CLINICAL AND TRANSLATIONAL RESEARCH

Insulin-Heparin Infusions Peritransplant

Substantially Improve Single-Donor Clinical Islet
Transplant Success
Angela Koh,1 Peter Senior,1 Abdul Salam,1 Tatsuya Kin,1 Sharleen Imes,1 Parastoo Dinyari,1
Andrew Malcolm,1 Christian Toso,1 Bo Nilsson,2 Olle Korsgren,2 and A. M. James Shapiro1,3

Background. Successful islet transplantation can result in insulin independence in many patients with type 1 diabetes
mellitus, but it often requires more than one islet infusion. The ability to achieve insulin independence with a single
donor is an important goal in clinical islet transplantation due to the limited organ supply.
Methods. We examined factors that may be associated with insulin independence after islet transplantation with islets
from a single donor, using univariate and multivariate analysis.
Results. Thirteen of 85 (15.3%) achieved insulin independence after single-donor islet transplantation. Using multi-
variate analysis, only the use of insulin and heparin infusions peritransplant was a significant factor associated with
insulin independence, with an adjusted odds ratio of 8.6 (95% confidence interval 2.0 –37.0). Patients who had received
insulin and heparin infusions peritransplant had greater indices of islet engraftment and a greater reduction in insulin
use (80.1%⫾4.3% vs. 54.2%⫾2.8%, P⬍0.001) even if insulin independence was not achieved.
Conclusions. Peritransplant intensive insulin and heparin enhances islet transplantation outcomes likely related in part
to mitigation of the effects of the instant blood-mediated inflammatory reaction, combined with islet rest and avoid-
ance of inflammation. It would be important to further investigate the effects of peritransplant insulin and heparin
infusions on islet engraftment.
Keywords: Islet transplantation, Single donor.
(Transplantation 2010;89: 465–471)

slet transplantation offers hope for patients with type 1 di-
I abetes mellitus who suffer from frequent hypoglycemia/
hypoglycemia unawareness and severe glycemic lability (1, 2).
Successful islet transplantation can result in insulin indepen-
dence of up to 80% at 1 year, although 5-year insulin inde-
pendence rates fall to approximately 20% with the Edmonton
Protocol. However, graft function persists in 80% of subjects
at 5 years, to a level that protects against recurrent hypogly-
cemia and improves HbA1C (2, 3).
A major limitation to the Edmonton Protocol has been
that the majority of recipients have required two or more islet
This work was supported by Juvenile Diabetes Research Foundation Islet
Transplant Center Grant, the National institute of Diabetes and Digestive
and Kidney Diseases of the National Institutes of Health (DK59101), the
Immune Tolerance Network, Capital Health and Alberta Health and
Wellness (Province Wide Services), the Roberts Family, the North Amer-
ican Foundation for the Cure of Diabetes, the Alberta Building Trades,
the Diabetes Research Institute Foundation Canada (DRIFCan), Na-
tional Medical Research Council-Singapore Totalisator Board Research
Fellowship grant (A.K.), and FS Chia PhD scholarship and fellowships
from the Swiss National Science Foundation and the Alberta Heritage
Foundation for Medical Research (C.T.).
The authors declare no conflict of interest.
1
Clinical Islet Transplant Program, University of Alberta, Edmonton, AB,
Canada.
2
Division of Clinical Immunology, Department of Oncology, Radiology and
Clinical Immunology, The Rudbeck Laboratory, Uppsala University,
Uppsala, Sweden.
infusions from multiple donors to achieve insulin indepen-
dence (2). Given the limited supply of organs, the ability to
achieve insulin independence after infusion of islets from a
single donor is an important goal that has been achieved by
Hering et al. (4) in carefully selected recipients given excellent
islet preparations, intensive peritransplant management, and
alternative induction immunosuppression. Because islets are
thrombogenic and can induce an immediate blood-mediated
inflammatory reaction, which may be deleterious to islet en-
graftment, the use of intravenous heparin in this series, along
with intravenous insulin infusion, which is postulated to have
anti-inflammatory actions, may have contributed to single
donor success.
3
Address correspondence to: A.M. James Shapiro, M.D., Ph.D., F.R.C.S.(Eng).,
F.R.C.S.C., Clinical Islet Transplant Program, University of Alberta, 2000
College Plaza, 8215-112 Street, Edmonton, AB, Canada T6G 2C8.
E-mail: amjs@islet.ca
A. Koh, P. Senior, A.M.J. Shapiro participated in performance of research,
data analysis, and writing of manuscript; A. Salam participated in data
analysis; T. Kin, S. Imes, P. Dinyari, A. Malcolm, C. Toso participated in
the performance of research and assisted in writing of manuscript; and B.
Nilsson, O. Korsgren participated in performance of research (TAT and
complement assays).
Received 5 March 2009. Revision requested 5 May 2009.
Accepted 20 September 2009.
Copyright © 2010 by Lippincott Williams & Wilkins
ISSN 0041-1337/10/8904-465
DOI: 10.1097/TP.0b013e3181c478fd

