Aim: The aim of this study was to establish the expression and the significance of angiogenic markers in T1 bladder cancer with concomitant carcinoma “in situ”. Introduction: It has been determined that overexpression of hypoxia-inducible factor 1-alpha (HIF-1alfa), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor1 (VEGFR1) correlates with tumor grade, disease progression and recurrence, as well as poor overall survival. Carcinoma in situ (CIS) is frequently seen in conjunction with bladder tumors and represents an additional obstacle for management of T1 bladder cancer patients. Material and Methods: The immunohistochemical expressions of HIF-1alfa, VEGF, and VEGFR1 were evaluated in 295 T1 bladder cancer samples, incorporated in tissue microarrays. HIF1-alpha was assessed through nuclear staining, while the angiogenic profile was estimated through cytoplasmatic positivity of the VEGF and VEGFR1. Microvessels were identified by immunostaining of endothelial cells for CD34 and microvessel density (MVD) was presented as the average number of counted microvesels. Results: After a mean followup of 53 months, we found that T1 bladder cancer patients, who had concomitant carcinoma in situ had worse overall survival (p<0.05), furthermore, those tumor samples less expressed HIF-1alfa (p<0.05), and VEGFR1 (p<0.05). Expression of VEGF, VEGFR1, HIF-1alfa, as well as MVD, did not have significant impact to survival rate and further outcome. Conclusion: Worse overall survival and decreased expression for angiogenic markers in samples with accompanying CIS were established. Estimation of HIF-1alfa and VEGFR1 expression emerged as potential diagnostic supplement, selecting the T1 bladder cancer patients that could require an intensive follow-up.

Aim: Here, we explored the role of NCAM/FGFR1 signaling and pro-fibrotic gene expression signatures as initiating or driving forces of EMT program in cultured human proximal tubular epithelial cells. Introduction: Epithelial-to-mesenchymal transition (EMT) contributes to maladaptive repair and parenchymal damage during renal fibrosis. Based on previous reports, neural cell adhesion molecule (NCAM) and fibroblast growth factor receptor 1 (FGFR1) are both considered to be mechanistically involved in the EMT process. Material and Methods: By using an established in vitro model of EMT of the human proximal tubular epithelial cells (HK-2 cells) in response to TGF-β1 (10ng/mL) exposure, NCAM/FGFR1 signaling responses were analyzed by light microscopy, immune-labelling, qRT-PCR and scratch assays. Modulation of FGFR1 was induced using PD173074 (100nM). Distribution of TGF-β1 downstream effectors was assessed in HK-2 cells of the EMT program as well as in 50 biopsies of different human kidney diseases to explore their in vivo correlation. Results: EMT associated with morphological changes started 48h after TGF-ß1 treatment of the HK-2 cells and was clearly apparent after 72 hours, associated with loss of CDH1 (encoding E-Cadherin) and transcriptional induction of SNAI1 (encoding SNAIL), SNAI2 (encoding SLUG), TWIST1, MMP2, MMP9, CDH2 (encoding N-Cadherin), ITGA5 (encoding integrin-α5), ITGB1 (encoding integrin-β1), ACTA2 (encoding α-SMA) and S100A4 (encoding FSP1). After 24 hours of TGF-β1 exposure at the early stage of SPECIJALNA SESIJA: SESIJA SPECIJALIZANATA 7 MATERIA MEDICA • Vol. 34 • Issue 1, suplement 1 • april 2018. EMT program, transcriptional induction of several NCAM isoforms along with FGFR1 was observed, implicating a mechanistic link between NCAM/FGFR1 signaling and induction of EMT. These assumptions were further supported by the inhibition of the EMT program after specific blocking of FGFR1 signaling responses by PD173074. Moreover, there was evidence for an in vivo TGF-β1 pathway activation with EMT response signal signatures in diseased human kidneys in correlation to impaired renal excretory functions. Conclusion: Modulation of NCAM/FGFR1 signaling blocks the EMT program in cultured human proximal tubular epithelial cells. NCAM/FGFR1 signaling appears to be involved in initial phases of TGF-ß1 initiated EMT of tubular cells and thus could contribute to maladaptive repair and parenchymal damage during renal fibrosis.

