Delayed diagnosis of parathyroid cancer manifested with pubic bone lesions and chronic kidney failure. Tricks and pitfalls of management.

INTRODUCTION: Parathyroid cancer (PTC) is an extremely rare malignancy with an incidence of 5.7 per 10 million people. The exact preoperative or intraoperative diagnosis is difficult, but of paramount importance, because resection with negative margins is the only effective treatment. CASE REPORT: A 46-years-old female was referred from another hospital with a diagnosis of "hyper-functioning thyroid nodule", based on the ultrasound showing a lesion of the right thyroid lobe and elevated FT4. At the admission, she had severe pain in the right inguinal area, fatigue, muscle weakness, and excessive diuresis. The blood assay demonstrated serum calcium of 4.02 mmol/l, parathyroid hormone of 1433.2 pg/ml, FT4 of 17.49 pmol/l, creatinine of 296 µmol/l. CT showed a tumor of the right thyroid lobe with a size of 2.5. A right lobectomy was performed. Right parathyroid glands were not found. Because of the constellation for hyperparathyroidism and suspicion of parathyroid malignancy ipsilateral and central lymph node dissection and partial removal of the right sternothyroid muscle were performed, which correlated with a significant intraoperative drop in the parathyroid hormone. Three months later, a re-resection was performed because of SPECT-CT evidence for residual parathyroid tissue. CONCLUSION: The timely diagnosis of PTC is a prerequisite for a good outcome. The best preoperative indicators are serum parathyroid hormone > 4 times above the upper limit, serum calcium > 14 mg/dL, a palpable neck mass, and a local invasion found intraoperatively. The only curative treatment is the complete removal of the tumor with a negative margin. KEY WORDS: Delayed diagnosis, Hyperparathyroidism, Parathyroid cancer, Surgery.

Correlation between Cytogenetic Findings and Spermatogenic Failure in Bulgarian Infertile Men.

The aim of our study was to determine the type and frequency of chromosomal aberrations and polymorphisms in men with different degrees of spermatogenic failure in comparison to men with normozoospermia, in order to find correlations between cytogenetic findings and the abnormal results of semen analysis. In our study, we performed cytogenetic analysis in 901 infertile men, divided into five groups according to semen analysis-normozoospermia (86), asthenozoospermia (394), oligoasthenozoospermia (182), severe male factor (100), and azoospermia (139). The frequency of polymorphisms was similar in all groups (11-16%, without significant differences). The frequency of numerical and structural aberrations increases with the degree of the spermatogenic failure (3.5% in normozoospermia, 5.6% in asthenozoospermia, 9.8% in oligoasthenozoospermia, 9% in severe male factor, and 13.5% in azoospermia). We found a significantly higher incidence of numerical chromosomal aberrations in severe male factor (7%) and azoospermia (9.3%). Oligoasthenozoospermia occured in 45% of cases with translocation, compared to 20% in the group with a normal karyotype. We revealed that chromosomal translocations are tightly associated with oligoasthenozoospermia, whereas numerical chromosomal aberrations-with severe male factor and azoospermia. The impact of chromosome polymorphisms on male infertility should be studied in greater detail.

Molecular pathogenesis of spontaneous abortions - Whole genome copy number analysis and expression of angiogenic factors.

OBJECTIVE: To study two major molecular alterations in spontaneous abortions (SA) with unexplained etiology - fetal genomic anomalies and the endometrial expression of main angiogenic factors VEGFA/VEGFR2 and chemokines SDF-1/CXCR4. MATERIALS AND METHODS: Whole genome copy number analysis by arrayCGH or Next Generation Sequencing (NGS) was applied for detection of fetal genomic imbalances. The abortive decidua of SA without fetal aneuploidies was further investigated for expression levels of the abovementioned factors using real time PCR analysis. A total of 30 abortive materials were collected from spontaneous abortions after exclusion of known predisposing factors. RESULTS: In 21 of 30 spontaneous abortions (70%), genomic anomalies were discovered by whole genome copy number analysis. Numerical anomalies were detected in 90% of aberrant cases, and in 10% - structural aberrations were revealed. An increased expression for essential factors of angiogenesis was identified in spontaneous abortions' tissues - 3.44 times for VEGFA and 10.29 times for VEGFR2. We found an average of 14 times increase in the expression levels of SDF-1 and 3.21 times for its receptor CXCR4. CONCLUSION: We could suggest the occurrence of increased angiogenesis in SA without fetal aneuploidies, compared to the control tissues, which could lead to increased oxidative stress and fetal loss.

