Olfactomedin 4, An Indicator of the Existence of Neutrophil Subsets.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3775-3775
Author(s):  
Stine Novrup Clemmensen ◽  
Sara Roervig ◽  
Jonathan Wren ◽  
Martin Illemann ◽  
Andreas Glenthoj ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mrna Expression ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Subcellular Fractionation ◽  
Human Neutrophils ◽  
Specific Granules ◽  
Blood Neutrophils

Abstract Abstract 3775 Olfactomedin 4 (OLFM4) was initially identified as a gene highly induced in myeloid stem cells by G-CSF treatment and independently as a gene highly expressed in colon cancers. OLFM4 was predicted in a bioinformatics analysis as associated with neutrophil specific granules. We analyzed the expression of OLFM4 mRNA in myeloid cells from normal human bone marrow and demonstrated that expression of OLFM4 mRNA is similar to the expression of LCN2 which codes for the specific granule protein NGAL (Figure 1), but distinct from expression of mRNA for myeloperoxidase and gelatinase which are marker proteins for azurophil granules and gelatinase granules, respectively. Subcellular fractionation of peripheral blood neutrophils demonstrated complete co-localization of OLFM4 with NGAL, and stimulation of neutrophils with fMLP or PMA resulted in co-release of NGAL and OLFM4, indirectly proving that OLFM4 is a genuine constituent of neutrophil specific granules. Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB. Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB. Interestingly, immunohistochemistry showed OLFM4 expression in only a subset of neutrophils (figure 2). We suspected that this might be dependent on the antibody, but two different commercial antibodies and an in-house antibody raised against a synthetic OLFM4 derived peptide, all polyclonal, showed similar patterns. Flow cytometry confirmed the existence of two populations of neutrophils, one expressing OLFM4 the other not. Figure 2. Immunohistochemistry of OLFM4 in neutrophils. Figure 2. Immunohistochemistry of OLFM4 in neutrophils. Immunohistochemistry of bone marrow cells showed that OLFM4 appears in myelocytes and is maintained in the cells during further maturation of the cells to segmented neutrophils. Again, only 30% of the neutrophil precursors from bone marrow stain positive for OLFM4 indicating, that different subsets of human neutrophils may exist. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Human Antibacterial Cathelicidin, hCAP-18, Is Synthesized in Myelocytes and Metamyelocytes and Localized to Specific Granules in Neutrophils

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2796-2803 ◽  
Author(s):  
Ole Sørensen ◽  
Kristina Arnljots ◽  
Jack B. Cowland ◽  
Dorothy F. Bainton ◽  
Niels Borregaard
Keyword(s):  
Electron Microscopy ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Subcellular Fractionation ◽  
Human Neutrophils ◽  
Major Protein ◽  
Equimolar Ratio ◽  
Binding Domains ◽  
Specific Granules ◽  
Immuno Electron Microscopy

hCAP-18 is the only human member of the antibacterial and endotoxin-binding family of proteins known as cathelicidins. The antibacterial and endotoxin binding domains reside in the C-terminal 37 amino acids of the protein (LL-37) and this is believed to be unleashed from the neutralizing N-terminus by proteases from peroxidase positive granules. In human neutrophils, peroxidase positive and peroxidase negative granules can be subdivided into granule subsets that differ in protein content and ability to be exocytosed. To determine the localization of hCAP-18, we performed high-resolution immuno-electron microscopy and subcellular fractionation on Percoll density gradients. Biosynthesis of hCAP-18 was investigated in isolated human bone marrow cells. hCAP-18 was found to colocalize and comobilize with lactoferrin, but not with gelatinase in subcellular fractions. This was confirmed by electron microscopy. hCAP-18 is synthesized at the same stage of myeloid cell maturation as lactoferrin, and is efficiently targeted to granules. Like the peroxidase negative granule's matrix metalloproteinases, collagenase and gelatinase, hCAP-18 is also stored in unprocessed form. hCAP-18 is a major protein of specific granules where it is present in equimolar ratio with lactoferrin.

The Ryk Receptor Regulates Hematopoietic Stem and Progenitor Cell Proliferation and Their Sensitivity To Myeloablative Agents

