Search

Background: Accurate quantitative detection of hepatitis C virus (HCV) RNA is critical for diagnosis of acute or chronic HCV infection, and for follow-up of virologic response during HCV targeted therapy. In the present study, traceability and reproducibility of a novel China-certified domestic Sansure HCV RNA diagnostic assay (Sansure, Changsha, Hunan, China) was evaluated and the clinical performance of this assay was also analyzed.Methods: Traceability of the Sansure HCV RNA assay to the WHO international standard for HCV (genotype 1a) was detected across multiple centers. Reproducibility, accuracy (the differences of observed average concentrations and expected concentrations) and precision were assessed using series dilutions of World HCV RNA performance panel WWHV303-02 (HCV-1b), WWHV303-04(HCV-2a), WWHV303-11(HCV-3a) and WWHV303-19 (HCV-6a). In addition, both Sansure HCV RNA and CAP/CTM HCV (Roche, Branchburg, NJ, USA) assays were used to detect HCV RNA in 346 EDTA anti-coagulated plasma samples from previous HCV-infected patients, during and after antiviral therapy.Results: The Sansure assay showed good traceability by agreeing with the HCV-1a WHO standard across all five concentrations tested (25, 50, 100, 1000, 10,000 IU/ml). The differences between observed average concentrations and expected concentrations were all within 0.2 log10 IU/ml. HCV WWHV303 standards across 4 HCV genotypes (1b, 2a, 3a and 6a) were used for evaluation of reproducibility and the accuracy of the test were all within 0.2 log10 IU/ml. The inter-assay variations across the above 4 HCV genotypes were all less than 0.03 on each evaluated concentration, indicating good precision of Sansure HCV RNA assay. In clinical practice, concordant results were determined in 99.42% (344/346) samples (215 positive and 129 negative samples). Two specimens with negative HCV RNA results by Sansure assay were detected positive by CAP/CTM HCV test. Correlation analysis indicated a significantly positive correlation in detected HCV RNA concentrations (r = 0.9439, P < 0.0001). HCV RNA levels in 95.35% (205/215) specimens were within mean difference ± 1.96 SD as tested by both assays.Conclusions: With the advantages of traceability, reproducibility and lower price, Sansure HCV RNA assay represented an alternative option for HCV RNA detection in hospital and medical institution in China. [ABSTRACT FROM AUTHOR]

The coagulation factor 9 gene (FIX) point mutation contributes to most hemophilia B cases, providing ideal gene correction models. Here we identified the frequent mutation G20519A (R226Q) in FIX , which resulted in many severe and moderate hemophilia B patients. This study aimed to investigate the effect of HDR and base editing in correcting FIX mutant. We first constructed HEK293 and liver-derived cell lines Huh7 cells stabling carrying mutated FIX containing G20519A (HEK293-FIXmut and Huh7-FIXmut). Then, CRISPR/Cas9-based homology-directed repair (HDR) and base editing were used for the correction of this mutated point. We used Cas9 nickase (nCas9) mediated HDR and the advanced base editor ABE8e to correct G20519A and then measured the concentration and activity of FIX. Furthermore, we used the star-shaped poly(lysine) gene nanocarriers to deliver the ABE8e correction systems into HEK293-FIXmut and Huh7-FIXmut stem cells to correct mutated FIX. As a result, we found that gRNAs directed inefficient HDR in correcting G20519A. The ABE8e corrected the mutation efficiently in both HEK293-FIXmut and Huh7-FIXmut stem cells. In addition, the star-shaped poly(lysine) carriers delivered non-viral vectors into stem cells efficiently. The nanocarriers-delivered ABE8e system corrected mutated FIX in stem cells, and the stem cells secreted active FIX in high concentration. In conclusion, our study provides a potential alternative for correcting mutated FIX in hemophilia B patients. • The point mutation G20519A (R226Q) in FIX results to lower concentration and lower activity of FIX. • ABE8e corrected mutated FIX more efficiently compared with HDR. • Bioinspired star shaped PLL polypeptides could deliver gene correction systems into stem cells. • PLL-delivered ABE8e system could correct mutated FIX efficiently in stem cells. [ABSTRACT FROM AUTHOR]

Dysfunction of natural killer (NK) cells has been implicated in the failure of antitumor immune responses in hepatocellular carcinoma (HCC) patients. However, the changes of NK profile in peripheral blood after surgery and tumor tissues of HCC patients, as well as the underlying reason and the significance are vague. Here, we observed that the frequencies of circulating NKG2D+CD56dimNK cells decreased significantly in HBV-related HCC and were negatively correlated with the levels of serum TGF-β and soluble MICA (sMICA).In vitroexperiments confirmed that the TGF-β and sMICA in tumor tissue homogenates, as well as sMICA in HCC cells culture supernatants could reduce the frequency of NKG2D+CD56dimNK cells. In addition, in HCC patients the lower frequency of circulating NKG2D+CD56dimNK cells was associated with larger tumor size and/or higher serum GGT. Noticeably, the frequency of NKG2D+CD56dimNK cells at one month after surgery usually failed to restore in early recurrent patients, and that frequency was negatively associated with early recurrence and shorter overall survival. These results suggest that declined frequency of NKG2D+CD56dimNK cells in HCC was associated with higher TGF-β and sMICA production, and low frequency of circulating NKG2D+CD56dimNK cells at one month after surgery may predict poor prognosis of HBV-related HCC patients accepting hepatectomy. [ABSTRACT FROM PUBLISHER]

