Commercial C-reactive Protein Research Articles

Some Acute Phase Proteins and Biochemistry Parameters of Female Dogs Affected with Pyometra

Some Acute Phase Proteins and Biochemistry Parameters of Female Dogs Affected with Pyometra

Impact of serum C-reactive protein level as a biomarker of cancer dissemination in canine lymphoid neoplasia.

C-reactive protein (CRP) is a highly sensitive but non-specific acute phase protein that has been widely used to predict the biological behavior of patients with cancer. This study aimed to examine the significance of the serum CRP biomarker in predicting the prognosis of dogs with lymphoma. Blood samples (5 mL) were collected from 34 lymphoma dogs and control healthy dogs. Canine lymphoma clinical staging was classified using the World Health Organization (WHO) criteria. All lymphoma dogs were reclassified into two groups based on the disease stage. Stages IV and V were designated as advanced stages, and Stages I-III were designated as other stages. The serum CRP level was then determined using a commercial canine CRP fluorescent immunoassay kit and routine hematological and biochemical analyses. C-reactive protein levels, circulating inflammatory parameters, such as neutrophil-to-lymphocyte ratio, lymphocyte-to-monocyte ratio, and platelet-to-lymphocyte ratio, and albumin levels were compared between advanced stages (IV and V) and Stages I to III using Mann-Whitney U tests. Receiver operating characteristic (ROC) curves were also generated to determine the cutoff value, diagnostic sensitivity, and specificity of the CRP level. A prospective study identified 34 dogs recently diagnosed with canine lymphoma. C-reactive protein levels were significantly higher in lymphoma dogs in advanced stages (IV and V) than in lymphoma dogs in Stages I-III. According to the ROC curve analysis, a CRP cutoff level of 54.1 mg/L indicates advanced-stage canine lymphoma, which can be used as a biomarker to predict cancer dissemination. Serum CRP concentrations can assist clinical decision-making on the WHO stage in lymphoma dogs in clinical applications. The limitations of this study include a small number of lymphomas and no survival analysis.

Immuno-battery: A single use self-powered immunosensor for REASSURED diagnostics

In this work, we present a novel self-powered approach totally independent from any external energy source. We have developed a self-powered paper-based immunosensor that generates energy in the presence of the biomarker in the sample. In particular, the device – which has been labeled as Immuno-Battery – makes use of magnesium as anode and the widely employed HRP-labeled antibody as cathodic catalyst to detect C-reactive protein (CRP) presence in artificial samples. Feasibility of self-powered sensing is proved by submitting the immuno-battery to a resistive load. In this regime, the sensor provides operation voltages above 1.55 V and maximum power densities from 40 to 571 μW cm−2 that allow for future implementation of an electronic readout circuit. We have demonstrated that sensitivity of the system is not compromised by the self-powered mode operation, as the LOD value delivered by our battery (20 ± 2 ng mL−1) is compliant with LOD values reported for protein detection in paper-based electrochemical immunoassays with chronoamperometric methods. Moreover, as a case study, a LOD of 269 ± 39 ng mL−1 is obtained for CRP detection, in accordance with available commercial high-sensitivity CRP detection kits. This proof-of-concept opens the path towards the development of digital diagnostic devices in a sustainable and affordable manner.

CRP and LOX-1: a Mechanism for Increasing the Tumorigenic Potential of Colorectal Cancer Carcinoma Cell Line.

