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O D Hegre

Publications and source records attributed to O D Hegre.

At least 19 recordsLinked to original sources
PubMed1998

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.

Animals↗
PubMed1997

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).

Animals↗
PubMed1994

Clonal insulinoma cell line that stably maintains correct glucose responsiveness.

A number of pancreatic beta-tumor cell (beta TC) lines have been derived from insulinomas arising in transgenic mice expressing the SV40 T antigen gene under control of the insulin promoter. Some of these lines secrete insulin in response to physiological glucose concentrations. However, this phenotype is unstable. After propagation in culture, these nonclonal lines become responsive to subphysiological glucose levels and/or manifest reduced insulin release. Here we report the use of soft-agar cloning to isolate single-cell clones from a beta TC line, which give rise to sublines that maintain correct glucose responsiveness and high insulin production and secretion for > 55 passages (over a year) in culture. One of these clonal lines, denoted beta TC6-F7, was characterized in detail. beta TC6-F7 cells expressed high glucokinase and low hexokinase activity, similarly to normal islets. In addition, they expressed mRNA for the GLUT2 glucose transporter isotype and no detectable GLUT1 mRNA, as is characteristic of normal beta-cells. These results demonstrate that transformed beta-cells can maintain a highly differentiated phenotype during prolonged propagation in culture, which has implications for the development of continuous beta-cell lines for transplantation therapy of diabetes.

Animals↗
PubMed1994

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 beta-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.

Biocompatible Materials↗
PubMed1993

Dendritic cell/lymphocyte clustering: morphologic analysis by transmission electron microscopy and distribution of gold-labeled MHC class II antigens by high-resolution scanning electron microscopy.

Dendritic cells (DCs) are potent antigen-presenting cells for a variety of immune responses; however, their mechanism of action has not been established. It is known that DCs can cluster with one another and with other cell types during in vitro immune responses, and clustering may be essential for the activation of resting lymphocytes. In this study, ultrastructural examination of clusters that form during extended culture of enriched rat splenic DCs (approximately 70% DCs) is reported. DCs were readily distinguished from other cell types, which included lymphocytes and macrophages. DCs displayed characteristic veils and/or dendritic processes that intertwined with processes of other cells within the cluster, or extended from the cluster periphery. Occasional DCs contained large vacuoles lined with small vesicles. A paramount feature of DCs is their constitutive expression of high levels of surface major histocompatibility complex class II antigens. The surface distribution of class II antigens on clustering DCs was examined using 10 nm immunogold labeling techniques and high-resolution scanning electron microscopy. DCs were readily distinguished by morphologic criteria, and examination of various surface membrane regions revealed a differential distribution of class II antigens. Gold label was frequently distributed in linear arrays and clusters, suggesting a cytoskeletal role in the recycling/redistribution of Class II antigens. These morphologic findings further an understanding of basic DC biology and their mechanism of action as antigen-presenting cells.

Animals↗
PubMed1993

Long-term survival and strain-specific tolerance induction in rat-to-mouse neonatal islet xenografts.

These studies were designed to determine (1) if culture-isolated, neonatal rat islets are capable of inducing xenogeneic tolerance in mice and (2) whether this tolerance is species- or strain-specific. We attempted to induce xenogeneic tolerance by transplanting culture-isolated neonatal FSH islets to 26 diabetic C57B1/6 recipients. These animals received one injection of ALS at the time of transplant. Fifteen (58%) animals remained reversed by xenotransplant for > 173 days. To assess the development of strain or species-specific tolerance, 14 of the animals bearing long-term surviving FSH grafts were divided into 3 treatment groups. Animals in group 1 were nephrectomized to remove the initial graft and then retransplanted with uncultured, adult FSH islets; animals in group 2 were retransplanted with uncultured, adult FSH islets without nephrectomy; and group 3 animals were nephrectomized and retransplanted with uncultured, adult third-party islets (WF). In naive controls, adult FSH islets were rejected in 9 +/- 2 days. The MST for adult FSH grafts transplanted to nephrectomized recipients was 104 +/- 54 days, with 4 out of 5 (80%) surviving until sacrifice 90-171 days posttransplant. The MST for FSH grafts transplanted to nonnephrectomized recipients was 120 +/- 70 days with 3 out of 4 (75%) surviving until sacrifice 143-154 days posttransplant. Thus, it appears that the initial neonatal FSH transplant induced the development of immune tolerance to highly immunogenic FSH islet tissue. In contrast, the MST for third-party adult WF grafts was 27 +/- 13 days compared with an MST of 36 +/- 24 days in naive controls. Thus, it appears that the xenogeneic tolerance induced by neonatal FSH islets was strain rather than species-specific. Factors such as the close evolutionary relationship between rats and mice, the neonatal condition of the initial graft, and its relative lack of donor APCs are included in a discussion of possible mechanisms of tolerance induction.

