pancreas pathology of latent autoimmune diabetes in ......pancreas pathology of latent autoimmune...
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Pancreas Pathology of Latent Autoimmune Diabetes in Adults (LADA) in Patients and in a LADA Rat Model Compared to Type 1 Diabetes Mellitus Anne Jörns, Dirk Wedekind, Joachim Jähne, and Sigurd Lenzen ,
Laser Capture Microdissection of Pancreatic Islets with and without Immune Cell Infiltration For laser capture microdissection islets from IDDM rats with the LADA form showing areas with and without islet infiltration and with T1DM form were collected from pancreatic sections. Under RNase-free conditions formaldehyde fixed paraffin embedded (FFPE) pancreatic sections of the IDDM rats were cut (4 µm thickness) and mounted on PEN-membrane coated slides (Arcturus Bioscience, Mountain View, CA). The FFPE sections were deparaffinized, de- and thereafter rehydrated without a specific staining. Using a cyanine 2 (Cy2) filter 50 to 100 islets were identified by the strong auto- fluorescence, marked, and microdissected from the exocrine parenchyma with an infrared laser from the automated Veritas System (Arcturus). The infrared laser is best suited for conservation of dissected tissue. After collecting tissue the RNA extraction was performed of around 50 islet equivalents with the Paradise Plus Extraction Kit (Arcturus) according to the manual. Quantification of RNA was performed with the NanoDrop instrument (VWR, Darmstadt, Germany). The starting amount of the RNA has been amplified with the Full Spectrum Complete Transcriptome RNA Amplification Kit (System Biosciences, Palo Alto, CA). The amplified RNA was used as a template for the qRT-PCR analyses.
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Supplementary Table 1. Data on the healthy human pancreas organ donors
Donor 1 Donor 2 Donor 3 Donor 4
Age (years) 38 67 29 40
Gender female (♀) male (♂) female (♀) male (♂)
Cause of pancreas removal
Donor organ
Donor organ
Donor organ
Pancreas resection
Blood glucose conc. (mmol/l) before organ collection
6.7 7.2 5.1 5.7
The pancreases from donors # 1 to 3 were obtained during 2003-2007 in Pisa and from patient # 4 in 2009 in Hannover. The pancreatic tissues were handled and processed according to the recommendations of the Pisa and Hannover Ethics Committees.
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Supplementary Table 2. Data on the human pancreas organ donor patients with T1DM
Donor 1 Donor 2 Donor 3 Donor 4
Age (years) 39 68 29 41
Gender female (♀) male (♂) male (♂) male (♂)
Cause of pancreas removal
Donor organ
Donor organ
Donor organ
Pancreas resection
Duration of diabetes (months)
23 33 8 4
Therapy insulin insulin insulin Insulin
Blood glucose conc. (mmol/l) before organ collection
26.7 12.2 7.1 15.7
The pancreases from donors # 1 to 3 were obtained during 2001-2005 in Pisa and from patient # 4 in 2009 in Hannover. The pancreatic tissues were handled and processed according to the recommendations of the Pisa and Hannover Ethic Committees.
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Supplementary Table 3. Data on the human pancreas organ donor patients with LADA
Donor 1 Donor 2 Donor 3 Donor 4
Age (years) 45 54 50 65
Gender female (♀) female (♀) female (♀) male (♂)
Cause of pancreas removal
Pancreas resection
Pancreas resection
Pancreas resection
Pancreas resection
Duration of diabetes (months)
3
10
12
10
Insulin independence (months)
7
6
5
5
Therapy insulin insulin insulin Insulin
Blood glucose conc. (mmol/l) before organ collection
10.7 10.8 8.9 12.8
GAD65 Auto-antibody
positive positive positive positive
The pancreases from patients 1-4 were obtained from 2010 in Hannover. The pancreatic tissues were handled and processed according to the recommendations of the Hannover Ethics Committee.
