Andreas Meyer zu Vilsendorf

Islet infiltration, cytokine expression and beta cell death in the NOD mouse, BB rat, Komeda rat, LEW.1AR1-iddm rat and humans with type 1 diabetes

Aims/hypothesisResearch on the pathogenesis of type 1 diabetes relies heavily on good animal models. The aim of this work was to study the translational value of animal models of type 1 diabetes to the human situation.MethodsWe compared the four major animal models of spontaneous type 1 diabetes, namely the NOD mouse, BioBreeding (BB) rat, Komeda rat and LEW.1AR1-iddm rat, by examining the immunohistochemistry and in situ RT-PCR of immune cell infiltrate and cytokine pattern in pancreatic islets, and by comparing findings with human data.ResultsAfter type 1 diabetes manifestation CD8+ T cells, CD68+ macrophages and CD4+ T cells were observed as the main immune cell types with declining frequency, in infiltrated islets of all diabetic pancreases. IL-1β and TNF-α were the main proinflammatory cytokines in the immune cell infiltrate in NOD mice, BB rats and LEW.1AR1-iddm rats, as well as in humans. The Komeda rat was the exception, with IFN-γ and TNF-α being the main cytokines. In addition, IL-17 and IL-6 and the anti-inflammatory cytokines IL-4, IL-10 and IL-13 were found in some infiltrating immune cells. Apoptotic as well as proliferating beta cells were observed in infiltrated islets. In healthy pancreases no proinflammatory cytokine expression was observed.Conclusions/interpretationWith the exception of the Komeda rat, the animal models mirror very well the situation in humans with type 1 diabetes. Thus animal models of type 1 diabetes can provide meaningful information on the disease processes in the pancreas of patients with type 1 diabetes.

Diabetes Prevention by Immunomodulatory FTY720 Treatment in the LEW.1AR1-iddm Rat Despite Immune Cell Activation

The prevention of diabetes by the immunomodulatory agent FTY720 (fingolimod) was studied in the LEW.1AR1-iddm (IDDM) rat, an animal model of human type 1 diabetes. Immune cell subtypes and cytokine profiles in pancreatic islets, secondary lymphoid tissue, and serum were analyzed for signs of immune cell activation. Animals were treated with FTY720 (1 mg/kg body weight) for 40 d starting on d 50 of life. Changes in gene and protein expression of cytokines, CD8 markers, monocyte chemoattractant protein-1, inducible NO synthase, and caspase 3 were evaluated. Treatment with FTY720 prevented diabetes manifestation and islet infiltration around d 60 of life, the usual time of spontaneous diabetes development. On d 120, 30 d after the end of FTY720 therapy, diabetes prevention persisted. However, six of 12 treated animals showed increased gene expression of IL-1beta, TNF-alpha, and CD8 markers in pancreas-draining lymph nodes, indicating immune cell activation. In parallel, serum concentrations of these proinflammatory cytokines were increased. These six animals also showed macrophage infiltration without proinflammatory cytokine expression in a small minority (2-3%) of islets. Interestingly, regulatory T lymphocytes were significantly increased in the efferent vessels of the pancreas-draining lymph nodes only in animals without signs of immune cell activation but not in the rats with immune cell activation. This provides an indication for a lack of protective capacity in the animals with activated immune cells. Thus, FTY720 treatment prevented the manifestation of diabetes by promoting the retention of activated immune cells in the lymph nodes, thereby avoiding islet infiltration and beta-cell destruction by proinflammatory cytokines.

Induction of heme oxygenase-1 improves the survival of pancreas grafts by prevention of pancreatitis after transplantation.

