Low-dose irradiation prior to bone marrow transplantation results in ATM activation and increased lethality in Atm-deficient mice.

Ataxia telangiectasia is a genetic instability syndrome characterized by neurodegeneration, immunodeficiency, severe bronchial complications, hypersensitivity to radiotherapy and an elevated risk of malignancies. Repopulation with ATM-competent bone marrow-derived cells (BMDCs) significantly prolonged the lifespan and improved the phenotype of Atm-deficient mice. The aim of the present study was to promote BMDC engraftment after bone marrow transplantation using low-dose irradiation (IR) as a co-conditioning strategy. Atm-deficient mice were transplanted with green fluorescent protein-expressing, ATM-positive BMDCs using a clinically relevant non-myeloablative host-conditioning regimen together with TBI (0.2-2.0 Gy). IR significantly improved the engraftment of BMDCs into the bone marrow, blood, spleen and lung in a dose-dependent manner, but not into the cerebellum. However, with increasing doses, IR lethality increased even after low-dose IR. Analysis of the bronchoalveolar lavage fluid and lung histochemistry revealed a significant enhancement in the number of inflammatory cells and oxidative damage. A delay in the resolution of γ-H2AX-expression points to an insufficient double-strand break repair capacity following IR with 0.5 Gy in Atm-deficient splenocytes. Our results demonstrate that even low-dose IR results in ATM activation. In the absence of ATM, low-dose IR leads to increased inflammation, oxidative stress and lethality in the Atm-deficient mouse model.

RUNX1/ETO blocks selectin-mediated adhesion via epigenetic silencing of PSGL-1.

RUNX1/ETO (RE), the t(8;21)-derived leukemic transcription factor associated with acute myeloid leukemia (AML) development, deregulates genes involved in differentiation, self-renewal and proliferation. In addition, these cells show differences in cellular adhesion behavior whose molecular basis is not well understood. Here, we demonstrate that RE epigenetically silences the gene encoding P-Selectin Glycoprotein Ligand-1 (PSGL-1) and downregulates PSGL-1 expression in human CD34+ and murine lin- hematopoietic progenitor cells. Levels of PSGL-1 inversely and dose-dependently correlate with RE oncogene levels. However, a DNA-binding defective mutant fails to downregulate PSGL-1. We show by ChIP experiments that the PSGL-1 promoter is a direct target of RE and binding is accompanied by high levels of the repressive chromatin mark histone H3K27me3. In t(8;21)+ Kasumi-1 cells, PSGL-1 expression is completely restored at both the mRNA and cell surface protein levels following RE downregulation with short hairpin RNA (shRNA) or RE inhibition with tetramerization-blocking peptides, and at the promoter H3K27me3 is replaced by the activating chromatin mark H3K9ac as well as by RNA polymerase II. Upregulation of PSGL-1 restores the binding of cells to P- and E-selectin and re-establishes myeloid-specific cellular adhesion while it fails to bind to lymphocyte-specific L-selectin. Overall, our data suggest that the RE oncoprotein epigenetically represses PSGL-1 via binding to its promoter region and thus affects the adhesive behavior of t(8;21)+ AML cells.

Activating c-KIT mutations confer oncogenic cooperativity and rescue RUNX1/ETO-induced DNA damage and apoptosis in human primary CD34+ hematopoietic progenitors.

