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D Brandhorst

Publications and source records attributed to D Brandhorst.

At least 37 records · Page 2Linked to original sources
PubMed1999

Glucose sensitivity of porcine and human islets in vitro.

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 heterogeneity 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 degrees 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.

Animals↗
PubMed1999

Large variability of the intracellular ATP content of human islets isolated from different donors.

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 degrees 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 degrees 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/microg IP (mean+/-SEM) corresponding to 20.7+/-6.3 pg/IEQ. After culture ATP content increased to 265.5+/-113.3 pg/microg 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/microg 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 degrees 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.

Adenosine Triphosphate↗
PubMed1999

Cytokine mRNA expression in peripheral blood cells of immunosuppressed human islet transplant recipients.

The macrophage derived cytokines interleukin-beta (IL-1beta), and tumor necrosis factor alpha (TNFalpha), and the T-cell derived cytokine interferon gamma (IFNgamma) 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-1beta 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-1beta mRNA could be detected. However, IL-1beta 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-1beta and TNFalpha.

Antilymphocyte Serum↗
PubMed1999

Critical islet mass for successful porcine islet autotransplantation.

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 microL/kg body weight) and a high (4.99+/-0.83 microL/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 microl/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 microL/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 microl/kg body weight.

Animals↗
PubMed1999

Viability and recovery of frozen-thawed human islets and in vivo quality control by xenotransplantation.

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.

Animals↗
PubMed1999

Isokinetic gradient centrifugation prolongs survival of pig islets xenografted into mice.

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), respectively, 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.

Animals↗
PubMed1999

Analysis of the cellular reaction towards microencapsulated xenogeneic islets after intraperitoneal transplantation.

Xenotransplantation of encapsulated islets of Langerhans is a possibility to overcome problems of human organ donor shortage in islet transplantation. Preexisting natural xenoantibodies are known to play a major role in the rejection of vascularized xenografts. Only little is known about the mechanism of rejection of non-vascularized cellular xenotransplants. In this study we introduce a method for the characterization of xenograft rejection of encapsulated islets by FACS analysis of peritoneal cells. Pig islets were transplanted intraperitoneally into non-diabetic Lewis rats either encapsulated or non-encapsulated. Animals receiving empty capsules and sham-operated animals served as controls. After 7 days a peritoneal lavage was performed. The total cell number and the viability of the cells were determined. Cells were analysed after staining with a panel of antibodies for the detection of T-lymphocytes, B-lymphocytes, macrophages, MHC class II molecules. Total cell number was highest after microencapsulated transplantation (149.4+/-30.1x10(6)) compared with empty capsules (41.4+/-19.7x10(6)) and non-encapsulated porcine islets (18.1+/-3.3x10(6)). The percentage of CD 3 positive T-lymphocytes rose to 44.5+/-11.5% in case of microencapsulated xenografts compared with 19.2+/-8.2% for non-encapsulated xenografts and 4.9+/-2.4% for empty controls. B-lymphocytes were detected in only small amounts. MHC class II expression on macrophages as activation marker was significantly increased after encapsulated transplantation (60.2+/-8.9% vs 15.2+/-7.0% for free islets and 4.9+/-1.2% for empty controls). The discrepancy between the macrophage activation due to encapsulated xenogeneic islets in comparison to empty capsules made from the same material clearly indicates that the reaction is not only material related but that a recognition of the encapsulated islet takes place despite the effective inhibition of a direct cell-to-cell contact. This recognition occurs on a T-cell level as well as on the macrophage level. 7 days after transplantation the reaction towards encapsulated xenografts is even more intense than to non-encapsulated xenografts. This might be due either to the time course of the rejection process or to a prolongation of the activation because antigen elimination is hindered by the capsule.

Alginates↗
PubMed1999

Relation between S-phase fraction of myeloma cells and anemia in patients with multiple myeloma.

