Improving Efficacy of Clinical Islet Transplantation with Iodixanol-Based Islet Purification,Thymoglobulin Induction,and Blockage of IL-1β and TNF-α

Abstract

Poor efficacy is one of the issues for clinical islet transplantation. Recently, we demonstrated that pancreatic ductal preservation significantly improved the success rate of islet isolation; however, two transplants were necessary to achieve insulin independence. In this study, we introduced iodixanol-based purification, thymoglobulin induction, and double blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression to improve the efficacy of clinical islet transplantation. Nine clinical-grade human pancreata were procured. Pancreatic ductal preservation was performed using ET-Kyoto solution in all cases. When the isolated islets met the clinical criteria, they were transplanted. We utilized two methods of immunosuppression and anti-inflammation. The first protocol prescribed daclizumab for induction, then sirolimus and tacrolimus to maintain immunosuppression. The second protocol used thymoglobulin for induction and tacrolimus and mycophenolate mofetil to maintain immunosuppression. Eternacept and anakinra were administered as anti-inflammatory drugs. The total amount of insulin required, HbA1c, and the SUITO index were determined to analyze and compare the results of transplantation. All isolated islet preparations (9/9) met the criteria for clinical transplantation, and they were transplanted into six type 1 diabetic patients. All patients achieved insulin independence with normal HbA1c levels; however, the first protocol required two islet infusions (N = 3) and the second protocol only required a single infusion (N = 3). The average SUITO index, at 1 month after a single-donor islet transplantation, was significantly higher in the second protocol (49.6 ± 8.3 vs. 19.3 ± 6.3, p < 0.05). Pancreatic ductal preservation, iodixanol-based purification combined with thymoglobulin induction, and blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression dramatically improved the efficacy of clinical islet transplantations. This protocol enabled us to perform successful single-donor islet transplantations. Further large-scale studies are necessary to confirm these results and clarify the mechanism of each component.

Keywords

Islet transplantationl Single donor Thymoglobulin Interleukin-1β (IL-1β)Tumor necrosis factor-α (TNF-α)

Introduction

Islet transplantation is a promising treatment for type 1 diabetic patients; however, several issues need to be resolved (12,24,25). Poor islet efficacy is one of the clinical issues of islet transplantation (11). The success rate of clinical islet isolation was about 50% in the majority of centers capable of utilizing the most advanced techniques (3,21). In addition, multiple islet transplantations are usually necessary to achieve insulin independence (24,25). Recently, we demonstrated that pancreatic ductal preservation significantly improved our success rate in clinical islet isolation; however, two islet transplantations were still necessary to achieve insulin independence using the Edmonton drug regimen (14).

One of the current advancements in islet transplantation seems the introduction of thymoglobulin to broadly suppress the immune system (8,26). The thymoglobulin induction increases regulatory T cells (26), which are important mediators in the prevention of type 1 diabetes (7) and allogeneic rejection (10). The University of Minnesota group demonstrated that 8 out of 8 patients became insulin independence after single-donor islet transplantation using combined thymoglobulin and daclizumab induction (8). However, a recent publication revealed that only 4 out of 8 patients became insulin independent after single-donor islet transplantations with thymoglobulin induction combined with efalizumab-based immunosuppression (22). More recently, the Emory University group demonstrated that 4 out of 4 patients became insulin independent at day 75 after single-donor islet transplantations with the daclizumab induction combined with an efalizumab-based immunosuppressive regimen (27). Thus, the protocol for successful single-donor islet transplantation is still contentious.

On the other hand, early graft loss immediately after the islet infusion, primarily due to inflammation, is another issue (5,6). The proinflammatory cytokine interleukin-1β (IL-1β) was shown to play a critical role in β-cell destruction at the onset of juvenile diabetes (2,9). Additionally, according to the collaborative islet transplant registry (CITR) report, tumor necrosis factor-α (TNF-α) blockage significantly improved clinical outcome of islet transplantation (1). These data supported the concept that inflammation significantly impacted on the engraftment of transplanted islet cells. Therefore, we hypothesize that thymoglobulin induction combined with potent anti-inflammatory drugs will improve the efficacy of isle engraftment.

