Large Surface Antigen Research Articles

Large HBV Surface Protein-Induced Unfolded Protein Response Dynamically Regulates p27 Degradation in Hepatocellular Carcinoma Progression.

Up to 50% of hepatocellular carcinoma (HCC) is caused by hepatitis B virus (HBV) infection, and the surface protein of HBV is essential for the progression of HBV-related HCC. The expression of large HBV surface antigen (LHB) is presented in HBV-associated HCC tissues and is significantly associated with the development of HCC. Gene set enrichment analysis revealed that LHB overexpression regulates the cell cycle process. Excess LHB in HCC cells induced chronic endoplasmic reticulum (ER) stress and was significantly correlated with tumor growth in vivo. Cell cycle analysis showed that cell cycle progression from G1 to S phase was greatly enhanced in vitro. We identified intensive crosstalk between ER stress and cell cycle progression in HCC. As an important regulator of the G1/S checkpoint, p27 was transcriptionally upregulated by transcription factors ATF4 and XBP1s, downstream of the unfolded protein response pathway. Moreover, LHB-induced ER stress promoted internal ribosome-entry-site-mediated selective translation of p27, and E3 ubiquitin ligase HRD1-mediated p27 ubiquitination and degradation. Ultimately, the decrease in p27 protein levels reduced G1/S arrest and promoted the progress of HCC by regulating the cell cycle.

Pre-S1 Mutations as Indicated by Serum Pre-S1 Antigen Negative is Associated with an Increased Risk of Hepatocellular Carcinoma in Chronic Hepatitis B Patients.

Pre-S1 antigen (pre-S1) is a component of hepatitis B virus large surface antigen (L-HBsAg). This study aimed to investigate the association between clinical pre-S1 antigen (pre-S1) status and adverse prognostic events in chronic hepatitis B (CHB) patients. This study retrospectively enrolled 840 CHB patients with comprehensive clinical data, including 144 patients with multiple follow-up of pre-S1 status. All patients were tested for serum pre-S1 and divided into pre-S1 positive and negative groups. Single factor and logistic multiple regression analyses were performed to explore the association between pre-S1 and other HBV biomarkers with the risk of hepatocellular carcinoma (HCC) in CHB patients. The pre-S1 region sequences of HBV DNA were obtained from one pre-S1 positive and two pre-S1 negative treatment-naïve patients using polymerase chain reaction (PCR) amplification followed by Sanger sequencing. The quantitative HBsAg level was significantly higher in the pre-S1 positive group than that in the pre-S1 negative group (Z=-15.983, P<0.001). The positive rate of pre-S1 increased significantly with the increase in HBsAg level (χ 2=317.963, P<0.001) and HBV DNA load (χ 2=15.745, P<0.001). The pre-S1 negative group had a higher HCC risk than the pre-S1 positive group (Z=-2.00, P=0.045, OR=1.61). Moreover, patients in the sustained pre-S1 negative group had a higher HCC risk (Z=-2.56, P=0.011, OR=7.12) than those in the sustained pre-S1 positive group. The sequencing results revealed mutations in the pre-S1 region from samples of pre-S1 negative patients, including frameshift and deletion mutations. Pre-S1 is a biomarker that indicates the presence and replication of HBV. Pre-S1 sustained negativity attributed to pre-S1 mutations in CHB patients may be associated with a higher risk of HCC, which has clinical significance and warrant further investigations.

Hepatitis B and Hepatitis D Viruses: A Comprehensive Update with an Immunological Focus.

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) are highly prevalent viruses estimated to infect approximately 300 million people and 12-72 million people worldwide, respectively. HDV requires the HBV envelope to establish a successful infection. Concurrent infection with HBV and HDV can result in more severe disease outcomes than infection with HBV alone. These viruses can cause significant hepatic disease, including cirrhosis, fulminant hepatitis, and hepatocellular carcinoma, and represent a significant cause of global mortality. Therefore, a thorough understanding of these viruses and the immune response they generate is essential to enhance disease management. This review includes an overview of the HBV and HDV viruses, including life cycle, structure, natural course of infection, and histopathology. A discussion of the interplay between HDV RNA and HBV DNA during chronic infection is also included. It then discusses characteristics of the immune response with a focus on reactions to the antigenic hepatitis B surface antigen, including small, middle, and large surface antigens. This paper also reviews characteristics of the immune response to the hepatitis D antigen (including small and large antigens), the only protein expressed by hepatitis D. Lastly, we conclude with a discussion of recent therapeutic advances pertaining to these viruses.

