Machine Learning of Cardiac Anatomy and the Risk of New-Onset Atrial Fibrillation After TAVR.

BACKGROUND: New-onset atrial fibrillation (NOAF) occurs in 5% to 15% of patients who undergo transfemoral transcatheter aortic valve replacement (TAVR). Cardiac imaging has been underutilized to predict NOAF following TAVR. OBJECTIVES: The objective of this analysis was to compare and assess standard, manual echocardiographic and cardiac computed tomography (cCT) measurements as well as machine learning-derived cCT measurements of left atrial volume index and epicardial adipose tissue as risk factors for NOAF following TAVR. METHODS: The study included 1,385 patients undergoing elective, transfemoral TAVR for severe, symptomatic aortic stenosis. Each patient had standard and machine learning-derived measurements of left atrial volume and epicardial adipose tissue from cardiac computed tomography. The outcome of interest was NOAF within 30 days following TAVR. We used a 2-step statistical model including random forest for variable importance ranking, followed by multivariable logistic regression for predictors of highest importance. Model discrimination was assessed by using the C-statistic to compare the performance of the models with and without imaging. RESULTS: Forty-seven (5.0%) of 935 patients without pre-existing atrial fibrillation (AF) experienced NOAF. Patients with pre-existing AF had the largest left atrial volume index at 76.3 ± 28.6 cm3/m2 followed by NOAF at 68.1 ± 26.6 cm3/m2 and then no AF at 57.0 ± 21.7 cm3/m2 (P < 0.001). Multivariable regression identified the following risk factors in association with NOAF: left atrial volume index ≥76 cm2 (OR: 2.538 [95% CI: 1.165-5.531]; P = 0.0191), body mass index <22 kg/m2 (OR: 4.064 [95% CI: 1.500-11.008]; P = 0.0058), EATv (OR: 1.007 [95% CI: 1.000-1.014]; P = 0.043), aortic annulus area ≥659 mm2 (OR: 6.621 [95% CI: 1.849-23.708]; P = 0.004), and sinotubular junction diameter ≥35 mm (OR: 3.891 [95% CI: 1.040-14.552]; P = 0.0435). The C-statistic of the model was 0.737, compared with 0.646 in a model that excluded imaging variables. CONCLUSIONS: Underlying cardiac structural differences derived from cardiac imaging may be useful in predicting NOAF following transfemoral TAVR, independent of other clinical risk factors.

IL-13 and IL-13-induced periostin levels are specifically decreased in patients following endoscopic sinus surgery for chronic rhinosinusitis.

BACKGROUND: Type 2 (T2) inflammation plays a pathogenic role in chronic rhinosinusitis (CRS). The effects of endoscopic sinus surgery (ESS) on T2 inflammation are unknown. OBJECTIVE: The aim of this study was to compare T2 inflammatory biomarkers from middle meatal (MM) mucus for distinguishing patients with CRS from CRS-free patients, identifying major phenotypes (CRS without nasal polyps [CRSsNP] and CRS with nasal polyps [CRSwNP]), assessing endotypic change, and establishing cross-sectional and longitudinal outcomes in patients undergoing ESS. METHODS: MM mucus samples were collected from patients with CRSsNP and patients with CRSwNP before and 6 to 12 months after ESS and compared with samples from CRS-free control patients. T2 biomarkers were evaluated both continuously and using threshold-based definitions of T2 endotype to identify relationships with patient-reported (based on the 22-Item Sinonasal Outcomes Test and Chronic Rhinosinusitis Patient-Reported Outcomes Measure) and clinician-reported (radiographic and endoscopic) severity. Linear mixed models were developed to analyze clinical variables associated with T2 biomarker levels. RESULTS: A total of 154 patients with CRS (89 with CRSsNP and 65 with CRSwNP) were enrolled, with a mean interval of 9 months between ESS and follow-up. An analysis of pre-ESS MM mucus samples revealed elevated levels of T2 mediators in patients with CRSwNP versus in patients with CRSsNP and CRS-free controls. Temporally stable correlations between levels of IL-13 and IL-5, levels of periostin and complement 5a, and levels of eosinophil cationic protein (ECP) and eotaxin-3 were observed. On this basis and on the basis of pathologic significance, levels of IL-13, periostin and ECP were further analyzed. After ESS, levels of IL-13 and periostin decreased significantly, whereas ECP levels remained unchanged. Across pre- and post-ESS evaluation, the T2 endotype was associated with radiographic severity but did not predict outcomes. CRSwNP status and African American race were associated with higher levels of IL-13 and periostin, whereas ECP level was higher in patients undergoing extensive surgery. CONCLUSION: ESS decreased levels of IL-13 and periostin in the middle meatus. T2 inflammation after ESS was correlated with patient- and clinician-reported severity across phenotypes. Pre-ESS T2 inflammation did not predict post-ESS outcomes.

Automated temporalis muscle quantification and growth charts for children through adulthood.

