BMP-2 (and partially GDF-5) coating significantly accelerates and augments bone formation close to hydroxyapatite/tricalcium-phosphate/brushite implant cylinders for tibial bone defects in senile, osteopenic sheep.

Bilateral defects (diameter 8 mm) in the medial tibial head of senile, osteopenic female sheep (n = 48; 9.63 ± 0.10 years; mean ± SEM) were treated with hydroxyapatite (HA)/beta-tricalcium phosphate (ß-TCP)/dicalcium phosphate dihydrate (DCPD; brushite) cylinders coated with BMP-2 (25 or 250 micrograms) or growth differentiation factor (GDF)-5 (125 or 1250 micrograms; left side); cylinders without BMP served as controls (right side). Three, 6, and 9 months post-operation (n = 6 each group), bone structure and formation were analyzed in vivo by X-ray and ex vivo by osteodensitometry, histomorphometry, and micro-computed tomography (micro-CT) at 3 and 9 months. Semi-quantitative X-ray evaluation showed significantly increasing bone densities around all implant cylinders over time. High-dose BMP-2-coated cylinders (3 and 9 months) and low-dose GDF-5-coated cylinders (3 and 6 months) demonstrated significantly higher densities than controls (dose-dependent for BMP-2 at 3 months). This was confirmed by osteodensitometry at 9 months for high-dose BMP-2-coated cylinders (and selected GDF-5 groups), and was again dose-dependent for BMP-2. Osteoinduction by BMP-2 was most pronounced in the adjacent bone marrow (dynamic histomorphometry/micro-CT). BMP-2 (and partially GDF-5) significantly increased the bone formation in the vicinity of HA/TCP/DCPD cylinders used to fill tibial bone defects in senile osteopenic sheep and may be suitable for surgical therapy of critical size, non-load-bearing bone defects in cases of failed tibial head fracture or defect healing.

GSK3 as a Master Regulator of Cellular Processes.

Since its initial purification and characterization as an enzyme negatively regulating glycogen synthase activity [...].

In Vitro Cartilage Regeneration with a Three-Dimensional Polyglycolic Acid (PGA) Implant in a Bovine Cartilage Punch Model.

Resorbable polyglycolic acid (PGA) chondrocyte grafts are clinically established for human articular cartilage defects. Long-term implant performance was addressed in a standardized in vitro model. PGA implants (+/- bovine chondrocytes) were placed inside cartilage rings punched out of bovine femoral trochleas (outer Ø 6 mm; inner defect Ø 2 mm) and cultured for 84 days (12 weeks). Cartilage/PGA hybrids were subsequently analyzed by histology (hematoxylin/eosin; safranin O), immunohistochemistry (aggrecan, collagens 1 and 2), protein assays, quantitative real-time polymerase chain reactions, and implant push-out force measurements. Cartilage/PGA hybrids remained vital with intact matrix until 12 weeks, limited loss of proteoglycans from "host" cartilage or cartilage-PGA interface, and progressively diminishing release of proteoglycans into the supernatant. By contrast, the collagen 2 content in cartilage and cartilage-PGA interface remained approximately constant during culture (with only little collagen 1). Both implants (+/- cells) displayed implant colonization and progressively increased aggrecan and collagen 2 mRNA, but significantly decreased push-out forces over time. Cell-loaded PGA showed significantly accelerated cell colonization and significantly extended deposition of aggrecan. Augmented chondrogenic differentiation in PGA and cartilage/PGA-interface for up to 84 days suggests initial cartilage regeneration. Due to the PGA resorbability, however, the model exhibits limitations in assessing the "lateral implant bonding".

Association of Human FOS Promoter Variants with the Occurrence of Knee-Osteoarthritis in a Case Control Association Study.

