Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression

Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The ex- pression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibi- tion on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.

Screening of Epstein-Barr Virus Early Antigen Expression Inducers from Chinese Medicinal Herbs and Plants

Ether extrilcls of 1693 Chinesc medicinal herbs and plilnts from 268 families werestudied for the induction of Epstcin-Barr viral (EBV ) early antigcn (EA ) expression in theRaji cell line. Fifty-two from 18 families were found to have inducing activity. Twenty-fiveand seven of them were from Euphorbiaccae and Thymclaeaceae, respectively. Some ofthem, such as Croton tiglium, Euphorbia kansui, Daphnc genkwa, Wikstrocmia chamacdaphen, Wikstroemia indica, Prunus mandshurica Koehne and Achyranthes bidentata arecommonly used drugs. The significance of these herbs in the activation of EBV in vivo andtheir relation to the development of nasopharyngeal carcinoma were discussed.

Effect of Ligustrazine on CD_(34) Antigen Expression of Bone MarrowCells in Immune-Induced Aplastic Anemia Mice

Objective:To explore the effect of ligustrazine on CD34 antigen expression of bone marrow cells in immune-induced aplastic anemia（AA）mice.Methods:The model of immune aplastic anemia mice was in duced by means of 6.0GY60γ-rayirr adi at ion and lym phocyte infusion through tail vein.Immuneinduce dAA micewere divided into 3 groups:thenormal group,the AA control group and the ligustrazine group.Miceof the ligustrazine group were fed by 4 mg of ligustrazine in jecti on twicea day bygastro gavage.On the 10 thday,CD34 an tigen expression intensity of bone marrow cell membrane was measured by flowcy tometer an alysis system.Results:CD34 antigen expressionin tensity of ligustrazine group was 77.6±6.5,with no statistic difference from that in normal group（80.0±2.6),while that of the control group was muchhigher（68.6±4.5,P＜0.05).Conclusion:Ligustrazine could promote proliferation of stem and progenitor cell of AA mice through in fluencing on bone marrowmicro-environment so as to increase the CD34antigen expression of bone marrow cells.

STRUCTURE AND EXPRESSION OF EPSTEIN-BARR VIRUS MEMBRANE ANTIGEN IN RECOMBINANT VACCINIA VIRUS

The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen was expressed under the control of a 7.5 K promoter of vaccinia virus. The antibody against the membrane antigen of EB virus was produced on rabbits vaccinated with recombinant vaccinia virus.

Effectof the Local Strain CN5 of Human Herpesvirus 6 on CD Molecule Expression and PHA-Induced Proliferation of Cord Blood or Peripheral Blood Mononuclear Cells

Cord blood mononuclear cells (CBMC's) and adult peripheral blood mononuclear cells (PBMCs), cultured with RPMI 1640 medium containing 20% fetal calf serum plus 5 mg/L PHA and 10 mg/L IL-Z, were inoculated with CN5 strain of human herpesvirus 6 (HHV 6 ) isolated from a Chinese patient with exanthem subitum and the CN5 infected cell lysates were added to cultures of CBMCs and PBMCs, so as to observe the effects of the local strain CN5 on expression of CD molecule or on proliferation of mononuclear cells by the methods of APAAP staining and MTT assay.The results were as follows: ①Expressions of some CD antigens of CBMCs and PBMCs could change after CN5 strain infection. In both cases, CD3 expresion was down-regulated while CD4 expression was up-regulated. There were no significant differences of CD2, CD8 and CD45RA expressions between the two groups with and without CN5 infection. But the ratio of CD4 to CD8 sigmificantly rose because of the increasing of CD4 positiveity. ②The lysates of CB5-infected CBMCs inhibited the liferation of PBMCs, not of CBMCs, in a protein concentration-dependent pattern. This inhibition was partially neutralized by specific antiserum to CN5, not by antisera to INF-α and TNF-α.

