Circulating Tumor Cells in Colorectal Cancer: Detection Systems and Clinical Utility.

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.

Profiling Colorectal Cancer in the Landscape Personalized Testing-Advantages of Liquid Biopsy.

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.

Cigarette smoke and extracellular Hsp70 induce secretion of ATP and differential activation of NLRP3 inflammasome in monocytic and bronchial epithelial cells.

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)-1ß mRNA expression, extracellular IL-1ß 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-1ß mRNA expression as well as IL-1ß 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.

Pro-inflammatory effects of extracellular Hsp70 on NCI-H292 human bronchial epithelial cell line.

Extracellular Hsp70 (eHsp70) exerts its biological actions via Toll-like receptors 2 and 4, and is increased in sera of chronic obstructive pulmonary disease (COPD) patients. The aim of this study was to explore the pro-inflammatory effects and cytotoxicity of eHsp70 alone and in combination with bacterial components lipoteichoic acid (LTA) and lipopolysaccharide (LPS) on NCI-H292 airway epithelial cells. NCI-H292 cells were treated with recombinant human Hsp70 protein (rhHsp70), LPS, LTA and their combinations for 4, 12, 24 and 48 hours. IL-6, IL-8 and TNF-α levels were measured by an ELISA method. Cell viability was determined by the MTS method, and caspase-3/7, caspase-8 and caspase-9 assays. rhHsp70 induced secretion of IL-6 and IL-8 in a concentration- and time-dependent manner, with the highest secretion at 24 hours. rhHsp70 combined with LTA had antagonistic and with LPS synergistic effect on IL-6 secretion, while the interactions between rhHsp70 and LPS or LTA on IL-8 were synergistic. TNF-α was not detected in the applied conditions. rhHsp70, LPS or LTA did not affect cell viability, and rhHsp70 even suppressed caspase-3/7 activities. We suggest that pro-inflammatory effects of eHsp70, together with other damaging molecules and/or COPD risk factors, might contribute to the aggravation of chronic inflammation in human bronchial epithelium.

Extracellular Hsp70 modulates the inflammatory response of cigarette smoke extract in NCI-H292 cells.

NEW FINDINGS: What is the central question of this study? Does extracellular heat shock protein 70 (eHsp70) alter cigarette smoke extract (CSE)-induced inflammatory responses in NCI-H292 bronchial epithelial cells? What is the main finding and its importance? eHsp70 modulates inflammatory responses and TLR2, TLR4 and Hsp70 gene expression, and protects NCI-H292 cells against CSE-induced cytotoxicity. eHsp70 might be implicated in development of inflammatory diseases affected by cigarette smoke, such as COPD. ABSTRACT: 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.

Valorization of Olive Pomace-Based Nutraceuticals as Antioxidants in Chemical, Food, and Biological Models.

Waste remaining after the production of olive oil (olive pomace) is known to contain significant amounts of phenolic compounds that exert different types of biological activities, primarily acting as antioxidants. In this work, a sustainable approach that combines ultrasound-assisted extraction with food-grade solvents and encapsulation with different types of cyclodextrins was used to prepare olive pomace-based polyphenol rich extracts that were tested as antioxidants in various chemical, food, and biological model systems. Encapsulation with cyclodextrins had a significant positive impact on the chemical composition of obtained extracts and it positively affected their antioxidant activity. Observed effects can be explained by an increased content of polyphenols in the formulations, specific physical properties of encapsulated compounds improving their antioxidant activity in complex food/physiological environment, and enhanced interaction with natural substrates. Depending on the applied model, the tested samples showed significant antioxidant protection in the concentration range 0.1⁻3%. Among the investigated cyclodextrins, hydroxypropyl-ß-cyclodextrin and randomly methylated-ß-cyclodextrin encapsulated extracts showed particularly good antioxidant activity and were especially potent in oil-in-water emulsion systems (1242 mg/g and 1422 mg/g of Trolox equivalents, respectively), showing significantly higher antioxidant activity than Trolox (reference antioxidant). In other models, they provided antioxidant protection comparable to commonly used synthetic antioxidants at concentration levels of 2⁻3%.

