Proangiogenic signature in advanced laryngeal carcinoma after microRNA expression profiling.

The aim of this study is to evaluate which dysregulated angiomiRs compose the specific proangiogenic microRNA signature of advanced laryngeal cancer and review the literature. Thirty-six samples from twelve patients with advanced laryngeal carcinoma were collected. Total RNA was extracted and microRNA global profiling was performed using Agilent Technologies Microarray Kit. Fifty-nine microRNAs were found to have significantly different expression levels. Eleven microRNAs from the whole group were sorted as regulators of tumor angiogenesis (angiomiRs): seven were up-regulated-miR-1246, miR-181b 5p, miR-18a 5p, miR-21 3p, miR-210 3p, miR-503 5p, miR-93 5p and four were down-regulated-miR148a 5p, miR-145 5p, miR-204 5p, miR-125b 5p. For none of those microRNAs we found heterogeneity in tumor tissue. We are the first to report the specific proangiogenic microRNA signature in advanced laryngeal carcinoma and we confirm and amplify findings from previous studies that expand our perception of a specific "molecular state" of angiogenesis that is distinctive only for laryngeal cancer.

Changes in gene expression of CXCR4, CCR7 and BCL2 after treatment of breast cancer cells with saponin extract from Tribulus terrestris.

UNLABELLED: Saponins are natural substances produced by a large number of plants, one of which is Tribulus terrestris L. (TT). They have been reported to possess an antitumor activity exerted by regulating various signaling pathways in the cell. Although the mechanisms of action of saponin extracts from various plants have been widely studied, limited data are available about TT. The present study aimed to analyze the impact of saponin extract from TT on cell processes in breast carcinoma cell lines. The variations in expression of a group of 32 selected genes were examined by real-time PCR after saponin treatment of MCF7 and MCF10A cell lines. Only three genes - CXCR4, CCR7 and BCL2, showed changes in their mRNA levels after the application of the herb extract. While CXCR4 expression was reduced in both cell lines, CCR7 and BCL2 levels decreased only in tumorigenic MCF7 cells, implying cell-specificity of the saponin action. Our results suggested that TT extract containing saponins was likely to affect the processes of apoptosis and metastasizing of cancer cells. Further in vivo studies will show its applicability as an anticancer therapeutic agent. KEYWORDS: saponins, Tribulus terrestris, breast cancer, CXCR4, CCR7, BCL2.

MBL2 polymorphisms - manifestations in Bulgarian patients with adult dermatomyositis and systemic lupus erythematosus.

Deficiency in some complement factors is known to cause both systemic lupus erythematosus (SLE) and dermatomyositis (DM). Mannose-binding lectin (MBL) is a recognition molecule of the lectin pathway, and its low levels are reported to influence some autoimmune diseases. Furthermore, MBL2 polymorphisms have been described associated with low MBL serum levels due to impaired MBL structure and function. This is a pilot study to investigate the role of MBL2-550G/C (H/L), -221G/C (Y/X), Arg52Cys (D), Gly54Asp (B), Gly57Glu (C) polymorphisms and MBL serum levels as a risk factor for a development of adult DM and SLE in Bulgarian patients. None of the studied MBL2 polymorphisms appeared associated with the diseases investigated. However, we have found an increased OR of MBL2-221XY genotype in the patients with SLE (OR 1.64, 95%CI 0.77-3.52). MBL2 polymorphisms seemed to affect MBL serum levels and to be associated with the clinical features although none of the associations remained statistically significant after Bonferroni correction. The-550L allele showed an association with electromyography findings in patients with DM. The-221XY genotype was associated with photosensitivity in patients with SLE. The 54AB genotype showed an association with malar rash in patients with SLE, but it appeared decreased among SLE patients with ANA. In conclusion, our results suggest that the MBL2 polymorphisms have rather a disease modifying role and they are not associated with the disease susceptibility in adult DM and SLE among Bulgarian patients.

Evaluation of the clinical value of the newly identified urine biomarker HIST1H4K for diagnosis and prognosis of prostate cancer in Bulgarian patients.

