Jun Hou

DNA-based vaccination against hepatitis B virus using dissolving microneedle arrays adjuvanted by cationic liposomes and CpG ODN

Abstract DNA vaccines are simple to produce and can generate strong cellular and humoral immune response, making them attractive vaccine candidates. However, a major shortcoming of DNA vaccines is their poor immunogenicity when administered intramuscularly. Transcutaneous immunization (TCI) via microneedles is a promising alternative delivery route to enhance the vaccination efficacy. A novel dissolving microneedle array (DMA)-based TCI system loaded with cationic liposomes encapsulated with hepatitis B DNA vaccine and adjuvant CpG ODN was developed and characterized. The pGFP expression in mouse skin using DMA was imaged over time. In vivo immunity tests in mice were performed to observe the capability of DMA to induce immune response after delivery of DNA. The results showed that pGFP could be delivered into skin by DMA and expressed in skin. Further, the amount of expressed GFP was likely to peak at day 4. The immunity tests showed that the DMA-based DNA vaccination could induce effective immune response. CpG ODN significantly improved the immune response and achieved the shift of immune type from predominate Th2 type to a balance Th1/Th2 type. The cationic liposomes could further improve the immunogenicity of DNA vaccine. In conclusion, the novel DMA-based TCI system can effectively deliver hepatitis B DNA vaccine into skin, inducing effective immune response and change the immune type by adjuvant CpG ODN.

Quorum sensing modulates transcription of cpsQ-mfpABC and mfpABC in Vibrio parahaemolyticus

Vibrio parahaemolyticus AphA and OpaR are the two master regulators of quorum sensing (QS) that are abundantly produced and operate at low cell density (LCD) and high cell density (HCD), respectively, with an outcome of reciprocally gradient production of these two proteins with transition between LCD and HCD. The cpsQ-mfpABC gene cluster is transcribed as two operons cpsQ-mfpABC and mfpABC in V. parahaemolyticus. MfpABC is a putative membrane fusion transporter that contributes to biofilm development. CpsQ is a c-di-GMP-binding regulator that activates the expression of capsular polysaccharide genes and mfpABC and, thus, induces biofilm development. As shown in this study, OpaR and AphA bind to the promoter region of mfpABC to enhance and repress its transcription, respectively. In contrast, the positive and negative regulation of cpsQ-mfpABC by AphA and OpaR, respectively, achieves probably through acting of AphA or OpaR on additional unknown regulator(s) of cpsQ-mfpABC. The transcriptional levels of cpsQ-mfpABC and mfpABC enhance gradually with transition from LCD to HCD due to the above reciprocal regulatory action of OpaR and AphA. Data presented here present a novel paradigm of combined action of the two master QS regulators in controlling expression of the QS regulon members.

Autoantibodies in Chinese patients with chronic hepatitis B: Prevalence and clinical associations

AIM To investigate the prevalence of autoantibodies and their associations with clinical features in Chinese patients with chronic hepatitis B (CHB). METHODS A total of 325 Chinese patients with CHB were enrolled in this retrospective, hospital-based study. Patients with chronic hepatitis C (CHC), autoimmune hepatitis (AIH), or primary biliary cirrhosis (PBC) were included, with healthy donors acting as controls. A panel of autoantibodies that serologically define AIH and PBC was tested by indirect immunofluorescence assay and line immunoassay. The AIH-related autoantibody profile included homogeneous anti-nuclear antibodies (ANA-H), smooth-muscle antibodies, anti-liver kidney microsome type 1, anti-liver cytosolic antigen type 1, and anti-soluble liver antigen/liver pancreas; the PBC-related antibodies were characterized by ANA-nuclear dots/membranous rim-like, anti-mitochondrial antibodies-M2 (AMA-M2), anti-BPO (recombinant antigen targeted by AMA-M2), anti-Sp100, anti-promyelocytic leukemia protein (anti-PML), and anti-gp210. The dichotomization of clustering was used to unequivocally designate the AIH or PBC profiles for each case. Anti-Ro52 antibodies were also tested. RESULTS The prevalence of any autoantibody in CHB amounted to 58.2%, which was similar to the 66.2% prevalence in CHC, significantly higher than the 6.7% in the healthy controls (P < 0.001), and lower than the 100% found in AIH and PBC (P = 0.004 and P < 0.001, respectively). There were more anti-PML and anti-gp210 antibodies among the CHB patients than the CHC patients (11.1% vs 0%, P = 0.003; 12.6% vs 0%, P < 0.001, respectively). The prevalence and titer of AMA, anti-BPO, anti-PML, and anti-gp210 were higher in PBC than in those with CHB. Among the CHB patients, the prevalence of ANA, especially ANA-H, was significantly lower in patients with compensated and decompensated cirrhosis compared with patients without cirrhosis. Thirty-eight cases of hepatocellular carcinoma (HCC) in CHB showed a significant difference compared with non-HCC patients in the prevalence of anti-PML (0% vs 12.5%, P = 0.013). Dichotomization of the autoantibodies revealed that the PBC profile was more prevalent in patients with CHB than in those with CHC, and that it was strongly correlated with both compensated and decompensated cirrhosis. In contrast, the prevalence of the AIH profile was significantly higher in non-cirrhosis patients with CHB than in those with compensated cirrhosis (18.5% vs 8.2%, P = 0.039). Moreover, the AIH profile was also closely associated with hepatitis B e-antigen positivity. CONCLUSION ANA-H could be an indicator of early-stage CHB. Dichotomizing the autoantibody profiles revealed that the PBC profile is strongly associated with cirrhosis in CHB.

