Equine Med- endotoxemia 2

wilsbach's version from 2016-02-23 15:19

More microscopic things

Question Answer
(babbled in class) so if a virus gets into the cell, how does innate immunity work from within?TLR3 or 7/8/9, it signals through complicated signalling processes which causing things to bind to the DNA which results in production of interferons (natural response to viral infections)
what is the end product of the TLR signalling which was triggered by the endotoxin? Is this good or bad?produce pro-inflammatory cytokines--> initially made to deal with the infection-- it leads to an inflammatory reaction. As long as that's contained, everything is fine. but if there is an OVERKILL of the production of pro-inflam. cytokines, the inflammatory reaction becomes deranged --> SYSTEMIC INFLAMMATORY RESPONSE SYNDROME (SIRS) and possibly multi-organ failure/dysfunction.
Overview of a variety of receptors which endotoxins and bacteria interact with (pic)
Chart that shows the receptors, their ligand (thing they bind to) and the origin of that ligand (pic-look it over)
we need to understand the down signalling pathway from the TLR receptor because the drugs we use in tx interact with this downsignalling. So, explain it a bit *part in brackets-- dont have to memorize this part of the cascade. The part below is important though. So: There is the I-kappa-B (inhibitory kappa B) that holds onto 2 different signalling proteins, and only when the I-k-B is phosphorylated, P50 and RelA are released (this is the 2 diff signalling proteins)-- the two of them together are Nuclear-factor-kappa-B (nf-k-b). So phosphorylation of I-k-B leads to release of nf-k-b which then binds to the DNA. And the release of nf-k-b results in it interacting with the DNA which leads to the production of pro-inflammatory cytokines
what is the NF-kappa-B pathway?Phosphorylate inhibitory kappa B to release the Nuclear-factor-kappa-B which binds to DNA and causes expression of pro-inflammatory cytokines.
why do we care about p38 and JNK, and ERK- what are these? these also activate pro-inflammatory cytokines, and we can inhibit these with drugs too.
explain what the NF-𝜅B pathway isaka Nuclear factor-𝜅B. This thing is Retained in the cytoplasm by Inhibitory-𝜅B (I-𝜅B), but Degradation (phosphorylation) of I-𝜅B allows translocation of NF-𝜅B to the nucleus, where it undergoes Binding to specific site on DNA: up-regulation of pro inflammatory genes
what are the inflammatory mediators which are created and released from the downstream signalling? what is the result of all of this?Cytokines (TNF, interleukins), Lipid-derived mediators, Tissue factor, Proteases, prostaglandins (NK-k-B actually signals to cell to turn on COX-2!) and histamine are all released due to the signals on the DNA to make pro-inflammatory cytokines. These products are what cause the clinical effects in the host


Question Answer
what are the 4 current approach methods to helping treat endotoxemia?(1) Eliminate the source of endotoxin (2) Prevent the interaction of endotoxin with cells (3) Prevent intracellular signalling (4) Prevent mediator synthesis (the higher up/earlier in the processes you are able to stop it, the more sig. of a diff you can make)
what are some things we can do to Eliminate the source of endotoxin?Identify the problem early, Remove devitalised intestine/ Remove the nidus of infection.
if you have a torsion which obstructs the BVs, and you think you might be able to save it (or the effected area is too large to be taken out) what do you need to know about releasing that torsion?you release torsion--> restoration of blood flow--> so all endotoxins that left the gut and are now in GI wall are going to be absorbed in systemic circulation, leading to a strong inflammatory response. (can lead to drop in BP, etc, bc of large dose of endotoxins into system) so need to be careful with that-- and warn athesthatist with that youre doing.
