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Jürgen Knobloch

Dr. Jürgen Knobloch
University of Cologne
Medical Clinic III
Department of Pneumology
Kerpener Strasse 62
D-50924 Cologne
Germany
phone: +49-221-4784191
juergen.knobloch@uk-koeln.de


Since 04/2007
Laboratory Manager
Medical Clinic III, Department of Pneumology, University of Cologne (Germany)
Head: A. Koch
Research: The molecular pathology of airway diseases


Postdoctoral Positions:

04/2003 - 03/2007
Institute for Animal Developmental and Molecular Biology (EMT), University of Düsseldorf (Germany)
Head: U. Rüther
Research: The molecular basis for thalidomide-induced birth defects
Awarded with the Ulrich-Hadding research prize 2006

10/2002 - 03/2003
Institute for Genetics, Department of Genetic Parasitology II, University of Düsseldorf (Germany)
Head: C. G. Grevelding
Research: Characterization of medically relevant signaling pathways of the parasitic and human-pathogenic trematode Schistosoma mansoni


PhD Thesis:

01/1999 - 09/2002
Institute for Genetics, Department of Genetic Parasitology I, University of Düsseldorf (Germany)
Head: W. Kunz
Project: Localization and inhibition of three protein tyrosine kinases and the isolation of a binding partner for the Src-like tyrosine kinase SmTK3 of Schistosoma
Awarded with a scholarship of the DÃœSSELDORF ENTREPRENEURS FOUNDATION

05/2001 - 10/2001
Institute de Pasteur, (Lille, France)
Project: Construction of a yeast-two-hybrid-library of Schistosoma


University Education:

10/1993 - 10/1998
Study of biology at the University of Düsseldorf (Germany)