Transplantation • Volume 89, Number 4, February 27, 2010 www.transplantjournal.com | 465

466 | www.transplantjournal.com
Since 1999, 97 patients have received islet transplanta-
tion(s) at the University of Alberta, a majority of whom required
more than one islet infusion to achieve insulin independence.
Only a small proportion (15.3%) of our patients became insulin
independent after infusion of islets isolated from a single donor.
Therefore, we studied our cohort of subjects who have under-
gone clinical islet transplantation (CIT) to examine by multivar-
iate analysis factors associated with insulin independence after a
single-donor islet infusion.
METHODS
Subjects
Ninety-seven subjects have undergone CIT for type 1
diabetes between March 1999 and August 2007. All subjects
provided informed consent, and the analysis of data was ap-
proved by the University of Alberta Health Research Ethics
Board. All subjects were C-peptide negative before islet trans-
plantation. We included all patients who have received islet
preparations isolated from a single donor in this analysis. Pa-
tients who had received islet preparations derived from mul-
tiple donors were excluded from this analysis because we were
primarily interested in factors associated with single-donor
success and because islet preparations from multiple donors
could result in more human leukocyte antigen mismatches,
which may impact transplantation success. A total of 85 sub-
jects were thus included. We only analyzed the first islet trans-
plant procedure, because the presence of functioning islet
graft would contribute to insulin independence success rate
with subsequent islet infusions. Indications for CIT were fre-
quent hypoglycemia, hypoglycemia unawareness, and severe
glycemic lability. Before 2003, these parameters were assessed
clinically and by the mean amplitude of glycemic excursion
score (2). After 2003, the hypoglycemic score and glycemic
lability index were used as selection criteria for CIT (2, 5).
Posttransplant Management
The transplant procedure has been described previ-
ously (2). In addition, from 2005 onward, every single patient
received both intravenous insulin and heparin infusions
posttransplant, similar to that developed by Hering et al. (4),
as part of our routine clinical practice. Intravenous insulin (at
a minimum of 1 unit/hr) was infused together with dextrose
10% to maintain blood glucose levels between 4.1 and 7.0
mmol/L for 48 hr after transplant. After discontinuing the
intravenous insulin infusion, subjects monitored capillary
blood glucose seven times per day and administered subcu-
taneous insulin aiming for preprandial glucose 4 to 6 mmol/L
and postprandial glucose 5 to 8 mmol/L. The decision to with-
draw insulin therapy was subsequently made if adequate glyce-
mic control could be maintained while insulin independent with
no fasting glucose more than 7.0 mmol/L and no postprandial
glucose more than 10.0 mmol/L. Before 2005, Neither posttrans-
plant heparin infusions nor insulin were given unless there was
hyperglycemia (glucose ⬎11.1 mmol/L).
Intravenous heparin was infused to keep the partial
thromboplastin time between 70 and 90 sec for 48 hr after
transplant. After the heparin infusion was discontinued, all
