Japan Agency for Medical Research and Development

ABSTRACTAlterations in the immune system, stemming from sleep/wakefulness disorders, increase the risk of inflammatory pathologies. Orexin, a hypothalamic neuropeptide, regulates sleep and wakefulness. However, the role of orexin in inflammatory responses—whether it is protective or pathological—is still unclear. In this study, our aim was to elucidate the role of orexin in sleep and inflammatory states through the examination of a lipopolysaccharide (LPS)‐induced systemic inflammatory model and the effects of daridorexant, a dual orexin receptor antagonist. Intraperitoneal LPS administration significantly decreased rapid eye‐movement (REM) sleep and wakefulness while increasing non‐REM sleep. Pretreatment with daridorexant enhanced REM sleep recovery in LPS‐induced systemic inflammation, evidenced by extended duration and increased episode frequency. Transcriptomic profiling demonstrated a rise in the expression of pro‐inflammatory cytokines (Cxcl1, Ccl2, Ccl7, and Tnf) within the hypothalamus of LPS‐challenged mice, which was mitigated by daridorexant administration. In addition, daridorexant mitigated LPS‐induced acute lung inflammation. These findings suggest that by reducing pro‐inflammatory cytokine expression, the inhibition of orexin activity mitigates the lethargy associated with systemic inflammation, while also improving sleep quality. This study explores the potential of orexin receptor antagonists as strategic options for inflammatory pathologies and their associated sleep disorders.

ABSTRACTThe endoplasmic reticulum (ER) stress‐response is an adaptive cellular mechanism activated by an accumulation of unfolded proteins within the ER. Although recent evidence shows that the ER stress‐response is activated in aged tissues, and therefore ER stress is considered a candidate driver of aging, the spatiotemporal regulation and roles of the ER stress‐response during aging remain unclear. To address this research gap, we introduced an Ire1‐Xbp1s ER stress‐response pathway‐sensitive reporter into the ultra‐short‐lived vertebrate Nothobranchius furzeri that allows for the analysis of its aging processes within a short period of time. Using this reporter in N. furzeri, we confirmed the previously reported age‐dependent activation of ER stress in various tissues and identified an unexpected role of the Ire1‐Xbp1s ER stress‐response pathway in regulating epidermal tissue homeostasis and aging. The Ire1‐Xbp1s ER stress‐response pathway is active in the young epidermal basal layer but declines with aging. Photo‐isolation chemistry‐based spatial transcriptomics and functional assays revealed that the Ire1‐Xbp1s pathway maintains young epidermal cell proliferation by activating the cell cycle regulator Vcp, whereas the age‐dependent decline in glucose metabolism reduces Ire1‐Xbp1s activity, consequently downregulating cell proliferation. Collectively, our study elucidates a previously unidentified role of the ER stress‐response in skin aging, which can offer insights into therapeutic targets for promoting healthy skin.

Reverse genetics enables the generation of recombinant viruses; however, conventional approaches using full-length cDNA cloned into bacterial artificial chromosomes or plasmids are time-consuming and technically challenging. While the circular polymerase extension reaction (CPER) has been applied to RNA viruses, it has not been used for DNA viruses. Here, we successfully applied CPER to generate infectious recombinant adenoviruses (AdVs) of two serotypes. The resulting viruses replicated comparably to parental strains, and replication was confirmed by immunostaining. These findings demonstrate that CPER is a rapid and efficient platform for reverse genetics of DNA viruses and may accelerate AdV research and therapeutic development.

Renal counterbalance, involving compensatory hypertrophy of the healthy kidney and atrophy of the injured one, remains incompletely understood, particularly at the glomerular level. In this study, we employed NEP25 mice, which selectively express human CD25 in podocytes, enabling precise induction of unilateral podocyte injury through the administration of LMB2, a CD25-targeted immunotoxin. Using a two-kidney, one-nephropathy (2K1N) model, we demonstrated that asymmetric changes in renal blood flow and proteinuria, with histological and transcriptomic analyses uncovering distinct pathological and molecular features between the injured and contralateral healthy kidneys. Notably, an imbalance in intrarenal angiotensin (Ang) II levels was observed, and angiotensin-converting enzyme inhibition ameliorated the glomerular damage and restored perfusion. These findings indicate that local Ang II dysregulation is a key factor in renal counterbalance. Our study provides the first glomerulopathy-based experimental platform to dissect asymmetric renal adaptation, offering fundamental insight into the homeostatic mechanisms of renal function in health and disease.

