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1.  MMP1 and MMP7 as Potential Peripheral Blood Biomarkers in Idiopathic Pulmonary Fibrosis 
PLoS Medicine  2008;5(4):e93.
Background
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease associated with substantial morbidity and mortality. The objective of this study was to determine whether there is a peripheral blood protein signature in IPF and whether components of this signature may serve as biomarkers for disease presence and progression.
Methods and Findings
We analyzed the concentrations of 49 proteins in the plasma of 74 patients with IPF and in the plasma of 53 control individuals. We identified a combinatorial signature of five proteins—MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A—that was sufficient to distinguish patients from controls with a sensitivity of 98.6% (95% confidence interval [CI] 92.7%–100%) and specificity of 98.1% (95% CI 89.9%–100%). Increases in MMP1 and MMP7 were also observed in lung tissue and bronchoalveolar lavage fluid obtained from IPF patients. MMP7 and MMP1 plasma concentrations were not increased in patients with chronic obstructive pulmonary disease or sarcoidosis and distinguished IPF compared to subacute/chronic hypersensitivity pneumonitis, a disease that may mimic IPF, with a sensitivity of 96.3% (95% CI 81.0%–100%) and specificity of 87.2% (95% CI 72.6%–95.7%). We verified our results in an independent validation cohort composed of patients with IPF, familial pulmonary fibrosis, subclinical interstitial lung disease (ILD), as well as with control individuals. MMP7 and MMP1 concentrations were significantly higher in IPF patients compared to controls in this cohort. Furthermore, MMP7 concentrations were elevated in patients with subclinical ILD and negatively correlated with percent predicted forced vital capacity (FVC%) and percent predicted carbon monoxide diffusing capacity (DLCO%).
Conclusions
Our experiments provide the first evidence for a peripheral blood protein signature in IPF to our knowledge. The two main components of this signature, MMP7 and MMP1, are overexpressed in the lung microenvironment and distinguish IPF from other chronic lung diseases. Additionally, increased MMP7 concentration may be indicative of asymptomatic ILD and reflect disease progression.
Naftali Kaminski and colleagues find increased levels of specific proteins in the bloodstream of individuals with idiopathic pulmonary fibrosis, and suggest that these proteins may ultimately provide a biomarker for the disease.
Editors' Summary
Background.
Idiopathic pulmonary fibrosis (IPF) is a serious disease in which the lungs become progressively scarred or thickened for unknown reasons. In healthy people, air is taken in through the mouth or nose and travels down the windpipe into tubes in the lungs called the airways. Each airway has many small branches that end in alveoli, tiny air sacs with thin walls that are surrounded by small blood vessels called capillaries. When air reaches the alveoli, the oxygen in it passes into the bloodstream and is taken to the organs of the body to keep them working. In IPF, the alveoli and the space around them (the “interstitial” area) gradually become scarred and thickened, which stops oxygen's movement into the bloodstream. When only small areas of the lung are scarred, IPF may cause no symptoms. But, as more of the lung becomes damaged, IPF eventually causes breathlessness, even when resting. There is no effective treatment for IPF, although steroids and drugs that suppress the body's immune system are often tried in an attempt to slow its progression. On average, half of the people with IPF die within three years of diagnosis, often from respiratory or heart failure.
Why Was This Study Done?
It can be difficult to diagnose IPF—there are many lung diseases with similar symptoms, including numerous other interstitial lung diseases—and currently, physicians can only follow the progression of IPF by repeatedly testing their patients' lung function or by doing multiple chest X-rays. If proteins could be identified whose level in blood indicated disease activity (so-called “peripheral blood biomarkers”), it would be easier to diagnose and monitor patients. In addition, the identification of such biomarkers might suggest new drug targets for the treatment of IPF. In this study, the researchers look for peripheral blood biomarkers in IPF by using a “multiplex analysis” system to measure the level of several proteins in patient blood samples simultaneously.
What Did the Researchers Do and Find?
The researchers measured the levels of 49 plasma proteins (plasma is the fluid part of blood) in 74 patients with IPF and 53 healthy people (controls) and used a technique called “recursive partitioning” to define a five-protein signature that distinguished patients from unaffected study participants (controls). Matrix metalloproteinase 7 (MMP7) and MMP1—the two plasma proteins whose levels were most increased in patients with IPF compared to controls—were key components of this signature. Concentrations of MMP7 and MMP1 were higher in bronchoalveolar lavage samples (fluid obtained by washing out the lungs with saline) and in lung tissue samples from patients with IPF than in similar samples taken from healthy individuals. Plasma concentrations of MMP7 and MMP1 were significantly higher in patients with IPF than in patients with hypersensitivity pneumonitis, an interstitial lung disease that mimics IPF, but not increased in patients with chronic obstructive pulmonary disease or sarcoidosis, two other lung diseases. In an independent validation group, patients with IPF and familial pulmonary fibrosis had increased plasma concentrations of MMP7 and MMP1 that correlated with the severity of their disease. In addition, MMP7 concentrations were raised in close relatives of people with familial pulmonary fibrosis who had normal lung function tests but some lung scarring.
What Do These Findings Mean?
These findings provide evidence for a protein signature in the blood for IPF and suggest MMP1 and MMP7 may be useful as biomarkers for IPF. These two matrix metalloproteinases have previously been suggested to be involved in the development of IPF. However, additional work is probably needed to confirm that increased plasma concentrations MMP7 and MMP1 are specific for IPF, since it may be that these markers will not distinguish IPF from other interstitial lung diseases.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050093.
Read a related PLoS Medicine Perspective article
The MedlinePlus Encyclopedia has a page on idiopathic pulmonary fibrosis (in English and Spanish) and on pulmonary fibrosis
The US National Heart Lung and Blood Institute and the British Lung Foundation also provide information on IPF for patients and relatives
Some of the researchers involved in this study provide more details about what might go wrong in IPF in a recent PLoS Medicine article
doi:10.1371/journal.pmed.0050093
PMCID: PMC2346504  PMID: 18447576
2.  High Resolution Multi-Detector CT Aided Tissue Analysis and Quantification of Lung Fibrosis 
Academic radiology  2007;14(7):772-787.
Rational and Objectives
Volumetric high-resolution scans can be acquired of the lungs with multi-detector CT (MDCT). Such scans have potential to facilitate useful visualization, characterization, and quantification of the extent of diffuse lung diseases, such as Usual Interstitial Pneumonitis or Idiopathic Pulmonary Fibrosis (UIP/IPF). There is a need to objectify, standardize and improve the accuracy and repeatability of pulmonary disease characterization and quantification from such scans. This paper presents a novel texture analysis approach toward classification and quantification of various pathologies present in lungs with UIP/IPF. The approach integrates a texture matching method with histogram feature analysis.
