High resolution isoelectric focusing of immunoprecipitated proteins under denaturing conditions. A simple analytical method applied to the study of complement component polymorphisms

A simple analytical method for the study of structural protein polymorphisms is described. It consists of the immunoprecipitation of non-radiolabeled proteins using monospecific polyclonal antibodies followed by isoelectric focusing (IEF) under completely denaturing conditions in vertical polyacrylamide slab gels. The method uses small amounts of sample (usually unfractionated plasma or serum), requires no sophisticated equipment and allows the screening of large numbers of samples with comparatively small effort. This method has been applied in the identification of 2 human complement-component polymorphisms, C4-binding protein (C4-bp) and factor H (beta 1H).     

Serum C-reactive protein and complement proteins in patients with acute myocardial infarction

C-reactive protein (CRP) and complement proteins levels were determined in 20 patients with acute myocardial infarction (AMI) and 20 controls. Blood was obtained from all subjects at admission, 6 hr and 12 hr later. Serum CRP levels were determined by ELISA and complement proteins by radial immunodiffusion. A statistically significant elevation of the mean CRP level was obtained at 12 hr postadmission. The mean complement proteins levels were 16-49% higher in AMI patients than the controls. It appeared that the alternate pathway was activated initially, followed by activation of the classical pathway. The increased levels of CRP and complement proteins are suggestive of their involvement in AMI.     

Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis

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Significant association between Helicobacter pylori infection and serum C-reactive protein

Background: Helicobacter pylori (H. pylori) infection in gastric mucosa may cause systemic inflammatory reaction. This study aimed to examine the association between the infection and serum high sensitivity C-reactive protein (hsCRP).Methods: Subjects were comprised of three groups; 453 health checkup examinees from Yakumo town inhabitants in Hokkaido, Japan (YTI, 153 males and 300 females), 449 health checkup examinees (ENUH, 273 males and 176 females), and 255 female patients of an infertility clinic (PIC), Nagoya University Hospital. Twenty participants with hsCRP more than 1 mg/dl were excluded from the analysis. Those with hsCRP more than 0.1mg/dl were defined as high hsCRP individuals. H. pylori infection status was examined with a serum IgG antibody test.Results: When the three groups were combined, the geometric mean of hsCRP concentration was significantly higher among the seropositives (0.047mg/dl) than among the seronegatives (0.035mg/dl); p<0.0001 by a t-test. The percentage of high hsCRP individuals was also higher in the seropositives than in the seronegatives among any group; 23.3% and 20.1% in YTI, 22.0% and 16.0% in ENUH, and 32.7% and 18.7% in PIC, respectively, although the difference was significant only in ENUH. The summary odds ratio of the high hsCRP for the seropositives relative to the seronegatives was 1.38 (95% confidence interval, 1.01-1.89), when age, sex, body mass index, smoking, and subject group were adjusted by a logistic model.Conclusions: In three groups, hsCRP was higher among the infected individuals. The summary odd ratio indicated that H. pylori infection could influence the serum hsCRP level.     

Complement regulation in innate immunity and the acute-phase response: inhibition of mannan-binding lectin-initiated complement cytolysis by C-reactive protein (CRP)

Mannan-binding lectin (MBL) is an acute-phase protein which activates complement at the level of C4 and C2. We recently reported that the alternative pathway also is required for haemolysis via this ‘lectin pathway’ in human serum. CRP is another acute-phase reactant which activates the classical pathway, but CRP also inhibits the alternative pathway on surfaces to which it binds. Since serum levels of both proteins generally increase with inflammation and tissue necrosis, it was of interest to determine the effect of CRP on cytolysis via the lectin pathway. We report here that although CRP increases binding of C4 to MBL-sensitized erythrocytes, which in turn enhances lectin pathway haemolysis, it inhibits MBL-initiated cytolysis by its ability to inhibit the alternative pathway. This inhibition is characterized by increased binding of complement control protein H and decreased binding of C3 and C5 to the indicator cells, which in turn is attributable to the presence of CRP. Immunodepletion of H leads to greatly enhanced cytolysis via the lectin pathway, and this cytolysis is no longer inhibited by CRP. These results indicate that CRP regulates MBL-initiated cytolysis on surfaces to which both proteins bind by modulating alternative pathway recruitment through H, pointing to CRP as a complement regulatory protein, and suggesting a co-ordinated role for these proteins in complement activation in innate immunity and the acute-phase response.     

