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Transplantation:
doi: 10.1097/TP.0b013e3182a7ab68
Abstracts

IPITA 2013 Abstracts Supplement

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217

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Islet Transplantation Improves Peripheral Neuropathy in Patients With Type 1 Diabetes: A 5 Year Prospective Study

Marie-Christine Vantyghem1, Delphine Quintin1, Wassila Karrouz1, Jean-François Hurtevent1, Violeta Raverdy1, Robert Caiazzo1, Christian Noel1, Julie Kerr-Conte1, Francois Pattou1

1INSERM UMR 859 Biotherapies for diabetes, Lille University Hospital, Lille, France.

Objective: Long-term benefit-risk ratio of islet transplantation remains unclear. We explored the evolution of peripheral and autonomic neuropathy during 5 years after islet transplantation with the Edmonton protocol in type 1 diabetic patients.

Patients and Methods: Twenty-one patients (13 islet-alone and 8 islet-after-kidney) were enrolled in this prospective cohort study. Islet transplantation consisted of 2 or 3 sequential infusions with IL2rAb / sirolimus - tacrolimus immunosuppression. All patients underwent biological evaluation, continuous blood pressure and continuous glucose monitoring (CGM), lower-limb electrophysiological testing and cardiovascular autonomic testing (R-R variation with paced breathing, Valsalva ratio, postural heart rate and blood pressure changes) before transplantation and yearly during 5 years. Outcomes were analyzed in intention to treat.

Results: At 5 years, islet remained functional in 18 patients (85%). Ten patients (48%) were insulin-independent with a median (IQR) HbA1c at 6.0 (5.8–6.7) % vs. 7.8 (6.9-8.3) % in those requiring insulin (p<0.001). The medians of sensory action potential (p<0.05) and both sensory and motor nerve conduction velocities (p<0.01) improved between 0 and 5 years. All 4 parameters significantly correlated negatively with mean glucose / CGM and all outcomes except sensory nerve conduction velocity correlated negatively with triglycerides (p≤0.01). Sensory conduction velocity correlated negatively with glucose variability (SD) / CGM (p<0.01). Tacrolimus levels negatively correlated with motor conduction parameters (p≤0.02). All four parameters correlated positively with ß score or post-prandial C-peptide level (p<0.05). Cardiovascular reflex testing did not change over the 5–year follow-up.

Conclusion: islet-alone or after-kidney transplantation improved significantly sensory nerve conduction parameters but not autonomic neuropathy after 5 years. Mean glucose was the main factor associated with this improvement.

(ClinicalTrial.gov: NCT00446264 / NCT01123187).

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218

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Primary Graft Function Predicts Retention of Insulin Independence After Islet Transplantation: A Collaborative Islet Transplant Registry (CITR) Study

Violeta Raverdy1, Marie-Christine Vantyghem1, Shari Messinger-Cayetano2, Konstantinos Rouskas1, Julie Kerr-Conte1, Rodolfo Alejandro3, Michael Rickels4, Franca B. Barton5, Francois Pattou1

1Faculty of Medicine, Department of General and Endocrine Surgery, Lille University Hospital, Lille, France; 2Division of Biostatistics, Department of Epidemiology and Public Health, Biostatistics Collaboration and Consulting Core, Miller School of Medicine, University of Miami, Miami, FL, United States; 3Metabolic Studies Core, Clinical Islet Transplantation, Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL, United States; 4Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; 5The Collaborative Islet Transplant Registry, The EMMES Corporation, Rockville, MD, United States.

Objective: Primary graft function (PGF) is an independent predictor of long term outcome in organ transplantation (1), and its role has been also suggested in islet transplantation (IT) (2). Here, we explored in a large, multicentric cohort, the influence of PGF on the long term outcome of allogenic IT in non uremic patients with type 1 diabetes (T1D).

Research Design and Methods: Participants in this retrospective analysis were T1D islet-alone transplant (IAT) recipients enrolled in the Collaborative Islet Transplant Registry ( www.CITRegistry.org), who received one to three sequential allogenic islet infusions. PGF was defined as Ryan’s ß-score, a previously validated index of islet graft function (3), ranging 0-8, and calculated from the last available fasting blood glucose, plasma C peptide, HbA1c, and daily exogenous insulin within 75 days post each of 1-3 infusions per recipient. PGF was considered as optimal / suboptimal / poor when ß-score was 7-8 / 5-6 / 0-4, respectively. The main study outcome was insulin independence post last infusion.

Results: A total of 460 IAT recipients with sufficient data (37%M/73%F, age 45±10 years, diabetes duration 29±11 years, A1c 7.7±1.3%, mean ±SD), received a total of 892 infusions. Time to first achievement of insulin independence after each infusion (censored at reinfusion or last follow-up) was significantly shorter with increasing PGF (p<0.001, Figure 1). Optimal PGF was more frequent after multiple infusions 17/460 (4%), 41/330 (12%) and 20/102 (19%) following one, two, or three infusions, respectively (p<0.001). Duration of insulin independence was significantly longer with increasing PGF (p<0.001, Figure 2), and this association was independent of the total number of infusions. The mean (95% CI) time to loss of insulin independence was 54 (44-64) / 41(35-47) / 37 (31-43) months after last infusion in patients with optimal / suboptimal / poor PGF, respectively (p=0.05).

Conclusion: PGF (ß score within 75 days post last infusion) predicted long term retention of insulin independance after IT in non uremic patients. These results suggest that 1. islet graft potency and/or engraftment should be optimized and 2. PGF may represent a simple and early end point for the clinical evaluation of new relevant strategies.

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Reference:

[1] Terasaki PI, Cecka JM, Gjertson DW, Takemoto S: High survival rates of kidney transplants from spousal and living unrelated donors. N Engl J Med 1995, 333:333–336.

[2] Vantyghem MC, Kerr-Conte J, Arnalsteen L, Sergent G, Defrance F, Gmyr V, Declerck N, Raverdy V, Vandewalle B, Pigny P, Noel C, Pattou F: Primary graft function, metabolic control, and graft survival after islet transplantation. Diabetes Care. 2009, 32(8):1473-8.

[3] Ryan EA, Paty BW, Senior PA, Lakey JR, Bigam D, Shapiro AM: Beta-score: an assessment of beta-cell function after islet transplantation. Diabetes Care. 2005, 28(2):343-7.

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Restoration of Glucose Counterregulation by Islet Transplantation in Long Standing Type 1 Diabetes

Michael Rickels1, Carissa Fuller1, Cornelia Dalton-Bakes1, Eileen Markmann2, Maral Palanjian2, Kevin Cullison1, Janice Tiao1, Shiv Kapoor3, Chengyang Liu2, Ali Naji2, Karen Teff1,4

1Medicine, Endocrine Division, University of Pennsylvania, Philadelphia, PA, United States; 2Surgery, Transplantation Division, University of Pennsylvania, Philadelphia, PA, United States; 3Medicine, Renal Division, University of Pennsylvania, Philadelphia, PA, United States; 4Monell Chemical Senses Center, Philadelphia, PA, United States.

Patients with long-standing type 1 diabetes (T1D) may exhibit defective glucose counterregulation and impaired hypoglycemia symptom recognition that substantially increase their risk for experiencing severe hypoglycemia. We sought to determine the effect of intrahepatic islet transplantation on glucose counterregulation and hypoglycemia symptoms in patients with long-standing T1D. Subjects prior to (PRE) and 6 months after (POST) intrahepatic islet transplantation (n=12) and normal controls (NL; n=6) underwent hyperinsulinemic (1 mU/kg/min) hypoglycemic (hourly steps ∼80, ∼65, ∼55 and ∼45 mg/dl) and euglycemic (∼90 mg/dl) clamps with infusion of 6,6-2H2-glucose (0.05 mg/kg/min) for measurement of endogenous glucose production (EGP) by the isotopic dilution method. Subjects had 29±4 years of T1D complicated by hypoglycemia unawareness (Clarke score 6.3±0.2; HYPO score 2564±715) and received 9,648±666 islet equivalents/kg by portal vein infusion resulting in 10/12 insulin-independent with reduction in HbA1c from 7.0±0.3 to 5.6±0.1% (P<0.01) and amelioration of problematic hypoglycemia. During the final hour of the hypoglycemic clamp, plasma glucagon was PRE 33±3, POST 60±7 and NL 81±9 pg/ml (P<0.001 both vs. PRE), epinephrine was PRE 116±18, POST 253±22 and NL 380±31 pg/ml (P<0.01 both vs. PRE and NL vs. POST), free fatty acids (FFA) were PRE 50±7, POST 161±37 and NL 95±14 μM (P<0.05 both vs. PRE), EGP was PRE 0.6±0.10, POST 1.2±0.1 and NL 1.4±0.2 mg/kg per min (P<0.01 both vs. PRE), and the autonomic symptom response was PRE 2.2±1.0, POST 5.3±1.0 and NL 5.8±1.8 (P<0.1 both vs. PRE). POST levels of glucagon, epinephrine, FFA, EGP and autonomic symptoms were greater in the final hour under hypo- vs. euglycemic conditions (P<0.01 for all). These results indicate that intrahepatic islet transplantation can restore glucose counterregulation and improve hypoglycemia symptoms in long-standing T1D, and support its consideration in patients with severe hypoglycemia unawareness.

This work was performed in part as a project of the Clinical Islet Transplantation Consortium, a collaborative clinical research program headquartered at the National Institutes of Health.

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220

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Long-term Outcomes of Clinical Islet Transplantation Using Donors After Cardiac Death: A Multicenter Experience in Japan

Takayuki Anazawa1,2, Akira Kenjo1,2, Takashi Kimura2, Kazuya Ise1,2, Junichiro Haga2, Sato Naoya2, Takao Tsuchiya1,2, Takuro Saito1,2, Mitsukazu Gotoh1,2

1The Japan Islet Transplantation Registry, Fukushima, Japan; 2Regenerative Surgery, Fukushima Medical University, Fukushima, Japan.

Objective: An obstacle to generally use of islet transplantation is an insufficient supply of cadaveric pancreas. In Japan, donors after cardiac death (DCD) are not deemed suitable for whole-organ pancreas transplantation and can provide a source of pancreas for islet transplantation. However, the long-term outcomes and the utility of DCD are still controversial in the clinical setting. Here, we summarize the long-term outcomes of islet transplantation employing DCD as reported to the Japan Islet Transplantation Registry.

Methods: Sixty-four islet isolations and 34 islet transplantations were performed in 18 type 1 diabetic patients under cover of immunosuppression with basiliximab, sirolimus and tacrolimus. All donors were DCD at the time of harvesting. The mean follow-up was 76 months.

Results: Factors influencing islet yield included duration of low blood pressure of the donor, cold ischemic time, and usage of the ETK solution for preservation. Of the 18 recipients, 8, 4, and 6 recipients received 1, 2, and 3 islet infusions, respectively. Overall graft survival defined as C-peptide level more than or equal to 0.3 ng/ml was 72.2, 44.4, and 22.2% at 1, 2, and 5 years, respectively, whereas corresponding graft survival after multiple infusions was 90.0, 70.0 and 30.0%, respectively (Figure). Three of these recipients achieved insulin independence in 14, 79, and 215 days. HbA1c levels and requirement of exogenous insulin were improved before loss of graft function. All recipients became free of severe hypoglycemia unawareness, however, at least 5 patients experienced a recurrence of severe hypoglycemia after the loss of graft function.

Conclusion: Islet transplantation employing DCD can relieve glucose instability and problems with hypoglycemia while the graft is functioning. However, islets from DCD might be associated with reduced long-term graft survival. Further improvements in the clinical outcome by modification of islet isolation/transplantation protocol are necessary to establish islet transplantation using DCD.


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221

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Cost Analysis of Whole Pancreas Transplant Versus Islet Transplant in Adults With Type One Diabetes (T1D).

Sara Moassesfar, Umesh Masharani, Lynda Frassetto, Greg Szot, Joan McElroy, Marissa Ramos, Kristina Johnson, Peter Stock, Andrew Posselt

1Pediatrics/Endocrinology, UCSF, San Francisco, CA, United States; 2Medicine/Endocrinology, UCSF, San Francisco, CA, United States; 3Medicine/Nephrology, UCSF, San Francisco, CA, United States; 4Surgery/Transplant, UCSF, San Francisco, CA, United States.

Background: Advances in manufacturing and immunosuppression have made islet transplantation alone (ITA) a viable alternative to pancreas transplant alone (PTA) in non-uremic patients with T1D. Compared to PTA, ITA is associated with additional manufacturing and processing costs; however, these expenses may be offset by a simpler procedure, shorter hospitalization, and fewer complications requiring readmission. Here, we compare costs of ITA vs. PTA at our institution in nonuremic patients with T1D.

Methods: A retrospective chart review was performed to compare total hospitalization costs for 10 ITA to 11 PTA performed during a similar period. Total costs included organ acquisition and preparation/processing, transplant procedure, initial hospitalization, and medications.

Results: The organ procurement fee in our region is $37,847/pancreas. 15 organs were processed to yield 14 clinically suitable islet preparations for transplantation into 10 recipients, giving an average clinical islet preparation cost of $32,571/organ. Mean total hospitalization, procedure and medication cost for ITA was $19,243/patient (average admission of 6 days). Thus, the total cost of each islet transplant was $89,661/ patient. Four of 10 patients required second transplants, thereby doubling the total cost for these patients to $179,322/patient. Together, the average total cost for this entire group was $125,525/patient. All ITA patients became insulin independent after final islet transplant. For PTA, the average total cost (organ procurement, transplant, hospitalization, and medications) was $127,050.50 (average admission of 12 days).

Conclusion: With improvements in islet processing and immunosuppression, insulin independence after one transplant is increasing, and thus, overall costs are reduced. In patients who are insulin independent after one islet transplant, the total cost is lower than PTA. In patients who require two islet infusions for insulin independence, the cost is substantially greater than that of PTA. Additional hospitalizations related to post-surgical complications and/or rejection may result in further expenses for PTA compared to ITA.

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222

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Islet Allotransplantation in Type 1 Diabetes: Phase 2 Pilot Study with CXCL8 Inhibitor (reparixin).

Paola Maffi1, Luisa Daffonchio2, Pier Adelchi Ruffini2, Marcello Allegretti2, Antonio Citro3, Paola Magistretti1, Raffaella Melzi3, Alessia Mercalli3, Rita Nano3, Valeria Sordi3, Antonio Secchi1, Lorenzo Piemonti3

1Internal Medicine - Transplant Unit- Diabetes Research Institute, Scientific Institute San Raffaele, Milan, Italy; 2Research and Development Department, Dompè s.p.a., L’ Aquila, Italy; 3Biology of beta cell - Diabetes Research Institute, Scientific Institute San Raffaele, Milan, Italy.

Background: Islet transplantation is actually considered a standard therapy for patients with type 1 diabetes who meet specific criteria of inclusion. The immunosuppressive therapy is by now supported by anti inflammatory strategies. CXCL8 inhibitor (reparixin), demonstrated in animal models efficacy in improving engraftment and delaying rejection after intrahepatic islet transplantation.

The aim of this study was to evaluate whether reparixin may improve clinical outcome of islet transplantation in humans.

Methods: Patients recruited for the study were randomly assigned to receive either no intervention (control-group) or reparixin treatment (Rep-group) by continuous i.v. infusion for 7 days starting 12 hours before islet infusion. The immunosuppressive regimen was: ATG (started 12 hours before islet infusion), FK506 for 3 months then sirolimus, micophenolate mophetile. The islets were infused in the liver.

Results: The results were analyzed in terms of islet function: early failure (c-peptide lost within 4 weeks); partial function (continuous c-peptide secretion ≥ 0.3 ng/mL); full function (insulin independence). Control-group: 3 patients were enrolled; islet equivalent/kg infused were 4529±398. In all cases early failure was observed after 35, 27, 26 days. Consequently the randomization was stopped and only Rep-group was completed. Rep-group: 6 patients were enrolled; islet equivalent/kg infused were 4911+897. 4/6 patients reached partial function, 2/6 patients had early failure, 28 and 32 days after islet infusion respectively. 3/4 patients who reached partial function became insulin independent after the 2nd infusion. They have been maintaining this condition since 16, 13, 5 months respectively, with glycaeted hemoglobin < 6.0%.

Conclusions: Only Rep-group patients, and not control-group, reached stable islet function after 1st infusion: thereafter, subjects who achieved insulin independence after 2nd infusion, kept that condition firm with excellent metabolic control.

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223

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Hepatic Steatosis After Intraportal Islet After Kidney (IAK), Islet Tranplantation Alone (ITA) and Islet Auto Tranplantation (IAT) in 108 Patients: Prognostic Value Of Ultrasound On Clinical Outcome

Massimo Venturini1, Querques Giulia1, Paola Maffi2, Giulia Agostini1, Lorenzo Piemonti3, Paola Magistretti2, Alessandro Del Maschio1, Antonio Secchi2

1Radiology, Scietific Institute San Raffaele, Milan, Italy; 2Internal Medicine - Transplant Unit, Scientific Institute San Raffaele, Milan, Italy; 3Biology of beta cell, Scientific Institute San Raffaele, Milan, Italy.

Engrafted islets produce insulin within the liver and can potentially restore euglycemia. Hepatic steatosis is a consequence of islet engraftment and can be determined by local insulin production, lipolysis inhibition, and lipogenesis stimulation. It was curiously found only in a limited number of patients and it’s meaning is controversial. The aim of this study was to assess steatosis at Ultrasound after islet allo- and auto-transplantation, investigating its relationship with graft function and its role in predicting clinical outcome.

Materials and Methods: From 1992 to 2012, 108 patients underwent islet transplantation (33 IAK, 50 ITA, 25 IAT). Ultrasound was performed at baseline, 6, 12, and 24 months, recording steatosis prevalence, first detection, duration, and distribution. Contemporaneously steatosic (S) and non-steatosic (NS) patients were compared for the following parameters: infused islet mass, insulin independence rate, ß-score, C-peptide, glycated hemoglobin, exogenous insulin requirement, and fasting plasma glucose.

Results: Steatosis was found in 21/108 patients: in 20/83 (24%) allotransplanted and 1/25 (4%) autotransplanted patients, mostly detected at 6 and 12 months (18 cases). Infused islet mass was significantly higher in S than NS patients (IE/kg: S=10.822; NS=6.138). When metabolic parameters were considered, S patients showed worse basal conditions (ß-score: S=1.7 ± 1.6; NS=2.8 ± 2.8), but better islet function at the time of steatosis first detection (ß-score: S=3.9 ± 2.0; NS=2.9 ± 2.3), after which progressive islet exhaustion, along with steatosis disappearance, was observed. Conversely, in NS patients these parameters remained stable in time.

Conclusions: Steatosis seems to be related to islets’ mass and local overworking activity. It precedes metabolic alterations and can predict graft’s dysfunction, addressing therapeutic decisions before islet exhaustion occurs. Therefore ultrasound can be considered an important tool in the diagnosis of graft exhaustion and its frequent application is recommended during follow-up.

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Long Term Outcomes of Total Pancreatectomy and Islet Auto Transplantation in Children

Srinath Chinnakotla1,2, Bellin Melena2, Sarah Jane Schwarzenberg2, Marie Cook1,2, David Radosevich1, Barbara Bland1, Josh Wilhelm1, Balamurugan Appakalai1, Bernhard Hering1, Ty Dunn1, Beilman Gregory1, Selwyn Vickers1, Martin Freeman3, Timothy Pruett1,2, David Sutherland1

1Surgery, University of Minnesota, Minneapolis, MN, United States; 2Pediatrics, University of Minnesota, Minneapolis, MN, United States; 3Medicine, University of Minnesota, Minneapolis, MN, United States.

Objectives: To evaluate long-term outcomes after Total-Pancreatectomy and Intra-portal Islet-Auto-Transplantation (TP-IAT) in children. Methods: Between 1989-2011, 54 children (ages 5-18yrs, mean=13yrs) underwent TP-IAT. All were narcotic dependent (mean ± SEM 8.0±0.6 years-of-pain) and had failed endoscopic-management and/or direct-pancreatic-surgery. Primary etiology of pancreatitis was hereditary (Genetic mutations-PRSS1). QoL was measured using the SF-36 in a subset (n=22).

Results: Post TP-IAT, 90% of the children were narcotic-free with sustained-pain-relief; over 50% were insulin independent (Figure 1). By multivariate-analysis, younger-recipients, and those transplanted with total-Islet-Equivalents/ Kg Body Weight >1500, were significantly more likely to achieve insulin-independence (Table I). Pre-procedure, >80% had frequent school-absences because of health; within 12 months of TP-IAT, 10% (p<.001). Post-procedure, there was also a significant decrease in # days per month that total activity was limited (p<.0.005). There was a significant improvement from baseline, by SF-36, in physical-and mental-component QoL scores (p <0.001), achieving normal-levels. None of the children developed pancreatic-cancer, or had direct complications due to the intra-portal-islet-location.

Conclusions: TP-IAT in children provides long-term pain-relief (90%) and preservation of beta-cell-function (>50%). Children with chronic-painful-pancreatitis due to genetic-mutations with a high-life-time risk of pancreatic cancer should be considered earlier for TP-IAT as younger-children are more likely to achieve insulin independence.


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225

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Early Treatment with Etanercept and Anakinra Prevents Islet Graft Damage in Clinical Islet Autotransplantation

Bashoo Naziruddin1, Morihito Takita2, Faisal Kunnathodi2, Rauf Shahbazov2, Kanak Mazhar3, Michael Lawrence2, Nicholas Onaca1, Marlon Levy1

1Baylor Annette C. and Harold C. Simmons Transplant Institute, Dallas, TX, United States; 2Islet Cell Laboratory, Baylor Research Institute, Dallas, TX, United States; 3Institute of Biomedical Studies, Baylor University, Waco, TX, United States.

Background: Total pancreatectomy followed by autologous islet transplantation (TP-AIT) is an effective treatment for refractory chronic pancreatitis (CP) patients. Peri-transplant inflammation can damage islet engraftment. We have employed blockage of TNF-α alone or in combination with IL-1β to improve islet engraftment in patients undergoing TP-AIT.

Methods: A total of 12 CP patients with TP-AIT were analyzed. Etanercept (TNFα antagonist; 25 mg subcutaneous injection [s.c.] on day 0, 1, 4 and 7) was given to 5 patients (single blockage: [SB] group). Another 7 patients were administered Anakinra (IL-1β blocker; 100 mg s.c. on day 0 to 7) additionally (double blockage [DB] group). There was no significant difference in pre-transplant basic characteristics (Table 1). Islet damage was assessed by proinsulin release along with serum proinflammatory cytokine profiles.

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Results: No significant adverse events were observed in both groups. Proinsulin levels in early post-AIT was higher in SB group compared to DB group suggesting islet damage (p = 0.05, Figure 1). DB group had lower TNF-α levels with significant difference at 3 hour-post IAT when compared to SB group. Proinflammatory cytokines of IP-10 and MCP-1, and anti-inflammatory IL-10 levels were inversely regulated between two groups during peri-transplant period.


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Conclusions: Double blockage of TNFα and IL-1β offers safe and better protection than blockade of TNFα alone from inflammatory reaction during the peri-transplant period in TP/AIT. This approach warrants further study on long-term auto-islet survival and function, and may have applications in allogeneic transplants as well.

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226

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Autoislet Cell Therapy Product Manufacturing with Intact Versus Truncated Collagenases I and II Combined with Neutral Protease

Mukesh Tiwari1, Josh J. Wilhelm1, Melena D. Bellin1, David M. Radosevich2, Gopalakrishnan Loganathan1, Sajjad M. Soltani1, Ty B. Dunn2, Srinath Chinnakotla2, Timothy L. Pruett2, Greg J. Beilman2, Bernhard J. Hering1,2, A.N. Balamurugan1,2

1Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN, United States; 2Department of Surgery, University of Minnesota, Minneapolis, MN, United States.

Background: Islet autotransplants are increasingly performed for the prevention of surgical diabetes in patients undergoing total pancreatectomy for the treatment of refractory pain associated with chronic pancreatitis. It will therefore be important to optimize and validate the quality of autoislet cell therapy products. Several new tissue-dissociating enzymes have become available in recent years for human islet isolation. Here we compare the effects of intact (IC) versus truncated (TC) Clostridium histolyticum (Ch) collagenases (C) I and II combined with the same Ch neutral protease on islet yield, islet viability, and post-transplant recipient serum C-peptide levels.

Methods: Islet yields and viability were studied in 290 autoislet products; 120 (41.4%) of which were processed with TC between 2007 and 2009 and 170 (58.6%) were manufactured with IC between 2009 and 2012 using a standard operating procedure for islet isolation. Posttransplant C-peptide (stimulated serum C-peptide responses 90 minutes after standardized mixed meal) were studied in recipients of autoislets processed with TC (n=85) and IC (n=53). Isolation yield and viability were analyzed by univariate analysis and the results presented as mean ± SEM. General and generalized mixed model methods were used for the clinical outcomes analysis of mean stimulated c-peptide values at different time points (3, 6, and 12 months).The values were adjusted for enzyme type, timing, and mutual interaction for statistical comparison.

Results: The pancreases were more fibrotic in the intact CI/II compared with the TC group (6.1 vs. 5.1, p<0.001); nevertheless pancreases processed with IC showed higher digest islet equivalents [IEQ] (306,373 vs. 239,674; p<0.001), % digested pancreas (84.1 vs. 81.2; p=0.030), digest IEQ/gm pancreas (4,487 vs. 3,908; p=0.064), transplanted IEQ (274,053 vs. 212,641; p<0.001), and transplanted IEQ/kg body weight (4,215 vs. 3,269; p<0.001) than pancreases dissociated with TC. The collagenase dose [Wunsch units/gm] used (27.2 vs. 35.8; p=0.002) and proportion of % embedded islet tissue (37.0 vs. 46.2; p=0.003) were lower in the IC compared to the TC group. Islet viability (FDA/PI) in the two groups was similar (89.6 vs. 90.8; p=0.196). The trajectory of change for mean C-peptide (ng/ml) values in both recipient groups at 3 months (2.4 vs. 2.7), 6 months (3.1 vs. 2.6) and 12 months (3.2 vs. 2.4) favored IC over TC (p=0.014) (see table and figure below).

Conclusion: These results suggest that higher autoislet yields and higher post-transplant C-peptide levels in autoislet recipients can be achieved with IC than with TC. More detailed analyses of posttransplant islet autograft functions are currently underway.

References:

Balamurugan AN, Loganathan G, Bellin MD, Wilhelm JJ, Harmon J, Anazawa T, Soltani SM, Radosevich DM, Yuasa T, Tiwari M, Papas KK, McCarthy R, Sutherland DE, Hering BJ. A new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products. Transplantation. 2012 Apr 15;93(7):693-702.


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Alpha Cells in Intraperitoneal, but not Intrahepatic, Autoislet Transplants Function Normally During Hypoglycemia in Humans After Total Pancreatectomy and Islet Autotransplantation

Melena Bellin1, Bernhard Hering2, A.N. Balamurugan2, Susan Parazzoli3, Elizabeth Oseid3, Ty Dunn2, Gregory Beilman2, David Sutherland2, R. Paul Robertson3

1Pediatrics, University of Minnesota, Minneapolis, MN, United States; 2Surgery, University of Minnesota, Minneapolis, MN, United States; 3Pacific Northwest Diabetes Research Institute, Seattle, WA, United States.

Successful intrahepatic autoislet transplantation after total pancreatectomy (TP-IAT) for chronic, painful pancreatitis can prevent diabetes and provide normal HbA1c levels for many years. However, our recent clinical experience has revealed a high prevalence of hypoglycemia after TP-IAT, especially following exercise and high carbohydrate meals. To examine whether intrahepatic alpha cell function is normal, we examined glucagon responses to intravenous arginine (AGRarg) and during hypoglycemic clamps (GLGNhypo) in TP-IATsubjects with a normal hemoglobin A1c level at 5.7 ± 5.1 years post-transplant (range 1.7-21 years).

All subjects had intact AGRarg (45 +/- 14, pg/ml, n=11, p<0.001 for stimulated vs basal level). However, the glucagon response to hypoglycemia was markedly defective in 11 intrahepatic autologous islet transplant recipients (GLGNhypo: 7 +/- 4, n=11 vs. Controls: 77 +/- 16, n=7; p<0.001). In marked contrast, 3/3 TP-IAT subjects who received autoislets intraperitoneally as well as intrahepatically had normal glucagon responses to hypoglycemia (72 +/- 14) at 4-7 years post-transplant. We also measured C-peptide levels during the clamps to ascertain whether the normal decline in beta cell secretion during hypoglycemia had occurred to provide the “switch-off” signal that is essential for hypoglycemia-induced glucagon secretion. All subjects with intrahepatic islets developed nearly non-detectable C-peptide levels that were equivalent to those observed in normal controls subjects undergoing the clamp.

These data in humans confirm previous observations in animals that the defective alpha cell response to hypoglycemia in autoislets is transplant site-specific for the liver. Transplanting a portion of the islets in an extra-hepatic site preserves the counter-regulatory glucagon response during hypoglycemia and may protect against post-transplant hypoglycemia. The preserved glucagon response >4 years post-transplant suggests the potential for longevity of islets in the peritoneal cavity.

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Robotic Total Pancreatectomy with Autologous Islet Transplant: The First 6 Cases

Carlos Alberto Galvani1,1,2,3, Julia Samame1,2,3, Horacio Rodriguez Rilo1,2,3, Rainer Gruessner1,2,3

1Surgery/Minimally Invasive & Robotic Surgery, University of Arizona, Tucson, AZ, United States; 2Surgery/Institute for Cellular Transplantation, University of Arizona, Tucson, AZ, United States; 3Surgery/Transplant, University of Arizona, Tucson, AZ, United States.

Background: Total pancreatectomy with autologous islet transplant (TP-AIT) is the rationale treatment for patients with chronic pancreatitis (CP) and chronic pain syndrome refractory to conservative management. Robotic assistance can help surgeons to overcome some of the technical challenges of complex pancreatic procedures using standard laparoscopy and may extend the acceptance and indication of TP-AIT.

Our objective is to report our initial experience of totally robotic TP-AIT and describe the surgical technique.

Method: This is a retrospective analysis of the first 5 cases of totally robotic TP-AIT at our institution from July 2012 and February 2013.

A 5 trocars technique was used. The pancreas was removed through a Pfannenstiel incision. The reconstruction entailed a hand-sewn end-to-side hepatico-jejunostomy; a side-to-side duodeno-jejunostomy with a modified Braun stapled jejuno-jejunostomy. The islets were infused with an 18-gauge laparoscopic needle in the stump of the splenic vein.

Results: There were 4 females and 2 males. The mean age of the patients was 41 (22-58) years; mean BMI was 23.9 (18.5-30.1) kg/m2. There were no conversions to either laparoscopic or open surgery. The mean time for robotic total pancreatectomy was 321±37min. The mean time for the reconstruction was 143±22min. Average operative time from incision to closure, including autoislet isolation process and infusion was 717±81.6min (612-835). Splenectomy was required in 2 of 5 patients. Estimated blood loss was 575±178mL (300-800). One patient requiered blood transfusion. No major procedure-related morbidity was observed. The median hospital stay was 11 days. Patients were successfully weaning off narcotics at an average of a month after surgery. Islet isolation results were: total autoislet count 141,167± 62,747; IE 140,570±70,191; IE/Kg 1848±1202. Discharge glargine insulin was 20±29 units/day.

Conclusion: This early experience demonstrates that fully robotic-assisted TP-AIT appears to be a feasible and safe technique for the treatment of chronic pancreatitis. The robotic approach is advantageous since allows for safe vascular dissection and digestive tract reconstruction. However, initially, longer operative times are expected due to the complexity of the procedure.

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Impact of Pancreatic Bacterial Colonization on Autologous Islet Transplantation

Linda Langman1, Nate Sarbin1, Farah Mukheef1, Preeti Chhabra1, Avinash Agarwal1, Brayman Kenneth1

1Surgery, University of Virginia, Charlottevsille, VA, United States.

Objective: Patients with chronic pancreatitis who have undergone stenting or drainage procedures may have bacterial colonization of the organ. Implications of chronic colonization in the setting of subsequent autologous islet transplantation are unclear. The objective of this analysis is to correlate pancreatic bacterial culture data with outcomes in patients receiving autologous islet transplants. Between 2007 and 2012, 19 patients underwent pancreatectomy with islet cell isolation for treatment of their chronic pancreatitis and associated chronic pain. Pancreata were surgically removed and sent to the islet processing facility. Islets were isolated using the standard Ricordi method, purified using Biocoll gradients, washed and loaded for transplant. Release criteria, including endotoxin testing and gram stains, were completed prior to transplantation. When positive cultures were reported, blood cultures were collected and appropriate antibiotics were given. Process safety was assured by confirming similar infectious agents in the transport solution and final product.

Results: Of 19 autologous isolations, six were identified with positive bacterial cultures within 24 hours post-transplant. No patient transplanted with contaminated islet preparations became septic and no hepatic sequelae were observed. However, pancreata which were colonized yielded significantly lower number of IEQs (105416± 103136 IEQs vs. 243,500±119862; p=0.02) 0 of 5 patients receiving islets from colonized pancreata became insulin free compared to 9 of 13 (69%) of the non-colonized cohort (p = 0.02) at mean follow up of 35±13 months. Average insulin requirement in the colonized group was 36units/day whereas the non-colonized group required 3 - 8units of insulin/day post-transplant.

Conclusions: The data suggest that despite the presence of bacterial colonization of the pancreas, no infectious complications followed autologous islet cell transplantation. The presence of bacteria correlated with poorer islet cell yield. Colonization of the pancreata most likely represents further progression of the disease state and increased loss of islet cell mass.

Focus to Cure Diabetes Foundation

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Results from 55 Open and Robot-assisted Pancreatectomies with Autologous Islet Transplantation for the Prevention of Surgically Induced Diabetes

Horacio Rilo, Monica Delbridge1, Manuel Beltran del Rio1, Galvani Carlos1, Abbas Rana1, Marian Porubsky1, Angelika Gruessner1, Rainer Gruessner1

1Surgery, University of Arizona, Tucson, AZ, United States.

55 (35F/20M) patients, diagnosed with chronic pancreatitis, underwent total pancreatectomy with autologous islet transplant (TP/AIT) at University of Arizona Medical Center between August 2009 and April 2013. Average age at the time of transplant was 42, (range 20-66). Preoperative BMI ranged from 16-50 (mean/median 26). 73% were diagnosed with idiopathic disease, 16% hereditary and 11% alcohol induced (all male). 17% of female patients and 15% of male patients presented with hereditary pancreatitis. Years between diagnosis and TP/AIT ranged from 1-36 (mean 7/median 9). 46 (84%) patients underwent total pancreatectomy, 8 patients underwent completion and 1 patient underwent partial pancreatectomy. Prior to TP/AIT, 52 (95%) presented with a surgical history of previous intervention that included 80% who presented with previous abdominal surgeries; 15% had undergone a Whipple procedure, and 56% had prior and often multiple ERCPs with stent placement. 62% of patients presented with severe systemic disease as defined by the American Society of Anesthesiologists (ASA) Physical Status Classification 3, 36% were ASA 2 and 2% ASA 1. Preoperatively, 53 patients presented with normal HbA1c (< 6.4%). HbA1c ranged from 4.6-7.3 (mean/median 5.6); however, preoperative continuous glucose monitoring (CGM) reflected abnormalities in 91% of patients despite normal HbA1c. Positive correlation was shown between CGM results and HbA1c (p < 0.01). Transplanted islet equivalents by body weight (IEQ/kg) ranged 10-17,770 (mean 3,227). Following TP/AIT 19% of patients were insulin-independent (between 1-24 months), 27% required ≤ 9 units insulin per day, 23% 10-25 and 31% ≥ 25. Postoperative length of hospitalization (LOS) ranged 6-26 days with 26% of patients’ LOS 11 (median). ICU days ranged 1-12 (mean/median 4). 27% of patients remained 5 days in ICU. 100% of patients experienced preoperative pain and were treated with opioid analgesics. Following AIT, 71% were pain-free and no longer required analgesics at 12 months postoperatively.

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The impact of HLA DR3/DR4 Matching on Outcome in T1DM Recipients

Angelika Gruessner1, Gruessner Rainer1

1University of Arizona, Tucson, AZ, United States.

As with most autoimmune diseases, type 1 diabetes mellitus (T1DM) is associated with genes within the major histocompatibility complex. The prevalence of class II antigens DR3, DR4, DQ2 and DQ8 is increased. We studied the outcome and possibility for recurrence of disease in recipients based on class II antigen matching.

8,292 primary deceased donor SPKs were analyzed. The diagnosis was T1DM in 93%. A multivariate Cox-regression analysis was performed to analyze outcome of pancreas and kidney graft function for T1DM and T2DM patients, adjusting for a multitude of recipient and donor risk factors and several immunosuppressive protocols.

The expression of DR3/DR4 and DQ2/DQ8 was significantly different between T1DM and T2DM transplant recipients: 86% of T1DM but only 63% of T2DM carried a DR3 and/or DR4 antigen (p<0.001). In addition a significant correlation between DR3 and DQ2 and DR4 and DQ8 was found.

The 5-year graft function rate in T1DM recipients was 81% for 0, 83% for 1, and 73% for 2 DR3/4 matches (p=0.001). For the simultaneous kidney graft, the respective 5-year survival rates were 79%, 81% and 88%.

Since we were looking for long-term outcome te reasons for failed matched outcomes was either ’chronic rejection’ or ’reason unknown’. Only in a few cases was the reason ’Recurrence of disease’ given.

The multivariate analysis confirmed that for the pancreas graft in T1DM recipients either one DR3 or one DR4 antigen match did not increase the relative risk for graft loss. In contrast, matching for both DR3 and DR4 increased the risk of graft loss by 77% (p=0.02). This finding was not noted in T2DM transplant recipients. In the simultaneous kidney graft, a negative impact of DR matching in both T1DM and T2DM transplant recipients was not observed. In contrast, there was a trend to better kidney graft function in DR matched patients.

Matching for both DR3 and DR4 in SPK recipients with T1DM should be avoided to improve pancreas graft survival. Histopathology studies are necessary to prove an increased risk of disease recurrence.

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Splenic Vein Thrombosis Following Pancreas Transplantation: Identification of Factors that Support Conservative Management

Jack Harbell1,2, Garrett Roll1,2, Tara Morgan1,2, Vickie A. Feldstein1,2, Andrew Posselt1,2, Sang-Mo Kang1,2, Sandy Feng1,2, Ryutaro Hirose1,2, Chris E. Freise1,2, Peter Stock1,2

1Department of Surgery, Division of Transplantation, University of California San Francisco, San Francisco, CA, United States; 2Department of Radiology, University of California San Francisco, San Francisco, CA, United States.

Introduction: Prophylaxis for graft venous thrombosis following pancreas transplant varies between institutions. Similarly, treatment of venous thrombosis ranges from urgent thrombectomy to conservative management with anticoagulation. In this study we wished to determine the prevalence of graft splenic vein (SV) thombosis, as well as the clinical significance of non-occlusive thrombus observed on routine imaging.

Methods: Records of 112 pancreas transplant recipients from January 1st, 2008 to December 31st, 2012 at a single center were reviewed. All patients received aspirin prior to surgery, and aspirin and dipyridimole post-operatively. Non-dialysis dependent patients also received a bolus of intravenous (IV) heparin (2000-4000 units) at the time of vascular anastomosis. Post-operative anticoagulation was surgeon specific, but usually included low dose heparin infusion (200-400 units/hr) for 24-48h after surgery. Venous thrombosis was defined as absence of flow or presence of thrombus identified in any part of the graft SV on ultrasound. Patients with SV thrombus were anticoagulated with IV heparin in addition to anti-platelet therapy, then transitioned to warfarin for 3-6 months or until documented thrombus resolution.

Results: 30 patients (27%) had thrombus or absence of flow in the SV on post-operative ultrasound. There were 4 graft losses in this group. All were due to arterial and venous thrombosis, and all occurred within 20 days of transplant. Graft losses occurred in dialysis dependent and independent recipients (2 SPK, 1 PAK, 1 PTA). Patients identified to have partial SV thrombus but normal arterial signal on ultrasound were all successfully treated conservatively with IV heparin followed by warfarin for 3-6 months, and remained insulin independent. Seven patients with ultrasound findings of graft swelling and abnormal arterial waveforms with reversal of diastolic flow were taken emergently to the operating room for thrombectomy. Two of those patients were found to have no thrombus at exploration, one patient underwent successful venous thrombectomy, one patient had a successful venous thrombectomy initially but lost the graft 19 days later due to arterial and venous thrombosis, and 3 were found to have graft necrosis and underwent graft pancreatectomy.

Conclusion: In the absence of graft swelling and arterial signal abnormalities on ultrasound, non-occlusive thrombosis of the pancreas graft SV can be successfully treated conservatively with anticoagulation. Findings of graft swelling and arterial signal abnormalities, such as reversal of diastolic flow within the graft, require urgent operative intervention, since this finding can be associated with more extensive thrombus that may lead to graft loss.

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Outcomes of Pancreas Transplantation from Marginal Donors in Japan

Toshinori Ito1,2

1The Japan Registry of Pancreas Transplantation, The Japan Society for pancreas & Islet transplantation, Suita, Osaka, Japan; 2Complementary & Alternative Medicine, Gastroenterological Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

Introduction: Absolute shortage of donors in Japan still continues even after the law allowing organ transplantation from brain-dead donors came into force in 1997. With the passage of the waiting period after registration for pancreas transplantation (PTx), both deaths and serious cases of diabetic complications necessitating withdrawal of the registration have undoubtedly increased. Therefore, as potential solutions, so-called “marginal donors (MD)” as well as living donors have been considered in Japan.

Methods: A total number of 148 PTx (119;SPK, 20;PAK, 9;PTA) was performed from deceased donors except for 2 non-heart-beating donors in Japan from 2000 to 2012. “MD” are defined as follows :1) >45 years old; 2) hemodynamically unstable at harvest using a high-dose dopamine (>10g) or > two vasopressors; 3) non-heart-beating status.

Results: The mean age of donors was 43.4 years (50% >45 years). The most common cause of brain death was CVA (58.9%) followed by trauma (18.9%). Thus, 108 donors (73.0%) meeting at least one of three conditions above were marginal. Pancreas grafts were lost in 15 cases during 3 months posttransplant (8;thrombosis, 3; sepsis, 2;rejection, 1;cardiogenic, 1;bleeding). Early graft loses were significantly higher in male donors and TCIT >12 hours, but not in marginal cases. Patient survival was 94.8% at 1, 3, and 5 years. Pancreas graft survival was 84.8%, 76.4% and 68.9% at 1, 3, and 5 years, respectively. Kidney graft survival in SPK recipients was 90.2%, 90.2%, and 82.8% at 1, 3, and 5 years, respectively. Pancreas graft survival in the marginal vs non-marginal group was 80.9% vs 92.5%, 73.2% vs 85.2%, and 66.0% vs 77.4% at 1, 2, and 5 years post-transplantation, respectively.

Conclusion: There was no significant difference of pancreas graft survival between marginal and non-marginal group, but further investigations are necessary to clarify factors in marginal donors that contribute to better outcomes.

The Japan Registry of Pancreas Transplantation

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Portal Versus Systemic Venous Drainage of Pancreatic Graft: The Effect on Glucose Metabolism in Pancreas and Kidney Transplant Recipients

Tereza Havrdova1, Saudek Frantisek1, Teodora Jedinakova1, Kvetoslav Lipar1, Matej Kocik1, Jelena Skibova1

1Institute for Clinical and Experimental Medicine, Prague, Czech Republic.

Background and Aims: Two different methods of graft venous drainage are used in pancreas transplantation: portal (PVD) and systemic (SVD). PVD is considered as more physiologic due to similarity to venous outflow of the native pancreas. The aim of our study was to compare glucose metabolism in Type 1 diabetic recipients of kidney and pancreatic grafts with PVD versus SVD.

Methods: We examined 28 insulin-independent patients after simultaneous pancreas and kidney transplantation: 14 recipients with PVD of the pancreatic graft and 14 ones with SVD after a mean post transplant period of 1 year. All recipients had a stable good function of the kidney graft. Fasting glycemia, insulin levels, HbA1c, a standard IVGTT with coefficient of glucose assimilation (KG) calculation were assessed. Insulin sensitivity and production were evaluated using the homeostasis model assessment (HOMA-IR, HOMA-B). Total C-peptide and insulin secretions were calculated as areas under the curves from the serum levels during the IVGTT.

Results: PVD and SVD groups did not differ in age, BMI, duration of post transplant period, fasting C-peptide level. We did not find any significant difference in response of IVGTT. In the PVD group 1 patient had an abnormal response to the glucose stimulus, 8 patients had an impaired glucose tolerance and 5 patients had a normal glucose tolerance. In the SVD group an abnormal response was present in none, the impaired glucose tolerance in 4 and the normal glucose tolerance in 10 recipients. Mean KG was 1.24 ± 0.5 %/min. in the PVD group and 1.55 ± 0.5 %/min. in the SVD group (NS). The remaining results are shown in the following table.

Conclusion: Though this was not a prospectively randomized trial, we conclude that the change of surgical technique from SVD to PVD did not lead to any substantial change in terms of glucose tolerance.

Image Tools

Supported by MH CZ - DRO (“Institute for Clinical and Experimental Medicine – IKEM, IN 00023001”)

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Similar Results with Solitary Pancreas Compared to Simultaneous Kidney-Pancreas Transplantation in the New Millennium

Jeffrey Rogers1, Alan Farney1, Samy Iskandar1, Giuseppe Orlando1, Umar Farooq1, Yousef Al-Shraideh1, Amber Reeves-Daniel1, Amudha Palanisamy1, William Doares1, Kaczmorski Scott1, Gautreaux Michael1, Robert J. Stratta 1

1Wake Forest University, School of Medicine, Wiston-Salem, United States.

Introduction: The majority (75%) of pancreas transplants (PTxs) are performed as simultaneous kidney-pancreas transplants (SKPT) whereas approximately 16% are performed as sequential PTx after kidney (PAK) and 9% as PTx alone (PTA). The latter two categories are usually combined together as solitary PTxs and historically have had inferior outcomes compared to SKPT. The purpose of this study was to analyze our single center outcomes according to PTx category in the new millennium.

Methods: We retrospectively studied 202 consecutive PTxs in 192 patients (pts) at our center. All pts received either r-ATG or alemtuzumab induction in combination with FK/MPA and tapered steroids or early steroid withdrawal. 179 PTxs (89%) were performed with portal-enteric and 23 with systemic-enteric drainage. All pts were both T and B cell negative by flow cytometry crossmatch. Solitary PTxs were managed with routine peri-operative anti-coagulation whereas SKPTs received selective anti-coagulation. Surveillance PTx biopsies were performed in solitary PTxs whereas clinical biopsies were prompted by biochemical and clinical parameters in all pts.

Results: From 11/01 to 3/13, we performed 162 SKPTs, 35 PAK and 5 PTA (40 solitary PTxs).

Demographic characteristics for SKPT vs solitary PTxs were mostly comparable; however, the solitary PTx group had fewer HLA mismatches (SKPT mean 4.5 ± 1.3 vs solitary PTx 3.0 ± 1.3, p<0.001), younger donors (SKPT mean 28 ± 12 vs solitary PTx 22 ± 7.6 years, p=0.004), fewer black recipients (SKPT 23% vs solitary PTx 7.5%, p=0.03), but more retransplants (SKPT 1.2% vs solitary PTx 35%, p<0.001). With a mean follow-up of 5.5 years, overall pt (87% SKPT vs 87.5% solitary PTx), kidney (74% SKPT vs 82.5% solitary PTx) and pancreas graft survival (both 65%) rates were comparable. Causes of PTx loss were also similar between SKPT and solitary PTx; the rates of early thrombosis were 8.6% and 5%, respectively. Cumulative clinical acute rejection rates were similar between groups (SKPT 29% vs solitary PTx 26%, p=NS).

Conclusions: In the setting of depleting antibody induction, flow crossmatch testing, HLA matching, careful donor and recipient selection, portal-enteric drainage, peri-operative anti-coagulation, FK/MPA immunosuppression, and PTx biopsy monitoring, equivalent outcomes can be achieved in SKPT and solitary PTxs in the new millennium.

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Excellent Outcomes can be Achieved in Young Pancreas Transplant Alone (PTA) Recipients by using Maintenance Therapy with Sirolimus/Tacrolimus/Mycophenolate Mofetil.

Marian Porubsky1, Angelika C Gruessner1, Abbas Rana1, Tun Jie1, Rainer WG Gruessner1

1Department of Surgery, Division of Transplantation, University of Arizona Medical Center, Tucson, AZ, United States.

Context: Pancreas transplant alone (PTA) has evolved as a viable treatment option for non-uremic patients with Type 1 DM. Historically, the results of PTA were inferior to simultaneous kidney/pancreas transplant due higher graft loss from rejection. With advances in immunosuppression (IS), the outcomes of PTA improved significantly with the exception of young PTA recipients. The more potent immune system in young recipients appears to play a key role.

Objective: To investigate PTA outcome according to recipient age using different maintenance IS regimens.

Methods: IPTR/UNOS information for 547 primary technically successful PTAs performed between 1/2003 and 12/ 2012 were analyzed. The recipients were divided into 3 age groups: 15 - <30 years (68 patients); 30 - <45 years (266 pts) and <45 years (213 pts). All patients received induction therapy with Thymoglobulin and steroids and were maintained on long-term low dose Prednisone. Three maintenance IS regimes were compared among each age group: (1) Tacrolimus (Tac)/Mycophenolate Mofetil (MMF); (2) Sirolimus (Srl)/Tac and (3) Srl/Tac/MMF. The Kaplan-Meier method was used to estimate graft function and the long-rank test for comparisons.

Results: In the 15 - <30 years group, the 3-year graft survival rates in IS groups 1, 2 and 3 were 68%, 18% and 87% (p= 0.033). In the 30 - <45 years group, the graft survival rates were 77%, 95% and 91% (p= 0.036). In the <45 years group, the graft survival rates were 88%, 86% and 80% (p=0.4).

Conclusion: The addition of Srl to the standard Tac/MMF treatment provided the best outcome in the youngest group. Standard Tac/MMF provided the best outcome in the oldest group. This proves that a more potent IS regimen is required in young (and immunologically more challenging) PTA recipients to achieve excellent outcomes.

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238

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The Physiologic Pancreas Transplant : Experience with 58 Portal-Duodenal Drained Pancreas Transplants

Marcelo Perosa1,2, Huda Noujaim1,2, Luiz Ianhez1,2, Rodrigo Azevedo1,2, Waldere Luconi1,2, Leonardo Mota1,2, Juan Branez1,2, Marcio Paredes1,2, Luciano Giacaglia1, Marcos Castro1,2, Tercio Genzini1,2

1Hepatology and Transplant, HEPATO, São Paulo, Brazil; 2Organ Transplantation, Bandeirantes Hospital, São Paulo, Brazil.

Systemic-enteric drainage is currently the most commonly used technique in pancreas transplantation (PT). Duodenal drainage represents a new technical alternative with potential physiological benefits and improved monitoring of the pancreatic graft. We describe experience with 58 PT ( 10 simultaneous pancreas-kidney, 40 pancreas after kidney and 8 pancreas transplant alone) using portal-duodenal drainage (PDD) over the last three years. A quadruple immunosuppression protocol with thymoglobulin was used in all cases.

This technique resulted in 1-year patient, kidney and pancreas survival of 100%,100% and 90% for SPK and 1-year patient and pancreas survival of 96% and 79% for solitary PT. There were two deaths in PAK recipients due to sepsis related to the urinary tract . There were 11 pancreas losses and the causes were : thrombosis(6), immunological(3) and death with functioning graft(2). In all cases requiring transplantectomy, a two-layer closure of the native duodenum was feasible. There was one fistula from the duodenal closure after transplantectomy and a partial gastrectomy with Roux-en-Y was performed.

Conclusions: The PDD technique in PT seems to achieve the same good results compared to other techniques already in use, with an advantage of easy accessibility to the pancreas graft by endoscopy.

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Key Components of the Human Islet Double Basement Membrane are Differentially Digested During Islet Isolation

Sarah Cross1, Abby Willcox1, Emma Pope1, James Johnson1, Elisa Maillar1, Paul Bateman1, Stephen Hughes1, Derek Gray1, Paul Johnson1

1Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom.

Background: We have an incomplete understanding of which components of the extracellular matrix (ECM) at the islet:exocrine interface are digested by collagenase blends during human islet isolation. The interface contains two basement membranes (BM), one associated with the islet capillary and the second integral to the islet itself. Maintaining integrity of BMs may be critical for optimal islet survival post-isolation. This study aimed to characterize digestion of key islet BM components in human pancreas and purified islets in culture prior to transplantation.

Methods: Human pancreases were retrieved with appropriate consent and ethical approval (n=10, age 38-59, BMI 20-32, CIT <10h). 0.5cm3 pancreatic tissue biopsies were snap-frozen, then cryosectioned. Sections were treated ± Serva collagenase-NB1 and neutral protease-NB at clinically-relevant concentrations for 2 or 5 minutes at 37°C. Following double immunolabelling for insulin and potential BM proteins, expression was semi-quantified by morphometry. Following islet isolation, islet samples were collected after purification and at 24h, 48h, 72h and 7 days in culture. Islet BM proteins were identified by immunofluorescence labelling and quantified by western blotting and ELISA.

Results: Collagen-IV, laminin and perlecan were identified as major islet BM components and were significantly digested after 5 minutes enzyme treatment (p <0.05 vs control). Complete dissolution of the laminin-511 isoform occurred within only 2 minutes. In purified islets laminin-511 and perlecan expression was absent. Collagen-IV and pan-laminin expression was present, yet markedly decreased during the first 24h of islet culture. Collagen-IV expression stabilised over the proceeding 48h and was present up to 7 days in culture, whereas laminin expression was lost after 72h.

Conclusions: Key components of the islet double BM (collagen-IV, laminin and perlecan) are substantially digested by clinically-used collagenase. Importantly, laminin-511 (the only laminin isoform found in both layers of the duplex BM) and perlecan are lost entirely, indicating extensive BM disruption resulting from the islet isolation procedure. Incomplete BM may compromise islet function and survival, as destruction of ECM can trigger integrin-mediated cell death, thereby contributing to the reduction in islet yield commonly seen following culture.

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Improving Purified Islet Viability by Continuous Quadrupole Magnetic Separation (QMS)

Bradley P Weegman1,2, Venkata SK Sajja3, Michael D Rizzari2, Thomas M Suszynski2, William E Scott, 3rd2, Jennifer P Kitzmann2, Kate R Mueller2, Thomas R Hanley3, David J Kennedy5, Paul W Todd5,6, A. N. Balamurugan2, Bernhard J Hering1, Kleachos K Papas2

1Center for Magnetic Resonance Dept. of Radiology, University of Minnesota, Minneapolis, MN, United States; 2Schulze Diabetes Institute Dept. of Surgery, University of Minnesota, Minneapolis, MN, United States; 3Chemical Engineering, Auburn University, Auburn, AL, United States; 4Department of Surgery, University of Arizona, Tucson, AZ, United States; 5IKOTECH LLC., New Albany, IN, United States; 6Techshot Inc., Greenville, IN, United States.

Islet transplantation (ITx) is a minimally-invasive alternative to whole pancreas transplant for patients with uncontrolled type 1 diabetes. The islet isolation and purification process requires enzymatic digestion, mechanical agitation and the use of damaging chemicals for density gradient separation (COBE), all of which inflict substantial damage reducing viable islet yield. Quadrupole magnetic sorting (QMS) during digestion has been explored as an alternative for islet purification to reduce warm ischemia, minimize enzyme exposure and eliminate the use of density gradients. We explored the use of QMS for islet purification and compared the viability of QMS and COBE purified islets. Porcine pancreata (n=3) were split into 2 parts; the combined connecting/duodenal lobe (CDL) and the splenic lobe (SPL). Islets were preferentially labeled using magnetic micro-particles (MMPs) that lodge within the islet micro-vasculature when infused into the pancreas. This allowed the continuous separation from the acinar tissue by QMS during the collection phase of the digestion process. An optimized dose [1] of MMPs (4.5 μm diameter) were infused into the splenic artery to label islets within the SPLs, which were then digested using the Ricordi method and then continuously purified by QMS. Unlabeled islets from the paired CDLs were isolated using the same method and purified using COBE. Oxygen consumption rate (OCR) normalized to DNA content (OCR/DNA) was used to compare the fractional viability of islets from both groups. Islets purified by QMS exhibited significantly improved viability and better morphology relative to control islets. The mean OCR/DNA of islets purified by QMS was higher than those purified by COBE (209±25 vs. 125±11 nmol/min/mg DNA; p=0.02 via two-tailed paired student’s t test). We conclude that continuous islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet isolation efficiency and ITx success.

Schulze Foundation and the Schulze Diabetes Institute, NIH (R44DK072647-03 ),The Carol Olson Memorial Diabetes Research Fund, The Iacocca, Schott, and Kettering Family Foundations, The Eunice Dwan Trust

Reference:

[1] Rizzari MD, Suszynski TM, Kidder LS, Stein SA, O’Brien TD, Sajja VSK, Scott WE, Kirchner VA, Weegman BP, Avgoustiniatos ES, Todd PW, Kennedy DJ, Hammer BE, Sutherland DER, Hering BJ, Papas KK: Surgical protocol involving the infusion of paramagnetic microparticles for preferential incorporation within porcine islets. Transplantation Proceedings 2010, 42:4209–4212

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Assessing the Viability of Human Pancreas Grafts using 31P MR Spectroscopy - a Pilot Study

Lina Sjöberg1, Alireza Biglarnia2, Håkan Ahlström1, Olle Korsgren3, Jan Weis1

1Department of Radiology, Oncology and Radiation Sciences, Uppsala University, Uppsala, Sweden; 2Department of Surgical Sciences, Uppsala University, Uppsala, Sweden; 3Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Introduction: In order to select pancreas suitable for transplantation a fast, objective, and noninvasive sterile method to assess graft viability is needed. Viability is to a large extent dependent on the metabolic status of the graft, which is reflected in its bioenergetic profile. 31P magnetic resonance spectroscopy (31P-MRS) has previously been applied in animal models to assess graft viability. The aim of this pilot study was to evaluate if 31P-MRS can be applied for viability assessment in human pancreas grafts prior to transplantation.

Methods: Pancreata from five human donors were included in the study. Immediately after removal from the donor each pancreas was perfused with histidine-tryptophan-ketoglutarate (HTK) solution and stored in hypothermic condition (4oC). 31P-MRS was performed on a 1.5 T clinical MR scanner. ISIS localization sequence (31P head coil, TR 3500 ms, BW 1500 Hz, 512 acquisitions) was completed with nuclear Overhauser enhancement and proton decoupling. Voxel size was ∼5×5×14 cm. The first spectra were acquired 6-9 hours after HTK perfusion of the pancreas. Subsequent spectra were obtained with a period of ∼2 hours. After 24 hours of cold preservation pancreas was exposed to room temperature during the next 24 hours and the last spectrum was measured. This spectrum served as a reference for non-viable tissue. The following metabolites were fitted: phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE), phosphocreatine (PCr), and adenosine triphosphate (ATP).

Results: γ-ATP and β-ATP lines decreased to the noise level within 2-4 hours after the start of 31P-MRS. PME, PDE and α-ATP levels gradually decreased and Pi increased. Non-viable pancreas tissue revealed dominant Pi and small distinguishable PME and PDE intensities.

Conclusion:

(γ-ATP+β-ATP)/Pi, PME/Pi and PDE/Pi spectral intensity ratios obtained by 31P-MRS are promising quantitative parameters for fast and objective noninvasive assessment of the viability of human pancreas grafts.

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Role of Insulin-Like Growth Factor 1 (IGF-1) in Preserving Pancreas for Islet Isolation

Keiko Omori1, Eiji Kobayashi2, Jeffrey Rawson1, Naoya Kasahara2, Masafumi Takahashi3, Ismail H Al-Abdullah1, Yoko Mullen1

1Southern California Islet Cell Resource Center, Department of Diabetes, Endocrinology and Metabolism, Beckman Research Institute of the City of Hope, Duarte, CA, United States; 2Division of Development Advanced Therapy, Center for Development of Advanced Medical Technology, Jichi Medical University, Tochigi, Japan; 3Division of Inflammation Research, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan.

Background: Islet yield is influenced by prolonged pancreas cold ischemia. However, the molecular mechanisms involved in islet destruction leading to low islet yield remains obscure. The objective of this study is to investigate the AKT and Extracellular signal-regulated kinase (ERK) signaling pathways during prolonged pancreas preservation and examine the effectiveness of IGF-1 treatment during pancreas preservation on isolation outcome.

Methods: Rat pancreata, harvested en bloc and perfused with cold UW solution containing IGF-1 (25nM) (UW+IGF-1) or UW solution alone through the aorta were preserved at 4°C for up to 18h. Biopsy samples were taken for western blot (n=3/ group). Subsequently, islets were isolated from en bloc pancreata preserved in 3 different groups; 1) 12h cold preservation with UW+IGF-1 (12h-UW+IGF-1), 2) 12h cold preservation with UW alone (12h-UW) or 3) without preservation (0h-Control), and islet number and post cultured islet viability were assessed (n=3/ group).

Results: The Western blot results showed that phosphorylation of AKT, ERK, and signal transducer and activator of transcription 3 (STAT3) was significantly decreased, while caspase-3 was significantly activated in the pancreata preserved for 18h in UW solution. The addition of IGF-1 in UW solution significantly reduced the dephosphorylation of AKT, ERK and STAT3 at 18h. Duration of cold preservation did not affect post isolation islet number, however, 12h cold preservation significantly decreased post cultured islet viability as compared to 0h control group (69.4±0.8 % in 12h-UW vs. 96.3±1.6 % in 0h-Control, p<0.001). This loss of viability was markedly improved by IGF-1 treatment (87.4±2.7 % in 12h-UW+IGF-1, p<0.05 vs. 12h-UW).

Conclusions: Our study demonstrated that IGF-1 protects islets from cold ischemia-rewarming injury promoting islet survival during culture and AKT, ERK and STAT3 are important signals during pancreas preservation maintaining viable islets.

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Insulin-Independent Reversal ff Type-1 Diabetes with Brown Adipose Tissue Transplants: Involvement Of IGF-1

Subhadra Gunawardana1, David Piston1

1Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, United States.

Current therapies for type 1 diabetes (T1D) involving insulin replacement or islet/pancreas transplantation have numerous limitations. The ultimate goal in treating T1D is to restore glucose homeostasis. Our recent work demonstrates that euglycemia can be achieved without insulin, using subcutaneous embryonic brown adipose tissue (BAT) transplants [1,2]. BAT transplants in Streptozotocin (STZ)-treated diabetic mice result in robust replenishment of white adipose tissue (WAT) and reversal of diabetes, accompanied by suppression of glucagon and progressive increases in adipokines. These effects are independent of insulin, and require new WAT to remain healthy and un-inflamed.

Objectives of this study were to identify the underlying mechanisms of insulin-independent glycemic regulation following BAT transplants, and to reproduce the results in auto-immune diabetic models similar to human T1D. In nonobese diabetic (NOD) mice, BAT transplants result in complete reversal of T1D associated with rapid and long-lasting euglycemia (Fig. 1). As with STZ-diabetic models, euglycemia is independent of insulin, and strongly correlates with decrease of inflammation and increase of adipokines. Mechanistic studies point to insulin-like growth factor-1 (IGF-1) as a major mediator in the new equilibrium. Plasma IGF-1 is elevated immediately following BAT transplant, prior to any increase in WAT-derived adipokines such as adiponectin and leptin. BAT transplant and surrounding WAT consistently express IGF-1 compared with little or no expression in the WAT of normal and diabetic controls. Plasma IGF-1 levels progressively increase following BAT transplants, in negative correlation with pro-inflammatory cytokines (Fig.1). IGF-1 has adipogenic and anti-inflammatory properties, and is likely to stimulate regeneration and maintenance of new healthy WAT which in turn can secrete adipokines to substitute for insulin. IGF-1 may also directly decrease blood glucose through activating the insulin receptor, since acute inhibition of insulin receptor impairs glucose tolerance in BAT transplant recipients who operate without insulin. These data suggest that IGF-1 plays several key functions in insulin-independent glucose regulation following BAT transplants.


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Cadherin Engagement Improves Insulin Secretion of Single Human Beta-Cells

Geraldine Parnaud1, Vanessa Lavallard1, Thierry Berney1, Domenico Bosco1

1Cell Isolation and Transplantation Center, Division of Surgical Research, Geneva University Hospital and University of Geneva, Geneva, Switzerland.

Aim: In this study we assessed whether cadherin-mediated adhesion of human islet cells was affected by insulin secretagogues and explored the role of cadherins in the secretory activity of beta-cells.

Methods: Experiments were carried out with single islet cells adherent to chimeric proteins made of functional E-, N- or P-cadherin ectodomains fused to Fc fragment of immunoglobulin (E-cad/Fc, N-cad/Fc and P-cad/Fc) and immobilized on an inert substrate. Adhesion of islet cells on these chimeric proteins was assessed in the absence of presence of insulin secretagogues and insulin secretion evaluated by reverse hemolytic plaque assay (RHPA). Beta- and alpha-cells were identified by immunofluorescence.

Results: On N-cad/Fc and E-cad/Fc, islet cells time- and secretagogue-dependently acquired a spreading form. This effect was inhibited using blocking cadherin antibodies. High glucose concentration increased the spreading of beta-cells and not that of alpha-cells. By RHPA, under basal condition (2.8 mmol/l glucose) single beta-cell insulin secretions were not affected by any of the cadherin peptides. In the presence of E-cad/Fc and after glucose stimulation (16.7 mmol/l), 5-10 % single beta cells were found with a spreading form. Whereas 52±8 % round beta cells, virtually all spreading beta cells (96±5 %) were surrounded by a hemolytic plaque, p<0.001. The mean plaque area of spreading cells was about three times higher compared to that of round cells. As a consequence, total insulin secretion was 6 times higher in spreading beta cells compared to round beta cells (928078±307022 vs. 163933±54204, spreading cells vs. round cells, respectively, p=0.04).

Conclusion: Our results show that adhesion of beta-cells to E- and N-cadherins is regulated by insulin secretagogues and that at least E-cadherin engagement promotes stimulated insulin secretion.

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NLRP3 Inflammasome is Expressed and Regulated in Human Islets

Vanessa Lavallard1, Géraldine Parnaud1, Philippe Morel1, Benoît Bedat1, Domenico Bosco1, Thierry Berney1

1Department of Surgery, Cell Isolation and Transplantation Center, Geneva University hospital and University of Geneva, Geneva, Switzerland.

Background and Aims: NRLP3 inflammasome is a protein complex playing an important role in innate immunity. This complex is activated in response to infection, inflammation and autoimmune processes and is involved in the maturation of IL1β by the cleavage of caspase-1. The real contribution of NRLP3 inflammasome in local islet production of IL1β has not been yet demonstrated. The aim of this study was to determine the expression and the regulation of the NRLP3 inflammasome in human islets.

Materials and Methods: Human islets were enzymatically isolated from cadaveric donor pancreases. Isolated islets were stimulated or not with 1μg/ml LPS for 4h and successively with 5mM ATP for 30 min in the presence or absence of 200μM glyburide, an inflammasome inhibitor. Secreted IL1β was quantified by ELISA. The NRLP3 and IL1β genes expressed were studied by real time PCR and caspase-1 and IL1β by western blot.

Results:NRLP3 and IL1β were found to be expressed in untreated isolated human islets. In response to the LPS plus ATP treatment, NRLP3 and IL1β increased 3.4±1.05 fold (p=0.048) and 50.4±1.9 fold (p=0.011), respectively. Glyburide prevented the augmentation of NLRP3 (2.1±0.7 fold, p=0.045) and IL1β (22.2±5.01 fold, p=0.023). The LPS plus ATP treatment induced the cleavage of caspase-1 and the increase of pro and mature IL1β protein expression. Human islets secreted 38.1±1.8 pg/ml IL1β (p=0.0097) in response to LPS plus ATP. Glyburide was shown to prevent the cleavage of caspase-1, decrease the pro and mature IL1β form and diminish IL1β secretion by 97.6% (0.92±0.28 pg/ml IL1β, p=0.0079).

Conclusion: These results show that the NRLP3 inflammasome is expressed and regulated in human islets in response to LPS plus ATP. The NRLP3 inflammasome could be a potential therapeutic target to prevent local IL1β production and the subsequent islet damages in both diabetes and islet transplantation.

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Metabolic Demand as a Barrier to Islet Allograft Tolerance Induction

Nicholas H. Bishop1, K. Scott Beard1, Ronald G. Gill1

1Surgery and Immunology, University of Colorado, Denver, Aurora, CO, United States.

Background: Several obstacles may prevent tolerance induction in transplant recipients. While pre-existing autoimmunity is a formidable barrier to tolerance induction in Type 1 diabetes, metabolic demand / hyperglycemia itself may be an under-appreciated barrier to tolerance induction. Therefore, the goal of this study was to determine whether the metabolic demand on islet transplants was an independent variable impacting tolerance resistance.

Methods: We studied the independent effect recipient metabolic demand on tolerance outcomes using the Akita mouse model of severe, chronic diabetes. The Akita phenotype is the result of a spontaneous polymorphism in the ins2 gene resulting in severe, lifelong diabetes. Importantly, this form of spontaneous diabetes is not associated with autoimmunity, C57Bl/6 (B6; H-2b) Akita uniformly accept syngeneic wild-type B6 islet grafts. To illustrate the role of metabolic demand on grafted islets in this model, Akita mice received a marginal mass of 200 islets. Costimulation blockade (anti-CD154 therapy) was used to induce long-term allograft survival. Temporary peri-transplant metabolic relief was provided by a subcutaneous implantation of a slow-release insulin pellet.

Results: 200 allogeneic BALB/c islets could engraft and normalize blood glucose for >100 days in 4/4 immune-deficient B6 Akita rag−/− recipients. However, the majority of BALB/c islet grafts failed to engraft in wild-type B6 Akita recipients with or without anti-CD154 therapy; only 1/9 allografts functioned > 60 days with anti-CD154 therapy. However, the provision of peri-transplant insulin treatment resulted in the engraftment of 9/9 allografts and long-term allograft survival in 4/6 animals (p < .01)

Conclusion: Results suggest an important interplay between metabolic demand and adaptive immunity during the peri-transplant period and the subsequent propensity for tolerance induction. We hypothesize that the degree of metabolic demand possibly related tissue distress in islet transplants can be an independent variable in determining islet allograft survival and can impact tolerance induction.

