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Lung epithelial cell death is a prominent feature of acute lung injury and acute respiratory distress syndrome (ALI/ARDS), which results from severe pulmonary infection leading to respiratory failure. Multiple mechanisms are believed to contribute to the death of epithelia; however, limited data propose a role for epigenetic modifiers. In this study, we report that a chromatin modulator protein arginine N-methyltransferase 4/coactivator-associated arginine methyltransferase 1 (PRMT4/CARM1) is elevated in human lung tissues with pneumonia and in experimental lung injury models. Here PRMT4 is normally targeted for its degradation by an E3 ubiquitin ligase, SCFFBXO9, that interacts with PRMT4 via a phosphodegron to ubiquitinate the chromatin modulator at K228 leading to its proteasomal degradation. Bacterial-derived endotoxin reduced levels of SCFFBXO9 thus increasing PRMT4 cellular concentrations linked to epithelial cell death. Elevated PRMT4 protein caused substantial epithelial cell death via caspase 3-mediated cell death signaling, and depletion of PRMT4 abolished LPS-mediated epithelial cell death both in cellular and murine injury models. These findings implicate a unique molecular interaction between SCFFBXO9 and PRMT4 and its regulation by endotoxin that impacts the life span of lung epithelia, which may play a key role in the pathobiology of tissue injury observed during critical respiratory illness.

Background/purpose Dental visits are a high risk activity during the COVID-19 pandemic. This study investigated the utilization of emergency dental services and clinical practical attitudes of dental residents in this period. Materials and methods Retrospective chart data from 13th November 2019 to 31st March 2020 in Kaohsiung Medical University Hospital, Taiwan were used. We obtained electronic medical records to review data from 515 patients who visited the emergency department with dental complaints and we contacted the 26 residents assigned to act as primary care providers to participate in this study. Results After the COVID-19 outbreak, 17% fewer patients had dental emergency utilization at a hospital emergency center relative to the previous period. A survey of residents also showed a decline in the number of patients. There were no significant differences of patients' problems and diagnoses between the two periods. After the COVID-19 outbreak, 61.5% of the residents were afraid of being infected by a patient's disease and the proportions of dentists wearing waterproof gowns, face shields, and surgical hair caps were 76.9%, 88.5%, and 76.3%, respectively. These variables increased significantly after the outbreak of COVID-19. Conclusion Despite the trend of a decreased number of patients, their utilization of dental emergency services seems to be similar before and after the COVID-19 outbreak, possibly related to strict hospital infection control policies and the relatively low number of COVID-19 confirmed patients internationally at that time.

We evaluated the effect of toothbrush/dentifrice brushing on the weight variation and surface properties of different denture bases. Four denture base materials (conventional heat cure, high impact, CAD/CAM, and polyamide resins) were subjected to toothbrushing abrasion (50,000 strokes). The weight value, surface roughness, and topography of each group were determined before and after toothbrushing. The hardness was measured by the Vickers hardness test. Data were analyzed using ANOVA and Bonferroni tests. After toothbrushing, the weight of the polyamide resin had significantly increased; significant weight losses were observed for conventional heat cure and high impact resins, but none for the CAD/CAM resin. The surface roughness of each group increased significantly owing to the wear caused by toothbrushing. The weight variation and surface roughness were not affected by the hardness. Our results suggested that denture base materials deteriorate after brushing with toothpaste, in which the polyamide resin exhibited lower levels of abrasion.

Pyroptosis is a pro-inflammatory type of regulated cell death (RCD) characterized by gasdermin D (GSDMD)-mediated membrane pore formation, cell swelling and rapid lysis, followed by the massive release of pro-inflammatory mediators such as interleukin-1β and interleukin-18. There are two main pathways of pyroptosis: the caspase-1-mediated canonical pathway and the caspase-4/5/11-mediated noncanonical pathway. However, the caspase-3-gasdermin E (GSDME) pathway and caspase-8-GSDMD pathway also induce pyroptosis. Pyroptosis can not only cause local inflammation but also lead to amplification of the inflammatory response. Recent studies have suggested that pyroptosis is closely related with cardiovascular disease (CVD); for example, in atherosclerosis, myocardial infarction, ischemia-reperfusion injury, heart failure, coronary calcification and aortic aneurysm, study results have promoted the development of inhibitors targeting the components related to pyroptosis, and some agents have been clinically proven to have cardiovascular benefits. In this review, we summarize emerging evidence to discuss the progressive understanding of pyroptosis and the pathways, effect and effectors of pyroptosis, as well as the role of pyroptosis in CVD. Additionally, we summarize pyroptosis-related pathway inhibitors and classic cardiovascular drugs targeting pyroptosis.

Motivation Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq), couples the measurement of surface marker proteins with simultaneous sequencing of mRNA at single cell level, which brings accurate cell surface phenotyping to single-cell transcriptomics. Unfortunately, multiplets in CITE-seq datasets create artificial cell types (ACT) and complicate the automation of cell surface phenotyping. Results We propose CITE-sort, an artificial-cell-type aware surface marker clustering method for CITE-seq. CITE-sort is aware of and is robust to multiplet-induced ACT. We benchmarked CITE-sort with real and simulated CITE-seq datasets and compared CITE-sort against canonical clustering methods. We show that CITE-sort produces the best clustering performance across the board. CITE-sort not only accurately identifies real biological cell types (BCT) but also consistently and reliably separates multiplet-induced artificial-cell-type droplet clusters from real BCT droplet clusters. In addition, CITE-sort organizes its clustering process with a binary tree, which facilitates easy interpretation and verification of its clustering result and simplifies cell-type annotation with domain knowledge in CITE-seq. Availability and implementation http://github.com/QiuyuLian/CITE-sort. Supplementary information Supplementary data is available at Bioinformatics online.

