Hematopoietic Progenitor Cell Colony Research Articles

The Optimal Storage Condition and Storage Time of Umbilical Cord Blood from Collection to Preparation

To explore the optimal storage condition and time of umbilical cord blood from collection to preparation. Collect cord blood samples from 30 healthy newborns, with each new born's umbilical cord blood was divided into two parts on average. One part was stored in cold storage (4 ℃) and the other was stored at room temperature (20-24 ℃). Samples were taken at 24, 36, 48, 60 and 72 h, respectively, total nucleated cells (TNC) count and TNC viability was analyzed. Flow cytometry was used to detect the ratio of viable CD34+ cells to viable CD45+ cells and viability of CD34+ cells, and colony-forming unit-granulocyte-macrophage (CFU-GM) count was performed by hematopoietic progenitor cell colony culture. The change trend of each index over time was observed, and the differences in each index was compared between cold storage and room temperature storage under the same storage time. The TNC count (r 4 ℃=-0.9588， r 20-24 ℃=-0.9790), TNC viability (r 4 ℃=-0.9941， r 20-24 ℃=-0.9970), CD34+ cells viability (r 4 ℃=-0.9932， r 20-24 ℃=-0.9828) of cord blood stored in cold storage (4 ℃) and room temperature storage (20-24 ℃) showed a consistent downward trend with the prolongation of storage time. The percentage of viable CD34+ cells (r 4 ℃=0.9169， r 20-24 ℃=0.7470) and CFU-GM count (r 4 ℃=-0.2537， r 20-24 ℃=-0.8098) did not show consistent trends. When the storage time was the same, the TNC count, TNC viability, CD34+ cells viability and CFU-GM count of cord blood stored in cold storage were higher than those stored at room temperature. Under the same storage time (24, 36, 48, 60 or 72 h), TNC viability in room temperature storage was significantly lower than that in cold storage (P <0.001), but TNC count, percentage of viable CD34+ cells and CFU-GM count were not significantly different between room temperature storage and cold storage. When stored at room temperature for 24 h and 36 h, the viability of CD34+ cells was significantly lower than that in cold storage (P <0.001, P <0.01), when the storage time for 48, 60 and 72 h, there was no significant difference in the CD34+ cells viability between room temperature storage and cold storage. It is recommended that cord blood be stored in cold storage (4 ℃) from collection to preparation, and processed as soon as possible.

Asperuloside alleviates cyclophosphamide-induced myelosuppression by promoting AMPK/mTOR pathway-mediated autophagy.

Cyclophosphamide (CTX) is a common anticancer chemotherapy drug, and myelosuppression is the most common serious side effect. Asperuloside (ASP), the active component of Hedyotis diffusa Willd., may have the effect of ameliorating chemotherapy-induced myelosuppression. This study aimed to explore the effect and possible mechanism of ASP on CTX-induced myelosuppression. Male SPF C57BL/6 mice were randomly divided into fivegroups: control group, CTX (25 mg/kg) group, CTX + granulocyte-macrophage-colony stimulating factor(GM-CSF) (5 μg/kg) group, CTX + high-dose ASP (50 mg/kg) group and CTX + low-dose ASP (25 mg/kg) group, with six mice in each group. The body weight of mice was monitored every other day, the hematopoietic progenitor cell colony number was measured by colony forming unit, and the relevant blood indicators were detected. Femoral bone marrow was observed by hematoxylin-eosin, C-kit expression was detected by immunohistochemistry, and autophagy and adenine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) pathway protein expressions were detected by immunohistochemistry and western blotting (WB). Then the AMPK inhibitor dorsomorphin was used to interfere with AMPK/mTOR pathway. Results showed that ASP significantly increased the body weight of CTX-induced mice, increased the number of hematopoietic progenitor cells, the expression of white blood cells, red blood cells, platelets, GM-CSF, thrombopoietin and erythropoietin in blood, and the expression of C-kit in bone marrow. In addition, ASP further promoted the expression of Beclin1 and LC-3II/I induced by CTX, and regulated the protein expressions in the AMPK/mTOR pathway. The use of dorsomorphin inhibited the alleviation effect of ASP on CTX-induced myelosuppression and the promotion effect of ASP on autophagy. In conclusion, ASP alleviated CTX-induced myelosuppression by promoting AMPK/mTOR pathway-mediated autophagy.

Further Characterization of Multi-Organ DEARE and Protection by 16,16 Dimethyl Prostaglandin E2 in a Mouse Model of the Hematopoietic Acute Radiation Syndrome.

