Anti-citrullinated peptide antibody (ACPA) is a highly specific serological marker for rheumatoid arthritis (RA).1,–,3 Different HLA-DRB1 alleles have been shown to be associated with the susceptibility to ACPA-positive RA.4 5 Former studies demonstrated that HLA-DRB alleles carrying a shared epitope (SE),6 consisting of a conserved amino acid motif at positions 70–74 of the HLA-DRβ chain, were strongly associated with ACPA-positive RA and with higher ACPA levels in European and Japanese populations.7,–,9 On the other hand, HLA-DRB1*09:01 was recently found to be negatively associated with ACPA levels in the Japanese.9 These observations imply that combinations of HLA-DRB1 alleles differentially influence ACPA levels in ACPA-positive RA.
To address this question, we conducted a genetic association study employing 2457 ACPA-positive Japanese RA patients. ACPA was quantified by MESACUP CCP ELISA kit (MBL Co Ltd, Nagoya, Japan) with a cut-off level of 4.5 U/ml. The patients were then divided into three groups …
To assess the effect of reproductive factors, especially hormone replacement therapy (HRT) and its interaction with HLA-DRB1 *01 and/or *04 alleles on the diagnosis of rheumatoid arthritis (RA) and the presence of anti-cyclic citrullinated peptide (CCP) antibodies in women included in the ESPOIR cohort (early arthritis cohort).
568 patients were included in the analyses, which were performed using logistic regression.
HRT reduced the risk of RA due to the HLA-DRB1 *01 and/or *04 alleles from OR 1.88 (95% CI 1.32 to 2.68, p<0.000) for HLA-DRB1 *01 and/or *04 alleles alone to OR 1.07 (95% CI 0.51 to 2.26, p=0.85) in women with HLA-DRB1 *01 and/or *04 alleles who received HRT. One explanation might be the protective effect of HRT on the presence of anti-CCP antibodies (OR 0.43, 95% CI 0.24 to 0.77, p<0.006). Other reproductive factors such as the number of pregnancies, menopause and age at menopause, age at menarche and a history of pregnancy with poor outcome were not associated with the diagnosis of RA and the presence of anti-CCP antibodies.
HRT may reduce the risk of RA due to HLA-DRB1 *01 and/or *04 alleles by protecting against the production of anti-CCP antibodies.
To study the influence MHC class II and TAP2 alleles exert on systemic lupus erythematosus (SLE) susceptibility and on the clinical and serological manifestations of the disease, in a cohort of Spanish patients.
HLA-DR serological typing and HLA-DQA, DQB, and TAP2 DNA sequence specific oligotyping, were carried out in 85 unrelated Spanish SLE patients and 186 healthy controls. Autoantibodies detection was carried out by indirect immunofluorescence and counter immunoelectrophoresis.
Total SLE group: the frequency of HLA-DR3 and HLA-DQA1*0501 is significantly increased in this group (pc < 0.005, delta = 0.34 and pc < 0.005, delta = 0.45, respectively) although the highest delta value (delta = 0.87) is obtained when the TAP2*01 alleles are considered. No DQB allele shows significant deviation from the control group. Renal damage: it mainly occurs in HLA-DR3 patients (pc < 0.0005 and delta = 0.72). HLA-DQA1*0501 (p < 0.05, delta = 0.57 and DQB1*0201 (pc NS, delta = 0.56) are weaker susceptibility factors. Ro+ (but not LA) group: this autoantibody response is associated with TAP2*01 alleles in homozygosity (p < 0.05, delta = 0.81). R0/La+ group: it has a different genetic background as HLA-DQA1*0501 (delta = 1) and HLA-DQB1*0201 (delta = 1) are the main susceptibility factors.
A differential association between HLA-DR, DQA1, and DQB1 alleles and SLE or its clinical and serological manifestations are found. Furthermore, the associations are different to the ones reported in other ethnic groups. Finally, TAP2*01 group of alleles are associated with the highest susceptibility to SLE (higher than HLA-DR3) and may influence Ro (but not La) autoantibodies production, whereas HLA-DQA1*0501 and DQB1*0201 mediates concomitant Ro and La productions.
To study collagen-induced arthritis in human leucocyte antigen (HLA)-DR1 transgenic mice lacking endogenous major histocompatibility complex class II molecules (MHC-II) and to determine T cell specificity against the arthritogenic CII(259-273) epitope of type II collagen either unmodified or post-translationally glycosylated at Lys(264).
Arthritis was induced by immunisation with human type II collagen in complete Freund's adjuvant and measured by footpad swelling, clinical score and histology. T cell responses were assessed by proliferation of spleen and lymph node cells and in antigen presentation assays, using T cell hybridomas specific for the glycosylated and non-glycosylated CII(259-273) epitope.
The incidence of arthritis was 50% in DR1-transgenic mice lacking endogenous MHC-II molecules. Recall T cell responses in draining lymph nodes and spleen were consistently greater against the non-glycosylated epitope than to the glycosylated CII(259-273). Most of the T cell hybridomas generated from CII-immunised mice recognised the non-glycosylated CII epitope and this form of the epitope was also presented with 100-fold higher efficiency and 1 h faster kinetics by both macrophages and dendritic cells.
This study shows that T cell responses to the non-glycosylated epitope of heterologous (human) CII are dominant in HLA-DR1 transgenic mice lacking MHC-II, which could contribute to the pathogenicity of autoimmune arthritis.
To evaluate HLA-DM alleles as markers for disease severity in rheumatoid arthritis (RA).
Two distinct cohorts of patients with RA were oligotyped for HLA-DB1 and HLA-DM genes using PCR amplified genomic DNA with sequence specific oligonucleotide probes. Cohort 1 comprised 199 unselected patients with RA (mean (SD) age 45.5 (13.5) years; disease duration 11.9(8.8) years), whose disease severity was assessed using Larsen score on hand and foot radiographs. Cohort 2 comprised 95 patients with severe RA and 70 patients with benign RA according to the Larsen method.
In cohort 1, after stratification according to DRB1 genotypes, patients positive for HLA-DMA*0103 and negative for HLA-DRB1*04 tended to have greater articular damage on hands and wrists (p = 0.07 by Mann-Whitney U test) and reached statistical significance for the Larsen score per year (p = 0.05). This association between HLA-DMA*0103 and articular damage was especially observed in patients with HLA-DRB1*01. Similarly, HLA-DMB*0104 positive patients had higher Larsen score on hands and wrists (p = 0.02). This association was even stronger in DRB1*04 positive patients (p = 0.005). In cohort 2, HLA-DMA*0103 was associated with severe RA in patients negative for HLA-DRB1*04 (OD = 5.4; p = 0.014). HLA-DMB*0104 allele frequency tended to be higher in patients with severe RA but without reaching significance.
This is the first study evaluating the role of HLA-DM genes in the severity of RA. Our results suggest that HLA-DMA*0103 and HLA-DMB*0104 alleles may represent new genetic markers of RA severity. The HLA-DMA*0103 allele tends to be associated with patients with RA negative for DRB1*04 and could predict a more severe form of disease especially in HLA-DRB1*01 positive patients. The HLA-DMB*0104 allele could have an additive effect in HLA-DRB1*04 patients. Combined determination of HLA-DM and HLA-DRB1 alleles could facilitate identification of patients likely to have a poor disease course.
To explore the in vivo effects of PD-0200347, an alpha(2)delta ligand of voltage gated Ca(2+) channels, on cell signalling in osteoarthritic (OA) chondrocytes from an experimental dog model, and examine the effect of PD-0200347 on the major signalling pathways involved in OA cartilage degradation.
OA was surgically induced in dogs by sectioning the anterior cruciate ligament. OA dogs were divided into three groups and treated orally with (a) placebo; (b) 15 mg/kg/day PD-0200347, or (c) 90 mg/kg/day PD-0200347. The animals were killed 12 weeks after surgery. Cartilage specimens from femoral condyles and tibial plateaus were processed for immunohistochemistry. Specific antibodies against the phosphorylated form of PKCalpha, Ras, c-Raf, the MAP kinases Erk1/2, p38, JNK, and the transcription factors, CREB and Elk-1, were used.
Levels of all the tested signalling mediators were increased in the placebo treated (OA) group compared with the normal group. PD-0200347 treatment significantly reduced the levels of the active forms of PKCalpha, c-Raf, Erk1/2, and Elk-1; however, the levels of the active forms of Ras, p38, JNK, and CREB were not affected by the PD-0200347 treatment.
The action of PD-0200347 on OA chondrocytes is probably mediated through the inhibition of Erk1/2 activation via a Ras independent mechanism. This effect is associated with reduction of the activation of transcription factors such as Elk-1, which leads to the inhibition of the induction of the major catabolic factors involved in the degradation process of OA cartilage.
