Granulocyte-macrophage Colony-forming Units Research Articles

Guideline on the management of primary resistant and relapsed classical Hodgkin lymphoma

Summary of key recommendations ● Repeat biopsy is generally recommended in Hodgkinlymphoma (HL) patients thought to have relapsed, andshould be considered in those who have residual fluoro-deoxyglucose (FDG)-avid lesions post-therapy (1C).● Positron-emission tomography/computerized tomogra-phy (PET-CT) is the preferred restaging modality aftersalvage therapy (1B).● The aim of salvage treatment should be to achieve anFDG-PET-negative remission (1B).● The choice of a first-line salvage regimen in patients eli-gible for autologous stem cell transplantation (ASCT)should be based on patient factors and familiarity ofthe treatment centre with the regimen (2C).● Regimens containing stem cell toxic agents (such as car-mustine and melphalan) should be avoided if possibleuntil stem cells have been successfully collected andcryopreserved if ASCT is planned (1B).● There is currently no evidence to support intensivesequential induction/consolidation strategies prior toASCT (1B).● Consider switching to an alternative non-cross-resistantsalvage regimen if there are residual FDG-avid lesionsafter first line salvage treatment and the intent is toproceed to ASCT (2B).● In patients not eligible for ASCT, combined modalitytherapy should be considered especially in early stagerelapse and in patients who have not received priorradiotherapy or who have relapsed outside of the initialradiotherapy field (2B).● In patients unlikely to tolerate the toxicities associatedwith more intensive regimens, palliation with either a sin-gle agent or with a multi-agent oral regimen with or with-out intravenous vinblastine should be considered (2C).● Early consideration of involvement of palliative care ser-vices is recommended, particularly in those not eligiblefor high dose therapy (1C).● ASCT is the standard treatment for patients withrelapsed disease who achieve an adequate response tosalvage therapy (1A).● ASCT is also the standard treatment for patients withprimary resistant disease who achieve an adequateresponse to salvage therapy (1B).● ASCT is not recommended in those failing to achievean adequate response (1B).Guideline

PRDM11 is dispensable for the maintenance and function of hematopoietic stem and progenitor cells

Hematopoietic stem cells (HSC)11Hematopoietic stem cell (HSC), hematopoietic stem and progenitor cell (HSPC), bone marrow (BM), bone marrow transplantation (BMT), acute myeloid leukemia (AML), peripheral blood (PB), multipotent progenitor (MPP), pre-megakaryocyte/erythroid (preMegE), megakaryocytic progenitor (MkP), pre-granulocyte/macrophage (preGM), granulocyte/macrophage progenitors (GMP), common lymphoid progenitors (CLP), colony forming unit erythroid (CFU-E), proErythroid (proE), colony forming unit megakaryocyte (CFU-Mk), colony forming unit granulocyte macrophage (CFU-GM), megakaryocyte (Mk), LSK (Lineage−, Sca1+, c-Kithi). supply organisms with life-long output of mature blood cells. To do so, the HSC pool size has to be maintained by HSC self-renewing divisions. PRDM3 and PRDM16 have been documented to regulate HSC self-renewal, maintenance and function. We found Prdm11 to have similar expression patterns in the hematopoietic stem and progenitor cell (HSPC) compartments as Prdm3 and Prdm16. Therefore, we undertook experiments to test if PRDM11 regulates HSC self-renewal, maintenance and function by investigating the Prdm11−/− mice. Our data shows that phenotypic HSPCs are intact in bone marrow (BM) of one-year-old Prdm11−/− mice. In addition, Prdm11−/− mice were able to fully regenerate the hematopoietic system upon BM transplantation (BMT) into lethally irradiated mice with a mild drop in lymphoid output only. Taken together, this suggests that PRDM11, in contrast to PRDM3 and PRDM16, is not directly involved in regulation of HSPCs in mice.

Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells

We evaluated the use of colony formation (colony-forming unit-granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], and colony-forming unit-granulocyte-erythroid-megakaryocyte-monocytes [CFU-GEMM]) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. The following compounds were added before (protection) or after (mitigation) ionizing irradiation: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor LY29400, triphenylphosphonium-imidazole fatty acid, the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propranolol, and the adenosine triphosphate-sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs were effective in murine assays; TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide but showed no significant protection or mitigation in human CB assays. These data support the testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, thus reducing the need for animal experiments.

Abstract 426: In-field head and neck irradiation radiosensitivity of FancD2-/- mice and bystander effect reduction of distant bone marrow hematopoietic progenitor cells.