Lean muscle mass (LMM) is an important aspect of human health. Temporalis muscle thickness is a promising LMM marker but has had limited utility due to its unknown normal growth trajectory and reference ranges and lack of standardized measurement. Here, we develop an automated deep learning pipeline to accurately measure temporalis muscle thickness (iTMT) from routine brain magnetic resonance imaging (MRI). We apply iTMT to 23,876 MRIs of healthy subjects, ages 4 through 35, and generate sex-specific iTMT normal growth charts with percentiles. We find that iTMT was associated with specific physiologic traits, including caloric intake, physical activity, sex hormone levels, and presence of malignancy. We validate iTMT across multiple demographic groups and in children with brain tumors and demonstrate feasibility for individualized longitudinal monitoring. The iTMT pipeline provides unprecedented insights into temporalis muscle growth during human development and enables the use of LMM tracking to inform clinical decision-making.

Evidence that oncostatin M synergizes with IL-4 signaling to induce TSLP expression in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Oncostatin M (OSM) may promote type 2 inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP) by inducing thymic stromal lymphopoietin (TSLP). OBJECTIVE: We sought to study the impact of OSM on TSLP synthesis and release from nasal epithelial cells (NECs). METHODS: OSM receptors, IL-4 receptors (IL-4R), and TSLP were evaluated in mucosal tissue and primary NECs from patients with CRSwNP by quantitative PCR and immunofluorescence. Air-liquid interface-cultured NECs were stimulated with cytokines, including OSM, and quantitative PCR, ELISA, Western blot, and flow cytometry were used to assess the expression of OSM receptors, IL-4R, and TSLP. RESULTS: Increased levels of OSM receptor ß chain (OSMRß), IL-4Rα, and TSLP were observed in nasal polyp tissues and primary epithelial cells from nasal polyps of patients with CRSwNP compared with control tissues or cells from control subjects. The level of expression of OSMRß in tissue was correlated with levels of both IL-4Rα and TSLP. OSM stimulation of NECs increased the expression of OSMRß and IL-4Rα. Stimulation with IL-4 plus OSM augmented the production of TSLP; the response was suppressed by a signal transducer and activator of transcription 6 inhibitor. Stimulation of NECs with IL-4 plus OSM increased the expression of proprotein convertase subtilisin/kexin 3, an enzyme that truncates and activates TSLP. CONCLUSIONS: OSM increases the expression of IL-4Rα and synergizes with IL-4 to induce the synthesis and release of TSLP in NECs. Because the combination of IL-4 and OSM also augmented the expression of proprotein convertase subtilisin/kexin 3, these results suggest that OSM can induce both synthesis and posttranslational processing/activation of TSLP, promoting type 2 inflammation.

Persistent discharge or edema after endoscopic sinus surgery in patients with chronic rhinosinusitis is associated with a type 1 or 3 endotype.

BACKGROUND: Patients with chronic rhinosinusitis (CRS) may have persistence of polyps, discharge, or edema after endoscopic sinus surgery (ESS). Inflammation in CRS can be classified into three endotypes, with the presence of polyps associated with the type 2 endotype. Here, we evaluate the endotypic underpinnings of discharge or edema without polyps after ESS. METHODS: At a visit 6-12 months post ESS, patients underwent endoscopy and completed the CRS-PRO and SNOT-22. Luminex analysis of middle meatal mucus obtained at that visit was performed for IFN-γ, ECP, and IL-17a. Type 1, 2, and 3 endotypes were defined as greater than the 90th percentile expression of each marker, respectively, in controls. Wilcoxon rank-sum and chi-squared tests were used to compare cytokine levels and endotype prevalence between those with and without endoscopic findings. RESULTS: A total of 122 CRS patients completed a clinical exam (median: 8.2 months post ESS). Of the 122 patients, 107 did not have polyps on endoscopy. Of these 107 patients, 48 had discharge, 44 had edema, and 46 had neither discharge nor edema. Compared with those patients without any findings, patients with discharge or edema reported significantly worse severity as measured by CRS-PRO (10.5 vs. 7.0, p = 0.009; 12.0 vs. 7.0, p < 0.001; respectively), and had higher post-ESS IFN-γ, ECP, and IL-17a. Patients with discharge had higher prevalence of only T1 and T3 endotypes, while patients with edema had higher prevalence of only the T3 endotype. CONCLUSIONS: Post-ESS discharge or edema in the absence of polyps was associated with higher patient-reported outcome severity and was more strongly associated with type 1 or 3 inflammation.

