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Search for: rheumatoid arthritis    methotrexate    autoimmune disease    biomarker    gene expression    GWAS    HLA genes    non-HLA genes   

ID PMID Title PublicationDate abstract
12475006 Drug-induced lupus following treatment with infliximab in rheumatoid arthritis. 2002 After introduction of infliximab for the treatment of rheumatoid arthritis (RA), there have been many reports of patients developing asymptomatic higher rate of antinuclear antibodies and anti-dsDNA antibodies than in non-infliximab-treated patients. However, only five clinical drug-induced lupus (DIL) cases have been documented following treatment with infliximab, in RA and in Crohn's diseases. We report a case of a 69-year-old female with a 5 year history of RA, whowas successfully treated with low-dose methotrexate (MTX) and infliximab (initially 3 mg/kg and from the fourth infusion 5 mg/kg) for 23 weeks. Before the sixth infusion, she was diagnosed with DIL by both clinical features (fever > 38 degrees C, recurrence of active synovitis, myalgia, erythematous rash and general malaise) and laboratory findings (antinuclear antibodies 1:160, anti-double-stranded DNA positive by ELISA assay, decreased serum complement C3 andC4, hypergammaglobulinaemia, increased erythrocyte sedimentation rate). After discontinuation of treatment and therapy with oral prednisone, lupus resolved within 8 weeks.
12858435 CD4+ PD-1+ T cells accumulate as unique anergic cells in rheumatoid arthritis synovial flu 2003 Jul OBJECTIVE: The PD-1 receptor, whose deficiency in mice causes autoimmune diseases such as arthritis, is considered to be a negative regulator of activated T cells and to play a crucial role in peripheral tolerance. To clarify the involvement of the PD-1 system in rheumatoid arthritis (RA), we investigated PD-1 expression on synovial fluid (SF) T cells from patients with RA. METHODS: FACS analysis for PD-1 was performed on SF T cells from 44 patients with RA and 6 with osteoarthritis (OA), and also on peripheral blood (PB) T cells from 12 RA patients and 7 healthy controls. Two-color analysis of cell surface PD-1 expression and the intracellular concentration of cytokine production was used to investigate CD4+ T cells from SF of patients with RA and PB from controls. RESULTS: Scarcely any PD-1 expression was detected on control PB T or OA SF T cells. In contrast, PD-1+ cells made up 20.9 +/- 8.6% (mean +/- SD) of RA SF T cells. In RA SF, PD-1 was expressed more predominantly on CD4+ T cells than on CD8+ T cells. As well as expressing CD45RO and CXCR3, CD4+ PD-1+ T cells were mostly CTLA-4 positive and CD26 negative, and were enriched in CD45RB(low) cells. Intracellular cytokine staining revealed that CD4+ PD-1+, but not CD4+ PD-1-, T cells produced interleukin 10 (IL-10), and that CD4+ PD-1+ T cells produced less IL-2 than CD4+ PD-1- T cells. CONCLUSION: PD-1+ T cells in RA SF are enriched, and phenotypic analysis suggests that these cells constitute a unique anergic T cell subset in RA SF.
12867574 Decreased levels of serum soluble complement receptor-II (CR2/CD21) in patients with rheum 2004 Feb OBJECTIVE: The soluble cluster of differentiation 21 (sCD21) represents the extracellular portion of the CD21 glycoprotein and is released by shedding from cell surfaces into plasma. Soluble CD21 binds complement fragments and activates monocytes through binding to membrane CD23. Elevated levels of sCD21 are found during Epstein-Barr virus EBV infections, B-cell lymphoma and other lymphoblastoid tumours. The present study was undertaken to investigate levels of sCD21 in rheumatoid arthritis. METHODS: A specific enzyme-linked immunoassay was developed using sCD21, biochemically purified to homogeneity from human plasma as a standard for the determination of sCD21 concentration in patient sera. Peripheral blood B and T lymphocytes were isolated from healthy donors and rheumatoid arthritis patients and cultured, and supernatants were analysed for CD21 shedding. RESULTS: The normal values of serum sCD21 in healthy individuals between 20 and 40 yr of age ranged from 100 to 477 ng/ml (median 292 ng/ml), decreasing with age but not differing with gender. In rheumatoid arthritis patients, sCD21 levels ranged from 50 to 300 ng/ml (median 182 ng/ml), did not differ with age and were independent of rheumatoid factor. CONCLUSIONS: In contrast to healthy donors, patients with rheumatoid arthritis have significantly lower sCD21 levels (P < 0.0001), independently of the age of the patients. Sorted B cells from rheumatoid arthritis patients released amounts of CD21 comparable with those of normal controls. Possible causes and consequences of the findings are discussed.
