Acute phase reactants, challenge in the near future of animal production and veterinary medicine

Abstract

The future of acute phase proteins (APPs) in science is discussed in this paper. Many functions and associated pathological processes of APPs are unknown. Extrahepatic formation in local tissues needs attention. Local serum amyloid A (SAA) formation may be involved in deposition of AA-amyloid induced by conformational change of SAA resulting in amyloid formation, having tremendous food safety implications. Amyloidogenesis is enhanced in mouse fed beta pleated sheet-rich proteins. The local amyloid in joints of chicken and mammary corpora amylacea is discussed. Differences in glycosylation of glycoproteins among the APPs, as has been shown for α1-acid glycoprotein, have to be considered. More knowledge on the reactivity patterns may lead to implication of APPs in the diagnostics and staging of a disease. Calculation of an index from values of several acute phase variables increases the power of APPs in monitoring unhealthy individuals in animal populations. Vaccinations, just as infections in eliciting acute phase response seem to limit the profitability of vaccines because acute phase reactions are contraproductive in view of muscle anabolism. Interest is focused on amino acid patterns and vitamins in view of dietary nutrition effect on sick and convalescing animals.

When inexpensive methodology such as liquid phase methods (nephelometry, turbidimetry) or protein array technology for rapid APP measurement is available, APPs have a future in routine diagnostics. Specific groups of patients may be screened or populations monitored by using APP.

INTRODUCTION

The systemic acute phase reaction known to occur on infection, inflammation, trauma, burns, malignancies and tissue damage in general, has been studied by scientists from various disciplines. In the last decade, emphasis has been laid on application of blood tests for acute phase reactants to monitor animal health in general, as well as for human patients suffering from specified classes of diseases (Gruys et al., 2005). However, basic mechanistic patterns associated with biological reaction mechanisms, such as local production of acute phase proteins by cells of organs involved in specific physiological mechanisms and disease processes which may be associated with defence functions and with development of, even worse, tissue alteration, still remain to be uncovered.

Precolostrum mammary tissue (McDonald et al., 2001) and mastitic mammary epithelium have been shown to form mammary serum amyloid A (mSAA) (Vivanco et al., 2003; Nguyen et al., 2003). Moreover, haptoglobin (Hp) and other acute phase reactants are found in the milk. These factors are supposed to have functions in regulating the inflammatory process and to be beneficial for the enteric milieu of the young mammal including protection of the gut mucosa by mucus formation (Gruys et al., 2005).

In birds with chronic arthritis the synovial cells may reveal SAA upregulation, SAA protein formation (Ovelgönne et al., 2001; Upragarin et al., 2002; 2005a) and amyloid formation (Landman, 1998; Upragarin et al., 2005b).

This paper on the expected future developments of acute phase protein (APP) science and APP-related disorders is based on (1) present scientific activities concerning basic mechanistic patterns not discovered sofar, (2) development of technology for quantitative measurement of APPs or the cellular upregulation of their formation, and (3) assessment of disorders, health and welfare with APP values. Some topics in these fields will be mentioned.

BASIC MECHANISTIC PATTERNS

Specific organs

Differentiated reactivity patterns of parenchymal cells in the organ involved, such as mammary gland, depend on locally active cytokines and are unraveled in this paper. Moreover, cell specific factors may be revealed. Just like enzymes known from clinical enzymology, specific cell proteins such as the fatty acid-binding gut protein (Niewold et al., 2004), may be discovered and have applications in diagnostics and therapy.

Functional aspects of acute phase reactants in milk and blood need further attention. Knowledge of the activities which activate or just mitigate the inflammatory reaction can lead to new ways for therapy and prevention of inflammatory processes in organs involved, such as lung or mammary gland.

Extrahepatic, local SAA formation (Landman, 1998) and its possible precipitation as amyloid (Upragarin, 2005) as has been found in avian amyloid arthropathy, needs further investigations. From the pathogenetic mechanisms preventive measures may be developed. Once the beta-pleated sheets of amyloid have been formed, the substance has tremendous food safety implications. In murine studies orally administered AA-amyloid appeared to enhance inflammation-/acute phase reaction-induced amyloidosis, where the administered material served as nidus for amyloidogenesis. This indicates that like prions, this pathological material should be banned for risk groups of consumers (Lundmark et al., 2002 & 2003). In mammary tissue, colostrum and fresh milk corpora amylacea may occur which contain amyloid (described to be derived from casein (Niewold et al., 1999)). Recently this amyloid was found to be positive for SAA (Toussaint et al., 2005) and this possibly originates from mammary epithelium after in situ hybridization results (Toussaint et al., 2005). By others the local formation of mSAA has been mentioned (Larson et al., 2005). The amyloidotic corpora should be regarded as unwanted beta pleated factors in colostrum and milk and the relationship to this mSAA is recommended for futher investigation.

