A rare PALB2 germline variant causing G2/M cell cycle arrest is associated with isolated myelosarcoma in infancy.

BACKGROUND: Isolated myelosarcoma of infancy is a rare presentation of acute myelogenous leukaemia (AML). Because of its rarity and early onset in infancy underlying genetic predisposition is potentially relevant in disease initiation. METHODS AND RESULTS: We report an oncologic emergency in an infant with thoracic and intraspinal aleukaemic myeloid sarcoma causing acute myelon compression and lower leg palsy. Whole-exome sequencing of the patient's germline DNA identified a rare PALB2 (OMIM 610355) variant (p.A1079S), which is located in a domain critical for the gene's proper function within the homology-directed repair pathway. In line with potential DNA damage repair defects mediated by the PALB2 deregulation, the patient's fibroblasts showed increased sensitivity towards radiation and DNA intercalating agents. CONCLUSION: Therefore, we suggest PALB2 p.A1079S as a pathogenic variant potentially contributing to the here observed patient phenotype.

Listening to the Whispers in Neuroimmune Crosstalk: A Comprehensive Workflow to Investigate Neurotrophin Receptor p75NTR Under Endogenous, Low Abundance Conditions.

Inflammatory conditions are critically influenced by neuroimmune crosstalk. Cytokines and neurotrophic factors shape the responses of both nervous and immune systems. Although much progress has been made, most findings to date are based on expression of recombinant (tagged) proteins. The examination of receptor interactions by immunoprecipitation (IP) at endogenous levels provides further insight into the more subtle regulations of immune responses. Here, we present a comprehensive workflow and an optimized IP protocol that provide step-by-step instructions to investigate neurotrophin receptor p75NTR at endogenous, low abundance levels: from lysate preparation and confirmation of receptor expression to antibody validation and successful detection of protein-protein interactions. We employ human melanoma cell line A375 to validate specific antibodies and IP conditions, and apply these methods to explore p75NTR interactions in human leukemic plasmacytoid dendritic cell line PMDC05 detecting 14-3-3ϵ:p75NTR interaction in this cell type. With p75NTR as an exemplary protein, our approach provides a strategy to detect specific interaction partners even under endogenous, low abundance expression conditions.

UTX - moonlighting in the cytoplasm?

The X-linked histone demethylase UTX has a pivotal role in cellular and developmental processes including embryogenesis, hematopoiesis and cancer. UTX removes di- and trimethyl groups on histone H3 lysine 27, thereby regulating gene expression. But there is growing evidence that UTX displays biological functions independent of its histone demethylase activity. To elucidate these novel functions, it is of great interest to define subcellular localizations of UTX. Here we show for the first time that native UTX is primarily localized in the cytoplasm whereas ectopic GFP and Flag-tagged UTX display nuclear and cytoplasmic localization. While its epigenetic function is exerted in the nucleus, its cytoplasmic localization points to a novel function.

Childhood primary large vessel CNS vasculitis: single-centre experience and review of the literature.

Ischaemic brain injuries are rare conditions in the paediatric age group. Main causes include non-arteriosclerotic arteriopathies, which in childhood usually result from primary vasculitis of large or small vessels and lead to impaired perfusion and subsequent ischaemic brain lesions. In accordance with the nomenclature of systemic forms, CNS vasculitis is subdivided into groups, based on the size of affected vessels: angiography-positive primary angiitis of medium-sized and large vessels (pPACNS), and angiography-negative angiitis of small vessels (svPACNS). We report the clinical presentation, diagnostic approach, and therapy of four children with progressive pPACNS. Patients were treated with high-dose corticosteroids and anticoagulation with unfractionated heparin in the acute phase, followed by immune modulatory treatment with mycophenolate mofetil (MMF) and dual antiplatelet therapy with acetylsalicylic acid and clopidogrel. In this manuscript, we illustrate the experience gained in our hospital, resulting in significantly faster diagnosis and treatment initiation, and discuss the applied immune modulating treatment regimen in the context of the literature. Based on our observations, we conclude that immune modulating therapy with initial high-dose corticosteroids, followed by steroid-sparing maintenance treatment with MMF, may be safe and effective in childhood progressive pPACNS.

