Rapid LC-MS/MS Evaluation of Collagen and Elastin Crosslinks in Human and Mouse Lung Tissue with a Novel Bioanalytical Surrogate Matrix Approach.

The distance-saturation product predicts mortality in idiopathic pulmonary fibrosis

RATIONALE: Fibrosis is a critical endpoint in numerous chronic diseases, including idiopathic pulmonary fibrosis (IPF). This study addresses the growing demand for new therapeutic targets in fibrotic diseases by focusing on the role of immunopeptidomics, specifically the MHC-I restricted antigen presentation, as a platform for developing effective treatments. The hypothesis posits that pathological condition in IPF alters MHC-I peptide presentation, offering the potential for identifying IPF-associated antigens (FAAs) for immunotherapy. METHODS: The study analyzed lung tissue from patients with IPF and bleomycin-induced mouse models using antibody-based immunoprecipitation and liquid chromatography-tandem mass spectrometry. Single-cell RNA sequencing (scRNA-seq) was conducted to profile immune cells in both human and mouse samples, and a scoring system was developed to identify peptides with therapeutic potential based on their correlation with pro-fibrotic cell populations and MHC-I binding characteristics. RESULTS: The immunopeptidomics analysis revealed an increased presence of MHC-I restricted peptides in IPF tissues compared to controls, with 56% of identified peptides being IPF-specific. The bleomycin-induced lung fibrosis in mice exhibited similar peptide characteristics to human IPF, with a strong overlap of antigen presentation profiles between species. A scoring system identified 2617 fibrosis-associated peptides, and the top-ranked peptides, including Tns3, Apbb2, and Maf, demonstrated significant therapeutic effects in the bleomycin mouse model, particularly in reducing IPF progression and inhibiting key pathological cells like macrophages and myofibroblasts. CONCLUSIONS: This study establishes the potential of immunopeptidomics in IPF as a robust platform for identifying therapeutic targets. It demonstrates that IPF-specific peptides can serve as effective vaccines, with the Maf116-124 peptide showing superior therapeutic effects in a pulmonary fibrosis model. The findings provide a new avenue for immunotherapy development in IPF, holding promise for clinical application.

Development of an isotope labeling ultra-high performance liquid chromatography mass spectrometric method for quantification of acylglycines in human urine

Background and Objectives: Pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF) and secondary pulmonary fibrosis (SPF), is a progressive lung disease that significantly impairs respiratory function. Accurate differentiation between IPF and SPF is crucial for effective management. This study explores the association between pulmonary fibrosis and hepatic conditions, evaluating the utility of various hemogram-derived ratios and hepatic fibrosis scores in distinguishing between IPF and SPF. Materials and Methods: We conducted a retrospective study involving patients diagnosed with IPF or SPF at the “Leon Daniello” Clinical Hospital of Pneumology in Cluj-Napoca, Romania. Pulmonary fibrosis was confirmed via imaging techniques, and hepatic steatosis and fibrosis were assessed using non-invasive scores. We analyzed clinical, laboratory, and pulmonary function data, focusing on hemogram-derived ratios and hepatic scores. Statistical analyses, including ROC curves, were used to evaluate the effectiveness of these biomarkers in differentiating IPF from SPF. Results: We included a total of 38 patients with IPF and 28 patients with SPF. Our findings revealed that IPF patients had a significantly higher FIB-4 score compared to SPF patients, suggesting increased hepatic fibrosis risk in IPF, as well as an increased RDW/PLT ratio. Conversely, SPF patients exhibited elevated PLR, PNR, and SII, reflecting a more pronounced inflammatory profile. PLR and PNR demonstrated the highest discriminatory ability between IPF and SPF, while traditional hepatic fibrosis scores showed limited differentiation capabilities. No significant differences in pulmonary function tests were observed across hepatic fibrosis risk categories. Conclusions: The study highlights the value of biomarkers like PLR and PNR in differentiating between IPF and SPF, offering additional diagnostic insights beyond traditional imaging. Integrating hepatic assessments into the management of pulmonary fibrosis could improve diagnostic accuracy and patient care.

