C Edmead Lec 3 & 4 Therapy Rationale Flashcards
Steroids are specific anti-______ agents unlike the DMARDs
Anti-inflammatory
Steroids decrease ______. Decrease the flow of WBC’s from blood vessels into tissue therefore less activation that can drive inflammatory response.
Extravasation
Steroids dampen down activation of cells in tissue e.g macrophages and decrease secretion of cytokines from _ cells.
Therefore fewer inflammatory cytokines produced.
T-cells
Steroids work at the gene level by altering ____ ______.
DNA encode genes - transcribed to proteins - function in the cell.
Want to decrease expression of inflammatory proteins and increase expression of anti-inflammatory proteins.
Glucocorticoids are lipophilic so diffuse across cell membrane and enters cell and binds receptors inside cell (intracellular). This steroid bound to the receptor (transcription factor) then translocates to _____ and effects which genes switched on and off by inhibit binding of other transcription factors (__-_ and ____).
- Try and target proteins that target pro-inflam gene transcriptions.
Gene transcription
Nucleus
AP-1 and NFKB
Idea is that glucocorticoids prevent transcription of __-_ and ___ and other inflammatory mediators and increase gene transcription of __-__ and __-_ that are anti-inflammatory cytokines.
IL-1 and TNF
IL-10 and IL-4
Side effects due to inability to get steroids that do not interfere with other effects of steroids e.g ________ (thinning of the bone)
Osteoporosis
Steroids decrease ____ synthesis. Which can be a good thing as we know the antigen is in collagen and new collagen can activate the immune synthesis even more! BUT we need to keep the joints working so need some of it!
Collagen
Osteoblasts lose function due to ______ use. And this use can also cause a reduction in _____ absorption which is needed to make strong bones.
Steroid
Calcium
Use of steroids can also increase ____ and also induce type 2 ____. This increases risk of obesity which can lead to stress on joints.
Appetite
Diabetes
Mineralocorticoid effects when using steroids can lead to __/__ retention. This can lead to hypertension, oedema and CV events.
Sodium/water retention
If too high a dose of steroids will block inflammation and WBCs (_____) can’t enter tissue. This can leave patient open to other opportunistic infections.
Leukocytes
TNF - Tumour Necrosis Factor
Is a ___-______ cytokine that can also cause _____
IL-1 is also a __-_____ cytokine that can stimulate NFKb transcription factor which switches on pro-inflammatory genes along with TNF.
TNF and IL-1 binding to receptors up regulates theirselves and each other,
Pro-inflammatory cytokine
Apoptosis
TNF and IL-1 activate the ____ immune system. This activates macrophages etc. This can then lead on through cytokines and presentation of antigens to activate the ____ ______
Innate immune system
Adaptive immune system
TNF and IL-1 are both produced by _ _ _ or endotoxic activated monocytes (in blood) become macrophages when enter tissue.
LPS
IL-1 is found in 2 forms.
Target IL-1 _ form as alpha is not involved.
BETA
Most proteins made in the __ whereas IL-1 synthesised in ________.
IL-1 synthesised as precursor and cleaved by IL-1 Converting Enzyme (ICE) - this is known as _____-1
Endoplasmic reticulum.
Microtubules.
Caspase-1
Targeting ICE inhibitors would be a good ___ ______ _____ but this has not been shown to be effective!
Release of enzyme facilitated by P2X7 receptor so interest in developing P2X7 ______ as an anti-inflammatory agent.
Anti inflammatory agent
Antagonist
- IL-1 initially localised effect but then circulates bloodstream ( _____ effect) and can cause fever (heat and swelling in joints).
Systemic
IL-1 production leads to increased synthesis of COX-2 (leads to _____ formation) and INOS (leads to ___ _____). These enzymes are only upregulated in inflammation.
Prostaglandins Nitric oxid (Inducable nitric oxide synthetase)
- IL-1 can lead to increased expression of VCAM and ICAM (selectins and _____ molecules) lead to extravasation of WBC into tissue.
Block this and WBC’s stay in blood vessels.
Adhesion
IL-1 increases bone erosion and activates RANK ligand and osteoclasts that digest _____ - can lead to osteoporosis.
Bone
IL-1 DOESN’T induce apoptosis but induces ___ which induces apoptosis (and septic shock)
TNF
IL-1 activation:
- Release of complement C5a due to activation of innate immune system (antigen/antibody driven)
- Complement can increase gene transcription of IL-1
- ______ feedback pathway
Positive
IL-1 release is increased in IFNg stimulated monocytes/_______.
T cells are main producers of IFNg (T cells activated, make more IFNg, feedback onto macrophages and produce more IL-1)
Macrophages
- CD40L activates ICE (interleukin 1 converting enzyme in endothelial cells and ___ ____ ___)
- CD40L is expressed on ___ cells.
Smooth muscle cells
TH2 cells (which activate B cells - lead to feedback and induces IL-1 release)
IL-1 receptors are members of the IG (________) superfamily.
