High-mobility group box 1 fragment ameliorates chronic pancreatitis induced by caerulein in mice.

BACKGROUND: Chronic pancreatitis (CP) is a progressive disease characterized by pancreatic fibrosis for which effective treatment options are lacking. Mesenchymal stem cells (MSCs) have shown potential for fibrosis treatment but face limitations in clinical application. The high-mobility group box 1 (HMGB1) fragment mobilizes MSCs from bone marrow into the blood and has emerged as a promising therapeutic agent for tissue regeneration in various pathological conditions. The aim of this study was to investigate the potential therapeutic effects of systemic administration of the HMGB1 fragment in a mouse model of CP. METHODS: A caerulein-induced CP mouse model was used, and the HMGB1 fragment was administered by tail vein injection. Parameters such as body weight, pancreatic tissue damage, fibrosis, inflammatory cytokine expression, and collagen-related gene expression were evaluated using various assays, including immunohistochemistry, real-time PCR, serum analysis, and single-cell transcriptome analysis. And the migration of MSCs to the pancreas was evaluated using the parabiosis model. RESULTS: Administration of the HMGB1 fragment was associated with significant improvements in pancreatic tissue damage and fibrosis. It suppressed the expression of inflammatory cytokines and activated platelet-derived growth factor receptor-α+ MSCs, leading to their accumulation in the pancreas. The HMGB1 fragment also shifted gene expression patterns associated with pancreatic fibrosis toward those of the normal pancreas. Systemic administration of the HMGB1 fragment demonstrated therapeutic efficacy in attenuating pancreatic tissue damage and fibrosis in a CP mouse model. CONCLUSION: These findings highlight the potential of the HMGB1 fragment as a therapeutic target for the treatment of CP.

Isolated massive pleural effusion as a manifestation of chronic graft versus host disease successfully treated with corticosteroid.

Isolated pleural effusion is a rare manifestation of chronic graft versus host disease (cGVHD) after hematopoietic stem cell transplantation (HSCT). We herein report a 58-year-old woman presenting with massive pleural effusion approximately 1 year after allogeneic HSCT, who was successfully treated with corticosteroid. She had discontinued tacrolimus approximately 1 month before she presented with pleural effusion, which was attributed to cGVHD after a thorough exclusion process. This case illustrates a unique manifestation of atypical cGVHD and highlights the need for prompt therapy initiation.

Neuropathies Following an Ultrasound-Guided Axillary Brachial Plexus Block.

Purpose: Ultrasound-guided brachial plexus block (UGBPB) has interscalene, supraclavicular, infraclavicular, and axillary approaches. The axillary block is considered to be the safest and with fewer adverse events compared to the interscalene (eg, phrenic nerve block, spinal cord or vertebral artery puncture) and supraclavicular (eg, pneumothorax). However, with regard to postoperative neurological symptoms (PONS), it is controversial whether its incidence after an axillary block was higher than that after non-axillary approaches". In this study, we investigated whether the incidence of a neuropathy after an axillary block was higher than that after non-axillary approaches. Patients and Methods: This was a single-center, retrospective cohort study. All UGBPBs were performed under general anesthesia between January 2014 and March 2020. The outcomes included the overall incidence of PONS and neuropathies for axillary and non-axillary approaches. The etiology, symptoms, and outcomes of patients were investigated. Results: Of the 992 patients, 143 (14%) and 849 (86%) were subjected to axillary and non-axillary approaches, respectively. Among 19 cases (19.2:1000; 95% confidence interval [CI], 18.2-20.1) of PONS, four (4.0:1000; 95% CI, 3.8-4.2) were neuropathies attributed to the UGBPB, three (21.0:1000; 95% CI, 18.1-23.8) to the axillary and one (2.8:1000; 95% CI, 2.6-3.1) to non-axillary approaches. The incidence of neuropathies after an axillary block was significantly higher than that after non-axillary approaches (P = 0.005). Conclusion: The incidence of neuropathies after US-guided axillary block under general anesthesia was significantly higher than that after non-axillary approaches.

