Suppression of Type I Interferon Signaling in Myeloid Cells by Autoantibodies in Severe COVID-19 Patients.

PURPOSE: Auto-antibodies (auto-abs) to type I interferons (IFNs) have been identified in patients with life-threatening coronavirus disease 2019 (COVID-19), suggesting that the presence of auto-abs may be a risk factor for disease severity. We therefore investigated the mechanism underlying COVID-19 exacerbation induced by auto-abs to type I IFNs. METHODS: We evaluated plasma from 123 patients with COVID-19 to measure auto-abs to type I IFNs. We performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from the patients with auto-abs and conducted epitope mapping of the auto-abs. RESULTS: Three of 19 severe and 4 of 42 critical COVID-19 patients had neutralizing auto-abs to type I IFNs. Patients with auto-abs to type I IFNs showed no characteristic clinical features. scRNA-seq from 38 patients with COVID-19 revealed that IFN signaling in conventional dendritic cells and canonical monocytes was attenuated, and SARS-CoV-2-specific BCR repertoires were decreased in patients with auto-abs. Furthermore, auto-abs to IFN-α2 from COVID-19 patients with auto-abs recognized characteristic epitopes of IFN-α2, which binds to the receptor. CONCLUSION: Auto-abs to type I IFN found in COVID-19 patients inhibited IFN signaling in dendritic cells and monocytes by blocking the binding of type I IFN to its receptor. The failure to properly induce production of an antibody to SARS-CoV-2 may be a causative factor of COVID-19 severity.

The USP7-STAT3-granzyme-Par-1 axis regulates allergic inflammation by promoting differentiation of IL-5-producing Th2 cells.

Uncontrolled type 2 immunity by type 2 helper T (Th2) cells causes intractable allergic diseases; however, whether the interaction of CD4+ T cells shapes the pathophysiology of allergic diseases remains unclear. We identified a subset of Th2 cells that produced the serine proteases granzyme A and B early in differentiation. Granzymes cleave protease-activated receptor (Par)-1 and induce phosphorylation of p38 mitogen-activated protein kinase (MAPK), resulting in the enhanced production of IL-5 and IL-13 in both mouse and human Th2 cells. Ubiquitin-specific protease 7 (USP7) regulates IL-4-induced phosphorylation of STAT3, resulting in granzyme production during Th2 cell differentiation. Genetic deletion of Usp7 or Gzma and pharmacological blockade of granzyme B ameliorated allergic airway inflammation. Furthermore, PAR-1+ and granzyme+ Th2 cells were colocalized in nasal polyps from patients with eosinophilic chronic rhinosinusitis. Thus, the USP7-STAT3-granzymes-Par-1 pathway is a potential therapeutic target for intractable allergic diseases.

A nonsense mutation in mouse Adamtsl2 causes uterine hypoplasia and an irregular estrous cycle.

The spontaneous mutation stubby (stb) in mice causes chondrodysplasia and male infertility due to impotence through autosomal recessive inheritance. In this study, we conducted linkage analysis to localize the stb locus within a 1.6 Mb region on mouse chromosome 2 and identified a nonsense mutation in Adamtsl2 of stb/stb mice. Histological analysis revealed disturbed endochondral ossification with a reduced hypertrophic chondrocyte layer and stiff skin with a thickened dermal layer. These phenotypes are similar to those observed in humans and mice with ADAMTSL2/Adamtsl2 mutations. Moreover, stb/stb female mice exhibited severe uterine hypoplasia at 5 weeks of age and irregular estrous cycles at 10 weeks of age. In normal mice, Adamtsl2 was more highly expressed in the ovary and pituitary gland than in the uterus, and this expression was decreased in stb/stb mice. These findings suggest that Adamtsl2 may function in these organs rather than in the uterus. Thus, we analyzed Gh expression in the pituitary gland and plasma estradiol and IGF1 levels, which are required for the development of the female reproductive tract. There was no significant difference in Gh expression and estradiol levels, whereas IGF1 levels in stb/stb mice were significantly reduced to 54-59% of those in +/+ mice. We conclude that Adamtsl2 is required for the development of the uterus and regulation of the estrous cycle in female mice, and decreased IGF1 may be related to these abnormalities.

The impact of a preoperative nurse-led orientation program on postoperative delirium after cardiovascular surgery: a retrospective single-center observational study.

