Search

Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are associated with abnormal immune cell functions. We combined manual and automated profiling in subpopulations of T-cells, B-cells and monocytes, in parallel to functional testing and clinical correlation.Using flow cytometry, we analysed the expression of CCR4, CCR6 and CXCR5 on helper and cyotoxic T-cells, CD32B and CD86 on naïve and memory B-cells, and CCR1, CCR2, CCR4 and CXCR4 on monocytes in chronic high-disease activity patients to identify peripheral blood subpopulations. Cell activation, proliferative capability and osteoclastogenic effects were tested in vitro. Comparison with synovial compartment, clinical data and anti-TNF treatment were added to peripheral blood analysis.PsA had lower double-negative T-cell frequency, while RA had lower double-positive T-cell frequency and expanded Th1-like and cytotoxic T-cell subsets. CD32B expression was increased on naïve and memory B-cells in AS and associated with disease activity. CCR6+ and CXCR5+ cytotoxic T-cells and CD32B+ naïve and memory B-cells were highly enriched within the synovial compartment. T-cells and B-cells from AS exhibited enhanced activation and proliferation in vitro, whereas T-cell conditioned medium from RA produced an increased osteoclastogenic effect. CCR1 and CXCR4 were upregulated on osteoclastogenic monocyte subsets of RA, AS and PsA patients. Bioinformatic Citrus analysis identified additional T-cell, B-cell and monocyte clusters specifically associated with each disease.By combining manual and automated data analysis, our study revealed several disease-specific immune cell subpopulations, particularly cytotoxic T-cell subsets in RA and memory B-cell subsets in AS, which may serve as an indicator of active disease or possible therapeutic target.

Background:Osteoclasts mediate periarticular and systemic bone loss in rheumatoid arthritis (RA). Osteoclast progenitor cells (OCPs) derived from the myeloid lineage are susceptible to regulation through Notch signaling. Murine bone marrow and splenic OCPs, identified as CD45+Ly6G-CD3-B220-NK1.1-CD11blo/+CD115+CCR2+ cells, are specifically increased in arthritis. We previously identified an increased frequency of OCPs expressing Notch receptors in arthritic mice.Objectives:Several studies suggested that Notch signaling modulation affects the course of experimental arthritis. We aimed to determine the effects of Notch receptor signaling inhibition on OCP activity and arthritis severity in murine collagen-induced arthritis (CIA).Methods:Male C57/Bl6 and DBA mice were immunized with chicken type II collagen and treated with i.p. injections of anti-Notch 1 neutralizing antibodies (1mg/kg). Notch receptor 1 through 4 expression on OCPs was analyzed by flow cytometry in periarticular bone marrow (PBM) and spleen (SPL). Gene expression of Notch receptors, ligands and transcription targets as well as osteoclast differentiation genes RANK, cFos and cFms was determined by qPCR from tissues and sorted OCPs. FACS sorted OCPs were stimulated by osteoclastogenic factors (M-CSF and RANKL), in control, IgG, Jagged (Jag)1 or Delta-like (DLL)1 coated wells, with or without anti-Notch 1 antibodies. Research was approved by the Ethics Committee.Results:We confirmed the expression of Notch receptors on OCPs by flow cytometry with Notch 1 and 2 being most abundantly expressed (around 25% and 40% positive OCPs in PBM and 35% and 20% in SPL respectively), with a significant increase of Notch 2 expression in arthritis. Seeding OCPs on DLL1 coated wells significantly increased while seeding on Jag1 coated wells significantly decreased osteoclastogenesis as reflected on the number of TRAP+ osteoclasts and expression of osteoclast differentiation genes. The addition of anti-Notch 1 antibodies to ligand-stimulated OCPs resulted in an increased number of TRAP+ osteoclasts, partially reversing Jag1 inhibition. In vivo treatment with anti-Notch 1 antibodies did not affect total OCP frequency, but increased expression of Notch 4 both in PBM and SPL as seen by flow cytometry and qPCR. Additionally, anti-Notch 1 treatment stimulated Notch transcription factors HES and HEY. Both PBM and SPL cultured OCPs from anti-Notch 1 treated mice produced a higher number of large TRAP+ osteoclasts, doubling the area covered with osteoclasts in the latter compared to untreated mice. Increased osteoclastogenesis in vitro was further confirmed by an increased expression of osteoclast differentiation genes in the treated group.Conclusion:Our results confirm that Notch signaling may represent an important therapeutic target for the regulation of osteoclast activity in arthritis. Both in vitro and in vivo anti-Notch 1 neutralizing antibodies enhanced osteoclastogenesis in CIA model, implying an inhibitory role of Notch 1 signaling in osteoclast differentiation. As Notch 2 expression is increased on OCPs of arthritic mice, we next plan to determine the effects of Notch 2 neutralization on osteoclast activity and arthritis severity.References:[1]Ikić Matijašević M, Flegar D, Kovačić N, Katavić V, Kelava T, Šućur A, et al. Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis. Clin Exp Immunol. 2016;186(3):321–35.[2]Šućur A, Filipović M, Flegar D, Kelava T, Šisl D, Lukač N, et al. Notch receptors and ligands in inflammatory arthritis – a systematic review. Immunology Letters 2020 Vol. 223, p. 106–14.Acknowledgements:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, UIP-2017-05-1965 and DOK-2018-09-4276.Disclosure of Interests:None declared.

