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Autoimmune Diseases 2 Flashcards

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1
Q

Non-genetic factors in autoimmunity: Infection

  • Some infections have been linked with the subsequent development of autoimmune disease
  • An immunological explanation is known as … …, in which epitopes relevant to the pathogen are shared with host antigens
A
  • Some infections have been linked with the subsequent development of autoimmune disease
  • An immunological explanation is known as molecular mimicry, in which epitopes relevant to the pathogen are shared with host antigens
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2
Q

Molecular mimicry

  • Viral infection: presentation of viral peptides to a CD4 T cell via MHC …, causing T cell activation
  • The viral peptides happen to be similar to a host-derived peptide; the T cell would normally recognise these peptides, but would not react to them
  • The activated T cell now reacts strongly to the self-peptide and initiates …
  • The process depends on having the correct MHC molecules to present this critical epitope that is common to both virus and host (inherited)
  • Also depends on having the correct T cell to recognise it (mainly bad luck)
A
  • Viral infection: presentation of viral peptides to a CD4 T cell via MHC 2, causing T cell activation
  • The viral peptides happen to be similar to a host-derived peptide; the T cell would normally recognise these peptides, but would not react to them
  • The activated T cell now reacts strongly to the self-peptide and initiates inflammation
  • The process depends on having the correct MHC molecules to present this critical epitope that is common to both virus and host (inherited)
  • Also depends on having the correct T cell to recognise it (mainly bad luck)
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3
Q

Molecular mimicry

  • Viral infection: presentation of viral peptides to a CD4 T cell via MHC 2, causing T cell activation
  • The viral peptides happen to be similar to a host-derived peptide; the T cell would normally recognise these peptides, but would not react to them
  • The activated T cell now reacts strongly to the self-peptide and initiates inflammation
  • The process depends on having the correct MHC molecules to present this critical epitope that is common to both virus and host (…)
  • Also depends on having the … to recognise it (mainly bad luck)
A
  • Viral infection: presentation of viral peptides to a CD4 T cell via MHC 2, causing T cell activation
  • The viral peptides happen to be similar to a host-derived peptide; the T cell would normally recognise these peptides, but would not react to them
  • The activated T cell now reacts strongly to the self-peptide and initiates inflammation
  • The process depends on having the correct MHC molecules to present this critical epitope that is common to both virus and host (inherited)
  • Also depends on having the correct T cell to recognise it (mainly bad luck)
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4
Q

Molecular mimicry

  • Viral …: presentation of viral peptides to a CD4 T cell via MHC 2, causing T cell activation
  • The viral peptides happen to be similar to a host-derived peptide; the T cell would normally recognise these peptides, but would not react to them
  • The activated T cell now reacts strongly to the self-peptide and initiates inflammation
  • The process depends on having the correct MHC molecules to present this critical epitope that is common to both virus and host (inherited)
  • Also depends on having the correct T cell to recognise it (mainly bad luck)
A
  • Viral infection: presentation of viral peptides to a CD4 T cell via MHC 2, causing T cell activation
  • The viral peptides happen to be similar to a host-derived peptide; the T cell would normally recognise these peptides, but would not react to them
  • The activated T cell now reacts strongly to the self-peptide and initiates inflammation
  • The process depends on having the correct MHC molecules to present this critical epitope that is common to both virus and host (inherited)
  • Also depends on having the correct T cell to recognise it (mainly bad luck)
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5
Q

Molecular mimicry

  • Viral infection: presentation of viral peptides to a CD4 T cell via MHC 2, causing T cell activation
  • The viral peptides happen to be similar to a host-derived peptide; the T cell would normally recognise these peptides, but would not react to them
  • The activated T cell now … strongly to the self-peptide and initiates …
  • The process depends on having the correct MHC molecules to present this critical epitope that is common to both virus and host (inherited)
  • Also depends on having the correct T cell to recognise it (mainly bad luck)
A
  • Viral infection: presentation of viral peptides to a CD4 T cell via MHC 2, causing T cell activation
  • The viral peptides happen to be similar to a host-derived peptide; the T cell would normally recognise these peptides, but would not react to them
  • The activated T cell now reacts strongly to the self-peptide and initiates inflammation
  • The process depends on having the correct MHC molecules to present this critical epitope that is common to both virus and host (inherited)
  • Also depends on having the correct T cell to recognise it (mainly bad luck)
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6
Q

Give 2 examples of molecular mimicry

A
  • Autoimmune haemolysis after Mycoplasma pneumoniae
    • Mycoplasma antigen has homology to ‘I’ antigen on red blood cells
    • IgM antibody to mycoplasma may cause transient haemolysis
  • Rheumatic fever: inflammatory disease occurring after streptococcal infection affecting heart, joints, skin and brain
    • Anti-streptococcal antibodies believed to cross-react with connective tissue
  • Even for these ‘best examples’ the target antigens are not well-defined; for other diseases the paradigm remains rather theoretical
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7
Q

Autoimmune haemolysis after Mycoplasma pneumoniae is an example of molecular …

A
  • molecular mimicry
    • Autoimmune haemolysis after Mycoplasma pneumoniae
    • Mycoplasma antigen has homology to ‘I’ antigen on red blood cells
    • IgM antibody to mycoplasma may cause transient haemolysis
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8
Q

