Barbara Olack

Automated Method for Isolation of Human Pancreatic Islets

We describe an automated method for the isolation of human pancreatic islets. The procedure meets the following requirements: 1) minimal traumatic action on the islets, 2) continuous digestion in which the islets that are progressively liberated can be saved from further enzymatic action, 3) minimal human intervention in the digestion process, and 4) high yield and purity of the isolated islets. After purification on Ficoll gradients, an average of 164,600 islets/pancreas was obtained (2279 islets/g), with an average purity of 78.5% islets. The average volume and average insulin content of the final islet preparation were 348 mm3 and 93.4 U, respectively. The islets were morphologically intact with a normal degree of β-granulation and responded to glucose stimulation with a fivefold increase of insulin secretion over basal levels. The procedure is now being used for the initiation of the second phase of clinical trials on human islet transplants.

Protection of Encapsulated Human Islets Implanted Without Immunosuppression in Patients With Type I or Type II Diabetes and in Nondiabetic Control Subjects

Human islets were macroencapsulated in permselective hollow fiber membrane devices and successfully allotransplanted subcutaneously with > 90% viability after 2 weeks in situ. Recipients were patients with type I or type II diabetes and normal control subjects; none was immunosuppressed. Between 150 and 200 islet equivalents were implanted in each of the nine patients. No adverse patient complications were observed. Biocompatibility of devices was excellent. Insulin-positive β-cells were confirmed in encapsulated islets recovered from the implanted devices in all patient populations including the type I diabetic patients. Glucose-stimulated insulin release could be demonstrated in vitro from recovered islets. These data demonstrate that macroencapsulated human islets can survive at the subcutaneous site and that permselective membranes can be designed to protect against both allogeneic immune responses as well as the autoimmune component of type I diabetes.

The Effect of Pancreatic Islet Transplantation and Insulin Therapy on Experimental Diabetic Autonomic Neuropathy

Rats with chronic streptozotocin (SZ) diabetes develop dilatation of the alimentary tract, loss of fecal consistency, and autonomic neuropathy involving unmyelinated axons of the extrinsic innervation of the small bowel. Diabetic autonomic neuropathy involving the ileal mesenteric nerves is characterized by modest to marked dilatation of axons by distinctive subcellular organelles identical to those described in experimental and clinical axonal dystrophies. Axonopathy is confined to the alimentary tract; examination of myelinated and unmyelinated axons of the sciatic (midthigh level) and distal somatic nerves of the tail of diabetic animals with prominent ileal axonopathy failed to demonstrate significant numbers of dystrophic axons. The prevention or reversal of diabetic autonomic neuropathy by a variety of experimental manipulations clearly indicates that the lesions we have demonstrated in chronically SZ-induced diabetic animals were produced by diabetes and were not the result of a direct neurotoxic effect of the diabetogenic agent streptozotocin. Animals did not develop axonopathy after simultaneous administration of SZ and nicotinamide, a procedure which prevents pancreatic beta-cell necrosis and induction of diabetes while exposing the nervous system to a possible neurotoxic agent. Selected animals that were given SZ, became diabetic, and subsequently received daily insulin therapy or pancreatic islet transplantation also did not develop axonopathy. Transplantation of pancreatic islets 6 mo after induction of diabetes, a time at which mesenteric axonopathy was well developed, quickly reestablished normoglycemia, and within 3 mo resulted in nearly complete resolution of the neuropathy. Mild chronic diabetes maintained for 5–6 mo failed to produce significant levels of axonopathy. Diabetes-induced alteration in the gross appearance and weight of portions of the digestive tract and stool consistency were completely or partially prevented by institution of diabetic control.

A significant role for histocompatibility in human islet transplantation

Background. In recent years, transplantation of islets and pancreas has become a viable option for patients debilitated with type I diabetes. The success of islet transplantation has been attributed to the ability to isolate high quality islets for transplantation and capacity to maintain the recipients immunosuppressive levels within a specific target range following transplantation. The purpose of this study was to determine the role of pretransplant sensitization to human leukocyte antigen (HLA) in islet transplantation. Methods. We retrospectively analyzed seven patients that were transplanted with islets under the auspices of the Juvenile Diabetes Research Foundation and Islet Cell Resource Center/National Institutes of Health. Humoral sensitization towards donor antigens both prior to and following islet transplantation was detected by FLOW panel reactive antibodies (PRA) and donor-specific cellular sensitization was detected by performing enzyme-linked immunospot assay analysis for cytokines interferon-γ and interleukin-2. Results. Our analysis demonstrates that humoral and cellular sensitization to histocompatibility antigens prior to and after islet transplantation are associated with the failure of transplanted islets Conclusion. Patient selection based on sensitization to donor HLA may be one of the factors crucial for the success of islet transplant. Further, in some patients, rejection of islets can be associated with sensitization to mismatched donor histocompatibility antigens.

