Visualizing bovine leukemia virus (BLV)-infected cells and measuring BLV proviral loads in the milk of BLV seropositive dams

International audienceAbstractBovine leukemia virus (BLV) infects cattle and causes serious problems for the cattle industry, worldwide. Vertical transmission of BLV occurs via in utero infection and ingestion of infected milk and colostrum. The aim of this study was to clarify whether milk is a risk factor in BLV transmission by quantifying proviral loads in milk and visualizing the infectivity of milk. We collected blood and milk from 48 dams (46 BLV seropositive dams and 2 seronegative dams) from seven farms in Japan and detected the BLV provirus in 43 blood samples (89.6%) but only 22 milk samples (45.8%) using BLV-CoCoMo-qPCR-2. Although the proviral loads in the milk tended to be lower, a positive correlation was firstly found between the proviral loads with blood and milk. Furthermore, the infectivity of milk cells with BLV was visualized ex vivo using a luminescence syncytium induction assay (LuSIA) based on CC81-GREMG cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) in response to BLV Tax and Env expressions when co-cultured with BLV-infected cells. Interestingly, in addition to one BLV-infected dam with lymphoma, syncytia with EGFP fluorescence were observed in milk cells from six BLV-infected, but healthy, dams by an improved LuSIA, which was optimized for milk cells. This is the first report demonstrating the infectious capacity of cells in milk from BLV-infected dams by visualization of BLV infection ex vivo. Thus, our results suggest that milk is a potential risk factor for BLV vertical spread through cell to cell transmission

Dataset for: A novel diet-induced murine model of steatohepatitis with fibrosis for screening and evaluation of drug candidates for non-alcoholic steatohepatitis

Many animal models of non-alcoholic steatohepatitis have been reported. While these models exhibit mild onset of hepatitis and fibrosis, induction is often slow. For faster screening of drug candidates, there is a compelling need for convenient animal models of steatohepatitis and non-alcoholic steatohepatitis in which fatty liver and hepatitis are stably induced within a short period. Here, we analyzed the hepatic lipid composition in non-alcoholic steatohepatitis, and used this information to successfully establish a murine model where steatohepatitis is induced within only 1 week using a novel diet (steatohepatitis-inducing high-fat diet, STHD-01) high in saturated fatty acids and cholesterol. After receiving STHD-01 for 1 week, normal mice (C57BL/6J) showed elevated markers of fatty liver and hepatitis, including hepatic triglycerides and plasma alanine aminotransferase; the administration of angiotensin receptor blockers reduced these symptoms. Furthermore, we confirmed that STHD-01 administration for 36 weeks induced not only sustained elevation of hepatic triglyceride and plasma alanine aminotransferase levels, but also fibrosis and tumor formation. Pretreatment with the carcinogen diethylnitrosamine accelerated tumor formation, and hepatic lesions were observed within 30 weeks of STHD-01 feeding following diethylnitrosamine pretreatment. Finally, branched-chain amino acids, known to reduce the risk for hepatocellular carcinoma in preclinical models, were effective in reducing the progression of liver fibrosis induced by STHD-01 feeding after diethylnitrosamine pretreatment. We concluded that STHD-01 administration successfully induces steatohepatitis within a short period of time. The proposed murine model is suitable for studying the long-term effects of pharmaceutical agents targeting steatohepatitis, fibrosis, and tumor formation

Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin

<div><p>Differentiation of cancer stem cells (CSCs) into cancer cells causes increased sensitivity to chemotherapeutic agents. Although inhibition of mammalian target of rapamycin (mTOR) leads to CSC survival, the effect of branched chain amino acids (BCAAs), an mTOR complex 1 (mTORC1) activator remains unknown. In this study, we examined the effects of BCAA on hepatocellular carcinoma (HCC) cells expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 5-fluorouracil (FU) on expression of EpCAM and other CSC-related markers, as well as cell proliferation in HCC cells and in a xenograft mouse model. We also characterized CSC-related and mTOR signal-related molecule expression and tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or overexpression of constitutively active rheb (caRheb). mTOR signal-related molecule expression was also examined in BCAA-treated HCC cells. <i>In-vitro</i> BCAA reduced the frequency of EpCAM-positive cells and improved sensitivity to the anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high doses of BCAA activated mTORC1. Knockdown and overexpression experiments revealed that inhibition of mTOR complex 2 (mTORC2) or activation of mTORC1 led to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance the sensitivity to chemotherapy by reducing the population of cscs via the mTOR pathway. This result suggests the utility of BCAA in liver cancer therapy. </p> </div

Treatment of Chronic Constipation using Elobixibat in a Real-World Setting: A Retrospective Cohort Study using an Electronic Medical Records Database in Japan