Rheumatic fever is an example of molecular …

A
  • molecular mimicry
    • It is an inflammatory disease occurring after streptococcal infection affecting heart, joints, skin and brain​
  • Anti-streptococcal antibodies believed to cross-react with connective tissue
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9
Q

Autoimmune haemolysis after Mycoplasma pneumoniae

  • Mycoplasma antigen has homology to ‘I’ antigen on red blood cells
  • Ig… antibody to mycoplasma may cause transient …
A
  • Mycoplasma antigen has homology to ‘I’ antigen on red blood cells
  • IgM antibody to mycoplasma may cause transient haemolysis
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10
Q

Rheumatic fever

  • It is an inflammatory disease occurring after … infection affecting heart, joints, skin and brain
    • Anti-… antibodies believed to cross-react with connective tissue
A
  • It is an inflammatory disease occurring after streptococcal infection affecting heart, joints, skin and brain
    • Anti-streptococcal antibodies believed to cross-react with connective tissue
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11
Q

Type 1 diabetes

  • Lack of insulin impairs cellular update of glucose, leading to polyuria, polydypsia, polyphagia and weight loss
  • Onset at any age, but typically …
  • Disease prevalence around …%; rising by around 5% per anum
  • Treatment by injection of insulin and diet
  • Important to differentiate from:
    • … diabetes - can present with a similar phenotype but requires different management
    • Type II diabetes mellitus = older onset, insulin secretion, ketoacidosis less likely and insulin not necessarily required
A
  • Lack of insulin impairs cellular update of glucose, leading to polyuria, polydypsia, polyphagia and weight loss
  • Onset at any age, but typically childhood
  • Disease prevalence around 0.8%; rising by around 5% per anum
  • Treatment by injection of insulin and diet
  • Important to differentiate from:
    • Monogenic diabetes - can present with a similar phenotype but requires different management
    • Type II diabetes mellitus = older onset, insulin secretion, ketoacidosis less likely and insulin not necessarily required
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12
Q

Type 1 diabetes

  • Lack of insulin impairs cellular update of glucose, leading to polyuria, polydypsia, polyphagia and weight …
  • Onset at any age, but typically childhood
  • Disease prevalence around 0.8%; rising by around …% per anum
  • Treatment by injection of insulin and diet
  • Important to differentiate from:
    • Monogenic diabetes - can present with a similar phenotype but requires different management
    • Type II diabetes mellitus = older onset, insulin secretion, ketoacidosis less likely and insulin not necessarily required
A
  • Lack of insulin impairs cellular update of glucose, leading to polyuria, polydypsia, polyphagia and weight loss
  • Onset at any age, but typically childhood
  • Disease prevalence around 0.8%; rising by around 5% per anum
  • Treatment by injection of insulin and diet
  • Important to differentiate from:
    • Monogenic diabetes - can present with a similar phenotype but requires different management
    • Type II diabetes mellitus = older onset, insulin secretion, ketoacidosis less likely and insulin not necessarily required
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13
Q

Immunology of type 1 diabetes

  • Evidence for … disease
    • Islet cell antibodies detectable for months to years before the onset of clinical disease
    • HLA associations
    • Mouse model
    • Early pancreatic biopsy shows … with CD4/ 8 T cells
    • Note that although antibodies present, they do not appear to be directly relevant to destruction of the pancreas
    • By the time patient has established diabetes, generally no active inflammation in pancreatic biopsy
A
  • Evidence for autoimmune disease
    • Islet cell antibodies detectable for months to years before the onset of clinical disease
    • HLA associations
    • Mouse model
    • Early pancreatic biopsy shows infiltration with CD4/ 8 T cells
    • Note that although antibodies present, they do not appear to be directly relevant to destruction of the pancreas
    • By the time patient has established diabetes, generally no active inflammation in pancreatic biopsy
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14
Q

Immunology of type 1 diabetes

  • Is there evidence for autoimmune disease?
A
  • Yes - Evidence for autoimmune disease
    • Islet cell antibodies detectable for months to years before the onset of clinical disease
    • HLA associations
    • Mouse model
    • Early pancreatic biopsy shows infiltration with CD4/ 8 T cells
    • Note that although antibodies present, they do not appear to be directly relevant to destruction of the pancreas
    • By the time patient has established diabetes, generally no active inflammation in pancreatic biopsy
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15
Q

This is a pancreatic biopsy showing what?

A

pre-diabetic pancreatic biopsy showing infiltration with CD8 T cells

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16
Q

Progression to type 1 diabetes

  • Fill in the blanks
A
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17
Q

Progression to Type 1 diabetes

  • By the time overt diabetes has developed, over …% of the pancreas has been destroyed
A
  • By the time overt diabetes has developed, over 90% of the pancreas has been destroyed​
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18
Q

Genetics and type 1 diabetes

  • Genetic background definitely important – conconcordance in monozygotic twins is close to 100% if they are observed for long enough (nb older texts underestimate this risk)
  • … class II alleles are the major defined genetic risk factor
    • DR3 or DR4 relative risk is 6
    • DR3 and DR4 relative risk is 15
  • Rather like coeliac disease, believed that these molecules are required to present relevant islet cell antigens to CD4 T cells
  • Autoimmune response may occur if appropriate T cell receptors are present, together with other … and … co-factors
A
  • Genetic background definitely important – conconcordance in monozygotic twins is close to 100% if they are observed for long enough (nb older texts underestimate this risk)
  • HLA class II alleles are the major defined genetic risk factor
    • DR3 or DR4 relative risk is 6
    • DR3 and DR4 relative risk is 15
  • Rather like coeliac disease, believed that these molecules are required to present relevant islet cell antigens to CD4 T cells
  • Autoimmune response may occur if appropriate T cell receptors are present, together with other genetic and environmental co-factors
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19
Q