Proteogenomic identification of Hepatitis B virus (HBV) genotype-specific HLA-I restricted peptides from HBV-positive patient liver tissues.

The presentation of virus-derived peptides by HLA class I molecules on the surface of an infected cell and the recognition of these HLA-peptide complexes by, and subsequent activation of, CD8+ cytotoxic T cells provides an important mechanism for immune protection against viruses. Recent advances in proteogenomics have allowed researchers to discover a growing number of unique HLA-restricted viral peptides, resulting in a rapidly expanding repertoire of targets for immunotherapeutics (i.e. bispecific antibodies, engineered T-cell receptors (TCRs), chimeric antigen receptor T-cells (CAR-Ts)) to infected tissues. However, genomic variability between viral strains, such as Hepatitis-B virus (HBV), in combination with differences in patient HLA alleles, make it difficult to develop therapeutics against these targets. To address this challenge, we developed a novel proteogenomics approach for generating patient-specific databases that enable the identification of viral peptides based on the viral transcriptomes sequenced from individual patient liver samples. We also utilized DNA sequencing of patient samples to identify HLA genotypes and assist in target selection. Liver samples from 48 HBV infected patients, primarily from Asia, were examined to reconstruct patient-specific HBV genomes, identify regions within the human chromosomes targeted by HBV integrations and obtain a comprehensive view of HBV peptide epitopes using our HLA class-I (HLA-I) immunopeptidomics discovery platform. Two previously reported HLA associated HBV-derived peptides, HLA-A02 binder FLLTRILTI (S194-202) from the large surface antigen and HLA-A11 binder STLPETTVVRR (C141-151) from the capsid protein were validated by our discovery platform, but both were detected at very low frequencies. In addition, we identified and validated, using heavy peptide analogues, novel strain-specific HBV-HLA associated peptides, such as GSLPQEHIVQK (P606-616) and variants. Overall, our novel approach can guide the development of bispecific antibody, TCR-T, or CAR-T based therapeutics for the treatment of HBV-related HCC and inform vaccine development.

Chemical-induced degradation of PreS2 mutant surface antigen via the induction of microautophagy

Naturally evolved immune-escape PreS2 mutant is an oncogenic caveat of liver cirrhosis and hepatocellular carcinoma (HCC) during chronic hepatitis B virus (HBV) infection. PreS2 mutant is prevalent in above 50% of patients with HCC. In addition, intrahepatic expression of PreS2 mutant large surface antigen (PreS2-LHBS) induces endoplasmic reticulum stress, mitochondria dysfunction, cytokinesis failure, and subsequent chromosome hyperploidy. As PreS2-LHBS has no enzymatic activity, the development of PreS2-specific inhibitors can be challenging. In this study, we aim to identify inhibitors of PreS2-LHBS via the induction of protein-specific degradation. We set up a large-scale protein stability reporter platform and applied an FDA-approved drug library for the screening. We identified ABT199 as a negative modulator of PreS2-LHBS, which induced the degradation of PreS2-LHBS without affecting the general cell viability in both hepatoma and immortalized hepatocytes. Next, by affinity purification screening, we found that PreS2-LHBS interacted with HSC70, a microautophagy mediating chaperone. Simultaneously, inhibitions of lysosomal degradation or microautophagy restored the expression of PreS2-LHBS, suggesting microautophagy is involved in ABT199-induced PreS2-LHBS degradation. Notably, a 24-hr treatment of ABT199 was sufficient for the reduction of DNA damage and cytokinesis failure in PreS2-LHBS expressing hepatocytes. In addition, a persistent treatment of ABT199 for 3 weeks reversed chromosome hyperploidy in PreS2-LHBS cells and suppressed anchorage-independent growth of HBV-positive hepatoma cells. Together, this study identified ABT-199 as a negative modulator of PreS2-LHBS via mediating microautophagy. Our results indicate that long-term inhibition of PreS2-LHBS may serve as a novel strategy for the therapeutic prevention of HBV-mediated HCC.

The role of hepatitis B virus surface proteins in regulating the maturation and secretion of complete and incomplete virions.