The role of mesenchymal estrogen receptor 1 in mouse uterus in response to estrogen.

Estrogens play important roles in uterine growth and homeostasis through estrogen receptors (ESR1 and ESR2). To address the role of ESR1-mediated tissue events in the murine uterus, we analyzed mice with a mesenchymal tissue-specific knockout of Esr1. Isl1-driven Cre expression generated Esr1 deletion in the uterine stroma and endometrium (Isl-Esr1KO). We showed that overall structure of the Isl1-Esr1KO mouse uterus developed normally, but estrogen responsiveness and subsequent growth were defective, suggesting that mesenchymal ESR1 is necessary for both epithelial and mesenchymal cell proliferation. Furthermore, RNA-seq analysis revealed that the majority of estrogen-induced genes were regulated by stromal ESR1. In control mice, E2 administration induced 9476 up-regulated differentially expressed genes (DEGs), whereas only 1801 up-regulated DEGs were induced by E2 in Isl1-Esr1KO mice. We further showed that stromal ESR1-regulated genes in the mouse uterus included several growth factors and cytokines, which are potential factors that regulate epithelial and stromal tissue interaction, and also genes involved in lipid homeostasis. Therefore, we infer that stromal ESR1 expression is indispensable for most estrogen actions in the mouse uterus and the current results provide new insights into estrogen-mediated homeostasis in female reproductive organs.

Postoperative Heart Failure with Preserved Ejection Fraction Induced by Flumazenil Administered for Remimazolam Antagonism.

Remimazolam is an ultrashort-acting benzodiazepine intravenous anesthetic characterized by rapid awakening after anesthesia. However, the method for administering remimazolam in clinical practice remains unclear. Here, we report a case of postoperative heart failure with preserved ejection fraction (HFpEF) after antagonizing remimazolam with flumazenil. An 82-year-old woman was scheduled to undergo lumbar laminectomy for lumbar spinal canal stenosis. Preoperative echocardiography revealed normal left ventricular systolic function, left atrial enlargement, and impaired left ventricular diastolic function. General anesthesia was induced with 10 mg/kg/h remimazolam and maintained with 0.8 mg/kg/h remimazolam intraoperatively. Before extubation, a total of 1.0 mg of flumazenil was administered. After extubation, the patient developed pulmonary edema due to HFpEF. When remimazolam is administered in elderly patients with cardiac dysfunction, the maintenance dose should be customized according to the patient's general condition to minimize the dosage of flumazenil.

Anti-Inflammatory Action of Dexmedetomidine on Human Microglial Cells.

Neuroinflammation, where inflammatory cytokines are produced in excess, contributes to the pathogenesis of delirium. Microglial cells play a central role in neuroinflammation by producing and releasing inflammatory cytokines in response to infection, tissue damage and neurodegeneration. Dexmedetomidine (DEX) is a sedative, which reduces the incidence of delirium. Thus, we hypothesized that DEX may alleviate delirium by exhibiting anti-inflammatory action on microglia. In the present study, we investigated the anti-inflammatory action of DEX on human microglial HMC3 cells. The results indicated that DEX partially suppressed the IL-6 and IL-8 production by lipopolysaccharide (LPS)-stimulated HMC3 cells as well as the phosphorylation of p38 MAPK and IκB and the translocation of NF-κB. Furthermore, DEX substantially suppressed IL-6 and IL-8 production by unstimulated HMC3 cells as wells as the phosphorylation of p38 MAPK and IκB and the translocation of NF-κB. These observations suggest that DEX exhibits anti-inflammatory action on not only LPS-stimulated but also unstimulated microglial cells via the suppression of inflammatory signaling and cytokine production.

Generation of a recessive dystrophic epidermolysis bullosa mouse model with patient-derived compound heterozygous mutations.

Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable genetic disease of the skin caused by mutations in the COL7A1 gene. The majority of patients with RDEB harbor compound heterozygous mutations-two distinct mutations on each chromosome-without any apparent hotspots in the COL7A1 mutation pattern. This situation has made it challenging to establish a reliable RDEB mouse model with mutations that accurately mimic the genomic background of patients. Here, we established an RDEB mouse model harboring patient-type mutations in a compound heterozygous manner, using the CRISPR-based genome-editing technology i-GONAD. We selected two mutations, c.5818delC and E2857X, that have frequently been identified in cohorts of Japanese patients with RDEB. These mutations were introduced into the mouse genome at locations corresponding to those identified in patients. Mice homozygous for the 5818delC mutation developed severe RDEB-like phenotypes and died immediately after birth, whereas E2857X homozygous mice did not have a shortened lifespan compared to wild-type mice. Adult E2857X homozygous mice showed hair abnormalities, syndactyly, and nail dystrophy; these findings indicate that E2857X is indeed pathogenic in mice. Mice with the c.5818delC/E2857X compound heterozygous mutation presented an intermediate phenotype between the c.5818delC and E2857X homozygous mice. Single-cell RNA sequencing further clarified that the intrafollicular keratinocytes in c.5818delC/E2857X compound heterozygous mice exhibited abnormalities in cell cycle regulation. The proposed strategy to produce compound heterozygous mice, in addition to the established mouse line, will facilitate research on RDEB pathogenesis to develop a cure for this devastating disease.

CAMK2G is identified as a novel therapeutic target for myelofibrosis.

Although JAK1/2 inhibition is effective in alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here, we established induced pluripotent stem cells (iPSCs) derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were used for chemical screening, and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutation recurrently detected in MF patients, were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 γ subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515L decreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes, such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.

PDGFRα-lineage origin directs monocytes to trafficking proficiency to support peripheral immunity.

Multiple embryonic precursors give rise to leukocytes in adults while the lineage-based functional impacts are underappreciated. Mesodermal precursors expressing PDGFRα appear transiently during E7.5-8.5 descend to a subset of Lin- Sca1+ Kit+ hematopoietic progenitors found in adult BM. By analyzing a PDGFRα-lineage tracing mouse line, we here report that PDGFRα-lineage BM F4/80+ SSClo monocytes/macrophages are solely Ly6C+ LFA-1hi Mac-1hi monocytes enriched on the abluminal sinusoidal endothelium while Ly6C- LFA-1lo Mac-1lo macrophages are mostly from non-PDGFRα-lineage in vivo. Monocytes with stronger integrin profiles outcompete macrophages for adhesion on an endothelial monolayer or surfaces coated with ICAM-1-Fc or VCAM-1-Fc. Egress of PDGFRα-lineage-rich monocytes and subsequent differentiation to peripheral macrophages spatially segregates them from non-PDGFRα-lineage BM-resident macrophages and allows functional specialization since macrophages derived from these egressing monocytes differ in morphology, phenotype, and functionality from BM-resident macrophages in culture. Extravasation preference for blood PDGFRα-lineage monocytes varies by tissues and governs the local lineage composition of macrophages. More PDGFRα-lineage classical monocytes infiltrated into skin and colon but not into peritoneum. Accordingly, transcriptomic analytics indicated augmented inflammatory cascades in dermatitis skin of BM-chimeric mice harbouring only PDGFRα-lineage leukocytes. Thus, the PDGFRα-lineage origin biasedly generates monocytes predestined for BM exit to support peripheral immunity following extravasation and macrophage differentiation.

Controlled induction of immune tolerance by mesenchymal stem cells transferred by maternal microchimerism.

Feto-maternal immune tolerance is established during pregnancy; however, its mechanism and maintenance remain underexplored. Here, we investigated whether mesenchymal stem/stromal cells (MSCs) as non-inherited maternal antigens (NIMAs) transferred by maternal microchimerism could induce immune tolerance. We showed that MSCs had a potential equivalent to hematopoietic stem and progenitor cells (HSPCs) to induce immune tolerance and that MSCs were essential to induce tolerance to MSC-specific antigens. Furthermore, we demonstrated that MSCs as NIMAs transferred by maternal microchimerism could induce robust immune tolerance that can be further enhanced using a drug. Our data shed light on induction of immune tolerance and serve as a foundation to develop new therapies using maternally derived cells for autoimmune or genetic diseases.