Immunoglobulin
IL-1 Type 1 receptors are present on ___ cells
- Not good drug target - side effects
- Leads to activation of kinases, NFKb and AP-1
IL-1 Type 2 receptors mainly on _ cells
- Decoy receptors - binds IL-1 and mops up - doesn’t signal
- Controls circulating levels
Both receptors can exist as _____ receptors. (increase numbers of these to mop up circulating IL-1)
Most
B cells
Soluble
IL-1 type 1 receptor = ____dimer.
- One molecule for type one receptor and accessory protein to dimerise to form receptor
- Activates cell signalling pathway, switches on gene transcription (NKFb and AP-1)
WANT to _____ this type of receptor.
Heterodimer
BLOCK
TNF belongs to a superfamily of cytokines which includes:
TNF-alpha (cell death), TNF-beta (Lymphotoxin), FAS-L (responsible for ______), CD40-L (responsible for ______ __ _ ___)
apoptosis
activation of B cells
TNF is a trimer.
Therefore _ molecules of TNF come together to activate the receptor.
3
TNF activates a signalling pathway which causes a functional response in cell - up regulation of __-inflammatory mediators.
Pro-Inflammatory
Bacteria, viruses, IL-1, IL-2 and TNF all increase levels of TNF transcription. This does not however, mean increased levels of protein as MRNA is ______.
LPS increases TNF for sure as it ______ the MRNA.
Unstable
Stabilises
TNF is often surface bound along with the receptor. So one cell will express TNF on it’s surface and one will express TNF receptor on it’s surface. So two cells come together to activate the receptor. ___’s can cleave TNF from a cells surface making soluble TNF.
SO need to target the membrane bound form and soluble form.
MMP’s (Matrix Metallo Proteinases)
Membrane and _____ forms of TNF both mediate cytotoxic (cell death) and inflammatory effects by cell to cell contact. (Binding to receptor)
Soluble
TNF has _ different receptors
TNF1 is present on all cells except ____ ____ ___.
Can cause growth through transcription AP-1 and NFKB
Can cause cell death through activation of _____ and apoptosis
TNF2 is inducible, upregulated in _______
Can cause cell growth through transcription AP-1 and NFKB
Don’t want too much cell growth due to synovial _______ blocking the joint and don’t want too much cell death as this damages the joint.
2 Red blood cells Caspases Inflammation Proliferation
In T cells, high levels of _ _ _ triggered apoptosis whereas low levels caused cell activation.
So TNF can exert either effect.
RIP
TNF binds to it’s receptor by trimerising. To block the TNF receptor you have to block at least __ TNF molecule(s).
C terminii engage _ receptors to signal
2
3
If TNF binds to a soluble receptor then it gets ______ __. But if it binds to a membrane bound receptor then this activates the cell and this is bad.
Mopped up
TNF causes the recruitment of ______ which release ROS that cause damage to the tissue.
TNF also causes the recruitment of monocytes which become _____ in the tissue which secrete pro inflammatory cytokines.
So need to block recruitment of these cells to reduce tissue damage.
Neutrophils
Macrophages
TNF increases expression of ______ molecules which increases extravasation.
Stimulates EC and macrophages to secrete _____ which draw cells to site of attack and infection.
Stimulates mononuclear ______ to secrete IL-1 which causes bone erosion.
Adhesion
Chemokines
Phagocytes
If TNF is released in large enough quantities it can enter the bloodstream and have _____ effects.
It can act on the ______ to induce fever (PGs)
Induces hepatocytes to release APPs (____ ___ ____) which are inflammatory ______.
Prolonged exposure can lead to _____ ____ (Muscle wasting, cardiac issues).
Systemic
Acute Phase Proteins
Mediators
Septic shock
Why might making more soluble TNF receptors in an attempt to get them to ‘mop up’ TNF, not work?
- The soluble TNF receptors might just bind the TNF and then deliver the TNF to membrane bound receptors instead, where they can then exert their effects.
THIS does not happen as much in IL-1 with soluble IL-1 receptors.
How do anti-TNF therapies work?
Antibodies that circulate round the body, bind to TNF and then like other antigens, the antigen bound to TNF will be removed from the body by endocytosis from macrophages.
‘Mop up TNF to prevent receptor engagement’
Antibodies are designed to specifically bind to one protein. They therefore have fewer ___ _____.
Side effects due to their specificity and high binding affinity.
Raise an antibody against a human protein e.g human TNF.
If you put human TNF into a mouse, the mouse will make antibodies against it as it will see it as foreign. Serum can then be harvested from this animal which will contain a range of antibodies against different epitopes of the protein (___clonal)
If you isolate one particular B cell which makes one type of antibody then this is ___clonal.
Polyclonal
Monoclonal
Can design antibodies against cellular adhesion molecules. If you block these molecules with an antibody then cells can’t ____ and pass from blood into the tissues.
