Initial Results of Screening of Nondiabetic Organ Donors for Expression of Islet Autoantibodies

Article (PDF Available)inJournal of Clinical Endocrinology & Metabolism 91(5):1855-61 · June 2006with17 Reads
DOI: 10.1210/jc.2005-1171 · Source: PubMed
Abstract
Type 1A diabetes is characterized by a long prodromal phase during which autoantibodies to islet antigens are present. Nevertheless, we lack data on the pancreatic pathology of subjects who are positive for islet autoantibodies (to islet autoantigens GAD65, insulin, and ICA512). In this manuscript, we describe a novel strategy in obtaining pancreata and pancreatic lymph nodes from islet autoantibody-positive organ donors that involves careful coordination among the laboratory and the organ donor provider organization. We developed a rapid screening protocol for islet autoantibodies measurement of organ donors to allow identification of positive subjects before organ harvesting. In this way we were able to obtain pancreata and pancreatic lymph nodes from subjects with and without islet autoimmunity. The organ donors used in this study were obtained from the general community. The population studied consisted of 112 organ donors (age range 1 month to 86 yr, mean age 39 yr). The main outcome measure of this study consisted of evaluating the pancreatic histology and identify T cells autoreactive for islet antigens in the pancreatic lymph nodes. To date we have identified three positive subjects and obtained the pancreas for histological evaluation from one of the autoantibody-positive donors who expressed ICA512 autoantibodies. Although this subject did not exhibit insulitis, lymphocytes derived from pancreatic lymph nodes reacted to the islet antigen phogrin. In summary, these results indicate that it is possible to screen organ donors in real time for antiislet antibodies, characterize pancreatic histology, and obtain viable T cells for immunological studies.

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Available from: Ronald Gill, Dec 28, 2013
Initial Results of Screening of Nondiabetic Organ Donors
for Expression of Islet Autoantibodies
R. Gianani, A. Putnam, T. Still, L. Yu, D. Miao, R. G. Gill, J. Beilke, P. Supon, A. Valentine, A. Iveson,
S. Dunn, G. S. Eisenbarth, J. Hutton, P. Gottlieb, and A. Wiseman
The Barbara Davis Center (R.G., A.P., T.S., L.Y., D.M., R.G.G., J.B., P.S., A.V., G.S.E., J.H., P.G., A.W.) and Department of
Pathology (R.G.), University of Colorado at Denver and Health Science Center, Aurora, Colorado 80010; Department of
Pathology (R.G.), The Children’s Hospital, Denver, Colorado 80218; and Donor Alliance (A.I., S.D.), Denver, Colorado 80246
Context: Type 1A diabetes is characterized by a long prodromal
phase during which autoantibodies to islet antigens are present. Nev-
ertheless, we lack data on the pancreatic pathology of subjects who are
positive for islet autoantibodies (to islet autoantigens GAD65, insulin,
and ICA512).
Objective: In this manuscript, we describe a novel strategy in ob-
taining pancreata and pancreatic lymph nodes from islet autoanti-
body-positive organ donors that involves careful coordination among
the laboratory and the organ donor provider organization.
Design: We developed a rapid screening protocol for islet autoanti-
bodies measurement of organ donors to allow identification of positive
subjects before organ harvesting. In this way we were able to obtain
pancreata and pancreatic lymph nodes from subjects with and with-
out islet autoimmunity.
Setting: The organ donors used in this study were obtained from the
general community.
Subjects: The population studied consisted of 112 organ donors (age
range 1 month to 86 yr, mean age 39 yr).
Main Outcome Measure: The main outcome measure of this study
consisted of evaluating the pancreatic histology and identify T cells
autoreactive for islet antigens in the pancreatic lymph nodes.
Results: To date we have identified three positive subjects and ob-
tained the pancreas for histological evaluation from one of the au-
toantibody-positive donors who expressed ICA512 autoantibodies.
Although this subject did not exhibit insulitis, lymphocytes derived
from pancreatic lymph nodes reacted to the islet antigen phogrin.
Conclusion: In summary, these results indicate that it is possible to
screen organ donors in real time for antiislet antibodies, characterize
pancreatic histology, and obtain viable T cells for immunological
studies. (J Clin Endocrinol Metab 91: 1855–1861, 2006)
D
URING THE PRODROME of type 1A diabetes, auto-
antibodies to islet antigens are usually detectable.
Currently autoantibodies to the islet autoantigens glutamic
acid decarboxylase (GAD)65, insulin, and ICA512 (IA-2) are
most commonly measured (1, 2). Multiple autoantibodies
and/or autoantibodies to IA-2 are detected in approximately
5% of first-degree relatives of affected individuals and 0.3%
of the general population (1, 2). The majority of nondiabetic
individuals expressing multiple and/or IA-2 autoantibodies
develop type 1A diabetes with prospective follow-up (1, 2).
