The role of citrullinated protein antibodies in predicting erosive disease in rheumatoid arthritis: a systematic literature review and meta-analysis.

Background. Autoantibodies to citrullinated peptides have been shown to be valuable in the diagnosis of rheumatoid arthritis (RA). The expanding repertoire of antibodies to citrullinated peptide antigens (ACPA) has been a topic of great interest in recent reviews and research studies, as has the ability of these autoantibodies to predict disease outcome. Objectives. The aim of this review was to provide an update on the relevance of ACPA as prognostic markers in RA. The ability to identify patients predisposed to an aggressive outcome at the time of initial diagnosis greatly facilitates the selection of appropriate and cost-effective treatment. Methods. A systematic review of the literature was carried out. Studies from 1967 up to June 2014 with data on prognostic value of ACPA were included. Quality assessment was done by using the modified Hayden list for prognostic studies. Meta-analysis was performed using BioStat software. Results. The results of 25 studies were selected for the final review. A total of 6421 patients with RA were included, mainly in inception cohorts, with follow-up duration ranging from one year to ten years. All studies carried prognostic data on all available isotypes of anticyclic citrullinated protein (CCP), while four had data on antimutated citrullinated vimentin (MCV). There was a single relevant study each on anticitrullinated enolase peptide 1 (CEP1) and antichimaeric fibrin/filaggrin citrullinated peptide 1 (CFFCP1). All studies showed ACPA to be strong predictors of joint erosions in RA. Other factors, particularly baseline erosions, showed an additive effect. Anti-MCV appeared to be a marker of a more aggressive form of disease. Ten studies had data on which a meta-analysis could be performed. This gave an overall odds ratio of 4.85 for ACPA (anti-CCP/MCV) positivity being predictive for the development of joint erosions. Two studies with data on anti-CEP1 and anti-CFFCP1 also showed this positive predictive role of ACPA for joint erosions. Conclusions. ACPA are strong predictors of severity in RA. Their use should be part of routine rheumatology practice.

Academic training in rheumatology in 2009: a UK trainee survey.

Significant changes to the structure and entry into specialist training continue to be implemented. This is likely to have had a long-term impact on rheumatology service provision and the proportion of trainees undertaking academic medicine. An online questionnaire was sent to all trainees on the Joint Royal Colleges Postgraduate Training Board (JRCPTB) database. Out of 211 trainees, 141 responded (66.8%). Of these, 33 (23%) were registered for, or had been awarded, an MD or PhD with a wide variety of funding sources. Mainstream funding sources included Arthritis Research UK, the Medical Research Council, the National Institute for Health Research and the Wellcome Trust, but a substantial number of trainees (n = 17, 51.5%) also utilised other sources of funding. The data from this study will be valuable in the planning of future rheumatology training and academic career pathways and provide useful comparative data for other medical specialties.

A randomised controlled trial of subcutaneous sodium salicylate therapy for osteoarthritis of the thumb.

BACKGROUND: Current treatment for osteoarthritis (OA) is limited. Many patients with OA of the hand have areas of tender subcutaneous thickening in the forearm and upper scapular region. A pilot study showed an improvement in pain from OA at the first carpometacarpal joint after injection of such areas with 0.5% sodium salicylate or saline, an inexpensive treatment that can be administered by general practitioners and nurses. The study indicated that a randomised, sham-controlled trial was justified. METHODS: 40 patients with OA of the first carpometacarpal joint were randomised to receive either injections of sodium salicylate into tender, thickened areas of subcutaneous tissue on the forearm (baseline) and upper scapular region (week 1) or sham injections consisting of pressure without skin penetration. Blinded assessments were made at weeks 3, 7 and 13 after baseline. RESULTS: Pain and tenderness during follow-up were both significantly lower in the active treatment group compared with the sham group: 19% and 14% greater reduction in mean visual analogue scale (VAS) score, respectively (p=0.007 and 0.02, baseline mean 5.65 and 5.35 cm, average difference in change from baseline VAS 1.9 and 1.4 cm, 95% CI 0.6 to 3.2 and 0.2 to 2.5). Active and sham injections were painful, the former significantly more so; however, there was no significant correlation between the pain of active injections and response. CONCLUSION: The data show that subcutaneous sodium salicylate injections are an effective symptomatic treatment for OA of the thumb. The results provide a basis for further physiological and therapeutic research in this area.