Journal articles

2012
Zeynep Mat, Barbara Grensemann, Yakup Yakin, Jürgen Knobloch, Andrea Koch (2012)  Effect of lipoteichoic acid on IL-2 and IL-5 release from T lymphocytes in asthma and COPD.   Int Immunopharmacol 13: 3. 284-291 Jul  
Abstract: Susceptibility to infections with gram-positive bacteria, which are an important trigger of exacerbations, is increased in COPD and asthma. Unraveling the underlying mechanisms may help developing therapeutic strategies to reduce exacerbation rates. The aim of this study was to evaluate the effects of lipoteichoic acid (LTA), a danger signal from gram-positive bacteria, on T cell cytokines related to bacterial infection defense in COPD and asthma. T cell populations within peripheral blood mononuclear cells (PBMCs) were ex-vivo activated towards T(H)2/T(C)2 subtypes and subsequently stimulated with LTA. IL-2 and IL-5 concentrations in cell culture supernatants were measured by ELISA comparative between non-smokers (NS), current smokers without airflow limitation (S), smokers with moderate to severe COPD and mild to moderate asthmatics (A) (each n=10). IL-2 and IL-5 baseline levels were without differences between the cohorts. After T cell activation, IL-2 and IL-5 releases were increased in all cohorts, however, for IL-2 this increase was significantly higher in S and by trend in COPD compared to the other groups. LTA time-dependently suppressed IL-2 release in NS, S and COPD but not in A. LTA reduced IL-5 release in COPD and A but not in NS and S. Summarized, LTA reduces T(H)2/T(C)2 cytokines indicating immunosuppressive effects, which are dysregulated in COPD and asthma. This implies a misguided response to gram-positive bacterial infections, which might help to explain the increased susceptibility to bacterial infections in COPD and asthma.
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2011
Jürgen Knobloch, David Jungck, Andrea Koch (2011)  Apoptosis Induction by Thalidomide: Critical for Limb Teratogenicity but Therapeutic Potential in Idiopathic Pulmonary Fibrosis?   Curr Mol Pharmacol 4: 26-61 Oct  
Abstract: Thalidomide is a powerful treatment for inflammatory and cancer-based diseases. However, its clinical use remains limited due to its teratogenic properties, which primarily affect limb development. A prerequisite for overcoming these limitations is to understand the cellular and molecular mechanisms underlying thalidomide teratogenicity, which involve induction of oxidative stress, suppression of ubiquitin-mediated protein degradation and disruption of angiogenesis. Here, we discuss the hypothesis that thalidomide-induced limb teratogenicity is primarily based on the generation of nuclear oxidative stress with subsequent induction of transient apoptosis in the outgrowing limb bud. To this end, we establish a model of the signaling network regulating cell proliferation, survival and endogenous apoptosis-induction required for correct limb outgrowth and patterning. We then summarize data showing how thalidomide interferes with this signaling network: thalidomide inhibits the activity of the redox-sensitive transcription factor NF-kB, shifts the balance of fibroblast growth factors and bone morphogenetic proteins (Bmps) towards pro-apoptotic Bmps, and suppresses Wnt/b-catenin- and Akt-dependent survival signaling in the limb bud. Consequently, prechondrogenic precursor cells that determine skeletal elements are eliminated leading to the development of truncated limbs. We further discuss the involvement of thalidomide effects on ubiquitin-mediated protein degradation and angiogenesis in the induction of apoptosis in the limb bud. Finally, we discuss the paradox that the embryonic molecular pathology induced by thalidomide suggests this drug as a candidate for therapeutic application in idiopathic pulmonary fibrosis (IPF), a chronic and fatal lung disease characterized by downregulation of Bmp signaling, increased Wnt and Akt activity, and apoptosis resistance.
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Katharina Schild, Jürgen Knobloch, Yakup Yakin, David Jungck, Katja Urban, Katja Müller, Andrea Koch (2011)  IL-5 release of CD4(+) non-effector lymphocytes is increased in COPD - modulating effects of moxifloxacin and dexamethasone.   Int Immunopharmacol 11: 4. 444-448 Apr  
Abstract: T-lymphocytes are crucial in chronic obstructive pulmonary disease (COPD) pathogenesis. Especially T(H)1-lymphocytes are involved in local and systemic inflammation in COPD, yet the role of T(H)2-mediated immune-responses in COPD pathogenesis is poorly understood. The objective of this study was to examine IL-5 expression in T(H)2-lymphocytes in smokers with and without COPD ex vivo compared with non-smokers and to evaluate the effects of bacterial endotoxin (lipopolysaccharide, LPS) as well as two drugs often used for treatment of COPD exacerbation, corticosteroids and moxifloxacin. CD4(+) lymphocytes were isolated from the peripheral blood of non-smokers (NS; n=11), current smokers without airflow limitation (S; n=11) and smokers with COPD (n=11). Baseline IL-5 release of CD4(+) T-lymphocytes was significantly increased in COPD compared to S and NS. After T-cell activation and differentiation into T(H)2-lymphocytes, IL-5 release increased without differences between the cohorts. LPS reduced IL-5 release of ex vivo generated T(H)2-lymphocytes without differences in all cohorts. Moxifloxacin and dexamethasone significantly reduced IL-5 release in T(H)2-lymphocytes in the absence and presence of LPS without differences between groups. In summary, our data indicate that IL-5 might contribute to systemic inflammation in smokers with COPD and that T(H)2-based immune responses might be suppressed in response to gram-negative bacterial infections independent from smoking and disease status. Dexamethasone and moxifloxacin both have T(H)2-immunmodulating effects.
