Roger F. L. James

Islet isolation assessment in man and large animals

SummaryRecent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity andin vitro andin vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 µ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason,in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers.

Interferon Expression in the Pancreases of Patients With Type I Diabetes

We have used a reverse transcriptase–polymerase chain reaction (RT-PCR) protocol to examine the expression of cytokines in the pancreases and islets of patients with type I diabetes. We detect a significant increase in the level of expression of interferon (IFN)-α in the pancreases of the diabetic patients as compared with the control pancreases. In contrast, IFN-β was detected at comparable levels in both groups, while IFN-γ was detected in three of four control pancreases and one of four pancreases from the diabetic individuals. The IFN-α cDNAs generated by the RT-PCR were cloned and sequenced to determine which α-subtypes were being expressed. We found that the repertoire of subtypes was quite limited in any one individual (diabetic or not), although each individual was different with respect to the pattern of subtypes expressed. We also examined these pancreases for the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-2, IL-4, and IL-6. We found no detectable expression of TNF-α or IL-2 in any pancreases, and the expression of the other cytokines was variable, with no pattern emerging from the comparison of the diabetic and nondiabetic individuals. We conclude that, of the cytokines examined, only IFN-α was significantly increased in the diabetic patients, a result that is consistent with the possibility that this cytokine is directly involved in the development of type I diabetes.

Functional studies of rat, porcine, and human pancreatic islets cultured in ten commercially available media

There have been no extensive studies investigating the effect of tissue culture media on the in vitro functional characteristics of rat, porcine and human Islets of Langerhans. We therefore aimed to compare ten commercially available tissue culture media on the basis of their ability to maintain islet viability. Following isolation, islets were cultured free-floating in the ten media (RPMI 1640-11mM glucose (control), RPMI 1640-2.2mM glucose, Dulbeccos MEM, TCM 199, CMRL 1066, Iscoves MEM, Waymouths MEM, Serum-Free medium, Ex-cell 300, Hams F-12) and viability was assessed after 24 hr, 3 days, and 7 days on the basis of macroscopic appearance, cell membrane integrity, and insulin secretion in response to glucose stimulation both by dynamic incubation and by perifusion. Each islet species demonstrated physiological insulin release characteristics in all media--however, it was possible to distinguish between the media by comparing the stimulation indices calculated from the insulin release studies. Significantly higher stimulation indices were produced in Iscoves MEM for rat islets, in Hams F-12 for porcine islets and in CMRL 1066 for human islets. Over the entire culture period a significant deterioration in function was observed in all species cultured in the control media, although this was reversed when islets were cultured in the optimal media. Furthermore, in the case of porcine and human islets a significant improvement in function over the seven-day period was noted in the optimal media. In conclusion, of the commercially available media, the optimal tissue culture medium for rat islets is Iscoves MEM, for porcine islets is Hams F-12, and for human islets is CMRL 1066.

Bovine serum albumin density gradient isolation of rat pancreatic islets

The use of a bovine serum albumin (BSA) density gradient for isolation of rat pancreatic Islets of Langerhans after collagenase digestion has been compared with the standard Ficoll separation technique. The criteria studied were islet yield (insulin extraction of the islet interfaces and pellet), purity of preparation (amylase content of the islet preparation), insulin release characteristics, and the result of isologous transplantation in diabetic rats. The islet interface of the BSA gradient contained 62.2% of the total insulin, whereas the corresponding interface of the Ficoll gradient contained only 36.6% (P less than 0.001). The amylase content of the Ficoll-separated islet preparation was 6133 U/L, as opposed to 1230 U/L with BSA (P less than 0.001). BSA-isolated islets gave similar insulin release characteristics to non-density-gradient-isolated islets, whereas Ficoll-separated islets showed suboptimal insulin release. Single-donor-single-recipient transplantation was successfully performed with BSA-isolated islets whereas multiple donors were required with Ficoll-separated islets. Thus significantly improved results were found with the bovine serum albumin density gradient separation in all criteria and consequently the use of this gradient represents an advance in islet isolation techniques.

