologic response was achieved in 10%, 48%, 72% and 78% of patients after treatment for 6, 12, 24 and 36 months respectively. The detection of V617F allele burden revealed that the molecular remission of patients (V617F%) was achieved in 41%, 76%, 89% and 89% after treatment for 6, 12, 24 and 36 months respectively. Molecular complete remission (JAK2V617F undetectable) was achieved in 4 patients, lasted from 6 to 12 months after IFN-α-2b discontinuation. The decrease of V617F% in patients with post-PV MF was significantly higher than that in patients without post-PV MF (53 ± 18% vs 32 ± 22%, respectively; p = 0.031) after treatment for 12 months. PV patients had a good tolerance to IFN-α-2b. It is concluded that IFN-α-2b can decrease the mutated V617F allele burden. Patients with PV, especially with post-PV MF, can achieve molecular remission after treatment with IFN-α-2b.
Adult ; Alleles ; Female ; Humans ; Interferon-alpha ; therapeutic use ; Janus Kinase 2 ; genetics ; Male ; Middle Aged ; Mutation ; Polycythemia Vera ; drug therapy ; genetics ; pathology ; Primary Myelofibrosis ; drug therapy ; genetics ; pathology ; Recombinant Proteins ; therapeutic use

OBJECTIVETo clarify the expression and clinical significance of metastasis-associated in colon cancer 1 (MACC1) mRNA in hepatocellular carcinoma (HCC).

METHODSThe expression and distribution of MACC1 were assessed by quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemical staining (IHC) in a cohort of hepatitis B virus-related HCC, including 138 in early (A), 96 in intermediate (B) and 120 in advanced stages (C). The association of MACC1 mRNA with disease progression and outcomes was analyzed by univariate and multivariate Cox analysis.

RESULTSThe intratumoral expressions of MACC1 mRNA in HCC stage I (0.001 76, range: 0.000 54 - 0.002 47), stage II (0.002 49, range: 0.000 55 - 0.006 78) and stage III (0.008 35, range: 0.006 86 - 0.009 88) were about 3-, 4- and 14-fold higher than that in the normal liver tissue (0.000 59, range: 0.000 57 - 0.000 60), respectively. Intratumoral expression of MACC1 mRNA increased with disease progression from stage I to stage III. HCC clinical staging classification, age, portal vein invasion and tumor differentiation were significantly associated with intratumoral high expression of MACC1 mRNA (All P < 0.05). Immunohistochemical staining showed that there was an increased MACC1 expression in cytoplasm of HCC cells and positive nuclear staining in some cases. Increased MACC1 mRNA expression could predict poor outcome and recurrence in stage A and B HCC postoperatively. The median tumor-free survival and total survival of patients with high MACC1 mRNA expression were 34.0 and 40 months, respectively, significantly lower than that in those with low expression (48.0 and 48.0 months) (all P < 0.01). Cox analysis showed that Child-Pugh grading and high expression of MACC1 mRNA were independent predictive factors, and high expression of MACC1 was an independent predictive factor affecting the tumor-free survival.

CONCLUSIONSMACC1 mRNA up-regulation is a feature of disease progression in HCC. MACC1 mRNA expression in the HCC may become an independent predictive factor for recurrence and survival in postoperative HCC patients.

Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; pathology ; virology ; DNA, Viral ; analysis ; Disease-Free Survival ; Female ; Follow-Up Studies ; Hepatitis B virus ; Humans ; Liver Neoplasms ; metabolism ; pathology ; virology ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Proportional Hazards Models ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Survival Rate ; Transcription Factors ; genetics ; metabolism ; Up-Regulation ; Young Adult

OBJECTIVETo study the characteristics of cell cycle and proliferation of CD34+ hematopoietic stem cells in patients with myelodysplastic syndromes (MDS).

METHODSPropidium iodide staining was used to examine cell cycle parameters (G(0)/G(1), S and G(2)/M) of bone marrow mononuclear cells (BMMNCs) while immunofluorescent double staining and FACS techniques were used to measure Ki67 expression in BM CD34+ cells from normal control, patients with MDS, acute myeloid leukemia preceded by MDS (MDS-AML) and primary AML.

RESULTSThere was a statistical up-tendency in G(0)/G(1) phase proportion of BMMNCs whereas a statistical down-tendency in S and G(2)/M phase proportions among normal control, MDS and primary AML. Compared to primary AML, MDS-AML had significantly higher ratios of S (P < 0.05), G(2)/M (P < 0.05) and S + G(2)/M (P < 0.05) phase cells while lower ratio of G(0)/G(1) phase cells (P < 0.05). The proportion of CD34+Ki67+ cells in MDS patients was significantly higher than that in normal control (P = 0.004). So were the percentages of CD34+Ki67+ cells in low-risk [(0.54 +/- 0.49)%, P < 0.05] and high-risk MDS patients [(1.69 +/- 1.66)%, P = 0.022]. Furthermore, there was statistical difference between low-risk and high-risk MDS (P < 0.05). Compared to normal control and primary AML, MDS-patients had the highest proportion of CD34+Ki67+ cells [(16.75 +/- 13.58)%, P < 0.05]. The proportion of CD34+Ki67+ cells in CD34+ cells in MDS patients [(48.50 +/- 20.49)%] was significantly higher than that in normal control [(27.71 +/- 16.04)%, P < 0.01]. So were the low-risk [(51.85 +/- 21.80)%, P = 0.002] and high-risk MDS [(43.93 +/- 18.57)%, P < 0.05]. The proportion of CD34+Ki67+ cells in CD34+ cells in MDS-AML patients [(60.92 +/- 30.12)%] was the highest, and was statistically higher than that in both normal control (P < 0.01) and primary AML patients [(17.01 +/- 15.93)%, P < 0.001]. The proportion of CD34+Ki67+ cells in Ki67+ cells in MDS patients [(4.91 +/- 4.68)%, P < 0.01] was significantly higher than that [(2.43 +/- 2.37)%] in normal controls. In the low-risk MDS group it was (4.11 +/- 3.94)%, (P > 0.05) and in high-risk MDS group it was (5.76 +/- 5.38)%, (P < 0.05).

