The role of genetic variants in CRP in radiographic severity in African Americans with early and established rheumatoid arthritis.

This study investigates the association of CRP (C-reactive protein) single-nucleotide polymorphisms (SNPs) with plasma CRP levels and radiographic severity in African Americans with early and established rheumatoid arthritis (RA). Using a cross-sectional case-only design, CRP SNPs were genotyped in two independent sets of African Americans with RA: Consortium for the Longitudinal Evaluation of African Americans with RA (CLEAR 1) and CLEAR 2. Radiographic data and CRP measurements were available for 294 individuals from CLEAR 1 (median (interquartile range (IQR) 25-75) disease duration of 1 (0.6-1.6) year) and in 407 persons from CLEAR 2 (median (IQR 25-75) disease duration of 8.9 (3.5-17.7) years). In CLEAR 1, in adjusted models, the minor allele of rs2808630 was associated with total radiographic score (incident rate ratio 0.37 (95% confidence interval (CI) 0.19-0.74), P-value=0.0051). In CLEAR 2, the minor allele of rs3093062 was associated with increased plasma CRP levels (P-value=0.002). For each rs3093062 minor allele, the plasma CRP increased by 1.51 (95% CI 1.15-1.95) mg dl(-1) when all the other covariates remained constant. These findings have important implications for assessment of the risk of joint damage in African Americans with RA.

C-reactive protein promotes diabetic kidney disease in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Although C-reactive protein (CRP) has been implicated as a risk factor in diabetes, its pathogenic importance in diabetic kidney disease (DKD) remains unclear. The present study investigated the potential role of CRP in DKD. METHODS: Diabetes was induced by streptozotocin in human CRP transgenic and wild-type mice for assessment of kidney injury at 24 weeks by real-time PCR, immunohistochemistry and western blot analysis. In vitro, the pathogenic effect of CRP was investigated using human kidney tubular epithelial cells cultured with high glucose and/or CRP. RESULTS: We found that CRP transgenic mice developed much more severe diabetic kidney injury than wild-type mice, as indicated by a significant increase in urinary albumin excretion and kidney injury molecule-1 abundance, enhanced infiltration of macrophages and T cells, and upregulation of pro-inflammatory cytokines (IL-1ß, TNFα) and extracellular matrix (collagen I, III and IV). Enhanced renal inflammation and fibrosis in CRP transgenic mice was associated with upregulation of CRP receptor, CD32a, and over-activation of the TGF-ß/SMAD and nuclear factor κB signalling pathways. In vitro, CRP significantly upregulated pro-inflammatory cytokines (IL-1ß, TNFα, monocyte chemoattractant protein-1 [MCP-1]) and pro-fibrotic growth factors (TGF-ß1, connective tissue growth factor [CTGF]) via CD32a/64. CRP was induced by high glucose, which synergistically promoted high glucose-mediated renal inflammation and fibrosis. CONCLUSIONS/INTERPRETATION: CRP is not only a biomarker, but also a mediator in DKD. Enhanced activation of TGF-ß/SMAD and nuclear factor κB signalling pathways may be the mechanisms by which CRP promotes renal inflammation and fibrosis under diabetic conditions.

C-reactive protein: a physiological activator of interleukin 6 receptor shedding.

The soluble interleukin 6 receptor (sIL-6R) circulates at elevated levels in various diseases. This suggests that inflammatory mediators control sIL-6R release. Through examination of human neutrophils, it was found that the acute phase reactant C-reactive protein (CRP) activates a threefold increase in sIL-6R production. Maximal release occurred after 30-60 min exposure to CRP (50 micrograms/ml), and was mimicked by peptides corresponding to amino acid residues 174- 185 and 201-206 of native CRP. A third peptide fragment (77-82) had no effect. Differential mRNA splicing did not account for the CRP-mediated release of sIL-6R, since this isoform was not detected in conditioned media. Furthermore, stimulation of neutrophils with CRP or with peptides 174-185 or 201-206 promoted a loss of membrane-bound IL-6R, suggesting release by proteolytic shedding. The metalloprotease inhibitor TAPI had only a marginal effect on CRP-mediated sIL-6R release, suggesting that shedding occurs via a mechanism distinct from that previously reported. It well established that IL-6 stimulates the acute phase expression of CRP. Our current findings demonstrate a novel relationship between these two mediators, since CRP may affect IL-6-mediated inflammatory events by enabling formation of the sIL-6R/IL-6 complex.

