Metabolomic Profiles for HBV Related Hepatocellular Carcinoma Including Alpha-Fetoproteins Positive and Negative Subtypes.

Background: Hepatocellular carcinoma (HCC) is very common globally prevalent cancer. Due to its poor clinical prognosis, increasing the diagnostic rate of HCC is urgently needed. Herein, we validate discovered metabolomic biomarkers to distinguish Hepatitis B virus (HBV)-related HCC, including alpha-fetoprotein (AFP) negative (AFP-) and positive (AFP+) individuals. Methods: We recruited 130 HCC subjects (independent case-control, randomized clinical cohorts) to our study. We separated the subjects randomly into two panels: (1) 58 individuals for the discovery panel; and (2) 72 individuals for the validation panel. For each panel, gender and age-matched hepatitis B group (HBG) and healthy group were included as controls. Plasma samples were collected for metabolic profiling by liquid chromatography-mass spectrometry-based metabolomics assays. We applied both non-targeted metabolomics analyses and targeted metabolomics analyses. Significantly changed metabolites (SCMs) were identified. The power of SCMs to discriminate HCC and HBG or healthy group was determined by receiver operating characteristic curve (ROC) analysis. Results: Ten SCMs were selected form the discovery panel, and further verified in the validation panel. ROC analyses indicated that 1 SCMs (LysoPC (24:0)) could discriminate HCC from HBG (AUC = 0.765). Further, 8 SCMs including (LysoPC (17:0), LysoPC (20:4(8Z,11Z,14Z,17Z)), LysoPC (22:0), LysoPC (24:0), PE (P-16:0/22:4(7Z,10Z,13Z,16Z)), SM (d18:1/22:1(13Z)), Creatinine, and L-Isoleucine) displayed a heightened ability to discriminate between HCC and healthy controls (AUC were more than 0.800). Most of these SCMs were important in lipid metabolism. Conclusions: LysoPC (24:0) could distinguished HCC from HBG, and 8 SCMs distinguished HCC from healthy controls. LysoPC and other metabolites have the potential to serve as non-invasive biomarkers for HBV related AFP- and AFP+ HCC.

Quality of Cryopreserved Peripheral Blood Mononuclear Cells Recovered from the Hepatitis/AIDS Biobank.

Background: Peripheral blood mononuclear cells (PBMCs) count among the most important samples in a biobank, and the quality of cryopreserved PBMCs is crucial for further research. This study evaluated the quality of PBMCs recovered from the Beijing Capital Medical University Hepatitis/AIDS Biobank after 2-11 years of cryopreservation. Materials and Methods: A total of 87 PBMC samples with different cryopreservation times (2006, 2007, 2013, and 2015) were thawed, and the cell number and cell viability were determined by acridine orange/propidium iodide staining. Then, DNA was extracted from the cryopreserved PBMCs and assessed for quantity on an ultramicrospectrophotometer. Results: The median cell viability rate was 73.58% for the 87 PBMC samples cryopreserved for 2-11 years. A rate of 80.98% was obtained for PBMCs collected in 2006, a value higher than those of other cryopreservation times (2007, 2013, and 2015). Similarly, more live and total cells were obtained in PBMCs cryopreserved since 2006 compared with other cryopreservation times (since 2007, 2013, and 2015, respectively). Nonparametric Spearman correlation analysis indicated positive associations of cell viability with live (r = 0.578, p < 0.0001) and total (r = 0.338, p = 0.0003) cell numbers. Meanwhile, DNA amounts increased with total cell number. Statistical analysis showed that 3.69 µg DNA was obtained from ∼1 × 106 cells. Conclusion: Cryopreservation time (2-11 years) has negligible effects on the quality of PBMCs. Meanwhile, the cell number is positively correlated with cell viability.

High Level Antibody Response to Pandemic Influenza H1N1/09 Virus Is Associated With Interferon-Induced Transmembrane Protein-3 rs12252-CC in Young Adults.

