M Krönke

Christian-Albrechts-Universität zu Kiel, Kiel, Schleswig-Holstein, Germany

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Publications (119)847.43 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Resting human T lymphocytes do not express receptors for interleukin-2, but expression is rapidly induced by exposure to PHA. After maximal expression 2–3 days after stimulation, a progressive decline in receptor number is observed. Receptor expression can be augmented by reexposure to PHA. In this study we show that activators of protein kinase C including phorbol diester, phospholipase C, and the diacylglycerol congener diC8 also increase IL-2 receptor expression. Moreover, 5-azacytidinc, which inhibits cytosine methyltransferase, and hydroxy-urea, which inhibits ribonucleotide reductase, also increased receptor number. These studies demonstrate that IL-2 receptor expression can be altered in vitro, and that IL-2 receptor number, in combination with IL-2 secretion, may contribute to the regulation of IL-2–dependent immune responses.
    Journal of Cellular Biochemistry 02/2004; 27(3):267 - 276. · 3.06 Impact Factor
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    ABSTRACT: In vitro, soluble MHC (sMHC) antigens modulate and induce apoptosis in alloreactive and antigen-specific T cells, demonstrating their potency to regulate T cell-mediated immune responses. However, their efficacy to regulate immunological responses in vivo remains unclear. Here, we report that repetitive intraperitoneal injection of recombinant Lewis rat-derived MHC class I antigens in Dark Agouti (DA) rats modulates alloreactivity. RT1.A1 (Lewis derived) genes were cloned into mammalian expression vectors, and RT1.Aa (DA derived) genes were used to transfect a rat myeloma cell line. RT1.A1 molecules were injected intraperitoneally in DA recipients that subsequently underwent transplantation with Lewis-derived cardiac allografts. Soluble class I antigens were secreted by the transfected cells and were shown to be heterodimeric, peptide-loaded, and conformationally folded. Injection of donor-derived soluble MHC significantly reduced the ability of recipient animals to mount a cytotoxic T-cell response to donor-derived tissue. More interestingly, this treatment significantly prolonged donor-graft survival and allowed 60% of treated animals to develop graft tolerance (>120 days), when donor sMHC were combined with a single subtherapeutic dosage of cyclosporine. Thymectomy of recipient animals before transplantation did not interfere with induction of peripheral tolerance. Donor-derived sMHC are potential tolerogens for down-regulating the cytotoxic T-cell response of animals that undergo transplantation. Thus, these data provide for the first time a rationale for the application of directly injected sMHC in vivo to down-regulate immunological responses and aid the induction of graft tolerance.
    Transplantation 12/2001; 72(12):1974-82. · 3.78 Impact Factor
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    ABSTRACT: TNF-alpha is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.
    Journal of Clinical Investigation 08/2001; 108(1):143-51. · 12.81 Impact Factor
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    ABSTRACT: The adhesion of immunomagnetic particles to surfaces of target cells based on ligand–receptor interactions was evaluated by counting the number of magnetically labeled cells using magnetophoresis and analyzed by the Langmuir adsorption theory. As few as 200 tumor necrosis factor (TNF) receptor molecules per cell could be detected with streptavidin-coated magnetic particles coupled to biotinylated TNF as ligand.
    Journal of Magnetism and Magnetic Materials 01/2001; 225(1):285-293. · 2.00 Impact Factor
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    ABSTRACT: Tumor necrosis factor (TNF) signals cell death and simultaneously induces generation of ceramide. To evaluate the contribution of ceramide to TNF-dependent cell death, we generated clones of the TNF-sensitive cell line L929 that constitutively overexpress human acid ceramidase (AC). Ceramidase, in concert with sphingosine kinase, metabolizes ceramide to sphingosine-1-phosphate (SPP), an inducer of proliferation. In response to TNF, parental L929 cells display a significant increase in intracellular ceramide correlated with an "atypical apoptosis" characterized by membrane blebbing, DNA fragmentation and degradation of poly(ADP-ribose) polymerase despite a lack of caspase activity. These features are strongly reduced or absent in AC-overexpressing cells. Pharmacological suppression of AC with N-oleoylethanolamine restored the accumulation of intracellular ceramide as well as the sensitivity of the transfectants to TNF, implying that an enhanced metabolization of intracellular ceramide by AC shifts the balance between intracellular ceramide and SPP levels towards cell survival. Correspondingly, inhibition of ceramide production by acid sphingomyelinase also increased survival of TNF-treated L929 cells.