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Acute Destruction of Islet Allografts in NOD Mice is Independent of Donor MHC Expression

Adam Burrack1, Joshua N. Beilke2, Tinalyn Kupfer1, Ronald G. Gill1

1Surgery and Immunology, University of Colorado, Denver, Aurora, CO, United States; 2Novo Nordisk Research Center, Seattle, WA, United States.

Background: There is ongoing debate regarding the relative contribution of islet-specific autoimmunity versus conventional transplant immunity in the destruction of islet allografts in autoimmune recipients. Specifically, it is unclear whether the pathways of autoimmune disease recurrence are similar to or distinct from allograft immunity. In this study, we determined whether donor MHC expression is required for disease recurrence versus allograft destruction in spontaneously diabetic NOD mice.

Methods: Spontaneously diabetic NOD mice were grafted with either syngeneic NOD or allogeneic C57Bl/6 (B6) islets that were genetically deficient in MHC class I (b2m−/−), MHC class II (NOD C2ta−/− or B6 I-Ab−/−), or both MHC class I and II by using appropriate donors. In some cases, NOD recipients were depleted of CD4 or CD8 T cells in vivo with GK1.5 or 116–13.1 antibody treatment, respectively.

Results: NOD rag−/− (n = 15) or MHC II-deficient (n =3) NOD islets grafts were acutely destroyed in NOD recipients. However, as shown by others, NOD MHC I-deficient or MHC I/II-deficient islets had greatly prolonged survival in untreated NOD mice (MST of 87 days; p < .01). In stark contrast, B6 islet allografts deficient in MHC class I (n=8), class II (n =6), or both (n = 6) were uniformly rejected in NOD mice in <17 days. Moreover, the destruction of B6 MHC I/II double-deficient islet allografts in NOD mice was CD4 T cell-dependent and CD8 T cell independent based on T cell depletion experiments. Interestingly, B6 MHC class I/II-deficient islets survived for >100 days in 7/7 non-autoimmune BALB/c recipients (p < .001), indicating that conventional islet allograft rejection does require donor MHC expression.

Conclusion: Results show a striking difference in the requirements for disease recurrence versus allograft destruction in autoimmune diabetic NOD mice. Disease recurrence requires donor MHC class I expression while allograft destruction is independent of donor MHC expression and requires CD4 T cells. We hypothesize that actual disease recurrence is primarily a CD8 T cell mediated response restricted by donor MHC class I molecules while ‘indirect’ CD4 T cell reactivity plays a predominant role in allograft rejection. In part, this exaggerated CD4 T cell response may contribute to the profound allograft tolerance resistance found in autoimmune NOD recipients.

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Liver Ischemia After Islet Transplantation - Rat Animal Model

Jan Kriz1, Eva Fabryova2, Andrea Galisova3, Milan Hajek3, Frantisek Saudek1,2

1Diabetes Center, Institute for Clinical and Experimental Medicine, Prague, Czech Republic; 2Center of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic; 3Department of MR spectroscopy, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.

Introduction: Currently the pancreatic islet (PI) transplantation (Tx) is mostly performed using the catheter inserted transcutaneously into the hepatic portal vein1. Islets embolize within peripheral branches of portal vein and can obstruct it causing ischemia of surrounding liver tissue2. This can cause necrosis of liver cells, which together with instant blood mediated inflammatory reaction (IBMIR), triggers the injury of islet cells3. The intensity of IBMIR and the size of liver ischemia correlate to the expression of tissue factor (TF) on islet cells4. The research of TF inhibition and subsequent reduction of IBMIR need the method for quantification of liver non-perfused tissue5. The main aim of this study was the proper modification of PI Tx model and the optimization of magnetic resonance imaging (MRI) protocol for detection of non-perfused regions of liver.

Methods: The Brown-Norway rats (n=15, 250-320g) served as PI or artificial particles recipients. While recipients of group-A remained intact, animals of group-B underwent ligation of hepatic arteries. 200/2000 of PI or 200/2000 artificial particles (heparin coated or non-coated; Corline, Sveden) were transplanted into the portal vein and spontaneously embolized within recipient liver. Two and 48 hours after Tx recipients were examined visually and by MRI. T1-weighted images were acquired by gradient echo sequence (resolution 234mm/234mm/1500mm) before and after the MultiHance® (60 ml) administration. Immediately after the first MRI, the PatentBlau® dye was injected into portal vein of two animals in both groups.

Results: The Patent-Blau® has confirmed the presence of non-perfused regions in periphery of the de-arterialized livers, considerably more frequent in recipient of 2000 PIs or particles. In animals of group-A the non-colored regions were not visible. Two hours after PI Tx the MRI detected clearly outlined hypointense regions (non-perfused liver tissue) in group-B. In these animals the non-perfused tissue, was barely detectable two days later. In group-A the MRI detected no changes.

Conclusion: In our model the MRI did not detect any ischemic tissue in healthy recipients. When the parallel liver perfusion via hepatic arteries was interrupted (group-B), the contrast agent cannot circumvent the islets and reach the tissue behind it. Then MRI became sensitive enough to detect non-perfused regions. The tested model opens a possibility to study the effect of clotting inhibitors on liver ischemia during early post-transplant period.

Supported by: The grant of the Czech Ministry of Health No. NT14240-3

MH CZ - DRO (“Institute for Clinical and Experimental Medicine – IKEM, IN 00023001”)

Supported by: 1) The grant of the Czech Ministry of Health No. NT14240-3 2) MH CZ - DRO („Institute for Clinical and Experimental Medicine – IKEM, IN 00023001“)

Reference:

[1] Shapiro, A.M., et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343, 230-238 (2000).

[2] Cabric, S., et al. Islet surface heparinization prevents the instant blood-mediated inflammatory reaction in islet transplantation. Diabetes 56, 2008-2015 (2007).

[3] Yin, D., et al. Liver ischemia contributes to early islet failure following intraportal transplantation: benefits of liver ischemic-preconditioning. Am J Transplant 6, 60-68 (2006).

[4] Johansson, H., et al. Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation. Diabetes 54, 1755-1762 (2005).

[5] Sakata, N., et al. MRI assessment of ischemic liver after intraportal islet transplantation. Transplantation 87, 825-830 (2009).

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Degradation of Laminin and Laminin-511 in the Human Peri-islet Extracellular Matrix is Targeted by Neutral Protease and Thermolysin, but not Collagenase

Paul A. Bateman1, Kevin J. Devereux-Cooke1, James D. Johnson1, Heide Brandhorst1, Daniel Brandhorst1, Derek W. R. Gray1, Sarah E. Cross1, Stephen J. Hughes1, Paul R. V. Johnson1

1Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom.

Objectives: We have previously reported that the enzymatic digestion of peri-islet laminin in the human pancreas is influenced by donor age and BMI. We have also reported that laminin-511, an isoform of laminin found in both the outer endocrine and inner vascular basement membrane of the intra-islet vascular channels, is susceptible to digestion. Here we investigate which components of digestion enzyme blends routinely used in human islet isolation are responsible for the breakdown of laminin and laminin-511.

Methods: Recombinant laminin-511 was incubated with individual enzyme components or with combinations of enzymes to mimic a commercially available collagenase blend, and then assessed for degradation by gel electrophoresis. Determination of laminin-511 cleavage sites was determined by proteomic tandem mass spectrometric methods. With appropriate consent and ethical approval, the digestion of peri-islet laminin and laminin-511 was measured in cryosections of human donor pancreases (n=6), following incubation with specific supplementary enzymes.

Results: Recombinant laminin-511 was digested by neutral protease and thermolysin, and to a lesser extent, clostripain. laminin-511 was not digested by Collagenase isoforms alone or in combination except in the presence of Neutral Protease. Cleavage sites of laminin-511 were localized to the “arms” of the individual peptide chains with thermolysin being the most aggressive enzyme. Additionally, thermolysin cleaved the laminin alpha5 chain in the C-terminal globular domain, which interacts with islet integrins and other cellular receptors. The degradation of both laminin and laminin-511 at the islet:exocrine interface was also observed in pancreatic sections incubated with neutral protease or thermolysin.

Conclusions: As peri-islet laminin increases with age, and its digestion by neutral protease is lower in younger donors, data from this study would suggest that substituting neutral protease with the more aggressive thermolysin as part of the enzyme combination used for human islet isolation, could potentially improve islet yields from younger donors.

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Restored Vascular Density and Blood Fow in Mouse and Human Islets Experimentally Transplanted to The Greater Omentum

Daniel Espes1, Joey Lau1, Gustaf Christoffersson1, My Quach1, Per-Ola Carlsson1,2

1Medical Cell Biology, Uppsala University, Uppsala, Sweden; 2Medical Sciences, Uppsala University, Uppsala, Sweden.

Islet transplantation is hampered by poor long-term results, which may relate to site-specific challenges of the liver. The highly vascularized greater omentum has been suggested as a possible alternative site, but its capability for islet vascular engraftment is unknown. The present study investigated the revascularization process, blood perfusion and oxygenation in mouse and human islets transplanted to the greater omentum of syngeneic or nude mice. Vascular density was assessed both in vivo for perfused blood vessels by CD31 and fluorescent dextran, and by immunohistochemistry. The attribution of donor endothelium was assessed by transplantation of flk-1GFP islets. Islet blood flow and oxygen tension was measured with laser-Doppler and Clark microelectrodes in vivo, and extent of islet hypoxia by the biochemical marker pimonidazole.

The islet vascular density was restored within 30 days (10.4±0.4%, n=5) when compared to native islets (8.5±0.6%, n=5), whereas at day 7 the vascular density was 3.0±0.6 % (n=4, p<0.001 vs. day 30 and native islets). Donor endothelium did not contribute in the revascularization process. A vascular network similar to that in native islets where all blood vessels were perfused was established within 14 days. Blood flow at 30 days was 22±3 TPU in transplanted mouse islets (n=7), 21±2 TPU in transplanted human islets (n=7), and 8±1 TPU in the gastric wall (reference organ; n=8). Oxygen tension in the transplanted tissue was also restored to values similar to those recorded in native islets; i.e. 35.4±5.1 mmHg in mouse islets and 34.8±3.7 mmHg in human islets (n=7 animals). Islet hypoxia in islet grafts was rapidly reversed (51±16%, 20±2% and 2.0±0.3% hypoxic cells at day 1, 4 and 7, respectively).

We conclude that islets transplanted to the greater omentum have a rapidly restored vascular density, blood flow and oxygen tension, and thereby represents an attractive alternative to the liver worthy of consideration.

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A Silk Based Encapsulation Platform for Pancreatic Islets Reduces Marginal Mass for Transplantation and Improves Islet function in Vivo

Magali Fontaine1, Diana Hamilton1, Hank Shih1, Richard Schubert1, David Kaplan2

1Pathology, Stanford University, Stanford, CA, United States; 2Biomedical Engineering, Tufts University, Medford, MA, United States.

Background: The success of pancreatic islet (PI) transplantation is challenged by PI functional damage and apoptosis during the peri-transplantation period. PI encapsulation in a silk hydrogel with mesenchymal stromal cells (MSCs) can support a three dimensional microenvironment enhancing engraftment. We evaluate in vivo the survival and function of PI transplanted in the same silk-based platform.

Methods: PI were isolated from C57BL/6J mice and transplanted in the epidydimal fat pad of streptozotocin induced diabetic syngeneic mice. Two preparations of 150 islet equivalents (IEQ) versus 200 IEQ were transplanted as follows: 1) Pelleted alone; 2) Encapsulated in silk 3) Co-encapsulated in silk with MSCs (1x106). Blood glucose levels were monitored and intraperitoneal glucose tolerance test (IPGTT) was performed on day 21 post-transplant. Grafts were removed on day 42 for immunohistochemistry analysis and on day 14 for cytokine analysis by Luminex assay.

Results: Recipients of 200 IEQ in silk returned to euglycemia at 4±2 days post transplant compared to 9.4±4 days with PI in silk with MSCs and to 34±7 days with pelleted PI. A marginal mass of 150 IEQ was sufficient to reverse to euglycemia only for PI in silk with MSCs. Tolerance to high glucose was significantly improved (IPGTT) with 200 IEQ in silk with MSCs, compared to PI in silk alone or pelleted (p< 0.05 ). Histology revealed widespread re-vascularization in both grafts of PI in silk with or without MSCs. Pro-inflammatory cytokines (IL-1β and TNFα) increased (5 fold) in PI grafts with silk alone but decreased significantly with islets co encapsulated with MSCs. Th2 cytokines (IL-4, IL-5, and IL-13), and pro-angiogenic factor (TGFβ, VEGF) were increased significantly in islets encapsulated in silk both with or without MSCs.

Conclusions: PI encapsulated in a silk scaffold with or without MSCs reversed hyperglycemia in association with increased levels of angiogenic factors and Th2 cytokines in the grafts. Further studies are ongoing to confirm silk biocompatibility for applications in allo- and xeno- islet transplantation.

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Survival Characteristics of the Inflammatory-Suppressed Islet Allograft: Reduced Immunosuppression and Prolongled Survival

Nathan W. Zammit1, Bernice M. Tan1, Stacey N. Walters1, David Liuwantara1, Jeanette E. Villanueva1, Elisabeth K. Malle1, Shane T. Grey1

1Immunology Department, Garvan Institute, Darlinghurst, Australia.

Islet-grafts can contribute to their own destruction via the elaboration of proinflammatory genes, many of which are transcriptionally regulated by NF-κB. Thus NF-κB constitutes an enticing gene therapy candidate to improve the success of islet transplantation. To test this hypothesis in vivo, we blocked NF-κB in BALB/c (H-2d) to C57/BL6 (H-2b) mouse islet allografts by genetically-engineering islets to express the NF-κB super-repressor, IκBα. Here we show by microarray and RTqPCR that islets exhibit an intrinsic early-immediate pro-inflammatory response; with the most highly up-regulated proinflammatory genes comprising the chemokines Cxcl1, Cxcl2, Cxcl10 and Ccl2; the cytokines Tnfα and Il6; and the adhesion molecule Icam1. Overexpression of IκBα inhibited the expression of these genes by 50-95 % in islets and MIN6 β-cells in vitro, by inhibiting NF-κB-dependent gene transcription. Histological and RTqPCR analysis at post-operative day (POD) 10 revealed that IκBα transduced islet allografts exhibited improved islet architecture and strong insulin-labelling with decreased Ccl2 and Il6 mRNA levels compared to GFP-transduced control grafts. Despite these protective effects, NF-κB blocked islet allografts were promptly rejected in our MHC mismatched mouse model. However, IκBα expressing grafts did harbour localized ’pockets’ of Foxp3+ mononuclear cells not evident in control grafts. This result suggested to us that the effect of NF-κB blockade might synergize with regulatory T-cell sparing Rapamycin. Indeed, combining intragraft IκBα expression with low-dose Rapamycin increased the mean survival time of islet allografts from 20 to 81 days; with 20 % of grafts surviving for greater than 100 days. In conclusion, Rapamycin unmasks the protective potential of intragraft NF-κB blockade, which can, in some cases, permit permanent allograft survival without continuous systemic immunosuppression.

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Prevention and Rescue of Diabetes by All-Trans Retinoid Acid and Exendin-4 in NOD Mice with and Without Islet Transplantation

Jyuhn-Huarng Juang1, Yang-Hau Van1, Chien-Hung Kuo1, Ying-Hsiu Liu1, Han-Yin Chang1

1Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, Taoyuan, Taiwan.

Type 1 diabetes usually develops due to an autoimmune destruction of insulin-producing beta cells of the pancreas. It has been shown that all-trans retinoid acid (ATRA), a potent derivative of vitamin A, hinders the development of autoimmune diabetes by inducing immune tolerance status. In addition, exendin-4, a glucagon-like peptide-1 receptor agonist, stimulates growth and differentiation of beta cells and exerts antiapoptotic effect on beta cells. Thus we hypothesized that the ATRA and exendin-4 therapy may prevent and rescue autoimmune diabetes in NOD mice with and without islet transplantation.

In experiment I, NOD/scid mice were intravenously transferred with splenocytes isolated from 12 week-old female NOD mice. After adopted transfer, mice were treated with ATRA (0.5 mg/mouse intraperitoneally qod), exendin-4 (3 mg/kg subcutaneously bid) or ATRA+exendin-4 for 6 weeks. In experiment II, NOD mice were treated with ATRA or exendin-4 after the onset of diabetes. In experiment III, diabetic NOD mice were transplanted with 300-600 islets from NOD/scid mice and then treated with ATRA or exendin-4.

In experiment I, all adopted transfer recipients treated with ATRA or exendin-4 developed diabetes before 8 weeks. However, the combination treatment of ATRA and exendin-4 delayed the onset of diabetes untill 10-12 weeks (p<0.05). In experiment II, all but one diabetic NOD mice treated with ATRA remained diabetic and the survival time was comparable in control (52±31 days), ATRA (45±19 days) and exendin-4 (62±34 days) groups. The mouse who achieved normoglycemia after ATRA treatment was alive for 366 days with periodic hyperglycemia. In experiment III, all mice remained diabetic after islet transplantation and the survival time was not significantly different among control (52±51 days), ATRA (101±101 days) and exendin-4 (48±19 days) groups.

In conclusion, the combination of ATRA and exendin-4 is effective in preventing autoimmune diabetes. However, ATRA or exendin-4 alone did not prevent or rescue diabetes in NOD mice with and without islet transplantation.

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Insulin-Like Growth Factor-II (IGF-II) Prevents Pro-Inflammatory Cytokine Induced Apoptosis and Significantly Improves Islet Survival Post-Transplantation

Amy Hughes1,3, Daisy Mohanasundaram1,2,3, Svjetlana Kireta1, Claire F Jessup4,5, Chris J Drogemuller1,2,3,5, P Toby H Coates1,2,3,5

1Renal and transplantation Immunobiology Laboratory, The Royal Adelaide Hospital, Adelaide, Australia; 2Australian Islet Consortium, Adelaide, Australia; 3School of Medicine, University of Adelaide, Adelaide, Australia; 4Department of Human Physiology, Flinders University, Adelaide, Australia; 5Centre for stem cell research, University of Adelaide, Adelaide, Australia.

Introduction: The early loss of functional islet mass (50-70%) due to apoptosis following clinical transplantation contributes to islet allograft failure. Insulin-like Growth Factor-II (IGF-II) is an anti-apoptotic protein that is highly expressed in β-cells during development, but rapidly decreases in post-natal life.

Methods: We used an Adenoviral (Ad) vector to over express IGF-II in isolated rat islets and investigated its anti-apoptotic action against exogenous cytokines IL-1β and IFN-γ induced islet cell death in vitro. Using an immunocompromised marginal mass islet transplant model, the ability of Ad-IGF-II transduced rat islets to restore euglycemia in NOD-SCID diabetic recipients was assessed.

Results: Ad-IGF-II transduction did not affect islet viability or function. Ad-IGF-II cytokine treated islets exhibited decreased cell death (40 ± 2.8%) vs. Ad-GFP and untransduced control islets (63.2 ± 2.5% and 53.6 ± 2.3%, respectively). Ad-IGF-II over expression during cytokine treatment resulted in a marked reduction in TUNEL positive apoptotic cells (8.3 ± 1.4%) vs. Ad-GFP control (41 ± 4.2%) and untransduced control islets (46.5 ± 6.2%). Western blot analysis confirmed that IGF-II inhibits apoptosis via activation of the PI3K/Akt signalling pathway. Transplantation of IGF-II over expressing islets under the kidney capsule of diabetic mice restored euglycemia in 78% of recipients, compared to 18% and 47% of Ad-GFP and untransduced control islet recipients, respectively (p<0.05 Log-rank (Mantel Cox)). Ad-IGF-II recipient mice stabilized and slightly increased their weight following transplantation, while this was not the case for mice receiving untransduced or Ad-GFP transduced islet grafts, p<0.0001 (1way ANOVA).

Conclusions: Anti-apoptotic IGF-II decreases apoptosis in vitro and significantly improved islet transplant outcomes in vivo. Anti-apoptotic gene transfer is a potentially powerful tool to improve islet survival post-transplantation.

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Preculture of Human Islets with Mesenchymal Stem Cells in a Direct Contact Configuration Enhances Islet Function in Vitro

Paramjeet Dhadda1, Chloe Rackham1, Aurélie Le Lay1, Alan Kerby1, Guo-Cai Huang1, Peter Jones1

1King’s College London, London, United Kingdom.

Objectives: We have previously shown that co-transplantation of mouse islets with mesenchymal stem cells (MSCs), or pre-transplant culture of islets with MSCs improves transplantation outcome in diabetic mice. The aims of this study were to investigate whether pre-treatment of human islets with human MSC populations improves islet function in vitro, to determine whether data generated in mouse models was translatable to clinically relevant human tissue.

Methods: Human islet preparations from three fully consented cadaveric donors were used for this study (received from King’s College Hospital). In all three experiments islets were cultured over a 4-day period either alone (IA) or directly upon a monolayer of bone-marrow MSCs (IBM), adipose MSCs (IAD) or pancreatic MSCs (IPA). Islets were subsequently assessed for insulin content and glucose stimulated insulin secretion (GSIS).

Results: At the end of the 4 day co-culture period, all MSC populations increased islet insulin content when compared to islets cultured alone (IA: 12.8 ± 1.1ng/islet; IBM: 18.9 ± 1.6, p<0.001; IAD: 19.1 ± 3.3, p<0.01; IPA: 19.4 ± 2.5, p<0.001, n=20 observations) in all three experiments. Pretreatment of islets with bone-marrow or adipose MSCs also enhanced glucose induced (20mM) insulin secretion over basal secretion (2mM) (IA: 12 ± 1 fold; IBM: 14 ± 3; IAD: 18 ± 3, p<0.05 vs. IA, n=10 observations). In contrast pancreatic MSCs did not improve GSIS (IPA: 10 ± 1, p>0.2, n=10). This trend was observed in all three experiments.

Conclusion: Pre-treatment of human islets with three different human MSC populations increased islet insulin content, however only bone marrow and adipose MSCs enhanced GSIS. Pre-treatment of isolated human islets on a monolayer of MSCs, prior to clinical transplantation, could serve as a viable option for the maintenance of islets in culture with potential to improve subsequent islet transplantation outcome.

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Vascular Heterogeneity Between Native Pancreatic Islets Determines Their Fate of Survival and Revascularization Posttransplantation

Sara Ullsten1, Joey Lau1, Nils Welsh1, Per-Ola Carlsson1,2

1Medical Cell Biology, Uppsala University, Uppsala, Sweden; 2Medical Sciences, Uppsala University, Uppsala, Sweden.

Highly blood perfused islets have been observed to be the most functional islets in the native pancreas. This study investigated the hypothesis that differences in vascular support of islets in donor pancreas influence their capacity of vascular engraftment and susceptibility to cellular stress after transplantation. Highly blood perfused islets in rat were identified by injection of microspheres into the ascending aorta, followed by retrieval of pancreas for islet isolation. Islets with or without microspheres were syngeneically transplanted beneath the renal capsule. Capacity for islet engraftment was evaluated 30 days posttransplantation with regard to blood perfusion (laser-Doppler flowmetry), oxygen tension (Clark microelectrodes) and vascular density (lectin staining). In vitro studies of susceptibility to cellular stress was performed on groups of 50 islets after exposure to cytokines (IL1-β, IFN-γ, TNF-α) or hypoxia (1.5% O2) through PI and bisbenzimide staining.

Microsphere-containing islets regained a higher blood perfusion and oxygen tension (14.1±0.6 TPU, 30.3±2.2 mmHg) when compared to control grafts of islets without microspheres (9.3±0.8 TPU, 8.7±1.3 mmHg; P<0.05, n=6 for both comparisons). The microsphere-containing islet grafts also had higher vascular density (4.2±0.3 % of insulin-positive area) compared to control grafts (2.7±0.1 %; P<0.05, n=6). In vitro data of islet viability indicated that microsphere-containing islets were more susceptible to cytokine-induced cell death (1.9±0.2 compared to 1.3±0.1 PI/bisbenzimide ratio; P<0.05, n=8) and, preliminary also, hypoxia-induced cell death.

Islets in the native pancreas which are highly blood perfused regain this feature after transplantation, which indicates a superior capacity for revascularization and posttransplant function. However, the same group of islets is more vulnerable to both cytokine- and hypoxia-induced cell death, factors of high importance for early survival posttransplantation. Preferential death of these most active islets may severely contribute to the high number of islets needed to provide cure with islet transplantation.

Supported by: SRC, NNF, SDF and Diabetes Wellness Sweden

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Adenoviral Vector-Mediated Anti-Apoptotic Gene Transduction for Engineering Functional Islet Cell Sheets

Hiroyuki Hanayama1, Kazuo Ohashi2, Rie Utoh2, Kazuya Ise1, Tatsuya Shimizu2, Masayuki Yamato2, Hiroyuki Mizuguchi3, Fuminori Sakurai3, Teruo Okano2, Mitsukazu Gotoh1

1Regenerative Surgery, Fukushima Medical University, Fukushima, Japan; 2Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan; 3Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.

Background: Toward the establishment of novel islet-based therapies, our group has recently established technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheet format of dispersed islet cells (islet cell sheets, Biomaterials 30: 5943, 2009, Transplantation 92: 1231, 2011). Islet cell sheets are made of single islet cells dispersed from islets. It has been known that dispersed single islet cells are occasionally susceptible to apoptotic process. To achive higher level of therapeutic values of the islet cell sheet-based therapy, it would be valuable if the dispersed islet cells could be protected from the apoptotic cell death process. This paper addresses the effect of fiber-modified adenoviral-mediated anti-apoptotic gene transduction to dispersed islet cells on their protective effects from apoptotic stimulation.

Methods and Results: Purified pancreatic islets were obtained from Lewis rats, followed by their dissociation into single islet cells. Cells were plated onto laminin-5-coated and temperature-responsive polymer poly(N-isopropylacrylamide)(PIPAAm)-immoblized plastic dishes. At day 1, islet cells were exposed by fiber-modified adenovirus vector (Ad-CA-K7-GFP) that harbors a polylysin (K7) peptide in the C-terminus of the fiber knob, for 1 hour. Gene Transduction efficiency and celluar cytotoxicity were evaluated 48 hours later. More than 95% of the cells were found to be transduced at MOI=10, without demonstrating detectable cellular cytotoxicities. We then transduced anti-apoptotic gene (mutant Bcl-xL gene, FNK) using Ad-K7-CA-FNK, then cells were incubalted with hydrogen peroxide for inducing apoptosis. There was higher cellular survival observed in the FNK-transduced islet cells compared with non-transduced cells. The FNK-transduced islet cells could be harvested as an intact cell sheet format.

Conclusions: These data suggest that adenoviral-mediated gene transduction is a valuable approach for providing anti-apoptotic molecules to the dispersed islet cells. Creation of highly functional islet tissues could be anticipated by transplanting islet cell sheets that are temporarily resistant to the apoptotic process.

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Autologous Islet Cell Transplantation for Treatment of Chronic Pancreatitis; The Five Year University of Virginia Experience

Avinash Agarwal1, Linda Langman1, Kenneth L Brayman1

1Department of Surgery, University of Virginia, Charlottesville, VA, United States.

Objective: To describe a single center clinical experience of autologous pancreatic islet transplants as a treatment for chronic pancreatitis.

Methods: Between January 2007 and December 2012, nineteen patients underwent an extended pancreatectomy for definitive treatment of chronic pancreatitis. Pancreata were surgically removed by the transplant division and sent to the islet processing facility. The islets were isolated using the Ricordi method and purified.

Results: Twelve patients underwent total pancreatectomy with seven cases of near-total pancreatectomy. Mean age was 41 years (range 15-62) with a male to female ratio of 8:11. Eighteen of nineteen patients received and tolerated autologous islet cell infusion. One patient did not receive islet infusion secondary to infectious concerns. The mean islet equivalents were 199,760± 129,881 Islet equivalents (IEQs) with mean IEQ/kg of 2,885±1876 IEQ/kg. One year and five year actuarial patient survival was 100% and 93%. There was one death secondary to sepsis. Overall, pancreatectomy with autologous islet cell transplantation was well tolerated with low morbidity. There were no cases of portal thrombosis. Surgical complications included one pancreatic leak and one SMA injury. No patients required insulin prior to surgery. At mean follow up of 35±13 months, nine patients (50%) remain insulin independent (two patients require oral hypoglycemics). Nine patients have a mean insulin requirement of 9± 5 U/day. At one month follow-up, 17 patients (94%) had detectable c-peptide (mean 1.3±1.1 ng/mL). None of the patients have reported hypoglycemic unawareness episodes post-transplant. Overall, all patients reported a significant increase in the quality of life represented by a decrease in pain and narcotic requirements.

Conclusions: Autologous islet transplantation after extensive pancreatic resection for chronic pancreatitis is a safe and successful procedure. It offers definitive treatment of their diseased pancreas without the morbidity of brittle diabetes. Ideally, patients should be offered this therapy earlier to decrease chronic abdominal pain and preserve endogenous endocrine function. The financial burden and poor health associated with diabetes can be successfully mitigated with pancreatectomy with autologous islet cell transplantation.

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Case of Successful Endoscopic Pancreatic Islets Auto-Transplantation Into Gastric Sub-Mucosa in Patient with Chronic Pancreatitis - Preliminary report

Michal Wszola1, Artur Kwiatkowski1, Andrzej Berman1, Lukasz Gorski1, Agata Ostaszewska1, Pawel Ziemianski1, Rafal Kieszek1, Piotr Domagala1, Krystian Pawelec2, Monika Krajewska3, Anna Lipinska4, Zuzanna Rymarczyk4, Jolanta Gozdowska5, Janusz Trzebicki6, Marcin Polkowski7, Marek Golebiowski8, Leszek Paczek3, Andrzej Chmura1

1Department of General and Transplantation Surgery, Warsaw Medical University, Warsaw, Poland; 2Department of Transplantology and General Surgery, Nicolaus Copernicus University, Bydgoszcz, Poland; 3Department of Immunology, Transplantology and Internal Diseases, Warsaw Medical University, Warsaw, Poland; 4Department of Internal Diseases and Cardiology, Warsaw Medical University, Warsaw, Poland; 5Department of Nephrology and Transplantology, Warsaw Medical University, Warsaw, Poland; 6First Department of Anesthesiology and Intensive Care, Warsaw Medical University, Warsaw, Poland; 7Department of Gastroenterology and Hepatology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland; 8Department of Clinical Radiology, Warsaw Medical University, Warsaw, Poland.

Chronic pancreatitis (CP) is a severe illness which may cause significant deterioration of patients well-being because of constant pain. Pancreatic cancer and diabetes are co-morbities which often follow CP. Pancreatic resection following pancreatic islets autotransplantation might be a successful therapeutic option for selected group of those patients. Unfortunately liver cirrhosis, portal vein thrombosis or hepatic virus infection (B or C) make standard transplantation into portal vein impossible. Alternative implementation sites might be an option for those patients. Successful islets transplantation into gastric sub-mucosa in pre-clinical study on pigs (1,2) makes gastric submucosa promising option for future. Aim of this study was to report a successful resection of the pancreas and endoscopic islets autotransplantation into gastric sub-mucosa in man.

Patient and Methods: A 53-year old man with alcohol related chronic pancreatitis for 15 years was operated on 14th of march 2013. In anamnesis: 3 months earlier patient was diagnosed with type 2 diabetes – only dietary treatment. Suffering with chronic pain treated with opioid analgesics, 16-years ago underwent antrectomy due to peptic ulcer perforation, also HBS-Ag positive and diagnosed with thrombosis of right portal vein. Patient underwent pancreatectomy and splenectomy modo Child. Pancreas was immediately flushed with 1000 ml of UW solution through splenic artery and vein and sent to isolation facility. Islets were isolated, Cobe procedure was not performed. Endoscopic gastric sub-mucosa transplantation was performed 6 hours later. Levels of fasting c-peptide was measured prior to procedure and 1,2,3,4,5,7,14 and 30 days post autotransplantation. C-peptide stimulation (CPS) test was performed prior to procedure and one month post autotransplantation. Fasting glicemia and oral glucose tolerance test (OGTT) was performed prior and 7 and 30 days post transplantation. Control gastroscopy and endoultrasonography (EUS) were performed within first week post-trasnplantation.

Results:Isolation: 250 000 IEq (unpurified) was isolated – 3700 IEq/kg of patients’ body weight. Pellets had 6 ml and was suspended in 60 ml of ringer solution. Transplantation: 18 injection was performed -3 to 5 ml of islets suspension was introduced into submucosa of gastric trunk and fundus. No complication during procedure was observed. Post-operative period: Patient did not require any insulin nor oral hypoglicemics post operation. In early post-operative patient underwent pneumonia and pulmonary embolism successfully treated with antibiotics and Low Molecular Weight Heparin. POST-procedure gastroscopy did not revealed any signs of inflammation or ulceration. EUS did not revealed any fluid collections within gastric wall. PRE-procedure fasting c-peptide was 1.23 ng/ml. POST-procedure C-peptide was(in 1,2,3,4,5,7,14,30 days): 1.11, 1.27, 0.55, 0.94, 1.08, 0.55, 0.69, 1.2, 1.09 ng/ml, respectively. PRE-procedure CPS-test in 0,5,10,15,30,60,120 min was: 1.22, 1.06, 1.35, 1.71, 4.39, 9.63,5.07 ng/ml, respectively. POST-procedure CPS-test was: 1.09, 1.13, 1.31, 3.9, 5.22, 3.82, 1.93 ng/ml, respectively. PRE-procedure OGTT in 0, 5, 10, 15, 30, 60, 120 min was: 85, 91, 98, 98, 167, 210, 76 mg/dl respectively. POST-procedure OGTT was: 96, 116, 127, 191, 224, 153, 68 mg/dl.

Conclusion: Preliminary results of endoscopic gastric sub-mucosa islets autotransplantation shows that it might be an alternative option for islets autotransplantation in case of patients with contraindication for transplantation into portal vein.

Acknowledgement for Fundation for Research and Science Development (Poland, www.fundacja-birn.pl) for its support

Reference:

[1] Wszola M, Berman A, Fabisiak M, Domagala P, Zmudzka M, Kieszek R, Perkowska-Ptasinska A, Sabat M, Pawelec K, Kownacki L, Piotrowska-Kownacka D, Ostrowski K, Januchta M, Klucinski W, Rowinski O, Kwiatkowski A, Chmura A. TransEndoscopic Gastric SubMucosa Islet Transplantation (eGSM-ITx) in pigs with streptozotocine induced diabetes - technical aspects of the procedure - preliminary report. Ann Transplant. 2009 Apr-Jun;14(2):45-50.

[2] Echeverri GJ, McGrath K, Bottino R, Hara H, Dons EM, van der Windt DJ, Ekser B, Casu A, Houser S, Ezzelarab M, Wagner R, Trucco M, Lakkis FG, Cooper DK. Endoscopic gastric submucosal transplantation of islets (ENDO-STI): technique and initial results in diabetic pigs. Am J Transplant. 2009 Nov;9(11):2485-96

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Evidence for Instant Blood Mediated Inflammatory Reaction After Autologous Islet Transplantation

Bashoo Naziruddin1, Shuichi Iwahashi2, Takeshi Itoh2, Mazhar Kanak3, Nicholas Onaca1, Morihito Takita2, Marlon Levy1

1Baylor Annette C. and Harold C. Simmons Transplant Institute, Dallas, TX, United States; 2Islet Cell Laboratory, Baylor Research Institute, Dallas, TX, United States; 3Institute of Biomedical Studies, Baylor University, Waco, TX, United States.

Background: Total pancreatectomy followed by autologous islet transplantation (TP/AIT) can effectively reduce the pain of refractory chronic pancreatitis while retaining endocrine function. Despite increased islet isolation efficiency, the insulin-independence rate after TP/AIT is less than 50%. We hypothesized that an instant blood-mediated inflammatory reaction (IBMIR) occurs during AIT, leading to loss of islets and tested the hypothesis in 24 patients and using an in vitro model.

Method: A total of 24 patients who underwent TP/AIT at our center were included in this study. Blood samples drawn during infusion and up to post-day 7 were analyzed for inflammatory markers and islet function. In the in vitro model, purified human islets from research-grade pancreata were mixed with autologous blood and gently agitated up to 6 hours; blood-only and islet-only controls were also tested. Soluble inflammatory markers and cell surface changes on the islets were examined by Luminex Multiplex Assay and Immunohistochemistry respectively.

Results: Patients who received TP/AIT experienced a significant and rapid increase of thrombin-antithrombin (TAT) and C-peptide during islet infusion, which persisted for up to 3 hours, along with a decreased platelet count (Fig. 1). A concomitant significant increase in IL-6, IL-8 and IP-10 was observed. In an in vitro model, only the islet plus autologous blood group had significantly increased levels of TAT (p < 0.05) and C-peptide (p < 0.05) compared to controls. After mixing islets and autologous blood, strong expression of tissue factor, which is a major trigger for IBMIR, was detected on islet surface.

{figure1}

Conclusion: AIT-induced elevation of TAT and destruction of islets suggests that IBMIR might occur during AIT. Modulating this process may help improve islet engraftment and the insulin-independence rate in TP/AIT patients.

FIGURE 1.
FIGURE 1.
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268

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Autologous Pancreas Islet Transplantation After Completion Pancreatectomy for Pancreatic Fistula After Hemipancreatoduodenectomy for Carcinoma

Matej Kocik1, Kvetoslav Lipar1, Frantisek Saudek2, Peter Girman2, Petr Boucek2, Milos Kucera1, Jiri Fronek1, Martin Oliverius1

1Transplant Surgery Department, IKEM, Prague, Czech Republic; 2Diabetes Center, IKEM, Prague, Czech Republic.

Objectives: Pancreas islet autotransplantation (IAT) has a potential to prevent brittle diabetes in patients after total pancreatectomy for chronic pancreatitis. Because of the fear of contamination of islet suspension with malignant cells and rapid development of liver metastases, IAT has rarely been used for treatment of patients with tumors. Data on long term outcomes of such patients are very limited. Here we report our experience with patients who underwent hemipancreatoduodenectomy for carcinoma and later completion pancreatectomy for pancreatic fistula with islet autotransplantation at our institution.

Methods: From August 2007 to December 2012 five patients underwent completion pancreatectomy for pancreatic fistula after hemipancreatoduodenectomy for carcinoma. Islets were isolated from pancreatic tail using digestion with collagenase (Serva). Non-purified islet suspension was infused into the portal vein intraoperatively. Before islet infusion, complete histology of the resected pancreatic head was obtained and negative resection margin proven.

Results: A median number of islets transplanted was 175000 ieq (range 70000 – 365000). One patient died postoperatively for reasons unrelated to IAT. One patient is off insulin 5 months after IAT and without tumor recurrence (fasting C-peptide 0.45 nmol/l, HbA1c 6.7 %). Other 3 patients had stable diabetes with partial graft function (fasting C-peptide levels 0.23, 0.61 and 0.22 nmol/l, HbA1c 5.8, 6.7 and 8.4 % at last follow-up). One patient with T3 pancreatic head carcinoma with perineural invasion is alive 25 months after IAT with lymph node and liver recurrence since 18 months after IAT. Second patient with gall bladder and distal bile duct carcinoma died 47 months after IAT with tumor recurrence. Third patient with carcinoma of the papilla of Vater with positive lymph nodes and angioinvasion died 12 months after IAT with local recurrence and solitary liver metastasis.

Conclusion: Autotransplantation of pancreatic islets isolated from the resudial pancreatic tissue in patients who previously underwent hemipancreatoduodenectomy for pancreatic cancer may provide stable glucose control thus improving quality of life. In our small series we did not observe early development of multiple liver metastases caused by islet suspension contamination with malignant cells.

Supported by the project (Ministry of Health, Czech Republic) for development of research organization 00023001 (IKEM, Prague, Czech Republic) – Institutional support.

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Comparison of the Use of Insulin and C-Peptide as Measures of Beta Cell Function After Autoislet Transplantation

Melena Bellin1,4, Bernhard Hering2,4, A.N. Balamurugan2,4, Susan Parazzoli3, Elizabeth Oseid3, Ty Dunn2, Gregory Beilman2, David Sutherland2,4, R. Paul Robertson3

1Pediatrics, University of Minnesota, Minneapolis, MN, United States; 2Surgery, University of Minnesota, Minneapolis, MN, United States; 3Pacific Northwest Diabetes Research Institute, Seattle, WA, United States; 4Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN, United States.

C-peptide is the most commonly used measure of beta cell function for assessment of success after total pancreatectomy and intrahepatic islet autotransplantation (TP-IAT). Our studies were designed to determine whether plasma levels of insulin, the major beta cell secretory product, or C-peptide, a surrogate marker for insulin secretion, more accurately reflects functional beta cell mass.

The method of glucose potentiation of arginine-induced insulin secretion (GPAIS) was used in 10 recipients of TP-IAT with normal HbA1c levels. The responses to intravenous arginine were calculated as baseline acute insulin responses (AIRarg) and acute C-peptide responses (ACRarg) before the glucose infusion and also as maximal responses to intravenous arginine during the glucose infusion (AIRargMAX and ACRargMAX). These responses were plotted as a function of the number of autoislets transplanted and correlation coefficients were calculated.

The correlation coefficients for AIRarg and AIRargMAX vs. number of islets transplanted were 0.80 (p < 0.01)and 0.91 (p < 0.001), respectively. The correlation coefficients for ACRarg and ACRargMAX vs. number of islets transplanted were 0.40 (p = ns) and 0.74 (p < 0.02) respectively. The higher correlation coefficients for insulin indicate that measures of insulin secretion more accurately reflect functional beta cell mass of transplanted islets. The close correlation between the insulin responses and the number of transplanted islets was used as a rationale to normalize individual insulin responses to the number of islets transplanted (insulin response/islet number X 106). They were compared with data from 11 normal non-diabetic control subjects with the conventional assumption that the controls had 106 islets in their pancreata. Insulin was expressed as % baseline insulin values to adjust the data for insulin resistance intrinsic to obesity. Responses (mean+/- SE) for Controls, TP-IAT, and TP-IAT/islet#, respectively, were:


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These results indicate that the transplanted islets performed generally as well as islets in normal control subjects. Conclusion: determination of beta cell responses to arginine can be used to estimate functional beta cell mass in autoislet recipients and have the potential to be used to estimate beta cell mass prior to performance of pancreatectomy and autoislet transplantation.

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Longevity of Insulin Independence After Islet Autotransplant is more Common in Young Patients with a Short Duration of Disease

Melena Bellin1,3, Gregory Beilman2, Ty Dunn2, Srinath Chinnakotla2, Timothy Pruett2, Bernhard Hering2,3, Joshua Wilhelm2,3, A.N. Balamurugan2,3, David Sutherland2,3

1Pediatrics, University of Minnesota, Minneapolis, MN, United States; 2Surgery, University of Minnesota, Minneapolis, MN, United States; 3Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN, United States.

Background: Achievement of insulin independence after total pancreatectomy and islet autotransplantation is more common with high islet mass transplanted (> 5,000 IEQ/kg), in young children, and in those with a surgically naïve pancreas. However, little is known about what factors are important for longevity of insulin independence in this population.

Methods: We reviewed all total pancreatectomy (TP) and islet autotransplant (IAT) recipients undergoing surgery in the modern era (between 2000 and April 2010) at a single institution, and identified 77 patients reporting insulin independence after IAT and for whom > 3 years of follow up was available. We compared those patients who achieved insulin independence but resumed insulin therapy at < 3 years post-transplant (short-term II, n = 23) to patients who sustained insulin independence at > 3 years post-IAT (long-term II, n = 54). Demographic characteristics and baseline metabolic data were compared between the two groups.

Results: There was no difference in gender, BMI, etiology of disease, or prior surgical history between the short-term II and long-term II groups, although prior surgeries were rare in patients achieving insulin independence, occurring in only 9 patients (7 with Whipple procedure, 1 with Puestow, and 1 with distal pancreatectomy). Mean transplanted islet mass transplanted was statistically similar between the groups (5,258 ± 2562 in long-term II vs 4,572 ± 2278 in short-term II, p = 0.28). There was no difference in pre-TPIAT hemoglobin A1c, basal, or stimulated C-peptide levels. Notably, those with long-term II were younger (29 ± 15 vs 38 ± 15 years, p = 0.03) and with a shorter duration of disease prior to surgery (4.2 ± 5.7 vs 7.6 ± 7.6 years, p = 0.04) compared to those with short-term II. Duration of disease was weakly correlated with age in this population, such that younger patients achieving insulin independence were also more likely to have a shorter duration of disease (r = 0.24, p = 0.03). In patients younger than 21 years of age at the time of transplant, when insulin independence was achieved, it was sustained at > 3 years post-IAT in 86% of cases; in contrast, in patients ≥ 21 years of age, when insulin independence was achieved, it was sustained for > 3 years post-transplant in only 64% (p = 0.05).

Conclusion: Young TPIAT recipients with a short duration of disease appear to have superior maintenance of insulin independence. Children under 21 years of age who achieve insulin independence frequently sustain insulin independence for > 3 years post-IAT despite growth and development.

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Direct Evidence of Insulin Secretion by Autologous Islets Autotransplanted in the Muscle After Extended Pancreatectomy (For Benign Pancreas Diseases)

Adrien Sterkers1,2, Gregory Baud1,2, Robert Caiazzo1,2, Violeta Raverdy1, Laurence Quintane1, Fanelly Torres1,2, Emmanuelle Leteurte3, Thomas Hubert1, Valery Gmyr1, Julie Kerr-Conte1, Marie Christine Vantyghem1,4, Francois Pattou1,2

1Biotherapies for diabetes, Lille University, Lille, France; 2Chirurgie générale et endocrinienne, CHRU Lille, Lille, France; 3Institut de pathologie, CHRU Lille, Lille, France; 4Endocrinologie et metabolisme, CHRU Lille, Lille, France.

Background: Intra muscular transplantation (IMT) offers attractive prospects for islet transplantation. The clinical relevance of this non-invasive alternative to the intraportal route remains not clearly established. The aim of this study was to evaluate the early experience of clinical IMT and tested its clinical relevance for islet autotransplantation.

Methods: Islet autograft was considered after 50% to 80% partial distal pancreatectomy for sporadic benign pancreatic lesion (insulinoma and other benign tumors, chronic pancreatitis or pancreatic trauma). Islets were isolated with standard automated technique after retrieval of tumor from the pancreatic tissue. Islets were injected under local anesthesia in the brachioradialis muscle. Three month after the graft, acute arginine insulin response was repeated before and after vascular exclusion of the graft (Casanova test). Those tests were repeated each year if primary graft function was efficient.

Results: Six patients (40 ± 17 years) were graft from November 2009 to September 2012. Pancreatectomy was considered for benign pancreatic tumors in 4 cases (including three insulinoma), 1 pancreatic traumas and 1 chronic pancreatitis. Islets isolation allows the graft of 75300 IEQ (min = 11500, max = 135000 IEQ) with 41 ± 13 % purity. Clinical islet IMT was uneventful in all cases. Direct evidence graft function could be documented in 4/6 patients. Three out of four patients with a follow up of more than one year had a relevant graft function 2 and 3 years after the graft

Conclusion: Conclusion: Intramuscular islets autograft can limit the metabolic consequences of pancreatectomy with a low morbidity. This alternative site is attractive for direct graft function follow up.

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272

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Viable Islet Dose Based on Oxygen Consumption Rate is Highly Predictive of Clinical Islet Auto-Transplant Outcome

Klearchos Papas1,2,3, Melena D Bellin2,3, David ER Sutherland2,3, Jennifer P Kitzmann1,2,3, Efstathios S Avgoustiniatos2,3, Angelika C Gruessner1, Katheryn R Mueller1,2,3, Appakalai N Balamurugan2,3, Phillip R Rozak2,3, Gopal Loganathan2,3, Thomas M Suszynski2,3, Josh J Wilhelm2,3, Dajun Qian4, Joyce C Niland4, Bernhard J Hering2,3

1Surgery, University of Arizona, Tucson, AZ, United States; 2Surgery, University of Minnesota, Minneapolis, MN, United States; 3Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN, United States; 4Information Science, City of Hope, Duarte, CA, United States.

There is a critical need for real-time in vitro islet characterization assays that are predictive of clinical transplant outcomes (CTO), but none is available so far. Previously published intraportal islet auto-transplant (IAT) data from the University of Minnesota [1] has shown that islet equivalent (IE) dose (IEtx/kg BW) has a correlation with CTO but it is not highly predictive of CTO [n = 59; area under the receiver operating characteristic (ROC) curve (AUC): 0.817; figure 1a]. In this paper we report on various islet characterization methods of IAT, which is an attractive model for evaluating the relationship between these assays and CTO due to the absence of confounding factors, such as auto-, allo-, and xeno-immunity or immunosuppressive drug toxicity. Islet cell membrane integrity (FDA/PI), the oxygen consumption rate normalized to DNA (OCR/DNA), IEtx/kg BW, and the viable IE dose (OCRtx/kg BW; IE dose multiplied by viability as measured by OCR/DNA) were assessed in islet autograft products with purities ranging from 10% to 95% (n = 35). Recipients with follow ups from 6-12 months were included in the analysis. IAT recipients with fasting blood glucose (BG) < 126 mg/dL, 2-hour postprandial BG < 180 mg/dL, and HbA1c ≤ 6.5% without administration of exogenous insulin were considered II. Relationships with outcomes were examined using ROC curve analysis and the AUC was examined. OCR/DNA and FDA/PI were not predictive of CTO (AUC: 0.580, 0.492 respectively) while IEtx/kg BW and OCRtx/kg BW were highly predictive of CTO (AUC: 0.964, 0.944; figure 1b,c). However, OCRtx/kg BW helped correctly classify CTO where IEtx/kg BW was high but II was not achieved or IEtx/kg BW was low and II was achieved. The data presented suggests that OCRtx/kg BW may be useful for the prospective evaluation of the quality of islet preparations prior to clinical transplantation.


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Reference:

[1] Anazawa T, Balamurugan AN, Bellin M et al: Human islet isolation for autologous transplantation: comparison of yield and function using SERVA/Nordmark versus Roche enzymes. Am J Transplant 2009, 9(10):2383-91.

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273

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Islet Size Index as a Predictor of Outcomes in Clinical Islet Autotransplantation

Thomas M Suszynski1, Joshua J Wilhelm1, David M Radosevich1, Appakalai N Balamurugan1, David ER Sutherland1, Gregory J Beilman1,3, Ty B Dunn1,3, Srinath Chinnakotla1,3, Timothy L Pruett1,3, Selwyn M Vickers1, Bernhard J Hering1, Klearchos K Papas1,2, Melena D Bellin1,3

1Surgery, University of Minnesota, Minneapolis, MN, United States; 2Surgery, University of Arizona, Tucson, AZ, United States; 3Pediatrics, University of Minnesota, Minneapolis, MN, United States.

The islet size distribution in a preparation may contribute to islet transplant outcomes. Diffusion limitations worsen in proportion to the islet radius, and thus β-cells located near the center of larger islets have the poorest access to the oxygen and nutrient supply required for insulin secretion [1].

To test this hypothesis, we studied the impact of islet size index (ISI) and other islet product characteristics on outcomes following islet autotransplant (IAT) in recipients receiving a marginal islet dose (2000-4999 islet equivalents (IEs) per kg body weight (BW) from 1/1/2009-6/11/2012 at the University of Minnesota (n = 58). ISI was defined as the number of IE divided by the number of islet particles (IPs) in a preparation; an ISI < 1 indicates a mean islet diameter that is < 150-μm. The primary post-IAT outcome was 6-month insulin use status. We hypothesized that the ISI would be predictive of insulin use status post-IAT, and that islet preparations with lower ISI would be associated with lower post-IAT insulin requirements than those with higher ISI.

Logistic regression analysis indicated that IPs/kg (p = 0.001), IEs/kg (p = 0.019), total IPs transplanted (p = 0.040) and ISI (p = 0.074) were most strongly correlated with the primary outcome. The ISI (mean ± standard error) was lower for recipients achieving insulin independence at 6-months (0.71 ± 0.05) versus those partially (0.83 ± 0.05) or completely (1.00 ± 0.07) insulin dependent. There were clear correlations between the daily insulin requirements (in terms of the number of units of insulin) at 6 months post-IAT, and both IPs/kg and ISI. Lower insulin requirements were associated with higher IPs/kg (ρ =-0.50) and a lower ISI (ρ = 0.51) (Figure 1). In contrast, IEs/kg exhibited poor correlation with insulin requirements at 6 months post-IAT (ρ =-0.08). The combination of islet dose (expressed as units IPs/kg) and ISI exhibited a sensitivity of 75% and specificity of 74% in predicting insulin independence in this population of patients.

In conclusion, IAT recipients of a marginal islet mass (at a similar IE dose per kg BW) were more likely to achieve insulin independence when transplanted with a greater number of smaller islets as opposed to a smaller number of larger islets.


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Reference:

[1] Dionne KE, Colton CK, Yarmush ML. Effect of hypoxia on insulin secretion by isolated rat and canine islets of Langerhans. Diabetes 1993;42(1):12-21.sss

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275

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Impact of Recipient Body Mass Index in Short and Long Term Survival of Pancreatic Grafts

Benoît Bédat1, Nadja Niclauss1, Anne-Sophie Jannot1, Axel Andres1, Christian Toso1, Philippe Morel1, Thierry Berney1

1Division of Visceral and Transplantation Surgery, University Hospital of Geneva, Geneva, Switzerland.

Introduction: The impact of recipient body mass index (BMI) on pancreatic grafts and patient survival is unknown. Method: Using the Scientific Registry of Transplant Recipients (SRTR), we analyzed data from all individuals who underwent a first pancreas transplant between years 1987 and 2011. Recipients were categorized into BMI classes, as defined by World Health Organization. Patient and graft survival were analyzed by recipient BMI classes using Kaplan-Meyer estimates and hazard ratios (HR) by BMI class were estimated using Cox proportional hazard models. Results: 21,075 adult recipients were included in the analysis. Median follow-up was 4 years. Subjects were overweight or obese in 39%. 90-day pancreatic graft loss censored for death increased with recipient BMI (p < 0.001). Obesity was associated with increased early mortality (p = 0.003 in obese class I, p = 0.009 in obese class II) but not in overweight. Increased BMI had an impact particularly in SPK transplantation. From 90 days to 5 years, obesity was associated with graft loss (p = 0.01). Underweight was associated with an important increased of death in long term (p < 0.001). Conclusion: increased BMI was associated with an increased risk of early graft loss and early mortality. Obesity was associated with an increased incidence of post transplant diabetes mellitus. Underweight is associated with an increased risk of death in long term.



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276

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Are the Outcomes of Pancreas Transplant in Small Centers Defensible?

Mark R. Laftavi1, Sunil K. Patel1, Rebecca Russell1, Lin Feng1, Meriem Said1, Mareena Zachariah2, Brian Murray2, Oleh Pankewycz2

1Surgery, State University of New York at Buffalo, Buffalo, NY, United States; 2Medicine, State University of New York at Buffalo, Buffalo, NY, United States.

Pancreas transplant (PTX) offers euglycemic status and may prevent severe complications of DM. Currently, the long-term advantage of PTX is debated, particularly in the recipients (R) of pancreas after kidney (PAK). Furthermore, UNOS requires that transplant centers perform a minimum number of PTXs to remain an active pancreas transplant center. In this study, we report short and long-term outcomes in a small center performing 2-9 PTX/year. Between 9-2001 and 11-2012, 45 PTX (24 SPK & 21 PAK) were performed in our center. Donor and recipient demographics are shown in Table 1&2. Eighty-two percent of donors were outside of our OPO region and organs were procured by their local surgeons.

TABLE 1
TABLE 1
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TABLE 2
TABLE 2
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All patients were induced with rATG (3-5 mg/kg, total) and received tacrolimus, MMF and prednisone maintenance immunosuppression. Patients and graft survival are shown in Figures 1 & 2. Five patients (11%) died (1 due to trauma, 1 brain lymphoma, 1 ruptured aneurysm & 1 unknown etiology). Two patients (1 SPK, 1 PAK), 4% lost their graft due to thrombosis on post op day 3 and 5 in 2002. No graft thrombosis occurred since 2002. Seven patients (15%) required re-operation (4 bleeding, 2 anastomotic leak, 1 small bowel perforation). Two patients (4%) developed PTLD. Five patients (11%) experienced CMV antigenemia that responded well to antiviral therapy. The short and long-term outcomes were similar in the SPK and PAK groups.

We conclude that compared to diabetic patients on dialysis, current SPK and PAK short and long-term outcomes are encouraging even in small PTX centers. Thus, PTX should be offered to all type 1 DM with ESRD or after kidney transplant.



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277

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Comparison of Insulin Resistance Post Transplant among Type 2 Diabetics Receiving SPK Transplant to Type 1 Diabetics Receiving SPK and to Non Diabetics Receiving Kidney alone

Harini Chakkera1, Raymond Heilman1, David Mulligan1, Adyr Moss1, Nitin Katariya1, Hasan Khamash1, Janna Huskey1, Winston Hewitt1, Senaida Behmen1, Kunam Reddy1

1Mayo Clinic in Arizona, Phoenix, AZ, United States.

Background: Simultaneous pancreas and kidney transplantation (SPKTx) is a well accepted therapeutic option for patients with type 1 diabetes mellitus (T1DM) and end-stage renal disease (ESRD). Few centers such as ours offer SPKTx in “select” patients with type 2 Diabetes Mellitus (T2DM) and ESRD. At our center we use the following selection criteria to identify “select” T2DM for SPKTx : Presence of C-peptide, absence of diabetic ketoacidosis, use of oral hypoglycemics during the course of disease, BMI < 30 kg/m2 and use of < 1 unit/kg of insulin/day.

Methods: To assess insulin resistance post-transplant (Tx) in these select T2DM we sought to compare distribution of homeostasis model assessment of insulin resistance (HOMA-IR) at 1, 4 and 12 months post Tx comparing T2DM receiving SPKTx to T1DM receiving SPKTx, and to nondiabetics receiving kidney Tx alone. We also report patient survival, pancreas and kidney allograft function, follow up spanned between 1-5 years post transplant.



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* Mean ± standard deviation, §P < 0.05

* Mean standard deviation

Patient mortality: 1 patient from group 1(cardiac arrest), 2 patients in group 2 (cancer and cardiac arrest) and 1patient in group 3 (sepsis).

Kidney graft loss: 1 patient in group 2 returned to dialysis, groups 1 and 3 had no loss in functioning allograft

Pancreas graft survival: 100% in groups 1 and 2 - non required insulin. No patients in group 3 developed NODAT

Conclusion: Our pilot demonstrates that select patients with T2DM receiving SPKTx demonstrate comparable measures of insulin resistance in comparison to the T1DM receiving SPKTx and nonDM receiving kidney transplant alone in the short-term post transplant. Longer follow-up on a larger cohort is needed to determine consistent trends long-term

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280

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Late Pancreas Retransplantation

Jonathan A. Fridell1, Richard S. Mangus1, John A Powelson1, Michelle L Goble1, Muhammad A. Mujtaba2, Tim E. Taber2

1Surgery, Indiana University School of Medicine, Indianapolis, IN, United States; 2Medicine, Indiana University School of Medicine, Indianapolis, IN, United States.

Late pancreas retransplantation (excluding immediate retransplantation for graft thrombosis) is a technically treacherous operation with the added challenges of adhesions from the prior transplant and difficulties identifying usable recipient vessels. The goal of this study was to review our single center experience with late pancreas retransplantation.

Methods: Charts for all pancreas transplant recipients between Jan 2003 and April 2013 were reviewed. All transplants were performed through midline laparotomy with systemic venous and enteric exocrine drainage. Immunosuppression consisted of rabbit antithymocyte globulin induction, steroid withdrawal and tacrolimus and sirolimus (+/- mycophenolate) maintenance. All patients recieved prophylaxis for CMV and PJP. Charts were reviewed for demographics, graft and patient survival, length of stay (LOS), readmissions and technical complications.

Results: Out of 473 pancreas transplants, excluding twelve immediate retransplants, there were 20 late pancreas retransplants (30%SPK, 55%PAK and 15% panc alone) compared to 441 first transplants (55% SPK, 19% PAK and 26% Panc alone). There were no significant differences in donor age, race, gender, BMI or cause of brain death or recipient age, gender, race or BMI. Median ischemia times were similar at 9 hours for late retransplantation and 8 hours for first transplants. There was no significant difference in graft or patient survival (see figures). The mean and median length of stay were 22 and 9 days respectively (range 5-175 days) and 11 recipients required readmission within the first 3 months post-transplant. Five patients were reexplored in the early postoperative period: one for an enteric leak at the site of allograft pancreatectomy for the original graft, one for abscess and hemorrhage after placement of a percutaneous gastrostomy tube and three negative laparotomies for hyperglycemia.

Conclusion: Pancreas retransplantation is technically challenging but can be safely performed with graft and recipient survival comparable to primary transplants.



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281

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No Difference in Early Graft and Patient Survival for Pancreas Transplant Recipients with a History of Smoking

Jonathan Fridell1, Richard Mangus1, Michelle Goble1, John Powelson1

1Surgery, Indiana University School of Medicine, Indianapolis, IN, United States.

Introduction: Smoking is associated with post-transplant complications such as cardiopulmonary events, vascular complications, and de novo cancer, as well as graft and patient survival. This study is a retrospective chart review of a large number of pancreas transplant recipients to determine the impact of tobacco use on long-term outcomes including 10-year graft and patient survival.

Methods: The records of 473 consecutive pancreas transplant recipient with a median follow up of 59 months (from 2003 to 2013) were reviewed. Tobacco use was extracted from the medical history. Patients were categorized as current, previous, or never smokers at listing for pancreas transplant, and total pack-year exposure was calculated. Kaplan-Meier methodology was utilized to evaluate long-term survival.

Results: Smoking history included: never smoker 325 (69%), previous smoker 98 (21%), and current smoker at transplant 50 (11%). For smokers, pack-years included 1-19 (16%), 20-29 (8%), and >= 30 years (8%). There were no differences among the groups for graft loss at 7- or 90-days (0.96 and 0.76), or 1-year patient and graft survival (p = 0.98 and 0.75). Kaplan-Meier patient and graft survival failed to demonstrate a survival difference based upon smoking status (log rank p = 0.59 and 0.39). Long-term survival for the group with > 30 pack-years of smoking approached statistically worse survival beginning around 5 years post-transplant (Figure; log rank p = 0.07).

Conclusions: These results suggest that appropriately chosen patients with a smoking history can successfully undergo pancreas transplantation with a reasonable expectation for long-term survival. The group with the greatest smoking history does begin to experience worse long-term survival after 5 years.


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282

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Management of BK Viremia and Nephropathy in Pancreas After Kidney Transplantation and Pancreas Transplantation Alone

Avinash Agarwal1, Bartholemew J Kane1, Kenneth L Brayman1

1Department of Surgery, University of Virginia, Charlottesville, VA, United States.

Objective: Pancreas after kidney transplantation represents a unique setting of sequential intense immunosuppression which could pose significant risk for infectious complications. It could be challenging to balance reduced immunosuppression for BK reactivation and sufficient for the pancreas allograft. There is limited understanding of BK viremia (BKV) and BK nephropathy (BKN) in pancreas after kidney transplantation (PAK) or pancreas transplantation alone (PTA).

Methods: Between January 2005 and February 2013, thirty one patients underwent PAK and ten underwent PTA. The pancreata underwent systemic venous drainage and duodenal drainage was determined by intra-operative surgeon’s preference. All patients received Thymoglobulin induction (6 mg/kg) and standard triple immunosuppression (prednisone, mycophenolate mofetil, and tacrolimus). Prior to 2011, plasma BK virus PCR was performed for clinical indications; subsequently, a prospectively monitoring protocol was implemented. BK nephropathy was defined as BK plasma viral load > 4 log copies/mL and a > 20% rise in serum creatinine. Pancreatic graft loss was defined as vascular compromise or loss of detectable c-peptide.

Results: 31 patients underwent PAK transplantation (20 bladder drainage; 11 enteric drainage) and 10 patients had PTA (8 bladder drainage; 2 enteric drainage). At a median follow up of 48 months, one and three year actuarial patient survival for PAK was 94% (etiologies: cardiac arrest, intracerebral hemorrhage) and 91%. Patient survival for PTA was 100%. One and three year actuarial PAK graft survival was 90% (2 deaths, 1 vascular thromboses) and 86% (one chronic rejection). One and three year graft survival for PAT is 80% and 63% (2 vascular thromboses and one chronic rejection). Among 31 PAK patients, six patients (19%) developed BKV within 191 ± 130 days post PAK while three (9%) progressed to biopsy proven BKN. None of the PTA demonstrated BK infection. BK viremia led to reduction in tacrolimus and mycophenolate mofetil. All three BK nephritic patients were treated cidofovir and IVIG therapy. All BKV patients and two BKN experienced clinical improvement. Two BKN patients (6%) progressed to dialysis dependence. Age, gender, kidney donor type was not associated with BK infection. All cases of BK viremia occurred in those with duodenocystostomy (p = 0.06). Pancreas graft rejection was not observed with the immunosuppression modifications. Prospective monitoring has allowed for earlier detection of viral reactivation (241 ± 124 days vs. with monitoring: 90 ± 80 days).

Conclusions: This study highlights the significant consequence of BK nephropathy in PAK transplantation. The data would also suggest there is interplay between a bladder drainage and BK reactivation. Potentially, earlier screening and more refined therapy would preserve renal and pancreas allograft survival.

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283

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Pancreas Transplantation Offers Definite Survival Benefit when Compared with Live Donor Kidney Transplantation in Diabetic Patients

Zia Moinuddin1, Judith Worthington1, Hussein Khambalia1, Bence Forgacs1, Titus Augustine1, Afshin Tavakoli1, David van Dellen1

1Transplant Surgery, Manchester Royal Infirmary, Manchester, United Kingdom.

Introduction: Pancreas transplantation for patients with end-stage renal failure (ESRF) due to type 1 diabetes mellitus improves quality of life and stabilises diabetic complications. Survival is also improved compared to equivalent control patients on the waiting list. Clinician reticence has persisted due to perceptions that these benefits are secondary to the kidney transplant (especially with live donation) rather than the potential benefits of diabetic cure from the pancreas allograft. However, cardiac risk burden remains significant in the patients who have kidney transplants alone. We therefore aimed to establish mortality risk across these disparate groups.

Methods: Retrospective analysis was performed of patients with end-stage renal failure (ESRF) due to type 1 diabetes mellitus undergoing pancreas and/or kidney transplantation in a single centre over 12 years (2001-12) (Simultaneous pancreas kidney (SPK)= 203, pancreas after kidney (PAK)= 36, kidney transplant alone (KTA)= 66 [live donor = 22, deceased donor = 44]). These groups were compared to each other and to a control group accepted contemporaneously onto the waiting list. The primary endpoint was patient mortality. Confounding factors (cardiac, demographic and waiting time) were also analysed.

Results: Mortality on the waiting list was 30.6% (56/183) compared with 7.3%(15/203) post-SPK (p < 0.0001, Fisher’s exact test), 2.7%(1/36) post-PAK (p < 0.0001, Fisher’s exact test) and 7.5%(5/66) post-KTA (p < 0.0001, Fisher’s exact test). There was no significant difference in mortality despite increased morbidity with more major surgery associated with the SPK. However, in the KTA group, the mortality post- deceased donor transplantation was 2.3%(1/44) compared with 18.18%(4/22) post-live donor transplantation (p = 0.039, Fisher’s exact test). Pancreas allograft survival was 83.7%(170/233) & 80.3%(163/203) in the SPK group compared with 69%(25/36) & 47.22%(17/36) in the PAK group at one and five years respectively (p = 0.05; p < 0.0001, Fisher’s exact test). There was no difference in kidney survival between the SPK and KTA groups. Confounding factors other than duration of diabetes were equivalent.

Conclusion: Patients with complicated type1 diabetes mellitus are at significant risk of cardiac mortality. Waiting list death accounts for the attrition burden of a caseload with significant co-morbidities. Transplantation offers significant survival benefit, despite surgical and immunosuppressive risks. SPK transplantation offers equivalent mortality risk to KTA and better pancreatic graft survival than PAK transplantation for patients with ESRF due to type 1 diabetes mellitus. Benefits from uraemic cure in KTA does not offset mortality risk possibly due to progression of cardiac disease from ongoing diabetes. Simultaneous pancreas and kidney transplantation therefore provides optimal treatment for this group of patients.

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284

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Recipient BMI Does not Negatively Affect Pancreas Graft Survival

Max Marquez1, Markus Selzner1, Fateh Bazerbachi1, Ian D. McGilvray1, Andrea Norgate1, Jeffrey Schiff1, Markus Boehnert1, John Seal1, Mark S. Cattral1

1Surgery, Toronto General Hospital, UHN, Toronto, ON, Canada.

Obesity has been associated with higher morbidity post pancreas transplantation. The impact of body mass index (BMI) on graft and patient survival is controversial.

Aim: To determine the impact of recipient BMI on outcome of pancreas transplantation.

Methods: We performed a retrospective review of 358 pancreas transplants (262 kidney-pancreas and 96 pancreas after kidney) performed at a single institution from 1998 to 2013. Clinical data was analyzed using Chi-square, Student’s t test or Mann-Whitney U test. Graft and patient survival was calculated using Log-Rank.