Aim: The goal was to examine the effects of direct exposure of stomach tissue to  acrylamide. Introduction: Acrylamide is a toxic chemical substance discovered in foods rich in starch, prepared at high temperatures. Material and Methods: The research was carried out 6 groups of 5 experimental animals (Wistar rats). Two control groups orally implicated distilled water. Two experimental groups orally administrated PLENARNE USMENE PREZENTACIJE 21 MATERIA MEDICA • Vol. 34 • Issue 1, suplement 1 • april 2018. acrylamide in a daily dose of 25 mg/kg, and two dose of 50 mg/kg. Three groups were sacrificed after 24h and three after 72h, On histological gastric tissue material is applied qualitative and semi-quantitative histological analysis, stereological measurements of individual compartments of the stomach wall, linear measuring the number of mast cells in the mucosa and submucosa. Results: Slight damage of the surface epithelium, accompanying mild inflammatory reaction and degranulation of mast cells are results of histological damages in the stomach tissue. Reconstruction of the epithelium follows directly toxic effect on epithelium, and it is confirmed by the presence of immature form of mucoproductive cells. Examined inflammatory and degenerative parameters show a positive correlation with respect to dose and/or a time of exposition to acrylamide. Conclusion: Understanding the mechanism of action of acrylamide allows to apply adequate prevention and make an appropriate choice of therapeutic methods.

Aim: The aim of the study is to investigate the effect of prenatal treatment of metronidazole on the process of cerebellar foliation in the guinea pig. Introduction: The use of metronidazole in pregnancy still occupies very different attitudes among doctors who prescribe them. Neurotoxicity is a common side effect in the use of metronidazole, but there is still no clear animal study. Materials and Method: We studied 42 guinea pig fetuses obtain from 12 pregnant guinea pig dams.  Dams were separated in experimental (6) and control6) group(K). Experimental group(E) received from 42-49 day of gestation at every PLENARY ORAL PRESENTATION 22 MATERIA MEDICA • Vol. 34 • Issue 1, suplement 1 • april 2018. 12 hour (at 8 a.m. and 8 p.m.) subcutaneous injection od saline solution of metronidazole in concentration of 28 mg/kg.  Control group received using same time protocol just saline solution. At 50th day of gestation all dams were sacrified and fetuses were used for further analysis. In each  fetuses (22 in E, and 20 K group). Firstly we make gross picture of the brain and secondary we separated vermis region for further histological and immunohistochemical analysis. Results: An analysis of macroscopic images of the dorsal side of the cerebellum in the experimental group of individuals revealed malformations in 15 out of 22 fetuses (68.2%). The most affected folia was VII and characterized with aberrant diagonal positioning along the entire dorsal side of the vermis. Other folia were partial presence, with micro, and agyria changes. Conclusion: Prenatal use of metronidazole in guinea pigs during the 42-49th day of gestation cause disturbance in cerebellar foliation.

Aim: To compare sensitivity, specificity and accuracy of bronchial brushing (BB) and transbronchial needle aspiration (TBNA) cytology in diagnosing lung cancer and to correlate and compare them with corresponding histopathology. Introduction: Lung cancer is a leading cause of death worldwide in both genders. Various cytological techniques such as BB and aspirate, TBNA, bronchoalveolar lavage, transthoracic and pleural puncture can aid in early diagnosis of lung malignancies. Material and Methods: One-year retrospective study included 359 patients with suspected lung cancer who underwent bronchoscopy. During bronchoscopy, cytopathological samples were obtained for smears using BB and TBNA as well as biopsy for histopathological examination that was considered the  gold standard .  All the samples were microscopically examined and statistically analyzed using descriptive methods and non-parametric Kendal-tau correlation coefficient (the level of significance p<0.05). Results: Sensitivity of BB and TBNA cytology comparing to histopathology was 97.17% and 98.32% respectively whereas specificity was 97.26% and 97.75 % respectively. Positive predictive value was 97.14% in BB and 99.66% in TBNA and negative predictive value was 93.23% in BB and 98.77% in TBNA. The accuracy of BB was 96.51% and 99.14% of TBNA cytology. Discordance of BB cytological and histopathological diagnosis was in 3.21%, whereas discordance of TBNA was in 2.03% cases. There was no statistically significant difference neither between BB (p=0.550) nor between TBNA (p=0.602) cytology and histopathological diagnosis. Conclusion: Cytology is valuable and useful in establishing lung cancer diagnosis, which yields almost the same information as histopathology no matter which method of cytological sampling is used.

