Zrinka Jajić

Primary hypertrophic osteoarthropathy: clinical, radiologic, and scintigraphic characteristics.

BACKGROUND Our aim was to establish clinical and imaging characteristics of primary hypertrophic osteoarthropathy (PHO). METHODS A group of 76 patients (71 men and 5 women) with primary hypertrophic osteoarthropathy was examined. Extensive history and status of the locomotor system were determined in all patients. Radiologic and scintigraphic examinations of the bones, thermography, capillaroscopy, gastroscopy, and histologic analysis were also performed. RESULTS Family history was positive in all 55 patients (100%) summoned for a check-up. Finger clubbing of the hands and feet as well as positive Schamroth test were found in all patients, while hyperhydrosis was found in 65 (85.5%), seborrhea in 39 (51%), and involvement of the joints in 52 (68.5%) patients. Histologic analysis in 18 (23.7%) patients predominantly showed periarticular edema and moderate cellular activity. Periostosis of the long bones was also found in all patients, while scintigraphy, performed in 44 patients, was positive in 18 (33.5%) patients in the active phase of the disease. Thermography, performed in 10 patients, ranged from hypothermia to thermal amputation of feet and hands. Capillaroscopy of hands and feet in 47 (61.8%) patients showed changes in small blood vessels. Gastroscopy indicated abnormalities (ulcer and hypertrophic gastritis) in 21 patients (27.6%). CONCLUSIONS PHO has special clinical and radiographic manifestations (finger clubbing and periostosis of the long bones) that are associated with some minor manifestations. Development of the disease is gradual (to 20 years) and requires a special diagnostic assessment.

Peripheral Blood Expression Profiles of Bone Morphogenetic Proteins, Tumor Necrosis Factor-superfamily Molecules, and Transcription Factor Runx2 Could Be Used as Markers of the Form of Arthritis, Disease Activity, and Therapeutic Responsiveness

Objective. To assess whether different forms of arthritis and disease activity could be distinguished by peripheral blood expression profiles of bone-regulatory factors including tumor necrosis factor (TNF)-superfamily [TNF-related apoptosis-inducing ligand (TRAIL), the Fas ligand (FasL), and the ligand for herpesvirus entry mediator (LIGHT)] and bone morphogenetic protein (BMP)-family members (BMP-2, BMP-4, BMP-6) as well as osteoblast differentiation gene Runx2. Methods. Blood cells from healthy controls (n = 25) and patients at different disease stages with rheumatoid arthritis (RA; n = 49), osteoarthritis (OA; n = 17), or spondyloarthritis, including ankylosing spondylitis (AS; n = 27) or psoriatic arthritis (PsA; n = 23), were processed for quantitative polymerase chain reaction. Gene expression was assessed in comparison with control samples, correlated with clinical data of different forms of arthritis, and analyzed for discriminative efficacy between groups by receiver-operation characteristic (ROC) curves. Results were confirmed on diagnostic RA (n = 5) and AS (n = 8) samples. Results. BMP-4, BMP-6, and Runx2 expressions were significantly decreased in patients with RA and OA versus controls. Patients with RA also had decreased FasL and LIGHT expression, while patients with AS had increased Runx2 expression. Negative correlation with disease activity was found for BMP-4, FasL, and Runx2 in RA and for Runx2 in PsA, while positive correlation was found for BMP-4 in PsA. Gene expression was higher in the therapy-resistant form of AS (for BMP-4, LIGHT, and Runx2) and in methotrexate-treated patients in RA (for BMP-2 and LIGHT). ROC curve analysis confirmed discrimination between groups, particularly decreased LIGHT and Runx2 for RA and increased Runx2 for AS. Conclusion. Our study identified BMP and Runx2 as possible biomarkers of bone metabolism in several forms of arthritis, while lower FasL and LIGHT were associated with RA. Correlation between gene expression and disease activity may be clinically useful in assessing therapeutic effectiveness and disease monitoring.