    Journal of Experimental Medicine 10/2000; 192(5):601-12. · 13.21 Impact Factor
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    ABSTRACT: The generation of proinflammatory eicosanoids in response to tumor necrosis factor (TNF) involves the activation of cytosolic phospholipase A(2) (cPLA(2)), presumably by phosphorylation through extracellular signal-regulated kinases (ERK). Earlier results had suggested that a pathway involving the p55 TNF receptor (TNF-R55), neutral sphingomyelinase (N-SMase), and c-Raf-1 activates ERK and cPLA(2). We have previously shown that a cytoplasmic region of TNF-R55 distinct from the death domain regulates the activation of N-SMase through binding of the adapter protein FAN. Analysis of embryonal fibroblasts from FAN knockout mice revealed that TNF-induced activation of both ERK and cPLA(2) occurs without involvement of FAN. Furthermore, we provide evidence that the TNF-dependent activation of ERK and cPLA(2) requires the intact death domain of TNF-R55. Finally, we demonstrate that in murine fibroblasts cPLA(2) is phosphorylated in response to TNF solely by ERK, but not by p38 mitogen-activated protein kinase, suggesting a signaling pathway from TNF-R55 via the death domain to ERK and cPLA(2).
    Biochemical and Biophysical Research Communications 09/2000; 274(2):506-12. · 2.28 Impact Factor
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    ABSTRACT: The generation of discriminative, monospecific anti-HLA antibodies used to be a difficult endeavor. Phage display technology, using single-chain antibody fragments (scFv) offers a powerful alternative obtaining target-specific, genetically stable reagents. Most of scFv obtained to date have been enriched by panning phage libraries to solid-phase coupled antigens. In the present study, HLA-C-specific scFv were isolated using a synthetic phage library in combination with a Cw*0602 overexpressing cell line. ScFv from this procedure precipitated HLA-Cw*0602 heavy chains from whole cell lysates. Flow cytometry analysis revealed that scFv stained HLA-Cw*0602-positive cells, but not cells expressing HLA alleles Cw*0302, Cw*0802, A*0201, B*2705, or Gm1*01011, indicating the specificity of scFv. Similarly they showed an ability to discriminate Cw*0602-positive from Cw*0602-negative peripheral blood lymphocytes (PBL). The results of our study demonstrate the feasibility to genetically engineer single-chain HLA-class I-specific antibodies, by phage display technology. This approach might be a valuable tool to develop a broad range of novel monospecific antibodies against HLA-class I specificities.
    Tissue Antigens 08/2000; 56(1):1-9. · 2.93 Impact Factor
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    ABSTRACT: Liver allografts transplanted between MHC-disparate mice, rats, and swine are spontaneously accepted in most strain combinations without requirement for immunosuppression. The underlying mechanism has, however, remained elusive. Here, we demonstrate that co-transplantation of donor-derived hepatocytes protect Lewis (RT1.A1) cardiac allografts from acute and chronic rejection in DA (RT1.Aa) recipients indefinitely. Livers of donor Lewis rats were harvested and the hepatocytes separated from hepatic leukocytes by collagenase digestion and gradient separation. DA recipient animals were transplanted Lewis cardiac allografts and simultaneously intraportally infused either Lewis-derived hepatocytes or hepatic leukocytes. Recipient animals were either not further treated or received a single dose of 15 mg/kg cyclosporine. Donor hepatocytes alone significantly protected syngeneic cardiac allografts from rejection, whereas hepatic leukocytes failed to influence graft survival. In combination with cyclosporine, recipient cardiac allografts were indefinitely protected from rejection. Graft-infiltrating cells in tolerant animals presented as clusters of CD4+ T cells and stained mostly positive for interleukin-4, whereas graft-infiltrating cells in rejected allografts were predominantly positive for interferon-gamma. Adoptive transfer of splenocytes derived from tolerant animals protected Lewis cardiac allografts from rejection in DA recipients without immunosuppression. In contrast, hepatic leukocytes protected only 50% of the allografts from rejection. We propose that donor hepatocytes induce permanent engraftment of syngeneic allografts by establishing a Th2 type alloresponse that is transferable to new graft recipients. The results of this study demonstrate that liver parenchymal cells significantly mediate spontaneously liver-induced tolerance.
    Transplantation 07/2000; 69(12):2538-46. · 3.78 Impact Factor
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    ABSTRACT: The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.