Background Combined emphysema and pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF), is a distinct disorder described with upper-lobe emphysema and lower-lobe fibrosis on chest computed tomography. Smoking appears to be the predominant risk factor for this disorder. We aimed to compare clinical features, smoking history, physiological and radiological findings between IPF with and without emphysema. Methods A sample of 125 IPF patients over a period of 48 months were evaluated. High resolution CT scans were reviewed blinded to clinical data. The IPF patients with or without emphysema were classified accordingly. Results The prevalence of emphysema in this IPF sample was 70/125. IPF with emphysema was significantly associated with smoking status (OR 63; 95% CI 4.4 to 915; P=0.002) and smoking pack year (OR 1.1; 95% CI 1.05 to 1.13; P=0.000). The patients with IPF and emphysema had a higher decrease in carbon monoxide diffusing capacity adjusted for alveolar volume ((58±19)% pred vs. (66±21)% pred; P=0.021) and a higher prevalence of pulmonary hypertension (24/70 vs. 7/55; P=0.006). The two groups of patients had similar forced and residual volumes. No significant differences were found in cell differentials of bronchoalveolar lavage or the scores of fibrosis on chest CT. Survival of the patients with emphysema was significantly less than that of patients with IPF alone. Conclusions Cigarette smoking induces IPF combined with emphysema. Emphysema further impairs physiological function and increases the prevalence of pulmonary hypertension that leads to poor prognosis. The inclusion of the patients with combined pulmonary fibrosis and emphysema in IPF clinical trials may lead to under evaluation of the effect of treatment in patients.

Transcription factor TFAP2A drives EMT progress by activating BDKRB1 transcription: The potential mechanism by which TFAP2A promotes idiopathic pulmonary fibrosis.

Transforming growth factor-beta (TGF-β) is a major mediator of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). However, therapeutic global inhibition of TGF-β is limited by unwanted immunosuppression and mitral valve defects. We performed an extensive literature search to uncover a little-known connection between TGF-β signaling and casein kinase (CK) activity. We have examined the abundance of CK1 delta and epsilon (CK1δ/ε) in lung tissue from IPF patients and non-diseased controls, and investigated whether inhibition of CK1δ/ε with PF670462 inhibits pulmonary fibrosis. CK1δ/ε levels in lung tissue from IPF patients and non-diseased controls were assessed by immunohistochemistry. Anti-fibrotic effects of the CK1δ/ε inhibitor PF670462 were assessed in pre-clinical models, including acute and chronic bleomycin mouse models and in vitro experiments on spheroids made from primary human lung fibroblast cells from IPF and control donors, and human A549 alveolar-like adenocarcinoma-derived epithelial cells. Increased expression of CK1δ and ε in IPF lungs compared to non-diseased controls was accompanied by increased levels of the product, phospho-period 2. In vitro, PF670462 prevented TGF-β-induced epithelial-mesenchymal transition. The stiffness of IPF-derived spheroids was reduced by PF670462 and TGF-β-induced fibrogenic gene expression was inhibited. The CK1δ/ε inhibitor PF670462 administered systemically or locally by inhalation prevented both acute and chronic bleomycin-induced pulmonary fibrosis in mice. PF670462 administered in a ‘therapeutic’ regimen (day 7 onward) prevented bleomycin-induced lung collagen accumulation. Elevated expression and activity of CK1 δ and ε in IPF and anti-fibrogenic effects of the dual CK1δ/ε inhibitor, PF670462, support CK1δ/ε as novel therapeutic targets for IPF.

Autotaxin Emerges as a Therapeutic Target for Idiopathic Pulmonary Fibrosis

A rapid and sensitive high-performance liquid chromatography and electrospray tandem mass spectrometry method was developed and validated for estimation of fulvestrant in rabbit plasma using liquid-liquid extraction. The separation and quantification of fulvestrant were achieved by reverse-phase chromatography on a Sunfire C18 column (50 x 2.1. i.d., 3.5 microm) with isocratic elution at a flow rate of 300 microL/min using norethistrone as an internal standard from 500 microL plasma sample. The method was validated over the concentration range from 0.092 to 16.937 ng/mL with a lower limit of detection of 0.023 ng/mL. The intra-day and inter-day accuracy and precision were within 10%. The recovery was 85 and 90% for fulvestrant and norethistrone respectively. The chromatographic run time was only 2.5 min.