No effect of C-reactive protein on early atherosclerosis development in apolipoprotein E*3-leiden/human C-reactive protein transgenic mice.

OBJECTIVE: C-reactive protein (CRP) has been associated with risk of cardiovascular disease. It is not clear whether CRP is causally involved in the development of atherosclerosis. Mouse CRP is not expressed at high levels under normal conditions and increases in concentration only several-fold during an acute phase response. Because the dynamic range of human CRP is much larger, apolipoprotein E*3-Leiden (E3L) transgenic mice carrying the human CRP gene offer a unique model to study the role(s) of CRP in atherosclerosis development. METHODS AND RESULTS: Atherosclerosis development was studied in 15 male and 15 female E3L/CRP mice; E3L transgenic littermates were used as controls. The mice were fed a hypercholesterolemic diet to induce atherosclerosis development. Cholesterol exposure did not differ between E3L/CRP and E3L mice. Plasma CRP levels were on average 10.2+/-6.5 mg/L in male E3L/CRP mice, 0.2+/-0.1 mg/L in female E3L/CRP mice, and undetectable in E3L mice. Quantification of atherosclerosis showed that lesion area in E3L/CRP mice was not different from that in E3L mice. CONCLUSIONS: This study demonstrates that mildly elevated levels of CRP in plasma do not contribute to the development of early atherosclerosis in hypercholesterolemic E3L/CRP mice.

Single-nucleotide polymorphisms in the C-reactive protein (CRP) gene promoter that affect transcription factor binding, alter transcriptional activity, and associate with differences in baseline serum CRP level.

To investigate whether functional polymorphisms exist in the C-reactive protein (CRP) gene, i.e., ones that contribute directly to differences in baseline CRP among individuals, we sequenced a 1,156-nucleotide-long stretch of the CRP gene promoter in 287 ostensibly healthy people. We identified two single-nucleotide polymorphisms (SNPs), a bi-allelic one at nucleotide -409 (G-->A), and a tri-allelic one at -390 (C-->T-->A), both resident within the hexameric core of transcription factor binding E-box elements. Electrophoretic mobility shift assays confirmed that the SNP within the sequence (-412)CACGTG(-407) (E-box 1) modulates transcription factor binding, and that the one within (-394)CACTTG(-389) (E-box 2) supports transcription factor binding only when the -390 T allele is present. The commonest of four E-box 1/E-box 2 haplotypes (-409G/-390T) identified in the population supported highest promoter activity in luciferase reporter assays, and the rarest one (-409A/-390T) supported the least. Importantly, serum CRP in people with these haplotypes reproduced this rank order, i.e., people with the -409G/-390T haplotype had the highest baseline serum CRP (mean +/- SEM 10.9 +/- 2.25 microg/ml) and people with the -409A/-390T haplotype had the lowest (5.01 +/- 1.56 microg/ml). Furthermore, haplotype-associated differences in baseline CRP were not due to differences in age, sex, or race, and were still apparent in people with no history of smoking. At least two other SNPs in the CRP promoter lie within E-box elements (-198 C-->T, E-box 4, and -861 T-->C, E-box 3), indicating that not only is the quality of E-box sites in CRP a major determinant of baseline CRP level, but also that the number of E-boxes may be important. These data confirm that the CRP promoter does encode functional polymorphisms, which should be considered when baseline CRP is being used as an indicator of clinical outcome. Ultimately, development of genetic tests to screen for CRP expression variants could allow categorization of healthy people into groups at high versus low future risk of inflammatory disease.

