Unsolved mystery of Fas: mononuclear cells may have trouble dying in patients with Sjögren's syndrome.

BACKGROUND: Patients with Sjögren's syndrome, like other patients with autoimmune disorders, display dysregulation in the function of their immune system. Fas and Fas Ligand (FasL) are among the dysregulated proteins. METHODS: We studied Fas and FasL on IL-2Rα+ cells and in serum of patients with Sjögren's syndrome (n = 16) and healthy individuals (n = 16); both from same ethnic and geographical background. We used flow cytometry and enzyme-linked immunosorbent for this purpose. We also measured the expression of Bcl-2 and Bax by reverse transcription quantitative real-time PCR (RT-qPCR) and percentage of apoptotic and dead cells using Annexin V and 7-AAD staining in lymphocytes. RESULTS: FasL was increased in patients' T and B cells while Fas was increased in patients' monocytes, T and B cells. No signs of increased apoptosis were found. sFas and sFasL in patients' serum were increased, although the increase in sFasL was not significant. We suspect an effect of non-steroidal anti-inflammatory therapy on B cells, explaining the decrease of the percentage Fas+ B cells found within our samples. In healthy individuals, there was a noticeable pattern in the expression of FasL which mutually correlated to populations of mononuclear cells; this correlation was absent in the patients with Sjögren's syndrome. CONCLUSIONS: Mononuclear cells expressing IL-2Rα+ had upregulated Fas in Sjögren's syndrome. However, the rate of apoptosis based on Annexin V staining and the Bcl-2/Bax expression was not observed in mononuclear cells. We suspect a functional role of abnormal levels of Fas and FasL which has not been cleared yet.

The relationship of uremic toxin indoxyl sulfate and intestinal elimination mechanisms in hemodialysis patients.

INTRODUCTION: High indoxyl sulfate (IS) concentration is a serious problem for patients with CKD increasing the risk of cardiovascular diseases and CKD progression. Thus, the methods of decreasing the toxin concentrations are highly desired. The study aimed to discover the role of selected intestine related factors on IS concentration. METHODS: We evaluated the impact of ABCG2 and ABCC2 polymorphisms influencing activity and protein intake by normalized protein catabolic rate. Additionally, we examined the relation of IS and uric acid (UA), that can share common elimination transporters. A monocentric, prospective, open cohort pilot study was performed on 108 patients undergoing dialysis treatment. RESULTS: The positive effect of residual diuresis on the reduction of IS levels was confirmed (p = 0.005). Also, an increase in IS depending on the dietary protein intake was confirmed (p = 0.040). No significant correlation between ABC gene polymorphisms was observed either, suggesting the negligible role of ABCG2 and ABCC2 in the elimination of IS in small bowel. The significant difference was observed for UA where ABCG2 421C>A (rs72552713) gene polymorphism was higher (505.3 µmol/L) in comparison with a wild type genotype (360.5 µmol/L). Discussion/ Conclusion: No evidence of bowel elimination pathway via ABCC2 and ABCG2 transporters was found in renal replacement therapy patients.

Aging in psoriasis vulgaris: female patients are epigenetically older than healthy controls.

BACKGROUND: Psoriasis vulgaris is a skin autoimmune disease. Psoriatic patients have significantly lowered life expectancy and suffer from various comorbidities. The main goal of the study was to determine whether psoriatic patients experience accelerated aging. As accelerated aging might be the reason for the higher prevalence of comorbidities at lower chronological ages, we also wanted to investigate the relationship between aging and selected parameters of frequent psoriatic comorbidities - endocan, vascular endothelial growth factor and interleukin-17. Samples were obtained from 28 patients and 42 healthy controls. Epigenetic age measurement was based on the Horvath clock. The levels of endocan, vascular endothelial growth factor and interleukin-17 were analyzed using standardized ELISA methods. RESULTS: The difference between the epigenetic age and the chronological age of each individual subject did not increase with the increasing chronological age of patients. We cannot conclude that psoriasis causes accelerated aging. However, the epigenetic and chronological age difference was significantly higher in female patients than in female controls, and the difference was correlated with endocan (r = 0.867, p = 0.0012) and vascular endothelial growth factor (r = 0.633, p = 0.0365) only in female patients. CONCLUSIONS: The findings suggest a possible presence of pathophysiological processes that occur only in female psoriatic patients. These processes make psoriatic females biologically older and might lead to an increased risk of comorbidity occurrence. This study also supports the idea that autoimmune diseases cause accelerated aging, which should be further explored in the future.