Materials and Methods
Patients with moderate UIP/IPF were scanned on a Lightspeed 8-detector GE CT scanner (140kVp, 250mAs). Images were reconstructed with 1.25mm slice thickness in a high-frequency sparing algorithm (BONE) with 50% overlap and a 512 × 512 axial matrix, (0.625 mm3 voxels). Eighteen scans were used in this study. Each dataset is pre-processed which includes segmentation of the lungs and the broncho-vascular trees. Two types of analysis were performed, first an analysis of independent volume of interests (VOIs) and second an analysis of whole lung datasets.
1.) Fourteen of the eighteen scans were used to create a database of independent 15×15×15 cubic voxel VOIs. The VOIs were selected by experts as having greater than 70% of the defined class. The database was composed of the following: Honeycombing (# of VOIs 337), Reticular (130), Ground glass (148), Normal (240), and Emphysema (54). This database was used to develop our algorithm. Three progressively challenging classification experiments were designed to test our algorithm. All three experiments were performed using a 10-fold cross validation method for error estimation. Experiment 1 consisted of a two class discrimination: Normal and Abnormal. Experiment 2 consisted of a four class discrimination: Normal, Reticular, Honeycombing, and Emphysema. Experiment 3 consisted of a five class discrimination: Normal, Ground glass, Reticular, Honeycombing, and Emphysema.
2.) The remaining four scans were used to further test the algorithm on new data in the context of a whole lung analysis. Each of the four datasets was manually segmented by three experts. These datasets included Normal, Reticular and Honeycombing regions and did not include Ground glass or Emphysema. The accuracy of the classification algorithm was then compared with results from experts.
Results
Independent VOIs: 1.) Two class discrimination problem (sensitivity, specificity): Normal versus Abnormal (92.96%,93.78%). 2.) Four class discrimination problem: Normal (92%,95%), Reticular (86%,87%), Honeycombing (74%,98%), and Emphysema (93%,98%). 3.) Five class discrimination problem: Normal(92%,95%), Ground glass (75%,89%), Reticular (22%,92%), Honeycombing (74%,91%), and Emphysema (94%,98%).
Whole lung datasets: 1.) William's Index shows that algorithm classification of lungs agrees with the experts as well as the experts agree with themselves. 2.) Student-T test between overlap measures of algorithm and expert (AE) and expert and expert (EE) : Normal (t=-1.20, p = 0.230), Reticular (t=-1.44, p = 0.155), Honeycombing (t=-3.15, p = 0.003). 3.) Lung Volumes Intra-class correlation: Dataset 1 (ICC = 0.9984, F = 0.0007); Dataset 2 (ICC = 0.9559, F = 0); Dataset 3 (ICC = 0.8623, F= 0.0015); Dataset 4 (ICC = 0.7807, F = 0.0136).
Conclusions
We have demonstrated that our novel method is computationally efficient and produces results comparable to expert radiologic judgment. It is effective in the classification of normal versus abnormal tissue and performs as well as the experts in distinguishing among typical pathologies present in lungs with UIP/IPF. The continuing development of quantitative metrics will improve quantification of disease and provide objective measures of disease progression.
doi:10.1016/j.acra.2007.03.009
PMCID: PMC2701291  PMID: 17574128
Multi-Detector CT; Lung imaging; Tissue Classification; Quantitative Lung Analysis; Texture Analysis
3.  Acute exacerbations in patients with idiopathic pulmonary fibrosis 
Respiratory Research  2013;14(1):86.
Idiopathic pulmonary fibrosis (IPF) is a chronic, fibrosing interstitial lung disease that primarily affects older adults. Median survival after diagnosis is 2–3 years. The clinical course of IPF may include periods of acute deterioration in respiratory function, which are termed acute exacerbations of IPF (AEx-IPF) when a cause cannot be identified. AEx-IPF may represent a sudden acceleration of the underlying disease process of IPF, or a biologically distinct pathological process that is clinically undiagnosed. An AEx-IPF can occur at any time during the course of IPF and may be the presenting manifestation. The incidence of AEx-IPF is hard to establish due to variation in the methodology used to assess AEx-IPF in different studies, but AEx-IPF are believed to occur in between 5 and 10% of patients with IPF every year. Risk factors for AEx-IPF are unclear, but there is evidence that poorer lung function increases the risk of an AEx-IPF and reduces the chances of a patient surviving an AEx-IPF. The presence of comorbidities such as gastroesophageal reflux disease (GERD) and pulmonary hypertension may also increase the risk of an AEx-IPF. AEx-IPF are associated with high morbidity and mortality. Patients who experience an AEx-IPF show a worsened prognosis and AEx-IPF are believed to reflect disease progression in IPF. Current treatments for AEx-IPF have only limited data to support their effectiveness. The latest international treatment guidelines state that supportive care remains the mainstay of treatment for AEx-IPF, but also give a weak recommendation for the treatment of the majority of patients with AEx-IPF with corticosteroids. There is emerging evidence from clinical trials of investigational therapies that chronic treatment of IPF may reduce the incidence of AEx-IPF. Additional clinical trials investigating this are underway.
doi:10.1186/1465-9921-14-86
PMCID: PMC3765544  PMID: 23964926
Acute exacerbation; Idiopathic pulmonary fibrosis (IPF); Impact; Management; Prevention; Treatment
4.  Idiopathic pulmonary fibrosis 
Idiopathic pulmonary fibrosis (IPF) is a non-neoplastic pulmonary disease that is characterized by the formation of scar tissue within the lungs in the absence of any known provocation. IPF is a rare disease which affects approximately 5 million persons worldwide. The prevalence is estimated to be slightly greater in men (20.2/100,000) than in women (13.2/100,000). The mean age at presentation is 66 years. IPF initially manifests with symptoms of exercise-induced breathless and dry coughing. Auscultation of the lungs reveals early inspiratory crackles, predominantly located in the lower posterior lung zones upon physical exam. Clubbing is found in approximately 50% of IPF patients. Cor pulmonale develops in association with end-stage disease. In that case, classic signs of right heart failure may be present. Etiology remains incompletely understood. Some environmental factors may be associated with IPF (cigarette smoking, exposure to silica and livestock). IPF is recognized on high-resolution computed tomography by peripheral, subpleural lower lobe reticular opacities in association with subpleural honeycomb changes. IPF is associated with a pathological lesion known as usual interstitial pneumonia (UIP). The UIP pattern consists of normal lung alternating with patches of dense fibrosis, taking the form of collagen sheets. The diagnosis of IPF requires correlation of the clinical setting with radiographic images and a lung biopsy. In the absence of lung biopsy, the diagnosis of IPF can be made by defined clinical criteria that were published in guidelines endorsed by several professional societies. Differential diagnosis includes other idiopathic interstitial pneumonia, connective tissue diseases (systemic sclerosis, polymyositis, rheumatoid arthritis), forme fruste of autoimmune disorders, chronic hypersensitivity pneumonitis and other environmental (sometimes occupational) exposures. IPF is typically progressive and leads to significant disability. The median survival is 2 to 5 years from the time of diagnosis. Medical therapy is ineffective in the treatment of IPF. New molecular therapeutic targets have been identified and several clinical trials are investigating the efficacy of novel medication. Meanwhile, pulmonary transplantation remains a viable option for patients with IPF. It is expected that, during the next decade, considerable progress will be made toward the understanding and treatment of this devastating illness.
