The interaction between apolipoprotein serum amyloid A and high-density lipoprotein

Serum amyloid A (SAA) is a small apolipoprotein that binds to high-density lipoproteins (HDLs) via its N-terminus. The murine isoform SAA2.2 forms a hexamer in solution and the N-terminus is shielded from the solvent. Therefore, it is unclear how the SAA2.2 hexamer might bind HDL. In this study, the binding of SAA2.2 to murine HDL was investigated by glutaraldehyde cross-linking and polyacrylamide gel electrophoresis. The hexamer did not bind HDL significantly at 20 degrees C. However, at temperatures between 25-30 degrees C, SAA2.2 became destabilized and its monomeric form bound to HDL. SAA2.2 binding did not significantly replace Apo A-I in HDL particles. At 37-45 degrees C SAA2.2 binds less to HDL, suggesting that its binding is weak and sensitive to physiological and pathological temperatures, and thereby, potentially modulated, in vivo, by other factors.     

Usefulness of C-reactive protein, serum amyloid A component, and haptoglobin determinations in bitches with pyometra for monitoring early post-ovariohysterectomy complications

The aim of this study was to assess changes in serum concentrations of C-reactive protein (CRP), serum amyloid A component (SAA), and haptoglobin (Hp) in bitches with pyometra undergoing ovariohysterectomy that developed postoperative wound infection-related complications. The study revealed that difficulties in postoperative wound healing were induced by infections with Escherichia coli and Staphylococcus spp. leading to re-increased levels of CRP and SAA immediately after surgery and persistently high Hp levels throughout the experiment. Our results indicate that acute-phase proteins in bitches undergoing surgery because of pyometra are useful markers for monitoring the postoperative period. Moreover, they enable prompt therapeutic management once complications develop.     

Concentrations of C-reactive protein,serum amyloid A,and haptoglobin in uterine arterial and peripheral blood in bitches with pyometra

Pyometra is a life-threatening reproductive disorder that affects the uterus of female dogs. This study was designed to identify the possible indicators of uterine inflammation by comparing C-reactive protein (CRP), serum amyloid A (SAA), and haptoglobin (Hp) concentrations in uterine arterial and peripheral venous blood in bitches with open- and closed-cervix pyometra. CRP, SAA, and Hp concentrations were higher in bitches with closed-cervix pyometra irrespective of the site of blood collection. Higher acute-phase protein concentrations were observed in peripheral compared with uterine arterial blood in bitches with closed-cervix pyometra, whereas the levels were comparable in dogs with open-cervix pyometra. Our results indicate that mean acute-phase protein concentrations differ according to pyometra type/severity and blood source and suggest the possible use of peripheral blood levels of CRP, SAA, and Hp to monitor inflammation during the course of pyometra.     

A study of extracellular matrix-cell adhesion peptidic epitopes related to human serum amyloid A (SAA)

Summary Serum amyloid A (SAA), an acute-phase protein, exists normally in the serum while complexed with high-density lipoprotein 3 (SAA-HDL₃). Its levels increase markedly during inflammatory diseases. The pentapeptide Tyr-Ile-Gly-Ser-Asp (YIGSR-like) and the tripeptide Arg-Gly-Asn (RGD-like), related to the cell adhesion domains of laminin and fibronectin, respectively, exist in SAA within close proximity (YIGSDKYFHARGNY; amino acid residues 29–42). A structure-function study of linear and head-to-tail cyclic peptides, related to the amino acid residues 29–42) and 70–76 (GRGAEDS) of human SAA, was performed in order to evaluate their ability to inhibit adhesion of human T-lymphocytes to surfaces coated with extracellular matrix purified from bovine corneal cells.     

A study of extracellular matrix-cell adhesion peptidic epitopes related to human serum amyloid A (SAA)

Serum amyloid A (SAA), an acute-phase protein, exists normally in the serum while complexed with high-density lipoprotein 3 (SAA- HDL3). Its levels increase markedly during inflammatory diseases. The pentapeptide Tyr-Ile-Gly-Ser-Asp (YIGSR-like) and the tripeptide Arg-Gly-Asn (RGD-like), related to the cell adhesion domains of laminin and fibronectin, respectively, exist in SAA within close proximity (YIGSDKYFHARGNY; amino acid residues 29–42). A structure-function study of linear and head- to-tail cyclic peptides, related to the amino acid residues 29–42 and 70–76 (GRGAEDS) of human SAA, was performed in order to evaluate their ability to inhibit adhesion of human T-lymphocytes to surfaces coated with extracellular matrix purified from bovine corneal cells.     

