External validation of a referral rule for axial spondyloarthritis in primary care patients with chronic low back pain

Objectives To validate and optimize a referral rule to identify primary care patients with chronic low back pain (CLBP) suspected for axial spondyloarthritis (axSpA). Design Cross-sectional study with data from 19 Dutch primary care practices for development and 38 for validation. Participants Primary care patients aged 18-45 years with CLBP existing more than three months and onset of back pain started before the age of 45 years. Main Outcome The number of axSpA patients according to the ASAS criteria. Methods The referral rule (CaFaSpA referral rule) was developed using 364 CL

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Het serum amyloïd A-chemokine netwerk

Bovine serum is frequently used as a supplement (10%) for cell culture media since it encloses numerous nutritional components promoting cell growth, proliferation and survival (e.g. growth factors, albumin, amino acids, lipids, hormones). For this reason, bovine serum is also regularly added to buffers used in biological experiments, such as chemotaxis assays. On top of elements beneficial for cell growth, bovine serum contains chemokines and other unknown chemotactic factors that may interfere within these experiments. Chemokines are small chemotactic cytokines that attract specific leukocyte subsets to sites of inflammation via interaction with G protein-coupled receptors (GPCRs). Chemokines also play a major role in various physiological processes, like basal homing of leukocytes, hematopoiesis and angiogenesis. In order to discover yet unidentified chemotactic factors, we aimed to biochemically and biologically characterize novel chemotactic factors from bovine newborn calf serum. Based on monocyte chemotactic activity, these factors were purified via a four-step chromatographical procedure, subsequently involving adsorption to silicic acid, heparin-Sepharose chromatography, cation-exchange chromatography and reversed phase high performance chromatography. The factors were biochemically identified via SDS-PAGE, mass spectrometry and Edman degradation and chemotactically characterized by determining their various target cells, receptor usage and interactions with chemokines. As a result, a novel inflammatory chemokine, bovine CCL3 isoform 2, was discovered. Bovine CCL3 isoform 2 was previously only described at the gene level. This novel chemokine was found to chemoattract monocytes and immature (but not mature) monocyte-derived dendritic cells, acting via CCR1 and CCR5, which are the classical human CCL3 receptors. Bovine CCL3 isoform 2, present at about 5 ng/ml in bovine serum, attracted monocytes and dendritic cells from a concentration of 0.1 ng/ml onwards, reaching a maximal chemotactic response at 0.3 to 3 ng/ml. This means that bovine CCL3 isoform 2 present in bovine serum could for sure interfere with cell behavior and migration during biological tests. Along the same line of research, another monocyte chemotactic factor was purified from bovine serum corresponding to the acute phase protein serum amyloid A (SAA). Serum concentrations of this highly conserved acute phase protein rise up to 1000-fold during inflammatory events. Despite being studied quite a lot in the past decades, its exact biological functions are still not fully understood. Several pro- and anti-inflammatory properties have been ascribed to human SAA. One of the pro-inflammatory functions of SAA is its chemotactic activity for neutrophils, monocytes, T cells and mast cells through interaction with the GPCR formyl peptide receptor 2 (FPR2). We confirmed its activity on monocytes and neutrophils and discovered that human SAA1α was chemotactic for immature monocyte-derived dendritic cells as well. In addition, we demonstrated that the chemotactic activity of SAA1α on monocytes and immature dendritic cells was in part mediated by rapid (within 2-3 h) induction of chemokines (such as CCL3 and CXCL8) that subsequently cooperated which each other in an autocrine way to attract these leukocytes. The synergy between SAA1α and CXCL8 was found to occur via the corresponding receptors of these chemoattractants, which are FPR2 and CXCR2, respectively. In addition to interacting with FPR2 to exert its direct chemotactic effect, SAA1α-induced CXCL8 production in monocytes was mediated by toll-like receptor (TLR)2. In contrast to monocytes and immature dendritic cells, SAA1α did not stimulate neutrophils to produce CCL3 and CXCL8 within 2 h, but synergized with these chemokines in a paracrine way. In vivo, human SAA1α also synergized with the paracrinely (e.g. in peritoneal macrophages) induced CXCL6 (which is the murine counterpart of human CXCL8) to greatly enhance intraperitoneal neutrophil recruitment. In summary, we studied the presence and role of yet unidentified and unrelated chemotactic factors (the chemokines CCL3 isoform 2 and the acute phase protein SAA) in bovine calf serum, widely used as a source of nutritional factors to promote cell growth. Our findings provide further evidence for the chemotactic potency and spectrum of target cells of SAA and allowed to unravel the mode of action of SAA to exert its chemotactic activity. Hence, during the inflammatory response to infection, exogenous inflammatory mediators, such as bacterial LPS and viral dsRNA, induce (via TLR4 and TLR3, respectively) chemokines and cytokines, such as IL-1 and IL-6. These cytokines subsequently induce more chemokines in various cell types and other endogenous inflammatory mediators, such as SAA, in the liver. The latter further stimulates the production of chemokines in leukocytes and other cell types to prolong and enhance the inflammatory response. The induced chemokines cooperate with each other or synergize with SAA to attract leukocytes. Taken together, the exogenous and endogenous inflammatory mediators generate a substantial cytokine-SAA-chemokine network, allowing to develop an appropriate inflammatory response.nrpages: 135status: publishe

