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Early identification of idiopathic pulmonary fibrosis (IPF) patients with poor prognosis remains a significant challenge. We Background: hypothesized that gene expression profiles in peripheral blood mononuclear cells (PBMC) may serve as predictors for IPF progression. The testing and validation cohorts consisted of 45/16 and 22/10 IPF/healthy individuals, respectively. The average follow-up time Method: was 18 months in testing and 36 months in validation cohort. Total RNA isolated from PBMC were hybridized on Affymetrix Exon 1.0ST array. Gene expression levels were summarized from exon intensity using dChip software. The R package WGCNA was used to parse genes in the array into 7 co-expressed modules designated with different colors. The module property was determined as the first component of principle component analysis (PCA) and used to calculate the association with individual clinical traits. Death after IPF diagnosis Result: was significantly correlated with the property of green-module containing 156 genes and anti-correlated with the property of turquoise-module containing 1252 genes. The turquoise-module property was also simultaneously correlated with the forced vital capacity (FVC) and diffusion capacity of lung (DLCO) and anti-correlated with composite progression index (CPI). Gene Ontology analysis revealed enrichment of green-module genes in defense response to bacterium and xenobiotic metabolic process (adjusted p-value 9.2x10-9 and 2.4x10-4), and enrichment of turquoise-module genes in DNA repair, mitosis and cell division (adjusted p-value 0.005, 0.01 and 0.0016, respectively). In the validation dataset, Receiver-Operating-Characteristic analysis for the prediction of death revealed area-under-curve (AUC) of 0.75 with green-module and 0.82 with turquoise-module. Moreover, turquoise-module demonstrated AUC of 0.81 for the diagnosis of IPF stability prior to PBMC sampling. The sample scatter plot by PCA accurately distinguished all deceased IPF patients from healthy individuals, with living IPF scattered across both groups. Two gene co-expression modules correlated Conclusion: with pulmonary function and prognosis of IPF were identified and validated in two large cohorts. Simultaneous correlation of multiple clinical traits with gene modules can greatly facilitate the elucidation of the relationships between individual clinical parameters along with the underpinning molecular signaling pathways. These findings indicate that genomic strategy may be a powerful tool in the prediction and mechanism exploration of the progression of chronic lung diseases.

Background: Idiopathic Pulmonary Fibrosis (IPF) is a chronic and fatal lung disease with lung transplant as the only available therapy. The course of IPF is variable and unpredictable. In this study we aimed to identify and validate a PBMC gene expression signature indicative of outcome in IPF. Methods: Microarray analysis of PBMC from patients with IPF was performed in two independent cohorts and two different microarray technologies: a derivation cohort of 75 patients at the University of Pittsburgh and a replication cohort of 45 patients at the University of Chicago. Mortality was the major outcome studied. After blood draw and PBMC isolation, RNA was extracted, labeled and hybridized to human gene expression microarrays (Agilent – Pittsburgh, Affymetrix – Chicago). Data was analyzed using BRB Array Tools. A Cox regression model was used to evaluate individual gene association with survival followed by Hierarchical clustering of these genes. Survival gene set analysis was performed to identify pathways associated with survival in both cohorts. For PCR validation of prognostic genes we designed a multi-sample high-throughput qRT-PCR SmarChip (Wafergen) that allowed us to measure their transcripts in the combined cohort of 139 patients. PCR results were adjusted to age, sex and FVC. Results: 2595 genes were associated with survival in the derivation cohort (p

