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Objectives: A subset of human circulating FoxP3+ regulatory T cells expresses CD39 (cTreg39+) and hydrolyses pro-inflammatory adenine nucleotides released at inflammatory foci, rendering the anti-inflammatory agent adenosine. Methotrexate (MTX), inhibiting ATIC, enhances the extrusion of adenine nucleotides and may help Treg39+ cells control inflammation. Therefore, we examined the relation of cTreg39+ cells with the effect of MTX in early Rheumatoid Arthritis (eRA)., Methods: Freshly isolated peripheral blood lymphocytes from 98 untreated eRA patients and 98 healthy controls (HC) were examined by cytometry. Twelve months (12m) after initiating MTX, 82 patients were clinically re-evaluated and cytometry was repeated in 40 of them. The effect of MTX on Treg cell potency was assessed in Treg/Tresp cocultures., Results: The baseline (0m) cTreg39+ cell frequency was elevated in eRA above HC levels. Patients who reached low disease activity at 12 months (12m-LDA, DAS28-ESR≤ 3.2, n = 51) had presented with a significantly higher 0m cTreg39+ frequency vs those who did not (n = 31). The 0m cTreg39+ cutoff for attaining 12 m-LDA was 42.0% (Sensitivity=90.4%/Specificity=96.8%). At 12m, the cTreg39+ frequency was no longer elevated but its association with disease activity remained: it was still significantly higher in patients who had reached LDA vs those who had not. In vitro, MTX augmented the Treg39+ cell potency but had no effect on Treg39- cells., Conclusion: MTX cooperates with Treg39+ cells and the baseline cTreg39+ frequency predicts the response to MTX in eRA. In addition, the transiently elevated baseline cTreg39+ frequency in eRA may provide a slot for prompt MTX initiation., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: CD4+CXCR5+PD-1hi follicular helper T (Tfh) cells dwell in the germinal centers (GCs) of lymphoid organs and participate in Rheumatoid Arthritis (RA) pathogenesis; the frequency of their circulating counterparts (cTfh-frequency) is expanded in RA and correlates with the pool of GC Tfh cells. Our objective was to study the effect of abatacept (ABT) or TNF blockers (TNFb) on the cTfh-frequency in RA., Methods: Peripheral blood was drawn from seropositive-longstanding RA patients chronically receiving csDMARDS (n = 45), TNFb (n = 59), or ABT (n = 34), and healthy controls (HC) (n = 137). Also, patients with an incomplete response to csDMARDS (n = 41) who initiated TNFb (n = 19) or ABT (n = 22), were studied at 0 and 12 months. The cTfh-frequency was examined by cytometry., Results: As compared with HC, an increased cTfh-frequency was seen in seropositive-longstanding RA chronically receiving csDMARDs or TNFb but not ABT. After escalating from csDMARDs, the cTfh-frequency did not vary in patients who were given TNFb but decreased to HC levels in those given ABT. In the ABT group, the baseline cTfh-frequency was higher for patients who attained 12M remission (12Mr), vs those who remained active (12Ma): 0m cutoff for remission >0.38% (Sens. 92%, Sp. 90%), OR 25.3. Conversely, in the TNFb group, the baseline cTfh-frequency was lower for 12Mr vs 12Ma: 0m cutoff for non-remission >0.44% (Sens. 67%, Sp. 90%), OR 8.5., Conclusion: ABT but not TNFb, is able to curtail the cTfh-frequency in RA. A higher baseline cTfh-frequency predicts a good response to ABT but a poor response to TNFb., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In rheumatoid arthritis (RA), the identification of biomarkers to adjust treatment intensity and to correctly diagnose the disease in early stages still constitutes a challenge and, as such, novel biomarkers are needed. We proposed that autoantibodies (aAbs) against CD26 (DPP4) might have both etiological importance and clinical value. Here, we perform a prospective study of the potential diagnostic power of Anti-CD26 aAbs through their quantification in plasmas from 106 treatment-naïve early and undifferentiated AR. Clinical antibodies, Anti-CD26 aAbs, and other disease-related biomarkers were measured in plasmas obtained in the first visit from patients, which were later classified as RA and non-RA according to the American College of Rheumatology criteria. Two different isotype signatures were found among ten groups of patients, one for Anti-CD26 IgA and other for Anti-CD26 IgG and IgM isotypes, both converging in patients with arthritis (RA and Unresolved Undifferentiated Arthritis: UUA), who present elevated levels of all three isotypes. The four UUA patients, unresolved after two years, were ACPA and rheumatic factor (RF) negatives. In the whole cohort, 51.3% of ACPA/RF seronegatives were Anti-CD26 positives, and a similar frequency was observed in the seropositive RA patients. Only weak associations of the three isotypes with ESR, CRP and disease activity parameters were observed. Anti-CD26 aAbs are present in treatment-naïve early arthritis patients, including ACPA and RF seronegative individuals, suggestive of a potential pathogenic and/or biomarker role of Anti-CD26 aAbs in the development of rheumatic diseases., (© 2022. The Author(s).)

