Molecular mechanisms of pathogenesis of chronic obstructive pulmonary disease

Kronična opstrukcijska plućna bolest (KOPB) najučestalija je kronična respiracijska bolest s progresivnim tijekom. Temeljni uzroci KOPB-a uključuju pušenje cigareta, čimbenike okoline te gensku predispoziciju. Dosadašnje spoznaje o razvoju upale, oksidacijskom stresu i neravnoteži proteaza i antiproteaza nisu dostatne za objašnjenje nastanka i progresije bolesti. Ovo istraživanje pokazalo je da ekstrakt dima cigareta u A549 alveolarnim epitelnim stanicama uzrokuje oksidacijska oštećenja staničnih lipida i proteina, što bi moglo voditi do gubitka važnih funkcija stanice, rezultirajući smrću stanice. Nakupljanje oštećenih proteina u stanicama vodi do aktivacije ER stresnog odgovora u vidu povećanja razine fosforiliranog eIF2α i smanjenja de novo sinteze proteina. Smanjena sinteza proteina vjerojatno pridonosi povećanju koncentracije slobodnih aminokiselina unutar stanica zajedno s razgradnjom proteina oštećenih dimom cigareta putem ubikvitin-proteasomalnog sustava. Iako se ER stresni odgovor obično povezuje s povećanom aktivnosti proteasoma, dim cigareta uzrokuje značajno smanjenje kaspazne, tripsinske i kimotripsinske aktivnosti proteasoma, što ih čini nedovoljno učinkovitima u procesu uklanjanja oštećenih proteina. Kao dio staničnog odgovora na stres dolazi i do porasta ekspresije šaperonskih proteina HSP32 i HSP27, što nismo pokazali i za HSP70. Također dolazi do promjena u MAPK signalnim putovima: značajno raste razina aktivirane p38, ne dolazi do aktivacije ERK, a aktivaciju JNK nismo uspjeli detektirati. Nadalje, rezultati su pokazali da A549 stanice ne sadrže MMP-9, ali pod utjecajem ekstrakta dima cigareta s vremenom u medij pojačano otpuštaju MMP-2, a TIMP-1 otpuštaju u manjoj količini, što bi moglo pridonijeti ukupnoj neravnoteži proteaza i antiproteaza. U bazalnim uvjetima polimorfonuklearni leukociti (PMN) bolesnika s KOPB-om otpuštaju značajno više MMP-9 u odnosu na zdrave kontrole. Stimulacijom PMN zdravih kontrola ekstraktom dima cigareta povećava se otpuštanje MMP-9, dok ista stimulacija u bolesnika s KOPB-om ne izaziva dodatno povećanje visoke razine otpuštanja MMP-9, što dokazuje kroničnu aktivaciju PMN bolesnika i njihovu moguću ulogu u razgradnji izvanstaničnog matriksa u plućima. Sistemska neravnoteža proteaza i antiproteaza u bolesnika s KOPB-om očituje se povećanom koncentracijom MMP-9 u plazmi. Analiza ROC krivulja pokazala je vrlo dobru dijagnostičku točnost za koncentraciju MMP-9, što je čini dobrim biljegom za razlikovanje bolesnika i zdravih osoba. Ispitivanje učestalosti polimorfizama MMP9 gena (-1562 C/T i (CA)n dinukleotidna ponavljanja u promotorskoj regiji te Q279R u egzonu 6) pokazalo je da nema značajnih razlika između bolesnika s KOPB-om i zdravih ispitanika, kao niti povezanosti koncentracije MMP-9 s genotipovima ispitanika. U serumu bolesnika s KOPB-om, kod kojih je dokazana sistemska upala (povećani ukupni leukociti, neutrofili i CRP), nalazimo smanjenu aktivnost DPPIV, proteaze koja bi mogla pridonositi razvoju upale. S obzirom na dokazanu vrlo dobru dijagnostičku točnost, ovaj enzim bi se također mogao koristiti kao učinkovit biokemijski pokazatelj KOPB-a.Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory disease with a progressive course. The principal causes of COPD include cigarette smoking, environmental factors and genetic predisposition. Current knowledge about the development of inflammation, oxidative stress and protease-antiprotease imbalance is not sufficient to explain the origin and progression of the disease. In this study we found that cigarette smoke extract causes oxidative damage to cellular lipids and proteins in A549 alveolar epithelial cells, which could lead to the loss of important cell functions, eventually resulting in cell death. The accumulation of damaged proteins in cells leads to the activation of ER stress response in the form of increased levels of phosphorylated eIF2α and reduced de novo protein synthesis. It is likely that reduced protein synthesis contributes to the increase in the level of free amino acids in the cells, together with the degradation of proteins damaged by cigarette smoke inside the ubiquitin-proteasome system. ER stress response is usually associated with increased proteasomal activity; however, cigarette smoke causes a significant reduction in caspase-, trypsin- and chymotrypsin-proteasomal activities, making the proteasome inefficient in the process of removing damaged proteins. As a part of the cellular stress response, we observed an increase in the expression of chaperone proteins HSP32 and HSP27, which was not shown for HSP70. Likewise, we noticed the changes in MAPK signaling pathways: a significant increase in the level of activated p38, no activation of ERK and undetectable activation of JNK. Furthermore, the results showed that A549 cells do not produce MMP-9, but after a while treatment with the cigarette smoke extract increases the release of MMP-2 in the medium and decreases the release of TIMP-1, which probably contributes to the overall protease-antiprotease imbalance. In basal conditions polymorphonuclear leukocytes (PMN) of patients with COPD release significantly much more MMP-9 compared to healthy controls. Stimulation of PMN of healthy controls with cigarette smoke extract increases the release of MMP-9, while in patients with COPD no further increase over the high secretion rate was found, suggesting the chronic activation of PMN of patients with COPD and their possible role in the degradation of extracellular matrix in the lungs. Systemic protease-antiprotease imbalance in plasma of patients with COPD is manifested by increased concentration of MMP-9. ROC curve analysis showed a very good diagnostic accuracy for concentration of MMP-9, making it a good biomarker for distinguishing patients from healthy individuals. Testing the frequency of MMP9 gene polymorphisms (-1562 C/T and (CA)n dinucleotide repeat in the promoter region, and Q279R in exon 6) showed no significant difference between patients with COPD and healthy subjects, and no correlation of MMP-9 concentration with subjects’ genotypes. In sera of patients with COPD, with the observed systemic inflammation (increased total leukocytes, neutrophils and CRP), we found a decrease in activity of DPPIV, a protease that could be contributing to the development of inflammation. As showing a very good diagnostic accuracy, this enzyme could also be used as an effective biochemical marker of COPD

