Hepatocyte transformation is induced by laminin γ2 monomer.

Serum laminin-γ2 monomer (Lm-γ2m) is a potent predictive biomarker for hepatocellular carcinoma (HCC) onset in patients with hepatitis C infection who achieve a sustained virologic response with liver cirrhosis (LC) and for the onset of extrahepatic metastases in early-stage HCC. Although Lm-γ2m involvement in late-stage cancer progression has been well investigated, its precise roles in HCC onset remain to be systematically investigated. Therefore, we analyzed an HCC model, human hepatocytes and cholangiocytes, and surgically resected liver tissues from patients with HCC to understand the roles of Lm-γ2m in HCC onset. Ck-19- and EpCAM-positive hepatic progenitor cells (HPCs) in the liver of pdgf-c transgenic HCC mouse model with ductular reaction showed ectopic expression of Lm-γ2m. Forced expression of Lm-γ2m in hepatocytes adjacent to HPCs resulted in enhanced tumorigenicity, cell proliferation, and migration in immortalized hepatocytes, but not in cholangiocytes in vitro. Further, pharmacological inhibition of epidermal growth factor receptor (EGFR) and c-Jun activator JNK suppressed Lm-γ2m-induced hepatocyte transformation, suggesting the involvement of EGFR/c-Jun signaling in the transformation, leading to HCC development. Finally, immunohistochemical staining of HCC tissues revealed a high level of Lm-γ2 expression in the HPCs of the liver with ductular reaction in normal liver adjacent to HCC tissues. Overall, HPC-derived Lm-γ2m in normal liver with ductular reaction acts as a paracrine growth factor on surrounding hepatocytes and promotes their cellular transformation through the EGFR/c-Jun signaling pathway. Furthermore, this is the first report on Lm-γ2m expression detected in the normal liver with ductular reaction, a human precancerous lesion of HCC.

Long-term course of a case with a novel homozygous kyphoscoliosis peptidase variant.

We herein report a case with a novel homozygous variant in the kyphoscoliosis peptidase (KY) gene. A 58-year-old Japanese female was referred to our hospital with a gait disturbance that gradually worsened after the age of 50. She had bilateral equinus foot deformity since early childhood. Neurological examination revealed moderate weakness of the neck, trunk, femoral, and brachial muscles, mild respiratory failure, and areflexia. Whole-exome sequencing revealed a novel homozygous frameshift variant of the KY gene, NM_178554.6:c.824del p.(Glu275Glyfs*53). Our case demonstrated that KY-associated neuromuscular disease can present with extremely slow progressive muscle weakness and respiratory failure over a long natural course.

Endoscopic treatment of bile duct stones with benign choledochojejunal anastomotic stenosis.

BACKGROUND AND AIMS: Endoscopic interventions for bile duct stones (BDSs) with benign choledochojejunal anastomotic stenosis (bCJS) are challenging. Therefore, we investigated endoscopic interventions for BDSs with bCJS. METHODS: Seventeen patients with BDSs with bCJS were retrospectively analyzed. Patient characteristics, technical success, adverse events (AEs), and recurrence were evaluated. RESULTS: In 17 patients, the median diameters of the bile duct and BDSs were both 8 mm. The median number of BDSs was 3. The technical success rate was 94% (16/17). Ten patients underwent balloon dilation at the choledochojejunal anastomotic site (CAS), the median diameter of balloon dilation was 10.5 mm, and waist disappearance was achieved in 2. Six patients had fully covered self-expandable metal stents (FCSEMSs) with a diameter of 10 mm placed at the CAS. BDSs were removed after balloon dilation or FCSEMS removal, and 6 of 16 patients were treated with a combination of lithotripsy and 5 with peroral direct cholangioscopy (PDCS). Regarding AEs, perforation at the CAS by balloon dilation occurred in 1 patient. The median follow-up was 3701 days. Nine of 16 patients (56%) had recurrence. The patients treated with a combination of PDCS at BDS removal (P = .022) and waist disappearance at the CAS by balloon dilation (P = .035) had significantly fewer recurrences. CONCLUSIONS: Endoscopic interventions for BDSs with bCJS are useful and relatively safe; however, long-term follow-up showed frequent recurrences. Recurrence was common in patients not treated with the combination of PDCS at BDS removal and those without waist disappearance at the CAS by balloon dilation.

Current status of the cost burden of first-line systemic treatment for patients with advanced hepatocellular carcinoma in Japan, 2021-22.

