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Keywords:

  • Islet transplantation;
  • inflammation;
  • coagulation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Instant blood mediated inflammatory reaction (IBMIR) occurs when islets are exposed to blood and manifests clinically as portal vein thrombosis and graft failure. The aim of this study was to determine the impact of recombinant human activated protein C (rhAPC) and platelet inhibition on IBMIR in order to develop a better targeted treatment for this condition. Five thousand human islet cell equivalents (IEQ) were mixed in a PVC loop system with 7 mL of ABO compatible human blood and incubated with rhAPC, either alone or in combination with tirofiban. Admixing human islets and blood caused rapid clot formation, consumption of platelets, leukocytes, fibrinogen, coagulation factors and raised d-dimers. Islets were encased in a fibrin and platelet clot heavily infiltrated with neutrophils. Tirofiban monotherapy was ineffective, whereas rhAPC monotherapy prevented IBMIR in a dose-dependent manner, preserving islet integrity while maintaining platelet and leukocyte counts, fibrinogen and coagulation factor levels, and reducing d-dimer formation. The combination of tirofiban and low-dose rhAPC inhibited IBMIR synergistically with an efficacy equal to high dose rhAPC. Tirofiban and rhAPC worked synergistically to preserve islets, suggesting that co-inhibition of the platelet and coagulation pathways’ contribution to thrombin generation is required for the optimal anti-IBMIR effect.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Allogeneic islet cell transplantation for type I diabetes mellitus has become a clinical reality with the Edmonton protocol (1,2). However, the success of the procedure is limited by the profound islet attrition that occurs in the immediate posttransplant period, leaving a final islet cell survival of around 20–40% of a healthy nondiabetic subject (3). This necessitates multiple transplants to reach insulin independence, thus reducing the efficiency of utilization of already scarce donor organs and often resulting in the transplantation of a marginal β-cell mass (4,5).

A major cause of early islet attrition is Instant blood mediated inflammatory reaction (IBMIR) which occurs rapidly when isolated islets are exposed to human blood following infusion in the portal vein. It is characterized by activation of complement, platelets and the coagulation pathway, as well as inactivation of the anti-inflammatory islet response. This inflammatory thrombosis is driven by tissue factor and manifests clinically with the formation of macro- and microthrombi despite the routine use of intravenous unfractionated heparin (1,5,6).

Given that IBMIR involves activation of the coagulation, platelet and inflammatory pathways, we proposed that a dual strategy blocking both coagulation and platelet activation might be more efficacious at preventing IBMIR. To evaluate this, we used an ex vivo model of IBMIR and tested the efficacy of rhAPC and tirofiban, alone and in combination to prevent IBMIR. Drotrecogin alfa activated or rhAPC is a naturally occurring anti-inflammatory, antithrombotic and antiapoptotic protein. It is known to down-regulate the release of islet toxic cytokines such as TNFα and interleukin 6 through inhibition of the protein C pathway (7). Treatment with rhAPC has shown improved survival in trials of severe sepsis (8). A recent trial of murine APC administered at the time of intraportal transplantation demonstrated improved graft function and islet cell survival in treated mice (9). Tirofiban is a platelet glycoprotein IIb-IIIa inhibitor (αIIB3), whose safety and efficacy has been established in clinical trials of acute coronary syndrome (10,11). It inhibits the final common pathway of platelet activation and further prevents the platelet contribution to thrombin generation through the tissue factor pathway (12).

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Human islet cell isolation

Human islet cells were procured from deceased donor pancreases as part of the clinical islet transplantation program at Westmead Hospital using previously described methods involving Liberase (Roche) over a Phicoll gradient with a Cobe refrigerated centrifuge (6). Islets were used within 1–3 days following isolation. The project was approved by the Human Research Ethics Committee of Sydney West Area Health Services.

