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Abstract We recently reported a method for recovering and quantifying residual proteins bound to surfaces of various medical instruments via thermal coagulation under neutral pH and room temperature. The method effectively recovered and solubilised coagulated proteins at high temperatures in dry and humid conditions, with a protein recovery rate of > 90%. This study validated the previous method by comparing residual protein recovery from test samples using a conventional extraction solution (1% SDS, [pH 11.0]) and proposed solution (1% SDS, 10 mM TCEP, and 10 mM HEPES [pH 7.0]). To mimic soiled medical equipment, pseudo-blood-contaminated stainless steel plates were prepared. Residual protein was recovered using conventional and proposed solutions under varying temperature and humidity conditions. Quantitative protein recovery limits were determined at nine facilities. Compared with the conventional solution, the proposed solution recovered proteins more effectively from samples processed at temperatures > 60 °C. However, low recovery rates were observed for samples processed at 95 °C, possibly owing to differences in protein adhesion due to sample and plate-surface properties. Our findings present a method for quantifying residual proteins on medical instruments exposed to high temperatures during use or disinfection. Further studies should standardise test soiling conditions, materials, and solutions to evaluate cleaning methods.

We started a registry for cases of immunoglobulin (Ig)G4-related disease (IgG4-RD) in December 2019 to clarify the clinical profile of IgG4-RD. In this study, clinical information from 854 cases registered by February 16, 2024 was analyzed from multiple perspectives. Diagnosis of IgG4-RD was made in 808 cases, comprising 638 definite, 38 probable, and 132 possible. The mean ± SD age at time of enrollment of the 808 cases was 67.9 ± 11.3 years, with 68.8% being male. The pancreas was the most frequently affected organ (49.8%), followed by the submandibular glands (46.2%) and lacrimal glands (30.6%). This study reconfirmed the pancreas and head-and-neck region as major affected areas in IgG4-RD. Clinically, submandibular adenitis and autoimmune pancreatitis often occur together in the same patient, but no association between the two organs was observed in our analysis. Regarding diagnosis, the comprehensive diagnostic criteria were most commonly used (63.6%). Storiform fibrosis and phlebitis obliterans were detected at different frequencies in different organs. In summary, this registry study identified clinical, imaging, hematologic, and pathologic findings in 808 Japanese patients with IgG4-RD. The frequency of affected organs and their characteristic pathological findings will be particularly useful for future practice.

Abstract Objective We evaluated the effect of CHA2DS2‐VASc score and prior use of oral anticoagulants (OACs) on endovascular treatment (EVT) in patients with acute ischemic stroke and atrial fibrillation (AF). Methods Patients with AF who received EVT in 353 centers in Japan (2018–2020) were included. The outcomes were symptomatic intracerebral hemorrhage (sICH), in‐hospital mortality, functional independence, and successful and complete reperfusion. The effects of CHA2DS2‐VASc score, its components, and prior use of OACs were assessed via a multiple logistic regression model. Results Of the 6984 patients, 780 (11.2%) used warfarin and 1168 (16.7%) used direct oral anticoagulants (DOACs) before EVT. Based on the CHA2DS2‐VASc score, 6046 (86.6%) presented a high risk (≥2 for males and ≥3 for females) while 938 (13.4%) had intermediate to low risks. Higher CHA2DS2‐VASc scores were associated with increased sICH, in‐hospital mortality, and decreased functional independence, regardless of prior OACs. For patients with a high‐risk category, prior DOACs increased the odds of successful and complete reperfusion (adjusted odds ratio [95% confidence interval (CI)], 1.27 [1.00–1.61] and 1.30 [1.10–1.53]). For those with integrated intermediate to low risks, neither prior warfarin nor DOAC affected the outcomes. Regardless of total CHA2DS2‐VASc scores, patients with congestive heart failure or left ventricular dysfunction, hypertension, age >75 years, or female benefited similarly from prior DOAC use. Interpretation Prior DOAC use for patients with high‐ and selected intermediate‐risk CHA2DS2‐VASc scores increased prevalence of successful and complete reperfusion. These findings may provide supplemental evidence to introduce preventive DOAC for patients with AF.