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 796-796
Author(s):  
Benjamin Povinelli ◽  
Michael Nemeth
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Peripheral Blood Cells ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract The molecular mechanisms that control the balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) are critical for maintaining life-long hematopoiesis. In a recent study (Povinelli, et al. Stem Cells, In Press, 2013) we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Related to Receptor Tyrosine Kinase (Ryk). Expression of Ryk on HSPCs in vivo was associated with a decreased rate of proliferation. Following treatment with fluorouracil (5-FU), the percentage of Ryk+ HSPCs increased at the expense of Ryk-/low HSPCs. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. To test this hypothesis, we injected 6-8 week old C57BL/6 mice with 150 mg/kg of 5-FU and analyzed bone marrow 48 hours later for the presence of apoptotic HSPCs, defined as lineage negative (Lin-), Sca-1+, CD48- cells positive for active caspase-3. There was a 2.5-fold decrease in the percentage of apoptotic Ryk+ HSPCs (12.9 ± 1.7%) compared to Ryk-/low HSPCs (32.4 ± 5.3%, p < 0.001, n = 3). To test whether this effect was limited to 5-FU, we performed a similar study in which we irradiated C57BL/6 mice with 3 cGy of total body irradiation (TBI) and analyzed bone marrow 72 hours later for apoptotic HSPCs (for this experiment, defined by a Lin-, c-kit+, Sca-1+, CD150+, CD48- immunophenotype or LSK, SLAM). Comparable to the effects of 5-FU, there was a significant 3.0-fold reduction in the percentage of apoptotic Ryk+ HSPCs (3.1 ± 0.2%) compared to Ryk-/low HSPCs (9.2 ± 1.5%, p < 0. 001, n = 3) in mice receiving 3 cGy TBI. These results demonstrated an association between Ryk expression and survival of HSPCs following myeloablative injury. To determine whether in vivo targeting of the Ryk receptor would increase the sensitivity of HSPCs to myeloablative injury, we utilized a neutralizing rabbit anti-Ryk antibody (α-Ryk). We injected C57BL/6 mice with 5 mg/kg α-Ryk or rabbit IgG isotype for 2 consecutive days. Twenty-four hours after the second dose, we determined the frequency and cell cycle status of LSK SLAM cells. Treatment with α-Ryk significantly increased the percentage of LSK SLAM cells in the S/G2/M phases compared to control (α-Ryk: 17.8 ± 2.2%; isotype IgG: 11.6 ± 2.7%, p < 0.05, n = 3). This was associated with a decrease in the percentage of LSK, SLAM cells in G1 following treatment with α-Ryk (α-Ryk: 40.5 ± 3.2%, isotype IgG: 51.3 ± 2.2; p < 0.01, n = 3). The percentage of G0 LSK SLAM cells was unchanged (α-Ryk: 37.9 ± 2.6, isotype IgG: 35.7 ± 3.1% n = 3) indicating that inhibiting Ryk promoted the exit of LSK SLAM cells from G1. Treatment with α-Ryk also increased the percentage of whole bone marrow cells expressing the LSK SLAM phenotype by 1.4-fold compared to controls (p < 0.05, n = 3). To determine if α-Ryk treatment altered HSPC function, we transplanted whole bone marrow cells from C57BL/6 mice treated with two days of α-Ryk or isotype IgG at a 1:1 ratio with whole bone marrow from untreated Ubc-GFP transgenic mice into lethally irradiated B6.SJL mice. Four weeks after transplant, we analyzed peripheral blood cells for the percentage of CD45.2+ GFP- cells. There was no difference in engraftment by transplanted bone marrow cells from mice treated with α-Ryk or isotype IgG (α-Ryk: 61.6 ± 6.1% n = 4, isotype IgG: 52.8 ± 13.6%, n = 5), indicating that the neutralizing antibody does not inhibit short-term HSPC function on its own. We then tested whether blocking Ryk function resulted in greater sensitivity of HSPCs to 5-FU. We treated B6.SJL mice with 5 mg/kg α-Ryk or isotype IgG for 2 consecutive days, followed by 150 mg/kg of 5-FU. Forty-eight hours after 5-FU treatment, we transplanted 2x106 C57BL/6 whole bone marrow cells into treated B6.SJL mice without additional conditioning. Four weeks after transplant, we determined the percentage of donor-derived CD45.2+ peripheral blood cells. Treatment of recipient mice with α-Ryk prior to 5-FU treatment resulted in increased engraftment of donor bone marrow by 3.6-fold compared to isotype (p < 0.05, n = 5), suggesting that inhibition of Ryk resulted in increased elimination of host HSPCs by 5-FU. Collectively, these data suggest a model in which inhibition of the Ryk receptor results in increased proliferation of HSPCs, rendering them more sensitive to the effects of myeloablative agents such as chemotherapy or TBI. Disclosures: No relevant conflicts of interest to declare.

