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A refratariedade é um problema recorrente no tratamento de pacientes trombocitopênicos que impede a restauração dos níveis normais de plaquetas circulantes após transfusões. Sua principal causa é a incompatibilidade entre os antígenos HLA Classe I das plaquetas transfundidas e os anticorpos do paciente. A produção de plaquetas in vitro a partir de células-tronco editadas geneticamente pode se tornar futuramente uma fonte de plaquetas universais. A linhagem de células-tronco/estromais mesenquimais derivadas de tecido adiposo (ASCL), ou adipócitos desdiferenciados (DFAT), são geradas a partir da desdiferenciação de adipócitos por meio do método “Ceiling Culture”, podem ser obtidas em abundância e diferenciar em megacariócitos e plaquetas em condições adequadas. Os antígenos HLA Classe I são fixados na superfície das células humanas por meio da ligação à proteína Beta-2 Microglobulina (B2M), expressa pelo gene B2M. Neste trabalho realizamos edição gênica em ASCL, obtendo populações carentes de antígenos HLA classe I, por meio de nocaute do gene B2M usando a ferramenta CRISPR/Cas9. Um RNA guia (sgRNA) foi projetado e sintetizado especificamente para quebra de DNA no éxon 2 do gene B2M. O sgRNA clonado no plasmídeo pL-CRISPR.EFS.GFP, foi sequenciado pelo método de sequenciamento automático de Sanger para confirmação. O plasmídeo clonado, juntamente com outros dois (segunda geração) para montagem de partículas lentivirais, foram transfectados em células HEK 293T e os lentivírus resultantes foram coletados e armazenados a -80°C até o uso. Diferentes lotes de lentivírus produzidos foram agrupados e utilizados em transduções de células K562 para a estimativa da produção lentiviral, quantificada através da detecção de proteína verde fluorescente (GFP) por citometria de fluxo. Células HEK 293T também foram usadas para testes iniciais de transdução e eficiência do nocaute usando três sgRNAs clonados e seus respectivos lentivírus, isoladamente ou em combinação. Os lentivírus portadores do plasmídeo clonado com sgRNA “guide#2”foram os mais eficazes na redução dos níveis de HLA Classe I na superfície das células HEK 293T e então escolhidos para a continuação do estudo. As transduções de ASCL, com MOI de aproximadamente 13,5 resultaram em populações de células viáveis e GFP positivas, sem antígenos HLA classe I. A maior redução foi observada no dia 7 pós-transdução, resultando em 96,4% de células negativas. Mais de 80% das células permaneceram negativas quando avaliadas nos dias 21 e 28. ASCL não editadas foram diferenciadas in vitro, originando megacariócitos e plaquetas, que foram caracterizados por microscopia óptica, citometria de fluxo normal e de imagem (anticorpos CD41, CD42b e CD61 e marcador nuclear Hoechst) e microscopia eletrônica de transmissão. No entanto, experimentos adicionais estão em andamento para comprovar a funcionalidade das plaquetas produzidas. A atual eficiência de transdução e nocaute do gene B2M usando CRISPR/Cas9, bem como a redução drástica dos níveis de antígenos HLA Classe I detectados na superfície das células ASCL nos permitirão armazenar as células modificadas em estado criogênico para posterior indução da diferenciação em plaquetas. Portanto, esta abordagem pode ser uma boa estratégia para gerar plaquetas funcionais, sem antígenos HLA Classe I.

Abstract Introduction Pirfenidone, an antifibrotic drug, slows-down the disease progression in idiopathic pulmonary fibrosis (IPF) over 12 months, however limited data on the decline of lung function and overall survival (OS) in real-world cohorts on longer follow-up exists. Patients/methods Of the enrolled Czech IPF patients (n = 841) from an EMPIRE registry, 383 (45.5%) received pirfenidone, 218 (25.9%) no-antifibrotic treatment and 240 (28.5%) were excluded (missing data, nintedanib treatment). The 2- and 5-yrs OS and forced vital capacity (FVC) and diffusing lung capacity for carbon monoxide (DLCO) were investigated at treatment initiation and 6, 12, 18 and 24 months’ follow-up. Results During a 2-yr follow-up, less than a quarter of the patients progressed on pirfenidone as assessed by the decline of ≥10% FVC (17.0%) and ≥ 15% DLCO (14.3%). On pirfenidone, the DLCO (≥10%) declines at 6, 12, 18 and 24 months’ and DLCO (≥15%) declines at 6, 18 and 24 months’ follow-up were associated with increased mortality. The DLCO decline showed higher predictive value for mortality than FVC decline. In patients with no-antifibrotics, FVC and DLCO declines were not predictive for mortality. Pirfenidone increased 5-yrs OS over no-antifibrotic treatment (55.9% vs 31.5% alive, P = 0.002). Conclusion Our study observed the 2-yrs sustained effect of pirfenidone on the decline of lung function and survival in the real-world patient’s IPF cohort. DLCO decline of ≥10% shows a potential as a mortality predictor in IPF patients on pirfenidone, and should be routinely evaluated during follow-up examinations.

