Objective To construct the recombinant plasmid pYES6-S and express and purify spike protein of severe acute respiratory syndrome(SANS)coronavirus in Saccharomyces cerevisiae. Methods DNA fragments of SANS coronavirus were obtained by reverse transeription.Four over- lapped fragments of spike protein genes were amplified by polymerase chain reaction(PCR)and ligated into an integral spike protein gene by restriction enzyme digestion.The spike protein gene recombined with pYES6 and cloned into E.coll.The recombinant plasmid pYES6-S was induced and expressed in Saccharomyces cerevisiae(INVScl)by galactose.Results The recombinant plasmid pYES6-S was confirmed that inserted fragment was right in length,direction and base matching by restriction enzyme digestion and sequencing.The purified protein encoded by the whole spike protein gene was about Mr 110?10~3 identified by electrophoresis.Conclusion The whole spike protein gene of SARS coronavirus is cloned into E.coli and the protein is expressed in Saccharomyces cerevisiae successful ly.which can be helpful in SARS vaccine research.

Objective: To investigate the outcomes of anti-CD19 CAR-T cell for relapsed and refractory B cell malignancies. Method: Ten patients with relapsed and refractory B cell acute lymphocytic leukemia (B-ALL) and non-Hodgkin’s lymphoma (NHL), diagnosed in the Department of Hematology of Peking University third Hospital from December 2015 to July 2017, were treated with anti-CD19 CAR-T cell therapy, and the efficacy and safety were analyzed. Results: Efficacy was assessed on the 28th day after cell infusion, including 66.7% (4/6) of complete remission (CR) for patients with ALL, 16.7% (1/6) of partial remission (PR), and 83.3% (5/6) of overall response rate (ORR). For NHL patients, CR was 33.3% (1/3) and most of the lesions disappeared in a patient with mantle cell lymphoma, but residual lesion presented persistent state. After infusion of anti-CD19 CAR-T cells, the main side effect was cytokine release syndrome (CRS) and fever. One patient presented with aphasia and the other one had multiple organ failure, which were improved after treatment. No patients died of CRS. Conclusion anti-CD19 CAR-T cell for relapsed and refractory B cells hematological malignancies is safe, and the most problematic side effect is CRS, which can be controlled by therapy.

Objective: To investigate the outcomes of anti-CD19 CAR-T cell for relapsed and refractory B cell malignancies. Method: Ten patients with relapsed and refractory B cell acute lymphocytic leukemia (B-ALL) and non-Hodgkin's lymphoma (NHL), diagnosed in the Department of Hematology of Peking University third Hospital from December 2015 to July 2017, were treated with anti-CD19 CAR-T cell therapy, and the efficacy and safety were analyzed. Results: Efficacy was assessed on the 28th day after cell infusion, including 66.7% (4/6) of complete remission (CR) for patients with ALL, 16.7% (1/6) of partial remission (PR), and 83.3% (5/6) of overall response rate (ORR). For NHL patients, CR was 33.3% (1/3) and most of the lesions disappeared in a patient with mantle cell lymphoma, but residual lesion presented persistent state. After infusion of anti-CD19 CAR-T cells, the main side effect was cytokine release syndrome (CRS) and fever. One patient presented with aphasia and the other one had multiple organ failure, which were improved after treatment. No patients died of CRS. Conclusion: anti-CD19 CAR-T cell for relapsed and refractory B cells hematological malignancies is safe, and the most problematic side effect is CRS, which can be controlled by therapy.
Antigens, CD19 ; B-Lymphocytes ; Cell- and Tissue-Based Therapy ; Humans ; Leukemia, B-Cell ; Receptors, Antigen, T-Cell ; Recurrence ; T-Lymphocytes

BACKGROUNDEndobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) can sample the enlarged mediastinal lymph nodes which are unreachable by conventional bronchoscopy. It is a relatively simple and safe method to see beyond the bronchial tree. We describe and discuss its initial application and our experience.

METHODSFrom July 2009 to December 2009, 52 patients with undiagnosed enlarged mediastinal lymph nodes were accessed with EBUS-TBNA in the People's Liberation Army General Hospital. Conventional bronchoscopy was performed before EBUS-TBNA, and patients with endobronchial lesions were excluded from this study. Smears fixed in 95% alcohol and histological specimens fixed in formalin were sent to Department of Pathology.

