ROMANIAN JOURNAL of MORPHOLOGY and EMBRYOLOGY

Vol. 52 No. 3 Suppl., 2011
This supplement was not sponsored by Outside Organizations.

1. Pseudobenign prostate carcinomas: causes of false-negative biopsy results

Alis Dema, Sorina Taban, Elena Lazar, Angela Borda, Codruta Lazureanu, Diana Herman, Anca Muresan, Marioara Cornianu, Denisa Anderco, Andrada Loghin

Prostate carcinomas are continuously surprising the pathologists through their multitude of variants and histological subtypes, some of them being recently described and characterized. Among these are individualized: atrophic carcinoma, foamy gland, pseudohyperplastic, microcystic, certain subtypes of ductal adenocarcinoma and hormone-treated adenocarcinoma, which because of minimal architectural and/or cytological atypia are often under-diagnosed, especially in small tissue fragments. This paper presents the morphological criteria, including information provided by some immunohistochemical markers for positive and differential diagnosis of these variants/subtypes of prostate adenocarcinoma with which the pathologist should be familiar and avoid their confusion with a series of similar histological structures or benign/premalignant lesions.

Corresponding author: Alis Dema, Professor, MD, PhD, e-mail: dema_alis@yahoo.com

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2. New insights on Alzheimer's disease diagnostic

Ioana Gabriela Fita, Ana-Maria Enciu, B. P. Stanoiu

One of the most common problems encountered in a world characterized by demographic ageing is Alzheimer's disease (AD) with an estimated number of 35.6 million people affected in 2010 to 65.7 million in 2030. Under recognition and delayed diagnose create problems for people diagnosed with dementia, for their families and for entirely health system. Although there have been many breakthroughs and new insights into AD etiopathogeny in the last two decades, few steps have been made toward an accurate diagnosis but all steps point into one major direction namely "personalized medicine" that could represent a future perspective for AD patients. Starting with a more accurate diagnosis not of the clinical syndrome, but of underlying molecular defects, that may eventually lead to a personalized, more effective treatment.

Corresponding author: Bogdan Stanoiu, Lecturer, MD, PhD, e-mail: stanoiu.bogdan@yahoo.com

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3. Macrophages and mast cells are involved in carotid plaque instability

A. Marzullo, M. M. Ciccone, Claudia Covelli, Gabriella Serio, D. Ribatti

The aim of this study was to evaluate the role of macrophages and mast cells and of microvascular density in atherosclerotic plaques collected from 63 consecutive symptomatic and asymptomatic patients undergoing carotid endarterectomy for carotid disease. Results have shown no statistically significant differences between the two groups as concerns: (i) the degree of stenosis; (ii) the extention of the lipidic core; (iii) the thickness of the fibrous cup; (iv) the inflammatory infiltrate; (v) the degree of calcification; (vi) the intraplaque hemorrhage. Otherwise, statistically significant difference was found in microvascular density, in the number of CD68-positive macrophages and tryptase-positive mast cells in plaques from symptomatic patients, as compared to asymptomatic patients. Overall, this study indicate that although advanced symptomatic and asymptomatic carotid plaques present similar histomorphological characteristics, the degree of macrophage and mast cell infiltration and differences in microvascular density could help to discriminate between symptomatic and asymptomatic patients.

Corresponding author: Domenico Ribatti, Professor, e-mail: ribatti@anatomia.uniba.it

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4. The utility of CD44, CD117 and CD133 in identification of cancer stem cells (CSC) in oral squamous cell carcinomas (OSCC)

Cl. Margaritescu, D. Pirici, Cristiana Simionescu, A. Stepan

One of the theories regarding oral carcinogenesis is that the tumor growth is dependent on cancer stem cells (CSCs) that have the capacity of self-renewal and of giving rise to more differentiated tumor cells, like the stem cells do in normal tissues. The most used methods of CSCs isolation are based on their identification based on the expression of different cell surface markers. The markers qualified for this purpose have been described originally in studies involving hematopoietic or embryonic stem cells. Thus, we were interested to study the expression of the most used CSCs surface markers for formalin fixed paraffin embedded tissue samples from oral squamous cell carcinoma (OSCC). We investigated by immunohistochemistry thirty tissue samples of OSCCs with different degrees of differentiation and different oral locations. We were interested to establish the tissular localization pattern for cells expressing CD44, CD133 and CD117 in tumoral samples. The results indicated that with the exception of CD44, the other two surface markers were expressed only in tumoral stromal cells. When we looked at their origin (by double immunohistochemistry for cytokeratins AE1-AE3, vimentin and CD34) we concluded that they are of mesenchymal nature. Also, we proved that some of these cells also co-expressed CD44 but were negative for CK5/6. Moreover, some of the stromal cells that were positive to CD133 and especially for CD117 also had reactivity to tryptase showing their mast cell nature. In conclusion, our study proved that CD44 has limited utility in identifying oral CSCs, while CD117 and CD133 expression appears to be limited more in identifying mesenchymal stem cells.

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

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5. Investigation of P213S SELL gene polymorphism in type 2 diabetes mellitus and related end stage renal disease. A case-control study

Monica Stavarachi, N. M. Panduru, C. Serafinceanu, E. Mota, Maria Mota, D. Cimponeriu, Daniela Adriana Ion

SELL (L-selectin) is a candidate gene for several complex diseases including diabetes mellitus and renal failure. Our aim was to investigate the involvement of P213S SELL gene polymorphism (rs2229569) in type 2 diabetes mellitus (T2DM) and related end stage renal disease (ESRD). Type 2 diabetes mellitus patients without ESRD (n=250) or with ESRD (n=90), ESRD patients without diabetes (n=119) and sex and age matched healthy subjects (n=459) were analyzed in this study. DNA samples from all these subjects were genotyped for the P213S polymorphism by PCR-RFLP technique. Statistical analysis indicated that SELL P213S genotypes and alleles were similar distributed in the patients and control groups (ORSS=0.37, CI 95%: 0.131>0.372>1.06, p=0.05, Yate's correction p=0.09, for T2DM patients without ESRD, ORSS=2.04, CI 95%: 0.365>2.047>1.465, p=0.4, Yate's correction p=0.67, for T2DM patients with ESRD and ORSS=1, CI95%: 0.198>1>5.057, p=1, Yate's correction p=0.67, for non-diabetic with ESRD patients). Also, no significant differences were noticed when we compared the ESRD subjects with diabetes vs. non-diabetic ones (OR=1.798, CI 95%: 0.392>1.798>8.245, p=0.44, Yate's correction p=0.7). No statistically significant results were found in order to sustain the hypothesis of association between SELL gene P213S polymorphism, type 2 diabetes mellitus and end stage renal disease.

