Acute phase proteins

acute phase proteins, classification, diagnosis

1. ACUTE PHASE PROTEINS (APP)

2. ACUTE PHASE REACTANTS/PROTEINS (APP) • Definition: These are the class of proteins whose plasma concentration increase or decrease in response to inflammation

3. Proteins that change their serum concentration by 50-1000 folds in response to inflammatory cytokines (IL-1, IL-6, TNFα ) The acute-phase response is considered part of the innate immune system APPs play role in mediating systemic effects as fever leukocytosis increased cortisol decreased thyroxine decreased serum iron and many others

4. Positive APPs Negative APPs C-reactive protein  (CRP) Albumin Serum Amyloid A  (SAA) Transferrin Haptoglobin (Hp) Transthyretin Ceruloplasmin Retinol-binding protein α2- Macroglobulin Adiponectin α1- Acid glycoprotein (AGP)   Fibrinogen   Complement (C3, C4)

5. A major APP Low concentration in the serum of healthy animals: 0.1 μ g/dl (1 μ g/ liter ) On stimulation concentration will increase 100 or 1000 fold Reach peak 24 to 48 hours after the insult and falling rapidly during recovery A moderate APP on stimulation the concentration will increase 5 to 10 fold Reach a peak concentration 2 to 3 days after stimulation, and decrease more slowly than the major APP Positive APPs are further categorized as major, moderate or minor, depending on the degree of increase A Minor APP Increase above resting levels at a gradual rate.

6. ACUTE PHASE PROTEINS (APP)

7. The rapidity and magnitude of the increase in each acute phase protein varies depending on the species. Species Major APP Moderate APP Cat SAA AGP, Hp Dog CRP, SAA Hp, AGP, Cp Horse SAA Hp Cow Hp, SAA AGP Pig CRP, Pig-MAP Hp, Cp Mouse SAA Hp, AGP Rat α2- macroglobulin Hp, AGP The rapidity and magnitude of the increase in each APP varies depending on the species.

8. Protein Main function Alpha-1-acid glycoprotein (cat) Antiinflammatory and immunomodulatory agent: has antineutrophil and anticomplement activity and increases macrophage secretion of IL-1 receptor antagonist. Binds to lipophilic and acidic drugs. Ceruloplasmin Copper transport (for wound healing, collagen formation and maturation) Antioxidant Reduces the number of neutrophils attaching to endothelium Haptoglobin (Cow, Pig) Binds free hemoglobin (limiting Hb iron availability for bacterial growth) Natural antagonist for receptor- ligand activation of the immune system. Inhibition of granulocyte chemotaxis and phagocytosis Functions of the positive APPs

9. Protein Main function Serum amyloid A (Horse, pig) Chemotactic recruitment of inflammatory cells to sites of inflammation Induction of inflammatory cytokines Inhibition of myeloperoxidase  release and lymphocyte proliferation Involved in lipid metabolism and transport immunomodulatory (via the inflammasome ) C-reactive protein (Dog, Pig, Human) On bacteria, it promotes the binding of complement, facilitating phagocytosis . Induction of cytokines Inhibition of chemotaxis and modulation of neutrophil function Neutralizes deleterious effects of histones

10. Negative acute phase proteins Decrease in plasma concentration by greater than 25% in response to inflammation This reduction can occur rapidly (within 24 hours) or may decrease gradually over a period of days The mechanism by which their concentrations decrease is likely including decreased production by the liver in response to inflammatory cytokines possibly increased loss or increased proteolysis.