Cell Density- and Quorum Sensing-Dependent Expression of Type VI Secretion System 2 in Vibrio parahaemolyticus

Background Vibrio parahaemolyticus AphA and OpaR are the two master quorum sensing (QS) regulators that are abundantly expressed at low cell density (LCD) and high cell density (HCD), respectively, with a feature of reciprocally gradient production of them with transition between LCD and HCD. The type VI secretion system 2 (T6SS2) gene cluster can be assigned into three putative operons, namely VPA1027-1024, VPA1043-1028, and VPA1044-1046. T6SS2 contributes to adhesion of V. parahaemolyticus to host cells. Methodology/Principal Findings OpaR box-like sequences were found within the upstream promoter regions of all the above three operons, while none of AphA box-like elements could be identified for them. The subsequent primer extension, LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays disclosed that OpaR bound to the promoter regions of these three operons to stimulate their transcription, while AphA negatively regulated their transcription most likely through acting on OpaR. This regulation led to a gradient increase of T6SS2 transcription with transition from LCD to HCD. Conclusions/Significance V. parahaemolyticus OpaR and AphA positively and negatively regulate T6SS2 expression, respectively, leading to a gradient elevation of T6SS2 expression with transition from LCD to HCD. T6SS2 genes are thus assigned as the QS regulon members in V. parahaemolyticus.

Immunologic changes during Pandemic (H1N1) 2009, China.

We analyzed changes in immunologic values over time for 28 hospitalized patients with pandemic (H1N1) 2009. Levels of interleukin-6, interferon-y, and interleukin-10 increased 1 day after illness onset and then decreased to baseline levels. Levels of virus-specific antibody were undetectable 1 day after illness onset and peaked 36 days later.

Dissemination of KPC-2-Encoding IncX6 Plasmids Among Multiple Enterobacteriaceae Species in a Single Chinese Hospital

Forty-five KPC-producing Enterobacteriaceae strains were isolated from multiple departments in a Chinese public hospital from 2014 to 2015. Genome sequencing of four representative strains, namely Proteus mirabilis GN2, Serratia marcescens GN26, Morganella morganii GN28, and Klebsiella aerogenes E20, indicated the presence of blaKPC-2-carrying IncX6 plasmids pGN2-KPC, pGN26-KPC, pGN28-KPC, and pE20-KPC in the four strains, respectively. These plasmids were genetically closely related to one another and to the only previously sequenced IncX6 plasmid, pKPC3_SZ. Each of the plasmids carried a single accessory module containing the blaKPC-2/3-carrying ΔTn6296 derivatives. The ΔTn6292 element from pGN26-KPC also contained qnrS, which was absent from all other plasmids. Overall, pKPC3_SZ-like blaKPC-carrying IncX6 plasmids were detected by PCR in 44.4% of the KPC-producing isolates, which included K. aerogenes, P. mirabilis, S. marcescens, M. morganii, Escherichia coli, and Klebsiella pneumoniae, and were obtained from six different departments of the hospital. Data presented herein provided insights into the genomic diversity and evolution of IncX6 plasmids, as well as the dissemination and epidemiology of blaKPC-carrying IncX6 plasmids among Enterobacteriaceae in a hospital setting.

