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Resistance in glucocorticoid-induced apoptosis is associated with poor prognosis for long term survival in childhood acute lymphoblastic leukemia (ALL). As Smac mimetics have been shown to reactivate apoptosis by antagonizing Inhibitor of Apoptosis (IAP) proteins, we investigate the potential of the Smac mimetic BV6 to overcome glucocorticoid-resistance in ALL. This study shows that BV6 synergistically cooperates with glucocorticoids to trigger apoptosis and to suppress clonogenic growth of pediatric ALL cells. Of note, the BV6/glucocorticoid combination treatment also induces cell death in cells having defects in the apoptotic signaling cascade by inducing a switch from apoptotic to necroptotic cell death. The clinical relevance of our novel combination treatment is underscored by parallel experiments in primary pediatric ALL samples, in which glucocorticoids and BV6 act together to induce cell death in a synergistic manner. Importantly, the addition of BV6 enhances the anti-leukemic effects of glucocorticoids in an in vivo mouse model of pediatric ALL without causing substantial side effects, highlighting the potency of a BV6/glucocorticoid combination treatment. In contrast, BV6 does not increase cytotoxicity of glucocorticoids against several non-malignant cell types of the lympho-hematopoietic system. Furthermore, we have identified the novel underlying mechanism of BV6/glucocorticoid-induced apoptosis by showing that BV6 and glucocorticoids synergistically act together to promote assembly of the ripoptosome, a RIP1/FADD/caspase-8-containing cell death complex. Ripoptosome assembly is critically required for BV6/Dexamethasone-induced cell death, since genetic silencing of its members, i.e. RIP1, reduces ROS production, caspase activation and most importantly cell death induction. BV6/glucocorticoid combination treatment promotes ripoptosome assembly by inhibition of both of its negative regulators, IAP proteins and cFLIP. Thus, we identify that BV6 and glucocorticoids cooperate together to reduce cIAP1, cIAP2 and XIAP protein levels and cFLIP expression. Ripoptosome formation occurs independently of autocrine/paracrine loops of death receptor ligands, since blocking antibodies for TNFα, TRAIL or CD95L or genetic silencing of their corresponding receptors fail to rescue BV6/glucocorticoid-induced cell death. In summary, this study shows that the Smac mimetic BV6 sensitizes for glucocorticoid-induced apoptosis by promoting ripoptosome assembly with important implications for the treatment of childhood ALL.
After the mass-vaccination campaign during the influenza A (H1N1) 2009 pandemic, a significant increase in narcolepsy incidence was observed initially in Scandinavia, later in other European countries and recently also in Canada. Narcolepsy is a sleep disease caused by the loss of hypocretin-producing cells in the hypothalamus. Almost all narcolepsy patients carry the HLA-DQB1*0602 allele, giving a link to an autoimmune-mediated process.
Most of the observed narcolepsy cases were correlated to the vaccination with Pandemrix, the most frequently used vaccine in the EU, and a slight connection to Arepanrix was also detected, which was distributed in Canada. Both vaccines were adjuvanted with AS03, suggesting a possible link between AS03 and narcolepsy. No narcolepsy cases were detected with MF59-adjuvanted or non-adjuvanted influenza vaccines. Recent studies reported differences between Pandemrix and Arepanrix and suggested the vaccine rather than the adjuvant as a suspect for narcolepsy development following vaccination. In addition, in China an increase of narcolepsy cases was reported to occur in absence of vaccination. Possible factors and potential additive effects that may have triggered narcolepsy after the pandemic vaccination are being reviewed in this paper.
Biomedical data obtained during cell experiments, laboratory animal research, or human studies often display a complex distribution. Statistical identification of subgroups in research data poses an analytical challenge. Here were introduce an interactive R-based bioinformatics tool, called “AdaptGauss”. It enables a valid identification of a biologically-meaningful multimodal structure in the data by fitting a Gaussian mixture model (GMM) to the data. The interface allows a supervised selection of the number of subgroups. This enables the expectation maximization (EM) algorithm to adapt more complex GMM than usually observed with a noninteractive approach. Interactively fitting a GMM to heat pain threshold data acquired from human volunteers revealed a distribution pattern with four Gaussian modes located at temperatures of 32.3, 37.2, 41.4, and 45.4 °C. Noninteractive fitting was unable to identify a meaningful data structure. Obtained results are compatible with known activity temperatures of different TRP ion channels suggesting the mechanistic contribution of different heat sensors to the perception of thermal pain. Thus, sophisticated analysis of the modal structure of biomedical data provides a basis for the mechanistic interpretation of the observations. As it may reflect the involvement of different TRP thermosensory ion channels, the analysis provides a starting point for hypothesis-driven laboratory experiments.
Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function.
Allergy against birch pollen is among the most common causes of spring pollinosis in Europe and is diagnosed and treated using extracts from natural sources. Quality control is crucial for safe and effective diagnosis and treatment. However, current methods are very difficult to standardize and do not address individual allergen or isoallergen composition. MS provides information regarding selected proteins or the entire proteome and could overcome the aforementioned limitations. We studied the proteome of birch pollen, focusing on allergens and isoallergens, to clarify which of the 93 published sequence variants of the major allergen, Bet v 1, are expressed as proteins within one source material in parallel. The unexpectedly complex Bet v 1 isoallergen composition required manual data interpretation and a specific design of databases, as current database search engines fail to unambiguously assign spectra to highly homologous, partially identical proteins. We identified 47 non-allergenic proteins and all 5 known birch pollen allergens, and unambiguously proved the existence of 18 Bet v 1 isoallergens and variants by manual data analysis. This highly complex isoallergen composition raises questions whether isoallergens can be ignored or must be included for the quality control of allergen products, and which data analysis strategies are to be applied.
The muscarinic M2 receptor (M2R) acts as a negative feedback regulator in central cholinergic systems. Activation of the M2 receptor limits acetylcholine (ACh) release, especially when ACh levels are increased because acetylcholinesterase (AChE) activity is acutely inhibited. Chronically high ACh levels in the extracellular space, however, were reported to down-regulate M2R to various degrees. In the present study, we used the PRiMA knockout mouse which develops severely reduced AChE activity postnatally to investigate ACh release, and we used microdialysis to investigate whether the function of M2R to reduce ACh release in vivo was impaired in adult PRiMA knockout mice. We first show that striatal and hippocampal ACh levels, while strongly increased, still respond to AChE inhibitors. Infusion or injection of oxotremorine, a muscarinic M2 agonist, reduced ACh levels in wild-type mice but did not significantly affect ACh levels in PRiMA knockout mice or in wild-type mice in which ACh levels were artificially increased by infusion of neostigmine. Scopolamine, a muscarinic antagonist, increased ACh levels in wild-type mice receiving neostigmine, but not in wild-type mice or in PRiMA knockout mice. These results demonstrate that M2R are dysfunctional and do not affect ACh levels in PRiMA knockout mice, likely because of down-regulation and/or loss of receptor-effector coupling. Remarkably, this loss of function does not affect cognitive functions in PRiMA knockout mice. Our results are discussed in the context of AChE inhibitor therapy as used in dementia.
To overcome poor treatment response of pediatric high-risk acute lymphoblastic leukemia (ALL), novel treatment strategies are required to reactivate programmed cell death in this malignancy. Therefore, we take advantage of using small-molecule antagonists of Inhibitor of apoptosis (IAP) proteins, so called Smac mimetics such as BV6, which are described to overcome apoptosis resistance and thereby sensitize tumor cells for several apoptotic stimuli. To address the question whether redox alterations can sensitize leukemic cells for Smac mimetic-mediated cell death, we interfered with the cellular redox status in different ALL cell lines. Here, we show for the first time that redox alterations, mediated by the glutathione depleting agent Buthioninesulfoximine (BSO), prime ALL cells for BV6-induced apoptosis. Besides ALL cell lines, BV6/BSO cotreatment similarly synergizes in cell death induction in patient-derived primary leukemic samples. In contrast, the combination treatment does not exert any cytotoxicity against peripheral blood lymphocytes (PBLs) or mesenchymal stroma cells (MSCs) from healthy donors, suggesting some tumor selectivity of this treatment. We also identify the underlying molecular mechanism of the novel synergistic drug interaction of BSO and BV6. We demonstrate that both agents act in concert to increase reactive oxygen species (ROS) production, lipid peroxidation and finally apoptotic cell death. Enhanced ROS levels in the combination treatment account for cell death induction, since several ROS scavengers, like NAC, MnTBAP and Trolox attenuate BSO/BV6-induced apoptosis. BSO/BV6-induced ROS can be mainly classified as lipid peroxides, since the vitamin E derivate α-Tocopherol as well as Glutathione peroxidase 4 (GPX4), which both specifically reduce lipid-membrane peroxides, prevent lipid peroxidation, caspase activation and cell death induction. Vice versa, GPX4 knockdown and pharmacological inhibition of GPX4 by RSL3 or Erastin enhance BV6-induced cell death. Importantly, cell death induction critically depends on the formation of a complex consisting of RIP1/FADD/Caspase-8, since all complex components are required for ROS production, lipid peroxidation and cell death induction. Taken together, we demonstrate that BSO and BV6 cooperate to induce ROS production and lipid peroxidation which are eventually required for caspase activation and cell death execution. Collectively, findings of this study indicate that BV6-induced apoptosis is mediated via redox alterations offering promising new treatment strategy to overcome apoptosis resistance in ALL.
The transcription factor Tal1 is a critical activator or repressor of gene expression in hematopoiesis and leukaemia. The mechanism by which Tal1 differentially influences transcription of distinct genes is not fully understood. Here we show that Tal1 interacts with the peptidylarginine deiminase IV (PADI4). We demonstrate that PADI4 can act as an epigenetic coactivator through influencing H3R2me2a. At the Tal1/PADI4 target gene IL6ST the repressive H3R2me2a mark triggered by PRMT6 is counteracted by PADI4, which augments the active H3K4me3 mark and thus increases IL6ST expression. In contrast, at the CTCF promoter PADI4 acts as a repressor. We propose that the influence of PADI4 on IL6ST transcription plays a role in the control of IL6ST expression during lineage differentiation of hematopoietic stem/progenitor cells. These results open the possibility to pharmacologically influence Tal1 in leukaemia.
Inhibitor of Apoptosis (IAP) proteins are expressed at high levels in many cancers and contribute to apoptosis resistance. Therefore, they represent promising anticancer drug targets. Here, we report that small molecule IAP inhibitors at subtoxic concentrations cooperate with monoclonal antibodies against TRAIL receptor 1 (Mapatumumab) or TRAIL receptor 2 (Lexatumumab) to induce apoptosis in neuroblastoma cells in a highly synergistic manner (combination index <0.1). Importantly, we identify RIP1 as a critical regulator of this synergism. RIP1 is required for the formation of a RIP1/FADD/caspase-8 complex that drives caspase-8 activation, cleavage of Bid into tBid, mitochondrial outer membrane permeabilization, full activation of caspase-3 and caspase-dependent apoptosis. Indeed, knockdown of RIP1 abolishes formation of the RIP1/FADD/caspase-8 complex, subsequent caspase activation and apoptosis upon treatment with IAP inhibitor and TRAIL receptor antibodies. Similarly, inhibition of RIP1 kinase activity by Necrostatin-1 inhibits IAP inhibitor- and TRAIL receptor-triggered apoptosis. By comparison, over-expression of the dominant-negative superrepressor IκBα-SR or addition of the TNFα-blocking antibody Enbrel does not inhibit IAP inhibitor- and Lexatumumab-induced apoptosis, pointing to a NF-κB- and TNFα-independent mechanism. Of note, IAP inhibitor also significantly reduces TRAIL receptor-mediated loss of cell viability of primary cultured neuroblastoma cells, underscoring the clinical relevance. By demonstrating that RIP1 plays a key role in the IAP inhibitor-mediated sensitization for Mapatumumab- or Lexatumumab-induced apoptosis, our findings provide strong rationale to develop the combination of IAP inhibitors and TRAIL receptor agonists as a new therapeutic strategy for the treatment of human cancer.
