1,720,961 research outputs found
Idiopathic Pulmonary Fibrosis (IPF): tissue identification of crucial biomarkers by RNA-Sequencing approach.
Full text linkBackground. Idiopathic pulmonary fibrosis (IPF) is one of the most common idiopathic interstitial lung diseases characterized by progressive lung scarring and a very poor overall prognosis with a median survival of 2-3 years. Despite extensive research efforts, the etiopathogenesis and pathophysiology of IPF are still little understood and consequently only slight improvement has been made for appropriate management and effective therapies. Some advances have been made in understanding the multiple interrelated pathogenic pathways underlying IPF and the disease is now considered to result from complex interactions among genetic, epigenetic, transcriptional, post-transcriptional, metabolic and environmental factors. Thus, the discovery and validation of theranostic biomarkers are necessary to enable a more precise and earlier diagnosis of IPF and to improve the prediction of future disease behavior. Usual interstitial pneumonia (UIP) is the histopathological pattern of IPF characterized by spatial/temporal heterogeneous histological lesions. Fibroblastic focus (FF) areas include fibroblasts and myofibroblasts arranged in a linear fashion with a pale staining matrix, with metaplastic epithelial cells lining on top of them. They are usually detected in the transitional area from dense scarring to normal lung and are considered an “active” component in IPF pathogenesis.
Aim of the research. The main goal of the present PhD research project was the identification of crucial biomarkers in IPF pathogenesis by extracting RNA from FF areas (FF plus metaplastic epithelial cells lining FF). The main steps of the study were the following:
1) validation of a protocol for the isolation of FF areas by laser microdissection of formalin-fixed and paraffin-embedded (FFPE) tissues of IPF patients and controls;
2) total RNA extraction, quality and quantity evaluation;
3) creation of cDNA libraries starting from the extracted RNAs;
4) transcriptomic analysis by a Next-Generation Sequencing (NGS) approach (RNA-sequencing; RNA-Seq);
5) validation of the biomarker emerged from the transcriptomic analysis in a more extensive (retrospective) cases series using immunohistochemistry.
Material and Methods. Total RNA was extracted from fibroblastic focus areas isolated with laser microdissection in 10 FFPE IPF lung tissues: 8 from lung transplanted patients and 2 from surgical biopsy. Microdissected fibroblastic focus areas from 2 patients with recurrent pneumothorax were also analyzed and considered as controls. The RNA was extracted using a modified protocol which provides an overnight tissue incubation at 43°C with 10 μl proteinase K. RNA was then preserved by adding RNase inhibitors at the end of the extraction procedure. This was a custom-made protocol (RNeasy® FFPE kit; Qiagen, Hilden, Germany) with additional procedures optimized during my PhD research study. The final RNAs quality and quantity were valuated with an Agilent RNA 6000 Pico Kit using a 2100 Bioanalyzer instrument. Quality was expressed as DV200 (percentage of RNA fragments > 200 nucleotides). Libraries were obtained with the SMARTer Stranded Total RNA-Seq Kit pico input mammalian of Takara Bio. DNA library was sequenced with a paired-end sequencing 2x150 bp on a HiSeq 4000 System sequencer of Illumina. MUC5B immunohistochemistry (clone 4A10-H2; 1:200, Novus Biologicals, Centennial, Colorado, USA) was performed in 44 interstitial lung disease (ILD) cases (39 UIP/IPF and 5 ILD with no UIP pattern) and 6 controls (5 spontaneous pneumothorax and 1 emphysema) following the antibody manufacturer's protocol using a Leica Bond-III Autostainer.
Results. Considering the whole population the mean quantity of extracted RNA was 2992.8 pg/μl±2473 (mean ± SD), ranging from 840 pg/μl to 7530 pg/μl. Quality evaluation showed 42% of total cases with a medium/high quality (DV200>50%). In all cases molecular analyses were performed and final libraries were obtained with a concentration ranging from 3.4 to 22.6 ng/ul and a mean cDNA fragment length of 289 nucleotides. RNA-Seq analysis showed that 323 genes were differentially expressed in UIP/IPF cases than controls: 14 of them were up-regulated and 309 down-regulated. The most significant up-regulated gene was MUC5B, the other up-regulated genes were those involved in epithelial-to-mesenchymal transition (EMT) and epithelial carcinogenesis process. Gene Ontology Enrichment Analysis (GOEA) was performed to identify the most enriched Gene Ontology (GO) categories for the down-regulated genes. We found that extracellular matrix structure and organization were the principally down-regulated pathways.