Abstract Background: Irradiation induced normal tissue toxicity limits effective radiotherapy of individuals with DNA repair defects including Fanconi anemia. We quantitated in-field head and neck tissue response in irradiated Fanconi anemia knockout mice, FancD2-/-, compared to wild type, FancD2+/+ & heterozygous FancD2+/- mice. We also assessed bystander effect of head and neck irradiation on distant bone marrow hematopoietic progenitor cell numbers. Method: Adult 10-12wk old FevB background FancD2+/+, +/- and -/- mice were irradiated to the head & neck to 24Gy, 26Gy, 28Gy or 30Gy (n=3) shielding all tissue below the cervical spine. On day 2 or 5 post irradiation, mice were sacrificed and tongue tissue and one femur excised for histopathology. Bone marrow of the contralateral femur was plated for colony assay. Colony forming unit-granulocyte macrophage (CFU-GM), burst forming unit erythroid (BFU-E), and colony forming unit-granulocyte-erythroid-megakaryocyte-monocyte (CFU-GEMM) numbers were counted & each genotype compared to its unirradiated control. H&E stained tongue sections were scored for oral mucositis. Results: Both FancD2-/- & FancD2+/- mice were radiosensitive to lower doses compared to FancD2+/+ mice measured as percent ulceration of the squamous layer of tongue epithelium 5 days post irradiation. Both FancD2+/- and FancD2-/- mice had significantly higher ulceration after 26Gy (52.8±6.5%, p=0.0014 and and 59.4±3.4%, p=0.0001, respectively) compared to wild type FancD2+/+ (21.7±4.7%). At the lower dose of 24Gy, FancD2+/- and -/- had higher percent ulceration (p=0.0001 and p=0.0003) than FancD2+/+ mice irradiated to even higher doses of 26Gy and 28Gy. FancD2+/+ mice had high levels of ulceration after 28Gy (p=0.2355). While there was no difference in distant bone marrow cellularity between irradiated groups, both FancD2-/- & FancD2+/- showed bystander reduction in colony forming cells. CFU-GM were significantly reduced at 2 days after 26Gy in FancD2+/- mice compared to unirradiated controls (p=0.007). FancD2 -/- mice had decreased BFU-E after 24Gy (p=0.009) & CFU-GEMM after 26Gy (p=0.048) and reduced BFU-E numbers after 26Gy (p=0.031). FancD2-/- mice had significantly less CFU-GM after either 26Gy or 28Gy (p=0.017 and p=0.028, respectively). FancD2-/- & +/- mice, at 5 days after 26Gy, had less CFU-GM (p=0.001 and 0.012, respectively) and CFU-GEMM (p=0.014 and p=0.004, respectively) compared to unirradiated controls. There was no reduction in CFU-GM, BFU-E or CFU-GEMM in wild type FancD2+/+mice after 28 or 30Gy. Conclusion: Biallelic or monoallelic loss of the FA gene renders mice radiosensitive to in-field head and neck irradiation compared to wild type mice, and is accompanied by a distant femur marrow bystander effect including reduction of colony forming unit. Supported by NIAID U19-A 1068021 and FA Research Foundation Citation Format: Hebist Berhane, Julie Goff, Ronny Kalash, Michael W. Epperly, Tracy Dixon, Donna Shields, Darcy Franicola, Joel S. Greenberger. In-field head and neck irradiation radiosensitivity of FancD2-/- mice and bystander effect reduction of distant bone marrow hematopoietic progenitor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 426. doi:10.1158/1538-7445.AM2013-426

Effect of different cryopreservation time on quality of umbilical cord blood cells

This study was aimed to explore the effect of different cryopreservation time on recovery rate of cord blood stem cells, and analyze the influence of cord blood cells after thawing on the engraftment speed of cord blood cells in patients. 20 cord blood units were stored at -196°C for 1 - 10 years. The cell viability, content of total nucleated cell (TNC), CD34(+) cells and the colony forming units of granulocyte/macrophage (CFU-GM) were assessed after thawing, the impact of cell recovery on engraftment speed in patients was analyzed. The results showed that as compared with data provided by Umbilical Cord Blood Bark, the different cryopreservation time had no effect on yield of cord blood stem cells after thawing. The cell viability was (92.75 ± 2.55)% after thawing, the yields of TNC, CD34(+) cells and CFU-GM were 89.9%, 84.8% and 84.3%, compared with that of pre-freezing, their differences were statistically significant (P = 0.000), however, loss of cells had no effect on the time of neutrophils and platelets engraftment. The TNC and CD34(+)cell count after thawing correlated closely with that of pre-freezing (r = 0.954 and r = 0.931, P = 0.000), but CFU-GM content poorly correlated with that (r = 0.285, P = 0.223). It is concluded that cryopreservation and thawing process can damage the cord blood stem cells, leading to cell loss, but not affect transplant results.

