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.

Selection and identification of ligand peptides targeting a model of castrate-resistant osteogenic prostate cancer and their receptors.

We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand-receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer.

Synchronous down-modulation of miR-17 family members is an early causative event in the retinal angiogenic switch.

Six members of the microRNA-17 (miR-17) family were mapped to three different chromosomes, although they share the same seed sequence and are predicted to target common genes, among which are those encoding hypoxia-inducible factor-1α (HIF1A) and VEGFA. Here, we evaluated the in vivo expression profile of the miR-17 family in the murine retinopathy of prematurity (ROP) model, whereby Vegfa expression is highly enhanced at the early stage of retinal neovascularization, and we found simultaneous reduction of all miR-17 family members at this stage. Using gene reporter assays, we observed binding of these miRs to specific sites in the 3' UTRs of Hif1a and Vegfa. Furthermore, overexpression of these miRs decreased HIF1A and VEGFA expression in vitro. Our data indicate that this miR-17 family elicits a regulatory synergistic down-regulation of Hif1a and Vegfa expression in this biological model. We propose the existence of a coordinated regulatory network, in which diverse miRs are synchronously regulated to target the Hif1a transcription factor, which in turn, potentiates and reinforces the regulatory effects of the miRs on Vegfa to trigger and sustain a significant physiological response.

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.

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.

Characterization of clinicopathological features, treatment practices, and outcomes among Finnish advanced breast cancer patients in real-life clinical practice.

PURPOSE: In recent years, several new targeted therapies have emerged for advanced breast cancer (aBC). However, real-life data specific to aBC and different breast cancer subtypes are scarce. This retrospective cohort study was designed to describe the distribution of aBC subtypes, incidence, treatment patterns, survival, and PIK3CA hotspot mutation frequency. METHODS: The study included all patients in the Hospital District of Southwest Finland diagnosed with aBC between 2004 and 2013 and with a sample available in Auria Biobank. In addition to registry-based data collection, 161 HR+/HER2- aBCs were screened for PIK3CA mutations. RESULTS: Altogether, 54.7% of the 444 patients included in the study had luminal B subtype. The smallest representations were in HR-/HER2+ (4.5%) and triple-negative (5.6%) subgroups. The percentage of aBC among all diagnosed breast cancers increased until 2010, after which it remained stable. The triple-negative cancers were associated with shorter median overall survival (5.5 months) compared to other subgroups (16.5-24.6 months). Most (84%) triple-negative cancers also metastasized during the first two years, whereas this was more evenly distributed over time in other subgroups. Of the HR+/HER2- tumors, 32.3% harbored a PIK3CA hotspot mutation. These patients, however, did not have inferior survival compared to patients with PIK3CA wild-type cancers. CONCLUSION: This study described real-world aBC subgroups and indicated that the clinical outcomes of subgroups vary. Although PIK3CA hotspot mutations did not lead to inferior survival, they are relevant as possible treatment targets. Overall, these data could be utilized to further evaluate the subgroup-specific medical needs in breast cancer.

Bexmarilimab-induced macrophage activation leads to treatment benefit in solid tumors: The phase I/II first-in-human MATINS trial.

Macrophage Clever-1 contributes to impaired antigen presentation and suppression of anti-tumor immunity. This first-in-human trial investigates the safety and tolerability of Clever-1 blockade with bexmarilimab in patients with treatment-refractory solid tumors and assesses preliminary anti-tumor efficacy, pharmacodynamics, and immunologic correlates. Bexmarilimab shows no dose-limiting toxicities in part I (n = 30) and no additional safety signals in part II (n = 108). Disease control (DC) rates of 25%-40% are observed in cutaneous melanoma, gastric, hepatocellular, estrogen receptor-positive breast, and biliary tract cancers. DC associates with improved survival in a landmark analysis and correlates with high pre-treatment intratumoral Clever-1 positivity and increasing on-treatment serum interferon γ (IFNγ) levels. Spatial transcriptomics profiling of DC and non-DC tumors demonstrates bexmarilimab-induced macrophage activation and stimulation of IFNγ and T cell receptor signaling selectively in DC patients. These data suggest that bexmarilimab therapy is well tolerated and show that macrophage targeting can promote immune activation and tumor control in late-stage cancer.

