Search

Colorectal cancer (CRC) is the third most common cancer worldwide. The high mortality from CRC is mainly related to metastasis affecting distant organs and their function. Dissemination of tumor cells from the primary tumor and hematogeneous spread are considered crucial in the formation of tumor metastases. The analysis of circulating tumor cells (CTCs) and CTC clusters in the blood can be used for the early detection of invasive cancer. Moreover, CTCs have a prognostic significance in the monitoring of a malignant disease or the response to chemotherapy. This work presents an overview of the research conducted on CTCs with the aim of finding suitable detection systems and assessing the possibility of clinical applications in patients with CRC.

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.

During chronic obstructive pulmonary disease (COPD) exacerbations interplay between pathogen- associated molecular patterns (PAMPs ; e.g. lipopolysaccharide (LPS) and lipoteichoic acid (LTA)) and damage-associated molecular patterns (DAMPs ; e.g. extracellular heat shock protein 70 (eHsp70) and adenosine triphosphate (ATP)) might influence patient’s outcome. Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome might have a role in dysfunctional immune system in COPD. We hypothesized that LPS, LTA, eHsp70 and their combinations induce NLRP3 inflammasome activation and we aimed to explore this assumption. We used monocytic (monocyte-derived macrophages (MDMs) and THP-1) and bronchial epithelial cells (NHBE and NCI-H292) to represent systemic and local airway compartments that could be affected in COPD. Bacterial components and eHsp70 stimulated NLRP3 and interleukin (IL)-1β gene expression as well as IL-1β and ATP release from all cells compared to non-treated cells. LDH secretion was induced in cell lines only. eHsp70 had inhibitory (NCI-H292) or stimulatory (NHBE) effects on eATP levels compared to PAMP alone. Regarding NLRP3 inflammasome activation, eHsp70 had mostly antagonistic effects. We demonstrated that bacterial components and eHsp70 activate NLRP3 inflammasome and increase ATP secretion. We suggest that extracellular Hsp70 might modulate immune responses provoked by bacterial infections and affect COPD patients’ outcome during acute exacerbations.

One of the major risk factors for development of chronic obstructive pulmonary disease (COPD) is cigarette smoke. Extracellular Hsp70 (eHsp70) is increased in sera of COPD patients, and can act as damage-associated molecular pattern (DAMP). In this study, we explored inflammatory parameters (cytokine concentrations, Toll-like receptor (TLR) 2 and 4 and Hsp70 expression, mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) activation, and cytotoxicity) after exposure of bronchial-epithelial NCI-H292 cells to cigarette smoke extract (CSE) alone (2.5 and 15%) and in combinations with recombinant human (rh) Hsp70 (0.3, 1 and 3 μg ml-1 ). We applied specific MAPKs, NF-κB and Hsp70 inhibitors to elucidate rhHsp70 inflammation-associated responses. CSE alone and combinations of 15% CSE with rhHsp70 stimulated IL-1α, IL-6 and IL- 8 release. However, rhHsp70 applied with 2.5% CSE decreased secretion of cytokines indicating antagonistic effects. Individual and combined treatments with 2.5% CSE suppressed TLR2 expression. CSE at 15% induced TLR2 and TLR4 gene expression, whereas rhHsp70 abolished that effect. rhHsp70 and 15% CSE alone reduced, while their combination increased, intracellular Hsp70 mRNA level. CSE alone and in combination with rhHsp70 activated extracellular signal-regulated kinase and p38 MAPKs, while inhibition of MAPKs, NF-κB and Hsp70 attenuated IL-6 and IL-8 secretion. CSE at 15% reduced cell viability and induced apoptosis, as shown by MTS and caspases-3/7 assays. CSE at 2.5% alone stimulated lactate dehydrogenase release, but cellular membrane integrity remained intact in co-treatments with rhHsp70. rhHsp70 might modulate the inflammatory response of CSE and could also protect NCI-H292 cells against CSE cytotoxicity. Those effects are implemented via MAPK and NF-κB signalling pathways.