BackgroundWe aimed to clarify the association between intraoperative P2Y12 inhibitor administration during EVT and clinical outcomes in patients with anterior circulation TO stroke.MethodsAmong consecutive patients with acute ischemic stroke (AIS) enrolled in the Recovery by Endovascular Salvage for Cerebral Ultra-acute Embolic and Atherothrombotic Stroke with Large Vessel Occlusion Registry from 2016 to 2019, those with anterior circulation TOs who underwent EVT were analyzed. These patients were categorized into the following groups: those who received P2Y12 inhibitors during the perioperative period and those who did not receive P2Y12 inhibitors. The outcomes included good functional outcomes, as indicated by a modified Rankin Scale score of 0–2 at 90 days, and the incidence of symptomatic intracranial hemorrhage (SICH) was compared between the two groups. Multivariate logistic regression models were used to assess the association of outcomes with perioperative P2Y12 inhibitor administration. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using the group that did not receive P2Y12 inhibitors as the reference. The perioperative period included the period in which antithrombotic therapy was administered immediately before EVT and during the operative period.ResultsWe enrolled 242 patients with AIS with anterior circulation TOs (42 females [17.4%]; median age, 76 [interquartile range, 69–81] years). Patients who received P2Y12 inhibitors during the perioperative period (n = 131) showed a higher frequency of carotid artery stenting than those who did not receive perioperative P2Y12 inhibitors (n = 111; 86.3% vs. 42.3%, p

BackgroundSjögren’s disease (SjD) is an autoimmune disease marked by lymphocytic infiltration of salivary and lacrimal glands, leading to glandular dysfunction, where CD4-positive helper T (Th) cells and their cytokines are crucial in the pathogenesis. Recent studies have demonstrated that Toll-like receptors (TLRs), particularly those recognizing immune complexes containing DNA and RNA, contribute to Th cell activation in various autoimmune diseases. This study explores the expression and function of these TLRs in SjD.MethodsDNA microarray analysis of salivary gland tissue from six SjD patients and real-time PCR (n = 32) was used to identify overexpressed TLRs. Single-cell RNA sequencing (scRNA-seq) was performed using tissue lesions and integrated with published scRNA-seq data from tissues and peripheral blood mononuclear cells to examine gene expression in macrophages and monocytes. Finally, multi-color immunofluorescence staining was conducted to confirm TLR8 expression and function in SjD lesions (n = 19).ResultsDNA microarray analysis revealed the up-regulation of TLR8, along with other TLRs and innate immune response genes in SjD. Real-time PCR showed significant up-regulation of TLR7 and TLR8. TLR8 up-regulated in both analyses. In scRNA-seq analysis, the TLR8-expressing cluster comprised macrophages and monocytes, which also produced T cell activation genes like CD86. TLR8-positive macrophages infiltrated inflammatory sites and frequently expressed CD86 in quantitative imaging approaches.ConclusionsThese results suggest that infiltrating monocytes and macrophages may produce cytokines and chemokines through TLR8 stimulation, potentially enhancing B7 molecule expression, promoting the adaptive immune response, and contributing to SjD pathogenesis.

Background The benefits of endovascular thrombectomy (EVT) for distal medium vessel occlusions (DMVOs) are not well established. This study aimed to determine the superiority of EVT over intravenous tissue‐type plasminogen activator (IV tPA) in the treatment of DMVOs. Methods This study analyzed data from the Kanagawa Intravenous and Endovascular Treatment of Acute Ischemic Stroke Registry, a prospective, multicenter, observational registry of acute ischemic stroke patients treated with EVT or IV tPA. The study evaluated patients with acute DMVOs who were treated with EVT and/or IV tPA. DMVOs was defined as occlusions in M2–M3 segment of the middle cerebral artery, anterior cerebral artery, or posterior cerebral artery. The analysis included primary DMVOs and excluded secondary DMVOs, such as distal embolism after recanalization of proximal vessel occlusion. Propensity score‐matched analysis was conducted to compare the outcomes between EVT and IV tPA alone. A good outcome was defined as a modified Rankin Scale score 0–2 or no worsening at 90 days. An excellent outcome was defined as an modified Rankin Scale score 0–1. Results The study included 1148 patients with DMVOs, of whom 816 were treated with EVT and 332 were IV tPA alone. Before propensity score matching, the incidence of good and excellent outcomes was significantly lower in EVT group (good outcomes: EVT 50.3% versus IV tPA 68.0%; P

