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ABSTRACT: : Accumulated evidence has shown that insulin producing beta-cells in pancreatic islets have the limited potential to regenerate. Adenoviruses have been widely employed to deliver genes of interest into pancreatic islets. This study was aimed at investigating whether adenovirus infection has any impact on the potential of beta-cell proliferation.
: Human adenovirus type 5 (Ad5) encoding rat insulin promoter driven reporter genes were used to infect freshly isolated pancreatic islets. Western blotting assays were performed to evaluate the expression and activation of key molecules involved in cell survival and proliferation following Ad5 infection. Immunofluorescence staining was employed to identify proliferating cells after culturing the infected and control islets in the presence of BrdU, an analog of thymidine that can be incorporated into the genome of proliferating cells.
: Ad5 infection of the islets resulted in expression and activation of Akt1, a key molecule in the PI3 kinase signaling pathway. Accordingly, a higher frequency of islet cell proliferation was detected in Ad5-infected islets than in control islets.
: These data suggest adenovirus infection can activate beta-cell survival and proliferation machinery, in particular operating through the PI3K/Akt signaling pathway. This information has significant ramification for the use of adenovirus as a gene delivery vehicle for pancreatic islet cells.
Transplantation 04/2009; 87(6):821-4. · 4.00 Impact Factor
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ABSTRACT: Background. Accumulated evidence has shown that insulin producing β-cells in pancreatic islets have the limited potential to regenerate. Adenoviruses have been widely employed to deliver genes of interest into pancreatic islets. This study was aimed at investigating whether adenovirus infection has any impact on the potential of β-cell proliferation.
Methods. Human adenovirus type 5 (Ad5) encoding rat insulin promoter driven reporter genes were used to infect freshly isolated pancreatic islets. Western blotting assays were performed to evaluate the expression and activation of key molecules involved in cell survival and proliferation following Ad5 infection. Immunofluorescence staining was employed to identify proliferating cells after culturing the infected and control islets in the presence of BrdU, an analog of thymidine that can be incorporated into the genome of proliferating cells.
Results. Ad5 infection of the islets resulted in expression and activation of Akt1, a key molecule in the PI3 kinase signaling pathway. Accordingly, a higher frequency of islet cell proliferation was detected in Ad5-infected islets than in control islets.
Discussion. These data suggest adenovirus infection can activate β-cell survival and proliferation machinery, in particular operating through the PI3K/Akt signaling pathway. This information has significant ramification for the use of adenovirus as a gene delivery vehicle for pancreatic islet cells.
Transplantation 03/2009; 87(6):821-824. · 4.00 Impact Factor
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ABSTRACT: Pancreatic resection can alleviate pain in properly selected patients with severe chronic pancreatitis (CP), although the apancreatic state causes "brittle" diabetes. Islet auto-transplantation (IAT) after resection can decrease diabetes-related morbidity. Twenty-six consecutive patients with CP who underwent 27 pancreatic resections with IAT from April 2005 to December 2007 were evaluated in this retrospective case control study. Data were collected by chart and operative note reviews and query of hospital databases. Subgroup analysis was performed on 21 cases of total pancreatectomy and six cases of pancreaticoduodenectomy (PD). Mean age was 43.8 years and 46.2 per cent of patients were female. The most common etiology of CP was alcoholism (34.6%), followed by idiopathic causes (30.8%) and pancreatic divisum (23.1%). There was no mortality and the complication rate was 56 per cent. Islet equivalents infused and islet equivalents/gram of pancreas were 82,094 and 2,739 respectively. Mean discharge insulin dose was 10.7 units/day. Mean follow-up was 6.5 months. At 6 months, 80 per cent of patients reporting had decreased or eliminated their use of narcotic medication and all total pancreatectomy patients required insulin (mean 23 units/day). In appropriately selected patients, pancreatic resection with IAT is safe and effective for the treatment of intractable pain associated with CP.
