IL-17A and TNF synergistically drive expression of proinflammatory mediators in synovial fibroblasts via IκBζ-dependent induction of ELF3.

OBJECTIVE: IL-17A and TNF act in synergy to induce proinflammatory mediators in synovial fibroblasts thus contributing to diseases associated with chronic arthritis. Many of these factors are regulated by transcription factor E74-like factor-3 (ELF3). Therefore, we sought to investigate ELF3 as a downstream target of IL-17A and TNF signalling and to characterize its role in the molecular mechanism of synergy between IL-17A and TNF. METHODS: Regulation of ELF3 expression by IL-17A and TNF was studied in synovial fibroblasts of RA and OA patients and RA synovial explants. Signalling leading to ELF3 mRNA induction and the impact of ELF3 on the response to IL-17A and TNF were studied using siRNA, transient overexpression and signalling inhibitors in synovial fibroblasts and HEK293 cells. RESULTS: ELF3 was marginally affected by IL-17A or TNF alone, but their combination resulted in high and sustained expression. ELF3 expression was regulated by the nuclear factor-κB (NF-κB) pathway and CCAAT/enhancer-binding protein ß (C/EBPß), but its induction required synthesis of the NF-κB co-factor IκB (inhibitor of NF-κB) ζ. siRNA-mediated depletion of ELF3 attenuated the induction of cytokines and matrix metalloproteinases by the combination of IL-17A and TNF. Overexpression of ELF3 or IκBζ showed synergistic effect with TNF in upregulating expression of chemokine (C-C motif) ligand 8 (CCL8), and depletion of ELF3 abrogated CCL8 mRNA induction by the combination of IκBζ overexpression and TNF. CONCLUSION: Altogether, our results establish ELF3 as an important mediator of the synergistic effect of IL-17A and TNF in synovial fibroblasts. The findings provide novel information of the pathogenic mechanisms of IL-17A in chronic arthritis and implicate ELF3 as a potential therapeutic target.

Serum Epstein-Barr virus DNA, detected by droplet digital PCR, correlates with disease activity in patients with rheumatoid arthritis.

OBJECTIVES: To study the prevalence of asymptomatic activation of Epstein-Barr virus (EBV) in patients with rheumatoid arthritis (RA) and to analyse the correlation of serum EBV DNA with the disease activity. METHODS: The level of EBV DNA was determined by droplet digital PCR assay from the serum of 46 DMARD naive early RA (ERA) and 22 chronic RA (CRA)-patients at study onset. Follow-up samples from 31 ERA and 16 CRA patients were obtained after starting or modifying the anti-rheumatic treatment. EBV DNA was also measured from 33 healthy controls and 9 patients with adult onset Still's disease (AOSD). Disease activity was assessed by the disease activity score (DAS28). RESULTS: At baseline, EBV DNA was detected in the serum of 7 of the 46 ERA patients all of whom had moderate or high disease activity. In the follow-up samples, 11 of 31 patients were EBV DNA positive. At baseline EBV positive patients had significantly higher disease activity (p=0.036) and the concentration of EBV DNA correlated significantly with DAS28 (rs=0.333, p=0.024). EBV DNA was detected in 3 of 22 CRA patients at study onset and in 8 of 16 in the follow-up samples. At follow-up EBV positive patients had significantly higher DAS28 (p=0.027) and the concentration of EBV DNA correlated significantly with DAS28 (rs=0.724, p=0.002). Only one of the healthy controls and none of the AOSD patients were positive for EBV DNA. CONCLUSIONS: Active RA is associated with a lytic EBV infection which may have a role in the pathogenesis of RA.

DEC2 Blocks the Effect of the ARNTL2/NPAS2 Dimer on the Expression of PER3 and DBP.

