The rat C-reactive protein — isolation and response to experimental inflammation and tissue damage

A new isolation procedure of rat C-reactive protein (CRP) by affinity chromatography on aminocoupled phosphorylcolamine-Sepharose is described. CRP serum level of rats injured by turpentine injection or two different arthritis models was determined to test the potency of this protein as an marker of inflammation. The influence of repeated adminstration of carbon tetrachloride as a hepatotoxic agent was investigated in the same way.It was shown that CRP determination is a useful method to observe the course of experimental inflammations and other tissue injuries as well.     

C-reactive protein in acute viral infections

A sensitive solid-phase enzyme immunoassay procedure was used to determine the concentrations of C-reactive protein (CPR) in the acute and convalescent phase sera of patients with verified rubella, herpes simplex, cytomegalo, influenza A or B, enterovirus, or mycoplasma infection. In all infection groups about 90% (80% for influenza) elevated CRP values were observed in the acute phase sera (mean values in the different groups 16-57 micrograms/ml), the highest values exceeding or approaching 100 micrograms/ml. The serum CRP values were highest in all groups before the specific serum antibodies were detectable and decreased approaching the upper limit or normal controls (2 microgram/ml) within 2 weeks. Notable individual variation in the CRP production was seen. We conclude tha serum CRP determination should not be used as a reliable criterion to distinguish bacterial and viral infections.     

Relationship of granulocyte colony stimulating factor with other acute phase reactants in man

The non-specific acute phase response in mice is associated with increased resistance to bacterial infection, which is critically mediated by granulocyte colony stimulating factor (G-CSF), but the behaviour of G-CSF in the human acute phase response is not known. Cardiothoracic surgery is a powerful acute phase stimulus and we show here that this procedure caused increased production of G-CSF, in addition to increases in the circulating concentrations of the proinflammatory cytokine interleukin (IL)-6 and the acute phase plasma proteins C-reactive protein (CRP) and serum amyloid A protein (SAA). Values of G-CSF correlated positively with IL-6 concentrations and circulating neutrophil counts, but not with CRP values. These results confirm that G-CSF is a physiological component of the acute phase response in humans that shares some of the same regulatory controls as IL-6, but its downstream effects are on neutrophils, not hepatic acute phase protein synthesis. Our observations are compatible with a protective role against bacterial infection for G-CSF in the human acute phase response, and support investigation of the prophylactic use of G-CSF in at-risk patients.     

The rat female protein,a pentraxin with lectinic properties

The C-reactive protein is the major acute phase protein (APP) in humans which binds lectin-like to different membraneous structures and exerts an important function in non-specific defense. Because of a pentameric molecular symmetry CRP as well as serum amyloid P component (SAP) and hamster female protein (FP) was merged into a special protein family named pentraxins. In rats a protein was found referred to as rat FP which was close related to hamster FP with respect to hormonal regulation and APP nature as well. Based on this conformity the molecular structure of rat FP was analyzed and as the results a pentameric structure could be demonstrated for rat FP, too. Furthermore, the response of rat CRP and FP on injection of adrenal hormones, agents being involved in acute phase reaction, was investigated. Epinephrine administration led to an increase in CRP and a decrease in FP serum concentration. Dexamethasone has the same effect in case of FP and changed the CRP concentration in a biphasic way with a maximum at about 0.01 mg/kg, a minimum at 0.6 mg/kg and a return to control values at 1.8 mg/kg. Thus, the results indicate a neuroendocrine control of CRP and FP but probably in a different way. Using FITC-labelled lectin the exposition of galactose-containing membraneous structures could be demonstrated in carbon tetrachloride-injured liver tissue in contrast to controls. These binding sites are in accordance with increased FP-binding shown by immunofluorescence histochemistry. Thus, lectin-like properties may be ascribed to rat FP comparable to CRP and SAP activity. The results are discussed with respect to findings from literature that also the acetylcholine receptor seems to have a pentameric structure.     

Isolation and characterization of C-reactive protein from the dog.

Using calcium-dependent affinity chromatography on Sepharose-bearing, covalently-coupled pneumococcal C-polysaccharide, a protein was isolated from the serum of dogs that had undergone general anaesthesia and major surgery. This protein was confirmed as the canine analogue of C-reactive protein (CRP) in other species by virtue of its electron microscopic appearance, subunit composition and behaviour as an acute phase reactant. Dog CRP had an apparent molecular weight of approximately 100,000 and was composed of five subunits of approximately 20,000 MW each. Two of the five subunits in each molecule were glycosylated. Negatively stained preparations had the typical cyclic pentameric disc-like structure of proteins of the pentraxin family, and in some preparations had a tendency to form stacks. Serum from normal healthy dogs of various strains usually contained less than 5 mg/l of CRP but, following the stimulus of major surgery, an increase in the CRP concentration was first detected at 4 hr.     