Whereas autoantibodies are a clear marker of autoimmu-
nity, autoreactive T cells to islet autoantigens are most likely
responsible for islet destruction in type 1A diabetes. How-
ever, the identification and quantification of T cell antigen-
specific responses in the peripheral blood of diabetic or pre-
diabetic subjects has been difficult. Previous studies have
demonstrated a T cell response to the islet autoantigen IA-2
(phogrin) in prediabetic subjects (3, 4). In particular, the
authors showed T cell response to two distinct epitopes of
IA-2
designated peptide 2 and peptide 7, which are also
targeted by diabetogenic CD4 T cell clones in the NOD
mouse. IA-2
is structurally related to the tyrosine phos-
phatase IA-2 molecule, and the two molecules are both tar-
gets of diabetes-associated autoantibodies with overlapping
specificities (5).
Several reports in animal models of islet destruction
and/or inflammation suggest that loss of insulin-producing
cells may be accompanied by formation of new
-cells (re-
generation) through either replication of preexisting
-cells
(proliferation) or differentiation of nonendocrine precursors
(neogenesis) (6 8). A recent report by Dor et al. (9) with fate
marking indicated proliferation but not neogenesis of
-cells.
Because autoantibodies to islet antigens can be present sev-
eral years before the development of diabetes, we assumed
that during this phase, one might identify pancreatic histo-
logical changes. Cadaveric organ donors represent a poten-
tial source of pancreatic specimens from islet autoantibody-
positive subjects. In a preliminary retrospective study of
stored sera of 777 organ donors, we identified autoantibodies
reacting with either insulin or GAD65 in 23 of 777 donors (10,
11), and two additional donors had multiple islet autoanti-
bodies (to GAD65 and IA-2). Islet antibody screening was
performed with a rapid assay that we hypothesized would
allow prospective rapid research analysis.
In the current study, we screened organ donors who be-
came available for pancreas donation with biochemical islet
First Published Online February 14, 2006
Abbreviations: ELISPOT, Enzyme-linked immunospot; GAD, glu-
tamic acid decarboxylase; HLA, human leukocyte antigen; IA-2, islet
autoantigen 2; ICA512, islet cell antigen 512; IFN, interferon; LCA, leu-
kocyte common antigen.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the en-
docrine community.
0021-972X/06/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 91(5):1855–1861
Printed in U.S.A. Copyright © 2006 by The Endocrine Society
doi: 10.1210/jc.2005-1171
1855
autoantibody radioassays. To date, we have prospectively
screened 112 organ donors for antibodies to GAD65, IA-2,
and insulin and identified three subjects to be autoantibody
positive. We obtained the pancreas of one IA-2 autoantibody-
positive individual and 14 antibody-negative subjects.
For a subset of subjects (one antibody positive and three
antibody negative subjects), we analyzed pancreatic lymph
node-derived lymphocytes by Enzyme-Linked Immunospot
(ELISPOT) assay to determine the presence of IA-2
reactive
T cells and assess whether viable T lymphocytes could be
recovered from pancreatic lymph nodes of cadaveric organ
donors.
Materials and Methods
Procurement of sera from organ donors
To date, we have obtained serum specimens from 112 nondiabetic
cadaveric organ donors for islet antibody screening.
The cadaveric donors were obtained through a collaborative arrange-
ment with our local donor provider organization (Donor Alliance). Al-
though all the sera were screened for autoantibodies, procurement of the
organ was dependent on final clinical donation of organs as well as
availability from the islet isolation laboratory. Consent for research use
was a prerequisite for both serum and organ analysis.
Screening of organ donors with biochemical
autoantibody assays
After institutional review board protocol review, serum specimens
for autoantibody measurements were obtained after organ donor cer-
tification and evaluation. Autoantibody assays for GAD65, IA-2, and
insulin were performed on receipt of the sera. The GAD65, IA-2, and
insulin autoantibody assays are based on immunoprecipitation of ra-
dioactively labeled molecules, and they have been previously described
(12). For these three assays the levels were expressed as an index with
positivity set above the usual cutoff [0.2 for GAD65 autoantibodies,
0.1 for IA-2 autoantibodies, and 0.02 for insulin antibodies of the 99th
percentile to enhance specificity (99th percentile index of 0.032 for
GAD65 autoantibodies, 0.049 for IA-2 autoantibodies, and 0.01 for in-
sulin antibodies)]. One hundred twelve organ donors were screened for
autoantibodies to IA-2, GAD65, and insulin.
Preparation of tissue for histological studies
Blocks of pancreatic tissue from one autoantibody-positive and 14
control antibody-negative organ donor subjects were obtained imme-
diately after surgical removal. Tissue blocks (n 22) were prepared from
the pancreas of the IA-2 autoantibody-positive organ donor and paraffin
embedded after fixation in 10% buffered formalin. Seven-micrometer
tissue sections were obtained for histological and immunohistochemical
studies. For each tissue block at least one hematoxylin and eosin-stained
section was obtained. Control pancreata were obtained from 14 anti-
body-negative (six male and eight females) organ donors ranging in age
between 18 and 52 yr (mean age 39.6 yr). Three donors were matched
with the antibody-positive donors for whom the pancreas was available
for both sex and age (mean age 54.28 yr). For 102 of 112 subjects, the
human leukocyte antigen (HLA) DR type (provided by Donor Alliance)
was available.