subjects received subcutaneous low molecular weight heparin
(enoxaparin 30 mg twice daily) for 7 days and aspirin 81 mg
daily for 14 days.
Transplantation • Volume 89, Number 4, February 27, 2010
Immunosuppression
The majority of patients (n⫽71) were transplanted
under the Edmonton protocol with daclizumab induction
and maintenance immunosuppression of sirolimus and
low dose tacrolimus (2). Fourteen patients received ale-
mtuzumab (30 mg) for induction followed by either main-
tenance sirolimus or tacrolimus and cellcept therapy (and
will be reported elsewhere).
Metabolic Endpoints
The primary endpoint studied was insulin independence
after CIT for at least 4 consecutive weeks with fewer than two
episodes of fasting glucose more than 10.0 mmol/L per week
during the period of no insulin use. Subjects were requested to
self-monitor blood glucose at least four times per day.
Most patients also underwent an intravenous arginine
stimulation test at 1 month after islet transplant. The acute
insulin response to intravenous arginine (AIRa) and acute
c-peptide response to intravenous arginine (ACRa) were cal-
culated as previously described (2). The engraftment index
was calculated by dividing the ACRa at 1 month by the islet
equivalents/kg transplanted⫻104. The secretory unit of islet
transplant objects (SUITO) index was also calculated from
fasting c-peptide and fasting glucose results from posttrans-
plant days 3 to 30 (6).
Thrombin-Antithrombin Complex and
Complement
Forty subjects had measurements of thrombin-
antithrombin complex (TAT) and C3a performed pre-
transplant, within the first hour, then 3, 6, 12, and 24 hr
after islet infusion. Plasma concentrations of TAT com-
plexes and the complement activation product C3a were
quantified as previously described (7).
Statistical Analysis
Descriptive statistics were presented as mean⫾standard
error, median with range for quantitative variables, and n (%)
for qualitative variables. Independent sample t test was used
to compare the means for all quantitative variables that satis-
fied the assumption of t test between subjects who became
insulin independent versus those who were insulin depen-
dent. Nonparametric Mann-Whitney U test was used instead
of t test if the assumptions were not met. Similarly, these tests
were used for comparing all the quantitative variables be-
tween subjects who received insulin heparin versus those who
did not as appropriate. Pearson’s chi-square test was used to
compare the proportion of each qualitative variable between
two groups (insulin independent vs. insulin dependent after
single-donor islet transplant) if the sample size was sufficient
in each cell. If 25% of the cell has an expected count of less
than 5, then Fisher’s exact test was used instead of Pearson
chi-square test. The univariate tests mentioned earlier were
used to identify the factors that are associated with insulin
independence as shown in Table 1. Multivariate logistic re-
gression was used to confirm the association of significant
factors with insulin independence identified by univariate
analysis after controlling for confounders. All the variables
with P less than 0.25 in univariate analysis and those which
are clinically important were entered into the multivariate
© 2010 Lippincott Williams & Wilkins Koh et al. 467