Results: A BMI cut-off value of 30 was used to create two groups of patients: 82.6% (285) had a BMI ≤ 30 and 17.4% (60) had a BMI ≥ 30; 13 cases had missing BMI information and were excluded from analysis. Both groups had similar pre-transplant characteristics with respect to male sex (62% vs. 70% p = 0.3), donor age (28.2 ± 10.3 vs. 25.7 ± 10.0 yrs p = 0.22), recipient age (43 ± 8 vs. 43 ± 7 yrs p = 0.78), CMV mismatch (31% vs. 30% p = 0.81), pancreas cold ischemic time (CIT) (9.9 ± 3 vs. 9.7 ± 2 h p = 0.54) and kidney CIT (8.7 vs. 8.1 h p = 0.55). Total length of hospital stay (12.7 ± 9.6 vs. 10.7 ± 6 days p = 0.13) and rejection rate for any organ (33% vs. 29% p = 0.7) were similar between groups. There was no difference in the rate of rejections, re-laparotomy, or infections (p = NS). Pancreas graft losses due to thrombosis, pancreatitits and leaks were lower in the BMI ≥ 30 group (10% vs. 2% p = 0.01) while death with a functioning graft was more frequent (3% vs. 12% p = 0.01). Deaths due to cardiovascular events were more frequent in the BMI ≥ 30 group (2% vs. 8% p = 0.01). Graft function as measured by serum creatinine and c-peptide levels were similar (p = NS). HbA1C was lower in the BMI ≥ 30 at 1 and 3 years (0.56 vs. 0.51, 0.56 vs. 0.52 respectively p = 0.01); and comparable beyond 5 years (0.58 vs. 0.56 p = 0.45). Kidney graft survival was similar at 1, 3, and 5 years (p = NS). Pancreas graft survival at 1, 3 and 5 year was comparable between BMI ≤ 30 and BMI ≥ 30 groups at (88% vs. 95%, 83 vs. 93% and 78 vs. 87% respectively p = 0.26). 5 year patient survival was comparable at 96% vs. 93%. (p = 0.09).

Conclusion: Pancreas transplantation in recipients with a BMI ≥ 30 offers comparable short-term survival and similar rate of postoperative complications. BMI may affect long term patient survival due to increased risk of cardiovascular events.

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285

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Portal Drainage - Why are We not Using It?

Angelika Gruessner1, Rainer Gruessner1

1University of Arizona, Tucson, AZ, United States.

Enteric drainage of a pancreas graft is now the standard technical method in all 3 major transplant categories all over the world. While the vascular drainage into the portal vein is the preferred technique in many countries outside the US, it did not find the acceptance in the US because of early technical problems; a decade ago, it was associated with an increased rate of pancreas graft thrombosis. In the USA in 2012, this technique was used in around 10% of primary enteric drained solitary transplants and in 24% of SPK transplants.

A comparison of systemic versus portal drainage in 3,555 US primary enteric drained transplants performed between 2007 and 2012 was done. Only transplants with anti-T-cell induction and a maintenance protocol of Tacrolimus and MMF were included. Of those transplants 2,852 were SPK (80%), 429 PAK (12%), and 274 PTA (8%). 28% of SPK, 12% of PAK and 9% of PTA were portal drained.

Patient survival was in all 3 categories and for both vascular managements techniques excellent and no differences could be detected (p > 0.50). Overall, 3-year pancreas graft function resulted for SPK in 83% for systemic and 80% for portal drainage (p = 0.23), in PAK for 72% versus 84% (p = 0.21), and in PTA in 72% versus 62% (p = 0.05). So only in PTA was the difference significant. No difference in technical failures between the 2 techniques could be found anymore in all 3 transplant categories (p > 0.3). The rate of immunologic graft loss was significantly higher in portal drained SPK with 8.1% versus 3.8% at 3 years (p = 0.0009). The same could be seen in PTA with 22% in portal drained versus 17% in systemic drained transplants (p = 0.4). In contrast, in PAK was the rejection rate at 3 years 8.9% for portal versus 14.7% for systemic drained transplants (p = 0.54).

In a multivariate model adjusted for recipient and donor factors, most differences between the 2 techniques vanished when the impact of the transplant center was included. While the relative risk in SPK and PTA for portal drained pancreas transplants was still slightly increased, it was decreased for PAK without reaching statistical significance.

The outcomes of the more physiological portal drainage in the US improved and show excellent results especially in centers with more experience in this technique.

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286

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Simultaneous Pancreas and Kidney Transplantation leads to Early Nerve Regeneration, as Evaluated by Corneal Confocal Microscopy

Hussein Khambalia1, Mitra Tavakoli2, Zia Moinuddin1, David van Dellen1, Rayaz Malik2, Titus Augustine1

1Department of Transplant Surgery, Manchester Royal Infirmary, Manchester, United Kingdom; 2Division of Cardiovascular Medicine, University of Manchester and Wellcome Trust Clinical Research Facility, Manchester, United Kingdom.

Introduction: Simultaneous pancreas-kidney transplantation (SPKT) cures Type 1 diabetes and improves long-term cardiovascular outcomes, retinopathy and nephropathy, but with minimal impact on neuropathy. We have evaluated the utility of the novel non-invasive ophthalmic technique of corneal confocal microscopy (CCM) for the assessment of an improvement in neuropathy in diabetic patients following SPKT.

Methods: A prospective controlled study undertook detailed assessment of neuropathy using CCM and FDA approved measures of neuropathy in Type 1 diabetic patients at baseline and 12 months after SPKT.

Results: 15 Type 1 diabetic patients (age: 47.0 ± 3.0 yrs., duration of diabetes: 27.0 ± 3.5 yrs.) and 15 control subjects were recruited. HbA1c (7.4 ± 0.8%) improved into the normal range (5.9 ± 0.4) after SPKT. The neuropathy symptom profile (P = 0.005), McGill Pain index (P = 0.01) and neuropathy disability score (P = 0.003) were significantly increased at baseline and did not improve after SPK. Vibration perception threshold (P = 0.01), cold sensation threshold (P = 0.004) and warm sensation threshold (P = 0.005) were significantly increased at baseline and showed no improvement at 12 months (P = 0.6, P = 0.5, P = 0.4). Heart rate variability was significantly reduced at baseline (P = 0.01) and did not change at 12 months (P = 0.8). Peroneal nerve conduction velocity and amplitude (P = 0.0001) were significantly reduced at baseline and did not change at 12 months (P = 0.3, P = 0.2). Intraepidermal nerve fibre density (IENFD) from skin biopsies was significantly reduced (P < 0.0001) in diabetic patients and did not change at 12 months (P = 0.9). Corneal nerve fibre density, branch density and nerve fibre length were significantly reduced in diabetic patients compared to control subjects at baseline (P < 0.0001) but improved significantly (P = 0.02, P = 0.008, P = 0.03) at 12 months.

Conclusions: SPKT normalises glycaemia, and CCM has been shown to be the most sensitive measure to detect a significant improvement in nerve morphology within 12 months of SPKT. Long term longitudinal studies are required to confirm whether this early morphologic improvement is followed by improvement in the other FDA accepted measures of neuropathy.

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287

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Short Cold Ischaemic Times Improve Cytokine, Diabetic and Clinical Markers in Pancreas Transplantation

Hussein Khambalia1, Zia Moinuddin1, David van Dellen1, Mahesh Nirmalan2, Yvonne Alexander3, Titus Augustine1

1Department of Transplant Surgery, Manchester Royal Infirmary, Manchester, United Kingdom; 2Department of Anaesthesia and Critical Care, Manchester Royal Infirmary, Manchester, United Kingdom; 3Cardiovascular Research Unit, University of Manchester, Manchester, United Kingdom.

Introduction: Prolonged cold ischaemic time (CIT) correlates to poor immediate outcomes following pancreas transplantation, possibly due to increased effects of ischaemia-reperfusion injury, resulting in an increased peri-operative inflammatory response and higher morbidity. The biochemical nature and resultant effects of this response on pancreas function are not established.

Methods: A prospective cohort study was performed investigating the relationship of CIT on pro- and anti- inflammatory cytokines in the peri-operative period and subsequent outcomes following pancreas transplantation. Inflammatory markers (Interleukin (IL) -6, IL-10) and endocrine pancreatic markers (Insulin and C-peptide) were measured (pre-operatively, at reperfusion, and 30 minutes, 6, 12, 24, 48 and 72 hours post-perfusion). Repeated measures ANOVA was used to compare biomarker levels at these time points. Clinical Outcomes (length of critical care stay and morbidity) were correlated (Chi-squared analysis).

Results: 18 consecutive simultaneous pancreas and kidney transplant recipients (9, CIT less than 14 hours and 9, CIT greater than 14 hours; p < 0.001 (t-test)); matched for donor and recipient demographics, body mass index, length of procedure and immunological risk were included.

Patients in the shorter CIT group had higher levels of IL-6 up to 48 hours post-surgery (significant at 12 hours (p = 0.007)) and higher levels of IL-10 up to 72 hours post-surgery (significant at 12, 24, 48 and 72 hours (p = 0.023, 0.023, 0.020 and 0.048 respectively)). Patients in the shorter CIT group demonstrated increased C-peptide levels, from pancreas perfusion until 72 hours post-surgery (p = 0.023) and higher levels of insulin from 12 to 72 hours post-surgery (significant at 48 and 72 hours (p = 0.004 and p = 0.003 respectively)).

Patients in the shorter CIT group had lower risk of developing post-operative respiratory complications and were less likely to require further surgery (0 vs. 5 (p = 0.029) and 2 vs. 7 (p = 0.022) (Short CITvs. Long CIT respectively)). These patients also had shorter surrogate markers of recovery including time to mobilisation from bed and to tolerating per-oral diet (3.11 days vs. 6 days (p = 0.008) and 5.89 days vs.12.44 days (p = 0.036) (Short CIT vs. Long CIT respectively). There were no statistically significant differences in length of critical care stays.

Conclusions: Shorter CIT appears to offer protective benefits to the pancreas allograft due to reduced ischaemia-reperfusion injury, reflected in increased IL-6 and consequently IL-10, which has potent anti-inflammatory effects. This correlates with subsequent graft function and an improved post-operative morbidity profile. These findings appear to highlight delayed graft function in longer CIT pancreas allografts, an under recognised phenomenon in pancreas transplantation. This could ultimately translate to reduced long-term graft function.

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288

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Long-Standing Functional Outcomes After Whole Organ Pancreas Transplantation Show Considerable Heterogeneity in Glucose Tolerance and Differ to Conventionally Accepted Normal Measures

Shruti Mittal1,2,3, Rachel Franklin5, Rachel Craven-Todd5, Edward Sharples1,4, Fredrik Karpe1,4, Peter Friend1,2, Stephen Gough1,4,5

1Oxford Transplant Centre, Oxford Radcliffe NHS Trust, Oxford, United Kingdom; 2Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom; 3Biomedical Research Centre, NIHR, Oxford, United Kingdom; 4Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom; 5Clinical Research Unit, Oxford Centre for Diabetes, Endocrinology and Diabetes, University of Oxford, Oxford, United Kingdom.

Aim: Despite good early function, graft attrition rates after pancreas transplantation remain high. Patients with graft dysfunction usually present requiring a return to insulin treatment. We aimed to determine glucose tolerance after pancreas transplantation to identify graft dysfunction prior to failure.

Method: Frequently-sampled oral glucose tolerance tests (FSOGTTs) were performed in 91 systemically-drained pancreas transplant recipients with longstanding pancreas graft function and insulin-independence. Profiles were compared to 16 non-diabetic non-transplant controls, and correlated to clinical data.

Results: A high proportion of insulin-independent pancreas transplant recipients displayed impaired (23/91, 25.3%) or diabetic (6/91, 6.6%) glucose tolerance. Glucose and insulin profiles in the pancreas transplant group differed from those in the control group with higher systemic insulin (mean area under the curve 8464 v 3914 mU/l, p = 0001) and delayed insulin secretion (peak by 30 min 10% v 44%, p = 0.05). Within the pancreas transplant group considerable heterogeneity in glucose and insulin profiles was found. Recipients with abnormal glucose tolerance had higher iAUC glucose compared to those with normal glucose tolerance (414 mmol/l v 221 mmol/l, p = 0.0001) but there was no significant difference in iAUC insulin (6169 mU/l v 6128 mU/l, p = ns). Pancreas transplant recipients with abnormal glucose tolerance showed delayed insulin peaks compared to those with normal glucose tolerance. Additionally, a finding of abnormal glucose tolerance was independent of time post-transplant, change in BMI since transplantation and donor/recipient factors.

Conclusion: Metabolic measures including glucose tolerance are frequently outside the conventionally accepted normal range in pancreas transplant patients. Glucose intolerance emerging after pancreas transplantation appears unrelated to graft duration and recognised risk factors for pancreas graft failure. Whilst abnormal glucose tolerance may represent a marker of future graft failure, longitudinal studies are required to fully understand the significance of these findings.

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289

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Clinical Predictors of Early Pancreas Graft Loss After Whole Organ Transplantation Differ to Established Risk Factors for Long-Term Outcome

Shruti Mittal1,2,3, Lisa Mumford4, Fangjann Lee1, Dave Collett4, Sanjay Sinha1, Anil Vaidya1, Edward Sharples1, Rutger Ploeg1,2, Peter Friend1,2

1Oxford Transplant Centre, Oxford Radcliffe NHS Trust, Oxford, United Kingdom; 2Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom; 3Biomedical Research Centre, NIHR, Oxford, United Kingdom; 4NHS Blood and Transplant, Bristol, United Kingdom.

Aim: Many publications have described risk factors associated with long-term pancreas graft survival. However, it is early graft failure that is associated with both the highest rates of graft loss and more importantly the greatest morbidity for patients, often associated with life-threatening complications. Predictors of 90-day graft loss may be different to those for longer-term outcomes and identification of donor pancreases at high risk of early graft failure would lead to more efficient organ utilisation.

Method: Data were obtained from the UK Transplant Registry on 1265 deceased donor, whole pancreas transplant recipients between 1st April 2004 and 1st July 2011. The dataset was randomly divided in modelling and validation datasets. The modelling dataset was used to investigate donor factors potentially influencing 90-day graft survival using Cox regression, adjusting for significant recipient and transplant factors. A risk index was derived and validated.

Results: Significant recipient variables predictive of 90-d graft loss included recipient BMI (p = 0.038), PAK transplant (p = 0.049) and transplant centre (p = 0.023). Other recipient characteristics including calculated Reaction Frequency and degree of mismatch were not significant. In a multivariate model, significant donor factors predicting poor pancreas graft outcome were donor type (DCD HR 2.395, p = 0.024), donor BMI (HR 1.049, p = 0.117) and donor ALT (> 50 mmol/l HR 2.506, p = 0.003). Donor age, cold ischaemia time and other donor factors (donor gender, ethnicity, BMI, biochemistry, serology, past medical history) were not significant predictors of 90-day graft outcome.

Conclusion: A model including donor type, donor BMI and donor ALT identifies donor pancreases with high risk of 90-d graft failure, and most likely relates to the effects of pancreas parenchymal fat, ischaemia-reperfusion injury and graft pancreatitis. Consideration of these factors at the time of organ offers will be of greater utility to both patient and physician than consideration of factors determining longer-term outcome.

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290

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Early Bacterial Infections in Simultaneous Kidney and Pancreas Transplantation: Impact of Preservation Fluid Contamination

Déborah Chaintreuil1, Thomas Benet2, Marie-Elisabeth Reverdy3, Maria Brunet1, Fanny Buron1, Cécile Chauvet1, Sameh Daoud1, Olivier Thaunat1, Lionel Badet4, Philippe Vanhems2, Emmanuel Morelon1

1Transplantation, Néphrologie et Immunologie clinique, Hospices Civils de Lyon, Lyon, France; 2Hygiène et Epidémiologie Médicale, Hospices Civils de Lyon, Lyon, France; 3Bactériologie, Hospices Civils de Lyon, Lyon, France; 4Urologie et Chirurgie de la Transplantation, Hospices Civils de Lyon, Lyon, France.

Contamination of preservation fluid (PF) has been associated with donor-transmitted infection in renal transplantation. Despite the infectious morbidity in simultaneous kidney-pancreas transplantation (SPKT), the role of PF contamination has never been reported. The aim of the study was to analyse the impact of PF contamination in bacterial infections of SPKT recipients.

We retrospectively analysed 75 SPKT performed in our centre, whose PF were systematically analysed. Our analysis focused on the first bacterial infectious episode in the three months post transplantation. A multivariate cox survival model was used to determine the impact of contaminated PF on infection risk.

A total of 30/75 (40%) patients presented at least one infection. Three month infection incidence was 6.6 per 1000 patient-day. Infection sites were mostly intra-abdominal and urinary (30% and 26.7%, respectively). The bacteria that caused the infection were mostly of digestive origin (44.4%). PF cultures were positive in 47 (62.7%) patients. In multivariate analysis, only pancreatic fistula were significantly associated with early bacterial infection (HR = 3.95, IC 95% [1.66-9.04], p = 0.002). No association was found between positive PF and early bacterial infection (HR = 0.99 IC 95% [0.47-2.08], p = 0.97).

In SPKT, positive PF did not have an impact on early bacterial infections; the main risk factor was post transplant pancreatic fistula.

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295

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Islet Preparation Purity is Overestimated and Less Pure Fractions have Lower Post-Culture Viability Prior to Clinical Allo-Transplantation

Jennifer P Kitzmann1,2,3, Kathryn R Mueller1,2,3, Efstathios Avgoustiniatos2,3, Angelika C Gruessner1, Appakalai Balamurugan2,3, Melena D Bellin2,3, Bernhard J Hering2,3, Klearchos Papas1,2,3

1Surgery, University of Arizona, Tucson, AZ, United States; 2Surgery, University of Minnesota, Minneapolis, MN, United States; 3Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN, United States.

Replacement of β-cells via isolated islet allo-transplantation (ITx) is an emerging therapy for type 1 diabetics with hypoglycemia unawareness. The current standard protocol calls for a 36-72 hour culture period prior to ITx. We examined 13 clinical islet preparations with at least two purity fractions to determine the effect of culture on viability. After islet isolation and purification, pure islet fractions (≥ 70% purity by dithizone stain) were placed at 37°C with 5% CO2 for 12-24 hours and subsequently moved to 22°C while less pure (30-55%) fractions were cultured at 22°C for the entire duration. Culture density was targeted at a range of 100-200 islet equivalents (IE)/cm2 adjusted for purity (i.e. 50-100 IE/cm2 of 50% pure) in a T-175 flask. Islets were assessed for purity (dithizone staining), quantity (pellet volume and DNA), and viability [oxygen consumption rate normalized to DNA content (OCR/DNA) and membrane integrity]. Results indicated that purity was typically grossly overestimated, especially in the less pure fractions. This was evidenced by the significantly larger pellet sizes [2ml of settled tissue volume is typically expected for 1 million IE, therefore a max pellet volume of 4ml would be expected for 1 million IE at 50% purity [1]; Figure 1a,b] and tissue amount as quantitated by a dsDNA assay when available (n = 9; 1IE = 10.4ngDNA, Figure 1c,d). Less pure fractions showed significantly lower OCR/DNA and membrane integrity compared to pure (Figure 1e,f). The difference in viability between the two purity fractions may be caused by a variety of reasons, including hypoxia, nutrient deficiency, toxic metabolite accumulation, and/or proteolytic enzymes released by acinar tissue impurities that are not neutralized by human serum albumin. In conclusion, current clinical islet culture protocols should be examined further, especially for less pure fractions, to ensure the maintenance of viability prior to transplant. Even though relatively small, the difference in viability is important as the amount of total and thus dead tissue introduced in recipients may be dramatically increased, especially with less pure preparations.


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Schulze Diabetes Institute islet isolation team

Reference:

[1] Colton CK, Papas KK, Pisania A et al: Characterization of islet preparations. In: Halberstadt C, Emerich DF, eds. Cell Transplantation from Laboratory to Clinic. New York: Elsevier, 2007, 35-132.

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296

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Automated Digital Image Analysis of Islet Cell Mass During Human Islet Isolation

Valery Gmyr1, Caroline Bonner1, Bruno Lukowiak1, Nathalie Dellaleau1, Valerie Pawlowski1, Rimed Ezzouaoui1, Sandrine Belaich1, Isanga Aluka1, Ericka Moerman1, Julien Thevenet1, Francois Pattou1, Julie Kerr-Conte1

1INSERM UMR 859 Biotherapies for diabetes, Lille University Hospital, Lille, France.

Background: Reliable assessment of islet viability, mass and purity must be met prior to transplanting anislet preparation into patients with type 1 diabetes. The standard method for quantifyinghuman islet preparations is by direct microscopic analysis of dithizone-stained islet samples, but this technique may be susceptible to inter / intra observer variability, which may inducefalse positive / negative islet counts. Here we describe a simple, reliable, automated digitalimage analysis (ADIA) technique, for accurately quantifying islets into total islet number, islet equivalent number (IEQ), and islet purity before islet transplantation.

Methods: Islets were isolated and purified from n = 42 human pancreata according to the automatedmethod of Ricordi et al. For each preparation, three islet samples were stained with dithizone,and expressed as IEQ number. Islets were analyzed manually by microscopy, or automaticallyquantified using Nikon’s inverted Eclipse Ti microscope, with built in NIS-ElementsAdvanced Research (AR) software.

Results: The AIDA method significantly enhanced the number of islet preparations eligible forengraftment compared to the standard manual method (P < 0.001). Comparisons of individualmethods showed good correlations between mean values of IEQ number (r2 ≤ 0.91), and totalislet number (r2 ≤ 0.88), and thus, increased to (r2 ≤ 0.93) when islet surface area was estimatedcomparatively with IEQ number. The ADIA method showed very high intra-observerreproducibility compared to the standard manual method (P < 0.001). However, islet purity wasroutinely estimated as significantly higher with the manual method vs. the ADIA method(p < 0.001). The ADIA method also detected small islets between 10-50 μm in size.

Conclusion: Automated digital image analysis utilizing the Nikon Instruments (Nikon) software is anunbiased, simple, and reliable teaching tool to comprehensively assess the individual size of each islet cell preparation prior to transplantation. Implementation of this technology toimprove engraftment may help to advance the therapeutic efficacy and accessibility of islettransplantation across centers.

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297

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Cryobanking of Pancreas as a Source of Viable Islets by Non-Enzymatic Cryo-Isolation: Preliminary Studies using Juvenile Porcine Pancreas Stored at -135°C for > 6months

Michael Taylor1,2, Simona Baicu1, Zhen Chen1, Elizabeth Greene1

1Cell and Tissue Systems, N. Charleston, SC, United States; 2Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, United States.

Objective: Cryo-isolation of therapeutic cells such as pancreatic islets has been proposed as a novel new alternative to conventional enzymatic digestion of the pancreas [1,2]. Apart from avoiding the expense, inconsistencies and toxicity associated with the collagenase-digestion process, cryo-isolation embodies inherent advantages including the convenience of biobanking for storage and shipping. This study investigates the feasibility of obtaining viable islets from porcine pancreas after storage at -135°C for > 6 months using the cryo-isolation technique.

Methods: Pancreases were procured from juvenile pigs using approved procedures and processed using the Cryo-isolation protocol involving in situ cryopreservation of islets and concurrent freeze-destruction of acinar tissue. The entire pancreas was frozen solid and stored at -135°C. After storage for 1 day to 8 months, pancreases were further processed by mechanical pulverization into small fragments and then thawed, filtered and washed with RPMI 1640 culture medium to remove the cryoprotectant. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets and with Syto13/PI for fluorescence viability testing and glucose-stimulated insulin secretion (GSIS) assessment.

Results: The cryo-isolate from pancreata stored for > 6 months contained pancreatic fragments comprising discrete dithizone-positive islets embedded in a shell of dead amorphous material. The cryo-isolate product was similar to that obtained from frozen pancreases stored for 1 day in that there was an abundance of largely intact and viable (> 90%) islets.{figure1} Moreover, the cryo-isolated islets were typically larger (range 50-500μm) than their counterparts isolated from juvenile pigs using conventional collagenase digestion techniques and demonstrated GSIS indices (> 3), which was not significantly different from controls (fresh islets from collagenase digestion).

Conclusion: An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a new and novel method that avoids the problems associated with conventional collagenase digestion methods and permits long-term biobanking of pancreases as a ready source of islets.

Funded in part by a grant from NIH/NIDDK # 1R43DK096773.

Reference:

[1] Taylor MJ, Baicu, S:Cryo-isolation: A novel method for Enzyme-Free Isolation of Pancreatic Islets Involving In Situ Cryopreservation of islets and Selective Destruction of Acinar Tissue. Transpl Proc 2011, 43:3181-3183

[2] Taylor MJ, Baicu, S: Non-enzymatic cryogenic isolation of therapeutic cells: Novel approach for enzyme-free isolation of pancreatic islets using In Situ cryopreservation of islets and concurrent selective freeze destruction of acinar tissue. Cell Transpl. (In Press).


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298

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Near Infra-Red Fluorescence Imaging to Assess Pancreatic Intra-Arterial and Intra-Ductal Perfusion Efficiency Prior to Islet Cell Isolation

Stuart Williams1, Jeremy Touroo1, Robert Reed1, David Kennedy2, Paul Todd2, Michael Hughes1

1Cardiovascular Innovation Institute, Louisville, KY, United States; 2IKOTEC LLC, New Albany, IN, United States.

The efficient isolation of islets from the pancreas is influenced by the ability to optimally deliver enzymes to the extracellular matrix protein targets. The efficient and selective delivery of magnetic particles to the islet-specific microcirculation as part of magnetic separation techniques is, in a similar manner, dependent on optimal deliver through the arterial vasculature. We have utilized a near-infra-red fluorescence imaging technique with the fluorescent dye indocyanine green (ICG) to evaluate the efficiency of the cannulation of pancreatic intra-lobular ducts and pancreatic arterial blood vessels. This imaging modality is non-destructive and can be performed under sterile conditions during the processing of human pancreata as part of the islet isolation procedure. Studies were performed using donated human cadaveric pancreata within 24 hours of procurement. To assess duct cannulation efficiency intralobular ducts were identified, cannulated with an 18 g catheter and a solution of indocyanine green infused while the entire pancreas was visualized using a near infra-red camera system. Efficiency of arterial cannulation and islet microvascular perfusion was assessed by cannulating a grossly-identified major arterial vessel, infusion of indocyanine green containing 4.5 micrometer beads and NIR imaging. Real-time observations identified the efficiency of the acinar perfusion with identification of areas of intense fluorescence emission (good perfusion) and areas of limited to no perfusion (background fluorescence/poor perfusion). The arterial infusion of ICG resulted in a different fluorescent emission pattern suggestive of islet localization of the dye (Figure illustrates fluorescence image following ICG infusion){figure1}. Following sectioning of the arterial infused pancreata the magnetic particles were observed to be localized in the islet microcirculation. These observation indicate near-infrared fluorescence imaging can be utilized to assess efficiency of ductal and arterial cannulation to provide optimization of islet release and isolation using magnetic separation techniques.


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299

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Human Islet Viability and Function is Maintained During High Density Shipment on Silicone Rubber Membrane Vessels

Jennifer P Kitzmann1, Andrew R Pepper2, Boris G Lopez2, Rena Pawlick2, Tatsuya Kin2, Doug O’Gorman2, Kathryn R Mueller1, Angelika C Gruessner1, Greg L Szot3, Andrew M Posselt3, Peter G Stock3, John R Wilson4, AM James Shapiro2, Klearchos K Papas1

1Surgery, University of Arizona, Tucson, AZ, United States; 2Clinical Islet Transplant Program, University of Alberta, Edmonton, AB, Canada; 3Diabetes Center, University of California, San Francisco, CA, United States; 4Wilson Wolf Manufacturing Corporation, New Brighton, MN, United States.

The shipment of human islets from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function during transit is critically important. Transport of islets in gas-permeable silicon rubber membrane (SRM) vessels reduces the risk of hypoxia-induced death or dysfunction during high-density shipment. SRM vessels may offer additional advantages; they’re cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment). Human islet equivalents (IE) were isolated from two manufacturing centers and shipped on SRM vessels (10cm2 surface area) to a distant center following at least 2 days of culture (n = 6, purity range of 45-65%). Three conditions were examined: low density (LD; 487 ± 233 IE/cm2, mean ± SD), high density (HD; 6549 ± 3341 IE/cm2), and negative control (NC; 7797 ± 3070 IE in a 1.5mL tube). Statistical analysis was performed using the Friedman test. LD was designed to mimic the standard culture density for human islet preparations (200 IE/cm2). Upon receipt, islets were assessed for viability as measured by oxygen consumption rate normalized to DNA content (OCR/DNA) and quantity by DNA (10.4 ngDNA/IE) and when possible for potency and function with the dynamic glucose-stimulated insulin secretion (GSIS) and immunodeficient B6 rag mouse transplants. Post-shipment OCR/DNA was slightly higher in HD (p = 0.046) versus LD, and substantially reduced by 37 ± 30% in the NC (p = 0.028) condition. {figure1} HD islets exhibited normal GSIS and were able to reverse diabetes in B6 rag mice (2000 IE) post-shipment. Based on the data we conclude that entire islet isolations (up to 450,000 IE at 50% purity) may be shipped using a single SRM vessel (100 cm2) with no negative effect on viability or function. In comparison, it would take roughly 45 T-175 flasks seeded at standard density to accomplish the same result.


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300

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The Dark Duodenum in Pancreas Transplantation

Marcelo Perosa1,2, Huda Noujaim1,2, Luiz Ianhez1,2, Rodrigo Azevedo1,2, Waldere Luconi1,2, Leonardo Mota1,2, Juan Branez1,2, Marcio Paredes1,2, Tercio Genzini1,2

1Hepatology and Transplant, HEPATO, São Paulo, Brazil; 2Organ Transplantation, Bandeirantes Hospital, São Paulo, Brazil.

Duodenal graft complications may occur in some 20% of recipients after pancreas transplants(PT) and is often a result of preservation injury or vascular complications in the duodenal-cephalic area of the graft. After changing surgical technique of PT to portal-duodenal drainage, duodeno-duodenostomy has been our first option of drainage since the appearance of the graft duodenum is favorable after reperfusion. We sought to investigate all cases of non-duodenal drainage due to dark duodenum(DD) appearance after reperfusion and analyze its causes and outcomes. We also compared the results obtained with DD to a normal duodenum(ND) group. From February/2010 to April/2013, 75 PT were performed, being 12 SPK, 51 PAK and 12 PTA. In 11(14.7%) cases a DD occurred after reperfusion and this group was compared to 64 ND. In 4 out of 11 DD patients, there was some vascular issue during donor procurement or bench surgery (hepatomesenteric trunk in 1, replaced right hepatic artery from superior mesenteric artery in 2 and need of gastroduodenal revascularization in 1). The donor mean age and cerebrovascular event as cause of death were similar between the two groups. Total ischemia time was 14.9 hours for DD group and 13.4hs for ND group (p = 0.016). The need of reoperations was significantly higher in the DD group (63.6% vs 6.25%, p = 0.004) and it also showed a trend of higher graft loss (54.5% vs 20.3%), although statistically not significant. The 1-year patient survival was similar between the groups (100% in DD group and 95.3% in ND group).

Conclusions: The occurrence of DD in pancreas transplantation is relatively common and is associated to vascular issues during pancreas procurement or bench surgery and longer ischemia time. These events determine higher rates of surgical complication, reoperations and graft loss, although not compromising patient survival if promptly treated.

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301

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Hypothermal Machine Perfusion of Human Donor Pancreata Prior to Islet Isolation: A Pilot Study.

Marten Engelse1, Marjolein Leemkuil2, Maaike de Nijs1, Jan Ringers3, Dries Braat3, Henry Leuvenink2, Christina Krikke2, Eelco de Koning1,4

1Nephrology, Leiden University Medical Center, Leiden, Netherlands; 2Surgery, University Medical Center Groningen, Groningen, Netherlands; 3Surgery, Leiden University Medical Center, Leiden, Netherlands; 4Diabetes and Islet Neogenesis, Hubrecht Intitute, Utrecht, Netherlands.

Introduction: Because of the shortage of donor pancreata for islet transplantation using pancreas from extended criteria donors (ECD) is an option to increase the donor pool. Of the ECD donors, defined as DBD donors with age > 45 yrs and/or DCD donors, especially the DCD donors are associated with high susceptibility to hypoxic damage. Optimization of organ preservation methods may ameliorate hypoxic damage. The traditional cold storage (CS) method of pancreas preservation lead to hypoxia of the pancreatic tissue. We hypothesize that oxygenated hypothermic machine perfusion (HMP) of the pancreas results in less hypoxia, resulting in a better quality of the isolated pancreatic islets.

Methods: In a pilot study 5 pancreata from DCD donors (age 53 ± 15,8, BMI 24,6 ± 4,2, CIT 8:30 ± 3:30 hrs) were procured and transported in HTK/UW solution. Upon arrival in the islet laboratory the pancreata were split transversally, resulting in a head and tail section. One section was preserved on ice (CS), the other was subjected to 3 hours of additional arterio-venous HMP. After prolonged CS and HMP islets were isolated separately. Pairwise, HMP tissue samples were compared with the CS tissue samples with regard to edema, oxygenation, amylase, lipase, LDH, gene expression and ATP content, islet isolation parameters (tissue retrieval, islet yield and purity) and islet quality parameters (gene expression, islet survival, glucose-induced insulin secretion).

Results: No significant changes were observed in islet yield, survival or function. In the HMP perfusate increasing concentrations of amylase, lipase and LDH were detected, in line with the expected wash-out. Interstitial pO2 increased strongly from non-detectable levels to 80-90%. In general, mRNA expression was elevated in HMP tissue as compared to CS. The change in tissue ATP content after three hours HMP increased significantly.

Conclusion: Three hours of oxygenated HMP of the pancreas increases oxygenation status but does not increase islet yield, function or viability. Earlier initiation and/or longer duration of HMP may alter these results.

This study is supported by the Diabetes Cell Therapy Initiative (DCTI)

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302

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Human Pancreas Persufflation Ameliorates Hypoxia-Induced Impairment of Islet Function Post-Isolation

Kathryn R Mueller1,3, William E Scott, III1,2,3, Tun Jie1, Bradley P Weegman2,3, A N Balamurugan3, Jennifer P Kitzmann1,3, Helen Marshall4, James Shaw4, Derek Manas5, Shanta Persaud6, Guo Cai Huang6, Shirin E Khorsandi7, Linda Tempelman8, Rebecca L Pongratz9, Gary W Cline9, Angelika C Gruessner1, Rainer W Gruessner1, Klearchos Papas1,2,3

1Surgery, University of Arizona, Tucson, AZ, United States; 2Radiology, Center for Magnetic Resonance Imaging, University of Minesota, Minneapolis, MN, United States; 3Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN, United States; 4Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom; 5HPB & Transplant Surgery, Freeman Hospital, Newcastle Upon Tyne, United Kingdom; 6Division of Diabetes and Nutritional Sciences, School of Medicine, King’s College London, London, United Kingdom; 7Department of Surgery and Cancer, Imperial College London, London, United Kingdom; 8Giner, Inc., Newton, MA, United States; 9Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, United States.

Recent reports [1] demonstrate that isolated human islets exhibit a “hypoxia-response gene expression signature” that is linked to persistent dysfunction, confirmed by an impairment of glucose-stimulated insulin secretion (GSIS) in vitro and in vivo. Brief exposure of islets to hypoxia leads to an increase in hypoxia induced factor (HIF-1α) and is believed to activate the molecular cascade leading to islet dysfunction. In this paper we report on HIF-1α expression in human and porcine pancreata and GSIS on isolated human islets from pancreata preserved either on static cold storage (SCS) or persufflated (PSF) with cold, humidified oxygen-enriched (40% O2) gas. Our results show a dramatic increase in HIF-1α during SCS (by western blot and ELISA). This effect is seen as early as after 6 hours of SCS and may be prevented by PSF. In split lobe experiments, human islets isolated from lobes preserved with SCS had impaired GSIS (in perifusion assays) relative to those preserved with PSF. The impairment in GSIS was present in the absence of any substantial effect on viability as measured by standard membrane integrity tests, islet morphology, and oxygen consumption rate normalized to DNA (OCR/DNA) assay. We conclude that improved oxygenation during human pancreas preservation with PSF prior to isolation may facilitate better maintenance of islet function post isolation. This could have profound implications for clinical islet transplantation, as well as on research studies utilizing human islets for understanding fundamental aspects of insulin secretion and diabetes drug development.

Reference:

[1] Cantley J, Walters SN, Jung M-H, Weinberg A, Cowley MJ, et al: A pre-existent hypoxic gene signature predicts impaired islet graft function and glucose homeostasis. Cell Transplant 2012, Oct 31 [Epub ahead of print].

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303

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Implication of Preservation Solutions in Pancreatic Ductal Injection for Human Islet Isolation: A Randomized Comparison Study

Morihito Takita1, Takeshi Itoh1, Mazhar Kanak2, Masayuki Shimoda1, Rauf Shahbazov1, Faisal Kunnathodi1, Michael Lawrence1, Bashoo Naziruddin3, Marlon Levy3

1Islet Cell Laboratory, Baylor Research Institute, Dallas, TX, United States; 2The Institute of Biomedical Studies, Baylor University, Waco, TX, United States; 3Baylor Annette C. and Harold C. Simmons Transplant Institute, Dallas, TX, United States.

Background: Preservation of pancreas is a major factor influencing the results of islet isolation for transplantation. We have reported that pancreatic ductal injection (PDI) can improve islet isolation outcomes by preserving pancreatic ducts using ET-Kyoto solution [1,2]. However, ET-Kyoto solution is not commercially available in US. In the present study we have compared Cold Storage/Purification Stock Solution (CSPS) for PDI and report on the islet isolation outcome.

Methods: A total of 12 pancreatic islet isolations were randomly assigned to ET-Kyoto or CSPS for PDI (n = 6 for each group). The islet isolations were performed according to the modified Ricordi method. The outcomes of 5 consecutive islet isolations without PDI were included in the comparison with ET-Kyoto and CSPS.

Results: No significant differences in donor characteristics, including cold ischemia time, and trimmed pancreas weight were detected among the three groups. The proportion of TUNEL-positive cells in pancreatic islets before islet isolation was significantly lower in both PDI groups compared with the control (median [interquartile range]: 1.1 [0.0–2.8], 3.4 [1.6–6.3] and 16.9 [7.9–23.2] for % in ET-Kyoto, CSPS and control groups, respectively; p < 0.05). Marginally significant difference in islet yield per pancreas weight were seen before purification between control and PDI groups (4.1 [1.7–8.2], 9.0 [4.8–12.2] and 8.4 [5.9–14.7] × 103 IEQ/g in control, ET-Kyoto, and CSPS groups, respectively; p = 0.07). Total islet yield expressed as islet equivalents were significantly improved in both PDI groups when compared to control (median [interquartile range]: 6.7 [4.9–8.4], 7.4 [6.3–9.1] × 105 and 2.5 [4.1–3.8] in ET-Kyoto, CSPS and control respectively (P < 0.05, figure). In in vivo bioassay with diabetic nude mice, there was no significant difference between the three groups in curative rate as well as high-mobility group box protein 1 (HMGB1) release.

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Conclusions: PDI using the either extracellular-type preservation solutions ET-Kyoto and CSPS can improve outcomes in pancreatic islet isolations and CSPS efficiency is equivalent to that of ET-Kyoto solution.

Reference:

[1] Noguchi H, Ueda M, Nakai Y, Iwanaga Y, Okitsu T, Nagata H, Yonekawa Y, Kobayashi N, Nakamura T, Wada H, Matsumoto S. Modified two-layer preservationmethod (M-Kyoto/PFC) improves islet yields in islet isolation. Am J Transplant. 2006, 6(3):496–504.

[2] Matsumoto S, Noguichi H, Shimoda M, Ikemoto T, Naziruddin B, Jackson A, Tamura Y, Olson G, Fujita Y, Chujo D, Takita M, Kobayashi N, Onaca N, Levy M. Seven consecutive successful clinical islet isolations with pancreatic ductal injection. Cell Transplant 2010, 19:291–297.

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304

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The Early Loss of Transplanted Islets is Prevented by Targeting Na+/Ca2+ Exchanger of Donor Islets Prior to Transplantation

Takeshi Itoh1, Toshiyuki Mera1, Satomi Kita2, Shohta Kodama1, Daibo Kojima1, Hitomi Nishinakamura1, Takahiro Iwamoto2, Keiko Omori3, Junko Ono4, Hiroshi Watarai5, Masaru Taniguchi5, Yohichi Yasunami1

1Department of Regenerative Medicine & Transplantation, Fukuoka University, Fukuoka, Japan; 2Department of Pharmacology, Fukuoka University, Fukuoka, Japan; 3Department of Diabetes, Endocrinology and Metabolism, Beckman Research Institute of City of Hope, Duarte, CA, United States; 4Murakami Karindo Hospital, Fukuoka, Japan; 5Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan.

Pancreatic islet transplantation is an attractive therapy for the treatment of insulin-dependent diabetes mellitus patients. However, the low efficiency of this procedure necessitating sequential transplantations of islets with the use of 2–3 donors for a single recipient, mainly due to the early loss of transplanted islets, hampers its clinical application. Previously, we have shown in mice that a large amount of High-mobility group box 1 protein (HMGB1) is released from islets soon after their transplantation and that this triggers innate immune rejection with the activation of dendritic cells (DC), natural killer T (NKT) cells and neutrophils to produce IFN-gamma, ultimately leading to the early loss of transplanted islets. Thus, HMGB1 release plays an initial pivotal role in this process; however, its mechanism remains unclear. Here we demonstrate that release of HMGB1 from transplanted islets is due to hypoxic damage resulting from Ca2 + influx into b cells through the Na +/Ca2 + exchanger (NCX). Moreover, the hypoxia-induced b cell damage was prevented by pretreatment with an NCX-specific inhibitor prior to transplantation, resulting in protection and long-term survival of transplanted mouse and human islets when grafted into mice. These findings suggest a novel strategy with potentially great impact to improve the efficiency of islet transplantation in clinical settings by targeting donor islets rather than recipients.

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305

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In Human Islets the Intracellular Concentration of Sirolimus Diminish when Combined with Tacrolimus, In Vitro.

Kristine Kloster-Jensen1,2, Nils Tore Vethe3,4, Sara Bremer3,4, Aksel Foss1,2,5, Olle Korsgren6, Stein Bergan3,4,7, Hanne Scholz1,2

1Department of Transplantation medicine, Oslo University Hospital, Oslo, Norway; 2Institute for Surgical Research, Oslo University Hospital, Oslo, Norway; 3Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway; 4Department of Pharmacology, Oslo University Hospital, Oslo, Norway; 5Faculty of Medicine, University of Oslo, Oslo, Norway; 6Department of Clinical Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden; 7School of Pharmacy, University of Oslo, Oslo, Norway.

Background: Immunosuppressive drugs cause islet deterioration after islet transplantation. Knowledge of intracellular concentration of immunosuppressive drugs and membrane drug transporters is necessary to predict the drugs impact on β-cells and to better understand the mechanisms of action when islets are exposed to regimens of immunosuppressive drugs after islet transplantation.

Methods: Human islets were exposed for 24 and 48hours, to therapeutic or toxic doses of Tacrolimus (10 or 30μg/L), Sirolimus (10 or 30μg/L) or Methylprednisolone (100 or 1000ng/ml) alone or in combination (Sirolimus + Tacrolimus), (Sirolimus + Tacrolimus + Methylprednisolone). A toxic dose of CyclosporineA (5μg/ml) and Sirolimus + CyclosporinA at 24hours were also investigated. Intracellular concentrations of drugs were evaluated, using a quantification technique established in our lab, performed with LC-MS/MS using Micromass Quattro micro API MS/MS-instrument with electrospray ionization. The drug influx and efflux transporters SLCO1B1 and ABC1B, as well as the enzyme CYP3A4 mRNA were quantified using PCR. Presence of ABC1B protein in islets was verified with immune staining.

Results: Islets incubated with Sirolimus + Tacrolimus and Sirolimus + Tacrolimus + Methylprednisolone had 50% reduced intracellular concentration of Sirolimus (p < 0,0008) compared to islets incubated with Sirolimus alone, independent of dose and exposure time. This reduction was not observed in Sirolimus + CyclosporineA, nor found in intracellular concentrations of Tacrolimus. Intracellular concentration of Sirolimus significantly increased (p < 0,0008) when exposure time was prolonged from 24 to 48hours. We found expression of ABC1B, CYP3A4 and SLCO1B1 mRNA in human islets. Both ABC1B and SLCO1B1 mRNA expression increased when exposure time was prolonged to 48hours whereas CYP3A4 decreased.

Conclusions: Human islets exposed to Sirolimus + Tacrolimus show a considerable decrease in intracellular concentration of Sirolimus. This reduction in Sirolimus exposure may limit its toxicity to islets. Expression of membrane transporters (SLCO1B1, ABC1B) and the important metabolizing enzyme CYP3A4 were detected in human islets; these proteins may be involved in the observed interaction between Sirolimus and Tacrolimus. Intracellular drug concentrations in human islets have never previously been described and will be of importance to drug monitoring of clinical islet transplant patients.

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The Effects of Dimethyl Fumarate on Rodent Isolated Islet Cells

Shiri Li1, Lourdes Robles1, Chie Takasu1, Yuichi Masuda1, Kelly Vo1, Aimee Le1, Jefferson Chan2, Nosratola Vaziri3, Hirohito Ichii1

1Surgery, University of California,Irvine, Orange, CA, United States; 2Pathology & Laboratory Medicine, University of California,Irvine, Orange, CA, United States; 3Department of Nephrology and Hypertension, University of California,Irvine, Orange, CA, United States.

It has been shown that oxidative stress in insulin producing cells is one of critical factors for Type I diabetes. Nuclear factor erythroid-derived 2-related factor (Nrf2) is a transcription factor that mediates a broad-based set of adaptive responses to intrinsic and extrinsic cellular stresses. In responses to oxidative stress, Nrf2 promotes genes encoding antioxidant and detoxifying enzymes. It is well known that islet isolation procedure can cause significant oxidative stress to islet cells. In this study, we explored Nrf2-keap1 pathway on islet cell and examined the effect of dimethyl fumarate (DMF), one of Nrf2 activator using rodent islet isolation. Firstly, Nrf2 knockout mice were used for islet isolation to testify the effect of Nrf2-keap1 pathway on islet isolation. Islet yield of Nrf2 knockout mice (15.6 ± 4.9 IEQ/g b.w.) was significantly lower than wild-type (24.2 ± 1.9IEQ/g b.w., P < 0.05). Viability of islet of Nrf2 KO mice (84.5 ± 0.6%) also showed significantly lower than wild-type mice (93 ± 3.8%, P < 0.05). Basing on these results, DMF, one of Nrf2 activator was administered to normal Sprague Dawley rats in pre-treatment group for 2-days prior to isolation. After rat islet isolation, islet yield in pre-treatment group (2136 ± 620 IEQ) was significantly higher than that in control group (1174 ± 416 IEQ, P < 0.05). The dead cells of isolated islet in pre-treatment group (5.8 ± 1.6%) was significantly lower than that in control group (8.8 ± 1.9%, P < 0.05). The apoptotic cells of isolated islet in pre-treatment group (41.1 ± 3.7%) was also significantly lower than that in control group (67.8 ± 3.2%, P < 0.05). Increased mRNA levels of both NAD(P)H dehydrogenase, quinine 1(NQO1) and Glutamate-cysteine ligase catalytic subunit (GCLC) of isolated islets were detected in pre-treatment group by real-time PCR. Summarily, Nrf2 knockout mouse showed significantly worse isolation results than wild-type. Furthermore, administration of DMF significantly improved islet yield, viability after isolation through enhancing mRNA expression of NQO1 and GCLC in normal rat. Our results strongly indicate a potential clinical use of DMF in clinical islet transplantation and type 1 diabetes prevention.

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A Simple and Safe Clinical Procedure for Human Encapsulated Islet Transplantation in the Subcutaneous Tissue for Diabetes Treatment.

Denis Dufrane1, Michel Mourad2, Eric Goffin3, Najima Aouassar1, Pierre Gianello4, Bernard Vandeleene5

1Center of Tissue/Cell Therapy; Endocrine Cell Therapy, University clinical hospital St-Luc, UCL, Brussels, Belgium; 2Unit of abdominal transplantation, University clinical hospital Saint-Luc, UCL, Brussels, Belgium; 3Service of Nephrology, University clinical hospital St-Luc, UCL, Brussels, Belgium; 4Experimental surgery laboratory, Université Catholique de Louvain/IREC, Brussels, Belgium; 5Service of endocrinology, University clinical hospital St-Luc, UCL, Brussels, Belgium.

An allograft of encapsulated human islets in the “Monolayer Cellular Device” (MCD) [1] was performed in a patient (74 years old,49 years of T1DM course and transplanted with a kidney in 2000) with: (i) a blood glucose (BG) between 37-530 mg/dl, (ii) a HbA1C between 8-9%, (iii) a mean short-acting (Actrapid®)/long-acting insulin (Levemir®) of 20 (range:14-27 units/day) and 8 units/day (range: 5-11 units/day), respectively and (iv) 1 to 2 severe hypoglycaemia episodes/week (requiring assistance). The MCD was transplanted (in 15 min), under local anaesthesia, in a small abdominal subcutaneous tissue pocket (Fig. 1). No inflammation/no immunization was found against donor-HLA/beta cells (anti IA-2/anti-GAD) up to 361 days post-transplantation. Ultrasonography/magnetic resonance confirmed no fibrotic surrounding tissue, no modification of the subcutaneous adipose tissue and the preservation of the implant initial size (16 cm2) at 11 months post-transplantation. Diabetes control was obtained with: (i) a mean BG at 142 ± 35 mg/dl,(ii) a Fasting BG (FBG) of 119 mg/dl;(iii) a significant reduction of daily Actrapid®/Levemir® quantities by 22% and 58%,(iv) a mean HbA1C at 7.2% (with a minimum at 6.6% at 7 months) with a stable body weight of 99% of the initial weight after 1 year follow-up. The graft function was demonstrated by the correction of the FBG (without any exogenous insulin) at 93 ± 40 mg/dl with a basal insulin serum level at 12.5 ± 5.3 μU/ml (in contrast to a FBG at 345 mg/dl without any serum insulin pre-transplantation) during 11 months post-transplantation. A significant reduction of hypoglycaemic episodes (< 52 mg/dl with symptoms) of 61% was obtained after transplantation. The MCD was easily removed (after 361 days) and demonstrated the macroscopical integrity of the graft without any sign of inflammation and viable human islets found inside the graft with pro-insulin/insulin positive cells (negative staining for a cocktail of anti-glucagon, anti-CD3/CD68 antibodies). The aim of this study demonstrated that a subcutaneous macrodevice is (i) a simple procedure;(ii) safe and (iii) reversible. Encapsulated human islets can survive and improve blood glucose homeostasis for 1 year after implantation.

Reference:

Dufrane D, Goebbels RM, Gianello P: Alginate macroencapsulation of pig islets allows correction of streptozotocin-induced diabetes in primates up to 6 months without immunosuppression. Transplantation 2010; 90: 1054-62.


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Hypoxia in Islets After Intramuscular Transplantation can be Overcome by Co-Implantation of Polymerized Hemoglobin

Daniel Espes1, Joey Lau1, My Quach1, Andre F. Palmer3, Per-Ola Carlsson1,2

1Medical Cell Biology, Uppsala University, Uppsala, Sweden; 2Medical Sciences, Uppsala University, Uppsala, Sweden; 3Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, United States.

Muscle is a promising alternative site for islet transplantation due to rapid restoration of islet vascularity. However, the development of fibrosis in the grafts suggests massive cellular death post-transplantation. The present study tested the hypothesis that there is prominent hypoxia in intramuscular islet grafts early after transplantation, but that it can be overcome by co-implantation of polymerized hemoglobin (P-Hb).

200 islets were syngeneically transplanted intramuscularly to C57BL/6 mice with or without different concentrations of P-Hb. The extent of hypoxia was evaluated by the biochemical marker pimonidazole, and related to cellular death (caspase-3 staining).

Substantial hypoxia was observed in control islet grafts with a 77 ± 3% pimonidazole-positive area one day post-transplantation. The area of hypoxic cells then decreased to 36 ± 5% (P < 0.05 vs. day 1) four days after transplantation and to 17 ± 5% (P < 0.05 vs. both day 1 and 4, n = 6 in all groups) after seven days. Reversal of hypoxia coincided with revascularization and decreased apoptotic rates. In islets co-transplanted with P-Hb the hypoxic area was substantially decreased. One day post-transplantation the hypoxia area was 30.5 ± 4.8% in the low-dose group (0.03mg P-Hb/g bw, p < 0.05 vs. untreated); 16.3 ± 3.2% in the medium-dose group (0.1mg P-Hb/g bw, p < 0.05 vs. untreated) and 6.5 ± 3.5% (p < 0.05 vs. low-dose and untreated) in the high-dose group (0.3mg P-Hb/g bw, n = 3 in all groups). The half-life of P-Hb is 30 hours, but we did not observe any rebound hypoxia in the islet grafts. The islet vascular density was neither hampered by the P-Hb treatment, since no difference could be observed 14 days post-transplantation between control islets (7.1 ± 1.0%, n = 3), islets implanted with high-dose P-Hb (6.6 ± 1.9%, n = 3) or native islets (8.6 ± 1.4%, n = 3).

We conclude that islet hypoxia is substantial in the immediate phase post-transplantation. Co-transplantation with P-Hb can be used to effectively bridge this critical phase, and thereby provide possibilities for improved engraftment.

Acknowledgment: This work was supported by grants from EFSD/JDRF/Novo, SRC, SDF and NNF.

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Long Term Insulin Independence After Islet Transplant Alone (ITA) and Pancreas Transplant Alone (PTA) in Patients with Type 1 Diabetes (T1D) – A Single Institution Experience

Umesh Masharani1, Sara Moassesfar2, Lynda Frassetto1, Greg Szot3, Mehdi Tavakol3, joan McElroy3, Marissa Ramos3, Kristina Johnson3, Peter G. Stock3, Andrew Posselt3

1Department of Medicine, University of California, San Francisco, CA, United States; 2Department of Pediatrics, University of California, San Francisco, CA, United States; 3Department of Surgery, University of California, San Francisco, CA, United States.

Background: We describe ITA in patients with T1D using two novel immunosuppressive regimens based on the anti-LFA 1 antibody, efalizumab, or the costimulation blocking antibody, belatacept, that permit long-term islet allograft survival without need for corticosteroids or calcineurin inhibitors (CNI). We also asked whether ITA using these protocols could achieve outcomes comparable to PTA performed at our institution.

Methods: Ten T1D patients received ITA between 2007–2010. Insulin independence, renal function & adverse reactions were compared to 17 TID who received consecutive PTA between 2002–2011. All patients received thymoglobulin induction. Maintenance immunosuppression for ITA consisted of Efalizumab (n = 5) or Belatacept (n = 5), sirolimus, and mycophenolate mofetil (MMF). PTA patients received low-dose tacrolimus, MMF, sirolimus and prednisone. High insulin requirements and BMI > 30 were exclusion criteria for the ITA; and high cardiovascular risk is an exclusion criterion for PTA.

Results: Six patients received one and four received two islet transplants. All 10 became insulin independent after the final transplant for a mean of 46 months (25–64). Seven (70%) remain insulin independent at most recent follow-up (3.1 - 5.8yrs), and 3 resumed insulin use at 24, 34, 34 months (see Table 1). Mean duration of insulin independence in the 17 PTA recipients was 72.8 months (12–136). Thus after final transplant, all ITA (100%) were insulin independent at 1 year and 7/10 (70%) at 3 years. For PTA, 16/17 (94%) were insulin independent at 1 year and 13/17 (76.5%) at 3 years.

Significant complications in the ITA group included 1 partial portal vein thrombosis which resolved with anticoagulation, and 1 case of post-transplant lymphoproliferative disorder (PTLD) which resolved with therapy and did not result in graft loss. In the PTA group, there was 1 case of PTLD necessitating withdrawal of immunosuppression; 4 graft pancreatectomies for pancreatitis/rejection; 1 bowel obstruction; 3 incisional hernias; 1 soft tissue infection; and 2 conversions to enteric drainage. Renal function remained stable in 10/10 ITA and decreased in 5/17 PTA patients on CNI based regimens.

Conclusions: Long term insulin independence following ITA performed in selected patients receiving co-stimulation/adhesion blockade is similar to that observed for PTA at our institution. Although selection criteria for ITA versus PTA are different, these data demonstrate increasing options to achieve long term insulin-free survival for people undergoing beta cell replacement for T1D.

TABLE 1
TABLE 1
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Reduction of Donor-Reactive T Cells is a Prerequisite for Effective Control of Allograft Rejection Using Regulatory T Cell Therapy

Karim Lee1,2, Kyung-Mi Lee2, Vinh Nguyen1, Sang-Mo Kang1, Qizhi Tang1

1Division of Transplantation, UC San Francisco, San Francisco, CA, United States; 2Korea University, Seoul, Korea.

Introduction: Regulatory T cells (Treg) are essential for the establishment and maintenance of immune tolerance, suggesting a potential therapeutic role for Treg in transplantation. However, Treg administration alone is insufficient in inducing long-term allograft survival in normal hosts, likely due to the high frequency of alloreactive T cells. We hypothesized that a targeted reduction of alloreactive T effector cells (Teff) would allow a therapeutic window for Treg efficacy.

Methods: Donor reactive T cells were induced into cycle by a donor specific transfusion (DST) of 20 x 106 BALB/c splenocytes into C57BL/6 mice. Two days after DST, cyclophosphamide (CY, 200 mg/kg) was administered IV to kill cycling T cells. The frequency of donor specific T cells was assayed using a FACS based MLR assay, and by assessing the frequency of TCR-transgenic tracer T cells of known specificity.

Donor specific Tregs from TCR-transgenic mice or polyclonal Tregs from wild-type mice were sorted and expanded with CD3-CD28 beads and IL-2 prior to infusion.

Islet transplants (tx) were performed in streptozocin-induced diabetic mice or in spontaneously diabetic NOD mice. DST + CY treatment was initiated 7 days prior to tx. Tregs were infused 1 day prior to tx.

Results: Treatment of recipient mice with DST followed by CY treatment deleted 70-80% donor-reactive T cells, but failed to prolong islet allograft survival. The infusion of 5x106 donor specific Treg or 25x106 polyclonal Treg alone also had no effect on islet allograft survival. However, DST + CY plus infusion of either 5 x 106 donor reactive Tregs or 25 x 106 polyclonal Tregs led to indefinite survival of BALB/c islets in more than 70% of C57BL/6 recipients. Notably, protection of C3H islets in autoimmune diabetic NOD mice required infusion of islet autoantigen-specific Tregs together with polyclonal Tregs. Treg therapy led to a significant reduction of CD8 + T cells and a concomitant increase in endogenous Tregs among graft-infiltrating cells early after transplantation.

Conclusions: The infusion of large numbers of donor-specific or polyclonal Treg is unable to prolong islet allograft survival in normal hosts. “De-bulking” of donor-reactive Teff cells can significantly alter the donor-reactive Treg/Teff ratio and allow long-term survival of islet allografts. Importantly, autoimmune NOD mice required the addition of islet autoantigen-specific Treg. These findings have important implications for the design of Treg based immunomodulation in transplantation.

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Pancreatic Antigen Expression in Extrathymic Aire-Expressing Cells Prevents Autoimmune Diabetes

James Gardner, Todd Metzger, Mark Anderson

University of California, San Francisco, San Francisco, CA, United States.

Introduction: The AutoImmune REgulator (Aire) gene is essential to immune tolerance. Aire is expressed in the thymus, where it allows specialized medullary thymic epithelial cells (mTECs) to express a diverse range of otherwise tissue-restricted self-antigens like insulin and thyroglobulin, thus exposing developing T cells to a more complete picture of immunologic self. Recently we described a novel population of extraThymic Aire-expressing Cells (eTACs) in the secondary and tertiary lymphoid organs that express a range of self-antigens and are highly potent inducers of self-tolerance in the peripheral immune system. This study examines whether targeting expression of disease-relevant pancreatic antigens to eTACs can induce immunologic tolerance and prevent autoimmune diabetes in two mouse models of disease.

Methods: Two novel transgenic mouse strains were generated expressing the pancreatic antigens islet-specific glucose-6-phosphatase related protein (IGRP) and the Barbara Davis Center 2.5 antigen (BDC2.5) in eTACs to study basic eTAC biology and eTAC-mediated prevention of autoimmune diabetes by CD4 + and CD8 + T cells, respectively. Impact on autoimmune diabetes was studied using the nonobese diabetic (NOD) mouse in both intrinsic and adoptive transfer models of disease. The Aire-driven IGRP-GFP (Adig) mouse expresses a GFP-tagged copy of the islet antigen IGRP in eTACs and allows study of interaction between eTACs and the IGRP-specific CD8 + T-cell clone 8.3, and the Aire-driven BDC antigen (AdBDC) mouse permits study of eTAC interactions with islet-specific CD4 + T cell clone BDC2.5. Diabetes incidence was tracked in cohorts using serial blood glucose measurement. Functional analysis was done with immunofluorescent microscopy and flow cytometry, as well as in vitro cytokine release assays.

Results: These studies identify and define eTACs as a unique population of bone marrow-derived antigen presenting cells (APCs) related to dendritic cells (DCs), and show that targeted expression the pancreatic antigens IGRP or BDC peptide in eTACs entirely prevents T cell-mediated autoimmune diabetes in both models studied. Interaction between eTACs and CD4 + or CD8 + T cells causes deletion, functional inactivation, or conversion of those interacting T cells into a regulatory phenotype, but disease prevention does not require regulatory T cells. Further, unlike other putative tolerogenic peripheral APC populations, eTAC-mediated tolerance is uniquely stable and resistant to conversion from tolerance to activation in the presence of inflammatory stimuli.

Conclusions: Together these results suggest that eTACs play an important role in maintaining immunologic self-tolerance, and suggest this population as an attractive therapeutic target for the prevention and treatment of autoimmune diabetes, and potentially for diverse applications in autoimmunity and transplantation.

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Simultaneous Pancreas and Kidney Transplantation from Living Donors. Outcome of Recipients and Donors of Sixteen Consecutive Clinical Trials in a Single Institution

Kenmochi Takashi1,2, Taihei Ito1,2, Takehide Asano3, Mikiko Hayashi2, Naotake Akutsu3, Michihiro Maruyama3, Kiyotaka Hoshinaga4, Hisahiro Matsubara5

1Department of Organ Transplant Surgery, Fujita Health University, School of Medicine, Toyoake, Japan; 2Transplantation Supporting Unit, Fujita Health University Hospital, Toyoake, Japan; 3Department of Surgery, Chiba-East National Hospital, Chiba, Japan; 4Department of Urology, Fujita Health University, School of Medicine, Toyoake, Japan; 5Department of Academic Surgery, Chiba University, Graduate School of Medicine, Chiba, Japan.

Objectives: Based on a poor prognosis of type 1 diabetic patients due to end-stage renal disease (ESRD) and a severe shortage of deceased donors in Japan, we have ifirstly ntroduced a simultaneous pancreas and kidney transplantations from living donors (LDSPK) in 2004 and sixteen cases of LDSPKs have been performed in our institution (26 cases in Japan).

Patients and Methods: [Recipients] Sixteen type 1 diabetic patients with ESRD underwent LDSPKs. Age and gender were 34.2 ± 5.7 years and 6 males / 10 females. All patients showed frequent hypoglycemic unawareness and negative serum C-peptide level (< 0.03ng/ml). [Donors] Donors were nine mothers, three fathers and four brothers/sisters. Six donors were ABO-incompatible for the recipients. All donors showed both normal endocrine andrenal function and fulfilled the donor criteria for LDSPK of Japan Society for Transplantation. [Operations and immunosuppression] Donor operation was performed by open laparotomy (former 8 cases) or laparoscopic procedure (latter 8). LDSPK was performed using pancreatico-cystostomy. Immunosuppression was achieved by a quadruple therapy with tacrolimus, MMF, predonisolone, and basiliximab. For ABO-incompatible cases, desensitization with rituximab, three times of double filtrated plasmapheresis and plasma exchange was performed.

Results: Although a pancreatic fistula (Grade B) was developed in one donor, it disappeared by conservative therapy. Another donor developed a pancreatic pseudo-cyst at 6 months after surgery and needed aspiration of the cyst through the stomach by gastro-scope. Other 14 donors showed no complication including, diabetes and renal dysfunction from 2 to 9 years. However, long-term (5 years) follow-up data of the donor demonstrated that siginicant deterioration of insulin secretion and elevated HbA1C were observed. One recipient developed primary nonfunction of the pancreas graft. Another patient developed venous thrombosis 2days after transplantation. Other 14 patients achieved insulin independency immediately after transplantation. These patients are maintaining insulin independency with positive serum CPR(2-7ng/ml) and showed the normal endocrine function evaluated by HbA1C, HOMA-beta, HOMA-R and 75g-oral glucose tolerance test. All six patients from ABO incompatible donors achieved insulin independency (100%) and withdrawal from hemodialysis (100%) without an episode of antibody mediated rejection.

Conclusions: The results of the consecutive clinical trial in our institution demonstrated that LDSPK might be recommended as a potent tool of treatment for type 1 diabetic patients with ESRD especially in our country. However, long term care for the donors not to develop diabetes should be esential in LDSPK.

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Raising Program Awareness Through Public and Media Relations.

Andrea Norgate1

1Ambulatory Transplant, Toronto General Hospital, Toronto, ON, Canada.

Background of Problem: Analysis of pancreas transplant awareness and referral patterns indicated that only a fraction of the dialysis centres and endocrinologists were referring patients for K/P transplantation. As a result, many patients who would benefit from K/P or pancreas transplantation were not offered this opportunity. A lack of knowledge among nephrologists and endocrinologists about the progress in pancreas transplantation and subsequent success rates inhibited patient referral. Patients with type 1 diabetes, even those on dialysis, were either unaware of transplant options available to them – or believed that a transplant may make them worse.

Goals of Project: The goal of the campaign was to raise awareness of pancreas transplantation. We sought to enhance the quality of patient care by improving communication with referring centres and through public education using social and conventional media tools.

Interventions: Our program embarked on a campaign targeting 3 levels, physicians, dialysis nursing staff and patients. Our key components included a new brand identity using posters,/brochures, an e-blast targeting physicians and new templates for the program. Using social media such as twitter, blogging, Facebook and YouTube we sought to educate patients regarding options. Lunch and learn sessions were held in dialysis units to encourage advocacy for potential patients.

Outcomes: To date the media campaign has produced an increase in referrals to our program by over 400% over the initial six month period and has had continued rates of referrals as our campain continues. Our campaign has been highly successful and has significantly improved our communications with patients and referring centres. To maintain this success we have designed a long-term strategy to promote awareness for pancreas transplantation and to establish ourselves as a leading pancreas transplant centre.

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One Hundred Pancreas Transplantation in University of Sao Paulo - Brazil Analysis of the Recipients and Complications

Vinicius Santos1, Rafael Pecora1, Rubens Arantes1, Oscar Ferro1, Carlos Pantanali1, Andre David1, Eleazar Chaib1, Elias David-Neto1, Willian Nahas1, Luiz D’Albuquerque1

1Department of Gastroenterology, University of São Paulo School of Medicine, São Paulo, Brazil.

Introduction: Pancreas transplantation is usually performed in association with kidney transplant in patients with insulin-dependent diabetes mellitus type 1 and end-stage diabetic nephropathy[1]. Although the final objective of the pancreas transplantation is to overcome the complications of dialysis and diabetes, it has the highest morbidity of all routinely performed abdominal solid organ transplantation procedures morbidity[2,3].

Objective: We analyzed the pancreas transplantation performed at the Clinics Hospital of Sao Paulo University, in Sao Paulo, Brazil, in the last 12 years.

Methods: We analyzed retrospectively the patients underwent pancreas transplantation from January 2000 to February 2013.

Results: The total number of pancreas transplantation was 105, divided into 3 categories: 88% simultaneous pancreas-kidney (SPK), 11% pancreas-after-kidney (PAK) and 1% pancreas transplantation alone (PTA). The median age was 36.3 years (range 24 to 55), with 65 male and 40 female patients. The recipient median body mass index was 22.3kg/m2 (range 17.6 to 41.3). Before pancreas transplantation, the diabetes diagnosis was 23.3 years (range 13 to 45) and the mean time of preoperative dialysis 36.3 months (range 2 to 120). Seventy nine of them (75%) had retinopathy and 37 patients (35%) had neuropathy. There were 45.4% of postoperative complications: 7 (6.6%) pancreatic/duodenal leak, 7 (6.6%) bleeding, 19 (18%) graft pancreatitis and 15 (14.2%) pancreas graft thrombosis. Twenty seven patients (25.7%) needed transplant pancreatectomy. The pancreas graft survival at 1, 3 and 12 years post-transplant was 80%, 75% and 65%, respectively. The patient survival rate at 1, 3 and 12 years post-transplant was 85%, 78% and 71%, respectively.

Conclusion: Despite the considerable morbidity follow pancreas transplantation, it remains the main modality of treatment in type I diabetic patients with renal failure presenting long term pancreas graft survival above 65%.

Liver and Gastrointestinal Transplant Division

Reference:

[1]. Foltys DB, Kaths JM, Zimmermann T, Heise M, Hoppe-Lotichius M, Otto G. Ten years of simultaneous pancreas-kidney transplantation: a retrospective single-center analysis of prospectively obtained data. Transplant Proc 2011, 43:3267–3269.

[2]. Schenker P, Vonend O, Krüger B, Klein T, Michalki S, Wunsch A, Krämer BK, Viebahn R. Long-term results of pancreas transplantation in patients older than 50 years. Transplant International 2010, 24:136–142.

[3]. Humar A, Kandaswamy R, Granger D, Gruessner RW, Gruessner AC, Sutherland DER. Decreased surgical risks of pancreas transplantation in the modern era. Annals of Surgery 2000, 231:269–275.

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Lessons from a 13 Year Cohort of the First UK Pancreas Transplant Programme

Christine Jansen1, Victor Tswen-We Lee1, Lisa Mumford2, John J Casey1, Murat Akyol1, Gabriel C Oniscu1

1Scottish Pancreas Transplant Unit, Royal Infirmary, Edinburgh, United Kingdom; 2Statistics and Clinical Audit, NHS Blood and Transplant, Bristol, United Kingdom.

Introduction: The aim of this study was to review the 13 years activity of the first solid pancreas transplant programme in the UK with particular emphasis on the donor and recipient factors influencing early pancreatic graft loss.

Methods: Recipient and donor details, patient, graft survival and follow-up data for all pancreas transplants performed were prospectively extracted from the local database, electronic medical records, and the NHS Blood and Transplant database.