The identification of "trained immunity/tolerance" in myeloid cells has changed our perception of the performance of monocytes and macrophages during inflammatory and immune responses. Pemetrexed (PMX) and methotrexate (MTX) are blockers of the one-carbon metabolism (OCM) and commonly used therapeutic agents in cancer and rheumatoid arthritis (RA). We have previously showed that MTX promotes trained immunity in human macrophages. In the present manuscript, we have assessed the anti-inflammatory effects of PMX and MTX and found that OCM blockers alter the functional and gene expression profile of human macrophages and that OCM blockade reprograms macrophages towards a state of lipopolysaccharide (LPS) tolerance at the signaling and functional levels. Moreover, OCM blockade reduced macrophage LPS responsiveness by impairing the expression of membrane-bound and soluble CD14 (sCD14). The therapeutic relevance of these results was later confirmed in early RA patients, as MTX-responder RA patients exhibit lower sCD14 serum levels, with baseline sCD14 levels predicting MTX response. As a whole, our results demonstrate that OCM is a metabolic circuit that critically mediates the acquisition of innate immune tolerance and positions sCD14 as a valuable tool to predict MTX response in RA patients., Competing Interests: IG-A reports personal fees from Lilly and Sanofi; personal fees and non-financial support from BMS; personal fees and non-financial support from AbbVie; research support, personal fees, and non-financial support from Roche Laboratories; and non-financial support from MSD, Pfizer, and Novartis, not related to the submitted work. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fuentelsaz-Romero, Barrio-Alonso, García Campos, Torres Torresano, Muller, Triguero-Martínez, Nuño, Villalba, García-Vicuña, Jansen, Miranda-Carús, González-Álvaro and Puig-Kröger.)

