Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis.

Inhibition of mTOR is the standard of care for lymphangioleiomyomatosis (LAM). However, this therapy has variable tolerability and some patients show progressive decline of lung function despite treatment. LAM diagnosis and monitoring can also be challenging due to the heterogeneity of symptoms and insufficiency of non-invasive tests. Here, we propose monoamine-derived biomarkers that provide preclinical evidence for novel therapeutic approaches. The major histamine-derived metabolite methylimidazoleacetic acid (MIAA) is relatively more abundant in LAM plasma, and MIAA values are independent of VEGF-D. Higher levels of histamine are associated with poorer lung function and greater disease burden. Molecular and cellular analyses, and metabolic profiling confirmed active histamine signaling and metabolism. LAM tumorigenesis is reduced using approved drugs targeting monoamine oxidases A/B (clorgyline and rasagiline) or histamine H1 receptor (loratadine), and loratadine synergizes with rapamycin. Depletion of Maoa or Hrh1 expression, and administration of an L-histidine analog, or a low L-histidine diet, also reduce LAM tumorigenesis. These findings extend our knowledge of LAM biology and suggest possible ways of improving disease management.

Interleukin-1ß Modulation of the Mechanobiology of Primary Human Pulmonary Fibroblasts: Potential Implications in Lung Repair.

Pro-inflammatory cytokines like interleukin-1ß (IL-1ß) are upregulated during early responses to tissue damage and are expected to transiently compromise the mechanical microenvironment. Fibroblasts are key regulators of tissue mechanics in the lungs and other organs. However, the effects of IL-1ß on fibroblast mechanics and functions remain unclear. Here we treated human pulmonary fibroblasts from control donors with IL-1ß and used Atomic Force Microscopy to unveil that IL-1ß significantly reduces the stiffness of fibroblasts concomitantly with a downregulation of filamentous actin (F-actin) and alpha-smooth muscle (α-SMA). Likewise, COL1A1 mRNA was reduced, whereas that of collagenases MMP1 and MMP2 were upregulated, favoring a reduction of type-I collagen. These mechanobiology changes were functionally associated with reduced proliferation and enhanced migration upon IL-1ß stimulation, which could facilitate lung repair by drawing fibroblasts to sites of tissue damage. Our observations reveal that IL-1ß may reduce local tissue rigidity by acting both intracellularly and extracellularly through the downregulation of fibroblast contractility and type I collagen deposition, respectively. These IL-1ß-dependent mechanical effects may enhance lung repair further by locally increasing pulmonary tissue compliance to preserve normal lung distension and function. Moreover, our results support that IL-1ß provides innate anti-fibrotic protection that may be relevant during the early stages of lung repair.

Demographic and clinical profile of idiopathic pulmonary fibrosis patients in Spain: the SEPAR National Registry.

BACKGROUND: Little is known on the characteristics of patients diagnosed with idiopathic pulmonary fibrosis (IPF) in Spain. We aimed to characterize the demographic and clinical profile of IPF patients included in the IPF National Registry of the Spanish Respiratory Society (SEPAR). METHODS: This is a prospective, observational, multicentre and nationwide study that involved 608 IPF patients included in the SEPAR IPF Registry up to June 27th, 2017, and who received any treatment for their disease. IPF patients were predominantly males, ex-smokers, and aged in their 70s, similar to other registries. RESULTS: Upon inclusion, mean ± SD predicted forced vital capacity was 77.6% ± 19.4, diffusing capacity for carbon monoxide was 48.5% ± 17.7, and the 6-min walk distance was 423.5 m ± 110.4. The diagnosis was mainly established on results from the high-resolution computed tomography in the proper clinical context (55.0% of patients), while 21.2% of patients required invasive procedures (surgical lung biopsy) for definitive diagnosis. Anti-fibrotic treatment was prescribed in 69.4% of cases, 51.5% pirfenidone and 17.9% nintedanib, overall with a good safety profile. CONCLUSIONS: The SEPAR IPF Registry should help to further characterize current characteristics and future trends of IPF patients in Spain and compare/pool them with other registries and cohorts.

