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.

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.

Construct validity of ILAR and EULAR criteria in juvenile idiopathic arthritis: a population based incidence study from the Nordic countries. International League of Associations for Rheumatology. European League Against Rheumatism.

OBJECTIVE: New classification criteria (ILAR) have been proposed for juvenile idiopathic arthritis (JIA). They are more descriptive than those formerly used [American College of Rheumatology (ACR), European League Against Rheumatism (EULAR)], but require validation against classifications already in use. We validated the ILAR criteria in relation to the EULAR criteria in a prospective, incidence, and population based setting, and analyzed their feasibility. METHODS: Construct validity of ILAR and EULAR classification criteria refers to how closely the 2 instruments are related and how each of them operates in classifying subgroups/categories. Twenty doctors in 5 Nordic countries collected data from the incidence cases within their catchment areas during an 18 month period beginning July 1, 1997. Clinical and serological data from the first year of disease were collected. RESULTS: A total of 322 patients were included. Classification according to the ILAR criteria was possible in 321 patients; 290 patients had a disease duration > or = 3 months and were classified according to the EULAR criteria. One child could only be classified according to the EULAR criteria. Thus, 31/322 (9.6%) children were classified according to the ILAR criteria only. Forty-eight of 321 (15%) patients did not fit into any category and 6% (20/321) fulfilled criteria for2 categories. In the ILAR classification 5 out of 7 categories/subgroups have 2 to 5 specified exclusion criteria that highly discriminate the definition of each patient. In our study the exclusion criteria were fulfilled to only a small extent. CONCLUSION: The EULAR and ILAR criteria differ concerning the operational definitions of the subvariables involved, which complicates their comparison. By using ILAR rather than EULAR criteria the number of cases with juvenile arthritis increased by 10%, considering the first half-year after onset. The validity of the ILAR criteria is low since they often exclude patients from subgroup classification and the possibility of having more than one diagnosis is not negligible. The specified exclusion criteria for some of the subgroups are difficult to fulfill in clinical work and variables involved could be questioned with regard to their consistency.

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.

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 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.

A two-dimensional electrophoretic study of serum amyloid A and C-reactive protein in infants and children.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to analyze C-reactive - (CRP) and serum amyloid A protein (SAA) in infants and children. Five SAA isotypes were identified. CRP showed vertical streaking, and its optical density values correlated with immunoturbidimetric measurements. As evaluated by densitometry, both proteins showed an age-dependent variation. In more than 50% of the neonates, SAA was present in equal or higher amounts than CRP, and only SAA1alpha could be detected. In children, CRP was expressed in higher amounts than SAA, and both SAA1alpha and SAA2alpha were present. N-terminally modified forms of both isotypes were present regardless of age, including in premature infants. These results suggest that the overall synthesis of the gene products SAA1alpha and SAA2alpha is developmentally regulated, but at the same time that their N-terminal processing occurs independently of developmental factors. The presented data suggest that SAA has an important function in neonates, and that the role of SAA as an infection marker in this population should be investigated further.

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 apoSAA and apoSAA2/SAA(SJL/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-gamma) 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.

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.

Serum amyloid A gene expression in rabbit, mink and mouse.

The expression of serum amyloid A (SAA) protein, a major acute-phase reactant in most species, was examined by in situ hybridization in multiple organs of rabbit, mink and mouse. In livers of unstimulated mice and rabbits a heterogeneous pattern of SAA expression in hepatocytes was observed. In all three species, lipopolysaccharide (LPS) administration resulted in extensive uniform hybridization of SAA probes to hepatocytes and in the rabbit SAA transcripts were detected in cells in the white pulp of the spleen, the adrenal cortex and ovary as well as in the mucosa and lymphatic vessels of the small intestine. Examination of hybridizing SAA signals in the rabbit myocardium showed a speckled distribution in myocytes. The rabbit endocardium was strongly positive, and in the kidney rabbit SAA mRNA was mainly confined to epithelial cells of the proximal and distal convoluted tubules. In the unstimulated mouse, SAA mRNA was detected in the liver and epithelial cells of the small and large intestine. After stimulation of an acute-phase response with LPS a strong response was seen in these organs as well as in the convoluted tubules of the kidney. In extrahepatic organs of the mink, no SAA mRNA was detectable in unstimulated animals, while the convoluted tubules of the kidney and uterine endometrium were strongly positive after systemic LPS injection.

Isolation of serum amyloid A protein by small-scale hydrophobic interaction chromatography and two-dimensional electrophoresis.

A recently introduced technique to isolate serum amyloid A protein is hydrophobic interaction chromatography combined with two-dimensional electrophoresis with immobilized pH gradients. A modification of the original version of this technique is presented. Mouse serum was subjected to hydrophobic interaction chromatography on a small scale, and the eluate was applied directly to two-dimensional electrophoresis. Simple electropherogramss with optimal resolution of serum amyloid A protein were obtained. The presented technique facilitates isolation of serum amyloid A protein from small blood volumes, and might also be adapted to alternative applications.

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.

Characterization of human serum amyloid A protein isoforms separated by two-dimensional electrophoresis by liquid chromatography/electrospray ionization tandem mass spectrometry.

A detailed structural analysis of the serum amyloid A proteins (SAA) of an individual with highly active, chronic rheumatoid arthritis is reported. SAA isoforms were separated by high-resolution two dimensional (2-D) gel electrophoresis. Peptide mapping by reverse-phase chromatography/electrospray ionization tandem mass spectrometry was applied to correlate the protein(s) contained in each spot with their respective coding gene and to study the post-translational processing and modification events which might result in differential electrophoretic mobility. Nine protein spots were analyzed. The six major spots corresponded to the Arg and des-Arg forms of SAA1 alpha and SAA2 alpha, respectively, and to the glycosylated and nonglycosylated form of constitutive serum amyloid A protein (C-SAA). Two minor spots were identified as SAA1 alpha isoforms containing post-translational modifications. We suggest that these variants contained a gamma-N, N'-dimethylasparagine residue at position 83 and that one of them was additionally oxidized at Trp53 and Trp85. The ninth spot was shown to contain a mixture of SAA1 alpha and SAA2 alpha. To our knowledge, this is the first report in which analysis of peptides has been used to verify the presence of C-SAA in acute-phase serum. Furthermore, the data illustrate that extensive post-translational processing results in a structurally diverse class of acute-phase SAA proteins, which are derived from a small number of genes. Finally, the fast and conclusive technology used in this study promises to be generally useful for the comprehensive investigation of proteins at the level of the primary structure.

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.

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 lipopolysaccharide-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 identified 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 amyloidogenic in mink.

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)

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.

Interaction between circulating amyloid fibril protein precursors and extracellular tissue matrix components in the pathogenesis of systemic amyloidosis.

Amyloidosis is a heterogeneous group of diseases characterized by deposition of a fibrillar, proteinaceous material, amyloid, in various tissues and organs. Increasing knowledge about the different proteins that constitute the amyloid fibrils has made it possible to classify amyloidosis by the fibril protein, which appears more rational than the traditional classification by its clinical expression. A serum protein is the precursor of the amyloid fibril protein in the various systemic forms of amyloidosis. Although the chemical composition of amyloid is presently well known, the pathogenetic processes that convert such proteins into a fibrillar form and lay them down in the tissues are far from clarified. We suggest some pathogenetic mechanisms for amyloid deposition, involving different types of fibril protein, their precursors, the extra-fibrillar amyloid P component, glycosaminoglycans, proteoglycans, and calcium with special reference to experimental work from our research group.

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 polyacrylamide 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.