Results: From April 2000 until April 2013 433 patients were assessed for pancreas transplantation, 231 patients were registered on the transplant waiting list and 171 patients were transplanted [160 simultaneous pancreas-kidney (SPK), 7 pancreas after kidney transplants (PAK) and 4 pancreas transplants alone (PTA)]. There were 5 technical failures (2.9%); in addition there were 21 early pancreatic graft losses (12%) and 8 late pancreatic graft losses (4.6%). Patient survival at 1, 3 and 5 years was 97%, 92% and 89%; SPK graft survival 82%, 81%, and 81%, PTA / PAK graft 60%, 50% and 50% respectively. The incidence of rejection episodes in the first 3 months was 10.5 %. 28% required laporotomy, 48% were due to bleeding although overall graft loss due to bleeding was 4%. Multivariate analysis showed that recipient age and donor age have independent prognostic value for early pancreatic graft loss (P < 0.05).

Conclusions: The long term survival in our programme is favourable but patients had a higher risk of bleeding complications. The donor and recipient age impact on graft survival, whilst pre-existent cardio-vascular comorbidity does not preclude a successful transplant.

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A Novel Screening Test for Graft Thrombosis After Pancreas Transplantation Using a Contrast-Enhanced Ultrasonography (CEUS) with Sonazoid™.

Taihei Ito1, Takashi Kenmochi1, Michihiro Maruyama2, Mamoru Kusaka3, Hitomi Sasaki3, Takehide Asano2, Hisahiro Matsubara4, Kiyotaka Hoshinaga3

1Department of Organ Transplant Surgery, Fujita Health University, School of Medicine, Aichi, Japan; 2Department of Surgery, National Hospital Organization Chiba-East-Hospital, Chiba, Japan; 3Department of Urology, Fujita Health University, School of Medicine, Aichi, Japan; 4Department of Frontier Surgery, Chiba University, Graduate School of Medicine, Chiba, Japan.

Pancreas graft thrombosis is the main cause of nonimmunologic graft loss, with an incidence of from 5 up to 15% and thus a screening test for graft thrombosis after pancreas transplantation is important. However, an enhanced CT which should have the highest sensitivity for thrombosis can be hard to use as a screening test because of nephrotoxic contrast agent especially for SPK recipients account for more than 80% of pancreas transplantation. Although a Doppler ultrasonography is a common examination to evaluate graft blood flow after transplantation, intestinal gas and slow and lateral blood flow may disturb an ultrasound image of pancreas graft. Therefore, we have started screening test for graft thrombosis using a contrast-enhanced ultrasonography (CEUS) with Sonazoid™ after pancreas transplantation. Sonazoid™ is 2nd generation of ultrasound contrast agent and consists of stabilized gas microbubbles, perfluorobutane surrounded by a phospholipid shell without nephrotoxity. Our strategies for the prevention of venous thrombosis after pancreas transplantation are as follow. 1. Taking wide diameter of anastomosis and continuous inverting suture for prevention of venous anastomosis stenosis. The pancreas graft portal vein is usually elongated with venous graft for easier anastomosis and prevention of kinking splenic vein. 2. Heparinization (target ACT:180-200sec.) 3. Over hydration up to 110% from dry weight 4. Administration of Nafamostat Mesilate and PGE1 5. Reducing operative duration and TIT(concurrent pancreas back table procedure and kidney transplantation) 6. Screening for graft thrombosis using Doppler and contrast-enhanced ultrasonography 7. Careful following up of intravenous thrombus without a blood glucose elevation and diastolic back flow in the pancreas graft. We have performed 37cases of pancreas transplantation including 18 cases from living donor, so far. Only one case of a pancreas graft loss resulting from graft thrombosis was experienced and a incidence of pancreas graft thrombosis was 2.7%. CEUS with Sonazoid™ demonstrated the blood flow in splenic vein and parenchyma of pancreas graft in detail in spite of intestinal gas and slow and lateral blood flow right after transplantation.

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Analysis of Pre and Post-Transplant Diabetes-Related Auto Antibodies in Pancreas Recipients

Benita Book1, M Ahmad Mujtaba2, Jeanne Chen3, Mark Rigby2, Jonathan Fridell1, Tim Taber2

1Surgery, Indiana University, Indianapolis, IN, United States

2Medicine, Indiana University, Indianapolis, IN, United States

3Pharmacy, Indiana University Health, Indianapolis, IN, United States.

Introduction: Autoantibodies have long been associated with the development of Type 1 diabetes mellitus (DM1). The natural course of these autoantibodies following pancreas transplantation has not been well characterized.

Methods and Materials: Sera were prospectively collected from pancreas transplant recipients (simultaneous kidney and pancreas (SPK) 9, pancreas after kidney (PAK) 4 and pancreas alone (PTA 5)) enrolled in an IRB approved immune monitoring clinical trial and were tested for the presence of certain commonly identified diabetes-related autoantibodies (Insulin autoantibody-2 (IA-2A), zinc transporter 8 (ZnT8), glutamine decarboxylase isoform 65 ((GAD65) and insulin auto antibody (mIAA). All pancreas allografts were implanted with systemic venous and enteric exocrine drainage. Induction immunosuppression consisted of rabbit antithymocyte globulin (1 mg/kg x 5) and steroid withdrawal (total dosage 250 mg) with the addition of anti-CD20 8 recipients. Routine maintenance immunosuppression included tacrolimus and sirolimus with the addition of mycophenolate mofetil (500 mg bid) for PTA. Samples were collected immediately prior to and 6 and 12 months (mo) after pancreas transplantation. Samples were tested for IA-2A, ZnT8, GAD65 antibodies, by radio-immunoassay and mIAA by a micro-IAA assay. Statistical analysis was derived by Wilcoxson signed rank tests comparing medians, p ≥ 0.5.

Results: Sera from 18 recipients transplanted for DM1 were studied. Subjects included 15 males/3 females, 17Cauc/1 AA, age at transplant 44.5 years (range 37.5-61) Duration of time from onset of diabetes until transplantation was 31 ± 10 years. There was 100% graph and patient survival at 1 year. HbgA1C at one year was 5.3 ± 0.39. There was no difference from baseline compared to 6 mo or 12 mo for ZNT8, GAD65, or IA-2 antibodies. However, there was a difference when 6 mo 0.3 DK units was compared to 12 mo 1.5 DK units for IA-2A p = 0.0174.There was a significant decrease in MIAA from baseline 0.042 index compared to 6 month 0.005 index, p ≥ 0.001 or 12 months 0.010 index, p =,0.002.

Conclusion: With the exception of IA-2A, antibody levels either fell (MIAA) or were unchanged (ZnT8, GAD65) following pancreas transplantation during this study period. This is consistent with the relatively infrequent recurrence of type 1 DM following pancreas transplantation using modern immunosuppressive techniques.

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Incidence and Outcomes of Cytomegalovirus in Pancreas Transplantation with Steroid Free Immunosuppression

Jeanne M Chen1, Ashesh P Shah1, Michelle L Goble1, Muhammed A Mujtaba2, Tim E Taber2, John A Powelson1, Jonathan A Fridell1

1Surgery, Indiana University School of Medicine, Indianapolis, IN, United States; 2Nephrology, Indiana University School Medicine, Indianapolis, IN, United States.

Cytomegalovirus (CMV) is a commonly encountered opportunistic infection after solid organ transplantation. This is an under described entity in pancreas transplantation.

The records of 405 pancreas transplant recipients (226 simultaneous pancreas and kidney transplant (SPK), 86 pancreas transplant after kidney (PAK) and 93 pancreas transplants alone (PTA)) performed at a single center with at least 1-year follow-up were reviewed. Immunosuppression included rabbit anti-thymocyte globulin (5 mg/kg) induction, rapid steroid withdrawal and maintenance therapy of tacrolimus and sirolimus (+/- mycophenolate). PTA recipients also received a single dose of rituximab. All recipients received valganciclovir prophylaxis.

One-year graft and patient survival were 92% and 96%, respectively. Patients were stratified by CMV risk: 34% were Donor (D) +/ Recipient (R) -, 17% D +/R +, 19% D +/R- and 30% D-/R-. Overall CMV infection rate was 12%. CMV infection rates by group were observed 26% in the D +/R- group, 7% in the D-/R + group, 5% in the D +/R + group, and 2% in the D-/R- group. Infection rate by transplant type was SPK 7%, PAK 19% and PTA 15%. The majority of infections developed after 3 months post-transplant. The most common presentation was viremia, 24% demonstrated tissue level disease.

Most were treated with short course IV ganciclovir followed by oral valganciclovir. Immunosuppression was NOT reduced in 72% of patients with infections. Ganciclovir resistant CMV was observed in 4 patients. All of these had a complete response to foscarnet used in combination with IV ganciclovir. No patients died or lost their allografts in the study from any CMV-related infection.

A relatively low rate of CMV infection was found with our immunosuppressive regimen. In a majority of patients, CMV infection was successfully managed without automatic reduction of maintenance immunosuppression. We were able to successfully manage CMV infections after pancreas transplant without compromising patient and graft survival.

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Subcutaneous Alemtuzumab (Campath) Induction in Pancreas Transplantation: A Safe Alternative to Intravenous Administration

Gail Defries1, Stephanie Smith1, Andrew Butler1, Menna Clatworthy1, Afzal Chaudhry1, Nicholas Torpey1, Julia Ertner1, Paul Williams1, Christopher Watson1

1Transplant Unit, Cambridge University Hospitals NHS Trust, Cambridge, United Kingdom.

Introduction: Alemtuzumab is increasingly used as an induction agent in transplantation, usually being administered intravenously. Such administration is associated with a high incidence of adverse first-dose cytokine release reactions. This paper reviews a single centre experience of subcutaneous administration.

Methods: Since 1st November 2004 all patients undergoing transplantation received induction with subcutaneous alemtuzumab; prior to March 2012 one 30mg dose was given in theatre and one a week later; since March 2012 only the intraoperative dose was given. Patients also received mycophenolate and tacrolimus without steroids. The records of all patients were reviewed with respect to survival and occurrence of rejection, infection and cancer.

Results: 136 patients underwent combined kidney and pancreas transplantation (SPK) and two solitary pancreas transplants (1 PAK; 1 PASPK) in the study period; all received alemtuzumab. Two patients were inadvertently given a second dose intravenously; both developed “first dose” adverse reactions. No other systemic reactions to alemtuzumab were noted.

One year SPK actuarial patient, pancreas and kidney survivals were 98.5%, 91.1% and 99.3% respectively.

37 (27%) patients had 49 episodes of rejection, with 25 patients having at least one episode of rejection within the first year and 5 having more than one episode of rejection.

39 (28%) patients were admitted a total of 67 times for the treatment of sepsis in the study period (32 admissions were in the first year). Of the viral infections encountered, 31 (22%) patients developed cytomegalovirus viraemia, 6(4%) varicella/zoster, and 23 (16%) developed BK viraemia.

3 (2.2%) of patients developed a non-skin malignancy, with 1 PTLD, one gastric and one oesophageal cancer. “Autoimmune” recurrence of type 1 diabetes was seen in 4 (3%) patients. Two other patients developed de novo autoimmune disease, one thrombocytopaenia and one haemolytic anaemia.

Conclusion: Subcutaneous alemtuzumab is an effective induction treatment with good short and long term results.

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327

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Impact of Years of Diabetes on Outcome Following Pancreas Transplantation

Jonathan A. Fridell1, Richard S. Mangus1, John A Powelson1, Michelle L Goble1, Muhammad A. Mujtaba2, Tim E. Taber2

1Surgery, Indiana University School of Medicine, Indianapolis, IN, United States; 2Medicine, Indiana University School of Medicine, Indianapolis, IN, United States.

Diabetes is an independent major risk factor for cardiovascular and cerebrovascular disease which is dependant on the years of exposure and may impact long term outcome after pancreas transplantation. The goal of this study was to review our single center experience and examine the impact of years of diabetes on outcome following pancreas transplantation.

Methods: Charts for all pancreas transplant recipients between Jan 2003 and April 2013 were reviewed. All transplants were performed through midline laparotomy with systemic venous and enteric exocrine drainage. Immunosuppression consisted of rabbit antithymocyte globulin induction, steroid withdrawal and tacrolimus and sirolimus (+/− mycophenolate) maintenance. All patients received prophylaxis for CMV and PJP. Charts were reviewed for demographics and graft and patient survival,

Results: Out of 451 pancreas transplant recipients, 111 (25%) were diabetic from 1–20 years, 157 (35%) from 21–30 years and 183 (41%) for more than 30 years. The groups with longer standing diabetes tended to be older (median age 36, 40and 49 years respectively), were more likely to be male (50%, 62% and 65% respectively) and were more likely to require a kidney transplant (59%, 78% and 81% respectively). Donor demographics and ischemia times were comparable. There was no difference in 7 day (4%, 5% and 2%), 90 day (6%, 6%, 4%) graft loss or 1 year graft survival (92%, 91%, 94%) between the three groups. There was a significant difference in 1 year patient survival (98%, 99%, 94%). This finding was confirmed by Kaplan-Meier for survival using 10 year data (Log rank p-value = 0.05).

Conclusion: Pancreas transplantation can safely be performed on recipients with longstanding diabetes, although the duration of diabetes prior to transplantation seems to have a negative impact on longterm patient survival.


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Pancreas Allograft Thrombosis - Every Graft Deserves a Second Chance.

Harish Mahanty, William Bry, Assad Hassoun, Nikole Neidlinger, Steven Katznelson, Parul Patel

Transplant, California Pacific Medical Center, San Francisco, CA, United States.

Objective: Thrombotic complications after pancreas transplant are not uncommon and can result in a high rate of graft loss. We reviewed our 5-yr, single center experience of pancreas transplants to evaluate the nature of graft thrombosis to better understand the probability of graft salvage.

Methods: A retrospective review of our data was conducted tracking all pancreas transplants done at our center from 6/ 2007 to 3/2013 using standard electronic records. We identified patients who developed pancreas vascular compromise documented by radiographic imaging. The patients were categorized by onset of vascular compromise. Overall graft survival and graft salvage rates were reported.

Results: 114 pancreas transplants were performed at our center during the study period and comprised of 45 females and 69 males. 71 were Type I and 43 were Type II diabetics. Three were PAK’s (pancreas after kidney), four were PASPK’s (pancreas after simultaneous pancreas-kidney) and 107 were SPK’s (simultaneous pancreas-kidney) transplants (Table 1). Of the 114 transplants 23 had radiographic evidence of either partial or complete occlusion of the pancreatic allograft vasculature. Twenty occurred in the early perioperative period of which eight resulted in primary graft loss with transplant pancreatectomy. Of the remaining twelve, two were treated with operative thrombectomy, one with catheter-directed therapy and tPA, one with systemic anticoagulation with aspirin and persantine and seven with warfarin. All but one patient was maintained on warfarin. Only one secondary graft loss was noted during the follow up period. The three late thrombotic events were all treated with warfarin. One of the three patients returned to insulin requirement (Table 2).

Conclusion: Early thrombosis events can result in high risk of graft loss. However immediate recognition and intervention can mitigate graft loss and lead to long term graft survival.

TABLE 1
TABLE 1
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TABLE 2
TABLE 2
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329

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Nutritional Support in Simultaneous Pancreas and Kidney Transplantation: A Single Centre Experience

Je Song Shin1, Hussein Khambalia1, Zia Moinuddin1, Rauri Greer2, Rosalind Campbell3, Titus Augustine1, David van Dellen1

1Transplant Surgery, Manchester Royal Infirmary, Manchester, United Kingdom; 2Critical Care, Manchester Royal Infirmary, Manchester, United Kingdom; 3Dietetics, Manchester Royal Infirmary, Manchester, United Kingdom.

Background: Simultaneous pancreas and kidney transplantation (SPKT) provides insulin and dialysis independence. However, the recipient may have comorbidities contributing to malnutrition and a chronic catabolic state, strong predictors of adverse outcome. Current trends favour early enteral feeding. However, in SPK patients, concerns exist due to perceived risks of gastroparesis, ileus and a fragile enteric anastomosis. Total parenteral nutrition (TPN) is therefore routine therapy in some units. We aimed to assess outcomes of this nutritional modality.

Methods: A retrospective study was performed of SPKT recipients in a single centre over 2 years (11/10- 11/12) focusing on nutritional therapies and subsequent clinical outcomes. Nutritional parameters included TPN duration, time to enteral diet, and complications directly attributed to TPN. Clinical outcomes included, critical care and total hospital stay. Post-operative nutritional biomarkers were assessed.

Results: 52 patients were included (32 male, 20 female; mean age 42.9, (SD = 7.9); mean BMI 24.0 (SD = 2.8)). 77% started TPN on day 1 post-transplant and mean duration was 9.8 days (range 3–23; SD = 4.2). Mean ITU and total lengths of stay were 6 (SD = 6) and 30 days (SD = 26) respectively. Complications attributable to TPN included sepsis (5.8%), feeding catheter leak (3.8%), fluid overload (1.9%) and hyperglycaemia (9.6%) which all normalised after a TPN break. Serum albumin dropped by > 30% immediately and remained below normal in the first week post-surgery, regardless of nutritional modality. Alkaline phosphatase levels increased above normal range after 6 days with no long term sequelae.

Conclusions: TPN appears to provide a safe modality of nutritional support for early post-SPK nutrition with a low rate of reversible complications. However, its potential advantages remain unclear. Further randomised comparative studies against enteral nutrition are required to clarify the optimal approach in this challenging group of patients.

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Pregnancy After Simultaneous Pancreas-Kidney Transplantation

Joo Hee Jung1, Han Kyung Cho1, Byung Hyun Choi2, Sung Shin2, Young Hoon Kim2, Duck Jong Han2

1Department of Nursing, Asan medical center, Seoul, Korea; 2Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.

Introduction: Diabetes Mellitus is the leading cause of renal failure. End stage renal disease(ESRD) can often lead to menstrual irregularity. DM and ESRD are thought to be a barrier for successful pregnancy. The aim of this study is to review the experience of successful pregnancies following Simultaneous Pancreas-Kidney Transplantation(SPK).

Results: We experienced 4 cases of successful pregnancy in patients who previously underwent SPK for type I diabetes mellitus with ESRD. After modifications in immunosuppressive therapy (from tacrolimus and mycophenolate, the latter being swiched to azathioprine or discontinuation), successful delivery can be performed. The mean age of patients at the time of transplant was 32 years old and interval from transplant to pregnancy was average 25 months(17 ~ 30). The gestation period was ranged from 34 to 38 weeks. One patient delivered a female infant with body weight of 2170g, who required a care in incubator. During pregnancy, kidney and pancreas graft function were maintained properly in all cases. In all the cases, blood glucose levels and Hb A1c remained within normal limits, without requiring insulin treatment at any time point during pregnancy. The blood pressure were normal without pharmacological treatment, and no pathological proteinuria developed. Kidney graft function was stable and no rejection developed. The pregnancy went on without specific complication except for three episode; one urinary tract infection and two asymptomatic bacteriuria.

Discussion: Pregnancy after SPK can be a source of diverse problems such as miscarriage, preterm labor or fetal malformation as well as graft dysfunction. In order to achieve successful pregnancy, close monitoring with integrated multidisciplinary care by healthcare professionals including nephrologist, obstetrician, endocrinologist, and pediatrician are required.

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331

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Outcome of the Assessment Process for Simultaneous Pancreas Kidney Transplant in a National Transplant Unit

Christine Jansen1, Andrew Sutherland1, Jamie Traynor3, Wendy Metcalf2, Gabriel C Oniscu1

1Scottish Pancreas Transplant Unit, Royal Infirmary, Edinburgh, United Kingdom; 2Renal Unit, Royal Infirmary, Edinburgh, United Kingdom; 3Renal Unit, Monklands Hospital, Lanarkshire, United Kingdom.

Introduction: The aim of this study was to review the assessment process and long-term outcome of patients considered for simultaneous pancreas kidney (SPK).

Methods: All patients assessed for pancreas transplantation between 2002 and 2011 were included in this analysis. Socio-demographic data, renal, diabetic complications and comorbidity at the time of assessment were collected by linking the records of the Scottish pancreas transplantation unit and the Scottish Renal Registry (SRR). The long-term outcome analysis was carried out according to the outcome of assessment (list for SPK, list for kidney transplant (KTx) or not listed (NoTx)) on an intention to treat basis.

Results: 309 patients were assessed for 311 procedures: 277 (89%) SPK, 18 (6%) pancreas after kidney and 16 (5%) pancreas transplant alone. Of those assessed for SPK, 162 (58.5%) were listed for an SPK. Of 115 patients assessed but not listed for SPK, 51 were listed for a kidney transplant, 48 (17.3%) were deemed not suitable to be listed for transplantation and 16 (5.8%) were still being assessed or were not yet deemed in need of listing. Median follow-up from the time of assessment was comparable (SPK (5 yrs), KTx (6 yrs), NoTx(5 yrs)). 1 and 5 year survival following assessment was significantly higher (p < 0.0001, log-rank test) in those listed for SPK (95% and 89%) compared to those subsequently listed for kidney transplant (96% and 65%) and those not listed (87% and 59%). Although survival was higher in those listed for kidney transplant compared to those not listed, this was not significant (p < 0.16, log-rank test).

Conclusions: Long-term survival in patients listed for simultaneous pancreas kidney is significantly higher than patients subsequently listed for kidney transplant alone or patients who remain on dialysis. This long-term survival benefit is not seen in those listed for kidney transplant alone.

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Single Centre Experience of Cardiopulmonary Exercise Testing in Simultaneous Pancreas and Kidney Transplantation

Hussein Khambalia1, Zia Moinuddin1, Angella Bryan2, Afshin Tavakoli1, John Moore2, Dougal Atkinson2, Titus Augustine1, Iain Gall2, David van Dellen1

1Department of Transplant Surgery, Manchester Royal Infirmary, Manchester, United Kingdom; 2Department of Anaesthesia and Critical Care, Manchester Royal Infirmary, Manchester, United Kingdom.

Introduction: Cardiopulmonary exercise testing (CPET) identifies patients at increased risk of post-operative morbidity and mortality following major surgery. We present outcome data on the largest cohort of patients to date, undergoing CPET during assessment for simultaneous pancreas and kidney transplantation (SPKT).

Methods: A single centre, prospective cohort study was undertaken conducting CPET on patients referred for SPKT over an eight year period (2005–2013). Results were utilised in subsequent clinical decision making on transplant listing. Patients were divided by outcome (SPKT performed, active on waiting list, died on waiting list, kidney transplant performed and removed from active waiting list) and CPET results compared.

Results: 102 patients (43 female, 59 male; mean age at CPET 41.9 (24–60)) underwent CPET assessment. Table 1 summarises CPET analysis with significant differences between groups highlighted. One year mortality was 0% and 16.7% in the "SPKT performed" and "removed from active waiting list" groups respectively.

Peak VO2 was reduced in the whole cohort compared to predicted normal values. Significant differences were present in peak VO2, predicted peak VO2 and AT between the "SPKT performed"/ "active on waiting list" and the "removed from"/ "died on the waiting list" groups.

Conclusions: Poor functional capacity, measured by CPET, has previously been validated as a predictive marker of perioperative morbidity and mortality in major general surgery and liver transplantation. Outcome prediction in SPKT is notoriously difficult, due to the multifactorial nature of surgery. Sub-optimal peak VO2 and AT values may increase likelihood of death on the active list, removal from the active list or kidney transplant alone. CPET may be a useful clinical adjunct in clinical decision making in SPKT but requires further validation as part of a multivariate risk analysis.

Table 1
Table 1
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Management of Pancreas Allograft Head or Duodenal Ischaemia in Pancreas Transplantation

Hussein Khambalia1, Zia Moinuddin1, Ravi Pararajasingham1, Bence Forgacs1, Afshin Tavakoli1, David van Dellen1, Titus Augustine1

1Transplant Surgery, Manchester Royal Infirmary, Manchester, United Kingdom.

Introduction: Pancreatic head and duodenal ischaemia during implantation has traditionally resulted in allograft explant due to concerns over immediate viability and potential enteric or vascular complications leading to severe morbidity or mortality. However, these grafts could be salvaged with unconventional reconstructive surgical techniques to allow pancreas preservation. We aimed to establish outcomes in this cohort.

Methods: A retrospective analysis was performed of pancreas transplants performed at a single centre over 12 years (2001- 2013; 283 patients (SPK, 230; PAK, 39; PTA, 14)). Patients undergoing limited resections for pancreatic head or duodenal ischaemia at implantation were identified. Primary endpoints were graft and patient survival.

Results: Six patients (2.1%) underwent salvage resections (3 male, 3 female; mean age 44.8 (33- 57), mean cold ischaemic time 14hours 23mins). Three patients (1 PAK; 2SPK) had pancreatic head ischaemia requiring allograft head and duodenal resection. The pancreas was drained into neo-conduits (bladder, skin of the anterior abdominal wall, and pancreatico-distal ileal anasotmosis with proximal ileo-colic bypass.) The first graft thrombosed after 5 days, the second was lost to rejection after 258 days with the third still functioning (43 days). Three patients (all SPK) had isolated duodenal ischaemia necessitating limited duodenal resection with bladder drainage. Two patients underwent enteric conversion (223 and 669 days) and still have functioning grafts after > 5 years. The other graft had a venous thrombosis after 1 day. Overall graft survival in this group is 67% (4/6) and 40% (2/5) at 1 month and 1 year respectively, with no mortality at one year.

Conclusion: Ischaemia of the pancreatic head or duodenal segment, usually due to disruption of the pancreaticoduodenal vascular arcade has traditionally mandated graft explant to minimise morbidity. Approaches utilising limited resections with unconventional drainage techniques provide viable salvage options. This will result in increased organ utilisation and potentially improved patient outcome.

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Retroperitoneal Transplantation of the Pancreas with the Formation of Duodeno-Duodenal Anastamosis

Mogely Khubutia1, Alexey Pinchuk1, Ilya Dmitriev1, Roman Storojev1

1Kidney and Pancreas Transplantation, Scientific-Research Institute of Emergency Care named by N.V.Sklifosovsky, Moscow, Russian Federation.

Introduction: SPK – the preferred method of treatment for patients suffering from Type 1 Diabetes complicated by terminal stage diabetic nephropathy. Successful SPK allows us to reach the optimal level of medical and social rehabilitation for this category of patients: it eliminates uremia, achieves complete independence from insulin, stops or considerably slows the development of secondary diabetic complications, and greatly improves quality of life.

Purpose: Evaluation of the early postoperative period in patients following the retroperitoneal SPK involving the formation of duodeno-duodenal anastamosis.

Materials and Method: Retrospective analysis of twelve cases of SPK performed between October 2011 and February 2013 in which the method of removal of the exocrine pancreatic secretion out of the donor pancreas was retroperitoneal duodeno-duodenal anastamosis and where the recipient duodenum was connected into the duodenal stump of the donor pancreas. The condition of the pancreas were evaluated during the early postoperative period, along with any surgical and immunological complications and as biochemical markers indicative of the function of the transplanted organs.

Results: During the early period, the survival rate for patients and both transplants was 100%. In every case patients were completely insulin independent. Amylase levels in the blood averaged 147.6 ± 64.74 u/l, pancreatic amylase 133.5 ± 76.27 u/l, lipase 146.43 ± 82.42 u/l, C-peptide 4.37 ± 1.97 ng/ml, HbA1c 5.76 ± 1.19%, free insulin – 11,9 ± 7,25 mcIU/ml. In one case, four weeks following surgery, critical stenosis of the superior mesenteric anastamosis occured, which was successfully treated with an emergency endovascular procedure. Surgical complications requiring long-term drainage developed in two cases: in one case, resulting in part from the failure of the duodeno-duodenal anastamosis, and in the other case, resulting from the formation of a pancreatic fistula. In both cases, complications were successfully resolved. Complications requiring repeat surgical procedures were not observed in the evaluated group. Kidney and/or pancreas organ rejection requiring urgent therapy (GCs, ATG, repeat plasmopheresis), were also observed in two cases and were resolved in a timely manner.

Conclusions: Retroperitoneal SPK involving the formation of a duodeno-duodenal anastamosis is a far more physiological method of removal of exocrine secretion and is superior in comparison with classical variations of intestinal drainage.

Benefits: Enables endoscopic evaluation of the condition of the intestinal anastamosis and the mucosa of the donor duodenum, including opportunity to obtain material for biopsy;

provides potential for endoscopic transintestinal biopsy of the pancreatic parenchyma;

enables provision of endoscopic treatment in the area of the duodeno-duodenal anastamosis and main pancreatic duct.



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High Urine Amylase Level (UAMY) Predicts the Risk of Enteric Conversion (EC) in Pancreas Transplant (PTX) Recipients with Bladder Drainage (BD)

Aleksandra Kukla1, David Radosevich1, Eric Finger1, Ty Dunn1, Tim Pruett1, Raja Kandaswamy1

1University of Minnesota, Minneapolis, MN, United States.

Exocrine bladder drainage (BD) of solitary pancreas transplants in contrast to enteric drainage (ED), enables monitoring for rejection by measuring urine amylase (UAMY) levels, but is associated with a higher risk of long term metabolic and infectious complications, necessitating enteric conversion (EC) in about a third of patients. We hypothesize that hypersecreting pancreata, with high urine amylase levels, have a higher propensity to need EC as a result of symptoms related to elevated enzymatic effect on the urinary tract and increased fluid losses leading to metabolic complications.

Methods: Of 467 pancreas transplants (PTX) performed between 2002 and 2011 there were 312 BD PTX (66.8%). Of those, 147 were pancreas transplants alone (47.1%) and 165 pancreas after kidney (52.9%). Urine amylase at 30 days was used to stratify cohorts at risk for EC. Median f/u was 184.6 months (range 30.3, 288.1). Actuarial 10 yr data was used for cohort analysis stratified by UAMY to estimate risk of EC. Actual 3 yr data was used for ROC analysis.

Results: Median 30 day UAMY/hr was 1749 (quartile 1: <777, quartile 3 ≥3272) (range 3 to 30,139). Patients with the UAMY/hr <777 (Figure 1) had the lowest risk for EC, while those with UAMY/h ≥ 3272 had the highest risk. Of note, the high UAMY group continued to show increasing separation from the other cohorts in the long-term (> 5 yrs), rising to almost 50% at 10 yrs.


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Using ROC anlaysis, it was determined that UAMY of > 3272/h was most specific for predicting risk of EC, albeit with a low sensitivity. The LR+ confirmed that higher levels of urine amylase/h better discriminate between EC and non-EC recipients. (Figure 2)

Conclusion: High UMY PTX recips should be counseled about the the higher risk of EC and should be monitored for complications related to BD more closely.


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Surgical Complications in Simultaneous Pancreas Kidney Transplantation: Influence on Graft Survival

Xavier Martin1, Palmina Petruzzo1, Ricardo Codas1, Fanny Buron2, Maria Brunet2, Charles-Eric Ber3, Emmanuel Morelon2, Lionel Badet1

1Urologie et Chirurgie de la Transplantation, Hospices Civils de Lyon, Lyon, France; 2Transplantation, Néphrologie et Immunologie clinique, Hospices Civils de Lyon, Lyon, France; 3Réanimation, Hospices Civils de Lyon, Lyon, France.

Although simultaneous pancreas kidney transplantation (SPKT) is the best treatment option for type 1 diabetic patients with end-stage renal disease, pancreas transplantation has been associated with high surgical complication rate, which can worsen the prognosis.

Aim of the study was to investigate surgical complication rate (need for re-laparotomy within the first 3 months after transplantation) and their impact on patient and graft survival.

The study included 136 first SPK transplantations performed between 2005 and 2010. Systemic venous drainage and enteric drainage were performed in all the recipients. The induction therapy was based on anti-thymocyte globulins and the maintenance therapy on low dose steroids, tacrolimus and MMF.

Patient survival at 1 year was 97.8%, pancreas survival was 85.3% and kidney survival 96.3%. Two patients died in the early post-operative period (1 haemorrhagic shock and 1 septic shock). The most common surgical complication was bleeding (18.4%) which determined only one death and no graft loss; then, vessel thrombosis (7.3%), enteric leakage (5.1%), intestinal occlusion (3.7%) and pseudo-aneurysm (2.2%). Although the high rate of surgical complications, those significantly correlated to pancreas loss were only vessel thrombosis and pseudo-aneurysm development.

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Islet Donor Score (IDS) Improves Donor Pancreas Selection for Clinical Islet Transplantation: A Single Center Study

Ling-jia Wang1, Olivia Cochet1, Karolina Golab1, Xiaojun Wang1, Martin Tibudan1, Jakub Grzanka1, Omid Savari1, Randall Grose1, Michael Millis1, Piotr Witkowski1

1Department of Surgery, Division of Transplantation, University of Chicago, Chicago, IL, United States.

In order to maximize the islet yield for successful islet transplantation, the key task is to select an ideal donor pancreas. IDS was developed from comprehensive measurements (O’Gorman et al. 2000). By using it in the past years, we consistently obtained 50% transplant rate from selected pancreata. In this study, we summarized IDS application in 27 cases of islet isolations (2011 to 2013). The data included both research and transplant grade pancreata (Table 1).

Table 1
Table 1
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In summary, 60% of cases in IDS >60 group had pre-purification IEQ > 400K. BMI, itself, should not be used as a unique determinant for donor pancreas selection. However, IDS > 60 along with BMI >30 are important for predicting higher islet yield. Body surface are (BSA) is not a direct factor in IDS. In this study, BSA significantly correlated to pre-purification IEQ (r=0.5052, p 0.0072). IDS application has improved successful clinical islet transplantation in our center.

Reference:

O’Gorman D, Kin T, Murdoch T, Richer B, McGhee-Wilson D, Ryan EA, Shapiro JA, Lakey JR: The standardization of pancreatic donors for islet isolations. Transplantation 2005, 80(6): 801-806.

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Stable Kidney Function During/After Immunosuppressive Therapy in Islet Transplantation: Ten years Follow Up

Eduardo Moraes Leao Peixoto1, Arnau Alvaro1,2, Alessia Fornoni1 Lourdes Dinkins1 Valentina Delmonte1,3, Andrea Corrales1, Eva Herrada1, Camillo Ricordi1, Rodolfo Alejandro1

1Diabetes Research Institute, University of Miami, Miami, FL, United States; 2Servicio de Nefrología, Hospital Universitario Marqués de Valdecilla, Universidad de Cantabria. Fundación Marqués de Valdecilla-IFIMAV, Santander, Spain; 3Department of Biomedical Science for Health, University of Milan, Milan, Italy.

Objective: The aim is to determine clinical, laboratory and immunosuppressant-related risk factors associated with kidney dysfunction in islet transplant (IT) recipients before and after graft failure.

Research Design and Methods: A retrospective study in (n=12) IT recipients collecting demographic, anthropometrical, laboratory data, immunosuppressive and anti-hypertensive therapy. Kidney function assessed by estimated glomerular filtration rate (eGFR) calculated by Modification of Diet in Renal Disease formula (MDRD); iohexol clearance GFR and urinary albumin creatinine ratio (UACR) on spot urine samples at study time points: before first islet infusion; half-life of graft survival; graft failure and at last follow up.

Results: Mean age: 49.5±9.3years; type 1 diabetes duration: 34.9±14.7years; 41.6% males (n=5). HbA1c at each time point: 7.0±1.0 (53±10.9); 6.0±0.8 (64±8.7); 6.8±0.9 (51±9.8); 7.6±0.9% (60±9.8mmol/mol). Average graft survival: 2.7±1.6years and follow up: 9.8±0.7years. The eGFR was not different between time points (p=0.91), even when it was divided by time to exposure to immunosuppression (p=0.14). The ΔeGFR increased 12.1±13.6cc/min/1.73m2 from 1st to 9.8years post-transplant (CI95%,-26.9 to -0.5;P=0.02). Microalbuminuria prevalence was 16% at baseline; 42% at graft failure. Subjects are normoalbuminuric at last follow up (p=0.007). Albuminuria correlations were observed for triglycerides (R=0.38; P=0.008), BMI (R=-0.349;P=0.015) and HbA1c (R=0.43;P=0.002).

Conclusions: Subjects maintained stable renal function during study follow up and post-graft failure even after several years under immunosuppression therapy. We cannot exclude that the selection bias for IT patients may account for the stability of renal function. The discontinuation of immunosuppressive therapy might be responsible for the regression of microalbuminuria to normoalbuminuria. Comparisons with a T1D control group would provide further evidence to the impact of immunosuppression therapy on IT recipients.

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BK Virus Nephropathy After Islet Transplant Alone

Melena Bellin1,3, James Harmon2, Jayne Pederson3, Marnee Brandenburg3, Bernhard Hering2,3

1Pediatrics, University of Minnesota, Minneapolis, MN, United States; 2Surgery, University of Minnesota, Minneapolis, MN, United States; 3Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN, United States.

BK virus nephropathy is rarely reported in nonrenal solid organ transplant recipients, and to our knowledge, has never been observed in islet transplant alone. We report a case of BK virus nephropathy in an islet transplant alone recipient at 4 years posttransplant.

A 49 year old woman with type 1 diabetes complicated by hypoglycemia unawareness received a single donor islet infusion with 6081 IEQ/kg. Anti-thymocyte globulin and etanercept were administered for induction immunosuppression, followed by efalizumab and sirolimus maintenance. At 8 months post-transplant, immunosuppression was changed to low dose tacrolimus and mycophenolate mofetil because of voluntary withdrawal of efalizumab by the manufacturer. Sirolimus was later substituted for mycophenolate mofetil due to development of biopsy confirmed drug-induced colitis, later changed to azathioprine due to sirolimus intolerance. The patient was insulin-independent for 2 years post-transplant, and has since demonstrated good partial islet graft function. At 4 years post-transplant, renal insufficiency was noted with peak creatinine of 2.5 mg/dL, prompting renal biopsy. Biopsy revealed BK-virus nephropathy, confirmed by serum and urine BK levels of 5.4 and >8.6 log copies respectively. Tacrolimus was reduced by 50%, and the patient received cidofovir for 4 weeks, with minimal change in serum BK virus levels. Subsequently, azathioprine was stopped, the patient received IVIG, and tacrolimus was again reduced by >50%, resulting in clearing of BK virus. The patient’s creatinine stabilized at 1.8 mg/dL. Notably, despite minimal immunosuppression, the patient maintained partial graft function, requiring 10-11 units of insulin per day, with basal and stimulated C-peptide levels of 0.7 and 1.38 ng/mL.

We report an unusual case of BK virus nephropathy in an islet transplant alone recipient. Unique features in this recipient include history of treatment with efalizumab. Rapid onset of renal insufficiency in islet transplant alone recipients should prompt consideration of BK virus nephropathy.

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Islet Yields from Younger Pancreas Donors with Lower BMIs are Still Sub-Optimal Using Current Enzyme Methods

Paul A. Bateman1, Sarah E. Cross1, Derek W. R. Gray1, Stephen J. Hughes1, Paul R. V. Johnson1

1Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom.

Background: Human islet isolation remains a variable process with <50% of clinical islet isolations resulting in transplantable yields. A variety of donor factors have been reported to impact on isolation success, with many studies having identified donor age to be one of the key variables. However, these studies are mainly based on data using the previous generation of collagenase blends. More recent studies demonstrating successful isolation from younger donors have been associated with high donor BMIs. The aim of this study was to investigate whether islet isolation using more recent collagenase blends from younger donors with BMIs <35, are as successful as those from older donors with similar BMIs.

Method: With appropriate consent and ethical approval, 104 clinical grade pancreases (all DBD donors, age 18-60, BMI <35, CIT <10hrs with acceptable perfusion quality) were processed using Serva Collagenase blends at our Human Isolation Facility. Donor characteristics were compared with islet isolation criteria, and the impact of these data on isolation outcomes was determined by Spearman’s rank correlation or Mann-Whitney U test. Criteria for islet isolation success were >200K IEQ, >50% purity and >80% viability.

Results: 25% of isolations from donors ≤35 years of age (n=20, BMI 21-33) were successful compared with 44% from donors >35 years of age (n=84, BMI 20-35). We were unable to obtain sufficient yields from any donors < 30 years of age (n=8, BMI 21-31). For isolation from donors ≤35 years of age, the success rate was comparable between females (29%, n=7, BMI 21-33) and males (23%, n=13, BMI 21-29). However, for donors >35 year old, there was a significant gender effect, with a success rate and IEQ per gram of pancreas from female donors of 54% (n=39), compared with 36% from male donors (n=45) (p=0.007).

Conclusions: Our experience using currently available Serva enzyme blends has shown donor age to still have a substantial impact on islet isolation success from donors with lower BMIs. Islet isolation success improves using donors >35 years of age, and this success demonstrates a gender difference (female>male). However, we still have poor success obtaining transplantable yields of viable islets of >50% purity from younger donors with BMIs <35.

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Autologous Islet Transplantation After Total Pancreatectomy for Chronic Pancreatitis Into the Liver Affected by Primary Sclerosing Cholangitis

Ryosuke Misawa1, Ling-Jia Wang1, Sona Young2, Ruba Azzam2, Xiao-Jun Wang1, Karolina Golab1, Olivia Cochet1, Michael J Millis1, Jeffrey B Matthews2, Piotr Witkowski1

1Department of Surgery, University of Chicago, Chicago, IL, United States; 2Department of Pediatric Gastroenterology, University of Chicago, Chicago, IL, United States.

Purpose: Intraportal islet autotransplantation is an attractive procedure for patients requiring total pancreatectomy for benign disease. It may improve long-term glucose control or even prevent the development of postsurgical diabetes. A healthy liver has been considered the most optimal site for islet transplantation. We present for the first time successful autoislet transplantation into a liver affected by primary sclerosing cholangitis (PSC).

Methods: Total pancreatectomy and subsequent islet autotransplantation were performed in a 16-year-old man with intractable pain due to chronic pancreatitis. Patient also had a history of ulcerative colitis and PSC with multiple biliary strictures. Wedge hepatic (portal) pressure during the preoperative transjugular liver biopsy was 14 mmHg and biopsy revealed PSC with focal bridging fibrosis. During the procedure, the pancreas was surgically removed and digested, islets were isolated, highly purified, and infused intraportally as 1 ml tissue volume suspended in transplant media containing 70u/kg heparin.

Results: Opening portal pressure prior to islet infusion was as high as 19 mmHg, but did not further increase after completion of islet infusion. Postoperatively, liver function and portal flow were not affected by the islet autoransplant.

At one year follow up, patient had excellent glycemic control with HbA1c 5.9%, c-peptide 0.77 pmol/ml (N=0.3-2.3), and requiring only periodic short-acting insulin despite chronic oral steroid therapy (Budesonide EC 9 mg daily) to control his autoimmune gastritis. Liver function remains unchanged; serum albumin, INR and bilirubin have been within normal limits. His transaminases and alkaline phosphatase are also not significantly changed from baseline prior to procedure.

Conclusion: Pancreatic autoislet can be successfully transplanted into the liver with a hepatobiliary disease related to PSC without affecting liver or graft function. Durability of the procedure may be compromised in the future by the natural course of the liver injury caused by PSC.

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345

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Prediction of Post-Transplant Glycemic Control from Pre-Transplant Patient Characteristics in Autologous Islet Transplantation for Refractory Chronic Pancreatitis

Morihito Takita1, Rauf Shahbazov1, Faisal Kunnathodi1, Mazhar Kanak2, Michael Lawrence1, Peter Kim3, Nicholas Onaca3, James Burdick4, Bashoo Naziruddin3, Marlon Levy3

1Islet Cell Laboratory, Baylor Research Institute, Dallas, TX, United States; 2Institute of Biomedical Studies, Baylor University, Waco, TX, United States; 3Baylor Annette C. and Harold C. Simmons Transplant Institute, Dallas, TX, United States; 4Baylor University Medical Center, Dallas, TX, United States.

Background: A major benefit of autologous islet transplantation (AIT) is to retain good glycemic control after pancreatectomy for patients with refractory chronic pancreatitis. Predictability of islets yields and post-transplant glycemic control, however, remain poorly established.

Methods: A total of 37 CP patients who underwent AIT were included. Average [± S.E.] age, body mass index and follow-up period were 42±2 years, 26.1±0.9 kg/m2 and 9±2 months, respectively. Stimulated serum C-peptide levels were measured pre-operatively with 75g oral glucose tolerance test. Pre-transplant predictors for post HbA1c were determined with multivariate linear regression model after univariate assessments where variables with p < 0.1 were selected (Table).

Table 1
Table 1
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Results: Final multivariate model identified female (β = -0.447, p = 0.01) and ΔC-peptide (β =-0.443, p = 0.01) as independent predictors for post-AIT HbA1c (R2 = 0.356, p = 0.01). ΔC-peptide > 5 showed 100% specificity for post HbA1c< 6.5. Ancillary analyses revealed that 63 and 88% of recipients achieved post HbA1c < 6.5 when islet dose > 3,300 and > 7,500 IEQ/kg were infused, respectively (Figure).


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Conclusions: Pre-transplant ΔC-peptide can predict post-AIT glycemic control. Results seem more favorable in the female patient and higher islet dose. Continued efforts at identify pre-transplant predictors of post transplant performance are indicated.

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346

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The Global Benefit of Islet Autotransplantation Following Total Pancreatectomy in Patient Quality of Life

Morihito Takita1, Rauf Shahbazov1, Faisal Kunnathodi1, Mazhar Kanak2, Michael Lawrence1, Peter Kim3, James Burdick4, Nicholas Onaca3, Bashoo Naziruddin3, Marlon Levy3

1Islet Cell Laboratory, Baylor Research Institute, Dallas, TX, United States; 2Institute of Biomedical Studies, Baylor University, Waco, TX, United States; 3Baylor Annette C. and Harold C. Simmons Transplant Institute, Dallas, TX, United States; 4Baylor University Medical Center, Dallas, TX, United States.

Background: Total pancreatectomy followed by autologous islet transplantation (TP-AIT) is a promising treatment option to retain endocrine function and improve patients’ quality of life (QoL) for patients with refractory chronic pancreatitis. The main purpose of TP-AIT includes elimination of intractable abdominal pain, however, little information on the detail benefit of TP-AITin QoL exists.

Methods: A total of 16 CP patients who underwent AIT were included. Average [± S.E.] age, body mass index and follow-up period were 40.6 ± 2.3 years, 26.1 ± 1.4 kg/m2 and 11 ± 1 months, respectively. Pre-AIT endocrine function was 6.0 ± 0.2 % of HbA1c and 5.8 ± 0.8 ng/mL of stimulated serum C-peptide. An average of 5,035 ± 638 IEQ/kg of patient body weight was transplanted. A general QoL form of the European Organization for Research and Treatment (EORTC)-C30 allows extensive study to evaluate detailed symptoms and was implemented at pre- and post-IAT with 3-month interval. The scores between pre- and the last post-AIT were compared with paired t test.

Results: All functional scales in EORIC-C30 were significantly improved after AIT including general health status scored as 32.8 ± 5.5 and 71.9 ± 4.4 at pre- and post-AIT, respectively (Fig. 1). All symptoms scales were also ameliorated after AIT; not only pain scale as well as scales on fatigue, nausea, vomiting, insomnia, appetite loss and constipation were significantly improved (Fig. 2).

FIGURE 1.
FIGURE 1.
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Higher scores indicate better QoL. Average ± S.E. of each score is shown. Abbreviations; GHS: Global health status, PF: Physical funtioning, RF: Role functioning, EF: Emotional funtioning, CF: Cognitive funtioning, SF: Social funtioning. *p<0.05, **p<0.01

FIGURE 2.
FIGURE 2.
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Lower scores indicate better QoL. Average ± S.E. of each score is shown. Abbreviations; FA: Fatigue, NV: Nausea and vomiting, PA: Pain, DY: Dyspnea, SL: Insomnia, AP: Appetite loss, CO: Constipation, Diarrhea: DI, FI: Financial difficulties. *p<0.05, **p<0.01

Conclusions: TP-AIT can enhance QoL in intractable chronic pancreatitis patients for generous symptoms as well as severe pain. These global QoL benefits of TP-AIT should be considered for medical decision making.

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347

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Postoperative Cholangitis and Liver Dysfunction After Total Pancreatectomy with Biliary Reconstruction Impair the Function of Autotransplanted Islets

Tatsuo Hata1, Naoaki Sakata1, Takeshi Aoki1, Gumpei Yoshimatsu1, Haruyuki Tsuchiya1, Hiroki Hayashi1, FuyuhikoMotoi1, Masafumi Goto2, Yu Katayose1,3, Shinichi Egawa1,4, Michiaki Unno1,3

1Hepato-Biliary-Pancreatic Surgery, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan; 2New Industry Creation Hatchery Center, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan; 3Integrated Surgery and Oncology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan; 4International Cooperation for Disaster Medicine, International Research Institute of Disaster Science, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.

Background: Total pancreatectomy (TP) with islet autotransplantation (IAT) is a good therapeutic option for benign pancreatic diseases that require TP. Although current TP with IAT has usually been performed by injection of the islets via the portal vein into the liver, in which the biliary tract had been reconstructed, the influence of postoperative cholangitis and liver dysfunction on transplanted islets in the liver remains unclear. We examined the influence of postoperative cholangitis on transplanted islets using case report and clinical data.

Methods: We assessed cases of postoperative cholangitis in which biliary reconstruction after pancreatectomy was performed concerning the incidence frequency, causes, treatment and prevention. Furthermore, we encountered a patient who had postoperative cholangitis after TP with IAT, and also evaluated the influence of cholangitis on glucose tolerance in that case.

Results: From 2009 to 2011, 15 (8.2%) patients who received biliary reconstruction, were diagnosed as postoperative cholangitis. All 23 cumulative cases received antibiotics therapy. Although all cases were administered ursodeoxycholic acid (UDCA) after recovering from the initial episode of cholangitis, 6 of the 15 patients developed cholangitis several times.

Regarding the case report, a 59-year-old man who had pancreatic arteriovenous malformation (AVM) received TP with IAT. He had good blood glucose control for 6 months, but cholangitis attacks were seen at 6 and 13 months after transplantation. After the initial episode of cholangitis, his hemoglobin A1c (HbA1c) gradually increased and temporal elevation of C-peptide was detected. After the second attack, his daily insulin use and HbA1c level increased. His diabetic condition was becoming worse with higher HbA1c, higher daily insulin use and significant lower serum C-peptide level at 2 years after transplantation.

Conclusion: Postoperative cholangitis after biliary reconstruction may be harmful to the function or viability of autotransplanted islets.

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348

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Islet Engraftment After TP-IAT may be Compromised by Postoperative Stressors

Krupa Desai1, Hongjun Wang1, Stefanie Owczarshi1, Katherine Morgan1, David Adams1

1Medical University of South Carolina, Charleston, SC, United States.

Aim: We aim to determine if islet function is affected adversely by major complications post-TPIAT by evaluating postoperative islet function.

Methods: A retrospective analysis of prospectively collected data base of patients post-TP-IAT was undertaken including only non-diabetic patients with at least 2,000 islet equivalents per kg(ieu/kg) harvest. Patients were divided in 2 groups: with or without Clavien-Dindo Grade 2 or higher complications. Also, patients were divided in with or without Clavien-Dindo Grade 3 or higher complication groups. Islet function at 6mo, 12mo, and 24months postoperative from TP-IAT was compared between the two groups in each Clavien-Dindo Grade group, utilizing the metric of islet harvest divided by insulin daily use per kg(patient_weight). T-test was used for statistical analysis (significant p-value<0.05).

Results: The cohort had 37 women and 8 men with a mean age of 44 years. 18 patients had Clavien-Dindo Grade 2 or higher complications and 27 had NO complications. 5 patients had Clavien-Dindo Grade 3 or higher complications and 27 had NO complications. For Grade 2 or higher complications, islet function at 6mo, 1yo, and 2yo were 12,480,564.77+/-23,117,590.87ieu-kg/u; 10,146,837.54+/-14,998,983.02ieu-kg/u; and 14,863,831.36+/-30,415,063.97ieu-kg/u, respectively. For Grade 3 or higher complications, islet function at 6mo, 1yo, and 2yo were 8,055,157.00+/-10,487,415.61ieu-kg/u; 1,780,292.00+/-1,146,947.19ieu-kg/u; and 24,922,719.93+/-39,169,182.24ieu-kg/u, respectively. Islet function at 6mo, 1yo, and 2yo for patients WITHOUT major complications were 5,077,232.25+/-9,169,583.39ieu-kg/u; 8,574,644.84+/-27,278,304.24ieu-kg/u; and 4,161,797.35+/-8,897,958.42ieu-kg/u, respectively.

Conclusions: No significant difference of islet function was seen between the Clavien-Dindo Grade 2 or higher complication group in comparison to the no complication group in chronic pancreatitis patients after TP-IAT. Also, no significant difference of islet function was seen between the Clavien-Dindo Grade 3 or higher complication group versus the no complication group. Postoperative physiological stressors due to postoperative complications may not impair islet engraftment; however additional data is needed to confirm this observation.

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349

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Intraportal Autoislet Cell Transplantation (IAT): Changes in Portal Pressure and Flow and Associated Clinical Outcomes

Ty Dunn1, Melena Bellin2, Josh Wilhelm2, A Balamurugan2, Greg Beilman1, Srinath Chinnakotla1, Selwyn Vickers1, David Sutherland1, Timothy Pruettt1

1Surgery, University of Minnesota, Minneapolis, MN, United States; 2Schulz Diabetes Institute, University of Minnesota, Minneapolis, MN, United States.

IAT tissue volume (TV) delivered into the portal venous system can be associated with acute portal hypertension. We analyzed the relationships between transplanted TV, portal vein (PV) pressure and PV flow, as well as the timing and nature of complications.

Methods: Between 7/2011 and 11/2012 we studied 39 total pancreatectomy IAT patients who underwent pre- and post-infusion portal pressure and blood flow measurements. TV (ml), islet equivalents/kg, PV pressure and PV flow (pre-infusion, peak post infusion, and post-infusion) were evaluated for the cohort. Patients were evaluated for PV thrombotic complications. The correlations between changes in PV flow, pressure and tissue volume were evaluated.

Results: Transplant data for the cohort are shown in Table 1. We found correlation between the delta PV pressure (peak minus baseline) and PV final flow rates (r=-0.67, p<0.0001), as well as TV and PV flow (r=-.063), p<0.0001). 3 patients had transient perterbations in flow; reversal of flow or no flow in the left PV, detected within a week of transplant. However, these resolved within a few weeks. We observed no episodes of main PV thrombosis.

TABLE 1
TABLE 1
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Conclusion: Autoislet cell transplant is associated with sometimes significant acute portal hypertension. PV pressures and TV both correlate with final PV flow. No serious/persistent complications were observed. Real-time PV flow measurements may be more useful than intermittent PV pressure measurements during Tx.

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350

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Novel Derived Measures from Pre-Transplant Continuous Glucose Monitoring for Autologous Islet Isolation Outcome Prediction.

Horacio Rilo1, Manuel Beltran del Rio1, Monica Delbridge1, Brianna Grigsby1, Abbas Rana1, Marian Porubsky, Carlos Galvani1, Angelika Gruessner1, Rainer Gruessner1

1Department of Surgery, University of Arizona, Tucson, AZ, United States.

We developed and tested a “length-of-curve” (LoC) derived measurement of continuous glucose monitoring (CGM) to complement standard analysis of deviations from normoglycemia to try to estimate islet yield pre-transplant in patients undergoing pancreatectomy and autologous islet cell transplant (TP/AIT) for the treatment of chronic pancreatitis. This novel quantity was meant to account for brief yet broad glycemic excursions that would otherwise not contribute a change in either the area-under-the-curve or the time-over or time-under analysis. LoC (λ) was calculated as the normalized CGM data curve length defined as the piecewise sum of the euclidean distance between data points as plotted in unitary intervals, divided by the number of intervals. This quantity was readily comparable for any two sequences since it was normalized and bound by the left by 1. We calculated the λ parameter of the pre-transplant CGM readings from the 50 patients who underwent pancreatectomy and auto islet transplant and then compared it to the Islet Equivalent yield (IEQ). While λ could not be directly linked to IEQ in a one-to-one way, it did provide a statistically significant (P ≈ 0.008) measurement of the maximum expected yield; patients whose CGM showed a low λ value had unpredictable outcomes, while patients that had high l values always had low yields, with similar results for the intermediate values. This behaviour of λ suggests that the parameter could be linked to the efficacy of a fraction of the factors that determine IEQ. This and other novel readings from conventional CGM may provide a valuable assessment tool in the pre-transplant evaluation of autologous islet transplant candidates.

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351

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Human Beta-Cell Lines as Tools to Study Immune Protection Strategies in Diabetes

Cornelis van der Torren1, Gaby Duinkerken1, Paul Czernichow2, Philippe Ravassard3, Raphael Scharfmann4, Bart Roep1

1Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands; 2Endocells, Paris, France; 3CNRS UMR 7725, Université Pierre et Marie Curie, Paris, France; 4Inserm-U845, Université Paris Descartes, Paris, France.

Genetically engineered human beta-cell lines provide a novel tool to study the pathogenesis of type 1 diabetes and to develop alternative beta-cell replacement therapy[1]. The immune system is essentially involved in type 1 diabetes and beta-cell transplantation. We assessed the interaction of human beta cell lines with the immune system to resolve their potential for immune intervention protocol studies.

Human pancreatic beta-cell lines derived from virally tranduced fetal beta-cells were co-cultured with alloreactive cytotoxic T-cells, proinflammatory cytokines from activated autoreactive Th1-cells or NK-cells in presence or absence of alloreactive antibodies. Beta-cell lines were pre-conditioned with Th1 cytokines or supernatant or high glucose to mimic inflammatory and hyperglycemia-stressed conditions. Beta-cell viability was assessed by chromium release and propidium iodides staining.

Low HLA expression protected human beta-cell lines from adaptive immune destruction, but was associated with direct killing by activated NK-cells. Autoreactive Th1 cell inflammation, rather than glucose stress, induced increased beta-cell apoptosis and upregulation of HLA, increasing their vulnerability to killing by alloreactive cytotoxic T-cells and alloreactive antibody mediated killing through NK-cells.

Genetically engineered human beta-cell lines are useful targets to study diverse immune responses that may be involved in the pathogenesis of T1D in humans. The use of beta-cells in immune assays may assist to evaluate novel immune intervention tools to interfere in type 1 diabetes pathogenesis and beta-cell protection protocols for transplantation.

Submitted work has been funded by the European Union; BetaCellTherapy, 241883 in the EC FP7 program

Reference:

[1] Ravassard P, Hazhouz Y, Pechberty S, Bricout-Neveu E, Armanet M, Czernichow P, Scharfmann R: A genetically engineered human pancreatic beta cell line exhibiting glucose-inducible insulin secretion. J Clin Invest 2011, 121: 3589-3597

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352

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Permanent Survival of Islet Allografts and Delayed Rejection of Heart and Skin Allografts Without Exogenous Immunosuppression by Targeting TNF-Receptor Associated Factor 2

Jeanette Villanueva1, Stacey N Walters1, Eliana Marino1, Mitsuru Saito2, Stephen Alexander2, Robert Brink1, Shane T Grey1

1Immunology, Garvan Institute of Medical Research, Sydney, Australia; 2Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney, Australia.

Introduction: The signaling molecule, TNF-receptor associated factor 2 (TRAF2) integrates multiple TNF-family signaling pathways critical for T cell activation suggesting TRAF2 may be a potential target for promoting allograft survival.

Aim: To define the role of TRAF2 in the alloimmune response using T cell specific TRAF2 knockout mice (TRAF2TKO).

Methods: Islet, heterotopic heart, and skin allograft survival (H-2d -> H-2b); MLR; signaling; T cell activation; effector function; T cell differentiation.

Results: 77% of TRAF2TKO mice permanently (>100d MST) accepted their islet allograft (n=13); with delayed rejection of heart (MST=9.5d versus control MST=7d; n=10) and skin allografts (MST=19d versus control MST=14d; n=8-11). Further, TRAF2TKO failed to proliferate in an in vitro MLR – thus TRAF2-deficiency impairs the alloimmune response. Loss of TRAF2 resulted in diminished T cell activation as shown by reduced proliferation and decreased expression of activation markers CD44 and CD25 and effector molecules granzyme B and IFN-gamma in response to alloantigen. Further, under Th1 polarizing conditions, TRAF2TKO CD4 T cells showed abnormal expression of the Th2 cytokine IL-13. Furthermore, TRAF2TKO CD4 T cells more readily converted to a Treg phenotype in the presence of TGF-beta; and in vivo TRAF2TKO mice harboured increased Tregs. TRAF2 deficiency delayed activation of TNF-induced JNK and NF-kB in T cells. Thus, TRAF2 coordinates extracellular signals from TNF-family ligands to generate productive and lineage defined T cells responses.

Conclusion: T cells require TRAF2 to differentiate into fully functional effectors, without which they skew towards a regulatory phenotype. Loss of this pathway leads to prolonged allograft survival.

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353

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Tolerance Induction for Islet Transplantation Using Donor Apoptotic Cells as Negative Vaccine

Xiaomin Zhang1, Shusen Wang4, Jane Bryant2, Taba Kheradmand2, Xunrong Luo1,2,3

1Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL, United States; 2Division of Nephrology and Hypertension, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, United States; 3Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL, United States; 4Department of Surgery, Nankai University Affiliated Hospital People’s Hospital of Tianjin, Tianjin, People’s Republic of China.

Background: We have previously developed a robust regimen for induction of donor-specific tolerance for allogeneic islet cell transplant using pre- and post-transplant infusions of donor splenocytes treated with a chemical cross-linker ethylcarbodiimide (ECDI-SPs). The current study examines several important parameters of this tolerance regimen to overcome potential limitations for its clinical translation.

Methods: Diabetic C57BL/6 mice were transplanted with BALB/c islets on day 0. 1×108 BALB/c ECDI-SPs or 1×109 polystyrene particles ECDI-fixed with BALB/c SP lysates were injected i.v. on day -7 and +1. Blood glucose levels were monitored for islet graft function.

Results: We show in using fresh donor splenocytes for ECDI cross-linking, as few as one tenth of the original dose (1×107 cells per dose) is sufficient and equally efficacious for tolerance induction to islet allograft. However, frozen donor splenocytes are significant compromised in their ability to induce tolerance, due largely to the presence of necrotic cells generated by the freeze/thaw process. Interestingly, donor antigens can also be delivered in the form of donor cell lysates ECDI-coupled to recipient splenocytes or inert PLG microparticles as carriers, and such indirect tolerance induce equally robust donor-specific tolerance. While the spleen appears to be the primary site for uptaking and processing of the infused ECDI-SPs, splenectomized recipients can be readily tolerized and appear to employ liver kupffer cells for uptaking and processing of the ECDI-SPs. In contrast to tolerance regimen using donor-specific transfusion with co-stimulation blockade, tolerance induced by ECDI-SPs is completely independent of CD47 expression on the infused donor cells. Test of commonly used immunosuppression reveals that not all immunosuppressive drugs are compatible with tolerance induction using ECDI-SPs.

Conclusion: We conclude that infusion of donor ECDI-SPs is a robust tolerance regimen that has a high potential for adaptation for clinical human islet transplantation. However, choice of combinatorial immunosuppression, if used, should be carefully selected.

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354

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Biotin-PEG-SVA as a more Effective Linking Molecule in Comparison to Biotin-PEG-NHS for Coating of Pancreatic Islets with Regulatory T Cells (Tregs) to Create Local Immunoprotection - Optimization of the Method

Karolina Golab1, Seda Kizilel2, Tugba Bal2, Manami Hara3, Mark Zielinski3, Xiao-Jun Wang1, Jakub Grzanka1, Ling-Jia Wang1, Olivia Cochet1, Martin Tibudan1, Piotr Witkowski1

1Department of Surgery, University of Chicago, Chicago, IL, United States 2Department of Chemical and Biological Engineering, College of Engineering, Koç University, Istanbul, Turkey; 3Department of Medicine, University of Chicago, Chicago, IL, United States.

Introduction: T regulatory cells (Tregs) can potentially serve as an effective immunosuppressive therapy in transplantation. We have shown that Tregs can be attached to the surface of pancreatic islets providing local immunoprotection [1]. Further optimization of the method can improve coating efficiency, which may prolong graft survival. In this study, we compared two molecules to attach Tregs to pancreatic isletsin order to increase number of Tregs attached to islets surface without compromising islets viability and function.

Method: Cell surface of human Tregs and pancreatic islets was modified using biotin-polyethylene glycol (-PEG-) -N-hydroxylsuccinimide(-NHS) or -succinimidylvalericacid ester (-SVA) (1mg/ml and 0.6 mg/ml was used for Tregs and islets, respectively). Then, islets were incubated for 15 minutes in 37°C with 1mg/ml of streptavidin as islet/Tregs binding molecule. Subsequently 150 islets were combined with 50×106Tregs and incubated overnight. To check coating effectiveness, Tregs were stained with CellTracker™ CM-DiL dye and visualized using Laser Scanning Confocal Microscope. The number of Tregs attached per islets surface area was analyzed by Imaris software. The effect of coating on islet functionality was determined using Glucose-Stimulated Insulin Response (GSIR) assay.

Results: Coating procedure with biotin-PEG-SVA allowed attaching 40% more Tregs per 1 μm2 of islets surface. While viability was comparable, function of the islets after coating using biotin-PEG-SVA molecule was better preserved than with NHS molecule. GSIR was 62% higher for islets coated with biotin-PEG-SVA than biotin-PEG-NHS, and as high as in unmodified islets controls.

Conclusion: Coating of islets with Tregs using biotin-PEG-SVA improves effectiveness with better preservation of the islet function. Improvement of the method of coating pancreatic islets with Tregs, could further facilitate the effectiveness of this novel immunoprotective approach and translation into clinical settings.

Reference:

[1] Marek N, Krzystyniak A, Ergenc I, Cochet O, Misawa R, Wang LJ, Golab K, Wang X, Kilimnik G, Hara M, Kizilel S, Trzonkowski P, Millis JM, Witkowski P: Coating human pancreatic islets with CD4(+)CD25(high)CD127(-) regulatory T cells as a novel approach for the local immunoprotection.

Ann Surg.2011 Sep;254(3):512-8; discussion 518-9. doi: 10.1097/SLA.0b013e31822c9ca7.

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Access to Islet Transplantation at a Single Health Service Funded National Centre

Shareen Forbes, Kirsty Duncan, Janet Barclay, Debbie Anderson, Tammie McGilvray, Neil McGowan, John Casey

1Diabetes and Endocrinology, University of Edinburgh, Edinburgh, United Kingdom; 2Transplant Unit, Royal Infirmary Edinburgh, Edinburgh, United Kingdom; 3SNBTS, Edinburgh, United Kingdom.

Introduction: Scotland with a population of 5 million, has > 28,000 people with Type 1 diabetes, 10% have severely impaired awareness of hypoglycaemia (IAH) and may be eligible for islet transplantation. Our aims were to observe referral patterns, demographics and personal characteristics of subjects referred to our national islet transplant programme and to record waiting times from referral to listing for islet transplantation.

Methods: Patients referred for transplant from September 2009 until April 2013 were included. Data was extracted from a local database, electronic medical records and patient case notes. The referring clinician and distance of the referral centre to our transplant centre was recorded. The percentage of patients on insulin pump therapy versus basal bolus regimens were noted along with socioeconomic status, employment and driving status. Waiting times from referral to listing were recorded.

Results: 77 patients were assessed from September 2009 until 29 April 2013. 91% of these referrals were from Diabetologists. Distance of referring centre to transplant centre was (median(range)): 61.8 (3-173) miles. 44% were on insulin pump therapy and 66% were on basal bolus regimens. Most subjects referred were in the lower socioeconomic categories (patients with DEPCAT scores 1-3: 38%; DEPCAT scores 4-7: 62%). Only 33% of subjects were in full-time employment and 29% were currently driving.

Waiting time from referral to listing was 124 (80-275) days in those subjects with no other contraindications to islet transplantation. Delays in listing included: 13% new commencement of insulin pump, 13% cardiovascular investigations, 9% active eye disease, 5% renal dysfunction and 4% operable carcinoma. Of the patients assessed 25% didn’t meet the criteria for islet cell transplantation and 21% didn’t progress with the assessment through choice.

Of our 77 patients, 30% (23) were listed for transplant and 48% (12) have been transplanted so far. 9 recipients have had two islet grafts and 3 recipients have had one islet graft.

Conclusion: Islet transplantation in Scotland (as in the rest of the UK) is a health service funded programme. This study demonstrates that central funding of islet transplantation drives the referral and listing of suitable patients and allows equity of access of the referral population for islet transplantation.

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Direct Immune Cell-Cell Contact During Immune Responses in Non-Lymphoid Target Tissues

Midhat H. Abdulreda1, Jason Miska2, Priyadharshini Devarajan2, Jen Bon Lui2, Jun Suzuki4, Antonello Pileggi1,2, Per-Olof Berggren1,3, Zhibin Chen2

1Diabetes Research Institute, University of Miami, Miami, FL, United States; 2Microbiology and Immunology Department, University of Miami, Miami, FL, United States; 3The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden; 4Department of Rheumatology and Internal Medicine, Juntendo University school of Medicine, Tokyo, Japan.

Intravital imaging is revealing immunological phenomena not predicted by in vitro studies. Pioneering in vivo imaging studies in lymph nodes have established cellular mechanisms crucial during immune defense of the organism. Such findings have primed therapies targeted at immune cells in the lymph nodes. However, immune responses in the non-lymphoid target tissues offer equally valuable therapeutic targets. Therefore, a comprehensive understanding of immune responses during development of autoimmune diabetes is critical for diabetes treatment. Here we studied noninvasively the in vivo behavior of antigen-specific Teff cells against beta-cell in intraocular pancreatic islet grafts. Our studies suggest that antigen-specific CD4+ Tcells could kill target beta-cell through direct long-lasting contacts, a feature characteristic of cytotoxic CD8+ Teff cells (CTL). Interestingly, islet grafts lacking the specific-antigen that were “conjoined” to antigen-bearing islets were not subject to “bystander” damage; instead, they remarkably increased in size as the immune attack on the apposed antigen-bearing islets ensued. Our results also show that Foxp3+ regulatory T (Treg) cells were constantly present within antigen-bearing pancreatic islets and persistently contacted antigen-specific Teff cells, suggesting that suppression of effector immune function within target tissue may depend on local Treg - Teff interactions. This was further supported by selective depletion of Diphtheria toxin-sensitive Tregs which resulted in destruction of previously protected islet mass expressing the specific antigen. Together, our findings demonstrate the importance of contact-dependent killing by antigen-specific CD4+ Teff cells, and highlight an unprecedented potential for “rapid” islet regeneration under specific conditions. Our results also show the potential role of direct Treg - Teff interactions within non-lymphoid target tissues in local immune modulation and tolerance induction.