The ectoenzyme-side of matrix metalloproteinases (MMPs) makes inflammation by serum amyloid A (SAA) and chemokines go round

During an inflammatory response, a large number of distinct mediators appears in the affected tissues or in the blood circulation. These include acute phase proteins such as serum amyloid A (SAA), cytokines and chemokines and proteolytic enzymes. Although these molecules are generated within a cascade sequence in specific body compartments allowing for independent action, their co-appearance in space and time during acute or chronic inflammation points toward important mutual interactions. Pathogen-associated molecular patterns lead to fast induction of the pro-inflammatory endogenous pyrogens, which are evoking the acute phase response. Interleukin-1, tumor necrosis factor-α and interferons simultaneously trigger different cell types, including leukocytes, endothelial cells and fibroblasts for tissue-specific or systemic production of chemokines and matrix metalloproteinases (MMPs). In addition, SAA induces chemokines and both stimulate secretion of MMPs from multiple cell types. As a consequence, these mediators may cooperate to enhance the inflammatory response. Indeed, SAA synergizes with chemokines to increase chemoattraction of monocytes and granulocytes. On the other hand, MMPs post-translationally modify chemokines and SAA to reduce their activity. Indeed, MMPs internally cleave SAA with loss of its cytokine-inducing and direct chemotactic potential whilst retaining its capacity to synergize with chemokines in leukocyte migration. Finally, MMPs truncate chemokines at their NH2- or COOH-terminal end, resulting in reduced or enhanced chemotactic activity. Therefore, the complex interactions between chemokines, SAA and MMPs either maintain or dampen the inflammatory response.status: publishe

Serum amyloid A chemoattracts immature dendritic cells and indirectly provokes monocyte chemotaxis by induction of cooperating CC and CXC chemokines

Serum amyloid A (SAA) is an acute phase protein that is upregulated in inflammatory diseases and chemoattracts monocytes, lymphocytes, and granulocytes via its G protein-coupled receptor formyl peptide receptor like 1/formyl peptide receptor 2 (FPRL1/FPR2). Here, we demonstrated that the SAA1α isoform also chemoattracts monocyte-derived immature dendritic cells (DCs) in the Boyden and μ-slide chemotaxis assay and that its chemotactic activity for monocytes and DCs was indirectly mediated via rapid chemokine induction. Indeed, SAA1 induced significant amounts (≥5 ng/mL) of macrophage inflammatory protein-1α/CC chemokine ligand 3 (MIP-1α/CCL3) and interleukin-8/CXC chemokine ligand 8 (IL-8/CXCL8) in monocytes and DCs in a dose-dependent manner within 3 h. However, SAA1 also directly activated monocytes and DCs for signaling and chemotaxis without chemokine interference. SAA1-induced monocyte migration was nevertheless significantly prevented (60-80% inhibition) in the constant presence of desensitizing exogenous MIP-1α/CCL3, neutralizing anti-MIP-1α/CCL3 antibody, or a combination of CC chemokine receptor 1 (CCR1) and CCR5 antagonists, indicating that this endogenously produced CC chemokine was indirectly contributing to SAA1-mediated chemotaxis. Further, anti-IL-8/CXCL8 antibody neutralized SAA1-induced monocyte migration, suggesting that endogenous IL-8/CXCL8 acted in concert with MIP-1α/CCL3. This explained why SAA1 failed to synergize with exogenously added MIP-1α/CCL3 or stromal cell-derived factor-1α (SDF-1α)/CXCL12 in monocyte and DC chemotaxis. In addition to direct leukocyte activation, SAA1 induces a chemotactic cascade mediated by expression of cooperating chemokines to prolong leukocyte recruitment to the inflammatory site.status: publishe

COOH-terminal SAA1 peptides fail to induce chemokines but synergize with CXCL8 and CCL3 to recruit leukocytes via FPR2.