Genetics and type 1 diabetes

  • Is Genetic background definitely important?
A
  • yes - Genetic background definitely important – conconcordance in monozygotic twins is close to 100% if they are observed for long enough (nb older texts underestimate this risk)
  • HLA class II alleles are the major defined genetic risk factor
    • DR3 or DR4 relative risk is 6
    • DR3 and DR4 relative risk is 15
  • Rather like coeliac disease, believed that these molecules are required to present relevant islet cell antigens to CD4 T cells
  • Autoimmune response may occur if appropriate T cell receptors are present, together with other genetic and environmental co-factors
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20
Q

… class II alleles are the major defined genetic risk factor for T1 diabetes

A
  • HLA class II alleles are the major defined genetic risk factor
    • –DR3 or DR4 relative risk is 6
    • –DR3 and DR4 relative risk is 15
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21
Q

What is the major defined genetic risk factor for T1 diabetes?

A

HLA class II alleles are the major defined genetic risk factor for T1 diabetes

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22
Q

Precipitating events - T1 diabetes

  • … to islet cell antigens present for months-years before onset of clinical disease
  • Gap between initiation of disease and its presentation makes identification of triggers difficult
  • Much of the data is epidemiological
  • Some evidence for … virus
    • Stronger immune response to virus in cases compared to controls
    • Viral infection can cause pancreatitis in mice and humans, and precipitate autoimmune diabetes in mouse models
    • Protein 2C from Coxsackie virus has homology with islet cell antigen glutamic acid decarboxylase (GAD) (?molecular mimicry mechanism)
A
  • Autoantibodies to islet cell antigens present for months-years before onset of clinical disease
  • Gap between initiation of disease and its presentation makes identification of triggers difficult
  • Much of the data is epidemiological
  • Some evidence for Coxsackie virus
    • Stronger immune response to virus in cases compared to controls
    • Viral infection can cause pancreatitis in mice and humans, and precipitate autoimmune diabetes in mouse models
    • Protein 2C from Coxsackie virus has homology with islet cell antigen glutamic acid decarboxylase (GAD) (?molecular mimicry mechanism)
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23
Q

Precipitating events - T1 diabetes

  • Autoantibodies to islet cell antigens present for months-years before onset of clinical disease
  • Gap between … of disease and its … makes identification of triggers difficult
  • Much of the data is epidemiological
  • Some evidence for Coxsackie virus
    • Stronger immune response to virus in cases compared to controls
    • Viral infection can cause pancreatitis in mice and humans, and precipitate autoimmune diabetes in mouse models
    • Protein 2C from Coxsackie virus has homology with islet cell antigen glutamic acid decarboxylase (GAD) (?molecular mimicry mechanism)
A
  • Autoantibodies to islet cell antigens present for months-years before onset of clinical disease
  • Gap between initiation of disease and its presentation makes identification of triggers difficult
  • Much of the data is epidemiological
  • Some evidence for Coxsackie virus
    • Stronger immune response to virus in cases compared to controls
    • Viral infection can cause pancreatitis in mice and humans, and precipitate autoimmune diabetes in mouse models
    • Protein 2C from Coxsackie virus has homology with islet cell antigen glutamic acid decarboxylase (GAD) (?molecular mimicry mechanism)
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24
Q

