Protein arginine deiminase 4 inhibition is sufficient for the amelioration of collagen-induced arthritis.

Citrullination of joint proteins by the protein arginine deiminase (PAD) family of enzymes is recognized increasingly as a key process in the pathogenesis of rheumatoid arthritis. This present study was undertaken to explore the efficacy of a novel PAD4-selective inhibitor, GSK199, in the murine collagen-induced arthritis model of rheumatoid arthritis. Mice were dosed daily from the time of collagen immunization with GSK199. Efficacy was assessed against a wide range of end-points, including clinical disease scores, joint histology and immunohistochemistry, serum and joint citrulline levels and quantification of synovial autoantibodies using a proteomic array containing joint peptides. Administration of GSK199 at 30 mg/kg led to significant effects on arthritis, assessed both by global clinical disease activity and by histological analyses of synovial inflammation, pannus formation and damage to cartilage and bone. In addition, significant decreases in complement C3 deposition in both synovium and cartilage were observed robustly with GSK199 at 10 mg/kg. Neither the total levels of citrulline measurable in joint and serum, nor levels of circulating collagen antibodies, were affected significantly by treatment with GSK199 at any dose level. In contrast, a subset of serum antibodies reactive against citrullinated and non-citrullinated joint peptides were reduced with GSK199 treatment. These data extend our previous demonstration of efficacy with the pan-PAD inhibitor Cl-amidine and demonstrate robustly that PAD4 inhibition alone is sufficient to block murine arthritis clinical and histopathological end-points.

Smoking is not associated with autoantibody production in systemic lupus erythematosus patients, unaffected first-degree relatives, nor healthy controls.

OBJECTIVE: The objective of this paper is to examine whether smoking is associated with autoantibody production in systemic lupus erythematosus (SLE) patients, unaffected first-degree relatives (FDR) of individuals with SLE--a group at increased risk of developing SLE--or unaffected, unrelated controls. METHODS: Detailed demographic, environmental, clinical, and therapeutic information was collected by questionnaire on 1242 SLE patients, 981 FDRs, and 946 controls in the Lupus Family Registry and Repository; a blood sample was obtained. All sera were tested for multiple lupus autoantibodies by immunofluorescence and luminex bead-based assays. Generalized estimating equations, adjusting for age, gender, and ethnicity and accounting for correlation within families, were used to assess smoking status with the dichotomous outcome variables of positivity for SLE status, positivity of ANA by immunofluorescence (≥1:120), positivity for ≥1 autoantibody by the luminex assay, and positivity for each of the 11 autoantibodies. RESULTS: Current smoking was associated with being positive for ≥1 autoantibody (excluding ANA) (adjusted OR = 1.53, 95% CI 1.04-2.24) in our subjects with SLE. No association was observed in unaffected FDRs or healthy controls. Former smoking was associated with anti-Ro/SS-A60 in our unaffected FDRs. There was an increased association with anti-nRNP A seropositivity, as well as a decreased association with anti-nRNP 68 positivity, in current smokers in SLE subjects. CONCLUSIONS: No clear association between smoking status and individual autoantibodies was detected in SLE patients, unaffected FDRs, nor healthy controls within this collection. The association of smoking with SLE may therefore manifest its risk through mechanisms outside of autoantibody production, at least for the specificities tested.

Studies of Filipino patients with systemic lupus erythematosus: autoantibody profile of first-degree relatives.