Chronic inflammation and dyslipidemia are associated with an increase in the incidence of colorectal cancer (CRC). Serum C- reactive protein (CRP) and oxidized low-density lipoprotein (oxLDL), as Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) ligands, increase during inflammation and dyslipidemia, respectively. To evaluate the effects of CRP on the expression of important genes involved in the development of CRC, the CRC cell line, LS174T, was treated with the commercial CRP. Based on the Real-time PCR data, in the presence of CRP, LOX-1, CEA, MMP1, and MMP2 mRNA expression significantly increased, compared to the control group. Moreover, in the presence of CRP, secretion, and expression of CEA in the cell lysate and conditioned media increased in a concentration-dependent manner. The results of flow cytometry showed that expression of LOX-1 receptors at the cell surface increased significantly in the presence of 10mg/L of CRP. However, inhibition of LOX-1 receptors with a specific monoclonal antibody reduced the effects of CRP on protein/mRNA expression. In conclusion, Increased CRP level, can potentially elevate the expression of important genes in CRC by stimulating LOX-1 receptors.

A Conductive Nanowire‐Mesh Biosensor for Ultrasensitive Detection of Serum C‐Reactive Protein in Melanoma

AbstractDetection of extracutaneous melanoma is still challenging and is of importance in improving survival rate. In this report, an ultrasensitive biosensor is constructed where a C‐reactive protein (CRP) aptamers based molecular recognition core and a conductive polypyrrole (PPy) nanowire mesh based signal amplifier are developed. The conductive PPy nanowire (less than 10 nm in diameter) mesh architecture is uniformly dispersed within polymeric matrix via template‐free in situ synthesis. Serum CRP levels are quantitatively analyzed through monitoring the conductance change caused by polymeric network shrinkage upon the aptamer‐CRP binding. The limit of detection (LOD) of the polymeric sensor for human CRP sample can reach 7.85 × 10−19m. This CRP‐specific biosensor and a commercial CRP enzyme‐linked immunosorbent assay (ELISA) kit are used to perform side‐by‐side measurement of serum CRP in melanoma patients. The results indicate that this conductive polymeric senor is highly sensitive and selective in accurately discriminating melanoma patients from healthy controls using serum CRP as a biomarker, which is further validated by a commercial human CRP ELISA kit. Collectively, this novel ultrasensitive nanowire‐based polymeric biosensor may hold promise in biomarker detection and diagnosis of cancer.

Expression and characterization of 15N-labeled human C-reactive protein in Escherichia coli and Pichia pastoris for use in isotope-dilution mass spectrometry

Levels of C-reactive protein (CRP) in serum are correlated with inflammation and disease in humans. A higher level quantitative method, such as isotope-dilution mass spectrometry (ID–MS) is needed to compare and standardize the many commercial CRP assays. We compare the expression and purification of 15N-CRP from Escherichia coli and Pichia pastoris and show that the protein isolated from P. pastoris has native pentameric structure along with high isotopic enrichment as shown by software developed specifically for this purpose. When this preparation was mixed in various ratios with unlabeled CRP and tryptic peptides of the mixtures were analyzed by LC–MS/MS, the ratios of heavy and light peaks were tightly correlated with input amounts of each protein. In this report we confirm the suitability of 15N-rCRP as an internal standard in ID–MS. Standardization of CRP assays should help validate the relationship between CRP and human health.

A Functional Turbidimetric Method to Determine C-Reactive Protein in Horses

A turbidimetric method to determine serum C-reactive protein (CRP) concentration, based on soybean oil-phosphocholine interaction, was performed on horse serum samples to evaluate its potential diagnostic value in veterinary medicine. Intralipid 20% in 0.1 M Tris-calcium buffer (pH 7.5) was added to horse serum. After 30 min of incubation at 37 °C, the CRP-phosphocholine complexes were turbidimetrically, bichromatically (660 nm/700 nm) quantified on a commercial analyzer. Furthermore, comparison between CRP and other inflammatory markers, including white blood cell and neutrophil counts, was performed to evaluate the diagnostic value of both tests. Standardization of the assay was done using a commercial human CRP calibrator. The CRP measurements were performed on serum samples (296 patients and 34 controls). Reference values were found to be lower than 10 mg/l. The method was found to be linear between 1 and 400 mg/l. A moderate correlation was observed between CRP values and the relative neutrophil counts. Receiver-operating characteristics analysis demonstrated the area under the curve for CRP was 0.928, which was superior (P < 0.001) to the neutrophil count (0.804) and the leukocyte count (0.664) in detecting the presence of inflammation. This CRP assay showed reliable results as an acute phase test in horses, confirming its species-independent capability to detect CRP in various mammals, including horses.

