Noves aproximacions per a l'estudi del sistema HLA : de la tipificació dels loci HLA basada en la PCR a temps real al desenvolupament de tetràmers de classe II /

Consultable des del TDXTítol obtingut de la portada digitalitzadaEl Complex Principal de Histocompatibilitat (MHC) és la regió gènica que conté els loci més polimòrfics del genoma. Aquests loci estan implicats en els mecanismes de presentació antigènica als limfòcits T, defineixen la resposta immunitària en general, incloent-hi la histocompatibilitat entre donant i receptor en els trasplantaments d'òrgans. Tot i que existeixen diverses tècniques aplicades a la tipificació dels polimorfismes del MHC i a l'anàlisi de la funcionalitat d'aquestes molècules, les més utilitzades en els laboratoris d'histocompatibilitat presenten encara limitacions i són millorables. En aquest sentit aquesta tesi doctoral descriu la utilització de la PCR a temps real amb sondes d'hibridació com a metodologia per a millorar la tipificació dels loci HLA. En primer lloc s'ha desenvolupat una tècnica de tipificació basada en la PCR a temps real per la família al·lèlica HLA-B27. L'aproximació desenvolupada es basa en tres reaccions amb sondes d'hibridació FRET que permeten donar una resolució mitja-alta dels al·lels HLA-B27 en la població caucàsica, alhora que discriminen els al·lels no associats amb la Espondilitis Anquilopoiètica. Després d'haver comprovat la validesa de la metodologia de tipificació per PCR a temps real amb sondes FRET en una família HLA de polimorfisme reduït, s'ha desenvolupat una aproximació per a la tipificació de baixa resolució del locus HLA-B, el més polimòrfic de tots els loci HLA, basada en la mateixa metodologia. En tercer lloc s'ha comparat la utilització de les sondes de tipus FRET en front a les sondes de tipus Taqman per a la tipificació HLA, i per això s'ha desenvolupat un sistema per a la tipificació del locus de classe II HLA-DR amb aquesta segona metodologia de sondes. Les metodologies de tipificació basades en la PCR a temps real que s'han desenvolupat comparades amb les tècniques de tipificació basades en el DNA tradicionals, comporten diverses avantatges, les principals són en quant a temps total necessari per a realitzar la tècnica i eliminació de passos de processat post PCR. D'altre banda, la comparació de les tres aproximacions de tipificació per PCR a temps real desenvolupades ha demostrat que per a la resolució del polimorfisme HLA són més valides les sondes FRET. Per a millorar les tècniques d'anàlisi de la funcionalitat de les molècules HLA, en la present tesi doctoral s'han elaborat tetràmers de classe II dels loci no clàssics de DRB, en concret dels dos al·lels més freqüents de DRB3. L'aplicació d'aquesta metodologia ha permès millorar les eines disponibles per a l'estudi de la funcionalitat dels denominats loci secundaris de DRB, alhora que també ha permès valorar la magnitud de la resposta cel·lular T CD4+ que reconeix antígens en el context d'aquestes molècules, posant de manifest la rellevància d'aquest locus en la presentació antigènica.The Major Histocompatibility Complex (MHC) is the genetic region that contains the loci more polymorphic of the genome. These loci are involved in the antigenic presentation to the T lymphocytes, defining in general the immune response and specifically including the histocompatibility between donor and acceptor in organ transplantation. There are different techniques that are used for typing the polymorphism of the MHC and also for the analysis of the functionality of these molecules. But the main techniques used for typing in Histocompatibility laboratories have still some limitations that can be solved. In reference to this point this thesis describes the usage of real time PCR with hybridization probes as an assay to be used for HLA typing. As a first approach to prove the validity of the methodology, an HLA-B27 typing assay by real time PCR with FRET probes has been optimized. The described HLA-B27 typing protocol uses three reactions with FRET probes that can achieve a medium-high resolution of the alleles in the Caucasoid population, at the same time alleles non associated with Spondylarthropathies are differentiated. After proving the validity of the real time PCR with FRET probes for a reduced HLA polymorphism, an approach based in the same methodology for typing of the HLA-B locus with a low-medium resolution has been developed. In third place, for HLA typing resolution the usage of the FRET probes has been compared to the usage of Taqman probes. With this aim a typing approach for the HLA-DRB locus with Taqman probes has been developed. The typing methodologies based on real time PCR that have been developed show several advantages when compared to the traditional DNA based typing techniques, the main are on reference to the time required for the developing of the assays and to the elimination of all post-PCR steps. At the same time the evaluation of the three described typing techniques has shown that FRET probes work better to solve the HLA polymorphism. In this thesis MHC class II tetramers have been developed to improve the tools for the functional study of the non classical HLA-DRB loci, concretely of the more frequent alleles of DRB3. This methodology allowed the evaluation of the T CD4+ cell response that recognizes antigens in the context of these HLA molecules, showing the relevancy of this locus in the antigenic presentation

