1. Immunopathology of Sepsis
Gideon Daniel, DVM
Internal medicine resident
8/13/2013

2. Overview Immunology review Introduction to Sepsis/SIRS
Immunopathophysiology in Sepsis/SIRS
Summary

3. Quick immunology review
Two major defense systems
Innate (non-specific)
Adaptive (specific)
Antigens: substances that can initiate an immune response
Effective Ag’s are foreign, large, chemically complex, degradable
A single Ag may nave numerous epitopes
Haptens: small chemical groups that are unable to induce an immune response by themselves
chemically simple and small
Usually have to be conjugated to a complex carrier
Innate refers to all those elements w/ which an indiviudal is born and which are always prsent and avaliable at very short notice to protect the body from challenges. Relies ion immediate barriers and pattern recognition receptors--- main focus w/ sepsis
Adaptive or acquired- more specialized. Initial contact w/ foreign agent triggers a chain of events that leads to activation of certain cells and the syn of proteins which exhibit Sp reactivity against a foreign agent.
Requirements for a good Ag- foreign, high MW (> 6000 daltons), chemically cmoplex, degradible (i.e able to undergo antigen processing)
Haptens: fail to induce immune response in their native form d/t LM wt and chemical simplicity. Not immunogenic by themselves unless conjugated to higher MW wt complexes. Ex: vaccine rxns.

4. Surface barrier mechanisms and AMPs
Keratinized epithelium of dermis and mucosal lining of body cavities
Chemical compounds
Lysozymes (GIT)
Basic polypeptides (calprotectin, lactoferrin)
Complement
AMPs (antimicrobial peptides) or HDP (host-defense peptides)
Found in epithelial cells, inflammatory cells and others
Ex: Defensins and cathelicidins
Other mechanical actions
Innate immunity and host defense peptides in veterinary medicine. Linde, et al JVIM 2008.
- Pathogens are expelled from the lungs by cilary action, coughing/sneezing; flushing action of tears/saliva, urine also forces pathogens out as does sloughing off skin
- Cf to eukaryotic cells, bacti cell walls lack cholestrole and negatively charged phsopholipids (instead have anions-basic aa)- so attract cationic defensins, which carpet the microbialmembrane and institute channel formation. Large # of AMPs form part of the non-oxidative killing mch w/in phagolysosomes in cells (nO or macrophages), a growing # are thought to be actively secreted onto epi surfacse of the GIT, RT and UT. Also thought to be secreted by atypical cells like type II pneuoocytes, endothelial cells, and monocytes.

5. Complement Series of ~30 proteins Major functions
Lysis of target cells via formation of MAC
Opsonization of microbes and immune complexes
Inflammation and activation of WBCs

6. Complement
Classical pathway- initiation occrs when C1 binds to an activating substacne such as an Ag-Ab complex- requires one IgM bound to Ag or two IgG.  C3 convertase  C5 convertase
C1 activation is very tightly controlled by molecules like C1-esterase inhibitor and quickly cleared by the RES
Alternative pathway- initation requres the presence of preformed C3b. C3 is continually ticking over at a low level- which is hydrolyzed by water to for C3i –C3 convertrase and C5 convertase.
Terminal pathway- cleavage of C5 into C5a and C5b. C5a
C5 convertase splits C5 into C5a and b. C5b binds C6 & 7 to form C5b67 which attaches to the cell membrane, C8 binds to this complex and penetrates the membrane, where it polymerizes a # of C9 to form the MAC.
Clinical immunology of the dog and cat, 2nd edition. Day. Pages

7. Cells of the immune system
Effector cells of the innate system
Macrophages
Neutrophils
NK cells (large granular)
Antigen presenting cells
Histiocytes
B cells
Non-professional APCs
T cells
CD4 T helper cells (Th0, Th1, Th2, Th 17)
CD8 Cytotoxic T cells
ϒδT cells
Major role in acquired immunity
Neutrophils circulate about 10hrs before entering tissue (survive only for a few days). Phagocytosis may be enhanced if opsonized (Ig or compleement).
Phagocytozed particles are contained w/in phagosomes phagolysosome which are exposed to enzymes (lysozyme, acid hydrolyssis, neutral proteasis and cationic proteins). May also kill by respiratory burst.
During maturation, most T cells express receptors for Ag- which are mostly alpha/beta. However these less common forms are thought to be evaluatoinaly older form of TCRs. Generally do not express CD4 or CD8 and therefore not MHC restricted, but they may produce either TH1 or TH2 cytokines. Reported to comprise up to 1/3 of T cells w/in the spleen.

8. MHC
Plays a crucial role in the response of T cells to Ag. MHC molecules bind peptides dervied from protein Ag and present them to the appropriate T cell receptors.
Class I- present peptides dervied from protein Ag to CD8
Class II- CD4 T cells.
Are cell-surface molecules that are responsible for the phenomenomen of graft-rejection. Class I- found on all nucleated cells, Class II mostly immune cells.
W/in HC molecules, there are areas of variable and constant aa sequences and as the constant domains have homology w/ the Ig Fc region- they are considered part of the Ig superfam that include Cd4, Cd8 and TCR.
Class I consists of a transmembrane alpha chain w/ 3 external domains and an associated B2 microglobulin. There is conserved sequence in the alpha 3 domain (that has homology w/ constant region of Ig molecule). Alpha 1 and 2 have variable sequesnces.
Class II- two transmembrane chains alpha and beta- each w/ two external domains (alpha and beta ), each w/ two conservative sequencs (alpha2 and beta2) and variable (alpha1 and beta 1).
Endogenous peptides fragments associated w/ class 1 w/in the Erbut are prevented form associated w/ class II as the peptide binding site is occupied by the invariant chain which allows it to leave the ER. After passage thru the Golgi, class I loaded w/ the Ag passes directly to the cell surface. Class II are transferrd to the endosome compartments where the acidic and enzyme-rich enviroment results in displacement of the invariant chain and association w/ peptides derived from exogenous Ag’s.
Exogenous like bacteria. Endogenous like viral particles.
Clinical immunology of the dog and cat, 2nd edition. Day. Pages

9. Cytokines and chemokines
Are proteins, peptides and glycoproteins;
Transmembrane- EC binds cytokine and cytoplasic initiate IC signlaming
Are a grp of soluble mediators of the immuen and inflammatory respones. Released by on cells and generally have an effect on either the same cells (autocrine), paracrine or endocrine. Can be syn by and act on many diff cels and one cell can produce many different cytoines.
Are low MW proteins, producedy locally and transiently. Are potent at picomolar conc. Binds to Sp receptors which initates IC signaling pathways.
Roles: hemotopoeisis, development, actiavtion of T/B?NK and ancillary wbcs, leukocyte chemotaxis, cloncal expasion, T/B regulation, cytotoxicy and inflammation.

