Avaliação da atividade proliferativa celular das lesões bucais causadas pelo uso de próteses totais através da impregnação tecidual pela prata / Proliferative cellular activity evaluation of lesions caused by dentures through tissue silver impregnation

Introdução: As próteses totais visam conservar a função do sistema estomatognático do paciente totalmente edêntulo. Porém, na mucosa bucal podem aparecer manifestações cuja principal causa são as próteses totais mal adaptadas. Objetivo: o presente estudo objetiva investigar a proliferação tecidual das lesões causadas por próteses totais removíveis através do método de impregnação pela prata (AgNOR), com isso facilitando o tratamento e a determinação do prognóstico das lesões a serem estudadas. Metodologia: foram selecionados todos os casos das lesões bucais mais comumente associadas à utilização de próteses totais registradas no Serviço de Diagnóstico Histopatológico do ICB­UPF nos anos de 2012 e 2013, tendo sido encontrados 5 casos de granuloma piogênico, 5 casos de hiperplasia de fundo de sulco, 5 casos de fibroma de irritação e 2 casos de fibroma ossificante periférico. Os cortes histopatológicos das lesões foram impregnados pela prata (método AgNOR), tendo sido obtido, com auxílio do programa Image Tool®, o número de NORs de 100 células de cada caso, resultando numa média de NORs em cada grupo de lesões. Resultados: os resultados obtidos foram tabulados em planilha eletrônica e a comparação do número médio de NORs de cada grupo foi realizado por meio do teste estatístico ANOVA, 5% de significância. Resultados: o grupo das hiperplasias de fundo de sulco mostrou média de 2,41 NORs por núcleo, o grupo dos granulo mas piogênicos mostrou 2,44, o fibroma de irritação 2,22, e o fibroma ossificante periférico mostrou média de 1,89 NORs por núcleo celular, diferindo estatisticamente esta lesão das anteriormente mencionadas (p = 0,002). Conclusão: o fibroma ossificante periférico mostrou ser a lesão causada por prótese total removível com a menor atividade proliferativa celular. Tal estudo precisa ser complementado por futuros estudos clínicos.

Introduction: the total dentures are aimed at preserving the function of the stomatognathic system of the fully edentulous patient. However, in the oral mucosa may appear manifestations whose main cause are the totally unsuitable dentures. Objective: this study aims to investigate the proliferation of tissue lesions caused by removable dentures by impregnation method for silver (AgNOR), thereby facilitating the treatment and determining the prognosis of the lesions to be studied. Methodology: we selected all cases of oral lesions most commonly associated with the use of dentures recorded in Histopathological Diagnostic Service ICB-UPF in the years 2012 and 2013, having been found 5 cases of pyogenic granuloma, 5 cases of hyperplasia, 5 cases of irritation fibroma and 2 cases of peripheral ossifying fibroma. Histopathological lesions cuts were impregnated by silver (AgNOR method), having been obtained with the aid of the program Image Tool®, the number of NOR cells 100 in each case, resulting in an average NORs in every group of lesions. Results: the results were tabulated in a spreadsheet and comparing the average number of NORs of each group was conducted through ANOVA, 5% significance level. Results: The group of hyperplasias showed average of 2.41 NORs per nucleus, the group of pyogenic granulomas showed 2.44, the irritation fibroma 2.22, and peripheral ossifying fibroma showed average of 1.89 NORs for cell nucleus, differing significantly from that of the aforementioned lesions (p = 0.002). Conclusion: the peripheral ossifying fibroma proved the injury caused by removable dentures with lower cell proliferative activity. This study needs to be complemented by future clinical studies.

Adaptación de la técnica de impregnación argéntica de Llombart para la demostración de fibras nerviosas, en cualquier tejido en cortes por parafina / Adaptation of Llombart's silver impregnation technique for demonstration of nerve fibers in any tissue in paraffin sections

En la investigación biológica sigue siendo necesaria la demostración de la inervación periférica en numerosos tejidos y órganos. El objetivo de este trabajo fue rescatar y modernizar uno de los métodos más constantes que hemos probado para demostrar la inervación periférica. La técnica de Llombart para fibras nerviosas se adaptó en cortes por parafina de 7 µm en diferentes tejidos animales. La impregnación argéntica se hizo por goteo en cámara húmeda. Se demostraron en forma constante, precisa y seriada terminaciones nerviosas y corpúsculos sensoriales, neuronas y fibras nerviosas periféricas. A pesar de la alta especificidad para fibras nerviosas, la técnica no compromete el panorama tisular por lo que da bellas imágenes de conjunto. Sin ser una técnica para argentafinidad, demuestra claramente dos tipos de células argentafines en las glándulas adrenales. La adición de los reactivos metálicos en gotas y en cámara húmeda, ofrece una variante sumamente económica.