Correction: Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness.

[This corrects the article DOI: 10.1371/journal.pone.0132546.].

The Burden of Comorbidity and Complexity in Sarcoidosis: Impact of Associated Chronic Diseases.

PURPOSE: To evaluate comorbidity, complexity and poor outcomes in patients with sarcoidosis and to compare those scores with a control group. METHODS: 218 consecutive patients were diagnosed with sarcoidosis according to the ATS/ERS/WASOG criteria; extrathoracic involvement was evaluated using the 2014 WASOG organ assessment instrument. Sarcoidosis patients were compared with an age- and gender-matched control group of primary care outpatients without sarcoidosis. Comorbidities were assessed retrospectively using the Charlson Comorbidity Index (CCI); complexity was evaluated according to the classification into Clinical Risk Groups (CRG) and severity levels. RESULTS: The cohort included 142 women and 76 men; the mean age was 47.1 years at diagnosis of sarcoidosis and 55.9 years at the last visit. Patients with a CCI > 1 had a higher frequency of calcium/vitamin D abnormalities (p < 0.001), kidney involvement (p = 0.005) and a higher mortality rate (p < 0.001) compared with patients with a CCI ≤ 1. Patients with a CRG ≥ 6 had a higher frequency of extrathoracic involvement (p = 0.039), calcium/vitamin D abnormalities (p = 0.019) and treatment with glucocorticoids (p = 0.032) compared with patients with a CRG < 6. 11% patients died after a mean follow-up of 102.3 months. Country of birth, kidney involvement and extrathoracic disease were significantly associated with death. Patients with sarcoidosis had a higher frequency of liver (p < 0.001), pulmonary (p = 0.002) and autoimmune disease (p = 0.011) and cancer (p = 0.007) compared with the control group. CONCLUSION: We found higher rates of comorbidity and complexity in patients with sarcoidosis compared with a control group. Liver, pulmonary, autoimmune and neoplastic diseases were the main comorbidities found in patients with sarcoidosis.

Dysregulated Collagen Homeostasis by Matrix Stiffening and TGF-ß1 in Fibroblasts from Idiopathic Pulmonary Fibrosis Patients: Role of FAK/Akt.

Idiopathic pulmonary fibrosis (IPF) is an aggressive disease in which normal lung parenchyma is replaced by a stiff dysfunctional scar rich in activated fibroblasts and collagen-I. We examined how the mechanochemical pro-fibrotic microenvironment provided by matrix stiffening and TGF-ß1 cooperates in the transcriptional control of collagen homeostasis in normal and fibrotic conditions. For this purpose we cultured fibroblasts from IPF patients or control donors on hydrogels with tunable elasticity, including 3D collagen-I gels and 2D polyacrylamide (PAA) gels. We found that TGF-ß1 consistently increased COL1A1 while decreasing MMP1 mRNA levels in hydrogels exhibiting pre-fibrotic or fibrotic-like rigidities concomitantly with an enhanced activation of the FAK/Akt pathway, whereas FAK depletion was sufficient to abrogate these effects. We also demonstrate a synergy between matrix stiffening and TGF-ß1 that was positive for COL1A1 and negative for MMP1. Remarkably, the COL1A1 expression upregulation elicited by TGF-ß1 alone or synergistically with matrix stiffening were higher in IPF-fibroblasts compared to control fibroblasts in association with larger FAK and Akt activities in the former cells. These findings provide new insights on how matrix stiffening and TGF-ß1 cooperate to elicit excessive collagen-I deposition in IPF, and support a major role of the FAK/Akt pathway in this cooperation.

Epithelial contribution to the profibrotic stiff microenvironment and myofibroblast population in lung fibrosis.

