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LABORATORY OF HAEMOPOIESIS RESEARCHMedical School-University of Crete

Head: HELEN A. PAPADAKI, MD

BACKGROUND

The growth and differentiation of haemopoietic cells in the bone marrow (BM) is regulat-ed by complex interactions between the cells and their surrounding microenvironment alterna-tively called haemopoiesis supporting stroma. Anatomically, the BM microenvironment is a tri-dimensional structure consisting of cells, vessels, extracellular matrix (ECM) proteins such asadhesive glycoproteins and structural proteoglycans, extracellular fibres such as collagen andelastin and cytokines such as growth factors and inhibitors of haemopoiesis. In this tridimen-sional structure and especially, in highly organized compartments known as “niches”, complexcell-cell, cell-ECM, cell-cytokine interactions regulate the fate of haemopoitic stem cells interms of self-renewal, differentiation, proliferation or apoptosis. We currently know that themajority of the haemopoiesis supporting stromal cells derive from a distinct progenitor, differ-ent of the haemopoietic stem cell, the mesenchymal stem cell (MSC). This cell displays a highdegree of “plasticity” it terms of the potential to generate cells of varying lineages such as chon-drocytes, osteoblasts, adipocytes and muscle cells. This cell is a novel therapeutic tool in degen-erative medicine. And while the MSCs display immunosuppressive properties, periphery-derived, inappropriately activated immune cells such as monocytes and lymphocytes may set-tle in the BM microenvironment and may damage the growth and survival of haemopoietic cellsin the niches through cell-to-cell contact interaction or through production of inhibitorycytokines. Such intricate interactions between the immune and haemopoietic system mayresult in marrow failure affecting one or more haemopoietic lineages and may even acceleratethe process of clonal disorders of haemopoiesis. On the basis of this knowledge, current thera-peutic approaches for clonal haemopoietic diseases target elements of the BM microenviron-ment rather that the clonal cells per se. Overall, the haemopoietic and the immune systems aremutually related.

RESEARCH ACTIVITIES

The Haemopoiesis Research Laboratory of the Medical School of Crete is linked to theHaematology Department of the University Hospital of Heraklion. The researchers and stu-dents work under the direct supervision of Dr Helen Papadaki who shares the time between theresearch and clinical arms of Haematology and also delivers a regular series of core lectures inBasic Immunology, Haematology and Pathophysiology. The Translation Research performedin Dr Papadaki’s Laboratory is focused on the biologic characteristics of the haemopoietic stemcells and MSCs and the role of the BM microenvironment in immune-mediated and clonal BMfailure syndromes using highly sophisticated culture assays, molecular techniques, flow cytom-etry, cytogenetics, Fluorescence In Situ Hybridization (FISH) and immunohistochemistry.

1. Biology of Mesenchymal Stem CellsFrom bench to bedside BM: MSCs are being considered as potential therapeutic agents in

various inflammatory autoimmune diseases for their tissue-repair and anti-inflammatory tis-sue-protective properties. In this context, during the last two years we have investigated thereserves and function, the molecular and proteomic profile and the differentiation potential ofBM MSCs in patients with rheumatoid arthritis using flow cytometry, apoptosis and prolifera-tion studies, colony assays for cell differentiation towards the osteogenic, chondrogenic andadipogenic lineages and molecular and cytochemical techniques for cell identification. In col-

laboration with Reference Laboratories we have performed a proteomic and transcriptomeanalysis for identification of differences between patients and normal subjects.

Immune-mediated BM failure syndromes: MSCs normally display immune-suppressiveproperties. In immune-mediated BM failure syndromes such as hypoplastic MyelodysplasticSyndromes and hypoplastic neutropenias, MSCs may have a role in the pathophysiologicprocess of disease development by displaying defective immune-regulatory properties, by pro-ducing abnormal cytokines or even by participating in the abnormal clone. T-lymphocyte pro-liferation assays in the presence of MSCs from healthy subjects and patients with BM failure,evaluation of cytokines levels in MSC culture supernatants, study of the mean telomere lengthin normal and disease states, and comparative cytogenetic evaluation of haemopoietic andMSCs in patients with Myelodysplastic syndromes are projects under investigation.

2. Biology of Haemopoietic Stem CellsMechanisms regulating the survival/apoptotic characteristics of BM haemopoietic cells in

early and late stages of differentiation in normal and disease states are under investigation withparticular interest in the role of TNF/TNF-Receptor family members such as FasL/Fas andCD40L/CD40. In this context, we have defined the pathophysiologic basis of a previously“orphan” disease, the chronic idiopathic neutropenia by demonstrating increased Fas mediat-ed, TNF-induced apoptosis of the neutrophil early progenitor cells in the BM. We have also pro-posed as a new mechanism in the pathophysiology of anaemia of chronic inflammation, alsoknown as anaemia of chronic disease, the TNF·-mediated increased apoptosis of the BM ery-throid progenitor/precursor cells through downregulation of erythropoietin receptor.Translating these data into the clinic we have showed the beneficial effect of the in vivo anti-TNF· therapy in treating anaemia in patients with chronic inflammation and we have alsoinvestigated the effect of this treatment in patients with Myelodysplastic syndromes andincreased levels of TNF· in the BM. The effect of the mutual interactions between TNF/TNF-Receptor family members in the reserves of BM stem cells in patients with autoimmune disor-ders undergoing stem cell transplantation is also an interesting field of investigation.

