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J R Lakey

Publications and source records attributed to J R Lakey.

At least 19 recordsLinked to original sources
PubMed2006

Culture medium modulates proinflammatory conditions of human pancreatic islets before transplantation.

A portion of transplanted islets is lost during engraftment as a result of stressful events, involving hypoxia and production of proinflammatory molecules by islets. Two of these molecules (monocyte chemoattractant protein-1, CCL2/MCP-1 and tissue factor, TF) are directly correlated with reduced graft function. We evaluated which factors reduce islet proinflammatory conditions. In particular the effects of different culture media supplemented with proteins or antioxidant agents on CCL2/MCP-1 and TF human islet release were evaluated. We observed that human islets after culture in final wash culture medium (FW) significantly decreased CCL2/MCP-1 release and TF production compared with CMRL and M199. These effects were independent from the type of protein added to the media (human serum, human albumin, fetal calf serum). Glutathione in FW further decreased CCL2/MCP-1 in a dose-dependent manner. Culture conditions can modulate the proinflammatory state of islets, and could be used in clinical islet transplantation to reduce inflammation during engraftment.

Amino Acids↗
PubMed2001

Novel approaches to cryopreservation of human pancreatic islets.

BACKGROUND: Optimized conditions for survival and function of human islets must be defined if sufficient islets are to be recovered from a single human donor pancreas to reverse type-1 diabetes after isolation, cryopreservation, and transplantation. The objective of this study was to compare the cryoprotective effect of ethylene glycol (EG) with the standard cryoprotectant, dimethyl sulfoxide (DMSO), on isolated human islet survival and function. Furthermore, the effect of different addition protocols and equilibrium concentrations of the cryoprotectants were studied. METHODS: Islets were isolated from human pancreata by using standard techniques of collagenase digestion and discontinuous Ficoll gradient purification. Aliquots of freshly isolated human islets were cryopreserved in six groups by using DMSO or EG. Cryoprotectants were added stepwise to produce a final concentration of 1.5 or 2.0 M, or added in a single step to a concentration of 1.5 M. Islets were cryopreserved by using established protocols and cultured for 48 hr at 37 degrees C before assessment of percentage of recovery and in vitro viability. RESULTS: After cryopreservation, percentage of recovery of islets was significantly higher in the group treated with 1.5 M of DMSO added in a stepwise protocol (74+/-3%, mean+/-SEM) compared with the standard 2.0 M of DMSO (62+/-4%) (P<0.05, unpaired t test, n=6). There was no difference between the recovery of islets cryopreserved with either 1.5 M of DMSO stepwise (74+/-3%) or 1.5 M of DMSO one-step (69+/-3%). Islet recovery was higher in groups treated with DMSO compared with EG, regardless of concentration of cryoprotectant or addition protocol, although the difference was significant only when comparing DMSO and EG 1.5 M one-step. Furthermore, islets treated with 1.5 M of DMSO, added either stepwise (6.0+/-0.4) or in one-step (6.5+/-0.8), had significantly higher stimulation indices compared with islets treated with the standard cryoprotectant for human islets, 2.0 M of DMSO (4.5+/-0.5) (P<0.05). CONCLUSIONS: These results demonstrate that a lower concentration of DMSO (1.5 M) allows for the cryopreservation of human islets with superior survival and preservation of function post-culture compared with 2.0 M of DMSO and various concentrations of EG.

Cryopreservation↗
PubMed2001

Serine-protease inhibition during islet isolation increases islet yield from human pancreases with prolonged ischemia.

BACKGROUND: Islet isolation from the pancreatic tissue matrix remains highly variable. Recent evidence suggests that intrinsic human pancreatic proteases, including trypsin, may inhibit effective collagenase enzymatic activity during islet isolation, thereby impairing the isolation success. In this study we have hypothesized that serine protease inhibition applied during pancreatic digestion, could improve yield and/or functional viability of islets isolated from human pancreases. METHODS: Twelve organ donor pancreases with 12.9+/-0.6 hr cold storage (mean+/-SEM) were perfused via their ducts with Liberase-HI enzyme in the presence (n=6) or absence (n=6) of 0.4 mM Pefabloc. All were then gently dissociated and their purified islets separated with Ficoll density gradient centrifugation. RESULTS: Donor-related factors (age, gender, cold storage time, body mass index, and pancreas weight) did not differ significantly between the two experimental groups. Pefabloc supplementation did not affect the digestion time, islets remaining trapped in exocrine tissue, or final islet purity. Islet recovery was increased in the Pefabloc-treated group (mean+/-SEM yield 323.8+/-80.8 x 10(3) islet equivalents vs. 130.8+/-13.6 x 10(3) islet equivalents, P<0.05). Cellular composition, DNA and insulin content, and insulin secretory activity of the isolated islets was similar. CONCLUSIONS: Inhibition of intrinsic protease activity within pancreases after prolonged cold storage improves isolation of viable islets.