The expression of various forms of hepatitis B virus (HBV) surface proteins regulates the release of mature virion, but whether they affect the release of other incomplete viral particles, such as naked capsid, is not clear. Here, by stable overexpression of large or middle/small hepatitis B surface proteins (LHBs, M/SHBs) in HepAD38 cells, we evaluated their effects on the release of complete and incomplete viral particles. Overproduction of LHBs inhibited the release of all surface proteins, which increased the ratio of naked capsids/virions. This effect was accompanied by the elevated extracellular HBV RNA. On the other hand, overexpression of M/SHBs greatly improved the secretion of enveloped viral and subviral particles. In situ visualization of viral DNA and LHBs revealed intracellular retention of mature virions when LHBs were overexpressed. These results indicate that the molecular decision on secretion of enveloped or unenveloped viral particles is modulated by the intracellular ratio of large, middle and small surface antigens. This mechanism may be relevant in the progression and resolution of HBV-induced chronic liver disease.

Hepatitis B virus middle surface antigen loss promotes clinical variant persistence in mouse models

ABSTRACT Hepatitis B virus (HBV) middle surface antigen (MHBs) mutation or deletion occurs in patients with chronic HBV infection. However, the functional role of MHBs in HBV infection is still an enigma. Here, we reported that 7.33% (11/150) isolates of CHB patients had MHBs start codon mutations compared with 0.00% (0/146) in acute hepatitis B (AHB) patients. Interestingly, MHBs loss accounted for 11.88% (126/1061) isolates from NCBI GenBank, compared with 0.09% (1/1061) and 0.00% (0/1061) for HBV large surface antigen (LHBs) loss and HBV small surface antigen (SHBs) loss, respectively. One persistent HBV clone of genotype B (B56, MHBs loss) from a CHB patient was hydrodynamically injected into BALB/c mice. B56 persisted for >70 weeks in BALB/c mice, whereas B56 with restored MHBs (B56M+) was quickly cleared within 28 days. Serum cytokine assays demonstrated that CXCL1, CXCL2, IL-6 and IL-33 were significantly increased during rapid HBV clearance in B56M+ mice. Furthermore, the enhancers and promoters of B56 were proved to be required for B56 persistence in mice. Ablating MHBs expression improved the persistence of a new clone (HBV1.3, genotype B) which was recreated by using enhancers and promoters of B56. These data demonstrated that MHBs deletion can promote the persistence of specific HBV variants in a hydrodynamic mouse model. MHBs re-expression restored a rapid clearance of HBV, which was accompanied by cytokine responses including the elevation of CXCL1, CXCL2, IL-6 and IL-33.

713d Downregulation of Apolipoprotein H Induce Hepatocyte Steatosis to Promote Gut Microbiota Dysbiosis in Chronic Hepatitis B Infection

Apolipoprotein H (apoH) is produced by hepatocytes and also an acute phase protein in hepatitis B virus (HBV) infection. We found HBV and the large surface antigen upregulate apoH expression, which inhibits HBsAg secretion and then induces ER stress. ApoH is also found on several classes of lipoproteins, and is involved in lipid metabolism. However, the possible mechanisms behind the relationship between steatosis and HBV infection are unclear. Herein, we explore whether apoH regulates the development of fatty liver in chronic HBV infection.

Aerobic glycolysis supports hepatitis B virus protein synthesis through interaction between viral surface antigen and pyruvate kinase isoform M2.

As an intracellular pathogen, the reproduction of the hepatitis B virus (HBV) depends on the occupancy of host metabolism machinery. Here we test a hypothesis if HBV may govern intracellular biosynthesis to achieve a productive reproduction. To test this hypothesis, we set up an affinity purification screen for host factors that interact with large viral surface antigens (LHBS). This identified pyruvate kinase isoform M2 (PKM2), a key regulator of glucose metabolism, as a binding partner of viral surface antigens. We showed that the expression of viral LHBS affected oligomerization of PKM2 in hepatocytes, thereby increasing glucose consumption and lactate production, a phenomenon known as aerobic glycolysis. Reduction of PKM2 activity was also validated in several different models, including HBV-infected HepG2-NTCP-C4 cells, adenovirus mediated HBV gene transduction and transfection with a plasmid containing complete HBV genome on HuH-7 cells. We found the recovery of PKM2 activity in hepatocytes by chemical activators, TEPP-46 or DASA-58, reduced expressions of viral surface and core antigens. In addition, reduction of glycolysis by culturing in low-glucose condition or treatment with 2-deoxyglucose also decreased expressions of viral surface antigen, without affecting general host proteins. Finally, TEPP-46 largely suppressed proliferation of LHBS-positive cells on 3-dimensional agarose plates, but showed no effect on the traditional 2-dimensional cell culture. Taken together, these results indicate that HBV-induced metabolic switch may support its own translation in hepatocytes. In addition, aerobic glycolysis is likely essential for LHBS-mediated oncogenesis. Accordingly, restriction of glucose metabolism may be considered as a novel strategy to restrain viral protein synthesis and subsequent oncogenesis during chronic HBV infection.