Adhesion molecules (VCAM‐1 and ICAM‐1) and C‐reactive protein in women with history of preeclampsia

Several studies suggest that women with previous preeclampsia (PE) are at increased risk of cardiovascular disease (CVD). We examined circulating concentrations of adhesion molecules and C-reactive protein (CRP), markers for endothelial and inflammatory reactions, in addition to blood pressure and anthropometric measurements in 58 women with a history of PE and 49 control women with no pathology associated to pregnancy. Soluble adhesion molecules were measured by standard commercial ELISA methods and plasma CRP levels by automated enzymatic assays. Systolic and diastolic blood pressures and waist-to-hip ratios were significantly higher in women with history of PE than in control group. There were no significant differences in circulating levels of sICAM-1, sVCAM-1 and CRP in the study population. Women with a history of PE do not have a persistent inflammatory state that could induce overexpression of these molecules, which was supported by normal levels of CRP. The study supports the existence of common risk factors for PE and CVD, namely obesity and hypertension.

Significance of Serum TRACP in Rheumatoid Arthritis

Human serum contains two related isoforms of TRACP: TRACP 5a and TRACP 5b. Serum TRACP 5a protein is increased in about one third of rheumatoid arthritis (RA) sera. This study was undertaken to examine the significance of serum TRACP isoforms 5a and 5b as disease markers of inflammation and bone destruction in RA. One hundred eighteen patients were recruited including 50 with RA (25 with nodules), 26 with osteoarthritis (OA), and 42 with other rheumatic diseases. Twenty-six healthy adults served as controls. Serum TRACP 5a activity, TRACP 5a protein, and TRACP 5b activity were determined by in-house immunoassays. C-reactive protein (CRP) was determined by in-house immunoassay using commercial antibodies and CRP. Other commercial markers included bone-specific alkaline phosphatase (BALP), C-telopeptides of type-I collagen (ICTP), cartilage glycoprotein-39 (YKL-40), and IgM rheumatoid factors (IgM-RF). Mean TRACP 5a protein was significantly elevated only in RA compared with healthy controls and other disease groups. TRACP 5a protein correlated significantly only with IgM-RF in RA. Among RA patients, mean TRACP 5a protein and IgM RF were significantly higher in nodule formers. In contrast, TRACP 5b activity was slightly elevated in RA and correlated with BALP, ICTP, and YKL-40 but not with IgM-RF or CRP. Mean TRACP 5b activity was no different in RA patients with or without nodules. TRACP isoforms could be useful disease markers in RA; TRACP 5a protein may be a measure of systemic inflammatory macrophage burden and disease severity. TRACP 5b activity is a marker for osteoclast number and perhaps local or systemic bone destruction.

