Distribution and determinants of circulating complement factor H concentration determined by a high-throughput immunonephelometric assay.

BACKGROUND: Research on complement factor H (fH) in human disease is hampered by lack of an assay suitable for use in large-scale epidemiological studies. We describe the development and validation of a high throughput nephelometric assay for fH. METHODS: Reagents from a commercial radial immunodiffusion (RID) assay (The Binding Site) were adapted for use on the Siemens BNII high throughput nephelometric instrument. The assay was calibrated with a highly purified human fH preparation with rigorously determined concentration, and assay performance was comprehensively evaluated using samples from healthy human volunteers, with the commercial RID assay as a comparator. The distribution and determinants of circulating fH concentration in humans were then investigated in a large representative population sample. RESULTS: The nephelometric assay had recovery close to 100%, was reproducible with intra- and inter-assay CV's of 11% and 5-15% respectively, and had a wider operating range than the RID assay. fH values were unaffected after multiple freeze-thaw cycles demonstrating that it is evidently a stable analyte for immunoassay. fH concentration was unaltered by an acute inflammatory stimulus. The population study showed that plasma fH concentration is associated with circulating lipids and indices of body fat. CONCLUSION: We present the first high throughput assay for circulating fH; the assay is accurate and reliable with reproducible measures from stored samples. It has established the distribution of fH values at a population level and demonstrated important associations with circulating lipids and indices of body fat, thus providing an important reference for future clinical and epidemiological investigations.

Isolation and characterization of pharmaceutical grade human pentraxins, serum amyloid P component and C-reactive protein, for clinical use.

The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non-specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL-6 or IL-8, nor does SAP cause release of IL-1ß or IL-10. Furthermore neither of our preparations was pro-inflammatory in mice in vivo.

Antibodies to human serum amyloid P component eliminate visceral amyloid deposits.

Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one in a thousand deaths in developed countries. Localized amyloid can also have serious consequences; for example, cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present. There is therefore a major unmet need for therapy that safely promotes the clearance of established amyloid deposits. Over 20 different amyloid fibril proteins are responsible for different forms of clinically significant amyloidosis and treatments that substantially reduce the abundance of the respective amyloid fibril precursor proteins can arrest amyloid accumulation. Unfortunately, control of fibril-protein production is not possible in some forms of amyloidosis and in others it is often slow and hazardous. There is no therapy that directly targets amyloid deposits for enhanced clearance. However, all amyloid deposits contain the normal, non-fibrillar plasma glycoprotein, serum amyloid P component (SAP). Here we show that administration of anti-human-SAP antibodies to mice with amyloid deposits containing human SAP triggers a potent, complement-dependent, macrophage-derived giant cell reaction that swiftly removes massive visceral amyloid deposits without adverse effects. Anti-SAP-antibody treatment is clinically feasible because circulating human SAP can be depleted in patients by the bis-d-proline compound CPHPC, thereby enabling injected anti-SAP antibodies to reach residual SAP in the amyloid deposits. The unprecedented capacity of this novel combined therapy to eliminate amyloid deposits should be applicable to all forms of systemic and local amyloidosis.

A prospective study of the sensitivity, specificity and diagnostic performance of soluble intercellular adhesion molecule 1, highly sensitive C-reactive protein, soluble E-selectin and serum amyloid A in the diagnosis of neonatal infection.

BACKGROUND: Diagnosis of neonatal infection is difficult, because of it's non-specific clinical presentation and the lack of reliable diagnostic tests. The purpose of this study was to examine the potential diagnostic value of serum soluble intercellular adhesion molecule-1 (sICAM-1), soluble E-selectin (sE-selectin), highly sensitive C-reactive protein (hsCRP) and serum amyloid A (SAA) measurements, both individually and in combination in the setting of a neonatal intensive care unit. METHODS: 219 consecutive serum samples were taken from 149 infants undergoing sepsis work up in a neonatal intensive care unit. Clinical diagnosis was established in a prospective manner, blind to the results of the study measurements. Infants were classified by an experienced paediatrician as infected or not-infected, one week after presentation. Classification was based on clinical presentation, routine laboratory and radiological investigations and response to therapy. The infected group were sub-classified as (a) culture positive infection or (b) culture negative infection. sICAM-1, sE-selectin, hsCRP and SAA levels were determined from stored serum samples after diagnosis was established. Further sub-group analysis of results was undertaken according to early or late onset of infection and preterm or term status. Statistical analysis utilised Mann Whitney U test and ROC curve analysis. RESULTS: There were significantly increased serum levels of sICAM-1, hsCRP, E selectin (p < 0.001) and SAA (p = 0.004) in infected infants compared with non-infected. ROC curve analysis indicated area under the curve values of 0.79 (sICAM-1), 0.73 (hsCRP), 0.72 (sE-selectin) and 0.61 (SAA). ROC curve analysis also defined optimum diagnostic cut-off levels for each measurement. The performance characteristics of sICAM-1, hsCRP and sE-selectin included a high negative predictive value (NPV) for culture positive infection and this was enhanced by combination of all 4 measurements. Clinical subgroup analysis suggested particularly high NPV for early onset symptoms, however further studies are required to elucidate this finding. CONCLUSIONS: All four study measurements demonstrated some diagnostic value for neonatal infection however sICAM-1, hsCRP and sE-selectin demonstrated the highest NPV individually. The optimum diagnostic cut off level for hsCRP measurement in this study was much lower than currently used in routine clinical practice. Use of a combination of measurements enhanced diagnostic performance, demonstrating sensitivity of 90.3% and NPV of 91.3%. This study suggests there may be value in use of several of these markers, individually and in combination to assist in excluding neonatal infection. Further work is needed to confirm a specific role in the exclusion of early onset infection.

