Advanced Technologies and Treatments for Diabetes

Immunomodulation
Immune intervention for type 1 diabetes mellitus
J. S. Skyler
Division of Endocrinology, Diabetes and Metabolism; and Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, USA

The major form of type 1 diabetes (T1D) is characterised by immune-mediated pancreatic islet b-cell destruction, and Correspomdence to:
Jay S. Skyler,
has also been called type 1A diabetes to distinguish it from idiopathic forms of islet b-cell loss. Since the first demon- Division of Endocrinology, Diabetes, and Metabolism; and
stration of islet cell antibodies in 1974, the concept has been that this form of diabetes is autoimmune in nature. The Diabetes Research Institute, University of Miami Miller School
commonly accepted concept is that antibodies (representing the humoral arm of the immune system) do not mediate of Medicine, Miami, FL, USA
Tel.: 305-243-6146
the b-cell destruction but rather serve as markers of that destruction, while the cellular arm of the immune system,
Fax: 305-243-4484
specifically T-lymphocytes, mediate the b-cell destruction. Yet, the T-lymphocytes do not act alone. They receive help in Email: jskyler@miami.edu
initiating the response from antigen-presenting cells such as dendritic cells and macrophages, and appear to receive
help also from B-lymphocytes. In addition, the initial immune response engenders secondary and tertiary responses – Disclosures:
JSS is Chairman of Type 1 Diabetes TrialNet, a clinical trials
involving the whole immunological army – which collectively result in impairment of b-cell function, progressive network conducting multiple studies designed to interrupt the
destruction of b-cells, and consequent development of type 1A diabetes. The process is insidious and may evolve over type 1 diabetes disease process. Also, recipient of grant
many years, with the overt expression of clinical symptoms becoming apparent only when most b-cells have been support from Bayhill Therapeutics and Osiris Therapeutics. The
author serves on the Board of Directors of Amylin
destroyed. Yet, the process clearly evolves at different speeds – much more rapidly in young children, much more Pharmaceuticals, and chairs the Type 1 Diabetes Advisory Board
slowly in older individuals. And, although it has been thought that ultimately there is complete b-cell destruction, sev- for sanofi-aventis.
eral studies have now demonstrated some degree of persistent b-cell function or existence (at autopsy) in long-stand-
Endorsed by the International Conference on ATTD organized
ing T1D. A major focus of investigation in T1D is the preservation of b-cell function (and, it is hoped, of b-cells
by Kenes International.
themselves), in the expectation that continuing endogenous insulin secretion will contribute towards better glycaemic
control, reduce episodes of severe hypoglycaemia, and slow the development of complications such as retinopathy and
nephropathy. Thus, there have been many studies designed to interdict the T1D disease process, mostly by altering the
immune system, both during the stage of evolution of the disease and at the time of disease onset. This chapter of
the Yearbook of Advanced Technology and Treatments in Diabetes reviews the key papers that have appeared in this
field between July 2009 and June 2010. Articles selected were confined to studies in human beings. All immune inter-
vention studies reported in this time frame were included. In addition, the author selected other relevant articles
dealing with mechanisms, markers, triggers, and pathology of human type 1 diabetes.

1
IMMUNE INTERVENTIONS Four-year metabolic outcome of Diabetes Research Center and University
Hospital, Brussels Free University-VUB,
STUDIES IN HUMAN TYPE 1 a randomised controlled CD3- Brussels, Belgium, 2Hospital München-
DIABETES antibody trial in recent-onset Schwabing, Munich, Germany, 3Department of
type 1 diabetic patients Endocrinology, Katolieke Universiteit Leuven-
The first group of papers involves reports of
KUL, Leuven, Belgium, 4Department of
immune intervention studies in human type 1 depends on their age and Biostatistics and Medical Informatics, Brussels
diabetes, mostly in recent-onset T1D. baseline residual b-cell mass Free University-VUB, Brussels, Belgium,
5
Hôpital Erasme, Université Libre de Bruxelles-
B. Keymeulen,1 M. Walter,2 C. Mathieu,3
ULB, Brussels, Belgium, 6Department of
L. Kaufman,4 F. Gorus,1 R. Hilbrands,1
Diabetology, University Hospital Antwerp,
E. Vandemeulebroucke,1 U. Van de Velde,1
Edegem, Belgium, 7INSERM U580-IRNEM,
L. Crenier,5 C. De Block,6 S. Candon,7
Hôpital Necker, Paris, France, and 8Sir William
H. Waldmann,8 A. G. Ziegler,2 L. Chatenoud,7
Dunn School of Pathology, Oxford, UK
D. Pipeleers1
Diabetologia 2010; 53: 614–23

ª 2011 Blackwell Publishing Ltd Int J Clin Pract, February 2011, 65 (Suppl. 170), 61–70 61
62 Immunomodulation
Background: The authors previously
reported the results of their initial rando-
mised, controlled 18-month trial in which a
short course of the humanised Fc-mutated
recombinant aglycosylated anti-human CD3
antibody, ChAglyCD3 (now called ote-
lixizumab), was given for six consecutive days
shortly after diagnosis of T1D. In their earlier
report, they demonstrated preservation of b-
cell function – as measured by C-peptide –
and equivalent glycaemic control with lower
insulin doses. The current paper extends fol-
low-up to 4 years.
Methods: The original study included 80
subjects (40 treated with anti-CD3, 40 treated
with placebo), ages 12–39. Four-year follow-
up data are reported on 64 subjects (33 of the
anti-CD3 subjects, 31 of the placebo subjects).
Change in insulin dose over 48 months was
the primary end-point.
Results: Treatment with anti-CD3 delayed
the rise in insulin requirements of subjects
with recent-onset T1D and reduced the mag-
nitude of that rise over 48 months of follow-
up. Using multivariate analysis, this effect was
correlated with higher baseline b-cell function
and younger age.
Conclusions: Anti-CD3 therapy shortly
after onset of T1D has sustained effects for
48 months.
Treatment of patients with
new-onset type 1 diabetes with
a single course of anti-CD3 mAb
Teplizumab preserves insulin
production for up to 5 years
K. C. Herold,1,2 S. Gitelman,3 C. Greenbaum,6
J. Puck,3 W. Hagopian,7 P. Gottlieb,8
P. Sayre,10 P. Bianchine,9 E. Wong,4
V. Seyfert-Margolis,10 K. Bourcier,10
J. A. Bluestone,5,10 Group Immune Tolerance
Network ITN007AI Study
1 antibodies used – otelixizumab and tep-
Department of Immunobiology, Yale Uni-
versity, New Haven, CT, USA, 2Department of
Medicine, Yale University, New Haven, CT,
USA, 3Department of Pediatrics, University of
California at San Francisco, CA, USA,
4 important. Nonetheless, in the original trials
Department of Statistics, University of Califor-
nia at San Francisco, CA; PPD Inc., Wilming-
ton, NC, USA, 5Department of Medicine,
University of California at San Francisco, CA,
USA, 6Benaroya Research Institute, Seattle,
WA, USA, 7Department of Medicine, Pacific
Northwest Research Institute and University of
Washington, USA, 8Department of Medicine,
University of Colorado, Denver, CO, USA,
9
National Institute for Allergy Immunology and Cyclosporin and methotrexate
Infectious Diseases, Bethesda, MD, USA, and
10
Immune Tolerance Network, Bethesda, MD,
therapy induces remission in
USA type 1 diabetes mellitus
Clin Immunol 2009; 132: 166–73
D. O. Sobel, A. Henzke, V. Abbassi
Department of Pediatrics, Georgetown Uni-
Background: The authors previously versity Medical Center, Washington, DC, USA
reported the 1- and 2-year results of an open- Acta Diabetol 2010; 47: 243–50
label trial in which a short course of the
humanised Fc-mutated CD3-specific mono-
Background: Studies reported in the late
clonal antibody, hOKT3c1 (Ala-Ala) (now
1980s and early 1990s had shown that cyclo-
called teplizumab), was given for 14 consecu-
sporine appears to have beneficial effects in
tive days shortly after diagnosis of T1D. In
recent-onset T1D, as long as treatment is
the reports of that study, they demonstrated
maintained, but at the price of significant
preservation of b-cell function – as measured
adverse effects, particularly induction of renal
by C-peptide – and both better glycaemic
damage. The current study was designed as a
control and lower insulin doses. The current
pilot study to determine whether lower doses
paper reports on a smaller second trial that
of cyclosporine could avert the adverse effects,
had enrolment interrupted due to adverse
and if combined with methotrexate might still
events.
demonstrate the beneficial effects.
