Rituximab Selectively Suppresses
Specific Islet Antibodies
Trialnet-Yu et al Diabetes 2011
Islet autoantibodies can discriminate
maturity-onset diabetes of the young
(MODY) from Type 1 diabetes.
McDonald TJ, Colclough K, Brown R,
Shields B, Shepherd M, Bingley P, Williams
A, Hattersley AT, Ellard S.
Diabet Med 2011
GAD and IA-2 measured:
MODY (GCK=227, HNF1A=229, HNF4A=52 patients
5/508 (1%) positive, all GAD only
Type 1 (n=98)
82% Positive
Dietary Intervention in Infancy
and Later Signs of Beta-Cell Autoimmunity
Knip et al NEJM 2010
Development of Autoantibodies in the
TrialNet Natural History Study
Vehik et al Diabetes Care 2011
Genetic Analysis of Adult-Onset Autoimmune Diabetes
Joanna M.M. Howson,1 Silke Rosinger,2 Deborah J. Smyth,1 Bernhard O.
Boehm,2
the ADBW-END Study Group,* and John A. Todd1
Diabetes 2011
• DR3 associated with GAD antibody positivity of
adults with Type 1 diabetes but absence of IA-2
autoantibodies.
• DR4 associated with IA-2 positivity and younger
age of onset as was DR3/4 heterozygotes.
HIGH LEVELS INSULIN AUTOANTIBODIES DAISY STUDY PROGRESSION TO DM
FIRST DOT: AGE ANY ANTIBODY APPEARED/ SECOND: DM AGE
Steck et al Diabetes Care 2011
Age of Islet Autoantibody Appearance and Mean Levels of Insulin, but Not GAD or
IA-2 Autoantibodies, Predict Age of Diagnosis of Type 1 Diabetes
Diabetes Autoimmunity Study in the Young
LOW LEVELS INSULIN AUTOANTIBODIES DAISY STUDY PROGRESSION TO DM
FIRST DOT: AGE ANY ANTIBODY APPEARED/ SECOND: DM AGE
Time to DM from age 1st
Ab+
MEAN LOG IAA vs Time to DM from age Islet Ab first +
15
R2=.37 P<.0001
10
Steck et al
Diabetes Care
2011
5
0
-3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5
log10 Mean Insulin Abs
0.0
Steck… Diabetes Care 2011
GAD65 Levels with Years to Diabetes ICA512 Levels with Years to Diabetes
17.5
Steck… Diabetes Care 2011
17.5
15.0
15.0
12.5
12.5
10.0
10.0
7.5
7.5
5.0
5.0
2.5
2.5
0.0
0.0
-3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0
-4
Mean GAD65 Levels (Log10)
-3
-2
-1
Mean ICA512 Levels (Log10)
0
Steck et al Diabetes Care 2011
Age of Islet Autoantibody Appearance and Mean Levels of Insulin, but Not GAD or
IA-2 Autoantibodies, Predict Age of Diagnosis of Type 1 Diabetes
Diabetes Autoimmunity Study in the Young
Mean Insulin AutoAb levels by INS genotypes
mean IAA Levels
0.6
0.5
P=0.027
0.4
0.3
0.2
0.1
0.0
A/A
N = 25
Mean= 0.096
SD = 0.159
A/T or T/T
13
0.025
0.045
Steck et al
Diabetes Care 2011
Enhancing Prediction Diabetes with ZnT8
Autoantibody Determination
• N=88 Children DAISY prospective study
+ Only one AutoAb (of GAD/IA-2/Insulin)
>3 Yrs age and >1 year Follow-up
ZnT8 Antibodies measured first Ab+ sample
• 14% also ZnT8 Antibody Positive (Thus >=2Ab)
ZnT8+ 37% (7/19) Progressed to Diabetes
ZnT8- 7% (5/69, P<.003) Progressed
Wenzlau et al PNAS 104:17040-17045, 2007
DAISY subjects with only 1 Ab (GAD/IA-2/Insulin) vs + ZnT8
Autoantibodies
Wenzlau PNAS 2007
Receiver Operator Characteristics of the ZnT8 antibody assays
J.M. Wenzlau, H.W. Davidson, and J.C. Hutton
ZnT8 detects autoantibodies in patients who are negative
for ICA and gold standard biochemical antibodies.