Induction of osteoclast progenitors in inflammatory conditions: key to bone destruction in arthritis

The inflammatory milieu favors recruitment and activation of osteoclasts, and leads to bone destruction as a serious complication associated with arthritis and with other inflammatory processes. The frequency and activity of osteoclast progenitors (OCPs) correspond to arthritis severity, and may be used to monitor disease progression and bone resorption, indicating the need for detailed characterization of the discrete OCP subpopulations. Collectively, current studies suggest that the most potent murine bone marrow OCP population can be identified among lymphoid negative population within the immature myeloid lineage cells, as B220−CD3−CD11b–/loCD115+CD117+CX3CR1+ and possibly also Ter119−CD11c−CD135loLy6C+RANK−. In peripheral blood the OCP population bears the monocytoid phenotype B220−CD3−NK1.1−CD11b+Ly6ChiCD115+CX3CR1+, presumably expressing RANK in committed OCPs. Much less is known about human OCPs and their regulation in arthritis, but the circulating OCP subset is, most probably, comprised among the lymphoid negative population (CD3−CD19−CD56−), within immature monocyte subset (CD11b+CD14+CD16−), expressing receptors for M-CSF and RANKL (CD115+RANK+). Our preliminary data confirmed positive association between the proportion of peripheral blood OCPs, defined as CD3−CD19−CD56−CD11b+CD14+, and the disease activity score (DAS28) in the follow-up samples from patients with psoriatic arthritis receiving anti-TNF therapy. In addition, we reviewed cytokines and chemokines which, directly or indirectly, activate OCPs and enhance their differentiation potential, thus mediating osteoresorption. Control of the activity and migratory behaviour of OCPs as well as the identification of crucial bone/joint chemotactic mediators represent promising therapeutic targets in arthritis.

Radiological changes of short and flat bones in primary hypertrophic osteoarthropathy

Hypertrophic osteoarthropathy (HO) is defined as a syndrome characterised by finger clubbing and periostosis of long bones.1 It is classified into primary (PHO) and secondary hypertrophic osteoarthropathy (SHO).2 To establish the HO diagnosis, finger clubbing and periostosis of long bones must be present.2 Cases of PHO have not been frequently reported in the medical literature.3 HO cases have recently been more described, since HO was classified into the bone and cartilage group of diseases according to the international classification of rheumatic diseases.4 We formed a large group of patients in whom changes of flat bones were found. The analysis was based on a group of patients with PHO from 1970 to 1996. Over 26 years, a total of 76 patients …

Association of systemic and intra-articular osteoclastogenic potential, pro-inflammatory mediators and disease activity with the form of inflammatory arthritis.

PurposeWe aimed to assess osteoclastogenic potential of peripheral blood mononuclear cells (PBMC) and synovial fluid-derived mononuclear cells (SFMC) in different forms of arthritis and to correlate it with inflammatory mediators within intra-articular and circulatory compartments.MethodsPaired PBMC and SFMC samples of patients with rheumatoid arthritis (RA; n = 10) and psoriatic arthritis (PsA; n = 10), and PBMC of healthy controls were cultured to assess osteoclastogenic potential by the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts (OCs) and expression of OC-related genes (receptor activator of nuclear factor-κΒ (RANK), cFMS, and TRAP). Osteoclastogenesis was correlated with the arthritis-related inflammatory indicators in serum and synovial fluid (SF).ResultsNumber of OCs differentiated from PBMC was significantly higher in RA and PsA compared with control, with RA having more OCs compared with PsA. There was no difference in SFMC OC number between arthritic patients, but RANK expression in OCs differentiated from SFMC was higher in PsA compared with RA. SF of PsA patients more potently induced OC differentiation from control CD3-CD19-CD56-CD11b+CD115+ PBMC compared with RA, paralleled with higher RANK-ligand expression in PsA SFMC. Positive correlations of OC number with erythrocyte sedimentation rate, serum level of CCL2, and PBMC gene expression of interleukin-18 and Fas-ligand were observed.ConclusionOsteoclastogenic potential is systemically enhanced in patients with RA, paralleled by disordered systemic and local expression of proinflammatory mediators, whereas PsA involves specific deregulation in RANKL/RANK axis. Our study reveals arthritis-specific mediators associated with the form of arthritis, indicating clinical relevance for diagnosis and treatment.

