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.

Amyloid A in systemic amyloidosis associated with cancer.

Amyloid fibrils from two cases of cancer-associated, systemic amyloidosis with renal cell carcinoma and mesothelioma as the respective underlying disorders were studied. The immunochemical studies suggested strongly that amyloid A comprised a principal fibril component in both cases of cancer-associated amyloidosis. This was definitively proven by amino acid sequence analyses, which revealed structural homology between a purified subcomponent of the amyloid fibrils from both of the two cases of cancer-associated amyloidosis and previously sequenced amyloid A proteins. The chemical composition of the amyloid fibrils from systemic amyloidosis associated with cancer thus corresponded to that seen in amyloidosis reactive to inflammatory diseases and Hodgkin's disease. Amyloid proteins of immunoglobulin light chain type, which are found associated with myelomatosis, macroglobulinemia, and idiopathic (primary) amyloidosis, were not found in the two amyloid preparations. Renal cell carcinoma appears to be an effective stimulator of amyloid formation, while only one case of amyloidosis associated with mesothelioma has been reported previously.

Expression of serum amyloid A genes in mink during induction of inflammation and amyloidosis.

Serum amyloid A (SAA) is an acute phase protein and the precursor of amyloid protein A (AA) in deposits of secondary amyloidosis. Several isotypes exist in mink, but previous studies suggest that mink AA is derived from only one. To assess the effect of repeated episodes of inflammation and induction of amyloidosis, qualitative and quantitative changes in hepatic and extrahepatic SAA mRNA were studied. Young female mink received subcutaneous lipopolysaccharide injections for amyloid induction. Studies were performed using RNA probes and oligonucleotide probes specific for each of two SAA mRNA species. Northern blot hybridization showed that hepatic SAA1 and SAA2 mRNA levels increased dramatically after inflammatory stimulation, and were subsequently maintained at elevated levels, showing considerable interindividual variation, but only a slight decrease during repeated inflammatory stimuli and the early stages of amyloid deposition. No preferential accumulation of mRNA specifying a particular isotype was found during the experiment. Differential expression of mink SAA mRNA during repeated inflammatory stimulation does not seem to explain why only SAA2-derived AA is found in amyloid deposits. Extrahepatic SAA mRNA seemed to be independently regulated and may thus represent another, yet not characterized, SAA isotype.

Developmental regulation of expression of rabbit C-reactive protein and serum amyloid A genes.

Serum amyloid A (SAA) and C-reactive protein (CRP) are acute phase plasma proteins which increases 100- to 1000-fold after inflammatory stimuli. In this study pregnant rabbits were given lipopolysaccharide (LPS) or subjected to laparotomy with fetal injections of LPS at different stages of gestation. Newborn rabbits were given LPS or saline. SAA and CRP mRNA were studied using Northern blot analyses and scanning densitometry. In vitro transcribed RNAs were used as standards for quantitative mRNA analyses. A gradual increase in LPS-induced SAA and CRP mRNA levels was observed during development, but only SAA mRNA induction was seen at gestational day 19. Fetal SAA and CRP mRNA induction was not seen after maternal LPS stimulation. The constitutive level of SAA and CRP mRNA was significantly lower in fetal rabbits than in adults. The control level of SAA mRNA in one-day-old rabbits was higher than the normal adult level, while the neonatal CRP mRNA level was lower. SAA2 seemed to be the major acute phase reactant in both fetal, neonatal and adult rabbits, while relatively more SAA3 was found during early developmental stages. The study demonstrated that CRP and three SAA genes are differentially regulated during development.

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.

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.

Serum amyloid A protein in humans and four animal species: a comparison by two dimensional electrophoresis.

Two-dimensional electrophoresis and N-terminal analysis were used to study serum amyloid A protein (SAA) from humans, mink, fox, goat and rabbit. Previously uncharacterized SAA variants were demonstrated in fox, goat and rabbit, and considerable interspecies homology was seen. In rabbit, two novel SAAs were characterized, and SAA1 and SAA2 were demonstrated in mink and rabbit sera. The results confirm previous cDNA studies and indicate that SAA do possess an important function also in fox and goat.

