Ex vivo gene transfer in the years to come

Synovial fibroblasts (SFs) have become a major target for ex vivo gene transfer in rheumatoid arthritis (RA), but efficient transduction of RA-SFs still is a major problem. The low proliferation rate and heterogeneity of RA-SFs, together with their lack of highly specific surface receptors, have hampered a more extensive application of this technique. Improving transduction protocols with conventional viral vectors, therefore, as well as developing novel strategies, such as alternative target cells, and novel delivery systems constitute a major challenge. Recent progress in this field will lead to the achievement of high transgene expression, and will facilitate the use of gene transfer in human trials

Molekulare Signalwege der aseptischen Endoprothesenlockerung

Die Behandlung von immobilisierenden degenerativen und entzündlichen Gelenkerkrankungen mit der Implantation von Endoprothesen ist ein großer Erfolg und Fortschritt in der Medizin und hat stark zur Verbesserung der Lebensqualität der betroffenen Patienten beigetragen. Jährlich werden weltweit ca. 1,3 Mio. Endoprothesen implantiert, davon allein 500.000 in den USA. Dennoch sind die einmal implantierten Prothesen nicht von lebenslanger Dauer und unterliegen multiplen Einflüssen. Trotz immer neuer Entwicklungen müssen innerhalb der ersten 15 Jahre bis zu 10% der Implantate aufgrund vorzeitiger Prothesenlockerung gewechselt werden. Bei vorzeitiger Lockerung ohne Infekt oder Trauma spricht man von aseptischer Lockerung. Es ist allgemein bekannt, dass durch Abrieb entstandene Kleinstpartikel und aktivierte Makrophagen die Hauptrolle im Prozess der aseptischen Lockerung spielen. Die Pathophysiologie ist jedoch noch nicht vollständig erklärt. Die vorliegende Arbeit gibt eine Übersicht über die anerkannten molekularen Mechanismen und die Signalwege, die zur aseptischen Prothesenlockerung führen. Außerdem werden neue Therapieoptionen zur Vermeidung der aseptischen Lockerung diskutier

ATP Induced Brain-Derived Neurotrophic Factor Expression and Release from Osteoarthritis Synovial Fibroblasts Is Mediated by Purinergic Receptor P2X4

Brain-derived neurotrophic factor (BDNF), a neuromodulator involved in nociceptive hypersensitivity in the central nervous system, is also expressed in synoviocytes of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. We investigated the role of P2 purinoreceptors in the induction of BDNF expression in synovial fibroblasts (SF) of OA and RA patients. Cultured SF from patients with symptomatic knee OA and RA were stimulated with purinoreceptor agonists ATP, ADP, or UTP. The expression of BDNF mRNA was measured by quantitative TaqMan PCR. BDNF release into cell culture supernatants was monitored by ELISA. P2X4 expression in synovial tissue was detected by immunohistochemistry. Endogenous P2X4 expression was decreased by siRNA transfection before ATP stimulation. Kinase pathways were blocked before ATP stimulation. BDNF mRNA expression levels in OASF were increased 2 h and 5 h after ATP stimulation. Mean BDNF levels in cell culture supernatants of unstimulated OASF and RASF were 19 (±9) and 67 (±49) pg/ml, respectively. BDNF levels in SF supernatants were only elevated 5 h after ATP stimulation. BDNF mRNA expression in OASF was induced both by P2X receptor agonists ATP and ADP, but not by UTP, an agonist of P2Y purinergic receptors. The ATP-induced BDNF mRNA expression in OASF was decreased by siRNA-mediated reduction of endogenous P2X4 levels compared to scrambled controls. Inhibition of p38, but not p44/42 signalling reduced the ATP-mediated BDNF mRNA induction. Here we show a functional role of the purinergic receptor P2X4 and p38 kinase in the ATP-induced expression and release of the neurotrophin BDNF in SF

The role of membrane lipids in the induction of macrophage apoptosis by microparticles

