Nucleoside Analogs in ADAR Guide Strands Enable Editing at 5'-GA Sites.

Adenosine Deaminases Acting on RNA (ADARs) are members of a family of RNA editing enzymes that catalyze the conversion of adenosine into inosine in double-stranded RNA (dsRNA). ADARs' selective activity on dsRNA presents the ability to correct mutations at the transcriptome level using guiding oligonucleotides. However, this approach is limited by ADARs' preference for specific sequence contexts to achieve efficient editing. Substrates with a guanosine adjacent to the target adenosine in the 5' direction (5'-GA) are edited less efficiently compared to substrates with any other canonical nucleotides at this position. Previous studies showed that a G/purine mismatch at this position results in more efficient editing than a canonical G/C pair. Herein, we investigate a series of modified oligonucleotides containing purine or size-expanded nucleoside analogs on guide strands opposite the 5'-G (-1 position). The results demonstrate that modified adenosine and inosine analogs enhance editing at 5'-GA sites. Additionally, the inclusion of a size-expanded cytidine analog at this position improves editing over a control guide bearing cytidine. High-resolution crystal structures of ADAR:/RNA substrate complexes reveal the manner by which both inosine and size-expanded cytidine are capable of activating editing at 5'-GA sites. Further modification of these altered guide sequences for metabolic stability in human cells demonstrates that the incorporation of specific purine analogs at the -1 position significantly improves editing at 5'-GA sites.

Structural perspectives on adenosine to inosine RNA editing by ADARs.

Adenosine deaminases acting on RNA (ADARs) are enzymes that catalyze the hydrolytic deamination of adenosine to inosine. The editing feature of ADARs has garnered much attention as a therapeutic tool to repurpose ADARs to correct disease-causing mutations at the mRNA level in a technique called site-directed RNA editing (SDRE). Administering a short guide RNA oligonucleotide that hybridizes to a mutant sequence forms the requisite dsRNA substrate, directing ADARs to edit the desired adenosine. However, much is still unknown about ADARs' selectivity and sequence-specific effects on editing. Atomic-resolution structures can help provide additional insight to ADARs' selectivity and lead to novel guide RNA designs. Indeed, recent structures of ADAR domains have expanded our understanding on RNA binding and the base-flipping catalytic mechanism. These efforts have enabled the rational design of improved ADAR guide strands and advanced the therapeutic potential of the SDRE approach. While no full-length structure of any ADAR is known, this review presents an exposition of the structural basis for function of the different ADAR domains, focusing on human ADAR2. Key insights are extrapolated to human ADAR1, which is of substantial interest because of its widespread expression in most human tissues.

Measuring patient-reported experience of solid organ transplantation healthcare: A scoping review of condition- and transplant-specific measures.

BACKGROUND: Measures of patient experience are increasingly valued as key to healthcare quality assessment. We aimed to identify and describe publicly available measures assessing patient-reported experience of solid organ transplantation healthcare, and identify patient groups, healthcare settings, or aspects of patient experience underserved by existing measures. METHODS: We systematically searched MEDLINE, Embase, CINAHL, PsycINFO, Cochrane CENTRAL, Scopus and Web of Science from inception to 6th July 2023; supplemented with grey literature searches. Two reviewers independently screened search hits; outputs reporting patient-reported measures of multiple aspects of established solid organ transplantation healthcare were eligible. We abstracted measure context, characteristics, content (i.e., attributes of patient experience assessed), and development and validation processes. RESULTS: We identified nine outputs reporting eight measures of patient experience; these related only to kidney (n = 5) or liver (n = 3) transplantation, with no available measures relating to heart, lung, pancreas or intestinal transplantation. Of the identified measures, four were specific to solid organ transplant recipients. Measures sought to assess "patient satisfaction" (n = 4) and "patient experience" (n = 4) of healthcare. Measures mapped to between five and 16 of 20 attributes of patient experience, most often Information and education, Communication, and Access to care (all n = 7). Six measures reported a development process, only three reported a validation process. CONCLUSIONS: Publicly available patient-reported measures of organ transplantation healthcare experiences are limited to kidney and liver transplantation. There is heterogeneity in measure context, characteristics, and content, and insufficient clarity concerning how well measures capture the specific experiences of transplant recipients. Formalised measures of patient experience, specific to solid organ transplantation, with transparent reporting of development and validity are needed.