PRAME Gene Expression in Acute Leukemia and Its Clinical Significance

Objective To investigate the expression of the preferentially expressed antigen of melanoma （PRAME） gene in acute leukemia and its clinical significance. Methods The level of expressed PRAME mRNA in bone marrow mononuclear cells from 34 patients with acute leukemia （AL） and in 12 bone marrow samples from healthy volunteers was measured via RT-PCR. Correlation analyses between PRAME gene expression and the clinical characteristics （gender, age, white blood count, immunophenotype of leukemia, percentage of blast cells, and karyotype） of the patients were performed. Results The PRAME gene was expressed in 38.2% of all 34 patients, in 40.7% of the patients with acute myelogenous leukemia （AML, n=27）, and in 28.6% of the patients with acute lymphoblastic leukemia （ALL, n=7）, but was not expressed in the healthy volunteers. The difference in the expression levels between AML and ALL patients was statistically significant. The rate of gene expression was 80% in M~, 33.3% in M2, and 28.6% in M~. Gene expression was also found to be correlated with CDl5 and CD33 expression and abnormal karyotype, but not with age, gender; white blood count or percentage of blast cells. Conclusions The PRAME gene is highly expressed in acute leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute leukemia.

Elementary study on the antigenicity of SARS-CoV Mc region

To test the antigenic activity of M protein （Mc protein） in the inner membrane of SARS-CoV, SARS-CoV Me protein＇s bases locating inside the membrane were cloned, the His-fusion protein was expressed in E. coli and analyzed for its antigenic activity. Among those 7 clinically diagnosed patients＇ sera, there were 5 positive and 2 negative in reaction with His-fusion protein. All of the 20 healthy persons＇ sera and rabbit anti-OC43 and 229E were of negative reaction with His-fusion protein. The animals immunized with His-fusion protein have produced muhi-clonal antibody. The His-fusion protein could specially react with clinically diagnosed SARS patients＇ sera and the animals immunized with His-fusion protein could produce specifically multi-clonal antibody, but it could not react with the sera of healthy persons and the rabbit anti-OC43 and 229E.

A Sensitive and Specific IgM-ELISA for the Serological Diagnosis of Human Leptospirosis Using a rLipL32/1-LipL21-OmpL1/2 Fusion Protein

Objective To construct a lipL32//1-1ipL21-OmpL1//2 fusion gene and its prokaryotic expression system, and to establish an enzyme-linked immunosorbent assay （ELISA） using the rLipL32/1-LipL21-OmpL1/2 fusion antigen of Leptospira interrogans for sensitive and specific detection of IgM in the serum of patients with leptospirosis. Methods lipL32/1-1ipL21-OmpL1/2 fusion genes were constructed using a primer-linking PCFI. The target recombinant protein antigens, rLipL32/1, rLipL21, rOmpL1/2 and rLipL32/1-LipL21-OmpL1/2, were expressed and the purified antigens were then immobilized to the surface of microplate wells for ELISA-based detection of IgM in the sera of leptospirosis patients; Results Of 493 acute leptospirosis patients, 95.7% and 97.8% were positive by rLipL32/1-LipL21- OmpL1/2-1gM-ELISA using different serum dilutions, which was higher than the rLipL32/1-1gM-ELISA （93.1% and 90.3%）, rLipL21-1gM-ELISA （90.3% and 87.0%）, and rOmpLI-lgM-ELISA （85.6% and 81.1%） （P〈0.01）. All IgM-ELISAs tested negative against 56 non-leptospirosis patients with typhoid fever, hemorrhagic fever or dengue fever. Conclusion Trigeminal fusion antigen increases ELISA sensitivity and the rLipL32/1-LipL21-OmpL1/2- IgM-ELISA is a sensitive and specific serological diagnostic method for clinical leptospirosis.

