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The thesis entitled „Investigations on the significance of nucleo-cytoplasmic transport for the biological function of cellular proteins" aimed to unreveal molecular mechanisms in order to improve our understanding of the impact of nucleo-cytoplasmic transport on cellular functions. Within the scope of this work, it could be shown that regulated nucleo-cytoplasmic transport of a subfamily of homeobox transcription factors controlled their intra- and intercellular transport, and thereby influencing also their transcriptional activity. This study describes a novel regulatory mechanism, which could in general play an important role for the ordered differentiation of complex organisms. Besides cis-active transport Signals, also post-translational modifications can influence the localization and biological activity of proteins in trans. In addition to the known impact of phosphorylation on the transport and activity of STAT1, experimental evidence was provided demonstrating that acetylation affected the interaction of STAT1 with NF-kB p65, and subsequently modulated the expression of apoptosis-inducing NF-kB target genes. The impact of nucleo-cytoplasmic transport on the regulation of apoptosis was underlined by showing that the evolutionary conservation of a NES within the anti-apoptotic protein survivin plays an essential role for its dual function in the inhibition of apoptosis and ordered cell division. Since survivin is considered a bona fide cancer therapy target, these results strongly encourage future work to identify molecular decoys that specifically inhibit the nuclear export of survivin as novel therapeutics. In order to further dissect the regulation of nuclear transport and to efficiently identify transport inhibitors, cell-based assays are urgently required. Therefore, the cellular assay Systems developed in this work may not only serve to identify synthetic nuclear export and Import inhibitors but may also be applied in systematic RNAi-screening approaches to identify novel components of the transport machinery. In addition, the translocation based protease- and protein-interaction biosensors can be applied in various biological Systems, in particular to identify protein-protein interaction inhibitors of cancer relevant proteins. In summary, this work does not only underline the general significance of nucleo-cytoplasmic transport for cell biology, but also demonstrates its potential for the development of novel therapies against diseases like cancer and viral infections.
Suicide genes have been broadly used in gene therapy. They can serve as safety tools for conditional elimination of infused cells or for directed tumor therapy. To date, the Herpes simplex virus thymidine kinase/ ganciclovir (HSVtk/GCV) system is the most prominent and the most widely used suicidegene/prodrug combination. Despite its promising performance, the system displays limitations, which include relatively slow killing kinetics and toxicity of the prodrug GCV. Consequently, several groups have either developed new suicide-gene/prodrug combinations or attempted to improve the established HSVtk/GCV suicide system. The present study also aimed towards optimization of the HSVtk/GCV system. To do so, a novel, codon-optimized point mutant (A168H) of HSVtk was developed. The novel mutant was named TK.007. It was extensively tested for its efficiency in two relevant settings: (1) control of severe graft-versus-host disease (GvHD) after adoptive immunotherapy with Tlymphocytes, and (2) direct elimination of targeted tumor cells. TK.007 was compared to the broadly used wild-type, splice-corrected scHSVtk and to a codon-optimized HSVtk (coHSVtk) not bearing the above point mutation. (1) For experiments related to the adoptive immunotherapy approach, HSVtkvariants were expressed from a γ-retroviral MP71 vector as a fusion construct with the selection and marker gene tCD34. Expression levels for TK.007 in transduced lymphoid and myeloid cell lines were significantly higher at initial transduction and over a 12 week period compared to the commonly used scHSVtk and coHSVtk indicating reduced toxicity of TK.007. Killing kinetics of transduced cell lines (PM1 and K562) and primary human T cells were significantly faster for TK.007 in comparison to scHSVtk and coHSVtk in vitro. In vivo-functionality of TK.007 was assessed in an allogeneic transplantation model. T cells derived from C57BL/6J.Ly5.1 donor mice were transduced with MP71 vectors expressing scHSVtk or TK.007. Transduced cells were selected and transplanted into Balb/c Rag2-/- γ-/- immune-deficient recipient mice. Acute, severe GvHD occurred and was effectively abrogated in all mice transplanted with TK.007- transduced T cells, and in five out of six mice transplanted with scHSVtk-transduced cells. In a slightly modified quantitative allogeneic transplantation mouse model, significantly faster and more efficient in vivo killing was demonstrated for TK.007 as compared to scHSVtk, especially at low doses of GCV. (2) In order to assess TK.007 functionality in cells derived from solid tumors, HSVtk-variants were expressed from lentiviral gene ontology (LeGO) vectors in combination with an eGFP/neo-opt selection cassette. Transduced and selected tumor cell lines that derived from several tissues were eliminated at significantly lower GCV doses and to higher extents when transduced with TK.007 compared to scHSVtk. Moreover, a significantly stronger bystander effect of TK.007 was demonstrated. The superior in vitro efficiency of TK.007 was confirmed in an in vivo subcutaneous xenograft mouse model for glioblastoma in NOD/SCID mice. Mice transplanted with TK.007 transduced cells stayed tumor-free after treatment with different GCV-doses. On the contrary, mice of the scHSVtk group either demonstrated only transiently reduced tumor growth in the low-dose GCV group (10 mg/kg) compared to the control groups or suffered from relatively fast relapses after initial tumor shrinking in the standarddose (50 mg/kg) GCV group. As a result, all mice in the scHSVtk group died from vigorous tumor growth. In summary, in two different applications for suicide gene therapy the present study has demonstrated superior functional performance of the novel suicide gene TK.007 as compared to the broadly used wild-type scHSVtk. Differences became particularly pronounced at low doses of GCV. It can be concluded that the new TK.007-gene represents a promising alternative to the commonly used scHSVtk for gene therapeutic applications.
The endosteal bone marrow niche and vascular endothelial cells provide sanctuaries to leukemic cells. In murine chronic myeloid leukemia (CML) CD44 on leukemia cells and E-selectin on bone marrow endothelium are essential mediators for the engraftment of leukemic stem cells (LSC). We hypothesized that non-adhesion of CML-initiating cells to E-selectin on the bone marrow endothelium may lead to superior eradication of LSC in CML after treatment with imatinib than imatinib alone. Indeed, here we show that treatment with the E-selectin inhibitor GMI-1271 in combination with imatinib prolongs survival of mice with CML via decreased contact time of leukemia cells with bone marrow endothelium. Non-adhesion of BCR-ABL1+ cells leads to an increase of cell cycle progression and an increase of expression of the hematopoietic transcription factor and protooncogene Scl/Tal1 in leukemia-initiating cells (LIC). We implicate SCL/TAL1 as indirect phosphorylation target of BCR-ABL1 and as a negative transcriptional regulator of CD44 expression. We show that increased SCL/TAL1 expression is associated with improved outcome in human CML. These data demonstrate the BCR-ABL1-specific, cell-intrinsic pathways leading to altered interactions with the vascular niche via the modulation of adhesion molecules - a strategy therapeutically exploitable in future.
Acute myeloid leukemia (AML) is a clonal malignancy of hematopoietic stem cells (HSCs) characterized by expansion of myeloid blasts in the bone marrow. It has been shown that autophagy is a degradative process, which delivers cytoplasmic components to lysosomes to prevent malignant transformation by maintaining HSC integrity. Besides its function as a bulk degradation machinery to recycle cytoplasmic components during limited energy supply, autophagy also serves as an intracellular quality control mechanism. Selective autophagy requires autophagy receptors such as p62 to specifically bridge the targeted cargos into autophagosomes. p62 is known as a central signaling hub involved in pro-oncogenic signaling pathways and autophagic degradation pathways. However, little is known about the role of p62 as a selective autophagy receptor in AML. This study aims to elucidate the precise function of p62 as an autophagy receptor in leukemia development and maintenance.
