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Factors affecting in vitro and in vivo function of isolated porcine islets
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Automated large-scale isolation, in vitro function and xenotransplantation of porcine islets of Langerhans
No full textAbstract: To evaluate the potential of utilizing porcine islet tissue as an alternative to human islet tissue for transplantation, we developed a method for the isolation of large amounts of highly purified porcine islets, and assessed the in vitro and in vivo function of the isolated islets after 1, 4, and 7 days of culture. The pancreatic duct of the splenic lobe was cannulated and distended by, injection of Hanks' balanced salt solution containing 1.5 mg/ml collagenase. The pancreas was then processed by a modification of the automated digestion-filtration method developed in this laboratory, and with purification accomplished by Euro-Ficoll gradients (dialyzed Ficoll in Eurocollins solution), consisting of two layers of 1.108 and 1.091 g/cm3 density, topped with a layer of HBSS. The postpurification yield was 5203 +/- 645 (mean +/- SEM) islets per gram of pancreas with a number of islet equivalents (IE) per gram pancreas (islet equivalence: 150-mu-m-sized islets) of 3551 +/- 305, and a volume of 6.27 +/- 1.7 mm3 islet tissue per gram of pancreas. The islet purity exceeded 90%. Overnight-cultured, perifused porcine islets released 53.1 +/- 8.2 pM insulin/200 IE at 3.3 mM glucose, and 114.9 +/- 25.4 p.M insulin/200 IE at 16.7 mM glucose (P < 0.001 vs. basal output). When theophylline was added, insulin secretion increased to 264.2 +/- 63.2 pM/200 IE (P < 0.001 vs. basal secretion and P < 0.005 vs. secretion at 16.7 mM glucose). After 4 days of culture, the islets still responded to secretagogues. The functional integrity of the isolated islets was confirmed by reversal of diabetes in aL3T4 antibody-treated C57B/B6 diabetic mice: normoglycemia was promptly restored by transplanting 1000 over-night- or 7-day-cultured (24-degrees-C) islets under the kidney capsule. These results suggest that continued improvements of porcine islet isolation and culture could permit the use of porcine islets in immunoalteration and immunoisolation studies that may lead to eventual human transplantation
Cryogenic storage of isolated, purified porcine pancreatic islets
No full textAbstract: Highly purified islets of Langerhans were prepared in the present study from adult pigs by collagenase digestion and density gradient purification. After overnight culture, the tissue was equilibrated with DMSO at 25 degrees C, supercooled to -7.5 degrees C, nucleated, slowly cooled at 0.25 degrees C/min to -40 degrees C, and stored at -130 degrees C. Then, after variable periods of storage, the islets were rapidly thawed at 37 degrees C. Postthaw actual islet and islet equivalent (150-mu m sized islets) recovery were 75+/-7% and 66+/-4%, respectively. The frozen-thawed porcine islets maintained good morphology on histological staining by hematoxylin-eosin and aldehyde-fuchsin. Upon perifusion, basal insulin secretion was 43+/-10 and 67+/-18 pmol/L from noncryopreserved, control islets, and cryopreserved islets, respectively (P=0.2). Peak insulin release at 16.7 mmol/L glucose was 85+/-28 pmol/L from noncryopreserved islets and 157+/-48 pmol/L from the frozen-thawed islets (P=0.1). When 10 mmol/L theophylline was added to 16.7 mmol/L glucose, the secretion of the hormone peaked to 221+/-83 (control islets) and 479+/-140 pmol/L (cryopreserved islets, P=0.1). Total insulin secretion differed significantly for the noncryopreserved and the cryopreserved islets at both 16.7 mmol/L (1412+/-306 vs. 3756+/-764 pmol/L, respectively, P=0.007) and 16.7 mmol/L glucose plus 10 mmol/L theophylline (2161+/-371 vs. 7505+/-2075 pmol/L, respectively, P=0.011). Normoglycemia was restored within 7 days from implantation in temporarily immunosuppressed (aL3T4 antibody) mice with streptozotocin-induced diabetes by transplanting 1500-2000 cryopreserved porcine islets under the kidney capsule. Mean survival time of frozen-thawed islet xenografts (39+/-3 days) was similar to that of noncryopreserved islet xenografts (43+/-6 days). This study demonstrates that cryogenic storage is feasible of isolated porcine islets, with the frozen-thawed pancreatic endocrine tissue maintaining morphological integrity and both in vitro and in vivo viability. Further studies are needed to define the effect of cryopreservation on the immunogenic properties of porcine islets