Background: The C allele of the interferon-induced transmembrane protein-3 (IFITM3) SNP rs12252, a common allele in South East Asia and China, is strongly associated with severe influenza infection. However, despite the high occurrence of rs12252-CC genotype in Chinese population (~25%), severe influenza infection is rare. The aim of study is to determine whether rs12252-CC individuals have pre-existing antibody responses to previous seasonal influenza infections. Cohort and Method: A total 99 young healthy volunteers (18-20 years) were recruited and received an influenza seasonal Vaccination [A/Switzerland/9715293/2013(H3N2), A/California/7/2009 (pdm09H1N1) and B/Jeep/3073/2013-like virus (Flu-B)]. Plasma and gDNA was isolated from each volunteer before, and 14, 28, 180, 360, and 540 days after vaccination. Additionally, 68 elderlies (>65 years) were also recruited as a control group to compare the levels of antibodies at baseline between the young adults and the elderly. For each sample IFITM3 rs12252 genotype was determined and antibody levels in response to pdmH1N1, H3N2 and Influenza B infection were measured for each time point. Results: We found a significantly higher level of pre-existing antibodies to pandemic influenza H1N1/09 virus (pdm09H1N1) but not to H3N2 or FluB in CC donors in comparison with CT/TT donors prior to vaccination. No impact of IFITM3 genotype in boosting influenza specific antibodies in young adults within 1 year after receiving seasonal influenza vaccination was observed. In addition, there was no difference in pdm09H1N1 specific antibody levels observed in the elderly cohort between volunteers carrying different IFITM3 genotypes. Higher levels of antibodies to pdmH1N1 were observed in elderly CC carriers when compared to the young CC carriers, but this trend was not replicated in TT carriers. Conclusion:IFITM3-rs12252 CC carriers exhibit a high level of pre-existing immunity to pdm09H1N1 compared to TT carriers in the young cohort. This suggests that compensatory mechanisms exist which might contribute to viral control in patients carrying the rs12252-CC genotype who do not become sick after flu infection. However, such a potential compensatory effect appears to be lost overtime, as evidenced in the elderly cohort. If this compensatory mechanism is lost, it may make the CC carrying elderly more susceptible to severe influenza infection.

Development of an alpha-fetoprotein and Golgi protein 73 multiplex detection assay using xMAP technology.

AIM OF THE STUDY: Development of a new method to simultaneously detect Alpha-fetoprotein (AFP) and Golgi protein 73 (GP73) from peripheral blood. MATERIAL AND METHODS: Anti human AFP and GP73 monoclonal antibodies was used to develop a sandwich immunity reaction using xMAP technology for the simultaneous detection of plasma AFP and GP73. The assay evaluated the sensitivity, cross reactivity, range of detection, precision, recovery and linearity dilution effect. The assay utilized plasma samples and compared its performance with commercially available Enzyme Linked Immunosorbent Assay (ELISA) kits. RESULTS: The assay was successful in detecting AFP and GP73 simultaneously. Validation experiments demonstrated the limit of detection was AFP 0.006 µg/l and GP73 0.482 µg/l. The functional sensitivity was AFP 0.010 µg/l and GP73 0.640 µg/l. The range of detection was AFP 0.01-50 µg/l and GP73 0.64-100 µg/l. No cross reactivity was observed. The intra- and inter-assay variation for AFP was 0.19-3.46% and 3.1-5.8% and for GP73 was 1.5-3.2% and 1.1-7.6% respectively. The recovery for AFP was 96-106% and GP73 was 89-110%. 80 clinical plasma samples from healthy controls, and patients with liver cirrhosis and Hepatocellular Carcinoma (HCC) were evaluated. For healthy controls (n = 25), the AFP was 2.40 (1.55, 3.30) µg/l and GP73 was 42.60 (39.10, 57.40) µg/l. For patients with liver cirrhosis (n = 19), the AFP was 2.60 (1.70, 4.20) µg/l and GP73 was 136.10 (92.10, 261.70) µg/l, and for HCC patients (n = 36), the AFP was 13.65 (3.35, 158.88) µg/l and GP73 was 186.25 (96.73, 262.03) µg/l. The new assay demonstrated a good correlation with commercially available ELISA kits (correlation coefficients (r) were 0.997 (AFP, p < 0.001) and 0.959 (GP73, p < 0.001). CONCLUSIONS: The method demonstrated a sensitive, effective and accurate method for the simultaneous detection of AFP and GP73, and could be used clinically for routine detection and monitoring of patients with chronic hepatitis B.

