Renê Donizeti Ribeiro de Oliveira

Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis

Significance Methotrexate (MTX) is the first-line therapy for rheumatoid arthritis (RA). However, about 40% of patients are resistant to MTX. Furthermore, MTX resistance is only apparent after a prolonged continuous MTX treatment (>3 mo), by which time the disease of the nonresponders would have aggravated. Thus, there is a considerable unmet need for a biomarker to select MTX-resistant patients and place them immediately on alternative therapy. We found here that the low density of CD39 on peripheral regulatory T cells in RA patients is a rapid, convenient, and reliable (P < 0.01) biomarker for MTX resistance. Our findings also provide previously unrecognized information on aspects of immune regulation in RA and the mechanism of action of MTX. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39+CD4+CD25+FoxP3+ Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients.

Relationship between periodontitis and rheumatoid arthritis and the effect of non-surgical periodontal treatment

This study analyzed the association of periodontal disease (PD) and rheumatoid arthritis (RA). Seventy-five 35-60-year-old patients were assigned to 5 groups according to the presence (+) or not (-) of PD and RA and the treatment received (TR+) or not (TR-) for PD. Group 3 uses total prosthesis (TP). Clinical and laboratory evaluations were performed at baseline, 3 and 6 months of follow-up by probing pocket depth, bleeding on probing and plaque index for PD, HAQ, DAS28, SF-36 and laboratory: AAG, ESR, CRP for RA. Statistically significant differences for PD after 3 (p=0.0055) and after 6 months (p=0.0066) were obtained in Group 1 (RA+PD+TR+) and 2(RA+PD+TR-); significant reduction in the % of BOP after 6 months (p=0.0128) and significant reduction in the % of Pl after 3 (p=0.0128) and 6 months (p=0.0002) in Group 1. Statistically significant differences between Groups 1 and 3 (RA+TP) for DAS28 at baseline and after 3 months were observed, but not after 6 months. No other parameters for RA were significantly affected. The relationship between RA and PD disease activities is not clear, but the importance of periodontal treatment in the control of inflammation to avoid tooth extraction is evident.

CCR2 Expression in Neutrophils Plays a Critical Role in Their Migration Into the Joints in Rheumatoid Arthritis

Infiltration of neutrophils into the joints plays an important role in bone erosion and articular destruction in rheumatoid arthritis (RA). Neutrophil trafficking during inflammation is a process that involves activation of chemotactic receptors. Recent findings suggest that changes in chemotactic receptor patterns could occur in neutrophils under certain inflammatory conditions. The aim of this study was to evaluate the gain of responsiveness of neutrophils to CCL2 in RA patients and to assess the role of CCL2 in driving neutrophil infiltration into the joints.

Differential gene expression of peripheral blood mononuclear cells from rheumatoid arthritis patients may discriminate immunogenetic, pathogenic and treatment features

This study aimed to evaluate the association between the differential gene expression profiling of peripheral blood mononuclear cells of rheumatoid arthritis patients with their immunogenetic (human leucocyte antigen shared‐epitope, HLA‐SE), autoimmune response [anti‐cyclic citrullinated peptide (CCP) antibodies], disease activity score (DAS‐28) and treatment (disease‐modifying antirheumatic drugs and tumour necrosis factor blocker) features. Total RNA samples were copied into Cy3‐labelled complementary DNA probes, hybridized onto a glass slide microarray containing 4500 human IMAGE complementary DNA target sequences. The Cy3‐monocolour microarray images from patients were quantified and normalized. Analysis of the data using the significance analysis of microarrays algorithm together with a Venn diagram allowed the identification of shared and of exclusively modulated genes, according to patient features. Thirteen genes were exclusively associated with the presence of HLA‐SE alleles, whose major biological function was related to signal transduction, phosphorylation and apoptosis. Ninety‐one genes were associated with disease activity, being involved in signal transduction, apoptosis, response to stress and DNA damage. One hundred and one genes were associated with the presence of anti‐CCP antibodies, being involved in signal transduction, cell proliferation and apoptosis. Twenty‐eight genes were associated with tumour necrosis factor blocker treatment, being involved in intracellular signalling cascade, phosphorylation and protein transport. Some of these genes had been previously associated with rheumatoid arthritis pathogenesis, whereas others were unveiled for future research.