RESULTSEBUS-TBNA was diagnostic in 33 (63%) patients, with diagnosis of lung cancer in 23 patients (14 patients of small cell lung cancer, eight patients with adenocarcinoma, and one patient of squamous carcinoma). Four patients, who had negative EBUS-TBNA results, were later diagnosed with malignancy at thoracotomy. One patient with negative EBUS-TBNA results died of cancer cachexia. The sensitivity, specificity, and positive and negative predictive value of EBUS-TBNA for the diagnosis of neoplastic disease were 85%, 100%, 100%, and 50% respectively. Among the 16 sarcoidosis patients, who were diagnosed by a combination of the clinical and radiological information as well as pathological results obtained by EBUS-TBNA, nine of them had granulomas and benign lymphoid cells detected by EBUS-TBNA. The sensitivity, specificity, and positive and negative predictive value of EBUS-TBNA for the diagnosis of sarcoidosis were 56%, 100%, 100%, and 13%, respectively. Five patients with no definite diagnosis from EBUS-TNBA examination are under close follow-up.

CONCLUSIONSEBUS-TBNA can provide a safe and effective method to sample mediastinal leisions suspected of malignancy. It also adds pathological information needed to make the diagnosis of sarcoidosis.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Biopsy, Fine-Needle ; methods ; Bronchi ; diagnostic imaging ; pathology ; Endosonography ; methods ; Female ; Humans ; Lung Neoplasms ; diagnosis ; Lymphatic Diseases ; diagnosis ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; methods ; Young Adult

OBJECTIVETo study the clinical features of children with 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency and review the literature.

METHODClinical features and treatment of one Chinese infant with 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency confirmed by HSD3B7 gene mutation analysis were retrospectively reviewed, and 51 cases of 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency who were internationally reported since 2000 were also reviewed in this paper.

RESULT(1) A 3-month-old infant with neonatal cholestasis was admitted to our hospital because of hyperbilirubinemia and abnormal liver dysfunction (total bilirubin 110.7 µmol/L, direct bilirubin 74.5 µmol/L, γ-glutamyltransferase 24.4 IU/L, total bile acid 0.1 µmol/L).His jaundice disappeared within a few weeks, serum liver biochemistries improved and his growth in weight and height was excellent after oral cholic acid therapy.HSD3B7 gene analysis using peripheral lymphocyte genomic DNA from the patient identified compound heterozygous mutations. This child was confirmed as the most common inborn error of bile acid metabolism-3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency by molecular analysis.(2) Retrospective review of the literature showed that the clinical features of 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency included neonatal cholestasis, some patients progressed to severe liver disease and needed liver transplantation without effective therapy; however, serum biochemical characteristics of normal γ-glutamyltransferase activity, normal or low total bile acid concentrations were not consistent with cholestasis, the replacement treatment with cholic acid produced a dramatic improvements in symptoms, biochemical markers of liver injury; 31 cases were diagnosed by HSD3B7 gene mutation analysis.

CONCLUSIONThe clinical characteristics of 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency include neonatal cholestasis, normal serum γ-glutamyltransferase activity, and normal or low serum total bile acid concentration.Oral cholic acid replacement is an effective therapy; definitive diagnosis of 3β-hydroxy-Δ(5)-C27-steroid dehydrogenase deficiency can be identified by molecular genetic testing technology.

3-Hydroxysteroid Dehydrogenases ; deficiency ; genetics ; Administration, Oral ; Bile Acids and Salts ; biosynthesis ; blood ; Bilirubin ; blood ; Chenodeoxycholic Acid ; administration & dosage ; therapeutic use ; Cholestasis, Intrahepatic ; diagnosis ; drug therapy ; enzymology ; DNA Mutational Analysis ; Humans ; Infant ; Liver ; drug effects ; metabolism ; physiopathology ; Liver Function Tests ; Male ; Metabolic Diseases ; drug therapy ; physiopathology ; Molecular Sequence Data ; Mutation ; genetics ; Retrospective Studies

