Search

International audience; Innate Lymphoid Cells (ILC) are involved in homeostasis and immunity. Their dynamic differentiation and characterization depend on their tissue of residency and is adapted to their role within these tissues. Lymphoid Tissue inducer (LTi) cells are an ILC member and essential for embryonic lymph node (LN) formation. LNs are formed at pre-defined and strategic positions throughout the body and how LTi cells are initially attracted towards these areas is under debate. Besides their role in LN formation, LTi-like and the closely related ILC type 3 (ILC3) cells have been observed within the embryonic gut. New studies have now shown more information on their origin and differentiation within the embryo. This review will evaluate the embryonic LTi cell origin from a specific embryonic hemogenic wave, which has recently been described in mouse. Moreover, I will discuss their differentiation and similarities with the closely related ILC3 cells in embryo and adult.

While bioactive glass and ions released during its dissolution are known to stimulate osteoblast cells, the effect bioactive glass has on human stem cells is not clear. Here, we show that spherical monodispersed strontium containing bioactive nanoparticles (Sr-BGNPs) of composition 90.6 mol% SiO2, 5.0 mol% CaO, 4.4% mol% SrO (4.4%Sr-BGNPs) and 88.8 mol% SiO2, 1.8 mol% CaO, and 9.4 mol% SrO (9.4%Sr-BGNPs) stimulate bone marrow derived human stem cell (hMSC) differentiation down an osteogenic pathway without osteogenic supplements. The particles were synthesised using a modified Stӧber process and had diameters of 90 ± 10 nm. Previous work on similar particles that did not contain Sr (80 mol% SiO2, 20 mol% CaO) showed stem cells did not differentiate when exposed to the particles. Here, both compositions of the Sr-BGNPs (up to concentration of 250 μg/mL) stimulated the early-, mid-, and late-stage markers of osteogenic differentiation and accelerated mineralisation in the absence of osteogenic supplements. Sr ions play a key role in osteogenic stem cell differentiation. Sr-BGNP dissolution products did not adversely affect hMSC viability and no significant differences in viability were measured between each particle composition. Confocal and transmission electron microscopy (TEM) demonstrated that monodispersed Sr-BGNPs were internalised and localised within vesicles in the cytoplasm of hMSCs. Degradation of particles inside the cells was observed, whilst maintaining effective cations (Ca and Sr) in their silica network after 24 h in culture. The uptake of Sr-BGNPs by hMSCs was reduced by inhibitors of specific routes of endocytosis, indicating that the Sr-BGNPs uptake by hMSCs was probably via mixed endocytosis mechanisms. Sr-BGNPs have potential as injectable therapeutic devices for bone regeneration or treatment of conditions such as osteoporosis, because of their ability deliver a sustained release of osteogenic inorganic cations, e.g. calcium (Ca) or and strontium (Sr), through particle degradation locally to cells. Statement of Significance Here, we show that 90 nm spherical strontium containing bioactive nanoparticles of stimulate bone marrow derived human stem cell (hMSC) differentiation down an osteogenic pathway without the use of osteogenic supplements. While bioactive glass and its dissolution products are known to promote excellent bone regeneration in vivo and to stimulate osteoblast cells to produce bone matrix in vitro, their effect on human stem cells is not clear. Previously our nanoparticles that contained only SiO2 and CaO did not provoke human bone marrow or adipose derived stem cell differentiation.

Background: Chronic rhinosinusitis (CRS) is a heterogeneous disease, with patients having either a high or low type 2 inflammatory endotype. Whereas the type 2-high group is well characterized by IL-5 expression, the type 2-low group, consisting of approximately 20% of CRS with and 50% of CRS without nasal polyp patients, lacks a clear biomarker profile and thus specific therapeutic targets. Objective: The aim was to identify underlying molecular pathways of type 2-low CRS, as stratification of patients may allow improvement of personalized treatments. Methods: Luminex assays were performed to analyze proteins in nasal secretions and tissues of CRS patients. Immunostainings were analyzed for differences in neutrophils, granulocyte-colony stimulating factor (G-CSF), and its receptor in nasal tissue. Neutrophils were isolated from blood of healthy volunteers and stimulated with G-CSF. Effects on apoptosis and neutrophil activity were analyzed with flow cytometry. Results: G-CSF was significantly upregulated in nasal tissue and secretion fluid of type 2-low CRS patients compared to type 2-high patients. In nasal polyp tissue of type 2-low patients, a large infiltration of neutrophils expressing both G-CSF and its receptor was detected, suggesting the presence of a neutrophil-intrinsic autocrine survival mechanism. In response to G-CSF, neutrophils were in an activated state and were resistant to apoptosis, possibly contributing to a chronic inflammation. Of interest, type 2-high nasal polyp patients treated with IgE-blocking omalizumab had increased G-CSF concentrations compared to before treatment. Conclusion: G-CSF is an important cytokine regulating neutrophils in type 2-low CRS and has potential in the diagnosis and therapy of the disease.