Methods: The study enrolled 10 subjects
Methods: The study was an open non-
before being halted for adverse events that
randomised pilot study, in which seven
seemed to be a consequence of higher doses
subjects with recent-onset T1D were treated
of the drug used in this trial. The subjects
with cyclosporine plus methotrexate, and
were followed, nonetheless.
compared to 10 subjects not so treated. Only
Results: At 2 years, the treated subjects
the clinical course was evaluated, and b-cell
(n = 6) had lower insulin doses than the
function was not reported.
comparison subjects (n = 4), and a trend
Results: During the 1 year of follow-up,
towards better maintained C-peptide. The C-
during which treatment was maintained in
peptide levels in the treated subjects remained
the intervention group, that group had
fairly constant between 2 and 5 years of fol-
lower insulin doses, greater likelihood of
low-up. The authors concluded that the
discontinuing insulin, and at the 12-month
higher doses resulted in more adverse events
time point lower A1c. Adverse effects were
without increased efficacy.
said to be less than in earlier cyclosporine
Conclusion: This small study suggests that
studies.
anti-CD3 therapy shortly after onset of T1D
Conclusion: The combination of low-dose
may have prolonged effects with fairly con-
cyclosporine plus methotrexate may improve
stant C-peptide levels between 2 and 5 years.
the course of T1D over 1 year.
• Comment: These two studies demonstrate
• Comment: This study attempts to take
that relatively short courses of treatment (6
advantage of an oft utilised medical principle
or 14 days) with an anti-CD3 monoclonal
that using lower doses of two drugs may
antibody can have sustained effects on b-cell
obviate the adverse effects of higher doses,
function – as measured by C-peptide – for as
whilst still achieving efficacy. The study was a
long as 4 to 5 years. Both of the anti-CD3
very small pilot study in which only seven
subjects were treated. That small number
lizumab – are now being studied in full-scale
makes it impossible to assess the risk of
phase 3 trials for potential commercialisation
adverse effects. Also, the absence of measure-
for use in recent-onset T1D. Thus, the fact
ment of b-cell function makes it impossible
that there are long-term beneficial effects is
to compare the outcome with that in other
contemporary studies. Finally, most current
with both of these antibodies, there was pro-
studies in recent-onset T1D do not intention-
gressive decline in b-cell function, suggesting
ally attempt to reduce insulin doses to achieve
that there may be a need for repeated courses
‘remission’, but rather do so only to avert
of administration. Alternatively, the efficacy
hypoglycaemia, maintaining the highest insu-
of these antibodies might be improved if used
lin dose that does not result in significant
in combination therapy with another agent
hypoglycaemia. This makes the claim of
(or agents) acting synergistically to improve
achieving ‘remission’ because insulin was able
b-cell function.
to be discontinued difficult to interpret.
ª 2011 Blackwell Publishing Ltd Int J Clin Pract, February 2011, 65 (Suppl. 170), 61–70

Failure to preserve b-cell
function with mycophenolate
mofetil and daclizumab
combined therapy in patients
with new-onset type 1 diabetes
P. A. Gottlieb,1 S. Quinlan,2 H. Krause-Stein-
rauf,2 C. J. Greenbaum,3 D. M. Wilson,4
H. Rodriguez,5 D. A. Schatz,6 A. M. Moran,7
J. M. Lachin,2 J. S. Skyler,8 Type 1 Diabetes
TrialNet MMF ⁄ DZB Study Group
1 T1D. There will no doubt be other combi-
Barbara Davis Center for Childhood Diabe-
tes, University of Colorado at Denver, Aurora,
CO, USA, 2Biostatistics Center, George Wash-
ington University, Rockville, MD, USA, 3Bena-
roya Research Institute, Diabetes Clinical
Research, Seattle, WA, USA, 4Pediatric Endo-
crinology Department, Stanford University,
Stanford, CA, USA, 5Department of Pediatrics,
Indiana University, Indianapolis, IN, USA,
6 being compared to its use in combination.
Department of Pediatrics, University of
Florida, Gainesville, FL, USA, 7Department of
Pediatrics, University of Minnesota,
Minneapolis, MN, USA, and 8Diabetes
Research Institute, University of Miami, Miami,
FL, USA
Diabetes Care 2010; 33: 826–32
Background: The authors evaluated the
use of mycophenolate mofetil (MMF), either
alone or in combination with daclizumab
(DZB), in recent-onset T1D. MMF has
potent cytostatic effects on lymphocytes, and
has been effective in other autoimmune dis-
eases and in diabetic animal models. DZB is
a humanised monoclonal antibody that
binds to CD25, the a-subunit of the inter-
leukin-2 (IL-2) receptor expressed on the
surface of activated lymphocytes, and has
been effective in other autoimmune diseases
as well as in islet transplant protocols. The
combination of MMF and DZB had a bene-
ficial synergistic effect in diabetic animal
models.
Methods: The study included 126 subjects,
ages 8–45, randomised to MMF alone, MMF
plus DZB, or placebo. Two DZB (or placebo)
infusions were administered – one initially
and one 2 weeks later. MMF (or placebo) was
given continuously, with a planned study
end-point at 2 years.
Results: The Data Safety and Monitoring
Board halted the study early due to the pro-
jected futility of achieving a beneficial result.
There was a similar decline in b-cell func-
tion in all three groups – MMF alone, MMF
plus DZB, and placebo. In addition, A1c and
insulin dosage were similar in all three
groups.
Conclusions: MMF, either alone or in
combination with DZB, failed to prevent
ª 2011 Blackwell Publishing Ltd Int J Clin Pract, February 2011, 65 (Suppl. 170), 61–70
decline in b-cell function in recent-onset
T1D.
• Comment: This large, well-powered study
failed to demonstrate preservation of b-cell
function in recent-onset T1D. Although
many interventions have shown benefit in
recent-onset T1D, this study reminds us
that not all interventions which show prom-
ise in animal models will work in human
beings. The study does represent one of the
first, if not the first, fully powered study
using combination therapy in recent-onset
nation therapy trials. In designing them,
one might note one feature of this study –
half of the treated subjects received an infu-
sion of DZB, and half of the placebo sub-
jects received an infusion of DZB. This
maintained masking, while not requiring all
subjects to receive infusions. This is a
design that can be used if a single agent is
Another approach would be a factorial
design. Attention to design of combination
studies will be an important consideration
in future T1D trials.
Rituximab, B-lymphocyte
depletion, and preservation of
b-cell function
M. D. Pescovitz,1 C. J. Greenbaum,2
H. Krause-Steinrauf,3 D. J. Becker,4
S. E. Gitelman,5 R. Goland,6 P. A. Gottlieb,7
J. B. Marks,8 P. F. McGee,3 A. M. Moran,9
P. Raskin,10 H. Rodriguez,1 D. A. Schatz,11
D. Wherrett,12 D. M. Wilson,13 J. M. Lachin,3
J. S. Skyler,8 Type 1 Diabetes TrialNet
Anti-CD20 Study Group
1
Indiana University School of Medicine,
Indianapolis, IN, USA, 2Benaroya Research
Institute, Seattle, WA, USA, 3George Washing-
ton University Biostatistics Center, Rockville,
MD, USA, 4University of Pittsburgh, Pitts-
burgh, PA, USA, 5University of California, San
Francisco, CA, USA, 6Columbia University,
New York, NY, USA, 7University of Colorado
Barbara Davis Center for Childhood Diabetes,
Aurora, CO, USA, 8University of Miami Dia-
betes Institute, Miami, FL, USA, 9University of
Minnesota, Minneapolis, MN, USA, 10Univer-
sity of Texas Southwestern Medical School, Dal-
las, TX, USA, 11University of Florida,
Gainesville, FL, USA, 12Hospital for Sick Chil-
dren, University of Toronto, Toronto, ON,
Canada, and 13Stanford University, Stanford,
CA, USA
N Engl J Med 2009; 361: 2143–52
Background: There is growing evidence
that B-lymphocytes play a role in many
Immunomodulation 63
T-lymphocyte mediated diseases, by serving as
antigen-presenting cells or generating cryptic
peptides to which T-lymphocytes are not tol-
erant. This study used the anti-CD20 anti-
body, rituximab, to selectively deplete
B-lymphocytes in recent-onset T1D.
