Rauf Shahbazov

Evaluation of Microrna375 as a Novel Biomarker for Graft Damage in Clinical Islet Transplantation

Background Early and sensitive detection of islet graft damage is essential for improving posttransplant outcomes. MicroRNA 375 (miR375) has been reported as a biomarker of pancreatic &bgr;-cell death in small animal models. Methods The miR375 levels were measured in purified human islets, sera from patients with autologous and allogeneic islet transplantation as well as total pancreatectomy alone (nontransplanted group). The miR375 levels were also determined in a miniaturized in vitro tube model comprising human islets and autologous blood. Results The miR375 expression level in islets was dose-dependent (P < 0.001) and significantly elevated after islet damage in plasma in the in vitro model (P = 0.003). Clinical analysis revealed that circulating miR375 levels in both autologous and allogeneic islet recipients were significantly elevated for 7 days after islet infusion, compared with the nontransplanted group (P = 0.005 and <0.001, respectively). Furthermore, miR375 detected the difference in islet graft damage among 3 different anti-inflammatory protocols for clinical autologous transplantation (P < 0.01). Conclusions Circulating miR375 can be a reliable biomarker to detect graft damage in clinical islet transplantation because serum C-peptide and proinsulin levels are difficult to interpret due to the influence of multiple factors, such as &bgr;-cell stress and physiological response.

Alleviation of instant blood-mediated inflammatory reaction in autologous conditions through treatment of human islets with NF-κB inhibitors.

Background The instant blood-mediated inflammatory response (IBMIR) has been shown as a major factor that causes damage to transplanted islets. Withaferin A (WA), an inhibitor of nuclear factor (NF) &kgr;B, was shown to suppress the inflammatory response in islets and improve syngeneic islet graft survival in mice. We investigated how treating islets with NF-&kgr;B inhibitors affected IBMIR using an in vitro human autologous blood islet model. Methods Human islets were pretreated with or without NF-&kgr;B inhibitors WA or CAY10512 before mixing autologous blood in a miniaturized in vitro tube model. Plasma samples were collected at multiple time points and used for the measurement of C-peptide, proinsulin, thrombin-antithrombin (TAT) complex, and a panel of proinflammatory cytokines. Infiltration of neutrophils into islets was analyzed using immunohistochemistry. Results Rapid release of C-peptide and proinsulin was observed 3 hr after mixing islets and blood in the control group, but not in the NF-&kgr;B inhibitor–treated groups, whereas TAT levels were elevated in all three groups with a peak at 6 hr. Significant elevation of proinflammatory cytokines was observed in the control group after 3 hr, but not in the treatment groups. Significant inhibition of neutrophil infiltration was also observed in the WA group compared with the control (P<0.001) and CAY10512 (P<0.001) groups. Conclusions A miniaturized in vitro tube model can be useful in investigating IBMIR. The presence of NF-&kgr;B inhibitor could alleviate IBMIR, thus improving the survival of transplanted islets. Protection of islets in the peritransplant phase may improve long-term graft outcomes.

NFAT targets signaling molecules to gene promoters in pancreatic β-cells.

Nuclear factor of activated T cells (NFAT) is activated by calcineurin in response to calcium signals derived by metabolic and inflammatory stress to regulate genes in pancreatic islets. Here, we show that NFAT targets MAPKs, histone acetyltransferase p300, and histone deacetylases (HDACs) to gene promoters to differentially regulate insulin and TNF-α genes. NFAT and ERK associated with the insulin gene promoter in response to glucagon-like peptide 1, whereas NFAT formed complexes with p38 MAPK (p38) and Jun N-terminal kinase (JNK) upon promoters of the TNF-α gene in response to IL-1β. Translocation of NFAT and MAPKs to gene promoters was calcineurin/NFAT dependent, and complex stability required MAPK activity. Knocking down NFATc2 expression, eliminating NFAT DNA binding sites, or interfering with NFAT nuclear import prevented association of MAPKs with gene promoters. Inhibiting p38 and JNK activity increased NFAT-ERK association with promoters, which repressed TNF-α and enhanced insulin gene expression. Moreover, inhibiting p38 and JNK induced a switch from NFAT-p38/JNK-histone acetyltransferase p300 to NFAT-ERK-HDAC3 complex formation upon the TNF-α promoter, which resulted in gene repression. Histone acetyltransferase/HDAC exchange was reversed on the insulin gene by p38/JNK inhibition in the presence of glucagon-like peptide 1, which enhanced gene expression. Overall, these data indicate that NFAT directs signaling enzymes to gene promoters in islets, which contribute to protein-DNA complex stability and promoter regulation. Furthermore, the data suggest that TNF-α can be repressed and insulin production can be enhanced by selectively targeting signaling components of NFAT-MAPK transcriptional/signaling complex formation in pancreatic β-cells. These findings have therapeutic potential for suppressing islet inflammation while preserving islet function in diabetes and islet transplantation.

