ROMANIAN JOURNAL of MORPHOLOGY and EMBRYOLOGY

Vol. 52 No. 1, 2011

1. Evidence on the neural crest origin of PEComas

A. Fernandez-Flores

The perivascular epithelioid cell (PEC) has been proposed to be the proliferating cell type in a group of tumors known as PEComas. The histogenesis of PEComas is one of the most mysterious aspects of pathology. Hypothesis on its precursor are many, including a cell from blood vessel walls or the myoblast. In the current report, we review many morphologic, clinical, ultrastructural, molecular and genetic aspects that support the hypothesis of an origin of PEComas from the neural crest.

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

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2. Morphological identification of neuron types in the rat hippocampus

T. Szilagyi, K. Orban-Kis, Emoke Horvath, Julia Metz, Zsuzsanna Pap, Z. Pavai

The cerebral cortex ensures an optimal interaction of mammals, including humans, with their environment, by encoding, storing and combining information about the surrounding world and the internal milieu. Probably the simplest and the most popular region for studying the cortical network is the hippocampal CA1 area, because it has the least heterogeneous neuronal population, the somata and dendrites of principal neurons (pyramidal cells) are arranged into well defined layers and the extrinsic and intrinsic inputs are segregated. The relatively homogeneous pyramidal cell population is supported by a very heterogeneous GABAergic interneuron population, which provides not only general inhibition, but also regulates the precise timing of pyramidal cell activity. Interneurons usually innervate distinct domains of the surface of their target cell. The strategic placement of inhibitory synapses, indicate that GABAergic interneurons belonging to different classes serve distinct functions in the hippocampal network. Neuron types are usually defined according to various morphological, molecular and physiological features. Under typical experimental conditions only some of these parameters are available, therefore an important scientific question is: which partial measures are sufficient for correct recognition of a class of cell. By immunohistochemistry it is possible to stain all neurochemically identical neurons in a given brain region, therefore it is the most widely used method for identifying neuron classes. This review presents the neuron types identified so far in the area CA1 of the rat hippocampus with special emphasis on the immunocytochemical characterization of these cells.

Corresponding author: Tibor Szilagyi, Professor, MD, PhD, e-mail: szilagyi67@gmail.com

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3. Inflammation in COPD: pathogenesis, local and systemic effects

Cornelia Barbu, Mihaela Iordache, M. G. Man

COPD is a disease characterized by inflammation both in its stable phase and during exacerbations. Inflammation is present in the respiratory compartment, the inflammatory cells and different mediators of inflammation being present. Studies have shown that some mediators of inflammation have a high level at systemic level also, inducing a certain grade of systemic inflammation, mainly responsible for the systemic manifestation of the disease. It seems that the both local and the systemic inflammation are amplified during exacerbations. The purpose of this paper is to review the respiratory inflammatory reaction, to identify the main actors involved in the stable phase of the disease and during exacerbations, the systemic inflammation accompanying the local inflammation, and to find the possible interrelations between the systemic and local inflammation, on one hand, and the respiratory and extra-respiratory manifestations of COPD on the other hand. The understanding of the pathogenic mechanisms, which stay at the base of inflammation and of the possible interrelations shown, represents a theoretical interest and also a practical one, concerning the targets of different therapeutic agents which could be used in the management and in the control of the disease.

Corresponding author: Cornelia Barbu, MD, PhD, e-mail: barbucornelia@yahoo.com

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4. Clinico-pathological correlations in fatal ischemic stroke. An immunohistochemical study of human brain penumbra

D. Arsene, Florina Vasilescu, C. Toader, Adina Balan, C. Popa, Carmen Ardeleanu

Ischemic stroke is one of the most frequent pathologies with high invalidating potential and a leading cause of death. The brain tissue adjacent to the central necrotic core, defined as penumbra, was extensively characterized mostly by imaging techniques and in animal models. Our goal was to identify a large panel of molecules in this particular area on human brains harvested at autopsy. Twenty-one patients with ischemic stroke and seven control cases were taken into study. We used immunohistochemistry to characterize necrotic lesions. Metalloproteinases, mostly MMP-9, seem to be involved in brain ischemia, but as a protective and not as a deleterious factor. Apoptotic molecules are not increasingly expressed in stroke compared to control cases. Mast cell enzymes chymase and tryptase are described for the first time in neurons and glia, even with unclear significance. Microglia appears active in stroke and stimulating methods directed to it could be useful. Nitric oxide synthases and cyclooxygenase-2 were also involved in stroke cases but not in control ones. Other factors as VEGF and its receptors, PDGF, b-FGF or TNF-alpha showed no significant expression related to ischemic brain injury. Animal study of penumbra and human tissue findings are distinct and research should be focused on the latter approach in order to find valuable and safe therapeutic methods.

Corresponding author: Dorel Arsene, MD, PhD, e-mail: dorelarsene@yahoo.com

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5. Immunohistochemical evaluation of COX-2 expression in HPV-positive cervical squamous intraepithelial lesions

Raluca Balan, Cornelia Amalinei, Simona Eliza Giusca, D. Ditescu, V. Gheorghita, E. Crauciuc, Irina-Draga Caruntu