Possible familial Mediterranean fever in a Caucasian patient with systemic lupus erythematosus.

We report the case of a Caucasian man with systemic lupus erythematosus who had recurrent fevers and abdominal pain. He was later found to carry E148Q polymorphism of MEFV, the gene responsible for familial Mediterranean fever.

The role of antigen in the selection of the human V3-23 immunoglobulin heavy chain variable region gene.

The immune system mounts antibody responses using few of the available immunoglobulin variable region (IgV) genes with some, such as the V3-23 heavy chain gene, regularly over-represented in responses to many antigens. The reasons for the over-representation of some V genes have not been established; the process could be either stochastic or selective. We demonstrated previously that the V3-23 gene, which is over-represented in the primary B lymphocyte repertoire in humans, encodes antibodies with differing antigen-binding reactivities in transgenic mice that express the human V3-23 gene. The aim of the current study was to assess if V3-23 gene over-representation is stochastic or could be influenced by antigen exposure. Transgenic mice were immunized with human IgG-Fc (hIgG-Fc), bovine collagen type II (bCII) or tetanus toxoid (TT), and hybridomas secreting human mu chain-containing antibodies generated. These were tested for binding to the immunogens and a panel of self- and exogenous antigens. In hybridomas derived from hIgG-Fc-immunized mice, 53% secreted antibodies specific for hIgG-Fc. A similar proportion (54%) of hybridomas from bCII-immunized mice secreted antibody that bound to collagen. By contrast, only 21% of hybridomas from mice immunized with TT bound to tetanus toxoid. Intriguingly, chimaeric antibodies generated from mice immunized with bCII or TT were mainly polyreactive, similar to antibodies generated from naive transgenic mice. However, hybridomas generated from mice immunized with hIgG-Fc were mainly specific, reacting exclusively with hIgG-Fc. These results suggest that selection and eventual expansion of B lymphocytes expressing the V3-23 gene are likely to be determined by exposure to self- and/or environmental antigens.

Synthesis, characterization, and electrochemistry of cis-oxothio- and cis-bis(thio)tungsten(VI) complexes of hydrotris(3,5-dimethylpyrazol-1-yl)borate.

The complexes TpWO2X react with sulfiding agents such as B2S3 or P4S10 to give the oxothio- and bis(thio)tungsten(VI) complexes TpWOSX (X = Cl(-)) and TpWS2X [X = Cl(-), S2PPh2(-); Tp = hydrotris(3,5-dimethylpyrazol-1-yl)borate]. The reaction of TpWS2Cl with (i) PPh3 in pyridine and (ii) dimethyl sulfoxide affords TpWOSCl in good overall yield. The chloro complexes undergo metathesis with alkali metal salts to yield species of the type TpWOSX and TpWS2X [X = OPh(-), SPh(-), SePh(-), (-)-mentholate]. The diamagnetic complexes exhibit NMR spectra indicative of C(1) (TpWOSX) or C(s) (TpWS2X) symmetry and IR spectra consistent with terminal oxo and thio ligation (nu(W=O), 940-925 cm(-1); nu(W=S) or nu(WS2), 495-475 cm(-1)). Crystals of (R,S)-TpWOS[(-)-mentholate] are monoclinic, space group P2(1), with a = 11.983(2) A, b = 18.100(3) A, c = 13.859(3) A, beta = 91.60(2) degrees, V = 3004.6(8) A(3), and Z = 4. Crystals of TpWS2(OPh)-CH2Cl2 are orthorhombic, space group Pbca, with a = 16.961(4) A, b = 33.098(7) A, c = 9.555(2) A, V = 5364(2) A(3), and Z = 8. The mononuclear, distorted-octahedral tungsten centers are coordinated by a tridentate Tp ligand, an alkoxy or aryloxy ligand, and two terminal chalcogenide ligands. The average W=O and W=S distances are 1.726(7) and 2.125(2) A, respectively, and the O=W=S and S=W=S angles 102.9(3) and 102.9(1) degrees, respectively. The tungsten and sulfur X-ray absorption spectra of TpWOSCl and TpWS2Cl are consistent with the presence of terminal pi-bonded thio ligands in both complexes. The thio complexes generally undergo a reversible one-electron reduction at potentials significantly more positive than their oxo analogues. The chemical, spectroscopic, and electrochemical properties of the complexes are heavily influenced by the presence of W=S pi frontier orbitals.

