Optimización de cultivos primarios de células de carcinoma renal de células claras como modelo "in vitro" para estudios metabólicos y determinantes de progresión neoplásica / Optimization of primary cultures of clear cell renal carcinoma cells as an "in vitro" model for metabolic studies and determinants of neoplastic progression

El objetivo del presente estudio fue optimizar la implementación de cultivos primarios a partir de muestras de carcinoma renal de células claras (CRCC) para comprobar la conservación del fenotipo lipogénico contra cortes fijados del mismo origen. Se utilizaron muestras de pacientes con CRCC, evaluándose diversas metodologías y condiciones experimentales de digestión de muestras, adherencia y despegue celular, fenotipo lipogénico, potencial de clonación, proliferación y capacidad de migración. El mayor rendimiento y viabilidad celular se verificó mediante digestión con colagenasa. La adherencia inicial se logró a las 24 hs de incubación, utilizando placas plásticas de cultivo, recubiertas con colágeno comercial y gelatina 0,2% en la mayoría de las muestras analizadas (60% de los casos). Se obtuvieron monocapas, con potencial de migración, en un 40% de los casos, tras 5 ± 1 días de incubación. El promedio de subcultivos fue de 3 ± 1. Este estudio permitió estandarizar cultivos primarios de CRCC comprobándose la conservación de la fenotipia lipogénica, logrando de dicha manera una herramienta importante y útil para el estudio de la biología tumoral y el ensayo de nuevas terapéuticas

The aim of this study was to optimize the implementation of primary cultures from samples of renal clear cell carcinoma (CRCC) to check the conservation of the lipogenic phenotype. CRCC Patient samples were used, in order to evaluate different methodologies and the experimental conditions of sample digestion, cell adhesion and lipogenic phenotype, proliferation and migration ability. The highest yield in cell number and viability was assessed using collagenase digestion. The initial adhesion was achieved after 24 hours of incubation in plastic plates recoverd with commercial collagen or 0.2% gelatin (60% of cases). Monolayers, with migration potential, were obtained in 40% of all cases, after 5 ± 1 days of incubation. The subcultures average was 3 ± 1. This study allowed us to standardize primary cultures of CRCC and check the conservation of the lipogenic phenotyping, achieving in this way an important and useful tool to study the tumor biology.

O objetivo deste trabalho foi otimizar a implementação de culturas primárias de amostras de carcinoma de células claras renal (CRCC) para verificar conservação fenótipo lipogenic contra os cortes previstos a mesma origem. As amostras dos pacientes foram utilizados CRCC, avaliando diferentes metodologias e as condições experimentais da digestão de amostras, adesão celular e fenótipo clonagem potencial take-lipogenic, proliferação e capacidade de migração. O maior rendimento e a viabilidade celular foi avaliada por digestão com colagenase. A adesão inicial foi obtida após 24 horas de incubação com colagénio e gelatina comercial 0,2% em 60% dos casos. As monocamadas foram obtidos em 40% após 5 ± 1 dias de incubação com o potencial de migração. As subculturas média foi de 3 ± 1. Este estudo nos permitiu padronizar culturas primárias de CRCC são verificados quanto à conservação da fenotipagem lipogenic, conseguindo desta forma um importante e útil para o estudo da biologia do tumor e teste de nova ferramenta terapêutica

Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2'-bipyridine) ruthenium (II).

The paper shows the ability of the fluorochrome tris(2,2'-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent staining with Rubipy (50 micrograms/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd = (8.56 +/- 2.97) x 10(-5) M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC 4.2.2.7.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC 4.2.2.8.) degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate in multiple biological samples tested in parallel.

Litotricia extracorporea: experiencia inicial con el HT-2000 lithotripter / Extracorporeal shock wave lithrotripsy: initial experience with the HT-2000 lithotripter

Se comunican los 100 primeros casos de pacientes con litiasis de las vias urinarias tratados con el litotriptor HT-2000 Litotripter con generador electrohidráulico y focalizador fluoroscópico, en el esctor de Litrotricia Extracorpórea del servicio de urología del Hospital Británico de Buenos Aires, entre los meses de noviembre de 1996 y febrero de 1998. Se evalúan las características de las litiasis en lo que respecta a ubicación, tamaño y densidad radiológica, el número, la duración y demás datos de las seciones de litotricia . Los 100 casos incluyeron 46 litiasis piélicas, 17 litiasis calicilares y 37 litiasis uretrales. El tamaño de las litiasis fue menor a 1 cm de diámetro máximo en 54 casos, entre 1,1 y 2 cm en 34 casos, y mayor de 2 cm en 11 casos. Todos ellos tuvieron seguimiento posterior. La tasa de resultados positivos fue de l 86 por ciento, con 77 pacientes sis evidencia de litiasis residual, 9 con litiasis residual y 14 casos con resultado negativo. El porcentaje de fragmentación obtenido con el litotriptor HT-2000 litotripter se equipara con los resultados publicados en la literatura

Litotricia extracorporea: experiencia inicial con el HT-2000 lithotripter / Extracorporeal shock wave lithrotripsy: initial experience with the HT-2000 lithotripter