Occurrence of C-reactive protein in cryoglobulins

A previous case report described the formation of a complex between a monoclonal IgA with cryolabile properties and C-reactive protein (CRP). Our study provides the first evidence for the frequent occurrence of CRP in cryoglobulins (Cg) of all three types according to Brouet's classification. We performed a systematic immunochemical analysis of cryoglobulins from 18 patients by Western blotting and in 15 of 18 cryoprecipitates a single band (23 KD), immunoreactive with anti-CRP antibody, was demonstrable irrespective of the clonal composition of the cryoglobulins. This band was detectable in 4/5 of type I, in 6/8 of type II, and in 5/5 of type III cryoprecipitates, classified according to Brouet et al. In addition, the complement proteins C1q and C3 were present in nearly all CRP-containing cryoglobulins, presumably reflecting previous activation of the classical complement pathway at least. All three CRP-negative cryoprecipitates were derived from sera with low cryoglobulin content (1-2 g/l). Longitudinal investigation of 23 cryoprecipitates from seven patients confirmed that successful detection of CRP by Western blotting depends on the protein concentration of the cryoglobulins. Since complexed CRP was previously shown to be an effective activator of complement, via C1q binding, CRP may modulate pathophysiologic effects mediated by cryoglobulins in vivo.     

Isolation of human complement factors C3, C5 and H

An improved method for simultaneous purification of complement factors C3, C5 and H from human plasma has been developed. Using an initial batch separation technique with QAE-Sephadex, followed by chromatography on SP-Sephadex and gel filtration in Sephadex G-200, 600 mg of highly pure C3 can be prepared from 1600 ml of plasma. Simultaneously about 70 mg of highly pure factor H and 30 mg of C5 are obtained by chromatography of post SP-Sephadex material on DEAE-Sephacel. A small amount of C3 in the C5 pool is removed by anti-C3-Sepharose. By maleylation or citraconylation of reduced and alkylated C3, the constitutive polypeptide chains are modified in a way that made them separable by ion exchange chromatography.     

Childhood overweight and asthma symptoms,the role of pro‐inflammatory proteins

Background Systemic inflammation is suggested as a mechanism by which overweight might induce asthma. However, few studies have linked childhood overweight, inflammation and asthma. Objective To study the association between body mass index (BMI), asthma symptoms and pro‐inflammatory proteins. Methods High‐sensitivity C‐reactive protein (hs‐CRP), complement factor 3 (C3) and 4 (C4) concentrations, and body weight and height were available for 359 4‐year‐old children participating in the Prevention and Incidence of Asthma and Mite Allergy birth cohort study. Data on asthma symptoms were obtained by yearly questionnaires. Logistic regression and generalized estimating equations were used to analyse the cross‐sectional and prospective associations between BMI, asthma symptoms and pro‐inflammatory proteins. Results BMI was associated with asthma symptoms {odds ratio [OR] 1.43 [95% confidence interval (CI): 1.08–1.88] per BMI standard deviation scores [SDS]}. The inclusion of hs‐CRP, C3 and C4 in the statistical models did not change this association. C3 was cross‐sectionally associated with frequent asthma symptoms [OR per interquartile range of C3: 1.97 (95% CI: 1.20–3.24)] and prospectively with asthma symptoms [OR: 1.48 (95%CI: 1.04–2.09)], independent of BMI SDS. Conclusions and Clinical Relevance We showed no evidence for a role of hs‐CRP, C3 and C4 in the association between BMI and asthma symptoms. C3 concentrations were associated with (frequent) asthma symptoms, independent of BMI. Cite this as: M. B. M. Bekkers, B. Brunekreef, J. C. de Jongste, M. Kerkhof, H. A. Smit, D. S. Postma, U. Gehring and A. H. Wijga, Clinical & Experimental Allergy, 2012 (42) 95–103.     

