Immuno Flashcards
(150 cards)
1
Q
HIV transmission and epidemiology
A
> 37 M people living with HIV/AIDS (2018 Report).
• ~ 39 M people have died of AIDS.
• >5000 persons infected per day - >10% (600) of these are children.
• Most will die within 20 years if no access to treatment
• Transmission = sexual, infected blood, mother-to-child (vertical – breastfeeding, in utero,
intra partum)
2
Q
HIV Pathogenesis
A
HIV targets CD4 cells. T helper, dendritic and macrophages all express CD4 and therefore can be infected by HIV.
• The virus binds via gp120 (initial binding) and gp41 (conformational change) – on CD4+ T cells
• Most strains use* CCR5 (mac/tcel/dendritic) and CXCR4 (t cell only) chemokine co-receptors, which HIV binds to via gp120 also
• Once entered into the cell, HIV is converted from RNA to DNA in the cytoplasm via reverse transcriptase. It then goes and incorporates itself into the host DNA.
When there is a trigger (e.g. infection) the host cell transcribes its DNA and HIV proteins are transcribed** and these form HIV particles which then leave the cell and infect other cells. (This is the normal HIV form and so therefore is retroviral).
When HIV invades a macrophage it can be taken into the LN where there lots of CD4+ cells in close proximity.
- this refers to viral tropism as in what receptors the virus prefers to bind to
- *this stage is particularly prone to error and can result in HIV particles having varying csf proteins i.e. strains
3
Q
What is the immune response to HIV?
A
The Innate response
• Non-specific activation of Macrophages, NK cells and complement
• Stimulation of dendritic cells via TLR
• Release of cytokines and chemokines
Adaptive response
• Neutralising antibodies: anti-gp120 and anti-gp41
• Non-neutralising antibodies: anti-p24 gag IgG
• CD8+ T Cells can prevent HIV entry by producing chemokines MIP-1a, MIP-1b, and
RANTES which block co-receptors.
4
Q
How does HIV impair / defeat the immune response?
A
HIV remains infectious even when Ab coated
• Activated infected CD4+ Th are killed by CD8+ T cells
• Activated infected CD4+ Th are anergised (disabled)
• CD4 T-cell memory lost & failure to activate memory CTL
• Monocytes and dendritic cells are therefore not @ by the CD4+ T cells and cannot prime naïve CD8+ CTL (due to impaired antigen presenting functions)
• Infected monocytes and dendritic cells are killed by virus or CTL
• Quasispecies are produced due to error-prone reverse transcriptase = these escape from the i/response
• Effective immunity requires antibodies to prevent infection and neutralize virus, and sufficient CTL to eliminate latently infected cells
5
Q
What are the different types of ‘progressors’ in HIV pts?
A
- Median time from infection with HIV to development of AIDS is 8 - 10 years (These are the typical progressors, 85% of patients)
- Rapid progressors (10%) in 2 - 3 years.
- Long Term Non Progressors (<5%) stable CD4 counts and no symptoms after 10 years
- Initial viral burden (set
6
Q
HIV investgations
A
Screening Test: Detects anti-HIV Ab via ELISA
Confirmation Test: Detects Ab via Western Blot
A positive test requires the patient to have SEROCONVERTED (i.e. started to produce Ab)
This happens after ~10 weeks incubation period
After Diagnosis:
Viral Load – PCR is used to detect viral RNA (very sensitive)
CD4 Count – via FACS (flow cytometry), used to assess course of disease. Onset of AIDS correlates with diminuation in number of CD4+ T cells. AIDS <200cells/µL blood.
Resistance Testing – resistance to antiretrovirals:
• Phenotypic: Viral replication is measured in cell cultures under selective pressure of increasing concentrations of antiretroviral drugs – compared to wild-type
• Genotypic: Mutations determined by direct sequencing of the amplified HIV genome
7
Q
HIV Mx
A
BHIVA guidelines for patients with chronic infection: Commence immediately once diagnosis
confirmed
HAART (Highly Active Anti Retroviral Therapy) = 2NRTIs + PI (or NNRTI)
Example regimen: Emtricitabine + Tenofovir + Efavirenz
Available as 1 pill: Atripla
Pregnancy – Zidovudine:
Antepartum PO; For delivery IV, PO to newborn for 6/52 reduces transmission
8
Q
HIV Drug classes + examples
A
1) Fusion inhibitors - Enfurviritide
2) Attachment inhibitors - Maraviroc
3) RTI - Nucleoside RTI (NRTI)- Zidovudine, Lamivudine, Emtricitabine
4) RTI - Nucleotide RTI - Tenofovir
5) RTI - Nucleoside RTI (NNRTI) - Efavirenz
6) Integration inhibitors - Raltegravir
7) Protease inhibitors - Indinavir
9
Q
HIV Drug classes side effects
A
FI - Local reactions to injection (hypersensitivity 0.1%)
AI - unkwon
NRTI - Generally rare. (Zido - GI distrubance, fever, headache)
Tenofovir - Bone and renal toxicity
NNRTI - Efavirenz - CNS effects (others: rash and hepatitis)
II - unknown
PI - Hyperlipideemia, fat redistribution, diabetes
10
Q
How does Th1/17 and Th2 response differ?
A
Th1/Th17 are triggered by foreign structures i.e. microbial PAMPS.
Multicellular organisms don’t necessarily have conserved structures that the immune system can recognise. Therefore we recognise them by the damage they cause to our epithelial barriers. Worms/ Allergens/Venoms stress our epithelium and cause it to releases Cytokines e.g. TSLP*. These act on Th2, Th9 ILC2 cells and promote the secretion of IL4, IL5 and IL13. These trigger eosinophils /basophils to expel the allergen/worm. IL 4 then actually stimulates B cells to make IgE and IgG4 (bridges the innate and adaptive system).
11
Q
How does the mast cell act as a sensor?
A
There are several ways the immune system ‘senses’ pathogens. In Th2 response, the epithelium is the sensor. Mast cells can also be the sensor when an allergen causes IgE to cross link on the mast cell surface membrane. This results in the production of histamines, leukotrienes and prostaglandins.
12
Q
What is the mechanism behind oral tolerance to allergens?
A
Oral exposure to allergen promotes immune tolerance compared to skin/resp induces sensitisation. This is because the GI tract tends to immune suppress as the T reg cells play a role in dampening immune activity.
Oral tolerance to food antigen requires induction of CD4 T reg immune response within GI mucosa. They inhibit secretion of IL10 and IL35. They inhibit IgE plasma cell cytoloysis and dendritic APC function. They are associated with the resolution of food allergies.
NB tolerance is only built orally. (oral exposure promotes immune tolerance whereas skin and respiratory exposure induces IgE sensitisation)
13
Q
Which allergic diseases are common at which ages?
A
Infants o Atopic dermatitis o Food allergy (milk, egg, nuts) (8%) Childhood o Asthma (house dustmite, pets) o Allergic rhinitis Adults o Allergic Rhinitis (most common apparently) o Drug allergy o Bee allergy o Oral allergy syndrome o Occupational allergy (Food is 5% in adults)
14
Q
Theories for the rise in incidence in allergy
A
- Hygiene hypothesis (lack of childhood exposure to infectious agents/city living/ small family sizes increase suspectibility to allergic disease by suppressing the natural development of the immune system)
- Lack of Vit D in infancy is a rf for developing food allergy
- Reduced omega and fatty acids
- Rise in food allergy might be associated with high concentration of dietary advance glycation end products and pro-glycating sugars which immune system mistakenly detects as causing tissue damage and responds by creating IgE. This can be found in fast foods and soda drinks.
15
Q
What are some common drug targets in allergic disease?
A
IgE
IL-13
histamine
IL-5
16
Q
Presentation of an allergic response
A
Criteria: Within minutes – hours, usually 2 organs involved, reproducible, Sx can be triggered by cofactors (OHs, exs, NSAID, viral inf in childhood)
- Skin (angioedema, urticarial, flushing itch
- Resp (wheeze, SOB, discharge)
- GI tract D&V
- Vascular – Hypotension Sx (faint, dizzy)
- Sense of impending doom
17
Q
List the allergy tests
A
Elective Investigations
1. Skin prick and intradermal tests (Skin prick and blood tests are used to detect the presence/absence of IgE antibody against external proteins. A positive IgE test only demonstrates sensitisation NOT clinical allergy. Diagnosis requires clinical hx, blood tests, skin prick etc. Stop antihistamines 48hrs beforehand)
- Laboratory measurement of allergen-specific IgE (Allergen is bound to a sponge and the specific IgE (if present) will bind to the allergens.This is washed over with anti-IgE antibody which is tagged with a fluorescent label. Expensive. Good for people who can’t stop anthistamines, skin is damaged from allergic itch (dermatographism), extensive eczema, history of anaphylaxis or borderline skin prick test results)
- Component-resolved diagnostics
(A newer blood test to detect IgE to single protein components - instead of whole allergen. Useful for peanut and hazelnut allergy (may reduce need for food challenges)) - Basophil activation test - expose basophils to allergen extract. Not used clinically yet.