Adolescent↗
PubMed2001

Novel approaches toward early diagnosis of islet allograft rejection.

BACKGROUND: The inability to diagnose early rejection of an islet allograft has previously proved to be a major impediment to progress in clinical islet transplantation. The need to detect early rejection will become even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation. METHODS: (a) Canine islet allograft transplant recipients were immunosuppressed for 1 month, then therapy was withdrawn. Serum glutamic acid decarboxylase antigen (GAD65), an endogenous islet protein, was monitored daily with a CO2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (beta-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral-specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glucose tolerance tests were followed until graft failure occurred. RESULTS: (a) Although serum monitoring of GAD65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engineered rodent islets demonstrated normal insulin kinetics in vitro (insulin stimulation index 2.57+/-0.2 vs. 2.95+/-0.3 for control islets, P=ns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet "sentinel signal" could be detected serologically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demonstrated evidence of allograft dysfunction (decline in KG) with a 2-day lead time to hyperglycemia (2.58+/-0.3 vs. 1.63+/-0.2%/min, respectively, P<0.001), with an accuracy of 89%, sensitivity of 78%, and specificity of 95%. CONCLUSIONS: Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunction due to rejection.

Animals↗
PubMed2001

University of wisconsin solution with trypsin inhibitor pefabloc improves survival of viable human and primate impure islets during storage.

Background. Recent studies suggest that impure islets (islets which have not been isolated from exocrine tissue and other parts of the pancreas) have great potential for successful transplantation. The evidence that supports this view includes findings that embedded islets (islets surrounded by exocrine tissue) undergo less apoptosis, peripancreatic lymph nodes prevent recurrence of IDDM (insulin dependent diabetes mellitus), and that islet yields and insulin content decrease during the purification process. Improved protocols have also been developed to prevent allorejection of impure islets. Despite these promising results, the storage of impure islets remains difficult, and was a method sought to decrease storage losses.Methods. Storage methods of impure human and non-human primate islets were compared, using either culture media or University of Wisconsin solution (UW). The effects of trypsin inhibition using Pefabloc (Roche Molecular Biochemicals, Indianapolis, IN) during storage period were also examined.Results. Low temperature and inhibition of trypsin activity during storage of impure islets improved both islet yield and viability. It was found that using UW solution and trypsin inhibition allowed perfect preservation of viable impure islets up to 48 h. A functional assay by glucose stimulation test showed these impure islet responded to glucose stimulation after 24 h.Conclusion. The benefits of storing impure islets using UW solution and Pefabloc at low temperature have been established. This improved method of preserving impure islets makes this model of transplantation even more viable.

Journal Article↗
PubMed2001

Improved islet survival and in vitro function using solubilized small intestinal submucosa.

In vitro proliferation of isolated pancreatic islets has become an area of great interest given the scarcity of clinical islet donors and the islet mass requirements for clinical islet transplantation. Small intestinal submucosa (SIS), a naturally occurring extracellular matrix, has been investigated to promote wound healing, tissue remodeling and cell growth. This study evaluated recovery and function of isolated canine pancreatic islets following in vitro tissue culture. Pancreatic islets were isolated from mongrel dogs using standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation and EuroFicoll purification. Groups of purified islets were cultured in a humidified atmosphere of 95% air and 5% CO(2) for 48 hours in standard islet culture conditions of CMRL 1066 tissue culture media (Gibco) which had been supplemented with 25microM HEPES, penicillin/streptomycin and either 10% heat inactivated fetal calf serum (FCS, Gibco) or solubilized SIS solution (Cook Biotech, Inc., West Lafayette, IN). The mean recovery of islets following the culture period was determined by sizing duplicate counts of a known volume and viability was assessed by static incubation with low glucose (2.8 mM), high glucose (20 mM) and high glucose solution supplemented with 50 microm IBMX solution. Remaining islets were embedded histologically. From a consecutive series of six culture experiments, a significantly higher (p < 0.05) recovery of islets co-cultured with SIS was observed when compared to controls. Mean islet recovery was 84.5 +/- 2.9% (mean +/- SEM) from the SIS cultured group compared with 64.7 +/- 4.5% from the control group cultured in FCS (p < 0.05, n=6). Islets from the SIS treated group exhibited a significantly higher (p <, 0.05) insulin response to the high glucose stimulus than islets cultured in the standard FCS cultured solution. The calculated stimulation index was 12.3 +/- 3.4 for the SIS-treated group compared with 5.6 +/- 1.8 for the standard cultured group (p < 0.05). The overall mean numbers of islets recovered following in vitro culture was also higher in the SIS-treated group. The proportion of islets with a mean diameter >150 microm increased from 24% to 31% in the SIS-treated group, whereas the same proportion decreased to 18% from 22% in the control (FCS-treated) group. Histological evaluation of fixed tissue samples collected following the culture period identified insulin and glucagon-secreting cells in the SIS and FCS treated groups, however a higher frequency of insulin positive cells were detected consistently in the SIS treated group. A proliferation marker (PCNA) identified positive cells within both groups as well. This study suggests that co-culture of freshly isolated canine islets in medium supplemented with solubilized SIS can improve the post-culture recovery and in vitro islet function. Future investigations will focus on the cellular interactions of SIS, both in vitro and in vivo.

