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.

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.

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.

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.

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.

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.

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.

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.

Pro-inflammatory effects of extracellular Hsp70 and cigarette smoke in primary airway epithelial cells from COPD patients.

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.

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%.

PON1 gene polymorphisms in patients with chronic obstructive pulmonary disease.

AIMS: Chronic obstructive pulmonary disease (COPD) is characterised with oxidative stress. Paraoxonase 1 (PON1) is an enzyme, coded by PON1 gene, with distinctive antiatherogenic and antioxidative roles. We aimed to investigate the frequencies of Q192R, L55M and -108C>T polymorphisms and association of those polymorphisms with paraoxonase and arylesterase activities in patients with COPD. METHODS: PON1 genotype was determined by PCR-restriction fragment length polymorphism method. PON1 activity was measured by paraoxon and phenylacetate. RESULTS: Only -108C>T polymorphism resulted in significantly different distribution of genotypes and alleles, with higher frequency of TT genotype and T allele in patients compared with control subjects. Moreover, T allele (OR 2.29 (95% CI 1.54 to 3.41); p<0.001) as well as TT genotype (OR 5.00 (95% CI 2.19 to 11.43); p<0.001) showed an association with the disease. -108C>T polymorphism was suggested as a significant diagnostic predictor for the disease (OR (95% CI) 2.65 (1.53 to 4.59), p=0.001), with an area under the receiver operating characteristic curve of 0.90 (95% CI 0.84 to 0.93) and with 83.90% of correctly classified cases. CONCLUSIONS: Higher frequency of TT genotype and T allele could contribute to the observed reduction of PON1 activity in patients with COPD. T allele and TT genotype are associated with COPD, and the PON1-108C>T polymorphism could be a potential predictor of the disease.

Extracellular Hsp70 induces inflammation and modulates LPS/LTA-stimulated inflammatory response in THP-1 cells.

Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP) via Toll-like receptors TLR2 and TLR4, and stimulate immune and inflammatory responses leading to sterile inflammation and propagation of already existing inflammation. It was found elevated in the blood of patients with chronic obstructive pulmonary disease (COPD), who might suffer occasional bacterial colonizations and infections. We used a monocytic THP-1 cell line as a cellular model of systemic compartment of COPD to assess inflammatory effects of eHsp70 when present alone or together with bacterial products lypopolysaccharide (LPS) and lypoteichoic acid (LTA). THP-1 cells were differentiated into macrophage-like cells and treated with various concentrations of recombinant human Hsp70 protein (rhHsp70), LPS (TLR4 agonist), LTA (TLR2 agonist), and their combinations for 4, 12, 24, and 48 h. Concentrations of IL-1α, IL-6, IL-8, and TNF-α were determined by ELISA. Cell viability was assessed by MTS assay, and mode of cell death by luminometric measurements of caspases-3/7, -8, and -9 activities. rhHsp70 showed cell protecting effect by suppressing caspases-3/7 activation, while LPS provoked cytotoxicity through caspases-8 and -3/7 pathway. Regarding inflammatory processes, rhHsp70 alone induced secretion of IL-1α and IL-8, but had modulatory effects on release of all four cytokines when applied together with LPS or LTA. Combined effect with LPS was mainly synergistic, and with LTA mainly antagonistic, although it was cytokine- and time-dependent. Our results confirmed pro-inflammatory function of extracellular Hsp70, and suggest its possible implication in COPD exacerbations caused by bacterial infection through desensitization or inappropriate activation of TLR2 and TLR4 receptors.