Nonclinical Characterization of Bexmarilimab, a Clever-1-Targeting Antibody for Supporting Immune Defense Against Cancers.

Common lymphatic endothelial and vascular endothelial receptor-1 (Clever-1) is a multifunctional type-1 transmembrane protein that plays an important role in immunosuppression against tumors. Clever-1 is highly expressed in a subset of human tumor-associated macrophages and associated with poor survival. In mice, Clever-1 supports tumor growth and metastasis formation, and its deficiency or blockage induces T-cell-dependent killing of cancer cells. Therefore, targeting Clever-1 could lead to T-cell activation and restoration of immune response also in patients with cancer. This is studied in an on-going clinical trial [Macrophage Antibody To INhibit immune Suppression (MATINS); NCT03733990] in patients with advanced solid tumors where bexmarilimab, a humanized IgG4 antibody against human Clever-1, shows promising safety and efficacy. Here, we report the humanization and nonclinical characterization of physicochemical properties, biological potency, and safety profile of bexmarilimab. Bexmarilimab showed high affinity to Clever-1 on KG-1 cells and bound to Clever-1 on the surface of classical and intermediate monocytes derived from healthy human blood. Bexmarilimab inhibited the internalization of its natural ligand acetylated low-density lipoprotein into KG-1 cells and increased TNFα secretion from macrophages but did not impair phagocytic clearance. Bexmarilimab did not induce significant cytokine release in human whole-blood cultures, did not contain nonsafe immunogenic glycans, or show any significant binding to human Fcγ receptors or complement pathway component C1q. In vivo, bexmarilimab showed dose-dependent duration of monocyte Clever-1 receptor occupancy in cynomolgus monkeys but did not induce a cytokine storm up to a dose of 100 mg/kg. In conclusion, these data support the clinical development of bexmarilimab for the restoration of immune response in cancers.

Systemic Blockade of Clever-1 Elicits Lymphocyte Activation Alongside Checkpoint Molecule Downregulation in Patients with Solid Tumors: Results from a Phase I/II Clinical Trial.

PURPOSE: Macrophages are critical in driving an immunosuppressive tumor microenvironment that counteracts the efficacy of T-cell-targeting therapies. Thus, agents able to reprogram macrophages toward a proinflammatory state hold promise as novel immunotherapies for solid cancers. Inhibition of the macrophage scavenger receptor Clever-1 has shown benefit in inducing CD8+ T-cell-mediated antitumor responses in mouse models of cancer, which supports the clinical development of Clever-1-targeting antibodies for cancer treatment. PATIENTS AND METHODS: In this study, we analyzed the mode of action of a humanized IgG4 anti-Clever-1 antibody, FP-1305 (bexmarilimab), both in vitro and in patients with heavily pretreated metastatic cancer (n = 30) participating in part 1 (dose-finding) of a phase I/II open-label trial (NCT03733990). We studied the Clever-1 interactome in primary human macrophages in antibody pull-down assays and utilized mass cytometry, RNA sequencing, and cytokine profiling to evaluate FP-1305-induced systemic immune activation in patients with cancer. RESULTS: Our pull-down assays and functional studies indicated that FP-1305 impaired multiprotein vacuolar ATPase-mediated endosomal acidification and improved the ability of macrophages to activate CD8+ T-cells. In patients with cancer, FP-1305 administration led to suppression of nuclear lipid signaling pathways and a proinflammatory phenotypic switch in blood monocytes. These effects were accompanied by a significant increase and activation of peripheral T-cells with indications of antitumor responses in some patients. CONCLUSIONS: Our results reveal a nonredundant role played by the receptor Clever-1 in suppressing adaptive immune cells in humans. We provide evidence that targeting macrophage scavenging activity can promote an immune switch, potentially leading to intratumoral proinflammatory responses in patients with metastatic cancer.