Various proteins

In birds acute phase proteins such as (ovo)transferrin appear to have special characteristics differing from those of mammals. In mammals and possibly in avian species as well, some acute phase proteins may reveal differences in glycosylation patterns associated with different diseases and stages of those diseases, as had been shown for feline α1-acid glycoprotein (AGP) (Ceciliani et al., 2004; Pocacqua et al., 2005). Further analysis may unravel basic biological mechanisms and indicate specificity of the glycosylation patterns for disease and reveal new concepts for therapy.

Reaction of different analytes in various situations, separate and combined

Viral, bacterial, and protozoal infections may be associated with different patterns in cytokine release and acute phase reactivity. Inflammatory processes in internal organs appeared to result in more severe reactivity patterns than diseases of the skin and the enteric system (Alsemgeest, 1994). Specific knowledge of pattern details may lead to implication of the parameters in diagnostics and staging of the disease.

Calculation of an index from values of rapid- and slow-reacting positive and negative APPs had been reported (Gruys and Toussaint, 2001; Gruys, 2002; Toussaint et al., 1995; 2002; 2004; Niewold et al., 2003), because it appeared to increase statistical sensitivity and specificity for detecting unhealthy subjects. It covers a broad time span and includes changes in blood values resulting from the body’s reactivity as well as starvation. In layer chickens, on contact with Staphylococcus aureus or turpentine, an acute phase protein reaction was induced. Measurement of values for SAA, transferrin, serum albumin and apolipoprotein A-1 in blood samples of these birds (Upragarin, 2005; Upragarin et al., 2005b) showed that calculation of an acute phase index for this species offers promising results in this species. Outcome was as has been calculated for cows with various diseases (Toussaint et al., 1995) and for pigs with a Streptococcus suis infection (Toussaint et al., 2002).

Infections and vaccination

To prevent spontaneous disease frequent vaccination is recommended. It has been shown, however, that upon vaccination an acute phase protein reaction may develop. This appears to limit the advantages of vaccines, because acute phase reactions are known to be contraproductive in view of muscle anabolism. Future interest will be more on amino acid pattern differences in muscle and APPs (Table 1) and on the negative acute phase reactants.

Table 1.

Major amino acid composition differences between muscle protein and two major acute phase proteins after a gross amino acid composition list (in arbitrary units) (Reeds et al., 1994)

Amino acid	Muscle protein	CRP	SAA
Phe	40	105	103
Tyr	36	50	67
Trp	13	42	45
Arg	69	36	116
Ala	59	31	106

CRP: C-reactive protein; SAA: serum amyloid A

On starvation and negative energy balance associated with most diseases, muscle proteins are catabolised for amino acid supply of the hepatic APP formation and as source of energy. Especially for those APPs which rapidly and quantitatively increase in blood, their formation may have amino acid impact. An increased hindquarter protein catabolism exceeding the hepatic protein synthesis, and efflux of glutamine and alanine from the hindquarter was measured during a porcine induced endotoxemia study (Bruins et al., 2003). For growth during and after recovery from a disease, food requirements for amino acids thus may differ from the formula in ordinary food. Some pig studies indicate positive influences of additional dietary tryptophan (Le Floc’h et al., 2004) or L-arginine (Bruins et al., 2002).

Negative APPs may be associated with a change in concentration of bound compounds. A decrease of retinol-binding protein and of vitamin A values may be vice versa interrelated, vitamin A-deficiency being well known to decrease immune reactivity of children in developing countries. It is astounding to encounter a huge negative variation from normal blood vitamin A values of around 1~0.75 μmol/L to around <0.1 μmol/L in fattening pigs, as was revealed in a local investigation by a Dutch veterinarian (Hogendoorn, 2004).

Association of APPs with parturition, starvation and ketosis in cattle has been described. Rise in non-esterified fatty acids (NEFAs) occurs; their level increase might parallel those of some APPs. It is to be expected that due to negative APPs, blood vitamin A levels decrease as suggested for the pig. The NEFAs are toxic and have negative influence on metabolism. It is hypothesized that the NEFAs may decrease with increased muscular activity (walking). An inverse association with walking activity had been shown (Adewuyi, 2004).