The overexpressed gene MUC5B was validated by immunohistochemistry. MUC5B was expressed only in IPF/UIP and ILDs, never in control group. The MUC5B expression was mainly detected in metaplastic epithelial cells lining: a) honeycomb areas, b) other alveolar spaces and c) in the metaplastic epithelial cells lining FF. Interestingly, a gradient of MUC5B expression was detected both in IPF/UIP and ILDs samples where MUC5B was overexpressed in lower lobes. Interestingly, MUC5B was overexpressed in upper and middle lobes of IPF/UIP compared with the same lobes of other ILDs.
Conclusion. The principal results obtained from the present research study offer interesting insights into the complex molecular system signature of IPF:
1) adequate quantity and quality of RNA was extracted from microdissected FF areas of FFPE IPF lung tissues. The quantity/quality of RNA was suitable to create cDNA libraries for transcriptomic analysis by RNA-seq: this represents an important step forward in tissue molecular investigation of this disease characterized by high tissue heterogeneity. Only a very few papers in the literature have used lung FFPE tissue for molecular analysis, in particular, this molecular approach on specific affected IPF tissues has not previously used.
2) Comparative analysis performed on selected areas found an overexpression of epithelial proliferation/cancer progression and EMT transcripts: this highlights the crucial role of metaplastic epithelial cells that are the key actors also in the FF areas, considered the active injured lesion in IPF.
3) The up-regulated transcript MUC5B, validated also by immunohistochemistry, confirms the crucial role of this mucin in the disease. Indeed previous works, mainly performed in blood samples, had highlighted the importance of this gene in the disease. Selective regulation of MUC5B in experimental models could open up an entire line of investigation that could bring us closer to understanding regulation of MUC5B and providing novel therapeutic options
Idiopathic pulmonary fibrosis and antifibrotic treatments focus on experimental studies
No full textContext.— Idiopathic pulmonary fibrosis (IPF) is a progressive fatal disease that up to now has been associated with a poor outcome. Some advances have been made in understanding the multiple interrelated pathogenic pathways underlying IPF. The disease is now believed to result from complex interactions among genetic, epigenetic, transcriptional, posttranscriptional, metabolic, and environmental factors. The discovery and validation of theranostic biomarkers are necessary to enable a more precise and earlier diagnosis of IPF and to improve the prediction of future disease behavior. Two drugs recently approved by the US Food and Drug Administration, pirfenidone and nintedanib, have shown the ability to reduce the progression of the disease, although survival benefits are only minimal and neither drug prevents or reverses the disease.
Objective.— To provide a critical overview of the main experimental studies carried out for testing the principal effects of pirfenidone and nintedanib on IPF.
Data Sources.— Experimental (animal and in vitro) studies concerning both drugs were used.
Conclusions.— Pirfenidone has a longer history of preclinical experimental studies than nintedanib. Many studies have been reported more recently (after 2014) and some of them evaluated the association of both drugs, thus suggesting their combination in future therapeutic approaches. Future investigations focusing on targets at molecular, cellular, and tissue levels are necessary to have a better in-depth knowledge of the properties of these drugs and to explore the potential efficacy of both or other drug combinations
Idiopathic pulmonary fibrosis: Are any of the morphological-molecular markers useful in clinical management?
Full text linkIdiopathic pulmonary fibrosis (IPF), the most
common form of chronic interstitial lung disease, is a
severe progressive fibrotic disorder of unknown
aetiology. The disease has a heterogeneous clinical
course, with frequent poor prognosis, similar to
malignant disease. Correctly diagnosing IPF has become
particularly important in view of the availability of more
precise therapeutic indications, thus avoiding steroid
treatment and allowing new approaches with novel
drugs.
To date we have limited information about
biomarkers predictive of progressive disease and
associated complications. Efforts should be made in the
future to more appropriately study lung tissue and then
to extrapolate the most clinically fitting biomarkers.
This approach is already used in routine
management of many cancers and provides a potential
road map for more appropriate clinical care of IPF.
This review will mainly focus on histology and
etiopathogenesis highlighting some morphological and
molecular features that may influence the overall
management of IPF
Are There New Biomarkers in Tissue and Liquid Biopsies for the Early Detection of Non-Small Cell Lung Cancer?