The RUNX1/ETO (RE) fusion protein, which originates from the t(8;21) chromosomal rearrangement, is one of the most frequent translocation products found in de novo acute myeloid leukemia (AML). In RE leukemias, activated forms of the c-KIT tyrosine kinase receptor are frequently found, thereby suggesting oncogenic cooperativity between these oncoproteins in the development and maintenance of t(8;21) malignancies. In this report, we show that activated c-KIT cooperates with a C-terminal truncated variant of RE, REtr, to expand human CD34+ hematopoietic progenitors ex vivo. CD34+ cells expressing both oncogenes resemble the AML-M2 myeloblastic cell phenotype, in contrast to REtr-expressing cells which largely undergo granulocytic differentiation. Oncogenic c-KIT amplifies REtr-depended clonogenic growth and protects cells from exhaustion. Activated c-KIT reverts REtr-induced DNA damage and apoptosis. In the presence of activated c-KIT, REtr-downregulated DNA-repair genes are re-expressed leading to an enhancement of DNA-repair efficiency via homologous recombination. Together, our results provide new mechanistic insight into REtr and c-KIT oncogenic cooperativity and suggest that augmented DNA repair accounts for the increased chemoresistance observed in t(8;21)-positive AML patients with activated c-KIT mutations. This cell-protective mechanism might represent a new therapeutic target, as REtr cells with activated c-KIT are highly sensitive to pharmacological inhibitors of DNA repair.

[Correct preparation of a transfusion: Part 1]. / Korrekte Vorbereitung der Transfusion: Teil 1.

The administration of blood products is strictly regulated. Several weeks before the operation the preparation for transfusion begins with optimizing the patient's hematological and hemostaseological situation. In elective surgery blood group testing and antibody screening are performed soon after admission of the patient. The identification of the blood sample is important. Informed consent of the recipient has to be obtained. On the day before the operation a further blood sample is necessary for cross-matching if red blood cells are to be transfused. Usually blood products are issued for immediate administration. Before transfusion begins the blood product has to be checked, the identity of the patient must be controlled and in the case of red blood cell transfusions the AB0 bedside test has to be performed.

[Correct performance of transfusion]. / Korrekte Durchführung der Transfusion.

The administration of blood products is strictly regulated. Warming of blood components at body temperature is required only in rare cases. Addition of drugs to blood products is not allowed. During transfusion the monitoring of the patient is continued. In the case of an adverse event, exclusion of acute hemolysis is very important. As emergency transfusions have a higher risk than standard transfusions, their indications have to be restricted. When transfusion is completed the blood bag has to be preserved for 24 h. The effects of the blood transfusion have to be controlled. The administration of blood products must be documented to allow a possible cross-check from the recipient to the donor as well as from the donor to the recipient. The disposal of administered and of non-administered blood components is subject to the guidelines for hospital waste.

Therapeutic potential of intravenously administered human mesenchymal stromal cells.

Mesenchymal stem cells (MSC) represent a stem and progenitor cell population that has been shown to promote tissue recovery in pre-clinical and clinical studies. The study of MSC migration following systemic infusion of exogenous MSC is difficult. The challenges facing these efforts are due to a number of factors, including defining culture conditions for MSC, the phenotype of cultured MSC, the differences observed between cultured MSC and freshly isolated MSC. However, even if, MSC populations consist of a mixture of stem and more committed multipotent progenitors, it remains probable that these cell populations are still useful in the clinic as discussed in this review.

MCF7-MSC co-culture assay: approach to assess the co-operation between MCF-7s and MSCs in tumor-induced angiogenesis.

The multipotent stromal cells (MSCs) exhibit a broad differentiation potential. MSCs might participate in neovascularization through their ability to migrate and generate capillary-like structures. These processes were shown to be modulated by tumor angiogenic factors, such as Vascular Endothelial Growth Factor (VEGF). The aim of our study was to define the way the MCF-7 cell line (MCF-7s) influenced the MSCs' recruitment for the tumor-induced angiogenesis, and to assess the role of VEGF in this process. We tested the chemotactic potential of plasma or VEGF, but also of MCF-7s or their conditioned medium (CM) in the MSCs' transmigration. We compared the migratory potential of MSCs, MSCEs (MSCs cultured in endothelial cell growth medium) and HUVECs. Recombinant VEGF has been shown to chemoattract MSCs, although to a lesser degree than plasma or serum containing medium alone. Moreover, it changed the MSCs' morphology, stimulating the appearance of longer and thinner prolongations as compared to plasma. MCF-7s or their CM both directly induced migration of MSCs. Surprisingly, CM augmented with MCF-7s attracted less cells than the control medium itself, but CM augmented or not with MCF-7s changed the morphology of MSCs in a manner similar to VEGF. The migratory behavior of the MSCEs was comparable to that of HUVECs, while their morphology could be considered intermediate between MSCs and HUVECs, as they developed shorter prolongations than MSCs, but much longer than HUVECs in the corresponding wells. In conclusion, both tumor cells and VEGF alter the migration behavior of MSCs in a transmigration model, indicating a role of tumor cell-derived VEGF to modulate the recruitment of MSCs into sites of angiogenesis.