Influence of heparin on interactions between C-reactive protein and polycations

C-reactive protein (CRP) is a trace serum protein which elevates up to 1000-fold in concentration in association with inflammation and tissue necrosis. CRP binds with phosphocholinc and phosphate esters; initiates reactions of agglutination, opsonization and complement consumption; and binds to and influences the reactivities of platelets and lymphocytes. CRP also selectively and reversibly precipitates with protamine and synthetic polymers of lysine and arginine, and these reactivities are modulated by calcium and phosphocholine. We now report on the interactions of heparin with these polycations in the absence and presence of CRP, which show marked similarities to reactions between antigen and antibody. Heparin optimally precipitated with the polycations over a narrow range of reactant ratios, peaking at slight anion charge excess. The complexes formed did not dissociate in excess heparin; however, complex formation was significantly depressed when heparin was added in a single dose in excess of the amount required for optimal precipitation. Calcium did not affect the precipitation of heparin with polycation, and heparin did not precipitate with CRP. However, heparin did induce a rapid and efficient dissociation of CRP-polycation precipitates preformed at equivalence, with total release of CRP. Small amounts of heparin augmented precipitation under conditions of polycation excess to CRP. but as heparin levels were increased to amounts needed to reach equivalence with polycation. CRP was resolubilized in favor of the preferred heparin-polycation complexes. Chondroitin sulfate was similar to heparin in its effects upon the CRP-poly-l-arginine (PLA) interaction, while hyaluronic acid enhanced CRP-PLA precipitation at all concentrations tested and DNA had neither augmenting nor solubilizing effects. These data indicate that CRP-polycation interactions are significantly and selectively influenced in the presence of small amounts of heparin and certain other polyanions. This modulatory reactivity may be of importance in the biological reactivities of CRP during episodes of acute inflammation.     

妊娠期糖尿病与孕妇血清C-反应蛋白水平相关性研究

目的：探究妊娠期糖尿病与孕妇血清C-反应蛋白水平相关性。方法：选取2014年4月至2015年11月于我院接受孕检的孕妇190例,其中糖尿病孕妇97例,作为本研究实验组;另外93例健康状况良好,作为对照组。记录并比较两组孕妇年龄、平均孕周、孕前体重指数(BMI);检查并比较其血清C-反应蛋白(CRP)水平、胰岛素抵抗指数、空腹血糖水平、空腹胰岛素水平、孕期体重增长,并对CRP的相关影响因素进行Pearson相关分析以及多元线性回归分析。结果：对照组孕妇平均年龄为(29.13±2.18)岁,平均孕周为(25.36±3.23)周,孕前BMI为(20.27±1.72) kg/m2;实验组孕妇平均年龄为(28.24±1.97)岁,平均孕周(26.13±2.79)周,孕前BMI为(20.32±1.68) kg/m2,对比结果无统计学差异(P>0.05);对照组空腹血糖含量为(4.62±0.54) mmol/L,实验组为(5.21±0.81) mmol/L,对比结果无统计学意义(P>0.05);对照组空腹胰岛素水平为(8.93±1.76) mmol/L,实验组为(11.71±4.83) mmol/L,对比结果具有统计学意义(P<0.05);对照组孕妇血清CRP含量为(2.17±0.76) mg/L,胰岛素抵抗指数为1.76±0.69,孕期体重增长(13.47±2.01) kg,实验组为孕妇血清CRP含量为(4.12±0.73) mg/L,胰岛素抵抗指数为2.57±2.18,孕期体重增长(17.13±5.79) kg,对比结果具有统计学意义(P<0.05);Pearson分析显示血清CRP水平与胰岛素抵抗指数、孕期体重增长、孕前体重指数、空腹血糖呈正相关关系,其系数分别为0.369、0.319、0.289、0.268,均具有统计学意义(P<0.05),对CRP的多元线性回归分析,其方程为y(CRP)=0.319X1+0.09X2+0.239X3?3.879,r2=0.259(X1：胰岛素抵抗指数,X2：孕期体重增长,X3：孕前体重指数)。结论：对于妊娠期妇女,血清C-反应蛋白水平超标是引起糖尿病的重要因素之一,但独立影响尚不能被证实,建议临床上在孕期定期检查孕妇血清C-反应蛋白水平,有利于妊娠期糖尿病的有效防治。     