Paraoxonase 1 activity in patients with chronic obstructive pulmonary disease.

INTRODUCTION: Paraoxonase 1 (PON1) is an antioxidative enzyme manly associated with high density lipoproteins (HDL) in the peripheral blood. The aim of this study was to determine the PON1 paraoxonase and arylesterase activities in patients with chronic obstructive pulmonary disease (COPD). We also aimed to determine the concentration of reduced thiol groups as a marker of protein oxidation. MATERIALS AND METHODS: The study included 105 patients with stable COPD and 44 healthy controls. PON1 activities and thiols concentration were assayed in sera by spectrophotometry. RESULTS: PON1 basal (POX) and salt-stimulated paraoxonase activity (POX1) as well as arylesterase activity (ARE) were significantly reduced in COPD patients. In addition, concentration of reduced thiol groups was significantly decreased in COPD group. PON1 activities were similar in patients with different disease severity (GOLD stages). However, a significant reduction in POX, POX1 and ARE was observed already in GOLD II stage when compared to controls. POX and POX1 showed modest while ARE yielded very good power for discrimination between healthy subjects and COPD patients. Univariate and multivariate logistic regression analysis indicated that ARE is a good COPD predictor. CONCLUSION: Reduction of PON1 activity observed in COPD patients could be partly caused by oxidative environment. Lower concentrations of reduced thiol groups in COPD patients suggest that a decrease in PON1 activity could reflect oxidative changes of enzyme free cysteine residues. Furthermore, decreased PON1 arylesterase activity might indicate a down-regulation of PON1 concentration. Our results suggest that ARE could be considered as potential biomarker for COPD diagnosis.

Point of care testing in Croatia: a survey of the Working group for point of care testing of the Croatian society of medical biochemistry and laboratory medicine.

Introduction: The aim of this study was to investigate attitudes and routine procedures in point of care testing (POCT) among non-laboratory and laboratory healthcare professionals in Croatia. Materials and methods: The Working Group (WG) for POCT of the Croatian society of medical biochemistry and laboratory medicine has designed two anonymous surveys for laboratory staff and non-laboratory staff with a total of 44 questions/statements on POCT (27 questions for non-laboratory staff and 17 for laboratory staff). Surveys were sent to 184 medical biochemistry laboratory (MBL) managers, the Croatian medical chamber and the Croatian chamber of nurses. The survey was disseminated using the online survey platform SurveyMonkey. Results: A total of 112 non-laboratory healthcare professionals and 50 laboratories participated in the survey, which represents a response rate of 0.25% for non-laboratory professionals and 27% for MBLs. The majority of non-laboratory staff stated that POCT enables better medical care for the patient (90/112) and that the implementation of new POCT devices should be the responsibility of a POCT team comprising laboratory and clinical healthcare professionals. The great majority of responding MBLs (42/50) acknowledge that POCT is necessary for better patient care, and also realize that validation of POCT devices and comparison to the central laboratory is necessary before implementation (49/50). Conclusions: The majority of participants consider POCT as a medical tool that enables better patient care but there is still a lack of communication between laboratory and clinical staff. The study identified some critical spots that will help to create national guidelines to ensure high patient safety when using POCT devices.

Paraoxonase and arylesterase activity of paraoxonase 1 and oxidative stress parameters in cervical intraepithelial neoplasia.