Hematopoietic stem cells (HSCs) have the unique abilities of life-long self-renewal and multi-lineage differentiation. They are routinely used in BM or stem cell transplantations to reconstitute the blood system of patients suffering from malignant or monogenic blood disorders. For an adequate production of each blood cell lineage in homeostasis and under stress conditions, the fate choice of HSCs to either self-renew or to differentiate must be strictly controlled. The incomplete understanding of the molecular mechanisms that control this balance makes it still impossible to maintain or expand undifferentiated HSCs in culture for advanced regenerative medical purposes.
The aim of this thesis was the identification and molecular characterisation of mechanisms that control the decision of HSCs to self-renew or to differentiate, and how they are connected to extrinsic cytokine signaling control. Prior to this thesis, a screening for genes upregulated under self-renewal promoting thrombopoietin (TPO) signaling via the transcription factors STAT5A/B in HSCs was conducted, and Growth arrest and DNA damage inducible 45 gamma (Gadd45g) was one of the regulated genes. GADD45G was described as stress sensor, DNA-damage response and tumor suppressor gene, that is epigenetically silenced in many solid tumors and leukemia. Furthermore, Gadd45g is upregulated in aged HSCs with impaired multi-lineage reconstitution abilities, and it is induced by differentiation promoting cytokines in GM-committed cells. However, the function of GADD45G in LT-HSCs was unknown. All these points warrant further investigation to unravel the function of GADD45G on early cell fate decisions of HSCs in hematopoiesis.
The expression of Gadd45g was stimulated by hematopoietic cytokines TPO, IL3 and IL6 both in HSCs and MPPs, making GADD45G an interesting target to focus on. To simulate the cytokine-induced expression GADD45G was lentivirally transduced in HSCs. Surprisingly, GADD45G did not induce cell cycle arrest or cell death in hematopoietic cells neither in vitro nor in vivo, as reported in many cell lines. Instead GADD45G revealed an enhanced and markedly accelerated differentiation of HSCs into mainly myelomonocytic cells, similar as observed for IL3 and IL6 containing cultures. Also in vivo, GADD45G rapidly initiates the differentiation program in HSCs at the expense of self-renewal and long-term engraftment, as shown by serial HSC transplantation experiments. Along the same line, HSCs from Gadd45g-knock out mice exhibited an increased self-renewal. In vitro, Gadd45g-/- progenitors showed higher and prolonged colony formation potential and slower expansion after cytokine stimulation. The loss of Gadd45g increased HSC self-renewal and improved repopulation in secondary recipients, determined by serial competitive transplantations. Taken together, GADD45G could be identified as molecular link between differentiation-promoting cytokine signaling and rapid differentiation induction in murine LT-HSCs.
As presented in this thesis the differentiation induction of GADD45G was mediated by the activation of the cascade of MAP3K4 – MKK6 –p38 MAPK. Small molecule inhibition of p38, but not JNK, blocked the GADD45G-induced differentiation. GADD45G binds to MAP3K4 and releases its auto-inhibitory loop by a change in confirmation, initiating this cascade. Phosphoflow cytometry demonstrated the activation of p38 and a downstream kinase MK2 by GADD45G expression in MPPs. Furthermore, the expression of constitutive active MAP3K4 and MKK6 were able to phenocopy GADD45G-induced differentiation, which could be blocked by p38 inhibition.
The other two family members GADD45A and B also induced accelerated differentiation in LT-HSCs. Interestingly, only GADD45G suppressed the differentiation into megakaryocyte and erythrocyte (Mek/E) lineage cells suggesting a role of GADD45G in lineage choice. Long-term time-lapse microscopy-based cell tracking of single LT-HSCs and their progeny revealed that, once GADD45G is expressed, the development of LT-HSCs into granulocyte-macrophage-committed progeny occurred within 36 hours, and uncovered a selective lineage choice with a severe reduction in Mek/E cells. Furthermore, no megakaryocytic-erythroid progenitors (MEPs) could develop from HSPCs in BM 2 weeks after transplantation suggesting a very early selection against Mek/E cell fates. In line with these findings, GADD45G-transduced MEPs could not expand or form colonies in vitro, demonstrating that the differentiation program induced by GADD45G is not compatible with Mek/E lineage fate. Gene expression profiling of HSCs indicated that GADD45G promotes myelomonocytic differentiation programs over programs for self-renewal or megakaryo-/ erythropoiesis. The here identified differentiation induction potential of GADD45G is so strong that the expression of GADD45G in primary acute myeloid leukemia (AML) cells inhibited their expansion accompanied by enhanced differentiation and increased apoptosis.
The here presented work shows that IL3 and IL6 induce a differentiation program in HSCs via GADD45G and p38 closing the link of extrinsic cytokine signaling and differentiation induction. Since the loss of Gadd45g increased the self-renewal and slowed HSC differentiation, this may be utilized, i.e. by p38 inhibition, to ex vivo maintain and expand HSCs by preventing cytokine-induced differentiation. Furthermore, Re-expression of GADD45G may overcome the differentiation block in leukemia to eliminate these cells by driving them into terminal differentiation and apoptosis.
The endocannabinoids (EC), their synthetizing and metabolizing enzymes, and the cannabinoid (CB) receptors comprise the endocannabinoid system (ECS) that has been detected by Yasuo et al. (2010) in rodent and human brain areas essential for circadian rhythmic control and hormone secretion. The EC are secreted in the pars tuberalis formation (PT) of the pituitary gland and unfold their effect as ligands on cannabinoid receptors type 1 (CB1) in the pars distalis (PD). The CB1 is mostly expressed on folliculo-stellate (fs) cells of the PD. The fs cells execute regulative and supportive functions to adjacent hormone-producing cells (Allaerts and Venkelecom, 2005; Mitsuishi et al., 2013). The lipid and calcium binding protein Annexin A1 (Anx A1) and the cell membrane permeable compound nitric oxide (NO) have been detected in the fs cells (Woods et al., 1990; Devnath and Inoue, 2008). There are published findings indicating strong influence of Anx A1 and NO on hormone production (Taylor et al. 1993; Venkelecom et al, 1997). The hypothesis of this study is that the EC influence hormonal secretion by acting upon CB1 receptors on fs cells and thus activating or inhibiting Anx A1 and NO that directly affect adjacent glandular cells.
Prevalently cell models were used to carry out the experimental work. The TtT/GF and Tpit/F1 cell lines represent the fs cells, the AtT20/D16v stand for the ACTH-producing corticotroph (C) cells, and GH4C1 for the PRL-producing lactotroph (L) cells. Whenever comparison with an integrity model was possible tissue from C3H mice was used. Chemoluminescent and photometrical detection, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunoblot (IB), immunocyto- and immunohisto-chemical analysis (ICC, IHC), in situ hybridization (ISH), and (q) PCR methods were used as assaying tools to investigate CB1, Anx A1, the Anx A1 receptor - Fpr-rs1, NO, ACTH, and PRL.
CB1 was detected on the fs, C, and L cell models. The presence of fatty acid amide hydrolase (FAAH, an EC degrading enzyme) was confirmed in the fs cells. Incubations of the fs cells with CB1 agonists (2-AG, AEA, WIN) and antagonist (otenabant) were performed and resulting increase of Anx A1, and inhibition of NO were detected. Anx A1 binding sites, known as formyl peptide like receptor – related sequence 1 (Fpr-rs1) were identified on the C and L cells. The hormone-producing cells were treated with a 2-AG, Anx A1, and NO and the resulting changes in the levels of ACTH and PRL were detected. Anx A1 acted stimulatory on ACTH in the C AtT20/D16v cell and inhibitory on PRL in the L GH4C1 cell. NO inhibited both ACTH and PRL release. Additional analysis of the levels of expression of mRNA for Anx A1 and Fpr-rs1 in murine PD tissue demonstrated that while the expression of the first was not influenced by time, the expression of the latter was activated during the subjective day.
The here presented study shows that EC influence the ACTH release stimulatory through activating Anx A1 and inhibiting NO. As for PRL, the EC unfold an inhibition through activating Anx A1, and stimulation through inhibiting NO. A clear regulatory linkeage between the EC and ACTH and PRL control is revealed, involving the fs cells with possible time-dependence.
Many diseases have been described to be associated with inflammatory processes. The currently available anti-inflammatory drug therapy is often not successful or causes intolerable side effects. Thus, new anti-inflammatory substances are still urgently needed. Plants were the first source of remedies in the history of mankind. Since their chemical characterization in the 19th century, herbal bioactive compounds have fueled drug development. Also, nowadays, new plant-derived agents continuously enrich our drug arsenal (e.g., vincristine, galantamine, and artemisinin). The number of new, pharmacologically active herbal ingredients, in particular that of anti-inflammatory compounds, rises continuously. The major obstacle in this field is the translation of preclinical knowledge into evidence-based clinical progress. Human trials of good quality are often missing or, when available, are frequently not suitable to really prove a therapeutical value. This minireview will summarize the current situation of 6 very prominent plant-derived anti-inflammatory compounds: curcumin, colchicine, resveratrol, capsaicin, epigallocatechin-3-gallate (EGCG), and quercetin. We will highlight their clinical potential and/or pinpoint an overestimation. Moreover, we will sum up the planned trials in order to provide insights into the inflammatory disorders that are hypothesized to be beneficially influenced by the compound.
Simultaneous and dose dependent melanoma cytotoxic and immune stimulatory activity of betulin
(2015)
Conventional cytostatic cancer treatments rarely result in the complete eradication of tumor cells. Therefore, new therapeutic strategies focus on antagonizing the immunosuppressive activity of established tumors. In particular, recent studies of antigen-loaded dendritic cells (DCs) eliciting a specific antitumor immune response has raised the hopes of achieving the complete elimination of tumor tissue. Genistein, fingolimod and betulin have already been described as active compounds in different types of cancer. Herein, we applied an integrated screening approach to characterize both their cytostatic and their immune-modulating properties side-by-side. As will be described in detail, our data confirmed that all three compounds exerted proapoptotic and antiproliferative activity in different B16 melanoma cell lines to a given extent, as revealed by an MTT assay, CFSE and DAPI staining. However, while genistein and fingolimod also affected the survival of primary bone marrow (BM) derived DCs of C57BL/6 mice, betulin exhibited a lower cytotoxicity for BMDCs in comparison to the melanoma cells. Moreover, we could show for the first time, that only betulin caused a simultaneous, highly specific immune-stimulating activity, as measured by the IL-12p70 release of Toll-like receptor 4-stimulated BMDCs by ELISA, which was due to increased IL-12p35 mRNA expression. Interestingly, the activation of DCs resulted in enhanced T lymphocyte stimulation, indicated by increased IL-2 and IFN-γ production of cytotoxic T cells in spleen cell co-culture assays which led to a decreased viability of B16 cells in an antigen specific model system. This may overcome the immunosuppressive environment of a tumor and destroy tumor cells more effectively in vivo if the immune response is specific targeted against the tumor tissue by antigen-loaded dendritic cells. In summary, cytostatic agents, such as betulin, that simultaneously exhibit immune stimulatory activity may serve as lead compounds and hold great promise as a novel approach for an integrated cancer therapy.