The contribution of epithelial-to-mesenchymal transition (EMT) to the profibrotic stiff microenvironment and myofibroblast accumulation in pulmonary fibrosis remains unclear. We examined EMT-competent lung epithelial cells and lung fibroblasts from control (fibrosis-free) donors or patients with idiopathic pulmonary fibrosis (IPF), which is a very aggressive fibrotic disorder. Cells were cultured on profibrotic conditions including stiff substrata and TGF-ß1, and analyzed in terms of morphology, stiffness, and expression of EMT/myofibroblast markers and fibrillar collagens. All fibroblasts acquired a robust myofibroblast phenotype on TGF-ß1 stimulation. Yet IPF myofibroblasts exhibited higher stiffness and expression of fibrillar collagens than control fibroblasts, concomitantly with enhanced FAKY397 activity. FAK inhibition was sufficient to decrease fibroblast stiffness and collagen expression, supporting that FAKY397 hyperactivation may underlie the aberrant mechanobiology of IPF fibroblasts. In contrast, cells undergoing EMT failed to reach the values exhibited by IPF myofibroblasts in all parameters examined. Likewise, EMT could be distinguished from nonactivated control fibroblasts, suggesting that EMT does not elicit myofibroblast precursors either. Our data suggest that EMT does not contribute directly to the myofibroblast population, and may contribute to the stiff fibrotic microenvironment through their own stiffness but not their collagen expression. Our results also support that targeting FAKY397 may rescue normal mechanobiology in IPF.

Guidelines for the medical treatment of idiopathic pulmonary fibrosis. / Normativa sobre el tratamiento farmacológico de la fibrosis pulmonar idiopática.

Idiopathic pulmonary fibrosis is defined as chronic fibrosing interstitial pneumonia limited to the lung, with poor prognosis. The incidence has been rising in recent years probably due to improved diagnostic methods and increased life expectancy. In 2013, the SEPAR guidelines for the diagnosis and treatment for idiopathic pulmonary fibrosis were published. Since then, clinical trials and meta-analyses have shown strong scientific evidence for the use of pirfenidone and nintedanib in the treatment of idiopathic pulmonary fibrosis. In 2015, the international consensus of 2011 was updated and new therapeutic recommendations were established, prompting us to update our recommendation for the medical treatment of idiopathic pulmonary fibrosis accordingly. Diagnostic aspects and non-pharmacological treatment will not be discussed as no relevant developments have emerged since the 2013 guidelines.

Idiopathic pulmonary fibrosis. / Fibrosis pulmonar idiopática.

Idiopathic pulmonary fibrosis is a fibrosing interstitial pneumonia associated with the radiological and/or histological pattern of usual interstitial pneumonia. Its aetiology is unknown, but probably comprises the action of endogenous and exogenous micro-environmental factors in subjects with genetic predisposition. Its diagnosis is based on the presence of characteristic findings of high-resolution computed tomography scans and pulmonary biopsies in absence of interstitial lung diseases of other aetiologies. Its clinical evolution is variable, although the mean survival rate is 2-5 years as of its clinical presentation. Patients with idiopathic pulmonary fibrosis may present complications and comorbidities which modify the disease's clinical course and prognosis. In the mild-moderate disease, the treatment consists of the administration of anti-fibrotic drugs. In severe disease, the best therapeutic option is pulmonary transplantation. In this paper we review the diagnostic and therapeutic aspects of the disease.

Epidemiologic patterns of disease expression in sarcoidosis: age, gender and ethnicity-related differences.

OBJECTIVES: To evaluate the influence of age, gender and ethnicity in the clinical presentation of sarcoidosis in a cohort of Spanish patients. METHODS: We analysed 175 consecutive patients diagnosed with sarcoidosis between 1990 and 2014 in the Hospital Clinic of Barcelona, Spain. Sarcoidosis was diagnosed according to the 1999 WASOG criteria. Organ involvement was defined using the 2014 WASOG organ assessment instrument. RESULTS: There were 110 women and 65 men, with a mean age at diagnosis of 47.31 ± 15.46 years (range, 16-92); 23% of patients were born outside Spain. Women had a higher mean age (p=0.027), a higher frequency of cutaneous (OR=2.28) and musculoskeletal (OR=2.73) symptoms at diagnosis, and a lower frequency of cumulated WASOG involvements including renal involvement (OR=0.17), hypercalcaemia (OR=0.20) and raised ACE levels (OR=0.30). Patients aged ≥65 years had a lower frequency of cutaneous (OR=0.23) and musculoskeletal (OR=0.13) symptomatology at diagnosis and a higher frequency of cumulated WASOG involvements including renal involvement (OR=18.70) and calcium/vitamin D abnormalities (OR=5.31). According to ethnicity, non-Spanish-born patients had a lower mean age (40 vs. 49 years, p=0.001), a higher predominance of females (68% vs. 54%, p=0.036) and a higher frequency of radiographic stages I/II at diagnosis (97% vs. 78%, p=0.041) in comparison with Spanish-born patients. CONCLUSIONS: Using the new 2014 WASOG organ assessment instrument, we found that epidemiological features (age at diagnosis, gender and ethnicity) play a significant role in the presentation of sarcoidosis. Variations in these epidemiological features may aid early diagnostic suspicion, the search for histopathological confirmation and the prompt introduction of the appropriate therapy.