In silico analysis revealed that high p62 expression was significantly associated with poor overall survival of adult patients with de novo AML, suggesting that p62 may promote leukemia maintenance. To address the functional role of p62 in leukemia, genome editing by CRISPR/Cas9 was used to knockout p62 in four human AML cell lines. Importantly, p62 loss reduced cell proliferation in all four cell lines. This observation could be transferred to a murine leukemia cell model in which leukemic transformation of lineage-depleted bone marrow (ldMBM) cells was induced by overexpression of the human transcriptional coactivator MN1. Knockdown of p62 by shRNA in MN1-driven leukemia cells impaired proliferation and decreased colony forming ability without altering apoptosis. This indicates that p62 is crucial for leukemia proliferation in vitro. To further characterize the role of p62 in leukemia development and maintenance a murine AML transplantation model was established. Therefore, ldMBM cells isolated from WT and p62-/- mice were transduced with MN1 and transplanted into lethally irradiated mice. As expected, all mice developed fatal myeloid proliferation. Notably, p62 loss in MN1-driven leukemia significantly prolonged survival in mice and caused a more immature phenotype. Consistent with the in vitro results, ex vivo analysis of p62-/- leukemic cells displayed decreased colony-forming ability, although p62 loss did not affect composition and function of HSCs. Moreover, re-transplantation of primary MN1-driven leukemia cells attenuated leukemia progression upon p62 loss. These findings support a decisive role of p62 in leukemia development and maintenance.
To gain molecular insight into the function of p62 during myeloid transformation an interactome analysis of murine MN1-driven leukemia cells was performed. This revealed first that p62 predominantly interacts with mitochondrial proteins and second that inhibition of autophagic degradation causes accumulation of p62-bound mitochondria. This leads to the first assumption that loss of p62 may provoke mitochondrial accumulation with increasing mitochondrial damage and second that p62 may mediate degradation of mitochondria by mitophagy. Indeed, in the absence of p62, accumulation of dysfunctional mitochondria was detected by morphological changes of the mitochondria, increased mitochondrial ROS and impaired mitochondrial respiration capacity. Furthermore, induction of PINK1/Parkin-independent mitophagy revealed that loss of p62 caused impaired degradation of mitochondrial proteins and reduced translocation of damaged mitochondria into autophagosomes. Taken together, p62 is required for effective degradation of dysfunctional mitochondria by mitophagy in AML.
Due to the fact that p62 is a multifunctional protein, rescue experiments with different mutants of p62 were performed to clarify if p62-mediated mitophagy contributes to leukemia proliferation. Notably, the autophagy-deficient mutant (disabled to bind autophagosomes) reduced cell growth and colony-forming ability to the same extent as knockdown of p62, as the clustering-deficient mutant (disabled to form aggregates) displayed an intermediate phenotype. Strikingly, only the autophagy-deficient mutant failed to rescue mitophagy.
In conclusion, this study demonstrates the prominent role of p62 as a selective autophagy receptor for mitochondrial quality control which contributes to leukemia development and maintenance. Therefore, targeting selective autophagy opens new venues in the treatment of AML.
Imatinib (GleevecTM; GlivecTM; formerly STI571), a specific inhibitor of Abl tyrosine kinase, is efficacious in treating Philadelphiachromosomepositive (Ph+) leukaemias such as chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL) (Ottmann, Druker et al. 2002). Within a few years of its introduction to the clinic, Imatinib had dramatically altered the firstline therapy for CML, because it was found that most newly diagnosed CML patients in the chronic phase achieve durable responses when treated with Imatinib (Goldman and Melo 2003). However, a small percentage of these patients, as well as most advancedphase CML and Ph+ ALL patients, relapse on Imatinib therapy (Yokota, Kimura et al. 2006). Several mechanisms of refractoriness and relapse have been reported. These include point mutations within the Abl kinase domain, overexpression of BcrAbl mRNA (Hofmann, Jones et al. 2002), decreased intracellular drug levels mediated by Pglycoprotein (Pgp) (Hegedus, Orfi et al. 2002), and nonBcrAbl dependent mechanisms (activation of the SFKs) (Donato, Wu et al. 2003). In this research work, a possible means of overcoming resistance to Imatinib by the use of the specific dual Src/Abl kinase inhibitor AZD0530 has been investigated. The efficacy of AZD0530 in the treatment of Ph+ leukaemias, sensitive to or resistant to Imatinib, has been tested on cell lines, primary patient material and in vivo in transduction/transplantation mouse model of Imatinib sensitive or resistant BcrAbl dependent CML-like disease. Data with AZD0530 has been compared to cells treated with Imatinib. The potential of inhibiting both Src and Abl kinases while inducing growth arrest and apoptosis has been analysed. AZD0530 specifically inhibited the growth of CML and Ph+ ALL cells in a dosedependent manner, but has shown a marginal effect on Ph- ALL cells. Treatment of p185BcrAbl expressing Ba/F3 cells with AZD0530 has led to apoptosis induction and growth inhibition in these cells, while the untransformed Ba/F3 cells have remained unaffected. Resistance to Imatinib due to mutation in the Ba/F3MutY253F cells has been overcomed by this compound. The growth inhibitory effect of AZD0530 correlates with its induction of apoptosis. Combination of AZD0530 and Imatinib at low concentrations has shown an additive effect on the inhibition of proliferation of BV173 cells. The growth inhibition and apoptosis induction by AZD0530 have shown to be uncoupled to major changes in cell cycle. An exception is the CML blast crisis cell line BV173 which has shown a considerable G0/G1 arrest in the presence of AZD0530 and Imatinib as single agents. Immunoblotting of whole cell lysates from Imatinib or AZD0530 treated BV173, Ba/F3 expressing p185(BcrAbl) MutT253F cells and the WTSupB15 cells, for Src and BcrAbl clearly demonstrates that there is an ongoing transphosphorylation taking place between the SFKs and BcrAbl. This transphosphorylation synergizes and influences the aggressive nature of CML blast crisis and Ph+ ALL. Investigations have been carried out on downstream signaling events to determine how Src family members contribute to BcrAbl signaling. Specifically, Stat, Erk and PI3K/ Akt activation status have been characterised in Imatinib sensitive and resistant Ph+ cells. AZD0530 has significantly downregulated the activation of survival signaling pathways as shown by it’s inhibition of Stat5, Akt and Erk kinases in Ph+ cells, resistant or sensitive to Imatinib. The only exception to this has been the Imatinib resistant cell line RTSupB15, in which activated Akt kinase level has remained unaffected. AZD0530 has shown to be efficient in the treatment of cells isolated from three Ph+ leukaemic patients (resistant or sensitive to Imatinib), and has led to an induction of apoptosis. Equally, in the same patients, growth and survival pathways have been inhibited in vitro in the presence of AZD0530. An overall therapeutic effect of AZD0530 in vivo has been studied in mouse model of Imatinib sensitive and Imatinib resistant, BcrAbldependent desease. Mice with a BcrAbllike disease responded to Imatinib treatment but not to AZD0530. Using the CFU assay, an influence on the differentiation status of primary leukaemic blast stem cells have been tested. The in vivo studies as well as the CFU results have shown discrepancies to the effects of AZD0530 tested so far in this research work. These discrepancies have paralleled with the upregulation of BcrAbl in most AZD0530 treated cells. These are to be further analysed. These data elucidate the role of Src kinases in BcrAbl leukaemogenesis. Results gotten from this research work has shown that AZD0530 targets both Src and BcrAbl kinase activity and reduces the transforming potential of BcrAbl. It also shows that there is an ongoing transphosphorylation between SFKs and BcrAbl kinase. AZD0530 has proven effective in CML cell lines, Ph+ ALL cell lines and patient cells resistant to Imatinib. These have demonstrated that AZD0530 is a potential drug target which can be used to overcome Imatinib resistance.
The Hepatitis C virus (HCV) infects more than 170 million individuals worldwide and causes challenging HCV-related diseases. Unfortunately, there is no vaccine available. Therefore, a better understanding of the HCV life cycle is urgently needed to develop more effective and better tolerated therapies.
It has been reported that the secretory pathway plays an essential role for the release of HCV, and the SNARE complexes are a central factor controlling intracellular vesicular trafficking. Recently, our group observed that α-taxilin that binds to free syntaxin 4 prevents the SNARE complex formation and exerts an inhibitory effect on the release of HCV particles. Therefore, it was analyzed whether the t-SNARE protein syntaxin 4 is involved in the HCV life cycle.