The American surgeon 07/2008; 74(6):530-6; discussion 536-7. · 1.28 Impact Factor
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Judith M Thomas, Juan L Contreras,
Pei X Wang,
Devin E Eckhoff,
Clement Asiedu,
William J Hubbard,
Samuel Cartner,
Francis T Thomas,
Michelle L Robbin,
Steven Nadler,
William J Cook,
Joshua Sharff,
David M Neville,
Joseph Shiloach
Transplantation 04/2008; 85(6):920. · 4.00 Impact Factor
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Juan L Contreras
Xenotransplantation 04/2008; 15(2):99-101. · 2.33 Impact Factor
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ABSTRACT: Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector for a variety of diseases. Many target cells, however, express low levels of Ad5 native receptor, the Coxsackie-Adenovirus Receptor (CAR), and thus are resistant to Ad5 infection. The Protein Transduction Domain of the HIV Tat protein, namely PTD tat, has been shown to mediate protein transduction in a wide range of cells. We hypothesize that re-targeting Ad5 vector via the PTD tat motif would improve the efficacy of Ad5-mediated gene delivery.
In this study, we genetically incorporated the PTD tat motif into the knob domain of Ad5 fiber, and rescued the resultant viral vector, Ad5.PTD tat. Our data showed the modification did not interfere with Ad5 binding to its native receptor CAR, suggesting Ad5 infection via the CAR pathway is retained. In addition, we found that Ad5.PTD tat exhibited enhanced gene transfer efficacy in all of the cell lines that we have tested, which included both low-CAR and high-CAR decorated cells. Competitive inhibition assays suggested the enhanced infectivity of Ad5.PTD tat was mediated by binding of the positively charged PTD tat peptide to the negatively charged epitopes on the cells' surface. Furthermore, we investigated in vivo gene delivery efficacy of Ad5.PTD tat using subcutaneous tumor models established with U118MG glioma cells, and found that Ad5.PTD tat exhibited enhanced gene transfer efficacy compared to unmodified Ad5 vector as analyzed by a non-invasive fluorescence imaging technique.
Genetic incorporation of the PTD tat motif into Ad5 fiber allowed Ad5 vectors to infect cells via an alternative PTD tat targeting motif while retaining the native CAR-mediated infection pathway. The enhanced infectivity was demonstrated in both cultured cells and in in vivo tumor models. Taken together, our study identifies a novel tropism expanded Ad5 vector that may be useful for clinical gene therapy applications.
Virology Journal 02/2007; 4:103. · 2.34 Impact Factor
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ABSTRACT: Evidence is emerging that the endoplasmic reticulum (ER) participates in initiation of apoptosis induced by the unfolded protein response and by aberrant Ca(++) signaling during cellular stress such as ischemia/reperfusion injury (I/R injury). ER-induced apoptosis involves the activation of caspase-12 and C/EBP homologous protein (CHOP), and the shutdown of translation initiated by phosphorylation of eIF2alpha. Sodium 4-phenylbutyrate (PBA) is a low molecular weight fatty acid that acts as a chemical chaperone reducing the load of mutant or unfolded proteins retained in the ER during cellular stress and also exerting anti-inflammatory activity. It has been used successfully for treatment of urea cycle disorders and sickle cell disease. Thus, we hypothesized that PBA may reduce ER-induced apoptosis triggered by I/R injury to the liver.
Groups of male C57BL/6 mice were subjected to warm ischemia (70% of the liver mass, 45 minutes). Serum aspartate aminotransferase was assessed 6 hours after reperfusion; apoptosis was evaluated by enzyme-linked immunosorbent assays of caspase-12 and plasma tumor necrosis factor alpha, Western blot analyses of eIF2alpha, and reverse transcriptase-polymerase chain reaction of CHOP expression.
A dose-dependent decrease in aspartate aminotransferase was demonstrated in mice given intraperitoneal PBA (1 hour before and 12 hours after reperfusion), compared with vehicle-treated controls; this effect was associated with reduced pyknosis, parenchymal hemorrhages, and neutrophil infiltrates in PBA-treated mice, compared with controls. In a lethal model of total liver I/R injury, all vehicle-treated controls died within 3 days after reperfusion. In contrast, 50% survival (>30 days) was observed in animals given PBA. The beneficial effects of PBA were associated with a greater than 45% reduction in apoptosis, decreased ER-mediated apoptosis characterized by significant reduction in caspase-12 activation, and reduced levels of both phosphorylated eIF2alpha and CHOP. Significant reductions in plasma levels of tumor necrosis factor alpha and liver myeloperoxidase content were demonstrated after PBA treatment.