Use of intraoperative frontal sinus mometasone-eluting stents decreased interleukin 5 and interleukin 13 in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: Mometasone-eluting stents (MES) have demonstrated improvement in short-term endoscopic outcomes and reduce short- to medium-term rescue interventions. Their effect on the local inflammatory environment, longer-term patient-reported outcomes, and radiographic severity have not been studied. METHODS: Middle meatal mucus and validated measures of disease severity were collected before and 6 to 12 months after endoscopic surgery in 52 patients with chronic rhinosinusitis with nasal polyps (CRSwNPs). Operative findings, type 2 mediator concentrations, intraoperative variables, and disease severity measures were compared between those who did and those who did not receive intraoperative frontal MES. RESULTS: A total of 52 patients with CRSwNPs were studied; 33 received frontal MES and were compared with 19 who did not. Pre-endoscopic sinus surgery (ESS) middle meatus (MM) interleukin (IL) 13 and eosinophil cationic protein (ECP) were higher in the stented group (p < 0.05), but pre-ESS clinical measures of disease severity were similar as were surgical extent and post-ESS medical management. Intraoperative eosinophilic mucin was more frequent in the stented group (58% vs 11%, p = 0.001). IL-5 (p < 0.05) and IL-13 (p < 0.001) decreased post-ESS in the stented group, but this was not observed in the nonstented group. Post-ESS IL-4 and IL-13 were higher in the nonstented vs stented group (p < 0.05 for both). CONCLUSION: Although patients who received intraoperative frontal MES had significantly higher pre-ESS MM IL-13 and ECP, patients who received frontal MES had lower concentrations of IL-4 and IL-13 than those who did not at a median of 8 months post-ESS. However, these changes did not correspond to significantly different measures of symptomatic or radiographic disease severity.

CRS-PRO and SNOT-22 correlations with type 2 inflammatory mediators in chronic rhinosinusitis.

The 22-item Sino-Nasal Outcome Test (SNOT-22) and 12-item Patient Reported Outcomes in Chronic Rhinosinusitis (CRS-PRO) instrument are validated patient-reported outcomes measures in CRS. In this study we assess the correlation of these with type 2 (T2) biomarkers before and after endoscopic sinus surgery (ESS). METHODS: Middle meatal mucus data were collected and the SNOT-22 and CRS-PRO were administered to 123 patients (71 CRS without nasal polyps [CRSsNP], 52 CRS with nasal polyps [CRSwNP]) with CRS before and 6 to 12 months after undergoing ESS. Interleukin (IL)-4, IL-5, IL-13, and eosinophilic cationic protein (ECP) were measured using a multiplexed bead assay and enzyme-linked immunoassay. Pre- and post-ESS SNOT-22 and CRS-PRO were compared with T2 biomarkers. RESULTS: Before ESS neither PROM correlated with any biomarker. After ESS, CRS-PRO showed a correlation with 2 mediators (IL-5 and IL-13: p = 0.012 and 0.003, respectively) compared with none for the SNOT-22. For CRSwNP patients, pre-ESS CRS-PRO and SNOT-22 correlated with IL-4 (p = 0.04 for both). However, after ESS, CRS-PRO correlated with 3 biomarkers (IL-5, IL-13, and ECP: p = 0.02, 0.024, and 0.04, respectively) and SNOT-22 with 2 biomarkers (IL-5 and IL-13: p = 0.038 and 0.02, respectively). There were no significant relationships between any of the T2 biomarkers pre- or post-ESS among patients with CRSsNP. Exploratory analyses of the subdomains showed the SNOT-22 rhinologic and CRS-PRO rhinopsychologic subdomains correlated better with the T2 biomarkers. On individual item analysis, IL-13 correlated significantly post-ESS with 8 of 12 items on the CRS-PRO vs 6 of 22 items on the SNOT-22. CONCLUSION: The CRS-PRO total score showed a significant correlation with T2 biomarkers especially when assessed post-ESS and among CRSwNP patients.

Studies on activation and regulation of the coagulation cascade in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Increased activation of the coagulation cascade and diminished fibrinolysis combine to promote fibrin deposition and polyp formation in chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP). More information is needed concerning mechanisms of coagulation in CRSwNP. OBJECTIVE: We investigated the mechanisms as well as the initiation and regulation of coagulation cascade activation in CRS. METHODS: Samples were collected from 135 subjects with CRSwNP, 80 subjects with chronic CRS without nasal polyps (NP), and 65 control subjects. The levels of activated factor X (FXa), prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex, tissue factor (TF), and TF pathway inhibitor (TFPI) were monitored in CRS by real-time PCR, ELISA, immunohistochemistry, or immunofluorescence. Heteromeric complexes of TF with activated factor VII (FVII) and TF with activated FVII and FXa were assessed by coimmunoprecipitation and Western blotting. RESULTS: Increased levels of FXa, F1+2, and thrombin-antithrombin complex were detected in NP tissue compared to uncinate tissue from CRS and control subjects. Although free TF protein levels were not increased in NP, immunoprecipitation of TF in NP tissue revealed increased complexes of TF with FVII. Local expression of FVII was detected in sinonasal mucosa, and the ratio of TFPI to FXa was lower in NP tissue. CONCLUSION: The coagulation cascade is associated with NP compared to control and uncinate tissue from CRS patients, and TF and FVII are produced locally in sinonasal mucosa in patients. TF and FVII can activate the extrinsic coagulation pathway, suggesting that this pathway may activate fibrin deposition in CRSwNP. Reduced formation of the complex of FXa and TFPI in NP may reduce natural suppression of the extrinsic coagulation pathway in CRSwNP.

