ROMANIAN JOURNAL of MORPHOLOGY and EMBRYOLOGY

Vol. 62 No. 1, January-March 2021

1. Fabry disease - current data and therapeutic approaches

Ilie-Robert Dinu, Stefan-George Firu

Fabry disease represents an X-linked inherited disorder resulting in the accumulation of globotriaosylceramide (Gb3). This review explains the clinical manifestations and the possible therapies for this condition. Fabry disease is considered the second most frequent lysosomal storage disease. More than 1000 mutations of the galactosidase alpha (GLA) gene associated with this disorder have been identified. Pain, either episodic crises or chronic pain, is one of the earliest symptoms in Fabry disease. Gastrointestinal, ocular, ear or skeletal manifestations may complete the clinical picture. Cardiac and renal involvements are the most severe complications leading to organ failure and death. The cerebrovascular lesions may result in severe symptoms including stroke at younger ages. The diagnosis of Fabry disease may be put by enzymatic assays of the alpha-galactosidase A (AGAL-A) activity in plasma or leukocytes but genetic analysis remains the gold standard in identifying the precise mutation and even guiding the treatment. Enzyme replacement therapy (ERT) was the first step in treating subjects with Fabry disease. It proved important decrease of the number of sever clinical events and reduction of symptoms. Chemical chaperone therapy has many advantages including oral administration and was already approved in Europe and US, but it is suitable only for subjects with amenable mutations. Gene therapies (either ex vivo or in vivo) promise to represent a new era for many disorders including Fabry disease, the preliminary data being encouraging. Although many steps were taken in understanding the pathogeny of Fabry disease, future research is needed especially in the field of therapeutic approaches.

Corresponding author: Ilie-Robert Dinu, Teaching Assistant, MD, PhD; e-mail: drdinurobert@yahoo.com

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2. Association between burnout and immunological and endocrine alterations

Adela Magdalena Ciobanu, Ana Claudia Damian, Cristina Neagu

Background: Weather a psychological construct or a clinical entity, numerous studies have been focused on the biological link between stress, burnout, and biomarkers. Aim: The purpose of our study was to search the existing literature and summarize the immunological and endocrine alterations found in burnout patients and, also, to provide updated data for clinicians to use. Methods: We performed a literature search in PubMed database using specific terms. Results: The primary focus of the literature seems to be the hypothalamic-pituitary-adrenal (HPA) axis, which may be affected due to chronic stress, which can be investigated by measuring hormonal responsiveness [corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), cortisol, prolactin, thyroid hormones]. An important challenge that this field is faced with is the pulsatile and diurnal fluctuation of them, which may not always be considered and the heterogeneity of burnout measurements. Many studies have explored the linking pathways between the immune system and chronic stress, but only a few have specifically evaluated this process for future diagnostic or prognostic biomarkers. Conclusions: Burnout has cumulative effects on our body and stress does not affect us in a singular direction, on the contrary, significant clinical implications are found, not only microscopic, but affective symptoms leading to anxiety and depression.

Corresponding author: Ana Claudia Damian, MD, PhD Student; e-mail: clau_damian@yahoo.com

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3. Anatomical remodeling of the aortic wall in relation with the cause of death

Doru Adrian Seicaru, Mirela Albu, Razvan Mihail Plesea, Florentina Gherghiceanu, Ioan Cordos, Mircea Litescu, Dragos Ovidiu Alexandru, Iancu Emil Plesea, Valentin Titus Grigorean

Aim: The authors set out to evaluate the correlations between three of the main morphological aortic parameters (aortic diameter, intima, and media thickness) and the cause of death. Materials and Methods: Study group included 28 people died of a cardiovascular (CV) disease and 62 people died of a noncardiovascular (NCV) disease. Four aortic cross-sections (base, cross, thoracic, abdominal) were collected during autopsy from the selected cases, fixed in 10% buffered formalin and photographed together with a calibrating ruler. Then, they were processed using the classical histopathological (HP) technique (formalin fixation and paraffin embedding), stained with Hematoxylin-Eosin (HE) and Orcein, and the obtained histological slides were transformed into virtual slides. Aortic diameters were determined on calibrated photos using a custom-made software, developed in MATLAB (MathWorks, USA). Intima and media thicknesses were determined on virtual slides using a dedicated image analysis software. Results and Discussions: The most frequent CV causes of death were the ischemic heart diseases and the most frequent NCV causes of death were the inflammatory diseases. Aortic diameter decreased from the aortic origin till the aortic end, with larger values in women than in men and in CV diseases than in NCV diseases. The difference in the remodeling of the aortic diameter between the two groups is smaller towards the abdominal region. Intima thickness increased from the aortic origin till the aortic end and was larger especially in women died of CV diseases, whereas in men there were some shifts at the extremities of the aorta. The difference in the remodeling of the intimal thickness between the two groups is extremely variable. Media was thicker in almost all of its segments in CV group than in NCV. It was a divergent evolution of the correlation degree trends in the two groups. Conclusions: The three morphological parameters of the aorta (diameter, intima, and media thicknesses) are more or less influenced by the pathological status that caused patient s death by the patient s sex and by the topographic region where the measurement was made.

Corresponding author: Iancu Emil Plesea, Professor, MD, PhD; e-mail: emil.plesea@umfcd.ro

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4. HOXB13 and TFF3 can contribute to the prognostic stratification of prostate adenocarcinoma

Andrei Daniel Timofte, Simona Eliza Giusca, Ludmila Lozneanu, Mariana Bianca Manole, Iulian Prutianu, Bogdan Gafton, Andreea Rusu, Irina-Draga Caruntu

Homeobox B13 (HOXB13) and trefoil factor 3 (TFF3) are novel candidates for the classification of prostate cancer (PC) in molecular subtypes that could predict the clinical evolution of patients. The aim of our study was to analyze the possible associations between HOXB13 and TFF3 immunohistochemical (IHC) expression in sporadic prostate adenocarcinoma (PAC), the potential prognostic value in relation to the classical clinico-pathological parameters, as well as their role in defining distinct molecular subtypes of this malignancy. The study group comprised 105 patients diagnosed with PAC who underwent radical prostatectomy. IHC exam was performed using anti-HOXB13 and anti-TFF3 antibodies and a scoring system that permit the separation of the cases into two subgroups, with low and high immunoexpression, respectively. The statistical analysis evaluated the relationship between the two immunomarkers and clinico-pathological parameters. The Kaplan-Meier curves and log-rank Mantel-Cox test were used for assessing the prostate-specific antigen (PSA)-progression free survival. Four subgroups of PAC were defined based on the IHC overexpression and low immunoexpression of HOXB13 and TFF3. High HOXB13 and TFF3 immunoexpression was commonly identified in cases characterized by a Gleason score over 7, a G4 or G5 dominant pattern, a grade group of 3 or 4 and a preoperatory PSA serum level over 20 ng/mL. HOXB13 overexpression was also associated with pathological tumor-node-metastasis (pTNM) stage. The subgroup with both low HOXB13 and TFF3 immunoexpression had the highest PSA-progression free interval, whereas the subgroup with high HOXB13 immunoexpression and low TFF3 immunoexpression presented the lowest rate, but no statistically significant differences were registered. Our results sustain the role of HOXB13 and TFF3 in the stratification of PAC. Further investigations in larger cohorts are imposed to validate the clinical significance of these subgroups in the diagnostic and prognostic of PAC.

Corresponding author: Simona Eliza Giusca, Lecturer, MD, PhD; e-mail: simonaelizagiusca@gmail.com

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5. Research algorithm for the detection of genetic patterns and phenotypic variety of non-syndromic dental agenesis

Cristina-Crenguta Albu, Romina-Christiana Pavlovici, Marina Imre, Ana Maria Cristina Tancu, Ioana Andreea Stanciu, Adriana Vasilache, Stefan Milicescu, George Ion, Stefan-Dimitrie Albu, Mihaela Tanase

Introduction: Dental agenesis (DA), brings together the anodontia, oligodontia, hypodontia, characterized by a deficit in the development of a variable number of teeth. The objectives of the study were to illustrate the phenotypic variability of non-syndromic DA, to identify cases of DA with hereditary genetic transmission, and establish the mode of DA genetic pattern in these cases, together with the determination of DA prevalence in the population group study. Patients, Materials and Methods: The cross-sectional observational study was performed on a mixed population group, consisting of 861 Caucasian patients, between January 2018-December 2019. The clinical evaluation protocol of patients with DA, used to illustrate their phenotype, included the following stages: oral examination, photographic examination, and radiological examination. The evaluation protocol specific to the family genetic study of patients with DA, involved the following three stages: family survey, construction of the family tree and analysis of the pedigree structure. Results: The prevalence of DA in the population group was 2.78%. Regarding the phenotype, DA mainly affected the upper arch (50% of cases); bilateral DA had a significantly increased incidence (83.33% of cases) compared to unilateral form; in most cases (75%), a patient lacked one to two teeth, the lack of two teeth being the most common form (83.33% of cases); the upper lateral incisors were the teeth most frequently involved in DA (31.11% of the total missing teeth). Regarding the family genetic study, hereditary DA with autosomal dominant inheritance was present in 37.50% of cases. In the other cases (62.50%), isolated, sporadic forms of DA were registered, suggesting a spontaneous de novo mutation or a disorder of odontogenesis of a non-genetic nature. Conclusions: We consider that this study is of interest for current scientific research with applicability in dental medicine, by bringing actual information on the prevalence of non-syndromic DA in South-East Romania, the variety of phenotypic spectrum of DA for this geographic area, and the role of heredity in the DA genetic determinism in the studied population.