Our aim was to analyse (i) the presence of single nucleotide polymorphisms (SNPs) in the JUN and FOS core promoters in patients with rheumatoid arthritis (RA), knee-osteoarthritis (OA), and normal controls (NC); (ii) their functional influence on JUN/FOS transcription levels; and (iii) their associations with the occurrence of RA or knee-OA. JUN and FOS promoter SNPs were identified in an initial screening population using the Non-Isotopic RNase Cleavage Assay (NIRCA); their functional influence was analysed using reporter gene assays. Genotyping was done in RA (n = 298), knee-OA (n = 277), and NC (n = 484) samples. For replication, significant associations were validated in a Finnish cohort (OA: n = 72, NC: n = 548). Initially, two SNPs were detected in the JUN promoter and two additional SNPs in the FOS promoter in perfect linkage disequilibrium (LD). JUN promoter SNP rs4647009 caused significant downregulation of reporter gene expression, whereas reporter gene expression was significantly upregulated in the presence of the FOS promoter SNPs. The homozygous genotype of FOS promoter SNPs showed an association with the susceptibility for knee-OA (odds ratio (OR) 2.12, 95% confidence interval (CI) 1.2⁻3.7, p = 0.0086). This association was successfully replicated in the Finnish Health 2000 study cohort (allelic OR 1.72, 95% CI 1.2⁻2.5, p = 0.006). FOS Promoter variants may represent relevant susceptibility markers for knee-OA.

Cellular and Molecular Effects of High-Molecular-Weight Heparin on Matrix Metalloproteinase 9 Expression.

Blood sampling with different anticoagulants alters matrix metalloproteinase (MMP-) 9 expression, thus influencing its concentration and diagnostic validity. Here, we aimed to evaluate the effects of different anticoagulants on MMP-9 regulation. MMP-9 expression was assessed in response to ethylenediaminetetraacetic acid, citrate, and high-/low-molecular-weight heparin (HMWH, LMWH) in co-culture experiments using THP-1, Jurkat, and HT cells (representing monocytes, T, and B cells). Triple and double cell line co-culture experiments revealed that HMWH treatment of THP-1 and Jurkat led to a significant MMP-9 induction, whereas other anticoagulants and cell type combinations had no effect. Supernatant of HMWH-treated Jurkat cells also induced MMP-9 in THP-1 suggesting monocytes as MMP-9 producers. HMWH-induced cytokine/chemokine secretion was assessed in co-culture supernatant, and the influence of cytokines/chemokines on MMP-9 production was analyzed. These experiments revealed that Jurkat-derived IL-16 and soluble intercellular adhesion molecule (sICAM-) 1 are able to induce MMP-9 and IL-8 production by THP-1. As a consequence, the increased MMP-9 expression found in HMWH blood samples may be influenced by HMWH-dependent secretion of IL-16 and sICAM-1 by T cells resulting in an increased production of MMP-9 and IL-8 by monocytes. IL-8, in turn, may support MMP-9 and its own expression in a positive autocrine feedback loop.

Proteome and Phosphoproteome Analysis in TNF Long Term-Exposed Primary Human Monocytes.

To better understand the inflammation-associated mechanisms modulating and terminating tumor necrosis factor (TNF-)induced signal transduction and the development of TNF tolerance, we analyzed both the proteome and the phosphoproteome in TNF long term-incubated (i.e., 48 h) primary human monocytes using liquid chromatography-mass spectrometry. Our analyses revealed the presence of a defined set of proteins characterized by reproducible changes in expression and phosphorylation patterns in long term TNF-treated samples. In total, 148 proteins and 569 phosphopeptides were significantly regulated (103 proteins increased, 45 proteins decreased; 377 peptides with increased and 192 peptides with decreased phosphorylation). A variety of these proteins are associated with the non-canonical nuclear factor κB (NF-κB) pathway (nuclear factor κB (NFKB) 2, v-rel reticuloendotheliosis viral oncogene homolog (REL) B, indolamin-2,3-dioxygenase (IDO), kynureninase (KYNU)) or involved in the negative regulation of the canonical NF-κB system. Within the phosphopeptides, binding motifs for specific kinases were identified. Glycogen synthase kinase (GSK) 3 proved to be a promising candidate, since it targets NF-κB inhibiting factors, such as CCAAT/enhancer binding protein (C/EBP) ß. Our experiments demonstrate that both proteome and phosphoproteome analysis can be effectively applied to study protein/phosphorylation patterns of primary monocytes. These results provide new regulatory candidates and evidence for a complex network of specific but synergistically acting/cooperating mechanisms enabling the affected cells to resist sustained TNF exposure and resulting in the resolution of inflammation.