Animals↗
PubMed1992

Increased islet allograft survival after extended culture by a mechanism other than depletion of donor APCs. Lack of correlation between the elimination of donor MHC class II-positive accessory cells and increased transplantability.

Neonatal rat islets derived by nonenzymic (in vitro) isolation procedures from the Fischer-344 (F-344, Rt1lv1) strain have been shown to be freely transplantable across complete MHC barriers without the use of any form of immunosuppression. However, islets obtained by in vitro isolation from some donor strains, such as the ACI, retain a degree of immunogenicity upon transplantation. This study examined the immunogenic nature of these neonatal ACI islets, and correlated this with the presence of a residual population of MHC class II-positive antigen presenting cells within the islets. In addition, further reduction of immunogenicity of isolated neonatal ACI islets by extension of the culture period was examined. Islets were obtained from neonatal ACI donors by culture-isolation and placed in secondary culture for either 2 days (10 day total culture period, standard culture) or 42 days (50 day total culture period, extended culture). Standard-culture ACI islets were rejected 33% to 100% of the time in five different recipient strains (depending on recipient strain). Compared with control grafts of fresh pancreatic fragments, rejection rates were significantly lower in two strain combinations (ACI to BUF, P = 0.044 and ACI to F-344, P = 0.028), and for all recipient strains combined (P less than 0.001). Extended-culture ACI islets survived significantly longer in all five recipient strains tested (ACI to BN, P = 0.024; ACI to BUF, P = 0.005; ACI to LEW, P = 0.004; ACI to F-344, P = 0.008 and ACI to WF, P = 0.024) and for all strains combined (P less than 0.001). No MHC class II-positive cells were detected upon examination of sectioned neonatal ACI islets using peroxidase immunocytochemistry (OX-6 antigen) in either the standard-culture or extended-culture groups. These results suggest that the increased survival of grafts of extended culture ACI islets is due to a mechanism other than the elimination of class II-positive APCs from the islets. Although possible down-regulation of MHC class II expression on residual APCs during the culture process may make identification of these cells difficult, alternative mechanisms such as the down-regulation of MHC class I antigen, resulting in the reduction of available donor target, or the existence of an accessory cell that is not constitutively MHC class II-positive, may be responsible for this increased graft survival.

Animals↗
PubMed1992

Growth of neonatal islet transplants in the spontaneously diabetic BB/Wor rat.

We have previously shown that culture-isolated neonatal islets are able to survive both rejection and the recurrence of autoimmunity in the spontaneously diabetic BB/Wor rat. In trials designed to demonstrate the MHC restriction of the autoimmune response in this model, we discovered that neonatal islet grafts from diabetic BB rats appeared larger than grafts from nondiabetic controls. This study was undertaken to quantify the mass difference seen in this original study and to determine the characteristics of graft growth in more highly controlled trials. Grafts from diabetic animals in the original study were significantly larger than those from nondiabetic animals (81 +/- 36 vs. 238 +/- 216 micrograms, P = 0.01). These findings were supported by results from a second series of experiments, in which the mean growth index of grafts from diabetic animals was 7.25 +/- 4.91, whereas that from nondiabetic animals was 2.5 +/- 1.15 (P = 0.011). Three animals in this study were reversed of hyperglycemia: two had normal and one had a subdiabetic ip GTTs. These three rats received 97, 317, and 408 micrograms of islet tissue that increased in mass to 1790, 3270, and 4107 micrograms, respectively. Nuclear/total cell area percentages were the same in diabetic and nondiabetic grafts (P = 0.76), suggesting that the increase in mass was attributable primarily to proliferation rather than hypertrophy. Limited studies that use BrDU incorporation support this conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Maintenance of normoglycemia in diabetic mice by subcutaneous xenografts of encapsulated islets.

The goal of islet transplantation in human diabetes is to maintain the islet grafts in the recipients without the use of immunosuppression. One approach is to encapsulate the donor islets in permselective membranes. Hollow fibers fabricated from an acrylic copolymer were used to encapsulate small numbers of rat islets that were immobilized in an alginate hydrogel for transplantation in diabetic mice. The fibers were biocompatible, prevented rejection, and maintained normoglycemia when transplanted intraperitoneally; hyperglycemia returned when the fibers were removed at 60 days. Normoglycemia was also maintained by subcutaneous implants that had an appropriately constructed outer surface on the fibers.