Type 2 diabetes mellitus and cardiovascular risk; what the pharmacotherapy can change through the epigenetics.

Diabetes mellitus and cardiovascular diseases are part of the metabolic syndrome and share similar risk factors, including obesity, arterial hypertension, and dyslipidemia. Atherosclerosis and insulin resistance contribute to the development of the diseases, and subclinical inflammation is observed in both conditions. There are many proofs about the connection between epigenetic factors and different diseases, including diabetes and cardiovascular diseases. Interestingly, recent studies show that at least some anti-diabetic drugs, as well as blockers of the renin-angiotensin-aldosterone system (RAAS), exert epigenetic effects aside from their hypoglycemic and antihypertensive functions, respectively. More studies are needed to discover other positive effects of the medications established through epigenetic mechanisms and to find out more about the epigenetic role in the development of diabetes mellitus and cardiovascular diseases.

SDF-1/CXCR4 signalling is involved in blood vessel growth and remodelling by intussusception.

The precise mechanisms of SDF-1 (CXCL12) in angiogenesis are not fully elucidated. Recently, we showed that Notch inhibition induces extensive intussusceptive angiogenesis by recruitment of mononuclear cells and it was associated with increased levels of SDF-1 and CXCR4. In the current study, we demonstrated SDF-1 expression in liver sinusoidal vessels of Notch1 knockout mice with regenerative hyperplasia by means of intussusception, but we did not detect any SDF-1 expression in wild-type mice with normal liver vessel structure. In addition, pharmacological inhibition of SDF-1/CXCR4 signalling by AMD3100 perturbs intussusceptive vascular growth and abolishes mononuclear cell recruitment in the chicken area vasculosa. In contrast, treatment with recombinant SDF-1 protein increased microvascular density by 34% through augmentation of pillar number compared to controls. The number of extravasating mononuclear cells was four times higher after SDF-1 application and two times less after blocking this pathway. Bone marrow-derived mononuclear cells (BMDC) were recruited to vessels in response to elevated expression of SDF-1 in endothelial cells. They participated in formation and stabilization of pillars. The current study is the first report to implicate SDF-1/CXCR4 signalling in intussusceptive angiogenesis and further highlights the stabilizing role of BMDC in the formation of pillars during vascular remodelling.

LAMA2 Congenital Muscle Dystrophy: A Novel Pathogenic Mutation in Bulgarian Patient.

Congenital muscle dystrophies (CMD) are genetically and clinically heterogeneous hereditary myopathies mainly with autosomal recessive type of inheritance. The most common form worldwide is considered to be merosin-deficient muscle dystrophy type 1A, called MDC1A (due to laminin-α2 defects as a result of LAMA2 gene mutation), accounting for 30-40% of total cases of CMD. The exact molecular and clinical diagnoses, respectively, are a prerequisite for the most effective treatment; sometimes orphan drugs exist for some rare diseases. One of such drugs is Tarix, which was FDA approved and announced in 2016 for treatment of MDC1A. Here we present a patient diagnosed postmortem as having early-onset LAMA2-related muscular dystrophy as a result of mutations in LAMA2, identified by Sanger sequencing in his parents: a novel nonsense mutation c.4452T>A in exon 31, identified in the mother, and a known pathogenic nonsense mutation c.2901C>A in exon 21, detected in the father. The truncating nature of both nonsense mutations made the clinical presentation severe and the outcome fatal. Genetic analysis in such cases of muscle dystrophy is of utmost impact, because it makes the correct diagnosis with at least some specific options for treatment, makes the prognosis depending on the severity of mutation discovered, determines reproductive risk, and offers prophylaxis in the family by means of prenatal or preimplantation diagnostics.