E-cadherin Plays a Role in Hepatitis B Virus Entry Through Affecting Glycosylated Sodium-Taurocholate Cotransporting Polypeptide Distribution.

Hepatitis B virus (HBV) infection is a major cause of chronic liver disease and hepatocellular carcinoma. Current antiviral therapy does not effectively eradicate HBV and further investigations into the mechanisms of viral infection are needed to enable the development of new therapeutic agents. The sodium-taurocholate cotransporting polypeptide (NTCP) has been identified as a functional receptor for HBV entry in liver cells. However, the NTCP receptor is not sufficient for entry and other membrane proteins contribute to modulate HBV entry. This study seeks to understand how the NTCP functions in HBV entry. Herein we show that knockdown of the cell-cell adhesion molecule, E-cadherin significantly reduced infection by HBV particles and entry by HBV pseudoparticles in infected liver cells and cell lines. The glycosylated NTCP localizes to the plasma membrane through interaction with E- cadherin, which increases interaction with the preS1 portion of the Large HBV surface antigen. Our study contributes novel insights that advance knowledge of HBV infection at the level of host cell binding and viral entry.

Retraction for Sung and Lai, "Murine Retroviral Pseudotype Virus Containing Hepatitis B Virus Large and Small Surface Antigens Confers Specific Tropism for Primary Human Hepatocytes: a Potential Liver-Specific Targeting System".

Retraction for Sung and Lai, "Murine Retroviral Pseudotype Virus Containing Hepatitis B Virus Large and Small Surface Antigens Confers Specific Tropism for Primary Human Hepatocytes: a Potential Liver-Specific Targeting System".

Optimisation of an in vitro cell system using pseudoviruses to investigate HBV entry mechanisms

Aim To explore the roles of large (L), middle (M) and small (S) surface antigens in an in vitro model of hepatitis B virus (HBV) entry, and thereby to achieve an optimal in vitro cell system using pseudoviruses to investigate HBV entry mechanisms. Methods RNA encoding sodium taurocholate co-transporting polypeptide (NTCP) was extracted from human hepatocytes and cloned into the pHIV-EGFP expression vector. The resulting pHIV-NTCP-EGFP construct was delivered into Huh7 hepatoma cells with the aid of pCMVR87.4 (packaging vector) and pCMV.VSV.G (glycoprotein), subsequently, a cell line over-expressing NTCP was generated. Meanwhile, by silencing start codons at L, M or S, seven constructs were obtained, i.e. L + M -S-, L-M + S -, l -M-S+, L + M + S-, L-M + S +, L + M -S+, l -M-S-. A matrix with various amounts of the seven constructs was used to generate HBV pseudoparticles (HBVpp) using a Luciferase-based HIV (pNL4.3.luc.R-E-) pseudotype entry model system. The infectivity of the HBVpp was tested in NTCP naïve and NTCP over-expressing Huh7 hepatoma cells. Results The relative amounts of L, M and S were critical in determining the efficiency of entry of HBVpp into NTCP + ve Huh7 cells. Conclusions Creation of NTCP-over expressing cells together with optimisation of conditions to maximise HBVpp entry provides an important tool to investigate the entry step in the HBV life cycle, and may allow identification of non-NTCP-dependent viral entry pathways.

MiR-185-5p suppresses HBV gene expression by targeting ELK1 in hepatoma carcinoma cells

AimsTo investigate the role and underlying mechanism of miR-185-5p in hepatitis B virus (HBV) expression and replication. Main methodsThe relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay (ELISA). The HBV DNA copies in the cultures medium were measured by RT-qPCR. The HBV large surface antigen promoter (S1p) activity was analyzed by luciferase reporter assay. The target relationship between miR-185-5p and ELK1 was identified by bioinformatics analysis and EGFP fluorescent reporter assay. The ELK1 expression was determined by RT-qPCR and Western blot. Key findingsmiR-185-5p significantly decreased HBV large surface antigen promoter activity and subsequently the production of HBV proteins and HBV DNA copies in vitro. Further, we identified the ETS transcription factor ELK1 is a target of miR-185-5p. Overexpression and knockdown experiments showed overexpression of ELK1 stimulated HBV large surface antigen promoter activity and promoted the production of HBV proteins and HBV DNA copies, whereas knockdown of ELK1 has the opposite effects. Moreover, the rescue of ELK1 expression reversed the suppression of miR-185-5p on HBV replication and gene expression. Further mechanistic study showed that the ETS binding sites within the HBV large surface antigen promoter are required for the repression effect of miR-185-5p on HBV. SignificanceThere are few reports about the interaction between miRNAs and the transcription from HBV S1p, we found that miR-185-5p decreases HBV S1p activity by targeting ELK1, which may provide a promising therapeutic strategy for HBV infection.