Introduction: Paraoxonase 1 (PON1) is the enzyme that removes carcinogenic radicals from lipids. The aim of the study was to investigate the differences in PON1 activity and oxidation stress parameters between patients with cervical intraepithelial neoplasia (CIN) and healthy controls. Materials and methods: The study included 65 women with CIN and 109 healthy women. Lipid parameters were determined on Cobas Integra 400 plus (Roche, Mannheim, Germany). Tiols and reduced glutathione (GSH) were determined spectrophotometric using Eliman reagent. Activity of PON1 was assessed with two substrates, paraoxon and phenylacetate by spectrophotometric method. Malondialdehyde (MDA) was determined by high performance liquid chromatography (Shimadzu Corporation, Kyoto, Japan). Mann-Whitney-test, t-test, χ2-test, correlation and logistic regression was used in statistical analysis. P < 0.05 was considered statistically significant. Results: The basal (P = 0.929) and NaCl-stimulated (P = 0.985) PON1 activity and activities standardised on the concentration of high-density lipoprotein (HDL; P = 0.076; P = 0.065, respectively) and apolipoprotein AI (apo AI; P = 0.444; P = 0.499, respectively) as well as PON1 phenotypes (P = 0.842) did not differ significantly between the groups. The PON1 arylesterase activity (53±19 kU/L vs. 77±17 kU/L; P < 0.001) and HDL-standardized activity (37 (28-44) kU/mmol vs. 43 (37-50) kU/mmol; P < 0.001) and apoAI (29±11 kU/g vs. 44±11 kU/g; P < 0.001) was significantly reduced in the CIN group. The concentration of the thiol groups was similar (P = 0.519), of MDA was lower (0.39 (0.27-0.55) µmol/L vs. 0.76 (0.57-1.15) µmol/L; P < 0.001) and of GSH was higher (112.0 (66.0-129.6) µg/mL vs. 53.4 (34.8-134.4) µg/mL; P < 0.001) in the CIN group. Conclusion: Reduced PON1 arylesterase activity, lower MDA and higher GSH concentration were observed in CIN patients.

Can cranberry extract and vitamin C + Zn supplements affect the in vivo activity of paraoxonase 1, antioxidant potential, and lipid status?

BACKGROUND: The modern way of life exposes us to substantial oxidative stress, putting the focus on the research of antioxidant effects of dietary supplements. Recent studies have shown that the effectiveness of particular vitamins and herbal preparations might have an effect on paraoxonase activity. Paraoxonase 1 is an HDL associated enzyme which prevents the oxidation of LDL. Several studies have shown the beneficial effect of some dietary components to the activity of paraoxonase. The aim of this study was to analyze the effects of cranberry extract and vitamin C and zinc preparations (vitamin C + Zn) on serum paraoxonase 1 activity, antioxidant status, and glucose and lipid concentration. METHODS: The study included 31 healthy volunteers (median age 24 years). They were divided into 3 groups according to the intervention type and smoking status and exposed to commercially available preparations of the cranberry extract (2 g/day) and vitamin C + Zn (300 mg/day) during 4 weeks. RESULTS: The results have shown that there is a significant increase in the activity of the paraoxonase 1 in nonsmokers after the intervention with the cranberry extract as well as with vitamin C + Zn preparations. Also, total antioxidant status increased in the non-smokers subgroup after intervention with vitamin C + Zn. However, the lipid profile did not change significantly in response to antioxidant preparations. CONCLUSIONS: Our results show that antioxidant supplements can increase the antioxidant potential of an organism as well as paraoxonase 1 activity. This observation is pointing to the potential complementary role of dietary supplements in the primary prevention of atherosclerosis.

Effect of Alpha Lipoic Acid Supplementation on Oxidative Stress and Lipid Parameters in Women Diagnosed with Low-Grade Squamous Intraepithelial Lesions (LSILs): A Double-Blind, Randomized, Placebo-Controlled Trial.

Limited scientific evidence shows that alpha lipoic acid (ALA) can induce regression rates of low-grade squamous intraepithelial lesions (LSILs), but the mechanisms of these effects have not been elucidated. To gain a broader insight into its therapeutic potential and mechanisms of action, the effects of 3 months of supplementation with 600 mg of ALA on antioxidant and lipid status parameters in 100 patients with LSILs were investigated in a randomized, placebo-controlled study. The obtained results are discussed in terms of patients' initial metabolic status and diet quality (particularly nutritional intake of antioxidants). The obtained results showed that oxidative status biomarkers were not significantly affected by ALA supplementation. However, serum superoxide dismutase (SOD) activity was positively affected in the subgroup of patients with higher dietary antioxidant intake. Surprisingly, ALA supplementation resulted in a small but statistically significant increase in serum low density lipoprotein (LDL), and the observed effect was significantly affected by the initial lipid status of the participants. Larger studies are necessary to gain additional insights on the clinical significance of ALA as an antioxidant and hypolipemic agent and to optimize its potential application in LSIL treatment.