Mesenchymal Stem Cell-Dependent Modulation of Liver Diseases.

Acute liver failure and cirrhosis display sequential and overlapping severe pathogenic processes that include inflammation, hepatocyte necrosis, and fibrosis, carrying a high mortality rate. Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells with immunonodulatory characteristics. MSCs are considered to act through multiple mechanisms to coordinate a dynamic, integrated response to liver inflammation and fibrosis, which prevents the progressive distortion of hepatic architecture. Accordingly, MSCs as well as their products have been investigated as a novel therapeutic approach for the treatment of inflammatory and fibrotic liver diseases. In this review, we highlight the current findings on the MSC-based modulation of liver inflammation and fibrosis, and the possible use of MSCs in the therapy of immune-mediated liver pathology. We briefly describe the cellular and molecular mechanisms involved in MSC-dependent modulation of cytokine production, phenotype and function of liver infiltrated inflammatory cells and compare effects of engrafted MSCs versus MSC-generated conditioned medium (MSC-CM) in the therapy of acute liver injury. In order to elucidate therapeutic potential of MSCs and their products in modulation of chronic liver inflammation and fibrosis, we present the current findings regarding pathogenic role of immune cells in liver fibrosis and describe mechanisms involved in MSC-dependent modulation of chronic liver inflammation with the brief overview of on-going and already published clinical trials that used MSCs for the treatment of immune mediated chronic liver diseases. The accumulating evidence shows that MSCs had a significant beneficial effect in the treatment of immune-mediated liver diseases.

Molecular regulation and role of angiogenesis in reproduction.

Angiogenesis is an essential process for proper functioning of the female reproductive system and for successful pregnancy realization. The multitude of factors required for physiological angiogenesis and the complexity of regulation of their temporal-spatial activities contribute to aberrations in human fertilization and pregnancy outcomes. In this study, we reviewed the current knowledge of the temporal expression patterns, functions, and regulatory mechanisms of angiogenic factors during foliculogenesis, early implantation/placentation and embryo development, as well as recurrent spontaneous abortions. Angiogenic factors including vascular endothelial growth factors and angiopoietins have documented roles in the development of primordial follicles into mature antral follicles. They also participate in decidualization, which is accompanied by the creation of an extensive network of vessels in the stromal bed that support the growth of the embryo and the placenta, and maintain early pregnancy. During placentation angiogenic and angiomodulatory cytokines, T and B lymphocytes and macrophages affect angiogenesis in a context-dependent manner. Defects in angiogenesis at the maternal-fetal interface contribute to miscarriage in humans. The establishment of more polymorphisms in the genes involved in angiogenesis/vasculogenesis, and their pathological phenotype and expression could give opportunities for prediction, creating a therapeutic strategy, and treatment of diseases related to female reproductive health and problematic conception.

A family study of complex chromosome rearrangement involving chromosomes 1, 8, and 11 and its reproductive consequences.

Complex chromosome translocations are structural chromosomal rearrangements involving three or more chromosomes and more than two breakpoints. A complex chromosome rearrangement was detected in a phenotypically normal female patient that was referred to the hospital for genetic counseling due to reproductive failure. A cytogenetic evaluation was performed, according to standard method of chromosomal analysis, using G-banding technique. The patient's karyotype showed a balanced complex chromosome rearrangement (BCCR) involving chromosomes 1, 8, and 11 with three breakpoints 1p31, 8q13, and 11q23. The karyotype designed according to ISCN (2013), is 46,XX,t(1;8;11)(p31;q13;q23) (8qter→8q13::1p31→1qter;8pter→8q13::11q23→11qter;11pter→11q23::1p31→1pter). Additionally, the proband's mother and brother were tested, resulting in the same exact translocation. In this study, we describe all possible meiotic segregations regarding this translocation, as well as the clinical phenotypes which could arise, if unbalanced products of conception survive. This is a rare case of familial complex chromosome rearrangement, giving a view for its reproductive consequences.