Cyclooxygenase 2 (COX-2) regulates the prostaglandins production and it seems to have a role in the onset and progression of different malignant tumors, being overexpressed in numerous human malignancies and premalignant conditions. Some cellular elements from chronic inflammatory processes, together with stromal cells may be involved in neoplastic transformation of proliferative stem cells and in the process of tumor invasion. Cervical carcinoma, as a commonly pattern of different tumors, can express COX-2 in association with glutathione-S-transferase isoenzymes and can be considered as possible molecular targets in antitumoral therapy. The purpose of this study was to evaluate the expression of cyclooxygenase (COX)-2 in cervical squamous intraepithelial lesions of low-grade (LSIL) and high-grade (HSIL), with morphologic evidence of HPV infection. Immunostains with COX-2 antibodies were performed on formalin-fixed and paraffin-embedded tissue sections from 20 cervical biopsies: 10 with LSIL histopathologic diagnosis and 10 with HSIL histopathologic diagnosis. All LSIL biopsies and four HSIL cases (equivalent to CIN2) presented also intermediate squamous cells, with pathognomonic morphology of HPV infection (koilocytes). The Allred immunohistochemical score for the intensity of staining and the percent of cells stained was assigned. The slides were scored by three independent pathologists and compared across histological categories. Regarding the intensity of cytoplasmic COX-2 immunostaining, a weaker expression was observed in specimens with LSIL and a stronger one in those diagnosed with HSIL, the highest score being noted in HSIL corresponding to CIN3 lesions. The increase of COX-2 expression in cervical cancer precursors certifies that COX-2 may have a role in the development and progression of cervical squamous intraepithelial lesions.

Corresponding author: Irina-Draga Caruntu, Professor, MD, PhD, e-mail: irina_caruntu@yahoo.com

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6. Evaluation of women knowledge and attitude regarding cervical cancer early detection

Fl. Badulescu, Ileana Prejbeanu, Cornelia Rada, Anca Patrascu, Manuela Dragomir, Florina Carmen Popescu

At European level, Romania unfortunately comes first as far as mortality due to cervical cancer is concerned, mortality rate recording a continuous increase due to disease detection in advanced stages. In this context, we followed an assessment of women's cognitive and attitudinal fund in relation to early detection of cervical cancer, through the application of a questionnaire on a batch of 617 women with ages from 18 to 75-year-old, with various levels of education, residents of Craiova City and communes from Dolj County. Statistic processing of their answers indicates that 43.3% of the inquired subjects have not had a routine gynecological exam for at least seven years (or even never), which is more often found in the subgroup of women from the rural environment, with ages over 35 years and with an elementary educational level (p<0.001). 65.8% of the women have not done a Babes-Papanicolau test for seven years or even never, residence area, age and education level being once again discrimination factors between the subgroups. The reasons usually put forward as a justification for the failure to do the cytological test for the past three years are negligence (23.8%), the lack of information on the existence of such an investigation (18.2%), the absence of a genital pathology (12.8%) or of a medical recommendation (11.8%). Results suggest the need to provide uniform accessibility of the population to health services, by developing and implementing new strategies of educational and sanitary intervention mainly in rural communities.

Corresponding author: Ileana Prejbeanu, Professor, MD, PhD, e-mail: ileana.manuela@yahoo.com

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7. Expression of VEGF, VEGFR, EGFR, COX-2 and MVD in cervical carcinoma, in relation with the response to radio-chemotherapy

Viorica Magdalena Nagy, R. Buiga, Ioana Brie, N. Todor, Oana Tudoran, Claudia Ordeanu, Piroska Virag, Oana Tarta, Meda Rus, O. Balacescu

Introduction: Despite the improvement in the treatment results due to modern irradiation techniques and to the association of chemo-radiotherapy, cervical cancer remains an unsolved problem of oncology both due to the increased rate of local failures and of the distant metastasis. Efforts to implement new therapeutic strategies in order to obtain better results in patients with cervical cancer appear justified. Neovascularization is an important step in the tumor progression and the therapeutic targeting of the tumor blood vessels appears to be a good strategy to follow in the anti-cancer treatment. Thus, even in an incipient phase of the clinical research process, the combination between the anti-angiogenic aimed therapies and the current radio-chemotherapy seems to represent a new, feasible and promising approach. The aim of the present study was to determine the prognostic and/or predictive value of some biological markers of tumor angiogenesis and of their implication in increasing the efficacy of current treatments for this cancer. Materials and Methods: So far, 54 women were included in a prospective trial: 44 having an advanced cervical carcinoma and 10 healthy women, as controls. A tumor biopsy and a blood sample were obtained from each patient before the start of therapy. The density of microvascularization was assessed using CD34 monoclonal antibody (hot spot technique), the expression of angiogenic factors VEGFR, EGFR and COX-2 were determined in tumor biopsies by specific immunohistochemistry techniques, using primary antibodies anti-EGFR, anti-VEGF and anti-COX-2 respectively. The quantitative polymerase chain reaction (Real Time PCR) was employed for assessing the expression level of the genes involved. Serum VEGF was determined by quantitative ELISA technique. Results: Among the studied clinical and molecular factors, we found to be predictive for the type of response the following factors: tumor size at diagnosis (p=0.01), VEGFR2 expression (p=0.02) and a tendency to significance for patients' age (p=0.06). From the large panel of studied markers it was observed correlation between MVD expression with stromal COX-2 (p=0.01) and a tendency with epithelial COX-2 (p=0.06). Stromal COX-2 has higher correlation with VEGFR2 (p=0.01) and MVD (p=0.01) and also has a lower correlation with tumor size (p=0.08). Conclusions: Univariate analysis demonstrates that the response to radio-chemotherapy in cervical cancer is related to a set of clinical and molecular factors as: the tumor size, the expression of VEGFR2 as mRNA level and the patients' age. Unfortunately, the multivariate analysis by logistic model selects only VEGFR2 expression for prediction of tumor response. The interrelations between the different biomarkers demonstrate the complexity of the tumor progression process and the necessity of further studies to identify new therapeutic targets.