Identification of a four-gene methylation biomarker panel in high-grade serous ovarian carcinoma.

Background The lack of effective biomarkers for the screening and early detection of ovarian cancer (OC) is one of the most pressing problems in oncogynecology. Because epigenetic alterations occur early in the cancer development, they provide great potential to serve as such biomarkers. In our study, we investigated a potential of a four-gene methylation panel (including CDH13, HNF1B, PCDH17 and GATA4 genes) for the early detection of high-grade serous ovarian carcinoma (HGSOC). Methods For methylation detection we used methylation sensitive high-resolution melting analysis and real-time methylation specific analysis. We also investigated the relation between gene hypermethylation and gene relative expression using the 2-ΔΔCt method. Results The sensitivity of the examined panel reached 88.5%. We were able to detect methylation in 85.7% (12/14) of early stage tumors and in 89.4% (42/47) of late stage tumors. The total efficiency of the panel was 94.4% and negative predictive value reached 90.0%. The specificity and positive predictive value achieved 100% rates. Our results showed lower gene expression in the tumor samples in comparison to control samples. The more pronounced downregulation was measured in the group of samples with detected methylation. Conclusions In our study we designed the four-gene panel for HGSOC detection in ovarian tissue with 100% specificity and sensitivity of 88.5%. The next challenge is translation of the findings to the less invasive source for biomarker examination, such as plasma. Our results indicate that combination of examined genes deserve consideration for further testing in clinical molecular diagnosis of HGSOC.

Next-Generation Sequencing Approach in Methylation Analysis of HNF1B and GATA4 Genes: Searching for Biomarkers in Ovarian Cancer.

DNA methylation is well-known to be associated with ovarian cancer (OC) and has great potential to serve as a biomarker in monitoring response to therapy and for disease screening. The purpose of this study was to investigate methylation of HNF1B and GATA4 and correlate detected methylation with clinicopathological characteristic of OC patients. The study group consisted of 64 patients with OC and 35 control patients. To determine the most important sites of HNF1B and GATA4, we used next-generation sequencing. For further confirmation of detected methylation of selected regions, we used high-resolution melting analysis and methylation-specific real-time polymerase chain reaction (PCR). Selected regions of HNF1B and GATA4 were completely methylation free in all control samples, whereas methylation-positive pattern was observed in 32.8% (HNF1B) and 45.3% (GATA4) of OC samples. Evaluating both genes together, we were able to detect methylation in 65.6% of OC patients. We observed a statistically significant difference in HNF1B methylation between samples with different stages of OC. We also detected subtype specific methylation in GATA4 and a decrease of methylation in late stages of OC. The combination of unmethylated HNF1B and methylated GATA4 was associated with longer overall survival. In our study, we employed innovative approach of methylation analysis of HNF1B and GATA4 to search for possible epigenetic biomarkers. We confirmed the significance of the HNF1B and GATA4 hypermethylation with emphasis on the need of selecting the most relevant sites for analysis. We suggest selected CpGs to be further examined as a potential positive prognostic factor.

Aberrant methylation of tumour suppressor genes WT1, GATA5 and PAX5 in hepatocellular carcinoma.