doi:10.1186/1750-1172-3-8
PMCID: PMC2330030  PMID: 18366757
5.  Global Methylation Patterns in Idiopathic Pulmonary Fibrosis 
PLoS ONE  2012;7(4):e33770.
Background
Idiopathic Pulmonary Fibrosis (IPF) is characterized by profound changes in the lung phenotype including excessive extracellular matrix deposition, myofibroblast foci, alveolar epithelial cell hyperplasia and extensive remodeling. The role of epigenetic changes in determining the lung phenotype in IPF is unknown. In this study we determine whether IPF lungs exhibit an altered global methylation profile.
Methodology/Principal Findings
Immunoprecipitated methylated DNA from 12 IPF lungs, 10 lung adenocarcinomas and 10 normal histology lungs was hybridized to Agilent human CpG Islands Microarrays and data analysis was performed using BRB-Array Tools and DAVID Bioinformatics Resources software packages. Array results were validated using the EpiTYPER MassARRAY platform for 3 CpG islands. 625 CpG islands were differentially methylated between IPF and control lungs with an estimated False Discovery Rate less than 5%. The genes associated with the differentially methylated CpG islands are involved in regulation of apoptosis, morphogenesis and cellular biosynthetic processes. The expression of three genes (STK17B, STK3 and HIST1H2AH) with hypomethylated promoters was increased in IPF lungs. Comparison of IPF methylation patterns to lung cancer or control samples, revealed that IPF lungs display an intermediate methylation profile, partly similar to lung cancer and partly similar to control with 402 differentially methylated CpG islands overlapping between IPF and cancer. Despite their similarity to cancer, IPF lungs did not exhibit hypomethylation of long interspersed nuclear element 1 (LINE-1) retrotransposon while lung cancer samples did, suggesting that the global hypomethylation observed in cancer was not typical of IPF.
Conclusions/Significance
Our results provide evidence that epigenetic changes in IPF are widespread and potentially important. The partial similarity to cancer may signify similar pathogenetic mechanisms while the differences constitute IPF or cancer specific changes. Elucidating the role of these specific changes will potentially allow better understanding of the pathogenesis of IPF.
doi:10.1371/journal.pone.0033770
PMCID: PMC3323629  PMID: 22506007
6.  Lung mast cell density defines a subpopulation of patients with idiopathic pulmonary fibrosis 
Histopathology  2012;61(1):98-106.
Aims
The relationship of mast cells to the pathogenesis of lung fibrosis remains undefined despite recognition of their presence in the lungs of patients with pulmonary fibrosis. This study was performed to characterize the relationship of mast cells to fibrotic lung diseases.
Methods and results
Lung tissues from patients with idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (HP), systemic sclerosis (SSc)-related interstitial lung disease (ILD) and normal individuals were subjected to chymase immunostaining and the mast cell density quantified. Eosinophils were quantified by immunostaining for eosinophil peroxidase. Changes in lung function were correlated with mast cell density. Lung tissue obtained from IPF patients had a higher density of chymase-immunoreactive mast cells than that from patients with HP, SSc-related ILD or normal lungs. IPF lung tissue had a higher density of eosinophils than normal lung. There was no correlation between mast cell density and eosinophil density in IPF lung. IPF patients with high mast cell density had a slower rate of decline in forced vital capacity (FVC) than IPF patients with low mast cell density.
Conclusions
Mast cell density in IPF lungs is higher than in other fibrotic lung diseases and normal lungs. Increased mast cell density in IPF may predict slower disease progression.
doi:10.1111/j.1365-2559.2012.04197.x
PMCID: PMC3371307  PMID: 22394225
chloroacetate esterase; eosinophil; hypersensitivity pneumonitis; idiopathic interstitial pneumonia; systemic sclerosis
7.  Gene Expression Profiling as a Window into Idiopathic Pulmonary Fibrosis Pathogenesis 
Expression microarrays that provide genome-level, transcriptional, high-resolution profiles have been applied successfully to multiple diseases. Although microarrays provide information regarding thousands of genes, many investigators prefer to focus on a single gene and validate its role, an approach often supported by grant and journal reviewers. Only a minority of investigators focus on global changes in gene expression. Here, we describe and contrast two general approaches to the use of microarray data: the reductionist “cherry picking” approach and the more global, quantitative “systems” approach. We describe microarray analysis experiments relevant to idiopathic pulmonary fibrosis (IPF) in the context of these two approaches. Although it seems that the cherry-picking approaches have been successful in identifying new relevant genes in IPF, we suggest that to fulfill the discovery potential of microarrays in IPF and to create a working model of IPF, unbiased integrative systems approaches are required.
doi:10.1513/pats.200601-011TK
PMCID: PMC2658685  PMID: 16738198
FIZZ1; matrix metalloprotease; microarrays; osteopontin; systems biology
8.  Regulation of Transforming Growth Factor-β1–driven Lung Fibrosis by Galectin-3 
Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies.
Objectives: To examine the role of galectin-3 in pulmonary fibrosis.
Methods: We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF.
Measurements and Main Results: Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1–induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β–induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF.
Conclusions: This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.
doi:10.1164/rccm.201106-0965OC
PMCID: PMC3410728  PMID: 22095546
fibrosis; epithelial cells; fibroblasts
9.  Hepatitis C and pulmonary fibrosis 
Hepatitis Monthly  2011;11(2):71-73.