The expression of clock genes ARNTL2, NPAS2 and DEC2 are disturbed in rheumatoid arthritis, an autoimmune disease with circadian variation of symptoms. We have shown that TNF is a potent inducer of these genes. We investigated the regulation of ARNTL2 and NPAS2 by TNF and elucidated their effect on other clock gene expressions. Additionally, we studied the effect of DEC1 and DEC2 on ARNTL, ARNTL2 and NPAS2. Cultured primary human fibroblasts were stimulated with TNF and the effects on ARNTL2 and NPAS2 were studied with RT-qPCR and immunofluorescence staining. The role of NF-κB was analyzed using IKK-2 inhibitor IMD-0354. TNF promoted ARNTL2 localization into the nuclei. Similar to DEC2, the effects of TNF on ARNTL2 and NPAS2 expressions were mediated via NF-κB. Cloned ARNTL, ARNTL2, NPAS2, DEC1 and DEC2 were transfected into HEK293. The ARNTL2/NPAS2 dimer was a weaker inducer of PER3 and DBP than ARNTL/NPAS2. ARNTL2 and NPAS2 are regulated by TNF via the same mechanism as DEC2. Compared to their paralogs they have unique effects on other circadian components. Our data suggest that these genes are responsible, at least in fibroblasts, for the accurate adaptation of circadian timekeeping in individual cells during inflammation.

Activation of histamine H4 receptor inhibits TNFα/IMD-0354-induced apoptosis in human salivary NS-SV-AC cells.

Apoptosis is involved in the pathogenesis of Sjögren's syndrome (SS), an autoimmune disease affecting exocrine glands. Our recent studies revealed diminished histamine H4 receptor (H4R) expression and impaired histamine transport in the salivary gland epithelial cells in SS. The aim was now to test if nanomolar histamine and high-affinity H4R signaling affect apoptosis of human salivary gland epithelial cell. Simian virus 40-immortalized acinar NS-SV-AC cells were cultured in serum-free keratinocyte medium ± histamine H4R agonist HST-10. Expression and internalization of H4R were studied by immunofluorescence staining ± clathrin inhibitor methyl-ß-cyclodextrin (MßCD). Apoptosis induced using tumor necrosis factor-α with nuclear factor-κB inhibitor IMD-0354 was studied using phase contrast microscopy, Western blot, flow cytometry and polymerase chain reaction (qRT-PCR). HST-10-stimulated H4R internalization was inhibited by MßCD. Western blotting revealed diminished phosphorylated c-Jun N-terminal kinase JNK, but unchanged levels of phosphorylated extracellular signal regulated kinase pERK1/2 in H4R-stimulated samples compared to controls. qRT-PCR showed up-regulated expression of anti-apoptotic B cell lymphoma-extra large/Bcl-xL mRNAs and proteins, whereas pro-apoptotic Bcl-2-associated X protein/BAX remained unchanged in H4R-stimulated samples. H4R stimulation diminished cleavage of PARP and flow cytometry showed significant dose-dependent inhibitory effect of H4R stimulation on apoptosis. As far as we know this is the first study showing inhibitory effect of H4R activation on apoptosis of human salivary gland cells. Diminished H4R-mediated activation may contribute to loss of immune tolerance in autoimmune diseases and in SS in particular.

Neutrophils produce interleukin-17B in rheumatoid synovial tissue.

OBJECTIVE: T helper 17 (Th17) and mast cells produce IL-17A in RA and critically contribute to the pathogenesis of RA. However, the complete IL-17 cytokine profile in RA is unknown. The aim of the study was to systematically study the expression of IL-17 family cytokines in RA. METHODS: The expression of all IL-17 cytokines in RA synovium and pannus as well as in the synovium of OA was determined using quantitative RT-PCR (qRT-PCR). IL-17A and IL-17B were immunostained. Peripheral blood neutrophils were analysed for IL-17B. The effect of IL-17B alone or in combination with TNF-α was tested in vitro on fibroblasts and endothelial cells. RESULTS: In all tissues IL-17B was the most expressed IL-17 family cytokine, found in lining but most strongly expressed in human neutrophil elastase containing polymorphonuclear cells. This pattern was distinct from that of IL-17A, which was found in mast cell tryptase immunoreactive cells. Circulating neutrophils contained IL-17B, verifying the in vivo results. Fibroblasts up-regulated the expression of IL-17RB, a putative receptor of IL-17B, after TNF-α stimulation. IL-17B significantly enhanced TNF-α-induced production of G-CSF and IL-6 in fibroblasts. CONCLUSION: IL-17B, which is present in synovium, may contribute to the pathogenesis of RA. IL-17B can enhance the effects of TNF-α on the production of cytokines and chemokines that control immune cell trafficking and neutrophil homeostasis in the inflamed tissues.