Dynamics of the Developing Chick Chorioallantoic Membrane Assessed by Stereology, Allometry, Immunohistochemistry and Molecular Analysis.

The chick chorioallantoic membrane (CAM) is a widely used model for the study of angiogenesis, tumour growth, as well as drug efficacy. In spite of this, little is known about the developmental alteration from its appearance to the time of hatching. In the current study the CAM has been studied by classical stereology and allometry. Expression levels of selected angiogenesis-related molecules were estimated by RT-PCR and cell dynamics assessed by proliferation and apoptosis assays. Absolute CAM volume increased from a low of 0.47 ± 0.11 cm3 at embryonic day 8 (E8) to a high of 2.05 ± 0.27 cm3 at E18, and then decreased to 1.6 ± 0.47 cm3 at E20. On allometric analysis, three growth phases were identifiable. Between E8-13 (phase I), the CAM grew fastest; moderately in phase II (E13-18) but was regressing in phase III (E18-20). The chorion, the mesenchyme and the allantoic layers grew fastest in phase I, but moderately in phase II. The mesenchyme grew slowly in phase III while the chorion and allantois were regressing. Chorionic cell volume increased fastest in phase I and was regressing in phase III. Chorionic capillaries grew steadily in phase I and II but regressed in phase III. Both the chorion and the allantois grew by intrinsic cell proliferation as well as recruitment of cells from the mesenchyme. Cell proliferation was prominent in the allantois and chorion early during development, declined after E17 and apoptosis started mainly in the chorion from E14. VEGFR2 expression peaked at E11 and declined steadily towards E20, VEGF peaked at E13 and E20 while HIF 1α had a peak at E11 and E20. Studies targeting CAM growth and angiogenesis need to take these growth phases into consideration.

Characterization of genomic changes in the cervical pre-cancerous lesions and tumors induced by different types of human papillomaviruses.

Cervical carcinoma is the second most common malignancy among women in both incidence and mortality. Although much is known about the etiology and treatment of cervical cancer, the role of genetic alterations in the multistep pathway of cervical tumorigenesis is largely unknown. The aim of this study was to characterize the genomic changes in the cervical pre-cancerous lesions and tumors, induced by different types of human papillomaviruses. In this research was used the BlueGnome CytoChip oligo 2 × 105 K microarray for whole-genome oligo-array CGH. Microarray CGH analysis of 40 specimens was carried out-12 specimens from patients with early-stage squamous cell carcinomas; 19 specimens from patients with mild to moderate dysplasia and 9 with severe dysplasia. First we performed microarray CGH analysis of five DNA pools which contained the DNA from homogeneous groups of patients. The results revealed presence of micro chromosomal aberrations in chromosome region 14q11.2. According to the genome database these aberrations represent polymorphisms. Microarray analysis of DNA from 9 separate carcinoma lesions revealed a total of 26 aberrations in 14 chromosomes of nine patients. Our results showed the advantages of high-resolution chips in the clinical diagnosis of patients with cancerous and precancerous lesions caused by viral infection with HPV, but also highlight the need for extensive population studies revealing the molecular nature and clinical significance of different CNVs and the creation of detailed maps of variations in the Bulgarian population. This would facilitate extremely precise interpretation of specific genomic imbalances in the clinical aspect.

Cardiac and renal nitrosative-oxidative stress after acute poisoning by a nerve agent Tabun.

We hypothesized that Tabun poisoning, as well as other organophosphorous treatment, cause specific organs' oxidative changes that have not previously been substantiated investigated. In this regard, a marker for nitrosative-oxidative stress in the main haemodynamic organs (heart and kidney) could reveal the existence of such changes. In this study, for the first time we studied the nitrosative/oxidative stress in heart and kidney after acute Tabun (Ethyl N,N- Dimethylphosphoramidocyanidate) poisoning measuring by immunohistochemistry the expression of 3-nitrotyrosine--a marker for nitrosative-oxidative stress. We investigated nitrotyrozine expression in three different groups of animals (with at least 3 animals in each group): the first group was treated with 0.5 LD50 Tabun and organs were collected after 24 h; the second group received vehicle for the same period; in the third group a highly specific re-activator was applied immediately after Tabun application. Heart and kidney were collected after 24 h. The levels of nitrotyrozine production significantly increased (more than 3 times) in cardiomyocytes after Tabun. The application of re-activator slightly reduced these levels not reaching the basal heart levels. Nitrotyrozine expression in kidney increased more than 2 times after Tabun and application of re-activator did not change it significantly. In conclusion, our study evidently demonstrated that Tabun trigger oxidative-nitrosative stress in heart and kidney and these cellular effects should be protected by an additional anti-oxidant therapy, since acetylcholinesterase re-activator is not efficient in this manner.