Aim To evaluate the modifications of the apparent diffusion coefficient (ADC) in myelomatous lesions before and after induction treatment and the correlation with patient response to therapy according to International Myeloma Working Group (IMWG) criteria. Materials and methods A homogeneous group of 18 patients with a diagnosis of symptomatic multiple myeloma who underwent whole-body MRI with diffusion-weighted imaging (DWI-MRI) before and after bortezomib-based induction chemotherapy were evaluated prospectively. Quantitative analysis of ADC maps of myelomatous lesions was performed with the following pattern types: focal pattern, diffuse pattern (moderate and severe), and “salt and pepper” pattern. Lesions were evaluated by quantitative image analysis including measurement of the mean ADC in three measurements. Imaging results were compared to laboratory results as the clinical reference standard. Results A statistically significant increase in ADC values were found in the lesions of patients that responded to treatment. Interestingly, focal lesions showed a strongly significant increase in ADC values in responders, whereas no significant variation in ADC value in non-focal lesions (diffuse pattern and “salt and peppers” pattern) between responders and non-responders group was demonstrated. Conclusions DWI-MRI could provide additional quantitative information useful in monitoring early therapy response according to ADC changes of focal lesions.

Background and aims: The simultaneous measurement of many proteins is now possible using multiplex assays. In this pilot study we investigated a total of 124 proteins in plasma from chronic myeloid leukemia (CML) patients with the purpose of identifying proteins that are differently expressed at diagnosis and after tyrosine kinase inhibitor (TKI) treatment initiation. Methods: Samples were taken from 14 CML patients at diagnosis and after three months of TKI treatment (imatinib or dasatinib). Samples were analyzed by Mesoscale Discovery, Myriad RBM MAP technology and Olink Proseek. Results: Multiple plasma proteins were differentially expressed before and after initiation of TKI therapy. Protein patterns demonstrated a possible shift towards Th1-immunity and reduced angiogenic stimuli. Further, some plasma proteins were identified that can be of potential interest to study further for biologic, prognostic or therapeutic significance such as E-selectin, uPAR, growth hormone and carbonic anhydrase IX. Conclusions: Plasma proteomics seems feasible and useful in CML patients, both for studying patterns of protein expression and for identifying single proteins differentially expressed before and after treatment. Plasma proteomics may be useful to map disease activity and biological processes. Hence, plasma proteomics can be used to understand drug mechanisms and treatment responses in CML. (C) 2016 Elsevier Ltd. All rights reserved.

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection. Neutrophils display circadian oscillations in numbers and phenotype in the circulation. Adrover and colleagues now identify the molecular regulators of neutrophil aging and show that genetic disruption of this process has major consequences in immune cell trafficking, anti-microbial defense, and vascular health., This study was supported by Intramural grants from A∗STAR to L.G.N., BES-2013-065550 to J.M.A., BES-2010-032828 to M.C.-A, and JCI-2012-14147 to L.A.W (all from Ministerio de Economía, Industria y Competitividad; MEIC). Additional MEIC grants were SAF2014-61993-EXP to C.L.-R.; SAF2015-68632-R to M.A.M. and SAF-2013-42920R and SAF2016-79040Rto D.S. D.S. also received 635122-PROCROP H2020 from the European Commission and ERC CoG 725091 from the European Research Council (ERC). ERC AdG 692511 PROVASC from the ERC and SFB1123-A1 from the Deutsche Forschungsgemeinschaft were given to C.W.; MHA VD1.2/81Z1600212 from the German Center for Cardiovascular Research (DZHK) was given to C.W. and O.S.; SFB1123-A6 was given to O.S.; SFB914-B08 was given to O.S. and C.W.; and INST 211/604-2, ZA 428/12-1, and ZA 428/13-1 were given to A.Z. This study was also supported by PI12/00494 from Fondo de Investigaciones Sanitarias (FIS) to C.M.; PI13/01979, Cardiovascular Network grant RD 12/0042/0054, and CIBERCV to B.I.; SAF2015-65607-R, SAF2013-49662-EXP, and PCIN-2014-103 from MEIC; and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) to A.H. The CNIC is supported by the MEIC and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505).