Objective: The protagonism of regulatory B cells seems to vary along the course of the disease in murine models of inflammatory conditions. Decreased numbers of circulating regulatory CD19+CD24hiCD38hi transitional (cTr) B cells have been described in patients with long-standing RA, thus our objective was to examine the frequency and evolution of cTr B cells in the peripheral blood of early RA (ERA) patients., Methods: Freshly isolated peripheral blood mononuclear cells from 48 steroid- and DMARD-naïve ERA patients with a disease duration of <24 weeks and 48 healthy controls (HCs) were examined by flow cytometry. Co-cultures of isolated memory B cells were established with autologous T cells in the absence or presence of Tr B cells., Results: As compared with HCs, ERA patients demonstrated an increased frequency of cTr B cells. cTr B cells of ERA patients and HCs displayed an anti-inflammatory cytokine profile and were able to downregulate T cell IFN-γ and IL-21 production, together with ACPA secretion in autologous B/T cell co-cultures. Basal frequencies of cTr B cells above the median value observed in HCs were associated with a good EULAR response to MTX at 12 months [relative risk 2.91 (95% CI 1.37, 6.47)]. A significant reduction of cTr B cells was observed 12 months after initiating MTX, when the cTr B cell frequency was no longer elevated but decreased, and this was independent of the degree of clinical response or the intake of prednisone., Conclusion: An increased frequency of regulatory cTr B cells is apparent in untreated ERA and the baseline cTr B cell frequency is associated with the clinical response to MTX at 12 months., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To describe the frequency and causes for the presence of a halo sign on the ultrasound of patients without a diagnosis of GCA., Methods: In total, 305 patients with temporal artery colour Doppler ultrasound showing the presence of halo sign (intima-media thickness ≥0.34 mm for temporal arteries [TAs] and ≥1 mm for axillary arteries) were included, and their medical records were reviewed. The clinical diagnosis based on the evolution of the patient over at least one year was established as the definitive diagnosis., Results: Fourteen of the 305 (4.6%) patients included showed presence of the halo sign without final diagnosis of GCA: 12 patients in the TAs (86%), and two patients with isolated AAs involvement (14%). Their diagnoses were PMR (n = 4, 29%); atherosclerosis (n = 3, 21%); and non-Hodgkin lymphoma type T, osteomyelitis of the skull base, primary amyloidosis associated with multiple myeloma, granulomatosis with polyangiitis, neurosyphilis, urinary sepsis and narrow-angle glaucoma (n = 1 each, 7%)., Conclusion: The percentage of halo signs on the ultrasound of patients without GCA is low, but it does exist. There are conditions that may also show the halo sign (true positive halo sign), and we must know these and always correlate the ultrasound findings with the patient's clinic records., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: A novel population of B helper cells, phenotypically CD4+CXCR5-PD-1hi, has been described in the synovial tissues and peripheral blood of seropositive RA patients, and termed 'peripheral helper T' (Tph) cells. Contrary to CD4+CXCR5+PD-1hi follicular helper T (Tfh), Tph cells are not located in lymphoid organs but accumulate in inflamed tissues. Our objective was to study the frequency of circulating Tph (cTph) and circulating Tfh cell counterparts (cTfh) in patients with early RA (eRA)., Methods: Freshly isolated peripheral blood mononuclear cells from 56 DMARD-naïve eRA patients and 56 healthy controls were examined by flow cytometry. Autologous cocultures of naïve or memory B cells were established with isolated peripheral blood Tph or Tfh cells., Results: Seropositive (RF+ and/or ACPA+, n = 38) but not seronegative eRA patients (n = 18) demonstrated increased frequencies and absolute numbers of cTph and cTfh cells. cTph but not cTfh cells expressed CCR2. Those eRA patients who experienced a significant clinical improvement at 12 months demonstrated a marked decrease of their cTph cell numbers whereas their cTfh cell numbers remained unchanged. Both isolated Tph and isolated Tfh cells were able to induce maturation of memory B cells, whereas only Tfh cells could differentiate naïve B cells., Conclusion: Two populations of PD-1hiCD4 T cells with distinct phenotype and B cell helping capacity are increased in the peripheral blood of seropositive eRA patients. Whereas cTph cells are present only in patients with an active disease, cTfh cells seem to be constitutively elevated., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Our objective was to study the frequency of circulating CD19+CD24hiCD38hi B cells (Breg) in AS patients. To this end, peripheral blood was drawn from AS patients naïve for TNF blockers (AS/nb) (n = 42) and healthy controls (HC) (n = 42). Six patients donated blood for a second time, 6 months after initiating treatment with anti-TNFα drugs. After isolation by Ficoll-Hypaque, PBMCs were stained with antibodies to CD3, CD4, CD19, CD24, and CD38, and examined by cytometry. For functional studies, total CD19+ B cells were isolated from PBMCs of 3 HC by magnetical sorting. Breg-depleted CD19+ B cells were obtained after CD19+CD24hiCD38hi B cells were removed from total CD19+ cells by cytometry. Total CD19+ B cells or Breg-depleted CD19+ B cells were established in culture and stimulated through their BCR. Secretion of IFNγ was determined by ELISA in culture supernatants. When compared with HC, AS/nb patients demonstrated a significantly increased frequency of Breg cells, which was independent of disease activity. Anti-TNFα drugs induced a significant reduction of circulating Breg numbers, which were no longer elevated after six months of treatment. Functional in vitro studies showed that the secretion of IFNγ was significantly higher in Breg-depleted as compared with total CD19+ B cells, indicating that Breg can downmodulate B cell pro-inflammatory cytokine secretion. In summary, an increased frequency of circulating CD19+CD24hiCD38hi B cells is observed in AS/nb patients, that is not related with disease activity; anti-TNFα drugs are able to downmodulate circulating Breg numbers in AS.

