Islet isolation from human pancreas with extended cold ischemia time.

The general consensus among transplant centers is that a cold ischemia time (CIT) beyond 8 hours results in reduced yields and quality of human islets. We sought to optimize the isolation process and enzymes for pancreata with extended CIT. We processed 16 extended CIT pancreata (13.2 +/- 0.7 hours). Donors averaged 50.8 +/- 2.6 (standard error of the mean) years old with a body mass index of 28.6 +/- 1.5. Glands were shipped in cold organ preservation solution without oxygenated perfluorocarbon. Isolations were performed under a protocol optimized for digestion with the new cGMP collagenase from Roche. Purification used continuous Euroficoll/University of Wisconsin gradients. Islets were cultured in two types of Prodo cGMP islet culture media and/or in Miami 1A media. Glucose-stimulated insulin secretion assays were performed after 8 to 16 days of culture. Prepurification yield averaged 415 +/- 41 KIEQ postpurification, 359 +/- 29 KIEQ (purification loss 13.5%); and postculture 317 +/- 27 KIEQ (culture loss 11.7%). Our process liberated an average of 4278 IEQ/g of pancreas (97 +/- 5 g). Most islets were recovered in the purest fraction (purity 79.7% +/- 1.9%). Culture loss in our enhanced culture media was 11.7%. After 2 to 3 days in culture, viability was 92% +/- 1%. Islets exhibited compactness and dithizone staining. Glucose-stimulated insulin secretion assays performed after 3 to 23 days in our PIM(R) media resulted in a stimulation index of 6.8 +/- 1.7 (G50 to G350). We concluded that our human islet isolation process permitted the recovery of large numbers of high-quality human islets from extended CIT pancreata and that our cGMP islet culture media was superior to the current standard CMRL-based media.

In vitro and in vivo performance of porcine islets encapsulated in interfacially photopolymerized poly(ethylene glycol) diacrylate membranes.

The usefulness of interfacial photopolymerization of poly(ethylene glycol) (PEG) diacrylate at a variety of concentrations and molecular weights to form hydrogel membranes for encapsulating porcine islets of Langerhans was investigated. The results from this study show in vitro and in vivo function of PEG-encapsulated porcine islets and the ability of PEG membranes to prevent immune rejection in a discordant xenograft model. Encapsulated islets demonstrated an average viability of 85% during the first week after encapsulation, slightly but significantly lower than unencapsulated controls. Encapsulated porcine islets were shown to be glucose responsive using static glucose stimulation and perifusion assays. Higher rates of insulin release were observed for porcine islets encapsulated in lower concentrations of PEG diacrylate (10-13%), not significantly reduced relative to unencapsulated controls, than were observed in islets encapsulated in higher concentrations (25%) of PEG diacrylate. Perifusion results showed biphasic insulin release from encapsulated islets in response to glucose stimulation. Streptozotocin-induced diabetic athymic mice maintained normoglycemia for up to 110 days after the implantation of 5,000-8,000 encapsulated porcine islet equivalents into the peritoneal cavity. Normoglycemia was also confirmed in these animals using glucose tolerance tests. PEG diacrylate-encapsulated porcine islets were shown to be viable and contain insulin after 30 days in the peritoneal cavity of Sprague-Dawley rats, a discordant xenograft model. From these studies, we conclude that PEG diacrylate encapsulation of porcine islets by interfacial photopolymerization shows promise for use as a method of xenoprotection toward a bioartificial endocrine pancreas.

Use of mass vaccination with a reduced dose of REV 1 vaccine for Brucella melitensis control in a population of small ruminants.

Mass vaccination with reduced dose 1/50 Rev 1 strain live vaccine (1-2 10(9) colony forming units), administered subcutaneously, over a four and a half year period reduced the prevalence of Brucella melitensis in Kuwait's small ruminant population from 5.8% in 1993 to 2.02% in 1997. Serological test results using the Rose Bengal Plate Test, Rivanol Agglutination Test and Complement Fixation showed no evidence of persistence of positive serology in animals nine or more months after vaccination. Questionnaires and post-vaccination flock inspections found that the effects on gestation (abortions) were minimal--and not proven to be due to the vaccine. The conclusion from these findings is that mass vaccination with reduced dose Rev 1 administered by the subcutaneous route is a practical field strategy for control of Brucella melitensis.

Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels.

Hydrogel membranes formed by interfacially photopolymerizing poly(ethylene glycol) (PEG) diacrylate precursor solution were prepared from PEG diacrylate of molecular weights (MW) ranging from 2000 (2K) to 20000 (20K) with concentrations ranging from 10% to 30% w/w. The effects of PEG diacrylate MW and concentration in the membrane precursor solution upon the diffusivities of vitamin B12, myoglobin, ovalbumin, albumin, and IgG were determined. Regardless of the concentration of the PEG diacrylate in the precursor solution, hydrogels prepared with PEG 2K, 4K, and 8K diacrylate were impermeable to proteins with a size equal to or larger than myoglobin (22 kDa), while hydrogels prepared with PEG 20K diacrylate were impermeable to proteins with a size equal to or larger than ovalbumin (45 kDa). Similarities between hydrogels formed from PEG 2K, 4K, and 8K diacrylates were also seen in calculations of the molecular weight between crosslinks and the mesh size, with values in the range of 150-750 g/mol and 15-35 A, respectively, depending on PEG diacrylate concentration. In contrast, hydrogels formed from PEG 20K diacrylate had molecular weight between crosslinks ranging from 1150 to 2000 g/mol and mesh sizes ranging from 45-70 A, with larger values being observed in membranes polymerized from more dilute PEG diacrylate precursor.

A sensitivity study of the key parameters in the interfacial photopolymerization of poly(ethylene glycol) diacrylate upon porcine islets.

A method has been defined to interfacially photopolymerize poly(ethylene glycol) diacrylates (PEG diacrylates) to form a crosslinked hydrogel membrane upon the surfaces of porcine islets of Langerhans to serve as an immune barrier for allo- and xenotransplantation. A sensitivity study of six key parameters in the interfacial photopolymerization process was performed to aid in determination of the optimal encapsulation conditions, leading to the most uniform hydrogel membranes and viable islets. The key parameters included the concentrations of the components of the initiation scheme, namely eosin Y, triethanolamine, and 1-vinyl 2-pyrrolidinone. Other parameters investigated included the duration and flux of laser irradiation and the PEG diacrylate molecular weight. Each parameter was doubled and halved from the standard conditions used in the encapsulation process while holding all the remaining parameters at the standard conditions. The effects of changing each parameter on islet viability, encapsulation efficiency, and gel thickness were quantified. Islet viability was sensitive to the duration of laser illumination, viability significantly increasing as the duration was reduced. Encapsulation efficiency was sensitive to the concentrations of eosin Y, triethanolamine, and 1-vinyl 2-pyrrolidinone, to the laser flux, and to the PEG diacrylate molecular weight. Increasing the concentration of eosin Y significantly improved the encapsulation efficiency, while decreasing the concentration of 1-vinyl 2-pyrrolidinone and increasing the concentration of triethanolamine had the greatest effects in significantly reducing the encapsulation efficiency. Gel thickness was sensitive to the concentrations of triethanolamine and 1-vinyl 2-pyrrolidinone, to the duration of laser illumination, and to the PEG diacrylate molecular weight. Increasing the PEG diacrylate molecular weight significantly increased the gel thickness, while decreasing the concentration of 1-vinyl 2-pyrrolidinone and increasing the concentration of triethanolamine had the greatest effects in significantly reducing the gel thickness. From this sensitivity study, conditions were determined to encapsulate porcine islets, resulting in greater than 90% islet viability and greater than 90% encapsulation efficiency.

Immunoisolation of adult porcine islets for the treatment of diabetes mellitus. The use of photopolymerizable polyethylene glycol in the conformal coating of mass-isolated porcine islets.

Functional porcine islets, free of known pathogens, can serve as a source of insulin producing cells for the treatment of experimentally induced insulin dependent Diabetes Mellitus. Porcine islets can be conformally coated (microencapsulated) with a covalently linked, stable permselective membrane while maintaining islet viability and function. The PEG conformal coating is immunoprotective in a discordant xenograft animal model (porcine islets to rat).

Human peripheral blood lymphocyte-reconstituted, severe combined immunodeficient mice as a model for porcine islet xenograft rejection in humans.