PVC loop perfusion system

A loop perfusion system was adopted from Bennet et al (13) consisting of Carmeda-coated heparinized polyvinyl chloride cardiac bypass tubing (Medtronic, Minneapolis, MN) cut to 30 cm lengths and connected end-to-end with plastic interconnects (Unomedical, Sydney, Australia). The loops were filled with 7 mL of ABO compatible blood together with 5000 human islet cell equivalents (IEQs) in a volume of 200–250 μL of RPMI solution. The positive control loops contained blood and islets without drugs. The negative control contained blood and 200–250 μL of RPMI but no islets. The loops were incubated on a shaker at 37°C at the rate of 100 rpm to avoid stasis. After 60 min, the contents passed through a 70 μL cell strainer (BD Falcon) for clot retrieval and the sieved blood solution was collected for analysis. The blood specimens were examined for full blood counts, fibrinogen, heparin levels, coagulation studies (PT, APTT), factors V, VII, VIII, IX, X, XI, XII, proteins C and S, and von Willebrand factor. Approximations of relevant clinical dose regimens were based upon the previously published conclusion that in the loops model doses around five times higher than those published in clinical trials were required (14).

rhAPC (‘Xigris’ or drotrecogin alfa) was kindly donated by Elli-Lilly. Approximations of relevant clinical doses for the loop perfusion system were estimated from standard clinical dosing schedules from the PROWESS trial where an infusion of 24 μg/kg body weight/h was used (8). This was converted to a loop dose of 2.5–5.0 μg/7 mL blood/h incubation (assuming 4900 mL blood volume for a 70 kg adult).

Tirofiban (‘Aggrostat’) was obtained from Merck, Sharp and Dohme. An approximation of a clinically relevant dose of the drug for the loop system was calculated from the PRISM trial where a dose of 0.75 μg/kg body weight was used for the first hour (10). This was translated to a loop dose of 2.5 μg tirofiban where there was 7 mL of blood per loop over a 60-min incubation.

Immunohistochemistry

Three- to five-micrometer tissue sections of filtered clots from formalin-fixed paraffin blocks were stained for neutrophil elastase (Dako-Cytomation) and tissue factor. A polyclonal antitissue factor antibody was kindly donated by Dr Dana Abendschein (Washington), and the staining methodology was adapted from St. Pierre et al (15). The secondary horseradish peroxidase conjugated polymerase solution (Dako) was applied for 30 min. DAB chromagen (Dako) was applied for 5 min before counterstaining for 1 min 30 s using hematoxylin.

C4d immunohistochemistry was performed using a polyclonal C4d antibody from BioGenesis. Staining was performed using the Biogenex SuperSensitive staining kit after antigen retrieval.

Hematological studies

Full blood counts were performed by the clinical hematology unit at Westmead Hospital on loop specimens collected into EDTA tubes. Specimens collected into sodium citrate tubes were used to estimate factors V, VII, VIII, IX, X, XI and XII, vWF:Ag and vWF:CB using previously published methods (16,17). At the conclusion of each experiment, selected samples were assayed for heparin to exclude the possibility that heparin had leaked from the Carmeda tubing during the assays using an anti-Xa chromogenic commercial kit from Instrumentation Laboratory (Beckman-Coulter, Sydney, Australia). Fibrinogen was assayed using ‘Fibrinogen-C,’ a von-Clauss based commercial kit from Instrumentation Laboratory (Beckman-Coulter, Sydney, Australia). d-Dimer was assayed by an in-house ELISA, using antibodies, controls and standards obtained from Agen Biomedical Ltd., Qld, Australia.

Statistical methods

The statistical software package S-PLUS Version 6.2 (Insightful Corporation, Seattle, WA) was used to analyze the data. Two-tailed tests with a significance level of 5% were used throughout. Linear mixed effect models were used to investigate the impact of increasing rhAPC or tirofiban doses on the levels of the various outcome variables of interest. Continuous autoregressive models were used to account for the within experiment correlation between observations at consecutive doses. Tirofiban dose, rhAPC dose and experiment identifier were considered as random effects, and tirofiban dose, rhAPC dose and their interaction were treated as fixed effects. The presence of a statistically significant interaction between the effects of tirofiban dose and rhAPC dose on an outcome variable was evidence that the dose-response observed for this variable with rhAPC alone differed significantly from those observed in the presence of increasing doses of tirofiban. In order to visualize significant interaction effects on a variable at clinically relevant doses, the line of best fit for the variable doses of rhAPC was compared with that obtained when 2.5 μg tirofiban was added to varying doses of rhAPC (Figure 4).