Reduction in ER stress-induced hepatocellular injury was achieved by the administration of PBA. Targeting the ER-associated cell death pathway might offer a novel approach to reduce I/R injury to the liver.
Surgery 09/2005; 138(2):342-51. · 3.10 Impact Factor
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ABSTRACT: Functional poly(ethylene glycol) (PEG) derivatives, including monosuccinimidyl PEG (MSPEG) with molecular weight (MW) of 2000 (2 kDa) as well as 5 kDa and disuccinimidyl PEG (DSPEG) with MW of 3 and 6 kDa, were synthesized and characterized. They were used to modify the surface of adult porcine islets for cytoprotection. The islets were isolated, purified and modified with functional PEG. Untreated porcine islets were used as control. An in vitro human antibody/complement-mediated cytotoxicity test based on the release of intracellular lactate dehydrogenase was used to evaluate cytotoxicity of human serum to the modified islets. In vitro cell viability was assessed using membrane-integrity straining and islet metabolism in culture. In vitro islet functionality was evaluated by glucose-stimulated insulin release of islets in static incubation with human serum. In vivo islet functionality was evaluated by monitoring non-fasting blood glucose level in streptozotocin-induced diabetic (SCID) immunocompromized mice after intraportal transplantation of porcine islets. Results show that all the PEG derivatives used in the study showed significant in vitro and in vivo cytoprotections against cytotoxic effects elicited by human serum and diabetic SCID mice, respectively, to porcine islets. DSPEG derivatives combined with human albumin exhibited a better cytoprotection, as compared to MSPEG ones, due to the capacity of the succinimidyl groups to selectively react with amino groups of the albumin under physiological conditions. The effects of both MW and concentration of the PEG derivatives on cytoprotection were significant. It appears that this novel biotechnology will be an attractive approach for improved xenotransplantation of islets.
Biomaterials 03/2005; 26(4):403-12. · 7.40 Impact Factor
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Tyler O Kirby,
Angel Rivera,
Daniel Rein,
Minghui Wang,
Ilya Ulasov,
Martina Breidenbach,
Manjula Kataram, Juan L Contreras,
Carlos Krumdieck,
Masato Yamamoto,
Marianne G Rots,
Hidde J Haisma,
Ronald D Alvarez,
Parameshwar J Mahasreshti,
David T Curiel
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ABSTRACT: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy of conditionally replicative adenoviruses. A novel model system is needed that will provide insight into the anticipated therapeutic index of conditionally replicative adenoviruses preclinically. We endeavored to show a novel model system, which involves ex vivo evaluation of conditionally replicative adenovirus toxicity and therapeutic efficacy in thin, precision-cut slices of human primary tumor and liver.
The Krumdieck thin-slice tissue culture system was used to obtain and culture slices of tumor xenografts of ovarian cancer cell lines, human primary ovarian tumors, and human liver. We determined the viability of slices in culture over a period of 36 to 48 hours by ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl-2-(4-sulfophenyl)-2H-tetrazolium, inner salt)]) (MTS) assay. In vitro Hey cells, slices of Hey xenografts, and human ovarian tumor or human liver slices were infected with 500vp/cell of either replication competent wild-type adenovirus (Ad5/3wt), conditionally replicative adenovirus (Ad5/3cox-2), or the replication deficient adenovirus (Ad5/3luc1). At 12-, 24-, and 36-hour intervals, the replication of adenoviruses in these slices was determined by quantitative reverse transcription-PCR of adenoviral E4 copy number.
Primary tumor slices were able to maintain viability for up to 48 hours after infection with nonreplicative virus (Ad5luc1). Infection of Hey xenografts with Ad5/3cox-2 showed replication consistent with that seen in Hey cells infected in an in vitro setting. Primary tumor slices showed replication of both Ad5/3wt and Ad5/3cox over a 36-hour time period. Human liver slices showed replication of Ad5/3wt but a relative reduction in replication of Ad5/3cox-2 indicative of conditional replication "liver off" phenotype, thus predicting lower toxicity.
The thin-slice model system represents a stringent method of ex vivo evaluation of novel replicative adenoviral vectors and allows assessment of human liver replication relative to human tumor replication. This is the first study to incorporate this system for evaluation of therapeutic efficacy and replicative specificity of conditionally replicative adenoviruses. Also, the study is the first to provide a valid means for preclinical assay of potential conditionally replicative adenovirus-based hepatotoxicities, thus providing a powerful tool to determine therapeutic index for clinical translation of conditionally replicative adenoviruses.