Survivors of acute radiation exposure suffer from the delayed effects of acute radiation exposure (DEARE), a chronic condition affecting multiple organs, including lung, kidney, heart, gastrointestinal tract, eyes, and brain, and often causing cancer. While effective medical countermeasures (MCM) for the hematopoietic-acute radiation syndrome (H-ARS) have been identified and approved by the FDA, development of MCM for DEARE has not yet been successful. We previously documented residual bone marrow damage (RBMD) and progressive renal and cardiovascular DEARE in murine survivors of H-ARS, and significant survival efficacy of 16,16-dimethyl prostaglandin E2 (dmPGE2) given as a radioprotectant or radiomitigator for H-ARS. We now describe additional DEARE (physiological and neural function, progressive fur graying, ocular inflammation, and malignancy) developing after sub-threshold doses in our H-ARS model, and detailed analysis of the effects of dmPGE2 administered before (PGE-pre) or after (PGE-post) lethal total-body irradiation (TBI) on these DEARE. Administration of PGE-pre normalized the twofold reduction of white blood cells (WBC) and lymphocytes seen in vehicle-treated survivors (Veh), and increased the number of bone marrow (BM) cells, splenocytes, thymocytes, and phenotypically defined hematopoietic progenitor cells (HPC) and hematopoietic stem cells (HSC) to levels equivalent to those in non-irradiated age-matched controls. PGE-pre significantly protected HPC colony formation ex vivo by >twofold, long term-HSC in vivo engraftment potential up to ninefold, and significantly blunted TBI-induced myeloid skewing. Secondary transplantation documented continued production of LT-HSC with normal lineage differentiation. PGE-pre reduced development of DEARE cardiovascular pathologies and renal damage; prevented coronary artery rarefication, blunted progressive loss of coronary artery endothelia, reduced inflammation and coronary early senescence, and blunted radiation-induced increase in blood urea nitrogen (BUN). Ocular monocytes were significantly lower in PGE-pre mice, as was TBI-induced fur graying. Increased body weight and decreased frailty in male mice, and reduced incidence of thymic lymphoma were documented in PGE-pre mice. In assays measuring behavioral and cognitive functions, PGE-pre reduced anxiety in females, significantly blunted shock flinch response, and increased exploratory behavior in males. No effect of TBI was observed on memory in any group. PGE-post, despite significantly increasing 30-day survival in H-ARS and WBC and hematopoietic recovery, was not effective in reducing TBI-induced RBMD or any other DEARE. In summary, dmPGE2 administered as an H-ARS MCM before lethal TBI significantly increased 30-day survival and ameliorated RBMD and multi-organ and cognitive/behavioral DEARE to at least 12 months after TBI, whereas given after TBI, dmPGE2 enhances survival from H-ARS but has little impact on RBMD or other DEARE.

Metformin Inhibits STAT3 and MAPK Signaling through AMPK Activation in CSF3RT618I Cells

