Characteristics of Liver Function in Patients With SARS-CoV-2 and Chronic HBV Coinfection.

BACKGROUND & AIMS: Coronavirus disease 2019 (COVID-19) is a major global health threat. We aimed to describe the characteristics of liver function in patients with SARS-CoV-2 and chronic hepatitis B virus (HBV) coinfection. METHODS: We enrolled all adult patients with SARS-CoV-2 and chronic HBV coinfection admitted to Tongji Hospital from February 1 to February 29, 2020. Data of demographic, clinical characteristics, laboratory tests, treatments, and clinical outcomes were collected. The characteristics of liver function and its association with the severity and prognosis of disease were described. RESULTS: Of the 105 patients with SARS-CoV-2 and chronic HBV coinfection, elevated levels of liver test were observed in several patients at admission, including elevated levels of alanine aminotransferase (22, 20.95%), aspartate aminotransferase (29, 27.62%), total bilirubin (7, 6.67%), gamma-glutamyl transferase (7, 6.67%), and alkaline phosphatase (1, 0.95%). The levels of the indicators mentioned above increased substantially during hospitalization (all P < .05). Fourteen (13.33%) patients developed liver injury. Most of them (10, 71.43%) recovered after 8 (range 6-21) days. Notably the other, 4 (28.57%) patients rapidly progressed to acute-on-chronic liver failure. The proportion of severe COVID-19 was higher in patients with liver injury (P = .042). Complications including acute-on-chronic liver failure, acute cardiac injury and shock happened more frequently in patients with liver injury (all P < .05). The mortality was higher in individuals with liver injury (28.57% vs 3.30%, P = .004). CONCLUSION: Liver injury in patients with SARS-CoV-2 and chronic HBV coinfection was associated with severity and poor prognosis of disease. During the treatment of COVID-19 in chronic HBV-infected patients, liver function should be taken seriously and evaluated frequently.

[Analysis of genotype IV distribution of hepatitis E virus infections in Wuhan by sequencing the open reading frame 3 gene].

OBJECTIVE: To determine the distribution of genotype IV among hepatitis E virus (HEV) infections in Wuhan by sequencing the open reading frame (ORF) 3 gene of HEV clinical isolates. METHODS: Serum samples were collected from 103 individuals who tested positive for the anti-HEV IgM antibody, and total HEV RNA was extracted for targeted gene sequencing analysis. Reverse transcription-nested polymerase chain reaction (PCR) was used to amplify two fragments of the ORF3 gene (5020 to 5392 nt and 5347 to 5956 nt, EF570133). The two PCR products were sequenced and the sequences were stitched with the ContigExpress program and used to determine the HEV genotype. RESULTS: Both ORF3 gene fragments were amplified in 18 out of the 103 anti-HEV IgM-positive serum samples. These 18 HEV isolates shared 92.5% to 99.4% identity with each other at the nucleotide level. Nucleotide sequence homology analysis of the HEV genotypes I, II, III, and IV indicated the highest homology was with genotype IV; specifically, homology with genotype I was 83.5% to 86.7%, with genotype II was 83.2% to 85.2%, with genotype III was 84.6% to 87.2%, and with genotype IV was 92.0% to 96.5%. CONCLUSION: Targeted sequencing of the HEV ORF3 gene facilitated genotyping of clinical isolates. Using this method, it was determined that nearly 20% of HEV clinical isolates from Wuhan belong to genotype IV.

Clinical characteristics and risk factors of sporadic Hepatitis E in central China.

