Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit (ZAFES)
Refine
Year of publication
Document Type
- Article (92) (remove)
Has Fulltext
- yes (92)
Is part of the Bibliography
- no (92)
Keywords
- Inflammation (4)
- Pre-analytics (3)
- inflammation (3)
- neuropathic pain (3)
- pain (3)
- spinal cord (3)
- Gene Regulation (2)
- Interleukin (2)
- Lipidomics (2)
- MLL (2)
Institute
Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.
Biglycan, a nitric oxide-regulated gene, affects adhesion, growth, and survival of mesangial cells
(2003)
During glomerular inflammation mesangial cells are the major source and target of nitric oxide that pro-foundly influences proliferation, adhesion, and death of mesangial cells. The effect of nitric oxide on the mRNA expression pattern of cultured rat mesangial cells was therefore investigated by RNA-arbitrarily-primed polymerase chain reaction. Employing this approach, biglycan expression turned out to be down-regulated time- and dose-dependently either by interleukin-1beta-stimulated endogenous nitric oxide production or by direct application of the exogenous nitric oxide donor, diethylenetriamine nitric oxide. There was a corresponding decline in the rate of biglycan biosynthesis and in the steady state level of this proteoglycan. In vivo, in a model of mesangioproliferative glomerulonephritis up-regulation of inducible nitric-oxide synthase mRNA was associated with reduced expression of biglycan in isolated glomeruli. Biglycan expression could be normalized, both in vitro and in vivo, by using a specific inhibitor of the inducible nitric-oxide synthase, l-N6-(l-iminoethyl)-l-lysine dihydrochloride. Further studies showed that biglycan inhibited cell adhesion on type I collagen and fibronectin because of its binding to these substrates. More importantly, biglycan protected mesangial cells from apoptosis by decreasing caspase-3 activity, and it counteracted the proliferative effects of platelet-derived growth factor-BB. These findings indicate a signaling role of biglycan and describe a novel pathomechanism by which nitric oxide modulates the course of renal glomerular disease through regulation of biglycan expression.
Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1β. We demonstrate here that the stable ATP analog adenosine 5′-O-(thiotriphosphate) (ATPγS) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5′-promoter region showed weak responses to ATPγS but confered a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3′-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATPγS potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3′-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATPγS. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATPγS, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1β-inducibility. By confocal microscopy we demonstrate that ATPγS increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.
Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression.
5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.
Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice
(2007)
Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E2 synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE2 synthase-1 (mPGES-1) isomerizes COX-2-derived PGH2 to PGE2. Here, we evaluated the effect of mPGES-1-deficiency on the noci-ceptive behavior in various models of nociception that depend on PGE2 synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE2 synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE2 synthesis to PGD2, PGF2α and 6-keto-PGF1α (stable metabolite of PGI2). Since the latter prostaglandins serve also as mediators of noci-ception they may compensate the loss of PGE2 synthesis in mPGES-1-deficient mice.
Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction
(2008)
The small leucine-rich proteoglycan (SLRP) family has significantly expanded in the past decade to now encompass five discrete classes, grouped by common structural and functional properties. Some of these gene products are not classical proteoglycans, whereas others have new and unique features. In addition to being structural proteins, SLRPs constitute a network of signal regulation: being mostly extracellular, they are upstream of multiple signaling cascades. They affect intracellular phosphorylation, a major conduit of information for cellular responses, and modulate distinct pathways, including those driven by bone morphogenetic protein/transforming growth factor β superfamily members, receptor tyrosine kinases such as ErbB family members and the insulin-like growth factor I receptor, and Toll-like receptors. The wealth of mechanistic insights into the molecular and cellular functions of SLRPs has revealed both the sophistication of this family of regulatory proteins and the challenges that remain in uncovering the totality of their functions. This review is focused on novel biological functions of SLRPs with special emphasis on their protein cores, newly described genetic diseases, and signaling events in which SLRPs play key functions.
Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection
(2008)
Autoimmune liver diseases, such as autoimmune hepatitis (AIH) and primary biliary cirrhosis, often have severe consequences for the patient. Because of a lack of appropriate animal models, not much is known about their potential viral etiology. Infection by liver-tropic viruses is one possibility for the breakdown of self-tolerance. Therefore, we infected mice with adenovirus Ad5 expressing human cytochrome P450 2D6 (Ad-2D6). Ad-2D6–infected mice developed persistent autoimmune liver disease, apparent by cellular infiltration, hepatic fibrosis, “fused” liver lobules, and necrosis. Similar to type 2 AIH patients, Ad-2D6–infected mice generated type 1 liver kidney microsomal–like antibodies recognizing the immunodominant epitope WDPAQPPRD of cytochrome P450 2D6 (CYP2D6). Interestingly, Ad-2D6–infected wild-type FVB/N mice displayed exacerbated liver damage when compared with transgenic mice expressing the identical human CYP2D6 protein in the liver, indicating the presence of a stronger immunological tolerance in CYP2D6 mice. We demonstrate for the first time that infection with a virus expressing a natural human autoantigen breaks tolerance, resulting in a chronic form of severe, autoimmune liver damage. Our novel model system should be instrumental for studying mechanisms involved in the initiation, propagation, and precipitation of virus-induced autoimmune liver diseases.
FTY720 is a novel immunosuppressive drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. In its phosphorylated form FTY720 is a potent S1P receptor agonist. Recently it was also shown that FTY720 can reduce prostaglandin synthesis through the direct inhibition of the cytosolic phospholipase A2 (cPLA2). Since prostaglandins are important mediators of nociception, we studied the effects of FTY720 in different models of nociception. We found that intraperitoneal administration of FTY720 reduced dose-dependently the nociceptive behaviour of rats in the formalin assay. Although the antinociceptive doses of FTY720 were too low to alter the lymphocyte count, prostanoid concentrations in the plasma were dramatically reduced. Surprisingly, intrathecally administered FTY720 reduced the nociceptive behaviour in the formalin assay without altering spinal prostaglandin synthesis, indicating that additional antinociceptive mechanisms beside the inhibition of prostaglandin synthesis are involved. Accordingly, FTY720 reduced also the nociceptive behaviour in the spared nerve injury model for neuropathic pain which does not depend on prostaglandin synthesis. In this model the antinociceptive effect of FTY720 was similar to gabapentin, a commonly used drug to treat neuropathic pain. Taken together we show for the first time that FTY720 possesses antinociceptive properties and that FTY720 reduces nociceptive behaviour during neuropathic pain.