The Role of JARID2 in Normal and Malignant Hematopoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 793-793
Author(s):  
Hamza Celik ◽  
Kramer C Ashley ◽  
Martens Andy ◽  
Elizabeth Eultgen ◽  
Cates Mallaney ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Median Survival ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Kaplan Meier ◽  
Marrow Cells

Abstract Despite the increasing availability of targeted therapies for myeloproliferative neoplasms (MPNs), there remains a subset of these patients that transform to secondary acute myeloid leukemia (sAML). MPN patients who develop sAML have a dismal outcome, with a median survival of six months. The mechanisms and pathways that contribute to transformation from MPN to sAML have not been well delineated. The most commonly mutated genes found in the MPN initiating clones include JAK2, MPL and CALR. Transformation to sAML however requires acquisition of additional co-operating mutations such as TET2, IDH1/2, and NRAS. Recent genome sequencing studies identified deletions of JARID2, a gene associated with the Polycomb Repressive Complex 2 (PRC2) involved in implementing global H3K27me3 in post-MPN sAML. Mutations in JARID2 are found only in the blast phase of MPNs, but not in chronic phase of the disease. This data suggests that a JARID2 deletion could be a sAML-specific transforming event by acting as a tumor suppressor in HSCs. To investigate the role of Jarid2 as a tumor suppressor, we utilized an inducible mouse model of the prototypical MPN driver mutation Jak2V617F. We have established our model system by crossing Mx1-CRE:Jarid2fl/fl (Jarid2KO) or Mx1-CRE:Jarid2fl/+ (Jarid2HET) with JAK2V617F mice to generate a Mx1-CRE:Jarid2fl/fl Jak2V617F/+ or Mx1-CRE: Jarid2fl/+Jak2V617F/+ strain. Mx1-CRE mediates both activation of Jak2V617Fand deletion of Jarid2 simultaneously in adult hematopoietic compartment upon injection of the double-stranded RNA analog polyinosinic:polycytidylic acid (pIpC). In all cases, the absence of Jarid2 in Jak2V617F/+ background accelerated MPN progression, characterized by elevated hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, and splenomegaly compared to the control groups. Median survival of Jarid2KO-Jak2V617F/+ and Jarid2HET-Jak2V617F/+ strains also revealed a dose-dependence of Jarid2 on life expectancy with a median of 27 and 56 days post pIpC treatment, respectively (Figure 1). Together, these data suggest that loss of Jarid2 in Jak2V617F/+ background accelerates the progression of MPN. To more completely understand the tumor suppressor function of Jarid2 in MPN, we aimed to define its role in normal hematopoiesis. Jarid2 is highly expressed in myeloid-biased compared to lymphoid-biased HSCs, suggestive of a role in HSC subtype determination. Moreover, conditional knock-out studies show that each core component of PRC2 (EED, SUZ12 and EZH2) has distinct as well as overlapping functional properties in hematopoiesis. To study the function of Jarid2 in normal hematopoiesis, we crossed Jarid2fl/fl mice to the Vav-CRE strain to facilitate conditional inactivation of Jarid2 in hematopoietic cells. Vav1-CRE is expressed throughout life in definitive HSCs and all hematopoietic lineages starting at E10.5. Analysis of eight-week old Vav1-CRE:Jarid2fl/fl mice showed that complete loss of Jarid2 induced a significant compromise in hematopoiesis with a robust reduction in phenotypically-defined bone marrow HSCs, a defective B-cell generation in the bone marrow (BM), a differentiation block in T-cell development in thymus, and a significant reduction in peripheral blood counts. A competitive transplantation strategy was also employed to assess the stem cell potential of Jarid2-KO HSCs. 2.5 x 105 whole bone marrow cells isolated from adult mice were transplanted into lethally irradiated recipient mice along with 2.5x105 whole bone marrow cells from congenic wild-type mice. Peripheral blood analysis of these mice over the period of 16-weeks post-transplant has shown that the loss of Jarid2 disrupts HSC function, leading to enhanced myeloid and reduced lymphoid output. Collectively, these data illustrate that Jarid2 is required for hematopoietic hemostasis including appropriate lineage fate determination of HSCs. The loss of Jarid2 in a Jak2V617F background promotes acceleration of MPN and implicates Jarid2 as a hematopoietic tumor suppressor. Figure 1. Kaplan-Meier analysis of a cohort of Jarid2KO-Jak2V617F (n = 5) and Jarid2HET -Jak2V617F (n = 6) and littermate controls (n = 4-8 each). Figure 1. Kaplan-Meier analysis of a cohort of Jarid2KO-Jak2V617F (n = 5) and Jarid2HET -Jak2V617F (n = 6) and littermate controls (n = 4-8 each). Disclosures No relevant conflicts of interest to declare.

scholarly journals Poor-Risk AML with Complex, Monosomal Karyotype: In Vivo Therapeutic Efficacy of Decitabine (DAC) in Patient-Derived Xenograft (PDX) Mouse Models

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2644-2644
Author(s):  
Gabriele Greve ◽  
Julia Schüler ◽  
Eva Oswald ◽  
Michael Lübbert
Keyword(s):  
Bone Marrow ◽  
Median Survival ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Disease Onset ◽  
Leukemic Cells ◽  
Mutational Status ◽  
Pdx Models ◽  
Monosomal Karyotype

