The Role of CXC Chemokines in Pulmonary Fibrosis of Systemic Lupus Erythematosus Patients.

The inflammatory process in systemic lupus erythematosus (SLE) affects many organs including the lungs. CXC chemokines are suggested to play an important role in the pathogenesis of SLE and pulmonary fibrosis. To estimate the concentrations of CXCL9, CXCL10, CXCL11 in bronchoalveolar lavage fluid (BALF) of patients with and without pulmonary involvements of SLE to evaluate CXC chemokines role in the pathogenesis of pulmonary fibrosis in SLE. Twenty-six SLE patients and 31 healthy controls were evaluated using high-resolution computed tomography (HRCT), pulmonary function tests, the SLE Disease Activity Index (SLEDAI), assessing CXCL9, CXCL11, CXCL10 level in BALF (an enzyme-immunosorbent assay kit). The mean CXCL9 and CXCL11 concentrations in BALF were higher in SLE patients compared to healthy controls (34.09 ± 102.34 vs 10.98 ± 14.65 pg/mL, p < 0.001; 72.65 ± 112.89 vs 16.12 ± 83.75 pg/mL, p = 0.012, respectively). The disease activity scored by SLEDAI and the concentration of CXCL10 in BALF were significantly higher in the SLE patients with pulmonary fibrosis when compared with patients with normal HRCT (8.23 ± 3.19 vs 5.01 ± 2.41; 73.45 ± 34.12 vs 40.76 ± 41.65, respectively, in both p < 0.05). In SLE patients positive correlations were found between SLEDAI and the percentage of lymphocytes in BALF (r = 0.51, p < 0.05); CXCL9 and CXCL10 concentrations in BALF (r = 0.65, p < 0.001); CXCL9 and CXCL11 concentrations in BALF (r = 0.69, p < 0.001). In lupus patients with pulmonary manifestations positive correlations were found between CXCL11 concentration in BALF and SLEDAI (r = 0.55, p < 0.05), CXCL11 concentration and the percentage of neutrophils in BALF (r = 0.69, p < 0.05), CXCL10 concentration and the percentage of neutrophils in BALF (r = 0.57, p < 0.05). Our observations indicate that CXCL9 and CXCL11 play an important role in the pathogenesis of SLE but it needs further studies. These results suggest that CXCL10 and CXCL11 are associated with neutrophils accumulation in the alveolar space of SLE patients with pulmonary fibrosis and should be considered as potential factor of interstitial fibrosis.

Single nucleotide polymorphisms in the STAT3 gene influence AITD susceptibility, thyroid autoantibody levels, and IL6 and IL17 secretion.

STAT3 (signal transducer and activator of transcription 3) is an important cellular effector in the Jak/STAT signaling pathway, which plays a pivotal role in human immune system regulation, mediating the effect of different cytokines. In the present study, we assessed the correlation between STAT3 polymorphisms (rs3816769 C>T and rs744166 A>G) and risk of the autoimmune thyroid diseases (AITDs) Hashimoto's thyroiditis (HT) and Graves' disease (GD) in the Polish population. Moreover, we evaluated the association of polymorphisms with the thyroid autoantibody levels (TPOAb, TgAb, TRAb) and the correlation between circulating proinflammatory IL6 and IL17 cytokines and thyroid autoantibody levels. The study included 71 AITD patients with HT (n = 39) or GD (n = 32) and a control group (n = 40). DNA SNP genotyping was performed using TaqMan probes. Serum levels of thyroid autoantibodies, IL6 and IL17 were measured according to enhanced chemiluminescence (ECL) assay. Allele A of STAT3 SNP rs744166 A>G was significantly more frequent in both HT and GD patients, while allele G was significantly more frequent in the control group. Similarly, allele C and CC genotype of STAT3 SNP rs3816769 C>T were significantly more frequent in the control group in comparison to HT and GD patients. Significantly higher TgAb median values were associated with CT rs3816769 genotype in HT patients. Serum levels of IL6 and IL17 positively correlated with TPOAb in the HT group. Serum level of IL6 positively correlated with TPOAb in the AITD group. Both studied polymorphisms seem to play a significant role in susceptibility to AITD (HT and GD). STAT3 SNPs may influence TAb level in AITD patients.

