The availability of highly purified human islets has increased with the progressive improvement of isolation methods. This has provided opportunities to perform various in vitro studies on human islets. However, when islets are maintained in culture, the overgrowth of fibroblasts results in a reduced islet purity and often has an adverse effect on islet function. To reduce fibroblast growth and to maintain normal islet function, we have investigated a new three-dimensional culture technique using a noncoated transparent Biopore membrane insert (Millicell CM, Millipore). Islets were isolated from seven human pancreata and cultured for 2 months using this membrane insert. At various time intervals, the functional viability of islets was assessed by measurements of insulin released into the culture medium, static incubation assays of basal and stimulated insulin release, islet insulin contents, and insulin biosynthesis. Results were compared to those of islets cultured in hydrophobic plastic petri dishes, our standard procedure. We found that the non-coated membrane does not allow islet attachment to the surface and prevents fibroblast growth, so that islets maintain a three-dimensional structure and remain in a free-floating form. Islets cultured in a membrane insert showed a function similar to or better than that of islets cultured in plastic petri dishes.

Culture Media↗

PubMed1997

Absence of GLUT2 protein in near-term fetal rat pancreatic islets.

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Animals↗

PubMed1997

A two-step digestion process and LAP-I cold preservation solution for human islet isolation.

Pancreatic islet transplantation has a high potential for treating diabetes mellitus, but long-lasting insulin independence has not been achieved in type I diabetic patients. In order to obtain better results, improvement is needed in many areas. The first area is the islet isolation process. The requirements for an islet isolation method are: 1) to produce a maximum number of healthy islets without demanding a high quality of donor pancreas; 2) to be able to perform the procedure with fewer numbers of personnel who may be without extensive skills and expertise; and 3) to lower isolation costs. In order to achieve these objectives, we have made two important modifications to the isolation process. One is the development of a new preservation solution, LAP-I and the other is the use of a two-step process for pancreas digestion, involving a short warm collagenase digestion, followed by cold mechanical digestion without collagenase. We also use a clear plastic digestion chamber in order to visualize the process. The chamber cover is designed to facilitate frequent removal of digested tissue fragments. The overall procedure is simple and straightforward, requires less manpower and is cost effective. Our procedure is described in detail, and its advantages are discussed.

Adenosine↗

PubMed1996

Decreased alloreactivity to human islets secreting recombinant viral interleukin 10.

The objective of this study was to analyze allogeneic lymphocyte proliferative responses to cultured human pancreatic islets after gene transfer of viral interleukin (IL)-10 to the islets using replication-defective adenoviral vector. Human islets, either whole or dispersed into single cells, were cocultured with adenovector containing an expression cassette encoding the viral IL-10 gene under control of an SV40 promoter, this sequence replacing viral E1A and part of E1B early viral protein sequences. Subsequent production of recombinant protein by islets was determined by ELISA, and was found dependent on the multiplicity of infection (or ratio of vector to target cells). Protein was secreted by transfected islets at high levels 3-7 days after gene transfer. At high multiplicity of infection (100:1), islet viability was normal, but insulin secretion in response to glucose stimulation was blunted by 50%. Low-level recombinant viral IL-10 secretion by the islets was associated with increased allogeneic lymphocyte proliferation in mixed islet lymphocyte reactions. At protein levels in islet supernatant above 5 ng/ml, lymphocyte proliferation was significantly reduced. This pattern of viral IL-10 effect on lymphocyte proliferation correlated well with mixed lymphocyte reaction assays using purified protein. We conclude that transferred cytokine sequences are secreted by transfected islets as a function of the initial vector inoculum. The functional effect of the secreted cytokine viral IL-10 on allogeneic lymphocyte proliferation is dose dependent. Low-level recombinant protein secretion tended to augment lymphocyte proliferation, whereas high-level secretion significantly down-regulates this response.

Adenoviridae↗

PubMed1996

Prefabrication of a neo-endocrine organ: a rat model.

Previous work in the field of flap prefabrication has demonstrated that many tissues, including skin, bone, cartilage, muscle, and composite tissue, can be neovascularized with a carrier flap and transplanted to a distant site using microvascular technique. We have recently shown in a rat model that islets of Langerhans survive in large numbers when transplanted into a groin-based fasciovascular pedicled (FVP) flap. In the current study, we examined whether sufficient islet tissue can be transferred using microvascular free transfer of islet-containing flaps to reverse experimental diabetes. In the first phase of the experiment, islets from two Lewis rat donors were transplanted into the FVP flap of an isogeneic diabetic animal. Within 5 days, reversal of diabetes was noted in 4/4 experimental animals and in 0/4 control animals. In the second phase of the experiment, islet-FVP flaps were created in nondiabetic "carrier" animals. After 2 weeks the islet-containing flaps were harvested and transplanted to recipient diabetic Lewis rats using microvascular free transfer technique. Reversal of diabetes was noted within 10 days of free-flap transplant, and the diabetic state returned following removal of the flaps. Although preliminary, these results demonstrate that fasciovascular flaps can act as vehicles for the creation and transplantation of a functional neo-endocrine pancreas.