A Next-Generation Sequencing-Based Platform for Quantitative Detection of Hepatitis B Virus Pre-S Mutants in Plasma of Hepatocellular Carcinoma Patients

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Early diagnosis and treatment of HCC remain a key goal for improving patient survival. Chronic hepatitis B virus (HBV) infection is a major risk factor for HCC development. Pre-S mutants harboring deletions in HBV large surface antigen have been well demonstrated as HBV oncoproteins that dysregulate multiple signaling pathways in hepatocytes, leading to HCC formation. The presence of pre-S mutants in plasma represents important predictive and prognostic markers for HCC in patients with chronic HBV infection. However, the method to detect pre-S mutants remains to be optimized. In this study, we developed a platform, based on the next-generation sequencing (NGS) technology, for detection of pre-S mutants in plasma of HBV-related HCC patients. Compared to the current TA cloning-based analysis, the NGS-based analysis could detect pre-S deletion quantitatively, and the detection rate was significantly more sensitive in 49 plasma analyzed (McNemar’s paired proportion test, P value < 0.0001; simple kappa coefficient, κ = 0.29 (95% CI, 0.12 to 0.46)). Our data suggest that the NGS-based platform may hold a promise for improving the clinical application of pre-S mutants in serving as predictive and prognostic markers for HBV-related HCC.

Intrahepatic hepatitis B virus large surface antigen induces hepatocyte hyperploidy via failure of cytokinesis.

Hepatitis B virus (HBV) is an aetiological factor for liver cirrhosis and hepatocellular carcinoma (HCC). Despite current antiviral therapies that successfully reduce the viral load in patients with chronic hepatitis B, persistent hepatitis B surface antigen (HBsAg) remains a risk factor for HCC. To explore whether intrahepatic viral antigens contribute directly to hepatocarcinogenesis, we monitored the mitotic progression of HBV-positive cells. Cytokinesis failure was increased in HBV-positive HepG2.2.15 and 1.3ES2 cells, as well as in HuH-7 cells transfected with a wild-type or X-deficient HBV construct, but not in cells transfected with an HBsAg-deficient construct. We show that expression of viral large surface antigen (LHBS) was sufficient to induce cytokinesis failure of immortalized hepatocytes. Premitotic defects with DNA damage and G2 /M checkpoint attenuation preceded cytokinesis in LHBS-positive cells, and ultimately resulted in hyperploidy. Inhibition of polo-like kinase-1 (Plk1) not only restored the G2 /M checkpoint in these cells, but also suppressed LHBS-mediated in vivo tumourigenesis. Finally, a positive correlation between intrahepatic LHBS expression and hepatocyte hyperploidy was detected in >70% of patients with chronic hepatitis B. We conclude that HBV LHBS provokes hyperploidy by inducing DNA damage and upregulation of Plk1; the former results in atypical chromatin structures, and the latter attenuates the function of the G2 /M DNA damage checkpoint. Our data uncover a mechanism by which genomic integrity of hepatocytes is disrupted by viral LHBS. These findings highlight the role of intrahepatic surface antigen as an oncogenic risk factor in the development of HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hepatitis B virus surface gene pre‐S2 mutant as a high‐risk serum marker for hepatoma recurrence after curative hepatic resection

Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S2 mutant large HBV surface antigen (LHBS) is highly associated with HCC. This study analyzed the expression of the large form of surface protein in tumors and evaluated the LHBS with mutations within the pre-S2 region as a high-risk recurrence marker in HCC patients after curative hepatic resection. By analyses using immunohistochemical staining (n = 12) and western blotting (n = 22), the HBV surface protein, which is mainly comprised of the major form of HBV surface antigen, was greatly diminished in the tumors. However, LHBS was not significantly decreased in tumorous regions, suggesting that LHBS maintains its expression in cancer development. A cohort of 175 patients with HBV-related HCC who underwent curative hepatic resection was analyzed for pre-S gene mutations using Pre-S Gene Chip. Results of the multivariate regression analysis showed that the serum pre-S2 mutant level and the American Joint Committee on Cancer stage were the two main independent high-risk factors for recurrence. A Cox proportional hazards analysis also revealed a prediction model, which indicated the recurrence-free survival rate along with the time after surgery; this was developed and further validated in an independent HCC cohort. Receiver operating characteristic curve analysis revealed that the model showed close sensitivities in the main and validation cohorts (area under the curve values, 0.741 and 0.704, respectively). Conclusion: Unlike the major HBV surface antigen, LHBS is mostly expressed in the tumorous regions of HBV-induced HCC, indicating that it plays a unique role in tumor progression; the relative level of pre-S2 mutant in serum is, independently of tumor stage, an important high-risk marker for HCC recurrence after primary hepatic resection. (Hepatology 2018).