Population pharmacokinetics of peginterferon α2a in patients with chronic hepatitis B

There were significant differences in response and pharmacokinetic characteristics to the peginterferon α2a treatment among Chronic Hepatitis B (CHB) patients. The aim of this study is to identify factors which could significantly impact the peginterferon α2a pharmacokinetic characteristics in CHB patients. There were 208 blood samples collected from 178 patients who were considered as CHB and had been treated with peginterferon α2a followed by blood concentration measurement and other laboratory tests. The covariates such as demographic and clinical characteristics of the patients were retrieved from medical records. Nonlinear mixed-effects modeling method was used to develop the population pharmacokinetic model with NONMEM software. A population pharmacokinetic model for peginterferon α2a has been successfully developed which shows that distribution volume (V) was associated with body mass index (BMI), and drug clearance (CL) had a positive correlation with creatinine clearance (CCR). The final population pharmacokinetic model supports the use of BMI and CCR-adjusted dosing in hepatitis B virus patients.

Nucleoside analogs treatment delay the onset of hepatocellular carcinoma in patients with HBV-related cirrhosis

Whether Nucleoside analogs (NA) treatment can delay the onset of HCC remains unclear. We retrospectively analyzed the clinical data of patients with HBV-related cirrhosis and HCC from 2000 to 2012. Cox proportional hazards model was used to explore the association between NA treatment and postponement of HCC development, the dependent variable was time interval from cirrhosis treatment towards the onset of HCC, and the covariates included age, sex, family history, compensation status at baseline. A total of 1155 HCC patients treated with NAs (n = 528, lamivudine, adefovir, entecavir) and non NA (n = 627) for more than 24 months before the occurrence of HCC were incorporated into the cohort. Compared with the non-NA group, NAs therapy was associated with delaying the onset of HCC in patients with cirrhosis. Significant factors were: adefovir treatment (n = 181; p = 0.0072; HR: 0.792; 90% CI: 0.687-0.914), entecavir treatment (n = 83; p = 0.0068; HR: 0.716; 90% CI: 0.585-0.877), lamivudine switched to adefovir treatment (n = 95, p = 0.0808; HR: 0.822; 90% CI: 0.684 to 0.989). But Lamivudine monotherapy was not a significant factor (n = 102; p = 0.6877; HR: 1.045; 90% CI: 0.873-1.250). Long-term NA treatment (> 6 months, except for lamivudine monotherapy) can delay the onset of HCC in patients with HBV-related cirrhosis, and applying high barrier NA to resistance is important in these patients.Whether Nucleos(t)ide analogs(NA) treatment can delay the onset of HCC remains unclear. We retrospectively analyzed the clinical data of patients with HBV-related cirrhosis and HCC from 2000 to 2012. Cox proportional hazards model was used to explore the association between NA treatment and postponement of HCC development, the dependent variable was time interval from cirrhosis treatment towards the onset of HCC, and the covariates included age, sex, family history, compensation status at baseline. A total of 1155 HCC patients treated with NAs (n = 528, lamivudine, adefovir, entecavir) and non NA (n = 627) for more than 24 months before the occurrence of HCC were incorporated into the cohort. Compared with the non-NA group, NAs therapy was associated with delaying the onset of HCC in patients with cirrhosis. Significant factors were: adefovir treatment (n = 181; p = 0.0072; HR: 0.792; 90% CI: 0.687–0.914), entecavir treatment (n = 83; p = 0.0068; HR: 0.716; 90% CI: 0.585-0.877), lamivudine switched to adefovir treatment (n = 95, p = 0.0808; HR: 0.822; 90% CI: 0.684 to 0.989). But Lamivudine monotherapy was not a significant factor (n = 102; p = 0.6877; HR: 1.045; 90% CI: 0.873–1.250). Long-term NA treatment (> 6 months, except for lamivudine monotherapy) can delay the onset of HCC in patients with HBV-related cirrhosis, and applying high barrier NA to resistance is important in these patients.

Serum soluble CD40 is associated with liver injury in patients with chronic hepatitis B

Soluble cluster of differentiation 40 (sCD40) is proteolytically cleaved from membrane-bound CD40 and binds to CD154, thereby inhibiting CD40-CD154-mediated immune responses. The aim of the present study was to clarify the role of sCD40 in chronic hepatitis B (CHB). The sCD40 levels in sera from 132 patients with CHB and 33 healthy individuals were retrospectively measured. sCD40 concentrations in patients with CHB were higher than those in healthy controls, and sCD40 levels correlated positively with serum levels of the liver dysfunction biomarkers alanine transaminase (ALT) and aspartate transaminase (AST). sCD40 concentrations increased with a rise in the severity of liver necroinflammation and fibrosis. Patients with >75% liver tissue staining positive for hepatitis B virus (HBV) antigen expression showed significantly lower sCD40 levels than those who stained negative for the HBV antigen. The area under the receiver operating characteristic curve of sCD40 was greater than that of ALT and AST; thus, sCD40 levels have a high diagnostic accuracy for detecting severe liver inflammation in patients with CHB, and could serve as an immunological marker of hepatic tissue injury.