Background
Hepatitis C virus (HCV) is a hepatotropic and lymphotropic virus that causes hepatic and extrahepatic disease. Emerging clinical data suggest that chronic HCV infection can lead to many direct and indirect effects on the lung.
Objectives
This article discusses evidence on the relationship between HCV infection and pulmonary fibrosis to increase knowledge on this topic among clinicians and scientists and highlights the need for further study.
Methods
We searched the MEDLINE, ISI WEB OF KNOWLEDGE, OVID, ELSEVIER, and MDCONSULT databases and top respiratory journals, such as the American Journal of Respiratory and Critical Care, Chest, and Thorax for articles in English using the following keywords: hepatitis C, HCV infection, IPF, pulmonary fibrosis, and interstitial pneumonitis. We reviewed the reference lists of all identified studies.
Results
The evidence for a pathogenetic link between pulmonary fibrosis and HCV is: the higher frequency of HCV markers in IPF patients, an increase in lymphocyte and neutrophil numbers in bronchoalveolar lavage of chronic HCV infection patients, and the development of IPF in HCV-related chronic hepatitis that is treated with interferon. There is a discrepancy between studies on the frequency of HCV in IPF patients, which might be attributed to geographical differences of in the prevalence of HCV infection, selection bias in choosing the control group, and the HCV genome.
Conclusions
BAL studies in HCV infection are associated with increased counts of lymphocytes and neutrophils in BAL fluid. These studies show that HCV infection is associated with nonspecific pulmonary inflammatory reactions that are not compatible with IPF but that it can lead to pulmonary fibrosis. The other factor is interferon therapy. Interstitial pneumonia and sarcoidosis are well-documented complications of IFN therapy. More extensive cohort studies should be conducted to confirm an actual causal relationship between HCV infection and pulmonary fibrosis.
PMCID: PMC3206667  PMID: 22087122
Hepatitis C; Pulmonary fibrosis
10.  Aberrant innate immune sensing leads to the rapid progression of idiopathic pulmonary fibrosis 
Fibrogenesis & Tissue Repair  2012;5(Suppl 1):S3.
Novel approaches are needed to define subgroups of patients with Idiopathic pulmonary fibrosis (IPF) at risk for acute exacerbations and/or accelerated progression of this generally fatal disease. Progression of disease is an integral component of IPF with a median survival of 3 to 5 years. Conversely, a high degree of variability in disease progression has been reported among series. The characteristics of patients at risk of earlier death predominantly rely on baseline HRCT appearance, but this concept that has been challenged. Disparate physiological approaches have also been taken to identify patients at risk of mortality, with varying results. We hypothesized that the rapid decline in lung function in IPF may be a consequence of an abnormal host response to pathogen-associated molecular patterns (PAMPs), leading to aberrant activation in fibroblasts and fibrosis. Analysis of upper and lower lobe surgical lung biopsies (SLBs) indicated that TLR9, a hypomethylated CpG DNA receptor, is prominently expressed at the transcript and protein level, most notably in biopsies from rapidly progressive IPF patients. Surprisingly, fibroblasts appeared to be a major cellular source of TLR9 expression in IPF biopsies from this group of progressors. Further, CpG DNA promoted profibrotic cytokine and chemokine synthesis in isolated human IPF fibroblasts, most markedly again in cells from patients with the rapidly progressive IPF phenotype, in a TLR9-dependent manner. Finally, CpG DNA exacerbated fibrosis in an in vivo model initiated by the adoptive transfer of primary fibroblasts derived from patients who exhibited rapidly progressing fibrosis. Together, these data suggested that TLR9 activation via hypomethylated DNA might be an important mechanism in promoting fibrosis particularly in patients prone to rapidly progressing IPF.
doi:10.1186/1755-1536-5-S1-S3
PMCID: PMC3368762  PMID: 23259678
11.  Lung transplantation in idiopathic pulmonary fibrosis: a systematic review of the literature 
BMC Pulmonary Medicine  2014;14:139.
Background
Idiopathic pulmonary fibrosis (IPF) is a distinct form of interstitial pneumonia with unknown origin and poor prognosis. Current pharmacologic treatments are limited and lung transplantation is a viable option for appropriate patients. The aim of this review was to summarize lung transplantation survival in IPF patients overall, between single (SLT) vs. bilateral lung transplantation (BLT), pre- and post Lung Allocation Score (LAS), and summarize wait-list survival.
Methods
A systematic review of English-language studies published in Medline or Embase between 1990 and 2013 was performed. Eligible studies were those of observational design reporting survival post-lung transplantation or while on the wait list among IPF patients.
Results
Median survival post-transplantation among IPF patients is estimated at 4.5 years. From ISHLT and OPTN data, one year survival ranged from 75% - 81%; 3-year: 59% - 64%; and 5-year: 47% - 53%. Post-transplant survival is lower for IPF vs. other underlying pre-transplant diagnoses. The proportion of IPF patients receiving BLT has steadily increased over the last decade and a half. Unadjusted analyses suggest improved long-term survival for BLT vs. SLT; after adjustment for patient characteristics, the differences tend to disappear. IPF patients account for the largest proportion of patients on the wait list and while wait list time has decreased, the number of transplants for IPF patients has increased over time. OPTN data show that wait list mortality is higher for IPF patients vs. other diagnoses. The proportion of IPF patients who died while awaiting transplantation ranged from 14% to 67%. While later transplant year was associated with increased survival, no significant differences were noted pre vs. post LAS implementation; however a high LAS vs low LAS was associated with decreased one-year survival.
Conclusions
IPF accounts for the largest proportion of patients awaiting lung transplants, and IPF is associated with higher wait-list and post-transplant mortality vs. other diagnoses. Improved BLT vs. SLT survival may be the result of selection bias. Survival pre- vs. post LAS appears to be similar except for IPF patients with high LAS, who have lower survival compared to pre-LAS. Data on post-transplant morbidity outcomes are sparse.
doi:10.1186/1471-2466-14-139
PMCID: PMC4151866  PMID: 25127540
Idiopathic pulmonary fibrosis; Systematic review; Survival; Lung transplantation
12.  Patients with Idiopathic Pulmonary Fibrosis with Antibodies to Heat Shock Protein 70 Have Poor Prognoses 
Rationale: Diverse autoantibodies are present in most patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that specific autoantibodies may associate with IPF manifestations.
Objectives: To identify clinically relevant, antigen-specific immune responses in patients with IPF.
Methods: Autoantibodies were detected by immunoblots and ELISA. Intrapulmonary immune processes were evaluated by immunohistochemistry. Anti–heat shock protein 70 (HSP70) IgG was isolated from plasma by immunoaffinity. Flow cytometry was used for leukocyte functional studies.