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Supplementary Table 4. Data on the human pancreas organ donor patients with T2DM
Donor 1 Donor 2 Donor 3 Donor 4
Age (years) 75 65 54 66
Gender female (♀) male (♂) male (♂) female (♀)
Cause of pancreas removal
Pancreas resection
Pancreas resection
Pancreas resection
Pancreas resection
Duration of diabetes (months)
72 68 24 56
Therapy diet anti-diabetic drug anti-diabetic drug anti-diabetic drug
Blood glucose conc. (mmol/l) before organ collection
7.8 9.7 8.8 7.5
The pancreases from patients 1-4 were obtained from 2010 in Hannover. The pancreatic tissues were handled and processed according to the recommendations of the Hannover Ethics Committee.
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Supplementary Table 5. Blood glucose concentration, age of diabetes manifestation, and duration of insulin independence in T1DM and LADA form in the LEW.1AR1-iddm (IDDM) rat compared to the non-diabetic, healthy IDDM rats.
Group Parameter
IDDM rat non-diabetic
IDDM rat T1DM
IDDM rat LADA
N 8 8 8
Blood glucose concentration (mmol/l)
5.2 ± 0.2 17.4 ± 1.2 14.8 ± 1.1
Onset of diabetes (day)
0 59 ± 2 87 ± 4
Insulin independence(days)
whole period 1-3 45-180
The LEW.1AR1-iddm rats were obtained from the Hannover Medical School animal facility.
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Supplementary Table 6. Primary antibodies used for immunohistochemistry
Species Peptide/protein Target
Clone Manufacturer, catalog Species raised in
All species Insulin Agilent; A0564 guinea pig; polyclonal
All species CD45 Abcam; ab10558 rabbit; polyclonal
Rat CD68 ED1 Bio-Rad; MCA341R mouse; monoclonal
Human CD68 514H12 Bio-Rad; MCA1815 mouse; monoclonal
Rat CD3 1F4 Bio-Rad; MCA772 mouse; monoclonal
Human CD3 UCHT1 Bio-Rad; MCA463G mouse; monoclonal
Rat CD4 OX-35 Bio-Rad; MCA153GA mouse; monoclonal
Human CD4 1F6 Acris; DM119 mouse; monoclonal
Rat CD8 OX-8 Bio-Rad; MCA48R mouse; monoclonal
Human CD8 4B11 Bio-Rad; MCA1817 mouse; monoclonal
Rat CD161 10/78 Bio-Rad; MCA1427GA mouse; monoclonal
Human CD161 B199.2 Bio-Rad; MCA1855GA mouse; monoclonal
Rat CD20 (IgD) MARD-3 Bio-Rad; MCA190 mouse; monoclonal
Human CD20 7D1 Bio-Rad; MCA1807 mouse; monoclonal
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Supplementary Table 7. Sequences of primers used for in situ PCR analysis of rat pancreas
Gene F (forward) R (reverse)
Primer sequence
Actb F R
5’-ACAGCTGAGAGGGAAATCGT-3’ 5’-CTGCTTGCTGATCCACATCT-3’
Il1b F R
5’-GATGTTCCCATTAGACAGCTGCACTG-3’ 5’-CTTTTCCATCTTCTTCTTTGGGTATGGT-3’
Ifng F R
5’-GCTCTGCCTCATGGCCCTCTC-3’ 5’-TGTTGCTGATGGCCTGGTTGTC-3’
Tnf F R
5’-CTACTGAACTTCGGGGTGATCGGTC-3’ 5’-CTGGTATGAAGTGGCAAATCGGCT-3’
Casp3 (cpp32)
F R
5’-TGGTACCGATGTCGATGCAG-3’ 5’-TACCGCAGTCCAGCTCTGTACC-3’
Il10 F R
5’-TGCACCCACTTCCCAGTCAGC-3’ 5’-CACCTGCTCCACTGCCTTGC-3’
Insulin2 F R
