Towards Anticancer and Antibacterial Agents: Design and Synthesis of 1,2,3-Triazol-quinobenzothiazine Derivatives.

In this paper, we describe a new method for synthesizing hybrid combinations of 1,2,3-triazoles with a tetracyclic quinobenzothiazinium system. The developed approach allowed for the production of a series of new azaphenothiazine derivatives with the 1,2,3-triazole system in different positions of the benzene ring. In practice, the methodology consists of the reaction of triazole aniline derivatives with thioquinanthrenediinium bis-chloride. The structure of the products was determined by 1H-NMR, 13C-NMR spectroscopy, and HR-MS spectrometry, respectively. Moreover, the spatial structure of the molecule and the arrangement of molecules in the crystal (unit cell) were determined by X-ray crystallography. The anticancer activity profiles of the synthesized compounds were tested in vitro against human cancer cells of the A549, SNB-19, and T47D lines and the normal NHDF cell line. Additional tests of antibacterial activity against methicillin-sensitive and methicillin-resistant staphylococci, vancomycin-sensitive and vancomycin-resistant enterococci, and two mycobacterial strains were also performed. In fact, the dependence of anticancer and antibacterial activity on the substituent type and its position in the quinobenzothiazinium system was observed. Furthermore, the distance-guided property evaluation was performed using principal component analysis (PCA) and hierarchical clustering analysis (HCA) on the pool of the calculated descriptors. Finally, the theoretically approximated partition coefficients (clogP) were (inter-)correlated with each other and cross-compared with the empirically specified logPTLC parameters.

Identification of Sliding Hiatus Hernia by High-Resolution Manometry and Upper Gastrointestinal Endoscopy in Patients with Gastro-Oesophageal Reflux Disease.

BACKGROUND/AIM: The aim of this study was to compare high-resolution manometry (HRM) and upper gastrointestinal (GI) endoscopy as diagnostic utilities in detecting a sliding hiatus hernia in patients with gastro-oesophageal reflux disease (GORD) symptoms. MATERIAL AND METHODS: For both diagnostic modalities, the data obtained from 31 patients (20 females; mean age 48.2) who qualified for Nissen fundoplication were analysed using oesophageal pressure topography in line with the Chicago Classification. Confirmation of hiatus hernia during the surgery was considered the gold standard. HRM protocol involved 10 consecutive boluses of 10 mL of water. RESULTS: Sliding hiatus hernia was confirmed intraoperatively in 29 out of 31 patients. In 14 patients, hiatus hernia was detected in HRM, while 19 patients were found to have hiatus hernia by upper GI endoscopy before surgery. No false positive results were obtained in HRM, while 15 false negative results were shown. In upper GI endoscopy, false positive data were observed in 1 patient, while false negative results were found in 10 patients. Thus, the sensitivity of HRM in detecting hiatus hernia was 48% (95%CIs: 29-67%), and sensitivity of upper GI endoscopy was 66% (95%CIs: 46-82%). It was not possible to assess the specificity of HRM or upper GI endoscopy because only 2 of 31 patients had no hiatus hernia during fundoplication (gold standard). False negative results (sensitivity) were not significantly different between compared diagnostic modalities HRM and upper GI endoscopy (52% vs. 34%, respectively, p = 0.29). CONCLUSIONS: Due to poor sensitivity, both modalities, i.e., HRM and upper GI endoscopy, are not reliable tools to diagnose sliding hiatus hernia in patients with GORD symptoms.

Design, Synthesis and Antimicrobial Properties of New Tetracyclic Quinobenzothiazine Derivatives.

A new method for modifying the structure of tetracyclic quinobenzothiazinium derivatives has been developed, allowing introduction of various substituents at different positions of the benzene ring. The method consists of reacting appropriate aniline derivatives with 5,12-(dimethyl)thioquinantrenediinium bis-chloride. A series of new quinobenzothiazine derivatives was obtained with propyl, allyl, propargyl and benzyl substituents in 9, 10 and 11 positions, respectively. The structure of the obtained compounds was analyzed by 1H and 13C NMR (HSQC, HMBC) and X-ray analysis. All the compounds were tested against reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212, and representatives of multidrug-resistant clinical isolates of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). In addition, all the compounds were evaluated in vitro against Mycobacterium smegmatis ATCC 700084 and M. marinum CAMP 5644. 9-Benzyloxy-5-methyl-12H-quino [3,4-b][1,4]benzothiazinium chloride (6j), 9-propoxy-5-methyl-12H-quino[3,4-b][1,4]benzothiazinium chloride (6a) and 9-allyloxy-5-methyl-12H-quino[3,4-b][1,4]benzothiazinium chloride (6d) demonstrated high activity against the entire tested microbial spectrum. The activities of the compounds were comparable with oxacillin, tetracycline and ciprofloxacinagainst staphylococcal strains and with rifampicin against both mycobacterial strains. Compound 6j had a significant effect on the inhibition of bacterial respiration as demonstrated by the MTT assay. The compounds showed not only bacteriostatic activity, but also bactericidal activity. Preliminary in vitro cytotoxicity screening of the compounds performed using normal human dermal fibroblasts (NHDF) proved that the tested compounds showed an insignificant cytotoxic effect on human cells (IC50 > 37 µM), making these compounds interesting for further investigation. Moreover, the intermolecular similarity of novel compounds was analyzed in the multidimensional space (mDS) of the structure/property-related in silico descriptors by means of principal component analysis (PCA) and hierarchical clustering analysis (HCA), respectively. The distance-oriented structure/property distribution was related with the experimental lipophilic data.