Introduction: Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease (COPD). Exposure to cigarette smoke may stimulate inflammatory response and activate polymorphonuclear leukocytes (PMN) thus resulting in secretion of cellular proteases. The aim of our study was to explore the effect of cigarette smoke extract (CSE) on the release of matrix metalloproteinase-9 (MMP-9) from PMN. Methods: The study included 23 patients with stable COPD and 9 healthy controls. PMN were isolated from blood of all participants and exposed to 4 % CSE or basal culture medium (0 % CSE) for 20 h. MMP-9 concentration in PMN culture media was measured using the ELISA method. Results: Exposure of PMN to 4 % CSE did not cause cytotoxic effects, as determined by no changes in lactate dehydrogenase (LDH) activity in PMN culture media when compared to untreated PMN (P=0.689). In basal conditions PMN of COPD patients released significantly more MMP-9 compared with PMN of healthy controls (P=0.016). However, concentration ratio of MMP-9 released from PMN exposed to 4 % CSE or 0 % CSE of each participant was significantly higher for healthy subjects than for COPD patients (P=0.025). Conclusion: Cigarette smoke induces activation of PMN in healthy controls. However, chronically activated PMN in COPD patients could not be further stimulated by in vitro exposure to CSE. Constantly raised amount of MMP-9 released into the tissues may be involved in the degradation of extracellular matrix in the lungs as seen in COPD patients.

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.

Extracellular heat shock protein (eHsp) 70 is a damage-associated molecular pattern that could participate in inflammatory response in bronchial epithelial cells. We hypothesized that the inflammatory response to eHsp70 as well as its effects on cell survival are mediated via mitogen-activated protein kinase (MAPK) and NF-κB signalling pathways in 16HBE bronchial epithelial cell line. We aimed to investigate these mechanisms using the specific NF-κB and MAPK inhibitors. 16HBE cells were pre-treated with specific inhibitors (BAY11-7082 for NF-κB, PD98059 for ERK, SP600125 for JNK, and SB202190 for p38) for 1 h before adding recombinant human (rh) Hsp70 (3 μg/ml) for 24 h. IL-1α, IL-6 and TNF-α concentrations were determined in cell supernatants using ELISA method, while cell viability was assessed by MTS assay. IL-1α secretion was decreased after pre-treatment with ERK, JNK and p38 inhibitors compared to rhHsp70 alone. IL-6 and TNF-α secretion was decreased with all inhibitors. Inhibition of NF-κB and p38 pathways almost completely attenuated secretion of TNF-α and IL-6. rhHsp70 alone did not affect cell viability ; however, inhibition of JNK and p38 pathways significantly lowered it in 16HBE cells. In conclusion, the results support our hypothesis that the NF-κB signalling is an important factor in inflammation, while MAPK signalling pathways are involved in both inflammatory processes and cell survival in 16HBE cells. This could have a potential role in new therapeutic drug discoveries.

Extracellular Hsp70 (eHsp70) is elevated in blood of patients with inflammatory diseases (e.g. chronic obstructive pulmonary disease), prone to bacterial infections. It stimulates pro-inflammatory effects in cells via TLR2 and TLR4. The main hypothesis was that eHsp70 induces inflammation and alters inflammatory responses to lipopolysaccharide (LPS) and lipoteichoic acid (LTA). We assessed pro-inflammatory cytokines concentrations, TLR2, TLR4 and Hsp70 mRNA expressions, NF-κB and MAPKs activation, and viability after treatment of monocyte-derived macrophages (MDMs) and THP-1 cells with recombinant human (rh) Hsp70, LPS and LTA. rhHsp70, LPS, LTA and their combinations induced IL-6, IL-8 and TNF-α from MDMs. LPS, LTA, and combinations with rhHsp70 increased TLR2 and decreased TLR4 in both cells. rhHsp70 increased TLR2 and TLR4 in THP-1 cells, and TLR2 in MDMs. rhHsp70, LPS and their combinations up-regulated Hsp70 in MDMs, while down-regulated it in THP-1. LPS and its combinations with rhHsp70 lowered cell viability in THP-1 cells and activated caspases-8 and -9. Involvement of NF-κB and MAPKs in inflammatory and survival mechanisms stimulated by rhHsp70 was demonstrated. eHsp70 alone and in combination with bacterial components could provoke inflammation and affect viability of MDMs and THP-1 cells leading to disturbed inflammatory responses in diseases accompanied with bacterial colonisations or infections.