HLA-DRB1*03 is strongly associated with anti-Jo-1-positive idiopathic inflammatory myopathies (IIM) and there is now increasing evidence that Jo-1 antigen is preferentially expressed in lung tissue. This study examined whether smoking was associated with the development of anti-Jo-1 antibodies in HLA-DRB1*03-positive IIM.
IIM cases were selected with concurrent information regarding HLA-DRB1 status, smoking history and anti-Jo-1 antibody status. DNA was genotyped at DRB1 using a commercial sequence-specific oligonucleotide kit. Anti-Jo-1 antibody status was established using a line blot assay or immunoprecipitation.
557 Caucasian IIM patients were recruited from Hungary (181), UK (99), Sweden (94) and Czech Republic (183). Smoking frequency was increased in anti-Jo-1-positive IIM cases, and reached statistical significance in Hungarian IIM (45% Jo-1-positive vs 17% Jo-1-negative, OR 3.94, 95% CI 1.53 to 9.89, p<0.0001). A strong association between HLA-DRB1*03 and anti-Jo-1 status was observed across all four cohorts (DRB1*03 frequency: 74% Jo-1-positive vs 35% Jo-1-negative, OR 5.55, 95% CI 3.42 to 9.14, p<0.0001). The frequency of HLA-DRB1*03 was increased in smokers. The frequency of anti-Jo-1 was increased in DRB1*03-positive smokers vs DRB1*03-negative non-smokers (42% vs 8%, OR 7.75, 95% CI 4.21 to 14.28, p<0.0001) and DRB1*03-positive non-smokers (42% vs 31%, p=0.08). In DRB1*03-negative patients, anti-Jo-1 status between smokers and non-smokers was not significantly different. No significant interaction was noted between smoking and DRB1*03 status using anti-Jo-1 as the outcome measure.
Smoking appears to be associated with an increased risk of possession of anti-Jo-1 in HLA-DRB1*03-positive IIM cases. The authors hypothesise that an interaction between HLA-DRB1*03 and smoking may prime the development of anti-Jo-1 antibodies.
Genetic factors are likely to be important both in determining the overall susceptibility to systemic lupus erythematosus (SLE) and in influencing the remarkable clinical heterogeneity in disease expression found in affected subjects. The more common clinical features seen in patients with SLE include, skin and joint diseases, renal disease, neuropsychiatric complications, and also some haematological abnormalities. Genetic factors, together with environmental factors, strongly influence the development of SLE. Multiple loci within the major histocompatibility complex (MHC) have been implicated in susceptibility as have HLA class II alleles, complement components, and tumour necrosis factor (TNF) loci.
Currently it is believed that some HLA alleles are in genetic linkage disequilibrium with certain disease related genes and they regulate the immune responses. Since 1969, when the first case of SLE was reported from India, the disease has been extensively studied in different regions of the country—namely, Chennai, Calcutta, Mumbai, and New Delhi. A statistically significant clinical correlation comparing the clinical variables from other racial groups of the world has been reported in Indian patients with SLE.1 HLA association studies from Indian patients …
Auranofin is an oral gold preparation used in the treatment of rheumatoid arthritis (RA). During treatment, proteinuria has been reported in approximately 3% of patients.1 In a few cases in which renal biopsies were performed, a membranous nephropathy was shown to have developed.2
Several antirheumatic agents have been reported to induce renal manifestations or drug induced lupus reactions, or both, in patients with RA. In these subjects a genetic predisposition for HLA has previously been suggested.3-6
To determine whether similar genetic factors might be operative in auranofin induced nephropathy, we analysed the HLA-DRB1, DQA1, and DQB1 alleles using a polymerase chain reaction. Furthermore, we investigated if the renal side effects were associated with serological findings in accordance with systemic lupus erythematosus (SLE), thus indicating the development of a drug induced lupus reaction.
Six patients (three female, three male) who …
To study HLA class II association in reactive arthritis.
63 patients with reactive arthritis and 46 with rheumatoid arthritis were included in the study. HLA-DR alleles were determined by using a sequence specific PCR method. Oligonucleotide hybridisation was used for definition of DRB1*04 subtypes and DQB1 alleles. HLA-B27 was determined by standard microcytotoxity test or by PCR. HLA-B27 subtyping was made by sequencing.
46 (73%) of 63 patients with reactive arthritis were HLA-B27 positive and 24 (38%) were HLA-DRB1*04 positive. When haplotypes were inferred according to the known associations between DRB1 and DQB1 alleles, the frequency of DRB1*04-DQB1*0301 haplotype was found to be 13% (12/92) in HLA-B27 positive reactive arthritis patients, in contrast to 0% in HLA-B27 negative reactive arthritis (P = 0.04) and 1% in random controls (P = 0.0009). However, this combination was also found in 5% of 84 HLA-B27 positive control haplotypes, showing a linkage disequilibrium between B27 and this particular class II haplotype. HLA-DRB1*0408 subtype was found in 8/24 (33%) of the HLA-DRB1*04 alleles in patients with reactive arthritis, accounting for most DQB1*0301 haplotypes, but only in 5/55 (9%) of the DRB1*04 alleles in random controls (P = 0.017). All reactive arthritis patients with this subtype were positive for HLA-B27. DRB1*04-DQB1*0302 haplotype was increased in patients with rheumatoid arthritis (28/92, 30%) compared with reactive arthritis (12/126, 10%) or with the controls (12/100, 12%; P = 0.003). HLA-B*2705 was by far the dominant B27 subtype both in reactive arthritis patients with the particular DRB1*0408-DQB1*0301 haplotype and in controls. It was found in 11 out of 12 DR analysed patients, as well as in 10 out of 11 randomly selected B27 positive controls.
Although no single class II allele was found to be increased among patients with reactive arthritis, HLA-B27, DRB1*0408, and DQB1*0301 might exert a haplotypic effect in the pathogenesis of reactive arthritis, or they may be markers of a subset of B27 haplotypes conferring susceptibility.
To compare epicutaneous ketoprofen in Transfersome (ultra-deformable vesicles, IDEA-033) versus oral celecoxib and placebo for relief of signs and symptoms in knee osteoarthritis.
This was a multicentre, randomised, double-blind, controlled trial; 397 patients with knee osteoarthritis participated and 324 completed the trial. They were randomly assigned 110 mg epicutaneous ketoprofen in 4.8 g Transfersome plus oral placebo (n = 138), 100 mg oral celecoxib plus placebo gel (n = 132), or both placebo formulations (n = 127) twice daily for 6 weeks. Primary efficacy outcome measures were the changes from baseline to end of the study on the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index pain subscale, physical function subscale and patient global assessment (PGA) of response.
The mean WOMAC pain subscale scores in the intent to treat population were reduced by 18.2 (95% confidence interval -22.1 to -14.3), 20.3 (-24.3 to -16.2) and 9.9 (-13.9 to -5.8) in the IDEA-033, celecoxib and placebo groups, respectively, and the physical function subscale score by 14.6 (-18.1 to -11.0), 16.6 (-20.2 to -13.0) and 10.2 (-13.8 to -6.6), respectively. The mean PGA of response scores were 1.8 (1.6 to 2.1), 1.7 (1.5 to 1.9) and 1.3 (1.1 to 1.5), respectively. The differences in change between IDEA-033 and placebo were statistically significant for pain subscale (p<0.01) and PGA of response (p<0.01). Gastrointestinal adverse events for IDEA-033 were similar to placebo.
IDEA-033 is superior to placebo and comparable with celecoxib in relieving pain associated with an acute flare of knee osteoarthritis.
Background and objectives:
Vascular disease is common in systemic lupus erythematosus (SLE) and patients with antiphospholipid antibodies (aPL) are at high risk to develop arterial and venous thrombosis. Since HLA class II genotypes have been linked to the presence of pro-thrombotic aPL, we investigated the relationship between HLA-DRB1 alleles, aPL and vascular events in SLE patients.
665 SLE patients of Caucasian origin and 1403 controls were included. Previous manifestations of ischaemic heart disease, ischaemic cerebrovascular disease (ICVD) and venous thromboembolism (together referred to as any vascular events (AVE)) were tabulated. aPL were measured with ELISA. Two-digit HLA-DRB1 typing was performed by sequence-specific primer-PCR.
HLA-DRB1*04 was more frequent among SLE patients with ICVD compared to unaffected patients. This association remained after adjustment for known traditional cardiovascular risk factors. HLA-DRB1*13 was associated with AVE. All measured specificities of aPL-cardiolipin IgG and IgM, β2-glycoprotein-1 IgG, prothrombin (PT) IgG and a positive lupus anticoagulant test were associated with HLA-DRB1*04-while HLA-DRB1*13 was associated with IgG antibodies (β2-glycoprotein-1, cardiolipin and PT). In patients with the combined risk alleles, HLA-DRB1*04/*13, there was a significant additive interaction for the outcomes AVE and ICVD.