Radiosensitivity of human haematopoietic stem/progenitor cells

The haematopoietic system is regenerative tissue with a high proliferative potential; therefore, haematopoietic stem cells (HSCs) are sensitive to extracellular oxidative stress caused by radiation and chemotherapeutic agents. An understanding of this issue can help predict haematopoietic recovery from radiation exposure as well as the extent of radiation damage to the haematopoietic system. In the present study, the radiosensitivity of human lineage-committed myeloid haematopoietic stem/progenitor cells (HSPCs), including colony-forming unit–granulocyte macrophage, burst-forming unit–erythroid and colony-forming unit–granulocyte–erythroid–macrophage–megakaryocyte cells, which are contained in adult individual peripheral blood (PB) and fetus/neonate placental/umbilical cord blood (CB), were studied. The PB of 59 healthy individual blood donors and the CB of 42 neonates were investigated in the present study. HSPCs prepared from PB and CB were exposed to 0.5 or 2 Gy x-irradiation. The results showed that large individual differences exist in the surviving fraction of cells. In the case of adult PB, a statistically significant negative correlation was observed between the surviving fraction observed at a dose of 0.5 Gy and the age of the blood donors; however, none of these correlations were observed after 2 Gy x-irradiation. In addition, seasonal and gender variation were observed in the surviving fraction of CB HSPCs. The present results suggest that there are large individual differences in the surviving fraction of HSPCs contained in both adult PB and fetus/neonate CB. In addition, some factors, including the gender, age and season of birth, affect the radiosensitivity of HSPCs, especially with a relatively low-dose exposure.

Refinement and verification of test conditions for colony-forming unit assay in rat bone marrow cells with preservable colony specimens

We established test conditions for colony-forming unit assay using rat bone marrow cells in which the colony specimens can be preserved. In our test system, all colonies can be transferred from a petri dish to a slide glass with whole agar medium because the agar medium volume is small and movable, enabling the agar medium to be dried and then fixed on the slide glass. Appropriate conditions for the colony-forming unit granulocyte–macrophage assay were as follows: 10 to 30 ng/ml recombinant murine granulocyte and macrophage colony-stimulating factor, 1 × 105 cells/dish, and culture periods of 3 days for neutrophil colonies and 8 days for macrophage colonies. Appropriate conditions for the colony-forming unit erythroid assay were as follows: 2 IU/ml recombinant human erythropoietin, 0.25 to 1 × 105 cells/dish, and culture period of 2–3 days. We also examined responses of bone marrow toxicants 5-fluorouracil and doxorubicin in these test systems, finding that both compounds decreased the numbers of colonies in a concentration-dependent manner in both assays. These findings suggest that these test systems are useful tools in evaluating bone marrow toxicity of these particular compounds.

Studies in C4b-Deficient Mice Provide Further Evidence That Complement Cascade Orchestrates the Mobilization of Hematopoietic Stem/Progenitor Cells.