This study surveyed the frequency of autoantibodies among un-affected first-degree relatives (FDRs) of Filipino systemic lupus erythematosus (SLE) patients compared with healthy un-related Filipino controls. The sensitivity, specificity and predictive value of the autoantibodies for SLE diagnosis were also assessed in this Filipino cohort. Filipino patients included in the University of Santo Tomas (UST) Lupus Database and un-affected FDRs were recruited. Healthy controls included those with no known personal or family history of autoimmune disease. The following autoantibodies were tested in all subjects: anti-nuclear antibody (ANA), anti-dsDNA, anti-Ro/SSA, anti-chromatin, anti-thyroid microsome, and anti-cardiolipin antibodies. Participants included 232 SLE patients, 546 FDRs, and 221 healthy controls. Median age of patients was 27 (range 8-66) years with median disease duration of 27.5 (range 1-292) months. Median age of FDRs was 42.0 (range 5-87) years. Compared with healthy controls, there were significantly more FDRs with positive ANA at titers 1 : 40 to 1 : 160 (p < 0.001) and 1 : 320 (p = 0.003), anti-Ro/SSA (4.94% versus 0.45%, p = 0.003), and anti-dsDNA ≥ 5.0 IU/ml (4.58% versus 1.36%, p = 0.031). ANA titer ≥1 : 160, anti-dsDNA, anti-Ro/SSA and anti-chromatin had the highest predictive value for SLE diagnosis. These findings reinforce the role of genetic influence in SLE risk among Filipinos, with a significant proportion of un-affected FDRs of SLE patients testing positive for autoantibodies compared with healthy Filipino controls. A longitudinal observational study in this same cohort will determine which proportion of these un-affected FDRs will evolve into clinical SLE disease in the future.

Epithelial secretion of C3 promotes colonization of the upper urinary tract by Escherichia coli.

To assess the role of complement in renal infection, we studied a model of Escherichia coli-induced pyelonephritis in mice deficient in complement components C3 and C4. Renal infection occurred less frequently in C3- and C4-deficient mice compared with wild-type mice. In vitro, renal epithelial cells internalized fewer bacteria in the absence of C3 or in the presence of blockade of C3 bound to the bacteria. Moreover, upregulation of epithelial C3 production by stimulation with lipopolysaccharide enhanced bacterial internalization. Here we provide evidence that uropathogenic E. coli might use host C3 to invade the renal epithelium and that local complement production is sufficient for the bacteria to achieve this effect.

Supervised machine learning and logistic regression identifies novel epistatic risk factors with PTPN22 for rheumatoid arthritis.

Investigating genetic interactions (epistasis) has proven difficult despite the recent advances of both laboratory methods and statistical developments. With no 'best' statistical approach available, combining several analytical methods may be optimal for detecting epistatic interactions. Using a multi-stage analysis that incorporated supervised machine learning and methods of association testing, we investigated epistatic interactions with a well-established genetic factor (PTPN22 1858T) in a complex autoimmune disease (rheumatoid arthritis (RA)). Our analysis consisted of four principal stages: Stage I (data reduction)-identifying candidate chromosomal regions in 292 affected sibling pairs, by predicting PTPN22 concordance using multipoint identity-by-descent probabilities and a supervised machine learning algorithm (Random Forests); Stage II (extension analysis)-testing detailed genetic data within candidate chromosomal regions for epistasis with PTPN22 1858T in 677 cases and 750 controls using logistic regression; Stage III (replication analysis)-confirmation of epistatic interactions in 947 cases and 1756 controls; Stage IV (combined analysis)-a pooled analysis including all 1624 RA cases and 2506 control subjects for final estimates of effect size. A total of seven replicating epistatic interactions were identified. SNP variants within CDH13, MYO3A, CEP72 and near WFDC1 showed significant evidence for interaction with PTPN22, affecting susceptibility to RA.

Identification of an alternative polyadenylation site in the human C3b/C4b receptor (complement receptor type 1) transcriptional unit and prediction of a secreted form of complement receptor type 1.

