Studies of acute phase proteins and tumour necrosis factor receptors as inflammatory markers in the cat
The measurement of acute phase proteins is used by human clinicians to give valuable infonnation about a patient's inflammatory response, both when monitoring clinical disease and when assessing the effect of therapy. Levels of soluble receptors for the cytokine, tumour necrosis factor, also increase as a result of inflammatory stimuli and are useful prognostic markers over the asymptomatic phase of human immunodeficiency virus infection. The aim of the work presented in this thesis was to detennine whether these markers are of value when investigating feline disease. Reference ranges for two acute phase proteins, aI-acid glycoprotein (AGP) and haptoglobin were detennined by measuring their concentrations in serum samples from healthy cats. Analysis of samples from cats with feline infectious peritonitis (FIP) and from cats suffering from conditions with a similar clinical presentation revealed that measurement of AGP can be a useful adjunct to other laboratory tests when reaching a diagnosis. In contrast, measurement of haptoglobin was not found to be of value. Despite increases in the levels of pro-inflammatory cytokines in samples taken from cats during the asymptomatic phase of feline immunodeficiency virus (FIV) infection, no changes were detected in the levels of AGP and haptoglobin. It was concluded that these acute phase proteins are of no benefit as prognostic markers in FlY. The L929 bioassay was used to investigate anti-TNF-a activity in cell culture fluids from feline splenic cells. Cytotoxic activity was demonstrated in very few of the samples whilst anti-cytotoxic activity was detected in the majority of samples. This anti-cytotoxic activity was attributed to the presence of feline soluble TNF receptor type 1 (sTNFR-I) binding to and inhibiting the effects of TNF-a. This was not confinned because of the lack of specific neutralising antibody. Subsequent work was therefore directed towards the development of immune-based species-specific assays for feline soluble TNF receptors (sTNFRs). The polymerase chain reaction was used to amplify the sequences coding for feline sTNFRs. Most of the extracellular domain of feline TNFR-l and part of the intracellular domain of feline TNFR-2 were cloned and sequenced using this technique. The amplified regions demonstrated 85% and 77% homology at the nucleic acid level and 83% and 67% homology at the amino acid level to the corresponding regions of the human sequences for TNFR-l and 2 respectively. Feline sTNFR-l was expressed as a glutathione-S-transferase fusion protein. After purification, concentration and electrophoresis, the appropriate protein band was excised and used to inoculate a sheep. Antiserum taken from the sheep post-inoculation recognised the expressed protein by western blotting, but results were inconsistent and analysis of the antiserum was hampered by the very small amounts of expressed protein available. Two peptides were synthesised based on regions of antigenicity in feline sTNFR-l and were used to inoculate sheep. Antiserum to peptide A showed a strong reaction against peptide A in an ELISA and gave a positive result when used as the primary antibody to stain healthy feline liver tissue. In conclusion, both antiserum to expressed feline sTNFR-l and anti-peptide antibody based on a region of feline sTNFR-l have been raised in sheep and are available for the development of an assay for this protein. Further expression of feline TNFR-l will be required before these antisera can be analysed fully.