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  • 1
    ISSN: 1432-0428
    Keywords: Keywords Lentiviral vector ; retrovirus ; human islet beta-cell ; gene transfer ; transplantation.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Pancreatic islet cells are terminally differentiated endocrine cells and are refractory to stable infection by retroviral vectors, which require the breakdown of the nuclear membrane during cell division in order to insert the transgene into the host cell genome. Thus, attempts to render beta-cell allografts less immunogenic have had to rely on stable transfection of surrogate cells. Similarly, this problem has precluded the development of conditionally immortalized human beta cells for clinical allotransplantation. In this report, we demonstrate that adult human islet beta cells can be transduced by a new three-plasmid integrating lentiviral vector with an efficiency of 62 ± 1.8 % at a multiplicity of infection (MOI) of 2.5 in vitro. This work makes genetic engineering of adult human pancreatic beta cells possible for the first time, allowing strategies to render beta-cell allografts non-immunogenic to be optimized and to creating conditionally immortalized human beta cells for clinical transplantation. [Diabetalogia (1998) 41: 736–739]
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1440
    Keywords: Terminal deoxynucleotidyl transferase ; Leukemia ; Cell surface marker ; Terminale Deoxynucleotidyl Transferase ; Leukämie ; Zelloberflächenmarker
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Zusammenfassung Analysen von Zelloberflächen-Merkmalen und TdT-Bestimmungen wurden bei 31 Patienten durchgeführt. Die Diagnosen wurden in folgenden Kategorien gegliedert: Akute Leukämie (Gruppe I), Non-Hodgkin Lymphome (Gruppe II) und verschiedene Diagnosen (Gruppe III). In leukämischen Zellen von Patienten der Gruppe I betrug die TdT Konzentration 0–7.9 E/mg gefriergetrockneten Materials. Das entspricht 0–95 E/108 Zellen. Präparate mononukleärer Zellen des peripheren Blutes Gesunder wiesen einen TdT-Gehalt bis 0.88 E/mg (10.6 E/108 Zellen) auf. Eine hohe TdT-Aktivität wurde bei einem jugendlichen Patienten mit AML (M1 der FAB-Klassifikation) gefunden. Bei einem weiteren Patienten dieser Gruppe mit ALL (L1) führte die cytostatische Therapie zu einem Rückgang der TdT Aktivität auf normale Werte und gleichzeitig zu einem deutlichen Anstieg der anfangs praktisch aufgehobenen T-Zellfunktion im Blut gemessen an der E-Rosettenbildung. Derartige kombinierte Untersuchungen liefern zusätzliche und möglicherweise empfindlichere Parameter für die Definition einer Remission bzw. eines Rezidivs der AL als die ausschließliche cytologische Methode. Innerhalb der Patienten der Gruppe II wurden zwei Patienten mit TdT-Aktivitäten von 1.2 bzw. 1.28 E/mg beobachtet, deren Zuordnung nach cytologischen Kriterien problematisch war und die als Prolymphocyten-Leukämie bzw. unklassifizierbar beurteilt wurden. Bestimmungen der TdT Aktivität ermöglichen vielleicht eine Identifizierung derartiger Zellen. Patienten mit ALL weisen meistens eine wesentlich höhere TdT Aktivität auf. Ein weiteres Ergebnis dieser Studie war der Befund einer mäßigen TdT-Aktivität von 1.2 E/mg in Zellen, die aus dem Pleuraexsudat eines der beiden untersuchten Patienten mit M. Hodgkin gewonnen wurden. Zellen aus dem Pleuraexsudat eines Patienten mit malignem Melanom und eines weiteren Patienten mit Terato-Carcinom waren dagegen negativ. Blasten aus dem peripheren Blut und dem Knochenmark eines Patienten mit einer Blastenkrise bei CLM zeigten TdT-Aktivität in Höhe von 1.52 und 2.72 E/mg. Zwei weitere Patienten mit einer Blastenkrise bei CML waren TdT negativ. Keinerlei Aktivität der TdT wurde in leukämischen Plasmazellen gefunden. Die Untersuchungen zeigen, daß gefriergetrocknetes Zellmaterial für Bestimmungen der TdT-Aktivität verwendet werden kann. Dadurch wird die Durchführung von Multiparameter-Studien, die cytologische, Oberflächenmarker-technische und biochemische Analysen beinhaltet, wesentlich erleichtert.