10. Summary of cytokines Interleukin Cell origin Action Misc
IL1
Macrophages
Activates and proliferation of lymphocytes
Pyrogen
Induces APP
cachexia
IL2
Th1
Clonal expansion
Augmented action of NK cells & cytotoxic lymphocytes
VIP pathogenesis of lesigmanian and demodex
IL3
From stimulated bone marrow
Promotes mast cell growth
Enhances phagocytosis
IL4
TH2
- activates B cells to produce IgE
- Promotes B cell differentiation (IgG, IgM, IgE, IgA)
Inhibitor TH1 response
VIP Type I HS rxns
IL5
Promotes eosinophil production
Activates B cells to produced IgA
VIP feline asthma
IL6
Macrophages & TH2
Activates B cells to plasma cells
Activates T cells and hematopoietic precursors
Elevations in FIP and Shar Pei fever
Pyrogens: IL1, IL6, IL8, TNFa, IFNy
APP- IL 1, 6, TNFa
Causes cachexia: TNFa and IL1

11. Summary of cytokines Interleukin Cell origin Action Misc IL7
Bone marrow and thymus
IL 8 (now known as CXCL-8)
Macrophages
Neutrophilc chemotexis
VIP inflammatory lung dz
pyrogen
IL9
TH2
Activates mast cells
Ig production
IL10
Th2
anti-inflammatory in combo w/ IL4 and TGF-beta
Inhibits release of IL12
Inhibits Th1 response
IL12
Macrophages and monocytes
Most potent activator/inducer of tumor destruction by NK cells & cytotoxic lymphocytes
VIP in induction of Th1 immunity
Increases IFNy
IL13
- Stimulates goblet cell mucus
IL15
TH1
Same as IL2
IL17
Th17
- Plays a role in chronic inflammatory dz

12. Cytotoxic T cells
Killing involving a target by a cytotoxic lymphocytes invovles different mech-
Degranulates and relases pore forming molecules (perforin) and enzymes into the area adjacent to the target cell membrane. Death by osmotic imblances, entrance by other toxic substances and mediated apoptosis.
Cytokines releaed from the cytotoxic cell may bind to receptors on the target cell membrane and trigger death
Target cell apoptosis may occur ind of perforin and be mediated by direct interaction b/w Fas ligated and Fas molecule on target cell.
NK cells recognizes surface cabs via NK receptors. NK calls can be inhibited by the interaction of class I w/ the Killer cell inhibtor receptor (KIR)
Clinical immunology of the dog and cat, 2nd edition. Day. Pages

13. CD4 T helper cells
There are two major subsets of CD4 T cells that are char by a distinct pattern of cytokien production. Activation of TH1 cells resuls in CMI effects such as macropahge activation by INFy. The Th2 cells mediated the humoral effects of Ab syntehsis and regulation of mast cells and eosinophil differentation and mobility. Th2 cells produce Il10 which can render Th1 cells unable to respond to Ag (anergic). The Th2 cytokines IL4, IL13 are also negatively influence Th1 cells. TH1 cells directly inhibit the fn of Th2 via IFNy.
CD4 Th cells may be divided into different subsets by the profile of cytokines produced. The major effector populations are the Th1 and Th2 (which arise form Th0).
Th1 cells produce predom IL2, Ifny- responbisble for CMI and cytotoxicity, limited effect on Ab prodcution (but enhances production of IgG). Antagnosizes TH2 via IFNy.
TH2 mainly for humoral, particular IgE and IgG. Antagnoize Th1 via IL4, 10 and 13.
New subset- Th17 can produced IL17.
Clinical immunology of the dog and cat, 2nd edition. Day. Pages

14. Macrophages

15. Definitions
Bacteremia: presence of live bacterial organisms in the bloodstream
Sepsis: the clinical syndrome caused by infection and host’s systemic inflammatory response to it
Bacteria (g+ or g-), viral, protozoal or fungal
Severe sepsis: sepsis complicated by dysfunction of one or more organs
Septic shock: acute circulatory failure and persistent arterial hypotension (despite volume resuscitation) associated with sepsis
SIRS: the clinical signs of systemic inflammation in response to infectious or noninfectious (trauma, pancreatitis, burn, snake bites, neoplasia or heat stroke)
MODS: physiologic derangement of the endothelial, cardiopulmonary, renal, nervous, endocrine, GI systems with the progression of uncontrolled systemic inflammation and DIC

16. Incidence In humans 240-300 cases/100,000
10th most common cause of death in the US
Unknown in veterinary population
1-5% of feline cases
6-10% of canine cases
Sepsis in veterinary patients: what do we know and where do we go? – Otto JVECCS 2007.
Survey from 2002
Survey sent out ACVECC, VECCS, ACVIM, SVECCS
Mortality in cats 29-79% , 21-68% in dogs

17. Staging sepsis PIRO (2001 International Sepsis Definitions Conference)
Predisposition
Insult or infection
Response
Organ dysfunction
Breed specific pro-inflammatory cytokine production as a predisposing factor for susceptibility to sepsis in the dog – Nemzek, et al, JVECCS 2007
Not validated in veterinary spp yet.
P: recongized that certain individuals may experience a more severe response or worse outcome than the avergage invidual with a similar insult. I.e Nemzek JVECC- mean TNFa production were collected from healhty dobie’s, rotties and age-matched purebreeds. Blood was incubated w/ LPS and bioassays for TNA and IL6 were collected. Highest levels in Dobies and Rotties.
I: human sepsis that g+ outnumber g- in humans. CS of sepsis are highly influenced by the type of inf, location and extent of infection
R: attempt to char this respose lead to SIRS criteria. Scoring systems however are not able to predict outcome. Search for biomarkers are more to provide targets or guides for thereapeutic intervention.
O: i.e ARDS and need for mech ventilation.

