Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF ß1/Smad3 and NF-κB signaling pathways.

BACKGROUND: Hepatic fibrosis is a serious condition, and the development of hepatic fibrosis can lead to a series of complications. However, the pathogenesis of hepatic fibrosis remains unclear, and effective therapy options are still lacking. Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1 (NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis, but its role in diseases including hepatic fibrosis remains undefined. Therefore, additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment. AIM: To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1. METHODS: Twenty-four male C57BL/6 mice were randomized and separated into three groups, comprising the normal, fibrosis, and calcitriol treatment groups, and liver fibrosis was modeled by carbon tetrachloride (CCl4). To evaluate the level of hepatic fibrosis in every group, serological and pathological examinations of the liver were conducted. TGF-ß1 was administered to boost the in vitro cultivation of LX-2 cells. NS3TP1, α-smooth muscle actin (α-SMA), collagen I, and collagen III in every group were examined using a Western blot and real-time quantitative polymerase chain reaction. The activity of the transforming growth factor beta 1 (TGFß1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected. The statistical analysis of the data was performed using the Student's t test. RESULTS: NS3TP1 promoted the activation, proliferation, and differentiation of hepatic stellate cells (HSCs) and enhanced hepatic fibrosis via the TGFß1/Smad3 and NF-κB signaling pathways, as evidenced by the presence of α-SMA, collagen I, collagen III, p-smad3, and p-p65 in LX-2 cells, which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference. The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression, as shown by the luciferase assay. NS3TP1 inhibited the apoptosis of HSCs. Moreover, both Smad3 and p65 could bind to NS3TP1, and p65 increased the promoter activity of NS3TP1, while NS3TP1 increased the promoter activity of TGFß1 receptor I, as indicated by coimmunoprecipitation and luciferase assay results. Both in vivo and in vitro, treatment with calcitriol dramatically reduced the expression of NS3TP1. Calcitriol therapy-controlled HSCs activation, proliferation, and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice. Furthermore, calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1. CONCLUSION: Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique, prospective therapeutic target in hepatic fibrosis.

Efficacy of Biejiajian Pill on Intestinal Microbiota in Patients with Hepatitis B Cirrhosis/Liver Fibrosis: A Randomized Double-Blind Controlled Trial.

OBJECTIVE: To analyze the efficacy of Biejiajian Pill (BJJP) on intestinal microbiota in patients with hepatitis B cirrhosis/liver fibrosis, and explore its relationship with liver fibrosis. METHODS: This was a prospective, randomized double-blind controlled trial. Using the stratified block randomization method, 35 patients with hepatitis B liver cirrhosis/liver fibrosis were randomly assigned (1:1) to receive entecavir (0.5 mg/d) combined with BJJP (3 g/time, 3 times a day) or placebo (simulator as control, SC group, simulator 3 g/time, 3 times a day) for 48 weeks. Blood and stool samples were collected from patients at baseline and week 48 of treatment, respectively. Liver and renal functions as well as hematological indices were detected. Fecal samples were analyzed by 16S rDNA V3-V4 high-throughput sequencing, and intestinal microbiota changes in both groups before and after treatment were compared, and their correlations with liver fibrosis were analyzed. RESULTS: Compared with the SC group, there was no significant difference in liver function, renal function and hematology indices in the BJJP group, however, the improvement rate of liver fibrosis was higher in the BJJP group (94.4% vs. 64.7%, P=0.041). Principal coordinate analysis (PCoA) based on weighted Unifrac distance showed significant differences in intestinal microbiota community diversity before and after BJJP treatment (P<0.01 and P=0.003), respectively. After 48 weeks' treatment, the abundance levels of beneficial bacteria (Bifidobacteria, Lactobacillus, Faecalibacterium and Blautia) increased, whereas the abundance levels of potential pathogenic bacteria, including Escherichia coli, Bacteroides, Ruminococcus, Parabacteroides and Prevotella decreased, among which Ruminococcus and Parabacteroides were significantly positively correlated with degree of liver fibrosis (r=0.34, P=0.04; r=0.38, P=0.02), respectively. The microbiota in the SC group did not change significantly throughout the whole process of treatment. CONCLUSION: BJJP had a certain regulatory effect on intestinal microbiota of patients with hepatitis B cirrhosis/liver fibrosis (ChiCTR1800016801).

The peptide encoded by a novel putative lncRNA HBVPTPAP inducing the apoptosis of hepatocellular carcinoma cells by modulating JAK/STAT signaling pathways.