15150427 The role of interleukin-1 in bone resorption in rheumatoid arthritis. 2004 Jun Bone loss in RA includes juxta-articular osteopenia, erosions and systemic osteoporosis. In each case, synthesis of new bone matrix is unable to balance osteoclast-mediated bone resorption, resulting in net bone loss. IL-1, TNF and other proinflammatory cytokines stimulate osteoclast differentiation and activation, resulting in bone loss. In addition, these proinflammatory cytokines stimulate synovial fibroblasts and chondrocytes to produce proteinases that degrade cartilage. In animal arthritis models, blocking IL-1 significantly reduces bone erosions and cartilage degradation, whereas blocking TNF decreases synovitis. In patients with active RA, treatment with the TNF blockers etanercept and infliximab, as well as with anakinra, a recombinant human IL-1 receptor antagonist, significantly reduced erosions and joint space narrowing. It remains to be determined, however, whether slowing radiographic progression with these biological therapies will significantly improve long-term outcomes in RA.
12568118 Decreased IL-4 producing CD4+ T cells in patients with active systemic lupus erythematosus 2002 Sep The balance of interferon-gamma (IFN-gamma) and/or interleukin-4 (IL-4) producing T cells and interleukin-12 receptor (IL-12R) expression on T cells were evaluated in patients with active systemic lupus erythematosus (SLE). Assessment of intracellular IFN-gamma and/or IL-4 were conducted with cytoplasmic staining. IL-12R presenting T cells were also assessed by flowcytometry without in vitro stimulation. In SLE, the number of IFN-gamma producing CD4+ T cells was increased, and the absolute number of IL-4 producing CD4+ T cells was significantly decreased. Although the ratio of IL-12R presenting CD4+ T cells was significantly greater, the absolute number did not increase. The ratio of IFN-gamma/IL-4-producing CD4+ T cells correlated with the SLE disease activity index (SLEDAI) and was significantly higher among patients with lupus nephritis. Therefore, the imbalance of IFN-gamma/IL-4 producing CD4+ T cells was due to the decrease in IL-4 producing CD4+ T cells and may play an important pathogenic role in active SLE.
12492250 Leukocyte protein calprotectin and outcome in rheumatoid arthritis. A longitudinal study. 2002 OBJECTIVE: To determine if calprotectin is predictive for outcome in patients with rheumatoid arthritis (RA). METHODS: Fifty six RA in-patients with variable disease duration were prospectively followed for five years. Clinical and laboratory data were collected to assess disease activity. Health Assessment Questionnaire (HAQ) and radiographic scores (of hands and wrists) as described by Larsen were used as outcome measures. Plasma calprotectin levels were determined with ELISA technique. RESULTS: Significant correlations (r) were found cross-sectionally at follow-up between calprotectin concentration and other known parameters of disease activity and severity: CRP (r = 0.67). investigator's global assessment of disease activity (r = 0.57). Waaler titre (r = 0.50). HAQ score (r = 0.48) and number of swollen joints (r = 0.48). Calprotectin at baseline was not identified as an independent predictor for HAQ or radiographic progression in the multivariate analysis. CONCLUSION: The results confirm calprotectin as a good measure of disease activity and joint inflammation in RA. However, the level of calprotectin at baseline was not predictive for radiographic damage or functional impairment five years later.
