Classification of rheumatoid arthritis: is it time to revise the criteria?

Classification criteria have been developed for rheumatoid arthritis (RA) and other rheumatic diseases in order to gather a homogeneous patient population for clinical studies and facilitate the timely implementation of therapeutic measures. Although classification criteria are not intended to be used for diagnosis, they are frequently used to support the diagnostic process in clinical practice, including clinical decision-making. The 2010 American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) classification criteria for RA are capable of identifying the majority of symptomatic patients with RA already in the earliest stages of the disease who are not yet showing radiographic changes. These patients will also profit from the early implementation of therapy with disease-modifying antirheumatic drugs (DMARDs). However, the risk of misclassification is higher as compared with the former 1987 ACR criteria, which were considerably less sensitive to the recognition of patients with early RA. Of note, the presence of rheumatoid factors (RFs) and anticitrullinated protein antibodies (ACPAs) has been attributed equal weight in the 2010 ACR/EULAR criteria and may contribute up to 50% of the score needed for being classified as RA. However, while ACPAs have been proven to be the most specific serological markers of RA, the specificity of RF is moderate, especially at lower titres. This may lead to the misclassification of RF-positive patients and, consequently, the unjustified implementation of DMARD therapy. Therefore, issues arise on how comprehensive the criteria should be and whether they should be updated and adapted to findings from the past two decades that might increase both their specificity and sensitivity.

Should ACR/EULAR criteria be revised changing the RF and ACPA scores?

Current classification criteria for rheumatoid arthritis (RA) encompass clinical and immunological items and are capable of correctly identifying the majority of symptomatic RA patients. The presence of positive rheumatoid factor (RF) and/or and anti-cyclic citrullinated protein/peptide antibodies (ACPA) gaining increasing importance according to their serological titer eases the recognition of RA, yet the debate is open on whether this scoring system ought to be optimized by hierarchizing ACPA or the combination of ACPA and RF over single positivity, prioritizing specificity over sensitivity. The risk of misdiagnosis and misclassification are often entangled, yet they are not the same. In fact, while ideal diagnosis requires 100% sensitivity and specificity, classification criteria are conceived to gather a homogeneous patient population, favoring specificity over sensitivity. Nevertheless, as they are frequently summoned to support the diagnostic process in clinical practice, issues arise on how comprehensive those should be and on how frequently they should be updated in light of novel acquisitions regarding measurable RA-related abnormalities. In this viewpoint two different views on the topic are confronted, discussing the performance of available criteria and the potentiality and pitfalls of their refinement according to novel data on ACPA and RF contribution and emergence of newly discovered specificities.

Bona fide dendritic cells are pivotal precursors for osteoclasts.

OBJECTIVES: Osteoclasts (OCs) are myeloid-derived multinucleated cells uniquely able to degrade bone. However, the exact nature of their myeloid precursors is not yet defined. METHODS: CD11c-diphtheria toxin receptor (CD11cDTR) transgenic mice were treated with diphtheria toxin (DT) or phosphate buffered saline (PBS) during serum transfer arthritis (STA) and human tumour necrosis factor transgenic (hTNFtg) arthritis and scored clinically and histologically. We measured cytokines in synovitis by quantitative polymerase chain reaction (qPCR). We performed ovariectomy in CD11cDTR mice treated with PBS or DT. We analysed CD11cDTR, CD11c-Cre/CX3CR1-STOP-DTR and Zbtb46-DTR-treated mice with DT using histomorphometry and OC of CD11c and Zbtb46 fate reporter mice by fluorescent imaging. We sorted murine and human OC precursors and stimulated them with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) to generate OCs. RESULTS: Targeting CD11c+ cells in vivo in models of inflammatory arthritis (STA and hTNFtg) ameliorates arthritis by reducing inflammatory bone destruction and OC generation. Targeting CD11c-expressing cells in unchallenged mice removes all OCs in their long bones. OCs do not seem to be derived from CD11c+ cells expressing CX3CR1+, but from Zbtb46+conventional dendritic cells (cDCs) as all OCs in Zbtb46-Tomato fate reporter mice are Tomato+. In line, administration of DT in Zbtb46-DTR mice depletes all OCs in long bones. Finally, human CD1c-expressing cDCs readily differentiated into bone resorbing OCs. CONCLUSION: Taken together, we identify DCs as important OC precursors in bone homeostasis and inflammation, which might open new avenues for therapeutic interventions in OC-mediated diseases.