DAP12/TREM2 deficiency results in impaired osteoclast differentiation and osteoporotic features.

Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), Nasu-Hakola disease, is a globally distributed recessively inherited disease. PLOSL is characterized by cystic bone lesions, osteoporotic features, and loss of white matter in the brain leading to spontaneous bone fractures and profound presenile dementia. We have earlier characterized the molecular genetic background of PLOSL by identifying mutations in two genes, DAP12 and TREM2. DAP12 is a transmembrane adaptor protein that associates with the cell surface receptor TREM2. The DAP12-TREM2 complex is involved in the maturation of dendritic cells. To test a hypothesis that osteoclasts would be the cell type responsible for the bone pathogenesis in PLOSL, we analyzed the differentiation of peripheral blood mononuclear cells isolated from DAP12- and TREM2-deficient PLOSL patients into osteoclasts. Here we show that loss of function mutations in DAP12 and TREM2 result in an inefficient and delayed differentiation of osteoclasts with a remarkably reduced bone resorption capability in vitro. These results indicate an important role for DAP12-TREM2 signaling complex in the differentiation and function of osteoclasts.

Human osteoblasts produce cathepsin K.

Healthy bone is a rigid yet living tissue that undergoes continuous remodeling. Osteoclasts resorb bone in the remodeling cycle. They secrete H(+)-ions and proteinases to dissolve bone mineral and degrade organic bone matrix, respectively. One of the main collagenolytic proteinase in osteoclasts is cathepsin K, a member of papain family cysteine proteinases. Recently, it has been shown that osteoblasts may contribute to organic matrix remodeling. We therefore investigated their ability to produce cathepsin K for this action. Trabecular bone samples were collected from patients operated due to a fracture of the femoral neck. Part of the bone was decalcified and the rest was used for cell isolation. Sections from the decalcified bone were immunostained with antibodies against cathepsin K. Isolated cells were characterized for their ability to form mineralized matrix and subsequently analyzed for their cathepsin K production by Western blotting and quantitative RT-PCR. Osteoblasts, bone lining cells and some osteocytes in situ showed cathepsin K immunoreactivity and osteoblast-like cells in vitro produced cathepsin K mRNA and released both 42 kDa pro- and 27 kDa processed cathepsin K to culture media. Osteoblastic cathepsin K may thus contribute to collagenous matrix maintenance and recycling of improperly processed collagen I. Whether osteoblastic cathepsin K synthesis has consequences in diseases characterized by abnormal bone matrix turnover remains to be investigated.

Pseudosynovial fluid from loosened total hip prosthesis induces osteoclast formation.

Interface tissue between the bone and loosening total hip implant is acidic and highly osteolytic. It is characterized by the formation of cathepsin K positive foreign body giant cells. Similar structures to those found in the normal joint surround the artificial hip joint. Cells in synovial membrane of the artificial hip generate synovial fluid that is called pseudosynovial fluid. Interface tissue fibroblasts are able to produce receptor activator of NF-kappaB ligand (RANKL), which can induce osteoclastogenesis during the loosening process. Western blot analysis indicated that RANKL is present in the pseudosynovial fluid. Pseudosynovial fluid induced cultured peripheral blood mononuclear cells to form multinuclear TRAP positive giant cells. In the presence of osteoprotegerin, the soluble RANKL decoy receptor, the number of TRAP positive multinuclear cells was reduced to half (p < 0.05). The multinuclear cells induced with pseudosynovial fluid contained active cathepsin K protein and were capable of bone matrix resorption in vitro. The cells were shown to express osteoclast phenotype markers, such as mRNA for cathepsin K, TRAP, and calcitonin receptor. It is therefore apparent that pseudosynovial fluid from patients with aseptic loosening of total hip prosthesis contains a potent osteoclastogenic factor RANKL that further suggests a favorable environment for osteoclast formation in the peri-implant tissues. It is thus concluded that suppression of RANKL activity may be beneficial in terms of increasing the lifetime of total hip prostheses.

