Characterizing asthma from a drop of blood using neutrophil chemotaxis

Asthma is a chronic inflammatory disorder that affects more than 300 million people worldwide. Asthma management would benefit from additional tools that establish biomarkers to identify phenotypes of asthma. We present a microfluidic solution that discriminates asthma from allergic rhinitis based on a patient’s neutrophil chemotactic function. The handheld diagnostic device sorts neutrophils from whole blood within 5 min, and generates a gradient of chemoattractant in the microchannels by placing a lid with chemoattractant onto the base of the device. This technology was used in a clinical setting to assay 34 asthmatic (n = 23) and nonasthmatic, allergic rhinitis (n = 11) patients to establish domains for asthma diagnosis based on neutrophil chemotaxis. We determined that neutrophils from asthmatic patients migrate significantly more slowly toward the chemoattractant compared with nonasthmatic patients (P = 0.002). Analysis of the receiver operator characteristics of the patient data revealed that using a chemotaxis velocity of 1.55 μm/min for asthma yields a diagnostic sensitivity and specificity of 96% and 73%, respectively. This study identifies neutrophil chemotaxis velocity as a potential biomarker for asthma, and we demonstrate a microfluidic technology that was used in a clinical setting to perform these measurements.Asthma is a chronic inflammatory disorder of the lungs that is associated with airway hyperresponsiveness (AHR) and obstructed airflow (1), affecting more than 300 million people worldwide (2). Over the past 30 y, asthma prevalence has increased significantly in many populations, with some indications that prevalence may be reaching a plateau in the developed world. Significant progress has been made in identifying primary mediators involved in the pathophysiology of asthma. Several cell types, such as T helper cells (T_H1/T_H2), dendritic cells, mast cells, macrophages, eosinophils, and neutrophils play central roles in the pathology of asthma (4–7). Additionally, various cytokines that regulate the leukocyte trafficking, such as interleukins, IFN-γ, and TNF-α, have been identified and targeted in drug therapies. The recruitment of leukocytes to the lungs, particularly eosinophils and neutrophils, is central to the pathogenesis of asthma. Increased numbers of eosinophils are prominently observed in the lung tissue and bronchoalveolar lavage (BAL) fluid for most asthmatics (5). Neutrophils play a more critical role in severe asthma, where elevated counts of neutrophils are often observed in the BAL fluid (7). An overview of the role of neutrophils in asthma is shown in Fig. 1A. Although significant progress has been made in uncovering mediators in the pathology of asthma, these gains have not yet greatly improved our ability to define clinically relevant phenotypes of asthma in patients.Open in a separate windowFig. 1.Overview of different diagnostic techniques and the role of neutrophils in the pathology of asthma. (A) Summary of the role of neutrophils in the pathology of asthma, showing neutrophil adhesion and transendothelial migration; chemotaxis mediated by macrophages and T-helper cells; and neutrophilia in the lung tissue that leads to airway remodeling and airflow obstruction. (B) Proposed microfluidic method (more details in Fig. S1) for phenotyping asthma patients by measuring upstream of the asthma pathology with rapid neutrophil sorting on a P-selectin–coated surface (1); neutrophil chemotaxis monitored with high-throughput microscopy and automatically tracked with software (2); and asthma characterization on the basis of chemotaxis outputs (3). (C) Traditional clinical asthma diagnostic methods occur downstream of the asthma pathophysiology by measuring the effect of leukocyte inflammation on airway obstruction, nitric oxide output, or clinical symptoms.Asthma is diagnosed clinically by physicians, informed by the patient’s medical history, spirometry tests that measure lung function, reversibility of AHR, and several other potential metrics (8). These diagnostic techniques measure the effects of the inflammatory response in the lung by assessing airway constriction, nitric oxide production, and the resulting clinical symptoms. However, all of these diagnostic tests require patient compliance, which can be challenging when diagnosing children or the elderly (9). Additionally, many asthma diagnostic tests partially rely on the patient experiencing clinical symptoms that are variable during or around the visit to the physician. Perhaps these common characteristics of current diagnostic techniques contribute to difficulties in diagnosing asthma, particularly in certain subpopulations. For example, in a recent Canadian study involving ∼500 obese and nonobese subjects, Aaron et al. (10) found that ∼30% of the test subjects had been falsely diagnosed with asthma by physicians. Additionally, it is well established that the elderly are consistently underdiagnosed for asthma (11, 12). Therefore, additional tools are needed to improve the diagnosis of asthma. Furthermore, current asthma assessments do not inform the clinician of disease severity, expected clinical course, and risk of exacerbations.To improve characterization of asthma in the clinic, we have developed a handheld microfluidic chip that can identify functional measures of asthma from a drop of whole blood. Microfluidic systems have several characteristics that make them well-suited for clinical use, including low sample-volume requirements (13, 14); simple integration with automated fluid handling systems (15); and diffusion-dominant laminar fluidic phenomena that allow for precise control of a cell’s microenvironment (16–18). Indeed, microfluidic-based tools are increasingly being used in clinical research for diagnostic purposes (19–26). Neutrophils have been used to diagnose clinical conditions in human patients based on proteomic and genomic analysis (22) and chemotaxis behavior (23, 27), demonstrating that assays measuring cell function can be used for diagnostics. In this work, we assay the neutrophil chemotactic function in a blind study to identify quantitative domains that can be used to discriminate asthma from nonasthmatic allergic rhinitis. This approach of directly measuring the effector cell in the pathology of asthma differs from traditional diagnostic tests, which measure the variable effect of inflammation on airway constriction (Fig. 1 B and C and Table S1). Importantly, we developed methods to simplify the sample preparation, assay protocol, and data analysis that offer significant time savings over traditional macroscale (28–30) and microscale (18) chemotaxis techniques, allowing for the translation of the technology into the clinic. We analyzed 34 patients, and discovered that neutrophil chemotaxis can be used to discriminate asthma from nonasthmatic, allergic rhinitis patients with sensitivity and specificity of 96% and 73%, respectively. The results of the clinical application of our microfluidic device represent a first step demonstration of how asthma can potentially be diagnosed and managed based on cellular function, rather than largely by clinical observations.     