18. Diagnostics Criteria for Sepsis
Dogs1
Cats2
Criteria
Infx + 2 or more listed below
Infx + 3 or more listed below
Temp
< 100.4, > 102.2F
> 103.5, < 100 HR
> 120
> 225, < 140 RR
> 20
> 40
WBC
< 6K/uL, > 16K//uL or > 3% bands
> 19.5K/uL, < 5K/uL, > 5% bands
Adapted from Hautman Vet Surg 1997 paper
Adapted from severe sepsis Brady JAVMA 2000.
1Evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs. Hautman Vet Surg 1997.
2Severe sepsis in cats: 29 cases ( ). Brady JAVMA 2000.

19. Septic Foci Dogs: GI tract most common Cats: pyothorax, GI tract*
Other- salmonella, pneumonia, endocarditis, pyelo, pyo, bite wounds, non-bacterial (toxo), abscess (pancreatic, hepatic, etc)
36-71%
GI neoplasia, FB, dehiscence, NSAID ulcers, perforation of megacolon ors severe colitis
GIT- 52% of cases in dogs, 17-47% in cats
Pleural space dz in cats 24%
Common isolates being Ecoli, enterococcurs, strep, pseudomonas and clostridium
*Small animal critical care medicine. Silverstein, Hopper, 1st edition. Pages 46-49,

20. Sepsis vs SIRS Bone et al. Chest 1992
When talking about pathophysio of SIRS and Sepsis- the only difference is the inciting cause.
Sepsis- there is an infetious etiology vs in non-infectious SIRS there is damage to the cells like w/ AP, trauma, burns. The body cannot distingusig an infectious from a noninfectious and can ultimately respond the same way.
Bone et al. Chest 1992

21. Recognition of pathogens
PAMPs: pathogen-associated molecular patterns
MAMPs: microbial associated molecular patterns
DAMPs: danger-associated molecular patterns
Ifnx not only causes PAMPs but also causes tissue and cell damage w/ subsequent DAMPS releast. Similary, injry caused by truama not only leads to DAMP relase but also renders the patient more sus to infection and therefore PAMP release.

22. Examples of PAMPs/MAMPs/DAMPs
LPS (gram – bacteria)
Lipoteichoic acids (gram +)
Peptidoglycan
Flagellin
DNA or RNA
LPS consists of three parts: lipid A (inner most, toxic portion), core oligosacc, and O side chain. The lpid A serves to anchor LPS in the bacti cell wall and is the fragement that trigger the cellular response.

23. Toll like receptors
TLR are named b/c of their homology w/ the roll recepotr of Drosophila (where it was 1st found).
All have the same basic structure
Possess extracellular leuceine rich repeats, a singel transmembrane region and IC Toll/IL-1 receptor domain (TIR)
Are membranes of the superfam of IL-1 receptors
They differ in extra and intracelluar regions, recogonziign different ligands and giving rise to distinct resposnes.
Cytoplasmic portion described as TIR- interacts w/ a varitey of TIR domaining containing adapters- activates a chain of kianses in the IL-1R associated kinase (IRAK) fam, ultimately resutling in the activation of the inhibitor of K B kinase (IKK) enzyme complex and the mitogen activated protein kinase (MAPK) pathway. Wherase the connectino of TLR activaiton to the NFKB and MAPK is well recognized, a # of other pathways are also triggered by TLR stim.
EX: TLR- LTA, peptidoglycan, heamgglutin, polysacc, lipoprotein. TLR- LPS, viral protein enveloples

24. Example of PARR in action
LPS sensed via LBP-LPS complex
Then signaling through TLR-MD2 complex
However, other cell surface sense LPS- MSR-macrophage scavnger receptor
CD11b/CD18 and ion channels. IC signaling depends on bindin of the IC TLR domaining, TIR- a process that is facilitated by two adapter proteinds MDY88 and TIRAP; inhibited by Tollip.
There is a also an indepndent pathway by which TIRAP/MAI signals through an RNA dep kinase- PKR and IRF-3.
NOD: Common to all are leucine rich repat domain, a central NOD domain and an N terminal effector domain. NOD1&2 are known to recognize muropeptides dervied form peptidoglycan, a major structural componenet of both g+ and – bacterial cell walls.
MD2 is a secreted glycoptoein that is part of the cell mbmrane. It occurs in a soluble form or ain a complex w/ TLR4. TLR4 itself cannot recognize the LPS- MD2 can rec LPS alone but the TLR/MD2 complex has a 10fold higher affinity for LPS than w/ MD2 alone. Signals from CD14/TLR compl activate NFKb thur a series of phosphyralzation cascasdes triggered by MAPK.
Under norm situations, NFKB is confined to the cytoplasma in an inactive form, bound to inhibtory K B (IKB). The phos of IKB releases NFKB.
The immunopathogenesis of sepsis. Cohen. Nature 2002.

25. Inflammasomes
Just as another consequence of NLR ligation is acvtiation of the inflammasome, a macromolecular complex comprising pro-capsase-1 togther w/ various adapater proteins.
Caspase-1 is imp in the syn of active IL1-b and IL17 and induces a type of cell death- pyroptosis.
The Immunopathology of Sepsis: pathogen, recognition, systemic inflammation, the compensatory anti-inflammatory response and regulatory T cells.
DH Lewis, et al. JVIM 2012

26. Early pro-inflammatory cytokines
TNF alpha
Interleukin-1-beta( IL-1β)
Primncipal fnc is to recruit leukocytes to site of infection and activate them to eradicate the pathogen, signal amplification
Activated macrophage secrete large amts of TNF and the severity of shock correlatees w/ plasma TNF levels. Used to be called cachetin. Indues a transmembrane signaling via TNF receptors – TNFR1 and R2 (R1 in most cells, R2 immune cells). Causes apotosis via Fas receptor (via caspase 3)
IL1b increases the prodcution of other proinflammatory mediators, expression of adhesion molecules and plays a role in a the dev of organ dysfnc.