Defective IL-23/IL-17 Axis Protects p47phox-/- Mice from Colon Cancer.

In the colon, a sophisticated balance between immune reaction and tolerance is absolutely required. Dysfunction may lead to pathologic phenotypes ranging from chronic inflammatory processes to cancer development. Two prominent modulators of colon inflammation are represented by the closely related cytokines interleukin (IL)-12 and IL-23, which initiate adaptive Th1 and Th17 immune responses, respectively. In this study, we investigated the impact of the NADPH oxidase protein p47phox, which negatively regulates IL-12 in dendritic cells, on colon cancer development in a colitis-associated colon cancer model. Initially, we found that IL-12-/- mice developed less severe colitis but are highly susceptible to colon cancer. By contrast, p47phox-/- mice showed lower tumor scores and fewer high grade tumors than wild-type (WT) littermates. Treatment with toll-like receptor 9 ligand CpG2216 significantly enhanced colitis in p47phox-/- mice, whereas tumor growth was simultaneously reduced. In tumor tissue of p47phox-/- mice, the IL-23/IL-17 axis was crucially hampered. IL-23p19 protein expression in tumor tissue correlated with tumor stage. Reconstitution of WT mice with IL-23p19-/- bone marrow protected these mice from colon cancer, whereas transplantation of WT hematopoiesis into IL-23p19-/- mice increased the susceptibility to tumor growth. Our study strengthens the divergent role of IL-12 and IL-23 in colon cancer development. With the characterization of p47phox as a novel modulator of both cytokines our investigation introduces a promising new target for antitumor strategies.

Perioperative intravenous fluid therapy in children: guidelines from the Association of the Scientific Medical Societies in Germany.

This consensus- based S1 Guideline for perioperative infusion therapy in children is focused on safety and efficacy. The objective is to maintain or re-establish the child's normal physiological state (normovolemia, normal tissue perfusion, normal metabolic function, normal acid- base- electrolyte status). Therefore, the perioperative fasting times should be as short as possible to prevent patient discomfort, dehydration, and ketoacidosis. A physiologically composed balanced isotonic electrolyte solution (BS) with 1-2.5% glucose is recommended for the intraoperative background infusion to maintain normal glucose concentrations and to avoid hyponatremia, hyperchloremia, and lipolysis. Additional BS without glucose can be used in patients with circulatory instability until the desired effect is achieved. The additional use of colloids (albumin, gelatin, hydroxyethyl starch) is recommended to recover normovolemia and to avoid fluid overload when crystalloids alone are not sufficient and blood products are not indicated. Monitoring should be extended in cases with major surgery, and autotransfusion maneuvers should be performed to assess fluid responsiveness.

Novel Mutation in the DKC1 Gene: Neonatal Hoyeraal-Hreidarsson Syndrome As a Rare Differential Diagnosis in Pontocerebellar Hypoplasia, Primary Microcephaly, and Progressive Bone Marrow Failure.

Primary microcephaly and severe developmental delay are complex but unspecific signs pointing to various genetic or acquired diseases. A concomitant finding of hematological failure may lead to the differential diagnosis of rare genetic diseases such as chromosome breakage disorders or diseases associated with telomere dysfunction. X-linked Hoyeraal-Hreidarsson syndrome (HHS) is a rare heterogenic disorder characterized by severe neurological impairment and progressive bone marrow failure. The latter represents the main cause of mortality, usually in early childhood. We report on the clinical course of an infant with HHS due to a novel mutation in the DKC1 gene and the particular finding of pontocerebellar hypoplasia.

Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.