    Journal of Biological Chemistry 04/2000; 275(11):7641-7. · 4.65 Impact Factor
  • Methods in Enzymology 02/2000; 312:429-38. · 2.00 Impact Factor
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    ABSTRACT: The possibility that the sphingomyelin (SM)-ceramide pathway is activated by CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily and that plays a critical role in the regulation of immune responses has been investigated. We demonstrate that incubation of Epstein-Barr virus-transformed lymphoid cells with an anti-CD40 antibody acting as an agonist results in the stimulation of a neutral sphingomyelinase, hydrolysis of cellular SM, and concomitant ceramide generation. In addition, SM degradation was observed in acid sphingomyelinase-deficient cells, as well as after ligation by soluble CD40 ligand. The anti-CD40 antibody, as well as the soluble CD40 ligand induced a decrease in thymidine incorporation and morphological features of apoptosis, which were mimicked by cell-permeant or bacterial sphingomyelinase-produced ceramides. Stable expression of a dominant-negative form of the FAN protein (factor associated with neutral sphingomyelinase activation), which has been reported to mediate tumor necrosis factor-induced activation of neutral sphingomyelinase, significantly inhibited CD40 ligand-induced sphingomyelinase stimulation and apoptosis of transformed human fibroblasts. Transformed fibroblasts from FAN knockout mice were also protected from CD40-mediated cell death. Finally, anti-CD40 antibodies were able to co-immunoprecipitate FAN in control fibroblasts but not in cells expressing the dominant-negative form of FAN, indicating interaction between CD40 and FAN. Altogether, these results strongly suggest that CD40 ligation can activate via FAN a neutral sphingomyelinase-mediated ceramide pathway that is involved in the cell growth inhibitory effects of CD40.
    Journal of Biological Chemistry 01/2000; 274(52):37251-8. · 4.65 Impact Factor
  • M Krönke
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    ABSTRACT: Sphingomyelin (N-acylsphingosin-1-phosphorylcholine) is a phospholipid preferentially found in the plasma membrane of mammalian cells. Signaling through the sphingomyelin pathway is associated with generation of ceramide, which acts as a second messenger in activating a variety of cellular functions. Ceramide belongs to the group of sphingosine-based lipid second messenger molecules that are critically involved in the regulation of signal transduction of diverse cell surface membrane receptors. The emerging picture suggests that coupling of ceramide to specific signaling cascades is both stimulus- and cell type-specific and depends on the subcellular topology of its production. Following membrane receptor triggering, neutral and acid isoforms of sphingomyelinases are rapidly activated generating ceramide through sphingomyelin hydrolysis. Here the molecular mechanisms of TNF-induced activation of sphingomyelinases and the functional consequences of ceramide generation will be discussed.
    Chemistry and Physics of Lipids 12/1999; 102(1-2):157-66. · 2.59 Impact Factor
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    ABSTRACT: Corneodesmosin (CD) is thought to play a key role in corneocyte cohesion, and its proteolysis appears to be a major event in the process of desquamation. Recently it was shown that CD is encoded by the S-gene, which is located approximately 160 kb telomeric of HLA-C. In the present study, the role of CD in the genetics of psoriasis vulgaris was studied in greater detail. The second exon of the CD gene was sequenced in 86 HLA-typed individuals from 13 psoriasis multiplex families. A total of 11 silent dimorphisms and 7 variants resulting in amino acid substitutions were found. Pedigree analysis showed that these variants could be grouped into 7 alleles, encoding 6 different amino acid sequences. These alleles are in strong linkage disequilibrium with HLA-B and -C, indicating that the polymorphism of the CD gene is ancient and well conserved rather than sporadic. One allele at the CD locus, designated CD2, displayed strong linkage disequilibrium with HLA-Cw6, the HLA allele most prominently associated with psoriasis. CD2 demonstrated a greater relative risk than Cw6 (3.4 vs. 2.5, not significant) and higher significant transmission disequilibrium with psoriasis than any of the investigated HLA-alleles. Due to its biologic function, cellular location and disease association, the CD gene appears to be an excellent candidate gene for PSORS1, the HLA-linked determinant of psoriasis vulgaris.