Summary: The development of AID is multi-step

  • … background: Critical for some diseases in determining which peptides can be presented
  • T cell receptor …: critical in determining whether the peptide-MHC complex can be recognised. Note: the gene segments are inherited, but the receptors are produced randomly and will differ even in identical twins
  • Infection: may influence the activation of T cells and B cells that are potentially auto reactive
  • Likely to be myriad other genetic and environmental factors
A
  • MHC background: Critical for some diseases in determining which peptides can be presented
  • T cell receptor repertoire: critical in determining whether the peptide-MHC complex can be recognised. Note: the gene segments are inherited, but the receptors are produced randomly and will differ even in identical twins
  • Infection: may influence the activation of T cells and B cells that are potentially auto reactive
  • Likely to be myriad other genetic and environmental factors
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25
_Summary: The development of AID is multi-step_ * MHC background: Critical for some diseases in determining which peptides can be presented * T cell receptor repertoire: critical in determining whether the peptide-MHC complex can be recognised. Note: the gene segments are inherited, but the receptors are produced randomly and will differ even in identical twins * ...: may influence the activation of T cells and B cells that are potentially auto reactive * Likely to be myriad other ... and ... factors
* MHC background: Critical for some diseases in determining which peptides can be presented * T cell receptor repertoire: critical in determining whether the peptide-MHC complex can be recognised. Note: the gene segments are inherited, but the receptors are produced randomly and will differ even in identical twins * **Infection**: may influence the activation of T cells and B cells that are potentially auto reactive * Likely to be myriad other **genetic** and **environmental** factors
26
_Multi-system autoimmune diseases_ * What are some examples? (3)
* Rheumatoid arthritis – predominantly a joint disease, but also many extra-articular features; see symposium for more discussion * SLE * Sjogrens syndrome
27
Rheumatoid arthritis(predominantly a joint disease but also many extra-articular features), SLE and Sjogrens syndrome are all examples of what?
multi-system autoimmune diseases
28
_Systemic lupus erythematosis (SLE)_ * A true multi-system autoimmune disorder * Skin * Butterfly (‘...’ rash) * Photosensitivity * Hives * Serositis * Pleurisy, pleural ... * Pericarditis * Renal * Nephritis * ... fibrosis * Joint pain * Autoimmune cytopenias
* A true multi-system autoimmune disorder * Skin * Butterfly (‘**lupine**’ rash) * Photosensitivity * Hives * Serositis * Pleurisy, pleural **effusion** * Pericarditis * Renal * Nephritis * Pulmonary fibrosis * Joint pain * Autoimmune cytopenias
29
_Systemic lupus erythematosis (SLE)_ * A true ...-system autoimmune disorder * Skin * ... (‘lupine’ rash) * Photosensitivity * Hives * Serositis * Pleurisy, pleural effusion * Peri... * Renal * Nephritis * Pulmonary fibrosis * Joint pain * Autoimmune cytopenias
* A true **multi**-system autoimmune disorder * Skin * **Butterfly** (‘lupine’ rash) * Photosensitivity * Hives * Serositis * Pleurisy, pleural effusion * **Pericarditis** * Renal * Nephritis * Pulmonary fibrosis * Joint pain * Autoimmune cytopenias
30
_Systemic lupus erythematosis (SLE)_ * A true multi-system autoimmune disorder * Skin * Butterfly (‘lupine’ rash) * Photosensitivity * Hives * S... * Pleurisy, pleural effusion * Pericarditis * Renal * Nephritis * Pulmonary fibrosis * Joint pain * Autoimmune ...
* A true multi-system autoimmune disorder * Skin * Butterfly (‘lupine’ rash) * Photosensitivity * Hives * **Serositis** * Pleurisy, pleural effusion * Pericarditis * Renal * Nephritis * Pulmonary fibrosis * Joint pain * Autoimmune **cytopenias**
31
_Systemic lupus erythematosis (SLE)_ * What gender usually? * More commin in people of ... and ... decent * Associated with presence of what antibodies?
* Mostly **women of reproductive age,** rare in men * More common in people of **Asian and African** decent * Associated with presence of **anti-nuclear antibodies** – a collection of antibodies that react with cell nuclei and cell division apparatus; however, these don’t seem to directly cause disease and are probably an epi-phenomon * Some elements of disease probably caused by immune complex deposition; others may be explained by disordered apoptosis * Some patients have deficiency of classical complement components (C1, C4, C2)
32
Systemic lupus erythematosis (SLE) is more common in who?
* women of reproductive age - rare in men * also people of asian and african decent
33
SLE is associated with presence of what antibodies?
anti-nuclear antibodies - a collection of antibodies that react with cell nuclei and cell division apparatus; however, these don’t seem to directly cause disease and are probably an epi-phenomon
34
_Monogenic disorders and autoimmunity: classical complement deficiency_ * Immune complexes are cleared by phagocytes; process enhanced by phagocyte Fc receptors and ... receptors * Deficiency of C1q/ C2/ C4 predispose to ..., presumably because immune complexes cannot be cleared effectively
* Immune complexes are cleared by phagocytes; process enhanced by phagocyte Fc receptors and **C3b** receptors * Deficiency of C1q/ C2/ C4 predispose to **lupus**, presumably because immune complexes cannot be cleared effectively
35
Deficiency of C1q/ C2/ C4 predispose to ..., presumably because immune complexes cannot be cleared effectively
Deficiency of C1q/ C2/ C4 predispose to **lupus**, presumably because immune complexes cannot be cleared effectively
36
... protein is the most important and abundant protein in the complement system
**C3** protein is the most important and abundant protein in the complement system
37
_Autoimmune serology for diagnosis_ * Some autoantibodies have diagnostic value * In some cases the antibodies are ... * In others, they are simply a ... effect * Broadly three methods for detection: * Indirect immunofluorescence * Solid-phase immunoassay * Direct immunofluorescence
* Some autoantibodies have diagnostic value * In some cases the antibodies are **pathogenic** * In others, they are simply a **bystander** effect * Broadly three methods for detection: * Indirect immunofluorescence * Solid-phase immunoassay * Direct immunofluorescence
38