Study of breast cancer incidence in patients of lymphangioleiomyomatosis.

Molecular evidence has linked the pathophysiology of lymphangioleiomyomatosis (LAM) to that of metastatic breast cancer. Following on this observation, we assessed the association between LAM and subsequent breast cancer. An epidemiological study was carried out using three LAM country cohorts, from Japan, Spain, and the United Kingdom. The number of incident breast cancer cases observed in these cohorts was compared with the number expected on the basis of the country-specific incidence rates for the period 2000-2014. Immunohistochemical studies and exome sequence analysis were performed in two and one tumors, respectively. All cohorts revealed breast cancer standardized incidence ratios (SIRs) ≥ 2.25. The combined analysis of all cases or restricted to pre-menopausal age groups revealed significantly higher incidence of breast cancer: SIR = 2.81, 95 % confidence interval (CI) = 1.32-5.57, P = 0.009; and SIR = 4.88, 95 % CI = 2.29-9.99, P = 0.0007, respectively. Immunohistochemical analyses showed positivity for known markers of lung metastatic potential. This study suggests the existence of increased breast cancer risk among LAM patients. Prospective studies may be warranted to corroborate this result, which may be particularly relevant for pre-menopausal women with LAM.

Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited response to currently available therapies. Alveolar type II (ATII) cells act as progenitor cells in the adult lung, contributing to alveolar repair during pulmonary injury. However, in IPF, ATII cells die and are replaced by fibroblasts and myofibroblasts. In previous preclinical studies, we demonstrated that ATII-cell intratracheal transplantation was able to reduce pulmonary fibrosis. The main objective of this study was to investigate the safety and tolerability of ATII-cell intratracheal transplantation in patients with IPF. METHODS: We enrolled 16 patients with moderate and progressive IPF who underwent ATII-cell intratracheal transplantation through fiberoptic bronchoscopy. We evaluated the safety and tolerability of ATII-cell transplantation by assessing the emergent adverse side effects that appeared within 12 months. Moreover, pulmonary function, respiratory symptoms, and disease extent during 12 months of follow-up were evaluated. RESULTS: No significant adverse events were associated with the ATII-cell intratracheal transplantation. After 12 months of follow-up, there was no deterioration in pulmonary function, respiratory symptoms, or disease extent. CONCLUSIONS: Our results support the hypothesis that ATII-cell intratracheal transplantation is safe and well tolerated in patients with IPF. This study opens the door to designing a clinical trial to elucidate the potential beneficial effects of ATII-cell therapy in IPF.

Auscultation of Velcro Crackles is Associated With Usual Interstitial Pneumonia.