AbstractAbstract 2316 Introduction:Complement cascade (CC) and innate immunity have emerged as important modulators of hematopoietic stem/progenitor cell (HSPC) trafficking. We reported that the CC becomes activated in bone marrow (BM) during HSPC mobilization induced by G-CSF or AMD3100 and proposed that the generation of C5a and C5b-C9 (membrane attack complex; MAC) is required for optimal mobilization (Stem Cells 2007; 25:3093; Leukemia 2010; 24:976). While C5a induces a proteolytic microenvironment in BM that attenuates SDF-1-CXCR4 retention signals for HSPC and promotes egress of leucocytes, C5b-C9 (MAC) induces the release of a crucial chemoattractant, sphingosine-1 phosphate (S1P), from red blood cells and augments mobilization of HSPC from BM. On the other hand, C3 cleavage fragments attenuate mobilization by enhancing responsiveness of HSPC to SDF-1 retention signals thus promoting HSPC retention in BM. Thus our findings suggest that mobilization is differently regulated by the proximal and distal parts of CC (upstream and downstream of C3, respectively). We also demonstrated that C3-deficient mice (lacking C3 cleavage fragments) are easy mobilizers whereas C5-deficient mice (lacking C5 cleavage fragments and not generating MAC) mobilize HSPC very poorly (Leukemia 2009; 23:2052). C4 is part of the classical pathway of CC whose activation/cleavage is initiated by C1 and C2, releasing smaller (C4a and C2b) and larger (C4b and C2a) fragments. C2a binds with C4b to form an enzymatic complex termed C3 convertase that cleaves C3 into C3a anaphylatoxin and C3b. Aim of Study:In the present study we investigated the role of C4 in HSPC mobilization and hypothesized that C4-deficient mice are easy mobilizers, supporting the notion that the proximal part of CC is crucial for retention of HSPC in the BM microenvironment. Experimental Approach:We employed 6–8 week old C4b-deficient mice (strain B6.129S-C4btm1Crr/J from Jackson Laboratory, Bar Harbour, ME) and wild type (WT) littermates. Mice were injected subcutaneously with 250 μg/kg of human recombinant G-CSF (Amgen, Thousand Oaks, CA) or saline (control) daily for 3 days. At 6 h after the last G-CSF injection, the mice were sacrificed and blood was collected from the vena cavae. Mobilization was evaluated by determining the number of leukocytes (WBC) and colony-forming unit granulocyte-macrophages (CFU-GM) circulating in the peripheral blood (PB). We also measured the level of mouse terminal complement complex C5b-C9 (MAC) in plasma using ELISA (Kamiya) in G-CSF-mobilized and non-mobilized C4b-deficient and WT mice. Results:We found that C4b-deficient mice treated with saline have slightly higher WBC counts than WT mice and that G-CSF induced a greater increase in WBC counts in these mice than in WT mice. Based on CFU-GM counts after G-CSF mobilization, C4b-deficient mice mobilized significantly more HSPCs into PB than WT mice (p < 0.0065). These observations were consistent in all four experiments performed involving 18–20 mice per experiment. The G-CSF mobilization responses correlated with a greater increase in activation of the distal part of CC in C4b-deficient mice compared to WT mice, as evidenced by C5b-C9 levels evaluated by ELISA. Conclusions:Our data indicate that C4-deficient mice are easy mobilizers, thus supporting a role for the proximal part of CC in retention of HSPC in BM. Furthermore, mobilization of HSPC in C4b-deficient mice correlated with activation of the distal part of CC and generation of C5b-C9 (MAC) suggesting that activation of the distal part of CC is crucial for egress of HSPCs into PB. Moreover, activation of the distal part of CC in C4-deficient mice that have defective activation of the proximal part of CC indicates that C5 may be cleaved in a C3-independent manner by other proteolytic enzymes present in blood plasma. Finally, our data further support that mobilization is part of a more general immune response in which all complement components, including C4, play a significant role. Disclosures:No relevant conflicts of interest to declare.

CD123-Specific Chimeric Antigen Receptor Redirected T Cells Exhibit Potent Cytolytic Activity and Multiple Effector Functions Against Acute Myeloid Leukemia without Altering Normal Hematopoietic Colony Formation in Vitro

AbstractAbstract 950Current treatment regimes for acute myeloid leukemia (AML) achieve complete remissions in only a subset of individuals and most adult patients will relapse within 5-years, emphasizing the need for novel treatment alternatives. One such therapy may be the administration of T cells engineered to express chimeric antigen receptors (CARs) specific for AML-associated antigens. CARs are typically composed of a single chain variable fragment (scFv) from a monoclonal antibody fused to the CD3ζ signaling domain and may contain one or more costimulatory endodomains. When expressed in T cells, CARs redirect T cell specificity to surface antigens on target cells in an MHC-independent manner. The interleukin 3 receptor alpha chain (IL3Rα, CD123) is a cell surface receptor which is aberrantly over-expressed on multiple hematologic malignancies including AML. Previous work has demonstrated that CD123 is not expressed on all CD34+/CD38− hematopoietic stem cells and is restricted to cells of the myeloid lineage, making CD123 an attractive target for CAR T cell therapy. We have therefore generated two novel CD123-specific (CD123R) CARs using scFvs from previously characterized antibodies, designated 26292 and 32716, which bind two distinct epitopes on CD123. Here we demonstrate that T cells expressing CARs derived from either 26292 or 32716 effectively redirect T cell specificity against CD123+ cells. Healthy donor T cells (n=3) engineered to express the CD123R CARs efficiently lysed CD123+ cell lines LCL and KG1a while sparing the CD123− cell line K562 as demonstrated by a 4 hour chromium-51 (51Cr) release assay. Additionally, both of the CD123R CAR T cells produced similar levels of IFN-γ and TNF-α and displayed comparable levels of antigen-dependent proliferation following co-culture with CD123+ cell lines. The potent cytolytic activity and activation of our CD123-targeting T cells was not limited to tumor cell lines. Indeed, CD123R CAR T cells, but not donor-matched CD19-specific (CD19R) CAR T cells, robustly lysed panel of primary AML samples (n=6, 3 – persistent, 1 – relapsed, 2 - untreated) (* p<0.05, ** p<0.001 using the unpaired students’ t-test comparing 26292 or 32716 CAR T cells to donor-matched CD19R CAR T cells), and exhibited multiple effector functions for both CD4 and CD8 T cell subsets (ie CD107a degranulation, IFN-γ and TNF-α production, and antigen specific proliferation) when co-cultured with primary AML samples (n=3, 2 – relapsed, 1-persistent). To examine the effect our CD123-specific T cells have on normal and leukemic progenitor cells, we co-cultured CD123R CAR T cells, or donor-matched CD19-targeting T cells, with either CD34-enriched cord blood (CB, n=3) or primary AML samples (n=3, 2 – relapsed, 1 - untreated) for 4 hours (E:T 25:1) prior to plating in semisolid methylcellulose progenitor culture. CD123-targeting T cells did not significantly reduce the number of colony-forming unit granulocyte-macrophage (CFU-GM) or burst-forming unit erythroid (BFU-E) colonies from CB when compared to CD19R CAR T cells. Finally, while CD19-specific T cells had little impact on leukemic colony formation of primary AML samples, CD123-targeting T cells significantly reduced leukemic colony formation in vitro.Collectively, our data demonstrate that CD123-specifc CARs can be expressed in primary healthy donor T cells, distinguish between CD123+ and CD123− cells, and mediate robust anti-leukemic activity against a panel of poor-risk primary AML patient samples. Importantly, we demonstrate that CD123R CAR T cells have little impact on normal progenitor colony formation while significantly reducing the growth of clonogenic myeloid leukemic progentiors in vitro. Thus, CD123R CAR T cells are a promising candidate for future immunotherapy of AML. [Display omitted] Disclosures:Bhatia:Novartis: Consultancy, Honoraria. Jensen:ZetaRx: Equity Ownership, Patents & Royalties.