Corresponding author: Ana Maria Cristina Tancu, Associate Professor, DMD, PhD; e-mail: amctancu@yahoo.com; Stefan-Dimitrie Albu, Assistant Professor, DMD, PhD Student; e-mail: stevealbu@yahoo.com

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6. Assessment of programmed death-ligand 1 receptor immunohistochemical expression and its association with tumor-infiltrating lymphocytes and p53 status in triple-negative breast cancer

Mariana Deacu, Liliana-Ana Tuta, Madalina Bosoteanu, Mariana Aschie, Anca Florentina Mitroi, Antonela-Anca Nicolau, Manuela Enciu, Oana Cojocaru, Lucian Cristian Petcu, Gabriela Izabela Baltatescu

Breast cancer (BC) is the second most frequent type of cancer for both sexes combined, after lung cancer. Triple-negative BC (TNBC) molecular subtype is characterized by lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) immunoexpression or amplification and represent 10-20% of all BC cases. The issue of the present study was to analyze the associations between programmed death-ligand 1 (PD-L1) immunoexpression and distribution of stromal tumor-infiltrating lymphocytes (stTILs) combined with clinico-morphological features of patients with TNBC. Secondly, our research evaluated PD-L1 immunoexpression as a prognostic factor and its correlation with p53 immunoexpression. Thirty cases with primary TNBC without prior neoadjuvant therapy were included in this research. stTILs were identified in all cases, most of them with low distribution (66.7%). A positive immunoreaction for PD-L1 was observed in 40% of cases. The PD-L1 immunoexpression was statistically significant associated with age, pathological tumor size, lymphovascular invasion, stTILs level, the presence of cluster of differentiation 8-positive (CD8+) TILs and p53 immunoexpression. In the present study, a positive PD-L1 immunoexpression was associated with a worse distant metastasis free survival (DMFS). We also found not only that high stTILs level were associated with a better DMFS but also that there was a statistically significant association between stTILs level and PD-L1 immunoexpression. Our results bring new insights to the fine connections between tumor microenvironment and molecular changes of TNBC. It helps us to better understand these aggressive tumors to identify the more useful biomarkers for predicting the response to adjuvant therapy and can represent a method for selecting the most suitable patients for immunotherapy.

Corresponding author: Liliana-Ana Tuta, Professor, MD, PhD; e-mail: tutaliliana@yahoo.com; Gabriela Izabela Baltatescu, MD, PhD; e-mail: gabrielabaltatescu@yahoo.com

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7. Thymus pathology in myasthenia gravis with anti-acetylcholine receptor antibodies and concomitant Hashimoto s thyroiditis. A four-case series and literature review

Lidia Ionescu, Claudia Florida Costea, Radu Danila, Camelia Margareta Bogdanici, Dragos Viorel Scripcariu, Roxana Maria Livadariu, Gabriela Florenta Dumitrescu, Cristina Maria Gavrilescu, Raluca Alina Dragomir, Anca Sava, Andrei Ionut Cucu, Daniela Maria Tanase, Mihaela Dana Turliuc, Delia Gabriela Ciobanu Apostol

Objective: Identifying the morphological features of thymus in patients with myasthenia gravis (MG) with anti-acetylcholine receptor (AChR) antibodies and concomitant Hashimoto s thyroiditis (HT), which were recruited from a single surgical unit of a tertiary referral hospital located in the North-Eastern region of Romania, over a period of 11 years. Patients, Materials and Methods: We retrospectively reviewed clinical, imaging, laboratory, thymic pathology, and outcome data that were obtained from medical records of patients with MG and concomitant HT, to whom a thymectomy was performed for a suspected thymic lesion. All the surgical interventions were done in the Third Clinic of Surgery, St. Spiridon Emergency County Hospital, Iasi, Romania, for an 11 years period, i.e., from January 1, 2000 and December 31, 2010. Results: Four patients (three females and one male) were included. The mean age of the patients at the time of their thymectomy was 40.25 years. Of all patients, 75% had moderate or severe MG, 100% had anti-AChR antibodies, and an electromyographic decrement greater than 25%. All patients have been diagnosed with HT in their past medical history by a full thyroid panel [high thyroid-stimulating hormone (TSH) values, low free thyroxine (fT4) values, and the presence of the anti-thyroid antibodies] and all of them have been treated with Euthyrox. Our four patients expressed different MG subtypes, each of them being associated with different thymus pathology. Thoracic computed tomography (CT) scan revealed heterogeneous mediastinal masses and established the correct diagnosis only in 25% of cases. The pathological exams also revealed a heterogeneous pattern of thymic lesions. In contrast with other studies, our patients with MG with anti-AChR antibodies and concomitant HT presented atrophic thymus more frequently (50%), but with particular morphological changes of Hassall s corpuscles. Also, 25% of cases were diagnosed with thymic lympho-follicular hyperplasia (TLFH) associated with thymic epithelial hyperplasia. In B2 thymoma, neoplastic epithelial cells expressed cytokeratin 19 (CK19) immunoreactivity, high Ki67 labeling index and strong p63 immunopositivity. Conclusions: In our series, MG and HT occurred simultaneously, or one of them was diagnosed before the other, raising some new questions regarding the immune mechanism of these two autoimmune diseases. Due to the heterogeneous morphological changes of the thymus that we found in this study, we can hypothesize that thymus is involved in the pathogenic mechanism of MG with anti-AChR-antibodies and concomitant HT development.

Corresponding author: Claudia Florida Costea, Associate Professor, MD, PhD; e-mail: costea10@yahoo.com; Camelia Margareta Bogdanici, Professor, MD, PhD; e-mail: camelia.bogdanici@umfiasi.ro

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8. Assessment of the aortic wall histological changes with ageing

Mirela Albu, Doru Adrian Seicaru, Razvan Mihail Plesea, Oana Cristina Mirea, Florentina Gherghiceanu, Valentin Titus Grigorean, Ioan Cordos, Mircea Litescu, Iancu Emil Plesea, Mircea-Sebastian Serbanescu

Aim: The authors aimed to quantitatively assess the variation with age of three of the main components of the aortic wall, namely elastic fibers (FE) and collagen fibers [FC(COL)], and smooth muscle cells (SMCs). Materials and Methods: Four aortic cross sections (base, cross, thoracic, abdominal) were collected during autopsy from 90 cases of all ages, processed using the classical histopathological (HP) technique (formalin fixation and paraffin embedding) and stained with Orcein and Goldner s trichrome. The obtained histological slides were transformed into virtual slides. Quantitative measurements of the three components were made on identical regions of interest (ROIs) selected from two-paired slides stained with the above-mentioned techniques using custom-made software, developed in MATLAB (MathWorks, USA). Results and Discussions: FE revealed an obvious decreasing general trend with age, present at all four levels of investigation. Smooth muscle fibers (FM) density showed almost no variation with age regardless of the level at which the measurement was made. FC(COL) density had an obvious increasing trend with age, expressed in all four aortic regions FE densities and FM densities were higher in men than in women, while FC(COL) densities were higher in women than in men in three of the aortic regions excepting, in all cases, the cross region. Conclusions: There were differences between men and women concerning the composition of aortic wall cellular and extracellular compartments. FE and FC(COL) dominated the age-related remodeling process of the aortic wall. The process evolved in the same way in all regions of the aorta. Studies need to be continued to define more clearly this complex process of vascular wall remodeling with aging.