When the hepatitis B virus (HBV) enters target cells, there are complex trans-regulatory mechanisms involved in the interactions between the virus and the target cells. In the present study, a new gene screened from the hepatoblastoma cell line HepG2 using suppression subtractive hybridization, referred to as lncRNA HBVPTPAP, was used to study the trans-regulation of HBV DNA polymerase. According to the structural characteristics of the full-length sequences, it was classified as long non-coding RNA. However, a unique and complete open reading frame (ORF) was still present. Therefore, to further identify the lncRNA HBVPTPAP gene's encoding potential, this study used several online tools to analyze and verify its encoding polypeptide authenticity. On that basis, the effects of the lncRNA HBVPTPAP gene on the biological behaviors of HepG2 cells and its molecular regulatory mechanism were investigated. It was found that the lncRNA HBVPTPAP subcellular was mainly located in the cytoplasm, and possibly activated the downstream JAK/STAT signaling pathway through the interaction between the encoding polypeptide and PILRA intracellular domain. Then, the mitochondrial apoptosis pathway may have been initiated to induce apoptosis. These results provided a basis for further study of the biological functions of the lncRNA HBVPTPAP gene.

Elevation of Cerebrospinal Fluid Light and Heavy Neurofilament Levels in Symptomatic Neurosyphilis.

BACKGROUND: Although clinical manifestations of symptomatic and asymptomatic neurosyphilis are different, few laboratory tests could reflect the difference. METHODS: A total of 92 non-HIV-infected patients with syphilis were enrolled in this study, including 23 with symptomatic neurosyphilis, 51 with asymptomatic neurosyphilis, and 18 with latent syphilis, which were excluded neurosyphilis because they were found to have no symptom and normal cerebrospinal fluid (CSF) tests and served as the control group. The concentrations of neurofilament light subunit (NF-L) and phosphorylated neurofilament heavy subunit (pNF-H) in the CSF were measured and compared among these groups, as well as before and after treatment in the symptomatic and asymptomatic groups. RESULTS: The median concentrations of NF-L in the symptomatic neurosyphilis, asymptomatic neurosyphilis, and control groups were 5806, 218, and 266 pg/mL, respectively (P < 0.001), and the median concentrations of pNF-H were 986, 43, and 49 pg/mL, respectively (P < 0.001). A subgroup of 15 symptomatic neurosyphilis and 10 asymptomatic neurosyphilis patients were followed up and underwent CSF examination 6 months after the antineurosyphilis treatment. The median concentration of NF-L in the symptomatic neurosyphilis group decreased from baseline 6420 to 2914 pg/mL after the treatment (P = 0.03), and the median concentration of pNF-H in the symptomatic neurosyphilis group decreased from baseline 1399 to 246 pg/mL after the treatment (P = 0.03). CONCLUSIONS: Neurofilament light subunit and pNF-H were significantly elevated in the symptomatic neurosyphilis patients, not in asymptomatic neurosyphilis, which was an implication of the different pathogeneses in neurosyphilis.

Association of Cytokines with Alanine Aminotransferase, Hepatitis B Virus Surface Antigen and Hepatitis B Envelope Antigen Levels in Chronic Hepatitis B.