Table 1 summarizes the age, sex, body mass index, and HLA type for
the subjects whose pancreata were available for histological studies.
The hematoxylin and eosin sections of the pancreata of both the
antibody-positive subject and the controls (for which pancreata were
available) were evaluated for the distribution of the endocrine tissue,
evidence of insulitis, and presence of fibrosis.
Immunohistochemical staining for insulin and glucagon
In each case, sections were stained with antibodies directed to insulin
and glucagon. The pancreatic sections were simultaneously stained with
an antiglucagon mouse monoclonal antibody (Jackson Immunoresearch,
West Grove, Pa) and an anti-insulin guinea pig polyclonal antibody
(Jackson Immunoresearch) followed by detection with Texas Red-con-
jugated donkey antimouse IgGs (Jackson Immunoresearch) and amin-
omethylcoumarin acetate-conjugated donkey antiguinea pig IgGs (Jack-
son Immunoresearch).
Immunostaining for leukocyte common antigen (LCA), and
insulin
Each donor section was double stained with a mouse monoclonal
antibody to the LCA (CD45) and insulin (as described above). The CD45
staining was detected with an antimouse IgG alkaline phosphatase
conjugated and developed with Fast Red (Sigma, St. Louis, MO). The
sections were analyzed by immunofluorescence microscopy, allowing
simultaneous visualization of both the insulin-stained tissue and the
inflammatory cells (LCA positive cells).
Morphometric analysis
For each case we also determined the ratio between insulin and total
area and glucagon and total area (expressed as percentage) as well the
ratio between insulin and glucagon-positive areas in five randomly
selected microscopic fields. In each section, the stained area for insulin
and glucagon was quantified by computer-assisted image analysis (Im-
age Pro Plus, Silver Spring, MD). The number of CD45-positive cells in
the pancreas was determined for each donor by calculating the average
cell density (cells per square millimeter) in 10 randomly selected fields.
To evaluate for insulitis, we determined for each of the subjects the
average number of leukocytes that was present within the 10 islets
within the largest mononuclear infiltrate (if less than 10 islets contained
at least one leukocyte, the remaining islets were considered to contain
0 leukocytes). We compared both the density of pancreatic leukocytes
and the number of leukocytes within the islets between the antibody-
positive subject and each of the negative controls using the Whitney
Mann rank test.
The difference between the values obtained for the IA-2 antibody-
positive and each of the control donors for insulin to glucagon ratio,
insulin to total pancreatic area ratio, and glucagon plus insulin to total
pancreatic area was also analyzed for statistical significance using the
Whitney Mann rank test.
Immunohistochemical staining for cytokeratin 19
and chromogranin
Formalin-fixed paraffin-embedded sections were stained simulta-
neously with a polyclonal rabbit antibody to chromogranin A and B
(Abcam, Cambridge, UK) followed by detection with Texas Red-con-
jugated antimouse donkey (Jackson Immunoresearch) and a mouse
monoclonal to cytokeratin 19 (Abcam) followed by detection with the
TSA plus system (PerkinElmer, Wellesley, MA).
TABLE 1. Summary of age, sex, BMI, and DR HLA type of the
15 donors for whom pancreata were available for histological
examination
Donor no. Age (yr) Sex BMI (kg/m
2
)
HLA type
7 47 M 24.69 DR2; DR3
9 53 M 32.54 DR2; DR3
13 53 F 22.96 DR1; DR3
17 57 M 25.71 DR11; DR2
21 51 M 23.74 DR14; DR2
26 18 M 24.7 DR1; DR11
29 42 F 28.72 DR1; DR8
36 50 M 15.11 DR7; DR12
39 45 F 21.3 DR11; DR3
41 33 F 25.59 DR4; DR13
46 53 F 31.48 DR7; DR11
53 26 M 26.73 DR1; DR11
56 26 F 23.38 DR10; DR14
59 46 F 25.39 DR4; DR7
61 68 M 33.33 DR1; DR11
M, Male; F, female; BMI, body mass index.
1856 J Clin Endocrinol Metab, May 2006, 91(5):1855–1861 Gianani et al. Islet Autoantibodies in Organ Donors
Isolation of lymphocytes from pancreatic lymph nodes
Pancreatic lymph nodes could be obtained from one antibody-
positive (donor 7) and three antibody-negative control subjects.
Lymph nodes were removed from pancreatic fat and processed using
a tissue homogenizer. After centrifugation of the material, supernatants
were collected and strained to remove any residual fatty deposits. Cells
were harvested, counted, and frozen in a solution containing 30% RPMI
1640 (CellGro, Herndon, VA), 60% fetal calf serum (Hyclone, Logan,
UT), and 10% dimethylsulfoxide. Long-term storage in liquid nitrogen
preserved cells for use in the ELISPOT experiments.
