R Schwinzer

Hannover Medical School, Hanover, Lower Saxony, Germany

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Publications (117)352.27 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulin-like domains. Among peripheral blood cells, CD96 is expressed on T cells as well as NK cells and a subpopulation of B cells. A possible function of this receptor in NK cell-mediated killing activities was suggested recently. Moreover, CD96 was described as a tumor marker for T-cell acute lymphoblastic leukemia and acute myeloid leukemia. CD96 binds to CD155 (poliovirus receptor) and nectin-1, an adhesion receptor related to CD155. Here we report that human but not mouse CD96 is expressed in two splice variants possessing either an I-like (variant 1) or V-like (variant 2) second domain. With the notable exception of an AML tumor sample, variant 2 predominates in all the CD96-expressing cell types and tissues examined. Using chimeric human/murine CD96 receptors, we show that the interaction with its ligands is mediated via the outermost V-like domain. In contrast to mouse, however, the binding of human CD96 to CD155 is sensitive to the characteristics of the two downstream domains. This is illustrated by a significantly weaker CD96/CD155 interaction mediated by variant 1 when compared with variant 2. Moreover, recent evidence suggested that mutations in human CD96 correlate with the occurrence of a rare form of trigonocephaly. One such mutation causing a single amino acid exchange in the third domain of human CD96 decreased the capacity of both variants to bind to CD155 considerably, suggesting that a CD96-driven adhesion to CD155 may be crucial in developmental processes.
    Journal of Biological Chemistry 01/2009; 284(4):2235-44. DOI:10.1074/jbc.M807698200 · 4.60 Impact Factor
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    ABSTRACT: Natural killer (NK) cells participate in pig-to-primate xenograft rejection both by antibody-dependent and -independent mechanisms. A majority of human NK cells express the inhibitory receptor CD94/NKG2A, which binds specifically to human leukocyte antigen (HLA)-E, a trimeric complex consisting of the HLA-E heavy chain, beta2-microglobulin (beta2m), and a peptide derived from the leader sequence of some major histocompatibility complex class I molecules. To use this mechanism for protection of pig tissues against human NK cell-mediated cytotoxicity, we generated transgenic pigs by pronuclear microinjection of genomic fragments of HLA-E with an HLA-B7 signal sequence and of human beta2-microglobulin (hubeta2m) into zygotes. Three transgenic founder pigs were generated. Northern blot analysis of RNA from peripheral blood mononuclear cells revealed the presence of the expected transcript sizes for both transgenes in two of the three founders. The founder with the highest expression and his offspring were characterized in detail. Fluorescence-activated cell sorting (FACS) and Western blot analyses demonstrated consistent expression of HLA-E and hubeta2m in peripheral blood mononuclear cells. Immunohistochemistry revealed the presence of HLA-E and hubeta2m on endothelial cells of many organs, including heart and kidney. In vitro studies showed that lymphoblasts and endothelial cells derived from HLA-E/hubeta2m transgenic pigs are effectively protected against human NK cell-mediated cytotoxicity, depending on the level of CD94/NKG2A expression on the NK cells. Further, HLA-E/hubeta2m expression on porcine endothelial cells inhibited the secretion of interferon (IFN)-gamma by co-cultured human NK cells. This novel approach against cell-mediated xenogeneic responses has important implications for the generation of multitransgenic pigs as organ donors for clinical xenotransplantation.