Item does not contain fulltext Classical chemotherapeutic anti-cancer treatments induce cell death through DNA damage by taking advantage of the proliferative behaviour of cancer cells. The more recent approach of targeted therapy (usually protein-targeted) has led to many treatments that are currently available or are under development, all of which are designed to strike at the critical driving forces of cancer cells. The interaction of the cancer cells with their microenvironment is one of these fundamental features of neoplasms that could be targeted in such cancer treatments. Haematological and solid tumour cells interact with their microenvironment through membrane chemokine receptors and their corresponding ligands, which are expressed in the tumour microenvironment. Important representatives of this system are the chemokine ligand CXCL12 and its receptor chemokine receptor 4 (CXCR4). This interaction can be disrupted by CXCR4 antagonists, and this concept is being used clinically to harvest haematopoietic stem/progenitor cells from bone marrow. CXCR4 and CXCL12 also have roles in tumour growth and metastasis, and more recently their roles in cancer cell-tumour microenvironment interaction and angiogenesis have been studied. Our review focuses on these roles and summarises strategies for treating cancer by disrupting this interaction with special emphasis on the CXCR4/CXCL12 axis. Finally, we discuss ongoing clinical trials with several classes of CXCR4 inhibitors, and their potential additive value for patients with a (therapy resistant) malignancy by sensitising cancer cells to conventional therapy. 01 januari 2013

Cell therapy has the potential to offer novel treatment modalities for a number of diseases including cancer, and stem cells and in particular mesenchymal stem cells (MSCs) have been experimentally used to deliver therapeutic transgenes. However, conflicting reports have on the one side found that human MSCs can promote metastasis, while on the other hand other studies have shown that MSCs can stall the growth of metastatic lesions. In order to clarify the role of MSCs in metastasis development, we tested whether murine MSCs would behave similarly to human cells in mice. We found that the tissue distribution of human and mouse MSCs was nearly identical after intravenous injection. In mice with MDA-MB-231 mammary carcinoma xenografts we found that a fraction of MSCs infiltrated the primary tumor mass, but that the general tissue distribution of MSCs was unaffected by the tumor-burden. About half of the tumor-burdened animals that were treated with murine and human MSCs, respectively, harbored metastatic lesions with only 17% of controls showing metastatic nodules. Hence, both human and mouse MSCs possess metastasis-promoting activity raising concerns about the safe use of MSCs, but at the same time making the use of murine transgenic model systems feasible to study the role of MSCs in metastasis development and possibly finding ways of using them safely as cell therapeutic vehicles.

Chronic transplant dysfunction (CTD) is the leading cause for limited kidney graft survival. Renal CTD is characterized by interstitial and vascular remodeling leading to interstitial fibrosis, tubular atrophy and transplant vasculopathy (TV). The origin of cells and pathogenesis of interstitial and vascular remodeling are still unknown. To study graft-versus-recipient origin of interstitial myofibroblasts, vascular smooth muscle cells (SMCs) and endothelial cells (ECs), we here describe a new rat model for renal CTD using Dark Agouti kidney donors and R26 human placental alkaline phosphatase transgenic Fischer344 recipients. This model showed the development of CTD within 12 weeks after transplantation. In interstitial remodeling, both graft- and recipient-derived cells contributed to a similar extent to the accumulation of myofibroblasts. In arteries with TV, we observed graft origin of neointimal SMCs and ECs, whereas in peritubular and glomerular capillaries, we detected recipient EC chimerism. These data indicate that, within the interstitial and vascular compartments of the transplanted kidney, myofibroblasts, SMCs and ECs involved in chronic remodeling are derived from different sources and suggest distinct pathogenetic mechanisms within the renal compartments.