12102478 Mode of inheritance of HLA-DRB1 shared epitope in Japanese familial rheumatoid arthritis. 2002 May OBJECTIVE: To clarify the mode of genetic contribution of the HLA-DR shared epitope (SE) to the pathogenesis of familial cases of Japanese rheumatoid arthritis (RA). METHODS: Fifty-three unrelated Japanese RA families that had more than 2 affected sibs were selected. The HLA-DR shared epitope typing was carried out by the PCR method and PCR-SSCP (single stranded DNA conformation polymorphism) method. Affected sib pair analysis was carried out using the MAPMAKER/SIB 2.0 program. The mode of inheritance was also calculated based on the sharing of genes identical by descent (IBD) between siblings in each of the 53 affected sib-pairs (propositus and the 2nd affected sib). RESULTS: The maximum LOD score of HLA-DR was 0.437, and the sharing of 2 IBDs, 1 IBD, and no IBDs between affected sibs were 0.330, 0.500, and 0.170, respectively. The sharing distribution of IBD was confirmed to be compatible with the dominant or additive mode since the observed gene frequency of SE was 0.255. CONCLUSION: The HLA-DR shared epitope participated in the pathogenesis of familial cases of Japanese RA. The SE contributes to this pathogenesis in either the dominant or additive mode of inheritance.
12133815 Promoter competition assay for analyzing gene regulation in joint tissue engineering. 2002 Aug 1 We describe a new biochemical technique, "promoter competition assay," for examining the role of cis-acting DNA elements in tissue cultures. Recent advances in tissue engineering permit the culture of a variety of cells. Many tissues are engineered, however, without an appropriate understanding of molecular machinery that regulates gene expression and cellular growth. For elucidating the role of cis-acting regulatory elements in cellular differentiation and growth, we developed the promoter competition assay. This assay uses a transient transfer into cells of double-stranded DNA fragments consisting of cis-acting regulatory elements. The transferred DNA fragments act as a competitor and titrate the function of their genomic counterparts. Using synovial cells derived from a rheumatoid arthritis patient, we examined a role of NF-kappa B binding sites in the regulation of the expression of matrix metalloproteinase (MMP) genes. The results support a stimulatory role of NF-kappa B in transcriptional regulation of MMP-1 and MMP-13.
15468379 Vasculitis associated with the use of leflunomide. 2004 Oct Vasculitis as a complication of leflunomide therapy in the treatment of rheumatoid arthritis has been reported. We describe a case of acute necrotizing vasculitis following leflunomide therapy. Characteristics of this case and 4 cases in the literature suggest that vasculitis may be a rare but serious adverse effect of leflunomide therapy.
15565327 Correction of iron-deficient erythropoiesis in the treatment of anemia of chronic disease 2005 Mar Anemia of chronic disease (ACD) is a frequent complication of chronic inflammation in rheumatoid arthritis (RA). Recombinant human erythropoietin (rHuEpo) has been shown to be effective in correcting ACD, although with a variable rate of nonresponders. The first aim of this trial was to improve the response to rHuEpo by parenteral iron supplementation in cases of iron-deficient erythropoiesis (IDE). An additional goal was the evaluation of the zinc protoporphyrin content of erythrocytes (ZnPP), the soluble transferrin receptor (sTrfR) serum concentration, and the hemoglobin (Hb) content of reticulocytes (CHr) in stimulated erythropoiesis as diagnostic and prognostic parameters. Thirty RA patients with ACD were treated with subcutaneous 150 IU rHuEpo/kg body weight twice weekly. Intravenous iron supplementation (200 mg iron sucrose once weekly) was added in cases of IDE (n=23), which was defined by the presence of two of three criteria: saturation of transferrin (TrfS) < or =15%, hypochromic erythrocytes (HypoE) > or =10%, and a serum ferritin (Fn) concentration < or =50 microg/l. All 28 completers met the treatment goal, with an increase of the median Hb concentration from 10.3 g/dl to 13.3 g/dl. Epo treatment and iron supplementation was safe and well tolerated in all patients. Monitoring of Fn, TrfS, and HypoE every other week allowed a successful correction of anemia. Retrospective analysis of the evaluable parameters (CHr, sTrfR, and ZnPP) revealed no additional benefit for predicting or monitoring IDE in this setting, although the one or other may be advantageous in other therapeutic situations.