    Transplantation 01/2009; 87(1):35-43. DOI:10.1097/TP.0b013e318191c784 · 3.78 Impact Factor
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    ABSTRACT: The inability of porcine thrombomodulin (TM) to activate human anticoagulant protein C after pig-to-human xenotransplantation may lead to an aberrant activation of coagulation with microthrombosis and ultimately failure of the transplanted organ. Here, we describe the production of triple-transgenic pigs expressing hCD59/DAF and human thrombomodulin (hTM) and tested hTM-transgenic fibroblasts obtained from these pigs for their ability to activate human protein C in a new in vitro assay. Fibroblast cell cultures were established from a hCD59/DAF transgenic pig and transfected with a vector coding for hTM under transcriptional control of the CMV promoter. Transfected cells were analyzed for integration and expression of the hTM vector by PCR and RT-PCR. One cell clone was used as donor for somatic cell nuclear transfer to produce triple transgenic (CD59/DAF/hTM) pigs. Pigs were characterized in detail with regard to hTM integration and expression by PCR, RT-PCR, Northern blot, Western blot, immunostaining, and FACS analysis. Fibroblasts from hTM-transgenic pigs were analyzed in a new in vitro hTM coactivity assay to assess the production of activated protein C (aPC) and results were compared to those from wild-type controls. In total, 1040 cloned transgenic embryos were transferred to eight recipients. Five recipients remained pregnant and delivered 22 piglets. Expression of hTM was detected in all xenorelavant organs including heart, liver, kidney, lung, and pancreas. The lowest levels of expression were found in lung and liver while all animals showed a strong, but frequently patchy expression pattern of hTM in heart, kidney, and pancreas. The hTM cofactor activity (ranging on a scale from 5-18 U/10(5) cells/2h) was significantly higher in fibroblasts of hTM-transgenic clones compared to wild-type porcine fibroblasts (1.7 U/10(5) cells/2h). For the first time, healthy hTM-transgenic pigs could be successfully generated by somatic cell nuclear transfer. hTM can be expressed in porcine organs without perturbation of the porcine coagulation system. hTM-transgenic porcine fibroblasts showed elevated aPC production in an in vitro hTM coactivity assay. These findings warrant further work on the control of the xenogenic activation of coagulation by transgenic approaches.
    Xenotransplantation 01/2009; 16(6):486-95. DOI:10.1111/j.1399-3089.2009.00537.x · 1.78 Impact Factor
  • R Schwinzer, A Plege
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    ABSTRACT: Hyperacute and acute vascular rejection of xenografts are well defined barriers to clinical pig-to-human xenotransplantation. Enormous progress has been made in recent years to overcome these immunological barriers. For example, transgenic expression of human complement regulatory molecules (e.g. CD46, CD55) in pigs has been shown to be an effective strategy to prevent hyperacute rejection in pre-clinical models of xenotransplantation. Alpha1,3-galactosyltransferase knock-out pigs are available and provide a second possibility to avoid hyperacute rejection mediated by pre-existing antibodies. Furthermore, transfer of protective genes (e.g. A20, HO-1) to endothelial cells is expected to reduce their susceptibility to effector mechanisms leading to acute vascular rejection. In addition, the efficiency of strategies to avoid coagulation/thrombosis after pig-to-human xenotransplantation (e.g. transgenic expression of human thrombomodulin, CD39) is currently tested. Thus, for further development of clinical xenotransplantation immunological concepts are now required facilitating the control of human anti-pig cellular immune responses. Our group focuses on the inhibition of human anti-pig T cell responses by targeting "negative" costimulatory pathways. We tested the hypothesis that overexpression of the human negative costimulatory ligands PD-L1 and PD-L2 on pig antigen presenting cells will result in reduced human anti-pig T cell responses. The data so far show that (i) human CD4(+) T cells respond with reduced proliferation and cytokine synthesis to PD-L1/PD-L2 expressing pig cells, (ii) PD-L1/PD-L2 pig transfectants induce human regulatory T cells (T(reg)) which suppress the activation of conventional T cells, and (iii) PD-L1/PD-L2 expressing pig cells are protected from lysis mediated by CD8(+) human cells. Together these observations support the assumption that transgenic expression of human PD-L1 and/or PD-L2 in pig cells and tissues could be an approach to prevent T cell reactivity after pig-to-human xenotransplantation. Supported by the Deutsche Forschungsgemeinschaft (Transregio Forschergruppe "Xenotransplantation", FOR 535).
    Xenotransplantation 10/2008; 15(5):296. DOI:10.1111/j.1399-3089.2008.00488_5.x · 1.78 Impact Factor
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    ABSTRACT: Following either discordant transplantation of porcine organs into primate recipients or after ex vivo perfusion of porcine organs with human blood, a profound activation of the primate/human blood coagulation can be observed. This activation may result in thrombotic microangiopathy and changes of coagulation resembling disseminated intravascular coagulation with subsequent death of the recipient animals. There are several factors contributing to this pathology including endothelial cell activation, activation of human blood cells, thrombocyte activation and incompatibilities of molecules regarding to control coagulation. Several potentially important molecular incompatibilities between the porcine and the primate coagulation system have been noted: The inability of porcine tissue factor pathway inhibitor (TFPI) to adequately neutralize human factor Xa (FXa), the aberrant activation of both human prothrombin and FX by porcine endothelial cells and the failure of the porcine natural anticoagulant thrombomodulin to activate the anti-coagulant human protein C. Normal hemostasis is a complex balance between pro- and antithrombotic pathways. These pathways are balanced by inhibitory systems including the heparin-antithrombin interaction, TFPI, the generation of activated protein C by a proper thrombomodulin/thrombin interaction and the fibrinolytic system. To achieve normal haemostasis after xenotransplantation it is important to overcome the known molecular incompatibilities (i.e. by transgenic organs) and to reliably prevent the derangement of coagulation.