RationaleDifferences in the lung microbial community influence idiopathic pulmonary fibrosis (IPF) progression. Whether the lung microbiome influences IPF host defense remains unknown.ObjectivesTo explore the host immune response and microbial interaction in IPF as they relate to progression-free survival (PFS), fibroblast function, and leukocyte phenotypes.MethodsPaired microarray gene expression data derived from peripheral blood mononuclear cells as well as 16S ribosomal RNA sequencing data from bronchoalveolar lavage obtained as part of the COMET-IPF (Correlating Outcomes with Biochemical Markers to Estimate Time-Progression in Idiopathic Pulmonary Fibrosis) study were used to conduct association pathway analyses. The responsiveness of paired lung fibroblasts to Toll-like receptor 9 (TLR9) stimulation by CpG-oligodeoxynucleotide (CpG-ODN) was integrated into microbiome-gene expression association analyses for a subset of individuals. The relationship between associated pathways and circulating leukocyte phenotypes was explored by flow cytometry.Measurements and main resultsDown-regulation of immune response pathways, including nucleotide-binding oligomerization domain (NOD)-, Toll-, and RIG1-like receptor pathways, was associated with worse PFS. Ten of the 11 PFS-associated pathways correlated with microbial diversity and individual genus, with species accumulation curve richness as a hub. Higher species accumulation curve richness was significantly associated with inhibition of NODs and TLRs, whereas increased abundance of Streptococcus correlated with increased NOD-like receptor signaling. In a network analysis, expression of up-regulated signaling pathways was strongly associated with decreased abundance of operational taxonomic unit 1341 (OTU1341; Prevotella) among individuals with fibroblasts responsive to CpG-ODN stimulation. The expression of TLR signaling pathways was also linked to CpG-ODN responsive fibroblasts, OTU1341 (Prevotella), and Shannon index of microbial diversity in a network analysis. Lymphocytes expressing C-X-C chemokine receptor 3 CD8 significantly correlated with OTU1348 (Staphylococcus).ConclusionsThese findings suggest that host-microbiome interactions influence PFS and fibroblast responsiveness.

Abstract Background Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) play important roles in the turnover of extracellular matrix and in the pathogenesis of idiopathic pulmonary fibrosis (IPF). This study aimed to determine the utility of circulating MMPs and TIMPs in distinguishing patients with IPF from controls and to explore associations between MMPs/TIMPs and measures of disease severity in patients with IPF. Methods The IPF cohort (n = 300) came from the IPF-PRO Registry, an observational multicenter registry of patients with IPF that was diagnosed or confirmed at the enrolling center in the past 6 months. Controls (n = 100) without known lung disease came from a population-based registry. Generalized linear models were used to compare circulating concentrations of MMPs 1, 2, 3, 7, 8, 9, 12, and 13 and TIMPs 1, 2, and 4 between patients with IPF and controls, and to investigate associations between circulating levels of these proteins and measures of IPF severity. Multivariable models were fit to identify the MMP/TIMPs that best distinguished patients with IPF from controls. Results All the MMP/TIMPs analyzed were present at significantly higher levels in patients with IPF compared with controls except for TIMP2. Multivariable analyses selected MMP8, MMP9 and TIMP1 as top candidates for distinguishing patients with IPF from controls. Higher concentrations of MMP7, MMP12, MMP13 and TIMP4 were significantly associated with lower diffusion capacity of the lung for carbon monoxide (DLCO) % predicted and higher composite physiologic index (worse disease). MMP9 was associated with the composite physiologic index. No MMP/TIMPs were associated with forced vital capacity % predicted. Conclusions Circulating MMPs and TIMPs were broadly elevated among patients with IPF. Select MMP/TIMPs strongly associated with measures of disease severity. Our results identify potential MMP/TIMP targets for further development as disease-related biomarkers.