Background: Accurate quantitative detection of hepatitis C virus (HCV) RNA is critical for diagnosis of acute or chronic HCV infection, and for follow-up of virologic response during HCV targeted therapy. In the present study, traceability and reproducibility of a novel China-certified domestic Sansure HCV RNA diagnostic assay (Sansure, Changsha, Hunan, China) was evaluated and the clinical performance of this assay was also analyzed., Methods: Traceability of the Sansure HCV RNA assay to the WHO international standard for HCV (genotype 1a) was detected across multiple centers. Reproducibility, accuracy (the differences of observed average concentrations and expected concentrations) and precision were assessed using series dilutions of World HCV RNA performance panel WWHV303-02 (HCV-1b), WWHV303-04(HCV-2a), WWHV303-11(HCV-3a) and WWHV303-19 (HCV-6a). In addition, both Sansure HCV RNA and CAP/CTM HCV (Roche, Branchburg, NJ, USA) assays were used to detect HCV RNA in 346 EDTA anti-coagulated plasma samples from previous HCV-infected patients, during and after antiviral therapy., Results: The Sansure assay showed good traceability by agreeing with the HCV-1a WHO standard across all five concentrations tested (25, 50, 100, 1000, 10,000 IU/ml). The differences between observed average concentrations and expected concentrations were all within 0.2 log10 IU/ml. HCV WWHV303 standards across 4 HCV genotypes (1b, 2a, 3a and 6a) were used for evaluation of reproducibility and the accuracy of the test were all within 0.2 log10 IU/ml. The inter-assay variations across the above 4 HCV genotypes were all less than 0.03 on each evaluated concentration, indicating good precision of Sansure HCV RNA assay. In clinical practice, concordant results were determined in 99.42% (344/346) samples (215 positive and 129 negative samples). Two specimens with negative HCV RNA results by Sansure assay were detected positive by CAP/CTM HCV test. Correlation analysis indicated a significantly positive correlation in detected HCV RNA concentrations (r = 0.9439, P < 0.0001). HCV RNA levels in 95.35% (205/215) specimens were within mean difference ± 1.96 SD as tested by both assays., Conclusions: With the advantages of traceability, reproducibility and lower price, Sansure HCV RNA assay represented an alternative option for HCV RNA detection in hospital and medical institution in China.

The present study reveals an immunological characterization of circulating and tumor-infiltrating T follicular helper cells (Tfh), namely CXCR5+CD45RA-CD4+ T cells, and their related cytokines in hepatitis B virus-related hepatocellular carcinoma (HCC) patients. In HCC patients, circulating Tfh cells showed a CCR7+ and/or ICOS+ phenotype with increased Th2-like cells and decreased Th1-like and Th17-like subsets. Although the bulk frequency of circulating Tfh cells was not altered in HCC patients, the frequency of infiltrated CXCR5+CD45RA-CD4+ CD3+cells was higher in tumor than in para-tumor tissues, and Th1-like cells were the predominant phenotype. Circulating Tfh cells in HCC patients were defective in the production of IL-21 in vitro, which was in accordance with lower IL-21 levels in tumor tissues than in para-tumor tissues. Serum CXCL13 was increased in HCC patients and associated with recurrence-free survival after hepatectomy. This was confirmed in an additional HCC cohort of 111 patients with up to 5 years follow-up. Immunohistochemical staining indicated that the percentage of CXCR5+ or CXCL13+ cells was higher in poorly differentiated than in well-differentiated tumors. In conclusion, patients with HBV-related HCC showed altered phenotypes and impaired function of Tfh cells or subpopulations. CXCL13 could be a potential biomarker for predicting recurrence in HCC patients after hepatectomy.

Dysfunction of natural killer (NK) cells has been implicated in the failure of antitumor immune responses in hepatocellular carcinoma (HCC) patients. However, the changes of NK profile in peripheral blood after surgery and tumor tissues of HCC patients, as well as the underlying reason and the significance are vague. Here, we observed that the frequencies of circulating NKG2D + CD56 dim NK cells decreased significantly in HBV-related HCC and were negatively correlated with the levels of serum TGF-β and soluble MICA (sMICA). In vitro experiments confirmed that the TGF-β and sMICA in tumor tissue homogenates, as well as sMICA in HCC cells culture supernatants could reduce the frequency of NKG2D + CD56 dim NK cells. In addition, in HCC patients the lower frequency of circulating NKG2D + CD56 dim NK cells was associated with larger tumor size and/or higher serum GGT. Noticeably, the frequency of NKG2D + CD56 dim NK cells at one month after surgery usually failed to restore in early recurrent patients, and that frequency was negatively associated with early recurrence and shorter overall survival. These results suggest that declined frequency of NKG2D + CD56 dim NK cells in HCC was associated with higher TGF-β and sMICA production, and low frequency of circulating NKG2D + CD56 dim NK cells at one month after surgery may predict poor prognosis of HBV-related HCC patients accepting hepatectomy.