Which clinical variables have the most significant correlation with quality of life evaluated by SF-36 survey in Croatian cohort of patient with ankylosing spondylitis and psoriatic arthritis?

The aim of our study was to assess clinical variables with the best correlation to quality of life (QOL) assessed by medical outcome survey Short-Form 36 (SF-36) in patients with spondyloarthritides, including ankylosing spondylitis (AS) and psoriatic arthritis (PsA). We analyzed the cohort of 54 patients (22 patients with PsA and 32 patients with AS), who filled the Croatian version of SF-36. For each type of arthritis, patients were clinically evaluated using the extensive list of clinical variables categorized into subjective and objective group. For AS patients, subjective and objective variables (spinal mobility measurements, clinical assessment of spinal pain, patient assessments of disease activity and pain) correlated mainly with the physical functioning concept of SF-36. Patients assessments of fatigue correlated with the energy/fatigue subscale, whereas patient assessment of enthesial pain correlated with the pain subscale. Correlations between clinical variables and SF-36 concepts of PsA patients showed more diverse distribution than for AS. Objective variables (spinal mobility measurements, a 76-joint score, clinical assessment of spinal pain) correlated with concepts concerning physical health and pain. Several subjective patient assessments correlated with energy/fatigue, emotional well-being, pain and general health subscales. Both patient and physician assessment of PsA activity correlated with the role limitations due to emotional problems. Bath ankylosing spondylitis functional index (BASFI) had the strongest correlation with the physical functioning concept of SF-36 in both diseases. Our findings provide important information to help selecting the variables with strongest impact on QOL, for better planning the management strategies and achieving better rehabilitation results.

Chemokine signals are crucial for enhanced homing and differentiation of circulating osteoclast progenitor cells.

BackgroundThe peripheral blood (PB) monocyte pool contains osteoclast progenitors (OCPs), which contribute to osteoresorption in inflammatory arthritides and are influenced by the cytokine and chemokine milieu. We aimed to define the importance of chemokine signals for migration and activation of OCPs in rheumatoid arthritis (RA) and psoriatic arthritis (PsA).MethodsPB and, when applicable, synovial fluid (SF) samples were collected from 129 patients with RA, 53 patients with PsA, and 110 control patients in parallel to clinical parameters of disease activity, autoantibody levels, and applied therapy. Receptors for osteoclastogenic factors (CD115 and receptor activator of nuclear factor-κB [RANK]) and selected chemokines (CC chemokine receptor 1 [CCR1], CCR2, CCR4, CXC chemokine receptor 3 [CXCR3], CXCR4) were determined in an OCP-rich subpopulation (CD3−CD19−CD56−CD11b+CD14+) by flow cytometry. In parallel, levels of CC chemokine ligand 2 (CCL2), CCL3, CCL4, CCL5, CXC chemokine ligand 9 (CXCL9), CXCL10, and CXCL12 were measured using cytometric bead array or enzyme-linked immunosorbent assay. Sorted OCPs were stimulated in culture by macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, and they were differentiated into mature osteoclasts that resorb bone. Selected chemokines (CCL2, CCL5, CXCL10, and CXCL12) were tested for their osteoclastogenic and chemotactic effects on circulatory OCPs in vitro.ResultsThe OCP population was moderately enlarged among PB cells in RA and correlated with levels of tumor necrosis factor-α (TNF-α), rheumatoid factor, CCL2, and CCL5. Compared with PB, the RANK+ subpopulation was expanded in SF and correlated with the number of tender joints. Patients with PsA could be distinguished by increased RANK expression rather than total OCP population. OCPs from patients with arthritis had higher expression of CCR1, CCR2, CCR4, CXCR3, and CXCR4. In parallel, patients with RA had increased levels of CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10, with significant elevation in SF vs PB for CXCL10. The subset expressing CXCR4 positively correlated with TNF-α, bone resorption marker, and rheumatoid factor, and it was reduced in patients treated with disease-modifying antirheumatic drugs. The CCR4+ subset showed a significant negative trend during anti-TNF treatment. CCL2, CCL5, and CXCL10 had similar osteoclastogenic effects, with CCL5 showing the greatest chemotactic action on OCPs.ConclusionsIn our study, we identified distinct effects of selected chemokines on stimulation of OCP mobilization, tissue homing, and maturation. Novel insights into migratory behaviors and functional properties of circulatory OCPs in response to chemotactic signals could open ways to new therapeutic targets in RA.

