Evaluating Responses to Gluten Challenge: A Randomized, Double-Blind, 2-Dose Gluten Challenge Trial.

BACKGROUND & AIMS: Gluten challenge is used to diagnose celiac disease (CeD) and for clinical research. Sustained gluten exposure reliably induces histologic changes but is burdensome. We investigated the relative abilities of multiple biomarkers to assess disease activity induced by 2 gluten doses, and aimed to identify biomarkers to supplement or replace histology. METHODS: In this randomized, double-blind, 2-dose gluten-challenge trial conducted in 2 US centers (Boston, MA), 14 adults with biopsy-proven CeD were randomized to 3 g or 10 g gluten/d for 14 days. The study was powered to detect changes in villous height to crypt depth, and stopped at planned interim analysis on reaching this end point. Additional end points included gluten-specific cluster of differentiation (CD)4 T-cell analysis with HLA-DQ2-gluten tetramers and enzyme-linked immune absorbent spot, gut-homing CD8 T cells, interleukin-2, symptoms, video capsule endoscopy, intraepithelial leukocytes, and tissue multiplex immunofluorescence. RESULTS: All assessments showed changes with gluten challenge. However, time to maximal change, change magnitude, and gluten dose-response relationship varied. Villous height to crypt depth, video capsule endoscopy enteropathy score, enzyme-linked immune absorbent spot, gut-homing CD8 T cells, intraepithelial leukocyte counts, and HLA-DQ2-restricted gluten-specific CD4 T cells showed significant changes from baseline at 10 g gluten only; symptoms were significant at 3 g. Symptoms and plasma interleukin-2 levels increased significantly or near significantly at both doses. Interleukin-2 appeared to be the earliest, most sensitive marker of acute gluten exposure. CONCLUSIONS: Modern biomarkers are sensitive and responsive to gluten exposure, potentially allowing less invasive, lower-dose, shorter-duration gluten ingestion. This work provides a preliminary framework for rational design of gluten challenge for CeD research. ClinicalTrials.gov number, NCT03409796.

Hypoxic Pulmonary Vasoconstriction Does Not Explain All Regional Perfusion Redistribution in Asthma.

RATIONALE: Regional hypoventilation in bronchoconstricted patients with asthma is spatially associated with reduced perfusion, which is proposed to result from hypoxic pulmonary vasoconstriction (HPV). OBJECTIVES: To determine the role of HPV in the regional perfusion redistribution in bronchoconstricted patients with asthma. METHODS: Eight patients with asthma completed positron emission tomographic/computed tomographic lung imaging at baseline and after bronchoconstriction, breathing either room air or 80% oxygen (80% O2) on separate days. Relative perfusion, specific ventilation (sV), and gas fraction (Fgas) in the 25% of the lung with the lowest specific ventilation (sVlow) and the remaining lung (sVhigh) were quantified and compared. MEASUREMENTS AND MAIN RESULTS: In the sVlow region, bronchoconstriction caused a significant decrease in sV under both room air and 80% O2 conditions (baseline vs. bronchoconstriction, mean ± SD, 1.02 ± 0.20 vs. 0.35 ± 0.19 and 1.03 ± 0.20 vs. 0.32 ± 0.16, respectively; P < 0.05). In the sVlow region, relative perfusion decreased after bronchoconstriction under room air conditions and also, to a lesser degree, under 80% O2 conditions (1.02 ± 0.19 vs. 0.72 ± 0.08 [P < 0.001] and 1.08 ± 0.19 vs. 0.91 ± 0.12 [P < 0.05], respectively). The Fgas increased after bronchoconstriction under room air conditions only (0.99 ± 0.04 vs. 1.00 ± 0.02; P < 0.05). The sVlow subregion analysis indicated that some of the reduction in relative perfusion after bronchoconstriction under 80% O2 conditions occurred as a result of the presence of regional hypoxia. However, relative perfusion was also significantly reduced in sVlow subregions that were hyperoxic under 80% O2 conditions. CONCLUSIONS: HPV is not the only mechanism that contributes to perfusion redistribution in bronchoconstricted patients with asthma, suggesting that another nonhypoxia mechanism also contributes. We propose that this nonhypoxia mechanism may be either direct mechanical interactions and/or unidentified intercellular signaling between constricted airways, the parenchyma, and the surrounding vasculature.

An ALK2 inhibitor, BLU-782, prevents heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.

Quantification of Celiac Disease Severity Using Video Capsule Endoscopy: A Comparison of Human Experts and Machine Learning Algorithms.