Corresponding author: Viorica Magdalena Nagy, Professor, MD, PhD, e-mail: nagyviorica@yahoo.com

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8. The vasculogenesis - a possible histological identification criterion for the molar pregnancy

Liliana Novac, Mihaela Niculescu, Maria Magdalena Manolea, D. Iliescu, Claudia Valentina Georgescu, Al. Comanescu, N. Cernea, Andreea Enache

The benign hydatidiform mole (partial and complete) practically represents an associated pathology of the first trimester miscarriage; it is considered that it represents the initial pathology, which, evolutionally, may be followed by the appearance of invasive mole and choriocarcinoma. The authors have studied clinical and cytogenetical 61 cases selected according to some specific criteria for the suspicion of molar pregnancy or hydropic abortion and 21 cases that represented the control group made by patients with normal pregnancy which have been presented for legal abortion. In all cases has been made a histological exam and has been studied the vasculogenesis by immunohistochemistry labeling for CD31 and CD34. Following-up the results has been established a number of histological and immunohistochemical criteria for the evaluation and differentiation of various molar pregnancy types and the vasculogenesis characteristics. The most important differential diagnosis criteria between partial and complete mole is represented by the trophoblast disposition and the atypical trophoblast in the complete mole.

Corresponding author: Liliana Novac, Professor, MD, PhD, e-mail: liliana.novac@gmail.com

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9. B-cell transcription factors Pax-5, Oct-2, BOB.1, Bcl-6, and MUM1 are useful markers for the diagnosis of nodular lymphocyte predominant Hodgkin lymphoma

Rosemarie Herbeck, D. Teodorescu Brinzeu, Marioara Giubelan, Elena Lazar, Alis Dema, Hortensia Ionita

In some instances, the overlap in morphologic features and antigen expression between nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and classical Hodgkin lymphoma (cHL) can cause confusion in the diagnosis. In these cases, the transcription factors (TFs) B-cell specific activator protein (BSAP)/Pax-5, octamer binding protein-2 (Oct-2), B-lymphocyte-specific co-activator BOB.1/OBF.1, Bcl-6 protein and multiple myeloma-1/interferon regulatory factor-4 (MUM1/IRF-4) may aid in clarifying the diagnosis. Twenty-two cases of NLPHL were studied for the immunohistochemical expression of Pax-5, Oct-2, BOB.1, Bcl-6 protein and MUM1/IRF-4. Our results sustain the usefulness of the selected set of TFs to diagnose and distinguish NLPHL from cHL since Pax-5, Oct-2, BOB.1 and Bcl-6 are consistently expressed by lymphocyte predominant (LP) cells and reported by others to be often unexpressed in Hodgkin and Reed-Sternberg cells. By contrast, MUM1/IRF-4 protein scored negative in the majority of LP cells, but is reported to be expressed in almost all cases of cHL. Thus, although the expression of transcription factors is very heterogeneous, their simultaneous implementation for positive and differential diagnosis may be useful.

Corresponding author: Rosemarie Herbeck, MD, PhD candidate, e-mail: roseherbeck@yahoo.com

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10. Myofibroblast involvement in tubular basement membrane remodeling in type II diabetic nephropathy

E. Mandache, Mihaela Gherghiceanu, C. Serafinceanu, M. Penescu, G. Mircescu

Diabetic nephropathy is always accompanied by tubulointerstitial damage. The mechanisms and the cells involved are not entirely clarified. The damaged tubules may regenerate or undergo necrosis or apoptosis. The purpose of this work was to investigate the structural transformations of both interstitial cells and extracellular matrix of the kidney stromal area in patients with type II diabetes mellitus associated with diabetic nephropathy. Tubulointerstitial fibrosis is characterized by loss of renal tubules and interstitial capillaries and the accumulation of extracellular matrix proteins. Tubular basement membranes were found to be a target of the remodeling process of the stromal area. Thickening, splitting and duplication were the main lesions of these membranes. Much attention has been focused on the importance of myofibroblasts in the progression of renal fibrosis. The results represent strong arguments for a direct involvement of myofibroblasts in the process of renal interstitial remodeling, tubular basement membrane thickening, and stromal fibrosis in the late stages of diabetic nephropathy.

Corresponding author: Eugen Mandache, MD, PhD, e-mail: mandache.eugen@yahoo.com

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11. Gross morphometric reduction of rats' cerebellum by gamma irradiation was mitigated by pretreatment with Vernonia amygdalina leaf extract

O. Owoeye, E. O. Farombi, S. K. Onwuka

The methanolic extract of Vernonia amygdalina (M) or "bitter leaf" is known for its antioxidant activity, and antioxidants are noted to mitigate radiation damage in tissues. The aim of the present study was to observe the radioprotective effect of M on the cerebellum of gamma irradiated rats using alpha-tocopherol (TOCO) as a reference antioxidant. Forty-two male Wistar rats (n=42) weighing 200-240 g were taken for the study. The study comprised of seven groups, with each group comprising of six (n=6) rats i.e. control, M at 250, and 500 mg/kg/day, radiation only, radiation plus M at 250, and 500 mg/kg/day, and TOCO. After 14 days of treatment administered via oral gavage, rats were irradiated with a single dose of 2.0 Gy of gamma rays on the 15-th day and euthanized the next day. Rats cerebella were removed, fixed in 10% formalin saline, weighed and vernier caliper used to obtain cerebellar dimensions as follows: (i) maximum width, (ii) rostrocaudal dimension, and (iii) dorsoventral extent. Data were analyzed using ANOVA with post-test. Gamma radiation caused a statistically significant reduction of the relative weight of the rats' whole brain, relative weight of the cerebellum, the maximum width, rostrocaudal dimension, and dorsoventral extent of the cerebellum. However, pretreatment with M and TOCO significantly mitigated these effects. This study demonstrated that administration of M and TOCO before 2.0 Gy gamma irradiation reduced significantly the radiation induced gross morphometry changes in rats' cerebellum, suggesting that M may qualify for consideration as a medicinal radioprotector.

