Type 1 Diabetes (5)

Question 1

Limitations of studying T1D in humans

Answer 1

Not possible to take pancreatic biopsies from at-risk individuals prior to or once diagnosed with T1D
(whatever functional beta cells they have left - not a viable option to do surgery on the pancreas)

Question 2

Difficult to determine whats happening during development of T1D

Answer 2

Need access to key tissues and cells, but only access to:
>peripheral blood of living individuals
>cadaveric donor pancreatic specimens
(dont provide data as to whats happening during disease development)

Question 3

Difficult to identify and confirm genetic and environmental factors in human T1D

Answer 3

> need controlled inbreeding to test affects of genes and their alleles
need controlled isolation to test affects of geographical location, infection, etc

Question 4

Nonobese diabetic (NOD) mouse: A model of human T1D

Answer 4

Spontaneously develop diabetes

Higher rates in females than males (humans about same)

Has to do with sex hormones in mice, if you castrate males then they get higher rates similar to females, giving testosterone to females will lower rates

> lymphocytes mediate specific destruction of insulin-producing Beta cells

Question 5

NOD mice: natural history of T1D

Answer 5

Very consistent development of diabetes in NOD mouse

> day 20: immune cell invasion of pancreas
day 40-120: increasing insulitis and beta cell destruction, progressive loss of insulin
day 120-death (200): clinical diagnosis (hyperglycemia), residual to no insulin secretion

Question 6

Genetic factors: Similarities between mouse and human

Answer 6

Mouse genetic studies identified > 30 T1D susceptibility loci

Humans >40 alleles that increase risk of T1D

Many of the genetic susceptibility loci overlap between mouse and humans

We find more genes in heterogenous humans, because we could have different combinations of loci, mouse are all basically clones

Question 7

The NOD mouse represents a single case study with an unfortunate collection of susceptibility alleles at multiple genes

Answer 7

pretty much all clones of each other

Note: mouse T1D loci termed ldd, human T1D loci termed IDDM

Question 8

Genetic factor: Idd1 and IDDM1

Answer 8

Similar amino acid change for MHC/HLA Class II molecule associated with T1D
(present peptides to CD4+ T cells)

High risk allele genotype for beta chain
>human HLA (DQ2 or DQ8) = DQB1 *0201 or *0302
>Mouse MHC = NOD H2-Ag7

Beta chain position 57 (peptide cleft where beta cell is presented with B-cell receptor)
>human = code for alanine
>mouse = code for serine
>both associated with increased T1D risk

Question 9

NOD mice and Humans have similar amino acid change for MHC/HLA Class II molecule associated with T1D

Answer 9

Salt bridge DISRUPTED when aspartic acid at position 57 is changed to serine or alanine
>associated with susceptibility to T1DM

Salt bridge FORMED between aspartic acid at position 57 in beta chain and arginine on alpha chain
>associated with resistance to T1DM

Question 10

How to prove a.a. change affects development of T1D?

Answer 10

Use gene editing in NOD mouse
>CRISPR-Cas9 mutagenesis to change MHC amino acid

> show that changing 1 aa from serine to aspartic acid is protective

NOD MICE WITH ASPARTIC ACID ARE PROTECTED

Question 11

Environmental factors: Similarities between human and mouse

Answer 11

Many NOD mice breeding facilities around the world with different mouse populations

Different diabetes incidence rates for differnt countries - definitely has environmental factors that affect incidence

Recall: T1D varaince around the world, showed increase incidence further from equator (correlation =/= causation)

Question 12

Environmental Factors: Temperature

Answer 12

Higher temperature reduces incidence of diabetes in NOD mice

what does this mean for humans? temp does it affect? prob not cause weather is seasonal and doesnt always stay 24 degrees but** (add in)

Question 13

Environmental factors: Microbial Pathogens

Answer 13

Can move NOD mouse from a ‘dirty’ to a ‘clean’ environment

Most research places have 1 dirty place and other clean breeding areas

Mice in conventional dirty breeding facilities have lower incidence of diabetes
>T1D incidence in NOD mice is affected by microbial pathogens

