Hering BJ, Kandaswamy R, Ansite JD, Eckman PM, Nakano M, Sawada T, Matsumoto I, Ihm S, Zhang H, Parkey J, Hunter DW, Sutherland DER. Single-Donor, Marginal-Dose Islet Transplantation in Patients With Type 1 Diabetes. JAMA. 2005;293(7):830-835. doi:10.1001/jama.293.7.830
Author Affiliations: Diabetes Institute for
Immunology and Transplantation and Department of Surgery; and Department of
Radiology (Dr Hunter), University of Minnesota, Minneapolis.
Context Islet allografts from 2 to 4 donors can reverse type 1 diabetes. However,
for islet transplants to become a widespread clinical reality, diabetes reversal
must be achieved with a single donor to reduce risks and costs and increase
the availability of transplantation.
Objective To assess the safety of a single-donor, marginal-dose islet transplant
protocol using potent induction immunotherapy and less diabetogenic maintenance
immunosuppression in recipients with type 1 diabetes. A secondary objective
was to assess the proportion of islet transplant recipients who achieve insulin
independence in the first year after single-donor islet transplantation.
Design, Setting, and Participants Prospective, 1-year follow-up trial conducted July 2001 to August 2003
at a single US center and enrolling 8 women with type 1 diabetes accompanied
by recurrent hypoglycemia unawareness or advanced secondary complications.
Interventions Study participants underwent a primary islet allotransplant with 7271
(SD, 1035) islet equivalents/kg prepared from a single cadaver donor pancreas.
Induction immunosuppression was with antithymocyte globulin, daclizumab, and
etanercept. Maintenance immunosuppression consisted of mycophenolate mofetil,
sirolimus, and no or low-dose tacrolimus.
Main Outcome Measures Safety (assessed by monitoring the severity and duration of adverse
events) and efficacy (assessed by studying the recipients’ insulin requirements,
C-peptide levels, oral and intravenous glucose tolerance results, intravenous
arginine stimulation responses, glycosylated hemoglobin levels, and hypoglycemic
episodes) associated with the study transplant protocol.
Results There were no serious, unexpected, or procedure- or immunosuppression-related
adverse events. All 8 recipients achieved insulin independence and freedom
from hypoglycemia. Five remained insulin-independent for longer than 1 year.
Graft failure in 3 recipients was preceded by subtherapeutic sirolimus exposure
in the absence of measurable tacrolimus trough levels.
Conclusions The tested transplant protocol restored insulin independence and protected
against hypoglycemia after single-donor, marginal-dose islet transplantation
in 8 of 8 recipients. These results may be related to improved islet engraftment
secondary to peritransplant administration of antithymocyte globulin and etanercept.
These findings may have implications for the ongoing transition of islet transplantation
from clinical investigation to routine clinical care.
Type 1 diabetes remains a therapeutic challenge. The success rate of
islet transplants has recently been increased markedly by transplanting a
higher mean islet mass (11 547 [SD, 1604] islet equivalents [IEs]/kg)
prepared from 2 to 4 donor pancreases and using glucocorticoid-free immunosuppression.1- 6 However,
for islet transplants to become a widespread clinical reality, additional
advances are still needed. In particular, restoration of insulin independence
must be achieved with a single donor, as is also the case with pancreas transplants,
to reduce the risks and costs and increase the availability of islet transplantation.
We designed a protocol to limit ischemic injury of islets during pancreas
storage, permit initiation of potent immunotherapy prior to transplantation,
and minimize calcineurin inhibitor exposure.
Our study was a prospective, single-center, 1-year follow-up pilot trial
conducted from July 2001 to August 2003. The primary efficacy end point was
the proportion of recipients who achieve insulin independence in the first
year after a single-donor islet transplant. We defined recipients as insulin-independent
if they maintained fasting blood glucose levels below 126 mg/dL (7.0 mmol/L)
and 2-hour postprandial levels below 180 mg/dL (10.0 mmol/L) after discontinuation
A total of 8 patients (coincidentally all women) were enrolled. Eligibility
criteria are detailed in the Box and detailed recipient characteristics are shown in Table 1. Our study protocol was approved by the local institutional
review board, and written informed consent was obtained from all participants.
