Effect of Vitamin D3 Supplementation on Interleukin 6 and C-Reactive Protein Profile in Athletes.

Vitamin D3 has a preventive, anti-inflammatory effect. However, there are still few studies linking the effects of athlete training to vitamin D3 supplementation and the immune response. The study evaluated the impact of vitamin D3 supplementation on interleukin 6 (IL-6) release during physical exercise in relation to C-reactive protein (CRP) levels in healthy male athletes. Twenty-five soccer players were divided into two groups-with (GS) and without (GN) vitamin D3 supplementation in a dose of 20,000 IU twice a week for 8 wk (about 6,000 IU/d). At the baseline (T1) and at the end (T2) of the training cycle serum concentrations of 25-hydroxyvitamin D [25(OH)D], IL-6 and CRP were measured. In the GS group, we observed a significant increase in 25(OH)D concentration (p=0.004), and non-significantly increased levels (p>0.05) of IL-6 and CRP. At the baseline, CRP in the supplemented athletes who had suboptimal vitamin D3 concentration in T1 (GSO) was significantly higher than in those with an optimal baseline vitamin D3 level (GO) (p=0.028). However, in GO in T2, a non-significant trend of negative correlation (p=0.055) between 25(OH)D concentration and IL-6 level was found. In the total study group (TG), a statistically significant (p=0.021) negative correlation in T1 was observed between 25(OH)D and CRP. However, our results do not support the immune-modulatory effect of vitamin D3 supplementation in a dose of 6,000 IU/d in athletes, in relation to IL-6 production and its subsequent stimulatory effect on CRP releasing.

Helicobacter pylori and Epstein-Barr Virus Co-Infection in Polish Patients with Gastric Cancer - A Pilot Study.

The infectious agents may be the etiological factor of up to 15-20% of cancers. In stomach cancer, attention is paid to Helicobacter pylori and Epstein-Barr virus, both of which cause gastritis and can lead to tumor development. In co-infection, the inflammatory process is much more intense. We assessed the seroprevalence towards H. pylori and EBV in 32 patients with diagnosed gastric cancer. H. pylori antibodies were found in 69% patients, and anti-EBV - in all of them. The study confirmed that co-infection of H. pylori and EBV seems to be important in etiopathology of gastric cancer.

Diagnostic value of PPARδ and miRNA-17 expression levels in patients with non-small cell lung cancer.

The PPARδ gene codes protein that belongs to the peroxisome proliferator-activated receptor (PPAR) family engaged in a variety of biological processes, including carcinogenesis. Specific biological and clinical roles of PPARδ in non-small cell lung cancer (NSCLC) is not fully explained. The association of PPARα with miRNA regulators (e.g. miRNA-17) has been documented, suggesting the existence of a functional relationship of all PPARs with epigenetic regulation. The aim of the study was to determine the PPARδ and miR-17 expression profiles in NSCLC and to assess their diagnostic value in lung carcinogenesis. PPARδ and miR-17 expressions was assessed by qPCR in NSCLC tissue samples (n = 26) and corresponding macroscopically unchanged lung tissue samples adjacent to the primary lesions served as control (n = 26). PPARδ and miR-17 expression were significantly lower in NSCLC than in the control (p = 0.0001 and p = 0.0178; respectively). A receiver operating characteristic (ROC) curve analysis demonstrated the diagnostic potential in discriminating NSCLC from the control with an area under the curve (AUC) of 0.914 for PPARδ and 0.692 for miR-17. Significant increase in PPARδ expression in the control for current smokers vs. former smokers (p = 0.0200) and increase in miR-17 expression in control tissue adjacent to adenocarcinoma subtype (p = 0.0422) were observed. Overexpression of miR-17 was observed at an early stage of lung carcinogenesis, which may suggest that it acts as a putative oncomiR. PPARδ and miR-17 may be markers differentiating tumour tissue from surgical margin and miR-17 may have diagnostic role in NSCLC histotypes differentiation.