Interleukin (IL)-18, formerly known as interferon (IFN)-γ-inducing factor, is a crucial mediator of host defence and inflammation. Control of IL-18 bioactivity by its endogenous antagonist IL-18 binding protein (IL-18BP) is a major objective of immunoregulation. IL-18BP is strongly up-regulated by IFN-γ, thereby establishing a negative feedback mechanism detectable in cell culture and in vivo. Here we sought to investigate in D.L. Dexter (DLD) colon carcinoma cells molecular mechanisms of IL-18BP induction under the influence of IFN-γ. Mutational analysis revealed that a proximal γ-activated sequence (GAS) at the IL-18BP promoter is of pivotal importance for expression by IFN-γ-activated cells. Use of siRNA underscored the essential role of the signal transducer and activator of transcription (STAT)-1 in this process. Indeed, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis proved STAT1 binding to this particular GAS site. Maximal expression of IL-18BP was dependent on de novo protein synthesis but unaffected by silencing of interferon regulatory factor-1. Altogether, data presented herein indicate that direct action of STAT1 on the IL-18BP promoter at the proximal GAS element is key to IL-18BP expression by IFN-γ-stimulated DLD-1 colon carcinoma cells.
We have investigated the role of reactive oxygen species and thiol-oxidizing agents in the induction of cell death and have shown that adenocarcinoma gastric (AGS) cells respond differently to the oxidative challenge according to the signaling pathways activated. In particular, apoptosis in AGS cells is induced via the mitochondrial pathway upon treatment with thiol-oxidizing agents, such as diamide. Apoptosis is associated with persistent oxidative damage, as evidenced by the increase in carbonylated proteins and the expression/activation of DNA damage-sensitive proteins histone H2A.X and DNA-dependent protein kinase. Resistance to hydrogen peroxide is instead associated with Keap1 oxidation and rapid translocation of Nrf2 into the nucleus. Sensitivity to diamide and resistance to hydrogen peroxide are correlated with GSH redox changes, with diamide severely increasing GSSG, and hydrogen peroxide transiently inducing protein-GSH mixed disulfides. We show that p53 is activated in response to diamide treatment by the oxidative induction of the Trx1/p38(MAPK) signaling pathway. Similar results were obtained with another carcinoma cell line, CaCo2, indicating that these findings are not limited to AGS cells. Our data suggest that thiol-oxidizing agents could be exploited as inducers of apoptosis in tumor histotypes resistant to ROS-producing chemotherapeutics.
Macrophages ingesting apoptotic cells attenuate inflammatory responses, such as reactive oxygen species (ROS) generation. In atherosclerosis, ongoing inflammation and accumulation of apoptotic/necrotic material are observed, suggesting defects of phagocytes in recognizing or responding to dying cells. Modified lipoproteins such as oxidized LDL (oxLDL) are known to promote inflammation and to interfere with apoptotic cell clearance. Here, we studied the impact of cells exposed to oxLDL on their ability to interfere with the oxidative burst in phagocytes. In contrast to apoptotic cells, cells dying in response to or in the presence of oxLDL failed to suppress ROS generation despite efficiently being taken up by phagocytes. In addition, apoptotic cells, but not oxLDL-treated cells, inhibited phosphorylation of extracellular signal-regulated kinase, which is important for NADPH oxidase activation. oxLDL treatment did not interfere with activation of the antiinflammatory transcriptional regulator peroxisome proliferator-activated receptor gamma by apoptotic cells. Moreover, cells exposed to oxLDL failed to suppress lipopolysaccharide- induced proinflammatory cytokine expression, whereas apoptotic cells attenuated these phagocyte responses. Thus, the presence of oxLDL during cell death impaired the ability of apoptotic cells to act antiinflammatory with regard to oxidative burst inhibition and cytokine expression in phagocytes.
Background Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter which mainly mediates its effects on neurons via ionotropic (GABAA) and metabotropic (GABAB) receptors. GABAB receptors are widely expressed in the central and the peripheral nervous system. Although there is evidence for a key function of GABAB receptors in the modulation of pain, the relative contribution of peripherally- versus centrally-expressed GABAB receptors is unclear. Results In order to elucidate the functional relevance of GABAB receptors expressed in peripheral nociceptive neurons in pain modulation we generated and analyzed conditional mouse mutants lacking functional GABAB(1) subunit specifically in nociceptors, preserving expression in the spinal cord and brain (SNS-GABAB(1)-/- mice). Lack of the GABAB(1) subunit precludes the assembly of functional GABAB receptor. We analyzed SNS-GABAB(1)-/- mice and their control littermates in several models of acute and neuropathic pain. Electrophysiological studies on peripheral afferents revealed higher firing frequencies in SNS-GABAB(1)-/- mice compared to corresponding control littermates. However no differences were seen in basal nociceptive sensitivity between these groups. The development of neuropathic and chronic inflammatory pain was similar across the two genotypes. The duration of nocifensive responses evoked by intraplantar formalin injection was prolonged in the SNS-GABAB(1)-/- animals as compared to their control littermates. Pharmacological experiments revealed that systemic baclofen-induced inhibition of formalin-induced nociceptive behaviors was not dependent upon GABAB(1) expression in nociceptors. Conclusion This study addressed contribution of GABAB receptors expressed on primary afferent nociceptive fibers to the modulation of pain. We observed that neither the development of acute and chronic pain nor the analgesic effects of a systematically-delivered GABAB agonist was significantly changed upon a specific deletion of GABAB receptors from peripheral nociceptive neurons in vivo. This lets us conclude that GABAB receptors in the peripheral nervous system play a less important role than those in the central nervous system in the regulation of pain.
Diabetic nephropathy (DN) is a major cause of end-stage renal failure worldwide. Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN. In this study we present evidence, that CXCL16 is the main receptor in human podocytes mediating the uptake of oxLDL. In contrast, in primary tubular cells CD36 was mainly involved in the uptake of oxLDL. We further demonstrate that oxLDL down-regulated α3-integrin expression and increased the production of fibronectin in human podocytes. In addition, oxLDL uptake induced the production of reactive oxygen species (ROS) in human podocytes. Inhibition of oxLDL uptake by CXCL16 blocking antibodies abrogated the fibronectin and ROS production and restored α3 integrin expression in human podocytes. Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes. Importantly, in streptozotocin-induced diabetic mice an early induction of CXCL16 was accompanied by higher levels of oxLDL. Finally immunofluorescence analysis in biopsies of patients with DN revealed increased glomerular CXCL16 expression, which was paralleled by high levels of oxLDL. In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN.