The effects of a human IgM monoclonal anticardiolipin antibody on pregnancy in a transgenic mouse model.

The clinical features of antiphospholipid (or Hughes') syndrome (APS) are most commonly seen in individuals who have raised levels of IgG anticardiolipin antibodies. Most murine models of the syndrome have involved the administration of such antibodies to normal mice. However, APS can occur in the presence of raised levels of serum IgM anticardiolipin antibodies alone. The present study was designed to see if an IgM monoclonal antibody can induce changes in mice similar to those seen in human APS. This antibody, BH1, has previously been derived from a patient with primary APS. In its ligand-binding and idiotypic characteristics it is representative of antiphospholipid antibodies (aPL) found in the serum of patients with APS. In order to minimise the immune response to human IgM, we used transgenic mice (F15) which express, and are predicted to be tolerant of, human immunoglobulin mu chains. The features of APS may develop more readily in individuals who have an existing autoimmune disorder, such as systemic lupus erythematosus (SLE). We therefore crossed these transgenic mice with New Zealand Black (NZB, SLE-prone) mice, and used the progeny (F15 x NZB/F1) in our experiments. Twenty-four F15 x NZB/F1 mice were given BH1, or a control IgM antibody, (A5566) immediately preceding and then three times during pregnancy. There was a reduction in the mean number of foetuses in animals given BH1 compared with those given A5566 (8.6 vs 11.0; P < 0.05), and a similar reduction in mean total foetal weight per pregnancy (9.05 vs 12.73 g; P < 0.05). Two mice showed a marked reduction in platelet count. No evidence of thrombosis was detected macroscopically or histologically. Our results show a lower incidence of APS-type features compared to previous studies in which mice have been administered aPL. This may be because BH1 is an IgM antibody. Nevertheless, the data support the concept that IgM aPL of particular ligand-binding specificities may have a direct pathogenetic role in certain cases of human APS.

Rearrangement of the human heavy chain variable region gene V3-23 in transgenic mice generates antibodies reactive with a range of antigens on the basis of VHCDR3 and residues intrinsic to the heavy chain variable region.

To formulate a 'logic' for how a single immunoglobulin variable region gene generates antibodies with different antigen specificity and polyreactivity, we analysed chimeric antibodies produced in transgenic mice carrying the germ-line human V3-23 gene, multiple diversity (D) and joining (J) gene segments. Hybridomas producing antibodies encoded by the V3-23 gene in combination with different mouse Vkappa genes were obtained by fusion of splenocytes from transgenic mice. All antibodies had human mu-chains and mouse light chains, were multimeric in structure and expressed the human V3-23 gene. Nucleotide sequence analyses of genes encoding the heavy and light chains of 12 antibodies in relation to antigen specificity highlighted the importance of heavy chain variable region CDR3 in determining reactivity with different antigens. However, the results also suggest that non-CDR3 sequences intrinsic to the V3-23 gene itself may be involved in, or determine, the binding of the chimeric antibodies to some of the antigens tested in the current study.

Pharmacokinetics of the anticoagulant 14C-DX-9065a in the healthy male volunteer after a single intravenous dose.

1. The plasma pharmacokinetics, excretion and metabolism of DX-9065a were studied in the healthy male Caucasian volunteer after a single intravenous dose of 10 mg 14C-labelled DX-9065a. 2. At the end of a 1 h infusion, the mean plasma concentration of total radioactivity was 380 ng ml(-1) (equivalent to unchanged DX-9065). Thereafter, it decreased in a bi-exponential manner and was below the detection limit by 48 h after dosing. The half-life for the distribution phase was 6.93 h. 3. The total radioactivity recovered in urine and faeces by 336 h post-dose was 83.8% of the administered dose, with excretion ongoing at the end of the 14-day collection. The major route of excretion was via urine, accounting for a mean of 77.6% of the administered radioactivity. The urinary excretion profile was biphasic, consisting of rapid (0-24 h) and slow (24-336 h) phases. A large renal clearance suggested that renal tubular secretion might contribute to the excretion of DX-9065 via urine. 4. No metabolite peaks in the radio-HPLC chromatograms of urine samples were detected, indicating that biotransformation of DX-9065 does not play a significant role in the elimination of DX-9065 in man.