Background. Ischemia/reperfusion (I/R) injury after pancreas transplantation might result in graft pancreatitis. The role of heme oxygenase-1 (HO-1) in pancreas transplantation and prevention of graft pancreatitis is unknown. Method. We studied the impact of HO-1 induction with cobalt protoporphyrin (CoPP) in experimental pancreas transplantation with moderate (6 hr) and prolonged (20 hr) cold ischemic time (CIT). Donor animals received CoPP 5 mg/kg intraperitoneal at 48 hr or intraperitoneal saline injections in the corresponding control groups before procurement. Harvested grafts were perfused with HTK solution and stored at 4°C. Results. After prolonged CIT, graft survival was 100% with CoPP pretreatment in contrast to only 37.5% without pretreatment. CoPP-pretreated grafts demonstrated an unimpaired endocrine graft function at moderate and prolonged CIT. Serum lipase activity as a sign of exocrine preservation was significantly lower. In addition, morphological architecture was well preserved. CoPP pretreatment markedly increased HO-1 gene expression in donor pancreas (130-fold increase) by means of quantitative reverse transcriptase -polymerase chain reaction. Immunohistochemical examinations showed that the increase of HO-1 on the protein level was related to HO-1-positive donor macrophages in the pancreas grafts. HO-1 overexpression was accompanied by significant decrease of proinflammatory cytokines such as tumor necrosis factor-&agr;, interleukin (IL)-2, IL-6, interferon-y, and by significant increase of the anti-inflammatory cytokine IL-10 and less expression of adhesion molecules such as e- and p-selectins. Conclusions. HO-1 is highly inducible in the allograft rat pancreas and associated with a survival benefit and good graft function after transplantation. This study contributes to the beneficial potentials of HO-1 for the prevention of graft pancreatitis.

The L-arginine/NO pathway in end-stage liver disease and during orthotopic liver and kidney transplantation : Biological and analytical ramifications

The L-arginine/nitric oxide (L-Arg/NO) pathway is altered in liver and kidney diseases. However, the status of the L-Arg/NO pathway during and after orthotopic transplantation is insufficiently investigated and findings are uncertain because of analytical shortcomings. Also, most human studies have focused on individual members of the L-Arg/NO pathway such as nitrate or asymmetric dimethylarginine (ADMA). In the present article we report on a pilot study investigating extensively the status of the L-Arg/NO pathway before and during orthotopic liver transplantation (OLT). By using fully validated, highly sensitive and specific GC-MS and GC-MS/MS methods nitrite, nitrate, ADMA and its hydrolysis product dimethylamine (DMA), L-arginine and L-ornithine were measured in blood and urine. Our study gives strong evidence of the exceptional importance of hepatic dimethylarginine dimethylaminohydrolase (DDAH) activity for the elimination of systemic ADMA. In end-stage liver disease the synthesis of NO and ADMA as well as the DDAH activity are elevated. However, increase in DDAH activity is insufficient to efficiently eliminate overproduced ADMA. The transplanted liver graft is capable of clearing ADMA in a rapid and sufficient manner. In contrast to studies from other groups, our study shows that in OLT as well as in living donor kidney transplantation, the second study reported here, reperfusion of the graft does not cause drastic alterations to the L-Arg/NO pathway with regard to NO synthesis. In the OLT study the concentration of circulating L-arginine fell temporally dramatically, while L-ornithine levels increased diametrically, most likely due to elevation of arginase activity. However, the relatively long-lasting decrease in plasmatic L-arginine in OLT seems not to have affected NO synthesis after reperfusion. Our OLT study suggests that liver reperfusion is associated with greatly elevated activity of proteolytic and hydrolytic enzymes including DDAH and arginase. Suppression of proteolytic and hydrolytic activity in transplantation could be a useful measure to improve outcome and remains to be investigated in further studies on larger patient collectives. The importance of analytical chemistry in this area of research is also discussed in this article.

Preconditioning with the prostacyclin analog epoprostenol and cobra venom factor prevents reperfusion injury and hyperacute rejection in discordant liver xenotransplantation

Abstract: Liver xenografts transplanted from guinea pig to rat suffer from inadequate organ reperfusion and initial dysfunction, despite sufficient complement depletion using cobra venom factor (CVF). Reperfusion injury is prevented when complement depleted donors are treated with the prostacyclin analog epoprostenol. Histological analysis suggests that epoprostenol preconditioning prevents post‐reperfusion spasms of the intrahepatic branches of the portal vein and strongly reduces appearance of hepatocyte apoptosis shortly after transplantation. Cobra‐venom‐treated rats show breakdown of glucose metabolism and die in acute hypoglycaemia, whereas the additional application of epoprostenol restores gluconeogenesis. Consequently, recipient survival after epoprostenol and CVF treatment is significantly improved compared with animals receiving CVF only (5.1 ± 2.6 h vs. 17.9 ± 5.1 h). These data demonstrate that initial dysfunction of discordant liver grafts in the guinea‐pig‐to‐rat species combination, can be overcome by the application of epoprostenol combined with CVF. Using this pharmacologic regimen, the discordant guinea‐pig‐to‐rat model appears useful to study further questions concerning functional and immunological compatibility of a discordant liver xenograft.