Oral presentation from 4th International Conference of cGMP Generators, Effectors and Therapeutic Implications ; Regensburg, Germany. 19–21 June 2009 Background: An exaggerated pain sensitivity is the dominant feature of inflammatory and neuropathic pain both in the clinical setting and in experimental animal models. It manifests as pain in response to normally innocuous stimuli (allodynia), increased response to noxious stimuli (hyperalgesia) or spontaneous pain, and can persist long after the initial injury is resolved. Research over the last decades has revealed that several signaling pathways in the spinal cord essentially contribute to the pain sensitization. To test the contribution of cGMP produced by NO-sensitive guanylyl cyclase (NO-GC) to pain sensitization, we investigated the localization of NO-GC in the spinal cord and in dorsal root ganglia, and we characterized the nociceptive behavior of mice deficient in NO-GC (GC-KO mice). Results: We show that NO-GC (β1 subunit) is distinctly expressed in neurons of the mouse spinal cord, while its distribution in dorsal root ganglia is restricted to non-neuronal cells. GC-KO mice exhibited a considerably reduced nociceptive behavior in models of inflammatory or neuropathic pain, but their responses to acute pain were not impaired. Moreover, GC-KO mice failed to develop pain sensitization induced by spinal administration of drugs releasing NO. Surprisingly, during spinal nociceptive processing cGMP produced by NO-GC may activate signaling pathways different from cGMP-dependent protein kinase I (cGKI), while cGKI can be activated by natriuretic peptide receptor-B (NPR-B) dependent cGMP production. Conclusion: Taken together, our results provide evidence that NO-GC has a dominant role in the development of exaggerated pain sensitivity during inflammatory and neuropathic pain. Furthermore, beside the NO-mediated cGMP synthesis, cGMP produced by NPR-B contributes to pain sensitization by activation of cGKI.
The small leucine-rich proteoglycans (SLRPs) are biologically active components of the extracellular matrix (ECM), consisting of a protein core with leucine rich-repeat (LRR) motifs covalently linked to glycosaminoglycan (GAG) side chains. The diversity in composition resulting from the various combinations of protein cores substituted with one or more GAG chains along with their pericellular localization enables SLRPs to interact with a host of different cell surface receptors, cytokines, growth factors, and other ECM components, leading to modulation of cellular functions. SLRPs are capable of binding to: (i) different types of collagens, thereby regulating fibril assembly, organization, and degradation; (ii) Toll-like receptors (TLRs), complement C1q, and tumor necrosis factor-alpha (TNFα), regulating innate immunity and inflammation; (iii) epidermal growth factor receptor (EGF-R), insulin-like growth factor receptor (IGF-IR), and c-Met, influencing cellular proliferation, survival, adhesion, migration, tumor growth and metastasis as well as synthesis of other ECM components; (iv) low-density lipoprotein receptor-related protein (LRP-1) and TGF-β, modulating cytokine activity and fibrogenesis; and (v) growth factors such as bone morphogenic protein (BMP-4) and Wnt-I-induced secreted protein-1 (WISP-1), controlling cell proliferation and differentiation. Thus, the ability of SLRPs, as ECM components, to directly or indirectly regulate cell-matrix crosstalk, resulting in the modulation of various biological processes, aptly qualifies these compounds as matricellular proteins.
The manifestation of chronic back pain depends on structural, psychosocial, occupational and genetic influences. Heritability estimates for back pain range from 30% to 45%. Genetic influences are caused by genes affecting intervertebral disc degeneration or the immune response and genes involved in pain perception, signalling and psychological processing. This inter-individual variability which is partly due to genetic differences would require an individualized pain management to prevent the transition from acute to chronic back pain or improve the outcome. The genetic profile may help to define patients at high risk for chronic pain. We summarize genetic factors that (i) impact on intervertebral disc stability, namely Collagen IX, COL9A3, COL11A1, COL11A2, COL1A1, aggrecan (AGAN), cartilage intermediate layer protein, vitamin D receptor, metalloproteinsase-3 (MMP3), MMP9, and thrombospondin-2, (ii) modify inflammation, namely interleukin-1 (IL-1) locus genes and IL-6 and (iii) and pain signalling namely guanine triphosphate (GTP) cyclohydrolase 1, catechol-O-methyltransferase, μ opioid receptor (OPMR1), melanocortin 1 receptor (MC1R), transient receptor potential channel A1 and fatty acid amide hydrolase and analgesic drug metabolism (cytochrome P450 [CYP]2D6, CYP2C9).
Background: In this interdisciplinary project, the biological effects of heavy ions are compared to those of X-rays using tissue slice culture preparations from rodents and humans. Advantages of this biological model are the conservation of an organotypic environment and the independency from genetic immortalization strategies used to generate cell lines. Its open access allows easy treatment and observation via live-imaging microscopy. Materials and methods: Rat brains and human brain tumor tissue are cut into 300 micro m thick tissue slices. These slices are cultivated using a membrane-based culture system and kept in an incubator at 37°C until treatment. The slices are treated with X-rays at the radiation facility of the University Hospital in Frankfurt at doses of up to 40 Gy. The heavy ion irradiations were performed at the UNILAC facility at GSI with different ions of 11.4 A MeV and fluences ranging from 0.5–10 x 106 particles/cm². Using 3D-confocal microscopy, cell-death and immune cell activation of the irradiated slices are analyzed. Planning of the irradiation experiments is done with simulation programs developed at GSI and FIAS. Results: After receiving a single application of either X-rays or heavy ions, slices were kept in culture for up to 9d post irradiation. DNA damage was visualized using gamma H2AXstaining. Here, a dose-dependent increase and time-dependent decrease could clearly be observed for the X-ray irradiation. Slices irradiated with heavy ions showed less gamma H2AX-positive cells distributed evenly throughout the slice, even though particles were calculated to penetrate only 90–100 micro m into the slice. Conclusions: Single irradiations of brain tissue, even at high doses of 40 Gy, will result neither in tissue damage visible on a macroscopic level nor necrosis. This is in line with the view that the brain is highly radio-resistant. However, DNA damage can be detected very well in tissue slices using gamma H2AX-immuno staining. Thus, slice cultures are an excellent tool to study radiation-induced damage and repair mechanisms in living tissues.
Peroxisome proliferator-activated receptor γ (PPARγ) gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer is associated with impaired PPARγ expression. Considering that regulation of PPARγ expression during inflammation is largely unknown, we were interested in elucidating underlying mechanisms. To this end, we initiated an inflammatory response by exposing primary human macrophages to lipopolysaccharide (LPS) and observed a rapid decline of PPARγ1 expression. Because promoter activities were not affected by LPS, we focused on mRNA stability and noticed a decreased mRNA half-life. As RNA stability is often regulated via 3′-untranslated regions (UTRs), we analyzed the impact of the PPARγ-3′-UTR by reporter assays using specific constructs. LPS significantly reduced luciferase activity of the pGL3-PPARγ-3′-UTR, suggesting that PPARγ1 mRNA is destabilized. Deletion or mutation of a potential microRNA-27a/b (miR-27a/b) binding site within the 3′-UTR restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS exposure, partially reversed PPARγ1 mRNA decay, whereas miR-27b overexpression decreased PPARγ1 mRNA content. In addition, LPS further reduced this decay. The functional relevance of miR-27b-dependent PPARγ1 decrease was proven by inhibition or overexpression of miR-27b, which affected LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin (IL)-6. We provide evidence that LPS-induced miR-27b contributes to destabilization of PPARγ1 mRNA. Understanding molecular mechanisms decreasing PPARγ might help to better appreciate inflammatory diseases.
