ROMANIAN JOURNAL of MORPHOLOGY and EMBRYOLOGY

Vol. 56 No. 1, 2015

1. Dermatology facing autoinflammatory syndrome

Mihail Alecu, Gabriela Coman, Alina Musetescu, Marian Emanuel Cojoaca, Oana Andreia Coman

Cutaneous symptoms are characteristic for the autoinflammatory disorders (AIDs), both in the classical autoinflammatory phenotype and in most disorders included in this syndrome, but they are not specific and inconstant. Several skin disorders (pyoderma gangrenosum and pustular acne) may be encountered either isolate or associated with autoinflammatory symptoms, forming well-defined clinical entities within the autoinflammatory syndrome. The high prevalence of cutaneous manifestations is an important characteristic of AIDs. The presence of cutaneous symptoms in AIDs opens the perspective of understanding the contribution of innate immunity mechanisms involved in skin pathology. It is possible that many diseases present the alteration, in various degrees, of the innate immune mechanisms. Recently, dermatology faced two challenges connected to AIDs. The first involves the diagnosis of skin symptoms in a clinical autoinflammatory setting and the investigative approach to identify a disorder classified as AID. The second is to identify the altered mechanisms of inborn immunity among the pathogenetic mechanisms of known dermatological diseases (e.g., neutrophilic dermatoses). On the other hand, cutaneous symptoms are in certain cases regarded as a criterion to asses the efficacy of specific or non-specific therapies with monoclonal antibodies in disorders included in AIDs. Dermatology mostly benefits from the identification and knowledge of AIDs due to the role of innate immunity in skin pathogeny and also due to the large extent of clinical forms resulting from the association of skin symptoms with other disorders included in this group.

Corresponding author: Coman Gabriela, PhD; e-mail: noime85@yahoo.com

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2. Renal carcinogenesis - insights into signaling pathways

Elena Cojocaru, Ludmila Lozneanu, Simona Eliza Giusca, Irina-Draga Caruntu, Mihai Danciu

Renal cancer represents about 3% of all human malignancies, 96% of cases being sporadic forms and only 4% hereditary. Research in renal tumor pathogenesis is currently oriented on the genetic and proteins framework of the renal cell carcinoma, with the aim to translate the level of knowledge on tumor subtypes from histological to molecular issues, simultaneously with the deciphering of the manner in which the signaling pathways intervene in the pathogenic mechanism. Alterations identified in proto-oncogenes and tumor suppressor genes lead to abnormal and deficient transmission of signal in the signaling pathways, and initiate the carcinogenesis mechanism by increased proliferation of tumor cells. Although it seems obvious that the classic sequence of carcinogenesis is respected at the renal site, unfortunately, the manner in which signaling pathways are involved, in the specific context of renal tumors, is not extensively investigated. This paper assembles recent data in the mainstream regarding the dialogue opened between the molecules in Wnt/beta-catenin, PI3K/AKT/mTOR, and HGF/cMET signaling pathways. The review is also justified by the fact that these molecules may represent potential prognosis markers and/or therapeutic targets.

Corresponding author: Irina-Draga Caruntu, Professor, MD, PhD; e-mail: irinadragacaruntu@gmail.com

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3. Microglial voltage-gated sodium channels modulate cellular response in Alzheimer s disease - a new perspective on an old problem

Bogdan Catalin, Smaranda Mitran, Mihai Ciorbagiu, Eugen Osiac, Tudor-Adrian Balseanu, Laurentiu Mogoanta, Sorin Nicolae Dinescu, Carmen Valeria Albu, Cecil Sorin Mirea, Ionica Daniel Vilcea, Maria Iancau, Veronica Sfredel

Alzheimer s disease (AD) determines gradual loss of cognition and memory function, eventually leading to clinical manifest dementia. The pathogenic mechanisms of AD remain elusive and treatment options unsatisfactory, targeting only symptoms like memory loss, behavior changes, sleep disorders and seizures. These therapies are not stopping the disease s progression, at their best they can only delay it. Accumulating evidence suggests that AD is associated with a microglial dysfunction. Microglia are resident immune cells that provide continuous surveillance within the brain. When excessively activated, microglial response can also have detrimental effects via the exacerbation of inflammatory processes and release of neurotoxic substances. Recently, it was recognized that microglia express voltage-gated ion channels, in particularly voltage-gated sodium channels (VGSC). Pharmacological block of VGSC has been attempted symptomatically in AD to control the epileptic features often associated with AD, as well as to relieve detrimental behavioral and psychological symptoms of dementia. The success of VGSC treatment in AD was unexpectedly variable, ranging from very beneficial to plain detrimental. This variability could not be satisfactorily explained solely by the neuronal effects. This article will try to discuss possible implication of microglial VGSC dysfunction in AD according to available data, own personal experience of the authors and propose a new way to investigate its possible implications.

Corresponding author: Bogdan Catalin, MD, PhD; e-mail: bogdan.catalin@webmail.umfcv.ro

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4. An overview of permanent vascular access in hemodialyzed patients

Ionel Alexandru Checherita, Liliana Ana Tuta, Cristiana David, Ileana Peride, Andrei Niculae, Bogdan Florin Geavlete, Catalin Pricop, Daniela Adriana Ion

In the last decade, because of significant number of end-stage renal disease individuals in need of renal therapy replacement and permanent quest of nephrologist to optimize kidney disease patients quality of life, there is an increased interest in achieving a suitable permanent vascular access, essential for an efficient dialysis. Furthermore, it is of high importance to preserve arteriovenous fistula in optimal condition and therefore, it is vital to correctly understand the histopathology and pathophysiological mechanisms implicated in maturation and well function of dialysis vascular access.

Corresponding author: Catalin Pricop, Assistant Professor, MD, PhD; e-mail: bobopricop@yahoo.com, fizij@yahoo.com

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5. Morphological and quantitative study of collagen fibers in healthy and diseased human gingival tissues

Tatiane Almeida, Thalita Valverde, Paulo Martins-Junior, Heder Ribeiro, Gregory Kitten, Lorenza Carvalhaes

Objectives: This study aimed to evaluate types I, III and IV collagen in healthy gingival tissue and to compare them to gingival tissues suffering from chronic gingivitis and chronic periodontitis. Materials and Methods: Thirty-two man patients were selected. The patients belonged to three diagnostic categories: healthy gingiva (HG), chronic gingivitis (CG) and chronic periodontitis (CP), based on clinical and radiographical criteria. Gingival tissue samples were obtained from patients who underwent periodontal surgery procedures. Hematoxylin and Eosin (HE), Picrosirius red, indirect immunofluorescence by confocal microscopy and quantitative analyses were performed to identify the presence and location of types I, III and IV collagen. Statistical significance was verified using the Kruskal-Wallis test. Results: Samples from HG group showed thick collagen fibers arranged in a parallel pattern. Samples from CG group showed dilated blood vessels; collagen fibers and inflammatory cells were found dispersed throughout the tissue. Samples from CP group showed the extracellular matrix severely damaged, disorganized collagen fibers and large amount of inflammatory cells. The HG group showed an apparent higher expression of type I collagen, when compared to tissues with CG and CP, however no statistical differences were detected (p=0.064). The types III and IV collagen fibers showed no difference in expression in tissues with gingivitis and periodontitis. Conclusions: Following the periodontal disease there was a morphological destruction of the extracellular matrix with lower expression of collagen, which led to a change in tissue architecture that might compromise its functional capacity. There were differences in type I collagen expression among healthy, chronic gingivitis and chronic periodontitis tissue samples.

Corresponding author: Thalita Valverde, PhD student, MS; e-mail: thalitamarcolan@yahoo.com.br

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6. Combined enriched environment/atipamezole treatment transiently improves sensory functions in stroke rats independent from neurogenesis and angiogenesis

Kristina Kuptsova, Elisabet Kvist, Franziska Nitzsche, Jukka Jolkkonen

Functional recovery after cerebral ischemia may be enhanced by activation of the noradrenergic system and by environmental enrichment. The underlying mechanisms have remained elusive, but endogenous neurogenesis and perilesional angiogenesis have been speculated to contribute to the behavioral improvement. To address this question, neurogenesis in the subventricular zone (SVZ) and perilesional angiogenesis (RECA-1) were correlated with behavioral performance in forty Wistar rats subjected to transient middle cerebral artery occlusion (MCAO) or sham-operation. Atipamezole, an alpha2-adrenoreceptor antagonist (1 mg/kg, i.p.), was administered for 10 days together with housing of rats in an enriched environment. MCAO rats and sham-operated rats housed in single non-enriched cages were used as controls. Histological analysis after 28-day behavioral follow-up showed a massive increase in doublecortin (DCX)-positive cells in the SVZ both in MCAO rats housed in single cages and in the enriched environment together with atipamezole treatment whereas perilesional RECA-1 staining for new blood vessels was not altered. Time to the first contact and time to remove sticky stimuli from the forelimbs indicated improved sensory processing, which disappeared after cessation of atipamezole administration. Skilled forelimb use as measured by performance in Montoya s staircase test was not affected by the treatment. There were no correlations between behavioral measures and histology. Thus, sensory facilitation or reversal of hypometabolism by the combined therapy may be the mechanism accounting for the improved behavior after stroke independent from neurogenesis and angiogenesis.