how might you Prevent the interaction of cells with endotoxin?You can give Hyperimmune plasma or serum (anti-LPS antibodies), can use polymyxin B, Phospholipid emulsion, TLR-4 antagonists, TLR-2 antagonists
explain how Hyperimmune plasma or serum (anti-LPS antibodies) worksDirected against the conserved core region (or lipid A region)...since it is the conserved portion, you will get Cross protection against different endotoxins. However use of this is controversial (studies showing benefit and NOT benefit), it is expensive,
explain how polymyxin B works(Prevent the interaction of cells with endotoxin) this is a abx which works particularly good against G- bact... it binds with lipid A (one on one- so poly molecule to one LPS). this prevents the interaction with CD14. Although there are def side effects (think abx and flora) the dose is smaller than a regular abx dose would be. (studies have also not seen the renal side effects of poly when used in this way). They can also use the poly with conjugates of dextran-- dextran is a large molecule so if poly is bound to it, cant leave systemic circulation, stays in circulation-- less likely to cause renal damage. (but this tx doesnt do anything about the results of the LPS which are already bound to the TLR 4)
explain this graph about a study with polymyxin B So the circles/triangles are diff concentrations of polymyxin B. then they looked at one of the inflammatory factors, TNF-alpha. So with inc doses of poly, there are dec in TNFa
explain how Phospholipid emulsions workthese Bind endotoxin. like hyperimmune plasma and polyB, if there is already full blown clinical endotoxemia it becomes more difficult to use this-- because if there are all those CSs, there is already a high production of cytokines. however you can see Effects on clinical outcome, cytokine production, heart rate and body temperature. In vivo studies have shown there to be a dec in TNF-a
explain how TLR-4 antagonists work (what is the difficulty associated with them?)They Prevent the interaction off cells with endotoxin. however, there are species differences in lipid A recognition (MD-2/TLR-4 responsible for species differences). Examples of this: LPS from Rhodobacter sphaeroides is an antagonist in humans and mice, but an agonist in the horse and hamster. E5564 (Eritoran) is a potent antagonist in humans and horses (but didnt seem to help much in clinical trials).... so basically TLR4 antagonists are a potential tx but havent proven to be particularly useful yet. (an TLR-2 antagonists have not been beneficial in horses)
explain the species differences with the recognition of lipid Amany times the MD2 is different. and the MD2 rearranges the TLR4 dimer which then results in the sp diffs of the recognition of lipid A.
he said this is a nice overview of what happensIn upper L corner: LPS binds to LBP. LBP takes LPS to CD14 which sits on the cell surface (no intracellular domain), it moves LPS across cell memb to TLR4, TLR 4 requires MD2- and you can see that TLR4 is in a homodimer with another TLR4. there is intracellular signalling with I-k-B and NF-k-B --> ERK, JNK, p38 are then activate
what is the basic way in which TLR4 receptor antagonists work?they are lipid-type molecules which don't activate TLR4
how do glucocorticoids interact with the downstream signaling?interfere with NF-k-B. (also interact with ERK/p38/JNK)
how do NSAIDs interact with the downstream signaling?interfere with NF-k-B.
how do Beta-2 agonists interact with the downstream signaling? interfere with NF-k-B. (also interact with ERK/p38/JNK)
how do phosphodiesterase inhibitors interact with the downstream signaling?interfere with NF-k-B. (also interact with ERK/p38/JNK)
what are 5 Inhibition of NF-𝜅B signalling drugs/things we can use?Glucocorticoids, NSAIDs, Phosphodiesterase inhibitors, Natural compounds, Anti-inflammatory cytokines (alternative pathways)
explain thisThis is inhibition of cytokine production (Inhibition of MAPK (mitogen activated protein kinase) signalling) (TNFa and LI6 specifically looked at) so there is a pos control (no tx), and then 3 compounds used to try to inhibit the cytokines- RP= rolipram, a phosphodieserase inhibitor: you can see this has a strong effect on TNF-a, but no effect on IL6. CB= clenbuterol, a beta-2 agonist: dec TNF-a production. Dexmethasone dec both. The BOTTOM graph is showing specific p38 or JNK or ERK inhibitors. so, inhibition of these three mediators does have benefit on horses with endotoxemia.
explain why IL6 is a weird cytokineHe explains: IL6 is a strange cytokine and it takes a while for it to be produced. and depending on the concentration, it can be pro or anti inflammatory. Initially anti and eventually with inc conc becomes pro inflammatory).