Corresponding author: Monica Stavarachi, Research Assistant, PhD, e-mail: monica.stavarachi@gmail.com

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6. Pancreatic exocrine adult cells and placental stem cells co-culture. Working together is always the best way to go

S. Susman, D. Rus-Ciuca, Olga Soritau, C. Tomuleasa, R. Buiga, D. Mihu, V. I. Pop, Carmen Mihaela Mihu

Background and Aim: The progress made in the last few years have managed to come up withy the possibility of using different stem cell types in an endeavor to correct the alterations that appear in different degenerative diseases. The pancreas, an organ with extremely low regenerative capacity, both for the endocrine and for the exocrine component, is an organ perfect for cell therapy in the hope of restoring its function and cure diabetes mellitus or chronic pancreatitis. One main issue in the stem cell transplantation problem is represented by the influence of the cellular niche, formed by completely differentiated cells, on the phenotype and function of the transplanted cells. In this study, we challenge current knowledge in the field by evaluating the influence of exocrine pancreatic cells on placental stem-like cells using the co-culture technique. Methods: In our experiments, we used two different protocols in which adult pancreatic cells were cultured together with mesenchymal stem cells isolated from human placenta. In the case of the first protocol, we seeded pancreatic cells on a pre-adhered single-cell layer of mesenchymal stem cells and in the second one, the seeding of two cell populations in suspension was done at the same time, after passage. During the experiment, we evaluated the alteration of the morphology of the placental cells using and inverted phase microscope and reverse transcriptase-PCR. Results and Conclusions: Based on morphology, in both cases the interaction between epithelial pancreatic cells and placental ones have determined a change in phenotype from mesenchymal to epithelial-like. Taking into consideration the gene expression, placental stem cells have maintained pluripotency gene expression throughout the study. They also expressed pancreatic amylase. These experiments bring out the plasticity of placental stem cells, the cell microenvironment with a decisive part in phenotype and the level of gene expression. The results obtained in vitro can bring a new picture on the effects of the pancreatic stem cell niche.

Corresponding author: Sergiu Susman, MD, e-mail: sergiu.susman@umfcluj.ro

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7. Establishment of an embryonic stem cell line from blastocyst stage mouse embryos

Emoke Pall, I. Groza, M. Cenariu, Olga Soritau, Elen Gocza, C. Tomuleasa

Embryonic stem cells have the ability to remain undifferentiated and proliferate in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. The aim of the present study was to establish mouse ES lines from blastocyst stage embryos obtained after CD1/EGFP mice superovulation. We isolated, cultured and determined the characteristics of mouse embryonic stem cells in early passages, which were first described by Evans M and Kaufman M. Therefore, we evaluated the morphological criteria for the approval of ES cells in early expansion stage. Two cell lines were isolated (CDE1 and CDE2) and analyzed. They showed similar characteristics to those reported earlier for blastocyst-derived ES cell lines.

Corresponding author: Emoke Pall, DVM, PhD student, e-mail: pallemoke@gmail.com

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8. Pericytes and myofibroblasts reaction in experimental thermal third degree skin burns

Florina Carmen Popescu, Cristina Jana Busuioc, G. D. Mogosanu, O. T. Pop, H. Parvanescu, I. Lascar, Corina Ivona Nicolae, L. Mogoanta

Pericytes and myofibroblasts are two types of connective cells that appear in large quantities in reparative processes. In order to evaluate their response and any relationships between them, we have inflicted third degree skin burns to seven groups of five common Wistar rats each from which we sampled granulation tissue at three days interval for histological and immunohistochemical studies. Alpha-SMA and CD34 antibodies revealed that granulation tissue myofibroblasts do not express CD34 and do not arise from pericytes. The parallel arrangement of myofibroblasts in relation to the burned wound surface suggests that their main function is mechanical traction on granulation tissue and wound edges.

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

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9. The diagnostic value of EMA expression in the renal parenchyma tumors

Aurora Alexa, Flavia Baderca, Rodica Lighezan, Delia Elena Zahoi, D. Izvernariu

Renal parenchyma tumors are a heterogeneous group of malignancies that are difficult to diagnose and classify. Immunohistochemistry begun to be routinely used for the diagnosis of these tumors. Panels of antibodies are developed for the diagnostic assessment of these tumors, which include cytokeratins, epithelial membrane antigen and vimentin. Epithelial membrane antigen (EMA) is expressed by most of the tumor cell types. Forty-seven specimens of renal parenchyma tumors were studied immunohistochemically for the expression of EMA. In the majority of the cases, clear cells carcinoma was positive for EMA (25/33, 75.70%). All of the papillary carcinomas were positive, with different staining patterns between the two subtypes. The two cases of chromophobe cells carcinomas were intensely positive with a granular cytoplasmic staining pattern. The mixed epithelial-stromal tumor was negative for EMA in both of the components. Out of the three cases of sarcomatoid carcinomas, one was negative, one was weakly positive (+1) and the last was positive (+2). Intensely positive normal tubes were caught by the tumor proliferation in the negative case and in the negative stained areas of the weakly positive case.