- Challenge test (supervised exposure to the antigen) (Gold standard)
Patients are given increasing dosage of allergen. Has to occur under close supervision.
During Acute Episode
Evidence of mast cell degranulation:
• Serial mast cell tryptase
Mast cell tryptase is a biomarker for anaphylaxis and can be useful to measure when it’s unclear whether anaphylaxis has occurred e.g. if the pt can’t otherwise communicate it such as being under anaesthetic
• Blood and/or urine histamine
18
Q
Oral Allergy Syndrome
A
Is a Type 1 Hypersenstiivty (IgE mediated) food-allergy syndrome in patients who have hay fever. Existing IgE antibodies against pollen cross react with other structurally similar proteins found in botanically related plants. Symptoms occur within minutes of eating the food (tend to be raw food- allergens are heat labile). Symptoms are limited to oral cavity, swelling and itch; only 1-2% progress to anaphylaxis.
19
Q
Anaphylaxis incidence and definition and presentaiton
A
• DEFINITION: a severe potentially systemic hypersensitivity reaction. Rapid onset, life-threatening airway, breathing and circulatory problems which is usually but not always associated with skin and mucosal changes
• Incidence: 1.5-8/100,000
Skin is the most frequent organ involved (84%) (angioedema, urticaria)
Cardiovascular (collapse, syncope, drop in BP)
Respiratory compromise (SOB, wheeze, stridor)
20
Q
Management of anaphylaxis
A
IM ADRENALINE is the most important treatment for anaphylaxis
• Alpha 1 - causes peripheral vasoconstriction, reverses low BP and mucosal oedema
• Beta 1 - increases heart rate, contractility and BP
• Beta 2 - relaxes bronchial smooth muscle and reduces the release of inflammatory mediators
Supportive Treatments:
• Adjust body position
• 100% Oxygen
• Fluid replacement
• Inhaled bronchodilators
• Hydrocortisone 100 mg IV (prevent late phase response)
• Chlorpheniramine 10 mg IV
Further Management • Referral to allergy/immunology clinic • Investigate cause • Written information on: • Recognition of symptoms • Avoidance of triggers • Indications for self-treatment with an EpiPen • Prescription of emergency kit to manage anaphylaxis
21
Q
What are APCs?
A
These are cells that can present antigens to T cells to initiate an acquired immune response APCs include: o Dendritic cells o Macrophages o B lymphocytes
Types of macrophages include: o Langerhans cells o Mesangial cells o Kupffer cells o Osteoclasts o Microglia
22
Q
How are memory T cells different to other T cells?
A
- Longevity - Memory T cell persist for a long time in the absence of antigen by the continuous low level proliferation in response to cytokines
- Memory cells have a different pattern of expression of cell surface proteins involved in chemotaxis cell adhesion
- This allows memory cells to rapidly access non-lymphoid tissues (where microbes enter) - Rapid, robust response to subsequent antigen exposure -Lower threshold of activation than naïve cells
23
Q
Aims of a vaccine
A
- Generate protective, long-lasting immune response
- No adverse reactions
- Single shot
- Easy storage
24
Q
What is a haemaglutinin inhibition assay and why is it relevant?
A
Haemaglutinin (HA) is the receptor-binding and membrane fusion glycoprotein of influenza virus and HA is the target for antibodies.
• If you put normal red cells in a dish, they will clump at the bottom forming a red spot
• If you add the influenza virus to the red cells, the haemaglutinin will make the cells stick together and it will cause a diffuse coloration across the well
• If you add the serum of someone who has a lot of antibodies against HA with the virus and red cells, it will inhibit the HA from causing the above effect - this results in the cells clumping at the bottom as if the virus was not present
• The higher the dilution at which the inhibitory effect can be seen, the greater the level of antibodies the patient has against HA
• The higher the antibody level the lower the likelihood of infection
Antibody protection begins 7 days after vaccine and protection can last for around 6 months
25
What is the BCG
BCG is an attenuated strain of bovine tuberculosis
Provides some protection against primary infection, mainly provides protection against progression to @ TB
T cell response is important in protection
Mantoux Test:
•Inject a small amount of liquid tuberculin (aka purified protein derivativ (PPD)) intradermally
•The area of injection is examined 48-72 hours after tuberculin injection
•The reaction is an area of swelling around the injection site
Protection usually lasts for 10-15 years
26
Types of vaccination:
LIVE, attenuated strain e.g. MMR, BCG, oral typhoid, polio
Pros: Lifelong protection after one dose, broad response, more likely to protect against strains
Cons: can't give to I/C or pregnants, storage, rare reversion to virulence (1: 750,000)
INACTIVATED TOXINS / COMPONENT vaccinations
E.g. component: Hep B (HBsAg); HPV (capsid); Influenza (HA, neuraminidase). Inactivated Toxins: Diptheria and tet
Pros: No mutation or reversion, can be used in immunodeficient patients, Can lead to elimination of wild-type virus from the community, Easier storage, Lower cost
Cons: poor immunogenicity, need multiple injections, may require conjugates or adjuvants
CONJUGATE & ADJUVANT VACCINATIONS:
This is composed of a Polysaccharide + protein carrier
The polysaccharide alone induces a T cell-independent B cell response (transient). Addition of the protein carrier promotes T cell immunity which enhances the B cell/antibody response. E.g. Hib, Meningococcus and Pneumococcus.
Adjuvants:
Increases the immune response without altering its specificity, by mimicing the action of PAMPs on TLR and other PRRs E.g. Aluminium salts, Lipids - monophosphoryl lipid A, Freund's adjuvant (in animals)
27
How can we replace missing components of the immune system? When do we use each method?
1. Haematopoietic Stem Cell transplant (used in haem malginancies e.g. lymphomas, leukaemias, MMs or used in severe immunodeficiencies e.g. SCID, LAD)
2. Antibody replacement (a variety of Immunoglobulin from a pool of human donors. Given for primary antibody deficiency (X-linked agamma..., X-L hyper IgM, Common variable immunodefiency) or for secondary Ab deficiency (CLL, MM or after BMT).
3. Specific immunoglobulin replacement - human immunoglobulin used for post exposure prophylaxis
4. Cell transfer (4 different types: Virus specific T cells, CAR T cell therapy, TIL T cell therapy and TCR T cell therapy).
28
How can we boost the immune system by block immune check points?
By blocking immune check points we limit the regulation of the immune system.
Ipilimumab – antibody specific for CTLA4 – blocks downregulatory immune checkpoint and allows T cell activation; indications: metastatic melanoma
Pembrolizumab/Nivolumab – antibody specific for PD-1 - blocks down regulatory immune checkpoint and allows T cell activation; indications: advanced melanoma and metastatic renal cell cancer.
Both of these drugs have a disadvantage of causing autoimmunity e.g. diabetes, thyroidism.
29
How else can the immune system be boosted?
Cytokine Therapy- The aim is to modify the immune response, unfortunately they haven't been very effective.
IFN a - Hep B, Hep C, Kaposi sarcoma, CML, MM, Hairy cell leukaemia
IFN B - Behcet's disease
IFN Gamma - Chronic granulomatous disease
IL 2- Renal cell cancer
30
How can the immune response be suppressed?
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PICCASO
Plasmapharesis
INhibitors of cell signalling
Csf directed agents
CK directed agents
Antiproliferative Agents
Steroids
Oh dear..
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31
Plasmapharesis
Remove plasma and filter out pathogenic antibody. Either reinfuse plasma or reinfused albumin instead (PEX)
Issues: Rebound antibody production (because although you've got rid of the antibodies, the plasma cells are still there) limits efficacy. Therefore, it is usually given with an anti-proliferative agent.
Indications (severe antibody-mediated disease):
• Goodpasture's syndrome (to filter out anti-glomerular replacement antibodies and prevent pulmonary haemorrhage and renal failure)
• Severe acute myasthenia gravis
• Severe transplant rejection (Ab against donor HLA)
32
Inhibitors of cell signalling
1. Calcineurin inhibitors (e.g. ciclosporin & tacrolimus) will prevent T cell signalling. Therefore, it blocks IL2 production (IL2 normally acts on T cells and drives proliferation -> calcineurin inhibitors will prevent T cell activation and proliferation
Side Effects: Nephrotoxicity, HTN, Neurotoxicity, Dysmorphic features (ciclosporin)
2. JAK Inhibitors (JAK hangs out inside the cell attached to a transmembrane receptor. When a CK binds to that receptor, the JAK proteins come together phosphorylate downstream molecules. This inhibits gene transcription and production of inflammatory molecules. Tofacitinib (JAK1 and JAK3 inhibitor) is used in Rheumatoid and Psoriatic Arthritis.