TABLE 1. Univariate analysis of factors important for insulin independence with single-donor islet infusion
Single-donor insulin independence
Factor Yes (nⴝ13) No (nⴝ72) P
Age (yr) 48.6⫾2.5 42.9⫾1.1 0.051
Gender: male/female 7 (20%)/6 (12%) 28 (80%)/44 (88%) 0.313
Duration of diabetes (yr) 34.5⫾2.9 27.5⫾1.3 0.042
Pretransplant body mass index (kg/m2) 24.3⫾0.7 24.2⫾0.3 0.862
Pretransplant insulin dose (U/kg/d) 0.51⫾0.03 0.64⫾0.02 0.006
Pretransplant lability indexa 381.6⫾52.7 489.7⫾ 36.9 0.213
Pretransplant HYPO scorea 2 086⫾381 1 476⫾207 0.189
Pretransplant class I and II PRA
Either of one ⱕ15% 11 (15.3%) 61 (84.7%) 0.992b
Either of one ⬎15% 2 (15.4%) 11 (84.6%)
Donor age (yr) 44.2⫾3.5 44.4⫾1.4 0.943
Islet mass transplanted (IE/Kg) 5677 (3801–10,907) 5556 (3100–9852) 0.214
Cold ischemic time (hr) 6.37⫾0.86 7.16⫾0.39 0.429
Islet viability (%) 91 (75–97) 87 (70–96) 0.105
Islet purity (%) 65 (20–90) 71 (10–95) 0.194
Glucose-stimulated insulin release 2.8 (0.1–12.4) 3.0 (0.5–12.1) 0.625
(stimulation index)
Immunosuppression protocol
Edmonton 11 (15.5%) 60 (84.5%) 1.000b
Alemtuzumab 2 (14.3%) 12 (85.7%)
Trough sirolimus levelc (ug/L) 14.1⫾1.4 14.6⫾0.5 0.670
Trough tacrolimus levelc (ug/L) 6.4⫾0.7 4.8⫾0.3 0.036
Insulin heparin infusions
Yes 8 (42.1%) 11 (57.9%) ⬍0.001b
No 5 (7.6%) 61 (92.4%)
Engraftment index 1.38⫾0.20 0.86⫾0.09 0.019
SUITO index 50.5⫾5.4 29.1⫾2.1 ⬍0.001
Acute insulin response (mU/L) 18.1⫾2.2 8.0⫾1.1 ⬍0.001
Acute C-peptide response (nmol/L) 0.27⫾0.03 0.16⫾0.02 0.002
Peak TAT posttransplant 18.2 (4.8–106.0) 21.9 (4.1–118.0) 0.420
Peak C3a posttransplant 160.5 (107.3–391.8) 179.0 (91.8–2173.9) 0.319
Results are expressed as mean⫾SE, median with range and n (%).
a
See Ref. 2 for definitions of lability index and hypo score.
b
P value was calculated with the Fisher’s exact test.
c
Trough sirolimus and tacrolimus levels represent the average of values to 1 mo after transplant.
PRA, panel reactive antibodies; SUITO, secretory unit of islet transplant objects (index); TAT, Thrombin-antithrombin complex; HYPO, hypoglycemic
score; IE, islet equivalents.

logistic regression model. The use of insulin and heparin in-
fusions was the only factor independently associated with in-
sulin independence after controlling for confounders. The
final model showed only those variables that are statistically
significant and variables that are clinically important for in-
sulin independence. Kaplan-Meier estimates were made for
insulin independent survival after single-donor islet infusion
for subjects who either did or did not receive insulin and
heparin infusions peritransplant, and compared between the
two groups by the Log-rank chi-square test. A P value of less
than 0.05 was considered statistically significant, and all P
values reported were two-sided. All analyses were performed
in SAS 9.1 TS Level 1M3 for Windows.
RESULTS
Factors Influencing Insulin Independence With
Single-Donor Islet Transplant
Thirteen subjects (15.3%) achieved insulin indepen-
dence after infusion of islets from a single donor. Insulin ther-
apy was discontinued a median of 26 days (17–78 days) after
islet transplantation. The median duration of insulin inde-
pendence in this group was 18.1 months (12.1–24.9 months)
as of July 4, 2008.
Subjects who received insulin and heparin infusions
were significantly more likely to become insulin independent
(8/19 [42.1%] vs. 5/66 [7.6%], P⬍0.001 using Fisher’s exact
test, Fig.1A). Subjects who achieved insulin independence af-
ter a single-donor islet transplant had lower pretransplant
insulin requirements and had higher indices of islet engraft-
ment (engraftment index and SUITO index). Both the AIRa
and ACRa were also associated with insulin independence
(Table 1).
There were no differences in recipient body mass indi-
ces, induction protocol, donor age, islet mass transplanted, or
islet preparation characteristics between those who did or did
not achieve insulin independence.
Impact of Insulin/Heparin Infusion
Insulin independence over time was examined by
Kaplan-Meier survival analysis in subjects who became insu-
lin independent after a single-donor islet transplant and was
significantly improved in subjects who had received insulin
468 | www.transplantjournal.com Transplantation • Volume 89, Number 4, February 27, 2010