_Autoimmune serology for diagnosis_ * Some autoantibodies have diagnostic value * In some cases the antibodies are pathogenic * In others, they are simply a bystander effect * Broadly three methods for detection: * Indirect ... * ...-phase immunoassay * Direct ...
* Some autoantibodies have diagnostic value * In some cases the antibodies are pathogenic * In others, they are simply a bystander effect * Broadly three methods for detection: * Indirect **immunofluorescence** * **Solid**-phase immunoassay * Direct **immunofluorescence**
39
_Detecting antibodies in blood: indirect immunofluorescence_
40
Detecting antibodies in blood: ... ...
Detecting antibodies in blood: **indirect immunofluorescence**
41
_Detecting antibodies in blood: indirect immunofluorescence_ * Glass slide with tissue of interest, harvested from animal source * Patient serum containing (or not) relevant ... onto glass slide * Add detection antibody labelled with ... ... * Look for ... under microscope
* Glass slide with tissue of interest, harvested from animal source * Patient serum containing (or not) relevant **antibodies** onto glass slide * Add detection antibody labelled with **fluorescent marker** * Look for **fluorescence** under microscope
42
_Type 1 diabetes - Indirect Immunofluorescence_ * Indirect immunofluorescence with monkey pancreas – ... staining of pancreatic islets demonstrates the presence of islet cell autoantibodies
* Indirect immunofluorescence with monkey pancreas – **positive** staining of pancreatic islets demonstrates the presence of islet cell autoantibodies
43
_Why is it important to identify type 1 DM?_ * Risk of ... * Requires ... * ... diabetes and type 2 diabetes require a different approach
* Risk of **ketoacidosis** * Requires **insulin** * **Monongenic** diabetes and type 2 diabetes require a different approach
44
Distinction between type 1/ type 2 and ... diabetes not as straightforward as once thought on clinical grounds
Distinction between type 1/ type 2 and **monogenic** diabetes not as straightforward as once thought on clinical grounds
45
_Detecting antibodies in blood by immunoassay_ * What is this experiment?
* **ELISA** * .....being replaced by newer methods that are more automated eg particle bead suspension
46
_ELISA_ * Antigen put onto well * Then add the serum to well - specific antibody binds to antigen (if it's present) * ...-linked antibody binds to specific antibody * Substrate added and converted by enzyme into coloured product - rate of colour formation is proportional to the amount of specific antibody * **Must ... in between steps to ensure things that unbound**
* Antigen put onto well * Wash - Then add the serum to well - specific antibody binds to antigen (if it's present) * **Enzyme**-linked antibody binds to specific antibody * Substrate added and converted by enzyme into coloured product - rate of colour formation is proportional to the amount of specific antibody * **Must wash in between steps to ensure things that unbound**
47
_ELISA_ * Antigen put onto well * Wash - Then add the serum to well - specific antibody binds to antigen (if it's present) * Enzyme-linked antibody binds to specific antibody * Substrate added and converted by enzyme into coloured product - ... of ... formation is ... to the amount of specific antibody * **Must wash in between steps to ensure things that unbound**
* Antigen put onto well * Wash - Then add the serum to well - specific antibody binds to antigen (if it's present) * Enzyme-linked antibody binds to specific antibody * Substrate added and converted by enzyme into coloured product - **rate** of **colour** formation is **proportional** to the amount of specific antibody * **Must wash in between steps to ensure things that unbound**
48
_Introduction to ELISA tutorial_ * A classic ELISA system uses a ... well plastic plate * The first step is to coat each well with the ... of interest - for this example, let’s say that we’re looking for tTG antibody to diagnose coeliac disease * To do coat the plate, tTG solution is simply added to the wells; because it’s a protein, it sticks to the plastic. Excess tTG is then washed off
* A classic ELISA system uses a **96** well plastic plate * The first step is to coat each well with the **antigen** of interest - for this example, let’s say that we’re looking for tTG antibody to diagnose coeliac disease * To do coat the plate, tTG solution is simply added to the wells; because it’s a protein, it sticks to the plastic. Excess tTG is then washed off
49
_Introduction to ELISA tutorial_ * A classic ELISA system uses a ... well plastic plate * The first step is to coat each well with the antigen of interest - for this example, let’s say that we’re looking for tTG antibody to diagnose coeliac disease * To do coat the plate, tTG solution is simply added to the wells; because it’s a protein, it sticks to the plastic. Excess tTG is then ...
* A classic ELISA system uses a **96** well plastic plate * The first step is to coat each well with the antigen of interest - for this example, let’s say that we’re looking for tTG antibody to diagnose coeliac disease * To do coat the plate, tTG solution is simply added to the wells; because it’s a protein, it sticks to the plastic. Excess tTG is then **washed off**
50
A classic ... system uses a 96 well plastic plate
A classic **ELISA** system uses a 96 well plastic plate
51
_Introduction to ELISA tutorial_ * The first step is to coat each well with the ... of interest (e.g tTG) * tTG Solution is simply added to the wells; because it’s a protein, it sticks to the plastic. Excess is then ... * .. samples are added to the wells * The samples are ..., to allow time for tTG antibody in the samples to react with the tTG antibody * Excess antibody is... * If there was tTG antibody in the sample, it will now be captured by the immobilised tTG antigen (...) * A ... antibody is added * The secondary antibody is an anti-IgA antibody, which binds to IgA Fc regions * It’s covalently linked to an enzyme such as horse radish peroxidase, indicated as a red circle on the figure below * Excess secondary antibody is washed away * A trigger is now added, which reacts with the horse radish peroxidase that is bound to the secondary antibody, which in turn is bound to antibody tTG antibody that was present in the patient sample * This produces a colour change * The amount of colour change can be measured using a photocell