The effect of Helicobacter pylori eradication on C-reactive protein: results from a meta-analysis

IntroductionHelicobacter pylori is a bacterium that causes chronic gastroduodenal infection and affects various systemic diseases. An increase in the blood level of C-reactive protein (CRP; a systemic inflammatory marker), at a low-grade chronic inflammation level, is observed in cases of infection. However, the effect of H. pylori eradication on CRP remains undetermined. Therefore, we aimed to evaluate the circulating CRP levels in eradicated patients through a meta-analysis.Material and methodsThe PubMed database was searched from its inception to June 2020. Studies that described the CRP levels following H. pylori eradication were collected. A random-effects meta-analysis was then performed using inverse variance with standardized mean difference.ResultsA total of 10 eligible studies (642 subjects in total) were available. The median age in the studies was 49.9 years. The CRP level was 6.0 (median) mg/l before H. pylori eradication and 5.8 (median) mg/l after eradication. From the results of the overall meta-analysis, there was found to be a significant reduction in the CRP levels with H. pylori eradication (standardized mean difference: –0.64; 95% confidence interval: –1.02 to –0.27). The result was not similarly confirmed in a subanalysis of the available randomized controlled trials.ConclusionsWeak evidence exists regarding the effects of H. pylori eradication on CRP levels. Further research is called for.     

C-反应蛋白在动脉粥样硬化中的作用

C-反应蛋白（C-reactive protein,CRP）是反映炎症的敏感指标。近年的研究揭示超敏C-反应蛋白（high-sensitive CRP,hs-CRP）是心血管疾病的危险标志之一。同时还发现CRP自身可能直接参与了动脉继样硬化的过程,CRP可增加单核细胞向血管壁黏附、促进巨噬细胞摄取LDL,促使内皮细胞功能失调及促进平常细胞迁移和增生;CRP还与血栓形成和斑块的破裂有关。     

Immunohistochemical studies of C-reactive protein and apolipoprotein B in inflammatory and arterial lesions

Interactions in vivo between C-reactive protein (CRP) and apolipoprotein B (apo-B)-containing lipoproteins were sought in inflammatory lesions and atherosclerosis. CRP was demonstrated immunohistochemically on the surface of some muscle fibres in locally induced inflammatory lesions in the rabbit, but apoB was not detected in the same distribution. CRP was not detected in catheter-induced aortic endothelial injuries in the rabbit, in arterial lesions containing apoB from cholesterol-fed rabbits, in apoB-containing human fatty streaks or in advanced human atherosclerotic lesions.     

Targeted inhibition of complement using complement receptor 2-conjugated inhibitors attenuates EAE

Multiple sclerosis (MS) is the most common autoimmune demyelinating disease, affecting millions of individuals worldwide. In the last two decades, many therapeutic options for the treatment of MS have become available, however they are limited in terms of effectiveness and some remain plagued by safety issues. The currently available treatment options target relapsing remitting forms of MS and are not effective against the more progressive forms of the disease. These limitations highlight a significant unmet treatment need for MS. In experimental autoimmune encephalomyelitis (EAE) studies from our laboratory, we have previously shown, using a number of complement mutant and transgenic mice, that inhibition of the alternative complement pathway and the C3 convertase confers significant protection from disease. We report here that targeted inhibition of complement activation using complement receptor 2 (CR2)-conjugated inhibitors significantly attenuates EAE. Administration of CR2-Crry (blocks all complement pathways at C3 activation) and CR2-fH (specifically blocks the alternative pathway) just prior to and during the onset of EAE blocks progression of both acute and chronic disease. These data indicate that inhibition of complement may offer an effective therapeutic approach to treating both acute and chronic forms of demyelinating disease through blocking the alternative pathway or complement convertases.     

