Hyperactivation of succinate dehydrogenase promotes pyroptosis of macrophage via ROS-induced GSDMD oligomerization in acute liver failure.

Acute liver failure (ALF) is a life-threatening disease with high mortality. Given excessive inflammation is one of the major pathogenesis of ALF, candidates targeting inflammation could be beneficial in the condition. Now the effect of hyperactivated succinate dehydrogenase (SDH) on promoting inflammation in lipopolysaccharide (LPS)-treated macrophages has been studied. However, its role and mechanism in ALF is not well understood. Here intraperitoneal injection of D-galactosamine and LPS was conducted in male C57BL/6 J mice to induce the ALF model. Dimethyl malonate (DMM), which inhibited SDH activity, was injected intraperitoneally 30 min before ALF induction. Macrophage pyroptosis was induced by LPS plus adenosine triphosphate (ATP). Pyroptosis-related molecules and proteins including GSDMD oligomer were examined by ELISA and western blot techniques, respectively. ROS production was assessed by fluorescence staining. The study demonstrated SDH activity was increased in liver macrophages from ALF mice. Importantly, DMM administration inhibited ROS, IL-1ß, and pyroptosis-associated proteins levels (NLRP3, cleaved caspase-1, GSDMD-N, and GSDMD oligomers) both in the ALF model and in macrophages stimulated with LPS plus ATP. In vitro, ROS promoted pyroptosis by facilitating GSDMD oligomerization. Additionally, when ROS levels were increased through the addition of H2O2 to the DMM group, the levels of GSDMD oligomers were reverted. In conclusion, SDH hyperactivation promotes macrophage pyroptosis by ROS-mediated GSDMD oligomerization, suggesting that targeting this pathway holds promise as a strategy for treating ALF and other inflammatory diseases.

Comparison of the Efficacy and Safety of Different Doses of Linaclotide for Patients with Chronic Constipation: A Meta-Analysis and Bayesian Analysis.

BACKGROUND: It is ambiguous whether a higher dose of linaclotide provides higher efficacy for chronic constipation (CC) patients. The meta-analysis aimed to assess the efficacy and safety of linaclotide doses ranging from 62.5 µg to 600 µg for CC patients. METHODS: A comprehensive search was conducted, and STATA16 software was used for data analysis. RESULTS: Seven studies with 4,107 patients were eligible. A significantly enhanced number of completely spontaneous bowel movement (CSBM) responders were found in the extremely low-dose group (OR: 2.94; 95% CI: 1.98-4.34; p < 0.001), the low-dose group (OR: 3.24; 95% CI: 2.44-4.31; p < 0.001), the medium-dose group (OR: 3.08; 95% CI: 1.46-6.50; p=0.003), and high-dose group (OR: 4.79; 95% CI: 3.04-7.54; p < 0.001). Bayesian analysis showed the high-dose group obtained the maximum CSBM responder rate (OR: 4.94; 95% credible interval (CrI): 3.22-7.79; probability rank = 0.87) indirectly compared with extremely low-dose, low-dose, and medium-dose groups. However, no significant difference presented in the CSBM responder rate by pairwise comparisons of the different dose groups. Additionally, no more any adverse events occurred in the higher linaclotide dose group (RR: 0.91; 95% CrI: 0.60-1.38) indirectly compared with other dose groups. CONCLUSIONS: High dose of linaclotide could be more effective and safer for CC patients, which need more trials to confirm in the future.

A meta-analysis of the association between obesity and COVID-19.