The human C3b/C4b receptor or complement receptor type one (CR1) is an approximately 200-kD single chain membrane glycoprotein of human peripheral blood cells that mediates the binding, processing, and transport of C3b-bearing immune complexes and regulates the activity of the complement cascade. Analysis of partial cDNA clones has shown that the COOH terminus is composed predominantly of three tandemly repeated regions of 450 amino acids each (15). In this report, we present a cDNA sequence that encodes the NH2 terminus of CR1. It appears to have been derived from an alternatively processed transcript, caused by polyadenylation occurring at a site within an intron in the CR1 transcriptional unit. The resulting truncated messenger carries an open reading frame that would produce a short, secreted CR1 form. We present genomic sequences and Northern blots which support this hypothesis and we propose that the NH2-terminal end of CR1 is a likely location for active sites. In addition, we report evidence for a CR1-like sequence in the human genome and we present a model for the organization of CR1.

Distinct receptor and regulatory properties of recombinant mouse complement receptor 1 (CR1) and Crry, the two genetic homologues of human CR1.

The relationship between the characterized mouse regulators of complement activation (RCA) genes and the 190-kD mouse complement receptor 1 (MCR1), 155-kD mouse complement receptor 2 (MCR2), and mouse p65 is unclear. One mouse RCA gene, designated MCR2 (or Cr2), encodes alternatively spliced 21 and 15 short consensus repeat (SCR)-containing transcripts that crosshybridize with cDNAs of both human CR2 and CR1, or CR2 alone, respectively. A five SCR-containing transcript derived from a second unique gene, designated Crry, also crosshybridizes with human CR1. We have previously shown that the 155-kD MCR2 is encoded by the 15 SCR-containing transcript. To analyze the protein products of the other transcripts, which are considered the genetic homologues of human CR1, we have expressed the 21 and the 5 SCR-containing cDNAs in the human K562 erythroleukemia cell line. We demonstrate that cells expressing the 21 SCR transcript express the 190-kD MCR1 protein. These cells react with five unique rat anti-MCR1 monoclonal antibodies, including the 8C12 antibody considered to be monospecific for MCR1. In addition, these cells efficiently form rosettes with mouse C3b-bearing sheep erythrocytes. In contrast, cells expressing the five SCR-containing Crry transcript are strongly recognized by an anti-human CR1 antibody that also defines the mouse p65 protein. Using a functional assay that measures the surface deposition of C3 activated via the classical complement pathway, we show that Crry/p65-expressing cells have a markedly decreased amount of C3 deposited on them as compared with control cells expressing the antisense construct or cells expressing MCR1 or MCR2. This suggests that Crry has intrinsic complement regulatory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Herpes simplex virus glycoproteins gC-1 and gC-2 bind to the third component of complement and provide protection against complement-mediated neutralization of viral infectivity.

Cells infected with herpes simplex virus type 1 (HSV-1) form rosettes with C3b-coated erythrocytes, whereas cells infected with herpes simplex virus type 2 (HSV-2) or other herpes viruses do not. It was reported that glycoprotein C of HSV-1 (gC-1) mediates the binding of C3b-coated erythrocytes to infected cells and has regulatory (decay-accelerating) activity for the alternative pathway C3 convertase of human complement. We show here that solubilized gC-1 binds to iC3-Sepharose affinity columns. We also report that solubilized gC-2, the genetically related glycoprotein specified by HSV-2, binds to iC3-Sepharose. mAb specific for gC-1 or gC-2 and mutant viral strains were used to identify the C3-binding glycoproteins. In other experiments, HSV-1 mutant strains and recombinants, differing only in their expression of gC, were tested for sensitivity to neutralization by human complement in the presence or absence of antibodies specific for HSV gD. In either case the gC- strain was most sensitive. Expression of gC-1 or gC-2 by isogenic insertion mutants provided protection against complement-mediated neutralization. These results indicate that the genetically and structurally related gC-1 and gC-2 share the functional activity of binding to human C3 and enhance viral infectivity.

Structural gene for human membrane cofactor protein (MCP) of complement maps to within 100 kb of the 3' end of the C3b/C4b receptor gene.

The structural gene for membrane cofactor protein (MCP), a widely distributed C3b/C4b binding regulatory glycoprotein of the complement system, has been mapped to the same locus as the structural genes for CR1, CR2, DAF, and C4bp. The order of the genes within an approximately 800-kb DNA fragment on the long arm of chromosome 1 is MCP-CR1-CR2-DAF-C4bp. Further, the MCP gene maps to within 100 kb of 3' end of the CR1 gene.