Clinical Cancer Research 01/2005; 10(24):8697-703. · 7.74 Impact Factor
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Juan L Contreras,
Christopher Eckstein,
Cheryl A Smyth,
Guadalupe Bilbao,
Mario Vilatoba,
Sharman E Ringland,
Carlton Young,
J Anthony Thompson,
José A Fernández,
John H Griffin,
Devin E Eckhoff
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ABSTRACT: Clinical studies indicate that significant loss of functional islet mass occurs in the peritransplant period. Islets are injured as a result of detrimental effects of brain death, pancreas preservation, islet isolation, hypoxia, hyperglycemia, and immune-mediated events. In addition, recent studies demonstrated that islets are injured as a result of their exposure to blood and of activation of intrahepatic endothelial and Kupffer cells, resulting in inflammation and thrombosis. Activated protein C (APC) is an anticoagulant enzyme that also exerts anti-inflammatory and antiapoptotic activities by acting directly on cells. Here, we report that exogenous administration of recombinant murine APC (mAPC) significantly reduced loss of functional islet mass after intraportal transplantation in diabetic mice. Animals given mAPC exhibited better glucose control, higher glucose disposal rates, and higher arginine-stimulated acute insulin release. These effects were associated with reduced plasma proinsulin, intrahepatic fibrin deposition, and islet apoptosis early after the transplant. In vitro and in vivo data demonstrated that mAPC treatment was associated with a significant reduction of proinflammatory cytokine release after exposure of hepatic endothelial cells to islets. mAPC treatment also prevented endothelial cell activation and dysfunction elicited by intrahepatic embolization of isolated islets inherent to pancreatic islet transplantation (PIT). This study demonstrates multiple remarkable beneficial effects of mAPC for PIT and suggests that APC therapy may enhance the therapeutic efficacy of PIT in diabetic patients.
Diabetes 12/2004; 53(11):2804-14. · 8.29 Impact Factor
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Juan L Contreras,
Dong Xie,
Jimmy Mays,
Cheryl A Smyth,
Christopher Eckstein,
Firoz G Rahemtulla,
Carlton J Young,
J Anthony Thompson,
Guadalupe Bilbao,
David T Curiel,
Devin E Eckhoff
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ABSTRACT: Effective cytoprotection to xenoislets would circumvent the major tissue limitation for pancreatic islet transplantation (PIT). Cell-surface engineering with poly[ethylene glycol] (PEG) derivatives can successfully prevent antibody binding to the surface antigens. Gene transfer of the antiapoptotic Bcl-2 gene has been shown to decrease cytotoxicity mediated by xenoreactive natural antibodies and complement. In this study, we assessed survival and function of surface-engineered porcine islets genetically modified to overexpress Bcl-2.
Incorporation of PEG derivatives into the islet surface and adenovirus-mediated gene transfer of Bcl-2 (AdBcl-2) was accomplished within 24 hours post-isolation. Cytotoxicity induced by human xenoreactive natural antibodies was evaluated by islet intracellular lactate dehydrogenase release and microscopic analysis using membrane-integrity staining. Islet functionality was assessed by static incubation and after intraportal infusion (5000 IEQ) into diabetic NOD-SCID mice reconstituted with human lymphocytes (5 x 10 8 /intraperitoneally/15 days before PIT).
No significant change in islet viability, morphology, and functionality was demonstrated after the incorporation of PEG-mono-succimidyl-succinate (MSPEG), or PEG-di-succimidyl-succinate "end"-capped with albumin (DSPEG) with or without gene transfer of Bcl-2. Islets treated with MSPEG presented a significant reduction in lactate dehydrogenase release compared with controls (41.2 +/- 3 vs 72.1 +/- 7, respectively, P <.05). Further protection was accomplished by DSPEG or AdBcl-2. The maximal cytoprotection was achieved by DSPEG +AdBcl-2 (15.5 +/- 4.9%, P <.001). Nonfasting glucose >200 mg/dL was found in 100% of the animals given control islets (n = 6) within 48 hours post-transplant. In contrast, euglycemia was achieved in 100% of the animals given islets modified with DSPEG + AdBcl-2 during the observation time.