Little attention so-far has been paid to the influence of chronobiology on the processes of nanoparticle uptake and transport into the brain, even though this transport appears to be chronobiologically controlled to a significant degree. Nanoparticles with specific surface properties enable the transport across the blood–brain barrier of many drugs that normally cannot cross this barrier. A clear dependence of the central antinociceptive (analgesic) effects of a nanoparticle-bound model drug, i.e., the hexapeptide dalargin, on the time of day was observable after intravenous injection in mice. In addition to the strongly enhanced antinociceptive effect due to the binding to the nanoparticles, the minima and maxima of the pain reaction with the nanoparticle-bound drug were shifted by almost half a day compared to the normal circadian nociception: The maximum in the pain reaction after i.v. injection of the nanoparticle-bound dalargin occurred during the later rest phase of the animals whereas the normal pain reaction and that of a dalargin solution was highest during the active phase of the mice in the night. This important shift could be caused by an enhanced endo- and exocytotic particulates transport activity of the brain capillary endothelial cells or within the brain during the rest phase.
Immune cells are key players in several physiological and pathophysiological events such as acute and chronic inflammation, atherosclerosis and cancer. Especially in acute inflammation, macrophages are indispensable for the switch from the acute inflammatory phase to the resolution phase. Not only the phagocytosis of apoptotic cells, but especially the surrounding cytokines and mediators are able to switch macrophage polarization from inflammatory- to anti-inflammatory phenotypes. Within this cytokine environment, sphingosine-1-phosphate (S1P) plays an important role for immune cell activation, polarization and migration.
Epicutanoeus immunotherapy as a novel prophylactic and therapeutic strategy for birch pollen allergy
(2014)
The development of a convenient, effective and safe allergen-specific immunotherapy (SIT) for birch pollen allergy, one of the most prevalent allergic diseases in Northern Europe, North America and Northern Japan, is of crucial importance. Epicutaneous immunotherapy (EPIT) has gained attention as a safe and non-invasive alternative for subcutaneous immunotherapy, a conventional SIT. However, clinical studies showed a limited effcacy of EPIT, indicating the necessity of improvement of the treatment regime. In this study, we hypothesized that a combination of a hypoallergen with an appropriate adjuvant could be a strategy to improve EPIT. To verify this hypothesis, we aimed at investigating the efficacy of epicutaneous treatment with rBet v 1, the major birch pollen allergen, plus Toll-like receptor (TLR) agonists for prophylaxis and therapy of birch pollen allergy using a murine model of birch pollen-induced allergic asthma. Furthermore, the efficacy of rBet v 1B2, a hypoallergenic variant of Bet v 1, as a therapeutic allergen in EPI was pre-clinically investigated. TLRs recognize conserved microbial molecules (like PAMPs), and are known to promote the counter-regulation of TH2 responses by the induction of TH1-type and/or regulatory cytokines by immune cells. The hypoallergen Bet v 1B2 is a folding-variant of the wild-type allergen rBet v 1 with reduced allergenicity, but retained T-cell immunogenicity. The low allergenicity, could allow the application of hypoallergens in higher doses, and therefore provide a safer and more effective treatment to regulate T-cell immune responses. First, the expression and purification of recombinant Bet v 1 and Bet v 1B2 was optimized. Compared to natural proteins, recombinant proteins offer the possibility to use well-defined molecules with a consistent pharmaceutical quality. Using optimal Escherichia coli expression strains in combination with immobilized metal chelate affinity chromatography (IMAC) and size exclusion chromatography (SEC), we successfully prepared a large amount of rBet v 1 and rBet v 1B2 with a high purity. The allergenic potency of rBet v 1 and the hypoallergenic characteristics of rBet v 1B2 were confirmed by measurement of IgE reactivity and mediator release capacity using ELISA and basophil activation tests, respectively. In a second part, a murine model of birch pollen-induced allergic asthma was established. It was shown that intraperetoneal sensitization with an optimal dose of rBet v 1 and intranasal challenge with birch pollen extract induced elevated IgE levels, airway eosinophilia and pulmonary inflammation in BALB/c mice. The clinical features are comparable to those in patients with allergic asthma, indicating that sensitized and challenged mice could be used for a pre-clinical study to assess the efficacy of the treatment for birch pollen allergy. Next, we investigated the adjuvant effects of Polyadenylic:polyuridylic acid (Poly(A:U)), a TLR3 agonist, and R848 (resiquimod), a TLR7 agonist, in prophylactic EPI with rBet v 1 to intervene with birch pollen allergy. Here, we hypothesized that TLR3 and TLR7 could be possible target receptors to induce adjuvant effects in EPI, since these receptors are expressed in Langerhans cells and dermal dendritic cells, persistent antigen presenting cells in the cutaneous tissues. BALB/c mice received EPI with rBet v 1 alone, or plus Poly(A:U), or R848 on their depilated back using patches. Mice treated epicutaneously were then sensitized with rBet v 1 plus ALUM and intranasally challenged with birch pollen extract. We found that prophylactic EPI with rBet v 1 plus R848 inhibited the production of Bet v 1-specific IgE antibodies in sensitization, suppressed pulmonary inflammation and airway hyperreactivity upon challenge. In contrast to R848, no adjuvant effect of Poly(A:U) on suppression of asthmatic features was observed. Our results indicated that R848, but not Poly(A:U), could be a potential adjuvant for prophylactic EPI of birch pollen induced allergic asthma. Finally, the therapeutic potency of EPI with rBet v 1, or rBet v 1B2 alone, or plus R848 was assessed. After sensitization and challenge, mice received therapeutic EPI with rBet v 1 alone, or plus R848, and re-challenge with birch pollen extract. We found that therapeutic treatment with Bet v 1B2 reduced established Bet v 1-specific IgE antibodies, pulmonary inflammation and airway hyperreactivity upon re-challenge. Therapeutic treatment with the recombinant wild-type allergen does not influence these key characteristics of allergic asthma. In contrast to the findings in the prophylactic treatment with rBet v 1 plus R848,no therapeutic benefit was found upon combination with R848. This could be due to the high number of treatment days. Reduction of this number may lead to a beneficial effect. However, these findings indicate that Bet v 1B2 could be a potential therapeutic agent for the treatment of established birch pollen induced allergic asthma. In conclusion, this study demonstrates for the first time that prophylactic EPI with the recombinant form of Bet v 1 in combination with R848 could prevent and suppress asthmatic features in an established birch pollen allergy. Not only therapeutic, but also prophylactic applications of EPI could be of importance to prevent allergic sensitization, considering the high prevalence of allergic diseases. R848 could be a potential adjuvant for enhancing the prophylactic potential of EPI for the treatment of birch pollen allergy. Furthermore, the beneficial use of the hypoallergen Bet v 1B2 in therapeutic EPI was demonstrated by intervention of established asthmatic features. In the future, a combination of hypoallergens alone or together with adjuvants in EPIT could lead to a more convenient and effective therapeutic treatment of established birch pollen induced allergic asthma.
Background: Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens.
Method: Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1.
Results: We generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation.
Conclusion: Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.
5-Lipoxygenase (5LO) is a key enzyme in biosynthesis of leukotrienes (LTs), lipid mediators of inflammation. To study the roles of the 5LO accessory proteins coactosin-like protein (CLP) and 5LO-activating protein (FLAP), we knocked down these proteins in human monocytic cells. Our results show that expression of CLP was required for full cellular 5LO activity when cells were activated with Ca2+ ionophore, as well as with a physiological stimulus (lipopolysaccharide followed by N-formylmethionyl-leucyl-phenylalanine). During LT biosynthesis in stimulated cells, 5LO typically translocates to the nuclear membrane. This redistribution, from cytosolic to perinuclear, was clearly compromised in both CLP- and FLAP-deficient cells. Our results suggest that the CLP–5LO interaction may be a target for reduced LT production.
Our focus is the identification, characterisation and functional analysis of different MLL fusions. In general, MLL fusion proteins are encoded by large cDNA cassettes that are difficult to transduce into haematopoietic stem cells. This is due to the size limitations of the packaging process of those vector-encoded RNAs into retro- or lentiviral particles. Here, we present our efforts in establishing a universal vector system to analyse different MLL fusions. The universal cloning system was embedded into the backbone of the Sleeping Beauty transposable element. This transposon has no size limitation and displays no integration preference, thereby avoiding the integration into active genes or their promoter regions. We utilised this novel system to test different MLL fusion alleles (MLL-NEBL, NEBL-MLL, MLL-LASP1, LASP1-MLL, MLL-MAML2, MAML2-MLL, MLL-SMAP1 and SMAP1-MLL) in appropriate cell lines. Stable cell lines were analysed for their growth behaviour, focus formation and colony formation capacity and ectopic Hoxa gene transcription. Our results show that only 1/4 tested direct MLL fusions, but 3/4 tested reciprocal MLL fusions exhibit oncogenic functions. From these pilot experiments, we conclude that a systematic analysis of more MLL fusions will result in a more differentiated picture about the oncogenic capacity of distinct MLL fusions.
HIV vaccine preclinical testing is difficult because HIV’s only relevant hosts are humans and no correlates of protection are known. To this end, we are working on the humanization of different mouse strains with human peripheral blood mononuclear cells (PBMCs) as well as human hematopoietic stem cells (HSC) to generate a useful small animal model.
We generated immune deficient mice (NOD Scid IL2gc -/- /NOD Rag1-/- IL2gc -/-) expressing human MHC class II (HLA-DQ8) on a mouse class II deficient background (Ab-/-). Here, the human HLA-DQ8 should interact with the matching T cell receptors of transferred matching human PBMCs and therefore could support the functionality of the transferred human CD4+ cells in the mice.
Mice that were adoptively transferred with human HLA-DQ8 PBMCs only showed engraftment of CD3+ T cells. Surprisingly, the presence of HLA class II did not significantly change the repopulation rates in the mice. Also, the presence of HLA class II did not advance B cell engraftment, such that humoral immune responses were undetectable. However, the overall survival of DQ8-expressing mice was significantly prolonged, compared to mice expressing mouse MHC class II molecules, and correlated with an increased time span until onset of GvHD.
To avoid GVHD and to increase and maintain the level of human cell reconstitution over a long period of time, the same mouse strains were reconstituted with human HSC. Compared to PBMC-repopulated mice, HSC-reconstituted mice develop almost all subpopulations of the human immune system detectable at week 12 after HSC transfer. These mice developed adaptive immune responses after Tetanus Toxoide (TT) immunizations. In addition, we are testing the susceptibility of these humanized mice to different HIV strains with a detailed look at immune responses.
Gene transfer vectors such as lentiviral vectors offer versatile possibilities to express transgenic antigens for vaccination purposes. However, viral vaccines leading to broad transduction and transgene expression in vivo, are undesirable. Therefore, strategies capable of directing gene transfer only to professional antigen-presenting cells would increase the specific activity and safety of genetic vaccines. A lentiviral vector pseudotype specific for murine major histocompatibilty complex class II (LV-MHCII) was recently developed and the present study aims to characterize the in vivo biodistribution profile and immunization potential of this vector in mice. Whereas the systemic administration of a vector pseudotyped with a ubiquitously-interacting envelope led to prominent detection of vector copies in the liver of animals, the injection of an equivalent amount of LV-MHCII resulted in a more specific biodistribution of vector and transgene. Copies of LV-MHCII were found only in secondary lymphoid organs, essentially in CD11c+ dendritic cells expressing the transgene whereas B cells were not efficiently targeted in vivo, contrary to expectations based on in vitro testing. Upon a single injection of LV-MHCII, naive mice mounted specific effector CD4 and CD8 T cell responses against the intracelllular transgene product with the generation of Th1 cytokines, development of in vivo cytotoxic activity and establishment of T cell immune memory. The targeting of dendritic cells by recombinant viral vaccines must therefore be assessed in vivo but this strategy is feasible, effective for immunization and cross-presentation and constitutes a potentially safe alternative to limit off-target gene expression in gene-based vaccination strategies with integrative vectors.