Molecular mechanisms of pathogenesis of chronic obstructive pulmonary disease

Kronična opstrukcijska plućna bolest (KOPB) najučestalija je kronična respiracijska bolest s progresivnim tijekom. Temeljni uzroci KOPB-a uključuju pušenje cigareta, čimbenike okoline te gensku predispoziciju. Dosadašnje spoznaje o razvoju upale, oksidacijskom stresu i neravnoteži proteaza i antiproteaza nisu dostatne za objašnjenje nastanka i progresije bolesti. Ovo istraživanje pokazalo je da ekstrakt dima cigareta u A549 alveolarnim epitelnim stanicama uzrokuje oksidacijska oštećenja staničnih lipida i proteina, što bi moglo voditi do gubitka važnih funkcija stanice, rezultirajući smrću stanice. Nakupljanje oštećenih proteina u stanicama vodi do aktivacije ER stresnog odgovora u vidu povećanja razine fosforiliranog eIF2α i smanjenja de novo sinteze proteina. Smanjena sinteza proteina vjerojatno pridonosi povećanju koncentracije slobodnih aminokiselina unutar stanica zajedno s razgradnjom proteina oštećenih dimom cigareta putem ubikvitin-proteasomalnog sustava. Iako se ER stresni odgovor obično povezuje s povećanom aktivnosti proteasoma, dim cigareta uzrokuje značajno smanjenje kaspazne, tripsinske i kimotripsinske aktivnosti proteasoma, što ih čini nedovoljno učinkovitima u procesu uklanjanja oštećenih proteina. Kao dio staničnog odgovora na stres dolazi i do porasta ekspresije šaperonskih proteina HSP32 i HSP27, što nismo pokazali i za HSP70. Također dolazi do promjena u MAPK signalnim putovima: značajno raste razina aktivirane p38, ne dolazi do aktivacije ERK, a aktivaciju JNK nismo uspjeli detektirati. Nadalje, rezultati su pokazali da A549 stanice ne sadrže MMP-9, ali pod utjecajem ekstrakta dima cigareta s vremenom u medij pojačano otpuštaju MMP-2, a TIMP-1 otpuštaju u manjoj količini, što bi moglo pridonijeti ukupnoj neravnoteži proteaza i antiproteaza. U bazalnim uvjetima polimorfonuklearni leukociti (PMN) bolesnika s KOPB-om otpuštaju značajno više MMP-9 u odnosu na zdrave kontrole. Stimulacijom PMN zdravih kontrola ekstraktom dima cigareta povećava se otpuštanje MMP-9, dok ista stimulacija u bolesnika s KOPB-om ne izaziva dodatno povećanje visoke razine otpuštanja MMP-9, što dokazuje kroničnu aktivaciju PMN bolesnika i njihovu moguću ulogu u razgradnji izvanstaničnog matriksa u plućima. Sistemska neravnoteža proteaza i antiproteaza u bolesnika s KOPB-om očituje se povećanom koncentracijom MMP-9 u plazmi. Analiza ROC krivulja pokazala je vrlo dobru dijagnostičku točnost za koncentraciju MMP-9, što je čini dobrim biljegom za razlikovanje bolesnika i zdravih osoba. Ispitivanje učestalosti polimorfizama MMP9 gena (-1562 C/T i (CA)n dinukleotidna ponavljanja u promotorskoj regiji te Q279R u egzonu 6) pokazalo je da nema značajnih razlika između bolesnika s KOPB-om i zdravih ispitanika, kao niti povezanosti koncentracije MMP-9 s genotipovima ispitanika. U serumu bolesnika s KOPB-om, kod kojih je dokazana sistemska upala (povećani ukupni leukociti, neutrofili i CRP), nalazimo smanjenu aktivnost DPPIV, proteaze koja bi mogla pridonositi razvoju upale. S obzirom na dokazanu vrlo dobru dijagnostičku točnost, ovaj enzim bi se također mogao koristiti kao učinkovit biokemijski pokazatelj KOPB-a.Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory disease with a progressive course. The principal causes of COPD include cigarette smoking, environmental factors and genetic predisposition. Current knowledge about the development of inflammation, oxidative stress and protease-antiprotease imbalance is not sufficient to explain the origin and progression of the disease. In this study we found that cigarette smoke extract causes oxidative damage to cellular lipids and proteins in A549 alveolar epithelial cells, which could lead to the loss of important cell functions, eventually resulting in cell death. The accumulation of damaged proteins in cells leads to the activation of ER stress response in the form of increased levels of phosphorylated eIF2α and reduced de novo protein synthesis. It is likely that reduced protein synthesis contributes to the increase in the level of free amino acids in the cells, together with the degradation of proteins damaged by cigarette smoke inside the ubiquitin-proteasome system. ER stress response is usually associated with increased proteasomal activity; however, cigarette smoke causes a significant reduction in caspase-, trypsin- and chymotrypsin-proteasomal activities, making the proteasome inefficient in the process of removing damaged proteins. As a part of the cellular stress response, we observed an increase in the expression of chaperone proteins HSP32 and HSP27, which was not shown for HSP70. Likewise, we noticed the changes in MAPK signaling pathways: a significant increase in the level of activated p38, no activation of ERK and undetectable activation of JNK. Furthermore, the results showed that A549 cells do not produce MMP-9, but after a while treatment with the cigarette smoke extract increases the release of MMP-2 in the medium and decreases the release of TIMP-1, which probably contributes to the overall protease-antiprotease imbalance. In basal conditions polymorphonuclear leukocytes (PMN) of patients with COPD release significantly much more MMP-9 compared to healthy controls. Stimulation of PMN of healthy controls with cigarette smoke extract increases the release of MMP-9, while in patients with COPD no further increase over the high secretion rate was found, suggesting the chronic activation of PMN of patients with COPD and their possible role in the degradation of extracellular matrix in the lungs. Systemic protease-antiprotease imbalance in plasma of patients with COPD is manifested by increased concentration of MMP-9. ROC curve analysis showed a very good diagnostic accuracy for concentration of MMP-9, making it a good biomarker for distinguishing patients from healthy individuals. Testing the frequency of MMP9 gene polymorphisms (-1562 C/T and (CA)n dinucleotide repeat in the promoter region, and Q279R in exon 6) showed no significant difference between patients with COPD and healthy subjects, and no correlation of MMP-9 concentration with subjects’ genotypes. In sera of patients with COPD, with the observed systemic inflammation (increased total leukocytes, neutrophils and CRP), we found a decrease in activity of DPPIV, a protease that could be contributing to the development of inflammation. As showing a very good diagnostic accuracy, this enzyme could also be used as an effective biochemical marker of COPD