Background:Systemic and periarticular bone loss in rheumatoid arthritis (RA) is mediated by osteoclasts, multinucleated cells originating from the myeloid lineage. Recently, Notch signaling pathway has emerged as a potential regulator of osteoclast progenitor (OCP) differentiation and activation.Objectives:The exact role of Notch signaling in the context of arthritis is still unknown; however, its inhibition has beneficial effects in animal arthritis models. We aimed to determine the expression of Notch receptors and ligands on specific OCP subpopulations and define changes that occur in murine collagen-induced arthritis (CIA) and RA patients.Methods:Peripheral blood, synovial tissue and subchondral bone marrow were collected from RA patients, and periarticular bone marrow (PBM) and spleen (SPL) were harvested from male C57/Bl6 mice immunized with chicken type II collagen. Notch 1 to 4 receptor expression on OCPs was analyzed by flow cytometry. Gene expression of Notch receptors/ligands was determined by qPCR from tissues and sorted OCPs. Sorted OCPs were cultured, with addition of MCSF and RANKL, in control, IgG, Jagged (Jag) 1 or Delta (DLL) 1 coated wells. Immunohistochemistry (IHC) for Notch 1 and 2 was performed on sections of murine hind paws. Research was approved by Ethics committee.Results:We previously identified peripheral and periarticular subpopulations of murine and human OCPs, as CD45+CD3-B220-NK1.1-CD11blo/+CD115+CCR2+and CD45+CD3-CD19-CD56-CD11b+CD14+CCR2+respectively, specifically associated with arthritis. Flow cytometry revealed that majority of murine splenic and periarticular OCPs express Notch 2, whereas Notch 1 and 4 were expressed on approximately 10% of cells. In CIA, this highly osteoclastogenic population is expanded as is the expression of Notch 4 in PBM and Notch 3 in SPL. Majority of human peripheral-blood OCPs express Notch 2 and 4, with a specific increase in the expression of Notch 1 and 3 in RA. In contrast, RA synovial-derived OCPs mostly express Notch 1 to 3, whereas subchondral OCPs mostly express Notch 1 and 4. Notch ligands were analyzed at mRNA level and revealed expression of Jag1, Jag2 and DLL4 in murine sorted OCPs and Jag1 and DLL1 in human sorted OCPs. Expression of Notch 1 and 2 was confirmed by IHC on arthritic murine hind paws, with Notch 2 expressed by bone marrow, synovial tissue and chondrocytes and Notch 1 expressed by chondrocytes and synovial tissue. Increased expression of Notch 1, Notch 2 and Jag1 was also confirmed in murine arthritic periarticular tissue by qPCR. During osteoclastogenic culture, murine and human OCPs exhibit a similar gene expression pattern with higher initial expression of Notch 1 and 2, and increase in the expression of Notch 3 and 4 with differentiation. Osteoclasts were also differentiated under Notch-ligand stimulation. Coating with DLL1 results in a greater number of cells expressing osteoclast-specific TRAP, whereas Jag1 seemed to inhibit osteoclastogenesis.Conclusion:Our results indicate that murine and human OCPs express a distinct tissue-specific pattern of Notch receptors. Notch signaling in OCPs is increased in arthritis and may contribute to the osteoclastogenic potential and increased bone resorption. Our next aim would be to determine the effect of Notch inhibition on OCP activity and arthritis severity.Acknowledgments:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, IP-2014-09-7406 and DOK-2018-09-4276.Disclosure of Interests:None declared

Background: Autoimmunity is major driving force in pathogenesis of rheumatic diseases, including ankylosing spondylitis (AS). AS is associated with abnormal immune cell functions. Several recent studies stressed the role of T- cells and monocyte subpopulations in AS, but these mechanisms are signifi cantly less understood compared to other rheumatic diseases (1). Objectives: Th e aim was to compare the frequency of T-cell and monocyte subpopulations in AS patients and controls, and to correlate them with disease activity. Methods: Mononuclear cells (MNC) were isolated from blood of healthy controls (n=110) and AS (n=65) patients. T-cell and monocyte phenotype of MNC was determined using fl ow cytometry for the following markers: Th 1/2 (CD3+CD4+CCR6–), Th 17 (CD3+CD4+CCR4+CCR6+), Tfh (CD3+CD4+CXCR5+), Tc (CD3+CD8+), memory Tc (CD3+CD8+CCR4+) double- positive (dp) Tc (CD3+CD4+CD8+) ; and monocytes (CD3-CD19-CD56-CD11b+CD14+). On lymphocytes IL21R, CD25 and CD11b were analyzed ; while on monocytes chemokine receptors CCR1, CCR2, CCR4, CXCR4 and RANK. Frequencies of lymphocyte and monocyte subpopulations were correlated with ASDAS, BASDAI, VAS of pain in spine and peripheral joints, stiff ness intensity and duration, ESR and CRP. Results: Results showed decrease of memory Tc (p=0.022) and Tfh in AS (p=0.038) which correlated negatively with physician’s assessment of disease activity. Th e frequency of dp Tc was unchanged, but negatively correlated with BASDAI and pain assessment. Th e frequency of monocytes was decreased in AS (p

Background The 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). Methods PB 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. Results The 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. Conclusions In 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. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1337-6) contains supplementary material, which is available to authorized users.