Identification of two forms of TNF tolerance in human monocytes: differential inhibition of NF-κB/AP-1- and PP1-associated signaling.

The molecular basis of TNF tolerance is poorly understood. In human monocytes we detected two forms of TNF refractoriness, as follows: absolute tolerance was selective, dose dependently affecting a small group of powerful effector molecules; induction tolerance represented a more general phenomenon. Preincubation with a high TNF dose induces both absolute and induction tolerance, whereas low-dose preincubation predominantly mediates absolute tolerance. In cells preincubated with the high TNF dose, we observed blockade of IκBα phosphorylation/proteolysis and nuclear p65 translocation. More prominent in cells preincubated with the high dose, reduced basal IκBα levels were found, accompanied by increased IκBα degradation, suggesting an increased IκBα turnover. In addition, a nuclear elevation of p50 was detected in tolerant cells, which was more visible following high-dose preincubation. TNF-induced phosphorylation of p65-Ser(536), p38, and c-jun was inhibited, and basal inhibitory p65-Ser(468) phosphorylation was increased in tolerant cells. TNF tolerance induced by the low preincubation dose is mediated by glycogen synthesis kinase-3, whereas high-dose preincubation-mediated tolerance is regulated by A20/glycogen synthesis kinase-3 and protein phosphatase 1-dependent mechanisms. To our knowledge, we present the first genome-wide analysis of TNF tolerance in monocytic cells, which differentially inhibits NF-κB/AP-1-associated signaling and shifts the kinase/phosphatase balance. These forms of refractoriness may provide a cellular paradigm for resolution of inflammation and may be involved in immune paralysis.

Regulation of monocyte differentiation by specific signaling modules and associated transcription factor networks.

Monocyte/macrophages are important players in orchestrating the immune response as well as connecting innate and adaptive immunity. Myelopoiesis and monopoiesis are characterized by the interplay between expansion of stem/progenitor cells and progression towards further developed (myelo)monocytic phenotypes. In response to a variety of differentiation-inducing stimuli, various prominent signaling pathways are activated. Subsequently, specific transcription factors are induced, regulating cell proliferation and maturation. This review article focuses on the integration of signaling modules and transcriptional networks involved in the determination of monocytic differentiation.

Cyclophilin A is a ligand for RAGE in thrombo-inflammation.

AIMS: Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. METHODS AND RESULTS: Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. CONCLUSION: We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation.

CCAAT/enhancer-binding protein beta inhibits proliferation in monocytic cells by affecting the retinoblastoma protein/E2F/cyclin E pathway but is not directly required for macrophage morphology.

Monocytic differentiation is orchestrated by complex networks that are not fully understood. This study further elucidates the involvement of transcription factor CCAAT/enhancer-binding protein ß (C/EBPß). Initially, we demonstrated a marked increase in nuclear C/EBPß-liver-enriched activating protein* (LAP*)/liver-enriched activating protein (LAP) levels and LAP/liver-enriched inhibiting protein (LIP) ratios in phorbol 12-myristate 13-acetate (PMA)-treated differentiating THP-1 premonocytic cells accompanied by reduced proliferation. To directly study C/EBPß effects on monocytic cells, we generated novel THP-1-derived (low endogenous C/EBPß) cell lines stably overexpressing C/EBPß isoforms. Most importantly, cells predominantly overexpressing LAP* (C/EBPß-long), but not those overexpressing LIP (C/EBPß-short), exhibited a reduced proliferation, with no effect on morphology. PMA-induced inhibition of proliferation was attenuated in C/EBPß-short cells. In C/EBPß(WT) macrophage-like cells (high endogenous C/EBPß), we measured a reduced proliferation/cycling index compared with C/EBPß(KO). The typical macrophage morphology was only observed in C/EBPß(WT), whereas C/EBPß(KO) stayed round. C/EBPα did not compensate for C/EBPß effects on proliferation/morphology. Serum reduction, an independent approach known to inhibit proliferation, induced macrophage morphology in C/EBPß(KO) macrophage-like cells but not THP-1. In PMA-treated THP-1 and C/EBPß-long cells, a reduced phosphorylation of cell cycle repressor retinoblastoma was found. In addition, C/EBPß-long cells showed reduced c-Myc expression accompanied by increased CDK inhibitor p27 and reduced cyclin D1 levels. Finally, C/EBPß-long and C/EBPß(WT) cells exhibited low E2F1 and cyclin E levels, and C/EBPß overexpression was found to inhibit cyclin E1 promoter-dependent transcription. Our results suggest that C/EBPß reduces monocytic proliferation by affecting the retinoblastoma/E2F/cyclin E pathway and that it may contribute to, but is not directly required for, macrophage morphology. Inhibition of proliferation by C/EBPß may be important for coordinated monocytic differentiation.

MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitous protein mediating versatile effects in a variety of cell types, including actin crosslinking, signal transduction, and intracellular transport processes. MARCKS's functional role in monocyte/macrophages, however, has not yet been adequately addressed. Thus, the aim of this study was to further elucidate the impact of MARCKS on central cellular functions of monocytic cells. To address this topic, we generated monocytic THP-1 (Tohoku Hospital Pediatrics-1)-derived MARCKS wildtype and knockout (KO) cells using the CRISPR/Cas9 technique. Remarkably, in the absence of MARCKS, both total and intracellular reactive oxygen species (ROS) production were strongly suppressed but restored following transient MARCKS re-transfection. In contrast, proliferation, differentiation, cytokine expression, and phagocytosis remained unaltered. A complete inhibition of ROS production could also be achieved in THP-1-derived PKCß KO cells or in PKC inhibitor Staurosporine-treated primary human monocytes. MARCKS deficiency also involved reduced basal Akt phosphorylation and delayed re-phosphorylation. Further analyses indicated that long-term TNF pre-incubation strongly enhances monocytic ROS production, which was completely blocked in MARCKS and PKCß KO cells. Collectively, our study demonstrates that MARCKS is an essential molecule enabling ROS production by monocytic cells and suggests that MARCKS is part of a signal cascade involved in ROS formation.

Single Application of Low-Dose, Hydroxyapatite-Bound BMP-2 or GDF-5 Induces Long-Term Bone Formation and Biomechanical Stabilization of a Bone Defect in a Senile Sheep Lumbar Osteopenia Model.

Effects of hydroxyapatite (HA) particles with bone morphogenetic BMP-2 or GDF-5 were compared in sheep lumbar osteopenia; in vitro release in phosphate-buffered saline (PBS) or sheep serum was assessed by ELISA. Lumbar (L) vertebral bone defects (Ø 3.5 mm) were generated in aged, osteopenic female sheep (n = 72; 9.00 ± 0.11 years; mean ± SEM). Treatment was: (a) HA particles (2.5 mg; L5); or (b) particles coated with BMP-2 (1 µg; 10 µg) or GDF-5 (5 µg; 50 µg; L4; all groups n = 6). Untouched vertebrae (L3) served as controls. Three and nine months post-therapy, bone formation was assessed by osteodensitometry, histomorphometry, and biomechanical testing. Cumulative 14-day BMP release was high in serum (76-100%), but max. 1.4% in PBS. In vivo induction of bone formation by HA particles with either growth factor was shown by: (i) significantly increased bone volume, trabecular and cortical thickness (overall increase HA + BMP vs. control close to the injection channel 71%, 110%, and 37%, respectively); (ii) partial significant effects for bone mineral density, bone formation, and compressive strength (increase 17%; 9 months; GDF-5). Treatment effects were not dose-dependent. Combined HA and BMPs (single low-dose) highly augment long-term bone formation and biomechanical stabilization in sheep lumbar osteopenia. Thus, carrier-bound BMP doses 20,000-fold to 1000-fold lower than previously applied appear suitable for spinal fusion/bone regeneration and improved treatment safety.