Global lung health: the dangers of mild lung function impairment

The relationship between low lung function and increased mortality risk, especially in the context of ageing and exposure to noxious particles and gases, was established several decades ago. However, it was not until recently that low peak lung function in early adulthood (and thus the vital lung function trajectory that followed, independently of the ageing process) was associated with an increased prevalence, and about a decade earlier incidence, of respiratory and cardiovascular abnormalities, as well as premature death

Cellular Senescence in Lung Fibrosis

Fibrosing interstitial lung diseases (ILDs) are chronic and ultimately fatal age-related lung diseases characterized by the progressive and irreversible accumulation of scar tissue in the lung parenchyma. Over the past years, significant progress has been made in our incomplete understanding of the pathobiology underlying fibrosing ILDs, in particular in relation to diverse age-related processes and cell perturbations that seem to lead to maladaptation to stress and susceptibility to lung fibrosis. Growing evidence suggests that a specific biological phenomenon known as cellular senescence plays an important role in the initiation and progression of pulmonary fibrosis. Cellular senescence is defined as a cell fate decision caused by the accumulation of unrepairable cellular damage and is characterized by an abundant pro-inflammatory and pro-fibrotic secretome. The senescence response has been widely recognized as a beneficial physiological mechanism during development and in tumour suppression. However, recent evidence strengthens the idea that it also drives degenerative processes such as lung fibrosis, most likely by promoting molecular and cellular changes in chronic fibrosing processes. Here, we review how cellular senescence may contribute to lung fibrosis pathobiology, and we highlight current and emerging therapeutic approaches to treat fibrosing ILDs by targeting cellular senescence

Systemic inflammatory response to smoking in chronic obstructive pulmonary disease: evidence of a gender effect

Background Tobacco smoking is the main risk factor of chronic obstructive pulmonary disease (COPD) but not all smokers develop the disease. An abnormal pulmonary and systemic inflammatory response to smoking is thought to play a major pathogenic role in COPD, but this has never been tested directly. Methods We studied the systemic biomarker and leukocyte transcriptomic response (Affymetrix microarrays) to smoking exposure in 10 smokers with COPD and 10 smokers with normal spirometry. We also studied 10 healthy never smokers (not exposed to smoking) as controls. Because some aspects of COPD may differ in males and females, and the inflammatory response to other stressors (infection) might be different in man and women, we stratified participant recruitment by sex. Differentially expressed genes were validated by q-PCR. Ontology enrichment was evaluated and interaction networks inferred. Results Principal component analysis identified sex differences in the leukocyte transcriptomic response to acute smoking. In both genders, we identified genes that were differentially expressed in response to smoking exclusively in COPD patients (COPD related signature) or smokers with normal spirometry (Smoking related signature), their ontologies and interaction networks. Conclusions The use of an experimental intervention (smoking exposure) to investigate the transcriptomic response of peripheral leukocytes in COPD is a step beyond the standard case-control transcriptomic profiling carried out so far, and has facilitated the identification of novel COPD and Smoking expression related signatures which differ in males and females

Treatable traits in the NOVELTY study

Background and objective: Asthma and chronic obstructive pulmonary disease (COPD) are two prevalent and complex diseases that require personalized management. Although a strategy based on treatable traits (TTs) has been proposed, the prevalence and relationship of TTs to the diagnostic label and disease severity established by the attending physician in a real-world setting are unknown. We assessed how the presence/absence of specific TTs relate to the diagnosis and severity of ‘asthma’, ‘COPD’ or ‘asthma + COPD’. Methods: The authors selected 30 frequently occurring TTs from the NOVELTY study cohort (NOVEL observational longiTudinal studY; NCT02760329), a large (n = 11,226), global study that systematically collects data in a real-world setting, both in primary care clinics and specialized centres, for patients with ‘asthma’ (n = 5932, 52.8%), ‘COPD’ (n = 3898, 34.7%) or both (‘asthma + COPD’; n = 1396, 12.4%). Results: The results indicate that (1) the prevalence of the 30 TTs evaluated varied widely, with a mean ± SD of 4.6 ± 2.6, 5.4 ± 2.6 and 6.4 ± 2.8 TTs/patient in those with ‘asthma’, ‘COPD’ and ‘asthma + COPD’, respectively (p < 0.0001); (2) there were no large global geographical variations, but the prevalence of TTs was different in primary versus specialized clinics; (3) several TTs were specific to the diagnosis and severity of disease, but many were not; and (4) both the presence and absence of TTs formed a pattern that is recognized by clinicians to establish a diagnosis and grade its severity. Conclusion: These results provide the largest and most granular characterization of TTs in patients with airway diseases in a real-world setting to date

Characterization, localization and comparison of c-Kit+ lung cells in never smokers and smokers with and without COPD