27. Late pro-inflammatory cytokines
IL6*
CXCL 8 (IL8)
High mobility group box-1 (HMGB-1)
Macrophage migratory inhibitor factors (MIF)
HMGB1- product of macrophages tha tappears much later after LPS stim – non-histone chromosomal protein that is abundantly distributed and exits in nuclear, cytoplasmic and membrnaebound forms. Participates in stablizing nucleosomes, facilitates gene transictipon and modulates the activity of steroid hormone receptors
Inc 12-18hrs after TNF peak, passive relase from necrotic cells. Active secretion upon TNF activation. Signals via RAGE, TLR2 and TLR4 and amplifies inflammation.
MIF- mediate shock cz by g+ bacti like toxic shoc syndrome. Promotes expression of TNFa in response to infection, enhances expression of TLR4, plasma levels correlates w/ the severity of infection. Occurs immediately following the activation of macrophages and monocytes. Large amts stored in cytoplasmic vacules.
IL 6: produced by T, B cells, endothelium. Production of APP, several studies in humans and dogs correlate plasma conc w/ morbidity and mortality. One human study predicted outcome w/ > 80% accurary w/ IL6. Persistnt production seems to be more imp than initial or peak concentrations. Two vet studies used different assay- one used bioassy and another used ELISA.
IL8- produces IFN an imp antiviral cytokine and acts as a chemotatic agent for No and T cells.

28. Leukocyte recruitment and activation
Endothelium express a rnage of site-specific adhesion molecules (addressin family) which interact w/ specific ligands on the surface of the particular leukocyte (homing receptors).
During the tethering the leukocyte is slowed down by molecular interactions (i.e selectin and intergrin)
Signaling events mediated by surface molecules or chemokines that upregulate and trigger conformation chagne in leukocyte intergrin that binds to endothelial ICAM
EX: E selecting binding to CD15)  Intergrins/ICAM  PECAM assits w/ diapedsis

29. Activation of AA cascade
Leukotrienes- increase vascular permeability and contract airway smooth muscle. Leuktriene B4 causes adherence of neutrophils to EC and is a potent chemotactic agent. It also stim release of lysosomal enzymes and generation of superoide anion in neutrohpils.
Thromboxane- plt aggregating agent, VC
Prostocyclines promotes down regulation of TNF alpha production but also have widespread vasodilatory affects
PG- VD themselves
Anti-inflamm benefits of ibuprofen was studied in a prosepctive, randomized, double blinded placebo controlled trail. Survival benefit was not shown in humans w/ sepsis.
High dose steroids were commonly used before the 1990s- was d/c t/t failure to increase survival and in some studies, increased mortality.

30. Nitric oxide induced vasodilation
iL1, IL6, LPS, IFNg and TNFa upregulated iNOS production;
NO is ubiqutious biologic mediator thought to play an imp role in pathophysio in sepsis.
LPS and other PAMPS stim exuberant production of NO via upregulation of iNOS in many cells including Macrophages, hepatocytes and Ecs.
Pro-inflamm cytokines (TNF, IL1b and IFNy) have also been shown to induce diNOS in the endo, vascualr smooth muscles and macrophages of various parencyhmas cells.
NO also has profound immunomod fnc in the face of sys inf, thought its effect on leukocyte adhesion and activation, preventing normal leukocytoe-endo inteactions and No oxidative burst via direct effects on NADPH oxidase. NO also inhibits plt aggregation and formation of plt plugs.
Free radical w/ cytotoxic properties. 3 types: endo, brain and macrophage. Macroph form triggerd by endotoxin, IL1, TNFa or PAF. Key mediator of mycardia dyfnc, hypotension and vascular hyporeactivity. Dogs w/ sepsis or SIRS have higher NO concentrations.
Xcess iNOS production (precursors to NO relase) and poss def of vasopressin adds to loss of vascular tone
Plasma nitrate/nitrite concentrations were gerater in the sepsis grp than SIRS and control grp. No difference b/w SIRS and control. 66% Se and 75% Sp for differe sepsis vs SIRS.
Endotoxin
IL1
TNFa
PAF
Plasma nitrate/nitrite concentrations in dogs with naturally developing sepsis and non infectious forms of SIRS Osterbur K et al. Veterinary Record 2011

31. Neutrophil function in septic dogs - Webb, et al JVIM 2007
Prospective study- 13 dogs with sepsis and 12 control dogs
Flow cytometry (+ fluorescent markers) to assess
Phagolysosomal oxidative burst
Was lower in septic dogs
Phagocytosis
Increased in septic dogs
Intracellular concentration of glutathione
No difference
Immunoparalysis – friend or foe?
Leukocytes appear to enter a hypo-inflammatory stat in huns- may be a major cause of morbidity & moratlity. Or can be a proective role- prevent furhter inflammation.
Changes in neutrophil size, granularity and surface markers expression demo in dogs w/ sepsis, MODS.
Hypo-inflamm re’s part of the innate immuen reponse that may be amenable to pharm manipulation- lysophophatidylcholine and IFNg have been used to enhance neutrohpil and monocyte fnc in human patients w/ sepsis.
A reduced oxidative burst ind of phagocytosis is seen in No from septic foals and is improved after plasma.