Age 18 years or older
C-peptide–negative* type 1 diabetes for >5
years complicated by 1 of the following:
Advanced secondary complications including proliferative retinopathy or clinically significant macular edema
or photocoagulation, urinary albumin excretion >300 mg/d but proteinuria <3
g/d, or symptomatic autonomic neuropathy
Metabolic lability/instability (≥2 episodes of severe hypoglycemia or ≥2 hospital admissions for ketoacidosis
in the past year)
Hypoglycemia unawareness (≥4 “reduced” responses
in the Clark “hypoglycemia questionnaire”7)
Body weight >70 kg
Insulin requirements >40 U/d
Previous islet transplant
Abnormal renal function (creatinine clearance <60
mL/min [1.002 mL/s] per 1.73 m2)
Portal hypertension, abnormal liver enzyme test
results, or history of significant liver disease
*Defined as C-peptide level <0.2 ng/mL after administration of 5
g of intravenous arginine.
Eighteen consecutive donor pancreases were procured from cadaver donors
younger than 50 years with a body mass index (calculated as weight in kilograms
divided by square of height in meters) of 27 or greater; the pancreases were
preserved for 8 hours or less using the 2-layer method.8 ABO
compatibility and a negative serum crossmatch for T cells were required, but
HLA antigen matching was not. Islets were isolated as previously described.9 Briefly, preserved pancreases were perfused with cold
Liberase (Roche Diagnostics Corp, Indianapolis, Ind).10 Islets
were isolated by the automated method,11 purified
with continuous iodixanol density gradients in a Cobe 2991 cell separator
(Gambro BCT, Lakewood, Colo), and cultured free-floating in supplemented CMRL
1066 for 1 day at 37°C and 1 day at 22°C.9,12,13 Of
the 18 consecutive cadaver donor pancreases processed for this study, preparations
from 8 were transplanted (detailed donor and graft characteristics for those
8 are shown in Table 2). Islet preparations
from the remaining 10 donor pancreases were not transplanted because of inadequate
islet yield for single-donor islet transplantation. The mean islet yield of
7 of those 10 preparations was 301 428 (SD, 59 780) IEs.
After establishing access to the portal vein via minilaparotomy or percutaneous
transhepatic portal venous catheterization, we infused 7271 (SD, 1035) IEs/kg
of recipient body weight by gravity, along with heparin, 70 U/kg, on day 0
into 8 consecutive participants. Prophylactic anticoagulation was continued
with intravenous heparin (target partial thromboplastin time, 50-60 seconds)
for 48 hours, followed by enoxaparin (30 mg subcutaneously twice daily) through
Induction immunosuppression, initiated on day –2, consisted of
rabbit antithymocyte globulin (RATG) (0.5 mg/kg of recipient body weight [day
–2], 1.0 mg/kg [day –1], 1.5 mg/kg [days 0 through +2]),14 methylprednisolone (on day –2 only, 2 mg/kg),
daclizumab (5 doses of 1 mg/kg every 2 weeks starting on day 0), and etanercept
(50 mg intravenously 1 hour pretransplantation, followed by 25 mg subcutaneously
on days 3, 7, and 10). Premedication for RATG included acetaminophen and diphenhydramine
as well as pentoxifylline, which was extended through day 7 posttransplantation.
Maintenance immunosuppression was initiated with sirolimus (0.2 mg/kg starting
on day –2, followed by 0.1 mg/kg daily; target whole blood trough levels,
5-15 ng/mL, as tolerated) and reduced-dose tacrolimus (0.015 mg/kg twice daily,
starting on day 1; target whole blood trough levels, 3-6 ng/mL). At 1 month
posttransplantation, tacrolimus was gradually replaced with mycophenolate
mofetil (750-1000 mg, twice daily); tacrolimus was either discontinued or
dosed to a target trough level of less than 3 ng/mL. If target levels of sirolimus
could not be achieved or maintained, however, tacrolimus (target level, 3-6
ng/mL) was continued.
