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Papers accepted for publication

1. Determination of VEGFR-2 (KDR) -604A>G polymorphism in recurrent pregnancy loss

Lidia Boldeanu, Cristian Adrian Silosi, Vlad Padureanu, Anda Lorena Dijmarescu, Maria Magdalena Manolea, Maria Carmen Tabacu, Mihail Virgil Boldeanu, Mircea Vasile Popescu-Driga, Ioan Sabin Poenariu, Rodica Padureanu, Liliana Victoria Novac, Marius Bogdan Novac

Background: Placental angiogenesis and vascular adaptation during pregnancy, along with diminished placental trophoblastic vascular endothelial growth factor immunoreactivity, play an important role in the early stages of human pregnancy, being possible causes of recurrent pregnancy loss (RPL). Aims: Our focus was directed towards investigating a possible association between vascular endothelial growth factor receptor-2 (kinase insert domain receptor) VEGFR-2 (KDR) -604A>G (rs 2071559) gene polymorphism and RPL in the study area of Dolj County, Romania. Patients, Materials and Methods: In this study, 169 women, diagnosed with RPL, were included. They were hospitalized in the Clinics of Obstetrics and Gynecology, Filantropia Municipal Hospital, Craiova, during the following period: October 2009-October 2016. The control group consisted of 145 women. All subjects were genotyped by means of allelic discrimination TaqMan polymerase chain reaction assay with specific probes. Results: No statistically significant difference was observed between the RPL patients and the control group, when one genotype was compared to another [in a dominant model, -604 AG+GG vs. AA: odds ratio (OR) 1.71, 95% confidence interval (CI) 0.99-2.96, p=0.051]. While studying the overall risk of RPL by the genotype frequencies of KDR polymorphism between controls and RPL patients, which were stratified according to the number of consecutive pregnancy losses (PLs), the chi-square test showed a significant association between the presence of this polymorphism and the increased risk observed in patients with four or more consecutive PLs, to develop RPL (in a dominant model - G allele carriers, KDR -604 AG+GG vs. AA: OR 1.91, 95% CI 1.03-3.52, p=0.037). These results prove that G allele carriers have an increased risk of RPL about 1.91-fold higher than those with the AA genotype do. Although our results bear limited statistical significance, the study nonetheless represents a step forward in the evaluation of recurrent abortion, which has not yet been explored sufficiently. Conclusions: VEGFR-2 (KDR) polymorphism does not influence RPL susceptibility in the study area of Dolj County, Romania. Therefore, further studies, which include a larger sample size, are required in order to clarify the role of KDR polymorphism in RPL.

Corresponding author: Mihail Virgil Boldeanu, Lecturer, MD, PhD; e-mails: laborator.imunologie@umfcv.ro, boldeanumihailvirgil@yahoo.com

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2. Occupational exposure to urinary bladder carcinogens - risk factors, molecular mechanisms and biomarkers

Marina Ruxandra Otelea, Viorel Jinga, Alexandru Stefan Catalin Rascu, Iancu Emil Plesea, Amelia Nicoleta Petrescu, Luminita Elena Mitrache, Mihai Olteanu, Dan Bondari, Agripina Rascu

Bladder cancer (BC) is one of the most frequent forms of cancer, particularly in Caucasian population. Many environmental factors are recognized as carcinogenic in humans for this form of neoplasia and some of them are related to occupation. In order to illustrate these effects, we have selected several relevant cases with smoking and occupational exposure to carcinogens and their histopathological findings. We reviewed the most important research published in the field of environmental-genomic interaction in relation with the oncogenesis of BC. Three main directions have been identified and described in the article: the environmental factors involved in BC pathogenesis and evolution, the molecular mechanisms involved in cell mitosis control and xenobiotic metabolism related to the qualitative and quantitative exposure and, finally, the possible biomarkers of the tumor evolution. From the genomic and proteomic research, new biomarkers emerged that are in the validation process. Immunohistochemical methods open also new perspectives to the diagnostic algorithms and could serve as prognosis biomarkers.