Ultraviolet-B (UVB)-induced inflammation produces a dose-dependent mechanical and thermal hyperalgesia in both humans and rats, most likely via inflammatory mediators acting at the site of injury. Previous work has shown that the gene expression of cytokines and chemokines is positively correlated between species and that these factors can contribute to UVB-induced pain. In order to investigate other potential pain mediators in this model we used RNA-seq to perform genome-wide transcriptional profiling in both human and rat skin at the peak of hyperalgesia. In addition we have also measured transcriptional changes in the L4 and L5 DRG of the rat model. Our data show that UVB irradiation produces a large number of transcriptional changes in the skin: 2186 and 3888 genes are significantly dysregulated in human and rat skin, respectively. The most highly up-regulated genes in human skin feature those encoding cytokines (IL6 and IL24), chemokines (CCL3, CCL20, CXCL1, CXCL2, CXCL3 and CXCL5), the prostanoid synthesising enzyme COX-2 and members of the keratin gene family. Overall there was a strong positive and significant correlation in gene expression between the human and rat (R = 0.8022). In contrast to the skin, only 39 genes were significantly dysregulated in the rat L4 and L5 DRGs, the majority of which had small fold change values. Amongst the most up-regulated genes in DRG were REG3B, CCL2 and VGF. Overall, our data shows that numerous genes were up-regulated in UVB irradiated skin at the peak of hyperalgesia in both human and rats. Many of the top up-regulated genes were cytokines and chemokines, highlighting again their potential as pain mediators. However many other genes were also up-regulated and might play a role in UVB-induced hyperalgesia. In addition, the strong gene expression correlation between species re-emphasises the value of the UVB model as translational tool to study inflammatory pain.
Background: Acute leukemia in early age (EAL) is characterized by acquired genetic alterations such as MLL rearrangements (MLL-r). The aim of this case-controlled study was to investigate whether single nucleotide polymorphisms (SNPs) of IKZF1, ARID5B, and CEBPE could be related to the onset of EAL cases (<24 months-old at diagnosis).
Methods: The SNPs (IKZF1 rs11978267, ARID5B rs10821936 and rs10994982, CEBPE rs2239633) were genotyped in 265 cases [169 acute lymphoblastic leukemia (ALL) and 96 acute myeloid leukaemia (AML)] and 505 controls by Taqman allelic discrimination assay. Logistic regression was used to evaluate the association between SNPs of cases and controls, adjusted on skin color and/or age. The risk was determined by calculating odds ratios (ORs) with 95% confidence interval (CI).
Results: Children with the IKZF1 SNP had an increased risk of developing MLL-germline ALL in white children. The heterozygous/mutant genotype in ARID5B rs10994982 significantly increased the risk for MLL-germline leukemia in white and non-white children (OR 2.60, 95% CI: 1.09-6.18 and OR 3.55, 95% CI: 1.57-8.68, respectively). The heterozygous genotype in ARID5B rs10821936 increased the risk for MLL-r leukemia in both white and non-white (OR 2.06, 95% CI: 1.12-3.79 and OR 2.36, 95% CI: 1.09-5.10, respectively). Furthermore, ARID5B rs10821936 conferred increased risk for MLL-MLLT3 positive cases (OR 7.10, 95% CI:1.54-32.68). Our data do not show evidence that CEBPE rs2239633 confers increased genetic susceptibility to EAL.
Conclusions: IKZF1 and CEBPE variants seem to play a minor role in genetic susceptibility to EAL, while ARID5B rs10821936 increased the risk of MLL-MLLT3. This result shows that genetic susceptibility could be associated with the differences regarding MLL breakpoints and partner genes.
Conjugated vaccines consisting of flagellin and antigen activate TLR5 and induce strong innate and adaptive immune responses. Objective of the present study was to gain further insight into the mechanisms by which flagellin fusion proteins mediate their immune modulating effects. In a mouse model of Ova-induced intestinal allergy a fusion protein of flagellin and Ova (rflaA:Ova) was used for intranasal and intraperitoneal vaccination. Aggregation status of flaA, Ova and flaA:Ova were compared by light scattering, uptake of fluorescence labeled proteins into mDC was analyzed, processing was investigated by microsomal digestion experiments. Mechanism of DC-activation was investigated using proteasome and inflammasome inhibitors. Immune responses of wildtype, IL-10−/−, TLR5−/− mDCs and Ova-transgenic T cells were investigated. Mucosal and i.p.-application of rflaA:Ova were able to prevent allergic sensitization, suppress disease-related symptoms, prevent body weight loss and reduction in food uptake. Intranasal vaccination resulted in strongest suppression of Ova-specific IgE production. These protective effects were associated with increased aggregation of rflaA:Ova and accompanied by tenfold higher uptake rates into mDC compared to the mixture of both proteins. Microsomal digestion showed that stimulation with rflaA:Ova resulted in faster degradation and the generation of different peptides compared to rOva. rflaA:Ova-mediated activation of mDC could be suppressed in a dose-dependent manner by the application of both inflammasome and proteasome inhibitors. Using TLR5−/− mDC the rflaA:Ova induced IL-10 secretion was shown to be TLR5 dependent. In co-cultures of IL-10−/− mDC with DO11.10 T cells the lack of rflaA:Ova-mediated IL-10 secretion resulted in enhanced levels of both TH2 (IL-4, IL-5) and TH1 (IL-2 and IFN-y) cytokines. In summary, mucosal vaccination with flaA:Ova showed strongest preventive effect. Stimulation with rflaA:Ova results in strong immune modulation mediated by enhanced uptake of the aggregated fusion protein, likely resulting in a different processing by DC as well as stronger TLR5 mediated cell activation.
Lipid mediators have been referred as bioactive lipids, whose change in lipid levels resulted in functional or pathophysiological consequences. They are in the focus of biological research, nevertheless this is a late recognition due to the many difficulties of working with bioactive lipids due to their properties: hydrophobic, unstable and they occur in only in small quantities. Liquid chromatography and mass spectrometry have facilitated the work with them. Especially in this field, cardiovascular diseases and inflammatory mediated diseases and cancer are pathophysiological events where LMs are deregulated. Additionally, if the modulation of one LM pathway is not sufficient to overcome a disease, the combination of targeting two or more pathways could be effective. Needless to say, lipid signaling cascades are complicated pathways and possible shunting into other pathways when inhibiting or genetically deleting enzymes should be taken into consideration.
The first part of this work has focused on enzymes that metabolize eicosanoids, like mPGES-1 and 5-LO. mPGES-1 is an important enzyme metabolizing PGH2 and one of the key players of the AA cascade. Its product, PGE2 plays an important role in different inflammatory processes. Inhibition of the mPGES-1 might be a promising step to circumvent COX dependent side effects of NSAIDs. The class of quinazoline compounds around the lead structure FR20 has been investigated on isolated human and murine enzyme, in HeLa cells and in different human whole blood (HWB) settings to establish the possible effects of these compounds on eicosanoid profiling. Novel compounds with inhibitory activities in the submicromolar range (IC50: 0.13 µM - 0.37 µM on isolated enzyme) were obtained which were also effective in cells and HWB. Furthermore, pharmacological profiling of toxicity and lipid screening with LC/MS-MS revealed that compounds also reduce PGE2 levels in intact cells and whole blood; they do not impair cell viability but lack the ability to inhibit the murine mPGES-1 enzyme. This problem could be overcome by means of chemical synthesis varying the scaffold (quinoline, quinazoline) or introducing biosteric replacement in the phenyl moieties.
5-LO is a relevant enzyme that plays an important role in eicosanoid signaling in particular in leukotriene biosynthesis. Leukotrienes are involved in asthma, allergic rhinitis, glomerulonephritis, rheumatoid arthritis, sepsis, cancer and atherosclerosis. Moreover, genetic variants in the genes of the 5-LO pathway have been associated with the risk of development of acute myocardial infarction and stroke. Eicosanoids are increased in infectious exacerbations of chronic obstructive pulmonary disease (COPD). They are also elevated in the airways of stable COPD patients compared to healthy subjects. Therefore, 5-LO has attired the scientific community as a possible therapeutic target to treat the several disease conditions listed before. In this study an extensive evaluation of imidazo[1,2-a]pyridines as a suitable lead structure for novel 5-LO targeting compounds was presented Within the three publications, 5-LO inhibitory activity of synthesized compounds was investigated in intact PMNL, a cell-free assay, in human whole blood and rodent cells to both elucidate structure-activity relationships and compounds were in vitro pharmacological evaluated. Chemical modifications for lead optimization via straight forward synthesis were used to combine small polar groups (hydroxy, and methoxy groups) which led to a suitable candidate with desired in vitro pharmacokinetic profile in terms of solubility and intrinsic clearance without showing any cytotoxicity. More than 70 imidazo[1,2-a]pyridine derivatives have been synthesized, resulting in more than 50 active compounds. Although it was not possible to introduce a solubility group without impairing the 5-LO inhibitory activity, combination of small polar groups lead to a more favorable solubility and in vitro metabolic stability. Overall, the development of 5-LO inhibitors with high efficacy and selectivity in vivo will provide a possible treatment for patients having one of the diseases where leukotriene biosynthesis plays an important role.
Other types of 5-LO inhibitors have been synthesized during this work, NO-NSAIDs can be postulated as novel 5-LO inhibitors that could circumvent the undesired side-effects of inhibiting COX isoforms (ulcer perforation, gastrointestinal bleeding and in some cases death). It is suggested that NO group is released in situ or after compounds are metabolized. NO-NSAIDs maintain the same anti-inflammatory properties by inhibiting 5-LO in clinical relevant concentrations. NO-NSAIDs are currently under clinical trial for the treatment of diseases where inflammation plays an important role. Synthesis of NO-NSAIDs is straightforward and can be applied for most NSAIDs recently published. Among them, the most promising candidate is NO-sulindac that was able to inhibit 5-LO product formation in intact PMNL, purified 5-LO and HWB in micromolar concentration. Additional experiments regarding their mechanism are currently being performed.
The present study could show that dual inhibitors are an interesting approach that is practicable. It has been used in the recent years to overcome side-effects and diseases concerning more pathophysiological conditions. MetS is an example of a conjunction of symptoms: hyperglycemia, hypertriglyceridemia, hypertension and obesity. Due to its complex nature, the current treatment strategies of MetS require multiple pharmacological compounds regulating lipid and glucose homeostasis as well as blood pressure and coagulation. This study describes the first synthesis of dual sEH/PPAR modulators as potential agents for treatment of MetS. Following a combinatorial approach, an acidic head group known as a pharmacophore important for PPARα/γ dual agonistic activity was combined with different hydrophobic urea derivatives in order to introduce an epoxide mimetic (sEH pharmacophore). The resulting compounds yielded high inhibition of sEH and different patterns of PPAR agonistic activity. This study demonstrates that the pharmacophores of PPAR agonists and sEH inhibitors can be easily combined, resulting in a simplified blueprint of a dual sEH/PPAR modulator. Further in vivo pharmacological evaluation studies are needed in order to evaluate, which pattern of PPAR activation shows the most promising profile for treatment of metabolic syndrome.
Another example of dual pharmacology has been presented in this work. Natural products derived compounds were able to target sEH and exhibit promising antiproliferative properties. The principle of addressing multiple targets by natural products can be transferred to synthetic multi-target ligands. In conclusion, several (E)-styryl-1H-benzo[d]imidazoles were synthesized and evaluated on recombinant sEH after an initial hit (IPS) that lead to potent sEH inhibitors exhibiting antiproliferative activities. Following the natural product-inspired design, the desired biological activity from a bacterial secondary metabolite has been enhanced and transferred to a synthetic compound series. The resulting compounds were accessible via an easy synthetic route and offered a possibility to investigate the structure-activity relationships. The natural product inspired drug design extends the valuable role of natural products as drugs and drug precursors to templates for fully synthetic bioactive molecules. Simplification of natural products by means of chemical synthesis could lead to an interesting field in the treatment of cancer.