Corresponding author: Iancu Emil Plesea, Professor, MD, PhD; e-mail: emil.plesea@umfcd.ro

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9. Virtual autopsy and confirmation of normal fetal heart anatomy in the first trimester using three-dimensional (3D) reconstruction of histological sections

Dan Ruican, Ana-Maria Petrescu, Anda Laura Ungureanu, Marius Cristian Marinas, Daniel Pirici, Anca-Maria Istrate-Ofiteru, Gabriela-Camelia Rosu, Anne Marie Badiu, Cristiana Eugenia Simionescu, Mircea-Sebastian Serbanescu, George-Lucian Zorila, Smaranda Belciug, Dominic-Gabriel Iliescu

Objective: In this pilot study, we tested the feasibility of cardiac structures reconstruction from histological sections in 12-13 weeks normal fetuses. Conventional autopsy is hampered at this gestational age because of the small size of the heart anatomical structures, while alternative non-invasive methods for pathology examination of the fetus are expensive, rarely available and lack accuracy data regarding the confirmation of first trimester heart defects suspected by early prenatal ultrasound (US) scans. Materials and Methods: Normal hearts from fetuses aged 12-13 gestational weeks (GW) were harvested for histological preparation, virtual reconstruction, and cardiac structures analysis. The normalcy of heart structures was confirmed before pregnancy termination, using a detailed US scan protocol. The fetal heart was routinely processed for formalin fixation and paraffin embedding (FFPE) and 10 micrometer seriate sections have been cut until finishing the specimen. All sections have been scanned and a three-dimensional (3D) reconstruction of the whole organ has been rendered, based on computer-aided manual tracing. Using the 3D navigation software, the main cardiac structures were searched for a proper and confident visualization. Results: Five cases were investigated. Visualization of the normal heart cavities, including atrioventricular septum was very good in all fetuses. The entire course of right and left ventricle outflow tracts was confidently confirmed, along the branching pattern of aorta and pulmonary artery trunk. Regarding the veno-atrial connections, it was easy to identify the entrance of the inferior and superior caval veins into the right atrium, but a detailed review of the histological sections was necessary for the visualization of the left atrium venous openings. The inherent morphological deformation following heart block sectioning resulted in a lower resolution or quality of the reconstructed planes, but these distortions did not represent a significant impediment in any of the cases. The resources involved ordinary histology and information technology (IT) equipment. To further decrease the time involved by the protocol, many steps may be automated: cutting, coloring, and scanning. Conclusions: The results indicate that this method can be implemented to routine clinical practice. The use of 3D reconstruction of fetal heart histological sections in first trimester may serve as an important audit to confirm the normalcy of heart structures. Also, the histological and postprocessed information is retained, and this volume can be stored, reanalyzed, or sent online for a second opinion. The method involves relatively undemanding resources, i.e., hardware, software, competences, and time. The procedure could also benefit from refinements used in other imaging techniques to limit human-computer interactions, such as sections distortion.

Corresponding author: Anda Laura Ungureanu, MD; e-mail: ungureanu_anda@yahoo.com; Mircea-Sebastian Serbanescu, Lecturer, MD, PhD; e-mail: mircea_serbanescu@yahoo.com

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10. Inflammatory status of the pancreas in NOD mice that do not develop overt diabetes

Ana-Maria Catrina, Mirel Adrian Popa, Ana-Maria Vacaru, Ioana Madalina Fenyo

Type 1 diabetes (T1D) is an autoimmune disease in which immune cells target the pancreatic islets and destroy the beta-cells, resulting in hyperglycemia and decreased plasmatic insulin levels. The non-obese diabetic (NOD) mouse is the most used animal model for studying diabetes because it spontaneously develops T1D and shares similarities with the human disease. A hallmark feature of this model is the appearance of insulitis, defined as an inflammatory cell infiltration of the pancreatic islets. However, a small percentage of NOD mice do not develop overt diabetes even after 28-35 weeks of age. Thus, we questioned the status of the pancreatic islets in these non-diabetic NOD mice, with particular focus on islet inflammation and plasmatic insulin levels, in comparison to pre-diabetic (11 weeks old) and new-onset diabetic mice. Diabetes progression was evaluated by assessing blood glucose and pancreas histology. The inflammatory score was determined on Hematoxylin-Eosin (HE)-stained sections of pancreas. Plasma insulin was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that inflammation increased in an age-dependent manner in all mice, irrespective of their diabetic status. Mostly affected within the analyzed groups were the 28 weeks old non-diabetic NOD mice, in which insulin production was reduced and inversely correlated with the inflammatory status. We conclude that in NOD mice, pancreatic inflammation progresses independently of diabetes onset and clinical signs of disease. Most likely, the NOD females that do not develop overt diabetes preserve a small mass of functional beta-cells, which is able to provide the physiological insulin levels and avoid diabetes onset.

Corresponding author: Ioana Madalina Fenyo, PhD; e-mail: madalina.fenyo@icbp.ro

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11. Pathological and immunohistochemical study of colon cancer. Evaluation of markers for colon cancer stem cells

Daniel Sorin Ilie, George Mitroi, Ion Paun, Tiberiu Stefanita Tenea-Cojan, Carmen Neamtu, Bogdan Dan Totolici, Konstantinos Sapalidis, Stelian Stefanita Mogoanta, Adrian Murea

Colorectal cancer is a major public health problem worldwide with increasing morbidity and mortality. Numerous exogenous and endogenous factors are involved in colorectal carcinogenesis: age, sex, diet, smoking, alcohol consumption, exposure to harmful environmental factors, intestinal microbiota, bacterial and viral infections, the ability of the host immune system to respond, genetic factors, etc. The present study analyzed histopathologically and immunohistochemically a number of 36 cases of colorectal adenocarcinomas. The existence of an accentuated cell pleomorphism was noted, which corresponds to different clones of tumor cells, in the same tumor coexisting aspects of tubular adenocarcinoma, mucinous areas and even signet-ring cell. The tumor stroma was mainly of the desmoplastic type, but also of the lax type, more or less infiltrated with inflammatory cells. Evaluation of immunomarkers for cancer stem cells (CSCs) showed that none of the markers used alone [cluster of differentiation (CD)133, CD44, aldehyde dehydrogenase 1 family member A1 (ALDH1A1), CD24, CD26] show CSCs.

Corresponding author: Daniel Sorin Ilie, MD, PhD Student; e-mail: dr.daniel.ilie@gmail.com

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12. T4 retroperitoneal liposarcoma. Challenges of big size sarcomas surgical treatment

Codrut Cosmin Nistor-Ciurba, Ioan Cosmin Lisencu, Tiberiu Florin Tat, Alexandra-Ioana Andries, Bogdan Fetica, Calin Ioan Cainap, Dan Tudor Eniu

Our article presents some of the challenges of the surgical treatment of T4 (>15 cm) retroperitoneal liposarcomas (up to 65/56/30 cm, 25.5 kg) series of cases treated by the Department of Surgical Oncology, Prof. Dr. Ion Chiricuta Oncology Institute, Cluj-Napoca (IOCN), Romania, with illustrations, insisting on important blood vessels and nerves dissection and preservation and discussions of strategies with references to important articles from the last 10 years specialty literature. Challenges do not come only from intraoperative difficulties but also from establishing the right attitude from the extent of resection and oncological safety point of view, the role of the pathologist being very important because histological subtype and completeness of the resections are the most important prognostic factors for such tumors. Despite all today available aids in decision making, like nomograms or high-resolution imagery, sometimes this decision is to be taken intraoperative based on surgeon s expertise and skills. That is why is strongly advised that such cases to be treated in high-volume specialized tertiary centers of surgical oncology.

Corresponding author: Ioan Cosmin Lisencu, MD, PhD; e-mail: cosminlisencu@yahoo.com

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13. Immunohistochemical evaluation of D2-40, Galectin-3, Maspin and MCM7 expression in palate squamous cell carcinomas

Adrian Patru, Valeriu Surlin, Claudiu Margaritescu, Eduard Mihai Ciuca, Marius Matei, Daniela Dumitrescu, Adrian Camen

Squamous cell carcinoma (SCC) is the most frequent cancer in oral cavity and its prognosis has exhibited little improvement in the last decades. Although much less common palate SCCs manifests a higher local aggression invading very quickly the adjacent muscles and jawbones, thus being able frequently to lead to dysfunctions in chewing, swallowing, and speech. To elucidate what underlies such local aggression, we investigated the immunohistochemical expression in palate SCCs of Podoplanin (D2-40), Galectin-3 (Gal-3), mammary serine protease inhibitor (Maspin) and minichromosome maintenance complex component 7 (MCM7), markers that are known to be involved in tumor invasiveness. We found a progressive increase in reactivity for D2-40 and MCM7 from the normal epithelium toward dysplastic epithelium and respectively to SCC, which suggests the intervention of these markers in the early stages of squamous cell carcinogenesis in the palate. The highest D2-40, Gal-3 and MCM7 reactivity was observed in basaloid and in poorly differentiated (G3) palate SCCs, while for Maspin the well-differentiated (G1) palate SCCs were the most reactive. The first three markers mentioned above were most intensely expressed at the invasion front, while the Maspin reactivity was low or absent at this level. Statistically, we found significant stratification on localization, grading, muscle invasion, and survival for all investigated markers, but with very high direct correlations between D2-40, Gal-3, and MCM7 immunoreactive score (IRS) values, while between the Maspin and each of the previous markers there were very high inverse correlations. Overall, all these investigate markers proved to be responsible for the local invasiveness and regional lymph node metastasis, thus allowing a prognostic and therapeutic stratification of patients with palate SCCs.

