Proceedings of the Annual Meeting of the American College of Veterinary Pathologists and American Society for Veterinary Clinical Pathology - Tucson, Arizona 2006 - Reprinted in the IVIS website with the permission of the ACVP and ASVCP www.ivis.org
Acute Phase Proteins as Biomarkers of Disease in Companion and Laboratory Animals P. David Eckersall, BSc, MBA, PhD, FRCPath Division of Animal Production and Public Health, Faculty of Veterinary Medicine University of Glasgow, Bearsden Rd, Glasgow, G61 1QH, UK (p.d.eckersall@vet.gla.ac.uk) Introduction The acute phase proteins (APP) are blood proteins the measurement of which allows assessment of the innate immune system's systemic response to disease. 1-3 By definition, these proteins change their serum concentration by > 25% in response to inflammation, infection and trauma. Therefore as quantitative markers for disease they can be used for prognosis and monitoring responses to therapy, for general health screening as well as for diagnosis of disease. The APP are highly sensitive for the presence of pathological lesions although they have a low specificity for a particular disease being elevated in numerous conditions. There are major differences between species in the APP response in disease (Table 1). In any one species positive APP have been found that have either major, moderate or minor responses. A major APP has a low concentration being in the order of 1 Ìg/liter or less, in the serum of healthy animals but with the concentration increasing over 100 or 1000 fold on stimulation, reaching a peak 24-48 hours after the insult and falling rapidly during recovery. A moderate APP is present in the blood of healthy animals but on stimulation the concentration will increase 5-10 fold, reach a peak 2-3 days after stimulation and decrease more slowly than the major APP. A minor APP shows a gradual increase and only increases in concentration by 50-100% of the resting level. Negative APP have also been identified, which fall in concentration during the response, though apart from the hypoalbuminemia of infection and inflammation the measurement of these proteins has not yet reached the clinical pathology laboratory. Acute Phase Protein in Canine Medicine C-reactive protein is the major APP in man and has become one of the most frequently requested biochemical tests in human clinics. However, there is considerable species variation in the pathophysiology of CRP. In a number of species such as the dog, CRP is a major APP and its serum concentration can increase rapidly from < 5 mg/liter to over 100 mg/liter. A number of infectious diseases (Table 2) lead to an increase in CRP in the dog including babesiosis, Leishmaniosis, leptospirosis, parvovirosis, trypanosomiasis, and infection with Bordetella brochiseptica, Ehlichia canis and Escherichia coli sepsis. 1 Similarly large increases can be found in dogs with arthritis and lymphoma. Moderately raised level of CRP have been found in inflammatory bowel disease 4 and in hematologic diseases of the dog. 5 Elevated levels of canine CRP have been observed in serum from mid gestation of pregnant bitches with its appearance coinciding with the implantation of the embryo in the endometrium. 6,7 It is likely that sufficient damage is caused to the endometrium by this process to stimulate the acute phase response in the maternal circulation. Haptoglobin is a moderate APP in dogs responding to inflammatory and infectious disease. However canine Hp is particularly sensitive to corticosteroid and elevated levels of Hp are found both after treatment with corticosteroids and during naturally occurring hyperadrenocorticism. 8-10 This is a potential disadvantage in monitoring inflammatory disease with canine Hp as steroid treatment could interfere with interpretation, but full understanding of this process may reveal novel uses for the Hp assay, possibly as a screening method for Cushing s syndrome. The glycosylation of canine Hp alters during the acute phase reaction 11,12 but methodology is currently too cumbersome to allow such changes to be used for diagnostic purposes. In the dog the circulating concentration of SAA does increase during an acute phase response and has been observed in experimental parvovirus infection 13 and in Leishmaniasis. 14 However with CRP becoming the primary canine APP it is likely that SAA will be used in a secondary role in monitoring the acute phase response in this species. The relationship of serum concentration of SAA with familial amyloidosis as encountered in Chinese Sharp-ei dogs and also Siamese and Abyssinian cats remains to be fully elucidated 15,16 TABLE 1. Acute Phase Protein: Major and Moderate Responders in Various Animal Species Species Major APP Moderate APP Cat SAA AGP, Hp Dog CRP, SAA Hp, AGP, Horse SAA Hp, Cow Hp, SAA AGP Pig CRP, MAP, SAA Hp,
Table 2. Canine diseases where an acute phase response has been described. 1 Acute Phase Protein CRP Haptoglobin SAA AGP Disease/Condition Surgical trauma Rheumatoid arthritis Polyarthritis Intestinal obstruction Inflammatory bowel disease Lymphoma Acute pancreatitis Pyometra Pneumonia E. Coli endotoxaemia Babesiosis Bordetella bronchiseptica Ehrlichia canis Leishmaniosis Leptospirosis Parvovirus Trypanosomiasis Bacterial enteritis Surgical trauma Leishmaniosis Trypanosomiasis Cushing s syndrome Corticosteroid treatment Parvovirus Bordetella bronchiseptica Leishmaniosis Parvovirus Babesiosis Ehrlichia canis Lymphoma Carcinoma Sarcoma A major potential application for APP measurement for dogs and other companion animals would be by inclusion in panels of analytes during regular health checks. The ability of the APP to detect sub-clinical disease is becoming clearer and is one of the emerging uses of CRP analysis in human medicine. concentration may be useful in cats as it was shown to be the most rapidly responding APP in a variety of inflammatory and infectious conditions. The cat is a species where CRP does not show a major response. Haptoglobin is raised in the feline acute phase response though further investigation is required to determine the full diagnostic value of this protein. Acute Phase Proteins in Equine Medicine Equine SAA is a major APP with elevated levels found in serum from horses with infection or inflammation. 