Corresponding author: Flavia Baderca, Lecturer, MD, PhD, e-mail: flaviabaderca@yahoo.com

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10. Histological changes and immunohistochemical markers in the assessment of glomerulosclerosis in patients with glomerulonephritis

F. R. Bob, Gh. Gluhovschi, Diana Herman, Gh. Bozdog, Cristina Gluhovschi, Silvia Velciov, Elena Potencz, Ligia Petrica

Introduction: Glomerular cells (mesangial, endothelial, epithelial) are activated during glomerulonephritis, a process indicated by the expression of the immunohistochemical marker alpha-smooth muscle actin (SMA). Many growth factors participate in the above-mentioned processes, among them of great importance is the transforming growth factor beta (TGF-beta). The result of these changes is represented by active lesions (mesangial matrix increase, mesangial cell proliferation) and chronic fibrotic lesions (glomerulosclerosis). Methodology: We studied a group of 41 patients with primary and secondary glomerulonephritis (24 males, 17 females, mean age 45.5+/-12.9 years), which underwent kidney biopsies, processed in light microscopy. We performed immunohistochemistry procedures with monoclonal antibodies (performed with the LSAB2-HRP system: anti-alpha-SMA, and anti-TGF-beta), which were assessed using a semiquantitative score, that was correlated with the histological and biological data. In order to quantify the histological changes and to assess the extent of active-inflammatory and chronic-sclerotic/fibrotic lesions, we adapted a scoring system initially used only for lupus nephritis, and ANCA-associated vasculitis. Results: TGF-beta expression in glomerular endothelial cells correlated with mesangial matrix increase (r=0.28, p<0.05), total activity index (r=0.29, p<0.05) and total chronicity index (r=0.34, p<0.05). Glomerular epithelial cell TGF-beta correlates with mesangial proliferation (r=0.29, p<0.05), mesangial matrix increase (r=0.4, p<0.01) and total activity index (r=0.28, p<0.05). We observed a strong correlation between endothelial immunolabeling of SMA and the mesangial proliferation score (r=-0.96, p<0.005) and also an indirect correlation with the glomerulosclerosis score (r=-0.35, p<0.05) and the total chronicity index (r=-0.39, p<0.05). Concerning biological data there was a correlation between mesangial SMA expression and serum creatinine (r=0.60, p<0.001) and an indirect correlation with GFR (r=-0.37, p<0.05). Conclusions: We conclude that TGF-beta has a key role in determining glomerulosclerosis especially through mesangial matrix increase, but possibly also through mesangial cells proliferation. Another role of this growth factor is related to transdifferentiation, not only epithelial-mesenchymal, but also endothelial-mesenchymal.

Corresponding author: Flaviu Raul Bob, MD, e-mail: flaviu_bob@yahoo.com

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11. The importance of tumor proliferation markers in assessing lesions of the palatine tonsil

Carmen Aurelia Mogoanta, Daniela Adriana Ion, G. Stanciu, Elena Ionita, Adriana Bold, Garofita Olivia Mateescu, O. T. Pop, Irina Gheorghisor

Oropharyngeal cancer, and especially squamous cell carcinoma, is one of the most common cancers worldwide, and its incidence is increasing, with the palatine tonsil being one of the main locations. The etiopathogenic factors, together with its location as well as the available immunohistochemical methods, make this type of cancer an accessible one in terms of diagnosis. However, it is usually diagnosed in late stages. Therefore, we tried to elucidate the causes of treatment failures and development of local recurrence. For this, we reassessed the proliferative pattern of tonsil lesions using the anti-p53, anti-PCNA and anti-Ki67 antibodies on 73 tonsil fragments collected after curative surgery on adults aged between 28 and 86 years. Following the reevaluation of the histopathological examination using markers for cell proliferation, the diagnosis was modified in 16 cases, representing about 22% of the cases take into study. By using immunohistochemical markers in the histopathological examination the diagnosis is improved, leading to a more appropriate therapeutical approach.

Corresponding author: Carmen Aurelia Mogoanta, MD, PhD, e-mail: carmen_mogo@yahoo.com

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12. Conventional examination versus immunohistochemistry in the prediction of hormone profile of pituitary adenomas. An analysis on 142 cases

B. Balinisteanu, Raluca Amalia Ceausu, Anca Maria Cimpean, Ionela Baciu, Nicoleta Baculescu, M. Coculescu, M. Raica

The large majority of neoplasms located in the sella turcica are benign pituitary adenomas derived from cells of the adenohypophysis. Pituitary adenomas represent the third most common primary intracranial tumor in neurosurgical practice, outnumbered only by gliomas and meningiomas. Their biology of pituitary adenomas is complex and they can cause a variety of endocrine syndromes and disorders, based on hormone profile secreted by proliferating cells. The aim of this study was to evaluate the routine conventional methods and highly specific immunohistochemical methods in order to accurately predict the type of hormone secretion. Our study was investigated 142 cases admitted with pituitary adenomas and treated by open surgery. Sections from each case were stained with routine Hematoxylin and Eosin method for histopathologic evaluation. Immunohistochemistry was performed on additional slides in order to detect specific pituitary adenomas. Based on the immunohistochemical profile of pituitary adenomas we found following results: 37 growth hormone (GH)-secreting adenomas, 23 prolactin (PRL)-secreting adenomas, 15 mixed GH-cell/PRL-cell adenomas, three mammosomatotroph cell adenomas, two adrenocorticotrophic hormone (ACTH)-secreting adenomas, one thyrotrophic cell adenoma, 18 gonadotroph adenomas, 30 null cell adenomas and 13 plurihormonal adenomas. No correlation was found between conventional features and the immunohistochemical profile. The immunohistochemical profile of functional pituitary adenomas is mandatory for a correct diagnosis. We revealed that staining characteristics of the tumor cells, such as acidophilic, basophilic or chromophobe are nowadays outdated as main principle of classification, because they not identify specific adenoma types.