BACKGROUND: Epidemiological investigations, detections and vaccines of hepatitis E (HE) have been paid a focus of attention in prior studies, while studies on clinical features and risk factors with a large number of sporadic HE patients are scarce. RESULTS: Sporadic HE can occur throughout the year, with the highest incidence rate in the first quarter of a year, in central of China. Of the 210 patients, 85.2% were male, and the most common clinical symptoms were jaundice (85.7%), fatigue (70.5%) and anorexia (64.8%). Total bilirubin (TBil), blood urea nitrogen (BUN), and international normalized ratio (INR) were found as major risk factors for death of HE patients. There was an overall mortality of 10%, and the mortality in the cirrhotic and non-cirrhotic group was 25% and 6.47%, respectively. Moreover, hepatitis E virus (HEV) infected patients with liver cirrhosis had a higher mortality and incidence of complications. CONCLUSIONS: TBil, BUN, and INR are major risk factors of mortality for HE. Liver cirrhosis can aggravate HE, and lead to a higher mortality. HEV infection can cause decompensation in patients with cirrhosis, as evidenced by a worsening Child-Pugh score.

Timing and efficacy of transjugular intrahepatic portosystemic shunt in patients with pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome.

There is no specific treatment for pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome (PA-HSOS). It is not clear when transjugular intrahepatic portosystemic shunt (TIPS) should be implemented in PA-HSOS patients. This study aimed to evaluate the timing of TIPS using total bilirubin (TBIL) as a measure, and to investigate efficacy of TIPS. We retrospectively analyzed the medical records of 10 PA-HSOS patients, among whom 4 patients had received TIPS (TIPS group), and the remaining patients were assigned to the internal medicine group. In the TIPS group, the TBIL level before TIPS was 84.4 ± 45.2 µmol/L (> 3 mg/dL), and TBIL levels were increased to different degrees after TIPS. With the extension of time, serum TBIL levels gradually decreased, and no liver failure occurred. With regards to the short-term outcomes, 3 patients recovered, 1 developed chronic illness and 0 died in the TIPS group. Moreover, 0 patients recovered, 5 developed chronic illness and 1 died in the internal medicine group. The rank sum test of group design revealed significant differences in clinical outcomes (P = 0.02). It was suggested that when the internal medicine effect of PA-HSOS patients is poor, TIPS should be considered, which is no trestricted to the limit of 3 mg/dL TBIL. It was also found TIPS effectively promote the recovery of liver function and reduce the occurrence of chronicity.

Preparation and functional evaluation of monoclonal antibodies targeting Hepatitis B Virus Polymerase.

HBV pol plays a critical role in the replication of hepatitis B virus (HBV). Previous studies conducted on HBV pol have produced limited evidence on HBV pol expression due to the lack of effective detection methods. The present study used the HBV pol (159-406 aa) protein as a target to screen for specific monoclonal antibodies that recognize HBV pol and subsequently evaluate their diagnostic and therapeutic value. Four antibodies (P3, P5, P12, P20) against HBV pol were obtained. Among them, the P20 antibody indicated optimal binding with HBV pol as demonstrated by Western blotting (WB) in a cell model transfected with the HBV genome. We also expressed P5 and P12 antibodies in mouse liver cells by transfection and the results indicated significant antiviral effects caused by these two antibodies especially P12. In summary, the present study established an antibody which was denoted P20. This antibody can be used to detect HBV pol expression by four HBV genomes via WB analysis. In addition, the antibody denoted P12 could exert antiviral effects via intracellular expression, which may provide a promising approach for the treatment of chronic hepatitis B.

Role of Hedgehog signaling pathway in proliferation and invasiveness of hepatocellular carcinoma cells.