Cytokine-directed cellular cross-talk imprints synovial pathotypes in rheumatoid arthritis.

INTRODUCTION: Structural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a critical component driving synovial tissue remodelling by interacting with immune cells resulting in distinct synovial pathotypes of RA. METHODS: Automated high-content fluorescence microscopy of co-cultured cytokine-activated FLS and autologous peripheral CD4+ T cells from patients with RA was established to quantify cell-cell interactions. Phenotypic profiling of cytokine-treated FLS and co-cultured T cells was done by flow cytometry and RNA-Seq, which were integrated with publicly available transcriptomic data from patients with different histological synovial pathotypes. Computational prediction and knock-down experiments were performed in FLS to identify adhesion molecules for cell-cell interaction. RESULTS: Cytokine stimulation, especially with TNF-α, led to enhanced FLS-T cell interaction resulting in cell-cell contact-dependent activation, proliferation and differentiation of T cells. Signatures of cytokine-activated FLS were significantly enriched in RA synovial tissues defined as lymphoid-rich or leucocyte-rich pathotypes, with the most prominent effects for TNF-α. FLS cytokine signatures correlated with the number of infiltrating CD4+ T cells in synovial tissue of patients with RA. Ligand-receptor pair interaction analysis identified ICAM1 on FLS as an important mediator in TNF-mediated FLS-T cell interaction. Both, ICAM1 and its receptors were overexpressed in TNF-treated FLS and co-cultured T cells. Knock-down of ICAM1 in FLS resulted in reduced TNF-mediated FLS-T cell interaction. CONCLUSION: Our study highlights the role of cytokine-activated FLS in orchestrating inflammation-associated synovial pathotypes providing novel insights into disease mechanisms of RA.

Antibodies against citrullinated proteins of IgA isotype are associated with progression to rheumatoid arthritis in individuals at-risk.

OBJECTIVE: Events triggering disease outbreak in individuals at-risk for rheumatoid arthritis (RA at-risk) remain unclear, and the role of the various anticitrullinated protein antibody (ACPA) isotypes in this process is still to be established. We aimed to investigate the prevalence of IgA ACPA in RA at-risk individuals, their role in the transition from the RA at-risk status to RA and their dynamics during this transition. METHODS: Cross-sectional measurement of serum IgA1 and IgA2 ACPA levels was conducted in healthy controls, RA at-risk individuals and patients with RA and compared with the frequency of RA development in at risk individuals during a follow-up of 14 months. In addition, longitudinal measurements of serum IgA1 and IgA2 ACPA levels prior to, at and after the onset of RA were performed. RESULTS: Approximately two-thirds of RA at-risk individuals were positive for serum IgA1 and IgA2 ACPA in levels comparable to IgG ACPA positive patients with RA. IgA1, but not IgA2 ACPA positivity was associated with the transition from the RA at-risk state to RA within the following 14 months. Interestingly, during this transition process, IgA1 ACPA levels declined at RA onset and also thereafter during the early phase of RA. This decline was confirmed in a second, independent cohort. CONCLUSION: Both IgA1 and IgA2 ACPA are present in RA at-risk individuals, but only IgA1 ACPA are associated with the progression to RA. The observed decline in serum IgA1 ACPA levels before the onset of RA might indicate starting barrier leakiness prior to disease outbreak.

IgA rheumatoid factor in rheumatoid arthritis.

OBJECTIVES: Rheumatoid factor (RF) is a well-established marker for the diagnosis and classification of rheumatoid arthritis (RA). Most studies evaluated IgM RF or isotype-nonspecific total RF assays. We evaluated the added value of IgA RF in this context. METHODS: An international sample cohort consisting of samples from 398 RA patients and 1073 controls was tested for IgA RF with 3 commercial assays. For all RA patients and 100 controls essential clinical and serological data for ACR/EULAR classification were available. RESULTS: The sensitivity of IgA RF for diagnosing RA was lower than the sensitivity of IgM RF. Differences in numerical values between IgA RF assays were observed. With all assays, the highest IgA RF values were found in patients with primary Sjögren's syndrome. Double positivity for IgM RF and IgA RF had a higher specificity for RA than either IgM RF or IgA RF. The sensitivity of double positivity was lower than the sensitivity of either IgA RF or IgM RF. Single positivity for IgA RF was at least as prevalent in controls than in RA patients. Adding IgA RF to IgM RF and anti-citrullinated protein antibodies (ACPA) did not affect RA classification. However, combined positivity for IgA RF, IgM RF and IgG ACPA had a higher specificity and lower sensitivity for RA classification than positivity for either of the antibodies. CONCLUSIONS: IgA RF showed a lower sensitivity than IgM RF. Combining IgA RF with IgM RF and ACPA did not improve sensitivity of RA classification. Combined positivity (IgA-RF/IgM-RF/ACPA) increased specificity.

