Qunxing An

Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64 fusion protein and its immunoprophylactic potential in mouse model

The completion of Mycobacterium tuberculosis genome sequence has opened a new way for the identification and characterization of bacterial antigens, such as ESAT-6, CFP10, MPT64, and Ag85 complex, which are helpful for tuberculosis control. In this work, genes of ESAT-6 and MPT64 were fused and expressed in Escherichia coli in form of inclusion bodies with a histidine tag. The expressed fusion protein was purified by nitrilotriacetic acid (Ni-NTA) affinity chromatography under denaturing conditions, and the yield was 18mg/L of culture. In mice, the purified ESAT-6-MPT64 fusion protein elicited stronger humoral response, greater splenic lymphocyte stimulated index, and higher levels of IFN-gamma and IL-12 production than that of the single MPT64 inoculation group, and rendered modest protection on the experimental tuberculosis mouse models. In short, the ESAT-6-MPT64 fusion protein might be a potential candidate vaccine for tuberculosis.

Apogossypolone, a novel inhibitor of antiapoptotic Bcl-2 family proteins, induces autophagy of PC-3 and LNCaP prostate cancer cells in vitro.

Limited treatment options are available for aggressive prostate cancer. Gossypol has been reported to have a potent anticancer activity in many types of cancer. It can increase the sensitivity of cancer cells to alkylating agents, diminish multidrug resistance and decrease metastasis. Whether or not it can induce autophagy in cancer cells has not yet been determined. Here we investigated the antiproliferative activity of apogossypolone (ApoG2) and (-)-gossypol on the human prostate cancer cell line PC3 and LNCaP in vitro. Exposure of PC-3 and LNCaP cells to ApoG2 resulted in several specific features characteristic of autophagy, including the appearance of membranous vacuoles in the cytoplasm and formation of acidic vesicular organelles. Expression of autophagy-associated LC3-II and beclin-1 increased in both cell lines after treatment. Inhibition of autophagy with 3-methyladenine promoted apoptosis of both cell types. Taken together, these data demonstrated that induction of autophagy could represent a defense mechanism against apoptosis in human prostate cancer cells.

Inhibition of proliferation of prostate cancer cell line, PC-3, in vitro and in vivo using (-)-gossypol.

We investigated the antiproliferative activity of (-)-gossypol on the human prostate cancer cell line PC3 in vitro and in vivo to elucidate its potential molecular mechanisms. Cell growth and viability were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell apoptosis was detected by flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and electron microscopy. Expression of proliferating cell nuclear antigen (PCNA), Bcl-2, CD31, caspase-3 and caspase-8 in tumour tissue was determined by immunohistochemistry. The drug concentration that yielded 50% cell inhibition (IC(50) value) was 4.74 microg mL(-1). In the PC-3 tumour xenograft study, (-)-gossypol (> 5 mg kg(-1)) given once a day for 7 days significantly inhibited tumour growth in a dose-dependent manner. Immunohistochemical analysis revealed that (-)-gossypol enhanced caspase-3 and caspase-8 expression and decreased the expression of PCNA, Bcl-2 and CD31 in tumour tissues. It suggested that cell apoptosis and inhibition of angiogenesis might contribute to the anticancer action of (-)-gossypol.

A novel hepatitis C virus vaccine approach using recombinant Bacillus Calmette-Guerin expressing multi-epitope antigen

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. HCV infection is associated with high morbidity and has become a major problem in public health. Until now, there has been no effective prophylactic or therapeutic vaccine. BCG, a live vaccine typically used for tuberculosis prevention, has been increasingly utilized as a vector for the expression of recombinant proteins that will induce specific humoral and cellular immune responses. In this study, recombinant BCG (rBCG) was engineered to express a HCV multi-epitope antigen CtEm, and HLA-A2.1 transgenic mice were immunized with rBCG-CtEm. High levels of specific anti-HCV antibodies targeted to mimotopes of HVR1 were detected in the serum. HCV-specific lymphocyte proliferation assay, cytokine determination and cytotoxicity assay indicated that HCV epotope-specific cellular immune responses were elicited in vitro. The rBCG-CtEm immunization conferred protection against infection with the recombinant vaccinia virus (rVV-HCV-CNS) in vivo. These results suggest that rBCG expressing multi-epitope antigen may serve as an effective vaccine against HCV infection.

