Association of serum amyloid A levels with adipocyte size and serum levels of adipokines: Differences between men and women

The aim of this study was to characterize the association between adipocyte enlargement and circulating levels of serum amyloid A (SAA). Furthermore, we wanted to search for possible associations with measures of glycemic control and levels of circulating adipokines and/or inflammatory markers in men and women with a large range in body mass index. The study cohort consisted of 167 subjects, 114 non-diabetic and 53 with Type 2 diabetes. Adipocyte diameter as well as circulating levels of SAA, C-reactive protein (CRP), adiponectin, leptin, interleukin-6, tumor necrosis factor alpha, glucose and insulin were measured. Women had higher serum levels of SAA than men (p = 0.044). SAA levels were weakly but positively correlated with BMI (p = 0.043) and % body fat (p = 0.027) in all subjects as well as subcutaneous adipocyte diameter (p = 0.034) in women. Furthermore, in all subjects we found correlations between SAA levels and levels of CRP (p < 0.001), interleukin-6 (p < 0.001), leptin (p = 0.003), insulin (p = 0.006), HbA1c (p = 0.02) and HOMA-IR (p = 0.002). A majority of the correlations were strongest in women. In conclusion, serum levels of SAA are strongly correlated with serum levels of inflammatory markers as well as measures of glycemic control. There seems to be large sex differences in these associations suggesting that sex-specific factors need to be considered when analyzing SAA levels in relation to metabolic disease.     

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.     

The acute phase protein serum amyloid A primes neutrophils

We studied here the effect of the acute phase protein serum amyloid A (SAA) on the oxidative burst of neutrophils. Incubation of neutrophils with SAA increased the rate of oxygen uptake and the production of reactive oxygen species of neutrophils activated with opsonized zymosan (OZ). The increment in the neutrophil oxidative burst was dependent on SAA concentration in the range of 3-33 microg protein ml(-1) and was observed only in the presence of a relatively low amount of OZ (1 x 10(6) particles ml(-1)). SAA did not affect oxygen consumption and reactive oxygen production triggered by other stimuli, such as f-Met-Leu-Phe, phorbol myristate acetate or non-opsonized zymosan. Our finding points to a priming effect of SAA probably associated with mobilization of receptors for opsonized particles and strengthens the role of SAA as an effector of neutrophil functions in inflammation.     

Increased plasma levels of adipokines and inflammatory markers in older women with persistent HPV infection

We observed diminished lymphoproliferation to multiple stimuli in older women with persistent cervical human papillomavirus (HPV) infection. Adipokines are a class of inflammatory cytokines that are altered in some persistent infections. The objective was to compare the level of adipokines and inflammatory cytokines in heparinized plasma from women with persistent HPV cervical infection (Cases, N=50, oversampled for their weak lymphoproliferation responses) with women with no evidence of persistent HPV cervical infection (Controls, N=50, oversampled for their strong lymphoproliferation responses). Plasma samples were analyzed with multiplex assays for adipokines and inflammatory cytokines. Cases had significantly elevated plasma levels of resistin (p<0.0001) and sFas (p=0.0038) as compared to controls. Risk of persistent HPV infection increased significantly with increasing levels of resistin and 8Fas. This is the first study to demonstrate elevated levels of resistin and sFas in HPV persistently infected, older women with decreased immune function expanding the understanding of the systemic inflammation and immune alterations in individuals persistently infected with HPV. Further studies within a larger cohort are needed to define the generalities of these findings and any role adipokines have in persistent HPV infection.     

Cytokines and serum amyloid A in the pathogenesis of hepatitis C virus infection

Expression of the acute phase protein serum amyloid A (SAA) is dependent on the release of the pro-inflammatory cytokines IL-1, IL-6 and TNF-α during infection and inflammation. Hepatitis C virus (HCV) upregulates SAA-inducing cytokines. In line with this, a segment of chronically infected individuals display increased circulating levels of SAA. SAA has even been proposed to be a potential biomarker to evaluate treatment efficiency and the course of disease. SAA possesses antiviral activity against HCV via direct interaction with the viral particle, but might also divert infectivity through its function as an apolipoprotein. On the other hand, SAA shares inflammatory and angiogenic activity with chemotactic cytokines by activating the G protein-coupled receptor, formyl peptide receptor 2. These latter properties might promote chronic inflammation and hepatic injury. Indeed, up to 80 % of infected individuals develop chronic disease because they cannot completely clear the infection, due to diversion of the immune response. In this review, we summarize the interconnection between SAA and cytokines in the context of HCV infection and highlight the dual role SAA could play in this disease. Nevertheless, more research is needed to establish whether the balance between those opposing activities can be tilted in favor of the host defense.     