    Tissue Antigens 12/1999; 54(5):439-49. · 2.93 Impact Factor
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    ABSTRACT: Epidermal TNF expression increases in response to cutaneous permeability barrier disruption and wound healing. TNF signaling is mediated by acid and neutral sphingomyelinases (A- and N-SMase), which generate ceramide, an important regulator of proliferation, differentiation, and apoptosis. In the epidermis, ceramide is known to be an integral part of the extracellular stratum corneum (SC) lipid bilayers that constitute the permeability barrier of the skin. We show here that topical application of TNF after experimental injury to the SC of hairless mice (hr(-/-)) enhances barrier repair. In TNF receptor p55-deficient (TNF-R55-deficient) mice (hr(+/+)), cutaneous barrier repair was delayed compared with wild-type (hr(+/+)) or TNF-R75-deficient (hr(+/+)) animals. After barrier disruption in hairless (hr(-/-)) and wild-type (hr(+/+)), but not in TNF-R55-deficient (hr(+/+)) mice, the enzymatic activities of both A-SMase and N-SMase were significantly enhanced. Stimulation of SMase activities was accompanied by an increase in C(24)-ceramide levels. Most A-SMase activity in hairless mice (hr(-/-)) was found in the outer epidermal cell layers and colocalized in the lamellar bodies with A-SMase and sphingomyelin. Reduction of epidermal A-SMase activity by the inhibitor imipramine resulted in delayed permeability barrier repair after SC injury. Together, these results suggest that TNF-R55 signaling pathways contribute to cutaneous permeability barrier repair through SMase-mediated generation of ceramide.
    Journal of Clinical Investigation 12/1999; 104(12):1761-70. · 12.81 Impact Factor
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    ABSTRACT: Ceramide has been recognized as a common intracellular second messenger for various cytokines, growth factors and other stimuli, such as CD95, chemotherapeutic drugs and stress factors. To understand the role of ceramide during apoptosis and other cellular responses, it is critically important to characterize direct targets of ceramide action. In this paper, we show that ceramide specifically binds to and activates the endosomal acidic aspartate protease cathepsin D. Direct interaction of ceramide with cathepsin D results in autocatalytic proteolysis of the 52 kDa pre-pro cathepsin D to form the enzymatically active 48/32 kDa isoforms of cathepsin D. Acid sphingomyelinase (A-SMase)-deficient cells show decreased cathepsin D activity, which could be reconstituted by transfection with A-SMase cDNA. The results of our study identify cathepsin D as the first endosomal ceramide target that colocalizes with and may mediate downstream signaling effects of A-SMase.
    The EMBO Journal 11/1999; 18(19):5252-63. · 9.82 Impact Factor
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    ABSTRACT: Corneodesmosin (CD) is thought to play a key role in corneocyte cohesion, and its proteolysis appears to be a major event in the process of desquamation. Recently it was shown that CD is encoded by the S-gene, which is located approximately 160 kb telomeric of HLA-C. In the present study, the role of CD in the genetics of psoriasis vulgaris was studied in greater detail. The second exon of the CD gene was sequenced in 86 HLA-typed individuals from 13 psoriasis multiplex families. A total of 11 silent dimorphisms and 7 variants resulting in amino acid substitutions were found. Pedigree analysis showed that these variants could be grouped into 7 alleles, encoding 6 different amino acid sequences. These alleles are in strong linkage disequilibrium with HLA-B and -C, indicating that the polymorphism of the CD gene is ancient and well conserved rather than sporadic. One allele at the CD locus, designated CD2, displayed strong linkage disequilibrium with HLA-Cw6, the HLA allele most prominently associated with psoriasis. CD2 demonstrated a greater relative risk than Cw6 (3.4 vs. 2.5, not significant) and higher significant transmission disequilibrium with psoriasis than any of the investigated HLA-alleles. Due to its biologic function, cellular location and disease association, the CD gene appears to be an excellent candidate gene for PSORS1, the HLA-linked determinant of psoriasis vulgaris Note.
    Tissue Antigens 10/1999; 54(5):439 - 449. · 2.93 Impact Factor
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    M Krönke
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    ABSTRACT: Ceramides have been implied in intracellular signal transduction systems regulating cellular differentiation, activation, survival and apoptosis and thus appear capable of changing the life style of virtually any cell type. Ceramide belongs to the group of sphingosine-based lipid second messenger molecules that are critically involved in the regulation of diverse cellular responses to exogenous stimuli. The emerging picture suggests that coupling of ceramide to specific signaling cascades is both stimulus and cell-type specific and depends on the subcellular topology of its production. However, little is understood about the molecular mode of ceramide action. In particular, in lieu of a defined ceramide binding motif it is not clear how ceramide would directly interact with putative target signaling proteins. This article proposes two modes of ceramide action. First, a protruding alkyl chain of ceramide may interact with a hydrophobic cavity of a signaling protein providing a lipid anchor to attach proteins to membranes. Second, the generation of ceramide generally increases the volume of hydrocarbon chains within the lipid bilayer thereby enhancing its propensity of to form a hexagonal II phase (Hex II). Besides the generation of a hydrophobic interaction site for proteins local hexagonal phase II formation can also change the membrane fluidity and permeability, which may impinge on membrane fusion processes, solubilization of detergent-resistant signaling rafts, or membrane receptor internalization. Thus, ceramide production by sphingomyelinases (SMase) can play a pivotal signaling role through direct interaction with signaling proteins or through facilitating the formation and trafficking of signal transduction complexes.