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

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(-/lo)CD115(+)CD117(+)CX3CR1(+) and possibly also Ter119(-)CD11c(-)CD135(lo)Ly6C(+)RANK(-). In peripheral blood the OCP population bears the monocytoid phenotype B220(-)CD3(-)NK1.1(-)CD11b(+)Ly6C(hi)CD115(+)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.

To determine the frequency of advanced clinical and radiological features of AS with reference to gender, onset of symptoms and disease duration. Fifty-seven patients diagnosed with AS were included in this study. Functional evaluation of the musculoskeletal system detected advanced clinical features: rubber-ball phenomenon, flattening of the chest anterior wall, diastasis of rectus abdominis muscle, steel back phenomenon, umbilical extrusion, skiing posture. Conventional radiographs of sacroiliac joints, pelvis and axial skeleton were obtained in order to analyze signs of sacroiliitis, syndesmophytes, vertebral squaring and ligamentous ossification. Statistical significance is found in the distribution of particular advanced clinical and radiological features of AS between men and women: rubber-ball phenomenon (p = 0.002), flat chest (p = 0.002), diastasis of rectus abdominis muscle (p = 0.002), skiing position (p = 0.000), syndesmophytes (p = 0.009) and ligamentous ossification (p = 0.030) in thoracic and lumbar spine. Onset of first disease symptoms (20 years of age) is significantly associated with radiological changes in thoracic spine (ligamentous ossification, p = 0.015) and cervical spine (vertebral squaring, p = 0.032). Longer disease duration (10 years) is significantly associated with the appearance of particular clinical features: rubber-ball phenomenon, p0.01; rectus abdominis diastasis, p=0.042) and radiological changes of sacroiliac joints (grade IV sacroileitis, p = 0.012), thoracic and lumbar spine (syndesmophytes, p = 0.015; ligamentous ossification, p = 0.027). Our study shows that the occurrence of clinical and some radiological features of AS appears to be gender dependent. Furthermore, onset of first disease symptoms (20 years of age) and longer disease duration (10 years) are associated with the higher risk of developing particular clinical signs and radiological features in sacroiliac joints and axial skeleton.

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.

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).

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.

OBJECTIVE: The present study was designed to assess the effect of monthly ibandronate on health-related quality of life (HR-QoL) in patients with postmenopausal osteoporosis previously treated with weekly bisphosphonates. ----- METHODS: HR-QoL was assessed by Euroqol (EQ-5D) and Osteoporosis Targeted Quality of Life (OPTQoL) questionnaires. ----- RESULTS: The EQ-5D questionnaire showed significant improvement associated with ibandronate treatment, occurring in mobility (p < 0.01), usual activity (p < 0.01), pain/discomfort (p < 0.05), and anxiety/depression (p < 0.05). In addition, ibandronate treatment considerably improved patients' perceived health on a visual analog scale (p < 0.001). For the OPTQoL questionnaire, patients reported less physical difficulty (p < 0.001), fewer adaptations in their lives (p < 0.001), and less fear (p < 0.001) with ibandronate than with weekly bisphosphonates. ----- CONCLUSION: The study demonstrated that patients who were transferred from weekly bisphosphonates to a monthly ibandronate experienced improved HR-QoL.

Rheumatoid arthritis (RA) is marked by persistent inflammation and joint destruction involving infiltration of osteoclasts. Human osteoclast progenitors (OCPs) are contained among peripheral blood monocytes at low frequency, exhibit chemotaxis and, furthermore, synovial compartment of RA patients highly expresses different chemokines. The aim was to define these chemotactic signals. Mononuclear cells were isolated from peripheral blood of control and RA patients. The phenotype of OCPs (CD3-CD19-CD56-CD11b CD14 ) was determined using flow cytometry for receptors CCR1, CCR2, CCR4, CXCR4, C5AR1. Chemokine ligand concentrations (CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10) were measured in serum and synovial fluid of RA patients using cytometric bead assay. OCPs were sorted and cultured with M-CSF and RANKL. After two weeks, the cells were stained for TRAP enzyme and mature osteoclasts were counted. Human peripheral blood OCPs similarly expressed chemokine receptors in RA and healthy subjects. However, CCL4 and CXCL10 concentrations were significantly higher in synovial fluid compared to serum levels in RA, while CCL2, CXCL9 and CXCL10 serum levels were higher in RA patients compared to the control group. Cell culture revealed no significant differences in mature osteoclast count between groups. Although OCPs in RA have a differentiation potential similar to controls, levels of several chemokines are upregulated, indicating a possible chemotactic mechanism of OCP migration to affected joints. These results may help to reveal a migration mechanism of OCPs specifically associated with RA in order to develop more efficient therapeutic approaches.