Transcripts of NANOG and OCT4 have been recently identified in human t(4;11) leukemia and in a model system expressing both t(4;11) fusion proteins. Moreover, downstream target genes of NANOG/OCT4/SOX2 were shown to be transcriptionally activated. However, the NANOG1 gene belongs to a gene family, including a gene tandem duplication (named NANOG2 or NANOGP1) and several pseudogenes (NANOGP2-P11). Thus, it was unclear which of the NANOG family members were transcribed in t(4;11) leukemia cells. 5'-RACE experiments revealed novel 5'-exons of NANOG1 and NANOG2, which could give rise to the expression of two different NANOG1 and three different NANOG2 protein variants. Moreover, a novel PCR-based method was established that allows distinguishing between transcripts deriving from NANOG1, NANOG2 and all other NANOG pseudogenes (P2–P11). By applying this method, we were able to demonstrate that human hematopoietic stem cells and different leukemic cells transcribe NANOG2. Furthermore, we functionally tested NANOG1 and NANOG2 protein variants by recombinant expression in 293 cells. These studies revealed that NANOG1 and NANOG2 protein variants are functionally equivalent and activate a regulatory circuit that activates specific stem cell genes. Therefore, we pose the hypothesis that the transcriptional activation of NANOG2 represents a ‘gain-of-stem cell function’ in acute leukemia.
Background: A delta and C fibers are the major pain-conducting nerve fibers, activate only partly the same brain areas, and are differently involved in pain syndromes. Whether a stimulus excites predominantly A delta or C fibers is a commonly asked question in basic pain research but a quick test was lacking so far. Methodology/Principal Findings: Of 77 verbal descriptors of pain sensations, "pricking", "dull" and "pressing" distinguished best (95% cases correctly) between A delta fiber mediated (punctate pressure produced by means of von Frey hairs) and C fiber mediated (blunt pressure) pain, applied to healthy volunteers in experiment 1. The sensation was assigned to A delta fibers when "pricking" but neither "dull" nor "pressing" were chosen, and to C fibers when the sum of the selections of "dull" or "pressing" was greater than that of the selection of "pricking". In experiment 2, with an independent cohort, the three-descriptor questionnaire achieved sensitivity and specificity above 0.95 for distinguishing fiber preferential non-mechanical induced pain (laser heat, exciting A delta fibers, and 5-Hz electric stimulation, exciting C fibers). Conclusion: A three-item verbal rating test using the words "pricking", "dull", and "pressing" may provide sufficient information to characterize a pain sensation evoked by a physical stimulus as transmitted via A delta or via C fibers. It meets the criteria of a screening test by being easy to administer, taking little time, being comfortable in handling, and inexpensive while providing high specificity for relevant information.
Leukotrienes constitute a group of bioactive lipids generated by the 5-lipoxygenase (5-LO) pathway. An increasing body of evidence supports an acute role for 5-LO products already during the earliest stages of pancreatic, prostate, and colorectal carcinogenesis. Several pieces of experimental data form the basis for this hypothesis and suggest a correlation between 5-LO expression and tumor cell viability. First, several independent studies documented an overexpression of 5-LO in primary tumor cells as well as in established cancer cell lines. Second, addition of 5-LO products to cultured tumor cells also led to increased cell proliferation and activation of anti-apoptotic signaling pathways. 5-LO antisense technology approaches demonstrated impaired tumor cell growth due to reduction of 5-LO expression. Lastly, pharmacological inhibition of 5-LO potently suppressed tumor cell growth by inducing cell cycle arrest and triggering cell death via the intrinsic apoptotic pathway. However, the documented strong cytotoxic off-target effects of 5-LO inhibitors, in combination with the relatively high concentrations of 5-LO products needed to achieve mitogenic effects in cell culture assays, raise concern over the assignment of the cause, and question the relationship between 5-LO products and tumorigenesis. Keywords: leukotriene, apoptosis, cell proliferation, mitogenic effects, cytotoxicity
Background: Descending inhibitory pain control contributes to the endogenous defense against chronic pain and involves noradrenergic and serotonergic systems. The clinical efficacy of antidepressants suggests that serotonin may be particularly relevant for neuropathic pain conditions. Serotonergic signaling is regulated by synthesis, metabolisms, reuptake and receptors. To address the complexity, we used inbred mouse strains, C57BL/6J, 129 Sv, DBA/2J and Balb/c, which differ in brain serotonin levels. Results: Serotonin analysis after nerve injury revealed inter-strain differences in the adaptation of descending serotonergic fibers. Upregulation of spinal cord and midbrain serotonin was apparent only in 129 Sv mice and was associated with attenuated nerve injury evoked hyperalgesia and allodynia in this strain. The increase of dorsal horn serotonin was blocked by hemisectioning of descending fibers but not by rhizotomy of primary afferents indicating a midbrain source. Para-chlorophenylalanine-mediated serotonin depletion in spinal cord and midbrain intensified pain hypersensitivity in the nerve injury model. In contrast, chronic inflammation of the hindpaw did not evoke equivalent changes in serotonin levels in the spinal cord and midbrain and nociceptive thresholds dropped in a parallel manner in all strains. Conclusion: The results suggest that chronic nerve injury evoked hypernociception may be contributed by genetic differences of descending serotonergic inhibitory control.
Background: R-flurbiprofen, one of the enantiomers of flurbiprofen racemate, is inactive with respect to cyclooxygenase inhibition, but shows analgesic properties without relevant toxicity. Its mode of action is still unclear. Methodology/Principal Findings: We show that R-flurbiprofen reduces glutamate release in the dorsal horn of the spinal cord evoked by sciatic nerve injury and thereby alleviates pain in sciatic nerve injury models of neuropathic pain in rats and mice. This is mediated by restoring the balance of endocannabinoids (eCB), which is disturbed following peripheral nerve injury in the DRGs, spinal cord and forebrain. The imbalance results from transcriptional adaptations of fatty acid amide hydrolase (FAAH) and NAPE-phospholipase D, i.e. the major enzymes involved in anandamide metabolism and synthesis, respectively. R-flurbiprofen inhibits FAAH activity and normalizes NAPE-PLD expression. As a consequence, R-Flurbiprofen improves endogenous cannabinoid mediated effects, indicated by the reduction of glutamate release, increased activity of the anti-inflammatory transcription factor PPAR gamma and attenuation of microglia activation. Antinociceptive effects are lost by combined inhibition of CB1 and CB2 receptors and partially abolished in CB1 receptor deficient mice. R-flurbiprofen does however not cause changes of core body temperature which is a typical indicator of central effects of cannabinoid-1 receptor agonists. Conclusion: Our results suggest that R-flurbiprofen improves the endogenous mechanisms to regain stability after axonal injury and to fend off chronic neuropathic pain by modulating the endocannabinoid system and thus constitutes an attractive, novel therapeutic agent in the treatment of chronic, intractable pain.
