Lipid nanoparticle-encapsulated DOCK11-siRNA efficiently reduces hepatitis B virus cccDNA level in infected mice.

The hepatitis B virus (HBV) infects many people worldwide. As HBV infection frequently leads to liver fibrosis and carcinogenesis, developing anti-HBV therapeutic drugs is urgent. Therapeutic drugs for preventing covalently closed circular DNA (cccDNA) production, which can eliminate HBV infection, are unavailable. The host factor dedicator of cytokinesis 11 (DOCK11) is involved in the synthesis and maintenance of HBV cccDNA in vitro. However, the effectiveness of DOCK11 as a target for the in vivo elimination of HBV cccDNA remains unclear. In this study, we assess whether DOCK11 inhibitors suppress HBV cccDNA production in mouse models of HBV infection. The tocopherol-conjugate hetero- gapmer, a DNA/RNA duplex of gapmer/complementary RNA targeting the DOCK11 sequence, partially reduces the expression of DOCK11, but not that of HBV cccDNA, in the livers of HBV-infected human hepatocyte chimeric mice, along with weight loss and decreased serum human albumin levels. Lipid nanoparticle-encapsulated chemically modified siRNAs specific for DOCK11 suppress DOCK11 expression and decrease HBV cccDNA levels without adverse effects in the mice. Therefore, nucleic acid-based drugs targeting DOCK11 in hepatocytes are potentially effective anti-HBV therapeutics that can reduce HBV cccDNA levels in vivo.

High-Throughput Screening of Antiviral Compounds Using a Recombinant Hepatitis B Virus and Identification of a Possible Infection Inhibitor, Skimmianine.

We developed a novel hepatitis B virus (HBV) infection-monitoring system using a luminescent, 11-amino acid reporter (HiBiT). We performed high-throughput antiviral screening using this system to identify anti-HBV compounds. After the infection of primary human hepatocytes with the recombinant virus HiBiT-HBV, which contains HiBiT at its preS1, 1262 compounds were tested in a first screening using extracellular HiBiT activity as an indicator of viral infection. Following a second screening, we focused on the compound skimmianine, which showed a potent antiviral effect. When skimmianine was added at the same time as HiBiT-HBV infection, skimmianine inhibited HiBiT activity with EC50 of 0.36 pM, CC50 of 1.67 µM and a selectivity index (CC50:EC50 ratio) of 5,100,000. When skimmianine was added 72 h after HiBiT-HBV infection, the EC50, CC50 and selectivity index were 0.19 µM, 1.87 µM and 8.79, respectively. Time-lapse fluorescence imaging analysis using another recombinant virus, ReAsH-TC155HBV, with the insertion of tetra-cysteine within viral capsid, revealed that skimmianine inhibited the accumulation of the capsid into hepatocytes. Furthermore, skimmianine did not inhibit either attachment or internalization. These results imply that skimmianine inhibits the retrograde trafficking of the virus after internalization. This study demonstrates the usefulness of the recombinant virus, HiBiT-HBV, for high-throughput screening to identify anti-HBV compounds.

Beta-Hydroxyisovaleryl-Shikonin Eradicates Epithelial Cell Adhesion Molecule-Positive Liver Cancer Stem Cells by Suppressing dUTP Pyrophosphatase Expression.

Cancer stem cells (CSCs) play an essential role in tumorigenesis, chemoresistance, and metastasis. Previously, we demonstrated that the development of hepatocellular carcinoma (HCC) is dictated by a subset of epithelial cell adhesion molecule-positive (EpCAM+) liver CSCs with the activation of Wnt signaling. In this study, we evaluated the expression of dUTP pyrophosphatase (dUTPase), which plays a central role in the development of chemoresistance to 5-fluorouracil, in EpCAM+ HCC cells. We further evaluated the effect of beta-hydroxyisovaleryl-shikonin (ß-HIVS), an ATP-noncompetitive inhibitor of protein tyrosine kinases, on HCC CSCs. EpCAM and dUTPase were expressed in hepatoblasts in human fetal liver, hepatic progenitors in adult cirrhotic liver, and a subset of HCC cells. Sorted EpCAM+ CSCs from HCC cell lines showed abundant nuclear accumulation of dUTPase compared with EpCAM-negative cells. Furthermore, treatment with the Wnt signaling activator BIO increased EpCAM and dUTPase expression. In contrast, ß-HIVS treatment decreased dUTPase expression. ß-HIVS treatment decreased the population of EpCAM+ liver CSCs in a dose-dependent manner in vitro and suppressed tumor growth in vivo compared with the control vehicle. Taken together, our data suggest that dUTPase could be a good target to eradicate liver CSCs resistant to 5-fluorouracil. ß-HIVS is a small molecule that could decrease dUTPase expression and target EpCAM+ liver CSCs.