Surface-engineering with functionalized PEG derivatives in combination with genetic modification with Bcl-2 significantly reduced islet loss after PIT. Application of this novel technology may improve results in xenoislet transplantation.
Surgery 09/2004; 136(3):537-47. · 3.10 Impact Factor
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ABSTRACT: Current isolation techniques recover only 20% to 50% of the pancreatic islets. Brain death (BD) is characterized by activation of proinflammatory cytokines (PICs) with reduced islet yields and functionality. We previously reported that 17beta-estradiol (E2) induces cytoprotection to human islets exposed to PICs. Furthermore, inhibition of PIC release has been demonstrated after E2 treatment. In the present study, we evaluated if E2 treatment to BD donors would improve pancreatic islet recovery and functionality.
BD was induced in male, 250- to 350-g Lewis rats by inflation of a Fogarty catheter placed intracranially. Rats were mechanically ventilated for 6 hours. Only rats with mean arterial blood pressure > 75 mm Hg were used. Animals (n = 6) received E2 (1 mg/kg/iv immediately after BD induction), vehicle (V), or the combination of 17beta-estradiol and a selective estrogen receptor antagonist ICI 182,780 (ICI, 3 mg/kg/ip/1 hour before BD induction). Islet viability was determined by ethidium bromide-acridine orange. PICs were assessed by ELISA. Islet functionality was determined by static incubation and glucose disposal rate (Kg) after intraportal transplantation (3000 islet equivalent[IEQ]/syngeneic streptozotocin-induced diabetic rat).
A 2- to 3-fold reduction in TNF-alpha, IL-1beta, and IL-6 was demonstrated in BD donors given E2; this effect reversed by ICI 182,780. Pancreatic sections from control BD donors presented 26.5% +/- 4% TUNEL-positive beta-cells compared with 15.1% +/- 3% in 17beta-estradio-treated animals. Islet recovery was enhanced in E2-treated donors (1233.4 +/- 123 IEQ/pancreas) compared with controls (725 +/- 224 IEQ, P < .05). Islet viability was significantly enhanced by E2. Higher islet functionality was demonstrated in vitro and in vivo after transplantation in islets recovered from E2-treated BD donors.
Islet recovery and functionality in vitro and in vivo were significantly improved by 17beta-estradiol treatment to BD donors. These observations may lead to strategies to reduce the effects of BD on isolated islets and improve the results in clinical islet transplantation.
Surgery 08/2004; 136(2):336-45. · 3.10 Impact Factor
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ABSTRACT: Ischemia/reperfusion injury (I/R injury) of the liver remains a significant problem during liver surgery and transplantation. I/R injury is associated with liver apoptosis, which is mediated by death receptors such as Fas and tumor necrosis factor alpha (TNF-alpha), and/or mitochondrial dysfunction induced by cellular stress. Caspase-8 is presumed to be the apex of the death-mediated apoptosis pathway, whereas caspase-3 belongs to the "effector" proteases in the apoptosis cascade. Synthetic small interfering RNAs (siRNAs) specifically suppress gene expression by RNA interference. Therefore, we evaluated the therapeutic efficacy of caspase-8 and caspase-3 siRNA in a murine model of liver I/R injury.
In C57BL/6 mice, 45% or 70% of the liver mass was clamped for 90 minutes. For survival analysis, total hepatic ischemia was induced for 45 minutes. In vivo delivery of siRNA was performed via the portal vein by high-volume injection (0.5 nmol of siRNA in 1 mL containing 10% lipiodol) 60 minutes before ischemia. As a control, animals received either vehicle or non-sense siRNA (siRNA-scrambled).
Liver uptake of siRNA was analyzed in transgenic mice who express beta-galactosidase (beta-gal) (C57BL/6J-TgN(MTn-LacZ)204Bri) after administration of siRNA-LacZ. A 3- to 4-fold decrease in beta-gal activity was accomplished at 0.5 nmol. No significant change in beta-gal activity was demonstrated in mice receiving non-sense siRNA. Immunohistochemical studies found that 60% of the liver cells efficiently took up siRNA. Significant reduction in serum aspartate transaminase was found in animals treated with siRNA caspase-8 or caspase-3 compared with siRNA-scrambed or vehicle-treated controls. More than a 60% reduction in caspase-8 and caspase-3 gene expression and activities was accomplished after siRNA administration. Animals treated with siRNA presented lower infiltration of polymorphonuclear leukocytes and better preservation of the liver architecture compared with controls. All of the control mice subjected to total liver ischemia died within 5 days. In contrast, 30% of the animals given siRNA caspase-8 and 50% of those treated with siRNA caspase-3 survived indefinitely (>30 days).