Multicentre study to improve clinical interpretation of rheumatoid factor and anti-citrullinated protein/peptide antibodies test results.

BACKGROUND: Rheumatoid factor (RF) and anti-citrullinated protein/peptide antibodies (ACPA) are important biomarkers for diagnosis of rheumatoid arthritis (RA). However, there is poor harmonisation of RF and ACPA assays. The aim of this study was to refine RF and ACPA interpretation across commercial assays. MATERIALS AND METHODS: Six total RF isotype-non-specific assays, 3 RF IgM isotype-specific assays and 9 ACPA immunoglobulin G assays of 13 different companies were evaluated using 398 diagnostic samples from patients with RA and 1073 disease controls. RESULTS: Using cut-offs proposed by the manufacturer, there was a large variability in diagnostic sensitivity and specificity between assays. Thresholds of antibody levels were determined based on predefined specificities and used to define test result intervals. Test result interval-specific likelihood ratios (LRs) were concordant across the different RF and ACPA assays. For all assays, the LR for RA increased with increasing antibody level. Higher LRs were found for ACPA than for RF. ACPA levels associated with LRs >80 were found in a substantial fraction (>22%) of patients with RA. CONCLUSION: Defining thresholds for antibody levels and assigning test result interval-specific LRs allows alignment of clinical interpretation for all RF and ACPA assays.

Improving the measurement of oral health-related quality of life: Rasch model of the oral health impact profile-14.

OBJECTIVES: The 14-item Oral Health Impact Profile (OHIP-14) is the most frequently used instrument to measure oral health-related quality-of-life (OHRQoL) in adults. Despite its popularity, its psychometric properties have been predominantly investigated based on the classical test theory while the fundamental principles of measurement have not been fully assessed. Therefore, our aim was to investigate to what extent the OHIP-14 meets the fundamental requirments of measurements. METHODS: We used the Rasch model to explore person-item-targeting, unidimensionality, local independence of items, invariance (differential-item-functioning, DIF), and the order of thresholds between response-options in the German version OHIP-14. We used data from osteoarthritis patients because hand disabilities and joint pain might influence oral hygiene. Furthermore, osteoarthritis in the temporomandibular-joint directly affects oral functioning. RESULTS: Five-hundred sixteen patients were included (mean age 66.5 years [±10.2; ranging from 34 to 89]; 71.3% [368] females). The OHIP-14 median total score was 0 (interquartile-range from 0 to 4), indicating a right-skewed distribution because many patients reported good OHRQoL. The instrument was found unidimensional. However, there was strong evidence of local dependency, disordered thresholds between response-options, and age-related DIF for item 5. A revised scoring scheme with three instead of five answer-options in all items and eliminating two items resolving local dependency, the newly adapted OHIP-12, showed better reliability and item-fit to the Rasch model than the original OHIP-14. CONCLUSIONS: This study assesses, for the first time, the OHIP-14 in terms of fundamental principles of measurement and proposes an item-reduced OHIP-12 as a psychometrically more accurate version of the instrument. CLINICAL SIGNIFICANCE: The Rasch model is essential to ensure instruments' precision and clinical meaningfulness when measuring OHRQoL in clinical practice and research. The OHIP-12, derived from the OHIP-14 by deleting two items due to local dependency, with a revised scoring scheme for all items distinguishing three answer-options instead of five, represents a psychometrically improved version of the instrument.

Characterization of the Inducible and Slow-Releasing Hydrogen Sulfide and Persulfide Donor P*: Insights into Hydrogen Sulfide Signaling.