C-Reactive Protein and Reendothelialization

See related article, pages 1452–1459 Disruption of the endothelium and its subsequent dysfunction appears to set the stage for atherogenesis.1 The maintenance of vascular homeostasis depends in part on a balance between endothelium-derived relaxing and contracting factors. Alterations in this balance lead to inflammation and the formation of fatty streaks and fibrous plaques. The diminished production or availability of nitric oxide (NO) appears paramount to setting the stage for inflammation and vascular injury. In addition to its vasodilatory effect on the vasculature, endothelium-derived NO promotes endothelial cell growth, survival and migration,2 stimulates angiogenesis and neovascularization in part by endothelial progenitor cell (EPC) recruitment,3 inhibits leukocyte adhesion to the endothelium,4 maintains vascular smooth muscle in a nonproliferative state,5 and limits platelet aggregation and thrombosis.6 Maintenance of an intact and functional endothelium protects against vascular disease while its disruption is detrimental. The study by Schwartz et al,7 in this issue of Circulation Research , adds to our understanding of how C-reactive protein (CRP) works to promote vascular pathology by inhibiting NO synthesis. CRP, long regarded as an acute phase protein and an active participant in the innate immune system, has more recently been linked to the development of cardiovascular disease.8 On the basis of several prospective epidemiologic studies, circulating high sensitivity CRP has emerged as an independent predictor of cardiovascular disease risk in various subgroups of patients, including those without overt cardiovascular disease, patients with stable angina or acute coronary syndromes, and in those with the metabolic syndrome.9–12 In primary prevention, CRP confers additional prognostic value at all levels of Framingham risk score and blood pressure.13,14 However, whether or not CRP plays a direct biological role in the initiation and progression of atherosclerosis is controversial.15,16 …

The Factor H Variant Associated with Age-related Macular Degeneration (His-384) and the Non-disease-associated Form Bind Differentially to C-reactive Protein, Fibromodulin, DNA, and Necrotic Cells

Recently, a polymorphism in the complement regulator factor H (FH) gene has been associated with age-related macular degeneration. When histidine instead of tyrosine is present at position 384 in the seventh complement control protein (CCP) domain of FH, the risk for age-related macular degeneration is increased. It was recently shown that these allotypic variants of FH, in the context of a recombinant construct corresponding to CCPs 6-8, recognize polyanionic structures differently, which may lead to altered regulation of the alternative pathway of complement. We show now that His-384, corresponding to the risk allele, binds C-reactive protein (CRP) poorly compared with the Tyr-384 form. We also found that C1q and phosphorylcholine do not compete with FH for binding to C-reactive protein. The interaction with extracellular matrix protein fibromodulin, which we now show to be mediated, at least in part, by CCP6-8 of FH, occurs via the polypeptide of fibromodulin and not through its glycosaminoglycan modifications. The Tyr-384 variant of FH bound fibromodulin better than the His-384 form. Furthermore, we find that CCP6-8 is able to interact with DNA and necrotic cells, but in contrast the His-384 allotype binds these ligands more strongly than the Tyr-384 variant. The variations in binding affinity of the two alleles indicate that complement activation and local inflammation in response to different targets will differ between His/His and Tyr/Tyr homozygotes.

C-reactive protein and cardiovascular disease: Weighing the evidence

C-reactive protein (CRP) has been widely promoted as a strong, independent predictor of cardiovascular events and metabolic syndrome, both in general populations and in patients with clinical cardiovascular disease, and as a causal player in atherothrombosis. However, recent evidence shows that the association of CRP with cardiovascular events is weaker than previously thought, that it may be largely attributed to confounding by established causal risk factors, and that CRP is, therefore, probably not a clinically useful risk predictor. The lack of association of noncoding CRP gene polymorphisms (which determine different baseline CRP values) with coronary events or metabolic syndrome does not support a causal role for CRP, and most of the putatively proatherothrombotic in vitro effects claimed for CRP were caused by contaminants in commercial CRP preparations and not by CRP. Future clinical trials of specific CRP inhibitors now in development could directly test the contribution of CRP to pathogenesis of cardiovascular disease.

Lipopolysaccharide (LPS) Contamination Plays the Real Role in C-Reactive Protein–Induced IL-6 Secretion From Human Endothelial Cells In Vitro

To the Editor, In a recent article, Taylor et al1 have shown that C-Reactive Protein (CRP), per se, does not activate endothelial cells; rather, lipopolysacchride (LPS) and azide were responsible for the observed cellular activation. We would like to provide our data to further support the conclusion by Taylor and colleagues. We have tested >10 batches of commercial human CRP preparations from 3 companies (Calbiochem, TriChem, and Sigma). Human saphenous vein endothelial cells (HUVECs, passages 3 to 5) were grown …