This work was mainly supported by funds from the National Institute of Health (DP3DK085696/ F32DK083226) and the Diabetes Research Institute Foundation.

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Cyanidin-3-O-Glucoside Enhances the Viability and Function of Islet Transplant in Mice

Zhihao (James) Xu2, Baoyou Xu1, Ping Wu1, Yang Bin3, Kunsong Chen4, Yulian Wu3, Ray Rajotte1, Gina Rayat1

1Surgery, University of Alberta, Edmonton, AB, Canada; 2University of British Columbia, Vancouver, BC, Canada; 3Surgery, 2nd Affiliated Hospital of Medical College Zhejiang University, Hangzhou, People’s Republic of China; 4Fruit Quality Biology, Sci-Tech Division of Zhejiang University, Hangzhou, People’s Republic of China.

The widespread application of islet transplantation is hindered by the loss of islets during isolation and immediately after transplantation. Since oxidative stress and hypoxia are contributors to islet damage during isolation and transplantation, we determined if cyanidin-3-O-glucoside (C3G), a major type of anthocyanin found in the extracts of Chinese bayberry, Myrica rubra, could enhance viability and function of the islet transplant. Streptozotocin-induced diabetic B6 mice (n=5 per group) were transplanted with 100, 200 or 400 untreated or C3G-treated syngeneic B6 mouse islets under the kidney capsule or into the portal vein. Treated islets were incubated with 1.0μM C3G for 24 hours prior to transplantation. Blood glucose levels of recipient mice were monitored once a week for >7 weeks post-transplantation. Recipients of 100 untreated or C3G-treated islets remained diabetic at 7 weeks post-transplantation. However, the mean blood glucose levels (MBGL) of mice that received treated islets were 12.2 and 13.2 mmol/L compared to 24.2 and 24.1 mmol/L in mice that were transplanted with untreated islets under the kidney capsule or into the portal vein, respectively. Recipients of 200 treated islets transplanted under the kidney capsule achieved normoglycemia within 2 weeks post-transplantation while recipients of 200 untreated islets became normoglycemic within 4 weeks post-transplantation. Mice that were transplanted with 200 treated or untreated islets into the portal vein remained diabetic at 8 weeks post-transplantation (MBGL = 12.3 mmol/L and 22.1 mmol/L, respectively). Recipients of 400 treated or untreated islets transplanted under the kidney capsule achieved normoglycemia within 1 week and 3 weeks post-transplantation, respectively while those mice that were transplanted with treated or untreated islets into the portal vein achieved normoglycemia within 1 week and two weeks post-transplantation, respectively. Our data show that C3G enhances the viability and function of mouse islets transplanted under the kidney capsule or into the portal vein.

Faculty of Medicine and Dentistry/VP-Provost Office, University of Alberta

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Assessment of Human Islet Grafts in Frozen Sections of Recipient Liver for Molecular Analysis

Craig Hasilo1,2, Sarita Negi1,2, Marco Gasparrini1,2, Steven Paraskevas1,2

1Surgery, McGill University, Montreal, QC, Canada; 2Human Islet Transplant Laboratory, McGill University Health Centre, Montreal, QC, Canada.

Post-transplant islet graft monitoring is hampered by a lack of efficient methods to locate and analyze islets in vivo. We evaluated histological methods to rapidly locate islets within the liver parenchyma post-transplant using several staining strategies, prior to analysis using laser capture microdissection.

Methods: Human islets were isolated from brain dead, multi-organ donor pancreases under expressed research consent, at the MUHC Islet Transplant Laboratory. The average yield of islets from 13 human pancreases was 258 153±40 697 islet equivalents (IE) and 3 246±1 822 IE/g pancreas (84.5±8.6% and 95±5% average purity and viability, respectively; mean±SD). Three days post-isolation, islets were collected, quantified, assessed for purity (dithizone) and viability (SYTO-13 and ethidium bromide) for transplantation. Athymic nu/nu mice were rendered diabetic by streptozotocin i.p. injection (200mg/kg) and maintained with sustained release insulin pellets, until transplantation. Intraportal islet transplantation of ∼1000 IE/mouse was performed via the ileocecal vein. Frozen sections of liver containing human islets were prepared from specimens collected on day 0, 7 and 30 post-transplant. Every twentieth slide from serial sectioned liver was stained using a rapid, abbreviated protocol to determine if islets were present. Sections were fixed and stained for 2 minutes with either an anti-human insulin FITC-conjugated primary antibody, Newport Green, or dithizone (diphenylthiocarbazone).

Results: Islets were found mostly towards the liver periphery. Islets were readily localized using each technique. Dithizone, however, had the advantage of avoiding the background fluorescence present in liver specimens, but a more faint appearance in the 10μm-thick sections.

Conclusions: FITC-conjugated human anti-insulin, Newport Green and dithizone are all good candidates for a rapid, abbreviated islet staining protocol to evaluate human islet grafts in vivo.

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Expanding the Donor Pool in Islet Transplantation: Age Impacts Parameters Regulating Glucose Metabolism.

Afaf Sahraoui1, Merete Hoeyem1, Maria Soerhede Winzell2, Dave Smith2, Olle Korsgren3, Aksel Foss1, Hanne Scholz1

1Section for Transplantation Surgery and Institute for Surgical Research, Oslo University Hospital, Oslo, Norway; 2CVMD iMed Metabolism, AstraZeneca R&D, Mölndal, Sweden; 3Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.

Background: Donor shortage is a serious problem in transplantation medicine. Expanding the donor pool is therefore essential, especially in islet transplantation where insulin independence rarely is achieved after a single transplantation. Additionally, many centers have a strict cut-off limits concerning donor age for islets (45 years), resulting in fewer eligible donors. In this study we investigate the possibility of expanding the donor pool in a novel experimental animal model.

Methods: Alloxan-induced diabetic NMRI nu/nu mice were cured with a therapeutic mouse-islet graft under the left kidney capsule. Thereafter (2-4 weeks), the cured normoglycemic mice were transplanted with a second graft under the right kidney capsule consisting of a minimal mass of human islets (250 islets) from 8 independent donors. The donors were divided in two groups: above 60 years and below 60 years. After a 14-days recovery, left sided nephrectomy was performed and the mice now left with only minimal human islet grafts, were followed for 15 days before harvesting. Blood samples were drawn at the day of harvest for analysis of metabolic parameters (insulin, c-peptide, pro-insulin, active GLP-1 and glucagon). An oral glucose tolerance test (OGTT) was performed at day 12 during follow-up.

Results: Plasma levels of insulin, c-peptide, pro-insulin, active GLP-1, and glucagon were significantly decreased in mice transplanted with islets from donors > 60 years compared to donors < 60 years (p<0.05). AUC for glucose after the OGTT was significantly higher for donors over 60 years compared to those younger (p<0.05). There were no significant differences between the two groups with respect to non-fasting blood glucose during following up.

Conclusion: Engrafted human islets from donors < 60 years significantly improves the metabolic outcome in a novel diabetic mice model. These data are supportive of expanding the donor pool up to 60 years. Islets from donors older than 60 years are less capable of maintaining insulin independence.

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Optimizing the Islet Cell Counter-2 for IEQ Assessment

Jason Doppenberg1, Marten Engelse1, Eelco de Koning1,2

1LUMC, Leiden, Netherlands; 2Hubrecht Institute, Utrecht, Netherlands.

Introduction: Assessment of the islet equivalent (IEQ) is an important parameter after islet isolation and during culture. For this purpose, and for comparing data between centers, a quick, accurate, reliable and reproducible method of quantification of IEQ is desired. While current automated computer-assisted image analysis techniques of calculating IEQ are robust and accurate, they are also cumbersome and time-consuming [1,2,3,4]. To overcome these shortcomings, we optimized the Biorep® Islet Cell Counter (ICC)-2 method.

Materials: Polyethylene microspheres of 125-150 μm diameter of differing colors were used to calibrate and adjust the ICC-2 settings. Cultured human isolated islets (n=3) were stained in 100 μl concentrated dithizone (DTZ) for 1 minute before diluting the sample to 300 μl with Ringer acetate and were used for IEQ calculations and size and purity estimations.

Results: DTZ-red microspheres proved to be a suitable islet substitute achieving 91.2% ±5.7% (standard deviation, n=5) purity when fully segmented with optimized settings and without DTZ in the medium. Furthermore, 94% ±2% of the microspheres were placed in the correct size category. To investigate whether DTZ concentration interfered with proper IEQ quantification, the concentration was increased in the medium from 0.08 mg/ml to 0.17 mg/ml to 0.33 mg/ml. The calculated purity of non-DTZ colored (orange) beads rose accordingly from 2.8% ±0.9% to 6.5% ±3.0% to 19.1% ±4.3%. The purity and size estimations of islet samples were reproducible, with a pooled variance of 2.1%. Strong variances were observed in batches of DTZ (n=3), staining concentration 0.75 mg/ml, 0.34 mg/ml, 0.30 mg/ml); while islets were easily distinguishable visually, decreasing calculations from 24.1% ±6% to 3.5% ±2% were observed. Markedly, the time required for each new assessment was approximately 8 seconds.

Conclusion: By using colored plastic beads, we optimized the ICC-2 counting method. Tests with isolated human islets could be performed rapidly with a high degree of accuracy. Further experimentation will determine whether the ICC-2 could be a practical tool in an islet isolation and transplantation setting.

Reference:

  • [1] Girman, P., Berkova, Z., & Dobolilova, E. How to Use Image Analysis for Islet Counting. The Rev, 2008, 5(1), 38–46.
  • [2] Friberg, A. S., Brandhorst, H., Buchwald, P., Goto, M., Ricordi, C., Brandhorst, D., & Korsgren, O. Quantification of the islet product: presentation of a standardized current good manufacturing practices compliant system with minimal variability. Transplantation, 2011, 91(6), 677–83.
  • [3] Pisania, A., Papas, K. K., Powers, D. E., Rappel, M. J., Omer, A., Bonner-Weir, S., Weir, G. C., et al. Enumeration of islets by nuclei counting and light microscopic analysis. Laboratory investigation; a journal of technical methods and pathology, 2010, 90(11), 1676–86.
  • [4] Niclauss, N., Sgroi, A., Morel, P., Baertschiger, R., Armanet, M., Wojtusciszyn, A., Parnaud, G., et al. Computer-assisted digital image analysis to quantify the mass and purity of isolated human islets before transplantation. Transplantation, 2008, 86(11), 1603–9.
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A New Approach to Microencapsulation of Pancreatic Islets within Stable Ultra-Thin Membranes for Cell Transplantation

Yuji Teramura1, Madoka Takai1, Bo Nilsson2

1Department of Bioengineering, The University of Tokyo, Tokyo, Japan; 2Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

The encapsulation of islets of Langerhans (islets) and insulin-secreting cells within a semi-permeable membrane has been suggested as a safe and simple technique for islet transplantation to attenuate early graft loss and avoid immunosuppressive therapy. The total volume of these implants tends, however, to increase upon encapsulation of the islets and cells within the polymer membrane, limiting transport between encapsulated cells and the surrounding tissue. Ultra-thin membranes could potentially overcome these diffusion limitations to provide for clinically applicable implants. Here we propose a novel method to encapsulate islets and cells within a stable ultra-thin polymer membrane using poly(ethylene glycol)-conjugated phospholipid bearing a maleimide group (Mal-PEG-lipids) and multiple interactive polymers (e.g., 4-arm PEG-Mal and 8-arm PEG-SH). When Mal-PEG-lipids were added to islet and cell suspensions, spontaneous incorporation into a cell surface occurred from the micelles at an equilibrium state. The addition of 4-arm PEG-Mal and 8-arm PEG-SH to the mixture induced a substantial increase in the membrane thickness because a number of Mal-PEG-lipid micelles were involved in the membrane formation at the micrometer level. No appreciable increase in islet volume was observed after microencapsulation by this method. Microencapsulation of islets with the polymer membranes, which showed semi-permeability, did not impair insulin release in response to glucose stimulation, even after 7 days. The polymer membrane structure surrounding the islets and cells was well maintained for at least 30 days. We also evaluated the immunoreactive properties of the membrane by using tests of serum component permeation, hemolytic assays of the complement alternative pathway, and blood compatibility tests of islets in contact with human whole blood.The membranes formed showed less thrombogenicityand inhibited complement attack when exposed to human whole blood and serum. This cell surface modification significantly improves the graft survival after transplantation.

Reference:

Teramura Y, Oommen OP, Olerud J, Hilborn J, Nilsson B: Microencapsulation of cells, including islets, within stable ultra-thin membranes of maleimide-conjugated PEG-lipid with multifunctional crosslinkers. Biomaterials, 2013, 34: 2683-2693.


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Xenotransplantation of Encapsulated Co-Aggregates of Sertoli Cells and Islet Cells

Rei Kuwabara1, Naohiro Takemoto1, HIroo Iwata1

1Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

Transplantation therapy utilizing isolated, donor islets of Langerhans (islets) has been employed successfully to treat insulin-dependent diabetes mellitus. However, it remains an experimental procedure, and shortage of human donor is one of major obstacles to overcome. For improvement of a xenograft survival, we examined co-transplantation of islets with Sertoli cells which are known to have immunosuppressive ability. Co-aggregates of Sertoli cells and islet cells from Wistar rat that were prepared by the hanging drop method were encapsulated with agarose gel. In the agarose-encapsulated aggregates, the sertoli cells occupied the core part while islet cells engulfed the sertoli core aggregate (Figure 1). The Sertoli portion continuously released activin, and islet cells could regulate insulin release in response to glucose concentration changes, indicating that the encapsulated co-aggregates well maintained the functions of both Sertoli and islet cells. Agarose-encapsulated aggregates from Wistar rat were transplanted into each diabetic BALB/c mouse intraperitoneally, and their blood glucose levels were monitored. Insulin levels remained low after transplantation in the recipients, whereas sharply increased in recipient mice with agarose-encapsulated naïve islets.

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A Macroencapsulation Device to Protect Allogeneic Cells from Alloimmune-Mediated Rejection

Gaetano Faleo1, Karim Lee1, Vinh Nguyen1, Qizhi Tang1

1Surgery, UCSF, San Francisco, CA, United States.

Embryonic-stem-cell (ESC)-derived islets are a renewable source of tissue and can potentially overcome the scarcity of islet donors for transplantation. However, the immunogenicity of ESC-derived grafts and the potential for ESC-derived teratomas to form are barriers to clinical application. Encapsulation is a potential solution to both of these issues.

To determine the immunogenicity of ESC-derived islets, mouse embryonic pancreata (EP) were used as a source of pancreatic progenitor cells. EP were isolated from BALB/c embryos and transplanted under the kidney capsule of C57BL/6 recipients. Allogeneic EP grafts triggered proliferation of BALB/c-reactive CD4 T cells activated through the indirect but not the direct pathway and prompted CD4 and CD8 T cell infiltration to the grafts and graft rejection.

We then tested the ability of an encapsulation device to prevent T cell priming and destruction of islet grafts. The encapsulation device abolished proliferation of alloreactive CD4+ T cell inducted by allogeneic splenocytes. The device effectively blocked allogeneic MIN6 beta cell rejection in pre-sensitized BALB/c recipients. We also transplanted allogeneic islet progenitors expressing luciferase under the control of mouse insulin 1 promoter to determine if the device can preserve beta cell function in fully sensitized recipients. Luciferase intensity progressively decreased in the unencapsulated group, but the signal intensity of the encapsulated graft steadily increased. Lastly, we transplanted allogeneic MIN6 cells into spontaneously diabetic NOD to test whether the device can protect the graft against both alloimmune and recurring autoimmune responses. The encapsulated graft was preserved and within 2 to 3 weeks the encapsulated MIN6 cells were able to re-establish normoglycemia.

Together, our results demonstrate that ESC-derived islets are immunogenic. However, an encapsulation device can effectively prevent alloimmune priming and rejection of islet grafts in fully sensitized and autoimmune hosts.

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Skeletal Muscle Tissue as a Potential Transplantation Site of Pancreatic Islets Encapsulated in Ca2+/Ba2+ Alginate Microbeads

Matt Bochenek1, Meirigeng Qi1, Enza Marchese1, Maureen Davis1, Yong Wang1, Jose Oberholzer1

1Surgery/Transplant, University of Illinois at Chicago, Chicago, IL, United States.

Background: Transplantation of isolated pancreatic islets can render type I diabetic patients insulin-independent, though at the cost of systemic immunosuppression with potential side effects. Immunoprotection of islets by microencapsulation could circumvent this problem. Commonly, the microbeads containing islets are transplanted into the intraperitoneal (IP) cavity because of the large volume available for the islet graft. Nonetheless, the IP is a hypoxic environment with large diffusion distances and may not be ideal for encapsulated islet function. Skeletal muscle tissue is highly vascularized with a higher oxygen tension and shorter diffusion distances. Muscle tissue, therefore, may be a superior transplantation site for encapsulated islets in terms of islet function and response time.

Materials and Methods: Lewis rat leg muscles (gracilis major) were transplanted with Ca2+/Ba2+ alginate microbeads containing isogeneic islets (n=9), allogeneic islets (n=6), and empty microbeads as a control (n=9). The microbeads were harvested at 2 and 8 weeks to analyze islet viability and to histologically assess the immune response.

Results: Empty microbeads and isogeneic islet microbeads showed a minimum immune response and 5% microbead coverage by pericapsular tissue overgrowth at both 2 and 8 weeks. The isogeneic islets remained viable after 2 and 8 weeks. On the other hand, allogeneic islet microbeads exhibited 90% microbead coverage by pericapsular tissue overgrowth with no viable allogeneic islets after 8 weeks.

Conclusion: Skeletal muscle tissue may represent a potential site for encapsulated islet transplantation; however, the properties of the present microbeads may have to be modified to protect allogeneic tissue from rejection.

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Long-term Survival of Ultrathin Coated Islets within a Fully Mismatched Allograft Murine Model

Hernan Rengifo1, Jaime Giraldo1, Cherie Stabler1

1Biomedical Engineering/Diabetes Research Institute, cstabler@med.miami.edu, Miami, FL, United States.

Clinical islet transplantation (CIT) has emerged as a promising treatment for type 1 diabetes mellitus (T1DM); however, the anti-rejection drug regimen necessary to mitigate allograft islet rejection impose a significant burden to the patient. The use of polymeric coatings to immunocamouflage the transplant from host immune attack has great potential. We have recently developed alginate and poly(ethylene glycol) (PEG)-based polymers, functionalized with azide and phosphine, respectively, which form spontaneous and chemoselective cross-links via the bio-orthogonal Staudinger ligation scheme [1-3]. Herein, we explored the utility of these polymers to form immunoprotective, covalently stabilized, ultrathin coatings on murine islets. Resulting coatings were nontoxic and resulted in unimpeded islet function. Transplantation of coated BALB/c (H-2d) islets into streptozotozin-induced diabetic C57BL/6 (H-2b) resulted in a significant subset of animals (57%) exhibiting long-term (> 100 d) normoglycemia (Figure 1). Control islets rejected after 15 d (+/- 9 d). Results illustrate the ability of these chemoselectively functionalized polymers to form ultrathin coatings on islets, with no detrimental effect to the underlying cells, with resulting coatings exhibiting significant protective effects in an allograft murine model.

Reference:

  • [1] Gattas-Asfura KM, Stabler CL. Chemoselective cross-linking and functionalization of alginate via Staudinger ligation. Biomacromolecules. 2009;10:3122-9.
  • [2] Gattas-Asfura KM, Fraker CA, Stabler C. Covalent stabilization of alginate hydrogel beads via Staudinger ligation: Assessment of poly(ethylene glycol) and alginate cross-linkers. J Biomed Mater Res A. 2011;99:47-57.
  • [3] Hall KK, Gattas-Asfura KM, Stabler CL. Microencapsulation of islets within alginate/poly(ethylene glycol) gels cross-linked via Staudinger ligation. Acta Biomater. 2011;7:614-24.

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Design of a Biocompatible Extracorporeal Device for Applying Islets of Langerhans as a Bio-Artificial Pancreas

Sana Asif1,1, Yuji Teramura1, Jan Bohlin2, Jöns Hilborn2, Peetra Magnusson1, Bo Nilsson1

1Department of immunology, genetics & pathology (IGP), Rudbeck lab, Uppsala University, Uppsala, Sweden; 2Department of chemistry, Uppsala University, Uppsala, Sweden.

Introduction: Islet transplantation is a treatment option for individuals with diabetes. However, there are still challenges hampering success of this procedure such as shortage of donor organs, instant blood mediated inflammatory reaction (IBMIR), gradual loss of transplanted islets overtime and chronic need of immunosuppressives. Immunoisolation of islets with a semi permeable membrane is a promising alternative to conventional strategies in islet transplantation. Herein we have designed an extracorporeal device for islet transplantation aiming initially for extracorporel treatment. The islets are kept behind a semi permeable membrane in a device, which is perfused with blood. The efficiency of device has been evaluated by blood exposure in vitro.

Methods: Human islets (3000 IEQ) were added into the device and sealed with a polypropylene membrane (0,22μm). All parts of the device including the membrane were heparinized according to manufacture’s instructions (Corline Systems, Uppsala; Sweden). Human whole blood was continuously circulated through inlet and outlet ports and the membrane was exposed to blood (n=6). After 24 hours, samples were collected and accessed for hemocompatibility and islet functionality. A rotating falcon tube with 3000 IEQ islets in direct blood contact served as control. All data are presented as (mean±SEM) and were analyzed by Wilcoxon’s test.

Results: After 24 hours, a significant maintenance of platelet count (258.3±28 10*9/L vs. 7.2±1.8 10*9/L, p < 0.05), activation of complement as accessed by C3a and C5b-9 levels (p < 0.05) and coagulation, measured by Thrombin-anti thrombin ELISA, (p < 0.05) was observed in the device in comparison to control. Blood glucose levels were normoglycemic (3.1±0.1 mmol/L vs. 0.3±0.2 mmol/L, p < 0,05) and histological analysis showed morphologically intact islets in the device whereas in control islets were destroyed and clotting was observed.

Conclusion: Our device is biocompatible and provides protection against IBMIR and immunological reactions.

This study is supported by grants from the Swedish research council and Stem Therapy.

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Microwell Scaffolds for Extrahepatic Transplantation of Islets of Langerhans

Mijke Buitinga1, Marten Engelse3, Roman Trückenmuller2, Lorenzo Moroni2, Eelco de Koning3, Marcel Karperien1, Aart van Apeldoorn1

1Developmental Bioengineering, University of Twente, Enschede, Netherlands; 2Department of Tissue regeneration, University of Twente, Enschede, Netherlands; 3Department of Nephrology, Leiden University Medical Center, Leiden, Netherlands.

Intra hepatic allogeneic islet transplantation has the potential to restore normoglycemia in patients with type 1 diabetes. Nevertheless, the suboptimal microenvironment for islets in the liver is involved in the progressive islet dysfunction that is often observed post-transplantation. After transplantation islets are exposed to biomechanical stress, high concentrations of immunosuppressive drugs, hypoxia due to disrupted vasculature and an IBMIR. This study validates a novel microwell scaffold platform to be used for the extrahepatic transplantation of islets of Langerhans, which could potentially overcome these disadvantages. Scaffolds were fabricated from either a thin polymer film or an electrospun mesh of poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) block copolymer (composition: 4000PEOT30PBT70) and were imprinted with microwells, ~400 μm in diameter and ~350 μm in depth. The water contact angle and water uptake were 39 ± 2° and 52.1 ± 4.0 wt%, respectively. The glucose flux through electrospun scaffolds was three times higher than that for thin film scaffolds, indicating enhanced nutrient diffusion. Subsequent in vitro experiments showed that human islets cultured in microwell scaffolds preserved their native morphology, and insulin secretion was comparable to that of free-floating control islets, indicating that this novel scaffold platform does not hamper islet functionality. Our results reveal that the microwell scaffold platform prevents islet aggregation by the confinement of individual islets, preserves the islet’s native rounded morphology, and provides a protective environment without impairing islet functionality, making it a promising platform for use in extrahepatic islet transplantation.

This research is part of the Diabetes Cell Therapy Initiative (DCTI) and funded by the Dutch Diabetes fund and the ministry of economical affairs (FES program) of the Netherlands.

FIGURE 1.
FIGURE 1.
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Hybrid Polymer Hydrogel Scaffolds for the Improvement of Islets of Langerhans Revascularization After Transplantation

Giulia Marchioli1,2, Andrea Di Luca2, Marten Engelse3, Eelco De Koning3, Clemens Van Blitterswijk2, Marcel Karperien1, Aart Van Apeldoorn1, Lorenzo Moroni2

1Developmental BioEngineering, University of Twente, Enschede, Netherlands; 2Tissue Regeneration, University of Twente, Enschede, Netherlands; 3Department of Nephrology, Leiden University Medical Center, Leiden, Netherlands.

Although intra–hepatic injection of allogeneic islets of Langerhans is a promising treatment for type 1 diabetes‚ islets scarce functionality and survival rate after transplantation account for an overall low efficiency of the procedure.

The use of scaffolds is a promising strategy to create a more favorable environment for islets to reside in and ultimately improve the transplantation outcome in extra-hepatic strategies. The major challenge for scaffold–aided islets transplantation is to fabricate a scaffold design that can provide sufficient nutrients and oxygen supply to the islets, also in the most inner part of the scaffold.

This study presents an innovative strategy for an extra–hepatic islets transplantation platform consisting of an hybrid, three–dimensional polymer/hydrogel scaffold. A three-dimensional polycaprolactone (PCL) scaffold is fabricated using a 3D fiber deposition machine. The surface of the printed scaffold is functionalized by immobilizing heparin via EDC/NHS chemistry. Heparin can be exploited to bind and release vascular endothelial growth factor (VEGF) in a controlled manner to attract and stimulate the proliferation of endothelial cells and blood vessels and eventually accelerate the revascularization of the construct. Islets are embedded in the core of the scaffold in a 2% wt/v alginate hydrogel (Figure 1).

Results showed that heparin immobilization can be performed with a good yield and it improves the amount of VEGF retained by the construct, up to 3.6 folds compared to untreated PCL scaffolds. Heparin/VEGF functionalization stimulates human umbilical vein endothelial cells (HUVEC) migration towards the scaffold and into the pores when compared to untreated scaffolds. Islets embedded in the alginate core of the construct showed full functional response to glucose stimuli, comparable to free–floating islets, up to 7 days in culture.

This research is part of the Diabetes Cell Therapy Initiative (DCTI) and funded by the Dutch Diabetes fund and the Ministry of economical affairs (FES program) of the Netherlands.The work was also funded by the Dutch Technology foundation STW (OTP 11135).


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BIOSID Project-a Bioartificial Pancreas to Treat Type 1 Diabetes: Optimization of Cell Survival and Function in Preclinical and Clinical Phases

Richard BOUAOUN1, Séverine SIGRIST1

1DEFYMED, Strasbourg, France.

MAILPAN (MAcroencapsulation of PANcreatic Islets) is a prototype of bioartificial pancreas usable in the human designed to treat type 1 diabetic patients. The prototype was developed along different stages since 1996 and led to the creation of the SME called Defymed in 2011. Next step is now to bring the prototype to the pre-clinical and clinical phases necessary to the ensuing commercialization of MAILPAN whose ultimate goal is to improve the life of at least 20 million persons in the world while providing positive effects on healthcare management and expenses, the environment and the competitiveness of the biomaterials industry. In order to reach this goal, CeeD and Defymed gathered a consortium made of seven partners from academia, clinical/public health research sector and industry/SMEs from three different European countries –Belgium, France and UK. The expertise gathered include encapsulation techniques, islet isolation, cell engineering, islet transplantation, islet preconditioning, surgical implantation, and medium formulation; items that are complementary and necessary to the implementation of the present project. The proposal of a 36-month duration intends to bring the most modern and up to date improvements that the bioartificial pancreas still needs and can receive such as to enhance cells survival inside the device by formulating a new adapted cell culture medium, to further lower the rejection risk by studying the biocompatibility and anti-inflammatory mechanisms, to test the prototype in primates, and to validate its further use in humans. Safety, bio-compatibility and interoperability of MAILPAN device combined to the islets/pseudo-islets, will be assessed, in respect to the applied regulatory directives. BIOSID is a Collaborative Project supported at the level of 5 470 000 € through Cooperation Program of the European Community’s FP7, Grant agreement number HEALTH-F2-2012-305746. BIOSID addresses the topic HEALTH-2012-2.4.3-1: “Innovative approach to manage diabetes”.


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Macroencapsulated Human Islet Viability is Drastically Reduced In Vivo as the Number of Islets per Device is Increased

Klearchos K Papas1,3, Kathryn R Mueller1,3, Leah V Penrod2, Melissa A Davis2, Jennifer P Kitzmann1,3, Stephan G Gruessner1, Thomas M Suszynski3, Tun Jie1, Linda Tempelman4, Sean W Limesand2, Efstathios S Avgoustiniatos3

1Surgery, University of Arizona, Tucson, AZ, United States; 2School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, United States; 3Surgery, University of Minnesota, Minneapolis, MN, United States; 4Giner, Inc., Newton, MA, United States.

Widespread clinical application of ITx is currently hindered by the need for systemic immunosuppression and the limited supply of human islet tissue. The use of cell isolating devices may address both critical barriers by enabling the use of allogeneic (and ultimately xenogeneic or stem cell derived islets) with minimum or no immunosuppression. Such devices have been tested in small and large animal models and to a limited extent in humans with excellent biocompatibility and safety profiles. However, work with large-animal models showing efficacy is limited. We hypothesize that scale-up of these devices for human use has been severely hindered by the device size requirements (Fig. 1a) necessary for sufficient islet oxygenation to support islet viability and especially function (glucose stimulated insulin secretion, GSIS) which is affected at oxygen levels higher than those affecting viability. Fig. 1b depicts theoretical calculations, demonstrating this effect on islet viability and function at a fixed transplant site pO2 (30mmHg) for increasing islet loading per device (IE/cm2). These calculations assume a best case scenario (fully pre-vascularized device). To evaluate the validity of the calculations we investigated the effect of increasing IE/cm2 on islet viability using Theracyte devices containing 500, 2000, 4000, or 8,000 human IE/cm2, each transplanted subcutaneously in a nude rat model. The devices were explanted after 7 days and islet viability [oxygen consumption rate normalized to DNA (OCR/DNA)] and insulin secretion rate (ISR) were measured. Results obtained were consistent with theoretical predictions and demonstrated substantially decreased OCR/DNA and ISR from explanted devices at loadings above 500 IE/cm2(Fig. 1c). As expected, the experimental results demonstrated an even lower viability than modeling because the devices tested were not pre-vascularized. Preliminary experiments showed that continuous enhanced oxygen supply to transplanted devices enables maintenance of human islet viability and function for loadings up to 8000IE/cm2, suggesting that oxygen is the key limiting nutrient in vivo. We conclude that methods for supporting higher islet densities in macro-encapsulation devices (such as enhanced oxygen supply) are critically needed to ensure in vivo islet viability and function.


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Prolonged Islet Allograft Survival After Transplantation into Pre-Vascularized Subcutaneous Site without Immunosuppression

Luan Nguyen1, Hiroo Iwata1

1Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

The transplantation of islets of Langerhans is a promising method to treat patients with insulin-dependent diabetes mellitus. In current clinical practice, islets are transplanted into the liver via portal vein. However, exposure of the islets to blood during intraportal islet infusion triggers a thrombotic/inflammatory reaction and results in significant islet loss. Subcutaneous is an attractive alternative site for islet transplantation due to its ability to accommodate large graft volume, easy accessibility, minimally invasive surgical procedure and the transplanted graft can be easily recovered under local anesthesia. However, subcutaneous tissue is very poorly vascularized, hence it does not supply sufficient oxygen and nutrient to maintain islet graft viability and function. In this study, we engineer subcutaneous tissue into a well-vascularized pockets using bFGF releasing device. To evaluate the engraftment of islets in this implantation site, 1500 to 3000 allogeneic islets were transplanted in either non-treated or pre-vascularized subcutaneous space of streptozotocin-induced diabetic rats. No recipients demonstrated normoglycemia after islet transplantation into non-treated subcutaneous space. In contrast, the transplantation of islets into pre-vascularized subcutaneous space could normalize blood glucose level in all diabetic rats. Interestingly, blood glucose levels were maintained stably in 8 out of 9 recipients of allogeneic islets for over 100 days without immunosuppression. The removal of islet graft promptly causes the increase of blood glucose level. Histologic findings of the retrieved grafts after this follow-up time revealed many viable islets with a perfectly intact shape and clear expression of insulin. No mononuclear cell infiltration into islet graft was observed. At day 100, the intraportal transplantation of islets or intraperitoneal transplantation of donor spleenocytes causes the destruction of islets in subcutaneous space. In conclusion, we have successfully engineered subcutaneous into a well-vascularized tissue that could accommodate not only syngeneic but also allogeneic islet graft.

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376

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Immunoisolation using Silicon Nanopore Membranes (SNM) with Convective Transport

Shang Song1, Gaetano Faleo2, Torin Yeager1, Rishi Kant1, Tejal Desai1, Qizhi Tang2, Shuvo Roy1

1Department of Surgery, University of California, San Francisco, San Francisco, CA, United States; 2Department of Bioengineering and Therapeutic Science, University of California, San Francisco, San Francisco, CA, United States.

Purpose: Type 1 diabetes results from autoimmune destruction of insulin-producing β cells and currently b cell replacement therapy is the only cure. We aim to develop a device to protect transplanted b cells against immune attacks. Our silicon nanopore membranes (SNM) exhibit monodisperse pore size distribution with 1% variation, which enables superior selectivity relative to conventional polymeric membranes. In this study, we investigated the feasibility of using SNM as an immune barrier to prevent islets from cytokine-induced β cell death under convective transport.

Methods: A closed loop fluid circuit with SNM assembled in a flow cell device was used to recapitulate local blood circulation. Mouse islets loaded in the flow cell between SNM membranes with a pressure difference of 51.7 – 77.6 mmHg. A combination of b cell toxic cytokines, TNF-α (1000U/mL), IL-1 β (50U/mL), and IFN-γ (1000U/mL), was applied to the fluid circuit. After 6-hour exposure, islets from each group were stained with fluorescein diacetate (FDA) and propidium iodide (PI). Beta cell apoptosis was assessed using a confocal microscope and compared to that obtained from islets directly exposed to the cytokines.

Results: Direct exposure of islets to cytokines in static control cultures lead to significant 400% increase in b cell apoptosis. Islets encapsulated in SNM flow cells showed high viability when the fluid circuit was inoculated with cytokines, comparable to the viability seen in control static culture without cytokines and flow cell without cytokines.

Conclusion: This result demonstrated the feasibility of using SNM to protect islets from pro-inflammatory cytokines. SNM could potentially reduce or even eliminate the immunosuppresants required by current therapies and open the possibilities of using xenogeneic islets and stem-cell derived islets to overcome donor shortage for future Type 1 diabetes treatment.

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Hybrid Polymer Hydrogel Scaffolds for the Improvement of Islets of Langerhans Revascularization After Transplantation

Giulia Marchioli, Andrea Di Luca, Marten Engelse, Eelco De Koning, Clemens Van Blitterswijk, Marcel Karperien, Aart Van Apeldoorn, Lorenzo Moroni

1Developmental BioEngineering, University of Twente, Enschede, Netherlands; 2Tissue Regeneration, University of Twente, Enschede, Netherlands; 3Department of Nephrology, Leiden University Medical Center, Leiden, Netherlands.

Although intra–hepatic injection of allogeneic islets of Langerhans is a promising treatment for type 1 diabetes‚ islets scarce functionality and survival rate after transplantation account for an overall low efficiency of the procedure.

The use of scaffolds is a promising strategy to create a more favorable environment for islets to reside in and ultimately improve the transplantation outcome in extra-hepatic strategies. The major challenge for scaffold–aided islets transplantation is to fabricate a scaffold design that can provide sufficient nutrients and oxygen supply to the islets, also in the most inner part of the scaffold.

This study presents an innovative strategy for an extra–hepatic islets transplantation platform consisting of an hybrid, three–dimensional polymer/hydrogel scaffold. A three-dimensional polycaprolactone (PCL) scaffold is fabricated using a 3D fiber deposition machine. The surface of the printed scaffold is functionalized by immobilizing heparin via EDC/NHS chemistry. Heparin can be exploited to bind and release vascular endothelial growth factor (VEGF) in a controlled manner to attract and stimulate the proliferation of endothelial cells and blood vessels and eventually accelerate the revascularization of the construct. Islets are embedded in the core of the scaffold in a 2% wt/v alginate hydrogel (Figure 1).

Results showed that heparin immobilization can be performed with a good yield and it improves the amount of VEGF retained by the construct, up to 3.6 folds compared to untreated PCL scaffolds. Heparin/VEGF functionalization stimulates human umbilical vein endothelial cells (HUVEC) migration towards the scaffold and into the pores when compared to untreated scaffolds. Islets embedded in the alginate core of the construct showed full functional response to glucose stimuli, comparable to free–floating islets, up to 7 days in culture.

This research is part of the Diabetes Cell Therapy Initiative (DCTI) and funded by the Dutch Diabetes fund and the Ministry of economical affairs (FES program) of the Netherlands.The work was also funded by the Dutch Technology foundation STW (OTP 11135).


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Can Cardiopulmonary Exercise Testing Predict Outcome in Simultaneous Pancreas and Kidney Transplantation?

Hussein Khambalia1, Zia Moinuddin1, Angella Bryan2, Afshin Tavakoli1, John Moore2, Dougal Atkinson2, Titus Augustine1, David van Dellen1, Iain Gall2

1Transplant Surgery, Manchester Royal Infirmary, Manchester, United Kingdom; 2Anaesthesia and Critical Care, Manchester Royal Infirmary, Manchester, United Kingdom.

Background: Outcome prediction in simultaneous pancreas and kidney transplantation (SPKT) remains challenging without validated objective investigations informing risk stratification. Cardiopulmonary Exercise Testing (CPET) identifies patients at increased post-operative morbidity and mortality risk following major surgery. We aimed to establish CPET’s utility in patients undergoing SPKT.

Methods: A prospective study of all patients undergoing CPET assessment with a view to SPKT was performed over a 7 year period (2005- 2012). CPET data, using established thresholds for major vascular surgery (anaerobic threshold (AT) <10.2ml/kg/min, peak O2 <15ml/kg/min and ventilatory equivalents for CO2 (VE/VCO2) >34), were used as predictive comparators against outcome measures (one-year mortality, cardiac events, total inpatient length of stay (LOS) and Critical Care length of stay (CCLOS)). Univariate and multivariate regression analysis for outcome measures was modelled accounting for confounding factors.

Results: 56 patients (35 male, 21 female; mean age at CPET 41.8 (SD 7.8)) with mean AT 11.4ml/kg/min (SD 3.3), mean peak VO2 17.7ml/kg/min (SD 3.8), mean VE/VCO2 28.5 (SD 3.3) and mean % predicted peak VO2 57.1% (SD14.9) were included. CPET data informed decision making process for patient activation on the waiting list.

17 patients had AT <10.2ml/kg/min and 17 had VO2 <15ml/kg/min. An AT threshold of 10.2ml/kg/min demonstrated no differences in mean LOS and CCLOS (p= 0.54 and 0.79). Peak VO2 threshold of 15ml/kg/min also demonstrated no difference in mean LOS and CCLOS (p= 0.56 and 0.78). No patients had VE/VCO2 >34. 2 patients had cardiac events in the immediate post-operative period, both having AT >10.2ml/kg/min and VO2 >15ml/kg/min. There was no mortality at 1 year.

Conclusion: Established CPET thresholds do not identify patients at increased risk of mortality and morbidity in this cohort. Excellent operative outcomes and selection bias from non-blinding may limit conclusions but suboptimal CPET should not currently preclude transplant consideration. Further evaluation in a larger cohort is required for validation in SPKT.

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Simultaneous Pancreas and Kidney Transplantation in a Patient with Sickle Cell Trait

Parul Patel1, Lawrence Lu1, William Bry1, Marilyn Kaszpurenko1, Lawrence Bohannon1, Steven Katznelson1, Harish Mahanty1, Ram Peddi1, Kimi Ueda1, Assad Hassoun1, Ward Hagar2

1Department of Transplantation, Californa Pacific Medical Center, San Francisco, CA, United States; 2Department of Hematology/Oncology, Children’s Hospital and Research Center, Oakland, CA, United States.

A major cause of early pancreas allograft failure is venous thrombosis. Pancreas transplantation in a patient with sickle cell trait may carry greater risk, given a two to four fold increased risk of venous thromboembolism in patients with sickle cell trait [1].

A 51-year-old lady with non-transfusional dependent SC hemoglobin, a single alpha hemoglobin gene deletion, type 2 diabetes mellitus and end stage renal disease on peritoneal dialysis was listed as active for simultaneous pancreas and kidney (SPK) transplantation. We coordinated a preemptive strategy to reduce the potential risk of perioperative thrombosis, including banking of well matched blood products with appropriate timing of red cell exchanges.

When an SPK transplant offer became available, partial phenotypically matched for C, E, and Kell antibodies, sickle cell negative blood was requested from the blood bank. Immunosuppression included Thymoglobulin, mycophenolate, tacrolimus, and corticosteroids. Routine antiplatelet therapy included aspirin and dipyridamole. Intraoperatively the patient underwent a 3 unit red cell exchange. Anticoagulation was not administered. She had immediate kidney graft function and blood glucose levels remained well controlled. On postoperative day 1, a doppler ultrasound of the pancreas allograft was normal. On postoperative day 2, she received an additional 5 units red cell exchange. She required an additional 3 units PRBC for gradually declining hematocritthroughout the rest of her hospitalization. The rest of the hospital course remained unremarkable. The patient was discharged 11 days aftersurgery with a hematocrit of 29.2. At 2 and half months post transplant fasting glucose is 95, hemoglobin A1C has dropped from 11.2% on admission to 5.4%, and serum creatinine is 1.59. The hematocrit isstable at 29.1.

This case report reviews the first successful pancreas transplantation in a patient with sickle cell trait. A transfusion strategy with careful perioperative timing of red cell exchanges resulted in no major thrombotic complications.

Reference:

Sickle cell trait and the risk of venous thromboembolism among blacks. Austin H, Key NS, Benson JM, Lally C, Dowling NF, Whitsett C, Hooper WC Blood. 2007;110(3):908.

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11 Year Experience with Simultaneous Pancreas and Kidney Transplantation in Patients with Type 1 and Type 2 Diabetes at a Single Center

Parul Patel1, Lawrence Bohannon1, William Bry1, Assad Hassoun1, Lawrence Lu1, Steven Katznelson1, Marilyn Kaszpurenko1, Harish Mahanty1, Ram Peddi1, Kimi Ueda1

1Department of Transplantation, Californa Pacific Medical Center, San Francisco, CA, United States.

Objective: Simultaneous kidney and pancreas transplantation (SPK) is commonly offered to patients with end stage renal disease (ESRD) and type 1 diabetes mellitus. Our center has been performing SPK for patients with type 1 and type 2 diabetes since 2002. We compared patient and graft outcomes based on diabetes type 1 or 2 in patients who underwent SPK from 2002 through 2012.

Methods: All patients who underwent first SPK consecutively from 2002 through 2012 were identified using standard patient tracking records. Patients were divided into type 1 or type 2 diabetes based on clinical history (diabetes onset, length of insulin dependence, and C-peptide levels). We compared patient, kidney, and pancreas graft survival between the two groups. Pancreas graft failure was defined as return to insulin dependence and kidney graft failure was defined as return to dialysis dependence.

Results: Of 219 patients who underwent SPK, 164 had type 1 diabetes and 55 had type 2 diabetes. A total of 10 deaths occurred, 7 in the type 1 group and 3 in the type 2 group. In patients with type 1 diabetes, 4 died from sepsis, 1 from mucormycosis, and 2 were unknown. In patients with type 2 diabetes, the cause of death was pneumonia, sepsis, and unknown. With a mean follow up of 4.6 years, patient survival was 96% and 95% for type 1 and type 2 patients, respectively. Pancreas graft survival was 86% and 78% for type 1 and type 2 patients, respectively. Kidney graft survival was 96% and 95% for type 1 and type 2 patients, respectively.

Conclusions: Our review of 219 patients, 164 with type 1 diabetes and 55 with type 2 diabetes, who underwent first SPK from 2002 through 2010 shows excellent patient and graft survival rates.

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Surgical Salvage of Partial Pancreatic Allograft Thrombosis Presenting as Ruptured Pancreatic Cyst

Victor Tswen-Wen Lee1,2, Ho-Yee Tiong2, A Vathsala2, Krishnakumar Madhavan2

1Department of Hepatopancreatobiliary & Transplant Surgery, Singapore General Hospital, Singapore, Singapore; 2National University Centre for Organ Transplantation, National University Hospital, Singapore, Singapore.

Introduction: Vascular thrombosis is an important cause of pancreatic graft loss, and the vast majority is managed by graft pancreatectomy. There are limited reports and case series of successful salvage of the pancreas allograft. We describe a case of partial pancreatic allograft thrombosis successfully salvaged by a graft distal pancreatectomy.

Methods: We used descriptive retrospective analysis.

Results: A 29-year-old patient with Type 1 diabetes and end-stage renal failure underwent a simultaneous pancreas kidney transplant with immediate graft function. The cadaveric pancreas allograft was placed head-up in the right iliac fossa with enteric exocrine drainage and standard vascular anastomosis. He presented with compressive symptoms on his bladder 5 months later, and a CT showed a 4 cm cystic lesion in the body and tail of the pancreas allograft. Spontaneous rupture of the cyst occurred 3 weeks after the initial onset of symptoms with generalized abdominal pain. He underwent graft distal pancreatectomy with good recovery. He remains euglycemic, insulin-free with a normal renal function. Histology of the resected unhealthy graft showed an arterial thrombus with xanthogranulomatous inflammation and necrosis.

Conclusion: Surgical salvage with graft distal pancreatectomy is feasible for partial pancreatic allograft thrombosis.

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A Cautionary Tale: Bleeding Duodenal Segment Varices from Portosystemic Shunting Across a Bowel-Drained Simultaneous Kidney Pancreas Transplant in a Hepatitis C+ Recipient.

William Bry1, Marilyn Kaszpurenko1, Assad Hassoun1, Parul Patel1, Steven Katznelson1

1California Pacific Medical Center, San Francisco, CA, United States.

Simultaneous kidney pancreas transplantation (SPKT) is being offered to select hepatitis C positive patients [1]. We present an unusual case of variceal hemorrage from a systemically drained pancreas transplant. This diagnosis should be entertained in late GI bleeding in SPKT patients with liver disease.

This 43 year old type l diabetic presented at age 43 for SPKT. She was found to be hepatitis C antibody positive after deceased donor kidney transplantation 14 years earlier. The first kidney failed after 12 years; 2 years before her SPKT. The patient became infected either from the first kidney transplant or from briefly experimenting with IV drugs as a teenager.

Formal pre-SPKT testing demonstrated no fibrosis or portal inflammation on biopsy. She had 1,148,200 copies/ml of genotype 1A. Infection occurred somewhere between 14 and 24 years before her SPKT. Treatment with interferon was entertained but not pursued due to coronary artery disease, anemia and depression.

The patient underwent SPKT which, ironically, was complicated by a self limited GI bleed from the duodenal-ileal anastamosis postoperatively. Immunosuppression was with Thymoglobulin induction followed by Prograf, Cellcept and prednisone.

Four years later, the patient presented with fatigue, loose diarrhea, weight loss and thrombocytopenia. Progression of liver disease was diagnosed.

Two years after that, the patient presented with an upper GI bleed whose etiology defied diagnosis. Multiple upper and lower endoscopies, capsule endoscopies and tagged red cell scans were nondiagnostic. Hepatic vein wedge pressures were borderline with a gradient of 11 cm H2O. Mesenteric angiography suggested small vessel abnormalities and microaneurysms leading to a diagnosis of polyarteritis nodosum treated with plasmapheresis.

Nevertheless, the patient was readmitted with GI bleeding every couple of weeks. After a year and 61 transfusions, the patient was taken to the operating room in desperation for anticipated intraoperative endoscopy to localize the bleeding site. At surgery, the body of the pancreas transplant appeared normal, but the duodenal segment was covered with a Medusa’s head tangle of varices extending onto the ileal surface. The liver was firm and micronodular. The biopsy showed cirrhosis.

Extensive oversewing of the varices from both the serosal and mucosal surfaces of the bowel resulted in cessation of GI bleeding for 10 months, when the patient was readmitted with a recurrent GI bleed. Hepatic wedge pressure was now elevated to a gradient of 20 cm H20. TIPS could not be performed due to bilateral internal jugular vein occlusion. Liver transplantation or mesocaval shunting was entertained until the patient succumbed to a fatal myocardial infarction.

Theoretically, portal venous drainage of SPKT would avoid this complication. Recognition of this unusual complication earlier might have improved her outcome.

Reference:

  • [1] Miguel M, Sampaio MS, Kuo HT, Poommipanit N, Martin P, Bunnapradist S. Influence of preexisting hepatitis C virus antibody positivity in simultaneous pancreas-kidney transplant recipients. Transplantation. 2010 Jul 15;90(1):61–7.
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The Pancreas Transplant Program in ASAN Medical Center: Retrospective Analysis of Outcome from 1992 to 2013

Young Hoon Kim1, Duck Jong Han1, Joong-Yeol Park2, Sung Shin1, Byung Hyun Choi1, Joo Hee Jung1, Han Kyung Cho1

1Surgery, Asan Medical Center, Seoul, Korea; 2Endocrinology, Asan Medical Center, Seoul, Korea.

Pancreas transplantation (PT) is a treatment of choice for insulin-dependent diabetes. There has been considerable improvement in patient and graft survival by technical refinement, newer immunosuppressants, and better postoperative management. However pancreas transplantation has not been yet widely performed in Asia.

From July 1992 to April 2013, we have performed 203 pancreas transplants (124 SPKs, 60 PTAs, and 19 PAKs). We retrospectively analyzed graft and patient survival rates of our clinical cases using the Kaplan-Meier method.

One-, 5- and 10-year patient survival rate were 94.7%, 88.7% and 86.2% respectively. 1, 5, and 10 year pancreas graft survival rates were 84.6%, 71.0% and 61.8% respectively. In SPK 1, 5, and 10 year pancreas graft survival rates were 90.5%, 84.8% and 80.1%, and kidney graft survival rates were 94.7%, 89.4% and 85% respectively. In the PTA category graft survival were 73.3 % after 1 year and 47.8% after 5 years.

Since 2006 we have performed more than 10 PT annually. When we divided the PT recipients into two groups according to the time of operation (up to 2005 in 57 patients vs. 2006 and later in 146 patients], the patient and pancreas graft survival rate at 5 year had improved currently 93.2%, 83.7% vs. 80.6%, 47.4% retrospectively (P < .001).

Considering the improved quality of life and long-term patient and graft survival, PT can be an effective treatment strategy in diabetic patients requiring insulin.

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386

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Graft Arterial Anastomotic Rupture by Klebsiella Pnumoniae After Pancreas Transplantation

Young Hoon Kim1, Young Soo Chung2, Duck Jong Han1, Sung Shin1, Byung Hyun Choi1, Joo Hee Jung1, Han Kyung Cho1

1Surgery, Asan Medical Center, Seoul, Korea; 2Surgery, Pusan National Hospital, Busan, Korea.

A 37-year old female underwent pancreas transplantation on 28th, November, 2012. She was diagnosed with type-I DM at her age 15.

Donor was 28-year old male cadaver. During the pancreas procurement, duodenal irrigation was done with solution containing kanamycin and ampotericin B.

The superior mesenteric artery (SMA) and splenic artery were reconstructed to Y-graft by using donor bifurcated iliac artery. The portal vein and Y-graft were anastomosised to external iliac vein (EIV) and external iliac artery (EIA), respectively. Duodenojejunostomy was done after duodenal irrigation with antibiotic solution. Klebsiella Pneumoniae was detected in the duodenal content, postoperatively. Total ischemic time was 8 hours and 42 minutes.

After operation, blood sugar level was well controlled without using insulin.

On the postoperative 6th day, fever was developed. On drainage fluids, klebsiella pneumonia was detected which was the same with that of dudodenal culture study. Antibiotics had been started.

On the postoperative 10th day, massive bleeding occurred. At emergent laparotomy, the disruption at the anastomosis of SMA and Y-graft was founded. Direct repair was performed. At a ascitic fluid culture, klebsiella pneumoniae was detected again.

On the postoperative 20th day, there was re-bleeding. At this time the disruption site was SMA distal to anastomosis of Y-graft. Following graftectomy was done, iliac artery anastomosis site was repaired using cadaveric vein patch,

On the postoperative 26th day, vein patch site bleeding was occurred again. The external iliac artery ligated and femoro-femoral extraanatomic bypass was done. The vein patch tissue culture also showed klebsiella pneumonia.

After last operation, there was no more bleeding and the patient was discharged at postoperative 54th day.

As far as we know, this is the first case of anastomotic rupture by klebsiella pneumoniae in pancreas transplant recipient. As a result, we minimized the duodenal irrigation to avoid spillage of duodenal content during recipient operation in pancreas transplant.

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387

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Successful Repair of a Ruptured Arterial Mycotic Pseudoaneurysm Following Simultaneous Pancreas and Kidney Transplantation

Petros Yiannoullou1,2, David van Dellen1, Hussein Khambalia1, Bence Forgacs1, Afshin Tavakoli1, David Murray2, Titus Augustine1

1Department of Transplant Surgery, Manchester Royal Infirmary, Manchester, United Kingdom; 2Department of Vascular and Endovascular Surgery, Manchester Royal Infirmary, Manchester, United Kingdom.

Background: Arterial mycotic pseudoaneurysms are a rare, but potentially catastrophic complication of pancreas transplantation[1]. Life threatening haemorrhage results from rupture into the peritoneum, adjacent bowel or bladder and as such the condition is synonymous with mortality and transplant graft loss[2,3]. An extensive arterial defect within a contaminated surgical field on a background of immunosuppression complicate definitive management. Synthetic vascular grafts often fail to provide a long-term solution due to subsequent graft infection[4]. Primary repair of the defect is both difficult and results in arterial stenosis. Arterial ligation may be required to control exsanguination

Case: A 41 year old male, type 1 diabetic of 38 years with associated end stage renal failure, underwent successful simultaneous pancreas and kidney (SPK) transplantation with primary function of both organs. He presented, 9 months following transplantation, with copious fresh rectal bleeding secondary to a mycotic pseudoaneurysm located at the anastomosis between the native external iliac artery and arterial conduit to the graft pancreas. This was successfully managed with a bovine pericardial patch (BPP) repair of the arterial defect {figure1} and enteric diversion following graft pancreatectomy. He remains well with no vascular insufficiency 12 months following the procedure, without aneurysm formation or stenosis on subsequent imaging.

Conclusions: A ruptured mycotic pseudoaneurysm following transplantation carries a significant risk of mortality and represents a surgical challenge as conventional vascular or endovascular techniques utilising synthetic materials are likely to fail due to the contaminated field. A BPP offers good handling characteristics, excellent haemostatic properties and off the shelf availability[5]. Its use for arterial reconstruction within infected fields has been reported, suggesting a favourable profile of infection risk in comparison to synthetic grafts[5]. This case highlights its use as a treatment for a post transplant ruptured mycotic pseudoaneurysm in a patient with life threatening haemorrhage.

Reference:

Ciancio, G., Monte, A. L., Julian, J.F., Romano, M., Miller, J. and Burke, G.W. (2000), Vascular complications following bladder drained, simultaneous pancreas-kidney transplantation: the University of Miami experience. Transplant International, 13: S187–S190.

Lubezky N, Goykhman Y, Nakache R, Kessler A, Baruch R, Katz P, Kori I, Klausner JM, Ben-Haim M. Early and Late Presentations of Graft Arterial Pseudoaneurysm Following Pancreatic Transplantation. World J Surg. 2013 Mar 2.

Tan M, Di Carlo A, Stein LA, Cantarovich M, Tchervenkov JI, Metrakos P. Pseudoaneurysm of the superior mesenteric artery after pancreas transplantation treated by endovascular stenting. Transplantation. 2001 Jul 27;72(2):336–8.

Akhtar MZ, Jones A, Sideso E, Sinha S, Vaidya A, Darby C. Management of a ruptured mycotic pseudo-aneurysm following pancreas-kidney transplantation. Ann Transplant. 2011 Oct-Dec;16(4):122–5

McMillan WD, Leville CD, Hile CN. Bovine pericardial patch repair in infected fields. J Vasc Surg. 2012 Jun;55(6):1712–5.


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Chronic Kidney Disease Following Pancreas Transplant Alone: A Kidney Biopsy Study.

Michelle Goble1, Muhamad Mujtaba2, Jonathan Fridell1, Sohail Yaqub2, Asif Sharfuddin2, Tim Taber2

1Surgery, Indiana University School of Medicine, Indianapolis, IN, United States; 2Medicine, Indiana University School of Medicine, Indianapolis, IN, United States.

Successful pancreas transplant alone (PTA), through restoration of sustained normoglycemia, improves diabetic nephropathy in type 1 diabetics. However the pt with PTA may still develop chronic kidney disease(CKD). Although CKD in nonrenal solid organ transplant is multifactorial, calcineurin inhibitor (CNI) toxicity is thought to be a major contributor. CNIs can lead to both acute (acute arteriolopathy, toxic tubulopathy, TMA) and chronic nephrotoxicity (hyaline arteriosclerosis). There is a lack of kidney biopsy data in CKD pts with PTA.

Methods: Out of 65 PTA, 7 pts with CKD underwent kidney biopsy. IS therapy : tacrolimus (trough 7–10 ng/ml), rapamycin (trough < 5 ng/ml) and mycophenolate mofetil (500 mg BID). Indications for kidney biopsy were new proteinuria with urine protein/urine creatinine >1 on at least 2 spot urine sample or rise in serum creatinine >30% from the baseline after excluding acute kidney injury. All kidney biopsied were interpreted by a single pathologist.

Results: All pts had acceptable serum creatinine with eGFR>50ml/min/1.73m2 at time of transplant. Pt #2 and #5 had pre-transplant nephrotic range proteinuria. Two pts i.e. #1 and # 2 had pre and post transplant hypertension. Pt #1 also had Lupus. All these patients had normal A1C values. Kidney biopsy data showed moderate to severe hyaline arteriosclerosis in 6 pts, diabetic glomerulosclerosis in 5, mild to moderate arteriosclerosis in 3, severe arteriosclerosis in 2, chronic TMA and focal glomerulosclerosis with fi brotic crescents in 1 and IgA nephropathy in 1pt.

Conclusion: The above biopsy data suggests that CNIs exposure plays a signifi cant role in the development of progressive CKD in patients with PTA. A low CNIs exposure may help to slow these histological changes. More studies are needed to confirm our findings.


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Establishment of Follow-Up Protocol for the Patients on Waiting List of Pancreas Transplantation.

Mikiko Hayashi1, Kenmochi Takashi1,2, Taihei Ito1,2, Sachie Nishiyama1, Tomoko Nishimura1, Emiko Suzuki1, Kiyotaka Hoshinaga3

1Division of Transplantation Supporting Unit, Fujita Health University Hospital, Toyoake, Japan; 2Department of Organ Transplant Surgery, Fujita Health University, School of Medicine, Toyoake, Japan; 3Department of Urology, Fujita Health University, School of Medicine, Toyoake, Japan.

Objective: In pancreas transplantation, the recipients usually have many risks due to diabetic complications. I order to realize the safe procedure of pancreas transplantation, we have introduced a newly designed follow-up protocol for the patients on waiting list.

Patients and Methods: In our institution, 23 pancreas transplantations from deceased donors (19SPK, 2PAK, 2PTA) have, so far, been performed since 2010. Moreover, 32 patients are on the waiting list of Japan Organ Transplant Network. Most of the patients on waiting list of pancreas transplantation (SPK in particular) have high risks because of long-term history of diabetesand hemodialysis. Active retinopathy, ischemic heart disease and arteriosclerosis are especially major problems which influence the safety for transplantation surgery. For the purpose of the safety of the recipient, we have established and performed the newly designed follow-up protoco since April, 2012. The transplant surgeons, the medical doctors (diabetologist) and the recipient clinical coordinators see the patients every three or four months as the outpatients. Evaluation of retinopathy, cardiac function and arteriosclerosis of iliac arteries or other arteries are routinely performed. When the patients need treatment for retinopathy and iscemic heart disease, they are treated by the specialist before performing pancreas transplantation. Recipient coordinator focus the mental care for the patients in addition to medical problerms.

Results: Until March, 2012, two patients out of 17 patients who were nominated as the 1st candidate had to decline the pancreas transplantation due to the uncontrolled complications and three patients developed severe complications and died after transplantation. In contrast, after the establishment of the new follow-up protocol, all seven patients showed no mortal complications and discharged from the hospital at 30 to 40 days after transplantation with the functining pancreas and kidney grafts (Table 1).

Conclusions: Our new follow-up protocol is of use for the safe and smooth performance of pancreas transplantation from deceased donors.


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Laparoscopic Sleeve Gastrectomy in a Obese Patient with a Functioning Pancreatic Graft: Case Report

Carlo Socci1, Elena Orsenigo1, Paola Maffi2, Danilo Parolini1, Alessandro Saibene2, Francesca Muffatti1, Antonio Secchi2, Carlo Staudacher1

1Gastrointestinal surgery, ospedale s. Raffaele, Milano, Italy; 2ospedale s. Raffaele, Milano, Italy.

Background: Association between obesity and diabetes, metabolic syndrome, pancreas insufficiency and progression of chronic kidney disease is well established. Recent evidences showed that renal and pancreatic function improve after bariatric surgery. We report the case of sleeve gastrectomy performed in an obese patient with a functioning pancreatic graft.

Clinical case: A 49 year-old obese man of 118 Kg, BMI 35.5, who underwent pancreas transplantation in 2004 due to uncontrolled type 1 diabetes came to our attention. He was affected by mild chronic kidney disease (Serum creatinine: 1,39 mg/dl, Plasma filtration rate: 61ml/min) and evidence of microalbuminuria (55,5 mg/l), arterial hypertension, dyslipidaemia. Pancreatic graft alone was effective in controlling diabetes (Hb1Ac: 41 mmol/mol, fasting plasma glucose: 107mg/dl). The patient post transplant immunosuppressive regimen was mycophenolate and FK 506 (3,5 mg/day with a plasma level of FK506 of 6,5 ng/ml). Despite multiple attempts, control of body weight was not obtained after transplantation. In order to improve body weight control, optimize metabolic control and improve kidney function a laparoscopic sleeve gastrectomy was planned. Multiple pre-operatory assessments were performed before surgery. At abdominal ultrasound scan hepatic steatosis was found. The section of the stomach was started at 6 cm from pylorus, with a gastric calibration of 12,7 mm (42 French), the whole fondus of the stomach was then completely removed, with cardias distance from the end staple-line of 1,5 cm. Procedure was well tolerated and no major complications in the post-operatory period were reordered. 2 month after surgery there was a weight loss of 10 Kg, with a BMI of 32,2. Good metabolic control was conserved and the plasma level of Fk 506 was not dramatically modified after surgery (5.5 ng/ml) without any change in drug dose (3,5 mg/day).

Conclusion: Sleeve gastrectomy is a safe and feasible procedure in obese patients who previously underwent pancreas transplantation, in order to preserve graft function.

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A new Model of Hipothermic Pancreas Ischemia-Reperfusion in Rats

Vinicius Santos1, Oscar Ferro1, Carlos Pantanali1, Rubens Arantes1, Rafael Pecora1, Andre David1, Eleazar Chaib1, Luiz D’Albuquerque1

1Gastroenterology, Clinics Hospital, São Paulo, Brazil.

Introduction: The pancreatic ischemia-reperfusion (I-R) can lead to graft pancreatitis and may contribute to graft loss and kidney injury, thereby increasing morbidity in these patients[1,2]. Furthermore, prolonged cold ischemia and warm ischemia might induce or worsen graft pancreatitis and increase the risk for vascular thrombosis[3]. Few experimental models of pancreatic I-R have been proposed[1, 4]. However, most studies cover just the normothermic period of I-R. Thus, there isn’tso much knowledge about the hypothermia in the pancreatic I-R.

Objective: We proposed an experimental model of hypothermic pancreas I-R that allowed to evaluate the benefic effects of hypothermia.

Methods: After an upper midline abdominal laparotomy, the pancreatic tail was exposed and an ischemia injury was induced by a 2.5mm microvascular clamp for 1-hour, following 4-hour reperfusion, in adult male Wistar rats. They are divided in three groups. Group 1 (n = 12, control, rats without I-R injury): sham; Group 2 (n = 12): normothermic I-R; Group 3 (n = 12): hypothermic I-R. To achieve the desired temperature of approximately 4.5 degrees, small pieces of ice were placed around the exteriorized organ, that it was carefully protected by a thin sheet of plastic to avoid the direct pancreas tissue contact with the ice. Blood and tissue samples were collected after reperfusion.

Results: In pancreatic I-R groups, high serum levels of amylase, lipase, cytokine and high histological damage were observed. The cold pancreas ischemia significantly reduced the release of TNF-α (p = 0.024) and IL-6 (p = 0.001), but had no significant effect in serum levels of amylase (p = 0.291), lipase (p = 0.993) and histological injury (p = 0.201).

Conclusions: The experimental model of hypothermic pancreas I-R is feasible, simple and reproducible. The hypothermia really protects the pancreas against the release of inflammatory mediators during I-R.

Liver and Gastrointestinal Transplant Division

Reference:

  • [1]. Drognitz O, Michel P, Koczan D, Neeff H, Mikami Y, Obermaier R, Thiesen HJ, Hopt UT, Loebler M. Characterization of ischemia/reperfusion-induced gene expression in experimental pancreas transplantation. Transplantation. 2006, 81:1428–1434.
  • [2]. Woeste G, Wullstein C, Meyer S, Usadel KH, Hopt UT, Bechstein WO, von Dobschuetz E. Octreotide attenuates impaired microcirculation in postischemic pancreatitis when administered before induction of ischemia. Transplantation. 2008, 86: 961–967.
  • [3]. Farney AC, Rogers J, Stratta RJ. Pancreas graft thrombosis: causes, prevention, diagnosis, and intervention. Curr Opin Organ Transplant. 2012, 17:87–92.
  • [4]. Junior RF, Kubrusly MS, Bellodi-Privato M, Molan NA, Machado MC, D’Albuquerque LA. Beneficial effects of N-acetyl cysteine on pancreas and kidney following experimental pancreatic ischemia-reperfusion in rats. Clinics (Sao Paulo). 2010, 65:311–316.
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Pancreas Transplantation with Venous Drainage to IVC: Short and Long-Term Outcome.

Mark R. Laftavi1, Sunil K. Patel1, Ji Lunan1, Lin Feng1, Meriem Said1, Merril Dayton1, Oleh Pankewycz2

1Surgery, State University of New York at Buffalo, Buffalo, NY, United States; 2Medicine, State University of New York at Buffalo, Buffalo, NY, United States.

Pancreas transplant surgery is a complicated procedure. Graft thrombosis and pancreatitis are the most common complications of the PTX surgery. Despite using several surgical techniques, up to 30% of PTX recipients require re-operation. For the first time, we report short and long-term outcomes of PTX with inferior vena cava (IVC) venus drainage. In this technique, the IVC is dissected ~2 inches at the bifurcation, then the side of the IVC is partially clamped by a Stansky vascular clamp. Then, the donor portal vein is anastomosed end to side to the IVC at the IVC bifurcation by 6-0 continuous prolene sutures. The fashioned Y arterial graft is anastomosed to the right or left common iliac artery of the recipient. The pancreas is emplaced head down toward the pelvis. Then, two-layer hand sewn side to side anastomosis is performed between the donor duodenum and the nearby loop of the jejunum. Forty-two PTX (22 SPK & 20 PAK) were performed with this technique in our center. Donor and recipient demographics are shown in Table 1 & 2.

TABLE 1
TABLE 1
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TABLE 2
TABLE 2
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Patient and graft survival are shown in Figure 1 & 2. No graft thrombosis occurred with this technique. Six patients (14%) required re-operation (3 bleeding, 2 anastomotic leak, 1 small bowel perforation). No patient or graft was lost due to surgical complications. We conclude that this technique provides simple and fast dissection of the venous drainage to the PTX without the need of complete occlusion of venous outflow. Surgical complications were lower with this technique compared to other reported techniques.



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Sensitization After Pancreas Transplant

Marcelo Perosa1,2, Helcio Rodrigues3, Nicolas Panajotopoulos3, Huda Noujaim1,2, Luiz Ianhez1,2, Rodrigo Azevedo1,2, Waldere Luconi1,2, Leonardo Mota1,2, Juan Branez1,2, Marcio Paredes1,2, Alisson Trevisol1, Tercio Genzini1,2

1Hepatology and Transplant, HEPATO, São Paulo, Brazil; 2Organ Transplantation, Bandeirantes Hospital, São Paulo, Brazil; 3Immunology Laboratory, Heart Institute University of São Paulo, São Paulo, Brazil.

Previously transplanted patients are more likely to be sensitized, leading to prolonged waitlist times and decreased graft survival. This analysis investigates factors at the time of first pancreas transplant(PT) or post-transplant associated with increased sensitization. Patients submitted to a previous PT that evolved with graft loss and were relisted for a pancreas retransplant were included in this study. Those who increased PRA greater than 20% were included in the sensitized group (S) and patients who maintained the same level of PRA after the first PT were included in the non sensitized(NS) group. Several variables were compared between the two groups, including age, gender, race, category of PT, need of blood transfusion, cause of pancreas loss, time interval between PT and graft loss, time interval between pre and post-transplant PRA, use of induction(depleting and non depleting), exocrine drainage and ischemia time of the failed PT. From November/1996 to June/2012, 37 patients were studied and the categories of the first PT were : 18 SPK, 7 simultaneous pancreas living-donor kidney transplant(SPLK), 5 PAK and 7 PTA. Ten patients were included in the group S and 27 in the group NS. There was a higher prevalence of non-SPK patients(solitary PT + SPLK) in the S group(p=0.007) although these patients have received induction more frequently than NS group(p=0.01). All NS patients underwent pancreas retransplant while 40% of S patients did not and developed donor specific antibodies(DSA) in high titles(p=0.003). There was a tendency of longer ischemia time in the first PT among the S patients (14.6 vs 12.7 hours, p=0.06).