Affinity chromatography has been used to unravel unknown- and off-target effects which either contribute to the biological effect of the inhibitor or that counteract or lead to undesired side-effects. During this PhD work, two main projects related to this technique have been established. In the first one, related to an imidazo[1,2-a]pyridine inhibitor (EP6), it has been shown that epoxide-sepharose is a reliable material in order to couple compounds bearing an alcohol. Coupling of an analogue of EP6 to the sepharose has been accomplished and affinity towards 5-LO was demonstrated. The challenging step is to discern from unspecific protein binders and analysis via SDS-PAGE separation and mass spectrometry. Further experiments using other cell types or improving SDS-PAGE analysis (e.g. 2D gel analysis) should be useful to unravel EP6 off-target effect. During the second project related to off-target effects of celecoxib and DMC, the main problem was the coupling of the functional group to the sepharose. Affinity towards COX-2 could not be demonstrated pointing out the inefficient coupling method. Higher pH values during coupling reaction should be tested in further experiments. Nevertheless, affinity chromatography is a useful technique to unravel cellular mechanisms.
Sphingolipid metabolism is also a recent area that attired the attention of cancer researchers, due to their important roles in cell proliferation and apoptosis. Ceramide metabolism inhibitors were synthesized and evaluated on different assay systems in order to assess their efficacy on several cancer lines. Remarkably, 2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (32) was a useful scaffold to mimic the sphingoid base. This key intermediate was used to produce ceramide analogues that could enter the cell and target apoptosis machinery. EB143 (38) increased ceramide levels in an in vitro ceramide synthase assay in a dose-response manner meaning that ceramide synthase was not inhibited but the ceramide de novo synthesis was activated. This effect was due to the fact that EB143 is a cytotoxic compound with an interesting antiproliferative profile. Further chemical modifications should be carried out to modulate this effect.
COX and LO inhibitors are cancer-preventive not only by inhibiting specific antiapoptotic AA metabolites but also by facilitating accumulation of AA which promotes neutral SMase activity and increases the proapoptotic ceramide. Several 5-LO inhibitors have been evaluated on several cancer lines and sphingolipid levels were measured in order to obtain a relationship. A549, Capan-2 and MCF-7 cells line were incubated with synthetic 5-LO inhibitors and zileuton. Compounds were cytotoxic to all cancer cell lines except from A549. Needless to say, zileuton did not exhibit a cytotoxic profile. Synthetic 5-LO inhibitors were able to modify ceramide levels but were useless when coincubating with sphingolipid metabolism inhibitors (myoricin, amitryptiline etc.) and inconsistent results were obtained. On the contrary, zileuton selectively increased Cer-C16 levels and in less extend Cer-C24:1. When using a SPT inhibitor (myoricin) alone was able to reduce C24:1 and Cer-C16:0 levels below the control, a similar effect occurred when incubation the cells with zileuton and myriocin. Interestingly, treatment of zileuton together with either amitryptiline or desipramine led to a decrease in Cer-C24:1 and levels Cer-C16:0 but the inhibition was not complete indicating that probably the de novo pathway has an important role. Further investigations on mRNA level should be carried out in order to discern which CerS is activated.
The main objective of the present thesis was the synthesis of lipid signaling modulators and their evaluation in vitro as therapeutic strategy to overcome pathophysiological conditions (cancer, metabolic syndrome, etc). It has been accomplished on many relevant targets like 5-LO, mPGES-1, sEH and PPAR and these lipid signaling modulators could be used in the treatment of diseases conditions where lipid mediators play an important role.
B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral vector transduction.
Rho-family GTPases like RhoA and Rac-1 are potent regulators of cellular signaling that control gene expression, migration and inflammation. Activation of Rho-GTPases has been linked to podocyte dysfunction, a feature of chronic kidney diseases (CKD). We investigated the effect of Rac-1 and Rho kinase (ROCK) inhibition on progressive renal failure in mice and studied the underlying mechanisms in podocytes. SV129 mice were subjected to 5/6-nephrectomy which resulted in arterial hypertension and albuminuria. Subgroups of animals were treated with the Rac-1 inhibitor EHT1846, the ROCK inhibitor SAR407899 and the ACE inhibitor Ramipril. Only Ramipril reduced hypertension. In contrast, all inhibitors markedly attenuated albumin excretion as well as glomerular and tubulo-interstitial damage. The combination of SAR407899 and Ramipril was more effective in preventing albuminuria than Ramipril alone. To study the involved mechanisms, podocytes were cultured from SV129 mice and exposed to static stretch in the Flexcell device. This activated RhoA and Rac-1 and led via TGFβ to apoptosis and a switch of the cells into a more mesenchymal phenotype, as evident from loss of WT-1 and nephrin and induction of α-SMA and fibronectin expression. Rac-1 and ROCK inhibition as well as blockade of TGFβ dramatically attenuated all these responses. This suggests that Rac-1 and RhoA are mediators of podocyte dysfunction in CKD. Inhibition of Rho-GTPases may be a novel approach for the treatment of CKD.
TO THE EDITOR: We read an interesting paper by Palta et al. in a recent issue of the Korean Journal of Hematology titled, "ZBTB16-RARA variant of acute promyelocytic leukemia with tuberculosis: a case report and review of literature" [1]. We would like to add some comments to their article and suggest additional molecular methods to confirm variant translocations in acute promyelocytic leukemia (APL)....
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.
CD69 is a transmembrane lectin that can be expressed on most hematopoietic cells. In monocytes, it has been functionally linked to the 5-lipoxygenase pathway in which the leukotrienes, a class of highly potent inflammatory mediators, are produced. However, regarding CD69 gene expression and its regulatory mechanisms in monocytes, only scarce data are available. Here, we report that CD69 mRNA expression, analogous to that of 5-lipoxygenase, is induced by the physiologic stimuli transforming growth factor-β (TGF-β) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in monocytic cells. Comparison with T- and B-cell lines showed that the effect was specific for monocytes. CD69 expression levels were increased in a concentration-dependent manner, and kinetic analysis revealed a rapid onset of mRNA expression, indicating that CD69 is a primary TGF-β/1α,25(OH)2D3 target gene. PCR analysis of different regions of the CD69 mRNA revealed that de novo transcription was initiated and proximal and distal parts were induced concomitantly. In common with 5-lipoxygenase, no activation of 0.7 kb or ~2.3 kb promoter fragments by TGF-β and 1α,25(OH)2D3 could be observed in transient reporter assays for CD69. Analysis of mRNA stability using a transcription inhibitor and a 3′UTR reporter construct showed that TGF-β and 1α,25(OH)2D3 do not influence CD69 mRNA stability. Functional knockdown of Smad3 clearly demonstrated that upregulation of CD69 mRNA, in contrast to 5-LO, depends on Smad3. Comparative studies with different inhibitors for mitogen activated protein kinases (MAPKs) revealed that MAPK signalling is involved in CD69 gene regulation, whereas 5-lipoxygenase gene expression was only partly affected. Mechanistically, we found evidence that CD69 gene upregulation depends on TAK1-mediated p38 activation. In summary, our data indicate that CD69 gene expression, conforming with 5-lipoxygenase, is regulated monocyte-specifically by the physiologic stimuli TGF-β and 1α,25(OH)2D3 on mRNA level, although different mechanisms account for the upregulation of each gene.
Recent clinical data support the clinical use of oral lavender oil in patients suffering from subsyndromal anxiety. We identified the molecular mechanism of action that will alter the perception of lavender oil as a nonspecific ingredient of aromatherapy to a potent anxiolytic inhibiting voltage dependent calcium channels (VOCCs) as highly selective drug target. In contrast to previous publications where exorbitant high concentrations were used, the effects of lavender oil in behavioral, biochemical, and electrophysiological experiments were investigated in physiological concentrations in the nanomolar range, which correlate to a single dosage of 80 mg/d in humans that was used in clinical trials. We show for the first time that lavender oil bears some similarities with the established anxiolytic pregabalin. Lavender oil inhibits VOCCs in synaptosomes, primary hippocampal neurons and stably overexpressing cell lines in the same range such as pregabalin. Interestingly, Silexan does not primarily bind to P/Q type calcium channels such as pregabalin and does not interact with the binding site of pregabalin, the α2δ subunit of VOCCs. Lavender oil reduces non-selectively the calcium influx through several different types of VOCCs such as the N-type, P/Q-type and T-type VOCCs. In the hippocampus, one brain region important for anxiety disorders, we show that inhibition by lavender oil is mainly mediated via N-type and P/Q-type VOCCs. Taken together, we provide a pharmacological and molecular rationale for the clinical use of the oral application of lavender oil in patients suffering from anxiety.
Hepatitis C virus (HCV) assembly and production is closely linked to lipid metabolism. Indeed, lipid droplets (LD) have been shown to serve as a platform for HCV assembly. To investigate the effect of HCV on the host cell proteome, 2D-gelelectrophoresis with subsequent MALDI-TOF mass spectrometry of HCV replicating and the corresponding control cells were done. Based on this analysis, it was found out that HCV-replicating Huh7.5 cells revealed lower amounts of TIP47 (tail interacting protein of 47kD) compared to HCV-negative cells. TIP47, a cytoplasmic sorting factor, has been shown to be associated with lipid droplets. As it is known that HCV-replication and assembly takes place at the so called ”membranous web” that is composed of LDs and rearranged ER-derived membranes, it was tempting to investigate the role of TIP47 in HCV life-cycle. Western blot analysis did reveal that overexpression of TIP47 in HCV replicating Huh7.5 cells leads to decreased amounts of the HCV core protein while the levels of non-structural protein (NS)5A and intracellular HCVgenomes are increased. Moreover, in TIP47 overproducing cells higher amounts of infectious HCV particles are secreted. Vice versa, inhibition of TIP47 expression by siRNA results in a decreased level of intracellular NS5A, increased amounts of intracellular core and less infectious viral particles in the supernatant. In addition, complete silencing of TIP47 by lentiviral transduction abolishes HCV replication that can be restored by transfection of these cells with a TIP47 expression construct. It has been shown recently that apoE binds to NS5A and that this interaction plays an important role for the HCV life cycle (Benga et al., 2010). The C-terminal part of TIP47 harbours a 4 helix bundle motif and displays high homology to the N-terminus of apoE. Therefore, we investigated the interaction of NS5A and TIP47. Confocal double immunofluorescence microscopy revealed that a fraction of NS5A colocalizes with TIP47. Coimmunoprecipitation experiments and a yeast-two-hybrid screening confirmed the interaction between NS5A and TIP47 and deletion of the N-terminal-TIP47-PAT domain abolishes this interaction. From this we conclude that the TIP47-NS5A interaction is required for virus morphogenesis. Moreover, TIP47 can bind to Rab9 and this is relevant for targeting the viral particle out of the cell. In accordance to this, TIP47 was identified to be associated to the viral particle. Mutants of TIP47 that fail to bind Rab9 reveal lower amounts and a changed distribution of the HCV core protein. Furthermore, we could see that the core staining colocalizes with subcellular structures that were identified as autophagosomes using a p62-specific antibody which is a specific autophagosome-marker. Based on this, we hypothized that destruction of the Rab9 binding domain misdirects the viral particle towards the lysosomal compartment.
For the first time it could be shown that TIP47 interacts with NS5A and is associated to the viral particle, therefore plays a crucial role for the virus morphogenesis and secretion of the viral article.
Taken together, these results indicate that TIP47 is an essential cellular factor for the life cycle of HCV Abstract and might be used as target for antiviral treatment, e.g. by targeting the NS5A-TIP47 interaction, based on small molecules that mimic the NS5A-specific sequence that binds to TIP47 which might result in a competition of the TIP47/NS5A interaction.