Extracorporeal photopheresis (ECP) in the treatment of chronic lung allograft dysfunction (CLAD): a prospective, multicentre, open-label, randomised controlled trial studying the addition of ECP to standard care in the treatment of bilateral lung transplant patients with CLAD (E-CLAD UK).

BACKGROUND: Long-term survival after lung transplantation is limited compared with other organ transplants. The main cause is development of progressive immune-mediated damage to the lung allograft. This damage, which can develop via multiple immune pathways, is captured under the umbrella term chronic lung allograft dysfunction (CLAD). Despite the availability of powerful immunosuppressive drugs, there are presently no treatments proven to reverse or reliably halt the loss of lung function caused by CLAD. The aim of the E-CLAD UK trial is to determine whether the addition of immunomodulatory therapy, in the form of extracorporeal photopheresis (ECP), to standard care is more efficacious at stabilising lung function in CLAD compared with standard care alone. METHODS AND ANALYSIS: E-CLAD UK is a Phase II clinical trial of an investigational medicinal product (Methoxsalen) delivered to a buffy coat prepared via an enclosed ECP circuit. Target recruitment is 90 bilateral lung transplant patients identified as having CLAD and being treated at one of the five UK adult lung transplant centres. Participants will be randomised 1:1 to intervention plus standard of care, or standard of care alone. Intervention will comprise nine ECP cycles spread over 20 weeks, each course involving two treatments of ECP on consecutive days. All participants will be followed up for a period of 24 weeks.The primary outcome is lung function stabilisation derived from change in forced expiratory volume in one second and forced vital capacity at 12 and 24 weeks compared with baseline at study entry. Other parameters include change in exercise capacity, health-related quality of life and safety. A mechanistic study will seek to identify molecular or cellular markers linked to treatment response and qualitative interviews will explore patient experiences of CLAD and the ECP treatment.A patient and public advisory group is integral to the trial from design to implementation, developing material to support the consent process and interview materials. ETHICS AND DISSEMINATION: The East Midlands-Derby Research Ethics Committee has provided ethical approval (REC 22/EM/0218). Dissemination will be via publications, patient-friendly summaries and presentation at scientific meetings. TRIAL REGISTRATION NUMBER: EudraCT number 2022-002659-20; ISRCTN 10615985.

A Split-Lung Ex Vivo Perfusion Model for Time- and Cost-Effective Evaluation of Therapeutic Interventions to the Human Donor Lung.

With the ongoing shortage of donor lungs, ex vivo lung perfusion (EVLP) offers the opportunity for objective assessment and potential therapeutic repair of marginal organs. There is a need for robust research on EVLP interventions to increase the number of transplantable organs. The use of human lungs, which have been declined for transplant, for these studies is preferable to animal organs and is indeed essential if clinical translation is to be achieved. However, experimental human EVLP is time-consuming and expensive, limiting the rate at which promising interventions can be assessed. A split-lung EVLP model, which allows stable perfusion and ventilation of two single lungs from the same donor, offers advantages scientifically, financially and in time to yield results. Identical parallel circuits allow one to receive an intervention and the other to act as a control, removing inter-donor variation between study groups. Continuous hemodynamic and airway parameters are recorded and blood gas, perfusate, and tissue sampling are facilitated. Pulmonary edema is assessed directly using ultrasound, and indirectly using the lung tissue wet:dry ratio. Evans blue dye leaks into the tissue and can quantify vascular endothelial permeability. The split-lung ex vivo perfusion model offers a cost-effective, reliable platform for testing therapeutic interventions with relatively small sample sizes.

The use of extracorporeal photopheresis in solid organ transplantation-current status and future directions.

Prevention and management of allograft rejection urgently require more effective therapeutic solutions. Current immunosuppressive therapies used in solid organ transplantation, while effective in reducing the risk of acute rejection, are associated with substantial adverse effects. There is, therefore, a need for agents that can provide immunomodulation, supporting graft tolerance, while minimizing the need for immunosuppression. Extracorporeal photopheresis (ECP) is an immunomodulatory therapy currently recommended in international guidelines as an adjunctive treatment for the prevention and management of organ rejection in heart and lung transplantations. This article reviews clinical experience and ongoing research with ECP for organ rejection in heart and lung transplantations, as well as emerging findings in kidney and liver transplantation. ECP, due to its immunomodulatory and immunosuppressive-sparing effects, offers a potential therapeutic option in these settings, particularly in high-risk patients with comorbidities, infectious complications, or malignancies.