Conclusions: PT recipients are more likely to sensitize after non-SPK categories and with longer ischemia time in the first transplant. The NS patients easily get a retransplant while almost half of the S group will not and develop high titles of DSAs.

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Back to the Reinnervation of the Pancreas After Transplantation?

Dimitri Mikhalski1, Very Coulic2, Valery Novikov4, Dmitry Bilibin3

1Digestive surgery Hopital Erasme, Hopital Erasme, Brussels, Belgium; 2Laboratory of Experimental Medicine, ULB site Horta, Brussels, Belgium; 3Neurophysiology, People Friendship University, Moscow, Russian Federation; 4Central institute of transplantation, Moscow, Russian Federation.

Introduction: The significant functional decrease and morphological sclerosis of the graft can not only be related to a chronic rejection process. Nowadays clinical organ transplantation includes only revascularisation of the graft and reconstruction of the natural excretion ways. It is well known that any transplantation leads to the nervous isolation of the grafted organ. The question of nervous and lymphatic connection reconstruction was discussed but never applied in clinics. Many studies concerning spontaneous reinnervation of whole pancreas grafts and Langerhans islets implants published controversial results. Anyway, the spontaneous growth of the recipient nervous into the graft occurs always belated. The denervation may be an important cause of the graft dysfunction, that surgical directed reinnervation is possible, efficient for fast penetration of the recipient nerves into the graft and promotes normalization of the graft function and morphology. AIM OF THE STUDY was to prove the feasibility and pertinence of the surgical directed reinnervation (SDR) of denervated (NRI) or transplanted pancreas for the optimization of the pancreas transplant condition: On the base of the study the human and animal anatomy of the local pancreas innervation, to create the models of NRI of the pancreas from CNS and reinnervation (RNRI), to elaborate the method of SDR of different pancreatic grafts; to evaluate the NRI and RNRI influence on the endocrine pancreatic function and morphology;to evaluate the NRI and NRI+SDR influence on the pancreatic graft function and morphology; to justify the possibility of the surgical reinnervation of the human pancreas.

Materials and Methods: The anatomical part of the study of the nervous systems penetrated in the pancreas was done in 22 human cadaver pancreas, adult 8 dogs, 9 cats, and 8 Lewis rats. The surgical and physiological parts were carried out in different mammals included mongrel dogs (27), cats (28), Lewis rats (94) in fasted and non-fasted protocols. Neurophysiogical investigation of nervous conductivity between pancreas and CNS used to tested NRI and SDR. Load tests with glucose, adrenalin, insulin and amylase, lipase determination were performed to evaluate the influence NRI and SDR on pancreatic functions.The samples were collected at 0, 30, 60, 90, 120 and 180 minutes at 1 week, 1month and 3 months after the operations.

Results: 1.Anatomical studies have shown the theoretical feasibility of surgical reconstruction of the continuity of nervous plexus responsible for pancreas transplant/graft innervations in animals as well as in humans 2.Models of pancreatic tail NRI and surgical reconstitution of the interrupted nervous pathways SDR were created and successfully tested in cats, dogs and rats.3. Electrophysiological studies performed in the cat models of NRI and NRI+SDR of the pancreas tail have proved the efficiency of the proposed SDR 4.NRI or transplantation of the pancreas leads to an exaggerated reaction to usual stimulations, that may cause of the functional exhaustion of the graft in late delays.5.The glycemia courves after load tests differed betwing NRI and SDR (P<0.05) either as serum and exocrine amylase and lipase in all groups. 6.SDR shortens the period of NRI of the graft, contributes to a quick restoration of its normal function and prevents its late degradation, as confirmed by relatively early normalization of its histological structure in rat model but also in dogs.

Conclusion: The SDR is a simple surgical technique, easily and quickly performed after the graft surgical revascularization without any complication and its functional and morphological effects were shown to be positive. So SDR may be recommended to be used in human pancreas transplantation.

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Patient and Graft Outcomes After Whole Organ Pancreas Transplantation: A Single Centre Experience

Shruti Mittal1,3,4, Rachel Franklin2,3,4, James Gilbert1, Sanjay Sinha1, Anil Vaidya1, Edward Sharples1, Stephen Gough2,4, Rutger Ploeg1,3, Peter Friend1,3

1Oxford Transplant Centre, Oxford University Hospital NHS Trust, Oxford, United Kingdom; 2Oxford Centre for Diabetes, Endocrinology and Metabolism, Oxford University Hospital NHS Trust, Oxford, United Kingdom; 3Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom; 4Biomedical Research Centre, NIHR, Oxford, United Kingdom.

Aim: Since whole organ pancreas transplantation began there have been considerable advances in surgical techniques, perioperative care and immunosuppressive therapies. Existing publications describing graft outcomes include heterogeneous cohorts of patients transplanted over several decades. We review outcomes after pancreas transplantation performed in the modern era in a high-output single centre.

Method: 485 pancreas transplants were performed at the Oxford Transplant Centre between March 2002 and December 2012. 16 IP (isolated pancreas transplants) were performed using bladder-drainage, all other transplants were performed using enteric drainage. All recipients were managed with Campath induction immunosuppression and maintained on tacrolimus and mycofenolate mofetil within a steroid-free regimen. Patients were anticoagulated with aspirin, subcutaneous heparin and dextran infusion. Graft failure was defined as a return to insulin therapy.

Results: 364 SPK (simultaneous pancreas kidney transplants), 111 IP and 10 re-transplants were performed. 62 transplants (12.8%) were performed using donors after circulatory death (DCD), of which 39 (8.0%) were used in IP transplants. The median recipient was female, 43.3 years old and BMI 23.9 kg/m2. The median donor was a 43.5 years old with BMI 23.9 kg/m2. The mean CIT was 688 mins. Overall 46 recipients died (9.5%), with 1- and 3- year patient survival at 95% and 90% respectively in the SPK group and 95% and 92% in the IP group. Overall 81(16.7%) pancreas grafts failed with graft survival at 1- and 3 years at 88% and 86% in the SPK group, and 79% and 72% in the IP group. In a univariate analysis, donor DCD status was the only significant predictor of pancreas graft survival, with other factors reaching only borderline significance. However, in a multivariate analysis DCD status lost significance and recipient male gender emerged as the only predictor of pancreas graft failure (HR 2.00, p=0.016).

Conclusion: Our graft and patient outcomes are in line with those reported by OPTN. Careful practice in recipient assessment and donor selection has meant that commonly recognised risk factors have been controlled for appropriately. Further optimisation of pancreas graft outcomes will require the identification of novel markers and predictors of pancreas graft failure in the pre and post-transplant setting.


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Pancreas Transplantation in African American Compared to Non African American Recipients: Does Recipient Ethnicity Influence Outcomes?

Jeffrey Rogers1, Alan Farney1, Giuseppe Orlando1, Umar Farooq1, Yousef Al-Shraideh1, Amber Reeves-Daniel1, Amudha Palanisamy1, William Doares1, Scott Kaczmorski1, Samy Iskandar1, Michael Gautreaux1, Robert J. Stratta1

1Wake Forest University, School of Medicine, Wiston-Salem, United States.

Introduction: African American (AA) ethnicity is a risk factor for inferior outcomes following kidney transplantation. The purpose of this study was to analyze outcomes according to recipient ethnicity following pancreas transplantation (PTx).

Methods: We retrospectively studied 202 consecutive PTxs in 192 patients (pts) at our center. All pts received either r-ATG or alemtuzumab induction in combination with FK/MMF, and tapered steroids or early steroid withdrawal. 179 PTxs (89%) were performed with portal-enteric and 23 with systemic-enteric drainage.

Results: From 11/01 to 3/13, we performed 162 simultaneous kidney-PTx transplants (SKPT), 35 sequential PTx after kidney (PAK) and 5 solitary PTx alone (PTA). All but 4 pts received kidney and PTxs either simultaneously or sequentially. 186 PTxs (92%) were primary and 16 PTx retransplants. Indications for PTx were insulin-requiring diabetes with complications and the predicted ability to tolerate the procedure and requisite immunosuppression irrespective of C-peptide production. A total of 39 PTxs (36 SKPT, 2 PAK, 1 PTA) were performed in AA recipients and the remaining 163 in non AA recipients (161 Caucasian, 1 Asian, 1 Hispanic). The AA group had fewer solitary PTxs (8% AA vs 23% non AA, p=0.04), more PTxs performed with systemic-enteric drainage (23% AA vs 9% non AA, p=0.02), more pts with a current PRA ≥10% (28% AA vs 10% non AA, p=0.008), more pts with 5-6 HLA mismatches (64% AA vs 42% non AA, p=0.01), more pts with a body weight ≥80 kg (51% AA vs 24% non AA, p=0.001), more pts with detectable C-peptide levels (≥ 2.0 ng/ml) at the time of PTx (36% AA vs 14% non AA, p=0.001), and more pts with a shorter duration (≤18years) of pretransplant diabetes (38% AA vs 17% non AA, p=0.004). The latter 3 differences suggested that a type 2 diabetes phenotype was more prevalent in the AA group. With a mean follow-up of 5.5 years, overall pt (90% AA vs 86.5% non AA), kidney (67% AA vs 77% non AA, p=0.21) and pancreas graft survival (59% AA vs 67% non AA, p=0.35) rates were comparable. The rates of early PTx thrombosis were 10% and 7%, respectively, in the AA and non AA groups. Cumulative clinical acute rejection rates were similar between groups (33% AA vs 27% non AA). However, the incidence of death-censored dual graft loss was much higher in AA pts (22% AA vs 6% non AA, p=0.0095).

Conclusions: PTx in AA recipients is characterized by fewer solitary PTxs and PTxs with portal-enteric drainage, more patients with detectable HLA antibodies and C-peptide levels at the time of PTx, more HLA-mismatching, and more pts with a type 2 diabetes phenotype. Although survival rates and the incidences of early thrombosis and acute rejection are similar, AA pts are at a greater risk for dual graft loss in the absence of mortality.

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Deleterious Effect of Delayed Graft Function on Pancreas Graft Survival Following Pancreas Transplantation

Sung Shin1

1Department of surgery, Asan Medical Center, Seoul, Korea.

Objective: Compared with the impact of delayed graft function (DGF) in renal transplantation, that in pancreas transplantation has not been fully evaluated. In this study, we verified the impact of DGF in surgically successful cases on long-term pancreas graft survival.

Methods: Between January 1999 and April 2013, we have performed 174 technically successful primary pancreas transplants. DGF was defined as total, cumulative insulin requirement of > 50 U within postoperative day 20.

Results: Of these 174 recipients, 49 (28%) had DGF after pancreas transplants. The incidence of DGF was similar in the three transplant categories: simultaneous pancreas kidney (29.8%), pancreas after kidney (25%), and pancreas transplant alone (25%) (p = NS). By multivariate analysis, DGF was associated with donor age >30 years (odds ratio [OR] 4.33, 95% confidence interval [CI]: 2.05 – 9.12, p < 0.001) and recipient body mass index (BMI) >20 (OR 2.61, 95% CI: 1.22 – 5.59, P = 0.014). There were no significant differences between recipients with and without DGF in acute rejection and mortality rates. In recipients with DGF, pancreas graft survival at 1, 5, and 10 years posttransplantation was 89%, 67%, and 52% respectively, while it was 95%, 93%, and 75% respectively in those without DGF (p=0.003). DGF was also associated with a higher risk of death-censored graft failure (p=0.025).

Conclusions: Risk factors for DGF following pancreas transplantation were donor age >30 years and recipient BMI >20. DGF was associated with a greater risk of overall graft failure and death-censored graft failure.

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Pancreas Re-Transplantation Graft Survival is Comparable to Primary Pancreas After Kidney Transplantation

John Seal1, Markus Selzner1, Max Marquez1, Fateh Bazerbachi1, Markus Boehnert, Jeffrey Schiff1, Andrea Norgate1, Mark S. Cattral1

1Surgery, Toronto General Hospital, UHN, Toronto, ON, Canada.

Reports on the outcomes of pancreas re-transplantation are scarce in the modern era. We compared the outcome of pancreas re-transplantation with primary pancreas after kidney (PAK) transplantation at a high volume North American center.

Retrospective analysis of 70 consecutive pancreas only transplants performed at a North American center from 2003 to May 2012. Primary pancreas after kidney transplants (PAK; n=56) were compared to pancreas re-transplantation (PRT) after pancreas graft loss (n=14). Patient and graft survival were assessed at 1 and 3 year follow-up. Pancreas graft function was determined by HbA1c. Serum creatinine was used to determine kidney graft function. Graft survival, and patient survival were calculated by log rank analysis.

There was no statistical difference in recipient or donor characteristics between the 2 groups including recipient sex, age, weight, and donor age. Pancreas graft cold ischemic time (7.75 vs. 8.25 h p=0.76) and warm ischemic time (26.76 vs. 31 min p=0.51) were comparable. Pancreas graft survival at 1 and 3 year follow-up was similar between PAK (91%, 88%) and PRT (100%, 100%) respectively (p=NS). There were no graft failures in the PRT group during the study interval with a mean follow-up of 35.3 months. Causes of graft loss in the PAK group were rejection (n=7) duodenal leak (n=4) and graft thrombosis (n=3). There was no difference in post-operative length of stay (12.7±11.3 vs. 10.7±6.6 d; p=0.54). There was no difference in the rate of post-operative complications in the first 6 months, except for duodenal leaks, which was lower in the re-transplant group (p=0.03). At 3-years follow-up PAK vs PRT patients had comparable HBA1c (0.08 vs 0.05; p=0.8) and creatinine (116.6 v 131.7; p=0.09). Pancreas re-transplantation did not adversely affect kidney transplant function in the recipient.

In conclusion, pancreas re-transplantation is safe and effective regardless of the cause of prior graft loss and has comparable outcomes to primary pancreas after kidney transplantation.

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Elevated BMI does not Affect Outcome in Type II Diabetics Undergoing Whole Organ Pancreas Transplantation

William Bry1, Harish Mahanty1, Parul Patel1, V. Ram Peddi1, Assad Hassoun1, Nikole Neidlinger1, Lawrence Lu1, Lawrence Bohannon1, Nina Topic1, Steven Katznelson1

1California Pacific Medical Center, San Francisco, CA, United States.

Introduction: The UNOS Pancreas Transplant Committee is formulating allocation rules for whole organ pancreas transplantation. Controversy exists in the role of pancreas transplantation for Type II diabetics. The current plan is to limit transplantation in Type II diabetics to patients with a BMI less than 30.

Objective: Over the past six years, we have transplanted 44 Type II diabetics. The diagnosis was based on clinical history (diabetes onset, length of insulin dependence, and C-peptide levels). 41 were SPK’s; 3 were Pancreas after SPK: two for thrombosis and one for leak of the original SPK. This report compares the outcome of thirty-five patients with a BMI less than 30 to nine patients whose BMI exceeded 30.

Outcome: Demographics and Results at Mean f/u >3years


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Losses in the low BMI group all occurred in the first 2 months; losses in the high BMI group all occurred after the first year. One and three year graft survival in the high BMI group is 100% and 71%; in the low BMI group 83% and 83%. Importantly, HbA1C values in the high BMI patients with functioning grafts are all normal regardless of their pre transplant insulin dose. Only one kidney was lost in all 44 patients aside from the two patient deaths with function. None of these comparisons between the groups reached statistical significance. There was a trend towards more deep space infections in the Type ll patients.

Conclusion: Sampaio et al. [1] compared 582 Type ll diabetics to 6141 Type l recipients in the SRTR database from 2000 to 2007 and found no difference in patient and graft survivals. They reported an increased risk of pancreas graft loss with elevated BMI in both Type l and ll recipients. Our report has the advantage of being a single center study but lacks the numbers to make a definitive conclusion. Our data does not support a BMI cutoff of 30 for whole organ pancreas transplantation in type II diabetics

Reference:

  • [1] Marcelo Santos Sampaio, Hung-Tien Kuo, and Suphamai Bunnapradist: outcomes of simultaneous pancreas-kidney transplantation in type 2 diabetic recipients. Clin J Am SOC Nephrol 6: 1198-1206, 2011
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Outcomes of a new Category of Pancreas Transplantation; Pancreas After Islet Transplantation (PAI)

James Gardner1, Garrett Roll2, Bruncelia Hynson2, Andrew Posselt2, Sang Mo Kang2, Chris Freise2, Sandy Feng2, Ryutaro Hirose2, Peter Stock2

1Dept of Surgery, University of California San Francisco, San Francisco, CA, United States; 2Dept of Surgery, Division of Transplant, University of California San Francisco, San Francisco, CA, United States.

Introduction: There are an increased number of treatment options for beta cell replacement in type 1 diabetic patients. Unfortunately, there are no established protocols to achieve insulin independence via re-transplantation in patients with late failure of islets. Some patients become sensitized after islet infusions, theoretically making further transplantation efforts more challenging. This abstract shows the success of pancreas transplantation after islet infusion, even in highly sensitized recipients.

Methods: From 2007 to 2011, 5 pancreas after islet transplants (PAI) were performed in preuremic type 1 diabetic patients with a prior history of hypoglycemic unawareness. Pancreas transplants were performed with portal and enteric drainage. Immunosuppression included thymoglobulin induction (6mg/kg), maintenance with low dose tacrolimus (trough 5-7ng/ml), MMF (1 gm/d), low-dose sirolimus (trough 5-7 ng/ml), and prednisone 5 mg/d. Low dose tacrolimus was utilized to prevent nephrotoxicity. Protocol pancreas biopsies were performed between 3 and 4 months or for clinical indications.

Results: All 5 patients achieved insulin independence, and at a mean follow-up of 44 months remained insulin independent with normalized Hgb A1C (table 1). There was no deterioration of renal function in these preuremic type 1 diabetic recipients. There were 2 episodes of rejection. The average number of previous islet infusions was 1.8 batches.

Conclusion: Aggressive immunosuppression and close monitoring with protocol biopsies yield excellent results in this new category of PAI. Insulin independence was achieved in all prior islet recipients with failed grafts, despite the pre-sensitized state. Tacrolimus minimization prevented deterioration of native renal function. Pancreas after islet transplantation provides a strategy for achieving insulin independence in Type I diabetic patients with prior hypoglycemia unawareness and failed islet allografts.


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Evaluation of Pancreatic Graft Survival Using 11C-Methionine Positron Emission Tomography After Living Donor and Cadaveric Donor Pancreas Transplantation

Kazunori Otsuki1, Kyosan Yoshikawa2, Takashi Kenmochi3, Naotake Akutsu1, Kenichi Saigo1, Michihiro Maruyama1, Masayuki Hasegawa1, Hiromichi Aoyama1, Ikuko Matsumoto1, Takehide Asano1, Taihei Ito3, Yoshio Uchino4

1Surgery, National Chiba-East Hospital, Chiba, Japan; 2National Institute of Radiological Sciences, Chiba, Japan; 3Transplantation Surgery, Fujita Health University, Nagoya, Japan; 4PET imaging division, Chiba Ryogo Center, Chiba, Japan.

Objective: We have recently reported 11C-methionine positron emission tomography (PET) is clinical useful for the evaluation of the living donor pancreatic function.

The aim of this study is to evaluate the postoperative insulin independence of living donor (LD) and cadaveric donor (CD) pancreas transplantation in eighteen Type I diabetes mellitus using 11C-methionine PET.

Methods: This study included10 LD pancreas transplantation and 8 CD pancreas transplantation; 9 LD simultaneous pancreas kidney (SPK) transplantation, 1LD pancreas transplantation alone (PTA), 5 CD SPK, 2 CD PTA and 1 pancreas after kidney (PAK) transplantation. The pancreas graft excretion was managed to bladder drainage (BD) for all LD transplants and enteric drainage(ED) for all CD transplants. Systemic venous drainage was operated to all cases.

PET examination was performed at 6 months after surgery. PET was scanned at 30 minutes after 11C-methionin (370 to 740MBq) injection, 11C-methionine PET uptakes of pancreas was expressed as standardized uptake value (SUV).

Results: Patient survivals were 100% at 5 year in LD transplants and 100% at 2 year in CD transplants. Graft survivals (insulin independence) were was 60% (six cases) in LDs at 5 year and 75% (six cases) in CDs at 2 year, respectively. There were no major surgical complications such as vascular thrombosis, intra-abdominal abscess and graft pancreatitis. Insulin dependence of LD and CD pancreas transplantation was observed in 40% and 25%, respectively. The SUVs of LD and CD pancreas transplants with insulin independence were 7.2+/−1.8 and 11.0+/−2.6. The SUVs of LD and CD pancreas transplants with insulin dependence were 3.6+/−1.1 and 2.9+/−1.0.

The SUVs of insulin independence transplants were significantly higher than the SUVs of insulin dependence transplants (p<0.05). Furthermore, all cases with insulin dependence indicated less than 5 of SUVs after transplantation.

Conclusion: The recipients who showed less than 5of SUVs have high risks of insulin dependence andthe potential graft failure.To maintain insulin independence and fair glycemic controls, the higher levels of SUVs may be needed for a prolonged period. The 11C-methionine PET may become a potent modality to offer further information for the avoidance of pancreas graft failure.

Reference:

  • [1] Otsuki K, Yoshikawa K, Kenmochi T, et al: Evaluation of segmental pancreatic function using 11C-Methionine positron emission tomography for safe living donor operation of pancreas transplantation. Transplantation proceedings 2011,43:3273–6.
  • [2] Otsuki K, Yoshikawa K, Kenmochi T, et al: Evaluation of pancreatic function in normal pancreas as living-related donors and type I diaberic pancreas as recipients for pancreas transplantation using 11C-Methionine positron emission tomography. Pancreas 2010,39:418–419.
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The Metabolic Effects of Tacrolimus and Sirolimus on Insulin Secretion and Insulin Resistance in Pancreas-Kidney Transplant Recipients

Richard Knight1, Archana Sadhu2, Jennifer Devos3, Samir Patel3, Linda Moore1, Osama Gaber1

1Surgery, The Methodist Hospital, Houston, TX, United States; 2Medicine, The Methodist Hospital, Houston, TX, United States; 3Pharmacy, The Methodist Hospital, Houston, TX, United States.

Background: There is controversy regarding the best immunsuppressive regimen for glucose metabolism in pancreas transplant recipients. Both tacrolimus and sirolimus have known diabetogenic effects by reducing insulin secretion, promoting beta cell apoptosis and causing insulin resistance. We sought to determine the metabolic effects of tacrolimus and sirolimus on insulin secretion and resistance in pancreas-kidney recipients.

Methods: We studied pancreas-kidney transplant recipients, maintained on 3 different immunosuppression regimens. Tacrolimus with mycophenolate (target trough level 6-8 ng/ml), Sirolimus with mycophenolate (target trough level 8-10 ng/ml), and Sirolimus with reduce-dose tacrolimus (combined target trough level 8-10 ng/ml). All grafts had systemic venous and enteric exocrine drainage. Fasting glucose, insulin, pro-insulin, c-peptide, and hgbA1c levels were compared between groups. Additionally, the homeostatis model of assessment-insulin resistance (HOMA-IR) was calculated and compared between groups using the formula (glucose (mg/dl) X insulin (uIU/L) ÷ 405.

Results: Thirty-four recipients were studied at a median of 27 months post-transplant (range 9-104 months). Mean±SD values are shown in the table below. None of the recipients required insulin or oral-hypoglycemic agents.


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Percent of recipients with evidence of insulin resistance as defined by a HOMA index >3: tacrolimus 58%, sirolimus 60%, and TAC/Sir combination 42% (p=ns).

Conclusion: Tacrolimus, sirolimus, and a combination of both agents provided equivalent glucose metabolism outcomes. Despite good glycemic control, there was evidence of insulin resistance by HOMA-IR in all groups studied.

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Sonographic Findings Following Pancreas Transplantation

Tara Morgan1, Jack Harbell, Peter Stock, Vickie Feldstein1

1Radiology, UCSF, San Francisco, CA, United States.

Introduction: The rate of pancreas transplantation, often done simultaneously with renal transplant in patients with diabetes, is increasing. Ultrasound (US) has an important role in assessing pancreas transplants following surgery [1]. Possible post-operative complications include splenic vein (SV) thrombosis, often identified by Doppler US. This sonographic finding, its clinical relevance, and appropriate management require further investigation [2,3].

Materials & Methods: Institutional Review Board permission was obtained prior to this research. The 50 most recent pancreas transplant recipients from 2010-2012 were retrospectively reviewed. For each patient, their serial post-operative US were reviewed by 2 investigators (TM, VAF), who were blinded to the clinical outcomes. Findings including transplant edema, peri-pancreatic fluid collection, and vascular abnormalities, specifically absent flow, SV thrombus and/or high resistance arterial flow were noted. A grading scheme for SV thrombus was created based on length of involvement, with grade 1: <25%, grade 2: >25 and <50%, grade 3: >50 and <75%, and grade 4:>75% thrombus. Findings were then correlated with clinical outcomes.

Results: 7 PAK, 3 PTA, and 40 SPK were performed. 49/50 patients had at least one post operative US, 44 from POD 0-7. 8 studies were read as normal, 21 with transplant edema, 20 with peri-transplant collections, and 17 with SV thrombosis. 4 grade 1, 7 grade 2, 3 grade 3, 3 grade 4 SV thromboses were present. In 6 cases, arterial flow was also affected. 13/17 cases presented from POD 0-8, 4 presented in a delayed fashion, POD 28 or later. 16/17 cases of SV thrombosis were reimaged, 12 within 0-7 days. 8 SV thromboses were unchanged, 7 improved or resolved, 1 worsened, and 1 was lost to follow up. All patients with SV thombosis were anticoagulated. 10 patients had no subsequent significant hemorrhage. 4 patients developed GI bleed, 2 had hematomas requiring evacuation, and 5 patients required blood transfusion of 2 or more units. One patient had a GI bleed and hematoma requiring evacuation for which anticoagulation was stopped and afterwards developed SV thrombosis. One patient underwent a successful thrombectomy and one transplant was lost, removed by pancreatectomy 1 day after diagnosis of a Grade 4 SV thrombosis. All patients with hemorrhagic complications presented within the POD 0-7. 9 patients were discharged on Coumadin, none with subsequent significant hemorrhage.

Conclusion: SV thrombosis is a common complication following pancreas transplantation occurring in about 34%. The majority involve approximately half of the SV (Grade 2,3) and remain stable over time. Hemorrhagic complications from anticoagulation tend to occur in the immediate post-operative period. Further investigation regarding the clinical implications and optimal management of SV thrombosis is required.

Reference:

  1. Gimenez JM, Bluth EI, Simon A, Troxclair L. Evaluation of pancreatic allografts with sonography. J Ultrasound Med. 2012 Jul;31(7):1041-51.
  2. Garcia-Roca R, Samamé J, Garcia-Criado MA, Real MI, Ricart MJ. Preservation of pancreas graft function after complete venous thrombosis: report of four cases treated conservatively. Transplantation. 2012 Jan 27;93(2):214-8.
  3. Delis S, Dervenis C, Bramis J, Burke GW, Miller J, Ciancio G. Vascular complications of pancreas transplantation. Pancreas. 2004 May;28(4):413-20.
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5 Year Results of a Prospective Randomized Single Center Study of Alemtuzumab Compared to Rabbit Anti-Thymocyte Globulin Induction in Kidney-Pancreas Transplantation

Robert J. Stratta1, Jeffrey Rogers1, Lois Hart1, William Doares1, Scott Kaczmorski1, Amber Reeves-Daniel1, Giuseppe Orlando1, Umar Farooq1, Yousef Al-Shraideh1, Amudha Palanisamy1, Samy Iskandar1, Alan Farney1

1Wake Forest University, School of Medicine, Wiston-Salem, United States.

Introduction: Alemtuzumab (Alem) and rabbit anti-thymocyte globulin (rATG) are the two most commonly used antibody induction agents in simultaneous kidney-pancreas transplantation (SKPT). The purpose of this study was to analyze our 5 year single center outcomes in a prospective, randomized trial of Alem vs rATG induction in SKPT.

Methods: From 2/05 through 10/08, 46 SKPTs (45 with portal-enteric drainage) were prospectively randomized to receive either single dose Alem (30 mg intra-operatively) vs multiple dose rATG antibody induction (starting intra-operatively, minimum of 3 doses administered) in combination with FK/MMF maintenance immunosuppression; PRA>20%, re-transplant, or African Americans <40 years of age defined high immunologic risk and determined steroid maintenance (n=11) or early elimination (n=35).

Results: Of 222 kidney or SKPT patients (pts) enrolled in the study, 46 received SKPTs; 28 (61%) received Alem and 18 (39%) rATG induction. Follow-up in SKPT pts ranged from 54 to 98 months (mean 69 months). There were no significant differences between the 2 groups in 1 year (yr, 93% Alem vs 100% rATG) or 5 yr (82% Alem vs 89% rATG) pt survival; 1 yr (93% Alem vs 94% rATG) or 5 yr (79% Alem vs 72% rATG) kidney graft survival; or 1 yr (82% Alem vs 83% rATG) or 5 yr (64% Alem vs 56% rATG) pancreas graft survival rates (all p=NS). Actual death-censored kidney (90% Alem vs 75% rATG) and pancreas (71% Alem vs 56% rATG) graft survival rates were likewise comparable (both p=NS). The overall acute rejection (21% Alem vs 44% rATG, p=0.11) and major infection (39% Alem vs 67% rATG, p=0.13) rates were slightly lower in the Alem group. CMV infections were significantly lower (0 Alem vs 17% rATG, p=0.05), and bacterial and fungal infections were slightly lower in the Alem group. Post-operative bleeding (11% Alem vs 0 rATG, p=0.27) was slightly more common in the Alem group. There were no differences in early pancreas thromboses (3.6% Alem vs 11% rATG), other surgical complications, or readmissions between groups. In pts with functioning grafts, 5 yr mean serum creatinine (1.4 Alem vs 1.6 mg/dl rATG), calculated aMDRD GFR (55 Alem vs 52 ml/min rATG), glycohemoglobin (both 5.4%) and C-peptide (2.2 Alem vs 2.3 ng/ml rATG, all p=NS) levels were similar.

Conclusions: Single dose Alem and multiple dose rATG induction provide similar long-term pt, kidney, and pancreas graft survival rates and similar following SKPT. Alem induction may be associated with less acute rejection and infection long-term but more bleeding complications short-term.

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Single Center Analysis of Mortality in 202 Consecutive Pancreas Transplants

Robert J. Stratta1, Alan Farney1, Giuseppe Orlando1, Umar Farooq1, Yousef Al-Shraideh1, Amber Reeves-Daniel1, Amudha Palanisamy1, William Doares1, Scott Kaczmorski1, Jeff Rogers1

1Wake Forest University, School of Medicine, Wiston-Salem, United States.

Introduction: Pancreas transplantation (PTx) remains an important treatment option for selected insulin-requiring diabetic patients (pts) with “complicated” diabetes. For pts with end stage diabetic nephropathy, mortality on the waiting list may be as high as 10% annually.

Methods: We retrospectively reviewed survival outcomes in 202 consecutive PTxs performed in 192 pts at our center. All pts received either r-ATG or alemtuzumab induction in combination with FK, MMF, and tapered steroids or early steroid withdrawal.

Results: From 11/01 to 3/13, we performed 162 simultaneous kidney-PTxs (SKPT), 35 sequential PTxs after kidney (PAK), and 5 solitary PTx alone (PTA). All but 4 pts received kidney and PTxs either simultaneously or sequentially. With a mean follow-up of 5.5 years, overall pt survival was 86.5%; mortality was similar (13%) following SKPT or solitary PTx. Mortality rates were likewise similar following primary (13.5%) vs PTx retransplants (6.25%, p=NS). One-year mortality was 3% following SKPT vs 0 for solitary PTx (p=NS). In SKPT pts, 15/21 (71%) died with functioning grafts (DWFG) whereas 0/5 pts following solitary PTx (p=0.007) had both grafts functioning at the time of death (4 had prior kidney graft loss [GL] and 3 had prior PTx GL). Of the 26 total deaths, 15 were DWFGs, 3 died after kidney GL, 6 died after PTx GL, and 2 following asynchronous kidney and PTx GL. In the absence of either kidney or PTx retransplant, mortality rates following isolated kidney, isolated PTx, and KPTx GL were 33%, 24%, and 17%, respectively (p=NS). However, 6 pts underwent successful kidney and 11 PTx retransplant; mortality following either kidney or PTx retransplant or both was 5%. 3 SKPT pts died early (within 5 months) of infection secondary to technical issues. Of the remaining 23 deaths that occurred ≥6 months post-PTx (mean 53 months), 11 were cardiovascular, 7 infectious, 2 malignancy, and 3 miscellaneous causes. The proportion of pts age 50 or older at the time of PTx was higher in those who died (42%) compared to survivors (23%, p=0.05).

Conclusions: Mortality rates following SKPT, solitary PTx and PTx retransplant are similar, usually occur late, and correlate with older recipient age. Following SKPT, the most common mortality pattern is DWFGs whereas mortality following PAK or PTA is heralded by either kidney or PTx GL or both. The most common causes of death are cardiovascular and infection regardless of PTx category. Kidney or PTx GL are both predictive of subsequent mortality whereas retransplant of either organ appears to reduce mortality.

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426

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Autoantibody Monitoring in the Follow-Up of Pancreas Transplant Recipients

Fanny Buron1, Valérie Dubois2, Olivier Thaunat1, Lionel Badet3, Lucienne Chatenoud4, Emmanuel Morelon1

1Transplantation, Néphrologie et Immunologie clinique, Hospices Civils de Lyon, Lyon, France; 2Laboratoire HLA, Etablissement Français du Sang, Lyon, France; 4Unité 1013, INSERM, Paris, France; 3Urologie et Chirurgie de la Transplantation, Hospices Civils de Lyon, Lyon, France.

Introduction: Whether autoimmune recurrence plays an important role in pancreas graft failure remains a matter of debate, mainly because of the lack of reliable diagnostic tools. The destruction of beta cells is indeed mediated by the cellular arm of recipients’ immune response, which is difficult to explore. Although devoid of a pathogenic role, circulating autoantibodies could be a useful marker. The aim of this study is to analyze the value of circulating autoantibodies to predict pancreas graft outcome.

Patients and Methods: Recipients of technically successful first pancreas transplantations performed in Lyon between January 2000 and December 2004 were enrolled. The immunosupressive regimen consisted in anti-thymocyte globulin, mycophenolate mofetil, tacrolimus, and prednisolone. Autoantibodies to glutamic acid decarboxylase, insulinoma associated protein 2 and zinc transporter 8 antigen were measured before, at 1 year and then every 2 years after transplantation and at the time of graft dysfunction. Graft survivals were compared using a Log-rank test.

Results: Seventy-six patients (73 SPK and 3 PAK recipients) were included, with a median follow-up duration of 7.5 years (range: 0.4-11.8). Thirty-eight (50%) patients had one or more autoantibodies before transplantation with no difference in pancreas graft survival, versus patients without autoantibodies (5-year survival 83.6% vs 83.9% respectively, p = 0.90). Autoantibodies appeared or significantly increased in 10 (13%) patients with a median onset of 3 years (range: 0.5-7). Graft survival was significantly lower in these patients with 5-year survival at 67.5% vs 86.1% in the other 66 patients (p = 0.01). The median period between the appearance or increase of autoantibodies and graft loss was 1.4 years (range 0-2.4).

Conclusion: While the presence of autoantibodies before pancreas transplantation has no impact on graft survival, their appearance or increased titer after transplantation is associated with lower graft survival. Autoantibody monitoring seems to be useful in the follow-up of pancreas transplant recipients.

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Improvement in Insulin Sensitivity Following Human Islet Transplantation for Type 1 Diabetes: Comparison of Euglycemic Clamp and Minimal Model Approaches

Michael Rickels1, Stephanie Kong1, Carissa Fuller1, Cornelia Dalton-Bakes1, Jane Ferguson2, Muredach Reilly2, Karen Teff1,3, Ali Naji4

1Medicine, Endocrine Divison, University of Pennsylvania, Philadelphia, PA, United States; 2Medicine, Cardiovascular Division, University of Pennsylvania, Philadelphia, PA, United States; 3Monell Chemical Senses Center, Philadelphia, PA, United States; 4Surgery, Transplantation Division, University of Pennsylvania, Philadelphia, PA, United States.

Islet transplantation is an emerging cell therapy for type 1 diabetes (T1D). We sought to determine whether improved metabolic control post-transplant would mitigate an anticipated reduction in insulin sensitivity attributable to immunosuppression drugs. We assessed insulin sensitivity by hyperinsulinemic euglycemic clamps and insulin modified frequently sampled intravenous glucose tolerance (FSIGT) tests, and analyzed how the FSIGT minimal model derived estimate of insulin sensitivity (SI) compared to those derived from the clamp. Pre- (n = 21) and post-intrahepatic islet transplant (n = 12) subjects receiving tacrolimus and sirolimus were studied, with results compared to normal controls (n = 14 clamp; n = 11 FSIGT). The transplant recipients received 9648 ± 666 islet equivalents/kg with reduction in HbA1c from 7.0 ± 0.3 to 5.6 ± 0.1% (P < 0.01) and 10/12 were insulin-independent. Clamp derived total body insulin sensitivity (M/ΔInsulin) increased from 0.11 ± 0.01 to 0.15 ± 0.02 dl/min·kg per μU/ml, and by the isotopic dilution method with 6,6-2H2-glucose, peripheral [(Rd-EGP)/ΔInsulin] sensitivity increased from 0.08 ± 0.01 to 0.12 ± 0.02 dl/min·kg per μU/ml and hepatic (1/EGP*basal insulin) sensitivity increased from 2.3 ± 0.1 to 3.7 ± 0.4 × 102 (mg/kg/min)-1·(μU/ml)-1 (P < 0.05 for all). FSIGT derived SI improved from 1.76 ± 0.45 to 4.21 ± 0.34 x 10-4 (μU/ml)-1·min-1 (P < 0.05). All measures were less than normal in T1D (P ≤ 0.05) and not different from normal post-transplant. The predictive power (r2) between SI and measures of total and peripheral insulin sensitivity was 0.66 and 0.70 respectively (P < 0.001 for both). Islet transplantation resulted in improved total body insulin sensitivity mediated by effects at both the liver and periphery. That SI correlated highly to the clamp derived measures indicates that the FSIGT is an appropriate methodology for the determination of insulin sensitivity in T1D and islet recipients.

This work was performed in part as a project of the Clinical Islet Transplantation Consortium, a collaborative clinical research program headquartered at the National Institutes of Health.

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Islet Transplantation Outcomes Similar in Islet After Kidney vs. Islet Alone in T1D Adjusted for Disease: Results from the Collaborative Islet Transplant Registry (CITR)

Rodolfo Alejandro1, Franca B. Barton2, Sharri Messinger-Cayetano3, Thierry Berney4, Jon Odorico5, Antonio Secchi6, Francois Pattou7, Michael Rickels8

1Clinical Islet Transplantation, Metabolic Studies Core, Division of Transplantation, Miller School of Medicine, University of Miami, Miami, FL, United States; 2The Collaborative Islet Transplant Registry, The EMMES Corporation, Rockville, MD, United States; 3Division of Biostatistics, Department of Epidemiology and Public Health; Biostatistics Collaboration and Consulting Core, Miller School of Medicine, University of Miami, Miami, FL, United States; 4Divisions of Transplantation and Visceral Surgery, Geneva Hospital, Geneva, Switzerland; 5Division of Transplantation/Department of Surgery, University of Wisconsin - Madison, Madison, WI, United States; 6Strategic Research Program on Transplantation, Department of Medicine, San Raffaele Institute, Milan, Italy; 7Department of General and Endocrine Surgery, Lille University Hospital, Lille, France; 8Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.

Background and Purpose: Clinical outcomes of islet transplantation may vary according to previous kidney transplant. The purpose of this investigation is to determine if outcomes of islet transplantation differ between islet after kidney (IAK) and islet-alone (IA) patients.

Patients/Methods: IA (N = 342) and IAK (N = 83) recipients of 1-3 islet transplants reported to the CITR 1999-2012 with non-detectable C-peptide (< 0.3 ng/ml) or severe hypoglycemia were characterized for long-term retention of insulin independence post last infusion, adjusting for baseline recipient, donor, and islet characteristics and cumulative immunosuppression. The two groups were similar in all baseline characteristics except: serum creatinine (1.0 ± 0.5 vs. 1.7 ± 1.4, p < 0.001); CKDEpi-eGFR (85 ± 23 vs. 57 ± 24, p < 0.001); A1c (5.5 ± 0.4 vs. 5.2 ± 0.4, p < 0.01); weight (66.7 ± 10.7 vs. 61.6 ± 8.9, p < 0.001); induction with IL2RA (49% vs 79%, p < 0.001); maintenance with IMPDH (17% vs. 36%, p < 0.001); and severe hypoglycemic events (84% vs 54%).

Results: First achievement of insulin independence (II) is similar in the two groups (CITR7th Annual Report Figure 5.1, www.citregistry.org), but there is an apparent reduction of retention of II (Exhibit A) for the IAK/SIK group. Adjusted risk ratios estimated with a Cox model (Exhibit B) suggests that this apparent difference is accounted for by adjustment for other variables, namely age, insulin requirement, and induction immunosuppression.

Conclusion: Outcomes of islet transplantation can be expected to be similar in simultaneous or after-kidney compared to islet-alone recipients, and are similarly subject to patient selection criteria and management strategies.

FIGURE 1.
FIGURE 1.
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The Collaborative Islet Transplant Registry Investigators (CITR) Investigators

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Factors Predictive of Clinical Outcomes Following Single Islet Allograft Infusion in Type 1 Diabetes from the Collaborative Islet Transplant Registry

Bernhard Hering1, Franca B Barton2, Sharri Messinger-Cayetano3

1Division of Transplantation, Diabetes Institute, University of Minnesota, Minneapolis, MN, United States; 2The Collaborative Islet Transplant Registry, The EMMES Corporation, Rockville, MD, United States; 3Biostatistics Collaboration and Consulting Core, Department of Epidemiology and Public Health of the Miller School of Medicine, University of Miami, Miami, FL, United States;

Background: Most human islet allograft recipients have received more than one islet transplant. However,protocols that promote sustained islet allograft function after single transplants would increase the safety, cost effectiveness, and availability of cell replacement therapy in type 1 diabetes. To determine whether single islet allograft infusions can suffice to achieve and maintain long-term (LT, i.e., over all infusions) islet graft function and which factors are most predictive of long-term success with single islet allograft infusion, the Collaborative Islet Transplant Registry (CITR) analyzed donor, islet graft, and recipient characteristics and available outcome data in 321 CITR islet-alone allograft recipients who had C-peptide < 0.3ng/mL at baseline with or without severe hypoglycemia.

Methods: We modeled by Cox proportional hazards several outcomes –achievement and retention of of insulin independence, retention of C-peptide > 0.3 ng/mL, and protection from severe hypoglycemic events (SHE) – on all baseline recipient, donor and islet factors, plus reinfusion as a time-dependent covariate. We then generated event rates for two groups for each of these outcomes: one with all favorable and one with all unfavorable factors, observed from the data.

Results: Overall, 68% of CITR negative C-peptide, islet-alone recipients received islet re-infusion. The model estimates that 64% can achieve insulin independence after single infusion (censored at reinfusion) with IEQ ≥ 8,000/kg; recipient age ≥ 35 and donor BMI ≥ 25are also significant favorable factors. Over all follow-up including reinfusion, favorable vs unfavorable factors (age ≥ 35 HR = 2.38; IEQ/kg ≥ 5,200 HR = 1.48; p < 0.001) at first transplant yielded 84% vs. 65% insulin independence achievement at 40 months; reinfusion boosted this rate (HR = 4.08, p < 0.001; not shown). For LT retention of insulin independence through all follow-up including re-infusion (Exhibit A), favorable factors at first transplant included age ≥ 35 (HR = 2.04, p = 0.02) and induction with TCD with/without TNFaInh (HR = 1.75, 0.03); reinfusion appeared to be associated with lower retention of insulin independence (HR = 0.54, p = 006), but this result may misleading because reinfusion and loss of insulin independence are confounded. Importantly, for N = 27 patients ≥ 35 yo given TCD + TNFaInh and >= 5,200 IEQ/kg at first transplant showed that reinfusion did not increase retention of insulin independence (p = 0.78). For LT retention of C-peptide > 0.3 ng/mL (Exhibit B), 80% vs. 53% success at 5 years is achievable. For SHE, prior graft loss is the only significant predictor of loss of SHE protection, which exhibits sustainably high rates: 93% of those without prior graft loss vs. 75% with prior graft loss retained LT protection from SHE (Exhibit C).

Conclusion: Very favorable outcomes can be achieved and maintained with single islet allograft infusion in T1D recipients. Additional protocol refinements are needed to achieve comparable outcomes in recipients in whom not all identified favorable factors are met. These data might have important implications for donor pancreas allocation and islet transplant protocol refinement and stratification.


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433

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Long Acting GLP1 Therapy in Islet Transplant Recipients

Eduardo Moraes Leao Peixoto1, Leonor Mireles-Zavala1, Eva Herrada1, Camillo Ricordi1, Rodolfo Alejandro1

1Diabetes Research Institute, University of Miami, Miami, FL, United States.

Background: The Glucagon like Peptide-1(GLP-1) analogs delays gastric emptying, inhibits glucagon secretion and hepatic glucose production. Our center switched subjects from a short-acting (exenatide) to a long-acting GLP-1(liraglutide) in an attempt to improve metabolic outcomes, adherence and minimize side effects.

Methods: Insulin, glucose, C-peptide, acetaminophen, glucagon and GLP-1 responses to a 300min Mixed Meal Tolerance Test (MMTT) were measured at 0; 6 and 12months(m), in addition to weight and HbA1c in 6 islet transplant recipients. Subjects discontinued exenatide prior to 0m and commenced liraglutide up to 1.2mg daily. Glucagon response was compared to previous exenatide treatment data. Data was analyzed using ANOVA and multiple comparisons.

Results: GLP-1 means at 0, 6 and 12m were 8.2 ± 3.1; 22.9 ± 11; 14.9 ± 4.2pM/L respectively (P < 0.0001 and P = 0.0028 when 0m is compared to 6 and 12 m). Fasting GLP-1 fold increase at 6m and 12m were 24.4 ± 45.2 and 10.76 ± 17.88 respectively. HbA1c at 0; 6 and 12m was 6.0 ± 0.30; 6.3 ± 0.4; 6.6 ± 0.4%. Weight loss was observed in 67%(-3.6 ± 5.9lbs) at 6m and 33% at 12m (-1.38 ± 4.9lbs). Abnormal glucagon responses to MMTT persisted during Liraglutide treatment in contrast to the inhibition observed with Exenatide. There were no significant differences in glucose, C-peptide responses and acetaminophen absorption.

Conclusions: Liraglutide therapy was well tolerated; resulted in minimal weight loss, despite increase in appetite. It also increased fasting and post-MMTT GLP-1 levels. As expected, no delay in gastric emptying was observed. The glucose control remained stable; however, Liraglutide did not inhibit the abnormal glucagon response to MMTT. As seen with the gastric effect in native GLP1 continuous infusion studies, tachyphilaxis on glucagon receptors might be responsible for the absence of glucagon inhibition. Randomized, long term studies, comparing long and short-acting GLP1 agents are needed to corroborate these findings.

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Reversible, Moderate and Early Alteration of Kidney Function in Non-Uremic Type 1 Diabetic Islet Recipients under Tacrolimus-Mycophenolate Maintenance Therapy

Pieter Gillard1,2, Maria Rustandi1, DaHae Lee1,2, Achmad Efendi3, Zhidong Ling2, Robert Hilbrands2, Dirk Kuypers4, Chantal Mathieu1, Daniel Jacobs-Tulleneers-Thevissen2, Frans Gorus2, Daniel Pipeleers2, Bart Keymeulen2

1clinical and experimental medicine, University Hospital Leuven-KUL, Leuven, Belgium; 2Diabetes Research Center, University Hospital Brussels-VUB, Brussels, Belgium; 3I-Biostat, Biostatistical Centre, University of Leuven-KUL, Leuven, Belgium; 4Department of Nephrology and Renal Transplantation, University Hospital Leuven-KUL, Leuven, Belgium.

Transplant patients on Tacrolimus (tac) are known to exhibit reduced estimated glomerular filtration rate (eGFR). Dynamics and possible reversibility of this effect need to be assessed in light of type of graft and treatment protocol.

The present single center study is conducted in 48 non-uremic type 1 diabetic recipients of an intraportal islet-cell graft under maintenance immunosuppression with tac and mycophenolate mofetil (MMF). eGFR and albuminuria were followed up to 5 years posttransplantation (PT) during which immunosuppressants were stopped in 21 patients because of graft failure.

Mean eGFR values decreased by 19 ml/min/1.73m2 between PT week 1 and 2 (p < 0,0001) and then remained stable throughout the treatment period. The decrease was related to tac-levels and disappeared upon its discontinuation; it was associated to the presence of albuminuria at the time of transplantation. Tac treatment resulted in a reduction in the degree of albuminuria; this effect disappeared after discontinuation.

In non-uremic patients, use of tacrolimus in our islet-cell transplant protocol caused a 20% reduction in eGFR which appeared reversible following discontinuation within the 5 year follow-up period. Associated changes in albuminuria are consistent with tac-induced vasoconstriction. These observations support use of tac-MMF in this patient population.

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435

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Improved Glycaemic Control with Concurrent Reduction in Calorific Intake and Fat Mass Following Islet Transplantation in Subjects with Type 1 Diabetes from Islets Donated after Circulatory Death and Brain Death in the Scottish Islet Transplant Programme.

Shareen Forbes1,2,3, Debbie Anderson1, Kirsty Duncan2, Calum Gray3, Janet Barclay1, Tammie McGilvary2, Donna Mitchell4, Alan Timpson4, Lora Irvine4, Peter Henry4, Laura Bailey4, George Galea4, Neil McGowan4, John Casey2,3

1Diabetes and Endocrinology, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom; 2Transplant Unit, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom; 3Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom; 4Scottish National Blood Transfusion Service, Edinburgh, United Kingdom.

Introduction: We hypothesised equivalent metabolic outcomes in recipients with islets donated after brain death (DBD) versus after circulatory death (DCD) and that following islet transplantation in subjects with Type 1 diabetes with recurrent hypoglycaemia, improved glycaemic control would be associated with a reduction in fat mass (FM) secondary to the reduction in hypoglycaemia and calorific intake.

Methods: Islets were isolated under GMP conditions using Edmonton protocols. Subjects were assessed pre- and post-transplant. Hypoglycaemic histories, continuous glucose monitoring systems (CGMS), HbA1c and insulin dose along with dietary histories, body weight, body mass index (BMI), %FM (bio-electrical impedance), abdominal FM (Magnetic Resonance (MR) imaging) and waist circumference were recorded. The most recent clinic assessments (range 1-24 months) were recorded.

Results: Twelve c-peptide negative patients received 20 islet transplants. Data collated for n = 9 patients (1 recipient single, 8 recipients two grafts including 3 DCD isolations (mean ± SEM): 10,131 ± 904 IEQ/kg; no difference in islet yield in DCD vs. DBD (p > 0.05)) shown. Engraftment was successful in all patients (stimulated 90 minute c-peptide: 635 ± 122 pmol/l; - no difference DCD vs. DBD with 2 DCD recipients achieving insulin independence). Frequency of hypoglycaemia decreased: 7 ± 3 to 1 ± 1 episodes/week, with improved awareness of hypoglycaemia (p < 0.01). The mean amplitude of glycaemic excursions (MAGE), Hba1c and insulin dose decreased post-transplant: (pre- vs. post-transplant: MAGE: 6 ± 0.6 mmol/l vs. 4 ± 0.6 mmol/l; HbA1c: 63 ± 2 vs. 50 ± 1 mmol/mol and insulin dose: 0.52 ± 0.03 vs. 0.14 ± 0.05 units/kg (all p < 0.01).

Excess calorific intake attributable to hypoglycaemia diminished post-transplant: 553 ± 251 vs. 14 ± 19 kcals/day (p < 0.01). Body weight, BMI, %FM and waist circumference of the subjects decreased following islet transplantation (pre- vs. post-transplant: (weight 73.5 ± 3.4 vs. 66.9 ± 3.6 kg, BMI 26.5 ± 0.8 vs. 24.1 ± 0.8 kg/m2, FM 28 ± 3 vs. 25 ± 3%, waist circumference 85.1 ± 2.8 vs. 80.0 ± 3.2 cm) (all p < 0.05)). Of the n = 2 patients that had serial abdominal MR imaging, subcutaneous FM diminished over 3 months: 5.5 ± 1.0 to 4.0 ± 0.5 kg.

Conclusions: Following islet transplantation in subjects with Type 1 diabetes, we have observed improved glycaemic control with no difference in stimulated c-peptide in subjects receiving islets from DCD vs. DBD donors with insulin independence achieved in two recipients to date. Concomitantly there have been reductions in body weight, FM, and waist circumference secondary to a reduction in hypoglycaemia and reduced calorific intake. These anthropometric changes may impact favourably on long term graft function.

UK Islet Transplant Consortium,Diabetes UK,SNBTS

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436

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Allogeneic Pancreas for Islet Donor Assessment for High Islet Product Transplant Rate and Donor Pancreas Utilization in a Phase III Clinical Trial - A Preliminary Analysis

Joshua Wilhelm1, AN Balamurugan1, David Radosevich1, William Clarke2, Bernhard Hering1

1Department of Surgery - Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN, United States; 2CTSDMC, University of Iowa, Iowa City, IA, United States.

Background: Processing of deceased donor pancreases for clinical islet transplantation requires significant expertise and resources. Achieving a high transplant rate will demonstrate control and consistency in the manufacturing to regulatory bodies, increase pancreas utilization and therapy availability, and facilitate reimbursement. Previous studies have analyzed pancreas donor “quality” in the context of isolation yields, islet quality, and metabolic outcomes. This preliminary study at a single manufacturing site was undertaken to examine and weight factors leading to high transplant rates in the very controlled setting of a Phase III licensure trial and to examine the impact in terms of organ utilization.

Methods: A single center series of 42 organ donors/pancreases processed uniformly with a consistent enzyme combination for the NIH CIT multicenter Phase II/III trial were retrospectively analyzed to determine factors associated with transplant rate using univariate, multivariate, and variable clustering techniques. A subset of donors was identified with a high transplant rate and associated donor, pancreas, islet processing characteristics reported. A parsimonious multivariate model was constructed using forward step-wise linear regression and evaluated for discrimination and calibration.

Results: Donor factors associated with size (therefore pancreas weight and subsequent islet yield) were most strongly associated with transplant success rate (i.e.; male gender, height, weight) and with each other, with body surface area as the strongest factor. Anoxia was associated with poor transplant rates, as were abnormal laboratory values associated with organ dysfunctions. The multivariate model provided good discrimination (AUC of 0.878, 95% CI 0.772-0.984) and was well calibrated (Chi-Square = 4.89, DF = 8, p value = 0.769), providing a sensitivity of 68% and a specificity of 64%. A subset of donors consisting of 41.5% (n = 17) of the overall pool was transplanted at a 94.1% success rate with a post-purification yield of 637,081 ± 198,813 IE or 5,637 ± 1,864 IE per gram pancreas. Yielding > 435,000 IE resulted in a predicted transplant rate of 94.1% (96.2% sens/93.7% spec) even with variable intended recipient weights. This donor pool may not overlap significantly with that for whole pancreas transplantation, principally due to the emphasis on large donor size (recommended BSA > 2.2 m2).

Conclusions: Improved understanding of pancreas donor variation can greatly increase islet product transplant rates and organ utilization. Data from additional CIT manufacturing sites will be analyzed and reported at the IPITA congress. Applying this risk model to deceased donor pancreases could markedly improve pancreas allocation, preclude substantial costs associated with unsuccessful islet isolations, and thereby increase availability and cost effectiveness of islet transplantation.

Submitted on behalf of the National Institutes of Health Clinical Islet Transplantation Consortium Investigators

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437

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Central Retinal Artery (CRA) Flow Velocity: Color Doppler Ultrasound (CDUS) Assessment in Type 1 Diabetic Patients After Kidney-Pancreas (KP), Kidney-Alone (KA) and Islet Transplantation Alone (ITA)

Giulia Querques1, Massimo Venturini1, Paola Maffi2, Giulia Agostini1, Lorenzo Piemonti3, Alessandro Del Maschio1, Antonio Secchi2

1Radiology, Scientific Institute San Raffaele, Milan, Italy; 2Internal medicine - Transplant Unit, Scientific Institute San Raffaele, Milan, Italy; 3Biology of beta cell, Scientific Institute San Raffaele, Milan, Italy.

KP transplantation can determine diabetes resolution in long-term type 1 diabetic uremic patients. ITA has been recently introduced to prevent complications in diabetic non-uremic patients such as retinopathy, which is characterized by endothelial dysfunction conditioning a reduction of the CRA flow velocity. The aim of our study was to compare the CRA flow velocity variations in KP, KA, and ITA patients before and after transplantation.

Methods: CRA flow velocity was assessed in 10 KP, 10 KA, and 10 ITA patients with CDUS (ATL-Philips, IU22) performed by the same experienced operator before and 2 years after transplantation. Peak systolic (PSV) and end diastolic (EDV) velocities were measured for each CRA at the retrobulbar level and expressed as mean values for each eye.

Results: ITA mean age was significantly lower than KP and KA patients (ITA: 38.0 ± 2.2; KP: 41.1 ± 5.9, p = 0.002; KA: 41.6 ± 5.3, p = 0.002). KA Diabetes duration was significantly higher than ITA and KP patients (KA: 30.8 ± 4.7; ITA: 24.9 ± 2.3, p < 0.001; KP: 26.0 ± 3.8, p < 0.001). All groups showed similar CRA flow velocities at baseline. At 2 years a statistically significant increase of CRA flow velocity was not found in KP and KA (KP-PSV: 5.73 ± 1.75 vs 6.06 ± 1.44; KP-EDV: 1.68 ± 0.31 vs 2.0 ± 0.44; KA-PSV: 4.55 ± 1.42 vs 5.74 ± 2.06; KA-EDV: 1.68 ± 0.54 vs 1.57 ± 0.39, p = ns), but only in ITA patients with significantly higher values than KP and KA (ITA-PSV: 10.12 ± 1.20 vs 6.09 ± 0.46, p < 0.01; ITA-EDV: 2.99 ± 0.48 vs 1.65 ± 0.07, p = 0.02).

Conclusion: Despite diabetes resolution in both groups, significant improvement of the CRA flow velocity was evident in diabetic non-uremic patients only after ITA and not KP transplantation: probably reversal of the endothelial dysfunction in retinal microcirculation is less likely in patients with a longer history of diabetes and related uremia and microvascular complications. CDUS can accurately and non-invasively study retinal microcirculation, allowing quantitative and reproducible measurements of the CRA flow velocity.

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438

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Reassembly of Alpha and Beta Cells of Neo-Islet Tissues Engineered in the Subcutaneous Space by Islet-Cell Sheet Transplantation in Mice

Hirofumi Shimizu1, Rie Utoh2, Hiroyuki Hanayama1, Kazuya Ise1, Masayuki Yamato2, Teruo Okano2, Mitsukazu Gotoh1

1Department of surgery, Fukushima Medical University, Fukushima, Japan; 2Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Shinjyuku, Japan.

Background: Our previous study has demonstrated therapeutic effect of bioengineered islet-cell sheets as a novel approach of islet transplantation for type 1 diabetes mellitus. Transplantation of the islet-cell sheets fabricated from rats conducted diabetic SCID mice to euglycemia, and the therapeutic effect was confirmed for 300 days after transplantation. In the present study, we show the topographical arrangement of these engineered neo-islets in vivo as a function of time after islet cell sheet transplantation.

Materials and Methods: Temperature-responsive culture dishes were prepared by covalently immobilizing a temperature-responsive polymer poly(N-isopropylacrylamide) (PIPAAm) on plastic dishes. Dissociated islet cells obtained from Lewis rats were seeded onto the PIPAAm dishes which was coated with rat laminin-5. After 2 days of culturing, islet cells reached confluency and were harvested as uniformly spread islet-cell sheets by lowering culture temperature from 37°C to 20°C for 20 min. Harvested islet-cell sheets were transplanted into subcutaneous space of diabetic SCID mice.

Results: Normal glucose levels were observed after 4 or 5 days of transplantation. Histological analyses of sample on day 4 revealed that the alpha and beta cells within neo-islet tissues were showed as mixed composition or out of alignment, in contrast, analysis of sample after 14 days revealed that alpha cells were located predominantly in the peripheral region of these engineered tissues. A ratio of alpha and beta cells was maintained constantly in neo-islet tissues throughout the observation period upto 300 day.

Conclusions: Histological analysis demonstrated that the reassembly of alpha and beta cells occurred islets in the neo-islet tissues engineered by islet-cell sheet transplantation. These morphological changes of in the neo-islet tissues indicated that the sequence stability, similar to original rat pancreatic islets, may contribute to their functioning and longevity.

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439

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Failure to Achieve Normal Metabolic Response with ex vivo Electroporation of the Human Proinsulin Gene (p3MTChins) in Primary Murine Hepatocytes for Correction of NOD and STZ-Induced Diabetes.

Ryan Lee1, Vinh Nguyen1, Garrett Roll1, Holger Willenbring1, Qizhi Tang1, Sang-Mo Kang1, Peter Stock1

1UCSF, San Francisco, CA, United States.

Background: A recent study by Chen et. al. [1] described a therapy for diabetes that utilized electroporation of the human proinsulin cDNA (p3MTChins) into hepatocytes for autologous transplantation. Syngeneic and autologous direct hepatic injection of transfected hepatocytes into STZ-diabetic murine and porcine models led to long-term euglycemic survival, weight maintenance, and physiologic production and secretion kinetics of insulin and c-peptide [2].

These findings prompted us to investigate: 1) the response of transfected hepatocytes to glucose challenge, 2) survival outcomes after direct hepatic versus intrasplenic injection, and 3) the efficacy of this strategy on survival, glucose control, and c-peptide secretion of STZ-diabetic and NOD mice after syngeneic transplantation.

Methods: Hepatocytes were isolated from B6 mice and electroporated with p3MTChins (National Cancer Centre, Singapore). Transfected hepatocytes of > 70% purity were divided into aliquots of 1x10^6 cells, incubated, and replaced with low (30mg/mL) or high (300mg/mL) glucose concentrations. Insulin was assayed at 1m and 60m. Other aliquots were injected into the hepatic interstitium or distal spleen parenchyma of our mouse models. In vivo outcome measures included survival, glucose monitoring, and c-peptide release. Transplanted primary hepatocytes served as controls.

Results: Transfected hepatocytes secreted insulin, but at indistinguishable levels during glucose stimulation at 1m and 60m (figure 1). Like the control, B6 mice that underwent direct hepatic injection survived for ≤ 5d (n = 6). B6 mice that underwent intrasplenic injection survived for 50d (n = 4, figure 2) compared to control (9d), and had a mild but durable improvement in hyperglycemia that plateaued at 320mg/dL (n = 1). C-peptide levels in B6 mice that were intrasplenically transplanted were durably elevated compared to control (n = 3). The c-peptide levels of the NOD mouse (n = 1) were also elevated, but declined to baseline over 62d.

Conclusions: Hepatocytes transfected with p3MTChins produce insulin and c-peptide at detectable levels, but do not respond to varying degrees of glucose concentrations. After transplantation, diabetic mouse models remain hyperglycemic and can lose c-peptide production over time. Therefore, we were unable to replicate the results of Chen et. al. in both our in vitro and in vivo experiments. Cell therapy involving electroporation of p3MTChins to target diabetes does not seem feasible particularly because glucose stimulation in plated transfected hepatocytes does not result in the dynamic release of insulin, and ultimately, glucose tolerance. Without the ability to achieve in vitro and in vivo normal metabolic responses, we will not pursue this technology at this time.

Reference:

  • [1] Chen NK, Sivalingam J, Tan SY, Kon OL: Plasmid-electroporated primary hepatocytes acquire quasi-physiological secretion of human insulin and restore euglycemia in diabetic mice. Gene Ther 2005, 12:655-667.
  • [2] Chen NK, Wong JS, Kee IH, Lai SH, Thng CH, Ng WH, Ng RT, Tan SY, Lee SY, Tan ME, Sivalingam J, Chow PK, Kon OL: Nonvirally modified autologous primary hepatocytes correct diabetes and prevent target organ injury in a large preclinical model. PLoS ONE 2008, 5;3(3):e1734.


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440

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Functional Imaging of the Pancreatic Graft by Positron Emission Tomography

Olof Eriksson1, Lina Sjöberg2, Håkan Ahlström2, Gunnar Antoni2, Jens Sörensen2, Mark Lubberink2, Alireza Biglarnia3, Gunnar Tufvesson3, Olle Korsgren4

1Preclinical PET Platform, department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden; 2Department of Radiology, Oncology and Radiation Sciences, Uppsala University, Uppsala, Sweden; 3Department of Surgical Sciences, Uppsala University, Uppsala, Sweden; 4Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Introduction: The functional status of pancreatic grafts in diabetic patients is today assessed by indirect circulating markers such as c-peptide and HbA1C. The lack of direct, non-invasive assessments of beta cell function in these sensitive grafts is an obstacle for optimized patient management. Serotonin has been associated with insulin secretion [1-2] and beta cell proliferation [3], and is not facilitated in the exocrine pancreas. We therefore posited that the serotonergic biomarker [11C]5-Hydroxy-tryptophan ([11C]5-HTP) could be used for functional imaging of the islets of Langerhans in the pancreatic graft by Positron Emission Tomography (PET).

Methods: Patients with variable pancreatic graft function (insulin independent, partial graft function, non-functioning grafts) were examined by 60 minutes dynamic PET/CT following intravenous administration of [11C]5-HTP over the site of transplantation. Two of the patients were then examined over the native pancreas. The uptake of the radiotracer was expressed as graft-to-blood (G/B) ratio to enable within- and between-patient comparison.

Results: [11C]5-HTP displayed high uptake in patients with functional pancreatic grafts (G/B55min 4.3 and 5.6). In patients with failing graft function, the uptake of [11C]5-HTP was conversely markedly reduced (G/B55min 1.1 and 1.3). Low or negligible uptake was seen in the native non-functional pancreata. In this pilot group, there was a correlation between serotonergic facilitation as measured by [11C]5-HTP-PET and graft function.

Discussion: Serotonergic facilitation as measured by [11C]5-HTP-PET seems to correlate well with the functional status of the transplanted pancreas. In fact, the functional grafts had a dynamic uptake similar to that of native, healthy pancreas in non-diabetic subjects while the non-functional grafts had a dynamic uptake similar to that in pancreas in T1D subjects, indicating a correlation between transplanted beta cell function/mass and serotonergic facilitation.

Conclusion: [11C]5-HTP-PET is a promising non-invasive technique for assessment of the endocrine function of the transplanted pancreas.

Reference:

  • 1. Paulmann, N., et al., Intracellular serotonin modulates insulin secretion from pancreatic beta-cells by protein serotonylation. PLoS biology, 2009. 7(10): p. e1000229.
  • 2. Ohta, Y., et al., Convergence of the insulin and serotonin programs in the pancreatic beta-cell. Diabetes, 2011. 60(12): p. 3208-16.
  • 3. Kim H., et al., Serotonin regulates pancreatic beta cell mass during pregnancy. Nat Med. 2010 Jul;16(7):804-8.
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441

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Clinical Impact of Allogenic Islet Transplantation in 55 Patients under ATG Induction Therapy and Tacrolimus-MMF Maintenance Immunosuppression

DaHae Lee1,2, Laurent Crenier3, Johannes Ruige4, Robert Hilbrands2, Daniel Jacobs-Tulleneers-Thevissen2, Sam Heye5, Nico De Leu2, Zhidong Ling2, Christophe De Block6, Chantal Mathieu1, Daniel Pipeleers2, Bart Keymeulen2, Pieter Gillard1,2

1Clinical and Experimental Medicine, University Hospitals Leuven-KUL, Leuven, Belgium; 2Diabetes Research Center, Brussels Free University-VUB, Brussels, Belgium; 3Department of Diabetology, Brussels Free University-Erasme, Brussels, Belgium; 4Department of Endocrinology, Ghent University Hospital, Ghent, Belgium; 5Department of Interventional Radiology, University Hospitals Leuven-KUL, Leuven, Belgium; 6Department of Diabetology, University Hospital Antwerp-UA, Antwerp, Belgium.

Allogeneic islet transplantation reduces glycemic variability and severe hypoglycemia in non-uremic type-1 diabetic patients maintained on immune suppression. We now report the impact of ATG-induction followed by tacrolimus and mycophenolate mofetil (MMF) on acute and chronic diabetic complications and its associated side-effects.

Data were collected during one (n = 55) to five year (n = 22) post-transplantation (PT). ATG dose ranged from 12.6 to 29.4 mg/kg (median 21.4), MMF was given at 1.5 gram/day (IQR 1.2 to 1.7) and tacrolimus trough levels were 9.4 ng/dl (IQR 8.8 to 10.0) during the first year and 7.2 ng/dl (IQR 6.2 to 8.2) until year 5 PT. The percentage of fasting glycemia > 300 and < 50 mg/dl decreased from 10 and 5% pre-transplantation respectively, to 2 (p = 0.004) and 1% (p < 0.001) post-transplantation. Several patients suffered from at least one hyperglycemic ketoacidosis (n = 6) or hypoglycemic coma (n = 25) in the year before transplantation but not in the 5 years thereafter (n = 0, p = 0.02 and n = 1, p < 0.001 resp.). No progression of micro- and macro-vascular complications was seen. Lipid profiles and blood pressure remained unchanged.