3. PDE4 inhibitors - PDE4 metabolises cAMP. Apremilast inhibits PDE4 -> rise in cAMP. This activates PKA which prevents activation of TFs and results in decrease in CK production. Used in psoriatic arthritis and psoriasis.
33
Cell surface directed agents
1. Anti-thymocyte globulin
Thymocyte (lymphocytes from the thymus gland) from humans were injected into rabbits. The rabbits then produced antibodies against the thymocytes of varying specificities (e.g. antibodies to CD2, CD3, CD4, CD8 etc.)
The serum was then taken and injected into patients. This is very effective at targeting T cells, however it is very non-specific. The main aim is to cause T cell depletion (thereby reducing the activation and migration of T cells). This is useful in allograft rejection
2. Basiliximab
It targets part of the IL2 receptor. Specific for CD25, which causes inhibition of T cell proliferation. Used before and after transplant for prophylaxis of allograft rejection surgery.
3. Rituximab*
Against CD20 - expressed on mature B lymphocytes but not plasma cells and causes their depletion. Rheum, Lymphoma, SLE, ITP.
S/E: exacerbations cardio vascular disease
4. Abatacept*
This drug binds to receptors on APCs and prevents them from engaging with T cells -> reduced T cell @. (It blocks CD80 and CD86). Rheum Arthritis.
5. Natalizumab - inhibits leukocyte migration effective for relapsing-remitting MS
S/E: hepatotoxic
6. Tocilixumab*
Against IL6 receptor and leads to reduced activation of macrophages, T cells, B cells and neutrophils
Castleman's disease (IL6 producing tumour) & Rheumatoid arthritis
S/E Hepatotoxic, elevated lipids
34
List the cytokines have been targeted for cytokine directed agents?
1. anti TNF alpha
2. TNF alpha/beta receptor (Etanercept- Rheum, Ankspon, Psor)
3. IL12 (Ustekinumab - Psor & Crohn's)
4. anti IL 17A (Secukinumab- Psor & Ank Spon )
5. anti-RANK ligand antibody (Denosumab - Osteoporosis, can cause avascular necrosis of jaw)
35
What are the anti TNF alpha agents?
infliximab, adalimumab, certolizumab, golimumab
TNF-alpha is a critical molecule within the cytokine cascade that is responsible for the inflammatory response in inflammatory arthritis .
Indications: • Rheumatoid arthritis, Ankylosing spondylitis, Psoriasis and psoriatic arthritis, IBD
S/E:
• Infusion or injection site reactions
• Infection (TB, HBV, HCV)
• Lupus-like conditions
• Demyelination
• Malignancy
36
MoA of antiproliferative agents
Stop proliferation, immune cells proliferate more rapidly than other cells; therefore with correct dosing, they can be selective for immune cells.
Examples: Cyclophosphamide (most toxic), Mycophenolate, Azathioprine, Methotrexate
MoA: Inhibit DNA synthesis, targeting cells with rapid turnover
S/E: BM suppression, Teratogenic, malignancy and infection
37
Cyclophosphamide
MoA: Alkylating agent, alkylates the guanine base of DNA and this damages the DNA ensuring it wont replicate. Initially affects B cells more than T cells, but at high doses it targets all cells with high turnover.
Indication: Used to induce remission in short term, then move on to a less toxic drug. Anti-cancer, multisystem connective tissue disease, Lupus, GPA/Wegners
S/E:
Toxic to proliferating cells (BM Depression, hair loss, irreversibly sterility M>F
Toxic to bladder (Haemorrhagic cystitis caused by the toxic metabolite of cyclophosphamide called acrolein which is excreted in the urine) ( acrolein destroys the urothelium, the underlying detrusor smooth muscle and blood vessels become exposed to urine,causing further cell death)
Malignant (Bladder cancer, haem cancers, non-melanoma skin cancer)
Infection (Pneumocystitis jiroveci- yeast like fungus which causes pneumonia in the immunosuppressed particularly important in HIV discovery)
38
Azathioprine
MoA: Anti-metabolite. In liver, azathioprine is metabolised to 6-mercaptopurine, which is a purine analogue i.e. interferes with DNA production and inhibits proliferating cells. T>B cells, and affects innate cells too.
Indication: Widely used in transplantation, auto-immune disease and auto-inflamm.
S/E:
BM suppression (rapid turnover are effect)
1:300 individuals are v suscpetible to BM suppression due to a thiopurine polymorphism called TPMT polymorphism (Thiopurine methyltransferase). These pts are unable to metabolise azathioprine and result in huge huge BM suprression and neutropenia. Therefore always check TPMT activity or gene variants before starting and always check FBC after starting thearpy. Homozygous for abnormal allele = don’t use. Heterozygous = try half dose or consdier other dryg
Hepatotoxic (uncommon)
Infection (less common than cyclophosphamide)
39
Mycophenoalte
used in place of azathiopurine these days
MoA: Anti-metabolite purine, blodcks de novo guanosine nucleotide synthesis and prevents replcation fo DNA again T> B population
Indication: Widely used in transplantation instead of azathioprine and also used in AI disease and vasculitis instead of cyclophos
SE: BM suprresion, infection (particular herpes reactivation - due to mycolphenolates effect on t cells,also PML (Progressive multifocal leukocencelopathy cause dby JC virus - a normal common virus)
40
Steroids MoA
1) Effects on Prostaglandins
o Corticosteroids inhibit phospholipase A2, which converts phospholipids into AA which is then converted to eicosanoids (e.g. PG and leukotrienes) by COX
o By inhibiting phospholipase A2, corticosteroids will block AA and PG formation thereby reducing inflammation
2) Effects on Phagocytes
o Decrease traffic of phagocytes to inflamed tissue by:
• Reduce adhesion molecules on the endothelium
• They also block the signals that tell immune cells to move from the bloodstream into tissues
• This leads to a transient increase in neutrophil count
o Decreased phagocytosis
o Decreased release of proteolytic enzymes
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3) Effects on Lymphocyte Function
o Lymphopaenia (Sequestration of lymphocytes in lymphoid tissue. Affects: CD4 > CD8 > B cells)
o Blocks cytokine gene expression
o Decreased antibody production
o Promotes apoptosis
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SIDE EFFECTS:
Metabolic (Central obesity, Moon face, Diabetes, Lipid disorders, Osteoporosis, Hirstuitism, Adrenal suppression)
Cataracts, glaucoma, peptic ulceration, pancreatitis
Immunosuppression
NB - This all refers to prednisolone (it has NO mineralocorticoid activity just glucocorticoid)
41
Most commonly transplanted organ?
* Kidneys are the most commonly transplanted organs (lasts 12 years)
* 2nd most common is liver transplant
42
What are the most relevant protein variations in clinical transplantation?
1. ABO blood group
2. HLA (Coded on chromosome 6 by MHC )
• NOTE: sometimes HLA is used to refer to the proteins and MHC refers to the genes but they are often used interchangeably
3. There are some other minor histocompatibility genes
43
What are the two mechanisms of rejection?
1. T cell mediated
2. Antibody mediated
3. Both
44
What are the HLA classes?
HLA Class I (A, B and C) - expressed on ALL cells
HLA Class II (DR, DQ, DP) - expressed on antigen-presenting cells but can also be upregulated on other cells under stress
HLA are cell surface proteins. The presentation of foreign antigens on HLA molecules to T cells is a vital part of T cell activation.
45
Why are HLA classes such an issue?
They are highly polymorphic with hundreds of alleles for each locus (The areas of protein lining the peptide-binding groove are responsible for the high degree of variability between HLA)
The high variability has evolved so that we are able to present a wide variety of antigens in that peptide-binding groove to the cells of the immune system
The variability in antigens is an issue in transplantation because they provide a key difference that the immune system can react with i.e. different people's HLA will recognise different range of things as foreign. If this discrepancy is large - (high mismatch) it will increase the risk of rejection.
• A, B and DR are thought to be the MOST IMMUNOGENIC
• The number of mismatches is a major determinant of the risk of rejection
46
Describe T cell mediated rejection
T cells require presentation of the foreign HLA antigens by an APC (+ Costimulation) to initiate activation of alloreactive T cells- this tends to occur in the LNs. Activated T cellls (who are specific for that foreign HLA antigen) then produce CKs (IL2), help CD8+ cells, recruit phagocyte cells, provide help for AB production etc etc.
The inflammation caused by this process will lead to graft dysfunction (i.e. characterised by a raise in creatinine).