An increased intracellular amount of syntaxin 4 was found in HCV-positive cells, while the level of syntaxin 4-specific transcripts was decreased as observed in HCV-positive Huh7.5 cells and in HCV-infected primary human hepatocytes (PHH). Since in HCV-positive cells a significant longer half-life of syntaxin 4 was found, the decreased expression is overcompensated, leading to the elevated amount of syntaxin 4. Overexpression of syntaxin 4 increases the amount of secreted infectious viral particles, while silencing of syntaxin 4 expression decreases the number of released viral particles, which indicates that HCV could use the SNARE-dependent secretory pathway for viral release. Confocal immunofluorescence microscopy and co-immunoprecipitation experiments revealed that syntaxin 4 interacts with HCV core and NS5A. To identify the binding domain, various mutants of syntaxin 4 were generated. Based on these mutants, it was found that the H3 domain of syntaxin 4 interacts with core. These data show that the t-SNARE protein syntaxin 4 is an essential cellular factor for HCV morphogenesis and secretion.
HCV induces autophagy, and in HCV-infected cells a major fraction of the de novo synthesized viral particles is not released but intracellularly degraded. Syntaxin 17 is an autophagosomal SNARE required for the fusion of autophagosomes with lysosomes to form autolysosomes and thereby to deliver the enclosed contents for degradation. Therefore, we aim to investigate whether syntaxin 17 is a relevant factor for the HCV life cycle by regulating the fusion between autophagosomes and lysosomes. It was found that HCV-positive cells possess a decreased amount of syntaxin 17, and HCV reduces the intracellular level of syntaxin 17 by NS5A-mediated interruption of c-Raf signaling, which triggers the syntaxin 17 transcription, and by HCV-dependently induced autophagy. Overexpression of syntaxin 17 decreases the intracellular amount of viral particles and reduces the number of released infectious viral particles by favoring the formation of autolysosomes, in which HCV particles can be degraded. Vice versa, inhibition of syntaxin 17 expression by specific siRNAs results in an elevated amount of intracellular viral particles and increases the number of released viral particles by impaired autophagosome-lysosome fusion. Confocal immunofluorescence microscopy analyses show a fraction of core protein in autophagosomes as stained by lysotracker and the autophagy maker p62. These data identify syntaxin 17 as a novel factor controlling the release of HCV and reveal the autophagosome-autolysosome fusion as an essential step affecting the equilibrium between the release of infectious viral particles and lysosomal degradation of intracellular viral particles.
Taken together, these data identify the t-SNARE proteins syntaxin 4 and syntaxin 17 as essential cellular factors for HCV morphogenesis and secretion.
The majority of B-cell precursor acute leukemias in infants are associated with the chromosomal translocation t(4;11)(q21;q23), resulting in the fusion of the mixed-lineage leukemia (MLL) and ALL1-fused gene of chromosome 4 (AF4) genes. While the fusion protein MLL-AF4 is expressed in all t(4;11) patients and essential for leukemia progression, the distinct role of the reciprocal fusion protein AF4-MLL, that is expressed in only 50-80% of t(4;11) leukemia patients (Meyer et al., 2018), remains unclear. In addition, t(4;11) leukemia could so far exclusively be generated in vivo in the presence of AF4-MLL and independent of the co-expression of MLL-AF4 (Bursen et al., 2010).
In a multifactorial approach inhibiting histone deacetylases (HDACs) and expressing the dominant negative mutation of Taspase1 (dnTASP1), both MLL fusion proteins were targeted simultaneously to evaluate a possible cooperative effect between MLL-AF4 and AF4-MLL during the progression of leukemia. Of note, neither HDACi nor dnTASP1 expression negatively affect endogenous MLL, but rather endorse its function hampered by the MLL fusion proteins (Ahmad et al., 2014; Bursen et al., 2004; Zhao et al., 2019). The mere expression of dnTASP1 failed to induce apoptosis, whereas dnTASP1 could elevate apoptosis levels significantly in HDACi-treated t(4;11) cells underlining the therapeutic potential of co-inhibiting both MLL fusion proteins.
Next, the impact of inhibiting either MLL-AF4 or AF4-MLL in vivo was resolved using whole transcriptome analysis. In PDX cells obtained by the Jeremias Laboratory (Völse, 2020) that co-expressed both t(4;11) fusion proteins, the knock-down of MLL-AF4 revealed the down-regulation of pivotal hemato-malignant factors. The expression of dnTASP1 led to massive deregulation of cell-cycle genes in vivo. Considering that the inhibition of particularly MLL-AF4 but not AF4-MLL impaired leukemic cell growth in vivo (Völse, 2020), the results of this work suggest a cooperative effect between both fusion proteins, while the loss of AF4-MLL during leukemia progression appears not essential.
Thereafter, a possible short-term role of AF4-MLL during the establishment of t(4;11) leukemia was analyzed. For this purpose, an in vitro t(4;11) model was constructed to investigate the transforming potential of transiently expressed AF4-MLL in cells constitutively expressing MLL-AF4, putatively reflecting the situation in vivo. Due to the lack of a leukemic background of the applied cell line, the aim was to investigate the long-term potential of AF4-MLL to significantly alter the epigenome rather than mimicking the development of leukemia. Strikingly, short-term-expressed AF4-MLL in cooperation with MLL-AF4 exerted durable epigenetic effects on gene transcription and chromatin accessibility. The here obtained in vitro data suggest a clonal evolutionary process initiated by AF4-MLL in a cooperative manner with MLL-AF4. Importantly, no long-term changes in chromatin accessibility could be observed by the transient expression of either MLL-AF4 or AF4-MLL alone.
All in all, considering endogenous MLL, MLL-AF4 and AF4-MLL in a targeted treatment is a promising approach for a more tailored therapy against t(4;11) leukemia, and AF4-MLL is suggested to act in a cooperative manner with MLL-AF4 especially during the development of a t(4;11) leukemia.
Acute myeloid leukemia (AML) is a hematopoietic cell disorder characterized by a block in differentiation and increased proliferation and survival of malignant blasts. Expansion of the malignant cell clone effects the normal production of blood cells and – if left untreated – leads to death. Receptor tyrosine kinases (RTKs) play an important role in the pathogenesis of AML, as they are either often mutated or overexpressed. In normal hematopoiesis, RTK signal termination is tightly controlled, and involves ubiquitination, internalization, endocytosis and degradation. Cbl proteins are E3 ligases and have been shown to ubiquitinate several activated RTKs, including Flt3 and Kit, targeting them for degradation. Recently, several Cbl mutations have been identified: Cbl-R420Q was identified in an AML patient and Cbl-70Z was identified in a mouse lymphoma model. In this thesis work, the role of these Cbl mutants in Kit signaling and in a mouse transplantation model was studied. Cbl mutants (Cbl-R420Q, Cbl-70Z) have the ability to transform the myeloid 32D cell line in cooperation with Kit WT. Cbl mutants along with Kit promoted interleukin-3 (IL3)-independent proliferation and enhanced the cell survival of 32D cells. In contrast, expression of the Cbl mutants alone did not confer IL3-independent growth. Stem cell factor (SCF, the Kit ligand) dependent growth was enhanced in the presence of Cbl mutants and Cbl mutants promoted colonogenic growth in the presence of Kit. Furthermore, Cbl mutants inhibited the ubiquitination of the activated Kit receptor. In addition, Cbl mutants inhibited the endocytosis of the activated Kit receptor. Retroviral expression of Cbl mutants in transplanted bone marrow induced a generalized mastocytosis, a myeloproliferative disease and, in rare care cases, myeloid leukemia. Splenomegaly was observed in the presence of Cbl mutants. Furthermore, mast cells with variable range of infiltration were noticed in all the vital organs (spleen, liver, bone marrow, lung, kidney, heart) of Cbl (mutant) transplanted mice. Almost all recipients of bone marrow cells transduced with Cbl mutants developed a lethal hematologic disorder with a mean latency of 341 days in the Cbl-R420Q group and 395 days in the Cbl-70Z group. This is the first published report on a hematological disease with Cbl mutants in a mouse model. Co-immunoprecipitation studies indicated that Cbl-70Z binds to Kit, even in the absence of Kit ligand. Cbl-R420Q also bound to Kit in the absence of SCF, albeit to a lesser extent. Association of Cbl mutants to Kit was enhanced in the presence of SCF. Signaling studies demonstrated the constitutive activation of Akt and Erk in the presence of Cbl mutants and Kit. In addition, Cbl mutants enhanced the SCF-dependent Kit, Akt and Erk activation. Cbl-70Z, in association with kinase-dead Kit (Kit-KD) or kinase-dead Flt3 (Flt3-KD), conferred IL3-independent growth and survival to the myeloid 32D cell line. Cbl-R420Q provided only a slight growth advantage in the presence of Kit-KD. As demonstrated by pharmacological inhibition studies, Akt activation was necessary for the transformation mediated by Cbl-70Z and Kit-KD / Flt3-KD. Cbl mutants enhanced the Src family kinases (SFKs) activity. The pharmacological inhibition of SFK activity inhibited the proliferation and colonogenic growth. Interaction was found between Cbl-70Z, SFKs and Kit-KD. The SFK member Fyn was identified to bind to Cbl. In addition, kinase activity of SFKs was necessary for binding to Cbl, since SFKs inhibition by PP-2 abolished the binding between the complex-binding partners. Dasatinib and PP-2, both SFK inhibitors, inhibited the Cbl and Akt phosphorylation indicating that Fyn acts upstream of Akt. Inhibition of Kit with imatinib reduced the proliferation of cells overexpressing Kit WT and Cbl-70Z much stronger compared with cells expressing Kit-KD and Cbl-70Z, but much less than the dual KIT/SFK inhibitor dasatinib. This indicated that Kit kinase activity was required but not essential. The data presented in this thesis work implies that both RTK and SFK inhibition may have to be targeted, in order to effectively prevent transformation. In summary, the present thesis work indicates an important role of Cbl, Kit and SFKs in myeloid transformation and deregulated signal transduction.