Small interfering RNA targeted to caspase-8 and caspase-3 provided significant protection against I/R injury to the liver. This approach could be therapeutic in liver transplantation and other conditions associated with I/R injury to the liver.
Surgery 08/2004; 136(2):390-400. · 3.10 Impact Factor
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ABSTRACT: The recent success of "steroid-free" immunosuppressive protocols and improvements in islet preparation techniques have proven that pancreatic islet transplantation (PIT) is a valid therapeutic approach for patients with type 1 diabetes. However, there are major obstacles to overcome before PIT can become a routine therapeutic procedure, such as the need for chronic immunosuppression, the loss of functional islet mass after transplantation requiring multiple islet infusion to achieve euglycemia without exogenous administration of insulin, and the shortage of human tissue for transplantation. With reference to the first obstacle, stable islet allograft function without immunosuppressive therapy has been achieved after tolerance was induced in diabetic primates. With reference to the second obstacle, different strategies, including gene transfer of antiapoptotic genes, have been used to protect isolated islets before and after transplantation. With reference to the third obstacle, pigs are an attractive islet source because they breed rapidly, there is a long history of porcine insulin use in humans, and there is the potential for genetic engineering. To accomplish islet transplantation, experimental opportunities must be balanced by complementary characteristics of basic mouse and rat models and preclinical large animal models. Well-designed preclinical studies in primates can provide the quality of information required to translate islet transplant research safely into clinical transplantation.
ILAR journal / National Research Council, Institute of Laboratory Animal Resources 02/2004; 45(3):334-42. · 2.33 Impact Factor
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ABSTRACT: The influence of ethnic origin on organ donation and renal allograft survival after renal transplantation has been controversial. Several large studies have reported inferior renal allograft survival in black recipients, whereas others have reported equal survival. However, the issue of race as it relates to organ donation, patient referral, and patient selection in orthotopic liver transplantation has not been investigated. We retrospectively reviewed our results of organ donation, patient referral and selection, and orthotopic liver transplantation since 1989. Because of a concerted educational effort by this organ procurement organization, the percentage of black donors has increased from 6.1% in 1988 to 21.9% in 1996. Since the inception of the Liver Transplant Program in 1989, 844 patients have been referred to our transplant center for organ transplant evaluation. Disproportionately fewer black patients (119; 14.1%) were referred for liver transplantation than white patients (725; 85.9%) based on the prevalence of end-stage liver disease in these populations. The acceptance rate for listing for transplantation was similar between the two groups. The percentage of patient referrals who actually underwent transplantation was similar across racial lines (43% black v42% white patients). However, it appeared that black patients were referred for liver transplantation at a later stage and were more critically ill at the time of referral. Nevertheless, the patient and graft survival were similar between black and white patients. The 1- and 3-year survival rates in white recipients was 88% and 81%, respectively, versus 96% and 84% in black recipients. Within this organ procurement organization, black donation has increased over the past 10 years. Unfortunately, there may be a selection bias at the level of referral for liver transplantation. However, once patients are referred to this center for liver transplantation, the rate of transplantation and survival is similar between white and black patients.