This new clonal population of T cells will attack the graft
They will tether, roll and arrest on the endothelial cell surface. They then crawl into the interstitium and start attacking the tubular epithelium. This results in:
• Lymphocytic interstitial infiltration
• Ruptured basement membrane of tubular epithelium
• Tubulitis (inflammatory cells within the tubular epithelium that have been recruited)
• The inflammatory cells can also attack the endothelium of blood vessels leading to arteritis
[NB the above is for kidney transplant - but it can occur in any transplanted organ.]
47
Describe antibody mediated rejection
Antibodies will bind to antigens (HLA) on the endothelium of the blood vessels in the transplanted organ. These antibodies can then fix complement which assembles to form membrane attack complexes (MAC) resulting in endothelial cell lysis & recruit inflammatory cells to the microcirculation- one of the cardinal features of antibody-mediated rejection is the presence of inflammatory cells within the capillaries of the kidney which then cause endothelial injury (capillaritis)
These processes will result in procoagulant tendencies and closure of the microcirculation leading to graft fibrosis
Antibodies against graft endothelial epitopes can also cause damage by cross-linking the MHC molecules and activating them
48
Histology of antibody mediated rejection
The little capillaries in between the tubules contain inflammatory cells
This is inflammation of the microcirculation (whereas in T cell mediated rejection you get inflammatory cells in the tubular epithelium and the interstitial space)
Immunohistochemistry looking for fixation of complement fragments on the endothelial cell surface may also be useful
49
How can we reduce the difference between the transplant and donor?
Determine donor and recipient blood group and HLA type (esp. BM, kidney) . We sequence the HLA using PCR.
Check recipient’s pre-formed Ab against ABO and HLA – via CDC, FACS and Luminex. Before during and after the transplanation.
Cross match – via CDC and FACS. Tests if serum from recipient is able to bind/kill donor lymphocytes- positive crossmatch is contraindication for transplantation.
After transplant check again for new antibodies vs the graft
CDC - complement dependent cytotoxicity (Looks at whether the host's serum will kill the lymphocytes of the donor in the presence of complement. Positive crossmatch suggests that there is cell lysis)
FACS - Flow cytometry (Looks at whether the host's serum binds to the donor's lymphocytes irrespective of complement. Bound antibody is detected by fluorescently-labelled anti-human immunoglobulin)
50
How do we prevent rejection?
1 . Suppress T cells
Calcineurin Inhibitors (Tacrolimus and Cyclosporin)
Cell Cycle Inhibitors (MMF)
Target TCR (Anti-thymocyte globulin)
• Alemtuzumab is an anti-CD52 monoclonal antibody that causes lysis of T cells
• Daclizumab is an anti-CD25 monoclonal antibody which targets the cytokine signal
2. Suppress Ab mediated Rejection
•B cells can be depleted using rituximab (anti-CD20)
•BAFF inhibitors target cytokines that promote B cell activation and growth
•Proteasome inhibitors such as bortezemib can block the production of antibodies by plasma cells
•Complement binding to the surface of endothelial cells can be blocked using complement inhibitors such as eculizimab
51
Modern Transplant Immunosuppression
•Induction agent: e.g. OXT3/ATG, anti-CD52, anti-CD25
oThis is given at the time of transplantation or just before in order to prepare the patient to receive the foreign organ
•Baseline immunosuppression: calcineurin inhibitor + mycofenolat mofetil or azathioprine with or without steroids
•Treatment of episodes of acute rejection
oCellular: steroids, OKT3/ATG
oAntibody-mediated: IVIG, plasma exchange, anti-C5, anti-CD20
NOTE: IVIG can reduce antibody production through feedback and it can displace troublesome antibodies so that they cannot exert their harmful effects
52
How do we prevent GvHD in HSCT?
o During the process of SCT, the host immune system is eliminated (using total body irradiation and drugs)
o It is then replaced by own (autologous) or HLA-matched donor (allogeneic) bone marrow
o Allogeneic stem cell transplantation leads to reaction of donor lymphocytes against host tissues
o Related to a degree of HLA-incompatibility
o If there is a malignancy (e.g. leukaemia), the graft can help kill these cells (graft-versus-tumour)
o GVHD Prophylaxis- Methotrexate/cyclosporine
o It is thought that the damage to the GI tract by the irradiation and cytotoxic drugs before the transplant has an important role in liberating antigens which will subsequently be presented to the donor's immune cells
oSymptoms - Rash, GI (N&V&D, Jaundice(
oTreatment: STEROIDS
53
What are the issues post transplant?
Infections
• Increased risk of conventional infections
• Also increased risk of opportunistic infection:
o CMV
o BK virus
o PCP
Post-Transplantation Malignancy
• Viral-associated malignancies are much more common, such as:
o Kaposi sarcoma (HHV8)
o Lymphoproliferative disease (EBV)
• Skin cancer is 20 x more common
• Risk of other cancers is also increased
54
Complement deficiency one-liners /stereotypes
* C1q deficiency = severe childhood-onset SLE with normal levels of C3 and C4
* C3 deficiency with presence of a nephritic factor = membranoproliferative nephritis and abnormal fat distribution
* C7 deficiency = meningococcus meningitis with family history of sibling dying of the same condition aged 6
* MBL deficiency = recurrent infections when neutropaenic following chemotherapy but previously well
55
C1q deficiency
o C1q deficiency is an inherited form of complement deficiency that tends to present with SLE in childhood
o In patients with C1q deficiency, they will not be able to activate their classical pathway so CH50 will be low
56
functional complement deficiency
* Active lupus causes the persistent production of immune complexes
* This leads to the consumption of complement components resulting in a functional complement deficiency
* C3 and C4 can be measured and they tend to be low in severe active disease. In active disease C4 is low. Inactive both normal.
57
How do nephritic factors affect complement
* Nephritic factors are autoantibodies that are directed against components of the complement pathway
* Nephritic factors will stabilise C3 convertases (which break down C3) resulting in C3 activation and consumption
* Nephritic factors are often associated with GLOMERULONEPHRITIS (classically membranoproliferative)
* It may be associated with partial lipodystrophy
58
Properdin
This joins Bb to C3(H2O) in the alternative pathway.
Properdin is the only known positive regulator of complement activation that stabilizes the alternative pathway convertases.
59
Immunodeficiency signs
```
o TWO major OR ONE major + recurrent minor infections in one year
o Unusual organisms
o Unusual sites
o Unresponsive to treatment
o Chronic infections
o Early structural damage
```
60
How do phagocytes interact with other cells?
* Cells have pattern recognition receptors (e.g. Toll-like receptors) which recognise generic motifs known as pathogen-associated molecular patterns (PAMPs) such as bacterial sugars, DNA and RNA
* Cells have Fc receptors to allow the detection of immune complexes
* They also have phagocytic capacity meaning that they can engulf the pathogens
* Cells can secrete cytokines and chemokines to regulate the immune response
61
CD8 Cells
•Specialised cytotoxic cells
•Recognise peptides derived from intracellular proteins in association with HLA Class I
o HLA-A, HLA-B, HLA-C
•Kill cells directly
o Perforin (pore forming) and granzymes
o Expression of Fas ligand (which binds to Fas and induces apoptosis)
•Secrete cytokines (e.g. IFN-, TNF)
• Particularly important in defence against viral infections and tumours
62
CD4 Cells
• Recognise
o Peptides derived from extracellular proteins
o Presented on HLA Class II molecules
• HLA-DR, HLA-DP, HLA-DQ
63
Managmeent of immunodeficiency if you have T cell deficiency
• Aggressive prophylaxis/treatment of infection
• Haematopoietic stem cell transplantation
o To replace abnormal populations in SCID
o To replace abnormal cells (class II deficient APCs in BLS)
• Enzyme replacement therapy
o PEG-ADA for ADA SCID
• Gene therapy
o Stem cells are treated ex vivo with viral vectors containing missing components. Transduced cells have a survival advantage in vivo.
• Thymic Transplantation
o To promote T cell development in Di George syndrome
o Cultured donor thymic tissue transplanted into quadriceps muscle
64
Lymphodeficiency Stereotype
* X-linked SCID: severe recurrent infections from 3 months of age, CD4 and CD8 cells are absent, B cells present, immunoglobulin are low. Normal facial features and echocardiogram.
* IFN- receptor deficiency: young adult with chronic infection with Mycobacterium marinum
* 22q 11.2 deletion syndrome: recurrent infections in childhood, abnormal facial features, congenital heart disease, normal B cells, low T cells, low IgA and low IgG
* Bare lymphocyte syndrome type II: 6-month old baby with two recent serious bacterial infections. T cells present - but only CD8+. B cells present. IgM present but IgG low.