Association between coronary endothelial dysfunction and local inflammation of atherosclerotic coronary arteries

We have examined a possibility whether or not severity and extent of coronary atherosclerosis may associate with degree of local inflammation in relation to endothelial dysfunction as is indicated by reduced NO formation. Blood samples were obtained from aortic root (Ao) and coronary sinus (CS) of 39 patients who underwent coronary angiography. Plasma NOx levels (nitrite + nitrate, stable NO end-products) were evaluated by HPLC-Griess system, and markers of inflammation, C-reactive protein (CRP) and serum amyloid A protein (SAA), were measured by Latex Turbidimetric Immunoassay. To evaluate the changes of these substances through coronary circulation, the percentage changes of respective markers [(CS – Ao) × 100/Ao] were calculated. The extent and severity of atherosclerosis of left coronary arteries were evaluated with Gensini Score (GS). The GS correlated with the percentage changes of NOx (r = –0.35, p < 0.05) and that of SAA (r = 0.43, p < 0.05) across coronary circulation, but not with changes in CRP. Moreover, the percentage changes of NOx correlated with that of SAA (r = –0.36, p < 0.05). These results indicated that severity and extent of coronary atherosclerosis related to degree of local inflammation which has a possible association with coronary endothelial dysfunction.     

The interaction of C-reactive protein and serum amyloid P component with nuclear antigens

The pentraxins are a family of proteins characterized by cyclic pentameric structure, calcium-dependent ligand binding and sequence homology. The two main representatives of this family are the serum proteins, C-reactive protein (CRP) and serum amyloid P component (SAP). In man CRP is an acute phase reactant which increases up to 1000 fold during the acute phase response whereas SAP is a constitutive protein expressed at about 30 g/ml. These proteins activate complement through the classical pathway and participate in opsonization of particulate antigens and bacteria. In the past several years it has been determined that both of these pentraxins interact with nuclear antigens including chromatin and small nuclear ribonucleoproteins (snRNPs). Both CRP and SAP have nuclear transport signals which facilitate their entry into the nuclei of intact cells. Furthermore, these pentraxins have been shown to affect the clearance of nuclear antigens in vivo. It is now believed that one of the major functions of the pentraxins could be to interact with the nuclear antigens released from apoptotic or necrotic cells. This interaction could mitigate against deposition of these antigens in tissue and autoimmune reactivity.Abbreviations CRP C-reactive protein - HSA human serum albumin - PC phosphocholine - SAP serum amyloid P component - snRNP small nuclear ribonucleoprotein - SLE systemic lupus erythematosus     

Molecular regulation of autophagy in a pro-inflammatory tumour microenvironment: New insight into the role of serum amyloid A

Chronic inflammation, systemic or local, plays a vital role in tumour progression and metastasis. Dysregulation of key physiological processes such as autophagy elicit unfavourable immune responses to induce chronic inflammation. Cytokines, growth factors and acute phase proteins present in the tumour microenvironment regulate inflammatory responses and alter crosstalk between various signalling pathways involved in the progression of cancer. Serum amyloid A (SAA) is a key acute phase protein secreted by the liver during the acute phase response (APR) following infection or injury. However, cancer and cancer-associated cells produce SAA, which when present in high levels in the tumour microenvironment contributes to cancer initiation, progression and metastasis. SAA can activate several signalling pathways such as the PI3K and MAPK pathways, which are also known modulators of the intracellular degradation process, autophagy. Autophagy can be regarded as having a double edged sword effect in cancer. Its dysregulation can induce malignant transformation through metabolic stress which manifests as oxidative stress, endoplasmic reticulum (ER) stress and DNA damage. On the other hand, autophagy can promote cancer survival during metabolic stress, hypoxia and senescence. Autophagy has been utilised to promote the efficiency of chemotherapeutic agents and can either be inhibited or induced to improve treatment outcomes. This review aims to address the known mechanisms that regulate autophagy as well as illustrating the role of SAA in modulating these pathways and its clinical implications for cancer therapy.     