image

Figure 4. Synergistic effect of combined tirofiban and APC therapy. Tirofiban was used in combination with varying doses of APC. The graphs compare the results obtained where varying doses of APC were used with or without 2.5 μg of tirofiban. For statistical analysis of data, see Table 2. The shaded areas are 95% confidence intervals. Significantly better results were obtained for clot weight (p < 0.01), platelet count (p < 0.01), fibrinogen (p = 0.02) and factor VIII (p = 0.03), see Table 2. Data are based on nine individual experiments using islets from five donors and blood from four volunteers.

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Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Human islets exposed to ABO compatible blood triggered IBMIR

In order to confirm that exposure of human islets to ABO compatible blood resulted in IBMIR in the loop perfusion system, isolated human islets were incubated in closed heparin-coated PVC loops for 60 min as described. After 60 min, there was obvious clot formation and profound consumption of platelets, leukocytes, factors V, VIII, fibrinogen and elevation of d-dimer levels (Figure 1, Table 1). Noticeably unchanged were the levels of the contact pathway factors such as factors X and XI as well as Von Willebrand Factor (Table 1) suggesting that IBMIR was initiated through tissue factor expressed on islets rather than through contact with the PVC tubing. Immunohistochemistry confirmed that islets stained positive for tissue factor both in whole pancreas and following isolation (Figures 1A and B).

image

Figure 1. Histology and immunohistochemistry of human pancreatic islets removed from the PVC loop perfusion system after 1 h. (A) Donor pancreas with pancreatic islets stained positive for tissue factor; (B) isolated human islet prior to incubation in the PVC loop system staining positive for tissue factor; (C) H and E section of human islets encased in clot; (D) human islet encased in neutophils (neutral protease stain); (E) neutrophil infiltration of human islet following treatment with 5 μg APC; (F) neutrophil infiltration of human islet following 10 μg APC; (G) human islet staining positive for C4d following incubation in the PVC loop system; (H) C4d staining of human islets after treatment with 5 μg APC; (I) C4d staining of human islet after treatment with 10 μg APC and (J) C4d staining after treatment with 5 μg APC plus 2.5 μg triofiban.

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Table 1.  Hematological and clotting parameters after exposure of human islets to ABO compatible blood in the PVC loop perfusion system
ParameterBaseline data1After 1 h exposure to human blood2p-Value
  1. 1Data expressed as mean ± SD.

  2. 2Data obtained after exposure of islets to ABO compatible blood for 1 h in the PVC perfusion loop system.

Clot weight (g)00.68 ± 0.44<0.001
Platelets (× 109/L) 252 ± 42.55 31.8 ± 54  <0.001
Leukocytes (× 109/L)3.40 ± 1.15 2.4 ± 0.47<0.001
Neutrophils (× 109/L)4.74 ± 1.931.36 ± 1.24<0.001
Monocytes (× 109/L)0.42 ± 0.070.07 ± 0.13<0.001
d-Dimers (ng/mL)9.83 ± 8.3 193.43 ± 435.35NS
Fibrinogen (mg/dL)396.14 ± 101.1193.625 ± 108.17<0.001
VWB:Ag (%) 83.5 ± 25.2170.89 ± 32.75NS
Factor V (%)96.25 ± 28.3734.75 ± 25.320.018
Factor VII (%)94.75 ± 20.9383.75 ± 15.75NS
Factor VIII (%)74.29 ± 20.05 42.5 ± 35.48 0.057
Factor IX (%)  86 ± 15.7257.25 ± 30.91NS
Factor XI (%)  73 ± 19.2954.25 ± 30.91NS
Factor XII (%)84.67 ± 18.77 74.5 ± 28.29NS

Clots were filtered from the loops after 60 min and islets were shown to be surrounded by fibrin and platelets (Figure 1C). Immunohistochemisty showed that islets were infiltrated by neutrophils (Figure 1D), which was consistent with previous reports of a similar assay (18), and islets contained within the clots demonstrated widespread staining for C4d implicating complement-mediated cell lysis as a feature of IBMIR (Figure 1G).