Corresponding author: Claudiu Margaritescu, Professor, MD, PhD; e-mail: c_margaritescu2000@yahoo.com; Daniela Dumitrescu, Associate Professor, MD, PhD; e-mail: daniela.dumitrescu@gmail.com

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14. Immunohistochemical expression of chemokine receptor in neuroendocrine neoplasms (CXCR4) of the gastrointestinal tract: a retrospective study of 71 cases

Oana Popa, Sorina Maria Taban, Alis Liliana Carmen Dema, Andrei Dorel Plopeanu, Robert Alexandru Barna, Marioara Cornianu, Sorin Dema

Aim: C-X-C motif chemokine receptor 4 (CXCR4) is expressed in many tumor entities, including gastrointestinal neuroendocrine neoplasms (GI-NENs). However, the role of CXCR4 expression in GI-NENs has been less studied. Our objective was to investigate the expression of CXCR4 in a series of GI-NENs with various clinical and pathological features. Methods: The immunohistochemical (IHC) expression of CXCR4 (clone UMB2) was examined in 71 GI-NENs and a semiquantitative immunoreactivity score (IRS) was calculated taking into consideration the intensity of the IHC reaction and the percentage of the tumor cells which showed positive expression. Results were compared with several clinical and pathological prognostic factors. Results: High CXCR4 expression was noted in 31 (43.7%) cases. Low IRS values were more frequent in NENs from the small intestine (66.7%) and stomach (60%). Also, all appendix tumors had IRS value of zero. High CXCR4 expression was noticed in 52.5% of liver metastases, compared to 40.4% primary tumors. A significant relationship was observed between the CXCR4 expression and the tumor grade (p=0.0216), and high IRS value was correlated with clinical stages III and IV (p=0.0142) and lympho-vascular invasion (p=0.0129). 74.1% of G1 neuroendocrine tumors (NETs) had a low IRS, G3 NETs showed minor differences between low (42.9%) and high (57.1%) expression and 66.7% of neuroendocrine carcinomas (NECs) presented high expression of CXCR4. Conclusions: The present study highlighted that high CXCR4 expression is associated with high grade and advanced stage GI-NENs, as well as with metastatic cases. In these cases, high CXCR4 expression could serve as an important target for CXCR4 antagonists.

Corresponding author: Sorina Maria Taban, Associate Professor, MD, PhD; e-mail: sorinataban@yahoo.com

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15. Nanoparticle-functionalized dressings for the treatment of third-degree skin burns - histopathological and immunohistochemical study

Ilona Mihaela Liliac, Elena Leocadia Popescu, Ion Alexandru Vaduva, Daniel Pirici, George Dan Mogosanu, Costin Teodor Streba, Cristina Jana Busuioc, Ludovic Everard Bejenaru, Cornelia Bejenaru, Nicolae Craciunoiu, Ilie Dumitru, Hazim Elayan, Laurentiu Mogoanta

Skin burns are one of the most common injuries associated with increased morbidity and mortality, especially in the children and the elderlies. Severe burns, especially, result in a systemic immune and inflammatory response, which may reflect in multiple organ insufficiency, and a fast and effective local restorative process is essential for functionality recovering, as well as for interrupting the generalized systemic response. We have aimed here to assess the effect of different wound dressings in what it regards the morphology and clinical restoration after a skin burn. On a rat animal model, we have evaluated the macroscopic and histopathological features of controlled third degree skin burns in control animals versus treatments with local dressings of silver sulfadiazine (SDA) cream, simple gel (G), gel + silver nanoparticles (AgNPs) (G+NPS), gel + exosomes (G+EXO) and gel + AgNPs + exosomes (Gel+NPS+EXO), at 14 days and, respectively, 21 days after the lesion. Tissue fragments were harvested and processed for histopathology and immunohistochemistry. Immunofluorescence was utilized to evaluate the maturity of underlaying granulation tissue based on double stainings for smooth muscle actin (SMA) and cluster of differentiation 31 (CD31). Our study showed variability in what it regards the vessel density and immunoexpression of SMA between the treatments, and image analysis revealed that most SMA reduction and blood vessel density reduction in the maturing granulation tissue occurred for the G+NPS and G+NPS+EXO treatments. A complete re-epithelization was also observed for the G+NPS+EXO treatment. Overall, our results show that improved topic treatments promote faster re-epithelization and reparation of the dermis after skin burn lesions, providing thus an avenue for new treatments that aim both local recuperation and systemic infection prevention.

Corresponding author: Elena Leocadia Popescu, MD, PhD Student; e-mail: popescu.elena88@yahoo.com

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16. Protective effects of Rosemary extract and/or Fluoxetine on Monosodium Glutamate-induced hippocampal neurotoxicity in rat

Reham Mohammed Atef, Islam Omar Abdel Fattah, Omayma Mahfouz Mahmoud, Gamal Mohamed Abdel-Rahman, Noha Ahmed Salem

The use of Monosodium Glutamate (MSG) as a food flavor enhancer is increasing worldwide despite its neurotoxic effects. Fluoxetine (FLX) and Rosemary extract (RE) are known to have beneficial neuroprotective properties. Rats were divided into five groups: control group; MSG group, rats received 2 g/kg/day intraperitoneal (i.p.) injections of MSG for seven days; RE/MSG group, rats received 50 mg/kg/day of oral RE for 28 days starting prior to MSG; FLX/MSG group, rats received 10 mg/kg/day of oral FLX for 28 days beginning before MSG; and RE/FLX/MSG group, received combined treatments as mentioned above. Rats underwent the Barnes maze test, in addition to histopathological, immunohistochemical, morphometric and ultrastructural evaluations for their hippocampi. MSG increased the number of errors and escaped latency in the Barnes maze test that was significantly minimized in the three treatment groups. The MSG group exhibited pyramidal cell (PC) degeneration, shrunken glial cells and massive vascular dilatation that were improved with RE and/or FLX treatment. The number of glial fibrillary acidic protein (GFAP)-immunopositive cells were increased, and the number of PCs was decreased in the MSG group, while these values were significantly reversed with the three treatment groups with the most significant improvement at RE/FLX/MSG one. Ultrastructurally, PCs were shrunken with degenerated nuclei, dilated endoplasmic reticulum, swollen mitochondria, and vacuolations in the MSG group that were improved with RE and/or FLX. In conclusion, the combined RE and FLX treatment can ameliorate the toxic effect of MSG on rat hippocampus probably through its antioxidant and anti-inflammatory effects.

Corresponding author: Islam Omar Abdel Fattah, Lecturer; e-mail: islam_omar2007@yahoo.com

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17. Carvacrol treatment opens Kir6.2 ATP-dependent potassium channels and prevents apoptosis on rat testis following ischemia-reperfusion injury model

Cemre Nur Balci, Tulin Firat, Nuray Acar, Aysel Kukner

Testicular torsion is a urological problem that causes subfertility and testicular damage in males. Testis torsion and detorsion lead to ischemia-reperfusion (IR) injury in the testis. Testicular IR injury causes the increase of reactive oxygen species (ROS), oxidative stress (OS) and germ cell-specific apoptosis. In this study, we aimed to investigate whether Carvacrol has a protective effect on testicular IR injury and its effects on Kir6.2 channels, which is a member of adenosine triphosphate (ATP)-dependent potassium channels. In the study, 2-4 months old 36 albino Wistar rats were used. For experimental testicular IR model, the left testis was rotated counterclockwise at 720 degrees for two hours, and after two hours following torsion, detorsion was performed. Carvacrol was dissolved in 5% Dimethyl Sulfoxide (DMSO) at a dose of 73 mg/kg and half an hour before detorsion, 0.2 mL was administered intraperitoneally. In testicular tissues, caspase 3 and Kir6.2 immunoexpressions were examined. Serum malondialdehyde (MDA) and testosterone levels were measured. Apoptotic cells and serum MDA levels were significantly decreased and Kir6.2 activation was significantly increased in Carvacrol-administrated IR group. As a result of our study, Carvacrol may activates Kir6.2 channels and inhibits apoptosis and may have a protective effect on testicular IR injury.