In this study, we applied iodixanol-based purification, thymoglobulin induction combined with inflammatory blockade using anakinra (IL-1β blocker), and etanercept (TNF-α blocker) as well as sirolimus-free immunosuppression to achieve single-donor islet transplantation.

Materials and Methods

Ethical Guidelines

This study was approved by the institutional review boards and the application of investigational new drug of clinical islet transplantation is approved by U.S. Food and Drug Administration.

Human Islet Isolation and Evaluation

From February 2007 to November 2009, nine clinical-grade human pancreata were obtained through our two local organ procurement agencies (LifeGift, Fort Worth, TX and Southwest Transplant Alliance, Dallas, TX). Donor pancreata were procured by surgeons who belonged to the islet isolation team. Pancreatic ductal preservation was performed using ET-Kyoto solution (14), and the oxygen-charged static two-layer method (17) was used for transportation of procured pancreata in all cases.

Islets were isolated using the modified Ricordi method (15,16,23). Serva collagenase NB1 with neutral protease (SERVA Electrophoresis, Heidelberg, Germany) was used for the first eight cases and Liberase MTF with thermolysin (Roche Diagnostics GmbH, Penzberg, Germany) was used for the last case. Islets were purified using a continuous density gradient in a COBE 2991 cell processor (15). For density gradient solution, Ficoll was used in the first six cases and ET-Kyoto solution and iodixanol (15,19) was used in the last three cases.

The final preparation of islets was assessed by using dithizone staining (Sigma Chemical Co., St. Louis, MO) (2 mg/ml) to determine yield and purity. The islet yield was converted into a standard number of islet equivalents (IE, diameter standardized to 150 μm) in accordance with previous protocols (16,24,25). Islet viability after purification was measured with fluorescein diacetate (10 μM) and propidium iodide (15 μM) staining (16,24,25). The average viability of 50 islets was calculated. To assess in vitro functionality, the glucose-stimulated insulin release assay was performed (16,24,25). After overnight culture, triplicates of 150 IE islets were incubated with low (2.8 mM) and high (20.0 mM) concentrations of glucose dissolved in Functionality/Viability Medium CMRL1066 (Mediatech Inc, Manassas, VA) for 1 h at 37°C. Insulin concentrations were measured by an ELISA kit (ALPCO Diagnostics, Salem, NH) and a spectrophotometer (BioTek Instrument, Inc., Winooski, VT). The stimulation index was calculated by dividing the insulin concentration in the high-glucose solution by that in the low-glucose solution. The sterility of the final preparations was assessed by measuring the concentration of endotoxin with EndoPrep (BioDtech, Inc., Birmingham, AL) and performing the Gram staining. When an islet preparation met transplantation criteria based on the Edmonton protocol (16,24,25), the islets were transplanted. The criteria for transplantation are i) islet yield: more than 4,000 IEQ/kg patient body weight, ii) purity: greater than 30%, iii) viability: higher than 70%, iv) tissue volume: less than 10 ml, v) the Gram staining of the final preparation: negative, vi) endotoxin concentration of the final preparation: less than 5 EU/kg patient body weight.

Immunosuppression and Anti-Inflammatory Protocols

We used two immunosuppression and anti-inflammatory protocols. For the first protocol, we used daclizumab (Zenapax, Roche Pharmaceuticals, NJ) for induction and sirolimus (Rapamune, Wyeth Pharmaceuticals Inc. PA) and tacrolimus (Prograf, Astellas, IL) to maintain immunosuppression based on the Edmonton protocol (1,2). Etanercept (Enbrel, Immunex, WA) (50 mg) was given intravenously 1 h before transplantation and 25 mg was administered subcutaneously on days 3, 6, and 10 posttransplant as an anti-inflammatory treatment.

For the second protocol, we used rabbit anti-thymocyte globulin (ATG, thymoglobulin, Genzyme, MA) at a concentration of 1.5 mg/kg on days 0, 2, 4, and 6 posttransplant for induction immunosuppression. Anakinra (Kineret, Amgen, CA) (100 mg) was administered intravenously 1 h pretransplant, and subcutaneously for 7 days after the transplantation as an anti-IL-1β therapy. In addition, 50 mg of etanercept was delivered intravenously 1 h before transplantation. A reduced 25 mg dose of etanercept was given subcutaneously on days 3, 6, and 10 posttransplant as an anti-TNFα therapy. To maintain immunosuppression, we used mycophenolate mofetil (MMF, CellCept, Roche Laboratories, NJ) (2 g/day orally) and tacrolimus (Prograf, Astellas, IL) aiming for target trough level of 5–10 ng/ml.