A natural leukocyte chemoattractant was isolated from bovine serum by an established 4-step purification procedure. Based on its relative molecular mass of 7287 and NH2-terminal sequence, the protein was identified as a carboxy-terminal peptide of the acute phase protein serum amyloid A1 (SAA1). This SAA1(46-112) fragment and its human equivalent SAA1(47-104) were chemically synthesized. Unlike intact SAA1α, these SAA fragments failed to directly chemoattract neutrophils and monocytes, to induce chemokines, and to stimulate downstream extracellular signal-regulated kinase signaling in monocytes. However, the SAA fragments potently synergized with CCL3 to induce monocyte migration and with CXCL8 to stimulate neutrophil shape changes and chemotaxis. Unlike intact SAA1α, SAA1(46-112) did not induce CXCL6 ex vivo but provoked a cooperative intraperitoneal neutrophil recruitment in mice when coinjected with CXCL6 into the peritoneal cavity. Moreover, SAA1(47-104) desensitized the synergy between intact SAA1α and CXCL8 in neutrophil chemotaxis, suggesting that this peptide binds formyl peptide receptor 2 (FPR2). This was evidenced by a complete blockade of synergy between the COOH-terminal SAA1 fragments and CXCL8 or CCL3 in neutrophil and monocyte chemotaxis, respectively, by the FPR2 antagonist WRW4 Thus, SAA1 is degraded into fragments lacking chemokine-inducing capacity, while keeping synergy with cytokine-induced chemokines to sustain limited inflammation.status: publishe

The cytokine-serum amyloid A-chemokine network

Levels of serum amyloid A (SAA), a major acute phase protein in humans, are increased up to 1000-fold upon infection, trauma, cancer or other inflammatory events. However, the exact role of SAA in host defense is yet not fully understood. Several pro- and anti-inflammatory properties have been ascribed to SAA. Here, the regulated production of SAA by cytokines and glucocorticoids is discussed first. Secondly, the cytokine and chemokine inducing capacity of SAA and its receptor usage are reviewed. Thirdly, the direct (via FPR2) and indirect (via TLR2) chemotactic effects of SAA and its synergy with chemokines are unraveled. Altogether, a complex cytokine-SAA-chemokine network is established, in which SAA plays a key role in regulating the inflammatory response.publisher: Elsevier articletitle: The cytokine-serum amyloid A-chemokine network journaltitle: Cytokine & Growth Factor Reviews articlelink: http://dx.doi.org/10.1016/j.cytogfr.2015.12.010 content_type: article copyright: © 2015 Elsevier Ltd. All rights reserved.status: publishe

Induction of Chemokines by Hepatitis C Virus Proteins: Synergy of the Core Protein with Interleukin-1 beta and Interferon-gamma in Liver Bystander Cells

Chronic hepatitis C virus (HCV) infection accounts for a large proportion of hepatic fibrosis and carcinoma cases observed worldwide. Mechanisms involved in HCV-induced hepatic injury have yet to be fully elucidated. Of particular interest is the capacity of HCV to regulate inflammatory responses. Here, we reveal modulation of cytokine activity by the HCV proteins non-structural protein 3 (NS3), glycoprotein E2, and core protein for their ability to induce chemokine expression in various liver bystander cells. Chemokines sustain chronic liver inflammation and relay multiple fibrogenic effects. CCL2, CCL3, CCL20, CXCL8, and CXCL10 were differentially expressed after treatment of monocytes, fibroblasts, or liver sinusoidal microvascular endothelial cells (LSECs) with HCV proteins. In comparison to NS3 and glycoprotein E2, core protein was a stronger inducer of chemokines in liver bystander cells. Interferon-γ (IFN-γ) and interleukin-1β (IL-1β) synergized with core protein to induce CCL2, CCL20, CXCL8, or CXCL10 in fibroblasts or LSECs. These findings reveal new mechanisms of hepatic injury caused by HCV.status: publishe

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.status: publishe

External Validation of a Referral Rule for Axial Spondyloarthritis in Primary Care Patients with Chronic Low Back Pain

Objectives To validate and optimize a referral rule to identify primary care patients with chronic low back pain (CLBP) suspected for axial spondyloarthritis (axSpA). Design Cross-sectional study with data from 19 Dutch primary care practices for development and 38 for validation. Participants Primary care patients aged 18-45 years with CLBP existing more than three months and onset of back pain started before the age of 45 years. Main Outcome The number of axSpA patients according to the ASAS criteria. Methods The referral rule (CaFaSpA referral rule) was developed using 364 CLBP patients from 19 primary care practices and contains four easy to use variables; inflammatory back pain, good response to nonsteriodal anti-inflammatory drugs, family history of spondyloarthritis and a back pain duration longer than five years. This referral rule is positive when at least two variables are present. Validation of the CaFaSpA rule was accomplished in 579 primary care CLBP patients from 38 practices from other areas. Performance of the referral rule was assessed by c-statistic and calibration plot. To fit the final referral rule the development and validation datasets were pooled leading to a total study population of 943 primary care participants. Results The referral rule was validated in 579 patients (41% male, mean age 36 (sd7.0). The percentage of identified axSpA patients was 16% (n=95). External validation resulted in satisfactory calibration and reasonable discriminative ability (c-statistics 0.70 [95% CI, 0.64-0.75]). In the pooled dataset sensitivity and specificity of the referral rule were 75% and 58%. Conclusions The CaFaSpA referral rule for axSpA consists of four easy to use predictors for primary care physicians and has a good predictive value in this validation study. The referral rule has the potential to be a screening tool for primary care by identifying CLBP patients suspected for axSpA