Heide Brandhorst

Positron emission tomography : A real-time tool to quantify early islet engraftment in a preclinical large animal model

Background. Clinical islet transplantation is currently being explored as a therapeutic option for persons with type I diabetes and hypoglycemic unawareness. Techniques to monitor graft survival are urgently needed to optimize the procedure. Therefore, the objective of the present study was to develop a technique for imaging survival of transplanted islets in the peritransplant and early posttransplant phase. Methods. Isolated porcine islets were labeled in vitro with 2-deoxy-2[18F]fluoro-D-glucose ([18F]FDG) and infused intraportally into anesthetized pigs (n=10). Dynamic examination was performed on a positron emission tomography/computed tomography hybrid system. Results. More than 95% of the radioactivity was confined to the islets at the time of transplantation. The peak percentage of infused radioactivity within the liver, quantified at the end of the islet infusion, was only 54±5.1%. The distribution of the radioactivity in the liver was found to be heterogeneous. A whole-body examination showed no accumulation in the lungs or brain; extrahepatic radioactivity was, except urinary excretion, evenly distributed in the pig body. Conclusions. Our results imply that almost 50% of the islets were damaged to the extent that the FDG contained was release within minutes after intraportal transplantation. The distribution of radioactivity without accumulation in the brain indicates that the activity is released from lysed islet cells in the form of [18F]FDG-6P rather than native [18F]FDG. The presented technique shows promise to become a powerful and quantitative tool, readily available in the clinic, to evaluate initial islet engraftment and survival.

The effects of glucagon-like peptide-I (GLP-I) on hormone secretion from isolated human pancreatic islets

Glucagon-like peptide-I (GLP-I) is a potent in-cretin hormone that is now considered as a new therapeutic tool in the treatment of diabetes mellitus. In this study we characterized the effects of GLP-I on peptide hormone release from isolated human pancreatic islets. GLP-I stimulated insulin release in the presence of 10 mM glucose (2.8 mM glucose, 100%; 10 mM glucose, 166%; 10 mM glucose + 10 nM GLP-I, 222%) but had only a weak insulinotropic effect (128%) at 2.8 mM glucose. Glucagon release was inhibited by 10 mM glucose (2.8 mM glucose, 100%; 10 mM glucose, 72%) and by 10 nM GLP-I at 2.8 mM glucose (67%). Somatostatin secretion was increased by 10 mM glucose (2.8 mM glucose, 100%; 10 mM glucose, 166%). GLP-I stimulated somatostatin release in the presence of 2.8 mM glucose (172%). Pancreatic polypeptide (PP) secretion was enhanced by 10 mM glucose (2.8 mM glucose, 100%; 10 mM glucose, 236%). GLP-I induced PP release only in the presence of 2.8 mM glucose (184%).

The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation

Background. The isolation of islets from the human pancreas critically depends on an efficient enzyme blend. Previous studies have solely focused on the presence of collagenase and neutral protease/thermolysin. Despite improved characterization of these components, the lot-related variability in efficacy still persists suggesting that additional so far disregarded enzymes are required for efficient islet cleavage. Methods. Varying activities of a tryptic-like enzyme were identified within collagenase NB1 lots, which were selected according to a matched ratio between tryptic-like and collagenase activity (TLA-ratio). Rat and human pancreata were processed with current standard procedures. Results. Increasing the TLA-ratio from 1.3% to 10% reduced pancreas dissociation time in rats by 50% without affecting islet yield, viability, or posttransplant function in diabetic nude mice. Enhancing the TLA-ratio from 1.3% to 12.6% for human pancreas processing resulted in a significant reduction of recirculation time and increased incrementally human islet yield without affecting purity, in vitro function or recovery after culture. Optimized pancreas digestion correlated with a higher percentage of islet preparations fulfilling quality criteria for clinical transplantation. Conclusions. We conclude that TLA is an effective component that should be included in moderate amounts in enzyme blends for human islet isolation to optimize the efficiency and minimize the lot-related variability.

A New Oxygen Carrier for Improved Long-Term Storage of Human Pancreata Before Islet Isolation

Background. Pancreas oxygenation during cold storage has been established in islet isolation and transplantation to prevent ischemic tissue damage using perfluorodecalin (PFD) as hyperoxygen carrier. However, studies in humans and pigs provided conflicting results about the efficiency of PFD for pancreas oxygenation. The aim of this study was to compare PFD with a newly developed oxygen carrier composed of perfluorohexyloctane and polydimethylsiloxane 5 (F6H8S5) for long-term storage of human pancreata. Methods. After 24-hr storage in preoxygenated PFD or F6H8S5, pancreata were processed using Liberase HI for pancreas dissociation and a Ficoll gradient for islet purification. Islet quality assessment was performed measuring glucose-stimulated insulin release, viability, islet ATP content, and posttransplant function in diabetic nude mice. Results. Compared with PFD, F6H8S5 significantly increased the intrapancreatic partial oxygen pressure and islet ATP content. This corresponded to an increase of islet yield, recovery after culture, glucose stimulation index, viability, and improved graft function in diabetic nude mice. Conclusions. The present findings indicate clearly that F6H8S5 improves isolation outcome after prolonged ischemia compared with PFD. This observation seems to be related to the significant lipophilicity and almost pancreas-specific density of F6H8S5. Moreover, these characteristics facilitate pancreas shipment without using custom-made transport vessels as required for PFD.