BACKGROUND: Cytokines play an important role in occurrence and recovery of hepatitis B virus (HBV) infection. The aim of this study was to investigate the changes of cytokines concentration and its correlation to alanine aminotransferase (ALT), HBV deoxyribonucleic acid (HBV-DNA), hepatitis B envelope antigen (HBeAg), and HBV surface antigen (HBsAg) in the development of chronic hepatitis B (CHB). METHODS: Thirteen healthy individuals (HI), 30 chronic HBV-infected patients in immune tolerant (IT) phase, and 55 CHB patients were enrolled between August 2015 and May 2017. The peripheral blood samples were collected from all individuals. The levels of interferon (IFN)-α2, interleukin (IL)-10, transforming growth factor (TGF)-ß1, HBV-DNA, HBsAg, and HBeAg and liver function were measured. The quantitative determinations of cytokines levels, including IFN-α2, IL-10, and TGF-ß1 were performed using Luminex multiplex technology. The correlation of cytokines to ALT, HBV-DNA, HBsAg, and HBeAg was analyzed by linear regression analysis. RESULTS: IFN-α2 levels were similar between HI and IT groups (15.35 [5.70, 67.65] pg/ml vs. 15.24 [4.07, 30.73] pg/ml, Z = -0.610, P = 0.542), while it elevated significantly in CHB group (35.29 [15.94, 70.15] pg/ml vs. 15.24 [4.07, 30.73] pg/ml; Z = -2.522, P = 0.012). Compared with HI group (3.73 [2.98, 11.92] pg/ml), IL-10 concentrations in IT group (5.02 [2.98, 10.11] pg/ml), and CHB group (7.48 [3.10, 18.00] pg/ml) slightly increased (χ2 = 2.015, P = 0.365), and there was no significant difference between IT and CHB group (Z = -1.419, P = 0.156). The TGF-ß1 levels among HI (3.59 ± 0.20 pg/ml), IT (3.62 ± 0.55 pg/ml), and CHB groups (3.64 ± 0.30 pg/ml) were similar (χ2 = 2.739, P = 0.254). In all chronic HBV-infected patients (including patients in IT and CHB groups), the elevation of IFN-α2 level was significantly associated with ALT level (ß= 0.389, t = 2.423, P = 0.018), and was also negatively correlated to HBV-DNA load (ß = -0.358, t = -2.308, P = 0.024), HBsAg (ß = -0.359, t = -2.288, P = 0.025), and HBeAg contents (ß = -0.355, t = -2.258, P = 0.027). However, when both ALT level and cytokines were included as independent variable, HBV-DNA load, HBsAg, and HBeAg contents were only correlated to ALT level (ß = -0.459, t = -4.225, P = 0.000; ß = -0.616, t = -6.334, P = 0.000; and ß = -0.290, t = -2.433, P = 0.018; respectively). CONCLUSIONS: IFN-α2 elevation was associated with ALT level in patients with chronic HBV infection. However, in CHB patients, only ALT level was correlated to HBV-DNA, HBsAg and HBeAg contents.

Quantitation of Plasmacytoid Dendritic Cells in Chronic Hepatitis B Patients with HBeAg Positivity During PEG-IFN and Entecavir Therapy.

Plasmacytoid dendritic cells (pDCs) are crucial for control of chronic hepatitis B (CHB) virus infection. In this study, we evaluated the frequencies of pDCs and expression of functional molecules on pDCs in patients treated with PEG-IFN-α-2a or entecavir (ETV) and investigated changes during treatment. The mean fluorescence intensity of CD86 (CD86MFI) on the surface of pDCs and frequencies of pDCs and CD86+ pDCs in peripheral blood were measured. Compared with baseline, CD86+ pDC% and CD86MFI increased obviously after PEG-IFN-α-2a treatment for 12 and 24 weeks. For patients treated with ETV, only pDC% increased observably after treatment weeks 12 and 24 (P < 0.001) compared with baseline. Hepatitis B surface antigen (HBsAg) decline was significantly associated with elevated CD86+ pDC% (r = 0.348, P = 0.015) during PEG-IFN-α-2a treatment. In the HBsAg response group, CD86+ pDC% and CD86MFI (P < 0.001) increased observably after PEG-IFN-α-2a therapy, whereas only CD86MFI had a statistically significant difference after therapy compared with baseline (12 weeks versus 0 weeks, P = 0.022; 24 weeks versus 0 weeks, P = 0.015) in the HBsAg nonresponse group. CD86+ pDC% between the 2 groups had statistically significant differences at baseline (P = 0.001) and at the treatment time points of 12 and 24 weeks (P < 0.001), respectively. For patients receiving ETV therapy, pDC% increased observably, but CD86+ pDC% decreased significantly (P < 0.001) in the HBV DNA nonresponse group during early treatment with ETV. In CHB patients, HBsAg response in PEG-IFN-α-2a therapy correlated with the increase of CD86+ pDC% and HBV DNA nonresponse in ETV treatment correlated with the decrease of CD86+ pDC%.

Cell culture-adaptive mutations in hepatitis C virus promote viral production by enhancing viral replication and release.