Assessments of Clinical Outcomes

To assess the outcome of our islet transplantations, the total amount of insulin required and HbA1c were monitored. Islet functions were categorized as insulin independent when patients were insulin free, partial function when patients required insulin but islets secreted some insulin as determined by serum C-peptide levels, and failed function when islets failed to secret insulin.

To estimate the functional islet mass, the average secretory unit of islet transplant objects (SUITO) index was calculated during the first month after islet transplantation (12,13,18). The formula of the SUITO index is: [fasting C-peptide (ng/ml)]/[fasting glucose (mg/dl) – 63] x 1500 (12,18). The SUITO index reflects the functional islet mass; the SUITO index for normal healthy people is set at 100 (12,18). A SUITO index value greater than 10 is associated with reduced insulin requirement with excellent glycemic control (13) and greater than 26 is associated with insulin independence (12,18).

Statistical Analysis

Results were expressed as mean ± SE. Two groups were compared by the Student's t-test and values p < 0.05 were deemed statistically significant.

Results

Islet Characteristics

All of the nine final preparations were negative for Gram staining and below 5.0 (EU/kg body weight) for endotoxin concentrations. All nine isolated islet preparations met criteria for clinical transplantation (9/9) (Table 1).

Table 1.

Islet Characteristics

	Islet Yield (IE)	Purity (%)	Viability (%)	Tissue Volume (ml)	Stimulation Index
First protocol
Patient 1	514,467	49	90.5	9.0	—
Patient 1	872,174	85	99.1	1.5	2.8
Patient 2	442,667	43	99.8	9.5	2.3
Patient 2	538,784	56	96.9	7.5	7.0
Patient 3	559,153	48	96.8	9.0	6.9
Patient 3	385,581	47	99.8	9.0	6.0
Average	552,138 ± 69,233	55. ± 6	97 ± 1	7.6 ± 1.2	5.1 ± 1.0
Second protocol
Patient 4	543,225	60	96.8	5.0	—
Patient 5	927,227	60	98.8	5.0	2.9
Patient 6	691,114	50	98.0	4.5	5.4
Average	720,522 ± 111,826	57 ± 3	98 ± 1	4.8 ± 0.2	4.1 ± 1.3
p-Value	0.22	0.83	0.75	0.18	0.66

The first six isolated islet preparations were transplanted under the first protocol and the last three isolated islet preparations were transplanted under the second protocol (Table 1). There were no significant differences in total islet yield, purity, viability, tissue volume, or stimulation indices between the two protocols (Table 1). There was no significant difference in donor age between the two protocols; the average donor age was 37.5 ± 5.4 years old in the first protocol and 49.3 ± 6.7 years old in the second protocol. There was no significant difference in the body mass index (BMI) of the donors between the two protocols, and the average donor BMI was 30.4 ± 2.9 kg/m² in the first protocol and 31.1 ± 3.5 kg/m² in the second.

Patient Characteristics

The first three patients were transplanted under the first protocol and the last three patients were transplanted according to the second protocol (Table 2). There were no significant differences in the patients' age, body weight, BMI, pretransplant average daily insulin dose, or pretransplant glycosylated hemoglobin (HbA1c) between the two protocols (Table 2).

Table 2.