IL-17C and its receptor IL-17RA/IL-17RE identify human oral epithelial cell as an inflammatory cell in recurrent aphthous ulcer.

BACKGROUND: Recurrent aphthous ulcer (RAU) is an ulcerative disease of non-keratinized oral mucosa. Colon and bronchial epithelial cells produce interleukin-17C (IL-17C) upon stimulation of Toll-like receptor 2 (TLR2), TLR3 and TLR5, which are highly expressed in epithelial cells in RAU lesions. We therefore investigated the eventual presence and function of IL-17C in cultured human oral keratinocytes (HOK) and control biopsies compared to RAU lesions. METHODS: Expression of IL-17A, IL-17C, IL-17RA and IL-17RE was analysed in cultured HOK cells using quantitative real-time polymerase chain reaction (qRT-PCR). HOK cells were stimulated with IL-17C and analysed for IL-8 and tumour necrosis factor-α (TNF-α) using qRT-PCR. Control mucosa (n = 5) was immunostained for IL-17A, IL-17C, IL-8, TNF-α and mast cell tryptase and compared with RAU lesions (n = 5) using the mean grey scale value. RESULTS: IL-17C, but no IL-17A, mRNA was found in cultured HOK cells. Components of the heterodimeric IL-17RA/IL-17RE receptor for IL-17C were also highly expressed. Stimulation of HOK with IL-17C increased TNF-α mRNA (P = 0.03; IL-8 increase was not statistically significant). HOK in RAU lesions stained intensively for IL-17C compared to controls (P = 0.006). This was associated with increased epithelial immunostaining of TNF-α (P = 0.04) and IL-8 (P = 0.02). Most of the inflammatory cells which stained for IL-17A in control mucosa and RAU lesions were also mast cell tryptase positive. CONCLUSION: IL-17C is highly expressed in epithelial cells in RAU lesions, where it seems to stimulate oral keratinocytes via IL-17RA/IL-17RE to produce pro-inflammatory cytokines. Human oral epithelial cells are probably important inflammatory cells in RAU.

Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis.

In monogenic autoinflammatory diseases, mutations in genes regulating innate immune responses often lead to uncontrolled activation of inflammasome pathways or the type I interferon (IFN-I) response. We describe a mechanism of autoinflammation potentially predisposing patients to life-threatening necrotizing soft tissue inflammation. Six unrelated families are identified in which affected members present with necrotizing fasciitis or severe soft tissue inflammations. Exome sequencing reveals truncating monoallelic loss-of-function variants of nuclear factor κ light-chain enhancer of activated B cells (NFKB1) in affected patients. In patients' macrophages and in NFKB1-variant-bearing THP-1 cells, activation increases both interleukin (IL)-1ß secretion and IFN-I signaling. Truncation of NF-κB1 impairs autophagy, accompanied by the accumulation of reactive oxygen species and reduced degradation of inflammasome receptor nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein 3 (NLRP3), and Toll/IL-1 receptor domain-containing adaptor protein inducing IFN-ß (TRIF), thus leading to combined excessive inflammasome and IFN-I activity. Many of the patients respond to anti-inflammatory treatment, and targeting IL-1ß and/or IFN-I signaling could represent a therapeutic approach for these patients.

Lactate and ammonia measurements during cardiopulmonary exercise testing and its recovery phase-Consideration of age and sex in its interpretation.