Corresponding author: Alexandru Stefan Catalin Rascu, Assistant Professor, MD, PhD; e-mail: stefanrascu.sr@gmail.com

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3. The process of liver fibrosis in chronic hepatitis C - histological and immunohistochemical study

Nicoleta Laura Popescu, Octavian Ion Predescu, Oana Badea, Ionica Pirici, Carmen Pantis, Cristina Jana Busuioc, Bogdan-Virgil Cotoi, Laurentiu Mogoanta

Liver fibrosis is one of the most serious histopathological (HP) lesions that, together with the inflammatory process and the hepatocyte lesions, determine the change of the liver architecture, having as a clinical result the onset of liver failure phenomena. Hepatitis C virus represents one of the most frequent conditions leading to the onset of liver fibrosis and favors the progression of the disease towards hepatocellular carcinoma. We evaluated the HP and immunohistochemical (IHC) aspects on fragments of liver biopsies taken from 104 patients diagnosed with chronic hepatitis C and altered capacity of work. In our study, we observed a growth of the portal (Kiernan) spaces by the presence of a chronic inflammatory infiltrate, the presence of collagen fibers and conjunctive matrix. The density and dimensions of collagen fibers were correlated with the severity of the liver disease, in the severe forms being highlighted porto-portal and porto-central fibrous bridges. The IHC examinations highlighted the change of the phenotype of perisinusoidal dendritic cells, the growth of the myofibroblast cells in the portal spaces, the growth of the macrophage number in the inflammatory infiltrate and of the Kupffer cells in the liver parenchyma.

Corresponding author: Cristina Jana Busuioc, Associate Professor, MD, PhD; e-mail: dr_cristinab@yahoo.com; Bogdan-Virgil Cotoi, Lecturer, MD, PhD; e-mail: bogdan230376@yahoo.ro

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4. Looking beyond gut inflammation in inflammatory bowel disease

Anca Cardoneanu, Cristina Cijevschi Prelipcean, Mihai Danciu, Catalina Mihai, Mihaela Dranga, Otilia Gavrilescu, Elena Rezus

Patients diagnosed with inflammatory bowel disease (IBD) often develop one or more extraintestinal manifestations (EIM). We performed a prospective study that included 517 patients with IBD (Crohn s disease - CD, ulcerative colitis - UC or undifferentiated colitis - CN) diagnosed between 1975 and 2016 in the Northeastern region of Romania. The patients were extracted from the national database (IBD Prospect). UC cases predominated compared to CD cases (n=368 vs. n=135). Only 10 patients were diagnosed with CN. In the study group, 51 cases with IBD and EIM were identified, having a prevalence of 9.9%. Musculoskeletal manifestations were the most common EIM. Peripheral involvement - arthritis (n=26, 68.42%) predominated, followed by axial damage - sacroiliitis/ankylosing spondylitis (SI/AS) (n=12, 31.58%) (p=0.001). Patients with CD had a 3.48-fold greater risk of developing joint manifestations [p<0.001, odds ratio (OR)=3.478, 95% confidence interval (CI) 1.779-6.801]. In both CD and UC patients, arthritis cases were the most frequent observed (68.42% vs. 31.58%). Patients with CD had a 5-fold higher risk of developing arthritis (p<0.001, OR=5.009, 95% CI 2.21-11.34). Neither CD, nor UC patients, had a confirmed risk of developing SI/AS (p=0.468, OR=1.565, 95% CI 0.463-5.293 for CD) (p=0.586, OR=0.714, 95% CI 0.211-2.413 for UC). Cases of arthritis and CD (n=16) mainly correlated with the colonic localization of inflammation (n=7, p=0.723) followed by ileo-colonic form of CD (n=7, p=0.321). Patients with arthritis and UC (n=10) initially correlated with pancolitis (n=5, p=0.072, OR=3.023, 95% CI 0.855-10.69) then with proctitis (n=3, p=0.392) and left-sided colitis (n=2, p=0.024, OR=0.196, 95% CI 0.041-0.938).

Corresponding author: Catalina Mihai, Lecturer, MD, PhD; e-mail: catalinamihai@yahoo.com; Mihai Danciu, Professor, MD, PhD; e-mail: mihai.danciu@umfiasi.ro

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5. Intraoperative assessment of sentinel lymph nodes in early-stage breast cancer

Andrei Ionut Costin, Ion Paun, Flore Varcus, Marian Madalin Racareanu, Ion Lomaca, Adelina-Gabriela Costin, Larisa Iovan, Lucretiu Radu, Gabriela-Marcelina Mihai

The assessment of axillary lymph node (ALN) status provides heavily weighing prognostic indicators in deciding on breast carcinoma treatment. In the 6th and 7th editions of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual are evaluated the nodal metastases based on size and taking into account the number of metastatic cells. According to these Manuals, a positive node is equated to metastasis whose size reaches at least 0.2 mm or amounting to more than 200 tumor cells. The clinical significance and the therapeutic optimum of the presence of a minimal nodal involvement after axillary sentinel lymph nodes (SLNs) biopsy remain controversial. The need for further axillary treatment (ALN dissection or axillary radiation) in clinical N0 patients with early-stage breast carcinoma and SLNs metastases remains unclear. In all likelihood, the delivery of the regular adjuvant treatment in association with systemic treatment and radiation therapy results in survival rates similar to axillary treatment completion. This review also presents several assessment methods related to the SLNs at the surgical stage, such as cytological, histological, immunohistochemical and molecular diagnostic techniques, evaluating the advantages and disadvantages of each of them. More studies including larger groups of breast patients are needed to confirm which of them is the most reliable method for the evaluation of the SLNs.