Journal Article↗
PubMed2001

Hepatitis C virus replication in mice with chimeric human livers.

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

Animals↗
PubMed2001

Peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta cells.

The proinflammatory cytokines, interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma), are cytotoxic to pancreatic islet beta cells, possibly by inducing nitric oxide and/or oxygen radical production in the beta cells. Peroxynitrite, the reaction product of nitric oxide and the superoxide radical, is a strong oxidant and cytotoxic mediator; therefore, we hypothesized that peroxynitrite might be a mediator of cytokine-induced islet beta-cell destruction. To test this hypothesis we incubated islets isolated from human pancreata with the cytokine combination of IL-1beta, TNFalpha, and IFNgamma. We found that these cytokines induced significant increases in nitrotyrosine, a marker of peroxynitrite, in islet beta cells, and the increase in nitrotyrosine preceded islet-cell destruction. Peroxynitrite mimicked the effects of cytokines on nitrotyrosine formation and islet beta-cell destruction. L-N(G)-monomethyl arginine, an inhibitor of nitric oxide synthase, prevented cytokine-induced nitric oxide production but not hydrogen peroxide production, nitrotyrosine formation, or islet beta-cell destruction. In contrast, guanidinoethyldisulphide, an inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite, prevented cytokine-induced nitric oxide and hydrogen peroxide production, nitrotyrosine formation, and islet beta-cell destruction. These results suggest that cytokine-induced peroxynitrite formation is dependent upon increased generation of superoxide (measured as hydrogen peroxide) and that peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta cells.

Cell Death↗
PubMed2001

Pancreatic islet transplantation in the treatment of diabetes mellitus.

This chapter reviews current developments and future directions in clinical islet transplantation. With the recent introduction of glucocorticoid-free immunosuppressive therapies and improved methods for islet isolation, the success of the procedure has increased substantially. Challenges ahead include progress with international multicentre trials, development of single donor protocols, progress in clinical tolerance based therapies to lower overall risk of immunosuppression, and ultimately finding an alternative source to provide effective therapy for more patients with diabetes. Recent advances in stem cell biology and xenotransplantation may soon provide this opportunity.

Diabetes Mellitus, Type 1↗
PubMed2001

Modulation of JNK and p38 stress activated protein kinases in isolated islets of Langerhans: insulin as an autocrine survival signal.

OBJECTIVE: The objective of this study was to determine the effects of islet isolation and cytokine exposure on e-JUN NH2 terminal kinase (JNK) and p38 activation and whether insulin or the p38 inhibitor PD169316 could modify the response. SUMMARY BACKGROUND DATA: Islet transplantation exposes the cells of the graft to a variety of stressful stimuli that could promote beta-cell death and lead to graft failure. METHODS: Islets from canine (n = 12) and cadaveric human (n = 6) pancreata were isolated and purified. Islets were cultured in CMRL 1066 with and without 100 ng/ml insulin. The response to cytokine stimulation with tumor necrosis factor (TNF)alpha and IL-1 beta and the p38 inhibitor PD169316 was also observed. Islet lysates were analyzed by Western blotting for total and phosphorylated JNK and p38 content. Apoptosis was assessed by TdT-mediated dUTP nick end labeling (TUNEL) assay and by a specific cell death enzyme-linked immunosorbant assay (ELISA). RESULTS: In unstimulated islets, JNK activity was highest immediately following isolation, declining over 3 days to a low baseline level. The activity of p38 was lowest immediately after isolation, increasing progressively with time. The addition of insulin resulted in a more rapid decline in JNK activity, as opposed to p38, which showed no decrease in phosphorylation in response to insulin. In the cytokine stimulation studies, IL-1 beta stimulated p38 activation in a dose dependent manner, while JNK was relatively unaffected. PD169316 (100 microg/ml) was able to inhibit p38 activation in response to the isolation procedure as well as cytokine stimulation. Apoptotic activity was highest 24 hours after isolation, and was significantly reduced when islets were maintained in insulin-supplemented medium. CONCLUSIONS: Inhibition of the stress-activated protein kinase (SAPK) pathways may be important for the maintenance of islet cell survival following islet isolation for transplantation. This study supports an autocrine role of insulin in this process.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol.