Contribution of PDGFRα lineage cells in adult mouse hematopoiesis.

Platelet-derived growth factor receptor alpha (PDGFRα) is a dominant marker of mesodermal mesenchymal cells in mice. Previous studies demonstrated that PDGFRα-positive (PDGFRα+) mesodermal cells develop not only into mesenchymal cells but also into a subset of total hematopoietic cells (HCs) in the limited period during mouse embryogenesis. However, the precise characteristics of the PDGFRα lineage positive (PDGFRα Lin+) HCs in adult mouse hematopoiesis are largely unknown. In this study, we systematically evaluated the characteristics of PDGFRα Lin+ HCs in the bone marrow and peripheral blood using PDGFRα-CRE; ROSAtdTomato mice. Flow cytometry analysis revealed that PDGFRα Lin+ HCs accounted for approximately 20% of total HCs in both the bone marrow and peripheral blood in adult mice. Compositions of myeloid and lymphoid subpopulations among CD45+ mononuclear cells were almost identical in both PDGFRα Lin+ and PDGFRα Lin- cells. Single-cell RNA-sequencing analysis also demonstrated that the transcriptomic signatures of the PDGFRα Lin+ HCs in the peripheral blood largely overlapped with those of the PDGFRα Lin- HCs, suggesting equivalent functions of the PDGFRα Lin+ and PDGFRα Lin- HCs. Although pathophysiological activities of the PDGFRα Lin + HCs were not evaluated, our data clearly demonstrate a significant role of the PDGFRα Lin + HCs in physiological hematopoiesis in adult mice.

Yokukansan, a Japanese Herbal Medicine, Suppresses Substance PInduced Production of Interleukin-6 and Interleukin-8 by Human U373 MG Glioblastoma Astrocytoma Cells.

BACKGROUND: Yokukansan is a traditional Japanese herbal medicine that has an antiallodynic effect in patients with chronic pain. However, the mechanisms by which yokukansan inhibits neuropathic pain are unclear. OBJECTIVE: This study aimed to investigate the molecular effects of yokukansan on neuroinflammation in U373 MG glioblastoma astrocytoma cells, which express a functional high-affinity neurokinin 1 receptor (substance P receptor), and produce interleukin (IL)-6 and IL-8 in response to stimulation by substance P (SP). METHODS: We assessed the effect of yokukansan on the expression of ERK1/2, P38 MAPK, nuclear factor (NF)-κB, and cyclooxygenase-2 (COX-2) in U373 cells by western blot assay. Levels of IL-6 and IL-8 in conditioned medium obtained after stimulation of cells with SP for 24 h were measured by enzyme-linked immunosorbent assay. All experiments were conducted in triplicate. Results were analyzed by one-way ANOVA, and significance was accepted at p < 0.05. RESULTS: Yokukansan suppressed SP-induced production of IL-6 and IL-8 by U373 MG cells, and downregulated SP-induced COX-2 expression. Yokukansan also inhibited phosphorylation of ERK1/2 and p38 MAPK, as well as nuclear translocation of NF-κB, induced by SP stimulation of U373 MG cells. CONCLUSION: Yokukansan exhibits anti-inflammatory activity by suppressing SP-induced production of IL-6 and IL-8 and downregulating COX-2 expression in U373 MG cells, possibly via inhibition of the activation of signaling molecules, such as ERK1/2, p38 MAPK, and NF-κB.

Comparison of two point of care whole blood coagulation analysis devices and conventional coagulation tests as a predicting tool of perioperative bleeding in adult cardiac surgery-a pilot prospective observational study in Japan.

