RESUMO
Visualising vascular endothelial cell function in individual blood microvessels allows elucidation of molecular interactions at the vascular wall, the first barrier between blood-borne therapeutic agent and its target. Functional analysis in situ requires sub-micrometer spatial resolution and tagged molecules generating contrast in living blood vessels. Light microscopy fulfills these requirements, particularly if fluorescent tags deliver the contrast. However, vascular arborisations in living organs defy morpho-functional analysis, filling tissues with closely meshed three-dimensional networks which are inaccessible to optical imaging. We protocol here successful morpho-functional analysis of microvascular processing in a living organ, the human placental cotyledon. Fluorescence-tagged tracer was positionally fixed by snap-freezing, frozen sections were cut, freeze-dried and heat-fixed. A brief histochemical procedure then labelled all vascular elements in the sections, providing fluorescence contrast in two colour channels. Mosaic monochromatic images acquired in both channels delivered high-resolution maps of centimeter-wide tissue areas. Quantitative analysis of the images' greyscale histograms defined objectifiable, reproducible thresholds, used to reduce the images to colour-coded wide-area functional maps tracking placental vascular processing of the tagged molecules. Rapid positional fixing of tracer with reduction of images to maps was combined with ultrastructural tracking to elucidate vascular processing at scales of nanometres and seconds.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Placenta/irrigação sanguínea , Placenta/ultraestrutura , Feminino , Angiofluoresceinografia , Humanos , Microscopia Eletrônica de Transmissão , Microvasos/fisiologia , Microvasos/ultraestrutura , GravidezRESUMO
Ochratoxin A (OA) is a nephrotoxic and nephrocarcinogenic mycotoxin which is predominantly produced by the two ubiquitous fungal genera, Aspergillus and Penicillium. OA is found in foodstuffs, predominantly in cereals but also in coffee beans. Inconsistent results have been published regarding the influence of roasting on the OA content in roasted beans and the transfer into the coffee brew. In the present study an HPLC method was used for the detection of OA in green and roasted coffee beans as well as in the coffee brew. For qualitative confirmation and quantification of low OA levels in roasted coffee beans and coffee brew an additional clean-up step by immunoaffinity column was applied before HPLC analysis. In green coffee beans OA was detected in 13 out of 25 commercial samples analysed (detection limit, 0.5 micrograms OA/kg). Roasting (250 degrees C, 150 sec) of naturally contaminated green beans or beans inoculated with A. ochraceus resulted only in a small reduction in the OA level. OA was also found to be eluted into the brew. Of 40 coffee brews prepared from commercially available samples OA was detected in 18 brews by HPLC and/or additional immunoaffinity column clean-up in the range of 0.4 to 7.8 micrograms OA/kg equivalent ground coffee. Our preliminary results suggest, therefore, that regular coffee consumption may contribute to exposure of humans to OA.
Assuntos
Café/química , Ocratoxinas/análise , Cromatografia Líquida de Alta Pressão , Contaminação de Alimentos/análise , Manipulação de Alimentos , Cromatografia Gasosa-Espectrometria de Massas , Temperatura Alta , Humanos , Medição de RiscoRESUMO
The purpose of the present study was the evaluation of ultrastructural characteristics of the enterochromaffin-like (ECL) cells in the fundic mucosa of three different rat strains without treatment and after treatment with the H+, K(+)-ATPase inhibitor pantoprazole. In the study, 20 one year old female Sprague Dawley (SD), Fischer 344 (F) and Wistar (W) rats each were treated orally for three months with 4 mg pantoprazole/kg/d or with the vehicle only. The control animals showed close conformity of ECL cell density and morphology in all three strains. Treatment with pantoprazole led to a significant increase in serum gastrin concentration and GPC density in all strains. However, the electron microscopically determined ECL cell density was markedly increased in the SD strain only. Ultrastructurally all treated rats showed activation of the ECL cells, and enhanced histamine release. The SD and F strains had an enhanced proportion of large ECL cell granules, with the F rats also showing an increased granule density. In contrast, the treated W rats were found to have a lower granule density and a higher proportion of small and medium sized granules compared to their controls.
Assuntos
Benzimidazóis/farmacologia , Células Enterocromafins/efeitos dos fármacos , Células Enterocromafins/ultraestrutura , Mucosa Gástrica/citologia , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Ratos Wistar , Sulfóxidos/farmacologia , 2-Piridinilmetilsulfinilbenzimidazóis , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Contagem de Células , Feminino , Mucosa Gástrica/efeitos dos fármacos , Microscopia Eletrônica , Omeprazol/análogos & derivados , Pantoprazol , Ratos , Especificidade da EspécieAssuntos
Terapia Psicanalítica , Psicoterapia , Adulto , Ansiedade , Terapia Comportamental , Condicionamento Clássico , Feminino , Humanos , MasculinoRESUMO
In the present study, female Sprague-Dawley rats were treated orally over 2, 14, and 29 days with 50 mg/kg/day of the H+, K(+)-ATPase inhibitor B8301-078, a substituted benzimidazole. The endocrine cells in the fundic mucosa were quantified by light microscopy with the aid of Grimelius silver staining and examined by electron microscopy. After 2 days of treatment, the number of argyrophilic Grimelius-positive cells (GPC) was clearly reduced compared to the controls, whereas after 14 and 29 days, hyperplasia was observed. Electron microscopy showed that there was only an apparent decrease in GPC density after 2 treatments. This reduction was due to the massive degranulation of the enterochromaffin-like (ECL) cells and the resultant decrease in stainability with silver. After 14 and 29 days the granular depletion was less pronounced. The cells were, therefore, detectable again by light microscopy.
Assuntos
Benzimidazóis/farmacologia , Células Enterocromafins/efeitos dos fármacos , Mucosa Gástrica/enzimologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Células Enterocromafins/enzimologia , Feminino , Ácido Gástrico/metabolismo , Mucosa Gástrica/citologia , Mucosa Gástrica/efeitos dos fármacos , Gastrinas/sangue , Microscopia Eletrônica , Ratos , Ratos Sprague-Dawley , Coloração pela PrataRESUMO
The mycotoxin ochratoxin A (OTA) is a rodent carcinogen produced by species of the ubiquitous fungal genera Aspergillus and Penicillium. OTA is found in a variety of food items and as a consequence is also found in human plasma (average concentrations found in this study: 0.1-1 ng OTA/ml plasma). To improve the scientific basis for cancer risk assessment the toxicokinetic profile of OTA was studied in one human volunteer following ingestion of 395 ng 3H-labeled OTA (3.8 microCi). A two-compartment open model consisting of a central compartment was found to best describe the in vivo data. This two-compartment model consisted of a fast elimination and distribution phase (T1/2 about 20 h) followed by a slow elimination phase (renal clearance about 0.11 ml/min.) and a calculated plasma half-life of 35.55 days. This half-life was approximately eight times longer than that determined previously in rats. In addition, the intraindividual fluctuation of OTA plasma levels was investigated in eight individuals over a period of 2 months. The concentrations determined ranged between 0.2 and 0.9 ng OTA/ml plasma. The plasma levels in some individuals remained nearly constant over time, while others varied considerably (e.g. increase of 0.4 ng/ml within 3 days, decrease of 0.3 ng/ml within 5 days) during the observation period. This intraindividual fluctuation in OTA plasma levels, which may represent differences in OTA exposure and/or metabolism, as well as the large difference in plasma half-life in humans compared to rats must be taken into consideration when the results of rat cancer study data are extrapolated to humans for risk assessment purposes.