The Hedgehog (Hh) pathway has been confirmed a contributor to the carcinogenesis and progression of various tumor types. To investigate the Hh signaling activity in human hepatocellular carcinoma (HCC), we detected the expression levels of Hh pathway components (Shh, Ptch1 and Gli2) in 57 samples of HCC and corresponding adjacent-tumor liver tissues. The Hh pathway was overexpressed in cancer tissues compared with non-cancer tissues and correlated closely with histologic differentiation and portal venous invasion of HCC. To elucidate the relationship between Hh signaling and HCC progression, we performed a further study in vitro. First, the expression levels of the signaling were detected in a subset of hepatoma cell lines and SMMC-7721 cells selected with high level of Hh signaling expression. Next, we employed KAAD-cyclopamine (a specific inhibitor of Hedgehog pathway) to block the Hh pathway in SMMC-7721 cells and assessed the changes of their biological behaviors. The results showed that the blockade of Hh signaling pathway by KAAD-cyclopamine induced reduction of DNA synthesis leading to marked cell growth inhibition and also caused significant attenuation in invasiveness and motility of HCC cells. Collectively, our data demonstrated that the Hh pathway plays an important role in HCC development and invasion. Blockade of the Hh signaling pathway may be a potential target of new therapeutic strategy for HCC.

Clinical significance of anti-HEV IgA in diagnosis of acute genotype 4 hepatitis E virus infection negative for anti-HEV IgM.

Anti-HEV IgM is a diagnostic for recent or ongoing HEV infection. However, some patients with acute hepatitis E (AHE) negative for anti-HEV IgM in acute period were often observed in clinical practice. In this study, we constructed the anti-HEV IgA indirect ELISA assay to evaluate the significance of anti-HEV IgA. The specificity of anti-HEV IgA was 99.6%. Among 245 AHE patients, 84 samples from 84 patients were positive for HEV RNA. The positive rate of anti-HEV IgA, anti-HEV IgM and anti-HEV IgG in 84 samples positive for HEV RNA was 96.3, 97.6, and 88.1%, respectively, and no sample was negative for anti-HEV IgA and anti-HEV IgM simultaneously. Among 245 AHE patients, we found nine samples collected from nine patients in acute period were negative for anti-HEV IgM but positive for anti-HEV IgA and two samples were positive for HEV RNA. Detection of anti-HEV IgA can be a useful supplement for diagnosis of acute HEV infection especially in patients negative for anti-HEV IgM.

Expression of toll-like receptor 9 in peripheral blood mononuclear cells from patients with different hepatitis B and C viral loads.

The aim of the present study was to investigate the expression of toll-like receptors (TLR) 9 in peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B and C with different virus copies. The study group included 90 patients (60 with chronic hepatitis B, and 30 with chronic hepatitis C), and 20 healthy people served as control group. The protein and mRNA levels of TLR9 were detected by using flow cytometry and real-time PCR. The serum viral copies of HBV and HCV were measured in all patients, and the correlation between HBV-DNA copies or HCV-RNA copies and the TLR9 expression was analyzed. Our results demonstrated that HBV or HCV infection led to a decreased expression of TLR9 mRNA and protein compared to the control group (P<0.05). The TLR9 protein and mRNA levels were negatively correlated with serum viral copies of HBV and HCV (r=-0.632, r=-0.909, P<0.01). It was concluded that TLR9 mRNA and protein are down-regulated in PBMC of HBV-infected or HCV-infected patients, and they are negatively correlated with serum viral copies and play an important role in detecting viral replication of HBV and HCV.

Effects of endotoxin on liver Smac apoptosis channel.

