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ABSTRACT: Exenatide is an analog of the incretin hormone glucagon-like peptide (GLP-1) that is used for the treatment of T2D for their metabolic effects. In addition to its insulinotropic effects, Exenatide increases functional islet mass and improves their survival. Improved outcomes have been reported in recent clinical islet transplantation trials for the treatment of type 1 diabetes. The purpose of this study was to investigate whether Exenatide has anti-inflammatory properties in human islets. Exenatide treatment improved islet function, significantly reduced content of inflammation-related molecules (tissue factor, IFN- γ, IL-17, IL-1β and IL-2) and caspase 3 activation, whereas increased phosphorylation of ERK1/2, STAT3 and Akt in vitro. Immunostaining showed expression of GLP-1R in β-cells but not in α-cells. IL-1β colocalized with GLP-1R in β-cells. Induction of serine proteinase inhibitor 9 (PI-9) was detected after exposure of human islets to Exenatide in vitro and after transplantation into immunodeficient mice. GLP-1 induced PI-9 in vitro but to a lower extent than Exenatide. This effect was partially blocked by the antagonist Exendin-9 in vitro. As assessed by immunostaining PI-9 is mostly expressed in β-cells but not in α-cells. In conclusion, we describe anti-inflammatory and cytoprotective properties of Exenatide in human islets. Exenatide-mediated PI-9 expression, the only known Granzyme B inhibitor, unveils potential immunoregulatory properties.
Cell Transplantation 06/2011; · 5.13 Impact Factor
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ABSTRACT: We used a rat model of pancreas cold preservation to assess its effects on islets. Glands were surgically retrieved and stored in University of Wisconsin (UW) solution for 3 hours (Short) or 18 hours (Long) cold ischemia time (CIT). Islet yield was significantly lower in the Long-CIT than the Short-CIT group, as well as islet recovery after overnight culture (P < .01). Islet cell viability after isolation was significantly reduced in the Long-CIT group (P < .05). Reversal of diabetes following transplantation of suboptimal islet grafts occurred earlier in the Short-CIT group than the Long-CIT. All animals in the Short-CIT group and 80% in the Long-CIT group achieved euglycemia. Freshly isolated islets showed a significant increase of JNK and p38 (P < .05) phosphorylation in Long-CIT compared with Short-CIT. Histopathological assessment of the pancreas showed a significantly higher injury score. Proteomic analysis of pancreatic tissue led to identification of 5 proteins consistently differentially expressed between Short-CIT and Long-CIT. Better understanding of the molecular pathways involved in this phenomenon will be of assistance in defining targeted interventions to improve organ use in the clinical arena.
Transplantation Proceedings 06/2009; 41(5):1808-9. · 1.00 Impact Factor
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A. Pileggi,
M. M. Ribeiro,
A. R. Hogan,
R. D. Molano,
J. Embury,
L. Cobianchi,
H. Ichii,
L. Inverardi,
A. Fornoni,
C. Ricordi, R. L. Pastori
American Journal of Transplantation. 01/2009; 9:671-672.
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A. R. Hogan,
M. Doni,
R. D. Molano,
L. Cobianchi,
J. Molina,
E. Zahr,
A. Szeto,
M. Ribeiro,
A. Mendez,
C. Ricordi, R. L. Pastori,
A. Pileggi
American Journal of Transplantation. 01/2009; 9:670-671.
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ABSTRACT: CD40 expression on non-haematopoietic cells is linked to inflammation. We previously reported that CD40 is expressed on isolated human and non-human primate islets and its activation results in secretion of IL-8, macrophage inflammatory protein 1-beta (MIP-1beta) and monocyte chemoattractant protein-1 (MCP-1) through nuclear factor-kappaB and extracellularly regulated kinases 1/2 pathways. The objective of this study was to identify the pattern of gene expression, and to study viability and functionality affected by CD40-CD40 ligand (CD40L) interaction in human islets. Furthermore, we have studied the CD40-mediated cytokine/chemokine profile in pancreatic ductal cells, as they are always present in human islet transplant preparations and express CD40 constitutively.
CD40-CD40L gene expression modulation was studied by microarray on islet cells depleted of ductal cells. Selected genes were validated by quantitative RT-PCR. The cytokine profile in purified ductal cells was evaluated by Luminex technology, based on the use of fluorescent-coated beads, known as microspheres, and capable of multiplex detection of proteins from a single sample. Glucose-stimulated insulin secretion and islet viability were assessed by perifusion and 7-aminoactinomycin D membrane exclusion, respectively.