Introduction. Rheumatoid arthritis (RA) is characterized by chronic inflammatory response as well as enhanced bone destruction. Development of bone erosions is critically dependent on osteoclasts, which are highly specialized bone cells capable of resorbing the mineralized matrix. Osteoclast progenitors (OCPs), contained among myeloid hematopoietic lineage, could be found in the peripheral blood and synovial tissue of patients with RA, mediating bone loss locally, in the form of bone erosions and joint osteolysis, and systemically, with loss of skeletal bone density. Within the affected joints, inflamed synovial tissue, with increased vascularity, hyperplasia and accumulation of immune cells (i.e. macrophages, plasma cells, T and B lymphocytes), invade adjacent structures (articular cartilage, cortical bone surface and underlying bone marrow) leading to typical symptoms of RA (joint pain, stiffness, swelling and structural changes). Under pathological conditions associated to arthritis, the process of osteoclastogenesis is markedly enhanced by various proinflammatory factors released by these infiltrating cells. The accumulation of inflammatory cells within the synovial compartment involves chemokine signals. It is known that synovial tissue and synovial fluid from RA patients highly express several chemokines and, furthermore, that OCPs exhibit chemotaxis as well. The aim of our study was to define these chemotactic signals by analyzing expression of several chemokines and chemokine receptors on T lymphocytes and OCPs in the peripheral blood of RA patients, to measure the levels of their respective ligands in serum and synovial fluid of RA patients, and to assess the osteoclastogenic potential of OCPs. Methods. Mononuclear cells were isolated from peripheral blood of healthy controls and RA patients. The phenotype of isolated mononuclear cells was determined using flow cytometry. OCPs (CD3-CD19- CD56-CD11b+CD14+) were analyzed for the expression of the following chemokine receptors: C5AR1, CCR1, CCR2, CCR4, CXCR4. T lymphocytes (CD3+CD4+ or CD3+CD8+) were analyzed for expression of CXCR5, CCR4, CCR6 chemokine receptors. Chemokine ligand concentrations (MIP-1α/CCL3, MIP-1β/CCL4, MCP- 1/CCL2, RANTES/CCL5) were measured in serum and synovial fluid of RA patients using flow cytometry bead based array. OCPs were sorted and plated into cell culture with M-CSF and RANKL. After two weeks, the cells were stained for TRAP enzyme and positive, mature, osteoclasts were counted. Results. Human peripheral blood OCPs similarly expressed chemokine receptors CCR1, CCR2, CCR4 and CXCR4 in RA and healthy subjects. However, MCP1/CCL2, MIP1a/CCL3 and MIP1b/CCL4 concentrations were significantly higher in synovial fluid (and blood for CCL2 and CCL4). Cell culture revealed no significant differences in mature osteoclast count between RA and control group. The proportion of T lymphocytes expressing CCR4 was two-fold higher in RA patients compared to controls. T lymphocyte expression of CXCR5 and CCR6 was similar between RA and control group. Conclusions. Although OCPs in RA have a differentiation potential similar to controls, levels of several chemokines are upregulated, indicating a possible chemotactic mechanism of OCP migration to affected joints. These results may help to reveal migration mechanism of T lymphocytes and OCPs specifically associated with RA in order to develop more efficient therapeutic approaches. This work has been supported by Croatian Science Foundation (project number 5699).

To determine the frequency of advanced clinical and radiological features of AS with reference to gender, onset of symptoms and disease duration. Fifty-seven patients diagnosed with AS were included in this study. Functional evaluation of the musculoskeletal system detected advanced clinical features: rubber-ball phenomenon, flattening of the chest anterior wall, diastasis of rectus abdominis muscle, steel back phenomenon, umbilical extrusion, skiing posture. Conventional radiographs of sacroiliac joints, pelvis and axial skeleton were obtained in order to analyze signs of sacroiliitis, syndesmophytes, vertebral squaring and ligamentous ossification. Statistical significance is found in the distribution of particular advanced clinical and radiological features of AS between men and women: rubber-ball phenomenon (p=0.002), flat chest (p=0.002), diastasis of rectus abdominis muscle (p=0.002), skiing position (p=0.000), syndesmophytes (p=0.009) and ligamentous ossification (p=0.030) in thoracic and lumbar spine. Onset of first disease symptoms (> 20 years of age) is significantly associated with radiological changes in thoracic spine (ligamentous ossification, p=0.015) and cervical spine (vertebral squaring, p=0.032). Longer disease duration (>10 years) is significantly associated with the appearance of particular clinical features: rubber-ball phenomenon, p20 years of age) and longer disease duration (>10 years) are associated with the higher risk of developing particular clinical signs and radiological features in sacroiliac joints and axial skeleton.