Hypothalamic hamartoma causing precocious puberty treated by surgery: case report.

A 6-year-old girl was treated for precocious puberty secondary to a hypothalamic hamartoma by resection of the tumor. When she was six months old, her parents noticed incipient pubic hair and menses accompanied by breast development. Computed tomography was judged as normal. The girl was treated with monthly gonadotropin-releasing hormone analogue injections until 6 years of age, when magnetic resonance imaging (MRI) demonstrated a pedunculated isodense mass below the tuber cinereum. The hamartoma was totally removed using microsurgery. The symptoms and signs of precocious puberty disappeared after surgery. Follow-up MRI 1 year later showed no remaining tumor.

Dynamics in serum of the inflammatory markers serum amyloid A (SAA), haptoglobin, fibrinogen and alpha2-globulins during induced noninfectious arthritis in the horse.

Despite the importance of noninfectious joint diseases in equine medicine, little is known about the acute phase response which may be elicited if the local inflammatory process of noninfectious arthritis is sufficiently strong, Therefore the aim of this study was to monitor the systemic inflammatory response during experimentally-induced noninfectious arthritis by studying the dynamics in serum of the acute phase proteins serum amyloid A (SAA), haptoglobin, fibrinogen and alpha2-globulins. Twenty-four Standardbred horses, age 3-7 years, found healthy on thorough clinical, radiological, haematological and serum biochemical examination, were injected aseptically into the right midcarpal joint with amphotericin B. Blood samples were drawn before induction of arthritis (0 h), and at 8, 16, 24, 36 and 48 h postinduction and then on Days 3, 4, 5 and 15 postinduction. All horses developed lameness with joint effusion and joint heat as well as increased respiratory rate, heart rate and body temperature. The lameness started to decline after 24-36 h and, in most animals, systemic signs disappeared on Day 2 postinjection. The concentration of the acute phase proteins increased following induction of arthritis. The SAA concentrations were higher than baseline concentrations from 16 h postinduction and were maximal at 36-48 h (227 times baseline concentration). The haptoglobin concentrations were higher than baseline concentrations from 24 h and were maximal at 48-96 h (1.14 times baseline concentration). The maximal concentrations of fibrinogen were seen between 36-72 h postinjection and increased on average 0.87 times from baseline concentrations. The fibrinogen concentrations were higher than baseline concentrations from 24 h postinjection. Alpha2-globulins concentrations showed a minor increase and increased 0.55 times from baseline concentrations. The markers had returned to baseline concentrations by Day 15. Our results demonstrate that amphotericin B-induced arthritis in a single joint gives rise to a systemic acute phase response measurable as increased concentrations in serum SAA, haptoglobin, fibrinogen and alpha2-globulins during the first 2 weeks of the condition and, thereby, that such an increase need not be indicative of infectious arthritis. Further research should be aimed at determining whether chronic noninfectious arthritis in the horse gives rise to increased acute phase protein concentrations in serum.

The acute phase protein serum amyloid A (SAA) as an inflammatory marker in equine influenza virus infection.

The acute phase protein serum amyloid A (SAA) has proven potentially useful as an inflammatory marker in the horse, but the knowledge of SAA responses in viral diseases is limited. The aim of this study was to evaluate SAA as a marker for acute equine influenza A2 (H3N8) virus infection. This is a highly contagious, serious condition that inflicts suffering on affected horses and predisposes them to secondary bacterial infections and impaired performance. Seventy horses, suffering from equine influenza, as verified by clinical signs and seroconversion, were sampled in the acute (the first 48 h) and convalescent (days 11-22) stages of the disease, and SAA concentrations were determined. Clinical signs and rectal temperature were recorded. Secondary infections, that could have influenced SAA concentrations, were clinically suspected in 4 horses. SAA concentrations were higher in the acute stage than in the convalescent stage, and there was a statistically positive relationship between acute stage SAA concentrations and clinical signs and between acute stage SAA concentrations and maximal rectal temperature. Horses sampled early in the acute stage had lower SAA concentrations than those sampled later, indicating increasing concentrations during the first 48 h. There was a statistically positive relationship between convalescent SAA concentrations and degree of clinical signs during the disease process. The results of this investigation indicate that equine SAA responds to equine influenza infection by increasing in concentration during the first 48 h of clinical signs and returning to baseline within 11-22 days in uncomplicated cases.