Expression of STAT5, COX-2 and PIAS3 in correlation with NSCLC histhopathological features.

Signal transducers and activators of transcription (STATs), their inhibitors and cyclooxygenase-2 (COX-2) participate in transformations of many various types of cancers. The aim of the present study was to evaluate the relationship between STAT5A/B, COX-2, and PIAS3 mRNA expression and tumor staging, metastasis status, and histopathological subtype in 71 patients with confirmed non-small cell lung cancer (NSCLC) diagnosis. Total RNA was isolated from NSCLC tissue samples and the expression of the studied genes was assessed using TaqMan probes in real-time PCR assay. The expression levels of STAT5A, STAT5B, and COX-2 genes were increased in 69%, 79%, and 71% NSCLC samples respectively, while PIAS3 expression was decreased in the majority (69%) of the studied tissues. Statistically significant differences were observed between STAT5 isoforms (P = 0.0008), with higher expression of STAT5B. We found statistically significant positive correlation between STAT5B and COX-2 (rho = 0.045), and significant negative correlation between STAT5B and PIAS3 (rho = -0.049). The negative correlation between STAT5B and PIAS3 (rho = -0.43) was also observed in T2a+T2b tumor group. Additionally, STAT5B and COX-2 expression levels were significantly different between T1a+T1b and T2a+T2b tumors (P = 0.002 and P = 0.041, respectively), with higher expression of both genes in T2 tumor stage. PIAS3 expression was significantly lower in NSCC subtype as compared with SCC subtype (P = 0.017). Also, STAT5A and STAT5B immunoexpression was assessed, and the results indicated significantly higher protein levels in NSCLC patients as compared with controls (P = 0.048 and P = 0.034, respectively). High STAT5B immunoexpression was positively correlated with STAT5B gene expression in tumors (rho = 0.755). STAT5B protein level was also significantly higher in T2a+T2b tumors, reflecting high STAT5B gene expression in this group. There was no statistically significant association between mRNA and protein expression levels of the studied genes and patients' characteristics: age, gender, smoking. The obtained results highlight the importance of the genes STAT5B and COX-2 in lung cancer progression.

Exhaled IL-8 in systemic lupus erythematosus with and without pulmonary fibrosis.

The purpose of this study is to evaluate the relationship between the concentration of interleukin-8 (IL-8) in exhaled breath condensate (EBC) and bronchoalveolar lavage fluid (BALF) with the disease activity score and pulmonary function of systemic lupus erythematosus (SLE) patients with and without pulmonary fibrosis. Thirty-four SLE patients and 31 healthy controls were enrolled and evaluated using high-resolution computed tomography (HRCT), pulmonary function tests, systemic lupus activity measure (SLAM), assessing BALF and EBC. IL-8 levels in BALF and EBC samples were measured with an enzyme-immunosorbent assay kit. The mean (±SEM) IL-8 concentrations in BALF and EBC were higher in SLE patients compared to healthy controls (34.84 ± 95.0 vs. 7.65 ± 21.22 pg/ml, p < 0.001; 3.82 ± 0.52 pg/m vs. 1.7 ± 1.7 pg/ml, p < 0.001, respectively). SLE patients had increased percentage of neutrophils in BALF when compared with control group (1.00 ± 5.99 vs. 0.00 ± 0.56 %, p = 0.0003). Pulmonary fibrosis in HRCT was found in 50 % of SLE patients. The disease activity scored by SLAM was significantly higher and total lung capacity was significantly lower in SLE patients with pulmonary fibrosis (8.00 ± 3.17 vs. 6.00 ± 2.31, p = 0.01; 88.00 ± 28.29 vs. 112.00 ± 21.08 % predicted, p = 0.01, respectively). In SLE patients with pulmonary fibrosis, correlations were found between SLAM and IL-8 concentration in BALF, forced expiratory volume in 1 s and forced vital capacity (r = 0.65, p = 0.006; r = -0.53, p = 0.035; r = -0.67, p = 0.006, respectively). Our results indicate that IL-8 plays an important role in the pathogenesis of SLE. An increased concentration of IL-8 according to BALF could be considered as a useful biomarker of SLE activity and pulmonary fibrosis in SLE.