Objectives: Methotrexate (MTX) functions as an antiproliferative agent in cancer and an anti-inflammatory drug in rheumatoid arthritis (RA). Although macrophages critically contribute to RA pathology, their response to MTX remains unknown. As a means to identify MTX response markers, we have explored its transcriptional effect on macrophages polarised by GM-CSF (GM-MØ) or M-CSF (M-MØ), which resemble proinflammatory and anti-inflammatory macrophages found in RA and normal joints, respectively., Methods: The transcriptomic profile of both human macrophage subtypes exposed to 50 nM of MTX under long-term and short-term schedules were determined using gene expression microarrays, and validated through quantitative real time PCR and ELISA. The molecular pathway involved in macrophage MTX-responsiveness was determined through pharmacological, siRNA-mediated knockdown approaches, metabolomics for polyglutamylated-MTX detection, western blot, and immunofluorescence on RA and normal joints., Results: MTX exclusively modulated gene expression in proinflammatory GM-MØ, where it influenced the expression of 757 genes and induced CCL20 and LIF at the mRNA and protein levels. Pharmacological and siRNA-mediated approaches indicated that macrophage subset-specific MTX responsiveness correlates with thymidylate synthase (TS) expression, as proinflammatory TS + GM-MØ are susceptible to MTX, whereas anti-inflammatory TS low/- M-MØ and monocytes are refractory to MTX. Furthermore, p53 activity was found to mediate the TS-dependent MTX-responsiveness of proinflammatory TS + GM-MØ. Importantly, TS and p53 were found to be expressed by CD163 + /TNFα + GM-CSF-polarised macrophages from RA joints but not from normal synovium., Conclusions: Macrophage response to MTX is polarisation-dependent and determined by the TS-p53 axis. CCL20 and LIF constitute novel macrophage markers for MTX responsiveness in vitro., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Chemokine receptor CCR7 directs mature dendritic cells (mDCs) to secondary lymph nodes where these cells regulate the activation of T cells. CCR7 also promotes survival in mDCs, which is believed to take place largely through Akt-dependent signaling mechanisms. We have analyzed the involvement of the AMP-dependent kinase (AMPK) in the control of CCR7-dependent survival. A pro-apoptotic role for AMPK is suggested by the finding that pharmacological activators induce apoptosis, whereas knocking down of AMPK with siRNA extends mDC survival. Pharmacological activation of AMPK also induces apoptosis of mDCs in the lymph nodes. Stimulation of CCR7 leads to inhibition of AMPK, through phosphorylation of Ser-485, which was mediated by G(i)/Gβγ, but not by Akt or S6K, two kinases that control the phosphorylation of AMPK on Ser-485 in other settings. Using selective pharmacological inhibitors, we show that CCR7-induced phosphorylation of AMPK on Ser-485 is mediated by MEK and ERK. Coimmunoprecipitation analysis and proximity ligation assays indicate that AMPK associates with ERK, but not with MEK. These results suggest that in addition to Akt-dependent signaling mechanisms, CCR7 can also promote survival of mDCs through a novel MEK1/2-ERK1/2-AMPK signaling axis. The data also suggest that AMPK may be a potential target to modulate mDC lifespan and the immune response., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Introduction: Circulating CD4 T cells expressing CXCR5, ICOS and/or PD-1 are counterparts of follicular helper T cells (Tfh). There are three subpopulations of circulating Tfh (cTfh): CXCR5 + CXCR3 + CCR6- (Tfh-Th1), CXCR5 + CXCR3-CCR6- (Tfh-Th2) and CXCR5 + CXCR3-CCR6+ (Tfh-Th17). Our objective was to study the B cell helping capacity of cTfh subsets, and examine their frequency in Rheumatoid Arthritis (RA) patients, together with the frequency of circulating plasmablasts (CD19 + CD20-CD38high)., Methods: Peripheral blood was drawn from RA patients with active disease (RA-a, DAS28 >2.6) (n = 17), RA in remission (RA-r, DAS28 <2.6) (n = 17) and healthy controls (HC) (n = 34). cTfh and plasmablast frequencies were determined by flow cytometry. Cocultures of sorted CD4 + CXCR5+ T cell subpopulations were established with autologous CD19 + CD27- naïve B cells of HC, and concentrations of IgG, A and M were measured in supernatants., Results: Isolated Tfh-Th2 and Tfh-Th17 but not Tfh-Th1 cells, induced naïve B cells to secrete IgG and IgA. The frequency of CXCR5+ cells gated for CD4+ T cells was not different among HC, RA-a and RA-r. In contrast, both RA-a and RA-r patients demonstrated an increased frequency of CD4 + CXCR5 + ICOS+ T cells and augmented (%Tfh-Th2 + %Tfh-Th17)/%Tfh-Th1 ratio as compared with HC. In addition, RA-a but not RA-r patients, showed an increased frequency of circulating plasmablasts., Conclusion: Both RA-a and RA-r patients demonstrate an increased frequency of cTfh and overrepresentation of cTfh subsets bearing a B cell helper phenotype, suggesting that altered germinal center dynamics play a role in RA pathogenesis. In contrast, only RA-a patients show an increased proportion of circulating plasmablasts.

Follicular helper T cells (Tfh), localized in lymphoid organs, promote B cell differentiation and function. Circulating CD4 T cells expressing CXCR5, ICOS and/or PD-1 are counterparts of Tfh. Three subpopulations of circulating CD4+CXCR5+ cells have been described: CXCR3+CCR6- (Tfh-Th1), CXCR3-CCR6+ (Tfh-Th17), and CXCR3-CCR6- (Tfh-Th2). Only Tfh-Th17 and Tfh-Th2 function as B cell helpers. Our objective was to study the frequencies of circulating Tfh (cTfh), cTfh subsets and plasmablasts (CD19+CD20-CD27+CD38high cells), and the function of cTfh cells, in patients with Ankylosing Spondylitis (AS). To this end, peripheral blood was drawn from healthy controls (HC) (n = 50), AS patients naïve for TNF blockers (AS/nb) (n = 25) and AS patients treated with TNF blockers (AS/b) (n = 25). The frequencies of cTfh and plasmablasts were determined by flow cytometry. Cocultures of magnetically sorted CD4+CXCR5+ T cells with autologous CD19+CD27- naïve B cells were established from 3 AS/nb patients and 3 HC, and concentrations of IgG, A and M were measured in supernatants. We obseved that AS/nb but not AS/b patients, demonstrated decreased frequencies of circulating CD4+CXCR5+ICOS+PD-1+ cells and plasmablasts, together with a decreased (Tfh-Th17+Tfh-Th2)/Tfh-Th1 ratio. The amounts of IgG and IgA produced in cocultures of CD4+CXCR5+ T cells with CD19+CD27- B cells of AS/nb patients were significantly lower than observed in cocultures established from HC. In summary, AS/nb but not AS/b patients, demonstrate a decreased frequency of cTfh and plasmablasts, and an underrepresentation of cTfh subsets bearing a B helper phenotype. In addition, peripheral blood CD4+CXCR5+ T cells of AS/nb patients showed a decreased capacity to help B cells ex vivo.