Objectives Oral submucous fibrosis (OSMF) is a chronic inflammatory disease and a potentially malignant oral disorder. However, the best therapeutic treatment for OSMF remains uncertain. Our previous study showed that photobiomodulation (PBM) therapy and forskolin could reduce arecoline‐induced fibrosis reactions via the cAMP pathway. The present study aimed to establish an animal model of areca nut extract (ANE)‐induced OSMF and to evaluate the therapeutic potential of PBM and forskolin for ANE‐induced OSMF. Subjects and methods The mice were divided into five groups. The buccal tissues were harvested for histomorphological analysis and immunoblotting. Results Our results showed that PBM significantly reduced the development of ANE‐induced OSMF, quantified by changes in submucosal layer thickness and collagen deposition. Additionally, PBM could extensively reduce the protein expression of the fibrotic marker genes alpha‐smooth muscle actin (α‐SMA) and connective tissue growth factor (CTGF) in buccal submucous lesions. However, forskolin treatment significantly decreased the protein expression of fibrotic marker genes but slightly decreased the observed histomorphological changes. Conclusions We established an ANE‐induced OSMF mouse model, which also provided a model for the development of a therapeutic treatment for OSMF. The anti‐fibrotic effects of PBM and forskolin may be useful for clinical interventions.

Background Guided endodontics technique has been introduced for years, but the accuracy in different types of teeth has yet to be assessed. The aim of this study is to evaluate the accuracy of three dimensional (3D)-printed endodontic guides for access cavity preparation in different types of teeth, and to evaluate the predictive ability of angular and linear deviation on canal accessibility ex vivo. Method Eighty-four extracted human teeth were mounted into six jaw models and categorised into three groups: anterior teeth (AT), premolar (P), and molar (M). Preoperative cone beam computed tomography (CBCT) and surface scans were taken and matched using implant planning software. Virtual access cavity planning was performed, and templates were produced using a 3D printer. After access cavities were performed, the canal accessibility was recorded. Postoperative CBCT scans were superimposed in software. Coronal and apical linear deviations and angular deviations were measured and evaluated with nonparametric statistics. The receiver operating characteristic (ROC) curve was used to evaluate the predictive ability of angular and linear deviation for canal accessibility in SPSS v20. Results A total of 117 guided access cavities were created and 23 of them were record as canal inaccessibility, but all canals were accessible after canal negotiation. The average linear deviation for all groups was 0.13 ± 0.21 mm at coronal position, 0.46 ± 0.4 mm at apical position, and 2.8 ± 2.6° in angular deviation. At the coronal position, the linear deviations of the AT and P groups were significantly lower than M group deviation (P P Conclusions In conclusion, this study demonstrated that the accuracy of access cavity preparation with 3D-printed endodontic guides was acceptable. The linear and angular deviations in the M group were significantly higher than those in the other groups, which might be caused by the interference of the opposite teeth. Angular deviation best discriminated the canal access ability of guided access cavity preparation. Graphical Abstract

Acute cellular rejection is common after lung transplantation and is associated with an increased risk of early chronic rejection. We present combined single cell RNA and T cell receptor sequencing on recipient derived T cells obtained from the bronchoalveolar lavage of three lung transplant recipients with acute cellular rejection and compare them with T cells obtained from the same three patients after clinical treatment of rejection with high-dose, systemic glucocorticoids. At the time of acute cellular rejection, we find an oligoclonal expansion of cytotoxic CD8+T cells, that all persist as tissue resident memory T cells following successful treatment. Persisting CD8+allograft-resident T cells have reduced gene expression for cytotoxic mediators following therapy with glucocorticoids. This clonal expansion is discordant with circulating T cell clonal expansion at the time of rejection, suggesting in-situ expansion. These findings pose a potential biological mechanism linking acute cellular rejection to chronic allograft damage.

A subunit mucosal vaccine conferred protection against heterologous Klebsiella pneumoniae strains, via IL-17R signaling in lung fibroblasts.

Bronchoalveolar lavage (BAL) samples from Severe Asthma Research Program (SARP) patients display suppression of a module of genes involved in cAMP-signaling pathways (BALcAMP) correlating with severity, therapy, and macrophage constituency. We sought to establish if gene expression changes were specific to macrophages and compared gene expression trends from multiple sources. Datasets included single-cell RNA sequencing (scRNA-seq) from lung specimens including a fatal exacerbation of severe Asthma COPD Overlap Syndrome (ACOS) after intense therapy and controls without lung disease, bulk RNA sequencing from cultured macrophage (THP-1) cells after acute or prolonged β-agonist exposure, SARP datasets, and data from the Immune Modulators of Severe Asthma (IMSA) cohort. THP monocytes suppressed BALcAMP network gene expression after prolonged relative to acute β-agonist exposure, corroborating SARP observations. scRNA-seq from healthy and diseased lung tissue revealed 13 cell populations enriched for macrophages. In severe ACOS, BALcAMP gene network expression scores were decreased in many cell populations, most significantly for macrophage populations ( P < 3.9e-111). Natural killer (NK) cells and type II alveolar epithelial cells displayed less robust network suppression ( P < 9.2e-8). Alveolar macrophages displayed the most numerous individual genes affected and the highest amplitude of modulation. Key BALcAMP genes demonstrate significantly decreased expression in severe asthmatics in the IMSA cohort. We conclude that suppression of the BALcAMP gene module identified from SARP BAL samples is validated in the IMSA patient cohort with physiological parallels observed in a monocytic cell line and in a severe ACOS patient sample with effects preferentially localizing to macrophages.