HPV16 E6*II gene expression in intraepithelial cervical lesions as an indicator of neoplastic grade: a pilot study.

Integration of the HPV genome into a host cell DNA leads to the deregulated overexpression of the viral E6 and E7 oncoproteins, and this is a key factor for progression from low-grade cervical lesions to high-grade lesions and invasive cervical cancer. The aim of our study was to analyze the expression levels of HPV E6*I/E6*II and E7 genes in cervical neoplasia of different grades. The analysis involved 10 low-grade squamous intraepithelial lesions (CIN1), 15 high-grade lesions (CIN2 and CIN3), as well as normal cytology samples (n=10). HPV genotyping was done using RealLine HPV 16/18 kit. The expression analysis was performed in real-time PCR assay using gene-specific primers and SYBR Green. HPV16 DNA was found in 65.71% patients, including also normal cytology samples. The increased expression level of E6*I was observed in 12 (34.3%) patients. The expression of E6*II was increased in 10 (28.6%) samples, and E7 overexpression was found in 14 (40%) patients. Significant positive correlation was observed between the amount of HPV16 DNA and the levels of E6*I and E6*II expression. There were no statistically significant differences in expression levels of the studied genes between the groups (CIN1 vs. CIN2/CIN3 vs. normal cytology). Statistically significant differences were found in CIN2/CIN3 group, with the higher expression of E6*II as compared with E6*I. We suggest that the expression level of E6*II gene might be used as an indicator of cervical cancer severity, in patients with high-grade cervical neoplasia, but these observations need to be confirmed in a larger patient cohort.

Allelic imbalance in 1p, 7q, 9p, 11p, 12q and 16q regions in non-small cell lung carcinoma and its clinical association: a pilot study.

In lung cancer pathogenesis, genetic instability, i.e., loss of heterozygosity (LOH) and microsatellite instability (MSI) is a frequent molecular event, occurring at an early stage of cancerogenesis. The presence of LOH/MSI in non-small cell lung carcinoma (NSCLC) was found in many chromosomal regions, but exclusive of 3p their diagnostic value remains controversial. In this study we focused on other than 3p regions-1p31.2, 7q32.2, 9p21.3, 11p15.5, 12q23.2 and 16q22-the loci of many oncogenes and tumour suppressor genes. To analyze the potential role of LOH/MSI involved in NSCLC pathogenesis we allelotyped a panel of 13 microsatellite markers in a group of 56 cancer specimens. Our data demonstrate the presence of allelic loss for all (13) analyzed markers. Total LOH/MSI frequency in NSCLC was the highest for chromosomal region 11p15.5 (25.84 %), followed by 9p21.3 and 1p31.2 (19.87 and 16.67 % respectively). A statistically significant increase of total LOH/MSI frequency was detected for the 11p15.5 region (p = 0.0301; χ(2) test). The associations of total LOH/MSI frequency: 1) increase in 11p15.5 region (p = 0.047; χ(2) test) and 2) decrease in 7q32.2 region (p = 0.037; χ(2) test) have been statistically significant in AJCC III (American Joint Committee on Cancer Staging). In Fractional Allele Loss (FAL) index analysis, the correlation with cigarette addiction has been statistically significant. The increased amount of cigarettes smoked (pack years) in a lifetime correlates with increasing FAL (p = 0.024; Kruskal-Wallis test). These results demonstrate that LOH/MSI alternation in studied chromosomal regions is strongly influenced by tobacco smoking but do not seem to be pivotal NSCLC diagnostic marker with prognostic impact.

Ctla-4 expression and polymorphisms in lung tissue of patients with diagnosed non-small-cell lung cancer.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a potent immunoregulatory molecule that downregulates T-cell activation and thus influences the antitumor immune response. CTLA-4 polymorphisms are associated with various cancers, and CTLA-4 mRNA/protein increased expression is found in several tumor types. However, most of the studies are based on peripheral blood mononuclear cells, and much less is known about the relationship between CTLA-4 expression, especially gene expression, and its polymorphic variants in cancer tissue. In our study we assessed the distribution of CTLA-4 two polymorphisms (+49A/G and -318C/T), using TaqMan probes (rs231775 and rs5742909, resp.), and CTLA-4 gene expression in real-time PCR assay in non-small-cell lung cancer (NSCLC) tissue samples. The increased CTLA-4 expression was observed in the majority of NSCLC patients, and it was significantly correlated with TT genotype (-318C/T) and with tumor size (T2 versus T3 + T4). The presence of G allele and GG genotype in cancer tissue (+49A/G) was significantly associated with the increased NSCLC risk. Additionally, we compared genotype distributions in the corresponding tumor and blood samples and found statistically significant differences. The shift from one genotype in the blood to another in the tumor may confirm the complexity of gene functionality in cancer tissue.