Vitamin B9, commonly known as folate, is an essential cofactor for one-carbon metabolism that enters cells through three major specialized transporter molecules (RFC, FR, and PCFT), which differ in expression pattern, affinity for substrate, and ligand-binding pH dependency. We now report that the expression of the folate transporters differs between macrophage subtypes and explains the higher accumulation of 5-MTHF-the major folate form found in serum-in M2 macrophages in vitro and in vivo. M1 macrophages display a higher expression of RFC, whereas FRβ and PCFT are preferentially expressed by anti-inflammatory and homeostatic M2 macrophages. These differences are also seen in macrophages from normal tissues involved in folate transit (placenta, liver, colon) and inflamed tissues (ulcerative colitis, RA), as M2-like macrophages from normal tissues express FRβ and PCFT, whereas TNF-α-expressing M1 macrophages from inflamed tissues are RFC+. Besides, we provide evidences that activin A is a critical factor controlling the set of folate transporters in macrophages, as it down-regulates FRβ, up-regulates RFC expression, and modulates 5-MTHF uptake. All of these experiments support the notion that folate handling is dependent on the stage of macrophage polarization., (© 2014 Society for Leukocyte Biology.)

Objective: To examine the frequency and phenotype of Th17 cells in the peripheral blood of patients with early non-radiographic axial SpA (early nrSpA)., Methods: CD4(+) T cells were isolated from the peripheral blood of 30 early nrSpA patients, 11 AS patients and 41 age- and sex-matched healthy controls by Ficoll-Hypaque gradient and magnetic negative selection. After polyclonal stimulation, the frequency of Th17 and Th1 cells and of cells producing TNF-α or IL-10 was determined by cytometry and concentrations of IL-17, IL-22, IFN-γ, TNF-α, IL-10 and IL-4 were measured by ELISA., Results: Early nrSpA but not AS patients demonstrated a significantly lower percentage of circulating Th17, Th1 and Th17/Th1 cells, together with lower CD4-derived IL-17 and IFN-γ secretion, as compared with controls. In contrast, the percentage of circulating cells producing IL-10 or TNF-α, and the secretion of CD4-derived IL-10, TNF-α, IL-22 and IL-4 in early nrSpA were not different from controls. All Th17 cells were CD45RO(+)CD45RA(-) and CCR6(+). The frequency of circulating Th17, Th1 and Th17/Th1 was negatively correlated with BASDAI, BASFI, ASDAS-CRP, ASDAS-ESR, AS quality of life (ASQOL) and patient's global assessment in HLA-B27(+) but not in HLA-B27(-) early nrSpA patients. A positive correlation between circulating Th17 cells and BASDAI was observed in AS., Conclusion: A decreased percentage of Th17, Th1 and Th17/Th1 cells is apparent in peripheral blood CD4(+) T cells from early nrSpA. Th17, Th1 and Th17/Th1 cell numbers are related to disease activity indices in HLA-B27(+), but not in HLA-B27(-), early nrSpA patients.

Objective: To examine the frequency and phenotype of Th17 cells in the peripheral blood of early RA (eRA) patients., Methods: CD4+ T cells were isolated from the peripheral blood of 33 eRA patients, 20 established RA patients and 53 healthy controls (HC), and from the synovial fluid of 20 established RA patients (RASF), by ficoll-hypaque gradient and magnetical negative selection. After polyclonal stimulation, the frequency of Th17 and Th1 cells was determined by flow cytometry and concentrations of IL-17, IFN-γ, TNF-α and IL-10 were measured by ELISA in cell-free supernatants., Results: When all of our eRA patients were analyzed together, a significantly lower percentage of circulating Th17 cells and a lower CD4-derived IL-17 secretion were observed in comparison with HC. However, after stratifying by anti-CCP antibody status, circulating Th17 cells were decreased in anti-CCP(+) but not in anti-CCP(-)-eRA. All Th17 cells were CD45RO+CD45RA- and CCR6+. Dual Th17/Th1 cells were also exclusively decreased in anti-CCP(+)-eRA. Circulating Th17 and Th17/Th1 cells were negatively correlated with anti-CCP titres. When anti-CCP(+)-eRA patients were retested one year after initiating treatment with oral methotrexate, their circulating Th17 frequency was no longer different from HC. Of note, the percentage of circulating Th1 cells and the secretion of CD4-derived IFN-γ, TNF-α and IL-10 were not different between eRA patients and HC. In established RA patients, circulating Th17 and T17/Th1 cell frequencies were comparable to HC. In RASF, both Th17 and Th1 cells were increased when compared with blood of eRA patients, established RA patients and HC., Conclusion: Decreased circulating Th17 levels in eRA seem to be a marker of anti-CCP seropositivity, and return to levels observed in healthy controls after treatment with methotrexate.

