Sepsis

Question 1

Define bacteremia, endotoxemia and sepsis

Answer 1

• Bacteremia is defined as live micro-organism in circulation
• Endotoxemia is the presence of LPS in the circulation - may or may not be associated with live organisms
• Sepsis represents the clinical signs associated with a systemic inflammatory response to an infectious agent

Note that bacteremia is not always associated with a systemic inflammatory response

Question 2

What is the initial triggering event in sepsis?

Answer 2

• Sepsis is triggered when circulating bacteria or endotoxin are recognised by the immune system is sufficient numbers
• Pathogen associated molecular patterns (PAMPs) are recognized by and interact with the innate immune system via TLR’s
• TLRs activation initiates a complex local inflammatory response to upregulate systemic host defences
• Sepsis results when these inflammatory systems are excessive or become dysregulated

Question 3

List the typical initial clinical signs of sepsis?

Answer 3

• Fever
• Tachycardia
• Tachypnoea
• Neutropenia/neutrophilia

This combination of signs are referred to as SIRS - systemic inflammatory response syndrome or sepsis when triggered by an infectious agent

Question 4

List the potential triggers of systemic inflammatory response syndrome, both endogenous and pathogen mediated

Answer 4

Danger associated molecular pathogens (DAMPs) - from damaged cells

• Pancreatitis
• Trauma
• Hypoxia

Pathogen associated molecular pathogens (PAMPs)

• Endotoxin (lipid A)
• Flagellin
• Bacterial or viral DNA
• Gram positive bacterial products
• Fungal products

Question 5

What processes most commonly lead to sepsis in dogs?

What common infections / conditions have most been associated with the development of sepsis

Answer 5

• Sepsis in dogs is most often seen following disruption to the normal mucosal layer (often in the GIT) and movement of bacterial into the circulation or tissues
• Peritonitis and pneumonia are the most common causes of sepsis in dogs
• Numerous other risk factors for endotroxemia and sepsis have been reported
  
  • Canine parvoviral enteritis
  • GDV
  • Pyometra
  • Mastitis
  • Gram negative infection
  • Heat stroke
  • Advanced liver disease / hepatic insufficiency
  • Obstructive biliary disease (bile salts bind endotoxin)
  • Chemotherapy

Question 6

Briefly describe the usual mechanisms that prevent endotoxin release into the systemic circulation

Answer 6

• Endotoxin is released primarily from Enterobacteriacae within the gut lumen when they lyse. This occurs with rapid replication or after administration of antibacterial agents
• Most endotoxin is bound by bile salts within the gut lumen
• The mucosal surface provides a barrier to endotoxin entry
• Small amounts absorbed into the portal system is cleared by the hepatic macrophages
• Endotoxin that enters the GIT lymphatic system is cleared within the local lymph nodes

Question 7

Describe the molecular pathway that leads to increased cytokine expression after exposure to lipopolysaccharide

Answer 7

• LPS binds to LPS binding protein, an acute phase protein
• The LPS-LBP complex binds with CD14 on the macrophage
  
  • This LPS-LBD-CD14 complex can be shed from the macrophage and is soluble in the blood
• This complex then interacts with TLR-4 on the monocytes / macrophages (predominantly)
• Binding to TLR-4 activates the transcription factor NF-kb
• Upregulation of NF-kb leads to increased expression of many pro-inflammatory cytokines
  
  • Note that the NF-kb pathway can also be activated by many other products via other TLRs
• The major cytokines released include IL-1b, TNF-a,
  
  • TNF-a further stimulates release of IL-1 and IL-6

Question 8

Describe the initial macrophage response following binding of endotoxin (+LBP and CD14) to TLR-4 and activation of NF-kb

Answer 8

• The initial response involves relase of multiple pro-inflammatory cytokines
  
  • TNF-a, IL-1b are the most relevant
• TNF-a levles are increased by 15 minutes following exposure, peak at 2 hours and then return to baseline by 4 hours ⇒ before any overt clinical signs
• TNF-a stimulates release of IL-6 and both macrophage colony stimulating factor and GM-CSF

Question 9

Describe the major mechanisms of action of IL-1 during the initial stages of sepsis

Answer 9

• IL-1 is a major co-stimulator of TH-2 cells
• Stimulates the acute phase response
• Interacts with the hypothalamus and thermoregulatory centre to stimulate a fever
• Stimulate hyperalgesia
• Increased expression of adhesion factors on the endothelial surface to enable diapedesis
• IL-1 also causes vasodilation and hypotension

Question 10

The cytokine casecade during early sepsis stimulates the release of many additional substances.