C-reactive protein: structural biology, gene expression, and host defense function.

Over the past few years substantial insight was gained into the biology and biochemistry of human C-reactive protein (CRP). X-ray crystallography in conjunction with mutational analyses, generated the three-dimensional structure of the protein and indicated the topology and structure of ligand-binding sites. Using human CRP transgenic mice infected with Streptococcus pneumoniae, we obtained data that clearly established CRP as an important host defense molecule. Studies using the same mice revealed a previously unknown testosterone-dependence of constitutive expression of human CRP. In this article we provide a brief overview of these recent findings.

Intestinal pathology and site specificity of the acanthocephalan Neoechinorhynchus carpiodi Dechtiar, 1968, in quillback, Carpiodes cyprinus (Lesueur).

Intestinal pathology and site specificity of Neoechinorhynchus carpiodi Dechtiar, 1968, in quillback, Carpiodes cyprinus (Lesueur), from Dauphin Lake, Manitoba, was investigated. Neoechinorhynchus carpiodi elicits the formation of a nodule at the point of proboscis attachment. Over 98% of the worms were attached in sections 6-8 of the intestine. Fish had clusters of 1-8 nodules, cluster size increased with worm burden, and the volume of nodules increased with worm numbers. Nodule number, size, and pathology was related to intensity of infection and to depth of proboscis penetration. Three types of pathology were noted. Type I nodules had few worms and the proboscides were attached in the lamina propria; Type II nodules harbored more worms and the proboscides penetrated to, but not through, the stratum compactum; and Type III nodules had the greatest numbers of N. carpiodi and proboscides penetrated through the stratum compactum. Hyperplasia, vascularization, and collagen deposition were extensive, especially in Type III nodules. An epithelium-lined channel was present in most nodules. Type III nodules were most anterior (75% of the time), harbored the greatest proportion of immature worms regardless of cluster size, and had more gravid females. The largest nodules (Type III pathology) were well vascularized, long-lasting, and appeared to be the preferred microhabitat for the parasite.

Proteocephalus ambloplitis and Contracaecum sp. from largemouth bass (Micropterus salmoides) stocked into Boundary Reservoir, Saskatchewan.

Nonresident (introduced) largemouth bass (Micropterus salmoides) from Boundary Reservoir, Saskatchewan were examined for helminths. Four species of parasites were found (Diplostomum sp., Proteocephalus ambloplitis, Pomphorhynchus bulbocolli, and Contracaecum sp.). Contracaecum sp. larvae were absent in age-0 and age-1 bass, but prevalence and mean intensity increased with age for bass age-2 or older. Similarly, the prevalence and mean intensity of P. ambloplitis plerocercoids in bass were low until age-2; older bass harbored significantly more plerocercoids. Analysis of stomach contents indicates that this pattern of recruitment for Contracaecum sp. and P. ambloplitis is probably due to increased feeding by largemouth bass on aquatic insects and cannibalism after age-2, respectively. Although Contracaecum sp. may have been established in the reservoir prior to the introduction of bass, we are certain that P. ambloplitis was introduced via stocking with infected fingerlings.

Evaluation of gill nets, fyke nets, and mark-recapture methods to estimate the number of Hirudinea and Crustacea on fish.