Non-human primates, such as cynomolgus monkeys, are invaluable experimental models for understanding human biology and disease. Their close genetic relationship to humans makes them essential for studying fundamental human developmental processes and disease progression. Although lentiviral methods for generating transgenic monkeys exist, several inherent technical difficulties limit their utility. To solve this problem, here we establish a non-viral method for generating transgenic cynomolgus monkeys using the piggyBac transposon system. After optimizing our protocol in mice, we show that the co-injection of piggyBac components with sperm into metaphase II-stage oocytes successfully generates transgenic monkeys expressing transgenes throughout their whole bodies. Transgene expression is observed in all examined tissue types, including germ cells, although the levels of expression vary. Insertion analysis further confirms the successful integration of the transgene. We propose that our method will be a practical non-viral protocol for generating transgenic non-human primates.

Patients with hematologic diseases have experienced coronavirus disease 2019 (COVID-19) with a prolonged, progressive course. Here, we present clinical, pathological, and virological analyses of three cases of prolonged COVID-19 among patients undergoing treatment for B-cell lymphoma. These patients had all been treated with anti-CD20 antibody and bendamustine. Despite various antiviral treatments, high severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) levels persisted for >4 weeks, and two of them succumbed to COVID-19. The autopsy showed bronchopneumonia, interstitial pneumonia, alveolar hemorrhage, and fibrosis. Overlapping cytomegalovirus, fungal and/or bacterial infections were also confirmed. Sequencing of SARS-CoV-2 showed accumulation of mutations and changes in variant allele frequencies over time. NSP12 mutations V792I and M794I appeared independently in two cases as COVID-19 progressed. In vitro drug susceptibility analysis and an animal experiment using recombinant SARS-CoV-2 demonstrated that each mutation, V792 and M794I, was independently responsible for remdesivir resistance and attenuated pathogenicity. E340A, E340D, and F342INS mutations in the spike protein were found in one case, which may account for the sotrovimab resistance. Analysis of autopsy specimens indicated heterogeneous distribution of these mutations. In summary, we demonstrated temporal and spatial diversity in SARS-CoV-2 that evolved resistance to various antiviral agents in malignant lymphoma patients under immunodeficient conditions caused by certain types of immunochemotherapies. Strategies may be necessary to prevent the acquisition of drug resistance and improve outcomes, such as the selection of appropriate treatment strategies for lymphoma considering patients' immune status and the institution of early intensive antiviral therapy.

Endosalpingiosis occurs in relatively young women. The incidence of endosalpingiosis exceeds that of other diseases affecting female tissues. As endosalpingiosis is a benign tumor, several women with endosalpingiosis are asymptomatic. Endosalpingiosis is a lesion characterized by the presence of ectopic glandular tissues lined with epithelium similar to the tubal-type epithelium. Therefore, the development of endosalpingiosis is frequently incidentally discovered during abdominal surgery for other diseases. A definitive diagnosis of endosalpingiosis is made by pathological diagnosis and pathological findings on the surgically removed tissue. In some cases, glandular structures comprising a single layer of columnar to cuboidal epithelial cells are observed in surgically removed tissues of the uterine body, bilateral peritoneal tissues, omental tissues, and even lymph node tissues. Recently, our medical staff has experienced cases of endosalpingiosis with a history of breast cancer, which rarely has clinically significant consequences. Elucidating the mechanism of endosalpingiosis will facilitate understanding of the origin of primary tumors in the ovaries and peritoneum. Furthermore, to prevent unnecessary additional surgical treatments (e.g., chemotherapy), differentiating endosalpingiosis from ovarian borderline (low-grade) tumors or dissemination of malignant tumors and from endosalpingiosis and primary peritoneal mesothelioma or serous tumors is essential. We here discuss the historical and medical aspects of tubal endometriosis, which was discovered as an intraabdominal mass and incidentally diagnosed.