Auscultation of Velcro crackles has been proposed as a key finding in physical lung examination in patients with interstitial lung diseases (ILDs), especially in idiopathic pulmonary fibrosis (IPF). However, no studies have been carried out to assess the association of Velcro crackles with other clinical variables.We evaluated a cohort of 132 patients, prospectively and consecutively included in our ILD diagnostic program at a tertiary referral center. All patients were auscultated during the physical examination. The patients were divided into 2 groups: "presence" or "nonpresence" of bilateral Velcro crackles.Of all patients assessed, 83 (63%) presented Velcro crackles in the respiratory auscultation. Patients with Velcro crackles usually had more frequently cough and dyspnea at the moment of diagnosis. Forced vital capacity (P = 0.002) and lung diffusion capacity for carbon monoxide (P = 0.04) was lower in these patients. The ILD-GAP index was higher in the group with Velcro crackles (P = 0.01). All patients with usual interstitial pneumonia (UIP) in high-resolution computed tomography and all patients with final IPF diagnosis presented Velcro crackles. In multivariate analysis, the presence of Velcro crackles was independently associated with an UIP pattern.In patients suspected of having ILD, the auscultation of Velcro crackles was associated with UIP, a possibility which must be taken into consideration in early ILD detection in primary care.

Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness.

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-ß3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.

Cryobiopsy in the diagnosis of diffuse interstitial lung disease: yield and cost-effectiveness analysis.

BACKGROUND: Assessment of patients with suspected interstitial lung disease (ILD) includes surgical lung biopsy (SLB) when clinical and radiological data are inconclusive. However, cryobiopsy is acquiring an important role in the ILD diagnostic process. The objective of this study was to evaluate the diagnostic yield, safety and economic costs of the systematic use of cryobiopsy in the assessment of patients with suspected ILD. METHODS: This was a retrospective observational study of patients who had undergone transbronchial cryobiopsy for evaluation of ILD from January 2011 to January 2014. The procedures were performed with a video bronchoscope using a cryoprobe for the collection of lung parenchyma specimens, which were analyzed by pathologists. Diagnostic yield, complications and economic costs of this technique were analyzed. RESULTS: Criobiopsy specimens from a total of 33 patients were included. A specific diagnosis was obtained in 26, producing a diagnostic yield of 79%. In 5 patients, SLB was required for a histopathological confirmation of disease, but the procedure could not be performed in 4, due to severe comorbidities. The most frequent complications were pneumothorax (12%) and gradei (9%) or gradeii (21%) bleeding. There were no life-threatening complications. The systematic use of cryobiopsy saved up to €59,846. CONCLUSION: Cryobiopsy is a safe and potentially useful technique in the diagnostic assessment of patients with ILD. Furthermore, the systematic use of cryobiopsy has an important economic impact.

Lung fibrotic tenascin-C upregulation is associated with other extracellular matrix proteins and induced by TGFß1.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive parenchymal lung disease of unknown aetiology and poor prognosis, characterized by altered tissue repair and fibrosis. The extracellular matrix (ECM) is a critical component in regulating cellular homeostasis and appropriate wound healing. The aim of our study was to determine the expression profile of highlighted ECM proteins in IPF lungs. METHODS: ECM gene and protein expression was analyzed by cDNA microarrays, rt-PCR, immunohistochemistry and western-blot in lungs from idiopathic pulmonary fibrosis (IPF), hypersensitivity pneumonitis (HP), categorized as chronic (cHP) and subacute (saHP), and healthy lung tissue. Primary fibroblast cultures from normal subjects and fibrotic patients were studied to evaluate tenascin-C (TNC) synthesis. RESULTS: A total of 20 ECM proteins were upregulated and 6 proteins downregulated in IPF. TNC was almost undetected in normal lungs and significantly upregulated in fibrotic lungs (IPF and cHP) compared to saHP. Furthermore, it was located specifically in the fibroblastic foci areas of the fibrotic lung with a subepithelial gradient pattern. TNC levels were correlated with fibroblastic foci content in cHP lungs. Versican and fibronectin glycoproteins were associated with TNC, mainly in fibroblastic foci of fibrotic lungs. Fibroblasts from IPF patients constitutively synthesized higher levels of TNC than normal fibroblasts. TNC and α-sma was induced by TGF-ß1 in both fibrotic and normal fibroblasts. TNC treatment of normal and fibrotic fibroblasts induced a non-significant increased α-sma mRNA. CONCLUSIONS: The difference in ECM glycoprotein content in interstitial lung diseases could contribute to the development of lung fibrosis. The increase of TNC in interstitial areas of fibrotic activity could play a key role in the altered wound healing.