Previously we established human peripheral blood lymphocyte-reconstituted severe combined immunodeficiency (SCID) (hu-PBL-SCID) mice as a model for human islet allograft rejection. The function of xenografted hu-PBL was confirmed to reject human alloislets in hu-PBL-SCID mice. In this study, we modified this model as a porcine islet xenograft to study porcine islet rejection in humans. Chimeric mice were used as the recipients of porcine islets to reveal the mechanisms of xenograft rejection in humans. SCID mice were reconstituted with 30 x 10(6) of hu-PBL initially, and 10 x 10(6) of antihuman CD3-primed PBL was injected intraperitoneally 2 days later as a booster. An additional booster injection provided greater possibility (86.7%, n = 15) of chimera establishment as well as a higher human immunoglobulin concentration in SCID mice than the single injection group. In an in vitro assay, sera from hu-PBL-SCID mice were found to recognize porcine islets by FACS staining. In an in vivo study, immunofluorescent analysis of a frozen section showed that human immunoglobulins adhered to the xenografted porcine islet under the kidney capsule of hu-PBL-SCID mice. Although no mouse immunoglobulins were detected on sections, mouse complement (C3) was shown to adhere to the xenografted porcine islet. Thus, hu-PBL-SCID mice provide a useful model for investigating the real-life situation of porcine islet xenograft rejection in humans.

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.

Continued insulin dependence despite normal range insulin sensitivity and insulin connecting peptide levels in a kidney/islet transplant patient.

OBJECTIVE: The majority of islet transplant recipients remain insulin-requiring, although many have near-normal connecting peptide (CP) levels. Insulin resistance may be one possible cause of the continuing need for exogenous insulin in islet transplant recipients. To assess this, we have studied the insulin sensitivity index (S1) in one patient with near-normal CP levels after islet transplant who remained insulin-requiring. RESEARCH DESIGN AND METHODS: The islet transplant recipient is a 36-year-old woman with no residual CP who received a kidney transplant, followed 7 days later by an islet transplant. The islets were infused into the liver via the umbilical vein. Induction immunosuppression consisted of OKT3, prednisone, cyclosporin A, and azathioprine, with maintenance on the latter three. RESULTS: Maximum CP levels after a standardized Sustacal meal were 2.09, 1.18, 0.85, and 0.81 nmol/l at 1,6,18, and 24 months posttransplant, respectively. Insulin requirements at the same times were 0.27, 0.45, 0.49, and 0.62 U.kg(-1).d(-1), while S1 was 36.3, 53.3, and 13.2 min (-1).nmol(-1).ml at 6,18, and 24 months, respectively. This compares with S1 values of 43.3+/- 10.0 min (-1).nmol(-1).ml for normal subjects. CONCLUSIONS: This patient had near-normal S1 and CP levels, but she was unable to discontinue insulin therapy, suggesting that other factors are critical. Despite this, she maintained normal or near-normal glycated hemoglobins, indicating metabolic benefit from the islet transplant.

Albumin improves islet isolation: specific versus nonspecific effects.

1. BSA-containing solutions improve islet yields using standard collagenase digestion techniques. 2. The BSA effect on islet isolation is independent of source and lot of collagenase. 3. The BSA effect on islet isolation is not due solely to its colloid action, as HES failed to achieve the same level of improvement seen with albumin. 4. BSA can protect islets from warm ischemic injury, and the protective action appears to be unique to albumin, as HES was not as effective.

Insulin inhibits its own secretion from isolated, perifused human pancreatic islets.