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Jürgen Knobloch, Katharina Schild, David Jungck, Katja Urban, Katja Müller, Elke K H Schweda, Jan Rupp, Andrea Koch (2011)  The T-Helper Cell Type 1-Immune Response to Gram-Negative Bacterial Infections is Impaired in COPD.   Am J Respir Crit Care Med 183: 204-214 Aug  
Abstract: RATIONALE: The increased susceptibility to bacterial infections in chronic obstructive pulmonary disease (COPD) is critical for exacerbations. Toll-like receptor-4 (TLR4) detects bacteria via lipopolysaccharide (LPS) and induces interferon gamma- (IFNgamma-) based immune responses. The direct responsiveness of TH1-lymphocytes to LPS is disputed as they lack surface expression of the TLR4 co-receptor CD14. OBJECTIVE: We hypothesized that the TH1-mediated adaptive immune response to bacterial infections is impaired in COPD. METHODS: LPS-induced TLR4-expression and IFNgamma-release in/from ex vivo-generated TH1-cells was compared between non-smokers (n=14), smokers without (n=13) and with COPD (n=25) via quantitative reverse transcription polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. TLR4 transfection experiments were performed to functionally link receptor- to IFNgamma-dysregulation in COPD. MEASUREMENTS AND MAIN RESULTS: Short-chain LPS from Salmonella species and nontypeable Haemophilus influenzae (NTHi) and NTHi whole-cell extract all induced TLR4-expression via TLR4/MyD88/IRAK/MAP-kinase signaling and IFNgamma-release via TLR4/TRIF/IKKepsilon/TBK1 signaling in TH1-cells of non-smokers. These effects were all impaired in smokers with and without COPD. The LPS-responses were partially dependent on soluble CD14 and correlated positively to lung-function parameters but negatively to cigarette smoking (pack years). Endogenous MyD88/IRAK signaling antagonists were up-regulated in TH1-cells of smokers and COPD, and TLR4 over-expression in TH1-cells of COPD restored LPS-dependent IFNgamma-release. CONCLUSION: TH1-cells directly respond to short-chain LPS. Cigarette smoking suppresses TH1-mediated immune responses to gram-negative bacterial infections by interfering with MyD88/IRAK signalling thereby reducing LPS-induced TLR4-expression. This can explain the increased susceptibility to bacterial infections in COPD. Targeting TLR-signalling might be useful to reduce exacerbation rates.
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Jürgen Knobloch, Haitham Hag, David Jungck, Katja Urban, Andrea Koch (2011)  Resveratrol Impairs the Release of Steroid-resistant Cytokines from Bacterial Endotoxin-Exposed Alveolar Macrophages in COPD.   Basic Clin Pharmacol Toxicol Mar  
Abstract:   Airway inflammation in chronic obstructive pulmonary disease (COPD) is believed to be insensitive to corticosteroids. However, corticosteroids are recommended in COPD (GOLD stages III, IV) with frequent exacerbations. Resveratrol has anti-inflammatory properties and could be an alternative to corticosteroids in COPD therapy. We investigated the effect of dexamethasone versus resveratrol on the release of COPD-related inflammatory mediators (IL-6, IL-8, GM-CSF, MCP-1) and matrix-metalloprotease-9 (MMP-9) from alveolar macrophages exposed to gram-negative bacterial endotoxin (lipopolysaccharide, LPS). We compared never-smokers (NS), current smokers without airway obstruction (S) and current smokers with COPD. The cytokines and MMP-9 were measured in cell culture supernatants with ELISA. The release of IL-8 and MMP-9 from LPS-exposed alveolar macrophages was increased in COPD, the release of GM-CSF and IL-6 was decreased in COPD and the release of MCP-1 was without differences between the cohorts. Dexamethasone impaired the release of all cytokines and MMP-9 from LPS-exposed alveolar macrophages of all cohorts, but for IL-8 and GM-CSF this effect was reduced in COPD. In alveolar macrophages of COPD, there was an almost complete reduction of IL-6 release but only a partial reduction of IL-8, GM-CSF, MCP-1 and MMP-9 release demonstrating a partial corticosteroid-insensitivity. In contrast, resveratrol almost completely reduced the release of all cytokines and MMP-9 without significant differences between the cohorts. Our data provide evidence for a corticosteroid resistance of alveolar macrophage-dependent inflammatory responses induced by gram-negative bacteria in COPD and thus question the utility of corticosteroids in COPD therapy. Instead, resveratrol may prove an alternative.
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2010
Jürgen Knobloch, Bernhard Sibbing, David Jungck, Yingfeng Lin, Katja Urban, Erich Stoelben, Justus Strauch, Andrea Koch (2010)  Resveratrol impairs the release of steroid-resistant inflammatory cytokines from human airway smooth muscle cells in chronic obstructive pulmonary disease.   J Pharmacol Exp Ther 335: 3. 788-798 Dec  
Abstract: Chronic obstructive pulmonary disease (COPD) therapy is complicated by corticosteroid resistance of the interleukin 8 (IL-8)-dependent and granulocyte macrophage-colony stimulating factor (GM-CSF)-dependent chronic airway inflammation, for whose establishment human airway smooth muscle cells (HASMCs) might be crucial. It is unclear whether the release of inflammatory mediators from HASMCs is modulated by cigarette smoking and is refractory to corticosteroids in COPD. Resveratrol, an antiaging drug with protective effects against lung cancer, might be an alternative to corticosteroids in COPD therapy. Vascular endothelial growth factor (VEGF) might offer protection from developing emphysema. We tested the following hypotheses for HASMCs: 1) smoking with or without airway obstruction modulates IL-8, GM-CSF, and VEGF release; and 2) corticosteroids, but not resveratrol, fail to inhibit cytokine release in COPD. Cytokine