Prognostic factors for polyp recurrence in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps is frequently managed with endoscopic sinus surgery (ESS). Prior studies describe individual clinical variables and eosinophil density measures as prognostic for polyp recurrence (PR). However, the relative prognostic significance of these have not been extensively investigated. OBJECTIVES: We sought to evaluate the impact of PR on measures of disease severity post-ESS and quantify the prognostic value of various clinical variables and biomarkers. METHODS: Ninety-four patients with chronic rhinosinusitis with nasal polyps and prospectively biobanked polyp homogenates at the time of ESS were recruited 2 to 5 years post-ESS. Patients were evaluated with patient-reported outcome measures and endoscopic and radiographic scoring pre- and post-ESS. Biomarkers in polyp homogenates were measured with ELISA and Luminex. Relaxed least absolute shrinkage and selection operator regression optimized predictive clinical, biomarker, and combined models. Model performance was assessed using receiver-operating characteristic curve and random forest analysis. RESULTS: PR was found in 39.4% of patients, despite significant improvements in modified Lund-Mackay (MLM) radiographic and 22-item Sinonasal Outcomes Test scores (both P < .0001). PR was significantly associated with worse post-ESS MLM, modified Lund-Kennedy, and 22-item Sinonasal Outcomes Test scores. Relaxed least absolute shrinkage and selection operator identified 2 clinical predictors (area under the curve = 0.79) and 3 biomarkers (area under the curve = 0.78) that were prognostic for PR. When combined, the model incorporating these pre-ESS factors: MLM, asthma, eosinophil cationic protein, anti-double-stranded DNA IgG, and IL-5 improved PR predictive accuracy to area under the curve of 0.89. Random forest analysis identified and validated each of the 5 variables as the strongest predictors of PR. CONCLUSIONS: PR had strong associations with patient-reported outcome measures, endoscopic and radiographic severity. A combined model comprised of eosinophil cationic protein, IL-5, pre-ESS MLM, asthma, and anti-double-stranded DNA IgG could accurately predict PR.

Anti-phospholipid antibodies are elevated and functionally active in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Polyps from patients with chronic rhinosinusitis with nasal polyps (CRSwNP) contain increased levels of autoreactive antibodies, B cells and fibrin deposition. Anti-phospholipid antibodies (APA) are autoantibodies known to cause thrombosis but have not been implicated in chronic rhinosinusitis (CRS). OBJECTIVE: To compare APA levels (anti-cardiolipin, anti-phosphatidylethanolamine (anti-PE), and anti-ß2 -glycoprotein (anti-B2GP)) in nasal polyp (NP) tissue with tissue from control and CRS without nasal polyp (CRSsNP) patients, we tested whether NP antibodies affect coagulation, and correlate APAs with anti-dsDNA IgG and markers of coagulation. METHODS: Patient specimens were assayed for APA IgG, anti-dsDNA IgG and thrombin-anti-thrombin (TaT) complex by ELISA. Antibodies from a subset of specimens were tested for modified activated partial thromboplastin time (aPTT) measured on an optical-mechanical coagulometer. RESULTS: Anti-cardiolipin IgG in NP was 5-fold higher than control tissue (p < .0001). NP antibodies prolonged aPTT compared to control tissue antibodies at 400 µg/mL (36.7 s vs. 33.8 s, p = .024) and 600 µg/mL (40.9 s vs. 34.7 s, p = .0037). Anti-PE IgG antibodies were increased in NP (p = .027), but anti-B2GP IgG was not significantly higher (p = .084). All APAs correlated with anti-dsDNA IgG levels, which were also elevated (R = .77, .71 and .54, respectively, for anti-cardiolipin, anti-PE, and anti-B2GP; all p < .001), but only anti-cardiolipin (R = .50, p = .0185) and anti-PE (R = 0.45, p = .037) correlated with TaT complex levels. CONCLUSIONS: APA IgG antibodies are increased in NP and correlate with autoreactive tissue antibodies. NP antibodies have in vitro anti-coagulant activity similar to those observed in anti-phospholipid syndrome, suggesting that they may have pro-coagulant effects in polyp tissue.

Clinical Value of Surveillance Biopsies in Pediatric Liver Transplantation.