    Xenotransplantation 10/2008; 15(5):303. DOI:10.1111/j.1399-3089.2008.00488_11.x · 1.78 Impact Factor
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    ABSTRACT: Introduction: The hyperacute rejection after porcine-to-human xenotransplantation can now be reliably overcome either by transgenic expression of complement regulating factors (done in this study) or by knocking out the gene for alpha1,3-galactosyltransferase in pigs. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation. Human hemeoxygenase-I (hHO-1) is known to have anti-apoptotic and cell protective properties. Thus, the expression of hHO-1 on porcine endothelial cells could have beneficial effects in a xenotransplantation setting. Here, we describe the generation and characterization of pigs transgenic for hHO-1. Methods: Fibroblasts were obtained by an ear punch from a CD55 transgenic female pig and were cultured in vitro (Kues WA et al. 2005 Biol Reprod 72: 1020-1028). Cells reaching confluency of 70 to 80%, were detached with EDTA/trypsin and subsequently transfected by electroporation at 450 V/350 muF with a vector coding for hHO-I driven by the SV40 promoter. Transfected cells were selected for resistance against G418 (800 mug/ml) for 14 days. Resistant cell clones were screened for integration of the vector by PCR. One positive cell clone was selected for somatic nuclear transfer. In total, 205 reconstructed embryos were transferred to two synchronized peripuberal German Landrace gilts which gave birth to nine live piglets, all with normal birth weights. The expression of the transgenes (CD55 and hHO-1) was determined by flow cytometry and Western blot in endothelial cells and peripheral blood lymphocytes. The expression pattern of the transgenes in different organs was shown by immunostaining, RT-PCR and Northern blot. Organ survival and XAC following the contact of human blood with porcine endothelium, were determined by using an ex-vivo perfusion circuit based on low-dose heparin mediated anticoagulation. Porcine kidneys were recovered following in situ cold perfusion with HTK organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled human AB blood. Results: PCR and Southern blot analyses revealed that all of the offspring had integrated the vector in their genome. Six transgenic animals were sacrificed for further characterization. In these animals, the expression of CD55 and hHO-1 in peripheral blood lymphocytes and cultured endothelial cells was shown by flow cytometry and Western blot. In addition, albeit all animals were cloned from the same cell clone, variations in the expression pattern and levels could be observed in immunostaining, RT-PCR and Northern Blot. The animals showed only weak expression of hHO-1 in most of the xenorelevant organs like heart, kidney and liver. Average survival of wildtype pig kidneys during organ perfusion with human blood and addition of heparin was 42 +/- 26 min. Application of soluble complement inhibition (C1-inhibitor) prolonged organ survival to 126 +/- 72 min. XAC was observed with significantly elevated concentrations of d-Dimer and thrombin antithrombin complex (TAT) combined with consumption of fibrinogen and antithrombin. The histological analyses revealed numerous microthrombi. In contrast, the perfusion of the CD55/hHO1 transgenic porcine kidneys was feasible for more than 240 min in all perfusion experiments (with and without C1-inh). Although elevated levels of d-Dimer and TAT were measured, no significant consumption of fibrinogen and antithrombin occurred. In addition, no microthrombi were detectable histologically. Conclusion: These results show that hHO-1 in combination with CD55 can be expressed in transgenic pigs. Somatic cell nuclear transfer is a powerful tool for the generation of transgenic animals for xenotransplantation. These results are encouraging and warrant further studies on endothelial cell activation and the biological function of hemeoxygenase-I in the context of xenotransplantation. This study was funded by the Deutsche Forschungsgemeinschaft Ni 256/ 22-1, -2, -3.