Assessment of OPG/RANK/RANKL gene expression levels in peripheral blood mononuclear cells (PBMC) after treatment with strontium ranelate and ibandronate in patients with postmenopausal osteoporosis.

CONTEXT: Recent research results have confirmed the high significance of the OPG/RANK/RANKL system in the development of bone diseases. AIM: The aim of the reported study was to assess gene expression levels of the OPG/RANK/RANKL system in peripheral blood mononuclear cells (PBMCs) after strontium ranelate (SR) and ibandronate administered to patients with postmenopausal osteoporosis. PATIENTS AND METHODS: A total of 89 postmenopausal women, aged 51 to 85 years, patients of the Outpatient Clinic of Osteoporosis of the Military Teaching Hospital in Lodz, were enrolled into the study. The patients were randomly assigned to different medical therapies: ibandronate and SR. Patients of the control group received only calcium and vitamin D3 supplements. Patient visits were repeated after 3 and 6 months. Measurements of serum alkaline phosphatase concentrations and of RNA expression in PBMCs as well as of total serum calcium and phosphate levels and of their 24-hour urine excretion rates were carried out in material, collected at baseline and after 3 and 6 months of the therapy. Densitometry of the left hip and of the lumbar spine was done at the baseline visit and after 6 months. RESULTS: The differences in gene expressions of RANKL and RANK were not significant during the study period and did not differ between the groups in a statistically significant manner. No OPG gene expression was observed in PBMCs of patients in any of the studied groups and at any time point. The tendency of correlation (P = .07) was observed between decreasing RANK gene expression and increasing bone mineral density in the patients treated with SR. CONCLUSIONS: Both ibandronate and SR do not seem to cause any significant changes in gene expression levels of OPG/RANK/RANKL in PBMCs during the first 6 months of treatment.

Exhaled cytokines in systemic lupus erythematosus with lung involvement.

INTRODUCTION: An inflammatory process in systemic lupus erythematosus (SLE) affects many organs including the lungs. Interleukin (IL) 6 and IL­10 are suggested to play an important role in the pathogenesis of SLE. OBJECTIVES: The aim of the study was to evaluate IL­6 and IL­10 levels in the exhaled breath condensate (EBC) and bronchoalveolar lavage fluid (BALF) of patients with and without pulmonary involvement in SLE. PATIENTS AND METHODS: The study included 34 patients with SLE and 31 healthy controls evaluated using high-resolution computed tomography, pulmonary function tests, the Systemic Lupus Activity Measure (SLAM), and IL-6 and IL-10 measurement (by an enzyme­linked immunosorbent assay) in the BALF and EBC. RESULTS: The mean IL­6 and IL­10 concentrations in the BALF and the IL­10 concentration in the EBC were higher in patients with SLE compared with healthy controls (4.03 ±8.3 vs. 0.62 ±1.2 pg/ml, P <0.0001; 5.54 ±1.85 vs. 0.00 ±1.82 pg/ml, P <0.0001; 8.28 ±2.7 vs. 0.00 ±1.68 pg/ml, P <0.0001, respectively). The IL­10 level in the EBC correlated with SLE activity (r = -0.40, P = 0.019). The SLAM was significantly higher and the total lung capacity was significantly lower in patients with pulmonary manifestation of SLE compared with those without such complications (8.00 ±3.17 vs. 6.00 ±2.31, P = 0.01; 88.00 ±28.29 vs. 112 ±21.08 % predicted, P = 0.01; respectively). In patients with pulmonary involvement, correlations were observed between the IL­10 level in the EBC and the percentage of lymphocytes in the BALF (r = -0.5, P = 0.04). CONCLUSIONS: Our results indicate that IL­6 and IL­10 are involved in the pathogenesis of SLE. The measurement of IL­10 in the EBC may be a useful biomarker of SLE activity. It is likely that IL­10 protects against pulmonary manifestations of SLE.