Although pediatric liver transplantation (LT) results in excellent long-term outcomes, a high incidence of early acute cellular rejection and late graft fibrosis persists. Routine measurement of allograft enzymes may not reliably detect rejection episodes, identify candidates for immunosuppression minimization, or indicate allograft fibrosis. Surveillance biopsies (SBs) can provide valuable information in this regard, but their role in pediatric LT is not fully established. A retrospective cohort of 236 pediatric LT recipients from a high-volume center was studied to characterize the risks and benefits of SB versus for-cause biopsies (FCBs). The study population was 47.1% male and 54.7% Hispanic, and 31% received living donor grafts. Our data suggest that patients in the SB group had better transplant outcomes (rejection-free, graft, and patient survival) compared with patients who had FCBs or who never underwent biopsy. Among 817 biopsies obtained from 236 patients, 150 (18.4%) were SBs. Only 6 patients had a biopsy-related complication, and none were observed in the SB subset. Graft biochemical blood tests did not accurately predict rejection severity on biopsy, with aspartate aminotransferase area under the receiver operating characteristic curve (AUROC) 0.66, alanine aminotransferase AUROC 0.65 (very poor predictions), and gamma-glutamyltransferase AUROC 0.58 (no prediction). SBs identified subclinical rejection in 18.6% of biopsies, whereas 63.3% of SBs had evidence of fibrosis. SBs prompted changes in immunosuppression including dose reduction. Our experience suggests that SB in pediatric LT is safe, offers valuable information about subclinical rejection episodes, and can guide management of immunosuppression, including minimization. Improved outcomes with SB were likely multifactorial, potentially relating to a more favorable early posttransplant course and possible effect of management optimization through SB. Further multicenter studies are needed to examine the role of SBs on long-term outcomes in pediatric LT.

Studies of the role of basophils in aspirin-exacerbated respiratory disease pathogenesis.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by the triad of chronic rhinosinusitis with nasal polyps (CRSwNP), asthma, and intolerance to cyclooxygenase-1 enzyme inhibitors. The underlying mechanisms contributing to AERD pathogenesis are not fully understood, but AERD is characterized by an enhanced type 2 inflammatory phenotype. Basophils are potent type 2 effector cells, but their involvement in AERD pathophysiology remains unclear. OBJECTIVE: We sought to characterize the systemic and local basophil responses in patients with AERD compared with patients with CRSwNP. METHODS: Sinonasal tissues including inferior turbinate and/or nasal polyps (NPs) and peripheral blood were collected from controls, patients with AERD, and patients with CRSwNP. Expression of cell surface (CD45, FcεRI, CD203c), activation (CD63), and intracellular (2D7) markers associated with basophils was characterized using flow cytometry. Clinical data including Lund-Mackay scores and pulmonary function were obtained. RESULTS: The mean number of basophils (CD45+CD203c+FcεRI+CD117-) detected in AERD NPs (147 ± 28 cells/mg tissue) was significantly elevated compared with that detected in CRSwNP NPs (69 ± 20 cells/mg tissue; P = .01). The number of circulating basophils was significantly elevated in patients with AERD (P = .04). Basophils in NPs had significantly higher CD203c and CD63 mean fluorescence intensity compared with blood in both conditions (P < .01). Basophils from AERD NPs had lower expression of the granule content marker 2D7 compared with those from matched blood (P < .01) or NPs of patients with CRSwNP (P = .06), suggesting ongoing degranulation. Basophil 2D7 mean fluorescence intensity significantly correlated with pulmonary function (r = 0.62; P = .02) and inversely correlated with sinonasal inflammation (r = -0.56; P = .004). CONCLUSIONS: Increased basophil numbers and extent of ongoing degranulation in NPs of patients with AERD compared with patients with CRSwNP may contribute to the exaggerated disease pathogenesis and severity unique to AERD.

Responsiveness and convergent validity of the chronic rhinosinusitis patient-reported outcome (CRS-PRO) measure in CRS patients undergoing endoscopic sinus surgery.

BACKGROUND: The chronic rhinosinusitis patient-reported outcome (CRS-PRO) measure is a 12-item measure with previously demonstrated validity in chronic rhinosinusitis (CRS) patients receiving medical therapy. This study establishes the factor structure, responsiveness, and convergent validity of the CRS-PRO following endoscopic sinus surgery (ESS). METHODS: Northwestern CRS Subject Registry patients had pre-ESS, 3-month (n = 111; CRS without nasal polyps [CRSsNP] = 60, CRS with nasal polyps [CRSwNP] = 51), and 6-month (n = 86; CRSsNP = 47, CRSwNP = 39) post-ESS assessments where patients completed the CRS-PRO, 22-item Sino-Nasal Outcome Test (SNOT-22), and four Patient-Reported Outcomes Measurement (PROM) Information System (PROMIS) short forms (general health measures). Patients had pre-ESS objective testing (endoscopic and radiographic assessment). Factor analysis was conducted using principal axis factoring with varimax rotation on the baseline CRS-PRO. The clinically important difference (CID) was estimated using both distribution-based and anchor-based methods. RESULTS: Factor analysis found the CRS-PRO comprised the "rhino-psychologic," "facial discomfort," and "cough" factors, which were responsive to ESS and correlated with the other PROMs. The changes observed in the CRS-PRO at 3 months had strong correlation with the corresponding changes in SNOT-22 (r = 0.792, p < 0.0001) and moderate correlations with changes in PROMIS fatigue and sleep domains. These changes had a very large effect size (Cohen's d 1.44) comparable to the longer SNOT-22 (Cohen's d 1.41) with slightly larger effect sizes observed in CRSwNP compared to CRSsNP patients. Similar convergent validity and responsiveness were observed in the 6-month data. The CRS-PRO CID was estimated to be between 5.0 and 7.5 (midpoint 6.0) using distribution-based and anchor-based methods. CONCLUSION: This study demonstrates the validity and responsiveness of the CRS-PRO in subjects receiving ESS.