Islet transplantation offers the prospect of good glycemic control without major surgical risks. After our initial report of successful islet transplantation, we now provide further data on 12 type 1 diabetic patients with brittle diabetes or problems with hypoglycemia previous to 1 November 2000. Details of metabolic control, acute complications associated with islet transplantation, and long-term complications related to immunosuppression therapy and diabetes were noted. Insulin secretion, both acute and over 30 min, was determined after intravenous glucose tolerance tests (IVGTTs). The median follow-up was 10.2 months (CI 6.5-17.4), and the longest was 20 months. Glucose control was stable, with pretransplant fasting and meal tolerance-stimulated glucose levels of 12.5+/-1.9 and 20.0+/-2.7 mmol/l, respectively, but decreased significantly, with posttransplant levels of 6.3+/-0.3 and 7.5+/-0.6 mmol/l, respectively (P < 0.006). All patients have sustained insulin production, as evidenced by the most current baseline C-peptide levels 0.66+/-0.06 nmol/l, increasing to 1.29+/-0.25 nmol/l 90 min after the meal-tolerance test. The mean HbA1c level decreased from 8.3+/-0.5% to the current level of 5.8+/-0.1% (P < 0.001). Presently, four patients have normal glucose tolerance, five have impaired glucose tolerance, and three have post-islet transplant diabetes (two of whom need oral hypoglycemic agents and low-dose insulin (<10 U/day). Three patients had a temporary increase in their liver-function tests. One patient had a thrombosis of a peripheral branch of the right portal vein, and two of the early patients had bleeding from the hepatic needle puncture site; but these technical problems were resolved. Two patients had transient vitreous hemorrhages. The two patients with elevated creatinine levels pretransplant had a significant increase in serum creatinine in the long term, although the mean serum creatinine of the group was unchanged. The cholesterol increased in five patients, and lipid-lowering therapy was required for three patients. No patient has developed cytomegalovirus infection or disease, posttransplant lymphoproliferative disorder, malignancies, or serious infection to date. None of the patients have been sensitized to donor antigen. In 11 of the 12 patients, insulin independence was achieved after 9,000 islet equivalents (IEs) per kilogram were transplanted. The acute insulin response and the insulin area under the curve (AUC) after IVGTT were consistently maintained over time. The insulin AUC from the IVGTT correlated to the number of islets transplanted, but more closely correlated when the cold ischemia time was taken into consideration (r = 0.83, P < 0.001). Islet transplantation has successfully corrected labile type 1 diabetes and problems with hypoglycemia, and our results show persistent insulin secretion. After a minimum of 9,000 IEs per kilogram are provided, insulin independence is usually attained. An elevation of creatinine appears to be a contraindication to this immunosuppressive regimen. For the subjects who had labile type 1 diabetes that was difficult to control, the risk-to-benefit ratio is in favor of islet transplantation.

Adult↗
PubMed2001

Islet cryopreservation using intracellular preservation solutions.