A protease-sensitive site in the proposed Ca(2+)-binding region of human serum amyloid P component and other pentraxins.

Serum amyloid P component (SAP) is a decamer of 10 identical 25.5-kDa subunits. Limited proteolysis of SAP with alpha-chymotrypsin cleaves the subunit into two fragments of 18 and 7.5 kDa, although the fragments stay together in the decamer under nondenaturing conditions. Proteolysis does not occur in the presence of Ca2+ (10 mM). Cleavage with alpha-chymotrypsin prevents the Ca(2+)-dependent binding of SAP to zymosan extract, nucleosomes, and DNA. The alpha-chymotrypsin cleavage site identified is in a region of SAP that is highly conserved in members of the human C-reactive protein (CRP) family of proteins (pentraxins) to which SAP belongs and is similar to the Ca(2+)-binding site in calmodulin and related Ca(2+)-binding proteins (Nguyen, N.Y., Suzuki, A., Boykins, R.A., & Liu, T.-Y., 1986, J. Biol. Chem. 261, 10456-10465). Treatment of SAP with other proteases (trypsin, Pronase, and Nagarse protease) yields fragmentation patterns upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that are similar to those obtained with alpha-chymotrypsin. Two other members of the pentraxin family of proteins, hamster female protein and rabbit CRP, also exhibit similar fragmentation patterns on SDS-PAGE when treated with the various proteases. Recently, it has been shown that the homologous protein, human CRP, is cleaved in the same homologous position as cleavage of SAP by alpha-chymotrypsin, resulting in the loss of Ca(2+)-binding (as shown by equilibrium dialysis) and Ca(2+)-dependent binding reactivities (Kinoshita, C.M., Ying, S.-C., Hugli, T.E., Siegel, J.N., Potempa, L.A., Jiang, H.J., Houghten, R.A., & Gewurz, H., 1989, Biochemistry 28, 9840-9848).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-18 induces serum amyloid A (SAA) protein production from rheumatoid synovial fibroblasts

Interleukin-18 (IL-18) is a novel proinflammatory cytokine that was recently found in synovial fluids and synovial tissues from patients with rheumatoid arthritis (RA). To investigate the role of IL-18 in rheumatoid synovitis, the levels of IL-18 and serum amyloid A (SAA) were measured in synovial fluids from 24 patients with rheumatoid arthritis (RA) and 13 patients with osteoarthritis (OA). The levels of IL-18 and SAA in the synovial fluids were elevated in RA patients. In contrast, the levels of IL-18 in synovial fluids from OA patients were significantly lower compared to those of RA patients. SAA was not detected in synovial fluids from OA patients. The expression of SAA mRNA in rheumatoid synovial cells was also examined. SAA4 mRNA, which was constitutively expressed by rheumatoid synovial cells, was not affected by IL-18 stimulation. Although acute phase SAA (A-SAA, SAA1 + 2) mRNA was not detected in unstimulated synovial cells, its expression was induced by IL-18 stimulation. By immunoblot, we demonstrated that IL-18 induced the SAA protein synthesis from rheumatoid synovial cells in a dose-dependent manner. These results indicate a novel role for IL-18 in rheumatoid inflammation through the synovial SAA production.     

Kinetics of c-reactive protein (CRP) and serum amyloid A protein (SAA) in patients with community-acquired pneumonia (CAP), as presented with biologic half-life times

Context: In management of community-acquired pneumonia (CAP), excellent biomarkers for inflammation would be helpful in our practice.

Objectives: Kinetics of c-reactive protein (CRP) and serum amyloid A (SAA) was characterized, using their biologic half-life times.

Materials and methods: Time course of CRP and SAA levels in the successfully treated 36 CAP patients were investigated and their half-life times were determined and compared.

Results & Discussions: SAA and CRP declined in an exponential mean and the biologic half-life times of SAA levels was 34.9?±?28.7?h, significantly shorter than that of CRP, 46.4?±?21.7?h (p?=?0.0014). Conclusion: The kinetic evidence, presented as biologic half-life times of CRP and SAA, helps us make a clinical assessment of CAP patients.     