Owing to limited data, we conducted a meta-analysis to re-evaluate the relationship between obesity and coronavirus-2019 (COVID-19). Literature published between 1 January 2020 and 22 August 2020 was comprehensively analysed, and RevMan3.5 was used for data analysis. A total of 50 studies, including data on 18 260 378 patients, were available. Obesity was associated with a higher risk of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV2) infection (odds ratio (OR): 1.39, 95% confidence interval (CI) 1.25-1.54; P < 0.00001) and increased severity of COVID-19 (hospitalisation rate: OR: 2.45, 95% CI 1.78-3.39; P < 0.00001; severe cases: OR: 3.74, 95% CI 1.18-11.87; P: 0.02; need for intensive care unit admission: OR: 1.30, 95% CI 1.21-1.40; P < 0.00001; need for invasive mechanical ventilation: OR: 1.59, 95% CI 1.35-1.88; P < 0.00001 and mortality: OR: 1.65, 95% CI 1.21-2.25; P: 0.001). However, we found a non-linear association between BMI and the severity of COVID-19. In conclusion, we found that obesity could increase the risk of SARS-CoV2 infection and aggregate the severity of COVID-19. Further studies are needed to explore the possible mechanisms behind this association.

C5a/C5aR pathway is essential for up-regulating SphK1 expression through p38-MAPK activation in acute liver failure.

AIM: To investigate the role of the complement 5a (C5a)/C5a receptor (C5aR) pathway in the pathogenesis of acute liver failure (ALF) in a mouse model. METHODS: BALB/c mice were randomly assigned to different groups, and intraperitoneal injections of lipopolysaccharide (LPS)/D-galactosamine (D-GalN) (600 mg/kg and 10 µg/kg) were used to induce ALF. The Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) levels, at different time points within a 1-wk period, were detected with a biochemistry analyzer. Pathological examination of liver tissue was performed 36 h after ALF induction. Serum complement 5 (C5), C5a, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, high-mobility group protein B1 (HMGB1) and sphingosine-1-phosphate levels were detected by enzyme-linked immunosorbant assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of C5aR, sphingosine kinase 1 (SphK1), p38-MAPK and p-p38-MAPK in liver tissue, peripheral blood mononuclear cells (PBMCs) and peritoneal exudative macrophages (PEMs) of mice or RAW 264.7 cells was analyzed by western blotting. C5aR mRNA levels were detected by quantitative real-time PCR. RESULTS: Activation of C5 and up-regulation of C5aR were observed in liver tissue and PBMCs of mice with ALF. Blockade of C5aR with a C5aR antagonist (C5aRa C5aRa) significantly reduced the levels of serum ALT, inflammatory cytokines (TNF-α, IL-1ß and IL-6) and HMGB1, as well as the liver tissue damage, but increased the survival rates (P < 0.01 for all). Blockade of C5aR decreased SphK1 expression in both liver tissue and PBMCs significantly at 0.5 h after ALF induction. C5aRa pretreatment significantly down-regulated the phosphorylation of p38-MAPK in liver tissues of ALF mice and C5a stimulated PEMs or RAW 264.7 cells. Moreover, inhibition of p38-MAPK activity with SB203580 reduced SphK1 protein production significantly in PEMs after C5a stimulation. CONCLUSION: The C5a/C5aR pathway is essential for up-regulating SphK1 expression through p38 MAPK activation in ALF in mice, which provides a potential immunotherapeutic strategy for ALF in patients.

Inhibition of sphingosine kinase 1 ameliorates acute liver failure by reducing high-mobility group box 1 cytoplasmic translocation in liver cells.