___ is an example of an adhesion molecule. Anti ICAM (Enlinomab) blocks _ cell migration and decreases levels of IL-_.
Extravasate.
T cell
IL-6
ICAM
Murine monoclonal antibodies eventually become immunogenic in humans. So needed a way to make these less immunogenic. This lead to the development of _____ antibodies. This is a mix of ____ antibody and ____ antibody sequence.
Needed to replace everything except ____ ____ ____ (Fab)
INFLIXIMAB (example of chimeric anti-TNF)
Chimeric
Mouse
Human
Antigen binding region
Antibodies are proteins so they have to be administered by ______.
Injection
If tolerated INFLIXIMAB is given with ______.
Methotrexate
The ends of the antibody on INFLIXIMAB are still murine so eventually an immugenic response may occur (inflammation at injection site). This is a reason for switching the ______ ___.
Injection site
Murine monoclonal antibodies THEN Chimeric monoclonal antibodies THEN \_\_\_\_\_ monoclonal antibodies.
These antibodies only contain murine ______ regions. All conserved regions within the ligand binding region are replaced.
e.g Certolizumab
Less chance of inducing an _________ reaction.
Humanised
Hypervariable
Immunogenic
Murine monoclonal antibodies THEN Chimeric monoclonal antibodies THEN Humanised monoclonal antibodies THEN \_\_\_ \_\_\_\_\_\_ monoclonal antibodies
These antibodies are made by replacing IG genes in mouse with human genes. (Find locus of IG genes that code for different region on antibody and alter to make human sequence antibodies)
Cost wise these are ______.
e.g ______ (Humira), Golimumab
Fully humanised monoclonal antibodies
Expensive
Adalimumab
Patients are often only started on Humira once they have failed on a _____ and other anti-TNF.
This is due to cost.
DMARD
Rather than increasing the number of antibodies you can increase the number of soluble receptors that circulate e.g in IL-1, TNF (but beware of passing in TNF). These will ____ __ cytokines.
Mop up
What are some issues with creating soluble receptors to mop up free cytokines? (4)
1) Soluble receptors are cleared rapidly from the body
2) Soluble receptors may facilitate signal transduction by passing the ligand
3) Homotrimeric TNF can still crosslink membrane receptors with soluble receptor bound
Body makes IL-1 receptor antagonist which competes with IL-1 for the receptor. If make more soluble IL-1 receptor (___-__) then could be mopping up more IL-1Ra.
This would be detrimental as less IL-Ra to block IL-1Rs
sIL-1R
In an attempt to increase the half-life of the circulating soluble receptors. They combined the soluble receptor with an _______ structure.
So bottom half is the ______ (Fc portion) and instead of Fab region of _____, have the protein sequence of the _____.
Whole complex is large protein and can bind ___ molecules of TNF. TNF binds as a ____, but if block two then ____ hinderance will prevent it engaging with the receptor.
Antibody x3
Receptor
2
Trimer
Steric
The structure and large size of Chimeric _____ proteins, extends the T1/2 so have to give it less often to mop up TNF.
Lenercept and ______ (Enbrel) - Chimeric ____ Proteins
Fusion
Etanercept
Fusion
Enbrel can be used in ______ arthritis and is given as a ___ weekly SC injection
Juvenile
Twice weekly SC injection
______ is a problem with repeated injections as the foreign protein can still cause an immune response. This is often dampened down with subsequent treatment with ______.
Tachyphalaxis (decreased response to drug after repeated admin)
Methotrexate
Monoclonal Abs can ____ disease progression but doesn’t cure.
Slows
Cytokine therapies:
Could increase levels of anti-inflammatory cytokines e.g __-, __- , IL-13
By injection or by ___ ____ (increase endogenous expression)
However, these have a narrow therapeutic window. Don’t want to _______ patients.
IL-4, IL-10, IL-13
Gene therapy
Immunosuppress
Gene therapy to increase levels of anti-inflammatory cytokines at required site.
Use tissue _____ _____ to switch on gene transcription in these areas (macrophages/lysosymes). This way you can concentrate the therapy to this specific area/joint rather than causing _____ circulation of the cytokines.
This way we can dampen down the response of say macrophages in joints.
Tissue specific promotors
Systemic
A disadvantage of gene therapy to increase levels of anti-inflammatory cytokines is that we need to be able to switch these genes off if ______ occurs.
This can be achieved by creating ____ promotors or by introducing _____ motifs
Infection
Inducible
Suicide
Gene therapy of cytokines can also be used to inhibit NFKb to block __-______ cytokine production. OR to inhibit FADD (Fas ______ _____ ____) which drives apoptosis and cell destruction.
Pro-Inflammatory
Fas Associated Death Domain
Anakinra - recombinant form of IL-Ra
- In patients with chronic inflam see _____ levels of this circulating ______.
Reduce bone destruction as IL-1 activates _____ which causes ______ of bone.
Inject into joints using a viral vector and synovial cells.
Decreased
Antagonist
Osteoclasts
Resorption