By the quantitative dermatoglyphic analysis of digitopalmar ridge count in fifty male psoriatic spondylitis patients were researched 25 dermatoglyphics traits: number of epidermal ridges on the all ten fingers, their sum for five and ten fingers, four traits on the both palms, i.e. between a-b, b-c, c-d and a-d triradii, and atd angles and their bilateral sum in degrees. The data obtained were compared with those recorded in a control group of 200 pairs of imprints of phenotypically healthy males from the Zagreb area. Statistically significant differences to control were found in 13 variables in decreased ridge count in second, third, fourth and fifth finger on the right palm, and in their sum on the all five fingers, than in second, third, fourth and fifth finger on the left palm, and in their sum in the all fingers, and in the all ten fingers. Atd angle was decreased on the left palm, and on the both palm together. Accordingly a polygenetic system identical in some loci to polygenetic system predisposing to male psoriatic spondylitis susceptibility might be found responsible for dermatogliyphic pattern development.

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.

PURPOSE: We 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. ----- METHODS: Paired 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). ----- RESULTS: Number 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. ----- CONCLUSION: Osteoclastogenic 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.

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 naive 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 naive 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 naive 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 naive 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

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: naive (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+ naive (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+ naive 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

Kvantitativna analiza dermatoglifa digitopalmarnog kompleksa u pedeset bolesnica s psorijatičnim monoartritisom

A prospective, open-labelled, multicentre 6-month study was designed to assess three categories that have high impact on Health-Related Quality of Life (HR-QoL). These categories were: satisfaction, preference and drug tolerability in postmenopausal patients with osteoporosis in Croatia, at first treated with weekly oral bisphosphonates, followed by monthly oral ibandronate. Three hundred eighty-five postmenopausal women who were treated with one of the weekly bisphosphonates for at least 6 months were included into the study and after they had signed written informed consent, the therapy was changed to monthly ibandronate. Satisfaction with the treatment was assessed with the Osteoporosis Patient Satisfaction Questionnaire (OPSAT-Q). Patients completed OPSAT-Q at the baseline visit before the change of therapy (visit 1) and 6 months after the change of therapy (visit 2). Following 6 months ibandronate therapy, the values in all four domains of the OPSAT-Q (convenience, confidence with daily activities, overall satisfaction, side effects) as well as in the Composite Satisfaction Score were higher in visit 2 (p

Kvantitativna analiza dermatoglifa digitopalmarnog kompleksa u dvadesetorice bolesnika s psorijatičnim oligoartritisom

Kvantitativna analiza dermatoglifa digitopalmarnog kompleksa u dvadesetorice bolesnika sa simetričnim psorijatičnim poliartritisom

Kvantitativna analiza dermatoglifa digitopalmarnog kompleksa u dvadeset bolesnica s mutilirajućim psorijatičnim artritisom

Quantitative dermatoglyphic analysis ofdigitopalmar ridge count was used to research psoriatic symmetrical polyarthritis in fifty women. Analyzed were 25 dermatoglyphics traits: number of epidermal ridges on all ten fingers, their sum for five and ten fingers, four traits on both palms, i.e., between a-b, b-c, c-d and a-d triradii, and atd angles and their bilateral sum. The data obtained were compared with those recorded in a control group of 200 pairs of imprints of phenotypically healthy females from Zagreb area. Statistically significant differences were found in 13 variables in decreased ridge count in all ten fingers, their sum in five and ten fingers separately. Accordingly, a polygenetic system identical in some loci to polygenic system predisposing to women psoriatic symmetrical polyarthritis susceptibility might be found responsible for the dermatoglyphic pattern development.

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.

Quantitative analysis of digitopalmar ridge count was performed in forty male patients with rheumatoid arthritis to evaluation of genetic factors in that disease. Twenty five variables (ridge count on each of ten fingers, their sum on five and ten fingers, four traits on each palm, i. e. ridge count between a-b, b-c and c-d triradii, their sum on each and both palm and at angle on two palms and their bilateral sum) were determined. The data thus obtained were compared with digitopalmar prints of 200 healthy men who served as a control group. A significant difference from the control group was found in eight variables. Ridge count was increased on the first and fifth finger bilaterally, on the fourth right finger tip, and their sum on each, and both fists. Accordingly, a polygenic system identical in some loci to the polygenic system predisposing to rheumatoid arthritis susceptibility might be found responsible for the dermatoglyphic pattern development. That means that they could used, and that is the aim of this study, as a diagnostic tool in rheumatic diseases., Kvantitativna analiza digitopalmarnih dermatoglifa provedena je u četrdesetorice muškaraca s reumatoidnim artritisom zbog procjene udjela genetičkih čimbenika u toj bolesti. Ispitano je 25 varijabli (broj kožnih grebenova na svakom od deset prstiju, njihov zbroj na pet i deset prstiju, četiri značajke na dlanovima: broj grebenova između a-b, b-c i c-d triradijusa, zatim njihov zbroj na svakom dlanu i obostrano, te atd kutovi na svakom dlanu i njihov obostrani zbroj). Dobiveni podaci uspoređeni su s dvjesto pari otisaka zdravih muškaraca koji su poslužili kao kontrolna skupina. Statistički značajne razlike u odnosu na kontrolnu skupinu, u smislu povećanog broja grebenova, nađene su na prvom i petom prstu obostrano, te četvrtom desno, zatim njihovu zbroju na svakoj šaci posebno i obje zajedno. Prema tome, moguće je pretpostaviti kako je poligenetički sustav odgovoran za razvoj dermatoglifa identičan s nekim lokusima za oboljevanje od reumatoidnog artritisa u muškaraca, što znači kako bi se dermatoglifi mogli iskoristiti kao dijagnostičko sredstvo u reumatskim bolestima.

