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Abstract

Although islet transplantation is effective in reducing severe hypoglycemia events and controlling blood glucose in patients with type 1 diabetes, maintaining islet graft function long-term is a significant challenge. Islets from multiple donors are often needed to achieve insulin independence, and even then, islet function can decline over time when metabolic demand exceeds islet mass/insulin secretory capacity. We previously developed a method that calculated the islet graft function index (GFI) and a patient’s predicted insulin requirement (PIR) using mathematical nonlinear regression. Both PIR and GFI could be used by physicians as tools to monitor islet graft function and to guide supplementing the patient with exogenous insulin to prevent beta-cell exhaustion. This study investigates the factors relating to the islet preparation process, as well as donor and recipient characteristics, and assessed their associations with PIR and GFI after transplantation. The goal is to determine the most relevant factors that influence islet graft function after transplantation. We examined the effects of donor and recipient characteristics, and islet processing factors on posttransplanted PIR and GFI. The PIR and GFI at 3 months were calculated using patients’ baseline insulin intake, posttransplant 2-h postprandial blood glucose, and glucagon-stimulated C-peptide. Thirteen transplants that resulted in progressive decline in patients’ weekly averaged insulin intake over the initial weeks after transplant (assuming constant glucose level) with available 3-month PIR and GFI data were chosen for the investigation. Univariate analyses were performed to assess the effects of donor and recipient characteristics and islet processing factors on islet graft function as reflected by PIR and GFI. The PIR and GFI were treated as continuous response variables in separate linear regression models. Shorter digestion time of isolated donor islets were associated with lower PIR (P = 0.014) and a higher GFI (P = 0.027) after transplantation. Islet injury related to digestion enzyme exposure influenced islet function as estimated using PIR and GFI post-transplantation.

Keywords

collagenase islet transplantation functional islet mass beta cell function

Introduction

Type 1 diabetes (T1D) is a chronic autoimmune disease in which pancreatic β-cell destruction by the patient’s immune system leads to insulin deficiency and chronic hyperglycemia. There is no cure, and lifelong insulin injections are needed to achieve glycemic control¹. For most patients, insulin therapy is sufficient to achieve glycemic control; nevertheless, a subset of T1D patients experience life-threatening hypoglycemic episodes despite insulin therapy²; currently, solid pancreas and allogeneic islet transplantation are the only therapeutic option for these patients. Islet transplant is a simple procedure for the treatment of these individuals, and if successful, it can result in 50% of patients having insulin independence after 5-years post-transplantation, with a majority of patients not experiencing severe hypoglycemic events 1 year after islet transplantation^3,4. However, successful insulin independence frequently requires multiple islet transplantations with major increase in cost and inconvenience to recipients. Consequently, there is a clinical need to identify optimal conditions to increase islet transplantation success rate that promote insulin independence after single transplantation to eliminate the need for multiple islet dosing.

Donor and recipient-specific characteristics, and/or post-isolation conditions of islets may influence the transplantation outcomes. We previously developed a mathematical nonlinear regression model that predicts patient’s insulin requirement (PIR) and calculates the islet graft function index (GFI) using patient’s baseline insulin intake, post-transplant 2-h postprandial blood glucose, and glucagon-stimulated C-peptide http://www.cityofhope.org/diabetes/ClinicalTools/MITRIS⁵. PIR and GFI are tools for physicians to monitor patients’ insulin requirements and transplanted islet function. In this study, we examined crucial islet preparation steps, as well as donor and recipient characteristics that may influence the PIR and GFI at 3 months to assess transplantation outcomes. The objective of this study is to identify factors that will guide the selection of donor, recipient, and islet preparation processes to improve the transplanted islet graft functions and reduce exogenous insulin requirement post-transplantation.