In an attempt to define the relation among anemia, tumor mass, and proliferative activity of tumor cells in vivo, we measured the proportion and cell cycle distribution of erythropoietic cells and myeloma cells in the bone marrow of patients with multiple myeloma using four-parameter flow cytometry. Forty-three bone marrow samples from 33 patients with stage II or III disease and normal renal function at diagnosis (n = 9), in partial remission (n = 9), and in progression or relapse after chemotherapy (n = 25) were evaluated. Early and late erythropoietic cells were discriminated based on published light scatter properties in combination with CD71 expression. Myeloma cells were detected by exploiting their strong CD38 positivity and light scatter characteristics. Cell cycle distribution of the three cell populations was determined by propidium iodine staining. In the whole group of patients, hemoglobin (Hb) concentration was inversely correlated with beta2-microglob-ulin (p = 0.03), percentage of marrow CD38++ cells (p = 0.008), and percentage of CD38(++) cells in S phase (S-CD38++; p < 0.001). Partial correlation analysis revealed S-CD38++ to be the only independent predictor of Hb concentration (p < 0.001). No correlation was found between Hb concentration and the S-phase fraction of erythropoietic cells. In the subgroup of patients with moderate to severe anemia, defined as Hb concentration <11 g/dL, Hb level correlated negatively only with S-CD38++ (p < 0.001) but not with beta2-microglobulin and percentage of marrow CD38++ cells. In addition, Hb and the S-phase proportion of early erythropoietic cells correlated positively (p = 0.029). The strong inverse correlation between Hb concentration and percentage of myeloma cells in S phase suggests that in multiple myeloma, tumor proliferative activity may have a more important impact on the development of anemia than tumor mass. The S-phase fraction of tumor cells appears to be the most important pathogenic factor, especially in anemic patients. In these patients, the positive relation between Hb concentration and the S-phase fraction of erythropoietic progenitors indicates that development of anemia is associated with inhibition of erythropoiesis.

ADP-ribosyl Cyclase↗
PubMed1999

Incompatibility between human blood and isolated islets of Langerhans: a finding with implications for clinical intraportal islet transplantation?

The remarkable difference in success rates between clinical pancreas transplantation and islet transplantation is poorly understood. Despite the same histocompatibility barrier and similar immunosuppressive treatments in both transplantation procedures, human intraportal islet transplantation has a much inferior success rate than does vascularized pancreas transplantation. Thus far, little attention has been directed to the possibility that islets transplanted into the blood stream may elicit an injurious incompatibility reaction. We have tested this hypothesis in vitro with human islets and in vivo with porcine islets. Human islets were exposed to nonanticoagulated human ABO-compatible blood in surface-heparinized polyvinyl chloride tubing loops. Heparin and/or the soluble complement receptor 1 (sCR1) TP10 were tested as additives. Adult porcine islets were transplanted intraportally into pigs, and the liver was recovered after 60 min for immunohistochemical staining. Human islets induced a rapid consumption and activation of platelets. Neutrophils and monocytes were also consumed, and the coagulation and complement systems were activated. Upon histological examination, islets were found to be embedded in clots and infiltrated with CD11+ leukocytes. Furthermore, the cellular morphology was disrupted. When heparin and sCR1 were added to the blood, these events were avoided. Porcine islets retrieved in liver biopsies after intraportal islet allotransplantation showed a morphology similar to that of human islets perifused in vitro. Thus, exposure of isolated islets of Langerhans to allogenic blood resulted in significant damage to the islets, a finding that could explain the unsatisfactory clinical results obtained with intraportal islet transplantation. Because administration of heparin in combination with a soluble complement receptor abrogated these events, such treatment would presumably improve the outcome of clinical islet transplantation by reducing both initial islet loss and subsequent specific immune responses.

Adult↗
PubMed1998

Successful islet auto- and allotransplantation in diabetic pigs.