BACKGROUND: Cardiopulmonary exercise testing with lactate and ammonia samples is used in the diagnostics of metabolic myopathies. As the effect of age and sex on the exercise lactate and ammonia levels are incompletely characterized for clinical associations, our aim was to assess the effects of these factors on healthy subjects to improve the test's interpretation. METHODS: Seventy-three subjects (34 men and 39 women; age < 35 years, n = 26, 35-50 years, n = 23 and >50 years, n = 24) performed cardiopulmonary exercise tests with venous blood gases, plasma lactate and ammonia analyses at rest, during exercise, and 2, 4, 6, 10, 20 and 30 min into recovery. RESULTS: The lactate (p = 0.021-0.044) and ammonia values (p = 0.002-0.038) differed between men and women measured during recovery and between three age groups point-by-point in maximal exercise and the recovery phase and also longitudinally, most notably between <35- and >50-year-groups (lactate p = <0.001-0.040, ammonia p = 0.002-0.03). In the linear model, the yearly reduction of lactate was maximally -0.119 mmol/L and that of ammonia -1.514 µmol/L. The yearly reduction of lactate was greater in women than in men (-0.131 vs.-0.099 2 min into recovery), but for ammonia, the results were not as clear. CONCLUSIONS: Plasma lactate and ammonia concentrations measured during cardiopulmonary exercise were lower in older age groups, and their yearly reduction was also influenced by sex. These data give new information on lactate and ammonia levels and the effect of aging on them during exercise and recovery and may help assess cardiopulmonary exercise testing results.

Verification of UriSed 3 PRO automated urine microscope in regional laboratory environment.

BACKGROUND AND AIMS: Ten UriSed 3 PRO automated microscopes (77 Elektronika, Hungary) were verified for nine HUSLAB laboratories with 160 000 annual urine samples. MATERIALS AND METHODS: Particle counting of the primary UriSed 3 PRO instrument (77 Elektronika, Hungary) was verified against reference visual microscopy with 463 urine specimens, and against urine culture on chromogenic agar plates with parallel 396 specimens. Nine secondary instruments were compared pairwise with the primary instrument. RESULTS: Relative imprecisions compared to Poisson distribution, R(CV), were estimated to be 1.0 for white blood cell (WBC) and 1.5 for red blood cell (RBC) counts, respectively. Spearman's correlations against visual microscopy were rS = 0.94 for WBC, rS = 0.87 for RBC, and rS = 0.82 for squamous epithelial cell (SEC) counts. Agreement with visual microscopy (Cohen's weighted kappa) was 0.94 for WBC, 0.89 for RBC, 0.88 for SEC, 0.59 for combined casts, and 0.49 for non-squamous epithelial cells (NEC). Bacteria were detected with a sensitivity of 90% and specificity of 39 against culture at 107 CFB/L (104 CFU/mL). Created flagging limits allowed automated reporting for 70-75% of patient results. CONCLUSIONS: UriSed 3 PRO instruments were adopted into routine use after acceptance of the verification.

Native and oxidised lipoproteins negatively regulate the serum amyloid A-induced NLRP3 inflammasome activation in human macrophages.

OBJECTIVES: The NLRP3 inflammasome plays a key role in arterial wall inflammation. In this study, we elucidated the role of serum lipoproteins in the regulation of NLRP3 inflammasome activation by serum amyloid A (SAA) and other inflammasome activators. METHODS: The effect of lipoproteins on the NLRP3 inflammasome activation was studied in primary human macrophages and THP-1 macrophages. The effect of oxidised low-density lipoprotein (LDL) was examined in an in vivo mouse model of SAA-induced peritoneal inflammation. RESULTS: Native and oxidised high-density lipoproteins (HDL3) and LDLs inhibited the interaction of SAA with TLR4. HDL3 and LDL inhibited the secretion of interleukin (IL)-1ß and tumor necrosis factor by reducing their transcription. Oxidised forms of these lipoproteins reduced the secretion of mature IL-1ß also by inhibiting the activation of NLRP3 inflammasome induced by SAA, ATP, nigericin and monosodium urate crystals. Specifically, oxidised LDL was found to inhibit the inflammasome complex formation. No cellular uptake of lipoproteins was required, nor intact lipoprotein particles for the inhibitory effect, as the lipid fraction of oxidised LDL was sufficient. The inhibition of NLRP3 inflammasome activation by oxidised LDL was partially dependent on autophagy. Finally, oxidised LDL inhibited the SAA-induced peritoneal inflammation and IL-1ß secretion in vivo. CONCLUSIONS: These findings reveal that both HDL3 and LDL inhibit the proinflammatory activity of SAA and this inhibition is further enhanced by lipoprotein oxidation. Thus, lipoproteins possess major anti-inflammatory functions that hinder the NLRP3 inflammasome-activating signals, particularly those exerted by SAA, which has important implications in the pathogenesis of cardiovascular diseases.