Corresponding author: Cemre Nur Balci, MSc; e-mail: balcicemrenur@gmail.com

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18. Current opportunities and new horizons into the genetic study of infertility

Elena-Silvia Nada, Dinu-Florin Albu, Anca Patrascu, Stefan-Dimitrie Albu, Alexandru Marian Goganau, Cristina-Crenguta Albu

Introduction: An estimated 12.5% of couples experiencing fertility problems and almost 12% of reproductive age women have turned to health services at least once due to infertility. First trimester miscarriage is the most common clinical manifestation of infertility associated with a genetic cause. Patients, Materials and Methods: The scientific research was conducted at A.S. Medical Center in Bucharest, Romania, between January 2016 and December 2018, on a representative group of 1264 Caucasian patients diagnosed with infertility, from which the study group was selected, consisting of 273 patients who were further genetically investigated. Results: Chromosomal instability, identified in 14% of patients, has been encountered most frequently in women (7%), and least often in fetuses (2%), unlike other chromosomal anomalies, identified in 55% of patients, which were more common in fetuses (27%) and least frequently in men (9%). Recurrent pregnancy loss due to genetic causes was identified in 53% of cases, being determined by chromosomal instability in 16% of cases and by other chromosomal anomalies in 37% of cases. Infertility due to a genetic cause was identified in 83% of cases, being determined by chromosomal instability in 17% of cases and by other chromosomal anomalies encountered in 66% of cases. In genetic risk pregnancies in evolution, fetal chromosomal anomalies were detected in 94% of cases, the most frequent being aneuploidy and polyploidy. Cytogenetic studies carried out on tissue fragments taken from aborted products of conception revealed the presence of a genetic cause in 57% of cases, an abnormal chromosome number being the most common (36%). The analysis of microdeletions of the long arm of the Y chromosome indicated that 5.5% of men with infertility are affected by this condition. Conclusions: Although genetic tests are considered complex and expensive laboratory investigations, they are crucial in identifying the etiology of over 40% of infertility cases associated with genetic factors, as well as in the correct and effective management of infertility.

Corresponding author: Cristina-Crenguta Albu, Associate Professor, MD, PhD; e-mail: crenguta.albu@yahoo.com; Stefan-Dimitrie Albu, DMD, PhD Student; e-mail: steve.albu@yahoo.com

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19. The immunoexpression of epidermal growth factor receptor in cutaneous squamous cell carcinoma

Mirela Marcela Nichita, Calin Giurcaneanu, Mara Madalina Mihai, Mihaela Ghigulescu, Cristina Beiu, Silvius Ioan Negoita, Liliana Gabriela Popa

Although cutaneous squamous cell carcinomas (cSCCs) account for only 20-25% of non-melanoma skin cancers (NMSCs), they are responsible for most deaths attributable to NMSCs. Apart from SCC seric level, which increases in late-stage disease, no other predictive biomarker for cSCC exists. Epidermal growth factor receptor (EGFR) serves as a predictive biomarker and therapeutic target in numerous malignancies. EGFR immunoexpression is highly elevated in head and neck mucosal SCC. However, its immunoexpression pattern, its relationship with prognosis and survival, and the effect of EGFR targeted therapy in advanced cSCC have not been clarified. We assessed EGFR immunoexpression in 18 cases of cSCC and correlated our findings with the clinicopathological features. Immunohistochemical stainings with anti-EGFR monoclonal antibodies were practiced and the membrane and cytoplasmic immunostaining intensity and quality in the tumors and the non-lesional epithelium were analyzed. Membrane EGFR immunoexpression within the tumors increased with the tumor grade. EGFR overexpression was more frequently found in head and neck cSCCs. We did not find a direct relationship between cytoplasmic EGFR immunoexpression and clinicopathological findings and prognosis. Our results confirm that increased EGFR immunoexpression correlates with aggressive cSCC phenotypes and underline the need for novel treatments for these patients.

Corresponding author: Mara Madalina Mihai, Assistant Professor, MD, PhD; e-mail: mara.mihai@umfcd.ro

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20. Histopathological and immunohistochemical study of periodontal changes in chronic smokers

Gabriel Valeriu Popa, Adrian Costache, Oana Badea, Melania Olimpia Cojocaru, George Mitroi, Adela Cristina Lazar, Daniel-Alin Olimid, Laurentiu Mogoanta

Periodontal disease is a chronic inflammatory, multifactorial condition, that, in the absence of an early and adequate treatment, may lead to a progressive damaging of the alveolar tissues that support the teeth (periodontal ligament, cement and alveolar bone) followed by teeth mobility and, subsequently, their loss. Periodontal disease is one of the most common inflammatory disease affecting adult individuals all over the world, being considered a real worldwide pandemic. This disease may influence the progression of certain systemic diseases: diabetes mellitus, cardiovascular diseases, ischemic cardiomyopathy, myocardial infarction, stroke, neurodegenerative diseases, chronic kidney diseases, cancer, etc. The association between smoking and periodontal disease was described in numerous clinical and epidemiological studies, suggesting that products derived from tobacco burning may change the clinical aspects and the disease progression. The present study analyzed microscopically and immunohistochemically 58 periodontal fragments, from 50 patients, chronic smokers, clinically diagnosed with severe periodontitis. There were highlighted major changes in the gingival epithelium (epithelium thickening, acanthosis, intraepithelial edema, infiltrates of neutrophils or lymphocytes, epithelial necrosis), in the periodontal conjunctive tissue (more or less intense inflammatory infiltrates, microhemorrhages, vascular congestion, intense immunohistochemical expression for some matrix metalloproteinases). The periodontal changes may be the expression of both toxic factors present in tobacco smoke and due to the changes caused by tobacco in the microbial flora of the oral cavity.

Corresponding author: Laurentiu Mogoanta, Professor, MD, PhD; e-mail: laurentiu_mogoanta@yahoo.com

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21. Preclinical and histological study of boron-containing compounds hydrogels on experimental model of periodontal disease

Ioana Mitrut, Melania Olimpia Cojocaru, Ion Romulus Scorei, Andrei Bita, George Dan Mogosanu, Mihai Popescu, Daniel-Alin Olimid, Horia Octavian Manolea

Periodontitis is a disease that affects a wide group of people, and there has been an increased interest in the research of finding useful materials that help reduce inflammation and the further loss of tissue. In this study, we have tested a boron-containing compound (BCC) Calcium Fructoborate (CaFB) and Boric Acid (BA) hydrogels on the gingival level on Wistar rats. First, we have induced the periodontal disease at the lower incisors, we have applied the hydrogels and after a week, we have euthanized the rats. Next, the oral soft tissue reaction was clinically and then histologically investigated. Our study has shown good clinical response of the oral tissue, and we have noticed lower levels of inflammation on the experimental groups treated with the BCCs hydrogels. Despite the generally good response of the biological structures to the presence of BA and CaFB on periodontal level, more scientifically proved information is needed to obtain the desired biological responses in all clinical situations.

Corresponding author: Ion Romulus Scorei, Professor, Biochem, PhD; e-mail: romulus_ion@yahoo.com

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22. The sinus septi nasi and other minor pneumatizations of the nasal septum

Alexandru Nicolae Muresan, Carmen Aurelia Mogoanta, Ruxandra Stanescu, Mugurel Constantin Rusu

The osseous nasal septum (NS) consists of the perpendicular plate of the ethmoid bone (PPE) and the vomer bone. Few studies evaluated the possibilities of septal pneumatization of the PPE, or adjacent to it. We aimed to observe the anatomical possibilities of NS pneumatizations. A retrospective lot of cone-beam computed tomography (CBCT) files was used. One hundred seventy-one CBCT files from 51 males and 120 females were documented. There were found 46 files that were null for septal pneumatization. The other cases (73.1%) had different septal pneumatizations extended from neighboring air spaces. Pneumatized crista galli (CG) exclusively extended from a frontal sinus was found in 7.01% of cases. The frontal sinuses had minor extensions anterior to the PPE in 7.6% of cases. Unique or double pneumatizations of the sphenoidal rostrum extending within the posterior part of the PPE were detected in 71.34% of cases. In six cases were found ethmoidal pneumatizations of the PPE, either from an anterior ethmoid cell, or from a posterior one, or from a pneumatized CG. In this last case was found a sinus septi nasi of 25.37 mm sagittal size. The supra-septal recesses of the ethmoid air cells were roofing the respective nasal fossa. As all the morphological possibilities of NS pneumatization involve the upper part of the PPE, they should be adequately discriminated anatomically, as well as when the NS and the cribriform plate of the ethmoid bone are approached surgically.