Improved liquid chromatography tandem mass spectrometry method for the determination of phenolic compounds in virgin olive oil

Interstitial lung disease (ILD) comprises a large number of chronic lung disease characterized by varying degrees of inflammation and fibrosis. Mostly they are idiopathic including idiopathic pulmonary fibrosis (IPF), which is a specific disorder characterized by progressive fibrosis leading commonly to end-stage lung disease, respiratory failure, and fatal outcome. IPF and many of these fibrotic ILDs lack effective therapy despite recent approval of two drugs to slow progression in certain IPF patients. Because there are no natural models for IPF, the use of animal models that reproduce key known features of the disease is warranted. Thus, different animal models have been developed to investigate key mechanisms underlying pathogenesis of pulmonary fibrosis and identify potential therapeutic targets for IPF. While no animal model can recapitulate all features of human disease, several are available to address select features of IPF and other fibrotic ILDs. Historically, among the first to be developed and used widely is the bleomycin model, which is the best-characterized and currently most extensively used animal model due to its ability to reproduce many aspects of IPF and other fibrotic ILDs, good reproducibility, and ease of induction. Studies using the bleomycin model have identified many of the cellular and molecular mechanisms now recognized as being important in pathogenesis of IPF and other fibrotic ILDs, as well as novel therapies for these diseases, including two recent drugs approved for treatment of IPF. This chapter will describe commonly used techniques for induction of the model by endotracheal administration of bleomycin through surgical and nonsurgical (transoral instillation).

The aim of this study was to evaluate New York Heart Association (NYHA) class and systolic pulmonary artery pressure (sPAP) as survival predictors in major interstitial lung diseases (ILD) including idiopathic pulmonary fibrosis (IPF), non-specific interstitial pneumonia (NSIP) and hypersensitivity pneumonitis (HP) and in other ILD like granulomatosis with polyangiitis (GPA). We analyzed survival, NYHA class, sPAP, and Octreoscan uptake index (UI) in 104 ILD patients (59 IPF, 19 NSIP, 10 HP and 16 GPA; median age 60.5 years) all referred to a single centre. Median survival was 68 months, with 1- and 2-year survival of 91% and 78%, respectively. Survival was lower among IPF and NSIP vs. HP and GPA patients (p=0.01). NYHA class 3-4 was more frequent among IPF (76.3%) vs. NSIP patients (31.6%; p<0.001). HP and GPA had NYHA class 1-2. NYHA class was negatively associated with survival (class 1=90.3 months vs. class 3=18.3 months and class 4=5.1 months; p=0.001). sPAP was >55 mmHg in 76.3% of patients with IPF and 35-55 mmHg in 63.2% of patients with NSIP. Patients with HP and GPA had sPAP < 55 mmHg. Among patients with IPF, NYHA and sPAP were negatively associated with survival (p<0.01) both showed a parallel trend. High-resolution computed tomography and survival were worse among IPF and NSIP vs. HP and GPA patients (p<0.001). Octreoscan UI was <10, 10-12, and >12 in IPF, NSIP, HP and GPA, respectively. Octreoscan UI was negatively associated with survival (p=0.002). NYHA class and sPAP are comparable ILD survival predictors. NYHA class is correlated with worse prognosis for IPF and NSIP vs. HP and GPA patients.

The study was conducted to evaluate the applicability of ultra high-performance liquid chromatography - tandem mass spectrometry method (UHPLC-MS/MS), establish the MS/MS detection parameters and determine the validation characteristics for the analysis of residual content of avermectins in milk. The UHPLC-MS/MS method has proved to be accurate, practical and universal. This was confirmed by Decision limit (CCα) data: abamectin - 12.56 μg/kg, doramectin - 17.74 μg/kg, eprinomectin - 24.02 μg/kg, ivermectin - 12.53 μg/kg, moxidectin - 44.69 μg/kg, recovery is 88.7-110%. The data obtained for assessing the suitability, accuracy and reproducibility of the results meet the requirements of the European Directive (2002/657/EC). The efficient ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method that was developed and adopted for routine use by the laboratories of veterinary medicine, allows to detect residual quantities of 5 avermectins used in animal breeding for the prevention of helminthiases in food products including milk.