BACKGROUND: Postoperative delirium in intensive care is common and associated with mortality, cognitive impairment, prolonged hospital stays and high costs. We evaluate whether a nurse-led orientation program could reduce the incidence of delirium in the intensive care unit after cardiovascular surgery. METHODS: In this retrospective cohort study, we enrolled patients admitted to the intensive care unit for planned cardiovascular surgery between January 2020 and December 2021. A nurse-led orientation program based on a preoperative visit was routinely introduced from January 2021. We assessed the association between these visits and postoperative delirium in the intensive care unit. We also assessed predictors of postoperative delirium with baseline and intraoperative characteristics. RESULTS: Among 253 patients with planned cardiovascular surgery, 128 (50.6%) received preoperative visits. Valve surgery comprised 44.7%, coronary surgery 31.6%, and aortic surgery 20.9%. Cardiopulmonary bypass use and transcatheter surgery were 60.5% and 12.3%, respectively. Incidence of delirium was lower in patients that received preoperative visits, and median hospital stay was shorter than in those without visits (18 patients [14.1%] vs 34 patients [27.2%], P < 0.01; 14 days vs 17 days, P < 0.01). After adjusting predefined confounders, preoperative visits were independently associated with decreased incidence of delirium (adjusted odds ratio [aOR] 0.45; 95% confidence interval [95% CI] 0.22-0.84). Other predictors of delirium were higher European System for Cardiac Operative Risk Evaluation II score and lower minimum intraoperative cerebral oxygen saturation. CONCLUSIONS: A preoperative nurse-led orientation program was associated with reduction of postoperative delirium and could be effective against postoperative delirium after cardiovascular surgery. Trial registration UMIN Clinical Trial Registry no. UMIN000048142. Registered 22, July, 2022, retrospectively registered, https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000054862 .

Thioredoxin-interacting protein is essential for memory T cell formation via the regulation of the redox metabolism.

CD4+ memory T cells are central to long-lasting protective immunity and are involved in shaping the pathophysiology of chronic inflammation. While metabolic reprogramming is critical for the generation of memory T cells, the mechanisms controlling the redox metabolism in memory T cell formation remain unclear. We found that reactive oxygen species (ROS) metabolism changed dramatically in T helper-2 (Th2) cells during the contraction phase in the process of memory T cell formation. Thioredoxin-interacting protein (Txnip), a regulator of oxidoreductase, regulated apoptosis by scavenging ROS via the nuclear factor erythroid 2-related factor 2 (Nrf2)-biliverdin reductase B (Blvrb) pathway. Txnip regulated the pathology of chronic airway inflammation in the lung by controlling the generation of allergen-specific pathogenic memory Th2 cells in vivo. Thus, the Txnip-Nrf2-Blvrb axis directs ROS metabolic reprogramming in Th2 cells and is a potential therapeutic target for intractable chronic inflammatory diseases.

Conventional and pathogenic Th2 cells in inflammation, tissue repair, and fibrosis.

Type 2 helper T (Th2) cells, a subset of CD4+ T cells, play an important role in the host defense against pathogens and allergens by producing Th2 cytokines, such as interleukin-4 (IL-4), IL-5, and IL-13, to trigger inflammatory responses. Emerging evidence reveals that Th2 cells also contribute to the repair of injured tissues after inflammatory reactions. However, when the tissue repair process becomes chronic, excessive, or uncontrolled, pathological fibrosis is induced, leading to organ failure and death. Thus, proper control of Th2 cells is needed for complete tissue repair without the induction of fibrosis. Recently, the existence of pathogenic Th2 (Tpath2) cells has been revealed. Tpath2 cells produce large amounts of Th2 cytokines and induce type 2 inflammation when activated by antigen exposure or tissue injury. In recent studies, Tpath2 cells are suggested to play a central role in the induction of type 2 inflammation whereas the role of Tpath2 cells in tissue repair and fibrosis has been less reported in comparison to conventional Th2 cells. In this review, we discuss the roles of conventional Th2 cells and pathogenic Th2 cells in the sequence of tissue inflammation, repair, and fibrosis.

Elevated Myl9 reflects the Myl9-containing microthrombi in SARS-CoV-2-induced lung exudative vasculitis and predicts COVID-19 severity.

