Abstracts of the 20th Symposium of the Croatian Society of Medical Biochemists

Sanja Popović-Grle

 

Chronic obstructive pulmonary disease (COPD) is a term for diseases previously named chronic bronchitis and emphysema. The most commonly used definition today for the COPD is given by the Initiative started by World Health organization (WHO) and National Heart, Blood and Lung Institute (NHLBI) - Global Initiative for Chronic Obstructive Lung Diseases (GOLD): “Chronic obstructive pulmonary disease (COPD) is preventable and treatable disease with some significant extra pulmonary effect that may contribute to the severity in individual patient. Its pulmonary component is characterized by airflow limitation for more than 6 months not fully reversible to bronchodilators. This obstruction is usually progressive and associated with abnormal inflammatory response to noxious particles and gases”.

COPD is one of the most under diagnosed diseases in the world, both in patients and doctors. About 75% of patients do not have established diagnosis, most of them in mild degree, but also 4% in severe and 1% in very severe degree of COPD. The reason for that is slow progression of symptoms as cough and excersize intolerance, as well as development of disease in elderly. COPD in one of diseases which prevalence is increasing the most – through 40 years it has increased for + 163%. By mortality the COPD is on 4th place in the world, after myocardial infarction, malignant diseases and cerebrovascular insults. There are around 600 billion patients with COPD today in the world, double than diabetics.

It is usually accepted that COPD patients have decreased values of spirometry parameters, with forced expiratory volume in first second (FEV1) under 80% of reference value, and FEV1/ FVC (index Tiffeneau) under 0,70 (or 70%).According to the GOLD there are 4 degrees of COPD. Mild degree (GOLD 1) is present in smokers who have symptoms, but still have normal lung function (FEV1 > 80%). Moderate degree of COPD (GOLD 2) is present in patients with FEV1 between 50-80% from reference values for that individual, severe degree of COPD (GOLD 3) is present in patients with FEV1 between 30-50%, while very severe degree of COPD (GOLD 4) is present in patients with FEV1 between < 30% with respiratory insufficiency.

 

József Petrik

 

Oxidative stress and inflammation are the major hallmarks of chronic obstructive pulmonary disease (COPD). Reactive oxygen species (ROS) and reactive nitrogen species (RNS) contribute to normal physiological functions such as cellular differentiation, cell signalling, apoptosis, and bactericidal activities, but in other hand, the enhanced concentrations of oxidants cause lipid peroxidation, protein oxidation, and DNA damage. Cells are protected against this oxidative attack by well developed enzymatic and non enzymatic antioxidant system.

ROS, either directly or via the formation of lipid peroxidation products, such as 4-hydroxy-2-nonenal and F2-isoprostanes, may play a role in enhancing the inflammation through the activation and phosphorylation of mitogen-activated protein kinases and redox-sensitive transcription factors such as nuclear factor- κB and activator protein-1 in COPD.

Cells and tissues are continuously exposed to oxidants which can be either generated endogenously by metabolic reactions (mitochondrial respiration, the NADPH oxidase system that is present in phagocytes and in epithelial cells, xanthine/xanthine oxidase), or exogenously such as inhaled from air pollutants or cigarette smoke. Whereas, cigarette smoke contains over 4700 chemical compounds, and both the tar and gas phases contain numerous free radicals and other oxidants, thus cigarette smoking becomes the major risk factor of COPD. One cigarette puff contains more than 1014 free radicals. ROS such as superoxide anion and the hydroxyl radical are unstable molecules with unpaired electrons, capable of initiating oxidative processes. Consequences of oxidative stress in COPD can be measured by biomarkers such as: a) hydrogen peroxide in exhaled breath, b) release of ROS from peripheral blood neutrophils, eosinophils and macrophages, c) MPO, EPO and xanthine/xanthine oxidase activities in bronchoalveolar lavage fluid, and d) F2-isoprostane, lipid peroxide (TBARS), CO and NO levels in exhaled breath.

 

A.J.M. van Oosterhout

 

Cigarette smoking and genetic susceptibility are the main risk factors for the development of Chronic Obstructive Pulmonary Disease (COPD). Three hypotheses have been postulated to underlie the tissue destruction and chronic airway inflammation characteristic for COPD patients: (i) an imbalance between oxidants and anti-oxidants, (ii) an imbalance between proteases and anti-proteases, and (iii) an adaptive immune response characterized by CD8+ T-cells and B-cells. Airway epithelial cells are the first line of defence against harmful inhaled substances including cigarette smoke. Therefore, we studied the effects of cigarette smoke (CS) on human airway epithelial cells in vitro. It appeared that CS disturbs mitochondrial function by inhibition of the electron transfer chain, leading to decreased mitochondrial membrane potential, oxygen consumption and ATP production and increased generation of intracellular reactive oxygen species (ROS). Moreover, loss of ATP generation induces airway apoptotic epithelial cells into necrosis. It is well-know that necrotic cell death generates danger signals that lead to activation of immune- and inflammatory cells. In order to translate these observations into an in vivo mouse model, BALB/c mice were exposed to CS or ROS-depleted CS for five consecutive days. Exposure to CS, but not ROS-depleted CS, induced massive neutrophil influx into the airways associated with increased levels of pro-inflammatory cyto- and chemokines. This model will be useful to further dissect the pathways leading to acute CS induced inflammation.