Corresponding author: Anca Maria Cimpean, Associate Professor, MD, PhD, e-mail: ancacimpean1972@yahoo.com

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13. Potassium level changes - arrhythmia contributing factor in chronic kidney disease patients

I. A. Checherita, Cristiana David, V. Diaconu, Al. Ciocalteu, I. Lascar

As the renal function progressive decline is often correlated to diuresis impairment, potassium level changes represent a major pathophysiological factor in monitoring chronic kidney disease. Even more, potassium level imbalance could lead to life-threatening situations with the risk of severe rhythm disorders appearance. The aim of the study was to determine in which degree the serum potassium changes are implicated in arrhythmias development in CKD patients. Patients and Methods: We included 678 CKD patients (predialysis and dialysed patients) to whom we recorded biohumoral and clinical features in correlations with the possibility of arrhythmias genesis. Results: we noticed, in our predialysis group, an important correlation between hyper-/hypokalemia and arrhythmias appearance, more frequent during hypokalemia episodes (OR=4.04, respectively OR=7.5). The same situation was observed in chronic dialysis group. Conclusions: Hypokalemia is a stronger risk factor than hyperkalemia, but all together, any minimal changes in serum potassium levels could determine arrhythmia in CKD patients.

Corresponding author: Ionel Alexandru Checherita, University Teaching Assistant, MD, PhD, e-mail: fizij@yahoo.com

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14. Expression pattern of CK7 and CK20 in nasal polyps, at patients with chronic rhinosinusitis with nasal polyposis

Mihaela Mitroi, Alina Capitanescu, Claudia Valentina Georgescu, Carmen Aurelia Mogoanta, Camelia Popescu, Monica Georgescu, G. Mitroi, Elena Ionita

We investigated the expression of cytokeratins 7 (CK7) and 20 (CK20) in nasal polyps on a group of 106 patients with chronic rhinosinusitis with nasal polyposis (45 women - 42% and 61 men - 58%) who benefited from surgical procedures. Harvested biological material was analyzed in the pathology laboratory through two methods: histopathological and immunohistochemical analysis. Classical histopathological method of processing the tissues initially fixed in 10% formalin was used. The tissues were then processed by paraffin impregnation, sectioned and stained with Hematoxylin-Eosin. The immunohistochemical method was based on soluble immunoenzymatic complexes - LSAB/HRP (labeled Streptavidin Biotin) method. We used DAKO LSAB 2 System HRP (Universal DAKO Labeled Streptavidin Biotin 2 System Horseradish Peroxidase). The expressions of CK7 and CK20 in nasal polyps were analyzed.

Corresponding author: Mihaela Mitroi, Associate Professor, e-mail: mhlmitroi@yahoo.com

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15. Ductal invasive mammary carcinoma - clinicopathological prognostic factors related to immunohistochemical expression of hormonal receptors and Her2/neu oncoprotein

Felicia Recareanu, Cristiana Simionescu, Claudia Valentina Georgescu, Elena Pirici

We analyzed 75 cases of invasive ductal mammary carcinoma type NOS and focused on comparative investigation of hormonal receptors (estrogen receptor ER and progesterone receptor PR) and Her2/neu oncoprotein expression, according to which we ranked the cases in molecular classification subtypes, determining certain correlations between them and morphoclinical prognostic factors. 73.4% of cases were ER+ and 26.6% were ER-. PR was present in 62.6% of cases and absent in 37.4%. Phenotype ER+PR+ (58.6%) had the highest incidence, followed by ER-PR- (22.8%) and ER+PR- (14.6%). Phenotype ER-PR+ (4%) registered the lowest incidence. 14.8% of tumors were Her2/neu + score 3+, 4% had equivocal score 2+ and 81.3% were negative Her2/neu scored 0 and 1+. 9.5% of cases Her2/neu positive scored 3+ were ER+PR+ and 88.5% of cases Her2/neu negative scored 0-1 were ER+PR+. In terms of the correlation among the status of ER, PR and Her2/neu, we determined a molecular classification of the cases, obtaining the following incidences: luminal A 70% of cases, basal 14.7% of cases, luminal B 8.3%, the lowest incidence being registered at Her2, 7% of cases. Luminal A and basal subtypes were associated with patients aged over 50 years (82% for luminal A and 90% for basal), whereas luminal B and Her2 subtypes were registered mostly at patients aged under 60-year-old (83% for luminal B and 100% for Her2). Luminal A subtype was characterized by small tumors (92% of cases were T1-T2), well and moderately differentiated tumors (58% of cases were G1-G2). 83.3% of cases in luminal B subtype had tumors with dimensions ranked T2-T3, all cases being moderately and low differentiated. Her2 subtype had T2-T3 tumors in 60% of cases, which were G3 low differentiated in a percent of 80%. The basal subtype mostly had tumors larger than 5 cm (91% of cases were T2-T3), out of which only a case (9%) presented well-differentiated G1.

Corresponding author: Felicia Recareanu, PhD candidate, e-mail: feliciarecareanu@yahoo.com

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16. A comparison regarding antiproliferative action between soy total extract and genistein

Corina Tiulea, Camelia Peev, Diana Brezovan, Cristina Dehelean, A. Motoc

The aim of this study is to make a comparison between the action of genistein and total soy extract regarding anticancer action on two different in vivo models: phytobiological test and animal model, and to see which of the two tested samples present a greater antiproliferative effect. Soybean seeds were grounded and a solvent formed of DMSO-ethanol-water in rapport 5-70-25 v/v/v was prepared. The extraction was made using an ultrasonic bath (Falc LCD Series) for 30 minutes, 59 kHz. The solvent was evaporated with a rotary evaporator at 50 degrees C. Genistein was acquired from Extrasynthese (France), hydroxypropyl-gamma-cyclodextrin (HPGCD) from Cyclolab Hungary, 7,12-dimethylbenz[a]anthracene (DMBA), dimethylsulfoxide (DMSO), and 12-O-tetradecanoylphorbol-13-acetate (TPA) from Sigma Aldrich, Germany. Because of the poor water solubility, genistein was prepared in a complex with hydroxypropyl-gamma-cyclodextrin in a molar ratio 1:2 by kneading method and total soy extract in a mass ratio 1:4 also by kneading method. Phytobiological test indicated an inhibition index over 50% in case of solutions of concentration between 8-33% in both samples, suggesting a possible antiproliferative action at a superior level. Study on C57BL/6J mice was made on which it was induced cancer with physical agents like DMBA, and it was promoted with TPA. Mice where divided in four groups: Group A - blank group, Group B - mice who received total soy extract, Group C - mice who received genistein, Group D - untreated mice. Results on animal model show that both soy total extract and genistein inhibited the initiation and promotion of chemically-induced skin tumorigenesis, but genistein had a greater success in recovering skin lesions type experimental malignant melanoma.