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.

Effect of Alpha-Lipoic Acid Supplementation on Low-Grade Squamous Intraepithelial Lesions-Double-Blind, Randomized, Placebo-Controlled Trial.

Low-grade squamous intraepithelial lesion (SIL) is a cytologic diagnosis etiologically related to human papilloma virus (HPV) infection that leads to the release of inflammation mediators, the formation of reactive oxygen species (ROS) and decreased levels of antioxidants in tissues, which is why antioxidants might be considered effective against SIL progression. This randomized double-blind placebo-controlled study aimed to investigate the effectiveness of alpha-lipoic acid (ALA) supplementation (600 mg/day) on the regression of low-grade SIL in 100 patients. Low-grade SIL was determined after the cytological screening, colposcopic examination and targeted biopsy and histological confirmation of cytological−colposcopic diagnosis. Inflammation parameters and the presence of HPV were determined by standard laboratory methods. Dietary and lifestyle habits were investigated using a standardized and validated semi-quantitative food questionnaire (FFQ). ALA supplementation significantly reduced the proportion of patients with low-grade cytological abnormalities, in comparison to placebo. Given the obtained level of significance (p < 0.001), the presented results indicate that short-term ALA supplementation shows a clinically significant effect on cervical cytology. Future studies should focus on the use of innovative formulations of ALA that might induce bioavailability and therapeutic efficiency against HPV infection and the investigation of synergistic effects of concurrent dietary/lifestyle modification and ALA supplementation in both low-grade and high-grade SIL.

Pathogen-associated molecular patterns and extracellular Hsp70 interplay in NLRP3 inflammasome activation in monocytic and bronchial epithelial cellular models of COPD exacerbations.

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)-1ß gene expression as well as IL-1ß 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.

Trauma and Antimicrobial Resistance Are Independent Predictors of Inadequate Empirical Antimicrobial Treatment of Ventilator-Associated Pneumonia in Critically Ill Patients.

Background: We aimed to assess independent risk factors for inadequate initial antimicrobial treatment (IAT) in critically ill patients with ventilator-associated pneumonia (VAP) treated in intensive care units (ICU) and to determine whether IAT is associated with adverse outcomes in patients with VAP. Patients and Methods: A prospective cohort study was performed and included 152 patients with VAP treated in an ICU for more than 48 hours. The main outcomes of interest were all-cause ICU mortality and VAP-related mortality. Other outcomes considered were: intra-hospital mortality, VAP-related sepsis, relapse, re-infection, length of stay in ICU (ICU LOS), and number of days on mechanical ventilation (MV). Results: One-third of patients (35.5%) received inadequate antimicrobial therapy. Trauma (odds ratio [OR], 3.55; 95% confidence interval [CI], 1.25-10.06) and extensively drug-resistant (XDR) causative agent (OR, 3.09; 95% CI, 1.23-7.74) were independently associated with inadequate IAT. Inadequate IAT was associated with a higher mortality rate (OR, 3.08; 95% CI, 1.30-7.26), VAP-related sepsis (OR, 2.39; 95% CI, 1.07-5.32), relapse (OR, 3.25; 95% CI, 1.34-7.89), re-infection (OR, 6.06; 95% CI, 2.48-14.77), and ICU LOS (ß 4.65; 95% CI, 0.93-8.36). Acinetobacter spp., Pseudomonas aeruginosa and Klebsiella/Enterobacter spp. were the most common bacteria in patients with IAT and those with adequate antimicrobial therapy. Conclusions: This study demonstrated that inadequate IAT is associated with a higher risk of the majority of adverse outcomes in patients with VAP treated in ICUs. Trauma and XDR strains of bacteria are independent predictors of inadequate IAT of VAP in critically ill patients.