Hydrogen peroxide- and peroxynitrite-induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells.

The mechanisms by which reactive species (RS) participate in the development of atherosclerosis remain incompletely understood. The present study was designed to test the hypothesis that RS produced in the vascular environment cause mitochondrial damage and dysfunction in vitro and, thus, may contribute to the initiating events of atherogenesis. DNA damage was assessed in vascular cells exposed to superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite. In both vascular endothelial and smooth muscle cells, the mitochondrial DNA (mtDNA) was preferentially damaged relative to the transcriptionally inactive nuclear beta-globin gene. Similarly, a dose-dependent decrease in mtDNA-encoded mRNA transcripts was associated with RS treatment. Mitochondrial protein synthesis was also inhibited in a dose-dependent manner by ONOO(-), resulting in decreased cellular ATP levels and mitochondrial redox function. Overall, endothelial cells were more sensitive to RS-mediated damage than were smooth muscle cells. Together, these data link RS-mediated mtDNA damage, altered gene expression, and mitochondrial dysfunction in cell culture and reveal how RS may mediate vascular cell dysfunction in the setting of atherogenesis.

Effect of age on the expression of Pex (Phex) in the mouse.

Pex is a newly discovered gene (also called Phex) whose mutation is the cause of X-linked hypophosphatemia. Other members of this gene family encode endopeptidases that activate or inactivate endocrine and paracrine factors. Though embryonic bone expresses mRNA for the Pex gene at relatively high levels, we have found Pex expression to be widespread in adult organs and to be poorly expressed in adult bone. This led to the hypothesis that Pex mRNA expression changes with age. To test this, genetically normal mice of the B6C3H hybrid strain were studied at 0 (newborn), 2, 3, 10, and 72 weeks of age. Organs known to express Pex were collected, and RNA was extracted from them. Following reverse transcription, cDNA was amplified by the polymerase chain reaction with primers for Pex and G3PDH, a housekeeping gene. The amplimers were separated by electrophoresis, blotted onto nylon membranes, and hybridized with radioactively labeled internal oligonucleotide probes. The radioactivity was quantified, and the data were analyzed as the Pex/G3PDH ratio. The brain samples had high levels of Pex mRNA expression that rose slightly with age. Calvaria, kidney, and lung samples had the highest Pex mRNA expression at birth. In these organs Pex mRNA expression fell with age to undetectable or barely detectable levels. Thymus, heart, and skeletal muscle samples had low Pex mRNA expression at birth that did not change with age. Some organs showed a decline in G3PDH levels with age, but Pex expression decreased more, leading to a reduced Pex/G3PDH ratio. The widespread expression of mRNA for Pex suggests a role beyond that of phosphate homeostasis. The high level of expression in newborn animals suggests a role in growth and development. This seems to occur in addition to its role for the endocrine regulation of phosphate homeostasis by as yet unknown humoral agents that must occur throughout life. In summary, Pex mRNA expression is high in brain and bone at birth. Expression remains high in brain with age but falls with age in bone, kidney, and lung.

Generation and biomimetic chemistry of tungsten-dithiolene complexes containing the hydrotris(3,5-dimethylpyrazol-1-yl)borate ligand.

