Exploring the genetic diversity of the Japanese population: Insights from a large-scale whole genome sequencing analysis.

The Japanese archipelago is a terminal location for human migration, and the contemporary Japanese people represent a unique population whose genomic diversity has been shaped by multiple migrations from Eurasia. We analyzed the genomic characteristics that define the genetic makeup of the modern Japanese population from a population genetics perspective from the genomic data of 9,287 samples obtained by high-coverage whole-genome sequencing (WGS) by the National Center Biobank Network. The dataset comprised populations from the Ryukyu Islands and other parts of the Japanese archipelago (Hondo). The Hondo population underwent two episodes of population decline during the Jomon period, corresponding to the Late Neolithic, and the Edo period, corresponding to the Early Modern era, while the Ryukyu population experienced a population decline during the shell midden period of the Late Neolithic in this region. Haplotype analysis suggested increased allele frequencies for genes related to alcohol and fatty acid metabolism, which were reported as loci that had experienced positive natural selection. Two genes related to alcohol metabolism were found to be 12,500 years out of phase with the time when they began to increase in the allele frequency; this finding indicates that the genomic diversity of Japanese people has been shaped by events closely related to agriculture and food production.

Potential of Mac-2-binding protein glycan isomer as a new therapeutic target in pancreatic cancer.

Pancreatic cancer (PC) is a challenging malignancy to treat. Mac-2-binding protein glycan isomer (M2BPGi) is a novel serum marker of liver fibrosis and hepatocellular carcinoma and is secreted by hepatic stellate and stroma cells. Serum M2BPGi levels are upregulated in PC patients. We measured the expression of M2BPGi in the serum of 27 PC patients and determined whether M2BPGi affects the malignant potential of PC cells in vitro. We also examined the effect of M2BP on PC tumor growth and gemcitabine sensitivity in vivo. Serum M2BPGi levels in PC patients were higher compared with those of healthy subjects. M2BPGi extraction in cancer-associated fibroblasts (CAFs) was higher compared with that of PC cells. M2BPGi treatment promoted the proliferation and invasion of PC cells. The suppression of galectin-3, which binds to M2BPGi, did not affect the proliferation-promoting effect of M2BPGi in PC cells. The suppression of M2BP reduced tumor growth and enhanced gemcitabine sensitivity in PC-bearing xenograft mice. CAF-derived M2BPGi promotes the proliferation and invasion of PC cells. Targeting M2BPGi may represent a new therapeutic strategy to circumvent refractory PC.

Human Gut Microbiota and Its Metabolites Impact Immune Responses in COVID-19 and Its Complications.

BACKGROUND & AIMS: We investigate interrelationships between gut microbes, metabolites, and cytokines that characterize COVID-19 and its complications, and we validate the results with follow-up, the Japanese 4D (Disease, Drug, Diet, Daily Life) microbiome cohort, and non-Japanese data sets. METHODS: We performed shotgun metagenomic sequencing and metabolomics on stools and cytokine measurements on plasma from 112 hospitalized patients with SARS-CoV-2 infection and 112 non-COVID-19 control individuals matched by important confounders. RESULTS: Multiple correlations were found between COVID-19-related microbes (eg, oral microbes and short-chain fatty acid producers) and gut metabolites (eg, branched-chain and aromatic amino acids, short-chain fatty acids, carbohydrates, neurotransmitters, and vitamin B6). Both were also linked to inflammatory cytokine dynamics (eg, interferon γ, interferon λ3, interleukin 6, CXCL-9, and CXCL-10). Such interrelationships were detected highly in severe disease and pneumonia; moderately in the high D-dimer level, kidney dysfunction, and liver dysfunction groups; but rarely in the diarrhea group. We confirmed concordances of altered metabolites (eg, branched-chain amino acids, spermidine, putrescine, and vitamin B6) in COVID-19 with their corresponding microbial functional genes. Results in microbial and metabolomic alterations with severe disease from the cross-sectional data set were partly concordant with those from the follow-up data set. Microbial signatures for COVID-19 were distinct from diabetes, inflammatory bowel disease, and proton-pump inhibitors but overlapping for rheumatoid arthritis. Random forest classifier models using microbiomes can highly predict COVID-19 and severe disease. The microbial signatures for COVID-19 showed moderate concordance between Hong Kong and Japan. CONCLUSIONS: Multiomics analysis revealed multiple gut microbe-metabolite-cytokine interrelationships in COVID-19 and COVID-19related complications but few in gastrointestinal complications, suggesting microbiota-mediated immune responses distinct between the organ sites. Our results underscore the existence of a gut-lung axis in COVID-19.