Corresponding author: Mugurel Constantin Rusu, Professor, Dr.Med., Dr.Biol., Dr.Hab.; e-mail: mugurel.rusu@umfcd.ro

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23. Clinical, histopathological and immunohistochemical features of glomus tumor of the nail bed

Mihaela Pertea, Vladimir Poroch, Natalia Velenciuc, Mihaela Mitrea, Sorinel Lunca, Cristina Terinte, Alexandru Filip, Ovidiu Alexa, Bogdan Veliceasa

Purpose: Glomus tumors account for 1-4% of benign hand tumors. In 65% of cases, it is located in the nail bed. Its rarity makes misdiagnosis problems relatively common. Symptomatology is characterized by the hallmark symptomatic triad. Imaging investigations may guide the diagnosis, but the diagnosis is made by pathological examination doubled by immunohistochemical (IHC) markers. Patients, Materials and Methods: We studied a group of seven female patients, aged 28 to 56 years. Clinical examination revealed the presence of the characteristic symptomatic triad. Ultrasound imaging tests were performed. Results: Anatomopathological examination made a diagnosis of glomus tumor in all seven cases. IHC staining showed that tumor cells were positive for alpha-smooth muscle actin (alpha-SMA) and h-caldesmon in all seven cases and negative for cluster of differentiation 34 (CD34) in 72.14%. IHC stainings for p63, S100, cytokeratin (CK) AE1/AE3 were negative in all cases. The clinical diagnosis completed by ultrasonography was histopathologically confirmed in all cases. Conclusions: Although the glomus tumor is a rare lesion, we need to be familiar with it because a diagnostic delay also implies a treatment delay which will lead to amplified suffering and even real disability due to the high-intensity pain in these cases.

Corresponding author: Sorinel Lunca, Professor, MD, PhD; e-mail: sdlunca@yahoo.com; Mihaela Mitrea, Assistant, MD, PhD; e-mail: mihaela.mitrea77@yahoo.com

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24. Histopathological and immunohistochemical changes of the marginal periodontium in patients with Turner syndrome

Smaranda Adelina Preda, Anca Mihaela Predescu, Loredana Elena Stoica, Dana Maria Albulescu, Nina Ionovici, Narcis Mihaita Bugala, Mihaela Popescu, Mihaela Jana Tuculina

Turner syndrome (TS) is characterized by partial or complete loss of a sexual chromosome, resulting in an incomplete development of the body, gonadic failure, cardiac and renal abnormalities, oro-dental changes, etc. In our study, we proposed to perform a histological and immunohistochemical (IHC) study of the periodontium changes in patients with TS. The biological material under study was represented by fragments of gingival mucosa harvested from 18 patients with TS who presented advanced periodontal lesions and required dental extractions. The fragments of gingival mucosa were processed by the classical histological technique of paraffin inclusion, subsequently the obtained sections being stained by the Hematoxylin-Eosin (HE) and examined under the optical microscope. For the IHC study, there were performed serial sections incubated with anti-cluster of differentiation (CD) 3, anti-CD20 and anti-CD68 antibodies for highlighting immune cells, as well as with anti-matrix metalloproteinase (MMP) 2 and anti-MMP8 antibodies for highlighting MMPs (MMP2 and MMP8) involved in the periodontal tissue lesions. In the present study, during the histological examination, there were observed morphological changes, both in the epithelium and in the gingival mucosa chorion. Epithelial changes consisted in the onset of acanthosis processes, in the thickening of the epithelium due to the increase of the spinous layer, as well as in the parakeratosis phenomenon. In the chorion, there was observed the presence of inflammatory infiltrates in various stages, presence of fibrosis (extended in some cases) and the presence of an important vascularization in some cases, with a high number of immunocompetent cells involved in the inborn immune response, but also in the adaptive one, as well as a more or less intense immunoexpression of MMP2 and MMP8. Our study suggests that TS may contribute to the development of some inflammatory processes in the marginal periodontium.

Corresponding author: Anca Mihaela Predescu, MD, PhD; e-mail: medpredes@yahoo.com; Narcis Mihaita Bugala, MD, PhD Student; e-mail: bugala.mihai@yahoo.com

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25. CD4+ CD25+ regulatory T-cells role in tumor microenvironment of the squamous cell carcinoma

Andrei Vasile Pascalau, Cornel Dragos Cheregi, Mihai Stefan Muresan, Mircea Ioan Sandor, Carmen Anca Huniadi, Zoran Nikin, Claudia Teodora Judea Pusta, Florian Dorel Bodog, Calin Ionescu, Ovidiu Laurean Pop

Introduction: Squamous cell carcinoma (SCC) is the most common skin cancer with a high rate of death. Different lymphocyte populations play an important role in modulating the immune response in the tumor microenvironment. The increase in the proportion of cluster of differentiation (CD)4+ CD25+ regulatory T-cell (Treg) lymphocytes is associated, in different studies, with the increase of the cell multiplication rate. Aim: To analyze the Treg lymphocyte subpopulations and to correlate the results with the presence of the CD8+ cytotoxic T-cell (Tc) lymphocyte population. Materials and Methods: Sixty primary skin SCC specimens were incubated with anti-CD8 (clone SP57) rabbit monoclonal antibody and anti-CD25 (clone 4C9) mouse monoclonal antibody. Results: The ratio of the intratumoral/peritumoral CD4+ CD25+ forkhead box protein p3 (Foxp3) lymphocytes was 0.46, emphasizing that at tumor margins, where tumor aggressiveness is higher, these lymphocytes subpopulations facilitate tumor progression. The comparative analysis of the tumor microenvironment profile revealed that in the case of intratumoral immune response, the number of Tc-type lymphocytes (CD8+) was 3.34 times higher compared to Treg lymphocytes (p<0001). In the peritumoral area, the number of Tc lymphocytes was 5.05 times higher compared to Treg lymphocytes (p<0001). Conclusions: Treg lymphocytes inhibition may cause the suppression of the antitumoral cell immune response in the tumor environment. We believe that Treg lymphocytes should represent a focus of interest for a new personalized therapy. New studies are needed to better understand the immune response in the tumor microenvironment.

Corresponding author: Florian Dorel Bodog, Professor, MD, PhD; e-mail: fbodog@gmail.com; Claudia Teodora Judea Pusta, Associate Professor, MD, PhD; e-mail: claupustaml@yahoo.com

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26. A software approach for identifying the effect of dental caries on dentin-enamel junction

Bogdan Mihai Galbinasu, Horia Octavian Manolea, Ioana Matei, Mihai Andrei, Mihnea Ioan Nicolescu

Dental decay is the most prevalent oral disease worldwide since more than 2.4 billion people suffer from caries of permanent teeth. Therefore, any details about its progression into the hard-dental tissues could contribute to unravelling the mechanisms underlying this process. We have analyzed dental tissue sections with and without caries in order to detect structure differences corelating them with clinical aspects observable from the tooth surface. Our working hypothesis was based on finding a link between the process of tertiary dentin laying (as a response to coronal caries) and the subsequent obliteration of dentin tubules. We have selected N=10 extracted teeth with/without coronal caries, resin-embedded and sectioned them. A specific software was used to digitally quantify the density of unobliterated dentin tubules reaching the dentin-enamel junction (DEJ), considering as positive threshold criteria the presence of the odontoblast process inside the analyzed tubule. This study showed the differences between the healthy and carious-affected hard-dental coronal tissues. More odontoblast processes reached the DEJ in unaffected teeth. Using specific software, we have quantified their density decrease near a lesion. We have studied the dynamics of the carious study and measured the consequent structural modifications of the dentin. In conclusion, there is a significant difference between the number of dentin tubules containing odontoblast processes that reach the DEJ in healthy/altered tissues. The tooth reacts not only by production of tertiary dentin to protect the pulp chamber, but also by obliteration of dentin tubules, thus reducing the number of odontoblast processes reaching the DEJ. This pilot study could serve as the starting point in developing a dedicated software that could deliver a personalized pattern for decay progression by analyzing one single tooth and extrapolate the result to all the patient s remaining ones.

Corresponding author: Horia Octavian Manolea, Professor, DMD, PhD; e-mail: horia.manolea@umfcv.ro

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27. Comparing PRP and bone marrow aspirate effects on cartilage defects associated with partial meniscectomy: a confocal microscopy study on animal model

Adrian Emil Lazarescu, Adrian Ovidiu Vaduva, Gheorghe Bogdan Hogea, Cristian Croicu, Jenel Marian Patrascu Jr, Pompiliu Horatiu Petrescu, Bogdan Corneliu Andor, Mirela Danina Muntean, Jenel Marian Patrascu

Aim: The aim of our study was to assess the therapeutic effects of platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMAC) in an animal knee lesion complex associating a large osteochondral defect and meniscal defect resulted from partial meniscectomy, a clinical situation that occurs quite often in orthopedic practice. Materials and Methods: Twenty-one male rabbits were included in the study, and all underwent initial surgery on the right knee to create the osteochondral defect on the internal femoral condyle, and remove the anterior horn of the internal meniscus, simulating a clinical situation. Rabbits were separated in three study groups: control, PRP group, in which three PRP injections were administered, and BMAC group, in which one single BMAC injection was administered. At the end of the six months follow-up period, knees were harvested and further analyzed using confocal microscopy and three-dimensional (3D) reconstruction of the articular surface. Results: Therapeutic groups had better results concerning articular surface remodeling and joint degeneration indicators in comparison to trauma group. Conclusions: Our results suggest that using post-operative regenerative therapies does improve final results concerning surface contact remodeling that was investigated using confocal microscopy and should be considered a valid treatment adjuvant in managing patients with this type of lesion complex, as it improves global joint outcome.