AIM: To explore hepatitis C virus (HCV) adaptive mutations or combinations thereof responsible for enhanced viral production and investigate the underlying mechanisms. METHODS: A series of plasmids with adaptive mutations were constructed. After the plasmids were transfected into Huh7.5 cells, we determined the infectious HCV particle titers by NS5A immunofluorescence assays, and detected HCV RNA replication by real-time PCR and protein expression by Western blot. Then we carried out immunoblotting of supernatants and cell lysates with anti-NS3 to analyze the virus release level. In addition, co-localization of lipid droplets (LDs) with NS5A was measured using confocal laser scanning microscopy. The ratio between the p56 and p58 phosphoforms of NS5A was analyzed further. RESULTS: The plasmids named JFH1-mE2, JFH1-mp7, JFH1-mNS4B, JFH1-mNS5A, JFH1-mE2/NS5A, JFH1-mp7/NS5A, JFH1-mNS4B/NS5A, JFH1-mE2/p7/NS5A, and mJFH1 were constructed successfully. This study generated infectious HCV particles with a robust titer of 1.61 × 106 focus-forming units (FFUs)/mL. All of the six adaptive mutations increased the HCV particle production at varying levels. The NS5A (C2274R, I2340T, and V2440L) and p7 (H781Y) were critical adaptive mutations. The effect of NS5A (C2274R, I2340T, and V2440L), p7 (H781Y), and NS4B (N1931S) on infectious HCV titers was investigated by measuring the HCV RNA replication, protein expression, and virion release. However, the six adaptive mutations were not required for the LD localization of NS5A proteins or the phosphorylation of NS5A. CONCLUSION: In this study, we generated infectious HCV particles with a robust titer of 1.61 × 106 FFUs/mL, and found that the viral replication and release levels could be enhanced by some of the adaptive mutations.

Plasmacytoid Dendritic Cell Function and Cytokine Network Profiles in Patients with Acute or Chronic Hepatitis B Virus Infection.

BACKGROUND: Plasmacytoid dendritic cells (pDCs) and cytokines play an important role in occurrence and recovery of hepatitis B virus (HBV) infection. The aim of this study was to explore the frequency and function of pDC and serum cytokine network profiles in patients with acute or chronic HBV infection. METHODS: The healthy individuals (HI group), hepatitis B envelope antigen (HBeAg)-positive chronic HBV patients in immune tolerance (IT) phase (IT group), HBeAg-positive chronic HBV patients (CHB group), and acute HBV patients (AHB group) were enrolled in this study. The frequency of cluster of differentiation antigen 86 (CD86) + pDC and the counts of CD86 molecular expressed on surface of pDC were tested by flow cytometer. The quantitative determinations of cytokines, including Fms-like tyrosine kinase 3 ligand (Flt-3L), interferon (IFN)-α2, IFN-γ, interleukin (IL)-17A, IL-6, IL-10, transforming growth factor (TGF)-ß1 and TGF-ß2, were performed using Luminex multiplex technology. RESULTS: In this study, there were 13 patients in HI group, 30 in IT group, 50 in CHB group, and 32 in AHB group. Compared with HI group, HBV infected group (including all patients in IT, CHB and AHB groups) had significantly higher counts of CD86 molecular expressed on the surface of pDC (4596.5 ± 896.5 vs. 7097.7 ± 3124.6; P < 0.001). The counts of CD86 molecular expressed on the surface of pDC in CHB group (7739.2 ± 4125.4) was significantly higher than that of IT group (6393.4 ± 1653.6, P = 0.043). Compared with IT group, the profile of cytokines of Flt-3L, IFN-γ, and IL-17A was decreased, IFN-α2 was significantly increased (P = 0.012) in CHB group. The contents of IL-10, TGF-ß1, and TGF-ß2 in AHB group were significantly increased compared with IT and CHB groups (all P < 0.05). CONCLUSIONS: This study demonstrated that the function of pDC was unaffected in HBV infection. The enhanced function of pDC and IFN-α2 might involve triggering the immune response from IT to hepatitis active phase in HBV infection. Acute patients mainly presented as down-regulation of the immune response by enhanced IL-10 and TGF-ß.

Ratios of T-helper 2 Cells to T-helper 1 Cells and Cytokine Levels in Patients with Hepatitis B.