BACKGROUND: Video capsule endoscopy (VCE) is an attractive method for diagnosing and objectively monitoring disease activity in celiac disease (CeD). Its use, facilitated by artificial intelligence- based tools, may allow computer-assisted interpretation of VCE studies, transforming a subjective test into a quantitative and reproducible measurement tool. OBJECTIVE: To evaluate and compare objective CeD severity assessment as determined with VCE by expert human readers and a machine learning algorithm (MLA). METHODS: Patients ≥ 18 years with histologically proven CeD underwent VCE. Examination frames were scored by three readers from one center and the MLA, using a 4-point ordinal scale for assessing the severity of CeD enteropathy. After scoring, curves representing CeD severity across the entire small intestine (SI) and individual tertiles (proximal, mid, and distal) were fitted for each reader and the MLA. All comparisons used Krippendorff's alpha; values > 0.8 represent excellent to 'almost perfect' inter-reader agreement. RESULTS: VCEs from 63 patients were scored. Readers demonstrated strong inter-reader agreement on celiac villous damage (alpha=0.924), and mean value reader curves showed similarly excellent agreement with MLA curves (alpha=0.935). Average reader and MLA curves were comparable for mean and maximum values for the first SI tertile (alphas=0.932 and 0.867, respectively) and the mean value over the entire SI (alpha=0.945). CONCLUSION: A novel MLA demonstrated excellent agreement on whole SI imaging with three expert gastroenterologists. An ordinal scale permitted high inter-reader agreement, accurately and reliably replicated by the MLA. Interpreting VCEs using MLAs may allow automated diagnosis and disease burden assessment in CeD.

Regional Ventilation and Aerosol Deposition with Helium-Oxygen in Bronchoconstricted Asthmatic Lungs.

BACKGROUND: Theoretical models suggest that He-O2 as carrier gas may lead to more homogeneous ventilation and aerosol deposition than air. However, these effects have not been clinically consistent and it is unclear why subjects may or may not respond to the therapy. Here we present 3D-imaging data of aerosol deposition and ventilation distributions from subjects with asthma inhaling He-O2 as carrier gas. The data are compared with those that we previously obtained from a similar group of subjects inhaling air. METHODS: Subjects with mild-to-moderate asthma were bronchoconstricted with methacholine and imaged with PET-CT while inhaling aerosol carried with He-O2. Mean-normalized-values of lobar specific ventilation sV* and deposition sD* were derived and the factors affecting the distribution of sD* were evaluated along with the effects of breathing frequency (f) and regional expansion (FVOL). RESULTS: Lobar distributions of sD* and sV* with He-O2 were not statistically different from those previously measured with air. However, with He-O2 there was a larger number of lobes having sV* and sD* closer to unity and, in those subjects with uneven deposition distributions, the correlation of sD* with sV* was on average higher (p < 0.05) in He-O2 (0.84 ± 0.8) compared with air (0.55 ± 0.28). In contrast with air, where the frequency of breathing during nebulization was associated with the degree of sD*-sV* correlation, with He-O2 there was no association. Also, the modulation of f on the correlation between FVOL and sD*/sV* in air, was not observed in He-O2. CONCLUSION: There were no differences in the inter-lobar heterogeneity of sD* or sV* in this group of mild asthmatic subjects breathing He-O2 compared with patients previously breathing air. Future studies, using these personalized 3D data sets as input to CFD models, are needed to understand if, and for whom, breathing He-O2 during aerosol inhalation may be beneficial.

A method for mapping regional oxygen and CO2 transfer in the lung.

This paper presents a novel approach to visualizing regional lung function, through quantitative three-dimensional maps of O2 and CO2 transfer rates. These maps describe the contribution of anatomical regions to overall gas exchange and demonstrate how transfer rates of the two gas species' differ regionally. An algorithm for generating such maps is presented, and for illustration, regional gas transfer maps were generated using values of ventilation and perfusion imaged by PET/CT for a healthy subject and an asthmatic patient after bronchoprovocation. In a sensitivity analysis, compartment values of gas transfer showed minor sensitivity to imaging noise in the ventilation and perfusion data, and moderate sensitivity to estimation errors in global lung input values, chiefly global alveolar ventilation, followed by cardiac output and arterial-venous O2 content difference. Gas transfer maps offer an intuitive display of physiologically relevant lung function at a regional level, the potential for an improved understanding of pulmonary gas exchange in health and disease, and potentially a presurgical evaluation tool.

What Causes Uneven Aerosol Deposition in the Bronchoconstricted Lung? A Quantitative Imaging Study.