Cryopreservation of islets adds great flexibility to clinical islet transplant programs. Methods of islet cryopreservation have traditionally utilized permeating cryoprotectants contained within isotonic solutions without specifically addressing issues of ionic balances, buffering capacity, or oxygen free radicals that occur during hypothermic stresses. These factors may become significant issues during low-temperature storage and during the freezing and thawing process. Since its development in the early 1980s, the University of Wisconsin (UW) organ preservation solution has become the standard vascular flush and preservation solution. Recently, Hypothermosol preservation solution (HTS) was developed as a hypothermic blood substitute. The unique characteristics and composition of these preservation solutions may be important when developing solutions specific for the cryopreservation of cells and tissues. It was the aim of this study to evaluate these two hypothermic preservation solutions as the media used in cryopreservation of islets. Groups of canine islets [5000 islet equivalents (IE)/group] were cryopreserved using the standard protocol of stepwise addition of dimethyl sulfoxide (DMSO) to 2 M, controlled nucleation, slow cooling (0.25 degrees C/min), and rapid thawing (200 degrees C/min). The cryopreservation solutions were made with 1) UW solution, 2) HTS solution, or 3) Medium 199 solution with 10% fetal calf serum (FCS). Additional control groups included islets cryopreserved using 4) HTS, 5) UW solution, and 6) Medium 199 alone, without DMSO. Recovery of islets immediately following thawing was equivalent between the groups with the exception of the islets cryopreserved without DMSO (groups 4-6, p < 0.05). After 48 h of postcryopreservation tissue culture, islet recovery was highest in the groups frozen with UW and HTS (mean +/- SEM) (79.8 +/- 1.9% and 82.5 +/- 1.5%, p < 0.05 vs. group 3, 69.1 +/- 3.3%, p < 0.05, ANOVA). Less than 15% of the islets were recovered when they were cryopreserved without the cryoprotectant DMSO (groups 4-6). Functional viability was assessed by measuring the glucose-stimulated insulin secretion during static incubation after 48-h culture. The stimulation indexes were 4.6 +/- 1.0, 4.2 +/- 0.8, 3.6 +/- 1.2, 0.6 +/- 0.5, and 0.4 +/- 0.2 for islets in groups 1-5, respectively. This study demonstrates that postcryopreservation survival can be improved using intracellular-based preservation solutions, including UW or HTS, in conjunction with DMSO.

Animals↗
PubMed2001

Osmotic and cryoprotectant permeation characteristics of islet cells isolated from the newborn pig pancreas.

The development of effective protocols for the low-temperature banking of pancreatic islets is an important step in islet transplantation for the treatment of type I diabetes mellitus. We have been exploring the use of islets from the newborn pig as an alternative source of tissue for transplantation. Current cryopreservation protocols are empirically derived, but may be optimized by modeling osmotic responses during the cryopreservation process. This study determined the osmotic and cryoprotectant permeability parameters of cells isolated from the pancreas of newborn pigs. Key parameters are: the osmotically inactive fraction of cell volume, hydraulic conductivity, the permeability coefficients of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) at varying temperatures, and the activation energies of these transport processes. Newborn pig islets were dispersed into single cells and kinetic and equilibrium cell volumes were recorded during osmotic excursions using an electronic particle counter interfaced to a computer. Data were fitted to theoretical descriptions of the osmotic responses of cells, based on the Kedem-Katchalsky approach. The hydraulic conductivity (Lp) in the absence of cryoprotectant was calculated as 0.050 +/- 0.005, 0.071 +/- 0.006, and 0.300 +/- 0.016 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees C, respectively (mean +/- SEM, n = 7, 6, or 9). These values give an activation energy value of 16.69 kcal/mol when put into an Arrhenius plot. The solute permeability (Ps) values for 1 M DMSO were 0.89 +/- 0.12, 1.86 +/- 0.28, and 5.33 +/- 0.26 microm/min at 4 degrees C, 10 degrees C, and 22 degrees C, respectively (n = 11, 8, or 10) giving an activation energy of 15.98 kcal/mol. The Lp values for cells exposed to 1 M DMSO were 0.071 +/- 0.006, 0.084 +/- 0.008, and 0.185 +/- 0.014 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees C, respectively. The activation energy for these values was 8.95 kcal/mol. The Ps values for 2 M DMSO were 1.11 +/- 0.13, 1.74 +/- 0.19, and 7.68 +/- 0.12 microm/min for the same temperatures, with a calculated activation energy of 17.89 kcal/mol. The Lp values in the presence of 2 M DMSO were 0.070 +/- 0.006, 0.085 +/- 0.008, and 0.192 +/- 0.009 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees C, respectively, with an activation energy of 9.40 kcal/mol. Solutions of 1 M EG gave Ps values of 1.01 +/- 0.13, 1.45 +/- 0.25, and 4.90 +/- 0.48 microm/min at the three test temperatures. The resulting activation energy was 14.60 kcal/mol. The corresponding Lp values were 0.071 +/- 0.007, 0.068 +/- 0.006, and 0.219 +/- 0.012 microm/min/atm with an activation energy of 10.96 kcal/mol. The solute permeabilities in the presence of 2 M EG for newborn pig islet cells were 1.03 +/- 0.15, 1.42 +/- 0.23, and 5.56 +/- 0.22 microm/min; the activation energy was 15.70. The Lp values for cells in the presence of 2 M EG were 0.068 +/- 0.008, 0.071 +/- 0.006, and 0.225 +/- 0.010 microm/min/atm; the activation energy for these values was 11.49 kcal/mol. These key cryobiological parameters permit the mathematical modeling of osmotic responses of intact islets during the cryopreservation process, which may lead to further improvements in the low temperature storage of islets from newborn pigs.