Background Chronic obstructive pulmonary disease (COPD) is an inflammatory disease mainly caused by smoking. Cigarette smoke damages airway epithelium and activates lung macrophages, causing inflammatory responses. It was suggested that nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome might have an important role in COPD development. Study aimed to explore whether cigarette smoke extract (CSE), extracellular heat shock protein 70 (eHsp70) or their combinations induce adenosine triphosphate (ATP) release and NLRP3 inflammasome activation. Methods We detected NLRP3 and interleukin (IL)-1β mRNA expression, extracellular IL-1β and ATP concentrations as well as lactate dehydrogenase (LDH) activity. We used bronchial epithelial (NCI-H292, 16HBE and NHBE) and monocytic cells (monocyte-derived macrophages (MDMs) and THP-1) as representative of local airway and systemic compartments that could be affected in COPD. Results CSE and eHsp70 increased NLRP3 and IL-1β mRNA expression as well as IL-1β and ATP secretion in all cells compared to untreated cells. Lytic cell death was observed in cell lines, especially those of bronchial epithelium origin, but not in primary cells (NHBE, MDMs). Regarding LDH activity, eHsp70 did not modulate CSE effects, except in NCI-H292 cell line. However, eHsp70 significantly affected CSE-provoked NLRP3 inflammasome activation by causing mostly antagonistic effects in airway epithelial cells and synergistic effects in MDMs. Conclusion We demonstrated that both CSE and eHsp70 induce ATP secretion and differential activation of NLRP3 inflammasome in bronchial epithelial and monocytic cells. We suggest that these mechanisms might be involved in pathophysiology of COPD by contributing to the propagation of inflammation.

Introduction: Chronic obstructive pulmonary disease (COPD) is a condition with underlying systemic inflammation and haemostatic changes. Aims and objectives: Assessment of the platelet indices - platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW) and platelet to lymphocyte ratio (PLR), evaluation of their diagnostic value and association with well-known inflammatory markers. Methods: 109 patients with stable COPD and 95 healthy controls were included. Blood samples were collected in different tubes depending on the analysis. The results were analyzed by Mann-Whitney Rank Sum test performed by MedCalc statistical software. Results: Among platelet indices, PLT (P=0.007) and PLR (P=0.005) were significantly increased, while MPV (P

Introduction: Patients with chronic obstructive pulmonary disease (COPD) are prone to bacterial colonizations and infections. They have elevated extracellular Hsp70 (eHsp70) in plasma, which exerts its biological actions via Toll-like receptors (TLR) 2 and 4 leading to the development of inflammation. Aims and Objectives: The aim of this study was to explore the effects of eHsp70 alone and in combination with bacterial components lipoteichoic acid (LTA) and lipopolysaccharide (LPS) on TLR2 and TLR4 mRNA expression in monocyte-derived macrophages (MDMs). Methods: MDMs were treated with recombinant human Hsp70 (rhHsp70), LPS, LTA, and their combinations for 24 h. RNA was isolated by Trizol/chlorophorm method, cDNA was synthesized followed by qPCR. Results: 3 and 10 µg/ml rhHsp70 alone, as well as 0.1 µg/ml LPS and 1 µg/ml LTA increased TLR2 expression compared to non-treated MDMs. Combinations of 1 µg/ml rhHsp70 with LPS or LTA increased, while combinations with 3 µg/ml rhHsp70 did not affect TLR2 expression. In contrast, LPS, LTA and their combinations with rhHsp70 decreased TLR4 expression compared to non-treated MDMs. Treatment of the cells with rhHsp70 alone did not change TLR4 expression. Conclusions: Pro-inflammatory effect in MDMs could be potentiated by rhHsp70, LPS and LTA via elevated TLR2 expression. It seems that rhHsp70 does not have an effect on TLR4 expression in MDMs, but LPS, LTA and their combinations with rhHsp70 supress it. This might lead to altered capacity to properly respond to the noxious stimuli, such as bacteria or viruses, thus leading to adverse consequences for patients with COPD.