In Biological research is still necessary for the demonstration of the peripheral innervation in numerous tissues and organs. The aim of this study was to rescue and modernize one of the most consistent methods that we have tried to demonstrate peripheral innervation. Llombart's technique for nerve fibers was adapted by paraffin cuts of 7 µm in different animal tissue. The silver impregnation was done by dripping in a moist chamber. It was demonstrated in a constant, precise and serial form, nerve terminations, and sensorial corpuscles, neurons, and peripheral nerve fibers. Despite being highly specific to nerve fibers, the technique does not sacrifice tissue panorama so it gives beautiful images set. Without being a technique to argentaffin structures, it clearly shows two types of argentaffin cells in the adrenal glands. The addition of the metal reactive in droplets and in a humid chamber provides a very economical variant.

Significance of AgNOR score in benign and malignant soft tissue tumours.

AgNOR staining was employed on FNAC and histopathological sections obtained from patients with soft tissue tumours. The study comprised of 20 normal appearing soft tissues, 74 benign and 36 malignant soft tissue tumours. The slides were stained with AgNOR in order to differentiate between benign and malignant soft tissue tumours. The mean AgNOR count in normal appearing soft tissues, benign lesions and malignant lesions was 1.04+/-0.10 (0.94-1.14), 1.51+/-0.21 (1.1-2.1) and 4.96+/-1.33 (2.57-7.21) respectively. The mean AgNOR count was found to be higher in benign soft tissue tumours as compared to normal appearing soft tissues and the difference was found to be statistically significant. The mean AgNOR count in soft tissue sarcomas was found to be higher as compared to both normal appearing soft tissues and benign soft tissue tumours and the results were found to be statistically significant. The increased AgNOR score in both benign and malignant soft tissue tumours as compared to normal appearing soft tissues indicates high proliferative activity. Thus AgNOR staining is a simple and useful method for estimating tumour cell proliferation thereby differentiating normal appearing soft tissues from benign and malignant soft tissue tumours.

Reticulin morphometry and collagen fibers optical anisotropy as assessed by image analysis after silver impregnation

A new method for the impregnation of tissue sections with ammoniacal silver carbonate in terms of single-step reaction, no gold toning, no oxidation step and silver impregnation after treatment with pancreatic (alfa)-amylase is described. This method yields silver nanocrystalloids with 18.40 nm in average diameter [16]. Natural carbohydrate radicals probably acting as natural reducing agents for silver are assumed to occur. Despite the fact that the method is not specific, reticulin fibers (type III colagen) were clearly distinguished from other collagen fibers types and easily and accurately segmented by image analysis (IA) systems, when using this procedure. Data from myocardial reticulin fibers are presented. Collagen bundles (type I collagen rich structures) exhibited intense linear dichroism (LD) interms of gray averages measured by IA (non-impregnated, natural, collagen fibers do not show LD) Anomalous dispersion of birefringence was also detected, highlighting typical morphologies, including crimp, which allow us to propose the methodo for molecular order studies and to assess collagen bundles as chiral objects, especially together with LD data.

Evaluation of AgNORs in pulmonary lesions: a cyto-histopathological correlation.

Silver nucleolar organizer regions (AgNORs) were studied in one hundred specimens of endobronchial brushings. Bronchial biopsies were also obtained from these one hundred cases and subjected to AgNOR staining. These included twenty histologically normal cases, ten cases of inflammatory pathology and seventy cases of pulmonary neoplasms. Fixed cytologic smears and three micron thick sections of paraffin embedded tissue were subjected to AgNOR staining. The mean AgNOR counts in bronchial tissue showed a progressive and statistically significant increase from normal, through inflammatory to neoplastic lesions in both cytologic smears and histopathologic specimens. The AgNOR technique is simple, inexpensive and a useful adjunct to routine histopathology, to evaluate pulmonary lesions. The counts, however, cannot be standardized for a particular lesion as there are inter-laboratory variations.