Serum Extracellular Vesicle-Derived miRNAs in Patients with Non-Small Cell Lung Cancer-Search for Non-Invasive Diagnostic Biomarkers.

The aim of the study was a search for diagnostic and/or prognostic biomarkers in patients with non-small cell lung cancer (NSCLC) patients, based on circulating microRNAs (miRs: miR-23a, miR-361, miR-1228 and miR-let7i) in extracellular vesicles (EVs). Serum EVs were isolated from NSCLC patients (n = 31) and control subjects (n = 21). RNA was isolated from EVs and reverse transcription reaction was performed. Relative levels of miR-23a, miR-361, miR-1228 and miR-let7i were assessed in real-time qPCR using TaqMan probes. Analysis was based on the 2-ΔΔCT method. Statistically significant lower levels of miR-23a and miR-let7i were observed among NSCLC patients vs. control group: miR-23a, 0.054 vs. 0.107; miR-let7i, 0.193 vs. 0.369 (p = 0.003, p = 0.005, respectively). A receiver operating characteristic (ROC) curve analysis demonstrated the diagnostic potential of each individual serum EV-derived miRNA with an area under the curve AUC = 0.744 for miR-23a (p = 0.0003), 0.733 for miR-let7i (p = 0.0007). The decreased level of miR-23a in patients correlated with metastasis to lymph nodes and with AJCC tumor staging system. The results demonstrate that miR-23a and miR-let7i may prove clinically useful as significant, non-invasive markers in NSCLC diagnosis. Additionally, changing profile level of miR-23a that correlates with cancer development may be considered as an NSCLC progression marker.

Type of training has a significant influence on the GH/IGF-1 axis but not on regulating miRNAs.

The growth hormone (GH)/insulin-like growth factor-1 axis is responsible for glucose homeostasis. In the present study we assessed the expression levels of miRNA-124, miRNA-210 and miRNA-375 and immunoexpression of IGFBP-3 in relation to the concentrations of IGF-1 and glucose in athletes performing different types of effort. Sixty-six young male athletes (age 25.4±4.1 years) were divided into: group EN (33 male athletes; age 25.6±4.4 years) with endurance-type efforts (disciplines: triathlon, long distance running, cycling) and group ST (33 male athletes; age 25.2±3.9 years) with strength-type efforts (disciplines: weightlifting, body building, CrossFit). The control group consisted of 28 non-training men (age 29.1±4.7 years). Statistically significantly higher IGF-1 concentration and lower glucose concentration (P<0.05) in serum were observed in the group of athletes (vs. controls). Immunoexpression of IGFBP-3 was higher in athletes (vs. controls), and a higher value of immunoexpression was obtained in athlete group ST vs. group EN (P>0.05). Levels of expression of miRNA-210 and miRNA-375 were higher in athletes vs. controls (P>0.05). The obtained data confirmed the importance of the somatotropic axis in the regulation of metabolic adaptation to physical exercise. The detected variation in the concentrations and expression levels of the studied molecules involved in the somatotropic axis in athletes confirmed the role of the somatotropic axis in adaptation to physical effort. Statistically significant reduction of glucose concentration and the highest expression of IGF-1in serum in athletes suggest the anabolic effect of IGF-1 through insulin receptors on many tissues under the influence of moderate physical exercises (mainly during resistance training).

Evaluation of the relationship between the IL-17A gene expression level and regulatory miRNA-9 in relation to tumor progression in patients with non-small cell lung cancer: a pilot study.