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.

Effect of cyclical intermittent etidronate therapy on circulating osteoprotegerin levels in patients with rheumatoid arthritis.

OBJECTIVE: To evaluate the role of serum osteoprotegerin (OPG) as a biochemical marker for disease activity assessment and drug monitoring in patients with rheumatoid arthritis (RA) treated with cyclical etidronate. DESIGN: Forty patients (35 women and 5 men) with RA of <5 years duration were randomized to receive intermittent cyclical etidronate therapy in conjunction with anti-rheumatic therapy or anti-rheumatic therapy alone (without etidronate) in a 2-year, open-label protocol. METHODS: Radiographs of hands and feet and serum samples for the determination of OPG, amino terminal propeptide (PINP), cross-linked C-telopeptide (ICTP) and amino terminal telopeptid of type I collagen were obtained at baseline and at 24 months. RESULTS: Etidronate treatment had no effect on circulating OPG levels, although the significant decline in PINP and ICTP (P=0.001 and P=0.04 respectively) reflected the efficacy of the anti-resorptive therapy. At baseline and at study termination, serum OPG correlated significantly with age (r=0.45; P=0.003 and r=0.56; P=0.0002 respectively). OPG was not related to biochemical markers of bone metabolism, indices of disease activity or radiographic disease progression. At baseline, the mean serum OPG was higher in patients receiving 5-10 mg/day prednisone (82.8+/-4.0 pg/ml, n=16) compared with those receiving <5 mg/day or with no prednisone (69.7+/-4.7 pg/ml, n=23) (P=0.05). CONCLUSIONS: Our results suggested that serum OPG measurement, perhaps because of the complexity of the regulation of the OPG, may be difficult to utilize in the evaluation of anti-resorptive therapy. Moreover, low dose corticosteroid-associated osteoporosis is probably not mediated by inhibition of OPG.

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.

Circadian timekeeping is disturbed in rheumatoid arthritis at molecular level.

INTRODUCTION: Patients with rheumatoid arthritis (RA) have disturbances in the hypothalamic-pituitary-adrenal (HPA) axis. These are reflected in altered circadian rhythm of circulating serum cortisol, melatonin and IL-6 levels and in chronic fatigue. We hypothesized that the molecular machinery responsible for the circadian timekeeping is perturbed in RA. The aim of this study was to investigate the expression of circadian clock in RA. METHODS: Gene expression of thirteen clock genes was analyzed in the synovial membrane of RA and control osteoarthritis (OA) patients. BMAL1 protein was detected using immunohistochemistry. Cell autonomous clock oscillation was started in RA and OA synovial fibroblasts using serum shock. The effect of pro-inflammatory stimulus on clock gene expression in synovial fibroblasts was studied using IL-6 and TNF-α. RESULTS: Gene expression analysis disclosed disconcerted circadian timekeeping and immunohistochemistry revealed strong cytoplasmic localization of BMAL1 in RA patients. Perturbed circadian timekeeping is at least in part inflammation independent and cell autonomous, because RA synovial fibroblasts display altered circadian expression of several clock components, and perturbed circadian production of IL-6 and IL-1ß after clock resetting. However, inflammatory stimulus disturbs the rhythm in cultured fibroblasts. Throughout the experiments ARNTL2 and NPAS2 appeared to be the most affected clock genes in human immune-inflammatory conditions. CONCLUSION: We conclude that the molecular machinery controlling the circadian rhythm is disturbed in RA patients.

Interaction between retinoid acid receptor-related orphan receptor alpha (RORA) and neuropeptide S receptor 1 (NPSR1) in asthma.