ELISPOT assay
Ninety-six-well nitrocellulose-backed plates were coated with anti-
human interferon (IFN)-
mAb (Endogen, Inc., Cambridge, MA) or
antihuman IL-5 (Endogen) overnight at 4 C. The plates were washed,
and blocking buffer (PBS/BSA) was added to the wells for 1 h. Plates
were then washed again, and 300,000-thawed pancreatic lymph node
cells/well were plated in the presence of antigen. Candidate antigens
included were B9 –23 peptide of insulin, recombinant human insulin,
GAD peptides 271–285 and 556 –581, peptide 7 of the human IA2
molecule (the structure of this peptide, highly conserved between hu-
man and mouse, is KNRSLAVLTYDHSRI), and the mitogen phytohe-
moagglutinin as a positive control.
The lymphocytes from each subject were run in triplicate. After 16 60
h incubation at 37 C, 5% CO
2
cells were washed and stained with
antihuman IFN
(Endogen) or antihuman IL-5 (BD PharMingen, San
Diego CA), a secondary biotinylated antibody (Pierce, Rockford, IL), and
peroxidase-conjugated avidin (Dako, Carpinteria, CA). The reaction was
then developed with N, N-dimethylformamide (Pierce) and 3-amino-
9-ethyl carbazole (Pierce). Spots derived from cytokine-producing cells
were quantified using the Series-1 immunospot and satellite analyzers
(CTL Inc., Cleveland, OH). For each well the results were expressed as
the difference between the spots counted and the background for that
particular subject (i.e. the number of spots counted in the absence of the
stimulated peptide). The values obtained from the antibody-positive
subject and the three negative controls were compared using the Whit-
ney Mann rank test.
Results
To date, 112 sera of cadaveric organ donors have been
analyzed for autoantibodies reacting with GAD65, IA-2, and
insulin by radioassay. Fifty-nine males and 53 females with
a mean age of 39.14 yr (sd 18. 6 yr), a median age of 42.1 yr,
and an age range of 1 month to 86 yr composed this popu-
lation. Seventeen percent of the these organ donors were
younger than 18 yr of age The cause of death in this popu-
lation of organ donors was represented by cerebrovascular
events (38%), trauma (22%), and gunshot wounds (9%). The
remainder of the donors succumbed to meningitis, heart
attack, brain tumor, and drug overdose.
None of these donors had a clinical diagnosis of pancreatic
disease (i.e. chronic or acute pancreatitis or pancreatic can-
cer). At the time of the hospitalization, the mean glucose level
in this group was 191 mg/ml (with a sd of 80) and the median
level was 169 mg/ml. As shown in Fig. 1, one donor had
autoantibodies reacting with IA-2 (donor 7) (autoantibodies
reacted with multiple IA-2 constructs including IA-2 iC,
ICA512 BDC, and full length IA-2); one had autoantibodies
reacting to GAD65 (donor 66); and a third donor had auto-
antibodies to both GAD65 and IA-2 (donor 37). The pancreas
of donor 7 was obtained, whereas the pancreata of donors 37
and 66 could not be obtained, in one case because of meta-
bolic decompensation preceding donation of the organs and
in the other because of surgical unavailability. Among the
102 organ donor antibody positive for whom HLA DR typing
FIG. 1. Autoantibody levels [for GAD65,
IA-2 (ICA512), and insulin autoanti-
bodies] in all 112 organ donors studied.
The solid line representing 3 SD above
the mean of normal controls indicates
the cut-off for positivity. One subject
was positive for both GAD65 and IA-2
(ICA512) antibodies. mIAA, Micro IAA.
Gianani et al. Islet Autoantibodies in Organ Donors J Clin Endocrinol Metab, May 2006, 91(5):1855–1861 1857
was available, only five subjects were positive for the type 1A
diabetes high-risk genotype DR3/DR4, whereas 43% were
DR4 or DR 3 positive. The DR HLA genotype of the IA-2
antibody-positive subject (donor 7) was DR2/DR3 (having
one high risk allele and the protective DR2 allele), whereas
the DRs of the other two antibody-positive subjects, donors
37 (positive for GAD65 and IA-2 antibodies, age 63 yr) and
66 (positive for GAD65 antibodies, age 23 yr), were DR8/
DR14 and DR4/DR3, respectively. Thus, one of the donors
positive for islet antibodies was one of five with the high-risk
DR4/3 genotype.
Donor 7 was a 47-yr-old male with no clinical history of
diabetes or pancreatic disease. The major feature of this pan-
creas was represented by the presence of multiple areas of
fibrosis scattered throughout the organ. These multifocal
areas of fibrosis were also present in two of 14 antibody-
negative organ donors (donors 21 and 41).
The pancreas of donor 7 contained numerous small and
medium-size islets that were often associated with ducts
(islet ductal complexes) (Fig. 2A) as well as clusters of glu-
cagon or insulin-positive cells immediately adjacent to the
epithelium lining the ducts (Fig. 2B). Furthermore, in several
ducts the epithelial cells lining the ducts exhibited strong
positivity for both cytokeratin 19 (a marker of ductal cells)
and chromogranin (a marker of endocrine cells) [Fig. 3, A
(cytokeratin 19) and B (chromogranin)]. Cytokeratin 19-
chromogranin double-positive cells were not seen in any of
the control adult pancreata (n 14) from antibody-negative
subjects but were seen in fetal pancreas [Fig. 3, C (cytokeratin
19), and D (chromogranin)].
