concepts of endotoxemia/septicemia are cross-species
blah blah blah
What is endotoxemia?
presence of bacterial endotoxins (Gram- positive and Gram-negative) in the blood (wide variety of product produced and released by bacteria which enter the bloodstream)
what is septicemia?
systemic bacterial infection (bacteria in the blood)
endotox versus septi?
ENDO= THE TOXINS IN THE BLOOD. SEPTICEMIA= THE BACT IN THE BLOOD
what are examples of gram-negative endotoxins? (2)
lipopolysaccharide, bacterial DNA
what are examples of gram-positive endotoxins? (4)
(he says they are endotoxins but please remember that only G- bact have endotoxins bc G- have a wall (why they are G- bc wall is deflecting stain) and the endotoxins come from the wall.... G+ dont have a wall, so they have EXOtoxins) lipoproteins, lipoteichoic acid, peptidoglycan, bacterial DNA
what is shock?
systemic inﬂammation caused by endotoxemia or sepsis leading to SIRS and/or MOF
which dzs in the horse commonly lead to endotoxemia/septicemia? (examples)
Occurs in diseases with the highest mortality rates!! Acute abdominal disease, Adynamic post-operative ileus, Laminitis, Exertion, Failure of passive transfer, Neonatal septicaemia, Recurrent airway obstruction, diarrhea (other examples: pyometra in dogs, GDV in dogs, toxic mastitis)
where do most endotoxins come from in the body?
GI tract. (specifically talking about G- lipopolysaccharides) (since endotoxins are made by bact, a severe bact infection of organs can lead to a endotoxemia-- such as colic)
**what is a sign you can see on your PE that basically screams endotoxemia at you?
The purple line around teeth (toxic rim/toxic line) [almost pathognomonic]
how common is endotoxemia in colic cases?
40% of colic cases test positive for some sort of endotoxin in the blood
what are some Clinical signs of endotoxemia, and what is very important you know about the CSs of endotoxemia?
they are Non-speciﬁc clinical signs: overshadowed by signs of the primary disease... however you might see: Mild fever (one of the first things you see), Mild to moderate abdominal pain, Depression, yawning, Anorexia, Hyperaemic mucous membranes, Prolonged capillary reﬁll time, Decreased GI sounds, Tachycardia and tachypnea, Sweating, Dehydration, (said in class: lactate goes up in blood/peritoneal fluid), the "toxic line"
is there a test to test for endotoxins?
yes, Limulus amebocyte lysate (LAL) (horseshoe crab has amebocytes in it which are very sensitive to endotoxins) but it is not used in clinical practice
are most gut bact in horse positive or negative?
Gram positive! (in cattle most rumen microbes are G- so don't confuse these) [endotoxins of G- bact cause a bigger prob]
Prolonged or severe endotoxaemia can lead to what clinical signs?
Signs of cardiovascular collapse and multiple organ failure... Stuporous, Cold extremities, Profuse sweating, Weak peripheral pulse, Muscular tremors, Diffusely purple and congested mucous membranes, Prolonged jugular reﬁll time, Dependent pitting edema, Oliguria or anuria, Abdominal distension from ileus, Signs of disseminated intravascular coagulopathy
explain a little bit about lipopolysaccharides
has 2 portions that are highly conserved (all LPSs have that porton) and then there is a large tail that varies between the bact. but in essence all G- bact have LPSs on their membrane. (in cases of colic, often all diff kinda bact releasing diff kinda endotoxins)
where are LPSs in the bacteria, and how are they released?
in the cell membrane- which means, the only times they release them are during (1) lysis/death (2) rapid proliferation (mitosis)
what does a CBC look like in an endotoxemia case?
Leukopenia, characterised by neutropenia, increased numbers of bands and toxic morphology of the neutrophils
what happens to neutrophils in endotoxemia?
See Cytoplasmic vacuolation, Toxic granulation, Dohle bodies (thought to be remnants of the rough endoplasmic reticulum)
what are some non-specific clinpath findings endotoxemia can cause?
Hemoconcentration, azotemia, metabolic acidosis, hypoglycaemia, increased ion gap, hypocalcemia, thrombocytopenia, APTT and PT prolongation
if all horses have about a gram of endotoxin in their gut, why dont they have endotoxemia?
they have their mucosal barrier intact
example of how endotoxins affect the horse in colic-- say it is a strangulation from a lipoma. what is happening to the gut that is strangulated?
Purple gut is ischemic.. microscopically, there is endothelial damage (damage to cells) so there are all kinds of issues with the mucosal barrier. And there are already endotoxins just naturally in the gut of the horse. So once mucosal barrier is damaged, endotoxins can leak out into the rest of the body.
so if mucosal/tight junction damage allows endotoxins to seep from gut into abd-- what do they do next?
taken up by peritoneum--> lymph drainage from peritoneum--> lymph vessels to heart. (there is a little bit of neutralization in your lymph system as well)
how does liver deal with endotoxins?
there are hepatocytes ofc, but also kupfer cells which are extremely good at neutralizing endotoxins. (but with inc number they can get overwhelmed--> cant break them down--> leads to inflammation and problems in the liver)
sit in outer cell wall of G- bact. sticks out a little bit. When in cell wall cause no problems, when cell wall damaged, they are released to go wreck havoc.
the complex puzzle of endotoxemia--> explain these puzzle piece factors (6)
(1) Intestinal mucosal barrier function (2) Endotoxins: source and structure (3) Interactions with plasma proteins and cell surface receptors (4) Intracellular signal transduction pathways (5) Synthesis and release of inﬂammatory mediators (6) Effects of inﬂammatory mediators
Explain what is happening in this pic
after 1 hr ischemia, part of GI (like fimbriae) is totally gone. very early on in a toxic insult, there is damage to your tight jxn-- and so as those go, leaves gap between cells, then relatively large endotoxin molecules can seep through and go into abd cavity.
what are the protective mechanisms of the mucosal barrier? What other things back the mucosal barrier up on its functions?