The role of the Ca2+-dependent protease calpain in the diabetes-associated platelet hyperreactivity
(2012)
Platelets from diabetic patients are characterised by hyperreactivity resulting in exaggerated adhesion, aggregation and thrombus formation which contribute to the development of cardiovascular complications known to be one of the main causes of diabetes-related mortality. One of the mechanisms suggested to be involved in the diabetes-related platelet hyperactivation is the increased [Ca2+]i which leads to the overactivation of Ca2+-dependent proteases, the calpains. Among the calpain isoforms expressed in platelets the two ubquitiously expressed μ- and m-calpain are thought to play an important role in physiological and pathophysiological processes. Particularly μ-calpain is known to be involved in many steps of physiological platelet activation such as aggregation, adhesion, secretion, and signalling. However, we could show that diabetes was associated with an enhanced activation of both μ- and m-calpain in platelets
In the first part of the study we focussed on the characterization of the molecular mechanism regulating calpain activity. Indeed, although Ca2+ is considered to be the main regulator of the proteolytic activity of the conventional calpains, other mechanisms such as the presence of phospholipids and phosphorylation have been reported to affect their activity. Since most studies reported the phosphorylation of m-calpain we were interested to see whether μ-calpain activity might be also affected by phosphorylation. We could show that the activity of μ-calpain was enhanced by the PKC activator PMA suggesting its possible regulation by phosphorylation. However, whether PKC directly targeted μ-calpain remains unclear. Given that substrate recognition is important for a protease to process its substrate and since no common consensus could be attributed to calpain substrates, our next interest was to understand the mechanism regulating the recognition of its substrates by calpain. Since phosphorylation has been reported to protect different proteins from calpain degradation we investigated whether the calpain substrate CD31 could be phosphorylated in platelets and whether this could affect its recognition by calpain. Although we could show that the tyrosine phosphorylation of CD31 was increased after activation of platelets by thrombin and that this effect was attenuated in platelets from diabetic patients, tyrosine phosphorylation of CD31 seemed to have no effect on its sensitivity to calpain-mediated proteolysis.
After the analysis of the mechanism regulating calpain activity as well as its interaction with its substrates, our next interest was the identification of new calpain substrates in platelets. Since a previous study from our group showed that PPARγ agonists could indirectly reverse the diabetes-associated calpain activation we performed DIGE analysis of platelet samples from diabetic patients before and after PPARγ agonist treatment. Using this approach we could identify four novel calpain substrates in platelets: Integrin-linked kinase (ILK), α parvin, CLP36 and septin-5. Next, we assessed the effect of calpain-mediated cleavage on the function of these newly identified proteins. We could show that μ-calpain was essential for the dissociation of ILK from the IPP complex and its activation while m-calpain-mediated cleavage led to its cleavage and inactivation. Functionally, we also showed that μ-calpain was involved in platelet adhesion while m-calpain was important for spreading.
The next protein we analysed was septin-5, a small GTPase known to regulate platelet degranulation by association with other septins and syntaxin-4. We found that the interaction between septin-5 and syntaxin-4 was inhibitory for platelet degranulation. We could demonstrate that the μ-calpain-mediated cleavage dissociated septin-5 from syntaxin 4 and led to increased secretion of platelet α-granules. Next, we investigated the in vivo role of calpain in the diabetes-associated platelet hyperreactivity. We induced diabetes in mice and could reproduce calpain activation in platelets such as that found in human. Indeed, calpain activation in murine platelets also led to the cleavage of several calpain substrates including ILK and septin-5. Moreover, platelets from diabetic mice demonstrated an increased aggregation and thrombus formation in vivo. Treatment of the animals with the calpain inhibitor A-705253 (30 mg/kg/day for 10 days) significantly restored platelet function and substrate cleavage. In conclusion, in this part of the study, we could show that the increased calpain-dependent α-granule secretion and platelet adhesion may account for the enhanced vascular proliferation and thrombus formation in diabetes and calpain inhibition represents a promising way to prevent atherothrombosis development.
In the last part of the study we analysed another enzyme known to play a crucial role in diabetes, the AMPK which is an energy-sensing kinase known to be impaired in diabetes. We could show that the two catalytic subunits AMPK α1 and α2 are expressed in platelets. The AMPKα2 seemed to be the subunit involved in platelet activation since AMPKα2-deficient mice demonstrated a defect in clot retraction and the stabilization of the thrombus while the animals showed a normal bleeding time. Mechanistically, we showed in platelets that the upstream kinase of AMPKα2 is LKB1 which was activated by thrombin stimulation via a PI-3K-dependent pathway. AMPKα2 then phosphorylated the Src-family kinase Fyn, which is responsible for the phosphorylation of its substrate β3 integrin on Tyr747. These data indicate that AMPKα2, by affecting Fyn phosphorylation and activity, plays a key role in platelet αIIbβ3 integrin signalling, leading to clot retraction and thrombus stability. Although the effect of diabetes in the AMPK-dependent pathway could not be investigated we assume that the dysregulation of this pathway may account for the thrombus destabilization and enhanced embolization encountered in diabetes.
In the scientific literature, the use of a surfactant is recommended for both designing quality control tests for water insoluble or sparingly water soluble drugs and for predicting the bioavailability of drugs from various types of formulations. Since the number of poorly soluble drugs is increasing, the selection of adequate dissolution test for these becomes more and more important. The aim of the present study was to develop predictive and discriminatory test methods based on surfactants that are recommended in the literature. Particular respect was given to the use of sodium lauryl sulfate and Tween 80, the two most commonly used surfactants for this purpose. Tamoxifen was used as a model drug. Dissolution experiments were performed using various concentrations of the two surfactants in buffer media typically used to prepare biorelevant test media. Results were then compared with those deriving from the same test formulations in biorelevant and simplified “biorelevant” media. Results from this study indicate that the concentration of surfactant has a huge impact on both the rate and extent of drug release from the formulation and also on the discriminatory power of the test. However, they also indicate that a well designed and validated test medium containing SLS or Tween 80 can be useful in terms of establishing a discriminatory test medium that possibly could also be used to assure batch to batch bioequivalence. Therefore, the approach described in the present paper might be very helpful for developing predictive and discriminatory methods in early formulation development for poorly soluble drugs and which could also be adopted for QC.
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.
Interleukin-22 predicts severity and death in advanced liver cirrhosis: a prospective cohort study
(2012)
Background: Interleukin-22 (IL-22), recently identified as a crucial parameter of pathology in experimental liver damage, may determine survival in clinical end-stage liver disease. Systematic analysis of serum IL-22 in relation to morbidity and mortality of patients with advanced liver cirrhosis has not been performed so far.
Methods: This is a prospective cohort study including 120 liver cirrhosis patients and 40 healthy donors to analyze systemic levels of IL-22 in relation to survival and hepatic complications.
Results: A total of 71% of patients displayed liver cirrhosis-related complications at study inclusion. A total of 23% of the patients died during a mean follow-up of 196 +/- 165 days. Systemic IL-22 was detectable in 74% of patients but only in 10% of healthy donors (P <0.001). Elevated levels of IL-22 were associated with ascites (P = 0.006), hepatorenal syndrome (P <0.0001), and spontaneous bacterial peritonitis (P = 0.001). Patients with elevated IL-22 (>18 pg/ml, n = 57) showed significantly reduced survival compared to patients with regular ([less than or equal to]18 pg/ml) levels of IL-22 (321 days versus 526 days, P = 0.003). Other factors associated with overall survival were high CRP ([greater than or equal to]2.9 mg/dl, P = 0.005, hazard ratio (HR) 0.314, confidence interval (CI) (0.141 to 0.702)), elevated serum creatinine (P = 0.05, HR 0.453, CI (0.203 to 1.012)), presence of liver-related complications (P = 0.028, HR 0.258 CI (0.077 to 0.862)), model of end stage liver disease (MELD) score [greater than or equal to]20 (P = 0.017, HR 0.364, CI (0.159 to 0.835)) and age (P = 0.011, HR 1.047, CI (1.011 to 1.085)). Adjusted multivariate Cox proportional-hazards analysis identified elevated systemic IL-22 levels as independent predictors of reduced survival (P = 0.007, HR 0.218, CI (0.072 to 0.662)).
Conclusions: In patients with liver cirrhosis, elevated systemic IL-22 levels are predictive for reduced survival independently from age, liver-related complications, CRP, creatinine and the MELD score. Thus, processes that lead to a rise in systemic interleukin-22 may be relevant for prognosis of advanced liver cirrhosis.
BACKGROUND: The endocannabinoid 2-arachidonoyl glycerol (2-AG) acts as a retrograde messenger and modulates synaptic signaling e. g. in the hippocampus. 2-AG also exerts neuroprotective effects under pathological situations. To better understand the mechanism beyond physiological signaling we used Organotypic Entorhino-Hippocampal Slice Cultures (OHSC) and investigated the temporal regulation of 2-AG in different cell subsets during excitotoxic lesion and dendritic lesion of long range projections in the enthorhinal cortex (EC), dentate gyrus (DG) and the cornu ammonis region 1 (CA1).
RESULTS: 2-AG levels were elevated 24 h after excitotoxic lesion in CA1 and DG (but not EC) and 24 h after perforant pathway transection (PPT) in the DG only. After PPT diacylglycerol lipase alpha (DAGL) protein, the synthesizing enzyme of 2-AG was decreased when Dagl mRNA expression and 2-AG levels were enhanced. In contrast to DAGL, the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MAGL) showed no alterations in total protein and mRNA expression after PPT in OHSC. MAGL immunoreaction underwent a redistribution after PPT and excitotoxic lesion since MAGL IR disappeared in astrocytes of lesioned OHSC. DAGL and MAGL immunoreactions were not detectable in microglia at all investigated time points. Thus, induction of the neuroprotective endocannabinoid 2-AG might be generally accomplished by down-regulation of MAGL in astrocytes after neuronal lesions.
CONCLUSION: Increase in 2-AG levels during secondary neuronal damage reflects a general neuroprotective mechanism since it occurred independently in both different lesion models. This intrinsic up-regulation of 2-AG is synergistically controlled by DAGL and MAGL in neurons and astrocytes and thus represents a protective system for neurons that is involved in dendritic reorganisation.
5-Lipoxygenase contributes to PPAR [gamma] activation in macrophages in response to apoptotic cells
(2012)
Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle.
Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression.
Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells.
Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages.
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.
Dendritic cells (DCs) play a pivotal role in the development of cutaneous contact hypersensitivity (CHS) and atopic dermatitis as they capture and process antigen and present it to T lymphocytes in the lymphoid organs. Recently, it has been indicated that a topical application of the sphingolipid sphingosine 1-phosphate (S1P) prevents the inflammatory response in CHS, but the molecular mechanism is not fully elucidated. Here we indicate that treatment of mice with S1P is connected with an impaired antigen uptake by Langerhans cells (LCs), the initial step of CHS. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Our results indicate that S1P inhibits macropinocytosis of the murine LC line XS52 via S1P2 receptor stimulation followed by a reduced phosphatidylinositol 3-kinase (PI3K) activity. As down-regulation of S1P2 not only diminished S1P-mediated action but also enhanced the basal activity of LCs on antigen capture, an autocrine action of S1P has been assumed. Actually, S1P is continuously produced by LCs and secreted via the ATP binding cassette transporter ABCC1 to the extracellular environment. Consequently, inhibition of ABCC1, which decreased extracellular S1P levels, markedly increased the antigen uptake by LCs. Moreover, stimulation of sphingosine kinase activity, the crucial enzyme for S1P formation, is connected not only with enhanced S1P levels but also with diminished antigen capture. These results indicate that S1P is essential in LC homeostasis and influences skin immunity. This is of importance as previous reports suggested an alteration of S1P levels in atopic skin lesions.