Ivacaftor is a drug that was recently approved by the U.S. Food and Drug Administration for the treatment of patients with cystic fibrosis (CF) and at least one copy of the G511D mutation in the CFTR (CF transmembrane conductance regulator) gene. The transcriptomic effect of ivacaftor in patients with CF remains unclear. Here, we sought to examine whether and how the transcriptome of patients is influenced by ivacaftor treatment, and to determine whether these data allow prediction of ivacaftor responsiveness. Our data originated from the G551D Observational Study (GOAL). We performed RNA sequencing (RNA-seq) on peripheral blood mononuclear cells (PBMCs) from 56 patients and compared the transcriptomic changes that occurred before and after ivacaftor treatment. We used consensus clustering to stratify patients into subgroups based on their clinical responses after treatment, and we determined differences between subgroups in baseline gene expression. A random forest model was built to predict ivacaftor responsiveness. We identified 239 genes (false discovery rate < 0.1) that were significantly influenced by ivacaftor in PBMCs. The functions of these genes relate to cell differentiation, microbial infection, inflammation, Toll-like receptor signaling, and metabolism. We classified patients into "good" and "moderate" responder groups based on their clinical response to ivacaftor. We identified a panel of signature genes and built a statistical model for predicting CFTR modulator responsiveness. Despite a limited sample size, adequate prediction performance was achieved with an accuracy of 0.92. In conclusion, for the first time, the present study demonstrates profound transcriptomic impacts of ivacaftor in PBMCs from patients with CF, and provides a pilot statistical model for predicting clinical responsiveness to ivacaftor before treatment.

Lymphopenia is common in severe COVID-19 disease, yet the mechanisms are poorly understood. In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4+ lymphopenia predominated, with lower CD4+/CD8+ ratios in severe COVID-19 compared to recovered, mild disease (p+ T cell responses to Spike-1(S1) produced increased in vitro TNF-α, but impaired proliferation and increased susceptibility to activation-induced cell death (AICD). CD4+TNF-α+ T cell responses inversely correlated with absolute CD4+ counts from severe COVID-19 patients (n=76; R=-0.744, P+ proliferation and abrogated S1-AICD in severe COVID-19 patients (P+ cells with infliximab treatment. Lung CD4+ T cells in severe COVID-19 were reduced and produced higher TNF-α versus PBMC. Together, our findings show COVID-19-associated CD4+ lymphopenia and dysfunction is autocrine TNF-α/TNFRI-dependent and therapies targeting TNF-α may be beneficial in severe COVID-19.One Sentence SummaryAutocrine TNF-α/TNFRI regulates CD4+ T cell lymphopenia and dysfunction in severe COVID-19 disease.

Chronic lung allograft dysfunction (CLAD) remains the major complication limiting long-term survival among lung transplant recipients (LTRs). Limited understanding of CLAD immunopathogenesis and a paucity of biomarkers remain substantial barriers for earlier detection and therapeutic interventions for CLAD. We hypothesized the airway transcriptome would reflect key immunologic changes in disease. We compared airway brush-derived transcriptomic signatures in CLAD (n =24) versus non-CLAD (n=21) LTRs. A targeted assessment of the proteome using concomitant bronchoalveolar lavage (BAL) fluid for 24 cytokines/chemokines and alloimmune T cell responses was performed to validate the airway transcriptome. We observed an airway transcriptomic signature of differential genes expressed (DGEs) in CLAD marked by Type-1 immunity and striking upregulation of two endogenous immune regulators: indoleamine 2, 3 dioxygenase 1 (IDO-1) and tumor necrosis factor receptor superfamily 6B (TNFRSF6B). Advanced CLAD staging was associated with a more intense airway transcriptome signature. In a validation cohort using the identified signature, we found an area under the curve (AUC) of 0.77 for CLAD LTRs. Targeted proteomic analyses revealed a predominant Type-1 profile with detection of IFN-γ, TNF-α, and IL-1β as dominant CLAD cytokines, correlating with the airway transcriptome. The airway transcriptome provides novel insights into CLAD immunopathogenesis and biomarkers that may impact diagnosis of CLAD.

Immune dysfunction is an important factor driving mortality and adverse outcomes after trauma but remains poorly understood, especially at the cellular level. To deconvolute the trauma-induced immune response, we applied single-cell RNA sequencing to circulating and bone marrow mononuclear cells in injured mice and circulating mononuclear cells in trauma patients. In mice, the greatest changes in gene expression were seen in monocytes across both compartments. After systemic injury, the gene expression pattern of monocytes markedly deviated from steady state with corresponding changes in critical transcription factors, which can be traced back to myeloid progenitors. These changes were largely recapitulated in the human single-cell analysis. We generalized the major changes in human CD14+ monocytes into 6 signatures, which further defined 2 trauma patient subtypes (SG1 vs. SG2) identified in the whole-blood leukocyte transcriptome in the initial 12 hours after injury. Compared with SG2, SG1 patients exhibited delayed recovery, more severe organ dysfunction, and a higher incidence of infection and noninfectious complications. The 2 patient subtypes were also recapitulated in burn and sepsis patients, revealing a shared pattern of immune response across critical illness. Our data will be broadly useful to further explore the immune response to inflammatory diseases and critical illness.

Identifying and removing multiplets are essential to improving the scalability and the reliability of single cell RNA sequencing (scRNA-seq). Multiplets create artificial cell types in the dataset. We propose a Gaussian mixture model-based multiplet identification method, GMM-Demux. GMM-Demux accurately identifies and removes multiplets through sample barcoding, including cell hashing and MULTI-seq. GMM-Demux uses a droplet formation model to authenticate putative cell types discovered from a scRNA-seq dataset. We generate two in-house cell-hashing datasets and compared GMM-Demux against three state-of-the-art sample barcoding classifiers. We show that GMM-Demux is stable and highly accurate and recognizes 9 multiplet-induced fake cell types in a PBMC dataset.