The use of cellular therapy in the treatment of dermal wounds is currently an active area of investigation. Multipotent adult progenitor cells (MAPC) are an attractive choice for cytotherapy because they have a large proliferative potential, the ability to differentiate into different cell types and produce a variety of cytokines and growth factors important to wound healing. Whole bone marrow (BM) was one of the initial attempts to treat impaired wounds. While it has shown some promise, the low frequency of progenitor cell populations in BM and the large number of inflammatory cells make it less attractive. Multipotent mesenchymal stromal cells (MSC) and endothelial progenitor cells are populations of BM-derived progenitor cells that have been isolated and used to treat chronic wounds with some success. Skin-derived MAPC are another heterogeneous population of progenitor cells present in the skin with the potential to differentiate into skin elements and participate in wound healing. All of these progenitor cell populations are potential sources for cytotherapy of wounds. This review focused on the contribution of adult progenitor cell populations to dermal wound healing and their potential for use in cytotherapy.

SummaryHeterogeneity in the differentiation behavior of hematopoietic stem cells is well documented but poorly understood. To investigate this question at a clonal level, we isolated a subpopulation of adult mouse bone marrow that is highly enriched for multilineage in vivo repopulating cells and transplanted these as single cells, or their short-term clonal progeny generated in vitro, into 352 recipients. Of the mice, 93 showed a donor-derived contribution to the circulating white blood cells for at least 4 months in one of four distinct patterns. Serial transplantation experiments indicated that two of the patterns were associated with extensive self-renewal of the original cell transplanted. However, within 4 days in vitro, the repopulation patterns subsequently obtained in vivo shifted in a clone-specific fashion to those with less myeloid contribution. Thus, primitive hematopoietic cells can maintain distinct repopulation properties upon serial transplantation in vivo, although these properties can also alter rapidly in vitro.

Objective. To model human leukemogenesis by transduction of human hematopoietic stem cells (HSC) with genes associated with leukemia and expressed in leukemic stem cells.Methods. Constitutive activation of Flt3 (Flt3-ITD) has been reported in 25 to 30% of patients with acute myeloid leukemia (AML). Retroviral vectors expressing constitutively activated Flt3 and STAT5A were used to transduce human cord blood CD34(+) cells and HSC cell self-renewal and differentiation were evaluated.Results. We have demonstrated that retroviral transduction of Flt3 mutations into CD34(+) cells enhanced HSC self-renewal as measured in vitro in competitive stromal coculture and limiting-dilution week-2 cobblestone (CAFC) assays. Enhanced erythropoiesis and decreased myelopoiesis were noted together with strong activation of STAT5A. Consequently, transduction studies were undertaken with a constitutively active mutant of STAT5A (STAT5A[1*6]) and here also a marked, selective expansion of transduced CD34(+) cells was noted, with a massive increase in self-renewing CAFC detectable at both 2 and 5 weeks of stromal coculture. Differentiation was biased to erythropoiesis, including erythropoietin independence, with myeloid maturation inhibition. The observed phenotypic changes correlated with differential gene expression, with a number of genes differentially regulated by both the Flt3 and STAT5A mutants. These included upregulation of genes involved in erythropoiesis and downregulation of genes involved in myelopoiesis. The phenotype of week-2 self-renewing CAFC also characterized primary Flt3-ITD+ AML bone marrow samples. Isolation of leukemic stem cells (LSC) with a CD34(+), CD38(-), HLA-DR- phenotype was undertaken with Flt3-ITD+ AML samples resulting in co-purification of early CAFC. Gene expression of LSC relative to the bulk leukemic population revealed upregulation of homeobox genes (HOXA9, HOXA5) implicated in leukemogenesis, and hepatic leukemia factor (HLF) involved in stem cell proliferation.Conclusion. Myeloid leukemogenesis is a multi-stage process that can involve constitutively activated receptors and downstream pathways involving STAT5, HOX genes, and HLF, (c) 2007 International Society for Experimental Hematology. Published by Elsevier Inc.