The HLA-DRB1*04 and HLA-DRB1*13 alleles are associated with vascular events and an aPL positive immune-phenotype in SLE. Results demonstrate that a subset of SLE patients is genetically disposed to vascular vulnerability.
To determine HLA-DR4 and DR1 allele frequencies in a series of patients with newly diagnosed early inflammatory arthritis.
HLA-DR1 and DR4 frequencies were determined by oligonucleotide typing of 208 patients classified as having either rheumatoid arthritis (RA) or undifferentiated inflammatory polyarthritis.
The frequency of occurrence of DR4 in these patients with RA did not differ significantly from that in controls in the United Kingdom (42 v 37%). HLA-DR1 was increased in the group with inflammatory polyarthritis (25 v 18%).
The frequency of DR4 is not increased in newly diagnosed community based patients with RA. This supports the hypothesis that DR4 is less important as a marker for susceptibility to RA than it is for disease persistence or severity.
Apoptosis or programmed cell death is one of the regulation mechanisms of cell homeostasis.
Fas is a transmembrane receptor protein which transmits a cell death signal when cross linked with an antibody or with its physiological ligand—Fas ligand (Fas L).1 Fas and Fas L have a pivotal role in regulating lymphocyte apoptosis and maintaining lymphocyte homeostasis.
Soluble forms of Fas and Fas L may be detectable and measured in the serum,2 and may reflect the activation of this pathway. Moreover, soluble forms of Fas regulate Fas/Fas L mediated apoptosis.3 Raised levels of soluble Fas (sFas) have been shown in various chronic inflammatory rheumatic diseases, systemic lupus erythematosus, Sjogren's syndrome,1 4 5 and in the synovial fluid of rheumatoid arthritis.6 These diseases are autoimmune diseases …
To test whether HLA-DR alleles influence the production of particular autoantibodies in rheumatoid arthritis (RA) patients, we screened synovial proteins with sera of RA patients homozygous for different HLA-DR alleles by using 2D blots. We found that sera of RA patients homozygous for HLA-DRB1*0404 recognised a 100-kDa synovial protein identified as calpastatin. We studied B and T cell epitopes on calpastatin and their association with HLA-DRB1*0404.
The frequency of positive sera in patients expressing different RA-associated HLA-DR allele combinations was calculated by inhouse ELISA using purified synovial calpastatin or calpastatin peptides encompassing the entire calpastatin protein as immunosorbent. Interaction between calpastatin peptides and HLA-DR alleles was tested by a direct binding assay. T cell responses to calpastatin were measured in RA patients and controls.
We found that RA-associated HLA-DR alleles are associated with presence of autoantibodies to synovial calpastatin in RA patients' sera. HLA-DRB1*0404 is strongly associated with antisynovial calpastatin in RA sera. One linear B cell epitope is preferentially associated with HLA-DRB1*0404. Multiple peptides from calpastatin bind every tested HLA-DR allele associated or not with RA. Peptides from domain 1 and 4 of calpastatin are the best HLA-DR allele binders. The T cell response to calpastatin is frequent in RA patients and independent of the HLA-DR background.
HLA-DRB1*0404 is strongly associated with anticalpastatin antibodies in rheumatoid arthritis.
To identify the association of HLA-DR4 subtypes with rheumatoid arthritis (RA) in Koreans.
Ninety five patients with RA and 118 normal control subjects were examined for HLA-DR antigens by serology. Subtypes of HLA-DR4 were determined by allele specific oligonucleotide typing.
The phenotype frequency of HLA-DR4 in RA patients was significantly greater than that in controls (60.0% versus 31.4%, odds ratio (OR) 3.28, 95% confidence interval (CI) 1.79 to 6.02 (p < 0.001)), but HLA-DR6 was decreased in RA patients (15.8% versus 32.2%, OR 0.39, 95% CI 0.19 to 0.81 (p < 0.001)). When DR4 was excluded from analysis of patients and controls, the allele frequency of DR1 was significantly increased in the patients compared with controls (11.3% versus 4.5%, OR 2.73, 95% CI 0.87 to 5.95 (p < 0.001)). Forty two of 57 DR4 positive patients (73.7%) possessed DRB1*0405, which was strongly associated with RA (44.2% of patients, versus 11.9% of controls: OR 5.88, 95% CI 2.81 to 12.47 (p < 0.001)). DRB1*0403 was not found in the patients, but was present in 8.5% of controls. Examining the third hyper-variable region at position 70-74 in the DRB1*04 chain by oligotyping, we found that 52 of 57 DR4 positive patients (91.2%) carried one of the conserved amino acid sequences QRRAA or QKRAA, known to be the epitope conferring predisposition to RA.
This study confirms that RA is strongly associated with DR4, especially with DRB1*0405, and that the presence of the inferred QRRAA sequence may be important in susceptibility to RA in Koreans.
Uveitis and retinal vasculitis are sight-threatening manifestations of Behçet's disease with limited treatment options. This pilot study aimed to evaluate the safety, pharmacokinetics and clinical activity of XOMA 052 (gevokizumab), a recombinant humanised anti-interleukin 1β antibody, in Behçet's disease patients with uveitis.
Patients with acute posterior or panuveitis, and/or retinal vasculitis, resistant to azathioprine and/or ciclosporin, and receiving 10 mg/day or less of prednisolone, were enrolled into the 98-day study. Immunosuppressive agents were discontinued at baseline. Patients received a single infusion of XOMA 052 (0.3 mg/kg). The safety and uveitis status and pharmacokinetics of XOMA 052 were evaluated.
Seven patients enrolled and completed the study. No treatment-related adverse event was observed. XOMA 052 treatment was associated with rapid and durable clinical response in all patients. Complete resolution of intraocular inflammation was achieved in 4-21 days (median 14 days), with a median duration of response of 49 days (range 21-97 days); one patient remained exacerbation free throughout the study.
Well tolerated, XOMA 052 resulted in a rapid onset and sustained reduction in intraocular inflammation in patients with resistant uveitis and retinal vasculitis. Moreover, the effect was observed despite discontinuation of immunosuppressive agents and without the need to increase corticosteroid dosages.
Psoriasis of early onset (type I; age of onset <or=40 years) is associated with HLA-Cw*06 while the shared epitope (SE) is associated with rheumatoid arthritis susceptibility. Our aim was to investigate the role of HLA-Cw*06 and HLA-DRB1 genes (including SE) with psoriatic arthritis (PsA) susceptibility.
In a case-control association study, HLA-Cw*06 phenotype frequencies were compared between patients with PsA (n = 480), psoriasis alone (n = 611) and healthy controls (n = 166). Similarly, at the HLA-DRB1 locus, phenotype and SE frequencies were compared in patients with PsA (n = 480), early undifferentiated inflammatory arthritis alone (n = 1621) and healthy controls (n = 537).
The HLA-Cw*06 phenotype was associated with type I psoriasis (OR 6.9, 95% CI 4.4, 11.1, p = 2.2 x 10(-21)) and with patients with PsA having type I psoriasis (OR 5.0, 95% CI 3.2, 7.9, p = 4.39 x 10(-13)), but not with patients with PsA having type II psoriasis (age of onset >40 years). HLA-DRB1*07, in linkage disequilibrium with HLA-Cw*06, was also associated with patients with PsA having type I psoriasis (OR 2.7, 95% CI 2.1, 3.7, p<0.00001). HLA-DRB1*04 alleles and the SE were associated with undifferentiated inflammatory arthritis but not with PsA.
The SE is not a PsA susceptibility locus. HLA-Cw*06 and HLA-DRB1*07 are associated with patients with PsA having type I psoriasis, suggesting that the primary association is with age of onset of psoriasis. Patients with PsA having type I psoriasis, therefore, have a genetic background different to those with type II psoriasis, and adjustment for this is necessary in future studies that investigate the genetic susceptibility of PsA.
To examine the association between three modifiable risk factors (obesity, smoking, and alcohol consumption) and reported joint pain.
Cross sectional data were collected on 858 people aged 58 years living in the West of Scotland and on the same individuals four years later, aged 62 years.
There was a positive relation between obesity and reported pain in the hips, knees, ankles, and feet. The strongest relation was with knee pain (odds ratio = 2.42 (95% confidence interval, 1.65 to 3.56)). There were no strong consistent associations between smoking habits and pain in any joint after adjusting for sex, alcohol consumption, body mass index, social class, and occupational exposures. Similarly, alcohol was not consistently related to pain in any joint in the fully adjusted models.
Obesity had consistent and readily explained associations with lower limb joint pain. The data suggest that smoking behaviour and alcohol consumption are not consistently associated with joint pain across the body.