Corresponding author: Olatunde Owoeye, Lecturer, MB, BS, MSc, PhD, e-mail: oowoeye2001@yahoo.com, o.owoeye@mail.ui.edu.ng

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12. HPV and cervical squamous intraepithelial lesions: clinicopathological study

Lavinia Mihaela Corneanu, Diana Stanculescu, Cecilia Corneanu

The aim of our study is to determine the evidence of HPV infection, by either HPV-DNA test or histopathological examination, in patients with abnormal PAP-smear, for further clinical management. In 6-month period, we retrospectively selected a number of 103 patients in 16-54 years range of age that were investigated by complex diagnostic techniques supporting the evidence of HPV infection. Initially, these patients were evaluated cytological using the Bethesda system for PAP-smear interpretation along with colposcopic examination. A number of these patients were orientated to HPV testing (PCR) or to biopsy with histopathological diagnostic intent. The net predominance of LSIL over HSIL cytodiagnostic class parallels with that of condyloma and CIN1 over CIN2 and CIN3 histodiagnostic and suggests that progression to high-grade intraepithelial squamous lesion is rare and the HPV infection remain mainly transitory. On the other side, the incidence and distribution of HPV types in cervical infections are high variable and may change over time, the cytological screening of sexual active female population remaining the most practical tool in detection of genital HPV infection.

Corresponding author: Lavinia Mihaela Corneanu, MD, e-mail: laviniadobrescu@ymail.com

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13. Atrial structural remodeling in patients with atrial chronic fibrillations and in animal models

D. Laky, Liliana Parascan, V. Candea

Arrhythmia's atrium fibrillation (AF) is the most often met in clinical setting and it is associated with an increased in mortality risk. For profound the structural changes in chronic AF, we are studied the morphological changes of atrium biopsies to be effected at 175 patients. With sustained AF malformative and valvular acquired cardiac diseases operated under extracorporeal circulation. Similar studies we are effected to 11 dogs with partial coronary obstructions to a made periodical EKG investigations. The morphological changes mainly concern accommodation (dedifferentiation) of cardiomyocytes (particularly at experimental model) and mal-accommodation (degeneration of cells with fibrosis replacement features) particularly in acquired valvular diseases. These changes were often interfered. Over study, maintain the hypothesis that the structural changes to be an accommodation more than degenerative response to AF.

Corresponding author: Liliana Parascan, MD, PhD, e-mail: liliana_parascan@yahoo.com

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14. Clinical and histoenzymatic interrelations of the edentulous ridge's mucosa

Monica Mihaela Craitoiu, Mihaela Pauna, Stefania Craitoiu, Veronica Mercut

The edentulous ridge's mucosa presents various histological aspects and different histoenzymatic equipment. These aspects are determined by constitutional factors (genetic, nutritional), as well as by age and by the associated pathology. The purpose of this paper is to evidentiate, through histochemical techniques, the aerobe (NADH2-cytochrome-c reductase) and anaerobe (lactatdehydrogenase) oxidoreducing enzymatic activities, as well as the hydrolytic enzyme's activity (ATP-ases, pH 9.4), corroborated with the histological aspects of the edentulous ridge's mucosa. The enzymatic activity and the morphological alterations present various aspects, depending on the metabolic type (aerobe oxidative, anaerobe oxidative, hydrolytic), according to the mucosal area (epithelium, chorion), to the different cell types, or even within the same cellular type (especially at the level of the connective lamina propria cells). They also vary from one edentulous patient to another, depending on the existence of a previous prosthetic treatment and on the type of this treatment. The local clinical examination, sustained by a histoenzymatic investigation, reveals the importance of the structural alterations and they can offer guidance for the proper prosthetic treatment and for the achievement of functional prosthetic restorations that provide the oral mucosa its necessary trophicity for as long as possible.

Corresponding author: Monica Mihaela Craitoiu, Assistant Professor, DMD, PhD candidate, e-mail: mcraitoiu@yahoo.com

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15. Involvement of the muscle-tendon junction in skeletal muscle atrophy: an ultrastructural study

L. de Palma, M. Marinelli, M. Pavan, C. Bertoni-Freddari

Background: The muscle-tendon junction (MTJ) is a physiologically vital tissue interface and a highly specialized region in the muscle-tendon unit. It is the weakest point in the muscle-tendon unit, making it susceptible to strain injuries. Nonetheless, knowledge of the pathological changes affecting this region and of its response to the atrophy process is very limited. The aim of the study was to examine MTJ ultrastructural morphology in patients with different conditions that induce skeletal muscle atrophy and to attempt a grading of the atrophy process. Materials and Methods: Fifteen patients undergoing amputation in the distal or proximal third of the lower leg due to chronic or acute conditions were divided into two groups. Specimens of gastrocnemius muscle collected at the time of surgery were analyzed by histology and electron microscopy. The contact between muscle and tendon was measured using a dedicated software that calculated semi-automatically the base (B) and perimeter (P) of muscle cell finger-like processes at the MTJ. Results: Electron microscopy. The cells in the atrophic muscle of the chronic group were shallow and bulky. In the acute group, the myotendinous endings differed significantly in their structure from those of the chronic group. In atrophic muscle, the contact between muscle and tendon was reduced by quantitative and qualitative changes in the myotendinous endings. The B/P ratio allowed definition of three grades of myotendinous ending degeneration. Discussion: It is unclear whether degenerative changes induced by immobilization in muscle and, specifically, the MTJ are temporary and reversible or permanent. Conclusions: This preliminary study suggested a classification of ultrastructural MTJ changes into grade 0, reflecting a quite normal MTJ; grade 1, an intermediate process that might lead to irreversible atrophy or to recovery, spontaneously or with drug therapy; and grade 2, irreversible process with complete structural alteration.

