Dr Edwardson - Immunosuppression II Flashcards
What are the cells of the adaptive immune system?
Antigen presenting cells
B cells
Th1 and Th2 cells
Memory B and T cells
What is the effect do glucocorticoids have on the adaptive immune system?
Powerfully suppress the cascade process
Give some examples of disease which involve Th1 cells?
Insulin-dependent diabetes mellitus, MS, aplastic anaemia and rheumatoid arthritis
Also implicated in allograft rejection
Give some examples of disease which involve a Th2 cell response?
Predominates in allergic conditions such as asthma
Draw the pathway of glucocorticoid synthesis and the exogenous glucocorticoids that can act upon it
eeofine
Draw a diagram depicting the interaction of glucocorticoids with their receptor
efoi
How does the glucocorticoid receptor bind to DNA?
Through the use of a zinc finger motif
Binds as a dimer
What are the anti-inflammatory genes glucocorticoids upregulate?
Lipocortin-1
Secretory leukocyte inhibitory protein
IL-1 receptor antagonist
IκB
What are the inflammatory genes downregulated by glucocorticoids?
IL-1
TNFα
iNOS
COX-2
What is Cushing’s syndrome? What causes it?
Syndrome characterised by:
- hypertension
- thinning of skin
- thin arms and legs
- easily bruised
- increased abdominal fat
Caused by excess endogenous or exogenous steroid
Hydrocortisone
Short acting corticosteroid (<24hrs)
Used in the treatment of asthma, rheumatoid arthritis and in immunosuppression
Prednisone
Short acting corticosteroid (<24hrs)
Prodrug of prednisolone
Used in the treatment of asthma, rheumatoid arthritis and in immunosuppression
Prednisolone
Short acting corticosteroid (<24hrs)
Active form of prednisolone
Used in the treatment of asthma, rheumatoid arthritis and in immunosuppression
Triamcinolone
Intermediate acting corticosteroid (24-48hrs)
Used in the treatment of asthma, rheumatoid arthritis and in immunosuppression
Dexamethasone
Long acting corticosteroid (>48hrs)
Used in the treatment of asthma, rheumatoid arthritis and in immunosuppression
What are the characteristic of asthma?
Variable airflow
Airway hyper-responsiveness to various stimuli
Episodic bronchoconstriction
What are the features of an asthmatic attack?
Narrowed airway
Tightened muscles constrict airway
Inflamed/thickened airway wall
Mucus
What is the time-course of an asthmatic attack?
Intermediate asthmatic response (Reduced pulmonary function for ~1hr)
Late asthmatic response = 2-6hrs
What are the inflammatory event in an asthmatic attack?
Allergen activates T cells to release cytokines
These stimulate B cells cause production of IgE = Mast cell degranulation (release of histamine, leukotrienes, cytokines)
Also stimulates eosinophils to release leukotrienes chemotactic factors
What are the treatments for asthma?
Inhaled glucocorticoids
Selective β(2) receptor agonist eg salbutamol or salmeterol
Mast cells stabilizers eg cromolyn sodium
Blockers of leukotriene synthesis or receptors eg zileuton and zafirlukast
Muscarinic antagonists eg ipratropium
Theophylline (PD inhibitor)
Cromolyn sodium
Used in treatment of asthma
Mast cell stabilizer - however this is unlikely to be its MODA as other mast cell stabilizers don’t work
More likely that it acts by inhibiting the response of sensory C fibres, inhibiting local axon reflexes and may inhibit the release of T cell cytokines
(Garland 1991)
May be via inhibtion of chloride channels
Zileuton
Blocks leukotriene synthesis through inhibition of 5-lipoxygenase
Used in the treatment of asthma
Zarfirlukast
Leukotriene receptor antagonist used in the treatment of asthma
How do PDE inhibits help in the treatment of asthma?
Prevent the breakdown of cAMP
cAMP => relaxation
How do muscarinic receptor antagonists help in the treatment of asthma?
Reduce stimulation of IP3 through M(3) receptor = reduced Ca+ release and reduced contraction
How do β(2) agonists help in the treatment of asthma?