Functional involvement of endothelial lipase in hepatitis B virus infection.

BACKGROUND: HBV infection causes chronic liver disease and leads to the development of HCC. To identify host factors that support the HBV life cycle, we previously established the HC1 cell line that maintains HBV infection and identified host genes required for HBV persistence. METHODS: The present study focused on endothelial lipase (LIPG), which binds to heparan sulfate proteoglycans (HSPGs) in the cell membrane. RESULTS: We found HBV infection was impaired in humanized liver chimeric mouse-derived hepatocytes that were transduced with lentivirus expressing short hairpin RNA against LIPG. Long-term suppression of LIPG combined with entecavir further suppressed HBV replication. LIPG was shown to be involved in HBV attachment to the cell surface by using 2 sodium taurocholate cotransporting peptide (NTCP)-expressing cell lines, and the direct interaction of LIPG and HBV large surface protein was revealed. Heparin and heparinase almost completely suppressed the LIPG-induced increase of HBV attachment, indicating that LIPG accelerated HBV attachment to HSPGs followed by HBV entry through NTCP. Surprisingly, the attachment of a fluorescently labeled NTCP-binding preS1 probe to NTCP-expressing cells was not impaired by heparin, suggesting the HSPG-independent attachment of the preS1 probe to NTCP. Interestingly, attachment of the preS1 probe was severely impaired in LIPG knockdown or knockout cells. Inhibitors of the lipase activity of LIPG similarly impaired the attachment of the preS1 probe to NTCP-expressing cells. CONCLUSIONS: LIPG participates in HBV infection by upregulating HBV attachment to the cell membrane by means of 2 possible mechanisms: increasing HBV attachment to HSPGs or facilitating HSPG-dependent or HSPG-independent HBV attachment to NTCP by its lipase activity.

Super-Resolution Microscopy Analysis of Hepatitis B Viral cccDNA and Host Factors.

Infection with hepatitis B virus (HBV) cannot be cured completely because of the persistence of covalently closed circular DNA (cccDNA). We previously found that the host gene dedicator of cytokinesis 11 (DOCK11) was required for HBV persistence. In this study, we further investigated the mechanism that links DOCK11 to other host genes in the regulation of cccDNA transcription. cccDNA levels were determined by quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) in stable HBV-producing cell lines and HBV-infected PXB-cells®. Interactions between DOCK11 and other host genes were identified by super-resolution microscopy, immunoblotting, and chromatin immunoprecipitation. FISH facilitated the subcellular localization of key HBV nucleic acids. Interestingly, although DOCK11 partially colocalized with histone proteins, such as H3K4me3 and H3K27me3, and nonhistone proteins, such as RNA Pol II, it played limited roles in histone modification and RNA transcription. DOCK11 was functionally involved in regulating the subnuclear distribution of host factors and/or cccDNA, resulting in an increase in cccDNA closely located to H3K4me3 and RNA Pol II for activating cccDNA transcription. Thus, it was suggested that the association of cccDNA-bound Pol II and H3K4me3 required the assistance of DOCK11. DOCK11 facilitated the association of cccDNA with H3K4me3 and RNA Pol II.