    Chemistry and Physics of Lipids 09/1999; 101(1):109-21. · 2.59 Impact Factor
  • Biochemical Society Transactions 09/1999; 27(4):393-9. · 2.59 Impact Factor
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    ABSTRACT: The WD-40 repeat protein FAN binds to a distinct domain of the p55 receptor for tumor necrosis factor (TNF) and signals the activation of neutral sphingomyelinase (N-SMase). To analyze the physiological role of FAN in vivo, we generated FAN-deficient mice by targeted gene disruption. Mice lacking a functional FAN protein do not show any overt phenotypic abnormalities; in particular, the architecture and cellular composition of lymphoid organs appeared to be unaltered. An essential role of FAN in the TNF-induced activation of N-SMase was demonstrated using thymocytes from FAN knockout mice. Activation of extracellular signal-regulated kinases in response to TNF treatment, however, was not impaired by the absence of the FAN protein. FAN-deficient mice show delayed kinetics of recovery after cutaneous barrier disruption suggesting a physiological role of FAN in epidermal barrier repair. Although FAN exhibits striking structural homologies with the CHS/Beige proteins, FAN-deficient mice did not reproduce the phenotype of beige mice.
    The EMBO Journal 06/1999; 18(9):2472-9. · 9.82 Impact Factor
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    ABSTRACT: The 55-kDa receptor for tumor necrosis factor (TR55) triggers multiple signaling cascades initiated by adapter proteins like TRADD and FAN. By use of the primary amine monodansylcadaverine (MDC), we addressed the functional role of tumor necrosis factor (TNF) receptor internalization for intracellular signal distribution. We show that MDC does not prevent the interaction of the p55 TNF receptor (TR55) with FAN and TRADD. Furthermore, the activation of plasmamembrane-associated neutral sphingomyelinase activation as well as the stimulation of proline-directed protein kinases were not affected in MDC-treated cells. In contrast, activation of signaling enzymes that are linked to the "death domain" of TR55, like acid sphingomyelinase and c-Jun-N-terminal protein kinase as well as TNF signaling of apoptosis in U937 and L929 cells, are blocked in the presence of MDC. The results of our study suggest a role of TR55 internalization for the activation of select TR55 death domain signaling pathways including those leading to apoptosis.
    Journal of Biological Chemistry 05/1999; 274(15):10203-12. · 4.65 Impact Factor

Publication Stats

9k Citations
847.43 Total Impact Points

Institutions

  • 1995–2001
    • Christian-Albrechts-Universität zu Kiel
      • • Sektion für Molekulare Onkologie
      • • UKSH Institut für Immunologie
      Kiel, Schleswig-Holstein, Germany
  • 2000
    • University of Bonn
      • Kekulé Institute of Organic Chemistry and Biochemistry
      Bonn, North Rhine-Westphalia, Germany
  • 1999–2000
    • University of Cologne
      • Institute for Medical Microbiology, Immunology and Hygiene
      Köln, North Rhine-Westphalia, Germany
  • 1998
    • Memorial Sloan-Kettering Cancer Center
      New York City, New York, United States
  • 1991–1996
    • Technische Universität München
      • Institut für Medizinische Mikrobiologie, Immunologie und Hygiene
      München, Bavaria, Germany
  • 1988–1992
    • Max Planck Society
      München, Bavaria, Germany
  • 1986
    • Georg-August-Universität Göttingen
      Göttingen, Lower Saxony, Germany
    • Universitätsmedizin Göttingen
      Göttingen, Lower Saxony, Germany
  • 1984
    • Johannes Gutenberg-Universität Mainz
      • Division of Medical Biometry I
      Mayence, Rheinland-Pfalz, Germany