Ann Rheum Dis

Objective This study was conducted with sera from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and arthritis and lupus-like disease animal models to identify innate immune system-dependent and -independent autoantigens

Influence of peptidylarginine deiminase type 4 genotype and shared epitope on clinical characteristics and autoantibody profile of rheumatoid arthritis.

Background: Recent evidence suggests that distinction of subsets of rheumatoid arthritis (RA) depending on anticyclic citrullinated peptide antibody (anti-CCP) status may be helpful in distinguishing distinct aetiopathologies and in predicting the course of disease. HLA-DRB1 shared epitope (SE) and peptidylarginine deiminase type 4 (PADI4) genotype, both of which have been implicated in anti-CCP generation, are assumed to be associated with RA. Objectives: To elucidate whether PADI4 affects the clinical characteristics of RA, and whether it would modulate the effect of anti-CCPs on clinical course. The combined effect of SE and PADI4 on autoantibody profile was also analysed. Methods: 373 patients with RA were studied. SE, padi4_94C.T, rheumatoid factor, anti-CCPs and antinuclear antibodies (ANAs) were determined. Disease severity was characterised by cumulative therapy intensity classified into ordinal categories (CTI-1 to CTI-3) and by Steinbrocker score. Results: CTI was significantly associated with disease duration, erosive disease, disease activity score (DAS) 28 and anti-CCPs. The association of anti-CCPs with CTI was considerably influenced by padi4_94C.T genotype (C/C: ORadj=0.93, padj=0.92; C/T: ORadj=2.92, padj=0.093; T/T: ORadj=15.3, padj=0.002). Carriage of padi4_94T exhibited a significant trend towards higher Steinbrocker scores in univariate and multivariate analyses. An association of padi4_94C.T with ANAs was observed, with noteworthy differences depending on SE status (SE2: ORadj=6.20, padj,0.04; SE+: ORadj=0.36, padj=0.02) and significant heterogeneity between the two SE strata (p=0.006). Conclusions: PADI4 genotype in combination with anti- CCPs and SE modulates clinical and serological characteristics of RA

Interaction between rheumatoid arthritis and pregnancy: correlation of molecular data with clinical disease activity measures

Objective. The factors that induce remission of RA during pregnancy and the relapse occurring after delivery remain an enigma. In a previous study, we investigated gene-expression profiles of peripheral blood mononuclear cells (PBMC) in patients with RA and healthy women in late pregnancy and postpartum. Profiles of samples from both groups were similar in late pregnancy with elevated monocyte and decreased lymphocyte signatures. Postpartum, in RA PBMC the high level of monocyte transcripts persisted. Further increase was observed in adhesion, migration and signalling processes related to monocytes but also in lymphocytes despite similar clinical activity due to intensified drug treatment. This prompted us to investigate correlations between clinical parameters of disease activity and gene profiles. Methods. Transcriptome data were correlated with RADAI, CRP, monocyte and lymphocyte counts. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations, monocytes and lymphocytes signatures were used as reference information. Results. Comparative analysis of PBMC expression profiles from RA patients during and after pregnancy with RADAI and CRP revealed a correlation of these disease activity parameters predominantly with monocyte transcripts. Genes related to cellular programs of adhesion, migration and response to infections were upregulated. Comparing clinically active and not-active RA patients postpartum revealed a cluster of 19 genes that could also identify active disease during pregnancy. Conclusion. The data suggest that an increase of the RADAI and an elevation of CRP is a consequence of molecular activation of monocytes. Furthermore, they indicate that molecular activation of T lymphocytes may remain clinically unrecognized postpartum. It is conceivable that a set of 19 genes may qualify as molecular disease activity marke

A customized monocyte cDNA microarray for diagnosis of rheumatoid arthritis and prognosis of anti-TNF-α therapy