Introduction: In the light of the discovery of the CSF3R mutation in chronic neutrophilic leukemia (CNL) patients, the T618I mutation demonstrated to be a molecular marker and a possible target for new therapies due to the JAK/STAT signaling pathway activation [N Engl J Med (2013) 368(19):1781-90]. Since no curative treatment options are available for patients diagnosed with CNL, it is of interest the study new therapeutical approaches for these patients. In this regard, the treatment of JAK2V617F murine models with metformin, an FDA-approved drug for type 2 diabetes, reduced splenomegaly, extramedullary hematopoiesis, and colony formation of HSPC [Cell Death Dis. 2018;9(3):311]. Hence, we characterized the preclinical cellular and molecular effects of metformin in CSF3RT618I models. Methods: The in vitro experiments were performed in Ba/F3 cells transduced retrovirally with CSF3RT618I overexpression (OE) model. Cells were treated with metformin (1-20 mM) to access cell viability and survival. Protein expression was evaluated by western blot. For in vivo evaluation, eight-week-old female NSG mice were transplanted with 2x106 Ba/F3 CSF3RT618I cells, resuspended in Matrigel: PBS (1:1 ratio). After the tumor formation (2 days), mice were treated for 10 days by intraperitoneal injection of vehicle (PBS) or metformin (125 mg/kg, n=10/group). Tumor volume was measured using the formula (V = W2 × L × 0.52), where W and L are the smallest and largest diameters, respectively. Ex vivo primary cells from a patient diagnosed with CNL harboring the CSF3RT618I mutation were used to assess the effects of metformin treatment. The primary bone marrow mononuclear cells were evaluated regarding cell survival and clonogenic capacity upon metformin treatment. All experiments were approved by the Animal and Human Ethics Committee of the Institution. Statistical analyses were performed by Mann-Whitney or ANOVA test and Bonferroni post-test. A p-value < 0.05 was considered. Results: After 24 and 48 hours, Ba/F3 CSF3RT618I treated with 1, 5, 10 and 20 mM of metformin presented a reduction in cell viability in a dose- and time-dependent manner (p < 0.01). The IC50 for metformin treatment for 24, 48, and 72 hours was 17.44, 1.3, and 3.2 mM, respectively. Increased frequency of apoptotic cells was observed at 5, 10, and 20 mM of metformin at times of 24, 48, and 72 hours (p < 0.01). Molecularly, CSF3RT618I cells displayed reduced STAT3 and ERK phosphorylation and increased AKT/mTOR activation, upon metformin 5 mM treatment for 24 hours. AMPK activation was observed in cells treated with 1-20 mM of metformin compared to vehicle. In vivo, metformin treatment decreased the tumor burden of CSF3RT618I transplanted mice after 9 days of treatment (vehicle versus metformin mean ± SEM: 731.9±90.14 vs. 481.6±38.14; p = 0.035). At the protein level, we observed inhibition in Cyclin D1 and the presence of caspase 3 cleavage in the isolated tumor from the metformin-treated mice. Ex vivo metformin treatment of the primary cells isolated from the CNL patient harboring CSF3RT618I resulted in increased apoptosis, with 51.94% (p < 0.05), 52.40% (p < 0.05), and 63.1% (p < 0.001), of apoptosis induction upon treatment with 5, 10 and 20 mM. Metformin ex vivo treatment strongly reduced hematopoietic progenitor cells colony formation on primary CNL CSF3RT618I peripheral blood (83.5% at 10 mM and 100% at 20 mM) and bone marrow mononuclear cells (100% at 10 mM and 98% at 20 mM). Conclusions: We provide evidence that metformin exerts anticancer activity in CSF3RT816I cells, by downregulation of STAT3 and MAPK signaling through AMPK activation. These results support the idea that metformin can be repurposed in the CNL clinics, as a therapeutical option.

Environmental radiation on large Japanese field mice in Fukushima reduced colony forming potential in hematopoietic progenitor cells without inducing genomic instability

Purpose To study the environmental radiation effects of wild animals after the Fukushima Dai-ichi nuclear power plant accident, we assessed effects on hematopoietic progenitor cells (HPCs) in large Japanese field mice (Apodemus speciosus). Materials and methods A. speciosus were collected from three contaminated sites and control area. The air dose-rates at the control and contaminated areas were 0.96 ± 0.05 μGy/d (Hirosaki), 14.4 ± 2.4 μGy/d (Tanashio), 208.8 ± 31.2 μGy/d (Ide), 470.4 ± 93.6 μGy/d (Omaru), respectively. We investigated possible DNA damage and pro-inflammatory markers in the bone marrow (BM) cells. The colony-forming potential of BM cells was estimated by the number of HPC colony-forming cells. Radiation-induced genomic instability (RIGI) in HPCs was also analyzed by quantifying delayed DNA damage in CFU-GM clones. Results Although no significant differences in DNA damage and inflammation markers in BM cells from control and contaminated areas, the number of HPC colonies exhibited an inverse correlation with air dose-rate. With regard to RIGI, no significant differences in DNA damage of CFU-GM clones between the mice from the control and the three contaminated areas. Conclusions Our study suggests that low dose-rate radiation of more than 200 Gy/d reduced HPCs, possibly eliminating genomically unstable HPCs.

3D collagen matrices modulate the transcriptional trajectory of bone marrow hematopoietic progenitors into macrophage lineage commitment

Physical signals provided by the extracellular matrix (ECM) are key microenvironmental parameters for the fate decision of hematopoietic stem and progenitor cells (HSPC) in bone marrow. Insights into cell-ECM interactions are critical for advancing HSC-based tissue engineering. Herein, we employed collagen hydrogels and collagen-alginate hydrogels of defined stiffness to study the behaviors of hematopoietic progenitor cells (HPCs). Three-dimensional (3D) collagen hydrogels with a stiffness of 45 Pa were found to promote HPC maintenance and colony formation of monocyte/macrophage progenitors. Using single-cell RNA sequencing (scRNA-seq), we also characterized the comprehensive transcriptional profiles of cells randomly selected from two-dimensional (2D) and 3D hydrogels. A distinct maturation trajectory from HPCs into macrophages within the 3D microenvironment was revealed by these results. 3D-derived macrophages expressed high levels of various cytokines and chemokines, such as Saa3, Cxcl2, Socs3 and Tnf. Furthermore, enhanced communication between 3D-macrophages and other hematopoietic clusters based on ligand-repair interactions was demonstrated through bioinformatic analyses. Our research underlines the regulatory role of matrix-dimensionality in HPC differentiation and therefore probably be applied to the generation of specialized macrophages.