Activation of GSK3 Prevents Termination of TNF-Induced Signaling.

BACKGROUND: Termination of TNF-induced signaling plays a key role in the resolution of inflammation with dysregulations leading to severe pathophysiological conditions (sepsis, chronic inflammatory disease, cancer). Since a recent phospho-proteome analysis in human monocytes suggested GSK3 as a relevant kinase during signal termination, we aimed at further elucidating its role in this context. MATERIALS AND METHODS: For the analyses, THP-1 monocytic cells and primary human monocytes were used. Staurosporine (Stauro) was applied to activate GSK3 by inhibiting kinases that mediate inhibitory GSK3α/ß-Ser21/9 phosphorylation (eg, PKC). For GSK3 inhibition, Kenpaulone (Ken) was used. GSK3- and PKC-siRNAs were applied for knockdown experiments. Protein expression and phosphorylation were assessed by Western blot or ELISA and mRNA expression by qPCR. NF-κB activation was addressed using reporter gene assays. RESULTS: Constitutive GSK3ß and PKCß expression and GSK3α/ß-Ser21/9 and PKCα/ßII-Thr638/641 phosphorylation were not altered during TNF long-term incubation. Stauro-induced GSK3 activation (demonstrated by Bcl3 reduction) prevented termination of TNF-induced signaling as reflected by strongly elevated IL-8 expression (used as an indicator) following TNF long-term incubation. A similar increase was observed in TNF short-term-exposed cells, and this effect was inhibited by Ken. PKCα/ß-knockdown modestly increased, whereas GSK3α/ß-knockdown inhibited TNF-induced IL-8 expression. TNF-dependent activation of two NF-κB-dependent indicator plasmids was enhanced by Stauro, demonstrating transcriptional effects. A TNF-induced increase in p65-Ser536 phosphorylation was further enhanced by Stauro, whereas IκBα proteolysis and IKKα/ß-Ser176/180 phosphorylation were not affected. Moreover, PKCß-knockdown reduced levels of Bcl3. A20 and IκBα mRNA, both coding for signaling inhibitors, were dramatically less affected under our conditions when compared to IL-8, suggesting differential transcriptional effects. CONCLUSION: Our results suggest that GSK3 activation is involved in preventing the termination of TNF-induced signaling. Our data demonstrate that activation of GSK3 - either pathophysiologically or pharmacologically induced - may destroy the finely balanced condition necessary for the termination of inflammation-associated signaling.

The Inverse Spacer-A Novel, Safe, and Cost-Effective Approach in Routine Procedures for Revision Knee Arthroplasty.

BACKGROUND: A major disadvantage of current spacers for two-stage revision total knee arthroplasty (R-TKA) is the risk of (sub-) luxation during mobilization in the prosthesis-free interval, limiting their clinical success with detrimental consequences for the patient. The present study introduces a novel inverse spacer, which prevents major complications, such as spacer (sub-) luxations and/or fractures of spacer or bone. METHODS: The hand-made inverse spacer consisted of convex tibial and concave femoral components of polymethylmethacrylate bone cement and was intra-operatively molded under maximum longitudinal tension in 5° flexion and 5° valgus position. Both components were equipped with a stem for rotational stability. This spacer was implanted during an R-TKA in 110 knees with diagnosed or suspected periprosthetic infection. Postoperative therapy included a straight leg brace and physiotherapist-guided, crutch-supported mobilization with full sole contact. X-rays were taken before and after prosthesis removal and re-implantation. RESULTS: None of the patients experienced (sub-) luxations/fractures of the spacer, periprosthetic fractures, or soft tissue compromise requiring reoperation. All patients were successfully re-implanted after a prosthesis-free interval of 8 weeks, except for three patients requiring an early exchange of the spacer due to persisting infection. In these cases, the prosthetic-free interval was prolonged for one week. CONCLUSION: The inverse spacer in conjunction with our routine procedure is a safe and cost-effective alternative to other articulating or static spacers, and allows crutch-supported sole contact mobilization without major post-operative complications. Maximum longitudinal intra-operative tension in 5° flexion and 5° valgus position appears crucial for the success of surgery.