PURPOSE: Searching for diagnostic and prognostic biomarkers for prostate cancer (PC) is main public health priority. DNA methylation in body fluids is a stable, easily detectable and promising PC biomarker. The major advantages of urine-based assays are their noninvasive nature and the ability to monitor PC with heterogeneous foci. The aim of this study was to determine the diagnostic value of the recently identified candidate PC biomarker HIST1H4K. METHODS: We investigated DNA methylation of HIST1H4K in urine samples from 57 PC patients, 29 controls with benign prostatic hyperplasia (BPH) and 50 young asymptomatic men (YAM) by MethyLight real-time PCR. RESULTS: The frequency of HIST1H4K promoter hypermethylation significantly discriminated PC patients from YAM (AUC =0.763; 95% CI 0.672-0.839; p<0.0001), but did not show any statistical difference between PC patients and BPH controls (AUC=0.513, 95% CI 0.402-0.622; p=0.8255). HIST1H4K could not outperform the prostatic specific antigen (PSA) in our sample (AUC=0.785; 95% CI 0.679-0.870; p<0.0001). Methylation of HIST1H4K showed significant correlation with aging (r=0.5418; p<0.0001), but with no other clinicopathological characteristics. CONCLUSION: The results suggest that the promoter hypermethylation of HIST1H4K is rather due to aging than related to prostate carcinogenesis. To elucidate this observation analysis of larger samples is needed.

Microsatellite instability and promoter hypermethylation of MLH1 and MSH2 in patients with sporadic colorectal cancer.

PURPOSE: Inactivation of the genes involved in DNA mismatch repair (MMR) is associated with microsatellite instability (MSI) and loss of heterozygosity (LOH). The aim of the current study was to assess the presence of MSI and promoter hypermethylation of MLH1 and MSH2 in Bulgarian PATIENTS WITH SPORADIC COLORECTAL CANCER (CRC) AND TO ANALYZE THEIR POSSIBLE EFFECT ON THE DEVELOPMENT, PROGRESSION AND PROGNOSIS OF THE DISEASE. METHODS: We examined MSI in 126 patients with sporadic CRC and the methylation status of the MLH1 and MSH2 promoter regions in the cases with MSI/LOH by using a panel of 5 microsatellite markers (BAT26, D5S346, D18S35, D2S123 and FGA) and methyl-specific PCR (MSP) of bisulfite converted DNA. RESULTS: MSI/LOH was found in 36 (28.6%) patients. Among them, 30 were analyzed for promoter hypermethylation of MLH1 and we detected hypermethylation in 15 (50%) of them, whereas promoter hypermethylation of MSH2 was observed only in one case. The presence of MSI/LOH was associated with younger age (p=0.002), more advanced stage (III/IV stage) (p=0.029), lower degree of differentiation (p=0.001), and right-sided tumor localization (p=0.0002), but not with overall survival (log rank, p=0.566). CONCLUSION: Our data suggest that sporadic CRCs with MSI/LOH are more aggressive, develop earlier and progress faster to more advanced stage. The most frequent cause of failure of DNA MMR system appeared to be the hypermethylation of CpG islands of the promoter region of MLH1, whereas the methylation of MSH2 was a rare event.

Establishment of the first stool bank in an Eastern European country and the first series of successful fecal microbiota transplantations in Bulgaria.

OBJECTIVE: For safe implementation and broader application of fecal microbiota transplantation (FMT), quality controlled stool banking is a must. Establishing a stool bank is a complex, time-consuming, and expensive process, making it a real challenge in an Eastern European country. We aimed to establish the first stool bank in Eastern Europe - in Bulgaria. SUBJECTS AND METHODS: A multidisciplinary team of gastroenterologists, microbiologists, infectionists, and geneticists was set up. We used a questionnaire based on the First European FMT Consensus in order to recruit possible stool donors. Laboratory blood and stool tests were performed on all potential donors. RESULTS: Between October 2018 and April 2019, 112 donor volunteers completed a questionnaire; 70 (62.5%) were excluded, mainly because of age above 50, an unhealthy BMI, and risk behavior. Fourty-two (37.5%) donor candidates were invited for laboratory testing of blood and feces, of which 12 (28.6%) passed this screening. Of 12 donors, 4 (33%) failed at the following screening test, which is performed every 3-6 months. Finally, 8 (7.14%) active donors were enrolled. Ten successful FMTs were performed on patients with recurrent Clostridium difficile infection. CONCLUSIONS: Even though we found many healthy volunteers, only a low percentage (7.14%) of them were suitable to become feces donors. Establishing a stool bank in an Eastern European country is essential for making FMT safe and more popular as a treatment method, finding further implementation and regulation of FMT and supporting physicians offering this treatment to their patients.