Corresponding author: Jukka Jolkkonen, PhD; e-mail: jukka.jolkkonen@uef.fi

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7. Heterogeneity of collagen secreting cells in gingival fibrosis - an immunohistochemical assessment and a review of the literature

Eugen Ionut Pascu, Catalina Gabriela Pisoschi, Ana-Marina Andrei, Maria Cristina Munteanu, Anne-Marie Rauten, Monica Scrieciu, Oana Taisescu, Mihai Surpateanu, Ileana Monica Banita

Aim: In this work, we compared the histological features of the gingival lesions clinically diagnosed as fibrotic overgrowths due to various etiologic factors as well as an immunohistochemical assessment of fibroblasts phenotypic heterogeneity using the specific labeling for vimentin, alpha-smooth muscle actin (alpha-SMA) and fibroblast specific protein-1 (FSP1). Materials and Methods: Tissue samples were obtained from 12 patients clinically diagnosed with fibrotic gingival overgrowth, divided in four groups. Fragments of gingiva were processed for paraffin embedding. Serial sections were used for routine staining Hematoxylin-Eosin, trichromic Masson and Goldner-Szekely, and for immunohistochemical reactions to label vimentin, alpha-SMA and FSP1 using for signal amplification several techniques (EnVision, LSAB, ABC). Results: Storage of collagen fibers, increase of fibroblast number and frequent presence of inflammatory infiltrate are histological issues of all fibrotic gingival overgrowth. The incidence of granulation tissue varies but the frequency of its presence point the attention to the involvement in collagen metabolism imbalance. Immunostaining for vimentin showed a difference between its expression in samples from different groups. Except the cases of fibrosis induced by orthodontic devices, cells positive for alpha-SMA were rare. FSP1-positive fibroblasts were the most frequent in all cases from all the groups selected for this study. Conclusions: The phenotype of fibroblasts is different in gingival fibrosis in relation to the risk factor, at present the most common being vimentin-positive and FSP1-positive fibroblasts. Myofibroblasts are rare in gingival fibrosis, the most numerous being in local lesions caused by wearing orthodontic devices and in syndromic fibromatosis. Further studies are required to elucidate the manner in which the active fibroblasts are recruited in relation to the etiologic factor of gingival overgrowth.

Corresponding author: Catalina Gabriela Pisoschi, Professor, PharmD, PhD; e-mail: c_pisoschi@yahoo.com

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8. Study regarding the microscopic aspects of pilo-sebaceous units after antiandrogen treatment in hirsute women

Maria Rotaru, Ioan Gheorghe Totoianu, Anca Ileana Sin, Laura Gheuca Solovastru

Aim: To analyze the morphological data of pilo-sebaceous units in hirsute women before and 12 months after the antiandrogen treatment with Cyproterone acetate (CPA) 100 mg/day. Materials and Methods: Fourteen female patients with idiopathic hirsutism that followed an antiandrogen treatment with CPA were biopsied from the androgen-dependent area of the chin before and 12 months after the treatment. Routine sections were stained with Hematoxylin-Eosin, Masson, Van Gieson, Sirius red and picric-indigocarmine, while additional sections were immunostained for S100 protein and vimentin. Electron microscopy was performed in two cases with Langerhans cell hyperplasia. Results: On biopsies-stained sections, an increased number of hair follicles, the deeper part of the epithelial sheath of the hair follicle with epithelial buds, hyperplasia of sebaceous glands, and no inflammatory infiltrate were noticed. Langerhans cells identified with S100 protein and vimentin were normal in terms of numbers and distribution. After the administration of the treatment, atrophy of the pilo-sebaceous units was visible in nine (64.2%) cases, while inflammatory infiltrate and cells included in the vacuoles of the basal layer of the epidermis became apparent. In six of the cases treated with antiandrogens, a marked hyperplasia of Langerhans cells was noticed. In conclusion, the benefit of antiandrogen treatments is supported by atrophy of the hair follicle and the sebaceous glands. The activation of Langerhans cells associated with inflammatory infiltrate in the dermis and hair follicles could be considered as a local consequence of the involution process of hair follicles after the administration of the treatment.

Corresponding author: Maria Rotaru, Associate Professor, MD, PhD; e-mail: mrotaru07@gmail.com

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9. In vitro evaluation of curcumin effects on breast adenocarcinoma 2D and 3D cell cultures

Hussam Abuelba, Carmen Elena Cotrutz, Bogdan Alexandru Stoica, Laura Stoica, Doinita Olinici, Tudor Petreus

Breast adenocarcinoma cell line MDA-MB-231, even if it expresses low levels of E-cadherin, still readily form multicellular aggregates of cells, namely spheroids. Curcumin is a diarylheptanoid antitumoral drug while it significantly inhibits cell migration, invasion, and colony formation in vitro and reduces tumor growth and liver metastasis in vivo. Curcumin photoactivation may enhance antiapoptotic role against cancer cells. Aim: To evaluate the effect of low curcumin concentrations, ranged from 1.9 to 15 micro-M, with and without photoactivation, using a manufactured 670 nm LED-matrix. A secondary aim was to evaluate the ideal method to produce easy-to-use tumor cell spheroids, comparing two low adherence plate supports. Materials and Methods: Breast adenocarcinoma cell line MDA-MB-231 were cultured according to 2D monolayer and 3D spheroid models then submitted to normal and photoactivated curcumin in micromolar concentrations. MTT assay was used to evaluate cell viability following curcumin application on cells. On 2D cell cultures, curcumin inhibits cell tumor development and proliferation at concentrations of 15 micro-M, with a viability of 65.7% at 48 hours incubation time. A decreased viability up to 25% for a concentration of 15 micro-M was recorded following photoactivation and cytotoxic action on breast cancer tumor cell line continued at concentrations of 7.5 and 3.75 micro-M. Curcumin photoactivation increases pro-apoptotic effects in both 2D and 3D tumor cell culture models and also responsiveness to curcumin is slightly reduced in spheroid-like structures. Thus, 3D tumor cell culture systems appear to be the ideal environment for in vitro assays regarding anticancer drug effects on cell viability.

Corresponding author: Carmen Elena Cotrutz, Professor, MD, PhD; e-mail: cotrutz@yahoo.com

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10. Effects of therapy with two combinations of antibiotics on the imbalance of MMP-2/TIMP-2 in chronic periodontitis

Marilena Bataiosu, Citto Iulian Taisescu, Catalina Gabriela Pisoschi, Eugen Ionut Pascu, Mihaela Jana Tuculina, Luminita Daguci, Constantin Daguci, Ileana Monica Banita

Periodontitis represents a chronic bacterial infection that induces immuno-inflammatory conditions affecting gingiva and tooth-supporting tissues. The role of some biological mediators in periodontal disease was widely investigated, especially that of MMP-8 and MMP-9. Recently, MMP-2 was also considered to be an appropriate therapeutic target for prevention of periodontal disease progression. However, effects of the combination of metronidazole with amoxicillin or spiramycin on the release and activation of MMP-2 and the balance MMP-2/TIMP-2 were rarely studied. This study was designed to assess the influence of two combinations of antibiotics used for treatment of periodontitis on the balance MMP-2/TIMP-2. Gingival samples obtained from patients with no pharmacological treated chronic periodontitis and those receiving either the association between amoxicillin-metronidazole and spyramicin-metronidazole were processed for paraffin embedding and then used to perform immunohistochemical reactions in order to detect MMP-2 and TIMP-2. All subjects were evaluated clinically and radiographic at the first visit and after treatment completed, the Loe and Silnees gingival index at six sites per tooth for the whole mouth being recorded. Statistical analysis was performed using non-parametrical techniques. Gingiva samples from untreated chronic periodontitis patients revealed a diffuse positive reaction for MMP-2 in the epithelium and also in fibroblasts and macrophages from the lamina propria. For gingiva samples from patients treated with antibiotics, MMP-2 positive reaction was restricted to deep epithelial layers and few cells of the connective tissue. No significant difference was observed for TIMP-2 expression. The clinical indexes were in accordance with immunohistochemical results. After treatment, gingival index values were significantly lower then before (p<0.001) in both groups treated with antibiotics. Conclusions: The two combinations of antibiotics tested in our study seem to have a dual ability to reduce inflammation as well as to inhibit MMP-2 activity.