The liver as the biggest endocrine gland of the human body plays a central role in many metabolic pathways such as detoxification, storage of carbohydrates and distribution of lipids. As the liver receives blood supply from the gut by the portal vein, liver cells are often challenged with high concentrations of nutrients and components of our commensal microbiota. Therefore, the immune system of the liver induces a tolerant state, meaning no or low inflammatory reactions to those constant stimuli. Yet, as various pathogens target the liver, the hepatic immune system also needs the capability to induce strong immune responses quickly. Chronical damage to the liver, which can be caused by alcohol, pathogens or toxins, might lead to liver cirrhosis, where the amount of functional liver tissue is decreased dramatically. This pathology can worsen and lead to acute-on-chronic liver failure, whose high mortality is due to high inflammation and multi-organ failure. Interleukin-7 is a cytokine known for its pro-survival functions especially in lymphopoiesis. However, it is also very important for maintenance of mature immune cells in the liver. As mouse experiments have demonstrated an induction of Interleukin-7 in the liver as a response to bacterial lipopolysaccharide, we aimed to characterize the role of Interleukin-7 in hepatic immunoregulation in both health and disease.
The experiments were mostly based on in vitro approaches. Induction of Interleukin-7 in liver cells was analyzed using ELISA, quantitative PCR, and Immunoblotting. Knockdown of signal transduction components was performed by siRNA transfection. Primary immune cells isolated from healthy donor buffy coat were studied for their ability to respond to Interleukin-7. Activation of downstream signal transduction was assessed by Immunoblotting. Functional consequences of Interleukin-7 signaling, such as alterations in cellular metabolism, cellular survival and endotoxin tolerance, were studied in monocyte-derived macrophages. Finally, serum concentrations of Interleukin-7 and frequencies of Interleukin-7 receptor positive immune cells were quantified in patients with compensated or decompensated liver cirrhosis or acute-on-chronic liver failure.
Interleukin-7 expression could be observed in human hepatic cell lines and primary hepatic sinusoidal endothelial cells when stimulated with IFNα or IFNγ, but not IFNλ. IRF-1 was identified as a key regulator of Interleukin-7 expression, as its transcription, translation and nuclear translocation were induced and enhanced upon IFNα or IFNγ, but not IFNλ treatment. We identified LPS-primed macrophages as innate immune target cells of Interleukin-7, which responded by an inhibitory phosphorylation of GSK3. This signal transduction led to enhanced production of pro-inflammatory cytokines and abolished endotoxin tolerance. In parallel, cellular fitness was reduced as demonstrated by reduced intracellular ATP concentration and intracellular WST-1 staining. Finally, we could identify components of the in vitro signal transduction also in liver cirrhosis patients. However, Interleukin-7 serum concentrations were significantly in liver cirrhosis patients compared to healthy controls. In addition, the frequencies of Interleukin-7 receptor positive immune cell populations differed in patients and controls.
We identify Interleukin-7 as a pro-inflammatory cytokine in hepatic immunoregulation. It is part of a cascade where its induction is regulated by type I and type II Interferons and mainly restricted by the presence of IRF1. We demonstrate the importance of Interleukin-7 also for innate immune cells, where the abolishment of endotoxin tolerance may provide an interesting strategy of liver cirrhosis patients. In addition, reduced viability of macrophages in response to Interleukin-7 is a striking contrast to the well-described survival functions in lymphocytes. The decrease of serum Interleukin-7 levels and alterations of Interleukin-7 receptor positive immune cell populations suggest an important role for Interleukin-7 also in the diseased liver. Due to the identified mechanisms of action, Interleukin-7 may be an interesting candidate for immunotherapeutic approaches of liver cirrhosis and acute-on-chronic liver failure.
Leukemia is a cancer of the blood and bone marrow characterized by an uncontrolled proliferation and accumulation of abnormal white blood cells. Leukemia can be classified based on the course of the disease (acute or chronic) and the blood cell type involved (myeloid or lymphocytic), leading to four main subtypes: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Leukemia represents 2.5% of all new cancer cases per year, and survival rates in some leukemias remain low at 40%.
The bone marrow microenvironment (BMM) is a system within the bone marrow comprising cellular and acellular components, all of which play a major role in hematopoiesis, providing the physical space where hematopoietic stem cells (HSCs) reside. The BMM interacts with HSCs, offering a “niche” for those cells and in case of leukemia, the BMM has a supportive role in disease maintenance and progression by supporting Leukemia stem cells (LSCs). One of the components of the BMM are calcium ions. Calcium is the most abundant mineral in the body, a key component of bones and is released by parathyroid hormone (PTH) induced bone remodeling. Calcium ions play a role in the localization, engraftment and adhesion of normal HSC to extracellular matrix (ECM) proteins in the BMM via the calcium sensing receptor (CaSR), thereby maintaining normal hematopoiesis. In addition of a major regulator of calcium homeostasis, CaSR contribute to the development of different cancers, functioning as either tumor suppressor or oncogene, depending on the involved tissue. However, the role of CaSR and its associated pathways in the local BMM for the development of leukemia is poorly understood. We hypothesized that calcium ions released from bone, subject to a fine balance between osteoblasts and osteoclasts, and/or CaSR, contribute to development, progression and response to therapy.
We have shown that the local calcium concentration forms a gradient in the bone marrow niche and in mice with CML is similarly low as in control mice, but significantly higher in mice suffering from BCR ABL1 driven B ALL or MLL AF9 driven AML. Similarly, the calcium concentration in the human BMM was found to be higher in AML than in other leukemias. Regarding the function of calcium in leukemia cells, we found that AML and CML cells respond differently to calcium exposure, with AML cells exhibiting regulation of cellular processes such as adhesion to the ECM protein fibronectin and migration toward CXCL 12, whereas CML cells remained mostly unaltered. Using genetic deletion or overexpression of CaSR in murine models of leukemia, we observed that CaSR acts as tumor suppressor in BCR-ABL1 driven CML and B ALL and as oncogene in AML.