Significant frequency of allelic imbalance in 3p region covering RARß and MLH1 loci seems to be essential in molecular non-small cell lung cancer diagnosis.

The aim of the study was to investigate the influence of allelic imbalance (AI) in several loci of tumor suppressor genes in 3p region on the non-small cell lung cancer (NSCLC) development. We evaluated the frequency of loss of heterozygosity and/or microsatellite imbalance (LOH/MSI) and assessed their association with patients' characteristics (age, gender, tobacco addiction) and NSCLC classification according to TNM/AJCC staging. To analyze the potential role of AI involved in NSCLC pathogenesis, we allelotyped a group of 74 NSCLC patients using 7 microsatellite markers. The highest frequency of LOH/MSI, however, not statistically significant, was observed in RARß and MLH1 (p = 0.104 and p = 0.216, respectively) loci. The association between high LOH/MSI frequency in 3p region with male gender (p = 0.041) as well as with age (especially >60 years) for RARß and MLH1 genes (p = 0.0001 and p = 0.020, respectively) was documented. Statistically significant increased frequency of MLH1 allelic loss in squamous cell carcinoma (SCC) versus non-squamous cell carcinoma (non-SCC) was observed (p = 0.01). Significant increase in LOH/MSI frequency in 3p region (mainly in FHIT and MLH1 loci) in correlation with cigarette addiction in a lifetime (≥40 years and ≥40 Pack Years) was also documented (p < 0.05). The highest LOH/MSI was revealed in RARß locus in IA tumors (p = 0.0001), while the similarly high allelic loss of MLH1 correlated with III A/B tumors (p = 0.0002), according to AJCC staging. The obtained results demonstrate that AI is influenced by tobacco smoking and seems to be vital in the molecular diagnosis of NSCLC, especially of SCC subtype.

Diagnostic value of DNA alteration: loss of heterozygosity or allelic imbalance-promising for molecular staging of prostate cancers.

The biological behavior of prostate cancer is uncertain, and therefore, search for molecular prognostic markers associated with disease progression seems to be essential. We performed microsatellite allelotyping of DNA isolated from primary prostate tumors biopsies (prostate adenocarcinoma, PCa). We evaluated the frequency of allelic imbalance (AI), including loss of heterozygosity and/or microsatellite imbalance (LOH/MSI) as well as the association of these DNA alterations with clinicopathological variables. We assessed the significance of LOH/MSI alterations in selected imprinted and non-imprinted chromosomal regions (IR and NIR) in PCa. A total of 50 biopsies of prostate tumor (containing >75 % tumor cells) were histologically examined confirming prostate carcinoma. Microsatellite allelotyping using 16 microsatellite markers linked to the following chromosomal regions: 1p31.2, 3p21.3-25.3, 7q32.2, 9p21.3, 11p15.5, 12q23.2, and 16q22.1 was performed. The incidence of LOH/MSI alterations in prostate tumor cells was the highest for chromosomal regions 7q32.2 and 16q22.1 (31.25 and 26.60 %, respectively), followed by 1p31.2 and 3p21.3-25.3 (26.50 and 17.40 %, respectively). Statistically significant increase in LOH/MSI alterations has been observed for markers: D1S2137 (1p region; p = 0.00032), D9S974 (9p region; p = 0.0017), and D16S3025 (16q region; p = 0.0017). Statistically significant differences in frequency of LOH/MSI alterations in particular chromosomal regions have been found for 1p31.2, 7q32.2 and 16q22.1 (p = 0.027, p = 0.012 and p = 0.031, respectively). We documented statistically significant association between Fractional Allele Loss (FAL) index and advanced tumor stage (p < 0.05). We suggest that genomic instability of LOH/MSI type is a frequent event in prostate carcinogenesis and assessed as FAL index has clinical value for the molecular staging of prostate cancer in (TRUS)-guided prostate biopsy material.

Association between the c.*229C>T polymorphism of the topoisomerase IIß binding protein 1 (TopBP1) gene and breast cancer.