Corresponding author: Citto Iulian Taisescu, Lecturer, MD, PhD; e-mail: taisescu@yahoo.com

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11. E-cadherin/CD44 immunophenotype in the epithelial-mesenchymal transition of bladder urothelial carcinomas

Alex Emilian Stepan, Daniel Pirici, Maria Balasoiu, Marius Bogdan Novac, Andrei Ioan Drocas, Raluca Niculina Ciurea, Desdemona Stepan, Dan Ionut Gheonea, Cristiana Eugenia Simionescu

The alteration of epithelial stability, which includes changes in the expression of E-cadherin and CD44, is one of the complex biomolecular mechanisms involved in the tumoral epithelial-mesenchymal transition (EMT) process. We followed the E-cadherin/CD44 immunophenotype by single and double detections in 25 cases of bladder urothelial carcinomas. Our study investigated simultaneously the differences in expression of the two markers, in different tumoral compartments, according to the prognostic parameters of the lesions. The study indicated significant differences in the expression of E-cadherin in relation to tumor grade, depth of invasion, tumor stage and Ki-67 proliferation index (PI), both intratumoral and at the advancing edge. For CD44, expression differences were found between the tumor grades in intratumoral sites, while for both intratumoral and advancing edge compartments the differences occurred for the depth of tumor invasion, tumor stage and Ki-67 PI. The only differences in the expression of the two markers in relation with the presence of lymph node metastasis were for E-cadherin at the advancing edge. In this study, the intratumoral E-caderin-/CD44- immunophenotype, respectively E-caderin-/CD44+ at the advancing edge were associated with the tumor aggressiveness analyzed parameters. The maintenance of CD44 expression at the advancing edge represents a negative prognostic factor for bladder urothelial carcinoma and supports the implication of EMT process, through the existence at this level of a cell population with particular properties.

Corresponding author: Dan Ionut Gheonea, MD, PhD, MSc; e-mail: digheonea@gmail.com

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12. Expression of TLR4 protein is reduced in chronic renal failure: evidence from an experimental model of nephron reduction

Ina Maria Kacso, Gabriel Mircea Borza, Catalin C. Ciuce, Andrei Birsan, Raluca Cristina Apostu, George Calin Dindelegan, Cosmina Ioana Bondor, Alina Ramona Potra, Mihai G. Netea, Cornel Catoi

Toll-like receptor 4 (TLR4) signaling is involved in various acute and chronic renal lesions and contributes to inflammation and fibrosis in several organs; the latter are important determinants to the progression of chronic kidney disease (CKD). We aimed to assess TLR4 expression in progressive CKD and relate it to severity of kidney damage, using an experimental nephron reduction model. Male Wistar rats were subjected to subtotal nephrectomy using the ligation technique, after 12 weeks of observation, serum creatinine and proteinuria were determined, animals were sacrificed, glomerulosclerosis and interstitial scarring were quantified histologically, and TLR4 expression was assessed by immunohistochemistry. Sham-operated rats served as controls. Case animals had significantly higher creatinine, proteinuria, glomerulosclerosis and tubulointerstitial involvement. TLR4 expression was prominent in proximal tubes, less staining was observed on infiltrating inflammatory cells. Percentage of TLR4-positive tubes was reduced in the subtotal nephrectomy animals, when compared to controls (0.67+/-0.09 versus 0.79+/-0.07, p=0.003). Percentage of TLR4-positive tubes correlated inversely to markers of kidney damage: to proteinuria (r=-0.55, p=0.02), serum creatinine (r=-0.53, p=0.01); percentage of glomeruli with glomerulosclerosis (r=-0.54, p=0.01) and tubulointerstitial score (r=-0.36, p=0.01). As TLR4 staining appears in tubular casts only in nephrectomy animals, shedding from damaged tubular cells is a very likely explanation for the reduced TLR4 expression in the kidneys of subjects with experimental nephron reduction.

Corresponding author: Ina Maria Kacso, Associate Professor, MD, PhD; e-mail: inakacso@yahoo.com

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13. TNF-alpha evaluation in tonsil cancer

Adriana Turculeanu, Carmen Aurelia Mogoanta, Elena Ionita, Carmen Silvia Avramescu, Mihai-Catalin Afrem, Adrian Costache

Squamous cell tonsil carcinoma is the most frequent form of oropharyngeal cancer, representing 70-80% of the total of head and neck malignant tumors. Poor clinical symptoms make that 60-80% of patients with squamous cell tonsil carcinoma have a late diagnosis, in the third and fourth stages, when the tumor exceeds the organ limits, invading the pharyngeal wall or the tongue base, being associated with metastases in the laterocervical lymphatic ganglions. The tumor necrosis factor alpha (TNF-alpha) represents an important inflammation mediator associated to carcinogenesis and even to tumor progression. We evaluated the seric values of TNF-alpha in a group of patients with tonsil cancer in comparison to a group of patients with chronic tonsillitis, as well as the reaction of mastocytes and macrophages in the two types of tonsil lesions. Seric levels of TNF-alpha in squamous cell tonsil carcinoma were quite high, varying from 1000 to 2000 pg/mL, and in four patients, with poorly differentiated tonsil carcinoma in the fourth stage, the TNF-alpha values varied from 2000 to 4000 pg/mL. In the patients undergoing radiotherapy, the TNF-alpha seric levels were within normal limits. In chronic tonsillitis, the TNF-alpha seric level varied from 10 to 200 pg/mL. There were not observed any significant differences between the two types of tonsil lesions, regarding the macrophages and mast cells density on the surface unit.

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

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14. Histone acetylation regulates the expression of HoxD9 transcription factor in endothelial progenitor cells

Florin Iordache, Andrei Constantinescu, Eugen Andrei, Horia Maniu

The homeobox (Hox) genes encode transcription factors that are involved in the morphogenesis of body. Recent data showed that the HoxD transcription factors control the cardiovascular system development, by modulation of endothelial cell proliferation and differentiation. For our knowledge, the role of histone acetylation in expression of HoxD9 has not been studied to date; therefore, the aim of this study was to investigate the expression of HoxD9 in endothelial progenitor cells after treatment with valproic acid (VPA), a histone deacetylase inhibitor. Our results showed that VPA inhibits the histone deacetylases leaving chromatin in an acetylated state corresponding to a decondensate conformation. qRT-PCR and Western blot assays showed that the expression of HoxD9 in endothelial progenitor cells treated with VPA was increased at both gene and protein level, suggesting that acetylation regulates HoxD9 expression. Furthermore, flow cytometry analysis revealed that the expression of endothelial specific markers such as CD31, CD105, CD117 and VEGFR2 was decreased in the presence of acetylating agent, VPA. The capacity of endothelial progenitor cells to form vascular networks on Matrigel was also reduced in the presence of VPA. In conclusion, investigating the role of histone acetylation in the regulation of accessibility of transcription factors to genes involved in differentiation can contribute to understanding epigenetic mechanisms underlying the commitment of stem cells.

Corresponding author: Florin Iordache, PhD; e-mail: floriniordache84@yahoo.com

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15. Study of restorative processes in brain laceration in the first seven days after traumatic brain injury

Charoula Florou, Bogdan Catalin, Oana Badea, Tudor-Adrian Balseanu, Cristina Eugenia Vasilescu, Laurentiu Mogoanta, Florin Grosu, Marius Matei, Adriana Turculeanu

Traumatic brain injuries represent the main cause of death and invalidity all over the world. Persons surviving a severe traumatic brain injury often present long-term disabilities, sensitive and motor deficits, cognitive, vegetative or mental disorders. Brain injuries are directly caused by the traumatic agent, and indirectly caused by the action of cells involved in the restorative process. The main cells involved in the restorative process are microglias and astrocytes. By using an experimental model, we investigated the reaction of these cells in the first week after a severe brain injury, followed by brain laceration. Of the two cell types, the most rapid and intense reaction was held by the macroglias, also known as resident macrophages of the central nervous system. Alongside the activation of local microglias, in the restorative process there were also involved blood monocytes that turned into macrophages. 24 hours after the injury, the number of macrophage cells/mm(2) at brain wound level increased 2-4 times, after three days - 10-12 times, and after seven days - over 20 times. The astrocyte reaction was slower, their activation being signaled no sooner than three days from injury, when their number in the perilesional brain parenchyma increased approximately two times, while after seven days - approximately 4-5 times. Both astrocytes and macrophages (microglias), besides their beneficial effects in restoring traumatic brain injuries, may have unfavorable effects upon the nervous cells in the immediate proximity of the injury. Destruction of vascular network by the traumatic agent, and the extremely slow restore of vascularization, partially explain brain neurons death on extend areas.