Sleep apnea (SA) is prevalent among patients with heart failure (HF) and contributes to a poor prognosis. Sodium-glucose cotransporter 2 (SGLT2) inhibitors have demonstrated efficacy in reducing the risk of serious clinical events in patients with HF. Additionally, SGLT2 inhibitors may reduce the risk of incident SA and mitigate its severity in patients with cardiovascular disease and T2DM. We aimed to investigate whether the SGLT2 inhibitor tofogliflozin reduced the severity of SA, as assessed using the apnea-hypopnea index (AHI), in patients with HF and T2DM and whether a decrease in AHI correlates with changes in body composition and cardiorenal function parameters. This is a single-arm, prospective pathophysiologic study involving patients with HF, T2DM, and SA, defined as having an AHI of 15 events/h and more. SA was assessed using polysomnography. Changes in AHI before and 6 months after starting oral administration of tofogliflozin (20 mg) were assessed. Additionally, body composition and cardiorenal functions were assessed before and 6 months after tofogliflozin administration. Ten patients with HF, T2DM, and SA were finally enrolled (60% men, 66.9 ± 13.4 years). Tofogliflozin reduced AHI from 43.2 [30.2] to 35.3 [13.1] events/h (p = 0.024) at 6 months. Hemoglobin A1c, body weight, and body water content decreased significantly. However, no significant changes were observed in the cardiorenal function parameters. A linear relationship was observed between the changes in body water content and AHI (r = 0.642, p = 0.045). Tofogliflozin reduced AHI, possibly associated with a reduction in body water content.

Pulmonary hypertension (PH) is a devastating disease characterized by vascular remodeling, resulting in right ventricular failure and death. Dysregulation of energy metabolism is linked to PH pathogenesis. Trimetazidine (TMZ), a selective long-chain 3-ketoacyl coenzyme A thiolase inhibitor, is critical in maintaining energy metabolism. Despite the indicated TMZ’s inhibitory effect on pulmonary vascular remodeling in PH development, the integrated evaluation of the changes in biomolecules, such as metabolites and transcripts, that TMZ induces in the lung and heart tissues is largely unknown in vivo. For an improved understanding of the molecular mechanism involving the effects of TMZ on PH development, we performed a comprehensive analysis of the changes in cardiac metabolites and pulmonary transcripts of SU5416-Hypoxia (Su/Hx) rats treated with TMZ. Metabolomic analysis of the Su/Hx-induced PH hearts demonstrated that TMZ reduced the long-chain fatty acid concentration. Additionally, TMZ alleviated PH degree and excessive strain on the right heart functions in rats with Su/Hx-induced PH. We identified the candidate target genes for TMZ treatment during PH development. Interestingly, the mRNA levels of the fatty acid transporters were substantially downregulated by TMZ administration in the lungs with Su/Hx-induced PH. Notably, TMZ suppressed excessive proliferation of human pulmonary artery smooth muscle cells under hypoxic conditions. Our study suggests that TMZ ameliorates PH development by involving energy metabolism in the lungs and heart.

Endosalpingiosis occurs in relatively young women. The incidence of endosalpingiosis exceeds that of other diseases affecting female tissues. As endosalpingiosis is a benign tumor, several women with endosalpingiosis are asymptomatic. Endosalpingiosis is a lesion characterized by the presence of ectopic glandular tissues lined with epithelium similar to the tubal-type epithelium. Therefore, the development of endosalpingiosis is frequently incidentally discovered during abdominal surgery for other diseases. A definitive diagnosis of endosalpingiosis is made by pathological diagnosis and pathological findings on the surgically removed tissue. In some cases, glandular structures comprising a single layer of columnar to cuboidal epithelial cells are observed in surgically removed tissues of the uterine body, bilateral peritoneal tissues, omental tissues, and even lymph node tissues. Recently, our medical staff has experienced cases of endosalpingiosis with a history of breast cancer, which rarely has clinically significant consequences. Elucidating the mechanism of endosalpingiosis will facilitate understanding of the origin of primary tumors in the ovaries and peritoneum. Furthermore, to prevent unnecessary additional surgical treatments (e.g., chemotherapy), differentiating endosalpingiosis from ovarian borderline (low-grade) tumors or dissemination of malignant tumors and from endosalpingiosis and primary peritoneal mesothelioma or serous tumors is essential. We here discuss the historical and medical aspects of tubal endometriosis, which was discovered as an intraabdominal mass and incidentally diagnosed.