Xiqin Yang

A novel and simple preparative method for uniform-sized PLGA microspheres: Preliminary application in antitubercular drug delivery.

Particle size has been demonstrated as a key parameter influencing the phagocytosis of drug-loaded PLGA microspheres (MS) by the target cells. However, the current preparative methods were either insufficient in controlling the homogeneity of the produced MS, or requires sophisticated and costly equipment. This study aimed to explore a simple and economical method for uniform PLGA MS preparation. Based on the heterogeneous emulsification of routine mechanical stirring, we designed an adjuvant strategy to enhance the homogeneity of MS. By using glass beads as adjutant, the dispersion produced during mechanical stirring was much more homogeneous in the solution. The particles produced were much smaller and the size distribution was much narrower as compared with those produced using the routine mechanical stirring method under the same condition. After enrichment by selective centrifugation, about 60% of the particles of similar size were obtained, providing further evidence for the efficiency of the novel method in controlling particle homogeneity. Further, the method was applied to prepare rifampicin-loaded PLGA MS of the optimized size for macrophage uptake. The functional evaluation showed that the prepared PLGA MS could efficiently deliver an antitubercular drug into macrophages and maintain a higher intracellular concentration by controlled release, suggesting the potential application of the method in PLGA MS-based drug delivery. Collectively, the study provided a simple and economical method for preparing uniform-sized PLGA MS with potential of widespread applications.

Hepatitis C virus core protein promotes the migration and invasion of hepatocyte via activating transcription of extracellular matrix metalloproteinase inducer

The chronic infection of hepatitis C virus (HCV) becomes a main factor evoking hepatocellular carcinoma, where the HCV core protein plays a central role in hepatocarcinogenesis. Whether the core protein directly contributes to metastasis of hepatocytes still remains to be reported in literature. Transwell chamber migration assay, Boyden chamber invasion assays and scanning electron microscopy observations were performed to determine the prometastatic ability of HCV core protein when expressed in human hepatocyte L02 cells. In addition, western blots, dual-luciferase assays, and chromatin immunoprecipitation assays were used to elucidate HCV core protein dependent pathways that promote metastasis in hepatocytes. Our investigation suggests that HCV core protein markedly enhances the capability of migration and invasion in L02 clones expressing HCV core proteins. The metastasis-promoting effect of the core protein is, in part, highly dependent on its effect on promoting the binding of transcription factor Sp1 to the extracellular matrix metalloproteinase inducer promoter. The effect of Sp1 binding resulted in an increase in extracellular matrix metalloproteinase inducer expression and progression of metastasis. Thus, we report that the expression of HCV core protein contributes to the metastasis of hepatocyte cells through activating transcription of extracellular metalloproteinase inducer.

Non-invasive detection of hepatocellular carcinoma serum metabolic profile through surface-enhanced Raman spectroscopy

The present study aims to identify distinctive Raman spectrum metabolic peaks to predict hepatocellular carcinoma (HCC). We performed a label-free, non-invasive surface-enhanced Raman spectroscopy (SERS) test on 230 serum samples including 47 HCC, 60 normal controls (NC), 68 breast cancer (BC) and 55 lung cancer (LC) by mixing Au@AgNRs with serum directly. Based on the observed SERS spectra, discriminative metabolites including tryptophan, phenylalanine, and etc. were found in HCC, when compared with BC, LC, and NC (P<0.05 in all). Common metabolites-proline, valine, adenine and thymine were found in HCC, BC and LC with compared to NC group (P<0.05). Importantly, Raman spectra of HCC serum biomarker AFP were firstly detected to analyze the HCC prominent peak. Orthogonal partial least squares discriminant analysis was adopted to assess the diagnostic accuracy; area under curve value of HCC is 0.991. This study provides new insights into the HCC metabolites detection through Raman spectroscopy.

Identification of Novel RD1 Antigens and Their Combinations for Diagnosis of Sputum Smear−/Culture+ TB Patients

Rapid and accurate diagnosis of pulmonary tuberculosis (PTB) is an unresolved problem worldwide, especially for sputum smear− (S−) cases. In this study, five antigen genes including Rv3871, Rv3874, Rv3875, Rv3876, and Rv3879 were cloned from Mycobacterium tuberculosis (Mtb) RD1 and overexpressed to generate antigen fragments. These antigens and their combinations were investigated for PTB serodiagnosis. 298 serum samples were collected from active PTB patients, including 117 sputum smear+ (S+) and sputum culture+ (C+) cases, 101 S−/C+ cases, and 80 S−/C− cases. The serum IgG levels of the five antigens were measured by ELISA. Based on IgG levels, the sensitivity/specificity of Rv3871, Rv3874, Rv3875, Rv3876, and Rv3879 for PTB detection was 81.21%/74.74%, 63.09%/94.78%, 32.21%/87.37%, 62.42%/85.26%, and 83.56%/83.16%, respectively. Furthermore, the optimal result for PTB diagnosis was achieved by combining antigens Rv3871, Rv3876, and Rv3879. In addition, the IgG levels of Rv3871, Rv3876, and Rv3879 were found to be higher in S−/C+ PTB patients than in other PTB populations. More importantly, combination of the three antigens demonstrated superior diagnostic performance for both S−/C+ and S−/C− PTB. In conclusion, the combination of Rv3871, Rv3876, and Rv3879 induced higher IgG response in sputum S−/C+ PTB patients and represents a promising biomarker combination for diagnosing of PTB.