A pro-inflammatory cytokine, IL-17A, is associated with increased risk of developing numerous cancers, including non-small cell lung cancer (NSCLC). IL-17A is a target gene for miR-9. This encouraged us to analyze these two genes in terms of their usefulness as prognostic markers in NSCLC. The expression levels of IL-17A gene and miR-9 was assessed in 26 NSCLC tissue samples and 26 unchanged lung tissue adjacent to lung tumors (control tissue), using qPCR. In both tissue groups, a decreased expression of IL-17A was observed in 100% of samples. Increased expression of miRNA-9 was observed in 92% of tumor samples, and in 100% of control samples. Neither statistical differences in the level of expression IL-17A depending on the patient's age, gender, smoking status, nor histopathology of the cancer was found. Regarding the presence of nodule metastasis ('N' value in TNM classification), significantly lower expression level of IL-17A was observed in cN2 as compared with cN1 group. Additionally, statistically lower IL-17A expression was found in III versus II tumor stage (cAJCC classification). Significant negative correlation between both studied genes was revealed in SCC subgroup. This leads to the conclusion that miRNA-9 can regulate the expression of IL-17A as an IL-17A mRNA antagonistic mediator. Inhibition of proinflammatory action of IL-17A in correlation with tumor progression can be related to various activity of Th17 cells on cancer development according to its immunogenicity, and also may suggest suppressive role of IL-17A in tumor progression. However, because of low number of analyzed samples, further studies on the functional role of IL-17A in development and/or progression NSCLC seem warranted.

Relationships between the Expression of the ACTN3 Gene and Explosive Power of Soccer Players.

Muscle strength and maximal speed are factors determining athlete's results during competition. Their association with ACTN3 gene activity has been documented. The purpose of this study was the analysis of ACTN3 gene expression during a 2 month training cycle of soccer players and its correlation with the countermovement jump (CMJ) and squat jump (SJ). The study group consisted of 22 soccer players (aged 17-18). The study material included peripheral blood lymphocytes. The relative expression (RQ) of the ACTN3 gene was analyzed by qPCR and performed before and after the two-month training cycle. Before the training cycle low expression levels of ACTN3 (median RQ = 0.95) were observed, yet after the training cycle they were elevated (median RQ = 1.98) ( p = 0.003). There was an increase in performance of both jumps: SJ (p = 0.020) and CMJ (p = 0.012) at the end of the training cycle. A simultaneous increase in the ACTN3 gene expression level and height in both jump tests was observed in 73% of athletes (p > 0.05). There were no significant relationships between the ACTN3 gene expression level and the results of the CMJ and SJ. However, explosive strength is a complex feature shaped by many different factors and it could be the reason why we did not observe correlations between these variables.

An assessment of the relationship between the expression of CCR7/CCL19 axis and selected regulatory miRNAs in non-small cell lung cancer.

CC chemokine receptor type 7 (CCR7) and its ligands has been implicated in the occurrence and progression of NSCLC. Previous studies have revealed that the diagnostic value of CCR7/CCL19 axis in lung tumorigenesis remains controversial. The present study evaluates the relationship between the mRNA expression of CCR7/CCL19 axis and selected regulatory miRNAs in NSCLC patients. It analyzes the expression level of CCR7 mRNA and its ligand in tumor tissue in relation to expression level of two miRNAs: miR let-7a and miR-335, as transcriptional regulators of study genes. Twenty-seven patients (n = 27) were enrolled. The expression of the studied genes and miRNAs was evaluated by qPCR. Tumour tissue fragments, adjacent macroscopically-unchanged lung tissue (control) and patient serum were used as biological material for study. Elevated expression of CCR7 and CCL19 mRNA was observed in patients with metastasis to lymph nodes. We noticed upregulated miR-335 expression and downregulated miR let-7a expression in patient serum with regard to AJCC tumor staging. Higher miR-335 expression and lower miR let-7a expression level was observed in patients with metastasis to lymph node. The presence of changes observed in the expression level of miR-335 and miR let-7a in the serum of NSCLC patients in relation to lymph node metastases and tumor stage may serve as a non-invasive molecular biomarker of tumor progression; however, this observation requires further investigation.

Overexpression of chitotriosidase and YKL-40 in peripheral blood and sputum of healthy smokers and patients with chronic obstructive pulmonary disease.