14611107 Influence of hypoxia on the expression of matrix metalloproteinase-1, -3 and tissue inhibi 2003 Sep OBJECTIVE: The rheumatoid synovium is a hypoxic environment, and hypoxia has been implicated as a factor in the pathogenesis of rheumatoid arthritis (RA). The purpose of this study was to investigate the effect of hypoxia on the expression of matrix metalloproteinase (MMP)-1, -3 and tissue inhibitor of metalloproteinase (TIMP)-1 in rheumatoid synovial fibroblasts. METHODS: Synovial fibroblasts obtained from RA patients were cultured for 48 h under normoxic or hypoxic conditions. Assays included western blot analysis and enzyme-linked immunosorbent assay (ELISA) for MMP-1, -3 and TIMP-1, and northern blot analysis to measure TIMP-1 mRNA levels. RESULTS: Compared with normoxic culture, hypoxia increased MMP-1 and MMP-3 expression in rheumatoid synovial fibroblasts. Hypoxia decreased TIMP-1 expression in rheumatoid synovial fibroblasts, as measured by both protein and mRNA levels. CONCLUSION: These results suggest that microenvironmental conditions, such as hypoxia, may directly contribute to joint destruction in RA by increasing the ratio of MMP-1, -3 to TIMP-1 production in synovial fibroblasts.
14498767 CPH 82. 2003 CPH 82 [Reumacon] is a purified semi-synthetic lignan glycoside derivative of the medicinal herb Podophyllum emodi which inhibits microtubules. It has been jointly developed by the Swedish company Conpharm and the Swiss company Analytecon SA. It has potential for the treatment of rheumatoid arthritis. The Swedish company Meda is licencing the drug and has taken over responsibility from Conpharm for development and documentation. Meda has obtained exclusive marketing rights for Reumacon worldwide, except in China and some other Asian countries. Meda plans to form partnerships with other companies in the marketing of Reumacon.
14668929 Role of adalimumab, a novel TNF antagonist in advancing rheumatoid arthritis control. 2003 Adalimumab was approved by the U.S. Food and Drug Administration in 2002 for the treatment of moderate to severe rheumatoid arthritis and was granted approval from the European Medicines Evaluation Agency (EMEA) in September 2003. The standard dose is 40 mg given subcutaneously every other week, and the drug can be used alone or in combination with disease-modifying antirheumatic drugs such as methotrexate. The efficacy of adalimumab has been demonstrated in clinical trials with more than 2400 patients. In these trials, adalimumab has acted rapidly to reduce the signs and symptoms of disease, with responses sustained over the long term. The significant inhibition of disease progression seen with adalimumab has led to significant improvements in physical function and health-related quality of life. Data from the clinical safety database in nearly 2500 patients have also shown that the drug is generally safe and well-tolerated.
15280823 A bullous neutrophilic dermatosis in a patient with severe rheumatoid arthritis and monocl 2004 Aug A 35-year-old man presented with a 2-month history of itchy grouped vesiculobullae with crusted papules and confluent plaques, distributed symmetrically on the thighs, knees, and elbows. The man had experienced severe disabling seropositive rheumatoid arthritis for 18 years previously. Histologically, subepidermal vesicles were noted, and a dense, diffuse neutrophilic infiltration of the dermis. Direct immunofluorescence studies did not detect IgA deposits. The patient responded well to dapsone (100 mg/d) but not to systemic steroids. This patient's eruption might have been an unusual manifestation of rheumatoid neutrophilic dermatitis.
12595627 Development of rheumatoid arthritis is not associated with two polymorphisms in the Crohn' 2003 Feb INTRODUCTION: It has been proposed that genetic susceptibility loci for rheumatoid arthritis (RA) may be shared with other autoimmune/inflammatory diseases. Recently, common variation in the CARD15 (NOD2) gene on chromosome 16q12 has been associated with Crohn's disease (CD) in several independent populations. CARD15 is an excellent functional and positional candidate gene for RA. METHODS: Genomic DNA was obtained from 392 RA cases and 471 ethnically matched healthy controls. All samples were genotyped for two polymorphisms in CARD15, 1007fs and R702W, using 5' nuclease reporter assays. Allele frequencies were compared between cases and controls using the chi(2) test. Estimated haplotype frequencies across the two mutations were determined using the EH program. RESULTS: The allele frequency of the 1007fs variant in RA cases was 1.8% compared with 1.6% in normal controls (not significant). The frequency of the R702W variant was 4.0% in both cases and controls. Haplotypes carrying either of the two mutations accounted for 5.6% of possible haplotypes. A haplotype carrying both mutations was rare, with estimated frequency <0.01%. This study provided high power to detect an association of similar magnitude to that in Crohn's disease. These data therefore exclude the possibility that the contribution of these mutations to RA is comparable to that seen in CD. CONCLUSION: Within defined statistical parameters, we excluded a role for the CARD15 1007fs and R702W variants in RA susceptibility. These data do not preclude a role for other polymorphisms in the CARD15 gene in RA susceptibility. Results from other autoimmune and inflammatory diseases will reveal whether the CARD15 gene is in fact a common autoimmune susceptibility locus.