Note the important substances released via the cytokines released during sepsis

Answer 10

1. Inflammatory phospholipid derived mediators
   
   • prostacyclin, PAF, thromboxane
2. Coagulation factors
3. Complement
4. Reactive oxygen species
5. Nitric oxide
6. Endothelin-1
7. Beta-endorphin
8. Histamine
9. Serotonin
10. Vasopresin
11. Angiotensin II
12. Catecholamines

Question 11

Numerous mediators are released during early sepsis.

Note the biological effect during early sepsis (before decompensation and shock)

Answer 11

• Increased neutrophil chemotaxis, activation and aggregation
• Platelet activation and aggregation
• Activation of coagulation proteins incl. increased TF
  
  • Hypercoagulability
• Increased plasma protease production
• Vasodilation ⇒ hypotension
• Generalised endothelial inflammation and increased vascular permeability
• GI mucosal sloughing / ulceration
• Cellular lysosymes deplete fibronectin and activate coagulation - can lead to DIC

Question 12

Describe the effects of early sepsis on the coagulation proteins and cascade

Answer 12

Sepsis promotes coagulation, inhibits anticoagulation and impairs fibrinolysis

• Widespread endothelial inflammation exposes tissue factor
  
  • TF binds with F VII to form extrinsic tenase activates factor X which eventually lead to thrombin formation
• Platelets are activation by endothelial inflammation and exposure of vWF
  
  • Activated platelets release thromboxane, serotonin, vWF which further promote coagulation
  • Platelets become consumed
• The three anti-coagulation protein systems are derranged during sepsis
  
  • ATIII
  • Tissue factor pathway inhibitor
  • Protein C
    
    • TFPI and PC are produced by endothelial cells which are damaged during sepsis
    • TFPI has impaired binding to endothelial cells and is less effective at inhibiting extrinsic tenase

Question 13

Describe the mechanisms of action of Protein C

Answer 13

• Cleavage and inactivation of F Va and F VIIIa
  
  • Reduction in intrinsic tenase and therefore thrombin generation
• Inhibition of neutrophil - endothelial interactions
  
  • Reduced neutrophil chemotaxis
  • Down regulation of vascular adhesion molecules
• Reduced pro-inflammatory cytokine release
• Protects endothelial barrier function

Question 14

Describe the activation of protein C and how this is altered during sepsis

Answer 14

• Proetin C is an inactive zymogen that is present within the normal ciculating plasma
• Protein C is activated by interaction with the thrombin / thrombomodulin complex present on endothelial cells
• In sepsis, thrombomodulin production is down regulated by inflammatory cytokines
  
  • Especially IL-1 and TNF-a
• Reduced thrombomodulin leads to reduced activation of Protein C
• During sepsis, there is also up-regulation of progoagulant factors and increased fibrin generation.
  
  • Activated Protein C is consumed more rapidly during the inactivation of FVa and FVIIIa

Question 15

Describe the mechanism of release and action of plasminogen and plasmin.

How is plasminogen activity altered during sepsis

Answer 15

• Plasminogen is an inactive zymogen released into the ciurculation by the liver
• Plasminogen activators are primarily release by endothelial cells during cellular injury as seen in sepsis
• In early sepsis or with mild endothelial damage, tissue PA is released cleaving plasminogen to plasmin
• Plasmin causes fibrinolysis together with other actions:
  
  • vWF cleavage
  • Activation of collagenases
  • Cleavage of thrombospondin
  • Activates certain components of complement
• Cytokines during sepsis stimulate production of tissue PA inhibitors - this reduces the production of the active enzyme
• Plasmin is consumed leading to a reduced concentration

Question 16

Describe the three pathways that can lead to activation of the complement cascade

Answer 16

1. Classic Pathway
   
   • Antibody/antigen complexes on cells, apoptotic cells, some bacteria and viruses
2. Mannose binding lectin pathway
   
   • Antibody independent - binds bacterial mannose residues
3. Alternate pathway
   
   • Endotoxin and other bacterial surface molecules

All three pathways lead to the eventual cleavage of C3 to C3a (anaphylatoxin) and cleavage of C5 to C5a and C5b (chemotactic protein and anaphylatoxin)