Chronic obstructive pulmonary disease (COPD) is an inflammatory disease that is associated with increased risk of lung cancer. Pseudomonas aeruginosa (PA) infections are frequent in patients with COPD, which increase lung inflammation and acute exacerbations. However, the influences of PA-induced inflammation on lung tumorigenesis and the efficacy of immune checkpoint blockade remain unknown. In this study, we initiated a murine model of lung cancer by treating FVB/NJ female mice with tobacco carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alone or in combination with PA-lipopolysaccharide (LPS). LPS-mediated chronic inflammation induced T-cell exhaustion, increased the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis, and enhanced NNK-induced lung tumorigenesis through an immunosuppressive microenvironment characterized by accumulation of myeloid-derived suppressive cells (MDSC) and regulatory T cells. Anti–PD-1 antibody treatment reduced tumors in NNK/LPS-treated mice with a 10-week LPS treatment but failed to inhibit tumor growth when LPS exposure was prolonged to 16 weeks. Anti-Ly6G antibody treatment coupled with depletion of MDSC alone reduced tumor growth; when combined with anti–PD-1 antibody, this treatment further enhanced antitumor activity in 16-week NNK/LPS-treated mice. Immune gene signatures from a human lung cancer dataset of PD-1 blockade were identified, which predicted treatment responses and survival outcome and overlapped with those from the mouse model. This study demonstrated that LPS-mediated chronic inflammation creates a favorable immunosuppressive microenvironment for tumor progression and correlates with the efficacy of anti–PD-1 treatment in mice. Immune gene signatures overlap with human and mouse lung tumors, providing potentially predictive markers for patients undergoing immunotherapy. Significance: This study identifies an immune gene signature that predicts treatment responses and survival in patients with tobacco carcinogen–induced lung cancer receiving immune checkpoint blockade therapy.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal complication of prematurity. Using suspension and imaging mass cytometry coupled with single-cell RNA sequencing, we demonstrate severe inflammation in patients with NEC. NEC mucosa could be subtyped by an influx of three distinct neutrophil phenotypes (immature, newly emigrated, and aged). Furthermore, CD16+CD163+ monocytes/Mϕ, correlated with newly emigrated neutrophils, were specifically enriched in NEC mucosa, found adjacent to the blood vessels, and increased in circulation of infants with surgical NEC, suggesting trafficking from the periphery to areas of inflammation. NEC-specific monocytes/Mϕ transcribed inflammatory genes, including TREM1, IL1A, IL1B, and calprotectin, and neutrophil recruitment genes IL8, CXCL1, CXCL2, CXCL5 and had enrichment of gene sets in pathways involved in chemotaxis, migration, phagocytosis, and reactive oxygen species generation. In summary, we identify a novel subtype of inflammatory monocytes/Mϕ associated with NEC that should be further evaluated as a potential biomarker of surgical NEC and a target for the development of NEC-specific therapeutics.

Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq), couples the measurement of surface marker proteins with simultaneous sequencing of mRNA at single cell level, which brings accurate cell surface phenotyping to single cell transcriptomics. Unfortunately, multiplets in CITE-seq datasets create artificial cell types and complicates the automation of cell surface phenotyping. We propose CITE-sort, an artificial-cell-type aware surface marker clustering method for CITE-seq. CITE-sort is aware of and is robust to multiplet-induced artificial cell types. We benchmarked CITE-sort with real and simulated CITE-seq datasets and compared CITE-sort against canonical clustering methods. We show that CITE-sort produces the best clustering performance across the board. CITE-sort not only accurately identifies real biological cell types but also consistently and reliably separates multiplet-induced artificial-cell-type droplet clusters from real biological-cell-type droplet clusters. In addition, CITE-sort organizes its clustering process with a binary tree, which facilitates easy interpretation and verification of its clustering result and simplifies cell type annotation with domain knowledge in CITE-seq.

Wedge-shaped defects are frequently observed on the cervical region of the human tooth. Previously, most studies explained that improper tooth-brushing causes such defects. However, recent clinical observation suggested that the repeated stress due to occlusal force may induce the formation of these wedge-shaped defects. In this study, therefore, two-dimensional human tooth models are considered with and without a wedge-shaped defect by applying the finite element method. To evaluate large stress concentrations accurately, a method of analysis is discussed in terms of the intensity of singular stress fields appearing at the tip of the sharp wedge-shaped defect. The effects of the position and direction of occlusion on the intensity of singular stress fields are discussed before and after restoration with composite resins.

Purpose Chronic lung allograft dysfunction (CLAD) in lung transplant recipients (LTRs) limits long-term survival compared to other solid organ transplants. Underlying mechanisms of CLAD are poorly understood. To address the pathogenesis of CLAD at the molecular level, we performed RNA-seq analysis of airway brush samples in LTRs with and without CLAD. Methods LTRs with and without CLAD were evaluated for inclusion. CLAD was staged using ISHLT criteria. RNA was extracted from airway brush samples and sequenced to obtain bulk RNA-seq data. These were subsequently analyzed for differential gene expression (DGE) profiling using false-discovery rate (FDR) p-value of 0.05 and absolute log2 fold change > 2 as thresholds for significance. Gene enrichment and gene and cell ontology analyses of DGE profile was completed to predict canonical pathways and cellular upstream regulator of activated signal pathways where p Results 24 CLAD and 21 stable control LTRs were included for RNA-seq analysis. The majority of cells identified from brushing were epithelial cells (54%). 293 genes were deferentially expressed between CLAD and non-CLAD. Gene enrichment analyses revealed activation TNF- α (p Conclusion Transcriptome analyses of bronchial brush samples revealed activation of Type-1 immune responses. Furthermore, this signature appears to strengthen over time as patients progress to CLAD and in later stages of CLAD. Further analyses may provide the rationale for testing select immune targets in CLAD and uncover distinct immune endotypes within CLAD.

