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 investigate the effects of two histone deacetylase (HDAC) inhibitors, valproic acid (VPA) and TSA, on the expression of vascular endothelial growth factor (VEGF) and its receptor KDR of the leukemia cell line Kasumi-1 cells, and to explore their potential mechanism in leukemia angiogenesis.

METHODKasumi-1 cells were treated with VPA and TSA at different concentrations for 3 days. The mRNA and protein expression levels of VEGF and KDR were determined by semi-quantitative RT-PCR and Western blot, and the bFGF mRNA by semi-quantitative RT-PCR.

RESULTSAs compared with that of control groups, VPA at 3 mmol/L downregulated the VEGF mRNA expression level for VEGF(121) from 0.632 ± 0.014 to 0.034 ± 0.004 and for VEGF(165) from 0.526 ± 0.021 to 0.015 ± 0.001, for KDR mRNA from 0.258 ± 0.034 to 0.038 ± 0.000, and for bFGF mRNA from 0.228 ± 0.017 to 0.086 ± 0.015. TSA downregulated the VEGF mRNA and KDR mRNA at concentration of 100 nmol/L, but its effect on bFGF mRNA only at higher concentration.

CONCLUSIONHDAC inhibitors might inhibit the leukemia angiogenesis by regulating the expression of VEGF and its recptor.

Angiogenesis Inducing Agents ; Cell Line ; Histone Deacetylase Inhibitors ; pharmacology ; Humans ; RNA, Messenger ; genetics ; Valproic Acid ; pharmacology ; Vascular Endothelial Growth Factor A

To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.
Apoptosis ; drug effects ; Blotting, Western ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Enzyme Inhibitors ; pharmacology ; Flow Cytometry ; Histone Deacetylase Inhibitors ; Humans ; Hydroxamic Acids ; pharmacology ; Leukemia, Myeloid ; metabolism ; pathology