CHEK2 I157T and colorectal cancer in Bulgaria.

PURPOSE: Germline variants of the CHEK2 gene have been shown to act as low-penetrance cancer susceptibility alleles for a wide range of human malignancies. CHEK2 I157T has particularly been linked to colorectal cancer (CRC) risk. We aimed at establishing the population frequency and contribution of this variant to colorectal carcinogenesis in Bulgaria. METHODS: We have genotyped 802 population controls and 343 CRC patients from Bulgaria for the CHEK2 I157T variant. RESULTS: Heterozygous were 9 of 343 patients (2.62%, odds ratio/OR=1.0, 95% confidence interval/CI = 0.42 - 2.33, p=0.99% and 21 of 802 controls (2.62%). Higher frequencies were found among patients with multiple polyposis (2/40, 5%, p=0.28) and the rarer mucinous histology (1/11, 9.09%, p= 0.26). CONCLUSION: We conclude that CHEK2 I157T is not relevant for CRC risk in Bulgaria, but studies on a larger scale might help evaluate its possible significance in respect to disease characteristics.

Genetic heterogeneity and minor CYP1B1 involvement in the molecular basis of primary congenital glaucoma in Gypsies.

Primary congenital glaucoma (PCG) is a genetically heterogeneous disorder of autosomal recessive inheritance, with mutations in the cytochrome P450 1B1 (CYP1B1) gene detected in an average of approximately 50% of cases worldwide. The Roma/Gypsies are considered to be a rare example of a single founder CYP1B1 mutation, E387K (identified in the Slovak Roma), accounting for 100% of disease alleles. Contrary to this concept, unusual genetic heterogeneity was revealed in this study of 21 Gypsy PCG patients from Bulgaria and 715 controls from the general Gypsy population. In our small sample of affected subjects, we identified five different CYP1B1 mutations - four known (E229K, R368H, E387K and R390C) and one novel and potentially pathogenic (F445I), which together accounted for approximately 30% of disease alleles. E387K was rare in both the patient and the control group, indicating that its high frequency in the Slovak Roma is the product of local founder effect not representative of the overall molecular pattern of PCG in the Gypsy population. Data on other Mendelian disorders and on the population genetics of the Gypsies suggest that a true founder mutation is likely to exist and has remained undetected. Our analysis of another candidate gene, MYOC, and the GLC3B and GLC3C loci did not provide support for their involvement. The molecular basis of PCG in the Gypsies is thus unresolved, and diagnostic analyses should be extended beyond the E387K mutation.

Rise of multidrug-resistant Streptococcus pneumoniae clones expressing non-vaccine serotypes among children following introduction of the 10-valent pneumococcal conjugate vaccine in Bulgaria.

OBJECTIVES: Pneumococcal conjugate vaccines (PCVs) have reduced the incidence of pneumococcal disease, but non-vaccine serotypes are of concern, particularly if antimicrobial-resistant. This study retrospectively evaluated the serotype-specific clonality of paediatric multidrug-resistant (MDR) invasive and non-invasive Streptococcus pneumoniae isolates collected following PCV10 introduction (2011-2017) in Bulgaria. METHODS: Capsular types, drug resistance patterns and multilocus sequence typing (MLST) of the most common MDR S. pneumoniae serotypes sampled from children were determined. RESULTS: Overall, the rate of MDR pneumococci was 44.6% (107/240). The most common serotypes among MDR strains were 19F (25.2%), 19A (19.6%), 6C (13.1%), 6A and 23A (6.5% each) and 15A (4.7%), contributing 75.7% of all MDR strains. With the exception of serotype 19F, the remaining serotypes were non-PCV10 types. Among MDR pneumococci, the most frequently found sequence types were ST320 (30.4%; 19A and 19F), ST386 (12.7%; 6C and 6A) and ST8029 (5.1%; 23A). The majority of MDR STs (74.7%) belonged to PMEN clonal complexes, of which the most common were CC320 (Taiwan19F-14), CC315 (Poland6B-20) and CC180 (Netherlands3-31), accounting for 43.0%, 13.9% and 5.1%, respectively. In the post-vaccine period, a shift in the genetic structure of serotype 19A was found, with a significant increase of PMEN-14 (CC320) and a concurrent decrease of the major clone Denmark14-32 (CC230) observed prior to PCV10 introduction in Bulgaria. CONCLUSIONS: Clonality was found behind the wide distribution of MDR capsular types 19A, 6C, 23A and 3 following vaccine introduction, and a highly multiresistant and virulent clone Taiwan19F-14/ST320 has emerged as a common pathogen in children.