Stimulate the β2 receptor to stimulate adenylyl cyclase
= ^cAMP and inhibition of MLCK
What are the stimuli that cause histamine release from mast cells? How do these stimuli work?
Cross-linking of IgE bound to FCεR1
Complement components C3a and C5a
Basic drugs eg morphine, tubocurarine and codeine
Mechanical damage
All cause increase in Ca2+ concentration
What are the stimuli that act to inhibit the release of histamine from mast cells?
How do these work?
β-agonists
Phosphodiesterase inhibitors
Sodium cromoglycate
Increase cAMP levels
What are the airway changes in COPD?
Disrupted alveolar attachments,
Mucosal inflammation, fibrosis
Mucus hypersecretion
What are the cell types involved in COPD?
Neutrophils
T-lymphocytes (CD8+)
Macrophages
What do neutrophils secreted in COPD?
IL-6, IL-8, GM-CSF, TNFα, PGE(2), NE, cathepsins and MMPs
What do CD8+ Tcells secrete in COPD?
Perforins
Granzymes
Cyc-LTs
What do macrophages secrete in COPD?
TNFα, IFNγ, GROα, MMPs and LTB(4)
What are the drug target in the treatment of COPD?
Stop smoking
Immunosuppressants
Mediator antagonists
Neutrophil inhibitors
Protease inhibitors
Mucoregulators
What is multiple sclerosis?
An autoimmune disease in which their is autoimmune attack of oligodendrocytes
Which joints are most affected in rheumatoid arthritis? What else does it affect?
Affects proximal phalanges but also feet, spine etc
Effects on heart, lung, kidney, skin, sclera of the eye
What are glucocorticoids?
Endogenous steroids regulating glucose metabolism
What are corticosteroids?
Artificial steroids used in therapy
Includes endogenous glucocorticoids
Describe the activation of the glucocorticoid receptor
Glucorticoid receptor (major form = GCRα) is present in the cytoplasm complexed with other proteins eg HSP90
On binding the ligand, the receptor dissociates and is translocated to the nucleus where it acts as a transcriptional regulator that increases/decrease gene expression
What are the three ways in which the glucocorticoid receptor can alter gene expression when activated?
Directly by activating transcription
Indirectly by enhancing the action of a transcription factor by binding to it
By enhancing the action of a transcription factor by interacting with it cooperatively
What is the important effect of prolonged treatment with corticosteroids?
Shuts down endogenous production of glucocorticoids
Type I diabetes
Immune attack on pancreatic beta cells
Hyper and hypothyroidism
Graves disease and Hashimoto’s thyroiditis respectively
Vitiligo
Immune attack on melanocytes, causing uneven skin pigmentation
Behcet’s disease
Immune attack on blood vessels and anterior chamber of the eye
Ulcerative colitis and Crohn’s disease
Immune attack on large bowl
What is the major downside to the use of corticosteroids in immunosuppression?
Requires high dose treatment
Large number of side effects
Cyclophosphamide
Used for aggressive short-term treatment of sever autoimmune diseases
Prodrug from which the active compound (a nitrogen mustard alkylating agent) is released in the liver (by cyt p450)
Adds an alkyl group to guanosine and so preventsDNA replication, causing apoptosis of rapidly dividing cells (eg T and B cells)
Side effects = hair loss, haemorrhagic cystitis and infertility
What are the sites of action of immunosuppressive drugs?
Resting T lymphocytes - Early and late activation
IL-2
IL-2R
Proliferation
What is calcineurin?
A protein phosphatase that activates nuclear factor of activated T cells (NFATc) by dephosphorylation
NFATc = upregulation of IL-2R
How do calcineurin inhibitors act as an immunosuppressant?
Calcineurin activates NFATc by dephosphoylation causing an upregulation of IL-2R
Therefore inhibition of calcineurin prevents upregulation of IL-2R
Cyclosporin
Calcineurin inhibitor used as an immunosuppressant
Prevents upregulation of IL-2R by preventing the dephosphorylation of NFATc by calcineurin
Binds to cyclophilin to inhibit calcineurin
Tacrolimus
Calcineurin inhibitor used as an immunosuppressant
Prevents upregulation of IL-2R by preventing the dephosphorylation of NFATc by calcineurin
Binds to FK-binding protein
How does cyclosporin interact with calcineurin?