Antimicrobial and antiviral prophylaxis with imipenem, vancomycin, trimethoprim/sulfamethoxazole,
and valganciclovir was administered. Glycemic control was achieved with intravenous
insulin from day –2 to day +2 relative to transplant and with subcutaneous
insulin for at least 3 additional weeks.
Safety of the transplant protocol was assessed by monitoring the severity
and duration of procedural complications, serious infections, or islet- or
immunosuppression-related adverse events.
Self-measured blood glucose concentrations (5 or more times daily),
hypoglycemic episodes, basal C-peptide levels, and levels of glycosylated
hemoglobin (by high-performance liquid chromatography with reference range
4.3%-6.0%; monthly through day 90 posttransplantation, then quarterly) were
recorded throughout the study. Recipients also underwent oral glucose tolerance
testing (75 g, 2 hours), intravenous glucose tolerance testing, and intravenous
arginine stimulation. The acute insulin and C-peptide responses to intravenous
arginine (AIR-arginine, ACR-arginine) and to intravenous glucose (AIR-glucose,
ACR-glucose) were defined as the mean of the 3 highest poststimulus values
between 2 and 5 minutes after the start of the stimulus administration minus
the mean of 2 prestimulus values.15 Human C-peptide
levels were measured by double antibody radioimmunoassay (Diagnostic Products
Corp, Los Angeles, Calif) (interassay coefficient of variation [CV], 7.9%
at a mean C-peptide level of 0.56 ng/mL and 9.1% at a mean of 1.6 ng/mL; intra-assay
CV, 9.9% at a mean of 0.08 ng/mL and 2.1% at a mean of 0.73 ng/mL). Human
insulin levels were measured by chemiluminescent immunoassay (Immulite; Diagnostic
Products Corp) (interassay CV, 6.4% at a mean insulin level of 7.5 mU/L and
7.6% at a mean of 47.7 mU/L; intra-assay CV, 3.3% at a mean of 14.3 mU/L and
6.4% at a mean of 46.5 mU/L). Results were compared with those obtained in
control individuals without diabetes, matched for age and body mass index.
Islet graft loss was defined by the absence of basal and arginine-stimulated
Anti-GAD65 antibody, anti-ICA512 antibody, and anti-insulin antibody
titers were measured with radiobinding assays.16
Data are presented as mean (SD) unless otherwise stated. Comparisons
were performed using the 2-tailed t test. Analyses
were performed using SAS version 9.1 (SAS Institute Inc, Cary, NC); P<.05 was used to determine statistical significance.
We did not observe procedural complications; serious infections; or
serious, unexpected, and islet- or immunosuppression-related adverse events.
Expected adverse events included lymphopenia and transient neutropenia requiring
short-term (<1 week) administration of granulocyte colony-stimulating factor
in 5 recipients. Intermittent oral aphthous ulcers were observed in all participants.
No clinically significant changes in creatinine clearance or urinary albumin
excretion were observed.
All 8 recipients became insulin-independent, with glycosylated hemoglobin
levels within reference range and freedom from hypoglycemia. Of the 8 recipients,
5 have remained insulin-independent for longer than 1 year and 3 were insulin-independent
for 121, 76, and 7 days (Table 3). Metabolic
tests performed 180 or more days posttransplantation in the 5 recipients with
sustained insulin independence showed a mean AIR-glucose level of 16.7 (SD,
5.5) μU/mL (30% [SD, 10%] of controls), an ACR-glucose of 1.23 (SD, 0.46)
ng/mL (40% [SD, 15%] of controls), an AIR-arginine of 15.5 (SD, 3.7) μU/mL
(53% [SD, 13%] of controls), and an ACR-arginine of 1.07 (SD, 0.15) ng/mL
(59 [SD, 8%] of controls). For 4 of these 5 recipients, results of oral glucose
tolerance testing at 180 or more days posttransplantation revealed 2-hour
plasma glucose levels below 140 mg/dL (7.8 mmol/L). The 2-hour plasma glucose
level in the fifth recipient was 208 mg/dL (11.5 mmol/L). These 5 recipients
received daily mycophenolate mofetil doses of 1.5 to 2.0 g. They either achieved
and maintained sirolimus trough levels greater than 9 ng/mL, with tacrolimus
trough levels of 0 to less than 3 ng/mL, or achieved tacrolimus trough levels
of 3 to 6 ng/mL in the absence of target sirolimus trough levels. The 3 recipients
who resumed exogenous insulin therapy had received 1.5 g/d or more of mycophenolate
mofetil but had subtherapeutic sirolimus trough levels (<9 ng/mL) in the
absence of measurable tacrolimus trough levels (<3 ng/mL). For additional
information, see Table 3 for metabolic
monitoring and Table 4 for exposure
to immunosuppressive drugs.