22,23 Increased SAA concentrations have been observed in horses (Table 3) following surgery, with aseptic inflammation or arthritis, septicemia, enteritis, pneumonia and diarrhea. 3 Measurement of the SAA concentration was found to be of value in diagnosis of horses with colic especially where inflammation was the primary component of the pathogenesis. 24 Experimental infections with equine herpes virus and influenza virus have also revealed an increase in the SAA concentration. 23,25 In horses Hp has been found elevated in animals with systemic inflammatory responses, alimentary laminitis and with grass sickness. 26-28 Table 3. Equine diseases where an acute phase response has been described. Acute Phase Disease/Condition Protein Haptoglobin Arthritis Grass sickness (equine dysautonomia) Noninfectious laminitis Equine influenza virus (strain 2) SAA Aseptic arthritis Surgical trauma Septicemia & focal inflammation (foals) Enteritis Pneumonia Diarrhea Fever Equine herpes virus serotype 1 Equine influenza virus serotype 2 Acute Phase Proteins in Feline Medicine There have been less investigations of the feline acute phase response than for the canine response. However, the measurement of AGP in feline serum and peritoneal fluid has become a recognized differential test for the identification of feline infectious peritonitis. 17,18 Raised levels of AGP have also been reported in tumor bearing cats 19 and those with lymphomas 20 though in the latter study the AGP concentration was not prognostic for survival. Determination of the SAA Implementation of Acute Phase Protein Analysis A critical mass has been developing in veterinary clinical pathology from investigation of APP such that there is sufficient knowledge of the pathophysiology of the response to support the valuable diagnostic information can be obtained by measuring their serum concentration. It is likely that over the next few years these analytes will become an important component of clinical
pathology requests. In the longer term, the analysis of APP among other serum proteins may be incorporated into the multiplexed assays on new laboratory equipment that is being developed to exploit the potential of proteomic investigation. Indeed it is a salutary lesson that a common outcome of serum proteomic investigation to discover biomarker for cancer is that the most prominent changes are in the APP. 29 Acute Phase Proteins in Laboratory Animals The profile of acute phase protein changes in laboratory animals was established in experimental studies of the acute phase reaction. 30 In rats the Hp and AGP are moderate APP while α 2 macroglobulin (A2M) has been reported as being the primary major APP to monitor in this species. In mice SAA, Hp and serum amyloid P (SAP) show moderate/major responses. In both species differences between strains in the profile of the acute phase response have been recognized (Table 4). Stimulation of the rat APP with Freund s complete adjuvant showed a 7-fold increase in Hp with no change in albumin or CRP following a short increase in concentrations of interleukin 1 and interleukin 6. 31 The APP have been used to assess inflammatory damage caused by phophodiesterase (PDE) inhibitor based drugs. In a study 32 of the gastrointestinal toxicologic response to a PDE4 inhibitor serum Hp concentration was found to mirror histological damage, as did the concentration of interleukin 6. However IL-6 levels among the affected animals were highly variable while Hp proved to be an early and sensitive biomarker of drug induced toxicity. It has also been found that A2M and interleukin 6 may herald early onset of drug-induced vascular inflammation in a study of PDE inhibitor cardiovascular injury induced in spontaneously hypertensive rats. 33 The acute phase response in mice has been used more as an end-point biomarker in models of disease. Thus Hp, SAA and SAP have been used in studies on trypanosomiasis, malaria and colitis 34-36 while a proteomic investigation using 2 dimension electrophoresis with Table 4. Acute phase proteins in rats and mice. 30 Acute Phase Protein Response Response in mice in rats C-reactive protein +/- + Serum amyloid A ++++* - Serum amyloid P +++ - Haptoglobin +++* +++ α1-acid glycoprotein ++ +++ α2-macroglobulin +/- ++++* * strain variation reported albumin depleted serum 37 revealed that murine hemopexin and inter α-trypsin inhibitor also displayed APP pathophysiology. It is therefore possible that the use of APP as biomarkers of inflammatory damage and especially of subclinical inflammation will have a role in the clinical pathology of drug safety testing. Indeed with a developing pharmaceutical interest in drugs designed to affect the immune reaction it may become essential to test new drugs for such side-effects. The recent incident in the UK where a clinical trial of monoclonal antibody TGN1412 caused a severe septic shock reaction in human volunteers illustrates the potential hazard of overstimulation of the cytokine driven host reactions and the requirement of species specific tests for this side-effect. Acknowledgements Reseach has been supported by BBSRC and EU funding. Many colleagues have contributed to the research of the Acute Phase Laboratory of the Faculty of Veterinary Medicine, University of Glasgow www.gla.ac. uk/vet/research/apph/genesandproteins/acutephaselab/. The assistance of Garscube Diagnostics is appreciated www.gla.ac.uk/garscubediagnostics/. References 1. J. J. Ceron, P. D. Eckersall and S. Martinez-Subiela, Acute phase proteins in dogs and cats; current knowledge and future perspectives. Vet Clin Path 34, 85-99 (2005). 2. H. Murata, N. Shimada and M. Yoshioka, Current research on acute phase proteins in veterinary diagnosis: an overview. 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