Identifying and removing multiplets from downstream analysis is essential to improve the scalability and reliability of single cell RNA sequencing (scRNA-seq). High multiplet rates create artificial cell types in the dataset. Sample barcoding, including the cell hashing technology and the MULTI-seq technology, enables analytical identification of a fraction of multiplets in a scRNA-seq dataset.We propose a Gaussian-mixture-model-based multiplet identification method, GMM-Demux. GMM-Demux accurately identifies and removes the sample-barcoding-detectable multiplets and estimates the percentage of sample-barcoding-undetectable multiplets in the remaining dataset. GMM-Demux describes the droplet formation process with an augmented binomial probabilistic model, and uses the model to authenticate cell types discovered from a scRNA-seq dataset.We conducted two cell-hashing experiments, collected a public cell-hashing dataset, and generated a simulated cellhashing dataset. We compared the classification result of GMM-Demux against a state-of-the-art heuristic-based classifier. We show that GMM-Demux is more accurate, more stable, reduces the error rate by up to 69×, and is capable of reliably recognizing 9 multiplet-induced fake cell types and 8 real cell types in a PBMC scRNA-seq dataset.

Rationale: Infection with Pneumocystis, an opportunistic fungal pathogen, can result in fulminant pneumonia in the clinical setting of patients with immunosuppression. In murine models, Pneumocystis has previously been shown to induce a CD4+ T cell–dependent eosinophilic response in the lung capable of providing protection.Objectives: We sought to explore the role of Pneumocystis in generating asthma-like lung pathology, given the natural eosinophilic response to infection.Methods: Pneumocystis infection or antigen treatment was used to induce asthma-like pathology in wild-type mice. The roles of CD4+ T cells and eosinophils were examined using antibody depletion and knockout mice, respectively. The presence of anti-Pneumocystis antibodies in human serum samples was detected by ELISA and Western blotting.Measurements and Main Results: Pneumocystis infection generates a strong type II response in the lung that requires CD4+ T cells. Pneumocystis infection was capable of priming a Th2 response similar to th...

Purpose Chronic lung allograft dysfunction (CLAD) significantly limits long-term survival in lung transplant recipients (LTRs) and episodes of acute cellular rejection (ACR) are the major risk factor for CLAD. To study the immune mechanisms leading to ACR/CLAD, we have characterized donor-specific alloreactive effector lung T (bronchoalveolar lavage; BAL) cells and peripheral blood mononuclear cells (PBMC) via indirect/direct allorecognition responses in LTRs with/without histologic evidence of ACR and into those who developed CLAD. Understanding these mechanisms may lead to new therapies to prevent CLAD. Methods To assess alloeffector T cell responses from LTRs cells in BAL and PBMC, we used an ex vivo flow cytometric assay and donor alloantigen. LTRs with/without ACR (median 4 months), and a subset who developed CLAD (median 20 months) were studied. The effector responses (IFN-γ, TNF-α, CD107a, IL-17a, IL-13, IL-2 and the costimulation surface molecule, CD154) to donor alloantigen were measured using either donor lysate or irradiated cells in a 6 h in vitro re-stimulation assay. We also used single cell RNAseq analysis to analyze the transcriptome in response to alloantigen in isolated BAL T cells from LTRs with CLAD. Results The predominant BAL alloeffector responses during active ACR were similar between CD8+ and CD4+ T cells, with a Type-1 effector profile and IFN-γ responses predominant. The CD4+ T cells alloeffector responses were higher in BAL>PBMC (p Conclusion Type-1 alloeffector responses during ACR are similar between CD8+ and CD4+ T cells, with the latter predominant in BAL. Progression into CLAD is associated with persistently elevated CD4+ alloeffector responses with a potential cytotoxic CD4+ subset playing an important role.

Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (-/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.

IL-17A is purported to help drive early pathogenesis in acute respiratory distress syndrome (ARDS) by enhancing neutrophil recruitment. Although IL-17A is the archetypal cytokine of T-helper 17 cells, it is produced by a number of lymphocytes, the source during ARDS being unknown.To identify the cellular source and the role of IL-17A in the early phase of lung injury.Lung injury was induced in wild-type (C57BL/6) and IL-17 knockout (KO) mice with aerosolized LPS (100 μg) or Pseudomonas aeruginosa infection. Detailed phenotyping of the cells expressing RORγt, the transcriptional regulator of IL-17 production, in the mouse lung at 24 hours was performed by flow cytometry.A 100-fold reduction in neutrophil infiltration was observed in the lungs of the IL-17A KO compared with wild-type mice. The majority of RORγt(+) cells in the mouse lung were the recently identified group 3 innate lymphoid cells (ILC3s). Detailed characterization revealed these pulmonary ILC3s (pILC3s) to be discrete from those described in the gut. The critical role of these cells was verified by inducing injury in recombinase-activating gene 2 KO mice, which lack T cells but retain innate lymphoid cells. No amelioration of pathology was observed in the recombinase-activating gene 2 KO mice.IL-17 is rapidly produced during lung injury and significantly contributes to early immunopathogenesis. This is orchestrated largely by a distinct population of pILC3s. Modulation of the activity of pILC3s may potentiate early control of the inflammatory dysregulation seen in ARDS, opening up new therapeutic targets.