Proapoptotic, Antimigratory, Antiproliferative, and Antiangiogenic Effects of Commercial C-Reactive Protein on Various Human Endothelial Cell Types In Vitro

Recent experimental studies suggest C-reactive protein (CRP) may be a potential mediator of atherosclerosis and its complications. However, there is growing criticism of in vitro CRP studies that use commercial CRP preparations containing biologically active contaminants. The effects of commercial CRP, dialyzed commercial CRP (dCRP) to remove azide, and sodium azide (NaN3) alone at equivalent concentrations to the undialyzed preparation were tested at varying concentrations on human umbilical vein endothelial cells (HUVEC), circulating endothelial outgrowth cells (EOC), and endothelial progenitor cells (EPC) in vitro. CRP and NaN3 alone exhibited equivalent concentration-dependent, proapoptotic effects on HUVEC, EOC, and EPC (P<0.01 versus control), whereas dCRP had no such effect. Similarly, CRP and NaN3 alone caused equivalent concentration-dependent decreases in migration, proliferation, and matrigel tube formation (P<0.01 versus control) in EOC and HUVEC, whereas dCRP had absolutely no effect on these biological functions at any of the concentrations used. We conclude that proapoptotic, antiproliferative, antimigratory, and antiangiogenic effects of this commercial CRP preparation on a number of endothelial cell phenotypes in culture may be explained by the presence of sodium azide in this preparation. This study has implications for interpretation of in vitro studies using CRP preparations containing azide at equivalent or higher concentrations.

CRP—Marker or Maker of Cardiovascular Disease?

C-reactive protein (CRP) has emerged as an interesting novel and potentially clinically useful marker for increased cardiovascular risk.1,2 This is an attractive concept because atherosclerosis is a disease characterized by chronic arterial inflammation3,4 and suggests the possibility that subclinical states of atherosclerosis can be identified by an increase in circulating markers of inflammation before acute events occur. Based on data obtained primarily from in vitro studies it has also been proposed that CRP in itself is actively contributing to disease progression and that it should be considered as true risk factor and consequently as a target for intervention. However, in the present issue of Arteriosclerosis, Thrombosis, and Vascular Biology , two independent reports demonstrate that transgenic overexpression of CRP does not affect the development of atherosclerosis in mice, suggesting that this is not the case. See pages 1635 and 1641 The association between moderately elevated CRP levels and an increased risk for development of cardiovascular disease is well established. Although the increase in risk may have been overestimated in initial studies, recent meta-analysis comprising >7000 patients with coronary events shows that subjects with CRP in the upper tertile have a 50% increased risk for development of acute cardiovascular events.2 There are several possible explanations for this association (Figure). (1) The inflammatory process in arterial tissue affected by atherosclerosis results in release of cytokines into the circulation that subsequently activates expression of CRP in the liver. Plasma CRP levels would then reflect the severity of atherosclerosis and, as a consequence, the risk for development of clinical events. If CRP in addition …

T05-P-005 Sodium azide and lps in commercial C-reactive protein (CRP) preparations are responsible for reported in vitro effects attributed to CRP

T05-P-005 Sodium azide and lps in commercial C-reactive protein (CRP) preparations are responsible for reported in vitro effects attributed to CRP

C-Reactive Protein–Induced In Vitro Endothelial Cell Activation Is an Artefact Caused by Azide and Lipopolysaccharide