11. Protein Main function Albumin Reduced production of albumin allows greater increase in the amount of amino acids available for positive APP production Albumin concentration falls gradually and more noticeable in chronic inflammatory disease Transferrin Usually measured to assess iron status Ovotransferrin is the avian analog, but it is a positive acute phase protein Adiponectin Produced in adipose tissue , and promotes energy usage through increasing sensitivity to insulin, has anti-inflammatory properties. Decreased concentrations may be seen in obese animals or animals with diabetes mellitus Functions of the negative APPs

12. C-reactive protein (CRP) A major acute-phase protein produced by the liver in response to tissue damage CRP was the first acute phase protein to be recognized CRP is a beta-globulin and has a molecular weight of 115-140 kD • CRP Gene- Chromosome 1, Short arm • Cytokines especially, IL-6 and IL-1 regulate CRP at transcritional level • Normal range: Less than 10mg/L

13. CRP • C-reactive protein (CRP) belongs to the pentraxin family of proteins, which has five identical subunits It was first discovered in 1930 by Tillet and Francis • Plasma levels begin increasing within 4-6 hours following acute inflammatory stimulus Half life of CRP is 18-20 h the level of CRP in the blood is regulated solely by its own synthesis.

14. C- REACTIVE PROTEIN Its name derives from its pattern recognition activity: C-reactive protein binds to the C-polysaccharide cell-wall component found on a variety of bacteria and fungi This binding activates the complement system, resulting in increased clearance of the pathogen either by complement-mediated lysis or by a complement mediated phagocytosis • It has highest affinity for, – Phosphocholine on bacteria – Mixture of sphingomyeline and phosphatidylcholine in eukaryotic membranes

15. FUNCTIONS OF CRP 1. Anti Infective  Opsonise particles for phagocytosis  Activate Complement via classical pathway 2. Anti Inflammatory actions  CRP helps in preventing systemic inflammation  CRP aids in the release of neutrophils from blood vessels  Stimulate release of anti-inflammatory molecules from monocytes 3. Scavenging actions  bind to cells that are undergoing apoptosis or necrosis (not bind to normal cell membranes)

16. CRP begins to rise within 4-6 h of stimulus, peaks within 36-50 hours, and returns to normal 3-7 days following resolution ESR shows a much slower response, taking up to a week to peak, and up to several weeks to return to normal. CRP and ESR patterns of response

17. • CRP is more sensitive than ESR to subtle changes in the acute phase response • There are distinct ranges of normal and abnormal in CRP reference ranges, without variations for age and gender • CRP is not affected by conditions such as pregnancy, intercurrent drug use, anaemia and plasma protein variations. CRP is a better measure of acute phase response 10

18. CLINICAL IMPORTANCE OF CRP Provides fast and adequate information of the actual clinical situation Useful for monitoring the effect of treatment Early detection of postoperative complications or intercurrent infections • Elevated CRP levels: – Osteoarthritis – Proinflammatory or prothrombotic effects ( Cerebral vein or sinus thrombosis ) _An extremely elevated suggestive of a possible bacterial infection _Elevated levels were seen in majority of kidney or heart transplant • Mild elevation of CRP levels: – SLE – Scleroderma – Sjogren syndrome – Dermatomyositis / Polymyositis

19. Pathophysiology There is considerable species variation in the pathophysiology of CRP Dog: (infectious diseases - babesiosis , leishmaniasis , leptospirosis , parvoviruses , trypanosomiasis , infection with Bordetella bronchiseptica, Ehrlichia canis , and Escherichia coli sepsis a nd in hematological and neoplastic diseases of the dog ) Pig: CRP concentration increases following aseptic Inflammation and in experimental infection with Actinobacillus pleuropneumoniae Bovine : respiratory syncytial virus, multiple myeloma, mastitis, lymphatic neoplasia .

20. hs -CRP • Definition: – High sensitivity C-reactive protein ( hs -CRP). – Similar to CRP, it is also an Acute Phase Reactant – More sensitive than CRP especially for cardiovascular injury – Normal range: less than 0.3 mg/ dL hs -CRP Uses • Performing risk assessment for cardiovascular diseases: – Independent risk factor for CVD, Stroke and Peripheral vascular disease. – It also adds to the predictive value of total cholesterol and HDL cholesterol – hs -CRP has been reported as a risk factor for hypotension

21. METHODS OF ESTIMATION OF APP

22. SERUM ELECTROPHORESIS The acute-phase proteins migrate in the α- (mostly) and β- regions of the electrophoretogram As many acute-phase proteins are α globulins , an increase in concentration of α1 or α2 globulins is a sign of an acute-phase response Detectable soon after the onset of inflammation, injury, or infection and may persist until the inciting stimulus has resolved