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

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16. Evaluation of the implantation site morphology in spontaneous abortion

Maria Magdalena Manolea, Anda Lorena Dijmarescu, Florina Carmen Popescu, Marius Bogdan Novac, Damian Ditescu

The aim of this study was the characterization of the implantation site through histological and immunohistochemical exams and the evaluation of the changes that appear in the pregnancies ended by spontaneous abortion compared to normal pregnancies ended by requested abortion. One hundred eight patients were divided in two groups: the study group that included 58 patients with spontaneous abortion and the control group that included 50 patients with requested abortion. There has been made uterine curettage in all the cases after a complete preoperative evaluation and the obtained product was sent for histopathological evaluation and immunohistochemical study using a VEGF antibody. Studying the histological sections, we noticed the vasculogenesis stages chronology and then according to the histological aspects of normal pregnancy we noticed the histological changes that occurred at the site of implantation in the cases with pathological pregnancies ended by miscarriage. Our results from this study seem to indicate a correlation between decidual vascular changes and the appearance of miscarriage. In pregnancies ended by miscarriage, we found delays in the trophoblast development according to the gestational age at which the event abortifacient happened. The study emphases the temporal differentiation of utero-placental angiogenesis comparing to villous vasculogenesis and angiogenesis in the first trimester miscarriage and normal pregnancy. At the control group, VEGF expression was positive in 88% of cases, while in the study group, pregnancies ended by spontaneous abortion, positive expression of VEGF was present in only 31% of cases. Our data suggest vascular disorders and are in concordance with other histological and ultrasound studies postulating the idea of a link between miscarriage and placental vascular bed pattern changes.

Corresponding author: Maria Magdalena Manolea, University Assistant, MD, PhD; e-mail: manoleamaria@gmail.com

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17. The immunoprofile of interstitial Cajal cells within adenomyosis/endometriosis lesions

Isabela-Magdalena Draghici, Liviu Draghici, Manole Cojocaru, Carmen-Loredana Gorgan, Camelia Doina Vrabie

Adenomyosis and endometriosis are lesions which have aroused the interest for the investigation of antibodies specific to the structures from the composition, but also for the cause behind the appearance of these lesions in completely different structures. The impact they have on fertility is not known entirely, for they are difficult to diagnose. Endometriosis causes infertility and it is a hard to treat lesion. The research performed in the last years has been focused on the so-called linkage analysis, or reverse genetics. It refers to identifying the genes which are prone to developing this affection. We investigated clinically 40 female inpatients (n=40) who had underwent genital surgery and received a variegate diagnosis in the Sf. Ioan Emergency Hospital, Bucharest, Romania, between January-September 2014 and also their histopathology and immunohistochemistry. We proceeded with the histopathology examination in order to establish a diagnosis in respect to the admission diagnosis and then, using the ABC (Avidin-Biotin complex) method, we analyzed the immunohistochemistry of the following markers: S100 protein (for detection of ganglia and nerve cells), CD117/c-kit (selective detection of interstitial Cajal cells - ICC), desmin and vimentin (intermediary filaments for detecting ICC-like cells, which cohabit with uterine myocytes and are not contractile cells) and CD10 (a sensitive and useful immunomarker in the diagnosis of endometrial stroma and, in some cases, of neoplasia). Our study, regarding the immunoprofile of some markers of adenomyosis/endometriosis lesions, supports the hypothesis that the interstitial Cajal cells are non-reactive, they are not in relationship with investigated lesions, but CD10 is a very useful marker to highlight the endometrial stroma in query cases.

Corresponding author: Liviu Draghici, Assistant Professor, MD, PhD; e-mail: liviudraghici@hotmail.com

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18. Cell populations involved in the processes of local mucosal defense in extended partially edentulous and completely edentulous patients. Clinical and immunohistochemical study

Monica Mihaela Craitoiu, Claudiu Margaritescu, Alma Florescu, Roxana Pascu, Alina Monica Picos, Andrei Picos, Mariana Sabau, Vasile Nicolae

Considering the fact that extended partial edentation and complete edentation have high incidence rates worldwide, the necessity for correct prosthetic treatment is very important. We performed a clinical study on 37 extended partially edentulous patients and completely edentulous patients, who were divided into three groups. We also performed a morphological study using classic techniques of histology and immunohistochemistry methods on sections obtained from oral mucosa fragments collected from these patients and processed by paraffin embedding technique. To identify the cell populations present in the inflammatory processes, we used the CD20, CD8, CD3 and CD68 markers. In the studied cases, we found the presence of changes that have interested both the epithelium and lamina propria. Epithelium showed in particular epithelial hyperplasia aspects, with orthokeratinization and parakeratinization areas and, in some sections, areas of ulceration. We found the inflammatory process present in the lamina propria to be chronic and it consists in particular of lymphocytes, plasma cells and macrophages. This process was differentiated in intensity from one case to another, but varied even within the same case, from one area to another. Inflammation was determined by the local microbial flora enhanced by the action of prosthetic appliances or by the prolonged edentulous state. We observed more intense changes in denture wearers patients. The inflammatory response indicates the reactivity of the edentulous mucosa in response to local aggression, the specific defense mechanism coexisting with the nonspecific defense mechanism, with predominance of cellular immune defense.

Corresponding author: Monica Mihaela Craitoiu, Lecturer, MD, PhD; e-mail: mcraitoiu@yahoo.com

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19. Identification of the anatomical elements used in periodontal diagnosis on 40 MHz periodontal ultrasonography

Radu Chifor, Mindra Eugenia Badea, Mihaela Hedesiu, Ioana Chifor

Gingival inflammation is highly prevalent among adult populations in all regions of the world. High rates of chronic periodontitis have been reported worldwide. The methods for assessing the gingival inflammation and periodontal disease would need more precision and less user-dependence. The aim of this study consists in identifying the information for diagnosis and staging of periodontal disease using 40 MHz periodontal ultrasonography. Our in vivo study has been made on 49 teeth of 10 patients with marginal periodontal disease. Standard clinical and radiological periodontal examinations were performed. Afterwards the results were compared with the information obtained from images recorded with Ultrasonix SonoTouch used at 40 MHz. On the ultrasound images, were performed very accurate measurements between the cortical bone and the cement-enamel junction or the root wall compared with the measurements made on intraoral digital radiographs. Those measurements could be used to diagnose the bone resorption. In order to monitor the gingival inflammation could be recorded the width of the attached gingival mucosa and the height of the gingival margin on ultrasound images. 40 MHz periodontal ultrasonography is a reliable imagistic method for identifying the necessary anatomical elements in order to make an accurate periodontal diagnosis for the examined area.

Corresponding author: Mindra Eugenia Badea, Professor, MD, PhD; e-mail: mebadea@umfcluj.ro

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20. The loss of Ivy cells and the hippocampal input modulatory O-LM cells contribute to the emergence of hyperexcitability in the hippocampus

Karoly Orban-Kis, Timea Szabadi, Tibor Szilagyi

Epilepsy is a frequent neurological disorder that affects directly 0.5-1.5% of the world s population. Despite advances regarding therapy, about 30% of patients cannot be relieved of seizures, mainly because the pathophysiological mechanisms are still not elucidated completely. Basket, axo-axonic, bistratified, oriens-lacunosum moleculare (O-LM) and Ivy cells exert spatially and temporary different inhibition on principal neurons. Our aim was to evaluate the alterations of these interneuron populations during epileptogenesis. We induced status epilepticus in male Wistar rats using intraperitoneal pilocarpine injection, which was followed, after a latency period, by spontaneous recurrent seizures (SRS). Nissl staining was used for the analysis of gross morphological changes, whereas triple immunofluorescent-labeled sections (parvalbumin, somatostatin, neuropeptide-Y) were used for differentiation of the selected interneuron types. Putative interneurons identified by their neurochemical contents were quantified, and the cell density was calculated. Although animals developing SRS showed similar behavior, the degree of hippocampal sclerosis was different. In animals with hippocampal sclerotic cell death pattern the density of perisomatic inhibitory neurons was higher, but not significantly. The dendritic inhibitory bistratified cells were preserved, whereas the number of O-LM cells showed a significant decrease. A substantial loss was observed in the number and density of Ivy cells. We suggest that the loss of hippocampal input modulatory O-LM cells, and overall excitation controlling Ivy cells, has a role in the emergence of hyperexcitability. In the same time, alterations of output controlling interneurons might contribute to the propagation of the pathological synchronization to the cortex.

Corresponding author: Karoly Orban-Kis, Lecturer, MD, PhD; e-mail: orbank@gmail.com

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21. Macrophage response in patients diagnosed with aseptic necrosis of the femoral head presenting different risk factors

Diana Kamal, Rodica Traistaru, Constantin Kamal Kamal, Dragos Ovidiu Alexandru, Daniela Adriana Ion, Dan Cristian Grecu

Aseptic necrosis of the femoral head is a condition caused by partial or total interruption of blood supply to the femoral head. The diminished blood supply causes necrosis of the cellular elements and of the bone marrow, followed by the collapse of the bone structure, events that ultimately lead to the destruction of the bone tissue, the appearance of local pain and loss of function in the affected coxofemoral joint. The importance of this condition is that it mainly affects young adults aged 30-50 years, active from a socio-professional standpoint, and increased life expectancy. The material studied to achieve CD68 immunostaining was represented by bone fragments from the area of necrosis and from the adjacent areas of the femoral heads, harvested from 39 patients when performing hip arthroplasty surgery. The patients were diagnosed with aseptic necrosis of the femoral head and hospitalized in the Clinic of Orthopedics and Traumatology, Emergency County Hospital of Craiova, Romania, from June 2014 to January 2015. The 39 patients included in the study were divided into four categories according to presented risk factors (alcohol, alcohol and smoking, trauma, corticosteroids). All the 39 cases had positive immunostaining for CD68, macrophage being highlighted both in the area of necrosis and in the adjacent areas. We noted significant differences in the number and arrangement of macrophages in patients presenting different risk factors. The highest number of macrophages was present in patients presenting a risk factor corticosteroids, and the lowest number of macrophages was found in patients who had trauma as the main risk factor.

