Relação da expressão de fatores de crescimento celular (IGF-1) e (SCF) com fatores prognósticos e o alvo da rapamicina em mamíferos (m-TOR) em mastocitomas cutâneos caninos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Histopathologic Changes Induced by Intense Pulsed Light in the Treatment of Poikiloderma of Civatte

Background The clinical efficacy of intense pulsed light (IPL) in the treatment of poikiloderma of Civatte (PC) is well documented, but little is known about microscopic changes. Objective To analyze histopathologic findings on the necks of individuals with PC after IPL therapy. Materials and Methods Fourteen patients with PC on the neck underwent three monthly sessions of IPL. Biopsies and clinical photographs were taken before and 60 similar to days after treatment. A dermatopathologist analyzed histopathologic slides stained with hematoxylin and eosin, Masson's trichrome, Verhoeff-van Gieson and Fontana-Masson or processed for CD-34 immunohistochemistry. The slides also underwent digital image analysis. Clinical results were based on the analysis of the pictures by three dermatologists and on patient satisfaction. Results Intense pulsed light treatment resulted in more-homogeneous melanin distribution; a greater number of fibroblasts and nonfragmented elastic fibers; and greater density (p similar to=similar to.01), color intensity (p similar to=similar to.02), number and thickness of the collagen bundles. No significant changes in vessels number or diameters were observed. Clinical results were positive in 92.9% of the cases. Conclusion IPL treatment of PC induced a more-homogeneous distribution of melanin and increased nonfragmented elastic fibers, collagen density, and intensity. These changes were related to clinical improvement.

Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa

The aim of this investigation was to determine the presence of abnormal sperm chromatin packaging in spermatozoa with large nuclear vacuoles (LNV) selected via high magnification by analysing the pattern of chromomycin A3 (CMA3) staining. A prospective observational study was designed to analyse semen samples obtained from 66 men undergoing infertility diagnosis and treatment. The numbers of cells with normal (dull yellow staining of the sperm head/CMA3-negative) and abnormal (bright yellow fluorescence of the sperm head/CMA3-positive) chromatin packaging were determined on slides with normal and LNV spermatozoa. The presence of bright yellow fluorescence (CMA3-positive) was significantly higher (p < 0.0001) in spermatozoa with LNV than in normal spermatozoa (719/1351; 53.2% vs. 337/835; 40.3%, respectively), reflecting a higher percentage of abnormal chromatin packaging in spermatozoa with large LNV. Our data support the hypothesis that the presence of LNV reflects the presence of abnormal chromatin packaging, which may facilitate sperm DNA damage. As sperm nuclear vacuoles are evaluated more precisely at high magnifications using motile sperm organelle morphology examination (MSOME), the present results support the use of high-magnification sperm selection for intracytoplasmic sperm injection (ICSI).

Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-stranded or normal double-stranded DNA in spermatozoa with large nuclear vacuoles (LNV) selected by high magnification. Fresh semen samples from 30 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and LNV were selected at x8400 magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation in spermatozoa with LNV (29.1%) was significantly higher (P < 0.001) than in spermatozoa with NN (15.9%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono- and oligonucleosomes), and single-strand breaks (nicks) in high molecular weight DNA occur more frequently in spermatozoa with LNV. Similarly, the percentage of denatured-stranded DNA in spermatozoa with LNV (67.9%) was significantly higher (P < 0.0001) than in spermatozoa with NN (33.1%). The high level of denatured DNA in spermatozoa with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibres. The results show an association between LNV and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high magnification for ICSI.

Significance of extruded nuclear chromatin (regional nuclear shape malformation) in human spermatozoa: implications for ICSI

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-strand or normal double-strand DNA in spermatozoa with extruded nuclear chromatin (ENC) selected by high magnification. Fresh semen samples from 55 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and ENC were selected at 8400x magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation was not significantly different (p = 0.86) between spermatozoa with ENC (19.6%) and those with NN (20%). However, the percentage of spermatozoa with detectable denatured-stranded DNA in the ENC spermatozoon group (59.1%) was significantly higher (p < 0.0001) than in the NN group (44.9%). The high level of denatured DNA in spermatozoa with ENC suggests premature decondensation and disaggregation of sperm chromatin fibres. The results show an association between ENC and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high-magnification intracytoplasmic sperm injection.