Current status of the lateral flow immunoassay for the detection of SARS-CoV-2 in nasopharyngeal swabs

Early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and diagnosis of coronavirus disease 2019 (COVID-19) are priorities during the pandemic. Symptomatic and suspected asymptomatic individuals should be tested for COVID-19 to confirm infection and to be excluded from social interactions. As molecular testing capacity is overloaded during the pandemic, rapid antigen tests, such as lateral flow immunoassays (LFIAs), can be a useful tool as they allow greater test availability and obtain results in a very short time. This short review aims to present the analytical properties of LFIAs in the detection of SARS-CoV-2 in nasopharyngeal swabs. Lateral flow immunoassay is a method that combines thin-layer chromatography and indirect immunochemical sandwich method and allows the detection of a specific SARS-CoV-2 antigen in nasopharyngeal swabs. Swab specimens should be adequately collected and tested as soon as possible. Users should pay attention to quality control and possible interferences. Antigen tests for SARS-CoV-2 show high sensitivity and specificity in cases with high viral loads, and should be used up to five days after the onset of the first symptoms of COVID-19. False positive results may be obtained when screening large populations with a low prevalence of COVID-19 infection, while false negative results may happen due to improper specimen collection or insufficient amount of antigen in the specimen. So as to achieve reliable results, a diagnostic accuracy study of a specific rapid antigen test should be performed

Terapijski pristup liječenju kronične opstrukcijske plućne bolesti u vrijeme pandemije COVID-19

Chronic obstructive pulmonary disease (COPD) is a common lung disease that is characterized by long-term respiratory symptoms and airflow limitation. It is important to investigate whether patients with COPD are at the increased risk of infection with SARS-Co V -2 and what are the clinical outcomes of coronavirus disease 2019 (COVID-19). Since both diseases affect the respiratory tract, the purpose of this review was to determine whether there is an interrelationship between COPD and COVID-19. Currently, it is not clear whether COPD patients are at higher risk of SARS-Co V-2 infection; however, it is confirmed that, if they are affected by COVID-19, there is a higher rate of hospitalisation and death. COPD patients should continue prescribed COPD therapy even if they have COVID-19 infection because it is not confirmed that taking these medications changes the clinical outcomes of COVID-19 infection. Protective measures are very important in times of higher prevalence of COVID-19 since they reduce the risk of infection and help COPD patients maintain their health

Terapijski pristup liječenju kronične opstrukcijske plućne bolesti u vrijeme pandemije COVID-19

Chronic obstructive pulmonary disease (COPD) is a common lung disease that is characterized by long-term respiratory symptoms and airflow limitation. It is important to investigate whether patients with COPD are at the increased risk of infection with SARS-Co V -2 and what are the clinical outcomes of coronavirus disease 2019 (COVID-19). Since both diseases affect the respiratory tract, the purpose of this review was to determine whether there is an interrelationship between COPD and COVID-19. Currently, it is not clear whether COPD patients are at higher risk of SARS-Co V-2 infection; however, it is confirmed that, if they are affected by COVID-19, there is a higher rate of hospitalisation and death. COPD patients should continue prescribed COPD therapy even if they have COVID-19 infection because it is not confirmed that taking these medications changes the clinical outcomes of COVID-19 infection. Protective measures are very important in times of higher prevalence of COVID-19 since they reduce the risk of infection and help COPD patients maintain their health

Platelet indices in stable chronic obstructive pulmonary disease – association with inflammatory markers, comorbidities and therapy

Introduction: Chronic obstructive pulmonary disease (COPD) is a complex inflammatory condition that can affect haemostasis. This study aimed to determine differences in platelet-related parameters between controls and COPD subjects. The hypothesis was that platelet indices are disturbed in COPD patients, and this would be accompanied by increased C-reactive protein (CRP), fibrinogen (Fbg) and white blood cells (WBC). Therefore, platelet count (Plt), platelet-related parameters – mean platelet volume (MPV), platelet distribution width (PDW), plateletcrit (Pct), their ratios (MPV/Plt, MPV/Pct, PDW/Plt, PDW/Pct), platelet to lymphocyte ratio (PLR), Plt index as well as CRP, Fbg and WBC were assessed. Materials and methods: Study included 109 patients with stable COPD and 95 control subjects, recruited at Clinical Department for Lung Diseases Jordanovac, University Hospital Centre Zagreb (Zagreb, Croatia). Complete blood count was performed on Sysmex XN-1000, CRP on Cobas c501, and Fbg on BCS XP analyser. Data were analysed with MedCalc statistical software. Results: Platelet (P = 0.007) and PLR (P = 0.006) were increased, while other platelet indices were decreased in COPD patients compared to controls. Combined model that included PLR, PDW and WBC showed great diagnostic performances, and correctly classified 75% of cases with an AUC of 0.845 (0.788 – 0.892), P < 0.001. Comorbidities (cardiovascular or metabolic diseases) had no effect on investigated parameters, while inhaled corticosteroids/long-acting β2-agonists (ICS/LABA) therapy increased MPV and PDW values in COPD patients. Conclusion: Platelet indices were altered in COPD patients and they could be valuable as diagnostic markers of COPD development, especially if combined with already known inflammatory markers