To study the effect of endotoxin on liver apoptosis, L02 liver cells were cultured and passaged in vitro, and then stimulated by endotoxin at 10 mg/mL for 4, 8, 16 and 24 h respectively. Liver apoptosis was flow cytometrically and fluorescently detected. Immunohistochemistry was used to detect the delivery of smac and caspase9. The delivery of liver cell smac and the activity of caspase3 were measured by caspase3 assay kit. The hepatic failure models of rats were established by using D-galactosamine. The blood serum and liver tissues were collected for the detection of the liver function, the level of endotoxin and the activity of caspase3 by using chromogenic substrate limulus amebocyte lysate method (LAL) and caspase3 active assay kit. The expression of smac and caspase9 in liver cells was detected by Western blotting. With in vitro study, the L02 cells stimulated by LPS condensed into conglobation and formed apoptotic bodies. After those cells were stained by hoechst, the apoptotic cells displayed blue color under the fluorescent microscope. The apoptosis rate was increased over time and the apoptosis was mainly of advanced stage. Meanwhile, the rate of smac delivery and activity of caspase9 and caspase3 were increased on L02 cell membrane. In vivo, hepatic failure and obvious endotoxemia were induced by injection of more than 200 mg/kg D-GalN. Hepatic mitochondria smac was reduced with dosage of D-GalN and, on the contrary, the activity of caspase3 was increased. D-GalN at 200 mg/kg increased Caspase9 while D-GalN at 300 mg/kg decreased caspase9. Mitochondria signal channel plays an important role in the endotoxin-induced apoptosis of hepatic cells by promoting the release of smac from mitochondria to cytoplasm and activating caspase9 and caspase3 in its low-level channel.

The effect of Gankang Suppository on duck hepatitis B virus, serum biochemistry and liver histology in ducklings.

To examine the effect of Gankang Suppository on duck hepatitis B virus (DHBV), the serum biochemistry and hepatic histology in an animal model of DHBV infection, a model of DHBV infection was established by infecting 1-day-old Yingtaogu ducklings with DHBV-positive serum. The successful model was confirmed by PCR assay and 48 ducklings infected with DHBV were randomly divided into 3 groups: a Gankang Suppository treatment group, an acyclovir (ACV) group and a DHBV model group (control), with each group having 16 animals. All the animals were given the medicines for 4 weeks in a row. The serum of the animals was taken 14 and 28 days after the medication and 7 days after drug discontinuation. Real-time PCR was performed to detect the copy numbers of DHBV DNA in the serum. ALT and AST were dynamically monitored. The ducklings were sacrificed on the 7th day after the discontinuation of the treatment and livers were harvested and examined for inflammation and degeneration of liver cells by using HE staining. The results showed that on day 14, 28 after the treatment and day 7 after the withdrawal, the logarithmic values (log) of DHBV DNA copy numbers in ducklings of Gankang Suppository treatment group were significantly lower than that before the treatment (P=0.0092, P=0.0070, P=0.0080, respectively). Compared with DHBV model control group, the ALT level was significantly decreased (P=0.0020, P=0.0019, respectively) on day 28 after the treatment and on day 7 after the withdrawal. The AST level was also reduced on day 14 after the treatment (P=0.0298). Compared with the ACV control group, the level of ALT was lower on day 7 after the withdrawal (P=0.0016). Histologically, the hepatocyte swelling, vacuolous degeneration and acidophilic degeneration in Gankang Suppository treatment group were alleviated 7 days after the withdrawal as compared with model control group (P=0.0282, P=0.0084, P=0.0195, respectively). It is concluded that Gankang Suppository can effectively suppress DHBV replication, reduce the levels of serum ALT and AST and improve hepatic histology.

Effects of hepatitis E virus infection on interferon production via ISG15.

AIM: To assess the effects of hepatitis E virus (HEV) on the production of type I interferons (IFNs) and determine the underlying mechanisms. METHODS: We measured the production of interferon (IFN)-alpha and -beta (-α/ß) in genotype 3 HEV-infected C3A cells at different time points (0, 8, 12, 24, 48, 72 and 120 h) by enzyme-linked immunosorbent assay (ELISA). The expression levels of IFN-stimulated gene (ISG)15 in HEV-infected C3A cells at different time points were tested by western blotting. The plasmid-expressing open reading frame 3 (ORF3) or control plasmids (green fluorescent protein-expressing) were transfected into C3A cells, and the levels of IFN-α/ß and ISG15 were evaluated, respectively. Furthermore, the plasmid-expressing ISG15 or small interfering RNA-inhibiting ISG15 was transfected into infected C3A cells. Then, the production of IFN-α/ß was also measured by ELISA. RESULTS: We showed that genotype 3 HEV could enhance the production of IFN-α/ß and induce elevation of ISG15 in C3A cells. HEV ORF3 protein could enhance the production of IFN-α/ß and the expression of ISG15. Additionally, ISG15 silencing enhanced the production of IFN-α/ß. Overexpression of ISG15 resulted in the reduction of IFN-α/ß. CONCLUSION: HEV may promote production of IFN-α/ß and expression of ISG15 via ORF3 in the early stages, and increased ISG15 subsequently inhibited the production of IFN-α/ß.