Twenty species of fishes (n = 20,759) were collected from Dauphin Lake, Manitoba, Canada, to determine the types and numbers of ectoparasites they harbored. Counts of ectoparasites on fishes collected with different gear were compared to evaluate different methods of collection and to estimate rates of recruitment of ectoparasites by fishes. Ectoparasites were found on 11 species of fishes and the majority of these were parasitic leeches (Myzobdella moorei, Cystobranchus verilli, and Placobdella montifera) and parasitic Crustacea (Argulus appendiculosus and Lernaea cyprinacea). Some fishes also were infested by neascus-type metacercariae (blackspot) or had tumors (lymphocystis). The prevalence of ectoparasites was correlated with the abundance, feeding habits, and spatial distribution of fish species. Argulus appendiculosus and blackspot were more prevalent on benthic fishes, whereas M. moorei and tumors were more prevalent on limnetic fishes. Mark-recapture records showed that fishes occupying shallow (less than or equal to 1.5 m) water had a higher prevalence of infestation and 28 of 29 infected fishes caught by gill nets were captured in shallow water. Placobdella montifera was the only ectoparasite found on fishes from deep (1.5-3.5 m) water and the only species that was acquired by fishes previously released with no ectoparasite (2 of 239 fishes). The littoral zone (less than or equal to 1.5 m) comprises only 14% of the surface area and 3% of the volume of Dauphin Lake, yet 72% of all gill-netted fishes harboring ectoparasites were collected there. Intensities of ectoparasites estimated from gill net and pound net samples were similar, but prevalence of ectoparasites estimated from samples obtained with gill nets was lower.

Use of confidence ellipses to detect effects of parasites on the growth of yellow perch, Perca flavescens.

Determining the causes of mortality in populations of fish is inherently difficult. To simplify the determination of whether parasite-induced mortality occurs, parasitologists have relied on 3 types of subjective analyses of graphs. Peaked host age-parasite intensity curves concomitant with a decrease in the degree of dispersion (measured by variance-to-mean ratio) of parasites in older age-classes of fishes, a slope of less than 2.0 for a log-log graph of variance versus mean intensity of infection, and differences between truncated and nontruncated forms of a theoretical frequency distribution for the parasite are considered indicators of parasite-induced mortality in fishes. The nematode Raphidascaris acus causes significant parasite-induced mortality in natural populations of yellow perch (Perca flavescens) in Dauphin Lake, Manitoba, Canada. Using this fish-parasite system we present a comparison of some of the graphical techniques used by parasitologists to detect parasite-induced mortality and show how confidence ellipses based on the parameters beta 0 and beta 1 of a linear model for growth of yellow perch (weight = beta 0 + beta 1 x age) can be used to compare many growth curves simultaneously. When plotted in a bivariate fashion (beta 0 vs. beta 1), vertical displacement of confidence ellipses along the ordinate (beta 1) are due to sublethal effects on growth of fishes in response to parasites, whereas lateral shifts along the abscissa (beta 0) are suggestive of parasite-induced mortality.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in numbers and growth of Ligula intestinalis in the spottail shiner (Notropis hudsonius), and their roles in transmission.

Eighteen species of fishes were collected during the open water (1985-1987) and winter seasons (1985-1986) from Dauphin Lake, Manitoba, Canada (51 degrees 17'N, 99 degrees 48'W) and examined for plerocercoids of Ligula intestinalis (L.). Plerocercoids were most prevalent (5.3%) in spottail shiners (Notropis hudsonius), the major fish host for Ligula in Dauphin Lake. Detailed analysis of the spottail shiner-Ligula host-parasite system revealed that the number of plerocercoids differed between years and among habitats but there was no statistically significant seasonal pattern; recruitment of new worms was highest in young fish and decreased with age, and infected spottails had reduced gonad development. Analysis of host and parasite growth revealed that the soma of male spottails infected with Ligula weighed more but had a reduced growth rate. Growth rate of females was unaffected by Ligula but somatic weight was slightly increased. This appears to be related to the greater metabolic stress on infected males. Based on increased mean somatic weight and skewness of the weight distribution for infected fish, we propose that infected spottails are subjected to size-selective mortality. Some of the contradictions in the literature may be attributed to underestimated prevalence due to increased numbers of spawning fish in the spring, mixed age-classes of Ligula in separate age-classes of fish, and differential effects on growth due to infection in male versus female hosts.

Helminths of stocked rainbow trout (Salmo gairdneri) with special reference to Clinostomum complanatum.