RNAi profiling of primary human AML cells identifies ROCK1 as a therapeutic target and nominates fasudil as an antileukemic drug.

Acute myeloid leukemia (AML) is characterized by a marked genetic heterogeneity, which complicates the development of novel therapeutics. The delineation of pathways essential within an individual patient's mutational background might overcome this limitation and facilitate personalized treatment. We report the results of a large-scale lentiviral loss-of-function RNA interference (RNAi) screen in primary leukemic cells. Stringent validation identified 6 genes (BNIPL1, ROCK1, RPS13, STK3, SNX27, WDHD1) whose knockdown impaired growth and viability of the cells. Dependence on these genes was not caused by mutation or overexpression, and although some of the candidates seemed to be rather patient specific, others were essential in cells isolated from other AML patients. In addition to the phenotype observed after ROCK1 knockdown, treatment with the approved ROCK inhibitor fasudil resulted in increased apoptosis and decreased viability of primary AML cells. In contrast to observations in some other malignancies, ROCK1 inhibition did not foster growth of immature malignant progenitors but was toxic to this cell fraction in feeder coculture and xenotransplant experiments, indicating a distinct effect of ROCK1 inhibition on leukemic progenitors. We conclude that large-scale RNAi screens in primary patient-derived cells are feasible and can complement other methods for personalized cancer therapies, such as expression and mutation profiling.

Cellular reporter systems for high-throughput screening of interactions between bioactive matrices and human mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) and factors secreted by them are essential components of the hematopoietic stem cell (HSC) niche within the bone marrow microenvironment. It has been shown that the extracellular matrix (ECM) can influence HSC-supportive potential of MSC and is a prerequisite for the proper signaling of morphogens. Therefore, we aimed at the identification of ECM components and candidate morphogens capable of enhancing the expression of HSC-supportive proteins in human MSC, namely, angiopoietin-1 (Ang-1) and stromal cell-derived factor 1 (SDF-1). For this purpose, highly sensitive secreted dual reporter constructs for Ang-1 and SDF-1 were established. These newly designed dual reporter systems enable continuous monitoring of the Ang-1 and SDF-1 promoter activity in an immortalized human MSC line cultured on ECM/morphogen microarrays. Reporter arrays showed that Ang-1 and SDF-1 expression can be induced by different ECM/morphogen combinations. In addition, continuous monitoring of promoter activity allows delineating time-dependent effects of the ECM and morphogens. Thus, we identified that collagen I and vitronectin in combination with Wnt3a favored SDF-1 expression over time, while only transiently inducing the expression of Ang-1. Taken together, the newly developed reporter systems allow for the monitoring of Ang-1 and SDF-1 promoter activity induced by morphogens and the ECM in a combinatorial and high-throughput manner. This technology might therefore be helpful to optimize culture conditions, which favor the activity of MSC as feeder cells for various types of stem and progenitor cells.

Overexpression of Jagged-1 and its intracellular domain in human mesenchymal stromal cells differentially affect the interaction with hematopoietic stem and progenitor cells.

Mesenchymal stromal cells (MSC) are an important component of the bone marrow microenvironment. Notch ligands expressed by MSC are known to play a regulatory role for hematopoietic stem and progenitor cells (HSPC) and in support of bone marrow homeostasis. While the role of Notch signaling in HSPC, their progeny, and MSC has been relatively well studied, little is known about the Notch-independent regulatory impact of Notch ligands on MSC themselves. In the present study, we used genetically engineered bone marrow-derived human MSC to study the function role of Jagged-1 and the Jagged-1 intracellular domain (JICD) with regard to the interaction with HSPC. We demonstrate that Jagged-1 in human MSC undergoes cleavage to produce an intracellular domain that translocates into the nucleus. JICD but not Jagged-1 overexpression was associated with an increased expression of stromal cell-derived factor-1. Short-term co-culture (7 days) of HSPC with JICD-overexpressing MSC, but not with Jagged-1-overexpressing MSC, led to increased proliferation of CD34(+) progenitors. In contrast, long-term co-culture of HSPC with Jagged-1-overexpressing MSC (up to 6 weeks) led to a significantly better support of cobblestone area-forming cells and long-term culture-initiating cells (LTC-ICs) compared with JICD-overexpressing MSC. Taken together, results of this study indicate that full-length Jagged-1 and JICD have differential effects on MSC and on their interaction with HSPC ex-vivo. JICD-overexpressing MSC induce proliferation of HSPCs in short-term culture at the expense of immature precursors (LTC-ICs), whereas Jagged-1 overexpressing MSC favor LTC-IC formation.