Impact of Disease-Associated Mutations on the Deaminase Activity of ADAR1.

The innate immune system relies on molecular sensors to detect distinctive molecular patterns, including viral double-stranded RNA (dsRNA), which triggers responses resulting in apoptosis and immune infiltration. Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I), serving as a mechanism to distinguish self from non-self RNA and prevent aberrant immune activation. Loss-of-function mutations in the ADAR1 gene are one cause of Aicardi Goutières Syndrome (AGS), a severe autoimmune disorder in children. Although seven out of the eight AGS-associated mutations in ADAR1 occur within the catalytic domain of the ADAR1 protein, their specific effects on the catalysis of adenosine deamination remain poorly understood. In this study, we carried out a biochemical investigation of four AGS-causing mutations (G1007R, R892H, K999N, and Y1112F) in ADAR1 p110 and truncated variants. These studies included adenosine deamination rate measurements with two different RNA substrates derived from human transcripts known to be edited by ADAR1 p110 (glioma-associated oncogene homologue 1 (hGli1), 5-hydroxytryptamine receptor 2C (5-HT2cR)). Our results indicate that AGS-associated mutations at two amino acid positions directly involved in stabilizing the base-flipped conformation of the ADAR-RNA complex (G1007R and R892H) had the most detrimental impact on catalysis. The K999N mutation, positioned near the RNA binding interface, altered catalysis contextually. Finally, the Y1112F mutation had small effects in each of the assays described here. These findings shed light on the differential effects of disease-associated mutations on adenosine deamination by ADAR1, thereby advancing our structural and functional understanding of ADAR1-mediated RNA editing.

Outcomes of listing for lung and heart-lung transplantation in pulmonary hypertension: comparative experience in France and the UK.

Background: Lung or heart-lung transplantation (LT/HLT) for severe pulmonary hypertension (PH) as the primary disease indication carries a high risk of waiting list mortality and post-transplant complications. France and the UK both have coordinated PH patient services but with different referral pathways for accessing LT services. Methods: We conducted a comparative analysis of adult PH patients listed for LT/HLT in the UK and France. Results: We included 211 PH patients in France (2006-2018) and 170 in the UK (2010-2019). Cumulative incidence of transplant, delisting and waiting list death within 3 years were 81%, 4% and 11% in France versus 58%, 10% and 15% in the UK (p<0.001 for transplant and delisting; p=0.1 for death). Median non-priority waiting time was 45 days in France versus 165 days in the UK (p<0.001). High-priority listing occurred in 54% and 51% of transplanted patients respectively in France and the UK (p=0.8). Factors associated with achieving transplantation related to recipients' height, male sex, clinical severity and priority listing status. 1-year post-transplant survival was 78% in France and 72% in the UK (p= 0.04). Conclusion: Access to transplantation for PH patients is better in France than in the UK where more patients were delisted due to clinical deterioration because of longer waiting time. High rates of priority listing occurred in both countries. Survival for those achieving transplantation was slightly better in France. Ensuring optimal outcomes after transplant listing for PH patients is challenging and may involve early listing of higher risk patients, increasing donor lung utilisation and improving allocation rules for these specific patients.

Distinct lung cell signatures define the temporal evolution of diffuse alveolar damage in fatal COVID-19.