Gene expression profiles for the human pancreas and purified islets in Type 1 diabetes: new findings at clinical onset and in long-standing diabetes

Type 1 diabetes (T1D) is caused by the selective destruction of the insulin‐producing β cells of the pancreas by an autoimmune response. Due to ethical and practical difficulties, the features of the destructive process are known from a small number of observations, and transcriptomic data are remarkably missing. Here we report whole genome transcript analysis validated by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) and correlated with immunohistological observations for four T1D pancreases (collected 5 days, 9 months, 8 and 10 years after diagnosis) and for purified islets from two of them. Collectively, the expression profile of immune response and inflammatory genes confirmed the current views on the immunopathogenesis of diabetes and showed similarities with other autoimmune diseases; for example, an interferon signature was detected. The data also supported the concept that the autoimmune process is maintained and balanced partially by regeneration and regulatory pathway activation, e.g. non‐classical class I human leucocyte antigen and leucocyte immunoglobulin‐like receptor, subfamily B1 (LILRB1). Changes in gene expression in islets were confined mainly to endocrine and neural genes, some of which are T1D autoantigens. By contrast, these islets showed only a few overexpressed immune system genes, among which bioinformatic analysis pointed to chemokine (C‐C motif) receptor 5 (CCR5) and chemokine (CXC motif) receptor 4) (CXCR4) chemokine pathway activation. Remarkably, the expression of genes of innate immunity, complement, chemokines, immunoglobulin and regeneration genes was maintained or even increased in the long‐standing cases. Transcriptomic data favour the view that T1D is caused by a chronic inflammatory process with a strong participation of innate immunity that progresses in spite of the regulatory and regenerative mechanisms.

Islet glutamic acid decarboxylase modified by reactive oxygen species is recognized by antibodies from patients with type 1 diabetes mellitus

The generation of an autoimmune response against islet beta‐cells is central to the pathogenesis of type 1 diabetes mellitus, and this response is driven by the stimulation of autoreactive lymphocytes by components of the beta‐cells themselves. Reactive oxygen species (ROS) have been implicated in the beta‐cell destruction which leads to type 1 diabetes and may modify beta‐cell components so as to enhance their immunogenicity. We investigated the effects of oxidation reactions catalysed by copper or iron on the major beta‐cell autoantigen glutamic acid decarboxylase (GAD). Lysates of purified rat islets were exposed to copper or iron sulphate with or without hydrogen peroxide or ascorbic acid. Immunostaining showed that these treatments generated high molecular weight covalently linked aggregates containing GAD. These are not formed by intermolecular disulphide bonds between cysteine residues since they cannot be resolved into monomeric form when electrophoresed under extreme reducing conditions. There was no modification of insulin or pro‐insulin by ROS. The same oxidative changes to GAD could be induced in viable islet cells treated with copper sulphate and hydrogen peroxide, and thus the modifications are not an artefact of the catalysed oxidation of cell‐free lysates. Sera from patients with type 1 diabetes and stiffman syndrome containing GAD antibodies reacted predominantly with the highest molecular weight modified protein band of GAD: normal human sera did not precipitate GAD. Thus, oxidatively modified aggregates of GAD react with serum antibodies of type 1 diabetes patients and some SMS patients: this is consistent with oxidative modifications of autoantigens being relevant to the pathogenesis of type 1 diabetes.

The effect of varying fibronectin concentration on the attachment of endothelial cells to polytetrafluoroethylene vascular grafts

Endothelial cell seeding onto untreated polytetrafluoroethylene vascular prostheses is inefficient. In an effort to improve cell attachment, numerous investigators have used fibronectin as a coating material to pretreat the luminal surfaces of these prostheses. The concentrations of fibronectin used have varied enormously, and no one has yet determined the most efficient concentration in terms of cell attachment and cost. Using endothelial cells labeled with indium 111 oxine we have studied the effect of varying fibronectin concentration on the attachment of these cells onto polytetrafluoroethylene vascular grafts. Seeding efficiency was significantly better in all groups of coated grafts, at all times (10, 30, 60, and 120 minutes), compared to uncoated controls (p less than 0.01). Overall, fibronectin at a concentration of 20 micrograms/ml was found to be the most efficient in terms of cell attachment and cost since any further increase in concentration was not accompanied by increased cell attachment. We now routinely use fibronectin at a concentration of 20 micrograms/ml to coat our grafts before endothelial cell seeding.

