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  • 1
    Electronic Resource
    Electronic Resource
    Viability and recovery of frozen-thawed human islets and in vivo quality control by xenotransplantation (1999)
    Springer
    Journal of molecular medicine 77 (1999), S. 172-174 
    Details
    ISSN: 1432-1440
    Keywords: Key words Islets of Langerhans ; Cryopreservation ; Vitrification ; Transplantation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Cryopreservation of islets of Langerhans offers advantages for the transplantation into diabetic patients. In this study two different methods of cryopreservation were compared with respect to islet viability and recovery after cryostorage. It was also investigated whether human islet survival in mice was affected by cryopreservation. Aliquots of human islets were cryopreserved conventionally or vitrified, respectively. After rapid thawing, islet viability and islet equivalent (IEQ) recovery rate were determined. Aliquots of freshly isolated or conventionally cryopreserved islets were transplanted beneath the kidney capsule of non-diabetic C57BL/6 mice. After three days renal insulin content was determined. Islet cell viability was 17.3±8.0% for vitrified and 51.8±3.0% for conventionally cryopreserved islets; the recovery rate was 84.8±12.2% and 92.8±12.4%, respectively. Insulin recovery after transplantation was 25.6±7.3% for fresh and 24.1±7.4% for cryopreserved islets. This study suggests that the conventional method of cryopreservation is superior to vitrification with respect to islet viability after thawing. We found no significant difference between fresh and cryopreserved islets with respect to insulin recovery after transplantation into mice.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001090050330
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  • 2
    Electronic Resource
    Electronic Resource
    Cytokine mRNA expression in peripheral blood cells of immunosuppressed human islet transplant recipients (1999)
    Springer
    Journal of molecular medicine 77 (1999), S. 115-117 
    Details
    ISSN: 1432-1440
    Keywords: Key words Islet allotransplantation ; Immunosuppressive induction therapy ; Cytokine expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The macrophage derived cytokines interleukin-1 beta (IL-1β), and tumor necrosis factor alpha (TNFα), and the T-cell derived cytokine interferon gamma (IFNγ) have been implicated to play an important role in early attack on islet cells during human islet transplantation (ITx). Therefore, the aim of this study was to investigate the influence of the current immunosuppressive induction therapy in clinical islet transplantation on mRNA expression of these cytokines in blood cells, compared to lipopolysaccharide (LPS) induced cytokine release in vitro and to plasma levels. The cytokine release correlated to lymphocyte counts and significantly decreased after ATG, and partially recovered 2 weeks after ITx. Unexpectedly, there was no correlation between mRNA expression for IL-1β in total blood and the number of lymphocytes and monocytes remaining after anti thymocyte globulin (ATG)-therapy. Even when the blood was nearly totally depleted from mononuclear cells, high amounts of IL-1β mRNA could be detected. However, IL-1β secretion could not be stimulated in vitro. Our results show that application of ATG during ITx might contribute to graft survival during the early posttransplant period by suppression of the synthesis of monocyte derived cytokines IL-1β and TNFα.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001090050315
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  • 3
    Electronic Resource
    Electronic Resource
    Oxygen radical production in human mononuclear blood cells is not suppressed by drugs used in clinical islet transplantation (1999)
    Springer
    Journal of molecular medicine 77 (1999), S. 121-122 
    Details
    ISSN: 1432-1440
    Keywords: Key words Monocyte activation ; Immunosuppressive drugs ; IL-1β ; TNFα ; Superoxide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Inflammatory islet damage mediated by cytokines and oxygen radicals may limit the success of clinical islet transplantation for treatment of insulin-dependent diabetes mellitus. In this study, we investigated whether drugs such as currently used in islet-transplanted patients inhibit the release of IL-1β, TNFα, and superoxide from mononuclear blood cells in vitro. Methylprednisolone (10 µg/ml) inhibited the release of IL-1β and TNFα, but had no effect on superoxide generation. Both pentoxifylline (66 µg/ml) and cyclosporin A (300 ng/ml) slightly inhibited TNFα release without affecting IL-1β or superoxide generation. Nicotinamide (0.25 mM) did not interfere with the generation TNFα or superoxide and only slightly inhibited IL-1β production. A combination of methylprednisolone, pentoxifylline, cyclosporin A, and nicotinamide (concentrations for each substance as described above) inhibited TNFα generation by 74±6% (mean value±SEM, mononuclear blood cells from seven diabetic patients) without affecting IL-1β or superoxide generation. These data show that standard immunosuppressive therapy in islet transplanted patients may partially inhibit cytokine release but does not affect the generation of potentially islet-toxic superoxide from mononuclear cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001090050317
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  • 4
    Electronic Resource
    Electronic Resource
    Critical islet mass for successful porcine islet autotrasplantation (1999)
    Springer
    Journal of molecular medicine 77 (1999), S. 126-129 
    Details
    ISSN: 1432-1440