Pancreatic Beta Cell-derived IP-10/CXCL10 Isletokine Mediates Early Loss of Graft Function in Islet Cell Transplantation.

Pancreatic islets produce and secrete cytokines and chemokines in response to inflammatory and metabolic stress. The physiological role of these “isletokines” in health and disease is largely unknown. We observed that islets release multiple inflammatory mediators in patients undergoing islet transplants within hours of infusion. The proinflammatory cytokine interferon-γ–induced protein 10 (IP-10/CXCL10) was among the highest released, and high levels correlated with poor islet transplant outcomes. Transgenic mouse studies confirmed that donor islet–specific expression of IP-10 contributed to islet inflammation and loss of β-cell function in islet grafts. The effects of islet-derived IP-10 could be blocked by treatment of donor islets and recipient mice with anti–IP-10 neutralizing monoclonal antibody. In vitro studies showed induction of the IP-10 gene was mediated by calcineurin-dependent NFAT signaling in pancreatic β-cells in response to oxidative or inflammatory stress. Sustained association of NFAT and p300 histone acetyltransferase with the IP-10 gene required p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) activity, which differentially regulated IP-10 expression and subsequent protein release. Overall, these findings elucidate an NFAT-MAPK signaling paradigm for induction of isletokine expression in β-cells and reveal IP-10 as a primary therapeutic target to prevent β-cell–induced inflammatory loss of graft function after islet cell transplantation.

Pancreatic Ductal Perfusion at Organ Procurement Enhances Islet Yield in Human Islet Isolation

Objective Pancreas preservation is a major factor influencing the results of islet cell transplantation. This study evaluated the effects of 2 different solutions for pancreatic ductal perfusion (PDP) at organ procurement. Methods Eighteen human pancreases were assigned to 3 groups: non-PDP (control), PDP with ET-Kyoto solution, and PDP with cold storage/purification stock solution. Pancreatic islets were isolated according to the modified Ricordi method. Results No significant differences in donor characteristics, including cold ischemia time, were observed between the 3 groups. All islet isolations in the PDP groups had more than 400,000 islet equivalence in total islet yield after purification, a significant increase when compared with the control (P = 0.04 and P < 0.01). The islet quality assessments, including an in vivo diabetic nude mice assay and the response of high-mobility group box protein 1 to cytokine stimulation, also showed no significant differences. The proportion of terminal deoxynucleotidyl transferase dUTP nick-end labeling–positive cells showing apoptosis in islets in the PDP groups was significantly lower than in the control group (P < 0.05). Conclusions Both ET-Kyoto solution and cold storage/purification stock solution are suitable for PDP and consistently resulted in isolation success. Further studies with a larger number of pancreas donors should be done to compare the effects of the PDP solutions.

Essential phospholipids prevent islet damage induced by proinflammatory cytokines and hypoxic conditions

The pancreatic islet damage that occurs through an inflammatory response and hypoxia after infusion is a major hurdle in islet transplantation. Because essential phospholipids (EPL) have been shown to exhibit anti‐inflammatory properties in liver disease, we analysed their protective effect on islets in inflammatory or hypoxic conditions.