Oxidative stress sensor Keap1 recognizes HBx protein to activate the Nrf2/ARE signaling pathway, thereby inhibiting hepatitis B virus replication.

IMPORTANCE: The Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is one of the most important defense mechanisms against oxidative stress. We previously reported that a cellular hydrogen peroxide scavenger protein, peroxiredoxin 1, a target gene of transcription factor Nrf2, acts as a novel HBV X protein (HBx)-interacting protein and negatively regulates hepatitis B virus (HBV) propagation through degradation of HBV RNA. This study further demonstrates that the Nrf2/ARE signaling pathway is activated during HBV infection, eventually leading to the suppression of HBV replication. We provide evidence suggesting that Keap1 interacts with HBx, leading to Nrf2 activation and inhibition of HBV replication via suppression of HBV core promoter activity. This study raises the possibility that activation of the Nrf2/ARE signaling pathway is a potential therapeutic strategy against HBV. Our findings may contribute to an improved understanding of the negative regulation of HBV replication by the antioxidant response.

Incidences of new hepatitis B infection and anti-hepatitis B core-negative occult hepatitis B infection among Japanese blood donors in relation to anti-hepatitis B surface antigen levels.

A transfusion-transmitted hepatitis B virus (HBV) infection caused by blood only positive for anti-hepatitis B surface antigen (anti-HBs) was reported. Occult HBV infection (OBI) with sole anti-HBs among blood donors is an issue. The incidence of HBV infection among repeat blood donors was investigated with a detailed HBV infection phase, focusing on the influence of anti-HBs level. This study followed 3 435 653 donors for HBV DNA conversion over 4 years and 9 months. Infection phase was determined based on marker changes over DNA conversion. This study identified 115 hepatitis B surface antigen (HBsAg) conversions, 72 DNA-only conversions, and 15 DNA plus anti-hepatitis B core (anti-HBc) conversions among donors all negative for HBV DNA, HBsAg, and anti-HBc. Total incidence was 2.38/100 000 person-years (PY). None of these 202 new HBV infections arose in the group with anti-HBs titer ≥ 10 mIU/mL. In total, 30 anti-HBc-negative OBIs were identified (incidence; 0.35/100 000 PY); 7 showed typical secondary anti-HBs response, and 23 showed stable anti-HBc and anti-HBs levels at DNA conversion. The HBV infection-protective ability of anti-HBs ≥ 10 mIU/mL was reinforced. In addition to new infections, the blood donor population includes anti-HBc-positive- and negative OBI with immune reactions or abortive HBV infection.

Hepatic Mac2-BP expression depends on liver fibrosis and inflammation due to fat accumulation in patients with metabolic dysfunction-associated steatotic liver disease.

AIM: Data on the upregulation of Mac-2 binding protein (M2BP) expression associated with fat accumulation in the liver are limited. Therefore, we aimed to assess the relationship between hepatic M2BP expression and changes in the liver microenvironment due to fat accumulation in patients with metabolic dysfunction associated steatotic liver disease (MASLD). METHODS: Liver specimens obtained from 46 patients with MASLD were subjected to immunohistochemical staining to visualize M2BP expression in the liver. The staining intensity in the hepatocytes and sinusoidal cells was classified as high or low grade. First, the correlation between hepatic M2BP expression and microenvironmental changes caused by fat accumulation was examined. Then, the influence of hepatic M2BP expression on serum M2BP glycosylation isomer levels in patients with MASLD was evaluated. RESULTS: The staining grade of M2BP was higher in the sinusoidal cells than in the hepatocytes (p = 0.015). The patients with high staining grade in their hepatocytes had more severe lobular inflammation than those with low staining grade (p = 0.037). Additionally, the patients with high staining grade in their sinusoidal cells presented more severe fibrosis than those with low staining grade (p = 0.018). The staining grade in the hepatocytes correlated positively with serum M2BP glycosylation isomer levels (p = 0.023), whereas no correlation was observed between sinusoidal staining grade and serum M2BP glycosylation isomer levels (p = 0.393). CONCLUSIONS: Fat accumulation in patients with MASLD leads to M2BP expression in hepatocytes due to liver inflammation and that in sinusoidal cells due to fibrosis.