Methods: The study randomised 87 sub-
jects (57 assigned to anti-CD20, 30 assigned
to placebo), ages 8–45. Intervention consisted
of four weekly infusions. The primary end-
point was b-cell function – as measured by
C-peptide – at 1 year, with 78 subjects
included in the analysis.
Results: At 1 year, the mean level of C-
peptide was significantly higher in the ritux-
imab group than in the placebo group, and
declined at a slower rate. The rituximab
group also had significantly lower A1c levels
and required less insulin. Adverse effects were
minimal and mostly occurred during the first
rituximab infusion.
Conclusions: The anti-CD20 antibody rit-
uximab showed beneficial effects on b-cell
function in recent-onset T1D, suggesting a
potential role for B-lymphocytes in T1D path-
ogenesis.
• Comment: This study demonstrates that a
relatively short course of treatment (weekly
infusions over 4 weeks) with an anti-CD20
monoclonal antibody can have beneficial
effects on b-cell function – as measured by
C-peptide – at 1 year. Nonetheless, there is
progressive decline in b-cell function, as in
virtually all intervention trials in recent-onset
T1D, again suggesting that there may be a
need for repeated courses of administration,
or, alternatively, for combination therapy with
another agent. An important feature of this
study is the demonstration that B-lympho-
cytes contribute to the pathogenesis of T1D.
This may have important implications for the
design of combination therapy regimens for
evaluation in T1D.
Six months of diazoxide
treatment at bedtime in newly
diagnosed subjects with type 1
diabetes does not influence
parameters of b-cell function
and autoimmunity but improves
glycaemic control
M. A. Radtke,1,2 I. Nermoen,3 M. Kollind,4
S. Skeie,5 J. I. Sørheim,6 J. Svartberg,7,8
I. Hals,1 T. Moen,9 G. H. Dørflinger,1
V. Grill1,2
1
Department of Cancer Research and Molec-
ular Medicine, Norwegian University of Science
and Technology, Trondheim, Norway, 2Depart-
ment of Endocrinology, St Olavs Hospital ⁄
64 Immunomodulation
University Hospital of Trondheim, Trondheim,
Norway, 3Department of Endocrinology, Akers-
hus University Hospital, Lørenskog, Norway,
4
Endocrinology Unit, Department of Internal
Medicine, Levanger Hospital, Levanger, Nor-
way, 5Section of Endocrinology, Division of
Medicine, Stavanger University Hospital,
Stavanger, Norway, 6Section of Endocrinology,
Department of Medicine, Haukeland University
Hospital, Bergen, Norway, 7Section of Endocri-
nology, Division of Internal Medicine, Univer-
sity Hospital of North Norway, Tromsø,
Norway, 8Institute of Clinical Medicine,
University of Tromsø, Tromsø, Norway, and
9
Department of Laboratory Medicine,
Children’s and Women’s Health, Faculty of
Medicine, Norwegian University of Science and
Technology, Trondheim, Norway
Diabetes Care 2010; 33: 589–94
Background: Diazoxide provides b-cell rest
by reversibly suppressing glucose-induced
insulin secretion. Previous studies have shown
that diazoxide may preserve b-cell function in
recent-onset T1D, but with frequent adverse
effects consisting of lanugo hair growth,
oedema and hypotension. The current study
was designed to test whether a lower dosage
of diazoxide would eliminate side effects and
still exert beneficial effects.
Methods: The study randomised 41 sub-
jects (22 to diazoxide, 19 to placebo), ages
18–40, with recent-onset T1D. Subjects
received 6 months of treatment with placebo
or 100 mg diazoxide at bedtime, and were
followed for a subsequent 6 months. Out-
come measures were C-peptide (fasting and
glucagon stimulated), A1c and insulin dose.
Results: A1c levels at both 6 months and
1 year were lower in the diazoxide group, but
there was no difference in insulin dose,
C-peptide levels (either actual levels or
C-peptide ⁄ glucose ratios), or in proportion of
Tregs (regulatory T-lymphocytes). There was
more weight gain in the placebo group.
Insulin sensitivity, as assessed by HOMA-S%
(homeostatic model assessment for insulin
sensitivity), increased in the diazoxide group
but remained stable in the placebo group.
Conclusions: Low dose diazoxide averted
adverse effects but failed to improve outcome
in recent-onset T1D.
• Comment: Diazoxide, at higher doses, has
previously been shown to have beneficial
effects in preserving b-cell function in recent-
onset T1D. However, its use has been limited
due to intolerability from side effects. In the
current randomised controlled trial, a lower
dose was better tolerated but failed to demon-
strate preservation of b-cell function. Interest-
ingly, there were differences in A1c, which
was lower in the diazoxide group. This may
have been a consequence of greater insulin
sensitivity and less weight gain in the diazox-
ide group. The authors propose that low-dose
diazoxide could serve as a component in a
combination therapy regimen. Indeed, that
may be worth exploring.
No effect of the altered peptide
ligand NBI-6024 on b-cell
residual function and insulin
needs in new-onset type 1
diabetes
M. Walter,1 A. Philotheou,2 F. Bonnici,2
A. G. Ziegler,1 R. Jimenez,3 NBI-6024 Study
Group
1
Diabetes Research Institute, Forschergruppe
Diabetes e.V., Munich, Germany, 2Diabetes
Clinical Trials Unit, Faculty of Health Sciences,
University of Cape Town, Cape Town, South
Africa, and 3Clinical Development, Neurocrine
Biosciences, San Diego, CA, USA
Diabetes Care 2009; 32: 2036–40
Background: Insulin B (9–23) peptide is
thought to be an important antigen of T-lym-
phocytes in autoimmune diabetes in animals
and in human beings. It is possible to target
specific autoreactive T-lymphocyte activation
using a soluble altered peptide ligand (APL)
to block or change that activation. NBI-6024
is an APL that contains two amino acid
substitutions in the (9–23) sequence of the
B-chain of insulin: alanine is substituted for
tyrosine at position 16, which is a key contact
site at the T-lymphocyte receptor; and alanine
also is substituted for cysteine at position 19.
The resulting APL (Ala16,19) (also known as
NBI-6024) does not activate insulin B (9–23)
reactive murine or human T-lymphocytes.
Methods: The study randomised 188 sub-
jects, ages 10–35, to three different doses of
the APL (50 subjects to 0.1 mg, 48 subjects to
0.5 mg, 43 subjects to 1.0 mg) or to placebo
(47 subjects). Intervention consisted of subcu-
taneous injections at randomisation, 2 weeks,
4 weeks and monthly until 24 months. The
primary end-point was b-cell function – as
measured by C-peptide – at 2 years.
Results: At 2 years, the mean peak C-pep-
tide concentration showed no significant dif-
ference between any of the treated groups and
the placebo group. C-peptide declined linearly
in all groups.
Conclusions: The insulin B (9–23) APL
(Ala16,19) (NBI-6024) failed to show benefit in
any of three doses tested in a large dose-
ranging study.
• Comment: Antigen-specific therapy is
thought to be a highly desirable strategy to
ª 2011 Blackwell Publishing Ltd Int J Clin Pract, February 2011, 65 (Suppl. 170), 61–70
interrupt the immune processes that result in
T1D. Such therapies are generally quite safe,
are specific for T1D, and are not expected to
alter generalised immune responses. The insu-
lin B (9-23) APL (Ala16,19) (NBI-6024) was
shown to alter the course of diabetes in the
NOD mouse and prevented activation of both
murine and human T-lymphocytes in the lab-
oratory. Preliminary studies demonstrated
that there were no safety issues. Thus, the
current dose-ranging study was awaited with
considerable enthusiasm. The study was well
designed and carefully conducted. Unfortu-
nately, the APL had no impact on b-cell func-
tion. A critical question is whether an
adequate dose was given. It is impossible to
measure blood levels of the APL or a marker
that would indicate that a given dose was
altering some biological response. This is a
vexing question that has hampered other
studies with antigen-specific interventions.
Extrapolation of effective doses from mice to
human beings is always a game of roulette. It
is unlikely that this particular APL will be
further pursued. That is unfortunate.