Of the 3 participants with graft failure, 2 tested positive for anti-GAD65
and anti-ICA512 in the pretransplantation period. In contrast, none of the
5 who remained insulin-independent tested positive for anti-GAD65 and anti-ICA512.
Graft failure was followed by allosensitization in 2 recipients.
Our results mark a distinct advance in islet transplant efficacy. We
not only achieved insulin independence using islets from only 1 donor pancreas
(as compared with 2 to 4 in the Edmonton trial1),
we also achieved superior glycemic control (as evidenced by normal results
of oral glucose tolerance testing in 4 of 5 recipients with sustained insulin
independence) using significantly fewer islets (7271 [SD, 1035] IEs/kg vs
11 547 [SD, 1604] IEs/kg; P<.001). We had
previously achieved insulin independence in 4 of 6 participants with type
1 diabetes who received an islet mass of 10 302 (SD, 2594) IEs/kg from
1 donor pancreas.9 However, transplantation
of such an islet mass is only available from a limited number of donor pancreases
and obscures assessment of the ability of a given protocol to permit reversal
of diabetes with a lower islet mass retrievable from a larger subgroup of
Determining the reasons for our high success rates with a lower islet
mass from a single donor pancreas will have important ramifications for the
advancement of the field. Previous studies by us and others have suggested
that excluding pancreases from donors older than 50 years, limiting cold storage
to less than 8 hours and using the 2-layer preservation method, avoiding use
of Ficoll during islet purification, and culturing islets pretransplantation
could conceivably preserve the potency of transplanted islets.13,17- 22 Since
pancreas procurement, preservation, islet processing, and culture protocols
in the 2 studies were all identical, we assume that the potency was the same
and therefore interpret the high efficacy of single-donor, marginal-dose islet
transplants in our current trial as preliminary evidence of improved engraftment.
In this study, induction therapy was with RATG, combined with daclizumab and
etanercept. The resistance of islet-directed autoimmune responses to conventional
immunosuppressive drugs23- 27 and
the immediate exposure of intraportally transplanted islets to primed autoreactive,
islet beta cell–directed T cells have also provided a strong rationale
for pretransplant initiation of RATG, which is known to cause selective depletion
of activated T cells and dose-dependent depletion of resting T cells.28 Many of these effects are shared with the anti-CD3
monoclonal antibody, hOKT3γ1 (Ala-Ala), used in our previous trial.9 Thus, they may not sufficiently explain the ability
of the protocol used in our current trial to facilitate reversal of diabetes
after single-donor, marginal-dose islet transplants. Therefore, the results
are possibly related to the peritransplant administration of the soluble tumor
necrosis factor receptor etanercept. Tumor necrosis factor α is cytotoxic
to human islet beta cells.29 In murine models,
selective inhibition of tumor necrosis factor α in the peritransplant
period has promoted reversal of diabetes after marginal-mass islet transplants.30 Etanercept administration is a new addition to our
protocol and distinguishes this trial from our previous trial,9 so
it could have been a major factor allowing consistent diabetes reversal with
a low islet dose.