Background: DNA-hypomethylating agents (HMAs) show an encouraging but not yet well-understood activity in AML/MDS patients (pts) with adverse cytogenetics, such as -7/del(7q) embedded in a complex, monosomal karyotype (MK). We have noted that MK pts with multiple monosomies responded better to DAC treatment than pts with only one monosomal autosome (Blum, Greve and Lübbert, Curr Opin Hematol. 2017). This important characteristic of HMAs differentiates them from cytarabine (AraC, lacking DNMT-inhibitory activity) as shown in randomized studies with DAC (Wierzbowska et al., Am. J. Hematology 2018) and AZA (Döhner et al., Leukemia 2018). The mechanism of action is currently under investigation and may involve preferential gene reactivation on monosomic chromosomes by DAC (Greve et al., ASH Annual Meeting 2017, #2612). In order to address this important difference between DAC and AraC, we compared their antileukemic activity in PDX models representing cytogenetically normal (CN) AML and adverse cytogenetics AML (complex, monosomal karyotypes including del(7q) and 1 or more monosomies). Materials and Methods: PDX models were generated for 5 CN and 3 MK AML pts with a median age of 68 years (range 55-80), a median WBC of 3.0 x 103/µl (range 1.4-139.7) and a median of 42% bone marrow (BM) blasts (range 5-97). 7/8 pts were sampled at diagnosis, i.e. prior to first-line treatment (3 pts received DAC, 3 pts induction and 1 pt upfront allografting); 1/8 pt had had multiple relapses. T-cell depleted peripheral blood or bone marrow cells (3x106 cells/mouse) were injected into NSG (NOD/Shi-scid/IL-2Rγnull) mice. Leukemic cell engraftment was determined by flow cytometry (FC) in BM, peripheral blood (PB) and spleen during the course of the experiment and at the end of a study. Leukemic cells were transplanted serially at least 4-5 times to propagate and ensure full leukemic potential of the cells. Mice were treated with low-toxic concentrations of DAC (1 mg/kg/day given intraperitoneally), AraC (15 mg/kg/day given intravenously) or vehicle (PBS) for 5 consecutive days (one cycle). Drug doses were titrated to be equitoxic and as potent as possible without causing side effects. Treatment commenced upon disease onset, as evaluated by FC positivity for human CD33, increased WBC in PB and weight loss. Overall survival served as the read-out. The study was carried out in accordance with the recommendations by the Society of Laboratory Animal Science in an AAALCA accredited animal facility. The animal experiments were approved by the regional council (Regierungspräsidium Freiburg, ref. 35, permit no. G-12/86). Results: We successfully established well-characterized PDX from pts with CN (n = 5), and with MK (n = 3) with a median of 2 autosomal monosomies (range 1-6; of chromosomes 4, 6, 7 [2x], 15, 17 [2x], 18, 21). TP53 (mutational status available for 6/8 pts) was wildtype in 4 CN pts and 1 MK pt, and mutated in 1 MK pt. All models were treated with either DAC, AraC or vehicle. Across both cytogenetic cohorts, DAC treatment significantly prolonged median survival by 36 days when compared to vehicle administration (p = 0.0002, see Fig 1A). Specifically, DAC increased median survival of CN PDX by 33 days (p = 0.0104, range 61-105 days with DAC, compared to 43-65 days with vehicle, Fig 1B), and in MK PDX by 40 days (p = 0.0197; range 72-105 days with DAC, compared to 39-50 days with vehicle, Fig 1C). Notably, disease progression in two DAC-treated PDX (one CN, one MK with 6 monosomies) was delayed to 105 days (Fig 1, marked with ⋆). AraC treatment also resulted in longer median survival across both cohorts compared to vehicle, albeit not as pronounced as with DAC, by an average of 20 days (p = 0.0066). In more detail, AraC increased median survival of CN PDX by 19 days (p = 0.0025, range 53-82 days), and, though not significantly, of MK PDX by 15 days (p = 0.2048, range 46-92 days). Conclusion: In this PDX study recapitulating two different cytogenetic risk groups of AML, DAC showed encouraging activity also in models with complex, monosomal karyotype, even in the presence of 6 autosomal monosomies, and was superior to AraC in extending survival. These data lend further support to the treatment of such adverse genetics AML patients with HMAs. Disclosures No relevant conflicts of interest to declare.