release from HASMCs exposed to tumor necrosis factor α (TNFα), dexamethasone, and/or resveratrol was measured via enzyme-linked immunosorbent assay and compared between nonsmokers (NS), smokers without COPD (S), and smokers with COPD (all n = 10). In response to TNFα, IL-8 release was increased, but GM-CSF and VEGF release was decreased in S and COPD compared with NS. Dexamethasone and resveratrol inhibited concentration-dependently TNFα-induced IL-8, GM-CSF, and VEGF release. For IL-8 and GM-CSF efficiency of dexamethasone was NS > S > COPD. That of resveratrol was NS = S = COPD for IL-8 and NS = S < COPD for GM-CSF. For VEGF the efficiency of dexamethasone was NS = S = COPD, and that of resveratrol was NS = S > COPD. All resveratrol effects were partially based on p38 mitogen-activated protein kinase blockade. In conclusion, smoking modulates cytokine release from HASMCs. Corticosteroid refractoriness of HASMCs in COPD is cytokine-dependent. Resveratrol might be superior to corticosteroids in COPD therapy, because it more efficiently reduces the release of inflammatory mediators and has limited effects on VEGF in COPD.
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Oliver Lieven, Jürgen Knobloch, Ulrich Rüther (2010)  The regulation of Dkk1 expression during embryonic development.   Dev Biol 340: 2. 256-268 Apr  
Abstract: During embryogenesis, the Dkk1 mediated Wnt inhibition controls the spatiotemporal dynamics of cell fate determination, cell differentiation and cell death. Furthermore, the Dkk1 dose is critical for the normal Wnt homeostasis, as alteration of the Dkk1 activity is associated with various diseases. We investigated the regulation of Dkk1 expression during embryonic development. We identified nine conserved non-coding elements (CNEs), located 3' to the Dkk1 locus. Analyses of the regulatory potential revealed that four of these CNEs in combination drive reporter expression very similar to Dkk1 expression in several organs of transgenic embryos. We extended the knowledge of Dkk1 expression during hypophysis, external genitalia and kidney development, suggesting so far to unexplored functions of Dkk1 during the development of these organs. Characterization of the regulatory potential of four individual CNEs revealed that each of these promotes Dkk1 expression in brain and kidney. In combination, two enhancers are responsible for expression in the pituitary and the genital tubercle. Furthermore, individual CNEs mediates craniofacial, optic cup and limb specific Dkk1 regulation. Our study substantially improves the knowledge of Dkk1 regulation during embryonic development and thus might be of high relevance for therapeutic approaches.
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2009
Jürgen Knobloch, Helena Peters, David Jungck, Katja Müller, Justus Strauch, Andrea Koch (2009)  TNFalpha-induced GM-CSF release from human airway smooth muscle cells depends on activation of an ET-1 autoregulatory positive feedback mechanism.   Thorax 64: 12. 1044-1052 Dec  
Abstract: BACKGROUND: There is an urgent need to inhibit endothelin-1 (ET-1) induced chronic inflammatory processes in early stages of lung diseases in order to prevent untreatable irreversible stages often accompanied by lung fibrosis and pulmonary hypertension. Nothing is known about the airway inflammation-inducing and/or maintaining role of ET-1 in human airway smooth muscle cells (HASMCs). OBJECTIVE: ET-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in response to tumour necrosis factor alpha (TNFalpha) and ET-1 stimulation was investigated, and the impact of mitogen-activated protein kinase (MAPK) pathways in this context was studied. To elucidate the anti-inflammatory properties of the dual endothelin receptor antagonist bosentan that targets both endothelin receptor subtypes A (ET(A)R) and B (ET(B)R), its effect on the TNFalpha/ET-1/GM-CSF network was investigated. METHODS: ET-1 and GM-CSF expression and activation of MAPKs were investigated via quantitative reverse transcription-PCR (RT-PCR), western blotting and ELISA. MAIN RESULTS: Both TNFalpha and ET-1 activated p38(MAPK) and extracellular signal-regulated kinase (ERK)-1/-2 signalling. ET-1 expression was induced by TNFalpha and by ET-1 itself. Both effects were inhibited by bosentan and by specific ET(A)R or p38(MAPK) blockade. ET-1- and TNFalpha-induced GM-CSF expression were both reduced by bosentan as well as by specific inhibition of either ET(A)R, ET(B)R, p38(MAPK) or ERK-1/-2. CONCLUSION: TNFalpha activates an ET(A)R- and p38(MAPK)-dependent ET-1 autoregulatory positive feedback loop to maintain GM-CSF release from HASMCs. Since bosentan impairs ET-1 autoregulation and TNFalpha-induced ET-1 release, as well as TNFalpha- and ET-1-induced GM-CSF release, the present data suggest therapeutic utility for bosentan in treating particularly the early stages of chronic inflammatory airway diseases.
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Thomas Quack, Jürgen Knobloch, Svenja Beckmann, Jérome Vicogne, Colette Dissous, Christoph G Grevelding (2009)  The formin-homology protein SmDia interacts with the Src kinase SmTK and the GTPase SmRho1 in the gonads of Schistosoma mansoni.   PLoS One 4: 9. 09  