Whole genome microarray analysis in non-small cell lung cancer.

Lung cancer is a serious health problem, since it is one of the leading causes for death worldwide. Molecular-cytogenetic studies could provide reliable data about genetic alterations which could be related to disease pathogenesis and be used for better prognosis and treatment strategies. We performed whole genome oligonucleotide microarray-based comparative genomic hybridization in 10 samples of non-small cell lung cancer. Trisomies were discovered for chromosomes 1, 13, 18 and 20. Chromosome arms 5p, 7p, 11q, 20q and Хq were affected by genetic gains, and 1p, 5q, 10q and 15q, by genetic losses. Microstructural (<5 Mbp) genomic aberrations were revealed: gains in regions 7p (containing the epidermal growth factor receptor gene) and 12p (containing KRAS) and losses in 3p26 and 4q34. Based on high amplitude of alterations and small overlapping regions, new potential oncogenes may be suggested: NBPF4 (1p13.3); ETV1, AGR3 and TSPAN13 (7p21.3-7p21.1); SOX5 and FGFR1OP2 (12p12.1-12p11.22); GPC6 (13q32.1). Significant genetic losses were assumed to contain potential tumour-suppressor genes: DPYD (1p21.3); CLDN22, CLDN24, ING2, CASP3, SORBS2 (4q34.2-q35.1); DEFB (8p23.1). Our results complement the picture of genomic characterization of non-small cell lung cancer.

Avian area vasculosa and CAM as rapid in vivo pro-angiogenic and antiangiogenic models.

Angiogenesis, the development of new blood vessels from preexisting ones, is driven by coordinated signaling pathways governed by specific molecules, hemodynamic forces, and endothelial and periendothelial cells. The processes involve adhesion, migration, and survival machinery within the target endothelial and periendothelial cells. Factors that interfere with any of these processes may therefore influence angiogenesis either positively (pro-angiogenesis) or negatively (antiangiogenesis). The avian area vasculosa (AV) and the avian chorioallantoic membrane (CAM) are two useful tools for studying both angiogenesis and antiangiogenesis since they are amenable to both intravascular and topical administration of target, agents, are relatively rapid assays, and can be adapted very easily to study angiogenesis-dependent processes, such as tumor growth. Both models provide a physiological setting that permits investigation of pro-angiogenic and antiangiogenic agent interactions in vivo.

New generation genomic platforms in investigation of complex diseases and BEN.

New generation genomic platforms enable us to decipher the complex genetic basis of complex diseases and Balkan Endemic Nephropathy (BEN) at a high-throughput basis. They give valuable information about predisposing Single Nucleotide Polymorphisms (SNPs), Copy Number Variations (CNVs) or Loss of Heterozygosity (LOH) (using SNP-array) and about disease-causing mutations along the whole sequence of candidate-genes (using Next Generation Sequencing). This information could be used for screening of individuals in risk families and moving the main medicine stream to the prevention. They also might have an impact on more effective treatment. Here we discuss these genomic platforms and report some applications of SNP-array technology in a case with familial nephrotic syndrome.

Next-generation sequencing of BRCA1 and BRCA2 in breast cancer patients and control subjects.

