[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.

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.

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.

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.

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.

Biological and clinical advances in stem cell expansion.

One of the most exciting areas of hematological research is the ex vivo expansion of peripheral blood progenitor cells (PBPC). Several groups clearly demonstrated that the ex vivo expansion of hematopoietic progenitor cells is possible in the presence of various cytokine combination and/or different feeder layer models. This minireview summarizes recent developments in ex vivo expansion systems as well as first clinical applications.

Screening for expression of cytokines with hematopoietic growth factor activity by permanent human B-cell lines.

Using colony assays in semi-solid media, several investigators have shown that supernatants (SN) of normal and malignant human B-cells can stimulate the growth of granulocyte-macrophage (GM) progenitor cells. So far macrophage colony-stimulating factor (M-CSF) and interleukin-6 (IL-6) have been identified as potential colony-stimulating activity (CSA) present in B-cell SN. However, other CSAs such as GM-CSF, G-CSF, IL-1-beta, IL-3, and IL-4 may also be candidates in this respect. Several human B-cell lines (CL) were screened for the expression of the respective genes at the mRNA and protein level. Constitutive production of GM-CSF was detected in the lymphoblastoid CL Wi-L2-729-HF2 and in the Burkitt line Raji. The signal intensity of specific transcripts and the amount of protein being secreted increased upon exposure to the phorbol ester PMA. The hybridoma line HB-564 also expressed the GM-CSF gene, but required prior stimulation with PMA. 3H-thymidine incorporation of Raji and Wi-L2-729-HF2 cells was unchanged in the presence or absence of a specific neutralizing sheep anti-GM-CSF serum, suggesting that GM-CSF did not serve as an extracellular autocrine growth factor. The expression of the GM-CSF gene was independent of the proliferative state (log phase growth versus plateau phase growth) and of the presence of serum in cultures of the respective CL. The expression of G-CSF, IL-1-beta, IL-3, and IL-4 genes was not detectable in the CL at the mRNA level.

The human lysozyme gene undergoes stepwise demethylation during phagocyte maturation.

The lysozyme (LZM) gene provides a very useful model for studies of phagocyte maturation, because its protein synthesis is increased during myelopoiesis and thus most abundant in terminally differentiated and activated phagoyctes. LZM gene expression and DNA methylation were examined in various normal and transformed hematopoietic cells. Two shifts toward LZM gene demethylation coincided with upregulation of expression: activation of expression in myeloid precursor cells was associated with significant demethylation at a CpG dinucleotide within an Alu repeat in the 5' flanking region; high-level expression in different types of mature phagocytic cells was associated with complete demethylation at two additional, intragenic CpG sites contained in Alu sequences. The possibility that methylation changes occurring within the 5' region of the human lysozyme gene could be involved in the transcription of this gene is discussed, as well as a possible role for demethylation in the maintenance of distinct maturation stages during phagocyte development.

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.

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.

Efficient serum-free retroviral gene transfer into primitive human hematopoietic progenitor cells by a defined, high-titer, nonconcentrated vector-containing medium.

Defined serum-free conditions have great conceptual advantages for the biological safety and standardization of clinical gene transfer into hematopoietic stem cells. In the only study reported to date, Sekhar et al. achieved low serum conditions by a complex concentration procedure of a retroviral supernatant initially containing 10% fetal bovine serum. The high cost, small volume, possible coenrichment of serum-derived pathogens, limited recovery of vector particles, and low titer of the final diluted medium restrict the clinical application of this procedure. Transduction of primitive hematopoietic progenitor cells was not demonstrated. In the present study, a defined serum-free medium containing high titers of the pseudotyped retroviral vector PG13/LN was generated from PG13/LN producer cells without requiring a physical enrichment procedure. The transduction of committed hematopoietic progenitor cells in the serum-free vector-containing medium was efficient, and similar to that occurring under serum-containing control conditions. The number of primitive human hematopoietic long-term culture-initiating cell-derived colonies (LTC-IC-derived colonies) generated from CD34+ and CD34+/HLA-DRlo peripheral blood progenitor "stem" cells (PBSCs) increased during 7 days of treatment in this vector-containing medium in the presence of IL-3, SCF, and flt-3 ligand. The described procedure allowed efficient transduction of LTC-IC-derived colonies generated from CD34+, CD34+/HLA-DRlo, and CD34+/CD38lo PBSCs. This is the first report to demonstrate an increase in primitive peripheral blood LTC-IC-derived colonies in vitro as well as their efficient transduction in a high-titer, serum-free vector-containing medium that can be produced exclusively from defined pharmaceutical-grade components, making it ideally suited for applications in clinical gene therapy.

Umbilical cord blood: an alternative to the transplantation of bone marrow stem cells.

Recent in vitro analyses of human UCB have demonstrated the potential of UCB as a source for haematopoietic stem and progenitor cell harvest. Clinical data have further indicated that UCB can be given in vivo to fully and partially HLA-matched siblings or non-familial recipients for marrow reconstitution in genetic disorders as well as malignancies. In comparison to adult peripheral blood, UCB displayed decreased immune responses to alloantigens and was enriched in the numbers of CD34+ progenitor cells with high proliferative and long-term marrow reconstituting potential. Cord blood banks now store large transplantable resources of UCB that are analysed with respect to immunological parameters. Cryopreserved UCB cells may fill the gap in finding a stem-cell transplant for patients who lack a matched related or unrelated donor when a bone marrow transplant is needed.