    Xenotransplantation 10/2008; 15(5):301. DOI:10.1111/j.1399-3089.2008.00488_9.x · 1.78 Impact Factor
  • Transplantation 01/2008; 86. DOI:10.1097/01.tp.0000331060.27749.d9 · 3.78 Impact Factor
  • Transplantation 01/2008; 86:236-237. DOI:10.1097/01.tp.0000332203.73954.56 · 3.78 Impact Factor
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    ABSTRACT: The C77G polymorphism in exon A of the human CD45 gene occurs with low frequency in healthy individuals. An enhanced frequency of C77G individuals has been reported in cohorts of patients suffering from multiple sclerosis, systemic sclerosis, autoimmune hepatitis, hepatitis C and human immunodeficiency virus (HIV)-1. C77G individuals overexpress CD45RA isoforms on activated/memory T cells. We have shown previously that aberrant expression of CD45RA isoforms enhances the intensity of T cell receptor (TCR) signalling. Here we report that the C77G polymorphism also influences the responsiveness of T cells to cytokines and alters their adhesion properties. When stimulated by interleukin (IL)-2, C77G T cells proliferated more strongly than wild-type controls and showed accelerated phosphorylation of Janus kinase (Jak1). Furthermore, C77G T cells exhibited a higher tendency to form homotypic aggregates in culture which could be enhanced significantly by antibody-mediated triggering of the variant CD45RA molecules. These data indicate that the changes in CD45 isoform combination resulting from C77G may not only affect TCR signalling but also cytokine-driven T cell responses and cellular adhesion. Altered immune responsiveness may enhance susceptibility of C77G carriers for certain diseases.
    Clinical & Experimental Immunology 01/2008; 150(3):509-17. DOI:10.1111/j.1365-2249.2007.03508.x · 3.28 Impact Factor
  • Transplantation 01/2008; 86. DOI:10.1097/01.tp.0000331104.64081.9c · 3.78 Impact Factor
  • Xenotransplantation 07/2007; 14(4):371 - 371. DOI:10.1111/j.1399-3089.2007.00418_10.x · 1.78 Impact Factor
  • Xenotransplantation 07/2007; 14(4):371-372. DOI:10.1111/j.1399-3089.2007.00418_12.x · 1.78 Impact Factor
  • Annegret Plege, Katja Borns, Reinhard Schwinzer
    Xenotransplantation 07/2007; 14(4):371-371. DOI:10.1111/j.1399-3089.2007.00418_11.x · 1.78 Impact Factor
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    ABSTRACT: CD45, the leucocyte common antigen, is a haematopoietic cell specific tyrosine phosphatase. Human polymorphic CD45 variants are associated with autoimmune and infectious diseases and alter the phenotype and function of lymphocytes, establishing CD45 as an important regulator of immune function. Here we report four patients with diverse diseases with unusual clinical features. All four have the C77G polymorphism of CD45 exon 4, which alters the splicing and CD45RA/CD45R0 phenotype of lymphocytes. We suggest that C77G may be a contributing factor in these unusual cases.
    Clinical & Experimental Immunology 01/2007; 146(3):448-54. DOI:10.1111/j.1365-2249.2006.03230.x · 3.28 Impact Factor
  • Xenotransplantation 11/2006; 13(6):576-587. DOI:10.1111/j.1399-3089.2006.00343.x · 1.78 Impact Factor
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    ABSTRACT: The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single-step transduction to introduce an autoregulative tetracycline-responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue-independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology.
    The FASEB Journal 07/2006; 20(8):1200-2. DOI:10.1096/fj.05-5415fje · 5.48 Impact Factor
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    ABSTRACT: The 77C-->G mutation in exon A of the human CD45 gene occurs with low frequency in healthy individuals. An enhanced frequency of 77C-->G individuals has been reported in cohorts of patients suffering from multiple sclerosis, systemic sclerosis, autoimmune hepatitis, and HIV-1. To investigate the mechanisms by which the variant allele may contribute to disease susceptibility, we compared T cell reactivity in heterozygous carriers of the mutation (healthy individuals and multiple sclerosis patients) and wild-type controls. In vitro-generated T cell lines and freshly isolated CD4+CD45R0+ primed/memory T cells from 77C-->G individuals aberrantly expressed CD45RA isoforms and showed enhanced proliferation and IL-2 production when stimulated with anti-TCR/CD3 mAb or Ag. Mutant T cell lines contained a more active pool of p56lck tyrosine kinase and responded with increased phosphorylation of Zap70 and TCR-zeta and an enhanced Ca2+ flux to TCR/CD3 stimulation. These data suggest that 77C-->G may act as a risk factor for certain diseases by increasing the intensity of TCR signaling.