Hydrogen sulfide (H2S) is an important mediator of inflammatory processes. However, controversial findings also exist, and its underlying molecular mechanisms are largely unknown. Recently, the byproducts of H2S, per-/polysulfides, emerged as biological mediators themselves, highlighting the complex chemistry of H2S. In this study, we characterized the biological effects of P*, a slow-releasing H2S and persulfide donor. To differentiate between H2S and polysulfide-derived effects, we decomposed P* into polysulfides. P* was further compared to the commonly used fast-releasing H2S donor sodium hydrogen sulfide (NaHS). The effects on oxidative stress and interleukin-6 (IL-6) expression were assessed in ATDC5 cells using superoxide measurement, qPCR, ELISA, and Western blotting. The findings on IL-6 expression were corroborated in primary chondrocytes from osteoarthritis patients. In ATDC5 cells, P* not only induced the expression of the antioxidant enzyme heme oxygenase-1 via per-/polysulfides, but also induced activation of Akt and p38 MAPK. NaHS and P* significantly impaired menadione-induced superoxide production. P* reduced IL-6 levels in both ATDC5 cells and primary chondrocytes dependent on H2S release. Taken together, P* provides a valuable research tool for the investigation of H2S and per-/polysulfide signaling. These data demonstrate the importance of not only H2S, but also per-/polysulfides as bioactive signaling molecules with potent anti-inflammatory and, in particular, antioxidant properties.

CCR6 controls autoimmune but not innate immunity-driven experimental arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, characterized by synovial infiltration of various inflammatory cells. Chemokines are involved in controlling the recruitment of different cell types into the synovial membrane. The role of CCR6 in the development of arthritis so far remains unclear. In this study, we investigated the role of CCR6 in the pathogenesis of arthritis using three different murine arthritis models. Compared to WT animals, CCR6-/- mice developed less clinical signs of arthritis in the collagen-induced arthritis model but not in the K/BxN serum transfer arthritis model and in the human tumour necrosis factor transgenic arthritis model, suggesting a defect in adaptive effector functions but intact innate effector functions in the development of arthritis in CCR6-/- animals. In line with this, anti-collagen antibody levels were significantly reduced in CCR6-/- mice compared with WT mice. Moreover, we demonstrate enhanced osteoclastogenesis in vitro in CCR6-/- mice compared with WT mice. However, we did not detect differences in bone mass under steady state conditions in vivo between WT and CCR6-deficient mice. These data suggest that CCR6 is crucially involved in adaptive but not in innate immunity-driven arthritis. CCR6 or its chemokine ligand CCL20 might represent a possible new target for the treatment of RA.

Contribution of Genetic Factors to Lower DHEAS in Patients with Rheumatoid Arthritis.

OBJECTIVE: Lower production of adrenal androgens has been confirmed in females with rheumatoid arthritis (RA); however, the mechanisms of this finding are not completely understood. The aim of our study was to assess the contribution of genetic factors associated with variability of dehydroepiandrosterone sulfate (DHEAS) levels to lower DHEAS in female RA patients. METHODS: 448 RA and 648 healthy controls were genotyped for single-nucleotide polymorphisms (SNPs) in genes ZKSCAN5 (rs11761528), SULT2A1 (rs2637125), HHEX (rs2497306), and ARPC1A (rs740160). Serum DHEAS concentrations were measured in 112 RA patients and 91 healthy women. RESULTS: The allele frequencies in DHEAS-related loci were similar in RA and controls. RA patients had significantly lower serum DHEAS concentrations compared to healthy women. The cumulative number of alleles associated with lower DHEAS within genes ZKSCAN5, SULT2A1, HHEX, and ARPC1A present in each individual negatively correlated with DHEAS levels in RA patients, but not in controls. Linear regression analysis showed significant effect of polymorphisms in genes ZKSCAN5 and ARPC1A on serum DHEAS levels in female RA patients but not in the control group. CONCLUSION: Our findings suggest that complex interactions exist between genotype and adrenal androgen hypofunction in RA.

Chloroquine inhibits human CD4+ T-cell activation by AP-1 signaling modulation.