Acrylic Resins↗
PubMed1991

Comparative analysis of potency of splenic dendritic and adherent cells (macrophages) as alloantigen presenters in vivo.

Dendritic cells and macrophages have been attributed with stimulatory capacity for in vivo and in vitro immune responses. However, the relative contribution of each of these cell types has long been in dispute. Therefore, the differential ability of dendritic cells and macrophages (splenic adherent cells [SACs]) to stimulate pancreatic islet allograft rejection in reversed alloxan-induced diabetic rats was examined. Rats bearing established allografts were challenged with various dosages of donor-strain dendritic cells or SACs, and graft rejection was assessed by analysis of plasma glucose levels and/or histological criteria. Marked differences in the ability to stimulate allograft rejection were observed at the 10(5)-cell dosage; 10(5) dendritic cells induced graft rejection in five of six rats (1 rat required 2 injections), whereas 10(5) SACs failed to induce rejection in four of four rats (P less than 0.10, chi 2 test). Challenge stimuli consisting of less than or equal to 10(5) SACs or less than or equal to 10(4) dendritic cells failed to induce graft rejection. These findings indicate that dendritic cells are potent stimulator cells for in vivo immune responses. Previous studies indicated that as few as 10(3) dendritic cells initiate allograft rejection in nondiabetic recipients. That more dendritic cells were required to stimulate rejection in reversed diabetic recipients compared with nondiabetic recipients suggests that other factors, such as the diabetic state and the production of a tolerant status achieved by larger amounts of grafted tissue, may influence graft survival.

Animals↗
PubMed1990

Cultured neonatal islet transplants in alloxan-treated and spontaneously diabetic rats.

Neonatal Fischer-344 (FSH) islets isolated by a nonenzymatic method have been shown to survive indefinitely in Wistar/Furth (WF) recipients. We have applied this islet isolation method to six different donor strains and transplanted the resulting islets across 20 different strain combinations. We report the variable results obtained, with FSH being the most consistently successful donor strain and ACI being the best recipient strain. Based on the complete success of culture-derived FSH (Rt1lv1) and BN (Rt1n) transplants to WF (Rt1u) recipients, we used this system to test the MHC-restriction of autoimmune beta cell destruction in the BB/Wor rat (Rt1u). Culture-derived FSH and/or WF islets were transplanted to pre-diabetic BB/Wor recipients. Animals were sacrificed at the onset of disease or up to 33 days after disease onset. No immune response developed in FSH and WF grafts in non-diabetic animals. In diabetic animals, all FSH grafts and 12/14 WF grafts survived intact, although some grafts exhibited mild-moderate infiltration which was more pronounced in MHC-matched grafts. In two animals which developed severe hyperglycemia and ketosis, the FSH grafts were found to be intact while the WF grafts were destroyed by disease recurrence. In addition, FSH and BN islets were transplanted to diabetic BB/Wor recipients. Three of four FSH islet recipients and two of four BN islet recipients were reversed of their disease. Neither these nor the non-reversed animals showed signs of disease recurrence in the MHC-mismatched grafts. Therefore, in these studies, MHC-mismatched cultured grafts survived well and were capable of reversing the diabetic syndrome in spontaneously diabetic BB/Wor recipients.

Animals↗
PubMed1990

Growth of neonatal islet graft following transplantation to the BB/Wor rat.

Culture-derived neonatal Fischer-344 (Rtllvl) rat islets have reduced immunogenicity and have been shown to be fully transplantable into Wistar Furth (Rtlu) recipients. In studies designed to test the MHC-restriction of the autoimmune disease process in the BB/Wor rat, MHC-matched (Wistar Furth) and MHC-mismatched (Fischer-344) neonatal islets were isolated by a nonenzymatic tissue culture procedure and transplanted to the renal subcapsular site of BB/Wor rats before the onset of disease. All MHC-mismatched and most MHC-matched grafts survived intact. In this study we report the growth of MHC-matched and mismatched islet tissue at the graft site. Grafts in diabetes resistant animals averaged 60 +/- 26 micrograms, a comparable mass to that which was transplanted. Grafts in diabetic animals were significantly increased in size, averaging 176 +/- 156 micrograms (P = 0.01). Analysis of nuclear to cytoplasmic volume ratios indicates that the increase in mass was primarily due to islet hyperplasia rather than hypertrophy. Factors contributing to the growth of islet tissue in situ are discussed.

Animals↗