BACKGROUND: A previous PET-CT imaging study of 14 bronchoconstricted asthmatic subjects showed that peripheral aerosol deposition was highly variable among subjects and lobes. The aim of this work was to identify and quantify factors responsible for this variability. METHODS: A theoretical framework was formulated to integrate four factors affecting aerosol deposition: differences in ventilation, in how air vs. aerosol distribute at each bifurcation, in the fraction of aerosol escaping feeding airways, and in the fraction of aerosol reaching the periphery that is exhaled. These factors were quantified in 12 of the subjects using PET-CT measurements of relative specific deposition sD*, relative specific ventilation sV* (measured with dynamic PET or estimated as change in expansion between two static HRCTs), average lobar expansion FVOL, and breathing frequency measured during aerosol inhalation fN. RESULTS: The fraction of the variance of sD* explained by sV* (0.38), by bifurcation effects (0.38), and by differences in deposition along feeding airways (0.31) were similar in magnitude. We could not directly estimate the contribution of aerosol that was exhaled. Differences in expansion did not explain any fraction of the variability in sD* among lobes. The dependence of sD* on sV* was high in subjects breathing with low fN, but weakened among those breathing faster. Finally, sD*/sV* showed positive dependence on FVOL among low fN subjects, while the dependence was negative among high fN subjects. CONCLUSION: The theoretical framework allowed us to analyze experimentally measured aerosol deposition imaging data. When considering bronchoconstricted asthmatic subjects, a dynamic measurement of ventilation is required to evaluate its effect on aerosol transport. The mechanisms behind the identified effects of fN and FVOL on aerosol deposition need further study and may have important implications for aerosol therapy in subjects with heterogeneous ventilation.

Analysis of three-dimensional aerosol deposition in pharmacologically relevant terms: beyond black or white ROIs.

BACKGROUND: This article presents a novel methodological approach to evaluate images of aerosol deposition taken with PET-CT cameras. Traditionally, Black-or-White (BW) Regions of Interest (ROIs) are created to cover Anatomical Regions (ARs) segmented from the high-resolution CT. Such ROIs do not usually consider blurring effects due to limited spatial resolution or breathing motion, and do not consider uncertainty in the AR position within the PET image. The new methodology presented here (Grayscale) addresses these issues, allows estimates of aerosol deposition within ARs, and expresses the deposition in terms of Tissue Dosing (in the lung periphery) and Inner Surface Concentration (in the larger airways). METHODS: Imaging data included a PET deposition image acquired during breathing and two CT scans acquired during breath holds at different lung volumes. The lungs were segmented into anatomically consistent ARs to allow unbiased comparisons across subjects and across lobes. The Grayscale method involves defining Voxel Influence Matrices (VIMs) to consider how average activity within each AR influences the measured activity within each voxel. The BW and Grayscale methods were used to analyze aerosol deposition in 14 bronchoconstricted asthmatics. RESULTS: Grayscale resulted in a closer description of the PET image than BW (p<0.0001) and exposed a seven-fold underestimation in measures of specific deposition. The Average Tissue Dosing was 2.11×10(-6) Total Lung Dose/mg. The average Inner Surface Concentration was 45×10(-6) Total Lung Dose/mm(2), with the left lower lobe having a lower ISC than lobes of the right lung (p<0.05). There was a strong lobar heterogeneity in these measures (COV=0.3). CONCLUSION: The Grayscale approach is an improvement over the BW approach and provides a closer description of the PET image. It can be used to characterize heterogeneous concentrations throughout the lung and may be important in translational research and in the evaluation of aerosol delivery systems.

Pendelluft in the bronchial tree.

Inhomogeneous inflation or deflation of the lungs can cause dynamic pressure differences between regions and lead to interregional airflows known as pendelluft. This work first uses analytical tools to clarify the theoretical limits of pendelluft at a single bifurcation. It then explores the global and regional pendelluft that may occur throughout the bronchial tree in a realistic example using an in silico model of bronchoconstriction. The theoretical limits of pendelluft volume exchanged at a local bifurcation driven by sinusoidal breathing range from 15.5% to 41.4% depending on the relative stiffness of the subtended regions. When nonsinusoidal flows are considered, pendelluft can be as high as 200% inlet tidal volume (Vin). At frequencies greater than 10 Hz, the inertia of the air in the airways becomes important, and the maximal local pendelluft is theoretically unbounded, even with sinusoidal breathing. In a single illustrative numerical simulation of bronchoconstriction with homogenous compliances, the overall magnitude of global pendelluft volume was <2% of the tidal volume. Despite the small overall magnitude, pendelluft volume exchange was concentrated in poorly ventilated regions of the lung, including local pendelluft at bifurcations of up to 13% Vin. This example suggests that pendelluft may be an important phenomena contributing to regional gas exchange, irreversible mixing, and aerosol deposition patterns inside poorly ventilated regions of the lung. The analytical results support the concept that pendelluft may be more prominent in diseases with significant heterogeneity in both resistance and compliance.