Upstream elements bestow T-cell and haemopoietic progenitor-specific activity on the granzyme B promoter.

Cytotoxic T cells and early haemopoietic progenitors share the expression of a number of specific genes. Of these, granzyme B has attracted particular interest because of its role in inducing apoptosis during cytotoxic T cell-mediated target cell killing, and its potential role in the mobilisation and homeostasis of haemopoietic stem cells. Studies of granzyme B regulation should therefore yield valuable information concerning the molecular control of these processes, and also identify elements capable of directing gene expression to two cell types of relevance to gene therapy. Here we show that proximal regulatory elements already known to direct promoter activity in T cells are similarly active in haemopoietic progenitors. However, this activity is not strictly specific, since the promoter regions also direct low levels of reporter gene expression in fibroblasts. More importantly, we also report the presence of two previously unidentified clusters of DNaseI hypersensitive sites upstream from the murine granzyme B gene, and show that these regions impart both increased transcriptional activity and the appropriate cell type specificity on the granzyme B promoter. These upstream regulatory regions are therefore likely to play a key role in the coordination of granzyme B expression in vivo.

Regulation of gene expression of macrophage-colony stimulating factor in human fibroblasts by the acute phase response mediators interleukin (IL)-1 beta, tumor necrosis factor-alpha and IL-6.

Fibroblasts constitute a major element of the bone marrow stroma. They play a pivotal role in blood cell development by providing the scaffolding required for cellular organization and tissue cohesion and by producing soluble molecules including colony stimulating factors (CSFs) and various interleukins regulating hematopoiesis. Our data demonstrate that the acute phase response mediators interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha and IL-6 which are abundantly produced by activated monocytes, enhance levels of macrophage-colony stimulating factor (M-CSF) in fibroblasts by both transcriptional and post-transcriptional mechanisms. The action of these proteins to induce M-CSF transcript levels was dependent on synthesis of new proteins and was not mediated by protein kinase C (PKC) stimulation as depletion of cellular PKC pools by prolonged exposure of fibroblasts to phorbolester TPA did not prevent factor induced synthesis of M-CSF transcripts. However, blockade of PKC by the isoquinoline sulfonamide derivative H7 and thus inhibition of phosphorylation was associated with augmentation of the fibroblasts response to TNF-alpha and IL-6.

Expression of functional receptors for interleukin-6 by human polymorphonuclear leukocytes. Downregulation by granulocyte-macrophage colony-stimulating factor.

Surface interleukin-6 receptors were identified on human polymorphonuclear leukocytes (PMNL) by monoclonal anti p80-chain antibody MT 18 Cytoplasmic RNA harvested from PMNL also contained IL-6-R transcripts. Binding of recombinant human (rh) interleukin-6 (IL-6) to IL-6-R bearing PMNL was identified by flow cytometry using phycoerythrin (PE)-conjugated ligand. Treatment of PMNL with rh granulocyte-macrophage colony-stimulating factor (GM-CSF) led to the inability of PMNL to bind MT 18 monoclonal antibody (moAb) and to display binding sites for PE-conjugated rh IL-6. Levels of IL-6-R transcripts in PMNL exposed to GM-CSF were about 5-fold below those of PMNL cultured in medium only. Though a definitive role for IL-6 to modulate the function of PMNL was not found, treatment of PMNL with rh IL-6 clearly resulted in an enhancement of transcript levels of the early response genes c-fos and c-jun in these cells, thus indicating that IL-6 binding is followed by signal transduction.

Differential regulation of interleukin-6 expression in human fibroblasts by tumor necrosis factor-alpha and lymphotoxin.

The treatment of human diploid fibroblasts with tumor necrosis factor (TNF)-alpha and with lymphotoxin (LT) is associated with induction of interleukin-6 (IL-6) transcripts with TNF-alpha being 10-fold more potent than LT. Here we report on the TNF-alpha/LT-induced signaling mechanisms responsible for the regulation of IL-6 gene expression in these cells. Run-on assays demonstrated that both TNF-alpha and LT increase IL-6 mRNA levels by transcriptional activation of this gene. Stability studies of IL-6 transcripts in fibroblasts showed that TNF-alpha delayed IL-6 mRNA decay but not LT. The induction of IL-6 transcripts by TNF-alpha and LT was not inhibited by the isoquinoline sulfonamide derivative H7. Similarly, depletion of protein kinase C (PKC) by 12-O-tetradecanoyl-phorbol 13-acetate (TPA) did not change the ability of TNF-alpha and LT to induce IL-6 transcripts, demonstrating that stimulation by these agents may not be mediated by activation of PKC. Stimulation of IL-6 transcripts in fibroblasts did also not require new protein synthesis as exposure to the protein synthesis inhibitor cycloheximide (CHX) enhanced accumulation of IL-6 mRNA in the presence or absence of TNF-alpha or LT.