BACKGROUND: Aberrant hypermethylation of tumour suppressor genes (TSGs) occurring in hepatocellular carcinoma (HCC) could provide a mean of molecular characterisation of this cancer. The aim of this study was to investigate promoter methylation and gene expression of selected TSGs in HCC to identify candidate genes for further validation as potential biomarkers. METHODS: Methylation-specific multiplex ligation-dependent probe amplification method was used to measure the methylation status of 25 TSGs in 49 HCC samples and 36 corresponding non-cancerous liver tissue samples. Relative expression of the differentially methylated genes was assessed at the mRNA level using quantitative PCR. RESULTS: We observed a significantly higher methylation in genes WT1, PAX5, PAX6, PYCARD and GATA5 in HCC compared with control samples. The expression of PAX5 was significantly decreased by methylation; conversely methylation of WT1 was associated with higher mRNA levels. Methylation of GATA5 was significantly associated with overall survival and methylation of WT1 and PAX5 significantly varied between patients with ALBI score 1 vs. 2+3. Moreover, PAX5 was significantly more methylated in patients with tumour grade 2+3 vs. grade 1, and methylation of the PAX5 correlated with the patient's age at the time of diagnosis. CONCLUSIONS: HCC evince aberrant promoter methylation of WT1, PAX5, PAX6, PYCARD and GATA5 genes. Correlation between GATA5, WT1 and PAX5 methylation and clinical/histological parameters is suggestive of applicability of these markers in non-invasive (epi)genetic testing in HCC.

[The Role of microRNA in head and neck cancer focusing on sinonasal carcinoma]. / Role mikroRNA u nádoru hlavy a krku se zamerením na sinonazální karcinom.

MicroRNAs are small (18-25 nt) noncoding RNA molecules that are part of gene expression regulation and influence tumorigenesis. They could possibly be used as biomarkers and therapeutic targets in cancer in the future.Head and neck cancer is the sixth most common malignancy worldwide. These also include sinonasal carcinoma, a rare disease arising in the epithelium of respiratory tract, which is very poorly studied from the molecular perspective.MicroRNAs that have influence on pathogenesis of head and neck tumors have been divided into three categories: microRNAs associated with invasiveness and metastatic processes, microRNAs operating as oncogenes and microRNAs associated with HPV status and smoking. We expect that the described microRNAs could be part of regulatory mechanisms also in sinonasal carcinoma.

Importance of promoter methylation of GATA4 and TP53 genes in endometrioid carcinoma of endometrium.

BACKGROUND: Epigenetic changes are considered to be a frequent event during tumor development. Various methylation changes have been identified and show promise as potential cancer biomarkers. The aim of this study was to investigate promoter methylation of GATA4 and TP53 genes in endometrioid carcinoma of endometrium. METHODS: To search for promoter methylation of GATA4 and TP53 genes we used methylation-specific PCR (MSP) to compare the methylation status of 54 patients with endometrioid carcinoma of endometrium and 18 patients with normal endometrial tissue. RESULTS: In our study MSP revealed GATA4 promoter methylation in 44 of 54 in the carcinoma group (81.5%), and in none of the control group. No methylation was observed in TP53 gene. CONCLUSIONS: In conclusion, our study showed that there is significantly higher methylation in GATA4 gene in the endometrial cancer group compared with samples of non-neoplastic endometrium. The finding suggests the importance of hypermethylation of this gene in endometrial carcinogenesis and could have implications for future diagnostic and therapeutic strategies for endometrial cancer based on epigenetic changes.

Telomere length, oxidative and epigenetic changes in blood DNA of patients with exacerbated psoriasis vulgaris.

BACKGROUND: The pathogenesis of psoriasis vulgaris involves changes in DNA molecules, genomic instability, telomere attrition, and epigenetic alterations among them. These changes are also considered important mechanisms of aging in cells and tissues. OBJECTIVE: This study dealt with oxidation damage, telomere length and methylation status in DNA originating from peripheral blood of 41 psoriatic patients and 30 healthy controls. METHODS: Oxidative damage of serum DNA/RNA was determined immunochemically. Real-time PCR was used for the analysis of the telomere length. ELISA technique determined levels of 5-methylcytosine in blood cells' DNA. RESULTS: Oxidative damage of serum DNA/RNA was higher in patients than in controls (median, 3758 vs. 2286pg/mL, p<0.001). A higher length of telomeres per chromosome was found in patients whole-cell DNA than in controls (3.57 vs. 3.04 kilobases, p=0.011). A negative correlation of the length of telomeres with an age of the control subjects was revealed (Spearman's rho=-0.420, p=0.028). Insignificantly different levels of 5-methylcytosine in patients and controls were observed (33.20 vs. 23.35%, p=0.234). No influences of sex, smoking, BMI, PASI score, and metabolic syndrome on the methylation status were found. STUDY LIMITATIONS: i) A relatively small number of the participants, particularly for reliable subgroup analyses, ii) the Caucasian origin of the participants possibly influencing the results of the parameters determined, and iii) Telomerase activity was not directly measured in serum or blood cells. CONCLUSION: The study demonstrated increased levels of oxidized DNA/RNA molecules in the serum of patients with exacerbated psoriasis vulgaris. The results were minimally influenced by sex, the presence of metabolic syndrome, or cigarette smoking. In the psoriatic blood cells' DNA, the authors observed longer telomeres compared to healthy controls, particularly in females. Insignificantly higher global DNA methylation in psoriasis cases compared to the controls indicated marginal clinical importance of this epigenetic test performed in the blood cells' DNA.