Activation of trout macrophages and production of CRP after immunization with Vibrio anguillarum

To examine the mechanism of the protection of rainbow trout (Salmo gairdneri) against Vibrio anguillarum in the early stage of immunization, the activation of macrophages and production of C-reactive protein (CRP) were investigated. Fish immunized with formalin-killed bacteria emulsified in Freund's complete adjuvant (FCA) resisted intraperitoneal challenge with living bacteria seven and ten days after immunization. The activation of macrophages was demonstrated by a significant increase of the chemiluminescent (CL) response and phagocytic activity. These fish also showed a significant increase of the CRP level in sera. Fish immunized with V. anguillarum alone or injected with FCA, however, did not resist the challenge. Though FCA itself increased CRP level and the sera enhanced phagocytic activity, increase of CL activity was weak. These results indicated that the increase of CL activity and opsonising effect of CRP on the phagocytosis of specifically activated macrophages concern to host defense in the early stage of infection.     

Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. The effects of CRP on primary human monocyte adhesion molecule expression and interaction with the endothelium have not been studied. Herein, we describe an investigation into the phenotypic and functional consequences of CRP binding to peripheral blood monocytes ex vivo. Peripheral whole blood was collected from healthy, non-smoking males. Mononuclear cells (MNC) and monocytes were isolated by differential centrifugation using lymphoprep and Dynal negative isolation kit, respectively. Cells were exposed to CRP from 0 to 250 micro g/ml for 0-60 min at 37 degrees C and analysed for (a) CD11b, PECAM-1 (CD31) and CD32 expression by flow cytometry and (b) adhesion to LPS (1 micro g/ml; 0-24 h) treated human umbilical vein endothelial cells (HUVEC). CD14+ monocyte expression of CD11b increased significantly up to twofold when exposed to CRP, compared to controls. There was no significant difference in CD32 expression, whereas CD31 expression decreased after exposure to CRP. CRP treatment of monocytes inhibited their adhesion to early LPS-activated HUVEC (0-5 h). However, the adhesion of CRP-treated monocytes to HUVEC was significantly greater to late activation antigens on HUVEC (24 h, LPS) compared to controls. We have shown that CRP can affect monocyte activation ex vivo and induce phenotypic changes that result in an altered recruitment to endothelial cells. This study provides the first evidence for a further role for C-reactive protein in both monocyte activation and adhesion, which may be of importance during an inflammatory event.     

Purification of human C-reactive protein by barium sulfate and preparative agarose electrophoresis

A procedure is described for the purification of C-reactive protein (CRP) from human serum. The methods described take advantage of the barium sulfate adsorption property of CRP and the unique biophysical property of CRP migration during electrophoresis in agarose gels containing Ca2+. The purified CRP had an apparant molecular weight of 28,000 as determined by migration of the reduced protein during SDS polyacrylamide gel electrophoresis. The described procedure has the advantage of not requiring either molecular sieve or affinity chromatography for purification of homogenous CRP from human sera.     

Radiometric ligand binding assay for C-reactive protein. Complexed C-reactive protein is not detectable in acute phase serum.

A radiometric ligand binding assay for human C-reactive protein (CRP) was established using pneumococcal C polysaccharide (CPS) coupled to magnetizable cellulose particles as the solid phase ligand. Competition for binding to the solid phase between 125I-CRP and unlabelled CRP permitted detection of 30 micrograms/l of CRP and the precise assay of concentrations up to 3000 micrograms/l. Identical results were obtained when the assay was used to quantitate isolated pure CRP and pure CRP added to normal human serum. However in vitro addition of known ligands for CRP to acute phase serum resulted in lowering of the apparent CRP concentration in this assay and addition of as little as 1 microgram/l of free CPS or 1 mg/l of lecithin was demonstrable in this way. A combination of the ligand binding assay and the standard electroimmunoassay for CRP was therefore used to test acute phase sera for the presence of CRP complexed in vitro. No evidence of complexed CRP was detected among sera containing between 1-319 mg/l of CRP from patients with Hodgkin's disease (10), rheumatoid arthritis (10), Crohn's disease (19) and various microbial infections (11), including six with subacute bacterial endocarditis. Since it is likely that CRP does form complexes with its ligands in the plasma these results suggest that complexed CRP is rapidly cleared from the circulation.     

Serum amyloid A protein concentration in bone marrow transplantation for beta thalassaemia.