BACKGROUND: It is widely accepted that Point-of Care Test (PoCT) devices are useful in the detection of coagulopathies in situations of massive bleeding such as major cardiac surgery. These devices contribute to the reduction of blood transfusion. However, their implementation remains limited in Japan because of their cost and lack of health insurance support. STUDY DESIGN AND METHODS: Conventional coagulation tests and thromboelastography (TEG)/Sonoclot values were measured in 50 consecutive cardiac surgery cases. Clinical background information such as operative procedures was obtained from electronic medical records, and the theoretical perioperative total blood loss was calculated by measuring the hemoglobin content and total red blood cell transfusion volume. The correlation between perioperative total blood loss and the measured laboratory values or clinical parameters was evaluated by a multivariate linear regression analysis. The risk factors of the total amount of platelet transfusion and postoperative drain bleeding volume were similarly evaluated. RESULTS: No significant association between the estimated perioperative total blood loss (eTBL) and the laboratory measurements including conventional coagulation tests, TEG and Sonoclot was observed. On the other hand, postoperative drain bleeding volume was significantly associated with postoperative Sonoclot CR (p = 0.039) as well as preoperative use of oral anticoagulants and cell saver treated blood volume. Platelet transfusion amount was significantly associated with post-CBP PF and time to peak value of Sonoclot (p = 0.014 and 0.001, respectively). CONCLUSION: Sonoclot measurements may be useful to estimate the risks of postoperative bleeding and platelet transfusion in cardiac surgeries in Japan.

Transcriptionally distinct mesenchymal stem/stromal cells circulate in fetus.

Umbilical cord blood contains mesenchymal stem/stromal cells (MSCs) in addition to hematopoietic stem cells, serving as an attractive tool for regenerative medicine. As umbilical cord blood originates from fetus, abundant MSCs are expected to circulate in fetus. However, the properties of circulating MSCs in fetus have not been fully examined. In the present study, we aimed to analyze circulating MSCs, marked by the expression of platelet-derived growth factor receptor α (PDGFRα), during fetal development. Using PDGFRα GFP knock-in mice, we quantified the number of circulating PDGFRα positive MSCs during development. We further performed whole transcriptome analysis of circulating MSCs at single cell levels. We found that abundant PDGFRα positive cells circulate in embryo and diminish immediately after birth. In addition, single cell RNA-sequencing revealed transcriptional heterogeneity of MSCs in fetal circulation. These data lay a foundation to analyze the function of circulating MSCs during development.

Fixation of intracapsular fracture of the femoral neck using combined peripheral nerve blocks and transthoracic echocardiography in a patient with severe obstructive hypertrophic cardiomyopathy: a case report.

BACKGROUND: Hypertrophic obstructive cardiomyopathy (HOCM) is a type of hypertrophic cardiomyopathy associated with left ventricular outflow tract stenosis. The increased pressure gradients across the left ventricular outflow tract in patients with HOCM could lead to circulatory collapse. We describe our experience with perioperative management under femoral nerve block (FNB), lateral femoral cutaneous nerve block (LFCNB), and transthoracic echocardiography (TTE) monitoring during open reduction and internal fixation of a femoral neck fracture in a patient with severe HOCM. CASE PRESENTATION: A 72-year-old man, who was indicated to undergo open reduction and internal fixation of an intracapsular femoral neck fracture, had a history of treatment for hypertension and HOCM. He had heart failure for 4 years and was hospitalized several times. He was resuscitated after ventricular fibrillation and received an implantable cardioverter-defibrillator at that time. He also had severe physical limitations (New York Heart Association class III). We selected FNB and LFCNB as the methods for anesthesia and injected 0.25% levobupivacaine (20 mL) around the femoral nerve and 0.25% levobupivacaine (10 mL) into the lateral femoral nerve region. He underwent TTE during the perioperative period, which enabled us to perform hemodynamic and morphological evaluations of the heart. The intraoperative TTE findings remained stable from before the induction of anesthesia to the patient's exit from the operating room. Postoperatively, his hemodynamic parameters continued to remain stable. CONCLUSIONS: In this case, FNB and LFCNB contributed to hemodynamic stability during non-cardiac surgery. Additionally, TTE was useful for the perioperative evaluation of cardiac hemodynamics and morphology in our patient with severe HOCM.