Fifty-six serious adverse events (SAEs) were reported. One patient died from cerebral hemorrhage at PT month 4 and one from liver metastasized gastric adenocarcinoma, diagnosed at PT month 27. Five patients developed CMV infections, successfully treated with intravenous (IV) ganciclovir. Most frequent SAE were gastro-intestinal infections (18/56), resolving with antibiotics and IV hydration.

Less severe adverse events were pyrosis (n = 12), insomnia (n = 12) and headache (n = 10), occurring mainly during the first year, respiratory infections (n = 23) and musculoskeletal pain (n = 18) with more stable presentation. Memory impairment was seen in 6 patients, mostly with later onset. Anemia, leucopenia, neutropenia were observed in > 50% of patients during the first 6 months PT, but decreased with time.

Our observations indicate that ATG-MMF–Tacrolimus regimen is safe, but is associated with expected side effects, requiring close monitoring for the occasional SAEs. The list of side effects should help select immune therapy protocols for further trials of islet transplantation.

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445

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Microfluidic Array with Integrated Oxygenation Control for Real-Time Live-Cell Imaging: Effect of Hypoxia on Physiology of Microencapsulated Pancreatic Islets

Yong Wang1, Mohammad Nourmohammadzadeh1, Joe Lo1, Matt Bochenek1, Joshua Mendoza-Elias1, Ze Li1, Feng Feng1, Meirigeng Qi1, David Eddington1, José Oberholzer1

1Surgery/Transplant, University of illinois at Chicago, Chicago, IL, United States.

Introduction: Islet transplantation requires immunosuppression. Islet microcapsulation is a promising to avoid immunosuppression. However, hypoxic stress is one of main factors for the function loss of microencapsulated islets and immunoisolation failure. Studies on encapsulated islets under hypoxia are limited. A microfluidic array was developed allowing studying pathophysiology of microencapsulated islets under hypoxia.

Methods: (1) A three-layer microfluidics was designed with integrated O2. controller. Top layer is flow controller and islet trapping array holding up to 400 microcapsules. Middle layer is PDMS membrane for controlling O2. Bottom layer is a gas channel. (2) Diffused and dissolved O2 on the array was evaluated by a Neofox oxygen sensor. (3)The arrayed microcapsulated islets were investigated under various O2.using simultaneous multiparametric assay (calcium influx, mitochondrial potentials, and NAD(P)H) response to glucose and KCL.

Results: (1) In both diffusive and dissolved modes, the device was capable of creating targeted O2.less than 40s and maintaining with high consistency. (2) The device can load 100 encapsulated islets less than 1min with 99% efficacy. (3) In response to 25mM glucose under normoxia, [Ca ++] changes of the microcapsulated islets were shown to be heterogeneous, with a mean of 5.91% ± 2.98 (max:13.2% and min:3.03%). In response to 30mM KCI,[Ca ++] also displayed a heterogeneous pattern with a mean of 6.25%± 1.68 (max:9.14% and min:4.14%). The changes of NAD(P)H for encapsulated islets in response to 25mM glucose also showed a heterogeneous response with a mean of 3.24%± 2.10 (max:6.91% and min:0.62%).(4) [Ca ++] of microencapsulated islets to 25mM glucose were O2.-dependent and were inhibited by hypoxia. Hypoxic concentrations decreased [Ca ++]:8.19%± 2.5 in 10%O2, 3.57%± 1.18% in 5%O2, and 1.70%± 0.64 in 1%O2.(p < 0.01 when 21% vs. 5% and 1%, as well as p < 0.01 when 10% vs. 5% and 1%). Similarly, mitochondrial potential changes were also inhibited in O2.-dependent manner (21-10-5-1%):17.23%± 3.13,8.83%± 3.53,6.40%± 2.56,and4.09%± 1.37(p < 0.01 when comparing 21% vs.10, 5,and 1%; p < 0.01 when comparing 10% vs. 5% and 1%). (5) The mean NAD(P)H change increase in response to 25mM glucose was 6.43%± 4.05. Under 1%O2, the NAD(P)H level was significantly inhibited (1.87% ± 1.75; p < 0.01). Moderate hypoxia (10 and 5%O2) also depressed NAD(P)H levels (10%O2: 4.65% ± 2.17 and 5%O2: 4.79% ± 2.84), but not significant.

Conclusion: We demonstrate that hypoxia impairs function of microencapsulated islets and showed a heterogeneous pattern. Our approach shows an improvement over conventional hypoxia chambers through the integration of dynamic and multiparametric analysis of key insulin stimulator-secretion coupling factors. This work demonstrates the feasibility of array-based cellomics analysis and opens up new opportunities to conduct informative analysis and cell-based screening for microencapsulated pancreatic islets.



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446

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Characterization of a NanoGland for the Auto-Transplantation of Human Pancreatic Islets

Omaima Sabek1, Alessandro Grattoni2, Silvia Ferrati2, Daniel Fraga1, A.Osama Gaber1

1surgery, The Methodist Research Institute, houston, TX, United States; 2Nanomedicine, The Methodist Research Institute, houston, TX, United States.

Despite the clinical success of pancreatic islet autotransplantation, graft function is frequently lost over time due to islet dispersion, lack of neovascularization, and loss of physiological architecture. To address these, islets encapsulation strategies including scaffolds and devices have been developed, which produced encouraging results in preclinical models. However, islets loss from such architectures has been reported and could represent a significant limitation to clinical use. Here, we developed and characterized a novel islet encapsulation silicon device, the NanoGland, to overcome islets loss, while providing a physiological-like environment for long term islet viability and revascularization. NanoGlands, microfabricated with channel size ranging from 3.6 nm to 60 μm, were mathematically modeled to predict the kinetics of response of encapsulated islets to glucose stimuli, based on different channel sizes, and to rationally select membranes for further testing. The model was validated in vitro using static and perifusion testing, during which insulin secretion and viability were demonstrated for over 30-days. In vitro testing also showed 70-83% enhanced islet retention as compared to porous scaffolds, here simulated through a 200 μm channel membrane. Finally, evidence of in vivo viability of human islets subcutaneously transplanted within NanoGlands was shown in mice for over 120 days. In this context, mouse endothelial cell infiltration, suggesting neovascularization from the host, were identified in the retrieved grafts. The NanoGland represents a novel promising approach for the autotransplantation of human islets.

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447

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Effect of Minimal Immunomodulation on the Biocompatibility of Alginate Microbeads Implanted into the Peritoneal Cavity of Cynomolgus Monkeys

Meirigeng Qi1, Enza Marchese1, Matt A Bochenek1, James McGarrigle1, Yong Wang1, Kirstie K Danielson1, Maureen Davis1, Igor Lacik2, Berit L Strand3, Jose Oberholzer1

1Surgery/Transplant, University of Illinois at Chicago, Chicago, IL, United States; 2Department of Special Polymers and Biopolymers, Polymer Institute of the Slovak Academy of Sciences, Bratislava, Slovakia (Slovak Republic); 3Department of Biotechnology, University of Science and Technology (NTNU), Trondheim, Norway.

Purpose: In this study, we tested the effect of T-cell directed immunosuppression and TNF-a blocker (Etanercept) on the biocompatibility of empty alginate microbeads after implantation.

Methods: Twenty naïve cynomolgus monkeys were transplanted laparoscopically with empty Ca2 +/Ba2 +-alginate microbeads. G1 (n = 7): treated with an immunosuppressive regimen (Tacrolimus, Sirolimus, Basiliximab, and Etarnercept) for a period of two weeks; G2 (n = 4): G1 regimen for 2 w + Etanercept only for another 6 w; G3 (n = 4): Etanercept alone for -9 d to 8 w; G4 (n = 5): No treatment. The microbeads were retrieved at 2 w (G1 and G4) and 4 w (G1, G2 and G3). Cellular overgrowth on the microbeads and serum biomarkers were assessed.

Results: At two weeks post implantation, a gross inflammatory reaction and clumps of microbeads were observed in 4 out of 5 animals in untreated G4. In contrast, a minimal inflammatory reaction and no clumps were found in the peritoneal cavity of 4 out of 5 animals in immune suppressed G1. The fold changes (prior to implantation to 1 week post-implantation; log scale) of the following serum biomarkers were significantly decreased in immune suppressed G1 compared to untreated G4: MIF (-1.17 vs. 0.10, p = 0.047); MDC (0.28 vs. 1.69, p = 0.04); G-CSF (-2.80 vs. 0.43, p = 0.04), respectively. At 4 weeks post implantation, there was no obvious improvement in terms of cellular overgrowth in both G2 and G3.

Conclusion: T-cell directed immunosuppression prevents foreign body reactions at the early stages of microbead implantation in cynomolgus monkeys. However this effect and Etanercept treatment alone do not last beyond the time immunosuppression was given.

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448

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The Effect of Cell-Matrix Interaction on Encapsulated Human Islets

Sophia Liao1, Keiko Omori1, Jeffrey Rawson, Davoud Mozhdehi2, Indu Nair1, Sergio Ayala, Ivan Todorov1, Zhibin Guan2, Yoko Mullen1

1Southern California Islet Cell Resrouces Center, Department of Diabetes, Endocrinology and Metabolism, Beckman Research Institute of City of Hope, Duarte, CA, United States; 2Department of Chemistry, University of California, Irvine, CA, United States.

Background: The extracellular matrix (ECM) is important in regulating multiple aspects of islets physiology. In the pancreas, each islet is surrounded by native ECM that helps maintain its heterogeneously-organized micro-structure. However, in isolated islets ECM is largely destroyed and may prolong engraftment following transplantation. Incorporation of short matrix-derived adhesive peptide (MDAP) sequences has been shown to increase insulin secretion in β-cell line. However, given the many architectural and organizational differences between β-cell lines and islets, there is a need to elucidate the effect of MDAP in human islets. We have previously reported islet encapsulation using novel saccharide-peptide (SP) hydrogel with improved islet function in extrahepatic site [1]. Given the positive attributes of SP hydrogel, MDAP-functionalized hydrogels were tested for their synergistic effects to maintain human islets in three-dimensional culture.

Method: MDAPs were synthesized using automatic peptide synthesizer. INS-1 cells and human islets were encapsulated in SP hydrogel for the evaluation of cell viability, function, ECM re-establishment and ECM gene expression using live/dead stain, insulin ELISA, immunohistochemistry and RT-PCR, respectively.

Results: MDAPs were successfully synthesized using peptide synthesizer as confirmed with mass spectrometry. Different MDAPs were functionalized onto SP hydrogel through Michael-type addition chemistry. MDAP improved viability of cultured INS-1 cells tested on day 7. Human islets cultured in vitro in SP hydrogel with MDAPs maintained 15% higher islet number than hydrogel without MDAPs. Quantitative analysis indicated increased collagen IV gene expression in the presence of MDAP than without MDAPs (3 folds) in as short as 3 days.

Conclusions: We have shown that successful incorporation of different MDAP can improve islet function and help ECM recovery to re-establish the native microenvironment. Therefore, combined with the ability of the SP hydrogel to facilitate extrhepatic site transplantation, the added islet-matrix interaction has great potential and applicability for clinical islet transplantation.

Reference:

  • 1. Liao S W, Rawson J, Omori K, Ishiyama K, Mozhdehi D, Oancea A, Ito T, Guan Z, Mullen Y: Maintaining functional islets through encapsulation in an injectable saccharide–peptide hydrogel. Biomaterials 2013, 34: 3984-3991.
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Thin-Film Encapsulation Devices for Pancreatic Islets

Crystal Nyitray1, Gaetano Faleo3, Qihizi Tang3, Tejal Desai1,2, Ryan Chang2

1Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, CA, United States; 2Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, United States; 3Transplant Surgery, University of California, San Francisco, San Francisco, CA, United States.

Islet transplantation in micro or macroencapsulation devices can be used to provide physiologic glucose-responsive insulin delivery. However creating a proper microenvironment to support islet secretory function within the macroencapsulation device is crucial for the success of this approach. To achieve this goal, we are developing: (1) a thin film polycaprolactone (PCL) macro-encapsulation device that protects the islets from immune destruction and (2) an engineered islet microenvironment to better maintain islet function. PCL is a polymer with tunable degradation properties that is commonly used in implanted biomedical devices. We have fabricated a thin flexible PCL device that can be controlled in terms of porosity (size and distribution) and cell loading. Current islet macro-encapsulation strategies include a large cell reservoir allowing the cells to obtain nutrients, however reservoir size largely hinders rapid diffusion and effective nutrient exchange. Our thin film device, with a thickness of less than 100 microns, seeks to overcome the challenges of immune isolation and limited nutrient exchange.

With our device we have confirmed comparable in vitro insulin kinetics between encapsulated islets and free islets suggesting that nutrient exchange is preserved in our devices. Our data also demonstrates engraftment and alloimunity after a period of one month. No such engraftment was achieved with free islets, which show no signal at one month’s time. This data indicates our film structure is effective in permitting diffusion of small molecules and proteins through the membrane while restricting immune cell infiltration and attack.

By using a cell-based treatment approach, it is hoped that the complications associated with poor blood glucose regulation can be reduced. Moreover, this research will further the advancement of other cell-encapsulation devices by outlining the requirements necessary for maximizing cellular exchange while providing critical immuno-isolation and an optimal islet microenvironment.

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Co-Encapsulation of Glucagon-Like Peptide-1 Secreting L Cells Improves Microencapsulated Islet Function

Alan Kerby1, Peter Jones1, Aileen King1

1Diabetes Group, King’s College London, London, United Kingdom.

Objective: L cells secrete glucagon-like peptide-1 (GLP-1) which can increase insulin secretion, insulin synthesis, β cell proliferation and neogenesis, and protect β cells against apoptosis. The aim of the study was to investigate if GLP-1 secreting L cells could improve microencapsulated islet function.

Methods: GLP-1 production from GLUTag and STC-1 L cell lines was measured. L cells were microencapsulated in alginate and cell growth was measured. Mouse islets were microencapsulated alone and incubated in 1x106 L cell conditioned medium or transferred into 1x106 L cell cultures. Islets were also co-encapsulated with 100 GLUTag cells per capsule. Islet function was measured by insulin secretion. Preliminary in vivo experiments were carried out using a minimal mass of 250 microencapsulated islets ± GLUTag cells transplanted intraperitoneally into streptozotocin diabetic mice.

Results: The L cells survived microencapsulation and grew within the capsules before reaching a plateau at 7 and 3 days for STC-1 and GLUTag cells respectively. Both L cell lines secreted GLP-1 and were responsive to glucose stimulation, however secretion from GLUTag cells was greater than STC-1 cells (111 vs. 51 fmol GLP-1/1x106 cells, P < 0.001). Neither L-cell conditioned media nor co-culture improved islet function using either cell line. However when GLUTag cells were co-encapsulated with islets, glucose stimulated insulin secretion was increased at day 3 (1.1 vs 1.6 insulin (ng/islet/h), P < 0.05). The average blood glucose at 56 days post-transplantation was lower in the GLUTag co-encapsulated group compared to the islet alone group (10.25 vs. 20.15 mM glucose, n = 2).

Conclusions: L-cell conditioned media or co-culture did not improve islet function which suggests pre-culture of microencapsulated islets with L cells prior to transplantation is unlikely to be successful. However L cells were able to improve islet function when co-encapsulated. Preliminary in vivo data indicate that transplantation outcome may be improved by co-encapsulation with L cells.

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Re-wiring the Molecular Circuitry of the Allograft to Promote Tolerance

Nathan W. Zammit1, Stacey N. Walters1, Jeannette E. Villanueva1, Shane T. Grey1

1Immunology, Garvan Institute, Darlinghurst, Australia.

Presentation of graft-derived antigens with correct co-stimulation drives the allo-immune response. Without these factors graft-specific tolerance may result. For DC’s, the A20 gene is an antigen-presentation attenuator; increased A20 levels negatively regulate DC maturation. We examined whether A20’s inhibitory capacity could be harnessed to promote graft-specific tolerance. BALB/c(H-2d) islets were transplanted under the renal capsule of MHC-mismatched diabetic C57BL/6(H-2b) recipients. A20 was over-expressed using a rAd.A20 construct. Control grafts (n>10) were rapidly rejected, whereas ~45% (n>27) of A20-expressing grafts were permanently accepted (POD>200). RTqPCR analysis at POD10 revealed that A20-transduced grafts exhibited reduced mRNA levels of CXCL10, IFNγ, IL6 and IL17, but increased levels for IL10, CTLA4 and TGFβ. In-vitro experiments demonstrated that A20 suppressed cytokine-induced NF-κB and JNK/AP1 activation. A20s inhibitory action on JNK activation preserved islet metabolic function via pERK and FOXO1. Conversely, introducing a point-mutation into the OTU domain of A20 results in deregulated NF-κB and JNK/AP1 activation, leading to the increased expression of inflammatory factors, as well as a loss of first-phase insulin response under stress. Consequently, A20 mutant islet grafts exhibited a significantly faster rejection tempo compared to control grafts. Long-term surviving A20-transduced grafts exhibited Foxp3+ cells at the islet/kidney parenchyma border. To assess the importance of these cells, purified T-cells were adoptively transferred into immune-deficient RAG−/− mice pre-transplanted with either a BALB/c or third-party CBA(H-2k) allograft. ~70% of BALB/c grafts were accepted, whereas, all third-party grafts were rejected. Next, effector T-cells (CD25-depleted) were adoptively transferred. Consequently, all grafts were rejected. Thus A20-expression induced graft-specific tolerance, dependent upon CD25+T-cells. To determine if CD25+T-cells were necessary early, recipients were administered mAb-PC61 prior to transplantation of A20-overexpressing BALB/c islet grafts. In this case all A20-transduced grafts were rejected. Therefore, A20 re-programmed intra-graft inflammatory circuits, generating a local milieu supporting graft function and T-reg mediated tolerance.

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Combined Anti-CD154 and CD8 T Cell Depletion Significantly Prolongs Islet Isograft Survival in Diabetic NOD Mice

Adam Burrack1, Tinalyn Kupfer1, K. Scott Beard1, Marilyne Coulombe1, Ronald Gill1

1Surgery and Immunology, University of Colorado, Denver, Aurora, CO, United States.

Type 1 diabetes (T1D) results in hyperglycemia due to an autoimmune attack on insulin-producing beta cells. Similar to human patients, non-obese diabetic (NOD) mice develop insulitis and beta cell destruction. In the NOD transplant recipient, islet allograft destruction is significantly more rapid than isograft destruction, despite the fact that NOD mice harbor self-MHC-restricted memory T cells specific for islet-derived peptides. We hypothesized that depletion of CD25+ regulatory T cells would accelerate isograft destruction, which we have observed. Memory T cells are thought to be relatively independent of co-stimulatory signals for activation. However, we observed that co-stimulation blockade significantly delays disease recurrence, measured by both days to transplant destruction (11 days to 22 days) and ex vivo T cell cytokine production (IFN-γ and TNF-α). Co-stimulation blockade had less dramatic, but still significant, effects on islet allograft rejection (5.5 days to 10 days). Because (1) CD4+ regulatory T cells are thought to be required for transplantation tolerance, (2) autoimmune CD8+ T cells can destroy beta cells, and (3) co-stimulation blockade had less effect on CD8+ T cells, we hypothesized that transient CD8+ T cell depletion in combination with co-stimulation blockade treatment would promote long-term isograft and allograft survival. Results demonstrate long-term transplant function in autoimmune NOD isograft recipients (>50 days) but little benefit beyond co-stimulation blockade for allograft recipients (median 12 days). These results suggest a therapeutic approach that promotes prolonged islet isograft function in the autoimmune recipient and highlight a crucial difference in allograft destruction by the autoimmune recipient.

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NK Cells are Required to Restrain CD8 T Cells During Tolerance Induction

Joshua N. Beilke3, Jon Buhrman2, Ronald G. Gill1

1Surgery and Immunology, University of Colorado, Denver, Aurora, CO, United States; 2Immunology, University of Colorado, Denver, Aurora, CO, United States; 3Novo Nordisk Research Center, Seattle, WA, United States.

Background: NK cells are surprisingly required for successful islet allograft tolerance induced with costimulation blockade (anti-CD154 antibody therapy). However, it is unclear what aspect of the host response is regulated by such NK reactivity. CD8 T cells are known to be important mediators of primary islet allograft rejection and can contribute to costimulation-blockade resistant rejection. Therefore, we determined whether CD8 T cells themselves were important targets of NK cell reactivity during tolerance induction.

Methods: BALB/c islet allografts (H-2d) were grafted under the kidney capsule of streptozotocin-induced diabetic mice of the following strains: wild-type control C57Bl/6 (B6, H-2b), B6 CD8 deficient (CD8 KO), or NKT cell-deficient B6 CD1 knockout (CD1KO) mice. Recipients were anti-CD154 treated (MR1) and/or treated with anti-NK1.1 (HB191) or anti-CD8 (2.43) during the peri-transplant period.

Results: Anti-CD154 therapy resulted in long-term allograft survival (>100 day) in 10/13 B6 wild-type recipients versus 0/9 in untreated controls (p< .001). Allografts also survived long-term in anti-CD154-treated B6 CD8KO (6/6) and B6 CD1KO (3/3) recipients. Anti-NK1.1 treatment alone did not impact acute, unmodified rejection of any of these strains. However, the addition of anti-NK1.1 to anti-CD154 treatment prevented long-term allograft survival in both B6 wild-type (0/8) and B6 CD1KO (0/4) recipients (p <.01). Importantly, anti-NK1.1 treatment did not prevent long-term allograft survival in 7/9 anti-CD154 treated B6 CD8 KO recipients (p < .01). Moreover, NK1.1 positive cells were not required for long-term allograft survival in wild-type B6 mice that also were depleted of CD8 T cells.

Conclusions: Taken together, results indicate that NK cells are required for long-term allograft survival induced with anti-CD154 therapy. Importantly, CD8 T cells appear to be a primary target of such NK cell regulation since the removal of CD8 T cells abrogates the NK cell requirement for tolerance induction.

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Tolerance Promoting Therapies Result in Early Restraint of T Cell Reactivity rather than Conversion to a Regulatory Phenotype

Szu-I (Selena) Wang1, Michelle Nelsen2, K. Scott Beard2, Marilyne Coulombe2, Ronald Gill2

1Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada; 2Surgery and Immunology, University of Colorado, Denver, Aurora, CO, United States.

Background: CD8+ T cells are primary mediators of acute islet allograft rejection and thus are potentially key targets of tolerance-inducing therapies. While monoclonal antibody therapy targeting LFA-1 (adhesion) or CD154 (costimulation) can independently induce allograft tolerance, their respective impact on graft-reactive CD8+ T cells has remained unclear. Therefore, we determined the impact of anti-LFA-1 or anti-CD154 therapy on the fate of antigen-specific CD8+ T cells and islet allograft survival in vivo.

Methods: To assess CD8 T cell reactivity in vivo, OT-I T cell receptor transgenic CD8 T cells were stimulated with ovalbumin-expressing mAct-OVA transgenic APCs. CD45.1 OT-I T cells were transferred into CD45.2 C57BL/6 (B6) hosts and challenged locally (footpad) with mAct-OVA APCs. Primary responses (day 3) were assessed with or without anti-LFA-1 (KBA) or anti-CD154 (MR1) therapy. For monitoring secondary responses, OT-I T cells initially stimulated as above were restimulated in vitro three weeks after primary challenge.

Results and Discussion: Anti-LFA-1 dramatically restrained the overall number of OT-I T cells responding in the draining (popliteal) lymph during primary stimulation. However, the proportion of OT-I T cells converting to an antigen-experienced CD44hiCD62Llo phenotype was comparable between anti-LFA-1 treated and control animals. Thus, LFA-1 perturbation did not prevent initial CD8+ T cell priming. Importantly, OT-I T cells from challenged, anti-LFA-1 treated animals remained reactive in secondary responses in vitro, indicating that cells were not inactivated. In contrast, anti-CD154 treatment had very limited impact on either the quantitative or qualitative response of OT-I T cells. Importantly, we did not find a significant early direct induction of antigen-specific regulatory T cells (Foxp3 or IL-10 expression). Thus, the early phase of tolerance induction appears to largely lead to immune inhibition rather than conversion to a regulatory phenotype. If so, this early ‘metastable’ phase of tolerance should be vulnerable to disruption by innate immune stimulation. Consistent with this concept, treatment with the TLR-3 agonist Poly I:C treatment at either the time of transplant (day 0–2) or at day 21 post transplant prevented islet allograft tolerance and prevented the restraint of antigen-specific T cells. However, by day +60, Poly I:C treatment no longer impacted long-term islet allograft survival, suggesting that active regulatory tolerance had become established.

Conclusion: LFA-1 and CD154 directed therapies inhibit but do not intrinsically tolerize antigen-specific T cells or directly convert such cells to a regulatory phenotype. Rather, active regulatory tolerance appears to develop over time. As such, this early ‘metastable’ phase of tolerance (< 3 wks) is especially vulnerable to disruption to immune insults, such as with pathogen-associated innate stimulatory signals.

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Tacrolimus Inhibits the Revascularization of Isolated Pancreatic Islets.

Ryuichi Nishimura1, Sho Nishioka2, Ikuma Fujisawa2, Hitoshi Shiku2, Miki Shimada3, Satoshi Sekiguchi1, Keisei Fujimori1, Akira Ushiyama4, Tomokazu Matsue5, Noriaki Ohuchi1, Susumu Satomi1, Masafumi Goto1,6

1Division of Advanced Surgical Science and Technology, Tohoku University, Sendai, Japan; 2Graduate School of Environmental Studies, Tohoku University, Sendai, Japan; 3Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan; 4Department of Environmental Health, National Institute of Public Health, Wako, Japan; 5WPI Advanced Institute for Materials Research, Tohoku University, Sendai, Japan; 6New Industry Creation Hatchery Center, Tohoku University, sendai, Japan.

Aims: Immunosuppressive drugscould be crucial factors for a poor outcome after islet allotransplantation. Unlike rapamycin, the effects of tacrolimus, the current standard immunosuppressant used in islet transplantation, on graft revascularization remain unclear. We examinedthe effects of tacrolimus on islet revascularization using a highly sensitive imaging system, and analyzed the gene expression in transplanted islets by introducing laser microdissection techniques.

Methods: Islets isolated from C57BL/6-Tg (CAG-EGFP) mice were transplanted into the nonmetallic dorsal skinfold chamberon the recipients. Balb/c athymic mice were used as recipients and were divided into two groups: including a control group (n=9) and tacrolimus-treated group (n=7). The changes in the newly-formed vessels surrounding the islet grafts were imaged and semi-quantified using multi-photon laser-scanning microscopy and a Volocity system. Gene expression in transplanted isletswas analyzed by the BioMark dynamic system.

Results: The revascularization process was completed within 14 days after pancreatic islet transplantation at subcutaneous sites. The newly-formed vascular volume surrounding the transplanted islets in the tacrolimus-treated group was significantly less than that in the control group (p<0.05){figure1}{figure2}. Although the expression of Vegfa(p<0.05) and Ccnd1(p<0.05) was significantly upregulated in the tacrolimus-treated group compared with that of the control group, no differences were observed between the groups in terms of other types of gene expression.

Conclusions: The present study demonstrates that tacrolimus inhibits the revascularization of isolated pancreatic islets without affecting the characteristics of the transplanted grafts. Further refinements of this immunosuppressive regimen, especially regarding the revascularization of islet grafts, could improve the outcome of islet allotransplantation.

The authors thank Megumi Goto, Toshiki Yanagi, and Masako Osawa for their excellent technical assistance. The authors also thank the Biomedical Research Core of Tohoku University Graduate School of Medicine and TAMRIC (Tohoku Advanced Medical Research and Incubation Center)

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HSP25/27 Plays a Key Function in Prolactin-Induced Cytoprotective Effects on Beta-Cells

Rosangela A. M. Wailemann1,3, Leticia F. Terra1,2, Talita C. Oliveira1, Ancély F. Santos1, Mari C. Sogayar1,2, Leticia Labriola1

1Chemistry Institute - Biochemistry dept., University of São Paulo, São Paulo, Brazil; 2Nucleo de Terapia Celular e Molecular, University of São Paulo, São Paulo, Brazil; 3Scholl of arts, Sciences and Humanities (EACH), University of São Paulo, São Paulo, Brazil.

Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type1 diabetes, however, it is severely limited by the shortage of organs. Ex-vivo expansion of islet cell cultures appears as an attractive but still elusive approach for curing type 1 diabetes. We have shown that recombinant human prolactin (rhPRL) inhibits beta-cell apoptosis. Moreover, we have recently reported PRL-induced up-regulation of the anti-apoptotic HSP25/27 in human islets. Since the function of HSP25/27 on beta-cells has not been directly studied, we set out to explore the role of HSP25/27 in prolactin-induced beta-cell cytoprotection. For this purpose, we used wild type or HSP25 knocked-down Min6 cells. Our data showed that upon cytokines and rhPRL treatment, the proportion of fragmented nuclei was increased in HSP25 silenced cells (p<0.05) when compared to control cells. In addition, the inhibition of cytokine-induced capase-3 activity as well as caspase-9 and Bax protein levels mediated by rhPRL in wild type cells were all significantly reverted in knocked-down cells (p<0.05). Moreover, the kinetics of HSP 25/27 and HSTF1 expression levels were studied in primary cultures of human pancreatic islets which were serum starved and treated with rhPRL for short periods of time (from 10 min to 2h). The results showed that while HSTF1 presented a significant increase (p<0.01) in protein expression level after 10 min of rhPRL treatment, HSP27 reached its maximum expression level upon 2h of hormonal treatment. Additionally, a significant (p<0.05) increase in STAT1 phosphorylation level was detected after 10min of rhPRL treatment reaching the highest levels upon 30 min (p<0.001) of hormonal treatment.

We demonstrated herein a key role of HSP25/27 on rhPRL-induced cytoprotective effects, since the lack of this protein completelyabolished the beneficial effects induced by PRL on beta-cell death. Moreover, we provided for the first time, evidence for the co-regulation of HSP27 and HSTF1 inducedby rhPRL in human pancreatic cells which could be mediated by activated STAT1. Collectively, our results could lead to the mitigation of beta-cell death through the up-regulation of an endogenous protective pathway in an immune system independent manner.

FAPESP, CNPq, FINEP, BNDES, MCT, MS-DECIT, PRP-USP.

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The Effect of Osteocalcin on Human Islet Function

Omaima Sabek1, Daniel Fraga1, Solmaz Afshar1, A. Osama Gaber1

1Surgery, The Methodist Research Institute, houston, TX, United States.

Osteocalcin modulation of β-cell biology has emerged over the last decade as a novel mechanism in sugar homeostasis and energy metabolism, and the osteoblast-specific protein hormone osteocalcin was identified and proposed as the main mediator of this pathway. Specifically, osteocalcin favors β-cell proliferation, insulin secretion, and insulin sensitivity, and therefore, this hormone may have utility in therapy of pathologies characterized by progressive loss of β-cell mass and function, namely type 1 and perhaps more effectively type 2 diabetes. However, to date all of the data supporting a role of osteocalcin in β-cell biology were generated in mouse models, and it is well documented that insights from mouse experiments cannot necessarily be extrapolated to human β-cell biology. In this study, we tested three types of osteocalcin for its ability to enhance human β-cell function and proliferation in culture as well as a well established in vivo system for the study of human β-cell biology, i.e. transplantation of human islets into NOD-scid mice. We found that osteocalcin increases insulin content of cultured human β-cells in a dose dependent manner, with D-osteocalcin being most active this was paralleled by increased synthesis of the b-cell marker SUR1 and SCG5. We also found that all three osteocalcin significantly enhanced human β-cell proliferation in culture. Finally we showed that continuous exposure to osteocalcin strongly enhances the metabolic response of human islets to glucose challenge, in terms of insulin and C-peptide secretion, in vivo, following transplantation into NOD-scid mice.

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Oxygen Consumption Rate of Porcine Pancreatic Acinar and Islet Tissue in Culture

Thomas M Suszynski1, Kathryn R Mueller1,2, Angelika C Gruessner2, Papas Klearchos K1,2

1Surgery, University of Minnesota, Minneapolis, MN, United States; 2Surgery, University of Arizona, Tucson, AZ, United States.

Assays based on measuring the oxygen consumption rate (OCR) have exhibited promise in predicting islet transplant (IT) outcomes. However, no studies have been done comparing the viability based on OCR of islet and acinar tissue, of particular importance for impure islet preparations.

In this study, we compare the OCR normalized to DNA (OCR/DNA), and the OCR and DNA recoveries from porcine islet and acinar tissue originating from the same pancreas digests for a number of isolations (n=16, paired). OCR/DNA is a measure of fractional viability in units nmol/min/mg DNA, and OCR and DNA recoveries measure viable and total tissue recovery, respectively [1-3]. These parameters were examined from the point of isolation and up to ~1 week of culture. The objective was to determine if any of these parameters change disproportionately for islet or acinar tissue with time in culture.

Paired comparisons were done to quantify differences in OCR/DNA between islet and acinar tissue from the same preparation, at specified time points during culture; the mean (± SE) OCR/DNA was 74.0 (±11.7) units lower for islet (vs. acinar) tissue on the day of isolation (n=16, p<0.0001), but 25.7 (±9.4) units higher after 1 day (n=8, p=0.03), 56.6 (±11.5) units higher after 2 days (n=12, p=0.0004), and 43.3 (±31.7) units higher after 8 days (n=5, p=0.2) in culture (Figure 1). OCR and DNA recoveries decreased disproportionately for acinar versus islet tissue over 6–9 days in culture; OCR recovery was reduced to 20±7% of original (day of isolation) values for acinar, whereas it remained virtually unchanged at 100±14% for islets (p=0.0007); DNA recovery reduced to 21±7% for acinar and 75±6% for islets (p=0.01).

Differences in the OCR profiles of acinar versus islet tissue must be considered when characterizing porcine preparations prior to IT, and the timing of assessment may have a substantial impact on the interpretation of a result.

Reference:

  • 1. Papas KK, Pisania A, Wu H, Weir GC, Colton CK. A stirred microchamber for oxygen consumption rate measurements with pancreatic islets. Biotechnol Bioeng. 2007; 98(5): 1071-1082.
  • 2. Papas KK, Colton CK, Nelson RA, Rozak PR, Avgoustiniatos ES, Scott WE 3rd, Wildey GM, Pisania A, Weir GC, Hering BJ. Am J Transplant. 2007; 7(3): 707-713.
  • 3. Papas KK, Suszynski TM, Colton CK. Islet assessment for transplantation. Curr Opin Organ Transplant. 2009; 14(6): 672-682.
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Label-Free Detection of Insulin and Glucagon within Islets of Langerhans using Raman Spectroscopy

Janneke Hilderink1, Cees Otto4, Cees Slump3, Aufried Lenferink4, Marten Engelse2, Clemens van Blitterswijk5, Eelco de Koning2, Marcel Karperien1, Aart van Apeldoorn1

1Developmental Bioengineering, University of Twente, Enschede, Netherlands; 2Department of Nephrology, Leiden University Medical Center, Leiden, Netherlands; 3Department of Signals and Systems, University of Twente, Enschede, Netherlands; 4Department of Medical Cell BioPhysics, University of Twente, Enschede, Netherlands; 5Department of Tissue Regeneration, University of Twente, Enschede, Netherlands.

Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require sample preparation. As an alternative, we have investigated the use of Raman spectroscopy, which is a non-destructive and label-free technique, that allows continuous real-time monitoring of tissues and cells[1,2]. Raman spectroscopic measurements on purified insulin and glucagon showed that the 520 cm-1 band assigned to disulfide bridges in insulin, and the 1552 cm-1 band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used for the label-free distinction between both hormones. High-resolution hyper spectral Raman imaging for these specific bands shows the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlates with the location of insulin and glucagon as revealed by conventional immunohistochemistry in INS1E cells and sections of human islets. Average Raman fingerprints of MIN6, INS-1E and alpha-TC6 cells were determined and multivariate data analysis was applied to the dataset to objectively assess subtle differences in the fingerprint region of the different cell spectra. Hierarchical cluster analysis (HCA) classified the various cell types in separate clusters and subsequent principal component analysis revealed that Raman bands designated to unsaturated fatty acids[3] caused the subtle differences. These initial results indicated that confocal Raman imaging could potentially be used to separate alpha from beta cells. In order to further investigate this potential, an assessment of the amount of overlap between immuno-labeling and confocal Raman imaging on sections of human donor islets was performed resulting in Dice coefficients of 0.36 for insulin and 0.19 for glucagon. These findings indicate that high-resolution Raman imaging, using sub-micrometer steps, for specific for disulfide bridges between cysteine groups (520 cm-1) and tryptophan (1552 cm-1) enable local detection of high concentrations of insulin and glucagon in human pancreatic islet cells. This method provides information on composition and structural organization of pancreatic endocrine tissue in a label-free manner, and may help to accurately monitor islet quality before transplantation in patients suffering from type 1 diabetes in the future.

This research is part of the Diabetes Cell Therapy Initiative (DCTI) and funded by the Dutch Diabetes fund and the Ministry of economical affairs (FES program) of the Netherlands.

Reference:

  • 1. Ellis DI, Goodacre R (2006) Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy. Analyst 131: 875-885.
  • 2. Notingher I, Hench LL (2006) Raman microspectroscopy: a noninvasive tool for studies of individual living cells in vitro. Expert Rev Med Devices 3: 215-234.
  • 3. van Manen HJ, Kraan YM, Roos D, Otto C (2005) Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes. Proc Natl Acad Sci U S A 102: 10159-10164.
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A Small Molecular GPR119 Agonist Stimulates Beta Cell Replication in Human Islets

Jie Gao1, A Ansarullah2, Colette Free2, Jenica Christopherson2, Quanhai Chen2, Friederike Gedge2, Neil Karjalainen2, Chengyang Liu3, Ali Naji3, Alex Rabinvitch2, Zhiguang Guo2

1Department of Hepatobiliary Surgery, People’s Hospital of Peking University, Beijing, People’s Republic of China; 2Sanford Project, Sanford Research/USD, Sioux Falls, SD, United States; 3Department of Surgery, University of Pennsylvania, Philadelphia, PA, United States.

G protein-coupled receptor 119 (GPR119) is expressed in β cells and enteroendocrine L cells. Activating GPR119 can stimulate mouse β cell regeneration in vitro and in vivo. In this study, we investigated whether PSN632408, a small molecular GPR119 agonist, can stimulate human β cell regeneration in vitro and in vivo. To determine β cell replication in vitro, human islets isolated from a 14 year-old donor were cultured for 4 days, with and without 10 μM PSN632408. Culture medium was changed daily and BrdU was added on day 3 for overnight. Insulin and BrdU double immunofluorescence staining was performed on intact islets. Insulin+and BrdU+ β cells were accounted using confocal microscope. To determine β cell replication in vivo, 1000 IE human islets isolated from a 16 year-old donor were transplanted under the left kidney capsule of each streptozotocin induced-diabetic NOD.scid mouse. These recipient mice were given BrdU and with or without PSN632408 at 10 mg/kg/day. At 4 weeks, islet grafts were removed for insulin and BrdU double immunofluorescence staining. In cultured islets, BrdU+ and insulin+ β cells in each cultured islet were 6.4±0.5 cells without treatment and 15.8±1.3 cells with PSN632408 treatment (P<0.01). At 2 weeks post-transplantation, the nonfasting blood glucose levels were 466±24 mg/dL in vehicle treated mice and 322±35 mg/dL in PSN632408 treated mice (P<0.05). At 4 weeks, 25% recipient achieved normoglycemia in PSN632408 treated mice. In contrast, none achieved normoglycemia in recipients without treatment. The percentage of insulin+ and BrdU+ positive β cells in islet grafts was 2.8±0.7% in vehicle treated mice and 5.0±0.9% in PSN632408 treated mice (P<0.05). Our data demonstrated that GPR119 agonist can stimulate β cell proliferation in human islets in culture and in immunodeficient mice with diabetes. Activating GPR119 is a new therapeutic approach to stimulate β cell replication in human islets from younger donors before and after islet transplantation.

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Human Pancreas-Derived Mesenchymal Stem Cells Improve Function of Transplanted Islets and Native Pancreas to Reverse Diabetes

Faisal Kunnathodi1, Rauf Shahbazov1, Nofit Borenstein1, Mazhar Kanak2, Morihito Takita1, Marlon Levy3, Bashoo Naziruddin3, Michael Lawrence1

1Islet Cell Laboratory, Baylor Research Institute, Dallas, TX, United States; 2Institute of Biomedical Studies, Baylor University, Waco, TX, United States; 3Baylor Annette C. and Harold C. Simmons Transplant Institute, Dallas, TX, United States.

Islet transplantation represents a viable cell therapy treatment to improve blood glucose control after loss of pancreatic beta cells in type 1 diabetes and in chronic pancreatitis (CP) patients undergoing total pancreatectomy. However, an insufficient supply of donor pancreatic tissue and the loss of islet cells upon transplantation due to inflammatory stress remain major hurdles to the success of islet transplantation. Recently, mesenchymal stem cells have been shown to have the capacity to protect islet cells from inflammation and immune assault. In this study, we isolated human mesenchymal stem cells from pancreatic tissue obtained from organ donors and CP patients undergoing pancreatectomy. The pancreatic-derived mesenchymal stem cells (PMSCs) were positive for expression of surface markers CD29, CD73, CD90, and CD105; negative for CD14, CD34, and CD45; and capable of adipocyte differentiation. PMSCs migrated toward islets and produced IL-1b and IL-6 when exposed to proinflammatory cytokines IL-1b, TNF-a, and IFN-g. PMSC migration and expression of IL-1b and IL-6 was blocked by calcineurin inhibitor and immunosuppressant FK506. Upon co-transplantation of a marginal dose (1500 IEQ) of human islets into kidney capsules of streptozotocin (STZ)-treated diabetic nude mice, PMSCs restored normal blood glucose concentrations for up to 30 days. Surprisingly, upon nephrectomy, and thus removal of the human islet graft, the mice remained normoglycemic and maintained an intraperitoneal glucose tolerance test profile comparable to non-STZ treated controls. Overall, the data indicate that PMSCs are activated and recruited to islets by cytokine signaling and calcineurin-dependent mechanisms to 1) enhance human islet graft function during co-transplantation and 2) restore islet function in the native damaged/inflamed mouse pancreas. Thus, PMSCs may not only have potential use for improving islet cell function in human islet transplantation, but also for repairing/restoring impaired native endocrine pancreas function to its original state.

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The Survival of Free and Encapsulated Islets Xenograft Transplanted in the Mouse Bone Marrow

Raphael Meier1, Jörg Seebach2, Philippe Morel1, Redouan Mahou3, Sophie Borot1, Laurianne Giovannoni1, Geraldine Parnaud1, Domenico Bosco1, Christine Wandrey3, Thierry Berney1, Leo Bühler1, Yannick Müller1,2

1Transplant Surgery, University Hospital of Geneva, Geneva, Switzerland; 2Division of Clinical Immunology and Allergology, University Hospital of Geneva, Geneva, Switzerland; 3Institut d’Ingénierie Biologique et Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

Objectives: Bone marrow (BM) has been recently shown to be an alternative and potentially immune-privileged site for islet transplantation. The aim of the study was to assess the survival of free and encapsulated xenogeneic islets transplanted into the BM of C57BL/6 mice, and to characterize the rejection.

Methods: Free rat or human islets were transplanted into the medullary cavity of the femur or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. Due to the immunosuppressive effect of the streptozotocin, every other experiment was performed in non-diabetic animals. Bones were harvested for immunofluorescence and quantification of CD4+, CD8+, and F4/80 expression by flow cytometry; splenocytes were isolated for in vitro proliferation assays. Rat islets were encapsulated in Calcium alginate beads. The survival and fibrotic reaction were assessed by insulin and Masson staining one month after transplantation in the BM or under the kidney capsule.

Results: Free rat/human islets had a median survival of 9/7 and 14/10 days after transplantation respectively into the BM and under the kidney capsule. Seven days after transplantation, increased numbers of CD8+ cells were present in femurs of transplanted mice compared to contralateral femur or to naïve mice femur. CD4+, CD8+, macrophages were recruited around the islets in the BM. Splenocytes of transplanted mice specifically proliferated in contact to donor stimulators. Encapsulated rat islets transplanted in the BM survived over one month and triggered similar fibrotic reaction when compared to encapsulated islet transplanted under the kidney capsule.

Conclusion: We successfully established an animal model for xenogeneic islets transplantation into the BM. There was no evidence for an immune-privileged environment. The xenorejection in the BM is cellular-dependent with antigen recognition in the spleen. Nevertheless, the BM may represent an adequate site for encapsulated xenogeneic islets transplantation.

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Bio-Artificial PLGA Device Prevascularized with Hypoxia-Conditioned Mesenchymal Stem Cells for Subcutaneous Islet Transplantation

Jung Sik Kim1, Hyun-Ju Lim1, Jun-Shup Shin1, Sang-Joon Kim1, Chung-Gyu Park1

1Department of Microbiology and Immunology, Translational Xenotransplantation Research Center, Seoul, Korea.

Subcutaneous site in clinical islet transplantation has been suggested strongly because of its surgical advantages. This site could minimize the unwanted blood-mediated inflammatory reactions which occasionally arose in intra-portal delivery of islets. However, clinical success of this site is limited by poor vascularization potential. To overcome this barrier, we constructed PLGA-based bioartificial device for promoting prevascularization in the subcutaneous site using hypoxia-preconditioned mesenchymal stem cells. PLGA device coated with hypoxia-preconditioned mesenchymal stem cells (HPLGA) supported the formation of vascularized structure inside and outside after the subcutaneous implantation, which was superior to PLGA device coated with normoxia-preconditioned mesenchymal stem cells (NPLGA). Maximum vascularization was observed 4 weeks after implantation. When porcine islet mass (5,000 IEQs) was transplanted in the prevascularized-PLGA device, only HPLGA supported complete normoglycemic control. The amount of marginal mass which reverse hyperglycemia by 50% of transplanted-mouse was around 2000 IEQs, which was comparable to those in kidney capsule. Therefore, a prevascularized PLGA device might give a useful modality for the success of clinical subcutaneous islet transplantation.

Reference:

  • 1. Shapiro, A. M., J. R. Lakey, E. A. Ryan, G. S. Korbutt, E. Toth, G. L. Warnock, N. M. Kneteman, and R. V. Rajotte: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230-238, 2000.
  • 2. Shapiro, A. M., C. Ricordi, B. J. Hering, H. Auchincloss, R. Lindblad, R. P. Robertson, A. Secchi, M. D. Brendel, T. Berney, D. C. Brennan, E. Cagliero, R. Alejandro, E. A. Ryan, B. DiMercurio, P. Morel, K. S. Polonsky, J. A. Reems, R. G. Bretzel, F. Bertuzzi, T. Froud, R. Kandaswamy, D. E. Sutherland, G. Eisenbarth, M. Segal, J. Preiksaitis, G. S. Korbutt, F. B. Barton, L. Viviano, V. Seyfert-Margolis, J. Bluestone, and J. R. Lakey: International trial of the Edmonton protocol for islet transplantation. N Engl J Med 355:1318-1330, 2006.
  • 3. Moberg, L., H. Johansson, A. Lukinius, C. Berne, A. Foss, R. Kallen, O. Ostraat, K. Salmela, A. Tibell, G. Tufveson, G. Elgue, K. Nilsson Ekdahl, O. Korsgren, and B. Nilsson: Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet 360:2039-2045, 2002.
  • 4. Biarnes, M., M. Montolio, V. Nacher, M. Raurell, J. Soler, and E. Montanya: Beta-cell death and mass in syngeneically transplanted islets exposed to short- and long-term hyperglycemia. Diabetes 51:66-72, 2002.
  • 5. Kim, H. I., J. E. Yu, C. G. Park, and S. J. Kim: Comparison of four pancreatic islet implantation sites. J Korean Med Sci 25:203-210, 2010.
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Pretreatment of Donor Pigs with a Diet Rich in Soybean Oil Increases the Yield of Isolated Porcine Islets

Gopalakrishnan Loganathan1, Melanie L Graham1, Tom Spizzo2, Mukesh Tiwari1, Sajjad M Soltani1, Joshua J Wilhelm1, A.N. Balamurugan1, Bernhard J Hering1

1Surgery, Schulze Diabetes Institute, Spring Point Project, Minneapolis, MN, United States; 2Spring Point Project, Minneapolis, MN, United Kingdom.

Background: Islet beta cell expansion has been reported in rodents and in our previous preliminary studies in pigs [1] exposed to high fat diet (HFD). In this study, we examined the influence of high fat diet enriched in soybean oil on porcine islet mass and yield after islet isolation.

Materials and Methods: Post weaning, starting at day 70 and ending on day 250, pigs were fed with standard diet (control group; n=5) or high-fat ration supplemented with 15% w/w soybean oil (HFD group; n=6). Metabolic parameters including fasting blood glucose levels, cholesterol, AST, ALT, Kg [glucose disposal rata] and acute C-peptide response [ACRg] to intravenous glucose were monitored before pancreas procurement. The pretreated donor pigs were masked, after procurement, the retrieved pancreas from control and pretreated donor pigs were stained with dithizone and assessed for number of islets using dithizone scoring method [2]. To determine the yield between control and HFD donor pigs, islets were isolated randomly using a standard isolation protocol [3] using new Roche C1, C2 collagenases in combination with BP-protease. Islet isolation factors and islet quality were assessed in both groups and the results were compared between the two groups [Mann-Whitney U Test and Spearman Rank correlation between continuous variables]

Results: The results are summarized in the table and figure. There were no significant differences in the donor characteristics [age, body weight, Kg, ACRg, cholesterol, AST, ALT] except fasting blood glucose between control and treatment groups [84±6 vs 99±12]. The stimulated insulin and C-peptide levels between the groups were similar. However, the dithizone score was slightly higher in the HFD compared to the control group (95.4±38.5 vs 62.6±23.9; p=0.1208). Digestion time, digested pancreas weight, pellet volume and the fragility index were similar in both groups. However, the average islet count (IEQ/gram pancreas) at digest level was significantly higher in the HFD than in the control group (1,578±994 vs 738±202; p=0.0344). The digestion profile using new Roche enzyme was comparable to old Liberase-PI. The functional viability of 2 and 7 day-cultured islets - as assessed by oxygen consumption rate corrected for DNA - was similar in both groups.

Conclusion: Pretreatment of pigs with HFD enriched in soybean oil could potentially be used to improve the islet yield in donor pigs. Further studies are needed to optimize the use of HFD for the purpose of increasing islet yield from young donor pigs.

Reference:

  • 1. Brandhorst D, Hering BJ, Brandhorst H, Kirchhof N, Dzapo V, Federlin K, Bretzel RG. Dietary treatment with soybean oil improves porcine islet culture and reduces islet immunogenicity. Transplant Proc. 1994 Apr; 26(2):613.
  • 2. Anazawa T, Balamurugan AN, Matsumoto S, Lafreniere SA, O’Brien TD, Sutherland DE, Hering BJ. Rapid quantitative assessment of the pig pancreas biopsy predicts islet yield. Transplant Proc. 2010 Jul-Aug; 42(6): 2036-9.
  • 3. Anazawa T, Balamurugan AN, Papas KK, Avgoustiniatos ES, Ferrer J, Matsumoto S, Sutherland DE, Hering BJ. Improved method of porcine pancreas procurement with arterial flush and ductal injection enhances islet isolation outcome. Transplant Proc. 2010 Jul-Aug; 42(6): 2032-5.

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Noninvasive Quantification of Vascular Hemodynamics in Subcutaneous Transplantation of Biomaterial Encapsulated Islets.

Rahul Krishnan1, Rajan Arora2, Morgan Lamb1, Sean M. White2,3, Rick Storrs4, Randy Dorian4, Scott King4, Clarence E. Foster, III1, Elliot Botvinick1,2,3, Bernard Choi1,2,3, Jonathan RT Lakey1,3

1Surgery, University of California, Irvine, Orange, CA, United States; 2Beckman Laser Institute and Medical Clinic, University of California, Irvine, Irvine, CA, United States; 3Biomedical Engineering, University of California, Irvine, Irvine, CA, United States; 4Islet Sheet Medical, San Francisco, CA, United States.

Although encapsulation has been demonstrated to prolong islet survival, insufficient oxygen delivery via the surrounding microcirculation after subcutaneous transplantation results in suboptimal islet function and early graft death as early as the first week after transplantation. We employ the mouse dorsal window model, Laser Speckle Imaging (LSI) and Wide-field Functional Imaging (WiFI) to noninvasively quantify changes in relative blood flow (RBF) and hemoglobin oxygen saturation (HOS) to study the vascular changes following subcutaneous transplantation of alginate-encapsulated xenogeneic islets over a seven day period post transplantation.

Subcutaneous dorsal window chambers were installed on C57BL/6 albino mice and implanted with a blank alginate sheet, a sheet containing young porcine islets isolated from pre-weaned Landrace pigs (18-22 days old) or no implant (negative control). Images obtained using LSI and WiFI were analyzed using algorithms to quantify the changes in RBF and HOS respectively. At the conclusion of the experiment, the implants were extracted and analyzed for islet viability and function.

Arterial and venous dilatation, peri-implant neovascularization and increase in the RBF and arterial HOS were noted in both groups by day 7. On further quantification, the increase in HOS from 90.03+0.69 to 98.77±0.40 and RBF from 0.88 to 1.62 and by day 7 in the porcine islet group was statistically significant (p=0.03) while the increase in the control group (96.09±1.05 to 99.02±0.43;HOS, 0.97 to 0.98; RBF) was not (p=0.67). The transplanted islets maintained viability (79.08±3.28; Day 0, 79.35±0.84; Day7) and remained functional (SI = 2.16±0.09; Day 0, 1.98±0.08; Day7).

Our results suggest that implanted islets induce vascular changes, while remaining functional and maintaining viability. Future experiments will attempt to study the role played by islet secreted VEGF in increasing blood flow and arterial oxygenation in the implant-adjacent vasculature while also extending the study period for up to 2 weeks post transplantation.

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Prolonged Euglycemia Following Intraperitoneal Xenotransplantation of Encapsulated Young Porcine Islets.

Morgan Lamb1, Michael Alexander1, Rahul Krishnan1, Tom Spizzo3, Michael Martin3, Tianyi Zhou1, Remick Stahl1, Jonathan RT Lakey1,2

1Surgery, University of California, Irvine, Orange, CA, United States; 2Biomedical Engineering, University of California, Irvine, Irvine, CA, United States; 3Spring Point Project, Minneapolis, MN, United States.

Limitations of islet transplantation include scarcity of quality organ donors, isolation inconsistency and need for chronic immunosuppression. The development of a stable, chemically purified, biologically inert alginate microcapsule containing viable porcine islets would address these key issues. The aim of this study was to demonstrate the function of encapsulated porcine islet xenotransplants in the absence of immunosuppression.

Athymic nude and C57BL/6 mice (8 weeks old, 10M/10F per group) were rendered diabetic (streptozotocin, 150 mg/kg, IV). Diabetes was confirmed (3 days of hyperglycemia (BG > 350 mg/dL) after which, mice were maintained on insulin (Lantus, 1-2 u/day SC). Control, non-transplanted mice (Nude and C57BL/6) were maintained on insulin for 28 days. Pancreatic tissue from pre-weaned Landrace piglets (22±0.5 days old) was cultured after partial enzymatic digestion. After 7 days, islets were encapsulated in 3% UPLVM alginate. Groups of diabetic mice were then transplanted with encapsulated porcine islets into the peritoneal cavity. No immunosuppression was administered to any mice.

Mean blood glucose was 552±24mg/dL (Nude) and 505±43 in C57BL/6 mice (mean±sem). All mice became euglycemic after xenotransplantation. Non-encapsulated controls (C57Bl/6) became hyperglycemic after 4 days of euglycemia whereas, non-encapsulated islet xenotransplants under the kidney capsule in diabetic nude mice maintained euglycemia until nephrectomy. Encapsulated transplant groups; mean blood glucose levels at 30 days (137±23 mg/dL, Nude; 141±24mg/dL, C57BL/6), at 60 days (119±25mg/dL, Nude; 126±12 mg/dL, C57BL/6) and at 90 days (122±8 mg/dL, C57BL/6) post transplant. Explanted encapsulated islets collected at the conclusion of the study (60 days) were viable (77±3% Newport Green/PI) and functional as demonstrated by GSIR, SI=2±0.4; (n=10) within the alginate capsules.

Piglet islets, isolated using a novel protocol and encapsulated in ultrapure alginate microcapsules, can survive and function in diabetic mice. Data is critical to design needed primate studies and ultimately clinical trials using this promising technology.

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Islet Sheets in Diabetic Yucatan Pigs: Initial Experiences.

Rick Storrs2, Morgan Lamb1, Lourdes Robles1, Tianyi Zhou1, Michael Alexander1, Remick Stahl1, Randy Dorian2, Scott King2, Jonathan RT Lakey1,3

1Surgery, University of California, Irvine, Orange, CA, United States; 2Islet Sheet Medical, San Francisco, CA, United States; 3Biomedical Engineering, University of California, Irvine, Irvine, CA, United States.

Encapsulating islets, preventing contact with the host’s cellular immune system would allow transplantation of cells without the need for immunosuppressive drugs. The aim of this study was to evaluate in vivo transplantation of allogenic piglet islets encapsulated in alginate sheets.

Alginate sheets (25 cm2 x 0.5mm) were made from 3% high-guluronate alginate gelled with CaCl­2 and exchanged with BaCl­2, with and without islets isolated from 21 day-old (pre-weaned) Yorkshire piglets. Sheets were implanted into Yucatan pigs (20-50kg). Four sheets containing a total of 360–390 x103 IE were sutured IP into each of two Yucatan pigs previously rendered diabetic by Streptozotocin (STZ, 150mg/kg IV). Blank sheets, without islets, were implanted in subcutaneous pockets (SC) or on the abdominal wall (IP) of non-diabetic Yucatan pigs. No immunosuppression was administered.

Diabetes was confirmed by hyperglycemia (2 days >350 mg/dL), after which insulin glargine was given twice a day. Prior to transplantation insulin requirements were on average 0.8 U/kg/day to maintain average glucose levels of 336 mg/dl and did not change post transplantation. Sheets containing islets were transplanted without surgical complications and were removed after 48 days. Islets within encapsulated sheets remained both dithizone positive and viable (Newport green/PI). Although the intraperitoneal sheets containing islets developed significant adhesions, viable islets (78.2 ±4%) were observed after an extended study period.

After 2-5 weeks blank sheets, implanted IP, were recovered intact and without evidence of cellular adhesion or overgrowth. Subcutaneous implants developed seromas within days of surgery in all locations, including the flank, back and chest, despite occlusive dressings and topical antibiotics.

This demonstrates immunoprotection by the alginate sheet and suggests that the young piglet islets are resistant to hypoxia. Further work will seek to improve the disposition of cell-containing sheets by pre-implant culture and improving the stiffness of the device.

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Declining Number of Pancreas Transplants but Significant Improvement in Outcome

Angelika Gruessner1, Rainer Gruessner1

1University of Arizona, Tucson, AZ, United States.

While outside the US the number of whole pancreas transplants are increasing, in the US the number declined significantly over the recent 9 years. The largest decrease could be found in pancreas after kidney (PAK) transplants. Between 2003/04 and 2011/12, the rate of primary PAKs declined by 63%, PTA by 27% and SPK by 11%. The rate of PAK retransplants declined by 55%. While the number of centers performing SPK and PAK remained stable, only 60% of centers continued to perform PAK. With the decline in numbers a significant improvement in outcome of patient survival as well as pancreas graft function can be found.

A comparison of outcomes of 2003/04 versus 2010/11 primary transplants showed that 1- year patient survival improved from 95% to 99% for PAK (p=0.002), from 95% to 98% for SPK (p=0.0004) and from 94% to 97% for PTA (p=0.16). While overall, early technical failure rates and acute rejection episodes did not change significantly, pancreas graft function improved significantly. For the same time periods 1-year pancreas graft function improved from 78% to 86% in PAK (p=0.01), from 71% to 82% in PTA (p=0.01), from 85% to 89% for the SPK pancreas (p=0.001)and from 93% to 96% for the simultaneous SPK kidney (p=0.002).

In 2010/11 in all 3 categories, recipients were significantly older at transplant and the duration of diabetes was longer, more younger trauma donor were used and preservation time was significantly shorter. More SPK recipients received their kidney preemptively and the kidney function in solitary transplants was significantly better. The rate of HLA mismatches remained the same. More patients received Tacrolimus and MMF without steroids as maintenance immunosuppression.

It seems that better pancreas donor and recipient selection may have been responsible for the improved outcome in the recent transplants.

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Duodenal and Pancreatic Graft Biopsies as Immunological Monitoring After Portal-Duodenal Drained Pancreas Transplants

Marcelo Perosa1,2, Evandro Mello5, Denise Malheiros6, Huda Noujaim1,2, Luiz Ianhez1,2, Rodrigo Azevedo1,2, Leonardo Mota1,2, Juan Branez1,2, Marcio Paredes1,2, Adriana Costa4, Alexandre Fonoff3, Leonardo Oliveira1, Tercio Genzini1,2

1Hepatology and Transplant, HEPATO, São Paulo, Brazil; 2Organ Transplantation, Bandeirantes Hospital, São Paulo, Brazil; 3Endoscopy, Bandeirantes Hospital, São paulo, Brazil; 4Endoscopy, Santa Helena Hospital, São Paulo, Brazil; 5Pathology, Oswaldo Cruz Hospital, São Paulo, Brazil; 6Pathology, Bandeirantes Hospital/Diagnostika, São Paulo, Brazil.

Portal-duodenal drainage(PDD) in pancreas transplantation(PT) is a new surgical alternative and opens a wide possibility of monitoring and intervention of the graft. We have performed protocol endoscopic graft duodenum biopsies(EGDB) and pancreatic biopsies(PB) percutaneously or by echoendoscopy combined to EGDB in cases of graft dysfunction. From February/2010 to April/2013 we analyzed 55 graft biopsies in 30 patients, being 24 PAK, 5 PTA and 1 simultaneous pancreas and living-donor kidney(SPLK) transplant. There were 46 EGDB and 9 PB performed simultaneously(in cases of echoendoscopy) or soon after EGDB if percutaneously. The specimens were blindly analyzed by different experienced pathologists, one for duodenal biopsies(DB), and another for PB. Most of biopsies were performed in the first year post-transplant(46=84%); 37(67%) were protocol biopsies(33 EGDB alone and 2 EGDB + PB) and 18(33%) were indicated for dysfunction(4 EGDB alone and 7 EGDB + PB). Two silent acute rejections were found among the 35 protocol EGDB(5.7%). There were 7 (23%)episodes of acute rejection, 5(71%) of which were diagnosed in DB. Among the 9 cases of duodenal and pancreatic graft biopsies, 7(78%) showed concordant results and in 2 cases rejection was confirmed only in PB. Comparing the 46 EGDB findings to immunological outcome(presence of rejection or immunological loss),5 positive DB were strongly correlated(80%) to rejection episodes and 41(95%) negative DB to a rejection-free outcome(p=0.002). One immunological loss (3.3%) occurred in this series.

Conclusion: EGDB showed a good correlation(78%) with PB and was able to detect silent rejection in almost 6% of protocol biopsies. The low incidence of immunological loss in this series of solitary PT and SPLK, which are categories of PT admittedly more immunogenic, suggests that protocol EGDB combined (or not) to PB in cases of dysfunction may become a helpful monitoring tool in a new scenario of PT with PDD technique.

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Increased Recipient Age (>55 years) does not Affect Outcome After Pancreas Transplantation

Max Marquez1, Markus Selzner1, Ian D. McGilvray1, Fateh Bazerbachi1, Andrea Norgate1, Jeffrey Schiff1, Markus Boehnert1, John Seal1, Mark S. Cattral1

1Surgery, Toronto General Hospital, UHN, Toronto, ON, Canada.

Age limits applied as selection criteria for pancreas transplant recipients are based on subjective center specific criteria rather than objective data. We analyzed the impact of recipient age (≥55 yrs) on outcome of pancreas transplantation from a single North American center.

Methods: 382 pancreas transplants (286 SPK, 96 PAK) were performed at our institution from 1995 until 2013. 30 recipients were ≥55 yrs (55-64 yrs) at the time of transplant while 352 patients were <55 yrs (26.5-54 yrs). Pancreas and kidney graft function were determined by HbA1c and serum creatinine. Postoperative complications, rejection, as well as graft and patient survival were assessed.

Results: Patients ≥ 55 yrs vs. <55 yrs had similar baseline characteristics regarding immunosuppression, length of hospital stay (12±8 vs. 15±14, p=0.06), recipient BMI (25.9±3.9 vs. 25.5±3.8), donor age (26.9±9 vs. 27.8±10), CMV mismatch (28% vs. 31%, p=NS), pancreas cold ischemia time (10.7±3.5h vs. 10.1±2.8 h), and kidney cold ischemia time (10.8±3.8 vs. 8.7±2.6, p=NS). The frequency of re-transplants was similar amongst the groups (4% vs. 3%). Patient survival at 5 years was 93% vs. 95% (p=0.39). The incidence of infectious complications (26% vs. 36% p=NS), including pneumonias (3.3% vs. 6.7%, p=NS), and soft tissue infections (2.9% vs. 4.6%, p=NS) were comparable. The overall rejection rate (13% vs. 33%, p=0.03) was lower in the ≥ 55 yr group. Time to first rejection (168 days (1-4648) vs. 540 days (12–4580), p=0.18) was similar in both groups. Pancreas graft survival at 1, 3 and 5 years was 76% vs. 91%, 76% vs. 86% and 71% vs. 81% respectively (p=0.09). Pancreas graft loss during the first 3 months occurred more frequently in the > 55 yr group; the causes included duodenal leak (37% (n=3) vs. 14% (14) (p=0.11)) and pancreatitis (25% (2) vs. 5% (5) (p=0.08)). Serum creatinine levels at 1-, 3-, and 5-years were similar in both groups (116±21 vs.121±42, 128±29 vs.123±44, 128±40 vs.127±51, respectively p=NS). HbA1c values at 1-, 3-, and 5-years were also similar (.059+.01 vs. 055+.01, .056+.01 vs. 057+.01, .058+.01 vs. 055+.01; p=NS).

Conclusion: Excellent outcome after pancreas transplantation can be achieved in carefully selected patients above 55 yrs of age. Age by itself should not be considered a contraindication for pancreas transplantation.

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HALS-Nephrectomy and Distal Pancreatectomy in Living Donor Operation for Simultaneous Pancreas and Kidney Transplantation: Summary of 10 cases in a Single Institution

Naotake Akutsu1, Michihiro Maruyama1, Kenichi Saigo1, Masayuki Hasegawa1, Kazunori Otsuki1, Hiromichi Aoyama1, Ikuko Matsumoto1, Takehide Asano1, Taihei Ito2, Takashi Kenmochi2

1Surgery, Chiba-East National Hospital, Chiba, Japan; 2Transplantation Surgery, Fujita Health University, School of Medicine, Toyoake, Japan.

Living donor operation for simultaneous pancreas and kidney transplantation is thought to be generally invasive. We have to improve this procedure less invasive and safer for donor benefit, so, we introduced hand-assisted laparoscopic (HALS) approach to donor operation and summarize the results in this presentation.

We have performed 16 cases of living donor simultaneous pancreas kidney transplantation (LD-SPKTx). With the last 10 cases, we introduced HALS-nephrectomy and distal pancreatectomy (LDNP) for donor operation. Three working ports were set (one hand port (7cm); above umbilicus, two 12mm ports; (left lateral abdomen and left lower abdomen)). First, we performed left nephrectomy after dividing ureter with double clipping and finally dividing renal artery and vein by ENDO GIA™. Next, distal pancreatectomy was performed. Pancreatic tail and spleen was dissected from retroperitoneal tissue until found celiac artery and portal vein. Then we cut the pancreatic body above the left side edge of portal vein with LCS and harvested pancreas and spleen.

Total operation time was 451±65 minutes. An estimated blood loss was 519±270 ml. A warm ischemia time (WIT) of kidney was 174±88 seconds and that of pancreas was 238±94 seconds. As compared with open approach (6 cases), HALS approach took more operation time and WIT of pancreas, but less WIT of kidney and blood loss. Graft functions of kidney were so good that urine was recognized immediately and all recipients obtained withdrawal of hemodialysis. As pancreas function, eight recipients got normalizing of blood sugar without insulin. Donors discharged the hospital without remarkable complications.

We demonstrated that HALS-LDNP would have advantages of safeness for donor and of transplant organ function in LD-SPKTx. Long-term, prospective follow-up is warranted to determine the impact of this procedure on LD-SPK benefit.

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The United Kingdom National Pancreas and Islet Allocation Scheme

Alex J Hudson1, Lisa L Mumford1, Christopher JE Watson2

1Statistics and Clinical Audit, NHSBT, Bristol, United Kingdom; 2University Department of Surgery and the NIHR Cambridge Biomedical Research Centre, Addenbrooke’s Hospital, Cambridge, United Kingdom.

Background: In 2010, the United Kingdom (UK) introduced a new National Pancreas Allocation Scheme. Pancreases from donors after brain death (DBD) and circulatory death (DCD) are allocated to patients listed nationally for either a vascularised pancreas (VP) or pancreatic islet (PI) transplant.

Methods: A points scoring system is used to prioritise patients: with prolonged waiting times; with higher levels of HLA sensitisation; on dialysis; local to the donor hospital; avoiding poor HLA mismatch and with minimal donor-recipient age differences. Donor body mass index (BMI kg/m2) is used to decide whether a pancreas is preferentially allocated to a patient listed for a VP (lower BMI) or PI graft (higher BMI).

Results: In the first two years islet transplant activity increased from 11 to 37 grafts with significant improvements in the rate of conversion from isolation to transplant (13% to 38%). VP transplant activity remained stable (~200). The proportion of patients on the waiting list ≥ 2 years decreased from 21% to 7%. The proportion of sensitised patients (reaction frequency ≥ 75%) listed increased (12% to 20%), largely due to an excess of long waiting patients awarded higher priority; these patients have now been transplanted. Despite increased organ transportation, cold ischaemia times remained unchanged (VP median ~12 hours; PI median ~7 hours). The proportion of poor HLA mismatched transplants (5 or 6 HLA A-B-DR mismatches) decreased from 34% to 16%. Compared to the year before the scheme started, three-month pancreas graft survival is unchanged: simultaneous pancreas/kidney (93% vs 90%, p=0.4), isolated pancreas (89% vs 75%, p=0.2). Islet recipients have noted significant reductions in hypoglycaemic events and HbA1c.