Corresponding author: Luigi de Palma, Professor, MD, PhD, e-mail: l.depalma@univpm.it

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16. Value of immunohistochemical investigation in the diagnosis of neuromuscular diseases in children

D. Matei, I. Gherghina, Eliza Cinteza, A. Matei, Marilena Alexianu

Neuromuscular diseases represent an important group in pediatric pathology. Immunohistochemistry together with clinical examination and morphologic exam are very important in the diagnosis of neuromuscular disorders. Patients and Methods: One hundred children diagnosed with neuromuscular disorders were included in a prospective-retrospective study in 25 years. Results: There were 58 neurogenic diseases and 42 muscular dystrophies. In positive and differential diagnosis very important were the morphologic and immunohistochemical investigations. Seventy-three percent had positive family history and 27% of dystrophic patients had "de novo" mutations. The most part of the neurogenic disorders were spinal motor atrophies, 91.38%. Conclusions: Neurogenic muscular diseases were the most common neuromuscular diseases in our patient group. The immunohistochemical investigation was very useful in diagnosing some of these cases.

Corresponding author: Dumitru Matei, Professor, MD, PhD, e-mail: drmateidumitru@yahoo.com

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17. Is there a correlation between the CEAP score and the histopathological findings in varicose disease?

A. Mironiuc, Laura Palcau, Liliana Rogojan, Sanda Micula, Claudia Gherman

Background: Varicose disease continues to represent an interesting subject. The factors triggering and maintaining vascular and cutaneous tissues alterations in chronic venous insufficiency are not fully known. Patients and Methods: This is a prospective, statistical analysis study, performed in a consecutive series of 40 patients with varicose disease admitted and treated at the Surgical Clinic II, Cluj-Napoca. The aim of the paper is to evidence histopathological (HP) changes in the vein wall, as well as to correlate histopathological findings, classified into stages, with the clinical stage of chronic venous insufficiency (CEAP classification). Statistical analysis was performed using the Fischer F-test for the comparison of the variances of two selections and the Student t-test for the comparison of the means. For correlation, Pearson's simple correlation coefficient was used. The software used was Excel and Matlab 7. Results: Following the statistical analysis, the mean of CEAP values was found to be higher than the mean of the histopathological stage values in all patients included in the study and in the different risk groups. The values of Pearson's linear correlation coefficient between CEAP values and histopathological stage values did not generally show a statistically significant correlation. Conclusions: CEAP classification remains the main pillar in the diagnosis and the treatment of varicose disease, even if there are some dissimilarities between the clinical appearance and the histopathological results. The presence of a correlation between histological and clinical aspects in varicose disease remains uncertain.

Corresponding author: Aurel Mironiuc, Professor, MD, PhD, e-mail: aurelmironiuc@yahoo.com

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18. Photodynamic diagnosis of non-muscle invasive bladder cancer using hexaminolevulinic acid

O. Dragoescu, P. Tomescu, A. Panus, M. Enache, C. Maria, L. Stoica, I. E. Plesea

Bladder cancer (BC) is the most common tumor of the urinary tract. White light cystoscopy (WLC) is currently considered the standard investigation for diagnosis of bladder tumors. Recent studies suggest that using exogenous fluorescence (photodynamic diagnosis, PDD) can improve its diagnostic sensitivity and specificity. Objective: Our study aims to analyze the value of using fluorescent cystoscopy (PDD) in the diagnosis and treatment of non-muscle invasive bladder cancer (NMIBC). Patients and Methods: The study designed as a prospective randomized clinical trial was conducted over a 12 months period and included 44 patients with primitive NMIBC diagnosed and treated in our department in 2009. Twenty-two patients were included in the study group (PDD), while 22 patients were diagnosed and treated by conventional methods (WLC). Results: There were no statistically significant differences between the two groups regarding age, sex, place of origin, smoking history, clinical symptoms or presence of urological history as well as tumor size, location or number. Fluorescence cystoscopy examination identified 25.8% more tumors than the conventional examination (p=0.004). We demonstrated a significant reduction of tumor recurrence rates at 3, 6, 9 and 12 months by using PDD (HR=0.3271, 95% CI 0.1091-0.9809; p=0.0461). Conclusions: The use of PDD in patients with NMIBC results in significant improvement of the efficiency of their initial diagnosis cystoscopy (by over 25%). We demonstrated improved patient prognosis and quality of life following conservative TUR treatment of these tumors by significantly reducing the tumor recurrence rate (by 9-27%) in the first year of follow-up.

Corresponding author: Octavian Dragoescu, Assistant, MD, PhD candidate, e-mail: pdragoescu@yahoo.com

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19. Etio-pathogenic and morphological correlations in congenital hydronephrosis

Madalina Bosoteanu, C. Bosoteanu, Mariana Deacu, Mariana Aschie, P. Bordei

Hydronephrosis, "distension in varying degrees of pelvis and calyces, accompanied by progressive atrophy of renal parenchyma due to obstruction in urinary flow", is an apparently simple reno-urinary disease, but, in reality, by association of its own characters with those conferred by causative lesions, becomes of a significant complexity. The purpose of this paper is to demonstrate the plurivalent character of this entity on a batch of cases with congenital hydronephrosis, by identification of lesional features correlated with cause of disease. The etiology of hydronephrosis in analyzed cases was characterized by heterogeneity: polar inferior artery, horseshoe kidney, extrarenal pelvis, transverse valves of pelviureteral junction (PUJ), adhesion of ureter to PUJ, intrinsic stenosis of PUJ, vesico-ureteral reflux, posterior urethral valves, stenosis of urethral meatus. The way of intervention of urinary obstruction and the uni- or bilateral character of damage were definitory for the macroscopical appearance of the hydronephrotic kidney, renal pelvis demonstrating its role of expansion room for kidney protection. In analyzed cases of congenital hydronephrosis, correlation specific cause-pelvic lesion evidenced histopathological differences related to etiology. Anatomical preparations obtained by injection followed by corrosion have revealed that renal vessels appear elongated, distanced from each other and even reduced in density, which explains the appearance of ischemia accompanying pathogenetic changes of obstructive uropathy. Regardless of etiology, all cases of congenital hydronephrosis were characterized by varying degrees of fibrosis in chorion of renal pelvis, accompanied by active chronic inflammation, observation that support the idea of connection between the two pathological changes.