Improved method for the isolation and purification of human islets of Langerhans using Liberase™ enzyme blend

To determine the effects of procedural modifications, 23 human islet isolations were analyzed. Isolations were divided into two groups based on the enzyme used. The influence of Liberase, with an improved method of mechanical disassociation of pancreas, was compared to an automated method using Sevac collagenase. Pancreases were processed within 10 h of cross clamping. Following ductal injection of the enzyme, tissue was placed in the digestion chamber for disassociation. Purification was accomplished using a COBE 2991 cell processor and continuous gradients of 1Hypaque EuroFicoll. Isolations in Group I (Sevac) had an average yield of 138,602 +/- 128,364 islet equivalents (IE) (2083 +/- 1679 IE/g) with a purity of 85 +/- 11%. Group II (Liberase) showed an average yield of 389,586 +/- 191,161 IE (5,958 +/- 3,083 IE/g) with a purity of 90 +/- 6.8%. Viability was confirmed by fluorescein diacetate and propidium iodide staining, static incubations, and perifusions. In conclusion, the combination of the enzyme blend, Liberase, and a more gentle system of disassociation has proven to be a more productive method of islet isolation with higher purity than the previously published methods.

Pulsatile Insulin Secretion from Isolated Human Pancreatic Islets

Insulin secretion from the pancreas is pulsatile. The precise site and function of the pacemaker that regulates insulin periodicity in humans have not been determined. We isolated human pancreatic islets from five cadaver organ donors by collagenase digestion and density gradient purification. After 24 h of culture in CMRL-1066 medium at 37°C, aliquots of 200 islets were perifused (1 ml/min for 120 min) with glucose and other secretagogues in oxygenated Krebs-Ringer bicarbonate solution at 37°C. Samples for insulin measurement were taken every minute, and insulin secretion was analyzed by the Clifton and Steiner cycle detection technique. With 3.3 mM glucose (n = 17), insulin oscillations were demonstrated with a periodicity of 9.8 ± 0.1 min (means ± SE), mean amplitude was 16.8 ± 1.8 pM, and overall mean insulin release was 43.8 ± 4.2 pM. With 16.7 mM glucose (n = 14), no change of insulin periodicity was observed (10.2 ± 0.9 min), mean amplitude was 41.4 ± 10.2 pM (P < 0.01 vs. 3.3 mM glucose), and mean insulin release was 118.2 ± 19.2 pM (P < 0.01 vs. 3.3 mM glucose). Both at 3.3 and 16.7 mM glucose, the addition of 1.4 mM glucagon (n = 4), 15 mM arginine (n = 4), or 100 µg/ml tolbutamide (n = 4) caused no change of insulin periodicity but enhanced mean amplitude and mean insulin release compared with glucose alone. These results show that a pacemaker is located within the islets that regulates pulsatile insulin secretion in humans; the pacemaker is remarkably stable, because its periodicity is not affected by factors altering insulin secretion.

Sensitization to HLA antigens in islet recipients with failing transplants

A QUESTION that has plagued islet transplantation since its inception is whether islet failure is the result of exhaustion or rejection. Attempts have been made to immunomodulate islet preparations prior to transplantation’ as well as improve immunosuppression’,’ to lessen the risk of rejection. However, clinical islet transplantation has not achieved the desired success rate and the cause of failure still remains unanswered. We have retrospectively analyzed seven of our islet recipients for anti-donor HLA sensitization. Our data suggest that there is a strong correlation between islet allograft failure, documented by a decrease in Sustacal Stimulation Indices (SSI), and a rise in anti-donor HLA sensitization as detected by panel reactive antibody (PRA) testing. Furthermore, the data imply that donor-specific T cells are present in these patients and may be responsible for loss of islet function.

HLA antibodies present in the sera of sensitized patients awaiting renal transplant are also reactive to swine leukocyte antigens.