Molecular dissection of Alzheimer's disease neuropathology by depletion of serum amyloid P component.

New therapeutic approaches in Alzheimer's disease are urgently needed. The normal plasma protein, serum amyloid P component (SAP), is always present in cerebrospinal fluid (CSF) and in the pathognomonic lesions of Alzheimer's disease, cerebrovascular and intracerebral Abeta amyloid plaques and neurofibrillary tangles, as a result of its binding to amyloid fibrils and to paired helical filaments, respectively. SAP itself may also be directly neurocytotoxic. Here, in this unique study in Alzheimer's disease of the bis(d-proline) compound, (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), we observed depletion of circulating SAP and also remarkable, almost complete, disappearance of SAP from the CSF. We demonstrate that SAP depletion in vivo is caused by CPHPC cross-linking pairs of SAP molecules in solution to form complexes that are immediately cleared from the plasma. We have also solved the structure of SAP complexed with phosphothreonine, its likely ligand on hyperphosphorylated tau protein. These results support further clinical study of SAP depletion in Alzheimer's disease and potentially other neurodegenerative diseases.

Serum amyloid A, C-reactive protein, and retinal microvascular changes in hypertensive diabetic and nondiabetic individuals: an Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) substudy.

OBJECTIVE: To study the association of the inflammatory markers serum amyloid A (SAA) and C-reactive protein (CRP) with retinal microvascular parameters in hypertensive individuals with and without type 2 diabetes. RESEARCH DESIGN AND METHODS: This cross-sectional analysis was a substudy in 711 patients (159 with and 552 without diabetes) of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) based on digital 30-degree images of superior and inferior temporal retinal fields. RESULTS: SAA was associated with arteriolar length-to-diameter ratio positively in nondiabetic patients (P(trend)= 0.028) but negatively in diabetic patients (P(trend)= 0.005). The difference was unlikely to be a chance finding (P = 0.007 for interaction). Similar results were found for the association of SAA with arteriolar tortuosity (P = 0.05 for interaction). Associations were less pronounced for CRP and retinal parameters. CONCLUSIONS: Inflammatory processes are differentially involved in retinal microvascular disease in diabetic compared with nondiabetic hypertensive individuals.

Transgenic human CRP is not pro-atherogenic, pro-atherothrombotic or pro-inflammatory in apoE-/- mice.

The pathogenic significance, if any, of the epidemiological association between baseline C-reactive protein (CRP) values and future atherothrombotic events is not known. We therefore investigated spontaneous atherosclerosis and atherothrombosis, and systemic markers of inflammation (acute phase proteins), in aged, normal diet-fed, male apolipoprotein E deficient (apoE(-/-)) mice with and without transgenic expression of human CRP. At 18 months of age, aortic atherosclerosis was extensive but with no significant difference in plaque size between C57BL/6apoE(-/-) mice with (apoE(-/-)-hCRP(+)) and without transgenic human CRP (apoE(-/-)). Atherosclerotic lesions in brachiocephalic arteries were typically complex and layered, with extensive fibrotic-cholesterol deposits, calcification and occasional recent intraplaque haemorrhage and thrombus, but with no significant overall differences between apoE(-/-) and apoE(-/-)-hCRP(+) animals. Concentrations of mouse serum amyloid P component (SAP) were essentially normal throughout and did not differ between apoE(-/-) and apoE(-/-)-hCRP(+) mice, or between wild-type (apoE(+/+)) and apoE(-/-) mice, regardless of human CRP expression. Mouse serum amyloid A protein (SAA), and human CRP concentrations were modestly but significantly higher in apoE(-/-)-hCRP(+) than in apoE(+/+)-hCRP(+) animals, but mouse SAA values were unaffected by transgenic expression of human CRP in either background. Thus, there was no evidence in this 18 month study of apoE(-/-), and control apoE(+/+) mice, that transgenic human CRP was pro-atherogenic, pro-inflammatory or pro-atherothrombotic.