[Conventional vs pathogen-inactivated platelet concentrates for the treatment of perioperative coagulopathy. A prospective cohort study]. / Konventionelle vs. pathogeninaktivierte Thrombozytenkonzentrate bei perioperativer Koagulopathie. Eine prospektive Kohortenstudie.

BACKGROUND: The aim of the present study was to assess ex-vivo function of pathogen-inactivated versus conventional platelet concentrates (PC) in the perioperative setting. MATERIAL AND METHODS: A total of 30 patients who underwent cardiac surgery and who postoperatively depended on the transfusion of two platelet concentrates were enrolled into this study. Of the patients 15 received conventional buffy coat PC (conv. PC) and 15 received pathogen-inactivated PC (PI-PC). Age, volume and platelet content of each PC were recorded. Before (T0) and 30 min after PC transfusion (T1), blood samples were taken and platelet function analyses (MEA) and conventional laboratory coagulation analyses were performed. The transfusion-associated increment of platelet concentration (increment) and the corrected count increment (CCI) were assessed at timepoint T1. RESULTS: There were no significant group differences between the groups in MEA analyses or conventional laboratory at T0 or T1. The platelet content per PC was significantly higher in the PI-PC group [3.3 (3.1/3.5)× 10(11) platelets per PI-PC versus 3 (2.9/3)× 10(11) platelets per conv. PC, p<0.001]. Platelet increment (42±27×10(9)/l versus 69.4±29×10(9)/l, p=0.013) was significantly lower in the PI-PC group. CONCLUSION: Whereas ex-vivo analyses of platelet function did not show any group differences at T1, a significantly lower increment was seen in the pilot study after transfusion of PI-PC as compared to conventional PC.

Human but not murine multipotent mesenchymal stromal cells exhibit broad-spectrum antimicrobial effector function mediated by indoleamine 2,3-dioxygenase.

Human multipotent mesenchymal stromal cells (MSCs) exhibit multilineage differentiation potential, support hematopoiesis, and inhibit proliferation and effector function of various immune cells. On the basis of these properties, MSC are currently under clinical investigation in a range of therapeutic applications including tissue repair and immune-mediated disorders such as graft-versus-host-disease refractory to pharmacological immunosuppression. Although initial clinical results appear promising, there are significant concerns that application of MSC might inadvertently suppress antimicrobial immunity with an increased risk of infection. We demonstrate here that on stimulation with inflammatory cytokines human MSC exhibit broad-spectrum antimicrobial effector function directed against a range of clinically relevant bacteria, protozoal parasites and viruses. Moreover, we identify the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) as the underlying molecular mechanism. We furthermore delineate significant differences between human and murine MSC in that murine MSC fail to express IDO and inhibit bacterial growth. Conversely, only murine but not human MSC express inducible nitric oxide synthase on cytokine stimulation thus challenging the validity of murine in vivo models for the preclinical evaluation of human MSC. Collectively, our data identify human MSC as a cellular immunosuppressant that concurrently exhibits potent antimicrobial effector function thus encouraging their further evaluation in clinical trials.

How much blood is needed?