BACKGROUND: Although recent advances in systemic therapies for hepatocellular carcinoma (HCC) have led to prolonged patient survival, the high costs of the drugs place a heavy burden on both patients and society. The objectives of this study were to examine the treatment regimens used as first-line systemic treatment for patients with advanced HCC in Japan and to estimate the treatment costs per regimen. METHODS: For this study, we aggregated the data of patients who had received first-line systemic treatment for advanced HCC between July 2021 and June 2022. The treatment cost per month of each regimen was estimated based on standard usage, assuming an average weight of 60 kg for male patients. The data were categorized by the treatment regimen, and the treatments were categorized based on the cost into very high-cost (≥1 000 000 Japanese yen [JPY]/month), high-cost (≥500 000 JPY/month) and other (<500 000 JPY/month) treatments. RESULTS: Of the total of 552 patients from 24 institutions whose data were analyzed in this study, 439 (79.5%) received atezolizumab plus bevacizumab, 98 (17.8%) received lenvatinib and 15 (2.7%) received sorafenib as the first-line treatment. The treatment cost per month for each of the above regimens was as follows: atezolizumab plus bevacizumab, 1 176 284 JPY; lenvatinib, 362 295 JPY and sorafenib, 571 644 JPY. In total, 82.2% of patients received high-cost regimens, and the majority of these patients received a very high-cost regimen of atezolizumab plus bevacizumab. CONCLUSIONS: Advances in systemic therapies for HCC have led to prolonged patient survival. However, the treatment costs are also increasing, imposing a burden on both the patients and society.

BMP9-ID1 Pathway Attenuates N6-Methyladenosine Levels of CyclinD1 to Promote Cell Proliferation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly lethal malignant neoplasm, and the involvement of bone morphogenetic protein 9 (BMP9) has been implicated in the pathogenesis of liver diseases and HCC. Our goal was to investigate the role of BMP9 signaling in regulating N6-methyladenosine (m6A) methylation and cell cycle progression, and evaluate the therapeutic potential of BMP receptor inhibitors for HCC treatment. We observed that elevated levels of BMP9 expression in tumor tissues or serum samples from HCC patients were associated with a poorer prognosis. Through in vitro experiments utilizing the m6A dot blotting assay, we ascertained that BMP9 reduced the global RNA m6A methylation level in Huh7 and Hep3B cells, thereby facilitating their cell cycle progression. This effect was mediated by an increase in the expression of the inhibitor of DNA-binding protein 1 (ID1). Additionally, using methylated RNA immunoprecipitation qPCR(MeRIP-qPCR), we showed that the BMP9-ID1 pathway promoted CyclinD1 expression by decreasing the m6A methylation level in the 5' UTR of mRNA. This occurred through the upregulation of the fat mass and obesity-associated protein (FTO) in Huh7 and Hep3B cells. In our in vivo mouse xenograft models, we demonstrated that blocking the BMP receptor with LDN-212854 effectively suppressed HCC growth and induced global RNA m6A methylation. Overall, our findings indicate that the BMP9-ID1 pathway promotes HCC cell proliferation by down-regulating the m6A methylation level in the 5' UTR of CyclinD1 mRNA. Targeting the BMP9-ID1 pathway holds promise as a potential therapeutic strategy for treating HCC.

Influence of oligonucleotides structures for separation of diastereomers by capillary electrophoresis method using polyvinylpyrrolidone 1,300,000.

In the field of oligonucleotides drug discovery, phosphorothioate (PS) modification has been recognized as an effective tool to overcome the nuclease digestion, and generates 2n of possible diastereomers, where n equals the number of PS linkages. However, it is also well known that differences in drug efficacy and toxicity are caused by differences in stereochemistry of oligonucleotides. Therefore, the development of a high-resolution analytical method that enables stereo discrimination of oligonucleotides is desired. Under this circumstance, capillary electrophoresis (CE) using polyvinylpyrrolidone (PVP) is considered as one of the useful tools for the separation analysis of diastereomers. In this study, we evaluated the several oligonucleotides with the structural diversities in order to understand the separation mechanism of the diastereomers by CE. Especially, five kinds of 2'-moieties were deeply examined by CE with PVP 1,300,000 polymer solution. We found that different trend of the peak shapes and the peak resolution were observed among these oligonucleotides. For example, the better peak resolution was observed in 6 mer PS3-DNA compared to the rigid structure of 6 mer PS3-LNA. As for this reason, the computational simulation revealed that difference of accessible surface area caused by the steric structure of thiophosphate in each oligonucleotide is one of the key attributes to explain the separation of the diastereomers. In addition, we achieved the separation of sixteen peak tops of the diastereomers in 6 mer PS4-DNA, and the complete separation of fifteen diastereomers in 6 mer PS4-RNA. These knowledge for the separation of the diastereomers by CE will be expected to the quality control of the oligonucleotide drugs.

