Development and application of proteomics as aid for unraveling smoke-induced COPD

De doelstelling van dit proefschrift was om potentiële biomarkers voor Chronic Obstructive Pulmonary Disease (COPD) te identificeren met behulp van een proteomics benadering. Om de allereerste processen bij het ontstaan van COPD te identificeren, hebben we niet alleen oudere personen met een aangetoond COPD vergeleken met gezonde leeftijdsgenoten, maar ook jongere personen die nog geen COPD hebben, maar het mogelijk wel kunnen ontwikkelen op latere leeftijd. Voordat wij onze proefpersonen blootstelden aan het roken van 3 sigaretten hebben wij voorbereidend onderzoek gedaan naar de haalbaarheid van proteomics analyse in de vloeistoffilm die de luchtwegen bedekt (de zogenaamde Epithelial Lining Fluid of ELF). Daarbij hebben we aandacht geschonken aan zowel de bemonstering als proteomics analyse. Met behulp van chemische stable isotope labeling (iTRAQ®-8Plex) werden significante verschillen in de aanwezigheid van vier eiwitten aangetoond tussen ELF van COPD-patiënten en gezonde controles. Al deze eiwitten spelen een rol bij twee belangrijke processen die bijdragen aan het ontstaan van COPD, namelijk ontsteking en oxidatieve stress. Zoals eerder aangegeven vormt roken (in de westerse wereld) de belangrijkste risicofactor voor COPD, terwijl slechts een gedeelte van de rokers COPD ontwikkelt. De genetische predispositie was voor ons de directe aanleiding van het onderzoek, en mede de reden om de gevoeligheid voor COPD op jonge leeftijd in te schatten op basis van de familieanamnese. Samenvattend toont onze studie aan dat personen met een positieve familiegeschiedenis voor COPD op jonge leeftijd anders reageren op sigarettenrook dan jonge personen met een negatieve familiegeschiedenis. Vooral Serpine B3 en Uteroglobine bleken eiwitten die een cruciale rol zouden kunnen spelen in de ontwikkeling van COPD

Susceptibility to COPD:Differential Proteomic Profiling after Acute Smoking

Cigarette smoking is the main risk factor for COPD (Chronic Obstructive Pulmonary Disease), yet only a subset of smokers develops COPD. Family members of patients with severe early-onset COPD have an increased risk to develop COPD and are therefore defined as "susceptible individuals". Here we perform unbiased analyses of proteomic profiles to assess how "susceptible individuals" differ from age-matched "non-susceptible individuals" in response to cigarette smoking. Epithelial lining fluid (ELF) was collected at baseline and 24 hours after smoking 3 cigarettes in young individuals susceptible or non-susceptible to develop COPD and older subjects with established COPD. Controls at baseline were older healthy smoking and non-smoking individuals. Five samples per group were pooled and analysed by stable isotope labelling (iTRAQ) in duplicate. Six proteins were selected and validated by ELISA or immunohistochemistry. After smoking, 23 proteins increased or decreased in young susceptible individuals, 7 in young non-susceptible individuals, and 13 in COPD in the first experiment; 23 proteins increased or decreased in young susceptible individuals, 32 in young non-susceptible individuals, and 11 in COPD in the second experiment. SerpinB3 and Uteroglobin decreased after acute smoke exposure in young non-susceptible individuals exclusively, whereas Peroxiredoxin I, S100A9, S100A8, ALDH3A1 (Aldehyde dehydrogenase 3A1) decreased both in young susceptible and non-susceptible individuals, changes being significantly different between groups for Uteroglobin with iTRAQ and for Serpin B3 with iTRAQ and ELISA measures. Peroxiredoxin I, SerpinB3 and ALDH3A1 increased in COPD patients after smoking. We conclude that smoking induces a differential protein response in ELF of susceptible and non-susceptible young individuals, which differs from patients with established COPD. This is the first study applying unbiased proteomic profiling to unravel the underlying mechanisms that induce COPD. Our data suggest that SerpinB3 and Uteroglobin could be interesting proteins in understanding the processes leading to COPD

Recycling wastes from Rubus idaeus by-products: sourdough bread production as a new end-use of exhausted seeds still containing active compounds