* The first step is to coat each well with the **antigen** of interest (e.g tTG) * tTG Solution is simply added to the wells; because it’s a protein, it sticks to the plastic. Excess is then **washed off** * **Patient** samples are added to the wells * The samples are **incubated**, to allow time for tTG antibody in the samples to react with the tTG antibody * Excess antibody is **washed off** * If there was tTG antibody in the sample, it will now be captured by the immobilised tTG antigen (**invisible**) * A **secondary** antibody is added * The secondary antibody is an anti-IgA antibody, which binds to IgA Fc regions * It’s covalently linked to an enzyme such as horse radish peroxidase, indicated as a red circle on the figure below * Excess secondary antibody is washed away * A trigger is now added, which reacts with the horse radish peroxidase that is bound to the secondary antibody, which in turn is bound to antibody tTG antibody that was present in the patient sample * This produces a colour change * The amount of colour change can be measured using a photocell
52
_Introduction to ELISA tutorial_ * The first step is to coat each well with the antigen of interest (e.g tTG) * tTG Solution is simply added to the wells; because it’s a protein, it sticks to the plastic. Excess is then washed off * Patient samples are added to the wells * The samples are incubated, to allow time for tTG antibody in the samples to react with the tTG antibody * Excess antibody is washed off * If there was tTG antibody in the sample, it will now be captured by the immobilised tTG antigen (invisible) * A ... ... is added * The ... is an anti-IgA antibody, which binds to IgA ... regions * It’s covalently linked to an enzyme such as horse radish peroxidase, indicated as a red circle on the figure below * Excess secondary antibody is washed away * A ... is now added, which reacts with the horse radish peroxidase that is bound to the secondary antibody, which in turn is bound to antibody tTG antibody that was present in the patient sample * This produces a colour change * The amount of colour change can be measured using a photocell
* The first step is to coat each well with the antigen of interest (e.g tTG) * tTG Solution is simply added to the wells; because it’s a protein, it sticks to the plastic. Excess is then washed off * Patient samples are added to the wells * The samples are incubated, to allow time for tTG antibody in the samples to react with the tTG antibody * Excess antibody is washed off * If there was tTG antibody in the sample, it will now be captured by the immobilised tTG antigen (invisible) * A **secondary antibody** is added * The **secondary antibody** is an anti-IgA antibody, which binds to IgA **Fc** regions * It’s covalently linked to an enzyme such as horse radish peroxidase, indicated as a red circle on the figure below * Excess secondary antibody is washed away * A **trigger** is now added, which reacts with the horse radish peroxidase that is bound to the secondary antibody, which in turn is bound to antibody tTG antibody that was present in the patient sample * This produces a colour change * The amount of colour change can be measured using a photocell
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_Introduction to ELISA tutorial_ * The first step is to coat each well with the antigen of interest (e.g tTG) * tTG Solution is simply added to the wells; because it’s a protein, it sticks to the plastic. Excess is then washed off * Patient samples are added to the wells * The samples are incubated, to allow time for tTG antibody in the samples to react with the tTG antibody * Excess antibody is washed off * If there was tTG antibody in the sample, it will now be captured by the immobilised tTG antigen (invisible) * A secondary antibody is added * The secondary antibody is an anti-IgA antibody, which binds to IgA Fc regions * It’s covalently linked to an enzyme such as horse radish peroxidase, indicated as a red circle on the figure below * Excess secondary antibody is washed away * A trigger is now added, which reacts with the horse radish peroxidase that is bound to the secondary antibody, which in turn is bound to antibody tTG antibody that was present in the patient sample * This produces a ... change * The amount of ... change can be measured using a ...
* The first step is to coat each well with the antigen of interest (e.g tTG) * tTG Solution is simply added to the wells; because it’s a protein, it sticks to the plastic. Excess is then washed off * Patient samples are added to the wells * The samples are incubated, to allow time for tTG antibody in the samples to react with the tTG antibody * Excess antibody is washed off * If there was tTG antibody in the sample, it will now be captured by the immobilised tTG antigen (invisible) * A secondary antibody is added * The secondary antibody is an anti-IgA antibody, which binds to IgA Fc regions * It’s covalently linked to an enzyme such as horse radish peroxidase, indicated as a red circle on the figure below * Excess secondary antibody is washed away * A trigger is now added, which reacts with the horse radish peroxidase that is bound to the secondary antibody, which in turn is bound to antibody tTG antibody that was present in the patient sample * This produces a **colour** change * The amount of **colour** change can be measured using a **photocell**
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What must you do in between steps of ELISA?
Washings! ensures anything unbound is washed away
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In the ELISA test, what are we looking for? (what does it tell us)
colour change - meausred using a photocell - rate of colour formation is proportional to the amount of specific antibody
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_Photocell - ELISA_ * To provide a value for the samples, they need to be compared with something * This involves setting up wells containing known ... of tTG antibody * The photocell readings from each well are used to create a standard ... * Positive and negative controls are also run to show that the assay has worked correctly
* To provide a value for the samples, they need to be compared with something * This involves setting up wells containing known **concentrations** of tTG antibody * The photocell readings from each well are used to create a standard **curve** * Positive and negative controls are also run to show that the assay has worked correctly
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_Photocell - ELISA_ * To provide a value for the samples, they need to be compared with something * This involves setting up wells containing known concentrations of tTG antibody * The photocell readings from each well are used to create a standard curve * ... and ... controls are also run to show that the assay has worked correctly