Corresponding author: Corina Tiulea, e-mail: corina_tiulea@yahoo.com

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17. MCF7-MSC co-culture assay: approach to assess the co-operation between MCF-7s and MSCs in tumor-induced angiogenesis

S. Comsa, Felicia Ciuculescu, R. Henschler, M. Raica

The multipotent stromal cells (MSCs) exhibit a broad differentiation potential. MSCs might participate in neovascularization through their ability to migrate and generate capillary-like structures. These processes were shown to be modulated by tumor angiogenic factors, such as Vascular Endothelial Growth Factor (VEGF). The aim of our study was to define the way the MCF-7 cell line (MCF-7s) influenced the MSCs' recruitment for the tumor-induced angiogenesis, and to assess the role of VEGF in this process. We tested the chemotactic potential of plasma or VEGF, but also of MCF-7s or their conditioned medium (CM) in the MSCs' transmigration. We compared the migratory potential of MSCs, MSCEs (MSCs cultured in endothelial cell growth medium) and HUVECs. Recombinant VEGF has been shown to chemoattract MSCs, although to a lesser degree than plasma or serum containing medium alone. Moreover, it changed the MSCs' morphology, stimulating the appearance of longer and thinner prolongations as compared to plasma. MCF-7s or their CM both directly induced migration of MSCs. Surprisingly, CM augmented with MCF-7s attracted less cells than the control medium itself, but CM augmented or not with MCF-7s changed the morphology of MSCs in a manner similar to VEGF. The migratory behavior of the MSCEs was comparable to that of HUVECs, while their morphology could be considered intermediate between MSCs and HUVECs, as they developed shorter prolongations than MSCs, but much longer than HUVECs in the corresponding wells. In conclusion, both tumor cells and VEGF alter the migration behavior of MSCs in a transmigration model, indicating a role of tumor cell-derived VEGF to modulate the recruitment of MSCs into sites of angiogenesis.

Corresponding author: Marius Raica, Professor, MD, PhD, e-mail: raica@umft.ro

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18. Does FK506 reduce the size of the watershed area after vascular injury of the sciatic nerve?

Arzu Hizay, B. M. Demirel, Guzide Gokhan, L. Sarikcioglu, N. Demir

Aim: FK506 (also known Tacrolimus, Prograf) is an immunosuppressant drug which is used to prevent rejection after organ transplantation. Although there are several studies on neuroprotective effect of FK506 on brain ischemia, few reports on effects of FK506 after peripheral nerve ischemia have been reported. In the present study, we examined the size of watershed area after stripping of the epineurial vessels and studied the effect of FK506 on reduction of the size of watershed area. Materials and Methods: Forty-eight adult female rats were used and randomly divided into four groups as control, sham, FK506-treated and vehicle-treated. In FK506-treated and vehicle-treated groups epineurial vessels around the sciatic nerve (vasa nervorum) were stripped. Additionally, FK506-treated group were received subcutaneous injection of 5 mg/kg FK506. Percent of watershed area (100 x total watershed areas / total nerve area) after stripping and FK506 treatment was calculated. Results: We found no significant difference in comparison of the total size of watershed areas in FK506 and vehicle-treated groups or even the percent of the watershed area in both groups. Conclusions: We think that this study will be helpful to understand neuroprotective effect of FK506 and will give an insight into sparing of the nerve fibers from vascular injuries of the peripheral nerve.

Corresponding author: Levent Sarikcioglu, Associate Professor, PhD, e-mail: levent@akdeniz.edu.tr, sarikcioglul@yahoo.com

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19. Ultrasonographical and histological cross-sectional study of the human superficial branch of the radial nerve

S. Chakravarthy Marx, S. Dhalapathy, C. Anitha Marx, M. Satish Babu, T. Pulakunta, Vasanthakumar

Purpose: The superficial branch of radial nerve (SBRN) is one of the cutaneous neves biopsied for diagnosis of peripheral neuropathy. The purpose of this study is to find out the cross sectional microanatomy of the (SBRN) at antecubital fossa for changes in the fascicular, non-fascicular components with respect to age and also to find cross sectional area of SBRN in healthy volunteers. Materials and Methods: Sixty SBRN were harvested bilaterally from 30 fresh human bodies (25 males and five females) with ages of 37 to 88 years. Samples were divided into three age groups (G1, 37-55 years; G2, 55-70 years; G3, >=71 years) and studied at different magnifications after histological processing for morphometric analysis - total cross-sectional (Asc), fascicular (Af), and non-fascicular area (Anonf). Fifteen volunteers SBRN cross sectional area was measured by ultrasonography (US). Results: The SBRN was found to have 2-6 fascicles. The differences in the adipose tissue (FAT) areas, between G1 and G2 (p=0.233) and between G2 and G3 (p=0.458) were not statistically significant. However, a significant difference was seen between G3 and G1 (p=0.021). The differences in the non-adipose tissue (FAT) areas, between G1 and G2 (p=0.110), between G2 and G3 (p=0.950) and between G3 and G1 (p=0.210) were not statistically significant. The amount of adipose tissue (FAT) in the epifascicular and interfascicular region increased in elderly individuals (age >=71 years). Ultrasonographic cross-sectional area ranged from 2 to 3 mm(2). Conclusions: Our study revealed comparatively more adipose tissue in human SBRN with advanced age (>=71 years). Findings may help to explain poor prognosis with advancing age following repair. SBRN ultrasonographical Asc was comparable to histological Asc in ages between 37-55 years.