AB0026 Chemokine signals are critical for homing and enhanced differentiation of circulating osteoclast progenitor cells

Background Peripheral blood (PB) monocyte pool contains cells capable of differentiating into osteoclasts (OCs). These osteoclast progenitors (OCPs) contribute to osteoresorption in inflammatory arthritides under influence of the cytokine milieu and chemokine mediated trafficking. Objectives Our study aimed to define chemokine receptor profile of peripheral OCPs in rheumatoid arthritis (RA), with comparison to psoriatic arthritis (PSA), as well as their susceptibility to chemotactic signals. Methods 129 RA, 53 PSA and 110 control patients were enrolled after Ethical approval. PB samples and synovial fluid (SF) samples, with clinical data of disease activity, inflammation and autoantibody levels were collected. Patients starting anti-TNF therapy were followed up 6 months. TNF-α and CTX serum levels were measured by ELISA. Frequency of OCP-rich subpopulation (CD3-CD19-CD56-CD11b+CD14+), expression of OC differentiation (CD115, RANK) and chemokine (CCR1, CCR2, CCR4, CXCR4) receptors was assessed by flow cytometry. OCPs were sorted using FACS, cultured with M-CSF and RANKL, stained for TRAP enzyme and mature OCs counted. Levels of CCL2, CCL3, CCL4, CCL5, CXCL9 and CXCL10 were measured using cytometric bead array, and of CXCL12 by ELISA. Osteoclastogenic effects of CCL2, CCL5 and CXCL10 were analyzed in cell culture, and chemotactic effects on OCPs were studied by cell migration assay using Transwell, with count of number of migrated cells and subsequently differentiated mature osteoclasts. Results OCP population was moderately enlarged in PB, further expanded in SF and correlated with TNF-α and rheumatoid factor (RF) levels in patients with RA. However, sorted OCPs generated similar number of mature OCs as control. RANK+ subpopulation was enlarged in SF vs PB and correlated with number of tender joints. In PSA, the OCP population was not enlarged, but had a higher RANK expression. OCPs in RA and PSA had higher expression of CCR1, CCR2, CCR4, CXCR4, and all except CCR4 showed positive PB-to-SF gradient. RA had higher levels of CCL2, CCL3, CCL4, CCL5, CXCL9 and CXCL10, with a positive PB-to-SF gradient for all except CCL5 and CXCL9. OCP frequency correlated with levels of CCL2 and CCL5. Subset expressing CXCR4 was associated with TNF-α, CTX and RF levels and was lower in patients treated with DMARD, who at the same time had lower osteoresorption (CTX). Subset expressing CCR4 showed significant negative trend during anti-TNF treatment. CCL2, CCL5 and CXCL10 showed significant osteoclastogenic effect. CCL5 showed greatest chemotactic effect, attracting the highest number of cells in the migration assay. At the same time, attracted cells possessed greater osteoclastogenic potential. Conclusions Our study provides evidence of the specific importance of certain chemokine signals for stimulation of OCP mobilization, subsequent tissue homing, and maturation, explaining local as well as generalized bone loss seen in RA. Novel insights in regards to migratory behavior and functional properties of PB OCPs in response to chemotactic signals could open way to new therapeutic targets in RA. Acknowledgements This work was supported by a grant from the Croatian Science Foundation (project number 5699). Disclosure of Interest None declared