Laparoscopic Versus Finger-Assisted Open Donor Nephrectomy Techniques: A Safe Alternative

Background Advances in minimally invasive surgery for live kidney donor nephrectomy have led to the use of the laparoscopic and robotic technique as the standard of care. The aim of the study was to compare an open finger-assisted open donor nephrectomy (FAODN) versus standard laparoscopic donor nephrectomy (LDN). Methods Retrospective demographic and surgical data were analyzed using the electronic medical records at the University of Virginia and Imperial College Hospital. The analysis included 95 consecutive donors in each center undergoing donor nephrectomy. LDN and FAODN were compared using Fishers Exact Test and Likelihood Ratio Chi-Square. Results Overall, donor demographics and clinical characteristics were similar between groups. The FAODN group had less females donors (70.5% vs. 29.5%, p=0.003) with similar median body mass index (BMI) (28 vs. 26, p=0.032). The LDN group had longer operative duration (3.5 vs. 1.2 hours, p<0.001), longer combined length of incision (6 vs. 5 cm. p=0.001), with shorter median hospital length of stay (LOS) (3 vs. 4 days, p<0.001). Left nephrectomy was preferred in both groups. Minor postoperative complications occurred less often in the FAODN group (14.7% vs. 31.6%, p=0.0094) while rates of hernia, operative blood transfusions and postoperative bleeding episodes were similar between groups. Recipients of LDN donors demonstrated a higher creatinine (1.1 vs. 0.9 mg/dl, p<0.001), and lower recipient GFR at 1 year (60 vs. 89 ml/min/1.73m2, p<0.001) post-donation. Conclusions Our study demonstrates that FAODN is a successful alternative to LDN. It appears to provide kidney donors with favorable outcomes in terms of complications and outcomes, and recipients with excellent renal function at 1 year post-donation.

Hemarthrosis: Concurrent acute presentation of pyrophosphate dehydrate and uric acid crystals in an elderly patient with a history of rheumatoid arthritis diagnosed with septic arthritis -

Concomitant septic arthritis in the presence of crystalline disease is a rare presentation of acute hemarthrosis and knee pain. Literature review showed that co-occurrence of these entities is an infrequent phenomenon but it needs to be acknowledged that these studies are few in number and were done on small patient population. This case challenges the notion that presence of crystals in the synovial fluid rules out septic arthritis even in the setting of low synovial WBC count. Additionally, the presence of pseudogout in patients suffering from gout is a rare entity as well. These findings in literature are described in case reports dispersed over the past three decades. We present a case where concurrent treatment of gout, pseudogout, and septic arthritis in a patient who presented with acute hemarhtrosis.

The Endoscopic Pancreas Function Test Is Useful to Detect Pancreatic Exocrine Insufficiency and Predict Results in Autologous Islet Transplantation for Chronic Pancreatitis.: Abstract# C1699