Background: Despite the absence of endogenous chitin in humans, chitinases are present in the serum of healthy subjects and their levels are increased in a variety of chronic inflammatory conditions. It has been shown that chitotriosidase and structurally related chitinase-like protein-YKL-40 contribute to the pathogenesis of COPD. However, details regarding the relation of their systemic and local airways levels remain unknown. Objectives: To examine peripheral blood and sputum chitotriosidase and YKL-40 expression in smokers and patients with COPD. Methods: Forty patients with COPD, 20 healthy smokers and 10 healthy never-smokers were studied. Serum and induced sputum chitotriosidase protein and activity levels, YKL-40 concentrations, and their gene expression in sputum cells and peripheral blood mononuclear cells (PBMC) were evaluated. Results: Both chitotriosidase protein levels and activity were higher in sputum obtained from COPD subjects compared to healthy never-smokers (P<0.05 and P<0.01, respectively). A similar pattern was observed for PBMC chitotriosidase mRNA expression (P<0.001). YKL-40 serum concentrations were elevated in healthy smokers and COPD subjects compared to healthy never-smokers (P<0.001 and P<0.01, respectively). In sputum, YKL-40 levels were increased in COPD compared to healthy never-smokers (P<0.01). PBMC YKL-40 mRNA expression was increased in COPD and healthy smokers compared to healthy never-smokers (P<0.0001). No associations were found between chitotriosidase or YKL-40 peripheral blood levels and sputum levels. Conclusions: Our results demonstrate that chitotriosidase and YKL-40 are overexpressed in peripheral blood and airways in both healthy smokers and COPD subjects which may indicate smoking-related activation of macrophages, neutrophils, and epithelial cells.

Serum levels and gene expression of pentraxin 3 are elevated in COPD.

PURPOSE: Pentraxin 3 (PTX-3) is an acute phase protein that belongs to the pentraxin superfamily. It is synthesized locally at the site of inflammation and its levels are related to the damage of blood vessels. There are only a few studies examining the relationship between PTX-3 and chronic obstructive pulmonary disease (COPD). The aim of this study was to evaluate the serum levels of PTX-3 and relative PTX-3 gene expression in COPD patients and their correlations with cigarette smoking history and lung function. MATERIALS/METHODS: A total number of 34 participants were enrolled into this study. Only stable patients without comorbidities were recruited. After obtaining written informed consent all planned procedures were performed (pre- and post-bronchodilator spirometry, blood samples for PTX-3 serum levels and PTX-3 gene expression measurements, demographical data, medical history, COPD patients were also asked for CAT and MMRC questionnaires). RESULTS: PTX-3 serum levels were significantly higher in the COPD group (29.22 (5.47) ng/ml vs. 14.64 (3.64) ng/ml). PTX-3 gene relative quantification (RQ) values were also significantly higher in the COPD group (0.15 (1.33) vs. -2.80 (1.99)). No differences in CRP serum levels were found between the control group and the COPD group. CONCLUSIONS: Our study demonstrates that serum levels of PTX-3 and the relative expression values of its gene are elevated in COPD, and can be related to cigarette smoking history.

The Expression Levels of IL-4/IL-13/STAT6 Signaling Pathway Genes and SOCS3 Could Help to Differentiate the Histopathological Subtypes of Non-Small Cell Lung Carcinoma.

BACKGROUND: The interleukin (IL)-4/IL-13/signal transducer and activator of transcription (STAT) 6 signaling pathway and the SOCS3 gene, one of its main regulators, constitute an important link between the inflammation process in the epithelial cells and inflammatory-related tumorigenesis. The present study is the first to evaluate IL-4, IL-13, STAT6, and SOCS3 mRNA expression in non-small cell lung carcinoma (NSCLC) histopathological subtypes. METHODS: Gene expression levels were assessed using TaqMan® probes by quantitative reverse transcription PCR (qRT-PCR) in lung tumor samples and unchanged lung tissue samples. RESULTS: Increased expression of IL-4, IL-13, and STAT6 was observed in all histopathological NSCLC subtypes (squamous cell carcinoma [SCC], adenocarcinoma [AC], and large cell carcinoma [LCC]). Significantly higher expression of IL-13 and STAT6 (p = 0.019 and p = 0.008, respectively) was found in SCC than in LCC. No statistically significant differences were found for IL-4. Significantly higher SOCS3 expression was found in LCC than in AC (p = 0.027). A negative correlation (rho = -0.519) was observed for the STAT6 and SOCS3 genes in SCC (p = 0.005). No associations were found between gene expression and tumor staging (post-operative Tumor Node Metastasis [pTNM], American Joint Committee on Cancer [AJCC]), patients' age, sex, or history of smoking. CONCLUSIONS: As the number of LCC cases in our study was quite low, the statistically significant results obtained should be confirmed in a larger group of patients, particularly as the relationships identified between increased IL-4, IL-13, and STAT6 mRNA expression and decreased SOCS3 expression suggest that these genes may serve as potential diagnostic markers for differentiating between NSCLC histopathological subtypes.