Abstract: BACKGROUND: Schistosomiasis (bilharzia) is a parasitic disease of worldwide significance affecting human and animals. As schistosome eggs are responsible for pathogenesis, the understanding of processes controlling gonad development might open new perspectives for intervention. The Src-like tyrosine-kinase SmTK3 of Schistosoma mansoni is expressed in the gonads, and its pharmacological inhibition reduces mitogenic activity and egg production in paired females in vitro. Since Src kinases are important signal transduction proteins it is of interest to unravel the signaling cascades SmTK3 is involved in to understand its cellular role in the gonads. METHODOLOGY AND RESULTS: Towards this end we established and screened a yeast two-hybrid (Y2H) cDNA library of adult S. mansoni with a bait construct encoding the SH3 (src homology) domain and unique site of SmTK3. Among the binding partners found was a diaphanous homolog (SmDia), which was characterized further. SmDia is a single-copy gene transcribed throughout development with a bias towards male transcription. Its deduced amino acid sequence reveals all diaphanous-characteristic functional domains. Binding studies with truncated SmDia clones identified SmTK3 interaction sites demonstrating that maximal binding efficiency depends on the N-terminal part of the FH1 (formin homology) domain and the inter-domain region of SmDia located upstream of FH1 in combination with the unique site and the SH3 domain of SmTK3, respectively. SmDia also directly interacted with the GTPase SmRho1 of S. mansoni. In situ hybridization experiments finally demonstrated that SmDia, SmRho1, and SmTK3 are transcribed in the gonads of both genders. CONCLUSION: These data provide first evidence for the existence of two cooperating pathways involving Rho and Src that bridge at SmDia probably organizing cytoskeletal events in the reproductive organs of a parasite, and beyond that in gonads of eukaryotes. Furthermore, the FH1 and inter domain region of SmDia have been discovered as binding sites for the SH3 and unique site domains of SmTK3, respectively.
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2008
Jürgen Knobloch, Kerstin Reimann, Lars-Oliver Klotz, Ulrich Rüther (2008)  Thalidomide resistance is based on the capacity of the glutathione-dependent antioxidant defense.   Mol Pharm 5: 6. 1138-1144 Nov/Dec  
Abstract: Thalidomide as an effective treatment for multiple myeloma and leprosy has also caused birth defects in thousands of children five decades ago particularly in Europe. Thus its use in humans remains limited. The rapid and fatal approval of thalidomide at that time ultimately was a consequence of the sole use of thalidomide-insensitive species in animal toxicity tests. Here, we aimed at elucidating the molecular basis for the resistance of mice to thalidomide teratogenicity. By using hydroethidine staining we demonstrate that thalidomide induces the formation of superoxide in embryonic fibroblasts of thalidomide-sensitive species but not in those of mice. As determined by trypan blue staining, scavenging of superoxide prevents thalidomide-induced apoptosis, a marker for thalidomide teratogenicity. Mouse embryonic fibroblasts are found to have higher glutathione levels than those of sensitive species and can be sensitized for thalidomide by glutathione depletion with diethyl maleate or diamide. Accordingly, experimental increase of glutathione levels in human embryonic fibroblasts by adding N-acetyl cysteine or glutathione ethyl ester to the culture medium counteracts thalidomide-induced apoptosis. Finally, we show that thalidomide-induced molecular pathology downstream of superoxide is essentially identical in human and sensitized mouse embryonic fibroblasts. In conclusion, thalidomide-resistance is based on the capacity of the glutathione-dependent antioxidant defense. We provide a basis to pharmacologically overcome the limitations of thalidomide use at humans and describe substantial differences between human and mouse embryonic cells regarding the protection against oxidative stress.
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Jürgen Knobloch, Ulrich Rüther (2008)  Shedding light on an old mystery: Thalidomide suppresses survival pathways to induce limb defects. [Review]   Cell Cycle 7: 9. 1122-1128 May  
Abstract: Many hypotheses have been proposed to explain the molecular mechanism of thalidomide teratogenicity, in particular regarding to limb defects. Most experimental evidence in vivo has been provided for a model that suggests the generation of oxidative stress by thalidomide with subsequent down-regulation of Wnt and Akt survival pathways. As a consequence apoptosis is induced during early embryonic limb development resulting in limb truncations. Here we summarize and discuss the relevant data supporting this hypothesis. We extend this model by presenting new data demonstrating an involvement of the transcription factors Tbx5 and Sall4 in thalidomide-induced molecular pathology. Finally, we discuss a possible participation of other stress-responsive and/or pro-apoptotic transcription factors in the mechanism of thalidomide teratogenicity.
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Jürgen Knobloch, Ingo Schmitz, Katrin Götz, Klaus Schulze-Osthoff, Ulrich Rüther (2008)  Thalidomide induces limb anomalies by PTEN stabilization, Akt suppression, and stimulation of caspase-dependent cell death.   Mol Cell Biol 28: 2. 529-538 Jan  
Abstract: Thalidomide, a drug used for the treatment of multiple myeloma and inflammatory diseases, is also a teratogen that causes birth defects, such as limb truncations and microphthalmia, in humans. Thalidomide-induced limb truncations result from increased cell death during embryonic limb development and consequential disturbance of limb outgrowth. Here we demonstrate in primary human embryonic cells and in the chicken embryo that thalidomide-induced signaling through bone morphogenetic proteins (Bmps) protects active PTEN from proteasomal degradation, resulting in suppression of Akt signaling. As a consequence, caspase-dependent cell death is stimulated by the intrinsic and Fas death receptor apoptotic pathway. Most importantly, thalidomide-induced limb deformities and microphthalmia in chicken embryos could be rescued by a pharmacological PTEN inhibitor as well as by insulin, a stimulant of Akt signaling. We therefore conclude that perturbation of PTEN/Akt signaling and stimulation of caspase activity is central to the teratogenic effects of thalidomide.
