Summary Background A major goal of asthma therapy is to achieve disease control, with maintenance of lung function, reduced need for rescue medication, and prevention of exacerbation. Despite current standard of care, up to 70% of patients with asthma remain poorly controlled. Analysis of serum and sputum biomarkers could offer insights into parameters associated with poor asthma control. Objective To identify signatures as determinants of asthma disease control, we performed proteomics using Olink proximity extension analysis. Methods Up to 3 longitudinal serum samples were collected from 23 controlled and 25 poorly controlled asthmatics. Nine of the controlled and 8 of the poorly controlled subjects also provided 2 longitudinal sputum samples. The study included an additional cohort of 9 subjects whose serum was collected within 48 hours of asthma exacerbation. Two separate pre‐defined Proseek Multiplex panels ( INF and CVDIII ) were run to quantify 181 separate protein analytes in serum and sputum. Results Panels consisting of 9 markers in serum ( CCL 19, CCL 25, CDCP 1, CCL 11, FGF 21, FGF 23, Flt3L, IL ‐10Rβ, IL ‐6) and 16 markers in sputum ( tPA , KLK 6, RETN , ADA , MMP 9, Chit1, GRN , PGLYRP 1, MPO , HGF , PRTN 3, DNER , PI 3, Chi3L1, AZU 1, and OPG ) distinguished controlled and poorly controlled asthmatics. The sputum analytes were consistent with a pattern of neutrophil activation associated with poor asthma control. The serum analyte profile of the exacerbation cohort resembled that of the controlled group rather than that of the poorly controlled asthmatics, possibly reflecting a therapeutic response to systemic corticosteroids. Conclusions and Clinical Relevance Proteomic profiles in serum and sputum distinguished controlled and poorly controlled asthmatics, and were maintained over time. Findings support a link between sputum neutrophil markers and loss of asthma control.

Diagnostic certainty of idiopathic pulmonary fibrosis (IPF) is critical to determine prognosis and therapeutic options. The U.S. Food and Drug Administration–approved antifibrotic agents pirfenidone and nintedanib are currently only indicated for IPF, and immunosuppressants frequently used to treat other interstitial lung diseases (ILD) have been associated with worse outcomes when used for IPF (1–4). According to current guidelines, a diagnosis of IPF requires identification of a usual interstitial pneumonitis (UIP) pattern by high-resolution computed tomography (HRCT) and/or microscopic tissue examination (1). The diagnosis can be made by HRCT alone in z50% of cases, when all UIP criteria are met, including honeycombing (1). However, the inability to visualize microscopic honeycombing (,2–3 mm) or distinguish honeycomb from traction bronchiectasis is a frequent cause of diagnostic uncertainty (5, 6). When HRCT is inconclusive, surgery is required to obtain wedge biopsies for microscopic evaluation (1), which is associated with morbidity/mortality risks in patients with IPF (7–9). This mandates a cautious approach to employing surgical biopsy for diagnosis, and precludes its repeated use to assess disease progression. We demonstrate the use of in vivo endobronchial optical coherence tomography (OCT) as a minimally invasive method to microscopically assess and diagnose UIP/IPF without tissue removal. OCT provides rapid, three-dimensional imaging with microscopic resolution (,10 mm) well beyond HRCT capabilities, and can assess significantly larger tissue volumes (z1003) than can be assessed microscopically from surgical biopsies (10). OCT is performed by passing a narrow catheter through a standard bronchoscope working channel out to the peripheral lung and conducting helical scanning with pullback lengths up to 10 cm. Some of the results of these studies have been previously reported in the form of an abstract (11–13). Methods We conducted a pilot study in five sequential patients with ILD undergoing video-assisted thoracic surgical biopsy for diagnosis at Massachusetts General Hospital. Endobronchial OCT was performed before each biopsy. Imaging locations were selected based on sites of abnormalities identified on HRCT, with four to six OCT pullbacks obtained per patient. The total time needed to collect all OCT data was ,6 minutes. OCT imaging was interpreted independently of biopsy. The OCT images in Figures 1 and 2 are representative still frames selected from larger volumetric datasets. Patients consented to participate in the study (approved by the Partners Human Research Institutional Review Board, #2015-P001345). A summary of the cases is presented in Table 1.

Background Fibrotic diseases including systemic sclerosis (SSc) or scleroderma are characterized by fibroblast differentiation into myofibroblasts and extracellular matrix deposition. The mechanisms driving fibroblast activation are not fully known, and the identification of new profibrotic mediators will hopefully lead to the development of new rational anti- fibrotic treatments. Microarray studies show that ephrin-B2 is overexpressed in SSc fibroblasts, suggesting that this cell- membrane-anchored ligand may be a novel profibrotic mediator. Objectives To identify the role of ephrin B2 in skin and lung fibrosis. Methods Mice were injected subcutaneously with recombinant mouse ephrin-B2/Fc (100μg/kg/mouse) daily for two weeks and assessed for the development of skin fibrosis. Fibroblast-specific ephrin-B2 knockout (KO) mice were generated and assessed for the development of lung and skin fibrosis induced by intratracheal or subcutaneous injection of bleomycin respectively, by histology, hydroxyproline levels, q-PCR and western blot. Ephrin-B2 levels were determined by western blot and ELISA in broncoalveolar lavage (BAL) fluids from these mice, and from humans with SSc. Cultured mouse and human skin fibroblasts were treated with recombinant ephrin-B2-Fc (0.1-5 mg/mL) and the expression of profibrotic genes was assessed by qPCR and western blot. 96-Multiwell Insert Systems (BD Biosciences) were used to measure the chemotaxis of mouse skin fibroblasts from mice treated with either PBS or bleomycin for 7 days. Fibroblasts in these chemotaxis experiments were transfected with 20nM siRNA (Dharmacon) targeting EphB2, EphB3 or EphB4 receptors. Non-targeting siRNA was used as a control. Results Treatment of normal human skin fibroblasts with recombinant ephrin-B2 in vitro induced myofibroblast differentiation, as indicated by increased stress fiber formation, increased expression of α-SMA and collagen type I. Mice treated sub-cutaneously with recombinant mouse ephrin-B2/Fc exhibited significant skin fibrosis indicated by increased dermal thickness, collagen deposition, hydroxyproline content and α-SMA-expressing myofibroblasts. Fibroblast-specific ephrin-B2 KO mice were significantly protected from bleomycin-induced lung and skin fibrosis, as indicated by significant reductions in hydroxyproline and TGF-b levels. Q-PCR and immunohistochemical analyses showed that ephrin-B2 expression was elevated in SSc fibroblasts and skin sections from IPF patients respectively. Soluble ephrin-B2 levels determined by ELISA and western blot were increased in BAL from SSc patients and bleomycin-challenged mice. Soluble ephrin-B2 induced fibroblast chemotaxis (through EphB3/EphB4 but not EphB2 receptors), suggesting that pathological shedding of soluble ephrin-B2 might account for the profibrotic effect of this ligand in lung and skin fibrosis. Conclusions Our study identifies ephrin-B2 as a novel mediator of fibrogenesis, and suggests that targeting ephrin-B2 ligand or its binding receptors EphB3/EphB4 could potentially be a new therapeutic strategy in SSc. Disclosure of Interest None declared