A simplified method to quantitatively predict the effect of lenvatinib on hepatocellular carcinoma using contrast-enhanced ultrasound with perfluorobutane microbubbles

Contrast-enhanced computed tomography (CECT) is generally used to evaluate the response to treatment of hepatocellular carcinoma (HCC); however, CECT is unsuitable for the early prediction of therapeutic effects and frequent monitoring. We aimed to investigate the usefulness of our simplified method for the quantification of tumor vascularity using contrast-enhanced ultrasound (CEUS) with perfluorobutane microbubbles [Sonazoid® (GE Healthcare, Oslo, Norway)] to predict the therapeutic effect of lenvatinib. Among the 13 patients studied, nine who had more than a 20% reduction in tumor vascularity within 2 weeks of starting treatment experienced complete response or partial response at 8-12 weeks as assessed by CECT. In contrast, three patients without reductions and one patient with only a slight decrease in tumor vascularity had a poor response to lenvatinib. Quantitative assessment of tumor vascularity by our simplified CEUS-based method could be a useful predictor of therapeutic responses to lenvatinib in patients with HCC

DNA methyltransferase 3B plays a protective role against hepatocarcinogenesis caused by chronic inflammation via maintaining mitochondrial homeostasis

Most hepatocellular carcinomas (HCCs) develop on the basis of chronic hepatitis, but the mechanism of epigenetic regulation in inflammatory hepatocarcinogenesis has yet to be elucidated. Among de novo DNA methyltransferases (DNMTs), DNMT3B has lately been reported to act specifically on actively transcribed genes, suggesting the possibility that it plays a role in the pathogenesis of cancer. We confirmed that DNMT3B isoforms lacking its catalytic domain were highly expressed in HCCs compared with non-tumorous liver tissue. To elucidate the role of DNMT3B in hepatocarcinogenesis, we generated a genetically engineered mouse model with hepatocyte-specific Dnmt3b deletion. The liver of the Dnmt3b-deficient mice exhibited an exacerbation of thioacetamide-induced hepatitis, progression of liver fibrosis and a higher incidence of HCC compared with the liver of the control mice. Whole-genome bisulfite sequencing verified a lower CG methylation level in the Dnmt3b-deficient liver, demonstrating differentially methylated regions throughout the genome. Transcriptome analysis revealed decreased expression of genes related to oxidative phosphorylation in the Dnmt3b-deficient liver. Moreover, primary hepatocytes isolated from the Dnmt3b-deficient mice showed reduced mitochondrial respiratory capacity, leading to the enhancement of oxidative stress in the liver tissue. Our findings suggest the protective role of DNMT3B against chronic inflammation and HCC development via maintaining mitochondrial homeostasis

Single-molecular real-time deep sequencing reveals the dynamics of multi-drug resistant haplotypes and structural variations in the hepatitis C virus genome

While direct-acting antivirals (DAAs) for hepatitis C virus (HCV) have dramatically progressed, patients still suffer from treatment failures. For the radical eradication of HCV, a deeper understanding of multiple resistance-associated substitutions (RASs) at the single-clone level is essential. To understand HCV quasispecies and their dynamics during DAA treatment, we applied single-molecule real-time (SMRT) deep sequencing on sera from 12 patients with genotype-1b HCV infections with DAA treatment failures, both pre- and post-treatment. We identified >3.2 kbp sequences between NS3 and NS5A genes of 187, 539 clones in total, classifying into haplotype codes based on the linkage of seven RAS loci. The number of haplotype codes during the treatment, per sample, significantly decreased from 14.67 ± 9.12 to 6.58 ± 7.1, while the number of nonsynonymous codons on the seven RAS loci, per clone, significantly increased from 1.50 ± 0.92 to 3.64 ± 0.75. In five cases, the minority multi-drug resistant haplotypes at pre-treatment were identical to the major haplotypes at relapse. Moreover, various structural variations (SVs) were detected and their dynamics analysed. These results suggest that SMRT deep sequencing is useful for detecting minority haplotypes and SVs, and to evaluate the dynamics of viral genomes at the single-clone level

Evolutional transition of HBV genome during the persistent infection determined by single-molecule real-time sequencing

BACKGROUND: Although HBV infection is a serious health issue worldwide, the landscape of HBV genome dynamics in the host has not yet been clarified. This study aimed to determine the continuous genome sequence of each HBV clone using a single-molecule real-time sequencing platform, and clarify the dynamics of structural abnormalities during persistent HBV infection without antiviral therapy. PATIENTS AND METHODS: Twenty-five serum specimens were collected from 10 untreated HBV-infected patients. Continuous whole-genome sequencing of each clone was performed using a PacBio Sequel sequencer; the relationship between genomic variations and clinical information was analyzed. The diversity and phylogeny of the viral clones with structural variations were also analyzed. RESULTS: The whole-genome sequences of 797, 352 HBV clones were determined. The deletion was the most common structural abnormality and concentrated in the preS/S and C regions. Hepatitis B e antibody (anti-HBe)-negative samples or samples with high alanine aminotransferase levels have significantly diverse deletions than anti-HBe-positive samples or samples with low alanine aminotransferase levels. Phylogenetic analysis demonstrated that various defective and full-length clones evolve independently and form diverse viral populations. CONCLUSIONS: Single-molecule real-time long-read sequencing revealed the dynamics of genomic quasispecies during the natural course of chronic HBV infections. Defective viral clones are prone to emerge under the condition of active hepatitis, and several types of defective variants can evolve independently of the viral clones with the full-length genome