Reactions of bis(thio)-W(VI) complexes, LWS2X (L = hydrotris (3,5-dimethylpyrazol-1-yl)borate, X = monoanion), with alkynes produce dithiolene complexes, LWX(dithiolene). The synthesis and characterization of orange LW(OPh){S2C2(CO2Me)2} (1) and burgundyred LW(SePh) {S2C2(Ph)(2-quinoxalinyl)} (2) and the X-ray crystal structure of 1.0.5CH2Cl2, are described in detail. Crystals of 1.0.5CH2Cl2 are orthorhombic, space group Pbcn, with a = 29.826(6), b = 13.291(4), c = 16.078(4) A, V = 6373(5) A3, and Z = 8. The six-coordinate, distorted-octahedral complex features a tridentate L ligand, a monodentate phenoxide ligand, and a bidentate dithiolene ligand. The short W-S bonds (2.267(4) and 2.279(4) A) and the parameters associated with the phenoxide ligand (W-O = 1.850(8) A, W-O-C = 146(1) degree), point to a considerable degree of W-ligand multiple bonding in the [W(OPh)(dithiolene)]+ unit. For 2, W-Se and average W-S distances of 2.49(2) A and 2.30(2) A, respectively, have been obtained from EXAFS studies. The formation of yellow 3,3'-dithiobis[2-(phenyl)thieno[2,3-b]quinoxaline] (3) upon oxidation of 2 supports the likely generation of urothione upon oxidative degradation of molybdopterin-containing tungsten enzymes from hyperthermophilic organisms.

The BH1 idiotype defines a population of anticardiolipin antibodies closely associated with the antiphospholipid syndrome.

BACKGROUND: A human IgM monoclonal anticardiolipin antibody - BH1 - has previously been described, which has characteristics typical of antiphospholipid antibodies in the serum of patients with antiphospholipid syndrome (APS). It appears to be idiotypically distinct from other human monoclonal autoantibodies of different or overlapping ligand-binding specificities derived from patients with related conditions. AIM: To determine whether the idiotype of BH1 is expressed on particular populations of antibodies (antiphospholipid and anti-beta2-glucoprotein I) in the serum of patients with APS and other conditions. METHODS: Sera from patients with APS (9), systemic lupus erythematosus without APS ('uncomplicated SLE' -9), and rheumatoid arthritis (RA 15), and from normal controls (15) were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with cardiolipin, beta2 glycoprotein I (beta2GPI), and a polyclonal anti-idiotype raised against BH1 (RIdBH1). Absorption experiments were subsequently performed on selected sera using micelles of cardiolipin or phosphatidyl choline. RESULTS: Eight out of nine patients with APS were positive for binding to RIdBH1 (IgG and/or IgM), while only one patient with uncomplicated SLE and none of the patients with RA or the healthy controls were positive. Although all of the patients with APS were positive for binding to beta2GPI, there was poor correlation between these results and levels of binding to cardiolipin and RIdBH1. Absorption of sera from patients with APS by cardolipin micelles resulted in a median reduction in IgG anticardiolipin and anti-beta2GPI activity of 81.6% and 6.3% respectively. For those sera positive for IgG reactivity with RIdBH1 the median reduction in this activity was 79.4%. Antibodies eluted from selected micelles showed activity against cardiolipin, beta2GPI and RIdBH1. Three anticardiolipin-positive sera from patients with RA were similarly absorbed; however the eluted antibodies failed to bind to RIdBH1. Absorption of all these sera with phosphatidyl choline resulted in no significant reduction in any of these activities. CONCLUSIONS: The BH1 idiotype defines a population of serum antibodies associated with features of APS. The antibody response in this condition, though diverse, may include the expression of a restricted group of variable region genes.

Chimaeric monoclonal antibodies encoded by the human VH26 gene from naïve transgenic mice display a wide range of antigen-binding specificities.

To elucidate the molecular basis for the ability of antibodies encoded by the human VH26 heavy-chain variable region gene to react with diverse antigens, we have generated 34 hybridomas secreting chimaeric monoclonal antibodies (human mu heavy chain/mouse light chains) from transgenic mice. The transgenic mice carry an immunoglobulin minilocus containing the human VH26 gene, human DH and JH gene segments, and genes encoding the human C mu region. The minilocus in these animals undergoes functional rearrangement resulting in the production of chimaeric antibodies in which human mu heavy chains utilizing the VH26 gene are paired with mouse kappa or lambda light chains. The hybridomas described in this study were generated from naïve animals and were selected solely on the basis of human mu-chain expression. The antibodies described have covalently attached mouse light chains and are multimeric in structure. The binding properties of the antibodies were examined using a panel of both self- and foreign antigens using enzyme-linked immunosorbent assays, agglutination or radio-immunoprecipitation assays and immunofluorescence. Chimaeric immunoglobulins from 21 of the 34 hybridoma clones (61.7%) reacted with one or more antigens, of which 13 (38.2%) reacted with more than two antigens. These studies demonstrate that the VH26 gene, in combination with human DH and JH gene segments, and mouse light-chain genes, is able to encode antibodies with a wide range of ligand-binding specificities. These findings have important implications in the context of the possible origins of autoantibodies encoded by VH26 which may play a role in the pathogenesis of a number of autoimmune conditions.