The aim of this study was to develop a bioactive membrane for inducing bone regeneration. The membrane was composed of polylactic acid, collagen, recombinant human bone morphogenetic protein-2 (rhBMP-2). The PLA + collagen + rhBMP-2 membrane was fabricated by solvent-casting and cool-drying. The mechanic properties of this compound membrane were tested. The two surfaces of membrane were observed by SEM. Degradability of PLA was evaluated by SEM observation and molecular weight measure in vitro and in vivo. The compound membranes were implanted in rabbit muscles. The samples were obtained when animals were sacrificed at different periods: 2 weeks, 1, 2, 3, 6 months after surgery. The biodegradability and biocompatibility of the membrane were evaluated. The heterotopic bone inducing activity of BMP was identified. The results indicated that the strength at extension to failure of the compound membrane was 36.4MPa at 2.3% strain. The compound membrane was found bearing active factor on its coarse side, which can induce bone regeneration. After implantation in vivo, the membrane maintained the structure for three months and degraded in 6 months. Based on histological analysis, there was no obvious inflammation. Heterotopic bone was induced. We could conclude that the PLA + collagen + rhBMP-2 membrane is an absorbable compound membrane that possesses good biocompatibility, adequate mechanic properties and excellent property of bone induction. It could be applied as an ideal membrane for inducing bone regeneration.
Animals ; Biocompatible Materials ; Biodegradation, Environmental ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; administration & dosage ; pharmacology ; Bone Regeneration ; drug effects ; Collagen ; administration & dosage ; pharmacology ; Guided Tissue Regeneration ; Lactic Acid ; administration & dosage ; pharmacology ; Male ; Membranes, Artificial ; Polyesters ; Polymers ; administration & dosage ; pharmacology ; Rabbits ; Transforming Growth Factor beta ; administration & dosage ; pharmacology

OBJECTIVEThis review focuses on the state-of-the-art of CXCL12/CXCR4 signaling axis in pancreatic cancer and its role in tumor progression.

DATA SOURCESRelevant articles published in English were identified by searching in Pubmed from 1997 to 2013, with keywords "CXCL12", "CXCR4" and "pancreatic cancer". Important references from selected articles were also retrieved.

STUDY SELECTIONArticles about CXCL12/CXCR4 signaling axis in pancreatic cancer and relevant mechanisms were selected.

RESULTSPancreatic cancer has been one of the most lethal human malignancies, with median survival less than one year and overall 5-year survival only 6%. Tumor cells from pancreatic cancer express high level of CXCR4. CXCL12, the ligand for CXCR4, is extensively secreted by neighboring stromal cells and other distant organs. CXCL12 primarily binds to CXCR4, induces intracellular signaling through several divergent pathways, which are involved in progression and metastasis of pancreatic cancer.

CONCLUSIONSCXCL12/CXCR4 signaling axis may play an important role in the communication between pancreatic cancer cells and their microenvironment, which may have effect on tumor proliferation, invasion, angiogenesis, metastasis and chemoresistance. CXCL12/CXCR4 signaling axis may serves as a novel therapeutic target for pancreatic cancer.

Chemokine CXCL12 ; genetics ; metabolism ; Humans ; Pancreatic Neoplasms ; genetics ; metabolism ; Receptors, CXCR4 ; genetics ; metabolism ; Signal Transduction ; genetics ; physiology

OBJECTIVETo investigate the distribution of hepatitis B virus genotype in Hubei province (China) and its clinical significance.

METHODSSerum samples from 190 HBV DNA positive patients with chronic HBV infection,including 52 asymptomatic HBV carriers (ASC), 56 chronic hepatitis (CH), 32 fulminant hepatic failure (FHF), 22 liver cirrhosis (LC), and 28 hepatocellular carcinoma (HCC) patients were collected and tested for HBV genotypes by type-specific primers.

RESULTSA simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes of hepatitis B virus (HBV). Of the 190 patients, 140 (73.7%) were genotype B and 42 (22.1%) were genotype C. Genotype B was more prevalent in the FHF and HCC patients than in the ASC patients; the ALT value was significantly higher in genotype B than in genotype C patients. The rate of anti-HBe was significantly higher in genotype B than in genotype C except in the patients of the ASC group.

CONCLUSIONThe system we used seems to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys. Genotype B, genotype C and BC combination exist in Hubei province, and genotype B is the major genotype in this area especially in FHF and HCC patients.

Adult ; Carcinoma, Hepatocellular ; virology ; Carrier State ; virology ; China ; Female ; Genotype ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; complications ; virology ; Humans ; Liver Cirrhosis ; virology ; Liver Failure, Acute ; virology ; Liver Neoplasms ; virology ; Male ; Middle Aged

OBJECTIVETo evaluate the efficacy and safety of pegylated interferon alpha 2a (PEG-IFN alpha-2a) in treating patients with chronic hepatitis B.

METHODSeventy-two patients with chronic hepatitis B were assigned to a PEG-IFN alpha-2a (experimental) group (n=42) and an interferon alpha (control) group (n=30) randomly. Each patient in the experimental group received 180 microg PEG-IFN alpha-2a every week. Each patient in the control group received 500 MU interferon alpha every day. All the patients were treated for 48 weeks, and then were followed for another 48 weeks with no treatment.