Summary: The UK’s patient-based allocation scheme for all deceased donor pancreases has increased islet transplant activity and reduced waiting time for a whole organ transplant. In addition pancreas utilisation in PI transplants has increased.

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Living Donor Pancreas Transplantation- Single Center Experience of 18 Cases

Young Hoon Kim1, Duck Jong Han1, Joong Yeol Park2, Sung Shin1, Byung Hyun Choi1, Joo Hee Jung1, Han Kyung Cho1

1Surgery, Asan Medical Center, Seoul, Korea; 2Endocrinology, Asan Medical Center, Seoul, Korea.

Living donor pancreas transplantation (LDPT) is not so popular due to high technical failure rate and elevated chance of donor morbidity including development of diabetes. However advantages of LDPT include shorter waiting time especially in patients waiting simultaneous pancreas and renal transplantation, lower probability of rejection, and chance of pre-conditioning in sensitized condition.

From October1992 to Apr 2013, we have performed 18 living donor PT (6 PTA, 12 SPK) including one ABO incompatible patient. We retrospectively reviewed our clinical cases.

Preoperatively all the donors have been studied extensively by endocrinologist. Mean donor age was 42.0 year old (±9.8) and a mean BMI 22.2 (±2.3). Relation of donors was parents in 7, siblings in 5, and spouse in 6. Mean recipient age was 30.7 year old (±8.2). Type I DM was 16 patients, and mean BMI 20.5 (±1.5). Overall patient survival was 100%. Pancreas graft survivals at 1 year, 3year, and 5 year were 91.6 %, 83.3 % and 83.3 % respectively in SPK recipients, and 50%, 33.3% and 16.7% in PTA recipients. Causes of graft failure in SPK were acute rejection and one of whom associated with thrombosis, while in PTA recipients those were 2 cases of non-compliance, two cases of thrombosis, and one chronic rejection. According to pancreatic exocrine drainage, one out of 8 grafts in bladder drained group survived while all the grafts in enteric drained group survived.

Minor pancreatic juice leak was the most common complication (7/18) in donors. Most of donors were normoglycemic, however diabetes developed in 2 donors that was treated with oral hypoglycemic agent.

According to our experience of living donor pancreas transplantation, the donor surgical risk is acceptable. The surgical procedure of LDPT seems to be justified is SPK recipient with enteric drainage. However considering the poor graft survival in PTA in our small series, LDPT in PTA recipient should be performed cautiously.

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Long Term Follow Up for Beta Cell Replacement Therapy in HIV Infected Patients with Renal Failure Secondary to Type I Diabetes Mellitus

Garrett Roll1, Jack Harbell2, Andrew Posselt1, Chris Freise1, Sang Mo Kang1, Ryutaro Hirose1, Greg Szot1, Sandy Feng1, Peter Stock1

1Dept of Surgery, Division of Transplantation, University of California San Francisco, San Francisco, CA, United States; 2Dept of Surgery, University of California San Francisco, San Francisco, CA, United States.

Introduction: The approach to transplantation (tx) in HIV+ patients (pts) has been conservative for fear of exacerbating an immune compromised condition. Short term insulin independence has been shown in a small series of patients, but long term outcomes have not been described. We report a pilot of islet or pancreas tx in HIV+ type I diabetic patients, with an average follow up 3.8 years.

Methods: HIV+ pts with CD4 counts >200 cells/ml, with undetectable HIV viral load on a stable anti-retroviral regimen underwent simultaneous pancreas-kidney tx (SPK), pancreas after kidney (PAK), or islet after kidney (IAK). Thymoglobulin (Thymo) induction (6 mg/kg) was used for pancreas transplant and one sensitized IAK pt. Maintenance immunotx: calcineurine inhibitor, Mycophenolate mofetil, and Pred. IAK were performed 6 (pt 6) and 7 (pt 7) years after living-donor kidney and deceased-donor kidney tx respectively for hypoglycemic unawareness.

Results: Thymo resulted in profound lymphocyte depeletion, with CD4 counts recovering to baseline in one year. Two opportunistic infections (BK viremia) occurred. After reduction of immune suppression BK viral loads are currently undetectable. A single opportunistic neoplasm occurred (squamous cell carcinoma); diagnosed 46 mo after transplantation. Two patients passed away from cardiovascular events, 48 months and 58 mo after therapy. 5/5 pancreas recipients, and 1/2 islet recipients have insulin independence, or were insulin independent when they died. One IAK recipient resumed insulin after presumed islet rejection. All patients are dialysis independent, or were dialysis independent when they died (Table 1, * = at time of death).

Conclusions: Type I diabetic recipients with well controlled HIV tolerate the intensive lymphocyte depleting induction therapy required for beta cell replacement. Adequate prophylaxis against opportunistic infections has been largely successful until the CD4+ counts return to baseline. Well controlled HIV+ type I diabetic patients with renal failure are candidates for beta cell replacement.

TABLE 1
TABLE 1
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Surface-Coating of Pancreatic Islets with Neural Crest Stem Cells Improves Islet Engraftment and Function After Intraportal Transplantation

Joey Lau1, Svitlana Vasylovska1, Elena Kozlova2, Per-Ola Carlsson1,3

1Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden; 2Department of Neurosciences, Uppsala University, Uppsala, Sweden; 3Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

Previous studies have shown that neural crest stem cells (NCSCs), obtained from dorsal root ganglia, improve beta-cell function and cause a potent stimulation of beta-cell proliferation when co-transplanted with mouse islets beneath the renal capsule. We aimed to develop techniques for surface-coating of islets with NCSCs in order to enable co-transplantation to the clinically used liver site, and then investigate engraftment and function intraportally of such bioengineered islets.

Mouse islets were incubated during gentle shaking for 1h with or without dispersed GFP-expressing mouse NCSCs to allow surface coverage. Islets were then transplanted into the portal vein of alloxan-diabetic mice. Blood glucose concentrations were monitored up to one month posttransplantation, and followed by an intravenous glucose tolerance test of the islet recipients. Islet grafts were then retrieved and processed for immunohistochemical evaluation for vascular density, beta-cell proliferation, nerves and glial cells.

Mice transplanted with NCSC bioengineered islets responded better to the glucose load than recipient mice of control islets (P<0.05). NCSCs remained present in the vicinity or had even migrated into many of the NCSC-coated islets, and an improved islet graft revascularization was observed (vascular density for NCSC-coated islets 2.31±0.29 % vs. control islets 0.64±0.11 %; P<0.05). Furthermore, NCSC-coated islet grafts had a better reinnervation (0.73±0.09 % vs. 0.28±0.02 % in control islet grafts; P<0.05). Transplanted NCSCs both differentiated into neurons and glial cells.

We conclude that bioengineering of islets with NCSCs for intraportal transplantation provides a possibility to improve islet engraftment and function. Pending successful establishment of protocols for expansion of NCSCs from e.g. human skin or bone marrow, this strategy may be applied to clinical islet transplantation.

Supported by: EFSD/JDRF/NN 2012, SRC, SDF, Diabetes Wellness Sweden, NNF and AFA.

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486

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Surface-Coating with Endothelial Progenitor Cells Improves Blood Flow, Oxygen Tension and Vascular Density in Experimentally Transplanted Human Pancreatic Islets

Liza Grapensparr1, Per-Ola Carlsson1,2

1Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden; 2Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

Endothelial progenitor cells (EPCs) are pro-angiogenic bone marrow-derived cells that promote neovascularization. The present study tested the hypothesis that EPCs of human derival could be coated to human islet surfaces and be used to promote islet vascular engraftment. Control or EPC bioengineered human islets were transplanted into the renal subcapsular space of NOD/SCID mice. Four weeks after transplantation, graft blood perfusion and oxygen tension was measured using laser-Doppler flowmetry and Clark microelectrodes, respectively. The vascular density and the respective contribution of human and recipient endothelium was assessed immunohistochemically by staining for human and mouse CD31. EPC-derived cells were tracked by luciferase transfection. Islet grafts with EPCs had a substantially higher blood flow and oxygen tension (5.5±0.6 TPU and 20.6±3.3 mmHg, respectively; n=9), than control transplants (2.5±0.3 TPU and 6.5±1.0 mmHg, respectively; n=10). Furthermore, analysis of the vascular network of the grafts revealed that EPC bioengineered islets had a much higher vascular density than transplanted control islets, with incorporated EPC-derived cells into functional blood vessels. We conclude that a simple procedure of surface-coating with EPCs provides a possibility to improve the vascular engraftment of transplanted human islets. Established protocols are easily applicable also for intraportal islet transplantation in order to obtain a novel directed cellular therapy at the site of implantation in the liver.

Supported by: EFSD/JDRF/Novo 2012, SRC, SDF, NNF, AFA and Diabetes Wellness Sweden

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487

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The Bone Marrow Cavity is Suitable for Islet Transplantation in Diabetic Rhesus Monkey

Chengshi Wang1, Xiaojiong Du1,2, Sirong He1, Younan Chen1, Jingping Liu1, Meimei Shi1, Gang Mai2, Guang Yang1, Li Wang3, Hongxia Li3, Guangneng Liao1, Pengfei Han4, James Kang4, Jingqiu Cheng1, Yanrong Lu1

1Laboratory of Transplant Engineering and Immunology, West China Hospital, Chengdu, People’s Republic of China; 2Department of General Surgery, West China Hospital, Chengdu, People’s Republic of China; 3National Center for Safety Evaluation of Traditional Chinese Medicine (Chengdu), Chengdu, People’s Republic of China; 4Regenerative Medicine Research Center, West China Hospital, Chengdu, People’s Republic of China.

Objective: The liver is currently the only approved site for clinical islet transplantation. Recent reports have revealed that the bone marrow cavity was an altered site for islet transplantation. In this study, we investigated whether the bone marrow cavity was better than liver for islet transplantation in diabetic rhesus monkeys.

Methods: Rhesus monkeys were used as recipients and made diabetic with streptozotocin at least 10 days before transplantation. Islets were isolated from rhesus monkeys and marked with SPIO. In this study, two groups were included: (1) control group (n=2), freshly isolated islets (11464 ± 2140 IEQ/kg) were transplanted into liver; (2) experimental group (n=5), same dose of islets were transplanted into bone marrow cavity of the tibia. An immunosuppression regimen was used to prolong allograft survival with Sou-Medrol, Lymphocyte Immune Globulin (ATG, 3 mg/kg/d for 4 days), Tacrolimus (0.2mg//kg/d) and Sirolimus (0.3mg/kg/d). An intravenous glucose tolerance (IVGTT) test was performed at 1 month after transplantation to evaluate the function of the grafted islets. The fasting blood glucose (FBG) levels were monitored 2 times per week, exogenous insulin was injected as fasting blood glucose greater than 11.1 mmol/l on two consecutive days.

Results: Five recipients in experimental group maintained normoglycemia without insulin therapy up to 195, 83, 174, 102 and 95 days (Fig 1), respectively. In contrast, two recipients in control group were only 39 and 58 days, respectively. The engrafted islets were observed at 3, 7, 30 days after transplantation using MRI. IVGTT results showed islet grafts were fully functional in both groups.

Conclusions: The operation of islets transplanted into bone marrow cavity is easier than into liver. The allogeneic islets in bone marrow cavity can survive and keep function for longer period than in liver in diabetic rhesus monkey. The bone marrow cavity is a better site for islet transplantation.

This study was supported by the National Program for High Technology Research and Development of China (No. 2012AA020702), Chinese National Science and Technology Project (No. 2011ZX09307-301) and Program of National Natural Science Foundation of China

FIGURE 1.
FIGURE 1.
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Polyclonal Serum Immunoglobulin M Antibody Therapy Prevents the Onset and Progression of Type 1 Diabetes, Induces the Development of β-cell Specific Hyporesponsiveness and Promotes Islet Transplantation Outcomes.

PREETI CHHABRA1, FARAH MUKHEEF1, KENNETH BRAYMAN1

1Surgery, Transplant division, University of Virginia, Charlottesville, VA, United States.

Purpose: To study the effect of polyclonal serum Immunoglobulin M (IgM) therapy in preventing the onset and progression of type 1 diabetes (T1D), inducing β-cell specific hyporesponsiveness (tolerance) and promoting islet transplantation.

Methods: 4–5 wks-old non-obese diabetic (NOD) mice received either 75μg IgM therapy biweekly or PBS/BSA intraperitoneal (IP) until 18 weeks of age, and blood glucose (BG) levels monitored. In adoptive transfer of tolerance or of disease experiments, 5–6 wks-old NOD-scid mice served as recipients of 1-2×107 splenocytes or 7.5×106 CD3 T-cells IP from tolerant mice (mice that remained non-diabetic for >25 weeks post-IgM therapy discontinuation) and/or from diabetic mice, with or without IgM therapy. Plasma insulin autoantibody (IAA) levels were measured by radioimmunoassay. 10–12 weeks-old female BALB/c mice served as donors in islet allotransplantation and retired female C57BL/6 mice in syngeneic transplantation experiments.

Results: When IgM therapy was initiated at 4–5 weeks of age, none of the IgM-treated mice (n=33) became diabetic. In contrast, 80-90% control mice (n=30) became diabetic by 18–20 weeks of age. Initiated at 11 weeks of age, only 20% (n=20) mice became diabetic (p<0.0001). Discontinuing therapy resulted in hyperglycemia in only 27% mice at 28 weeks post-discontinuation, indicating induction of β-cell specific hyporesponsiveness in the remaining animals. IAA production, a marker for the onset/progression of T1D pathogenesis, was significantly inhibited by IgM therapy (p<.001). IAA in the diabetic group (n=5) was either very high (2/5), moderate (1/5) or low (2/5), and moderate in the prehyperglycemic group (n=1). In contrast, IAA levels in the IgM-treated group (n=10) and tolerant mice (n=2) were 0. Adoptive transfer of CD3 T-cells from diabetic NOD mice into NOD-scid recipients resulted in hyperglycemia in 24.0±1.0days (n=7), which was significantly delayed by IgM therapy (53.0±19.1days; n=7) (p<0.001). Recipients of splenocytes from tolerant mice alone (n=5) remained permanently non-diabetic. When diabetic to tolerant splenocytes ratios were 1:1 (n=5) and 1:2 (n=3), the incidence of hyperglycemia was significantly delayed, while with 1:3 ratio (n=3), mice have remained non-diabetic. The mean survival time of BALB/c islet allografts was 41.2±3.3 days for IgM-treated C57BL6 recipients (n=4, fifth remained normoglycemic) vs. 10.2±2.6 days for controls (n=5,p<0.001). In syngeneic transplantation, time to return to normoglycemia was 15.4±3.6 days for IgM-treated recipients (n=5) and >35 days for controls (n=4).

Conclusion: IgM therapy prevents the onset and progression of T1D, induces β-cell specific hyporesponsiveness and promotes islet transplantation. IgM therapy has significant clinical translational potential in the prevention and cure of T1D and in promoting islet transplantation outcomes.

Funded by the Focus to Cure Diabetes Foundation.

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Soluble Human TNFRI-Fc and HO-1 Gene Transfection into Porcine Islet Can Prolong Islet Graft Survival in Humanized Mice

Hyojun Park1, Han Shin Lee2, Jae Berm Park1, Na Yoon Hur1, Sung Joo Kim1

1Transplantation division of general surgery, Samsung medical center, Seoul, Korea; 2Department of Molecular Medicine, Sungkyunkwan University School of Medicine, Seoul, Korea.

Background: Recent studies have also demonstrated that the main causes of xenograft rejections are tissue factor (TF) of recipient platelets and hypoxic conditions during the early stage of islet engraftment. Several studies have shown that HO-1 and anti-TNF-a antibodies can prevent xenograft rejection and improve islet function and survival by their strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. in this study, we investigated that soluble human TNFRI-Fc (shTNFRI-Fc) and Heme oxygenase 1 (HO-1) gene improve islet survival and function in diabetic humanized mice.

Method: For humanized mice, CD34+ stem cells obtained from cord blood and then injected in NOD/SCID/IL-2 gamma−/−(NSG) mice. After 16 weeks, human T and B cell population was analysed by flow cytometry and used for this study. ShTNFRI-Fc, HO-1 and ShTNFRI-Fc-hHO-1 gene expression vectors were constructed using adenovirus and transfected into porcine islets. ShTNFRI-Fc, HO-1 and ShTNFRI-Fc-hHO-1 gene expression in transfected porcine islets were identified by fluorescence microscopy and analysed by flow cytometry. Diabetes was induced with intraperitoneal injection of streptozocin (50mg/kg/day, 5days). transfected porcine islets (5000 IEQ) were transplanted under the kidney capsule of diabetic humanized mice. Blood glucose levels were monitored everyday during first 2weeks and every 2days after 2weeks. Islet survival and function was confirmed by blood glucose level and intraperitoneal glucose tolerance test (IPGTT). Finally all islet grafts were processed for histological analysis.

Results: After 16weeks, NOD/SCID/IL-2 gamma−/−(NSG) mice were engrafted with 20% ± 15% of human cells in peripheral blood (48.6 ± 22 of CD3+ T cells and 36.8 ± 18.7 of CD19+ B cells). Porcine islet transfection rate was 29% in shTNFRI-Fc, 32% in HO-1 and 45% in shTNFRI-Fc+HO-1. After islets transplantation (5000 EIN), blood glucose levels were decreased to nearly euglycemic level in most of transfected islet groups, Survival of islet grafts was markedly prolonged in transfected groups, especially HO-1 and double group compared to control group.

Conclusion: The results of this study revealed that Islet cell isolation and transfected porcine islet xenotransplantation to improve the survival and function of islet cells that are important in the treatment of diabetes. This also suggests the clinical potential of the future of diabetes treatment.

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Architecture of Islets After their Transplantation

Vanessa Lavallard1, Géraldine Parnaud1, Philippe Morel1, Raphaël Meier1, Laurianne Giovannoni1, Benoît Bedat1, Thierry Berney1, Domenico Bosco1

1Department of Surgery, Cell Isolation and Transplantation Center, Geneva University hospital and University of Geneva, Geneva, Switzerland.

Background: Human pancreatic islets have a unique architecture that favors interactions between beta- and alpha-cells. The aim of this study was to assess whether this architecture was maintained in human islets after their isolation and transplantation and to assess the role of vascularization in this phenomenon.

Method: Human islets were isolated and cultured for 1–4 days. Then, they were transplanted under the kidney capsule of Scid mice. In addition, isolated islets were dispersed into single cells and either transplanted under the kidney capsule of Scid mice or kept in culture for 2 weeks allowing cell aggregation and pseudoislet formation. Some transplanted mice were treated with bevacizumab (anti VEGF) to alter vascularization of grafts. Histology of islets and islet cells was analyzed two weeks after transplantation and compared to that of islets and pseudoislets after culture. Immunofluorescence for insulin, glucagon and CD31 was performed. Morphometric analysis of staining was performed with Metamorph software.

Results: By immunofluorescence and following quantification by Metamorph we showed that in cultured islets alpha- and beta-cells lost their specific arrangement and were intermingled. In pseudoislets formed after 2 weeks of culture, alpha- and beta-cells had an inverted core-mantle position with alpha-cells at the center surrounded by beta-cells. Two weeks after transplantation, islet cells reorganized forming structures similar to native pancreatic islets with alpha-cells surrounding beta-cells and vessels lining alpha-cells. Similar results were obtained when islet cells were transplanted instead of islets. After bevacizumab treatment, transplanted islet cells revealed an absence of vascularization but still displayed structure with alpha-cells surrounding beta-cells similar to native islets.

Conclusion: Islets and pseudoislets in vitro have a disorganized architecture and alpha- and beta-cells have the ability to reorganize in vivo after transplantation forming structures similar to pancreatic islets. Mechanism underlying this phenomenon still remains to be elucidated but is independent of vascularization.

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491

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Adiponectin Prevents Islet Ischemia-Reperfusion Injury through the COX-2—TNF-α—NF-κB Dependent Signal Transduction Pathway in Mice

Sirong He1, Xiaojiong Du2, Weiming Hu2

1Regenerative Medicine Research Center, West China Hospital, Sichaun University, Chengdu, People’s Republic of China; 2Department of Hepatobiliopancreatic Surgery, West China Hospital, Sichuan University, Chengdu, People’s Republic of China.

Islets are exceptionally susceptible to ischemia-reperfusion injury, an increased incidence of primary graft nonfunctionality, and β-cell death during a transplant procedure. Therefore, islets require protection during the early stages of the transplant procedure. Based on the beneficial vascular and anti-inflammatory activity of adiponectin, we hypothesize that the adiponectin protects islet cells against ischemia/reperfusion injury and graft dysfunction after transplantation. To examine the effects of adiponectin on the resistance of islet ischemia-reperfusion injury, we used the islet hypoxia-reoxygenation injury model and performed kidney subcapsular syngeneic islet transplants to assess the islets’ vitality and function (Fig1). Furthermore, we utilized lipopolysaccharide (LPS)-induced or tumor necrosis factor (TNF)-α–induced damage to islet cells to model the inflammation of post-transplant ischemia-reperfusion injury and transplanted islets in adiponectin-knockout (APN-KO) mice to explore whether the protective action of adiponectin is involved in TNF-α production and nuclear transcription factor-κB (NF-κB) activation. Adiponectin suppressed TNF-α production and IκB-α phosphorylation; decreased hypoxia-reoxygenation, LPS-induced and TNF-α–induced islet apoptosis; and improved islet function in vivo and in vitro (Fig2). Our results demonstrate that adiponectin protects the islet from injury. We show that islet protection occurs in response to ischemia-reperfusion and is dependent on the suppression of islet production by TNF-α through cyclooxygenase (COX)-2 and the inhibition of the TNF-α–induced NF-κB activation pathways.



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493

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Immunological Impact of Efalizumab Versus Belatacept in Islet Transplantation

Qizhi Tang1, Andrew Posselt1, Gregory L. Szot1, Kristin Melli1, Mariela Pauli1, Lynda Frasetto2, Umesh Masharani2, Lawrence Fong2, Peter G. Stock1

1Surgery, UCSF, San Francisco, CA, United States; 2Medicine, UCSF, San Francisco, CA, United States.

Background: We have treated islet transplant recipients with thymoglobulin induction and maintenance immunosuppression consisted of sirolimus or mycophenolate along with either efalizumab to block LFA-1 or belatacept to block CD28 costimulation. Dosing of sirolimus or mycophenolate was adjusted when efalizumab was withdrawn from the market. The immunological impact of these immunosuppressive regimens was compared in this study.

Methods: Peripheral blood mononuclear cells were collected and cryopreserved beginning at enrollment before transplantation. Donor spleens were banked at the time of transplant. Percentages of regulatory T cells (Tregs) were determined using flow cytometry. Proliferative responses of CD4, CD8, and Tregs to donor and polyclonal stimulations were determined in vitro using a CFSE dilution assay.

Results: All patients on efalizumab showed dramatic increase of percentage of Tregs from 5 to 10% among CD4+ T cells pre-transplant to peaks of 15 to 67% between 1 and 3 months post transplant. Treg percentages remained elevated while patients were on the regimen. T cell proliferation to donor and polyclonal stimulations was markedly reduced in patients on efalizumab and returned to pre-transplant levels 5 months after the cessation of efalizumab (Figure). In contrast, percentages of Tregs and T cell proliferative responses remained stable at all time points in patients on the belatacept-based regimen. These results demonstrate that efalizumab caused profound changes of patients’ immunological make-up when compared to belatacept and other conventional immunosuppression. Particularly, one of the most severely immunosuppressed efalizumab patient experienced recurrent infections that necessitated complete withdrawal of immunosuppression. Her anti-donor and polyclonal responses rebounded from non-detectable levels to her pre-transplant baseline after efalizumab withdrawal. Remarkably, she has remained insulin independent despite receiving no immunosuppression in the past 9 months.


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Conclusion: Efalizumab increases percentages of circulating Tregs and profoundly suppresses T cell reactivity. Short-term efalizumab treatment may promote transplantation tolerance.

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Treg Requires Transcription Factor T-Bet for Proper Migration to Prolong Islet Allograft Survival

Daiki Iwami1, Yanboa Xiong1, Colin Brinkman1, Jonathan Bromberg1

1School of Medicine, University of Maryland, Baltimore, MD, United States.

Introduction: Foxp3+ regulatory T cells (Treg) must sequentially migrate and properly position in allografts and in draining lymph nodes (dLN) to suppress alloimmunity and prolong graft survival. T-bet, a transcription factors essential for Th1 development, has been shown to regulate some aspects of Treg function during inflammation and autoimmunity. Recently, T-bet has been shown to regulate chemokine receptors and adhesion molecules such as CXCR3, CCR5, LFA-1, P-selectin ligand, and ICAM-1. We hypothesized that T-bet regulates Treg migration and protective function for graft survival.

Methods: Four hundred BALB/c islets were transplanted underneath the kidney capsule of streptozotocin-induced diabetic C57BL/6 recipients. 1x106 CD4+CD25+ Treg from wild type (WT) or T-bet knock out (KO) C57BL/6 were administered intravenously or locally together with the islets at the time of transplantation. Allograft function was monitored by blood glucose. Treg were assessed by flow cytometry and qRT-PCR for in vivo migration and expression of effector-related molecules, and by in vitro functional suppression and in vitro migration assays.

Results: Untreated recipients rejected islets rapidly (median survival time (MST) 10 days). WT Treg prolonged survival when administered intravenously (MST 30d, p<.01) and were more effective when administered locally (MST 39d, p<.01). In contrast, T-bet KO Treg prolonged graft survival only a few days when transferred either iv (MST 15d) or locally (MST 18d). Assessing in vivo migration, WT and T-bet KO Treg both migrated rapidly from blood to the islet grafts. However, while WT Treg migrated from blood to LN and from islet grafts to dLN, T-bet KO Treg were impaired in their ability to migrate to LN via blood or lymphatics. T-bet KO Treg recovered from islets expressed less CXCR3 compared to WT Treg, and T-bet KO Treg recovered from dLN expressed less CCR5 compared to WT Treg. CCR4 and CCR7 expression were similar in both strains of Treg in both islets and dLN. In vitro transmigration in response to CCL19, the ligand for CCR7, did not reveal a difference between Treg from either strain. Foxp3, CTLA-4 and granzyme B expression and in vitro suppressive function of Treg were also similar in both strains.

Conclusions: T-bet regulates the expression and function of several Treg chemokine receptors, and therefore the ability of Treg to migrate to lymphatics and dLN. In turn these functions are required for proper Treg migration, so that T-bet is essential for the ability of Treg to prolong islet allograft survival. Thus, Treg co-opt Th1 transcription factors in order to express Th1 related chemokine receptors and suppress Th1 effector cells. Therapies directed against Th1 pathways have likely failed in the past due to unintended consequences on Treg migration and function.


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495

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Graft of Microencapsulated Sertoli Cells for the Cell Therapy of Type 2 Diabetes Mellitus (T2DM)

Giovanni Luca2, Francesca Mancuso2, Mario Calvitti2, Iva Arato2, Giulia Falabella2, Mariella Bobo2, Ennio Beccetti2, Riccardo Calafiore1

1Internal Medicine, University of Perugia, Perugia, Italy; 2Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy.

T2DM is caused by an association of insulin resistance with beta-cell failure, both being linked to chronic inflammation of the visceral adipose tissue (VAT), which results in generation and release of noxious local and systemic proinflammatory cytokines and adipokines. Sertoli cells (SC) synthesize a vast array of growth factors and immunomodulatory molecules, already proven able to effectively target several autoimmune and inflammatory chronic disorders. In order to assess whether prepubertal porcine SC (pSC), upon immunoisolation in our ultrapure alginate-based microcapsules, prepared by our method, would reverse hyperglycemia in T2DM recipients, we transplanted microencapsulated pSCWe then grafted prepubertal porcine SC into the subcutaneous fat of db/db mice (M) with spontaneous, overt T2DM, exhibiting, at graft time, high blood glucose (BG) as well as HbA1c levels. We also have initiated a pilot trial of microencapsulated SC transplantation into the peritoneal cavity of rhesus monkeys (RM) with spontaneous insulin-dependent T2DM. Upon graft, daily BG and weekly HBA1c levels were measured. At 5 wk of transplant IPGTT and IVGTT (in M and RM respectively) were performed. 70% of the grafted M showed significant decline in HbA1c levels (5.0% vs 9.2%), and normalization of IPGTT . Significant increase of CD4+/CD25+/Foxp3+ (Treg) cells, and decline of IL17, upon anti-CD3 stimulation, in stromal VAT and lymphonode-retrieved lymphocytes as well as splenocytes, respectively, were observed, while pre- and post-prandial BG was normalized, as compared to untreated controls. RM, very preliminarily, showed significant decline in HbA1c levels, with significant decline, over mohths, of the daily exogenous insulin supplementation. The latter was transiently discontinued withdrawal in one recipient. Furthermore, the grafted RM showed an increase in peripheral Treg while assessment of splenocytes is being in progress. In summary, we observed that encapsulated SC grafts significantly impacted both M and RM with spontaneous T2DM, with normalization and significant improvement of metabolic as well as immune and inflammatory parameters. In conclusion,in light of these preliminary data, microencapsulated pSC might offer a novel potential avenue for the cure of T2DM.

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496

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PTF1A Drives PDX1 Expression and Potentiates Differentiation of Monohormonal Endocrine Cells from ESCs.

Gopika G Nair1, Robert K Vincent1, Jon S Odorico1

1Surgery, University of Wisconsin-Madison, Madison, WI, United States.

Efficient differentiation of embryonic stem cells (ESCs) to insulin-expressing β cells has remained elusive, due to incomplete understanding of the transcriptional networks and their timing of activation, and the difficulty in reproducing suitable niches in a dish. Current protocols produce immature multiple-hormone expressing cells, in vitro and an in vivo environment appears essential for their maturation and amelioration of the diabetic state[1–3]. Directed differentiation by induced expression of critical pancreatic genes could further our efforts in this regard. One such important transcription factor is pancreas-specific transcription factor 1a (PTF1a) that is essential for specification of the pancreas from the foregut endoderm, besides its role in exocrine differentiation later in development[4]. A study exploiting this early function of PTF1a to facilitate differentiation of ESCs to the pancreatic lineage has not been performed. To this end, we derived and characterized a mouse ESC line with tetracycline-inducible expression of PTF1a (tet-Ptf1a). We found that transient ectopic expression of PTF1a initiated the pancreatic program in differentiating ESCs causing cells to activate PDX1 expression in bud-like structures resembling pancreatic primordia in vivo. These bud-like structures also expressed progenitor markers including HNF6, FOXA2, HLXB9, SOX9 and NKX6.1 that are characteristic of the developing pancreatic epithelium. The epithelium differentiated to generate a wave of NGN3+ endocrine progenitors that were distinct from CPA1+ cells (potentially exocrine progenitors), and further formed cells of all three pancreatic lineages. Insulin+ cells in the cultures were monohormonal, and expressed PDX1 and NKX6.1 as expected for bona fide beta cells. PTF1a-induced cultures differentiated into significantly more endocrine and exocrine cells and the ratio of endocrine-to-exocrine cell differentiation could be regulated by retinoic acid (RA) and nicotinamide (Nic) signaling. Moreover, induced cultures treated with RA and Nic exhibited a modest glucose response. Thus, this tet-Ptf1a ESC-based in vitro system is a valuable new tool for interrogating the role of PTF1a in pancreas development and in directing differentiation of ESCs to endocrine cells.

Reference:

  • 1. Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S, et al. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotech. 2008;26(4):443–52.
  • 2. Rezania A, Bruin JE, Riedel MJ, Mojibian M, Asadi A, Xu J, et al. Maturation of Human Embryonic Stem Cell–Derived Pancreatic Progenitors into Functional Islets Capable of Treating Pre-existing Diabetes in Mice. Diabetes. 2012.
  • 3. Nostro MC, Sarangi F, Ogawa S, Holtzinger A, Corneo B, Li X, et al. Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells. Development. 2011;138(5):861–71.
  • 4. Kawaguchi Y, Cooper B, Gannon M, Ray M, MacDonald RJ, Wright CVE. The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nat Genet. 2002;32(1):128–34.
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497

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Functional Pancreatic Beta Cell Lines Derived from Human Pluripotent Stem Cells

Dodanim Talavera1,2, Soshana Svendsen1, Clive Svendsen1, Kira Chaiboonma1, Vaithilingaraja Arumugaswami1,2, Donald Dafoe1,2

1Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, United States; 2Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, United States.

Generation of pancreatic beta-cell lines derived from pluripotent stem cells (PSCs) is necessary to cover diabetic patient demands. However, these cell lines have not been expanded in vitro. We differentiated induced PSCs (iPSCs) to insulin-producing beta cells in a complex co-culture system with endothelial cells (ECs), extracellular matrix components, and growth factors. Then, we labeled the differentiated beta cells with a red fluorescent protein (mCherry) under the control of insulin promoter for isolation and expansion. Beta-cell marker expression was evaluated by immunocytochemistry and qRT-PCR. Quinacrine secretion assay and ELISA were used to evaluate secretion of C-peptide in vitro after a glucose challenge. Labeled cells were sorted by FACS and transplanted under the kidney capsule of SCID mice. Human C-peptide was measured in mouse blood at baseline and after glucose challenges. Grafted kidneys were analyzed by immunohistochemistry (IHC). Higher expression of beta cell markers (insulin, PDX-1, Nkx6.1, Kir6.2, Glut2, GKS, SUR1, PC1/3, PC2, and amylin) was found in differentiated and sorted cells compared to non-sorted cells (P <0.05). Sorted and labeled cells were expanded up to ten passages. Quinacrine secretion and human C-peptide was detected in the culture media of sorted cells at 0 mM (1.14 ± 1.3 pmol/L) or 17 mM (4.6 ± 2.2 pmol/L, P<0.05) glucose concentrations. The levels of C-peptide in mice blood samples at 60 days after transplantation indicated an increase in human C-peptide secretion after 30 (91.2 ± 3.9 pmol/L) and 60 (150 ± 10 pmoL/L) minutes following glucose challenge (basal = 58.2 ± 1.5 pmol/L). Harvested cells were positive for insulin after IHC analysis. These results indicate that beta cells can be derived from iPSCs and expanded in vitro and that these cells maintain their functional phenotype in vitro and in vivo.

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498

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Generation of Human Pancreatic Organoids in a Three-Dimensional Culture System as a Potential New Source for Beta Cells

Cindy Loomans, Nerys Williams, Leon van Gurp, Femke Ringnalda, Toshiro Sato, Marten Engelse, Ton Rabelink, Hans Clevers, Eelco de Koning

1Hubrecht Institute, Utrecht, Netherlands; 3Department of Medicine, Leiden University Medical Center, Leiden, Netherlands; 2Dept. of Gastroenterology, Keio University, Tokyo, Japan.

Beta cell replacement therapy for a wider group of patients with type 1 diabetes is hampered by a lack of donor organs. Here we explore the possibility of using human adult pancreatic progenitor cells as a source for generating beta-cells. Islet-depleted tissue, left over after islet isolation from human donor pancreas (n= 25), was cultured in a novel 3-dimensional (3D) matrigel-based culture system. In the expansion phase, small ducti grew extensively, producing budding structures with trunk and tip sections which resembled the ductal tree during embryogenesis. These structures, referred to as organoids, could be passaged several times without phenotypic changes. Expression of ductal marker Ck19 was present in 92,3%±5,4 of the cells. Tips of budding structures co-expressed progenitor markers Pdx1 and Sox9, and showed high proliferative activity. High expression of aldehyde dehydrogenase (ALDH) was found exclusively in tip cells. Sorted ALDH-high cells could initiate organoid growth, while the ALDH-low cells could not. Furthermore, high expression levels of Cpa1, Myc, Pdx1 and Ptf1a were present only in ALDH-high cells potentially indicating multipotency. During the differentiation phase, an increase in gene expression of endocrine progenitor marker Ngn3 was observed. No insulin-positive cells could be detected in culture. Next, organoids were transplanted under the kidney capsule in NOD-SCID mice (n=15). Insulin-positive cells appeared just 14 days after transplantation. More insulin-positive cells were present one month after transplantation (<2% of total graft). Circulating human C-peptide could be detected in the transplanted mice (227 ±113 pmol/l) but normoglycemia was not restored in diabetic animals.

In conclusion, we have developed a 3D culture system to generate human pancreatic organoids that have the capacity to differentiate into insulin-positive cells.

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Neonatal Porcine Islet-Mesenchymal Stem Cells Co-Transplantation in Type I Diabetes Rhesus Monkeys

Yanrong Lu1, Sirong He1, Xiaojiong Du2, Younan Chen1, Jiuming Zhao1, Bin Ye4, Chengshi Wang1, Bole Tian2, Huimin Lu2, Jingping Liu1, Guang Yang1, Li Wang3, Hongxia Li3, Jie Zhang1, Wei Wang4, Jingqiu Cheng1

1Laboratory of Transplant Engineering and Immunology, West China Hospital, Chengdu, People’s Republic of China; 2Department of General Surgery, West China Hospital, Chengdu, People’s Republic of China; 3National Center for Safety Evaluation of Traditional Chinese Medicine (Chengdu), Chengdu, People’s Republic of China; 4Cell Transplantation and Gene Therapy Institute, The Third Xiang Ya Hospital of Central South University, Changsha, People’s Republic of China.

Objective: Mesenchamal Stem Cell (MSC) has been demonstrated to play an important role in allo-islet graft survival in small animals. In order to explore effective and feasible strategy for pig-monkey islet xenotransplantation, we investigated the effects of monkey MSCs on neonatal porcine islets (NPI) survival and insulin secretion in vitro and in vivo.

Methods: NPI were cultured with or without MSCs, and exposed to hypoxia (O2 ≤ 1%, for 12h) /reoxygenation (for 48h) respectively. Apoptotic cells were quantified using Annexin V-FITC. The effect of MSC on the viability of NPI was detected by CCK8. Islet function was evaluated by GSIS assay. mRNA expression of hypoxia-resistant molecules: HIF-1α, HO-1, COX-2 and iNOS were detected by RT-PCR. 30,000 IEQ/kg NPI were infused into the mesenteric vein of diabetic monkeys with or without MSC (2×106/kg MSC were infused at 0, 1, 3, 5 and 7 weeks post-transplantation). ATG, tacrolimus and sirolimus administration were used to prevent islet rejection. The general conditions of animals were observed: exogenous insulin dose, hematological and serum biochemical parameters, C-peptide and multiple cytokines.

Results: When islets were cocultured with MSC in ratio of 20:1, MSC can decrease H/R-induced apoptotic cells (46.2 % vs. 64.3%, P<0.01), increase the islet metabolism activity at 38 % (Fig 1), maintained the stimulation index of GSIS at higher level (1.35 vs. 1.15), increase HIF-1a, HO-1, COX-2, and iNOS mRNA expression (Fig 2). In islet-MSC co-transplantation group (n=5), 4 recipients attained insulin independence with good glycemic control for 43.75±21.50 days, and then with reduced insulin injection for another 2-3 months. In the NPI transplantation alone group (n=6), one recipient had reduced insulin injection for one month. MSC control group (n=3), insulin dose had not been changed.

Conclusion: Monkey MSC can protect islet vitality and function from H/R-induced injury. NPI and MSC co-transplantation can improve the porcine islets functional differentiation and the success rate of pig-monkey islet transplantation.

This study was supported by the National Program for High Technology Research and Development of China (No. 2012AA020702), Chinese National Science and Technology Project (No. 2011ZX09307-301) and Program of National Natural Science Foundation of China.

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505

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Development of a Microfluidic-Based Multiparametric Real-Time Assay for Human Islet Function and Viability Prior to Transplant

Yong Wang1, Diana Gutierrez1, James McGarrigle1, Barbara Barbaro1, Kirstie Danielson1, Qian Wang1, Maureen Davis1, Marchese Enza1, Pilar Vaca1, Merigeng Qi1, José Oberholzer1

1Surgery/Transplant, University of illinois at Chicago, Chicago, IL, United States.

Introduction: Islet transplantation is an alternative therapy for T1DM. In vivo animal transplantation has certain degree of predictive correlation with human islet transplant outcomes while other standard tests including viability, islet score, and static glucose stimulation test for islet function have low predictive value. Performing the animal transplantation is technical challenge, time consuming, and expensive. It only brings retrospective information on in vivo graft function. Herein, we developed a microfluidic-based perifusion system (UIC-MPS) that can simultaneously measure intracellular calcium, mitochondrial potentials, and kinetics of insulin secretion and aimed to allowing the system to be real-time function assay with a strong predicative value for in vivo graft function.

Methods: 10 human islet preparations were systemically evaluated in response to 25 mM glucose and 30 mM KCL and compared to other in vitro standard tests and in vivo cure rate (CR). The 10 preparations were also physically stressed by pelleting for either 30 or 120 min to create hypoxic condition for further correlating the results derived from UIC-MPS modeling with the results of the in vitro and in vivo tests. Intracellular calcium and mitochondrial potentials were measured by Fura-2/AM and Rhodamine123. Insulin kinetics was measured by ELISA. Simple liner regression and multiple variable regression were used to determine correlation strengths.

Results: (1) Viability and islet score have no strong correlation with the CR (R2= 0.03% and 26.3%) while SI (stimulation index) has a strong correlation (R2= 50.6%, F=0.021). (2) Glucose-stimulated [Ca++] has a strong correlation with the CR (57.2%, F=0.012) while [insulin] has a moderate correlation (R2= 31.2%, F=0.089). However if combined together, it showed a strong correlation (R2=58.5%, F=0.048) with the CR. (3) KCl-stimulated [Ca++] or [Insulin] or combined has moderate correlations with the CR. (4) Combined glucose and KCL-stimulated [Ca++] and [Insulin] have a very strong correlation with the CR (R2= 60.8%). (5) Mitochondrial potentials have no strong correlation with the CR. (6) Further analyses of the hypoxic-treated islets show that the predicated cure rate model derived from this assay can predict in vivo transplant outcomes while the in vitro assays including SI did not predict in vivo outcomes.

Conclusion: UIC-MPS is multiparametric system measuring key insulin stimulator-release factors and provides more detail spatiotemporal information of islet function. It can be used to assess human islet function and viability. Since it does not involve islet fixing and dissociation that allows be performing as real-time assay and completing prior to islet transplant.

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506

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Islet Activity and Outcomes in the United Kingdom

Lisa L Mumford1, James AM Shaw2, Paul Johnson3, Gareth Jones4, Neil Parrott5, Martin K Rutter, Stephanie Amiel6, Richard M Smith7, Neil W A McGowan8, Stephen Hughes3, Guo Cai Huang6, John Casey8

1Statistics and Clinical Audit, NHSBT, Bristol, United Kingdom; 2Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom; 3Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, United Kingdom; 4Renal and Diabetes Units, Royal Free Hospital, London, United Kingdom; 5Department of Transplant Surgery, Central Manchester University Hospitals NHS Foundation Trust, Manchester, United Kingdom; 6Diabetes Research Group, King’s College London, London, United Kingdom; 7Richard Bright Renal Unit, Southmead Hospital, Bristol, United Kingdom; 8Transplant Unit, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom.

Introduction: On 1 April 2008 an integrated national islet transplant programme was commissioned in the United Kingdom (UK) for treatment of recurrent life-threatening hypoglycaemia. In the first year 10 islet infusions were performed in 6 recipients (0.1 pmp). In 2010, islet transplant recipients became part of the same organ allocation scheme as whole pancreas recipients resulting in a substantial increase in activity with 30 islet infusions performed in 27 recipients (0.4 pmp) in 2012/2013, making it one of the largest islets programmes in the world per capita. This review reports initial outcomes of islet transplantation in the UK.

Results: In total, 70 islet infusions were performed between 1 April 2008 and 31 March 2012 in 42 recipients. 18 of the 28 (64%) recipients receiving a second islet graft did so within six months (median 5.7 months, Inter-Quartile Range (IQR) 3–8 months). The median islet yield was 380,000 islet equivalents (IEQ) and 5,256 IEQ/kg (IQR 320,000-480,000 IEQ, 4,667-7,260 IEQ/kg) and this was similar for both grafts. One year follow-up was reported for 29 (69%) recipients. Graft function (c-peptide>=50pmol/l) was maintained in 86% (25) of recipients at 12 months. Recurrent severe hypoglycaemia was prevented in 72% (21) of recipients; pre: 24 episodes/year (IQR 6–50); 12 months: 0 episodes/year (IQR 0–1) p<0.001. Target HbA1c of <7.0% (<53 mmol/mol) was attained in 62% of recipients; pre: 8.2% (IQR 7.2-9.6%); 12 months: 6.5% (IQR 6.0-7.1%) p<0.001.

Conclusions: From the outset, the islet transplant program has realised its goal of allowing islet recipients equity of access to donor organs, preventing recurrent severe hypoglycaemia and improving overall glycemic control in suitable recipients throughout the UK.

On behalf of the NHSBT Pancreas Islet Task Force

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507

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Early Loss of Transplanted Allogeneic Islets Despite T-Cell Depleting Induction: The Role of Autoimmunity in Clinical Islet Transplantation

Morihito Takita1, Rauf Shahbazov1, Faisal Kunnathodi1, Cynthia Mueller2, Parvathi Kurup2, Mazhar Kanak3, Michael Lawrence1, Nicholas Onaca4, Hideki Ueno2, Bashoo Naziruddin4, Marlon Levy4

1Islet Cell Laboratory, Baylor Research Institute, Dallas, TX, United States; 2Baylor Institute for Immunology Research, Dallas, TX, United States; 3Institute of Biomedical Studies, Baylor University, Waco, TX, United States; 4Baylor Annette C. and Harold C. Simmons Transplant Institute, Dallas, TX, United States.

Immunosuppression protocol with T cell depletion (TCD) and TNF-α inhibition has been shown to enhance islet engraftment and prolong long-term graft survival. However, little information on autoimmune recurrence in TCD protocol has been documented for clinical allo islet transplantation. A total of 8 patients with type 1 diabetes (5 females) underwent allo islet transplants with anti-thymoglobulin (ATG) plus anti-inflammatory agents of etanercept and anakinra. Total islet dose was 15,001±2,417 IEQ/kg per recipient weight. Serum islet autoantibody levels of GAD-65, islet antigen (IA)-2, micro-insulin autoantibody (mIAA) and zinc transporter 8 (ZnT8) were measured on a monthly basis post-transplant. Peripheral blood mononuclear cells were tested using EpiMax assay to determine the T cell response to GAD65 overlapping peptides [1]. Five patients have achieved insulin independence after TCD protocol. Positive results on either one or more auto antibodies were seen in all patients after transplant while GAD65 and ZnT8 antibodies were newly detected in three (Patient 1,2 and 8) and one (Pt 5) recipients respectively (Fig. 1). Positive GAD65 antibody was observed before all events with graft dysfunction defined by fasting C-peptide ≤ 0.3 ng/mL (Month 18 in Pt 1 and M1 in Pt4,6 and 8). Strong CD4+ T cell response characterized by IFN-γ and IL-21 secretion to GAD65 peptide cluster 1 was observed in Patient 6 with acute graft dysfunction (Fig. 2). TCD with anti-inflammatory regimen can enhance islet engraftment and survival; however, autoimmune recurrence evident by positive islet auto antibodies, especially for GAD65, and specific T cell response is still a major concern to improve outcome of clinical allo islet transplantation.

Reference:

  • [1] Chujo D, Foucat E, Takita M, Itoh T, Sugimoto K, Shimoda M, Yagi K, Yamagishi M, Tamura Y, Yu L, Naziruddin B, Levy MF, Ueno H, Matsumoto S. Emergence of a broad repertoire of GAD65-specific T cells in type 1 diabetes patients with graft dysfunction after allogeneic islet transplantation. Cell Transplant. 2012, 21(12):2783–2795.
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Islet Oxygen Consumption Rate Predicts Clinical Islet Allo-Transplant Insulin Independence for First Transplants

Jennifer P Kitzmann1, Doug O’Gorman2, Tatsuya Kin2, Angelika C Gruessner1, Peter Senior2, Sharleen Imes2, Rainer W Gruessner1, AM James Shapiro2, Klearchos Papas1

1Surgery, University of Arizona, Tucson, AZ, United States; 2Clinical Islet Transplant Program, University of Alberta, Edmonton, AB, Canada.

Human islet allo-transplantation (ITx) is in phase III clinical registration trials in the US and standard of care in several other countries. Current clinical islet product release criteria include viability based on cell membrane integrity (>70%), glucose stimulated insulin release (GSIR; >1 stimulation index, SI), and islet equivalent (IE) dose based on counts (IE per kilogram body weight; >5,000). However, only a fraction of patients transplanted with islets that meet or exceed these release criteria become insulin independent. There is a need for more reliable assays that are predictive of clinical transplant outcome (CTO). Measurements of islet oxygen consumption rate (OCR) have been reported as highly predictive of transplant outcome in small and large animal models as well as clinical islet auto-transplantation. In this paper we report on the assessment of clinical islet allograft preparations using OCRtx/kg BW (a measure of transplanted viable IE – the product of total IE transplanted and islet viability measured by OCR/DNA) normalized to recipient body weight, and current product release assays in a series of 13 first transplant recipients. The predictive capability of each assay was examined using receiver operating characteristic (ROC) curve analysis. Successful graft function was defined as 100% insulin independence within 45 days post-transplant. All transplanted preparations met, or exceeded, current product release criteria. However, only 38% of transplanted recipients were insulin independent within 45 days. The results showed that OCRtx/kg BW was the measure most predictive of CTO [area under the curve (AUC): 1.000; figure 1a]. IE dose was also highly predictive (AUC: 0.975; figure 1b) while GSIR and membrane integrity stains were not (AUC: 0.825, 0.625 respectively; figure 1c,d). Interestingly, preparations with higher GSIR were less likely to reverse diabetes. In conclusion, OCRtx/kg BW can predict CTO with high specificity and sensitivity and is a useful tool for evaluating islet preparations prior to clinical ITx.

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This research was performed in part as a project of the Clinical Islet Transplantation Consortium, a collaborative clinical research project headquartered at the (NIH Institute).

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509

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Liver Micro-Vascular Response to Intra-Hepatic Islets Transplantation Influence the Clinical Success of the Graft: A Magnetic Resonance Imaging Study

Anna Palmisano2, Antonio Esposito2, Paola Maffi1, Maria Luisa Malosio3, Tamara Canu2, Rita Nano4, Lorenzo Piemonti4, Francesco De Cobelli2, Alessandro Del Maschio2, Antonio Secchi1

1Internal Medicine - Transplant Unit - Diabetes Research Institute, Scientific Institute San Raffaele, Milan, Italy; 2Radiology, Scientific Institute San Raffaele, Milan, Italy; 3Institute of Biomedical Technologies, Italian National Research Council, Milan, Italy; 4Biology of beta cell - Diabetes Research Institute, Scientific Institute, Milan, Italy.

Preclinical studies demonstrated that microvascular phenomena play a crucial role in the engraftment of transplanted islet immediately after intra–portal infusion. Furthermore it is well known that the success of engraftment strongly correlates with clinical outcome of the graft.

Aim: The aim of this study was to investigate in type 1 diabetic patients, undergoing intra-hepatic islet transplantation (islet-tx): the liver micro-vascular adaptation to intra-portal vein infusion of the tissue by using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI); the potential relationship among early vascular changes, post-transplantation islets loss and functional outcome of the graft

Methods: Six diabetic patients underwent DCE-MRI before and 7 days after islet-tx. Area under curve (AUC) and Volume Transfer Coefficient (Ktrans) were assessed as markers of liver perfusion. Among the six transplanted patients, three patients received iron-labeled islets and were monitored with an additional MRI follow-up based on “iron-sensitive” T2* sequences. The other three patients received unlabeled islets. β-score, transplant estimated function (TEF) and islet estimated function (IEF) were calculated during follow-up as indixes of graft function.

Results: Changes of AUC and Ktrans from pre-transplant to post-transplant (7 days after islets-tx) study resulted to be tightly correlated to mean values of β-score and IEF measured between 3 and 6 months after transplantation: ΔAUC0-7days - β-score: R=0,841, p<0,05; ΔAUC0-7days - IEF: R=0,886, p<0,01; ΔKtrans0-7days - β-score: R=0,899, p<0,01; ΔKtrans0-7days - IEF: R=0,943, p<0,01. Not significant correlation was found between DCE-MRI parameters and TEF. In the subset of the three patients transplanted with iron-labeled islets, an association between early changes in liver perfusion and the rate of islets loss in the first month was observed.

Conclusion: In patients who underwent intra-hepatic islets transplantation, DCE-MRI assessment of vascular events occurring in the first week of islets engraftment was associated to the subsequent clinical outcome of the graft.

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Use of NF-κB Blockade to Alleviate Instant Blood Mediated Inflammatory Reaction

Mazhar Kanak1, Shuichi Iwahashi2, Takeshi Itoh2, Morihito Takita2, Marlon Levy3, Bashoo Naziruddin3

1Institute of Biomedical Studies, Baylor University, Waco, TX, United States; 2Islet Cell Laboratory, Baylor Research Institute, Dallas, TX, United States; 3Baylor Annette C. and Harold C. Simmons Transplant Institute, Dallas, TX, United States.

Instant blood-mediated inflammatory reaction (IBMIR) is known to cause the loss of a significant amount of transplanted islets. IBMIR is characterized by activation of complement, coagulation and secretion of pro-inflammatory cytokines/chemokines followed by infiltration of leukocytes into the islets. Using a “miniaturized in vitro tube” model, we provide evidence that inhibition of the transcription factor NF-κB, results in significant reduction in pro-inflammatory cytokine production and neutrophil infiltration into islets. Purified human islets were mixed with heparinized autologous blood in an in vitro “tube model”. Withaferin A (WA) or CAY10512 (known NF-κB inhibitors) were added to islets in culture and also to blood-islet mix. Plasma samples and islet clots were retrieved at regular intervals to analyze cytokines and cellular infiltration respectively. Panel of inflammatory cytokines were measured using Luminex multiplex assay. Exposure of “naked” islets to autologous blood increased production of pro-inflammatory cytokines and caused severe islet damage shown by decrease in viability and elevated levels of C-peptide and pro-insulin in plasma. Treatment with WA or CAY10512 showed remarkable increase in islet viability post-IBMIR when compared to control. Presence of WA or CAY10512 significantly inhibited release of G-CSF (260.4 ± 65.6 vs. 33.3 ± 9.4 pg/mL vs. 58.6 ± 24.9; p<0.01), TNFα (27.9 ± 3.7 vs. 12.4 ± 1.1 pg/mL vs. 9.4 ± 1.9; p < 0.01), IP-10 (423.5 ± 38.9 vs. 236.1 ± 20.0 vs. 158.3 ± 6.2 pg/mL; p < 0.05) MCP-1 (10302.5 ± 130.8 vs. 2377.7 ± 147.5 vs. 8381.7 ± 665 pg/mL; p < 0.05). Immunohistochemistry of islets showed significant reduction in infiltration of neutrophils by WA treatment (8.13 ± 2.7 vs. 1.69 ± 0.8 neutrophils/islet; p < 0.001). WA was more potent than CAY10512 in protection of islets viability (40.9 ± 25.1 vs. 70.6 ± 9.7 vs. 41.9±10.1; p < 0.01). In summary, blockade of NF-κB activation could be a key strategy to minimize damage to islet transplants due to IBMIR and improve engraftment. Incorporation of inhibitor(s) of NF-kB as an adjunct to treatment regimen may improve outcomes in clinical islet transplantation.

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Contribution of Nutrient Deprivation to Islet Death After Transplantation

Gaetano Faleo1, Vinh Nguyen1, Qizhi Tang1

1Surgery, UCSF, San Francisco, CA, United States.

Islet transplantation is a proven effective minimally invasive therapy for type 1 diabetes, but often requires more than one donor to achieve insulin independence. While transplanting higher islet mass is beneficial to the recipients, it exacerbates donor shortage and limits broader application of the therapy. We hypothesize that the main reason for the demand of high islet mass is islet death shortly after transplant, leading to gradual attrition of the remaining islets due to overwork and exhaustion. In this study, we investigated factors that contributed to islet death in mouse models. We transplanted under kidney capsules islets expressing a firely luciferase in β-cells under the control of mouse insulin 1 promoter (MIP-LUC) so that functional islet mass could be monitored using bioluminescence imaging in the same animals over time. We found that the density of transplanted islets in the graft bed was directly proportional to the extent of islet death in syngeneic recipients. High-density transplant resulted in 80-90% islet death within the first 5 days, whereas spreading islets in wider areas led to significant improvement of islet survival with greater than 50% preservation of the islet mass. Islet death in vitro under normal atmospheric oxygen tension was also proportional to the culture density, suggesting factors other than hypoxia contributed to islet death. In high-density cultures, we detected increased LC3+ autophagosomes in β-cells, indicative of nutrient deprivation. We further investigated downstream cellular events that contributed to islet demise. We found increased XDP1 splicing and CHOP mRNA upregulation, consistent with triggering of irremediable endoplasmic reticulum stress. These results demonstrate a previous unrecognized factor of nutrient deprivation exacerbated by high-density transplant in peri-transplant islet death. Interventions that protect islets from nutrient deprivation may reduce the demand for high islet mass and improve therapeutic efficacy of islet transplantation.

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Utilization of Luciferase Transgenic “Firefly” Rats as a Marker for Subcutaneous Islet Graft Viability

Jeffrey Rawson1, Indu Nair1, Yoko Mullen1

1Diabetes, Endocrinology, and Metabolism, Beckman Research Institute of the City of Hope, Duarte, CA, United States.

Background: There is increasing evidence that the liver may not be the optimal site for islet transplantation and survival. Subcutaneous islet transplant has become an interesting option, due to the large space available for transplantation, as well as the ability to monitor the graft site, make multiple transplantations to the site, and remove the graft, should it become medically necessary. Previous studies have shown that islets transplanted subcutaneously fail to reverse diabetes, likely due to hypoxia caused by lack of vascularity. In this study, we have developed a method to monitor islet viability in vivo, even in cases where the islet graft fails to reverse diabetes.

Methods: Islets isolated from luciferase transgenic rats were transplanted subcutaneously on the abdomen of diabetic and non-diabetic, syngeneic animals. Islet viability was measured using the IVIS imaging system following I.V. or I.P. injection of 0.03mg/g luciferin. Images were taken at 2, 5, and 10 minutes after injection. At the conclusion of the experiment, abdominal sections were fixed, embedded in paraffin, and histology was prepared.

Results: Luciferase transgenic islets were able to be detected as long as 100 days after transplantation using the imaging system. Post-transplantation histology shows that the surviving islets maintain good morphology. However, insulin/glucagon staining shows that subcutaneously transplanted islets have a decrease in insulin positive cells, with an increase in glucagon positive cells.

Discussion In this study, we have shown that we are able to detect luciferase transgenic islets over 100 days after subcutaneous transplantation. The presence of luciferase positive cells, in conjunction with a decrease in insulin positive cells, indicates that β-cells are more susceptible to the hypoxic subcutaneous conditions. This suggests that additional steps are necessary to promote subcutaneous vascularization and transplanted islet survival.

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516

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Influence of Antigenicity upon the Instant Blood Mediated Inflammatory Reaction in a Dual Transplant Model of Islet Transplantation

Benjamin Martin1, Michael Lowe1, Peter Thompson1, Douglas Anderson1, Frank Leopardi1, Jose Cano1, Mingqing Song1, Elizabeth Strobert2, Alton Farris1, Christian Larsen1, Allan Kirk1, Kannan Samy1

1Transplantation, Emory University, Atlanta, GA, United States; 2Yerkes National Primate Research Center, Atlanta, GA, United States.

A significant proportion of intraportally infused islets are destroyed by the instant blood mediated inflammatory reaction (IBMIR) composed primarily of preformed antibody, complement, platelets, neutrophils, and macrophages. Further characterization of IBMIR is critical for the development of pharmacologic interventions and transgenic modifications designed to enhance islet engraftment. Therefore, utilizing the dual transplant model we sought to rigorously define the inflammatory response to alloislets and neonatal porcine xenoislets transplanted within the isolated hemi-livers of a single non-human primate recipient.

Anesthetized Rhesus macaques underwent liver hilum dissection including right and left portal venous branch isolation. Approximately 10,000 IEQ/kg of MHC-mismatched allogeneic islets or wild-type neonatal porcine xenoislets were infused into each hemi-liver through the corresponding portal venous inflow branch after vascular exclusion of the opposing side. Animals were sacrificed at 1-hour (n=4) for detailed immunohistochemical analysis. An additional three animals received an intraportal infusion of inert polyethylene microspheres to serve as embolic controls. Slides derived from predetermined anatomical locations within each hemi-liver were quantitatively analyzed with Aperio Imagescope software.

Microsphere transplantation induced low levels of IgM/IgG binding, C4d deposition, neutrophil/macrophage infiltration, and platelet aggregation. The intraportal infusion of allo- or xenogeneic islets significantly increased platelet aggregation (p=0.01) and complement deposition (p=0.05) around and within the islet clusters. There was also a trend towards increased neutrophilic infiltration (p=0.11). No statistically significant differences were noted between allo- or xenoislets for the IBMIR markers targeted, however, allogeneic islets demonstrated a trend towards increased insulin (p=0.08) and decreased caspase 3 (p=0.07) expression at the 1-hour time point studied.

After intraportal infusion inert emboli lodging within the hepatic parenchyma produce low levels of inflammation. Embolization of antigenic tissue, such as allo- or xenoislets, significantly increases platelet aggregation and complement deposition 1-hour post-transplant. We are currently extending the model to examine the inflammatory evolution at 24-hours.

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517

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Non-Invasive Tracking of Encapsulated Insulin Producing Cells Labelled with Iron Nanoparticles by Magnetic Resonance Imaging (MRI)

Vijayaganapathy Vaithilingam1,2, Mandy Yim2, Jayne Foster2, Timothy Stait-Gardner3, Bernie Tuch1,2

1Materials Science & Engineering, Commonwealth Scientific and Industrial Research Organization (CSIRO), North Ryde, Australia; 2Diabetes Transplant Unit, University of New South Wales, Sydney, Australia; 3Nanoscale Organisation and Dynamics Group, School of Science and Health, University of Western Sydney, Penrith, Australia;

Microencapsulating pancreatic islets is a strategy being explored as a treatment for type 1 diabetes which may overcome the immune-mediated rejection of the graft without toxic immunosuppression. Allo- and xeno- transplantation studies with microencapsulated islets have shown considerable promise but long term graft survival was limited and varied considerably. Microencapsulated cells are often injected free-floating into the peritoneal cavity, so the position of the grafts remains elusive after transplantation. The aim of this study was to assess magnetic resonance imaging (MRI) as a non-invasive means to track microencapsulated insulin-producing cells following transplantation.

Murine insulin-producing cells (MIN6) and human islets were labelled with fluorescent superparamagnetic iron oxide (SPIO) nanoparticles and encapsulated within barium alginate microcapsules. Viability and insulin secretion of encapsulated SPIO-labelled MIN6 and human islets were assessed. In vitro imaging of encapsulated SPIO-labelled cells was carried out using a clinical grade 3 T MRI. Encapsulated SPIO-labelled MIN6 were transplanted into the peritoneal cavity of immunocompetent (C57BL/6) mice and tracked non-invasively using both 3 T and 11.7 T MRI.

Fluorescent imaging demonstrated the uptake of SPIO nanoparticles by both MIN6 and human islets with no evident changes in cell morphology. SPIO-labelling affected neither the viability of encapsulated MIN6 and islets over 7 days in culture, nor their capacity to secrete insulin in response to glucose. Normalization of blood glucose levels was achieved when encapsulated SPIO-labelled MIN6 were transplanted into the peritoneal cavity of diabetic C57BL/6 mice. In vitro imaging demonstrated that clusters as well as single capsules of encapsulated SPIO-labelled MIN6 and islets could be visualised using the 3 T MRI (Figure 1). In vivo encapsulated SPIO-labelled MIN6 cells could be visualised within the peritoneal cavity as discrete hypointensities using the high power 11.7 T but not the clinical grade 3 T MRI (Figure 2).

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Imaging of Transplanted Islets by Ultrasonography

Naoaki Sakata1, Gumpei Yoshimatsu1, Haruyuki Tsuchiya1, Takeshi Aoki1, Masamichi Mizuma1, Fuyuhiko Motoi1, Yu Katayose1,2, Tetsuya Kodama3, Michiaki Unno1

1Division of Hepato-Biliary Pancreatic Surgery Department of Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan; 2Division of Integrated Surgery and Oncology, Tohoku University Graduate School of Medicine, Sendai, Japan; 3Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan.

Recently, experimental trials for visualizing transplanted islets by imaging modalities have been promoted by many groups. Some groups including ours have tried to visualize transplanted islets by ultrasonography (US).

We attempted to visualize transplanted islets with high frequency ultrasonography (HF-US), evaluating the correlations between HF-US findings and islet function. We transplanted syngeneic (BALB/c mice) and xenogeneic (Sprague–Dawley rats) islets into the subrenal capsular space of diabetic mice. After the transplantation, the mice were examined by HF-US (central frequency 35 MHz, axial resolution 50 μm, focal length 10 mm) at 3, 7, 14, 28 and 56 days after transplantation. In the syngeneic transplant model, a hyperechoic area was detected at the subrenal capsular space during the observation. On the other hand, transplanted islets were visualized as hypoechoic areas that reflected the damage to the islets due to rejection at 3 days after transplantation and completely disappeared at 28 days in the xenogeneic transplant model. The islet volume calculated by the HF-US device was correlated with numbers of transplanted islets, blood glucose and serum insulin. These experimental data indicated that US could be used for the visualization of transplanted islets, and for evaluating the endocrinal function and condition including rejection of the islets.

We also clarified that individual islets in the portal vein could be visualized by intraoperative US (central frequency was 7.5 MHz) in the clinic. We performed total pancreatectomy with islet autotransplantation via portal vein in a 39 year-old man who had chronic pancreatitis with pancreatic arteriovenous malformation. We examined the portal vein by US during the transplantation to check for portal thrombosis, and detected individual transplanted islets as hyperechoic clusters that flowed toward the periphery of the portal vein. This finding and our previously described experimental data clarified some speculations about US imaging for evaluating the islet condition. First, viable islets can be visualized as hyperechoic images not only in rodent but also in human.

It is conceivable that islet imaging in intraoperative US (especially echogenicity) can provide reliable information to predict the outcome of the islet transplantation. Islets can be visualized not only with high frequency US but also with the usual US used for the human abdomen in spite of their tiny structures (within 1 mm in diameter). Our data also suggest US could be an essential examination in islet transplantation.

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519

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Oxygen Sensitive Nanofibers for Spatiotemporal Oxygen Detection

Daniel T. Bowers1, Michael L. Tanes1, Nicole A. Keane1, Yong Lin1, Anusuya Das1, Cassandra L. Fraser2, Edward A. Botchwey3, Kenneth L. Brayman4

1Biomedical Engineering, University of Virginia, Charlottesville, VA, United States; 2Chemistry, University of Virginia, Charlottesville, VA, United States; 3Biomedical Engineering, Georgia Tech, Atlanta, GA, United States; 4Surgery, University of Virginia, Charlottesville, VA, United States.

Development of an ideal islet encapsulation material would involve the ability to monitor oxygen tension. Little is currently known about the oxygen concentrations in transplanted islet grafts, however an encapsulation device provides a structure to attach oxygen sensors where extracellular hypoxia is detrimentally affecting islet health while not requiring modification of the islets.

The objective of this study was to demonstrate construction and in vitro concept of function of an oxygen sensing nanofiber construct. Nanofibers were electrospun from an iodine substituted boron based organometallic dye that is conjugated to a 15kDa Poly-lactic Acid polymer (BF2dbm(I)PLA). The dye is responsive to oxygen tensions in the range of 0.5 to 10 parts per million (ppm) dissolved oxygen, a range which includes oxygen concentrations found in transplanted and native islet tissue[1]. The fibers were confirmed to be biocompatible by cell viability staining (Figure 1 A,B fluorescein diacetate and propidium iodide respectively). Preliminary experiments show that adherent cells seeded in monolayer onto the nanofibers create a measureable oxygen gradient (2.2ppm under the cell to 7.2ppm at 6 microns outside the cell) when a closed system is used. A trend of decreasing oxygen can also be observed with increasing time (8.2 ppm at 0 mins vs. 4.7 ppm at 20 mins). Furthermore, a macrogradient is visible (Figure 1c), where the region to the right of the dotted line was seeded with a monolayer of cells that are not shown.

In conclusion, these fibers are sensitive to oxygen concentrations which occur in monolayer cell culture closed systems. This sensitivity can be measured at the individual cell level which is expected to extend to islets considering the increased oxygen demand that a mass of cells exerts. Thus, the oxygen sensitive nanofibers investigated here are expected to be validated in vitro with islets and in mouse models.

Reference:

  • [1] Carlsson PO, Liss P, Andersson A, Jansson L: Measurements of oxygen tension in native and transplanted rat pancreatic islets. Diabetes 1998, 47(7):1027–32.
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Role of Oxidative Stress in The Pro-Angiogenic Effect of Liraglutide on Rat Pancreatic Islets

Allan Langlois1,2, Stephanie Dal-Ros1,2, William Bietiger1,2, Carole Mura1,2, Elodie Seyfritz1,2, Camille Dollinger1,2, Michel Pinget1,2,3, Nathalie Jeandidier2,3, Severine Sigrist1,2

1Centre européen d’étude du Diabète, Strasbourg, France; 2University of Strasbourg, Strasbourg, France; 3Service d’endocrinologie, diabète, maladies métaboloiques, Pôle NUDE, Hôpitaux universitaires de Strasbourg, Strasbourg, France.

Introduction The formation of microvascularization by capillary sprouting at site of pancreatic islet implantation is crucial for survival of the graft. Vascular Endothelial Growth Factor (VEGF), a major angiogenic factor, may be a key protein in modulating the angiogenesis of islets after transplantation. Increased of VEGF by Deferoxamine via HIF-1α pathway has been shown to be beneficial for islet survival. Previous in vitro study realized in the laboratory showed that liraglutide stimulated VEGF secretion significantly in islets and improved their function. Thus, this angiogenic property could explain the benefic effect of liragl