Sweet, Salty, and Umami Taste Sensitivity and the Hedonic Perception of Taste Sensations in Adolescent Females with Anorexia Nervosa.

OBJECTIVE: The aim of this study was to perform analysis of sensitivity to sweet, salty, and umami tastes based on three measurement methods and of the hedonic perception of taste sensations in adolescent females with anorexia nervosa (AN). The aim of the research was to confirm the results of other authors in terms of the perception of sweet and salty taste in patients with AN, and then develop knowledge about the perception of umami taste, which is still insufficiently studied. METHOD: A total of 110 females with an age ranging from 13 to 19 years, including 50 newly diagnosed patients with a restrictive subtype of AN and 60 healthy controls participated in gustatory research involving analyses of taste perception (recognition thresholds, ability to identify the taste correctly, taste intensity, and hedonic response) applying the sip and spit method. RESULTS: Females with AN showed reduced sensitivity to salty taste and increased sensitivity to umami taste and, more often than healthy controls, wrongly classified the taste of solutions with a low sucrose concentration. Patients with AN assessed the sodium chloride and monosodium glutamate tastes less negatively than did control participants, and they did not show differences in their hedonic assessment of sucrose. CONCLUSIONS: The taste sensitivity alterations in females with AN demonstrated in this paper do not entail decreased hedonic assessment of taste experiences. Based on our results, we cannot consider the observed variation in taste sensitivity in patients with AN to be a factor that increases their negative attitude toward food consumption.

Lack of berberine effect on bone mechanical properties in rats with experimentally induced diabetes.

Multidirectional health-promoting activities of some plant-derived substances make them candidates for drugs used in diabetes and its complications such as osteoporosis. Berberine is a compound for which both antidiabetic and antiosteoporotic effects have been documented. The aim of the study was to investigate the effects of berberine on the skeletal disorders induced by experimental type 1 diabetes in rats. The experiments were performed on 3-month-old female rats, divided into three groups: I - healthy control rats, II - diabetic control rats, III - diabetic rats receiving berberine. Diabetes was induced by a single streptozotocin injection. Berberine administration (50 mg/kg/day p.o.) started two weeks later and lasted four weeks. Serum bone turnover markers and other biochemical parameters, bone mass and mineralization, histomorphometric parameters and mechanical properties were studied. Diabetes induced strong disorders of bone turnover, bone microarchitecture, and strength of cancellous bone. Berberine counteracted the effect of diabetes on the bone formation marker (osteocalcin) concentration, the growth plate, and some parameters of cancellous bone microarchitecture, but did not improve bone mineralization and bone mechanical properties in the diabetic rats. The lack of effect of berberine on bone quality does not support its use in the prevention of diabetes-induced bone damage.

Effects of diosgenin on the skeletal system in rats with experimental type 1 diabetes.

There is a great interest in substances of plant origin, which may exert health-promoting activities in diabetes and its complications. Previous studies suggested that diosgenin may favorably affect both glucose metabolism and osteoporosis. The aim of the study was to investigate the effects of diosgenin on the skeletal disorders induced by experimental type 1 diabetes (T1D) in rats. The experiments were performed on 3-month-old female rats, divided into three groups: I - healthy control rats, II - streptozotocin-induced diabetic control rats, III - diabetic rats receiving diosgenin. T1D was induced by a single streptozotocin injection (60 mg/kg i.p.). Diosgenin administration (50 mg/kg/day p.o.) started two weeks later and lasted four weeks. Serum bone turnover markers and other biochemical parameters, bone mass and mineralization, mechanical properties and histomorphometric parameters were examined. Diabetes induced profound metabolic disturbances and disorders of cancellous bone microarchitecture and strength. Diosgenin did not favorably affect the serum bone turnover markers and other biochemical parameters, bone mass, mineralization and mechanical properties in the diabetic rats. However, it counteracted the effect of diabetes on the growth plate and cancellous bone microarchitecture in the distal femur, indicating some limited beneficial influence on the skeleton.