Corresponding author: Jenel Marian Patrascu Jr, Assistant Professor, MD, PhD; e-mail: patrascujenel@yahoo.com

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28. Analysis of breast cancer subtypes and their correlations with receptors and ultrasound

Gabriela Sechel, Liliana Marcela Rogozea, Nadinne Alexandra Roman, Daniel Ciurescu, Maria Elena Cocuz, Rosana Mihaela Manea

The study aim was to evaluate the ultrasound (US) signs of the mammary lesions classified in the Breast Imaging-Reporting and Data System (BI-RADS) score category 3, 4, and 5, corresponding to US BI-RADS. It also followed the correlation between US changes of lesions suggestive for malignancy with the histopathological results and evaluated the proper management of those lesions. There were correlations of breast cancer (BC) subtypes with the receptors [estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2)], and Ki67 index, and the signs of conventional ultrasonography and US elastography. We selected 108 female patients examined with US, mammography and fine-needle biopsy who presented suspicions for malignancy lesions. Following the immunohistochemical analysis, they were classified in one of the BC subtypes. According to chi-squared analysis of molecular cancer subtypes correlation to receptors and Ki67 index, we found significant associations between both luminal A and luminal B HER2-negative subtypes and hormone receptors (ER, PR). These have an inverse relationship with Ki67 index elevated values; luminal B HER2-positive subtype has a direct association with HER2 presence; HER2-enriched subtype was statistically significant associated to HER2 presence and elevated Ki67 index values but had an inverse relationship to hormone receptors (ER, PR); triple-negative subtype was strongly associated to Ki67 index values and inversely correlated to ER and PR. We found luminal A subtype as being the most common and luminal B HER2-positive subtype as having the fewer cases.

Corresponding author: Maria Elena Cocuz, Lecturer, MD, PhD; e-mail: maria_elenacocuz@yahoo.com

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29. Hemophagocytic lymphohistiocytosis in a child with human immunodeficiency virus infection - a case report

Lacramioara-Eliza Chiperi, Andra Diana Ionescu, Cristian Tiberiu Marcu, Corina Itu-Muresan, Cristina Pantelemon

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a rare condition and furthermore human immunodeficiency virus (HIV)-associated HLH is rarely reported in the literature. The most frequent presentation of secondary HLH is in association with infections, malignancies or drugs. In HIV-positive patients, the viral infection itself or the antiretroviral therapy (ART) could trigger HLH. Case presentation: A 14-year-old boy was admitted for persistent diarrhea, severe weight loss and chest burns. Laboratory tests revealed important neutropenia, which led to HIV infection diagnosis. ART in combination was started associated with granulocyte-colony stimulating factor. Viral copies declined after six weeks of treatment, but the cluster of differentiation 4 (CD4) T-lymphocytes and neutrophil counts remained very low. Infections and malignancies were ruled out. The bone marrow aspirate revealed hemophagocytosis which in association with fever, bicytopenia, hypofibrinogenemia and hypertriglyceridemia established HLH diagnosis. Cortisone therapy and intravenous immunoglobulins were added. Due to lack of response, HLH-2004 protocol was initiated in collaboration with pediatric hematologist. In the first six months of treatment, CD4 T-lymphocytes and neutrophil count remained low and then they showed significant increase simultaneously. During treatment, the patient developed spontaneous severe back pain. Magnetic resonance imaging showed vertebral compression, osteomalacia and a thoracic vertebral fracture. Conclusions: Having ruled out the usual associated infections and malignancies, this is a case of HIV-associated with HLH. In this case, only the ART and immunomodulatory therapy were not enough and failed to work. Systemic immunosuppression also worked only after a prolonged course which led to an unfortunate complication: a vertebral fracture.

Corresponding author: Lacramioara-Eliza Chiperi, MD; e-mail: lacramioara-eliza.pop@umfst.ro

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30. Large borderline phyllodes breast tumor related to histopathology, diagnosis, and treatment management - case report

Nicolae Ovidiu Penes, Anca Lucia Pop, Roxana Georgiana Bors, Valentin Nicolae Varlas

Breast phyllodes tumors (PTs) are uncommon tumors with a biphasic fibroepithelial component, characterized by rapid development in middle-aged women. A correct preoperative diagnosis after Tru-Cut biopsy allows for proper surgical planning. The treatment of choice remains surgery (wide local excision or mastectomy) with or without breast reconstruction, depending on the size and histopathological (HP) nature of the tumor. We reported a case of a 50-year-old woman with a large PT in her left breast, measuring 11/10 cm. Preoperative HP examination revealed biphasic proliferation, with the appearance of benign PT. The patient underwent a left mastectomy, with a favorable postoperative evolution. The final HP diagnosis was borderline PT, with areas of lipomatous metaplasia. After three months, breast reconstruction was performed. The therapeutic management of large PTs continues to be a challenge for pathologists and surgeons. Due to the increased frequency of local recurrence and HP progression to malignancies, the treatment of choice for these patients is mastectomy, without lymphadenectomy.

Corresponding author: Anca Lucia Pop, Lecturer, MD, PhD; e-mails: anca.pop@umfcd.ro, ancapop@hotmail.com

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31. Atypical posterior circulation strokes: a case-based review of rare anatomical variations involved

Carmen Corina Roman-Filip, Anca-Rafila Stingaciu, Maria-Gabriela Catana, Andreea-Alina Dan, Adrian Florian Balasa, Ciprian Juravle, Florin Grosu

The circle of Willis is a very important vascular mechanism of protecting against cerebral ischemia, especially when circulation within the main arteries irrigating the brain is somehow impeded. As result of congenital malformation arising early in embryonic development, the fetal-type posterior circle of Willis remains as such during the rest of one s life. Consequently, the posterior cerebral artery (PCA) becomes a branch of the internal carotid artery (ICA), rather than of the basilar artery (BA). Furthermore, the rest of collateral circulation, between the anterior and the posterior regions of the brain, is also negatively affected (e.g., leptomeningeal vessels). The anatomical variant represented by the artery of Percheron (AOP) has its origin on one of the PCAs, supplying singlehandedly both paramedian areas of the thalamus (right and left) and posterior regions of the midbrain. In the present study, we report a case of bilateral thalamic infarction with midbrain involvement, where the correct diagnosis was made retrospectively using computed tomography (CT) scan, magnetic resonance imaging (MRI), diffusion-weighted imaging (DWI) and three-dimensional time-of-flight magnetic resonance angiography (3D TOF MRA).

Corresponding author: Carmen Corina Roman-Filip, Associate Professor, MD, PhD; e-mail: corinaromanf@yahoo.com

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32. Spinal cord infarction caused by extensive aortic intramural hematoma

Rodica Nicoleta Diaconu, Adelina Oana Neagoe, Ionut Donoiu, Oana Cristina Mirea, Alexandru Claudiu Munteanu, Dumitru Sandu Ramboiu, Octavian Istratoaie

We report the case of a 62-year-old Caucasian man, an ex-smoker, who presented to the emergency room complaining of intense lower back pain followed by immediate bilateral loss of inferior limbs motor function. Clinical examination showed complete paralysis and paranesthesia in both legs, while pain and temperature sensory examination revealed a sensory level at dermatome T6, with normal touch, vibration, and position senses. His blood pressure was 190/100 mmHg. Computed tomography demonstrated dilated thoracic aorta (maximum diameter 44 mm) and abdominal aorta (maximum 58 mm), with extended intramural hematoma (IMH), thus establishing the diagnosis of type A aortic IMH complicated with paraplegia through spinal perfusion deficit. Due to the extension of the lesions, surgical intervention for aortic repair was considered at high risk while cerebrospinal fluid drainage was not recommended by the neurologist. The patient remained stable while hospitalized and was released from the hospital with mild improvement of neurological deficiencies.