What does this mean?
By bind to cyclophilin A
CsA-CypA binary complex lies at the base of the helical arm of the catalytic subunit of calcineurin (CnA) that binds the regulatory subunit (CnB)
CsA sits in a hydrophobic groove in intimate contact with both subunits at a region unique to calcineurin
Means that CsA has a high specificity
How do inhibitors of mTOR help to cause immunosuppression?
mTOR is a protein kinase that regulates cell growth/proliferation and is stimulated by activation of the IL-2R
There inhibits proliferation of T-cells/B-cells
Rapamycin
Macrocyclic triene from a soil bacterium
Binds to FKBP and inhibits mTOR (a serine/threonine protein kinase involved in cell proliferation following activation of IL-2R
Azathioprine
Active metabolite inhibits guanosine synthesis and so inhibits DNA synthesis in B, T cells
Other cell can obtain guanosine via the salvage pathway
Mycophenolate
Inhibits guanosine synthesis (IM dehydrogenase) and so inhibits DNA synthesis in B and T cells
Other cells can obtain guanosine via the salvage pathway
To which region of the antibody do antigens bind?
The hypervariable region at the tips of the light and heavy chains
What is a chimeric antibody?
One that has mice variable light and heavy chains
What is a humanized antibody?
One that has mouse complementarity determining regions
Infliximab
TNFα mAb used in the treatment of rheumatoid arthritis and Crohn’s disease
Adalimumab
TNFα mAb used in the treatment of rheumatoid arthritis and Crohn’s disease
Omalizumab
IgE mAb used in the treatment of asthma
Prevents binding of the IgE Fc domain to the Fc receptor and so inhibits release of inflammatory mediators from basophils and mast cells
Alemtuzumab
CD52 mAb used in the treatment of B cell leukaemia and MS (trials)
Resets acquired immunity to a naive state
Still functional innate system
What is the difference between drugs with the suffix -mab, -imab and -umab?
- mab = monoclonal antibody
- imab = chimeric monoclonal antibody
- umab = fully humanized antibody
Etanercept
Artificially construct of the TNFαR fused to an antibody Fc domain
What are the benefits of antibodies as therapeutic agents over chemicals?
High target affinity High target specificity Low off-target toxicity Blocks protein-protein Long serum half life Effector mechanisms
What are the benefits of chemicals as therapeutic agents over antibodies?
Access to small sites Extravascular targets Intracellular targets Oral route Immunogenicity
What are the new techniques to improve antibodies?
Bispecific antibodies Supercharged ADCC Drug conjugations Longer PL Shrinkage of the scaffold - even by mimicking binding domain
How do bispecific antibodies work?
Bind different targets on two cell types, bringing them into proximity of each other
Allows ADCC
Eg CD19 on B-lymphoma cell and CD3 on CD8+ Tcells
How do supercharged ADCC antibodies work?
Killing action of Abs can be improved by improving binding of AbFc to receptors on NKcells (FcγRIII) by:
Changes in Ab Fc protein
Changes in Ab Fc sugars (afucosylated glycoforms)
How do Ab drug conjugates work?
Conjugation of small highly toxic chemical drugs to Abs
Abs bind to target on cells and are taken up
Bound drug kills cells
MMAE (monomethyl auristatin E)
An antimitotic which inhibits cell division by blocking the polymerization of tubulin
Conjugated to Ab then cleaved in the cell by cathepsin
Maytansine
Inhibits microtubule assembly
Recently shown to improve survival when conjugated to trastuzamab
Calichaemycin
Binds to DNA in the minor groove and causes strand scission
What are the implication of improving the PK of antibodies? Give an example
Slow release formulation and long biological action could lead to injection of Abs once a year
eg Denusomab is given every 6 months
Denusomab
Binds to RANKL (receptor activator of NFκB ligand) a ligand for osteoprotegerin and functions as a key factor for osteoclast differentiation and activation
Inhibits bone loss in osteoporosis, RA and multiple myeloma