Question 14

Environmental Factors: Gut microbiota can affect development of T1D

Answer 14

Observed:
NOD males < NOD females for T1D incidence
NOD males have different gut microbiomes to NOD females

Hypothesis
>NOD females fed gut-derived microbes isolated from NOD males should reduce incidence of T1D

> > > Microbes in gut of NOD males provide protection against development of T1D (not complete protection)

Question 15

NOD mice: immune cell infiltration and insulin autoantibodies

Answer 15

same types of immune cells that invade humans also invade NOD mice
>macrophges, dendritic cells, CD8+ T cells (cytotoxic), CD4+ T cells (proinflammatory cytokines), B cells (autoantibodies)

> insulin autoantibodies can be detected in peripheral blood and precede T1D in NOD mice

Question 16

NOD mice exhibit more extensive insulitis than human with T1D

Answer 16

> invasion by immune cells starts at periphery (peri-insulitis)
(40-80 days)

> around 100 days, see insulitis with beta cell destruction

> at clinical diagnosis, see insulitis with islets lacking beta cells (might not even see the immune cells, destroyed everything then moved out)

Question 17

NOD mice: Autoreactive CD8+ T cells recognise and kill beta cells

Answer 17

CD8+ T cells interact with beta cells via TCR-MHC I (presenting autoantigen peptides)

> activated CD8+ T cells secrete perforin and granzymes to directly kill beta cells by inducing cell death

Question 18

Gene editing to delete the perforin gene in NOD mice

Answer 18

Protects against T1D
>insulitis still occurs, but development of T1D is significantly delayed and reduced
(perforin only involved in death, not involved in immune cell infiltration and not necessary going to prevent the other causes of cell death)

Question 19

Immunological abnormalities in NOD mice

Answer 19

Thymus
>impaired deletion of autoreactive T cells
>beta cell specific T cells escape into periphery

Macrophages
>less efficient at clearing dying cells, produce higher level of inflammatory cytokines (e.g. IL-1B, TNFa)
>increases inflammation in islet and exposure of beta cell antigens

Dendritic cells
>defective maturation and responses to certain stimuli, fail to eliminate autoreactive T cells in the periphery
>enhanced activation of Beta-cell specific T cells

T cells
>enhanced propensity to proliferate and produce IFNgamma
>Regulatory T cells are less effective at suppressing autoreactive CD4+ and CD8+ T cells in the periphery
>increased numbers and enhanced activation of beta-cell specific T cells

Question 20

NOD Mice: Effect of depleting immune cells

Answer 20

Macrophages
>delete by antibody treatment
>reduces T1D incidence

Dendritic cells
>delete by gene editing
>prevents T1D

B cells
>deplete B cells by gene editing or antibody treatment
>prevents T1D

B cells
>do not produce antibodies but can present antigen to T cells
>still develop T1D

B cells
>produce Ab but do not present antigen to T cells
>reduces T1D incidence

CD4+/CD8+ T cells
> deplete both T cells by gene editing or antibody treatment
> Prevent T1D

CD4+/CD8+ T cells
>Transfer both T cells from diabetic mouse to non-diabetic mouse
>cause T1D onset
>NEED BOTH to be transferred to cause onset, if not, doesnt cause T1D (indicates need defective cd4 and cd8)

Question 21

T1D: NOD mice vs Humans (not that impt, will add more later)

Answer 21

Sex bias
>female mice higher T1D incidence,
>humans equal incidence for gender

**

Question 22

Case study: some facts about T1D

Answer 22

Australia relatively high incidence of T1D

~7 new cases/day diagnosed in Aus (not very high, nurse or dr in emergency room isnt going to come across T1D very often)

Family history
>3-5% chance if you have parent with T1D
>8% chance if sibling has T1D, >50% if identical twin

90% of individuals with T1D do not have first degree relative with T1D

Question 23

3 classical signs of DM

Answer 23

polyuria (increased urine)
Polydipsia (increase thirst)
polyphagia (increase hunger)

Question 24

Case study: T1D presentation in ER

Answer 24

Urine sample test (glucose oxidase based test)
>UTI cause same symptoms

High glycosuria (glucose in urine) points to DM
>kidneys dont typically filter glucose from blood until BGL >10mmol/L

Question 25

TO confirm diagnosis of DM

Answer 25

Random venous blood sample taken >11.1mmol/L indicative of DM

sweet smelling breath > acetone (ketone body breakdown)

Test ketone body levels in blood to diagnose diabetic ketoacidosis

Question 26

Diagnosing T1D

Answer 26

95% diabetes diagnosed in children is T1D

Venous blood sample taken for radiobinding assay
>screen for islet cell autoantibodies

Question 27

Why has sally developed T1D?