Adult Kasabach-Merritt Syndrome due to Hepatic Giant Hemangioma

Cavernous hemangiomas are the most common benign tumors of the liver. They can reach enormous sizes and cause various complications. Kasabach-Merritt syndrome is a rare but serious complication characterized by consumptive coagulopathy caused by the hemangioma; mortality rate ranges between 10 and 37%. More than 80% of cases occur within the first year of life. Goals of the treatment are to control the coagulopathyand thrombocytopenia as well as to eradicate the hemangioma. Different nonsurgical treatment regimens are performed, includingsystemic corticosteroids, irradiation and various chemicals. Surgery should be limited to symptomatic or complicated cases. Although difficult, resection of the tumor is usually curative. Here we present a 44-year-old woman with giant hepatic hemangioma causing Kasabach-Merritt syndrome managed by enucleation.

Lack of influence of ω-3 fatty acid–enriched lipids on apoptosis and secondary necrosis of cultured human lymphocytes

Abstract Objective: The anti-inflammatory properties of parenteral nutrition might be improved by enrichment with ω-3 polyunsaturated fatty acids (PUFAs), which are responsible for the enhanced release of metabolites derived from eicosapentaenoic acid. Under physiologic conditions, lymphocyte populations are regulated by cellular mechanisms such as apoptosis. In contrast to cell death by necrosis, apoptosis does not induce an inflammatory response that might injure the host. Methods: Apoptosis and necrosis of cultured human blood lymphocytes were investigated in vitro after incubation for 48 and 72 h with three lipid emulsions containing 50% medium-chain triacylglycerols. The lipid emulsions differed in the percentage of long-chain triacylglycerols, which were replaced in part by different amounts of ω-3 PUFA (8%, 20%, or 40%). Rates of apoptosis and necrosis of lymphocyte subpopulations were analyzed with a sensitive annexin V flow cytometric assay. Results: After 48 and 72 h of incubation, time- and dose-dependent increases of apoptosis and necrosis, respectively, were found in all lymphocyte subsets regardless of the percentage of ω-3 PUFAs. Conclusions: Our results suggested that enrichment with ω-3 PUFAs in the tested lipid emulsions does not alter apoptosis and secondary necrosis of lymphocyte populations. Thus PUFAs may exert their functional effects through other mechanisms.

Reduced P-selectin expression on circulating platelets after prolonged cold preservation in renal transplantation

Abstract: The uremic state in patients with terminal renal insufficiency is accompanied by a bleeding tendency connected with platelet dysfunction. Prolonged cold ischemia and inflammatory interactions between leukocytes, platelets and endothelial cells contribute to ischemia‐/reperfusion (I/R) injury and may impair long‐term graft survival. We evaluated the influence of the duration of cold preservation time on the expression of platelet GPIIb/IIIa and P‐selectin and on the formation of leukocyte‐platelet complexes after kidney transplantation. Fourteen patients undergoing kidney transplantation were divided into group I with long preservation time (26.6 ± 1.9 h) and group II with short preservation time (8 ± 6.1 h). Five venous blood samples (3 ml) were taken before induction of anesthesia, 12 h, 2, 7 and 14 d after transplantation. Surface expression of the GPIIb/IIIa, P‐selectin and the percentage of platelet‐granulocyte complexes were quantified by flow cytometry. Additionally blood from seven healthy volunteers was analyzed. GPIIb/IIIa and P‐selectin expression on circulating platelets were significantly decreased in the long and the short‐term graft preservation group compared with healthy volunteers. A significantly reduced P‐selectin expression was found in the long‐term preservation group compared with the short‐term group. The percentage of platelet‐granulocyte complexes also decreased in both preservation groups in the first 2 d after reperfusion and remained in this state in the long‐term preservation group. Reduced expression of P‐selectin on circulating platelets may be an indicator of I/R injury after prolonged kidney graft preservation.