Ernst Holler, Günther Eissner, Joachim Hahn, Silvia Kirchner, Patricia Ewing-Bartz, Heike Bremm, Silvia Haffner, Simone Planke, Constanze Winter, Verena Burger

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.

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, investigation 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 excision 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

Selected references (1997 to date):

Holler E, Ertl B, Hintermeier-Knabe R, Roncarolo MG, Eissner G, Mayer F, Fraunberger P, Behrends P, Pfannes W, Kolb HJ. Inflammatory reactions induced by pretransplant conditioning - an alternative target for modulation of acute GvHD and complications following allogeneic bone marrow transplantation?
Leukemia & Lymphoma 25:217, 1997

Kolb HJ, Holler E. Adoptive immunotherapy with donor lymphocyte infusions.
Current Opinion in Oncology 9:139, 1997

Salat C, E Holler E, Kolb HJ, Reinhardt B, Pihusch R, W Wilmanns W, Hiller E. Plasminogen Activator Inhibitor-1 confirms the diagnosis of heaptic veno-occlusive disease in patients with hyperbilirubinemia after bone marrow transplantation.
Blood 89,6:2184, 1997

Lindner H, Holler E, Gerbitz A, Johnson JP, Bornkamm GW, Eissner G: Influence of bacterial endotoxin on radiation-induced activation of human endothelial cells in vitro and in vivo: 2. IL-10 protects against transendothelial migration.
Transplantation 64:1370, 1997

Eissner G, Multhoff G, Holler E: Influence of bacterial endotoxin on the allogenicity of human endothelial cells.
Bone Marrow Transplant 21:1286, 1998

Holler E, Kolb HJ, Eissner G, Wilmanns W. Cytokines in GvH and GvL.
Bone Marrow Transplant 22 (Suppl.4):S3, 1998

Holler E, Roncarolo MG, Hintermeier-Knabe R, Eissner G, Ertl B, Schulz U, Knabe H, Kolb HJ, Andreesen R, Wilmanns W: Prognostic significance of increased IL-10 production in patients prior to allogeneic bone marrow transplantation.
Bone Marrow Transplant 25:237, 2000

Eissner G, Kirchner S, Lindner H, Kolch W, Janosch P, Grell M, Scheurich P, Andreesen R, Holler E: Reverse signaling through transmembrane TNF (mTNF) confers resistence to LPS in human monocytes and macrophages (M/MF).
J Immunol 164:6193, 2000

Jarvis M., Schulz U., Dickinson A.M., Sviland L., Jackson G., Konur A., Wang X.N., Hromadnikova I., Kolb H.J., Eissner G., and Holler E. The Detection of Apoptosis in a Human in Vitro Skin Explant Assay for Graft Versus Host Reactions.
J Clin Pathol 55:127, 2001

Eissner G, Multhoff G, Gerbitz A, Kirchner S, Haffner S, Sondermann D, Bauer S, Andreesen R, Holler E: Fludarabine induces apoptosis, activation and allogenicity in human endothelial and epithelial cells, protective role of defibrotide.
Blood 100:334, 2002

Holler E. Cytokines, Viruses, and Graft-Versus-Host Disease.
Curr Opin Hematol 9:479, 2002