Huntingtin associated protein 1 regulates trafficking of the amyloid precursor protein and modulates amyloid beta levels in neurons

J. Neurochem. (2012) 122, 1010-1022. ABSTRACT: Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Aβ) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Aβ production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371-599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1-/- neurons. HAP1 deletion significantly alters APP endocytosis and reduces the re-insertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1-/- neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer's disease mouse model increases Aβ levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Aβ production in neurons.     

Interaction of serum amyloid A with human cystatin C—assessment of amino acid residues crucial for hCC–SAA formation (part II)

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life‐threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute‐phase‐reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86–104) and hCC(96–102), respectively. Here, we report further details concerning the hCC–SAA interaction. With the use of affinity tests and florescent ELISA‐like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser‐98 and Tyr‐102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC–SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

三项血清学检查联合应用对老年菌血症早期诊断的价值

目的 探讨C-反应蛋白（CRP）、降钙素原（PCT）和血清样淀粉蛋白A（SAA）对于老年菌血症早期诊断的价值。方法 选择2016年12月至2018年11月我院收治的老年感染患者140例（年龄≥65岁）为研究对象,按血培养结果分为菌血症患者（60例）和非菌血症患者（80例）;选择同期健康体检者60例（年龄≥65岁）为对照组,测定全部研究对象血清CRP、PCT和SAA水平及阳性率。结果 与对照组比较,菌血症和非菌血症患者血清CRP、PCT和SAA水平均显著升高（P<0.05）;且菌血症患者这3项指标水平高于非菌血症者（P<0.05）。在单项检测中,PCT的敏感性和特异性最高。3项联合检测的诊断价值高于单项检测。由G－菌引起的菌血症患者这3项指标水平高于G+菌菌血症患者（P<0.05）。结论 血清CRP、PCT和SAA这3项指标对老年菌血症的早期诊断具有一定价值。     

Interaction of serum amyloid A with human cystatin C—identification of binding sites

Serum amyloid A (SAA) is a multifunctional acute‐phase protein whose natural role seems to be participation in many physiologic and pathological processes. Prolonged increased SAA level in a number of chronic inflammatory and neoplastic diseases gives rise to reactive systemic amyloid A amyloidosis, where the N‐terminal 76‐amino acid residue‐long segment of SAA is deposited as amyloid fibrils. Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been described. Here, we report further evidence corroborating this interaction, and the identification of the SAA and hCC binding sites in the SAA–hCC complex, using a combination of selective proteolytic excision and high‐resolution mass spectrometry. The shortest binding site in the SAA sequence was determined as SAA(86–104), whereas the binding site in hCC sequence was identified as hCC(96–102). Binding specificities of both interacting sequences were ascertained by affinity experiments (ELISA) and by registration of mass spectrum of SAA–hCC complex. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of two novel amyloid A protein subsets coexisting in an individual patient of AA-amyloidosis

Amyloid A protein (AA), the major fibril protein in AA-amyloidosis, is an N-terminal cleavage product of the precursor protein, serum amyloid A (SAA). Using mass spectrometry and amino-acid sequencing, we identified and characterized two novel AA protein subsets co-deposited as amyloid fibrils in an patient having AA-amyloidosis associated with rheumatoid arthritis. One of the AA proteins corresponded to positions 2–76 (or 75) of SAA2α and the other corresponded to positions 2–76 (or 75) of known SAA1 subsets, except for position 52 or 57, where SAA1α has valine and alanine and SAA1β has alanine and valine in position 52 and 57, respectively, whereas the AA protein had alanine at the both positions. Our findings (1), demonstrate that not only one but two SAA subsets could be deposited together as an AA-amyloid in a single individual and (2), support the existence of a novel SAA1 allotype, i.e., SAA1^52,57Ala.     