J.M. Wenzlau, H.W. Davidson, and J.C. Hutton
Value of 4 antibodies
40
40
30
26
20
13
9
10
0
0
1
1
2
2
3
3
4
Number of Autoantibodies
Sera from 223 newly diabetic individuals were assayed for reactivity to insulin, GAD65, and IA-2 ± ZnT8. Nine individuals were negative for insulin,
IA-2, and GAD65 but positive for ZnT8 autoantibodies, increasing the percentage of sera positive for at least 1 autoantibody from 94% to 98%. In
addition 26 individuals (11.7%) were re-classified from single to multiple autoantibody positivity on the basis of ZnT8 autoreactivity.
J.M. Wenzlau, H.W. Davidson, and J.C. Hutton
Insulin autoantibodies
ICA
?
ZnT8
IA-2 (ICA512BDC)
GAA
mIAA
(GAD65)
JH4 Autoantibodies
1.1
Levels (index)
0.9
0.7
0.5
0.3
0.1
-0.1
Wenzlau et al PNAS 104:17041-17045, 2007
Zinc Transporter (Znt8) Autoantibodies
1.1
Pos = 63%
Levels (index)
0.9
Follow up analysis
(Wenzlau et al PNAS
104:17040, 2007)
new onset patients
0.7
0.5
0.3
0.1
0.022
-0.1
New Onsets
n=340
Controls
N=200
Znt8 Islet Autoantigen
• ZnT8 cation efflux transporter (Slc30A8)
• Approximately 60% of prediabetic and new onset
patients express autoantibodies to C-terminus (amino
acids 268-369) with fluid phase radioassay
• Beta cell specific molecular target
• Autoantibodies usually appear post mIAA and
GAD65 AA in children followed from birth to type 1
diabetes.
Wenzlau et al PNAS 104:17040-17045, 2007
Stages in Development of Type 1 Diabetes
GENETICALLY AT RISK
BETA CELL MASS
MULTIPLE ANTIBODY POSITIVE
LOSS OF FIRST PHASE
INSULIN RESPONSE
GENETIC
PREDISPOSITION
INSULITIS
BETA CELL INJURY
“PRE”DIABETES
DIABETES
TIME
J. Skyler
NEWLY DIAGNOSED DIABETES
Diabetes Classification
• Type 1A: Immune Mediated
Type 1B: Insulin deficient, no autoantibodies
• Type 2: No Autoantibodies, Can initially be
treated without insulin
• Other Specific forms of Diabetes
• Gestational Diabetes
BDC-July01
Cytoplasmic ICA kindly provided by the discoverer Franco Bottazzo
Inhibition of NOD Diabetes in Absence of
Transplacental Antibodies (Ab)
Greeley et al, Nature Med 8:399, 2002
80
70
60
50
40
30
20
10
0
IgM
Knockout
AntiHEL+KO
Control
DBA/2
Foster
Mother
No Maternal Ab
SCID
Mother
“Biochemical” Autoantibody Assays
• Insulin
• Glutamic Acid Decarboxylase
• ICA512 (IA-2)
BDC
Percent
100
ROC of GAD65 (DASP2002-BDC)
100 controls, 50 new onset DM
80
Sensitivity
60
Specificity
40
20
0
-50
450
ROC= Receiver Operator Curve
950
1450
IDS Units
1950
100
ROC: IA-2 Full Length (DASP2002- BDC)
100 Controls, 50 New Onset DM
Percent
80
Sensitivity
Specificty
60
40
20
0
-50
150
350
IDS Units
550
750
ROC of ICA512bdc (DASP2002-BDC)
Percent
100
80
Sensitivity
60
Specificity
40
20
0
-25
125
275
IDS Units
425
575
ROC of mIAA (DASP2002-BDC)
Percent
100
80
Sensitivity
60
Specificity
40
20
0
-0.050
0.050
ROC = Receiver Operator Curve
0.150
Index
0.250
ROC of mIAA (DASP2002-BDC)
Percent
100
80
Sensitivity
60
Specificity
40
20
0
-0.020
0.000