Generation of inducible immortalized dendritic cells with proper immune function in vitro and in vivo.

Dendritic cells are the professional antigen presenting cells of innate immunity and key players in maintaining the balance of immune responses. Studies with dendritic cells are mainly limited by their low numbers in vivo and their difficult maintenance in vitro. We differentiated bone marrow cells from transgenic mice expressing an inducible SV40 large T-antigen into dendritic cells. When immortalized by dexamethasone and doxycycline, these cells were stable in long-term culture. In the absence of dexamethasone and doxycycline (de-induction), dendritic cells displayed properties of primary cells, characterized by expression of classical dendritic cell surface markers CD11c, CD11b, MHCII, CD40 and CD86. Furthermore, de-induced lipopolysaccharide activated dendritic cells secreted IL-1ß, IL-6, TNFα and IL-12. De-induced, Ovalbumin-loaded dendritic cells polarize CD4(+) T cells into Th1, Th17 and Th2 cells, indicating their correct antigen presenting property. Consistent with intratracheal application of Ovalbumin-loaded primary dendritic cells into mice, the application of de-induced dendritic cells resulted in recruitment of lymphocytes to the lungs. In summary, we successfully expanded dendritic cells using conditional immortalization. The generated dendritic cells demonstrate the characteristic immunophenotype of primary dendritic cells and will facilitate further studies on immunomodulatory properties of dendritic cells.

The histone demethylase UTX regulates stem cell migration and hematopoiesis.

Regulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4-dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis.

Autotaxin is expressed in FLT3-ITD positive acute myeloid leukemia and hematopoietic stem cells and promotes cell migration and proliferation.

Autotaxin (ATX) has been reported to act as a motility and growth factor in a variety of cancer cells. The ATX protein acts as a secreted lysophospholipase D by converting lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which signals via G-protein-coupled receptors and has important functions in cell migration and proliferation. This study demonstrates that ATX expression is specifically upregulated and functionally active in acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) mutation of the FLT3 receptor gene. Moreover, ATX expression was also found in normal human CD34+ progenitor cells and selected myeloid and lymphoid subpopulations. Enforced expression of mutant FLT3-ITD by retroviral vector transduction increased ATX mRNA in selected cell lines, whereas inhibition of FLT3-ITD signaling by sublethal doses of PKC412 or SU5614 led to a significant downregulation of ATX mRNA and protein levels. In the presence of LPC, ATX expression significantly increased proliferation. LPA induced proliferation, regardless of ATX expression, and induced chemotaxis in all tested human leukemic cell lines and human CD34(+) progenitors. LPC increased chemotaxis only in cells with high expression of endogenous ATX by at least 80%, demonstrating the autocrine action of ATX. Inhibition of ATX using a small molecule inhibitor selectively induced killing of ATX-expressing cell lines and reduced motility in these cells. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration during hematopoiesis and that deregulation of ATX contributes to the pathogenesis of AML.

Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells.

The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

Impact of CXCR4 inhibition on FLT3-ITD-positive human AML blasts.