Background Idiopathic pulmonary fibrosis (IPF) is a progressive and medically refractory lung disease with a grim prognosis. Although the etiology of IPF remains perplexing, abnormal adaptive immune responses are evident in many afflicted patients. We hypothesized that perturbations of human leukocyte antigen (HLA) allele frequencies, which are often seen among patients with immunologic diseases, may also be present in IPF patients. Methods/Principal Findings HLA alleles were determined in subpopulations of IPF and normal subjects using molecular typing methods. HLA-DRB1*15 was over-represented in a discovery cohort of 79 Caucasian IPF subjects who had lung transplantations at the University of Pittsburgh (36.7%) compared to normal reference populations. These findings were prospectively replicated in a validation cohort of 196 additional IPF subjects from four other U.S. medical centers that included both ambulatory patients and lung transplantation recipients. High-resolution typing was used to further define specific HLA-DRB1*15 alleles. DRB1*1501 prevalence in IPF subjects was similar among the 143 ambulatory patients and 132 transplant recipients (31.5% and 34.8%, respectively, p = 0.55). The aggregate prevalence of DRB1*1501 in IPF patients was significantly greater than among 285 healthy controls (33.1% vs. 20.0%, respectively, OR 2.0; 95%CI 1.3–2.9, p = 0.0004). IPF patients with DRB1*1501 (n = 91) tended to have decreased diffusing capacities for carbon monoxide (DLCO) compared to the 184 disease subjects who lacked this allele (37.8±1.7% vs. 42.8±1.4%, p = 0.036). Conclusions/Significance DRB1*1501 is more prevalent among IPF patients than normal subjects, and may be associated with greater impairment of gas exchange. These data are novel evidence that immunogenetic processes can play a role in the susceptibility to and/or manifestations of IPF. Findings here of a disease association at the HLA-DR locus have broad pathogenic implications, illustrate a specific chromosomal area for incremental, targeted genomic study, and may identify a distinct clinical phenotype among patients with this enigmatic, morbid lung disease.

Idiopathic pulmonary fibrosis (IPF) remains the most common of the idiopathic interstitial pneumonias and portends a poor prognosis. Significant strides have been made in the approach to diagnosis and in the ability to predict outcome in the last few years. Advances in high-resolution CT (HRCT) scanning have allowed an accurate diagnosis obviating the need for surgical biopsy in many patients. Furthermore, HRCT scanning may aid in determining prognosis and identifying disease progression. The appropriate use of the HRCT scan requires a multidisciplinary iterative approach incorporating all available data to reach a final diagnosis. However, there remains great heterogeneity in disease progression. Pulmonary hypertension and acute exacerbations of IPF negatively influence prognosis and are increasingly a target of therapy. There has been an increase in the number of well-designed clinical trials of IPF that have focused on more specific targets. While no cure has yet been found, each trial expands our understanding regarding the natural course of the disease and the impact of targeted therapy. In the interim, lung transplantation, which appears to improve survival in a subset of IPF patients, remains the only intervention. The objective of this article is to review advances in the understanding of IPF and the evidence for the findings outlined above.

The natural history of idiopathic pulmonary fibrosis (IPF) has been characterized as a steady, predictable decline in lung function over time. Recent evidence suggests that some patients may experience a more precipitous course, with periods of relative stability followed by acute deteriorations in respiratory status. Many of these acute deteriorations are of unknown etiology and have been termed acute exacerbations of IPF. This perspective is the result of an international effort to summarize the current state of knowledge regarding acute exacerbations of IPF. Acute exacerbations of IPF are defined as acute, clinically significant deteriorations of unidentifiable cause in patients with underlying IPF. Proposed diagnostic criteria include subjective worsening over 30 days or less, new bilateral radiographic opacities, and the absence of infection or another identifiable etiology. The potential pathobiological roles of infection, disordered cell biology, coagulation, and genetics are discussed, and future research directions are proposed.