Subjects and Methods

Human Subjects

Seventeen adult type 1 diabetic (T1D) subjects with frequent episodes of hypoglycemia and/or hypoglycemia unawareness were transplanted with islets between 2004 and 2011 under the City of Hope’s Islet Transplant Alone protocol. Under this protocol, subjects received up to three separate islet infusions (except one patient who received four islet infusions) at a minimum of 2 weeks apart. Immunosuppression included basiliximab or daclizumab and etanercept at induction and a combination of tacrolimus, sirolimus, and/or MMF for maintenance. Out of a total of 40 transplants performed, nine were excluded due to insufficient insulin-to-glucose ratio data to access the trend or lack of appreciable decline in patients’ weekly averaged insulin over the initial weeks after transplant, resulting in 31 transplants (Table 1). Thirteen transplants in 12 subjects met the analysis objectives by having calculated GFI and PIR at 3 months post-transplant and demonstrated successful decline in the weekly averaged insulin intake over the first few weeks after transplant (which excluded patients with minimal insulin requirements prior to the final transplant who became insulin free) and therefore were investigated in this study. Comparisons of PIR and GFI to the beta 2 score were performed following the removal of a single anemic patient from the data set, due to the possibility of misleading estimates caused by extremely low HbA1c levels. Table 2 summarizes the donor and recipient characteristics, and islet preparation factors investigated in this study. Seven transplants were first infusions, two transplants were second, and four transplants were third infusions. Informed consents were obtained from all subjects, and study activities were approved by the City of Hope’s Institutional Review Board.

Table 1.

Sequence and Duration of Follow-up in 31 Transplants.

Initials	# of Tx received	Duration from Tx1 to Tx2	Duration from Tx2 to Tx3	Duration following final transplant
COH-001	3	3.3	1.5	24.4
COH-002	2	2.4	N/A	135.4
COH-003	3	2.6	2.8	24.4
COH-004	3	3.1	1.9	152.3
COH-006	2	5.4	N/A	24.9
COH-008	3	1.5	10	24
COH-010	1	N/A	N/A	4.3
COH-013	2	1.5	N/A	24.8
COH-014	3	6.6	2.7	62.3
COH-016	3	1.6	0.7	160.8
COH-018	3	2.5	4.4	15.6
COH-019	2	11	N/A	35
COH-020	1	N/A	N/A	53.2

Duration expressed in months

Table 2.

Donor, recipient, and islet preparation characteristics in 13 Transplants*.

Factors	Median(Range)†
Donor characteristics
Donor Age (yrs)	43 (29, 51)
Donor HbA1c	5.35 (4.95, 5.43)
Donor BMI	31.0 (27.2, 33.5)
Donor weight (lbs)	211 (185, 234)
Weight of the pancreas (g)	98 (90, 121)
Weight of the remaining tissue (g)	65 (58, 91)
Cold ischemic time (min)	428 (333, 541)
Sodium	139.0 (137.0, 140.0)
Amylase	74 (43, 187)
Lipase	26 (17, 49)
Hospitalization time (days)	2.00 (2.00, 3.75)
Islet preparation process
IEQ	307,100 (291,798, 340,359)
IEQ per Kg	3,063 (2,455, 3,774)
Culture time (hrs)	18.1 (16.6, 23.5)
Digestion time (min)	11.00 (10.00, 15.00)
Collagenase Concentration (U)	2,485 (2,414, 2,927)
Collagenase Per Pancreas (U/gram)	26 (25, 30)
Thermolysin Concentration (U)	66,061 (64,204, 166,845)
Thermolysin Per Pancreas (U/gram)	753 (674, 1,507)
% Purity of preparation	70 (63, 74)
Islet size index	0.90 (0.73, 1.00)
Recipient characteristics
Recipient BMI	24.33 (23.00, 27.33)
Recipient HbA1c post transplant	5.50 (5.33, 5.65)
Recipient HbA1c at baseline	7.00 (6.78, 7.20)

Three transplants have combined donor islets, and therefore were not included;

†

Median(IQR)

Islet Isolation and Transplantation

Human pancreata were procured from ABO-compatible, lymphocyte cross-matched negative cadaveric donors, and islets were isolated under cGMP conditions by the Southern California Islet Cell Resource Center at City of Hope using the modified Ricordi method⁶. Out of the 13 transplants, only one transplant used collagenase purchased from Vitacyte (Indianapolis, IN), while all other transplants used enzyme manufactured by Roche (Penzberg, Germany) to isolate the islets. Isolated islets were assessed for number and purity, then maintained in culture for up to 72 hours. Islets were then transplanted via the intraportal route with heparinized saline (35 u/kg recipient body weight) using the transhepatic percutaneous approach.

Insulin Data Collection

Subjects were asked to self-monitor blood glucose levels at least four times daily (before breakfast, before lunch, before dinner, and before bed) and record all insulin doses using a commercial blood glucose meter (FreeStyle or OneTouch UltraSmart). Insulin dose data were downloaded from the meters and summed each day to calculate the total daily insulin dose.