BACKGROUND: Lung damage in severe COVID-19 is highly heterogeneous however studies with dedicated spatial distinction of discrete temporal phases of diffuse alveolar damage (DAD) and alternate lung injury patterns are lacking. Existing studies have also not accounted for progressive airspace obliteration in cellularity estimates. We used an imaging mass cytometry (IMC) analysis with an airspace correction step to more accurately identify the cellular immune response that underpins the heterogeneity of severe COVID-19 lung disease. METHODS: Lung tissue was obtained at post-mortem from severe COVID-19 deaths. Pathologist-selected regions of interest (ROIs) were chosen by light microscopy representing the patho-evolutionary spectrum of DAD and alternate disease phenotypes were selected for comparison. Architecturally normal SARS-CoV-2-positive lung tissue and tissue from SARS-CoV-2-negative donors served as controls. ROIs were stained for 40 cellular protein markers and ablated using IMC before segmented cells were classified. Cell populations corrected by ROI airspace and their spatial relationships were compared across lung injury patterns. FINDINGS: Forty patients (32M:8F, age: 22-98), 345 ROIs and >900k single cells were analysed. DAD progression was marked by airspace obliteration and significant increases in mononuclear phagocytes (MnPs), T and B lymphocytes and significant decreases in alveolar epithelial and endothelial cells. Neutrophil populations proved stable overall although several interferon-responding subsets demonstrated expansion. Spatial analysis revealed immune cell interactions occur prior to microscopically appreciable tissue injury. INTERPRETATION: The immunopathogenesis of severe DAD in COVID-19 lung disease is characterised by sustained increases in MnPs and lymphocytes with key interactions occurring even prior to lung injury is established. FUNDING: UK Research and Innovation/Medical Research Council through the UK Coronavirus Immunology Consortium, Barbour Foundation, General Sir John Monash Foundation, Newcastle University, JGW Patterson Foundation, Wellcome Trust.

A precise swaying map for how promiscuous cellobiose-2-epimerase operate bi-reaction.

Promiscuous enzymes play a crucial role in organism survival and new reaction mining. However, comprehensive mapping of the catalytic and regulatory mechanisms hasn't been well studied due to the characteristic complexity. The cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) with complex epimerization and isomerization was chosen to comprehensively investigate the promiscuous mechanisms. Here, the catalytic frame of ring-opening, cis-enediol mediated catalysis and ring-closing was firstly determined. To map the full view of promiscuous CE, the structure of CsCE complex with the isomerized product glucopyranosyl-ß1,4-fructose was determined. Combined with computational calculation, the promiscuity was proved a precise cooperation of the double subsites, loop rearrangement, and intermediate swaying. The flexible loop was like a gear, whose structural reshaping regulates the sway of the intermediates between the two subsites of H377-H188 and H377-H247, and thus regulates the catalytic directions. The different protonated states of cis-enediol intermediate catalyzed by H188 were the key point for the catalysis. The promiscuous enzyme tends to utilize all elements at hand to carry out the promiscuous functions.

Momentum-Space Imaging of Ultra-Thin Electron Liquids in δ-Doped Silicon.

Two-dimensional dopant layers (δ-layers) in semiconductors provide the high-mobility electron liquids (2DELs) needed for nanoscale quantum-electronic devices. Key parameters such as carrier densities, effective masses, and confinement thicknesses for 2DELs have traditionally been extracted from quantum magnetotransport. In principle, the parameters are immediately readable from the one-electron spectral function that can be measured by angle-resolved photoemission spectroscopy (ARPES). Here, buried 2DEL δ-layers in silicon are measured with soft X-ray (SX) ARPES to obtain detailed information about their filled conduction bands and extract device-relevant properties. This study takes advantage of the larger probing depth and photon energy range of SX-ARPES relative to vacuum ultraviolet (VUV) ARPES to accurately measure the δ-layer electronic confinement. The measurements are made on ambient-exposed samples and yield extremely thin (< 1 nm) and dense (≈1014  cm-2 ) 2DELs. Critically, this method is used to show that δ-layers of arsenic exhibit better electronic confinement than δ-layers of phosphorus fabricated under identical conditions.

Lymphocyte Depleting and Modulating Therapies for Chronic Lung Allograft Dysfunction.

Chronic lung rejection, also called chronic lung allograft dysfunction (CLAD), remains the major hurdle limiting long-term survival after lung transplantation, and limited therapeutic options are available to slow the progressive decline in lung function. Most interventions are only temporarily effective in stabilizing the loss of or modestly improving lung function, with disease progression resuming over time in the majority of patients. Therefore, identification of effective treatments that prevent the onset or halt progression of CLAD is urgently needed. As a key effector cell in its pathophysiology, lymphocytes have been considered a therapeutic target in CLAD. The aim of this review is to evaluate the use and efficacy of lymphocyte depleting and immunomodulating therapies in progressive CLAD beyond usual maintenance immunosuppressive strategies. Modalities used include anti-thymocyte globulin, alemtuzumab, methotrexate, cyclophosphamide, total lymphoid irradiation, and extracorporeal photopheresis, and to explore possible future strategies. When considering both efficacy and risk of side effects, extracorporeal photopheresis, anti-thymocyte globulin and total lymphoid irradiation appear to offer the best treatment options currently available for progressive CLAD patients. SIGNIFICANCE STATEMENT: Effective treatments to prevent the onset and progression of chronic lung rejection after lung transplantation are still a major shortcoming. Based on existing data to date, considering both efficacy and risk of side effects, extracorporeal photopheresis, anti-thymocyte globulin, and total lymphoid irradiation are currently the most viable second-line treatment options. However, it is important to note that interpretation of most results is hampered by the lack of randomized controlled trials.