1,25-dihydroxyvitamin D3 inhibits directly human osteoclastogenesis by down-regulation of the c-Fms and RANK expression

Objective1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a key molecule to maintain calcium homeostasis and bone metabolism. It was recently reported that 1,25(OH)2D3 directly inhibited osteoclast differentiation in mouse bone marrow cells and human bone marrow-derived colony-forming unit granulocyte macrophage (CFU-GM) cells. However, the direct effects of 1,25(OH)2D3 and its affecting mechanisms on the osteoclast differentiation of human osteoclast precursors remain largely unknown. In this study, we examined the direct effects of 1,25(OH)2D3 on the osteoclastogenesis of human peripheral blood (PB) osteoclast precursors. MethodsIn vitro osteoclastogenesis assays were performed using osteoclast precursors from normal PB. The gene expressions were analyzed using real-time PCR. The cell surface proteins, including c-Fms and RANK, were measured by flow cytometry. Results1,25(OH)2D3 strongly inhibited osteoclast differentiation and it suppressed the expression of RANK in the human PB osteoclast precursors. One mechanism of RANK inhibition by 1,25(OH)2D3 is down-regulation of the M-CSF receptor c-Fms, which is required for the expression of RANK. In contrast to the previous reports on mouse osteoclast precursors, 1,25(OH)2D3 did not affect the expression of c-Fos. Parallel to the inhibition of osteoclastogenesis, 1,25(OH)2D3 increased the expression and phosphorylation of CCAAT enhancer-binding protein β (C/EBPβ), which is a recently discovered inhibitor of osteoclastogenesis. ConclusionsOur results show that 1,25(OH)2D3 inhibits human osteoclastogenesis by decreasing the RANK+ osteoclast precursors, and we suggest that 1,25(OH)2D3 may be a powerful therapeutic agent for treating inflammation-induced bone disease that shows excessive osteoclast activation.

Deletion of CD74, a putative MIF receptor, in mice enhances osteoclastogenesis and decreases bone mass

CD74 is a type II transmembrane protein that can act as a receptor for macrophage migration inhibitory factor (MIF) and plays a role in MIF-regulated responses. We reported that MIF inhibited osteoclast formation and MIF knockout (KO) mice had decreased bone mass. We therefore examined if CD74 was involved in the ability of MIF to alter osteoclastogenesis in cultured bone marrow (BM) from wild-type (WT) and CD74-deficient (KO) male mice. We also measured the bone phenotype of CD74 KO male mice. Bone mass in the femur of 8-week-old mice was measured by micro-computed tomography and histomorphometry. Bone marrow cells from CD74 KO mice formed 15% more osteoclast-like cells (OCLs) with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) (both at 30 ng/mL) compared to WT. Addition of MIF to WT cultures inhibited OCL formation by 16% but had no effect on CD74KO cultures. The number of colony forming unit granulocyte-macrophage (CFU-GM) in the bone marrow of CD74 KO mice was 26% greater than in WT controls. Trabecular bone volume (TBV) in the femurs of CD74 KO male mice was decreased by 26% compared to WT. In addition, cortical area and thickness were decreased by 14% and 11%, respectively. Histomorphometric analysis demonstrated that tartrate-resistant acid phosphatase (TRAP)(+) osteoclast number and area were significantly increased in CD74 KO by 35% and 43%, respectively compared to WT. Finally, we examined the effect of MIF on RANKL-induced-signaling pathways in bone marrow macrophage (BMM) cultures. MIF treatment decreased RANKL-induced nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and c-Fos protein in BMM cultures by 70% and 41%, respectively. Our data demonstrate that CD74 is required for MIF to affect in vitro osteoclastogenesis. Further, the bone phenotype of CD74 KO mice is similar to that of MIF KO mice. MIF treatment of WT cultures suppressed RANKL-induced activator protein 1 (AP-1) expression, which resulted in decreased osteoclast differentiation in vitro. We propose that CD74 plays a critical role in the MIF inhibition of osteoclastogenesis.