32. Mortality was greater in sepsis vs SIRS*
Clinical and immunologic assessment of sepsis and the systemic inflammatory response syndrome in cats – DeClue JAVMA 2011
% bands and albumin were significantly different in sepsis vs SIRS groups
Plasma TNF activity was significantly higher in sepsis group than control group
Mortality was greater in sepsis vs SIRS*
Moderate + correlations b/w non-survival and IL1b and IL6
Moderate – correlation b/w chloride and non-survival
Prospective case control study
Enrolled 19 enrolled in SIRS group
16 Sepsis group
8 control group
Plasma IL1b greater in sepsis than in SIRS or control (not SS)
IL6 not detectable in any of the control, detectable in 4/19 SIRS and 10/16 sepsis cats
CXCL8 < lower limit of detection in all control, 14/19 SIRS and 10/16 sepsis cats
Looking at both groups together- IL1b, total lymphocyte, Cl, TP correlations w/ non-survival
TNF, IL1b, IL6 and CXCL8
IL 6 and CXCL8 are invovled in maintenance of infalmmatino where IL1b and TNFa are imp in early proinflammatory phoase
TNF, IL6 and CXCL8 can be exp produced by admin of endotoxin in cats
2/4 SIRS criteria assiged to sepsis or SIRS group
TNF activity evaluated by cell kill bioasssay, IL1b activity measured by use of modified cytotoxicty biosassy, feline specific IL6 and fel specific CXCL8
Sepsis group had a significantly higher band cell % and lower eosinophil count cf SIRS or control
Plasma Na & Cl were signficnatly lower in the sepsis & SIRS group cf control
Sepsis group had lower albumin cf SIRS and control
Tca, ALT were lower and Tbili was higher in the Sepsis vs control
No difference in monocyte, hct, BG< BUN, Cr, K, TCO2, TP, Glob and Phos
Assoicated w/ sepsis- high bands, eosinpenia, hypoNa/Cl, hypoalb, hypocalcemia, hyperbiili. HypoNa/Cl in SIRS group too.
Sepsis had a higher band and lower alb cf SIRS (SIRS had a higher ALP concentrations) – but these were not associated w/ higher mortality
Sepsis had sig greater TNF activity than HP and were more likely to have IL6 detectable.
Plasma IL1b, IL6 and Cl were the only variables correlated w/ nonsurvival in sepsis group**
TNF- early phase proinflammatory mediated, activaties secodnary inflammatory cascads resultsing in prolonged inflammatory response, EC permeability, neutrohpil relase from BM, expression of endothelial leukocyte adhesion molecules, lymphocyte apoptosis, AP protein and pyrexia. TNF was NOT associated w/ poorer prognosis (vs in humans it is correlated w/ death). In dogs increasing plasma TNF activty over time is associated w/ death w/ parvovirus**
**therefore, serial evaluations than a value taken at 1 time point may be more predicitive in cats**
IL1B- increases the production of other proinflamm mediators and expression of adhesion moleucles and plays a role in the developmemtn of organ dysfnc. IL1b was geather in septic cats cf SIRS & HP- difference was not signficnatn. IL1b WAS correlated w/ nonsurvival in septic cats with higher conc of IL1b being associated w/ a poorer outcome. Thus IL1b may be a possible prongostic biomarker for sepsis in cats**
IL6 and CXCL8- 2 late phase inflammaory mediators in sepsis and are produced in response to early cytokines (TNF and IL1b) and bacti products (LPS).
IL6 induces fever, synthesis of APP, differentaiton of B cells to produced Ig, dysfn of GI barrier and hemostatic derangement.
CXCL- main role is to reruite and activate No.
High levels of CXCL8 and IL6 are associated w/ poor outcomes in huamns w/ sepsis. IL6 is directly correlatd w/ risk of death in ppl- predcits outcome w > 80% accuracy.**
Plasma IL6 is also predcitivty of poor outcome in dogs w/ sepsis.
IL6 had a moderate but sig association w/ nonsurvival in cats w/ sepsis; and septic cats were more likely to have detectable conc cf SIRS and HP.
Only a low # of cats had dectable CXCL- diff to come up w/ a conclusion.
Mortality rate & hosp was greater for cats w/ sepsis than SIRS- but not SS.
In all but 2 cats, hypoNa was concurrent w/ hypoCl.
In dogs, higher SIRS scores are associated w/ a poorer prognosis- but # of SIRS criteria fulfilled was not correlated w/ outcome in cats w/ sepsis. Similar to humns were higher SIRS scors are not predicitive of death.
Need to est appropriate cutoff values for inflamm mediator conc by use of reciever operatiing char curves to obtain Se and Sp and predictive values for these markers. Need to dev feline Sp assays w/ ggreater analytic Se.
THM: sepsis (not SIRS) more likely to have bands and lower albumin, had greater TNF activity and detectable IL6. IL1b, IL6 and Cl may be prognostic biomarkers for sepsis in cats.

33. 43 dogs in septic grp 36 in SIRS grp Mortality rate 48%
Plasma IL 6 response is predictive for severity and mortality in canine systemic inflammatory response sydnrome and sepsis – Rau, et al Vet Clin path 2007
43 dogs in septic grp 36 in SIRS grp
Mortality rate 48%
Higher plasma IL6 levels were noted on the day of admission- significantly correlated with a more severe degree of dz, increased mortality rate and earlier fatality
MR in dogs w/ sepsis ranges from 31%-50%.
IL 6 looked at as it has a longer plasma ½ life cf TNFa or IL1b.
K9 models that used e.coli, LPS turpentine oil have shown induction of high IL6 levels.
Statstical analysis showed a moderate correlction b/w IL6 on the day of admission and the # of abnormal SIRS criteria. On indiviudal basis only corrleated w/ total wbc’s
A higher IL6 conc was related to a higher proportion of sepsis/SIRS. Also a higher IL6 levels increased the probablity for death.
Higher IL6 levels were sig correlated w/ a more severe degree of dz, increasedMR and earlier fatility. Majority of dogs in study were euthanized instead of dying.
In humans, survivors were char by rapid decreases in IL6.
IL 6 levels were undetectable in 6d w/ sepsis and 2 w/ severe sepsis- may be explained by cytokine kinetics. - i.e sick for long time and other cytokines were higher.