Molecular cloning of a murine fibronectin receptor and its expression during inflammation. Expression of VLA-5 is increased in activated peritoneal macrophages in a manner discordant from major histocompatibility complex class II.

Human fibronectin receptor (VLA-5) alpha and beta chain probes were used to identify their mouse homologues in a thioglycollate-elicited peritoneal exudate cell cDNA library. Sequence analysis of both alpha and beta chain-related murine clones revealed approximately 90% homology to their human counterparts by both nucleotide and derived amino acid sequence comparisons. Detectable alpha chain transcripts were seen predominantly in total RNA of peritoneal macrophages. beta chain expression, however, was detected at higher levels in lung, heart, brain, and kidney, suggesting the presence of a large murine VLA family similar to the human family. Analysis of levels of expression comparing resting peritoneal macrophages with macrophages elicited using inflammatory stimuli indicated that alpha chain message and surface VLA-5 expression were significantly increased using thioglycollate or Listeria monocytogenes as stimuli to elicit cells. Interestingly, beta chain message was unaffected by these inflammatory stimuli, suggesting that VLA-5 expression is regulated by VLA-5 alpha chain message levels. These results indicate that macrophage VLA-5 expression can be modulated in vivo and may provide an important mechanism by which macrophages are recruited to or adhere to fibronectin in inflammatory foci.

Mouse complement regulatory protein Crry/p65 uses the specific mechanisms of both human decay-accelerating factor and membrane cofactor protein.

Normal host cells are protected from the destructive action of complement by cell surface complement regulatory proteins. In humans, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) play such a biologic role by inhibiting C3 and C5 convertases. DAF and MCP accomplish this task by specific mechanisms designated decay-accelerating activity and factor I cofactor activity, respectively. In other species, including mice, structural and/or functional homologues of these proteins are not yet well characterized. Previous studies have shown that the mouse protein Crry/p65 has certain characteristics of self-protecting complement regulatory proteins. For example, Crry/p65 is expressed on a wide variety of murine cells, and when expressed on human K562 erythroleukemic cells, it prevents deposition of mouse C3 fragments on the cell surface during activation of either the classical or alternative complement pathway. We have now studied factor I cofactor and decay-accelerating activities of Crry/p65. Recombinant Crry/p65 demonstrates cofactor activity for factor I-mediated cleavage of both mouse C3b and C4b. Surprisingly, Crry/p65 also exhibits decay-accelerating activity for the classical pathway C3 convertase strongly and for the alternative pathway C3 convertase weakly. Therefore, mouse Crry/p65 uses the specific mechanisms of both human MCP and DAF. Although Crry/p65, like MCP and DAF, contains tandem short consensus repeats (SCR) characteristic of C3/C4 binding proteins, Crry/p65 is not considered to be a genetic homologue of either MCP or DAF. Thus, Crry/p65 is an example of evolutionary conservation of two specific activities in a single unique protein in one species that are dispersed to individual proteins in another. We propose that the repeating SCR motif in this family has allowed this unusual process of evolution to occur, perhaps driven by the use of MCP and DAF as receptors by human pathogens such as the measles virus.

Transgenic mice overexpressing the complement inhibitor crry as a soluble protein are protected from antibody-induced glomerular injury.