Um der Erkennung durch das körpereigene Immunsystem entkommen, weisen Tumore Modifikationen in ihrer Mikroumgebung auf. Zu diesen gehören u. a. veränderte Sauerstoffkonzentrationen im Tumorkern und die Freisetzung biochemischer Faktoren aus Tumorzellen, welche die Funktion von Tumor-assoziierten Phagozyten, wie z.B. Dendritischen Zellen (DC) beeinflussen. DC sind professionelle Antigen-präsentierende Zellen, die eine Spezialisierung in verschiedene funktionale Subtypen aufweisen. Myeloische DC (mDC) sind besonders effizient in Hinsicht auf die Präsentation von Antigenen, wohingegen plasmazytoide DC (pDC) regulatorisch auf das Immunsystem einwirken. Beide Subtypen spielen eine wichtige Rolle bei der Karzinogenese.
Während humane mDC, zur therapeutischen Verwendung, ex vivo aus Monozyten hergestellt werden können, war dies für humane pDC bisher nicht möglich. Ein war deshalb ein erstes Ziel dieser Arbeit, ein Protokoll zur Generierung humaner pDC aus humanen Monozyten zu entwickeln. Diese wurden mittels des Wachstumsfaktors Fms-related tyrosine kinase 3 ligand (Flt3-L) zu pDC-Äquivalenten differenziert, welche als monocyte-derived pDC (mo-pDC) bezeichnet wurden. In der Tat zeigten mo-pDC ein für humane pDC charakteristisches Oberflächenmarkerprofil und wiesen, im Vergleich zu mDC, eine geringe Kapazität zur Induktion der Proliferation autologer T Zellen und zur Phagozytose apoptotischer Zellen auf. Mo-pDC erwarben im Verlauf ihrer Differenzierung aus Monozyten eine kontinuierlich erhöhte Expression des pDC-spezifischen Transkriptionfaktors E2-2 und seiner spezifischen Zielgene. Der wichtigste funktionale Parameter von pDC ist die Produktion großer Mengen von Interferon-α (IFN-α). Mo-pDC sezernierten, nach vorheriger Aktivierung mit Tumornekrosefaktor-α (TNF-α) oder wenn zu ihrer Differenzierung neben Flt3-L auch Vitamin D3 oder all-trans-Retinolsäure verwendet wurde, ebenfalls große Mengen IFN-α. Wurden mo-pDC unter Hypoxie, einem prominenten Faktor der Tumormikroumgebung, generiert, so waren die Expression des spezifischen Transkriptionsfaktors E2-2 und die Freisetzung von IFN-α stark vermindert. Diese Daten zeigten zunächst, dass mo-pDC für das Studium von Differenzierung und Funktion humaner pDC eingesetzt werden können.
Weiterhin lieferten sie Hinweise auf eine veränderte Differenzierung humaner pDC unter Hypoxie. In einem nächsten Schritt wurde folglich untersucht, ob Hypoxie auch die Differenzierung von pDC aus deren physiologischen Vorläufern beeinflusst. Wurden Knochenmarkszellen der Maus mit Flt3-L unter Normoxie oder Hypoxie kultiviert, so war die Differenzierung zu pDC unter Hypoxie in der Tat unterdrückt. Dies war abhängig von der Hypoxie-induzierten Aktivität des Hypoxie-induzierten Faktors 1 (HIF-1), da die Flt3-Linduzierte Differenzierung von murinen Knochenmarkszellen, in denen die Expression von HIF-1 in pDC-Vorläuferzellen ausgeschaltet war, unter Hypoxie normal verlief.
Zusammenfassend kann also gesagt werden, dass Hypoxie, durch Aktivierung von HIF-1, Differenzierung und Funktion von pDC unterdrückt. Dieser Mechanismus könnte zu ihrer beschriebenen Dysfunktion in humanen Tumoren beitragen.
Neben Hypoxie sind viele andere Faktoren an der Immunsuppression in Tumoren beteiligt.
Eine Komponente der Mikroumgebung in Tumoren ist das Vorhandensein apoptotischer Tumorzellen. Apoptose von Tumorzellen findet, im Kontrast zur generellen Sicht von Tumoren als Apoptose-resistente Entitäten, auch in unbehandelten Tumoren im Überfluss statt. Apoptotische körpereigene Zellen unterdrücken unter physiologischen Bedingungen das Immunsystem. Deshalb könnte das Freisetzen von apoptotischem Material oder die Sekretion von Faktoren aus sterbenden Tumorzellen einen starken Einfluss auf die Funktion von Tumor-assoziierten DC und die damit verbundene Aktivierung von tumoriziden Lymphozyten haben. Eine diesbezügliche Studie war das zweite Ziel der vorliegenden Arbeit. Humane mDC wurden zu diesem Zweck mit Überständen lebender, apoptotischer oder nekrotischer humaner Brustkrebszellen aktiviert und anschließend mit autologen T Zellen ko-kultiviert. Danach wurde das zytotoxische Potential der ko-kultivierten T Zellen analysiert. Interessanterweise unterdrückte die Aktivierung mit Überständen apoptotischer Tumorzellen die DC-vermittelte Generierung tumorizider T Zellen durch die Ausprägung einer Population von regulatorischen T Zellen (Treg), die durch die gleichzeitige Expression der Oberflächenmoleküle CD39 und CD69 charakterisiert war. Die Ausprägung der CD39-und CD69-exprimierenden Treg Zell-Population war abhängig von der Freisetzung des bioaktiven Lipids Sphingosin-1-Phosphat (S1P) aus apoptotischen Zellen, welches durch den S1P-Rezeptor 4 zur Freisetzung des immunregulatorischen Zytokins IL-27 aus mDC führte.
Neutralisierung von IL-27 in AC-aktivierten Ko-Kulturen von mDC und T Zellen blockierte die Generierung von CD39- und CD69-exprimierenden Treg Zellen und resultierte folglich in der Aktivierung zytotoxischer T Zellen. Weiterhin war die Bildung von Adenosin in den Ko-Kulturen für die Unterdrückung zytotoxischer T Zellen vonnöten. Erste Experimente lieferten Hinweise auf eine direkte Interaktion von CD69- und CD39-exprimierenden Treg Zellen mit CD73-exprimierenden zytotoxischen T Zellen. CD39 und CD73 werden für die Bildung von Adenosin aus ATP benötigt, weswegen die Interaktion von Treg Zellen und zytotoxischen T Zellen die Adenosin-Produktion fördern könnte.
Zusammenfassend zeigen die hier präsentierten Befunde wie Faktoren der
Tumormikroumgebung die Funktion von humanen DC Subtypen beeinflussen können. Ein Verständnis der zugrundeliegenden Mechanismen kann wertvolle Informationen für die Wahl effektiver Immuntherapien oder Chemotherapien liefern und so die Therapie humaner Tumore unterstützen.
Drug toxicity and viral resistance limit long-term efficacy of antiviral drug treatment for HIV
infection. Thus, alternative therapies need to be explored. Previously, group of “Prof. von Laer”
tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an
HIV entry inhibitory peptide (maC46). Gene-modified autologous T cells were infused into 10
HIV-infected patients with advanced disease and multidrug resistant virus during antiretroviral
combination therapy. T cell infusions were tolerated well with no severe side effects. A
significant increase of CD4 counts was observed post infusion. At the end of the one-year
follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified
cells could be detected in peripheral blood, lymph nodes and bone marrow throughout the oneyear
follow-up, whereby marking levels correlated with the cell dose. No significant changes of
viral load were observed during the first four months. Four of the seven patients that changed
their antiviral drug regimen thereafter responded with a significant decline in plasma viral load.
In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene
marking and may improve immune competence in HIV-infected patients with advanced disease
and multidrug resistant virus. However, the low level of gene marking and the lack of substantial
long-term in vivo accumulation of gene-protected cells observed in this trial clearly demonstrate
the requirement for new vectors with new strategy.
In this thesis self‐inactivating lentiviral vectors harboring internal promoters and RNA elements
were therefore evaluated for their potential use in a clinical gene‐therapy trial. The results from
this work provide the basis for the selection of a suitable candidate vector for extensive
preclinical testing. Apart from being capable of transducing non‐dividing cells, lentiviral vectors
incorporate a number of additional features that are of potential value for gene therapeutic
applications. These include a larger packaging capacity, higher titers than γ‐retroviral vectors
and, most importantly, a reduced risk of deregulating cellular genes due to its natural integration
profile. The use of internal promoters to drive expression of the therapeutic transgene maC46
should further improve the safety profile of these new‐generation vectors, while an additional
artificial splice acceptor (SA) into the 5‟UTR of the transgene over all elevate transgene
expression. The rationale for this is that hematopoietic stem and progenitor cells will be
Summary
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protected from enhancer‐mediated transactivation effects and also from potential side effects due
to the aberrant expression of maC46 while at the same time the full clinical benefit for the
patients is maintained.
In order to find a suitable candidate for preclinical studies, two candidate therapeutic vectors
harboring different regulatory elements were selected based on results from pilot experiments.
The internal promoters used to drive expression of codon optimized maC46 were the PGK
promoter and MPSV promoter. This work focuses on the transgene expression levels in
lymphoid cells and antiviral activity. The issues of long term expression, propensity to
methylation mediated silencing of the promoters, and genotoxicity were also touched. In a first
step the performance of different vectors was evaluated in the human T cell lines. Based on
promising data from ex vivo human peripheral blood mononuclear cells, the vector carrying the
MPSV promoter along with intron were selected for in vivo transplantation experiments.
In summary, the ex vivo data suggested the long term survival of lentiviral gene modified cells,
along with maintained expression of introduced genes. It was observed that the expression of
these constructs depends strongly on the activation and differentiation status of the targeted T
cells. This regulation was not linked to any specific promotor. In vivo study shows that maC46
can be introduced into murine multiple hematopoietic lineages via lentiviral vector and expressed
at high levels in their mulilineage progeny, without altering the hematopoiesis. There was no
sign of any kind of hematopoietic or lymphoid malignancies. Although gene-modified
lymphocytes persisted in-vivo, the downregulation of transgene expression was consistent with
the ex-vivo observation. In contrast to that the T cells transplanted group showed delayed
engraftment of donor cells and there was no expression of C46 in blood and lymphatic organs. .
In conclusion, when considering HIV gene therapy focusing CD4+ T cells, potential problems of
T cell activation status as related to the desired clinical effect must be addressed. These results
might open the way for a gene therapy targeting mainly or exclusively activated T cells and
could be exploited for immunostimulatory as well as suppressive approaches.
Gene therapy is a promising therapeutic strategy that emerged from the attractive idea of targeting therapy at the molecular level. For many patients who suffer from genetic and acquired diseases that cannot be effectively treated by conventional treatment approaches gene therapy remains a huge hope of cure in spite of the hurdles regarding efficacy and safety that need to be overcome. The development of efficient gene transfer vehicles, mainly retroviral vectors, led to the first successful gene therapy trial, to treat patients suffering from X-linked severe combined immunodeficiency syndrome (X-SCID) using gene modified stem cells (Hacein-Bey-Abina, Le Deist et al. 2002). Despite the success of this trial, it revealed the danger of retroviral insertional mutagenesis as a major adverse event of gene therapy using gene-modified stem cells (Hacein-Bey-Abina, von Kalle et al. 2003). In contrast to stem cells, T cells are relatively resistant to insertional mutagenesis and transformation even after transduction with potent oncogenes using retroviral vectors (Newrzela, Cornils et al. 2008). However, mature T cells can self-renew, proliferate and survive for long periods. These criteria are supposed to render T cells prone to transformation. Therefore, the questions of mature T cells transformability and the control mechanism limiting their transformation are still elusive.