Breast cancer is currently the most common type of cancer in females. The majority of the hereditary forms of breast cancer are caused by mutations in the BRCA1 and BRCA2 genes, whose main function is the DNA repair of double-strand breaks. Genetic testing of females with a family history of breast cancer is recommended to determine their hereditary predisposition for this type of cancer. The variants with no clear clinical significance may represent a diagnostic challenge when performing targeted resequencing. In this study, DNA samples were obtained from 24 breast cancer patients (mean age, 35±10 years) with a positive family history and from 71 age-matched healthy controls. Informed consent was obtained from all the subjects. Sequence-targeted BRCA1 and BRCA2 libraries were prepared using the TruSeq Custom Amplicon method and sequenced on the Illumina MiSeq system. A wide range of variants were identified in the BRCA1 and BRCA2 genes. Two pathological/presumably pathological variants were detected in the breast cancer patient group: a mutation in BRCA2 at the chromosomal (chr) position chr13:32890665, which affected the first position of the 5' splice region following exon 2; and a mutation in BRCA1 at chr17:41219635, causing an in-frame triple nucleotide deletion of valine 1688 (8.3%). In the patient and control groups, 7 likely polymorphic variants and 13 common variants were detected in the BRCA1 and BRCA2 genes. To the best of our knowledge, this study was the first to identify 3 common polymorphisms in BRCA2, characteristic solely of the Bulgarian population, including chr13:32973737, T/-, a single-nucleotide polymorphism (SNP) within the 3'-UTR of exon 27; chr13:32973280, A/-, a mononucleotide deletion within the 5'-UTR of exon 27; and chr13:32973924, T/-, a mononucleotide deletion downstream of the gene sequence. To the best of our knowledge, this study was the first to apply next-generation sequencing of the BRCA1 and BRCA2 genes in a Bulgarian population, prompting further investigation for local founder mutations and variants characteristic for this particular region.

Angiogenesis in cancer - general pathways and their therapeutic implications.

A vast amount of data shows that angiogenesis has a pivotal role in tumor growth, progression, invasiveness and metastasis. This is a complex process involving essential signaling pathways such as vascular endothelial growth factor (VEGF) and Notch in vasculature, as well as additional players such as bone marrow-derived endothelial progenitor cells. Primary tumor cells, stromal cells and cancer stem cells strongly influence vessel growth in tumors. Better understanding of the role of the different pathways and the crosstalk between different cells during tumor angiogenesis are crucial factors for developing more effective anticancer therapies. Targeting angiogenic factors from the VEGF family has become an effective strategy to inhibit tumor growth and so far the most successful results are seen in metastatic colorectal cancer (CRC), renal cell carcinoma (RCC) and non-small cell lung cancer (NSCLL). Despite the initial enthusiasm, the angiogenesis inhibitors showed only moderate survival benefit as monotherapy, along with a high cost and many side effects. Obviously, other important pathways may affect the angiogenic switch, among them Notch signaling pathway attracted a large interest because its ubiquitous role in carcinogenesis and angiogenesis. Herein we present the basics for VEGF and Notch signaling pathways and current advances of targeting them in antiangiogenic, antitumor therapy.

Gene expression in peripheral blood of patients with hypertension and patients with type 2 diabetes.

AIM: To evaluate the expression of atherosclerosis-associated genes in patients with hypertension and type 2 diabetes mellitus. MATERIAL AND METHODS: Twenty-seven patients (14 men, 13 women), mean age 43.26 ±â€Š11.69 years, were included in the study, which was divided into three groups: group 1 - patients with newly diagnosed hypertension and normal glucose tolerance (n = 9), group 2 - normotensive individuals with newly diagnosed type 2 diabetes (n = 9), and control group - normotensive individuals with normal glucose tolerance (n = 9). Gene expression analysis was performed with Human Atherosclerosis RT2 Profiler PCR Array. RESULTS: In patients with hypertension, we found eight genes with increased expression - FABP3, FAS, FN1, IL1R2, LPL, SERPINE1, TGFB1, and VCAM1. Decreased expression was observed for two genes - SELPLG and SERPINEB2. In patients with type 2 diabetes we found seven up-regulated genes - APOE, BAX, MMP1, NFKB1, PDGFB, SPP1, and TGFB2, whereas no specifically down-regulated genes were observed. Three genes - KLF2, PDGFRB, and PPARD were found to be expressed only in groups 1 and 2. CONCLUSION: Hypertension is associated with increased expression of FABP3, FAS, FN1, IL1R2, LPL, SERPINE1, TGFB1, and VCAM1 and decreased expression of SELPLG and SERPINEB2. The up-regulation of FAS, FN1, SERPINE1, TGFB1, and VCAM1 might be associated with an increased cardiovascular risk. Type 2 diabetes is associated with increased expression of APOE, BAX, MMP1, NFKB1, PDGFB, SPP1, and TGFB2. KLF2 and PPARD might be part of protective mechanisms that limit target organ damage in both disease conditions. Expression of PDGFRB might play an important role in the pathogenesis of both hypertension and type 2 diabetes.