The Transcription Factor IRF8 is a Key Transcription Factor for Basophil Development

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1197-1197
Author(s):  
Daisuke Kurotaki ◽  
Haruka Sasaki ◽  
Naoki Osato ◽  
Izumi Sasaki ◽  
Chika Kaneda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Promoter Regions ◽  
Retroviral Transduction ◽  
Pass Through ◽  
Myeloid Progenitors

Abstract Basophils are the rarest granulocytes circulating in the peripheral blood. They play critical roles in anti-parasite Th2-type immune responses and chronic allergic disorders. The developmental pathway for basophils has been recently demonstrated; myeloid progenitors pass through common myeloid progenitors, granulocyte-monocyte progenitors, granulocyte-committed progenitors (GPs), and basophil-committed progenitors (BaPs) in the bone marrow. BaPs then give rise to mature basophils. However, our understanding of how this pathway is regulated remains still elusive. Interferon Regulatory Factor-8 (IRF8), a hematopoietic cell-specific IRF transcription factor, is essential for the development of monocytes, dendritic cells, and eosinophils, while it inhibits neutrophil differentiation. Its role in the development of basophils has yet to be analyzed. In this study, we investigated whether IRF8 has any role in the development of the basophil lineage. We found that Irf8–/– mice displayed a severe reduction of basophil counts in the bone marrow, peripheral blood and spleen compared to wild-type (WT) mice. Irf8–/– mice retained GPs but lacked BaPs. Cell transfer experiments revealed that the defect of basophil development in Irf8–/– mice resides in bone marrow cells. We utilized IRF8-GFP chimera knock-in mice to examine IRF8 protein expression in the basophil lineage at a single cell level. We found that GPs, but not BaPs and mature basophils, expressed IRF8. Furthermore, purified Irf8–/– GPs failed to efficiently give rise to basophils in vitro. These results indicate that IRF8 acts at the stage of GPs in a cell-intrinsic manner. To understand the mechanism by which IRF8 promotes basophil development, we performed transcriptome analysis of purified GPs from WT and Irf8–/– mice by microarray. Because IRF8 is no more expressed in BaPs, we envisaged that IRF8 acts by inducing downstream transcription factors in GPs. The expression of several transcription factor genes such as Gata2 and Spib was reduced in Irf8–/– GPs compared to WT GPs. Analysis of DNA motifs in the promoter regions of genes downregulated in Irf8–/– GPs predicted that GATA transcription factor(s) may act downstream of IRF8. Indeed, retroviral transduction of GATA2, known to be essential for basophil development, into Irf8–/– hematopoietic progenitor cells rescued basophil differentiation in vitro. On the other hand, Spib–/– mice showed no obvious defects in basophil development. Taken together, these results suggest that the IRF8-GATA2 axis in GPs critically regulates basophil development. Disclosures: No relevant conflicts of interest to declare.

The Interferon Score Towards Interferon Alpha Tailored Therapy In Essential Thrombocythemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4073-4073
Author(s):  
Novella Pugliese ◽  
Novella Pugliese ◽  
Concetta Quintarelli ◽  
Biagio De Angelis ◽  
Luana Marano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mrna Expression ◽  
Interferon Alpha ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Jak2 V617f ◽  
Predictive Biomarker ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Operating Characteristics

Abstract Introduction Interferon-alpha 2 (IFN) is able to induce hematological response in about 70-80% of ET patients but some of them could be defined as bad responders. IFN binding its receptor results in tyrosine cross-phosphorylation and auto-phosphorylation of the JAKs proteins (Tyk2 and Jak1). These phosthyrosines recruit and activate STAT family member such as STAT1 and STAT3. These proteins induce the transcription of SOCSs, whose role is to extinguish cytokine signaling by inhibition of JAK kinase-activity directly through the KIR-domain, and indirectly promoting the proteasomal degradation of Jak2, by SOCS-box-motif. In summary, IFN induces the expression of SOCSs, which inhibit TPO mediated signaling through Jak2 double inhibition. This allows IFN-α and TPO pathway to cross-talks by means of the JAK-STAT-SOCS cascade. Aims To identify molecular markers that identify those patients who respond to IFN, we analyzed bone marrow cells transcript levels of specific genes involved in the IFN receptor pathway, whose signal cross-talks with the TPO dependent JAK-STAT pathway. In particular we investigated the mRNA expression of JAK1, TYK2, STAT1, STAT3, SOCS1 and SOCS3. Methods We analyzed 60 ET patients treated with 3 million units of IFN-α-2b 5 times a week as induction (3 months), and 3 times a week as maintenance. Responses were classified as follow: Good-Responders(R) (n=44), those who achieved complete response according to European Leukemia Net criteria, and Bad-Responders(NR) (n=17) who didn’t reach the criteria. The mRNA expression of genes of interest was measured in bone marrow samples from ET patients by RTq-PCR and tested for their predictive value using receiver operating characteristics (ROC) curves. Data were normalized as following: [mRNA normalized copy number (NCN)=mRNA target gene/mRNA GUSB]. An IFN score was calculated as an average in log2 of mRNA levels of genes differently expressed between Good-R and Bad-R. Results Main clinical characteristics were similar between the two groups of response. JAK2 V617F mutation was detected in 56,8% of Good-R and 58,8% of Bad-R (p=0,81) and no difference was found in JAK2V617F allele burden (p=0,17) and mRNA expression (p=0,2). Patients showed a median spleen volume of 500 ml in Good-R and 250 ml in Bad-R group (p=0.01). Bad-R compared with Good-R showed higher mRNA expression of JAK1 (13.4 vs 4.7; p<0.00001), STAT3 (2.7 vs 2.4; p=0.0002) and SOCS3 (1.1 vs 2; p=0,002). The AUC, using the normalized gene expression values, was 0.88 for JAK1, 0.81 for STAT3 and 0.7 for SOCS3. Average expression in log2 of these three genes was calculated and used as IFN score. The ROC curve AUC analysis for IFN-score revealed an AUC of 0.9 (95% CI:0.8-1.0). The score value with highest combined sensitivity (94.1%, 95% CI: 71.3-99.8) and specificity (88.6%, 95% CI: 75.4-96.2) was 4.74, with a likelihood ratio of 8.28. In our cohort, all Bad-R patients but one, showed IFN-score higher than the established cut off, and this further support the accuracy of our IFN-responsive score for ET patients. Conclusions We identified a set of three genes whose expression could be compounded into IFN score that showed a significant correlation with response in ET patients. The IFN score could represent a predictive biomarker for responsiveness to IFN and may likely become a substantial aid to the physician. Disclosures: No relevant conflicts of interest to declare.