Corresponding author: Rodica Traistaru, Associate Professor, MD, PhD; e-mail: rodicatraistru@hotmail.com

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22. Synovial inflammation in patients with different stages of knee osteoarthritis

Razvan Ene, Ruxandra Diana Sinescu, Patricia Ene, Monica Mihaela Cirstoiu, Florin Catalin Cirstoiu

The synovium is an intra-articular mesenchymal tissue and essential for the normal joint function. It is involved in many pathological characteristic processes and sometimes specific for this distinctive tissue. In this study, we refer to synovial proliferative disorders according to the stage of osteoarthritis (OA) disease. Forty-three patients with knee OA were treated in the Department of Orthopedics and Traumatology, Emergency University Hospital of Bucharest, Romania, in the last two years. In all cases, we used at least five criteria for the knee OA: knee pain, knee joint tenderness, no palpable warmth over the knee, stiffness, erythrocyte sedimentation rate and C-reactive protein levels. In all the cases the synovial tissue was selected by the orthopedic surgeon. X-ray examination was taken in every case of the affected joint. Patients who were considered to have early OA underwent arthroscopic synovial biopsy of the symptomatic joint. Synovial tissue samples from patients with late OA were obtained at the time of knee joint arthroplasty. Microscopic examination in early osteoarthritis revealed for more than half of patients with synovial biopsy through arthroscopic technique having synovitis lesions with mononuclear infiltrates, diffuse fibrosis, thickening of the lining layer, macrophages appearance and neoformation vessels also. The synovitis seen in advanced OA knees tends to be diffuse and is not mandatory localized to areas of chondral defects, although an association has been reported between chondral defects and associated synovitis in the knee medial tibio-femoral compartment. The overexpression of mediators of inflammation and the increased mononuclear cell infiltration were seen in early OA, compared with late OA.

Corresponding author: Razvan Ene, MD, PhD; e-mail: razvan77ene@yahoo.com

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23. Histochemical and immunohistochemical evidence of tumor heterogeneity in colorectal cancer

Ovidiu Mircea Zlatian, Maria Victoria Comanescu, Alexandra Floriana Rosu, Lucica Rosu, Mihai Cruce, Alice Elena Gaman, Cornelia Daniela Calina, Veronica Sfredel

Introduction: Intratumoral heterogeneity implies the existence of differences between tumor cells, which can best be shown by histochemical and immunohistochemical techniques. The histological study is a mandatory step in any research aimed at characterizing tumor heterogeneity. Immunohistochemistry (IHC) also plays an important role in the differentiation of tumor types, assessing aggressiveness. Materials and Methods: Investigated group consisted of 50 patients with colorectal adenocarcinoma, for each were recorded clinicopathological data and harvested samples intraoperatively, which were included in paraffin blocks. We perform Hematoxylin-Eosin staining for histological grade and other indices. IHC study used Avidin-Biotin-Peroxidase (ABC), with the markers: CK7, CK20, MUC1, MUC2, Ki-67, PCNA, p53, KRAS, BCL2, PTEN, EGFR. The resulting data were analyzed by statistical methods. Results: Most of colorectal adenocarcinoma studied had no special histological features and had G2 grade. IHC detected in most cases the CK20+/CK7- phenotype (78%) and MUC1 (74%) protein expression. The proliferation markers (Ki-67 and PCNA) were present in all tumor mass with a variable index, which shows high intratumoral heterogeneity, but p53 and KRAS were distributed more uniformly, showing low intratumoral heterogeneity. PTEN was expressed nuclearly in 86% of the cases and EGFR in 42%. Conclusions: The expression profiles of cytokeratins and mucins in the colorectal adenocarcinomas are useful in defining tumor phenotypes with different prognosis and therapy. We found a significant positive correlation between KRAS protein expression and BCL2 and TP53 expression. The study demonstrated the intratumoral and intertumoral heterogeneity, expressed at phenotypic level.

Corresponding author: Ovidiu Mircea Zlatian, University Assistant, MD, PhD; e-mail: ovidiu.zlatian@gmail.com

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24. The prevalence of the red cell morphology changes in patients with type 2 diabetes mellitus

Marius Cristian Neamtu, Stefania Craitoiu, Elena Taina Avramescu, Denisa Marilena Margina, Manuela Violeta Bacanoiu, Denisa Turneanu, Rucsandra Danciulescu Miulescu

Previous studies have shown that hematological alterations are a common finding in patients with diabetes. The aim of our study was to estimate the prevalence of the red cell morphology changes in diabetic patients and their correlation with markers of glycemic control. Thirty patients with type 2 diabetes mellitus were recruited for this study. Patient demographics, relevant concomitant illnesses and medical history were recorded. Anthropometric, biochemical parameters (fasting plasma glucose - FPG, glycated hemoglobin - HbA1c, glomerular filtration rate - GFR) and morphology of blood smear were assessed. Results were compared with the same measurements in 30 subjects without diabetes mellitus. The groups were similar in terms of age and gender but there were statistically significant differences for the recorded parameters in patients of study group and control subjects. Regarding the assessment of FPG, in the study group were recorded averages of 217.70+/-73.20 mg/dL compared with controls that compared with controls that had a blood glucose value of 90.03+/-6.59 mg/dL. In the study group, mean HbA1c was 7.95+/-1.99%. For the control group, the mean value of HbA1c was 5.65+/-0.32%. In the study group, GFR ranged between 47.70 and 118.90 mL/min./1.73 m(2). For the control group, GFR values were between 88.00 and 130.00 mL/min./1.73 m(2). In the analysis of blood cytology for the study group, there were changes in the smear type hypochromia, anisocytosis and poikilocyosis (20 patients - 66.66%). In terms of red cell morphology, changes were recorded anulocytes type, red cells in mark to the target fired (codocytes), bream (leptocytes), schizocytes, and red cells in drop (dacryocytes). We observed a high prevalence of the red cell morphology changes in diabetic patients compared with non-diabetic subjects. Our findings suggest the need of screening for routine hematological tests in type 2 diabetes mellitus.

Corresponding author: Marius Cristian Neamtu, Lecturer, MD, PhD; e-mail: drcristianneamtu@yahoo.com

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25. A pilot study on the role of fractal analysis in the microscopic evaluation of colorectal cancers

Liliana Streba, Mircea Catalin Fortofoiu, Carmen Popa, Daniela Ciobanu, Corina Lavinia Gruia, Stelian Stefanita Mogoanta, Costin Teodor Streba

Introduction: Colorectal cancer (CRC) represents one of the most common cancers worldwide; its growing incidence and prevalence quickly transforming it into a major health burden. Globally, survival varies from one country to another and constantly remains significantly low, despite increasing diagnostic efforts and tools. Fractal geometry and, specifically, fractal dimension (FD) are interesting tools to quantify cellular elements. In this paper, we aimed to identify and quantify by fractal analysis the elements obtained from medical images from pathological and immunohistochemical investigations of colonic biopsy fragments. Materials and Methods: We prospectively selected the study group between September 2014 and January 2015, from patients who underwent surgery for previously diagnosed CRC at the Emergency County Hospital, Craiova, Romania. We performed the histological and immunohistochemical studies by following standardized protocols. Anti-Ki67, anti-p53 and anti-VEGF-C antibodies were used for immunostaining. We performed the fractal analysis with an in-house tool and we performed statistical tests on the results. Results: We have included 41 (29 males) consecutive patients with different characteristics; after analyzing the FDs we found significant differences between adenocarcinomas and the other types of colonic cancers (p<0.001). However, we found no significant differences between most types of CRCs. We found significant statistical differences when compared well-differentiated tumors with all other stages (p<0.001). Conclusions: Fractal analysis with the calculation of FDs is a novel, interesting tool, for determining the pathologic diagnosis of CRCs and may further improve diagnostic and prognostic rates, thus improving patient care.