23. Acute-phase- electrophoretogram ACUTE-PHASE-ELECTROPHORETOGRAM Serum protein Electrophoretic region α1- Acid glycoprotein α 1 Serum amyloid A α Haptoglobin α 2 Ceruloplasmin α 2 Transferrin β 1 C-reactive protein γ

24. CRP is prominent among the acute-phase proteins It is a blood test marker for inflammation in the body Sample considerations Storage:  stable at -10º C for 3 months Anticoagulant:  Do not use citrate tube as levels are significantly lowered CRP : a direct and quantitative measure of the acute-phase reactions

25. C-REACTIVE PROTEIN Tests Slide/Rapid latex agglutination tests Turbidimetric immunoassay:  used in humans and has been adapted for automated biochemical analyzers . However, there is variation in cross-activity with different antihuman CRP antibodies. Hemolysis will interfere with immunoturbidimetric testing. ELISA :  a commercially available kit for canine CRP Time-resolved fluorometry (TRFIA):   recently developed for CRP assays in canine whole blood, saliva and effusions

26. • Rapid latex agglutination test: Principle Based on the reaction between patient serum containing CRP as the antigen & the corresponding antibody coated to the treated surface of latex particle. INTERPRETATION • A negative reaction is indicated by a uniform milky suspension with no agglutination as observed with the CRP Negative Control. • A positive reaction is indicated by any observable agglutination in the reaction mixture.

27. A positive CRP test may indicate Cancer Connective tissue disease Heart attack Infection Inflammatory bowel disease (IBD) Lupus Pneumococcal pneumonia Rheumatoid arthritis Rheumatic fever Tuberculosis Heart disease. Coronary heart disease (CHD) Cardiovascular disease Factors affecting CRP levels: Smoking Obesity Diabetic Sedentary lifestyle Increased cholesterol Hypertension and metabolic syndrome

28. Turbidimetric immunoassay for CRP This rapid, reliable equilibrium superior to radial immunodiffusion or nephelometry Involves a potent monospecific antibody Polyethylene glycol-6000 : accelerate immunoprecipitation reaction Tween-20 : surfactant to stabilize the sample blank values Spectrophotometer turbidimetrically at 340 nm can be used Values up to 220 mg/L (standard curve is linear)measured without sample dilution.

29. CRP- ELISA Kit

30.   The time-resolved fluorescent immunoassay (TRFIA) Uses a polyclonal antibody bound to immunomagnetic beads as capture antibody and the same antibody labeled with europium as the detection antibody

31. Ceruloplasmin Sample considerations Anticoagulant : concentrations are higher with heparin and lower with EDTA Tests There are problems with assays - lack of commercially available reference materials to standardize ceruloplasmin concentrations. Therefore, different arbitrary units based on increased absorbance per unit time have been used ( oxidase units UI/L).

32. Haptoglobin Sample considerations Storage : Values decrease in serum stored at -20ºC. Storage at -70°C Anticoagulants : concentration is increased with heparin Interferences : Dogs with high endogenous steroids or on exogenous steroid therapy will have increase Hp concentrations. Canine Hp concentrations in health or disease are significantly higher than other species. Tests Spectrophotometric assays Hemoglobin- haptoglobin complexes that alter the absorbance characteristic of Hb in proprotion of the concentration of Hb in serum Peroxidase acitivity at an acidic pH detected & quantifi ed Immunoassays Nephelometric assay: rate of precipitation of the Ag- Ab complex is measured.

33. Alpha-1 acid glycoprotein Tests: Estimation by   precipitation  of majority of serum proteins by perchloric acid and quantification of the remaining soluble proteins. Single radial immunodiffusion  on agarose gel impregnated with anti-species AGP rabbit serum. Dog and cat specific assays have been developed. Immunoturbidimetric assays  have been developed for canine and feline AGP measurement