In the past, the genetically diabetic-obese diabetes/diabetes (db/db) and obese/obese (ob/ob) mouse strains were used to investigate mechanisms of diabetes-impaired wound healing. Here we determined patterns of skin repair in genetically normal C57Bl/6J mice that were fed using a high fat diet (HFD) to induce a diabetes-obesity syndrome. Wound closure was markedly delayed in HFD-fed mice compared to mice which had received a standard chow diet (CD). Impaired wound tissue of HFD mice showed a marked prolongation of wound inflammation. Expression of vascular endothelial growth factor (VEGF) was delayed and associated with the disturbed formation of wound margin epithelia and an impaired angiogenesis in the reduced granulation tissue. Normal wound contraction was retarded and disordered. Wound disorders in obese C57Bl/6J mice were paralleled by a prominent degradation of the inhibitor of NFκB (IκB-α) in the absence of an Akt activation. By contrast to impaired wound conditions in ob/ob mice, late wounds of HFD mice did not develop a chronic inflammatory state and were epithelialized after 11 days of repair. Thus, only genetically obese and diabetic ob/ob mice finally developed chronic wounds and therefore represent a better suited experimental model to investigate diabetes-induced wound healing disorders.
If insufficiently treated, Lyme borreliosis can evolve into an inflammatory disorder affecting skin, joints, and the CNS. Early innate immunity may determine host responses targeting infection. Thus, we sought to characterize the immediate cytokine storm associated with exposure of PBMC to moderate levels of live Borrelia burgdorferi. Since Th17 cytokines are connected to host defense against extracellular bacteria, we focused on interleukin (IL)-17 and IL-22. Here, we report that, despite induction of inflammatory cytokines including IL-23, IL-17 remained barely detectable in response to B. burgdorferi. In contrast, T cell-dependent expression of IL-22 became evident within 10 h of exposure to the spirochetes. This dichotomy was unrelated to interferon-gamma but to a large part dependent on caspase-1 and IL-1 bioactivity derived from monocytes. In fact, IL-1beta as a single stimulus induced IL-22 but not IL-17. Neutrophils display antibacterial activity against B. burgdorferi, particularly when opsonized by antibodies. Since neutrophilic inflammation, indicative of IL-17 bioactivity, is scarcely observed in Erythema migrans, a manifestation of skin inflammation after infection, protective and antibacterial properties of IL-22 may close this gap and serve essential functions in the initial phase of spirochete infection.
Epoxyeicotrienoic acids (EETs) are cytochrome P450-dependent anti-hypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered reno-protective. EETs are degraded by the enzyme soluble epoxide hydrolase (sEH) and sEH inhibitors are considered treatment for chronic renal failure (CRF). We determined whether sEH inhibition attenuates the progression of CRF in the 5/6-nephrectomy model (5/6-Nx) in mice. 5/6-Nx mice were treated with a placebo, an ACE-inhibitor (Ramipril, 40 mg/kg), the sEH-inhibitor cAUCB or the CYP-inhibitor fenbendazole for 8 weeks. 5/6-Nx induced hypertension, albuminuria, glomerulosclerosis and tubulo-interstitial damage and these effects were attenuated by Ramipril. In contrast, cAUCB failed to lower the blood pressure and albuminuria was more severe as compared to placebo. Plasma EET-levels were doubled in 5/6 Nx-mice as compared to sham mice receiving placebo. Renal sEH expression was attenuated in 5/6-Nx mice but cAUCB in these animals still further increased the EET-level. cAUCB also increased 5-HETE and 15-HETE, which derive from peroxidation or lipoxygenases. Similar to cAUCB, CYP450 inhibition increased HETEs and promoted albuminuria. Thus, sEH-inhibition failed to elicit protective effects in the 5/6-Nx model and showed a tendency to aggravate the disease. These effects might be consequence of a shift of arachidonic acid metabolism into the lipoxygenase pathway.
The E3 ubiquitin ligase MYCBP2 negatively regulates neuronal growth, synaptogenesis, and synaptic strength. More recently it was shown that MYCBP2 is also involved in receptor and ion channel internalization. We found that mice with a MYCBP2-deficiency in peripheral sensory neurons show prolonged thermal hyperalgesia. Loss of MYCBP2 constitutively activated p38 MAPK and increased expression of several proteins involved in receptor trafficking. Surprisingly, loss of MYCBP2 inhibited internalization of transient receptor potential vanilloid receptor 1 (TRPV1) and prevented desensitization of capsaicin-induced calcium increases. Lack of desensitization, TRPV internalization and prolonged hyperalgesia were reversed by inhibition of p38 MAPK. The effects were TRPV-specific, since neither mustard oil-induced desensitization nor behavioral responses to mechanical stimuli were affected. In summary, we show here for the first time that p38 MAPK activation can inhibit activity-induced ion channel internalization and that MYCBP2 regulates internalization of TRPV1 in peripheral sensory neurons as well as duration of thermal hyperalgesia through p38 MAPK.
Correlations between personality traits and a wide range of sensory thresholds were examined. Participants (N = 124) completed a personality inventory (NEO-FFI) and underwent assessment of olfactory, trigeminal, tactile and gustatory detection thresholds, as well as examination of trigeminal and tactile pain thresholds. Significantly enhanced odor sensitivity in socially agreeable people, significantly enhanced trigeminal sensitivity in neurotic subjects, and a tendency for enhanced pain tolerance in highly conscientious participants was revealed. It is postulated that varied sensory processing may influence an individual's perception of the environment; particularly their perception of socially relevant or potentially dangerous stimuli and thus, varied with personality.
Accumulating evidence indicates that increased generation of reactive oxygen species (ROS) contributes to the development of exaggerated pain hypersensitivity during persistent pain. In the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin C and vitamin E in mouse models of inflammatory and neuropathic pain. We show that systemic administration of a combination of vitamins C and E inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by Complete Freund's Adjuvant. In contrast, vitamin C or vitamin E given alone failed to affect the nociceptive behavior in all tested models. The attenuated neuropathic pain behavior induced by the vitamin C and E combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. Moreover, the vitamin C and E combination ameliorated the allodynia induced by an intrathecally delivered ROS donor. Our results suggest that administration of vitamins C and E in combination may exert synergistic antinociceptive effects, and further indicate that ROS essentially contribute to nociceptive processing in special pain states.