AIMS: To investigate whether serum amyloid A protein (SAA) and C-reactive protein (CRP) concentrations could be used in the management of beta thalassaemic patients undergoing bone marrow transplantation (BMT). METHODS: Serum SAA and CRP concentrations were determined in paired samples from 66 patients with beta thalassaemia before and after BMT. Serum SAA concentrations were determined by an enzyme linked immunoassay (EIA); serum CRP concentrations were determined by a nephelometric assay. RESULTS: Serum SAA concentrations before transplantation were significantly higher in the group that subsequently rejected the transplant than the group without complications. SAA concentrations increased after BMT in acute graft versus host disease (GvHD) and rejection. No significant increase in SAA or CRP was found in chronic GvHD. Increases in serum in SAA and CRP concentrations were not related to concomitant infection episodes. CONCLUSIONS: The different acute phase response in acute GvHD and rejection compared with chronic GvHD suggests that different immunopathogenic mechanisms are responsible.     

In vivo turnover studies of C-reactive protein.

The in vivo plasma clearance rate of the acute phase reactant C-reactive protein (CRP) was studied in mice and rats. The clearance rate of 125I-human CRP in mice and 125I-rat CRP in rats showed a T1/2 of approximately 4 h. The T1/2 was independent of circulating levels of CRP and was not affected by the presence of C-polysaccharide (CPS), a ligand to which CRP binds. However, in mice receiving sufficient CPS, more radioactivity localized to the spleen compared to mice receiving 125I-CRP only. 125I-CPS was rapidly cleared at the same rate by normal mice and by mice undergoing an acute phase response while rats cleared 125I-CPS more slowly despite having high circulating CRP concentrations. These findings suggest that CRP does not provide a mechanism for extremely rapid clearance of its ligands from the circulation, although the handling and subsequent fate of these ligands may be affected.     

血清C反应蛋白和半乳糖凝集素-1对脑胶质瘤的诊断价值

目的 探讨血清C反应蛋白（CRP）和半乳糖凝集素1含量对于脑胶质瘤的诊断价值.方法 分别检测116例健康对照组、103例低级别脑胶质瘤组和138例高级别脑胶质瘤组中C反应蛋白和半乳糖凝集素1的含量.结果 与健康对照组比较,低级别脑胶质瘤组和高级别脑胶质瘤组中CRP和半乳糖凝集素-1含量均升高,且差异具有统计学意义;与低级别脑胶质瘤组比较,高级别脑胶质瘤组的CRP和半乳糖凝集素-1含量均升高,且差异具有统计学意义;CRP和半乳糖凝集素-1联合检测区分健康对照组和脑胶质瘤组的诊断灵敏度和特异性分别为91.29％和88.80％,优于单独检测;CRP和半乳糖凝集素-1联合检测区分低级别和高级别脑胶质瘤组的诊断灵敏度和特异性分别为87.55％和87.93％,优于单独检测.结论 CRP和半乳糖凝集素-1联合检测对脑胶质瘤的辅助诊断具有一定的潜在价值.     

Acute phase proteins as markers of inflammatory lesions

Acute phase proteins are liver-derived serum proteins, the concentration of which alters in response to infection or inflammation. Cytokines such as interleukin-6, interleukin-1 and tumour necrosis factor- stimulate the liver to produce haptoglobin (Hp), serum amyloid A (SAA), C-reactive protein (CRP), ₁-acid glycoprotein (AGP) and fibrinogen (Fb) but limit the production of negative acute phase proteins such as albumin. There are interspecies variations in the pattern of response of the acute phase proteins but they are valuable as markers of inflammatory lesions providing diagnostic information for veterinary medicine.Advances have been made in the study of acute phase protein in pigs, dogs and cattle. In the pig, it is evident that CRP is a major reactant whereas Hp is a moderate acute phase reactant. The use of CRP for the diagnosis of inflammatory disease in dogs has been confirmed with studies relating the canine serum CRP concentration to haematological determination of inflammation. CRP assay in canine serum has been shown to confirm primary infectious or inflammatory conditions but can also detect secondary conditions where the primary condition is non-inflammatory. In cattle, the use of Hp assay is now established as a major aid to the diagnosis of infectious and inflammatory disease. Additional benefit to the diagnosis of disease can be provided by analysis of a profile of acute phase proteins such as a combined analysis of SAA, Hp and AGP. The circulating concentration of AGP is maintained at an increased level for a more extended period than that of SAA or Hp in chronic inflammatory conditions.A major advance would be made in the analysis of animal acute phase protein if internationally recognised standards of the proteins in each species were to become available.Originally presented at ECCP 95.     

Agglutination of intravenous lipid emulsion (''Intralipid'') and plasma lipoproteins by C-reactive protein.

Isolated human C-reactive protein (CRP), or CRP in acute phase serum, produced in-vitro agglutination ('creaming') of the intravenously administered lipid suspension 'Intralipid'. CRP also produced similar agglutination of isolated normal very low density lipoproteins (VLDL). Agglutination in both cases was calcium-dependent and inhibitable by phosphoryl choline. These findings have important implications for patients receiving intravenous lipid suspensions and may be relevant to the pathogenesis of the fat embolism syndrome following trauma.     