Hepatitis B Virus Utilizes a Retrograde Trafficking Route via the Trans-Golgi Network to Avoid Lysosomal Degradation.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is difficult to cure owing to the persistence of covalently closed circular viral DNA (cccDNA). We performed single-cell transcriptome analysis of newly established HBV-positive and HBV-negative hepatocellular carcinoma cell lines and found that dedicator of cytokinesis 11 (DOCK11) was crucially involved in HBV persistence. However, the roles of DOCK11 in the HBV lifecycle have not been clarified. METHODS: The cccDNA levels were measured by Southern blotting and real-time detection polymerase chain reaction in various hepatocytes including PXB cells by using an HBV-infected model. The retrograde trafficking route of HBV capsid was investigated by super-resolution microscopy, proximity ligation assay, and time-lapse analysis. The downstream molecules of DOCK11 and underlying mechanism were examined by liquid chromatography-tandem mass spectrometry, immunoblotting, and enzyme-linked immunosorbent assay. RESULTS: The cccDNA levels were strongly increased by DOCK11 overexpression and repressed by DOCK11 suppression. Interestingly, DOCK11 functionally associated with retrograde trafficking proteins in the trans-Golgi network (TGN), Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with HBV capsid, to open an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. Clinically, DOCK11 levels in liver biopsies from patients with chronic hepatitis B were significantly reduced by entecavir treatment, and this reduction correlated with HBV surface antigen levels. CONCLUSIONS: HBV uses a retrograde trafficking route via EEs-TGN-ER for infection that is facilitated by DOCK11 and serves to maintain cccDNA. Therefore, DOCK11 is a potential therapeutic target to prevent persistent HBV infection.

Clinical Features and Resistance to Entecavir Monotherapy of Patients with Hepatitis B.

Aim: Hepatitis B virus (HBV) infection is a major public health concern worldwide. Entecavir (ETV), a first-line nucleos(t)ide analogue (NA) for HBV, has a low risk of resistance. We evaluated the efficacy of ETV monotherapy, ratio of ETV-resistant, and the clinical features of patients with ETV resistance. Methods: A total of 130 patients (72 males, 58 females; mean age, 61 ± 15 years) were divided into a NA-naïve group (n = 108) and NA-experienced group (n = 22). We examined the clinical outcomes of ETV monotherapy and associated factors. We also assessed the clinical features of 15 patients with resistance to ETV (mean, 51.0 ± 27.4 weeks). Results: Among the 130 patients, 94.1% achieved ALT normalization and 63.6% achieved serum HBV DNA negativity after ETV monotherapy for 96 weeks. Of the patients in the NA-naïve group, 93.1% and 60.4% achieved ALT normalization and HBV DNA negativity, respectively. Of the patients in the NA-experienced group, 100% and 74.9% achieved ALT normalization and HBV DNA negativity, respectively. Compared to patients on ETV continuously, 15 ETV-resistant patients had a higher baseline HBV viral load. There was a significant difference in the time to HBV DNA negativity, but not ALT normalization after ETV monotherapy in these groups. Rescue treatment with other NAs led to ALT normalization in all of these patients, but not HBV DNA negativity. Conclusions: ETV monotherapy has a long-term clinical efficacy. While some patients especially with HBV DNA high viral load developed ETV resistance, rescue treatment led to ALT normalization in these patients.

Imaging features and pathological evaluation by EUS-FNA enable conservative management in patient of lymphoepithelial cyst of the pancreas: a case report.

Pancreatic lymphoepithelial cysts (LECs) are rare cystic lesions filled with a keratinous substance and lined by squamous epithelium with underlying lymphoid tissue. Because pancreatic LECs are entirely benign, correct preoperative diagnosis is important to avoid unnecessary surgery. However, the imaging features of pancreatic LECs are not specific and preoperative diagnosis has proven difficult. A pancreatic mass was incidentally detected through abdominal ultrasonography in a 63-year-old male presenting without any symptoms. Computed tomography showed an exophytic cystic lesion in the pancreatic head. The lesion had heterogeneous high signal intensity with partial low intensity on T2-weighted magnetic resonance imaging (MRI) and high signal intensity on diffusion MRI. Endoscopic ultrasound (EUS) examination showed an encapsulated cystic lesion with relatively homogenous and highly echoic contents. EUS-guided fine-needle aspiration (EUS-FNA) revealed caseous appearance and rare fragments of apparently benign squamous epithelium on a background of keratinous debris, cyst contents, and scattered lymphocytes. We diagnosed a pancreatic LEC and opted for conservative management without surgery. Pathological evaluation based on images obtained through EUS-FNA showed macro- and microscopic features that were critical to determining the management strategy. In conclusion, the imaging and pathological features of pancreatic LECs can inform preoperative diagnosis, which may enable conservative management.

Clinical Features and Dynamics of T Cells-Related Markers in Immunocompetent Patients with Cytomegalovirus Hepatitis.