Serum HBcrAg and Hepatocellular Carcinoma in a Taiwanese Population Seronegative for HBsAg and Anti-HCV.

BACKGROUND & AIMS: Hepatitis B core-related antigen (HBcrAg) is a surrogate seromarker of intrahepatic hepatitis B virus covalently closed circular DNA quantity and activity and a predictor of hepatocellular carcinoma (HCC) in chronic hepatitis B patients. We assess association between HBcrAg and HCC in individuals seronegative for hepatitis B surface antigen and anti-hepatitis C virus (NBNC) in Taiwan. METHODS: A total of 129 newly developed HCC cases and 520 frequency-matched non-HCC controls were drawn from the REVEAL-NBNC cohort. Serum HBcrAg and other risk factors measured at recruitment were compared between cases and controls. Regression analysis was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: The proportion of baseline HBcrAg positivity (≥1000 U/mL) was significantly higher in HCC cases than in controls (12.4% vs 1.4%, P < .001). In multivariate analysis, HBcrAg positivity was associated with significantly higher risk of HCC (adjusted OR [95% CI]: 9.3 [3.3-26.4]; P < .001]. The HCC population attributable to HBcrAg positivity was 11.1% (95% CI: 9.7%-12.5%). CONCLUSIONS: Seropositivity of HBcrAg might identify a subset of the NBNC population at higher risk of HCC in hepatitis B virus endemic areas.

Population-level Metagenomics Uncovers Distinct Effects of Multiple Medications on the Human Gut Microbiome.

BACKGROUND & AIMS: Medication is a major determinant of human gut microbiome structure, and its overuse increases the risks of morbidity and mortality. However, effects of certain commonly prescribed drugs and multiple medications on the gut microbiome are still underinvestigated. METHODS: We performed shotgun metagenomic analysis of fecal samples from 4198 individuals in the Japanese 4D (Disease, Drug, Diet, Daily life) microbiome project. A total of 759 drugs were profiled, and other metadata, such as anthropometrics, lifestyles, diets, physical activities, and diseases, were prospectively collected. Second fecal samples were collected from 243 individuals to assess the effects of drug initiation and discontinuation on the microbiome. RESULTS: We found that numerous drugs across different treatment categories influence the microbiome; more than 70% of the drugs we profiled had not been examined before. Individuals exposed to multiple drugs, polypharmacy, showed distinct gut microbiome structures harboring significantly more abundant upper gastrointestinal species and several nosocomial pathobionts due to additive drug effects. Polypharmacy was also associated with microbial functions, including the reduction of short-chain fatty acid metabolism and increased bacterial stress responses. Even nonantibiotic drugs were significantly correlated with an increased antimicrobial resistance potential through polypharmacy. Notably, a 2-time points dataset revealed the alteration and recovery of the microbiome in response to drug initiation and cessation, corroborating the observed drug-microbe associations in the cross-sectional cohort. CONCLUSION: Our large-scale metagenomics unravels extensive and disruptive impacts of individual and multiple drug exposures on the human gut microbiome, providing a drug-microbe catalog as a basis for a deeper understanding of the role of the microbiome in drug efficacy and toxicity.

Metagenomic Identification of Microbial Signatures Predicting Pancreatic Cancer From a Multinational Study.

BACKGROUND & AIMS: To identify gut and oral metagenomic signatures that accurately predict pancreatic ductal carcinoma (PDAC) and to validate these signatures in independent cohorts. METHODS: We conducted a multinational study and performed shotgun metagenomic analysis of fecal and salivary samples collected from patients with treatment-naïve PDAC and non-PDAC controls in Japan, Spain, and Germany. Taxonomic and functional profiles of the microbiomes were characterized, and metagenomic classifiers to predict PDAC were constructed and validated in external datasets. RESULTS: Comparative metagenomics revealed dysbiosis of both the gut and oral microbiomes and identified 30 gut and 18 oral species significantly associated with PDAC in the Japanese cohort. These microbial signatures achieved high area under the curve values of 0.78 to 0.82. The prediction model trained on the Japanese gut microbiome also had high predictive ability in Spanish and German cohorts, with respective area under the curve values of 0.74 and 0.83, validating its high confidence and versatility for PDAC prediction. Significant enrichments of Streptococcus and Veillonella spp and a depletion of Faecalibacterium prausnitzii were common gut signatures for PDAC in all the 3 cohorts. Prospective follow-up data revealed that patients with certain gut and oral microbial species were at higher risk of PDAC-related mortality. Finally, 58 bacteriophages that could infect microbial species consistently enriched in patients with PDAC across the 3 countries were identified. CONCLUSIONS: Metagenomics targeting the gut and oral microbiomes can provide a powerful source of biomarkers for identifying individuals with PDAC and their prognoses. The identification of shared gut microbial signatures for PDAC in Asian and European cohorts indicates the presence of robust and global gut microbial biomarkers.