Patient Characteristics

	Age (Years)	Body Weight (kg)	BMI (kg/m²)	Insulin Dose (IU)	HbA1c (%)
First protocol
Patient 1	61	58	21.2	27	7.7
Patient 2	54	66	26.6	41	7.0
Patient 3	58	80	29.9	29	8.3
Average	58 ± 2	68 ± 7	26 ± 3	32 ± 5	7.7 ± 0.4
Second protocol
Patient 4	40	55	20.7	36	8.3
Patient 5	55	76	27.9	40	9.3
Patient 6	51	52	19.1	29	8.2
Average	49 ± 5	61 ± 8	23 ± 3	35 ± 3	8.6 ± 0.3
p-Value	0.14	0.53	0.42	0.61	0.14

Clinical Outcomes

All patients achieved insulin independence; however, the first protocol required two islet infusions and the second protocol required only a single infusion. The average SUITO index at 1 month after single islet transplantation was significantly higher in the second protocol (Table 3). The SUITO indices of all three patients were more than 26.0 at 1 month posttransplantation with the second protocol. One month after a second islet infusion using the first protocol, patients achieved an average SUITO value similar to the SUITO index value at 1 month reached by patients who received a single islet infusion with the second protocol (Table 3). The SUITO indices of all three patients were more than 26.0 after the second islet infusion in the first protocol.

Table 3.

Clinical Outcomes

	SUITO Index at 1 Month	SUITO Index at 1 Month After 2nd Infusion	Period for Insulin Free After Final Infusion (Days)	HbA1c at Insulin Free (%)
First protocol
Patient 1	30.6	63.1	48	6.0
Patient 2	18.3	36.0	37	5.2
Patient 3	8.9	42.6	36	6.0
Average	19.3 ± 6.3	47.3 ± 8.1	40 ± 4	5.7 ± 0.3
Second protocol
Patient 4	39.1	—	51	5.6
Patient 5	65.9	—	65	5.9
Patient 6	43.9	—	42	6.1
Average	49.6 ± 8.3	—	53 ± 7	5.9 ± 0.1
p-Value	<0.05		0.19	0.68

There was no significant difference of period for insulin free after final islet infusion between the two protocols (Table 3). There was no significant difference in average HbA1c between the two protocols at the time patients no longer required insulin (Table 3).

Long-Term Clinical Outcomes

Under the first protocol, the first patient has maintained insulin independence for 917 days and currently she is insulin independent (Fig. 1). Her recent HbA1c was 5.7% at postoperative day (POD) 1055. The second patient maintained insulin independence for 447 days; her HbA1c was 6.4% at POD 632 when she was insulin independent. This second patient lost islet function at POD 877, and the HbA1c became 9.3% at POD 847. The third patient maintained insulin independence for 353 days, and HbA1c was 6.6% at POD 411 when she was insulin independent. Currently, she has maintained partial islet function, and her recent HbA1c was 6.0% at POD 807.

Figure 1.

Long-term clinical outcomes after islet transplantations. The white bar indicates patients are insulin independent, the gray bar indicates islets have partial function, and the black bar indicates islet graft failure. The triangles indicate second islet infusions.

Under the second protocol, the fourth patient maintained insulin independence for 147 days after the first islet infusion. She lost partial islet function and received a supplemental islet infusion (467,478 IE) at POD 249. She became insulin independent again at POD 315, and she has maintained insulin independence for additional 382 days. Currently she does not need insulin and her HbA1c was 5.6% at POD 676. The fifth patient has maintained insulin independence for 636 days. The HbA1c was 6.4% at POD 671 at which time she had continued independence from insulin. The sixth patient is currently insulin independent and has maintained this status for 281 days; at POD 175, the HbA1c was 6.6%.

Discussion

In this study, we were able to achieve insulin independence with single-donor islet transplantations using pancreatic ductal preservation, iodixanol-based purification method, thymoglobulin induction, double blockage of IL-1β and TNF-α, and mycophenolate mofetil for maintenance immunosuppression. Recently, we demonstrated that the pancreatic ductal preservation significantly improved the success rate of islet isolations; however, two islet transplants were necessary to achieve insulin independence (14). On the other hand, the University of Minnesota group demonstrated single-donor islet transplantation was feasible (8), but their success rate for islet isolations to obtain transplantable islet preparation was still less than 50% with the current protocol (3). In this study, the islet isolation success rate was 100% (9/9). In addition, single-donor transplantations under the second protocol achieved a success rate of 100% (3/3). Therefore, the efficacy of islet transplantation was dramatically improved.