Activation of the 15-lipoxygenase pathway in aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), and an intolerance of medications that inhibit cyclooxygenase-1. Patients with AERD have more severe upper and lower respiratory tract disease than do aspirin-tolerant patients with CRSwNP. A dysregulation in arachidonic acid metabolism is thought to contribute to the enhanced sinonasal inflammation in AERD. OBJECTIVE: Our aim was to utilize an unbiased approach investigating arachidonic acid metabolic pathways in AERD. METHODS: Single-cell RNA sequencing (10× Genomics, Pleasanton, Calif) was utilized to compare the transcriptional profile of nasal polyp (NP) cells from patients with AERD and patients with CRSwNP and map differences in the expression of select genes among identified cell types. Findings were confirmed by traditional real-time PCR. Lipid mediators in sinonasal tissue were measured by mass spectrometry. Localization of various proteins within NPs was assessed by immunofluorescence. RESULTS: The gene encoding for 15-lipooxygenase (15-LO), ALOX15, was significantly elevated in NPs of patients with AERD compared to NPs of patients with CRSwNP (P < .05) or controls (P < .001). ALOX15 was predominantly expressed by epithelial cells. Expression levels significantly correlated with radiographic sinus disease severity (r = 0.56; P < .001) and were associated with asthma. The level of 15-oxo-eicosatetraenoic acid (15-Oxo-ETE), a downstream product of 15-LO, was significantly elevated in NPs from patients with CRSwNP (27.93 pg/mg of tissue) and NPs from patients with AERD (61.03 pg/mg of tissue) compared to inferior turbinate tissue from controls (7.17 pg/mg of tissue [P < .001]). Hydroxyprostaglandin dehydrogenase, an enzyme required for 15-Oxo-ETE synthesis, was predominantly expressed in mast cells and localized near 15-LO+ epithelium in NPs from patients with AERD. CONCLUSIONS: Epithelial and mast cell interactions, leading to the synthesis of 15-Oxo-ETE, may contribute to the dysregulation of arachidonic acid metabolism via the 15-LO pathway and to the enhanced sinonasal disease severity observed in AERD.

Modeling Mixed Vascular and Alzheimer's Dementia Using Focal Subcortical Ischemic Stroke in Human ApoE4-TR:5XFAD Transgenic Mice.

Subcortical white matter ischemic lesions are increasingly recognized to have pathologic overlap in individuals with Alzheimer's disease (AD). The interaction of white matter ischemic lesions with amyloid pathology seen in AD is poorly characterized. We designed a novel mouse model of subcortical white matter ischemic stroke and AD that can inform our understanding of the cellular and molecular mechanisms of mixed vascular and AD dementia. Subcortical white matter ischemic stroke underlying forelimb motor cortex was induced by local stereotactic injection of an irreversible eNOS inhibitor. Subcortical white matter ischemic stroke or sham procedures were performed on human ApoE4-targeted-replacement (TR):5XFAD mice at 8 weeks of age. Behavioral tests were done at 7, 10, 15, and 20 weeks. A subset of animals underwent 18FDG-PET/CT. At 20 weeks of age, brain tissue was examined for amyloid plaque accumulation and cellular changes. Compared with sham E4-TR:5XFAD mice, those with an early subcortical ischemic stroke showed a significant reduction in amyloid plaque burden in the region of cortex overlying the subcortical stroke. Cognitive performance was improved in E4-TR:5XFAD mice with stroke compared with sham E4-TR:5XFAD animals. Iba-1+ microglial cells in the region of cortex overlying the subcortical stroke were increased in number and morphologic complexity compared with sham E4-TR:5XFAD mice, suggesting that amyloid clearance may be promoted by an interaction between activated microglia and cortical neurons in response to subcortical stroke. This novel approach to modeling mixed vascular and AD dementia provides a valuable tool for dissecting the molecular interactions between these two common pathologies.

EZH2 and histone deacetylase inhibitors induce apoptosis in triple negative breast cancer cells by differentially increasing H3 Lys27 acetylation in the BIM gene promoter and enhancers.

