Protease-Activated Receptor 1 Enhances Poly I:C Induction of the Antiviral Response in Macrophages and Mice

The coagulation cascade is activated during viral infections as part of the host defense system. Coagulation proteases activate cells by cleavage of protease-activated receptors (PAR). Recently, we reported that activation of PAR-1enhanced interferon (IFN)-β and CXCL10 expression in cardiac fibroblasts and in the hearts of mice infected with Coxsackievirus B3. In this study, we used the double-stranded RNA mimetic polyinosinic:polycytidylic acid (poly I:C) to induce an anti-viral response in macrophages and mice. Activation of PAR-1 enhanced poly I:C induction of IFNβ and CXCL10 expression in the murine macrophage cell line RAW264.7, bone-marrow derived mouse macrophages (BMM) and mouse splenocytes. Next, poly I:C was used to induce a type I IFN innate immune response in the spleen and plasma of wild-type (WT) and PAR-1−/− mice. We found that poly I:C treated PAR-1−/− mice and WT mice given the thrombin inhibitor dabigatran etexilate exhibited significantly less IFNβ and CXCL10 expression in the spleen and plasma compared to WT mice. These studies suggest that thrombin activation of PAR1 contributes to the anti-viral response in mice

コンバラトキシンによる凝固亢進における単球由来組織因子陽性細胞外小胞の関与

Objectives: Convallatoxin (CNT) is a natural cardiac glycoside extracted from lily of the valley (Convallaria majalis). Although it is empirically known to cause blood coagulation disorders, the underlying mechanism remains unclear. CNT exerts cytotoxicity and increases tissue factor (TF) expression in endothelial cells. However, the direct action of CNT on blood coagulation remains unclear. Therefore, herein, we investigated the effects of CNT on whole blood coagulation system and TF expression in monocytes. Methods: Blood samples were collected from healthy volunteers to measure plasma thrombin–antithrombin complex (TAT) concentration using ELISA and to perform rotational thromboelastometry (ROTEM) and whole-blood extracellular vesicle (EV)-associated TF (EV-TF) analysis. The effects of CNT were also investigated using the monocytic human cell line THP-1. Quantitative real-time PCR and western blotting were performed, and PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, was used to elucidate the action mechanism of CNT-mediated TF production. Results: CNT treatment increased EV-TF activity, shortened the whole blood clotting time in rotational thromboelastometry analysis, and increased TAT levels, which is an index of thrombin generation. Furthermore, CNT increased TF mRNA expression in THP-1 cells and EV-TF activity in the cell culture supernatant. Therefore, CNT may induce a hypercoagulable state with thrombin generation, in which elevated EV-TF activity derived from monocytes might be involved. These procoagulant effects of CNT were reversed by PD98059, suggesting that CNT-induced TF production in monocytes might be mediated by the MAPK pathway. Conclusions: The findings of the present study have further clarified the procoagulant properties of CNT.本文は発行元が定める公開猶予期間終了後に公開

Microvesicle Tissue Factor Activity and Interleukin-8 Levels are Associated with Mortality in Patients with Influenza A/H1N1 Infection

To identify plasma biomarkers that can be early predictors of mortality in critically ill patients with primary influenza A/H1N1

Characterization of the MASCOT landing area by Hayabusa2

Context. After landing on C-type asteroid Ryugu, MASCOT imaged brightly colored, submillimeter-sized inclusions in a small rock. Hayabusa2 successfully returned a sample of small particles from the surface of Ryugu, but none of these appear to harbor such inclusions. The samples are considered representative of Ryugu. Aims. To understand the apparent discrepancy between MASCOT observations and Ryugu samples, we assess whether the MASCOT landing site, and the rock by implication, is perhaps atypical for Ryugu. Methods. We analyzed observations of the MASCOT landing area acquired by three instruments on board Hayabusa2: a camera (ONC), a near-infrared spectrometer (NIRS3), and a thermal infrared imager. We compared the landing area properties thus retrieved with those of the average Ryugu surface. Results. We selected several areas and landforms in the landing area for analysis: a small crater, a collection of smooth rocks, and the landing site itself. The crater is relatively blue and the rocks are relatively red. The spectral and thermophysical properties of the landing site are very close to those of the average Ryugu surface. The spectral properties of the MASCOT rock are probably close to average, but its thermal inertia may be somewhat higher. Conclusions. The MASCOT rock can also be considered representative of Ryugu. Some of the submillimeter-sized particles in the returned samples stand out because of their atypical spectral properties. Such particles may be present as inclusions in the MASCOT rock

microRNA-345の過剰発現は、MUC1およびTJP2の発現を抑制することにより、膵管腺癌細胞株の浸潤能に影響を及ぼす

The majority of pancreatic carcinomas are pancreatic ductal adenocarcinomas (PDAC), and the presence of non-invasive pancreatic intraepithelial neoplasia or intraductal papillary mucinous neoplasm, as an associated lesion, is considered important. These microscopic hyperplastic or grossly papillomatous lesions exhibit varying degrees of morphological atypia and may develop into invasive carcinomas. In this study, we investigated whether mucin-1 (MUC1) is involved in the progression of pancreatic carcinoma and examined the mechanisms by which microRNAs regulate MUC1 expression in vitro. In PDAC cell lines, suppression of MUC1 expression reduced cell proliferation and invasion; PDAC cell lines transfected with an miR-345 precursor suppressed the expression of MUC1, and reduced cell proliferation and invasion. Tight junction protein 2 (TJP2), a putative target of miR-345, is regulated by MUC1. The suppression of TJP2 expression reduced cell proliferation by inducing apoptosis. These results suggest that MUC1 and TJP2, the putative target molecules of miR-345, are critical in maintaining the invasive potential of pancreatic carcinoma cells, and regulating their expression may prevent the progression of non-invasive pancreatic intraductal lesions to invasive carcinomas. This study provides new insights for the development of novel molecular targeted therapies for pancreatic carcinomas.博士（医学）・甲第866号・令和5年3月15