Measurements and Main Results: HSP70 was identified as a potential IPF autoantigen in discovery assays. Anti-HSP70 IgG autoantibodies were detected by immunoblots in 3% of 60 control subjects versus 25% of a cross-sectional IPF cohort (n = 122) (P = 0.0004), one-half the patients with IPF who died (P = 0.008), and 70% of those with acute exacerbations (P = 0.0005). Anti-HSP70 autoantibodies in patients with IPF were significantly associated with HLA allele biases, greater subsequent FVC reductions (P = 0.0004), and lesser 1-year survival (40 ± 10% vs. 80 ± 5%; hazard ratio = 4.2; 95% confidence interval, 2.0–8.6; P < 0.0001). HSP70 protein, antigen–antibody complexes, and complement were prevalent in IPF lungs. HSP70 protein was an autoantigen for IPF CD4 T cells, inducing lymphocyte proliferation (P = 0.004) and IL-4 production (P = 0.01). IPF anti-HSP70 autoantibodies activated monocytes (P = 0.009) and increased monocyte IL-8 production (P = 0.049). ELISA confirmed the association between anti-HSP70 autoreactivity and IPF outcome. Anti-HSP70 autoantibodies were also found in patients with other interstitial lung diseases but were not associated with their clinical progression.
Conclusions: Patients with IPF with anti-HSP70 autoantibodies have more near-term lung function deterioration and mortality. These findings suggest antigen-specific immunoassays could provide useful clinical information in individual patients with IPF and may have implications for understanding IPF progression.
doi:10.1164/rccm.201203-0506OC
PMCID: PMC3678112  PMID: 23262513
B cells; T cells; adaptive immunity; interstitial lung disease
13.  Reactive Oxygen Species Are Required for Maintenance and Differentiation of Primary Lung Fibroblasts in Idiopathic Pulmonary Fibrosis 
PLoS ONE  2010;5(11):e14003.
Background
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal illness whose pathogenesis remains poorly understood. Recent evidence suggests oxidative stress as a key player in the establishment/progression of lung fibrosis in animal models and possibly in human IPF. The aim of the present study was to characterize the cellular phenotype of fibroblasts derived from IPF patients and identify underlying molecular mechanisms.
Methodology/Principal Findings
We first analyzed the baseline differentiation features and growth ability of primary lung fibroblasts derived from 7 histology proven IPF patients and 4 control subjects at different culture passages. Then, we focused on the redox state and related molecular pathways of IPF fibroblasts and investigated the impact of oxidative stress in the establishment of the IPF phenotype. IPF fibroblasts were differentiated into alpha-smooth muscle actin (SMA)-positive myofibroblasts, displayed a pro-fibrotic phenotype as expressing type-I collagen, and proliferated lower than controls cells. The IPF phenotype was inducible upon oxidative stress in control cells and was sensitive to ROS scavenging. IPF fibroblasts also contained large excess of reactive oxygen species (ROS) due to the activation of an NADPH oxidase-like system, displayed higher levels of tyrosine phosphorylated proteins and were more resistant to oxidative-stress induced cell death. Interestingly, the IPF traits disappeared with time in culture, indicating a transient effect of the initial trigger.
Conclusions/Significance
Robust expression of α-SMA and type-I collagen, high and uniformly-distributed ROS levels, resistance to oxidative-stress induced cell death and constitutive activation of tyrosine kinase(s) signalling are distinctive features of the IPF phenotype. We suggest that this phenotype can be used as a model to identify the initial trigger of IPF.
doi:10.1371/journal.pone.0014003
PMCID: PMC2982828  PMID: 21103368
14.  Immunohistochemical and morphometric evaluation of COX-1 and COX-2 in the remodeled lung in idiopathic pulmonary fibrosis and systemic sclerosis* ,** 
OBJECTIVE:
To study the expression of COX-1 and COX-2 in the remodeled lung in systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF) patients, correlating that expression with patient survival.
METHODS:
We examined open lung biopsy specimens from 24 SSc patients and 30 IPF patients, using normal lung tissue as a control. The histological patterns included fibrotic nonspecific interstitial pneumonia (NSIP) in SSc patients and usual interstitial pneumonia (UIP) in IPF patients. We used immunohistochemistry and histomorphometry to evaluate the expression of COX-1 and COX-2 in alveolar septa, vessels, and bronchioles. We then correlated that expression with pulmonary function test results and evaluated its impact on patient survival.
RESULTS:
The expression of COX-1 and COX-2 in alveolar septa was significantly higher in IPF-UIP and SSc-NSIP lung tissue than in the control tissue. No difference was found between IPF-UIP and SSc-NSIP tissue regarding COX-1 and COX-2 expression. Multivariate analysis based on the Cox regression model showed that the factors associated with a low risk of death were younger age, high DLCO/alveolar volume, IPF, and high COX-1 expression in alveolar septa, whereas those associated with a high risk of death were advanced age, low DLCO/alveolar volume, SSc (with NSIP), and low COX-1 expression in alveolar septa.
CONCLUSIONS:
Our findings suggest that strategies aimed at preventing low COX-1 synthesis will have a greater impact on SSc, whereas those aimed at preventing high COX-2 synthesis will have a greater impact on IPF. However, prospective randomized clinical trials are needed in order to confirm that.
doi:10.1590/S1806-37132013000600008
PMCID: PMC4075907  PMID: 24473763
Scleroderma, systemic; Idiopathic pulmonary fibrosis; Inflammation; Survival rate
15.  Regulation of the effects of TGF-β1 by activation of latent TGF-β1 and differential expression of TGF-β receptors (TβR-I and TβR-II) in idiopathic pulmonary fibrosis 
Thorax  2001;56(12):907-915.
BACKGROUND—Idiopathic pulmonary fibrosis (IPF) is characterised by subpleural fibrosis that progresses to involve all areas of the lung. The expression of transforming growth factor-β1 (TGF-β1), a potent regulator of connective tissue synthesis, is increased in lung sections of patients with IPF. TGF-β1 is generally released in a biologically latent form (L-TGF-β1). Before being biologically active, TGF-β must be converted to its active form and interact with both TGF-β receptors type I and II (TβR-I and TβR-II). TGF-β latency binding protein 1 (LTBP-1), which facilitates the release and activation of L-TGF-β1, is also important in the biology of TGF-β1.