The Human Antibacterial Cathelicidin, hCAP-18, Is Synthesized in Myelocytes and Metamyelocytes and Localized to Specific Granules in Neutrophils

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2796-2803 ◽  
Author(s):  
Ole Sørensen ◽  
Kristina Arnljots ◽  
Jack B. Cowland ◽  
Dorothy F. Bainton ◽  
Niels Borregaard
Keyword(s):  
Electron Microscopy ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Subcellular Fractionation ◽  
Human Neutrophils ◽  
Major Protein ◽  
Equimolar Ratio ◽  
Binding Domains ◽  
Specific Granules ◽  
Immuno Electron Microscopy

Abstract hCAP-18 is the only human member of the antibacterial and endotoxin-binding family of proteins known as cathelicidins. The antibacterial and endotoxin binding domains reside in the C-terminal 37 amino acids of the protein (LL-37) and this is believed to be unleashed from the neutralizing N-terminus by proteases from peroxidase positive granules. In human neutrophils, peroxidase positive and peroxidase negative granules can be subdivided into granule subsets that differ in protein content and ability to be exocytosed. To determine the localization of hCAP-18, we performed high-resolution immuno-electron microscopy and subcellular fractionation on Percoll density gradients. Biosynthesis of hCAP-18 was investigated in isolated human bone marrow cells. hCAP-18 was found to colocalize and comobilize with lactoferrin, but not with gelatinase in subcellular fractions. This was confirmed by electron microscopy. hCAP-18 is synthesized at the same stage of myeloid cell maturation as lactoferrin, and is efficiently targeted to granules. Like the peroxidase negative granule's matrix metalloproteinases, collagenase and gelatinase, hCAP-18 is also stored in unprocessed form. hCAP-18 is a major protein of specific granules where it is present in equimolar ratio with lactoferrin.

scholarly journals Expression of JAK2V617I Does Not Result in Overt Myeloproliferative Disorder, but Results in Cytokine Hypersensitivity

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3218-3218
Author(s):  
Stefan Brooks ◽  
Samuel B Luty ◽  
Hew Yeng Lai ◽  
Lacey R Royer ◽  
Sarah J Morse ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Spleen Weight ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Empty Vector ◽  
Increased Risk ◽  
Germline Expression