3. Interactions between Haemopoietic Stem Cells-BM microenvironment,Chronic Idiopathic Neutropenia: The pathophysiology of this disease remains our mainresearch interest. An inflammatory BM milieu has been shown to mainly contribute to thepathophysiology of the disease. Activated T-lymphocytes with myelosuppressive properties andpro-apoptotic mediators such as IFNÁ, TNF·, FasL, and TGF‚‚1 result in accelerated apoptosisof the granulocytic progenitor cells. The decreased levels of the anti-inflammatory cytokine IL-

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10 disturb further the balance between the survival and pro-apototic mediators. The role ofviruses, the implication of specific TLR expression, investigation for polymorphisms in inflam-matory cytokine genes and analysis of the CDR region of the TCR in association with specificHLA background, are projects under investigation.

Translational Research in Myelodysplastic syndromes: MyelodysplasticSyndromes comprise a heterogeneous group of haematological malignancies characterized bythe paradox of BM hyperplasia with blood cytopenias due to abnormal, clonal proliferation ofa pluripotent haemopoietic stem cell. Recent evidence suggests that an abnormal BM microen-vironment may be involved in the pathogenesis of the disease through inflammatory cytokineproduction, abnormal angiogenesis and increased level of apoptosis. The effect of the treatmentwith anticytokine and immune-modulating agents by combining clinical data and research datafrom the in vitro BM studies is of particular interest in our laboratory.

REPRESENTATIVE PUBLICATIONS

1. Koumaki V, Damianki A, Ximeri M, Pontikoglou C, Axioti F, Spanoudakis M, EliopoulosGD, Papadaki HA. Pro-inflammatory bone marrow milieu in patients with chronic idiopathicneutropenia is associated with impaired local production of interleukin-10. Br J Haematol.2006; 135:570-573.

2. Psyllaki M, Damianaki A, Gemetzi C, Pyrovolaki K, Eliopoulos GD, Papadaki HA.Impaired megakaryopoiesis in patients with chronic idiopathic neutropenia is associated withincreased transforming growth factor beta1 production in the bone marrow. Br J Haematol.2006; 134:624-631

3. Pontikoglou C, Liapakis G, Pyrovolaki K, Papadakis M, Bux J, Eliopoulos GD, PapadakiHA. Downregulation of erythropoietin receptors in bone marrow erythroid cells as an addition-al mechanism of anemia in patients with Chronic Idiopathic Neutropenia. Exp Hematol2006;34:1312-1322.

4. Boula A, Voulgarelis M, Giannouli S, Katrinakis G, Psyllaki M, Pontikoglou C, MarkidouF, Eliopoulos GD, Papadaki HA. Effect of cA2 anti-tumor necrosis factoralpha antibody thera-py on hematopoiesis of patients with myelodysplastic syndromes. Clin Cancer Res2006;15:3099-3108

5. Papapadaki HA, Stamatopoulos K, Damianaki A, Gemetzi C, Anagnostopoulos A,Eliopoulos AG, Papadaki T, Eliopoulos GD. Activated T-lymphocytes with myelosuppressiveproperties in patients with chronic idiopathic neutropenia. Brit J Haematol 2005; 128: 863-8766. Papadaki HA, Eliopoulos AG, Kosteas T, Gemetzi C, Damianaki A, Koutala H, Bux J,Eliopoulos GD. Impaired granulocytopoiesis in chronic idiopathic neutropenia is associatedwith increased apoptosis of bone marrow myeloid progenitor cells. Blood 2003; 101: 2591-2600

7. Papadaki HA, Kritikos HD, Valatas V, Boumpas DT, Eliopoulos GD. Anemia of chronicdisease in rheumatoid arthritis is associated with increased apoptosis of bone marrow ery-throid cells. Improvement following anti-tumor necrosis factor-· antibody therapy. Blood2002; 100: 474-482.

8. Papadaki HA, Kritikos HD, Gemetzi C, Koutala H, Marsh JCW, Boumpas DT,Eliopoulos GD. Bone marrow stem cell reserve and function and stromal cell function aredefective in rheumatoid arthritis. Evidence for a tumor necrosis factoralpha-mediated effect.Blood 2002; 99:1610-1619.

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GROUP MEMBERS

Helen Papadaki, MD, PhD, (Head)George Eliopoulos, MD, PhD, DSc, Emeritus ProfessorCharis Pontikoglou, BSc, MD, PhD, (Post-Doc)Mirjam Klaus, MD (Post-Doc)Claudia Gemetzi, TechnicianAthina Dmianaki, TechnicianMaria-Christina Kastrinaki, BSc (PhD student)Maria Ximeri, MD (PhD student)Maria Velegraki, MD (PhD student)Irene Mavroudi, BSc (PhD student)Katerina Pyrovolaki, MD (PhD student)Michael Spanoudakis, MD (PhD student)Dimitrios Eliopoulos, MD (PhD student)

FUNDING

Association for International Cancer Research, UK6th Framework Program, EUGeneral Secretary of Research and Technology, GreeceLenalidomide study on MDS, Genesis Pharmaceutical Company

COLLABORATORS

Pierre Charbord, University Francois Rabelais, Tours, France.Thomas Häupl, Charité Universitätsmedizin, Berlin, GermanyStephan Roche, Institut de Génétique Humaine, Montpellier, FranceJuergen Bux, Deutsches Rotes Kreuz Blood Service West, Hagen, Germany

CONDUCT INFORMATION

Helen A. PapadakiProfessor of HaematologyDepartment of HaematologyUniversity Hospital of HeraklionPO Box 1352, Heraklion, Crete, GreeceTel: +30 2810 394629, Fax: +30 2810 394632e-mail: epapadak@med.uoc.gr

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