Histological and immunohistochemical study of the expression of p53 and ki-67 proteins in the mucosa of the tongue, pharynx and larynx of rats exposed to cigarette smoke

Introduction: Head and neck cancers are linked to smoking. The most affected sites are the oral cavity, pharynx and larynx. Experimental studies show epithelial lesions caused by cigarette smoke. Objectives: To investigate in rats the effects of acute cigarette smoke exposure on the mucosa of the tongue, pharynx and larynx.Material and method: Wistar rats were allocated into two groups of 20 animals: CG (control) receiving food and water ad libitum and TG (Tobacco) exposed to the smoke of 40 cigarettes/day for 60 days. Biopsy of their tongues, pharynxes and larynxes were subjected to histopathological, histomorphometric and immunohistochemical studies of protein p53 and ki-67.Result: The histological analysis of tongue from the Tobacco group revealed epithelial hyperplasia (90%), basal cell hyperplasia (95%) and mild to moderate dysplasia (85%). In pharynx showed basal cell hyperplasia (85%), dysplasia (25%) and vascular congestion (95%). In larynx showed basal cell hyperplasia (70%), epithelial hyperplasia (55%), congestion (100%) and inflammatory infiltrate (25%). Morphometric analysis revealed that keratin layer thickness was greater in the tobacco group. P53 immunoexpression was negative in both groups. Ki-67 immunoexpression was positive in basal cell nuclei but in parabasal cell nuclei it was positive only in the Tobacco group.Conclusions: The exposure of animals to cigarette smoke for 60 days resulted in benign lesions. The duration of exposure was not enough to cause the development cancer, as confirmed by the negative expression of p53 protein in all slides examined. Analysis of ki-67 expression showed intense epithelial proliferation in response to damage.

Vocal Polyps: Clinical, Morphological, and Immunohistochemical Aspects

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Vocal Fold Nodules: Morphological and Immunohistochemical Investigations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hepatoprotective effect of water soluble extract of Coleus barbatus on cholestasis on young rats

OBJETIVO: Testar os efeitos do extrato aquoso de Coleus barbatus (EAB) na cirrose biliar secundária por obstrução das vias biliares extra-hepáticas em ratos jovens. MÉTODOS: Quarenta ratos Wistar machos com 21 dias de vida (P21), foram distribuídos em quatro grupos de 10 animais, submetidos a operação simulada ou dupla ligadura e ressecção do ducto biliar (S ou L) combinados EAB e a Água (B ou A). No P48, foi medido o tempo de sono com o pentobarbital (TS). No P49, foram submetidos a eutanásia para a determinação das atividades séricas do aspartato aminotransferase (AST) e da alanina aminotransferases (ALT); após a eutanásia foram avaliados o peso fresco do fígado (PFF) e, em cortes histológicos do fígado, a freqüência de mitoses (FM), o número de áreas de necrose (NN), a intensidade da fibrose (IF) e da proliferação ductal (IPD). Os efeitos da colestase, os do EAB e suas interações foram testados pela ANOVA com dois fatores, e as comparações múltiplas pareadas foram realizadas pelo teste de S.N.K ou teste de Wilcoxon. Também foi determinada a correlação linear de Pearson entre as variáveis histológicas duas a duas separadamente para os grupos LA e LD. O nível de significância estatística para os vários testes foi de p do erro alfa <0,05. RESULTADOS: A colestase aumentou significativamente o TS, a ALT, a AST, o PFF, a MI, o NN, a IF e a IPD. O EAB diminuiu o TS e a IM nos animais sem colestase (operação simulada). O EAB diminuiu o TS, a ALT, a AST, o PFF, a MI, o NN e IF na colestase. No grupo LA houve correlação positiva entre a IPD e a IF, correlação negativa entre a IPD e a FM e correlação negativa entre a IF a FM. No grupo LD houve correlação negativa entre o NN e a IPD. CONCLUSÕES: Na ausência de colestase o EAB encurta o tempo de sono e diminui a freqüência de mitoses. O EAB apresenta efeito hepatoprotetor no modelo de cirrose biliar secundária a obstrução biliar extra-hepática.

Perineural Invasion in Aggressive Skin Carcinomas of the Head and Neck

Introduction: Perineural invasion is a well-recognized form of cancer dissemination. However, it has been reported only in few papers concerning cutaneous carcinomas ( basal cell, BCC, and squamous cell, SCC). Moreover, the incidence is considered to be very low. Niazi and Lambert [Br J Plast Surg 1993; 46: 156-157] reported only 0.18% of perineural invasion among 3,355 BCCs. It is associated with high-risk subtypes, as morphea-like, as well as with an increased risk of local recurrence. No paper was found in the literature looking for perineural invasion in very aggressive skin cancers with skull base extension, with immunohistochemical analysis. Methods: This is a retrospective review, including 35 very advanced skin carcinomas with skull base invasion (24 BCCs and 11 SCCs, operated on at a single institution from 1982 to 2000). Representative slides were immunohistochemically evaluated with antiprotein S-100, in order to enhance nerve fibers and to detect perineural invasion. The results were compared to 34 controls with tumors with a good outcome, treated in the same time frame at the same Institution. Results: Twelve (50.0%) of the BCCs with skull base invasion had proven perineural invasion, as opposed to only 1 (4.6%) of the controls, and this difference was statistically significant (p < 0.001). Regarding SCCs, 7 aggressive tumors (63.6%) showed perineural invasion compared to only 1 (10.0%) of the controls, but this difference did not reach significance (p=0.08), due to the small number of cases. Conclusions: In this series, it was demonstrated that immunohistochemically detected perineural invasion was very prevalent in advanced skin carcinomas. In addition, it was statistically associated with extremely aggressive BCCs with skull base invasion. Copyright (c) 2008 S. Karger AG, Basel