It is still a controversial question whether insulin suppresses its own secretion. We prepared pure human islets from three pancreases by collagenase digestion and density gradient purification. Aliquots of 200 islet equivalents (IE, 150-microns sized-islets) were sequentially perifused at 37 degrees C with 3.3 mmol/l glucose (3.3G, 40 min), 16.7 mmol/l glucose (16.7G, 30 min) and again 3.3G (30 min) after 24 h, 37 degrees C culture in CMRL 1066 medium with or without the addition of either 200 or 400 microU/ml human insulin in the incubation medium (6 replicates each). Insulin secretion was assessed by C-peptide (Cp) measurement in the perifusate. Without added insulin (C) and with 200 (Ins200) or 400 (Ins400) microU/ml added insulin, basal Cp release was 0.12 +/- 0.03, 0.14 +/- 0.02 and 0.14 +/- 0.04 ng/ml, respectively. At 16.7G, the first-phase secretion peak (expressed as Cp value) was significantly lower with Ins200 (0.47 +/- 0.13 ng/ml, P < 0.02) and Ins400 (0.68 +/- 0.15 ng/ml, P < 0.05) than C (0.83 +/- 0.15 ng/ml). The second-phase secretion peak was also significantly (P < 0.05) reduced with added insulin (Ins200: 0.47 +/- 0.08 ng/ml; Ins400: 0.45 +/- 0.07 ng/ml) than in its absence (C: 0.65 +/- 0.09 ng/ml). Accordingly, total Cp secretion was lower with Ins200 (10.6 +/- 2.3 ng/ml, P = 0.03) and Ins400 (11.8 +/- 2.3 ng/ml) than with C (16.0 +/- 2.2 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

A selective decrease in the beta cell mass of human islets transplanted into diabetic nude mice.

Streptozocin-induced diabetic nude mice (blood glucose 493 +/- 14 mg/dl) received aliquots of 2000 human islet equivalents (IE) under the kidney capsule and were then followed for up to 30 days with measurement of blood glucose concentration and body weight. Characterization of islet aliquots before the implantation included the assessment of the endocrine beta cell and nonbeta cell mass, estimated by point counting morphometry of immunostained sections. Islet transplantation was followed by a rapid decrease in blood glucose levels and by a progressive increase in body weight; 15 days after transplantation mean glycemic levels were 102 +/- 11 mg/dl and further decreased to 70 +/- 3 mg/dl at 30 days. Despite the progressive improvement in the glucose levels, the beta cell mass of the islet grafts significantly decreased over time from 2.63 +/- 0.2 mg, at the time of transplantation, to 1.16 +/- 0.1 and 0.86 +/- 0.1 mg 15 and 30 days, respectively, after transplantation. In contrast, the endocrine nonbeta cell mass remained stable from before the implantation to 30 days after. Therefore, the endocrine nonbeta cell/beta cell ratio increased from 14% at the time of transplantation, to 35% and 37%, 15 and 30 days, respectively, after transplantation. The rate of replication of the transplanted beta cells was similar in the grafts harvested at 15 and 30 days, with the percentage of beta cells positive for bromo-2' deoxyuridine (BrdU) incorporation being in the range of approximately 0.1% 6 hr after the BrdU injection. These data demonstrate that an important decrease in beta cell mass takes place immediately after islet transplantation--the most dramatic decrease occurring in the first 15 days and persisting even after revascularization has occurred. However, endocrine nonbeta cell mass remained stable indicating that engrafted nonbeta cells are less likely to die than beta cells. The finding that the nonbeta/beta cell ratio of a human islet graft can increase over time, raises questions about whether such a change in islet structure could have an influence upon function.

Successful long-term cryopreservation of highly purified canine islets.

This study evaluated the ability of cryopreservation to store large quantities of canine islets for transplantation studies. Islets were isolated by automated screen methods and purified by Euro-Ficoll gradients. After overnight 37 degrees C incubation, islets were equilibrated with 2 M dimethylsulfoxide, cryopreserved at a cooling rate of 0.25 degree C/min and subjected to long-term storage in liquid nitrogen. Six months later, some islets were thawed at a rate of 180 degrees C/min. The viability of cryothawed islets was determined in vitro by comparing islet count, total insulin content, morphology and perifusion studies in both control and cryothawed islets, and in vivo by transplantation into diabetic nude mice. The percentages of recovery of both islet count and total insulin content were 74.17 +/- 4.46 and 70.66 +/- 14.08, respectively. Islet morphology after cryothaw by light and electron microscopy revealed structurally intact islets with varying degrees of granulation. The results of perifusion indicated that there was no significant difference (p > 0.1) in both total stimulated insulin secretion and stimulation index response to high-dose glucose plus 3-isobutyl-1-methylxanthine and carbachol of isolated islets as compared to those of cryothawed islets. Transplantation into nude mice proved that grafted islets can successfully reverse diabetes. In conclusion, these findings indicate that the majority of purified canine islets can survive the frozen-thawed insult while maintaining their secretory function and permitting mass storage of canine islets for further transplant studies.