Phospholipid binding specificities and idiotype expression of hybridoma derived monoclonal autoantibodies from splenic cells of patients with systemic lupus erythematosus.

OBJECTIVE: To analyse the phospholipid binding specificity, functional characteristics and idiotype expression of human hybridoma derived monoclonal autoantibodies (MAb) derived from the spleens of two patients with active systemic lupus erythematosus (SLE). METHODS: The IgM MAbs binding to phospholipids were generated from spleen cells of two patients (RSP and RT) with active SLE and their specificity of binding to neutral phospholipids (phosphatidyl ethanolamine, phosphatidyl choline, platelet activating factor, sphingomyelin) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, phosphatidyl inositol and cardiolipin (CL)) analysed. Binding specificity of cross reactive antibodies (those binding to CL and DNA) was confirmed by fluid phase inhibition assays. Lupus anticoagulant activity and beta 2-glycoprotein-1 (beta 2 GP-1) requirement for the antigen binding of these MAbs were detected using the modified dilute Russell's viper venom test and modified anti-CL enzyme linked immunosorbent assay (ELISA), respectively. Expression of idiotypes (Id) Id RT-84 and Id H3 was analysed using rabbit polyclonal and murine monoclonal anti-idiotype reagents, respectively. RESULTS: Twelve clones from the patient RSP and eight clones from patient RT were reactive with phospholipids. Marked differences in phospholipid binding of these MAbs were noted, varying from truly polyreactive (RT-72 bound to most phospholipids tested) to monospecific (RT-84 bound only to CL). Furthermore, MAbs RT-84, RT-129, and RSP-57 had lupus anticoagulant activity and required beta 2 GP-1 for CL binding. It was found that 75% of phospholipid binding antibodies from RT clones expressed RT-84 Id, but none from RSP clones did so, and that Id H3 was expressed only by the RT-83 antibody. CONCLUSION: These results show that human anti-phospholipid MAbs are heterogeneous with respect to phospholipid binding, functional characteristics, and Id expression.

Endothelium-derived haemostatic factors and the antiphospholipid syndrome.

The cause of thrombosis in the antiphospholipid syndrome (APS) is unknown. There have been reports of abnormalities in the antigenic levels or activity of endothelium-derived haemostatic factors, such as tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1); however the data from these studies are conflicting. We studied plasma from nine patients with APS; seven of them had a history of thrombosis, and three had systemic lupus erythematosus (SLE). We also studied nine matched control patients who had SLE without APS, and 14 healthy individuals. We measured t-PA, von Willebrand factor (vWF), anticardiolipin antibody (ACA) and anti-endothelial cell antibody (AECA) levels by enzyme-linked immunoassay (ELISA), PAI-1 activity by a parabolic-rate chromogenic assay, and lupus anticoagulant (LA) activity by a standard mixing test. For t-PA and PAI-1, measurements were made on morning and evening plasma samples. The two groups of patients did not differ significantly with respect to age, sex, plasma lipids or anti-inflammatory drugs. Most APS patients (7/9) but none of the controls were taking warfarin. Between the APS and the control patients no significant differences were detected in t-PA, PAI-1, vWF or AECA levels. When APS patients were considered alone, vWF levels correlated positively with IgG ACA levels (r = 0.81, P < 0.01) and negatively with platelet count (r = -0.68, P < 0.05). There was no correlation between levels of ACA or LA activity and t-PA, PAI-1 or AECA. Compared with healthy volunteers, the diurnal variation of t-PA and PAI-1 was blunted in the two patient groups.(ABSTRACT TRUNCATED AT 250 WORDS)