Human leucocyte antigen (HLA) genes predict disease severity in psoriasis (HLA-Cw6) and rheumatoid arthritis (shared epitope (SE)), but the situation is unclear for psoriatic arthritis (PsA).
To determine the association of the HLA-Cw6 and HLA-DRB1 gene with disease severity in a large UK cohort with PsA.
Genotyping of the HLA-Cw and HLA-DRB1 loci was undertaken in DNA samples from patients with PsA (n = 480). Stratification and regression analysis were used within the PsA cases to determine whether HLA-Cw6, HLA-DRB1 or the presence of the SE alleles predicted disease severity as measured by the Health Assessment Questionnaire score, the total number of damaged or involved joints adjusted for disease duration and disease-modifying antirheumatic treatments.
HLA-Cw6 was found to be in linkage disequilibrium with HLA-DRB1*07 (r(2) = 0.46). Patients with PsA who carried both HLA-Cw6 and HLA-DRB1*07 had fewer damaged or involved joints (41% fewer damaged (95% CI 23% to 55%, p = 0.02) and 31% fewer involved joints (95% CI 16% to 44%, p<0.001)) compared with those who carried neither HLA-Cw6 nor HLA-DRB1*07 alleles. Those who carried either HLA-Cw6 or HLA-DRB1*07 alleles alone had no evidence of a reduction in joint involvement. The SE, HLA-DRB1*03 and HLA-DRB1*04 alleles did not predict severity using these outcome measures.
Patients with PsA carrying both HLA-Cw6 and HLA-DRB1*07 alleles have a less severe course of arthritis. This suggests that a protective locus lies on a haplotype marked by these alleles. No association was detected with disease severity and SE status.
Anti-cyclic citrullinated peptide (anti-CCP) antibody is a highly specific biomarker for Rheumatoid Arthritis (RA), and recognized as a predictor of development of RA.[1,2] It had been demonstrated that some HLA-DRB1 alleles, such as shared epitope (SE) alleles, significantly contribute to the positivity of anti-CCP antibody and the susceptibility of anti-CCP antibody-positive RA.[3,4] However, the quantitative effect of HLA-DRB1 alleles on anti-CCP antibody levels in RA patients is controversial.[3,5,6] Therefore, we carried out a large-scale study to study the quantitative effects of HLA-DRB1 alleles on anti-CCP antibody levels in Japanese patients with RA.
Rheumatoid arthritis (RA) tends to remit during pregnancy, with more patients achieving remission in the third trimester, coinciding with an increase in levels of alpha-fetoprotein (AFP). In vitro and animal studies have shown that AFP has immunomodulatory properties. MM-093 is a non-glycosylated, recombinant version of human AFP.
To assess the safety, tolerability and clinical effects of MM-093 during a 12-week, randomised, double-blind, placebo-controlled study.
12 patients with RA, who had active disease and were on stable doses of methotrexate, received weekly subcutaneous injections of placebo or 21 mg of MM-093. Assessments were carried out at baseline and weekly thereafter.
Baseline characteristics were similar in both groups. There was one dropout in the placebo group, due to flare of disease. Treatment with MM-093 was well tolerated. No serious adverse event was observed. By day 85, MM-093 produced a significant mean improvement from baseline in Disease Activity Score 28 (DAS28; 0.913 vs 0.008, p = 0.033) and patient's global assessment (28.9% vs -36.3%, p = 0.02) compared with placebo.
This is the first randomised, controlled trial of MM-093, a recombinant version of human AFP, in patients with RA. MM-093 was well tolerated. Evidence of efficacy was observed, suggesting that MM-093 may have therapeutic potential in RA.
Twenty patients with severe symptomatic Paget's disease were treated with a series of 15 mg intravenous infusions of 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD). A regimen of either five consecutive days of treatment (regimen 1) or a course of 12 weekly infusions was administered (regimen 2). In five cases regimen 2 followed regimen 1 after a three month interval. Alkaline phosphatase levels fell in all patients and returned to the normal range in 12. All but one of the patients obtained symptomatic improvement. There was a median fall in alkaline phosphatase activity of 63%. Eight patients observed a transient increase in bone pain starting about 24 hours after the first infusion. Intravenous APD was well tolerated, and we conclude that it is an effective treatment for Paget's disease; this route of administration avoids the problem of poor and unpredictable gastrointestinal absorption seen when a bisphosphonate is given orally. The optimal dose and duration of APD therapy, frequency of relapse, requirement for further courses, and merits relative to other second generation bisphosphonates remain to be established.
Previous work has shown that renal metabolism of 25-dihydroxyvitamin D3 (25(OH)D3) to the active metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is stimulated by prostaglandin E2 and inhibited by acetylsalicylate (aspirin). As prostaglandins are primary inflammatory mediators and synovial fluid macrophages are known to synthesise 1,25(OH)2D3 in vitro, the effects of prostaglandin E1, prostaglandin E2, and aspirin on the metabolism of 25(OH)D3 by cells cultured from synovial fluid of patients with inflammatory arthritis were investigated. Most cultures contained non-proliferating macrophages which formed 1,25(OH)2D3; however, two of 13 cultures contained colonies of rapidly proliferating fibroblast-like cells which formed 24,25(OH)2D3 (24,25(OH)2D3). Prostaglandin E1 and prostaglandin E2 (0.01-10 mumol/l) induced marked inhibition of 1,25(OH)2D3 synthesis (up to 94%) in a dose dependent manner after preincubations of 24 hours but not over straightforward six hour incubations. Exposure of macrophages to aspirin (1 mumol/l-1 mmol/l) for 24 hours did not affect 1,25(OH)2D3 synthesis unless the cells had been pretreated with lipopolysaccharides, in which instance 1 mM aspirin increased 1,25(OH)2D3 synthesis. Lipopolysaccharide is a macrophage activating factor which stimulates macrophages to form 1,25(OH)2D3, and it also induces prostaglandin synthesis which would be inhibited by aspirin. Taken together these results suggest that prostaglandin E1 and prostaglandin E2 synthesised by macrophages may act in an autocrine manner to attenuate the ability of macrophage activating factors, such as lipopolysaccharide, to stimulate 1,25(OH)2D3 synthesis. Prostaglandins synthesised by other inflammatory cells may also inhibit 1,25(OH)2D3 synthesis in a paracrine manner. In contrast, prostaglandin E2 and aspirin had limited effects on fibroblast 24,25(OH)2D3 synthesis. This study shows that the effects of prostaglandin E1, prostaglandin E2, and aspirin in macrophages contrast with those previously reported for the renal 25(OH)D3-1alpha-hydroxylase, where prostaglandin E2 stimulated and aspirin inhibited enzyme activity. These results further emphasise that synthesis of 1,25(OH)2D3 in non-renal sites is independently regulated, which is consistent with it having an immunological role at a local level rather than playing a part in systemic calcium homeostasis.
We have previously compared 25-hydroxycholecalciferol levels in the serum of patients with osteoarthrosis and rheumatoid arthritis, finding no significant difference between the circulating levels of this hormone. We have now estimated 1,25-dihydroxycholecalciferol levels on stored sera from the same groups of patients and found no significant difference in the levels of this hormone between the 2 groups. The osteopenia that distinguishes rheumatoid arthritis from osteoarthrosis is not the result of altered levels of systemic 25OHD3 or of 1,25(OH)2D3. Local factors may be more important in its pathogenesis.
Synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown in cells from knee joint synovial fluid of 20 patients with inflammatory rheumatoid disease, reactive or psoriatic arthritis, or gout, all of which had high synovial fluid cell counts, and by cells from a patient with aseptic necrosis of a femoral condyle after short term (less than 24 hours) or long term (seven days) primary culture. Cells from 18 patients with inflammatory arthritis, five of which had low synovial fluid cell counts and cells from six patients with osteoarthritis were unable to synthesise this metabolite from 25-hydroxyvitamin D3 (25(OH)D3). Macrophages are believed to be the cells responsible for synthesising 1,25(OH)2D3 because these were significantly more numerous in samples that formed 1,25(OH)2D3; they were also the predominant cell type present in the aseptic necrosis sample and the only cell type present in preparations maintained for one week in monolayer culture.
T helper 17 (Th17) cells from patients with early rheumatoid arthritis (RA) induce a proinflammatory feedback loop upon RA synovial fibroblast (RASF) interaction, including autocrine interleukin (IL)-17A production. A major challenge in medicine is how to control the pathogenic Th17 cell activity in human inflammatory autoimmune diseases. The objective of this study was to examine whether tumour necrosis factor (TNF) blockade and/or 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) controls Th17-mediated synovial inflammation.
Peripheral CD4+CD45RO+CCR6+ Th17 cells of patients with early RA, Th17-RASF cocultures and synovial biopsy specimens were cultured with or without 1,25(OH)(2)D(3) and/or TNFα blockade. Intracellular cytokine expression was detected by flow cytometry. Cytokine and matrix metalloprotease (MMP) production was determined by ELISA.