Microsatellite instability in Bulgarian patients with endometrial cancer.

PURPOSE: Microsatellite instability (MSI) is a frequent event in different types of cancer. In several studies MSI was shown to have both clinical and prognostic value. The aim of our study was to determine the frequency of MSI in Bulgarian patients with endometrial cancer (EC) and the possible relation of this phenomenon to their clinicopathological characteristics. PATIENTS AND METHODS: A total of 33 histologically confirmed EC patients were analyzed for tumor MSI using a panel of 6 microsatellite markers. RESULTS: We identified MSI in 30% of endometrial cancer cases. Six of them had high degree of MSI (MSI-H), and 4 displayed low degree of MSI (MSI-L). CONCLUSION: The frequency of MSI in Bulgarian EC patients does not differ significantly from that reported in other European studies.

Strong Correlation Between mRNA Expression Levels of HIF-2α, VEGFR1, VEGFR2 and MMP2 in Laryngeal Carcinoma.

The hypoxia that arises due to the rapid proliferation of tumor cells is a fundamental driving force for the canonical pathway of neovascularization. In the current study we report a very strong correlation between mRNA expression levels of HIF-2α (but not HIF-1α), VEGFR-1, VEGFR-2 and MMP2 in ex vivo samples from laryngeal carcinoma. Sixty-three samples from patients with histopathologically verified carcinoma of the larynx were examined in this study. Total RNA was isolated from both normal and tumor fresh frozen tissues of each patient and real-time quantitative PCR reactions were performed. The mRNA expression levels of HIF-1α, HIF-2α, VEGFR1, VEGFR2 and MMP2 were acquired. We found strong positive correlations between mRNA expression levels of HIF-2α and VEGFR-1, r s (98) = .671, p < .0005; HIF-2α and VEGFR-2, r s (98) = .742, p < .0005; HIF-2α and MMP2, r s (98) = .566, p < .0005; VEGFR-1 and VEGFR-2, r s (98) = .791, p < .0005; VEGFR-1 and MMP2, r s (98) = .709, p < .0005; VEGFR-2 and MMP2, r s (98) = .793, p < .0005. Our results provide evidence for the regulatory connection between HIF-2α and VEGFR-1, VEGFR-2 and MMP2 in the light of ETS1/ HIF-2α regulatory axis on a non-in-vitro level in carcinoma tissue, uncover some of the differences between the homologues HIF-1α and HIF-2α and round up and support the results from different experimental models in this field.

Macrophageal infiltration and microvessel density in laryngeal carcinoma: study of 52 cases.

Angiogenesis is one of the six originally constituted hallmarks of cancer that has been extensively studied in the last two decades. The aim of our study is to assess the microvessel and macrophageal density in laryngeal carcinoma and its clinicopathological correlations. We immunohistochemically assessed microvessel density (CD34) and macrophage count (CD68) using microarray techniques and then looked for clinicopathological correlations. The mean micro-vessel density in the study group was 14.27 ± 12.92 vessels in a ×200 field with a mean macrophageal infiltration density of 5.19 ± 4.32. Median microvessel density was significantly higher in patients with metastasis than in patients without metastasis. Additionally, linear regression established that macrophageal infiltration density could predict microvessel density in laryngeal carcinoma. We found no association between either factor and recurrence rate or other clinical characteristics. Our study adds additional data to a problem that has been widely studied during the last two decades, even if controversies in this area still remain.