Dried blood spot-based host genome analysis technique targeting pathological associations with hepatitis B: Development and clinical application in the Cambodian population.

AIM: Reports of patients with hepatitis B have highlighted associations between polymorphisms in the human leukocyte antigen (HLA)-DPB1, CXCL13, and CXCR5 genes and disease pathology. Owing to its potential to contribute to the development of new diagnostic and therapeutic methods, we aimed to establish a reliable host genome analysis technique that can be used in countries with inadequate infrastructure. METHOD: We compared multiple commercially available kits for dried blood spot (DBS)-based sample collection to develop a basic DBS-based host genome analysis technique. We then collected blood samples from Cambodian patients with hepatitis  B and performed single-nucleotide polymorphism genotyping and HLA allele typing by the DBS system. RESULT: We were able to perform single-nucleotide polymorphism genotyping and HLA allele typing with host DNA samples obtained using a combination of a HemaSpot™ filter paper-based device and a SMITEST® EX-R&D DNA extraction kit. The accuracy of genotyping using samples obtained by this method was not inferior to one using samples obtained by venipuncture. In the Cambodian population, significant associations of HLA-DPB1*04:01 with protection against chronic hepatitis B virus (HBV) infection, and HLA-DPB1*05:01 and HLA-DPB1*13:01 with susceptibility to chronic HBV infection were identified. CONCLUSION: Based on the DBS system, we clarified the associations of HLA-DPB1 alleles with chronic HBV infection in the Cambodian population for the first time. Because the DBS is a low-cost, durable, transportable, and easy-to-handle modality, genetic analysis based on the DBS system is a feasible strategy for obtaining a deeper understanding of HBV epidemiology, especially in middle- or low-income countries.

Impact of liver fibrosis on the relative abundance of a urease-positive Streptococcus salivarius group from saliva in patients with chronic liver disease.

AIM: We performed genomic analysis to study the relative abundance of a urease-positive Streptococcus salivarius group isolated from the saliva of patients with chronic liver disease. METHODS: Male and female patients with chronic liver disease aged over 20 years were included. First, we assessed the frequency and type of the S. salivarius group isolated from oral saliva using molecular biology techniques based on 16S rRNA and dephospho-coenzyme A kinase gene sequencing. Next, we assessed the correlation between the urease positivity rate in the S. salivarius group isolated from oral saliva and liver fibrosis based on chronic liver disease. Urease-positive strains were identified by the urease test using urea broth (Difco, Franklin Lakes, NJ, USA). Liver fibrosis was evaluated by the liver stiffness measurement value based on magnetic resonance elastography. RESULTS: A total of 45 patients identified using the multiplex polymerase chain reaction for the 16S rRNA gene were tested using the multiplex polymerase chain reaction for the dephospho-coenzyme A kinase gene. Confirming the strains detected in each of the 45 patients, urease-positive S. salivarius was detected in 28 patients (62%), urease-negative S. salivarius in 25 patients (56%), and urease-positive Streptococcus vestibularis in 12 patients (27%). There was no patient with urease-negative S. vestibularis. The urease-positive rate of the S. salivarius group in the cirrhosis and non-cirrhosis groups were 82.2% and 39.2%, respectively. The liver cirrhosis group had a higher urease positivity rate than the non-cirrhotic group (p < 0.001). CONCLUSIONS: Liver fibrosis influences the frequency of a urease-positive S. salivarius group isolated from oral saliva.

Possible biological mechanisms of entecavir versus tenofovir disoproxil fumarate on reducing the risk of hepatocellular carcinoma.