Uric acid enhances T cell immune responses to hepatitis B surface antigen-pulsed-dendritic cells in mice.

AIM: To study the induction of T cellular immune responses in BALB/c mice immunized with uric acid and dendritic cells (DCs) pulsed with hepatitis B virus surface antigen (HBsAg). METHODS: DCs were generated from bone-marrow cells of BABL/c mice, and then pulsed or unpulsed with HBsAg protein (HBsAg-pulsed-DCs or unpulsed-DCs) in vitro. BABL/c mice were immunized with HBsAg-pulsed-DCs (1 x 10(6)) and uric acid, injected through the tail vein of each mouse. The mice in control groups were immunized with HBsAg-pulsed-DCs alone, unpulsed-DCs alone or 200 microg uric acid alone or PBS alone. The immunization was repeated 7 d later. Cytotoxic T lymphocytes (CTLs) in vivo were determined by the CFSE labeled spleen lysis assay. Spleen cells or spleen T cells were isolated, and re-stimulated in vitro with HBsAg for 120 h or 72 h. Production of IFN-gamma and IL-4 secreted by spleen cells were determined by ELISA method; proliferation of spleen T cells were detected by flow cytometry. RESULTS: The cytotoxicities of HBsAg-specific-CTLs, generated after immunization of HBsAg-pulsed-DCs and uric acid, were 68.63% +/- 11.32% and significantly stronger than that in the control groups (P < 0.01). Compared with control groups, in mice treated with uric acid and HBsAg-pulsed-DCs, the spleen T cell proliferation to HBsAg re-stimulation was stronger (1.34 +/- 0.093 vs 1.081 +/- 0.028, P < 0.01), the level of IFN-gamma secreted by splenocytes was higher (266.575 +/- 51.323 vs 135.223 +/- 32.563, P < 0.01) , and IL-4 level was lower (22.385 +/- 2.252 vs 40.598 +/- 4.218, P < 0.01). CONCLUSION: Uric acid can strongly enhance T cell immune responses induced by HBsAg-pulsed-DCs vaccine. Uric acid may serve as an effective adjuvant of DC vaccine against HBV infection.

Knockdown of survivin expression by siRNA induces apoptosis of hepatocellular carcinoma cells.

Survivin, a newly identified member of IAP family, is a powerful apoptosis-inhibiting factor. It is expressed in embryonic tissues as well as in the majority of human cancers, but not in most normal adult tissues. The cancer-specific expression of survivin makes it a potential target for cancer treatment. A survivin-specific small inhibitory RNA (siRNA) was introduced into hepatocellular carcinoma cells to investigate its effect on cancer cell apoptosis, growth and sensitivity to chemotherapeutic drugs. It was found that expressions of survivin protein and proliferation index (PI) in siRNA groups were significantly decreased, the apoptosis index (AI) of siRNA groups was significantly higher than those of others groups, and the growth inhibition rate (GIR) of chemotherapeutic drugs in siRNA groups were significantly higher than those of other groups. Our study suggests that the expression of survivin may be significantly decreased in hepG2 cell after siRNA transfection. siRNA targeting survivin could induce cell apoptosis, inhibit cell proliferation and sensitize hepatocarcinoma cells to chemotherapy. Our findings provide preliminary evidence for the therapeutic use of survivin-targeted RNA interference for human tumors that express high levels of this molecule.