Production and Characterization of Monoclonal Antibody Specific for NS3 Helicase of Hepatitis C Virus

Hepatitis C virus (HCV) infection is the major etiological agent of chronic hepatitis, which leads to liver cirrhosis and hepatocellular carcinomas. HCV NS3 helicase is a promising target of anti-virus therapy. In this report, we discuss a strategy to generate monoclonal antibodies (MAbs) of the HCV NS3 helicase, and investigate its potential characteristic. Our results showed the production of MAbs against NS3 helicase, which could specifically recognize the native NS3 helicase in transiently transfected cells in the immunofluorescence experiment. The resultant MAbs were used as the first antibody in Western blot analyses, and observed the specific band that defines the NS3 helicase. Likewise, one MAb could inhibit the NS3 helicase enzymatic activity distinctly in the NS3 helicase-mediated DNA-unwinding assay. To conclude, these antibodies may be useful to generate specific diagnostic tools for HCV infection and may also be developed for potential therapeutics.

Growth inhibition and apoptosis induction of human umbilical vein endothelial cells by apogossypolone.

AIMS AND BACKGROUND Prostate cancer is one of the most common malignant tumors in the male reproductive system, which causes the second most cancer deaths of males, and control of angiogenesis in prostate lesions is of obvious importance. This study assessed the effect of apogossypolone (ApoG2) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs). SUBJECTS AND METHODS HUVECs were treated with different concentrations of ApoG2. The survival rate of HUVECs were determined by MTT assay. Utrastructural changes of HUVECs were assessed with transmission electron microscopy. Apoptosis in HUVECs was analyzed by flow cytometry and cell migration by Boyden chamber assay. Matrigel assays were used to quantify the development of tube-like networks. RESULTS ApoG2 significantly inhibited HUVEC growth even at 24 h (P<0.05). The inhibitory effect of ApoG2 is more obvious as the concentration and the culture time increased (P<0.05). These results indicate that ApoG2 inhibits the proliferation of HUVECs in a time- and concentration-dependent manner with increase of the apoptosis rate. Besides, ApoG2 reduced the formation of total pseudotubule length and network branches of HUVECs. CONCLUSIONS The results suggest that ApoG2 inhibits angiogenesis of HUVECs by growth inhibition and apoptosis induction.

Inhibitory activity of apogossypol in human prostate cancer in vitro and in vivo.

Apogossypol, a gossypol derivative, is a novel small-molecule inhibitor of the Bcl-2 family proteins and has been demonstrated to have anti-tumor activities. Prostate cancer is the most common malignancy in males, for which chemotherapy is the usual treatment option in clinical practice. The aim of the present study was to investigate the growth inhibitory effects of apogossypol on prostate cancers in vitro and in vivo. An MTT assay and a colony formation assay were used to assess the anti-survival and anti-proliferation effects of apogossypol in LNCaP cells. Immunofluorescence was performed in order to detect the expression levels of apoptosis-associated proteins in xenograft tumors following apogossypol treatment. Apogossypol exerted strong anti-tumor effects on LNCaP cells in a dose-dependent manner. Furthermore, immunofluorescence revealed that apogossypol inhibited the growth and proliferation of prostate cancer cells by downregulating Bcl-2 protein expression and activating caspase-3 and -8. In addition, the in vivo study indicated that apogossypol significantly inhibited tumor growth in a dose-dependent manner with reduced toxicity compared with gossypol. In conclusion, the present study indicated that apogossypol effectively inhibited the growth and proliferation of prostate cancer cells and may be a potential agent for prostate cancer therapy.

