[Application of anti-HCV and HCV RNA detection in intravenous drug users].

Objective: To explore the diagnostic value of anti-HCV and HCV RNA so as to provide an accurate and efficient detection strategy for the diagnosis of HCV in intravenous drug users. Methods: 527 plasma samples from intravenous drug users were collected, and preliminary anti-HCV ELISA screening test was performed. A recombinant immunoblot assay (RIBA) was used as confirmatory assay for reactive antibody samples. All samples were tested for HCV RNA, followed by analysis of anti-HCV screening test, RIBA and HCV nucleic acid test results. Results: Anti-HCV ELISA results were reactive in 386 out of 527 intravenous drug users and non-reactive in 141. Among the 386 reactive antibody samples detected by RIBA, 370 cases were anti-HCV positive, 6 cases were anti-HCV indeterminate and 10 cases were anti-HCV negative. Anti-HCV ELISA and RIBA positive coincidence detection rate was 95.85% (370/386), and 70.21% (370/527) among intravenous drug users. HCV RNA was negative in all 10 anti-HCV RIBA non-reactive samples. 376 anti-HCV RIBA-positive and indeterminate samples were tested for HCV RNA, of which 56.93% (300/527) were current HCV infection, and 14.42% (76/527) were past HCV infection. Among 141 anti-HCV ELISA negative samples, the residual risk by anti-HCV ELISA screening for HCV RNA was 1.52% (8/527). HCV viral load distribution among intravenous drug users showed that the high viral load value (>10(7) IU/ml) and low viral load values (< 10(2) IU/ml) accounted for 1.95% and 2.27%, respectively, while the samples with viral load value of 1×10(2) ~ 1×10(7) IU/ mL accounted for 95.78% (295/308), and were mainly distributed in 1×10(5) ~ 1×10(6) IU/ml (37.99%). ELISA + RIBA + NAT assay detection strategies had differentiated 300 cases of current HCV infection, 76 cases of past HCV infection and 10 cases of false positive anti-HCV results, while ELISA+NAT assay detection strategies had only detected 300 cases of current HCV infection. However, of the 386 positive subjects screened for antibodies, 10 (2.59%) were undifferentiated false positives. Conclusion: Intravenous drug users are the high-risk population of HCV infection with high prevalence and high viral load. Anti-HCV screening for intravenous drug users will have a certain degree of residual risk. Therefore, anti-HCV ELISA screening and nucleic acid detection strategy can accurately diagnose the current infected patients; however, it cannot distinguish the false positive results of antibody screening.

The effect of platelet-derived microparticles in stored apheresis platelet concentrates on polymorphonuclear leucocyte respiratory burst.

BACKGROUND AND OBJECTIVES: Platelet-derived microparticles (PMPs) and other proinflammatory mediators, which are accumulated during the storage process, might induce transfusion adverse events. We hypothesized that the PMP primed polymorphonuclear neutrophil (PMN) respiratory burst after the transfusion, which could be linked to the transfusion-related acute lung injury (TRALI). MATERIALS AND METHODS: The PMPs were isolated by centrifugation of the platelet-free plasma from 10 apheresis platelet concentrates (A-PLTs) at 20,000×g for 1 h. The PMPs were counted by flow cytometric analysis, followed by Western blotting, that were performed on isolated PMPs. The soluble CD40 ligand (sCD40L, sCD154) was assayed with ELISA. The priming of the formyl-Met-Leu-Phe (fMLP)-activated PMN respiratory burst was measured with the hydrogen peroxide production. RESULTS: The PMP counts increased by 1·7-folds after 3 days of storage. Meanwhile, sCD40L also significantly increased in PMP fraction isolated from the 3-day stored A-PLTs. Furthermore, Western blotting indicated that sCD40L was involved and concentrated in PMP. The PMPs were able to effectively prime the fMLP-activated PMN respiratory burst, which was partly inhibited by CD154 monoclonal antibody or by filtration with 0·1 µM membrane. Significant relativity was existed between the PMP counts, sCD40L and priming activity during the A-PLT storage. CONCLUSION: The platelet-derived microparticles, which carried the sCD40L, accumulated in the apheresis platelet concentrates during the 5 days of storage. They primed the fMLP-activated PMN respiration burst, which might be relative to TRALI.

Introducing a dexamethasone mouse model for monitoring in vivo viability of stored human platelets.

OBJECTIVES: The aim of this study is to establish an available animal model which can evaluate in vivo viability of stored human platelets (HuPLTs). BACKGROUND: The viability in vivo of HuPLTs was usually evaluated by transfusing HuPLTs into animals before clinical trials. It is necessary to develop a method which may slow down rapid clearance of HuPLTs from circulation of the animal. METHODS: Carbon clearance tests were performed by treating mice with dexamethasone (DEX) to determine the phagocytic ability of the mice macrophages. HuPLTs in mice whole blood were detected by flow cytometric analysis with mouse anti-human CD41-fluorescein isothiocyanate monoclonal antibody. Recovery and survival of the HuPLTs stored at 22 °C for 1 day were evaluated after transfusing these HuPLTs into DEX-treated mice, and compared with those either stored at 22 °C for 5 days or at 4 °C for 1 day. RESULTS: Corrected phagocytic indexes of DEX-treated mice decreased significantly compared with those of control mice (P < 0.05). The recovery after 24 h and survival time of fresh HuPLTs in DEX-treated mice were much higher than those in control mice (P < 0.01). After transfused into the DEX-treated mice, HuPLTs stored either at 22 °C for 5 days or at 4 °C for 1 day showed decrease in recovery and survival compared with those stored at 22 °C for 1 day (P < 0.05). CONCLUSION: Dexamethasone slows down the rate of HuPLTs clearance efficiently in mouse circulation. And the DEX-treated mouse model was able to evaluate the in vivo viability of stored HuPLTs.