Abstract This study discusses safety confirmation systems and how Social Capital (SC) affects the resilience of the safety confirmation network for natural disasters in the U.S. and Japanese society. When a large-scale disaster occurs, people desire to inform their loved ones of their safety status and search for their loved one’s status in a chaotic environment. Safety confirmation is the action of confirming the current status of people in disaster areas by collecting safety, injury, missing, and death information and sending the contact request. First, this study summarized the safety confirmation resources. The U.S. approach focuses on the missing and dead, while the Japanese approach focuses on the survivors and tries to define the remaining people who are missing and dead. Second, the social background that formed the SC was compared. Third, how SC affects the safety confirmation network in and between communities was analyzed. The case areas were Honolulu County in the U.S. and Aichi prefecture in Japan, and 1,324 samples were analyzed by ordinal logistic regression. The safety confirmation network reflected the social and SC structure for each country. The ethnic diversity in the network provided high resilience. Honolulu’s network was more open beyond the community and connected more with others. Residents’ economic network was linked to the safety confirmation network. Aichi’s network was more closed in the community and connected with kinship or neighbor ties. Trust and interaction with others shaped the foundation of the safety confirmation network. Finally, the integrated safety confirmation system was discussed.

Abstract Oral lichen planus (OLP) is a chronic inflammatory disease associated with T cell infiltration. The crosstalk between oral epithelium and mucosal T cells was considered to be crucial in the pathogenesis of OLP. Here, we selectively extracted the normal epithelium (NE) and lesional epithelium (LE) of buccal mucosa specimens from three patients with OLP by laser capture microdissection due to identify the pathogenic factors. Cathepsin K (CTSK) was identified as one of common upregulated genes in the LE by DNA microarray. Immunohistochemically, CTSK was distinctly detected in and around the LE, while it was rarely seen in the NE. Recent studies showed that CTSK enhanced Toll-like receptor 9 (TLR9) signaling in antigen-presenting cells, leading to Th17 cell differentiation. TLR9 expression mainly co-localized with CD123+ plasmacytoid dendritic cells (pDCs). The number of RORγt-positive cells correlated with that of CTSK-positive cells in OLP tissues. CD123+ pDCs induced the production of Th17-related cytokines (IL-6, IL-23, and TGF-β) upon stimulation with TLR9 agonist CpG DNA. Moreover, single cell RNA-sequencing analysis revealed that TLR9-positive pDCs enhanced in genes associated with Th17 cell differentiation in comparison with TLR9-negative pDCs. CTSK could induce Th17-related production of CD123+ pDCs via TLR9 signaling to promote the pathogenesis of OLP.

Background Atrial fibrillation (AF) is known to be a strong risk factor for stroke. However, the risk of stroke recurrence in patients with cryptogenic stroke with AF detected after stroke by an insertable cardiac monitor (ICM) is not well known. We sought to evaluate the risk of ischemic stroke recurrence in patients with cryptogenic stroke with and without ICM‐detected AF. Methods and Results We retrospectively reviewed patients with cryptogenic stroke who underwent ICM implantation at 8 stroke centers in Japan. Cox regression models were developed using landmark analysis and time‐dependent analysis. We set the target sample size at 300 patients based on our estimate of the annualized incidence of ischemic stroke recurrence to be 3% in patients without AF detection and 9% in patients with AF detection. Of the 370 patients, 121 were found to have AF, and 110 received anticoagulation therapy after AF detection. The incidence of ischemic stroke recurrence was 4.0% in 249 patients without AF detection and 5.8% in 121 patients with AF detection (P=0.45). In a landmark analysis, the risk of ischemic stroke recurrence was not higher in patients with AF detected ≤90 days than in those without (hazard ratio, 1.47 [95% CI, 0.41–5.28]). In a time‐dependent analysis, the risk of ischemic stroke recurrence did not increase after AF detection (hazard ratio, 1.77 [95% CI, 0.70–4.47]). Conclusions The risk of ischemic stroke recurrence in patients with cryptogenic stroke with ICM‐detected AF, 90% of whom were subsequently anticoagulated, was not higher than in those without ICM‐detected AF.