[The effect of endotoxemia on gluconeogenesis in livers and kidneys during acute hepatic failure].

OBJECTIVES: To explore the effects of endotoxemia on gluconeogenesis in livers and kidneys during acute hepatic failure. METHOD: Twenty-four healthy male SD rats were randomly divided into four groups (6 rats in each group) and all of them were injected intraperitoneally with solutions: group I with normal saline, group II with 400 mg/kg of D-galactosamine (D-GaLN), group III with 400 mg/kg of D-GaLN plus 50 microg/kg lipopolysaccharide(LPS), and group IV with 400 mg/kg of D-GaLN plus 500 microg/kg LPS. At 6 hours after the administration of different solutions intraperitoneally, blood samples were collected to examine blood urea nitrogen (BUN) and serum creatinine. Realtime PCR was used to study the expression of phosphoenolpyruvate carboxykinase (PEPCK) in the livers and kidneys. RESULTS: No endotoxemia developed in group I or group II but it was evident in group III and group IV. The level of endotoxemia in group IV was higher than in group III (8.05+/-0.43, 3.50+/-2.25, P<0.05). After 6 hours of administration of LPS in group IV, hypoglycemia appeared, and blood glucose was normal in the other three groups. BUN and serum creatinine were all normal in the four groups, except that blood urea nitrogen was elevated in group IV. The mRNA of PEPCK in livers decreased gradually in all the four groups (2.54+/-1.32 vs 1.87+/-0.15 vs 0.91+/-0.13 vs 0.44+/-0.42, P<0.05). In the kidneys there was no change in the expression of PEPCK in group I and group II (0.75+/-0.03 and 0.77+/-0.04, P>0.05), but it increased in group III (0.75+/-0.03 vs 1.63+/-0.86, P<0.05), and decreased in group IV (0.75+/-0.03 vs 0.13+/-0.07, P<0.05). CONCLUSION: During acute hepatic failure severe endotoxemia would damage the function of gluconeogenesis in livers and kidneys by inhibiting transcription of PEPCK and this can induce hypoglycemia.

Significance of serum IgA in patients with acute hepatitis E virus infection.

AIM: To study the significance of serum anti-hepatitis E virus (HEV) IgA in patients with hepatitis E. METHODS: A new method was established to assay anti-HEV IgA, which could be detected in the middle phase of the infection. We compared anti-HEV IgA assay with anti-HEV IgM and anti-HEV IgG assay in sera from 60 patients with positive HEV-RNA. RESULTS: The 60 patients with positive HEV-RNA had both anti-HEV IgA and anti-HEV IgM and 410 patients with negative HEV-RNA were used as control. Periodic serum samples obtained from 60 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, anti-HEV IgA and anti-HEV IgG. Their HEV-RNA was detectable in the serum until 20 +/- 11 d. We used anti-HEV IgM and anti-HEV IgA assay to detect HEV infection and positive results were found in 90 +/- 15 d and 120 +/- 23 d respectively, the positive rate of anti-HEV IgA was higher than that of anti-HEV IgM and HEV-RNA (P < 0.05). CONCLUSION: The duration of anti-HEV IgA in serum is longer than that of anti-HEV IgM, and anti-HEV IgA assay is a good method to detect HEV infection.

Estimated Glomerular Filtration Rate Increases in Chronic Hepatitis B Patients Treated With Telbivudine Monotherapy and Combination Treatment.