Corresponding author: Ion Paun, Associate Professor, MD, PhD; e-mail: dripaun@gmail.com

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6. The role of glomerular morphometric features in pediatric podocytopathies - a single center study

Diana Derewicz, Valentin Tiberiu Moldovan, Leila Ali, Roxana Taras, Cosmin Florescu, Mihaela Balgradean, Maria Sajin

Podocytopathies represent a well-studied subgroup of glomerulopathies, being characterized by proteinuria due to damage or dysfunction of podocytes. Glomerular size in podocytopathies has been studied in different population, but only a few studies take in consideration the pediatric population. There are different methods to assess the glomerular size, but most of the studies report the maximal profile area as being the most accurate one. The aim of this study is to determine the range values of glomeruli in pediatric population with glomerulopathies and to establish a correlation between the measured size and several laboratory features. The patients that undergo renal biopsy in the Department of Nephrology, Maria Sklodowska Curie Clinical Emergency Hospital for Children, Bucharest, Romania, were divided into two groups: control vs. affected/patient group. The control group included children that require renal biopsy for renal impairments other than high-range proteinuria (most of them recurrent microscopic asymptomatic hematuria), while the affected group had nephrotic-range proteinuria. Thirty patients were selected to be part of the control group and 30 patients in the affected group. In control group, the mean value diameter was 166.23+/-13.04 micro-m, and the area of the glomerulus had a mean value of 19 126.86+/-3070.83 micro-m(2). In the affected group, we obtained the following results: the mean value diameter was 192.42+/-28.15 micro-m, while the glomerular cross-sectional area had a mean value of 23 535.55+/-6456.57 micro-m(2). Using the linear regression, we concluded that all the cases with increased-size glomeruli had more urinary protein loss compared with the ones that had small-size glomeruli and low-range proteinuria.

Corresponding author: Diana Derewicz, MD, PhD Student; e-mail: diana_costache195@yahoo.com

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7. Intestinal fatty acid-binding protein, as a marker of anastomotic leakage after colonic resection in rats

Gabriel Alexandru Popescu, Ioan Jung, Bogdan Andrei Cordos, Leonard Azamfirei, Adina Hutanu, Simona Gurzu

Aim: The aim of this experimental study was to determine if the type of termino-terminal anastomosis of the colon affect the process of healing of the intestinal mucosa and if the severity of the anastomotic leakage (AL) can be predicted based on the circulating level of intestinal fatty acid-binding protein (I-FABP). Materials and Methods: In 18 healthy Wistar rats, two types of open colon resection with termino-terminal anastomosis were performed: group A (n=9) - without inversing the vascularization and group B (n=9) - with inversing the vascularization. To assess the intestinal barrier function, circulating level of I-FABP was measured pre- and post-operatively. I-FABP tissue expression was immunohistochemically assessed in the anastomotic and perianastomotic colonic mucosa. The rats were sacrificed at three, five, and seven days after surgery. Results: In both groups, the post-operative serum level of I-FABP increased 3-4 times at 3-5 days and seven times in the seventh post-operative day. In the six cases that showed AL, the increased level was significantly higher: seven times at three days (n=2) or five days (n=2) and 30 times at seven days (n=2). The I-FABP epithelial expression was lost in all cases from group B (as result of prolonged ischemia) and in cases with AL from group A. The I-FABP was translocated in the intraglandular mucus. Conclusions: The post-operative level of I-FABP can be appreciated based on the pre-operative value only. A 7-8 increased value in first five days might indicate a risk of AL. After seven days, a seven times increased value is an indicator of a proper healing process but an increasing amount higher that 30 times might predict risk for AL, fistula, peritonitis and septic shock. The risk of AL does not depend on the anastomotic method, although the level of ischemia is higher in anastomoses that involve vascular damage.