    Keywords: Key words Pancreatectomy ; Porcine islets ; Autotransplantation ; Glucose tolerance test
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A major reason for the failure of clinical islet transplantations may be a limited islet mass. The aim of this study was to determine the critical islet mass necessary for normalization of glucose metabolism in a porcine model. Diabetes was induced by total pancreatectomy. The splenic lobe of the pancreas was intraductally distended with UW-solution containing 2.67–3.33 mg/ml collagenase, and the distended pancreas was digested in a continuous digestion filtration device. The islets were purified on a isoosmotic Ficoll-sodium-diatrizoate gradient. The survival period of the diabetic recipients in group 2 and 3 receiving, respectively, a low (2.14±0.39 µL/kg body weight) and a high (4.99±0.83 µL/kg body weight) islet mass was significantly prolonged compared to that of diabetic recipients in group 1 receiving no islet transplantation. However, the survival period of the recipients in group 2 was not significantly different to that in group 3. Three recipients of an islet mass of 〉5 µl/kg body weight became normoglycemic (fasting blood glucose 〈100 mg/dl) for more than two months. Furthermore, the glucose and insulin release reactions to the glucose challenge were comparable to that before pancreatectomy. Contrarily, another five diabetic recipients of an islet mass of 〈4 µL/kg body weight became a fasting blood glucose level of 〈200 mg/dl. The glucose and insulin release reactions to the glucose challenge were improved only, but not normalized compared to that before pancreatectomy. The data presented in this study demonstrate that metabolic normalization in pancreatectomized diabetic minipigs can be established by autotransplantation of an islet mass of 〉5 µl/kg body weight.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001090050319
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  • 5
    Electronic Resource
    Electronic Resource
    Evidence for a significant correlation of donor pancreas morphology and the yield of isolated purified human islets (1999)
    Springer
    Journal of molecular medicine 77 (1999), S. 87-89 
    Details
    ISSN: 1432-1440
    Keywords: Key Words Pancreas ; Islets of Langerhans ; Islet isolation ; Pancreas morphology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In clinical islet transplantation to patients with type 1 diabetes mellitus, the number of isolated and purified islet has been identified as a key determinant for functional success of the islet graft [1]. With improved isolation methods based on the original procedure published by Ricordi et al. [2] yield and function of isolated islets were considerably enhanced. However, there is still a large variance in the number, purity, viability and secretory capacity of islets isolated from brain-dead human donor pancreata, significantly hampering utilization of human islet preparations derived from a single donor for one diabetic recipient. The reasons for the limited success in islet isolation and purification have not been clarified in detail yet. Recent studies have indicated, that donor preconditions, and a number of technical factors during organ procurement and the islet isolation process itself are critical to successful islet isolation [3, 4]. This study aimed at identifying distinct morphological and histopathological characteristics of the donor pancreas as determinants for the outcome of human islet isolation and purification.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001090050308
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  • 6
    Electronic Resource
    Electronic Resource
    Glucose sensitivity of porcine and human islets in vitro (1999)
    Springer
    Journal of molecular medicine 77 (1999), S. 90-92 
    Details
    ISSN: 1432-1440
    Keywords: Key words Glucose sensitivity ; Human islet ; Porcine islet ; Islet culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Preliminary experiments about the suitability of different commonly used culture media in our laboratory indicated, that prolonged exposure to high glucose concentrations during low temperature culture (LTC) impairs the viability of long term cultured human islets. As a consequence of the heterogenity of tested media the present study was aimed to evaluate the influence of different glucose concentrations on survival, viability and in-vitro function of cultured human islets in order to optimize islet survival until transplantation and to compare species dependent differences in glucose sensitivity. Quantified aliquots of freshly isolated (digestion-filtration, ficoll gradient purification) islets from consecutively processed human (n=6) and porcine (n=11) pancreata were subjected to different glucose concentrations (human islets: 500, 750, 1000 and 2000 mg/l; porcine islets: 1000 and 2000 mg/l) in CMRL (22°C) for 8–10 days. After LTC survival, viability and glucose-stimulated insulin release of incubated tissue was assessed. A reduction of glucose concentration promotes survival and viability of human islets but impairs in vitro function at the same time, presumably due to a reduced glucose oxidation as expressed by the significantly reduced stimulation index. In contrast to these findings in the human, elevated glucose concentration in porcine islet culture increases survival but reduces the glucose-stimulated insulin release and the viability of cultured islets. The contradiction of the results in regard to islet survival related to islet viability are still unclear in the pig and needs further evaluation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001090050309
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  • 7