The authors show that the 1,25(OH)(2)D(3), but not TNFα blockade, significantly suppressed autocrine IL-17A production in Th17-RASF and synovial biopsy cultures. Combining 1,25(OH)(2)D(3) and TNFα blockade had a significant additive effect compared with single treatment in controlling synovial inflammation, indicated by a further reduction in IL-6, IL-8, MMP-1 and MMP-3 in Th17-RASF cocultures and IL-6 and IL-8 expression in cultures of RA synovial tissue.
These data show that TNF blockade does not suppress IL-17A and IL-22, which can be overcome by 1,25(OH)(2)D(3). The combination of neutralising TNF activity and 1,25(OH)(2)D(3) controls human Th17 activity and additively inhibits synovial inflammation. This indicates more valuable therapeutic potential of activation of Vitamin D receptor signalling over current TNF neutralisation strategies in patients with RA and potentially other Th17-mediated inflammatory diseases.
Frequencies of immunoglobulin G (Gm) allotypes were determined in 240 patients with ankylosing spondylitis (AS). The uncommon phenotype Gm(1,2;21) was increased in frequency in 55 patients with AS and peripheral arthritis (14.5% v 3.5% of healthy blood donors; p less than 0.05). In 16 patients with arthritis only of wrist/hand or ankle/forefoot, or both, the Gm(1,2;21) frequency was even higher (31.3%; p less than 0.0005). Patients with AS negative for the HLA antigen B27 (n = 28) differed from the B27 positive patients (n = 205) with regard to the frequency of the Gm(1,2,3;5,21) phenotype (39.3% v 9.3%; p less than 0.0005). These findings support the notion of genetic heterogeneity among patients with AS.
Patients with U1-nRNP antibodies (n = 35, 31 female, four male) were typed for HLA-A, -B, -C, and -DR antigens and IgG heavy chain allotypes G1m(1), -(2), -(3), G3m(5), and -(21). The patient group was clinically heterogeneous. Four met the American Rheumatism Association criteria for systemic lupus erythematosus, six for progressive scleroderma, and 14 for rheumatoid arthritis. Sicca syndrome was present in seven cases. Twenty three had overlapping features compatible with mixed connective tissue disease (MCTD). Healthy blood donors served as controls for HLA typing (n = 64), Gm typing (n = 228), or both (n = 56). Sixty six per cent of the patients with U1-nRNP antibodies were DR4 positive compared with 28% of the controls (relative risk = 4.9, p = 0.00053). The Gm(1,3;5,21) phenotype was found in 46% of the patients and 25% of the controls (relative risk = 2.47, p = 0.0247). Within the patient group Gm(1,3;5,21) was found only in DR4 positive individuals. The coincidence of HLA-DR4 and Gm(1,3;5,21) increases the relative risk values to 8.0 (compared with the group with neither risk factor). DR4 and Gm(1,3;5,21) primarily seem to be related to U1-nRNP antibody formation and not to disease expression. Patients with or without MCTD did not differ with respect to DR4 or Gm(1,3;5,21) frequency. Disease onset was earlier in patients with HLA-DR4/Gm(1,3;5,21) than in patients without both markers (mean 27.9 v 40.1 years; p less than 0.05).
The sex hormone prolactin (PRL) has immunomodulatory properties, can be produced by immune cells, and elevated PRL serum levels have been reported in rheumatoid arthritis (RA) patients. Here we examined synovial expression of PRL and PRL receptor (PRLR) in patients with inflammatory arthritis, their expression in polarised macrophages from patients and healthy donors, and the effects of PRL on macrophage activation.
PRL levels in paired synovial fluid (SF) and peripheral blood of RA (n = 19), psoriatic arthritis (PsA, n = 13) and gout (n = 11) patients were measured using an immunofluorescent metric assay. PRL mRNA expression was measured in synovial tissue (ST) of RA (n = 25), PsA (n = 11) and gout (n = 12) patients, and in macrophages differentiated in RA, PsA, spondyloarthritis (SpA) and gout SF by qPCR. PRLR protein expression was determined in ST of RA (n = 19), PsA (n = 15) and osteoarthritis (OA, n = 9) patients by immunohistochemistry and detected in specific cell types by immunofluorescence. IL-6 production by IFN-y and IL-10 -differentiated macrophages following stimulation with CD40L or TNF in the absence or presence of PRL was measured by ELISA.
PRL protein levels were similar in serum and SF of RA, PsA and gout patients, as was mRNA expression in RA, PsA and gout ST. Of interest, PRL mRNA expression significantly correlated with clinical disease parameters in PsA (DAS28, R = 0.729, P = 0.017) and RA (ESR, R = 0.424, P = 0.049). PRL expression was also detected in monocyte-derived macrophages from RA patients, and significantly higher (P≤0.01) in healthy donor macrophages differentiated in pooled SF of RA and PsA compared to SpA and gout SF. In RA SF-differentiated macrophages PRL production was increased by CD40L or IgG stimulation but not LPS or TNFα. Median (IQR) PRLR expression was significantly higher (P<0.05) in RA [0.06 (0.00-0.33)] and PsA [0.18 (0.00-1.67)] ST compared to OA [0.00 (0.00-0.02)], and there was no significant difference in PRLR expression between (pre/postmenopausal) females and males, independently of disease. PRLR expression was mainly colocalised with CD68(+) macrophages and vWF(+) endothelial cells. In vitro, PRLR was prominently expressed in IFN-y and IL-10 polarised monocyte-derived macrophages compared to macrophages polarised in GM-CSF, M-CSF or RA SF. In these macrophages, PRL stimulation significantly enhanced IL-6 production in response to TNFα or CD40L.
Our results provide the first evidence that PRL is produced locally in the synovium of patients with inflammatory arthritis, and contributes to the activation of macrophages in the presence of other inflammatory stimuli.
In autoimmune conditions, like rheumatoid arthritis (RA), the own immune system attacks the body and causes inflammation. One of the consequences of inflammation during autoimmune diseases is a changed glycosylation profile of IgG antibodies. RA is not only characterised by an altered N-glycan structure bound to the Fc portion of IgG molecules but also by circulating rheumatoid factors (RF) that recognizes the Fc portion of IgG as antigen. The aim of our work was to investigate the glycosylation of immune complexes circulating in the blood of patients with seropositive and seronegative RA.
Sera from 70 patients with RA (RF positive n = 43 and RF negative n = 27) and 8 healthy donors (control) have been used in the analysis. (I) Random serum IgG-complexes were captured with F(ab')2 fragments of anti-human-IgG and analysed by ELISA for their interaction with anti-human-IgG, anti-human-IgM, AAL lectin (fucose-specific) and LCA lectin (manose-specific). (II) The IgG complexes eluted from the ELISA-plates were analysed by Western Blots for the presence of IgG, IgM and AAL as well as LCA binding. (III) IgG affinity purified with AAL-, SNA- and jacalin-agarose columns was tested by ELISA as target for IgM rheumatoid factor.
(I) The accessibility of fucose residues for the AAL lectin of circulating IgG complexes was much higher in patients with RA when compared to healthy donors. This reactivity was independent of the presence of IgM-RF. The accessibility of mannose residues for the LCA lectin of circulating IgG complexes was much higher in patients with RA when compared to healthy donors. This reactivity was only to be observed in the presence of IgM-RF. (II) Western Blot confirmed an increased presence of IgM in IgG complexes from seropositive patients with RA. The fucosylation of IgG did not differ much between all groups. (III) No difference was detected in IgM-RF affinity towards positive and negative fractions of AAL- and SNA-reactive IgG. However, IgM-RF displayed a reduced binding to the minor IgG fraction isolated with jacalin-affinity chromatography.
In not-denatured IgG circulating in patients with RA the glycan shows a lower accessibility for the fucose-specific AAL lectin, independent of the presence of IgM-RF. In contrast, the increased binding of the LCA lectin is due to the presence of IgM complexed to IgG. IgM-RF did not discriminate between IgG reactive with AAL and SNA lectin. AAL - Aleuria aurantia lectin (α-1,6 core fucose) LCA - Lens culimaris agglutinin (α-mannose) SNA - Sambucus nigra agglutinin (α-2,6-sialic acid).
During pregnancy, gammadelta T cells expand at the fetomaternal interface where they induce a tolerogenic milieu. Patients with rheumatoid arthritis (RA) experience a spontaneous improvement of their disease during pregnancy and a postpartum aggravation. By contrast, pregnant patients with ankylosing spondylitis (AS) often experience persistent active disease. We hypothesised that the pregnancy related modulation of disease activity in RA patients versus AS patients is associated with numerical and functional changes of circulating gammadelta T cells.