34. Acute phase response
Produced under the influence of IL1b, TNFa, and esp IL6
Few hrs after injury and subsides w/in 24-48hrs
Associated with acute infection and inflammation
CRP- major acute phase protein in dogs
SAA- major acute phase protein in cats
Haptoglobin
Hepcidin
Char by fever, nuetrophilia, activation of acoagulation, complement (classical, alternative, mannose binding lectin pathwasy), serum Fe and n binding, ehnaced GNG, incrased muscle catabolism and alter lipid metabolisms
Type I- indued by IL1a, IL1b and TNFa
Type II- IL6
Dec in albumin, protein C, protein S and AT; recently- adiponectin, insulin-like growth factor 1
+ GCSF;

35. CRP
Released in response to TNFa, IL1b- peaks at 36-50hrs and has a ½ life of 19hrs. Documted in other inflammatory dz like trauma, sx, AP and MI.
May reflect severity of inflamm process but have not been shown to different survival and non-survival
Not Sp
Is a pentamaric structure w/ two faces.
One face binds to phosphacholine, a common side chain found in all cell membranes and many bacti & protazoa
The other face is responsible for binding to neutrophils thru the Ab receptors FCyRIand Iia and to the complement component C1q
Can thus proote phagocytosis and removal of damaged, dying or dead cells as well as microbes
CRP can bind to bacti polysacc and glycolipids and to healthy and damaged cells, where it can activate C1q and the classical complement pathway
Also has an anti-inflamm role since it inhibits neutrohpil superoxide productiona nd degranulation and blocks plt aggregation
May therefore promote healing by reducing damage and enhancing repair of damaged tissue

36. Older literature
Increasing plasma TNF activity over time is associated w/ death in dogs with parvovirus
Endotoxemia and TNF activity in dogs with naturally occurring parvoviral enteritis. Otto JVIM 1997.
CRP in dogs with naturally occurring pancreatitis- was elevated in all 16 dogs compared to the control group. CRP decreased in all dogs that were hospitalized for 5d.
C-reactive protein concentrations in canine acute pancreatitis- Holm, et al JVECCS 2004.
CRP associated with SIRS and prolonged hospitalization in a group of dogs with pyometra
C-reactive protein, tumor necrosis factor alpha, and interleukin-6 in dogs with pyometra and SIRS. Fransoon, et al JVECCS 2007
Pyo group divided into SIRS + (57% or negative 43%- total of 53 dogs). CRP was the only paramater that sig releated to SIRS apart from clinical criteria. MR was low (4%) and conclusion regarding associated w/ SIRS could not be drawn. A + SIRS status, high CRP and high body temp were variables that related to Inc morbidity reflected by length of hosp stay.
IL6 were not sig different b/w healthy and diseased dogs. TNFa were sig different b/w dogs w/ pyo and healthy dogs but was not able to differntiate b/w SIRS+ and SIRS – dogs so not related to severity of dz.

37. Surviving dogs had greater decrease in CRP than non- survivors
Use of C-reactive protein to predict outcome in dogs with systemic inflammatory respone syndrome or sepsis -Gebhardt C JVECC 2009
Surviving dogs had greater decrease in CRP than non- survivors
No relationship b/w survival and the initial CRP levels
Unable to differentiate SIRS and Sepsis with CRP
13 dogs had SIRS and 48 had sepsis. 14 day survival rate 61%
CRP- APP
Increased CRP associated w/ sx, imune d, panc, GI dz and sepsis
Changes over tiem suggested to predict survival
Binding ability to nuclear ribonucleoproteins facilitates clearance of nuclear mateiral of injured and necrotic tissues. Protective effect agsinst infx.
Also can activated IL6 causing a revovling effect

38. Prospective observational study using 79 dogs with parvo
Evaluation of the use of serum C-reactive protein concentration to predict outcome in puppies infected with canine parvovirus - McClure JAVMA 2013
Prospective observational study using 79 dogs with parvo
CRP was serially evaluated during hospitalization
CRP concentrations at 12 and 24hrs after admission were negatively associated with survival
Sensitivity and specificity of CRP to differentiate b/w survivors and non-survivors at 24hrs after admission- 87% and 79%
Still proving to be not a great predictor of outcome when used alone
Evalutaed at admission, then hrs for the 1st 48hrs, then every 24hrs until d/c.

39. Procalcitonin
Plasma procalcitonin appear to correlate w/ bacteremia and organ dysfunction in human clinical studies
Extrathyroidal procalcitonin gene expression documented in dogs w/ SIRS but not in healthy animals
Canine procalcitonin messenger RNA expression Kuzi J Vet Dian investigation 2008
Can may be used to decide when to d/c antibiotics.
Procalcitonin for reduced antibiotic exposure in the critical care setting: a systemic review and an economic evaluation – Heyland CCM 2011.
PCT- a precursor for calcitonin- has been investigated as a potential marker. Produced by thyroid C cells, PCT during sepsis though to orginate from mononuclear leukocytes following endotoxin and cytokine stimulation
Released 2-6 hrs after endotoxin release and peaks persists for up to 24hrs. Although exact role in spsis still ukn- thought to incrase iNOS
Studies doucmented elevated PCT levels in bacti infx complicated by ssy inflamm and lil to non changes in pCT in localized inf and in infalmmation by virus- so maybe a good marker to differ b/w bacti sepsis and SIRS
In some studies correlate w/ dz seveirty and may have prongnostic implications
Can also be used to decide when to d/c ab’s. Ref: Procalcitonin for reduced ab exposure in the critical care setting: a systemic review and an economic evaluaiton – Heyland CCM 2011.
Elisa cannot be used for dogs d/t difference in the carboxy terminus of the PCT molecule. One study was performed looking at PCT mRNA expression – greater concentration in sick dogs. Changes in the expression from one time pt to another were in agreement w/ the clincial evalatuion of patient imporveemnt or deteriation. Canine procalcitonin messanger RNA expression – Kuzi J Vet Dian investigaiton 2008