BACKGROUND To determine whether preformed HLA alloantibodies present in the sera of patients awaiting kidney transplantation will be detrimental to a potential porcine xenograft, we tested their cross-reactivity to swine leukocyte antigens (SLA). METHODS Sera obtained from patients with varying levels of HLA sensitization (high panel-reactive antibodies > 70%, n= 7; moderate panel-reactive antibodies 30-40%, n=2) were analyzed. Pooled normal human AB sera and sera from nonsensitized patients (n=3) served as negative control. IgG was purified by protein-G chromatography, and xenoreactive natural antibodies (XNA) were depleted by passing the IgG through a series of melibiose and thyroglobulin-agarose columns. The elimination of XNA from HLA IgG preparations was confirmed by GS-IB4 lectin blocking assay and by an ELISA. RESULTS IgG isolated from normal AB serum and three nonsensitized patients, which was depleted of XNA (HLA-IgG), did not react to human or porcine lymphocytes (peripheral blood mononuclear cells; PBMC) either by flow cytometry or by complement-dependent microcytotoxicity assays. However, HLA-IgG isolated from nine sensitized patients were reactive to a panel of porcine peripheral blood lymphocytes (n=6) by flow cytometry (>50 mean channel shift) and in complement-dependent microcytotoxicity assays in addition to their reactivity to human PBMC. The binding of HLA-IgG to porcine PBMC was significantly reduced by preabsorption with pooled human platelet concentrate. Further, the HLA IgG showed recognition of 45-kDa affinity-purified SLA class I on Western blots. CONCLUSIONS This study demonstrates that HLA antibodies present in the sera of sensitized individuals can cross-react with SLA. Thus, xenotransplantation of porcine organs into HLA-sensitized patients has the potential to be rejected by humoral mechanisms. Testing to avoid such cross-reactive antibodies should be considered.

Improved Islet Yields from Pancreas Preserved in Perflurocarbon Is Via Inhibition of Apoptosis Mediated by Mitochondrial Pathway

Islet transplantation is a treatment option for type I diabetic patients. Preservation of human pancreata prior to islet isolation using two‐layer method with perfluorocarbon (PFC) and University of Wisconsin solution (UW) results in twofold increase in islet yields. The objective of this study was to determine the mechanism by which islets undergo apoptosis and determine PFCs effects on this process. Gene array analysis was used to analyze the expression of pro‐ and anti‐apoptotic genes in islets isolated from pancreata preserved under varying conditions. A 12‐fold increase in the expression of inhibitor of apoptosis (IAP) and survivin was observed in islets isolated from pancreata preserved in PFC. This was accompanied by decreased expression of BAD (3.7‐fold), BAX (2.7‐fold) and caspases (5.2‐fold). Levels of activated caspase‐9 (77.98%), caspase‐2 (61.5%), caspase‐3 (68.3%) and caspase‐8 (37.2%) were also reduced. ‘Rescue’ of pancreata after storage (12 h) in UW by preservation using PFC also resulted in a down‐regulation of pro‐apoptotic genes and inhibition of caspase activation. Apoptosis observed in islets from all groups was mainly mitochondria‐dependent, mediated by change in redox potential initiated by hypoxia. We demonstrate that reduction in hypoxia of pancreata preserved using PFC leads to significant up‐regulation of anti‐apoptotic and inhibition of pro‐apoptotic genes.

Prolonged survival of discordant porcine islet xenografts

Purified porcine islets were prepared by collagenase digestion and density gradient purification, and transplanted under the kidney capsule of C57B/B6 mice with streptozotocin-induced diabetes which were receiving varying temporary immunosuppressive therapies. Islets that had been cultured for 1 day at 37 degree C were rejected after : 9+/-0.1 (mean+/-SE) days in control mice: 14+/-3 days in mice receiving mouse antilymphocyte serum (MLS) plus porcine antilymphocyte serum (PLS) on day of transplant (day 0); 43+/-6 days in mice treated for 1 week with anti-CD4 antibody (aCD4); 36+/-4 days in mice given aCD4 for 1 week plus PLS on days 0 and 7; 47+/-3 days in mice treated with aCD4 for 1 week plus MLS and PLS on day 21. Porcine islet survival in these latter three groups was significantly (P<0.01) and similarly longer than in the control and MLS plus PLS groups. Then, we transplanted islets that had been either cultured at 24 degrees C for 7 days or cryopreserved into 7-day aCD4-treated mice, to evaluate whether low temperature culture or the freezing-thawing procedure could affect survival. Neither 7-day, low temperature culture (mean survival time: 37+/-2 days) nor cryopreservation (mean survival time: 39+/-2 days) prolonged islets function further. Thus, the present study demonstrates that prolonged survival can be achieved with discordant porcine islet xenografts, and shows the greater efficacy of aCD4 treatment, which was not improved by additional immunosuppressive therapies we tested, nor by culture or cryopreservation of the islets.