* To provide a value for the samples, they need to be compared with something * This involves setting up wells containing known concentrations of tTG antibody * The photocell readings from each well are used to create a standard curve * **Positive** and **negative** controls are also run to show that the assay has worked correctly
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_Detecting antibodies bound to tissue: direct immunofluorescence_
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This is showing ... immunofluorescence
**Detecting antibodies bound to tissue: direct immunofluorescence**
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_Bullous skin disease: pemphigoid_ * ...-walled bullae, rarely on mucus membranes * Fulfils criteria for ...-... disease * Target is antigen at dermo-epidermal junction * Linear deposition of antibody, which activates complement producing skin dehiscence and tense blister
* **Thick**-walled bullae, rarely on mucus membranes * Fulfils criteria for **antibody-mediated** disease * Target is antigen at dermo-epidermal junction * Linear deposition of antibody, which activates complement producing skin dehiscence and tense blister
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_Bullous skin disease: pemphigoid_ * Thick-walled bullae, rarely on ... membranes * Fulfils criteria for antibody-mediated disease * Target is antigen at dermo-epidermal junction * Linear deposition of antibody, which activates ... producing skin dehiscence and tense blister
* Thick-walled bullae, rarely on **mucus** membranes * Fulfils criteria for antibody-mediated disease * Target is antigen at dermo-epidermal junction * Linear deposition of antibody, which activates **complement** producing skin dehiscence and tense blister
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What bullous skin diseases fulfil criteria for antibody-mediated disease?
pemphigus and pemphigoid
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_Bullous skin disease: pemphigus_ * ...-walled bullae on skin and mucus membranes, rupture easily * Fulfils critiera for antibody-mediated disease * Target is the intercellular cement protein desmoglein 3 in superficial skin layers
* **Thin**-walled bullae on skin and mucus membranes, rupture easily * Fulfils critiera for antibody-mediated disease * Target is the intercellular cement protein desmoglein 3 in superficial skin layers
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_Bullous skin disease: pemphigus_ * ...-walled bullae on skin and mucus membranes, ... easily * Fulfils critiera for antibody-mediated disease * Target is the intercellular cement protein desmoglein 3 in superficial skin layers
* **Thin**-walled bullae on skin and mucus membranes, **rupture** easily * Fulfils critiera for antibody-mediated disease * Target is the intercellular cement protein desmoglein 3 in superficial skin layers
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pemphigus vs pemphigoid - which is most severe?
* **pemphigus** * thin walled blisters - affects mucus membranes - nutrition and hydration difficult and also large areas of opened skin
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_Coeliac disease diagnosis_ * ... binding to the ... of smooth muscle fibres found to have a strong disease association with coeliac disease in the 1990s. * Subsequently target antigen found to be tissue ... (tTG), which is now expressed in recombinant systems to provide antigen for modern immunoassays * HLA typing also increasing utilised – absence of HLA DQ-2/ 8 makes coeliac disease very unlikely (ie high negative predictive value)
* **Antibodies** binding to the **endomysium** of smooth muscle fibres found to have a strong disease association with coeliac disease in the 1990s. * Subsequently target antigen found to be tissue **tranglutaminase** (tTG), which is now expressed in recombinant systems to provide antigen for modern immunoassays * HLA typing also increasing utilised – absence of HLA DQ-2/ 8 makes coeliac disease very unlikely (ie high negative predictive value)
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_Coeliac disease diagnosis_ * Antibodies binding to the ... of smooth muscle fibres found to have a strong disease association with coeliac disease in the 1990s. * Subsequently target antigen found to be tissue ... (tTG), which is now expressed in recombinant systems to provide antigen for modern immunoassays * ... typing also increasing utilised – absence of ... DQ-2/ 8 makes coeliac disease very unlikely (ie high negative predictive value)
* Antibodies binding to the **endomysium** of smooth muscle fibres found to have a strong disease association with coeliac disease in the 1990s. * Subsequently target antigen found to be tissue **tranglutaminase** (tTG), which is now expressed in recombinant systems to provide antigen for modern immunoassays * **HLA** typing also increasing utilised – absence of HLA DQ-2/ 8 makes coeliac disease very unlikely (ie high negative predictive value)
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_Pernicious anaemia_ * Vitamin ... absorbed in terminal ileum * Absorption requires a co-factor called ... FACTOR which is secreted by the gastric parietal cells * Pernicious anaemia describes ... destruction of the gastric parietal cells * Loss of intrinsic factor abrogates ... absorption * Liver stores around 2 years supply of ... * Once depleted, multiple possible manifestations: * Anaemia * Neurological * Subfertility
* Vitamin **B12** absorbed in terminal ileum * Absorption requires a co-factor called **INTRINSIC** FACTOR which is secreted by the gastric parietal cells * Pernicious anaemia describes **autoimmune** destruction of the gastric parietal cells * Loss of intrinsic factor abrogates **B12** absorption * Liver stores around 2 years supply of **B12** * Once depleted, multiple possible manifestations: * Anaemia * Neurological * Subfertility
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_Pernicious anaemia_ * Vitamin B12 absorbed in terminal ... * Absorption requires a co-factor called INTRINSIC FACTOR which is secreted by the ... ... cells * Pernicious anaemia describes autoimmune destruction of the ... ... cells * Loss of intrinsic factor abrogates B12 absorption * Liver stores around ... yearssupply of B12 * Once depleted, multiple possible manifestations: * Anaemia * Neurological * Subfertility
* Vitamin B12 absorbed in terminal **ileum** * Absorption requires a co-factor called INTRINSIC FACTOR which is secreted by the **gastric parietal** cells * Pernicious anaemia describes autoimmune destruction of the **gastric parietal** cells * Loss of intrinsic factor abrogates B12 absorption * Liver stores around **2** years supply of B12 * Once depleted, multiple possible manifestations: * Anaemia * Neurological * Subfertility