Whole genome methylation array analysis reveals new aspects in Balkan endemic nephropathy etiology.

BACKGROUND: Balkan endemic nephropathy (BEN) represents a chronic progressive interstitial nephritis in striking correlation with uroepithelial tumours of the upper urinary tract. The disease has endemic distribution in the Danube river regions in several Balkan countries.DNA methylation is a primary epigenetic modification that is involved in major processes such as cancer, genomic imprinting, gene silencing, etc. The significance of CpG island methylation status in normal development, cell differentiation and gene expression is widely recognized, although still stays poorly understood. METHODS: We performed whole genome DNA methylation array analysis on DNA pool samples from peripheral blood from 159 affected individuals and 170 healthy individuals. This technique allowed us to determine the methylation status of 27 627 CpG islands throughout the whole genome in healthy controls and BEN patients. Thus we obtained the methylation profile of BEN patients from Bulgarian and Serbian endemic regions. RESULTS: Using specifically developed software we compared the methylation profiles of BEN patients and corresponding controls and revealed the differently methylated regions. We then compared the DMRs between all patient-control pairs to determine common changes in the epigenetic profiles.SEC61G, IL17RA, HDAC11 proved to be differently methylated throughout all patient-control pairs. The CpG islands of all 3 genes were hypomethylated compared to controls. This suggests that dysregulation of these genes involved in immunological response could be a common mechanism in BEN pathogenesis in both endemic regions and in both genders. CONCLUSION: Our data propose a new hypothesis that immunologic dysregulation has a place in BEN etiopathogenesis.

Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells.

Notch is an intercellular signaling pathway related mainly to sprouting neo-angiogenesis. The objective of our study was to evaluate the angiogenic mechanisms involved in the vascular augmentation (sprouting/intussusception) after Notch inhibition within perfused vascular beds using the chick area vasculosa and MxCreNotch1(lox/lox) mice. In vivo monitoring combined with morphological investigations demonstrated that inhibition of Notch signaling within perfused vascular beds remarkably induced intussusceptive angiogenesis (IA) with resultant dense immature capillary plexuses. The latter were characterized by 40 % increase in vascular density, pericyte detachment, enhanced vessel permeability, as well as recruitment and extravasation of mononuclear cells into the incipient transluminal pillars (quintessence of IA). Combination of Notch inhibition with injection of bone marrow-derived mononuclear cells dramatically enhanced IA with 80 % increase in vascular density and pillar number augmentation by 420 %. Additionally, there was down-regulation of ephrinB2 mRNA levels consequent to Notch inhibition. Inhibition of ephrinB2 or EphB4 signaling induced some pericyte detachment and resulted in up-regulation of VEGFRs but with neither an angiogenic response nor recruitment of mononuclear cells. Notably, Tie-2 receptor was down-regulated, and the chemotactic factors SDF-1/CXCR4 were up-regulated only due to the Notch inhibition. Disruption of Notch signaling at the fronts of developing vessels generally results in massive sprouting. On the contrary, in the already existing vascular beds, down-regulation of Notch signaling triggered rapid augmentation of the vasculature predominantly by IA. Notch inhibition disturbed vessel stability and led to pericyte detachment followed by extravasation of mononuclear cells. The mononuclear cells contributed to formation of transluminal pillars with sustained IA resulting in a dense vascular plexus without concomitant vascular remodeling and maturation.