Tryptophan hydroxylase polymorphism and suicidality in unipolar and bipolar affective disorders: a multicenter association study.

BACKGROUND: Being the rate-limiting enzyme in the biosynthesis of serotonin, the tryptophan hydroxylase gene (TPH) has been considered a possible candidate gene in bipolar and unipolar affective disorders (BPAD and UPAD). Several studies have investigated the possible role of TPH polymorphisms in affective disorders and suicidal behavior. METHODS: The TPH A218C polymorphism has been investigated in 927 patients (527 BPAD and 400 UPAD) and their matched healthy control subjects collected within the European Collaborative Project on Affective Disorders. RESULTS: No difference of genotype distribution or allele distribution was found in BPAD or UPAD. No statistically significant difference was observed for allele frequency and genotypes counts. In a genotype per genotype analysis in UPAD patients with a personal history of suicide attempt, the frequency of the C-C genotype (homozygosity for the short allele) was lower in UPAD patients (24%) than in control subjects (43%) (chi(2) = 4.67, p =.03). There was no difference in allele or genotype frequency between patients presenting violent suicidal behavior (n = 48) and their matched control subjects. CONCLUSIONS: We failed to detect an association between the A218C polymorphism of the TPH gene and BPAD and UPAD in a large European sample. Homozygosity for the short allele is significantly less frequent in a subgroup of UPAD patients with a history of suicide attempt than in control subjects.

A European multicenter association study of HTR2A receptor polymorphism in bipolar affective disorder.

The available data on the role of 5-HT in a variety of behaviors support the hypothesis that a dysfunction in brain serotoninergic system activity contributes to vulnerability to major depression. The diversity in the electrophysiological actions of 5-HT in the central nervous system can now be categorized according to receptor subtypes and their respective effector mechanisms. In particular, the implication of central postsynaptic 5-HT2A receptor in affective disorders has been supported by findings consistent with the hypothesis of 5-HT2A receptor up-regulation in depression. For these reasons, the 5-HT2A receptor (HTR2A) gene can be considered as a candidate gene in bipolar affective disorder (BPAD). We tested the possible genetic contribution of the polymorphic DNA variation T102C in exon 1 of HTR2A (chromosome 13q14-21) gene in a large European multicentric case-control sample. Allele and genotype frequencies, as well as homo-heterozygote distributions were compared between the two groups of 309 bipolar affective disorder patients and 309 matched controls. No significant differences were observed in the allelic and genotypic (also for homo-heterozygote) distribution between BPAD and controls. These results indicate that, in our sample, the 5-HT2A receptor polymorphism studied is unlikely to play a major role in the genetic susceptibility to BPAD. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:136-140, 2000.

Tyrosine hydroxylase polymorphism and phenotypic heterogeneity in bipolar affective disorder: a multicenter association study.

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the metabolism of catecholamines, is considered a candidate gene in bipolar affective disorder (BPAD) and has been the subject of numerous linkage and association studies. Taken together, most results do not support a major gene effect for the TH gene in BPAD. Genetic and phenotypic heterogeneity may partially explain the difficulty of confirming the exact role of this gene using both association and linkage methods. Four hundred one BPAD patients and 401 unrelated matched controls were recruited within a European collaborative project (BIOMED1 project in the area of brain research, European Community grant number CT 92-1217, project leader: J. Mendlewicz) involving 14 centers for a case-control association study with a tetranucleotide polymorphism in the TH gene. Patients and controls were carefully matched for geographical origin. Phenotypic heterogeneity was considered and subgroup analyses were performed with relevant variables: age at onset, family history, and diagnostic stability. No association was observed in the total sample or for subgroups according to age at onset (n = 172), family history alone (n = 159), or high degree of diagnostic stability and a positive family history (n = 131). The results of this association study do not confirm the possible implication of TH polymorphism in the susceptibility to BPAD.

Social adjustment and self-esteem of bipolar patients: a multicentric study.