Abstract Background Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease for which diagnosis and management remain challenging. Defining the circulating proteome in IPF may identify targets for biomarker development. We sought to quantify the circulating proteome in IPF, determine differential protein expression between subjects with IPF and controls, and examine relationships between protein expression and markers of disease severity. Methods This study involved 300 patients with IPF from the IPF-PRO Registry and 100 participants without known lung disease. Plasma collected at enrolment was analysed using aptamer-based proteomics (1305 proteins). Linear regression was used to determine differential protein expression between participants with IPF and controls and associations between protein expression and disease severity measures (percent predicted values for forced vital capacity [FVC] and diffusion capacity of the lung for carbon monoxide [DLco]; composite physiologic index [CPI]). Multivariable models were fit to select proteins that best distinguished IPF from controls. Results Five hundred fifty one proteins had significantly different levels between IPF and controls, of which 47 showed a |log2(fold-change)| > 0.585 (i.e. > 1.5-fold difference). Among the proteins with the greatest difference in levels in patients with IPF versus controls were the glycoproteins thrombospondin 1 and von Willebrand factor and immune-related proteins C-C motif chemokine ligand 17 and bactericidal permeability-increasing protein. Multivariable classification modelling identified nine proteins that, when considered together, distinguished IPF versus control status with high accuracy (area under receiver operating curve = 0.99). Among participants with IPF, 14 proteins were significantly associated with FVC % predicted, 23 with DLco % predicted, 14 with CPI. Four proteins (roundabout homolog-2, spondin-1, polymeric immunoglobulin receptor, intercellular adhesion molecule 5) demonstrated the expected relationship across all three disease severity measures. When considered in pathways analyses, proteins associated with the presence or severity of IPF were enriched in pathways involved in platelet and haemostatic responses, vascular or platelet derived growth factor signalling, immune activation, and extracellular matrix organisation. Conclusions Patients with IPF have a distinct circulating proteome and can be distinguished using a nine-protein profile. Several proteins strongly associate with disease severity. The proteins identified may represent biomarker candidates and implicate pathways for further investigation. Trial registration ClinicalTrials.gov (NCT01915511).

Background: An association between idiopathic pulmonary fibrosis (IPF) and advancing age is suspected since IPF occurs primarily in patients over 60 years of age. Though, little is known about the disease in the elderly. The aim of this study was to characterize elderly IPF patients using data from the longitudinal, German-wide INSIGHTS-IPF registry.Methods: Patients were grouped into elderly (≥75 years) and nonelderly IPF (

Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition with poor survival times. We previously published a genome-wide meta-analysis of IPF risk across three studies with independent replication of associated variants in two additional studies. To maximise power and to generate more accurate effect size estimates, we performed a genome-wide meta-analysis across all five studies included in the previous IPF risk genome-wide association studies. We used the distribution of effect sizes across the five studies to assess the replicability of the results and identified five robust novel genetic association signals implicating mTOR (mammalian target of rapamycin) signalling, telomere maintenance and spindle assembly genes in IPF risk. [ABSTRACT FROM AUTHOR]

Background: Idiopathic pulmonary fibrosis (IPF) is a rare and serious condition that is associated with high health-care resource use. The goal of this study was to estimate hospital-related resource use and costs by using a national, prospective registry of patients who were diagnosed with IPF or who had their diagnosis confirmed at the enrolling center in the past 6 months in the United States.Methods: Participants enrolled between June 5, 2014, and April 12, 2016, in the ongoing Idiopathic Pulmonary Fibrosis Prospective Outcomes Registry were included (N = 300). Time to first hospitalization was analyzed by using Kaplan-Meier methods. Annualized costs were estimated for hospitalizations, ICU admissions, and ED visits.Results: At enrollment, most participants were male (75%), white (95%), commercially insured (64%), smokers (68%), had an FVC between 50% and 80% predicted (66%), and received antifibrotic drugs (55%). During the first 12 months of follow-up, participants averaged 0.11 ED visit, 0.42 hospitalization, 0.08 ICU admission, 2.18 hospital days, and 0.45 ICU day. Probability of hospitalization was 18% and 30% at 6 and 12 months, respectively, and was highest for those with FVC < 50% predicted/diffusing lung capacity for carbon monoxide < 30% predicted. Mean annual costs (95% CI) for ICU admission and inpatient care were $10,098 ($4,732-$16,662) and $13,975 ($8,482-$20,918), respectively, per patient.Conclusions: IPF is associated with a substantial economic burden incurred by patients requiring hospital care. Future research in IPF should focus on improving clinical outcomes while reducing cost of care in hospitals.Trial Registry: ClinicalTrials.gov; No.: NCT01915511; URL: www.clinicaltrials.gov. [ABSTRACT FROM AUTHOR]