Alkaptonuria is a rare metabolic disorder characterized by a deficiency of the homogentisic acid oxidase enzyme. The result is accumulation of homogentisic acid in collagenous structures throughout the body, especially in fibrous and cartilaginous tissue. This leads to gradual development of a phenomenon known as ochronosis. Characteristic features of ochronosis are urine darkening, progressive scleral pigmentation, subcutaneous cartilage pigmentation (for example, ear cartilage and nails) and degenerative ochronotic arthropathy resembling osteoarthritis. In addition, cardiovascular and genitourinary systems are also affected. Alkaptonuric ochronosis is particularly interesting because it can be detected based only on clinical signs and medical history. Herein we present a patient with typical signs and symptoms such as darkening of urine, pigmented sclerae, nails and ear cartilage, manifesting arthritis in his fifth decade. Additional clinical examination indicated alkaptonuria, which was unrecognized in childhood.

Spondyloarthritides (SpA) as a group are one of the most common rheumatic disorders with a predominant affection of the spine. Conventinal disease modifying antirheumatic drugs which are effective in rheumatoid arthritis have poor effect on spinal inflammation. Today there is confirmed efficacy ofbiologics in spondylitis. This therapy is expensive and potentially hazaradous. Croatian Society for Rheumatology set up recommendations for the use of TNF-alpha blockers in SpA. There are several important points to be considered before their use: diagnosis of Spa, duration and disease activity, previous therapy and it's efficacy, application and efficacy ofbiologics, contraindications and safety preacutions and finally a decision for continuous tretament with biologics.

The purpose was to determine the efficacy of therapeutic ultrasound in patients with chronic low back pain. Thirty-one patients, age 38-77, with low back pain lasting more than three months and the intensity of pain on visual analogue scale at least 50 mm, are randomly divided in two groups. Ultrasound is applied on the lumbar paravertebral muscle in 16 patients and in 15 patients the machine was not switched on. All patients also underwent kinesitherapy. Pharmacological treatment was not changed during the research (except the possibility of using paracetamol as the "rescue drug"). Following parameters were measured at the beginning and at the end of the research: pain intensity on the visual analogue scale/mm, modified Schober measure/cm, patient's and physician's global assessment of treatment efficacy (1-5 scale). The intensity of pain in the ultrasound group before the treatment was 82,7+/-14,0 and after the treatment 79,8+/-12,2 (p0,05). The intensity of pain in the placebo group before the treatment was 81,7+/-12,1 and at the end of the treatment 78,9+/-12,1 (p0,05). The value of the modified Schober measure for the ultrasound group were 5,7+0,8 cm vs. 5,8+/-0,9 cm (p0,05) and in the placebo group were 5,4+/-0,9 cm vs. 5,6+/-1,0 cm (p0,05). There was no significant statistical difference between ultrasound and placebo group regarding the efficacy of the treatment (patients p0,05, physicians p0,05). Therapeutic ultrasound was effective in decreasing the pain intensity in this research, but showed no improvement regarding the functional ability of the lumbar spine in patients with chronic low back pain.

Spondiloartritisi (SpA) su velika skupina reumatskih bolesti s dominantnom afekcijom aksijalnog skeleta u većine bolesnika. Klasični temeljni lijekovi za liječenje reumatoidnog artritisa su nedjelotovorni u spondilitisu dok biološki preparati imaju dokazan učinak kod svih vrsta spondilitisa. Kako je terapija inhibitorima TNF-α skupa i potencijalno opasna na sastanku Hrvatskog reumatološkog društva dogovoren je prijedlog za njihovu primjenu. Prijedlog sadrži nekoliko osnovnih točaka koje valja respektirati kod donošenja odluke o primjeni antagonista TNF-α, a to su: dijagnoza SpA, trajanje i aktivnost bolesti, vrsta i djelotvornost prethodne terapije, primjena i učinak bioloških lijekova, kontraindikacije i mjere opreza te konačna odluka o trajnoj primjeni antagonista TNF-α., Spondyloarthritides (SpA) as a group are one of the most common rheumatic disorders with a predominant affection of the spine. Conventinal disease modifying anti­rheumatic drugs which are effective in rheumatoid arthritis have poor effect on spinal inflammation. Today there is confirmed efficacy of biologics in spondylitis. This therapy is expensive and potentially hazaradous. Croatian Society for Rheumatology set up recommendations for the use of TNF-α blockers in SpA. There are several important points to be considered before their use: diagnosis of Spa, duration and disease activity, previous therapy and it’s efficacy, application and efficacy of biologics, contraindications and safety preacutions and finally a decision for continuous tretament with biologics.