The SUITO index clearly demonstrated that the second protocol significantly improved the functional islet mass when compared to the first protocol. Although the second protocol used a new islet purification method (20), there was no significant difference detected in either the quality or the quantity of isolated islets between the first and second protocols. Therefore, improvement in the functional islet mass should be mainly result of the immunosuppressive and anti-inflammatory protocol. Previously, we demonstrated that a SUITO index of more than 26.0 was associated with insulin independence (18). The SUITO indices of all three patients in the second protocol at 1 month after the first islet transplantation were more than 26.0, indicating that those patients had adequate number of functioning islets. The SUITO index provided important insights into the efficacy of islet engraftments according to the different protocols.

Previously, three protocols have been published describing successful single-donor islet transplantations (8,22,27). The first protocol used both daclizumab and thymoglobulin for the induction therapy and eternacept for anti-inflammation (8). This protocol claimed that 8 out of 8 patients became insulin independent after single-donor islet transplantations. The second protocol used daclizumab for its induction therapy and efalizumab for its induction and maintenance therapy (27). This protocol led to 4 out of 4 patients becoming insulin independent after single-donor islet transplantation without thymoglobulin induction. This protocol is comparable with the Edmonton protocol, which includes daclizumab induction. Therefore, the key of their success seems the introduction of efalizumab. The third protocol used thymoglobulin for induction and efalizumab for induction and maintenance therapy (22). However, only 4 out of 8 patients became insulin independent after single-donor islet transplantation. The combination of thymoglobulin and efalizumab was less effective when compared to a combination of thymoglobulin and daclizumab (8) or the combination of daclizumab and efalizumab (27). Thus, efalizumab may not be a critically important factor. In addition, efalizumab is currently unavailable due to safety concerns (22). Therefore, it is necessary to find a different protocol for successful single-donor islet transplantation.

In this study, our first protocol used daclizumab for induction therapy and eternacept for anti-inflammation, which required two islet transplants to achieve insulin independence. Our second protocol used thymoglobulin for induction therapy and eternacept and anakinra for anti-inflammation, which enabled us to achieve successful single-donor islet transplantations. Therefore, thymoglobulin induction with the addition of anakinra seems an important factor for successful transplantation of islets from a single donor. However, in the second protocol we also used iodixanol-based purification and mycophenolate mofetil instead of sirolimus for maintenance immunosuppressive drugs. The University of Miami group demonstrated that iodixanol-based purification method had anti-proinflammatory effects (19). Therefore, using iodixanol-based purification method should have positive effects to prevent inflammation-related islet loss on the second protocol. Recently, it was demonstrated that mammalian target of rapamycin 1 (mTOR1) activation regulates β-cell mass and proliferation (4). This suggested that the use of sirolimus could negatively impact the success of islet transplantation and the adaptation of β-cells to insulin resistance. Therefore, elimination of sirolimus also had possible positive impacts on the second protocol.

Under the first protocol, one patient still maintained insulin independence and another patient maintained partial islet function for more than 2 years, but one patient lost islet function after 2 years. It seems difficult to achieve constant long-term insulin independence under the first protocol. Under the second protocol, all patients are currently insulin independent, even though one patient required a supplemental islet infusion. Although these data are promising, the observation period is limited at most 2 years, so further follow-up study will be necessary.

This study was limited by small numbers of patients and increasing numbers should be the next step to prove the efficacy of this new protocol. Also, the role of thymoglobulin induction along with our anti-inflammatory strategy is unclear. Further studies including mechanistic research will be important to establish the validity of the new protocol.

In conclusion, pancreatic ductal preservation, iodixanol-based purification combined with thymoglobulin induction, double blockage of IL-1β and TNF-α, and elimination of sirolimus from maintenance immunosuppressive drugs dramatically improved the efficacy of clinical islet transplantation. This protocol enabled us to achieve single-donor islet transplantation. Further large-scale (multicenter) randomized clinical trials are necessary to confirm the benefit of this protocol.

Footnotes

Acknowledgments

This research is partially supported by All Saints Heath Foundation. Dr. Chujo has been supported by the proceedings of the W. W. Caruth Jr. Chair for Organ Transplantation Immunology to J. Banchereau, Ph.D. Authors acknowledge Ms. Yoshiko Tamura and Mr. Greg Olsen for their technical supports and Ms. Sara Shah for editing the manuscript. Footnotes. This research is partially supported by All Saints Health Foundation (Fort Worth, TX). The authors declare no conflicts of interest.

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