Chloroquine (CQ) is widely used as an anti-inflammatory therapeutic for rheumatic diseases. Although its modes of action on the innate immune system are well described, there is still insufficient knowledge about its direct effects on the adaptive immune system. Thus, we evaluated the influence of CQ on activation parameters of human CD4+ T-cells. CQ directly suppressed proliferation, metabolic activity and cytokine secretion of T-cells following anti-CD3/anti-CD28 activation. In contrast, CQ showed no effect on up-regulation of T-cell activation markers. CQ inhibited activation of all T helper cell subsets, although IL-4 and IL-13 secretion by Th2 cells were less influenced compared to other Th-specific cytokines. Up to 10 µM, CQ did not reduce cell viability, suggesting specific suppressive effects on T-cells. These properties of CQ were fully reversible in re-stimulation experiments. Analyses of intracellular signaling showed that CQ specifically inhibited autophagic flux and additionally activation of AP-1 by reducing phosphorylation of c-JUN. This effect was mediated by inhibition of JNK catalytic activity. In summary, we characterized selective and reversible immunomodulatory effects of CQ on human CD4+ T-cells. These findings provide new insights into the biological actions of JNK/AP-1 signaling in T-cells and may help to expand the therapeutic spectrum of CQ.

Cartilage damage and bone erosion are more prominent determinants of functional impairment in longstanding experimental arthritis than synovial inflammation.

Chronic inflammation of articular joints causing bone and cartilage destruction consequently leads to functional impairment or loss of mobility in affected joints from individuals affected by rheumatoid arthritis (RA). Even successful treatment with complete resolution of synovial inflammatory processes does not lead to full reversal of joint functionality, pointing to the crucial contribution of irreversibly damaged structural components, such as bone and cartilage, to restricted joint mobility. In this context, we investigated the impact of the distinct components, including synovial inflammation, bone erosion or cartilage damage, as well as the effect of blocking tumor necrosis factor (TNF) on functional impairment in human-TNF transgenic (hTNFtg) mice, a chronic inflammatory erosive animal model of RA. We determined CatWalk-assisted gait profiles as objective quantitative measurements of functional impairment. We first determined body-weight-independent gait parameters, including maximum intensity, print length, print width and print area in wild-type mice. We observed early changes in those gait parameters in hTNFtg mice at week 5 - the first clinical signs of arthritis. Moreover, we found further gait changes during chronic disease development, indicating progressive functional impairment in hTNFtg mice. By investigating the association of gait parameters with inflammation-mediated joint pathologies at different time points of the disease course, we found a relationship between gait parameters and the extent of cartilage damage and bone erosions, but not with the extent of synovitis in this chronic model. Next, we observed a significant improvement of functional impairment upon blocking TNF, even at progressed stages of disease. However, blocking TNF did not restore full functionality owing to remaining subclinical inflammation and structural microdamage. In conclusion, CatWalk gait analysis provides a useful tool for quantitative assessment of functional impairment in inflammatory destructive arthritis. Our findings indicate that cartilage damage and bone erosion, but not synovial inflammation, are the most important determinants for progressive functional impairment in this chronic erosive arthritis model.

In Vitro Study of a Liposomal Curcumin Formulation (Lipocurc™): Toxicity and Biological Activity in Synovial Fibroblasts and Macrophages.

BACKGROUND/AIM: The polyphenol curcumin is produced in the rhizome of Curcuma longa and exhibits potent anti-inflammatory, antioxidant, and chemopreventive activities. Due to the fact that curcumin is poorly soluble in water, many delivery systems have been developed to improve its solubility and bioavailability achieving optimum therapeutic application. In this study, we evaluated the biological effects of a liposomal curcumin formulation (Lipocurc™) on human synovial fibroblasts (SW982) and mouse macrophages (RAW264). MATERIAL AND METHODS: Cellular uptake of liposomes was studied using calcein-loaded liposomes. Effects of Lipocurc™ on cell viability and proliferation were determined with Celltox green cytotoxicity assay and 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, respectively. To induce cytokine/chemokine expression, the cells were stimulated with interleukin (IL)1ß or lipopolysaccharide (LPS). The release of IL6, IL8, and tumor necrosis factor-alpha (TNFα) was quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: Data showed that the liposomal curcumin formulation Lipocurc™ was significantly less toxic to synovial fibroblasts and macrophages compared to non-encapsulated, free curcumin. Furthermore, Lipocurc™ effectively reduced pro-inflammatory cytokine/chemokine expression in synovial fibroblasts as well as in macrophages without affecting cell viability, suggesting that this curcumin nanoformulation might be a promising tool for the treatment of inflammatory diseases.

A Combination of CD28 (rs1980422) and IRF5 (rs10488631) Polymorphisms Is Associated with Seropositivity in Rheumatoid Arthritis: A Case Control Study.