5’-CAACATGGCCCTGTGGA-3’ 5’-AGAGAGAGCAGATGCTGGTG-3’
Inos (Nos2) F R
5’-CCGCTTTGCCACGGAAGAGA-3’ 5’-TGTACCGGGCCCAATGAGGA-3’
Pcna F R
5’-CGTCTCACGTCTCCTTAGTGC-3’ 5’-GATCACCACAGCATCTCCAAT-3’
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Supplementary Table 8. Sequences of primers used for in situ PCR analysis of human pancreas
Gene F (forward) R (reverse)
Primer sequence
ACTB F R
5’-ACGGCCGAGCGGGAAATCGT-3’ 5’-CTGCTTGCTGATCCACATCT-3’
IL1B F R
5’-CTTCTTCGACACATGGGATAACGAG-3’ 5’-CACAGGACTCTCTGGGTACAGCTCT-3’
IFNG F R
5’-TGCAGAGCCAAATTGTCTCCTTTTAC-3’ 5’-GGATGCTCTTCGACCTCGAAACAGCA-3’
TNF F R
5’-CTTCTCCTTCCTGATCGTGGCA-3’ 5’-ATTGATCTCAGCGCTGAGTCGG-3’
CASP3 (CPP32)
F R
5’-TGGTACAGATGTCGATGCAGC-3’ 5’-TGCCACAGTCCAGTTCTGTACC-3’
IL10 F R
5’-GCACCCACTTCCCAGGCAAC-3’ 5’-TGCTCCACGGCCTTGCTCTT-3’
Insulin F R
5’-CCGGGAGGCAGAGGACCTG-3’ 5’-CCTGCGGGCTGCGTCTAGTT-3’
PCNA F R
5’-GTAAACCTGCAGAGCATGGA-3’ 5’-CGGCATATACGTGCAAATTC-3’
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Supplementary Table 9. Sequences of primers used for in qPCR analysis of rat pancreas
Gene F (forward) R (reverse)
Primer sequence Efficiency
Il1b F R
5’-CACCTCTCAAGCAGAGCACAG-3’ 5’-GGGTTCCATGGTGAAGTCAAC-3’
1.99
Ifng F R
5’-AAAAAGGATGCATTCATGAGC-3’ 5’-ACTCCTTTTCCGCTTCCTTAG-3’
1.99
Tnf F R
5’-CCAGGAGAAAGTCAGCCTCCT3’ 5’-TCATACCAGGGCTTGAGCTCA-3’
1.99
Casp3 (cpp32)
F R
5’-TTGGAACGAACGGACCTGTGG-3’ 5’-TGTCTCAATACCGCAGTCCAGCTC-3’
1.98
Il10 F R
5’-GGCTCAGCACTGCTATGTTGCC-3’ 5’-AGCATGTGGGTCTGGCTGACTG-3’
1.99
Insulin2 F R
5’-GCCCAGGCTTTTGTCAAACA-3’ 5’-TCCACGCTCCCCACACA-3’
1.98
Inos (Nos2) F R
5’-TCCTGCAGGCTCACGGTCAA-3’ 5’-TCGTACTTGGGATGCTCCATGG-3’
1.99
Pcna F R
5’-TGCCATCTGGTGAATTTGCA-3’ 5'-TCCCCACTCGCAGAAAACTT-3'
1.98
Actb F R
5’-TCCATCATGAAGTGTGACGTT-3’ 5’-CAGGGCAGTAATCTCCTTCTG-3’
1.99
18S rRNA F R
5’-GGACCAGAGCGAAAGCATTTGC-3’ 5’-CGCCAGTCGGCATCGTTTATG-3’
1.99
Rpl32 F R
5’-TGTCCTCTAAGAACCGAAAAGCC-3’ 5’-CGTTGGGATTGGTGACTCTGA-3’
1.99
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Supplementary Figure 1. Gene expression of cytokines, IL-1β, TNF-α, IFN-γ, IL-10 as well as iNOS, Casp3 and PCNA by in situ RT-PCR in the human pancreas from the non-diabetic control situation, from the T1DM and LADA form with and without immune cell infiltration. Representative pictures are shown for each gene and for the different groups. Scale bar 20 µm..
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1
Supplementary Figure 2. Gene expression of cytokines, IL-1β, TNF-α, IFN-γ, IL-10 as well as iNOS, Casp3 and PCNA by in situ RT-PCR in the rat pancreas from the non-diabetic control situation, from the T1DM and LADA form with and without immune cell infiltration. Representative pictures are shown for each gene and for the different groups. Scale bar 20 µm.
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©2020 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1