Gudmund Marhaug

Serum amyloid A (SAA): biochemistry, genetics and the pathogenesis of AA amyloidosis

Serum amyloid A (apoSAA) is a polymorphic protein encoded by a family of SAA genes in which new members continue to be identi3ed. Those isoforms or allelic forms that are precursors for the amyloidfibril protein A(AA) in reactive (secondary) amyloidosis, are among the acute phase or regulated apoSAA apoproteins complexed with high densig lipoprotein (HDL); other isoforms are expressed constitutively. The chieffunction of apoSAA, which is a well conserved protein found in birds and mammals, has probably not yet been disclosed, but is thought to be part of the host response to infections and tissue damage. Structural and functional aspects of apoSAA. and its role in AA amyloidosis are complex due to the presence of multiple isoforms. Most species appear to possess two main acute phase apoSAA isoforms of hepatic origin in their serum (apoSAA, and apoSAAJ and a third form that has predominant extrahepatic expression (apoSAAJ A single constitutive isoform, apoSAA,, has been identijied in human and apoSAA, has been described in BALB/c mice. Thus, apoSAA proteins can be regarded or subclassified as either acute phase reactants (regulated apoSAA also termed A-SAA) or constitutive proteins (C-SAA).

Mouse serum amyloid A (SAA) proteins isolated by two-dimensional electrophoresis: characterization of isotypes and the effect of separate and combined administrations of cytokines, dexamethasone and lipopolysaccharide (LPS) on serum levels and isotype distribution

Hydrophobic interaction chromatography and two‐dimensional electrophoresis were used to isolate and characterize mouse SAA, and to study the in vivo effect of separate or combined administrations of cytokines, dexamethasone (DEX) and LPS on mouse SAA. Four SAA spots containing partial amino acid sequence in accordance with mouse apoSAA1 and apoSAA2/SAASJL/J pI 5.9 were demonstrated in serum. One of these proteins represents a previously undescribed, acidic acute‐phase mouse SAA protein. Both DEX and interferon‐gamma (IFN‐γ) proved to be capable of increasing SAA serum levels. In contrast to what has been shown in previous in vivo studies, administration of IL‐6 did increase the SAA levels to nearly the same magnitude as IL‐1, and the effect of IL‐6 and LPS on SAA production was not significantly altered by the addition of DEX. Irrespective of the inflammatory stimuli that was administered, a non‐selective production of SAA1 and SAA2 was observed in most groups, including the group that received IL‐6. The results illustrate that data obtained about mouse SAA are highly dependent on which models, isolation and identification methods are used.

A non-competitive chemiluminescence enzyme immunoassay for the equine acute phase protein serum amyloid A (SAA) -- a clinically useful inflammatory marker in the horse.

A non-competitive chemiluminescence enzyme immunoassay for measuring serum amyloid A (SAA) in equine serum was developed. A polyclonal anti-equine-amyloid A antiserum specific for equine SAA was utilized, and the assay was standardized using highly purified equine SAA. An acute phase horse serum was calibrated against the purified SAA and was used as standard when running the assay. Serum SAA concentrations in the range of 3-1210 mg/l could be measured. The reference range of SAA in clinically healthy adult horses was <7 mg/l. The clinical validation of the assay comprised the SAA responses after surgery and experimentally induced aseptic arthritis, and those associated with viral and bacterial infections. The SAA response after surgery (castration) was consistent, with peak concentrations on day 2 and a return to normal SAA concentrations within eight days. The aseptic arthritis produced an SAA response with a pattern similar to that seen after surgery, with peak concentrations of SAA 36-48 h after induction. Seven horses showed a biphasic pattern, with a second rise in SAA concentrations on day 4 and 5. All animals had SAA levels <7 mg/l on day 15. All horses with viral and bacterial infections had SAA concentrations above 7 mg/l. The ranges of SAA concentrations following the different types of inflammation overlap, being consistent with the unspecific nature of the SAA response. This study revealed that SAA is a sensitive and unspecific marker for inflammation, and describes the dynamics of the SAA response after standardized and well defined tissue damage.