Statistical analysis of microarrays identified 30 genes exhibiting at least a 2.5-fold increase across all replicate arrays. The majority of them were related to oxidative stress/inflammation. Prominently upregulated were chemokine C-X-C motif ligand 1 (CXCL1), CXCL2 and CXCL3 belonging to the CXC family of chemokines related to IL-8. CD40-mediated CXCL1 secretion was confirmed by ELISA. The viability or in vitro function was not affected by CD40 activation. In addition to previously reported IL-8, MIP-1beta and MCP-1, CD40 stimulation in ductal cells produced IL-1beta, IFN-gamma, TNF-alpha, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.
CD40 activation in islets and ductal cells produces cytokines/chemokines with a broad-spectrum range of biological functions.
Diabetologia 10/2008; 51(10):1853-61. · 6.81 Impact Factor
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ABSTRACT: Activation of c-jun N-terminal kinase (JNK) has been described in islet isolation and engraftment, making JNK a key target in islet transplantation. The objective of this study was to investigate if JNK inhibition with a cell-permeable TAT peptide inhibitor (L-JNKI) protects functional beta cell mass in human islets and affects AKT and its substrates in islet cells.
The effect of L-JNKI (10 micromol/l) on islet count, mitochondrial membrane potential, glucose-stimulated insulin release and phosphorylation of both AKT and its substrates, as well as on reversal of diabetes in immunodeficient diabetic Nu/Nu mice was studied.
In vitro, L-JNKI reduced the islet loss in culture and protected from cell death caused by acute cytokine exposure. In vivo, treatment of freshly isolated human islets and diabetic Nu/Nu mice recipients of such islets resulted in improved functional beta cell mass. We showed that L-JNKI activates AKT and downregulates glycogen synthase kinase-3 beta (GSK-3B) in human islets exposed to cytokines, while other AKT substrates were unaffected, suggesting that a specific AKT/GSK-3B regulation by L-JNKI may represent one of its mechanisms of cytoprotection.
In conclusion, we have demonstrated that targeting JNK in human pancreatic islets results in improved functional beta cell mass and in the regulation of AKT/GSK3B activity.
Diabetologia 03/2008; 51(2):298-308. · 6.81 Impact Factor
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ABSTRACT: Despite differences in function and embryonic origin, pancreatic islet cells and neurons express proteins belonging to the tumour necrosis factor receptor superfamily. While neurons express the CD40 receptor, it is unknown whether islet cells also express it. We investigated CD40 expression in human and mouse pancreatic islets as well as in NIT-1 insulinoma cells.
CD40 expression was studied by reverse transcriptase polymerase chain reaction, flow cytometry, immunohistochemistry and western blot. Responses mediated by CD40 were assessed by a luciferase gene reporter assay following stimulation with a CD40 agonist antibody.
We found that CD40 is expressed in mouse and human pancreatic islet cells. CD40 is expressed by beta cells, and its expression is upregulated by proinflammatory cytokines (IL-1beta, IFN-gamma and TNF-alpha). CD40 signalling in NIT-1 insulinoma cells activates nuclear factor kappa-B, demonstrating that CD40 is functional.
We present evidence that, in addition to immune cell types, mouse and human pancreatic beta cells express CD40. Its expression is upregulated by proinflammatory stimuli, and signalling through this receptor activates NF-kappaB. We suggest that the effects of inflammatory stimuli that affect beta cell function and survival may be also mediated by signalling through the CD40 receptor. Thus, CD40 may have a role in processes associated with islet autoimmunity and transplantation.
Diabetologia 03/2005; 48(2):268-76. · 6.81 Impact Factor
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A Pileggi,
R D Molano,
T Berney,
P Cattan,
C Vizzardelli,
R Oliver,
C Fraker,
C Ricordi, R L Pastori,
F H Bach,
L Inverardi
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ABSTRACT: Transplantation of islets of Langerhans represents a viable therapeutic approach for the treatment of type 1 diabetes. Unfortunately, transplanted islets are susceptible to allogeneic recognition and rejection, recurrence of autoimmunity, and destruction by local inflammation at the site of implantation. The last of these phenomena might not only result in functional impairment and death of islet cells but could also contribute to amplifying the subsequent specific immune response. Induction of islet cell protection against inflammation could therefore be postulated to be a powerful means to improve overall graft fate. Heme oxygenase-1 (HO-1) has been described as an inducible protein capable of cytoprotection via radical scavenging and apoptosis prevention. The purpose of the present study was to analyze whether HO-1 upregulation in a beta-cell line and in freshly isolated murine islets could result in protection from apoptosis and improve in vivo functional performance. HO-1 upregulation was induced reproducibly with protoporphyrins and was correlated with protection from apoptosis induced in vitro with proinflammatory cytokines or Fas engagement. Furthermore, in vivo HO-1 upregulation resulted in improved islet function in a model of marginal mass islet transplantation in rodents. Strategies aimed at inducing HO-1 upregulation might result in improved success in islet transplantation.