Introduction: The purpose of this study was to examine the role of RA Synovial Fibroblast (RASFib) IL-15 expression on B cell survival., Methods: Magnetically sorted peripheral blood memory B cells from 15 healthy subjects were cocultured with RASFib., Results: RASFib constitutively expressed membrane IL-15. Survival of isolated B cells cultured for 6 days, below 5%, was extended in coculture with RASFib to 52+/-8% (p<0.001). IL-15 neutralizing agents but not isotype controls, reduced this rate to 31+/-6% (p<0.05). Interestingly, rhIL-15 had no effect on isolated B cells but significantly increased their survival in coculture with RASFib. In parallel, B cell IL-15R chains were upregulated in cocultures. BAFF and VCAM-1, that are expressed on RASFib, were tested as potential candidates involved in upregulating B cell IL-15R. Culture of B cells in the presence of rhBAFF or rhVCAM-1 resulted in significantly increased survival, together with upregulation of all three IL-15R chains; in parallel, rhIL-15 potentiated the anti-apoptotic effect of BAFF and VCAM-1. Both BAFF and VCAM-1 neutralizing agents downmodulated the effect of RASFib on B cell survival and IL-15R expression. In parallel, rhIL-15 had a lower effect on the survival of B cells cocultured with RASFib in the presence of BAFF or VCAM-1 neutralizing agents. Peripheral blood B cells from 15 early RA patients demonstrated an upregulated IL-15R and increased survival in cocultures., Conclusion: IL-15 expression on RASFib significantly contributes to the anti-apoptotic effect of RASFib on B cells. IL-15 action is facilitated by BAFF and VCAM-1 expressed on RASFib, through an upregulation of IL-15R chains.

Orally available small molecule compounds have recently been developed for the treatment of RA, and inhibitors of signalling cascades, specifically inhibitors of kinases, have reached advanced stages of clinical development. The p38 mitogen-activated protein kinase blockers have shown poor clinical response despite encouraging preclinical data. In contrast, inhibitors of the non-receptor tyrosine kinases, spleen tyrosine kinase and janus kinase 3, have demonstrated a significant clinical efficacy together with an acceptable safety profile. We herein present a review on published preclinical and clinical data on these new drugs.

We previously described that fibroblast-like cells from the synovium of rheumatoid arthritis patients (RASFib) constitutively express intracellular and surface IL-15, which induces activation of cocultured T cells. Our objective was to study the effect of RASFib IL-15 expression on the function of human CD4(+)CD25(+) regulatory T cells (Treg). RASFib, through their constitutive IL-15 expression, were able to induce the proliferation of human Tregs stimulated through their TCR, and at the same time potentiated their suppressive action on the cytokine secretion of CD4(+)CD25(-) responder T cells (Tresp). In parallel, constitutive RASFib IL-15 expression mediated an up-regulated response of Tresp. Subsequently, total CD4(+) T cells, containing natural proportions of Treg and Tresp, secreted an increased amount of pathogenic cytokines when cocultured with RASFib despite the presence of proliferating Treg with superior regulatory potency. In summary, RASFib IL-15 exerts a dual action on the equilibrium between Treg and Tresp by potentiating the suppressive effect of Treg while augmenting the proinflammatory action of Tresp; the result is a shift of the Treg/Tresp balance toward a proinflammatory state. This alteration of the Treg/Tresp equilibrium is not observed in the presence of osteoarthritis synovial fibroblasts or dermal fibroblasts, which do not constitutively express surface IL-15. Additionally, Treg with superior suppressive potency were present in the peripheral blood and the synovial fluid of RA patients, but this enhanced immunoregulatory activity was not able to overcome the increased secretion of pathogenic cytokines by RA-Tresp, indicating that rheumatoid arthritis patients demonstrate an altered Treg/Tresp equilibrium in vivo.