Acute phase protein changes in antigen-induced mono-articular arthritis in rabbits and mice.

Acute phase protein levels have been measured during the induction and progression of antigen-induced mono-articular arthritis in rabbits and mice. In rabbits there was a short lived elevation in serum C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) immediately following intra-articular injection which returned to baseline levels 10-12 days after the injection. In BALB/c mice, serum amyloid P-component (SAP) and the third component of complement (C3) were elevated after intra-articular injection, returning towards baseline levels 6 weeks after the injection. The levels of CRP and SAP correlated with the inflammatory changes in the joints during the acute phase of the arthritic response (7 days after intra-articular injection). During the chronic phase the levels of these acute phase proteins bore no relationship to the degree of connective tissue destruction.     

Impairment of acute protein reactivity in chronic renal failure.

The acute phase protein response was studied after elective surgery in 13 normal subjects and 9 patients with severe chronic renal failure. Total haemolytic complement reactivity (CH50) and serum concentrations of C1q, C1s, C4, C3, factor B, properdin, C5, C9, C-reactive protein (CRP), caeruloplasmin, alpha1-acid glycoprotein and haptoglobin were measured preoperatively and on days 2, 4 and 6 after operation. Abnormalities were seen in the group with chronic renal failure. Firstly, there was no significant acute phase response of C1s, C3, C5, C9 and CH50 and a significant reduction in the response of factor B. Secondly, CRP showed prolonged elevation in the post-operative period in contrast to the transient rise seen in the control group. With the possible exception of alpha1-acid glycoprotein, the behaviour of the non-complement proteins (caeruloplasmin and haptoglobin) was comparable for the two groups. These defects could impair the physiological response to infection in patients with severe chronic renal failure.     

Isolation and characterization of C-reactive protein and serum amyloid P component in the rat.

C-reactive protein (RP) and serum amyloid P component (SAP) have been identified for the first time in rat serum and isolated by calcium-dependent affinity chromatography. Rat CRP closely resembled human CRP in its amino acid composition, in having five subunits per molecule and in its electron microscopic appearance as a pentameric annular disc. It differed, however, from all other mammalian CRP's characterised hitherto in being a glycoprotein bearing a single complex oligosaccharide on each polypeptide subunit. Furthermore one pair of tis subunits per molecule was linked by a interchain disulphide bridges whereas in other animals the subunits of both CRP and SAP are all non-covalently associated. The serum concentration of CRP in normal healthy laboratory rats and in specific pathogen-free rats was 300-600 micrograms/ml which is much greater than has been described in any other species and exceeds even maximal acute phase levels of CRP in man. Following injections of casein or croton oil, serum CRP levels rose to a maximum of about 900 micrograms/ml. Rat CRP bound to pneumococcal C-polysaccharide (CPS( but, in marked contrast to the behaviour of CRP from man, rabbit and marine teleost fish, it did not precipitate with CPS solutions, agglutinate CPS-coated sheep erythrocytes or initiate complement activation. Rat SAP, like SAP of other species, was a glycoprotein but unlike them it was composed only of a single pentameric disc not two such discs interacting face-to-face. The normal level of SAP in rat serum was 20-50 micrograms/ml, very similar to the levels seen in man, and it did not behave as an acute phase reactant in response to casein or croton-oil injections. In this respect it resembled human SAP but differed from murine SAP which is a major acute phase reactant.     

Inappropriate responses to Mycobacterium leprae infections--C reactive protein in man and serum amyloid P in mice.

In a study of C-reactive protein (CRP) levels in the sera of 77 patients with leprosy, it was found that in the majority of newly diagnosed patients, the level was within the normal range for a healthy Malaysian population. Elevated levels did occur, but were usually found in patients with complications, and were more likely to occur in patients who had been receiving drug treatment for some time. This suggested that Mycobacterium leprae infection by itself does not stimulate CRP synthesis and could reflect a failure of synthesis by macrophages of interleukin-1, or related molecules. This was supported by the study of an analogous acute phase protein, serum amyloid P (SAP) in mice bearing M. leprae from human sources in their hind footpads. Such mice showed no significant difference in SAP levels from control mice.     

Role of C-reactive protein in HIV infection: a pilot study

Limited data on acute phase C-reactive protein (CRP) levels in human immunodeficiency virus (HIV) infection exist. The relationship of CRP with HIV was assessed in 119 HIV-positive patients and correlated with CD4 counts and mortality at 1 y. CRP was negatively correlated with CD4 counts with levels of CRP being highest in the group with CD4 counts below 200 cells/microL. It was an indicator of mortality and hence may serve as a useful and inexpensive predictor of HIV disease progression.