Atrioesophageal Fistula in the Era of Atrial Fibrillation Ablation: A Review

The purpose of this review is to understand the epidemiology, clinical features, etiopathogenesis, and management of atrioesophageal fistula (AEF) after atrial fibrillation (AF) ablation. The incidence of AEF after AF ablation is 0.015%-0.04%. The principal clinical features include fever, dysphagia, upper gastrointestinal bleeding, sepsis, and embolic strokes. The close proximity of the esophagus to the posterior left atrial wall is responsible for esophageal injury during ablation. Prophylactic proton pump inhibitors, esophageal temperature monitoring, visualization of the esophagus during catheter ablation, esophageal protection devices, and avoidance of energy delivery in close proximity to the esophagus play an important role in preventing esophageal injury. Early surgical repair or esophageal stenting are the mainstay of treatment. Eliminating esophageal injury during AF ablation is of utmost importance in preventing AEF. A high index of suspicion and early intervention is necessary to prevent fatal outcomes.     

Is There a Link Between Obesity and Asthma?

Increasing epidemiological data identify a link between obesity and asthma incidence and severity. Based on experimental data, it is possible that shared inflammatory mechanisms play a role in determining this linkage. Although controversial, the role of adipokines may be central to this association and the maintenance of the asthma phenotype. While leptin and adiponectin have a causal link to experimental asthma in mice, data in humans are less conclusive. Recent studies demonstrate that adipokines can regulate the survival and function of eosinophils and that these factors can affect eosinophil trafficking from the bone marrow to the airways. In addition, efferocytosis, the clearance of dead cells, by airway macrophages or blood monocytes appears impaired in obese asthmatics and is inversely correlated with glucocorticoid responsiveness. This review examines the potential mechanisms linking obesity to asthma.     

Pevonedistat and azacitidine upregulate NOXA (PMAIP1) to increase sensitivity to venetoclax in preclinical models of acute myeloid leukemia

Dysregulation of apoptotic machinery is one mechanism by which acute myeloid leukemia (AML) acquires a clonal survival advantage. B-cell lymphoma protein-2 (BCL2) overexpression is a common feature in hematologic malignancies. The selective BCL2 inhibitor, venetoclax (VEN) is used in combination with azacitidine (AZA), a DNAmethyltransferase inhibitor (DNMTi), to treat patients with AML. Despite promising response rates to VEN/AZA, resistance to the agent is common. One identified mechanism of resistance is the upregulation of myeloid cell leukemia-1 protein (MCL1). Pevonedistat (PEV), a novel agent that inhibits NEDD8-activating enzyme, and AZA both upregulate NOXA (PMAIP1), a BCL2 family protein that competes with effector molecules at the BH3 binding site of MCL1. We demonstrate that PEV/AZA combination induces NOXA to a greater degree than either PEV or AZA alone, which enhances VEN-mediated apoptosis. Herein, using AML cell lines and primary AML patient samples ex vivo, including in cells with genetic alterations linked to treatment resistance, we demonstrate robust activity of the PEV/VEN/AZA triplet. These findings were corroborated in preclinical systemic engrafted models of AML. Collectively, these results provide rational for combining PEV/VEN/AZA as a novel therapeutic approach in overcoming AML resistance in current therapies.