65
Do B Cells need T cells for activation?
YES AND NO!
NO-
When B cells encounter antigens, the early response tends to be an IgM response (this is NOT T cell=dependent)
They will differentiate to form IgM memory cells and plasma cells
YES -
They also undergo a T-cell dependent germinal centre reaction
1) Dendritic cell will prime the CD4+ T cells
2) Then, these CD4+ T cells will help B cell differentiation
(This interaction requires CD40 ligand expression on T cells, and CD40 expression on B cells)
3) Once they have received this help from CD4+ T cells, the B cells can proliferate and undergo somatic hypermutation (to refine their receptor to develop high affinity) and they can isotype switch (to IgA, IgG and IgE)
This process results in the production of much higher affinity memory cells and plasma cells
66
List the different types of macrophages
- Kupffer Cells (liver)
- Mesangial Cells (kidney)
- Bone (osteclast)
- Spleen (Sinusoidal lining cells)
- Lung (alveolar macrophage)
- Neural Tissue (microglia)
- Connective tissue (histiocyte)
- Skin (langerhans)
- joints (macrophage like synoviocytes)
67
What helps the phagocyte do its job?
- PRRs such as Toll like and Mannose receptors
- Fc receptors allows phagocytes to recognise immune complexes
- Complement receptors ..
- Attracted by chemokines to site of damage
- Promoted by acute phase proteins e.g. CRP
- Phagocytes has killing oxidative mechanisms that help it to kill the organism once it has engulfed it in the phagolysosome:
1) NADPH Oxidase
2) Myeloperoxidase
3) Hydrochlorous acid
Also non-oxidative mechanisms:
• Killing by the release of bacteriocidal enzymes such as lactoferrin and lysozyme into the phagolysosome
• Enzymes are present in granules
• Each has a unique antimicrobial spectrum
• This results in a broad range of cover against bacteria and fungi
68
NK cells
* These are a type of lymphocyte
* Present in blood and may migrate to inflamed tissue
* Express inhibitory receptors for self HLA which prevents inappropriate activation by normal self cells
* Express a range of activatory receptors including natural cytotoxicity receptors that recognise heparan sulphate proteoglycans
* Integrate signals from inhibitory and activatory receptors (usually the inhibitory signals will dominate)
* They are cytotoxic - kill 'altered self' cells (i.e. malignancy or virus-infected cells)
* Secrete cytokines to regulate inflammation and promote dendritic cell function
69
CD4
CD4+ T Cells (Helper T Cells)
oRecognise peptides derived from extracellular proteins
oThese peptides are usually presented on HLA Class II molecules (HLA-DR, HLA-DP, HLA-DQ)
oThey have important immunoregulatory functions via cell: cell interactions and the expression of cytokines
They provide help for the development of a full B cell response
They provide help for the development of some CD8+ T cell responses
Have a large list of subsets: Tfh, Th2, Treg, Th1, Th17
70
CD8 T cells
CD8+ Cytotoxic T Cells
oSpecialised cytotoxic cells
oRecognise peptides derived from intracellular proteins presented on HLA Class I (HLA-A, HLA-B and HLA-C)
oKills cells directly
•Perforin (pore-forming) and granzymes
•Expression of Fas ligand
oSecrete cytokines (e.g. IFN-gamma, TNF-alpha)
oParticularly important in defence against viral infections and tumours
71
Immunoglobulin
o Soluble proteins made up of 2 heavy chains + 2 light chains
o Heavy chain determines the antibody class (GAMED)
o There are subclasses of IgG and IgA
o Antigen is recognised by the antigen binding region (Fab) which is made up of both the heavy and light chains
o Effector function is determined by the constant region (Fc) of the heavy chain
72
CGD
-Excessive inflammation
- Persistent neutrophil/ macrophage accumulation
-Failure to degrade antigens
-Granuloma formation
-Lymphadenopathy and hepatosplenomegaly
-Susceptibility to bacteria esp. catalase positive bacteria i.e. PLACESS (Pseduomonas, Listeria, Aspergillus, Candida, E.Coli, Staph Aureus, Serratia)
Negative Nitro-Blue Tetrazolium test (NBT). NBT is a dye that changes colour from yellow to blue following interaction with hydrogen peroxide.
Dihydrorhodamine (DHR) flow cytometry test. DHR is oxidized to rhodamine, which is strongly fluorescent, following interaction with hydrogen peroxide.
73
What are you most susceptible to when you have a complement deficiency?
• Particularly increases susceptibility to encapsulated bacterial infections (NHS)
o Neisseria meningitidis
o Haemophilus influenzae
o Streptococcus pneumoniae
NOTE: susceptibility to N. meningitidis infection is particularly common in properidin deficiency and C5-9 deficiency
74
Secondary C3 Deficiency
Nephritic factors: auto-antibodies directed against parts of the complement pathway Nephritic factors stabilise C3 convertases resulting in C3 activation and consumption Often associated with glomerulonephritis (classically membranoproliferative glomerulonephritis- which is also associated with partial lipodystrophy)
75
How are complement and SLE linked?
In several ways:
- C2 deficiency is the most common complement deficiency and almost all of the patients have SLE. They have severe skin disease with the risk of infection.
- SLE also causes consumption of complement (specifically C3 and C4) due to persistence of immune complexes.
76
How do we investigate the complement pathway?
AP50 - alternative pathway (factor B, D, properidin, 5-9)
| CH50 - classical pathway (C1, 2, 4, C5-9)
77
Summary of complement deficiencies
- SLE induced c3/c4 consumption
- SLE associated c2 deficiency (have severe skin disease)
- nephritic induced c2 consumption (membranous glomerulonephritis, which is assoicated with partial lipodystrophy)
- Familial Angioedema (C1 esterase inhibitor is low)
- Properidin Deficiency
- C7 deficiency = meningococcus meningitis with family history of sibling dying of the same condition aged 6
- MBL deficiency = recurrent infections when neutropaenic following chemotherapy but previously well
78
Anisopoikilocytosis
Anisopoikilocytosis (in the context of a microcytic anaemia) is more associated with iron deficiency than thalassemia trait
79
Basophillic stipling
```
This appearance is due to the presence of aggregated ribosomal material
• Causes:
o Beta-thalassemia trait
o Lead poisoning
o Alcoholism
o Sideroblastic anaemia
```
80
Secondary causes of hyposplenism and blood film features
```
o Inflammatory bowel disease
o Coeliac disease
o Sickle cell disease
o SLE
Howell Jolly bodies (only hyposplenism causes these) and Target cells (aka codocytes) (also appear in IDA, thalassemia and liver disease)
```
81
Crohn's
• THREE mutations of this gene have been found to be associated with Crohn's disease
• NOD2 gene mutations are present in about 30% of patients (i.e. it is NOT a necessary mutation to develop Crohn's) It is expressed in the cytoplasm of myeloid cells (macrophages, neutrophils and dendritic cells) & acts as a microbial sensor (recognises muramyl dipeptide). This alongisde other environmental and genetic factors cause xs infammation -> crohn's
Histopathology: inflammation in/around crypts, granulomata formation, tissue damage with mucosal ulceration.