AIM: To determine the therapeutic potential of sphingosine kinase 1 (Sphk1) inhibition and its underlying mechanism in a well-characterized mouse model of D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver failure (ALF). METHODS: Balb/c mice were randomly assigned to different groups, with ALF induced by intraperitoneal injection of D-GaIN (600 mg/kg) and LPS (10 µg/kg). The Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at different time points within one week were determined using a multi-parametric analyzer. Serum high-mobility group box 1 (HMGB1), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, IL-10, and sphingosine-1-phosphate were detected by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after acute liver injury induction were assessed by hematoxylin and eosin staining. HMGB1 expression in hepatocytes and cytoplasmic translocation were detected by immunohistochemistry. Expression of Sphk1 in liver tissue and peripheral blood mononuclear cells (PBMCs) was analyzed by Western blot. RESULTS: The expression of Sphk1 in liver tissue and PBMCs was upregulated in GalN/LPS-induced ALF. Upregulated Sphk1 expression in liver tissue was mainly caused by Kupffer cells, the resident macrophages of the liver. The survival rates of mice in the N,N-dimethylsphingosine (DMS, a specific inhibitor of SphK1) treatment group were significantly higher than that of the control group (P < 0.001). DMS treatment significantly decreased the levels of serum ALT and AST at 6, 12, and 24 h compared with that of the control group (P < 0.01 for all). Serum HMGB1 levels at 6, 12, and 24 h, as well as serum TNF-α, IL-6, and IL-1ß levels at 12 h, were significantly lower in the DMS treatment group than in the control group (P < 0.01 for all). Furthermore, hepatic inflammation, necrosis, and HMGB1 cytoplasm translocation in liver cells were significantly decreased in the DMS treatment group compared to the control group (43.72% ± 5.51% vs 3.57% ± 0.83%, χ(2) = 12.81, P < 0.01). CONCLUSION: Inhibition of SphK1 ameliorates ALF by reducing HMGB1 cytoplasmic translocation in liver cells, and so might be a potential therapeutic strategy for this disease.

Liuweiwuling tablets attenuate acetaminophen-induced acute liver injury and promote liver regeneration in mice.

AIM: To explore the mechanism of protection against acetaminophen-induced acute liver injury by Liuweiwuling tablets. METHODS: Intraperitoneal injections of acetaminophen (250 mg/kg) were used to induce acute liver injury in male C57BL/6 mice. A total of 24 healthy mice were randomly assigned to two groups: an acute liver injury group (control group) and a Liuweiwuling tablet group. Mice were given Liuweiwuling tablets or a vehicle (PBS) orally prior to the administration of acetaminophen. Serum alanine aminotransferase (ALT) and aspartate aminotransaminase (AST) levels were measured at different time points within one week, and pathological examinations of liver tissues were performed 36 h after induction of acute liver injury. Serum inflammatory cytokines, such as high mobility group box protein B1 (HMGB1), tumor necrosis factor (TNF)-α and interleukin IL-1ß, were detected using an ELISA method according to the manufacturer's instructions. Hepatic morphological changes at 36 h were assessed by hematoxylin and eosin staining. Expression of proliferating cell nuclear antigen (PCNA) in liver tissue was determined by Western blot analysis. The mRNA levels of hepatocyte proliferation markers (PCNA, CyclinD1 and p21) were detected by real-time quantitative reverse transcription-polymerase chain reaction. RESULTS: The levels of ALT/AST in the Liuweiwuling tablet group were decreased significantly at 6, 12 and 24 h compared to that of the control group (654.38 ± 120.87 vs 1566.17 ± 421.64, 1154.18 ± 477.72 vs 4654.84 ± 913.71 and 935.13 ± 252.34 vs 4553.75 ± 727.37, P < 0.01). Serum HMGB1 levels at 6 and 12 h for the Liuweiwuling tablet group were significantly lower than those of the control group (23.49 ± 3.89 vs 58.6 ± 3.65, 61.62 ± 13.07 vs 27.32 ± 5.97, P < 0.01). Furthermore, serum TNF-α and IL-1ß levels at 12 h in the Liuweiwuling tablet group were also significantly lower than those of the control group (299.35 ± 50.61 vs 439.03 ± 63.59, 57.42 ± 12.98 vs 160.07 ± 49.87, P < 0.01). Centrilobular necrosis was evident in liver tissue of mice with acetaminophen-induced acute liver injury, but was almost abolished in the Liuweiwuling tablet group. The expression levels of PCNA and CyclinD1 were up-regulated in liver tissue in the Liuweiwuling tablet group (321.08 ± 32.87 vs 157.91 ± 21.52, 196.37 ± 25.39 vs 68.72 ± 11.27, P < 0.01); however, expression of p21 in liver tissue was down-regulated compared to that of the control group (40.26 ± 9.97 vs 138.24 ± 13.66, P < 0.01). CONCLUSION: Liuweiwuling tablets can attenuate acute liver injury by decreasing inflammatory cytokine (HMGB1, TNF-α and IL-1ß) levels and promoting liver regeneration.