C1701 The Development of Gold Nanoparticles as an Ideal Non-Viral Nanoscale Delivery System for Islet Transplantation. D. Gutierrez,1,2 Y. Wang,2 J. Oberholzer.1,2 1Bioengineering, University of Illinois at Chicago, Chicago; 2Surgery, University of Illinois at Chicago, Chicago. Islet transplantation is a promising therapy for T1DM, but shows variable success. A long-standing goal is to develop an effi cient system for the delivery of molecular cargos to islets in order to improve islet transplant outcomes. Common viral and non-viral systems can only deliver molecular cargos to the periphery cells of an islet. Most of these systems alternate islet function, pose potential oncogenic risks, and increase immunogenicity. Gold nanoparticles are macromolecular nanostructures with high biocompatibility containing void spaces in their interior regions and modifi able surface groups that allow conjugation of therapeutic drugs and proteins. Gold nanoparticles of 12 nm in size have demonstrated a high effi cacy to enter cells without auxiliary agents and minimal cytotoxicity. We hypothesize that gold nanoparticles could be used as a unique vehicle to deliver functional molecular cargos for islets. Previous studies performed in the lab indicated the ability to use Cy5 labeled gold nanoparticles conjugated with a specifi c oligonucleotide sequence. A new spherical nanoparticle tagged with Cy5 was tested for penetration effi cacy. Gold nanoparticles were conjugated with mRNA and tagged with green fl uorescent protein (GFP) in order to further determine the stability and time required for the mRNA to be delivered and expressed. Gold nanoparticle transfection effi cacy was tested in human islets using confocal microscopy and optimal concentration was determined. Islet function was evaluated by measuring the calcium infl ux and insulin secretion in response to glucose. (i) Confocal images showed high uptake of gold nanoparticles including islet cores. (ii) 12 nM of gold nanoparticles conjugated with Cy5, Cy5/Oligonucleotide and GFP/mRNA were tested for 24 hours, 48 hours and 72 hours of incubation in order to determine the optimal condition. (iii) The functionality of gold nanoparticle-treated islets was well preserved after 72 hours. There was no signifi cant difference in the intracellular calcium concentration of the control group versus the islets treated with gold nanoparticles (p>0.05). Mitochondrial potential indices demonstrated similarity between the control group and islets treated with gold nanoparticles (p>0.05). With the ability to use gold nanoparticles as a delivery system to islets with high transfection effi cacy will allow the possibility to conjugate a specifi c protein or siRNA. Abstract# C1702 Antigen-Specific Therapy With Human Proinsulin and IL10 in Combination With Short-Course Monoclonal CD3 Antibody in Preclinical Models of Islet Transplant. J. Monteiro Carvalho Mori da,1 T. Takiishi,1 T. Van Belle,1 H. Korf,1 P. Rottiers,2 L. Steidler,2 C. Gysemans,1 C. Mathieu.1 1Clinical and Experimental Endocrinology, KULeuven, Leuven, Belgium; 2Actogenix Nv, Zwijnaarde (Ghent), Belgium. Beta-cell replacement into a diabetic patient with pre-existing immunity to islet auto-antigens (Ags) results in autoimmunity recurrence after islet transplantation. Anti-CD3 monoclonal antibodies remain the most promising immune therapy for reversing type 1 diabetes but lack antigen-specifi city. Induction of Ag-specifi c tolerance by auto-Ag peptides/proteins delivered to the gastrointestinal tract by genetically-modifi ed food-grade lactic acid bacterium Lactococcus lactis (L. lactis) represents an attractive alternative approach but its therapeutic effi cacy in islet transplantation remains to be determined. The present study investigated whether a short-course sub-therapeutic dose of monoclonal CD3 antibody (anti-CD3) alone C1702 Antigen-Specific Therapy With Human Proinsulin and IL10 in Combination With Short-Course Monoclonal CD3 Antibody in Preclinical Models of Islet Transplant. J. Monteiro Carvalho Mori da,1 T. Takiishi,1 T. Van Belle,1 H. Korf,1 P. Rottiers,2 L. Steidler,2 C. Gysemans,1 C. Mathieu.1 1Clinical and Experimental Endocrinology, KULeuven, Leuven, Belgium; 2Actogenix Nv, Zwijnaarde (Ghent), Belgium. Beta-cell replacement into a diabetic patient with pre-existing immunity to islet auto-antigens (Ags) results in autoimmunity recurrence after islet transplantation. Anti-CD3 monoclonal antibodies remain the most promising immune therapy for reversing type 1 diabetes but lack antigen-specifi city. Induction of Ag-specifi c tolerance by auto-Ag peptides/proteins delivered to the gastrointestinal tract by genetically-modifi ed food-grade lactic acid bacterium Lactococcus lactis (L. lactis) represents an attractive alternative approach but its therapeutic effi cacy in islet transplantation remains to be determined. The present study investigated whether a short-course sub-therapeutic dose of monoclonal CD3 antibody (anti-CD3) alone

Implication of IFNγ Production in Clinical Allogeneic Islet Transplantation in Contrast to Autologous Transplantation.: Abstract# 599