Background In rheumatoid arthritis (RA) macrophages (Mf) play a pivotal role. They become highly activated in synovitis and at the cartilage–pannus junction. Furthermore, therapeutic neutralization of molecules produced by activated Mf lead to clinical improvement in RA, and circulating monocytes (MO) of the peripheral blood in patients with RA spontaneously express proinflammatory genes (IL-1β, IL-6, TNF). Methods A custom RA-MO cDNA microarray was generated using differentially expressed genes obtained from gene subtraction and from comparative whole genome wide U133A analysis in normal donors, active and anti-TNF-α created RA patients. Genes were selected using MAS 5.0, multtest and PAM. The custom microarray consists of 313 genes including guide dots, and positive (housekeeping genes and spike controls) and negative controls for image and statistical analysis. Each probe was spotted in 16 replicates. Results The RA-MO chipset-II was validated using the following: non-stimulated and LPS, PMA, Vit.D3+LPS, PMA+LPS stimulated U937 cells; nonstimulated and LPS stimulated healthy donor MO; MO from normal donors (n = 3) and RA patients before and during anti-TNF-α treatment (n = 5 each); and synovial tissue from normal individuals (n = 2) and RA patients (n = 2). Not only LPS/PMA regulated genes but also RA specific and anti-TNF-α regulated genes were validated. In addition, we could clarify whether these genes are differentially transcribed only in MO or whether they can also be found in RA tissue Mf. Our data indicate a high degree of reproducibility that is sufficient for diagnostic applications and therapy monitoring. Conclusion The RA-MO chipset-II microarray is competitive and flexible for enlargement of the number of genes. The current gene selection will contribute to validating the role of monocytes in disease activity, to therapeutic interventions, and may improve the knowledge on the regulation of pathways in activated monocytes in chronic inflammation

Quaternary structure of the European spiny lobster (Palinurus elephas) 1 x 6-mer hemocyanin from cryoEM and amino acid sequence data

Arthropod hemocyanins are large respiratory proteins that are composed of up to 48 subunits (8 x 6-mer) in the 75 kDa range. A 3D reconstruction of the 1 x 6-mer hemocyanin from the European spiny lobster Palinuris elephas has been performed from 9970 single particles using cryoelectron microscopy. An 8 Angstrom resolution of the hemocyanin 3D reconstruction has been obtained from about 600 final class averages. Visualisation of structural elements such as a-helices has been achieved. An amino acid sequence alignment shows the high sequence identity (>80%.) of the hemocyanin subunits from the European spiny lobster P. elephas and the American spiny lobster Panulirus interruptus. Comparison of the P. elephas hemocyanin electron microscopy (EM) density map with the known P. interruptus X-ray structure shows a close structural correlation, demonstrating the reliability of both methods for reconstructing proteins, By molecular modelling, we have found the putative locations for the amino acid sequence (597-605) and the C-terminal end (654-657), which are absent in the available P. interruptus X-ray data. (C) 2002 Elsevier Science Ltd. All rights reserve

Proteasome alpha-type subunit C9 is a primary target of autoantibodies in sera of patients with myositis and systemic lupus erythematosus

Autoantibodies occur in low frequencies among patients with myositis characterizing only distinct subsets of this disease. Most of these known antibodies are directed to enzymatically active complexes. The 20S proteasome represents an essential cytoplasmatic protein complex for intracellular nonlysosomal protein degradation, and is involved in major histocompatibility complex class I restricted antigen processing. In this study we investigated whether the 20S proteasome complex is an antibody target in myositis and in other autoimmune diseases. 34 sera of poly/dermatomyositis patients were assayed for antiproteasomal antibodies using enzyme-linked immunosorbent assay, immunoblot, and two-dimensional non-equilibrium pH gradient electrophoresis (NEPHGE). Sera was from patients with systemic lupus erythematosus (SLE), mixed connective tissue disease, and rheumatoid arthritis; healthy volunteers served as controls. In 62% (21/34) of the cases sera from patients with myositis and in 58% (30/52) of the cases sera from patients with SLE reacted with the 20S proteasome. These frequencies exceeded those of sera from patients with mixed connective tissue disease, rheumatoid arthritis, and healthy controls. The alpha-type subunit C9 of the 20S proteasome was determined to be the predominant target of the autoimmune sera in myositis and SLE. Lacking other frequent autoantibodies in myositis, the antiproteasome antibodies are the most common humoral immune response so far detected in this disease entity

CpG Methylation in the Hexamerin 110 Gene in the European Honeybee, Apis mellifera

The European honeybee, Apis mellifera L. (Hymenoptera: Apidae), has a full set of machinery for functional CpG methylation of its genome. A recent study demonstrated that DNA methylation in the honeybee is involved in caste differentiation. In this study, the expression and methylation of the hexamerin 110 gene (Hex110), which encodes a storage protein, was analyzed. High levels of the Hex110 transcript were expressed in both worker and queen larvae. Low levels of this transcript were also detected in adult fat bodies, and the expression level was higher in the queen than in the worker. Bisulfite sequencing revealed that the Hex110 gene is overall methylated at a low level, with a limited number of CpG sites methylated at relatively high levels. These highly methylated sites were exclusively located in the exon regions. The average methylation rate of the Hex110 gene was higher in the adult stage than in the larval stage. Furthermore, several CpG sites were differentially methylated between the worker and queen larvae. These observations suggest that the methylation of the Hex110 gene is regulated at the developmental stage and in a caste-dependent manner