BACKGROUND: Hepatitis B is an immune response-mediated disease. The aim of this study was to explore the differences of ratios of T-helper (Th) 2 cells to Th1 cells and cytokine levels in acute hepatitis B (AHB) patients and chronic hepatitis B virus (HBV)-infected patients in immune-tolerance and immune-active phases. METHODS: Thirty chronic HBV-infected patients in the immune-tolerant phase (IT group) and 50 chronic hepatitis B patients in the immune-active (clearance) phase (IC group), 32 AHB patients (AHB group), and 13 healthy individuals (HI group) were enrolled in the study. Th cell proportions in peripheral blood, cytokine levels in plasma, and serum levels of HBV DNA, hepatitis B surface antigen, and hepatitis B e antigen were detected. RESULTS: The Th1 cell percentage and Th2/Th1 ratio in the HBV infection group (including IT, IC, and AHB groups) were significantly different from those in HI group (24.10% ± 8.66% and 1.72 ± 0.61 vs. 15.16% ± 4.34% and 2.40 ± 0.74, respectively; all P < 0.001). However, there were no differences in the Th1 cell percentages and Th2/Th1 ratios among the IT, IC, and AHB groups. In HBV infection group, the median levels of Flt3 ligand (Flt3L), interferon (IFN)-γ, and interleukin (IL)-17A were significantly lower than those in HI group (29.26 pg/ml, 33.72 pg/ml, and 12.27 pg/ml vs. 108.54 pg/ml, 66.48 pg/ml, and 35.96 pg/ml, respectively; all P < 0.05). IFN-α2, IL-10, and transforming growth factor (TGF)-ß2 median levels in hepatitis group (including patients in AHB and IC groups) were significantly higher than those in IT group (40.14 pg/ml, 13.58 pg/ml, and 557.41 pg/ml vs. 16.74 pg/ml, 6.80 pg/ml, and 419.01 pg/ml, respectively; all P < 0.05), while patients in hepatitis group had significant lower Flt3L level than IT patients (30.77 vs. 59.96 pg/ml, P = 0.021). Compared with IC group, patients in AHB group had significant higher median levels of IL-10, TGF-ß1, and TGF-ß2 (22.77 pg/ml, 10,447.00 pg/ml, and 782.28 pg/ml vs. 8.66 pg/ml, 3755.50 pg/ml, and 482.87 pg/ml, respectively; all P < 0.05). CONCLUSIONS: Compared with chronic HBV-infected patients in immune-tolerance phase, chronic HBV-infected patients in immune-active phase and AHB patients had similar Th2/Th1 ratios, significantly higher levels of IFN-α2, IL-10, and TGF-ß. AHB patients had significantly higher IL-10 and TGF-ß levels than chronic HBV-infected patients in immune-active phase.

HCBP6 Modulates Triglyceride Homeostasis in Hepatocytes Via the SREBP1c/FASN Pathway.

Hypertriglyceridemia leads to liver steatosis, cardiovascular disease, and type 2 diabetes. Although HCBP6 (hepatitis C virus core-binding protein 6) was previously shown to be an HCV (hepatitis C virus) core-binding protein, its biological function remains unclear. Here, we demonstrate that HCBP6 negatively regulates intracellular triglyceride (TG) levels in hepatocytes. We found that bidirectional manipulation of hepatocyte HCBP6 expression by knockdown or overexpression results in increased or decreased TG accumulation, respectively. In addition, HCBP6 mRNA and protein levels exhibited significant time- and dose-dependent increases in a cellular model of lipid-overload hepatic steatosis. Furthermore, TG levels are regulated by HCBP6-sterol regulatory element binding protein 1c (SREBP1c)-mediated fatty acid synthase (FASN) expression. We also demonstrate that HCBP6 mRNA and protein expression is inhibited by microRNA-122 (miR-122), and miR-122 overexpression elicited more robust translational repression of luciferase activity driven by the full 3'-UTR of HCBP6. Taken together, our results provide new evidence that miR-122-regulated HCBP6 functions as a sensor protein to maintain intrahepatocyte TG levels.

MicroRNA-185-5p mediates regulation of SREBP2 expression by hepatitis C virus core protein.

AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus (HCV) core protein in HepG2 cells. METHODS: HCV genotype 1b core protein was cloned and expressed in HepG2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2 (SREBP2) and the rate-limiting enzyme in cholesterol synthesis (HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, microRNA (miRNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular miRNA. RESULTS: HCV core protein expression in HepG2 cells increased the total intracellular cholesterol level (4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR mRNA levels (P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism study revealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity (P = 0.004). In addition, miR-185-5p expression was tightly regulated by the HCV core protein (P = 0.041). Moreover, overexpression of miR-185-5p repressed the SREBP2 mRNA level (P = 0.022) and protein expression. In contrast, inhibition of miR-185-5p caused upregulation of SREBP2 protein expression. miR-185-5p was involved in the regulation of SREBP2 expression by HCV core protein. CONCLUSION: HCV core protein disturbs the cholesterol homeostasis in HepG2 cells via the SREBP2 pathway; miR-185-5p is involved in the regulation of SREBP2 by the core protein.

Complete genome sequence of a new recombinant echovirus 25 strain isolated from a neonatal patient with hand, foot, and mouth disease complicated by encephalitis in Beijing, China.