UriSed 3 PRO automated microscope in screening bacteriuria at region-wide laboratory organization.

BACKGROUND AND AIMS: We assessed the possibility to rule out negative urine cultures by counting with UriSed 3 PRO (77 Elektronika, Hungary) at Helsinki and Uusimaa Hospital District. MATERIALS AND METHODS: Bacteria counting of the UriSed 3 PRO automated microscope was verified with reference phase contrast microscopy against growth in culture. After acceptance into routine, results of bacteria and leukocyte counting from 56 426 specimens with eight UriSed 3 PRO instruments were compared against results from parallel samples cultured on chromogenic agar. Laboratory data including preanalytical details were accessed through the regional database of the Helsinki and Uusimaa Hospital District. RESULTS: A combined sensitivity of 87-92% and a negative predictive value of 90-96% with a specificity of 54-50% was reached, depending on criteria. Preanalytical data (incubation time in bladder) combined with the way of urine collection would improve these figures if reliable. CONCLUSIONS: Complex patient populations, regional logistics and data interfases, and economics related to increased costs of additional particle counts against costs of screening cultures of all samples, did not support adaptation of a screening process of urine cultures. This conclusion was made locally, and may not be valid elsewhere.

Enhancement of silicon using micro-patterned surfaces of thin films.

Micro-textured biomaterials might enhance cytocompatibility of silicon-based micro-electro-mechanical system (bio-MEMS) dummies. Photolithography-physical vapour deposition was used to produce diamond-like carbon (DLC) or Ti squares and circles on silicon, and also their inverse replicas; then DLC and Ti were compared for their guiding potential, using a SaOS-2 cell model. Scanning electron microscopy at 48 hours indicated cells were well-spread on large-sized patterns (several cells on one pattern) and assumed the geometrical architecture of underlying features. Medium-sized patterns (slightly smaller than solitary indicator cells) were inhabited by singular cells, which stretched from one island to another, assuming longitudinal or branching morphologies. On small-sized patterns (much smaller than individual cells;rpar; cells covered large micro-textured areas, but cellular filopodia bypassed the bare silicon. Immunofluorescence and confocal laser scanning microscopy indicated that the actin cytoskeleton and vinculin-containing adhesion junctions were present on the patterned areas, but not on the bare silicon. Cell density/coverage disclosed a 3.4-3.7-fold preference for the biomaterial patterns over silicon substrate (p 0.001). Differences in the cellular response between materials were lost at 120 hours when cells were confluent. The working hypothesis was proven; enhancement by micro-patterning depends on the pattern size, shape and material and can be used to improve biocompatibility during the initial integration phase of the device.

Tumor necrosis factor primes and metal particles activate the NLRP3 inflammasome in human primary macrophages.