Association between C-reactive protein and pulmonary function in postmenopausal women

Objectives

The objective of this study is to investigate the association of CRP and impaired pulmonary function in postmenopausal women.

Methods

The study was carried out in Gangnam Severance Hospital in Korea between March 2006 and October 2008. A total of 5978 healthy women subjects (age range, 20–75 years) were recruited from Seoul. We performed a cross-sectional study to evaluate the association of CRP with impaired pulmonary function, especially the restrictive pattern in 1555 nonsmoking postmenopausal women. We evaluated CRP as a categorical variable and constructed three groups (a very low risk group, CRP < 0.5 mg/dl; a low risk group, 0.5–1.0 mg/dl; a medium and high risk group, 1.0–10.0 mg/dl). The odds ratios (ORs) for a low FVC were calculated across all three groups.

Results

CRP levels are negatively associated with forced expiratory volume (FEV₁)/forced vital capacity (FVC) and FVC after adjustment for confounding variables. The adjusted ORs (95% CIs) for a low FVC according to three groups were 1.00 (reference), 2.08 (1.03–4.20), and 2.55 (1.31–4.98) in postmenopausal women after adjusting for confounding variables.

Conclusions

In summary, increased C-reactive protein (CRP) levels are strongly and independently associated with impaired pulmonary function and more frequent a low FVC in postmenopausal women.     

Consumption of C4b-binding protein (C4BP) during in vivo activation of the classical complement pathway.

C4BP has a central role in regulating the classical complement (C') pathway, but it is still uncertain whether or not it is consumed during in vivo complement activation. Attempts to demonstrate changes in C4BP plasma levels in systemic lupus erythematosus and essential mixed cryoglobulinaemia have failed, probably due to up-regulation of this protein during the inflammatory reaction. We have studied one patient with severe post-transfusion complement-mediated anaphylaxis (CMA), and 67 patients with hereditary C1 inhibitor deficiency (hereditary angioedema (HAE)). The first of these two conditions is characterized by the absence of systemic inflammatory reaction and the second by acute and chronic activation of the C' classical pathway. C4BP, C4BP-C4b complex, and soluble terminal C' complex (sC5b-9) were measured in the patients' plasmas by ELISA techniques and C3a and C4a by radioimmunoassays. In CMA, 15 min after the transfusion, there was a massive C' activation, with increases in C4a, C3a, sC5b-9, C4BP-C4b complexes and decreases in C4, C3 and C4BP. All parameters reverted to preinfusion values within 24 h. Depletion of C4 was correlated with that of C4BP. In patients with HAE, the median value of C4BP (83% range 54-165) was significantly lower (P < 0.0001) than in normal controls (99% range 70-159), with no difference between patients in remission or during acute attacks. C4BP-C4b complexes could not be detected in HAE patients. The results of this study indicate that C4BP is consumed in vivo during acute, and possibly during chronic activation of the C' classical pathway, and that this protein, after interaction with C4b, not longer circulates in plasma.     