Animals↗

PubMed1996

Modulating autoimmune responses to GAD inhibits disease progression and prolongs islet graft survival in diabetes-prone mice.

In nonobese diabetic (NOD) mice, beta-cell reactive T-helper type 1 (Th1) responses develop spontaneously and gradually spread, creating a cascade of responses that ultimately destroys the beta-cells. The diversity of the autoreactive T-cell repertoire creates a major obstacle to the development of therapeutics. We show that even in the presence of established Th1 responses, it is possible to induce autoantigen-specific anti-inflammatory Th2 responses. Immune deviation of T-cell responses to the beta-cell autoantigen glutamate decarboxylase (GAD65), induced an active form of self-tolerance that was associated with an inhibition of disease progression in prediabetic mice and prolonged survival of syngeneic islet grafts in diabetic NOD mice. Thus, modulation of autoantigen-specific Th1/Th2 balances may provide a minimally invasive means of downregulating established pathogenic autoimmune responses.

Adoptive Transfer↗

PubMed1996

Establishment of an islet bank and its future perspectives.

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Adolescent↗

PubMed1996

Islet protective effect of pravastatin from nonspecific inflammation in mouse pancreatic islet isografts.

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Animals↗

PubMed1995

Fetal pancreas transplantation in miniature swine. V. The functional and immunodulatory effects of ultraviolet light on fetal pig islets.

We have used the pig as a large animal model for studies of fetal pancreas transplantation. Fetal pig pancreas (FPP) has also been proposed as a potential source of endocrine cells for the treatment of diabetes mellitus. Among the approaches to prevent rejection, the irradiation of donor islets with ultraviolet B light has been used for its immunomodulating properties. Our goal was to study in vitro the effects of UV-B irradiation of FPP on the function and immunogenicity of the tissue. FPP were collagenase-digested and cultured for 1-29 days prior to UV-B irradiation. Static incubation tests were used to measure glucose-theophylline stimulated insulin release. Data obtained at 300 J/m2 revealed no impairment of insulin release (78% to 129% of controls, P = ns). At 500 J/m2, a significant reduction of glucose-theophylline stimulated insulin release was observed with 50-60-day-old FPP (35% to 66% of controls, P < 0.05), but not with 80-day-old FPP (93% of controls, P = ns). At both doses, prolonged observation in culture did not show any alteration of the growth and proliferation of islet cell clusters. UV-irradiated (300 J/m2) adult and fetal pig islet allografts released C-peptide and survived > 200 days. The immunogenicity of irradiated tissues was determined in vitro with allogeneic mixed islet-lymphocyte cultures (MILC). Proliferative responses of allogeneic lymphocytes to UV-irradiated FPP were very significantly decreased by 52-91% at both 300 and 500 J/m2 doses. This effect was observed from 1 to 10 days following UV irradiation and was not modulated by exposure of the tissues to gamma-interferon. We conclude that UVB-irradiation of FPP at a dose of 300 J/m2 does not alter its endocrine function and growth and is effective in reducing tissue immunogenicity. This treatment may be a useful approach for fetal islet transplantation in large animal models.

Animals↗

PubMed1995

Efficient gene transfer to pancreatic islets mediated by adenoviral vectors.

Genetic manipulation of pancreatic islets before transplantation has the potential to alter cellular immunity as well as islet function. The purpose of this study was to examine the feasibility of gene transfer to islets, using replication-defective adenoviral vectors. Newborn mouse islets were infected with AdHCMVsp1LacZ vector encoding Escherichia coli beta-galactosidase (beta-gal). Islets were cocultured with vector, at virus-to-target cell ratios of 10:1, for 1 hr. Gene transfer was assessed by specific histochemical stain for beta-gal (X-gal). Islet DNA and RNA were analyzed by Southern and PCR for beta-gal and adeno sequences, and recombinant protein production by western and ONPG assays. Islet integrity after gene transfer was assessed by static incubations and transplantation to nondiabetic and to diabetic mice. Southern analysis and PCR confirmed the presence of E coli beta-galactosidase and the E4 adeno DNA in infected islets, but not in controls. Reverse-transcription PCR and western analysis demonstrated expression and protein production of inserted E coli beta-galactosidase, but not E4 message. Insulin release in response to static incubations was unimpaired in infected islets. Syngeneic islet grafts stained positively for insulin for up to 7 days. Transplanted, genetically manipulated islets functioned similarly to control islets in reversing murine drug-induced diabetes. Thus, gene transfer into islets can be accomplished using adenovirus-based vectors. The capacity of this virus to infect non-dividing cells allows insertion of cDNA into pancreatic islets, with potential application to the transplant setting.