Corresponding author: Costin Teodor Streba, Teaching Assistant, MD, PhD; e-mail: costinstreba@gmail.com

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26. CT characters versus morphopathological characters in pharyngeal squamous cell carcinoma

Ana Magdalena Bratu, Iulia Alecsandra Salcianu, Daniel Alin Cristian, Constantin Zaharia, Garofita Olivia Mateescu, Marius Matei, Serban Vifor Gabriel Bertesteanu, Gabriela Lica, Elena Carmen Niculescu

Pharyngeal squamous cell carcinoma is a rare neoplasm, whose incidence increases with age. Computed tomography (CT) imaging is an easy way to explore the pharyngeal region, having the advantage of being able to highlight and characterize the existence of a tumor in this region, and to determine its local extension and lymphatic metastasis. In this group were included a total of 27 patients, who, following the histopathological findings were diagnosed with pharyngeal squamous cell carcinoma and who have previously received a CT scan. CT examination protocol included a native scan and post-intravenous administration of contrast medium, in both the arterial phase and in parenchymal and venous phase. The scan was made with 2 mm thin sections, subsequently were performed coronal and sagittal reconstructions. The examination plan included the thoracic region down to the aperture. The paper tries to establish correlations between the morphological appearance and semiological computed tomography characters of the lesions.

Corresponding author: Ana Magdalena Bratu, MD, PhD; e-mail: anaconst@clicknet.ro

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27. Relevant infrastructural alterations in invasive pancreatic ductal adenocarcinoma

Vlad Denis Constantin, Gabriel-Valeriu Mirancea, Ana-Maria Morosanu, Nicolae Mirancea

In this study, we focus our interest on some peculiar infrastructural abnormalities detected in a pancreatic cancer case. Our electron microscopic observations underline the high plasticity of the pancreatic parenchyma cells. Tumor pancreatic exocrine lesions are represented by putative ductal and acinar cells, which proliferate and grow in a haphazard pattern, detrimental to endocrine counterpart. The tumor cells do not exhibit neither a pure ductular or ductal nor a pure acinar phenotype, but tumor lesions represented by neoplastic ductal cells with invasive growth are by far prevalently. In our pancreatic cancer case, electron microscopic investigation clearly shows that a plethora of the epithelial cells from the tumor lesions contain large areas of autophagy leading to the pleomorphic inclusions represented by fibrillary/filamentous inclusions frequently associated with hyaline-amorphous material, and secondary lysosomes. One of the mostly striking and important finding in this report for a case of pancreatic cancer is the high fragility (extensive dissolutions) of plasma membrane of tumor cells leading to pseudo-syncytia formation. Desmosomal junctions are severely altered, almost missing. Plasma membranes showed shedding membrane vesicles. Extravasated inflammatory cells contribute to the dramatic and extensive destructive areas of epithelial cells as well as tumor-stroma counterpart, including the basement membrane. All above severe infrastructural abnormalities, especially down regulation of cell-cell and cell-extracellular matrix adhesions might result in aberrant cell behavior and, consequently, much care should be taken for the postoperatory patient evolution.

Corresponding author: Nicolae Mirancea, Professor, PhD, Senior Scientist Grade I; e-mail: nick_mirancea@yahoo.com

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28. The use of cone beam computed tomography in the diagnosis and management of internal root resorption associated with chronic apical periodontitis: a case report

Paula Perlea, Cristina Coralia Nistor, Mihaela Georgiana Iliescu, Alexandru Andrei Iliescu

Internal root resorption is a consequence of chronic pulp inflammation. Later on, the pulp necrosis followed by a chronic apical periodontitis is installed. Hence, usually, in clinical practice, both lesions have to be simultaneously managed. Conventional periapical radiograph is mandatory in diagnosis. Improving the diagnosis and management of both lesions, cone beam computed tomography proves to be more reliable than conventional radiography.

Corresponding author: Paula Perlea, Senior Lecturer, DMD, PhD; e-mail: paula.perlea@gmail.com

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29. Silent sinus syndrome - report of a case

Daniela Dumitrescu, Bogdan Fanuta, Alex Emilian Stepan, Adrian Ioan Fronie, Cristiana Iulia Dumitrescu, Marius Cristian Martu, Petra Surlin, Valeriu Surlin, Mihai Popescu

Introduction: The silent sinus syndrome is a rare entity that was first described in 1964 and given this name 30 years later. Although it is well described both from clinically and radiologically point of view we consider that its rarity still makes it a subject for report. Case presentation: A 46-year-old patient was admitted for facial asymmetry, diplopia, unilateral left enophthalmos, and inferior displacement of the eye globe, and decreased occlusal pressure in left dentate region. CT scan revealed interior bulging of all left maxillary sinus walls with osteolysis and intense opacification, enlargement of the left middle meatus especially in the posterior part and lateralization of the uncinate process. Nasal endoscopy with 00 rigid scope visualized mild deviation to the right of the nasal septum, enlargement of the left middle meatus by the lateral deviation of the left intersinusal septum and uncinate process. Surgery was scheduled and performed a left maxillary sinus antrostomy. Histopathological examination on the biopsies revealed inflammation. A complete study was performed to assess the elements of inflammation. Postoperative course was simple. Follow-up visit at three and six months, showed significant regression of diplopia and improved facial aspect. Conclusions: Silent sinus syndrome is a well-defined clinical entity with characteristic imagistic findings. Surgical intervention that restores sinus drainage will interrupt the pathogenesis of the disease and lead to its progressive regression. Topographic associations and density of inflammatory elements analyzed in relation with neoforming vessels suggest their implication in reparatory angiogenesis characteristic to chronic inflammation. Modulating activity in the frame of inflammatory process, of the T-lymphocytes and especially of T-lymphocytes may represent a target for the therapeutic management. Surgery can and should be performed by an endoscopic approach.

Corresponding author: Valeriu Surlin, Associate Professor, MD, PhD; e-mail: vsurlin@gmail.com

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30. Giant gastrointestinal stromal tumor of the stomach

Sever Ionescu, Emil Barbu, Calin Ionescu, Adrian Costache, Maria Balasoiu

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal malignancies of the digestive tract. Gastric localization is the most frequent. The aim of this study is to evaluate the importance of immunohistochemical factors (CD117, CD34, alpha-SMA, vimentin, p53, Ki67) in diagnostic and size tumor and mitotic activity as prognostic factors for these tumors. We present the case of a 66-year-old male patient with a giant gastric GIST. Like in the vast majority, the symptomatology in this patient has long been faint, despite the large tumor size, and when it became manifest, it was nonspecific. Imagery wise, the computer tomography (CT) scan was the most efficient, showing the origin of the tumor from the greater curvature of the stomach, its dimensions, as well as the relations with the other abdominal viscera. Surgery in this patient was en-bloc, according to the principles of GIST. The histological aspect is characterized by a proliferation of spindle cells positive for CD117 and CD34. Despite complete microscopic resection, the size of the tumor (25x20x27 cm) and the mitotic activity (21/5 mm(2)) remains important relapse factor.

Corresponding author: Calin Ionescu, Lecturer, MD, PhD; e-mail: ionescu_calincj2002@yahoo.com

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31. A case with tumor of left parotid gland and denture stomatitis

Veronica Mercut, Daniel Iorgulescu, Sanda Mihaela Popescu, Claudiu Margaritescu, Razvan Mercut, Monica Mihaela Craitoiu, Monica Scrieciu

The article discuss the case of an edentulous patient presented to the dental clinic for pain and burning sensation of oral mucosa and diagnosed subsequently with oxyphilic adenoma. A 58-year-old patient, with maxillary edentulism, treated with complete denture, presented to the Clinic of Prosthetic Dentistry of the Faculty of Dental Medicine, University of Medicine and Pharmacy of Craiova, Romania, for pain of oral mucosa and bad stability of denture. The diagnosis of erythematous denture stomatitis as consequence of salivary flow reduction was establish after patient s clinical examination, and by unstimulated sialometry. Clinical exam suggested hyposalivation could be a consequence of a tumor situated at the level of the left parotid gland. The echography confirmed the diagnosis of left parotid gland tumor. The tumor was surgical removed and histopathological exam confirmed oxyphilic adenoma. Education of patient for wearing new denture while maintaining a strict oral hygiene and dealing with low salivary flow was the treatment for denture stomatitis. General dentist has an important role in finding and diagnosing patients with systemic diseases, especially salivary tumors.

Corresponding author: Sanda Mihaela Popescu, Associate Professor, MD, PhD; e-mail: sm_popescu@hotmail.com

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32. Pathological aspects underlying pancreatogenous hyperinsulinemic hypoglycemia - report of three cases

Bogdan Mircea Mihai, Cristina Mihaela Lacatusu, Lidia Iuliana Arhire, Mariana Graur, Viorel Scripcariu, Maria Gabriela Anitei, Iulian Radu, Dan Ferariu, Mihai Danciu

Pancreatogenous hyperinsulinemic hypoglycemia (PHH) is a rare disorder determined by an abnormally high secretion of insulin in the pancreas, in the absence of other medical or pharmacological factors. Either beta-cell tumors (insulinomas) or beta-cell hyperplasia (nesidioblastosis) can determine this pathology. Most publications on insulinomas or nesidioblastosis approached these subjects from a clinical point of view. This paper aims to analyze pathological aspects underlying pancreatogenous hyperinsulinemic hypoglycemia. We present two cases of insulinomas with unusual pancreatic localization and size, one of them showing amyloid deposits in the stroma. In both cases, immunohistochemistry confirmed the clinical and imagistic supposition. The third reported case refers to a 57-year-old patient with nesidioblastosis with isolated disposition of endocrine cells and areas of focal organization, both morphological aspects being extremely rare in adults. Although clinical and laboratory data are usually identical in the two forms of PHH, histopathological and immunohistochemical diagnosis is essential in differentiating insulinomas from nesidioblastosis, as the surgical management is different: enucleation for insulinomas and total or subtotal pancreatectomy for nesidioblastosis.