BACKGROUND: Several studies have reported a renoprotective effect of telbivudine during the treatment of patients for chronic hepatitis B (CHB). OBJECTIVES: This longitudinal retrospective study aimed to examine the effects of telbivudine monotherapy and combination therapy (adefovir plus telbivudine) on renal function. PATIENTS AND METHODS: This study included 336 Chinese CHB patients, who were selected from outpatients in Tongji Hospital. 44, 122, 66, 58, and 46 of these patients had been orally taking adefovir, telbivudine, entecavir, adefovir plus telbivudine, and adefovir plus lamivudine, respectively, for at least 24 months. RESULTS: The estimated glomerular filtration rate (eGFR) in the telbivudine and adefovir plus telbivudine groups increased by 5.14 mL/min (P < 0.001) and 6.19 mL/min (P = 0.005), respectively. The patients taking the five drug regimens were further grouped into the following three subpopulations: those with compensated hepatic cirrhosis, those aged 50 or more years, and those with baseline eGFR values of 50 - 90 mL/min. The three subgroups that received telbivudine monotherapy exhibited eGFR increases of 6.38, 6.74, and 10.82 mL/min, respectively. The three subgroups that received combination therapy of adefovir plus telbivudine exhibited eGFR increases of 18.31, 14.73, and 16.59 mL/min, respectively (P < 0.05). The predictive factors for the change in eGFR levels over time were analyzed by means of two linear mixed effects models for the three monotherapy regimens and two combination regimens. Age, gender, and medication are predictive factors of eGFR changes. In addition, abnormal creatinine kinase (CK) levels in the telbivudine group were not correlated with eGFR changes (P = 0.992). CONCLUSIONS: These findings indicate that telbivudine, used in both monotherapy and combination therapy, improves the renal function of patients with CHB. The improvements are particularly significant in patients at high renal risk.

α-Mannosidase I Protein Expression in Peripheral Blood Mononuclear Cells Is Upregulated During Hepatitis B Virus Infection.

Hepatitis B virus (HBV) has been reported to be recognized by dendritic cell-specific ICAM-3-grabbing nonintegrin in the presence of the α-mannosidase I inhibitor kifunensine, whereas native HBV is not. The aim of our study was to determine whether changes in α-mannosidase I expression in peripheral blood mononuclear cells (PBMCs) occur in patients with HBV infection. Peripheral blood was collected from 90 HBV-infected patients (grouped into immune tolerance, chronic hepatitis B, or inactive carrier group based on their clinical states) and 30 healthy donors. Expression of the three α-mannosidase I subtypes, MAN1A1, MAN1A2, and MAN1C1, was measured using western blot analyses. Compared with the healthy controls, significant increases in the MAN1A1, MAN1A2, and MAN1C1 expression levels were observed in the three HBV-infected groups, among whom the immune tolerance group showed the largest increase. For the patients in the immune tolerance phase, the expression levels of both MAN1A1 and MAN1A2 were linearly and positively correlated with the hepatitis B e antigen (HBeAg) titer and HBV DNA level, although a positive correlation was only found between MAN1C1 expression and the HBeAg titer. These results indicate that increased α-mannosidase I expression in PBMCs may play an important role in HBV immune escape and that its expression level is closely related to viral replication activity.

A novel platform designed by Au core/inorganic shell structure conjugated onto MTX/LDH for chemo-photothermal therapy.

A novel platform making up of methotrexate intercalated layered double hydroxide (MTX/LDH) hybrid doped with gold nanoparticles (NPs) may have great potential both in chemo-photothermal therapy and the simultaneous drug delivery. In this paper, a promising platform of Au@PDDA-MTX/LDH was developed for anti-tumor drug delivery and synergistic therapy. Firstly, Au NPs were coated using Layer-by-Layer (LbL) technology by alternate deposition of poly (diallyldimethylammonium chloride) (PDDA) and MTX molecules, and then the resulting core-shell structures (named as Au@PDDA-MTX) were directly conjugated onto the surface of MTX/LDH hybrid by electrostatic attraction to afford Au@PDDA-MTX/LDH NPs. Here MTX was used as both the agent for surface modification and the anti-tumor drug for chemotherapy. The platform of Au@PDDA-MTX/LDH NPs not only had a high drug-loading capacity, but also showed excellent colloidal stability and interesting pH-responsive release profile. In vitro drug release studies demonstrated that MTX released from Au@PDDA-MTX/LDH was relatively slow under normal physiological pH, but it was enhanced significantly at a weak acidic pH value. Furthermore, the combined treatment of cancer cells by using Au@PDDA-MTX/LDH for synergistic hyperthermia ablation and chemotherapy was demonstrated to exhibit higher therapeutic efficacy than either single treatment alone, underscoring the great potential of the platform for cancer therapy.