Generation of monoclonal antibodies against MGA and comparison of their application in breast cancer detection by immunohistochemistry.

Mammaglobin A (MGA) is an organ specific molecular biomarker for metastatic breast cancer diagnosis. However, there are still needs to develop optimal monoclonal antibodies (mAbs) to detect MGA expression in breast carcinoma by immunohistochemistry. In this study, we first generated mAbs against MGA. Then, we used epitope prediction and computer-assisted structural analysis to screen five dominant epitopes and identified mAbs against five epitopes. Further immunohistochemical analysis on 42 breast carcinoma specimens showed that MHG1152 and MGD785 had intensive staining mainly in membrane, while CHH11617, CHH995 and MJF656 had more intensive staining within the cytoplasm. MGA scoring results showed that MJF656 had the highest rate (92.8%) of positive staining among five mAbs, including higher staining intensity when compared with that of MHG1152 (p < 0.01) and CHH995 (p < 0.05) and the highest the mean percentage of cells stained among mAbs. Furthermore, we analyzed the relationship of positive staining rate by mAbs with patient clinical characteristics. The results suggest that MJF656 was able to detect MGA expression, especially in early clinical stage, low grade and lymph node metastasis-negative breast carcinoma. In conclusion, our study generated five mAbs against MGA and identified the best candidate for detection of MGA expression in breast cancer tissues.

Construction, Expression, Purification and Biotin Labeling of a Single Recombinant Multi-Epitope Antigen for Double-Antigen Sandwich ELISA to Detect Hepatitis C Virus Antibody

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

Hepatitis C Virus Genotype Analyses in Chronic Hepatitis C Patients and Individuals With Spontaneous Virus Clearance Using a Newly Developed Serotyping Assay.

We developed a novel HCV serotyping assay and detected the genotypes in chronic hepatitis C (CHC) patients and individuals with spontaneous viral clearance (SVC).

Anti-hMAM monoclonal antibodies evaluated in breast and non-breast tissues for differential diagnosis implication

Purpose Human mammaglobin (hMAM) is a breast tissue-specific marker that may have potency for the diagnosis of breast cancer. However, there is a lack of commercialization of anti-hMAM antibody made in China. Methods Immunoreactivities of 2 self-made monoclonal anti-hMAM antibodies, MEF521 and MDA822, were evaluated by immunohistochemistry staining and compared with imported monoclonal antibody ab81611. A total of 48 cases of primary breast cancers, 36 cases of benign or normal breast tissues, 52 cases of lymph nodes or organ metastases from breast cancer, and 90 cases of non-breast primary carcinoma tissues were analyzed. Results All 3 anti-hMAM antibodies showed high positive expression of hMAM in primary breast cancers, benign, and normal breast tissues. The positive ratio for MEF521 (33.3%) or MDA822 (44.4%) was much higher than that of ab81611 (16.7%) in lymph node metastasis from breast cancer (p = 0.038). There was no correlation between hMAM expression and clinicopathologic features of breast cancer in the 3 groups of antibodies. In 90 cases of non-breast primary carcinoma tissues, no hMAM-positive ones were observed in the MEF521 or MDA822 group, but 48 (53.3%) in the ab81611 group were positive, indicating that breast tissue specificity of the 2 self-made anti-hMAM monoclonal antibodies much higher than that of ab81611 (p<0.001). Conclusions Our results showed that MDA822 and MEF521 are more specific to breast cancer as measured by means of immunohistochemistry. Therefore, the 2 self-made anti-hMAM antibodies may have good prospects for clinical application in the differential diagnosis of breast tumor and breast cancer metastases.

Construction of an immunostimulatory plasmid, pUCpGs10, and research on its immune adjuvant effect.

In order to overcome the instability of CpG ODN in vivo, sequence diversity, and individual differences, eleven CpG ODN fragments were meticulously selected and linked to form a Multi-CpG, which were repeatedly inserted into the cloning vector pUC19 for constructing the recombinant plasmid pUCpGs10 containing ten of Multi-CpG. Using the multi-genotype HCV E1 and multi-epitope complex HCV-T as immunogens, and plasmid pUCpGs10 as the immune adjuvant, Balb/c mice were immunized through nasal and subcutaneous immunization. Strong-specific humoral and cellular immune response were induced, which can obviously inhibit the growth of homograft expressing HCV antigen. The immune adjuvant effect of pUCpGs10 closely matched that of Freund’s complete adjuvant. The plasmid pUCpGs10 can significantly improve IgA content in serum and different mucosal extract and systematical T-cell response via intranasal immunization. In conclusions, the newly constructed immunostimulatory plasmid pUCpGs10 is able to effectively activate the humoral and cellular immune activity, and possesses activation on mucosal immune response.