Performance of Calcium Phosphate Cements in the Augmentation of Sheep Vertebrae-An Ex Vivo Study.

Oil-based calcium phosphate cement (Paste-CPC) shows not only prolonged shelf life and injection times, but also improved cohesion and reproducibility during application, while retaining the advantages of fast setting, mechanical strength, and biocompatibility. In addition, poly(L-lactide-co-glycolide) (PLGA) fiber reinforcement may decrease the risk for local extrusion. Bone defects (diameter 5 mm; depth 15 mm) generated ex vivo in lumbar (L) spines of female Merino sheep (2-4 years) were augmented using: (i) water-based CPC with 10% PLGA fiber reinforcement (L3); (ii) Paste-CPC (L4); or (iii) clinically established polymethylmethacrylate (PMMA) bone cement (L5). Untouched (L1) and empty vertebrae (L2) served as controls. Cement performance was analyzed using micro-computed tomography, histology, and biomechanical testing. Extrusion was comparable for Paste-CPC(-PLGA) and PMMA, but significantly lower for CPC + PLGA. Compressive strength and Young's modulus were similar for Paste-CPC and PMMA, but significantly higher compared to those for empty defects and/or CPC + PLGA. Expectedly, all experimental groups showed significantly or numerically lower compressive strength and Young's modulus than those of untouched controls. Ready-to-use Paste-CPC demonstrates a performance similar to that of PMMA, but improved biomechanics compared to those of water-based CPC + PLGA, expanding the therapeutic arsenal for bone defects. O, significantly lower extrusion of CPC + PLGA fibers into adjacent lumbar spongiosa may help to reduce the risk of local extrusion in spinal surgery.

Increase of α-dicarbonyls in liver and receptor for advanced glycation end products on immune cells are linked to nonalcoholic fatty liver disease and liver cancer.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver with a very poor prognosis and constantly growing incidence. Among other primary risks of HCC, metabolic disorders and obesity have been extensively investigated over recent decades. The latter can promote nonalcoholic fatty liver disease (NAFLD) leading to the inflammatory form of nonalcoholic steatohepatitis (NASH), that, in turn, promotes HCC. Molecular determinants of this pathogenic progression, however, remain largely undefined. In this study, we have focussed on the investigation of α-dicarbonyl compounds (α-dC), highly reactive and tightly associated with overweight-induced metabolic disorders, and studied their potential role in NAFLD and progression toward HCC using murine models. NAFLD was induced using high-fat diet (HFD). Autochthonous HCC was induced using transposon-based stable intrahepatic overexpression of oncogenic NRASG12V in mice lacking p19Arf tumor suppressor. Our study demonstrates that the HFD regimen and HCC resulted in strong upregulation of α-dC in the liver, heart, and muscles. In addition, an increase in α-dC was confirmed in sera of NAFLD and NASH patients. Furthermore, higher expression of the receptor for advanced glycation products (RAGE) was detected exclusively on immune cells and not on stroma cells in livers of mice with liver cancer progression. Our work confirms astable interplay of liver inflammation, carbonyl stress mediated by α-dC, and upregulated RAGE expression on CD8+ Tand natural killer (NK) cells in situ in NAFLD and HCC, as key factors/determinants in liver disease progression. The obtained findings underline the role of α-dC and RAGE+CD8+ Tand RAGE+ NK cells as biomarkers and candidates for a local therapeutic intervention in NAFLD and malignant liver disease.

ITD- and FL-induced FLT3 signal transduction leads to increased C/EBPbeta-LIP expression and LIP/LAP ratio by different signalling modules.