Focusing on AML, our data shows that deficiency of CaSR on LICs leads, on one hand to increased apoptosis, and on the other hand to reduced cell cycle, reactive oxygen species (ROS) production and DNA damage in vivo, which may explain the observed prolongation of survival of mice. Complementary, in vitro experiments demonstrated that cells overexpressing CaSR have a distinct, cancer promoting phenotype compared to wildtype cells. Overexpression of CaSR led to an increase in proliferation, cell cycle, ROS production, DNA damage and reduced apoptosis. We have identified CaSR mediated pathways in AML and shown that CaSR enhances leukemia progression by activating MAPK/ERK and Wnt β catenin signaling. In addition, the CaSR interacting protein filamin A (FLNA) was shown to contribute to aggressive disease in vitro and in vivo. Furthermore, the mechanism underlying the role of CaSR in AML pathogenesis and possible regulation of LSCs was studied. Our findings demonstrated that CaSR ablation reduces myeloid progenitor function and proved that CaSR is required for maintenance of LSC pool by regulating its frequency and function. Further supporting the role of CaSR in LSC maintenance, genes associated with AML stemness and self renewal capacity were upregulated when CaSR was overexpressed and downregulated when CaSR was depleted. Given the role of CaSR in AML, the CaSR antagonist NPS 2143 was tested in vivo. The combination treatment of NPS 2143 with the standard of care, ara C, significantly reduced the tumor burden and prolonged the survival of mice with AML in syngeneic and xenotransplantation experiments. Based on the finding that CaSR functions as a tumor suppressor in CML, treatment of mice with the CaSR agonist cinacalcet in combination with imatinib prolonged survival of mice with CML compared to treatment with the mice given vehicle.
Our results suggest that calcium ions stemming from the calcium-rich BMM via CaSR strongly and differentially influence leukemia progression. As an adjunct to existing treatment therapies, targeting of CaSR with specific pharmacologic antagonists may prolong survival of patients with AML.
Bei ca. 95% der chronisch myeloischen Leukämie (CML) und 20-30% der akuten lymphatischen Leukämie (ALL) des Erwachsenen liegt eine reziproke Chromosomentranslokation t(9;22)(q34;q11) vor, in deren Rahmen das BCR (Breakpoint Cluster Region) Gen auf Chromosom 22 mit dem ABL (Abelson-Leukämie-Virus) Gen auf Chromosom 9 fusioniert. Auf Chromosom 22 gibt es zwei verschiedene Bruchpunkte, die somit zur Bildung von unterschiedlichen Fusionsgenen führen. Bei der CML findet man den sogenannten „großen“ Bruchpunkt (M-bcr), während bei der Ph+ ALL der sogenannte „kleine“ Bruchpunkt (m-bcr) vorkommt. Das hybride Fusionsgen auf Chromosom 22q+ (Philadelphia-Chromosom) kodiert für das jeweilige BCR/ABL Protein, während das Fusionsgen auf Chromosom 9q+ für das reziproke ABL/BCR Protein kodiert. Das ABL-Protein ist eine Nicht-Rezeptor Tyrosinkinase, die eine wichtige Rolle in der Signaltransduktion und der Regulation des Zellwachstums spielt. Im BCR/ABL Fusionsprotein wird die Kinase-Aktivität von ABL, die im Normalfall streng reguliert ist, durch die Fusion mit BCR konstitutiv aktiv. Dadurch kommt es zur Deregulierung intrazellulärer Signalwege, welche die maligne Transformation hämatopoetischer Zellen verursacht. Eine zielgerichtete Inhibierung von BCR/ABL mittels ABL-Kinase-Inhibitoren induziert Apoptose in BCR/ABL transformierten Zellen, was eine komplette Remission im größten Teil Ph+ Leukämie Patienten zur Folge hat.
Chromosomal translocations (CTs) are a genetic hallmark of cancer. They could be identified as recurrent genetic aberrations in hemato-malignancies and solid tumors. More than 40% of all "cancer genes" were identified in recurrent CTs. Most of these CTs result in the production of oncofusion proteins of which many have been studied over the past decades. They influence signaling pathways and/or alter gene expression. However, a precise mechanism for how these CTs arise and occur in a nearly identical fashion in individuals remains to be elucidated. Here, we performed experiments that explain the onset of CTs: proximity of genes able to produce prematurely terminated transcripts, which leads to the production of transspliced fusion RNAs, and finally, the induction of DNA double-strand breaks which are subsequently repaired via EJ repair pathways. Under these conditions, balanced chromosomal translocations could be specifically induced.
Cardiac progenitor cells hold great potential for regenerative therapies in heart disorders. However, the molecular mechanisms regulating cardiac progenitor cell expansion and differentiation remain poorly defined. Here we show that the multi- adaptor protein Ldb1, which mediates interactions between different classes of LIM domain transcription factors, is a multifunctional regulator of cardiac progenitor cell differentiation. Ldb1-deficient embryonic stem cells (ESCs) show a markedly decreased expression of second heart field (SHF) marker genes and subsequently impaired cardiomyocyte differentiation. Conditional ablation of Ldb1 in the early SHF using an Isl1-Cre driver led to embryonic lethality at Embryonic day (E)10.5 with cardiac abnormalities including a significantly smaller right ventricle and a shortened outflow tract, supporting a crucial role of Ldb1 in the SHF. Mechanistically we show that the importance of Ldb1 for SHF development is two-fold: On the one hand, Ldb1 binds to Isl1 and protects it from proteasomal degradation, as a consequence of which Ldb1-deficiency leads to an almost complete loss of Isl1+ cardiovascular progenitor cells. On the other hand the Isl1/Ldb1 complex promotes long-range promoter-enhancer interactions at the loci of the core cardiac transcription factors Mef2c and Hand2. Chromosome conformation capture followed by sequencing (3C- seq) identified specific Ldb1-mediated interactions of the Isl1/Ldb1 responsive Mef2c anterior heart field enhancer with genes which play key roles in cardiac progenitor cell function and cardiovascular development. These interactions are of critical importance to regulate the expression of the downstream target genes since their expression levels are strongly dependent on the Ldb1/Isl1 levels. Overexpression of an Ldb1 mutant, which contains the LIM interaction domain and thereby can protect Isl1 protein from degradation, but lacks the dimerization domain and thus cannot promote long-range interactions, does not collaborate with Isl1 to regulate the expression of their common targets and results in defects in Isl1+ cardiac progenitor differentiation. In this thesis we show one of the first examples of genome-wide chromatin reorganization mediated by a developmental regulated, cell type specific, transcription complex. Ldb1 in concert with Isl1 promotes long range promoter- enhancer and enhancer-enhancer interactions in order to create active chromatin hub where gene important for heart development can be co-regulated. Moreover, Isl1 and Ldb1 genetically interact during heart development, as Isl1/Ldb1 haplodeficient embryos show various cardiac anomalies. The dosage-sensitive interdependence between Isl1 and Ldb1 in the expression of these key factors in cardiogenesis, further supports a key role of the Isl1/Ldb1 complex in coordinating a three dimensional genome organization, upstream of a regulatory network driving cardiac differentiation and heart development.
In conclusion, the Isl1/Ldb1 complex orchestrate a genome-wide three dimensional chromatin reorganization resulting in a transcriptional program responsible for the differentiation of multipotent cardiac progenitor cells into cardiomyocytes.