Clinical Features: Abdominal pain and tenderness, Diarrhoea (blood, pus and mucus), Fever. Malaise
Treatment
o Corticosteroids
o Azathioprine
o Anti-TNFa antibodies
o Anti-IL12/23 antibodies
82
Graves'
* Excessive production of thyroid hormones
* Mediated by IgG antibodies that stimulate the TSH receptor
* This is a type II hypersensitivity reaction (however, it is stimulatory rather than destructive)
83
Hashimoto's
• MOST COMMON cause of hypothyroidism in iodine-replete areas
• May present with a goitre - enlarged thyroid infiltrated by T and B cells
• Associated with anti-TPO antibodies
o Their presence correlates with thyroid damage and lymphocyte inflammation
o Some have been shown to induce damage to thyrocytes
• Associated with the presence of anti-thyroglobulin antibodies
• This is a type II and type IV hypersensitivity reaction
• We don't tend to measure anti-thyroid antibodies to diagnose hypothyroidism because these antibodies are present in a lot of normal people who do not have hypothyroidism
84
Diabetes type 1
CD8+ T cell infiltration of the pancreas
• The T cell clones have specificity for islet antigens
• The CD8+ lymphocytes bind to peptides presented by MHC Class I molecules on the beta-cells of the pancreas
• These autoantigens include GAD and IA2
• The presence of antibodies against these antigens will pre-date the development of the disease
o Anti-islet cell
o Anti-insulin
o Anti-GAD
o Anti-IA2
o NOTE: individuals with 3-4 of the above are highly likely to develop type 1 diabetes
• IMPORTANT: detection of antibodies does NOT currently play a role in diagnosis
85
Pernicious Anaemia
• In pernicious anaemia, patients develop antibodies against intrinsic factor which leads to failure of absorption of vitamin B12
• Vitamin B12 deficiency can also lead to subacute degeneration of the spinal cord
o This involves the posterior and lateral columns
• Other neurological features include peripheral neuropathy and optic neuropathy
• Antibodies against gastric parietal cells or intrinsic factor are useful in diagnosis
86
Myasthenia Gravis
• In myasthenia gravis the patient develops antibodies against the nicotinic acetylcholine receptor
• This lead to a failure of depolarisation
• Myasthenia gravis is characterised by fluctuating weakness
• Ptosis is a common feature
• EMG studies are usually abnormal
• Tensilon test is used to confirm the diagnosis
o This involves administering a very short-acting anticholinesterase (edrophonium bromide), which will cause a rapid improvement in symptoms
• Anti-acetylcholine receptor antibodies are present in 75% of patients and so they are useful in diagnosis
• Offspring of affected mothers may experience transient neonatal myasthenia
• This is a type II hypersensitivity reaction
87
Goodpasture's
* Also known as anti-glomerular basement membrane disease
* Leads to lung and kidney damage
* The deposition of antibodies along the basement membrane gives a smooth linear appearance
* They are detected using fluorescein conjugated anti-human immunoglobulin
88
Rheum genetic predispositions
```
o HLA DR4
o HLA DR1
o PTPN22
o Polymorphisms affecting TNF, IL1, IL6 and IL10
o PAD2 and PAD4 polymorphisms
```
89
Pathophsyiology behind PAD2 and PAD4 polymorphisms causing Rheum A
o These are enzymes that are involved in the deamination of arginine to form citrulline
o Polymorphisms in these genes are associated with increased citrullination leading to a high load of citrullinated peptides
90
Environmental factors that can cause Rheum A
o Smoking is associated with the development of erosive disease (due to increased citrullination)
o Gum infection with Porphyromonas gingivalis is associated with rheumatoid arthritis
• It is the only bacterium known to express PAD, thereby promoting citrullination
91
Rheum Arthritis antibodies
1. Anti-Cyclin Citrullinated Peptide Antibodies
• Bind to peptides in which arginine has been converted to citrulline by PAD
• Around 95% specific for rheumatoid arthritis
• 60-70% sensitive for the diagnosis of rheumatoid arthritis
2. Rheumatoid Factor
• An antibody directed against the Fc region of human IgG
• IgM anti-IgG antibody is the most commonly tested although you can get IgA and IgG variants
92
What type of hypersensitivity is Rheum Arthritis?
Type 2, 3, and 4
o Type II
• Antibodies bind to citrullinated peptides leading to activation of complement, macrophages and NK cells
o Type III
• Immune complexes form and get deposited
• This leads to complement activation
Type 4
T cell involvement in Rheumatoid Arthritis Pathology
APCs will present (self, unknown specific) peptides to CD4 cells which will then lead to the production of IFN-gamma and IL17
These cytokines act on fibroblasts and macrophages
o This, in turn, leads to the productions of:
• MMPs
• IL-1
• TNF-alpha
This destroys the joint
93
Rheum ARthritis joint pathology
o In rheumatoid arthritis, the synovium becomes very inflamed forming a pannus
o This invades the articular cartilage and adjacent bone
o There is also an increase in synovial fluid volume
94
How do we test for ANA Ab?
* These are a group of antibodies that bind to nuclear proteins
* They are tested by staining Hep-2 (human epidermoid cancer line) cells
* VERY COMMON - sometimes present in normal individuals
95
What are theorised mechanisms behind the genetic predisposition for SLE?
1. Abnormalities in clearing apoptotic cells
• Polymorphisms in genes encoding complement, MBL and CRP
2. Abnormalities in cellular activation
• Polymorphisms in genes encoding/controlling expression of cytokines, chemokines, co-stimulatory molecules and intracellular signalling molecules
3. B cell hyperactivity and loss of tolerance
4. Antibodies directed particularly at intracellular proteins
96
How does the immunoflorescence in goodpastures vary with lupus?
Goodpastures -> antibody against basement membrane -> immunoflorescne is smooth and continuous with the membrane
Lupus -> granular, "lumpy bumpy" pattern
97
Types of ANA, their staining, what diseases do they arise in and what functional purpose can they have?
ANA: Anti-dsDNA and ENAs (extractable nuclear antigens)
AntidsDNA is specific for SLE and gives a homogenous immunoflorescence pattern. They will be present in 60-70% of pts at some point. It is good for tracking disease treatment monitoring (rises can precede relapse) and disease severity (high titres associated with CNS or renal involvement).
ENAs e.g. AntiRo, Anti La, Sm and U1RNP produce a speckled immunoflorescence pattern as these stain some ribonucleitide proteins. They are not helpful in disease monitoring.
Anti-Ro and Anti La for Sjogrens
Scl70 - diffuse cutaneous systemic sclerosis
98
Antiphospholipid syndrome
• TRIAD
o Recurrent venous or arterial thrombosis
o Recurrent miscarriage
o Thrombocytopaenia
• May occur alone (primary) or in conjunction with autoimmune disease (secondary)
• TWO major types of antibody test:
1. Anti-cardiolipin antibody
• Immunoglobulins directed against phospholipids and b2 glycoprotein-1
2. Lupus Anticoagulant
• Prolongation of phospholipid-dependent coagulation tests
• Cannot be assessed if the patient is on anticoagulant therapy
o NOTE: 40% of patients have discordant antibodies - if there is clinical suspicion of anti-phospholipid syndrome, both tests should be performed
99
Why does APS cause thrombosis?
Anti-ApoH and a subset of anti-cardiolipin antibodies bind to ApoH, which in turn inhibits Protein C, a glycoprotein with regulatory function upon the common pathway of coagulation (by degrading activated factor V).
Lupus anticoagulant (LAC) antibodies bind to prothrombin, thus increasing its cleavage to thrombin, its active form.
In APS there are also antibodies binding to Protein S, which is a co-factor of protein C. Thus, anti-protein S antibodies decrease protein C efficiency.[7]
Annexin A5 forms a shield around negatively charged phospholipid molecules, thus reducing their availability for coagulation. Thus, anti-annexin A5 antibodies increase phospholipid-dependent coagulation steps
100
Systemic Sclerosis (types)
SS:
Inflammation with Th2 and Th17 cells predominating
Cytokines lead to activation of fibroblasts and the development of fibrosis
Cytokines lead to activation of endothelial cells and contribute to microvascular disease
Loss of B cell tolerance to nuclear antigens
```
Limited Cutaneous Systemic Sclerosis (CREST)
Skin involvement does NOT progress beyond forearms (although it may involve perioral skin)
• Calcinosis
• Raynaud's phenomenon
• Esophageal dysmotility
• Sclerodactyly
• Telangiectasia
• (also primary pulmonary hypertension)
ANTI CENTROMERE
```
Diffuse Cutaneous Systemic Sclerosis
oSkin involvement DOES progress beyond the forearms
o CREST features
o More extensive gastrointestinal disease
o Interstitial pulmonary disease
o Scleroderma kidney/renal cysts
ANTI TOPOISOMERASE AKA ANTISCL70
101
Types of ANCA staining and their usefulness
These antibodies are useful in diagnosis and monitoring disease activity
cANCA
o Cytoplasmic fluorescence
o Associated with antibodies to enzyme proteinase 3 (PR3)
o Occurs in > 90% of patients with granulomatous polyangiitis with renal involvement
pANCA
o Perinuclear staining pattern
o Associated with antibodies to myeloperoxidase
o Less sensitive and specific than cANCA
o Associated with microscopic polyangiitis and eosinophilic granulomatosis with polyangiitis
102
What is ANCA?
Pathogenesis: there is a trigger event (e.g. infection) which causes neutrophils to upregulate their pro-inflammatory cytokines such as PR3 or MPO. Floating autoantibodies then bind to these csf proteins and activate the neutrophil causing the neutrophils to bind to the endothelium wall, activate complement cascade, ROS etc. This can result in a vasculitis.
Type II hypersensitivity
103
Idiopathic inflammatory Myopathy
• Dermatomyositis
o Within muscle - perivascular CD4 T cells and B cells
o Immune complex mediated vasculitis (type III response)
• Polymyositis
o Within muscle - CD8 T cells surround HLA Class I expressing myofibres
o CD8 T cells kill myofibres via perforin/granzyme - type IV response
• Positive ANA (in some patients)
o Dermatomyositis: Anti-aminoacyl tRNA synthetase antibody (e.g. Jo-1) (cytoplasmic)
o Polymyositis: Anti-signal recognition peptide antibody (nuclear and cytoplasmic)
o Dermatomyositis > Polymyositis: Anti-Mi2 (nuclear)
104
HIV 2
- HIV-2 infection is predominantly found in West African nations, such as Guinea-Bissau, The Gambia, Senegal, Cape Verde, Cote d'Ivoire, Mali, Sierra Leone, and Nigeria.