Corresponding author: Oana Cristina Mirea, Teaching Assistant, MD, PhD; e-mail: oana.munteanu@umfcv.ro; Dumitru Sandu Ramboiu, Lecturer, MD, PhD; e-mail: sandu.ramboiu@umfcv.ro

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33. A dedifferentiated rare primary breast liposarcoma - case report and literature review

Valentin Nicolae Varlas, Yassin Rhazi, Oana Maria Ionescu, Luminita Gentiana Micu, Anca Lucia Pop, Nicolae Bacalbasa, Bogdana Adriana Nasui, Nicolae Ovidiu Penes

Liposarcoma of the breast is a very rare soft tissue malignant tumor arising in the fat cells, with a prevalence of 0.3% of all malignant breast tumors, clinically manifested as a palpable breast mass mimicking a primary breast cancer. In the present paper, we had two objectives: (i) to report the first liposarcoma case in our Clinic and (ii) to screen the scientific literature on the topic. Our report presents an unusual case of a 56-year-old female with symptomatic left breast mass initially histopathologically diagnosed as a mesenchymal lesion. Four months later, the tumor was histopathologically identified as a grade 3 dedifferentiated liposarcoma (DDLPS) Federation Nationale des Centres de Lutte Contre le Cancer (FNCLCC; French Federation of Cancer Centers). We present the histopathological, immunohistochemical, and radiological features of the case and outcomes. Secondly, we performed a systematic search on liposarcoma on the PubMed/Medline and Web of Science databases, using the keyword primary breast liposarcoma (all-time topic). Due to the small number of cases found in the literature, the best treatment choice and determination of prognosis are difficult to make. Our patient underwent breast radical surgery, received adjuvant treatment, continuously monitored, being disease-free after five years of follow-up.

Corresponding author: Anca Lucia Pop, Lecturer, MD, PhD; e-mails: anca.pop@umfcd.ro, ancapop@hotmail.com

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34. ETV6-NTRK3-positive parotid mammary analogue secretory carcinoma: a case report

Juan Rodriguez Cobo, Servando Lazuen Fernandez, Marta Maria Mayorga Fernandez, Javier Gomez Roman, Javier Freire Salinas, Maria Soledad Rodriguez Duque, Carmen Vallejo Secadas, Javier Alvarez Gama, Eduardo Ibaseta Fidalgo, Julia Medina del Valle

Introduction: Mammary analogue secretory carcinoma (MASC) is a recent discovered entity of salivary glands tumors, reported for first time in 2010. The presence of a translocation encodes the ETS variant transcription factor 6-neurotrophic tyrosine receptor kinase (ETV6-NTRK3) gene fusion differences MASC from other tumors. Case presentation: A 68-year-old male showed a non-painful right parotid enlargement, came from dermatology service, and followed by some facial squamous cell carcinomas. A computed tomography (CT) scan showed a 1.7x1.6 cm right parotid enlargement in superficial lobe. The patient underwent a right superficial parotidectomy. The final pathology confirmed the presence of ETV6-NTRK3-positive MASC. Complete right deep parotidectomy and functional cervical emptying were performed. Discussions and Conclusions: It is necessary to establish an appropriated differential diagnosis between salivary gland tumors. MASC is a low-grade malignancy cancer that sometimes can evolve to a high-grade tumor that might produce local and distance dissemination. Most times, these tumors are only treated by surgical resection and evaluating by a multidisciplinary team the need of more treatments. In our case, the patient showed a primary parotid tumor, removed surgically with free edges, and being identified as MASC. We decided to underwent neck dissection and discovered a second MASC focus on cervical salivary gland; however, there was no nodal dissemination. The patient remains disease-free after 14 months from last surgery. It is important to keep studying genetic therapy targets to ETV6-NTRK3 to obtain a new therapy line to treat those cases that require.

Corresponding author: Juan Rodriguez Cobo, MD; e-mail: juan.rodriguezcobo@gmail.com

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35. Colonic tubular adenoma with incidental oxyntic gastric heterotopia

Jose-Fernando Val-Bernal, Maria Luisa Cagigal, Marta Maria Mayorga, Marta Maria Cuadrado, Carlos Rodriguez-Escaja

Oxyntic gastric heterotopia (GH) in the colon is not common. Its presence in a colon tubular adenoma is even rare. A 73-year-old woman with a history of resected colon carcinoma underwent periodical colonoscopies for the removal of tubular adenomas for 12 years. In the last colonoscopy, a sessile, non-ulcerated polyp, centrally depressed, with a smooth surface, measuring 20 mm, located at 50 cm from the anal verge was excised. A histological study identified a tubular adenoma with focal low-grade dysplasia and ectopic gastric oxyntic epithelium. The GH, composed of parietal and chief cells, and was found incidentally. Oxyntic GH in a tubular adenoma is extraordinarily rare. To the best of our knowledge, there is only one previously published case. The main possible difficulties and/or errors in the diagnosis include a tissue floater or a cross-contaminant. Precise diagnosis of oxyntic GH is basic for appropriate management. Diagnosis relies on histopathological examination. The immunohistochemical study for mucin 6 (MUC6) can confirm the nature of the epithelium. Oxyntic GH has the potential to produce serious complications including tumor development. However, GH is considered a benign disease and adenocarcinoma rarely occurs in the heterotopic mucosa. The optimal treatment of oxyntic GH associated with a tubular adenoma is endoscopic complete polypectomy.

Corresponding author: Jose-Fernando Val-Bernal, Professor, MD, PhD; e-mail: fernando.val@unican.es

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36. Legal approach regarding dental malpractice in Romania

Adela Cristina Lazar, Dan Buhatel, Ovidiu Muresan, Liana Todor, Mariana Pacurar

In Romania, the term dental malpractice is relatively new compared to medical malpractice. The common premises that underlie these iatrogenics have made doctors more vigilant about treatment and achieving the desired results. At the same time, the conditions of medical liability are consistent with those of civil liability, namely: damage, wrongdoing, guilt, and causation. To counteract these effects, public attention has been focused in recent years on patients rights (involving, causes, effects, informed consent of the patient regarding the collection of personal data, informing the patient about the work itself, and possible complications which may occur postoperatively). As a result, the number of reported cases of malpractice has increased markedly in recent years.

Corresponding author: Dan Buhatel, Lecturer, DMD, PhD; e-mail: dbuhatel@yahoo.com; Liana Todor, Associate Professor, DMD, PhD; e-mail: liana.todor@gmail.com

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37. Heritage of Leopold Auerbach in the field of morphology of nervous system

Andrzej Wincewicz, Piotr Woltanowski

Leopold Auerbach (April 27, 1828-September 30, 1897) belongs to world famous figures in medicine, who were born, spent most of their lifetimes and died in Wroclaw (Breslau). Auerbach reported for the first time in literature about existence of plexus myentericus (plexus Auerbachi) including ganglion cells between circular and longitudinal layers of tunica muscularis propria in intestinal wall, in 1862. With his publication on muscular hypertrophy, dated on 1871, he provided ground for another eponym: Friedreich-Auerbach disease, that refers to facial hemihypertrophy. He was raised in Jewish family, which lived for generations in Wroclaw. His elaborative scientific work was his struggle for human dignity and safe social status, which was shared with many other members of the community at the time of Jewish emancipation thorough the whole XIXth century in Germany. The great value of Leopold Auerbach for the Wroclaw University - his Alma Mater -, which was founded by an Austrian Emperor Leopold I, is even metaphorically coded by the fact, that AL are not only initials for Academia Leopoldina but also curiously for the name and surname of this brilliant scientist, who led rather a calm and unspectacular life. This paper is the last one from the series of our biographical papers, in which we focused on his output in the field of vascular system, morphology of invertebrates and - in only short one page-long note until now - on the topic of nervous system, so we decided to present a full text report on the latter but the most famous area of his activity.

Corresponding author: Andrzej Wincewicz, Associate Professor, MD, PhD; e-mail: ruahpolin@yahoo.com, andwinc@gmail.com

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38. George Gershwin (1898-1937) - genius composer, malignant brain tumor patient. Malignant glioma: an irritating/stimulating element in triggering geniality?

Alexandru Vlad Ciurea, Razvan Onciul, Aurel George Mohan, Mircea Vicentiu Saceleanu, Andrei Alexandru Marinescu, Corneliu Toader

George Gershwin (1898-1937) was one of the most influential American composers of the 20th century. Likewise his swift, comet-like, career progress from a high school dropout to an iconic composer of the 20th century: The man who said he had more notes in his head than he could write down in a hundred years died suddenly … in Hollywood. His malignant glioma presented symptoms only in his last year of life and even then, the symptoms were masked by other hectic events in his personal life, which typical accompany the life of a famous and handsome composer from Hollywood. Considering new, emerging studies regarding the relationship between gliomas and brain activity, the authors explore the following questions: (i) Is intense neuronal, intellectual activity a cause that can trigger the development of a malignant brain tumor, especially a glioblastoma? (ii) Is glioblastoma and its connections with normal nerve cells, with noble tissue (a fact demonstrated in recent years), a cause that can trigger geniality, a different thought process from most people with a normal-functioning brain?

Corresponding author: Razvan Onciul, MD; e-mail: razvan.onciul@gmail.com

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