Complement receptor 1-related gene/protein y (Crry) is a potent murine membrane complement regulator that inhibits classical and alternative pathway C3 convertases. In nephrotoxic serum (NTS) nephritis, injected antibodies (Abs) bind to glomeruli, leading to complement activation and subsequent glomerular injury and albuminuria. To study the phenotypic effects of continuous complement pathway blockade, transgenic mice were created that express recombinant soluble (rs) Crry directed by the broadly active and heavy metal-inducible metallothionein-I promoter. One transgenic line expressing high levels of rsCrry was propagated. Serum rsCrry levels were 18.7 +/- 2.7 microg/ml (n = 5) at basal level and increased to 118.1 +/- 20.6 microg/ml 4 d after addition of zinc to the drinking water. By reverse transcription polymerase chain reaction (RT-PCR), transgene messenger (m)RNA was present in liver, kidney, brain, lung, and spleen, but not in heart. By in situ RT-PCR analysis of kidneys, transgene mRNA was widely expressed both in renal glomeruli and tubules. Urinary excretion of rsCrry was 113.4 +/- 22.4 microg/ml with a fractional excretion relative to creatinine of 13.2 +/- 2.7%, consistent with local renal production of rsCrry and secretion into urine. The founder and all transgene positive adult animals have remained healthy with no mortality or apparent phenotypic abnormalities, including infection or immune complex disease. To determine whether rsCrry blocked complement-mediated injury, NTS nephritis was induced by injection of NTS immunoglobulin (Ig)G, followed by an 18-h urine collection to quantitate the excretion of albumin as a measure of glomerular injury. In transgene-negative littermates (n = 15), transgene-positive animals (n = 10), and transgene-positive animals fed zinc (n = 10), albuminuria was 4,393 +/- 948, 1,783 +/- 454, and 1,057 +/- 277 microg/mg creatinine, respectively (P < 0.01 by ANOVA). Glomerular C3 was evident by immunofluorescence staining in 12/15 transgene-negative animals, but in none of the transgene-positive animals fed zinc. Thus, we have produced the first transgenic animals that overexpress a soluble C3 convertase inhibitor. rsCrry expression markedly ameliorates an Ab-induced disease model in vivo. These results support the hypothesis that continuous complement inhibition at the C3 convertase step is feasible and effective in complement-mediated injury states.

The gene encoding decay-accelerating factor (DAF) is located in the complement-regulatory locus on the long arm of chromosome 1.

Delay-accelerating factor (DAF) protects host cells from complement-mediated damage by regulating the activation of C3 convertases on host cell surfaces. Using a panel of hamster-human somatic cell hybrids, the DAF gene was mapped to human chromosome 1. In situ hybridization studies using human metaphase cells further localized the gene to bands 1q31-41, with the largest cluster of grains at 1q32. This establishes the close linkage of the DAF gene to genes for four other proteins (C3b/C4b receptor or complement receptor 1, C3d receptor or complement receptor 2, factor H, and C4-binding protein) that share 60-amino-acid homologous repeats as well as complement-regulatory or -receptor activity, thereby enlarging the complement-regulatory gene family on the long arm of human chromosome 1.

Complement activation pathways in murine immune complex-induced arthritis and in C3a and C5a generation in vitro.

The alternative pathway (AP) of complement alone is capable of mediating immune complex-induced arthritis in the collagen antibody-induced arthritis (CAIA) model in mice. Whether the classical pathway (CP) or lectin pathway (LP) alone can mediate CAIA is not known. Using mice genetically deficient in different complement components, our results reported herein establish that the CP and LP alone are each incapable of mediating CAIA. A lower level or absence of C3 and/or C5 activation by the CP may be possible explanations for the importance of the AP in CAIA and in many murine models of disease. In addition, other investigators have reported that CP C5 convertase activity is absent in mouse sera. To address these questions, we employed an in vitro system of adherent immunoglobulin (Ig)G-induced complement activation using plates coated with murine anti-collagen monoclonal antibody (mAb). These experiments used complement-deficient mouse sera and wild-type mouse or normal human sera under conditions inactivating either the CP (Ca(++) deficiency) or the AP (mAb inhibitory to factor B). Robust generation of both C3a and C5a by either the AP or CP alone were observed with both mouse and human sera, although there were some small differences between the species of sera. We conclude that neither the CP nor LP alone is capable of mediating CAIA in vivo and that mouse sera exhibits a high level of IgG-induced C5a generation in vitro through either the CP or AP.