    The Journal of Immunology 01/2006; 176(2):931-8. DOI:10.4049/jimmunol.176.2.931 · 5.36 Impact Factor
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    ABSTRACT: Major histocompatibility complex class II (MHC II) peptide complexes can associate with lipid rafts, and this is a prerequisite for their recruitment to the immunological synapse and for efficient T cell stimulation. One of the most often used criterion for raft association is the resistance to extraction by the detergent Triton X-100 (TX-100) at low temperature. For MHC II, a variety of detergents have been used under different conditions, leading to variable and often conflicting conclusions about the association of MHC II with detergent-resistant membranes (DRMs). To clarify whether these inconsistencies were caused by variations in the isolation protocols or reflect different biochemical properties of MHC II lipid complexes, we used two standardized procedures for the isolation of membranes resistant to TX-100, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or Brij 98. Our results suggest that some of the reported variations in the association of MHC II with DRMs are caused by differences in the methods. We also show that in our hands, specific and efficient flotation of MHC II and the MHC II-associated invariant chain from mouse B-lymphoma cells was only achieved with Brij 98, but not with TX-100 and CHAPS. We furthermore used DRMs prepared from hen egg lysozyme-fed B-lymphoma cells to activate the T cell hybridoma 3A9. In agreement with our biochemical data, T cell activation could only be achieved with Brij 98- but not with TX-100-resistant membranes. Thus, MHC II and also the invariant chain belong to a set of proteins comprising the T cell receptor, prominin, and the prion protein, which reside in membrane environments distinct from conventional lipid rafts.
    Journal of Leukocyte Biology 12/2005; 78(5):1097-105. DOI:10.1189/jlb.0405189 · 4.30 Impact Factor
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    ABSTRACT: Xenotransplantation of porcine organs into human recipients is a potential option for overcoming the dramatic shortage of suitable donor organs. To date, transgenic modification of pig organs has achieved partial or temporal reduction of xenograft rejection by inhibition of hyperacute rejection. Expression of human tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) in transgenic pigs might be a strategy for controlling posthyperacute rejection mechanisms mediated by cellular components of the immune system. The objective of this study was generation of a transgenic pig model to evaluate the potential of this strategy for xenotransplantation. Transgenic pigs were generated by microinjection of an expression vector for human TRAIL under control of the murine H-2K promoter. Expression of the transgene was analyzed by Western blot and immunohistochemistry. Biologic activity of TRAIL on transgenic porcine lymphocytes was evaluated in co-culture experiments using Jurkat and Hut 78.2 cells as targets. In three lines of transgenic pigs, human TRAIL protein was detected in the membrane fractions of various tissues. Highest expression levels were observed in spleen and lung. Human TRAIL expression on porcine lymphocytes was augmented on activation of cells. Transgenic pig lymphoblasts induced apoptosis in Jurkat and Hut 78.2 cells, which was inhibited by neutralizing anti-TRAIL antibodies, demonstrating a TRAIL-specific effect. Ubiquitous expression of human TRAIL was achieved in transgenic pigs without detrimental side effects. Pigs expressing biologically active human TRAIL will be used for future xenotransplantation experiments to modulate primate anti-pig cellular immune responses.
    Transplantation 08/2005; 80(2):222-30. DOI:10.1097/01.TP.0000164817.59006.C2 · 3.78 Impact Factor
  • The Journal of Heart and Lung Transplantation 02/2004; 23(2). DOI:10.1016/j.healun.2003.11.132 · 5.61 Impact Factor

Publication Stats

1k Citations
352.27 Total Impact Points

Institutions

  • 1990–2014
    • Hannover Medical School
      • • Clinic for General, Abdominal and Transplant Surgery
      • • Department of Gastroenterology, Hepatology and Endocrinology
      Hanover, Lower Saxony, Germany
  • 2011
    • Friedrich Loeffler Institute
      • Institute of Farm Animal Genetics
      Griefswald, Mecklenburg-Vorpommern, Germany
  • 2005
    • Ludwig-Maximilian-University of Munich
      • Chair for Molecular Animal Breeding and Biotechnology
      München, Bavaria, Germany
  • 1987–1991
    • Hochschule Hannover
      Hanover, Lower Saxony, Germany