Increased HSP70 and TLR2 Gene Expression and Association of HSP70 rs6457452 Single Nucleotide Polymorphism with the Risk of Chronic Obstructive Pulmonary Disease in the Croatian Population.

Heat shock protein 70 (Hsp70) engages Toll-like receptors (TLR) 2 and 4 when found in the extracellular compartment and contributes to inflammation in chronic obstructive pulmonary disease (COPD). Since there is growing evidence for the genetic risk factors for COPD, the gene expression of HSP70, TLR2 and TLR4 was determined, as well as the association between HSP70, TLR2 and TLR4 single nucleotide polymorphisms, (SNPs) and COPD. The gene expression was assessed in peripheral blood cells of 137 COPD patients and 95 controls by a quantitative polymerase chain reaction (qPCR), while a total of nine SNPs were genotyped by TaqMan allelic discrimination real-time PCR. HSP70 and TLR2 gene expression was increased in COPD patients compared to the controls, regardless of the disease severity and smoking status of participants. The rs6457452 SNP of HSP70 was associated with COPD, indicating the protective role of the T allele (OR = 0.46, 95% CI = 0.24-0.89, p = 0.022). Furthermore, COPD C/T heterozygotes showed a decreased HSP70 mRNA level compared to COPD C/C homozygotes. In conclusion, HSP70 and TLR2 may have a role in the pathogenesis of COPD, and the HSP70 rs6457452 variant might influence the genetic susceptibility to COPD in the Croatian population.

Association of Plasma Heat Shock Protein 70 with Disease Severity, Smoking and Lung Function of Patients with Chronic Obstructive Pulmonary Disease.

Extracellular heat shock protein 70 (eHsp70) might modulate immune responses in chronic obstructive pulmonary disease (COPD). The aim of the study was to explore eHsp70 concentration in stable COPD, its association with disease severity and smoking status as well as its diagnostic performance in COPD assessment. Plasma samples were collected from 137 COPD patients and 95 healthy individuals, and concentration of eHsp70 was assessed by commercially available enzyme-linked immunosorbent assay (ELISA) kit (Enzo Life Science, Farmingdale, NY, USA). COPD patients were subdivided regarding airflow obstruction severity and symptoms severity according to the Global Initiative for COPD (GOLD) guidelines. eHsp70 concentration increased in COPD patients when compared to controls and increased with the severity of airflow limitation as well as symptoms burden and exacerbation history. eHsp70 concentration did not differ among COPD patients based on smoking status, yet it increased in healthy smokers compared to healthy nonsmokers. In addition, eHsp70 negatively correlated with lung function parameters forced expiratory volume in one second (FEV1) and FEV1/ forced vital capacity (FVC), and positively with COPD multicomponent indices BODCAT (BMI, airflow obstruction, dyspnea, CAT score), BODEx (BMI, airflow obstruction, dyspnea, previous exacerbations), CODEx (Charlson's comorbidity index, airflow obstruction, dyspnea, previous exacerbations) and DOSE (dyspnea, airflow obstruction, smoking status, previous exacerbations) With great predictive value (OR = 7.63) obtained from univariate logistic regression, eHsp70 correctly classified 76% of cases. eHsp70 is associated with COPD prediction and disease severity and might have the potential for becoming an additional biomarker in COPD assessment.

Extracellular adenosine triphosphate is associated with airflow limitation severity and symptoms burden in patients with chronic obstructive pulmonary disease.

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 < 0.001). Moreover, it was increasing with disease severity (GOLD 2-4) as well as symptoms burden and exacerbations history (GOLD A-D) (P < 0.05). eATP in healthy smokers differed from healthy non-smokers (P < 0.05) but was similar to GOLD 2 and GOLD A patients. eATP showed great diagnostic performances (OR = 12.98, P < 0.001) and correctly classified 79% of study participants. It demonstrated association with FEV1 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.

Effects of extracellular Hsp70 and cigarette smoke on differentiated THP-1 cells and human monocyte-derived macrophages.

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.