Sphingosine kinase 1 dependent protein kinase C-δ activation plays an important role in acute liver failure in mice.

AIM: To investigate the role of protein kinase C (PKC)-δ activation in the pathogenesis of acute liver failure (ALF) in a well-characterized mouse model of D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced ALF. METHODS: BALB/c mice were randomly assigned to five groups, and ALF was induced in mice by intraperitoneal injection of D-GaIN (600 mg/kg) and LPS (10 µg/kg). Kaplan-Meier method was used for survival analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at different time points within one week were determined using a multiparameteric analyzer. Serum levels of high-mobility group box 1 (HMGB1), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-10 as well as nuclear factor (NF)-κB activity were determined by enzyme-linked immunosorbent assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of PKC-δ in liver tissue and peripheral blood mononuclear cells (PBMCs) was analyzed by Western blot. RESULTS: The expression and activation of PKC-δ were up-regulated in liver tissue and PBMCs of mice with D-GalN/LPS-induced ALF. Inhibition of PKC-δ activation with rottlerin significantly increased the survival rates and decreased serum ALT/AST levels at 6, 12 and 24 h compared with the control group (P < 0.001). Rottlerin treatment also significantly decreased serum levels of HMGB1 at 6, 12, and 24 h, TNF-α, IL-6 and IL-1 ß at 12 h compared with the control group (P < 0.01). The inflammatory cell infiltration and necrosis in liver tissue were also decreased in the rottlerin treatment group. Furthermore, sphingosine kinase 1 (SphK1) dependent PKC-δ activation played an important role in promoting NF-κB activation and inflammatory cytokine production in ALF. CONCLUSION: SphK1 dependent PKC-δ activation plays an important role in promoting NF-κB activation and inflammatory response in ALF, and inhibition of PKC-δ activation might be a potential therapeutic strategy for this disease.

Heterogeneous virulence of pandemic 2009 influenza H1N1 virus in mice.

BACKGROUND: Understanding the pathogenesis of influenza infection is a key factor leading to the prevention and control of future outbreaks. Pandemic 2009 Influenza H1N1 infection, although frequently mild, led to a severe and fatal form of disease in certain cases that make its virulence nature debatable. Much effort has been made toward explaining the determinants of disease severity; however, no absolute reason has been established. RESULTS: This study presents the heterogeneous virulence of clinically similar strains of pandemic 2009 influenza virus in human alveolar adenocarcinoma cells and mice. The viruses were obtained from patients who were admitted in a local hospital in China with a similar course of infection and recovered. The A/Nanchang/8002/2009 and A/Nanchang/8011/2009 viruses showed efficient replication and high lethality in mice while infection with A/Nanchang/8008/2009 was not lethal with impaired viral replication, minimal pathology and modest proinflammatory activity in lungs. Sequence analysis displayed prominent differences between polymerase subunits (PB2 and PA) of viral genomes that might correlate with their different phenotypic behavior. CONCLUSIONS: The study confirms that biological heterogeneity, linked with the extent of viral replication, exists among pandemic H1N1 strains that may serve as a benchmark for future investigations on influenza pathogenesis.

Genetic and clinical assessment of 2009 pandemic influenza in southern China.