The frequency of surface markers and the intracellular cytokine profile of freshly isolated gammadelta T cells from RA (n = 54) and AS (n = 26) patients and healthy controls (n = 40) were analysed at each trimester during pregnancy and 6-8 weeks postpartum by flow cytometry.
Very discrete changes of Vdelta1 or Vdelta2 frequency were seen during pregnancy and postpartum in healthy controls and AS patients. In RA, however, the frequency of Vdelta2 cells decreased in the third trimester when disease activity was low. Low percentages of Vdelta 2 cells were also found in non-pregnant RA patients with active arthritis, yet only pregnant RA patients showed reduced percentages of Vdelta2 cells positive for the activation marker CD69 and the intracellular cytokine TNFalpha. Similarly, Vdelta1 + TNFalpha + cells were lower in pregnant RA patients compared to non-pregnant RA patients. The percentage of Vdelta2 + TNFalpha + cells, Vdelta2+ CD69+ and Vdelta1+ CD69+ cells correlated with disease activity in RA. As for the receptors which modulate cytotoxicity, RA patients showed a rise of the anti-cytotoxic receptor NKG2A on Vdelta1 cells in the 2(nd) trimester and a decrease postpartum. Since the pro-cytotoxic receptor NKG2D remained unchanged, the NKG2D/NKG2A ratio on Vdelta1 cells was reduced in RA patients during pregnancy. In AS patients, persistent disease activity during pregnancy was reflected by an increased frequency of Vdelta2+ CD69+ cells and an unchanged frequency of Vdelta2+ TNFalpha+ cells. In addition, pregnant AS patients showed an increased frequency of Vdelta1+CD161+ cells.
Disease amelioration of RA during pregnancy correlates with changes of cell activation, pro-inflammatory cytokines and anti-cytotoxic receptors of gammadelta T cells. By contrast, active disease during pregnancy as found in AS is associated with unchanged inflammatory responses of gammadelta T cells. Since gammadelta T cells remain unchanged in healthy pregnant controls, the modulation of gammadelta T cells in RA rather seems to be an effect of improved disease than of pregnancy itself.
In established RA, autoantibodies (AAB) to human citrullinated fibrinogen (AhFibA) were demonstrated to be mainly composed of two subfamilies of AAB directed to immunodominant epitopes borne by the fibrin peptides α36-50Cit38,42 and β60-74Cit60,72,74,respectively. Serum reactivity toward those peptides defines subgroups of patients. In the present study, we investigated whether AhFibA, anti-α36-50Cit38,42 and β60-74Cit60,72,74 AAB, have different predictive values for disease outcome in very early RA. We also analysed whether these AAB differentially associate with SE and tobacco exposure.
The French ESPOIR cohort is comprised of very early RA and of undifferentiated arthritides (UA) of less than 6-month duration. AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB were assayed by ELISA at baseline. After 3-year follow up, 701 patients were diagnosed RA according to the ACR/EULAR 2010 criteria. Relationships between SE HLA-DR alleles, tobacco exposure and the 3 AAB were analysed on those patients. Disease activity (DAS28, HAQ) and radiographic damage (SHS) were evaluated at baseline, and after 2- or 3-year follow up. Associations with clinical parameters and predictive value of each AAB were investigated.
AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB were detected in 349/701 (50%), 203/701 (29%), and 257/701 (37%), RA sera, respectively. Their positive predictive values for RA (72%, 82%, and 79%, respectively) were not significantly different. The presence and titre of each AAB were associated with SE HLA-DR alleles without significant additional effect of tobacco exposure. When 2 vs 0 copies of SE alleles were present, the odds ratios for AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB presence reached 8.0, 6.1 and 9.5, respectively. Neither the presence nor the titres of AhFibA, α36-50Cit38,42 and β60-74Cit60,72,74 AAB were associated with DAS28 or HAQ at baseline and after 2 years. However, for the 3 AAB, patients whose sera contained one or several AAB had a progression of SHS during the first 3 years (medians from 6 to 8 depending on the subgroup), significantly higher than the AhFibA-negative patients (median = 4). Nevertheless, no significant correlation was observed between the titres of AAB and radiographic progression.
Not only AhFibA but also their anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB subfamilies, are prognostic markers for bone erosion in RA. Moreover, AhFibA, anti-α36-50Cit38,42 and anti-β60-74Cit60,72,74 AAB are similarly associated to HLA-DR and to tobacco exposure.
Biomarkers for the early diagnosis of RA are still needed despite the improvement brought about by ACR/EULAR-2010 criteria which include the ACPA biomarker. T-cell subset dys-regulation was shown to occur early in the RA disease continuum (Blood, 2002), to predict response to MTX (ARD, 2013) and safe discontinuation of biological when in clinical remission (ARD 2010). The aim of the current study is to determine whether T-cell subset phenotyping adding novel cell surface markers for TLR2, TLR4, IL-6R and 4 chemokine receptors (CCR3, CXCR4 CCR5 and CCR6) can discriminate between patients who are clearly RA from those with other (inflammatory) rheumatic diseases in an early arthritis clinic.
46 patients with <12 months IA were enrolled; age, CRP, joint counts, symptom duration, SE and ACPA were recorded. 6-8 colour flowcytometry was performed using standard protocols. 95 healthy controls (HC) were included to calculate age-corrected naïve cell frequency.
23 of the 46 patients were diagnosed with RA. 9 had undifferentiated arthritis and 14 other rheumatic diseases (AS, SPA, gout and self-limiting). Not surprisingly, as part of the EULAR-2010 criteria, ACPA positivity (P<0.0001), CRP (P = 0.004) and swollen joint count (P = 0.003) were associated with RA. Naïve CD4+ T-cells tended to be reduced (p = 0.093) in RA and reached significance when RA and UA were compared to other diagnosis (p = 0.008). TLR4 expression was increased in RA (p = 0.100) and again significant in RA+ UA (P = 0.038). CXCR4 expression appeared increased in RA but a number of outliers reduced significance. CXCR4 expression was then analysed in relation with other chemokine receptors. The number of subset generated prevented classic statistical analysis so we used a clustering software which separated 2 groups of patients, one being associated with RA (p = 0.070) and ACPA-positivity (P = 0.072). Clustering was driven by increased CD4+ T-cell subsets positive for CXCR4 and expressing CCR5, CCR6 or IL-6R. This group was clearly associated with RA as well as ACPA-positivity. The second group was also characterised by increased CXCR4+ but on CD8+ T-cells with expression of CCR5, CCR6 or IL-6R and included mixed RA (mostly ACPA-negative) and non-RA patients.
There are clear T-cell related immunological differences in patients with RA: loss of naïve T-cell, increased TLR4 + and combination of chemokine expression on CD4+ T-cell. We will therefore need to evaluate these biomarkers in larger number of patients with a high risk of progression towards RA to establish their definite value.
Previous studies from our laboratory have supported the concept that BM can actively participate in the pathogenesis of RA by TLR triggered B cell activation and distinct subpopulations of mature lymphocytes present in BM of osteoarthritis (OA) and RA patients. In the present study we analysed presence and functional activity of CD4(+)CD25(++) Tregs isolated from RA and OA bone marrow samples.
Bone marrow mononuclear cells (BMMC) were obtained from BM of RA and OA patients undergoing hip bone replacement surgery. The Tregs phenotype and number was assessed by FACS analysis immediately after isolation. After in vitro 72h culture of BMMC with or without IL-15, CD4(+)CD25(+++) Tregs were isolated by cell sorter for functional activity measured in proliferation assays and TNF and IL-17 production.
As reported previously, CD4(+)Foxp3(+) regulatory T cells are present in OA BM at higher number than in RA patients (4.7% vs 2.6% of CD4(+), p = 0.0075). Tregs from both OA and RA BM suppress CD4(+)CD25(-) T cells proliferation (respectively 52.4%; p = 0.0001 in OA and 52.3%; p = 0.0001 in RA). After stimulation of BMMC by IL-15, suppression was similar (48.5%; p = 0.00006 in OA and 47.3%; p = 0.002 in RA). Tregs from OA patients suppress TNF production by CD25(-) cells independently on IL-15 stimulation (respectively 26.3%; p = 0.0001 in control and 30.2%; p = 0.0001 after IL-15 stimulation). In contrast to OA, Tregs isolated from RA patients suppress TNF production only in control (30.7%; p = 0.00005) but not after IL-15 stimulation (79.8%; ns). IL-15 does not trigger increase of CD4(+)Foxp3(+)IL-17(+) cell number (respectively 0.5% vs 0.5% in OA and 0.3% vs 0.1% in RA), although stimulates IL-17 production by these cells (8904.8 vs 3525.5; p = 0.008 in OA and 7522.0 vs 3901.8; ns in RA).