Enhancer of zeste homolog 2 (EZH2), a subunit of polycomb repressive complex 2, is a histone methyl-transferase and is considered to work cooperatively with histone deacetylases (HDACs) in the same protein complex to mediate gene transcription repression by increasing histone H3 Lys27 trimethylation (H3K27me3), in particular in the nucleosome (s). EZH2 is overexpressed in numerous types of cancer, including triple negative breast cancer (TNBC), a subtype of breast cancer, which there are no effective treatment options for. Thus, inhibition of EZH2 may be harnessed for targeted therapy of this disease. The present study demonstrated that co-treatment with an EZH2 inhibitor and a HDAC inhibitor additively induced apoptosis in two TNBC cell lines, namely MDA-MB-231 and MDA-MB-436. The increased rate of cell death was associated with an elevation of B cell lymphoma-2 like 11 (BIM) expression level, a pro-apoptotic protein at the protein and mRNA expression levels in these two cell lines. The expression of forkhead box O1 (FOXO1), a known upstream transcriptional activator of BIM, was upregulated in both cell lines by the HDAC inhibitor, and the effect was more pronounced in MDA-MB-436 cells with higher phosphorylation levels of protein kinase B, a negative regulator of FOXO1, compared with MDA-MB-231 cells. Conversely, FOXO1 expression was inhibited following treatment with the EZH2 inhibitor, suggesting that EZH2 and HDAC inhibitors induced BIM expression via a FOXO1-independent mechanism. The present study further revealed that the EZH2 inhibitor, but not the HDAC inhibitor, induced high levels of H3K27 acetylation (H3K27ac) in the BIM promoter. By contrast, compared with the effect of the EZH2 inhibitor, HDAC inhibitor treatment resulted in an increase in H3K27ac at two BIM enhancers. Collectively, the results of the present study indicated that EZH2 and HDACs act differentially on H3K27ac levels in the nucleosome at the promoter and enhancer regions of the BIM gene. Through the upregulation of BIM, co-treatment with EZH2 and HDAC inhibitors had a pronounced therapeutic effect on TNBC cells, suggesting that co-targeting EZH2 and HDAC proteins represents a viable therapeutic option for the treatment of TNBC.

A prospective analysis evaluating tissue biopsy location and its clinical relevance in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) has a high propensity for recurrence. Studies suggest that eosinophilia influences disease severity and surgical outcomes, but the selection of sinonasal site for measuring eosinophilia has not been examined. The aim of this study was to investigate how region-specific tissue eosinophilia affects radiographic severity, comorbidity prevalence, and polyp recurrence risk following sinus surgery. METHODS: Eosinophil cationic protein (ECP) levels in uncinate tissue (UT) and nasal polyp (NP) homogenates from 116 CRSwNP patients were measured using enzyme-linked immunosorbent assay (ELISA). Clinical history, radiographic severity, and time to polyp recurrence were obtained from electronic health records. The correlations between baseline Lund-Mackay scores and comorbidities were compared between UT and NP ECP levels. Cox regression and Kaplan-Meier analysis were then performed to assess whether UT or NP ECP better predicted recurrence. Censoring occurred at 4 years or at last follow-up if there was no endoscopic diagnosis of recurrent polyps. RESULTS: Lund-Mackay scores were significantly correlated with UT and NP ECP (r = 0.46 and 0.26 respectively, p < 0.05). UT but not NP ECP was significantly higher in patients with asthma (p < 0.01) and aspirin-exacerbated respiratory disease (AERD) (p < 0.05). Polyp recurrence risk was only significantly higher for patients with eosinophilic UT tissue (hazard ratio [HR] = 2.84, p = 0.025). When measured in NP, eosinophilia did not predict recurrence. CONCLUSION: Although ECP in NP was higher than in UT tissue, eosinophilia in UT tissue was a more clinically coherent biomarker of baseline radiographic severity, comorbid asthma and AERD, and prospective polyp recurrence risk than NP eosinophilia.

Evidence for altered levels of IgD in the nasal airway mucosa of patients with chronic rhinosinusitis.

BACKGROUND: IgD is an enigmatic antibody isotype best known when coexpressed with IgM on naive B cells. However, increased soluble IgD (sIgD) levels and increased IgD+IgM- B-cell populations have been described in the human upper respiratory mucosa. OBJECTIVE: We assessed whether levels of sIgD and IgD+ B cell counts are altered in nasal tissue from patients with chronic rhinosinusitis (CRS). We further characterized IgD+ B-cell populations and explored clinical and local inflammatory factors associated with tissue sIgD levels. METHODS: sIgD levels were measured by means of ELISA in nasal tissues, nasal lavage fluid, sera, and supernatants of dissociated nasal tissues. IgD+ cells were identified by using immunofluorescence and flow cytometry. Inflammatory mediator levels in tissues were assessed by using real-time PCR and multiplex immunoassays. Bacterial cultures from the middle meatus were performed. Underlying medical history and medicine use were obtained from medical records. RESULTS: sIgD levels and numbers of IgD+ cells were significantly increased in uncinate tissue (UT) of patients with chronic rhinosinusitis without nasal polyps (CRSsNP) compared with that of control subjects (4-fold, P < .05). IgD+ cells were densely scattered in the periglandular regions of UT from patients with CRSsNP. We also found that IgD+CD19+CD38bright plasmablast numbers were significantly increased in tissues from patients with CRSsNP compared with control tissues (P < .05). Among numerous factors tested, IL-2 levels were increased in UT from patients with CRSsNP and were positively correlated with tissue IgD levels. Additionally, supernatants of IL-2-stimulated dissociated tissue from patients with CRSsNP had significantly increased sIgD levels compared with those in IL-2-stimulated dissociated control tissue ex vivo (P < .05). Tissue from patients with CRS with preoperative antibiotic use or those with pathogenic bacteria showed higher IgD levels compared with tissue from patients without these variables (P < .05). CONCLUSION: sIgD levels and IgD+CD19+CD38bright plasmablast counts were increased in nasal tissue of patients with CRSsNP. IgD levels were associated with increased IL-2 levels and the presence of pathogenic bacteria. These findings suggest that IgD might contribute to enhancement mucosal immunity or inflammation or respond to bacterial infections in patients with CRS, especially CRSsNP.