Estimates of Islet Graft Function

GFI, which provided a scoring assessment of functional islet mass ranging from 1 (poor islet function with higher risk of islet exhaustion over time) to 5 (good islet function with negligible islet exhaustion risk), and Predicted Insulin Requirements (PIR), reported as U/kg body weight were calculated as previously described⁵. In essence, a Model for Insulin Therapy and Islet Scoring (MITRIS) was used to describe the relationship between indicators of potential islet efficacy (glucagon-stimulated c-peptide, 2-h postprandial glucose, and baseline insulin requirements) to the patient’s reported post-transplant insulin intake. To ensure that the level of reported insulin intake reflected times when the patient was properly insulinized, insulin intake data were limited to periods of time where HbA1C was below 6.5% in the absence of hypoglycemic events. The result is a proxy representing an estimate of the appropriate level of required insulin therapy and functional state of the engraftment.

Statistical Analysis

The donor islet preparation considered in this study were: donor’s age, donor’s HbA1c, donor’s body mass index (BMI), weight of donor, weight of pancreas, weight of digested pancreas, islet size index, cold ischemia time, sodium, amylase, lipase, hospitalization time, islet equivalent (IEQ), IEQ per kg of recipient, culture time, digestion time, purity, viability; recipient factors included BMI and HbA1c at baseline and 90 days post-transplant. Two transplants had combined islets from two donors, and therefore, the donor characteristics of these two transplants were not included in the analysis. The PIR and GFI at 3 months were treated as continuous response variables in separate linear regression models. P values <0.05 were considered statistically significant. Statistical analyses were performed using the Dplyr and Gtsummary packages in R.

Results

Shorter digestion time during islet isolation was associated with a reduction in PIR (P = 0.014) and an increase in GFI (P = 0.027) in the 3 months following transplantation (Tables 3 and 4). PIR was also shown to increase in patients with elevated post-transplant HbA1c (P = 0.019) and was associated with a reduction in beta score^7,8(Table 3). Other measures of enzyme exposure, such as concentration of collagenase and thermolysin per gram weight of pancreas, were not shown to be significantly associated with either digestion time (R = −0.097, P = 0.75 and R = −0.34, P = 0.25, respectively) or graft function in our cohort (Tables 3 and 4). In addition to digestion time, patients with higher GFI also had lower post-culture islet purity when measured prior to transplantation (P = 0.03, Table 4). We have found that when considering all digests used for transplant, islets exposed to dissociative enzyme for 15 minutes or longer had significantly higher purity than those below 15 minutes (P < 0.001, n = 59), and this may be the case for the cohort targeted in this study as well (P = 0.058, n = 13). Purity was also shown to be significantly associated with enzyme exposure based on collagenase concentrations used (R = 0.35, P = 0.012), whereas thermolysin concentration did not show a significant correlation to islet purity (R = 0.022, P = 0.88). Although the patient’s baseline insulin intake alone did not predict transplant outcome (P = 0.2, 0.09 for PIR and GFI, respectively), the association may potentially be clinically relevant (Tables 3 and 4).

Table 3.

Association of donor, recipient, islet preparation with PIR (U/100kg) in 12 patients at post-transplant month 3 (13 transplants)^†.

Characteristic	Estimate	95% CI¹	p-value
Donor characteristics
Donor Age (yrs)	-0.001	-0.004, 0.002	0.6
Donor BMI	0.005	-0.004, 0.013	0.3
Donor weight (lbs)	0.000	-0.001, 0.001	0.5
Donor HbA1c	0.031	-0.057, 0.118	0.5
Weight of the pancreas (g)	0.001	-0.001, 0.003	0.5
Weight of the remaining tissue (g)	0.001	0.000, 0.003	0.071
Cold ischemic time (min)	0.000	0.000, 0.000	0.7
Sodium	0.001	-0.009, 0.010	0.9
Amylase	0.000	0.000, 0.000	0.5
Lipase	0.001	0.000, 0.001	0.14
Hospitalization time (days)	-0.013	-0.053, 0.026	0.5
Islet preparation process
IEQ	0.000	0.000, 0.000	0.8
IEQ per Kg	0.000	0.000, 0.000	0.6
Culture time (hrs)	0.001	-0.003, 0.006	0.6
Digestion time (min)	0.016	0.005, 0.027	0.014
Collagenase Per Pancreas (U/gram)	0.000	-0.003, 0.002	0.8
Thermolysin Per Pancreas (U/gram)	0.000	0.000, 0.000	>0.9
% Purity of preparation	0.003	0.000, 0.007	0.081
Islet size index	-0.052	-0.182, 0.079	0.5
Beta Score	-0.040	-0.065, -0.015	0.017
Beta 2 Score‡	-0.015	-0.022, -0.007	0.010
Recipient characteristics
Recipient BMI	0.000	-0.017, 0.017	>0.9
Recipient HbA1c post-transplant	0.073	0.023, 0.124	0.019
Baseline insulin intake	0.289	-0.097, 0.674	0.2