Serum activities of adenosine deaminase, dipeptidyl peptidase IV and prolyl endopeptidase in patients with fibromyalgia: diagnostic implications

Fibromyalgia (FM) is a chronic pain syndrome with number of symptoms that present challenge in terms of diagnosis and treatment. Patients with FM show abnormal profile of purines in plasma. In this work, we measured serum activities of enzymes involved in purine metabolism, namely total adenosine deaminase (ADE) and its isoforms (ADE1 and ADE2), ecto- ATPase, and 5′- nucleotidase (5′-NT). We also measured activity of dipeptidyl peptidase IV (DPPIV) and prolyl endopeptidase (PEP). Spectrophotometric and fluorometric methods were used for enzyme activity determinations. Enzyme activities were measured in sera of 24 patients with FM that were not undergoing pharmacological treatment during the study. Control group comprised 32 healthy control subjects. Significantly higher activities of total ADE (P = 0.025) and ADE2 (P = 0.011) were observed in FM patients, while no significant differences in ADE1, ecto-ATPase, and 5′-NT activities (P > 0.05) were found when compared to healthy controls. Moreover, increase in the activity of DPPIV (P = 0.015) and lower activity of PEP (P = 0.011) were also found in the FM group. ROC analysis pointed to different diagnostic sensitivities/specificities for individual enzyme activities measured as follows: ADE (50.0/87.5), ADE2 (41.7/90.6), DPPIV (62.5/71.9), and PEP (83.3/62.5). ADE2 and PEP were shown to be independent predictors of FM, while combination of the two gives AUC of 0.786 (95 % confidence interval of 0.656–0.885, P < 0.05). Our results are showing that serum activities of ADE2 and PEP could be useful as biomarkers for FM diagnosis. However, relatively low diagnostic sensitivity of ADE2 and specificity of PEP must be taken into account

Extracellular Hsp70 modulates 16HBE cells' inflammatory responses to cigarette smoke and bacterial components lipopolysaccharide and lipoteichoic acid

Cigarette smoke is a major risk factor for chronic obstructive pulmonary disease (COPD), leading to chronic inflammation, while bacterial components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are often present in airways of COPD patients, especially during exacerbations. We hypothesised that extracellular heat shock protein 70 (eHsp70), a damage-associated molecular pattern elevated in serum of COPD patients, induces inflammation and alters cigarette smoke and LPS/LTA-induced inflammatory effects in the airway epithelium. We used 16HBE cells exposed to recombinant human (rh)Hsp70 and its combinations with cigarette smoke extract (CSE), LPS or LTA to investigate those assumptions, and we determined pro-inflammatory cytokines’ secretion as well as TLR2 and TLR4 gene expression. rhHsp70 and CSE alone stimulated IL-6, IL-8 and TNF-α secretion. CSE and rhHsp70 had antagonistic effect on IL-6 secretion, while combinations of LPS or LTA with rhHsp70 showed antagonistic effect on TNF-α release. By using specific inhibitors, we demonstrated that effects of rhHsp70 on cytokines’ secretion were mediated via NF-κB and/or MAPK signalling pathways. rhHsp70 increased, and CSE decreased TLR2 gene expression compared to untreated cells, but their combinations increased it compared to CSE alone. LPS and rhHsp70 combinations decreased TLR2 gene expression compared to untreated cells. TLR4 expression was not induced by any of the treatments. In conclusion, we demonstrated that extracellular Hsp70 modulates pro-inflammatory responses of human airway epithelial cells to cigarette smoke and bacterial components LPS and LTA. Simultaneous presence of those compounds and their interactions might lead to inappropriate immune responses and adverse consequences in COPD