40. Coagulation and sepsis
Hypercoag state- induced by cytokine medated tissue factor expression on the surface of the wbcs leads to fibrin depsotiion in the microvascular and is thought to contribute to organ failrue in proinflammatory state
Endogenous anticoag systems like AT, protein C and tissue factor pathway inhibitors are overwhelmed
Thrombin stim wbcs activation and further production of cytokines
TF is enhanced leading ito increased production of prothrombin that is converted to thrombin and in turns generates more fibin form fibrinogen.
Simutalneously, levels of plasmingen-activator inhbitor-1 are increased, resulting in imparied production
Normally, there is a fine balance b/w coagulation and fibrinolysis to prevent hemorrhage and disseminated thrombosis. The homeostatsic state is disrupted by inflammation which leads to shift the balance toward a procoagulant antifibrinolytic environment.
Prothrombitc systems- plt activation, coaguatlion cascard and anti-fibrinolysis
vs antithrombitc systems- tissue factor pathway inhibitor, AT, protein C and fibrinolysis
Plt production in response to infalmation are more thrombogenic w/ an increased Se to plt agonists. The activated plt aggregates provide the negatively charged phospholipid surface neceassry for secondary homeostasis which results in thrombin formation. Also activated plts can syn IL1b which targets EC to enhance their adhesive proprtiers.
Dysregulated coagulation
TF is stimulated by LPS, TNF alpha, IL-1, IL-6, activated complement, immune complexes. Tissue factor enhancement leads to increased production of thrombin and fibrin.
Tissue factor pathway inhibitor inactivates both factor Xa and the TF-VII complex. Levels can be low, normal, or elevated in septic patients. In sepsis there is an increase in TF without a corresponding increase in TFPI
There is downregulation of protein C pathway through inhibition of thrombomodulin and endothelial cell protein C receptor transcription.
Early in the course of inflammation fibrinolysis is enhanced through increased release of stored plasminogen activators. Inflammation impairs the fibrinolytic system through enhanced production of plasminogen activator inhibitor which is a potent inhibitor of TPA.
Combination of decreased fibrinolysis, widespread activation of coagulation, and excessive production and release of inflammatory mediators contribute to development of hypercoagulability. In late stages of inflammation, consumption of platelets and prolongation of clotting times ultimately results in DIC.
The immunopathogenesis of sepsis. Cohen. Nature 2002.

41. Serial evaluation of Protein C and Antithrombin in dogs with sepsis
Serial evaluation of Protein C and Antithrombin in dogs with sepsis. AM de Laforcade JVIM 2008.
12 dogs with sepsis were enrolled into study
Protein C and AT levels decreased over time with sepsis
Non survivors had lower protein C and AT levels
Identified a signficnat decrease in PC activity from days 1 and 2, followed by a gradual increase in PC activity over time.
Protien C- conerted to the activated form when thrombin complexes w/ thrombomudulin.
Anticoagulant, anti-inflammation and antiapoptotic effects.
Since decreased- replacement may have therapeutic value.
Prwoness trail- resulted in 20% decrease in the risk of death
Interesting this did seem to help where other anticoagulants (AT, TFPI) failed. Was approved but w/d from the market.
Has marked Sp specificity and requires 15-20x fold higher dose in dogs which is
Adverse effect of bleeding. $6800/person

42. Others
14 dogs w/ sepsis (11 control)- median vWF Ag concentration in dogs w/ sepsis was significantly higher than healthy dogs, however no difference b/w survivors and non-survivors were noted.
Von Willebrand factor antigen concentration in dogs with sepsis. CL Rogers, et al JVIM 2010.
Reduced activity of AT and aPC were identified in septic dogs, but no correlations w/ outcome
Hemostatic changes in dogs with naturally occurring sepsis. De LaForcarde, et al JVIM 2003.
Hypercoagubility noted via assessment of TEG, AT. 5/9 dogs had clinical evidence of thrombosis.
Evidence of hypercoagulability in dogs with parvoviral enteritis. Otto JVIM 2000.
Dogs with SIRS had more PNA (platelet-neutrophil aggregate) formation, large neutrophil size and less granularity relative to control dogs. However no difference were found b/w dogs that had sepsis, SIRS or DIC.
Platelet-neutrophil aggregate formation in blood samples from dogs with systemic inflammatory disorders. Dircks, et al AJVR 2012.
Plt are invovled in the initation and propagation of inflammotory nad immune porcesses via scretion of IL-1beta and beta-thromboglobulin. Plt activation leads to expression of adesion molecules on their surface, mediatoing plt aggregate and interection w/ other cells like EC and wbcs. Once activated plt release and express p-selectin and allows binding to p-selectin glycoprotein ligand-1 expressed on the surrface of all lekuocytes, thus promoting the initial adhesion b/w cells. The interection of neutrophil w/ plts appears to mediate neutrophil accumulation and emigration into inflammatory tissue. This interaction may also enhacne activaiton, secertion and aggreation of plt and therefore amplyfly the fnc of both cells reciprocally. Therefore, PNA provide an impt link b/w thrombosis and inflammation and their hinhibition might be target for phar intervetion in prothrombotic conditions. Higher than typical #s of PNSA have been found in humasn w/ yeloproliferative disorders, coranry artery dz, DM and sepsis. Therefore, this interaction b/w plt and No may be a Se means of detecting plt activation.
In humans, those w/ uncomplicated sepsis had a higher degree of PNA formation and those w/ septic shock had a considerably lower degree- implying a negative correlation b/w the severity of sepsis and formation of PNAs in humans. This study had a sm number of dogs w/ sepsis for them to try to differentiate them based on severity.
A higher degree of plt activation w/ PNAs have been id in septic humasn cf nonseptic crticically ill pts- which might be d/t PNAs adhering to endothelium.
Dogs w/ DIC had higher #s of PNAs than did control dogs but no difference was found b/w dogs w/ SIRS/DIC vs SIRS w/o DIC.
LPS can induce plt p-selectin expression, which plays a impt role in plt-neutrophil interaction.

