Facilitated Engraftment of Isolated Islets Coated With Expanded Vascular Endothelial Cells for Islet Transplantation.

BACKGROUND: Diabetes is complex disease, which involves primary metabolic changes followed by immunological and vascular pathophysiological adjustments. However, it is mostly characterized by an unbalanced decreased number of the ß-cells unable to maintain the metabolic requirements and failure to further regenerate newly functional pancreatic islets. The objective of this study was to analyze the properties of the endothelial cells to facilitate the islet cells engraftment after islet transplantation. METHODS: We devised a co-cultured engineer system to coat isolated islets with vascular endothelial cells. To assess the cell integration of cell-engineered islets, we stained them for endothelial marker CD31 and nuclei counterstained with DAPI dye. We comparatively performed islet transplantations into streptozotocin-induced diabetic mice and recovered the islet grafts for morphometric analyses on days 3, 7, 10, and 30. Blood glucose levels were measured continuously after islet transplantation to monitor the functional engraftment and capacity to achieve metabolic control. RESULTS: Cell-engineered islets showed a well-defined rounded shape after co-culture when compared with native isolated islets. Furthermore, the number of CD31-positive cells layered on the islet surface showed a direct proportion with engraftment capacities and less TUNEL-positive cells on days 3 and 7 after transplantation. CONCLUSIONS: We observed that vascular endothelial cells could be functional integrated into isolated islets. We also found that islets that are coated with vascular endothelial cells increased their capacity to engraft. These findings indicate that islets coated with endothelial cells have a greater capacity of engraftment and thus establish a definitely vascular network to support the metabolic requirements.

Colour and pulsed Doppler ultrasonographic study of the canine testis.

This study was performed to characterize the normal blood flow of the canine testis and to measure the peak systolic velocity (PSV), end-diastolic velocity (EDV), resistive index (RI) and pulsatility index (PI) of testicular arteries weekly during a period of 6 months in five healthy Beagle dogs and to evaluate whether there were any change along this time. The ultrasonographic exams were made with an 11 MHz linear transducer. The vessels of the testes were subdivided into three categories: supratesticular arteries, marginal artery and intratesticular vessels. At the supratesticular arteries, two measurements were recorded at the cranial and the looping parts. No significant differences in any of the parameters studied were observed for the 6 months that the study was performed. The cranial part of the supratesticular artery showed a flow pattern of high-resistive vessel, whereas in the looping part of the supratesticular artery, marginal and intratesticular arteries, the flow showed a low-resistance pattern. PSV, RI and PI values were higher at the cranial part of the supratesticular artery, followed by the looping part of the supratesticular artery, marginal and intratesticular vessels. EDV measurements were higher in the looping part of the supratesticular artery.

Endothelial cells promote pancreatic stem cell activation during islet regeneration in mice.

OBJECTIVES: Diabetes is the clinical consequence of the loss of the majority of the ß-cell population and failure to regenerate new pancreatic ß cells. The current therapies based on ß-cell replacement have failed to achieve ß-cell renewal and thus, long-term insulin freedom. We have hypothesized that early rejection of endothelial elements within the islet grafts may seriously hamper islet regeneration in both native and islet grafts. METHODS: In the present study, we analyzed the role of endothelial cells to activate pancreatic stem cells during islet regeneration. Mice were pretreated with or without endothelial pharmacological ablation of endothelial cells, followed by an acute ß-cell injury using a single intraperitoneal injection of streptozotocin. We performed comparative morphometric analyses of recovered pancreata on days 3, 7, 10, and 30 after streptozotocin injury, staining with bromodeoxyuridine (BrdU) for representative cell types, ß cells, endothelial elements, and stem cells. Blood glucose levels were measured continuously after the injury to monitor the capacity for metabolic control. RESULTS: Morphometric analyses revealed an increasing number of cells over time to be stained with a stem cell and BrdU markers among animals only injured with streptozotocin but not with endothelial ablation. Notably, on day 10, stem cell markers were dramatically decrease nearly to basal levels, with appearance of numerous insulin-positive cells. Intact vessels with cobblestone-shaped endothelial elements were observed in direct proportion to the better outcomes, both by morphometric and by metabolic parameters. In contrast, fewer insulin-positive cells were observed in pancreata that had been ablated of endothelial cells showing extensive collapse of endocrine functions. CONCLUSIONS: We observed that endothelial elements promoted stem cell proliferation and islet regeneration after a ß-cell insult. We believe that preservation of endothelial cells positively affects the process of pancreatic regeneration.

Determination of phthalates in wine by headspace solid-phase microextraction followed by gas chromatography-mass spectrometry. Use of deuterated phthalates as internal standards.

This study proposes the use of deuterated phthalates as internal standards for the accurate determination of phthalates in wine by headspace solid-phase microextraction followed by gas chromatography-mass spectrometry. Unlike other internal standards proposed previously such as benzyl benzoate, deuterated phthalates enabled matrix-error free determinations to be performed without standard addition because statistically equal slopes were obtained for synthetic, white, rose and red wines. The relative standard deviation values under intermediate precision conditions ranged from 0.24 to 4.6%, and detection limits below 35 ng L(-1) were obtained. Recovery values were around 100% in most of cases and the method provided similar results to standard addition. Finally, the method was used to screen phthalate levels in 10 wine samples.

Determination of phthalates in wine by headspace solid-phase microextraction followed by gas chromatography-mass spectrometry: fibre comparison and selection.

This paper describes the development of a headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) method for determining phthalates in wine. The HS-SPME conditions were thoroughly studied: first, the performance of six fibres at three temperature values and two sample volumes was surveyed by means of a 6 x 3 x 2 multi-factor categorical experimental design. From this study, three fibres - carbowax-divinylbenzene (CW-DVB), polyacrylate (PA) and polydimethylsiloxane-divinylbenzene (PDMS-DVB) - were selected. Then, temperature, sample volume and sodium chloride concentration were optimised using a central composite design and the overall desirability function for each fibre. The optimal values were 70 degrees C, a NaCl concentration of 2.6, 3.6 and 5.5M for PA, CW-DVB and PDMS-DVB fibres, respectively, and sample volumes of 4.0, 3.5 and 3.0 mL. Next, the performance characteristics of the three fibres were obtained and compared. PDMS-DVB fibre showed the best repeatability values followed by CW-DVB. PA fibre was not suitable for diethylhexylphthalate extraction and showed poor repeatability for the heavier phthalates, and was therefore discarded. Finally, the performance of CW-DVB and PDMS-DVB fibres was checked for red, white and rosé wines.