Hepatitis B virus (HBV) is a life-threatening infectious virus associated with the risk of liver failure and hepatocellular carcinoma (HCC). Regarding HBV treatment, the recent development of nucleoside/nucleotide analogs (NUC), HBV reverse transcriptase inhibitors, enabled favorable viral control as well as improved prognosis in patients with chronic hepatitis B. However, NUC fails to clear HBV because the formation of covalently closed circular DNA or HBV surface antigen occurs upstream of the point of action of NUC. Recently, we found that acyclic nucleoside phosphonates (ANP) such as adefovir or tenofovir, but not lamivudine or entecavir, induced IFN-λ3 productions in the gastrointestinal tract and modulated lipopolysaccharide (LPS)-mediated cytokine profiles in peripheral blood mononuclear cells, such as interleukin (IL)-12p70 induction and IL-10 inhibition, which are immunologically favorable cytokine profiles for HBV elimination. Furthermore, IFN-α, in combination with ANP, showed additional and synergistic effects on IFN-λ3 and IL-12p70 production, respectively, while not affecting IL-10 levels. Mechanistic analyses of the cytokine modulation by ANP revealed that ANP blocked the mammalian target of the rapamycin (mTOR) pathway by inhibiting Akt translocation to the plasma membrane, thereby inhibiting Akt phosphorylation. As it has been reported that IFN-λ inhibits tumor growth directly or indirectly and the mTOR pathway is generally activated in most cancer cells, ANP might have potential anti-HCC effects. Our in vitro and ex vivo findings might stir the debate on whether types of NUC affect the risk of HBV-related HCC incidence.

Regulation of MEK inhibitor selumetinib sensitivity by AKT phosphorylation in the novel BRAF L525R mutant.

BACKGROUND: Oncogenic mutations in BRAF genes are found in approximately 5-10% of colorectal cancers. The majority of BRAF mutations are located within exons 11-15 of the catalytic kinase domains, with BRAF V600E accounting for more than 80% of the observed BRAF mutations. Sensitivity to BRAF- and mitogen-activated protein kinase (MEK) inhibitors varies depending on BRAF mutations and tumor cell types. Previously, we newly identified, BRAF L525R-mutation, in the activation segment of the kinase in colorectal cancer patient. Here, we characterized the function of the BRAF L525R mutation. METHODS: HEK293 cells harboring a BRAF mutation (V600E or L525R) were first characterized and then treated with cetuximab, dabrafenib, and selumetinib. Cell viability was measured using WST-1 assay and the expression of proteins involved in the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways was evaluated using western blot analysis. RESULTS: The MEK inhibitor selumetinib effectively inhibited cell proliferation and ERK phosphorylation in BRAF L525R cells but not in BRAF V600E cells. Further studies revealed that AKT phosphorylation was reduced by selumetinib in BRAF L525R cells but not in BRAF V600E cells or selumetinib-resistant BRAF L525R cells. Moreover, the AKT inhibitor overcame the selumetinib resistance. CONCLUSIONS: We established a model system harboring BRAF L525R using HEK293 cells. BRAF L525R constitutively activated ERK. AKT phosphorylation caused sensitivity and resistance to selumetinib. Our results suggest that a comprehensive network analysis may provide insights to identify effective therapies.

Prediction Model with HLA-A*33:03 Reveals Number of Days to Develop Liver Cancer from Blood Test.

The development of liver cancer in patients with hepatitis B is a major problem, and several models have been reported to predict the development of liver cancer. However, no predictive model involving human genetic factors has been reported to date. For the items incorporated in the prediction model reported so far, we selected items that were significant in predicting liver carcinogenesis in Japanese patients with hepatitis B and constructed a prediction model of liver carcinogenesis by the Cox proportional hazard model with the addition of Human Leukocyte Antigen (HLA) genotypes. The model, which included four items-sex, age at the time of examination, alpha-fetoprotein level (log10AFP) and presence or absence of HLA-A*33:03-revealed an area under the receiver operating characteristic curve (AUROC) of 0.862 for HCC prediction within 1 year and an AUROC of 0.863 within 3 years. A 1000 repeated validation test resulted in a C-index of 0.75 or higher, or sensitivity of 0.70 or higher, indicating that this predictive model can distinguish those at high risk of developing liver cancer within a few years with high accuracy. The prediction model constructed in this study, which can distinguish between chronic hepatitis B patients who develop hepatocellular carcinoma (HCC) early and those who develop HCC late or not, is clinically meaningful.

Hepatic Stellate Cells in Hepatocellular Carcinoma Promote Tumor Growth Via Growth Differentiation Factor 15 Production.