Superior turbinate eosinophilia correlates with olfactory deficit in chronic rhinosinusitis patients.

OBJECTIVE: To evaluate if molecular markers of eosinophilia in olfactory-enriched mucosa are associated with olfactory dysfunction. STUDY DESIGN: Cross-sectional study of tissue biopsies from 99 patients, and an additional 30 patients who underwent prospective olfactory testing prior to sinonasal procedures. METHODS: Tissue biopsies were processed for analysis of inflammatory markers using quantitative real time polymerase chain reaction (qRT-PCR). Ipsilateral olfactory performance was assessed using the Sniffin' Sticks (Burghart, Wedel, Germany) threshold component and the University of Pennsylvania Smell Identification Test (Sensonics, Haddon Heights, NJ). Age-adjusted data was correlated with inflammatory marker expression and clinical measures of obstruction from computed tomography and endoscopy. RESULTS: Gene expression of the eosinophil marker CLC (Charcot Leyden crystal protein) was elevated in superior turbinate (ST) tissue in chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) compared to ST and inferior turbinate tissue in CRS without nasal polyps (CRSsNP) and control patients (all P < 0.001, respectively). CLC in ST tissue was correlated with IL-5 and eotaxin-1 expression (all P < 0.001; P = 0.65, and 0.49, respectively). CLC expression was strongly correlated with eosinophilic cationic protein levels (P < 0.001; r = -0.76), and ST CLC expression was inversely related to olfactory threshold (P = 0.002, r = -0.57) and discrimination scores (P = 0.05, r = -0.42). In multiple linear regression of CLC gene expression, polyp status, and radiographic and endoscopic findings with olfactory threshold, CLC was the only significantly correlated variable (P < 0.05). CONCLUSION: Markers of eosinophils are elevated in the ST of patients with CRSwNP and correlate with olfactory loss. These findings support the hypothesis that olfactory dysfunction in CRS correlates local eosinophil influx into the olfactory cleft. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:2210-2218, 2017.

Classical complement pathway activation in the nasal tissue of patients with chronic rhinosinusitis.

BACKGROUND: Complement plays a major role in inflammatory diseases, but its involvement and mechanisms of activation in patients with chronic rhinosinusitis (CRS) are not known. OBJECTIVES: After earlier studies discovering autoantibodies in patients with CRS, we sought to investigate the nature, extent, and location of complement activation in nasal tissue of patients with CRS. Specifically, we were interested in whether antibody-mediated activation through the classical pathway was a major mechanism for complement activation in patients with CRS. METHODS: Nasal tissue was obtained from patients with CRS and control subjects. Tissue homogenates were analyzed for complement activation products (ELISA-C5b-9, C4d, activated C1, and C5a) and major complement-fixing antibodies (Luminex). Tissue sections were stained for C5b-9, C4d, and laminin. Antibodies were purified with protein A/G columns from nasal polyps (NP), matching patient serum, and control serum and assayed for basement membrane binding by means of ELISA. RESULTS: C5b-9 levels were significantly increased in NP tissue compared with uncinate tissue (UT) of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and those with chronic rhinosinusitis without nasal polyps (CRSsNP; P < .01). Similarly, C4d levels were increased in NPs compared with UT of patients with CRSwNP, patients with CRSsNP, and control subjects (P < .05). Activated C1 levels were also increased in NP tissue compared with UT of patients with CRSsNP and control subjects (P < .05) and correlated with levels of C5a (P < .01), local immunoglobulins (especially IgM, P < .0001), and anti-double-stranded DNA IgG (P < .05). Immunofluorescence showed that C5b-9 and C4d deposition occurred linearly along the epithelial basement membrane. NP tissue extracts had significantly more anti-basement membrane antibodies than sera from patients with CRSwNP and control subjects (P < .0001). CONCLUSION: Levels of C5b-9, C4d, and activated C1 were significantly increased locally in NP tissue. C5b-9 and C4d were almost universally deposited linearly along the basement membrane of NP tissue. Furthermore, activated C1 levels were best correlated with local immunoglobulin and C5a levels. Together, these data suggest that the classical pathway plays a major role in complement activation in patients with CRS.