Tirofiban monotherapy failed to inhibit IBMIR

In order to evaluate the effect of platelet inhibition alone on IBMIR, tirofiban was added to the ex vivo loop model. Tirofiban alone failed to inhibit IBMIR (Table 2 and Figures 2A and B). In fact, there was an increase in clot size in a dose-dependent manner (Table 2 and Figure 1A) and a null effect on other IBMIR parameters (Table 2). Even at doses that were 2 logs in excess of those used clinically, tirofiban could not inhibit IBMIR (Figure 2).

Table 2.  The effect of rhAPC and/or tirofiban on hematological and clotting factors after exposure of human islets to human blood in the PVC loop perfusion system.
ParameterTreatmentChange from intercept valueDFt-Valuep-Value
  1. 1Intercept = point where the dose response curve intercepts the y-axis. For example, with the effect of APC alone or in combination with tirofiban on clot weight the curve intercepts the y-axis at 0.461 (see Figure 3). This means that the clot weight when the APC dose is zero is 0.461 g.

  2. 2Effect of escalating doses of APC on the clot weight was calculated by the following: Clot weight = intercept + (−0.021 × APC dose).

  3. 3Effect of escalating doses of tirofiban on the clot weight was calculated by the following: Clot weight = intercept + (0.016 × tirofiban dose).

  4. 4Effect of APC:tirofiban combination treatment on the clot weight was calculated by the following:

  5.  Clot weight = intercept + tirofiban dose [0.016 + (−0.02 × APC dose)]. The result was compared to using rhAPC alone. A p-value <0.05 confirms an additional effect of tirofiban + rhAPC compared to rhAPC alone.

Clot weightIntercept value10.461 ± 0.096944.82<0.0001
APC2− 0.021 ± 0.004  94−4.31<0.0001
Tirofiban30.016 ± 0.006942.570.012
APC:Tirofiban4− 0.02 ± 0.007 94−2.770.0068
PlateletsIntercept value48.928 ± 16.391932.990.004
APC4.271 ± 0.904934.72<0.0001
Tirofiban1.006 ± 1.022930.980.33
APC:Tirofiban5.452 ± 1.150934.740.007
NeutrophilsIntercept value2.133 ± 0.707933.020.003
APC0.104 ± 0.020935.11<0.0001
Tirofiban−0.007 ± 0.023 93−0.330.73
APC:Tirofiban0.009 ± 0.026930.340.73
MonocytesIntercept value0.125 ± 0.050932.470.015
APC0.011 ± 0.002935.72<0.001
Tirofiban0.001 ± 0.002930.350.72
APC:Tirofiban0.003 ± 0.002931.310.19
d-DimersIntercept value172.0 ± 95.1 721.810.07
APC −6.8 ± 2.7  72−2.480.016
Tirofiban −1.8 ± 3.4  72−0.540.59
APC:Tirofiban  7.3 ± 4.3  721.680.10
FibrinogenIntercept value150.5 ± 49.6 783.030.003
APC3.5 ± 1.1783.090.003
Tirofiban−0.04 ± 1.4  78−0.020.98
APC:Tirofiban4.25 ± 1.8 782.370.02
Factor VIIIIntercept value43.4 ± 12.8793.390.001
APC2.3 ± 0.7793.390.001
Tirofiban−0.5 ± 0.8  79−0.640.52
APC:Tirofiban2.3 ± 1.1792.220.03
image

Figure 2. Clotting and lymphocyte parameters after treatment with tirofiban. Treatment with tirofiban alone made no difference to neutrophil or platelet count. There was a modest increase in clot weight with higher doses of tirofiban. For statistical analysis of data presented, see Table 2. Data are based on nine individual experiments using islets from five donors and blood from four volunteers.