A majority of Brazilian patients with rheumatoid arthritis HLA-DRB1 alleles carry both the HLA-DRB1 shared epitope and anti-citrunillated peptide antibodies

The objective of the present study was to evaluate the contribution of the shared epitope (SE), the rheumatoid arthritis (RA) protection model, and the occurrence of anti-cyclic citrullinated peptide (anti-CCP) antibodies in RA patients from a genetically diverse population. One hundred and forty Brazilian RA patients and 161 matched controls were typed for HLA-DRB1 alleles using amplified DNA hybridized with sequence-specific oligonucleotide probes or primers. Patients were stratified according to the presence or absence of SE (DRB1*0401, *0404, *0405, *0101, *1001, and *1402), of the DERAA alleles (DRB1*0103, *0402, *1102, *1103, *1301, *1302, and *1304), and X (all other alleles). Anti-CCP antibodies were measured by ELISA. The combined frequency of SE-positive alleles was significantly greater (76.4 vs 23.6%, P < 0.0001) than the controls. The SE/SE and SE/X genotypes were over-represented (P < 0.0001, OR = 6.02) and DERAA/X was under-represented in RA patients (P < 0.001, OR = 0.49), whereas the frequencies of the SE/DERAA, X/X and X/DERAA genotypes were not significantly different from controls. The frequency of anti-CCP antibodies was higher in SE-positive patients than in SE-negative patients (64.6 vs 44.7%, P = 0.03; OR = 2.25). Although the Brazilian population is highly miscegenated, the results of this study support the findings observed in most genetically homogeneous populations with RA; however, they are not mutually exclusive but rather complementary. The participation of DRB1-DERAA alleles in protection against RA was also observed (OR = 0.4; 95%CI = 0.23-0.68).

Share Epitope, Citrullinated Cyclic Peptide Antibodies and Smoking in Brazilian Rheumatoid Arthritis Patients

The events involved in the pathogenesis of rheumatoid arthritis (RA) still remain unclear, but certainly the etiology is multifactorial. Shared epitope (SE) of HLADRβ1 is the most important genetic risk factor. Environmental risk factors are less understood. Smoking is a candidate, associated with the rising of citrullinated cyclic peptide antibodies (anti-CCP). Anti-CCP antibodies are highly specific for RA. In this study, we investigated whether the association between anti-CCP production and smoking was influenced by carriage of SE in a highly miscegenated population of patients with RA. One hundred Brazilian patients were inquired about cigarette smoking. For all of them, DNA for HLA typing and serum to anti-CCP antibodies quantification were obtained. Forty-two were smokers and 58 were nonsmokers. The SE was present in 61 patients and the anti-CCP was positive in 71 patients. We found that, among smokers, 25 were SE-positive, 22 presented with anti-CCP and 3 without anti-CCP, and 17 were SE-negative, 9 presented with anti-CCP and 8 without anti-CCP (OR 6.5, 95% CI 1.40 to 30.20). These results suggest that environmental factors contribute to the raising of anti-CCP in individuals with HLA background to RA, smoking being a strong candidate.

Differential Gene Expression Profiles May Differentiate Responder and Nonresponder Patients with Rheumatoid Arthritis for Methotrexate (MTX) Monotherapy and MTX plus Tumor Necrosis Factor Inhibitor Combined Therapy

Objective. We aimed to evaluate whether the differential gene expression profiles of patients with rheumatoid arthritis (RA) could distinguish responders from nonresponders to methotrexate (MTX) and, in the case of MTX nonresponders, responsiveness to MTX plus anti-tumor necrosis factor-α (anti-TNF) combined therapy. Methods. We evaluated 25 patients with RA taking MTX 15–20 mg/week as a monotherapy (8 responders and 17 nonresponders). All MTX nonresponders received infliximab and were reassessed after 20 weeks to evaluate their anti-TNF responsiveness using the European League Against Rheumatism response criteria. A differential gene expression analysis from peripheral blood mononuclear cells was performed in terms of hierarchical gene clustering, and an evaluation of differentially expressed genes was performed using the significance analysis of microarrays program. Results. Hierarchical gene expression clustering discriminated MTX responders from nonresponders, and MTX plus anti-TNF responders from nonresponders. The evaluation of only highly modulated genes (fold change > 1.3 or < 0.7) yielded 5 induced (4 antiapoptotic and CCL4) and 4 repressed (4 proapoptotic) genes in MTX nonresponders compared to responders. In MTX plus anti-TNF non-responders, the CCL4, CD83, and BCL2A1 genes were induced in relation to responders. Conclusion. Study of the gene expression profiles of RA peripheral blood cells permitted differentiation of responders from nonresponders to MTX and anti-TNF. Several candidate genes in MTX non-responders (CCL4, HTRA2, PRKCD, BCL2A1, CAV1, TNIP1, CASP8AP2, MXD1, and BTG2) and 3 genes in MTX plus anti-TNF nonresponders (CCL4, CD83, and BCL2A1) were identified for further study.