†

Three transplants have combined donor islets, and therefore were not included;

‡

One patient with anemia was excluded from this analysis

CI = Confidence Interval

Table 4.

Association of donor, recipient, islet preparation with GFI in 12 patients at post-transplant month 3 (13 transplants)^†.

Characteristic	Estimate	95% CI¹	p-value
Donor characteristics
Donor Age (yrs)	0.028	-0.020, 0.077	0.3
Donor BMI	-0.066	-0.210, 0.079	0.4
Donor weight (lbs)	-0.008	-0.026, 0.011	0.4
Donor HbA1c	-0.608	-2.112, 0.897	0.4
Weight of the pancreas (g)	-0.009	-0.044, 0.027	0.6
Weight of the remaining tissue (g)	-0.025	-0.049, -0.002	0.058
Cold ischemic time (min)	0.000	-0.004, 0.005	0.9
Sodium	-0.039	-0.198, 0.120	0.6
Amylase	-0.002	-0.006, 0.003	0.5
Lipase	-0.006	-0.018, 0.005	0.3
Hospitalization time (days)	0.042	-0.641, 0.725	>0.9
Islet preparation process
IEQ	0.000	0.000, 0.000	>0.9
IEQ per Kg	0.000	-0.001, 0.001	>0.9
Culture time (hrs)	0.004	-0.080, 0.087	>0.9
Digestion time (min)	-0.253	-0.447, -0.059	0.027
Collagenase Per Pancreas (U/gram)	-0.003	-0.046, 0.039	0.9
Thermolysin Per Pancreas (U/gram)	0.000	0.000, 0.000	0.7
% Purity of preparation	-0.069	-0.123, -0.014	0.030
Islet size index	0.591	-1.638, 2.821	0.6
Beta Score	0.541	-0.025, 1.107	0.10
Beta 2 Score‡	0.280	0.178, 0.383	0.002
Recipient characteristics
Recipient BMI	0.035	-0.190, 0.260	0.8
Recipient HbA1c post-transplant	-1.091	-2.224, 0.042	0.092
Baseline insulin intake	-5.887	-12.04, 0.270	0.090

†

Three transplants have combined donor islets, and therefore were not included;

‡

One patient with anemia was excluded from this analysis

CI = Confidence Interval

Discussion

Insulin independence and the elimination of severe hypoglycemic episodes in a subset of T1D patients often require multiple islet transplantations. We identified critical factors associated with insulin requirement and islet graft function post-transplantation to guide the selection of the donor, recipient, and islet preparation parameters to improve transplant outcomes and reduce the need for multiple transplantations. Our results, which were based primarily on the use of Roche enzyme, suggested that shorter digestion time during the islet isolation process played critical roles in reducing PIR and improving GFI at 3 months post-transplant. For instance, every 5-minute increase in dissociative enzyme exposure during islet isolation resulted in a 3 U/day increase in PIR post-transplant in patients, using the average weight of 44 kg found in our cohort.

Multiple factors influence islet graft function post-transplantation; published studies examined the donor characteristics, pancreas preservation, and islet isolation process that may impact the success of islet transplantation^9–13. Preparations that consist of highly purified islets are not always predictive of a favorable clinical outcome, particularly if the purity is the result of over digestion. Despite the poor clinical outcome associated with long duration of enzyme exposure, we have found that islet exposed to dissociative enzyme for 15 or more minutes had a purity of 71% compared to 62% for exposure below 15 minutes when considering all islets preparations used for transplant. This suggests a narrow window for duration of enzyme exposure during the isolation process, with shorter exposure time leading to trapped islets, and excessive exposure leading to poor in vivo function. Factors such as pancreas quality during the organ procurement process was also positively associated with higher islet yield^11,12 and suggest that fast and efficient preservation of the donor pancreas is a critical factor in obtaining greater islet mass and quality after isolation, and ultimately, influence transplantation outcomes.