Autoantigen-specific regulatory
T cells induced in patients with
type 1 diabetes mellitus by
insulin B-chain immunotherapy
T. Orban,1 K. Farkas,1 H. Jalahej,1 J. Kis,1,2
A. Treszl,1,3 B. Falk,4 H. Reijonen,4
J. Wolfsdorf,5 A. Ricker,1 J. B. Matthews,6
N. Tchao,6 P. Sayre,6 P. Bianchine7
1
Joslin Diabetes Center, Boston, MA, USA,
2
Polyclinic of the Hospitaller Brothers, Buda-
pest, Hungary, 3Zentrum für Experimentelle
Medizin Institut für Medizinische Biometrie
und Epidemiologie, Hamburg, Germany,
4
Benaroya Research Institute at Virginia
Mason, Seattle, WA, USA, 5Children’s Hospital
Boston, MA, USA, 6Immune Tolerance Net-
work, University of California, San Francisco,
CA, USA, and 7National Institute of Allergy
and Infectious Diseases, Bethesda, MD, USA
J Autoimmun 2010; 34: 408–15
Background: Insulin B-chain contains the
insulin B (9–23) peptide, which is a major
epitope recognised by the immune system
and thought to be a diabetes-specific antigen.
The insulin B-chain fragment is metabolically
inactive. Incomplete Freund’s adjuvant (IFA)
has been used in a number of vaccines as a
delivery system that promotes regulatory
immune responses. The current report
describes a pilot study testing insulin B-chain
administered with IFA in T1D.
Methods: The study randomised 12 sub-
jects (six to 2 mg insulin B-chain in IFA, six

to placebo, IFA alone), ages 18–35, with
recent-onset T1D. Subjects received a single
intramuscular injection of test substance, and
were followed for 2 years. Outcome measures
were b-cell function – as measured by C-pep-
tide – and insulin-specific humoral and
T-lymphocyte responses.
Results: There was no statistical difference
in b-cell function, measured every 6 months,
between arms. All patients in the experimen-
tal group who received insulin B-chain, but
none who received placebo, developed robust
insulin-specific humoral and T-lymphocyte
responses, including insulin B-chain-specific
CD4+ T-lymphocytes that were cloned and
showed characteristics of regulatory T-lym-
phocytes.
Conclusions: Insulin B-chain, given
together with IFA, may beneficially impact
immune response in T1D.
• Comment: This is another test of an anti-
gen-specific therapy in T1D. It was only a
very small pilot study involving 12 subjects,
six who received a single injection of insulin
B-chain in IFA, and six who received IFA
alone. Although claimed to be safe, it is
impossible to assess safety in such a small
study. Also impossible to assess in a small
study is b-cell function, which here was care-
fully measured with meal tolerance tests.
Although there was no statistical difference
between groups, the C-peptide levels were
persistently higher in the placebo group. Had
the results been reversed, it would not be sur-
prising for the authors to claim a beneficial
‘trend’. This illustrates the difficulty in inter-
pretation of small pilot studies. Intriguingly,
the cloned T-lymphocytes from the experi-
mental group had characteristics of regulatory
T-lymphocytes. But, what if there was also
activation of effector T-lymphocytes that was
not demonstrated. Could that have resulted
in a subtle greater loss of b-cell function?
Despite these questions, the results are suffi-
ciently provocative to support further studies
with insulin B-chain. Questions that can be
raised, however, are whether 2 mg is the right
dose and whether a single intramuscular vac-
cination is the right number of vaccinations.
Only full-scale dose-ranging studies can
answer these questions. These preliminary
data suggest that such studies are warranted.
ª 2011 Blackwell Publishing Ltd Int J Clin Pract, February 2011, 65 (Suppl. 170), 61–70
No effect of the 1a,25-
dihydroxyvitamin D3 on b-cell
residual function and insulin
requirement in adults with
new-onset type 1 diabetes
M. Walter,1 T. Kaupper,1 K. Adler,1 J. Foersch,1
E. Bonifacio,2 A. G. Ziegler1
1 Pediatrics, Tampere University Hospital,
Diabetes Research Institute, Forschergruppe
Diabetes e.V., Munich, Germany, and
2 Adolescents and Folkhälsan Research Center,
Deutsche Forschungsgemeinschaft Center for
Regenerative Therapies, Dresden, Germany
Diabetes Care 2010; 33: 1443–8
Background: Mechanistic studies show that
1a,25-dihydroxyvitamin D3 [1,25(OH)2D3]
modulates dendritic cell maturation in vitro
and in vivo and facilitates a shift from a Th1
to a Th2 immune response. Studies in the
NOD mouse show that 1,25(OH)2D3 reduces
the incidence of insulitis and diabetes. Thus,
the current study was conducted to determine
whether 1,25(OH)2D3 is safe and improves b-
cell function in patients with recent-onset
T1D.
Methods: Two studies were conducted – a
safety study in 25 subjects, all treated with
1,25(OH)2D3 and followed for 18 months,
and an efficacy study in which 40 subjects,
ages 18–39, with recent-onset T1D, were
randomised either to 1,25(OH)2D3 (n = 22)
or to placebo (n = 18). The primary end-
point was b-cell function – as measured by
C-peptide – at 18 months. Careful safety
assessments were performed.
Results: There was a similar decline in b-
cell function in both groups – 1,25(OH)2D3
and placebo. In addition, A1c and insulin
dosage were similar in both groups. The
treatment was safe.
Conclusion: 1,25(OH)2D3 was shown to be
safe, but did not have a beneficial effect in
adults with recent-onset T1D.
Maternal intake of vitamin D
during pregnancy and risk of
advanced b-cell autoimmunity
and type 1 diabetes in offspring
L. Marjamäki,1 S. Niinistö,2,3 M. G. Kenward,4
L. Uusitalo,1,2 U. Uusitalo,2 M. L. Ovaskainen,2
C. Kronberg-Kippilä,2 O. Simell,5 R. Veijola,6
J. Ilonen,7,8 M. Knip,9,10 S. M. Virtanen1,2,9,11
1
Tampere School of Public Health, Univer-
sity of Tampere, Tampere, Finland, 2Depart-
ment of Lifestyle and Inclusion, National
Institute for Health and Welfare, Helsinki,
Finland, 3Department of Public Health, Uni-
versity of Helsinki, Helsinki, Finland, 4Depart-
ment of Epidemiology and Population Health,
Immunomodulation 65
Medical Statistics Unit, London School of
Hygiene and Tropical Medicine, London, UK,
5
Department of Pediatrics, University of
Turku, Turku, Finland, 6Department of
Pediatrics, University of Oulu, Oulu, Finland,
7
Immunogenetics Laboratory, University of
Turku, Turku, Finland, 8Department of
Clinical Microbiology, University of Eastern
Finland, Kuopio, Finland, 9Department of
Tampere, Finland, 10Hospital for Children and
University of Helsinki, Helsinki, Finland, and
11
Research Unit, Tampere University Hospital,
Tampere, Finland
Diabetologia 2010; 53: 1599–607
Background: Maternal intake of vitamin D
from food or use of vitamin-D-containing
supplements during pregnancy was weakly
associated with decreased risk of early b-cell
autoimmunity in two cohort studies and with
clinical T1D in one case–control study.
Methods: Mothers of 3723 infants born
between 1997 and 2002 completed a 181-item
food frequency questionnaire, including ques-
tions on dietary supplements. Offspring were
observed at 3–12 month intervals for appear-
ance of T1D-associated autoantibodies and
for the development of T1D.
Results: Maternal mean daily intake of
vitamin D was 5.1 lg from food and 1.3 lg
from supplements. Maternal intake of vitamin
D, either from food or from supplements,
was not associated with the risk of b-cell au-
toimmunity or with the risk of T1D in off-
spring.
Conclusions: Maternal intake of vitamin D
did not appear to influence the T1D process
in offspring.
• Comment: For some time, the potential
role of vitamin D as a preventative interven-
tion for T1D has been raised. There are
clearly effects of vitamin D in vitro and some
beneficial effects in animal models, particu-
larly in vitamin-D-deficient animals. In
human beings, cohort studies and meta-analy-
ses of studies done in infants have suggested
potential benefit. Thus, the use of vitamin D
remains an attractive hypothesis for preven-
tion of T1D, the best case being for its use in
infants. In the two studies described above,
there was no support for this hypothesis. The
intervention study with 1,25(OH)2D3 was
conducted in adults after the development of
T1D. Although a small study, it failed to
show a beneficial effect. Intervention with
vitamin D at that stage might not be expected
to have beneficial effects. The Finnish study
evaluated maternal intake of vitamin D and
whether it correlated with b-cell autoimmuni-
ty or with the risk of T1D in offspring. There
66 Immunomodulation
are two important points to note, however.
First, this was not an intervention study,
which might better have permitted assessment
of the impact of vitamin D. Second, although
the food questionnaire has been validated, it
is difficult to assess how well it reflected vita-
min D intake, and whether the range of vita-
min D intake was sufficient to allow
assessment of its impact.