Serum amyloid A enhances plasminogen activation: implication for a role in colon cancer

We have recently reported that the acute phase protein serum amyloid A (SAA), is locally and differentially expressed in neoplastic tissues of human colon. In the present study, we demonstrate that SAA enhances the plasminogen activation (PA)-activity of HT-29 colon cancer cell line. Cell-associated PA-activity was measured following the plasminogen-dependent ability of the cells to cleave the chromogenic substrate S-2251. The SAA-enhanced PA-activity was inhibited by anti-SAA antibodies. These antibodies also decreased the basal PA-activity of HT-29 cells and neutralized their cytokines (Interleukin-1β + Interleukin-6)—enhanced PA-activity. Using specific chromogenic substrates and the fibrin clot-lysis assay, we found that SAA enhances also the PA-activity mediated by purified urokinase- and tissue-type plasminogen activators. Together, the data indicate that SAA enhances plasminogen activation and suggest its possible role in plasmin(ogen)-mediated colon cancer progression.     

Cellular mechanism of fibril formation from serum amyloid A1 protein

Serum amyloid A1 (SAA1) is an apolipoprotein that binds to the high‐density lipoprotein (HDL) fraction of the serum and constitutes the fibril precursor protein in systemic AA amyloidosis. We here show that HDL binding blocks fibril formation from soluble SAA1 protein, whereas internalization into mononuclear phagocytes leads to the formation of amyloid. SAA1 aggregation in the cell model disturbs the integrity of vesicular membranes and leads to lysosomal leakage and apoptotic death. The formed amyloid becomes deposited outside the cell where it can seed the fibrillation of extracellular SAA1. Our data imply that cells are transiently required in the amyloidogenic cascade and promote the initial nucleation of the deposits. This mechanism reconciles previous evidence for the extracellular location of deposits and amyloid precursor protein with observations the cells are crucial for the formation of amyloid.     

Impact of serum amyloid A on high density lipoprotein composition and levels

Serum amyloid A (SAA) is an acute-phase protein mainly associated with HDL. To study the role of SAA in mediating changes in HDL composition and metabolism during inflammation, we generated mice in which the two major acute-phase SAA isoforms, SAA1.1 and SAA2.1, were deleted [SAA knockout (SAAKO) mice], and induced an acute phase to compare lipid and apolipoprotein parameters between wild-type (WT) and SAAKO mice. Our data indicate that SAA does not affect apolipoprotein A-I (apoA-I) levels or clearance under steady-state conditions. HDL and plasma triglyceride levels following lipopolysaccharide administration, as well as the decline in liver expression of apoA-I and apoA-II, did not differ between both groups of mice. The expected size increase of WT acute-phase HDL was surprisingly also seen in SAAKO acute-phase HDL despite the absence of SAA. HDLs from both mice showed increased phospholipid and unesterified cholesterol content during the acute phase. We therefore conclude that in the mouse, SAA does not impact HDL levels, apoA-I clearance, or HDL size during the acute phase and that the increased size of acute-phase HDL in mice is associated with an increased content of surface lipids, particularly phospholipids, and not surface proteins. These data need to be transferred to humans with caution due to differences in apoA-I structure and remodeling functions.     

Frame-shifted amyloid precursor protein found in Alzheimer's disease and Down's syndrome increases levels of secreted amyloid beta40

Frame-shifted amyloid precursor protein (APP(+1)), which has a truncated out-of-frame C-terminus, accumulates in the neuropathological hallmarks of patients with Alzheimer's disease pathology. To study a possible involvement of APP(+1) in the pathogenesis of Alzheimer's disease, we expressed APP695 and APP(+1) in the HEK293 cell-line and studied whether the processing of APP695 was affected. APP(+1) is a secretory protein, but high expression of APP695 and APP(+1) results in the formation of intracellular aggregate-like structures containing both proteins and Fe65, an adaptor protein that interacts with APP695. APP(+1) is shown to interact with APP695, suggesting that these structures consist of functional protein complexes. Such an interaction can also be anticipated in post-mortem brains of young Down's syndrome patients without any sign of neuropathology. Here we observed APP(+1) immunoreactivity in beaded fibres. Additional support for functional consequences on the processing of APP695 comes from a 1.4-fold increase in levels of secreted amyloid beta40 in cells co-expressing APP695 and APP(+1), although APP(+1) itself does not contain the amyloid beta sequence. Taken together, these data show that co-expression of APP695 and APP(+1) affects the processing of APP695 in a pro-amyloidogenic way and this could gradually contribute to Alzheimer's disease pathology, as has been implicated in Down's syndrome patients.