Markers of progression to rheumatoid arthritis: discriminative value of the new ACR/EULAR rheumatoid arthritis criteria in a Portuguese population with early polyarthritis.

OBJECTIVES: Our goal was to test the performance of the new American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria for the classification of rheumatoid arthritis (RA) in a cohort of patients with very recent onset polyarthritis. PATIENTS: Untreated polyarthritis patients with less than 6 weeks of duration were enrolled. All patients were followed-up in order to establish a definitive diagnosis. RESULTS: Thirty-seven patients were included. During the follow up 57% of the patients evolved to RA. The median age of the RA-group patients was simi­lar to the median age of the non-RA group [median (IQR) 47 (31-58.5) vs 43 (34-69) years, p=0.74]. At the initial visit the DAS 28 in the RA group was significantly higher than in the non-RA group, as well as the visual analogue scale (VAS), the HAQ and the number of swollen joints. Among the 21 RA patients, 43% presented RF and 28.6% presented anti-citrullinated protein antibody (ACPA) in the first visit. RF and ACPA were not detectable in any of the patients who did not evolve to RA. According to the new ACR/EULAR criteria, the mean total score of the RA group at baseline was significantly higher than the non-RA group [median (IQR) 6 (4.5-8) vs 4.5 (2.2-6), p=0.007]. CONCLUSION: In our cohort high DAS28, swollen joint count, VAS and HAQ and the presence of RF or ACPA were eventually associated with the evolution into RA. The new ACR/EULAR criteria for the classification of RA seem to perform well in very early RA.

Alterations on peripheral blood B-cell subpopulations in very early arthritis patients.

OBJECTIVE: To characterize circulating B-cell subpopulations of arthritis patients with <6 weeks of disease duration. METHODS: Peripheral blood samples were collected from very early untreated polyarthritis patients, with <6 weeks of disease duration, for flow cytometric evaluation of B-cell subpopulations. Samples from patients who were later diagnosed as RA [very early RA (VERA)] were also collected 4-6 weeks after starting a low dose of prednisone (5-10 mg) and 4 months after reaching the minimum effective dose of MTX. A matched healthy group was used as a control. RESULTS: VERA patients have a lower percentage of total peripheral blood memory B cells (CD19(+)CD27(+)) and a significant decrease in the frequency of circulating pre-switch memory B cells (CD19(+)IgD(+)CD27(+)) as compared with controls. Therapy with corticosteroids or MTX was unable to restore the normal frequencies of these B-cell subpopulations. A significant decrease in peripheral pre-switch memory B cells is equally observed in other early arthritis patients. Furthermore, no significant differences are found in the frequencies of CD4(+) and CD8(+) T cells in all patient groups. CONCLUSIONS: In very early polyarthritis patients, there is a reduction in circulating pre-switch memory B cells. The reasons that may account for this effect are still unknown. Short-term corticosteroids and MTX do not seem to have a direct effect on circulating B-cell subpopulations in VERA patients.

B cells from the bench to the clinical practice.

B cells develop in bone marrow and undergo antigen-induced activation and terminal differentiation in germinal centres of secondary lymphoid organs. Each B cell is a clone which means that an individual B cell has a unique genetic code and produces only one type of antibody when stimulated by antigen being able to multiply itself and originate several B cells with the same antigen specificity (clonal selection theory). However their important role in adaptive immune responses is supported by the remarkable capacity of recognizing an unlimited array of antigens due to mechanisms of antibody diversity such as V(D)J recombination class switching and somatic hypermutation. B cells can also function as antigen presenting cells that can activate T cells improving the effectiveness of the immune response. Immune B cell tolerance surveillance through clonal deletion anergy and receptor editing is also necessary to avoid pathological conditions like autoimmune diseases. B cells can contribute to autoimmunity by autoantibody production cytokine synthesis antigen presentation T cell activation and ectopic lymphogenesis.

Contribution for new genetic markers of rheumatoid arthritis activity and severity: sequencing of the tumor necrosis factor-alpha gene promoter.