AB0115 Differently Associated Frequency of B Lymphocyte Subpopulations with Disease Activity in Ankylosing Spondylitis Compared To Rheumatoid Arthritis

Background Both rheumatoid arthritis (RA) and ankylosing spondylitis (AS) belong to the group of chronic rheumatic diseases with the important role of autoimmune pathogenic mechanisms. However, these forms differ in the major target tissues as well as the intensity of bone and cartilage destruction, with RA being the prototype of “destructive” arthritis affecting peripheral joints and AS being the prototype of “remodeling” arthritis predominantly of the axial skeleton. Objectives Both forms of arthritis are associated with abnormal immune cell functions, including B lymphocytes. Although several recent studies stressed the role of B lymphocyte subpopulations and autoantibodies in AS, these mechanisms in the AS pathogenesis are significantly less understood compared to RA and other rheumatic autoimmune diseases.The aim of our study was to compare the frequency of B lymphocyte subpopulations between RA and AS patients, and to correlate them with the disease activity. Methods Mononuclear cells were isolated from peripheral blood of healthy controls (n=30), RA (n=33) and AS (n=18) patients, after obtaining Ethical approval and informed consent from patients. The B lymphocyte phenotype of peripheral-blood mononuclear cells was determined using flow cytometry for the following surface markers: CD19, CD27, IgD, CD32 and CD38. In addition, statistical correlation was assessed to test the association between the frequency of selected B lymphocyte subpopulations and the disease-activity score, DAS28 (Disease Activity Score 28) for RA and BASDAI (Bath Ankylosing Spondylitis Disease Activity Index) for AS. Results Gating strategy applied for flow-cytometry data was set to discriminate between naïve B lymphocytes (CD19+ IgD+ CD27-), unswitched memory B lymphocytes (CD19+ IgD+ CD27+), class-switched memory B lymphocytes (CD19+ IgD- CD27-) and plasmablasts (CD19+ IgD+ CD27hi CD38+). In addition, expression of CD32 (FcgRII receptor) and CD86 (B7-2 co-stimulator), associated with the maturation and activation of B lymphocytes, within these B lymphocyte subsets were assessed. Data analysis showed expanded CD32+ subset among naïve and memory B lymphocytes in RA (16.4±11.6% and 9.8±8.2%) compared to control (4.3±2.7% and 7.6±3.5%) and AS (5.6±2.6% and 5.8±1.9%). Similarly, there were more CD86+ cells among naïve and unswitched memory B lymphocytes in RA (9.0±8.2% and 18.2±8.0%) compared to control (2.8±1.3% and 11.7±5.5%) and AS (4.9±5.9% and 11.6±6.2%). Class-switched memory B lymphocytes were negatively associated with disease activity score in RA (ρ=-0.45) but positively in AS (ρ=+0.39), whereas the associations were reversed for the population of naïve B lymphocytes (ρ=+0.32 for RA and ρ=-0.38 for AS). Conclusions Our results indicate that B lymphocyte-mediated immune response may be important for both RA and AS, but with distinct effector mechanisms. Therefore it is reasonable to suggest that B lymphocyte subpopulations may represent promising cellular targets for the therapeutic interventions in different forms of arthritis. Acknowledgement This work was fully supported by Croatian Science Foundation (project nr. 5699). Disclosure of Interest None declared