Aseptic loosening of total joint replacements is driven by a macrophage-mediated inflammatory reaction to implant-derived wear particles. Phagocytosis of implant debris has been suggested to activate the NLRP3 inflammasome leading to secretion of interleukin (IL)-1ß. However, factors and molecular mechanisms driving the particle-induced inflammasome activation are yet to be fully elucidated. In this study, we investigated the inflammasome response of human primary macrophages to titanium, chromium, and molybdenum particles in vitro. We observed that particles alone were not sufficient to induce IL-1ß secretion, but an additional priming signal-such as bacterial lipopolysaccharide (LPS)-was required to license the inflammasome activation. By using specific inhibitors against the inflammasome signaling pathway, we demonstrate that the particle-induced IL-1ß secretion depended upon activation of the NLRP3 inflammasome. We further hypothesized that tumor necrosis factor (TNF) could substitute for LPS as a priming signal, and found that particle stimulation together with preceding TNF treatment resulted in inflammasome-dependent IL-1ß production as well. Our results show that the NLRP3 inflammasome mediates wear particle responses in human primary macrophages, and its activation does not necessarily require the presence of bacterial components, but can be induced under aseptic conditions by TNF priming. STATEMENT OF SIGNIFICANCE: This study was conducted to elucidate the molecular mechanisms of metal particle-induced IL-1ß secretion in human primary macrophages. Production of this pro-inflammatory mediator from wear particle-activated macrophages has been associated with increased bone loss around total joint replacements-a condition eventually requiring revision surgery. Our results confirm that together with a co-stimulatory priming signal, particles of common implant metals elicit macrophage-mediated IL-1ß secretion through activation of the NLRP3 inflammasome pathway. We also present a concept of TNF priming in this context, demonstrating that the particle-related IL-1ß secretion can take place in a truly sterile environment. Thus, inhibition of inflammasome signaling appears a means to prevent wear particle-induced inflammation and development of peri­prosthetic osteolysis.

Correlations between macrophage polarizing cytokines, inflammatory mediators, osteoclast activity, and toll-like receptors in tissues around aseptically loosened hip implants.

Aseptic loosening and osteolysis of joint replacements are driven by macrophage-mediated inflammatory reactions to implant-derived wear debris, but many aspects of these events remain poorly characterized. To better understand the relationships among inflammatory and chemotactic mediators, macrophage phenotype and polarizing cytokines, osteoclast activity, and Toll-like receptors (TLRs) in the pathogenesis of aseptic loosening, we determined how the relative expressions of these factors in the peri-implant tissues correlate to each other and to the life span of the implants using Pearson correlation. The expression of pro-inflammatory mediators and chemokines showed positive correlations among themselves, and with TLR4. Furthermore, M1-polarizing IFN-γ showed positive correlations with a number of pro-inflammatory and chemotactic mediators, whereas M2-polarizing IL-4 showed no such association. IL-8 expression significantly correlated with early time to revision. Similar trends were observed for TNF-α, IFN-γ, and CCL3, while the opposite was detected for IL-4. However, none of the inflammatory mediators correlated with the markers of osteoclast activity or the RANKL/OPG ratio. The results highlight the importance of the inflammatory mediators, IFN-γ and TLR4, in the pathogenesis of aseptic loosening; increased pro-inflammatory status was associated with early time to revision, whereas IL-4 correlated with longer implant survival. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 454-463, 2017.

Hemin and Cobalt Protoporphyrin Inhibit NLRP3 Inflammasome Activation by Enhancing Autophagy: A Novel Mechanism of Inflammasome Regulation.

Inflammasomes are intracellular protein platforms, which, upon activation, produce the highly proinflammatory cytokines interleukin (IL)-1ß and IL-18. Heme, hemin and their degradation products possess significant immunomodulatory functions. Here, we studied whether hemin regulates inflammasome function in macrophages. Both hemin and its derivative, cobalt protoporphyrin (CoPP), significantly reduced IL-1ß secretion by cultured human primary macrophages, the human monocytic leukemia cell line and also mouse bone marrow-derived and peritoneal macrophages. Intraperitoneal administration of CoPP to mice prior to urate crystal-induced peritonitis alleviated IL-1ß secretion to the peritoneal cavity. In cultured macrophages, hemin and CoPP inhibited NLRP3 inflammasome assembly by reducing the amount of intracellular apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). The reduction of ASC was associated with enhanced autophagosome formation and autophagic flux. Inhibition of autophagy prevented the CoPP-induced depletion of ASC, implying that the depletion was caused by increased autophagy. Our data indicate that hemin functions as an endogenous negative regulator of the NLRP3 inflammasome. The inhibition is mediated via enhanced autophagy that results in increased degradation of ASC. This regulatory mechanism may provide a novel approach for the treatment of inflammasome-related diseases.