C-reactive protein (CRP) has been proposed to be an independent risk factor for cardiovascular disease. In vitro studies investigating the mechanism behind this have used purified commercial CRP (cCRP) and endothelial cells. We investigated the role of contaminants in cCRP preparations. Human umbilical vein endothelial cells and the human endothelial cell line EA.hy926 were incubated with Escherichia coli-derived cCRP, in-house-generated azide-free recombinant, and ascites-purified CRP, azide, or lipopolysaccharide (LPS) equivalent to the concentration present in cCRP preparations. Cells were investigated for change in cell proliferation, morphology, apoptosis, and expression of endothelial NO synthase and intercellular adhesion molecule-1. Cell supernatants were assessed for monocyte chemoattractant protein-1 (MCP-1), interleukin-8, von Willebrand factor secretion, and pH change. Only cCRP was able to induce all activation events analyzed; however, this ability was lost on extensive dialysis, suggesting that low molecular weight contaminants were responsible for these events. Indeed, the effects of cCRP were mirrored by azide or LPS. We investigated a wide range of effects on endothelial cells ascribed to CRP; however, azide and LPS, but never CRP itself, were responsible for the cell activation events. We conclude that CRP, per se, does not activate endothelial cells.

Evidence for Sodium Azide as an Artifact Mediating the Modulation of Inducible Nitric Oxide Synthase by C-Reactive Protein

C-reactive protein (CRP) is an acute-phase protein identified as a cardiovascular risk marker. In recent years, an increasing number of studies have investigated the possible direct effects of CRP on the vasculature, using mainly commercial CRP. In the present work, a potential role for CRP as a modulator of inducible nitric oxide synthase (iNOS) induction was explored. Cultured human aortic vascular smooth muscle cells (HASMC) were stimulated for 18 hours with 10 ng/mL interleukin-1beta (IL-1beta), resulting in a marked increase of iNOS levels and NO production, as determined by Western blotting and nitrite measurement, respectively. Commercial CRP (1 to 100 microg/mL) concentration-dependently inhibited the effects elicited by IL-1beta. Unexpectedly, similar results were observed when the commercial CRP solution was replaced by the corresponding vehicle medium containing growing concentrations of sodium azide. The inhibitory effects of commercial CRP or vehicle medium were lost on sodium azide removal by dialysis. In conclusion, sodium azide from the commercial CRP solution, but not CRP itself, mainly accounts for the inhibitory effect on IL-1beta-evoked iNOS induction and NO release. Care should be taken before attributing any biologic role to commercial CRP containing sodium azide.

Letter Regarding Article by Li et al, “C-Reactive Protein Upregulates Complement-Inhibitory Factors in Endothelial Cells”

To the Editor: Recently, Li et al reported an increase in decay accelerating factor (DAF) expression on human endothelial cells on incubation with C-reactive protein (CRP).1 Using our in-house–generated recombinant CRP,2 we were unable to reproduce these effects on human umbilical vein endothelial cells or the endothelial cell line EA.hy926. Commercial CRP (recombinant, Calbiochem), however, increased DAF expression on both cell types. Commercial CRP preparations contain 0.05% to 0.1% azide …

C-reactive protein-induced in vitro vasorelaxation is an artefact caused by the presence of sodium azide in commercial preparations.

Although C-reactive protein (CRP) is increasingly recognized as an independent risk factor for acute myocardial events, recent evidence suggests that it can directly induce vasorelaxation. This study aimed to investigate the mechanism of this CRP-induced response. Isometric tension recordings were used to measure endothelium-dependent and endothelium-independent vascular smooth muscle relaxation in isolated rabbit aortic rings. CRP generated in-house by genetic engineering and expressed in Chinese hamster ovary cells, CRP purified from ascites, and CRP obtained from commercial sources were assessed for vasorelaxing properties. Only the commercial CRP preparation induced vasorelaxation; more than half maximal relaxation was observed at 0.025 microg/mL and maximum relaxation attained at 0.25 microg/mL. Commercial CRP contains high levels of sodium azide, a well-known vasorelaxant. Removal of this agent by dialysis abolished the vasodilatory effect of commercial CRP. Sodium azide alone at concentrations equivalent to that present in the commercial CRP produced a near-identical relaxation pattern to the undialyzed commercial product. CRP has no vasorelaxant properties per se, and the reported vasorelaxant ability of CRP is an artifact caused by sodium azide present in commercial preparations of this agent.