Although human echovirus 25 (E-25), a type of the enterovirus B species, is implicated in aseptic meningitis, information on its gene structure, evolution, and virulence are limited. We report here the complete genome sequence of a novel recombinant E-25 strain (E25/2010/CHN/BJ) isolated from a neonate with hand, foot, and mouth disease complicated by encephalitis in Beijing, China in 2010. The complete viral genome consists of 7429 nucleotides (nts), including a 6585-nt open reading frame. Phylogenetic dendrogram based on VP1 gene regions revealed that this strain belonged to subgroup D4, which contains the other E-25 strains isolated from China in recent years. The difference in the amino acid sites (P130S, K/T135I) of the VP1 region may affect its immunogenicity. SimPlot and Bootscan analyses suggested that E25/2010/CHN/BJ is a recombination result of E-25 and Coxsackievirus B3 (CVB-3) strains. Our results would facilitate the study of the origin, evolution, and molecular epidemiology of E-25.

[Dendritic cell subsets and function in newborns from mothers of different HBV infection status].

OBJECTIVE: The aim of this study was to explore the frequency of mDC and pDC and expression of surface markers of the neonates and to discuss the effect of different status of HBV infection of mother on biological characteristics of DC. METHODS: Umbilicus cord blood in neonates of HBeAg positive HBV infected mother, HBeAg negative HBV infected mother, and normal mother were collected respectively; peripheral blood of healthy adults were selected as control group. Flow cytometry was employed to detect frequency of the mDC and its expression of CD86, frequency of pDC and its expression of CD80, CD83, CD86, and FlowJo software was used to compare these indicators among the groups. RESULTS: Compared with control group, the frequency of mDC of cord blood (0.29 +/- 0.16 vs 0.81 +/- 0.17), CD86 positive rate of mDC (10.72 +/- 10.01 vs 32.13 +/- 7.46), the frequency of pDC (0.15 +/- 0.07 vs 0.30 +/- 0.07), and CD86/CD83 positive rate of pDC (31.61 +/- 12.81 vs 74.96 +/- 9.78; 42.66 +/- 20.83 vs 82.00 +/- 6.94) were lower (t = -7.86, P = 0.00; t = -5.36, P = 0.00; t = -5.43, P = 0.00; t = -8.49. P = 0.00; t = -4.90, P = 0.00). CONCLUSIONS: The frequency of mDC and pDC in umbilical cord blood was lower than the peripheral blood of healthy adult, which was the possible mechanism of newborns easier to chronicity after the infection of hepatitis B virus. A significant correlation was found between different status of HBV infection and costimulatory molecule CD86 positive rate of mDC, but not for the frequency of mDC and pDC, and the expression of pDC molecules.

[Involvement of dendritic cells frequency and function in outcomes of interferon-alpha therapy for chronic hepatitis B patients].

OBJECTIVE: To elucidate the change in frequencies and functions of myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC) before and after interferon-alpha therapy for chronic hepatitis B (CHB) patients, and its correlation with virological and biochemical data. METHODS: Thirty patients with HBeAg-positive CHB who underwent IFN-alpha therapy were examined. Frequencies and expression of CD86 of mDC and pDC of peripheral blood were measured at baseline and treatment week (TW) 12 by flow cytometry. According to biochemical and virological parameters, the 30 patients were divided into ALT normalized group, ALT non-normalized group and virological responder group, virological non-responder group respectively. Statistical analysis of DC changes among different groups at baseline and TW12 was proceeded. RESULTS: (1) In the ALT normalized group, the pDC frequency at TW12 (0.25 +/- 0.14%) was higher than that at baseline (0.18 +/- 0.09%) (P = 0.023); in the ALT non-normalized group, the mDC frequency (0.58 +/- 0.34%) and its surface CD86 expression (61.80 +/- 22.52%) decreased significantly as compared with baseline (0.88 +/- 0.51%, 79.92 +/- 25.94%, respectively), (P = 0.025, P = 0.036, respectively). (2) In the virological responder group, the CD86 expression on pDC at TW12 (46.86 +/- 12.22%) was higher than that at baseline (29.42 +/- 15.16%) (P = 0.002); in the virological non-responder group, the mDC frequency (0.51 +/- 0.22%) and its surface CD86 expression (59.63 +/- 22.94% ) decreased significantly as compared with baseline (0.94 +/- 0.58%, 80.11 +/- 29.34%, respectively), (P = 0.006; P = 0.049, respectively). CONCLUSION: In IFN-alpha therapy for CHB patients, the increments of pDC frequency and function were related to biochemical and viral response, and decreases of mDC frequency and function were related to non-biochemical and non-viral response.

[The cytotoxic effect of CD8+ T subsets plays an important role in the chronic hepatitis B].