 

Tihana Žanić Grubišić

 

COPD is chronic, slowly progressive disease characterized with accelerated increase in airflow limitation resulting from hiperactive local inflammatory response to inhaled particles and gases. The current paradigm for the pathogenesis of COPD is involving 1) an abnormall inflammatory response in the lung, 2) increased levels of cytokines and chemokines, 3) accumulation and activation of neutrophils, macrophages, monocytes and CD8+ T lymphocytes in airways, 4) increased levels of inflammatory mediators: TNF-α, cytokines IL-6 and IL-8 in sera of patients, in the stable phase and especially during the exacerbations, 5) deregulation of protease - antiprotease balance resulting in an impaired processing of inflammatory mediators, 6) development of tissue and systemic oxidative stress, 7) increased concentrations of redox-sensitive transcription factors and 8) development of systemic pro-oxidative and inflammatory conditions leading to cardiovascular diseases, changes in the bone masse, progressive muscle waist and loss of muscle function. Intensity of these changes is connected with progressive destruction of pulmonary parenchyma. Deregulation in the activity of proteases, metalloproteinases MMP8 and MMP9 and dipeptidyl peptidase IV released by cells, in response to inflammation, cause breakdown of the extracellular matrix generating peptide fragments still active on leukocytes, which in turn release cytokines and more proteases, leading to further leukocyte infiltration and disease progression.

Great heterogeneity in the rate of progression and clinical manifestations of COPD could not be successfully monitored because of the lack of reliable markers for the severity of the disease.. The specific biochemical markers for the progression of inflammation in COPD have not been proposed. In our work we try to evaluate reliable markers that could be assayed in samples obtained by non-invasive methods – blood serum and cells polymorphonucleares, lymphocytes, erythrocytes, urine and possibly in the expired breath condensate.

 

Karmela Barišić

 

Increasing prevalence of chronic obstructive pulmonary disease (COPD) is an important public health concern. COPD is a clinical entity which includes a collection of disorders that share the common physiological features of expiratory airflow limitation. The etiology of COPD involves a complex interplay between genetic and environmental factors.

COPD is associated with an abnormal inflammatory response of the lung to noxious particles or gases, most commonly tobacco smoke. Active cigarette smoking contributes to the origin of COPD in more than 90% of subjects who are susceptible. However, in Caucasians only 10-20% of heavy cigarette smokers develop COPD. This fact suggests that genetic factors are important in developing of COPD.

Several genes that are involved in COPD pathogenesis have been identified and classified into following groups:

1. genes encoding xenobiotic metabolizing enzymes (microsomal epoxide hydrolase, EPHX1; glutathione S-transferase, GST; superoxide dismutase, SOD),

2. genes encoding inflammatory mediators (interleukin-1 family, IL-1; tumor necrosis factorα, TNF-α; transforming growth factor β, TGF-β),

3. genes encoding proteases/antiproteases (protease inhibitor, PI; matrix metalloproteinases, MMPs; elastin, ELN; tissue inhibitor of metalloproteinase, TIMP),

4. novel genes (disintegrin and metalloprotease, ADAM).

Moreover, increased frequency of microsatellite DNA instability (MSI) has been detected in COPD patients. MSI has been correlated with a high somatic mutation rate and is associated with a defective DNA mismatch repair system. MSI indicates destabilisation of the genome at various loci. COPD patients positive for G29802, D13S71 and D14S588 markers presented an increased exacerbation frequency. The significant association between MSI and COPD exacerbations indicates possible involvement of somatic mutations in pathogenesis of disease.

 

Sanja Popović-Grle

 

According to current knowledge inhaled corticosteroids are indicated in the therapy with severe and very severe degree, when FEV1 is less than 50% and in COPD patients with frequent exacerbations (more than 3 annually). The advantage of inhaled therapy is that drug could be delivered directly in the affected organ, where it has high concentration, while avoiding systemic adverse effects. It is proven that inhaled steroids acts the best when they are combined with long-acting 2-agonists, in Croatia powerful combine drugs Seretide® i Symbicort® are available. Application of these drugs increases quality of life in patients with COPD, decreases mortality, especialy by decreasing frequency of COPD exacerbations. Acute exacerbation of COPD could be life threating, where the mortality rate is the same as in acute coronary incident. Because of that COPD exacerbations should be energically treated with antibiotics, usually from the beta lactam group or cinolons, with increased bronchodilators doses, parenteral or oral steroids and oxygeotherapy. The most severe COPD patients have the last oppurtunity for improved health status in lung transplantation, usually unilateral, with survival rate 50% in 5 years after lung transplantation.