Macrophage migration inhibitory factor drives neutrophil accumulation by facilitating IL-1β production in a murine model of acute gout.

This study evaluated the role of macrophage migration inhibitory factor in inflammation caused by monosodium urate crystals. The concentration of macrophage migration inhibitory factor was increased in synovial fluid of patients with acute gout, and there was a positive correlation between intra‐articular macrophage migration inhibitory factor and IL‐1β concentrations. In mice, the injection of monosodium urate crystals into the knee joint increased the levels of macrophage migration inhibitory factor in macrophages and in inflamed tissue. The injection of recombinant macrophage migration inhibitory factor into the joint of mice reproduced the inflammatory response observed in acute gout, including histologic changes, the recruitment of neutrophils, and increased levels of IL‐1β and CXCL1. Importantly, the accumulation of neutrophils and the amount IL‐1β in the joints were reduced in macrophage migration inhibitory factor‐deficient mice when injected with monosodium urate crystals. We observed a similar effect when we blocked macrophage migration inhibitory factor with (S,R)‐3‐(4‐hydroxyphenyl)‐4,5‐dihydro‐5‐isoxazole acetic acid or anti‐macrophage migration inhibitory factor. In addition, the blockade of IL‐1R and CXCR2 reduced recombinant macrophage migration inhibitory factor‐induced neutrophil recruitment. Mechanistically, recombinant macrophage migration inhibitory factor is important for the synthesis of il1β mRNA in vivo and in isolated macrophages. Altogether, macrophage migration inhibitory factor promotes neutrophil accumulation and is important for IL‐1β production, which are 2 crucial events contributing to the pathogenesis of acute gout.

Postural control parameters in elderly female fallers and non-fallers diagnosed or not with knee osteoarthritis.

OBJECTIVES To compare stabilometric parameters of elderly female fallers and non-fallers associated or not with knee osteoarthritis (OA). METHODS Fifty-six elderly female fallers and non-fallers diagnosed or not with unilateral or bilateral knee OA were divided into the following groups: FOA (n = 10), elderly female fallers with knee OA; FNOA (n = 11), elderly female fallers without knee OA; NFOA (n = 14), elderly female non-fallers with knee OA; and NFNOA (n = 21), elderly female non-fallers without knee OA. For analyzing semi-static balance on a force platform with the elderly females standing, the following parameters were assessed in four conditions: center of pressure (COP), anterior-posterior and mediolateral displacements (APD and MLD, respectively); and COP anterior-posterior and mediolateral displacement velocities (APV and MLV, respectively). The following conditions were assessed: 1) standing on a firm wooden surface with eyes open (WSEO); 2) standing on a firm wooden surface with eyes closed (WSEC); 3) standing on a foam surface with eyes open (FSEO); 4) standing on a foam surface with eyes closed (FSEC). RESULTS The elderly females with knee OA showed greater APD in all four conditions assessed (P < 0.05), while the elderly female fallers showed greater MLD (P < 0.05). No difference between the groups was observed for APV and MLV (P > 0.05). CONCLUSIONS Knee OA per se increases APD of the COP, while the history of falls, regardless of the presence of knee OA, hinders postural control in the ML direction.

Evaluation of postural control and quality of life in elderly women with knee osteoarthritis

OBJECTIVE To assess the balance in dynamic tasks and in the quality of life in elderly women with and without knee osteoarthritis. METHODS Elderly women were divided into Group 1 (n = 12), consisting of participants with bilateral knee osteoarthritis (Kellgreen-Lawrence grade 1 and 2), and Group 2 (n = 12), consisting of controls. A force plate (EMG System do Brazil) was used to assess postural sway in dynamic tasks, whereas the quality of life was assessed by using the WHOQOL-Bref questionnaire. RESULTS Students t-test showed no statistical difference during sitting down and standing up from the chair (p > 0.05). However, stair ascent revealed difference in displacement speed (p < 0.05), whereas stair descent showed differences in both displacement speed and amplitude (p < 0.05). In the questionnaire, Group 1 showed values lower than those in the control group regarding physical domain (p < 0.05). CONCLUSION Elderly women with knee osteoarthritis seemed to have more difficulty on stair descent task and had perception of worst physical domain. These findings were observed in OA group, even in the early stages of the disease, which shows the importance of even earlier interventions.