Corresponding author: Madalina Bosoteanu, Lecturer, MD, PhD, e-mail: mbosoteanu@yahoo.com

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20. Immunohistochemical study of stellate cells in patients with chronic viral hepatitis C genotype 1

Larisa Sandulescu, I. Rogoveanu, T. Ciurea, Maria Victoria Comanescu, C. T. Streba, A. G. Ionescu, Alice Oproaica, M. Ene

Introduction: Hepatic stellate cells (HSC) are key-players in the pathogenesis of liver fibrosis, inducing collagen deposition and abnormal extracellular matrix remodeling. Aim: The purpose of this study was to identify the stellate cells using immunohistochemical techniques and to establish if there is a correlation between the expression of stellate cells and the clinical and histological parameters in patients with chronic viral hepatitis C. Materials and Methods: The studied group included 30 patients with chronic viral hepatitis C genotype 1, in whom a liver biopsy was performed previous to the antiviral treatment. After the histological analyze, the biopsy was stained with an anti-SMA antibody (Dako, Carpinteria, CA). The amount of positive stained area was determined using an arbitrary semiquantitative score from 1 to 4. Results: Our observations suggest that there is a strong correlation between the stellate cells activity, evaluated using a semiquantitative score, and the stage of liver fibrosis (rs=0.76, p<0.001). Also, our study revealed a direct correlation, but less intense, with the necro-inflammatory activity (rs=0.39, p=0.03), the steatosis degree (rs=0.428, p=0.01) and the value of alanine aminotransferase (rs=0.4, p=0.03). The age and the viremia level were not correlated with the activity of the stellate cells. Conclusions: This study suggests that the transition of stellate cells from inactivated state to the state of highly fibrogenic cell is influenced mainly by the histological liver modifications (necroinflammatory activity and steatosis) and less by clinical parameters (age, sex) or the viremia level.

Corresponding author: Larisa Sandulescu, MD, PhD, e-mail: larisasandulescu@yahoo.com

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21. How accurate replicates the Thermafil System the morphology of the apical endodontic space? An ex vivo study

S. I. Stratul, Andreea Didilescu, Mihaela Grigorie, Emilia Ianes, D. Rusu, Luminita Nica

Aim: To evaluate the morphology of the root canal in its apical third and the capacity of the Thermafil System to reproduce the entire morphology of the cleaned and shaped root canal. Materials and Methods: Thirty-two roots of periodontally compromised teeth were prepared using the ProTaper System to an apical size 30 and filled with the Thermafil obturation technique and sealer. The roots were surgically amputated and prepared for metallographic evaluation by incremental reductions of 0.5 mm each, starting with the apical foramen. Photomicrographs of each section were taken at a magnification of 500x and 100x. The images were analyzed and processed. The position of the apical foramen with respect to the anatomical apex was identified and marked. Additional morphological details as lateral canals and recesses were also recorded. The cross-sectioned area of the canal and gutta-percha, the total perimeter, the shaped perimeter and the filled perimeter were recorded for each sample and the results were expressed as percentages. Multiple images of successive sections were used to create a 3D reconstruction of the apical anatomy of the tooth. The ANOVA test was performed to assess mean differences between evaluations of perimeters/areas at different levels. Results: The anatomical apical foramen was found at the tip of the root in 50% of the evaluated samples. In the remaining samples, the foramen was located between 0.5 and 2.5 mm from the centre of the apex. Lateral canals, which opened in accessory foramens, were recorded in 25% of the evaluated samples. Statistical significant differences (p<0.05) were found between different levels of preparation and obturation. Conclusions: The complex morphology of the apical third of the root canal is satisfactory microstructurally replicated by the Thermafil System. Moreover, polarized light microscopy and the 3D reconstruction offered a discriminative vision of morphological details as lateral canals, recesses, the gutta-percha and debris.

Corresponding author: Stefan-Ioan Stratul, Senior Lecturer, DMD, PhD, MS, MDiv, e-mail: sbs@online.ro

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22. Pterygium: histological and immunohistochemical aspects