Identification of Biomphalaria havanensis and Biomphalaria obstructa populations from Cuba using polymerase chain reaction and restriction fragment length polymorphism of the ribosomal RNA intergenic spacer

In Cuba, several Biomphalaria species have been reported such as B. orbignyi, B. schrammi, B. helophila, B. havanensis and B. peregrina; only the latter three are considered as potential hosts of Schistosoma mansoni. The specific identification of Biomphalaria species is based on anatomical and morphological characters of genital organs and shells. The correct identification of these snails is complicated by the high variation in these characters, similarity among species and in some cases by the small size of the snails. In this paper, we reported the classical morphological identification, the use of PCR and RFLP analysis of the internal transcribed spacer region of the ribosomal RNA genes for molecular identification of seven snail populations from different localities in Cuba. Using morphological and molecular analysis, we showed that among the studied Cuban Biomphalaria populations only B. havanensis and B. obstructa species were found

Genetic variability in Brazilian populations of Biomphalaria straminea complex detected by simple sequence repeat anchored polymerase chain reaction amplification

Biomphalaria glabrata, B. tenagophila and B. straminea are intermediate hosts of Schistosoma mansoni, in Brazil. The latter is of epidemiological importance in the northwest of Brazil and, due to morphological similarities, has been grouped with B. intermedia and B. kuhniana in a complex named B. straminea. In the current work, we have standardized the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) technique, using the primers (CA)8RY and K7, to study the genetic variability of these species. The similarity level was calculated using the Dice coefficient and genetic distance using the Nei and Li coefficient. The trees were obtained by the UPGMA and neighbor-joining methods. We have observed that the most related individuals belong to the same species and locality and that individuals from different localities, but of the same species, present clear heterogeneity. The trees generated using both methods showed similar topologies. The SSR-PCR technique was shown to be very efficient in intrapopulational and intraspecific studies of the B. straminea complex snails

Determinación de microproteínas urinarias en orinas sin concentrar / Urinary low molecular weight proteins on unconcentrated urine

Se realizó la determinación cuali y cuantitativa de las microproteínas urinarias (MU): Alfa-1 microglobulina (A1m), proteína transportadora del retinol (RBP) y Beta-2 microglobulina (B2m), en 138 orinas de 24 h de recolección con pH superiores o iguales a 5.5, en pacientes adultos con distintos cuadros uroproteicos: 38 fisiólogicos, 59 glomerulares, 19 tubulares y 22 mixtos. Se describe la metodología analítica para optimizar la detección de las MU mediante la técnica de electroinmunofijación (EIF) con coloración argéntica, en orinas sin concentrar. El rango de sensibilidad para las distintas MU fue de 3-100mg/l. La cantidad de antisuero monoespecífico empleado por cm2 de gel fue: anti-A1m (4µl/cm2), anti-RBP (4µl/cm2) y antiB2m (2µl/cm2). La determinación cuantitativa se relizó por inmunodifusión radial (IDR) para RBP y B2m, y por electroinmunodifusión (EID) para A1m. La detección de MU en orinas sin concentrar mediante esta metodología de alta sensibilidad y la cuantificación de las mismas permitieron efectuar un correcto diagnóstico de proteinuria tubular y mixta superando las eventuales pérdidas o desnaturalización protéica que acarrean los clásicos métodos de concentración. La detección elevada de A1m (15-65 mg/l) en 12 de 59 orinas con proteinuria glomerular, plantea un interrogante sobre la cuantificación aislada de la MU para definir un patrón tubular

Improvement of silver impregnation technique (Protargol) to obtain morphological features of protists ciliates, flagellates and opalinates

The research on ciliates, flagelates and opalinates have been widespread by the utilization of techniques employing silver impregnation (Protargol), modified by several authors. However, these are time consuming and its results are variable. The present work is a variant of the technique described by Tuffrau (1964, 1967) showing some adaptations made in our laboratory. The organisms can be preserved by different fixatives (alcoholic Bouin, Stieve's fluid, 2.5 percent glutaraldehyde and others) and then rinsed in destilled water followed by a fast clarification by 3 percent sodium hypochloride. If the organism is very sensitive to hypochloride, 4 percent sodium lauryl sulfate may be used and then washed 3 times in distilled water. The protista can be adhered to the glass slides with Mayer's glycerinated-albumin (1 glycerin vol. to 1 or 2 albumin vol.), diluted in water at a proportion of 1:10 Cv/v., or with 1 percent polylysine followed by fast washes with distilled water. After the slide preparation, they were covered with a layer of 0,8 percent Silver proteinate. Right after that, the slide has to be placed in a glass tray lined with moist tissue and covered to prevent the proteinate to dry. The tray was placed in a incubator at 40º-50ºC for 30 minutes. The slides are rinsed for 1 minute. with warm (35ºC) distilled water. The development of the material should be done with 0.4 percent hydroquinone with a maximum incubation time of 1 minute. It should be developed gradually, controlling the silver impregnation intensity by observation under optical microscope. Next, rinse in distilled water for 1 minute, and then, fix in 2,5 percent Sodium thiosulfate. Rinse the slide for two minutes before dehydrating it in an alcoholic serial 50-100º. Finally rinse the slides in xylene. Mount the slides with Entellan MerckTM or Canada balsam