Mucosal epithelial cells, their secretions, tight junctions and resident bacteria--> Restrict endotoxin and bacteria to the intestinal lumen. Backed up by Kupffer-cells, circulating antibodies, and binding proteins
what is this pic showing
as ischemic damage takes place, the tight junctions are disrupted and gaps are formed which the endotoxins can get through
explain the structure of the LPS and what each part does?
(remember this is the G- endotoxin) there is the lipid A region, which is at the bottom of the pic. This area is highly conserved and normally sits within the bacterial cell wall. HIGHLY conserved= same in a variety of G- bacteria. Between 2 white lines is the core region and this is also highly conserved. The part at the top is the O-chain, which is specific to the type of bacteria it came from.
what are we looking at here?
This is the G+ endotoxins-- specifically, lipoteichoic acid the purple oval-shaped structures attached to one another. They are important in a variety of things for the host-- phage binding, host recognition (which is why they lead to endotoxemia and the CSs of endotoxemia). It sits in the G+ cell membrane
explain what is going on in this pic
Top picture (a) has techoic acid, lipotechoic acid, peptidoglycan and all of them are recognized by the receptors that lead to CSs endtoxemia. (so 3 endotoxins recognizable with g+) (b) is the G- bacteria, which has a cytoplasmic membrane and an outer membrane and the LPSs are in the outer membrane.
which endotoxin is most toxic?
lipopolysaccharide. (so although G+ and G- both cause endotoxemia, LPS is the strongest)
what does LPS do once it gets into the bloodstream? (pathophys of it from bloodstream to receptor on cell)
First they aggregate and form what are calle "medusa heads" with lipid A portion to the center (lipid=hydrophobic,so, makes sense). Then, there are lipopolysaccharide binding proteins, which are acute phase proteins (produced by liver, and produced by the liver in higher amounts when there is an inflammation-- Endotoxemia goes hand in hand with inflammation) so as soon as the first endotoxin molecules enter the blood, and the blood goes to the liver--> liver makes more LPS-binding proteins (LBPs). however, LBPs binding to LPSs make them WAY WAY MORE TOXIC (1000x). Why? bc it transports it to the particular endotoxin targets. The LBP is also able to break off LPSs from the medusa head in the blood and then it transports it to the cell surface (of WBCs/endothelial cells) where there is a protein CD14(cluster of differentiation antigen), when LPS binds to CD14 the LBP goes away. Then the CD14 transports LPSs across the cell surface to it's receptor (so also facilitates tox of LPS).
what are the receptors that the LPSs bind to on the cell membrane?
Toll-like receptors (TLRs) first discovered as receptor required for dorso-ventral polarity of the developing Drosophila aka fruit fly embryo. In 1981: it was discovered the TLRs were responsible for Characterisation of inducible antibacterial peptides (so stim of toll receptor results in production of antibacterial peptides. So toll is part of innate immunity of the fruit fly). in mammals there are 10 types of TLRs.
what is the toll-like-receptor that is the endotoxin signalling transducer in the horse?
what is the CD14? what does it do?
Cell surface receptor on mononuclear phagocytes. Lacks transmembrane and intracellular domain (which is why LPS still needs to go to a diff receptor-- ie Requires additional signalling components) what it does is Enhances reactivity to endotoxin: transports endotoxin to its receptor
Explain this mess
so TLR-4 (receptor involved in endotoxin signal transducing) can't signal on its own- it needs to dimerize by binding to another TLR (so needs 2 TLRs that is what dimerizing is). But it ALSO needs MD-2, a co-receptor, which sits inside the TRL receptor. So top picture shows that there are two TLRs bound together, and then they are both bound to a MD-2....and this is when signalling occurs. (without MD2, without dimerization, there is no signal) Why does this matter? This is the basis for species differences-- not only does the dimerization have sp differences, but the MD2 in the dimerized TLR, also accounts for species differences. This is why in certian species there are endotoxins that are recognized and you see an inflammatory response, whereas in other species it is an antagonist of TLR4 and we don't see an inflammatory response. (all TLR receptors need to dimerize! but doesnt always dimerize with the same things)
what does TRL4 recognize?
LPS (lipopolysaccharide, the G- endotoxin)
what should you know about the action of the signal from a TLR receptor?
receptor interacted with--> INTRACELLULAR PROCESSES---> then signal (there is downstream signalling in the cell)
why do we care that there is downstream signalling after a TLR is activated?
this is where we can try to start treating the endotoxemia problems
which TLRs recognize G+ endotoxins?
TLR2 in a heterodimer with TLR1 or TLR6 (1+2 or 2+6)
flagellin comes from both G+ and G-...which TLR recognizes this?
which TLRs recognize G- endotoxins?
7 of the 10 most important TLRs are where on the cell?
outside of the cell
which TLRs are in the cell?
9, 7, 8, 3
(slide about toll receptors) so what are they? what happens when a ligand (like LPS) binds to them? what things play a central role in this process?
TLRs are Transmembrane receptors with IL-1 like cytoplasmic domain. Ligand binding induces dimerisation, which causes Activation of signal transduction cascade. NF-𝜅B and MAP kinases play a central role in this signalling (ultimately results in pro-inflammatory cytokine release)
why would TLR9, 7, 8, and 3 be on the INSIDE of the cell?
recognize portions of bact and viruses that are able to enter the cell (like DNA and RNA from viruses and bacteria)