Extracellular DEK Regulates Hematopoiesis through CXCR2-Mediated Intracellular Signaling That Does Not Require Its Translocation into the Nucleus or Binding to DNA

DEK is a highly conserved, ubiquitously expressed and structurally unique DNA-binding nuclear protein involved in transcription, DNA replication, and DNA repair implicating a role for DEK in gene regulation. Interestingly, hematopoietic cells, following stimulation with inflammatory mediators, can actively secrete DEK, which can then be taken up by cells via binding to heparan sulfate proteoglycans (HSPGs), translocating to the nucleus, and mediating its normal function of gene regulation. Previous work from our group indicates that both endogenous, intracellular DEK and secreted, extracellular DEK regulates normal hematopoiesis by decreasing numbers of hematopoietic progenitor cells (HPCs) and increasing numbers of hematopoietic stem cells (HSCs) suggesting that DEK plays a role in regulating HSC/HPC function and/or cell fate. Extracellular DEK-mediated regulation of hematopoiesis occurs in a HSPG-, CXCR2-, Gai protein-dependent manner. Neutralizing these pathways inhibits extracellular DEK regulation of HSCs and HPCs. However, what was not known is whether the HSPG-dependent internalization of DEK is required for the extracellular DEK-mediated regulation of hematopoiesis as HSPGs are also required for maximum binding efficiency of ELR-CXC chemokines to CXCR2. To address this unknown, two mutant forms of recombinant (r)DEK were utilized: DEK that lacks its nuclear translocation sequence (NTS) and DEK that lacks its DNA-binding motif (DBM). First we examined whether NTS- or DMB-rDEK could alter HPC or HSC numbers in vitro / ex vivo . We had previously shown that C57Bl/6 bone marrow (BM) cells when in culture with rDEK inhibited HPC colony formation in a dose-dependent manner, with the minimum dose of WT rDEK required for inhibition being 12.5 nM (p=0.002). Both NTS- and DMB-rDEK also inhibited HPC colony formation in a dose-dependent manner starting at 12.5 nM (p=0.03 and p=0.02 respectively) suggesting that neither the ability of DEK to get into the nucleus or bind to DNA is required to regulate HPC numbers in BM. To examine whether NTS- or DMB-rDEK can still enhance HSC numbers, ex vivo HSC expansion assays were performed. C57Bl/6 lineage negative BM cells were cultured in SCF, TPO, and Flt3L with vehicle control, WT-rDEK, NTS-rDEK, or DMB-rDEK then examined for HSC expansion on days 4 and 7. On both days, WT-, NTS-rDEK and DMB-rDEK significantly expanded long-term HSC numbers when compared to vehicle control, suggesting that like DEK-mediated suppression of HPCs, DEK-mediated expansion of HSCs does not require DEK to translocate to the nucleus or bind to DNA. To examine whether extracellular NTS- and DMB-rDEK can still mediate one of DEK's known nuclear functions, heterochromatin stability, DEK-/- lineage negative BM cells were incubated for 12 hours with WT rDEK, NTS-rDEK or DMB-rDEK then examined for H3K9 trimethylation in HSCs and HPCs by ImageStream flow cytometry. Neither NTS- or DMB-rDEK were able to enhance the trimethylation of H3K9 in DEK-/- HSCs or HPCs. However, incubating DEK-/- lineage negative BM with WT rDEK, increased H3K9me3 in the nucleus of the cells by ~26%. When utilizing WT BM, incubating cells for 12 hours with WT, NTS- or DMB-rDEK did not result in changes in H3K9Me3 that was detectable by ImageStream technology suggesting that extracellular DEK-mediated changes in chromatin remodeling in HSCs and HPCs only occurs when the cells lack endogenous DEK. Co-culturing C57Bl/6 BM cells with WT rDEK mediated AKT and ERK1/2 phosphorylation in HSCs and ERK1/2 in HPCs which is blocked when BM cells are pretreated with a neutralizing antibody against CXCR2 or when preteated with heparin neutralizing HSPGs. Both NTS- and DMB-rDEK were able to mediate similar activation of AKT and ERK1/2 in HSCs and HPCs as WT rDEK but not when the BM was pretreated with anti-CXCR2 antibody or heparin. Taken together, these data suggest that extracellular DEK functions to regulate HSC and HPC numbers/function by binding to CXCR2 to mediate intracellular signaling events in C57Bl/6 BM cells. The fact that HSPGs are required for extracellular DEK's effect, but DEK does not have to get into the nucleus and bind DNA, also suggests that internalization of DEK is not required to regulate hematopoiesis in cells with normal levels of endogenous DEK. Therefore, like the ELR-CXC chemokines, DEK requires HSPGs for its CXCR2-mediated signaling processes in both HSCs and HPCs. DisclosuresNo relevant conflicts of interest to declare.