Epstein-Barr virus in peripheral blood is associated with response to rituximab therapy in rheumatoid arthritis patients.

Autoreactive B cells infected by Epstein-Barr virus (EBV) are suspected to be involved in the etiology of various human chronic autoimmune diseases. This motivated us to study the relationship between peripheral blood EBV load at baseline and treatment response to B cell-depleting therapy in rheumatoid arthritis (RA) patients. Thirty-five RA patients who started treatment with rituximab (RTX) in a routine clinical setting were assessed for baseline disease activity using disease activity score using 28 joint counts (DAS28) (erythrocyte sedimentation rate [ESR]). Treatment response was evaluated 3-7 months after RTX. EBV load in baseline whole blood (WB) samples was determined using quantitative PCR. EBV DNA was detected in 16/35 (46 %) of the WB samples. In these 16 EBV-positive patients, the median viral load was 3.15 (2.68-4.00) log copies/ml. Good/moderate European League Against Rheumatism (EULAR) response was observed in 16/16 of the EBV DNA-positive vs 13/19 EBV DNA-negative patients, p = 0.022. Significant response (DAS28 change >1.2) was observed in 14/16 of the EBV DNA-positive vs 10/19 EBV DNA-negative patients, p = 0.035. The decline in DAS28 after RTX was 2.10 (1.03-4. 78) in the EBV DNA-positive vs 1.47 (-0.7-4.70) in the EBV DNA-negative patients, p = 0.13. EBV load at baseline significantly correlated with change in DAS28 after RTX (τB = -0.261, p = 0.042). Our results suggest that the presence of EBV genome in WB could serve as a predictive marker to RTX therapy in RA.

Differentially Expressed in Chondrocytes 2 (DEC2) Increases the Expression of IL-1ß and Is Abundantly Present in Synovial Membrane in Rheumatoid Arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) have altered circadian rhythm of circulating serum cortisol, melatonin and IL-6, as well as disturbance in the expression of clock genes ARNTL2 and NPAS2. In humans, TNFα increases the expression ARNTL2 and NPAS2 but paradoxically suppresses clock output genes DPB and PER3. Our objective was to investigate the expression of direct clock suppressors DEC1 and DEC2 (BHLHE 40 and 41 proteins) in response to TNFα and investigate their role during inflammation. METHODS: Cultured primary fibroblasts were stimulated with TNFα. Effects on DEC2 were studied using RT-qPCR and immunofluorescence staining. The role of NF-κB in DEC2 increase was analyzed using IKK-2 specific inhibitor IMD-0354. Cloned DEC2 was transfected into HEK293 cells to study its effects on gene expression. Transfections into primary human fibroblasts were used to confirm the results. The presence of DEC2 was analyzed in (RA) and osteoarthritis (OA) synovial membranes by immunohistochemistry. RESULTS: TNFα increased DEC2 mRNA and DEC2 was mainly detected at nuclei after the stimulus. The effects of TNFα on DEC2 expression were mediated via NF-κB. Overexpression, siRNA and promoter activity studies disclosed that DEC2 directly regulates IL-1ß, in both HEK293 cells and primary human fibroblasts. DEC2 was increased in synovial membrane in RA compared to OA. CONCLUSION: Not only ARNTL2 and NPAS2 but also DEC2 is regulated by TNFα in human fibroblasts. NF-κB mediates the effect on DEC2, which upregulates IL-1ß. Circadian clock has a direct effect on inflammation in human fibroblasts.

Identification of histamine receptor subtypes in skeletal myogenesis.