The islet isolation process from the pancreas also impacted the insulin requirement and the graft function post-transplantation; shorter digestion time positively associates with the transplantation outcome at 3 months and is consistent with Kim et al.¹⁴. Although, exact mechanisms not known, over-digestion during the isolation process may lead to the destruction of integrin receptors and/or extracellular matrix (ECM) necessary for the islet survival and function. Islets are isolated from the pancreas with collagenase; however, collagenase degrades the interstitial and peri-insular ECM and destroys the islet microvasculature required for islet cell survival and function^15,16. Restoring islet-ECM attachments following isolation improve islet quality¹⁷; islet β-cells express integrin receptors that bind to ECM proteins contain Arg-Gly-Asp (RGD) motifs and induce signaling cascades involved in β-cell survival and insulin secretion^18–22. Moreover, the addition of ECM molecules or RGD-containing elastin-like polypeptides to grafted islets enhances their function and survival^23,24. In addition, others showed that enzyme quality and types could influence the islet isolation yield and quality^9,10,13. Caballero-Corbalán et al.²⁵ demonstrated that digestion time is reduced with Liberase MTF-S compared with other blends, which could be associated with improvements in clinical outcomes, although we did not test this directly. Other centers showed promising results in producing clinical grade islet using the Serva enzyme²⁶. Because our clinical dataset contained only a single preparation using the Vitacyte enzyme, and none with Serva, meaningful group comparisons of clinical outcomes between manufacturer enzymes was not possible.

Although PIR and GFI are inversely correlated, the GFI represents a range of ordinal values which rank the entire range of islet function on a scale of 1 to 5. Because most of the patients in our study were classified as having a GFI of either 1, 2, or 3, the use of such a scale as a correlate to the Beta score⁷ (range 0–8) may have lacked the necessary precision to show significance. Conversely, the beta score 2 represents a more sensitive assessment of beta cell function⁸, expressed as a range from 0 to 42 but is also susceptible to extreme estimates, such as when a patient has exceptionally low HbA1c due to anemia. As such, we justified the removal of the single outlier when comparing beta 2 score results in our analysis.

In conclusion, we found that digestion enzyme exposure is a determinant of islet quality that can influence clinical outcomes by 3 months following islet transplantation. We have also observed that islets digested for long periods of time resulted in higher islet purity, and this could be associated with poor islet function. Our data suggest that protecting islet integrity during organ procurement and islet isolation may enhance islet graft function and improve clinical outcomes in transplant patients.

Footnotes

Acknowledgements

The authors thank the City of Hope Islet Transplant team and the Arthur Riggs Diabetes and Metabolism Research Institute for their contributions to the study.

Author Contributions

C.W and C.O. were involved in data acquisition, conceptualization, data analysis and visualization, and drafting of the manuscript; F.K. was involved in investigation, conceptualization, methodology, drafting and critical revision of the manuscript; J.S. was involved in data acquisition, interpretation, and critical discussion; M.S., K.O. and M.Q. were involved in investigation

Ethical Approval

Results reported from Islet Transplantation Alone (ITA) in Patients With Difficult to Control Type I Diabetes Mellitus Using a Glucocorticoid-free Immunosuppressive Regimen study (registration identifier: NCT00706420).

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

Informed consent was obtained from all individuals, and studies were approved by the Institutional Review Board Committee (COH IRB# 1083). Study name: “Islet Transplantation Alone (ITA) in Patients With Difficult to Control Type I Diabetes Mellitus Using a Glucocorticoid-free Immunosuppressive Regimen”; ClinicalTrials.gov ID: NCT00706420. Study site: .

Data Availability Statement

Datasets generated for this study are available upon request from the corresponding author.

Declaration of Conflicting Interests

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work is supported by the National Institutes of Health (National Center for Research Resources, National Institute of Diabetes and Kidney Diseases, U42-RR16607).

ORCID iD

Christopher Orr

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Shorter Digestion Times of Donor Islets Is Associated With Better Islet Graft Function After Islet Transplantation

Abstract

Keywords

Introduction

Subjects and Methods

Human Subjects

Islet Isolation and Transplantation

Insulin Data Collection

Estimates of Islet Graft Function

Statistical Analysis

Results

Discussion

Footnotes

Acknowledgements

Author Contributions

Ethical Approval

Statement of Human and Animal Rights

Statement of Informed Consent

Data Availability Statement

Declaration of Conflicting Interests

Funding

ORCID iD

References