In the past few years, in light of the European Commission’s directives on environmental sustainability, the food industries and academic research institutes focused their attention on the use of plant waste and by- products as ingredients to improve the nutritional value of foods. In this work, the agro-industrial waste from cold pressing of Rubus ideaus seeds (WRS) was explored as a valuable food ingredient in bread production. Bread making trials were performed under laboratory conditions using a mix of wheat flour and semolina (1/1) and sourdough as fermenting agent. Two experimental bread productions (5-WRS and 10-WRS) were obtained replacing wheat flour/semolina with WRS at 5 and 10 % (w/w), while control production (CTR) was WRS free. WRS did not negatively affect sourdough LAB starter development and their cell densities reached almost 108 CFU/g at the end of fermentation. Illumina technology identified 14 taxonomic groups, and lactobacilli constituted the major group of the mature sourdough (75.91 % of relative abundance (RA)) and doughs (82.08 – 88.76 % RA). WRS decreased the volume and increased crust and crumb redness of the final breads. The replacement of wheat flour/semolina with WRS at 5 and 10 % (w/w) significantly increased the functional value of breads, in term of content of polyphenolic compounds, proanthocyanidins content and antioxidant activity. In particular, these functional properties increased until 25 % in bread, confirming the thermal stability of WRS. The addition of WRS did not spoil the sensory traits of breads, but the highest values of overall satisfaction were displayed by 5 % (w/w) WRS enriched breads. This work clearly indicated that the addition of WRS in bread production represents a promising strategy to increase the antioxidant activity in cereal-based fermented products

Acute and chronic inflammatory responses induced by smoking in individuals susceptible and non-susceptible to development of COPD:from specific disease phenotyping towards novel therapy. Protocol of a cross-sectional study

<p>Introduction: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease with pulmonary and extra-pulmonary manifestations. Although COPD is a complex disease, diagnosis and staging are still based on simple spirometry measurements. Different COPD phenotypes exist based on clinical, physiological, immunological and radiological observations. Cigarette smoking is the most important risk factor for COPD, but only 15-20% of smokers develop the disease, suggesting a genetic predisposition. Unfortunately, little is known about the pathogenesis of COPD, and even less on the very first steps that are associated with an aberrant response to smoke exposure. This study aims to investigate the underlying local and systemic inflammation of different clinical COPD phenotypes, and acute effects of cigarette smoke exposure in individuals susceptible and non-susceptible for the development of COPD. Furthermore, we will investigate mechanisms associated with corticosteroid insensitivity. Our study will provide valuable information regarding the pathogenetic mechanisms underlying the natural course of COPD.</p><p>Methods and analysis: This cross-sectional study will include young and old individuals susceptible or non-susceptible to develop COPD. At a young age (18-40 years) 60 'party smokers' will be included who are called susceptible or non-susceptible based on COPD prevalence in smoking family members. In addition, 30 healthy smokers (age 40-75 years) and 110 COPD patients will be included. Measurements will include questionnaires, pulmonary function, low-dose CT scanning of the lung, body composition, 6 min walking distance and biomarkers in peripheral blood, sputum, urine, exhaled breath condensate, epithelial lining fluid, bronchial brushes and biopsies. Non-biased approaches such as proteomics will be performed in blood and epithelial lining fluid.</p><p>Ethics and dissemination: This multicentre study was approved by the medical ethical committees of UMC Groningen and Utrecht, the Netherlands. The study findings will be presented at conferences and will be reported in peer-reviewed journals.</p><p>Trial registration: ClinicalTrials.gov, NCT00807469 (study 1) and NCT00850863 (study 2).</p>

Characteristics of the participating subjects.

<p>Characteristics of the participating subjects.</p

Immunohistochemistry of aldehyde dehydrogenase 3A1.

<p>Panel A: immunostaining of a COPD patient current smoker. Panel B: immunostaining of a healthy control current smoker. Panel C: immunostaining of a COPD patient ex-smoker. Panel D: immunostaining of a healthy control non-smoker. All COPD patients are GOLD STAGE II. The red arrows indicate epithelial cells and blue arrows indicate macrophages, more or less positive for ALDH3A1.</p

ELISA results of individual epithelial lining fluid (ELF) samples of young susceptible individuals, young non-susceptible individuals, and established COPD patients, before and after acute smoking.

<p>Results are given in box-plots with medians and interquartile ranges. *: p<0.05 before vs after smoking, ∧: p<0.05 vs young susceptible individuals at baseline.</p