Natural history and outcome in systemic AA amyloidosis.

BACKGROUND: Deposition of amyloid fibrils derived from circulating acute-phase reactant serum amyloid A protein (SAA) causes systemic AA amyloidosis, a serious complication of many chronic inflammatory disorders. Little is known about the natural history of AA amyloidosis or its response to treatment. METHODS: We evaluated clinical features, organ function, and survival among 374 patients with AA amyloidosis who were followed for a median of 86 months. The SAA concentration was measured serially, and the amyloid burden was estimated with the use of whole-body serum amyloid P component scintigraphy. Therapy for inflammatory diseases was administered to suppress the production of SAA. RESULTS: Median survival after diagnosis was 133 months; renal dysfunction was the predominant disease manifestation. Mortality, amyloid burden, and renal prognosis all significantly correlated with the SAA concentration during follow-up. The risk of death was 17.7 times as high among patients with SAA concentrations in the highest eighth, or octile, (>or=155 mg per liter) as among those with concentrations in the lowest octile (<4 mg per liter); and the risk of death was four times as high in the next-to-lowest octile (4 to 9 mg per liter). The median SAA concentration during follow-up was 6 mg per liter in patients in whom renal function improved and 28 mg per liter in those in whom it deteriorated (P<0.001). Amyloid deposits regressed in 60% of patients who had a median SAA concentration of less than 10 mg per liter, and survival among these patients was superior to survival among those in whom amyloid deposits did not regress (P=0.04). CONCLUSIONS: The effects of renal dysfunction dominate the course of AA amyloidosis, which is associated with a relatively favorable outcome in patients with SAA concentrations that remain in the low-normal range (<4 mg per liter).

Normal circulating serum amyloid P component concentration in systemic sclerosis.

OBJECTIVE: The observation of reduced circulating concentrations of the constitutive plasma pentraxin protein, serum amyloid P component (SAP), in serum samples obtained from a small number of patients with systemic sclerosis (SSc) has been reported as confirmation of an antifibrotic role of this protein. Because neither sustained SAP depletion in humans nor SAP deficiency in mice is associated with fibrosis, we sought to establish rigorously the serum SAP concentration in well-characterized patients with SSc. METHODS: Serum concentrations of SAP were measured by electroimmunoassay in a cross-sectional cohort of 20 patients with diffuse cutaneous SSc and 12 patients with limited cutaneous SSc, and in a separate 12-month longitudinal cohort of 13 patients with diffuse disease and 37 patients with limited disease. The extent and severity of disease were characterized in detail at the time of serum sampling. Serum concentrations of the classic acute-phase reactants, C-reactive protein and serum amyloid A protein, were measured by immunonephelometric assays. RESULTS: SAP values were entirely within the normal range, regardless of the extent and severity of disease, apart from a very few isolated raised values associated with acute intercurrent complications causing major acute-phase responses. CONCLUSION: We observed no reduced circulating concentrations of SAP in patients with SSc, nor any evidence of an association between SAP levels and the extent or severity of fibrosis.

Phenotype, genotype, and sustained response to anakinra in 22 patients with autoinflammatory disease associated with CIAS-1/NALP3 mutations.