A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction

Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors.

Vaccines targeting preS1 domain overcome immune tolerance in hepatitis B virus carrier mice

These results suggest that preS1 can function as a therapeutic vaccine for the control of CHB. (Hepatology 2017;66:1067-1082).

Abstract 3820: Hepatitis B virus gene pre-S2 mutant as a high risk serum marker for hepatoma recurrence after a hepatic resection

Abstract Background: Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S2 mutant large HBV surface antigen (LHBS) is highly associated with HCC. This study aimed to evaluate the serum pre-S2 mutant LHBS as a high-risk recurrence marker in HCC patients after a hepatic resection. Methods: The pre-S gene chip was used to analyze 175 patients with HBV-related HCC who underwent a hepatic resection between 2008 and 2012. Thirty independent pre-S clones in every patient were analyzed for a semi-quantitative detection. Results: Multivariate regression analysis showed that the serum pre-S2 mutant level (≥ 5%) and the American Joint Committee on Cancer (AJCC) tumor stage were two independent high-risk factors for HCC recurrence. A Cox proportional hazards analysis also showed that the combined evaluations of the AJCC stage and serum pre-S2 mutant level provided a reliable predictive marker for HCC recurrence after primary surgery. We developed a Cox prediction model, which indicated the recurrence-free survival rates along with the time after surgery. This model was validated in an independent HCC cohort. Receiver operating characteristic curve analysis revealed that the model showed close sensitivities in the main and validation cohorts (AUC values: 0.741 and 0.704, respectively). Conclusions: The relative level of pre-S2 mutant in serum is, independently of tumor stage, an important predictive high-risk marker for HCC recurrence after a primary hepatic resection. The combined evaluations of the AJCC tumor stage and serum pre-S2 mutant level potentially serve as a convenient high-risk predictive marker for HCC prognosis. Citation Format: Wen-Ya Huang. Hepatitis B virus gene pre-S2 mutant as a high risk serum marker for hepatoma recurrence after a hepatic resection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3820. doi:10.1158/1538-7445.AM2017-3820

Effect of Histophilus somni on Heart and Brain Microvascular Endothelial Cells.

Histophilus somni is a pathogenic gram-negative bacterium responsible for pneumonia and septicemia in cattle. Sequelae include infectious thrombotic meningoencephalitis (ITME), myocarditis, arthritis, and abortion. These syndromes are associated with widespread vasculitis and thrombosis, implicating a role for endothelium in pathogenesis. Histopathologic and immunohistochemical investigation of 10 natural cases of bovine H. somni myocarditis and 1 case of ITME revealed intravascular H. somni in large biofilm-like aggregates adherent to the luminal surface of microvascular endothelium. Ultrastructurally, bacterial communities were extracellular and closely associated with degenerating or contracted endothelial cells. Histophilus somni was identified by bacterial culture and/or immunohistochemistry. Western blots of the bacterial isolates revealed that they expressed the immunodominant protective 40 kDa OMP and immunoglobulin-binding protein A (IbpA) antigens. The latter is a large surface antigen and shed fibrillar antigen with multiple domains. The cytotoxic DR2Fic domain of IbpA was conserved as demonstrated by polymerase chain reaction. Treatment of endothelial cells in vitro with IbpA in crude culture supernatants or purified recombinant GST-IbpA DR2Fic (rDR2) cytotoxin induced retraction of cultured bovine brain microvascular endothelial cells. By contrast, no retraction of bovine endothelium was induced by mutant rDR2H/A with an inactive Fic motif or by a GST control, indicating that the cytotoxic DR2Fic motif plays an important role in endothelial cell retraction in vasculitis. The formation of biofilm-like aggregates by H. somni on bovine microvascular endothelium may be fundamental to its pathogenesis in heart and brain.