METHODS—Open lung biopsy samples from patients with IPF and normal controls were examined to localise TβR-I, TβR-II, and LTBP-1. Alveolar macrophages (AM) and bronchoalveolar lavage (BAL) fluid were examined using the CCL-64 bioassay to determine if TGF-β is present in its active form in the lungs of patients with IPF.
RESULTS—Immunoreactive L-TGF-β1 was present in all lung cells of patients with IPF except for fibroblasts in the subepithelial regions of honeycomb cysts. LTBP-1 was detected primarily in AM and epithelial cells lining honeycomb cysts in areas of advanced IPF. In normal lungs LTBP-1 immunoreactivity was observed in a few AM. AM from the upper and lower lobes of patients with IPF secreted 1.6 (0.6) fmol and 4.1 (1.9) fmol active TGF-β, respectively, while AM from the lower lobes of control patients secreted no active TGF-β (p⩽0.01 for TGF-β in the conditioned media from AM obtained from the lower lobes of IPF patients v normal controls). The difference in percentage active TGF-β secreted by AM from the lower lobes of patients with IPF and the lower lobes of control patients was significant (p⩽0.01), but the difference between the total TGF-β secreted from these lobes was not significant. The difference in active TGF-β in conditioned media of AM from the upper and lower lobes of patients with IPF was also not statistically significant. BAL fluid from the upper and lower lobes of patients with IPF contained 0.7 (0.2) fmol and 2.9 (1.2) fmol active TGF-β, respectively (p⩽0.03). The percentage of active TGF-β in the upper and lower lobes was 17.6 (1.0)% and 78.4 (1.6)%, respectively (p⩽0.03). In contrast, BAL fluid from control patients contained small amounts of L-TGF-β. Using immunostaining, both TβR-I and TβR-II were present on all cells of normal lungs but TβR-I was markedly reduced in most cells in areas of honeycomb cysts except for interstitial myofibroblasts in lungs of patients with IPF. TGF-β1 inhibits epithelial cell proliferation and a lack of TβR-I expression by epithelial cells lining honeycomb cysts would facilitate repair of the alveoli by epithelial cell proliferation. However, the presence of both TβRs on fibroblasts is likely to result in a response to TGF-β1 for synthesis of connective tissue proteins. Our findings show that biologically active TGF-β1 is only present in the lungs of patients with IPF. In addition, the effects of TGF-β1 on cells may be further regulated by the expression of TβRs.
CONCLUSION—Activation of L-TGF-β1 and the differential expression of TβRs may be important in the pathogenesis of remodelling and fibrosis in IPF.


doi:10.1136/thorax.56.12.907
PMCID: PMC1745982  PMID: 11713352
16.  IPF Fibroblasts Are Desensitized to Type I Collagen Matrix-Induced Cell Death by Suppressing Low Autophagy via Aberrant Akt/mTOR Kinases 
PLoS ONE  2014;9(4):e94616.
Idiopathic pulmonary fibrosis (IPF) is a chronic, lethal interstitial lung disease in which the aberrant PTEN/Akt axis plays a major role in conferring a survival phenotype in response to the cell death inducing properties of type I collagen matrix. The underlying mechanism by which IPF fibroblasts become desensitized to polymerized collagen, thereby eluding collagen matrix-induced cell death has not been fully elucidated. We hypothesized that the pathologically altered PTEN/Akt axis suppresses autophagy via high mTOR kinase activity, which subsequently desensitizes IPF fibroblasts to collagen matrix induced cell death. We found that the autophagosome marker LC3-2 expression is suppressed, while mTOR activity remains high when IPF fibroblasts are cultured on collagen. However, LC3-2 expression increased in response to IPF fibroblast attachment to collagen in the presence of rapamycin. In addition, PTEN over-expression or Akt inhibition suppressed mTOR activity, thereby increasing LC3-2 expression in IPF fibroblasts. Furthermore, the treatment of IPF fibroblasts over-expressing PTEN or dominant negative Akt with autophagy inhibitors increased IPF fibroblast cell death. Enhanced p-mTOR expression along with low LC3-2 expression was also found in myofibroblasts within the fibroblastic foci from IPF patients. Our data show that the aberrant PTEN/Akt/mTOR axis desensitizes IPF fibroblasts from polymerized collagen driven stress by suppressing autophagic activity, which produces a viable IPF fibroblast phenotype on collagen. This suggests that the aberrantly regulated autophagic pathway may play an important role in maintaining a pathological IPF fibroblast phenotype in response to collagen rich environment.
doi:10.1371/journal.pone.0094616
PMCID: PMC3984186  PMID: 24728102
17.  miR-199a-5p Is Upregulated during Fibrogenic Response to Tissue Injury and Mediates TGFbeta-Induced Lung Fibroblast Activation by Targeting Caveolin-1 
PLoS Genetics  2013;9(2):e1003291.
As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.
Author Summary
Fibrosis is the final common pathway in virtually all forms of chronic organ failure, including lung, liver, and kidney, and is a leading cause of morbidity and mortality worldwide. Fibrosis results from the excessive activity of fibroblasts, in particular a differentiated form known as myofibroblast that is responsible for the excessive and persistent accumulation of scar tissue and ultimately organ failure. Idiopathic Lung Fibrosis (IPF) is a chronic and often rapidly fatal pulmonary disorder of unknown origin characterized by fibrosis of the supporting framework (interstitium) of the lungs. Given the poor prognosis of IPF patients, new insights into the biology of (myo)fibroblasts is of major interest to develop new therapeutics aimed at reducing (myo)fibroblast activity to slow or even reverse disease progression, thereby preserving organ function and prolonging life. MicroRNAs (miRNAs), a class of non-coding RNA recently identified, are associated with normal cellular processes; and deregulation of miRNAs plays a causative role in a vast array of complex diseases. In this study, we identified a particular miRNA: miR-199a-5p that governs lung fibroblast activation and ultimately lung fibrosis. Overall we showed that miR-199a-5p is a major regulator of fibrosis with strong therapeutic potency to treat fibroproliferative diseases such as IPF.
doi:10.1371/journal.pgen.1003291
PMCID: PMC3573122  PMID: 23459460
18.  Meta-Analysis of Genetic Programs between Idiopathic Pulmonary Fibrosis and Sarcoidosis 
PLoS ONE  2013;8(8):e71059.
Background
Idiopathic pulmonary fibrosis (IPF) and pulmonary sarcoidosis are typical interstitial lung diseases with unknown etiology that cause lethal lung damages. There are notable differences between these two pulmonary disorders, although they do share some similarities. Gene expression profiles have been reported independently, but differences on the transcriptional level between these two entities have not been investigated.