Drug-specific therapeutic approaches for colorectal cancer (CRC) have contributed to significant improvements in patient health. Nevertheless, there is still a great need to improve the personalization of treatments based on genetic and epigenetic tumor profiles to maximize the quality and efficacy while limiting cytotoxicity. Currently, CEA and CA 19-9 are the only validated blood biomarkers in clinical practice. For this reason, laboratories are trying to identify new specific prognostics and, more importantly, predictive biomarkers for CRC patient profiling. Thus, the unique landscape of personalized biomarker data should have a clinical impact on CRC treatment strategies and molecular genetic screening tests should become the standard method for diagnosing CRC. This review concentrates on recent molecular testing in CRC and discusses the potential modifications in CRC assay methodology with the upcoming clinical application of novel genomic approaches. While mechanisms for analyzing circulating tumor DNA have been proven too inaccurate, detecting and analyzing circulating tumor cells and protein analysis of exosomes represent more promising options. Blood liquid biopsy offers good prospects for the future if the results align with pathologists’ tissue analyses. Overall, early detection, accurate diagnosis and treatment monitoring for CRC with specific markers and targeted molecular testing may benefit many patients.

Introduction: Cigarette smoking is the main etiologic factor for chronic obstructive pulmonary disease (COPD), characterized by chronic airway inflammation. Extracellular ATP (eATP) is a damage-associated molecular pattern (DAMP) molecule that may induce activation of inflammatory cells. Aims and objectives: The aim of this study was to assess eATP concentration in stable COPD patients in relation to the severity of the disease and smoking status. Methods: We obtained blood samples from stable COPD patients (N=137) and healthy volunteers (N=95), matched by age and sex. COPD patients were divided into subgroups according to GOLD (Global Initiative for Chronic Obstructive Lung Disease) or "ABCD" classifications, by both mMRC (modified Medical Research Council) and CAT (COPD assessment test) questionnaires. Plasma eATP concentrations were determined using the commercially available luminescence assay. Nonparametric statistics using the MedCalc software was used for data analysis. Results: Plasma eATP concentration was significantly increased in COPD patients compared to healthy controls (P

Extracellular adenosine triphosphate (eATP)-driven inflammation was observed in chronic obstructive pulmonary disease (COPD) but was not investigated in patients’ blood. Therefore, this study aimed to investigate eATP concentration in plasma of COPD patients and its association with disease severity and smoking. Study included 137 patients with stable COPD and 95 control subjects. eATP concentration was determined in EDTA plasma by luminometric method, and mRNA expression of eATP receptors P2X7R and P2Y2R was analysed by quantitative polymerase chain reaction (qPCR). eATP concentration was increased in COPD patients compared to controls (P 1 and multicomponent indices (ADO, BODEx, BODCAT, CODEx, DOSE). Regarding gene expression, P2Y2R was increased in the blood of COPD patients. Plasma eATP could become a diagnostic and/or prognostic biomarker in COPD, as it seems to be associated with patients’ condition, quality of life and disease progression.