- The modes of transmission for HIV-2 are the same as those for HIV-1, namely sexual contact, blood-borne exposure (blood transfusion, shared needles), and perinatal transmission. However, HIV-2 has a lower infectivity than HIV-1
- HIV-2 infection is notable for a longer asymptomatic phase and slower progression to AIDS than HIV-1 infection
- HIV-2 infection is characterized by higher CD4 cell counts and lower viral RNA levels than that seen in HIV-1 infection
105
Mechanism behind the long term non progressors of HIV?
```
Genetic:
- Potentially mutation in CCR5 receptor
- Slow progressor HLA profile
- MBL alleles
- TNF alleles
Immune factors:
- Secretion of effectors that block HIV co receptors CCR5/ CXCR4. (RANTES and MIP 1a or MIP1b - block CCR5. SDF-1 blocks CXCR4.
- Effective CTL response
- Higher levels of IL 16, CD8
```
106
How do we test for HIV resistance?
1. Phenotypic
o Viral replication is measured in cell cultures under selective pressure of increasing concentrations of antiretroviral drugs (compared to wild-type)
2. Genotypic
o Mutations detected by direct sequencing of the amplified HIV genome (so far limited to sequencing RT and P)
107
WHen do we start HIV treatment?
* All symptomatic patients
* All CD4+ count < 200 cells/microL
* CD4 count 200-350 cells/microL
* All offered IMMEDIATE treatment
108
Megaloblastic anaemia
Hypersegmented neutrophils due to B12 deficiency, folate deficiency and drugs
109
Manifestations of coeliac disease/crohn's
Malabsorption causes:
- Vitamin D deficiency causing myalgia and hypocalcaemia
- Iron deficiency
- B12 / Folate deficiency
- Hyposplenism (xs loss of lymphocytes through the damaged GI tract or related to the mucosal lesion)
110
Is coealiac an autoimmune/inflam poly/mono disease?
Polygenic Autoimmune disease it has sever mutations affecing many proteins resulting in abnormalities in the adaptive response (primarily T cells)
111
Which mutations are involved in coeliac?
• 90% of coeliac disease patients have HLA DQ2 (vs 20% of non-coeliac)
o DQA1*0501 and DQB1*02 alleles
• Other patients carry HLA DQ8
112
Coeliac pathology
• Peptides from gluten (i.e. gliadin) are deamidated by tissue transglutaminase
• The deamidated gluten is taken up by antigen-presenting cells
• It is then presented, via HLA molecules (DQ2 or DQ8) to CD4+ T cells
• CD4+ T cell activation results in secretion of IFN-gamma and may directly lead to increased IL-15 secretion
• The cytokines promote activation of the intra-epithelial lymphocytes
• These intra-epithelial lymphocytes kill epithelial cells via the NKG2D receptor (which normally recognises the stress protein MICA)
In other words, the intraepithelial lymphocytes cause damage to the gut wall and lead to the presentation of coeliac disease
113
The role of B cells in coeliac pathology?
* There may also be B cells whose antibodies recognise gliadin
* The B cells will process the antigens and present them via the HLA molecule (DQ2 and DQ8) to the CD4+ T cell
* These primed T cells will then be able to provide help (via CD40L-CD40) to the B cells that are trying to undergo germinal centre reactions
* The B cells will then undergo isotype switching and affinity maturation to become memory cells and plasma cells which will produce antibodies that are specific for gliadin
114
investigationscoeliac
serology:
IgA anti-TTG is as specific and slighlty more sensitive than IgA anti-endomysial antibody (antiendomysial antibody binds to tTG expressed on endomysial cells)
BUT- An IgA test needs to be done to make sure they aren't igA deficiency
NB Anti-gliadin antibody is not spec or sens
if positive -> duodenal biopsy as gold standard
(need at least 4 biopsies) which will show:
1. Villous atrophy with crypt hyperplasia (normal is 2-4V:1C) - it is reversed in coeliac;s
2. Intra-epithelial lymphocytes (>20 lymphocytes* per 100 epithelial cells)
*These lymphocytes are Gamma-Delta T cells (NOTE: most T cells are alpha-beta T cells)
115
Other causes of villous atrophy
* Giardiasis
* Tropical sprue
* Crohn's disease
* Radiation/chemotherapy
* Nutritional deficiencies
* Graft-versus-host disease
* Microvillous inclusion disease
* Common variable immunodeficiency
116
Complications of coealic
```
o Malabsorption
o Osteomalacia and osteoporosis
o Neurological disease
• Epilepsy
• Cerebral calcification
o Lymphoma (MOST DANGEROUS- Causes a multi-focal T cell lymphoma which is very difficult to treat. Inidicated by a HIGH TTG level (NB this can be due to lymphoma or because they have been eating gluten inadvertently)
o Hyposplenism
```
117
Disorders associated with coeliac
Dermatities herpetiforms (100% prevalence)
T1DM
AI Thyroid disease
Down' syndrome
118
What are HLA?
* HLA Class I (A, B and C) - expressed on ALL cells
* HLA Class II (DR, DQ, DP) - expressed on antigen-presenting cells but can also be upregulated on other cells under stress
* They are highly polymorphic with hundreds of alleles for each locus
* The areas of protein lining the peptide-binding groove are responsible for the high degree of variability between HLA
* The high variability has evolved so that we are able to present a wide variety of antigens in that peptide-binding groove to the cells of the immune system
* The variability in antigens is an issue in transplantation because they provide a key difference that the immune system can react with
* A, B and DR are thought to be the MOST IMMUNOGENIC
* The number of mismatches is a major determinant of the risk of rejection
119
Transplant rejection overview
```
Transplant rejection is the immune system mounting a response to a foreign antigen as it should
3 Stages:
1. Recognition →
2. Activation →
3. Effector Function
```
Mechanisms (2):
1. T cell mediated
2. Antibody related
But also 2 types of recognition.
1. Direct (DONOR apc presenting antigen and/or MHC to recipient T cells. Acute rejection is often this.)
2. Indirect (RECIPIENT apc presenting donor antigen to recipient T cell i.e. how the immune systems fight foreigners. Chronic is often this)
120
Isograft
Allograft
XenoGraft
Split Graft
Isograft - transplant from a twin
Allograft - from same species
Xenograft - different species
Split graft - shared by two recipients
121
Types of allograft
Deceased donor
Solid organs: kidney, heart, pancreas, lungs, liver,
Others: cornea, heart valves, bone, skin, composite
Living donor – bone marrow, kidney, liver
122
Diapedesis
the passage of blood cells through the intact walls of the capillaries, typically accompanying inflammation.
123
Effector phase of graft rejection
Characterised by antibodies binding to graft ENDOTHELIUM and cause endothelium cell lysis due to complement @ and MAC formation- as well as the recruitment of inflammatory cells causing....
oOften resulting in graft infiltration by alloreactive CD4 cells,
- cyototoxic T cells which kill target cells via perforin, fas lignad and release of toxin granzyme B,
- macrophages (phagocytosie and release proteolytic enzymes)
These result in procoagulant tendences and closure of microcirculation of the graft leading to graft fibrosis.
124
Graft dysfunction markers
raised creatinine, raised LFTs
125
How do we prevent graft rejection?
DNA sequence people using PCR
| Screen for blood group and anti-HLA antibodies using cytotoxic assays, flow cyto and solid phase assays.
126
What are cytotoxic assays?
It looks at whether the recipient's serum will kill the lymphocytes of the donor in the presence of complement
Positive crossmatch suggests that there is cell lysis
127
How does flo cyto help in preventing transplant rejection?
* Looks at whether the recipient's serum binds to the donor's lymphocytes
* Bound antibody is detected by fluorescently-labelled anti-human immunoglobulin
* This is broad and looks at whether the antibodies bind antigen irrespective of whether they bind complement
128
How does solid phase assays help?
* This uses a series of beads containing all the possible HLA epitopes
* The recipient's serum is mixed with these beads and fluorescently-labelled immunoglobulin is used to determine which HLA epitopes the antibodies bind to
* It can also give an indication of the strength of the reaction
* NOTE: patients that have antibodies to lots of different types of antibodies are regarded as highly sensitised
129
What immunosuppression can be used in a transplant?