Aim: Cytomegalovirus (CMV) can cause hepatitis, encephalomyelitis, and pneumonitis in immunocompromised patients. In contrast, CMV infection of immunocompetent patients can lead to the development of infectious mononucleosis and is typically self-limiting; severe complications are rare. We evaluated the pathophysiology and immunological aspects of CMV hepatitis in recently immunocompetent adult patients. Methods: We examined the clinical features and outcomes of 47 adult immunocompetent patients with CMV hepatitis (29 men, 18 women; mean age, 34 ± 11 years) from January 2005 to August 2019 treated in our hospital. We also assayed T-cell activation to evaluate the immune responses in these patients. Results: Fever (74.5%), hepatosplenomegaly (74.5%), sore throat (36.2%), headache (31.9%), abdominal pain (27.7%), lymphadenopathy (23.4%), and skin rash (6.4%) were present at admission. Complications included gastrointestinal injury (25.5%), neuropathy (4.3%), thrombocytopenia (2.1%), and splenic infarction (2.1%). All patients had a good clinical course without liver failure or transition to chronic liver injury. The time to recover from liver injury ranged from 12 to 142 days (mean, 43.4 ± 28.7 days). The serum sIL-2R level, which reflects T-cell activation, was transiently elevated and correlated with the extent of hepatic inflammation. Conclusions: CMV hepatitis in immunocompetent individuals has a satisfactory outcome, but occasionally results in complications in other organs. The sIL-2R level has potential as a surrogate marker of hepatic inflammation in immunocompetent patients with CMV hepatitis.

Tumor lysis syndrome in a patient with metastatic melanoma treated with nivolumab.

A 79-year-old man with metastatic melanoma of the right maxillary sinus and multiple liver metastases received a single dose of nivolumab. Eight days later, he experienced impaired consciousness, accompanied by abnormal laboratory and electrocardiographic findings. He was therefore diagnosed with tumor lysis syndrome (TLS). Laboratory and electrocardiographic findings improved immediately after continuous hemodiafiltration; however, he died 22 days after receiving nivolumab. Autopsy revealed massive tumor necrosis in the liver. There are few case reports of TLS associated with immune checkpoint inhibitors, indicating that we should be prepared to manage especially in a patient with liver involvement of high tumor burden.

Phase II clinical trial of second-line weekly paclitaxel plus trastuzumab for patients with HER2-positive metastatic gastric cancer.

BACKGROUND: Combination therapy with fluorouracil, platinum, and trastuzumab (Tmab) is the first-line treatment for human epidermal growth factor receptor 2 (HER2)-positive gastric cancer, and there is currently no established second-line therapy. We evaluated the efficacy and safety of weekly paclitaxel plus Tmab as second-line chemotherapy for HER2-positive gastric cancer patients. PATIENTS AND METHODS: Eligible patients were older than or equal to 20 years, had histologically confirmed gastric adenocarcinoma that was HER2 positive (immunohistochemistry 3+ or immunohistochemistry 2+ and fluorescence in-situ hybridization positive or dual color in-situ hybridization positive), and had been treated previously with chemotherapy (pretreated or not with Tmab). Patients received weekly paclitaxel plus Tmab as the second-line chemotherapy. The primary endpoint was the overall response rate (ORR; threshold ORR=20% and expected ORR=35%). RESULTS: Twenty-eight patients were enrolled. ORR was 21.4%. The median progression-free survival (PFS) was 4.6 months. The median overall survival (OS) was 9.6 months. No significant differences were observed in ORR, PFS, or OS between the Tmab beyond progression (TBP) group (n=20) and the non-TBP group (n=8). However, in the TBP group, a therapeutic effect was associated with the duration of PFS in the first-line Tmab treatment [≥6 months PFS in the first-line Tmab treatment (n=10) vs. <6 months (n=10); ORR: 40 and 10%, P=0.303, PFS: 6.2 and 2.8 months, P=0.005, OS: 15.8 and 6.5 months, P=0.006, respectively]. CONCLUSION: Weekly paclitaxel plus Tmab was not superior as second-line chemotherapy for HER2-positive gastric cancer patients, but may be effective for patients who showed better responses to Tmab-combined chemotherapy in the first-line treatment.