14558086 Bone morphogenetic proteins 2 and 6, expressed in arthritic synovium, are regulated by pro 2003 Oct OBJECTIVE: To examine the expression, regulation, and potential roles of bone morphogenetic proteins (BMPs) in arthritic synovium. METHODS: Expression of BMPs in arthritic synovium from patients with rheumatoid arthritis (RA) or spondylarthropathy (SpA) and in noninflamed synovium from patients undergoing diagnostic or therapeutic arthroscopies was studied by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, immunohistochemistry, and 2-color immunofluorescence. In vitro regulation of gene expression in fibroblast-like synoviocytes (FLS) was determined by real-time quantitative RT-PCR and immunohistochemistry. We used (3)H-thymidine incorporation after serum deprivation-induced growth arrest to examine effects on FLS proliferation. FLS apoptosis was evaluated by flow cytometry cell cycle analysis. Apoptotic cells in synovial tissue were detected by TUNEL staining. RESULTS: Transcripts of different BMPs, most strikingly BMP-2 and BMP-6, were detected in synovial tissues. By Western blot, BMP-2 and BMP-6 precursor protein was found in RA and SpA synovial tissue extracts, but not in extracts of noninflamed synovial tissue. By immunohistochemistry, BMP-2 and BMP-6 were detected in the hyperplastic lining and the sublining layer of synovium from RA and SpA patients, both in CD90+ cells (FLS) and in some CD68+ cells (macrophages). Proinflammatory cytokines, such as interleukin-1beta and tumor necrosis factor alpha, but not interferon-gamma, enhanced the expression of BMP-2 and BMP-6 transcripts in FLS in vitro. Neither BMP-2 nor BMP-6 affected FLS proliferation. BMP-2 promoted FLS apoptosis, whereas BMP-6 protected against nitric oxide-induced FLS apoptosis. BMP-2-positive apoptotic cells were found in arthritic synovium. CONCLUSION: BMP-2 and BMP-6 are expressed in arthritic synovium and are strongly up-regulated by proinflammatory cytokines. Although BMP signaling has been proposed to be involved in cartilage and bone repair in arthritis, this pathway may be equally important in modulating FLS cell populations in inflamed synovium.
12534638 Population pharmacokinetic analysis of meloxicam in rheumatoid arthritis patients. 2003 Jan AIM: To perform a nonlinear mixed effect modelling (NONMEM) population pharmacokinetic analysis of meloxicam plasma concentrations in rheumatoid arthritis (RA) patients participating in three clinical trials, and to evaluate the effects of age, weight, gender and concomitant medications on meloxicam pharmacokinetics. METHODS: Meloxicam was administered to RA patients once daily for 3 weeks or 6 months at doses between 7.5 and 60 mg. Plasma samples were obtained at least 7 days after the first dose and meloxicam plasma concentrations were quantified by h.p.l.c. RESULTS: NONMEM analysis was conducted on plasma samples derived from 586 patients. A one-compartmental model was found to describe the data adequately. For a typical subject in the population, a clearance of 0.377 l h-1 (95% confidence interval (CI) 0.0304-0.449) in males and 0.347 l h-1 (95% CI 0.274-0.419) in females was obtained. The volume of distribution was estimated to be 14.9 l. The findings were corroborated by subsequent analysis using WinBUGS. Analysis of covariates showed that age and gender both significantly (P < 0.005) affected clearance. The effect of age was relatively small and a dose adjustment of <10% was deemed unnecessary. Differences between males and females were attributed to differences in weight. No clinically relevant drug-drug interactions were found, although sulphasalazine and glucocorticoids both significantly (P < 0.005) affected meloxicam clearance (+19% and - 12%, respectively). The mechanisms by which these agents affect meloxicam clearance remain to be elucidated. CONCLUSIONS: The population pharmacokinetic meloxicam data from patients with RA gave similar results to those obtained from phase I trials. However, uncommon drug interactions may not be detected in phase I trials because of the small number of observations made.