A B 1.0
No Insulin/Heparin
Insulin + Heparin
100

0.8

80
% Insulin Independent

Insulin Free Survival

0.6
60

40
0.4

20

0.2

0
Insulin + Heparin No Insulin/Heparin
0.0
0 5 10 15 20 25 30 35

Time in Months

FIGURE 1. Impact of insulin and heparin on single-donor islet transplant success. (A) Percentage of subjects insulin
independent after a single-islet infusion with or without insulin and heparin infusions peritransplant, 42.1% vs. 7.6%, P less
than 0.001 using Fisher’s exact test. (B) Survival analysis for insulin independence over time for subjects who were insulin
independent after a single-islet infusion, depending on whether insulin and heparin infusions were given peritransplant
(n⫽8, dashed line) or not (n⫽5, solid line), P⫽0.046.

TABLE 2. Univariate analysis of important factors related to insulin and heparin use
Insulin heparin
Factor Yes (nⴝ19) No (nⴝ66) P

Pretransplant insulin dose (U/kg/d) 0.54⫾0.03 0.65⫾0.02 0.010

Islet mass transplanted (IE/kg) 5584 (4098–7522) 5561 (3100–10,907) 0.813
Islet viability (%) 89 (75–96) 88 (70–97) 0.626
Islet purity (%) 63 (35–88) 73 (10–95) 0.153
Glucose-stimulated insulin release 1.7 (0.1–12.4) 3.2 (0.7–12.1) 0.009
(stimulation index)
Posttransplant insulin dosea (U/kg/d) 0.12⫾0.03 0.30⫾0.02 ⬍0.001
% Change in insulin posttransplant 80.1⫾4.3 54.2⫾2.8 ⬍0.001
Engraftment index 1.29⫾0.15 0.81⫾0.10 0.011
Acute insulin response (AIRa) (mU/L) 16.1⫾2.0 7.0⫾1.0 ⬍0.001
Acute C-peptide response (nmol/L) 0.23⫾0.02 0.16⫾0.02 0.02
SUITO index 45.5⫾5.5 28.6⫾2.0 0.003
Peak TAT 18.7 (4.8–106) 21.8 (4.1–118) 0.948
Peak C3a 184.3 (109.2–794.3) 154.9 (91.8–2173.9) 0.371
Results are expressed as mean⫾SE and n (%).
a
Posttransplant insulin dose was averaged from 2.5 to 3.5 mo after first islet infusion, or the average of 3 d before second procedure if the subject received
a second islet infusion within 3 mo of the first islet infusion.
PRA, panel reactive antibodies; SUITO, secretory unit of islet transplant objects (index); TAT, Thrombin-antithrombin complex; IE, islet equivalents.