Methods/Results
All expression data of lung tissue samples for IPF and sarcoidosis were from published datasets in the Gene Expression Omnibus (GEO) repository. After cross platform normalization, the merged sample data were grouped together and were subjected to statistical analysis for finding discriminate genes. Gene enrichments with their corresponding functions were analyzed by the online analysis engine “Database for Annotation, Visualization and Integrated Discovery” (DAVID) 6.7, and genes interactions and functional networks were further analyzed by STRING 9.0 and Cytoscape 3.0.0 Beta1. One hundred and thirty signature genes could potentially differentiate one disease state from another. Compared with normal lung tissue, tissue affected by IPF and sarcoidosis displayed similar signatures that concentrated on proliferation and differentiation. Distinctly expressed genes that could distinguish IPF from sarcoidosis are more enriched in processes of cilium biogenesis or degradation and regulating T cell activations. Key discriminative network modules involve aspects of bone morphogenetic protein receptor two (BMPR2) related and v-myb myeloblastosis viral oncogene (MYB) related proliferation.
Conclusions
This study is the first attempt to examine the transcriptional regulation of IPF and sarcoidosis across different studies based on different working platforms. Groups of significant genes were found to clearly distinguish one condition from the other. While IPF and sarcoidosis share notable similarities in cell proliferation, differentiation and migration, remarkable differences between the diseases were found at the transcription level, suggesting that the two diseases are regulated by overlapping yet distinctive transcriptional networks.
doi:10.1371/journal.pone.0071059
PMCID: PMC3743918  PMID: 23967151
19.  Evolving Concepts of Apoptosis in Idiopathic Pulmonary Fibrosis 
Idiopathic pulmonary fibrosis (IPF) is a chronic, relentlessly progressive fibrosing disease of the lung of unknown etiology. Significant progress has been made in recent years in elucidating key aspects of the pathobiology of IPF. Insights into disease pathogenesis have come from studies of cell biology, growth factor/cytokine signaling, animal models of pulmonary fibrosis, and human IPF cells and tissue. A consistent finding in the ultrastructural pathology of IPF is alveolar epithelial cell injury and apoptosis. Another consistent finding in the histopathology of human IPF, described as usual interstitial pneumonia, is the accumulation of aggregates of myofibroblasts in fibroblastic foci. The extent or profusion of fibroblastic foci in lung biopsies is strongly correlated with increased mortality in patients with IPF. There is emerging evidence that myofibroblasts in IPF/usual interstitial pneumonia, both in the in vivo microenvironment and during the process of differentiation in vitro, acquire resistance to apoptosis. Here, we review the current evidence and mechanisms for this apparent “apoptosis paradox” in the pathogenesis of IPF.
doi:10.1513/pats.200601-001TK
PMCID: PMC2231523  PMID: 16738200
epithelial cells; fibroblasts; oxidative stress; transforming growth factor β; wound healing
20.  Evolving Concepts of Apoptosis in Idiopathic Pulmonary Fibrosis 
Idiopathic pulmonary fibrosis (IPF) is a chronic, relentlessly progressive fibrosing disease of the lung of unknown etiology. Significant progress has been made in recent years in elucidating key aspects of the pathobiology of IPF. Insights into disease pathogenesis have come from studies of cell biology, growth factor/cytokine signaling, animal models of pulmonary fibrosis, and human IPF cells and tissue. A consistent finding in the ultrastructural pathology of IPF is alveolar epithelial cell injury and apoptosis. Another consistent finding in the histopathology of human IPF, described as usual interstitial pneumonia, is the accumulation of aggregates of myofibroblasts in fibroblastic foci. The extent or profusion of fibroblastic foci in lung biopsies is strongly correlated with increased mortality in patients with IPF. There is emerging evidence that myofibroblasts in IPF/usual interstitial pneumonia, both in the in vivo microenvironment and during the process of differentiation in vitro, acquire resistance to apoptosis. Here, we review the current evidence and mechanisms for this apparent “apoptosis paradox” in the pathogenesis of IPF.
doi:10.1513/pats.200601-001TK
PMCID: PMC2231523  PMID: 16738200
epithelial cells; fibroblasts; oxidative stress; transforming growth factor β; wound healing
21.  Multiplex protein profiling of bronchoalveolar lavage in idiopathic pulmonary fibrosis and hypersensitivity pneumonitis 
Annals of Thoracic Medicine  2013;8(1):38-45.
CONTEXT:
Idiopathic pulmonary fibrosis (IPF) and chronic hypersensitivity pneumonitis (HP) are diffuse parenchymal lung diseases characterized by a mixture of inflammation and fibrosis, leading to lung destruction and finally death.
AIMS:
The aim of this study was to compare different pathophysiological mechanisms, such as angiogenesis, coagulation, fibrosis, tissue repair, inflammation, epithelial damage, oxidative stress, and matrix remodeling, in both disorders using bronchoalveolar lavage (BAL).
METHODS:
At diagnosis, patients underwent bronchoscopy with BAL and were divided into three groups: Control (n = 10), HP (n = 11), and IPF (n = 11), based on multidisciplinary approach (clinical examination, radiology, and histology): Multiplex searchlight technology was used to analyze 25 proteins representative for different pathophysiological processes: Eotaxin, basic fibroblast growth factor (FGFb), fibronectin, hepatocyte growth factor (HGF), interleukine (IL)-8, IL-12p40, IL-17, IL-23, monocyte chemotactic protein (MCP-1), macrophage-derived chemokine (MDC), myeloperoxidase (MPO), matrix metalloproteinase (MMP)-8, MMP-9, active plasminogen activating inhibitor 1 (PAI-1), pulmonary activation regulated chemokine (PARC), placental growth factor (PlGF), protein-C, receptor for advanced glycation end products (RAGE), regulated on activation normal T cells expressed and secreted (RANTES), surfactant protein-C (SP-C), transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1), tissue factor, thymic stromal lymphopoietin (TSLP), and vascular endothelial growth factor (VEGF).
RESULTS:
All patients suffered from decreased pulmonary function and abnormal BAL cell differential compared with control. Protein levels were increased in both IPF and HP for MMP-8 (P = 0.022), MMP-9 (P = 0.0020), MCP-1 (P = 0.0006), MDC (P = 0.0048), IL-8 (P = 0.013), MPO (P = 0.019), and protein-C (P = 0.0087), whereas VEGF was decreased (P = 0.0003) compared with control. HGF was upregulated in HP (P = 0.0089) and active PAI-1 was upregulated (P = 0.019) in IPF compared with control. Differences in expression between IPF and HP were observed for IL-12p40 (P = 0.0093) and TGF-β1 (P = 0.0045).