Interstitial lung diseases (ILD) include idiopathic pulmonary fibrosis (IPF), a progressive fibrotic lung disease affecting around 3 million people globally, and a manifestation of systemic sclerosis (SSc-ILD) characterized by progressive pulmonary fibrosis and declining lung function. Oncostatin M (OSM), a member of the gp130 cytokine family that also includes IL-6 and LIF, acts as a mediator between immune cells and stromal cells to regulate a diverse set of physiological processes contributing to the maintenance of homeostasis and tissue repair. Transcriptional and proteomic analyses showed that OSM is dysregulated in IPF and SSc-ILD subjects, and that OSM can drive fibrotic responses in human fibroblasts. Lung damage and fibrosis are reduced in Osm-/- mice as well as WT mice treated with OSM blocking mAbs in the bleomycin model of lung injury, inflammation and fibrosis. These observations, along with a substantial body of literature implicating OSM in fibrogenic responses, led to the hypothesis that OSM contributes to tissue remodeling and development of pulmonary fibrosis. There is also evidence that OSM is dysregulated in chronic inflammatory diseases such as human inflammatory bowel disease (IBD) where elevated OSM and OSMR expression is associated with greater disease severity and resistance to anti-TNFɑ therapies; blocking OSMR signaling in Helicobacter/anti-IL10R mAb-treated mice reduces colonic inflammation. We hypothesized that OSMR+ fibroblasts would differ in their profibrotic and proinflammatory programs between ILD and IBD. To address this question, we collected clinical tissue samples from patients with IPF, SSc-ILD, and IBD and profiled fibroblast diversity using scRNA-seq. OSMR expression is observed on fibroblasts in both ILD and IBD (Figure 1A). However, while OSMR+ stroma was observed across indications, ILD and IBD displayed marked differences in the abundance (Figure 1B) and profibrotic and proinflammatory phenotype (Figure 1C) of these cells, raising the possibility of tissue- and disease- specific responses to OSMR blockade. Vixarelimab, an anti-human OSMR antagonist, is currently being tested in the clinic for SSc-ILD, IPF and UC. Acknowledgements: The authors would like to acknowledge the Mark S. Wilson lab for their contributions to this research.

BackgroundRecent evidence has underscored the role of hypoxia and angiogenesis in the pathogenesis of idiopathic fibrotic lung disease. Inhibitor of growth family member 4 (ING4) has recently attracted much attention as a tumor suppressor gene, due to its ability to inhibit cancer cell proliferation, migration and angiogenesis. The aim of our study was to investigate the role of ING4 in the pathogenesis of pulmonary fibrosis both in the bleomycin (BLM)-model and in two different types of human pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF) and cryptogenic organizing pneumonia (COP).MethodsExperimental model of pulmonary fibrosis was induced by a single tail vein injection of bleomycin in 6- to 8-wk-old C57BL/6mice. Tissue microarrays coupled with qRT-PCR and immunohistochemistry were applied in whole lung samples and paraffin-embedded tissue sections of 30 patients with IPF, 20 with COP and 20 control subjects.ResultsA gradual decline of ING4 expression in both mRNA and protein levels was reported in the BLM-model. ING4 was also found down-regulated in IPF patients compared to COP and control subjects. Immunolocalization analyses revealed increased expression in areas of normal epithelium and in alveolar epithelium surrounding Masson bodies, in COP lung, whereas showed no expression within areas of active fibrosis within IPF and COP lung. In addition, ING4 expression levels were negatively correlated with pulmonary function parameters in IPF patients.ConclusionOur data suggest a potential role for ING4 in lung fibrogenesis. ING4 down-regulation may facilitate aberrant vascular remodelling and fibroblast proliferation and migration leading to progressive disease.