Corresponding author: Cristina Mihaela Lacatusu, Lecturer, MD, PhD; e-mail: cristina.lacatusu@umfiasi.ro, cmlacatusu@yahoo.co.uk

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33. Microscopic morphological changes of the tooth surface in relation to fixed orthodontic treatment

Cristina Teodora Preoteasa, Dan Florin Nitoi, Elena Preoteasa

Orthodontic treatment has, as any other medical intervention, in addition to its benefits, side effects, some of them being perceived as unavoidable. The aim of this case series was to microscopically evaluate the changes of the tooth surface in relation to fixed orthodontic treatment. A case series study was implemented by the usage of four extracted first maxillary premolars, from patients with previous orthodontic treatment, of 12 and 23 months. Analysis was performed using the high precision stereomicroscope (Axiovert, Carl Zeiss, Germany), at magnifications from 10x to 50x. The tooth surface corresponding to the bracket bonding area registered numerous disorderly grooves and cracks, with various directions and depths, and was flattened, having lower convexity compared to teeth surfaces where brackets were not bonded. Root resorption lacunae were more frequently observed in teeth under orthodontic treatment, these having various depths, and sizes considerably larger than those observed in teeth without orthodontic treatment. Following orthodontic treatment, teeth exhibit changes that can be perceived as being directly linked to this medical intervention. These teeth changes usually have low or moderate severity, which can be influenced at some degree by the clinical conduct of the orthodontic treatment. The stereomicroscope proved to be a high sensitivity tool for the analysis of morphological changes of teeth in relation to the fixed orthodontic treatment.

Corresponding author: Cristina Teodora Preoteasa, Assistant Professor, DMD, PhD; e-mail: cristina_5013@yahoo.com

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34. Extrarenal retroperitoneal angiomyolipoma with unusual evolution

Ionica Daniel Vilcea, Radu Victor, Cecil Sorin Mirea, Cristiana Eugenia Simionescu, Stelian Stefanita Mogoanta, Emil Moraru, Ioana Manuela Vasile, Mihai Calin Ciorbagiu, Cosmin Vasile Obleaga, Antoanela Valentina Crafciuc, Ion Vasile, Alina Maria Vilcea, Cristian Gheonea

The aim of this paper is to present the case of an extrarenal retroperitoneal angiomyolipoma with unusual evolution, due to the herniation through the inguinal canal, determining an extraperitoneal hernia. A ureteral duplicity and associated hydronephrosis contributed to the peculiarity of the case. The case was operated (en block tumor and right kidney removal), the postoperative evolution being favorable at seven years after the surgery.

Corresponding author: Ionica Daniel Vilcea, MD, PhD; e-mail: id.vilcea@yahoo.com

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35. Gestational choriocarcinoma after term pregnancy: a case report

Elvira Bratila, Cringu Antoniu Ionescu, Camelia Teodora Vladescu, Monica Mihaela Cirstoiu, Costin Berceanu

Choriocarcinoma coexisting with or after a normal pregnancy has an incidence of one per 160 000 pregnancies. In case of choriocarcinoma after term pregnancy, early diagnosis by histopathological examination of the placenta is very important, the precocity of the diagnosis influencing the prognosis and tumor response to chemotherapy. In this paper, we report the case of a 29-year-old woman gravidity 2, parity 2, with metastatic choriocarcinoma after term pregnancy, diagnosed at four months after the delivery of a healthy baby. An episode of abundant vaginal bleeding occurred after four months from delivery. The local exam revealed a vaginal tumor whose pathological examination on biopsy samples was inconclusive. Subsequently, she was admitted in our clinic with abundant vaginal bleeding, severe anemia and fever. Abdominal ultrasonography revealed an intracavitary uterine tumoral mass with signs of myometrial invasion to the uterine serosa, strong Doppler signal and moderate ascites. Pulmonary X-ray and computed tomography scan excluded extrapelvic tumoral masses. The pretreatment human chorionic gonadotropin (HCG) level was 31 030 IU/mL and her FIGO risk factor score was 8 (high-risk group). Total hysterectomy with bilateral salpingo-oophorectomy and omentectomy was performed as an optimal cytoreduction. Postoperative remaining lesions were represented by the metastasis located in the lower two-thirds of the vagina. Histopathological examination revealed uterine choriocarcinoma with ovarian metastasis. Postoperative was initiated four courses of polychemotherapy. Case evolution was favorable, with the normalization of the betaHCG value in two months postoperative and complete remission of the vaginal metastasis in six weeks postoperative.

Corresponding author: Elvira Bratila, MD; e-mail: elvirabarbulea@gmail.com

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36. Vagus nerve schwannoma in the parapharyngeal space: surgical, histological and immunohistochemical aspects. A case report

Vlad Andrei Budu, Ioan Alexandru Bulescu, Cristiana Gabriela Popp, Bogdan Constantin Mocanu, Carmen Aurelia Mogoanta

Tumors of the parapharyngeal space are rare accounting approximately for 0.5% of all head and neck tumors. In the retrostyloid space, schwannomas are a more common finding, in contrast to other tumors. Usually, they present with a variety of slight symptoms until they grow in size and compress surrounding organs. Surgical treatment of parapharyngeal space tumors is difficult; due to the anatomical complex area, they develop in, and include several approaches, according to its size and relations. In this paper, we present a case of a 63-year-old female with a vagus nerve schwannoma in the parapharyngeal space. Beside the surgical difficulties, the resected tumor had a peculiar histopathological aspect (large areas of degeneration and atypia and little typical palisading) that compelled a thorough histological and immunohistochemical evaluation for positive and differential diagnosis.

Corresponding author: Ioan Alexandru Bulescu, MD, PhD candidate; e-mail: ibulescu@gmail.com

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37. Analyzing the morphology and intensity of occlusal contacts in implant-prosthetic restorations using T-Scan system

Ana Maria Cotruta, Cristina Steliana Mihaescu, Livia Alice Tanasescu, Ruxandra Margarit, Oana Cella Andrei

The dentists must have solid knowledge in occlusology in order to obtain predictable long-term results for the implant-prosthetic treatment. The aim of our study was to verify if, with methods and materials currently used in any dental office for occlusal equilibration, the dentist can successfully integrate the implant-prosthetic restoration in the habitual occlusion of the patient. Using the T-Scan II system, we analyzed occlusion of three mandibular class III Kennedy edentulous male patients, from a private practice in Bucharest (Romania), all missing the first mandibular molar and all wearing a metal-ceramic crown on implant. In all these cases, the occlusion of the crowns was adjusted using articulating paper and vinyl polysiloxane biting material. The analysis of occlusion with the T-Scan II system in those three cases showed that the crowns were successfully integrated in the habitual occlusion. This study proves that those traditional methods, which are most frequently used in practice, are good enough to adjust the morphology and intensity of the occlusal contacts, if they are correctly and conscientiously used.

Corresponding author: Ruxandra Margarit, University Assistant, MD, PhD; e-mail: ruxandra.margarit@gmail.com

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38. Anatomopathological findings in scars: comparative study between different specimens

Andrada Despina Munteanu, Stefan Iulian Bedereag, Cristian Nitescu, Ioan Petre Florescu

In spite of the remarkable progress science and medicine have experienced, many facts concerning healing processes and pathological scars are still unknown or incompletely explained. This paper is part of a larger study (research for a PhD thesis) concerning new approaches in the prevention and treatment of pathological post-burn scars. We present and analyze the cases of some patients who developed abnormal scars in order to understand and point out the characteristics, that different types of pathological scars have in common and how we can differentiate them. Knowing what issue to address is the key to any successful therapy. Thus, the information we obtained will help us in applying more appropriate and efficient methods of treatment and in our further research: comparing the efficiency of newer therapies to that of older ones.