The aim of the present study was to investigate impairment in social adjustment and self-esteem of bipolar patients (n=144) in remission for at least 3 months. Patients were recruited among four different centres: Sofia, Athens, Jerusalem and Milan, and were individually matched to control subjects in relation to sex, age and geographical origin. Subjects completed the Rosenberg self-esteem scale (SES) and the self-report version of the social adjustment scale (SAS). Bipolar patients reported to experience more difficulties in social adjustment than controls, specifically for leisure and work activities. Further, our results show that bipolar patients have significantly lower self-esteem compared to controls, even after remission.

Linkage of mood disorders with D2, D3 and TH genes: a multicenter study.

BACKGROUND: It has been suggested that the dopaminergic system is involved in the pathophysiology of mood disorders. We conducted a multicenter study of families with mood disorders, to investigate a possible linkage with genes coding for dopamine receptor D2, dopamine receptor D3 and tyrosine hydroxylase (TH). METHODS: Twenty three mood disorder pedigrees collected within the framework of the European Collaborative Project on Affective Disorders were analyzed with parametric and non-parametric linkage methods. Various potential phenotypes were considered, from a narrow (only bipolar as affected) to a broad (bipolar+major depressive+schizoaffective disorders) definition of affection status. RESULTS: Parametric analyses excluded linkage for all the candidate genes, even though small positive LOD (Limit of Detection) scores were observed for TH in three families. Non-parametric analyses yielded negative results for all markers. CONCLUSION: The D2 and D3 dopamine receptors were, therefore, not a major liability factor for mood disorders in our sample, whereas TH may play a role in a subgroup of patients.

Effects of dexamethasone in rat neonatal model of axotomy-induced motoneuronal cell death.

In this study the effect of dexamethasone on the motoneuronal cell death and the nuclear and somatic morphology changes occurring after peripheral nerve transection in the neonatal rats has been determined. The study was performed on 3 day old Wistar rats. Animals were divided into 3 groups--control, axotomised, and axotomised and dexamethasone-treated. The nerve transection was performed bilaterally. A dose of 0.5 mg/kg/24h dexamethasone, administered i.p., was used. On day 7 after the operation the animals were sacrificed and the motoneurons in segments L4 and L5 in the spinal cord were counted and their morphology was analysed. 25. 88% cell loss was found in the axotomised group (p<0.001 vs. control) versus 43.33% cell loss in the dexamethasone-treated and axotomised animals (p<0.01 vs. control). Dexamethasone significantly decreased the number of the surviving motoneurons (p<0.05 vs. axotomised). The axotomised group showed enlargement of the somatic area and the maximal and minimal diameters of the cell while the dexamethasone-treated and axotomised group showed soma shrinkage and decrease in the minimal cell diameter. Our results propose a possible hazard towards the application of dexamethasone in the treatment of new-borns with concomitant nerve injuries.

Performance enhancing genetic variants, oxygen uptake, heart rate, blood pressure and body mass index of elite high altitude mountaineers.

AIM: To analyse and compare the ACE (angiotensin-converting enzyme), ACTN3 (actinin-3) and AMPD1 (adenosine monophosphate deaminase 1) genetic variants, oxygen uptake (VO2max), heart rate (HR), blood pressure (BP) and body mass index (BMI) of elite high altitude mountaineers and average athletes. METHODS: Elite Bulgarian alpinists (n = 5) and control group of athletes (n = 72) were recruited. VO2max was measured using a treadmill graded protocol. HR, BP and BMI were recorded. Genotyping was done by polymerase chain reaction (PCR) amplification followed by agarose gel electrophoresis. Chi2-test and Fisher's exact test were used for statistical analysis. RESULTS: Alpinists showed significantly higher frequencies of 60% ACE I allele (p = 0.002), 50% ACTN3 X allele (p = 0.032) and 30% AMPD1 T allele (p = 0.003) compared to controls - 39%, 36%, 13%, respectively. ACE ID genotype prevalence and null DD genotype were observed in mountaineers. Higher absolute VO2max, but no differences in VO2max ml kg-1 min-1, HR, oxygen pulse, blood pressure and BMI were found. CONCLUSIONS: The ID genotype and higher frequencies of ACE I allele could contribute to successful high altitude ascents in mountaineers. The genetic make-up of the two mountaineers who made the summit of Mt Everest was distinctive, revealing ACE ID genotype, mutant ACTN3 XX and AMPD1 TT genotypes.