FLT3 receptor-associated signalling plays a role in proliferation and leukaemia. The transcription factor C/EBPbeta may be involved in malignancy with its alternative translation product C/EBPbeta-LIP. We investigated a potential connection between FLT3 signalling and the C/EBPbeta system in FLT3-internal tandem duplication (ITD)-positive leukaemia cells and FLT3-ITD- or FLT3-wild type (WT)-transfected 32D cells. In FLT3-ITD-positive cells or when ITD sequences were inserted into the FLT3-WT receptor, significant LIP levels, increased LIP/LAP ratios, and enhanced proliferation rates were detected, which were reduced by FLT3 inhibition. In FLT3-WT cells, incubation with FLT3 receptor ligand (FL) also elevated LIP, LIP/LAP, and proliferation, albeit to a lesser extent. CEBPB-directed siRNA decreased both LIP and proliferation rates in FLT3-ITD-positive and FL-stimulated FLT3-WT-positive cells. PI3K inhibition affected ITD-associated and FL-induced LIP levels. Rapamycin, an inhibitor of mTOR involved in CEBPB translation, completely blocked the increase in LIP in FL-stimulated FLT3-WT- but not FLT3-ITD-positive cells. In contrast, the ITD-associated LIP elevation was mediated by p(90)-ribosomal-S6-kinase. This is the first report showing a LIP increase in the presence of ITD or following FL exposure. Our data suggest fundamental differences in the signalling cascades activated via ITD mutations or following FL stimulation, indicating the need for adapted molecular therapy.

GSK3: A Kinase Balancing Promotion and Resolution of Inflammation.

GSK3 has been implicated for years in the regulation of inflammation and addressed in a plethora of scientific reports using a variety of experimental (disease) models and approaches. However, the specific role of GSK3 in the inflammatory process is still not fully understood and controversially discussed. Following a detailed overview of structure, function, and various regulatory levels, this review focusses on the immunoregulatory functions of GSK3, including the current knowledge obtained from animal models. Its impact on pro-inflammatory cytokine/chemokine profiles, bacterial/viral infections, and the modulation of associated pro-inflammatory transcriptional and signaling pathways is discussed. Moreover, GSK3 contributes to the resolution of inflammation on multiple levels, e.g., via the regulation of pro-resolving mediators, the clearance of apoptotic immune cells, and tissue repair processes. The influence of GSK3 on the development of different forms of stimulation tolerance is also addressed. Collectively, the role of GSK3 as a kinase balancing the initiation/perpetuation and the amelioration/resolution of inflammation is highlighted.

Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue.

In rheumatoid arthritis (RA), the expression of many pro-destructive/pro-inflammatory proteins depends on the transcription factor AP-1. Therefore, our aim was to analyze the presence and functional relevance of mutations in the coding regions of the AP-1 subunits of the fos and jun family in peripheral blood (PB) and synovial membranes (SM) of RA and osteoarthritis patients (OA, disease control), as well as normal controls (NC). Using the non-isotopic RNAse cleavage assay, one known polymorphism (T252C: silent; rs1046117; present in RA, OA, and NC) and three novel germline mutations of the cfos gene were detected: (i) C361G/A367G: Gln121Glu/Ile123Val, denoted as "fos121/123"; present only in one OA sample; (ii) G374A: Arg125Lys, "fos125"; and (iii) C217A/G374A: Leu73Met/Arg125Lys, "fos73/125", the latter two exclusively present in RA. In addition, three novel somatic cjun mutations (604-606ΔCAG: ΔGln202, "jun202"; C706T: Pro236Ser, "jun236"; G750A: silent) were found exclusively in the RA SM. Tansgenic expression of fos125 and fos73/125 mutants in NIH-3T3 cells induced an activation of reporter constructs containing either the MMP-1 (matrix metalloproteinase) promoter (3- and 4-fold, respectively) or a pentameric AP-1 site (approximately 5-fold). Combined expression of these two cfos mutants with cjun wildtype or mutants (jun202, jun236) further enhanced reporter expression of the pentameric AP-1 construct. Finally, genotyping for the novel functionally relevant germline mutations in 298 RA, 288 OA, and 484 NC samples revealed no association with RA. Thus, functional cfos/cjun mutants may contribute to local joint inflammation/destruction in selected patients with RA by altering the transactivation capacity of AP-1 complexes.