Regeneration of insulin
production by autologous bone
marrow blood
autotransplantation in patients
with type 1 diabetes
E. Esmatjes,1,6,7 X. Montaña,2 M. I. Real,2
J. Blanco,1 I. Conget,1,6,7 R. Casamitjana,3,6,7
M. Rovira,4,6 R. Gomis,1,6,7 P. Marin5,6
1
Diabetes Unit, Hospital Clinic Universitari,
Barcelona, Spain, 2Interventional Angioradiolo-
gy Unit, Hospital Clinic Universitari, Barce-
lona, Spain, 3Biochemistry and Molecular
Genetics Department, Hospital Clinic Universi-
tari, Barcelona, Spain, 4Bone Marrow Trans-
plant Unit, Hospital Clinic Universitari,
Barcelona, Spain, 5Hemotherapy Unit, Hospital
Clinic Universitari, Barcelona, Spain, 6Institut
d’Investigacions Biomèdiques August Pii Sunyer
(IDIBAPS), Barcelona, Spain, and 7CIBER de
Diabetes y Enfermedades Metabólicas Asociadas
(CIBERdem), Barcelona, Spain
Diabetologia 2010; 53: 786–9
Background: Some experimental reports
suggest that the bone marrow harbours cells
capable of differentiation into b-cells, or as a
facilitator of b-cell regeneration, or as an
immune modulator, and thus use of bone
marrow cells has been proposed as treatment
for T1D.
Methods: A pilot study was conducted in
three subjects with established T1D and
absent b-cell function. Autologous bone mar-
row was harvested and subsequently injected
via selective arteriography as an intra-
pancreatic infusion.
Results: Data were available for two
of the three subjects. There was no evidence
of b-cell function during 6 months of
follow-up.
Conclusions: Intrapancreatic autologous
bone marrow infusion had no impact in two
subjects with long-standing T1D.
Autologous umbilical cord blood or more probably to the effects of the pro-
found immunosuppression given at the out-
transfusion in very young set with AHSCT serving to rescue the patient
children with type 1 diabetes from death from destruction of their
immune system. Thus, in human beings there
M. J. Haller,1 C. H. Wasserfall,2
is no clear evidence yet of beneficial effects
K. M. McGrail,2 M. Cintron,1 T. M. Brusko,3
of any cellular treatment per se in T1D (other
J. R. Wingard,4 S. S. Kelly,5 J. J. Shuster,6
than pancreatic or islet transplantation). The
M. A. Atkinson,2 D. A. Schatz1
1 two studies described above also failed to
Department of Pediatrics, University of
show beneficial effects. These negative out-
Florida, Gainesville, FL, USA, 2Department of
comes, however, should not be cited as a rea-
Pathology, University of Florida, Gainesville,
son to halt research of cellular therapies. In
FL, USA, 3Diabetes Center, University of Cali-
animal models and laboratory experiments,
fornia at San Francisco, San Francisco, CA,
USA, 4Department of Medicine, University of
there is clearly much promise for cellular
based therapies. Eventually, success will be
Florida, Gainesville, FL, USA, 5Department of
achieved.
Pediatrics, University of Texas, Houston, TX,
USA, and 6Department of Epidemiology and
Health Policy Research and the General Clini- Developing combination
cal Research Center, University of Florida,
immunotherapies for type 1
Gainesville, FL, USA
Diabetes Care 2009; 32: 2041–6 diabetes: recommendations
from the ITN-JDRF Type 1
Background: Umbilical cord blood con-
Diabetes Combination Therapy
tains a population of immature unprimed
functional regulatory T-lymphocytes. These Assessment Group
cells could, in theory, limit inflammatory J. B. Matthews,1 T. P. Staeva,2 P. L. Bernstein,1
cytokine responses and anergize effector M. Peakman,4,5 M. von Herrath,3 and
T-lymphocytes, which are thought to mediate ITN-JDRF Type 1 Diabetes Combination
cellular autoimmune processes. Therapy Assessment Group
Methods: Children aged > 1 year who 1
Immune Tolerance Network, San Francisco,
developed T1D and had banked umbilical CA, USA, 2Juvenile Diabetes Research Founda-
cord blood at an approved centre were tion International, New York, NY, USA, 3La
recruited into this study. Fifteen subjects Jolla Institute for Allergy and Immunology, La
enrolled in an open-label phase 1 study using Jolla, CA, USA, 4Department of Immunology,
autologous umbilical cord blood infusion. King’s College London, UK, and 5NIHR Bio-
They were followed for 1 year. medical Research Center at Guy’s and St Tho-
Results: There was no evidence at 1 year of mas’ NHS Foundation Trust and King’s
maintenance of preservation of b-cell function College London, London, UK
– as measured by C-peptide – while insulin Clin Exp Immunol 2010; 160: 176–84
doses increased over time. No changes were
observed in autoantibody titres, regulatory T-
• Comment: This paper describes the results
lymphocyte numbers, or other immune
of a workshop conducted by the Immune
parameters. There were no significant adverse
Tolerance Network (ITN) and the Juvenile
events.
Diabetes Research Foundation (JDRF) to
Conclusions: Autologous umbilical cord
review strategies for developing combination
blood infusion was safe, but had no beneficial
therapies for T1D. The advantages of a com-
effect in children with T1D.
bination strategy include the ability to mini-
• Comment: Many investigators believe that
mise toxicities and realise synergies to
cellular therapies for T1D hold substantial
enhance and prolong efficacy. The paper
promise. A study from Brazil (1) discussed in
notes that a clear framework must be devel-
last year’s Yearbook evaluated the effects of
oped that specifies the type and quality of
non-myeloablative autologous haematopoietic
preclinical data, including which animal mod-
stem cell transplantation (AHSCT) in recent-
els are acceptable, as well as toxicology and
onset T1D. In that study, 20 of 23 subjects
pharmacodynamic data expectations that will
became insulin free, some for protracted
be required for a combination to meet
periods of time. Although many have attrib-
acceptable safety standards to justify human
uted the results to the infused cells, it is not
trials. It points out that there is a major need
possible to distinguish whether the putative
for surrogate end-points based upon benefi-
effects of AHSCT were due to immune
cial immunological responses that manifest
reconstitution or otherwise altering the
soon after treatment, which would facilitate
immune-mediated b-cell destruction that
rapid assessment and prioritisation of any
eventuates in T1D, to regeneration of b-cells,
ª 2011 Blackwell Publishing Ltd Int J Clin Pract, February 2011, 65 (Suppl. 170), 61–70

individual T1D therapeutic and would be par-
ticularly important for selection of potential
combinations. The paper notes that biomar-
kers could speed clinical assessments by pro-
viding surrogate end-points, permit
stratification of analysis of trial data, and
facilitate personalised medicine by informing
treatment decisions. The authors gave priority
rankings of combination immune-based ther-
apies for T1D, favouring approved agents,
favouring combination of immune modula-
tors with antigen-specific therapies, and limit-
ing initial proposals to two agents in
combination. The paper is cognizant of regu-
latory hurdles and the need for negotiations
with commercial sponsors. It is a solid paper
worth reading by anyone conducting investi-
gations in the field.
MECHANISMS, MARKERS,
TRIGGERS, PATHOLOGY
The second group of papers includes reports
of studies in human T1D that focus on mech-
anisms, markers, triggers and pathology.
Progression to diabetes in
relatives of type 1 diabetic
patients: mechanisms and mode
of onset
E. Ferrannini,1 A. Mari,2 V. Nofrate,2
J. M. Sosenko,3 J. S. Skyler,3 DPT-1 Study
Group
1
Department of Medicine, University of Pisa
School of Medicine, Pisa, Italy, 2C.N.R. Insti-
tute of Biomedical Engineering, Padua, Italy,
and 3Division of Endocrinology, University of
Miami, Miami, FL, USA
Diabetes 2010; 59: 679–85
Background: The Diabetes Prevention Trial
– Type 1 (DPT-1) studied relatives of T1D
patients at enhanced risk of developing T1D.
This study explored the mode of onset of hy-
perglycaemia and how insulin sensitivity and
b-cell function contribute to the progression
of the disease.
Methods: The study analysed results from
328 non-diabetic relatives from the control
arms of the DPT-1 studies. Subjects had
sequential oral glucose tolerance tests per-
formed at baseline, every 6 months, and an
average of 2.7 years later, when 115 subjects
became diabetic. b-cell glucose sensitivity
(slope of the insulin secretion ⁄ plasma glucose
dose–response function) and insulin sensitiv-
ity were obtained by mathematical modelling
of the oral glucose tolerance test glucose ⁄ C-
peptide responses.