Extracellular Hsp70 (eHsp70) can activate immune cells via Toll-like receptors (TLR) 2 and 4, and induce cytokine synthesis. The aim of this study was to explore inflammation-associated effects of eHsp70 alone and in combination with cigarette smoke extract (CSE) in primary bronchial epithelial cells.We assessed IL-6 and IL-8 concentrations, TLR2, TLR4 and Hsp70 mRNA expressions, and mitogen-activated protein kinases (MAPKs) activation induced by recombinant human (rh) Hsp70, CSE or their combinations in normal human bronchial epithelial cells (NHBE) obtained commercially, and primary bronchial epithelial cells isolated from non-COPD lung donors (PBEC) or COPD patients (PBEC COPD).Baseline levels of IL-6 and IL-8 were significantly higher in PBEC COPD than in non-COPD PBECs. Upon rhHsp70 stimulation, IL-6 and IL-8 were significantly increased, with the strongest response in COPD-derived PBECs. CSE alone elevated cytokine secretion in all examined cells. rhHsp70 and CSE had antagonistic interactions on IL-8 release in PBECs from COPD patients, while the addition of rhHsp70 further increased CSE-induced IL-6 secretion in NHBE cells. rhHsp70 and CSE alone decreased TLR2 and TLR4 mRNA expression in COPD-derived PBECs. In non-COPD PBECs, combined treatments decreased only TLR2 mRNA expression. Hsp70 mRNA expression, as indicator of intracellular Hsp70, which may have anti-inflammatory effects, was reduced in COPD-derived cells upon exposure to CSE and rhHsp70 alone, but not with their combinations. Contrary to this, in PBECs from lung donors only combined treatments supressed Hsp70 gene expression. CSE activated JNK and p38 MAPKs, while rhHsp70 increased activation of c-Jun kinase in NHBE cells.Collectively, both eHsp70 and CSE induce pro-inflammatory responses in PBECs from non-COPD as well as COPD donors, but in combination antagonistic effects were observed in COPD-derived cells. These effects may be related to the regulation of TLR2/4 and might lead to modulation of inflammation with possible deleterious consequences for COPD patients. (C) 2018 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Extracellular Hsp70 (eHsp70) can act as pro- inflammatory mediator and is elevated in blood of chronic obstructive pulmonary disease (COPD) patients. Most of those patients are smokers, and it was suggested previously that cigarette smoke might induce Hsp70 secretion from the circulating cells. Therefore, we aimed to explore inflammation- associated effects of cigarette smoke extract (CSE) and its combinations with eHsp70 in monocyte-derived macrophages (MDMs) and THP-1 cell line, used as systemic component models of COPD We hypothesized that eHsp70 induces inflammation, but that it can also modulate cigarette smoke extract (CSE)- stimulated inflammatory responses. We assessed IL-8 secretion, TLR2, TLR4 and Hsp70 expressions, MAPKs and NF-κB activation, and cytotoxicity after treating the cells with CSE (2.5 and 5%) and its combinations with low-endotoxin recombinant human (rh) Hsp70, used to mimic eHsp70 effects. CSE induced IL-8 secretion from both cell types, but its combinations with rhHsp70 increased IL-8 release compared to CSE alone only from MDMs. In THP-1, combinations of rhHsp70 with 2.5% CSE induced TLR2 and TLR4 mRNA, while 5% CSE decreased TLR2 expression. In MDMs, CSE alone attenuated TLR2, while rhHsp70 increased TLR2 and lowered TLR4 gene expression. Hsp70 mRNA expression was suppressed in THP-1 with rhHsp70 and CSE ; however, the same treatments increased its level in MDMs. CSE had cytotoxic effect only on MDMs, but cytotoxicity was reduced in co-treatments with rhHsp70, which also triggered apoptosis. CSE and rhHsp70 activated p38 and JNK, while ERK was activated only by rhHsp70 in MDMs. In THP-1, 2.5% CSE activated ERK, and 5% CSE activated p38. Inhibition of NF-κB and JNK in MDMs, and ERK and JNK in THP-1 cells, attenuated IL-8 release after rhHsp70 treatment. In conclusion, rhHsp70 provoked pro- inflammatory effects and could also modulate inflammatory response to CSE on protein and gene expression levels in THP-1 cells and MDMs, which suggests that eHsp70 might be implicated in systemic inflammation induced by cigarette smoke.