The association between airway eosinophilic inflammation and IL-33 in stable non-atopic COPD.

BACKGROUND: Interleukin(IL)-33 is an epithelial alarmin important for eosinophil maturation, activation and survival. The aim of this study was to examine the association between IL-33, its receptor expression and airway eosinophilic inflammation in non-atopic COPD. METHODS: IL-33 concentrations were measured in exhaled breath condensate (EBC) collected from healthy non-smokers, asthmatics and non-atopic COPD subjects using ELISA. Serum and sputum samples were collected from healthy non-smokers, healthy smokers and non-atopic COPD patients. Based on sputum eosinophil count, COPD subjects were divided into subgroups with airway eosinophilic inflammation (sputum eosinophils > 3%) or without (sputum eosinophils ≤3%). IL-33 and soluble form of IL-33 receptor (sST2) protein concentrations were measured in serum and sputum supernatants using ELISA. ST2 mRNA expression was measured in peripheral mononuclear cells and sputum cells by qPCR. Hemopoietic progenitor cells (HPC) expressing ST2 and intracellular IL-5 were enumerated in blood and induced sputum by means of flow cytometry. RESULTS: IL-33 levels in EBC were increased in COPD patients to a similar extent as in asthma and correlated with blood eosinophil count. Furthermore, serum and sputum IL-33 levels were higher in COPD subjects with sputum eosinophilia than in those with a sputum eosinophil count ≤3% (p < 0.001 for both). ST2 mRNA was overexpressed in sputum cells obtained from COPD patients with airway eosinophilic inflammation compared to those without sputum eosinophilia (p < 0.01). Similarly, ST2 + IL-5+ HPC numbers were increased in the sputum of COPD patients with airway eosinophilia (p < 0.001). CONCLUSIONS: Our results indicate that IL-33 is involved in the development of eosinophilic airway inflammation in non-atopic COPD patients.

Immunoexpression analysis of selected JAK/STAT pathway molecules in patients with non- small-cell lung cancer.

INTRODUCTION    Signal transducer and activator of transcription (STAT) proteins are critically involved in tumorigenesis in various cancers, including lung cancer. OBJECTIVES    The aim of the study was to analyze the immunoexpression levels of 3 STAT proteins: STAT3, STAT5, and STAT6 in their phosphorylated forms (pSTATs), STAT inhibitors PIAS3 and SOCS3, and additionally cyclooxygenase 2 (COX­2), as potential diagnostic and prognostic markers in lung cancer. PATIENTS AND METHODS    The study included 71 patients diagnosed with non- small-cell lung cancer (NSCLC). The immunoexpression levels of the proteins were assessed in lung tissue samples, using an enzyme­linked immunosorbent assay. Tumors were staged using the postoperative TNM classification. RESULTS    All studied STATs were overexpressed in 54% to 55% of NSCLC specimens. Significantly higher STAT3 and STAT6 immunoexpression levels were observed in squamous cell carcinoma. Significant differences between NSCLC samples and controls were found for STAT5. Significantly higher STAT5 levels were observed in pT2 tumors. The COX­2 overexpression was observed in 55% of NSCLC specimens and was significantly higher in T2 tumors. STAT inhibitors were underexpressed in 56% to 58% of NSCLC specimens. The PIAS3 immunoexpression was significantly lower in non-squamous cell carcinoma. The SOCS3 level was significantly lower in smaller tumors (pT1). Negative correlations between STAT5 and PIAS3 levels, as well as between STAT6 and SOCS levels, and a positive correlation between STAT5 and COX-2 levels were observed. CONCLUSIONS    The deregulated expression of the studied pSTATs and their inhibitors may be involved in the development and progression of lung cancer. The observed differences between the histotypes suggest the potential usefulness of STAT proteins as diagnostic markers. Our results may contribute to the search for targets in lung cancer therapy.