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... anaemia describes autoimmune destruction of the gastric parietal cells
**Pernicious** anaemia describes autoimmune destruction of the gastric parietal cells
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Vitamin B12 absorbed in ... ..., absorption requires a co-factor called ... FACTOR which is secreted by the gastric parietal cells
Vitamin B12 absorbed in **terminal ileum**, absorption requires a co-factor called **INTRINSIC** FACTOR which is secreted by the gastric parietal cells
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Vitamin B12 absorbed in terminal ileum, absorption requires a co-factor called INTRINSIC FACTOR which is secreted by the ... ... cells
Vitamin B12 absorbed in terminal ileum, absorption requires a co-factor called INTRINSIC FACTOR which is secreted by the **gastric parietal** cells
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Liver stores around ... years supply of B12
Liver stores around **2** years supply of B12
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_Treatment of AID: manage the consequences_ * Often preferable to treating the immunology * ... drugs are toxic * By the time the disease is overt, the damage may already have been done and ... may be unhelpful * Examples: * Thyroxine for under-active thyroid * Carbimazole, surgery or drugs for thyrotoxicosis * Insulin for diabetes * B12 for pernicious anaemia * However, in some AID, particularly non organ-specific, treatment of the immune system is the best option
* Often preferable to treating the immunology * **Immunosuppressive** drugs are toxic * By the time the disease is overt, the damage may already have been done and **immunosuppression** may be unhelpful * Examples: * Thyroxine for under-active thyroid * Carbimazole, surgery or drugs for thyrotoxicosis * Insulin for diabetes * B12 for pernicious anaemia * However, in some AID, particularly non organ-specific, treatment of the immune system is the best option
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_Treatment of AID: manage the consequences_ * Often preferable to treating the immunology * Immunosuppressive drugs are toxic * By the time the disease is overt, the damage may already have been done and immunosuppression may be unhelpful * Examples: * ... for under-active thyroid * Carbimazole, surgery or drugs for thyrotoxicosis * ... for diabetes * ... for pernicious anaemia * However, in some AID, particularly non organ-specific, treatment of the immune system is the best option
* Often preferable to treating the immunology * Immunosuppressive drugs are toxic * By the time the disease is overt, the damage may already have been done and immunosuppression may be unhelpful * Examples: * **Thyroxine** for under-active thyroid * Carbimazole, surgery or drugs for thyrotoxicosis * **Insulin** for diabetes * **B12** for pernicious anaemia * However, in some AID, particularly non organ-specific, treatment of the immune system is the best option
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_Treatment of AID: manage the consequences_ * Often ... to treating the immunology * Immunosuppressive drugs are ... * By the time the disease is ..., the damage may already have been done and immunosuppression may be unhelpful * Examples: * Thyroxine for under-active thyroid * Carbimazole, surgery or drugs for thyrotoxicosis * Insulin for diabetes * B12 for pernicious anaemia * However, in some AID, particularly non organ-specific, treatment of the immune system is the best option
* Often **preferable** to treating the immunology * Immunosuppressive drugs are **toxic** * By the time the disease is **overt**, the damage may already have been done and immunosuppression may be unhelpful * Examples: * Thyroxine for under-active thyroid * Carbimazole, surgery or drugs for thyrotoxicosis * Insulin for diabetes * B12 for pernicious anaemia * However, in some AID, particularly non organ-specific, treatment of the immune system is the best option
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_Treatment of AID: manage the consequences_ * Often preferable to treating the immunology * Immunosuppressive drugs are toxic * By the time the disease is overt, the damage may already have been done and immunosuppression may be unhelpful * Examples: * Thyroxine for under-active thyroid * Carbimazole, surgery or drugs for thyrotoxicosis * Insulin for diabetes * B12 for pernicious anaemia * However, in some AID, particularly ... ...-specific, treatment of the immune system is the best option
* Often preferable to treating the immunology * Immunosuppressive drugs are toxic * By the time the disease is overt, the damage may already have been done and immunosuppression may be unhelpful * Examples: * Thyroxine for under-active thyroid * Carbimazole, surgery or drugs for thyrotoxicosis * Insulin for diabetes * B12 for pernicious anaemia * However, in some AID, particularly **non organ-**specific, treatment of the immune system is the best option
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_Some drugs used for immunomodulation_ * Used particularly for ‘...-system’ autoimmune diseases * Systemic ... * Small molecule immunosuppressive drugs (eg methotrexate, azathioprine, ciclosporin) * Increasing interest in ‘biologics'
* Used particularly for ‘**multi**-system’ autoimmune diseases * Systemic **corticosteroids** * Small molecule immunosuppressive drugs (eg methotrexate, azathioprine, ciclosporin) * Increasing interest in ‘biologics'
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_Some drugs used for immunomodulation_ * Used particularly for ‘multi-system’ autoimmune diseases * Systemic corticosteroids * Small molecule ... drugs (eg methotrexate, azathioprine, ciclosporin) * Increasing interest in ‘...'
* Used particularly for ‘multi-system’ autoimmune diseases * Systemic corticosteroids * Small molecule **immunosuppressive** drugs (eg methotrexate, azathioprine, ciclosporin) * Increasing interest in ‘**biologics**'
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_Plasmapharesis_ * Plasmapharesis removes ... from the ... therefore may be useful in ...-mediated diseases
* Plasmapharesis removes **antibodies** from the **bloodstream** therefore may be useful in **antibody**-mediated diseases
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What is plasmapharesis?
Plasmapheresis is a process in which the liquid part of the blood, or plasma, is separated from the blood cells (Plasmapharesis removes antibodies from the bloodstream therefore may be useful in antibody-mediated diseases)
Year 2 Medicine
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