Partial dependence of (overproduced in RA BM) IL-15 triggered Foxp3 expression, suggests that immunosuppression is an active process that takes part in bone marrow. However, lower number and limited functionality of Tregs present in RA bone marrow in comparison to OA may account for less efficient inhibition of T-cell activation in RA.
RA is a very heterogeneous disease, recently highlighted by distinct differences in the genetic contribution to APCA(+) and ACAP(-) disease suggesting two divergent pathogenic models, with different rates of progression and response to treatment. Synovial tissue studies showed that the cellular composition of synovial membrane is relatively consistent however the size of the inflammatory cell infiltrate is variable in direct relation to local levels of inflammation and sometimes highly organised with germinal centre like structures (GCL). The role of IL-7 as an essential factor in tissue organisation has recently been recognised and we hypothesize that the synovial tissue architecture (TA) may be driven by IL-7 expression in RA.
Synovial tissue biopsies were obtained during arthroscopy (n = 93, distributed between studies). Histology, IHC and two TaqMan gene-arrays were used. Tissues were classified as diffuse infiltration, aggregates and GCL-structures (defined B and T-cell zones). Clustering was used to analyse gene expression data.
IL-7 was detected with large variation between samples. There was association between% of IL-7(+) cells and local inflammation (n = 30, rho = 0.620, p<0.0001), synovitis score (rho = 0.556, p = 0.001) but only in the sublining layer, as well as with increased TA-complexity (p = 0.001), CD3(+) cells (rho = 0.599, p = 0.002) and CD20(+) (rho = 0.605, p = 0.001) but not with CD68(+) cells. IL-7 gene expression was measured in 29 biopsies. IL-7 mRNA expression was significantly higher in tissue presenting aggregates over diffuse infiltration (p = 0.019) and GCL structure (p = 0.037). A first TaqMan-arrays (48-genes) of 44 RA biopsies generated a 2 groups samples hierarchy clearly associated with TA (p = 0.002) separating genes associated with B-cell biology from those associated with stroma, T cells and macrophages. IL-7 surprisingly clustered with the B-cell-group however its expression was highly correlated with CD4 (rho = 0.520, p<0.0001). A second array (96-genes) of 29 biopsies also showed association with TA (p = 0.032) and clustered B-cell maturation genes (CD80, CD86, CD40L, RAG-1, BLIMP, AIOLOS, XBP-1, all Ig-genes) but not CD19/CD20 therefore suggesting a plasma cell specific signature. CD68 and CD4 were excluded from this gene signature. IL-7 was included in the plasma-cell cluster and was closely associated with CD38 (rho = 0.381, p = 0.042) and CD31 (Rho = 0.381, p = 0.042). A regulatory loop between IL-6/IL-7 was also highlighted by the gene dendrogram.
Our data suggest that IL-7 could orchestrate the synovial tissue architecture providing a niche for B-cell maturation. Blocking IL-7 may therefore be of therapeutic value as was recently demonstrated in several animal models of auto-immune diseases.
Interleukin (IL)-33 is a new member of the IL-1 family that exerts pleiotropic activities in innate and adaptive immunity. With its receptor ST2, they have newly emerged as key molecules strongly involved in several inflammatory and autoimmune disorders. Recent evidence suggests that the IL-33/ST2 axis is strongly involved in the pathophysiology of rheumatoid arthritis (RA). However in RA models, the role of IL-33 and its receptor is still controversial. We aimed at deciphering IL-33 mode of action after administration in an experimental model of RA, namely collagen-induced arthritis (CIA).
CIA was induced by immunisation of C57Bl/6 mice with type 2 collagen. IL-33 was ip administrated in CIA mice and cells were analysed by flow cytometry on day 28 after CIA induction.
We show a previously unshown dramatic inhibition of mouse collagen-induced arthritis (CIA) development after repeated administration of IL-33. This therapeutic effect was related to an enhanced type-2 immune response, including the expansion of eosinophils, Th2 cells, innate type 2 lymphoid cells (ILC2, defined as CD25(+) c-Kit(+) Lin(-) Sca-1(+)ST2L(+)) and an increase in Th2 cytokines levels in the serum of treated mice. Moreover, our work brings out the interplay between Treg and IL-33. Since IL-33 acts directly on Treg via ST2L, we showed that IL-33 treatment of CIA majors Treg frequency and increases the suppressive capacities of those cells. IL-33 also induces the emergence of a CD39(+/high) Treg population in a ST2L dependant manner.
In the light of our present study, IL-33 can exert powerful anti-inflammatory properties in CIA, integrating the establishment of a type-2 immune response, the expansion and the activation of Treg. Our study reveals an undescribed mechanism by which IL-33 inhibits arthritis development, thus updating and strengthening the crucial role of IL-33 in RA.
Women, having two X chromosomes, are more predisposed than men to autoimmune diseases. The X chromosome contains many genes linked to immunity which may contribute to this gender bias. In a mouse model, a duplication of the innate immunity X-linked toll like receptor 7 (Tlr7) gene has been shown to potentiate autoimmunity in males. We then proposed to investigate whether TLR7 gene and its neighboring paralog TLR8 could have variations in their copy numbers, contributing to the pathogenesis of rheumatoid arthritis (RA) in men.
A real-time quantitative PCR protocol was developed to assess copy number variation (CNV) of TLR7 and TLR8 gene, using sensitive and optimised ΔΔCt and standard curve methods, in DNA from peripheral blood mononuclear cells of 60 patients with RA (including 49 men) and 64 healthy controls (including 42 men). Among them, 31 men with RA and 18 healthy men were further screened for TLR7/8 CNV in 4 subpopulations: B cells, T cells, granulocytes and the depleted fraction of the former 3.
TLR7/8 copy numbers significantly increased with age in PBMCs from all men (P < 0.0001, Spearman's rank correlation test), whether they had RA or not. The increase had mean amplitude of 20%, spanning from the age of 20 until 80, according to the linear-regression-curve's best fit. This age-dependent and disease-independent CNV increase was also observed in all cell subsets. Interestingly, such increase was not observed in women, healthy or with RA, but rather an opposite trend.
For the first time we showed an increased CNV in TLR7 and TLR8 genes which is age and sex-mediated. Several hypotheses could explain such phenomenon. For example, somatically acquired duplications can affect some cells over time and result in an increase with age in men. In parallel, X chromosome monosomy, previously described in aging women could account for the opposite trend in those. Another explanation for men can be due to the presence of feminine microchimerism, arising from feto-maternal or twin sister exchange during in utero life, as previously described. Such cells would carry 2 X chromosomes and contribute to the increased pool of X-linked genes among XY host cells. Investigating these hypotheses would provide better understanding of age-associated X-linked genetic modifications and the role of the X chromosome in gender differences in health and disease. Abstract topicOther.
Rheumatoid arthritis (RA) and Psoriasis are chronic inflammatory diseases, both characterised by an immune cell infiltrate, notably Th17 cells and a migration of blood derived mononuclear cells that interact with fibroblasts (synoviocytes or skin fibroblasts) and this promotes cell proliferation. In this context, the aim of our work is to study and compare the interactions between fibroblasts and peripheral blood mononuclear cells (PBMC) on cytokine production with focus on the effect of cell contact on IL-17 pathway, in order to differentiate intracellular expression from secretion of IL-17.
A co-culture system with synoviocytes or skin fibroblasts and PBMC was developed. Fibroblasts were cultured overnight in 96 well plate and normal PBMC were seeded at a 1:5 ratio for 24 h, in the presence or not of activation with PHA. Transwell system was used to study cell-cell contact effect. Supernatants were collected and IL-6, IL-1β and IL-17 production measured by ELISA. Extracellular (CD3, CD4) and intracellular (IL-17) staining of PBMC was analysed by flow cytometry.
Interaction between synoviocytes or skin fibroblasts and PBMC was sufficient to induce IL-6 and IL-1β production. Flow cytometry analysis showed no difference in the percentage of Th17 cells in activated-PBMC alone or cultured with synoviocytes (3.21% vs. 4.70%, respectively, p = 0.42), indicating that Th17 differentiation requires only T cell activation but not cell-cell contact. Conversely IL-17 production was highly increased only in co-cultures (activated-PBMC in co-cultures (338.31 pg/ml) vs. activated-PBMC alone (26.49 pg/ml, p = 0.0002). Transwell experiments confirm that contact between fibroblasts and PBMC was critical for IL-17 secretion, as no detection was observed in transwell system. Therefore IL-17 secretion, in addition to T cell activation, requires interaction between cells. T cell activation is required to Th17 differentiation but the interaction between cells promotes IL-17 secretion.
The interaction between PBMC and fibroblasts (synoviocytes or skin fibroblasts) alone induces IL-6 and IL-1β production. T cell activation is required for Th17 differentiation but both PBMC activation and cell interaction with fibroblasts are needed to have high IL-17 secretion. This applies in particular to the pathogenesis of RA and psoriasis.