Topoisomerase IIß binding protein 1 (TopBP1) is involved in cell survival, DNA replication, DNA damage repair and cell cycle checkpoint control. The biological function of TopBP1 and its close relation with BRCA1 prompted us to investigate whether alterations in the TopBP1 gene can influence the risk of breast cancer. The aim of this study was to examine the association between five polymorphisms (rs185903567, rs116645643, rs115160714, rs116195487, and rs112843513) located in the 3'UTR region of the TopBP1 gene and breast cancer risk as well as allele-specific gene expression. Five hundred thirty-four breast cancer patients and 556 population controls were genotyped for these SNPs. Allele-specific TopBP1 mRNA and protein expressions were determined by using real time PCR and western blotting methods, respectively. Only one SNP (rs115160714) showed an association with breast cancer. Compared to homozygous common allele carriers, heterozygous and homozygous for the T variant had significantly increased risk of breast cancer (adjusted odds ratio = 3.81, 95% confidence interval: 1.63-8.34, p = 0.001). Mean TopBP1 mRNA and protein expression were higher in the individuals with the CT or TT genotype. There was a significant association between the rs115160714 and tumor grade and stage. Most carriers of minor allele had a high grade (G3) tumors classified as T2-T4N1M0. Our study raises a possibility that a genetic variation of TopBP1 may be implicated in the etiology of breast cancer.

The significance of epigenetic alterations in lung carcinogenesis.

Lung cancer is recognized as a leading cause of cancer-related death worldwide and its frequency is still increasing. The prognosis in lung cancer is poor and limited by the difficulties of diagnosis at early stage of disease, when it is amenable to surgery treatment. Therefore, the advance in identification of lung cancer genetic and epigenetic markers with diagnostic and/or prognostic values becomes an important tool for future molecular oncology and personalized therapy. As in case of other tumors, aberrant epigenetic landscape has been documented also in lung cancer, both at early and late stage of carcinogenesis. Hypermethylation of specific genes, mainly tumor suppressor genes, as well as hypomethylation of oncogenes and retrotransposons, associated with histopathological subtypes of lung cancer, has been found. Epigenetic aberrations of histone proteins and, especially, the lower global levels of histone modifications have been associated with poorer clinical outcome in lung cancer. The recently discovered role of epigenetic modifications of microRNA expression in tumors has been also proven in lung carcinogenesis. The identified epigenetic events in lung cancer contribute to its specific epigenotype and correlated phenotypic features. So far, some of them have been suggested to be cancer biomarkers for early detection, disease monitoring, prognosis, and risk assessment. As epigenetic aberrations are reversible, their correction has emerged as a promising therapeutic target.

STAT3 rs3816769 polymorphism correlates with gene expression level and may predispose to nonsmall cell lung cancer: a preliminary study.

INTRODUCTION:  The STAT3 gene functions as both the oncogene and the regulator of immunity. Despite its important role in cancer development and regulation of the immune cells, studies of single nucleotide polymorphisms (SNPs) of the STAT3 gene and the associated risk of lung cancer are sparse. OBJECTIVES:  In the present study, we evaluated the association of SNPs (rs744 166 [AG] and rs3 816 769 [CT]) with predisposition to nonsmall cell lung cancer (NSCLC) development and their potential effect on STAT3 expression. PATIENTS AND METHODS:  DNA and RNA, isolated from lung tissue samples, were obtained from patients with diagnosed NSCLC (n = 71) and those without NSCLC, included in a control group (n = 104). STAT3 SNP genotyping and relative expression were performed using TaqMan® probes.  RESULTS:  STAT3 CC (rs3 816 769) and AA genotypes (rs744 166) were associated with lower lung cancer risk, whereas TT (rs3 816 769) and GG genotypes (rs744 166) were found to be associated with significantly elevated lung cancer risk. In the NSCLC group, odds ratio analysis showed that allele A was rare and might be linked with decreased while allele G with increased lung cancer risk. We demonstrated that overexpression of STAT3 positively correlated with TT genotype (rs3 816 769) in NSCLC patients (P = 0.0464). Moreover, the differences in STAT3 gene expression between squamous cell carcinoma and large cell carcinoma histopathological subtypes were observed. CONCLUSIONS:  It has been shown that rs3816769 STAT3 gene polymorphisms are associated with NSCLC susceptibility and might be regarded as having a significant functional and diagnostic value.