The mortality of coronavirus disease 2019 (COVID-19) is strongly correlated with pulmonary vascular pathology accompanied by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-triggered immune dysregulation and aberrant activation of platelets. We combined histological analyses using field emission scanning electron microscopy with energy-dispersive X-ray spectroscopy analyses of the lungs from autopsy samples and single-cell RNA sequencing of peripheral blood mononuclear cells to investigate the pathogenesis of vasculitis and immunothrombosis in COVID-19. We found that SARS-CoV-2 accumulated in the pulmonary vessels, causing exudative vasculitis accompanied by the emergence of thrombospondin-1-expressing noncanonical monocytes and the formation of myosin light chain 9 (Myl9)-containing microthrombi in the lung of COVID-19 patients with fatal disease. The amount of plasma Myl9 in COVID-19 was correlated with the clinical severity, and measuring plasma Myl9 together with other markers allowed us to predict the severity of the disease more accurately. This study provides detailed insight into the pathogenesis of vasculitis and immunothrombosis, which may lead to optimal medical treatment for COVID-19.

Novel aerosol treatment of airway hyper-reactivity and inflammation in a murine model of asthma with a soluble epoxide hydrolase inhibitor.

Asthma currently affects more than 339 million people worldwide. In the present preliminary study, we examined the efficacy of a new, inhalable soluble epoxide hydrolase inhibitor (sEHI), 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), to attenuate airway inflammation, mucin secretion, and hyper-responsiveness (AHR) in an ovalbumin (OVA)-sensitized murine model. Male BALB/c mice were divided into phosphate-buffered saline (PBS), OVA, and OVA+TPPU (2- or 6-h) exposure groups. On days 0 and 14, the mice were administered PBS or sensitized to OVA in PBS. From days 26-38, seven challenge exposures were performed with 30 min inhalation of filtered air or OVA alone. In the OVA+TPPU groups, a 2- or 6-h TPPU inhalation preceded each 30-min OVA exposure. On day 39, pulmonary function tests (PFTs) were performed, and biological samples were collected. Lung tissues were used to semi-quantitatively evaluate the severity of inflammation and airway constriction and the volume of stored intracellular mucosubstances. Bronchoalveolar lavage (BAL) and blood samples were used to analyze regulatory lipid mediator profiles. Significantly (p < 0.05) attenuated alveolar, bronchiolar, and pleural inflammation; airway resistance and constriction; mucosubstance volume; and inflammatory lipid mediator levels were observed with OVA+TPPU relative to OVA alone. Cumulative findings indicated TPPU inhalation effectively inhibited inflammation, suppressed AHR, and prevented mucosubstance accumulation in the murine asthmatic model. Future studies should determine the pharmacokinetics (i.e., absorption, distribution, metabolism, and excretion) and pharmacodynamics (i.e., concentration/dose responses) of inhaled TPPU to explore its potential as an asthma-preventative or -rescue treatment.

Contribution of reductase activity to quinone toxicity in three kinds of hepatic cells.

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle, and the arylation of intracellular nucleophiles. The redox cycle is catalyzed by intracellular reductases, and therefore the toxicity of redox cycling quinone is considered to be closely associated with the reductase activity. This study examined the relationship between quinone toxicity and the intracellular reductase activity using 3 kinds of hepatic cells; rat primary hepatocytes, HepG2 and H4IIE. The intracellular reductase activity was; primary hepatocyte >>HepG2>H4IIE. The three kinds of cells showed almost the same vulnerability to an arylating quinone, 1,4-naphthoquinone (NQ). However, the susceptibility to a redox cycling quinone, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) was; primary hepatocyte>HepG2>H4IIE. In addition, the cytotoxicity elicited by DMNQ was significantly attenuated in HepG2 cells and almost completely suppressed in primary hepatocytes by diphenyleneiodonium chloride, a reductase inhibitor. These data suggest that cells with a high reductase activity are susceptible to redox cycling quinones. This study provides essential evidence to assess the toxicity of quinone-based drugs during their developmental processes.

Technique for differentiating alveolar soft part sarcoma from other tumors in paraffin-embedded tissue: comparison of immunohistochemistry for TFE3 and CD147 and of reverse transcription polymerase chain reaction for ASPSCR1-TFE3 fusion transcript.