Using patient-derived iPSCs to develop humanized mouse models for chronic myelomonocytic leukemia and therapeutic drug identification, including liposomal clodronate.

Chronic myelomonocytic leukemia (CMML) is an entity of myelodysplastic syndrome/myeloproliferative neoplasm. Although CMML can be cured with allogeneic stem cell transplantation, its prognosis is generally very poor due to the limited efficacy of chemotherapy and to the patient's age, which is usually not eligible for transplantation. Comprehensive analysis of CMML pathophysiology and the development of therapeutic agents have been limited partly due to the lack of cell lines in CMML and the limited developments of mouse models. After successfully establishing patient's derived disease-specific induced pluripotent stem cells (iPSCs) derived from a patient with CMML, we utilized these CMML-iPSCs to achieve hematopoietic re-differentiation in vitro, created a humanized CMML mouse model via teratomas, and developed a drug-testing system. The clinical characteristics of CMML were recapitulated following hematopoietic re-differentiation in vitro and a humanized CMML mouse model in vivo. The drug-testing system using CMML-iPSCs identified a MEK inhibitor, a Ras inhibitor, and liposomal clodronate as potential drugs for treating CMML. Clodronate is a drug commonly used as a bisphosphonate for osteoporosis. In this study, the liposomalization of clodronate enhanced its effectiveness in these assays, suggesting that this variation of clodronate may be adopted as a repositioned drug for CMML therapy.

ADAM8 Is an Antigen of Tyrosine Kinase Inhibitor-Resistant Chronic Myeloid Leukemia Cells Identified by Patient-Derived Induced Pluripotent Stem Cells.

Properties of cancer stem cells involved in drug resistance and relapse have significant effects on clinical outcome. Although tyrosine kinase inhibitors (TKIs) have dramatically improved survival of patients with chronic myeloid leukemia (CML), TKIs have not fully cured CML due to TKI-resistant CML stem cells. Moreover, relapse after discontinuation of TKIs has not been predicted in CML patients with the best TKI response. In our study, a model of CML stem cells derived from CML induced pluripotent stem cells identified ADAM8 as an antigen of TKI-resistant CML cells. The inhibition of expression or metalloproteinase activity of ADAM8 restored TKI sensitivity in primary samples. In addition, residual CML cells in patients with optimal TKI response were concentrated in the ADAM8+ population. Our study demonstrates that ADAM8 is a marker of residual CML cells even in patients with optimal TKI response and would be a predictor of relapse and a therapeutic target of TKI-resistant CML cells.

Comparison of garenoxacin with levofloxacin as antimicrobial prophylaxis in acute myeloid leukemia.

OBJECTIVE: Levofloxacin is widely used as antimicrobial prophylaxis against high-risk chemotherapy-induced neutropenia. Garenoxacin, a fluoroquinolone developed in Japan, shows a stronger in vitro antimicrobial activity against Gram-positive bacteria than levofloxacin. METHODS: We retrospectively analyzed high-risk patients with acute myeloid leukemia who were administered garenoxacin (n = 36) or levofloxacin (n = 120) during chemotherapy. We compared the profiles of infections between these fluoroquinolones. RESULTS: Febrile events occurred in 31 (86%) and 93 (78%) cases in the garenoxacin and levofloxacin group, respectively (P = 0.35). Bloodstream infections by Gram-positive bacteria were recorded in one (3%) case in the garenoxacin group and 25 (21%) cases in the levofloxacin group (P < 0.01). In contrast, bloodstream infections by Gram-negative microorganisms were identified in five (4%) cases in the levofloxacin group and eight (22%) cases in the garenoxacin group (P < 0.01). CONCLUSIONS: These results indicate that there may be substantial differences in the antimicrobial spectrum between different fluoroquinolones. Although there are several biases due to rather small sample size and the retrospective nature, we should take the differences into consideration when we administer a prophylactic fluoroquinolone to a patient with hematological disease.