DNA synthesis and nucleolar organizer regions circadian rhythm in mouse regenerating liver hepatocytes

DNA synthesis and Nucleolar Organizer Regions (NORs) were studied in C3HS inbred mice standardized for periodicity analysis. Immunohistochemical detection of Bromodeoxyuridine (BrdU) incorporated into DNA with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections of regenerating liver. Tissue samples were obtained at different times after hepatectomy along a circadian span. The results showed a strong correlation of values between DNA synthesis (BrdU labelling index) and AgNOR numbers, with higher counts during the activity period of animals at 00:00/38 and 04:00/42 hours Time of Day/Hours Post-Hepatectomy (TD/HPH), being the differences with other time points highly significant. Our observations demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU incorporation and AgNOR numbers with a defined circadian rhythm in mouse regenerating hepatocytes.

Carcinoma epitelial mioepielial. Técnica para identificar células mioepiteliales / Epithelial-myoepithelial carcinoma. Method to mark myoepithelial cells

Se estudian dos casos de carcinoma epitelial-mioepitelial de glándula parótida de pacientes de sexo femenino de 48 y 80 años de edad, y tejido glandular normal. La técnica argéntica de Del Río Hortega- Polak impregna selectivamente las células mioepiteliales y resulta un marcador alternativo. Esta técnica puede ser usada en materiales incluidos en parafina o en cortes obtenidos por congelamiento

Detecting p53 gene mutation of breast cancer and defining differences between silver staining PCR-SSCP and immunohistochemical staining

This study detects and defines the patterns of p53 gene mutations in breast cancers. We analyse p53 gene mutations through comparing the results of single-strand-conformation-polymorphism (SSCP) and immunohistochemistry (IHC), and we try to define the differences between the results of SSCP and IHC. Twenty-seven fresh primary breast cancer tissues and eight normal breast tissues were studied. The IHC was done with the usual streptavidin-biotin peroxidase complement method by using monoclonal antibody DO-7. The results of staining was scored. The SSCP method was done by using Cold SSCP Electrophoresis System. Overexpressions of p53 protein were seven (25.9%) among 27 cancer cases on IHC. Four (57.1%) of seven cases were positive in SSCP. In SSCP, the mutations were detected in 10 (37%) among 27 cancer cases. The mutations were two in exon 5, one in exon 8, and seven cases in exon 7. All of 10 mutations were proved by sequencing analysis. Of them, only four (40%) were positive in IHC. We consider the IHC as a screening method for p53 gene mutations.

AgNORs in endometrial lesions.

Silver nucleolar organizer region (AgNOR) staining was employed in one hundred specimens of endometrium. These included fifteen normal controls (Proliferative + Secretory endometrium) and eighty five lesions. Endometrial lesions comprised of endometritis (15), endometrial hyperplasia (25) and endometrial carcinoma (45). Three micron thick sections of paraffin embedded tissue were subjected to AgNOR staining as described by Crocker and Smith with a little modification of 0.01% safranin counterstain--The mean AgNOR scores were found to increase steadily from normal to endometritis to endometrial hyperplasia and carcinoma--The observations revealed statistically significant differences in values between atypical hyperplasia and carcinoma also. AgNOR staining and scoring is simple, inexpensive and a useful adjunct to routine histopathology to evaluate endometrial lesions especially to differentiate borderline lesions. Though scores cannot be standardized and fixed for a particular lesion as there are intralaboratory variations.

Microwave-enhanced of a method of silver impregnation applied in dermatohistopathology

An experimental development based on the combination of microwaves action with one of the methods of silver staining by Del RÝo Hortega is presented. Materials from normal and pathological tissues were studied. The stuff were fixed in 10 formalin and received a paraffined embedding. The impregnation was made with ammoniacal silver carbonate in a domestic microwave oven during twelve seconds at 375 w. As a result of this, a time shortage of the technique, the high degree of definition of the nuclear, cytoplasmatic and extracellular matrix (collagenus fibbers with an unsteady reticular representation) details and the easy visualization of mitosis was observed. Also, omitting the reduction, they were clearly evidenced the argirophilics granules (melanin pigment)

Tumores con rabdomioblastos y neuroblastos / Tumors with rhabdomyoblasts and neuroblasts