BackgroundCancer immunotherapy targeting CD8+ T cells has made remarkable progress, even for oral squamous cell carcinoma (OSCC), a heterogeneous epithelial tumor without a substantial increase in the overall survival rate over the past decade. However, the therapeutic effects remain limited due to therapy resistance. Thus, a more comprehensive understanding of the roles of CD4+ T cells and B cells is crucial for more robust development of cancer immunotherapy.MethodsIn this study, we examined immune responses and effector functions of CD4+ T cells, CD8+ T cells and B cells infiltrating in OSCC lesions using single-cell RNA sequencing analysis, T cell receptor (TCR) and B cell receptor (BCR) repertoire sequencing analysis, and multi-color immunofluorescence staining. Finally, two Kaplan-Meier curves and several Cox proportional hazards models were constructed for the survival analysis.ResultsWe observed expansion of CD4+ cytotoxic T lymphocytes (CTLs) expressing granzymes, which are reported to induce cell apoptosis, with a unique gene expression patterns. CD4+ CTLs also expressed CXCL13, which is a B cell chemoattractant. Cell–cell communication analysis and multi-color immunofluorescence staining demonstrated potential interactions between CD4+ CTLs and B cells, particularly IgD- CD27- double negative (DN) B cells. Expansion of CD4+ CTLs, DN B cells, and their contacts has been reported in T and B cell-activated diseases, including IgG4-related disease and COVID-19. Notably, we observed upregulation of several inhibitory receptor genes including CTLA-4 in CD4+ CTLs, which possibly dampened T and B cell activity. We next demonstrated comprehensive delineation of the potential for CD8+ T cell differentiation towards dysfunctional states. Furthermore, prognostic analysis revealed unfavorable outcomes of patients with a high proportion of CD4+ CTLs in OSCC lesions.ConclusionOur study provides a dynamic landscape of lymphocytes and demonstrates a systemic investigation of CD4+ CTL effects infiltrating into OSCC lesions, which may share some pathogenesis reported in severe T and B cell-activated diseases such as autoimmune and infectious diseases.

Abstract Sjögren’s syndrome is a chronic autoimmune disorder whose pathogenesis is poorly understood and that lacks effective therapies. Detailed quantitative and spatial analyses of tissues affected by Sjögren’s syndrome were undertaken, including the quantitation of the frequency of selected cell–cell interactions in the disease milieu. Quantitative analyses of CD4+ T cell subsets and of CD8+ T cells in the labial salivary glands from untreated patients with primary Sjögren’s syndrome revealed that activated CD8+ cytotoxic T cells (CD8+CTLs) were the most prominent T cells in these infiltrates. An accumulation of apoptotic glandular epithelial cells, mainly ductal and acinar cells, was observed, consistent with the impaired salivary secretion often observed in patients with this disease. FasL expressing activated CD8+ T cells were seen to accumulate around Fas expressing apoptotic epithelial cells. Quantitative analyses of apoptotic cell types and of conjugates between cytotoxic T cells and epithelial cells undergoing apoptosis suggest that Sjögren’s syndrome is primarily driven by CD8+CTL mediated execution of epithelial cells mainly represented by ductal and acinar cells.

Summary: Although therapeutic B cell depletion dramatically resolves inflammation in many diseases in which antibodies appear not to play a central role, distinct extrafollicular pathogenic B cell subsets that accumulate in disease lesions have hitherto not been identified. The circulating immunoglobulin D (IgD)−CD27−CXCR5−CD11c+ DN2 B cell subset has been previously studied in some autoimmune diseases. A distinct IgD−CD27−CXCR5−CD11c− DN3 B cell subset accumulates in the blood both in IgG4-related disease, an autoimmune disease in which inflammation and fibrosis can be reversed by B cell depletion, and in severe COVID-19. These DN3 B cells prominently accumulate in the end organs of IgG4-related disease and in lung lesions in COVID-19, and double-negative B cells prominently cluster with CD4+ T cells in these lesions. Extrafollicular DN3 B cells may participate in tissue inflammation and fibrosis in autoimmune fibrotic diseases, as well as in COVID-19.