Subacute toxicity of nano‐selenium compared to other selenium species in mice

Sixteen groups of mice were fed diets containing different selenium species to compare their toxicity. Inorganic sodium selenate and sodium hydroselenite, elementary nanoSe, organic Sel-Plex, and Lacto-MicroSelenium were administered for 14 d at concentrations of 0.5, 5, and 50 ppm Se, equivalent to 0.5, 5, and 50 mg Se/kg food, corresponding to an estimated 4, 40, and 400 µg/kg body weight/d Se uptake, respectively. At the end of the treatment, body, liver, spleen, kidney, heart, and brain weights were measured, mice were subjected to necropsy, and histological examinations were performed on the liver. At lower Se doses (0.5 and 5 ppm) a moderate reduction was observed in the number of bone marrow and white blood cells and in granulocyte-macrophage colony-forming units (GM-CFUs) relative to the untreated control group of mice. A comparison of lowest toxic doses of sodium selenite in mice (0.5 ppm) and mallard (10 ppm) indicates that birds are more resistant to Se than rodents. In mice, a small but measurable weight loss was observed after 5 ppm selenate and LactoMicroSe treatment. The most significant changes took place after 50-ppm administration in body and spleen weight, hematology, and liver histology. Toxicity was more pronounced when inorganic Se was applied than after subacute application of Sel-Plex, nanoSe, or LactoMicroSe. To summarize the effects, the authors' 14-d murine subacute toxicity study showed that the toxicity of Se species decreased in the following order: selenate > selenite > nanoSe > Sel-Plex > LactoMicroSe.

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

Circulating hematopoietic stem cell count is a valuable predictor of prematurity complications in preterm newborns

BackgroundThe frequency of preterm labour has risen over the last few years. Hence, there is growing interest in the identification of markers that may facilitate prediction and prevention of premature birth complications. Here, we studied the association of the number of circulating stem cell populations with the incidence of complications typical of prematurity.MethodsThe study groups consisted of 90 preterm (23–36 weeks of gestational age) and 52 full-term (37–41 weeks) infants. Non-hematopoietic stem cells (non-HSCs; CD45-lin-CD184+), enriched in very small embryonic-like stem cells (VSELs), expressing pluripotent (Oct-4, Nanog), early neural (β-III-tubulin), and oligodendrocyte lineage (Olig-1) genes as well as hematopoietic stem cells (HSCs; CD45+lin-CD184+), and circulating stem/progenitor cells (CSPCs; CD133+CD34+; CD133-CD34+) in association with characteristics of prematurity and preterm morbidity were analyzed in cord blood (CB) and peripheral blood (PB) until the sixth week after delivery. Phenotype analysis was performed using flow cytometry methods. Clonogenic assays suitable for detection of human hematopoietic progenitor cells were also applied. The quantitative parameters were compared between groups by the Mann–Whitney test and between time points by the Friedman test. Fisher’s exact test was used for qualitative variables.ResultsWe found that the number of CB non-HSCs/VSELs is inversely associated with the birth weight of preterm infants. More notably, a high number of CB HSCs is strongly associated with a lower risk of prematurity complications including intraventricular hemorrhage, respiratory distress syndrome, infections, and anemia. The number of HSCs remains stable for the first six weeks of postnatal life. Besides, the number of CSPCs in CB is significantly higher in preterm infants than in full-term neonates (p < 0.0001) and extensively decreases in preterm babies during next six weeks after birth. Finally, the growth of burst-forming unit of erythrocytes (BFU-E) and colony-forming units of granulocyte-macrophage (CFU-GM) obtained from CB of premature neonates is higher than those obtained from CB of full-term infants and strongly correlates with the number of CB-derived CSPCs.ConclusionWe conclude that CB HSCs are markedly associated with the development of premature birth complications. Thus, HSCs ought to be considered as the potential target for further research as they may be relevant for predicting and controlling the morbidity of premature infants. Moreover, the observed levels of non-HSCs/VSELs circulating in CB are inversely associated with the birth weight of preterm infants, suggesting non-HSCs/VSELs might be involved in the maturation of fetal organism.