THU0011 CD32+ B Lymphocytes and IL21R+ T Lymphocytes Are Associated with Disease Activity and Increased Levels of Proinflammatory Cytokines in Patients with Rheumatoid and Psoriatic Arthritis

Background Autoimmunity is presumed to be a major driving force in the pathogenesis of chronic rheumatic diseases, including rheumatoid arthritis (RA) and psoriatic arthritis (PSA). Although these forms of arthritis differ in their clinical features, both are marked by persistent inflammation and osteoresorption underpinned by aberrant lymphocyte populations and disturbed cytokine network. Objectives Various subpopulations of both T and B lymphocytes have been implied in RA and PSA, but their relevance to disease onset and progression remains largely unclear. The aim of our study was to define the association of T, B and NK cell subpopulations with cytokine levels and clinical parameters in RA and PSA patients. Methods Mononuclear cells were isolated from peripheral blood of healthy controls (n=35), RA (n=36) and PSA (n=13) patients, after obtaining Ethical approval and informed consent. Flow cytometry was applied to discriminate between T lymphocyte subpopulations: Th1/2 (CD3+CD4+CCR6-), Th17 (CD3+CD4+CCR4+CCR6+), Tfh (CD3+CD4+CXCR5+), Tc (CD3+CD8+) and memory Tc (CD3+CD8+CCR4+); B lymphocyte subpopulations: naïve (CD19+IgD+CD27-), unswitched memory (CD19+IgD+CD27+), class-switched memory (CD19+IgD-CD27-) and plasmablasts (CD19+IgD+CD27hiCD38+); and NK cells (CD3-CD19-CD56+). Markers of lymphocyte maturation (CD32), activation (CD86, IL21R, CD25) and migration (CD11b) were also analyzed. Frequencies of lymphocyte subpopulations were correlated with clinical parameters, including DAS28 (for RA and PSA) and BASDAI (for PSA with spondylitis). Finally, serum levels of various cytokines (TNF, IL4, IL6, IL10, IL17, CCL2, CCL3, CCL4, CCL5) were measured by flow cytometry bead based assay. Results Several subpopulations were found to be significantly expanded: CD32+ naïve (Ctrl 1.8%, RA 5.8%, PSA 6.0%) and memory B lymphocytes, both class-switched (Ctrl 1.8%, RA 5.3%, PSA 3.8%) and unswitched (Ctrl 4.0%, RA 16.8%, PSA 13.7%); memory Tc lymphocytes (Ctrl 4.8%, RA 6.8%, PSA 9.1%); and CD11b+ B lymphocytes (Ctrl 15.1%, RA 20.9%, PSA 20.2%). Significant correlations between lymphocyte subpopulations and clinical parameters included: positive association of IL21R+ T lymphocytes with DAS28 in RA (ρ=0.45) and negative with BASDAI in PSA (ρ=-0.86); negative association of class-switched memory B lymphocytes with DAS28 in RA (ρ=-0.52), but positive with BASDAI in PSA (ρ=0.71). Correlation of lymphocyte subpopulations with cytokine levels showed: significant positive association of CD32+ naïve B lymphocytes with TNF and CCL5 (ρ=0.55 and 0.54); CD32+ class-switched and unswitched memory B cells with TNF (ρ=0.47 and 0.55) and CCL4 (ρ=0.47 and 0.54); CD11b+ B lymphocytes with CCL4 (ρ=0.47); and IL21R+ T lymphocytes with TNF, CCL3 and CCL4 (ρ=0.42, 0.47 and 0.49). Conclusions Our results indicate novel T and B lymphocyte subpopulations induced in both RA and PSA. CD32+ B lymphocytes, as well as IL21R+ T lymphocytes, may be of particular interest as possible therapeutic targets, since their frequency is associated with disease activity and increased levels of proinflammatory and proresorptive cytokines. Acknowledgement The work has been fully supported by Croatian Science Foundation (project no. 5699). Disclosure of Interest None declared