Chronische Entzündungen und die daraus resultierenden Morbiditäten gehören zu den häufigsten Ursachen für einen frühen Tod beim Menschen. Einer der Hauptfaktoren für die Verschlechterung des Gesundheitszustands bei Patienten mit chronischen-entzündlichen Erkrankungen ist die pathologische Infiltration von Leukozyten in gesundes Gewebe, die zu Gewebeschäden und dem Fortschreiten der Krankheit führt. Das vaskuläre Endothel, das die Innenseite der Blutgefäße auskleidet, spielt eine entscheidende Rolle bei der Entzündungsreaktion, da es als Schnittstelle für die Interaktion mit Leukozyten fungiert, um die Extravasation von Leukozyten aus dem Blutstrom in das Gewebe zu ermöglichen. Die Adhäsion von Leukozyten an die Zellen des Endothels wird dabei hauptsächlich durch die von Zytokinen ausgelösten pro-inflammatorischen NFκB- und AP-1-Signalkaskaden ermöglicht, die die Hochregulierung der wichtigsten endothelialen Adhäsionsmoleküle – ICAM-1, VCAM-1 und E-Selektin – bewirken. Eine Klasse von Wirkstoffen, die für ihre entzündungshemmenden Eigenschaften und ihren Nutzen bei der Behandlung chronischer Entzündungskrankheiten bekannt sind, sind die Mikrotubuli-bindenden-Substanzen (microtubule-targeting-agents; MTAs), die nachweislich auch den Entzündungszustand in den Zellen des Endothels und die Leukozyten-Adhäsionskaskade beeinflussen können. MTAs lassen sich in Mikrotubuli-Destabilisatoren, die eine Depolymerisation des Mikrotubuli-Zytoskeletts bewirken, und Mikrotubuli-Stabilisatoren, die die Depolymerisation der Mikrotubuli verhindern, unterteilen. Die zugrundeliegenden biomolekularen Vorgänge und Wirkungen, die die MTAs auf die Zellen des Gefäßendothels haben, und wie sie die Adhäsionskaskade der Leukozyten beeinflussen, sind jedoch weitgehend unbekannt.
Ziel dieser Studie war es, die Auswirkungen des neuartigen Mikrotubuli-Destabilisators Prätubulysin, eines Vorläufers der Tubulysine, die ursprünglich in Stämmen des Myxobakteriums Angiococcus disciformis entdeckt wurden, auf die entzündlichen Prozesse zu untersuchen, die die Leukozyten-adhäsion in TNF-aktivierten primären Endothelzellen aus der menschlichen Nabelschnurvene (HUVECs) ermöglichen. Zusätzlich wurden auch die Auswirkungen der bereits klinisch etablierten Mikrotubuli-Destabilisatoren Colchicin und Vincristin sowie des Mikrotubuli-Stabilisators Paclitaxel untersucht.
Das entzündungshemmende Potenzial von Prätubulysin wurde daher zunächst in vivo in einem Imiquimod-induzierten psoriasiformen Dermatitis-Mausmodell getestet, wobei sich zeigte, dass Prätubulysin den Entzündungszustand deutlich verringert. Um zu beweisen, dass der entzündungshemmende Effekt mit einer verringerten Interaktion von Leukozyten mit dem Endothel zusammenhängt, wurde die Wirkung von Prätubulysin in vivo mittels Intravitalmikroskopie des TNF-aktivierten Kremaster-Muskels der Maus untersucht. Dabei zeigte sich, dass die Behandlung mit Prätubulysin zu einer signifikant verringerten Adhäsion von Leukozyten an die Zellen des Gefäßendothels führte. Die verringerte Adhäsion von Leukozyten an Endothelzellen wurde auch in der in vitro Umgebung bestätigt, indem die Adhäsion von Leukozyten unter Flussbedingungen getestet wurde. Mittels Durchflusszytometrie, Western-Blot-Analyse, sowie qRT-PCR-Analyse der jeweiligen mRNA-Level konnte gezeigt werden, dass die verringerten Leukozyten-Interaktionen auf der verringerten Expression der Zelladhäsionsmoleküle ICAM-1 und VCAM-1 sowie teilweise von E-Selektin nach Behandlung mit Prätubulysin, Vincristin und Colchicin beruhen, wobei Paclitaxel keine signifikanten hemmenden Auswirkungen hatte. Weitere Untersuchungen des Einflusses von Prätubulysin auf die NFκB- und AP-1-Signalübertragung zeigten, dass diese intrazellulären Signalkaskaden durch Prätubulysin nicht behindert werden, wobei NFκB und AP-1 weitgehend in den Promotoren der Zelladhäsionsmoleküle angereichert waren, wie durch Chromatin-Immunpräzipitation nachgewiesen wurde. Darüber hinaus induzierte die Behandlung mit Prätubulysin die Aktivität der NFκB-induzierenden Kinase IKK und führte zu einem signifikanten Anstieg der Aktivität der AP-1 Upstream-Kinase JNK, wie eine Western Blot Analyse ergab. Die Prüfung der Transkriptionsaktivität von NFκB und AP-1 in Reportergen Assays zeigte, dass insbesondere die Mikrotubuli-Destabilisatoren die Promotoraktivität dieser Transkriptionsfaktoren in einer konzentrationsabhängigen Weise verringerten. Weitere Tests zur Abhängigkeit der durch Prätubulysin induzierten Hemmung der Zelladhäsionsmoleküle von der Aktivität der JNK zeigten, dass die Hemmung empfindlich auf die Aktivität dieser Kinase reagiert. Es konnte gezeigt werden, dass die Inhibition der Aktivität der JNK die Expression der Zelladhäsionsmoleküle durch die Behandlung mit Prätubulysin auf mRNA und Proteinebene wiederherstellt. Mit Hilfe der Chromatin-Immunpräzipitation konnte weiterhin gezeigt werden, dass die Behandlung mit Prätubulysin zunächst die Assoziation des Bromodomänen-enthaltenden Proteins 4 mit den Promotoren/Genen von ICAM-1 und VCAM-1 erhöhte, aber zu einem behandlungszeitabhängigen Rückgang der Anreicherung führte. Darüber hinaus wurde durch die Behandlung mit Prätubulysin auch der Abbau dieses Proteins leicht erhöht. Durch den Einsatz eines JNK Inhibitors konnte gezeigt werden, dass die Verdrängung des Bromodomänen-enthaltenden Proteins 4 von icam-1 und vcam-1, sowie der erhöhte Abbau dieses Faktors auch von der Aktivität der JNK abhängig sind. Die Verdrängung des Bromodomänen-enthaltenden Proteins 4 induzierte auch das Vorhandensein von repressiven Chromatinmarkierungen in den Genen von ICAM-1 und VCAM-1. Die Prüfung der Anreicherung der RNA-Polymerase II an den Promotoren/Genen von ICAM-1 und VCAM-1 zeigte jedoch auch eine behandlungszeitabhängige differentielle Anreicherung dieser Polymerase, wobei die Anreicherung nach kurzen Behandlungszeiten reduziert war, sich nach mittleren Behandlungszeiten erholte und nach längeren Behandlungszeiten wieder stark reduziert war. Die anschließende Prüfung der Bedeutung des Bromodomänen-enthaltenden Proteins 4 für die Expression von ICAM-1 und VCAM-1 durch Knock-down-Experimente ergab, dass das vcam-1 Gen durch Knock-down dieses Proteins unterdrückt, das icam-1 Gen jedoch induziert wird. Dies deutet auf das Vorhandensein zusätzlicher Faktoren hin, die auch auf die Aktivität der JNK reagieren und neben dem Bromodomänen-enthaltenden Proteins 4 die Transkriptionsverlängerung des icam-1 Gens bewirken.
At the beginning of the 1980s, an increased frequency of immune deficiency was discovered in a population of homosexual men, which is nowadays known as the Acquired Immune Deficiency Syndrome (AIDS). A few years later, the retro virus Human Immunodeficiency Virus 1(HIV-1) has been discovered as the cause of AIDS. Since the beginning of the pandemic, more that 74 million people have become infected and more than 32 million people died. In 2018, it was estimated that 38 million people where living with HIV-1 of which 24.5 million had access to Highly Active Antiretroviral Therapy (HAART), which blocks viral replication and prevents the progression towards AIDS. In the most cases an HIV-1 infection leads to the patient’s death within a few years Without HAART.