599 Implication of IFNγ Production in Clinical Allogeneic Islet Transplantation in Contrast to Autologous Transplantation. M. Takita,1 R. Shahbazov,1 F. Kunnathodi,1 M. Kanak,2 M. Lawrence,1 H. Ueno,3 B. Naziruddin,4 M. Levy.4 1Islet Cell Laboratory, Baylor Research Institute, Dallas, TX; 2Institute of Biomedical Studies, Baylor University, Waco, TX; 3Baylor Institute for Immunology Research, Dallas, TX; 4Baylor Annette C. and Harold C. Simmons Transplant Institute, Dallas, TX. Introduction: We have shown that the autoimmune response to GAD65 peptide is a crucial cause of graft dysfunction in clinical islet transplantation for type 1 diabetes and a signifi cant IFNγ release from CD4+ or CD8+ T cells which corresponded with the autoimmune response. In this study, the serum IFNγ levels in allogeneic islet recipients were assessed, in comparison with autologous recipients. Methods: Six allogeneic and 6 autologous islet recipients were included. Patients in both groups matched in basic characteristics. There was no difference in transplanted islet dose between both groups: 9,393±707 and 7,949±672 IEQ/kg of body weight, respectively (p=0.17). All recipients were administered with both etanercept and anakinra from Day 0 to 10. Autoimmune response was investigated with EpiMax assay after stimulation of peripheral blood mononuclear cells by GAD65 peptide clusters. Cytokine levels were measured using Luminex assay. Results: Signifi cant IFNγ production from both CD4+ and CD8+ T cells was observed at peri-transplant in allogeneic islet recipients (Fig. A). Signifi cant elevation of serum IFNγ levels were also seen immediately after islet infusion in allo-recipients and the elevation were sustained for 7 days (Fig. B). Autologous recipients, however, did not show signifi cant increase in IFNγ levels. Conclusions: Signifi cant difference in serum IFNγ levels was observed between allogeneic and autologous islet recipients even when similar dose of islets were transplanted. Further characterization of IFNγ production may help to determine the role of autoimmune response in clinical islet transplantation. Figure. An example of IFNγ production in EpiMax assay in an allogeneic recipient (A) and serum IFNγ levels in islet transplantation (B) are shown. Abstract# 600 The Instant Blood Mediated Inflammatory Reaction in a Dual Transplant Model of Islet Transplantation. K. Samy,1 B. Martin,1 M. Lowe,1 P. Thompson,1 A. Anderson,1 J. Cano,1 M. Song,1 F. Leopardi,1 E. Strobert,2 A. Kirk.1 1Emory Transplant Center; 2Yerkes National Primate Research Center, Atlanta, GA. Purpose: Signifi cant islet loss following intraportal infusion is attributed to a poorly defi ned Instant Blood Mediated Infl ammatory Reaction (IBMIR). We developed a nonhuman primate (NHP) dual transplant model to study factors contributing to early posttransplant alloand xeno-islet loss. Methods: Isolated islets were infused intraportally into rhesus macaques, with comparator islet preps separated into the right and left hemiliver. We assessed rhesus alloislets(AI) vs wild-type neonatal pig islets(WT)(n=4) and α-1,3-galactosyl transferase knockout neonatal pig islets(GKO) vs inert microspheres(MS)(n=6). Digital immunohistological analysis using Aperio Imagescope® software staining quantifi cation was performed on protocol liver sections taken at 1 and 24 hours post-transplant. Results: Comparing AI vs WT islets, galactose-α-1,3-galactose staining was distinct at 1h and 24h(p<0.05) indicating successful islet type segregation. C4d, IgG, IgM, macrophages, neutrophils, platelets, and caspase 3 were similar between islet phenotypes at 1 and 24 hours. Complement and antibody deposition unexpectedly increased over 24 hours in AI but not WT islets. Macrophage staining increased over 24 hours in WT(p=0.02) and AI(p=0.07) with increasing infi ltration observed on histology. Interestingly, inert MS vs GKO at one hour had no difference in staining of C4d, IgG, IgM, macrophages, or neutrophils(p>0.05). At 24 hours, GKO islets stained stronger than MS for C4d(p=0.06), neutrophils(p=0.04), macrophages(p=0.02), and platelets(p=0.02). Conclusions: These data suggest that complement is a signifi cant component of both alloand xeno-specifi c responses. Similarly, antibody deposition occurs in both and is initially non-specifi c. Macrophage infi ltration also plays a signifi cant role in the response to both islet phenotypes. The embolic phenomenon in the portal sinusoids also contributes to IBMIR, although this may be self-contained as infused cell antigenicity seemed to propagate the reaction. Thus, early IBMIR is likely a largely innate response initially driven by non-specifi c features of portal vein embolization, with subsequent infl ammatory responses common to both alloand xeno-islet preps. Differential alloor xeno-specifi c interactions likely manifest beyond 24 hours.