Effect sizes in experimental pain produced by gender, genetic variants and sensitization procedures
(2011)
Background: Various effects on pain have been reported with respect to their statistical significance, but a standardized measure of effect size has been rarely added. Such a measure would ease comparison of the magnitude of the effects across studies, for example the effect of gender on heat pain with the effect of a genetic variant on pressure pain. Methodology/Principal Findings: Effect sizes on pain thresholds to stimuli consisting of heat, cold, blunt pressure, punctuate pressure and electrical current, administered to 125 subjects, were analyzed for 29 common variants in eight human genes reportedly modulating pain, gender and sensitization procedures using capsaicin or menthol. The genotype explained 0–5.9% of the total interindividual variance in pain thresholds to various stimuli and produced mainly small effects (Cohen's d 0–1.8). The largest effect had the TRPA1 rs13255063T/rs11988795G haplotype explaining >5% of the variance in electrical pain thresholds and conferring lower pain sensitivity to homozygous carriers. Gender produced larger effect sizes than most variant alleles (1–14.8% explained variance, Cohen's d 0.2–0.8), with higher pain sensitivity in women than in men. Sensitization by capsaicin or menthol explained up to 63% of the total variance (4.7–62.8%) and produced largest effects according to Cohen's d (0.4–2.6), especially heat sensitization by capsaicin (Cohen's d = 2.6). Conclusions: Sensitization, gender and genetic variants produce effects on pain in the mentioned order of effect sizes. The present report may provide a basis for comparative discussions of factors influencing pain.
Background: MicroRNA-21 (miR-21) is up-regulated in tumor tissue of patients with malignant diseases, including hepatocellular carcinoma (HCC). Elevated concentrations of miR-21 have also been found in sera or plasma from patients with malignancies, rendering it an interesting candidate as serum/plasma marker for malignancies. Here we correlated serum miR-21 levels with clinical parameters in patients with different stages of chronic hepatitis C virus infection (CHC) and CHC-associated HCC.
Methodology/Principal Findings: 62 CHC patients, 29 patients with CHC and HCC and 19 healthy controls were prospectively enrolled. RNA was extracted from the sera and miR-21 as well as miR-16 levels were analyzed by quantitative real-time PCR; miR-21 levels (normalized by miR-16) were correlated with standard liver parameters, histological grading and staging of CHC. The data show that serum levels of miR-21 were elevated in patients with CHC compared to healthy controls (P<0.001); there was no difference between serum miR-21 in patients with CHC and CHC-associated HCC. Serum miR-21 levels correlated with histological activity index (HAI) in the liver (r = −0.494, P = 0.00002), alanine aminotransferase (ALT) (r = −0.309, P = 0.007), aspartate aminotransferase (r = −0.495, P = 0.000007), bilirubin (r = −0.362, P = 0.002), international normalized ratio (r = −0.338, P = 0.034) and γ-glutamyltransferase (r = −0.244, P = 0.034). Multivariate analysis revealed that ALT and miR-21 serum levels were independently associated with HAI. At a cut-off dCT of 1.96, miR-21 discriminated between minimal and mild-severe necroinflammation (AUC = 0.758) with a sensitivity of 53.3% and a specificity of 95.2%.
Conclusions/Significance: The serum miR-21 level is a marker for necroinflammatory activity, but does not differ between patients with HCV and HCV-induced HCC.
Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.
Nerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess protein regulations in the spinal cord after injury of the sciatic nerve in the Spared Nerve Injury model (SNI) we performed a proteomic analysis using 2D-difference gel electrophoresis (DIGE) technology. Among approximately 2300 protein spots separated on each gel we detected 55 significantly regulated proteins after SNI whereof 41 were successfully identified by MALDI-TOF MS. Out of the proteins which were regulated in the DIGE analyses after SNI we focused on the carboxypeptidase A inhibitor latexin because protease dysfunctions contribute to the development of neuropathic pain. Latexin protein expression was reduced after SNI which could be confirmed by Western Blot analysis, quantitative RT-PCR and in-situ hybridisation. The decrease of latexin was associated with an increase of the activity of carboxypeptidase A indicating that the balance between latexin and carboxypeptidase A was impaired in the spinal cord after peripheral nerve injury due to a loss of latexin expression in spinal cord neurons. This may contribute to the development of cold allodynia because normalization of neuronal latexin expression in the spinal cord by AAV-mediated latexin transduction or administration of a small molecule carboxypeptidase A inhibitor significantly reduced acetone-evoked nociceptive behavior after SNI. Our results show the usefulness of proteomics as a screening tool to identify novel mechanisms of nerve injury evoked hypernociception and suggest that carboxypeptidase A inhibition might be useful to reduce cold allodynia.
IL-22 is an immunoregulatory cytokine displaying pathological functions in models of autoimmunity like experimental psoriasis. Understanding molecular mechanisms driving IL-22, together with knowledge on the capacity of current immunosuppressive drugs to target this process, may open an avenue to novel therapeutic options. Here, we sought to characterize regulation of human IL22 gene expression with focus on the established model of Jurkat T cells. Moreover, effects of the prototypic immunosuppressant cyclosporin A (CsA) were investigated. We report that IL-22 induction by TPA/A23187 (T/A) or αCD3 is inhibited by CsA or related FK506. Similar data were obtained with peripheral blood mononuclear cells or purified CD3(+) T cells. IL22 promoter analysis (-1074 to +156 bp) revealed a role of an NF-AT (-95/-91 nt) and a CREB (-194/-190 nt) binding site for gene induction. Indeed, binding of CREB and NF-ATc2, but not c-Rel, under the influence of T/A to those elements could be proven by ChIP. Because CsA has the capability to impair IκB kinase (IKK) complex activation, the IKKα/β inhibitor IKKVII was evaluated. IKKVII likewise reduced IL-22 induction in Jurkat cells and peripheral blood mononuclear cells. Interestingly, transfection of Jurkat cells with siRNA directed against IKKα impaired IL22 gene expression. Data presented suggest that NF-AT, CREB, and IKKα contribute to rapid IL22 gene induction. In particular the crucial role of NF-AT detected herein may form the basis of direct action of CsA on IL-22 expression by T cells, which may contribute to therapeutic efficacy of the drug in autoimmunity.
We among others have recently demonstrated that normal cells produce “fusion mRNAs”. These fusion mRNAs do not derive from rearranged genomic loci, but rather they are derived from “early-terminated transcripts” (ETTs). Premature transcriptional termination takes place in intronic sequences that belong to “breakpoint cluster regions”. One important property of ETTs is that they exhibit an unsaturated splice donor site. This results in: (1) splicing to “cryptic exons” present in the final intron; (2) Splicing to another transcript of the same gene (intragenic trans-splicing), resulting in “exon repetitions”; (3) splicing to a transcript of another gene (intergenic trans-splicing), leading to “non-genomically encoded fusion transcripts” (NGEFTs). These NGEFTs bear the potential risk to influence DNA repair processes, since they share identical nucleotides with their DNA of origin, and thus, could be used as “guidance RNA” for DNA repair processes. Here, we present experimental data about four other genes. Three of them are associated with hemato-malignancies (ETV6, NUP98 and RUNX1), while one is associated with solid tumors (EWSR1). Our results demonstrate that all genes investigated so far (MLL, AF4, AF9, ENL, ELL, ETV6, NUP98, RUNX1 and EWSR1) display ETTs and produce transpliced mRNA species, indicating that this is a genuine property of translocating genes.
Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy.