Modeling the Effects of IL-1ß-mediated Inflammation During Ex Vivo Lung Perfusion Using a Split Human Donor Model.

BACKGROUND: The association between interleukin-1ß (IL-1ß) concentrations during ex vivo lung perfusion (EVLP) with donor organ quality and post-lung transplant outcome has been demonstrated in several studies. The mechanism underlying IL-1ß-mediated donor lung injury was investigated using a paired single-lung EVLP model. METHODS: Human lung pairs were dissected into individual lungs and perfused on identical separate EVLP circuits, with one lung from each pair receiving a bolus of IL-1ß. Fluorescently labeled human neutrophils isolated from a healthy volunteer were infused into both circuits and quantified in perfusate at regular timepoints. Perfusates and tissues were subsequently analyzed, with perfusates also used in functional assays. RESULTS: Neutrophil numbers were significantly lower in perfusate samples collected from the IL-1ß-stimulated lungs consistent with increased neutrophil adhesion ( P = 0.042). Stimulated lungs gained significantly more weight than controls ( P = 0.046), which correlated with soluble intercellular adhesion molecule-1 (R 2 = 0.71, P = 0.0043) and von-Willebrand factor (R 2 = 0.39, P = 0.040) in perfusate. RNA expression patterns for inflammatory genes were differentially regulated via IL-1ß. Blockade of IL-1ß significantly reduced neutrophil adhesion in vitro ( P = 0.025). CONCLUSION: These data illustrate the proinflammatory functions of IL-1ß in the context of EVLP, suggesting this pathway may be susceptible to therapeutic modulation before transplantation.

Identification of a broadly fibrogenic macrophage subset induced by type 3 inflammation.

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in fibrosis progression and resolution. Identifying key macrophage populations found in human fibrotic tissues could lead to new treatments for fibrosis. Here, we used human liver and lung single-cell RNA sequencing datasets to identify a subset of CD9+TREM2+ macrophages that express SPP1, GPNMB, FABP5, and CD63. In both human and murine hepatic and pulmonary fibrosis, these macrophages were enriched at the outside edges of scarring and adjacent to activated mesenchymal cells. Neutrophils expressing MMP9, which participates in the activation of TGF-ß1, and the type 3 cytokines GM-CSF and IL-17A coclustered with these macrophages. In vitro, GM-CSF, IL-17A, and TGF-ß1 drive the differentiation of human monocytes into macrophages expressing scar-associated markers. Such differentiated cells could degrade collagen IV but not collagen I and promote TGF-ß1-induced collagen I deposition by activated mesenchymal cells. In murine models blocking GM-CSF, IL-17A or TGF-ß1 reduced scar-associated macrophage expansion and hepatic or pulmonary fibrosis. Our work identifies a highly specific macrophage population to which we assign a profibrotic role across species and tissues. It further provides a strategy for unbiased discovery, triage, and preclinical validation of therapeutic targets based on this fibrogenic macrophage population.

Identification of a protein expression signature distinguishing early from organising diffuse alveolar damage in COVID-19 patients.

Diffuse alveolar damage (DAD) is the histological expression of acute respiratory distress syndrome and characterises lung pathology due to infection with SARS-CoV-2, and other respiratory pathogens of clinical significance. DAD reflects a time-dependent immunopathological process, progressing from an early/exudative stage through to an organising/fibrotic stage, yet within an individual these different stages of DAD may coexist. Understanding the progression of DAD is central to the development of new therapeutics to limit progressive lung damage. Here, we applied highly multiplexed spatial protein profiling to autopsy lung tissues derived from 27 patients who died from COVID-19 and identified a protein signature (ARG1, CD127, GZMB, IDO1, Ki67, phospho-PRAS40 (T246) and VISTA) that distinguishes early DAD from late DAD with good predictive accuracy. These proteins warrant further investigation as potential regulators of DAD progression.