Successful Reversal of Diabetes in Nude Rats by Transplantation of Isolated Adult Human Islets of Langerhans

A method is described in which the viability of isolated adult human islets of Langerhans can be assessed in vivo. The Rowett nude rat, made diabetic with streptozocin (STZ), has been used as the islet recipient in these studies. Although these animals are athymic and are able to accept xenogeneic grafts for prolonged periods, they are very susceptible to dehydration and infection oncemade diabetic. Therefore, a considerably shortened diabetes induction period was used. The basis of the study was to prepare pure adult human pancreatic islets that were cultured for 48 h. Nude rats were given 80 mg/kg i.v. STZ during islet isolation and were transplanted with 800–1000 islets under the renal capsule at 48 h. To monitor islet function, animals were bled regularly for random blood glucose measurements and were given a glucose tolerance test at day 20. The kidney containing the graft was removed on day 21 to allow histological assessment of the graft and to confirm that glucose control was due to the transplanted islets and was not secondary to reversion of the animals own islets. Seven rats were transplanted, and five were deemed to have received viable human islets. Two rats that received islets from the same donor did not reverse their diabetes and were found by histology to have vacuolated islet structures with scant insulin-staining tissue under the kidney capsule. This method allows a definitive judgment of the ability of isolated adult human islets to reverse diabetes.

Suppression of human T-cell responses to beta-cells by activation of B7-H4 pathway.

B7-H4, a recently described member of the B7 family of cosignal molecules, is thought to be involved in the regulation of cellular and humoral immune responses through receptors on activated T and B cells. Human islet cells express positive B7-H4 mRNA in RT-PCR assays, but not B7-H4 protein on cell surface in flow cytometric analyses. To investigate the regulatory effects of activation of the B7-H4 pathway on the function of activated T cells of patients with type 1 diabetes (T1D), we have used our in vitro human experimental system, including human β-cell antigen-specific T-cell clones and human β-cell lines CM and HP62, as well as primary islet cells. B7-H4.Ig protein was purified from the culture supernatant of 293T cells transfected by a B7-H4.Ig plasmid (pMIgV, containing a human B7-H4 cDNA and a mouse IgG2a Fc cDNA). Our preliminary studies showed that immobilized fusion protein human B7-H4.Ig (coated with 5 μg/ml for 2 h at 37°C), but not control Ig, clearly inhibited the proliferation of activated CD4+ and CD8+ T cells of patients induced by anti-CD3 antibody in CFSE assays. B7-H4.Ig also arrested cell cycle progression of T cells in G0/G1 phase and induced T-cell apoptosis as measured by BrdU-7-AAD flow cytometric analysis. To determine the cytoprotective effects of B7-H4, we developed transfectants of human β-cell lines CM and HP62 and islet cells transfected with the B7-H4.Ig plasmid, using empty vector transfectants as controls. The results demonstrate that cell-associated B7-H4.Ig expressed on human β-cells clearly inhibits the cytotoxicity of the T-cell clones to targeted human β-cells in 51Cr release cytotoxicity assays. Activation of the B7-H4 pathway may represent a novel immunotherapeutic approach to inhibit T-cell responses for the prevention of β-cell destruction in T1D.

Non-heart-beating Organ Donors: A Potential Source of Islets for Transplantation?1

BACKGROUND The shortage of organ donors relative to the number of patients on transplant waiting lists has led to a renewed interest in the use of non-heart-beating (NHB) organ donors in many centers. The lack of donors is also a problem for islet transplantation. The disparity between donor organs and potential recipients is further exacerbated by the requirement to transplant a large number of islets to increase the chance of success and the high level of variability in islet isolation yield. Non-heart-beating (NHB) donors have not previously been assessed as a source of islets for transplantation, and it is unknown what affects the additional factor of warm ischemic injury associated with NHB organs may have on the success of islet isolation. METHODS This study assesses the yield and function of islets from NHB donors and compares the results with islets obtained from heart-beating brain-dead (HB) donors. RESULTS There were no differences in the yield of islets per gram of pancreas, 1788 (0-4620) NHB vs. 1580 (26-2544) HB (median, range). The secretory function was also similar in both groups, with stimulation indices of 0.71-3.49 for NHB vs. 0.30-3.57 for HB (overall range). There was no correlation between islet yield and warm ischemia time in the NHB donor group. CONCLUSIONS In conclusion, the study has demonstrated that it is possible to isolate large numbers of islets from NHB donor pancreata and that, where NHB donor programs exist, these could provide a significant addition to the number of potentially transplantable islets.