Small suitability of the DLEC1, MLH1 and TUSC4 mRNA expression analysis as potential prognostic or differentiating markers for NSCLC patients in the Polish population.

According to the latest data, lung cancer is one of the most common cancer worldwide, men contributing nearly 21.2% and women 8.6% of all diagnosed cancers. Late detection of tumour drastically reduces the chance for a cure. Thus, it is important to search for candidate biomarkers for screening of early stage nonsmall cell lung carcinoma (NSCLC). Tumour suppressor genes, DLEC1, TUSC4 and MLH1, localized on 3p21 are recognized to play a role in NSCLC carcinogenesis. The aim of this study was to assess the relationship between the DLEC1, TUSC4 and MLH1 mRNA expression, and clinical features of NSCLC patients, tobacco addiction, and tumour histopathological characteristics. The DLEC1, TUSC4 and MLH1 expression was analysed in lung tumour tissue samples obtained from 69 patients diagnosed with NSCLC: squamous cell carcinoma (n = 34), adenocarcinoma (n = 24), large cell carcinoma (n = 5), carcinoma adenosquamosum (n = 5). A decreased gene expression (RQ < 0.7) was observed for DLEC1 in 60.9% of tumour samples, for MLH1 in 50.7% and for TUSC4 in 26% of NSCLC samples. DLEC1 was decreased in more aggressive subtypes: large cell carcinoma and adenocarcinoma-squamous cell carcinoma. The simultaneous downregulation of two of the studied genes, DLEC1 andMLH1,was observed in 30.4% of NSCLCsamples, highlighting the importance of these two genes in lung carcinogenesis. We found no correlation between the DLEC1, TUSC4 and MLH1 gene expression and NSCLC patient characteristics (gender, age and smoking) or cancer histopathology. No significant differences in the gene expression among NSCLC subtypes indicate the weakness of DLEC1, TUSC4 and MLH1 expression analysis as potential differentiating markers of NSCLC subtypes in the Polish population.

Signaling pathways and their miRNA regulators involved in the etiopathology of idiopathic pulmonary fibrosis (IPF) and hypersensitivity pneumonitis (HP).

Idiopathic pulmonary fibrosis (IPF) and hypersensitivity pneumonitis (HP) belong to heterogenic group of interstitial lung diseases (ILD). For the reason that this group of diseases present with complex clinical non-specific features, they represent a diagnostic and therapeutic challenge. In this review we focus on several crucial signaling pathways participating in inflammation, fibrosis and EMT processes, so important in the course of ILD: TNF-α/NFκß, TGF-ß/SMAD, Wnt-ß-catenin and PI3K-Akt signaling. Moreover, this review summarizes the role of selected signaling pathways and some miRNAs which are their regulators during development and progression of IPF and HP. Recent advances indicate the potential role of miRNAs as a molecular markers differentiating clinical course of ILD.

Expression analysis of selected classes of circulating exosomal miRNAs in soccer players as an indicator of adaptation to physical activity.