The interrelationship of complement-activation fragments and angiogenesis-related factors in early pregnancy and their association with pre-eclampsia.

OBJECTIVE: To determine the interrelationships during early pregnancy of complement-activation fragments Bb, C3a and sC5b-9, and angiogenesis-related factors placental growth factor (PiGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and their associations with pre-eclampsia. DESIGN: Prospective cohort study. SETTING: Denver complement study (June 2005-June 2008). POPULATION: A total of 668 pregnant women with singleton gestations, recruited between 10 and 15 weeks of gestation. METHODS: Using univariable and multivariable logistic regression analysis, concentrations of complement-activation fragments and angiogenesis-related factors were compared between 10 and 15 weeks of gestation in women who subsequently did or did not develop pre-eclampsia. Interrelationships between these variables were tested using the non-parametric Spearman rank correlation coefficient. MAIN OUTCOME MEASURE: Pre-eclampsia. The association of complement-activation fragments and angiogenesis-related factors with obesity was also examined. RESULTS: The mean (+/-SD) levels of complement Bb in early pregnancy among women who did and did not develop pre-eclampsia were 0.84 (+/-0.26) microg/ml and 0.69 (+/-0.2) microg/ml, respectively (P = 0.001). Concentrations of PiGF were significantly (P = 0.01) lower (31 +/- 12 pg/ml) in early pregnancy in the pre-eclamptic group of women, as compared with the normotensive group (39 +/- 32 pg/ml). The adjusted odds ratio (AOR) of Bb and PiGF were 2.1 (CI = 1.4-3.1, P < 0.0003) and 0.2 (CI = 0.07-0.7, P = 0.01), respectively. There was no significant difference in the levels of C3a, sC5b-9, sFlt-1 and sEng in early pregnancy among women who developed pre-eclampsia, compared with women who remained normotensive during pregnancy. Higher levels of Bb (P = 0.0001) and C3a (P = 0.03), and lower levels of sFlt-1 (P = 0.0002) and sEng (P = 0.0001) were found among women with obesity, compared with non-obese controls. No meaningful relationships were found between the complement-activation fragments and the angiogenesis-related factors. CONCLUSIONS: In this cohort during early pregnancy, increased concentrations of complement-activation factor Bb and lower concentrations of PiGF were associated with the development of pre-eclampsia later in pregnancy.

Increased expression of activation-induced cytidine deaminase is associated with anti-CCP and rheumatoid factor in rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL-17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation-induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL-17. The results of immunohistochemical staining showed that organized AID(+) germinal centres were observed in six of the 12 RA synovial samples, and AID(+) cells were found almost exclusively in the B-cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID(+) cells. Significantly higher levels of AID mRNA (Aicda) detected by RT-PCR were found in the PBMCs from RA patients than PBMCs from normal controls (P < 0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10(+)IgM(+)CD20(+) B-cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls (P < 0.01). AID expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) (P

Complement receptor 3 (CR3, Mac-1, integrin alpha M beta 2, CD11b/CD18) is required for tyrosine phosphorylation of paxillin in adherent and nonadherent neutrophils.