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.

Fusion Imaging Objectively Demonstrates Improved Pancreas Visualization through Manipulation Techniques: A Prospective Interventional Study.

Objective Abdominal ultrasonography (AUS) is used to screen for abdominal diseases owing to its low cost, safety, and accessibility. However, the detection rate of pancreatic disease using AUS is unsatisfactory. We evaluated the visualization area of the pancreas and the efficacy of manipulation techniques for AUS with fusion imaging. Methods Magnetic resonance imaging (MRI) volume data were obtained from 20 healthy volunteers in supine and right lateral positions. The MRI volume data were transferred to an ultrasound machine equipped with a fusion imaging software program. We evaluated the visualization area of the pancreas before and after postural changes using AUS with fusion imaging and assessed the liquid-filled stomach method using 500 ml of de-aerated water in 10 randomly selected volunteers. Patients This study included 20 healthy volunteers (19 men and 1 woman) with a mean age of 33.0 (21-37.5) years old. Results Fusion imaging revealed that the visualization area of the entire pancreas using AUS was 55%, which significantly improved to 75% with a postural change and 90% when using the liquid-filled stomach method (p=0.043). Gastrointestinal gas is the main obstacle for visualization of the pancreas. Conclusion Fusion imaging objectively demonstrated that manipulation techniques can improve pancreatic visualization.

Potential utility of l-carnitine for preventing liver tumors derived from metabolic dysfunction-associated steatohepatitis.

BACKGROUND: Recent reports have unveiled the potential utility of l-carnitine to alleviate metabolic dysfunction-associated steatohepatitis (MASH) by enhancing mitochondrial metabolic function. However, its efficacy at preventing the development of HCC has not been assessed fully. METHODS: l-carnitine (2 g/d) was administered to 11 patients with MASH for 10 weeks, and blood liver function tests were performed. Five patients received a serial liver biopsy, and liver histology and hepatic gene expression were evaluated using this tissue. An atherogenic plus high-fat diet MASH mouse model received long-term l-carnitine administration, and liver histology and liver tumor development were evaluated. RESULTS: Ten-week l-carnitine administration significantly improved serum alanine transaminase and aspartate transaminase levels along with a histological improvement in the NAFLD activity score, while steatosis and fibrosis were not improved. Gene expression profiling revealed a significant improvement in the inflammation and profibrotic gene signature as well as the recovery of lipid metabolism. Long-term l-carnitine administration to atherogenic plus high-fat diet MASH mice substantially improved liver histology (inflammation, steatosis, and fibrosis) and significantly reduced the incidence of liver tumors. l-carnitine directly reduced the expression of the MASH-associated and stress-induced transcriptional factor early growth response 1. Early growth response 1 activated the promoter activity of neural precursor cell expressed, developmentally downregulated protein 9 (NEDD9), an oncogenic protein. Thus, l-carnitine reduced the activation of the NEDD9, focal adhesion kinase 1, and AKT oncogenic signaling pathway. CONCLUSIONS: Short-term l-carnitine administration ameliorated MASH through its anti-inflammatory effects. Long-term l-carnitine administration potentially improved the steatosis and fibrosis of MASH and may eventually reduce the risk of HCC.

Maintenance of R-loop structures by phosphorylated hTERT preserves genome integrity.

As aberrant accumulation of RNA-DNA hybrids (R-loops) causes DNA damage and genome instability, cells express regulators of R-loop structures. Here we report that RNA-dependent RNA polymerase (RdRP) activity of human telomerase reverse transcriptase (hTERT) regulates R-loop formation. We found that the phosphorylated form of hTERT (p-hTERT) exhibits RdRP activity in nuclear speckles both in telomerase-positive cells and telomerase-negative cells with alternative lengthening of telomeres (ALT) activity. The p-hTERT did not associate with telomerase RNA component in nuclear speckles but, instead, with TERRA RNAs to resolve R-loops. Targeting of the TERT gene in ALT cells ablated RdRP activity and impaired tumour growth. Using a genome-scale CRISPR loss-of-function screen, we identified Fanconi anaemia/BRCA genes as synthetic lethal partners of hTERT RdRP. Inactivation of RdRP and Fanconi anaemia/BRCA genes caused accumulation of R-loop structures and DNA damage. These findings indicate that RdRP activity of p-hTERT guards against genome instability by removing R-loop structures.