Aim: To determine the degree of correlation between TTF-1 (+) expression and EGFR mutation status in lung adenocarcinoma. Introduction: Adenocarcinoma of the lung is mainly diagnosed based on standard morphological criteria. The thyroid gland transcription factor (TTF-1) is currently the most commonly used immunohistochemical marker in the differentiation of invasive adenocarcinoma of the lung from another primary and metastatic carcinoma, has a prognostic significance and is a predictor of the EGFR mutation status. Material and Methods: This retrospective study enrolled 60 patients with histologically confirmed primary lung adenocarcinoma who underwent lung cancer surgery at Institute for lung disease Vojvodina between 2010 and 2015. Tumor specimens of these patients were investigated for TTF-1 expression and mutations in EGFR using immunohistochemistry and PCR analysis. Statistical analysis is in statistic software Statistica 12. Results: The study included 35 men and 25 women, with an average age of 61.8 ą 8.08 years. Of the 60 cases, TTF-1 ( ) expression was recorded in 52 (87%) (p <0.001), the statistical difference is not significant when comparing smoking habitsby gender, and tumor size among them. EGFR ( ) mutation status was found in 3/60 (5%) cases [egzon 21 (2) and exon 20 (1)], of which TTF-1 (+) expression was in two cases. Conclusion: There is a statistically significant difference between the TTF-1 (-) and TTF-1 (+) adenocarcinoma and a high degree of correlation between EGFR mutation status and TTF-1 (+) expression.

Aim: Pathohistological diagnostics (PD) of the Yolc Sac tumor (YST) of primary mediastinal localization. Introduction: YST are malignant germ cell tumors of primitive endodermal gonadal differentiation. In 1% -5% of cases their localization is extragonadal, including mediastinal in 50% -70% published cases. Case report: A 30-year-old man was admitted to the Thoracic Surgery Clinic for diagnosis and treatment of mediastinal tumor with lungs and liver metastases. Laboratory analyzes revealed high elevations in serum alpha-fetoprotein (AFP), in addition moderate elevation of beta-chorionic gonadotropin (ß-HCG). No pathological changes in testis and retroperitoneum have been found. An open biopsy of the mediastinal change was made for PD. Histologically, tumor tissue showed a significant degree of necrosis. There were reticular, microcystic and pseudopapillary forms of neoplastic cell growth, surrounded by myxomatosis stroma. Tumor cells cytoplasm was scarce and vacuolated with high nuclear polymorphism and hyperchromasia. Positive immunohistochemical (IMH) reaction was obtained for panCK, AFP, PLAP. Immunoreactivity for TTF-1 was found in about 50% of tumor cells, as well as the focal reaction for ß-HCG in rare multinuclear cells. Reactions for D2-40, CD5, CK-7, CK-20, p63, CD117, CD30, napsin A and calretinin were negative. Conclusion: The results of our analysis showed the expression of divergent endodermal linear markers in the mediastinal YST, especially TTF-1 expression. The diagnosis of YST in small biopsies can be difficult and requires using of a wide range of IMH markers in order to closer the differentiation of primary tumor localization and the application of appropriate chemotherapy