A short course of granulocyte–colony-stimulating factor to accelerate wound repair in patients undergoing surgery for sacrococcygeal pilonidal cyst: proof of concept

Stem cells, namely easily accessible bone marrow-derived cells (BMC), are reportedly capable of tissue repair in different damaged organs and might favor wound healing. The present study was undertaken to evaluate the feasibility and safety of BMC mobilization induced by granulocyte-colony-stimulating factor (G-CSF) in patients undergoing surgery for sacrococcygeal pilonidal cysts (SPC). To evaluate the possible clinical benefit of G-CSF in reducing the time to complete resolution, a comparison with a control group receiving surgery without G-CSF was performed. Eight patients with complex SPC were included in this prospective trial. Patients were treated with G-CSF (5 µg/kg b.i.d.) for 3 consecutive days; standard surgical exeresis of the pilonidal cyst was scheduled on day 2 of mobilization. Mobilization was assessed in terms of circulating CD34(+) cells and granulocyte-macrophage colony-forming unit (CFU-GM) progenitors. Mobilization of CD34(+) cells and CFU-GM occurred in all patients, along with a marked increase in white blood cells (median peak value 28 435/µL, day 3). G-CSF was well tolerated and no adverse events occurred. All patients received the planned surgical treatment without any complications. Interestingly, the G-CSF group patients had a median time to resolution (117 days, range 110-130) significantly shorter than control patients (145 days, range 118-168) (P = 0.034). G-CSF administration, along with BMC mobilization, is feasible and well tolerated in patients undergoing surgery for SPC; clinical results compare favorably with those observed in controls not receiving G-CSF; the results suggest the potential use of G-CSF as an additional treatment to accelerate wound healing in patients undergoing surgery.

Exercise Increases the Frequency of Circulating Hematopoietic Progenitor Cells, But Reduces Hematopoietic Colony-Forming Capacity

Circulating hematopoietic progenitor cells (CPCs) may be triggered by physical exercise and/or normobaric hypoxia from the bone marrow. The aim of the study was to investigate the influence of physical exercise and normobaric hypoxia on CPC number and functionality in the peripheral blood as well as the involvement of oxidative stress parameters as possibly active agents. Ten healthy male subjects (25.3±4.4 years) underwent a standardized cycle incremental exercise test protocol (40 W+20 W/min) under either normoxic (FiO2 ∼0.21) or hypoxic conditions (FiO2<0.15, equals 3,500 m, 3 h xposure) within a time span of at least 1 week. Blood was drawn from the cubital vein before and 10, 30, 60, and 120 min after exercise. The number of CPCs in the peripheral blood was analyzed by flow cytometry (CD34/CD45-positive cells). The functionality of cells present was addressed by secondary colony-forming unit-granulocyte macrophage (CFU-GM) assays. To determine a possible correlation between the mobilization of CPCs and reactive oxygen species, parameters for oxidative stress such as malondialdehyde (MDA) and myeloperoxidase (MPO) were obtained. Data showed a significant increase of CPC release under normoxic as well as hypoxic conditions after 10 min of recovery (P<0.01). Most interestingly, although CD34+/CD45dim cells increased in number, the proliferative capacity of CPCs decreased significantly 10 min after cessation of exercise (P<0.05). A positive correlation between CPCs and MDA/MPO levels turned out to be significant for both normoxic and hypoxic conditions (P<0.05/P<0.01). Hypoxia did not provoke an additional effect. Although the CPC frequency increased, the functionality of CPCs decreased significantly after exercise, possibly due to the influence of increased oxidative stress levels.

SPARC promotes the development of erythroid progenitors

Secreted protein acidic and rich in cysteine (SPARC) is a well-recognized regulator that affects cell proliferation, differentiation, and survival. Here, we investigated the impact of SPARC on erythropoiesis. Erythropoiesis in bone marrow (BM) from SPARC(-/-) mice was analyzed by flow cytometry and colony-forming assays. The mechanisms that affected erythropoiesis were investigated by BM transplantation experiments. CD34(+) cells from umbilical cord blood were isolated by MiniMACS, and the effect of SPARC on human erythropoiesis was assessed by colony-formation assay and erythroid differentiation culture. The hemoglobin level in peripheral blood (PB) was lower in SPARC(-/-) mice. Neither red blood cell count in PB nor the percentage of Ter119(+) erythrocytes in BM showed significant difference between SPARC(-/-) and WT mice. However, the ability of SPARC(-/-) bone marrow cells (BMCs) to form erythroid burst-forming units (BFU-E), as well as spleen colony-forming units (CFU-S8), was significantly decreased. The addition of exogenous SPARC could promote the formation of BFU-E from SPARC(-/-) BMCs in vitro. And the impaired ability of SPARC(-/-) BMCs to form BFU-E could be restored by transplanted into WT BM microenvironment, whereas the BFU-E formation of WT BMCs was impaired after transplanted into SPARC(-/-) recipients. Furthermore, exogenous SPARC promoted umbilical cord blood CD34(+) cells to form erythroid colonies and the hemoglobinization of erythroid, but did not affect the expression levels of glycophorin A and CD71. In conclusion, our results indicate that SPARC promotes the development of erythroid progenitors, but does not affect terminal erythroid differentiation.