[Advances in studies of HCV nucleic acid testing for clinical diagnosis].

HCV infection is of global concern with the characteristic of insidious onset. Laboratory testing stands indispensably for clinical diagnosis with antibody assay and nucleic acid testing the main methods being adopted. For early detection and diagnosis of HCV infection, it is important to choose the best detection method and diagnostic strategy. This paper reviews the HCV nucleic acid testing methods, as well as performance evaluation of reagents and current strategies.

[Progress in HIV-2 detection methodology].

Laboratory test is the routine method of diagnosis, monitoring and blood screening of HIV infection, and main basis for early diagnosis of AIDS. HIV is divided into HIV-1 and HIV-2 subtypes, HIV-1 infection is the major cause of AIDS pandemic, while HIV-2 infection occurs in limited areas in the world, mainly in West Africa. HIV-2 infection has been reported in China since 1998. They are sporadic cases, and mainly HIV-1/HIV-2 mixed infections. There are less concerns about HIV-2 detection in China at present, and domestic HIV-2 detection reagents have not come into the market. At present, the detection method of HIV-2 is mainly antibody test and nucleic acid test. The initial screening is through rapid test and other methods and the confirmation is depended on Western Blot and Line Immune Assay. According to the HIV antibody test results, HIV-2 infection is confirmed. With the rapid development of molecular biology, the diagnostic method of nucleic acid detection laboratory has made great progress.

Anti-tumour effects of exosomes in combination with cyclophosphamide and polyinosinic-polycytidylic acid.

We examined the anti-tumour activity of exosomes derived from dendritic cells (DCs) in combination with cyclophosphamide (CTX) and polyinosinic-polycytidylic acid sodium salt (poly I:C). DCs were pulsed with L1210 lymphocytic leukaemia cell antigen and lipopolysaccharide. The exosomes that the DCs secreted were purified. In vitro, the anti-tumour activity of exosomes was assessed by measuring their ability to induce spleen cell proliferation and the extent to which they induced spleen cells to kill L1210 cells. Poly I:C was able to induce DC maturation. DC-derived exosomes stimulated spleen cell proliferation and enhanced the cytotoxic effects of spleen cells in vitro. DC-derived exosomes, in combination with CTX and poly I:C, suppressed L1210 tumour growth in vivo and gave the greatest prolongation of survival time in tumour-bearing DBA2 mice. These findings suggest that this combination of a tumour vaccine, a conventional anti-cancer agent and a promoter of DC maturation might be a useful anti-cancer therapy.

CXCL13 inhibition induce the apoptosis of MDA-MB-231 breast cancer cells through blocking CXCR5/ERK signaling pathway.

OBJECTIVE: Treatment of the high-risk triple negative breast cancer (TNBC) is a critical clinical challenge. Here we aimed to explore a novel strategy for TNBC treatment by blocking the tumor-associated chemokine CXCL13 in the MDA-MB-231 TNBC cells. MATERIALS AND METHODS: MDA-MB-231 cells were treated with anti-CXCL13 antibodies (inhibition group), or phosphate-buffered saline (PBS) (control group), followed by determining the levels of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta-1 (TGF-ß1) with enzyme-linked immunosorbent assay (ELISA). The effects of CXCL13 inhibition on cell proliferation and apoptosis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Quantitative Real Time-PCR (qRT-PCR) and Western blot were used to compare the levels of CXCL13, CXCR5, extracellular signal-regulated kinase (ERK). The levels of cyclin D1 and cleaved caspase-9 were detected by Western blot. RESULTS: The levels of IL-1, TNF-α and TGF-ß1 in MDA-MB-231 cells treated with anti-CXCL13 antibodies were significantly downregulated (p<0.05). Meanwhile, CXCL13 blockade decreased the cell proliferation and increased the apoptosis rate of MDA-MB-231 cells. The inhibition of CXCL13 led to marked reduction in CXCL13 and CXCR5 mRNA and an increase in ERK mRNA. The inhibition of CXCL13 resulted in the downregulation of CXCL13, CXCR5, p-ERK/ERK, cyclin D1 and upregulation of cleaved caspase-9 proteins. CONCLUSIONS: CXCL13 blockade effectively suppressed the proliferation of MDA-MB-231 cells by promoting cell apoptosis. This effect is presumably associated with the downregulation of CXCL13 and suppression of the CXCR5/ERK signaling pathway.