0.020
Index
0.040
New Onset Children with Diabetes Seen at the Barbara Davis Center
P
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n
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e
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o
a
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y
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a
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Babu et al. 2,001
BDC
Caveats of Autoantibody Testing
CAVEAT
Assays Vary
sensitivity/specificity
SUGGESTION
Cytoplamsic ICA not used
Biochemical AutoAb in
Proficiency: spec>=99th
Insulin Abs induced by insulin Post "2 weeks" do not
injections
use insulin Ab assay
IAA children; GAD adults
Multiple Abs
Single Ab Low Risk
Multiple Abs
Subset No Ab
HLA/Insulin/Autoimmunity
Abs appear any age
Measurement over time
Transient Abs possible;
Check more than once
"Sera" mistakes
Similar “rules” Other Autoimmune
Disorders
• Addison’s Disease(DQ8/DQ2 DRB1*0404) 21hydroxylase Auotantibodies
30/2,100 type 1 pts (1.5%)
5/30 Addison’s first Test (1/400 patients)
• Celiac Disease (DQ2 or DQ8)
Transglutaminase Autoantibodies
98/847 type 1 pts (12%)
15/20 Biopsies Positive/Estimate 1/20celiac
1/3 DQ2/DQ2 Transglutaminase +
21-Hydroxylase Autoantibodies
Levels of autoantibodies
2
1.5
1
0.5
n= 241
Known
Healthy
Controls
Addison's
Yu et al, JCEM 84:328-335, 1999
n= 817
n= 13
Negatives
Positives
Type I Diabetics
Figure 2
Percent 21-OH Autoantibody
Positive/ Patients with type 1 DM
N=208
53
57
55
307
6
5
4
3
2
1
0
DQ2/DQ8
0501/0301:X
DQ2/DQ2
DQ8/DQ8
Other
Yu et al, JCEM 84:328-335, 1999
BDC
Prevalence of Transglutaminase
Autoantibodies by HLA-DR
Prevalence
IDDM
Relatives
Population
25%
20%
15%
10%
5%
HLA-DR
0%
DR3+
Bao et al, 13:143-148, 1999
DR3-
log FPIR (1+3' insulin)
FPIR in pre-diabetic relatives with initial FPIR
> 50mU/L
500
50
5
-8
-7
-6
-5
-4
-3
-2
-1
Years prior to diabetes
Melbourne Pre-Diabetes Study (Colman PG & Harrison LC)
0
Contrasting Insulin and GAD as
Primary Antigen type 1 DM
KNOCKOUT/Alter
GENE SEQUENCE
ANTI-SENSE
INSULIN
GAD
Prevent NOD
DM
N/A
GAD65 no effect
Decrease DM 2/6
Ab LEVEL correlate rate YES
to DM
High Affinity Ab-NOD
YES
NO
High Affinity Ab-Man
YES
YES
Early T-Cell NOD
YES
YES
Protection with Ag-NOD YES
YES
Induction Diabetes TCR YES
NO
T-Cell Response Man
YES
YES
Not Detected
BDC
125I-Insulin T1/2 in vivo in
man
Anti-Insulin Abs: Insulin Turnover
10000
1000
Not Insulin Rx
100
Not Resistant
Insulin Resistant
10
1
0.1
1
10
100
1000
10000
mU/ml Maximum Binding Titer
Davidson and DeBra, Immunologic Insulin Resistance: Diabetes 27:307-18, 1978
10000
Anti-insulin
autoantibodies
(nU/ml)
1000
100
10
1
5
10
15
20
Age (years)
25
30
Insulin Autoantibodies Versus Age of
Diabetes Onset
Diabetes Care 11:736-739, 1988
35
IAA assay
H
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r
INSULIN
BDC
Insulin Autoantibodies:
A Chain L13
BDC
Insulin
Receptor