Diabetes 10/2001; 50(9):1983-91. · 8.29 Impact Factor
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J Embury,
D Klein,
A Pileggi,
M Ribeiro,
S Jayaraman,
R D Molano,
C Fraker,
N Kenyon,
C Ricordi,
L Inverardi, R L Pastori
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ABSTRACT: The resounding success of a new immunosuppressive regimen known as the Edmonton protocol demonstrates that islet cell transplantation is becoming a therapeutic reality for diabetes. However, under the Edmonton protocol, a single donor does not provide enough islets to attain the insulin independence of a transplant recipient. This limitation is mainly caused by islet apoptosis triggered during isolation. In this study, we describe a highly efficient system of transiently transferring anti-apoptotic proteins into pancreatic islets, thus opening an exciting new therapeutic opportunity to improve the viability of transplantable islets. We fused beta-galactosidase to the 11-amino acid residues that constitute the protein transduction domain (PTD) of the HIV/TAT protein and transduced pancreatic islets ex vivo with this fusion protein in a dose-dependent manner with >80% efficiency. We observed that transduction of the anti-apoptotic proteins Bcl-X(L) and PEA-15 fused to TAT/PTD prevented apoptosis induced by tumor necrosis factor-alpha in a pancreatic beta-cell line, indicating that TAT/PTD anti-apoptotic proteins retained their biological activity. Finally, we demonstrated that TAT-fusion proteins did not affect the insulin secretion capability of islets, as determined by glucose static incubation and by reversion of hyperglycemia in diabetic immunodeficient mice.
Diabetes 09/2001; 50(8):1706-13. · 8.29 Impact Factor
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ABSTRACT: In this study we have designed and constructed an anti-Fas ribozyme and show that it can specifically cleave the Fas mRNA in vitro. Moreover, to test its efficacy ex vivo, we transfected the anti-Fas ribozyme into betaTC-3 insulinoma cells, using a RNA polymerase III promoter to drive its expression. Like pancreatic beta cells, betaTC-3 cells do not constitutively express Fas, but Fas expression can be induced with IL-1 and IFN-gamma. Transfected cells expressed an average of 5000 copies of anti-Fas ribozyme transcript per cell as assessed by reverse transcriptase-real-time PCR. After IL-1/IFN-gamma treatment, betaTC-3 cells transfected with the anti-Fas ribozyme expressed 80% less Fas compared with mock-transfected cells. In addition, the anti-Fas ribozyme also inhibited Fas expression in NIT-1 insulinoma cells and in primary cultures of dispersed pancreatic islet cells. Inhibition of de novo Fas expression in betaTC-3 cells expressing the anti-Fas ribozyme correlated with resistance to Fas-mediated apoptosis as determined by the number of cells exhibiting caspase 3 proteolytic activity. Hence, we have engineered a ribozyme capable of preventing Fas expression in the betaTC-3 pancreatic insulinoma cell line and conferring resistance to Fas-mediated apoptosis. We suggest that ribozymes may be potentially useful to engineer resistance to apoptosis in transplantable beta cells, a feature that may significantly improve the survival of islet cell grafts.
Human Gene Therapy 06/2000; 11(7):1033-45. · 4.22 Impact Factor
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BioTechniques 06/1999; 26(5):828-30, 832, 834. · 2.67 Impact Factor
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ABSTRACT: Graft-versus-host disease (GVHD) is a feared complication of allogeneic bone marrow transplantation. Research in rodent models has linked perforin and Fas ligand (FasL), two components of independent lytic pathways, with the induction of GVHD. In this study we characterized two hammerhead ribozymes that cleave their target perforin and Fas ligand RNAs with high efficiency in CTLL-2 cells. The perforin and Fas ligand ribozymes were expressed from a tRNA-directed RNA polymerase III promoter that was inserted in an episomal multicopy plasmid derived from papilloma virus. Chimeric anti-perforin and anti-FasL tRNA-ribozymes had sequences engineered in order to have specific secondary structure effects. These sequence modifications allow the formation of a 5' --> 3' stem structure and also place the ribozyme in a flexible bulge region that keeps the ribozyme separated from the tRNA domain. Northern and RT in situ PCR analyses showed high levels of transcription and efficient transportation to the cytoplasm. The expression of perforin and FasL in CTLL-2 cells was significantly reduced as assessed by RNA and protein analyses.
Human Gene Therapy 08/1998; 9(11):1551-60. · 4.22 Impact Factor
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Transplantation Proceedings 07/1997; 29(4):2224-5. · 1.00 Impact Factor
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ABSTRACT: Polyfunctional ribozymes targeting more than one RNA might be ideal for treatment of diseases caused by the expression of more than one mRNA. This is the case of Graft versus Host Disease, which in the mouse model is due to the action of two proteins responsible for different lytic pathways: perforin and fasligand. We have created a bifunctional ribozyme fusing two antiperforin and antifas-ligand hammerhead ribozymes with a stretch of CA dinucleotides. This bifunctional ribozyme is able to recognize and cleave in vitro perforin and fas-ligand mRNA in a specific and selective fashion, with a catalytic efficiency similar to that of its individual components.