Calcineurin inhibitors e.g. tacro and cyclosporine
MMF
Azathiopurine
o Anti-CD3 antibody (OKT3)
o Anti-thymocyte globulin
o These cause apoptosis of T cells
- Alemtuzumab is an anti-CD52 monoclonal antibody that causes lysis of T cells
- Daclizumab is an anti-CD25 monoclonal antibody which targets the cytokine signal
- B cells can be depleted using rituximab (anti-CD20)
• BAFF inhibitors target cytokines that promote B cell activation and growth
• Proteasome inhibitors such as bortezemib can block the production of antibodies by plasma cells
• Complement binding to the surface of endothelial cells can be blocked using complement inhibitors such as eculizimab
130
What immunosuppresion do we use in a transplant
• Induction agent: e.g. OXT3/ATG, anti-CD52, anti-CD25
o This is given at the time of transplantation or just before in order to prepare the patient to receive the foreign organ
• Baseline immunosuppression: calcineurin inhibitor + mycofenolat mofetil or azathioprine with or without steroids
• Treatment of episodes of acute rejection
o Cellular: steroids, OKT3/ATG
o Antibody-mediated: IVIG, plasma exchange, anti-C5, anti-CD20
• NOTE: IVIG can reduce antibody production through feedback and it can displace troublesome antibodies so that they cannot exert their harmful effects
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How do we prevent and treat GvHD for HSCT>
Reaction of donor lymphocytes against host tissues.
Prophylaxis with methotrexate and cyclosporine
Treat: Steroids
(Presnets with rash, nausea, vomiting, abdo pain, diarrhoea, jaundice)
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Post transplant complications
Post-Transplantation Infections
• Increased risk of conventional infections
• Also increased risk of opportunistic infection:
o CMV
o BK virus
o PCP
Post-Transplantation Malignancy
• Viral-associated malignancies are much more common, such as:
o Kaposi sarcoma (HHV8)
o Lymphoproliferative disease (EBV)
• Skin cancer is 20 x more common
• Risk of other cancers is also increased
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Dendritic cells - what ar ethey and what do they do
• Reside in peripheral tissues
• Express receptors for cytokines and chemokines (to detect inflammation)
• Express pathogen recognition receptors (to detect pathogens)
• Express Fc receptors for immunoglobulin (to detect immune complexes)
• Capable of phagocytosis
• Following phagocytosis, dendritic cells will:
o Upregulate expression of HLA molecules
o Express co-stimulatory molecules
o Migrate via lymphatics to lymph nodes (mediated by CCR7)
• Present processed antigen to T cells in lymph nodes to prime the adaptive immune system
• Express cytokines to regulate the immune response
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Thoracic duct
Carries lymphocytes from lymph nodes back into circulation
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What happens in the thymus?
T cell maturation- what does that entail?
Deletion of T cells that are too reactive to self or not reactive enough
Maturation of T cells into CD4 or CD8 lineage
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Difference between Tfh cells and T reg cells
Tfh - promote GC reactions and seroconversion of B cells
T reg cells are subsets of lymphocytes that express FocP3 and CD25. They negatively regulate T cells to ensure that autoantibodies aren't formed.
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How do CD4+ t cells help B cells differentiate?
Already primed T cells have CD40 receptor that binds to the CD40 ligand on the B cells- this promotes B cell prolfieration, somatic hypermutation and isotype switching.
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What part of antibodies recognise what part of pathogens?
immunoglobulins: Soluble proteins made up of 2 heavy chains + 2 light chains
o Heavy chain determines the antibody class (GAMED)
o There are subclasses of IgG and IgA
o Antigen is recognised by the antigen binding region (Fab) which is made up of both the heavy and light chains
o Effector function is determined by the constant region (Fc) of the heavy chain
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How is the alternative pathway triggered?
Alterative pathway is AMPLIFIED by C3 bind to the bacteria cell wall. Remember the C3 Hydrolysis occurs spontanoeusly
C3 convertase is directly triggered by binding of C3 to bacterial cell wall components
• E.g. lipopolysaccharide of Gram-negative bacteria
• E.g. teichoic acid of Gram-positive bacteria
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Once activated what does complement actually do?
o Increase vascular permeability and cell trafficking to sites of inflammation
o Opsonisation of immune complexes keeps them soluble
o Opsonisation of pathogens to promote phagocytosis
o Activation of phagocytes
o Promotes mast cells/basophil degranulation
o Punches holes in bacterial membranes
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Phenotype of SCID
• Unwell by 3 months of age
o For the first 3 months they are protected by maternal IgG
• Infections of all types
• Failure to thrive
• Persistent diarrhoea
• Unusual skin disease
o Colonisation of infant's empty bone marrow by maternal lymphocytes
o This is sort of like graft-versus-host disease because the maternal lymphocytes are in the baby's bone marrow
• Family history of early death
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CD8 vs CD4 cells
CD8
• Kill cells directly
o Perforin (pore forming) and granzymes
o Expression of Fas ligand (which binds to Fas and induces apoptosis)
• Secrete cytokines (e.g. IFN-, TNF)
• Particularly important in defence against viral infections and tumours
CD4+ Helper T Cells
• Recognise
o Peptides derived from extracellular proteins
o Presented on HLA Class II molecules
• HLA-DR, HLA-DP, HLA-DQ
• Immunoregulatory functions via cell: cell interactions and expression of cytokines
o Provide help for development of a full B cell response
o Provide help for development of some CD8+ T cell responses
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Homeostatic immune proliferation with age
Feature of Di George Syndrome
• This means that if T cells are in an empty compartment they will just keep proliferating, so, in the end, T cell numbers tend to increase with age
o Immune function is mildly impaired and tends to improve with age
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Bare Lymphocyte Syndrome
• CD4+ T cells are selected in the thymus based on their ability to recognise MHC Class II
• If you have a deficiency of MHC Class II, CD4+ T cells will NOT be selected, and so, you will have a CD4+ T cell deficiency
This is called Bare Lymphocyte Syndrome (BLS) Type 2
• This can occur if you have a defect in one of the regulatory proteins involved in the expression of Class II genes
o Regulatory factor X
o Class II transactivator
• Mutations in these genes leads to absent MHC Class II
This, in turn, leads to a profound deficiency of CD4+ cells
o CD8+ count is usually NORMAL
o B cell count is NORMAL
o LOW IgG or IgA antibody levels due to lack of CD4+ T cell help
BLS Type 1 is a similar condition that is caused by failure of expression of HLA Class I
• Clinical Phenotype of Bare Lymphocyte Syndrome
o Unwell by 3 months of age
o Infections of all types
o Failure to thrive
o Family history of early infant death
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Bruton's phenotype
o Boys present in the first few years of life (they are fine for the first few months)
o Recurrent bacterial infections
• Otitis media, sinusitis, pneumonia, osteomyelitis, septic arthritis, gastroenteritis
o Viral, fungal and parasitic infections
• Enterovirus, pneumocystis
o Failure to thrive
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Hyper IgM phenotype
o Boys present in the first few years of life
o Recurrent infections (bacterial)
o Subtle abnormality in T cell function predisposes to Pneumocystis jirovecii infection, autoimmune disease and malignancy
o Failure to thrive
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Functional Tests of B cells?
o Specific antibody responses to known pathogens
o Measure IgG antibodies against tetanus, H. influenzae B and S. pneumoniae
o If the specific antibody levels are LOW, immunise with the appropriate killed vaccine and repeat antibody measurement 6-8 weeks later
o Functional tests have generally superseded IgG subclass quantification
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How is protein electrophoresis useful in immunodeficiency investigation?
* When you run your proteins through the gel you will get a big peak for albumin
* Then you will also get alpha-1, alpha-2 and beta peaks which are showing the presence of various other proteins
* At the end, you get the gamma peak - this contains ALL THE ANTIBODIES
* A patient who is antibody deficient will have NO gamma peak (as shown below)
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B cell deficiency steroetypes
* Common variable immunodeficiency: adult with bronchiectasis, recurrent sinusitis and development of atypical SLE
* X-linked Hyper IgM syndrome due to CD40L mutation: recurrent bacterial infections as a child, episode of PCP, high IgM, absent IgA and absent IgG
* Bruton's X-linked Hypogammaglobulinaemia: 1 year old boy. Recurrent bacterial infections. CD4 and CD8 T cells present. B cells absent. IgG, IgA and IgM absent.
* IgA deficiency: recurrent respiratory tract infections, absent IgA, normal IgM and normal IgG
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How do we treat B cell defieincy?
• Aggressive prophylaxis/treatment of infection
• Immunoglobulin replacement if required
o Derived from pooled plasma from thousands of donors
o Contains IgG antibodies to a variety of common organisms
o Aim to maintain trough IgG levels within the normal range
o Treatment is lifelong
• Immunisation
o NOT worthwhile because they will NOT be able to produce any antibodies
o UNLESS it is a selective IgA deficiency