 

Lada Rumora

 

Chronic obstructive pulmonary disease (COPD) is characterized by chronic local and systemic inflammation, and increased oxidative stress.

Cigarette smoking is the major etiological factor responsible for COPD. Reactive oxygen species (ROS) generated by cigarette smoke and by activated lung and peripheral blood cells, may play a role in enhancing the inflammation through activation of redox-sensitive signalling molecules, including mitogen-activated protein kinases (MAPKs) and heat shock proteins (Hsps).

Three major mammalian MAPKs are extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. While ERK pathway usually induces cell proliferation, JNK and p38 pathways primarily induce apoptosis.

Hsps are implicated in many steps of apoptotic machinery and they act as molecular chaperones by assisting the correct folding of proteins, and by preventing their aggregation. Hsps could stimulate antioxidant defence of cells by decreasing intracellular levels of ROS and by neutralizing toxic effects of oxidized proteins.

We assessed expression and activation of MAPKs and expression of Hsp70 and Hsp27 in leukocytes of COPD patients and healthy individuals.

ERK was activated in non-smokers only. In contrast, strong induction of JNK and p38 phosphorylation was detected in COPD smokers and ex-smokers, but also in healthy smokers. In addition, Hsp70 and Hsp27 were decreased in COPD ex-smokers and healthy smokers, and especially in COPD smokers.

These results show that COPD and smoking affect intracellular signaling pathways. Understanding of the basic cellular and molecular mechanisms in COPD is essential for identification of molecules that may serve as targets for diagnosis and therapeutic interventions.

 

Anita Somborac

 

The mechanism by which cigarette smoking leads to development of chronic obstructive pulmonary disease is still unclear. Cigarette smoke contributes to oxidative damage of proteins inside the lung. One of the main systems to protect cells from damaged proteins is ubiquitin-proteasome pathway. Degradation of proteins via this pathway involves conjugation of multiple ubiquitin moieties and degradation of the tagged protein by proteasome into peptides which are rapidly hydrolyzed into amino acids by proteases and aminopeptidases. It was shown that cigarette smoke causes an increase of free amino acids inside the A549 airway epithelial cells. The question is weather the proteasome is involved in degradation of proteins damaged by cigarette smoke. We have exposed A549 cells to cigarette smoke and Epoxomicin (inhibitor of proteasomal activity) and we have shown, using LC-MS method, that proteasome is involved in the increase of free amino acids (P < 0,001). Western blot analysis has shown that cigarette smoke causes an increase of ubiquitin-protein conjugates. Proteasomal active sites were labelled by using the fluorescent probe MV151 and in general were not disturbed by cigarette smoke. Using the fluorogenic substrates we have shown that cigarette smoke changes all three kinds of proteasomal activities. Briefly, our data suggest that cigarette smoke induces damage to proteins which are subsequently ubiquitinated and tagged for proteasomal degradation. Proteasome is involved in elimination of proteins damaged by cigarette smoke. Although proteasomal active sites remain intact, cigarette smoke significantly disturbs proteasomal activity which may contribute to the aggregation of damaged proteins and enhanced cell death.

 

Mirela Matokanović

 

Heat shock proteins are intracellular proteins with ability to stabilize missfolded proteins and peptides, thereby promoting cell survival and preventing programmed cell death. Genes encoding members of the heat shock protein (Hsp70) family hsp70-1, hsp70-2 i hsp70-hom lie in the class III region of the human major histocompatibility complex (MHC). Relating to their location, polymorphisms of these genes were analyzed in association with occurence of different diseases with immune component. Relating to their chaperone role polymorphisms of these genes were also analyzed in association with an oxidative/antioxidative imbalance.

In the pathogenesis of chronic obstructive lung disease (COPD) oxidative/antioxidative pathways play important role. Therefore we studied hsp70-2 and hsp70-hom gene polymorphisms using restriction fragment length polymorphism – polymerase chain reaction method (RFLP-PCR). The obtained data showed that there were no statistically significant difference in A/G(+1267) polymorphism distribution in the coding region of hsp70-2, as well as in T/C (+2437) polymorphism distribution in the coding region of hsp70-hom between healthy individuals and patients with COPD.

Hsp32 gene (HMOX-1) encodes for small heat shock protein 32 (Hsp32 or heme oxygenase-1), enzyme that catalyzes the degradation of heme to biliverdin in a reaction which provides cells with protection in oxidative mediated injury. Polymorphism of this gene is presented in the number GT repeats in 5’-flanking region responsible for gene expression and plausible enzyme induction in oxidative stress conditions. The obtained data showed that there was no statistically significant difference in hsp32 gene polymorphism between between healthy individuals and patients with COPD.