    Notes: Summary Surface marker analyses and TdT assays were performed on cells from 31 patients. A variety of diagnoses were made and categorized as follows: acute leukemia (group I), non-Hodgkin lymphoma (group II) and diverse diagnoses (group III). Levels of TdT in the range from 0 to 7.9 U/mg lyophilized blasts from the peripheral blood were found in AL. This corresponds to 0–95 U/108 cells. Preparations of mononuclear cells from the peripheral blood of healthy donors showed TdT values up to 0.88 U/mg or 10.6 U/108 cells. High TdT activity was observed in a patient with AML, type M1 according to the FAB classification. In a patient with ALL (L1) cytostatic treatment effected the clearance of TdT activity from the peripheral blood cells and at the same time induced a significant increase of E rosette forming cells. Combined studies of the TdT activity and cell surface markers may enable us to define remissions and relapses of AL more precisely than it is possible by conventional cytological methods. Within the group II two patients with moderate TdT activities of 1.2 and 1.28 U/mg, respectively, were observed whose cells were of prolymphocytic or unclassifiable appearance, respectively. The TdT assay may be helpful to identify such cells of unknown origin and in addition may provide the means of discrimination between such cases and ALL patients who mostly show high TdT activities. Another result of our studies was the finding of moderate TdT activity of 1.2 U/mg with cells from the pleural effusion of a patient with Hodgkin's disease. Cells from malignant effusions from a patient with melanoma and a patient with teratoid carcinoma showed no TdT activity. Cells form the peripheral blood and from the bone marrow of a patient with blast crisis of CML showed TdT activity of 1.52 and 2.72 U/mg, respectively. Two other patients with blast crisis were negative. Not TdT activity was found in leukemic plasma cells. Our results show that lyophilized cells can be used for determinations of TdT activity. This greatly facilitates multi-parameter studies including cytological, cell surface marker and biochemical analyses.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular medicine 58 (1980), S. 585-587 
    ISSN: 1432-1440
    Keywords: SDS-Polyacrylamidgel-Elektrophorese ; Uroproteine ; SDS-Polyacrylamide-gel-Electrophoresis ; Uroproteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Samples of the urine of 10 myeloma patients with proteinuria were examined by SDS-PAGE. Light chain proteins of Bence Jones (B.J.) type were excreted by 7 patients as monomer (m.w. 20–26.5 × 103 Dalton, by 2 patients as a mixture of monomer and dimer and by one patient as dimer. By two-dimensional electrophoresis in SDS-PAG and subsequent electrophoresis in agarose containing kappa and lambda chain specific antibodies the immunological identity of monomeric and dimeric B.J. protein of one patient has been shown. The two-dimensional analysis has been proven a valuable procedure in cases with the excretion of complete monoclonal protein and B.J. protein at the same time.
    Notes: Zusammenfassung Harnproben von 10 Patienten mit multiplem Myelom und Proteinurie wurden in der SDS-PAGE untersucht. Leichtkettenproteine vom Bence Jones (B.J.) Typ wurden in 7 Fällen als Monomere (M.G. 20–26×103 Dalton), in 2 Fällen als Monomer-Dimer-Gemisch und in einem Fall als Dimer ausgeschieden. Durch SDS-PAGE in der 1. Dimension und nachfolgende Elektrophorese in Agarose, die kappa- und lambda-Ketten-spezifische Antikörper enthielt, wurde in einem Fall die immunologische Identität monomerer und dimerer B.J. Proteine nachgewiesen. Das Verfahren der SDS-PAGE hat sich als aufschlußreiche Methode in den Fällen von Myelom erwiesen, in denen neben einem B.J. Protein ein komplettes monoklonales Protein im Urin ausgeschieden wird.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular medicine 61 (1983), S. 1101-1103 
    ISSN: 1432-1440
    Keywords: Mycloma ; Tumor stem cell assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The human tumor stem cell assay (HTSCA) introduced by Hamburger and Salmon [2] was modified in several details. It was found that a short period of treatment with deoxyribonuclease (DNase) of the material aspirated from the patients bone marrow greatly enhances the production of single cell suspensions and thereby may improve the assay. Instead of the dextran sedimentation method, we used the density gradient centrifugation method according to Bøyum [1] for the isolation of mononuclear cells from the bone marrow. Another methodical modification introduced by us is the use of the same medium for culturing the cells before plating them and in the agar after plating. Under the conditions reported here the formation of colonies was observed in 8 of 11 samples from individual myeloma patients. The average plating efficiency was 0.024% with a range of 0.009% to 0.039% showing that possibly an improvement was achieved when compared to the results obtained with the original method showing an average plating efficiency of 0.014% [2].
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  • 5
    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
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  • 6
    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
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  • 7
    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
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  • 8
    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.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular medicine 77 (1999), S. 90-92 
    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.
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  • 10
    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
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