Background: c-Kit + lung stem cells have been described in the human healthy lung. Their potential relation with smoking and/or chronic obstructive pulmonary disease (COPD) is unknown. Methods: We characterized and compared c-Kit+ cells in lung tissue of 12 never smokers (NS), 15 smokers with normal spirometry (S) and 44 COPD patients who required lung resectional surgery. Flow cytometry (FACS) was used to characterize c-Kit+ cells in fresh lung tissue disaggregates, and immunofluorescence (IF) for further characterization and to determine their location in OCT- embedded lung tissue. Results: We identified 4 c-Kit+ cell populations, with similar proportions in NS, S and COPD: (1) By FACS, c-Kithigh/CD45 + cells (4.03 ± 2.97% (NS), 3.96 ± 5.30% (S), and 5.20 ± 3.44% (COPD)). By IF, these cells were tryptase+ (hence, mast cells) and located around the airways; (2) By IF, c-Kitlow/CD45+/triptase- (0.07 ± 0.06 (NS), 0.03 ± 0.02 (S), and 0.06 ± 0.07 (COPD) cells/field), which likely correspond to innate lymphoid cells; (3) By FACS, c-Kitlow/CD45-/CD34+ (0.95 ± 0.84% (NS), 1.14 ± 0.94% (S) and 0.95 ± 1.38% (COPD)). By IF these cells were c-Kitlow/CD45-/CD31+, suggesting an endothelial lineage, and were predominantly located in the alveolar wall; and, (4) by FACS, an infrequent c-Kitlow/CD45-/ CD34- population (0.09 ± 0.14% (NS), 0.08 ± 0.09% (S) and 0.08 ± 0.11% (COPD)) compatible with a putative lung stem cell population. Yet, IF failed to detect them and we could not isolate or grow them, thus questioning the existence of c-Kit+ lung stem-cells. Conclusions: The adult human lung contains a mixture of c-Kit+ cells, unlikely to be lung stem cells, which are independent of smoking status and/or presence of COPD

Multi-level immune response network in mild-moderate Chronic Obstructive Pulmonary Disease (COPD)

Background: Chronic Obstructive Pulmonary Disease (COPD) is associated with an abnormal pulmonary and systemic immune response to tobacco smoking. Yet, how do immune cells relate within and between these two biological compartments, how the pulmonary infiltrate influences the lung transcriptome, and what is the role of active smoking vs. presence of disease is unclear. Methods: To investigate these questions, we simultaneously collected lung tissue and blood from 65 individuals stratified by smoking habit and presence of the disease. The immune cell composition of both tissues was assessed by flow cytometry, whole lung transcriptome was determined with Affymetrix arrays, and we used Weighted Gene Co-expression Network Analysis (WGCNA) to integrate results. Results: Main results showed that: (1) current smoking and the presence of COPD were both independently associated with a reduction in the proportion of lung T cells and an increase of macrophages, specifically those expressing CD80 + CD163+; (2) changes in the proportion of infiltrating macrophages, smoking status or the level of airflow limitation were associated to different WGCNA modules, which were enriched in iron ion transport, extracellular matrix and cilium organization gene ontologies; and, (3) circulating white blood cells counts were correlated with lung macrophages and T cells. Conclusions: Mild-moderated COPD lung immune infiltrate is associated with the active smoking status and presence of disease; is associated with changes in whole lung tissue transcriptome and marginally reflected in blood

SARS-CoV-2 T-cell response in COVID-19 convalescent patients with and without lung sequelae

A specific T-cell response persists in the majority of COVID-19 patients 6 months after hospital discharge. This response is more prominent in those who required critical care during the acute COVID-19 episode but is reduced in patients with lung sequelae

Lung immune signatures define two groups of end-stage IPF patients

BackgroundThe role of the immune system in the pathobiology of Idiopathic Pulmonary Fibrosis (IPF) is controversial.MethodsTo investigate it, we calculated immune signatures with Gene Set Variation Analysis (GSVA) and applied them to the lung transcriptome followed by unbiased cluster analysis of GSVA immune-enrichment scores, in 109 IPF patients from the Lung Tissue Research Consortium (LTRC). Results were validated experimentally using cell-based methods (flow cytometry) in lung tissue of IPF patients from the University of Pittsburgh (n = 26). Finally, differential gene expression and hypergeometric test were used to explore non-immune differences between clusters.ResultsWe identified two clusters (C#1 and C#2) of IPF patients of similar size in the LTRC dataset. C#1 included 58 patients (53%) with enrichment in GSVA immune signatures, particularly cytotoxic and memory T cells signatures, whereas C#2 included 51 patients (47%) with an overall lower expression of GSVA immune signatures (results were validated by flow cytometry with similar unbiased clustering generation). Differential gene expression between clusters identified differences in cilium, epithelial and secretory cell genes, all of them showing an inverse correlation with the immune response signatures. Notably, both clusters showed distinct features despite clinical similarities.ConclusionsIn end-stage IPF lung tissue, we identified two clusters of patients with very different levels of immune signatures and gene expression but with similar clinical characteristics. Weather these immune clusters differentiate diverse disease trajectories remains unexplored