INTRODUCTION: South China has a proven role in the global epidemiology of previous influenza outbreaks due to its dual seasonal pattern. We present the virologic, genetic and clinical characterization of pandemic H1N1 influenza infection (pH1N1) in Shantou and Nanchang, cities in southern China, during the second wave of the 2009-2010 pandemic. METHODOLOGY: Nasopharyngeal swabs were collected from 165 individuals with influenza-like illness (ILI) who presented to the hospitals in Shantou and Nanchang. Laboratory diagnosis and characterization was performed by real-time PCR, virus isolation in embryonated chicken eggs, and sequencing. RESULTS: pH1N1 activity was sustained in three different temporal patterns throughout the study period. The overall positivity rate of pH1N1 was 50% with major distribution among young adults between the ages of 13 and 30 years. High fever, cough, expectoration, chest pain, myalgia, nasal discharge and efficient viral replication were observed as major clinical markers whereas a substantial number of afebrile cases (17%) was also observed. Rate of hospitalization and disease severity (39%) and recovery (100%) were also high within the region. Furthermore, severe complications were likely to develop in young adults upon pH1N1 infection. Genetic characterization of the HA and NA genes of pH1N1 strains exhibited homogenous spread of pH1N1 strains with 99% identity with prototypic strains; however, minor unique mutations were also observed in the HA gene. CONCLUSION: The study illustrates the detailed characteristics of 2009 influenza pandemic in southern parts of China that might help to strategize preparedness for future pandemics and subsequent influenza seasons.

[The effects of different PAP domains on hepatitis B virus replication].

OBJECTIVE: To investigate the effects of different PAP domains on hepatitis B virus replication. METHODS: The full length and two truncated PAP mutants were cloned into a eukaryotic expression plasmid, and were transfected into HepG2.2.15 cells using lipofectamine 2000. 3 days after transfection, the medium and cells were collected. HBsAg and HBeAg were measured using ELISA. The titers of HBV DNA were quantified using fluorogenic quantitative PCR (FQ-PCR). HepG2 cells were used to determine the cytotoxicity of the plasmids transfection by MTT assays. RESULTS: The inhibitory effect on HBV replication of the C-terminal 25 amino acids deleted PAP mutant (pXF3H-PAP14) was not significantly different from that of the full length PAP (pXF3H-PAP12) (Chi-square test = 0.5, 2.0, 0.02, probability value more than 0.05), however, the cytotoxicity of pXF3H-PAP14 was lower than that of pXF3H-PAP12 (Chi-square test = 7.7, probability value less than 0.01). Both N-terminal 69 amino acids deleted mutant and C-terminal 25 amino acids deleted mutant had no cytotoxicity and no antiviral activity. CONCLUSION: C-terminal 25 amino acid of PAP is related to cytotoxicity but not related to antiviral activity of PAP. N-terminal 69 amino acid of PAP is related to the anti-HBV effect of PAP.

[Study of the effect of hepatitis C virus core protein on interferon-induced antiviral genes expression and its mechanisms].

To study the effect of HCV core protein on the interferon-induced antiviral genes expression and its mechanisms. Methods HepG2 cells were transiently transfected with HCV core protein expression plasmid and the blank plasmid respectively. RT-PCR was used to analyze the effect of HCV core protein on PKR and 2'-5'OAS expression. The effect of HCV core protein on ISRE-medicated gene expression was detected by luciferase activity assay. Western-blot assay was performed to observe the change of mRNA and protein levels of SOCS3, STAT1 and p-STAT1 following HCV core expression. In the presence of HCV core protein, the transcription of PKR and 2'-5' OAS are down-regulated. ISRE-medicated reporter gene expression and STAT1 phosphorylation were inhibited. The transcription and expression of SOCS3 were induced compared with blank plasmid-transfected group. In HepG2 cells, HCV core protein can down-regulate the expression of some interferon-induced antiviral genes, which involves the induction of SOCS3 and the inhibition of STAT1 phosphorylation.

N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication.

AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA. The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.

[Inhibition of hepatitis B and duck hepatitis B virus replication by APOBEC3G].