Morphometric analysis revealed that there was no reduc-
tion of the
-cell area (or statistically significant increase in
the ratio of glucagon/insulin stained areas) in comparison
with the antibody-negative controls (and in particular in
comparison with the three controls matched for sex and age).
Interestingly, however, the pancreata containing multiple
areas of fibrosis (D7, D21, and D41) had as a group an in-
crease ratio of endocrine (insulin plus glucagon stained ar-
eas) vs. total pancreatic area (P 0.01) in relation to the
pancreata without fibrosis.
Table 2 summarizes the weight of each pancreas, the ratio
between insulin- and glucagon-stained tissue, and the ratio
between endocrine tissue (expressed as the sum of insulin
and glucagon stained areas) and total pancreatic area in each
case.
Immunostaining for LCA (CD45) revealed that all the pan-
creata examined contained scattered leukocytes. Statistical
analysis also revealed that the number of leukocytes outside
the islet tissue was not significantly higher in the antibody-
positive subject than in the controls. Further immunohisto-
chemical characterization of the inflammatory cells scattered
throughout the pancreas in the antibody-positive subject and
the control revealed that the majority of these cells were CD8
lymphocytes.
After examining multiple sections of donor 7 and all of the
control pancreata, we could not detect more than five CD45-
positive cells (up to four leukocytes found within the islets
of antibody negative subjects) within any of the islets exam-
ined. Furthermore, there was no statistical significance dif-
ference between the antibody-positive and control subjects in
the number of CD45-positive cells within the 10 islets with
the largest mononuclear infiltrate. Table 3 shows the average
number of leukocytes in the pancreatic tissue and the islets
for each case examined. Figure 4 shows double immuno-
staining of this pancreas and a control pancreas for LCA and
insulin.
The pancreatic lymph nodes from the antibody-positive
subject and three antibody-negative controls yielded be-
tween 60 million and 90 million viable lymphocytes. All the
donors tested responded to phytohemoagglutinin, confirm-
ing the functionality and viability of the thawed lymphocytes
obtained from the pancreatic lymph nodes. We then mea-
sured reactivity to islet-associated antigens using IFN
and
IL-5 productions in ELISPOT assay.
The cells from the antibody-positive donor responded
with IFN
production only to the IA-2
epitope tested (IFN
spots 19 3. 2/300,000 cells) but not to any of the other
antigens. None of the three controls responded to this pep-
tide (all 5 1 spots/300,000 cells) (P 0.05) or any of the
other antigens. We did not detect an IL-5 response to the islet
antigens tested in donor 7 or in the antibody-negative con-
trols. Figure 5 shows the ELISPOT measuring IFN
secretion
from the IA-2 autoantibody-positive organ donor lympho-
cytes in response to stimulation with peptide 7 of IA-2
.
FIG. 2. A, Area of fibrosis containing islets (arrows) adjacent to the ductal epithelium. B, Duct lined by cells positive for glucagon (in red,
indicated by the red arrow) and insulin (green, indicated by the green arrow).
1858 J Clin Endocrinol Metab, May 2006, 91(5):1855–1861 Gianani et al. Islet Autoantibodies in Organ Donors
Discussion
To date, we have screened 112 organ donors with the
identification of two subjects positive for IA-2 autoantibodies
and a third subject positive for GAD65 autoantibodies. There
was a wide age range of the subjects with 17% of the subjects
below the age of 18 yr. As expected, a significant percentage
of the subjects had either DR3 or DR4 alleles (43%), whereas
TABLE 2. Summary of the pancreatic weights, ratio between
glucagon and insulin stained areas (ratio G/I), and ratio between
the endocrine and total pancreatic tissue [ratio (I G)/total area]
in the donors studied (for each value, calculated as the average of
five microscopic fields, SEM is shown)
Donor
no.
Pancreatic
weight (g)
Ratio G/I
Ratio (I G)/total
area (%)
7
a
72 1.02 0.26 11.28 1.03
9 117 0.68 0.11 5.59 1.61
13 87 0.4 0.03 7.89 1.67
17 135 0.78 0.12 3.81 1.57
21
a
67 0.67 0.28 9.99 1.33
26 79 1.14 0.14 8.81 0.73
29 91 0.44 0.19 6.12 2.14
36 74 0.81 0.21 3.88 0.39
39 93 0.91 0.09 7.57 0.81
41
a
91 1.21 0.16 14.76 2.42
46 58 0.71 0.31 2.21 0.61
53 102 0.93 0.53 4.93 1.1
56 96 0.02 0.008 2.04 1.23
59 73 0.28 012 3.99 1.88
61 119 0.49 0.1 4.86 0.38
a
Donors with pancreatic fibrosis.