Previous studies have suggested a decreased need for the surgical biopsy of intrathoracic lymph nodes (LNs) due to improved diagnostic rates utilizing transbronchial needle aspiration (TBNA) with endobronchial ultrasound and endoscopic ultrasound. The goal of this study was to determine whether conventional TBNA using combined cytologic and histologic analysis of tissue specimens impacted the rates of surgical diagnostic biopsies of patients with intrathoracic lymphadenopathy.Retrospective review at a single academic center. All mediastinal and hilar tissue samples submitted for pathologic analysis over an 8.4-year period were analyzed. Patients were categorized into a "before" group and an "after" group based on two different time periods. The before group underwent only cytologic analysis of Wang needle (19-gauge or 21-gauge) aspirates. The after group had cytologic analysis of aspirates as well as histologic analysis of needle "core" (19 gauge) biopsy specimens. The groups were compared for the rate of intrathoracic LNs sampled by surgical means vs TBNA and the number of times that TBNA averted the need for a surgical diagnostic procedure.The success of TBNA increased significantly in the after group compared to that in the before group. The yield for the successful sampling of mediastinal and hilar LNs increased from 53 to 91% (p0.001) in the before group vs the after group. TBNA averted a surgical biopsy in 35% of the before cases compared to 66% of the after cases (p0.001).Conventional TBNA using large-bore needles with both cytology and surgical pathology evaluation decreases the need for surgical sampling of the mediastinum to diagnose thoracic lymphadenopathy.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease causing irreversible lung scarring and loss of pulmonary function. RNA analysis has shown that patients with IPF have reduced ICOS expression in peripheral blood mononuclear cells. We now show that ICOS surface expression on CD4 T cells positively correlates with lung function in patients with IPF. However, whether decreased ICOS is a result of disease progression or playing a role in the pathogenesis is unclear. Therefore, using bleomycin to induce pulmonary fibrosis in mice, we found that ICOS was decreased in peripheral CD4 T cells similar to our findings in patients. In contrast, ICOS was increased on lung CD4 T cells by 3d post-bleomycin and remained elevated through 21d at the height of collagen deposition. Compared to wild-type B6 mice, ICOS-deficient mice had increased weight loss and mortality after bleomycin challenge, suggesting a protective role for ICOS. Strikingly, ILC2s expanded in the lung at 3d post-bleomycin in B6 mice, but not in ICOS-deficient mice. In B6 mice, ILC2 expansion was associated with CD4 T cell infiltration at 5d and Th2 cytokine production in the draining lymph nodes at 21d. However, ICOS-deficient mice that survived the initial bleomycin insult failed to recruit T cells to the lungs and had less IL-13 in the draining lymph nodes. Overall, these results indicate that ICOS expression promotes lung tissue repair by promoting both innate and adaptive type-2 immune responses.

Summary Churg-Strauss syndrome is a rare diffuse vasculitis that is almost invariably accompanied by severe asthma. Although overall prognosis is good, and treatment with prednisone alone or in combination with immunosuppressive drugs is usually successful, severe asthma typically persists. Diffuse organ involvement of Churg-Strauss syndrome, especially cardiovascular and rare involvement of the CNS and renal system, suggests a poorer prognosis than usual, and can be fatal. The cause of Churg-Strauss syndrome is unknown, but its characteristic histological findings and association with asthma distinguish it from other vasculitides. Controversy surrounds the use of asthma drugs—especially antileukotrienes—and development of the disorder. We review the epidemiological evidence for an association of drug treatment with Churg-Strauss syndrome, the diverse diagnostic and pathological criteria for this syndrome, and treatment options.

From the Authors: We thank Drs. Bendstrup and Hilberg for their comments regarding the IPFnet ACE trial examining the use of warfarin in idiopathic pulmonary fibrosis (IPF), and agree with their premise for the need for further investigation of anticoagulant therapy in IPF (1). We agree that the ACE study addresses the use of only warfarin in IPF. Importantly, there are several differences between ACE and the previous study examining the utility of anticoagulation in IPF by Kubo and coworkers (2). In the prior study, much less physiologically impaired patients with IPF were enrolled at hospitalization and placed on warfarin and prednisolone in combination, as opposed to warfarin alone. In fact, the number of subjects on both agents in ACE was very small. Furthermore, dalteparin, a low-molecular-weight heparin, was added during subsequent hospitalizations for acute exacerbation events. By allowing sicker patients in ACE, we both enhanced event rates, which was the objective, but may not have allowed sufficient time for a treatment effect to occur in altering the course of fibrosis. There is a plethora of evidence demonstrating antifibrotic effects of anticoagulants, both dependent and independent of their anticoagulant actions (3). Factor VII activity has been demonstrated to be elevated in patients with interstitial lung disease, and specifically in IPF (3). Also recently demonstrated, factor VIIa stimulates proliferation of human lung fibroblasts and extracellular matrix in a PAR-2–dependent manner. This effect is factor Xa and thrombin independent (4). Additionally shown after ACE’s inception is that increased local expression of coagulation factor X contributes to the fibrotic response in human and murine lung injury (5). Furthermore, evidence supports a protective role for protein C, a vitamin K–dependent anticoagulant protein, on the endothelial cell barrier function in acute lung injury (6). This growing body of evidence supports the concept that anticoagulants targeting specific coagulation factors or their “fibrosis-signaling” receptors may exert antifibrotic effects independent of their anticoagulant activities. Heparin shares anticoagulant action with warfarin, but its coagulation factor targets only partially overlap with that of warfarin. Furthermore, different heparin size fractions, and biochemical modifications, differ in their potencies, anticoagulant targets (such as thrombin or Factor X), and in their antiinflammatory and antiproliferative capacities (7–9). There is much evidence in vitro and in vivo to suggest that all anticoagulants are not equal with respect to their antifibrotic therapeutic potential. Thus, we agree that the compelling basic science and animal model observations would provide a solid foundation upon which to implement additional trials of “anti-coagulants” in IPF. In conclusion, we have answered the question of the utility of warfarin in progressive IPF, but many questions remain.