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2007
Jürgen Knobloch, John D Shaughnessy, Ulrich Rüther (2007)  Thalidomide induces limb deformities by perturbing the Bmp/Dkk1/Wnt signaling pathway.   FASEB J 21: 7. 1410-1421 May  
Abstract: Thalidomide, a sedative originally used to treat morning sickness and now used to treat leprosy and multiple myeloma, is also a teratogen that induces birth defects in humans such as limb truncations and microphthalmia. However, the teratogenic mechanism of action of this drug remains obscure. Thalidomide induces limb and eye defects in the chicken embryo at an EC50 of 50 microg/kg egg wt and apoptosis in primary human embryonic fibroblasts (HEFs) at an EC50 of 8.9 microM. Using these model systems, we demonstrate by semiquantitative reverse transcriptase-polymerase chain reaction and whole-mount in situ hybridization that thalidomide-induced oxidative stress enhances signaling through bone morphogenetic proteins (Bmps). This leads to up-regulation of the Bmp target gene and Wnt antagonist Dickkopf1 (Dkk1) with subsequent inhibition of canonical Wnt/beta-catenin signaling and increased cell death as shown by trypan blue and terminal deoxynucleotidyl transferase-mediated nick end labeling staining. Thalidomide-induced cell death was dramatically reduced in HEFs and in embryonic limb buds by the use of inhibitors against Bmps, Dkk1, and Gsk3beta, a beta-catenin antagonist acting downstream of Dkk1 in the Wnt pathway. Most interestingly, blocking of Dkk1 or Gsk3beta dramatically counteracts thalidomide-induced limb truncations and microphthalmia. From this, we conclude that perturbing of Bmp/Dkk1/Wnt signaling is central to the teratogenic effects of thalidomide.
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Jürgen Knobloch, Svenja Beckmann, Cora Burmeister, Thomas Quack, Christoph G Grevelding (2007)  Tyrosine kinase and cooperative TGFbeta signaling in the reproductive organs of Schistosoma mansoni. [Review]   Exp Parasitol 117: 3. 318-336 Nov  
Abstract: Drug-induced suppression of female schistosome sexual maturation is an auspicious strategy to combat schistosomiasis since the eggs are the causative agent. The establishment of drug targets requires knowledge about the molecular mechanisms that regulate the development of the female reproductive organs, which include vitellarium and ovary. This review summarizes recent studies suggesting tyrosine kinases as important factors for the regulation of female gonad development. In this context, especially cytoplasmatic tyrosine kinases of the Src class seem to play dominant roles. Moreover, experimental data and theoretical concepts are provided supporting a crosstalk between tyrosine kinase and TGFbeta signaling in the production of vitellocytes. Finally, we take advantage from the schistosome genome project to propose a model for the regulation of vitelline-cell production and differentiation.
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Andrea Koch, Jürgen Knobloch, Cathrin Dammhayn, Maria Raidl, Andrea Ruppert, Haitham Hag, Dennis Rottlaender, Katja Müller, Erland Erdmann (2007)  Effect of bacterial endotoxin LPS on expression of INF-gamma and IL-5 in T-lymphocytes from asthmatics.   Clin Immunol 125: 2. 194-204 Nov  
Abstract: Epidemiological evidence, in vitro studies and animal models suggest that exposure to the bacterial endotoxin lipopolysaccharide (LPS) can influence the development and severity of asthma. Although it is known that signaling through Toll-like receptors (TLR) is required for adaptive T helper cell type 1 and 2 responses, it is unclear whether the LPS ligand TLR 4 is expressed on CD4(+) and CD8(+) T-lymphocytes and if so, whether LPS could modulate the T(H)1 or T(H)2 response in this context. The present authors have, therefore, examined the expression of TLR 4 on peripheral blood CD4(+) and CD8(+) T-lymphocytes using RT-PCR method and FACS analyses. Furthermore, the authors have studied the IL-12-induced expression of the T(H)1-associated cytokine INF-gamma and the IL-4-induced expression of the T(H)2-specific cytokine IL-5 in the presence of LPS using ELISA and compared nine atopic asthmatic subjects and eleven nonatopic normal volunteers. There was an increased anti-CD3/anti-CD28-induced IL-5 expression in T cells of asthmatics compared with normals (p<0.01). In the presence of IL-4 (10 ng/ml), there was an additional increase in IL-5 expression and this additional increase was greater in T cells of normals compared with asthmatics (p<0.05). There was an expression of INF-gamma in anti-CD3/anti-CD28-induced T-lymphocytes without differences between both groups (NS). In the presence of IL-12 (10 ng/ml), there was an increase in INF-gamma release without differences between normals and asthmatics (NS). In the presence of different concentrations of LPS (10 ng/ml, 1 mug/ml), there was a decrease in IL-4-induced IL-5 expression without differences in both groups, indicating an intact T(H)2 response to bacterial endotoxin LPS in asthma. Interestingly, LPS increased the IL-12-induced INF-gamma release in a concentration-dependent manner in T-lymphocytes of normals but this could not be found in T cells of asthmatics, indicating an impaired T(H)1 response to bacterial endotoxin LPS in asthma. In addition, there was a TLR 4 expression on CD4(+) T-lymphocytes of normals and to a lesser extent in asthmatics but this TLR 4 expression could not be found on CD8(+) T cells of both groups. In conclusion, there may be an impaired concentration-dependent LPS-induced T(H)1 rather than a T(H)2 response in allergic adult asthmatics compared with normal volunteers. One reason for this could be a reduced TLR 4 expression on CD4(+) T-lymphocytes of asthmatic subjects.