Question 17

Briefly describe the major effects of the complement factor C5a

Answer 17

• Chemotactic protein
• Activates endothelial cells
• Increases vascular permeability
• Pro-inflammatory - generally especially in sepsis
  
  • My have variable effects on inflammation depending on the concentration, the type of receptor it binds and the presence of pro- or anti-inflammatory cytokines

Question 18

Describe the potential structural and metabolic changes that can develop in the vascular endothelial cells during sepsis

Answer 18

• Adhesion molecules are upregulated
  
  • Leads to increased neutrophil chemotaxis and can lead to phagocytic cell accumulation in tissues
  • Increased platelet adhesion can lead to microclot formation
• Increased permeability
  
  • Can lead to increases protein transfer into the interstitial space
  • Increased interstitial oncotic pressure together with reduced oncotic pressure and increased capillary permeability leads to development of oedema
• With increased severity or persistance fo the vasodilation, the vascular endothelium may become unresponsive to pressor therapy
  
  • This condition is reffered to as catecholamine-refractory vasodilatory shock

Question 19

Describe why sepsis can lead to multiple organ dysfunction despite normalisation of the macrocirculation including systemic blood pressure

Comment of the changes seen in the microcirculation

Answer 19

• Decreased perfusion within the microcirculation and local ischaemia are major drivers of multiple organ failure.
• Normalisation of the macrocirculation and maintenance of normal blood pressure is often done at the expense of the microvascular bed
• Microvascular dysfunction can include
  
  • Constriction with reduced blood flow
    
    • Reduced oxygen delivery, reduced waste removal
    • Localised H⁺ buildup and lactic acidosis
    • Localised necrosis as a result of above
  • Micro-clots
    
    • Widespread coagulation and reduced anti-coagulation and fibrinolysis can lead to occlusion of tiny capillaries
    • Large numbers of tiny thrombi can lead to reduced blood flow and local infarction
  • Microvascular damage of the GIT leads to mucosal necrosis and can allow further translocation of endotoxin and bacteria

Question 20

Describe the changes in routine diagnostic laboratory tests that can help confirm a clinical suspicion of sepsis

Answer 20

• Haematological findings
  
  • Leukocytosis generally with bands +/- toxic change
  • Mild anaemia
    
    • Blood loss or haemolysis - immune mediated or due to oxidative damage
  • Thrombocytopenia
    
    • Vasculitis, consumption and sequestration
• Coagulation abnormalities
  
  • Initial hypercoagulability is difficult to detect in the clinical setting - TEG, sequential platelet and fibrin changes, clinical evidence of thrombosis
  • Hypocoagulability - increased PT and APTT together with thrombocytopenia
  • Increased D-dimer and FDPs
• Routine biochemistry tends to reflect either the inciting cause or the secondary results of sepsis, rather than indicating the clinical entity primarily
  
  • Hypo- or hyper-glycaemia
  • Hypoalbuminemia
  • Hyperbilirubinemia
• Positive cytology / culture of a suspected infected area

Question 21

List and briefly comment on the potential end-organ complications seen following sepsis

Answer 21

• The complications and pathological findings that arise due to sepsis are dependent on severity and the underlying trigger together with management
  
  • The primary pathological process is widespread vasculitis and endothelial injury together with thrombosis, neutrophil margination and oedema
• Vasculitis and coagulation abnormalities with thrombosis with impairment of fibrinolysis and anti-coagulant processes
  
  • This is clinically recognised as DIC
• Cardiovascular
  
  • Primarily due to inflammatory cytokine mediated mechanisms
    
    • Increased vascular permeability and vasodilation lead to hypovolemia
    • Impaired contractility
    • Loss of vasomotor tone and vasodilation
    • Altered intracellular calcium trafficking
• Respiratory System
  
  • ARDS and pulmonary thromboembolism
  • Alveolar endothelial damage leads to extravasation of protein rich fluid into the alveoli and reduced oxygen transport
• Renal System
  
  • Ischaemia and re-perfusion injury and free-radical poroduction
• Neurological
  
  • direct toxic effects / alterations in cerebral blood flow
  • Specific pathogenesis not well described in dogs/cats
• GIT / Hepatic
  
  • GIT mucosal endothelial injury, ischaemia and increased permeability
    
    • Leads to both fluid losses and increased bacterial translocation
  • Hepatic dysfunction due to increased exposure to endotoxin or altered blood flow
    
    • Particularly susceptible to reduced oxygen delivery given high metabolic rate