Programmed cell death-1 is involved with peripheral blood immune cell profiles in patients with hepatitis C virus antiviral therapy.

Mutations in the non-structural protein regions of hepatitis C virus (HCV) are a cause of a non-sustained virological response (SVR) to treatment with direct-acting antivirals (DAAs) for chronic hepatitis; however, there are non-SVR cases without these mutations. In this study, we examined immune cell profiles in peripheral blood before and after ombitasvir/paritaprevir/ritonavir treatment and screened for genes that could be used to predict the therapeutic effects of DAAs. Fluorescence-activated cell sorting analysis indicated that the median frequencies of programmed cell death-1-positive (PD-1+) effector regulatory T cells (eTregs), PD-1+CD8+ T cells, and PD-1+Helper T cells were decreased significantly in SVR cases, but without significant changes in non-SVR cases. The frequency of PD-1+ naïve Tregs was significantly higher in the SVR group than in the non-SVR group before and after treatment. Similar results were found in patients treated with other DAAs (e.g., daclatasvir plus asunaprevir) and supported an immune response after HCV therapy. RNA-sequencing analysis indicated a significant increase in the expression of genes associated with the immune response in the SVR group, while genes related to intracellular and extracellular signal transduction were highly expressed in the non-SVR group. Therefore, we searched for genes associated with PD-1+ eTregs and CD8+ T cells that were significantly different between the SVR and non-SVR groups and found that T-box transcription factor 21 was associated with the non-SVR state. These results indicate that PD-1-related signaling pathways are associated with a non-SVR mechanism after DAAs treatment separate from mutation-related drug resistance.

Partial Stent-in-Stent Method with an Uncovered Self-Expandable Metallic Stent for Unresectable Malignant Hilar Bile Duct Obstruction.

(1) Background: There is controversy regarding stent placement for unresectable malignant hilar biliary obstruction (UMHBO). We mainly use the partial stent-in-stent (PSIS) method with an uncovered self-expandable metallic stent (UCSEMS) based on the drainage area and patency period. In this study, we investigated the usefulness and safety of the PSIS method. (2) Methods: In total, 59 patients who underwent the PSIS method for UMHBO at our hospital were included in the study. The technical success rate, clinical success rate, time to recurrent biliary obstruction (TRBO) and overall survival (OS) from the first placement, factors affecting TRBO and OS, and early complications within 30 days after the procedure were evaluated retrospectively. (3) Results: The technical and clinical success rates were 100% and 96.6%, respectively, with a TRBO of 121 days [95% confidence interval: 82-231] and an OS of 194 days [95% confidence interval: 113-305] after the first placement. Early complications occurred in nine patients (15.3%), including five cases of cholangitis, three cases of pancreatitis, and one case of cholecystitis. (4) Conclusions: The PSIS method for UMHBO is safe and useful with high technical and clinical success rates.

Nucleos(t)ide analogs for hepatitis B virus infection differentially regulate the growth factor signaling in hepatocytes.

BACKGROUND: Recent clinical studies have suggested that the risk of developing HCC might be lower in patients with chronic hepatitis B receiving tenofovir disoproxil fumarate than in patients receiving entecavir, although there is no difference in biochemical and virological remission between the 2 drugs. METHODS: The effects of nucleoside analogs (NsAs; lamivudine and entecavir) or nucleotide analogs (NtAs; adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide) on cell growth and the expression of growth signaling molecules in hepatoma cell lines and PXB cells were investigated in vitro. The tumor inhibitory effects of NsAs or NtAs were evaluated using a mouse xenograft model, and protein phosphorylation profiles were investigated. The binding of NsAs or NtAs to the insulin receptor (INSR) was investigated by thermal shift assays. RESULTS: NtAs, but not NsAs, showed direct growth inhibitory effects on hepatoma cell lines in vitro and a mouse model in vivo. A phosphoprotein array revealed that INSR signaling was impaired and the levels of phosphorylated (p)-INSRß and downstream molecules phosphorylated (p)-IRS1, p-AKT, p-Gab1, and p-SHP2 were substantially reduced by NtAs. In addition, p-epidermal growth factor receptor and p-AKT levels were substantially reduced by NtAs. Similar findings were also found in PXB cells and nontumor lesions of liver tissues from patients with chronic hepatitis B. Prodrug NtAs, but not their metabolites (adefovir, adefovir monophosphate, adefovir diphosphate, tenofovir, tenofovir monophosphate, and tenofovir diphosphate), had such effects. A thermal shift assay showed the binding of NtAs to INSRß. CONCLUSIONS: NtAs (adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide), which are adenine derivative acyclic nucleotide analogs, potentially bind to the ATP-binding site of growth factor receptors and inhibit their autophosphorylation, which might reduce the risk of HCC in patients with chronic hepatitis B.