Background Rheumatoid arthritis (RA) is characterized by chronic inflammatory response as well as enhanced bone destruction. Development of bone erosions is critically dependent on osteoclasts, which are highly specialized bone cells capable of resorbing the mineralized matrix. Osteoclast progenitors (OCPs), contained among myeloid hematopoietic lineage, could be found in the peripheral blood and synovial tissue of patients with RA, mediating bone loss locally, in the form of bone erosions and joint osteolysis, and systemically, with loss of skeletal bone density. Objectives To define these chemotactic signals by analyzing expression of several chemokines and chemokine receptors on T lymphocytes and OCPs in the peripheral blood of RA patients, to measure the levels of their respective ligands in serum and synovial fluid of RA patients, and to assess the osteoclastogenic potential of OCPs. Methods Mononuclear cells were isolated from peripheral blood of healthy controls and RA patients. The phenotype of isolated mononuclear cells was determined using flow cytometry. OCPs (CD3-CD19-CD56-CD11b+CD14+) were analyzed for the expression of the following chemokine receptors: C5AR1, CCR1, CCR2, CCR4, CXCR4. T lymphocytes (CD3+CD4+ or CD3+CD8+) were analyzed for expression of CXCR5, CCR4, CCR6 chemokine receptors. Chemokine ligand concentrations (MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, RANTES/CCL5) were measured in serum and synovial fluid of RA patients using flow cytometry bead based array. OCPs were sorted and plated into cell culture with M-CSF and RANKL. After two weeks, the cells were stained for TRAP enzyme and positive, mature, osteoclasts were counted. Results Human peripheral blood OCPs similarly expressed chemokine receptors CCR1, CCR2, CCR4 and CXCR4 in RA and healthy subjects. However, MCP1/CCL2, MIP1a/CCL3 and MIP1b/CCL4 concentrations were significantly higher in synovial fluid (and blood for CCL2 and CCL4). Cell culture revealed no significant differences in mature osteoclast count between RA and control group. The proportion of T lymphocytes expressing CCR4 was two-fold higher in RA patients compared to controls. T lymphocyte expression of CXCR5 and CCR6 was similar between RA and control group. Conclusions Although OCPs in RA have a differentiation potential similar to controls, levels of several chemokines are upregulated, indicating a possible chemotactic mechanism of OCP migration to affected joints. These results may help to reveal migration mechanism of T lymphocytes and OCPs specifically associated with RA in order to develop more efficient therapeutic approaches. Acknowledgements This work has been supported by Croatian Science Foundation (project number 5699). Disclosure of Interest None declared

The patterns of the ridges of the skin of the fingers and palms were determined in sixty men with complex regional pain syndrome (type I) as a measure of disease prevention. The study included 25 dermatoglyphic traits: number of epidermal ridges on all ten fingers; their sum for five and ten fingers; four traits on both palms, i.e. between a-b, b-c and c-d triradii; atd angles: and their bilateral sum. The data obtained were compared with those recorded in a control group of 200 pairs of imprints of phenotipycally healthy male adults from the Zagreb area. Statistically significant difference from control values were found in 12 dermatoglyphic variables, including an increased sum of ridges on nine fingers (except for left second finger pad), and total sum for five and ten fingers. These findings suggested the polygenic system responsible for development of dermatoglyphics to be identical with some polygenic loci for the onset of algodystrophy syndrome, which might prove useful in disease prevention (e.g., taking fingerprints following a trauma and before rehabilitation), and to facilitate identification of risk groups, and thus the treatment for this longterm and yet obscure syndrome.