Se comunican los 100 primeros casos de pacientes con litiasis de las vias urinarias tratados con el litotriptor HT-2000 Litotripter con generador electrohidráulico y focalizador fluoroscópico, en el esctor de Litrotricia Extracorpórea del servicio de urología del Hospital Británico de Buenos Aires, entre los meses de noviembre de 1996 y febrero de 1998. Se evalúan las características de las litiasis en lo que respecta a ubicación, tamaño y densidad radiológica, el número, la duración y demás datos de las seciones de litotricia . Los 100 casos incluyeron 46 litiasis piélicas, 17 litiasis calicilares y 37 litiasis uretrales. El tamaño de las litiasis fue menor a 1 cm de diámetro máximo en 54 casos, entre 1,1 y 2 cm en 34 casos, y mayor de 2 cm en 11 casos. Todos ellos tuvieron seguimiento posterior. La tasa de resultados positivos fue de l 86 por ciento, con 77 pacientes sis evidencia de litiasis residual, 9 con litiasis residual y 14 casos con resultado negativo. El porcentaje de fragmentación obtenido con el litotriptor HT-2000 litotripter se equipara con los resultados publicados en la literatura(AU)

Cytochemical application of tris (2,2'-bipyridine) ruthenium (II): fluorescence reaction with sulfated polyanions of mast cell granules.

We describe the use of tris (2,2'-bipyridine) ruthenium (II) (Rubipy) as a cationic fluorochrome for cytochemical and histochemical studies. After staining with Rubipy, mast cell granules (MCGs) and lymphocyte nuclei (LN) from mouse peritoneal cavity and human breast carcinoma showed intense orange fluorescence and no fading under blue or blue-violet exciting light. Staining at low pH (< 2) or pre-treatment with Al3+ ions strongly diminished the fluorescence of LN, whereas that of MCG was less affected. Ca2+ and Ba2+ ions only diminished MCG fluorescence. Blots of DNA, pectic acid, heparin, and other sulfated polysaccharides stained with Rubipy showed high emission, which was reduced in DNA and pectic acid staining at low pH. Studies with chemically modified heparins suggested that O-sulfates were more important than N-sulfates in Rubipy-heparin interactions. These results are in agreement with an ionic binding mode between Rubipy and heparin. A very suitable method for mast cell detection was found with Mayer's hematoxylin before Rubipy staining, which could be of great value for histopathological studies. This procedure allowed visualization of the mast cells by fluorescence microscopy, and nuclei and tissue morphology were easily visualized under brightfield illumination.

[Isolation of Ancylostoma duodenale antigens and production of immune sera]. / Aislamiento de antígenos de Ancylostoma duodenale y produccion de inmunosueros.

Rabbits were immunized using intact larvae or homogenates from Ancylostoma duodenale. Antisera were tested by immunodiffusion. The homogenates promote the formation of antibodies but the intact worms were not able to induce them. The antisera were partially purified by precipitation with amonium sulphate 40% saturation and filtration through Sephadex G-200. The purified material was attached to Sepharose 6B and used as immunoabsorbent for the isolation of the antigens from the soluble extracts of parasites. The isolated antigens were used in order to obtain new antisera. These antisera were used for the preparation of more efficient immunoabsorbent which allow to isolate new antigens that gave three precipitation lines by immunodiffusion. The polyacrylamide gel electrophoresis of crude homogenate discriminate 12 components, and the electrophoresis of the isolated antigens gave only 3 bands.

Aislamiento de antígenos de Ancylostoma duodenale y produccion de inmunosueros. / [Isolation of Ancylostoma duodenale antigens and production of immune sera]

Rabbits were immunized using intact larvae or homogenates from Ancylostoma duodenale. Antisera were tested by immunodiffusion. The homogenates promote the formation of antibodies but the intact worms were not able to induce them. The antisera were partially purified by precipitation with amonium sulphate 40

saturation and filtration through Sephadex G-200. The purified material was attached to Sepharose 6B and used as immunoabsorbent for the isolation of the antigens from the soluble extracts of parasites. The isolated antigens were used in order to obtain new antisera. These antisera were used for the preparation of more efficient immunoabsorbent which allow to isolate new antigens that gave three precipitation lines by immunodiffusion. The polyacrylamide gel electrophoresis of crude homogenate discriminate 12 components, and the electrophoresis of the isolated antigens gave only 3 bands.

Aislamiento de antígenos de Ancylostoma duodenale y produccion de inmunosueros / [Isolation of Ancylostoma duodenale antigens and production of immune sera]

Rabbits were immunized using intact larvae or homogenates from Ancylostoma duodenale. Antisera were tested by immunodiffusion. The homogenates promote the formation of antibodies but the intact worms were not able to induce them. The antisera were partially purified by precipitation with amonium sulphate 40

saturation and filtration through Sephadex G-200. The purified material was attached to Sepharose 6B and used as immunoabsorbent for the isolation of the antigens from the soluble extracts of parasites. The isolated antigens were used in order to obtain new antisera. These antisera were used for the preparation of more efficient immunoabsorbent which allow to isolate new antigens that gave three precipitation lines by immunodiffusion. The polyacrylamide gel electrophoresis of crude homogenate discriminate 12 components, and the electrophoresis of the isolated antigens gave only 3 bands.