Corresponding author: Andrada Despina Munteanu, MD; e-mail: munteanu.andrada@gmail.com

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39. Biphasic synovial sarcoma in a 19-year-old pregnant woman: a case report

Madalina Olivia Adamesteanu, Razvan Scurtu, Ioan Lascar, Marek Valcu, Serban Arghir Popescu, Ioana Teona Sebe

Synovial sarcoma is a rare malignant tumor of mesenchymal multipotent cells. We hereby present a case of synovial sarcoma of the upper left thigh. A 19-year-old patient was referred to our clinic by another hospital in Bucharest, Romania, for a soft tissue mass in the left upper thigh. Local examination of the left thigh revealed a 15/13 cm, ovoid, painful upon touch, soft tissue mass occupying the proximal-medial aspect of the thigh. Bilateral inguinal nodes enlargement was noticed. Upon suspecting regional node involvement, the surgical team decided to perform left limb amputation due to tumor size and the proximity to major arterial and nervous trunks as well as the femoral shaft, making curative surgery and free of disease resection margins improbable. The patient refused the operation. The surgical team (plastic surgeon, orthopedic surgeon) decided to attempt limb-sparing surgery. After tumor resection, free-of-disease surgical margins were achieved. The pathological examination as well as the immunohistochemistry (IHC) diagnosed a large biphasic synovial sarcoma warranting oncologic treatment. The association between tumor growth and pregnancy poses important therapeutic problems, such as the use of preoperative chemotherapy, potential pregnancy termination, limb amputation versus limb salvage intervention and types of protocols of chemotherapy or radiotherapy indicated.

Corresponding author: Madalina Olivia Adamesteanu, Assistant Professor, MD; e-mail: madalinaadamesteanu@yahoo.de

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40. Thoracic endometriosis with a long delay in diagnosis

Roxana Maria Nemes, Cristian Paleru, Olga Danaila, Edith Simona Ianosi, Corina Silvia Pop, Damian Ditescu, Costin Teodor Streba, Mimi Floarea Nitu

This paper describes a case of thoracic endometriosis in 36-year-old woman with a long delay in diagnosis. At the admission in the hospital, the patient had a medical history of persistent dysmenorrhea since the age of 13, infertility and an episode of total right pneumothorax two months ago successfully resolved by minimum pleurotomy of the right hemitorax. She came with moderate pain on right hemithorax and dyspnea, which occurred on the first day of menstruation but she did not have any other respiratory symptoms such as hemoptysis, cough. Radiological imaging (chest radiography and computer tomography) at the time of admission confirmed recurrence of the right pneumothorax. She underwent surgical treatment of the right pneumothorax using a single-port video-assisted approach. Intraoperative macroscopic lesions were found catamenial pneumothorax characteristic diagnosis and biopsy material taken (parietal pleura) for histopathology. Immunohistochemical tests confirmed the diagnosis of thoracic endometriosis. The gonadotropin-releasing hormone analogue was received by the patient early after surgery and there was no clinical or radiological recurrence at a four months follow-up.

Corresponding author: Cristian Paleru, MD; e-mail: cpaleru@gmail.com

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41. Velamentous insertion of the umbilical cord vessels with vasa praevia - a case report

Mirela Anisoara Siminel, Cristian Gheonea, Mihail-Relu Stanescu, Alexandru Cristian Comanescu, Anda Lorena Dijmarescu, Simona Daniela Neamtu, Bogdan Virgil Cotoi, Ramona Mihaela Nedelcuta, Elena Carmen Niculescu

Among the abnormal cord insertion pathology, velamentous cord insertion associated with rupture of vasa praevia is the most severe condition related to the outcome of the newborn. In velamentous cord insertion, the fetal vessels run freely through the fetal membranes without protection from Wharton s jelly, umbilical vessels diverging as they traverse the membranes. When the membranes are ruptured, complete tearing of fetal vessels through the torn membranes or partial rupture near the site of membrane rupture may occur. Velamentous insertion occurs in approximately 1% of singleton gestations, but is observed in as many as 15% of monochorionic twin gestations. The risk of perinatal death was doubled in pregnancies with velamentous cord insertion relative to normal cord insertion. This condition can be diagnosed by ultrasonography with a sensitivity of 67% and specificity of 100% in the second trimester. We report a case of a newborn who came from a velamentous cord insertion condition associated with rupture of vasa praevia after the spontaneously membranes rupture. After a difficult resuscitation and stabilization, the newborn survived with a good outcome after the follow-up.

Corresponding author: Cristian Gheonea, Associate Professor, MD, PhD; e-mail: cgheonea@gmail.com Mirela-Anisoara Siminel, University Assistant, MD, PhD; e-mail: mirelasiminel@gmail.com

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42. Squamous cell carcinoma developed on chronic venous leg ulcer

Adelina Gabriela Sirbi, Marius Florea, Virgil Patrascu, Maria Rotaru, Dan Gabriel Mogos, Claudia Valentina Georgescu, Nicolae Dragos Margaritescu

Chronic venous leg ulcers (VLU), especially long-lasting non-healing ulcers, are among the risk factors for squamous cell carcinoma (SCC). Malignant transformation of a VLU is a rare finding and the relative risk of carcinomatous transformation is quite low (about 5.8). SCC arising in the context of a VLU has a particularly aggressive behavior. A 76-year-old male patient with no relevant medical familial history, with chronic venous insufficiency CEAP C6 for 10 years [recurrent leg ulcers with favorable outcome (healing) after specific local and systemic treatment], showing for about three years one ulcerated lesion located on the anterior upper third of the right calf non-responsive to specific treatment, which subsequently increased their size and merged. Biopsy sample was taken. Histopathology showed epidermal acanthosis, papillomatosis, intense parakeratosis, pseudoepitheliomatous hyperplasia, dysplasia and moderately differentiated squamous cell carcinoma with areas of acantholysis. Immunohistochemistry (Ki67, EMA, cytokeratin 34betaE12 and p63) was performed and all types of immunostaining were moderately to intense positive. Above-knee leg amputation and specific oncologic treatment were proposed as possible curative solutions but the patient refused. Ten months after diagnosis and discharge form the Department of Dermatology, the patient died. Patients with chronic venous leg ulcers and clinically suspicious lesions should be evaluated for malignant transformation of the venous lesion. When diagnosed, malignancy complicating a chronic venous leg ulcer requires a resolute treatment as it may be fatal.

Corresponding author: Marius Florea, MD, PhD; e-mail: dr.mariusflorea@gmail.com

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43. Pages from the history of the Anatomical Meeting from Cluj

Cristian Barsu

The Anatomical Meeting from Cluj was one of the most significant morpho-clinical societies from Romania during the interwar period. It was created in 1920 by Professor Titu Vasiliu (1885-1961). The purpose of our paper is to present the most important landmarks of this Society and to put into evidence its place in the Romanian series of morpho-clinical societies. Documentary Material: The activity reports of Anatomical Meeting from Cluj, summaries published in Medical Cluj journal referring to the presentations and discussions of the cases, which were held at different meetings of this Society. The activity of the Anatomical Meeting from Cluj increased, in a significantly and consistently way, the level of morphological and clinical training of physicians from Cluj. There are mentioned some of the most important presentations and the personalities which had special contributions in the evolution of this Society. Conclusions: The Anatomical Meeting was a scientific forum that propagated morpho-clinical concept in Romanian medicine, initially in Cluj, and then on a national level. It contributed to the collaboration between pathologists, clinicians and laboratory physicians from Cluj and favored scientific emulation between them. It also was a tribune from which were discussed a few new medical achievements.

Corresponding author: Cristian Barsu, Associate Professor, MD, PhD; e-mail: cristianbarsu@yahoo.com

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44. First year medical students attitude toward anatomical corpse dissection and its relationship with their personality

Mihai Horatiu Bob, Codruta Alina Popescu, Soimita Mihaela Suciu, Anca Dana Buzoianu

Introduction: Anatomy is an important and intense mandatory course offered during the first year of medical school. Corpse dissection is very important in Anatomy teaching, and first year students will encounter, most likely for the first time, a dead human body during Anatomy labs. Objectives: The purpose of this study was to evaluate the anxiety experienced by medical students with no previous corpse dissection background just before, after a week, and then after a month of dissection labs, and to investigate the relationship between students personality and their attitude towards dissection. Subjects and Methods: 138 first year English Section medical students from the Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania, completed a personality inventory, an anxiety scale and a questionnaire to assess their attitudes and reactions to anatomical dissection. Results: The level of anxiety reported by students increased from before the first dissection encounter to after one month of dissection labs. Conclusions: There is a relationship between the Five Factor model of personality and students attitudes towards dissection. Medical students could be better prepared for their first corpse dissection experience if the preparation before dissection would take in consideration their psychological traits.

Corresponding author: Codruta Alina Popescu, Lecturer, PhD; e-mail: cpopescu@umfcluj.ro

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45. Letter to the Editor: Dante and human reproduction: concept of embryogenesis in the Middle Ages

Luca Locatelli, Antonella Nespoli, Michele A. Riva

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Corresponding author: Michele A. Riva, MD; e-mail: michele.riva@unimib.it

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46. In Memoriam: Academician Vasile Anestiadi (1928-2014)

Eremia Zota, Laurentiu Mogoanta

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47. Conference Announcement: The XIIIth National Symposium on Microscopic Morphology with international participation, June 4-6, 2015, Craiova, Romania

Laurentiu Mogoanta

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Corresponding author: -

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