T. Golu, L. Mogoanta, C. T. Streba, D. N. Pirici, D. Malaescu, Garofita Olivia Mateescu, Gabriela Mutiu

Introduction: Pterygium represents a triangular conjunctivo-epithelial overgrowth, proliferating from the bulbar conjunctiva and covering the cornea, causing severe vision loss. It is an abnormal growth and differentiation of the conjunctive epithelial structures of the corneal limbus. Chronic exposures to solar ultraviolet radiation, heat and dust, as well as viral agents, are the most common pathogenic entities involved in its evolution. Recent studies linked pterygium with neoplastic proliferation, as ocular limbic stem cells and p53-protein expression are altered. Materials and Methods: Our study was conducted on 84 fragments of pterygium, collected after surgery from patients admitted between 2008 and 2009 in the Departments of Ophthalmology of the Emergency County Hospital of Craiova and Hospital of Rovinari. Histological studies were performed by staining with Hematoxylin-Eosin, light green trichromic (Goldner-Szekely technique) and PAS-Hematoxylin. Immunohistochemistry highlighted the T-lymphocytes by using the CD3 antibody, B-lymphocytes by using the CD20 antibody and cells of the macrophage system using the CD68 antibody. The slides were analyzed under a 55I Nikon microscope, resulting pictures being captured with a 5 MP digital camera and digitally retrieved and enhanced using the dedicated NIS-Elements software. Results: Histology showed the presence of a conjunctivo-epithelial structure, significantly different from the structure from which it developed. In 20% of the cases, the covering epithelium was similar to that of the bulbar conjunctival mucosa. In some cases, when pathogens were more active in the environment, the appearance of the covering epithelium of the membrane appeared pleomorphic, with dysplastic aspects, suggesting significant alteration of cell proliferation and differentiation. In approximately 75% of patients, we identified goblet cells in the surface epithelium. They appeared either isolated or associated in variable numbers, structures resembling intraepithelial glands. These particular cells synthesize and accumulate PAS-positive mucines rich in glycosaminoglycans, and are usually found in conjunctival epithelium. We observed a number of invaginations in the connective tissue underlying the epithelium, mostly formed by goblet cells, this giving the aspect of mucous glands, similar to the "glands of Henle". A highly developed vascular neoformation network, consisting of arterioles, venules and a very large number of capillaries can also be found in the connective tissue. Immunohistochemistry suggested that B-lymphocytes marginally take part in the immune response in pterygium. T-lymphocytes formed the majority of the mass of immune system cell present in connective tissue of the pterygium. Macrophage-type cells were distributed unevenly in the pterygium tissue, as the intensity of the inflammatory process varies depending on antigen levels. Conclusions: Pterygium shows significant changes both in the epithelium and in the underlying connective tissue. Immune cell infiltrate was diffuse, more abundant in areas with erosion of the covering epithelium.

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

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23. Protective effect of L-N-acetylcysteine against gentamycin ototoxicity in the organ cultures of the rat cochlea

Alma Maniu, Maria Perde-Schrepler, M. Cosgarea

The aim of this study was to test if L-N-acetylcysteine (L-NAC) can protect hair cells against gentamycin-induced damage in vitro. Mammalian auditory cells are unable to regenerate when affected by several toxic agents. Aminoglycosides are large-scale antibiotics, extremely useful for the treatment of several Gram-negative bacterial infections, but their use is limited by the extremely severe side effects like ototoxicity and nephrotoxicity. Materials and Methods: 1-4-day-old rat cochlea explants were exposed to different doses of gentamycin. Half of the cochleas were pretreated for 24 hours with different doses of L-NAC. The explants were fixed and stained with phalloidin, and the intact hair cells were counted. Results: GM treatment resulted in the loss of sensorial cells in the organ of Corti explants in a dose-dependent manner. All doses of L-NAC offered significant protection (p<0.001) when added in culture 24 hours prior to GM. There was no significant difference between the level of protection offered by the different doses of L-NAC, both in the outer and inner hair cells. Conclusions: Our results demonstrate that L-NAC can protect cochlear cells against gentamycin toxicity.

Corresponding author: Alma Maniu, Assistant Professor, MD, PhD, e-mail: almacjro@yahoo.com

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24. Micropathological changes in the sub-epidermal zone of normal appearing skin in leprosy

Rakhi Rastogi, Virendra Budhiraja, C. S. Ramesh Babu, Molly Madan, Arvind Krishna, A. K. Asthana

Leprosy is a chronic inflammatory disease caused by Mycobacterium leprae, which affects not only the peripheral nerves and skin but also various internal viscera through the hematogenous spread, especially in lepromatous cases. The micropathological changes in epidermis, nerves and skin appendages from lesioned skin reported by various authors but reports of involvement of apparently normal sites are few. We investigated skin biopsy material taken from 130 patients with clinically diagnosed leprosy. Biopsies were taken at least 10 cm away from site of lesion. Hematoxylin and Eosin staining and Harada's modified Allochrome method for acid-fast bacilli were applied for histological investigations. The pattern of leprosy among the patients were indeterminate in 53 cases (40.8%), tuberculoid in 29 cases (22.3%), borderline tuberculoid in 14 cases (10.8%), borderline leprosy in ten cases (7.7%), borderline lepromatous in nine cases (6.9%) and lepromatous leprosy in 15 cases (11.5%). The changes were seen in sub-epidermal zone of normal appearing skin in all type of leprosy, but involvement was greater at the lepromatous end of the spectrum compared to tuberculoid end. Acid-fast bacillus (AFB) was seen in subepidermal zone of normal appearing site. Presence of AFB is significant as far as dissemination and transmission of disease is concerned.

Corresponding author: Rakhi Rastogi, Associate Professor, MD, PhD, e-mail: rakhirastogi1207@gmail.com

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25. Aberrant TTF-1 expression in papillary high-grade urothelial neoplasm: case report and literature review

Maria J. Fernandez-Acenero, S. Cordova, C. Santonja

We herein report the case of a 48-year-old man who developed synchronous advanced tumors in the lung and the bladder. The most striking feature of our case is that the otherwise typical bladder urothelial carcinoma showed focal areas (comprising less than 5% of the tumor mass) of nuclear positivity for TTF-1 (thyroid transcription factor-1). The different pattern of cytokeratin expression led us to consider them two independent primary tumors. Several recent reports have indicated that the type of clone used can influence the results of TTF-1 staining and can explain positivity in extrapulmonary and extrathyroid tumors.

Corresponding author: Maria J. Fernandez-Acenero, MD, PhD, e-mail: mgg10167@gmail.com

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26. Diffuse polypoid lesions of the digestive tract in severe hypogammaglobulinemia

C. Jurcut, Mariana Jinga, Raluca Costache, Florina Vasilescu, Alina Stanescu-Popp, I. Copaci

We present a case report of diffuse polypoid lesions of the digestive tract in which multiple endoscopic techniques (upper endoscopy, colonoscopy, and capsule endoscopy) were performed in order to complete and refine the diagnosis in a patient with severe hypogammaglobulinemia with recurrent infections, bronchiectasies and diffuse nodular lymphoid hyperplasia of the digestive tract. In patients with hypogammaglobulinemia, the endoscopic evaluation of the digestive tract might be mandatory, even in asymptomatic patients. The complete assessment should include the upper endoscopy and colonoscopy with multiple biopsies and, if possible, capsule endoscopy.