OBJECTIVE: Internal tandem duplication (ITD) mutations of the FLT3 receptor are associated with a high incidence of relapse in acute myeloid leukemia (AML). Expression of the CXCR4 receptor in FLT3-ITD-positive AML is correlated with poor outcome, and inhibition of CXCR4 was shown to sensitize AML blasts toward chemotherapy. The aim of this study was to evaluate the impact of FLT3-ITD on cell proliferation and CXCR4-dependent migration in human hematopoietic progenitor cells and to investigate their response to CXCR4 inhibition. MATERIALS AND METHODS: We used primary blasts from patients with FLT3-ITD or FLT3 wild-type AML. In addition, human CD34(+) hematopoietic progenitor cells were transduced to >70% with retroviral vectors containing human FLT3-ITD. RESULTS: We found that FLT3-ITD transgene overexpressing human hematopoietic progenitor cells show strongly reduced migration toward stromal-derived factor-1 in vitro and display significantly reduced bone marrow homing in nonobese diabetic severe combined immunodeficient mice. Cocultivation of FLT3-ITD-positive AML blasts or hematopoietic progenitor cells on bone marrow stromal cells resulted in a strong proliferation advantage and increased early cobblestone area-forming cells compared to FLT3-wild-type AML blasts. Addition of the CXCR4 inhibitor AMD3100 to the coculture significantly reduced both cobblestone area-forming cells and proliferation of FLT3-ITD-positive cells, but did not affect FLT3-wild-type cells-highlighting the critical interaction between CXCR4 and FLT3-ITD. CONCLUSION: CXCR4 inhibition to decrease cell proliferation and to control the leukemic burden may provide a novel therapeutic strategy in patients with advanced FLT3-ITD-positive AML.

Notch signaling enhances osteogenic differentiation while inhibiting adipogenesis in primary human bone marrow stromal cells.

OBJECTIVE: The Notch signaling pathway has been shown to play a role in bone marrow-derived stromal cell differentiation, however, the precise outcome of Notch activation remains controversial. The aim of this study was to evaluate the effect of Notch signaling in primary human bone marrow-derived stromal cells (hBMSCs). MATERIALS AND METHODS: hBMSCs were transduced to >90% with lentiviral vectors containing either human notch1 intracellular domain (NICD), jagged1, or dominant negative mastermind1. Cells were exposed to adipogenic and osteogenic differentiation stimuli and differentiation was quantified by oil red or alizarin red staining, alkaline phosphatase liver/bone/kidney (ALPL) activity and expression of adipogenic or osteogenic marker genes. RESULTS: NICD and jagged1 transgene-expressing hBMSCs demonstrated enhanced mineralization, nodule formation, and ALPL activity in osteogenic differentiation media. These findings correlated with increased gene expression of bone morphogenetic protein 2 and ALPL. In contrast, NICD or jagged1 transgene expression strongly inhibited adipocyte formation and reduced peroxisome proliferator-activated receptor-gamma, fatty acid binding protein 4, and adiponectin precursor gene expression. Co-overexpression of dominant negative mastermind1 and NICD or jagged1 led to a partial rescue of the differentiation phenotypes. In addition, high endogenous jagged1 expression levels were observed in hBMSCs samples with strong ALPL activity compared to a group of samples with low ALPL activity. CONCLUSION: In summary, our data suggest that induction of Notch signaling enhances the osteogenic differentiation of hBMSCs while inhibiting the adipogenic fate.

Stromal cell-derived factor-1alpha-directed chemoattraction of transiently CXCR4-overexpressing bone marrow stromal cells into functionalized three-dimensional biomimetic scaffolds.