Binding
Region
PERCENT
100
90
80
70
60
50
40
30
20
10
0
Affin>10(9)
Multipe Abs
f/u
Diabetes
Proinsulin
High Risk
"False Positive"
Mature high-affinity immune responses to (pro)insulin anticipate the autoimmune cascade
that leads to type 1 diabetes. Achenbach et al, J.Clin Invest 2004, 114:589
96-Well Plate Micro-IAA Assay for
Mouse Serum
100
Micro-IAA Assay
(index)
10
1
0.1
0.01
0.001
0.0001
0
1
NOD
Yu et al. PNAS: 97:1701-1706, 2,000
2
3
Balb/c
4
5
6
C57/Bl6
BDC
96-Well filtration Plate IAA Radioassay
100
Levels of IAA (index)
10
1
0.1
0.01
0.001
0.0001
Normal Controls
Yu et al. PNAS: 97:1701-1706, 2,000
New Onset Patients
RC Curve of mIAA
Sensitivity (%)
90
80
0.006
0.009
70
0.010
0.008
60
50
40
30
20
10
0
0
0
0.001
0.004
0.005
0.003
10
20
Specificity (1-%)
Yu et al. PNAS: 97:1701-1706, 2,000
30
The Levels of mIAA in Prediabetic Children
Relatives (n= 3)
General Population (n= 2)
10
10
1
DM
DM
0.1
0.01
0.001
0.0001
Level of mIAA (index)
Level of mIAA (index)
DM
DM
1
0.1
DM
0.01
0.001
0.0001
0
2
4
Age (years)
Yu et al. PNAS: 97:1701-1706, 2,000
0
2
4
Age (years)
BDC
Anti-insulin
Autoantibodies
10
IAA (+) at 8wks
1
IAA (+) at 20wks or later
0.1
0.01
0.001
0.0001
0
4
8
12
16
20
24
28
32
36
40
glucose (mg%)
Age (weeks)
600
500
IAA (+) at 8wks
400
IAA (+) at 20wks or later
300
200
100
0
0
4
8
12
16
20
24
28
32
36
40
Age Diabetes
Onset
Age (weeks)
IAA(+)
35
>
IAA(-)
30
25
20
15
10
5
0
4
8
12
16
20
24
28
32
36
Weeks of first positive of IAA positive
Yu et al. PNAS: 97:1701-1706, 2,000
Percent Not DM
Age Insulin
NOD Mice Divided by IAA Appearance
100
Autoantibodies first
Appeared
P<.001
80
8 wks +
60
12,16 +
40
>=20+ or -
20
0
0
10
20
30
Age (w eeks)
40
BDC
Rapid induction of Insulin Autoantibodies by Insulin B:9-23
peptide immunization in Normal BALB/c mice
IAA (index)
10
1
0.1
0.01
0.001
3
4
5
6
7
8
9
10
11
12
13
weeks
B:9-23+ IFA
B:9-23+ IFA
BDC
Dynamic Changes GAD65 Autoantibody
epitope Specificities…
Schlosser et al, Diabetologia 48:922, 2005
• Analysis competition with recombinant monoclonals to
GAD of prediabetic children.
-- No difference epitopes initial sample
-- High risk children emergence of antibodies to
conformational N-terminus and middle region
--For high risk but not low risk children binding to Nterminus and middle region increases
Domains/Splice Variants
ICA512
1
LUMINAL DOMAIN
CYTOPLASMIC
576
979
PTP domain
577-600
Mini:930-978
Transmembrane
1
ICA512
979
256
979
ICA512bdc
556
630
Alternative Splice minus 557 to 629 (73 aa)
BDC
F1
ICA512 (IA2) Fragments
Positivity
1
577
256
DM Transient
600
556
979
630
(100%) (100%)
605
ICA512ic
1
979
TM
256
90%
30%
0%
0%
37%
30%
54%
10%
760
760
761
F2B
10%
979
ICA512bdc
F2A
98%
928
Modified from Miao et al. J. Autoimmunity 2002 with F1= Full Length IA-2
BDC
IA-2 mRNA Expression
370 bp, Regular mRNA
151 bp, Alternatively Spliced mRNA
Thymus fetal tissues (21-27 weeks), adult pancreas.
Pugliese et al.