TABLE 3. Summary of the average number of leukocytes in the
pancreas outside and inside the islets for each case
Donor
no.
Leukocytes in pancreatic
tissue outside the islets per
mm
2
of tissue, average (SEM)
Leukocytes within the 10 islets
with the largest mononuclear
infiltrate, average (
SEM)
7
a
0.01 (0.025) 0.4 (0.307)
9 0.05 (0.011) 0.1 (0.1)
13 0.02 (0.013) 0.4 (0.267)
17 0.01 (0.006) 0.0 (0)
21 0.01 (0.013) 1.1 (0.407)
26 0.02 (0.003) 0.1 (0.1)
29 0.01 (0.009) 0.1 (0.1)
36 0.01 (0.001) 0.2 (0.133)
39 0.01 (0.001) 0.9 (0.334)
41 0.02 (0.004) 0.4 (0.214)
46 0.01 (0.003) 0.1 (0.1)
53 0.01 (0.001) 2.1 (0.334)
56 0.02 (0.003) 0.3 (0.153)
59 0.04 (0.008) 0.2 (0.133)
61 0.02 (0.027) 0.0 (0.03)
For each value, calculated as the average of 10 microscopic fields
(for the leukocytes in the pancreatic tissue outside the islets) or 10
islets (for the leukocytes within the islets).
a
The donor positive for IA-2 antibodies.
FIG. 3. Staining of the pancreas of the IA-2 antibody-positive subject (A and B) and fetal pancreas (C and D) with a cytokeratin 19 antibody
(green, A and C) and chromogranin antibody (red) plus cytokeratin 19 antibody (B and D). The pancreas of the IA-2 antibody-positive subject
shows a duct lined by hyperplastic cells. A subset of these cells is cytokeratin 19 and chromogranin positive (in yellow, indicated by the red
arrow). The duct is surrounded by clusters of chromogranin-positive cytokeratin 19-negative cells (indicated by the red arrow). In the fetal
pancreas, there are cells budding from the ductal lumen that are stained with both antibodies (indicated by green arrow in D).
Gianani et al. Islet Autoantibodies in Organ Donors J Clin Endocrinol Metab, May 2006, 91(5):1855–1861 1859
four had the highest-risk DR3/DR4 genotype, including the
one subject positive for GAD65 autoantibodies. Consistent
with the present study, our previous retrospective study
indicated that only two of 777 sera from cadaveric donors
were positive for IA-2 autoantibodies (10).
Pancreas was obtained from one of three autoantibody-
positive subjects. The pancreatic histology in this IA-2 au-
toantibody-positive case for which the pancreas could be
obtained revealed alteration in the islet architecture.
The pancreas of this subject showed features that are found
in normal fetal but not adult pancreas. These included nu-
merous islet-like clusters associated with ducts and the pos-
itivity of some ductal cells for both chromogranin and cy-
tokeratin 19.
Morphometric analysis revealed that in this pancreas there
was no loss of
-cells. This may signify either that
-cell loss
did not occur in this subject or that if it did occur, it was
compensated by regeneration or new
-cell formation. In-
terestingly, morphometry also revealed that individuals
whose pancreata contained areas of fibrosis had an increased
ratio between area of endocrine tissue and total pancreatic
area. Because the pancreata with fibrosis had normal weight
(see Table 2), it is unlikely that this was due to a loss of
exocrine tissue (as seen in patients with chronic pancreatitis).
To our knowledge, this is the first report that describes the
histology of the pancreas in an islet autoantibody-positive
nondiabetic cadaveric donor. Given the pancreatic histology
in this subject’s pancreas, there was unusual endocrine tissue
potentially resulting from replication of duct-associated en-
docrine cells or neogenesis.
The results of the ELISPOT experiments indicate that the
pancreatic lymph node of the IA-2 autoantibody subject con-
tains IA-2
-reactive T cells producing IFN
. In this subject
we did not identify T cell reactivity (by ELISPOT) for insulin
and GAD65. A recent report (13) shows that insulin autore-
active T cells could be recovered from pancreatic lymph
nodes of diabetic subjects. It is possible that the lack of T cell
reactivity for insulin and GAD in this subject is related to his
lack of humoral immunity for these autoantigens. In addi-
tion, we did not detect insulitis, raising the possibility that the
presence of the single islet autoantibody was not specifically
related to pancreatic disease. This subject had a DR genotype
DR2/DR3 that has been associated with presence of auto-
antibodies but strong protection from progression to type 1
diabetes (14). Bottazzo and co-workers (15) similarly did not
find insulitis in two pancreata from autopsy specimens of
nondiabetic GAD65 autoantibody-positive subjects. We have
evaluated 22 pancreatic blocks and more than 44 sections
from this pancreas but still could have potentially missed
focal insulitis.
It is also likely that individuals expressing more than one
autoantibody (or diabetic subjects as in the study cited above)
will have a higher probability of insulitis with T cell reactivity
to multiple islet antigens.