CONCLUSIONHigh proportion of G(0)/G(1) cells and G(1) phase arrest occurred in MDS. High proliferation capacity of MDS clone, especially that derived from CD34+ cells, might play an important role in the clonal expansion, diseases deterioration and worse prognosis of MDS.

Acute Disease ; Adolescent ; Adult ; Aged ; Antigens, CD34 ; blood ; Bone Marrow Cells ; metabolism ; pathology ; Cell Cycle ; Cell Proliferation ; Female ; Flow Cytometry ; Fluorescent Antibody Technique ; Humans ; Ki-67 Antigen ; blood ; Leukemia, Myeloid ; blood ; Male ; Middle Aged ; Myelodysplastic Syndromes ; blood ; Young Adult

OBJECTIVETo study the quantity and ratio of Th1, Th2 cells in the bone marrow of myelodysplastic syndromes (MDS) patients, and to evaluate the correlation between the ratio of the blast cells and the number of the Th1 cells in the bone marrow of MDS patients.

METHODSBy FACS, the quantity and ratio of IFN-gamma producing CD4(+) T cell (Th1) and IL-4 producing CD4(+) T cell (Th2) cells in the bone marrow were detected in 21 MDS patients, 18 normal controls and 13 severe aplastic anemia (SAA) patients respectively. The karyotypes of 18 MDS patients and 15 normal controls were assayed. The correlation between the ratio of the blast cells in the bone marrow and the number of the Th1 cells in the MDS patients were analyzed.

RESULTSThe percentages of Th1 cells, Th2 cells and ratio of Th1/Th2 in the bone marrow of normal controls were (0.48 +/- 0.10)%, (0.24 +/- 0.19)% and 2.31 +/- 0.76 respectively, while those of the MDS patients were (0.36 +/- 0.11)%, (0.76 +/- 0.35)% and 0.51 +/- 0.13. The percentage of Th1 cells of patients with MDS was reduced and the Th1/Th2 ratio was significantly lower than that of normal controls (P < 0.01). Those of the patients with SAA were (4.75 +/- 0.49)%, (0.40 +/- 0.28)% and 26.5 +/- 8.79 respectively, their Th1 cells and Th1/Th2 ratio were markedly higher than those of normal controls (P < 0.01). In all of the 15 normal controls the karyotypes were normal, but that of MDS patients was (50.00 +/- 0.10)%. The lower ratio of the Th1 cells in the bone marrow of the patients with MDS and the AML which progressed from MDS was negatively correlated with the higher percentage of the blast cells (r = -0.563, P < 0.01).

CONCLUSIONS(1) The immune function of T lymphocytes in MDS is abnormal: the balance between Th1 and Th2 cells is broken. (2) With descending of the number of Th1 cells in the bone marrow of the MDS patients, the disease is progressing to leukemia.

Adult ; Aged ; Bone Marrow ; immunology ; Female ; Humans ; Karyotyping ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; immunology ; T-Lymphocytes, Helper-Inducer ; immunology

OBJECTIVESInvestigate the clinical efficacy of cryotherapy ablation treatment for advanced hepatocellular carcinoma. analyse the predictive factors of cryotherapy ablation treatment.

METHODSThere were 190 cases of hepatitis B-related HCC patients with advanced HCC from 2005 to 2008 in our hospital. By using clinical cohort method, they included cryoablation group (147 cases) and control group (43 cases), The median survival time and time to disease progression were compared. Evaluate clinical significance of age, gender, location of portal vein tumor thrombus, HBeAg, tumor histological grade, Child-Pugh classification, end-stage liver disease (MELD) score, advanced liver cancer prediction system (ALCPS) score and the Eastern Cooperative Oncology Group performance status (ECOG PS) score for predicting the efficacy of cryoablation. Group rates were compared with the x2 test, survival analysis by using Kaplan-Meier method, survival rates were compared by Log-rank analysis; multiple factor survival analysis by using Cox regression model.

RESULTSMedian survival time of cryoablation group and Control group was 7.5 (4.2 to 14.6) months and 3.2 (1.2 to 8.6) months, median TTP was 3.5 (2.5 to 4.5) months and 1.5 (1.0 to 3.5 months), the differences were statistically significant ( P less than 0.05 ). Median OS and TTP of advanced HCC patients who had Well-differentiated tumor, Child-pugh A-class and low score of MELD score, ALCPS score, ECOG PS score were significantly longer than the poorly differentiate, Child-Pugh B-class and the high those scores ( P less than 0.05). ECOG PS ( P less than 0.05, 95% CI 1.074 to 2.143) and ALCPS (P less than 0.05, 95% CI 1.005 to 2.121) were independent predictors for OS of advanced HCC.

CONCLUSIONCryoablation treatment can prolong median OS and TTP of advanced HCC; ECOG PS and ALCPS are important predictors for survival time of advanced HCC.

Carcinoma, Hepatocellular ; surgery ; Cryosurgery ; Female ; Humans ; Liver Neoplasms ; surgery ; Male ; Middle Aged ; Prognosis ; Proportional Hazards Models ; Survival Rate ; Treatment Outcome