Objective: To investigate the osteoclastogenic potential of T cells from the peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) on autologous monocytes, and to study the cytokines implicated in this process., Methods: T cells and monocytes were isolated from the PB of 20 healthy subjects and 20 patients with early RA, and from the SF of 20 patients with established RA. Autologous T cell/monocyte cocultures were established in the absence of exogenous cytokines or growth factors in order to examine spontaneous ex vivo osteoclast differentiation by tartrate-resistant acid phosphatase staining and calcified matrix resorption activity., Results: Surface RANKL was expressed on freshly isolated T cells from the PB of patients with early RA and the SF of patients with established RA. In addition, surface interleukin-15 (IL-15) was detected on freshly isolated T cells and monocytes from the PB of patients with early RA and the SF of patients with established RA. Autologous T cell/monocyte cocultures derived from the SF of patients with established RA and from the PB of patients with early RA, but not from the PB of healthy controls, resulted in osteoclast differentiation that was significantly inhibited by osteoprotegerin (OPG) and by neutralizing monoclonal antibodies to IL-15, IL-17, tumor necrosis factor alpha (TNFalpha), and IL-1beta. OPG, anti-TNFalpha, and anti-IL-1beta demonstrated a cooperative inhibitory effect. At 1-year followup, surface RANKL and IL-15 and ex vivo osteoclastogenesis were no longer observed on PB T cells or monocytes from patients with early RA in whom clinical remission had been achieved with treatment., Conclusion: T cells are important contributors to the pathogenesis of bone erosions in RA through interaction with osteoclast precursors of the monocyte/macrophage lineage.

Homeostatic proliferation of T cells in vivo is responsible for the maintainance of the T cell pool, and IL-15 is a pivotal cytokine implicated in this process. Known cell sources providing physiologically active IL-15 are monocytes/macrophages, dendritic cells, and stromal cells. T lymphocyte expression of functionally active IL-15 and its possible role in T cell biology have not been investigated. In this study, we demonstrate that human T cells constitutively express IL-15 that acts through autocrine/juxtacrine loops to promote ex vivo homeostatic T cell proliferation.

Objective: To examine fibroblasts grown from the synovial fluid of rheumatoid arthritis (RA) patients for TRAIL-R2 expression, and for susceptibility to apoptosis induced by an agonistic anti-TRAIL-R2 monoclonal antibody (mAb)., Methods: The expression of TRAIL-R2 (DR5) was determined by flow cytometry on fibroblasts grown from the synovial fluid of patients with RA, osteoarthritis (OA), seronegative arthritis, and unclassified monarthritis or oligoarthritis, and on fibroblasts from the synovial membrane of RA and OA patients. Susceptibility to apoptosis mediated by an agonistic anti-TRAIL-R2 mAb was determined by alamar blue bioassay, fluorescence microscopy (annexin V/propidium iodide staining), and caspase activation., Results: Fibroblasts grew from 35 of 50 RA synovial fluid samples, of which 26 were DR5(+) (mean [+/-SD] fluorescence intensity [MFI] 18.74 +/- 2.5). Fibroblasts also grew from 15 of 30 seronegative arthritis synovial fluid samples, 28 of 40 OA synovial fluid samples, and 8 of 20 unclassified monarthritis or oligoarthritis synovial fluid samples; all of these were DR5- (MFI 0.32 +/- 0.02). All 10 of the fibroblast lines from joint replacement surgery or synovectomy specimens of RA patients were DR5(+) (MFI 20.3 +/- 3.2). All fibroblast lines from the synovium of 10 OA patients were DR5-, as were fibroblasts from the skin of 5 healthy subjects. DR5(+) fibroblast cultures underwent apoptosis when treated in vitro with an agonistic anti-DR5 antibody., Conclusion: Fibroblasts grown from the synovial fluid and synovial membrane of RA patients express TRAIL-R2 that is functionally active. An agonistic anti-TRAIL-R2 antibody that does not induce hepatocyte toxicity may be an alternative strategy for treatment of RA.

To characterize the molecules responsible for synovial fibroblast-T lymphocyte (TL) cross-talk in rheumatoid arthritis (RA), synovial fibroblasts from patients with established RA (RASFibs) were cocultured with TLs from peripheral blood of early RA patients (RAPBTL). TLs from peripheral blood of healthy controls and from synovial fluid of RA served as controls. Adhesion molecules and cytokines were determined by flow cytometry, ELISA, and real-time PCR. RAPBTL (n = 20) induced an up-regulation of ICAM-1, intracellular IL-8, IL-6, IL-15, and surface IL-15 in cocultured RASFibs. In turn, RAPBTL showed an up-regulation of TNF-alpha, IFN-gamma, IL-17, CD25, and CD69 expression. Responses seen with TLs from peripheral blood of healthy controls (n = 20) were significantly lower, whereas responses with TLs from synovial fluid of RA (n = 20) were maximal. Blocking Abs to IL-15 and CD54, but not an isotype-control Ab, down-regulated the increased TL cytokine and activation marker expression. Abs to CD69, CD11a, IL-17, TNF-alpha, and IFN-gamma significantly decreased the up-regulation of RASFib cytokine and CD54 expression. Cocultures using 0.4- micro m inserts did not result in up-regulation of surface molecules or cytokines. Methotrexate significantly inhibited RASFib/TL cross-talk signals and decreased adhesion of TL to RASFibs. In summary, RASFib production of IL-15 induces the proinflammatory cytokines TNF-alpha, IFN-gamma, and IL-17 in cocultured TLs through a cell contact-dependent mechanism. In turn, these cytokines stimulate the expression of IL-15, IL-8, and IL-6 in RASFibs, thereby creating a feedback loop that favors persistent synovial inflammation. Methotrexate seems to disrupt this loop by decreasing cell adhesion.

