nPOD

Mark A. Atkinson

The University of Florida

nPOD Mission

• To obtain tissues from organ donors with T1D and

other disorders of related interest

• To distribute tissues to investigators in support of

investigator initiated research projects

• To promote and coordinate tissue and data

sharing, collaboration, manage project interactions

• To lead towards a comprehensive understanding

of human T1D and the identification of new

therapeutic targets

Current Operations

-Type 1 diabetes patients (new onset to long term)

- Autoantibody positive, non-diabetic subjects

- Others “of interest” (i.e., very young, pregnancy,

pancreatitis, type 2 diabetes, CFRD, etc.)

Obtain tissues from:

From these individual donors, obtain: - Pancreas - Spleen

- Pancreatic lymph nodes - Peripheral blood

- Non-pancreatic lymph nodes - Thymus

- Bone marrow - Skin

Processed as fresh cells, frozen/fixed slides, RNAlater,

etc.

Three Phases of nPOD Structure

• Tissue Recovery

– Partnerships with Organ Procurement Organizations

– Partnerships with Screening labs

• Distribution of Specimens

– to our network of investigators, approved and

managed by the Tissue Prioritization Committee and

the Path Core

• Data Collection and Analysis

Green - proposed for this year, 2013

Total nPOD Cases Recovered

Donor Type Count Age Range

Autoantibody Positive, No Type 1 Diabetes

21 2 mos. to 69

Type 1 Diabetes 61 4 to 50

Type 1 Diabetes Medalist 10 59 to 93

Type 2 Diabetes Fulminate Diabetes Transplant

20 1 3

18 to 76 14

38-50

Subtotal with Aab to Diabetes or with Diabetes

116

Other 5 15 to 62

No diabetes (Type 1 or Type 2), Autoantibody Negative

76 0.01 to 75

Pending 5 26 to 51

TOTAL 212

2,440,000 Total Deaths in U.S.

1,250,000 In-Hospital Deaths reported to OPOs

110,000 Potential Cornea Donors

50,000 Potential Tissue Donors

46,000 Cornea Donors recovered

25,000 Tissue Donors recovered

12,500 Potential Organ Donors (Medically Suitable)

6,776 Organ Donors recovered** (Down 20% in 2012)

U.S. Estimated Potential and

Actual Organ and Tissue Donors

Tissue Prioritization Committee

Approved nPOD Projects

There are now

a total of 85

nPOD

approved

studies as of

12/2012

14

24

31

54

63

0

10

20

30

40

50

60

70

2007 2008 2009 2010 2011

84

2012

Growing International Influence

How Do I Obtain nPOD Tissues?

nPOD Online Pathology Users

We believe the increase, to a large extent, has resulted from our continued

outreach programs…especially to the scientific community, as well as improving

name recognition

7

31

114

150

330

0

50

100

150

200

250

300

350

2007 2008 2009 2010 1st half 2011

nPOD Major New Initiatives

• nPOD-E (Europe) – Started June, 2011

• nPOD-T (Transplantation) – Started November, 2011

• nPOD-V (Viral Workgroup) – Organized May, 2012

KEY

QUESTIONS

AND TASKS

The Future - Pushing the Boundaries of

the Forefront of Research

• Obtain partial medical records of nPOD donors

• Develop networks to obtain more new onset

cases

• Establish novel reporting mechanisms for

sharing findings (LabKey) - DataShare

• Serve as a model for other

registries/biorepositories in T1D…encouraging

openness

• International “outposts”

15

nPOD Data Flow: The Future

Image used w/permission, Adam Rauch, LabKey Software™

Organizations are fine, but…

Why study human tissues AND what have we

learned from nPOD?

Diabetes 14:619-633, 1965

InVeldt

Pipeleers and Ling, Diabetes/Metabolism Reviews 8:209, 1992.

Gepts et al, 1965, 1978; ,Doniach et al, 1973; Klöppel et al, 1984; Bottazzo et al, 1985; Foulis et al, 1986; Hänninen et

al, 1992; Somoza et al, 1994; Lernmark et al, 1995; Shimada et al, 1999; Dotta et al, 2007; Uno et al, 2007; Butler et al,

2007, Gianani and Atkinson, 2010.

Duration of disease

≤1 week >1 week -

≤1 year

>1 year total

Onset childhood (0-

14 yrs)

22/23

37/42

3/32

62/97

Onset young adult

(15-39 yrs)

8/14

17/26

1/23

26/63

Meta-Analysis of Insulitis – T1D Patients Stratified to

Age at Onset and Duration of Disease

Need to Understand the Number of

Forms of Type 1 Diabetes

Age (years)

beta-cell

function

(%)

10 20 30 40

100

LADA

Adult Juvenile

Early

child-

hood

Disease Diversity - Medalists

Ki67+Insulin

Amyloid

Insulin+ Medalist (6065) 76 yo FC T1D for 56 yrs

Insulin- Medalist (6066) 78 yo MC T1D for 74 yrs

Islets are large and ~100% non-beta cells

Glucagon

Keenen H et al, Diabetes 2010

Genetic

susceptibility

% Isle

t C

ell

Mass

100

50

0

Inciting

Event(s)

“Brittle”

Diabetes

Time (years)

“Silent”

Cell Loss Diabetes

Onset

The Natural History of Type 1 Diabetes

Adapted from Eisenbarth, 1986

78 yr male Medalist (4 at onset) IA2+

Most islets have no insulin+ but half of sections have scattered insulin+ cells (small

clusters, singlets or doublets);

no TUNEL+ insulin+ cells seen.

insulin

TUNEL

C peptide

undetectable

Beta Cells – Long Standing Type 1

Keenen, Diabetes, 2012

Insulin- Insulitis+ T1D (6062) 10 yo MAA, T1D for 6 yrs

Genotype: DRB1 1/7, DQA1 1/3 Histopath: Ins- islets, CD3+ infiltrates.