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.

Clinicopathological Significance of Overall Frequency of Allelic Loss (OFAL) in Lesions Derived from Thyroid Follicular Cell.

BACKGROUND: Loss of heterozygosity (LOH) and microsatellite instability (MSI) are frequent molecular events in thyroid tumor etiopathogenesis occurring in several chromosomal critical areas, including 3p12-25.3, 7q21-31, 10q22-24, and 15q11-13, with loci of tumor suppressor genes. OBJECTIVE: We evaluated the usefulness of LOH/MSI as a diagnostic/prognostic biomarker in lesions derived from thyroid follicular cells: follicular thyroid carcinoma (FTC); follicular adenoma (FA), papillary thyroid carcinoma (PTC), and nodular goiter (NG). METHODS: We performed allelotyping (GeneMapper Software v. 4.0.) of ten microsatellite markers linked to the 1p31.2, 3p21.3, 3p24.2, 9p21.3, 11p15.5, and 16q22.1 region on DNA from 93 primary thyroid lesions then evaluated the LOH/MSI frequency and overall frequency of allelic loss (OFAL). RESULTS: We found regions with significantly increased frequency of LOH/MSI for specific histotypes: the 3p24.2 region for FA and 1p31.2 for FTC. LOH/MSI in 3p21.3 was significantly elevated in PTC and FTC. LOH/MSI in 3p21.3 was increased for small size tumors (T1a + T1b), tumors with no regional lymph node involvement (N0 + Nx), American Joint Committee on Cancer (AJCC) stage I tumors, and tumor diameter (Td) < 10 mm; in 1p31.2 for T2-3, N1, stage II-IV, and Td 10-30 mm; in 11p15.5 for T2-3, N1, stage II-IV, and Td > 30 mm. OFAL values were significantly higher in younger patients (< 40 years), in men, in those with T2-3 stage tumors, in those with increased Td, and in FA and FTC compared with NG and PTC. CONCLUSIONS: We confirmed the occurrence of LOH/MSI in 3p21.3 at an early stage of tumorigenesis and mapped 1p31.2 and 11p15.5 as characteristic for advanced-stage tumors. The results of our study may enable consideration of OFAL, defined as LOH/MSI coincidence in various chromosomal regions, as a tumor progression marker. OFAL values were significantly higher in follicular neoplasms (FA and FTC) than in PTC or NG; hence, increased OFAL values can be regarded as a characteristic feature of the follicular phenotype.

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.

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.

High-resolution Manometry in Patients with Gastroesophageal Reflux Disease Before and After Fundoplication.

BACKGROUND/AIMS: The study aimed to determine pre- and post-fundoplication esophagogastric junction (EGJ) pressure and esophageal peristalsis by high-resolution manometry (HRM) in patients with gastroesophageal reflux disease (GERD). METHODS: Pre-operative and post-operative HRM data from 25 patients with GERD were analyzed using ManoView version 2.0.1. with updated software for Chicago classification and pressure topography. The study involved swallowing water boluses of 10 mL in the upright position. RESULTS: Significant increase of mean basal EGJ pressure and minimal basal EGJ pressure was found in post-operative as compared with pre-operative patients (P < 0.05 and P < 0.001, respectively). Integrated relaxation pressure (IRP) reached higher values in post-operative patients than in pre-operative patients (P < 0.001). Intra-bolus pressure (IBP) was significantly higher (P < 0.05) and contractile front velocity (CFV) was slower (P < 0.01) in post-operative patients than in pre-operative patients. Moreover significant increase of distal contractile integral (DCI) was found in post-operative patients (P < 0.05). Hiatal hernia was detected by HRM in 11 pre-operative patients. Fifteen out of 25 post-operative patients complained of dysphagia. CONCLUSIONS: Fundoplication restores the antireflux barrier by reinforcing EGJ basal pressures, repairing hiatal hernias, and enhances peristaltic function of the esophagus by increasing DCI. However slight IRP elevation found in post-fundoplication patients may result in bolus pressurization and motility disorders.

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.

Alterations in taste perception as a result of hyperbaric oxygen therapy.

The present study evaluates the effect of hyperbaric oxygen therapy on taste sensitivity, hedonic perception of taste, and food preferences. The studied groups included 197 people in total (79 in the study group; 118 in the control group). All patients from the study group were treated with hyperbaric oxygen therapy due to chronic non-healing wounds. The control group consisted of healthy people, who did not receive hyperbaric oxygen therapy. The taste intensity, recognition thresholds, and hedonic perception were examined using gustatory tests. The aqueous solutions of sucrose for sweet, sodium chloride for salty, citric acid for sour, quinine hydrochloride for bitter, and monosodium glutamate for umami taste were used. The participants fulfilled the questionnaire to examine pleasure derived from eating certain types of dishes. Gustatory tests and analyses of the pleasure derived from eating in the study group were carried out before the first exposure to hyperbaric oxygen and then at the end of therapy, after at least 25 sessions of treatment. In the control group, examination of perception of taste sensations was conducted only once. The results of comparing patients with non-healing wounds with healthy people are characterized by reduced taste sensitivity. After participation in hyperbaric oxygen therapy, the improvement in perception of taste sensations and changes in hedonic evaluation have occurred among patients with non-healing wounds. In terms of food preference, a decreased desire for eating sweet desserts, chocolate, and crisps was observed in those patients who received hyperbaric oxygen therapy.