OBJECTIVE: To characterize the multisystem chronic inflammatory phenotype, dermatopathologic features, and response to therapy with interleukin 1 receptor antagonist (anakinra) in patients with mutations in the CIAS-1/NALP3 gene. DESIGN: Retrospective review of medical records and evaluation of histologic findings. SETTING: The National Amyloidosis Centre, London, and a tertiary referral clinic for urticaria. PATIENTS: Twenty-two individuals from 13 families with autoinflammatory disease associated with CIAS-1/NALP3 mutations. MAIN OUTCOME MEASURES: Phenotype, genotype, skin histologic findings, and response to treatment with anakinra. RESULTS: Five heterozygous missense mutations were identified in CIAS-1/NALP3. Skin histologic findings revealed marked vascular dilatation and neutrophilic infiltration involving small vessels and eccrine glands. Serologic evidence of intense inflammation was present in untreated patients, with median serum amyloid A protein and C-reactive protein levels of 141 and 38 mg/L, respectively. Fifteen patients received anakinra for up to 39 months, all of whom achieved serologic remission and complete resolution of fever, rash, conjunctivitis, and rheumatic symptoms, without any adverse effects. Six patients had AA (reactive systemic) amyloidosis, 2 of whom died of renal failure complications before interleukin 1-inhibiting therapy was available; 1 patient underwent renal transplantation and remains clinically well taking anakinra, and in the remaining 3 patients, anakinra therapy resulted in remission of their nephrotic syndrome. CONCLUSIONS: Anakinra therapy was well tolerated and has sustained efficacy on dermatologic and rheumatic manifestations in these patients with CIAS-1/NALP3 mutations. This treatment also resulted in resolution of AA amyloidosis-associated nephrotic syndrome in all affected patients.

Targeting C-reactive protein for the treatment of cardiovascular disease.

Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement, increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively. Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement. Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.

Proinflammatory effects of bacterial recombinant human C-reactive protein are caused by contamination with bacterial products, not by C-reactive protein itself.

Intravenous administration to human volunteers of a commercial preparation of recombinant human C-reactive protein (CRP) produced in Escherichia coli was recently reported in this journal to induce an acute phase response of serum amyloid A protein (SAA) and of CRP itself, and to activate the coagulation system. The authors concluded that CRP is probably a mediator of atherothrombotic disease. Here we confirm that this recombinant CRP preparation was proinflammatory both for mouse macrophages in vitro and for mice in vivo, but show that pure natural human CRP had no such activity. Furthermore mice transgenic for human CRP, and expressing it throughout their lives, maintained normal concentrations of their most sensitive endogenous acute phase reactants, SAA and serum amyloid P component (SAP). The patterns of in vitro cytokine induction and of in vivo acute phase stimulation by the recombinant CRP preparation were consistent with contamination by bacterial products, and there was 46.6 EU of apparent endotoxin activity per mg of CRP in the bacterial product, compared with 0.9 EU per mg of our isolated natural human CRP preparation. The absence of any proinflammatory activity in natural CRP for macrophages or healthy mice strongly suggests that the in vivo effects of the recombinant preparation observed in humans were attributable to proinflammatory bacterial products and not human CRP.

Transgenic human C-reactive protein is not proatherogenic in apolipoprotein E-deficient mice.

The association between circulating concentrations of C-reactive protein (CRP) and future atherothrombotic events has provoked speculation about a possible pathogenetic role of CRP. However, we show here that transgenic expression of human CRP had no effect on development, progression, or severity of spontaneous atherosclerosis, or on morbidity or mortality, in male apolipoprotein E (apoE)-deficient C57BL/6 mice up to 56 weeks, despite deposition of human CRP and mouse complement component 3 in the plaques. Although female apoE knockouts develop atherosclerosis more rapidly than males, the human CRP transgene is under sex hormone control and is expressed at human levels only in males. We therefore studied only male mice. The concentration of mouse serum amyloid P component, an extremely sensitive systemic marker of inflammation, remained normal throughout except for transient spikes in response to fighting in a few animals, indicating that atherogenesis in this model is not associated with an acute-phase response. However, among human CRP transgenic mice, the circulating CRP concentration was higher in apoE knockouts than in wild-type controls. The higher CRP values were associated with substantially lower estradiol concentrations in the apoE-deficient animals. Human CRP transgene expression is thus up-regulated in apoE-deficient mice, apparently reflecting altered estrogen levels, despite the absence of other systemic signs of inflammation. Extrapolation to human pathology from this xenogeneic combination of human CRP with apoE deficiency-mediated mouse atherosclerosis must be guarded. Nevertheless, the present results do not suggest that human CRP is either proatherogenic or atheroprotective in vivo.

Inflammation and endothelial function: direct vascular effects of human C-reactive protein on nitric oxide bioavailability.