Mutational spectrum of hepatitis C virus in patients with chronic hepatitis C determined by single molecule real-time sequencing

The emergence of hepatitis C virus (HCV) with resistance-associated substitution (RAS), produced by mutations in the HCV genome, is a major problem in direct acting antivirals (DAA) treatment. This study aimed to clarify the mutational spectrum in HCV-RNA and the substitution pattern for the emergence of RASs in patients with chronic HCV infection. HCV-RNA from two HCV replicon cell lines and the serum HCV-RNA of four non-liver transplant and four post-liver transplant patients with unsuccessful DAA treatment were analyzed using high-accuracy single-molecule real-time long-read sequencing. Transition substitutions, especially A>G and U>C, occurred prominently under DAAs in both non-transplant and post-transplant patients, with a mutational bias identical to that occurring in HCV replicon cell lines during 10-year culturing. These mutational biases were reproduced in natural courses after DAA treatment. RASs emerged via both transition and transversion substitutions. NS3-D168 and NS5A-L31 RASs resulted from transversion mutations, while NS5A-Y93 RASs was caused by transition substitutions. The fidelity of the RNA-dependent RNA polymerase, HCV-NS5B, produces mutational bias in the HCV genome, characterized by dominant transition mutations, notably A>G and U>C substitutions. However, RASs are acquired by both transition and transversion substitutions, and the RASs-positive HCV clones are selected and proliferated under DAA treatment pressure

１分子リアルタイムシーケンシングを用いたＢ型肝炎ウイルス持続感染下におけるウイルスゲノムの進化的変遷の解析

京都大学新制・課程博士博士(医学)甲第24841号医博第5009号新制||医||1068(附属図書館)京都大学大学院医学研究科医学専攻(主査)教授 朝長, 啓造, 教授 波多野, 悦朗, 教授 竹内, 理学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Expansion of viral variants associated with immune escape and impaired virion secretion in patients with HBV reactivation after resolved infection

HBV reactivation could be induced under immunosuppressive conditions in patients with resolved infection. This study aimed to clarify the viral factors associated with the pathogenesis of HBV reactivation in association with the immunosuppressive status. Whole HBV genome sequences were determined from the sera of 24 patients with HBV reactivation, including 8 cases under strong immunosuppression mediated by hematopoietic stem cell transplantation (HSCT) and 16 cases without HSCT. Ultra-deep sequencing revealed that the prevalence of genotype B and the ratio of non-synonymous to synonymous evolutionary changes in the surface (S) gene were significantly higher in non-HSCT cases than in patients with HSCT. Those non-synonymous variants included immune escape (6/16 cases) and MHC class II-restricted T-cell epitope variants (6/16 cases). Furthermore, reactivated HBV in 11 of 16 (69%) non-HSCT cases possessed substitutions associated with impaired virion secretion, including E2G, L77R, L98V, T118K, and Q129H in the S region, and M1I/V in the PreS2 region. In conclusion, virologic features of reactivated HBV clones differed depending on the intensity of the immunosuppressive condition. HBV reactivation triggered by immunosuppressive conditions, especially those without HSCT, was characterized by the expansion of variants associated with immune escape, MHC class II-restricted T-cell epitope alterations, and/or impaired virion secretion

Mediterranean mimicker

投稿時のタイトルは "Familial Mediterranean Fever with Colonic Involvement Mimicking Inflammatory Bowel Diseas

Treatment with Sotrovimab and Casirivimab/Imdevimab Enhances Serum SARS-CoV-2 S Antibody Levels in Patients Infected with the SARS-CoV-2 Delta, Omicron BA.1, and BA.5 Variants

Background: The neutralizing ability of sotrovimab and casirivimab/imdevimab against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is attenuated in the subvariant BA.5. However, the efficacy of sotrovimab in the clinical setting remains to be investigated. Methods: Patients admitted to Kishiwada City Hospital with COVID-19 delta, omicron BA.1, or BA.5 subvariants were evaluated retrospectively for serum SARS-CoV-2 S and N antibody levels using the Elecsys Anti-SARS-CoV-2 assay. Results: In patients with COVID-19 during the BA.5 wave of the COVID-19 pandemic, anti-SARS-CoV-2 S antibody titers (median [interquartile range]) increased from 2154.0 (864.0–6669.3) U/mL on day 0 to 21,371.0 (19,656.3–32,225.0) U/mL on day 3 in the group treated with sotrovimab (N = 40) and were significantly higher than in the group treated with remdesivir plus dexamethasone plus baricitinib (p < 0.001). Conclusion: Treatment with sotrovimab could prevent severe disease in high-risk patients infected with SARS-CoV-2 subvariant BA.5