Demographic changes in developed countries as their populations age lead to a steady increase in the consumption of standard blood components. Complex therapeutic procedures like haematopoietic stem cell transplantation, cardiovascular surgery and solid organ transplantation are options for an increasing proportion of older patients nowadays. This trend is likely to continue in coming years. On the other hand, novel aspects in transplant regimens, therapies for malignant diseases, surgical procedures and perioperative patient management have led to a moderate decrease in blood product consumption per individual procedure. The ageing of populations in developed countries, intra-society changes in the attitude towards blood donation as an important altruistic behaviour and the overall alterations in our societies will lead to a decline in regular blood donations over the next decades in many developed countries. Artificial blood substitutes or in vitro stem cell-derived blood components might also become alternatives in the future. However, such substitutes are still in early stages of development and will therefore probably not alleviate this problem within the next few years. Taken together, a declining donation rate and an increase in the consumption of blood components require novel approaches on both sides of the blood supply chain. Different blood donor groups require specific approaches and, for example, inactive or deferred donors must be re-activated. Optimal use of blood components requires even more attention.

The t(6;9) associated DEK/CAN fusion protein targets a population of long-term repopulating hematopoietic stem cells for leukemogenic transformation.

The t(6;9)-positive acute myeloid leukemia (AML) is classified as a separate clinical entity because of its early onset and poor prognosis. The hallmark of t(6;9) AML is the expression of the DEK/CAN fusion protein. The leukemogenic potential of DEK/CAN has been called into question, because it was shown to be unable to block the differentiation of hematopoietic progenitors. We found that DEK/CAN initiated leukemia from a small subpopulation within the hematopoietic stem cell (HSC) population expressing a surface marker pattern of long-term (LT) HSC. The propagation of established DEK/CAN-positive leukemia was not restricted to the LT-HSC population, but occurred even from more mature and heterogeneous cell populations. This finding indicates that in DEK/CAN-induced leukemia, there is a difference between 'leukemia-initiating cells' (L-ICs) and 'leukemia-maintaining cells' (L-MCs). In contrast to the L-IC cells represented by a very rare subpopulation of LT-HSC, the L-MC seem to be represented by a larger and phenotypically heterogeneous cell population.

Basic research and clinical applications of non-hematopoietic stem cells, 4-5 April 2008, Tubingen, Germany.

From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to present and discuss the latest proceedings in research on non-hematopoietic stem cells (NHSC). This report presents issues of basic research including characterization, isolation, good manufacturing practice (GMP)-like production and imaging as well as clinical applications focusing on the regenerative and immunomodulatory capacities of NHSC.

Accumulation of soluble inflammatory mediators between blood donation and pre-storage leucocyte depletion.

BACKGROUND AND OBJECTIVES: Leucocyte-derived cytokines accumulate in stored whole blood. Pre-storage leucocyte depletion has reduced cytokine levels and, consequently, febrile non-haemolytic transfusion reactions. As leucocyte filtration and component separation can be performed until 24 h after donation, we hypothesized that within this time, inflammatory cytokines might accumulate. MATERIALS AND METHODS: Serial plasma samples were collected 4, 10 and 20 h after donation and cytokine concentrations were measured. RESULTS: Interleukin-8 increased > 20-fold and soluble CD40 ligand > sixfold during the observation time, less pronounced changes for several other mediators were also observed. CONCLUSION: Leucocyte depletion within 10 h of blood donation will reduce the concentrations of pyrogenic mediators.

Analysis of leukocyte binding to depletion filters: role of passive binding, interaction with platelets, and plasma components.

Since limited knowledge exists on the mechanisms which regulate cell binding to leukocyte removal filter surfaces, we investigated the binding patterns of leukocytes to individual layers of leukocyte depletion filters. After passage of 1 unit of whole blood, blotting of isolated filter layers on glass slides or elution of cells from filter layers revealed that most leukocytes were located within the first 10 of a total of 28 filter layers, peaking at layers 6 to 8, with granulocytes binding on average to earlier filter layers than lymphocytes. Leukocytes preincubated with inhibitors of actin activation showed unchanged distribution between filter layers, suggesting that cytoskeletal activation does not significantly contribute to their binding. When leukocytes were directly incubated with single filter layers, binding of up to 30% of input cells was recorded in the absence of Ca(2+). Immunohistological analyses showed colocalization of platelets and leukocytes, with co-clustering of platelets and leukocytes. Monocytes and to some degree lymphocytes but not granulocytes competed with platelets for filter binding. Precoating of filter layers with individual plasma components showed that hyaluronic acid, plasma type fibronectin, and fibrinogen all increased the binding of leukocytes compared with albumin coating. In conclusion, leukocytes can bind passively to filters in a process which does not require Ca(2+), which is independent of cytoskeletal activation and which may depend on individual plasma components. These results are of importance when new selective cell enrichment or depletion strategies through specific filters are envisaged.