Corresponding author: Ciprian Jurcut, MD, PhD student, e-mail: cjurcut@gmail.com

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27. Talon cusp: a morphological dental anomaly

H. A. Balcioglu, N. Keklikoglu, Gulseren Kokten

Talon cusp is a rare developmental anomaly that occurs most commonly on the lingual side of the incisors. It may cause various clinical problems, such as occlusal interference, irritation of the tongue, pulpal necrosis, caries and periodontal problems. Genetics is thought to be a major cause for the occurrence of this anomaly. This article reports three cases of talon cusp in three members of the same family. Clinical and radiographic findings of talon cusp in two siblings and the mother are presented. Early diagnosis of talon cusp helps in selecting the correct treatment procedure and avoiding complications.

Corresponding author: Huseyin Avni Balcioglu, Assistant Professor, DDS, PhD, e-mail: habalci@istanbul.edu.tr

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28. Laryngocele associated with laryngeal carcinoma

Mihaela Mitroi, Alina Capitanescu, Florina Carmen Popescu, Camelia Popescu, Carmen Aurelia Mogoanta, G. Mitroi, C. Surlin

Laryngocele is an abnormal dilatation of Morgagni ventricle (saccule) in direct communication with the laryngeal lumen. Symptoms are not characteristic: hoarseness, dyspnea, foreign body sensation and cough. Sometimes it is presented as cervical swelling causing airway obstruction and need an emergency tracheotomy. In this paper, we report a case of upper airway obstruction due to laryngocele associated with a laryngeal carcinoma who was treated by emergency tracheotomy and, finally by total laryngectomy. A review of the literature is also presented.

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

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29. The value of the immunohistochemistry in a case of gastric neuroendocrine tumor and thyroid metastasis

Catalina Poiana, Mara Carsote, Carmen Ardeleanu, Dana Terzea, Elena Taina Avramescu, M. C. Neamtu, Rucsandra Danciulescu Miulescu

Thyroid metastasis is atypical. We present a 70-year-old female case that was first diagnosed as gastric cancer after surgical approach. Two years later a thyroidectomy was performed and the immunohistochemistry (IHC) profile revealed a neuroendocrine tumor (NET): poorly differentiated neuroendocrine carcinoma (with small cells), with positive reaction for SYN, CROMO, negative for calcitonin, TTF1 and thyreoglobulin. The Ki-67 index was 25%. Considering the unusual metastasis, the IHC exam of the stomach tumor was performed pointing the same features as the thyroid findings. This proved that the thyroid tumor was a metastasis from a primary gastric neoplasia. This is an unusual case of NET because of the thyroid involvement. Nevertheless, the IHC exam played the major role in elucidating the diagnosis and the prognosis of the case.

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

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30. Retroperitoneal seminoma as a first manifestation of a partially regressed (burnt-out) testicular germ cell tumor

O. Preda, Alina Nicolae, Andrada Loghin, Angela Borda, F. F. Nogales

Regressed (burnt-out) testicular germ cell tumors (TGCT) are rare clinical situations that are clinically difficult to recognize. This 43-year-old patient was admitted because of a suspicion of prostatic carcinoma, which eventually was followed by transrectal ultrasonography and a CT scan, both of which revealed a large retroperitoneal mass. Surgery showed extensive ureteral and vas deferens infiltration. Pathology was consistent with a classical seminoma. Eventually, testes were normal on palpation but ultrasonography only revealed areas of fibrosis and microcalcifications in the left testis, which was followed by a left orchidectomy. Microscopically, there were extensive areas of fibrosis and only a 2 mm area of seminoma was demonstrated. The few areas of uninvolved testicular tissue lacked lesions of intratubular germ cell neoplasia (IGCNU). Retroperitoneal germ cell tumors are rare in the male and consequently, an origin from an occult testicular tumor should always be discarded by image analysis and eventually a biopsy. Immunologic response may be responsible for tumor involution.

Corresponding author: Ovidiu Preda, MD, e-mail: ovidiupreda79@gmail.com

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31. Basal cell carcinoma miming cutaneous melanoma

Ina Ruxandra Petrescu, Maria Sajin, Mariana Costache, G. Simion

In the last decades, the incidence of skin cancer is in continuous growth, but the mortality remains at the same level thanks to the new imaging diagnosis methods and surgical treatment. A real problem regarding differential diagnosis between cutaneous melanoma and non-melanotic skin tumor appear, despite advanced technologies, which have a major impact on treatment management. We reported two cases with clinical and dermoscopic aspects of cutaneous melanoma, sustained by positive lymphoscintigraphy. The histopathological exams established that the skin tumor was a pigmented basal cell carcinoma. In such difficult cases, the accuracy of diagnosis is certificated only by the microscopical examination with clinical, treatment and prognosis changes.

Corresponding author: Ina Ruxandra Petrescu, MD, e-mail: inapetrescu@yahoo.com

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32. A rare case of communicating branch between the posterior femoral cutaneous and the sciatic nerves

S. Tunali, Neslihan Cankara, S. Albay

During routine dissection of a 75-year-old male cadaver, we observed a communicating branch between the posterior femoral cutaneous nerve and the sciatic nerve. The connection was 11 cm below the infrapiriform foramen and was 3 cm long. Excluding this communicating branch, the origin, course and distribution of the posterior femoral cutaneous nerve showed no variation. The other branches of the sacral plexus were as usual.

Corresponding author: Selcuk Tunali, MD, PhD, e-mail: tunali@hacettepe.edu.tr, selcuk@hawaii.edu

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33. Book Review: Maxillary Tumors and Pseudotumors

M. Raica

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