Recently studies have shown that, depending on the type of training and its duration, the expression levels of selected circulating myomiRNAs (c-miR-27a,b, c-miR-29a,b,c, c-miR-133a) differ and correlate with the physiological indicators of adaptation to physical activity. To analyse the expression of selected classes of miRNAs in soccer players during different periods of their training cycle. The study involved 22 soccer players aged 17-18 years. The multi-stage 20-m shuttle run test was used to estimate VO2 max among the soccer players. Samples serum were collected at baseline (time point I), after one week (time point II), and after 2 months of training (time point III). The analysis of the relative quantification (RQ) level of three exosomal myomiRNAs, c-miRNA-27b, c-miR-29a, and c-miR-133, was performed by quantitative polymerase chain reaction (qPCR) at three time points - before the training, after 1 week of training and after the completion of two months of competition season training. The expression analysis showed low expression levels (according to references) of all evaluated myomiRNAs before the training cycle. Analysis performed after a week of the training cycle and after completion of the entire training cycle showed elevated expression of all tested myomiRNAs. Statistical analysis revealed significant differences between the first and the second time point in soccer players for c-miR-27b and c-miR-29a; between the first and the third time point for c-miR-27b and c-miR-29a; and between the second and the third time point for c-miR-27b. Statistical analysis showed a positive correlation between the levels of c-miR-29a and VO2 max. Two months of training affected the expression of c-miR-27b and miR-29a in soccer players. The increased expression of c-miR-27b and c-miR-29 with training could indicate their probable role in the adaptation process that takes place in the muscular system. Possibly, the expression of c-miR-29a will be found to be involved in cardiorespiratory fitness in future research.

FHIT promoter methylation status, low protein and high mRNA levels in patients with non-small cell lung cancer.

FHIT is a tumor suppressor gene that is frequently silenced in non-small cell lung cancer (NSCLC) and also in preneoplastic lesions. Promoter hypermethylation was previously observed in NSCLC, and its epigenetic silencing, observed on mRNA or protein level, was proposed to predict NSCLC outcome. In the present study we evaluated the relationship between FHIT expression on mRNA level and promoter methylation, or immunoexpression level. The aim of this study was to analyze the usefulness of FHIT as early differentiating biomarker in NSCLC patients. Lung tissue specimens were obtained from 59 patients with diagnosed NSCLC (SCC=34, AC=20, LCC=5). FHIT promoter methylation was assessed in methylation-specific PCR. Relative expression analysis of FHIT was performed in real-time PCR (qPCR) and protein immunoexpression by ELISA assay. Significant differences in FHIT expression between NSCLC histopathological groups (SCC, AC, LCC) were observed (p=0.000009), with the lowest level in SCC. FHIT expression was significantly higher (p=0.034) in men vs. women. Methylated FHIT alleles were present both in NSCLC and control specimens. Mean MI value was higher in control tissue vs. neoplasm, and in men vs. women and it increased with patient age. Significant increase in MI level was observed in N0 group vs. N1 and N2, according to the TNM staging (p=0.0073). Differences in FHIT expression levels between AC, LCC and SCC indicated the usefulness of this gene as a diagnostic marker for NSCLC subtype differentiation. FHIT promoter hypermethylation both in cancer and control tissue indicated the presence of epigenetic alterations in early stage of NSCLC development. Differences in gene promoter methylation between cancer patients with and without node infiltration might be considered as a prognostic marker. Significantly lower FHIT protein immunoexpression was revealed in the group with long and intense history of smoking assessed as PYs (PY<40 vs. PY≥40, p=0.01). These results suggest the need of further study on FHIT as a potential biomarker.

Expression level and methylation status of three tumor suppressor genes, DLEC1, ITGA9 and MLH1, in non-small cell lung cancer.

Despite therapeutic advances, lung cancer remains one of the most common causes of cancer-related death in the world. There is a need to develop biomarkers of diagnostic and/or prognostic value and to translate findings in basic science research to clinical application. Tumor suppressor genes (TSGs) represent potential useful markers for disease detection, progression and treatment target. We tried to elucidate the role of three 3p21.3 TSGs: DLEC1, ITGA9 and MLH1, in non-small cell lung cancer (NSCLC). We assessed their expression pattern by qPCR in 59 NSCLC tissues and in the matched macroscopically unchanged lung tissues. Additionally, we analyzed gene promoter methylation status by methylation-specific PCR in NSCLC samples. We did not find significant correlations between gene expression and methylation. In case of DLEC1 and ITGA9, expression levels were decreased in 71-78 % of tumor samples and significantly different between tumor and normal tissues (P = 0.0001). It could point to their diagnostic value. ITGA9 could also be regarded as a diagnostic marker differentiating NSCLC subtypes, as its expression level was significantly lower in squamous cell carcinoma (P = 0.001). The simultaneous down-regulation of DLEC1 and ITGA9 was observed in 52.5 % of NSCLCs. MSPs revealed high frequencies of gene promoter methylation in NSCLCs: 84 % for DLEC1 and MLH1 and 57 % for ITGA9. Methylation indexes reflected moderate gene methylation levels: 34 % for ITGA9, 27 % for MLH1 and 26 % for DLEC1. However, frequent simultaneous methylation of the studied genes in more than 50 % of NSCLCs suggests the possibility of consider them as a panel of epigenetic markers.