The objective of this study was to assess whether clinical measures of rheumatoid arthritis activity and severity were influenced by tumor necrosis factor-alpha (TNF-alpha) promoter genotype/haplotype markers. Each patient's disease activity was assessed by the disease activity score using 28 joint counts (DAS28) and functional capacity by the Health Assessment Questionnaire (HAQ) score. Systemic manifestations, radiological damage evaluated by the Sharp/van der Heijde (SvdH) score, disease-modifying anti-rheumatic drug use, joint surgeries, and work disability were also assessed. The promoter region of the TNF-alpha gene, between nucleotides -1,318 and +49, was sequenced using an automated platform. Five hundred fifty-four patients were evaluated and genotyped for 10 single-nucleotide polymorphism (SNP) markers, but 5 of these markers were excluded due to failure to fall within Hardy-Weinberg equilibrium or to monomorphism. Patients with more than 10 years of disease duration (DD) presented significant associations between the -857 SNP and systemic manifestations, as well as joint surgeries. Associations were also found between the -308 SNP and work disability in patients with more than 2 years of DD and radiological damage in patients with less than 10 years of DD. A borderline effect was found between the -238 SNP and HAQ score and radiological damage in patients with 2 to 10 years of DD. An association was also found between haplotypes and the SvdH score for those with more than 10 years of DD. An association was found between some TNF-alpha promoter SNPs and systemic manifestations, radiological progression, HAQ score, work disability, and joint surgeries, particularly in some classes of DD and between haplotypes and radiological progression for those with more than 10 years of DD.

Identification of caspase-10 in human neutrophils and its role in spontaneous apoptosis.

In the present study, we investigated the molecular mechanisms of spontaneous and tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis of human polymorphonuclear neutrophils (PMN). Whereas TNF-alpha-mediated apoptosis was almost absent in the presence of the caspase-8 inhibitor Z-Ac-Ala-Glu-Val-Asp-7-fluoromethyl ketone (Z-AEVD-FMK), the inhibitor had no effect on spontaneous apoptosis, suggesting that spontaneous apoptosis was independent of caspase-8. Subsequently, we identified different isoforms of caspase-10 in human PMN and found high expression of caspase-10/b and/or -10/d and low expression of caspase-10/a and -10/c at the mRNA level. At the protein level, freshly isolated PMN showed high expression of caspase-10/b and -10/d as well as moderate expression of caspase-10/a and -10/c. Upon spontaneous apoptosis, caspase-10/b was down-regulated, which was accompanied by the appearance of a specific 47-kDa caspase-10/b cleavage product and an increased caspase-10 activity. In contrast, no down-regulation of caspase-10/a, -10/c, or -10/d was observed, suggesting that spontaneous apoptosis was associated with a differential activation of caspase-10/b. This was confirmed by the finding that spontaneous apoptosis was inhibited in the presence of Z-Ile-Glu-Thr-Asp (Z-IETD)-FMK, which blocks caspase-10. However, no down-regulation of caspase-10 isoforms was observed in the presence of TNF-alpha, suggesting that caspase-10 was not involved in TNF-alpha-induced apoptosis. Taken together, our study demonstrates that spontaneous and TNF-alpha-mediated apoptosis of PMN have different molecular requirements. Whereas TNF-alpha-mediated apoptosis depends on the activation of caspase-8, spontaneous apoptosis requires the activation of caspase-10/b. This finding may reveal that PMN apoptosis in different (patho-) physiological settings results from distinct molecular mechanisms.

A role for apoptosis in the control of neutrophil homeostasis in the circulation: insights from CD18-deficient mice.

The control of neutrophil turnover in the circulation is a key event in homeostasis and inflammation. Using CD18- deficient (CD18(-/-)) mice that show a 19-fold increase of blood neutrophil counts when compared with wild-type animals (CD18(+/+)), we found that apoptosis of peripheral neutrophils was significantly reduced from 27.4% in the wild-type to 4.8% in CD18(-/-) mice within 4 hours after isolation as measured by analysis of DNA content. This was confirmed by detecting CD16 expression, nuclear morphology, and internucleosomal DNA degradation. In contrast, no difference in apoptosis was observed in neutrophils derived from the bone marrow. Neutrophilia and delayed neutrophil apoptosis were also present in CD18(-/-)/interleukin 6 (IL-6(-/-)) double knockout mice. Moreover, plasma of CD18(-/-) mice was not able to delay apoptosis of CD18(+/+) neutrophils and plasma of CD18(+/+) mice did not augment apoptosis of CD18(-/-) neutrophils. However, CD18(-/-) neutrophils revealed an up-regulation of the antiapoptotic gene bcl-X(l) and a down-regulation of the proapoptotic gene bax-alpha compared with CD18(+/+) neutrophils suggesting that this delayed apoptosis. Accordingly, down-regulation of Bax-alpha using antisense technique delayed apoptosis and prolonged neutrophil survival. The replacement of the hematopoietic system of CD18(+/+) mice by a 1:1 mixture of CD18(+/+) and CD18(-/-) hematopoietic cells abolished the delay of apoptosis in peripheral CD18(-/-) neutrophils and prevented neutrophilia. Altogether, this suggests that a delay of neutrophil apoptosis in CD18(-/-) mice causes an alteration of neutrophil homeostasis, which may induce the massive increase of peripheral neutrophil counts. Thus, apoptosis seems to be critically involved in the control of neutrophil turnover in the circulation.