Moreover, replacing or minimizing tacrolimus at 1 month posttransplantation,
as we did in our current trial, may have enhanced the function of engrafted
islets. Low-dose calcineurin inhibitor therapy, with no or minimal doses of
steroids, has previously been associated with significantly reduced insulin
sensitivity and beta cell secretory reserve,31 suggesting
that even low-dose tacrolimus therapy may limit the ability of a reduced islet
mass to reverse diabetes. Maintenance immunosuppression with mycophenolate
mofetil and sirolimus, shown to be synergistic in experimental studies,32 is without diabetogenic or nephrotoxic adverse effects
and is sufficiently potent, provided induction immunosuppression is administered
and adequate sirolimus levels are achieved and maintained. In light of the
results on exposure to immunosuppressive drugs (Table 4), it seems likely that the islet graft failure experienced
by 3 recipients was caused by alloimmunity and/or recurrent autoimmunity.
The observation that 2 of 3 recipients with graft failure, but none of the
5 who remained insulin-independent, tested positive for both anti-GAD65 and
anti-ICA512 in the pretransplant period suggests a possible involvement of
autoimmunity in graft failure. More detailed studies in a larger series of
recipients will be needed to accurately ascribe islet graft loss to metabolic
or immunologic reasons.
In conclusion, potent induction immunotherapy as used in this study
may increase the ability of low-dose islet allografts to reverse diabetes
and may minimize nephrotoxicity and cardiovascular toxicity by sparing calcineurin
inhibitor dosing. While these findings may suggest a distinct advance in islet
transplantation, further study in a larger population with longer follow-up
will be critical to assess the risk-benefit ratio of this emerging therapeutic
Corresponding Author: Bernhard J. Hering,
MD, Department of Surgery, University of Minnesota, Mayo Mail Code 195, 420
Delaware St SE, Minneapolis, MN 55455 (email@example.com).
Author Contributions: Dr Hering had full access
to all of the data in the study and takes responsibility for the integrity
of the data and the accuracy of the data analyses.
Study concept and design: Hering, Kandaswamy,
Eckman, Matsumoto, Hunter, Sutherland.
Acquisition of data: Hering, Kandaswamy, Ansite,
Nakano, Sawada, Matsumoto, Ihm, Zhang, Parkey, Hunter.
Analysis and interpretation of data: Hering,
Drafting of the manuscript: Hering, Ansite,
Eckman, Nakano, Matsumoto, Ihm, Zhang.
Critical revision of the manuscript for important
intellectual content: Hering, Kandaswamy, Eckman, Sawada, Parkey, Hunter,
Statistical analysis: Sawada, Ihm.
Obtained funding: Hering.
Administrative, technical, or material support:
Hering, Kandaswamy, Ansite, Eckman, Nakano, Matsumoto, Zhang.
Study supervision: Hering, Sutherland.
All authors contributed to the preparation of the report.
Financial Disclosures: None reported.
Funding/Support: This study was supported by
grants from Roche Laboratories Inc (RO49272), the National Center for Research
Resources, National Institutes of Health (MO1-RR00400 and U42 RR016598-01),
and the Juvenile Diabetes Research Foundation (JDRF #4-1999-841). SangStat
provided rabbit anti-thymocyte globulin. Wyeth-Ayerst supplied sirolimus.
Role of the Sponsors: The sponsors had no role
in the design or conduct of the study; the collection, management, analysis,
and interpretation of the data; or the preparation, review, or approval of
Acknowledgment: We are indebted to Kathy Duderstadt,
Kathy Hodges, and Carrie Gibson for their invaluable contributions as study
nurse coordinators; to the staff members of the General Clinical Research
Center for excellent patient care; to Jeremy Oberbroeckling for his technical
expertise in the islet isolation laboratory; and to Robin Jevne, PhD, and
Dylan Zylla for data analysis and presentation. We appreciate the critical
manuscript review by Klearchos Papas, PhD. We are grateful to Richard Bergenstal,
MD, David Kendall, MD, and Lisa Fish, MD, for verifying participant eligibility;
to George Eisenbarth, MD, PhD, for measuring autoantibodies; and to Edmond
Ryan, MD, for assisting with metabolic studies. We thank LifeSource, other
organ procurement organizations, and Julianne Zabloski for their efforts in
pancreas procurement and Deborah Butterfield of Diabetes Portal for assistance
in participant recruitment. We thank Mary Knatterud, PhD, for editing the