ª 2011 Blackwell Publishing Ltd Int J Clin Pract, February 2011, 65 (Suppl. 170), 61–70
Results: Progressors to T1D and non-pro-
gressors had similar baseline insulin sensitiv-
ity, fasting insulin secretion and total post-
glucose insulin output. In contrast, b-cell glu-
cose sensitivity was impaired at baseline and
predicted development of diabetes. Moreover,
glucose sensitivity began to decline signifi-
cantly before any changes in plasma glucose,
while insulin secretion and insulin sensitivity
remained stable.
Conclusions: A defect in b-cell glucose
sensitivity is detectable in at-risk subjects
years before diagnosis, and anticipates plasma
glucose increments.
• Comment: The failure of the b-cell to
adequately respond to glucose – b-cell
‘blindness’ to glucose – is a characteristic
pathophysiological abnormality in both T1D
and type 2 diabetes. It results in a lack of
adequate early insulin secretory response
that eventuates in significant postprandial
hyperglycaemia. Here it is demonstrated that
this defect is present very early in the dis-
ease process, before other metabolic abnor-
malities. Teleologically, it may be a way the
injured b-cell manifests itself. Presumably,
in T1D, the b-cell has been injured via
immunological attack. Indeed, if humoral
anti-islet antibodies are a marker of the
immune attack, all of the subjects included
in DPT-1 already had that under way, as
an entry criterion was presence of antibod-
ies. It would be interesting to do similar
modelling in studies involving genetically
high risk individuals who do not yet have
anti-islet antibodies, as it is possible that a
defect in b-cell glucose sensitivity is geneti-
cally determined and may indicate that a
particular subject is more vulnerable to
immune-mediated damage.
Pancreatic islet autoantibodies
as predictors of type 1 diabetes
in the Diabetes Prevention
Trial – Type 1
T. Orban,1 J. M. Sosenko,2 D. Cuthbertson,3
J. P. Krischer,4 J. S. Skyler,2 R. Jackson,1
L. Yu,5 J. P. Palmer,6 D. Schatz,7
G. Eisenbarth,5 Diabetes Prevention Trial –
Type 1 Study Group
1
Joslin Diabetes Center, Boston, MA, USA,
2
Division of Endocrinology, University of
Miami, Miami, FL, USA, 3Pediatrics Epidemi-
ology Center, University of South Florida,
Tampa, FL, USA, 4Division of Informatics and
Biostatistics, University of South Florida,
Tampa, FL, USA, 5Barbara Davis Center for
Childhood Diabetes, Denver, CO, USA,
Immunomodulation 67
6
Division of Endocrinology ⁄ Metabolism, Uni-
versity of Washington, Seattle, WA, USA,
and 7Division of Endocrinology, University of
Florida, Gainesville, FL, USA
Diabetes Care 2009; 32: 2269–74
Background: In another report from the
DPT-1 studies, the prediction of T1D by spe-
cific types of pancreatic islet autoantibodies,
either alone or in combination, was explored.
Methods: The study included two cohorts
from DPT-1, both derived from initial screen-
ing for islet cell autoantibodies (ICAs). Also
measured were antibodies to GAD65, ICA512
and insulin. Participants were followed for
the occurrence of T1D. One cohort, called
‘Trials’, included 528 DPT-1 participants
(83.3% ICA+) who were randomised in the
DPT-1 studies. The other cohort, called
‘Questionnaire’, included 28,507 individuals
(2.4% ICA+) who did not enter the DPT-1
trials but responded to questionnaires as to
T1D status.
Results: In both cohorts autoantibody
number was highly predictive of T1D. In the
Questionnaire cohort, as single autoantibod-
ies, ICA (3.9%), GAD65 (4.4%) and ICA512
(4.6%) were similarly predictive of T1D,
whereas no subjects with only insulin autoan-
tibodies developed T1D.
Conclusions: The number of autoantibod-
ies is predictive of T1D. However, there are
differences in prediction based on antibody
type and titre.
• Comment: The finding that autoantibody
number predicts T1D is consistent with other
studies. However, this represents the largest
prospective study of relatives yet to be
reported. It confirms that an individual with
only a single antibody being positive is far less
likely to develop T1D than those with two or
more antibodies. It also finds that the particu-
lar type and titre of an autoantibody can
influence prediction. Over the years, our tools
for prediction of T1D have continued to
improve. This has been demonstrated in the
DPT-1 studies, in the ENDIT (European Nic-
otinamide Diabetes Intervention Trial) study
and many others. Yet, the focus of many
studies has been on relatives of individuals
with T1D. It is probably time to advance
screening and prediction efforts more widely
into the general population. This might best
be accomplished with screening at birth for
high risk HLA genotypes, as has been done in
studies such as DIPP (Diabetes Prediction
and Prevention), DAISY (Diabetes Autoim-
munity Study in the Young) and TEDDY
(The Environmental Determinants of Diabetes
in the Young).
68 Immunomodulation
Preservation of b-cell function
in autoantibody-positive youth
with diabetes
C. J. Greenbaum,1 A. M. Anderson,2
L. M. Dolan,3 E. J. Mayer-Davis,4 D. Dabelea,5
G. Imperatore,6 S. Marcovina,7 C. Pihoker,8
SEARCH Study Group
1 b-cell function after 5 years. This may have
Diabetes Research Program, Benaroya
Research Institute, Seattle, WA, USA, 2Wake
Forest University School of Medicine, Winston-
Salem, NC, USA, 3Cincinnati Children’s Hospi-
tal and Medical Center, Cincinnati, OH, USA,
4 currently being studied only in recent-onset
Department of Nutrition, University of North
Carolina at Chapel Hill, Chapel Hill, NC, and
the Department of Epidemiology and Biostatis-
tics, University of South Carolina, Columbia,
SC, USA, 5Department of Epidemiology, Colo-
rado School of Public Health, University of Col-
orado, Denver, CO, USA, 6Division of Diabetes
Translation, National Center for Chronic Dis-
ease Prevention and the Health Promotion
Centers for Disease Control and Prevention,
Atlanta, GA, USA, 7Department of Medicine,
University of Washington, Seattle, WA, USA,
and 8Department of Pediatrics, University of
Washington, Seattle, WA, USA
Diabetes Care 2009; 32: 1839–44
Background: SEARCH is a population-
based study conducted at six centres in the
USA, including existing (prevalent) and newly
diagnosed (incident) cases of diabetes in
youth. This report describes the extent of b-
cell function in youth with diabetes who have
GAD65 and ⁄ or IA2 autoantibodies.
Methods: Fasting C-peptide levels were
obtained from 2789 GAD65 and ⁄ or IA2 auto-
antibody-positive youth aged 1–23 years. Pre-
served b-cell function was defined on the
basis of cut points derived from the Diabetes
Control and Complications Trial (DCCT)
(fasting C-peptide ‡ 0.23 ng ⁄ ml) and from
the adolescent population of the National
Health and Nutrition Examination Survey
(NHANES) 5th percentile for fasting C-pep-
tide (‡ 1.0 ng ⁄ ml). The clinical characteristics
between those with and without preserved b-
cell function were compared.
Results: Within the first year of diagnosis,
82.9% of youth had a fasting C-peptide
‡ 0.23 ng ⁄ ml and 31.2% had values
‡ 1.0 ng ⁄ ml. Among those with 5 or more
years of diabetes, 10.7% had preserved b-cell
function based on the DCCT cutoff and 1.0%
were above the NHANES 5th percentile.
Conclusions: Early on, the vast majority of
antibody-positive youth with diabetes have
significant residual b-cell function, but by
5 years of diabetes, only 10% do.
• Comment: The SEARCH study data, as well
as the control groups in the intervention
studies discussed earlier, clearly demonstrate
that b-cell function is not rapidly lost after
diagnosis of diabetes, but rather that over
80% of youth have significant residual b-cell
function within the first year of diagnosis,
and 10% continue to have significant residual
important implications as successful interven-
tion strategies evolve, as those with significant
residual b-cell function may potentially bene-
fit from intervention, which in most trials is
T1D.