Hepatitis B virus upregulates host expression of α-1,2-mannosidases via the PPARα pathway.

AIM: To assess the effects of hepatitis B virus (HBV) on the expression of host α-1,2-mannosidases and determine the underlying mechanisms. METHODS: We measured the expression levels of MAN1A1, MAN1A2, MAN1B1, and MAN1C1 in cell lines HepG2.2.15, HepN10, HepAD38 and HepG2 by Western blot. Viral antigens (HBsAg and HBeAg) in the culture medium were measured using the chemiluminescence method. HBV DNA quantification assays were performed using a commercial real-time PCR kit. Protein levels of human liver tissue α-1,2-mannosidases were also evaluated by Western blot. Plasmids containing seven individual viral genes of HBV (PTT22-HBx, PTT22-HBs, PTT22-preS2, PTT22-preS1, PTT22-HBc, PTT22-HBe, and PTT22-HBp) or control plasmids (PTT22-vector) were transfected into HepG2 cells. MK886 (PPARα) and GW9662 (PPARγ) inhibitors were used to explore the effects of HBV on α-1,2-mannosidase expression after the PPARα and PPARγ pathways were blocked. RESULTS: We showed that the expression of α-1,2-mannosidases was higher in stably transfected HBV cells than in controls. The expression levels of α-1,2-mannosidase were higher in AD38 cells than those in ND10 cells, which were in turn greater than those in G2.2.15 cells, and positively correlated with the expression of HBsAg in all the cell lines. Levels of α-1,2-mannosidase in non-tumorous liver tissues of HBV-related HCC patients were also higher than in the tissues from non-HBV-related HCC patients. Moreover, transfecting HepG2 cells with a component of the HBV viral envelope also increased the expression of α-1,2-mannosidases. However, this envelope protein component could not induce MAN1C1 expression in the presence of a PPARα inhibitor, MK886. We also found that MK886 did not affect the expression of MAN1C1 in AD38 cells without tetracycline in the culture medium. This phenomenon was not observed in the case of GW9662. CONCLUSION: Our results indicate that HBV increases the expression of α-mannosidases both in vitro and in vivo via activation of the PPARα pathway by its envelope protein.

The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1.

Hepatitis E virus (HEV) genotype 1 infection is common and can emerge as outbreaks in developing areas, thus posing a threat to public health. However, due to the absence of feasible animal models, the mechanism of HE pathogenesis remains obscure. The HEV pathogenic mechanism has been suggested to be mediated by the immune system and not by direct viral duplication. We firstly discovered that the open reading frame 3 (ORF3) protein of genotype 1 HEV downregulates TLR3-mediated NF-κB signaling in Human A549 Lung Epithelial Cells (A549 cells) which were exposed to different TLR agonists associated with viral nucleic acids. Additionally, we identified the P2 domain of ORF3 as being responsible for this inhibition. Intriguingly, tumor necrosis factor receptor 1-associated death domain protein (TRADD) expression and receptor-interacting protein kinase 1 (RIP1) K63-ubiquitination were reduced in the presence of both ORF3 and Poly(I:C). Furthermore, we found that Lys377 of RIP1 acts as the functional ubiquitination site for ORF3-associated inhibition. Overall, we found that ORF3 protein downregulates TLR3-mediated NF-κB signaling via TRADD and RIP1. Our findings provide a new perspective on the cellular response in HEV infection and expand our understanding of the molecular mechanisms of HEV pathogenesis in innate immunity.