OBJECTIVE: To investigate the effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) mediated antiviral activity against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV). METHODS: Total RNA was extracted from peripheral blood mononuclear cells (PBMCs), RT-PCR product was cloned into the EcoR I/Hind III restriction sites of the CMV-driven expression vector fused with a hemagglutinin fusion epitope tag at its carboxyl terminal. Replication competent 1.3 fold over-length HBV was constructed with full-length HBV of ayw subtype. The mammalian hepatoma cell HepG2 was cotransfected with the replication competent 1.3 fold over-length HBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA, HBV DNA. RNA from intracellular core particles was examined using Northern and Southern blot analyses. Chicken hepatoma cell LMH was cotransfected with head-to-tail dimer of an EcoR I monomer of DHBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. DHBV DNA from intracellular core particles was examined using Southern blot analysis. RESULTS: CMV-driven expression vector encoding APOBEC3G-HA and replication competent 1.3 fold over-length HBV were constructed. There was a dose dependent decrease in the levels of intracellular core-associated viral (HBV and DHBV) DNA and extracellular production of HBsAg and HBeAg. Levels of intracellular core-associated viral RNA were also decreased, but the expression of HBcAg remained almost unchanged. CONCLUSION: APOBEC3G suppresses HBV and DHBV replication and also suppresses HBsAg and HBeAg expression.

Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo.

AIM: To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model. METHODS: The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. RESULTS: There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with in vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups. CONCLUSION: These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.

[Detection of mutants of the "a" determinant region of hepatitis B surface antigen S gene among Wuhan childhood patients].

OBJECTIVE: To investigate whether the presence of HBV mutant in vaccinees simply reflects the prevalence of HBV mutant in a specific geographic area or is indeed due to the immune pressure induced by vaccination. METHODS: HBV S genes were amplified by using polymerase chain reaction (PCR) and DNA sequence analysis of the "a" determinant was performed on sera from 30 childhood patients with immunoprophylaxis and 30 patients without vaccinations. RESULTS: Mutations of the "a" determinant were detected in 8 of the 60 patients. They were all of the adw subtype. The prevalence of amino acid substitutions as detected by direct sequencing was higher in those fully-vaccinated than of those not vaccinated. In all 8 vaccinated and also with detectable mutants, the mean age was older than the other vaccinated children. CONCLUSION: The prevalence of mutants is related to HBV subtypes and genotypes. Universal vaccination has accelerated an accumulation of HBsAg "a" determinant mutants with amino acid changes critical for immune escape in vaccinated children who became carriers. This suggests that new vaccination strategies should be considered.

[Distribution of hepatitis B virus genotypes in Hubei province and its clinical significance].

OBJECTIVE: To investigate the distribution of hepatitis B virus genotype in Hubei province (China) and its clinical significance. METHODS: Serum samples from 190 HBV DNA positive patients with chronic HBV infection,including 52 asymptomatic HBV carriers (ASC), 56 chronic hepatitis (CH), 32 fulminant hepatic failure (FHF), 22 liver cirrhosis (LC), and 28 hepatocellular carcinoma (HCC) patients were collected and tested for HBV genotypes by type-specific primers. RESULTS: A simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes of hepatitis B virus (HBV). Of the 190 patients, 140 (73.7%) were genotype B and 42 (22.1%) were genotype C. Genotype B was more prevalent in the FHF and HCC patients than in the ASC patients; the ALT value was significantly higher in genotype B than in genotype C patients. The rate of anti-HBe was significantly higher in genotype B than in genotype C except in the patients of the ASC group. CONCLUSION: The system we used seems to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys. Genotype B, genotype C and BC combination exist in Hubei province, and genotype B is the major genotype in this area especially in FHF and HCC patients.

Lethiferous effects of a recombinant vector carrying thymidine kinase suicide gene on 2.2.15 cells via a self-modulating mechanism.