Full text linkLung cancer is one of the most lethal malignancies worldwide, mainly due to its late diagnoses. The detection of molecular markers on samples provided from routine bronchoscopy including several liquid-based cytology tests (e.g., bronchoaspirate, bronchoalveolar lavage) and/or on easily obtained specimens such as sputum could represent a new approach to improve the sensitivity in lung cancer diagnoses. Recently growing interest has been reported for "noninvasive" liquid biopsy as a valuable source for molecular profiling. Unfortunately, a biomarker and/or composition of biomarkers capable of detecting early-stage lung cancer has yet to be discovered even if in the last few years there has been, through the use of revolutionary new technologies, an explosion of lung cancer biomarkers. Assay sensitivity and specificity need to be improved particularly when new approaches and/or tools are used. We have focused on the most important markers detected in tissue, and on several cytological specimens and liquid biopsies overall
Morphological and genetic heterogeneity in multifocal lung adenocarcinoma: The case of a never-smoker woman
No full textDiscrimination of multifocal primary lung cancers from lung metastases is crucial to allow for an appropriate clinical management. We report here a case of multifocal lung adenocarcinomas with different morphological and molecular patterns. Radical surgery of one lung nodule was performed at the time of diagnosis, and subsequently on two other lung nodules. At the time of distant relapse, biopsy was repeated for molecular characterization. The patient was treated with EGFR tyrosine kinase inhibitor according to the detection of EGFR exon 21 mutation in metastatic sample and in one of the three lung tumors, characterized by lower mutated allele frequency. The progression free survival was three months according to radiological criteria and the treatment was provided for six months, until clinical progression. Following the assessment of EGFR mutations by pyrosequencing, tumor samples were analyzed by a 30-gene next generation sequencing (NGS) panel, allowing to study intra-and inter-tumor heterogeneity and to confirm the three lung tumors as independent. Different molecular profiles of synchronous tumors and identical EGFR, PIK3CA and TP53 mutations in one of three primary lung tumors and the metachronous metastasis were identified.
In conclusion, morphological and molecular characterization of multiple lung nodules by NGS may help to define synchronous and metachronous adenocarcinomas, thus affecting surgical indication and systemic treatment. Intratumor heterogeneity may be associated with differential sensitivity to targeted treatment. (C) 2016 Elsevier Ireland Ltd. All rights reserved
Higher Risk of Acute Cellular Rejection in Lung Transplant Recipients with Cystic Fibrosis
No full textBACKGROUND Acute cellular rejection (ACR) affects up to 40% of recipients within the first year after lung transplant (LTx). The aim of this study was to determine the frequency of ACR and associated major risk factors in cystic fibrosis (CF) recipients. Bronchiolitis obliterans syndrome (BOS) and 1-year/long-term survival were also evaluated. MATERIAL AND METHODS ACR was reviewed in 643 scheduled biopsies from 44 CF (Group 1) versus 89 other recipients (Group 2). We performed univariate/multivariate analyses of risk factors for ACR and BOS, and survival analysis. RESULTS Group 1 showed higher ACR frequency, especially for ACR ≥ A2. Multivariable generalized linear models considering both native lung disease and age showed that higher values of ACR index were significantly related to the pretransplant diagnosis of CF. BOS and long-term survival were not influenced by the increased incidence of ACR. Poorer long-term survival was observed in Group 2. CONCLUSIONS CF recipients have a higher ACR risk, which may be due to enhanced immune activation related to a genetic disorder, and younger age
MDM2 and HIF1alpha expression levels in different histologic subtypes of malignant pleural mesothelioma: Correlation with pathological and clinical data
Full text linkMalignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited treatment options. Sarcomatoid/biphasic mesotheliomas are characterized by more aggressive behaviour and a poorer prognosis compared with the epithelioid subtype. To date prognostic and tailored therapeutic biomarkers are lacking. The present study analyzed the expression levels of MDM2 and HIF1alpha in different histologic subtypes from chemonaive MPM patients. Diagnostic biopsies of MPM patients from four Italian cancer centers were centrally collected and analyzed. MDM2 and HIF1alpha expression levels were investigated through immunohistochemistry and RT-qPCR. Pathological assessment of necrosis, inflammation and proliferation index was also performed. Molecular markers, pathological features and clinical characteristics were correlated to overall survival (OS) and progression free survival (PFS). Sixty MPM patients were included in the study (32 epithelioid and 28 non-epithelioid). Higher levels of MDM2 (p < 0.001), HIF1alpha (p = 0.013), necrosis (p = 0.013) and proliferation index (p < 0.001) were seen mainly in sarcomatoid/biphasic subtypes. Higher levels of inflammation were significantly associated with epithelioid subtype (p = 0.044). MDM2 expression levels were correlated with HIF1alpha levels (p = 0.0001), necrosis (p = 0.008) and proliferation index (p = 0.009). Univariate analysis showed a significant correlation of non-epithelioid histology (p = 0.04), high levels of necrosis (p = 0.037) and proliferation index (p = 0.0002) with shorter PFS. Sarcomatoid/biphasic and epithelioid mesotheliomas showed different MDM2 and HIF1alpha expression levels and were characterized by different levels of necrosis, proliferation and inflammation. Further studies are warranted to confirm a prognostic and predictive role of such markers and features
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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