Expression of the leukocyte (beta 2) integrins is required for many functions of activated neutrophils (PMN), even when there is no recognized ligand for any beta 2 integrin. To investigate the hypothesis that beta 2 integrins may be involved in a signal transduction pathway related to cytoskeletal reorganization, we examined whether beta 2 integrins have a role in tyrosine phosphorylation of the cytoskeletal protein paxillin. Treatment of PMN in suspension with phorbol esters, f-Met-Leu-Phe, and TNF-alpha resulted in paxillin tyrosine phosphorylation. However, treatment of beta 2-deficient (LAD) PMN failed to induce paxillin tyrosine phosphorylation. Normal PMN phosphorylated paxillin in response to adhesion to immune complexes, while the LAD PMN did not. Adhesion of phorbol ester activated-LAD PMN to the extracellular matrix proteins fibronectin, laminin, and vitronectin failed to induce paxillin tyrosine phosphorylation. Treatment of activated normal PMN with mAb directed against the beta 2 integrin alpha chains demonstrated that CR3 (alpha M beta 2) was required for paxillin phosphorylation. Transfection of the cell line K562 with CR3 confirmed that CR3 ligation resulted in paxillin tyrosine phosphorylation. As a control, K562 transfected with CR2 (CD21) which bound equally avidly to the same complement C3-derived ligand (C3bi) as the CR3 transfectants, showed no enhanced tyrosine phosphorylation of paxillin upon receptor ligation. While both CR2 and CR3 transfectants showed efficient adhesion to a C3bi-coated surface, only the CR3 transfectants spread during adhesion and phosphorylated paxillin. Together these data demonstrate that CR3 is required for paxillin phosphorylation during activation of both adherent and nonadherent PMN. Even PMN activated in suspension or by adhesion to immune complexes, when no CR3 ligand is apparent, still require CR3 for a signal transduction pathway leading to paxillin tyrosine phosphorylation. This pathway is likely to be important for PMN function in inflammation and host defense.

A critical role for murine complement regulator crry in fetomaternal tolerance.

Complement is a component of natural immunity. Its regulation is needed to protect tissues from inflammation, but mice with a disrupted gene for the complement regulator decay accelerating factor were normal. Mice that were deficient in another murine complement regulator, Crry, were generated to investigate its role in vivo. Survival of Crry-/- embryos was compromised because of complement deposition and concomitant placenta inflammation. Complement activation at the fetomaternal interface caused the fetal loss because breeding to C3-/- mice rescued Crry-/- mice from lethality. Thus, the regulation of complement is critical in fetal control of maternal processes that mediate tissue damage.

Structure of complement receptor 2 in complex with its C3d ligand.

Complement receptor 2 (CR2/CD21) is an important receptor that amplifies B lymphocyte activation by bridging the innate and adaptive immune systems. CR2 ligands include complement C3d and Epstein-Barr virus glycoprotein 350/220. We describe the x-ray structure of this CR2 domain in complex with C3d at 2.0 angstroms. The structure reveals extensive main chain interactions between C3d and only one short consensus repeat (SCR) of CR2 and substantial SCR side-side packing. These results provide a detailed understanding of receptor-ligand interactions in this protein family and reveal potential target sites for molecular drug design.

Duration of preclinical rheumatoid arthritis-related autoantibody positivity increases in subjects with older age at time of disease diagnosis.

OBJECTIVES: To investigate factors that may influence the prevalence and timing of appearance of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies during the preclinical phase of rheumatoid arthritis (RA) development. METHODS: 243 serial prediagnosis serum samples from 83 subjects with RA were examined for the presence of RF and anti-CCP antibodies. RESULTS: Of the 83 cases, 47 (57%) and 51 (61%) subjects had at least one prediagnosis sample positive for RF or anti-CCP, respectively. Gender and race were not significantly associated with the prevalence or timing of preclinical antibody appearance. Preclinical anti-CCP positivity was strongly associated with the development of erosive RA (odds ratio = 4.64; 95% confidence interval 1.71 to 12.63; p<0.01), but RF was not (p = 0.60). Additionally, as age at the time of diagnosis of RA increased the duration of prediagnosis antibody positivity for RF and anti-CCP increased, with the longest duration of preclinical antibody positivity seen in patients diagnosed with RA over the age of 40. In no subjects did symptom onset precede the appearance of RF or anti-CCP antibodies. CONCLUSIONS: The period of time that RF and anti-CCP are present before diagnosis lengthens as the age at the time of diagnosis of RA increases. This finding suggests that factors such as genetic risk or environmental exposure influencing the temporal relationship between the development of RA-related autoantibodies and clinically apparent disease onset may differ with age.