Taken together, this thesis shows that hematopoietic stem and progenitor cells harbor the prerequisites and characteristics to form an HIV-1 reservoir in vivo. The subsets of HSCs, MPPs and CD34+CD38+ progenitors harbor CD4 & CXCR4 double-positive cells as well as a lower amount of CD4 & CCR5 doublepositive cells. In addition, the susceptibility to X4-tropic HIV-1 is shown in vitro. Susceptibility to R5-tropic HIV-1 is only seen to a very low amount for CD34+CD38+ progenitors. The results also show that transduced HSPCs are capable to pass on integrated viral genomes via proliferation and differentiation during in vitro colony formation. More over the experiments provide evidence that this can take place for long time span as the outcome of the replating assays shows. Ex vivo analysis of HSPCs isolated from PLHIV also suggests that these cells are susceptible to HIV-1. Proviral DNA detection using a nested PCR showed infection of Lin- cells of a single donor with an R5-tropic subtype B HIV-1 clone. However, the assay could not detect infection of CD34+ cells. The
received results of this thesis are in agreement with previously published results. Albeit the obvious susceptibility to HIV-1 and existing reports of viral survival within HSPCs for several years, the low frequency of detected in vivo infected HSPCs could be related to the cytopathic effects of HIV-1 during replication resulting in cell death of potentially infected CD34+ cells. Other reasons could be associated with assay sensitivity or the small number of available patient samples. This makes hematopoietic stem and progenitor cells a target, which can be infected by HIV-1. The role and the clinical relevance of hematopoietic stem and progenitor cells in contribution to the latent viral HIV-1 reservoir within an HIV-1 infected patient needs to be further analyzed.
The small leucine-rich proteoglycan biglycan (Bgn) is a part of the extracellular matrix providing structure and enhancing fibril stability. In its soluble form, biglycan is able to bind and signal via the innate immune receptors Toll-like receptor (TLR) 2 and 4, thereby activating MAP-kinases and the NF-κB pathway. In macrophages soluble biglycan induces the secretion of several cytokines and chemokines, including TNF-α, CCL2, CXCL5 and CXCL13. A unique feature of biglycan is its ability to stimulate the secretion of mature IL-1β. By orchestrating TLR2 and 4 with the purinergic P2X4 and P2X7 receptor signalling biglycan triggers the activation of the NLRP3/ASC inflammasome, which in turn activates caspase-1 to cleave pro-IL-1β to mature IL-1β. Furthermore, in several inflammatory diseases an upregulated biglycan expression is found. Enhanced levels of biglycan could be measured in plasma and inflamed tissue. In mouse models of sepsis, lupus nephritis and renal ischemic reperfusion injury, biglycan-deficiency improved the disease outcome. Overexpression of soluble biglycan on the other hand increased immune cell infiltration into the kidney by inducing cytokine and chemokine expression in a TLR2/4-dependent manner. These studies emphasise its importance in inflammatory processes, especially in the kidney. Furthermore, the pro-inflammatory effects on macrophages and diseases established biglycan as a danger signalling molecule, yet its role as a soluble molecule in plasma was not further investigated.
Although an increase of soluble biglycan in the circulation could be seen in several inflammatory diseases, the source is not fully unravelled. Previously it could be shown that macrophages and dendritic cells secrete soluble biglycan after stimulation with IL-6 and TGF-β1. However, since these cell are resident in organs and do not circulate in the blood stream their contribution to soluble biglycan levels in plasma is likely minor. Therefore, monocytes as precursor of both macrophages and dendritic cells were investigated as a possible source of circulating biglycan. Analysis of blood from septic patients revealed elevated soluble biglycan levels as well as an increased number of monocytes. Isolated monocytes from healthy volunteers incubated with the inflammatory cytokines IL-1β, IL-6 and TGF-β1 displayed increased biglycan mRNA expression and secretion of soluble biglycan into the supernatant, revealing monocytes as a producer of soluble biglycan in blood. Therefore this work was directed to further investigate the influence of soluble biglycan on circulating monocytes, with regard to sepsis.
Monocytes can be classified into three subtypes, while the classical monocytes express CD14 (CD14++CD16low), intermediate monocytes express both CD14 and CD16 (CD14++CD16+) and non-classical monocytes express mainly CD16 (CD14lowCD16++). The intermediate and non-classical monocytes make up about 10 % of all monocytes and are referred to as CD16-positive subtypes. The CD16-positive monocytes express higher levels of TNF-α and IL-1β upon stimulation and display different migration behaviour. In most inflammatory diseases an expansion of CD16-positive monocytes is observed, especially an increased number of intermediate monocytes frequently correlate with disease severity and mortality. Since septic patients had increased circulating biglycan levels and augmented CD16-positive monocytes, a possible correlation between these two parameters was investigated. Using FACS analysis of biglycan-stimulated monocytes from healthy donors revealed a significant shift from classical to intermediate and non-classical monocytes. This shift was mediated by increased expression of CD14 and CD16 on mRNA and protein levels upon biglycan treatment. Furthermore, biglycan induced the mRNA expression of the adhesion molecules ICAM-1, VCAM-1 and ELAM-1 in CD14-positive monocytes. Four hours after biglycan stimulation an increased ICAM-1 protein expression on the cell surface of classical and intermediate monocytes was observed. Additionally, biglycan-treated CD14-positive monocytes rolled and attached to pre-stimulated endothelial cells to a greater extent compared to untreated monocytes. This demonstrates that biglycan not only triggers the expression of CD14 and CD16 but also induces a functional shift of monocytes. ...
Schätzungen zufolge sind weltweit etwa 71 Millionen Menschen chronisch mit dem Hepatitis-C-Virus (HCV) infiziert. Im Jahre 2016 sind rund 400.000 Menschen an einer HCV-bedingten Lebererkrankung gestorben, insbesondere aufgrund der Entwicklung von Leberzirrhose und Lebertumoren. Trotz der großen Unterschiede in den Prävalenzschätzungen und der Qualität der epidemiologischen Daten zeigt die jüngste weltweite Bewertung, dass die virämische Ausbreitung der HCV-Infektion (Prävalenz der HCV-RNA) in den meisten Industrieländern, einschließlich der USA, weniger als 1,0% beträgt (www .cdc.gov / Hepatitis / HCV). In einigen osteuropäischen Ländern wie Lettland (2,2%) oder Russland (3,3%) und bestimmten Ländern in Afrika, Ägypten (6,3%) und Gabun (7,0%) oder im Nahen Osten Syriens (3,0%) ist die Prävalenz bemerkenswert höher. In den USA und den am weitesten entwickelten Ländern gilt die gemeinsame Nutzung von Werkzeugezur Herstellung von Arzneimitteln und zur Injektion von Medikamenten (Nadeln) als die häufigste derzeitige Übertragungsart. Die vorherrschende Übertragungsart in Ländern, in denen die Ausbreitung von HCV-Infektionen im Vergleich zu den Industrieländern höher ist, beruht jedoch auf schlechten Methoden zur Infektionskontrolle und unsicherer Handhabung von Injektionsnadeln.
Wenn die chronische Infektion unbehandelt bleibt, kann sich im fortschreitenden Verlauf eine Zirrhose oder ein hepatozelluläres Karzinom bilden (Alter H. J. und Seef L. B. 2000). Die Doppeltherapie, bei der es sich um eine Kombination aus pegyliertem Interferon-α (PEG IFNα) und Ribavirin (riba) handelt, war in einigen Ländern der Dritten Welt bis vor kurzem der goldene Standard für die Behandlung von Patienten mit chronischer Hepatitis C und hat eine anhaltende virologische Reaktion erzielt. Mit nur 50% der mit HCV-Genotyp 1 infizierten Patienten (der häufigere) im Vergleich zu 80% mit Genotyp 2 oder 3, obwohl sie kostspielig und langwierig sind (z. B. 24-48 Wochen) und zahlreiche harte Nebenwirkungen aufweisen, die schwer zu bekämpfen sind tolerieren (Erklärung der National Institutes of Health Consensus Development Conference: Management von Hepatitis C: 2002 - 10.-12. Juni 2002 2002). Die Identifizierung des JFH1 (japanische fulminante Hepatitis Typ 1) -Isolats wurde in einigen in vitro-Studien zu HCV als wichtiger Durchbruch bei der HCV-Behandlung angesehen. Die Verwendung dieses Isolats führte nachfolgend zu einem besseren Verständnis des HCV-Lebenszyklus und der 3D-Strukturen der viralen Proteine. Basierend auf dieser Erkenntnis konnten die ersten direkt wirkenden antiviralen Mittel (DAAs) entwickelt werden, die spezifisch virale Proteine beeinflussen. Die beiden Proteasehemmer (PI) Telaprevir und Boceprevir hemmen die virale NS3-4A-Protease und wurden 2011 als Kombinationstherapie mit PEG IFNα und Ribavirin zugelassen, was die anhaltende virologische Reaktion auf 67-75% erhöhte (Pawlotsky et al. 2015).