AIM: To determine the lethiferous effects of a recombinant vector carrying thymidine kinase (TK) suicide gene on 2.2.15 cells and the possible self-modulating mechanism. METHODS: A self-modulated expressive plasmid pcDNA3-SCITK was constructed by inserting the fragments carrying hepatitis B virus antisense-S (HBV-anti-S) gene, hepatitis C virus core (HCV-C) gene, internal ribosome entry site (IRES) element of HCV and TK gene into the eukaryotic vector pcDNA3, in which the expression of TK suicide gene was controlled by the HBV S gene transcription. 2.2.15 cells that carry the full HBV genome and stably express series of HBV antigen were transfected with pcDNA3-SCITK or vector pcDNA3-SCI which was used as the mock plasmid. The HepG2 cells transfected with pcDNA3-SCITK were functioned as the negative control. All the transfected cells were incubated in DMEM medium supplemented with 10 microg/ml. of ganciclovir (GCV). The HBsAg levels in the supernatant of cell culture were detected by ELISA on the 1st, 3rd and 6th day post-transfection. Meanwhile, the morphology of transfected cells was recorded by the photograph and the survival cell ratio was assessed by the trypan blue exclusion test on the 6th day post-transfection. RESULTS: The structural accuracy of pcDNA3-SCITK was confirmed by restriction endonuclease digestion, PCR with specific primers and DNA sequencing. The HBsAg levels in the supernatant of transfected 2.2.15 cell culture were significantly decreased on the 6th day post-transfection as compared with that of the mock control (P<0.05). The lethiferous effect of pcDNA3-SCITK expression on 2.2.15 cells was initially noted on the 3rd day after transfection and aggravated on the 6th day post transfection, in which the majority of transfected 2.2.15 cells were observed shrunken, round in shape and even dead. With assessment by the trypan blue exclusion test, the survival cell ratio on the 6th day post transfection was 95% in the negative control and only 11% in the experimental group. CONCLUSION: The results indicate that suicide gene expression of pcDNA3-SCITK can only respond to HBV-S gene transcription, which may be potentially useful in the treatment of HBV infection and its related liver malignancies.

[Susceptibility of 570 Pseudomonas aeruginosa strains to 11 antimicrobial agents and the mechanism of its resistance to fluoroquinolones].

OBJECTIVE: To investigate the resistance of Pseudomonas aeruginosa (P. aeruginosa) to 11 antimicrobial agents and the mechanism of its resistance to fluoroquinolones (FQs). METHODS: The susceptibility of 570 strains of P. aeruginosa isolated clinically to 11 antimicrobial agents were detected by Kirby-Bauer disc diffusion method. The minimal inhibitory concentration (MIC) of 111 strains thereof to two FQs was determined by agar dilution method. 80 strains resistant to ciprofloxacin (MIC >or= 4 mg/L) were studied for the presence of point mutations in the gyrA and parC gene by direct sequencing and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method. RESULTS: The resistance rates of P. aeruginosa to cefepine, imipenem, amikacin, and ceftazidine, and aztreonam were 10.9%, 11.1%, 11.8%, 12.5%, and 16.6% respectively. The resistance rates to ciprofloxacin, levofloxacin, cefoperazone/sulbactam, and piperacillin were 29.2%, 32.9%, 23.5% and 33.7% respectively. The resistance rates to gentamycin and cefotaxime were 35.7% and 41.1% respectively. More than 60% of the ciprofloxacin resistant strains and the intensive care units (ICU) isolates were multidrug resistant. Among the 80 ciprofloxacin resistant strains, 66 (75%) had a mutation in gyrA codon: Thr83 (ACC)-->Ile (ATC), 52 strains (65%) had a mutation at parC codon: Ser87 (TCG)-->Leu (TTG), and 52 strains (65%) had both the above mentioned gyrA and parC mutations; while gyrA or parC mutation was not observed in the 31 ciprofloxacin susceptible strains. The MIC of ciprofloxacin for the 52 strains with mutations in both gyrA and parC genes was 19.80 mg/L +/- 2.11 mg/L, significantly higher than those for the 14 strains with mutation only in gyrA gene (11.88 mg/L +/- 2.73 mg/L, P < 0.05) and the 143 strains without gyrA and parC gene mutants (11.89 mg/L +/- 2.12 mg/L, P < 0.05). CONCLUSION: Resistance to antimicrobial agents of P. aeruginosa strains remains a problem. In particular, those ciprofloxacin resistant strains and ICU isolates are resistant to most of the antimicrobial agents. The resistance to fluoroquinolones of the clinical isolates of P. aeruginosa is mainly due to the mutations in gyrA and parC genes encoding the target enzyme of fluoroquinolones.