15841793 Gonadal and adrenal steroid hormones in plasma and synovial fluid of patients with rheumat 2004 Dec OBJECTIVES: Gonadal and adrenal steroids were shown to affect multiple immune processes including inflammatory response. These effects were documented, specifically, through an influence on local productions of cytokines and the functions of synovial cells at the site of inflammatory processes. The aim of this study was to investigate the levels of selected hormones in synovial fluid of knee joints of patients with rheumatoid arthritis (RA) and with osteoarthrosis (OS, control group). METHODS: The concentrations of cortisol (CORT), 17-beta-estradiol (ES), dehydroepiandrosterone (DHEA), testosterone (TE), progesterone (PRG), and aldosterone (ALD) were determined in plasma and synovial fluid. RESULTS: Significant positive correlations between the levels in plasma and synovial fluids were observed in hormones ES, PRG, TE, DHEA and ALD. In most hormones, the levels in synovial fluids were similar as in plasma; however, the content of ALD was higher in synovial fluid as compared to plasma. Higher levels of ES (in females), DHEA (in males), and ALD were observed in plasma and synovial fluids of RA patients as compared to OS patients. After adjustment to age, no significant RA vs. OS difference was noted in ES, TE, DHEA, PRG, and CORT in plasma and synovial fluid. Age-adjusted ALD concentration tended to be higher in synovial fluid of RA patients as compared to OS patients. The ratio of ES/TE concentrations in synovial fluid was significantly higher in male RA patients compared to OS group. Also the ES/CS and ES/DHEA ratios in synovial fluid were elevated in RA patients in comparison to controls. CONCLUSIONS: These results demonstrated the prevalence of pro-inflammatory hormones in synovial fluid of RA patients, suggesting the possible role of these steroid hormones in inflammatory processes.
12621554 [Enhancement by FK506 of triptolide-induced inhibition of expression of COX-2 and iNOS in 2003 Mar To explore the effects of FK506 on the inhibition by triptolide (TP) of cell proliferation and expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and their inducing products PGE2, NO in human rheumatoid arthritis synovial fibroblasts (RASF), and to study the mechanisms of combination of FK506 and TP in RA therapy, RASF used in the experiments were obtained from synovial tissue of patients with RA and were cultured. RASF were pretreated with FK506(10-1000 nmol/L)for 2 h, then the cells were stimulated with TNF alpha(20 microg/L) in the presence or absence of TP (10 microg/L). The RASF proliferation was determined by [(3)H]-TdR incorporation, and the productions of PGE2 and NO in culture supernatants of RASF were detected with competitive ELISA and enzyme reduction of nitrate. Expression of COX-2 and iNOS mRNA in RASF were analyzed by semi quantitative RT-PCR. Expressions of COX-2 and iNOS protein were estimated by Western blot method and cellular enzyme immunoassay in synovial fibroblasts. NF-kappa B activity in whole-cell extract of RASF was also measured by an ELISA-based method. Results showed that neither FK506 nor TP at lower concentration (10 microg/L) alone affected TNF alpha-induced COX-2, iNOS expression and production of PGE2, NO in synovial cells. Combined treatment of FK506 and a lower concentration of TP (10 microg/L) down-regulated COX-2 and iNOS mRNA and protein expression, and their inducing products PGE2 and NO of synovial fibroblasts. This effect was positively correlated with FK506 concentrations (10-1000 nmol/L). NF-kappa B activity in TNF alpha-stimulated synovial cells was suppressed more profoundly by FK506 plus TP (10 microg/L) treatment than those with TP (10 microg/L) alone. No change was observed in inhibition of proliferation of synovial cells after combined treatment of FK506 and TP. In conclusion, FK506 enhanced TP-mediated down-regulation of COX-2, iNOS and their inducing products PGE2, NO in human RASF by suppressing the activity of NF-kappa B.
15106716 Rheumatoid arthritis probably induced by pegylated interferon in a patient with chronic he 2004 Jan We report a 47-year-old woman who developed features of rheumatoid arthritis 20 weeks after initiation of treatment with pegylated interferon and ribavirin for chronic hepatitis C. She continued to have symptoms six months after discontinuation of the drugs which she received for 24 weeks with rival clearance.