Background: Performance benchmarks for the management of idiopathic pulmonary fibrosis (IPF) have not been established. We used data from the IPF-PRO Registry, an observational registry of patients with IPF managed at sites across the US, to examine associations between the characteristics of the enrolling sites and patient outcomes.Methods: An online survey was used to collect information on the resources, operations, and self-assessment practices of IPF-PRO Registry sites that enrolled ≥ 10 patients. Site variability in 1-year event rates of clinically relevant outcomes, including death, death or lung transplant, and hospitalization, was assessed. Models were adjusted for differences in patient case mix by adjusting for known predictors of each outcome. We assessed whether site-level heterogeneity existed for each patient-level outcome, and if so, we investigated potential drivers of the heterogeneity.Results: All 27 sites that enrolled ≥ 10 patients returned the questionnaire. Most sites were actively following > 100 patients with IPF (70.4%), had a lung transplant program (66.7%), and had a dedicated ILD nurse leader (77.8%). Substantial heterogeneity was observed in the event rates of clinically relevant outcomes across the sites. After controlling for patient case mix, there were no outcomes for which the site variance component was significantly different from 0, but the p-value for hospitalization was 0.052. Starting/completing an ILD-related quality improvement project in the previous 2 years was associated with a lower risk of hospitalization (HR 0.60 [95% CI 0.44, 0.82]; p = 0.001).Conclusions: Analyses of data from patients with IPF managed at sites across the US found no site-specific characteristics or practices that were significantly associated with clinically relevant outcomes after adjusting for patient case mix. Trial registration ClinicalTrials.gov, NCT01915511. Registered 5 August 2013, https://clinicaltrials.gov/ct2/show/NCT01915511. [ABSTRACT FROM AUTHOR]

Introduction: Chronic cough is a highly problematic symptom for patients with idiopathic pulmonary fibrosis (IPF); limited therapeutic options are available. We evaluated gefapixant, a P2X3 receptor antagonist, for the treatment of chronic cough in IPF. Methods: This randomized, double-blind, placebo-controlled, crossover study included subjects with IPF. Sequence A included gefapixant 50 mg BID (period 1; 14 days) followed by placebo (period 2; 14 days); sequence B had the opposite sequence of treatments. This regimen was specified in a protocol amendment that modified the original active treatment regimen of gefapixant 50 mg BID for 10 days and 150 mg BID for 4 days. Patients randomized to the original treatment regimen were excluded from efficacy analyses but included in safety assessments. The primary efficacy endpoint was change from baseline in awake cough frequency (coughs/hour) from periods 1 and 2 combined. Adverse events (AEs) were monitored throughout the study. Results: A total of 51 subjects were randomized, 44 of whom were randomized to treatment sequences evaluated in the primary efficacy analysis (i.e., 22 subjects in sequence A and 22 subjects in sequence B); seven subjects received the treatment assigned before the protocol amendment and were excluded from efficacy analyses. The change from baseline in awake cough frequency from periods 1 and 2 combined (mixed model for repeated measures analysis) did not demonstrate a significant reduction versus placebo in cough at day 14 (p = 0.90); in a post hoc analysis of log-transformed data p value for reduction versus placebo at day 14 was 0.07. The most common AEs were related to taste (dysgeusia and ageusia). Conclusions: Gefapixant was generally well tolerated but was not associated with a significant improvement in chronic cough in subjects with IPF as defined by the primary endpoint in this study. Trial Registration: NCT02502097. [ABSTRACT FROM AUTHOR]