Abstract BACKGROUND: A germline JAK2V617I mutation has recently been identified in a family with hereditary thrombocytosis (Mead et al, NEJM 2012). Like acquired MPN, family members with JAK2V617I have thrombocytosis and megakaryocytic hyperplasia in the marrow with increased risk of thrombosis. But unlike acquired MPN, individuals with this germline mutation do not develop a fibrotic bone marrow, splenomegaly, or transform to acute leukemia. Why germline JAK2V617I recapitulates some aspects but not others of the MPN phenotype in humans is unclear. To delineate the differences between JAK2V617F and JAK2V617I we compared the phenotype of mice with hematopoietic cells expressing JAK2V617F or JAK2V617I. METHODS AND RESULTS: Lethally irradiated C57B/6 mice were transplanted with bone marrow cells infected with retrovirus expressing JAK2V617F, JAK2V617I, or empty MSCV-IRES-GFP (MIG) vector. As expected, mice transplanted with JAK2V617F-expressing cells developed erythrocytosis and leukocytosis, whereas mice transplanted with JAK2V617I-expressing cells had peripheral blood counts similar to empty vector mice. Humans with germline JAK2V617I do not display constitutive activation of the kinase, but they do demonstrate cytokine hyper-responsiveness as evidenced by increased phosphorylation of STATs at low concentrations of ligand. We compared phosphorylated STAT5 in peripheral blood of mice transplanted with JAK2V617I, JAK2V617F, and MIG empty vector following stimulation with increasing concentrations of GM-CSF. At all concentrations of GM-CSF tested JAK2V617I and JAK2V617F-expressing cells had exaggerated phosphorylation of STAT5 as compared to MIG empty vector mice. We also measured phospho-STAT3 and STAT5 in unstimulated bone marrow and spleen from each mouse at time of euthanasia, there was no difference between JAK2V617I and MIG empty vector mice. JAK2V617F mice did demonstrate phosphorylation of STAT3 and STAT5 in the absence of GM-CSF, confirming the ability of JAK2V617F but not JAK2V617I to constitutively activate downstream signaling pathways. Next, to evaluate for histologic evidence of MPN and assess spleen size, all mice were euthanized at 120 days post-transplant. JAK2V617F mice had splenomegaly as expected, spleens from JAK2V617I mice appeared larger than empty vector mice, but spleen weight was not statistically different (p>0.05). While JAKV617I mice had increased cellularity of their marrow with increased numbers of megakaryocytes as compared to empty vector mice, this was not nearly to the extent of JAK2V617F mice. Mild fibrosis was seen in JAK2V617I mice, JAK2V617F mice had severe reticulin fibrosis in the marrow as expected. In the spleen architecture was preserved in the JAK2V617I mice, whereas in the JAK2V617F mice splenic architecture was disrupted by invasion of myeloid cells including megakaryocytes. To identify whether JAK2V617I affects the frequency of stem and progenitor cells or expands mature myeloid lineage cells we measured the frequency of hematopoietic stem cells, myeloid progenitors, and mature myeloid populations in JAK2V617F, JAK2V617I, and MIG empty vector. The bone marrow of JAK2V617I mice contained an increased percentage of GMP and MEP populations as compared to both the MIG empty vector and the JAK2V617F mice. Mature granulocyte (Gr-1+CD11b+) and erythroid (Ter119+) populations were expanded in the bone marrow and spleen of JAK2V617F but not JAK2V617I mice. CONCLUSIONS: These data demonstrate that the JAK2V617I mouse model recapitulates the effect of germline expression of JAK2V617I seen in humans: it results in cytokine hyper-responsiveness without the ability to constitutively activate downstream signals in the absence of ligand. Why JAK2V617F is so exquisitely conserved in acquired MPN is still unknown. Disclosures No relevant conflicts of interest to declare.

Mobilization with Cyclophosphamide Shifts Hematopoiesis From the Bone Marrow to the Spleen In Mouse.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1590-1590 ◽  
Author(s):  
Ludek Sefc ◽  
Ko-Tung Chang ◽  
Viktor Sykora ◽  
Emanuel Necas
Keyword(s):  
Bone Marrow ◽  
Mrna Expression ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Sublethal Dose ◽  
Hematopoietic Tissue ◽  
Hematopoietic Stem ◽  
Hematopoietic Organ ◽  
Total Body