Background and Aims: Chronic infection with the hepatitis B virus (HBV) is a major health issue worldwide. Recently, single nucleotide polymorphisms (SNPs) within the human leukocyte antigen (HLA)-DP locus were identified to be associated with HBV infection in Asian populations. Most significant associations were observed for the A alleles of HLA-DPA1 rs3077 and HLA-DPB1 rs9277535, which conferred a decreased risk for HBV infection. We assessed the implications of these variants for HBV infection in Caucasians.
Methods: Two HLA-DP gene variants (rs3077 and rs9277535) were analyzed for associations with persistent HBV infection and with different clinical outcomes, i.e., inactive HBsAg carrier status versus progressive chronic HBV (CHB) infection in Caucasian patients (n = 201) and HBsAg negative controls (n = 235).
Results: The HLA-DPA1 rs3077 C allele was significantly associated with HBV infection (odds ratio, OR = 5.1, 95% confidence interval, CI: 1.9–13.7; p = 0.00093). However, no significant association was seen for rs3077 with progressive CHB infection versus inactive HBsAg carrier status (OR = 2.7, 95% CI: 0.6–11.1; p = 0.31). In contrast, HLA-DPB1 rs9277535 was not associated with HBV infection in Caucasians (OR = 0.8, 95% CI: 0.4–1.9; p = 1).
Conclusions: A highly significant association of HLA-DPA1 rs3077 with HBV infection was observed in Caucasians. However, as a differentiation between different clinical courses of HBV infection was not possible, knowledge of the HLA-DPA1 genotype cannot be translated into personalized anti-HBV therapy approaches.
We present a computational method for the reaction-based de novo design of drug-like molecules. The software DOGS (Design of Genuine Structures) features a ligand-based strategy for automated ‘in silico’ assembly of potentially novel bioactive compounds. The quality of the designed compounds is assessed by a graph kernel method measuring their similarity to known bioactive reference ligands in terms of structural and pharmacophoric features. We implemented a deterministic compound construction procedure that explicitly considers compound synthesizability, based on a compilation of 25'144 readily available synthetic building blocks and 58 established reaction principles. This enables the software to suggest a synthesis route for each designed compound. Two prospective case studies are presented together with details on the algorithm and its implementation. De novo designed ligand candidates for the human histamine H4 receptor and γ-secretase were synthesized as suggested by the software. The computational approach proved to be suitable for scaffold-hopping from known ligands to novel chemotypes, and for generating bioactive molecules with drug-like properties.
Progranulin haploinsufficiency is associated with frontotemporal dementia in humans. Deficiency of progranulin led to exaggerated inflammation and premature aging in mice. The role of progranulin in adaptations to nerve injury and neuropathic pain are still unknown. Here we found that progranulin is up-regulated after injury of the sciatic nerve in the mouse ipsilateral dorsal root ganglia and spinal cord, most prominently in the microglia surrounding injured motor neurons. Progranulin knockdown by continuous intrathecal spinal delivery of small interfering RNA after sciatic nerve injury intensified neuropathic pain-like behaviour and delayed the recovery of motor functions. Compared to wild-type mice, progranulin-deficient mice developed more intense nociceptive hypersensitivity after nerve injury. The differences escalated with aging. Knockdown of progranulin reduced the survival of dissociated primary neurons and neurite outgrowth, whereas addition of recombinant progranulin rescued primary dorsal root ganglia neurons from cell death induced by nerve growth factor withdrawal. Thus, up-regulation of progranulin after neuronal injury may reduce neuropathic pain and help motor function recovery, at least in part, by promoting survival of injured neurons and supporting regrowth. A deficiency in this mechanism may increase the risk for injury-associated chronic pain.
Increasing evidence about the central nervous representation of pain in the brain suggests that the operculo-insular cortex is a crucial part of the pain matrix. The pain-specificity of a brain region may be tested by administering nociceptive stimuli while controlling for unspecific activations by administering non-nociceptive stimuli. We applied this paradigm to nasal chemosensation, delivering trigeminal or olfactory stimuli, to verify the pain-specificity of the operculo-insular cortex. In detail, brain activations due to intranasal stimulation induced by non-nociceptive olfactory stimuli of hydrogen sulfide (5 ppm) or vanillin (0.8 ppm) were used to mask brain activations due to somatosensory, clearly nociceptive trigeminal stimulations with gaseous carbon dioxide (75% v/v). Functional magnetic resonance (fMRI) images were recorded from 12 healthy volunteers in a 3T head scanner during stimulus administration using an event-related design. We found that significantly more activations following nociceptive than non-nociceptive stimuli were localized bilaterally in two restricted clusters in the brain containing the primary and secondary somatosensory areas and the insular cortices consistent with the operculo-insular cortex. However, these activations completely disappeared when eliminating activations associated with the administration of olfactory stimuli, which were small but measurable. While the present experiments verify that the operculo-insular cortex plays a role in the processing of nociceptive input, they also show that it is not a pain-exclusive brain region and allow, in the experimental context, for the interpretation that the operculo-insular cortex splay a major role in the detection of and responding to salient events, whether or not these events are nociceptive or painful.
Novel chalcone-based fluorescent human histamine H 3 receptor ligands as pharmacological tools
(2012)
Novel fluorescent chalcone-based ligands at human histamine H(3) receptors (hH(3)R) have been designed, synthesized, and characterized. Compounds described are non-imidazole analogs of ciproxifan with a tetralone motif. Tetralones as chemical precursors and related fluorescent chalcones exhibit affinities at hH(3)R in the same concentration range like the reference antagonist ciproxifan (hH(3)R pK(i) value of 7.2). Fluorescence characterization of our novel ligands shows emission maxima about 570 nm for yellow fluorescent chalcones and ≥600 nm for the red fluorescent derivatives. Interferences to cellular autofluorescence could be excluded. All synthesized chalcone compounds could be used to visualize hH(3)R proteins in stably transfected HEK-293 cells using confocal laser scanning fluorescence microscopy. These novel fluorescent ligands possess high potential to be used as pharmacological tools for hH(3)R visualization in different tissues.
Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFNγ-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.
Background: The transcription factor T-bet is pivotal for initiation of Th1-related immunoactivation. Identification of novel genes directly regulated by T-bet is crucial.
Results: Genome-wide analysis and subsequent experiments revealed that T-bet up-regulates IL-36γ/IL-1F9 in myeloid cells.
Conclusion: IL-1-related IL-36γ is a direct T-bet target in myeloid cells.