Biochemical and Biophysical Research Communications 10/1996; 226(3):595-600. · 2.48 Impact Factor
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ABSTRACT: Adhesion of lymphocytes to the endothelial venules inside the islets of Langerhans seems to initiate the infiltration of islets in NOD mice. An overexpression of the lymphocyte surface molecule CD44 in infiltrated NOD islets compared with peripheral blood lymphocytes was recently reported. The CD44 protein family includes a variety of molecules generated by alternative RNA splicing from 10 variant exons (v1-v10). By using reverse transcriptase-polymerase chain reaction followed by Southern blotting and hybridization to exon-specific cDNA probes, we investigated the expression of CD44 isoforms in highly purified islets of Langerhans from 4- and 10-week-old NOD mice. At least six CD44 isoforms were strongly overexpressed in NOD islets at 4 and 10 weeks when compared with age-matched BALB/c islets. Controls in different tissues indicate that these variants are specifically increased in the islets from the NOD strain. Islets from the NOD-scid/scid strain also expressed these variant exons. Splenocytes from BALB/c did not express CD44 isoforms, whereas splenocytes from 4-week-old NOD mice did express CD44 variants. Treatment with inflammatory mediators induced new isoforms; however, these transcripts have a different variant exon composition from that found in NOD mice islets. These results suggest that some isoforms are expressed very early in the development of insulitis by a component of the NOD islet itself and underscore a possible role of CD44 in islet infiltration.
Diabetes 07/1996; 45(6):718-24. · 8.29 Impact Factor
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Transplantation Proceedings 01/1996; 27(6):3409. · 1.00 Impact Factor
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ABSTRACT: Molecular cloning of the dog homologue of the human CD44 was achieved using RT/PCR. A 1055 bp cDNA has a deduced amino acid sequence of 351 residues, 338 of them correspond to the mature protein. Nine conserved cysteine residues were found. The extracellular region contains a single link superfamily domain on the N-terminal part and potential post-translational modification sites as: N- and O-linked glycosylation sites and chondroitin sulfate attachment sites. Three mRNAs of 2.2, 3.8 and 4.4 kb were identified on Northern blot analysis and Western blot hybridization revealed a 85-90 kDa protein expressed in lymph node tissue.
Biochimica et Biophysica Acta 06/1994; 1218(1):112-4. · 4.66 Impact Factor
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Immunogenetics 02/1994; 39(5):373. · 2.93 Impact Factor
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ABSTRACT: The gene of the CD44 cell surface glycoprotein consists of 20 exons. Ten exons known as variant exons are inserted by alternative RNA splicing between exon 5 and 16, thus generating diversity in the extracellular portion of the protein. We have cloned the dog cDNA homologue of the human variant exon region of CD44 and characterized its transcript expression in normal lymphatic tissues. Using PCR with primers complementary to regions contiguous to the insertion point and a variant exon, all ten variant exons of dog CD44 were detected in a high number of different isoforms. Transcripts containing the ten variable exons directly spliced to both sides of the insertion point were detected. We found an extensive usage of all variant exons from v1 to v10 in a high number of different combinations, some presenting a tissue-specific expression pattern. A similar complex profile of transcript expression was found in peripheral blood lymphocytes from rat and human.
Immunogenetics 02/1994; 40(6):437-44. · 2.93 Impact Factor
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ABSTRACT: Islet transplantation has become an accepted method to treat type 1 diabetes. To succeed and achieve normal levels of glucose in transplant recipients, the quality of the transplanted islets is of the utmost importance. Lack of oxygen during organ procurement, islet isolation, and subsequent culture triggers apoptosis or necrosis and loss of islet function, causing the yield and quality to diminish. A promising candidate for cytoprotection against oxygen deprivation is neuroglobin (Ngb). Ngb is a recently described member of globin family and is expressed in neurons, retina, and pancreatic islets. To overexpress this protein in the islets and study its ability to protect them, we utilized protein transduction. Protein transduction is achieved by fusing Ngb to the TAT/PTD transduction domain, a peptide originated from the HIV transcriptional transactivator protein. Our study proved that TAT-Ngb is an efficient fusion protein capable of protecting the human islets in culture from loss of cell mass and function, thus increasing the quality of transplantable islets. If the islets could be cultured for a longer period of time without suffering harmful effects, it would be possible to precondition the recipient and there would be more time to assess their quality and function before transplantation.
Transplantation Proceedings 37(1):237-40. · 1.00 Impact Factor