Apogossypolone inhibits the proliferation of LNCaP cells in vitro and in vivo

The aim of the present study was to investigate the anti-tumor effect of apogossypolone (ApoG2) on human LNCaP cells in vitro and in vivo. Cell viability was evaluated using an MTT assay. Cell autophagy and apoptosis were detected by flow cytometry and using a terminal deoxynucleotidyl transferase dUTP nick end labeling assay, respectively. Morphological autophagy alterations were observed by transmission electron microscopy. The formation of acidic vesicular organelles was assessed by acridine orange staining and fluorescence microscopy. Quantitative polymerase chain reaction (qPCR) was conducted to detect the expression levels of apoptosis-associated protein B-cell lymphoma 2 (Bcl-2) and Bak. The models of transplantation tumors in nude mice were established via subcutaneous injection of LNCaP cells. Growth of LNCaP cells was inhibited by ApoG2 treatment. Flow cytometry demonstrated that ApoG2 induced apoptosis in LNCaP cells. The Bcl-2 expression was decreased while Bak expression was increased. In addition, activation of cysteine aspartate protease (caspase)-3 and -8 was observed and 3-methyladenine (3-MA) enhanced apoptosis of LNCaP cells. Furthermore, nude mice treated with ApoG2 demonstrated a significant decrease in tumor volume and a significant increase in cell viability. Immunohistochemical analysis of tumor tissues demonstrated that ApoG2 enhanced caspase-3, -8, LC-3B and beclin-1 expression and reduced the expression of Bcl-2. ApoG2 was able to effectively suppress the growth of LNCaP cells through the induction of autophagy and apoptosis.

Mesenchymal Stromal Cells Directly Promote Inflammation by Canonical NLRP3 and Non-canonical Caspase-11 Inflammasomes

Mesenchymal stromal cells (MSCs) based therapy is a promising approach to treat inflammatory disorders. However, therapeutic effect is not always achieved. Thus the mechanism involved in inflammation requires further elucidation. To explore the mechanisms by which MSCs respond to inflammatory stimuli, we investigated whether MSCs employed inflammasomes to participate in inflammation. Using in vitro and in vivo models, we found that canonical NLRP3 and non-canonical caspase-11 inflammasomes were activated in bone-associated MSCs (BA-MSCs) to promote the inflammatory response. The NLRP3 inflammasome was activated to mainly elicit IL-1β/18 release, whereas the caspase-11 inflammasome managed pyroptosis. Furthermore, we sought a small molecule component (66PR) to inhibit the activation of inflammasomes in BA-MSCs, which consequently improved their survival and therapeutic potential in inflammation bowel diseases. These current findings indicated that MSCs themselves could directly promote the inflammatory response by an inflammasome-dependent pathway. Our observations suggested that inhibition of the proinflammatory property may improve MSCs utilization in inflammatory disorders.

Mesenchymal stromal cells can be applied to red blood cells storage as a kind of cellular additive

During storage in blood banks, red blood cells (RBCs) undergo the mechanical and metabolic damage, which may lead to the diminished capacity to deliver oxygen. At high altitude regions, the above-mentioned damage may get worse. Thus, more attention should be paid to preserve RBCs when these components need transfer from plain to plateau regions. Recently, we found that mesenchymal stromal cells (MSCs) could rescue from anemia, and MSCs have been demonstrated in hematopoietic stem cells (HSCs) transplantation to reconstitute hematopoiesis in vivo by us. Considering the functions and advantages of MSCs mentioned above, we are trying to find out whether they are helpful to RBCs in storage duration at high altitudes. In the present study, we first found that mice MSCs could be preserved in citrate phosphate dextrose adenine-1 (CPDA-1) at 4 ± 2°C for 14 days, and still maintained great viability, even at plateau region. Thus, we attempted to use MSCs as an available supplement to decrease RBCs lesion during storage. We found that MSCs were helpful to support RBCs to maintain biochemical parameters and kept RBCs function well on relieving anemia in an acute hemolytic murine model. Therefore, our investigation developed a method to get a better storage of RBCs through adding MSCs, which may be applied in RBCs storage as a kind of cellular additive into preservation solution.