CONCLUSIONS:
Using BAL, we demonstrated not only expected similarities but also important differences in both disorders, many related to the innate immunity. These findings provide new clues for further research in both disorders.
doi:10.4103/1817-1737.105718
PMCID: PMC3573557  PMID: 23440593
Bronchoalveolar lavage; enzyme-linked immunosorbent assay; hypersensitivity pneumonitis; interstitial lung disease; idiopathic pulmonary fibrosis
22.  Cleaved cytokeratin-18 is a mechanistically informative biomarker in idiopathic pulmonary fibrosis 
Respiratory Research  2012;13(1):105.
Background
Stress of the endoplasmic reticulum (ER) leading to activation of the unfolded protein response (UPR) and alveolar epithelial cell (AEC) apoptosis may play a role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our objectives were to determine whether circulating caspase-cleaved cytokeratin-18 (cCK-18) is a marker of AEC apoptosis in IPF, define the relationship of cCK-18 with activation of the UPR, and assess its utility as a diagnostic biomarker.
Methods
IPF and normal lung tissues were stained with the antibody (M30) that specifically binds cCK-18. The relationship between markers of the UPR and cCK-18 was determined in AECs exposed in vitro to thapsigargin to induce ER stress. cCK-18 was measured in serum from subjects with IPF, hypersensitivity pneumonitis (HP), nonspecific interstitial pneumonia (NSIP), and control subjects.
Results
cCK-18 immunoreactivity was present in AECs of IPF lung, but not in control subjects. Markers of the UPR (phosphorylated IRE-1α and spliced XBP-1) were more highly expressed in IPF type II AECs than in normal type II AECs. Phosphorylated IRE-1α and cCK-18 increased following thapsigargin-induced ER stress. Serum cCK-18 level distinguished IPF from diseased and control subjects. Serum cCK-18 was not associated with disease severity or outcome.
Conclusions
cCK-18 may be a marker of AEC apoptosis and UPR activation in patients with IPF. Circulating levels of cCK-18 are increased in patients with IPF and cCK-18 may be a useful diagnostic biomarker.
doi:10.1186/1465-9921-13-105
PMCID: PMC3547729  PMID: 23167970
Idiopathic interstitial pneumonia; idiopathic pulmonary fibrosis; lung fibrosis; ER stress; apoptosis
23.  The HLA Class II Allele DRB1*1501 Is Over-Represented in Patients with Idiopathic Pulmonary Fibrosis 
PLoS ONE  2011;6(2):e14715.
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.
doi:10.1371/journal.pone.0014715
PMCID: PMC3044131  PMID: 21373184
24.  Defective efferocytosis by alveolar macrophages in IPF patients 
Respiratory medicine  2012;106(12):1800-1803.
Summary
Rationale
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrosing interstitial pneumonia. The pathogenicity of IPF has been widely investigated but still remains to be clarified. Efferocytosis, the specialized recognition and ingestion of apoptotic cells by phagocytes, is essential for the resolution of inflammation in the lungs and repair of injured tissues. Impaired efferocytosis contributes to the pathogenesis of chronic lung diseases such as emphysema and cystic fibrosis. We hypothesized that efferocytosis would also be reduced in alveolar macrophages isolated from subjects with IPF.
Methods
Efferocytosis, was evaluated using Wright-Giemsa stained cell preparations isolated from the bronchoalveolar lavage (BAL) fluid of patients with IPF (n = 5), nonspecific interstitial pneumonitis (n = 6), cryptogenic organizing pneumonia (n = 4) and eosinophilic pneumonia (EP) (n = 5).
Results
Uningested apoptotic cells were significantly higher in BAL fluid from patients with IPF compared to other forms of interstitial lung disease. Macrophages isolated from patients with eosinophilic pneumonia had significantly fewer phagocytic ingestions than macrophages from the other three groups.
Conclusion
Efferocytosis by alveolar macrophages was significantly lower in subjects with IPF compared to subjects with other interstitial pneumonia. Dysregulated efferocytosis may contribute to the pathogenesis of IPF.
doi:10.1016/j.rmed.2012.08.020
PMCID: PMC4030720  PMID: 22999220
Idiopathic pulmonary fibrosis; Efferocytosis; BAL; Idiopathic interstitial pneumonia
25.  Plasma B-Lymphocyte Stimulator (BLyS) and B-cell Differentiation in Idiopathic Pulmonary Fibrosis Patients* 
We hypothesized B-cells are involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a progressive, restrictive lung disease that is refractory to glucocorticoids and other nonspecific therapies, and almost invariably lethal. Accordingly, we sought to identify clinically-associated B-cell-related abnormalities in these patients. Phenotypes of circulating B-cells were characterized by flow cytometry. Intrapulmonary processes were evaluated by immunohistochemistry. Plasma B-lymphocyte stimulating factor (BLyS) was assayed by ELISA. Circulating B-cells of IPF subjects were more antigen-differentiated, with greater plasmablast proportions (3.1±0.8%) than in normal controls (1.3±0.3%) (p<0.03), and the extent of this differentiation correlated with IPF patient lung volumes (r=0.44, p<0.03). CD20+ B-cell aggregates, diffuse parenchymal and perivascular immune complexes, and complement depositions were all prevalent in IPF lungs, but much less prominent or absent in normal lungs. Plasma concentrations of BLyS, an obligate factor for B-cell survival and differentiation, were significantly greater (p<0.0001) in 110 IPF (2.05±0.05 ng/ml) than among 53 normal (1.40±0.04 ng/ml) and 90 chronic obstructive pulmonary disease (COPD) subjects (1.59±0.05 ng/ml). BLyS levels were uniquely correlated among IPF patients with pulmonary artery pressures (r=0.58, p<0.0001). The 25% of IPF subjects with the greatest BLyS values also had diminished one-year survival (46±11%), compared to those with lesser BLyS concentrations (81±5%) (HR=4.0, 95%CI=1.8-8.7, p=0.0002). Abnormalities of B-cells and BLyS are common in IPF patients, and highly associated with disease manifestations and patient outcomes. These findings have implications regarding IPF pathogenesis, and illuminate the potential for novel treatment regimens that specifically target B-cells in patients with this lung disease.
doi:10.4049/jimmunol.1203476
PMCID: PMC3804013  PMID: 23872052
B-cells; Adaptive Immunity; Interstitial Lung Disease; COPD

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