DNA hypermethylation of CADM1, PAX5, WT1, RARß, and PAX6 genes in oropharyngeal cancer associated with human papillomavirus.

Recently, an increasing incidence of HPV-induced oropharyngeal squamous cell carcinoma (OPSCC) has been observed. Moreover, locoregionally advanced stages require a combined modal approach, and the prognosis is poor. Therefore, it is essential to find early diagnostic and prognostic biomarkers. DNA methylation changes play a crucial role in the process of carcinogenesis and are often investigated as promising biomarkers in many types of cancer. For analysis of DNA methylation levels of selected tumour suppressor genes in HPV-positive and HPV-negative samples (including primary tumours and corresponding metastases of metastasizing OPSCCs, primary tumours of non-metastasizing OPSCCs, and control samples), methylation-specific MLPA and methylation-specific high-resolution melting analyses were used. A significant difference in methylation between OPSCCs and the control group was observed in WT1, PAX6 (P < 0.01) and CADM1, RARß (P < 0.05) genes. CADM1 and WT1 hypermethylation was detected mostly in HPV-positive samples; all but one HPV-negative samples were unmethylated. Moreover, hypermethylation of PAX5 gene was observed in metastases compared with control samples and was also associated with shorter overall survival of all patients (P < 0.05). Associations described herein between promoter methylation of selected genes and clinicopathological data could benefit OPSCC patients in the future by improvement in screening, early detection, and prognosis of the disease.

[The most frequent methods used for DNA methylation analysis]. / Nejcasteji vyuzívané metodiky k analýze DNA metylacních zmen.

There is an important relationship between the degree of DNA methylation and gene activity in cancer Number of techniques of DNA methylation analysis has been reported. The aim of this study was to review methodologies that can be used to monitoring DNA methylation changes. We reviewed techniques important for monitoring of DNA methylation changes of CpG islands in gene promoter regions. Advantages, disadvantages and potential use of these techniques are described.

MicroRNA Deregulation in Papillary Thyroid Cancer and its Relationship With BRAF V600E Mutation.

BACKGROUND: MicroRNAs (miRNAs) are non-coding regulatory molecules 18-25 nucleotides in length that act as post-transcriptional regulators of gene expression. MiRNAs affect various biological processes including carcinogenesis. Deregulation of miRNAa expression has been described in a variety of tumors including papillary thyroid carcinoma (PTC). The aim of the present study was to investigate the role of selected miRNAs in PTC and find associations between miRNA expression and the BRAF (V600E) mutation. MATERIALS AND METHODS: The study group comprised a total of 62 patients with surgically treated PTC. The control group consisted of 30 patients with nodular goitre that were surgically treated in the same time period. The expression status of miR-146b, miR-181a, miR-187, miR-221 and miR-222 was determined using quantitative real-time PCR. BRAF mutation analysis was performed by PCR with reverse hybridization. RESULTS: MiR-146b, miR-181a, miR-187, miR-221 and miR-222 were up-regulated in PTC compared to normal thyroid gland tissue of the same patient. MiR-146b, miR-187, miR-221 and miR-222 were also up-regulated in PTC compared to nodular goitre. The recurrent tumors were statistically significantly associated with up-regulation of miR-221. The mutation V600E of BRAF gene was significantly associated with up-regulation of miR-146b and with down-regulation of miR-187. CONCLUSION: Over-expression of selected miRNAs in PTC compared to normal thyroid gland tissue and nodular goitre was found. Moreover, miR-221 may serve as a prognostic marker as its over-expression was significantly associated with recurrent tumors.