Adenoviruses, Human↗

PubMed1995

Abrogation of allospecific T lymphocyte responses in swine by ultraviolet light-B irradiation.

Immunomodulatory effects of ultraviolet-light B (UV-B) irradiation were tested in pigs, using peripheral blood lymphocytes (PBL), with special emphasis on the generation of cytotoxic T lymphocytes (CTL). Stimulator PBL were irradiated with 300-1100 Joules/m2 (J/m2) of UV-B (experimental group) and cultured for 24 h prior to being placed in mixed lymphocyte culture (MLC) to determine proliferative responses against alloantigens, or placed in bulk culture with allogeneic PBL to determine the induction of CTL. PBL that received 3000 rads gamma irradiation were used as controls. Proliferative alloresponses against gamma irradiated PBL (control group) were high, with stimulation indices (S.I.) ranging from 13.46 to 81.93. The generation of allospecific CTL activity was also high, ranging from 50.78% to 95.40%. In contrast, proliferative responses and generation of allospecific CTL were markedly inhibited (> 49.77%) against the same donor PBL irradiated with > or = 500 J/m2 UV-B. These results demonstrated that ultraviolet light treatment of pig PBL can reduce or prevent allospecific T cell proliferation as well as the generation of specific CTL, similar to results obtained with UV-B irradiation of lymphocytes in rodents.

Animals↗

PubMed1995

Ultraviolet light irradiation reduces human islet immunogenicity without altering islet function.

Allograft rejection is the major cause for failure in clinical islet transplantation for diabetic patients. A reduction of donor islet immunogenicity is potentially a useful approach for altering recipient's immune responses. Studies in animal models have shown the immunomodulatory properties of ultraviolet (UV)-B light that are beneficial for allograft survival. However, there is a narrow window between the doses required for immunomodulation and those toxic to beta-cells. In addition, this window varies between one species to another. Our study was designed to determine, in vitro, the UV-B dose for human islets that effectively reduces immunogenicity and maintains islet viability and normal function. Islets were isolated from donor pancreas by collagenase digestion and density gradient centrifugation on Euro-Ficoll. Static incubation and perifusion tests were used to measure glucose-stimulated insulin release. Viability was also assessed by histology and function of UV-irradiated islets transplanted under the renal capsule of athymic mice. The immunogenicity of UV-treated islets was determined in vitro with mixed islet lymphocyte culture using healthy human peripheral blood lymphocytes as responders. At a dose of 300 J/m2, both functional assays detected no impairment of insulin release. At 500 J/m2, a slight decrease of stimulated insulin release was observed only in the perifusion system. At the levels of 600 and 900 J/m2, a clear alteration was observed in both basal and stimulated insulin release. Islets irradiated at 300 J/m2 and transplanted into athymic mice stained strongly for insulin and responded to high glucose challenge in in vivo perfusion performed at two weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Essential fatty acid deficiency prevents autoimmune diabetes in nonobese diabetic mice through a positive impact on antigen-presenting cells and Th2 lymphocytes.

Protective effects of essential fatty acid deficiency (EFAD) on autoimmunity were shown in rodents. Our goal was to investigate the mechanisms of EFAD effects on autoimmune diabetes in nonobese diabetic (NOD) mice. Weanling female mice were randomized between a control diet group and an EFAD diet group, and the development of diabetes and immune response was determined over a 6-month period. The cumulative incidence of diabetes was significantly reduced in the EFAD group (20 vs 68.75% in the control group; p < 0.01), without affecting the insulitis process. Splenocyte reactivity to phytohemagglutinin and anti-CD3 antibody was significantly increased in EFAD-fed mice (p < 0.01). The EFAD group also exhibited a dramatic increase in baseline (29-fold) and antigen-presenting cell (APC)-stimulated (10-fold) T cell responses in syngeneic mixed leukocyte reaction. These responses were associated with a marked increase in splenocyte interleukin-4 (IL-4) production, a reduction in interferon-gamma production, and a down-regulation of CD45RB isoform expression. Macrophages in the EFAD group exerted a reduced suppressive effect on concanavalin A-induced splenocyte proliferation and were found to release increased amounts of tumor necrosis factor-alpha and IL-1 and reduced amounts of prostaglandin E2. These results clearly demonstrate that EFAD prevents diabetes in NOD mice. The data suggest an enhanced activity of Th2-like cells, as well as an effect on APC activity linked to alteration in eicosanoid metabolism.

Animals↗