    Electronic Resource
    Electronic Resource
    Large variability of the intracellular ATP content of human islets isolated from different donors (1999)
    Springer
    Journal of molecular medicine 77 (1999), S. 93-95 
    Details
    ISSN: 1432-1440
    Keywords: Key words Islets of Langerhans ; Tissue culture ; ATP ; Human islets
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Observations in experimental heart, liver, kidney and pancreas transplantation indicated that graft function and survival correlates significantly with ATP content of transplanted tissue. The ATP content of cells can be reduced by several factors i.e. the nutritional donor status, storage technique, warm ischemia and cold ischemia time. This study investigates the intracellular ATP content of isolated human islets for the first time. Quantified samples of freshly isolated (digestion-filtration, continuous ficoll gradient purification) and cultured (22°C, CMRL+10% FCS) islet equivalents (IEQ) of consecutively processed human pancreata from multiorgan donors (UW vascular flush) were shock frozen in liquid nitrogen and stored at –196°C until rapid thawing, sonification and subsequent luminometric determination of ATP (Luciferin-Luciferase-reaction) and assessment of islet protein (IP). The ATP content was analysed for freshly isolated and subsequently 5±1 days cultured islets (n=10). The ATP content of freshly isolated human islets was 130.4±53.4 pg/µg IP (mean ± SEM) corresponding to 20.7±6.3 pg/IEQ. After culture ATP content increased to 265.5±113.3 pg/µg IP (204.2±41.5%) corresponding to 43.7±15.3 pg/IEQ (216.1±34.9%; p〈0.05). The coefficient of variation was 129.5%, 96.5% (fresh) and 135.0%, 111.0% (cultured) for ATP/µg IP and ATP/IEQ, respectively. The present data show that: (1) the ATP content of freshly isolated human islets varies enormously; (2) intraislet ATP levels increase significantly during 22°C culture suggesting that the capacity to produce ATP is maintained despite hypothermic environment. More data are necessary to clarify the relevance of intraislet ATP content for graft function and survival after islet transplantation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001090050310
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  • 8
    Electronic Resource
    Electronic Resource
    Isokinetic gradient centrifugation prolongs survival of pig islets xenografted into mice (1999)
    Springer
    Journal of molecular medicine 77 (1999), S. 175-177 
    Details
    ISSN: 1432-1440
    Keywords: Key words Porcine islets of Langerhans ; Purification ; Islet xenograft rejection ; Islet transplantation ; Islet culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Highly purified porcine islets were prepared by isokinetic gradients performed subsequently to isopycnic gradients. This additional purification step separates ductal, vascular, and lymphoid tissue effectively from endocrine tissue. Although ductal, vascular, and lymphoid tissue comprises only a minor contamination of the islet suspensions, a significant prolongation of the survival of porcine islets xenografted into streptozotocin diabetic C57BL/6 mice can be achieved by the elimination of the non-endocrine tissue. Rejection after islet transplantation is delayed from 2.2±0.4 days (n=27) to 13.1±2.1 days (n=36), respectiveley, when conventionally purified and highly purified islets are compared. Irrespective of the purification state, pretreatment of islets by low temperature culture had no effect on xenograft survival.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001090050331
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  • 9
    Electronic Resource
    Electronic Resource
    Endotoxin-mediated activation of cytokine production in human PBMCs by collagenase and Ficoll (1999)
    Springer
    Journal of molecular medicine 77 (1999), S. 118-120 
    Details
    ISSN: 1432-1440
    Keywords: Key words Collagenase ; Ficoll ; Endotoxin ; Human mononuclear cells ; Interleukin-1β release
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Endotoxin-induced early inflammatory reactions may inhibit the function and survival of isolated cells or cell aggregates after transplantation. By the chromogenic Limulus amebocyte lysate assay we found rather high but variable endotoxin concentrations in the chemicals used for islet isolation, i.e. collagenase and Ficoll. Liberase, a special collagenase preparation from Boehringer, was nearly endotoxin-free. Correlating to the endotoxin content, collagenase and Ficoll had the capacity to induce interleukin-1β release from human peripheral blood mononuclear cells. Because collagenase and density gradient media are needed in most cell isolation procedures from solid organs, each lot of these chemicals should be tested for endotoxin contamination. In pancreatic islet transplantation, the use of endotoxin-free chemicals may diminish early local inflammatory reactions at the graft site and thereby reduce the number of islets needed for successful islet transplantation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001090050316
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  • 10
    Electronic Resource
    Electronic Resource
    Improved survival of intraportal pancreatic islet cell allografts in patients with type-1 diabetes mellitus by refined peritransplant management (1999)
    Springer
    Journal of molecular medicine 77 (1999), S. 140-143 
    Details
    ISSN: 1432-1440
    Keywords: Key words Islet isolation ; Clinical islet transplantation ; Type-1 diabetes mellitus ; Immunosuppression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001090050322
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