Corresponding author: Simona Gurzu, Professor, MD, PhD, Dr. habil.; e-mail: simonagurzu@yahoo.com

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8. Hand enchondroma - complete evaluation and rehabilitation

Magdalena Rodica Traistaru, Diana Kamal, Raluca Niculina Ciurea, Paraschiva Postolache, Dragos Ovidiu Alexandru, Otilia Constantina Rogoveanu, Kamal Constantin Kamal

Enchondroma, the most frequently reported primary tumor in the long bones of the hand, is a common benign cartilaginous tumor, defined more exactly as a benign mature hyaline cartilaginous tumor that is located into the medullar space of the tubular bones. Between 2012 and 2018, we performed an observational study on six patients (two males and four females), aged 16 to 49 years old (mean 29.5 years), who were diagnosed during routine physical examinations with isolated enchondroma in their hand. The tumor was more frequently located in their proximal phalanges (four patients), while two patients had middle phalange tumors. The most common digit involved was the little finger (five patients), followed by the ring finger (one patient). The symptoms, which prompted surgical interventions, were: enlargement without fracture (two patients), pain or limitation of mobility (four patients), pain without pathological fracture (six patients). Studied patients were completely assessed before the treatment program (surgical intervention and rehabilitation program). The measured parameters and scales used were Visual Analogue Scale (VAS), Health Assessment Questionnaire (HAQ) scale for functional status and histological exam. For both VAS and HAQ, we observed a statistically significant difference between the initial and final evaluations, with p=0.03 and p=0.035, respectively. Histological assessment and differential diagnosis are essential for the post-surgical treatment and rehabilitation program.

Corresponding author: Dragos Ovidiu Alexandru, Associate Professor, MD, PhD, MSc; e-mail: dragosado@yahoo.com; Diana Kamal, MD, PhD, MSc; e-mail: dianakamal84@gmail.com

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9. Immunoexpression of p53 and COX-2 in basal cell carcinoma

Andreea Oana Enache, Alex Emilian Stepan, Claudiu Margaritescu, Virgil Patrascu, Raluca Niculina Ciurea, Cristiana Eugenia Simionescu, Adrian Camen

Basal cell carcinoma (BCC) is a variety of cutaneous carcinoma associated with an excellent prognosis because it rarely metastasizes, but it can cause significant local destruction and morbidity if surgical excision is not made. In this study, we examined the immunohistochemical expression of p53 and cyclooxygenase-2 (COX-2) in 51 BCCs, nodular and infiltrative subtypes, with various Clark levels. The immunoexpression of p53 was identified in 74.5% BCC cases and COX-2 reactions in 88.2% of cases. The scores of p53 reactions revealed significant differences depending on Clark level and borderline significance with tumor type, the high positive scores being associated to infiltrative tumors and high Clark level. No differences were revealed between COX-2 scores with both Clark level and tumor type. The analysis of the percentage values of p53 and COX-2 indicated a positive linear correlation. The positivity of p53 and COX-2 in a large proportion of BCCs, regardless of histological type and of depth of invasion, supports the two markers involvement in tumor progression.

Corresponding author: Alex Emilian Stepan, Associate Professor, MD, PhD; e-mail: astepan76@yahoo.com; Cristiana Eugenia Simionescu, Professor, MD, PhD; e-mail: csimionescu2004@yahoo.com

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10. Acute limb ischemia in neonates: etiology and morphological findings - short literature review

Simona Cerbu, Florin Birsasteanu, Elena Rodica Heredea, Daniela Iacob, Emil Radu Iacob, Maria Corina Stanciulescu, Eugen Sorin Boia

The acute limb ischemia (ALI) in neonates is a rare phenomenon, but with serious consequences if undiagnosed or untreated. The purpose of this review is to briefly present the etiology of ALI and morphological findings in correlation with specific causes. Etiology can be classified into two main groups: prenatal (in utero compression, thrombosis and embolism) and postnatal (iatrogenic, thromboembolism and vascular malformations). The most common cause of ALI is catheter-related thrombosis (almost 90% of thrombosis cases are associated with catheter use), but other rare causes like vascular malformations should not be overlooked. Ultrasound represents a non-invasive, inexpensive and widely available imaging technique, which provides sufficient information to evaluate the situation and establish proper therapeutic strategies. Morphological tests do not represent the standard diagnostic procedure in ALI, but they can provide useful information. The findings depend on the etiology: intraluminal thrombi, vascular changes, placental pathological modifications. Every morphological result must be correlated with the clinical picture and imagistic findings. In conclusion, ALI in neonates is a rare condition, usually associated with catheter use in intensive care unit setting, with multiple risk factors and conditions that increase the risk of occurrence.