Se evalúa uma técnica de impregnación metálica, doble impregnación de Del Río Hortega, para evidenciar rabdomioblastos y neuroblastos, con recomendaciones sobre los tiempos de impregnación para obtener mejores resultados. Las imágenes obtenidas son muy demostrativas, tanto de los elementos embrionarios del mesénquima primitivo desde mioblasto, miotubo a célula acintada rabdomiobástica con estraciones transversales, hasta los elementos neopláticos de esta estripe. Y de los neuroblastos con sus prolongaciones. El material de estudio incluye un Tumor de Wilms renal con rabdomioblastos y neuroblastos, y un Neuroblastoma de cerebelo con componente rabdomioblástico. A estas lesiones se las considera desembrioplasias. Además se estudiaron 2 Rabdomiosarcomas embrionarios botrioides, uno de ellos de presentación inusual en una mujer menopáusica, 2 Tumores müllerianos mixtos de útero y trompa de Falopio, un Rabdomioma de faringe, y 3 embriones humanos de material de aborto entre 5 y 13 semanas. Destacamos la utilidad de la doble impregnación para estudiar rabdomioblastos y neuroblastos. En los Rabdomiosarcomas se pueden ver estructuras y elementos comparables con los de la etapa embrionaria: células raquetoides, acintadas, miotubos, rabdomioblastos. El Tumor de Wilms es un tumor disembrioplásico y está constituido por el blastema renal. En él hemos encontrado rabdomioblastos y neuroblastos. En el Neuroblastoma del cerebelo servamos rabdomioblastos con cierta organicidad (ectomeséquima). Y por último describimos un caso poco frecuente de Ragdomiosarcoma botrioide de cuello uterino en una mujer menopáusica.

Rapid and easy detection of Helicobacter pylori by in situ hybridization

Various in situ hybridization (ISH) methods have been used to identify Helicobacter pylori, a causative organism responsible for chronic gastritis and peptic ulcer disease, but they were hard to perform and time consuming. To detect H. pylori in a rapid and easily reproducible way, we developed synthetic biotinylated oligonucleotide probes which complement rRNA of H. pylori. Formalin-fixed and paraffin-embedded tissues from 50 gastric biopsy specimens were examined. Using a serologic test and histochemical stain (Warthin-Starry silver stain and/or Giemsa stain) as a standard, 40 of them were confirmed to be H. pylori-positive. Our ISH was quickly carried out within one hr and results were compared with those obtained from immunohistochemical stain. The ISH produced a positive reaction in 38 of 40 cases (95%). All H. pylori-negative cases failed to demonstrate a positive signal. The ISH has a sensitivity comparable to those of conventional histochemical and immunohistochemical stain, and has high specificity. In conclusion, ISH with a biotinylated oligonucleotide probe provides a useful diagnostic method for detecting H. pylori effectively in routinely processed tissue sections.

Quetotaxia cercariana de dos cepas venezolanas / Cercarial chaetotaxy of 2 Venezuelan strains of schistosoma mansoni

By means of the silver impregnation of two lots of Schistosoma mansoni cercariae belonging to the strains: JR (32 cercariae) and C5 (45 cercariac), the number and pattem of disposition of the argentophillic papillae were detertnined with the following results: -Average number of total dorsal papillae: 12.1 (JR strain) and 12.0 (C5 strain); the variation coefficients in this surface were less than 4 por ciento (JR strain) and bigger in JR than C5 strain. -Average number of total ventral papillac: 12.15 (JR), 11.97 (C5); maximum value of the variation coeficiente 6.4 por ciento (JR), higher in JR than C5 strain. -When the ventral surface was classified in four quadrants, the average number of papillae by quadrant was: quadrant A: 1. 15 (JR) and 1.06 (C5); B: 1.06 (JR) and 1 (C5); C: 4.97 (JR) and 4.96 (C5); D: 5.03 (JR) and 4.98 (C5). The variation coefficients were higher in the A and B ventral quadrants, reaching maximum values of 31.9 por ciento, and 61.0 por ciento for the posterior quadrants C and D. -When the ventral surface was divided in three equal parts for determining the position of the most variable papillae of this area, the greatest value of the variation coefficients obtained were for the 2nd thirds of the cercariae: 89.8 por ciento and 76.8 por ciento for C5 strain and 43.5 por ciento and 56.8 por ciento for JR strain. In relation to the total lateral papillac, the average numbers were: 17.0 (JR) and 16.6 (C5), with a maximum value of variation coefficient of (8. 1 por ciento (C5). The average total numbers of papillae of the tail were: 25.6 (JR) and 26.1 (C5) for the ventral surface and 20.72 (JR) and 21.33 (C5) for the dorsal papillae. The comparison between the percents of the cercariae of two S. mansoni strains with more than 1 papillae on the anterior ventral quadrants A or B (94 por ciento JR and 34 percent C5), resulted with significant diferentes (P < 0.05) (AU)##