BACKGROUND: Circulating concentrations of the sensitive inflammatory marker C-reactive protein (CRP) predict future cardiovascular events, and CRP is elevated during sepsis and inflammation, when vascular reactivity may be modulated. We therefore investigated the direct effect of CRP on vascular reactivity. METHODS AND RESULTS: The effects of isolated, pure human CRP on vasoreactivity and protein expression were studied in vascular rings and cells in vitro, and effects on blood pressure were studied in rats in vivo. The temporal relationship between changes in CRP concentration and brachial flow-mediated dilation was also studied in humans after vaccination with Salmonella typhi capsular polysaccharide, a model of inflammatory endothelial dysfunction. In contrast to some previous reports, highly purified and well-characterized human CRP specifically induced hyporeactivity to phenylephrine in rings of human internal mammary artery and rat aorta that was mediated through physiological antagonism by nitric oxide (NO). CRP did not alter endothelial NO synthase protein expression but increased protein expression of GTP cyclohydrolase-1, the rate-limiting enzyme in the synthesis of tetrahydrobiopterin, the NO synthase cofactor. In the vaccine model of inflammatory endothelial dysfunction in humans, increased CRP concentration coincided with the resolution rather than the development of endothelial dysfunction, consistent with the vitro findings; however, administration of human CRP to rats had no effect on blood pressure. CONCLUSIONS: Pure human CRP has specific, direct effects on vascular function in vitro via increased NO production; however, further clarification of the effect, if any, of CRP on vascular reactivity in humans in vivo will require clinical studies using specific inhibitors of CRP.

Obesity is an important determinant of baseline serum C-reactive protein concentration in monozygotic twins, independent of genetic influences.

BACKGROUND: C-reactive protein (CRP) values predict atherothrombotic cardiovascular disease and type 2 diabetes mellitus. Associations between CRP and obesity, predominantly assessed anthropometrically, may partly explain these observations. Previous studies have been unable to control for genetic influences on CRP and obesity. The aim of this study was to examine the relationship between CRP and accurately measured body fat, lipids, apolipoproteins, blood pressure, and environmental and behavioral factors, independent of genetic influences. METHODS AND RESULTS: One hundred ninety-four healthy female twins (age 57.2+/-7 years) were studied after excluding pairs with CRP values >10 mg/L. Total body fat and central abdominal fat (CAF) were measured by dual-energy x-ray absorptiometry. CRP concentration was strongly related to surrogate and direct measures of body fat (r=0.31 to 0.54, P<0.001), diastolic blood pressure (r=0.20, P=0.003), and lipid and apolipoprotein levels (r=0.21 to 0.51, P<0.008). Light-to-moderate alcohol consumers and nonusers of hormone replacement therapy (HRT) had lower CRP levels than abstainers and HRT users, respectively. In stepwise multiple regression analysis, CAF, triglycerides, apolipoprotein B, and HRT use explained 46% of the variance in circulating CRP. In analyses controlling for genetic influences in monozygotic twins, within-pair differences in CRP were associated with within-pair differences in total body fat (r=0.39, P<0.001), CAF (r=0.34, P=0.002), diastolic blood pressure (r=0.24, P=0.03), apolipoprotein AI (r=-0.33, P=0.01), HDL cholesterol (r=-0.42, P=0.001), and triglycerides (r=0.35, P=0.007). CONCLUSIONS: CRP was strongly related to total and central abdominal obesity, blood pressure, and lipid levels, independent of genetic influences. These relationships are likely to contribute significantly to prospective associations between CRP and type 2 diabetes and coronary events.

Genetic effects on baseline values of C-reactive protein and serum amyloid a protein: a comparison of monozygotic and dizygotic twins.

BACKGROUND: C-Reactive protein (CRP) and serum amyloid A protein (SAA) are exquisitely sensitive acute-phase reactants, but their baseline values are surprisingly constant in individuals in the general population. These values, especially of CRP, are associated with future atherothrombotic events, and the determinants of baseline CRP and SAA concentration are therefore of considerable interest. METHODS: CRP and SAA concentrations were measured by well-validated automated microparticle capture enzyme immunoassays, standardized on the respective WHO International Reference Standards, in serum from 146 monozygotic and 164 dizygotic healthy female UK twin pairs from the general population, with mean (range) ages of 58.0 (40-69.6) and 55.7 (40-70.3) years, respectively, who were also very closely matched for height, weight, body mass index, blood pressure, and lifestyle variables. Statistical modeling based on variance components analysis was used to estimate the genetic contribution to the observed values. RESULTS: As reported previously, CRP values were associated with body mass index, smoking, and hormone replacement therapy. After exclusion of the few samples with CRP concentrations >10 mg/L, which indicate an ongoing acute-phase response rather than baseline values, and inclusion of adjustments for all known confounding variables, there was significantly higher correlation of CRP and SAA results among monozygotic than among dizygotic twins. The estimated hereditability (95% confidence interval) of baseline values was 52% (40-62%) for CRP and 59% (49-67%) for SAA. CONCLUSION: There is a substantial genetic contribution to baseline serum concentrations of CRP and SAA.