Inhibition or prevention of mediator synthesis--> 3 things we can give in this category?TACE (TNF-𝛼 converting enzyme) inhibitors, IL-1ra (receptor antagonists), NSAIDs
Inhibition or prevention of mediator synthesis--> what can we give that does Prevention of cytokine release?TACE (TNF-𝛼 converting enzyme) inhibitors (enzyme essential in release of TNFa) inhibit TACE, inhibit release of TNF alpha.
Inhibition or prevention of mediator synthesis--> what can we give that does Prevention of cytokine binding to its receptor?IL-1ra (receptor antagonists)
what do NSAIDs do with this big pathway? (several things)Inhibit cyclo-oxygenase--> Prevent production of prostaglandines. But aside from that have Non-enzyme inhibiting effects: inhibition of the phosphorylation of I-𝜅B (so NK-k-B will not be released) Blck bars are DMSO- no effect. but high conc of carprofen has a sig effect on LPS changes in optical density (sthing you can measure this with) flunixin may or may not (conflicting studies)-- these are really high conc tho so not feasable in endotoxemic pts. BUt what we do know is that a low dose of flunixin prevents production of cytokines-- usually about a 1/3 of the dose (no analgesia but sufficient inhibition of cytokine release) it does NOT BIND ENDOTOXINS!! it stops the inflammatory response.
what is a time where NSAIDs can particularly have an adverse effect?when endotoxemic pt is dehydrated.
what do Phosphodiesterase inhibitors do?Inhibition or prevention of mediator synthesis-- Mechanism of action: inhibition of the breakdown of cAMP. have effect on cAMP binding protein in this pic under p38 there is CREB which is cAMP responsive element binding protein. This is important in the pathway, an PDE inhibitos effect this--> effect on CREB--> DEC in production of cytokines (slide says: cAMP molecular target: cAMP dependent protein kinase A (PKA), and PKA controls: intracellular calcium - cell proliferation - transcription)
what do beta-2 agonists do?Inhibition or prevention of mediator synthesis-- Mechanism of action:enhanced formation of cAMP. PDEinhibiors inhibit the breakdown, beta-2 enhance formation, so closely related in function. have effect on cAMP binding protein in this pic under p38 there is CREB which is cAMP responsive element binding protein. This is important in the pathway..... leads to dec release of cytokines (slide says: cAMP molecular target: cAMP dependent protein kinase A (PKA), and PKA controls: intracellular calcium - cell proliferation - transcription)
why do we need to be careful admin abx to pts with bacteria in their blood (sepsis)?bc bact death is when they release their endotoxins!!
** which abx causes the least amount of endotox activity? WHY?This is the endotoxin activity (endotox moleculeS) and then look at diff abx. ....amikacin causes the least activity because it kills them withouts lysis of bact
** which drug is the worst for causing endotoxins, and why?ceftiofur, a betalactam. In endotoxin activity AND TNF-a activity. ( on slide: Effects of β-lactams is likely due to filament formation and release of active biomass)
which two drugs are the best to tx septicemia without inducing a horrible endotoxemia?amikacin and ampicillin
so if you encounter a patient with endotox/septacemia, you are relatively late. why?see CSs bc there is already mediator production. makes tx options difficult.
mentioned in class: what other weird drug shows a dec in cytokine production?ketamine
why might use of glucocoroticoids be controversial in foals?diff balance of steroid hormones in them than in adults. adding more might not be the best thing? also might be dernagement of hypothalamic-pituitary axis.
what is the problem with blocking the intracellular pathways?if you block one, another one will just be used instead.
what is the really tricky part about inhibiting the inflammatory pathway?cant block the WHOLE path bc it's there for a reason..this is the hard part. (its just deranged in endotox, but cant just shut down whole path)
what are some future considerations for tx of endotoxemia?still need to be early to affect the initial interaction of endotoxins and receptors. but (1) Binding protein that does not recognise CD14 (2) Antagonism at the CD14 level (Antibody approaches, Naturally occurring non-toxic LPS) (3) Interfere at the TLR level (4) Post-TLR inhibition
The missing silver bullet…what are the things we still struggle with right now?No single lethal mediator is involved in clinical situations! Redundancy of mediator systems, Timing of illness and treatment is essential, Individual differences in the response to stimuli