The Primary Structure of Serum Amyloid A Protein in the Sheep: Comparison with Serum Amyloid A in Other Species

Serum amyloid A (SAA) protein was isolated from acute phase sheep sera by ultracentrifugation, gel filtration and ion‐exchange chromatography. The purified protein was characterized by sodium dodecylsulfate polyacrylamidc gel electrophoresis (SDS‐PAGE), isoelectric focusing, amino acid composition and Edman degradation. Protein SAA sheep consists of 112 amino acid residues and has a blocked N‐terminus. The amino acid sequence showed a high degree of homology with SAA proteins from other species, especially at positions 32 to 54, indicating that this particular part of the protein is important for its function. When compared to human protein SAA, nine inserted amino acids could be demonstrated, located in regions 69 to 77. Similar observations have been seen in cow, horse, dog, cat, and mink protein SAA. Heterogeneities were found in positions 28, 55, 63, 64, 66, 75, 77, 78, 80 and 89. Positions 63, 64, 66, 75, 77, 78 and 80 revealed the existence of a minor gene product of protein SAA sheep. The minor variant of protein SAA sheep is identical in these positions with the corresponding positions in protein SAA cow. By comparing the amino acid sequences of the different SAA proteins, two separate branches in the evolutionary pattern of protein SAA appear. One of the branches includes the species with the insertion which represents also one of the more heterogeneous part of the protein.

Characterization of amyloid protein AA and its serum precursor SAA in the horse.

Amyloid was extracted from the liver of a horse that had developed amyloidosis after being used for several years for the production of antibodies to bacterial antigens. The amyloid fibrils were shown to he of the AA type. Two AA proteins with molecular weights of 9000 and 11,000 and with identical partial N‐terminal amino acid sequences were identified. Marked structural homology with AA from other species including man was seen, although clear species‐related antigenic specificity was observed. SAA isolated from an acute phase (septic abortion) horse serum was identical to AA with respect to antigenicity and the 10 first N‐terminal amino acid residues that have been studied up to now. The hulk of SAA was present in the high‐density lipoprotein complex in serum. Also SAA was heterogeneous with respect to size, most molecules having a molecular weight of 11,000, and a minority 9000.

Differential Expression of Rabbit Serum Amyloid A Genes in Response to Various Inflammatory Agents

Serum amyloid A (SAA) is an acute‐phase plasma protein which increases up to 1000‐fold after an acute‐phase stimulus. Several SAA genes and corresponding protein isotypes exist in individual species. Liver is the main source of production, but extra‐hepatic SAA expression has been described. In this study inflammation was induced in rabbits with lipopolysaccharide, turpentine, or casein. Transcription of SAA mRNA was studied using Northern blot analysis with probes specific for three different rabbit SAA isotypes and analysed by scanning densitometry. In the stimulated liver slight variation in SAA mRNA transcription level was seen after stimulation with different inflammatory agents. After lipopolysacchar‐ide‐stimulation SAA gene expression was also seen in most of the extra‐hepatic organs. After turpentine stimulation SAA mRNA was seen in the liver, the ovary, and the small intestines, and after casein stimulation only in the liver and the ovary. SAA1 and SAA2 were induced exclusively in the liver, while SAA3 was induced mainly in the extra‐hepatic organs. This indicates that the SAA genes probably are independently regulated both in relation to stimulus, gene‐ and tissue‐specificity.

The acute phase serum amyloid A protein (SAA) in the horse: isolation and characterization of three isoforms

Serum amyloid A (SAA) from acute phase horse serum was isolated using hydrophobic interaction chromatography, gel filtration and ion exchange chromatography. Three SAA isoforms with different isoelectric points, i.e. SAA pI 8.0, SAA pI 9.0 and SAA pI 9.7, were identified by two-dimensional electrophoresis and further characterized with amino acid sequence analysis. These isoforms were found in similar concentrations in all animals investigated, with SAA pI 9.7 constituting about half of the total SAA content. Partial amino acid sequence analysis verified the previously published heterogeneous SAA sequence. SAA pI 8.0 was found to have isoleucine in Position 16, glutamine in Position 44 and glycine in Position 59. SAA pI 9.0 had leucine, glutamine and alanine in the corresponding positions. In SAA pI 9.7 leucine, lysine and alanine were detected. The three isoforms characterized in this study are all acute phase SAAs. SAA pI 9.0 and 9.7 correspond to amyloid A protein variants previously isolated from amyloid deposits of equine liver, while there are no reports on an amyloid A variant corresponding to SAA pI 8.0.