Novel truncated isoforms of constitutive serum amyloid A detected by MALDI mass spectrometry

The main focus of the serum amyloid A (SAA) family has been on the acute phase isoforms. However, the constitutive isoform (SAA4) may have a strong effect on the metabolism of human serum lipoproteins. In this study, the SAA4 protein was examined in the high-density lipoprotein fraction of both healthy and diseased individuals. Novel isoforms of SAA4 were detected using ultracentrifugation combined with solid-phase extraction and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Three truncated isoforms were identified as well as two glycosylated isoforms. Patterns of isoform distribution may be significant for assessment of cardiovascular risk as well as direction of patient treatment.     

Effect of amino acid variations in the central region of human serum amyloid A on the amyloidogenic properties

Human serum amyloid A (SAA) is a precursor protein of the amyloid fibrils that are responsible for AA amyloidosis. Of the four human SAA genotypes, SAA1 is most commonly associated with AA amyloidosis. Furthermore, SAA1 has three major isoforms (SAA1.1, 1.3, and 1.5) that differ by single amino acid variations at two sites in their 104-amino acid sequences. In the present study, we examined the effect of amino acid variations in human SAA1 isoforms on the amyloidogenic properties. All SAA1 isoforms adopted α-helix structures at 4 °C, but were unstructured at 37 °C. Heparin-induced amyloid fibril formation of SAA1 was observed at 37 °C, as evidenced by the increased thioflavin T (ThT) fluorescence and β-sheet structure formation. Despite a comparable increase in ThT fluorescence, SAA1 molecules retained their α-helix structures at 4 °C. At both temperatures, no essential differences in ThT fluorescence and secondary structures were observed among the SAA1 isoforms. However, the fibril morphologies appeared to differ; SAA1.1 formed long and curly fibrils, whereas SAA1.3 formed thin and straight fibrils. The peptides corresponding to the central regions of the SAA1 isoforms containing amino acid variations showed distinct amyloidogenicities, reflecting their direct effects on amyloid fibril formation. These findings may provide novel insights into the influence of amino acid variations in human SAA on the pathogenesis of AA amyloidosis.     

TLR3 activation evokes IL-6 secretion, autocrine regulation of Stat3 signaling and TLR2 expression in human bronchial epithelial cells

Human bronchial epithelial cells exposed to synthetic double-stranded RNA (poly I:C) exhibited increased IL-6 and RANTES secretion and TLR2 expression that was inhibited following TLR3 silencing. Increased NF-κB and Stat3 phosphorylation were detected after poly I:C exposure and pretreatment with neutralizing antibody targeting IL-6 receptor α (IL-6Rα -nAb) or blocking Jak2 and Stat3 activity inhibited Stat3 phosphorylation. TLR2 up-regulation by poly I:C was also reduced by IL-6Rα-nAb and inhibitors of Jak2, Stat3 and NF-κB phosphorylation, whereas RANTES secretion was unaffected, but abolished following NF-κB inhibition. Treatment with exogenous IL-6 failed to increase TLR2. These findings demonstrate that TLR3 activation differentially regulates TLR expression through autocrine signaling involving IL-6 secretion, IL-6Rα activation and subsequent phosphorylation of Stat3. The results also indicate that NF-κB and Stat3 are required for TLR3-dependent up-regulation of TLR2 and that its delayed expression was due to a requirement for IL-6-dependent Stat3 activation.     

Expression of TGF-beta signaling genes in the normal, premalignant, and malignant human trophoblast: loss of smad3 in choriocarcinoma cells

We had earlier shown that TGF-beta controls proliferation, migration, and invasiveness of normal human trophoblast cells, whereas premalignant and malignant trophoblast cells are resistant to TGF-beta. To identify signaling defects responsible for TGF-beta resistance in premalignant and malignant trophoblasts, we have compared the expression of TGF-beta signaling molecules in a normal trophoblast cell line (HTR-8), its premalignant derivative (RSVT2/C), and two choriocarcinoma cell lines (JAR and JEG-3). RT-PCR analysis revealed that all these cell lines expressed the mRNA of TGF-beta1, -beta2, and -beta3, TGF-beta receptors type I, II, and III, and post-receptor signaling genes smad2, smad3, smad4, smad6, and smad7 with the exception that TGF-beta2 and smad3 were undetectable in JAR and JEG-3 cells. Immunoblot analysis confirmed the absence of smad3 protein in choriocarcinoma cells. Treatment with TGF-beta1 induced smad3 phosphorylation and smad3 translocation to the nucleus in the normal and premalignant trophoblast cells. These results suggest that loss of smad3 may account for a functional disruption in the TGF-beta signaling pathway in choriocarcinomas, but not in the premalignant trophoblast.     

Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua

Large granular lymphocytes (LGL) are the most abundant cell type in first trimester human pregnancy decidua. We have shown previously that CD56-positive decidual LGL have cytotoxic activity against the natural killer (NK) target K562, and that this cytotoxicity is augmented by pretreatment with interleukin-2 (IL-2). We now report that flow cytometrically purified populations of CD56-positive decidual LGL have no cytotoxic activity against either the BeWo choriocarcinoma cell line or freshly isolated term trophoblast. Incubation of unfractionated decidual cells with IL-2 induced cytotoxicity against BeWo, but term trophoblast remained resistant to lysis. Both BeWo and trophoblast showed much lower binding frequencies to decidual or peripheral blood cells than K56 targets, and excess trophoblast did not inhibit cytotoxic activity against K562. This suggests that the resistance of trophoblast to lysis by either decidual or peripheral blood LGL is due to the lack of accessible NK target structures on the surface of trophoblast.     

The acute-phase protein serum amyloid A3 is expressed in the bovine mammary gland and plays a role in host defence

The serum amyloid A protein is one of the major reactants in the acute-phase response. Using representational difference analysis comparing RNA from normal and involuting quarters of a dairy cow mammary gland, we found an mRNA encoding the SAA3 protein (M-SAA3). The M-SAA3 mRNA was localized to restricted populations of bovine mammary epithelial cells (MECs). It was expressed at a moderate level in late pregnancy, at a low level through lactation, was induced early in milk stasis, and expressed at high levels in most MECs during mid to late involution and inflammation/mastitis. The mature M-SAA3 peptide was expressed in Escherichia coli, antibodies made, and shown to have antibacterial activity against E. coli, Streptococcus uberis and Pseudomonas aeruginosa. These results suggest that the mammary SAA3 may have a role in protection of the mammary gland during remodelling and infection and possibly in the neonate gastrointestinal tract.     

Serum insulin-like growth factor-I,iron, C-reactive protein,and serum amyloid A for prediction of outcome in dogs with pyometra

Pyometra, accumulation of pus in the uterus, is a bacterial infection that frequently initiates systemic inflammation. The disease may have lethal consequences when the systemic effects are severe or complications occur. Markers for identifying high-risk patients and predicting outcome are therefore in high demand. The objective of this study was to measure serum concentrations of insulin-like growth factor-I (IGF-I), iron, C-reactive protein (CRP), and serum amyloid A (SAA) in bitches with pyometra and to explore the possible value of these variables for detection of increased morbidity. In total, 31 bitches were diagnosed with pyometra and destined for surgical treatment (ovariohysterectomy) and 17 healthy bitches were included in the study. Concentrations of IGF-I and iron were lower in the pyometra group (mean concentration 221.2 ± 22.5 ng/mL and 16.9 ± 1.6 μmol/L, respectively) compared with the healthy control group (mean concentration 366.7 ± 46.2 ng/mL and 38.1 ± 2.7 μmol/L, respectively). In contrast, concentrations of CRP and SAA were significantly higher in bitches with pyometra (mean concentrations 212.9 ± 17.3 mg/L and 119.9 ± 8.5 mg/L, respectively) compared with the control group (<5 mg/L and <10 mg/L, respectively). None of the explored variables were associated with morbidity as measured by duration of postoperative hospitalization. In conclusion, IGF-I and iron concentrations were decreased in pyometra, whereas SAA and CRP concentrations were increased in the disease. Although unspecific, measurement of these variables may be valuable as adjunctive markers for prognosis in cases of pyometra.     

Modulation of cytokine expression in human macrophages by endocrine-disrupting chemical Bisphenol-A

Exposure to environmental endocrine-disrupting chemical Bisphenol-A (BPA) is often associated with dysregulated immune homeostasis, but the mechanisms remain unclear. In the present study, the effects of BPA on the cytokines responses of human macrophages were investigated. Treatment with BPA increased pro-inflammation cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production, but decreased anti-inflammation cytokines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) production in THP1 macrophages, as well as in primary human macrophages. BPA effected cytokines expression through estrogen receptor α/β (ERα/β)-dependent mechanism with the evidence of ERα/β antagonist reversed the expression of cytokines. We also identified that activation of extracellular regulated protein kinases (ERK)/nuclear factor κB (NF-κB) signal cascade marked the effects of BPA on cytokines expression. Our results indicated that BPA effected inflammatory responses of macrophages via modulating of cytokines expression, and provided a new insight into the link between exposure to BPA and human health.     

Expression and sequence analyses of serum amyloid A in the Syrian hamster

Reactive amyloidosis occurs during chronic inflammation and involves deposition of amyloid A (AA) fibrils in many organs. Amyloid A is derived by proteolysis from serum amyloid A component (SAA), a major acute-phase reactant in many species. Since spontaneous amyloidosis occurs commonly in Syrian hamsters, we have studied the structure and expression of SAA genes during inflammation in these animals. Two cDNA clones and one genomic clone were sequenced, suggesting that Syrian hamster SAA is encoded by at least two genes. Hepatic mRNA analyses showed that SAA was inducible in many hamster organs during acute inflammation. These studies also demonstrated that SAA mRNA for one isotype is maximally expressed at a site of local tissue damage.