Cilj istraživanja bio je odrediti učinkovitost primjene terapijskog ultrazvuka u liječenju bolesnika s kroničnom križoboljom. Trideset jedan bolesnik u dobi 38-77 godina, s križoboljom koja traje najmanje tri mjeseca i u kojih je stupanj boli prema vrijednosti na vizualnoj analognoj skali (VAS) bio iznad 50 mm, metodom su slučajnog izbora podijeljeni u dvije skupine. U 16 je bolesnika ultrazvuk primijenjen paravertebralno u području slabinske kralježnice, a u ostalih 15 bolesnika ultrazvučni aparat nije bio uključen. Svi su bolesnici provodili medicinsku gimnastiku, a medikamentna terapija tijekom istraživanja nije mijenjana (osim mogućnosti uzimanja paracetamola kao “lijeka za spas”). Prije i nakon provedenih deset terapija mjereni su sljedeći parametri: stupanj boli primjenom vizualne analagone skale (VAS/mm), modificirana Schoberova mjera/mm, bolesnikova i liječnikova globalna ocjena učinka liječenja (ljestvica 1-5). Stupanj boli je za UZV skupinu prije početka liječenja bio 82,7±14,0 mm, a nakon liječenja 79,8±12,2 mm (p0,05). Nije bilo statistički značajne razliku u vrijednosti modificirane Schoberove mjere u skupini na aktivnom liječenju i u placebo skupini prije i nakon liječenja (u oba slučaja p>0,05). Između dvije skupine bolesnika (UZV i placebo) nije bilo statistički značajne razlike niti u bolesnikovoj i liječnikovoj ocjeni učinka liječenja (p>0,05, p>0,05). U ovom je istraživanju primjena terapijskog ultrazvuka bila učinkovita u smanjenju boli, ali ne i u poboljšanju funkcijskog statusa slabinske kralježnice kao niti ocjeni bolesnika ili liječnika u bolesnika s kroničnom križoboljom., The purpose was to determine the efficacy of therapeutic ultrasound in patients with chronic low back pain. Thirty-one patients, age 38-77, with low back pain lasting more than three months and the intensity of pain on visual analogue scale at least 50 mm, are randomly divided in two groups. Ultrasound is applied on the lumbar paravertebral muscle in 16 patients and in 15 patients the machine was not switched on. All patients also underwent kinesitherapy. Pharmacological treatment was not changed during the research (except the possibility of using paracetamol as the “rescue drug”). Following parameters were measured at the beginning and at the end of the research: pain intensity on the visual analogue scale/mm, modified Schober measure/cm, patient’s and physician’s global assessment of treatment efficacy (1-5 scale). The intensity of pain in the ultrasound group before the treatment was 82,7±14,0 and after the treatment 79,8±12,2 (p0,05). The value of the modified Schober measure for the ultrasound group were 5,7±0,8 cm vs. 5,8±0,9 cm (p>0,05) and in the placebo group were 5,4±0,9 cm vs. 5,6±1,0 cm (p>0,05). There was no significant statistical difference between ultrasound and placebo group regarding the efficacy of the treatment (patients p>0,05, physicians p>0,05). Therapeutic ultrasound was effective in decreasing the pain intensity in this research, but showed no improvement regarding the functional ability of the lumbar spine in patients with chronic low back pain.

Većina bolesnika s reumatoidnim artritisom ima radiološke promjene na zglobovima šaka i/ili koljena u smislu osteoartritisa. Raspodjela tih promjena je skoro podjednaka: na šake, koljena, šake i koljena. Osteoartritis se češće nađe u starijih bolesnika s reumatoidnim artritisom, dok nije nađena povezanost s trajanjem reumatoidnog artritisa.

The patterns of the ridges of the skin of the fingers and palms were determined in sixty men with complex regional pain syndrome (type I) as a measure of disease prevention. The study included 25 dermatoglyphic traits: number of epidermal ridges on all ten fingers; their sum for five and ten fingers; four traits on both palms, i.e. between a-b, b-c and c-d triradii; atd angles: and their bilateral sum. The data obtained were compared with those recorded in a control group of 200 pairs of imprints of phenotipycally healthy male adults from the Zagreb area. Statistically significant difference from control values were found in 12 dermatoglyphic variables, including an increased sum of ridges on nine fingers (except for left second finger pad), and total sum for five and ten fingers. These findings suggested the polygenic system responsible for development of dermatoglyphics to be identical with some polygenic loci for the onset of algodystrophy syndrome, which might prove useful in disease prevention (e.g., taking fingerprints following a trauma and before rehabilitation), and to facilitate identification of risk groups, and thus the treatment for this longterm and yet obscure syndrome., U svrhu prevencije, u ovom se genetičkom ispitivanju, istražio broj kožnih grebenova na prstima i dlanovima šezdesetorice muškaraca s kompleksnim regionalnim sindromom Tip I (Syndroma algodystrophicum). Ispitivanje je provedeno u 25 dermatoglifskih značajki: broju epidermalnih grebenova na svih deset prstiju, njihovu sveukupnom zbroju na pet i deset prstiju, četirima svojstvima na oba dlana - između triradijusa a-b, b-c i c-d, te atd kutovima, i njihovom obostranom zbroju. Dobiveni podaci su uspoređeni s kontrolnom skupinom - 200 pari otisaka odraslih i fenotipskih zdravih osoba Zagrebačke regije. U dvanaest dermatoglifskih varijabli pronađene su statistički značajne razlike prema kontroli: u povišenom broju grebenova na devet prstiju (izuzev jagodice drugog prsta lijevo), te u ukupnom zbroju na pet i deset prstiju zajedno. Iz toga se može pretpostaviti, kako je poligenetički sustav odgovoran za razvoj dermatoglifa identičan s nekim lokusima za oboljevanje od algodistrofičnog sindroma, a što bi se moglo iskoristiti u preventivne svrhe (uzimanjem otisaka ruku nakon traume, odnosno prije rehabilitacije), te formiranjem rizičnih skupina, a time i usmjeriti liječenje tog dugotrajnog i tajanstvenog poremećaja.