Introduction: Cigarette smoke is one of the major risk factors for development of chronic obstructive pulmonary disease (COPD), which is characterized by chronic inflammation. Extracellular Hsp70 is increased in sera of COPD patients, and can act as damage-associated molecular pattern. Aims and objectives: We explored the pro-inflammatory effects of cigarette smoke extract (CSE) alone and in combination with recombinant human Hsp70 (rhHsp70) on bronchial-epithelial NCI-H292 cell line. Methods: NCI-H292 cells were treated with 2.5% or 15% CSE alone or in combination with 0.3, 1.0 or 3.0 µg/mL rhHsp70 for 24 h. IL-1α, IL-6 and IL-8 were determined in cell supernatants by ELISA method. Results: Compared to the non-treated cell, secretion of IL-1α, IL-6 and IL-8 was significantly increased after treatment with 2.5% CSE, but not in combination with rhHsp70. Combination of rhHsp70 and CSE resulted in different secretion of IL-1α, IL-6 and IL-8 compared to the cells treated with CSE alone, and theirinteractions were antagonistic. Concentrations of IL-1α, IL-6 and IL-8 were different after treatment of cells with 15% CSE or combination of CSE and rhHsp70 compared to the non-treated cells, but their concentrations did not differ after combined treatment compared to CSE alone.

Introduction: The main etiologic factor for chronic obstructive pulmonary disease (COPD), which is characterized by chronic airway inflammation, is cigarette smoking. Patients with COPD are susceptible to bacterial infections causing acute exacerbations. Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern and activate immune cells via Toll-like receptors 2 and 4. Aims and objectives: The aim of this study was to examine the effects of recombinant human Hsp70 (rhHsp70) when present alone or together with bacterial components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) as well as cigarette smoke extract (CSE) on activation of mitogen-activated protein kinases (MAPKs) signalling pathways in bronchial-epithelial NCI-H292 cell line. Methods: NCI-H292 cells were treated either with rhHsp70 (0.3, 1.0 or 3.0 µg/mL), 0.1 µg/ml LPS, 1 µg/ml LTA and CSE (2.5% or 15%) alone, or with their combinations, for 30 min, 2 h, 8 h and 24 h. Expression and activation of MAPKs (ERK, JNK and p38) were determined by Western blotting method. Results: rhHsp70, LPS, LTA and their combinations caused activation of ERK, JNK and p38 MAPK, while rhHsp70 together with CSE caused activation of ERK and p38 MAPK in NCI-H292 cells, compared to the control cells. Extent of activation was dependant on concentration and time of treatment. Conclusions: eHsp70 as well as bacterial components (LPS and LTA) and CSE activate MAPK signalling pathways in NCI-H292 cells. Combined presence of eHsp70, LPS, LTA and/or CSE might lead to worsening of COPD patients’ condition.