Abstract CUX2 gene encodes a transcription factor that controls neuronal proliferation, dendrite branching and synapse formation, locating at the epilepsy-associated chromosomal region 12q24 that we previously identified by a genome-wide association study (GWAS) in Japanese population. A CUX2 recurrent de novo variant p.E590K has been described in patients with rare epileptic encephalopathies and the gene is a candidate for the locus, however the mutation may not be enough to generate the genome-wide significance in the GWAS and whether CUX2 variants appear in other types of epilepsies and physiopathological mechanisms are remained to be investigated. Here in this study, we conducted targeted sequencings of CUX2, a paralog CUX1 and its short isoform CASP harboring a unique C-terminus on 271 Japanese patients with a variety of epilepsies, and found that multiple CUX2 missense variants, other than the p.E590K, and some CASP variants including a deletion, predominantly appeared in patients with temporal lobe epilepsy (TLE). The CUX2 variants showed abnormal localization in human cell culture analysis. While wild-type CUX2 enhances dendritic arborization in fly neurons, the effect was compromised by some of the variants. Cux2- and Casp-specific knockout mice both showed high susceptibility to kainate, increased excitatory cell number in the entorhinal cortex, and significant enhancement in glutamatergic synaptic transmission to the hippocampus. CASP and CUX2 proteins physiologically bound to each other and co-expressed in excitatory neurons in brain regions including the entorhinal cortex. These results suggest that CUX2 and CASP variants contribute to the TLE pathology through a facilitation of excitatory synaptic transmission from entorhinal cortex to hippocampus.

Abstract Background Anomalous systemic arterial supply to normal basal lung segments is a rare congenital malformation, in which aberrant arteries arising from the systemic circulation flow into the basal segment of the lung and return to normal pulmonary veins without abnormal bronchial branching. It presents a left-to-right shunt, resulting in volume overload of the pulmonary circulation, and consequently, pulmonary hypertension. Therefore, nearly all cases require surgery. Herein, we present a case, in which indocyanine green was used to demarcate the lung segment perfused by an anomalous systemic artery. Case presentation A 15-year-old boy was diagnosed with an anomalous artery originating from the celiac artery and supplying the right dorsobasal lung segment (S10). Via three-port video-assisted thoracoscopic surgery, the anomalous artery was ligated and processed with an auto-stapler. Indocyanine green was injected intravenously to identify the tissue perfused by the anomalous artery, and the lung was resected. Conclusions With anomalous systemic arterial supply to normal basal lung segments, indocyanine green can be particularly helpful in identifying the boundaries of the perfused area. Then, the affected tissue can be resected by thoracoscopic surgery.

BackgroundParoxysmal atrial fibrillation (AF) is a probable cause of cryptogenic stroke (CS), and its detection and treatment are important for the secondary prevention of stroke. Insertable cardiac monitors (ICMs) are clinically effective in screening for AF and are superior to conventional short-term cardiac monitoring. Japanese guidelines for determining clinical indications for ICMs in CS are stricter than those in Western countries. Differences between Japanese and Western guidelines may impact the detection rate and prediction of AF via ICMs in patients with CS. Available data on Japanese patients are limited to small retrospective studies. Furthermore, additional information about AF detection, including the number of episodes, cumulative episode duration, anticoagulation initiation (type and dose of regimen and time of initiation), rate of catheter ablation, role of atrial cardiomyopathy, and stroke recurrence (time of recurrence and cause of the recurrent event), was not provided in the vast majority of previously published studies. ObjectiveIn this study, we aim to identify the proportion and timing of AF detection and risk stratification criteria in patients with CS in real-world settings in Japan. MethodsThis is a multicenter, prospective, observational study that aims to use ICMs to evaluate the proportion, timing, and characteristics of AF detection in patients diagnosed with CS. We will investigate the first detection of AF within the initial 6, 12, and 24 months of follow-up after ICM implantation. Patient characteristics, laboratory data, atrial cardiomyopathy markers, serial magnetic resonance imaging findings at baseline, 6, 12, and 24 months after ICM implantation, electrocardiogram readings, transesophageal echocardiography findings, cognitive status, stroke recurrence, and functional outcomes will be compared between patients with AF and patients without AF. Furthermore, we will obtain additional information regarding the number of AF episodes, duration of cumulative AF episodes, and time of anticoagulation initiation. ResultsStudy recruitment began in February 2020, and thus far, 213 patients have provided written informed consent and are currently in the follow-up phase. The last recruited participant (May 2021) will have completed the 24-month follow-up in May 2023. The main results are expected to be submitted for publication in 2023. ConclusionsThe findings of this study will help identify AF markers and generate a risk scoring system with a novel and superior screening algorithm for occult AF detection while identifying candidates for ICM implantation and aiding the development of diagnostic criteria for CS in Japan. Trial RegistrationUMIN Clinical Trial Registry UMIN000039809; https://tinyurl.com/3jaewe6a International Registered Report Identifier (IRRID)DERR1-10.2196/39307