BACKGROUND & AIMS: Although the tumor microenvironment plays an important role in tumor growth, it is not fully understood what role hepatic stellate cells (HSCs) play in the hepatocellular carcinoma (HCC) microenvironment. METHODS: A high-fat diet after streptozotocin was administered to HSC-specific Atg7-deficient (GFAP-Atg7 knockout [KO]) or growth differentiation factor 15 (GDF15)-deficient (GFAP-GDF15KO) mice. LX-2 cells, a human HSC cell line, were cultured with human hepatoma cells. RESULTS: In the steatohepatitis-based tumorigenesis model, GFAP-Atg7KO mice formed fewer and smaller liver tumors than their wild-type littermates. Mixed culture of LX-2 cells and hepatoma cells promoted LX-2 cell autophagy and hepatoma cell proliferation, which were attenuated by Atg7 KO in LX-2 cells. Hepatoma cell xenograft tumors grew rapidly in the presence of LX-2 cells, but Atg7 KO in LX-2 cells abolished this growth. RNA-sequencing revealed that LX-2 cells cultured with HepG2 cells highly expressed GDF15, which was abolished by Atg7 KO in LX-2 cells. GDF15 KO LX-2 cells did not show a growth-promoting effect on hepatoma cells either in vitro or in the xenograft model. GDF15 deficiency in HSCs reduced liver tumor size caused by the steatohepatitis-based tumorigenesis model. GDF15 was highly expressed and GDF15-positive nonparenchymal cells were more abundant in human HCC compared with noncancerous parts. Single-cell RNA sequencing showed that GDF15-positive rates in HSCs were higher in HCC than in background liver. Serum GDF15 levels were high in HCC patients and increased with tumor progression. CONCLUSIONS: In the HCC microenvironment, an increase of HSCs that produces GDF15 in an autophagy-dependent manner may be involved in tumor progression.

N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture-Generated Hepatitis B Virus.

BACKGROUND AND AIMS: An efficient cell-culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against antiviral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture. APPROACH AND RESULTS: We found that an 11-amino-acid deletion (d11) in the preS1 region enhances the infectivity of cell-culture-generated HBV (HBVcc) to sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was ~10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pregenomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the HBV large surface protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection. CONCLUSIONS: This system provides a powerful tool for studying the infection and propagation of HBV in cell culture and also for developing the antiviral strategy against HBV infection.

Establishment of monoclonal antibodies broadly neutralize infection of hepatitis B virus.

Antibodies against hepatitis B virus S protein can protect against hepatitis B virus (HBV) infection. Therefore, hepatitis B immunoglobulin (HBIG), which contains HBsAb, is used clinically as a therapy for HBV infection. In this study, a series of monoclonal antibodies that recognize multiple HBV genotypes was obtained. All the antibodies recognized conformational epitopes of S protein, but not linear epitopes. Several antibodies neutralized HBV infection and exhibited strong affinities and neutralizing activities. Antigenic epitope analysis demonstrated that they recognized residue Ile152 of S protein, which is localized outside the "a" determinant. Ile152 is highly conserved, and a mutation in this residue resulted in reduced expression of large hepatitis B surface proteins (L protein), suggesting that the amino acid at this position is involved in the expression of L protein. In addition, the antibodies neutralized the infection of hepatitis D virus possessing a Gly145 mutation to Arg in S protein, which is a well-known escape mutation against HBIG treatment. Using mouse monoclonal antibodies, a humanized antibody possessing affinities and neutralizing activities similar to those of the original mouse antibody was successfully established. The antibodies generated in this study may have the potential for use in alternative antibody therapies for HBV infection.

Effects of nucleos(t)ide analogs on hepatitis B surface antigen reduction with interferon-lambda 3 induction in chronic hepatitis B patients.