NEW FINDINGS What is the central question of this study? What is the effect of cigarette smoke on cell death, oxidative damage, expression of heat shock proteins (HSPs) and activation of mitogen-activated protein kinases (MAPKs) in A549 alveolar epithelial cells? What is the main finding and its importance? Cigarette smoke induces cytotoxicity and oxidative damage to A549 cells, increases expression of different HSPs and activates MAPK signalling pathways. This could be related to inflammatory response and apoptosis observed in lungs of patients with smoking-related diseases. ABSTRACT Cigarette smoking is one of the main risk factors for development of chronic obstructive pulmonary disease (COPD). We previously reported that cigarette smoke (CS) induces damage to proteins and their ineffective degradation. Here, we hypothesize that CS could induce oxidative stress and cytotoxicity in lung epithelial cells through alterations of heat shock protein (HSP) expression and mitogen-activated protein kinase (MAPK) signalling pathways. We exposed A549 alveolar epithelial cells to various concentrations of cigarette smoke extract (CSE). Higher concentrations of CSE caused apoptosis of A549 cells after 4 h, while after 24 h cell viability was decreased, and lactate dehydrogenase in cell culture medium was increased as well as the number of necrotic cells. Concentrations of malondialdehyde (MDA) were elevated, while total thiol groups were decreased. Changes in the expression of HSPs (HSP70, HSP32 and HSP27) were time-dependent. After 6 h, CSE caused an increase in the expression of HSP70 and HSP32, while after 8 h all examined HSPs were up-regulated and remained increased up to 48 h. Treatment of A549 cells with CSE stimulated phosphorylation of extracellular signal-regulated kinase and p38 in a dose-dependent manner, while c-Jun N-terminal kinase activation was not detected. By using specific inhibitors, we demonstrated that MAPKs and HSPs interplay in CSE effects. In conclusion, our results show that MAPKs and HSPs are involved in the mechanism underlying CSE-induced cytotoxicity and oxidative damage to A549 alveolar epithelial cells. These processes could be related to inflammatory response and apoptosis observed in lungs of patients with smoking-related diseases, such as COPD.

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.

Objectives The objective of this study was to measure soluble dipeptidyl peptidase IV (sDPPIV) activity in sera of patients with stable chronic obstructive pulmonary disease (COPD) in comparison to healthy controls. The main goal was to assess changes in the enzyme activity in relation to severity of the disease, age and smoking history and to evaluate diagnostic accuracy for prediction of COPD by level of serum sDPPIV activity. Design and methods The study included 106 patients with stable COPD (GOLD II–GOLD IV stages) and 38 healthy controls. Serum sDPPIV activity as well as some inflammatory markers (CRP, total and differential leukocyte counts) was measured. Multivariate logistic regression models were applied to analyze association of sDPPIV activity and inflammatory markers in risk estimation for COPD development. Results sDPPIV activity in COPD patients was significantly reduced when compared to healthy controls. Decrease was observed already in GOLD II stage. Age and smoking history did not influence sDPPIV activity. Very good diagnostic accuracy (AUC = 0.833; sensitivity and specificity of 85.7% and 78.9%, respectively) for GOLD II and good diagnostic accuracy (AUC = 0.801; sensitivity and specificity of 65.1% and 86.8%, respectively) for total cohort of COPD patients were found. The multivariate logistic regression model showed that the use of sDPPIV in combination with CRP and lymphocyte proportion improved diagnostic strength and gave an AUC of 0.933. Conclusions sDPPIV activity is decreased in COPD patients as early as in GOLD II stage. Very good diagnostic accuracy of sDPPIV activity suggests it as a candidate biomarker for early diagnosis of COPD.

New findings • What is the central question of this study?The endoplasmic reticulum stress response caused by cigarette smoke may lead to excessive apoptosis with disruption of the epithelial barrier, thus contributing to chronic obstructive pulmonary disease. One way of promoting cell survival is to facilitate degradation of cigarette smoke-induced protein damage through the ubiquitin–proteasome pathway. Direct effects of gas-phase cigarette smoke on proteasomal activities have not been demonstrated previously. • What is the main finding and what is its importance?We show that cigarette smoke induces protein damage and triggers the endoplasmic reticulum stress response in human alveolar epithelial cells. A significant reduction of all three proteasomal activities was found. Ineffective degradation of damaged proteins could lead to a sustained epithelial stress response and development of chronic obstructive pulmonary disease. Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease. Cigarette smoke (CS) causes oxidative stress and severe damage to proteins in the lungs. One of the main systems to protect cells from the accumulation of damaged proteins is the ubiquitin–proteasome pathway. In the present study, we aimed to find out whether exposure of alveolar epithelial cells to CS induces an endoplasmic reticulum (ER) stress response by accumulation of damaged proteins that are inefficiently degraded by the proteasomes. The hypothesis was tested in a human alveolar epithelial cell line (A549) exposed to gas-phase CS. Exposure to gas-phase CS for 5 min caused an increase in the amount of ubiquitin–protein conjugates within 4 h. Cigarette smoke exposure also induced the ER stress response marker eIF2α, followed by a significant reduction of nascent protein synthesis and increase in the level of free intracellular amino acids. Moreover, CS exposure significantly reduced all three proteasomal activities (caspase-, trypsin- and chymotrypsin-like activity) within 4 h, which was still present after 24 h. It can be concluded that gas-phase CS induces ER stress in A549 alveolar epithelial cells, leading to inadequate protein turnover caused by an accumulation of damaged proteins, reduction in nascent protein synthesis and inhibition of the proteasome. We suggest that prolonged ER stress may lead to excessive cell death with disruption of the epithelial barrier, contributing to development of chronic obstructive pulmonary disease.