Despite the near universal association of congenital heart block and maternal Abs to SSA/Ro and SSB/La, the intracellular location of these Ags has made it difficult to substantiate their involvement in pathogenicity. To define whether components of the SSA/Ro-SSB/La complex, which translocate during apoptosis, are indeed accessible to extracellular Abs, two approaches were taken: immunoprecipitation of surface biotinylated proteins and scanning electron microscopy. Human fetal cardiocytes from 16-24-wk abortuses were cultured and incubated with staurosporine to induce apoptosis. Surface biotinylated 48-kDa SSB/La was reproducibly immunoprecipitated from apoptotic, but not nonapoptotic cardiocytes. Surface expression of SSA/Ro and SSB/La was further substantiated by scanning electron microscopy. Gold particles (following incubation with gold-labeled sera containing various specificities of anti-SSA/Ro-SSB/La Abs and murine mAb to SSB/La and 60-kDa SSA/Ro) were consistently observed on early and late apoptotic cardiocytes. No particles were seen after incubation with control antisera. To evaluate whether opsonized apoptotic cardiocytes promote inflammation, cells were cocultured with macrophages. Compared with nonapoptotic cardiocytes or apoptotic cardiocytes incubated with normal sera, apoptotic cardiocytes preincubated with affinity-purified Abs to SSB/La, 52-kDa SSA/Ro, or 60-kDa SSA/Ro increased the secretion of TNF-alpha from cocultured macrophages. In summary, apoptosis results in surface accessibility of all SSA/Ro-SSB/La Ags for recognition by circulating maternal Abs. It is speculated that in vivo such opsonized apoptotic cardiocytes promote an inflammatory response by resident macrophages with damage to surrounding conducting tissue.

To correlate the arrhythmogenic effects of maternal autoantibodies with the genesis of congenital heart block, female BALB/c mice were immunized with human recombinant 48-kDa SSB/La, 60-kDa SSA/Ro, 52-kDa SSA/Ro (52alpha), and 52beta (amino acids 169-245 deleted) as well as with murine recombinant 52-kDa SSA/Ro. Control animals received beta-galactosidase or a polypeptide encoded by pET-28 alone. Following primary immunization and two boosters, high titer responses to the respective Ags were established by ELISA, immunoblotting, and immunoprecipitation. Sera from mice immunized with either human 52alpha or 52beta immunoprecipitated murine 52Ro. mRNA and protein expression of 52Ro was demonstrated in the newborn murine heart. A spectrum of atrioventricular nodal conduction abnormalities was identified by electrocardiogram. First-degree block was detected in 7% of 27 pups born to mothers immunized with 48La, 20% of 54 pups born to 60Ro-immunized mothers, 6% of 56 pups born to 52alpha-immunized mothers, 7% of 86 pups born to 52beta-immunized mothers, and 9% of 22 pups born to mothers immunized with murine 52Ro. Advanced conduction abnormalities were only identified in offspring of 52alpha- or 52beta-immunized mice. In the 52alpha group, one pup had complete block and another had second-degree block (Wenckebach type); in the 52beta group, five pups had complete block. Maternal Abs to the primary immunogens were detected in the pups. No control had any conduction abnormalities. This Ab-specific animal model provides strong evidence for a pathogenic role of anti-SSA/Ro-SSB/La Abs, particularly 52Ro, in the development of congenital heart block. The range and frequency of conduction defects suggest that additional factors promote disease expression.

We report a patient with a chronic intramedullary spinal cord abscess who suffered an episode of acute meningitis due to rupture of the abscess into the subarachnoid space.

Left adrenal pheochromocytoma and mesenteric paraganglioma were diagnosed in a 52-year-old female with diabetes mellitus (fasting blood glucose 14.5 mmol/l), hypertension and myocardial asymmetric septal hypertrophy (septal/left ventricular free wall thickness 1.31). Carbohydrate metabolism and cardiac disease returned to normal after the resection of both tumors (fasting blood glucose 5.2 mmol/l, septal/left ventricular free wall thickness 1.10). This is the first patient reported in the Spanish literature in whom asymmetric septal hypertrophy has been correlated with the hypersecretion of catecholamines.