Liver Transplantation 12/2003; 4(6):499 - 505. · 3.39 Impact Factor
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Juan L Contreras,
Christopher Eckstein,
Cheryl A Smyth,
Marty T Sellers,
Mario Vilatoba,
Guadalupe Bilbao,
Firoz G Rahemtulla,
Carlton J Young,
J Anthony Thompson,
Irshad H Chaudry,
Devin E Eckhoff
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ABSTRACT: Although approximately 1 million islets exist in the adult human pancreas, current pancreas preservation and islet isolation techniques recover <50%. Presently, cadaveric donors remain the sole source of pancreatic tissue for transplantation. Brain death is characterized by activation of proinflammatory cytokines and organ injury during preservation and reperfusion. In this study, we assessed the effects of brain death on islet isolation yields and functionality. Brain death was induced in male 250- to 350-g Lewis rats by inflation of a Fogarty catheter placed intracranially. The rats were mechanically ventilated for 2, 4, and 6 h before removal of the pancreas (n = 6). In controls, the catheter was not inflated (n = 6). Shortly after brain death induction, a significant increase in serum tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 was demonstrated in a time-dependent manner. Upregulation of TNF-alpha, IL-1beta, and IL-6 mRNA was noted in the pancreas. Brain death donors presented lower insulin release after glucose stimulation assessed by in situ perfusion of the pancreas. Islet recovery was reduced in brain death donors compared with controls (at 6 h 602.3 +/- 233.4 vs. 1,792.5 +/- 325.4 islet equivalents, respectively; P < 0.05). Islet viability assessed in dissociated islet cells and in intact cultured islets was reduced in islets recovered from brain death donors, an effect associated with higher nuclear activities of NF-kappaB p50, c-Jun, and ATF-2. Islet functionality evaluated in vitro by static incubation and in vivo after intraportal transplantation in syngeneic streptozotocin-induced diabetic rats was significantly reduced in preparations obtained from brain death donors. In conclusion, brain death significantly reduced islet yields and functionality. These observations may lead to strategies to reduce the effects of brain death on pancreatic islets and improve the results in clinical transplantation.
Diabetes 12/2003; 52(12):2935-42. · 8.29 Impact Factor
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ABSTRACT: The c-Jun N-terminal kinase (JNK) activation occurs after islet isolation, oxidative stress, and proinflammatory cytokine (PIC) exposure to beta-cells. Previous studies demonstrated that 17beta-estradiol modulates the activity of JNK; therefore we assessed the effects of 17beta-estradiol on JNK activation on islet survival and function after transplantation.
Isolated human pancreatic islets were incubated with PIC and 17beta-estradiol. Viability was analyzed by a colorimetric assay, islet mass by DNA content, JNK activity by Western blots, AP-1 nuclear activity with a promoter-Luciferase AP-1 responsive construct, and c-Fos, Jun-D, and ATF-2 nuclear activities by an enzyme-linked immunosorbent assay. Islet functionality was evaluated after transplantation in streptozotocin-induced diabetic NOD-SCID mice.
The 17beta-estradiol enhanced islet viability and islet mass after exposure to PIC. A significant reduction in JNK activation occurred in islets treated with 17beta-estradiol, compared with controls, an effect partially dependent on estrogen receptors. The 17beta-estradiol induced a significant reduction in nuclear AP-1, c-fos, Jun-D, and ATF-2 activities. Animals that received 17beta-estradiol-treated islets had better islet functionality compared with saline solution-treated controls.
The 17beta-estradiol improved isolated human pancreatic islets survival after PIC exposure by inhibition of JNK. These effects were associated with reduction in JNK targets, including the nuclear activities of transcription factors AP-1, c-Jun, c-Fos, Jun-D and ATF-2, involved in apoptosis in beta-cells. The 17beta-estradiol therapy may improve the results in clinical transplantation.
Surgery 09/2003; 134(2):169-79. · 3.10 Impact Factor
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ABSTRACT: Pancreatic islet transplantation has been validated as a treatment for type 1 diabetes. However, a high number of islets is required to establish euglycemia. Transplantation of islets leads to loss of islet vasculature, which requires revascularization to ensure adequate survival. Islet vascular density in transplanted islets is markedly decreased compared with endogenous islets. The feasibility of revascularization of ischemic tissues by mobilizing endothelial progenitor cells or angioblasts has been demonstrated. Therefore, we investigated the therapeutic potential of angioblast mobilization for stimulation of islet revascularization and therefore engraftment after transplantation.
FVB/NJ mice underwent bone marrow transplantation from transgenic mice constitutively expressing beta-galactosidase encoded by LacZ under regulation of the endothelial cell-specific promoter TIE-2 (FEV/NJ-TIE-2-LacZ). Three weeks after reconstitution, animals received an intrahepatic islet syngeneic infusion (FVB/NJ donors). The contribution of angioblasts into sites of islet revascularization was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), beta-galactosidase (beta-gal) activity, and immunohistochemistry. Islet vascular density was assessed morphometrically followed by in situ BS-1 lectin staining and functional islet mass after transplantation by metabolic studies. Angioblasts were mobilized with murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.5 microg/day/7 days).