To determine human leucocyte antigen-class II (HLA-class II) (DRB1, DQB1, DQA1 and DPB1) alleles, haplotypes and shared epitopes associated with scleroderma (systemic sclerosis (SSc)) and its subphenotypes in a large multi-ethnic US cohort by a case-control association study.
1300 SSc cases (961 white, 178 black and 161 Hispanic subjects) characterised for clinical skin forms (limited vs diffuse), SSc-specific autoantibodies (anticentromere (ACA), anti-topoisomerase I (ATA), anti-RNA polymerase III (ARA), anti-U3 ribonucleoprotein (fibrillarin)) and others were studied using molecular genotyping. Statistical analyses in SSc itself by ethnicity, gender, skin type and autoantibodies were performed using exact logistic regression modelling for dominant, additive and recessive effects from HLA.
The strongest positive class II associations with SSc in white and Hispanic subjects were the DRB1*1104, DQA1*0501, DQB1*0301 haplotype and DQB1 alleles encoding a non-leucine residue at position 26 (DQB1 26 epi), while the DRB1*0701, DQA1*0201, DQB1*0202 haplotype and DRB1*1501 haplotype were negatively correlated and possibly protective in dominant and recessive models, respectively. These associations did not discriminate between limited and diffuse SSc. SSc in black subjects was associated with DRB1*0804, DQA1*0501, DQB1*0301 alleles. DPB1*1301 showed the highest odds ratio for ATA (OR = 14). Moreover, it showed no linkage disequilibrium or gene interaction with DR/DQ. ACA was best explained by DQB1*0501 and DQB1*26 epi alleles and ARA by DRB1*0404, DRB1*11 and DQB1*03 alleles in white and Hispanic subjects but DRB1*08 in black subjects.
These data indicate unique and multiple HLA-class II effects in SSc, especially on autoantibody markers of different subphenotypes.
To identify the main causes of morbidity and mortality in patients with antiphospholipid syndrome (APS) during a 5-year period and to determine clinical and immunological parameters with prognostic significance.
The clinical and immunological features of a cohort of 1000 patients with APS from 13 European countries who had been followed up from 1999 to 2004 were analysed.
200 (20%) patients developed APS-related manifestations during the 5-year study period. Recurrent thrombotic events appeared in 166 (16.6%) patients and the most common were strokes (2.4% of the total cohort), transient ischaemic attacks (2.3%), deep vein thromboses (2.1%) and pulmonary embolism (2.1%). When the thrombotic events occurred, 90 patients were receiving oral anticoagulants and 49 were using aspirin. 31/420 (7.4%) patients receiving oral anticoagulants presented with haemorrhage. 3/121 (2.5%) women with only obstetric APS manifestations at the start of the study developed a new thrombotic event. A total of 77 women (9.4% of the female patients) had one or more pregnancies and 63 (81.8% of pregnant patients) had one or more live births. The most common fetal complications were early pregnancy loss (17.1% of pregnancies) and premature birth (35% of live births). 53 (5.3% of the total cohort) patients died. The most common causes of death were bacterial infection (21% of deaths), myocardial infarction (19%) and stroke (13%). No clinical or immunological predictor of thrombotic events, pregnancy morbidity or mortality was detected.
Patients with APS still develop significant morbidity and mortality despite current treatment (oral anticoagulants or antiaggregants, or both).
The novel synergistic drug candidate CRx-102 comprises dipyridamole and low dose prednisolone and is in clinical development for the treatment of immunoinflammatory diseases. The purpose of this clinical study was to examine the efficacy and safety of CRx-102 in patients with hand osteoarthritis (HOA).
The study was conducted as a blinded, randomised, placebo-controlled trial at four centres in Norway. Eligibility criteria included being of age 30-70 years, at least one swollen and tender joint, a Kellgren-Lawrence (K-L) score of 2 or higher on radiographs, and a score of at least 30 mm pain on the Australian/Canadian Osteoarthritis Hand Index (AUSCAN) visual analogue pain scale (VAS). The primary endpoint was a reduction in pain from baseline to day 42 on the AUSCAN pain subscale. Two-sided p values for the differences in least squares (LS) means adjusted for baseline are presented.
The mean age of the 83 patients with HOA was 60 years and 93% were females. CRx-102 was statistically superior to placebo at 42 days for changes in AUSCAN pain (LS mean -14.2 vs -4.0) and for clinically relevant secondary endpoints (joint pain VAS (-18.6 vs -6.3), patient global VAS (-15.9 vs -4.2)) in the intention to treat population. The most frequently reported adverse event during the study was headache (52% in CRx-102 vs 15% in the placebo group).
The novel synergistic drug candidate CRx-102 demonstrated efficacy by statistically reducing pain compared to placebo in HOA and was generally well tolerated.
The heteropolymer technology was developed to remove pathogens from the circulation.
To evaluate the safety and tolerability of a single administration and to establish proof of principle for ETI-104 in normal healthy volunteers (NHV) and patients with systemic lupus erythematosus (SLE) METHODS: The drug was given intravenously to 11 NHV and six patients with SLE. Over 28 days, vital signs were noted, a haematological and chemical analysis of blood and urine was carried out, and adverse events were recorded. CR1 receptor numbers, the ability of antigen based heteropolymers to bind to red blood cells (RBCs), and the clearance of high avidity and total anti-dsDNA antibodies were measured by Farr assays and FACS analysis.
No safety measure differed significantly from normal in both groups; no drug related serious adverse events occurred. ETI-104 rapidly bound to RBCs in NHV and patients with SLE. Binding of the drug to RBCs of patients with SLE also caused a rapid reduction of circulating anti-dsDNA antibodies in the plasma 15 minutes after administration, with a maximum reduction of 55% (range 43-62). At 28 days statistically significant decreases were maintained in three patients, while in the other three the values had returned to baseline levels.
These clinical trials established the safety and the proof of principle of the new immunoconjugate ETI-104. This provides the basis for further development of this technology for numerous indications-for example, therapeutic options for autoimmune diseases or viral and bacterial infections.
To assess the efficacy and safety of golimumab over 104 weeks in patients with active ankylosing spondylitis.
At baseline, patients with active ankylosing spondylitis (n=356) were randomly assigned (1:1.8:1.8) to subcutaneous injections of placebo (group 1), golimumab 50 mg (group 2) or golimumab 100 mg (group 3) every 4 weeks. At week 16, patients in groups 1 and 2 with <20% improvement in total back pain and morning stiffness entered early escape to 50 or 100 mg, respectively. At week 24, patients still receiving placebo crossed over to golimumab 50 mg. Findings through week 24 were previously reported; those through week 104 are presented herein.
At week 104, 38.5%, 60.1% and 71.4% of patients in groups 1, 2 and 3, respectively, had at least 20% improvement in the Assessment in SpondyloArthritis international Society response criteria (ASAS20); 38.5%, 55.8% and 54.3% had an ASAS40 response and 21.8%, 31.9% and 30.7% were in ASAS partial remission. Mean Bath Ankylosing Spondylitis Disease Activity Index and Bath Ankylosing Spondylitis Functional Index scores were <3 at week 104 for all the treatment regimens. Golimumab safety through week 104 was similar to that through week 24.
Clinical response that was achieved by patients receiving golimumab through 24 weeks was sustained through 52 and 104 weeks. The golimumab safety profile appeared to be consistent with the known safety profile of tumour necrosis factor inhibitors.
Systemic sclerosis (SSc) is a generalised autoimmune disease, causing morbidity and a reduced life expectancy, especially in patients with rapidly progressive diffuse cutaneous SSc. As no proven treatment exists, autologous haematopoietic stem cell transplantation (HSCT) is employed as a new therapeutic strategy in patients with a poor prognosis. This study reports the effects on survival, skin and major organ function of HSCT in patients with severe diffuse cutaneous SSc.
A total of 26 patients were evaluated. Peripheral blood stem cells were collected using cyclophosphamide (4 g/m2) and rHu G-CSF (5 to 10 microg/kg/day) and were reinfused after positive CD34+ selection. For conditioning, cyclophosphamide 200 mg/kg was used.
After a median follow-up of 5.3 (1-7.5) years, 81% (n = 21/26) of the patients demonstrated a clinically beneficial response. The Kaplan-Meier estimated survival at 5 years was 96.2% (95% CI 89-100%) and at 7 years 84.8% (95% CI 70.2-100%) and event-free survival, defined as survival without mortality, relapse or progression of SSc, resulting in major organ dysfunction was 64.3% (95% CI 47.9-86%) at 5 years and 57.1% (95% CI 39.3-83%) at 7 years.
This study confirms that autologous HSCT in selected patients with severe diffuse cutaneous SSc results in sustained improvement of skin thickening and stabilisation of organ function up to 7 years after transplantation.