This review summarizes recent progress in understanding the pathogenesis of IgG4-related disease (IgG4-RD), with a focus on fibrosis. Several studies reported that CD4+ T cells with cytotoxic activity promoted by the secretion of granzyme and perforin, cytotoxic CD4+ T cells (CD4+CTLs), and disease-specific activated B cells, infiltrated inflamed tissues and cooperated to induce tissue fibrosis in autoimmune fibrotic diseases such as IgG4-RD, systemic sclerosis, and fibrosing mediastinitis. An accumulation of cells undergoing apoptotic cell death induced by CD4+CTLs and CD8+CTLs followed by macrophage-mediated clearing and finally tissue remodeling driven by cytokines released by CD4+CTLs, activated B cells, and M2 macrophages may contribute to the activation of fibroblasts and collagen production. In IgG4-RD, this process likely involves the apoptosis of non-immune, non-endothelial cells of mesenchymal origin and subsequent tissue remodeling. In summary, CD4+CTLs infiltrate affected tissues where they may cooperate with activated B cells, CD8+CTLs, and M2 macrophages, to induce apoptosis by secreting cytotoxic cytokines. These immune cells also drive fibrosis by secreting pro-fibrotic molecules in IgG4-RD.

Introduction JTR-161 is a novel allogeneic human cell product consisting of dental pulp stem cells isolated from the extracted teeth of healthy adults. It is currently under development as a cell-based therapy for ischaemic stroke. The aim of this study is to evaluate the safety and efficacy of JTR-161 in patients with acute ischaemic stroke when given as a single intravenous administration within 48 hours of symptom onset.Methods and analysis This is a first-in-human, randomised, double-blind, placebo-controlled, multicentre, phase 1/2 clinical trial to be conducted in Japan (from January 2019 to July 2021). Patients with a clinical diagnosis of anterior circulation ischaemic stroke with a National Institutes of Health Stroke Scale (NIHSS）score of 5–20 at baseline were enrolled. Patients previously treated with recombinant tissue-type plasminogen activator and/or endovascular thrombectomy were allowed to be enrolled. The study consists of three cohorts: cohorts 1 and 2 (each eight patients) and cohort 3 (60 patients). Subjects were randomly assigned to receive either JTR-161 or placebo in a 3:1 ratio in cohorts 1 and 2, and in a 1:1 ratio in cohort 3. The number of cells administered was increased sequentially from 1×108 (cohort 1) to 3 x 108 (cohort 2). In cohort 3, the higher tolerated dose among the two cohorts was administered. The primary endpoint is the proportion of patients who achieve an excellent outcome as defined by all of the following criteria at day 91 in cohort 3: modified Rankin Scale ≤1, NIHSS ≤1 and Barthel Index ≥95.Ethics and dissemination The protocol and informed consent form were approved by the institutional review board at each participating study site. A manuscript with the results of the primary study will be published in a peer-reviewed journal.Trial registration number NCT04608838; JapicCTI-194570 and Clinical Trials. gov.

An alternative source of mesenchymal stem cells has recently been discovered: dental pulp stem cells (DPSCs), including deciduous teeth, which can thus comprise potential tools for regenerative medicine. DPSCs derive from the neural crest and are normally implicated in dentin homeostasis. The clinical application of mesenchymal stem cells (MSCs) involving DPSCs contains various limitations, such as high cost, low safety, and cell handling issues, as well as invasive sample collection procedures. Although MSCs implantation offers favorable outcomes on specific diseases, implanted MSCs cannot survive for a long period. It is thus considered that their mediated mechanism of action involves paracrine effects. It has been recently reported that secreted molecules in DPSCs-conditioned media (DPSC-CM) contain various trophic factors and cytokines and that DPSC-CM are effective in models of various diseases. In the current study, we focus on the characteristics of DPSC-CM and their therapeutic potential against various disorders.