BDC
GAD and ICA512 Abs:
71,000 DPT Screening Samples
6
Percent Positive
5
4
GAD
ICA512
1 Ab+
2 Ab+
3
2
1
0
Sibling
n=27,128
Yu et al, NYAS 2002
Offspring
n=17063
Parent
n=15,561
BDC
Percent Positive
GAD and ICA512 Abs:
71,000 DPT Screening Samples
9
8
7
6
5
4
3
2
1
0
GAD
ICA512
1 Ab+
2 Ab+
2 DM Parents
n=105
Yu et al, NYAS 2002
One DM Parent
n=16,901
BDC
Percent Positive
GAD and ICA512 Abs:
71,000 DPT Screening Samples
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
GAD
ICA512
1 Ab+
2 Ab+
1st Degree Relative
n=59,752
Yu et al, NYAS 2002
2nd Degree Relative
n=9,856
BDC
DPT-1: Percent GAD or ICA512+
High % Eligible or Diabetic Biochemical Ab+ of
Cytoplasmic ICA+ Relatives
80
70
60
50
40
30
20
10
0
E
le
b
i
lig
P
en
r
a
al
r
e
t
El
le
b
i
ig
al
r
O
A
IC
at
e
ep
R
g.
e
N
ti c
e
ab
i
D
2+
0
06
Yu et al, Diabetes 50,2001
Percent of 71,148 DPT Screening Samples
GAD/ICA512/Cytoplasmic ICA+
2.5
2.2
95%-/-/-
2
1.7
1.5
1
GAD65 only
ICA512 only
GAD and ICA512
Neg. GAD/512
0.8
0.78
0.5
0.15
0.25 0.19
0
ICA +
ICA Negative
Yu et al. Diabetes 50: 2001
DAISY Study Population
General population families
screened = 21,000
patients
enrolled = 108
Families with type 1 diabetes
Diabetic
Non-diabetic
infants
high risk
- DR3/3
96
15
545
moderate risk - DR3/x, 3/4
607
230
553
low risk
650
415
1,353
660
1,206
All
Rewers et al
DAISY Interviews and Clinical
Interviews:
B 3m
6m
9m
diet
infections
immunizations
allergies
stress
1y
15m
2y
3y
Visits
Clinical Visits: blood sample for GAA, IAA, ICA512, ICA
DNA
throat and rectal swabs
Rewers et al
saliva sample
BDC
The Age at Autoantibody Conversion in DAISY
AAb+ Siblings
12
10
Age
AAb+
(yrs)
8
6
4
2
Robles et al,
0
A1, A2 +
A1, A2 -
Clin Immunol
102:217, 2002
Percent BabyDiab (Offspring) Autoantibody
Positive at age 5 years
HLA and Insulin Gene VNTR
30
25
20
INS I/I
INS I/III III/III
15
10
5
0
DR3-4 DQ8
4-4 DQ8
Other HLA
Walter et al, Diabetologia (2003) 46:712-720
Progression to Diabetes vs Number of Autoantibodies
(GAD, ICA512, Insulin)
Percent not Diabetic
100
80
3 Abs
2 Abs
1 Ab
60
40
20
0
0
2.5
5
7.5
10
12.5
15
Years of Follow-up
3 Ab n = 41
2 Abs n = 44
1 Abs n = 93
17
27
23
8
15
14
Verge et al, Diabetes 45:926-933, 1996
1
4
10
2
6
1
4
BDC
Lack of Progression to DM of ICA+
0602+ Relatives
Percent Not Diabetic
0602+
0602-
100
75
50
25
0
0
Pugliese et a.
2
4
6
8
10
12
Years of Follow up
BDC
Islet Autoantibodies are rapid responders to stimulus
- rises in GADA immediately post-islet tx
100
1
10
0.1
1
0
20
40
60
80
200 400 600 800
1000
10
1
100
0.1
10
1
0
20
40
60
80
200 400 600 800
Days post islet transplant
10
1
100
0.1
10
1
1000
10
100
1
0.1
10
C-peptide (ng/ml)
1000
Patient D
x
Patient C
GADA
IA2A
IA
x
Antibody units
10
x
x
1000
C-peptide (ng/ml)
Patient B
Patient A
1
0
20
40
60
80
100 120 140 160
0
300 600 900 1200
Days post islet transplant
1800
2400
Bosi et al, Diabetes, 2001