and heparin infusions peritransplant compared with those
who did not (Log-rank statistic 3.99, P⫽0.046, Fig.1B).
Among these patients, there was no difference in glycemic
control between subjects who received insulin and heparin
infusions peritransplant and those who did not (HbA1c at 3
months: 5.9%⫾0.2% vs. 6.2%⫾0.2%, P⫽0.389).
Univariate analysis revealed a significantly greater per-
centage reduction in insulin requirement posttransplant in
subjects who received the insulin and heparin infusions than
the subjects who did not (80.1%⫾4.3% vs. 54.2%⫾2.8%,
P⬍0.001). Furthermore, the engraftment index, SUITO in-
dex, AIRa, and ACRa were all significantly higher in subjects
who received insulin and heparin infusion posttransplant
(Table 2). However, there was no difference in the islet mass
transplanted, islet purity, or viability in patients who received
insulin and heparin infusions versus those who did not. In-
© 2010 Lippincott Williams & Wilkins
deed, the stimulation index was lower in the former group
(Table 2). Thus, improvement in islet preparation character-
istics cannot explain the better outcomes in subjects who have
received insulin and heparin infusions.
For subjects who received insulin infusions peritrans-
plant, there was no difference between those who became
insulin independent and those who did not in terms of mean
glucose during the period of insulin infusion (6.7⫾0.3
mmol/L vs. 6.4⫾0.2 mmol/L, P⫽0.308), insulin infusion rate
(1.0 [0.1–1.1] vs. 1.0 [0.7–1.0] units/hr, P⫽0.589), dextrose
infusion rate (35⫾12 vs. 55⫾9 mL/hr, P⫽0.214), or the du-
ration of insulin infusion (46.3⫾5.7 hr vs. 37.8⫾5.1 hr,
P⫽0.288).
TAT and C3a
There was no significant difference in peak TAT and
C3a measurements (Table 1) or at all time points between
subjects who became insulin independent (n⫽8) and those
who did not (n⫽32). Neither was there a difference between
subjects who received insulin-heparin infusions (n⫽19) and
those who did not (n⫽21; Table 2).
Adverse Events
Periprocedural bleeds occurred in 4 of the 85 subjects
(4.7%) and was not different between subjects who received
posttransplant heparin and those who did not (1/19 [5.3%]
vs. 3/66 [4.5%], P⫽1.000 by Fisher’s exact test). Therefore,
the addition of therapeutic posttransplant heparin did not
increase the risk for posttransplant bleeding complications.
There were three episodes of branch vein thrombosis in this
series, with no difference between subjects who received post-
transplant heparin and those who did not (1/19 [5.3%] vs.
2/66 [3.0%], P⫽0.537 by Fisher’s exact test). Aggressive anti-
coagulation could further reduce this potential risk.
Multivariate Logistic Regression of Factors
Influencing Insulin Independence
On multivariate logistic regression, only the use of in-
sulin and heparin infusion remained a significant factor asso-
ciated with insulin independence, with an adjusted odd ratio
of 8.6 (95% confidence interval 2.0 –37; Table 3).
DISCUSSION
In this study, we found that following the implementa-
tion of peritransplant infusions of insulin and heparin, the
rates of single-donor insulin independence rates were signif-
icantly enhanced from 7.6% to 42.1%. The impressive odds
ratios of 8.6 on multivariate logistic regression for the effect of
insulin heparin infusions compared with the modest (and
nonsignificant) effects of traditional predictors of islet trans-
plant success (such as higher islet mass and lower pretrans-
plant insulin use) suggests that this intervention was a key
factor associated with an enhancement of single-donor insu-
lin independent rates.
Careful patient selection continues to be important.
From the univariate analysis, donor age, diabetes duration,
and insulin use pretransplant were associated with insulin
independence after a single-donor islet transplant. However,
these factors were not found to be significant in the multiple
logistic regression; the only factor that was significantly asso-
Koh et al. 469
TABLE 3. Factors associated with insulin independence
resulting from a multivariate logistic regression
Odds ratio (95%
Factor confidence interval)
Insulin heparin
No 1
Yes 8.6 (2.0–37)
Duration of diabetes (yr) 1.1 (0.97–1.1)
Pretransplant BMI 1.05 (0.8–1.4)
Pretransplant insulin U/kg/d
⬎0.6 1
ⱕ0.6 2.4 (0.5–11.4)
Pretransplant class I and II PRAa
Either of one ⬎15% 1
Either of one ⱕ15% 1.4 (0.2–11.2)
Donor age (yr) 0.99 (0.94–1.1)
Islet equivalents/kga
ⱕ5670 1
⬎5670 1.7 (0.4–7.1)
Cold ischemic time (hr) 0.92 (0.75–1.14)
a
Pretransplant Insulin use, pretransplant class I and II PRA, and islet