犬モデルにおける ex vivo および in vivo 遺伝子治療のための代替遺伝子導入技術の開発

Introduction: Gene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused. Methods: Green fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections. Results: No major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime. Conclusions: Two types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.博士（医学）・乙第1517号・令和3年12月21日© 2021, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/)

Repression of factor VIII inhibitor development with apoptotic factor VIII-expressing embryonic stem cells

Development of factor VIII (fVIII)-neutralizing antibodies, called inhibitors, is a challenging problem in the management of hemophilia A patients. We explored the possibility of pretreatment with apoptotic fVIII-expressing embryonic stem (ES) cells to prevent the development of fVIII inhibitors. Murine ES cells integrated with the human F8 gene were differentiated into embryoid bodies, dissociated to a single cell suspension, subjected to hypo-osmotic shock to induce apoptosis, and intraperitoneally injected into hemophilia A mice. Inhibitors were induced by periodic intraperitoneal injections of recombinant human fVIII (rhfVIII). In the groups in which intraperitoneal injections of rhfVIII began at 1-3 weeks after pretreatment, the titers of inhibitors were significantly lower after the third administration of rhfVIII compared with that in the control group in which apoptotic Ainv18 ES cells (without the human F8 gene) were used for pretreatment, and continued to show lower levels until the sixth administration of rhfVIII. These results suggest that pretreatment with apoptotic hfVIII-expressing ES cells might be promising for the prevention of fVIII inhibitor development in hemophilia A patients

Hepatocyte tissue factor contributes to the hypercoagulable state in a mouse model of chronic liver injury

Patients with chronic liver disease and cirrhosis have a dysregulated coagulation system and are prone to thrombosis. The basis for this hypercoagulable state is not completely understood. Tissue factor (TF) is the primary initiator of coagulation in vivo. Patients with cirrhosis have increased TF activity in white blood cells and circulating microparticles. The aim of our study was to determine the contribution of TF to the hypercoagulable state in a mouse model of chronic liver injury

Incidence of endocrine-related immune-related adverse events in Japanese subjects with various types of cancer

BackgroundImmune checkpoint inhibitors (ICIs), such as cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitors, programmed cell death protein 1 (PD-1) inhibitors, and programmed cell death protein 1 ligand 1 (PD-L1) inhibitors, are often used to treat a variety of malignancies. ICIs are known to cause endocrine-related immune-related adverse events (irAEs), but the incidence varies among reports and/or agents. This study evaluated the incidence of endocrine-related irAEs in patients who were treated with ICIs in Japan.MethodThis single-center, retrospective, observational study examined the incidence and clinical characteristics of endocrine-related irAEs in 466 participants who were treated with ICIs at Kawasaki Medical School Hospital.ResultThe mean age of participants with and without endocrine-related irAEs was 69.1 ± 1.8 years and 68.1 ± 1.1 years, respectively, with no difference between them. The overall incidence of any endocrine-related irAEs among the participants was 25.5%. Hypothyroidism was prevalent in 24.3%, hypoadrenocorticism in 3.2%, hypopituitarism in 0.9%, and insulin-dependent diabetes mellitus in 1.1%. Participants receiving combination therapy with CTLA-4 and PD-1 inhibitors had a significantly higher incidence of endocrine-related irAEs than those receiving monotherapy.ConclusionEndocrine-related irAEs correlated significantly with survival and mean observation period. There was substantial difference in the incidence of endocrine-related irAEs among various types of ICIs and types of cancer. We should bear in mind that endocrine testing is necessary during the treatment with ICIs

Risk factors for acute exacerbation following bronchoalveolar lavage in patients with suspected idiopathic pulmonary fibrosis: A retrospective cohort study

Introduction: Bronchoalveolar lavage (BAL) is useful for diagnosing diffuse lung disease and excluding other conditions. However, acute exacerbations (AEs) are recognized as important complications of BAL in patients with idiopathic pulmonary fibrosis (IPF). This study aimed to identify risk factors for BAL-induced AEs in patients with IPF.Material and methods: We retrospectively analyzed the data of 155 patients with suspected IPF who had undergone BAL between January 2013 and December 2018. BAL-related AE was defined as the development of AE within 30 days after the procedure. We compared clinical features and parameters between patients with AE (AE group) and without AE (non-AE group). We also reviewed the relevant reported literature.Results: Among the 155 patients, 5 (3.2%) developed AE within 30 days after BAL. The average duration from BAL to AE onset was 7.8 days (2–16 days). Results from the univariate analysis revealed PaO2 < 75 mm Hg (p = 0.036), neutrophil content in BAL ≥ 7% (p = 0.0061), %DLCO < 50% (p = 0.019), Gender-Age-Physiology (GAP) stage III (p = 0.034), and BAL recovery rates < 30% (p < 0.001) as significant risk factors for post-BAL AE. All five patients who developed AE recovered and were discharged.Conclusions: Disease severity, high neutrophil levels in BAL, and poor BAL recovery rates may be risk factors for BAL-induced AE