Objective. Busulfan (BU) is often used in conditioning regimens prior to bone marrow transplantation, but its mechanism of action remains to be resolved. We have examined the possibility that BU may exert part of its toxic effects via DNA alkylation at the Oh position of guanine as this might provide an approach to improving the conditioning regimen. Methods. Survival of LAMA-84 and RJKO cells was assessed by colony-forming assay and cell counting, respectively. O-6-alkylguanine-DNA alkyltransferase (ATase) activity was assayed by transfer of radioactivity from [H-3]-methylated DNA, Colony-forming potential of normal human bone marrow cells (BMC) was measured in the presence of appropriate growth factors as the formation of both granulocyte-macrophage colony-forming units (CFU-GM) or burst-forming unit erythroids (BFU-E) within the same assay. Murine hematopoietic precursors were grown under a bone marrow stromal cell line to allow measurement of the frequency of cobblestone area-forming cells (CAFC) that correspond to CFU-GM, spleen colony-forming units (CFU-S), and the primitive stem cells with long-term repopulating ability. Results. Inactivation of ATase by O-6-benzylguanine (O-6-BeG) sensitized a human erythromegakaryocytic cell line (LAMA-84) and normal human bone marrow progenitors to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) but not to BU toxicity, BCNU, but not BU, inactivated ATase in LAMA-84 cells. Overexpression of human ATase in cDNA transfected Chinese hamster cells attenuated the toxicity of BCNU but not BU, Finally, the in vivo treatment of mire showed that the depletion of primitive stem cells by BU as measured in the CAFC assay was not affected by addition of O-6-BeG. O-6-BeG did, however, dramatically potentiate BCNU toxicity in all CAFC subsets, leading to depletion of more than 99% stem cells, Conclusion. These data suggest that BU does not elicit toxicity via alkylation at the O-6 position of guanine in DNA in a way that can be influenced by ATase modulation, (C) 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.

To investigate whether the type of programmed cell death of myelodysplastic erythroid cells depends on their cellular context, we performed studies on cells from patients with low-risk myelodysplastic syndromes. We compared erythroid cells (and their precursor cells) from the mononuclear cell fraction with those from the hematon fraction, which are compacted complexes of hematopoietic cells surrounded by their own micro-environment. In directly fixed materials, erythroblasts exhibited signs of autophagy with limited apoptosis (3%) based on ultrastructural characteristics and immunogold labeling for activated caspase-3. After 24 h in culture, myelodysplastic erythroblasts exhibited a significant increase in apoptosis (22 ± 7% vs. 3 ± 2%, p = 0.001). In contrast, the myelodysplastic erythroblasts from the hematon fraction did not exhibit an increased tendency toward apoptosis after culture (7 ± 3.3% vs. 1.8 ± 2.3%), which was in line with results for normal bone marrow cells. The same dependency on the micro-environment was noted for immature erythroid progenitor cells. Myelodysplastic hematons exhibited distinct numbers of erythroid burst-forming units in association with an extensive network of stromal cells, whereas small numbers of erythroid burst-forming units were generated from the myelodysplastic mononuclear cells compared with normal mononuclear cells (10.2 ± 9 vs. 162 ± 125, p0.001). Co-culture of erythroid myelodysplastic cells in the presence of growth factors (vascular endothelial growth factor, leukemia inhibitory factor) or on the MS-5 stromal layer did not restore the expansion of erythroid precursor cells. These data indicate that surviving myelodysplastic erythroid progenitors become more vulnerable to programmed cell death when they are detached from their own micro-environment.

Histone deacetylase inhibitors (HDIs) have been shown to enhance hematopoietic stem and progenitor cell activity and improve stem cell outcomes after ex vivo culture. Identification of gene targets of HDIs is required to understand the full potential of these compounds and can allow for improved stem cell culturing protocols. The molecular process that underlies the biological effects of valproic acid (VPA), a widely used HDI, on hematopoietic stem/progenitor cells was investigated by studying the early-response genes of VPA. These genes were linked to VPA-induced enhancement of cell function as measured by in vitro assays. Genome-wide gene expression studies revealed over-representation of genes involved in glutathione metabolism, receptor and signal transducer activity, and changes in the hematopoietic stem/progenitor cells surface profile after short, 24-hour VPA treatment. Sca-1, a well-known and widely used stem cell surface marker, was identified as a prominent VPA target. We showed that multiple HDIs induce Sca-1 expression on hematopoietic cells. VPA strongly preserved Sca-1 expression on Lin(-)Sca1(+)ckit(+) cells, but also reactivated Sca-1 on committed progenitor cells that were Sca-1(neg), thereby reverting them to the Lin(-)Sca1(+)ckit(+) phenotype. We demonstrated that reacquired Sca-1 expression coincided with induced self-renewal capacity as measured by in vitro replating assays, while Sca-1 itself was not required for the biological effects of VPA as demonstrated using Sca-1-deficient progenitor cells. In conclusion, our results show that VPA modulates several genes involved in multiple signal transduction pathways, of which Sca-1 was shown to mark cells with increased self-renewal capacity in response to HDIs. (c) 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.