Rainbow trout (Salmo gairdneri) stocked into a small prairie dug-out were examined for helminths at harvest time. Five species of helminths were found (Diplostomum sp., Diplostomulum scheuringi, Clinostomum complanatum, Crepidostomum farionis and Pomphorhynchus bulbocolli); C. farionis and P. bulbocolli mature in fish while the remaining species utilize fish-eating birds as definitive hosts. Examination of stomach contents indicated that all invertebrate hosts required for the life-cycles of these helminths were present in the dug-out. The most conspicuous and prevalent helminth was C. complanatum as fish were "wormy" and unmarketable due to the presence of high numbers of metacercariae. Metacercariae of C. complanatum were recovered from most organs of rainbow trout. The low survival of stocked fish suggests that C. complanatum may cause some host mortality, but the condition of infected and uninfected fish was similar. It appears that a community of ichthyoparasites can become established in a population of stocked fish in a single growing season in north temperate regions.

C-reactive protein induces expression of tissue factor and plasminogen activator inhibitor-1 and promotes fibrin accumulation in vein grafts.

BACKGROUND: C-reactive protein (CRP) promotes tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) expression in vitro, and an elevated plasma CRP concentration is associated with an increased risk of vein graft (VG) thrombosis after coronary artery bypass surgery. However, little is known about the effects of CRP on VG TF and PAI-1 expression in vivo, or on VG thrombosis. OBJECTIVES: We studied transgenic (Tg) mice expressing human CRP in a VG model to explore in vivo cause-and-effect relationships between CRP and TF, PAI-1, and VG thrombosis. METHODS: Vein segments from wild-type (WT) and CRP-Tg donors were transplanted into carotid arteries of WT and CRP-Tg recipients. VGs were analyzed 1-4 weeks later. RESULTS: Human CRP accumulated in VGs during the first 4 weeks after surgery, but appeared to originate exclusively from systemic sources, rather than local production. Human CRP significantly increased TF gene expression, protein concentration and activity in VGs. Human CRP also increased PAI-1 concentrations in VGs, although only in vascular endothelial cells. Human CRP stimulated macrophage migration, invasion into VGs, and TF expression. Fibrin deposition was significantly greater in VGs of CRP-Tg mice than in WT controls. CONCLUSIONS: CRP accumulates in VGs early after surgery, originating from systemic sources rather than local synthesis. Human CRP promotes TF and PAI-1 expression in VGs, although with different expression patterns. Human CRP stimulates macrophage invasion and fibrin deposition within VGs. These results suggest that CRP induces pathologic changes in VGs that contribute to early VG occlusion.

Overexpression of innate immune response genes in a model of recessive polycystic kidney disease.

Defects in the primary cilium/basal body complex of renal tubular cells cause polycystic kidney disease (PKD). To uncover pathways associated with disease progression, we determined the kidney transcriptome of 10-day-old severely and mildly affected cpk mice, a model of recessive PKD. In the severe phenotype, the most highly expressed genes were those associated with the innate immune response including many macrophage markers, particularly those associated with a profibrotic alternative activation pathway. Additionally, gene expression of macrophage activators was dominated by the complement system factors including the central complement component 3. Additional studies confirmed increased complement component 3 protein levels in both cystic and non-cystic epithelia in the kidneys of cpk compared to wild-type mice. We also found elevated complement component 3 activation in two other mouse-recessive models and human-recessive PKD. Our results suggest that abnormal complement component 3 activation is a key element of progression in PKD.

Systemic lupus erythematosus in a multiethnic US Cohort (LUMINA). XXX: association between C-reactive protein (CRP) gene polymorphisms and vascular events.

