Type 1 Diabetes (5) Flashcards
Mouse models vs human studies of T1D
Limitations of studying T1D in humans
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)
Difficult to determine whats happening during development of T1D
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)
Difficult to identify and confirm genetic and environmental factors in human T1D
> need controlled inbreeding to test affects of genes and their alleles
need controlled isolation to test affects of geographical location, infection, etc
Nonobese diabetic (NOD) mouse: A model of human T1D
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
NOD mice: natural history of T1D
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
Genetic factors: Similarities between mouse and human
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
The NOD mouse represents a single case study with an unfortunate collection of susceptibility alleles at multiple genes
pretty much all clones of each other
Note: mouse T1D loci termed ldd, human T1D loci termed IDDM
Genetic factor: Idd1 and IDDM1
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
NOD mice and Humans have similar amino acid change for MHC/HLA Class II molecule associated with T1D
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
How to prove a.a. change affects development of T1D?
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
Environmental factors: Similarities between human and mouse
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)
Environmental Factors: Temperature
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)
Environmental factors: Microbial Pathogens
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
Environmental Factors: Gut microbiota can affect development of T1D
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)
NOD mice: immune cell infiltration and insulin autoantibodies
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
NOD mice exhibit more extensive insulitis than human with T1D
> 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)
NOD mice: Autoreactive CD8+ T cells recognise and kill beta cells
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
Gene editing to delete the perforin gene in NOD mice
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)
Immunological abnormalities in NOD mice
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
NOD Mice: Effect of depleting immune cells
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)
T1D: NOD mice vs Humans (not that impt, will add more later)
Sex bias
>female mice higher T1D incidence,
>humans equal incidence for gender
**
Case study: some facts about T1D
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
3 classical signs of DM
polyuria (increased urine)
Polydipsia (increase thirst)
polyphagia (increase hunger)
Case study: T1D presentation in ER
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
TO confirm diagnosis of DM
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
Diagnosing T1D
95% diabetes diagnosed in children is T1D
Venous blood sample taken for radiobinding assay
>screen for islet cell autoantibodies
Why has sally developed T1D?
Autoimmune disease
>insulitis
>lead to activated autoreactive T cells
>mediate destruction of Insulin-producing beta cells
Recap what happens in beta cells
>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
So what happens when there are beta cells after insulin released?
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
Overview of T1D: destruction of beta cells involves complex interaction between immune cells and the beta cells
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
Overview of T1D: Beta cell death Mechanism
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
Overview of T1D: Treatment
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
HLA locus genotyping
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
HbA1c levels
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%
T1D overview: Long term complications
diabetic renopathy
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
Examining Pancreas of T1D PT post-mortem
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