Prevention of early islet graft failure by selective inducible nitric oxide synthase inhibitors after pig to nude rat intraportal islet transplantation.

Background. Clinical and experimental data indicate that early failure of intraportally grafted islets is caused by inflammation including secretion of cytokines and nitric oxide. Direct inducible nitric oxide synthase suppression may avoid detrimental effects associated with steroid administration. We compared the efficiency of selective and unselective inducible nitric oxide synthase inhibitors with dexamethasone to suppress nitric oxide generation after intraportal islet xenotransplantation into nude rats. Methods. Nonfasting serum glucose levels were daily evaluated after intraportal transplantation of 4000 freshly isolated pig islets into diabetic nude rats (85 mg/kg streptozotocin) either sham-treated with saline (n=21) or continuously infused for 7 days with L-NG-monomethyl-arginine (n=7), S-methyl-isothiourea (n=15), or S-(2-aminoethyl)-isothiourea (n=19) in a dosage of 240, 100, or 50 mg/kg/day, respectively. Dexamethasone was injected i.p. twice as a daily bolus of 20 mg/kg (n=10) starting 1 day pretransplant. The nitrate/nitrite serum level was quantified colorimetrically 0, 24, and 48 hr posttransplant. Results. Saline treatment partially resulted in graft function (4/21) throughout the observation period (21 days). L-NG-monomethyl-arginine-treated rats showed sustained hyperglycemia (0/7) not different from diabetic controls. Normoglycemia was observed after treatment with dexamethasone (6/10, P <0.05 versus saline and L-NG-monomethyl-arginine), S-methyl-isothiourea (10/15, P <0.01), or S-(2-aminoethyl)-isothiourea (15/19, P <0.001). Graft function was associated with complete suppression of nitric oxide generation after S-methyl-isothiourea and S-(2-aminoethyl)-isothiourea treatment (P <0.001 versus saline) and partial suppression after dexamethasone treatment (P <0.05). Conclusions. Our observation of long-term function of xenogeneic islets in an inflammatory environment without interference of reactive T cells revealed the potency of highly selective isothioureas to completely suppress inducible nitric oxide synthase making reduction of islet-toxic immunosuppression feasible.

The ratio between collagenase class I and class II influences the efficient islet release from the rat pancreas.

Background. Previous studies indicated different roles of collagenase class I, class II and neutral protease in the enzymatic islet release from pancreatic tissue. Because no information has been available, this study was aimed to investigate the isolation efficiency of different ratios between collagenase class II and I (C-ratio) in the rat pancreas serving as model for the human pancreas without being restricted by the large variability observed in human donors. Methods. Rat pancreata were digested using a marginal neutral protease activity and 20 PZ-U of purified collagenase classes recombined to create a C-ratio of 0.5, 1.0, or 1.5. Collagenase efficiency was evaluated in terms of isolation outcome and posttransplantation function in diabetic nude mice. Results. The highest yield of freshly isolated islets was obtained using a C-ratio of 1.0. Purity and fragmentation of freshly isolated islets were not influenced by the C-ratio. After 24-hr culture performed for quality assessment, a marginal but significant reduction of viability was observed in islets isolated by means of a C-ratio of 0.5 and 1.5. Islet in vitro and posttransplantation function revealed no negative effect mediated by different C-ratios. Conclusions. The present study demonstrates that the C-ratio is of significant relevance for the outcome after enzymatic rat islet isolation. The data indicate further that purified collagenase class I or class II does not damage islet tissue even if used in excess. The present study can serve as a start for subsequent experiments in the human pancreas.

Pancreas storage in oxygenated perfluorodecalin does not restore post-transplant function of isolated pig islets pre-damaged by warm ischemia

Abstract:  Background:  Cold storage in oxygenated perfluorodecalin (PFD) restores transplant function of ischemically damaged dog pancreata and reduces the impact of cold ischemia on recovery of isolated human islets. Whether PFD storage can improve islet isolation from pancreata exposed to significant warm ischemia (WI) is unclear yet. The present study aimed to clarify this question in adult pigs.

Vitacyte collagenase HA: a novel enzyme blend for efficient human islet isolation.