Corresponding author: S. Chakravarthy Marx, PhD, e-mail: marxmanipal@gmail.com

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20. Mast cells contribute to the angiogenesis in non-Hodgkin lymphoma. An immunohistochemical study based on the relationship with microvessel density

Adina Octavia Duse, Raluca Amalia Ceausu, T. Mezei, Anca Maria Cimpean, Pusa Gaje, Hortensia Ionita, I. Jung

Only few data are available in the literature concerning angiogenesis in hematological malignancies. Non-Hodgkin lymphoma classified on the base of molecular profile is frequently characterized by unpredictable behavior that seems to be related to tumor cells and also to the tumor microenvironment. The tumor microenvironment contains blood vessels and a large variety of cells that can play an important role to the progression of angiogenesis and tumor growth. From these, mast cells have been shown to be a source of angiogenic factors. The aim of this work was to investigated the relationships between mast cells and blood vessels in non-Hodgkin lymphoma and reactive lymphoid tissue from three different anatomical sites. Using double immunostaining method CD34/mast cell tryptase we noticed that mast cell density was significantly lower in the follicular lymphoma than in diffuse type lymphoma. The morphology of vessels, the presence of pillars and splitting suggested that intussusceptions is the main mechanism of angiogenesis. In the cases with primary lymphoma of the spleen, we found few mast cells and a high number of blood vessels. Our data suggest that lymphoma-associated angiogenesis is driven in part by the tumor microenvironment, and particularly, by mast cells. On the other hand, our results support the organ-specific tumor-associated angiogenesis in malignant non-Hodgkin lymphoma.

Corresponding author: Anca Maria Cimpean, Associate Professor, MD, PhD, e-mail: ancacimpean1972@yahoo.com

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21. Morphological investigation of cranial sutures in Indian human adult skulls

B. V. Murlimanju, Chandni Gupta, D. Samiullah, Latha V. Prabhu, Mangala M. Pai, Chettiar Ganesh Kumar, M. S. Somesh

Objectives: The goal of the study was to evaluate the gross morphology of the coronal, sagittal and lambdoid sutures in human adult dried skulls and to determine if any difference exists in terms of patency. Materials and Methods: The study included 78 human dry skulls of Indian population. The coronal, sagittal and lambdoid sutures were analyzed using the modified grading scale (Sabini RC and Elkowitz DE, 2006) for quantifying the sutural patency. An open suture was graded as 0, a fused suture as 1 and an obliterated suture as 2, 3 or 4, depending on the extent of obliteration. Results: In coronal suture, the grade 1 suture was seen in 3.9%, grade 2 in 55.1%, grade 3 in 33.3%, and grade 4 in 7.7% of the cases. The sagittal sutures had grade 1 in 2.6%, grade 2 in 46.1%, grade 3 in 37.2%, and grade 4 in 14.1% of the cases. In contrast, the lambdoid suture showed 23.1% grade 1, 55.1% grade 2, 16.7% grade 3, and 5.1% grade 4 sutures. The grade 0 suture morphology was not observed in any of the skulls. Conclusions: When compared with the coronal and sagittal sutures, the lambdoid suture was more likely to be patent. The prolonged patency of the lambdoid suture may be due to external forces acting on it. The greater number of muscles acting on the lambdoid suture compared to coronal and sagittal sutures may be considered as the cause. We believe that, these findings may be helpful to the researchers who are interested in biomedical science and osteopathic manipulative medicine. The findings are also enlightening for the neuroscientists, morphologists, anthropologists and clinicians.

Corresponding author: B. V. Murlimanju, Assistant Professor, MD, e-mail: flutesnowmm@gmail.com, murali.manju@manipal.edu

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22. Human epidermal growth factor receptor 2 (HER2/neu) supraexpression in the mammary tumors

M. Moise, A. Motoc, Andreea Raducan, S. Raducan, V. Niculescu, M. Niculescu

A series of morphological and molecular factors can be used in order to appreciate the prognostic of mammary cancer. A multitude of molecular factors has been investigated in order to identify a prognostic factor, one of the most studied factors being HER2 (human epidermal receptor growth factor). For this purpose, we have carried out an immunohistochemical research, in order to determine the correlation between the presence of morphological factors in tumor formation, different stages of evolution, and the response to therapy. We have correlated HER2 expression with the histological type of carcinomas, the histological differential degree, ER, PR immunohistochemical expression, as well as the available clinical data. Within the studied carcinomas, we have identified a number of 98/156 (62.8%) carcinomas HER2/neu negative, out of which 74/156 (47.4%) did not reveal the presence of HER2/neu and 24/156 (15.4%) have been quantified as being 1+. The HER2/neu supraexpression was encountered in 58/156 (37.17%) cases: in 34/156 (21.8%) cases the immunostaining was interpreted as being 2+ and in 24/156 (15.4%) cases it was interpreted as being 3+. HER2/neu supraexpression should be routinely determined through immunohistochemical methods in all primary mammary tumors, especially in those poorly differentiated.

Corresponding author: Andrei Motoc, Professor, MD, PhD, e-mail: amotoc@umft.ro

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23. E-cadherin in gastric carcinomas related to histological prognostic parameters

Diana Stanculescu, Cl. Margaritescu, A. Stepan, Anca Oana Mitrut

Aim: The immunohistochemical study of E-cadherin in gastric carcinomas, related to tumor aggressiveness factors (invasive and metastatic potential). Materials and Methods: The studied material was gastric resection specimens taken from of 60 patients with gastric cancer, during 2009. The tissue was processed using standard histopathological technique, which allowed the assessment of the well-known morphological parameters of prognostic value. Later on the specimens has undergone immunohistochemical processing for E-cadherin (NCH-38 clone), to evaluate its expression in relation with these prognostic parameters. Results: E-cadherin was positive in 65% from gastric carcinomas, with highest positivity index for well (80% cases) and moderate (17.64% cases) differentiated intestinal type tumors, while a large number of poorly differentiated tumors (55.55%) were E-cadherin negative. Among diffuse type carcinomas, the majority of advanced stage tumors (50% of serosal invasive tumors and 100% of peritoneal disseminated tumors) and also a high number of tumors with vascular and lymphatic invasion (50% and respective 80% cases) represented the E-cadherin negative category (54.54%). The E-cadherin staining was also negative in 75% of lymph node positive diffuse type carcinomas and in all metastatic tumors. Conclusions: We found that irrespective of histologic type, the E-cadherin expression was reduced to negativity in advanced stages of gastric carcinoma.