The NS5B protein of the hepatitis C virus (HCV) is a RNA-dependent RNA polymerase, which is the key enzyme for viral replication. It is recognized as one of the promising targets for antiviral intervention within the new HCV treatment approach of direct-acting antivirals (DAA). However, several of the known non-nucleoside HCV polymerase inhibitors (NNIs) identified by screening approaches show limitations in the coverage of all six major HCV genotypes (GT). Genotypic profiling therefore has to be implemented early in the screening cascade to discover new broadly active NNIs. This implies knowledge of the specific individual biochemical properties of polymerases from all GTs which is to date limited to GT 1 only. The work submitted here gives a comprehensive overview of the biochemical properties of HCV polymerases derived from all major GTs 1 - 6. Biochemical analysis of polymerases from 38 individual sequences revealed that the optima for monovalent cations, pH and temperature were similar between the GTs, whereas significant differences concerning concentration of the preferred cofactor Mg2+ were identified. Implementing the optimal requirements for the polymerases from each individual GT led to significant improvements in their enzymatic activities. However, the specific activity was distributed unequally across the GTs and could be ranked in the following descending order: 1b, 6a > 2a, 3a, 4a, 5a > 1a. Furthermore, the optimized assay conditions for GT profiling were confirmed by testing the inhibitory activity of four known prototype NNIs, each addressing one of the four NNI binding sites. Additionally, a novel NNI chemotype - identified by screening - is described, the substituted N-phenyl-benzenesulphonamides (SPBS). This inhibitor class showed reversible inhibition of NS5B from HCV 1b Con1 with IC50 values up to 39 nM. Based on the decreased inhibitory activity against a recombinant NS5B protein carrying the mutation L419M, it was assumed that the SPBS inhibitors bound to the thumb site II as it has been described for the carboxy thiophene inhibitors. The postulated binding site was consequently confirmed by analysing a provided co-crystal structure of NS5B in complex with a SPBS analogue. Notably, the two SPBS analogues SPBS-1 and SPBS-2 reported here revealed significant differences in addressing the NH-group of the main chain Y477 by hydrogen-bonds, watermediated or directly, which provoked a shift of the carboxyphenyl group of the inhibitors towards the H475 position for the water-mediated binding mode. Interestingly, the differences observed in the binding mode led to a different cross resistance profile at positions M423 and I482. Using the previously optimized biochemical primer-dependent transcription assay, inhibitory activity of the SPBS could be demonstrated against polymerases from HCV GTs 1a and 1b whereas the inhibitor class failed to inhibit any of the non-GT 1 polymerases. Furthermore, initial antiviral activity for SPBS was demonstrated against the subgenomic replicons of HCV GTs 1a and 1b, respectively, and no considerable cytotoxic potential against a panel of ten different cell types. Finally, concerning a possible future treatment without PEG-IFN α or ribavirin, the SPBS analogues were found to display additive to synergistic effects in combination with the benzothiadiazine, the benzofuran and the indole - representative inhibitors for the binding sites palm I, palm II and thumb I, repectively - in the biochemical assay. Within the same binding site as the SPBS, the reference compound hydroxydihydropyranone displayed additive interactions only with the benzothiadiazine (palm I) in the biochemical assay as well as in cell culture. Hence it could be concluded that, having characterized one individual NNI, no universal predication is possible concerning the combinatory behaviour of NNIs binding to the same binding site. As synergistic, antagonistic or additive interactions are inhibitor-dependent (not binding sitedependent) each novel NNI has to be characterized individually in one-to-one combinations.
Decorin, a small leucine rich proteoglycan (SLRP) of the extracellular matrix (ECM) is a biologically active molecule with signaling capabilities modulating diverse cellular functions 1. In this report, we explore the role of the matrix proteoglycan decorin in the regulation of inflammation and apoptosis and the resultant biological significance in cancer and diabetic nephropathy. The mechanisms linking immunity and inflammation with tumor development are not well defined. Here we report a novel finding that the soluble form of decorin could autonomously trigger the synthesis of TNFα and IL-12 in macrophages through TLR2 and TLR4 in a p44/42- and p38-dependent manner. In the presence of LPS, decorin enhanced the effects of LPS by signaling additionally via TLR2. Further, decorin could enhance PDCD4 protein expression with subsequent inhibition of LPS-mediated IL-10 protein synthesis by two mechanisms: i) by TLR2/TLR4-dependent stimulation of PDCD4 synthesis and ii) by inhibition of the TGFβ1-induced increase of miR-21, a posttranscriptional suppressor of PDCD4 protein synthesis. Enhanced PDCD4, a translational inhibitor of IL-10, downregulated this anti-inflammatory cytokine, thereby further driving the cytokine profile towards a proinflammatory phenotype.
Importantly, these mechanisms appear to operate in a broad biological context linking pathogen-mediated with sterile inflammation as shown here for sepsis and growth retardation of established tumor xenografts. In sepsis, decorin is an early response gene evoked by inflammation and is markedly elevated in plasma of septic human patients and in plasma and tissues of septic mice. Our findings suggested that in vivo decorin alone mimics the effects of LPS by enhancing the plasma and tissue levels of pro-inflammatory TNFα, IL-12 and PDCD4 but when administered together with LPS, it potentiated the proinflammatory response of this PAMP by inhibiting active TGFβ1, miR-21 and hence the LPS mediated IL-10 production. In vivo, overexpression of decorin in tumor xenografts resulted in decorin/TLR2/4-driven synthesis of PDCD4, TNFα, IL-12 and decorin/TGFβ1/miR-21-mediated inhibition of PDCD4 suppression shifting the immune response to a pro-apoptotic and proinflammatory axis with strong anti-tumorigenic effects resulting in increased apoptosis and growth retardation of solid tumor. Thus, decorin signaling boosts inflammatory activity in sepsis and tumor. In contrast to the proinflammatory and proapoptotic role of decorin in tumor, decorin deficiency in diabetic kidneys led to enhanced apoptosis and increased mononuclear cell infiltration indicating that decorin might give rise to distinct biological outcomes depending on the cell type and biological context. Accordingly, in this study, we used a model of streptozotocin-induced diabetes type 1 in wild-type (Dcn+/+) and decorin-deficient- (Dcn-/-) mice to further elucidate the role of decorin in diabetic nephropathy. In this model, decorin was overexpressed in the mesangial matrix of the glomerulus and in the tubulointerstitium both at the mRNA and protein level in early stages of diabetic nephropathy which declined as the disease further progressed supporting the concept that decorin might act as a part of a natural response to hyperglycemia and to damage caused there from. These observations correlate with the data obtained in renal biopsies from patients at various stages of diabetic nephropathy 15, suggesting clinical relevance of our findings for the human disease. In the diabetic kidney, decorin deficiency was associated with: i) glomerular and tubular overexpression of p27Kip1 and enhanced proteinuria, ii) enhanced expression of TGFβ1 and CTGF resulting in increased accumulation of ECM, iii) overexpression of biglycan and elevated infiltration of mononuclear cells, iv) enhanced apoptosis of tubular epithelial cells despite overexpression of tubular IGF-IR. We further discovered that decorin binds to the IGF-IR in tubular epithelial cells and conveys protection against high glucose-mediated apoptosis providing evidence for a protective role of decorin during diabetic nephropathy development.
Thus, future therapeutic approaches that would either enhance the endogenous production of decorin or deliver exogenous decorin to the diseased solid tumors and/or diabetic kidney might improve the prognosis of these chronic diseases.
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.
TRPC channels are a family of nonselective cation channels that regulate ion homeostasis and intracellular Ca2+ signaling in numerous cell types. Important physiological functions such as vasoregulation, neuronal growth, and pheromone recognition have been assigned to this class of ion channels. Despite their physiological relevance, few selective pharmacological tools are available to study TRPC channel function. We, therefore, screened a selection of pharmacologically active compounds for TRPC modulating activity. We found that the synthetic gestagen norgestimate inhibited diacylglycerol-sensitive TRPC3 and TRPC6 with IC50s of 3–5 µM, while half-maximal inhibition of TRPC5 required significantly higher compound concentrations (>10 µM). Norgestimate blocked TRPC-mediated vasopressin-induced cation currents in A7r5 smooth muscle cells and caused vasorelaxation of isolated rat aorta, indicating that norgestimate could be an interesting tool for the investigation of TRP channel function in native cells and tissues. The steroid hormone progesterone, which is structurally related to norgestimate, also inhibited TRPC channel activity with IC50s ranging from 6 to 18 µM but showed little subtype selectivity. Thus, TRPC channel inhibition by high gestational levels of progesterone may contribute to the physiological decrease of uterine contractility and immunosuppression during pregnancy.
Background: After focal neuronal injury the endocannabinioid system becomes activated and protects or harms neurons depending on cannabinoid derivates and receptor subtypes. Endocannabinoids (eCBs) play a central role in controlling local responses and influencing neural plasticity and survival. However, little is known about the functional relevance of eCBs in long-range projection damage as observed in stroke or spinal cord injury (SCI).
Methods: In rat organotypic entorhino-hippocampal slice cultures (OHSC) as a relevant and suitable model for investigating projection fibers in the CNS we performed perforant pathway transection (PPT) and subsequently analyzed the spatial and temporal dynamics of eCB levels. This approach allows proper distinction of responses in originating neurons (entorhinal cortex), areas of deafferentiation/anterograde axonal degeneration (dentate gyrus) and putative changes in more distant but synaptically connected subfields (cornu ammonis (CA) 1 region).
Results: Using LC-MS/MS, we measured a strong increase in arachidonoylethanolamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) levels in the denervation zone (dentate gyrus) 24 hours post lesion (hpl), whereas entorhinal cortex and CA1 region exhibited little if any changes. NAPE-PLD, responsible for biosynthesis of eCBs, was increased early, whereas FAAH, a catabolizing enzyme, was up-regulated 48hpl.
Conclusion: Neuronal damage as assessed by transection of long-range projections apparently provides a strong time-dependent and area-confined signal for de novo synthesis of eCB, presumably to restrict neuronal damage. The present data underlines the importance of activation of the eCB system in CNS pathologies and identifies a novel site-specific intrinsic regulation of eCBs after long-range projection damage.
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.
Introduction: Despite the excellent anti-inflammatory and immunosuppressive action of glucocorticoids (GCs), their use for the treatment of inflammatory bowel disease (IBD) still carries significant risks in terms of frequently occurring severe side effects, such as the impairment of intestinal tissue repair. The recently-introduced selective glucocorticoid receptor (GR) agonists (SEGRAs) offer anti-inflammatory action comparable to that of common GCs, but with a reduced side effect profile.
Methods: The in vitro effects of the non-steroidal SEGRAs Compound A (CpdA) and ZK216348, were investigated in intestinal epithelial cells and compared to those of Dexamethasone (Dex). GR translocation was shown by immunfluorescence and Western blot analysis. Trans-repressive effects were studied by means of NF-κB/p65 activity and IL-8 levels, trans-activation potency by reporter gene assay. Flow cytometry was used to assess apoptosis of cells exposed to SEGRAs. The effects on IEC-6 and HaCaT cell restitution were determined using an in vitro wound healing model, cell proliferation by BrdU assay. In addition, influences on the TGF-β- or EGF/ERK1/2/MAPK-pathway were evaluated by reporter gene assay, Western blot and qPCR analysis.
Results: Dex, CpdA and ZK216348 were found to be functional GR agonists. In terms of trans-repression, CpdA and ZK216348 effectively inhibited NF-κB activity and IL-8 secretion, but showed less trans-activation potency. Furthermore, unlike SEGRAs, Dex caused a dose-dependent inhibition of cell restitution with no effect on cell proliferation. These differences in epithelial restitution were TGF-β-independent but Dex inhibited the EGF/ERK1/2/MAPK-pathway important for intestinal epithelial wound healing by induction of MKP-1 and Annexin-1 which was not affected by CpdA or ZK216348.
Conclusion: Collectively, our results indicate that, while their anti-inflammatory activity is comparable to Dex, SEGRAs show fewer side effects with respect to wound healing. The fact that SEGRAs did not have a similar effect on cell restitution might be due to a different modulation of EGF/ERK1/2 MAPK signalling.