OBJECTIVE: The aim of this study is to explore the cytotoxic effect of CD8+ T subsets in peripheral blood with chronic hepatitis B subjects who are at immune tolerance phase and immune clearance phase (before and after three months' treatment with interferon-alpha), further to investigate their impact in the pathogenesis of chronic hepatitis B and antiviral therapy. METHODS: The subjects of chronic hepatitis B, including 20 subjects of immune tolerance phase and 20 subjects of immune clearance phase, are enrolled in the study. And we use flow cytometry to detect Lysosome-associated membrane protein-1 (LAMP-1, CD107a) and Granzyme B (GrB) expression of CD8(high) and CD8(low) T cells in peripheral blood with chronic hepatitis B subjects. RESULTS: (1) At immune clearance phase, the CD8+ T subsets expressing GrB and CD107a are higher than counterpart of immune tolerance phase. (2) At immune tolerance phase and immune clearance phase in HBV infection, the CD8(low) T cells expressing GrB and CD107a are higher than that of CD8(high) T cells. (3) After three months' treatment with interferon-a, except for GrB+ CD107a+ CD8(high) T cells, CD8(high) T cells expressing GrB and CD107a present a tendency of ascensus at the same time with that of CD8(low) T cells a tendency of descensus except for GrB(-)CD107a+ CD8(low) T cells. (4) The CD8+ T cell expressing GrB and CD107a, correlate positively with HBV DNA load at immune tolerance phase, but correlated negatively at immune clearance phase. CONCLUSIONS: (1) GrB and CD107a molecule expressed by different CD8+ T cell subsets play an important role in disease evolution and antivirus therapy of chronic hepatitis B, the cytotoxic effect of CD8(high) T cell subset became more and more stronger during the treatment of IFN-alpha, and the cytotoxic effect of CD8(low) T cell subset couldn't be neglected before antivirus therapy. (2) To some degree, the correlation between HBV-DNA load and the expression of GrB and CD107a at different CD8+ T cell subsets, could hint the relationship between virus and immune response.

[Studies about the level of CD4+ CD25+ regulatory T cells and relation between expression of Foxp3 and CD127 in peripheral blood of chronic HBV infection].

OBJECTIVE: To investigate the level of CD4+ CD25+ Foxp3+ regulatory T cells and observe relation between expression of Foxp3 and CD127 in peripheral blood of chronic HBV infection. METHODS: CD4+ CD25+ Foxp3+ and CD4+ CD25+ CD127low Treg in peripheral blood from 34 patients of immune tolerance stage, 26 patients of immune clearance stage and 31 patients of non-active status were quantitatively analyzed by flow cytometry. RESULTS: Immune tolerance group presented a higher fraction of CD4+ CD25+ Foxp3+ and CD4+ CD25+ CD127low Treg than non-active group in chronic HBV infection (Z = -2.693, P = 0.007 and t = 3.251, P = 0.002), and HBV positive group also presented a higher fraction than non-active group (t = 2.266, P = 0.026 and t = 3.208, P = 0.002), But ALT normal group is similar to ALT abnormal group (P > 0.05). In this study, the relation between expression of CD127low and Foxp3+ from CD4+ CD25+ regulatory T cells was observed, and CD4+ CD25+ CD127low Treg presented a higher fraction than CD4+ CD25+ Foxp3+ Treg. CONCLUSION: Peripheral Treg in HBV active replication group is higher than HBV negative group of chronic HBV infection. Expression of CD127low is consistent with Foxp3+ in CD4+ CD25+ regulatory T cells, but the former is significantly higher than the latter.

[Relationship of CD4+ CD25hi regulatory T (Treg) cells to disease progression in HIV-infected patients].

OBJECTIVE: Analyzing the relationships between peripheral blood CD4+ CD25hi regulatory T (Treg) cells and peripheral blood immune status or plasma HIV-lviral load in HIV-infected individuals,so as to determine whether Treg were related to the progression of HIV-infected disease. METHODS: 116 HIV-infected patients in different stages and 21 healthy control individuals were included in this study. The CD4+ and CD8+ T cell counts were determined by a standard 4-color flow cytometry technique. The Treg cells were examined with 3-color immune staining flow cytometry. The plasma HIV-1 viral load was detected by real time PCR. RESULTS: The frequencies of Treg cells decreased in HIV-infected individuals with high CD4+ T cell counts( > 300/microl) compared with normal controls. With the progression of disease the frequencies of Treg cells were raised gradually, until were increased in HIV-infected individuals with low levels of CD4+ T cell counts ( < 100/microl). In addition, the frequencies of Treg cells were inversely related to CD4+ T cell counts and CD4+ /CD8+ ratio, data showed a statistically significant (respectively, r = -0.564, P < 0.001; r = -0.377, P < 0.001). Furthermore, the proportions of Treg cells were closely related to plasma HIV-1 RNA viral load (r = 0.514, P < 0.001). CONCLUSION: CD4 CD25hi Treg cells should be a kind of important cells participating the immunopathogenesis of AIDS. It may play different roles in different stages of HIV-infected disease. The exact mechanism of Treg cells in the progression of the HIV-infected disease needs to be investigate further.