Development of a high sensitivity C-reactive protein immunoassay and comparison with a commercial kit

C-reactive protein (CRP) is intricately sensitive marker of inflammation, infection, and tissue damage. Role in the prognosis of heart diseases has been recently discovered. This study aimed to develop a cost-effective and high-sensitivity CRP immunoassay for use in cardiac risk assessment. Assay was optimized for coating, blocking of capturing antibody, dilution, and reaction time of the conjugate and sample volume. For normal reference range, CRP was determined in serum samples from apparently healthy volunteers. For clinical validation, CRP was determined in samples of acute coronary syndrome patients by in-house and commercial assays. The lower detection limit of in-house assay was 0.16 µg/L. Intra and inter assay imprecision was 4.39%, 4.6% and 8.6%, 9.3%, respectively. The correlation between the CRP levels by the two assays was r = 0.861. Sensitivity, specificity, predictive value for a positive test, and a negative test of in-house assay was 95.3%, 92.8%, 95.3%, and 92.8%, respectively. At lower-end CRP levels of both kits correlated very well but showed variation at upper end. In-house assay showed high sensitivity and reliability at lower end and it is hoped that will help to evaluate cardiac risk assessment (after improvement at upper end) in clinically poor settings.     

Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP)

The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (E_Man) was examined. While the C4BP concentration for inhibiting 50% (IC₅₀) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05 nM), 3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81 nM) and E_Man (42.66 nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000–431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven α-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79 ± 0.60) when the C4b density on E_Man was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a 7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.     

Mast cells and complement system: Ancient interactions between components of innate immunity

The emergence and evolution of the complement system and mast cells (MCs) can be traced back to sea urchins and the ascidian Styela plicata, respectively. Acting as a cascade of enzymatic reactions, complement is activated through the classical (CP), the alternative (AP), and the lectin pathway (LP) based on the recognized molecules. The system's main biological functions include lysis, opsonization, and recruitment of phagocytes. MCs, beyond their classic role as master cells of allergic reactions, play a role in other settings, as well. Thus, MCs are considered as extrahepatic producers of complement proteins. They express various complement receptors, including those for C3a and C5a. C3a and C5a not only activate the C3aR and C5aR expressing MCs but also act as chemoattractants for MCs derived from different anatomic sites, such as from the bone marrow, human umbilical cord blood, or skin in vitro. Cross talk between MCs and complement is facilitated by the production of complement proteins by MCs and their activation by the MC tryptase. The coordinated activity between MCs and the complement system plays a key role, for example, in a number of allergic, cutaneous, and vascular diseases. At a molecular level, MCs and complement system interactions are based on the production of several complement zymogens by MCs and their activation by MC-released proteases. Additionally, at a cellular level, MCs act as potent effector cells of complement activation by expressing receptors for C3a and C5a through which their chemoattraction and activation are mediated by anaphylatoxins in a paracrine and autocrine fashion.     

Effects of tibolone and combined 17beta-estradiol and norethisterone acetate on serum C-reactive protein in healthy post-menopausal women: a randomized trial

BACKGROUND: Serum C-reactive protein (CRP) is an independent risk factor for the development of cardiovascular diseases in healthy post-menopausal women. Oral unopposed and progestin-combined 17beta-estradiol (E(2)) increase serum CRP in post-menopausal women. The aim of this study was to compare the effects of tibolone, a steroid with estrogenic, androgenic or progestogenic properties, with a combination of E(2) and norethisterone acetate (E(2) + NETA) on serum CRP levels in healthy post-menopausal women. METHODS: A total of 139 post-menopausal women (mean age: 55 years, range 44-48) was randomly assigned to receive tibolone 1.25 mg/day (n = 52), tibolone 2.5 mg/day (n = 39) or E(2) 2 mg/day plus NETA 1 mg/day (n = 48) for 2 years. Serum CRP was measured at baseline and at 6, 12 and 24 months. RESULTS: Both doses of tibolone and E(2) + NETA increased serum CRP by a similar extent as soon as 6 months with a sustained effect over the 24 month treatment period. For example, after 6 months of treatment, serum CRP increased by a median of +106% (P < 0.001), +89% (P < 0.05) and +139% (P < 0.001) for tibolone 1.25 mg/day, tibolone 2.5 mg/day and E(2) + NETA respectively. CONCLUSIONS: Tibolone and E(2) + NETA significantly increase serum CRP levels in healthy post-menopausal women to a comparable extent. Relationships between induced elevated CRP levels with tibolone and E(2) + NETA and cardiovascular events require further studies.