DEVELOPMENT OF TECHNOLOGY TO QUANTATIVELY MEASURE PROTEINS OR CELLULAR UPREGULATION OF THEIR FORMATION

Several groups, after many laboratories used radio-immunoassays (RIA) and enzyme-linked methodology (ELISA) for APP measurement in particular of C-reactive protein (CRP) in human hospitals, are developing methods for rapid measurements of APP values. Multiple immunoassays with simultaneous analysis of various different serum analytes have been recommended (Lukacs et al., 2005; Ray et al., 2005; Petrou et al., 2002). Different nephelometry systems are compared during application for human CRP (Maggiore et al., 2005). Turbidimetry is developed for APP in the dog (Kjelgaard-Hansen et al., 2003), horse (Jacobsen et al., 2005a; 2005c) and for the cat (Kjelgaard-Hansen et al., 2005). Other methods of liquid phase analysis with the option of recycling of the sample (Philips, 2001), such as those possible with Surface Plasmon Resonance (Biacore® system), have been tested for APP in bovine milk (Ǻkerstedt et al., 2005). Two-dimensional electrophoresis with mass spectrometry has been shown to be applicable to animal samples with the aim to measure acute phase reactants (Miller et al., 2004; 2005a) as has been done by others for analytes of human cancer growth and activity (Mian et al., 2005; Wang et al., 2005). A protein chip has been developed for measurement of haptoglobin and SAA in human patients (Tolson et al., 2004). Protein microarray methodology on slides has been proposed for APP in pigs (Toussaint et al., 2004). Preliminary experiments with a monoclonal anti porcine CRP and pig acute phase sera using methodology as described (Timmerman et al., 2004), offered the possibility to measure more than 1000 pig blood sample spots on a single slide.

Indirectly, acute phase protein formation may be measured in biopsies by methods to assess upregulation of protein synthesis (quantitative PCR). Especially the technique may be applied on samples after slaughter, or in histopathology and together with assessment of cytokines.

These technological developments may have crucial importance in the future if done rapidly, and at low costs, many samples can be handled, the APPs have a good future in diagnostics. This technique is for general assessment just as the erythrocyte sedimentation rate is used in internal medicine, but more sensitive, and for special groups of patients such as horses after castration (Jacobsen et al., 2005c) or laparotomy (Miller et al., 2005b).

ASSESSMENT OF DISORDERS, HEALTH AND WELFARE

Specific groups (Petersen et al., 2004; Murata et al., 2004), such as castrated horses (Alsemgeest, 1994; Jacobsen et al., 2005b), cows with inflammatory processes including mastitis (Alsemgeest, 1994; Eckersall et al., 2001), peri-parturient cattle (Alsemgeest et al., 1993; Alsemgeest, 1994; Koets et al., 1998), periparturient sows (Zhu et al., 2005), or dogs and cats with infectious disorders (Ceron et al., 2005) benefited from acute phase reactant measurement. In milk samples of cows (McDonald et al., 2001; Eckersall et al., 2001; Jacobsen et al., 2005d) and sheep and goats (Winter et al., 2005) mastitis may be monitored.

Combination of various APP values in an index increases the diagnostic power of the APPs to discern between normal animals in a population and non-healthy ones. After it was shown that an index combining values of fast and slow, positive and negative APPs can be used to assess human cancer patients (Ingenbleek and Young, 1994), such an index has been mentioned to assess health in populations of cattle, pig, dog and chicken (Toussaint et al., 1995; 2000; Martinez-Subiella and Ceron, 2005; Upragarin et al., 2005a; 2005b; Upragarin, 2005). Not only blood or milk samples can be used, meat juice (Petersen et al., 2005) and other body fluids such as saliva (Parra et al., 2005), are tested and found useful.

Involvement of multi-analysis technology in measuring the APPs and when coupled with pattern recognition software, the power of selective diagnostics of the APPs can increase.

Finally, because with respect to reaction of acute phase analytes in slaughter pigs (Heegaard et al., 2005) and in cows with experimental induction (Jacobsen et al., 2005d) individual variability is striking, in the future the APP reactivity pattern of an individual breeding animal may be used for selection on robustness.

CONCLUSION

Future possibilities for acute phase reactants depend on basic new mechanistic findings of known proteins, new discoveries such as organ specific components, and on technological possibilities for rapid immunological or chemical multi-analyses coupled with computer analysis of the patterns found. A shared cost European Union (EU) project (No. QLK5-2001-02219) on porcine acute phase proteins has helped to spread the knowledge to scientists of member states involved, and to develop a base for practical applications. For other species such as cattle, horse, dog and cat, but also chicken and even human, a similar field is open for development of clinical and health management applications. Finally, new fields of research and application are in the negative metabolic influences of acute phase processes and their relationship with growth and nutrition.