Nicotinamide phosphoribosyltransferase and intra-amniotic inflammation in preterm prelabor rupture of membranes.

Introduction: The amniotic fluid nicotinamide phosphoribosyltransferase (NAMPT) levels have not been compared among women with preterm prelabor rupture of membranes (PPROM) comorbid with intra-amniotic infection, sterile intra-amniotic inflammation (IAI), colonization, or without IAI and microbial invasion of the amniotic cavity (MIAC). Therefore, the main aim was to quantify the amniotic fluid NAMPT in women with PPROM complicated by intra-amniotic infection, sterile IAI, or colonization. The second aim was to characterize the diagnostic indices of NAMPT to reveal IAI. The third aim was to determine whether the cervical fluid and maternal serum NAMPT quantitation might be of value in the identification of intra-amniotic inflammatory complications in PPROM.Methods of study: NAMPT levels in amniotic fluid, cervical fluid, and maternal serum were assessed in three independent cohorts of women with singleton pregnancies complicated by PPROM between 24+0 and 36+6 weeks of gestation consisting of 88, 121, and 88 women, respectively. Amniotic fluid samples were obtained by transabdominal amniocentesis, cervical fluid samples were obtained using a Dacron polyester swab and maternal blood was obtained by venipuncture of the cubital vein. The NAMPT levels were measured by an enzyme-linked immunosorbent assay. Testing for MIAC and IAI was performed on all women, who were then categorized into four subgroups: intra-amniotic infection (MIAC and IAI), sterile IAI (IAI alone), colonization (MIAC alone), and without MIAC and IAI.Results: Women with intra-amniotic infection and women with sterile IAI had higher NAMPT levels than did women with colonization and women without MIAC and IAI (intra-amniotic infection: median 73.6 ng/mL, sterile IAI: median 55.5 ng/mL, colonization: median 12.1 ng/mL, without MIAC and IAI: 10.6 ng/mL; p < .0001). An amniotic fluid NAMPT level of 37 ng/mL was the best value for the detection of intra-amniotic infection in women with PPROM. Cervical fluid (p = .51) and maternal serum (p = .50) NAMPT levels did not reflect intra-amniotic inflammatory complications in women with PPROM.Conclusions: Intra-amniotic infection and sterile IAI are associated with higher NAMPT levels in amniotic fluid but not in cervical fluid or maternal serum in women with PPROM. Amniotic fluid NAMPT might be a marker for invasive identification of IAI in PPROM.

Importance of Cadherins Methylation in Ovarian Cancer: a Next Generation Sequencing Approach.

Epigenetic aberrations are well known to play an important role in carcinogenesis, and also have a great potential to serve as biomarkers in many types of cancers, including ovarian cancer in which sensitive and specific biomarkers and detection methods are critically needed. The aim of this study was to investigate methylation of cadherin genes CDH10, CDH13 and CDH18 in ovarian cancer tissue by comparison with control tissue. The study group consisted of 38 patients with ovarian cancer and 25 control patients. For detection of epigenetic events we used next generation sequencing, the most important data were confirmed using high-resolution melting analysis and real-time PCR. We observed significantly higher methylation in CDH13, sporadic methylation in CDH10 and loss of methylation in CDH18 in the ovarian cancer group compared with the control group. These observations suggest that changes in methylation of cadherin genes may be one of the major mechanisms associated with ovarian cancer progression. In addition, because of the high frequency of methylation of the CDH13 gene in the early stages of ovarian cancer, the analyzed CpG sites might be good targets for next study of potential ovarian cancer screening biomarkers.

The effect of sodium butyrate and cisplatin on expression of EMT markers.