An islet dose-dependent increase in beta-gal was demonstrated after transplantation. These results were confirmed by RT-PCR and immunohistochemistry. GM-CSF increased the number of peripheral angioblasts and their localization into sites of islet revascularization. A significant increase in islet vascular density was observed in animals treated with GM-CSF versus controls. Higher functional islet mass was demonstrated in animals treated with GM-CSF.
Augmentation of angioblasts in the peripheral circulation resulted in higher islet vascular density and engraftment. This novel strategy may improve the results in clinical islet transplantation.
Surgery 09/2003; 134(2):390-8. · 3.10 Impact Factor
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ABSTRACT: New strategies for improving durable functional islet mass will be instrumental in facilitating islet transplantation as a cure for type 1 diabetes mellitus. The ability to transfer immunoregulatory or cytoprotective genes into pancreatic islets may enhance survival. Adenoviral vectors (Ad5) have been used widely to deliver therapeutic genes to different tissues. Limitations associated with the use of Ad5 for gene therapy are related to the reliance of the virus on the presence of its primary receptor, the transient nature of the transgene expression, and the immediate inflammatory and immune response elicited by the infection. Because the arginine-glycine-aspartame (RGD) and polylysine (pK7) motifs have been shown to enhance Ad5 infection through an Ad5 receptor-independent pathway, we hypothesized that they could act additively to improve infectivity and reduce toxicity to isolated human pancreatic islets (IHPI).
Hand-picked IHPI were infected with nonmodified Ad5, single-modified Ad5 with RGD (Ad5RGD) or pK7 (ad5pK7), and Ad5RGDpK7. Transfection efficiency was evaluated by green fluorescent protein and luciferase expression. Apoptosis was assessed using a quantitative assay, activation of caspase 3 by a colorimetric assay, nuclear factor (NF)-kappaB nuclear translocation using a promoter-luciferase NF-kappaB responsive construct, regulated on activation normal T-cell expressed and secreted (RANTES) by enzyme-linked immunosorbent assay. In vivo functionality was evaluated after transplantation into diabetic nonobese diabetic severe combined immunodeficiency mice.
Compared with unmodified and singly-modified Ad5 vectors, Ad5RGDpK7 demonstrated the highest infectivity. After the infection of IHPI with adenoviral vectors using the minimal dose required to infect greater than 80% of the islet cells (Ad5, 500 viral particles [VP]/cell; Ad5RGD and Ad5pK7, 10 VP/cell; Ad5RGDpK7, 0.1 VP/cell), islets infected with Ad5RGDpK7 presented a significant reduction in apoptosis, NF-kappaB nuclear translocation, RANTES expression, and higher glucose disposal rate; reduced Ad5-driven specific Th1 and antibody response were also observed.
Ad5RGDpK7 exhibited higher transfection efficiency, allowing a significant reduction in the viral dose required to infect greater than 80% of the islet cells. The reduction in the viral dose was associated with reduced toxicity, inflammation, and immune responses related to Ad5 infection. This strategy may thus be used to successfully modify isolated pancreatic islets.
Transplantation 08/2003; 76(1):252-61. · 4.00 Impact Factor
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ABSTRACT: A variety of toxic insults can result in endoplasmic reticulum (ER)-stress that ultimately leads to apoptosis. beta-cells have a highly developed ER due to a great commitment to insulin production. The present study was carried out to determine the role of ER-stress in isolated human pancreatic islet apoptosis, and the potential protective effects of Bcl-2. Isolated human islets were infected with an adenoviral vector encoding Bcl-2 and then exposed to brefeldin-A, tunicamycin, A23187 and pro-inflammatory cytokines. Activation of caspase-12 was analyzed by means of Western blots. Apoptosis was evaluated using a commercial quantitative assay. ER-stress-inducers promoted caspase-12 activation and apoptosis, effect reversed by overexpression of Bcl-2. Co-localization of caspase-12 and Bcl-2 in the microsomal islet fractions were demonstrated by means of Western blots. We can conclude that the current studies highlight the importance of Bcl-2 as an anti-apoptotic protein, and shed new light on the mechanisms underlying its cytoprotective effects on pancreatic islets.
Transplant International 08/2003; 16(7):537-42. · 2.92 Impact Factor