Microparticles are membrane-derived vesicles that are released from cells during activation or cell death. These particles can serve as mediators of intercellular cross-talk and induce a variety of cellular responses. Previous studies have shown that macrophages undergo apoptosis after phagocytosing microparticles. Here, we have addressed the hypothesis that microparticles trigger this process via lipid pathways. In these experiments, microparticles induced apoptosis in primary macrophage cells or cell lines (RAW 264.7 or U937) with up to a 5-fold increase. Preincubation of macrophages with phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)BP) reduced the microparticle-induced apoptosis in a dose-dependent manner. PtdIns(3,5)BP is a specific inhibitor of the acid sphingomyelinase and thus can block the generation of pro-apoptotic ceramides. Similarly, the pre-incubation of macrophages with PtdIns(3,5)BP prevented microparticle-induced upregulation of caspase 8, which is a major target molecule of ceramide action in the apoptosis pathway. PtdIns(3,5)BP, however, had no effect on the spontaneous rate of apoptosis. To evaluate further signaling pathways induced by microparticles, the extracellular signal regulated kinase (ERK-) 1 was investigated. This kinase plays a role in activating phospholipases A2 which cleaves membrane phospholipids into arachidonic acid; microparticles have been suggested to be a preferred substrate for phospholipases A2. As shown in our experiments, microparticles strongly increased the amount of phosphorylated ERK1/2 in RAW 264.7 macrophages in a time-dependent manner, peaking 15 min after co-incubation. Addition of PD98059, a specific inhibitor of ERK1, prevented the increase in apoptosis of RAW 264.7 macrophages. Together, these data suggest that microparticles perturb lipid homeostasis of macrophages and thereby induce apoptosis. These results emphasize the importance of biolipids in the cellular cross-talk of immune cells. Based on the fact that in clinical situations with excessive cell death such as malignancies, autoimmune diseases and following chemotherapies high levels of circulating microparticles might modulate phagocytosing cells, a suppression of the immune response might occur due to loss of macrophage

DREAM is reduced in synovial fibroblasts of patients with chronic arthritic pain: is it a suitable target for peripheral pain management?

INTRODUCTION: The endogenous pain-relieving system depends in part on the regulation of nociceptive signals through binding of opioids to the corresponding opioid receptor. Interfering with the trans-repression effect of downstream regulatory element antagonist modulator (DREAM) on the transcription of the opioid dynorphin-encoding prodynorphin (pdyn) gene might enhance pain relief in the periphery. METHODS: Expression levels were measured in osteoarthritis (OA) synovial fibroblast-like cells (SFLCs) (n = 8) and in peripheral blood mononuclear cells (PBMCs) from OA patients (n = 53) and healthy controls (n = 26) by real-time polymerase chain reaction. Lysed OA SFLCs were analyzed by immunoprecipitation. Translation of DREAM mRNA was inhibited by small interfering RNAs (siRNAs). Expressions of DREAM, pdyn, and c-fos mRNAs were measured at 24, 48, and 72 hours after transfection. RESULTS: The expression of DREAM mRNA was shown in both healthy and OA SFLCs as well as PBMCs. Inhibiting transcription using siRNAs led to a marked reduction in DREAM expression after 24, 48, and 72 hours. However, no significant changes in c-fos and pdyn expression occurred. In addition, DREAM mRNA expression was significantly reduced in OA patients with chronic pain (pain intensity as measured by a visual analog scale scale of greater than 40), but no pdyn expression was detectable. CONCLUSION: To our knowledge, this is the first report showing the expression of DREAM in SFLCs and PBMCs on the mRNA level. However, DREAM protein was not detectable. Since repression of pdyn transcription persists after inhibiting DREAM translation, DREAM appears to play no functional role in the kappa opioid receptor system in OA SFLCs. Therefore, our data suggest that DREAM appears not to qualify as a target in peripheral pain management

Molecular profile of synovial fibroblasts in rheumatoid arthritis depends on the stage of proliferation

The aim of this study was to explore the molecular profile of proliferating rheumatoid arthritis synovial fibroblasts (RA-SF). Total RNA was extracted from two cultures of RA-SF (low-density [LD] proliferating cells and high-density [HD] nonproliferating cells) and suppression subtractive hybridization was performed to compare differential gene expression of these two cultures. Subtracted cDNA was subcloned, and nucleotide sequences were analyzed to identify each clone. Differential expression of distinct clones was confirmed by semiquantitative RT-PCR. The expression of certain genes in synovial tissues was examined by in situ hybridization. In both LD and HD cells, 44 clones were upregulated. Of the 88 total clones, 46 were identical to sequences that have previously been characterized. Twenty-nine clones were identical to cDNAs that have been identified, but with unknown functions so far, and 13 clones did not show any significant homology to sequences in GenBank (NCBI). Differential expression of distinct clones was confirmed by RT-PCR. In situ hybridization showed that certain genes, such as S100A4, NFAT5, unr and Fbx3, were also expressed predominantly in synovial tissues from patients with RA but not from normal individuals. The expression of distinct genes in proliferating RA-SF could also be found in RA synovium, suggesting that these molecules are involved in synovial activation in RA. Most importantly, the data indicate that the expression of certain genes in RA-SF depends on the stage of proliferation; therefore, the stage needs to be considered in any analysis of differential gene expression in SF