Animals↗
PubMed2001

Nonviral transfection of intact pancreatic islets.

Ex vivo gene transfer offers a potential means to introduce genes into cells, which may play an important role in preventing graft rejection and inducing graft tolerance. This study examined the efficiency and toxicity of several lipid-based transfection reagents (LipofectAMINE, DOTAP, and DOSPER) in intact pancreatic islets. Isolated islets were transfected with a pCMV-beta-galactosidase plasmid using several DNA/liposome ratios (1:12) of liposomes (3-72 microl) and DNA (3 and 6 microg). Transfection efficiency was quantified by microscopic evaluation of beta-galactosidase gene expression in whole intact islets. Functionality of the transfected islets was measured by insulin response to glucose solutions. All transfection reagents evaluated in this study transfected cells within the islets. As expected, untransfected controls and transfected islets with DNA alone did not express beta-gal. The highest transfection efficiency and functional viability were obtained following a 48-h incubation after exposure to the transfection mixtures as follows: 3 microl DNA and 18 microl DOTAP/ml (1:6 ratio), 6 microg DNA and 12 microl DOSPER/ml (1:2 ratio), or 6 microg DNA and 12 microl Lipofect-AMINE/ml (1:2 ratio). The highest rate of transfected cells per islet was obtained using DOTAP. In vitro functionality was not significantly different between DOTAP and nontreated controls. However, optimal transfection efficiency doses of LipofectAMINE and DOSPER significantly reduced the stimulated insulin response of the transfected islets (p < 0.05, ANOVA). The calculated stimulation index (SI) was 7.8+/-0.6 (mean +/- SEM) for DOTAP-transfected islets compared with 8.4+/-0.5 for nontransfected control islets (p = ns). The SI of DOSPER- and LipofectAMINE-transfected islets was significantly lower (6.1+/-0.5 and 3.4+/-0.5, respectively, p < 0.05). Lipid-based transfection using DOTAP at a DNA/lipid ratio of 1:6 provides an effective means of ex vivo gene delivery without compromising in vitro functionality of the transfected islets.

Animals↗
PubMed2000

Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.

BACKGROUND: Registry data on patients with type 1 diabetes mellitus who undergo pancreatic islet transplantation indicate that only 8 percent are free of the need for insulin therapy at one year. METHODS: Seven consecutive patients with type 1 diabetes and a history of severe hypoglycemia and metabolic instability underwent islet transplantation in conjunction with a glucocorticoid-free immunosuppressive regimen consisting of sirolimus, tacrolimus, and daclizumab. Islets were isolated by ductal perfusion with cold, purified collagenase, digested and purified in xenoprotein-free medium, and transplanted immediately by means of a percutaneous transhepatic portal embolization. RESULTS: All seven patients quickly attained sustained insulin independence after transplantation of a mean (+/-SD) islet mass of 11,547+/-1604 islet equivalents per kilogram of body weight (median follow-up, 11.9 months; range, 4.4 to 14.9). All recipients required islets from two donor pancreases, and one required a third transplant from two donors to achieve sustained insulin independence. The mean glycosylated hemoglobin values were normal after transplantation in all recipients. The mean amplitude of glycemic excursions (a measure of fluctuations in blood glucose concentrations) was significantly decreased after the attainment of insulin independence (from 198+/-32 mg per deciliter [11.1+/-1.8 mmol per liter] before transplantation to 119+/-37 mg per deciliter [6.7+/-2.1 mmol per liter] after the first transplantation and 51+/-30 mg per deciliter [2.8+/-1.7 mmol per liter] after the attainment of insulin independence; P<0.001). There were no further episodes of hypoglycemic coma. Complications were minor, and there were no significant increases in lipid concentrations during follow-up. CONCLUSIONS: Our observations in patients with type 1 diabetes indicate that islet transplantation can result in insulin independence with excellent metabolic control when glucocorticoid-free immunosuppression is combined with the infusion of an adequate islet mass.

Adult↗