Three-dimensional (3D) bone substitute material should not only serve as scaffold in large bone defects but also attract mesenchymal stem cells, a subset of bone marrow stromal cells (BMSCs) that are able to form new bone tissue. An additional crucial step is to attract BMSCs from the surface into deeper structures of 3D porous bone substitute scaffolds. Here we show that transient overexpression of CXCR4 in human BMSCs induced by mRNA transfection enhances stromal cell-derived factor-1alpha (SDF-1alpha)-directed chemotactic capacity to invade internal compartments of porous 3D bone substitute scaffolds in vitro and in vivo. In vitro native BMCSs invaded up to 500 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSCs invaded up to 800 mum within 5 days. In addition, 60% downregulation of endogenous SDF-1 transcription in BMSCs by endoribonuclease-prepared siRNA before CXCR4 mRNA transfection enhanced SDF-1alpha-directed migration of human BMSCs by 50%. Implantation of SDF-1alpha-releasing scaffolds seeded with transiently CXCR4-overexpressing BMSCs resulted in an increase of invasion into internal compartments of the scaffolds in a mouse model. In vivo native BMCS invaded up to 250 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSC invaded up to 500 mum within 5 days. Thus, the SDF-1alpha/CXCR4 chemoattraction system can be used to efficiently recruit BMSCs into SDF-1alpha-releasing 3D scaffolds in vitro and in vivo.

Serum albumin strongly influences SDF-1 dependent migration.

Stem cell migration is largely regulated by the chemokine SDF-1 and its receptor CXCR4. In the present study, we analyzed the effect of protein on SDF-1 dependent chemotaxis using CXCR4 expressing primary CD34+ hematopoietic progenitor cells for transwell migration assays. We show that migration towards SDF-1 is abolished in the absence of protein, while addition of serum albumin rescues SDF-1 dependent migration. Acid hydrolyzation or tryptic digest of protein eliminates its migration supporting effect, showing that the intact protein is necessary. We demonstrate that gradients of human serum albumin (HSA) that are physiologically present in vivo between human plasma and interstitial fluid (bone marrow) greatly influence SDF-1 dependent migration of hematopoietic progenitor cells. While SDF-1 dependent migration is strongly enhanced in the presence of a HSA gradient from 4% (plasma) towards 1% (interstitial fluid), reversion of the protein concentrations inhibits SDF-1 dependent chemotaxis. Furthermore, migration is induced to lower serum albumin concentrations in the presence of equal SDF-1 concentration, while albumin gradients in the absence of SDF-1 have no effect. Our results suggest that physiological gradients of serum albumin between blood and bone marrow support SDF-1 dependent homing of hematopoietic progenitor cells to the stem cell niche.

S1P(1) overexpression stimulates S1P-dependent chemotaxis of human CD34+ hematopoietic progenitor cells but strongly inhibits SDF-1/CXCR4-dependent migration and in vivo homing.

The CXC chemokine receptor 4 (CXCR4) and its ligand stromal derived factor 1 (SDF-1) regulate egress and homing of hematopoietic stem cells. Activation of sphingosine-1-phosphate (S1P) receptors (S1P(1-5)) modulates chemokine-induced migration of lymphocytes and hematopoietic stem cells. To analyze the influence of S1P(1) on SDF-1-dependent chemotaxis and trafficking, we overexpressed S1P(1) in CD34+ mobilized peripheral blood progenitor cells (PBPCs). Using a gamma-retroviral vector, transgene overexpression was achieved in more than 90% of target cells. S1P(1) transgene positive PBPCs showed enhanced chemotaxis towards S1P. S1P(1) overexpression resulted in reduced CXCR4 surface expression levels and strong inhibition of SDF-1-dependent ERK1/2 phosphorylation and Ca(2+) flux. Furthermore, SDF-1-dependent migration of S1P(1) overexpressing PBPCs or Jurkat cells was reduced up to 10-fold. Sublethally irradiated NOD/SCID mice were transplanted with 6-day cultured PBPCs overexpressing either S1P(1)-IRES-GFP or GFP alone. Screening for GFP positive human cells in the mouse bone marrow 20h after transplantation revealed an eightfold reduction in bone marrow homing of S1P(1) transgene expressing cells. Our data suggest that S1P(1) acts as an inhibitor of CXCR4-dependent migration of hematopoietic cells to sites of SDF-1 production.