Photodynamic therapy with talaporfin sodium for endoscopically unresectable gastric cancer using a novel simultaneous light-emitting method.

We describe a case of gastric cancer treated by photodynamic therapy (PDT) with talaporfin sodium using a novel simultaneous light-emitting method. An 82-year-old man was diagnosed with gastric cancer near the cardia with suspected deep submucosal invasion. Surgical resection was deemed high-risk owing to an underlying pulmonary disease. After ruling out endoscopic procedures due to intense fibrosis resulting from the scarring, PDT with talaporfin sodium was chosen. PDT was successfully conducted using an endoscope with simultaneous light emission. The patient experienced a complete response to the treatment and showed no signs of recurrence during follow-up. This case highlights the potential of PDT with talaporfin sodium as a viable alternative for challenging cases, particularly in patients unsuitable for surgery and endoscopic resection. Furthermore, the novel simultaneous light-emitting method may improve the efficiency of the procedure. This case demonstrates the potential of PDT in gastric cancer treatment, especially for high-risk patients.

Novel Utility of Leucine-Rich Alpha-2-Glycoprotein as a Biomarker in Ulcerative Colitis: A Predictor of Endoscopic Remission Independent of Symptoms.

Introduction: Leucine-rich alpha-2-glycoprotein (LRG) is a potential biomarker for disease activity and reflects mucosal healing in patients with ulcerative colitis (UC). However, only a few studies have described a detailed sensitivity analysis of LRG in predicting mucosal healing in patients. This study aimed to evaluate the association between LRG and the endoscopic activity of UC and its predictability for mucosal healing and explore the utility and clinical application of LRG. Methods: The diagnostic accuracy of biomarkers, including LRG, in predicting the endoscopic activity of UC was evaluated. All consecutive patients who underwent total colonoscopy between April 2021 and September 2022 were included. The Mayo endoscopic subscore (MES) was used for assessing endoscopic activity. Furthermore, endoscopic remission was defined as an MES of ≤1. Clinical activity was evaluated based on stool frequency and bloody stool. Receiver operating characteristic curve analysis and binary logistic regression were performed to assess the diagnostic accuracy of the biomarkers. We evaluated LRG trends and treatment response in patients with MES ≥2 who underwent induction therapy. Results: This study comprised 214 patients. The proportions of endoscopically and clinically active patients were 33.6% and 49.1%, respectively. LRG had an area under the curve (AUC) of 0.856, with a higher diagnostic accuracy than other biomarkers, such as C-reactive protein, leukocyte, neutrophil, platelet, and albumin. The cutoff value for LRG was 15.6 µg/mL (sensitivity, 72.2%; specificity, 86.6%). Using the MES, patients with higher scores had higher LRG levels than those with lower scores. The cutoff value, AUC, sensitivity, and specificity varied with a higher AUC for left-sided colitis and pancolitis than for proctitis. Logistic regression analysis showed that LRG was an independent predictor of endoscopic remission using multivariate analysis, even with the factor of clinical activity. The change ratio of LRG pre- and post-treatment was statistically significant in the higher LRG group. Conclusion: LRG reflected endoscopic activity independently, regardless of clinical symptoms. An LRG below the cutoff value could indicate a significantly low probability of endoscopic activity in asymptomatic patients, and follow-up endoscopy (not for cancer screening) may be unnecessary. Furthermore, a higher LRG level might be more useful as an indicator of treatment efficacy.

Origin of Transthyretin in Cerebral Amyloid Angiopathy.

This case series evaluates the correlation between transthyretin and cerebral amyloid angiopathy progression in individuals with hereditary transthyretin amyloidosis.