The diagnosis of alveolar soft part sarcoma is commonly based on characteristic histology and distinctive periodic acid-Schiff-positive crystals; however, the characteristic crystals may not always be observed, rendering the diagnosis difficult. Three important characteristics of alveolar soft part sarcoma, the presence of ASPSCR1-TFE3 fusion transcript, nuclear immunoreactivity for TFE3, and immunoreactivity for monocarboxylate transporter 1 and CD147, have recently been reported. To identify the best marker for alveolar soft part sarcoma in formalin-fixed, paraffin-embedded tissues, we evaluated the sensitivity and specificity of the detection of the ASPSCR1-TFE3 fusion transcript along with the immunoreactivity for TFE3 and CD147 in 24 alveolar soft part sarcomas and 23 non-alveolar soft part sarcoma tumors, including 5 granular cell tumors, 5 paragangliomas, 3 clear cell sarcomas, and 10 clear cell renal cell carcinomas. The ASPSCR1-TFE3 fusion transcript was detected in 24 of 24 alveolar soft part sarcomas (7 type 1, 17 type 2), and TFE3 immunoreactivity was observed in 22 of 24 alveolar soft part sarcomas. In non-alveolar soft part sarcoma tumors, the ASPSCR1-TFE3 fusion transcript was not detected; however, the TFE3 immunoreactivity was observed in 2 of 5 granular cell tumors. CD147 immunoreactivity was demonstrated in 20 of 24 alveolar soft part sarcomas, 3 of 5 granular cell tumors, and 8 of 10 clear cell renal cell carcinomas. Our results demonstrate that the most sensitive marker of alveolar soft part sarcoma was the presence of the ASPSCR1-TFE3 fusion transcript. Thus, detection of the ASPSCR1-TFE3 fusion transcript was considered applicable for formalin-fixed, paraffin-embedded tissues with superior sensitivity as compared with TFE3 immunohistochemical staining. In alveolar soft part sarcomas with unusual locations or histology, we consider that the detection of the ASPSCR1-TFE3 fusion transcript would be the highly effective diagnostic technique.

Oral nuclear factor-κB decoy oligonucleotides delivery system with chitosan modified poly(D,L-lactide-co-glycolide) nanospheres for inflammatory bowel disease.

Chitosan (CS)-modified poly(D,L-lactide-co-glycolide) (PLGA) nanospheres (NS) were developed and evaluated for use with a nuclear factor kappa B (NF-κB) decoy oligonucleotide (ODN) oral delivery system in an experimental model of ulcerative colitis (UC). Decoy ODN-loaded PLGA NS were prepared by an emulsion solvent diffusion method, and the physicochemical properties of NS were investigated. CS-modified PLGA NS (CS-PLGA NS) showed positive zeta potential, while unmodified PLGA NS (plain-PLGA NS) were negatively charged. Decoy ODN uptake studies with Caco-2 cells using confocal laser scanning microscopy (CLSM) indicated that CS-PLGA NS were more effectively taken up by the cells than plain-PLGA NS. Decoy ODN-loaded CS-PLGA NS were able to improve the stability of ODN against DNase I or an acidic medium, such as gastric juice. Daily oral administration of CS-PLGA NS in a rat model significantly improved dextran sulfate sodium-induced diarrhea, bloody feces, shortening of colon length, and myeloperoxidase activity. Furthermore, decoy ODN-loaded CS-PLGA NS were specifically deposited and adsorbed on the inflamed mucosal tissue of the UC model rat. These results suggested that CS-PLGA NS provide an effective means of colon-specific oral decoy ODN delivery in UC.

Diagnostic utility of dual-color break-apart chromogenic in situ hybridization for the detection of rearranged SS18 in formalin-fixed, paraffin-embedded synovial sarcoma.

Pathological diagnosis of synovial sarcoma is often problematic due to its broad spectrum of histology. Because synovial sarcoma consistently carries a specific chromosomal translocation, t(X;18), and its derivative chimeric gene, either SS18-SSX1 or SS18-SSX2, detecting these abnormalities by reverse transcription polymerase chain reaction or fluorescence in situ hybridization has been recognized as a powerful aid for diagnosis. Recently, chromogenic in situ hybridization, which enables simultaneous visualization of both genomic abnormality and the morphology of tumor cells, has gained attention. This study investigated the diagnostic utility of dual-color break-apart chromogenic in situ hybridization as a novel method for detecting SS18 rearrangement in synovial sarcoma. Formalin-fixed, paraffin-embedded tissue samples from 16 cases of synovial sarcoma and 10 cases of 5 other types of soft tissue sarcoma were collected. Dual-color break-apart probes were designed against the genomic region adjacent to SS18. Fluorescence and chromogenic in situ hybridization studies were performed using the same sections. In both assays, the number of signals was counted for sixty nuclei per sample. Scoring ratios (unpaired signals/paired signals) were calculated. Subsequently, SS18-SSX1 and SS18-SSX2 were examined by reverse transcription polymerase chain reaction. The results of chromogenic in situ hybridization, fluorescence in situ hybridization, and reverse transcription polymerase chain reaction were correlated. Unpaired signals were clearly observed in all the synovial sarcoma samples, which mostly indicated rearranged SS18. Synovial sarcoma and non-synovial sarcoma samples were clearly distinguished from each other by the scoring ratios. Reverse transcription polymerase chain reaction demonstrated SS18 chimeric gene transcripts in all the synovial sarcoma cases, while no fusion genes were detected in the non-synovial sarcoma cases. Taken together, unpaired signals in synovial sarcoma reflected rearranged SS18. The present chromogenic in situ hybridization-based SS18 rearrangement detection system provides a highly sensitive and specific method for the diagnosis of synovial sarcoma. Chromogenic in situ hybridization-based methods have great potential for routine use in the diagnosis of synovial sarcoma.