To date, conventional and/or novel histamine receptors (HRs) have not been investigated in mouse skeletal myogenesis. Therefore, the present study aimed to investigate the HR­subtypes in skeletal myogenesis. The myogenesis of C2C12 skeletal myoblasts was evaluated using desmin, myogenin and myosin heavy chain (Myh) as early, intermediate and late differentiation markers, respectively. Reverse transcription­quantitative polymerase chain reaction and immunostaining were performed and the messenger RNA (mRNA) expression levels of the HR­subtypes and markers were determined. H1R mRNA was found to be highly expressed in myoblasts at day 0; however, the expression levels were reduced as differentiation progressed. By contrast, H2R mRNA expression remained constant, while H3R mRNA expression increased by 28­, 103­ and 198­fold at days 2, 4 and 6 compared with the baseline level (day 0), respectively. In addition, Myh expression increased by 7,718­, 94,487­ and 286,288­fold on days 2, 4 and 6 compared with the baseline expression level (day 0). Weak positive staining of the cells for H3R protein was observed on day 2, whereas highly positive staining was observed on days 4 and 6. HR expression during myogenesis was, in part, regulated by the stage of differentiation. These results along with previous findings indicated possible involvement of H1R in the regulation of progenitor cell mitogenesis and of H2R in the relaxation of acetylcholine­stimulated contraction of muscle cells, following the activation of professional histamine­producing cells, including mast cells. By contrast, H3R may participate in the regulation of specialized myocyte functions, potentially by maintaining the relaxed state under the influence of constitutive H3R activity and low histamine concentrations, locally produced/released by non­professional histamine­producing cells.

Characterization of macrophage polarizing cytokines in the aseptic loosening of total hip replacements.

Aseptic loosening of hip replacements is driven by the macrophage reaction to wear particles. The extent of particle-induced macrophage activation is dependent on the state of macrophage polarization, which is dictated by the local cytokine microenvironment. The aim of the study was to characterize cytokine microenvironment surrounding failed, loose hip replacements with an emphasis on identification of cytokines that regulate macrophage polarization. Using qRT-PCR, the expression of interferon gamma (IFN-γ), interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-13, and IL-17A was low and similar to the expression in control synovial tissues of patients undergoing primary hip replacement. Using immunostaining, no definite source of IFN-γ or IL-4 could be identified. IL-17A positive cells, identified as mast cells by double staining, were detected but their number was significantly reduced in interface tissues compared to the controls. Significant up-regulation of IL-10, M-CSF, IL-8, CCL2-4, CXCL9-10, CCL22, TRAP, cathepsin K, and down regulation of OPG was seen in the interface tissues, while expression of TNF-α, IL-1ß, and CD206 were similar between the conditions. It is concluded that at the time of the revision surgery the peri-implant macrophage phenotype has both M1 and M2 characteristics and that the phenotype is regulated by other local and systemic factors than traditional macrophage polarizing cytokines.

The response of macrophages to titanium particles is determined by macrophage polarization.

Aseptic loosening of total joint replacements is driven by the reaction of macrophages to foreign body particles released from the implant. It was hypothesized that the macrophages' response to these particles is dependent, in addition to particle characteristics and contaminating biomolecules, on the state of macrophage polarization as determined by the local cytokine microenvironment. To test this hypothesis we differentiated M1 and M2 macrophages from human peripheral blood monocytes and compared their responses to titanium particles using genome-wide microarray analysis and a multiplex cytokine assay. In comparison to non-activated M0 macrophages, the overall chemotactic and inflammatory responses to titanium particles were greatly enhanced in M1 macrophages and effectively suppressed in M2 macrophages. In addition, the genome-wide approach revealed several novel, potentially osteolytic, particle-induced mediators, and signaling pathway analysis suggested the involvement of toll-like and nod-like receptor signaling in particle recognition. It is concluded that the magnitude of foreign body reaction caused by titanium particles is dependent on the state of macrophage polarization. Thus, by limiting the action of M1 polarizing factors, e.g. bacterial biofilm formation, in peri-implant tissues and promoting M2 macrophage polarization by biomaterial solutions or pharmacologically, it might be possible to restrict wear-particle-induced inflammation and osteolysis.