CD3+Glucagon

Insulitis Can Persist Long After Disease Onset (but rare)

Time Course of Beta Cell Loss

Linear, Chronic Model Eisenbarth (NEJM 1986, 314:1360)

Benign:Malignant Model Lafferty (J Aut 1997, 10:261)

Random Loss Model Palmer (Diabetes 1999, 48:170)

Age

Age

Beta Cell Mass

Beta Cell Mass

Age

Benign Malignant

Beta Cell Mass

Courtesty BDC T1D site

TYPE 1A DIABETES: VITILIGO OF THE

PANCREAS?

Eisenbarth,

Diabetes

2010

Pancreatic pathology (JDRF nPOD) suggests:

• Sporatic islet destruction (lobular)

• Perhaps a disease of relapse/remission ?

Insulin and Ki67 Staining

The Elusive Insulitis Lesion

Rare in multiple AA+ (IA-2A+?)

Extremely rare in single AA+

Campbell-Thompson, JAMA.

2012

Smaller Pancreas in the Natural History of Type 1

Diabetes

Need to better understand when animal models are

good…and not so good, for studies of type 1 diabetes

?

Cure?

Regeneration?

Immune

responses?

Islets

Mouse versus Man

• Islet cell number/ratio

• Islet cell arrangement

• Basement membrane

• Proliferative capacity

• Ability to increase islet

mass

• Less vascularized

Structure/Growth

• MafB expression

• Components of GSIS

like GLUT2

• Glucose-regulated

gene expression (GSIS

and transcription

factors)

• Amyloid formation

Gene Expression

• Basal insulin secretion

• Fold glucose-

stimulated secretion

• Ca++ oscillations

• Susceptibility to STZ

Function

Courtesy of Al Powers, Vanderbilt Univ

Need to understand beta cells can replicate and if so,

under what conditions

Pregnancy?

Obesity?

Incretins?

Injury?

Rhodes, JCEM, 2012

?

Stages of

Insulitis –

Islet Level

or

Pancreas

Staged at Islet

Level

Present/absent;

number islets

Islet Immune Cells – the Story in Type 1 diabetes

Lymphocyte

CD45+

B cell

CD20+

CD8+

T cells

CD4+

T cells

Insulin+ Macrophage

CD68+

12 year-old male with 1 year of T1D, died of DKA

ICA512+ mIAA+ IA2ic+, Cpep 0.18, BMI 20.3

Ins- islets; Insulitis, esp. peri-islet, in several blocks;

Heterogenous distribution of normal to atrophic islets,

wide variation in sizes, most lobules have numerous islets.

Ins+ islets: Head: none/Body: none/Tail: 3

Zones in Ins- samples are HLA high (stain for GLU).

Tail: ins+ islets are HLA high.

All sections contain considerable amounts of CD8’s,

ins+ zones in the tail are insulitic

Insulin HLA-ABC

DAPI

Insulin CD8

DAPI

6052 tail region

Massive HLA Class I upregulation in islets from recent-onset individuals

Ken Coppieters J Exp Med, 2012

IA2:797-805

IGRP:265-273 IGRP:265-273

In Situ Tetramer Staining (patient 6052)

CD8-APC + anti-APC/biotin

+ Avidin-HRP

NOTHING + anti-APC/biotin

+ Avidin-HRP

Negative controls

Screened for:

Insulin B10-18

IA-2 797-805

IGRP 265-273

PPI 15-23

GAD65 114-

123

ppIAPP 5-13

Ken Coppieters J Exp Med, 2012

Beta Cells

“Downregulate”

Metabolic

Activities in Pre-

diabetes

Courtesy, Clayton Mathews,

nPOD

Therapeutic

opportunities for:

• Beta cell rest?

• Incretins?

Deaf1-EGFP + DF1-VAR

Deaf1

DF1-VAR

(insertion)

(nuclear localization signal)

Deaf1-EGFP only Insulin mRNA DF1-VAR mRNA

Fathman Lab, Stanford University Linda Yip et al. (Nature Immunology, 2009)

Alternative Splicing Produces a Non-functional Deaf1 in T1D that Inhibits Canonical Deaf1

THANK YOU

• Roberto Gianani & George Eisenbarth

• Teo Stavea, Marie Nierras, Dick Insel, Helen Nickerson & the JDRF for their support

• nPOD leadership (Executive Committee members, Chairs, Committee members)

• Scientists

• Anastasia Albanese-O'Neil, John Kaddis, Desmond Schatz, Jayne Moraski, Alberto Pugliese, Suzanne Ball

• Martha Campbell Thompson & Jim Crawford

• Our Cores and their staff

• Our partners (NDRI, IIAM, Kronus, OPO)

• George King, Susan Bonner-Weir, Al Powers

• Organ donors & their families