Pancreas Pathology of Latent Autoimmune Diabetes in ......Pancreas Pathology of Latent Autoimmune Diabetes in Adults (LADA) in Patients and in a LADA Rat Model Compared to Type 1 Diabetes

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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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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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Supplementary Table 2. Data on the human pancreas organ donor patients with T1DM


Age (years) 39 68 29 41

Gender female (♀) male (♂) male (♂) male (♂)


Donor organ

Donor organ

Donor organ

Pancreas resection

Duration of diabetes (months)

23 33 8 4

Therapy insulin insulin insulin Insulin


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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Supplementary Table 3. Data on the human pancreas organ donor patients with LADA


Age (years) 45 54 50 65

Gender female (♀) female (♀) female (♀) male (♂)


Pancreas resection

Pancreas resection

Pancreas resection

Pancreas resection


3

10

12

10

Insulin independence (months)

7

6

5

5

Therapy insulin insulin insulin Insulin


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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Supplementary Table 4. Data on the human pancreas organ donor patients with T2DM


Age (years) 75 65 54 66

Gender female (♀) male (♂) male (♂) female (♀)


Pancreas resection

Pancreas resection

Pancreas resection

Pancreas resection


72 68 24 56

Therapy diet anti-diabetic drug anti-diabetic drug anti-diabetic drug


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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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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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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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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Supplementary Table 8. Sequences of primers used for in situ PCR analysis of human pancreas


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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Supplementary Table 9. Sequences of primers used for in qPCR analysis of rat pancreas


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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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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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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Pancreas Pathology of Latent Autoimmune Diabetes in ......Pancreas Pathology of Latent Autoimmune Diabetes in Adults (LADA) in Patients and in a LADA Rat Model Compared to Type 1 Diabetes