Rationale: Sarcoidosis is a granulomatous disease of unknown etiology, although M. tuberculosis may play a role in the pathogenesis. The traditional view holds that inflammation in sarcoidosis is compartmentalized to involved organs.

In this study, a single-nucleotide polymorphism in the mucus-forming gene MUC5B was strongly associated with the presence of idiopathic pulmonary fibrosis (IPF). These results replicate the findings of other investigators, also published in this issue.

The role of the adaptive immune response in idiopathic pulmonary fibrosis (IPF) has been an area of debate. Several studies have shown that patients with IPF had increased numbers of T cells in their lungs, suggesting that these cells may play a role in disease pathogenesis. We have found that decreased expression of an important co-inhibitory molecule, B- and T- lymphocyte attenuator (BTLA) on peripheral blood CD4+ T cells is correlated with IPF patients most likely to rapidly progress in their disease course. Based on these findings, the objective of our study was to determine the role of BTLA on CD4+ T cells in the development of pulmonary fibrosis by utilizing a mouse model of bleomycin-induced pulmonary fibrosis. We found that, similar to our patient studies, bleomycin treated WT mice express decreased levels of BTLA on CD4+ T cells compared to untreated WT mice. BTLA-/- mice exhibited significantly increased weight loss and pulmonary fibrosis compared to WT mice. However, CD4+ depletion in the BTLA-/- mice prior to bleomycin challenge protected these mice, as they lost less weight had decreased lung collagen deposition. These findings support a significant role for the loss of BTLA on CD4+ T cells in the pathogenesis of fibrotic disease progression.

Main Outcomes and Measures The primary end point was all-cause mortality. Results ThenumbersofpatientsintheGG,GT,andTTgenotypegroupswere148(34%), 259 (59%), and 31 (7%), respectively, in the INSPIRE cohort and 41 (28%), 98 (66%), and9(6%),respectively,intheChicagocohort.Themedianfollow-upperiodwas1.6years for INSPIRE and 2.1 years for Chicago. During follow-up, there were 73 deaths (36 GG, 35GT,and2TT)amongINSPIREpatientsand64deaths(26GG,36GT,and2TT)among Chicagopatients.Theunadjusted2-yearcumulativeincidenceofdeathwasloweramong patientscarrying1ormorecopiesoftheIPFriskallele(T)inboththeINSPIREcohort(0.25 [95% CI, 0.17-0.32] for GG, 0.17 [95% CI, 0.11-0.23] for GT, and 0.03 [95% CI, 0.000.09] for TT) and the Chicago cohort (0.50 [95% CI, 0.31-0.63] for GG, 0.22 [95% CI, 0.13-0.31] for GT, and 0.11 [95% CI, 0.00-0.28] for TT). In the INSPIRE cohort, the TT andGTgenotypes(riskforIPF)wereassociatedwithimprovedsurvivalcomparedwithGG (hazardratios,0.23[95%CI,0.10-0.52]and0.48[95%CI,0.31-0.72],respectively;P.001). ThisfindingwasreplicatedintheChicagocohort(hazardratios,0.15[95%CI,0.05-0.49] and0.39[95%CI,0.21-0.70],respectively;P.002).TheobservedassociationofMUC5B withsurvivalwasindependentofage,sex,forcedvitalcapacity,diffusingcapacityofcarbon monoxide, MMP-7, and treatment status. The addition of the MUC5B genotype to the survivalmodelssignificantlyimprovedthepredictiveaccuracyofthemodelinboththeINSPIRE cohort (C=0.71 [95% CI, 0.64-0.75] vs C=0.68 [95% CI, 0.61-0.73]; P.001) and the Chicago cohort (C=0.73 [95% CI, 0.62-0.78] vs C=0.69 [95% CI, 0.59-0.75]; P=.01). ConclusionsandRelevance AmongpatientswithIPF,acommonriskpolymorphism in MUC5B was significantly associated with improved survival. Further research is necessarytorefinetheriskestimatesandtodeterminetheclinicalimplicationsofthesefindings.