Imuno-histoquímica para identificação de células neoplásicas no infiltrado ativo de melanomas finos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection of bovine Herpesvirus type 5 in formalin-fixed, paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue-based diagnostic test of bovine encephalitis

The aim of this study was to evaluate the application of PCR technique for the detection of BoHV-5 in routinely formalin-fixed, paraffin-embedded brain tissues in 20 naturally infected calves affected by fatal meningoencephalitis. Brains were divided into two halves, one kept fresh for virus isolation and PCR assay, targeting the glycoprotein C gene from BoHV-5 genome. The other half brain, corresponding to posterior cortex region, was submitted to formalin fixation and embedded into paraffin blocks for microscopic evaluation and total DNA isolation. Most of the slides showed severe multifocal non-supurative encephalitis with neuronal degeneration, neurophagia, and no acidophilic intranuclear inclusions could be found in neurons and glial. The 20 fresh samples were confirmed, by virus isolation and PCR assay, as having the BoHV-5 virus and, respective glicoprotein C sequence, while 15 of 20 formalin-fixed, paraffin-embedded samples were considered positive for the same analysis. The results revealed the first description of PCR efficiency, applied to formalin-fixed, paraffin-embedded brain collected from naturally infected calves, improving the detection of BoHV-5 from archival samples in South America. (c) 2007 Published by Elsevier B.V.

Detection of turkey coronavirus in commercial turkey poults in Brazil

Poult enteritis complex has been incriminated as a major cause of loss among turkey poults in other countries. We have observed this in Brazil, associated with diarrhoea, loss of weight gain and, commonly, high mortality In this study, we have used the reverse transcriptase polymerase chain reaction (RT-PCR) to detect turkey coronavirus (TCoV) in sick poults 30 to 120 days of age from a particular producer region in Brazil. The RT-PCR was applied to extracts of intestine tissue suspensions, and the respective intestinal contents, bursa of Fabricius, faecal droppings and cloacal swabs. Primers were used to amplify the conserved 3' untranslated region of the genome, and the nucleocapsid protein gene of TCoV Histo pathological and direct immunohistochemical examinations were performed to detect TCoV antigen in infected intestine and bursa slides. All the results from stained tissues revealed lesions as described previously for TCoV infection. The direct immunohistochemical positive signal was present in all intestine slides. However, all bursa of Fabricius tissues analysed were negative. RT-PCR findings were positive for TCoV in all faecal droppings samples, and in 27% of cloacal swabs. Finally, the best field material for TCoV diagnosis was faecal droppings and/or intestine suspensions.

Histomorphometric analysis and immunolocalization of RANKL and OPG during the alveolar healing process in female ovariectomized rats treated with oestrogen or raloxifene

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hybrid layer thickness and resin tag length of a self-etching adhesive bonded to sound dentin

Objectives: the purpose of this study is to employ optical microscopy to measure the thickness of the hybrid layer and the penetration (tags) of an aggressive self-etching adhesive system into sound dentin.Methods: occtusat cavities were prepared in 40 extracted human posterior teeth. The prepared teeth were randomly assigned to four experimental groups with 10 specimens each. The self-etching adhesive system Adper Prompt L-Pop was applied to the dentin surface as follows: Group 1: cavosurface enamel was etched for 60 s and dentin for 20 s with 35% phosphoric acid get, immediately followed by application of the self -etching adhesive with a brush to the entire cavity for 15 s; Groups 2, 3, and 4: no pre-etching was performed, and the self -etching adhesive was applied to both enamel and dentin for 15, 30 and 45 s, respectively. After curing, the cavities were fitted with composite resin Fittek Z250. Afterwards, the teeth were decalcified and the restorations were carefully removed for later embedding in paraffin. The specimens were serially sectioned at 6 mu m of thickness and sequentially mounted in glass slides. These sections were stained with Brown and Brenn staining for posterior analysis and measurement of the hybrid layer and resin tags on a tight microscope with a micrometric ocular 40/075. The results were submitted to analysis of variance at the 5% level.Results: whenever there was significance, the Tukey test was applied at the 5% level. The specimens receiving application of acid etching before the selfetching. adhesive displayed a larger thickness of the hybrid layer; on the other hand, specimens receiving only application of the self -etching adhesive on dentin for 15, 30 and 45 s exhibited similar thickness of the hybrid layer. As regards the resin tags, no statistically significant differences could be found between the study groups.Conclusions: it could be concluded that the increase in the time of application of the self-etching adhesive Adper Prompt L-Pop did not significantly influence the formation and thickness of hybrid layer, as well as its penetration into the sound dentin surface. (c) 2005 Elsevier Ltd. All rights reserved.