OBJECTIVES: To determine if a polymorphic GTn repeat in the intron of the C-reactive protein (CRP) gene associates with occurrence of vascular arterial events in systemic lupus erythematosus (SLE). METHODS: We performed a nested case-control study on the LUMINA cohort of 546 Hispanic, African-American and Caucasian SLE patients. Twenty-five patients who developed vascular arterial events (i.e. myocardial infarction, angina, coronary artery bypass graft surgery, stroke, claudication, gangrene or significant tissue loss and/or arterial peripheral thrombosis) after enrolment were selected as cases and 32 ethnically matched patients with no previous vascular arterial events served as controls. Their CRP gene GTn polymorphism and plasma CRP was determined. RESULTS: Patients with vascular events had more severe SLE and were more likely to have plasma CRP in the highest quintile of measured values. The overall distribution of GTn alleles for patients with vascular events had a greater number of the GT20 variant compared with controls [26.0% of alleles (13/50) vs 15.6% (10/64)]. This greater number of GT20 in patients with vascular events was observed for African-Americans [29.2% (7/24) vs 21.0% (8/38)] and Hispanics [33.0% (4/12) vs 0% (0/16)] but not for Caucasians [14.3% (2/14) vs 20.0% (2/10)]. For African-Americans and Hispanics combined (45 patients), the frequency of GT20 in those with vascular events (30.6%, 11/36) was significantly higher than in those without them (14.8%, 8/54) (P<0.05, one-tailed test for difference in proportions). When patients were categorized according to the number of GT20 alleles they carried (thus GT20/GT20, GT20/GTx or GTx/GTx, where x is any allele other than GT20), for both African-Americans and Hispanics the likelihood of vascular arterial events increased in proportion with the GT20 dose, and all GT20-homozygous patients developed vascular arterial events. CONCLUSIONS: The CRP GT20 variant is more likely to occur in African-American and Hispanic SLE patients than in Caucasian ones, and SLE patients carrying the GT20 allele are more likely to develop vascular arterial events.

Chelation affects the conformation, lability and aggregation of channel catfish (Ictalurus punctatus) phosphorylcholine-reactive protein (PRP).

1. Phosphorylcholine-reactive protein (PRP) affinity-purified from channel catfish (Ictalurus punctatus) serum on phosphorylcholine-Sepharose, eluted from Bio-Gel A-5M as a 94.6 +/- 2.4 kDa protein when the gel filtration column buffer (Tris-saline) contained 25mM ethylenediaminetetraacetic acid (EDTA). 2. PRP chelated with EDTA immediately after affinity purification and gel-filtered in Tris-saline-EDTA, eluted as a 75.5 +/- 2.67 kDa protein referred to as fast-PRP (F-PRP). 3. PRP and F-PRP were identical on SDS-PAGE. Both resolved as a broad band of protein (ca 86-100 kDa) on non-reducing gels or as a ca 100 kDa protein after reduction with 2-mercaptoethanol (2-ME). 4. After gel-filtration in Tris-saline-EDTA, nearly complete reduction of 100 kDa PRP was achieved on SDS-PAGE. However, the protein regained its resistance to reduction upon storage at -60 degrees C. 5. SDS-PAGE and native PAGE also revealed that during storage, PRP and F-PRP combined to form 3 different aggregates referred to as aggregated-PRP (aggPRP). These aggregates are readily dissociated in the presence of 2-ME, suggesting a covalent interaction between adjacent pentamers comprising decameric aggPRPs. 6. PRP, F-PRP, and aggPRP have similar amino acid compositions.

Human C-reactive protein is protective against fatal Streptococcus pneumoniae infection in transgenic mice.

C-reactive protein (CRP) is an acute phase protein with a well known association with infection and other inflammatory conditions. Studies with use of purified CRP in in vitro assays provided early evidence that CRP has antibacterial activity. Subsequently it was shown that passively administered human CRP can protect mice from lethal infection with Streptococcus pneumoniae. In this study, we extend these observations to an in vivo model of host resistance by using human CRP transgenic mice. CRP transgenic mice experimentally infected with S. pneumoniae lived longer and had significantly lower mortality than their nontransgenic littermates. This increased resistance to infection was associated with q 10- to 400-fold reduction of bacteremia. Furthermore, male transgenics exhibited longer survival time than females, and this difference could be attributed to increased expression of CRP by males, which was mediated by testosterone. This study provides the first unequivocal evidence that CRP plays an important role in vivo in host defense against pneumococcal infections, and shows that sex hormones can affect expression of the human CRP transgene in mice.