Amelioration of radiation-induced hematopoietic and gastrointestinal damage by Ex-RAD(R) in mice

The aim of the present study was to assess recovery from hematopoietic and gastrointestinal damage by Ex-RAD®, also known as ON01210.Na (4-carboxystyryl-4-chlorobenzylsulfone, sodium salt), after total body radiation. In our previous study, we reported that Ex-RAD, a small-molecule radioprotectant, enhances survival of mice exposed to gamma radiation, and prevents radiation-induced apoptosis as measured by the inhibition of radiation-induced protein 53 (p53) expression in cultured cells. We have expanded this study to determine best effective dose, dose-reduction factor (DRF), hematological and gastrointestinal protection, and in vivo inhibition of p53 signaling. A total of 500 mg/kg of Ex-RAD administered at 24 h and 15 min before radiation resulted in a DRF of 1.16. Ex-RAD ameliorated radiation-induced hematopoietic damage as monitored by the accelerated recovery of peripheral blood cells, and protection of granulocyte macrophage colony-forming units (GM-CFU) in bone marrow. Western blot analysis on spleen indicated that Ex-RAD treatment inhibited p53 phosphorylation. Ex-RAD treatment reduces terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay (TUNEL)-positive cells in jejunum compared with vehicle-treated mice after radiation injury. Finally, Ex-RAD preserved intestinal crypt cells compared with the vehicle control at 13 and 14 Gy. The results demonstrated that Ex-RAD ameliorates radiation-induced peripheral blood cell depletion, promotes bone marrow recovery, reduces p53 signaling in spleen and protects intestine from radiation injury.

Relative recovery of haematopoietic stem cell products after cryogenic storage of up to 19 years

There is an increasing trend towards long-term frozen storage of haematopoietic stem cells. For such stem cells, harvested from peripheral blood (PB) or BM, it is not known if stem cell viability decreases with time. In this study, 31 separate bags of stem cell product (SCP) stored for 11-19 years (median 15 years) were assessed for total nucleated cell (TNC) count, colony forming unit-granulocyte/macrophage (CFU-GM), CD34⁺ cell count and cell viability. The results were compared with the initial results obtained for the products at the time of stem cell harvest, and the percentage recovery of each parameter was plotted against time. Recovery of TNC, CD34⁺ cell count and cell viability decreased with time (P=<0.01) but CFU-GM did not. This study shows that SCPs harvested from PB and BM do deteriorate with long-term storage. This could have an impact on rates of engraftment.

Leukemia-related chromosomal loss detected in hematopoietic progenitor cells of benzene-exposed workers

Benzene exposure causes acute myeloid leukemia, and hematotoxicity, shown as suppression of mature blood and myeloid progenitor cell numbers. As the leukemia-related aneuploidies monosomy 7 and trisomy 8 previously had been detected in the mature peripheral blood cells of exposed workers, we hypothesized that benzene could cause leukemia through the induction of these aneuploidies in hematopoietic stem and progenitor cells. We measured loss and gain of chromosomes 7 and 8 by fluorescence in situ hybridization in interphase colony-forming unit-granulocyte-macrophage (CFU-GM) cells cultured from otherwise healthy benzene-exposed (n=28) and unexposed (n=14) workers. CFU-GM monosomy 7 and 8 levels (but not trisomy) were significantly increased in subjects exposed to benzene overall, compared to levels in the control subjects (p=0.0055 and p=0.0034, respectively). Levels of monosomy 7 and 8 were significantly increased in subjects exposed to <10 ppm (20%, p=0.0419 and 28%, p=0.0056, respectively) and ≥10 ppm (48%, p=0.0045 and 32%, p=0.0354) benzene, compared with controls, and significant exposure-response trends were detected (ptrend=0.0033 and 0.0057). These data show that monosomies 7 and 8 are produced in a dose-dependent fashion in the blood progenitor cells of workers exposed to benzene and may be mechanistically relevant biomarkers of early effect for benzene and other leukemogens.