Plasma quality after whole-blood filtration depends on storage temperature and filter type.

The study evaluated the quality of plasma obtained after whole-blood filtration with four different polyester filters and one polyurethane filter. The activities of coagulation factors and proteinase inhibitors were not or only negligibly affected by filtration, in all experiments. Filtration did not increase markers of clotting and fibrinolysis. Only a strong neutrophil and complement activation was observed, which depended on the type of filter and whole-blood storage conditions. However, as neutrophil elastase-specific degradation products did not increase and the complement-derived anaphylatoxin C3a was found in its inactivated form, C3a-desArg, these filtration-dependent changes apparently have little impact on the therapeutic quality of whole-blood-filtered fresh frozen plasma for transfusion.

A mouse model to study organ homing behaviour of haemopoietic progenitor cells reveals high selectivity but low efficiency of multipotent progenitors to home into haemopoietic organs.

To study the homing behaviour of an enriched multipotent primitive haemopoietic progenitor cell (HPC) population in mice, undifferentiated murine factor-dependent multipotent HPCs (FDCP-mix), stably transfected with the green fluorescence protein gene, were intravenously injected into congenic mice. After 2 or 24 h, cell suspensions were prepared from bone marrow, spleen, lung, liver, muscle, colon, kidney, brain or blood of the mice and analysed by flow cytometry. Using direct quantifiable determination of total HPC numbers homed per organ and a method to estimate the degree of organ contamination by HPC that were present in blood vessels within the organs before preparation, the highest absolute numbers of HPC were detected in the liver and lungs at 2 h but this was sharply decreased at 24 h, whereas HPC selectively accumulated in the bone marrow and spleen at 24 h after transplantation. Only a few HPC were detected in other organs. The seeding efficiency of homed FDCP-mix HPC to the bone marrow and spleen was approximately 1.5% and ranged between that of primary whole bone marrow cells and lineage-depleted freshly isolated bone marrow cells. Pretreatment of HPC with inhibitors of signal transduction indicated that short-term homing of multipotent HPC into haemopoietic organs is an active process requiring co-ordinated intracellular signalling through Rho family small GTPases and protein kinases. Thus, short-term homing of FDCP-mix HPC into haemopoietic organs is of low efficiency but high selectivity, and provides a system to analyse the mechanisms and manipulation of primitive HPC which saves large numbers of donor animals.

Rho family small GTPases control migration of hematopoietic progenitor cells into multicellular spheroids of bone marrow stroma cells.

Seeding of hematopoietic progenitor cells (HPC) into the bone marrow requires a complex interaction between cell membrane and adhesion systems and cell signaling pathways. We established a multicellular, spheroid coculture model to study HPC migration in a three-dimensional stromal environment. Here, entry of primary CD34(+) cells into stroma cell spheroids was independent of the integrins very late antigen (VLA)-4, VLA-5, lymphocyte function-associated antigen-1, and the chemokine receptor CXCR4. Experiments using a panel of bacterial toxins selectively targeting key regulators of cellular locomotion, the Rho family small GTPases Rho, Rac, and Cdc42, revealed a considerable reduction or even abrogation of TF-1 cell migration without an increase of apoptosis or impairment of proliferation. Pertussis toxin, an inhibitor of Galpha(i) proteins, showed a similar effect. In some in vitro invasion assays, phosphatidylinositol-3 kinase (PI-3K) was shown to mediate Rac- and Cdc42-induced cell motility and invasion. However, inhibition of the PI-3K pathway by LY294002 did not impair TF-1 cell migration in our three-dimensional model system.