Die Optimierung der gegenwärtigen Arzneimittelregime, die Einschränkung des Problems der Mutationsresistenz, die Gestaltung einer individualisierten Therapie, der Zugang zu diesen therapeutischen antiviralen Arzneimitteln und ihr hoher Preis bleiben weiterhin eine Herausforderung (Pawlotsky 2016; Pawlotsky et al. 2015; Sarrazin 2016). Die Entwicklung eines Impfstoffs wird jedoch als größte Herausforderung für die weltweite Kontrolle von HCV angesehen (Bukh 2016). Aus diesem Grund ist es wichtig, weiterhin mehr über den HCV-Lebenszyklus und die Faktoren zu erfahren, die sich auf die Replikation und den gesamten Lebenszyklus auswirken können, um effiziente, qualitativ hochwertige und vor allem leicht zugängliche Behandlungen für alle Menschen weltweit zu entwickeln.
Der Lipidstoffwechsel und insbesondere das Cholesteringleichgewicht werden durch die HCV-Infektion beeinflusst. Die Korrelation zwischen Lipidstoffwechsel und HCV wurde klinisch seit langem beobachtet. In den Leberbiopsien von mit HCV infizierten Patienten wurde ein Anstieg der in den Lipidtröpfchen im Cytosol akkumulierten neutralen Lipide festgestellt (Dienes et al. 1982). Das Hepatitis-C-Virus wurde auch von Hypobetalipoproteinämie, Hypocholesterinämie und Lebersteatose begleitet (Schaefer und Chung 2013). Die Leber ist der primäre Ort für die Synthese, Speicherung und Oxidation von Lipiden und anderen Makromolekülen. Daher ist der Fettstoffwechsel in der Leber für die Aufrechterhaltung der systemischen Nährstoffhomöostase von wesentlicher Bedeutung. Eine Dysregulation des Leberlipidstoffwechsels ist ein Kennzeichen mehrerer Krankheiten wie Diabetes, alkoholische und nichtalkoholische Fettlebererkrankungen sowie parasitäre und virale Infektionen, einschließlich einer HCV-Infektion. (Erklärung der National Institutes of Health Consensus Development Conference: Management von Hepatitis C: 2002 - 10.-12. Juni 2002 2002; Fon Tacer und Rozman 2011; Chen et al. 2013; Reddy und Rao 2006; Visser et al. 2013; Wu und Parhofer 2014)
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Chronic inflammation is considered to be a cause of the autoimmune diseases such as rheumatoid arthritis, Alzheimer’s disease, multiple sclerosis, etc. The search for effective compounds with anti-inflammatory properties to combat these diseases is still ongoing. Natural compound narciclasine, derived from plants of Narcissus species, demonstrated its anti-inflammatory activity in in vivo arthritis models. Further investigation of narciclasine’s anti-inflammatory activity together with its impact on the interaction between leukocytes and endothelial cells was the main focus of this PhD thesis.
Narciclasine reduced the infiltration of monocytes and neutrophils to the abdomen and the concentration of the pro-inflammatory cytokines TNF, IL-6 and IL-1β. Together with this, it reduced acute visceral pain caused by zymosan injection. Narciclasine interfered with leukocyte-endothelial cell interaction in both in vivo and in vitro models. In vivo microscopy revealed that the compound reduced rolling, adhesion and transmigration of leukocytes in the vessels of an injured murine cremaster muscle. This observation was confirmed in the in vitro models for adhesion and transmigration where narciclasine reduced the level of leukocyte’s interaction with HUVECs. Narciclasine demonstrated profound anti-inflammatory properties based on its interference with leukocyte-endothelium interaction by downregulation of endothelial cell adhesion molecules expression (ICAM-1, VCAM-1, E-selectin, CX3CL1) and shutdown of NF-κB pathway. All these effects were a result of the TNF receptor 1 protein translation blocking by narciclasine.
In this work the ability of the compound to reduce visceral pain, downregulate the expression of the endothelial cell adhesion molecules and to interfere with the interaction between leukocytes and endothelial cells was demonstrated for narciclasine for the first time. Obtained results open a promising insight into the understanding of narciclasine’s anti-inflammatory properties and justify further investigation of its potential for treatment of inflammatory diseases.
B-cell acute lymphoblastic leukaemia (B-ALL) is characterized by the overproduction of lymphoblasts in the bone marrow (BM), and it is the most common cancer in children while being comparatively uncommon in adults. On the other hand, in chronic myeloid leukaemia (CML), 70% of cases are found in patients older than 50 years, making it uncommon in children. All CML cases and up to 3% of paediatric B- ALL (and 25% of adult B-ALL) cases are due to fusion gene BCR-ABL1, which gives rise to the cytoplasmatic, constitutively active oncoprotein, tyrosine kinase BCR-ABL1 through a reciprocal translocation between chromosomes 9 and 22. The constitutively active BCR-ABL tyrosine kinase leads to deregulation of different signal transduction pathways such as cell growth, proliferation and cell survival. The role of the bone marrow microenvironment (BMM) can mediate disease initiation (only in mice), progression, therapy resistance, and relapse, as has been increasingly recognized over the last two decades. In general, the BMM is a very complex arrangement of various cell types such as osteoblasts, osteoclasts, endothelial cells, adipocytes, mesenchymal stromal cells, macrophages and several others. In addition, the BMM is composed of multiple chemical and mechanical factors and extra cellular matrix (ECM) proteins which contribute to the BMM’s features influencing leukaemia behaviour. Considering the incidence of B-ALL and CML in children and in adults respectively, we hypothesized that the young and/or an aged BMM might also play a previously unrecognized role in the aggressiveness of B-ALL and CML. We proposed that BM, transduced with BCR-ABL1-expressing retrovirus in the murine transduction/transplantation model of B-ALL, transplanted into young versus old recipient mice would lead to a more aggressive disease in young mice, and similarly CML would be more aggressive in old recipient mice. In close recapitulation with the human incidence, induction of CML led to a significantly shorted survival in old recipient mice. On the other hand, induction of B-ALL showed a shortened survival in young compared to old syngeneic mice, as well as in a xenotransplantation model. Among the highly heterogenous composition of the BMM, we implicate young BM macrophages as a supportive niche for B-ALL cells. The results were found to be mostly due to potential soluble factors differentially secreted from young and old macrophages. Therefore, we hypothesized that the chemokine CXCL13, which has been demonstrated to play a role in B cell migration and act as a diagnostic marker in the cerebrospinal fluid of patients with neuroborreliosis, might be responsible for the observed phenotype. CXCL13 was found to be more highly expressed in healthy and leukaemic young mice as well as in conditioned medium of young macrophages. Using a variety of in vitro experiments, CXCL13 showed to significantly increase the proliferation and the migration of leukaemia cells when exposed to young macrophages, and the phenotype was rescued while using a CXCL13 neutralizing antibody. The CXCL13 role was also confirmed in vivo, since macrophage ablation led to a prolongation of survival in young mice and a reduction of CXCL13 levels. The use of an additional mouse model, leukaemia cells with CXCR5 deficiency, led to a significant prolongation of survival of young mice, confirming the importance of the CXCL13-CXCR5 axis in B-ALL. In line with our murine results, we found that human macrophages and CXCL13 levels were higher in pediatric B-ALL patients than in adults. Consistent with our murine data, the expression level of CXCR5 may act as a prognostic marker in B-ALL, as well as a predictive marker for central nervous system relapse in human B-ALL. The overall findings show that a young BMM, and in particular macrophages, influences B-ALL progression. We specifically identified CXCL13, secreted by young macrophages, as a promoter of proliferation of B-ALL cells, influencing survival in B-ALL via CXCR5. The CXCR5-CXCL13 axis may be relevant in human B-ALL, and higher CXCR5 expression in human B-ALL may act as a predictive marker.
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.