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2006
Jürgen Knobloch, Werner Kunz, Christoph G Grevelding (2006)  Herbimycin A suppresses mitotic activity and egg production of female Schistosoma mansoni.   Int J Parasitol 36: 12. 1261-1272 Oct  
Abstract: The eggs of the endoparasite Schistosoma are the causative agent of schistosomiasis, an important disease of humans, which is endemic in (sub-) tropical regions. The absence of a vaccine with sufficient protective qualities and increasing resistance to approved and established drugs like praziquantel, justify the exploration of novel ways to fight schistosomes. Our strategy is based on interference with the sexual maturation of the female. Prerequisites for gonad development in adult females are a continuous pairing contact with the male and significantly increased mitotic activity. In this study we show that the male governs sexual maturation of the female, as the separation of couples causes a clear reduction of female mitotic activity and, consequently, egg production. We demonstrate that treatment of schistosomes with Herbimycin A, an inhibitor of protein tyrosine kinases (PTKs), mimics the separation of couples as the drug blocks mitotic activity and egg production of paired females. However, the synthesis of the eggshell precursor protein p14 is elevated. Furthermore, we show for the first time in invertebrates that Herbimycin A decreases tyrosine phosphorylation and PTK stability in schistosomes. Summarised, our data provide evidence that PTKs have key functions in regulating gonad development, eggshell gene expression and, consequently, egg production. Therefore, we suggest envisaging schistosome PTKs as novel targets for strategies to combat schistosomiasis.
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G Schramm, A Gronow, Jürgen Knobloch, Volker Wippersteg, Christoph G Grevelding, J Galle, H Fuller, R G Stanley, P L Chiodini, H Haas, M J Doenhoff (2006)  IPSE/alpha-1: a major immunogenic component secreted from Schistosoma mansoni eggs.   Mol Biochem Parasitol 147: 1. 9-19 May  
Abstract: During infection with Schistosoma mansoni the egg stage of this parasite modulates the initial T helper (Th1) response into a Th2 response. This suggests that schistosome eggs contain factors responsible for that effect. We have recently described a glycoprotein (IPSE) from S. mansoni eggs that has a potent IL-4-inducing effect on human basophils. Here we demonstrate that IPSE is identical to a previously described molecule, the S. mansoni egg antigen alpha-1. We furthermore show that the expression of IPSE/alpha-1 at the level of both mRNA and protein is restricted to the egg stage. IPSE/alpha-1 is produced in and released from the subshell area of the egg and comes into close contact with inflammatory cells recruited to the vicinity of the egg surface. In line with this IPSE/alpha-1 is one of three major S. mansoni egg glycoproteins that induce pronounced antibody responses. Its IL-4-inducing capacity, moreover, suggests that IPSE/alpha-1 plays a role in initiating the Th2 response induced by patent S. mansoni infections.
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2005
Ingo Ebersberger, Jürgen Knobloch, Werner Kunz (2005)  Cracks in the shell--zooming in on eggshell formation in the human parasite Schistosoma mansoni.   Dev Genes Evol 215: 5. 261-267 May  
Abstract: Schistosomiasis, currently the second most common parasitic disease of humans in tropical regions is caused by the eggs of trematode worms of the genus Schistosoma. Understanding egg formation and specifically the synthesis of the eggshell comprises, consequently, a promising starting point to cure and prevent the disease. To shed light on the genetics of the latter process, we analysed the three known S. mansoni eggshell proteins P14, P19 and P48 against the background of the species' inferred proteome and of eggshell proteins identified in other trematode species. Our results suggest that eggshell formation in Schistosoma involves a multitude of different proteins organised in currently three distinct protein families (P14, P48 and P34 eggshell protein family). The first two families are of simple structure. Their respective members share a substantial degree of sequence similarity and are, to date, observed only in the genus Schistosoma. In contrast, the P34 family of eggshell proteins is complex. Its in part highly diverged members share only a conserved motif of 67-aa length on average and are detected in various trematode species. The resulting widespread occurrence of this protein motif suggests an important role during eggshell formation in trematodes. Screening more than 7,000 putative proteins of S. mansoni, we could identify six new members of the P34 protein family that are likely to be involved in eggshell formation in this species.
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2004
Katja Kapp, Jürgen Knobloch, Peter Schüssler, Stefan Sroka, Reiner Lammers, Werner Kunz, Christoph G Grevelding (2004)  The Schistosoma mansoni Src kinase TK3 is expressed in the gonads and likely involved in cytoskeletal organization.   Mol Biochem Parasitol 138: 2. 171-182 Dec  