43. Compensatory anti-inflammatory response syndrome and cell death
Compensatory anti-inflammatory response syndrome (CARS)-
Adaptive response
Usually controlled, but might be lost in sepsis
Programmed cell death
Type I (apoptosis)
Type II (autophagy)
Pyropoptosis
CARS is char by release of anti-inflam mediates like Il10, TGFb and IL13
Produciton of soluble recepotrs and recptor antagnosits for cytokines such as TNFa
Excessive stim of comp anti-inflamm response may contribute to immunoparalysis and increase susptibiity to nosoocomial infx in the late stages of sepsis (2nd hit)
addaptive response to excessive proinflammatory process in SIRS and sepsis
Usually controlled, but lost in sepsis which leads to uncontrolled proinflamm rxn to inf, result in organ dysfnc, undesirable compromise of the immuen system permitting opportunisitic infx (second hit) or combo
Autophagy, also mediate type II PCD and interacts w/ apoptosis
Pyroptosis- capsase-1 mediated PCD- distinct from death mediated by apoptotic capases 3, 6, 8
Char by rapid plasma membrane rupture and release of proinflamm IC contents (some that can act as DAMPs)
Apoptosis- type I PCD
Autophagy- cytoplasmic clean up, assists in delivery of proteins to APC. Mediates type II PCD and inteacts with apoptosis.
Pyroptosis- caspase-1 mediated PCD.

44. Regulatory T cells
Naturally occuring Tregs are known to inteact w/ innate and adaptive immune sys to suppressive fnc to prevent the dev of autoaggresive response to maintain a popualtion of peripheral CD4 T cells.
MOA: cell contact, induction of death and secretion of cytokines (IL10 and TGFb).
Regultory activity of T cells reside primary w/ CD4. T cells regulation include cross-regluation of Th1 vs Th2. The action of TGFb secreting Th3 cells that are likely ip in mediated mucosal Ag tolerance. Presence of CD4/26 natural suppressor cells that mediated suppression by direct contact. And actions of IL10 that inhigt the fun of target cells via cytokine.
Clinical immunology of the dog and cat, 2nd edition. Day. Pages

45. Targeted therapy Anti-endotoxin strategies
Antibodies
Polymyxin B
Mediator blocking strategies
Anti-tumor necrosis factor (TNF)
Soluble TNF receptor
IL-1 receptor antagnosits
PAF receptor antagonist
NO synthase inhibitor
Bradykinin inhibitor
PG inhibition
High dose GC
Immune enhancement
GCSF
Coagulation inhibitors AT
TFPI
In animals- feline IFN for enahnced antiviral activity- randomized double blinded placebo control study from france showed improvement w/ tx. A similar study failed to show benefit in the US although lower dose was used and overall survival was higher.
Anti-endotoxin agents- (Bactericial permeability increasing protein)- failed to demonstrate a benefit to morbidity or mortality.
GCSF- no benefit on wbc cell count or outcome was identified.

46. Ideal Biomarker Detects the presence of infection
Provide information about dz progression
Monitor response to treatment
provide prognostic index
In human medicine- three of the most studeited are CRP, IL6 and procalcitonin- but there ar emany that are being looked at (thromobudulin, adhresion molecules). Procalcitonin is a presuc

47. Summary
Tyep of response detemined by many factors including viruelnce, size of incoculum, pt’s co-exsiting conditiosn, nutrional status, age and polymophrism in cytokine genes or other immune effector molecules.
Measureemetns of inflamm mediators may prove to be useful in evaluating the stage of sepsis and in tailoring tx- i.e are they at a hyper or hypoimmune phase.
Disparity b/w animal models and sepsis and clinical treatment of septic humans highlight several key issues.

48. References
The Immunopathology of Sepsis: pathogen, recognition, systemic inflammation, the compensatory anti-inflammatory response and regulatory T cells. DH Lewis, et al. JVIM 2012
The immunopathogenesis of sepsis. Cohen. Nature 2002.
Immunotherapy of sepsis. Van der Poll. Infectious disease 2001.
The pathophysiology and treatment of Sepsis. Hotchkiss. NEJM 2003.
Breed specific pro-inflammatory cytokine production as a perdisposing factor for susceptibility to sepsis in the dog. Nemzek, et al JVECCS 2007.
Clinical and immunology assessment of sepsis and the systemic inflammatory response syndrome in cats. Declue et al. JAVMA 2011
Clinical trails in spontaneous disease in dogs: a new paradigm for investigaiton of sepsis. Otto. JVECCS 2007
Evaluation of the sensitivity and specifitiy of diagnostic criteria for sepsis in dogs. Hautman Vet Surg 1997.
Hemostatic changes in dogs with natrually occuring sepsis. AM de Laforcade JVIM 2003.
Innate immunity and host defense peptides in veterinary medicine. Linde, et al JVIM 2008.
Neutrophil Function in septic dogs. Webb JVIM 2007.
Regulation of Immune response by T cells. Jiang NEJM 2006.

49. The sepsis-coagulant axis: a review. Weiss JVIM 1998.
Plasma IL-6 response is predictive for severity and mortality in canine systemic inflammatory response syndrome and sepsis. Rau Vet Clin Path 2007.
Platelet-neutrophil aggregate formation in blood samples from dogs with systemic inflammatory disorders. Dircks, et al AJVR 2012.
The sepsis-coagulant axis: a review. Weiss JVIM 1998.
Sepsis in veterinary patients: what do we know and where do we go? Otto JVECCS 2007.
Comparison of dogs with septic peritonitis: vs Bentley, et al. JVECCS 2007.
Serial evaluation of Protein C and Antithrombin in dogs with sepsis. AM de Laforcade JVIM 2008.
Severe sepsis in cats: 29 cases ( ). Brady JAVMA 2000.
Underlying cause, pathophysiologic abnormalities and response to treatment in cats with septic peritonitis: 51 cases ( ). Costello JAVMA 2004.
Use of C reactive protein to predict outcome in dogs with systemic inflammatory response syndrome or sepsis. Gebhardt, et al JVECCs 2009.
Von Willebrand factor antigen concentration in dogs with sepsis. CL Rogers JVIM 2010.
Evidence of hypercoagulbility in dogs with parvoviral enteritis. Otto JVIM 2000.
Clinical immunology of the dog and cat, 2nd edition. Day. Pages
Small animal critical care medicine. Silverstein, Hopper, 1st edition. Pages 46-49,
The systemic inflammatory response syndrome, sepsis and multiple organ failure. Brady, Otto, VCNA SAP 2001.

50. Questions?