[Immune response for phase I clinical trial of a hepatitis B immunogenic complex therapeutic vaccine, YIC].

OBJECTIVE: A hepatitis B immunogenic complex therapeutic vaccine, yeast-derived recombinant HBsAg combined with human anti-HBs immunoglobulin (YIC), was evaluated for safety and immune response in phase I clinical trial. METHODS: The subtypes IgG1, IgG2, IgG3 and IgG4 of serum anti-HBs collected from 20 immunized subjects were analyzed by ELISA. The lymphocyte proliferation assay was carried out in five subjects and was analyzed by 3H-thymidine incorporation. The assays for IFNgamma, IL-2, IL-4, IL-6, IL-10 and TNFalpha were measured using Human Cytometric Bead Array Kit with FACSCalibur. RESULTS: The results showed that the subtypes of anti-HBs antibodies induced by 30, 60 and 90 microg YIC-immunized groups among all of the adult volunteers (20/20) were IgG1 and IgG3. The level of IgG1 was higher than that of IgG3 in each volunteer but the strength was different from each other. The rHBsAg-stimulated lymphocyte proliferation induced by three injections of 90 microg of YIC showed that the stimulation index was more than 2.0 in four out of the five individuals (4/5), ranging from 2.70 to 4.75. PHA-stimulated lymphocyte proliferation was not related to rHBsAg-stimulated lymphocyte proliferation. In the 60 microg YIC-immunized group there was no significant difference between the levels of IFNgamma, IL-2, IL-4, IL-6 and IL-10 at day 0 and day 42. At day 71, in comparison to day 0, the level of IFNgamma was higher in all eight subjects studied (P = 0.015) and the level of IL-2 was also increased in seven out of eight subjects (P = 0.002). In contrast, the levels of IL-4, IL-6, IL-10 and TNFalpha showed no significant difference in all the subjects (P-values: 0.298, 0.976, 0.202 and 0.996). CONCLUSION: Our results indicate that this hepatitis B immunogenic complex therapeutic vaccine (YIC) can induce a potent anti-HBs response.

[Inhibition of HBsAg and HBeAg secretion by RNA interference of the polymerase gene sequence of hepatitis B virus: an experimental study].

OBJECTIVE: To investigate the effects of small interfering RNA (siRNA) targeting the polymerase (P) gene sequence of hepatitis B virus (HBV) on the replication and antigen secretion of HBV. METHODS: From the 29 base sequences of the HBV in the HepG2.2.15 cells that accord with the demands of siRNA designing five sequences targeting the P gene of HBV were selected and cloned into the siRNA expressing vector pGE-1. Then the plasmid pGE-HBVP was transfected into the cultured HepG2.2.15 cells. Chemiluminescent immunoassay was used to determine the levels of HBsAg and HBeAg in the supernatant of culture medium 24, 48, 72, and 96 hours after the transfection and the expression of HBsAg in the 2.2.15 cells 24 hours after the transfection so as to observe the inhibitory effects. Untransfected cells and cells transfected with blank pGE-1 vector were used as controls. RESULTS: Five vectors expressing the siRNAs targeting the HBV P region, pGE-HBVP1-pGE-HBV5 were successfully constructed. The efficiency of transfection of the vectors into the 2.2.15 cells were 30% to 40%. 24, 48, 72, and 96 hours after the transfection of pGE-HBVP2, the strongest inhibitor among the five, the inhibitory rates of HBsAg secretion in the supernatant were 28.88%, 32.28%, 29.10%, and 18.42% respectively; and the inhibitory rates of HBeAg secretion were 38.33%, 27.50%, 33.41%, and 12.60% respectively. In view of the transfection efficiency of 30%-40%, the actual inhibitory rate of HBV antigen secretion might reach 80% and over. 24 hours after the transfection the expression rate of HBsAg in the 2.2.15 cells transfected with pGE-HBVP2 was 50%, significantly lower than that in the cells transfected with the blank vector pGE-1 (82%). CONCLUSION: siRNAs targeting the HBV P gene effectively prevent the HBV gene expression and replication and may play an important role in the clinical anti-viral treatment.