Breastfeeding patterns of
mothers with type 1 diabetes:
results from an infant feeding
trial
S. Sorkio,1 D. Cuthbertson,2 S. Bärlund,1
A. Reunanen,3 A. M. Nucci,4 C. L. Berseth,5
K. Koski,6 A. Ormisson,7 E. Savilahti,8
U. Uusitalo,2 J. Ludvigsson,9 D. J. Becker,10
J Dupré,11 J. P. Krischer,2 M. Knip,8,12,13
H. K. Akerblom,6 S. M. Virtanen,1,14,15 TRIGR
Study Group
1
Nutrition Unit, National Institute for
Health and Welfare, Helsinki, Finland,
2
Pediatrics Epidemiology Center, University of
South Florida, Tampa, FL, USA, 3National
Institute for Health and Welfare, Helsinki,
Finland, 4Division of Nutrition, School of
Health Professions, College of Health and
Human Sciences, Georgia State University,
Atlanta, GA, USA, 5Bristol-Myers Squibb,
Evansville, IN, USA, 6Institute of Clinical
Medicine, University of Helsinki, Helsinki,
Finland, 7Department of Paediatrics, University
of Tartu, Tartu, Estonia, 8Hospital for Children
and Adolescents, University of Helsinki,
Helsinki, Finland, 9 Division of Pediatrics,
Department of Health and Environment,
Faculty of Health Sciences, University of
Linköping, Linköping, Sweden, 10Children’s
Hospital of Pittsburgh, Pittsburgh, PA, USA,
11
Robarts Research Institute, London, ON,
Canada, 12Folkhlsan Research Center,
University of Helsinki, Helsinki, Finland,
13
Department of Pediatrics, Tampere University
Hospital, Tampere, Finland, 14Tampere School
of Public Health, University of Tampere,
Tampere, Finland, and 15Research Unit,
Tampere University Hospital, Tampere, Finland
Diabetes Metab Res Rev 2010; 26: 206–11
Background: It had previously been
reported that mothers with T1D are less
ª 2011 Blackwell Publishing Ltd Int J Clin Pract, February 2011, 65 (Suppl. 170), 61–70
likely to breastfeed their children or breast-
feed for a shorter period of time than non-
diabetic mothers. The aim of this study was
to prospectively examine the breastfeeding
patterns among mothers with and without
T1D.
Methods: Families from an infant feeding
study (TRIGR) included 2160 babies; 1096
were born to women with T1D and 1064 to
unaffected women. Information on infant
feeding was acquired by frequent prospective
dietary interviews.
Results: Most (> 90%) of the infants were
initially breastfed, regardless of the mother’s
T1D status. However, breastfeeding rates
declined more steeply among mothers with
T1D than without T1D, being 50% and 72%
at 6 months, respectively. After adjusting for
age, educational status, timing of delivery and
caesarean section rate, all factors associated
with the termination of breastfeeding, there
was no difference in the duration of breast-
feeding among mothers with and without
T1D.
Conclusions: Maternal diabetes status was
not itself associated with shorter breastfeed-
ing. Shorter duration of breastfeeding in
mothers with T1D could be explained by
their more frequent caesarean sections, earlier
delivery and lower age and education.
• Comment: Although mothers with T1D
breastfed for a shorter duration, this did not
seem to be related to T1D itself, but rather to
other confounding factors, i.e. differences in
the mode of delivery, length of gestation,
parental age and education. Breastfeeding has
substantial health advantages and thus is
desirable for all infants, and should be
encouraged. It also may impact the frequency
of development of T1D. Yet, the TRIGR study
(2), from which this study is derived, is eval-
uating the content of infant formula – cow’s
milk based versus casein hydrolysate – in
order to ascertain whether early introduction
of cow’s milk may serve as an environmental
trigger for T1D. It will be several years before
the results of this important study become
available. In the meantime, breastfeeding
defers the introduction of any formula, and
prolonged breastfeeding may be beneficial in
terms of forestalling T1D. It would be unfor-
tunate if the presence of maternal T1D
directly led to shorter duration of breastfeed-
ing. This study also highlights a separate
issue. Namely, one has to wonder whether
greater efforts should be made to have
women with T1D progress to spontaneous
labour and vaginal delivery in contrast to the
not uncommon practice of early delivery by
caesarean section.

Prevalence of enteroviral capsid
protein vp1 immunostaining in
pancreatic islets in human type
1 diabetes
S. J. Richardson,1 A. Willcox,1 A. J. Bone,2
A. K. Foulis,3 N. G. Morgan1
1
Institute of Biomedical and Clinical Sci-
ences, Peninsula Medical School, Plymouth,
UK, 2School of Pharmacy and Biomolecular
Sciences, University of Brighton, Brighton, UK,
and 3Department of Pathology, Royal Infir-
mary, Glasgow, UK
Diabetologia 2009; 52: 1143–51
Background: Much evidence suggests that
enterovirus infection may be an important
environmental trigger of T1D. This study
examined pancreatic islets in autopsy speci-
mens for evidence of enteroviral infection.
Methods: Pancreatic autopsy specimens
were examined, including 72 from recent-
onset T1D (mean age 12.65 ± 1.1 years, range
1–42 years, and a mean time since diagnosis
of 8.2 ± 4.1 months, range 0–6 years). A large
number of control specimens were examined,
including 25 pancreases from adult patients
with type 2 diabetes. Serial sections were
stained for insulin, glucagon, enteroviral cap-
sid protein vp1, protein kinase R (PKR)
(which is unregulated in islets with enterovi-
rus infection), MHC class 1 and CD45. Dou-
ble immunofluorescence staining was
performed. Islets were classified as insulin-
containing islets (ICIs), presumably residual
b-cells, or insulin-deficient islets (IDIs). (In
the T1D pancreases, 60% of islets are IDIs.)
Results: The majority (44 of 72, 61%) of
recent-onset T1D cases were positive for
enteroviral vp1 antigen. Enteroviral capsid
protein vp1 immunostaining was restricted to
ICIs, and double immunofluorescence demon-
strated that this is restricted to b-cells. Other
markers of infection (PKR, MHC-1) were also
present. Enteroviral capsid protein vp1 was
detected in a few cells in the pancreases of
three of 39 (7.7%) non-diabetic paediatric
cases. A total of 40% of type 2 diabetic
pancreases also stained for enteroviral capsid
protein vp1.
Conclusions: Enterovirus staining is com-
mon in pancreases from recent-onset T1D.
Interestingly, it is also seen in some pancre-
ases from individuals with type 2 diabetes.
• Comment: These findings provide further
support for a potential role of enterovirus
infection as an environmental factor leading
to T1D. There is a growing body of evidence
supporting this concept, which is being stud-
ied in detail by the TEDDY Study Group (3).
Identification of environmental factors that
ª 2011 Blackwell Publishing Ltd Int J Clin Pract, February 2011, 65 (Suppl. 170), 61–70
are important in the aetiology or pathogenesis
of T1D may facilitate the development of
intervention strategies to reduce or eliminate
the impact of such factors, and thus may be
important in the eventual prevention of T1D.
Recurrence of type 1 diabetes
after simultaneous pancreas–
kidney transplantation, despite
immunosuppression, is
associated with autoantibodies
and pathogenic autoreactive
CD4 T-cells
F. Vendrame,1 A. Pileggi,1,2 E. Laughlin,3
G. Allende,1 A. Martin-Pagola,1 R. D. Molano,1
S. Diamantopoulos,1 N. Standifer,3,4
K. Geubtner,3 B. A. Falk,3 H. Ichii,1,2
H. Takahashi,2 I. Snowhite,1 Z. Chen,5
A. Mendez,1,6 L. Chen,2 J. Sageshima,2
P. Ruiz,2 G. Ciancio,2 C. Ricordi,1,2,5,6
H. Reijonen,3 G. T. Nepom,3 G. W. Burke,1,2
A. Pugliese1,5,6
1
Diabetes Research Institute, Leonard Miller
School of Medicine, University of Miami,
Miami, FL, USA, 2Department of Surgery,
Division of Transplantation, Leonard Miller
School of Medicine, University of Miami,
Miami, FL, USA, 3Benaroya Research Institute,
Seattle, WA, USA, 4Clinical Immunology, Am-
gen Inc., Seattle, WA, USA, 5Department of
Microbiology and Immunology, Leonard Miller
School of Medicine, University of Miami,
Miami, FL, USA, 6Department of Medicine,
Division of Endocrinology and Metabolism,
Leonard Miller School of Medicine, University
of Miami, Miami, FL, USA
Diabetes 2010; 59: 947–57
Background: Recurrent autoimmunity was
first described in pancreas transplants from
identical twin donors, who did not need
immunosuppression to prevent graft rejec-
tion. The current study explores the nature
of recurrent diabetes in pancreas–kidney
transplant recipients who were receiving
immunosuppression therapy.