Three assays for the characterization and quantitation of human serum amyloid A.

Two different radioimmunoassays (RIA) and an enzyme-linked immunosorbent assay (ELISA) were developed for the quantitation and antigenic characterization of amyloid A (AA) and serum amyloid A (SAA) proteins, and the three assays were evaluated and compared with each other. Sensitivity, reproducibility, effect of denaturation and storage of serum and range of determination were considered. All three assays were found useful, but for different purposes. The most suitable method for the determination of SAA in whole serum was a second antibody precipitation RIA with purified SAA as labelled tracer and standard, and polyclonal rabbit anti-SAA as first antibody. This assay provided SAA concentrations in absolute amounts (mg/l) and acceptable reproducibility without need for prior denaturation of serum. Both advantages and disadvantages of ELISA using monoclonal antibodies to SAA and a solid-phase RIA using AA, SAA, anti-AA and anti-SAA were observed. The three assays were found suitable for antigenic studies of AA and SAA.

Idiopathic juvenile osteoporosis.

Osteoporosis is the most common bone disease in adults, but rarely occurs in children. A seven year old boy with idiopathic juvenile osteoporosis is reported. X-ray investigation revealed moderate generalized osteoporosis with compression fractures and wedging of the thoracic and lumbar vertebral bodies. Clinical examination and biochemical investigations ruled out the known causes of osteoporosis in childhood. During the following four years he complained of back pain, but no new fractures appeared. Entering puberty he improved both clinically and radiologically without treatment.

Characterization of human amyloid-related protein SAA as a polymorphic protein: association with albumin and prealbumin in serum.

Human amyloid-related protein SAA has been prepared and purified by gel filtration, ion-exchange and affinity chromatography techniques. It was shown that SAA, even after extensive purification, is an electrophoretically heterogeneous protein. In addition, prealbumin and fragments of albumin were detected in the SAA preparation. Most of the SAA molecules and the fragments of albumin were present in a free form, but some SAA was also found to be complexed with albumin fragments.

Fragments of IgE antibodies in human feces.

The gel filtration profile of immunoglobulin E (IgE) in extracts of feces from 2 children was compared with IgE myeloma protein which had been exposed to proteolytic digestion by chymotrypsin. The peak of the chymotrypsin-digested IgE myeloma protein was found to be similar to that of fecal IgE after an elution volume between those of albumin and myoglobin, corresponding to a molecular weight of approximately 40,000 daltons. In the fractions where the peak of fecal IgE was found, no evidence for the presence of specific IgE antibodies (measured by RAST) could be detected. Fecal IgE could be purified by using an immunosorbent column to which rabbit antihuman IgE was coupled. Sufficient amounts of fecal IgE could thus be obtained and used in autoradiographic experiments. The IgE-containing fractions could also be detected with 125I-labelled second antibodies to visualize the IgE precipitates.

Amyloid-related serum protein (SAA) during and after pregnancy in healthy women and women with rheumatic disease.

The usefulness of amyloid-related serum protein (SAA) as an indicator of disease activity has been evaluated in 11 patients with rheumatoid arthritis (RA), 2 patients with psoriatic arthritis (PA) and 13 patients with ankylosing spondylitis (AS) prospectively studied during and after pregnancy. For comparison, SAA levels were recorded serially during and after pregnancy in 28 healthy pregnant women. SAA levels were unaltered by gestation and thus within the normal range during normal pregnancy, but were raised in healthy pregnant women with episodes of intercurrent infections. In RA and AS patients, SAA concentrations correlated to disease activity during and after pregnancy. Serial levels of SAA and C-reactive protein in healthy women and patients paralleled each other with the most pronounced inflammatory response displayed by SAA. We conclude that SAA is a sensitive and reliable indicator of inflammatory events both in the pregnant and non-pregnant state.