Abstract Abstract 1590 Introduction: Spleen is an active hematopoietic tissue in adult mice. Spleen niches supporting hematopoiesis were not extensively studied yet. Spleen does not contain osteoblastic niches which are considered to be essential for long-term hematopoiesis, and splenic hematopoiesis is assumed to originate mainly from progenitors with short-term repopulating ability. Normal spleen is primarily a lymphoid organ but it also contributes to myelopoiesis by containing approximately 5% of total body multipotent progenitors CFU-S. We have studied the effect of mobilization with cyclophosphamide on trafficking of hematopoietic stem/progenitor cells (HSPC) from the bone marrow to the spleen, and dependence of normal splenic hematopoiesis on continual supply of HSPC from the bone marrow. Method: Murine congenic model C57Bl/6 Ly5.1/Ly5.2, and C57Bl/6 GFP, was used in the experiments. Kinetics of HSPC in the bone marrow and the spleen after a sublethal dose of cyclophosphamide (135 mg/kg) was measured by clonal assays (CFU-GM and CFU-S – progenitors) and by competitive repopulation assay (short- and long-term repopulating cells, STRC and LTRC). Parabiotic pairs were established from control and cyclophosphamide-treated partners in order to demonstrate HSPC exchange between hematopoietic organs. Selective irradiation of either spleen with bone marrow shielded, or irradiation of bone marrow with spleen shielded, was used to measure significance of HSPC migration from the spleen to the bone marrow during hematopoietic regeneration. Selected cytokine expression in the bone marrow and in the spleen was determined by real-time PCR. Result: Bone marrow HSPC rapidly but transiently regenerated from cyclophosphamide damage. Secondary decrease in HSPC between 5 and 7 days after cyclophosphamide in the bone marrow was accompanied with significant reduction of SDF-1 mRNA expression, mobilization of HSPC into peripheral blood and their rapid accumulation in the spleen. Spleen thus transiently became the main hematopoietic organ in the mouse by containing 69% of total body CFU-S, and also contained significantly more LTRC compared to the femur (Fig). Surprisingly, there was no increase in SDF-1 mRNA expression during homing of HSPC in the spleen. Spleens of parabiotic partners were rapidly colonized by partner cells including HSPC within two weeks. Bone marrow also contained small but significant amount of partner HSPC one and 2 weeks after parabiosis establishment. In pairs made from control and cyclophosphamide treated mice, mobilized HSPC preferentially engrafted in both own and partner spleens. Spleen cellularity and progenitor content was not affected by selective spleen irradiation and was restored from shielded bone marrow 6 days after irradiation. Expansion of extramedullary splenic hematopoiesis after cyclophosphamide occurred even when HSPC were destroyed in the spleen 1 day after cyclophosphamide, i.e. the spleen expansion was dependent on the bone marrow. Irradiation of bone marrow with spleen shielded led to abruption of splenic hematopoiesis in both control and cyclophosphamide treated mice. Conclusion: There is a quantitatively significant trafficking of HSPC by means of the circulation in mice. The splenic hematopoiesis appears to be almost fully dependent on a continuous supply of HSPC from the bone marrow. This trafficking of HSPC can be enhanced by hematopoietic cell mobilization. In mice, damage caused by cyclophosphamide results in a transient, however quantitatively remarkable relocation of HSPC, including LTRC, from the bone marrow to the spleen. This accumulation of HSPC in spleen is not accompanied with increased expression of SDF-1 mRNA. HSPC circulating in peripheral blood also significantly colonize the bone marrow and actively participate in its hematopoiesis. Support: Projects LC06044 and MSM 0021620806 of the Ministry of Education, Youth and Sports of the Czech Republic. Disclosures: No relevant conflicts of interest to declare.

scholarly journals THE LOCALIZATION OF LIPIDS IN HUMAN BLOOD AND BONE MARROW CELLS

Blood ◽  
1950 ◽  
Vol 5 (1) ◽  
pp. 79-88 ◽  
Author(s):  
MARVIN L. BLOOM ◽  
GEORGE B. WISLOCKI
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Ground Substance ◽  
Metabolic Factors ◽  
Baker’S Method ◽  
Sudan Black B ◽  
Specific Granules ◽  
Neutrophilic Leukocytes ◽  
Extraction Test ◽  
Supravital Staining

Abstract By Baker’s method for the staining of lipids, various types of cytoplasmic rods and granules in leukocytes give a positive acid hematein test but are negative after the pyridine-extraction test. According to Baker,8 this combination of results indicates the presence of phospholipids. Some of the rods and granules which are stained appear to be mitochondria, judging from their shape, size and distribution. as well as from the fact that they coincide in all respects with mitochondria demonstrable by supravital staining. Baker8 pointed out that in a great variety of cells his method stained mitochondria, although he did not report having examined leukocytes. In addition to mitochondria, Baker’s method stains the specific granules of the three varieties of granular leukocytes. In the neutrophilic leukocytes, the granules appear gray or black; in some of the eosinophilic leukocytes, they are intensely black, while in others they are unstained although surrounded by stained ground substance; and in the basophilic leukocytes, some of the granules of individual cells are stained while others are not. Provided the method is chemically specific, it follows that the specific granules of these leukocytes contain phospholipids. The appearance of the eosinophils indicates that many of them possess granules composed of protein surrounded by phospholipid, although some of them contain granules with lipid in their interiors. These differences in the eosinophils suggest possible functional stages, related perhaps to the age of the cells or some other metabolic factors. In the monocytes, besides a general stippling of the cytoplasm corresponding to mitochondria, the region of the attraction sphere often contains numerous deeply stained granules. Sudan black B stains both the mitochondria and the specific leukocytic granules in a manner similar to Baker’s method. However, certain slight differences were noticed. Platelets were brought out moderately distinctly by the acid hematein procedure, whereas in sudan black preparations they were barely visible. A variability was noted in the staining of the granules of the eosinophilic leukocytes in Baker’s method, in contrast to the uniformity of their staining with sudan black. This difference should perhaps be interpreted as indicating that the eosinophilic granules contain other lipid substances besides phospholipids. Mitochondnia, in whatsoever type of leukocyte they are observed, appear to stain more deeply and distinctly by Baker’s method than with sudan black, a circumstance suggesting that mitochondria are very rich in phospholipids.

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