RESULTSAt the end of the 12th week, the rate of HBeAg negative cases was 30% in the PEG-IFN alpha-2a group, which was much higher than in the control group (x2 = 4.162, P < 0.05). The values of HBeAg and the log value of HBV DNA in the PEG-IFN alpha-2a group were much lower than the values before the treatment (t = 2.689, t = 4.080, P <0.01), but there was no difference between before and after treatment in the control group ( t = 1.229, t = 1.009, P > 0.05). At the end of the 24th week, the rate of HBeAg negative cases in the PEG-IFN alpha-2a group was much higher than that in the control group (x2=6.190, P < 0.05). The value of HBeAg and the log value of HBV DNA in the PEG-IFN alpha-2a group were much lower than in the control group (t=2.215, t=2.122, P < 0.05). At the end of the 48th week, besides the reduction mentioned above, the rate of cases with HBeAg/antiHBe seroconversion and normalization of ALT and complete responsiveness in the PEG-IFN alpha-2a group were all much higher than those in the control group (x2=5.771, x2=5.617, x2=5.308, P < 0.05). At the end of 48 weeks with no treatment, all the parameters mentioned above in the PEG-IFN alpha-2a group were much better than those in the control group and they remained so, but they were different in the control group (x2=11.943, t=3.439, t=6.111, x2=9.930, x2=9.522, x2=7.920, P < 0.01). Nine patients in the PEG-IFN alpha-2a group had liver biopsies before their treatment and also at the end of their treatment. The expressions of HBsAg and HBcAg were decreased at the end of the treatment. The rate of expression of HBsAg in the liver tissues before the treatment was 88.9% but only 22.2% at the end of the treatment (x2=8.001, P < 0.01). The rate of expression of HBcAg in the livers before treatment was 66.7% but only 33.3% at the end of the treatment. Before and at the end of the PEG-IFN alpha-2a treatment, there were no significant changes in the degrees of inflammation and fibrosis and the quantity of collagen in the liver tissues. Three patients in the PEG-IFN alpha-2a group (10%) were HbsAg negative. Two of them were found so at the end of 32 weeks with treatment and one patient was found at the end of 24 weeks with no treatment, but there were no HBsAg negative patients in the control group. The adverse reactions that occurred in the PEG-IFN alpha-2a and in the control groups were similar.

CONCLUSIONPEG-IFN alpha-2a was effective in inhibiting HBV replication. The effect of PEG-IFN alpha-2a was lasting. PEG-IFN alpha-2a was well tolerated during our treatment.

Adolescent ; Adult ; Antiviral Agents ; therapeutic use ; Female ; Hepatitis B, Chronic ; drug therapy ; Humans ; Interferon-alpha ; therapeutic use ; Male ; Middle Aged ; Polyethylene Glycols ; therapeutic use ; Recombinant Proteins ; Young Adult

OBJECTIVETo investigate the effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) mediated antiviral activity against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV).

METHODSTotal RNA was extracted from peripheral blood mononuclear cells (PBMCs), RT-PCR product was cloned into the EcoR I/Hind III restriction sites of the CMV-driven expression vector fused with a hemagglutinin fusion epitope tag at its carboxyl terminal. Replication competent 1.3 fold over-length HBV was constructed with full-length HBV of ayw subtype. The mammalian hepatoma cell HepG2 was cotransfected with the replication competent 1.3 fold over-length HBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA, HBV DNA. RNA from intracellular core particles was examined using Northern and Southern blot analyses. Chicken hepatoma cell LMH was cotransfected with head-to-tail dimer of an EcoR I monomer of DHBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. DHBV DNA from intracellular core particles was examined using Southern blot analysis.

RESULTSCMV-driven expression vector encoding APOBEC3G-HA and replication competent 1.3 fold over-length HBV were constructed. There was a dose dependent decrease in the levels of intracellular core-associated viral (HBV and DHBV) DNA and extracellular production of HBsAg and HBeAg. Levels of intracellular core-associated viral RNA were also decreased, but the expression of HBcAg remained almost unchanged.

CONCLUSIONAPOBEC3G suppresses HBV and DHBV replication and also suppresses HBsAg and HBeAg expression.

APOBEC-3G Deaminase ; Cytidine Deaminase ; genetics ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B Virus, Duck ; physiology ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; physiology ; Humans ; RNA, Messenger ; genetics ; Virus Replication