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.

Altered miRNA expression in pulmonary sarcoidosis.

BACKGROUND: miRNAs control important cellular functions including angiogenesis/angiostasis or fibrosis and reveal altered expression during pathological processes in the lung. METHODS: The aim of the study was to investigate the expression of selected miRNAs (miR-let7f, miR-15b, miR-16, miR-20a, miR-27b, miR-128a, miR-130a, miR-192 miR-221, miR-222) in patients with pulmonary sarcoidosis (n = 94) and controls (n = 50). The expression was assessed by q-PCR in BALF cells and peripheral blood lymphocytes (PB lymphocytes). For statistical analysis, the Kruskal-Wallis test, Mann-Whitney U- test, Neuman-Keuls' multiple comparison test, and Spearman's rank correlation were used. RESULTS: In BALF cells, significantly higher expression of miR-192 and miR-221 and lower expression of miR-15b were found in patients than controls. MiR-27b, miR-192 and miR-221 expression was significantly higher in patients without parenchymal involvement (stages I) than those at stages II-IV. Patients with acute disease demonstrated significantly higher miR-27b, miR-192 and miR-221 expression than those with insidious onset. For PB lymphocytes, patients demonstrated significantly greater miR-15b, miR-27b, miR-192, miR-221 and miR-222 expression, but lower miR-let7f and miR-130a expression, than controls. Stage I patients demonstrated significantly higher miR-16 and miR-15b expression than those in stages II-IV, and patients with the acute form demonstrated higher miR-130a and miR-15b expression. In BALF cells, miR-16 and miR-20a expression was significantly higher in patients with lung volume restriction, and miR-let7f was higher in the PB lymphocytes in patients with obturation. Several correlations were observed between the pattern of miRNA expression, lung function parameters and selected laboratory markers. CONCLUSION: The obtained results suggest that the studied miRNAs play a role in the pathogenesis of sarcoidosis, and that some of them might have negative prognostic value.

Immunoexpression of TGF-ß/Smad and VEGF-A proteins in serum and BAL fluid of sarcoidosis patients.

BACKGROUND: The chronic course of pulmonary sarcoidosis can lead to lung dysfunction due to fibrosis, in which the signalling pathways TGF-ß/Smad and VEGF-A may play a key role. METHODS: We evaluated immunoexpression of TGF-ß1, Smad2, 3, and 7, and VEGF-A in serum and bronchoalveolar lavage (BAL) fluid of patients (n = 57) classified according to the presence of lung parenchymal involvement (radiological stage I vs. II-III), acute vs. insidious onset, lung function test (LFT) results, calcium metabolism parameters, percentage of BAL lymphocytes (BAL-L%), BAL CD4(+)/CD8(+) ratio, age, and gender. Immunoexpression analysis of proteins was performed by ELISA. RESULTS: The immunoexpression of all studied proteins were higher in serum than in BAL fluid of patients (p >0.05). The serum levels of TGF-ß1 (p = 0.03), Smad2 (p = 0.01), and VEGF-A (p = 0.0002) were significantly higher in sarcoidosis patients compared to healthy controls. There were no differences within the sarcoidosis group between patients with vs. without parenchymal involvement, acute vs. insidious onset, or patients with normal vs. abnormal spirometry results. In patients with abnormal spirometry results a negative correlation was found between forced vital capacity (FVC) % predicted value and TGF-ß1 immunoexpression in BAL fluid, and positive correlations were observed between the intensity of lung parenchymal changes estimated by high-resolution computed tomography (HRCT scores) and Smad 2 level in serum. CONCLUSIONS: TGF-ß/Smad signalling pathway and VEGF-A participate in the pathogenesis of sarcoidosis. BAL TGF-ß1, and Smad 2 in serum seem to be promising biomarkers with negative prognostic value, but further studies are required to confirmed our observations.

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients.

BACKGROUND: Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation. METHODS: Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene. RESULTS: The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38-76%, depending on the gene. The highest MI value was found for RASSF1A (52%) and the lowest for NPRL2/G21 (5%). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71% tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = -0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75-92% NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found. CONCLUSIONS: The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn't seem to be a critical determinate of its promoter hypermethylation.