Corresponding author: Diana Stanculescu, PhD candidate, e-mail: seddiana@yahoo.com

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24. The OPG/RANKL system and zinc ions are promoters of bone remodeling by osteoblast proliferation in postmenopausal osteoporosis

Camelia Vidica Gurban, O. Mederle

RANKL and its decoy receptor osteoprotegerin (OPG) is a mediator system involved in bone resorption and may be responsible for the homeostatic mechanism of normal bone remodeling. The serum levels of both OPG and soluble RANKL (sRANKL), the level of RANKL in primary cultures of osteoblasts, and the bone level of Zn(2+) were measured in six women with postmenopausal osteoporosis and three women without osteoporosis (control group). As compared to control cases, patients with less than 15 years of estrogenic deprivation (cohort 1, n=3) presented increased levels of OPG (109.82%, p<0.002), sRANKL (229.13%, p<0.001) and RANKLOBL (272.35%, p<0.001), and decreased levels of Zn(2+) (67.81%, p<0.001), whereas patients with more than 15 years of estrogenic deprivation (cohort 2, n=3) showed decreased levels of OPG (70.44%, p<0.003), and Zn(2+) (61.41%, p<0.001), and increased levels of sRANKL (181.69%, p<0.002) and RANKLOBL (201.1%, p<0.002). The significantly increased levels of sRANKL and RANKLOBL in postmenopausal osteoporosis demonstrate osteoclastogenesis activation. According to the length of the estrogenic deprivation period, postmenopausal women with osteoporosis presented either increased (cohort 1) or decreased (cohort 2) OPG levels demonstrating osteoblast activation and osteoblast apoptosis stimulation, respectively. The bone levels of Zn(2+) were significantly decreased showing limited proliferation and differentiation of the osteoblasts.

Corresponding author: Ovidiu Mederle, MD, PhD, e-mail: ovidiu.mederle@gmail.com

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25. This article has been retracted

The article has been retracted

This article has been retracted

Corresponding author: The article has been retracted

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26. Importance of early complex evaluation in high-risk pregnancy associated to diabetes mellitus. Case presentation and review of the literature

Lavinia Gheorman, D. Iliescu, Iuliana Ceausu, Daniela Paulescu, I. E. Plesea, V. Gheorman

We report and analyze a case of pregnancy associated with pre-existent diabetes mellitus and fetal congenital anomalies involving neural tube defect (NTD) and congenital heart defect (CHD). We discuss the early antenatal management of such high-risk pregnancies. The clinical course, maternal paraclinic profile and morpho-sonographic investigation of the fetus are described. A 28-year-old pregnant woman with pre-existing diabetes and a pre-pregnancy BMI 31 kg/m(2), without preconception counseling for optimization of glycemic control was evaluated in our center for first trimester genetic screening at 12 weeks of gestation. Considering a high-risk pregnancy, careful fetal morphological assessment by ultrasound was performed; the extensive examination using high-resolution probes, both by transabdominal and transvaginal approach, found hypoplastic left heart syndrome (HLHS) and open spina bifida (OSB). Both anomalies present important difficulties regarding first trimester diagnostic. The couple was informed and chose termination of pregnancy (TOP). We consider that an anomaly scan at 12-13 + 6 gestational weeks by expert operators should be offered to high-risk pregnancies, because it provides the chance to detect the majority of fetal anomalies. This offer for couples the option of an early decision about the management of pregnancy in cases of severe fetal anomalies; postnatal treatment could be discussed as well as TOP and if the latter is chosen, the maternal risk and the potential psychological burden are lowered, as compared with TOP performed in the mid-second trimester.

Corresponding author: Dominic Iliescu, Teaching Assistant, MD, PhD student, e-mail: dominic.iliescu@yahoo.com

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27. Adjusting dento-alveolar morphology with orthodontic mini-implants (miniscrews). A clinical case report

Cristina Dana Bratu, R. V. Pop, Silvia-Izabella Pop, Em. A. Bratu

Mini-implants are increasingly popular for creating skeletal anchorage in clinical orthodontics. The aim of this article is to present and discuss the clinical uses, benefits and drawbacks of the miniscrew implants used to reorder and adjust the dento-alveolar morphology of the overerupted maxillary molars. The loss of the lower premolars and molars very often leads to overeruption of the opposing maxillary teeth, combined with insufficient space for prosthetic restorations. The available treatment options are either a significant reduction of the maxillary teeth, often associated with endodontic treatment, or a complex orthodontic treatment. In the previous years, different cases of orthodontic intrusion with mini-implants were described. In this report, the authors describe a case of a young patient who needed a maxillary molar intrusion in order to get sufficient prosthetic space for an implant supported fixed restoration in the third quadrant. This treatment type preserved maximum tooth structures and allowed a successful mandibular restoration.

Corresponding author: Cristina Dana Bratu, Assistant Professor, e-mail: danacristinabratu@yahoo.com

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28. Isolated neurosarcoidosis

Bianca Szabo, Doinita Crisan, I. Tompa, I. Szabo

Sarcoidosis is an inflammatory granulomatous disease affecting multiple organ systems. Neurosarcoidosis (central nervous system involvement) is seen in approximately 25% of patients with systemic sarcoidosis, although it is subclinical in most of these cases. Clinical presentations and imaging findings in nervous system were varied. Cranial nerve abnormalities were the most common clinical presentation and involvement of the optic nerve in particular was associated with a poor prognosis for visual recovery. A patient is described who presented with decreased visual acuity, hypoesthesia of the face and multiple tumors of the eyelids on both eyes. Initial biopsy of one of the tumor of the eyelids revealed a non-caseating granulomatous inflammatory process with nodular infiltrates made up of epithelioid cells, Langhans-type giant multinucleated cells and mononuclear cells; the diagnosis of sarcoidosis was suspected. After two years of clinical and MRI (Magnetic Resonance Imaging) follow up, the diagnosis of isolated neurosarcoidosis was confirmed by histology. In this study, we analyzed clinical and radiologic records of this patient with biopsy proven and clinically diagnosed neurosarcoidosis for the following reasons: (1) to assess the concordance between abnormalities noted on MRI with neurologic symptoms at presentation; (2) to correlate changes in imaging findings during follow-up with clinical worsening; and (3) to show up the characteristic feature of this case with no general sign/symptoms of sarcoidosis.

