EurotransplantBank

Regensburg

Research Activities of the Laboratory for Experimental Allogeneic BMT

Ernst Holler, Günther Eissner, Joachim Hahn, Silvia Kirchner, Patricia Ewing-Bartz, Heike Bremm, Silvia Haffner, Simone Planke, Constanze Winter, Verena Burger,Ute Schulz, Nalini Kumar Vudattu, Susanna Blüml, Matthias Grube, Stephanie Kareth

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For about 30 years allogeneic stem cell transplantation (SCT), among other curative approaches, is a well established method for the treatment of hematological malignancies. However, due to the disparity in major and minor histocompatibility antigens between donor and recipient, transplant-related complications (TRC), such as the graft-versus-host disease (GvHD) may arise. It is likely that the pathophysiology of GvHD consists of three phases. The first "toxic" phase is triggered by conditioning-mediated inflammation and recipient cell apoptosis with a contribution of proinflammatory cytokines. In the second "acute" ("lichenoid") phase one can find a predominance of Th1 cytokines, such as TNF-a and IFN-g accompanied by massive cellular infiltrates in the inflamed tissues. Thirdly, during progression to the third "chronic" ("sclerodermic") phase there is a cytokine switch to the Th2 cytokines IL-4 and IL-10.

Pathophysiology 

In our lab we are addressing the following questions using experimental models as well as translational research on clinical samples (clinical studies are not displayed here):

  • Pathophysiology of endothelial complications with a focus on cytokines and adhesion molecules
  • Monitoring of endothelial complications with functional assays of pts.´sera, isolation and cultivation of pt.-specific endothelial cells by magnetic bead separation and specialized culturing conditions
  • Influence of alternative conditioning regimens on the vitality, activation and allogenicity of endothelial and epithelial cells, inverstigation of protective reagents, such as Defibrotide
  • Animal models for allogeneic stem cell transplantation (SCT), focus on pulmonary and endothelial complications
  • Monitoring of organ-specific alloreactions in the lung (clinical samples); pathophysiology and pathogenesis of pulmonary complications in the late phase post SCT
  • In vitro monitoring for GvHD and GvL by the help of in vitro predictive tests (skin explant assay, cytokine secretions assay, cytokine gene polymorphisms, minor Hag typing)
  • Studies on tolerance induction by regulatory T cells and anergizing dendritic cells
  • Bidirectional cross-talk between monocytes and endothelial cells under normal and proinflammatory conditions, focus on signal transduction mechanisms of tumor necrosis factor alpha (TNF)
  • Assessment of donor-recipient chimerism by the help of an PCR approach for the detection of variable numbers of tandem repeats (VNTR)
  • Minor histocompatibility typing of HLA-A2 positive donors and recipients
  • Analysis of immune recontitution post SCT by the help of T cell receptor excsion circles (TREC) determination

Some of our key findings are:

  • Prognostic significance of TNF and interleukin 10 (IL-10) in the course of BMT
  • Balance between Th1 and Th2 cytokines in GvH and GvL
  • The endothelium is a target organ of BMT conditioning in vivo and in vitro with a crucial involvement of transmembrane TNF (mTNF)
  • mTNF is able to elicit reverse signals in monocytic cells that lead to LPS resistance, this type of anergy is caused by an exhaustion of the signal pathway of mitogen-activated protein kinases (MAPK)
  • Histopathological damage and keratinocyte apoptosis in the skin explant assay correlate with clinical outcome and the development of clincal GvHD in certain diagnostic subgroups
  • Correlation of keratinocyte apoptosis in the skin explant assay and clinical GvHD
  • Fludarabine as a compound of novel conditioning regimens induces activation and damage as well as allogenicity in human endothelial and epithelial cells, protective effect of Defibrotide
  • The presence of endothelial apoptosis inducing factor(s) in pts.´ sera correlates with episodes of clinical endothelial complications, including microangiopathy, and with acute GvHD
  • Single nucleotide loss of function mutations (SNP) in the NOD2/CARD15 gene in both donor and recipient are strongly associated with an increased transplant-related mortality.

                

 

 

Participant 4- U.REG- Günther Eissner, Ph.D., PD,

Department of Hematology and Oncology, University of Regensburg,

Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany

Tel: (49-941) 944-5552, Fax: (49-941) 944-5533

e-mail: guenther.eissner@klinik.uni-regensburg.de

 

Contractural links to other participants

Participant 9 is the Associate Contractor to Participant 4

 

 

Objectives -Functional studies- Use of the immortalised cells for patient monitoring post transplant GvH/GvL reactivity.The use of the cell lines for monitoring patients pre and post donor lymphocyte infusions will aid in the assessment of the effects of the therapy, and aid in improving regimens. The use of the cell lines for monitoring patients pre and post SCT will aid in early diagnosis of GvHD

 

Methodology and study materials Use of the cell lines for post transplant monitoring. Immortalised patient or donor cells will be used as target cells for monitoring patients reactivity to donor cells post transplant or post donor lymphocyte infusions. Standard in vitro cytotoxicity  and proliferation techniques will be used. Results will aid in defining graft versus host or graft versus leukaemia reactivity.

 

Collaborator- Professor Ernst Holler leads the allogeneic stem cell transplant unit in Regensburg and will allow the collection of patient and donor samples and aid in the correlation of results with clinical outcomes.

 

Among the patient-specific cell lines to be obtained an additional approach for the transdifferentiation of endothelial-like cells (ELC) from peripheral blood monocytes1,2 will be followed. As a source of monocytes CD14+-Bead-separated and elutriated monocytes will be used and diffentiated into dendritic cells or directly to endothelial cells according to the cell culturing system depicted in Fig. 1.

 

 

              

 

 

Fig. 1: Monocyte-Endothelial transdifferentiation as a possible source of patient-specific endothelial cells

 

 

First results with blood from healthy donors are promising. Indeed, peripheral blood monocytes can transdifferentiate into ELC, as shown by the positive staining for FactorVIII-related Antigen (vWF), a typical endothelial marker (Fig. 2).

 

 

           

 

 

 

Fig. 2: ELC are positive for endothelial cell markers.

 

Once these cells have been fully charaterized to be of ELC type, ex vivo immortalization procedures will be applied, using the Reverse Transcriptase of human telomerase (hTERT) as published previously.3 The resulting cell lines can then be used for tissue-specific monitoring studies (including apoptosis assays) as described above. This experimental approach gaines importance as data are accumulating that the vascular endothelium is the primary site of conditioning-mediated tissue damage.4

 

Reference List

 

     (1)        Fernandez PB, Lucibello FC, Gehling UM et al. Endothelial-like cells derived from human CD14 positive monocytes. Differentiation. 2000;65:287-300.

 

     (2)        Fernandez PB, Lucibello FC, Zuzarte M et al. Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells. Eur J Cell Biol. 2001;80:99-110.

 

     (3)        Toouli CD, Huschtscha LI, Neumann AA et al. Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit. Oncogene. 2002;21:128-139.

 

     (4)        Paris F, Fuks Z, Kang A et al. Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science. 2001;293:293-297.