Histone modifications play a key role in the epigenetic regulation of gene transcription in cancer cells. Histone acetylations are regulated by two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs are increased in ovarian carcinomas and they are involved in carcinogenesis and resistance to chemotherapeutic agents. In our study we investigated anticancer effect of HDAC inhibitor sodium butyrate (NaBu) on cisplatin-sensitive and cisplatin-resistant ovarian cell lines A2780 and A2780cis. A2780 and A2780cis were treated with NaBu alone or in combination with cisplatin (CP). NaBu inhibited the growth of both cell lines and enhanced cytotoxic effect of CP. Exposure to NaBu for 24 h induced cell cycle arrest. The expressions of EMT-related genes and proteins were further investigated by qPCR and western blot analysis. Loss of E-cadherin has been shown to be crucial in ovarian cancer development. We found that NaBu dramatically induce expression of E-cadherin gene (CDH1) and protein levels in A2780 and A2780cis. We investigated correlation between transcription and methylation of CDH1gene. Methylation level analysis in 32 CpG sites in CDH1 gene (promoter/exon1 regions) was performed using bisulfite NGS (Next Generation Sequencing). We found that cisplatin-resistant cell line A2780cis cells differ from their cisplatin-sensitive counterparts in the CDH1 methylation. Methylation in A2780cis cells is elevated compared to A2780. However, NaBu-induced expression of CDH1 was not accompanied by CDH1 demethylation. NaBu treatment induced changes in expression of EMT-related genes and proteins. Interestingly E-cadherin zinc finger transcriptional repressor SNAIL1 was upregulated in both cell lines. Mesenchymal marker vimentin was downregulated. Matrix metalloproteases (MMPs) are necessary for pericellular proteolysis and facilitate migration and invasion of tumour cells. NaBu induced mRNA expression of MMPs, mild changes in activities of gelatinases MMP2 and MMP9 were detected. Our data demonstrate that NaBu sensitizes cisplatin-resistant ovarian cancer cells, re-established E-cadherin expression, but it was not able to reverse the EMT phenotype completely.

Aberrant methylation of PCDH17 gene in high-grade serous ovarian carcinoma.

BACKGROUND: Aberrant DNA methylation of protocadherins (PCDHs) has been associated with development and progression of various types of cancer. It could represent possible direction in the search for critically needed tumor biomarkers for ovarian cancer. OBJECTIVE: To investigate methylation of δ2 group of non-clustered PCDHs in high-grade serous ovarian carcinoma (HGSOC) tissue in comparison with control tissue. METHODS: We used next-generation sequencing for detecting regions with the most altered methylation. For further confirmation of discovered alterations we used methylation-sensitive high-resolution melting analysis. RESULTS: PCDH17 methylation was detected in almost 70% of HGSOC patients without any methylation in the group of control samples and was found both in the late stage tumors as well as in the early stage ones. Other selected PCDHs did not show any relevant changes in methylation. Subsequent gene expression analysis of PCDH17 revealed decreased expression in all of the tumor samples in comparison to the control ones. Statistically significant negative correlation was found between methylation and levels of expression suggesting potentially methylation-based silencing. CONCLUSIONS: Methylation of PCDH17 could play an important role in development and progression of HGSOC and has potential to become a target in the search for new clinical biomarkers.

Elevated DNA methylation in malignant tumors of the sinonasal tract and its association with patient survival.

BACKGROUND: Epigenetic modifications have been recognized as an important mechanism underlying carcinoma progression. DNA methylation plays an important role in cancer biology and represents potentially heritable changes in gene expression that do not involve DNA sequence. The aim of this study was to investigate promoter methylation of selected genes in sinonasal carcinoma by comparison with noncancerous sinonasal tissue. METHODS: To search for epigenetic events (methylation in 25 tumor suppressor genes) we used MS-MLPA (Methylation-specific multiplex ligation-dependent probe amplification) to compare methylation status of 59 formalin fixed, paraffin embedded tissue samples of sinonasal carcinomas with 18 control samples. The most important changes in methylation were confirmed using MSP (Methylation specific PCR). Detected alterations in methylation were compared with clinicopathological characteristics. RESULTS: Using a 20% cut-off for methylation (MS-MLPA), we found significantly higher methylation in GATA5 (P=0.0005), THSB1 (P=0.0002) and PAX5 (P=0.03) genes in the sinonasal cancer group compared to the control group. Methylation in five or more genes was associated with impaired overall survival (P=0.017). CONCLUSION: These findings provide evidence that alterations in methylation profile may be one of the major mechanisms in sinonasal carcinogenesis. In addition, changes in methylation could potentially be used as prognostic factors of sinonasal carcinoma and may have implications for future individualized therapy based on epigenetic changes.