INTRODUCTION: The aim of the study was to analyse genetic architecture of RA by utilizing multiparametric statistical methods such as linear discriminant analysis (LDA) and redundancy analysis (RDA). METHODS: A total of 1393 volunteers, 499 patients with RA and 894 healthy controls were included in the study. The presence of shared epitope (SE) in HLA-DRB1 and 11 SNPs (PTPN22 C/T (rs2476601), STAT4 G/T (rs7574865), CTLA4 A/G (rs3087243), TRAF1/C5 A/G (rs3761847), IRF5 T/C (rs10488631), TNFAIP3 C/T (rs5029937), AFF3 A/T (rs11676922), PADI4 C/T (rs2240340), CD28 T/C (rs1980422), CSK G/A (rs34933034) and FCGR3A A/C (rs396991), rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPA) and clinical status was analysed using the LDA and RDA. RESULTS: HLA-DRB1, PTPN22, STAT4, IRF5 and PADI4 significantly discriminated between RA patients and healthy controls in LDA. The correlation between RA diagnosis and the explanatory variables in the model was 0.328 (Trace = 0.107; F = 13.715; P = 0.0002). The risk variants of IRF5 and CD28 genes were found to be common determinants for seropositivity in RDA, while positivity of RF alone was associated with the CTLA4 risk variant in heterozygous form. The correlation between serologic status and genetic determinants on the 1st ordinal axis was 0.468, and 0.145 on the 2nd one (Trace = 0.179; F = 6.135; P = 0.001). The risk alleles in AFF3 gene together with the presence of ACPA were associated with higher clinical severity of RA. CONCLUSIONS: The association among multiple risk variants related to T cell receptor signalling with seropositivity may play an important role in distinct clinical phenotypes of RA. Our study demonstrates that multiparametric analyses represent a powerful tool for investigation of mutual relationships of potential risk factors in complex diseases such as RA.

Antiproliferative and Pro-apoptotic Activities of a Novel Resveratrol Prodrug Against Jurkat CD4+ T-Cells.

BACKGROUND/AIM: Resveratrol, a natural polyphenol, possesses many beneficial health properties but its therapeutic application is limited due to its low water solubility and instability against oxidative processes. To improve the stability and lipophilicity of the natural compound, we synthesized a resveratrol prodrug, termed FEHH4-1. In the present study, we compared the antiproliferative and pro-apoptotic effects of resveratrol with FEHH4-1 on Jurkat T-cells. MATERIALS AND METHODS: Cell proliferation and viability were monitored by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, annexin-V/7-amino-actinomycin D staining and western blot. To induce interleukin-2 (IL2) expression, cells were stimulated with phorbol 12-myristate 13-acetate/phytohemagglutinin. IL2 production was quantified by enzyme-linked immunosorbent assay. IL2 promoter activity was studied by a Jurkat T-cell line containing an IL2 promoter luciferase reporter construct. RESULTS: Both polyphenols inhibited proliferation, induced apoptotic cell death and blocked IL2 synthesis in Jurkat T-cells. Most importantly, FEHH4-1 was three-to four-times more potent than resveratrol. CONCLUSION: FEHH4-1 had improved antiproliferative and pro-apoptotic potential against Jurkat T-cells compared to resveratrol.

Hydrogen sulfide inhibits endothelial nitric oxide formation and receptor ligand-mediated Ca(2+) release in endothelial and smooth muscle cells.

BACKGROUND: In the vascular system, ATP-sensitive K(+)-channels are a target for H2S. Recent evidence suggests that H2S may also modulate Na(+)- and Ca(2+)-permeable channels and intracellular Ca(2+) stores, but the influence of H2S on endothelial Ca(2+) dynamics and Ca(2+)-dependent activation of endothelial nitric oxide synthase (eNOS) is unclear. In this study, we investigated the effects of H2S on Ca(2+) signaling in endothelial and smooth muscle cells with special emphasis given to the role of H2S in modulating endothelial NO formation. METHODS: Experiments were performed with endothelial cells from porcine aorta, the human endothelial cell line HMEC-1, and smooth muscle cells from rat aorta and trachea. Mobilization of intracellular Ca(2+) and Ca(2+) entry was monitored with Fura-2. Activity of eNOS was determined as conversion of incorporated l-[(3)H]arginine into l-[(3)H]citrulline. RESULTS: Incubation of endothelial cells with the H2S donors sodium hydrogen sulfide (NaHS) and GYY4137 blocked activation of eNOS by the receptor agonist ATP but not by the Ca(2+) ionophore A23187. Data revealed that H2S inhibited ATP-induced release of Ca(2+) from intracellular stores indicating that H2S attenuates eNOS activity by blocking capacitative Ca(2+) entry. A similar inhibitory effect of H2S on ATP-induced Ca(2+) release and Ca(2+) entry was also observed in human microvascular endothelial cells and smooth muscle cells. CONCLUSIONS: H2S antagonized Ca(2+) mobilization by receptor agonists and store-operated Ca(2+) entry thereby limiting eNOS activation and NO formation. The effect of H2S on Ca(2+) stores was not restricted to endothelial cells but was also observed in vascular and tracheal smooth muscle cells.