Abstract: Cytoplasmic protein tyrosine kinases of the Src family play a pivotal role in the regulation of cellular processes including proliferation and differentiation. Among other functions, Src kinases are involved in regulating the cell architecture. In an approach to identify protein tyrosine kinases from the medically important parasite Schistosoma mansoni, we isolated the TK3 gene by degenerate primer PCR and cDNA library screening. Sequencing of the complete cDNA and data-base analyses indicated that TK3 is a Src family kinase. Its predicted size of 71 kDa was confirmed by Western blot analysis. Southern blot analysis showed that TK3 is a single-copy gene, and Northern blot and RT-PCR experiments indicated its expression in both sexes and throughout development. Localization studies by in situ hybridization and immunolocalization revealed that TK3 is predominantly expressed in the reproductive organs such as the testes of the male and the ovary as well as the vitellarium of the female. Its enzymatic activity was confirmed by functional analyses. In transient transfection experiments with HEK293 cells, TK3 phosphorylated the well-known Src-kinase substrate p130 Cas, an intracellular scaffolding protein. Yeast two-hybrid screenings in a heterologous invertebrate system identified dAbi, vinculin and tubulin as binding partners, representing molecules that fulfill functions in the cell architecture of many organisms. These findings suggest that TK3 may play a role in signal transduction pathways organizing the cytoskeleton in the gonads of schistosomes.
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Jürgen Knobloch, Alessandro Rossi, Ahmed Osman, Philip T LoVerde, Mo-Quen Klinkert, Christoph G Grevelding (2004)  Cytological and biochemical evidence for a gonad-preferential interplay of SmFKBP12 and SmTbetaR-I in Schistosoma mansoni.   Mol Biochem Parasitol 138: 2. 227-236 Dec  
Abstract: In eukaryotes, FK506-binding proteins with a molecular weight of 12 kDa (FKBP12s) influence a variety of signal transduction pathways that regulate cell division, differentiation, and ion homeostasis. Amongst these, TGFbeta signaling and calcineurin (CN) phosphatase activity is modulated by FKBP12 via binding to TGFbeta-family type I receptors (TbetaR-Is) or to the CN subunit A, respectively. In this work, we demonstrate the tissue-specific expression of the Schistosoma mansoni FKBP12 homologue (SmFKBP12) in the gonads of female parasites as well as in the tegument of both genders. Components of the TGFbeta pathway have been characterized in schistosomes and their roles in mediating host-parasite or male-female interactions proposed. We show that a schistosome TGFbeta-family type I receptor (SmTbetaR-I, SmRK-1) is expressed in the female gonads, suggesting that SmFKBP12 may regulate its activity in this tissue. This hypothesis is supported by yeast two-hybrid analyses showing a direct binding of SmFKBP12 and SmTbetaR-I, which was specifically inhibited by the drug FK506. Our data provide the first evidence for the activity of a transmembrane receptor in the vitellarium of schistosome females and indicate that FKBP12-meditated regulation of the TGFbeta pathway is evolutionarily conserved in a primitive metazoan such as Schistosoma. Furthermore, we show that the schistosome CN (SmCN) is not expressed in the female gonads, but co-localizes with SmFKBP12 only in the tegument. From these data we conclude an SmFKBP12/SmTbetaR-I, but not an SmCN/SmFKBP12 interplay in the female gonads.
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2003
Anja Michel, Jürgen Knobloch, Werner Kunz (2003)  P19: a female and tissue specifically expressed gene in Schistosoma mansoni, regulated by pairing with the male.   Parasitology 127: Pt 6. 519-524 Dec  
Abstract: A female-specific sequence was isolated from a cDNA library of Schistosoma mansoni and further characterized. Expression of the corresponding gene (p19) depends on pairing with a male. In situ hybridization and immunohistology experiments revealed exclusive expression of the gene in the cells of the vitellarium, suggesting a function in egg formation. In addition, experimental evidence for cross-linking of the protein under oxidative conditions supports the assumption that the p19 gene may code for an egg-shell precursor protein.
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2002
Jürgen Knobloch, Ralf Winnen, Marcus Quack, Werner Kunz, Christoph G Grevelding (2002)  A novel Syk-family tyrosine kinase from Schistosoma mansoni which is preferentially transcribed in reproductive organs.   Gene 294: 1-2. 87-97 Jul  
Abstract: The complete coding deoxyribonucleic acid for a novel tyrosine kinase (TK) of the human parasite Schistosoma mansoni has been cloned and characterized. The molecule was designated TK4. The sequence predicts a translation product of about 140 kDa containing two Src homology 2 domains and a tyrosine kinase domain. Data base analyses indicate that TK4 belongs to the Syk family of TKs which has not been identified in schistosomes or other Acoelomata yet. The presence of a member of the Syk family in this phylum supports previous findings demonstrating that TK subclasses were established early in evolution. Although Northern blot and reverse transcription polymerase chain reaction analyses show transcription of TK4 in larval stages and adult schistosomes of both genders, TK4 is more abundantly transcribed in males. In situ hybridization data demonstrate the gender-independent occurrence of TK4 transcripts in parenchymatic cells. Significant signals were detected in the oocytes of the female and in the spermatocytes of the male suggesting that TK4, among other functions, may play a role in germ cell development. This is an unexpected finding considering that Syk-family TKs of invertebrates and vertebrates described so far are not involved in the differentiation of the gonads.
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Jürgen Knobloch, Werner Kunz, Christoph G Grevelding (2002)  Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique.   Dev Growth Differ 44: 6. 559-563 Dec  
Abstract: The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4',6-diamidino-2-phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.
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Technical reports

2000

PhD theses

2002
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