Clinical and epidemiological evidence suggest that chronic infection and inflammation increase risk of lung cancer. Pseudomonas aeruginosa infection is frequently found in patients with chronic obstructive pulmonary disease (COPD) and is associated with increased lung inflammation and acute exacerbations. However, the mechanism of chronic bacterial infection-induced lung inflammation in promoting lung tumorigenesis remains unclear. To elucidate this mechanism, we established a murine lung cancer model by treating mice with or without recurrent lipopolysaccharides (LPS) from Pseudomonas aeruginosa in combination with nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Interestingly, combined LPS and NNK exposure significantly increased tumor number, tumor incidence, and tumor area compared to NNK treatment alone. In addition, the inflammatory cell counts in the bronchoalveolar lavage (BAL) including macrophages, neutrophils, and lymphocytes were significantly increased in the LPS/NNK treatment group. The BAL fluid of chemokines/cytokines, as analyzed by luminex assays, revealed higher levels of IL-17, CXCL10, GM-CSF, G-CSF, MIP-1a, and KC in LPS/NNK than in NNK treatment group. Flow cytometry analysis of the mouse lung tissue revealed that combined LPS and NNK exposure significantly increased CD4+ T cells including Th1, Th17, and Tregs and myeloid-derived suppressor cells recruitment in the lung. Real-time polymerase chain reaction of mouse lung tissue showed T cell exhaustion related genes, including Pdcd1, Ctla-4, Tim-3, Lag-3, and Foxp3, were significantly upregulated in the LPS/NNK treatment than NNK treatment. Moreover, immunohistochemical staining of LPS/NNK-exposed lung tumors showed higher PD-L1 expression than NNK-exposed lung tumors. Our data suggest that chronic LPS exposure-promoted and NNK-induced lung tumorigenesis is associated with immunosuppressive tumor microenvironment. The changes include recruitment of Tregs and MDSCs, increased T cell exhaustion, and upregulated PD-1/PD-L1 pathway, which may be used as the therapeutic target for chronic inflammation-associated lung cancer treatment. Citation Format: Chia-Hsin Liu, Kong Chen, Yuanpu Di. Lipopolysaccharide-induced chronic inflammation promotes lung tumorigenesis in the context of an immunosuppressive microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1094.

Purpose Chronic lung allograft dysfunction (CLAD) is the major limitation to long-term survival in lung transplant recipients (LTRs). The underlying biologic mechanisms that drive CLAD are poorly understood. To address the pathogenesis of CLAD at the molecular level, we performed RNA-seq analysis of airway brush samples and Meso Scale Discovery (MSD) multiplex cytokine and chemokine measurements in bronchial lavage (BAL) samples. Methods Total RNA was extracted from distal bronchial brush samples and sequenced using an Illumina instrument to obtain bulk RNA-seq data which were subsequently analyzed for differential gene expression (DGE) profiling with correction for a potential batch effect. Gene enrichment and gene and cell ontology analyses of DGE profile was done and used to predict canonical pathways and cellular upstream regular of activated signal pathways. False discovery rate (FDR) p Results 21 CLAD and 18 stable control LTRs were included for RNA-seq analysis. Corresponding MSD measurements were performed on 17 CLAD and 11 control BAL samples. 1031 DEGs were overrepresented in CLAD samples. Gene analyses revealed enrichment of the Type-1 adaptive immune response and the inflammasome. TNF- α (p Conclusion Transcriptome analyses and the lung allograft BAL immune proteome, provide novel insights into the immune activation pathways prevalent in CLAD versus controls. Further analyses may provide the rationale for testing select immune targets in CLAD and uncover distinct immune endotypes within CLAD.

This study focused on the application of novel finite-element analysis software for constructing a finite-element model from the computed tomography data of a human dentulous mandible. The finite-element model is necessary for evaluating the mechanical response of the alveolar part of the mandible, resulting from occlusal force applied to the teeth during biting. Commercially available patient-specific general computed tomography–based finite-element analysis software was solely applied to the finite-element analysis for the extraction of computed tomography data. The mandibular bone with teeth was extracted from the original images. Both the enamel and the dentin were extracted after image processing, and the periodontal ligament was created from the segmented dentin. The constructed finite-element model was reasonably accurate using a total of 234,644 nodes and 1,268,784 tetrahedral and 40,665 shell elements. The elastic moduli of the heterogeneous mandibular bone were determined from the bone density data of the computed tomography images. The results suggested that the software applied in this study is both useful and powerful for creating a more accurate three-dimensional finite-element model of a dentulous mandible from the computed tomography data without the need for any other software.

To study the mechanisms of gastric tumorigenesis, we have established CSN cell line from human normal gastric mucosa, and CS12, a tumorigenic and invasive gastric cancer cell line from CSN passages. Many stem cell markers were expressed in both CSN and CS12 cells, but LGR5 and NANOG were expressed only in CS12 cells. Increased expression of homeobox A13 (HoxA13) and its downstream cascades was significant for the tumorigenic activity of CS12 cells, and was associated with recruitment of E2F-1 to HoxA13 promoter accompanied with increased trimethylation of histone H3 lysine 4 (H3K4me3) at the hypomethylated E2F motifs. Knockdown of HoxA13 caused the downregulation of long non-coding RNA HOTTIP and insulin growth factor-binding protein 3 (IGFBP-3) genes, indicating that both were targets of HoxA13. Concurrent regulation of HoxA13-HOTTIP was mediated by the mixed lineage leukemia-WD repeat domain 5 complex, which caused the trimethylation of H3K4 and then stimulated cell proliferation. HoxA13 transactivated the IGFBP-3 promoter through the HOX-binding site. Activation of IGFBP-3 stimulated the oncogenic potential and invasion activity. Increased expression of HoxA13 (63.2%) and IGFBP-3 (28.6%) was detected in human gastric cancer tissues and was found in the gastric cancer data of The Cancer Genome Atlas. Taken together, the HoxA13-HOTTIP-IGFBP-3 cascade is critical for the carcinogenic characteristics of CS12 cells.