Growth inhibiting effects of antisense eukaryotic expression vector of proliferating cell nuclear antigen gene on human bladder cancer cells

Objective To explore the growth inhibiting effects on human bladder cancer by antisense RNA targeting the proliferating cell nuclear antigen (PCNA) gene. Methods The eukaryotic expression vector for antisense PCNA cDNA was constructed and transferred into a bladder cancer EJ cell line. The PCNA expression in the cancer cells was detected by RT-PCR and Western blotting assays. The in vitro proliferation activities of the transferred cells were observed by growth curve,tetrazolium bromide (MTT) colorimetry,tritiated thymidine ( 3H-TdR)incorporation, flow cytometry and clone formation testing,while its in vivo anti-tumor effects were detected on nude mice allograft models.Results After the antisense vector,pLAPSN,was transferred,cellular PCNA expression was inhibited at both protein and mRNA levels. The growth rates of EJ cells were reduced from 27.91% to 62.07% ( P <0.01),with an inhibition of DNA synthesis rate by 52.31% ( P <0.01). Transferred cells were blocked at G 0/G 1 phases in cell-cycle assay,with the clone formation ability decreased by 50.81% ( P <0.01). The in vivo carcinogenic abilities of the transferred cancer cells were decreased by 54.23% ( P <0.05). Conclusions Antisense PCNA gene transfer could inhibit the growth of bladder cancer cells in vitro and in vivo,which provided an ideal strategy for gene therapy of human cancers.

EXPRESSION OF PROLIFERATING CELL NUCLEAR ANTIGEN IN CUTANEOUS MALIGNANT LYMPHOMAS AND LYMPHOID INFILTRATES

Paraffin-embedded tissue of skin biopsy specimens taken retrospectively from 24 patients with cutaneous malignant lymphomas (CML) and 8 patients with cutaneous lymphoid infiltrates (CLI) and other dennatoses were studied retrospectively with PCIO immunostaining. The results show a statistical significant difference among PCIO indices for cutaneous genuine histiocytic lymphoma (CGHL), cutaneous germinal center cell-derived lymphomas (CGCCL), cutaneous peripheral T-cell lymphomas (CPTL), non-mycosis fungoides (MF) and Sezary's syndrome (SS), and MF when compared with those for CLI. There is a linear relationship between PCIO index and square root of PCIO density, both of which seem to have a parallel relationship to the severity of malignancy in CML. The nuclear volume of the positive tumor cell or lymphocyte with PCIO immunostaining may be also useful in differentiating CML from CLI.

Bortezomib improves progression-free survival in multiple myeloma patients overexpressing preferentially expressed antigen of melanoma

Background Significant efforts have been made to identify factors that differentiate patients treated with novel therapies,such as bortezomib in multiple myeloma （MM）.The exact expression pattern and prognostic value of the cancer/testis antigen preferentially expressed antigen of melanoma （PRAME） in MM are unknown and were explored in this study.Methods The transcript level of PRAME was detected in bone marrow specimens from 100 newly diagnosed MM patients using real-time quantitative polymerase chain reaction,and the prognostic value of PRAME was determined through retrospective survival analysis.PRAME expression higher than the upper limit of normal bone marrow was defined as PRAME overexpression or PRAME （＋）.Results Sixty-two patients （62.0％） overexpressed PRAME.PRAME overexpression showed no prognostic significance to either overall survival （n=100） or progression-free survival （PFS,n=96,all P ＞0.05） of patients.The patients were also categorized according to regimens with or without bortezomib.PRAME overexpression tended to be associated with a lower two-year PFS rate in patients treated with non-bortezomib-containing regimens （53.5％ vs.76.9％,P=0.071）.By contrast,it was not associated with the two-year PFS rate in patients with bortezomib-containing regimens （77.5％ vs.63.9％,P ＞0.05）.When the patients were categorized into PRAME （＋） and PRAME （-） groups,treatment with bortezomib-containing regimens predicted a higher two-year PFS rate in PRAME （＋） patients （77.5％ vs.53.5％,P=0.027） but showed no significant effect on two-year PFS rate in PRAME （-） patients （63.9％ vs.76.9％,P ＞0.05）.Conclusion PRAME overexpression might be an adverse prognostic factor of PFS in MM patients treated with non-bortezomib-containing regimens.Bortezomib improves PFS in patients overexpressing PRAME.