BACKGROUND: Because of its anatomical and physiological similarities to humans, the pig appears to be a suitable large animal model for preclinical studies of islet transplantation. The aim of this study was to investigate islet auto- and allotransplantation in a pig model with diabetes induced by total pancreatectomy. METHODS: Porcine islets were isolated by a continuous digestion-filtration device at 32 degrees C and purified by a discontinuous iso-osmolar Ficoll-sodium-diatrizoate gradient on a Cobe 2991. The purified islets were autografted into the liver or the renal subcapsular space. The liver appears to be a more suitable site for the islet grafts than the renal subcapsular space, and the minimal amount of islets for reversal of diabetes is >5 microl/kg of body weight. RESULTS: Persistent normoglycemia (fasting blood glucose level: 72.4+/-44.38 mg/dl) with a normal insulin secretion response to glucose stimulation was successfully achieved in five of six diabetic pigs by implanting a sufficient islet mass into the liver. Triple-drug immunosuppressive therapy with cyclosporine, azathioprine, and prednisolone did not prevent porcine islet allografts from experiencing early failure. However, the addition of 15-deoxyspergualin to the triple-drug immunosuppressive regimen significantly prolonged the function of the islet allografts. When antithymocyte globulin was added to the above-mentioned immunosuppressive drug regimen, the normoglycemic period was prolonged to more than 1 month (fasting blood glucose level: 75.4+/-17 mg/dl). CONCLUSION: We conclude that autotransplantation with a sufficient islet mass can induce normoglycemia with a normal insulin secretion response to glucose stimulation in pancreatectomized diabetic pigs and that allotransplantation can be successfully achieved when 15-deoxyspergualin and antithymocyte globulin are combined with the triple-drug immunosuppression described above. However, this immunosuppressive protocol results in a high rate of infectious complications.

Animals↗
PubMed1998

Improved glucose counterregulation and autonomic symptoms after intraportal islet transplants alone in patients with long-standing type I diabetes mellitus.

BACKGROUND: Defective glucose counterregulation and hypoglycemia unawareness are both well-recognized risk factors for recurrent episodes of severe hypoglycemia in patients with type I diabetes. At present, no conventional therapy is available to routinely overcome these acquired impairments in long-standing diabetes. METHODS: To test the hypothesis that successful intraportal islet transplantation could improve this syndrome, hormonal counterregulatory responses and symptoms were studied during stepped hypoglycemic clamp tests before and after intraportal islet transplantation in three patients with type I diabetes who were prone to severe hypoglycemia. RESULTS: As compared with matched nondiabetic control subjects, before islet transplantation, glucagon responses were absent while epinephrine and cortisol responses were either markedly decreased or absent in all diabetic subjects. One patient also had decreased norepinephrine and growth hormone responses. Autonomic warning symptoms were absent in all patients during hypoglycemia. One month after successful islet transplantation, there was no improvement in the glucagon response. However, glycemic thresholds and/or peak incremental responses of epinephrine, norepinephrine, and cortisol improved in all patients. Moreover, all patients had developed autonomic warning symptoms so that glycemic thresholds were detectable within the examined range. CONCLUSION: We conclude that intraportal islet transplantation does not restore hypoglycemia-induced glucagon secretion, but it improves the responses of most counterregulatory hormones and hypoglycemic warning symptoms even in long-standing type I diabetes.

Adult↗
PubMed1998

Transduction of non-dividing adult human pancreatic beta cells by an integrating lentiviral vector.

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.

Cell Division↗
PubMed1998

[Isolation of islands of Langerhans from human and porcine pancreas for transplantation to humans].

The enormous variability of donor factors and organ procurement related variables prevent a constant isolation success, thus reducing the potential number of clinical islet transplants. Since the availability of intact and viable pancreatic donor tissue intended for islet transplantation is limited, the porcine pancreas was selected as a potential source of xenogeneic islets for human recipients. The differences of islet histomorphology between porcine and human pancreas result in a higher intrinsic fragility of porcine islets during collagenase digestion. Nevertheless, if the isolation method is modified to inhibit factors potentially toxic to pig islets, reproducibility of isolation success is higher in the pig as in the human due to a lower variability in donor characteristics and the opportunity of preselection in regard to age and race. If xenograft rejection can be overcome and the risk of xenosis can be minimized, the logistic prerequisites for xenotransplantation of large amounts of viable pig islets into human recipients with insulin dependent diabetes are fulfilled.

Adult↗