Objectives. To investigate the correlation between frontal gaps and skeletal stability after intraoral vertical ramus osteotomy (IVRO) for correction of mandibular prognathism.Materials and Methods. Thirty-three patients with frontal gaps after IVRO-based mandibular prognathism correction were included. Three lateral and frontal cephalometric radiographs were obtained: preoperatively (T1), immediately postoperatively (T2), and 2 years postoperatively (T3). Two linear measurements (menton [Me] and frontal gap) were compared from T1 to T3 (T21: immediate surgical changes; T32: postoperative stability; T31: 2-year surgical change). Data were analyzed using Pearson’s correlation coefficient and multiple linear regression.Results. The T21 mean surgical horizontal change in the Me position was12.4±4.23 mm. Vertically, the mean downward Me movement was0.6±1.73 mm. The mean frontal gaps were4.7±2.68 mm and4±2.48 mm in the right and left gonial regions, respectively. Postoperative stability (T32) significantly correlated with the amount of setback. Frontal gaps did not have a significant effect on postoperative stability. However, multiple regression model (R2=0.341,P=0.017) showed value predictability, especially in the amount of setback.Conclusion. Frontal gaps occur after IVRO but have no significant effect on long-term postoperative skeletal stability. The primary risk factor for postoperative relapse remains the amount of mandibular setback.

During surgical correction of facial deformities, accurate prediction of the resulting facial profile is important for the patient and the surgeon. The purpose of the present study was to investigate profile changes after surgical treatment of mandibular prognathism. Thirty patients (20 females and 10 males; ages 17–28 years) with mandibular prognathism underwent vertical ramus osteotomy. Preoperative and postoperative cephalograms were analyzed; landmarks were identified and compared. The mean horizontal setback of the pogonion (Pog) was 11.7mm. The setback ratios of labrale inferius (Li)/incision inferius (Ii), labiomental sulcus (Si)/point B, and soft tissue pogonion (PogS)/pogonion (Pog) were 0.98, 0.99, and 0.95, respectively. There were no sex-related changes in soft tissue. These findings indicate that changes in soft tissue closely correlate with the amount of mandibular setback in the horizontal direction. Such information might facilitate more accurate prediction of the outcome of orthognathic surgery.

This paper presents a novel design of adjustable elliptical exerciser, which can adjust both stride and inclined angle. Firstly, we use a selected coupler curve of four-bar linkage to design a six-bar linkage. From the six-bar linkage, we investigate the parameters which affect the corresponding elliptical path and its inclined angle. By considering the way of adjusting the inclined angle, the final design becomes a 2-DOF seven-bar-eight-joint linkage. We use SolidWorks software to design this novel elliptical exerciser, and use CAE software to check the stresses and strains to ensure the strength of the members of this design. The prototype of this novel design of adjustable elliptical exerciser has been fabricated, and its feasibility has been confirmed.

OBJECTIVE: In the present study, we examined whether 2, 2-bis [4-(2-hydroxy-3-methacryloxypropoxy) phenyl] propane (Bis-GMA) has effects on LSC2 cells, human dental pulp cell line. MATERIAL AND METHODS: The viability, cell cycle, and morphology of LSC2 cells were analyzed after exposure to several different concentrations of Bis-GMA. The recovery of viability of Bis-GMA exposed cells was also analyzed in the condition without Bis-GMA. Further, penetration of Bis-GMA to dentin disc was examined using isocratic high-performance liquid chromatography. RESULTS: There was a concentration-dependent decrease in cell proliferation and an increase in cell number in the sub-G1 population after exposure to Bis-GMA. Furthermore, the cells showed typical characteristics of apoptotic cells after the exposure to high concentration of Bis-GMA. In contrast, cells exposed to lower concentrations of Bis-GMA recovered their viability after being cultured without Bis-GMA. We also found that Bis-GMA is capable of penetrating 1-mm-thick dentin discs, though the penetrated concentration was lower than that showing cytotoxicity. CONCLUSION: These results suggest that Bis-GMA has cytotoxic effects, though dental pulp exposed to lower concentrations is able to recover their viability when Bis-GMA is removed.

Background/Purpose Since the implementation of National Health Insurance in Taiwan, Emergency Department (ED) volume has progressively increased, and the current triage system is insufficient and needs modification. This study compared the prioritization and resource utilization differences between the four-level Taiwan Triage System (TTS) and the standardized five-level Canadian Triage and Acuity Scale (CTAS) among ED patients. Methods This was a prospective observational study. All adult ED patients who presented to three different medical centers during the study period were included. Patients were independently triaged by the duty triage nurse using TTS, and a single trained research nurse using CTAS with a computer support software system. Hospitalization, length of stay (LOS), and medical resource consumption were analyzed by comparing TTS and CTAS by acuity levels. Results There was significant disparity in patient prioritization between TTS and CTAS among the 1851 enrolled patients. With TTS, 7.8%, 46.1%, 45.9% and 0.2% were assigned to levels 1, 2, 3, and 4, respectively. With CTAS, 3.5%, 24.4%, 44.3%, 22.4% and 5.5% were assigned to levels 1, 2, 3, 4, and 5, respectively. The hospitalization rate, LOS, and medical resource consumption differed significantly between the two triage systems and correlated better with CTAS. Conclusion CTAS provided better discrimination for ED patient triage, and also showed greater validity when predicting hospitalization, LOS, and medical resource consumption. An accurate five-level triage scale appeared superior in predicting patient acuity and resource utilization.