equivalents/kg were used as binary instead of continuous variables in the
model for ease of interpretation by the reader.
PRA, panel reactive antibodies; BMI, body mass index.
ciated with single-donor insulin independence on multiple
logistic regression was the use of insulin and heparin infu-
sions. A more formal randomized controlled, blinded trial
would be required to confirm these findings.
The early time-point metabolic data (ACRa and AIRa)
imply that the combination of intensive insulin and heparin
in the peritransplant period enhances islet engraftment (8).
This may relate in part to islet protection from IBMIR, but
may also be due to the anti-inflammatory effects of high dose
insulin and ␤-cell rest in the peritransplant period.
It has been estimated that up to 70% of the transplanted
islet mass is lost in the early posttransplant period as a result
of IBMIR (9). Islets are transplanted in heparinized medium
to prevent coagulation. The additional heparinization there-
after could further reduce IBMIR by its anticoagulation ef-
fect. Indeed, when human islets were perifused with human
blood, the addition of heparin to blood reduced cellular changes,
prevented clotting, and decreased complement activation. How-
ever, heparin by itself was not sufficient to prevent platelet and
fibrin deposition (10), leukocyte infiltration, and islet morphol-
ogy disruption (11). More recently, the use of low molecular
weight dextran sulfate was found to block IBMIR to a greater
extent, possibly by its more potent inhibition of the comple-
ment system (10). The use of islet surface heparinization was
also shown to significantly attenuate IBMIR in vitro and in
vivo, without systemic side effects (7).
Furthermore, the anti-inflammatory properties of hep-
arin have been recognized and may also contribute to im-
proved islet survival. Some of the potential mechanisms by
which heparin modulates the inflammatory response include
inhibition of complement activation, reducing reactive oxy-
gen species generation and cytokine secretion, and inhibiting
470 | www.transplantjournal.com
nuclear factor ␬B (NF-␬B) (12). Indeed, in vitro studies sug-
gest that the addition of heparin to islet supernatant limits
macrophage migration and reduces the secretion of cytokines
(tumor necrosis factor-␣ and interleukin-1␤) (13).
Hyperglycemia impairs ␤-cell function (14), reduces
␤-cell mass (15) and has been shown to reduce blood perfu-
sion and delay revascularization of islet grafts (16). ␤-cell rest
induced by insulin therapy can reverse the effects of glucotox-
icity, thereby improving ␤-cell function and reducing apo-
ptosis (17). It has been found that insulin administration to
achieve normoglycemia at the time of and after islet trans-
plantation in rodent models enhances graft survival and re-
duces islet mass required to reverse hyperglycemia (18 –20).
The mechanisms by which insulin therapy enhance islet graft
survival are not elucidated. Insulin therapy peritransplant in
mice did not reduce apoptosis or ␤-cell loss in the early period
posttransplant when compared with animals that did not re-
ceive insulin. However, the prevention of hyperglycemia in
the short term resulted in improved long-term ␤-cell mass
recovery from a combination of enhanced ␤-cell replication
and reduced apoptosis after insulin withdrawal (20). Recent
work in patients with newly diagnosed type 2 diabetes sug-
gests that aggressive short-term insulin therapy early in the
course of the disease can lead to better preservation of ␤-cell
mass in the long term compared with oral hypoglycemic
agents, although similar correction of hyperglycemia was
achieved in both groups (21). This further suggests that the
beneficial effect of insulin therapy extend beyond the correc-
tion of hyperglycemia.
Insulin has been shown to reduce plasma concentrations
of tissue factor and plasminogen early activator inhibitor-1 (22,
23), thereby exerting an antithrombotic effect. It also exerts its
antiplatelet effect by activating platelet nitric oxide synthase and
releasing nitric oxide (24). Furthermore, insulin suppresses the
generation of reactive oxygen species and the binding of
NF-␬B (25). It also increases the expression of inhibitor ␬B ␣
by mononuclear cells (26). This could mitigate the induc-
tion of apoptosis by inflammatory cytokines released from
infiltrating leukocytes, which act mainly by tumor necrosis
factor-␣ signaling pathways that activate NF-␬B regulated
apoptotic genes (26).
The fact that neither TAT nor C3a levels were lower in
subjects receiving insulin-heparin infusions implies that the