Retinoid acid receptor-related Orphan Receptor Alpha (RORA) was recently identified as a susceptibility gene for asthma in a genome-wide association study. To investigate the impact of RORA on asthma susceptibility, we performed a genetic association study between RORA single nucleotide polymorphisms (SNPs) in the vicinity of the asthma-associated SNP (rs11071559) and asthma-related traits. Because the regulatory region of a previously implicated asthma susceptibility gene, Neuropeptide S receptor 1 (NPSR1), has predicted elements for RORA binding, we hypothesized that RORA may interact biologically and genetically with NPSR1. 37 RORA SNPs and eight NPSR1 SNPs were genotyped in the Swedish birth cohort BAMSE (2033 children) and the European cross-sectional PARSIFAL study (1120 children). Seven RORA SNPs confined into a 49 kb region were significantly associated with physician-diagnosed childhood asthma. The most significant association with rs7164773 (T/C) was driven by the CC genotype in asthma cases (OR = 2.0, 95%CI 1.36-2.93, p = 0.0003 in BAMSE; and 1.61, 1.18-2.19, p = 0.002 in the combined BAMSE-PARSIFAL datasets, respectively), and strikingly, the risk effect was dependent on the Gln344Arg mutation in NPSR1. In cell models, stimulation of NPSR1 activated a pathway including RORA and other circadian clock genes. Over-expression of RORA decreased NPSR1 promoter activity further suggesting a regulatory loop between these genes. In addition, Rora mRNA expression was lower in the lung tissue of Npsr1 deficient mice compared to wildtype littermates during the early hours of the light period. We conclude that RORA SNPs are associated with childhood asthma and show epistasis with NPSR1, and the interaction between RORA and NPSR1 may be of biological relevance. Combinations of common susceptibility alleles and less common functional polymorphisms may modify the joint risk effects on asthma susceptibility.

Role and regulation of VEGF and its receptors 1 and 2 in the aseptic loosening of total hip implants.

It was hypothesized that vascular endothelial growth factor (VEGF) in fibroblasts participates in aseptic loosening of total hip replacement (THR) implants. Therefore, osteoarthritic (OA) samples (n = 11) were compared with synovial membrane-like interface tissues from revision THR (n = 10). VEGF-A and its receptors were stained using streptavidin-immunoperoxidase method. Their regulation by hypoxia and cytokines were studied in cultured fibroblasts using quantitative real-time polymerase chain reaction (qRT-PCR). VEGFR1(+) lining cells (p < 0.01), stromal fibroblast-like cells (p = 0.001) and stromal macrophage-like cells (p < 0.05) were more numerous in rTHR than in OA. As to VEGFR2(+), only stromal fibroblast-like cells in rTHR outnumbered those found in OA (p < 0.05). VEGFRs in synovial fibroblasts were not affected by hypoxia, but VEGF increased 2.4-fold (p < 0.05). Interleukin-4 up-regulated VEGFR1 expression 23-fold. This is the first study to describe a difference between rTHR and OA in VEGF receptors, particularly VEGFR1. Hypoxia increased VEGF, but the VEGFR1 increase in the lining and stroma is probably IL-4 driven, in accordance with the M2-type macrophage dominance in interface tissues. VEGF/VEGFR system is also affected by hypoxia and may play a role in angiogenesis and bone pathology in aseptic loosening of total hip implants.

Three-dimensional tissue culture based on magnetic cell levitation.

Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.

Extracellular and intracellular mechanisms that mediate the metastatic activity of exogenous osteopontin.

BACKGROUND: Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. In this study, the authors used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS: Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo that were designed to test the extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS: In the extracellular environment, the results confirmed that soluble osteopontin is required for its prometastatic effects; this phenomenon is specific, arginine-glycine-aspartic acid (RGD)-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induced rapid tyrosine 418 (Tyr-418) dephosphorylation of the cellular homolog of the Rous sarcoma virus (c-Src), with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation was followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSIONS: The results of this study revealed a complex molecular interaction as well as a dual role for osteopontin in metastasis that depends on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to antimetastasis strategies.