Aim: Reporting of autopsy case of fatal accidental poisoning by colchicine from meadow saffron. Introduction: Meadow saffron (Colchicum autumnale) is a perennial herbaceous plant similar to the edible wild garlic (Allium ursinum). Toxic substance in meadow saffron is alcaloid colchicine. Colchicine poisoning is a very dangerous condition, which can lead to fatal outcome. Case report: A 50-years-old male was admitted to the hospital complaining of weakness, abdominal pain, nausea, vomiting and diarrhea without blood. Day before he ate two plants thinking they’re wild garlic and seven hours after ingestion he felt first symptoms. During the course of the hospital stay, he had gastroenterocolitis, acute renal failure, hepatic lesion and cardiorespiratory insufficiency with fatal outcome. Post-mortem examination reveled:brain oedema, lung oedema and congestion, heart weighing 700 grams, ventricular hypertrophy, myocardial fibrosis, liver congestion and steatosis, spleen congestion, pancreatic fibrosis. Organs sections were taken for pathohistological analysis. Body fluids and parts of organs were toxicologically analyzed. Pathohistological findings:brain oedema, diffuse perivascular and interstitial myocardial fibrosis, myocardial haemorrhage, lungs congestion and edema, micro- and macrovesicular liver steatosis, centrilobular liver necrosis, lymphocytic inflammatory infiltrate in liver portions, spleen’s red pulp congestion, kidney congestion and interstitial bleeding, coagulation necrosis of the proximal tubules of the kidney. Toxicological analysis showed colchicine in blood-0.011 mg/L, urine-0.051 mg/L, liver with gallbladder-0.007 mg/ kg, kidney-0.008 mg/kg. Conclusion: Ingestion of the meadow saffron can lead to poisoning with fatal outcome due to colchicine. Colchicine intoxication should be suspected in patients with gastrointestinal symptoms after consuming wild plants.

The design and construction of new biological systems in the way engineers design electronic or mechanical systems is the primary goal of synthetic biology. The ability to create and modify life forms and easy access to information to do so has raised a number of issues related to ethics and security. In the era of rapid development of biotechnology, and the perception of the consequent risks to the environment and health, the ethics of knowledge becomes a matter of practical significance. The concern about the misuse of knowledge from synthetic biology influences new risk reduction strategies, which can have significant effects on scientific progress. This paper will provide an overview of the main bioethical and biosafety issues of synthetic biology.