mechanisms by which this intervention improves single-donor
insulin independence rates are not solely due to mitigation of
IBMIR. Indeed, it would seem that the anti-inflammatory and
metabolic rest effects of insulin are at least as powerful as the
antithrombotic effects of heparin in protecting islets during the
engraftment phase. However, only a limited number of patients
had TAT and C3a measurements performed, and there may not
be sufficient statistical power to detect a small difference.
Further investigation of whether insulin or heparin in-
fusion influence the inflammatory milieu posttransplant
would provide greater insight into mechanisms by which
these infusions could impact transplant outcomes. The rou-
tine monitoring of C-peptide release from damaged islets at
early time points after islet infusion, coupled with TAT levels
and the measurement of markers of inflammation (such as
C-reactive protein and inflammatory cytokines), would be
valuable adjunctive mechanistic information to interpret the
clinical findings.
Transplantation • Volume 89, Number 4, February 27, 2010
Posttransplant anticoagulation did not increase the in-
cidence of periprocedural bleeds. Our routine practice of
plugging the portal tract with microfibrillar collagen (Avitene
paste; Davol, Cranston, RI) has eliminated the potential risk
of periprocedural bleeds. As an additional precaution, hepa-
rin is withheld if there is inadequate tract plugging (⬍3 cm in
length) until imaging confirms the absence of bleeding.
Although the average tacrolimus levels were higher
in insulin independent subjects, this is likely to be related
to protocol design, because there was a higher proportion
of subjects on therapeutic tacrolimus (target trough level
8 –10 ng/mL)⫹cellcept rather than sirolimus⫹low dose ta-
crolimus for maintenance immunosuppression among in-
sulin-heparin infusion recipients (results not shown).
However, the univariate and multivariate analyses did not
identify different immunosuppressive protocols as a sig-
nificant factor in single-donor islet transplant success in
our data.
A potential limitation of the current analysis is the se-
quential design, because time-bias affecting outcomes cannot
be excluded. The possibility remains that improved outcomes
were a result of increased experience with the islet transplan-
tation process. However, when we compared outcomes with
the Edmonton Protocol over time between 2000 and 2004
(before the institution of insulin and heparin infusions), we
could not find differences in insulin independence rates and
durability over time (data not shown). We also did not find
differences in islet preparation characteristics in patients who
received insulin and heparin infusions compared with those
who did not. Clearly, a randomized, controlled, blinded trial
would provide a stronger design for future studies.
Our observations confirm and extend the previous
published observations of Hering et al. (4). It was not possible
to identify the single factors or protocol variations in the pre-
vious report by Hering et al., because seven separate factors
had been modified in protocol refinement in that study com-
pared with the original Edmonton experience (27). A control
group in our series of patients transplanted at the same cen-
ter, and the use of multivariate analysis, has allowed us to
dissect the key factors associated with enhanced early single-
donor islet engraftment.
To conclude, the use of insulin and heparin infusions
peritransplant may be an important factor associated with
insulin independence posttransplant in our current study.
On the basis of our clinical observations, we cannot state cat-
egorically whether protection from IBMIR, metabolic rest or
the anti-inflammatory effects of insulin was the more domi-
nant factor leading to single-donor success and improved is-
let engraftment, but it is likely a combination of these three
factors. Mechanistic studies directly examining clotting path-
ways, the role of excessive metabolic demand, and the role of
inflammation and studies trying to dissect whether the bene-
ficial effects are due to insulin or heparin, or the combination,
would be valuable. This would help to establish a causal link
between insulin and heparin infusions with improved trans-
plant outcomes and may help us move toward increasing the
likelihood of single-donor islet transplant success.
ACKNOWLEDGMENTS
The authors thank the staff of the Clinical Islet Transplant
Program for their assistance and support.
© 2010 Lippincott Williams & Wilkins
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