Razvojem nanotehnologije metalnim nanočesticama pridodaje se sve veća važnost kao antimikrobno aktivnim tvarima jer dokazano djeluju na širok spektar bakterija i gljivica. Koloidne nanočestice metala i metalnih oksida sve se više razmatraju kod prevencije bakterijskih infekcija te kao moguća alternativa antibioticima, a osobito su popularne koloidne nanočestice srebra, titanova dioksida i cinkova oksida, koje imaju široku primjenu u biomedicini, industriji i biotehnologiji. U ovom radu je bio cilj ispitati antimikrobni učinak koloidnih nanočestica Ag (10 nm), ZnO (100 nm), TiO2 (150 nm) na vrstu Escherichia coli (E. coli ATCC 10536 i E. coli ESBL+ MFBF 12794) u uvjetima in vitro modificiranom metodom serijalnog i dvostrukog razrjeđenja [1]. Dobiveni rezultati korišteni su za računalno optimiranje smjese ispitivanih nanočestica (Design Expert®, State-Ease v9.1) i ispitivanje sinergizma putem FIK indeksa (engl. fractional inhibitory concentration indeks) [2], koja je impregnirana (sol-gel postupkom u 3D metaloksidnu mrežu) na tkaninu i čiji je antimikrobni učinak u vremenu ispitan metodom time kill tj. određivanjem kinetike stanične smrti [3]. Potentan antimikrobni učinak pokazale su sve ispitivane nanočestice (raspon minimalne inhibicijske koncentracije – MIK je bio od 2, 5 ppm do 40, 3 ppm, te minimalne baktericidne koncentracije – MBK je između 5, 0 ppm i 644.5 ppm), pri čemu je nano srebro imalo najizraženiji baktericidni učinak (MIK 2, 5 ppm ; MBK 5, 0 ppm). Pri ispitivanju optimirane nano smjese nije pokazana statistički značajna razlika antimikrobnog učinka između dva soja E. coli, a vrijednosti za indeks djelomične inhibicijske i baktericidne koncentracije (FIK i FBK indeks) nisu pokazali sinergizam. U time kill analizi pokazano je da kod oba soja dolazi do naglog pada vijabilnih bakterija između 6 i 12 h i do potpune redukcije mikrobnog rasta nakon 12 sati inkubacije inokulirane tkanine modificirane nano smjesom (0, 8 ppm Ag, 683, 2 ppm TiO2 i 16, 0 ppm ZnO), također bez statistički značajne razlike učinka između sojeva. Može se zaključiti da ispitivane nanočestice posjeduju značajan antimikrobni učinak na ispitivanom modelu E. coli te otvaraju mogućnost daljnjim istraživanjima razvoja antimikrobno aktivnog tekstila. Rezultati su dio diplomskog rada Filip Penava: Antimikrobni učinak koloidnih otopina nanočestica elementarnih metala i metalnih oksida na E. coli.