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rhAPC monotherapy prevented IBMIR in a dose-dependent manner

Next rhAPC was used to determine its effect on clot formation within the ex vivo loop system. Dose-dependent improvements in factor VIII levels confirmed that the enzymic cleavage of factors V and VIII by rhAPC was occurring (19) (Figure 3F). rhAPC demonstrated a linear dose-dependent reduction in all measured IBMIR parameters, including platelet, neutrophil, monocyte counts and fibrinogen levels. There was an inverse relationship between rhAPC dose and clot size (Figure 3A). At a clinically relevant dose of 2.5 μg/loop, rhAPC was effective at reducing clot weight, improving platelet, neutrophil and monocyte counts and improving fibrinogen and d-dimer levels (Figure 3 and Table 2). However, supraclinical doses (up to 40 μg/loop) were required to completely abolish clot formation and to normalize clotting and hematological parameters. Although rhAPC could reverse IBMIR, doses higher than those used clinically were required to fully inhibit the response.

image

Figure 3. Clotting and lymphocyte parameters after treatment with APC. Increasing doses of APC led to dose depended reduction in clot weight and a normalizing of both clotting and lymphocyte parameters. For statistical analysis of data presented, see Table 2. Data are based on nine individual experiments using islets from five donors and blood from four volunteers.

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Clots isolated from loops, which had been treated with rhAPC, were evaluated by immunohistochemistry for neutrophil aggregation, islet infiltration and for C4d binding. With increasing doses of rhAPC there was a substantial reduction in the neutrophil aggregation (Figures 1D–F) and corresponding reduction in C4d binding (Figures 1G–I). Taken together these results show that rhAPC reduces both the thrombotic and inflammatory consequences of IBMIR in a dose-dependent manner.

rhAPC and tirofiban act synergistically to inhibit IBMIR

To confirm whether inhibition of platelet activation in combination with rhAPC would result in a substantial reduction in the dose of rhAPC required, tirofiban was used in combination with varying doses (0–20 μg) of rhAPC and the effect on IBMIR in the PVC perfusion loop system was assessed. Whereas tirofiban alone was ineffective, the combination of 2.5 μg of tirofiban with rhAPC was found to be synergistic and more effective than using rhAPC alone (Table 2). Whereas tirofiban alone lead to a significant increase in clot weight (Table 2, Figure 2), the addition of tirofiban to rhAPC lead to a significant further decease in clot weight when compared to the use of rhAPC alone (Table 2, Figure 4). The effect was to permit the same reduction in clot size with half the dose of rhAPC (Figures 3 and 4). The combination of tirofiban and rhAPC was significantly more effective than rhAPC alone at preventing the usual steep drop in the platelet count. There was a trend (approaching significance) to preventing monocyte consumption (Table 2 and Figures 4B–D). In addition, fibrinogen and factor VIII levels were better preserved, and there was a trend to less d-dimer production when rhAPC was used in combination with tirofiban compared to rhAPC used alone (Figures 4E and F and Table 2). Analysis of immunohistochemistry showed that C4d staining of islets was substantially reduced when rhAPC and tirofiban were used in combination although small amounts of residual staining remained (Figure 1). These data confirm that platelet inhibition with tirofiban allowed significantly smaller doses of rhAPC to be used to prevent IBMIR.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In clinical islet transplantation, only a minority of islets survive to engraftment and the majority of islets are thought to be lost to an innate inflammatory response called IBMIR (3). The significance of IBMIR in vivo was confirmed in animal models where 50–70% of transplanted islets were lost in the immediate post transplant period (13). Its relevance to clinical islet transplantation in humans has been demonstrated in patient studies where thrombin–antithrombin and FVIIa–antithrombin complexes were identified within the first 60 min of islet infusion suggesting that the procedure precipitated an immediate thrombotic response in vivo (3). IBMIR is triggered by tissue factor expressed on islets. Activated tissue factor leads to the generation of thrombin which is a potent mediator of inflammation leading to activation of complement, platelets and granulocytes. Thrombin mediates platelet and neutrophil activation via up-regulation of P-selectin and P-selectin ligand and stimulates additional secretion of TF by neutrophils and monocytes which further amplifies the response (20).