Islet transplantation has found its niche in diabetes treatment. It has contributed to a better quality of life and better glycemic control of patients with diabetes suffering from severe hypoglycemia that are not eligible for vascularized pancreas transplantation. Islet isolation is a technically challenging procedure. The different studies within this doctoral thesis aim to improve and standardize different steps in the isolation procedure. They are in particular looking to improve human pancreas preservation during cold storage, to optimize islet release from the exocrine tissue and to assess whether the isolated islet yield can be predicted from a biopsy. We found that pancreas preservation with pre-oxygenated perfluorodecalin (two-layer method) did not improve the ischemic tolerance of the human pancreas as compared to cold storage with the University of Wisconsin (UW) solution. Furthermore, in pancreas with long cold ischemia time (CIT) (>10 hours), Histidine-Tryptophan-Ketoglutarate (HTK) had a limited preservation capacity as compared with the UW solution with respect to isolation outcome. We also found that during enzymatic pancreas digestion, Vitacyte HA was able to provide a similar islet yield and quality as Serva NB1 with less collagenase activity and shorter digestion time. We further describe the first experience with a new GMP manufactured enzyme called Liberase MTF-S for successful human islet isolation. Finally, we found that the isolated islet yield could not be predicted from a biopsy taken from the head of the pancreas concerning solely morphological parameters of the islets tissue. The improvement of pancreas preservation will allow for marginal organs with prolonged cold ischemia time to expand the donor pool. Better knowledge of how the pancreatic extracellular matrix is digested by collagenase will lead to a fast and predictable islet release from the exocrine tissue. By standardizing the isolation procedure and improving organ selection we will increase the success rate in human islet isolation, thereby making islet transplantation available for more patients.

Activation of NFκB dependent apoptotic pathway in pancreatic islet cells by hypoxia

Insulin producing β-cells are exposed to hypoxic stress in the early period after islet transplantation. Although it has been suggested that hypoxia leads to islet loss, the underlying mechanisms are unclear. In this study, the early transcriptional profiles of b cell response to hypoxia were determined by using the Affymetrix Murine Genome Array U74Av2 GeneChip (about 12,422 genes). In addition to the upregulation of genes associated with glycolysis, genes related to apoptosis and stress response were also upregulated. The downregulated genes on hypoxia are classified as transcription and inflammatory response. These findings were confirmed by TUNEL assay and real-time reverse transcription-PCR for mRNA of genes related to apoptosis. On the other hand, we have found that the transcription factor NFκB was activated by hypoxia in islet cells. The affected genes involved in the activated NFκB pathway were mainly categorized as apoptosis-related genes by quantitative real-time PCR array (84 genes). Our comprehensive analysis of transcriptional changes of islets by hypoxia may assist the development of strategies that protect islet grafts from early loss.

Quality of Isolated Pig Islets Is Improved Using Perfluorohexyloctane for Pancreas Storage in a Split Lobe Model

Pancreas transportation between donor center and islet production facility is frequently associated with prolonged ischemia impairing islet isolation and transplantation outcomes. It is foreseeable that shipment of pig pancreases from distant centralized biosecure breeding facilities to institutes that have a long-term experience in porcine islet isolation is essentially required in future clinical islet xenotransplantation. Previously, we demonstrated that perfluorohexyloctan (F6H8) is significantly more efficient to protect rat and human pancreata from ischemically induced damage compared to perfluorodecalin (PFD). To evaluate the effect of F6H8 on long-term stored pig pancreases in a prospective study, we utilized the split lobe model to minimize donor variability. Retrieved pancreases were dissected into the connecting and splenic lobe, intraductally flushed with UW solution and immersed alternately in either preoxygenated F6H8 or PFD for 8–10 h. Prior to pancreas digestion, the intrapancreatic pO2 and the ratio of ATP-to-inorganic phosphate was compared utilizing 31P-NMR spectroscopy. Isolated islets were cultured for 2–3 days at 37°C and subjected to quality assessment. Pancreatic lobes stored in preoxygenated F6H8 had a significantly higher intrapancreatic pO2 compared to pancreata in oxygen-precharged PFD (10.11 ± 3.87 vs. 1.64 ± 1.13 mmHg, p < 0.05). This correlated with a higher ATP-to-inorganic phosphate ratio (0.30 ± 0.04 vs. 0.14 ± 0.01). No effect was observed concerning yield and purity of freshly isolated islets. Nevertheless, a significantly improved glucose-stimulated insulin response, increased viability and postculture survival (57.2 ± 5.7 vs. 39.3 ± 6.4%, p < 0.01) was measured in islets isolated from F6H8-preserved pancreata. The present data suggest that F6H8 does not increase islet yield but improves quality of pig islets isolated after prolonged cold ischemia.