Downregulation of MALAT1 is a hallmark of tissue and peripheral proliferative T cells in COVID-19.

T cells play key protective but also pathogenic roles in COVID-19. We studied the expression of long non-coding RNAs (lncRNAs) in COVID-19 T-cell transcriptomes by integrating previously published single-cell RNA sequencing datasets. The long intergenic non-coding RNA MALAT1 was the most highly transcribed lncRNA in T cells, with Th1 cells demonstrating the lowest and CD8+ resident memory cells the highest MALAT1 expression, amongst CD4+ and CD8+ T-cells populations, respectively. We then identified gene signatures that covaried with MALAT1 in single T cells. A significantly higher number of transcripts correlated negatively with MALAT1 than those that correlated. Enriched functional annotations of the MALAT1- anti-correlating gene signature included processes associated with T-cell activation such as cell division, oxidative phosphorylation, and response to cytokine. The MALAT1 anti-correlating gene signature shared by both CD4+ and CD8+ T-cells marked dividing T cells in both the lung and blood of COVID-19 patients. Focussing on the tissue, we used an independent patient cohort of post-mortem COVID-19 lung samples and demonstrated that MALAT1 suppression was indeed a marker of MKI67+ proliferating CD8+ T cells. Our results reveal MALAT1 suppression and its associated gene signature are a hallmark of human proliferating T cells.

Lung transplantation for interstitial lung disease: evolution over three decades.

BACKGROUND: Interstitial lung disease (ILD) has emerged as the most common indication for lung transplantation globally. However, post-transplant survival varies depending on the underlying disease phenotype and comorbidities. This study aimed to describe the demographics, disease classification, outcomes and factors associated with post-transplant survival in a large single-centre cohort. METHODS: Data were retrospectively assessed for 284 recipients who underwent lung transplantation for ILD in our centre between 1987 and 2020. Patient characteristics and outcomes were stratified by three eras: 1987-2000, 2001-2010 and 2011-2020. RESULTS: Median patients' age at time of transplantation was significantly higher in the most recent decade (56 (51-61) years, p<0.0001). Recipients aged over 50 years had worse overall survival compared with younger patients (adjusted HR, aHR 2.36, 95% CI 1.55 to 3.72, p=0.0001). Better survival was seen with bilateral versus single lung transplantation in patients younger than 50 years (log-rank p=0.0195). However, this survival benefit was no longer present in patients aged over 50 years. Reduced survival was observed in fibrotic non-specific interstitial pneumonia compared with idiopathic pulmonary fibrosis, which remained the most common indication throughout (aHR 2.61, 95% CI 1.40 to 4.60, p=0.0015). CONCLUSION: In patients transplanted for end-stage ILD, older age and fibrotic non-specific interstitial pneumonia were associated with poorer post-transplant survival. The benefit of bilateral over single lung transplantation diminished with increasing age, suggesting that single lung transplantation might still be a feasible option in older candidates.

miRNA Expression in Fibroblastic Foci within Idiopathic Pulmonary Fibrosis Lungs Reveals Novel Disease-Relevant Pathways.

miRNAs are 22 nucleotides long and belong to a class of noncoding RNAs that plays an important role in regulating gene expression at a post-transcriptional level. Studies show aberrant levels of miRNAs to be associated with profibrotic processes in idiopathic pulmonary fibrosis (IPF). However, most of these studies used whole IPF tissue or in vitro monocultures in which fibrosis was artificially induced. The current study used laser microdissection to collect fibroblastic foci (FF), the key pathologic lesion in IPF, isolated miRNAs, and compared their expression levels with those found in whole IPF lung tissue and/or in vitro cultured fibroblast from IPF or normal lungs. Sequencing libraries were generated, and data generated were bioinformatically analyzed. A total of 18 miRNAs were significantly overexpressed in FF tissue when compared with whole IPF tissue. Of those, 15 were unique to FF. Comparison of FF with cultured IPF fibroblasts also revealed differences in miRNA composition that impacted several signaling pathways. The miRNA composition of FF is both overlapping and distinct from that of whole IPF tissue or cultured IPF fibroblasts and highlights the importance of characterizing FF biology as a phenotypically and functionally discrete tissue microenvironment.