CTLA-4 gene polymorphisms and their influence on predisposition to autoimmune thyroid diseases (Graves' disease and Hashimoto's thyroiditis).

INTRODUCTION: Autoimmune thyroid disease (AITD) is associated with both genetic and environmental factors which lead to the overactivity of immune system. Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) gene polymorphisms belong to the main genetic factors determining the susceptibility to AITD (Hashimoto's thyroiditis, HT and Graves' disease, GD) development. The aim of the study was to evaluate the relationship between CTLA-4 polymorphisms (A49G, 1822 C/T and CT60 A/G) and HT and/or GD in Polish patients. MATERIAL AND METHODS: Molecular analysis involved AITD group, consisting of HT (n=28) and GD (n=14) patients, and a control group of healthy persons (n=20). Genomic DNA was isolated from peripheral blood and CTLA-4 polymorphisms were assessed by polymerase chain reaction-restriction fragment length polymorphism method, using three restriction enzymes: Fnu4HI (A49G), BsmAI (1822 C/T) and BsaAI (CT60 A/G). RESULTS: Statistical analysis (χ(2) test) confirmed significant differences between the studied groups concerning CTLA-4 A49G genotypes. CTLA-4 A/G genotype was significantly more frequent in AITD group and OR analysis suggested that it might increase the susceptibility to HT. In GD patients, OR analysis revealed statistically significant relationship with the presence of G allele. In controls, CTLA-4 A/A genotype frequency was significantly increased suggesting a protective effect. There were no statistically significant differences regarding frequencies of other genotypes and polymorphic alleles of the CTLA-4 gene (1822 C/T and CT60 A/G) between the studied groups. CONCLUSIONS: CTLA-4 A49G polymorphism seems to be an important genetic determinant of the risk of HT and GD in Polish patients.

MSI and LOH in the development and prognosis of follicular cell-derived thyroid tumours.

Microsatellite instability (MSI) and loss of heterozygosity (LOH) represent molecular disorders acquired by the cell during neoplastic transformation. Both are associated with genetic instability. Functional silencing of tumour suppressor genes may be the consequence of genomic instability, particularly of the globally occurring LOH phenomenon. Numerous studies have confirmed the role of MSI/LOH at both the early and the late stages of thyroid tumourigenesis. This paper reviews the available study results on MSI/LOH significance and prevalence in thyroid neoplasms. Additionally, it summarises the knowledge regarding the practical usage of the study findings on MSI/LOH in aspects of cancer risk assessment as well as the development of prognostic markers for thyroid neoplasms.

Aberrant methylation as a main mechanism of TSGs silencing in PTC.

In the present study the role of tumour suppressor genes (TSGs) hypermethylation and genetic instability of LOH/MSI type in thyroid tumorigenesis was assessed. Expression, methylation status and presence of LOH/MSI were analyzed for 8 TSGs selected from imprinted (IR) and non-imprinted (NIR) chromosomal regions in papillary thyroid carcinomas (PTCs) and nodular goitres (NGs). The results show that methylation-induced gene silencing occurs at an early step of thyroid carcinogenesis and involves multiple genes. Genetic changes of LOH/MSI type are less frequent. In PTC samples, the lack of significant differences in the frequency of LOH in IR and NIR suggests that it is not a key mechanism changing the pattern of gene expression. Co-methylation observed both in NG and PTC raises a possibility that, in thyroid tissue, methylation-induced silencing may occur not only in malignant transformation but also in functional context. We did not recognize any of the studied TSGs - in regard to aberrant methylation status or LOH/MSI frequency - as a selective molecular marker in thyroid tumorigenesis.

Increased expression of PIN1 gene in papillary thyroid carcinoma.