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 lipopolysaccharide-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.

Serum amyloid A: an acute phase apolipoprotein and precursor of AA amyloid.

Serum amyloid A is an acute phase protein complexed to HDL as an apoprotein. The molecular weight is 11.4-12.5 kDa in different species and the protein has from 104 to 112 amino acids, without or with an insertion of eight amino acids at position 72. The protein is very well conserved throughout evolution, indicating an important biological function. The N-terminal part of the molecule is hydrophobic and probably responsible for the lipid binding properties. The most conserved part is from position 38 to 52 and this part is therefore believed to be responsible for the until now unknown biological function. The protein is coded on chromosome 11p in man, and chromosome 7 in mice, and found in all mammals until now investigated, and also in the Peking duck. In the rat a truncated SAA mRNA has been demonstrated, but no equivalent serum protein has been reported. Acute phase SAA is first of all produced in hepatocytes after induction by cytokines, but extrahepatic expression of both acute phase and constitutive SAA proteins have been demonstrated. Several cytokines, first of all IL-1, IL-6 and TNF are involved in the induction of SAA synthesis, but the mutual importance of these cytokines seems to be cell-type specific and to vary in various experimental settings. The role of corticosteroids in SAA induction is somewhat confusing. In most in vitro studies corticosteroids show an enhancing or synergistic effect with cytokines on SAA production in cultured cell. However, in clinical studies and in vivo studies in animals an inhibitory effect of corticosteroids is evident, probably due to the all over anti-inflammatory effect of the drug. Until now no drug has been found that selectively inhibits SAA production by hepatocytes. Effective anti-inflammatory or antibacterial treatment is the only tool for reducing SAA concentration in serum and reducing the risk of developing secondary amyloidosis. The function of SAA is still unclear. Interesting theories, based on current knowledge of the lipid binding properties of the protein and the relation to macrophages, in the transportation of cholesterol from damaged tissues has been advanced. A putative role in cholesterol metabolism is supported by the findings of SAA as an inhibitor of LCAT. The potential that SAA is a modifying protein in inflammation influencing the function of neutrophils and platelets is interesting and more directly related to the inflammatory process itself.(ABSTRACT TRUNCATED AT 400 WORDS)

The covalent structure of amyloid-related serum protein SAA from two patients with inflammatory disease.

The complete covalent structure of an amyloid-related serum protein SAA from a patient (Jen.) with severe rheumatoid arthritis, is presented. The structure was elucidated by N-terminal analyses of the protein as well as on peptides derived from tryptic digestion and after cleaving the protein with BNPS-skatole. The characterization of tryptic peptide T-9 revealed a polymorphism similar to that seen in protein AA. Structural studies performed on another protein SAA, isolated from a patient (Mik.) with acute systemic lupus erythematosus, indicated that this protein is homologous to that from patient Jen. The formation and deposition of the protein AA-containing amyloid fibrils is discussed.

Amyloid-related serum protein (SAA) as an indicator of lung infection in cystic fibrosis.

Amyloid-related serum protein (SAA) was analysed by radioimmunoassay in 32 patients with cystic fibrosis, and compared with other acute phase reactants and lung function. The level of SAA showed significant correlation with impaired lung function due to active Pseudomonas aeruginosa infection, and also to C-reactive protein. SAA seemed to correlate better to the presence of bacteria in sputum than C-reactive protein. Ten of the patients received extensive antibiotic treatment for their pulmonary infection, and falling serum levels of SAA paralleled the clinical response to treatment. Thus the concentration of SAA in these patients was a valuable guide for the selection of patients for antibiotic treatment as well as a good parameter of the response to therapy.