Prevention of contamination of isolated porcine islets of Langerhans
No full text[abstract non disponibile
Prolonged survival of discordant porcine islet xenografts
No full textAbstract: Purified porcine islets were prepared by collagenase digestion and density gradient purification, and transplanted under the kidney capsule of C57B/B6 mice with streptozotocin-induced diabetes which were receiving varying temporary immunosuppressive therapies. Islets that had been cultured for 1 day at 37 degrees C were rejected after: 9 +/- 0.1 (mean +/- SE) days in control mice; 14 +/- 3 days in mice receiving mouse antilymphocyte serum (MLS) plus porcine antilymphocyte serum (PLS) on day of transplant (day 0); 43 +/- 8 days in mice treated for 1 week with anti-CD4 antibody (aCD4); 36 +/- 4 days in mice given aCD4 for 1 week plus PLS on days 0 and 7; 47 +/- 3 days in mice treated with aCD4 for 1 week plus MLS and PLS on day 21. Porcine islet survival in these latter three groups was significantly (P<0.01) and similarly longer than in the control and MLS plus PLS groups. Then, we transplanted islets that had been either cultured at 24 degrees C for 7 days or cryopreserved into 7-day aCD4-treated mice, to evaluate whether low temperature culture or the freezing-thawing procedure could affect survival. Neither 7-day, low temperature culture (mean survival time: 37 +/- 2 days) nor cryopreservation (mean survival time: 39 +/- 2 days) prolonged islet function further. Thus, the present study demonstrates that prolonged survival can be achieved with discordant porcine islet xenografts, and shows the greater efficacy of aCD4 treatment, which was not improved by the additional immunosuppressive therapies we tested, nor by culture or cryopreservation of the islets
Insulin inhibits its own secretion from isolated, perifused human pancreatic islets
No full textAbstract: It is still a controversial question whether insulin suppresses its own secretion. We prepared pure human islets from three pancreases by collagenase digestion and density gradient purification, Aliquots of 200 islet equivalents (IE, 150-mu m sized-islets) were sequentially perifused at 37 degrees C with 3.3 mmol/l glucose (3.3G, 40 min), 16.7 mmol/l glucose (16.7G, 30 min) and again 3.3G (30 min) after 24 h, 37 degrees C culture in CMRL 1066 medium with or without the addition of either 200 or 400 mu U/ml human insulin in the incubation medium (6 replicates each). Insulin secretion was assessed by C-peptide (Cp) measurement in the perifusate. Without added insulin (C) and with 200 (Ins200) or 400 (Ins400) mu U/ml added insulin, basal Cp release was 0.12 +/- 0.03, 0.14 +/- 0.02 and 0.14 +/- 0.04 ng/ml, respectively. At 16.7G, the first-phase secretion peak (expressed as Cp value) was significantly lower with Ins200 (0.47 +/- 0.13 ng/ml, P < 0.02) and Ins400 (0.68 +/- 0.15 ng/ml, P < 0.05) than C (0.83 +/- 0.15 ng/ml). The second-phase secretion peak was also significantly (P < 0.05) reduced with added insulin (Ins200: 0.47 +/- 0.08 ng/ml; Ins400: 0.45 +/- 0.07 ng/ml) than in its absence (C: 0.65 +/- 0.09 ng/ml). Accordingly, total Cp secretion was lower with Ins200 (10.6 +/- 2.3 ng/ml, P = 0.03) and Ins400 (11.8 +/- 2.3 ng/ml) than with C (16.0 +/- 2.2 ng/ml). Thus, the addition for 24 h of either 200 or 400 mu U/ml insulin in the culture medium caused a significant decrease of insulin (as assessed by Cp measurement) secretion from perifused human islets, suggesting that feedback suppression of insulin release is at least in part due to a direct action of insulin on the islets
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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