Given that multiple islet preparations are required to achieve insulin independence following islet transplantation and the majority of those that achieve insulin independence loose function by 2 years, it is essential that we develop clinically relevant strategies to overcome this problem. One alternative strategy already proposed has been low-molecular-weight dextran. There is in vitro data showing that it inhibits coagulation and complement activation and prevents IBMIR in vitro (21). Our strategy has been to use a two-pronged attack on both coagulation and platelets. Not only are platelets essential for clot formation, they are also an important link for the recruitment of neutrophils and initiating the inflammatory response. To prevent platelet activation we used tirofiban, a IIbIIIa inhibitor that has been shown to inhibit platelets in the context of the acute coronary syndrome (10,11). We chose rhAPC to inhibit coagulation. This agent has several theoretical advantages over heparin. Not only does it inhibit coagulation by cleaving Factor VIIIa and factor Va but it also has anti-inflammatory actions and is cytoprotective (22). In particular, it has been shown to inhibit neutrophil activation and prevents the release of islet toxic cytokines such as IL-1, IL-6 and TNF-α and prevents TF release (8). In our model, there was reduced islet-induced neutrophil adhesion when rhAPC was used. However, this could have been due solely to its anticoagulant effects and consequent reduction in thrombin generation. Hence, we have not demonstrated fully that rhAPC or combination therapy was anti-inflammatory in our model of IBMIR.

Our data showed that rhAPC and tirofiban worked synergistically to prevent islet-induced clotting in an ex vivo loop system used to simulate IBMIR. The strategy of targeting both coagulation and platelets was superior to targeting each aspect alone. It is known that platelets make a contribution to the propagation phase of coagulation by generating a burst of thrombin in a positive feedback cycle. It is likely that rhAPC and tirofiban act together to inhibit synergistically this thrombin-driven positive feedback cycle, which in turn inhibits the expansion of the IBMIR response. Direct thrombin inhibitors have previously been shown to be effective in preventing IBMIR in a similar ex vivo model (14). Even though rhAPC acts on factors involved in the amplification phase of the coagulation cascade, relatively large concentrations of rhAPC were required to achieve the full effect. By contrast tirofiban alone had no effect on IBMIR and in particular had no effect on platelet consumption. If anything, the data suggested that increasing doses of tirofiban made the IBMIR response worse. This supports the hypothesis that platelet activation per se was not a major initiator of IBMR but rather had a downstream impact on the response. However, co-administration of Tirofiban with rhAPC allowed smaller doses of rhAPC to be used and resulted in a significant reduction in clot size, platelet and monocyte consumption, reduced d-Dimer levels and better preserved fibrinogen levels. Histological evaluation of the clots following rhAPC and tirofiban co-administration showed a marked reduction in neutrophil adhesion and better preservation of islet morphology.

Previous studies in a mouse model of IBMIR showed that rhAPC inhibited IBMIR after intraportal islet transplantation. The data suggested that in the presence of the moderating effects of endothelium, rhAPC prevented thrombosis and inflammation and preserved islet mass (9). Although in an in vitro system, our data confirmed the beneficial effects of a two pronged approach inhibiting platelet activation and coagulation by adding tirofiban to rhAPC. The advantage of our system was that it is a robust model of IBMIR and confirms the beneficial effects, using human islets and human blood. The problem with all anti-IBMIR strategies is the risk of bleeding. Certainly this would be the case of a combined antiplatelet strategy with rhAPC. However as these data suggest, the addition of tirofiban allows for a substantial reduction in the effective dose of rhAPC which should ameliorate this risk. Although the effect of rhAPC and tirofiban on islet function was not evaluated, the use of APC in animal studies showed improved insulin release and glucose control (9) and no adverse effects were identified when tirofiban was used in patients with diabetes (11). Furthermore, any clinical strategy to inhibit IBMIR need only be in place for the first 24–48 h. Hence, using a combination of rhAPC and tirofiban is clinically feasible and utilizes agents that are already in clinical practice. Hence, this study provides strong evidence in support of a clinical trial of these agents in clinical islet transplantation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported by grants from the Juvenile Diabetes Research Foundation and the Cecilia Kilkeary Foundation.

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  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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