Methods: In three patients with recurrent
diabetes after pancreas–kidney transplanta-
tion, there was serial monitoring of antibodies
and autoreactive T-lymphocytes, as well as
pancreas biopsy.
Results: Pancreas transplant biopsies were
taken after hyperglycaemia recurrence and
revealed b-cell loss and insulitis, and no evi-
dence of graft rejection. From the time of
biopsy and on further follow-up, both anti-
bodies and autoreactive T-cells were repeat-
edly demonstrated. Immune monitoring
Immunomodulation 69
during immunosuppressive therapy showed
that autoimmunity was not resolved by the
agents used.
Conclusions: Recurrent autoimmunity may
be responsible for hyperglycaemia recurrence
in pancreatic transplant recipients, despite
immunosuppressive therapy.
• Comment: Loss of function after time is a
common feature in recipients of pancreas or
islet transplants. There are many possible rea-
sons for this, one of which is the recurrence
of autoimmunity. Yet, it might be expected
that the immunosuppressive agents used to
prevent allograft rejection would also prevent
recurrent autoimmunity. In the patients
described here, that is clearly not the case, as
there was no evidence of rejection either in
the pancreas biopsy or in the function of the
transplanted kidney. Yet, biopsies revealed b-
cell loss and insulitis, and both autoantibodies
and autoreactive T-lymphocytes were demon-
strable. This causes one to conclude that the
mechanisms responsible for allograft rejection
and for autoimmunity are different, and may
require different forms of immune interven-
tion to control each. This has profound
importance in transplantation of these organs,
particularly because simultaneous pancreas
and kidney transplantation is the procedure
of choice for treatment of end stage renal
disease in individuals with T1D. It also offers
important insights into evolving appropriate
immune modulation strategies to interrupt
the T1D disease process, either for prevention
or to sustain b-cell function in recent-onset
T1D.
Dimorphic histopathology of
long-standing childhood-onset
diabetes
R. Gianani,1 M. Campbell-Thompson,2
S. A. Sarkar,1 C. Wasserfall,2 A. Pugliese,3
J. M. Solis,1 S. C. Kent,4 B. J. Hering,5
E. West,1 A. Steck,1 S. Bonner-Weir,6
M. A. Atkinson,2 K. Coppieters,7
M. von Herrath,7 G. S. Eisenbarth1
1
Barbara Davis Center for Childhood Diabe-
tes, University of Colorado Denver, Aurora,
CO, USA, 2Department of Pathology, Univer-
sity of Florida at Gainesville, Gainesville, FL,
USA, 3Diabetes Research Institute, Miami, FL,
USA, 4Center for Neurologic Diseases, Brigham,
and Women’s Hospital Harvard Medical
School, Boston, MA, USA, 5Schulze Diabetes
Institute, University of Minnesota, Minneapolis,
MN, USA, 6Joslin Diabetes Center, Harvard
Medical School, Boston, MA, USA, and
7
La Jolla Institute for Allergy and Immunology,
La Jolla, CA, USA
Diabetologia 2010; 53: 690–8
70 Immunomodulation
Background: Analysis of C-peptide in chil-
dren characterised at diabetes onset for au-
toantibodies shows heterogeneous
preservation of insulin secretion in long-
standing diabetes. This study sought to cha-
racterise pancreases of childhood-onset diabe-
tes in order to define the pathological basis of
that heterogeneity.
Methods: Pancreases were evaluated from
20 cadaveric organ pancreases from individu-
als with childhood-onset (mean age of onset
11.2 years, range 3–18 years) long-term
(mean duration 14 years, range 1–35 years)
T1D. Pancreatic histology, islet autoantibodies
and C-peptide of patients were analysed.
Results: Most (70%) of the pancreases had
only IDIs. C-peptide was not present in
patients lacking histological evidence of
b-cells. Of six patients with extant b-cells,
these could be divided into two patterns. In
pattern A, there were mostly insulin-deficient
islets (IDIs) and lobular retention of areas
with ‘abnormal’ b-cells; islets retaining b-cells
contained survivin and enhanced HLA class 1,
had low or no C-peptide, and were positive
for anti-islet autoantibody at time of death.
In pattern B, none had IDIs, with all islets
containing normal-appearing but quantita-
tively reduced b-cells, without survivin or
HLA class 1; they were antibody-negative with
relatively high C-peptide, and lacked high risk
HLA alleles.
Conclusions: The data suggest that
C-peptide secretion in long-standing diabetic
patients can be explained by two different
patterns of b-cell survival, one (pattern A)
that probably reflects autoimmune type 1A
diabetes, and the other possibly reflecting a
different subset of T1D.
• Comment: The investigators used pancre-
ases collected by the JDRF nPOD (Network
for Pancreatic Organ Donors with Diabetes)
consortium to evaluate the histology of
patients with long-standing childhood diabe-
tes. Although most patients had the expected
lack of b-cells and lack of C-peptide, 30%
showed persistent C-peptide, and these could
be divided into two patterns. One pattern was
consistent with classical type 1A diabetes pre-
sumably on an immune basis. The other pat-
tern appeared to be totally different, with
normal-appearing islets, relatively high C-pep-
tide, absence of high risk HLA, and lack of
antibodies at the time of death. All were said
to have had the typical clinical appearance of
T1D at the time of diagnosis. This suggests
that we still have a lot to learn about the evo-
lution of T1D, and that we need better tools
at diagnosis to better characterise individuals.
This may prove to be particularly important
if we are to appropriately select patients with
recent-onset T1D for newer immunological
interventions, either in the context of clinical
trials or for treatment when such agents are
approved by regulatory agencies.
OVERALL COMMENTARY
This year has been marked by a stunning
number of papers relating to immune inter-
vention of T1D. Longer term observations
suggest that there may be sustained effects of
a short course of anti-CD3 monoclonal anti-
body, which depletes T-lymphocytes and on
recovery seems to favour regulatory T-lym-
phocytes over effector T-lymphocytes. That
B-lymphocytes might be involved in T1D
pathogenesis was suggested by positive results
from a trial with an anti-CD20 monoclonal
antibody, rituximab. There were also provoca-
tive observations from an extraordinarily tiny
study with insulin B-chain given with IFA.
Yet, most intervention studies were negative –
mycophenolate mofetil given alone or in
combination with daclizumab, low-dose diaz-
oxide, vitamin D, an altered peptide ligand of
insulin B (9–23), autologous bone marrow
ª 2011 Blackwell Publishing Ltd Int J Clin Pract, February 2011, 65 (Suppl. 170), 61–70
blood injected into the pancreatic arterial sys-
tem, and umbilical cord blood transfusion.
The combination of low-dose cyclosporine
and methotrexate in a very small pilot study
was impossible to interpret, and is unlikely to
be pursued further anyway. That negative
studies continue to dominate the field, and
that the positive ones still show decline in b-
cell function over time, has led to more calls
for combination approaches. When I have
advanced such prospects at meetings of paedi-
atric diabetologists, I hear groans. Yet when
I have advanced these prospects at meetings
of immunologists and transplant surgeons,
I hear cheers. Hopefully, preclinical studies
will provide further guidance to support
combination therapies, and hopefully regula-
tory agencies will offer a path for evaluation
of these in human beings.
Meanwhile, a number of interesting studies
have offered further insights into the pathol-
ogy and pathogenesis of T1D, the pathways
by which it may unfold, and how better to
quantify who is at risk of developing T1D.
These studies have also suggested that T1D is
not the same in everybody who appears to
develop it. And, perhaps most importantly,
that autoimmunity may not be able to be
addressed with the same therapeutic agents
that are used to prevent transplant rejection.
It has been a very exciting year, indeed.
References
1 Couri CE, Oliveira MC, Stracieri AB et al. C-peptide levels
and insulin independence following autologous non-
myeloablative haematopoietic stem cell transplantation in
newly diagnosed type 1 diabetes mellitus. JAMA 2009; 301:
1573–9.
2 TRIGR Study Group. Study design of the Trial to Reduce
IDDM in the Genetically at Risk (TRIGR). Pediatr Diabetes
2007; 8: 117–37.
3 The TEDDY Study Group. The Environmental Determinants
of Diabetes in the Young (TEDDY) study: study design.
Pediatr Diabetes 2007; 8: 286–98.