Given the expected frequency of positive donors, it will be
important to expand similar screening to multiple sites and
screen a larger number of autoantibody-positive and nega-
tive subjects with the major aim of characterizing both nor-
mal pancreas and the pancreatic response to injury and ob-
tain islet infiltrating T lymphocytes.
Acknowledgments
Received May 25, 2005. Accepted February 8, 2006.
Address all correspondence and requests for reprints to: Roberto
Gianani, The Barbara Davis Center, 1775 North Ursula Street, Aurora,
Colorado 80010. E-mail: roberto.gianani@uchsc.edu.
This work was supported by Autoimmunity Prevention Center Grant
U19 AI50864, Diabetes Endocrine Research Center Grant P30 DK57516,
National Institutes of Health Grant DK62718, and Islet Cell Resource
Grant U42RR16599.
The authors have no conflict of interest.
FIG. 5. A, ELISPOT assay (staining for IFN
) of pancreatic lymph
nodes incubated with peptide 7 of phogrin. B, The same assay without
peptide.
FIG. 4. Double-immunofluorescence staining with insulin (blue) and LCA (red) of sections of pancreas from the autoantibody-positive subject
(A) and an antibody-negative control (B). In all sections examined (from both the antibody positive subject pancreas and the controls), the
majority of the inflammatory cells were outside the islets.
1860 J Clin Endocrinol Metab, May 2006, 91(5):1855–1861 Gianani et al. Islet Autoantibodies in Organ Donors
References
1. Verge CF, Gianani R, Kawasaki E, Yu L, Pietropaolo M, Jackson RA, Chase
HP, Eisenbarth GS 1996 Prediction of type I diabetes in first-degree relatives
using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies.
Diabetes 45:926 –933
2. Bingley PJ, Bonifacio E, Williams AJK, Genovese S, Bottazzo GF, Gale EAM
1997 Prediction of IDDM in the general population: strategies based on com-
binations of autoantibody markers. Diabetes 46:1701–1710
3. Achenbach P, Kelemen K, Wegmann DR, Hutton JC 2002 Spontaneous pe-
ripheral T-cell responses to the IA-2
(phogrin) autoantigen in young nonobese
diabetic mice. J Autoimmun 19:111–116
4. Kelemen K, Gottlieb PA, Putnam AL, Davidson HW, Wegmann DR, Hutton
JC 2004 HLA-DQ8-associated T cell responses to the diabetes autoantigen
phogrin (IA-2
) in human prediabetes. J Immunol 172:3955–3962
5. Kawasaki E, Hutton JC, Eisenbarth GS 1996 Molecular cloning and charac-
terization of the human transmembrane protein tyrosine phosphatase homo-
logue, phogrin, an autoantigen of type 1 diabetes. Biochem Biophys Res Com-
mun 227:440 447
6. Rafaeloff R, Barlow SW, Rosenberg L, Vinik AI 1995 Expression of Reg gene
in the Syrian golden hamster pancreatic islet regeneration model. Diabetology
38:906–913
7. Huang HP, Chu K, Nemoz-Gaillard E, Elberg D, Tsai MJ 2002 Neogenesis of
-cells in adult
2/NeuroD-deficient mice. Mol Endocrinol 16:541–551
8. O’Reilly LA, Gu D, Sarvetnick N, Edlund H, Phillips JM, Fulford T, Cooke
A 1997
-Cell neogenesis in an animal model of IDDM. Diabetes 46:599 606
9. Dor Y, Brown J, Martinez OI, Melton DA 2004 Adult pancreatic
-cells are
formed by self-duplication rather than stem-cell differentiation. Nature 429:
41–46
10. Maniatis AK, Yu L, Miao D, Nelson K, Eisenbarth GS 2001 Rapid assays for
detection of anti-islet autoantibodies: implications for organ donor screening.
J Autoimmun 16:71–76
11. Kawasaki E, Yu L, Gianani R, Verge CF, Babu S, Bonifacio E, Eisenbarth GS
1997 Evaluation of islet cell antigen (ICA) 512/IA-2 autoantibody radioassays
using overlapping ICA512/IA-2 constructs. J Clin Endocrinol Metab 82:375–
380
12. Ambrosini G, Adida C, Altieri DC 1997 A novel anti-apoptosis gene, survivin,
expressed in cancer and lymphoma. Nat Med 3:917–921
13. Kent SC, Chen Y, Bregoli L, Clemmings SM, Kenyon NS, Ricordi C, Hering
BJ, Hafler DA 2005 Expanded T cells from pancreatic lymph node of type 1
diabetic subjects recognize an insulin epitope. Nature 435:224–228
14. Pugliese A, Gianani R, Moromisato R, Awdeh ZL, Alper CA, Erlich HA,
Jackson RA, Eisenbarth GS 1995 HLA-DQB1*0602 is associated with domi-
nant protection from diabetes even among islet cell antibody-positive first-
degree relatives of patients with IDDM. Diabetes 44:608613
15. Wagner R, McNally JM, Bonifacio E, Genovese S, Foulis A, McGill M,
Christie MR, Betterle C, Bosi E, Bottazzo GF 1994 Lack of immunohistological
changes in the islets of nondiabetic, autoimmune, polyendocrine patients with
-selective GAD-specific islet cell antibodies. Diabetes 43:851– 856
JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the
endocrine community.
