High-throughput simultaneous genotyping of human platelet antigen-1 to -16 by using suspension array.

BACKGROUND: Comprehensive and accurate detection of human platelet antigens (HPAs) plays a significant role in diagnosis and prevention of the platelet (PLT) alloimmune syndromes and ensuring clinical safety of patients undergoing PLT transfusion. The majority of the available methods are incapable of performing high-throughput simultaneous detection of HPA-1 to -16, and the accuracy of many methods needs to be further enhanced. STUDY DESIGN AND METHODS: We have developed a new HPA-genotyping method for simultaneous detection of HPA-1 to -16 based on suspension array technology. A total of 216 samples from Chinese Han donors in Xi'an were genotyped using the developed method, and all the samples again were genotyped using polymerase chain reaction (PCR) sequence-based typing (PCR-SBT), which is considered the gold standard. RESULTS: All 216 samples were successfully genotyped for HPA-1 to -16 using both our method and PCR-SBT. Results showed that the genotype and allele frequencies obtained using our method were fully consistent with those obtained using PCR-SBT. CONCLUSION: Our method provides accurate, high-throughput, and simultaneous genotyping of HPA-1 to -16 and will serve as the foundation for large-scale clinical genotyping of HPAs and for the establishment of an HPA-typed PLT donor registry.

[A comparative study on the properties of trichosanthin before and after site-directed PEGylation].

OBJECTIVE: To construct PEGylated trichosanthin (TCS) mutein and analyze its bioactivities, immunogenicity, acute toxicity, and pharmacokinetics. METHODS: The potential antigenic determinant site YFF81-83 in the molecule of TCS was selected to undergo site-directed mutagenesis. Thus, a TCS mutein named TCS(YFF81-83ACS) was constructed and expressed in Escherichia coli of the line BL21 (DE3). Wild TCS (wTCS), TCSY(FF81-83ACS), and PEGylated TCS(YFF81-83ACS) (PEG- TCS(YFF81-83ACS)) of different concentrations were incubated with the supercoiled plasmid pUC19 to detect the DNAse activity, mixed with rabbit reticulocyte lysate to detect the ribosome inactivation activity, subcutaneously injected into 6 mice respectively to measure the serum IgG and IgE levels, intravenously injected into mice to observe the toxicity, and intravenously injected into SD rats to observe its -plasma half-life. RESULTS: The DNAse activity of the PEG-TCS(YFF81-83ACS) was similar to that of the wTCS. The ribosome inactivation activity of the PEG-TCS(YFF81-83ACS) was 1/9-1/8 of that of the wTCS (P < 0.05). The serum IgE and IgG levels of the PEG-TCS(YFF81-83ACS) were both significantly lower than those of the wTCS (both P < 0.05). The LD50 of the PEG-TCS(YFF81-83ACS) was 1.8 times that of the wTCS (P < 0.05). The mean residence time and plasma half-life of the PEG-TCS(YFF81-83ACS) were significantly increased and its plasma clearance was significantly decreased (all P < 0.05). CONCLUSION: Site-directed mutagenesis and PEGylation of TCS provide a new approach for reconstructing TCS.

Fixed rate of blood component improves the survival rate of patients in massive transfusion.

The aim of this study was to examine the manner in which varying proportions of serum and red blood cells (RBC) in massive blood transfusion affect the survival rates of patients with severe blood loss. Massive transfusion (MT) was determined as receiving ≥10 units of red blood cells in 24 h. The electronic medical records and blood transfusion information for the period January, 2002 to December, 2011 of patients with MT were examined. Moreover, we calculated the ratio of blood components and examined their correlation with survival. In total, 1,658 patients underwent MT during the period 2002-2011, with an overall of 28,030 units RBC, accounting for 2.8% of the total blood transfusion. In conclusion, fixing blood-component ratios has the potential to help improve survival rate in MT.

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.

The prevalence and trends of transfusion-transmissible infectious pathogens among first-time, voluntary blood donors in Xi'an, China between 1999 and 2009.

OBJECTIVES: The prevalence of infectious diseases is increasing in developing countries, and this may threaten the biological safety of donated blood. This study analyzed trends in the prevalence of transfusion-transmissible infectious pathogens among Chinese, first-time, voluntary blood donors from 1999 to 2009 to evaluate the potential for disease transmission. METHODS: From 1999 to 2009, all first-time donors at the Xi'an Blood Service (XBS) were screened for hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and syphilis infections using enzyme-linked immunosorbent assays (ELISA); results were confirmed using alternative commercial kits. The prevalence and temporal trends were analyzed using the Cochran-Armitage trend test and other appropriate methods. RESULTS: From 1999 to 2009, 263 299 first-time blood donors were analyzed. The overall prevalence rates were 1.16% for HBV, 0.51% for HCV, 0.02% for HIV, and 0.31% for syphilis. There was a significant decrease in the trend for HBV and HCV infections, while a significant increase was found for syphilis. The prevalence of HIV infection remained low and stable during the study period. CONCLUSIONS: These findings suggest that HBV infection is the primary threat to blood safety, while the increasing prevalence of syphilis might also be a potential threat.

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.

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.

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.