Significance: Observations suggest that IL-36γ , besides IFNγ, contributes to T-bet functions in immunopathology
By concerted action in dendritic (DC) and T cells, T-box expressed in T cells (T-bet, Tbx21) is pivotal for initiation and perpetuation of Th1 immunity. Identification of novel T-bet-regulated genes is crucial for further understanding the biology of this transcription factor. By combining siRNA technology with genome-wide mRNA expression analysis, we sought to identify new T-bet-regulated genes in predendritic KG1 cells activated by IL-18. One gene robustly dependent on T-bet was IL-36γ, a recently described novel IL-1 family member. Promoter analysis revealed a T-bet binding site that, along with a κB site, enables efficient IL-36γ induction. Using knock-out animals, IL-36γ reliance on T-bet was extended to murine DC. IL-36γ expression by human myeloid cells was confirmed using monocyte-derived DC and M1 macrophages. The latter model was employed to substantiate dependence of IL-36γ on endogenous T-bet in human primary cells. Ectopic expression of T-bet likewise mediated IL-36γ production in HaCaT keratinocytes that otherwise lack this transcription factor. Additional experiments furthermore revealed that mature IL-36γ has the capability to establish an inflammatory gene expression profile in human primary keratinocytes that displays enhanced mRNA levels for TNFα, CCL20, S100A7, inducible NOS, and IL-36γ itself. Data presented herein shed further light on involvement of T-bet in innate immunity and suggest that IL-36γ, besides IFNγ, may contribute to functions of this transcription factor in immunopathology.
IL-18 is an important mediator involved in chronic inflammatory conditions such as cutaneous lupus erythematosus, psoriasis and chronic eczema. An imbalance between IL-18 and its endogenous antagonist IL-18 binding protein (BP) may account for increased IL-18 activity. IL-27 is a cytokine with dual function displaying pro- and anti-inflammatory properties. Here we provide evidence for a yet not described anti-inflammatory mode of action on skin resident cells. Human keratinocytes and surprisingly also fibroblasts (which do not produce any IL-18) show a robust, dose-dependent and highly inducible mRNA expression and secretion of IL-18BP upon IL-27 stimulation. Other IL-12 family members failed to induce IL-18BP. The production of IL-18BP peaked between 48–72 h after stimulation and was sustained for up to 96 h. Investigation of the signalling pathway showed that IL-27 activates STAT1 in human keratinocytes and that a proximal GAS site at the IL-18BP promoter is of importance for the functional activity of IL-27. The data are in support of a significant anti-inflammatory effect of IL-27 on skin resident cells. An important novel property of IL-27 in skin pathobiology may be to counter-regulate IL-18 activities by acting on keratinocytes and importantly also on dermal fibroblasts.
Background: Antithrombotic treatment is a continuous therapy that is often performed in general practice and requires careful safety management. The aim of this study is to investigate whether a best practice model that applies major elements of case management, including patient education, can improve antithrombotic management in primary health care in terms of reducing major thromboembolic and bleeding events.
Methods: This 24-month cluster-randomized trial will be performed in 690 adult patients from 46 practices. The trial intervention will be a complex intervention involving general practitioners, health care assistants and patients with an indication for oral anticoagulation. To assess adherence to medication and symptoms in patients, as well as to detect complications early, health care assistants will be trained in case management and will use the Coagulation-Monitoring-List (Co-MoL) to regularly monitor patients. Patients will receive information (leaflets and a video), treatment monitoring via the Co-MoL and be motivated to perform self-management. Patients in the control group will continue to receive treatment-as-usual from their general practitioners. The primary endpoint is the combined endpoint of all thromboembolic events requiring hospitalization, and all major bleeding complications. Secondary endpoints are mortality, hospitalization, strokes, major bleeding and thromboembolic complications, severe treatment interactions, the number of adverse events, quality of anticoagulation, health-related quality of life and costs. Further secondary objectives will be investigated to explain the mechanism by which the intervention is effective: patients' assessment of chronic illness care, self-reported adherence to medication, general practitioners' and health care assistants' knowledge, patients' knowledge and satisfaction with shared decision making. Practice recruitment is expected to take place between July and December 2012. Recruitment of eligible patients will start in July 2012. Assessment will occur at three time points: baseline (T0), follow-up after 12 (T1) and after 24 months (T2).
Discussion: The efficacy and effectiveness of individual elements of the intervention, such as antithrombotic interventions, self-management concepts in orally anticoagulated patients and the methodological tool, case-management, have already been extensively demonstrated. This project foresees the combination of several proven instruments, as a result of which we expect to profit from a reduction in the major complications associated with antithrombotic treatment.
Because of its highly bioactive properties sphingosine 1-phosphate (S1P) is an attractive target for the treatment of several diseases. Since the expression of sphingosine kinases as well as S1P receptors was demonstrated in the kidney, questions about the physiological and pathophysiological functions of S1P in this organ have been raised. In this review, we summarize the current state of knowledge about S1P-mediated functions in the kidney. A special focus is put on S1P modulated signal transduction in renal glomerular and tubular cells and consequences for the development and treatment of several kidney diseases, diabetic nephropathy, glomerulonephritis, ischemia-reperfusion injury, as well as for Wilms tumor progression.
This study indicates that embryonic stem cells [ESCs] cultured with retinoic acid and activin A significantly upregulate the miRNA let-7e. This specific miRNA modulates the Wnt pathway and the expression of early nephrogenic markers under these differentiation conditions. The differentiation markers WT1, Pax2 and Wnt4 were downregulated when miRNA let-7e was silenced, thus indicating the role of miRNA let-7e in the differentiation process. PKCβ, GSK3β phosphorylation (GSK3βP) and β-catenin expression was reduced in differentiated cells and reversed by miRNA let-7e silencing. Addition of a PKCβ inhibitor to the miRNA let-7e silenced cells abolished let-7e-derived effects in differentiation markers, and reversed the increase in GSK3βP and β-catenin, thus indicating that miRNA let-7e is involved in differentiation via the modulation of GSK3β phosphorylation and β-catenin production.
The role of the mRNA-binding protein human antigen R (HuR) in stabilization and translation of AU-rich elements (ARE) containing mRNAs is well established. However, the trafficking of HuR and bound mRNA cargo, which comprises a fundamental requirement for the aforementioned HuR functions is only poorly understood. By administering different cytoskeletal inhibitors, we found that the protein kinase Cδ (PKCδ)-triggered accumulation of cytoplasmic HuR by Angiotensin II (AngII) is an actin-myosin driven process functionally relevant for stabilization of ARE-bearing mRNAs. Furthermore, we show that the AngII-induced recruitment of HuR and its bound mRNA from ribonucleoprotein particles to free and cytoskeleton bound polysomes strongly depended on an intact actomyosin cytoskeleton. In addition, HuR allocation to free and cytoskeletal bound polysomes is highly sensitive toward RNase and PPtase and structurally depends on serine 318 (S318) located within the C-terminal RNA recognition motif (RRM3). Conversely, the trafficking of the phosphomimetic HuRS318D, mimicking HuR phosphorylation at S318 by the PKCδ remained PPtase resistant. Co-immunoprecipitation experiments with truncated HuR proteins revealed that the stimulus-induced association of HuR with myosin IIA is strictly RNA dependent and mediated via the RRM3. Our data implicate a microfilament dependent transport of HuR, which is relevant for stimulus-induced targeting of ARE-bearing mRNAs from translational inactive ribonucleoprotein particles to polysomes.
Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18:1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18:1, 9-and 13-HODE and HETEs.