Sarcoidosis, a systemic granulomatous syndrome invariably affecting the lung, typically spontaneously remits but in ~20% of cases progresses with severe lung dysfunction or cardiac and neurologic involvement (complicated sarcoidosis). Unfortunately, current biomarkers fail to distinguish patients with remitting (uncomplicated) sarcoidosis from other fibrotic lung disorders, and fail to identify individuals at risk for complicated sarcoidosis. We utilized genome-wide peripheral blood gene expression analysis to identify a 20-gene sarcoidosis biomarker signature distinguishing sarcoidosis (n = 39) from healthy controls (n = 35, 86% classification accuracy) and which served as a molecular signature for complicated sarcoidosis (n = 17). As aberrancies in T cell receptor (TCR) signaling, JAK-STAT (JS) signaling, and cytokine-cytokine receptor (CCR) signaling are implicated in sarcoidosis pathogenesis, a 31-gene signature comprised of T cell signaling pathway genes associated with sarcoidosis (TCR/JS/CCR) was compared to the unbiased 20-gene biomarker signature but proved inferior in prediction accuracy in distinguishing complicated from uncomplicated sarcoidosis. Additional validation strategies included significant association of single nucleotide polymorphisms (SNPs) in signature genes with sarcoidosis susceptibility and severity (unbiased signature genes - CX3CR1, FKBP1A, NOG, RBM12B, SENS3, TSHZ2; T cell/JAK-STAT pathway genes such as AKT3, CBLB, DLG1, IFNG, IL2RA, IL7R, ITK, JUN, MALT1, NFATC2, PLCG1, SPRED1). In summary, this validated peripheral blood molecular gene signature appears to be a valuable biomarker in identifying cases with sarcoidoisis and predicting risk for complicated sarcoidosis.

IPF is an interstitial lung disease that affects over 128,000 people in the U.S., has a yearly mortality of 48,000, and has no effective treatment. Current theories on the etiology of IPF have not focused on the role of the immune system due to the inefficacy of steroid therapy. However, we have found in microarray studies that peripheral blood T cell co-stimulatory markers CD28, ICOS and BTLA, are associated and significantly predictive of early mortality in IPF patients. A significant association was found between decreased BTLA mRNA levels and decreased predicted diffusing capacity of carbon monoxide (DLCO) levels, a clinical measure of disease severity. Analysis of all samples demonstrated a significant and dramatically decreased BTLA surface expression on both peripheral CD4 and CD8 T cells, but not B cells, in rapidly progressing patients compared to controls or stable patients. Similar, but less dramatic decrease in surface expression is found for CD28 and ICOS. In a bleomycin-induced pulmonary fibrosis model, BTLA-/- mice, and to a lesser extent ICOS-/- mice, show increased numbers of lung CD4+ T cells and macrophages, significantly decreased weight, and more severe lung fibrosis when compared to controls. These results support the role of altered co-stimulatory molecules in the progression of IPF implying that these molecules are involved in the pathogenesis of disease progression.