Altered Cyclooxygenase-2 Expression in Pulmonary Sarcoidosis is not Related to Clinical Classifications.

Elevated COX-2 activity is associated with the development of chronic lung diseases leading to bronchial obstruction, including sarcoidosis. The aim of the study was to examine expression pattern of COX-2 messenger RNA (mRNA). Expression was performed by q-PCR method in bronchoalveolar lavage (BAL) cells and peripheral blood (PB) lymphocytes in sarcoidosis patients (n = 61) and control group (n = 30). Analysis of COX-2 mRNA expression level in BAL fluid and PB revealed downregulation in sarcoidosis and control groups. In PB lymphocytes, the statistically significant difference between patients and controls was observed (P = 0.003, Mann-Whitney U test), with higher expression in patients. There were no statistically significant differences between patients without and with parenchymal involvement (stages I vs. II-IV), between patients with acute vs. insidious onset of disease and between patients with abnormal vs. normal spirometry (P > 0.05, Mann-Whitney U test). Results suggest that expression of COX-2 mRNA in patients with pulmonary sarcoidosis is not related to clinical classifications.

Assessment of the frequency of genetic alterations (LOH/MSI) in patients with intraepithelial cervical lesions with HPV infection: a pilot study.

In the present study, we analyzed (1) the type of HPV infection and (2) the frequency of loss of heterozygosity and microsatellite imbalance (LOH/MSI) in normal cytology and cervical intraepithelial neoplasia (CIN1-3). The cytological material included: low-grade squamous intraepithelial lesions (CIN1, n = 11), high-grade lesions (CIN2 and CIN3, n = 13), and cytologically normal cells from non-neoplastic cervical samples (n = 8). HPV genotyping was done using RealLine HPV 16/18 kit. We used 20 microsatellite markers from: 1p31.2, 3p14.3, 3p21.3, 3p22.2, 3p24.2, 3p25.3, 7q32.2, 9p21.3, 11p15.5, 12q23.2, and 16q22.1. LOH/MSI was correlated with clinicopathological parameters. The presence of HPV DNA was revealed in 78.13 % samples, including normal cytology. LOH/MSI was the most frequent for: 3p25.3 (39 %), 3p22.2 (20.83 %), 3p24.2 (20 %), and 3p14.3 (16.67 %). It was demonstrated that D3S1234 (FHIT; 3p14.3), D3S1611 (MLH1; 3p22.2), D3S1583 (RARB; 3p24.2), D3S1317 and D3S3611 (VHL; 3p25.3) could differentiate patients with CIN2/CIN3 versus CIN1, showing significantly higher frequency in CIN2/CIN3. LOH/MSI frequency for other than 3p markers was lower, 10-22.2 %. The simultaneous occurrence of LOH/MSI for several markers (OFAL) was higher in CIN2/CIN3. Significant differences in OFAL were found between samples with versus without HPV infection. In HPV-positive patients, significant differences in OFAL were found between normal cytology, CIN1 and CIN2/CIN3. HPV infection influences the increase in LOH/MSI frequency, especially in tumor suppressor gene loci. Several studied microsatellite markers seem to be useful for CIN grading. Hopefully, the obtained results, if confirmed on larger patient cohort, would allow creating a panel of markers supporting clinical diagnosis in patients with HPV infection.