Corresponding author: Emil Radu Iacob, Lecturer, MD, PhD; e-mails: radueiacob@umft.ro, radueiacob@yahoo.com; Daniela Iacob, Associate Professor, MD, PhD; e-mail: danielariacob@yahoo.com

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11. Clear cell renal cell carcinomas - epithelial and mesenchymal immunophenotype

Alex Emilian Stepan, Claudiu Margaritescu, Lucian Eugen Stoica, Mioara-Desdemona Stepan, Cristiana Eugenia Simionescu

Clear cell renal cell carcinomas (CCRCCs) are the most common kidney tumors that despite current advances in diagnosis continue to have high rates of metastasis and mortality. In this study, we analyzed the cytokeratin (CK) AE1/AE3 and vimentin immunoexpression in 26 CCRCCs in relation to histopathological prognostic parameters. Immunoreactions were positive and heterogeneous in all analyzed cases. CK AE1/AE3 immunoexpression was associated with low grade and early stage lesions, while vimentin immunoexpression was associated with high grade and advanced lesions. The aspect may be used to determine the tumor heterogeneity and a better patients stratification for therapy.

Corresponding author: Claudiu Margaritescu, Professor, MD, PhD; e-mail: c_margaritescu2000@yahoo.com

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12. Evaluation of antioxidant capacity and clinical assessment of patients with chronic periodontitis treated with non-surgical periodontal therapy and adjunctive systemic antibiotherapy

Simina Boia, Stefan-Ioan Stratul, Marius Boariu, Sorin Ursoniu, Smaranda Laura Gotia, Eugen-Radu Boia, Claudia Borza

This study aims to evaluate the oxidative stress changes in patients with chronic periodontitis (CP) undergoing non-surgical periodontal therapy alone, compared with non-surgical periodontal therapy with adjunctive systemic antibiotic therapy. Sixteen patients with CP, randomly assigned into two equal groups, were treated either with scaling and root planing (SRP) + Amoxicillin + Metronidazole, each 500 mg, three times daily, for seven days (test group), or with SRP + placebo for seven days (control group). Venous blood and unstimulated saliva samples were collected. Non-surgical periodontal therapy was performed simultaneously with antibiotics administration. Oxidative stress balance was evaluated by measuring derivatives of reactive oxygen metabolites (d-ROMs) and the biological antioxidant potential (BAP) in plasma. After the microscopic evaluation of the pathological aspect of the epithelial cells (ECs), their number, viability and the presence of C-reactive protein (CRP) were reevaluated from saliva at seven days, while reduced glutathione (GSH) level, d-ROMs and BAP at three months. Wilcoxon and Kruskal-Wallis rank-tests were used for statistics. At three months, statistical significant reductions of mean periodontal pocket depth (PPD) and clinical attachment level (CAL) gains (both p=0.01) were found in test group. Full-mouth plaque score (FMPS) decreased statistically significant in control group (p=0.02), d-ROMs decreased statistically significant in test group (mean difference 116.24+/-107.6 U CARR, p=0.01). Mean GSH, BAP level, number of ECs, their viability and CRP were statistically non-significant. In test group patients, oxidative stress status changed from a very high level to a medium one, suggesting that adjunctive use of antibiotics could have contributed to the reduction of reactive oxygen metabolites, along with significant clinical improvements.

Corresponding author: Marius Boariu, Lecturer, DMD, PhD; e-mail: boarium@yahoo.com

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13. Ethical governance of the medical research: clinical investigation and informed consent under the new EU Medical Devices Regulation (2017/745)

Daniel-Alin Olimid, Anca Parmena Olimid, Feng Ifrim Chen

Purpose: The paper focuses on the ethical appraisal of the clinical investigations (CIs) and the informed consent within the new European Union (EU) legislation on medical devices (MDs). The Regulation (EU) 2017/745 of the European Parliament and of the Council was adopted on 5 April 2017 and entered into force on 25 May 2017, repealing the Council Directives concerning Medical Devices 93/42/EEC and the Active Implantable Medical Devices 90/385/EEC. Background: For the past thirty years, the EU legislation on MDs has been updated by several directives: Council Directive 90/385/EEC on Active Medical Devices (1990); Council Directive 93/42/EEC on Medical Devices (1993) and Council Directive 98/79/EC on In vitro Medical Devices (1998) aiming to frame the MDs market development. Content: From the ethical perspective, the present article investigates the new rules concerning the CIs of the MDs for human use and accessories for such devices conducted in the EU by highlighting new regulatory aspects: (1) the framework of the clinical evaluation and CI; (2) the relevant definitions; (3) the ethical principles related to CIs; (4) the informed consent; (5) the role of the national ethics committees. Conclusions: Although the new guidelines enable an extension of the definition of medical device and the harmonization of the rules for the placing on market and putting into service of the medical devices, it also regulates the MDs industry to ensure clinical benefits for patients and high standards of quality and safety.

Corresponding author: Daniel-Alin Olimid, Lecturer, PhD; e-mail: olimiddaniel@yahoo.com

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