Corresponding author: Bianca Szabo, Associate Professor, MD, PhD, e-mail: bianca.szabo@gmail.com

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29. Right accessory hepatic artery arising from the left gastric artery: a case report

Eleni Panagouli, D. Venieratos

During educational dissection of the abdomen in a female Caucasian cadaver, an unusual origin of an accessory right hepatic artery from the left gastric artery was observed. The left gastric artery was the first branch of the celiac trunk, but ended trifurcating into two abnormal large gastric branches and an accessory hepatic artery (d=1.27 cm) which entered the right hepatic lobe at the margin between the two lobes and close to the quadrate lobe. An aberrant hepatic artery branching from the left gastric supplies the left lobe of the liver in most of the cases. The irrigation of the right lobe described by us seems to be extremely rare. Nevertheless, this arterial anomaly can be enlightened by embryonic development. The knowledge of existence of aberrant hepatic arteries, either accessory or replacing, is important because they may influence surgical and interventional radiological procedures.

Corresponding author: Eleni Panagouli, Research and Teaching Associate, MD, e-mail: eleni72000@yahoo.gr

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30. "In situ" mantle cell lymphoma associated with hyaline-vascular Castleman disease

Camelia Dobrea, Mihaela Mihai, E. Danaila, Amelia Gaman, D. Coriu, Iulia Ursuleac

Mantle cell lymphoma (MCL) is a very rare non-Hodgkin B-cell lymphoma, with an aggressive clinical course and poor response to conventional therapy. Few cases of "in situ" MCL were reported in the last years. We present the case of a 31-year-old woman with a unique cervical lymphadenopathy. The morphologic findings are of hyaline-vascular Castleman disease (HV-CD). Immunohistochemical stain for cyclin D1 detects scattered cyclin D1+ cells within the mantle zones of few reactive-appearing lymphoid follicles, corresponding to the definition of "in situ" MCL. We also performed cyclin D1 in other 27 cases of CD (13 HV-CD and 14 plasma-cell CD) but the reported case was the only who associated "in situ" MCL. An adequate immunohistochemical panel, including a marker for cyclin D1, is required to differentiate this neoplasm from follicular hyperplasia. From our knowledge, this is the first reported case of "in situ" MCL associated with HV-CD.

Corresponding author: Camelia Dobrea, MD, PhD, e-mail: cameliadobrea@yahoo.com

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31. McKittrick-Wheelock syndrome: a rare etiology of acute renal failure associated to well-differentiated adenocarcinoma (G1) arising within a villous adenoma

Liliana Ana Tuta, Madalina Bosoteanu, Mariana Deacu, E. Dumitru

Introduction: Large villous adenomas or adenocarcinomas of the rectum can determine secretory diarrhea, associated with a depleting syndrome of prerenal acute renal failure, hyponatremia, hypokalemia, and hypoproteinemia, with favorable prognosis if early detected and properly treated. The syndrome is rare, with approximately 50 cases reported in the literature. Aim: Acute renal failure, caused by fluids and electrolytes hypersecretion, secondary to a malignant rectal villous adenoma is revealed in a 55-year-old patient, admitted with major hydro-electrolytic and acid-base disturbances to our Nephrology Department. Case Presentation: The 55-year-old male patient had a nine months history of mucous diarrhea, for which he was treated unsuccessfully by GP's and infectionists. The symptomatology aggravated progressively and the patient was admitted through ICU with oligoanuria, severe dehydration and hydro-electrolytic and acid-base disturbances. Rectosigmoidoscopy revealed a giant villous adenoma at the rectum. Conservative therapy initially improved, and finally normalized renal function and made possible surgical resection of the tumor, with an excellent evolution afterwards. Conclusions: The McKittrick-Wheelock syndrome is a rare, life-threatening condition that requires interdisciplinary medical diagnosis and treatment, but has a good prognosis if renal function is recovered in time and makes possible curative tumoral resection.

Corresponding author: Liliana Tuta, Associate Professor, MD, PhD, e-mail: tutaliliana@yahoo.com

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32. A case of connected superior and middle trunks of the brachial plexus or with abnormal upper trunk

Selda Yildiz, A. Comert, H. Ozan

During treatment of tumors of nerve sheaths, such as schwannomas and neurofibromas, neurosurgeons should be aware of variations and aberrant formations of brachial plexus. Variations of the branches of brachial plexus are common, but its variations in the level of the roots and trunks are rare. Variations regarding lower trunk were founded more frequent in previous studies. An unusual variant of the brachial plexus was found unilaterally during routine dissection of a 75-year-old male cadaver. It was observed that middle trunk was connected to superior trunk. Deep cervical artery originating from subclavian artery passed between C6 and C7 roots. Similar variations in the brachial plexus were not observed on the contralateral side. In available literature, only two similar bilateral cases were reported. The details of this variation and its clinical significance were discussed. Knowledge about these rare variations in the trunks is very useful in surgical practice and anesthesia.

Corresponding author: Selda Yildiz, MD, e-mail: seldayildiz@gata.edu.tr, aksoyselda@yahoo.com

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33. Conference Announcement: 1st Dermatopathology Course in El Bierzo "Controversies in Dermatopathology", July 19-20, 2012, Ponferrada, Spain

A. Fernandez-Flores

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Corresponding author: Angel Fernandez-Flores, MD, PhD, e-mail: gpyauflowerlion@terra.es

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