BACKGROUND: Peptidyl-prolyl cis/trans isomerase (Pin1), encoded by PIN1 gene with locus in chromosome 19p13, is an enzyme that catalytically induces conformational changes in proteins after phosphorylation on serine or threonine residues preceding proline (pSer/Thr-Pro motifs); in this way, it has an influence on protein interactions and intracellular localizations of proteins. The aim of the study were: 1) an assessment of PIN1 gene expression level in benign and malignant thyroid lesions; 2) the evaluation of possible correlations between gene expression and histopathological variants of papillary thyroid carcinoma (PTC) or tumour size, classified according to TNM classification of primary tumours (in case of PTC only); 3) the estimation of possible relationships between expression of the gene in question and patients' sex or age. METHODS: Seventy (70) tissue samples were analyzed: 32 cases of PTC, 7 cases of medullary thyroid carcinoma (MTC), 7 cases of follicular adenoma (FA), and 24 cases of nodular goitre (NG). In real-time polymerase chain reaction (real-time PCR), two-step RT-PCR (reverse transcriptase-polymerase chain reaction) in an ABI PRISM 7500 Sequence Detection System was employed. The PIN1 gene expression level was assessed, calculating the mean relative quantification rate (RQ rate) increase for each sample. RESULTS: The level of PIN1 gene expression (compared to that in macroscopically unchanged thyroid tissue) was higher in PTC group than those in FA, MTC and/or NG groups, but the statistical significance was noted for difference between PTC and NG groups only. On the other hand, the differences of RQ rate value between different PTC variants were statistically insignificant. No correlations were found between RQ values and tumour size, as well as between RQ values and patients' sex or age in PTC group. CONCLUSIONS: The PIN1 gene expression may have - in future - an important meaning in the diagnostics of PTC and in understanding its pathogenesis. However, our results - mostly due to the small number of cases - do not yet allow considering PIN1 gene as a prognostic molecular PTC marker.

A minireview: the role of MAPK/ERK and PI3K/Akt pathways in thyroid follicular cell-derived neoplasm.

The MAPK/ERK (mitogen - activated protein kinase/extracellular signal-regulated kinase signaling pathway) and PI3K/Akt (lipid kinase phoshoinositide-3-kinase signaling pathway) play an important role in transmission of cell signals through transduction systems (ligands, transmembrane receptors and cytoplasmic secondary messengers) to cell nucleus, where they influence the expression of genes that regulate important cellular processes: cell growth, proliferation and apoptosis. The genes, coding the signaling cascade proteins (e.g., RET, RAS, BRAF, PI3K, PTEN, AKT), are mutated or aberrantly expressed in thyroid cancer derived from follicular thyroid cell. Genetic and epigenetic alternations, concerning MAPK/ERK and PI3K/Akt signaling pathways, contribute to their activation and interaction in consequence of malignant follicular cell transformation. This review is focused mainly on genetic alterations in genes, coding signaling pathway proteins. Moreover, it is additionally pointed out that genetic, as well as epigenetic DNA changing via aberrant methylation of several tumour suppressor and thyroid-specific genes are associated with tumour aggressiveness, being a jointly responsible mechanism for thyroid tumorigenesis. The understanding of this molecular mechanism provides access to novel molecular therapeutic strategies for inhibiting oncogenic activity of signaling pathways.

Association of Trp64Arg polymorphism of beta3-adrenergic receptor with insulin resistance in Polish children with obesity.

AIM: To establish the influence of the Trp64Arg variant of the beta3-adrenergic receptor (Trp64Arg- beta3AR) on body mass index (BMI) and insulin resistance (IR) in obese children. METHODS: BMI, presence of the Trp64Arg mutation, plasma glucose and insulin concentrations during an oral glucose tolerance test (OGTT) and IR were determined in 60 obese and 33 normal weight children. RESULTS: The frequency of Trp64Arg was similar in normal weight and obese children. BMI, glucose and insulin concentrations during an OGTT in children with Trp64Argbeta3AR were not different from those with Trp64Trpbeta3AR. IR was confirmed in 42.8% of children with Trp64Argbeta3AR and in 45.6% of children with Trp64Trpbeta3AR (NS). CONCLUSIONS: 1. The similar frequency of the Trp64Argbeta3AR variant in normal weight and obese children suggests that it is not a susceptibility gene for obesity in Polish children. 2. The presence of the Trp64Argbeta3AR variant does not have an unfavourable influence on BMI, glucose or insulin concentrations during OGTT or on IR frequency in Polish obese children.