Enhanced Antiproliferative and Pro-apoptotic Activities of a Novel Curcumin-related Compound in Jurkat Leukemia T-Cells.

BACKGROUND/AIM: Inhibition of arachidonic acid metabolism by curcumin has been suggested to be a key mechanism for its anti-carcinogenic action. Recently, we reported on the synthesis of curcumin analogues and their evaluation as selective COX1 inhibitors. Two compounds (HP109/HP102) were selected for evaluation of their anti-proliferative and pro-apoptotic potential in Jurkat T-cells. MATERIALS AND METHODS: Jurkat T-cells were stimulated with phorbol 12-myristate 13-acetate/phytohemagglutinin (PMA/PHA) in the absence and presence of different concentrations of curcumin or HP109/HP102. Interleukin 2 (IL2) production and IL2 promoter activity were analyzed by enzyme-linked immunosorbent assay and a luciferase reporter assay, respectively. Proliferation and cell viability were monitored by 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide assay, annexin -V/7-amino-actinomycin D staining and western blotting. RESULTS: HP102 was about 10-times more effective in blocking IL2 synthesis compared to curcumin. Enhanced effects of HP102 were also observed in reducing the proliferation rate and cell viability. In contrast to HP102, HP109 did not exhibit enhanced effects compared to curcumin. CONCLUSION: The curcumin analog HP102 had strongly improved the anti-proliferative and pro-apoptotic potential in Jurkat T-cells compared to curcumin.

Inhibition of Inflammation and Bone Erosion by RNA Interference-Mediated Silencing of Heterogeneous Nuclear RNP A2/B1 in Two Experimental Models of Rheumatoid Arthritis.

OBJECTIVE: The nuclear protein heterogeneous nuclear RNP A2/B1 (hnRNP A2/B1) is involved in posttranscriptional regulation of gene expression. It is constitutively expressed in lymphoid organs and highly up-regulated in the synovial tissue of patients with rheumatoid arthritis (RA), who may also generate autoantibodies to this protein. This study was undertaken to investigate the potential involvement of hnRNP A2/B1 in the pathogenesis of autoimmune arthritis, by silencing hnRNP A2/B1 expression in 2 animal models of RA. METHODS: Collagen-induced arthritis (CIA) and the K/BxN serum-transfer model were used as animal models of RA. Efficient silencing of hnRNP A2/B1 was achieved using a liposome-based carrier system for delivery of small interfering RNAs. Expression of hnRNP A2/B1 was analyzed by flow cytometry, reverse transcription-quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. The number of osteoclasts was determined by tartrate-resistant acid phosphatase staining. Cytokine levels and anticollagen antibody levels were measured by enzyme-linked immunosorbent assay. RESULTS: Efficient silencing of hnRNP A2/B1 was achieved in all lymphoid organs. In both experimental models, the incidence and severity of arthritis were largely reduced and bone erosion was not detectable as compared to the control groups. Down-modulation of hnRNP A2/B1 significantly interfered with the production of proinflammatory cytokines from monocyte/macrophages, but not from T cells. Consistent with these findings, production of T cell cytokines was not impaired when cells were restimulated in vitro with type II collagen. Furthermore, levels of anticollagen antibodies were not affected by hnRNP A2/B1 silencing. CONCLUSION: Our findings suggest that hnRNP A2/B1 has an important role in regulation of the innate immune system, especially at the level of monocyte/macrophage activation. Therefore, down-modulation of hnRNP A2/B1 seems to affect primarily the effector phase of autoimmune arthritis.