Case 1: Clinical History -The patient was an 18-year-old woman with a history of primary amenorrhea. The physical examination revealed normal female external genitalia, moderate breast development, scarce pubic hair, and scant axillary hair. Pelvic examination revealed an absence of a uterus. The vagina ended in a blind pouch and measured 6cm in length. An abdominal ultrasonogram showed solid bilateral gonadal structures measuring 31x15mm right, and 30x 6mm left in the pelvic cavity without any evidence of a uterus. These findings were confirmed by MRI scan. Serum levels of estradiol and testosterone were 45.7pg/mL and 7.5nmol/L, respectively. FSH and LH levels were 8.46mlU/ml and 20.2mlU/ml. Cytogenetic analysis of peripheral blood lymphocytes revealed a 46,XY karyotype, with translocation t(13,14) inherited from her father. The molecular investigation confirmed active SRY with amplification of 6 nonpolymorphic loci on AZFa, AZFb, and AZFc regions on Y chromosome. Three months after her first referral the patient underwent bilateral prophylactic laparoscopic gonadectomy. The patient is being followed up regularly and she has no complaints after five years. Pathological Findings- Grossly, the right gonad was 4.3x1.8x1.8cm and the left gonad was 4.5x2.0x1.7cm. The right fallopian tube was 3.8cm long and 0.2cm in diameter, while the left one was 1.5cm long and 0.2cm in diameter. In addition, 4 cysts measuring 0.3- 1cm in diameter were found in the fat tissue on the surface of the left fallopian tube. The cut surface of the right gonad resembled testicular tissue showing two discrete, firm, well-demarcated, slightly bulging, homogenous, light grey-tan colored nodules, measuring 0.1 and 0.9cm in maximal dimension. Three similarly looking nodules were present in the left gonad measuring 0.4-0.5cm in diameter. In addition, a white whorled, firm, smooth muscle body fused to one pole of the gonad was found bilaterally measuring 1.2cm right, and 1.3cm left. Microscopically, both gonads were composed of varying proportions of small and solid, or less frequently larger, with early lumen formation seminiferous tubules lined by immature Sertoli cells, surrounded with fibrous, focally edematous stroma in which prominent Leydig cells were present. Rare tubules contained Sertoli cells with abundant cytoplasm filled with coarse, eosinophilic granules. The nodules were composed of solid immature tubules lined by cylindrical immature Sertoli cells separated by fibrous stroma containing fewer Leydig cells. Fallopian tubes were hypoplasic, while the cysts were lined by a single layer of cuboidal to columnar cells some of which had cilia. Thus, the clinical, laboratory, imaging, genetic and histological findings confirmed the diagnosis of complete androgen insensitivity syndrome with bilateral testicular hamartomas.Discussion Androgen insensitivity syndrome (AIS) is a disorder where there is resistance to androgen actions influencing both the morphogenesis and differentiation of androgen-responsive body structures. It is the most common type of male pseudohermaphroditism, characterized by an absence of androgen receptor activity due to a mutation at Xq11–q12 localization on the androgen receptor gene. the. In the largest series of 43 patients with the AIS published by Rutgers and Scully hamartomas were present in 63% of the cases, while Sertoli cell adenomas were reported in 23% and malignant tumors including two seminomas, one intratubular germ cell neoplasm with early stromal invasion and a malignant sex cord tumor in 9% of the cases. Case 2: Clinical History- The patient was a 17-year-old girl with primary amenorrhea. On external examination, she had an unambiguous female phenotype albeit with poor breast development. Her external genitalia had a normal appearance but internal examination showed a hypoplastic uterus. Ultrasonography failed to show follicular activity in the gonads. Her gonadotropins were high (FSH, 55.6mU/ml; LH, 18.3mU/ml) and estradiol was low (20.0pg/ml). Nonmosaic 46,XY karyotype was detected in patient’s leukocytes by cytogenetic analysis. Under the tentative clinical diagnosis of pure gonadal dysgenesis, a prophylactic bilateral laparoscopic gonadectomy with bilateral salpingectomy was performed. A biopsy specimens from the gonads were sent for cytogenetic analysis. The sequencing of the SRY gene of the proband revealed a C/G substitution at the first nucleotide of codon 133, leading to Arg/Gly replacement in the SRY protein. The mutation was also present in patient’s father, who is a phenotypically normal male. APSTRAKTI 77 MATERIA MEDICA • Vol. 34 • Issue 1, suplement 1 • april 2018. The patient is alive and well 12 years after the operation without any evidence of disease. Pathological Findings- Macroscopically, both fallopian tubes were 4cm long and 0.4cm in diameter, while the gonads were measuring 3x2.2x1.2cm the right, and 2.2x0.8x1cm the left. Upon dissection, both gonadal streaks had a similar, variegated appearance with multiple areas of microcalcification. The histological evaluation of the gonads confirmed a gonadoblastoma of 3cm in diameter in the right gonad and a predominantly “burnt-out” gonadoblastoma of 2.2cm in the left gonad with only a microscopic focus of recognizable gonadoblastoma less than 1mm in diameter. The tumors were composed of primordial germ cells intimately admixed with sex cord elements, which surrounded round spaces filled with eosinophilic basement membrane-like material. The atypical germ cells had abundant clear or pale, slightly granular cytoplasm and round vesicular nuclei with prominent nucleoli. The small, often fusiform sex cord element cells had uniform round or oval nuclei with indiscernible nucleoli and scant cytoplasm. The uninvolved gonadal tissue bilaterally had the morphology of a streak gonad composed largely of ovarian-type stroma, with extended calcification and small germinal inclusion cysts present, without any follicles. The fallopian tubes were infantile. Therefore, the histopathological findings confirmed the diagnosis of bilateral gonadoblastoma in streak gonads, FIGO Stage IB, in a patient with a XY gonadal dysgenesis (Swyer syndrome). Discussion: XY gonadal dysgenesis (GD) is a result of abnormal testis development in utero. Pure 46,XY GD should be considered when an adolescent presents as a phenotypic female with delayed puberty and primary amenorrhea. Rarely, they may present with a detectable abdominal or pelvic gonadoblastoma mass. When the presumptive diagnosis suggests GD, further criteria may strengthen the diagnosis, while ultimately, the most definitive diagnosis is through biopsy of the gonads. Bilateral streak gonads are seen in pure XY GD. The risk of otherwise undetected gonadoblastoma in XY GD patients is high, and prophylactic or therapeutic gonadectomy is therefore often indicated when XY GD is diagnosed.