In response to the essential role probe cards play in the semiconductor testing industry, a CMOS-MEMS force sensing devices capable of simultaneously monitoring the probe reacting force and electrical test signals is designed for probe cards. The probe reacting force can assist operators immediately to identify a broken, deformed, or worn-out probe and recognize that the measured electrical signals are erroneous. The debug time and cost of the IC test can therefore be effectively reduced. Array-type CMOS-MEMS force sensors are capable of monitoring the status of vertical probe cards on-line in the case of both die-level and wafer-level applications. The force sensor essentially is a Wheatstone-bridge-based piezoresistive force sensor with a small surface area and can be easily fabricated. It consists of a membrane composed of metal, silicon oxide, and a piezoresistive layer made of polycrystalline silicon. Moreover, as the conventional back-etch process can barely handle the increasingly smaller probe pitch, we use front etching to etch out a cavity under the membrane on the silicon substrate in order to deform the membrane during the post-process. For measuring the output signals of the sensors, on-chip circuits to scan and amplify the output signals of the sensors are integrated in the design process. Furthermore, a finite element method is adopted to analyze the structure of the sensors and to find the optimal piezoresistive sensor design. The TSMC 0.35 μm 2P4M process is used to fabricate the force sensors and the circuits. According to the measurement results, the designed sensor reports a sensitivity of 3.114 mV/N/V while a load-bearing force is 0.0294 N.

The aim of this study was to determine the correlation between pre- and postsurgical loss of blood and blood components among patients undergoing treatment of facial deformities by bilateral parasymphyseal osteotomy (BPsO).The pre- and postoperative values of blood components were determined in 30 facial deformity patients who underwent orthognathic surgery by hypotensive anesthesia. Correlations among the blood loss, sex, age, operation time, and reduced values of blood components were assessed by a correlation matrix. The mean blood loss and operation time were 437.5 (± 52.5) mL and 355.8 (± 209.42) minutes, respectively. Two patients included in this study had required blood transfusion. The mean reduced red blood cell (× 10/μL), hemoglobin (g/dL), and hematocrit (%) were -1.02, -2.98, and -9.18, respectively. There was no significant correlation between blood loss and other related factors (eg, age, operation time, and reduced blood components). All patients, however, showed significantly lower values of blood components after surgery. In conclusion, no significant factor was associated with blood loss and reduced blood components among patients undergoing BPsO. Furthermore, hypotensive anesthesia is a well-accepted method to reduce blood loss during orthognathic surgery.

Acinetobacter baumanniiis a significant cause of severe hospital-acquired infections with a recent rise in multidrug-resistant infections involving traumatic wounds of military personnel. The interleukin-17 (IL-17) pathway is essential for neutrophil recruitment in response to a variety of pathogens, while the control ofA. baumanniiinfection is known to be dependent on neutrophils. This suggests that IL-17 may play an important role inA. baumanniiinfection; however, this has yet to be studied. Here, we summarize the recent advances in understanding the host-pathogen interaction ofA. baumanniiand propose a potential role of the IL-17 pathway in generating a protective immune response.

Purpose. The aim of this study was to determine the correlation between the pharyngeal airway space and head posture after mandibular setback surgery for mandibular prognathism.Materials and Methods. Serial lateral cephalograms of 37 patients with mandibular prognathism who underwent intraoral vertical ramus osteotomy (IVRO) were evaluated before (T1) and immediately (T2), between 6 weeks and 3 months (T3), and more than 1 year (T4) after surgery. Pairedt-tests and Pearson’s correlation analysis were used to evaluate the postoperative changes in all cephalometric parameters, including the mandible, hyoid, head posture (craniocervical angle), and pharyngeal airway space.Results. The mandible and hyoid were set back by 12.8 mm and 4.9 mm, respectively, at T2. Furthermore, the hyoid showed significant inferior movement of 10.7 mm, with an 8 mm increase in the tongue depth. The upper oropharyngeal airway (UOP) shortened by 4.1 mm, the lower oropharyngeal airway (LOP) by 1.7 mm, and the laryngopharyngeal airway by 2 mm. The craniocervical angle showed a significant increase of 2.8°. UOP and LOP showed a significant correlation with the craniocervical angle at T2 and T4.Conclusions. Our findings conclude that the oropharyngeal airway space is significantly decreased and correlated with a change in the head posture after mandibular setback surgery.

Objective.To investigate the factors affecting intraoperative hemorrhage and postoperative sequelae after orthognathic surgery.Materials and Methods.Eighty patients with mandibular prognathism underwent surgical mandibular setback with intraoral vertical ramus osteotomy (IVRO). The correlation between the blood loss volume and postoperative VAS with the gender, age, and operating time was assessed using thet-test and Spearman rank correlation coefficient. The correlation between the magnitude of mandibular setback with the presence of TMJ clicking symptoms and lip sensation was also assessed.Results.The mean operating time and blood loss volume for men and women were 249.52 min and 229.39 min, and 104.03 mL and 86.12 mL, respectively. The mean VAS in men and women was 3.21 and 2.93, and 1.79 and 1.32 on the first and second postoperative days. There is no gender difference in the operating time, blood loss, VAS, TMJ symptoms, and lip numbness. The magnitude of mandibular setback was not correlated with immediate and long-term postoperative lip numbness.Conclusion.There are no gender differences in the intraoperative hemorrhage and postoperative sequelae (pain, lip numbness, and TMJ symptoms). In addition, neither symptom was significantly correlated with the amount of mandibular setback.

Wedge-shaped defects are frequently observed on the cervical region of the human tooth. Previously, most studies explained that improper toothbrushing causes such defects. However, recent clinical obsarvation suggested that the repeated stress due to occlusal force may induce the formation of these wedge-shaped defects. In this study, a two-dimensional human tooth model after a wedge-shaped defect is restored with the composite resin is analyzed by using the finite element method. To obtain the intensity of the singular stress field accurately, a method of analysis is discussed for calculating generalized stress intensity factors, which control the singular stress around the tip of the defect. Then, the relationships between the stress intensity and occlusion are discussed.