ABSTRACT: Background: Chronic constipation is a common condition affecting people of all ages; therefore, the socioeconomic burden of chronic constipation is nonnegligible. Elobixibat (ELO), an ileal bail acid transport inhibitor, was launched in Japan in 2018. However, evidence of its use in diverse populations is limited. Objectives: This study aimed to evaluate the prescription of ELO, risk factors associated with ELO discontinuation, and the continuation of stimulants or saline laxatives during ELO treatment in a real-world setting using an extensive electronic medical records database that primarily includes data from acute-care hospitals. Methods: Data of patients prescribed for ELO from April 1, 2018, to March 31, 2022, were extracted from the database. The discontinuation of ELO and stimulant or saline laxatives during ELO treatment was evaluated using the Kaplan-Meier method. The Cox proportional hazards model evaluated risk factors associated with laxative discontinuation. Results: In total, 11,062 patients were evaluated. The rate of ELO discontinuation within 360 days of initiation was 78.7%. Hospitalized at the ELO initiation, stage 5 chronic kidney disease, and diagnosis of constipation by departments of obstetrics and gynecology or by departments of malignant neoplasm were identified as risk factors for discontinuation. Diagnosis of constipation, diabetes mellitus, Parkinson's disease, and previous laxative treatment was associated with a lower risk of ELO discontinuation. The prescription rate of stimulants and saline laxatives markedly decreased after ELO initiation; furthermore, nearly half of patients who were continuously prescribed ELO discontinued these laxatives within 360 days. Conclusions: The discontinuation of ELO was associated with various factors and using ELO may be beneficial in the withdrawal of concurrent stimulants and saline laxatives. These findings may help effectively manage chronic constipation

Changes in the percentage of EpCAM-positive cells upon control medium (DMEM containing 10% FBS) with 4 mM BCAA stimulation or 100 nM rapamycin pretreatment and 4 mM BCAA stimulation (A) or liver cirrhosis modified medium (LC) containing 10% FBS stimulation (B) for 72 h in Huh7 by using the target activation protocol of array scan.

<div><p>The rate change of EpCAM-positive cells in 5000 cells with overexpression of caRheb or control plasmid cDNA (pc DNA) in control medium (DMEM containing 10% FBS) with and without 4 mM BCAA stimulation for 24 h in Huh7 (C).</p> <p>The detection of P70S6 kinase phosphorylation, a member of downstream mTOR signaling, in the presence of DMEM, BCAA treatment, pretreatment with rapamycin and BCAA treatment, or LC stimulation for 72 h in Huh7 (A,B). </p> <p>Tukey’s test **p < 0.01 vs. control, $$$p < 0.001 vs. BCAA, n = 8, mean ± SE (A).</p> <p>Student t-test *p < 0.05, ****p < 0.0001, n = 8, mean ± SE (B,C).</p></div

The rate change of EpCAM positive cell in 5000 cells in the presence of 100 nM rapamycin treatment for 1 h or 72 h (A).

<div><p>Dunnett's test, *p < 0.05, ***p <0.001 vs. control, n = 6, mean ± SE.</p> <p>The relative expressions of EpCAM, c-myc, and FOXO3a mRNA upon Raptor and Rictor knockdown (B-D).</p> <p>Dunnett's test, **p < 0.01, ***p < 0.001 vs. control n = 8, mean ± SE.</p></div

Changes in the percentage of EpCAM-positive cells by using the target activation array scan protocol (A), and CYP3A4 and Bmi mRNA levels (B) in HAK1-B cells cultured in RPMI1640 containing 10% FBS only, or with 1, 2, or 4 mM BCAA added for 72 h.

<div><p>Dunnett's test, *p < 0.05, **p < 0.01 n = 6, mean ± SE.</p> <p>The percentage of Annexin V-positive cells (C) and relative viable cell number (D) by array scan in HAK-1B cells cultured in RPMI1640 containing 10% FBS with or without 2 mM BCAA in the presence (1 or 2 µg/mL) or absence of 5-FU for 72 h by using target activation protocol of array scan. Student <i>t</i>-test, *p < 0.05, **p < 0.01, n = 7, mean ± SE.</p></div

Protein expression and phosphorylation in Huh7 cells under Knockdown conditions for 5 days and overexpression conditions for 1 day, and detection of phosphorylation after 4 mM BCAA treatment for 30 min.

<div><p>Rictor or Raptor Knockdown compared to negative control (NC), caRheb compared to control plasmid cDNA (pc DNA), BCAA treatment compared to DMEM (FBS 10%) only (Ctrl) (A). The average tumor volumes and tumorigenesis ratio at the 4^th week in a xenograft model with transplanted cells with negative control, knockdown of Raptor, Rictor for 5 days, or overexpression of control plasmid DNA, caRheb for 1 day (C), and tumorigenesis rate (B).</p> <p>Dunnett's test, *p<0.05, ***p < 0.001 vs. N.C. n = 5, mean ± SE.</p></div