Development and application of a fluorescence protein microarray for detecting serum alpha-fetoprotein in patients with hepatocellular carcinoma.

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC ( n = 65) and healthy control subjects ( n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC.

A Comprehensive Analysis of the Impact of HIV on HCV Immune Responses and Its Association with Liver Disease Progression in a Unique Plasma Donor Cohort.

OBJECTIVE: Human Immunodeficiency Virus (HIV) and Hepatitis C virus (HCV) co-infection is recognized as a major cause of morbidity and mortality among HIV-1 infected patients. Our understanding of the impact of HIV infection on HCV specific immune responses and liver disease outcome is limited by the heterogeneous study populations with genetically diverse infecting viruses, varying duration of infection and anti-viral treatment. METHODS: Viral-specific immune responses in a cohort of 151 HCV mono- and HIV co-infected former plasma donors infected with a narrow source of virus were studied. HCV and HIV specific T cell responses were correlated with clinical data. RESULTS: HIV-1 accelerated liver disease progression and decreased HCV specific T cell immunity. The magnitude of HCV specific T cell responses inversely correlated with lower HCV RNA load and reduced liver injury as assessed by non-invasive markers of liver fibrosis. HIV co-infection reduced the frequency of HCV specific CD4+ T cells with no detectable effect on CD8+ T cells or neutralizing antibody levels. CONCLUSION: Our study highlights the impact of HIV co-infection on HCV specific CD4+ T cell responses in a unique cohort of patients for both HCV and HIV and suggests a crucial role for these cells in controlling chronic HCV replication and liver disease progression.

[Influence of HIV infection on hepatitis C progress in patients co-infected with HIV/HCV].

OBJECTIVE: To understand the influence of HIV infection on hepatitis C progress in patients co-infected with HIV and hepatitis C virus (HCV) and related immune mechanism. METHODS: Twenty eight patients co-infected with HIV/HCV and 12 patients with simplex HCV infection were enrolled. The liver function and hepatic fibrosis progress were evaluated by detecting peripheral blood and with Fibro-Scan. The viral load of HCV was detected by using real time quantitative PCR. And the percentage of Treg/CD4⁺ T lymphocyte cell was tested by using flow cytometry. RESULTS: The levels of ALT and ALP in HIV/HCV co-infection group were (76.16 ± 81.248) U/L, (24.507 1 ± 8.194) g/L respectively, higher than those of simplex HCV infection group [(27.475 0 ± 13.985) U/L, (16.966 7 ± 7.189) g/L], the differences were statistical significant. P value was 0.012 and 0.009 respectively. The liver fibrosis index in HIV/HCV co-infection group was 5.950 0-5.825 0 Kpa, higher than that in simplex HIV infection group (5.150 0-1.050 0 Kpa), and the difference was nearly statistical significant (P = 0.077). The HCV viral load in HIV/HCV co-infection group was (6.476 8-5.343 4) lg copy/ml, higher than that in simplex HCV infection group [(1.699 0-2.681 5) lg copy/ml], and the rate of HCV clearance in HIV/HCV co-infection group was 32.14%, lower than that in simplex HCV infection group (75.00%). P value was 0.012 and 0.032 respectively. The percentage of Treg/CD4⁺ T lymphocyte cell in HIV/HCV co-infection group was (7.460 0%-2.287 5%), higher than that in simplex HCV infection group (5.965 0%-2.105 0%), and the difference was significant (P = 0.032). The percentage of Treg/CD4⁺ T lymphocyte cell was significantly related with HCV viral load (ρ = 0.350, P = 0.027), and HCV viral load was significantly related with the liver fibrosis index (ρ = 0.487, P = 0.001). CONCLUSION: HIV infection could accelerate the progress of hepatitis C, and Treg cells were involved in this progress.