Telomere length, oxidative and epigenetic changes in blood DNA of patients with exacerbated psoriasis vulgaris

Abstract Background The pathogenesis of psoriasis vulgaris involves changes in DNA molecules, genomic instability, telomere attrition, and epigenetic alterations among them. These changes are also considered important mechanisms of aging in cells and tissues. Objective This study dealt with oxidation damage, telomere length and methylation status in DNA originating from peripheral blood of 41 psoriatic patients and 30 healthy controls. Methods Oxidative damage of serum DNA/RNA was determined immunochemically. Real-time PCR was used for the analysis of the telomere length. ELISA technique determined levels of 5-methylcytosine in blood cells' DNA. Results Oxidative damage of serum DNA/RNA was higher in patients than in controls (median, 3758 vs. 2286 pg/mL, p < 0.001). A higher length of telomeres per chromosome was found in patients whole-cell DNA than in controls (3.57 vs. 3.04 kilobases, p = 0.011). A negative correlation of the length of telomeres with an age of the control subjects was revealed (Spearman's rho = -0.420, p = 0.028). Insignificantly different levels of 5-methylcytosine in patients and controls were observed (33.20 vs. 23.35%, p = 0.234). No influences of sex, smoking, BMI, PASI score, and metabolic syndrome on the methylation status were found. Study limitations i) A relatively small number of the participants, particularly for reliable subgroup analyses, ii) the Caucasian origin of the participants possibly influencing the results of the parameters determined, and iii) Telomerase activity was not directly measured in serum or blood cells. Conclusion The study demonstrated increased levels of oxidized DNA/RNA molecules in the serum of patients with exacerbated psoriasis vulgaris. The results were minimally influenced by sex, the presence of metabolic syndrome, or cigarette smoking. In the psoriatic blood cells' DNA, the authors observed longer telomeres compared to healthy controls, particularly in females. Insignificantly higher global DNA methylation in psoriasis cases compared to the controls indicated marginal clinical importance of this epigenetic test performed in the blood cells' DNA.

Methylation status as a predictor of intravesical Bacillus Calmette-Guérin (BCG) immunotherapy response of high grade non-muscle invasive bladder tumor.

BACKGROUND AND AIMS: Genetic and epigenetic alterations play an important role in urothelial cancer pathogenesis. Deeper understanding of these processes could help us achieve better diagnosis and management of this life-threatening disease. The aim of this research was to evaluate the methylation status of selected tumor suppressor genes for predicting BCG response in patients with high grade non-muscle-invasive bladder tumor (NMIBC). MATERIALS AND METHODS: We retrospectively evaluated 82 patients with high grade non-muscle-invasive bladder tumor (stage Ta, T1, CIS) who had undergone BCG instillation therapy. We compared epigenetic methylation status in BCG-responsive and BCG-failure groups. We used the MS-MLPA (Methylation-Specific Multiplex Ligation-Dependent Probe Amplification probe sets ME001 and ME004. The control group was 13 specimens of normal urotel (bladder tissue)). RESULTS: Newly identified methylations in high grade NMIBC were found in MUS81a, NTRK1 and PCCA. The methylation status of CDKN2B (P=0.00312**) and MUS81a (P=0.0191*) is associated with clinical outcomes of BCG instillation therapy response. CDKN2B and MUS81a unmethylation was found in BCG failure patients. CONCLUSION: The results show that the methylation status of selected tumor suppressor genes (TSGs) has the potential for predicting BCG response in patients with NMIBC high grade tumors. Tumor suppressor genes such as CDKN2b, MUS81a, PFM-1, MSH6 and THBS1 are very promising for future research.