Gianani et al. Islet Autoantibodies in Organ Donors J Clin Endocrinol Metab, May 2006, 91(5):1855–1861 1861
    • "It is somewhat better characterized today and we know that the most frequent cell types are CD8 lymphocytes, followed by macrophages, B cells and CD4 T cells (Willcox et al. 2009 ). However, only a few studies have been carried out in non-diabetic, autoantibody positive (Ab+) donors, with the majority of the donors showing no leukocytic infiltration or beta cell damage (Gianani et al. 2006; In't Veld et al. 2007; Wagner et al. 1994). The Network for Pancreatic Organ Donors with Diabetes (nPOD) has now opened up the unique possibility of investigating and characterizing the histopathological presentation of all the stages of the disease, from the pre-diabetic to the chronic state. "
    [Show abstract] [Hide abstract] ABSTRACT: Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells are destroyed in the islets of Langerhans. One of its main pathological manifestations is the hyper-expression of Major Histocompatibility Complex I (MHC-I) by beta cells, which was first described over 3 decades ago yet its cause remains unknown. It might not only be a sign of beta cell dysfunction but could also render the cells susceptible to autoimmune destruction; for example, by islet-infiltrating CD8 T cells. In this report, we studied pancreas tissue from a 22-year-old non-diabetic male cadaveric organ donor who had been at high risk of developing T1D, in which autoantibodies against GAD and IA-2 were detected. Pancreas sections were analyzed for signs of inflammation. Multiple insulin-containing islets were identified, which hyper-expressed MHC-I. However, islet density and MHC-I expression exhibited a highly lobular and heterogeneous pattern even within the same section. In addition, many islets with high expression of MHC-I presented higher levels of CD8 T cell infiltration than normal islets. These results demonstrate the heterogeneity of human pathology that occurs early during the pre-diabetic, autoantibody positive phase, and should contribute to the understanding of human T1D. © The Author(s) 2015.
    Article · Aug 2015
    • "During the last few years, many papers have reported novel findings based on the study of nPOD samples [ 1. A key question posed when nPOD was established was whether individuals with autoantibody (single or multiple ) would have insulitis in the pancreas [26, 48, 49]. To date, insulitis has been demonstrated in two of three nondiabetic donors with multiple autoantibodies but not in any of the 18 donors with a single autoantibody. "
    [Show abstract] [Hide abstract] ABSTRACT: The Juvenile Diabetes Research Foundation (JDRF) Network for Pancreatic Organ Donors with Diabetes (JDRF nPOD) was established to obtain human pancreata and other tissues from organ donors with type 1 diabetes (T1D) in support of research focused on disease pathogenesis. Since 2007, nPOD has recovered tissues from over 100 T1D donors and distributed specimens to approximately 130 projects led by investigators worldwide. More recently, nPOD established a programmatic expansion that further links the transplantation world to nPOD, nPOD-Transplantation; this effort is pioneering novel approaches to extend the study of islet autoimmunity to the transplanted pancreas and to consent patients for postmortem organ donation directed towards diabetes research. Finally, nPOD actively fosters and coordinates collaborative research among nPOD investigators, with the formation of working groups and the application of team science approaches. Exciting findings are emerging from the collective work of nPOD investigators, which covers multiple aspects of islet autoimmunity and beta cell biology.
    Full-text · Article · Oct 2014
    • "Development of IA-2A and/or ZnT8A confer a high and age-independent risk of approximately 50 % of developing the disease within a 5-year period. Studies on autoantibody-positive non-diabetic organ donors surprisingly showed only limited evidence of islet lesions and beta cell damage [29, 37,575859. The cumulative data from these studies show that only two out of the 72 autoantibody-positive subjects showed diabetes-related histopathological changes [37]. "
    [Show abstract] [Hide abstract] ABSTRACT: Human type 1 diabetes (T1D) is considered to be an autoimmune disease, with CD8+ T-cell-mediated cytotoxicity being directed against the insulin-producing beta cells, leading to a gradual decrease in beta cell mass and the development of chronic hyperglycemia. The histopathologically defining lesion in recent-onset T1D patients is insulitis, a relatively subtle leucocytic infiltration present in approximately 10 % of the islets of Langerhans from children with recent-onset (<1 year) disease. Due to the transient nature of the infiltrate, its heterogeneous distribution in the pancreas and the nature of the patient population, material for research is extremely rare and limited to a cumulative total of approximately 150 cases collected over the past century. Most studies on the etiopathogenesis of T1D have therefore focused on the non-obese diabetic (NOD) mouse model, which shares many genetic and immunological disease characteristics with human T1D, although its islet histopathology is remarkably different. In view of these differences and in view of the limited success of clinical immune interventions based on observations in the NOD mouse, there is a renewed focus on studying the pathogenetic process in patient material.
    Article · Jul 2014
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