Age-dependent inhibition of ectopic calcification: a possible role for fetuin-A and osteopontin in patients with juvenile dermatomyositis with calcinosis

OBJECTIVES To assess if age and/or age-dependent variations in the levels of two major calcification regulatory proteins, fetuin-A and osteopontin, could be associated with an increased risk of calcinosis in children with juvenile dermatomyositis (JDM). METHODS The frequency of calcinosis was derived from a national UK database of 212 cases of JDM. Serum fetuin-A and plasma osteopontin levels were determined using ELISA in 15 JDM patients with calcinosis and 15 JDM patients without calcinosis. Healthy controls were 19 age-matched children, 24 adolescents and 13 adults. Sixteen patients with juvenile idiopathic arthritis (JIA) were additional paediatric disease controls. RESULTS Of the 212 JDM cases 10% had calcinosis. Calcinosis patients had younger age of disease onset than those without calcinosis (mean age of 5.3 yrs vs 7.1 yrs, respectively, P = 0.016). No significant difference in fetuin-A or osteopontin could be detected between the two JDM groups. Fetuin-A levels in all groups of children and the adolescent group were much lower than described previously in adults, and there was a significant positive correlation between age and fetuin-A level, and also between osteopontin levels in plasma and serum fetuin-A. CONCLUSIONS Children who develop JDM at an younger age may have increased risk of developing calcinosis. Physiologically low levels of fetuin-A in young children combined with an additional negative acute-phase effect on fetuin-A due to chronic inflammation could explain in part the propensity to develop ectopic calcification observed in JDM patients, and why calcinosis is less frequent in adults with dermatomyositis.

Chorea in juvenile primary antiphospholipid syndrome : Reversible decreased circulation in the basal ganglia visualised by single photon emission computed tomography

Chorea was observed in a 12-year-old girl with primary antiphospholipid syndrome (APS). She developed severe chorea in a few weeks. On immunosuppressive treatment, including high doses of glucocorticoids and cyclophosphamide, she had a rapid clinical recovery. Single photon emission computed tomography (SPECT) of the brain showed decreased circulation in the basal ganglia and in the medial parts of both temporal lobes. One month after treatment, SPECT was completely normalised. APS in children has a variety of clinical manifestations, and should be suspected in cases of unexplained thromboembolic disease or obscure neurological symptoms.

SERUM AMYLOID A PROTEIN IN MINK DURING ENDOTOXIN INDUCED INFLAMMATION AND AMYLOIDOGENESIS

Two‐dimensional electrophoresis was used to study SAA and AA proteins in mink during lipopoly‐saccharide‐induced inflammation and amyloidogenesis. Three isotypes, SAA pI 6.8 and SAA pI 6.5 (both SAA1‐like), and SAA pI 6.0 (SAA1‐ and SAA2‐like), were identifled in serum after both single and multiple LPS injections. Total SAA serum levels were highest in the early phase of induction, followed by a decrease ranging from 1 to 50% of the peak value during the rest of the experiment. The variation in the total SAA levels correlated with the total SAA mRNA levels. Low total SAA levels were seen both in non‐amyloidotic and amyloidotic animals, and a general decrease of all isotypes was demonstrated. In hepatic amyloid fibrils, several AA isotypes, with amino acid sequence homologous exclusively to that of SAA2, were found. In the corresponding splenic material, fragments of histones H2A and H2B constituted most of the low molecular mass proteins, and no protein AA was detected. In spite of low serum levels and a non‐specific isotype removal, the results confirm that SAA2 is amyloidogenie in mink.