BACKGROUND & AIMS: Benefits of nucleos(t)ide analogs (NAs) on hepatitis B surface antigen (HBsAg) reduction and interferon-lambda3 (IFN-λ3) induction are still not known. This study aimed to investigate the effects of NAs on HBsAg reduction and association with serum IFN-λ3 levels in chronic hepatitis B (CHB) patients. METHODS: A total of 91 patients [51 treated with nucleoside analog entecavir hydrate (ETV) and 40 treated with nucleotide analog adefovir dipivoxil (ADV) or tenofovir disoproxil fumarate (TDF)] with clinically evident CHB (chronic hepatitis, 57; liver cirrhosis, 34) were enrolled in this study. Serum IFN-λ3 levels among patients receiving ETV and ADV/TDF were measured before the initiation of therapy and 1, 3, and 5 years post-therapy. RESULTS: The change (mean ± standard deviation) in serum HBsAg levels from baseline to year five was -0.38 ± 0.46 and -0.84 ± 0.64 log10 IU/ml in ETV and ADV/TDF groups, respectively (p = 0.0004). Higher serum IFN-λ3 levels were observed in ADV/TDF group compared with ETV group during treatment (p < 0.001). Serum IFN-λ3 levels showed negative correlation with HBsAg reduction in ADV/TDF group (r = -0.386, p = 0.038) at week 48. Nucleotide analogs (ADV/TDF) treatment has associated factors with -0.3 log HBsAg decline at 1 year, -0.5 log HBsAg decline at 3 years, and -0.8 log HBsAg decline at 5 years after NAs treatment on multivariate analysis. CONCLUSIONS: Nucleotide analog (ADV/TDF) treatment reduced HBsAg levels greater compared with nucleoside analog (ETV) in parallel with IFN-λ3 induction.

Impact of Immune Reconstitution-Induced Hepatic Flare on Hepatitis B Surface Antigen Loss in Hepatitis B Virus/Human Immunodeficiency Virus-1 Coinfected Patients.

BACKGROUND: Hepatitis B surface antigen (HBsAg) loss is an ideal goal for chronic hepatitis B patients. Antiretroviral therapy (ART) in hepatitis B virus/human immunodeficiency virus-1 (HBV/HIV-1)-coinfected patients can lead to hepatic flare (HF) caused by immune reconstitution-induced inflammatory syndrome (IRIS). Here, we investigated the impact of IRIS-HF on HBsAg loss. METHODS: This was a retrospective study of 58 HBV/HIV-1-coinfected subjects HBsAg-positive for ≥6 months before ART initiation and followed for ≥1 year (median 9.9 years) after ART initiation. We examined humoral factors in sera from healthy volunteers, HIV-monoinfected patients, and HBV/HIV-1-coinfected patients with IRIS-HF or acute hepatitis B infection. RESULTS: During ART, HBsAg loss was observed in 20 of 58 HBV/HIV-1-coinfected patients (34.5%). Of the 58 patients, 15 (25.9%) developed IRIS-HF within 12 months of ART initiation. HBsAg loss was more frequent among patients who developed IRIS-HF (11/15, 73.3%) than those who did not (9/43, 20.9%). Multivariate analysis showed IRIS-HF was an independent predictor of subsequent HBsAg loss. Younger age and higher baseline HBV DNA titer were associated with IRIS-HF. Elevation of sCD163, not CXCL9, CXC10, CXCXL11, or CXCL13, was observed at IRIS-HF. CONCLUSIONS: IRIS-HF was associated with HBsAg loss in HBV/HIV-1-coinfected patients.

Lenvatinib suppresses cancer stem-like cells in HCC by inhibiting FGFR1-3 signaling, but not FGFR4 signaling.

In hepatocellular carcinoma (HCC), a subset of cells defined by high CD44 and CD133 expression has been reported to possess cancer stem-like cell (CSC) characteristics and to be associated with a poor prognosis. Since the approval of the multikinase inhibitor, lenvatinib, for patients with unresectable HCC, two such inhibitors (sorafenib and lenvatinib) have been employed as first-line systemic chemotherapeutics for these patients. Based on differences in the kinase-affinity profiles between these two drugs, evidence has suggested that both exert different effects on HCC, although these differences are not fully characterized. In this study, using in vitro and a preclinical in vivo xenograft mouse model, we showed that lenvatinib alone (not sorafenib or the cytotoxic agent, 5-fluorouracil) diminished CD44High/CD133High CSCs in HCC. Furthermore, western blotting and reverse transcriptase-polymerase chain reaction analysis revealed that the expression of fibroblast growth factor receptor (FGFR)-1-4 differed between CD44High/CD133High CSCs and control cells. Analysis of the effects of selective FGFR inhibitors and FGFR small interfering RNAs on CSCs in HCC revealed that lenvatinib diminished CSCs in HCC by inhibiting FGFR1-3 signaling, however, FGFR4 signaling was not impacted. Finally, we showed that FGF2 and FGF19 were involved in maintaining CD44High/CD133High CSCs in HCC, potentially, via FGFR1-3. The findings provide novel mechanistic insights into the effects of lenvatinib on CSCs in HCC and provide clues for developing effective targeted therapies against CSCs in HCC.