Answer 27

Autoimmune disease
>insulitis
>lead to activated autoreactive T cells
>mediate destruction of Insulin-producing beta cells

Question 28

Recap what happens in beta cells

Answer 28

>increase BGL
>GLUT 1/2/3 on beta cell
>increase ATP:ADP ratio
>Inhibit K+ Atp dependent channel
>Buildup K+ in cell, depolarise membrane
>voltage-dependent Ca2+ channels open
>influx Ca2+
>trigger exocytosis of Insulin granules

Question 29

So what happens when there are beta cells after insulin released?

Answer 29

If autoimmune response alr started, will be no 1st phase insulin spike, or not enough to control BGL

> glucose-induced insulin secretion is characterised by a rapid first phase and a slower second phase of insulin released into the bloodstream

Question 30

Overview of T1D: destruction of beta cells involves complex interaction between immune cells and the beta cells

Answer 30

APCs activate autoreactive lymphocytes

Activated T cells mediate destruction of insulin-producing beta cells (through perforin/granzyme release)

Beta-cell destruction is exacerbated by release of proinflammatory cytokines and ROS from adaptive and innate immune cells

Activated B cells produce autoantibodies that serve as biomarkers for diagnosing T1D

Question 31

Overview of T1D: Beta cell death Mechanism

Answer 31

Activated CD8+ T cells secrete perforin and granzyme, directly kills beta cells by inducing cell death

Evidence in humans that this happens as well

Cannot delete perforin gene in humans because then PT would be immunosuppressed, higher risk of infections and disease

Question 32

Overview of T1D: Treatment

Answer 32

Exogenous Insulin

Goal: keep glucose conc within healthy range 4-6mmol/L
>minimise hyperglycaemia events
>prevent hypoglycaemia

Insulin injections:
>rapid acting before meals
>long acting in between meals and before sleep
>note that you still have glucagon that will cause BGL to increase

Question 33

HLA locus genotyping

Answer 33

Results for DQ genes
>DQ2 and DQ8 are high risk alleles, but doesnt mean you will develop T1D

What additional tests can be done to better determine if individual is likely to develop T1D?
>autoantibody testing
>testing for insulin response

Question 34

HbA1c levels

Answer 34

8-9% means 8-9% of RBCs have glucopse attached

HbA1c is measurement of glycated hemoglobin
>represents 3 month average of BGL
>higher HbA1c correspnds to higher average BGL

> Person without diabetes HbA1c <5.7%
HbA1c >8% = poor glucose control
Regular high HbA1c levels = increased risk for long-term diabetes-related complications (diabetic neuropathy/retinopathy)
Common target for person with diabtes HbA1c <7%

Question 35

T1D overview: Long term complications

diabetic renopathy

Answer 35

Microvascular complications

• hyperglycaemia = primary risk factor
• intensive management of BGL associated with reduced microvascular disease

Diabetic retinopathy
>new BV formed but poorly supported at back of eye
>can burst and cause haemorrhages
>80% of individuals with T1D develop retinopathy
>can lead to blindness if not managed properly

Question 36

Examining Pancreas of T1D PT post-mortem

Answer 36

Pancreas stained for insulin, glucagon, immune cell markers

> most islets atrophic, little to no insulin staining
(i.e. devoid of beta cells)
when insulin-positive cells are observed, they are scattered as single cells or small clusters in a few isolated parts of the pancreas
a few CD3+ cells were in insulin-positive islets, insulitis not as prevalent as in a person with recent onset T1D

Very little insulin in PTs >50 years of diagnosis

*c-peptide can often still be detected in urine and blood