Detection of SYT and EWS gene rearrangements by dual-color break-apart CISH in liquid-based cytology samples of synovial sarcoma and Ewing sarcoma/primitive neuroectodermal tumor.

To improve cytologic diagnostic accuracy for translocation-associated sarcomas, we explored dual-color break-apart (dc) chromogenic in situ hybridization (CISH) on liquid-based cytology (LBC) samples of 2 prototypic sarcomas: synovial sarcoma (SS) and Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET). LBC samples of 10 cases of SS and 9 cases of ES/PNET were subjected to dc-CISH using probes for the specifically rearranged genes in each tumor entity: SYT in SS and EWS in ES/PNET. Rearranged SYT was successfully detected in all SSs but not in any ES/PNETs. In contrast, EWS rearrangement was identified in all ES/PNETs but not in any SSs. These results were validated by dc-fluorescence in situ hybridization and reverse transcription-polymerase chain reaction. dc-CISH on LBC samples is a reliable modality to detect gene rearrangements in sarcomas. This system has a clear advantage over other methods, enabling simultaneous visualization of the genetic abnormality and well-preserved, nonoverlapping cytomorphologic features with clear background under bright-field microscope.

Approval of new biopharmaceuticals 1999-2006: comparison of the US, EU and Japan situations.

Biopharmaceuticals, defined as either proteins derived from recombinant DNA technology (rDNAs) or therapeutic monoclonal antibodies (mAbs), have become the therapeutics of significance in the 21st century. This article identifies the new biopharmaceuticals approved in the three major pharmaceutical markets (US, EU and Japan) and analyzes the so-called "drug lag" in said regions. Between 1999 and 2006, a total of 65 new biopharmaceuticals were approved. Of this total, 59 (90.8%) were approved in the US, 52 (80.0%) in EU and 22 (33.8%) in Japan. The mean approval lag was 3.7 months in the US, 7.5 months in EU and 52.6 months in Japan. The US was ahead of the two other regional markets in approvals of biopharmaceuticals, while there was a significant drug lag in Japan. The authors also found that US companies were the licensors of 42 out of 65 new biopharmaceuticals, followed by European companies with 21 licensors and Japanese companies with only 2 licensors. These figures suggest that Japanese companies are still weak in biopharmaceuticals innovation and licensing, and this weakness appears to be a major contributing factor to the drug lag in the country.

Transcriptional regulation of SLP-76 family hematopoietic cell adaptor MIST/Clnk by STAT5.

SLP-76-related adaptor protein MIST (also called Clnk) is expressed in a variety of cytokine-dependent hematopoietic cell lines of myeloid and lymphoid origin as well as some cytokine-independent mast cell lines. To understand the molecular mechanisms underlying the MIST gene expression, we have characterized the 5'-flanking region of the mouse MIST gene. We have identified an enhancer region (-773 to -709), which is active in P815 mast cells expressing the endogenous MIST gene, but not in EL-4 T cells lacking MIST expression. Outside of this enhancer region, one STAT element present in the MIST promoter (-44 to -36) was found to bind STAT5A when IC-2 mast cells were stimulated with IL-3. Mutation of this STAT element did not affect basal MIST promoter activity in P815 mast cells, but was required for STAT5-mediated activation of the MIST promoter. Furthermore, endogenous MIST gene expression was induced in mast cells by a constitutively activated form of STAT5A, but not by an active mutant of c-Kit receptor. These findings suggest that STAT5 is involved in cytokine-mediated up-regulation of MIST gene expression, probably in collaboration with other lineage-specific transcription factors that promote basal MIST expression in mast cells.