Five Active Components Compatibility of Astragali Radix and Angelicae Sinensis Radix Protect Hematopoietic Function Against Cyclophosphamide-Induced Injury in Mice and t-BHP-Induced Injury in HSCs.

Although the compatibility of Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) has favorable effect on promoting hematopoiesis in traditional Chinese medicine (TCM), the main active components and pharmacological mechanism are unknown. We investigated the five active components and its mechanisms in vitro and in vivo. Five active components of Astragalus glycosides (AST), Formononetin (FRM), Ferulic acid (FRA), Calycosin (CAL), and Calycosin-7-glucoside (CLG), which could be absorbed in intestinal tract, were detected in this study. The peripheral blood, hematopoietic growth factors (HGFs), and hematopoietic progenitor cells (HPCs) colony were observed to evaluate the effect of these five active components promoting hematopoiesis. Furthermore, hematopoietic stem cell (HSC) proliferation, aging, cycle, and related proteins were detected to explore the mechanism of these five components promoting HSC proliferation. i) The in vivo experiments showed that the combination of the five active components could remarkably increase the number of RBCs, WBCs, PLTs, and content of Hb in peripheral blood and the area of bone marrow hematopoietic tissue, as well as thrombopoietin (TPO), erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), and colony of CFU-GM, CFU-MK, CFU-E, and BFU-E in serum. Each of these five components promoted the recovery of RBCs and Hb, and increased TPO, CFU-MK, and CFU-E. All components except for AST increased the CFU-GM. FRA increased the number of WBCs, the area of bone marrow hematopoietic tissue, and BFU-E. FRA and AST promoted PLT recovery. FRA and CAL improved the content of GM-CSF. FRA, CAL, and CLG improved the content of EPO. ii) The in vitro experiments showed that FRA, FRM, and AST significantly promoted cell proliferation, reduced the positive rate and G0/G1 cells, and increased G2/M + S cells and the expression of cyclin D1 and CDK4 proteins in aging HSCs. Furthermore, the combination of five components had the best effect. Taken together, the five active components of AST, FRM, FRA, CAL, and CLG were the main pharmacodynamic substances of the AR-ASR compatibility, which promoted hematopoiesis. The combination of them had a synergistic effect. The mechanism of promoting hematopoiesis may be relevant to regulating cyclin-related proteins, promoting cell cycle transformation, and promoting HSC proliferation.

Phosphatidylinositol transfer proteins regulate megakaryocyte TGF-β1 secretion and hematopoiesis in mice

AbstractWe hypothesized that megakaryocyte (MK) phosphoinositide signaling mediated by phosphatidylinositol transfer proteins (PITPs) contributes to hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) regulation. Conditional knockout mice lacking PITPs specifically in MKs and platelets (pitpα−/− and pitpα−/−/β−/−) bone marrow (BM) manifested decreased numbers of HSCs, MK-erythrocyte progenitors, and cycling HPCs. Further, pitpα−/−/β−/− BM had significantly reduced engrafting capability in competitive transplantation and limiting dilution analysis. Conditioned media (CM) from cultured pitpα−/− and pitpα−/−/β−/− BM MKs contained higher levels of transforming growth factor β1 (TGF-β1) and interleukin-4 (IL-4), among other myelosuppressive cytokines, than wild-type BM MKs. Correspondingly, BM flush fluid from pitpα−/− and pitpα−/−/β−/− mice had higher concentrations of TGF-β1. CM from pitpα−/− and pitpα−/−/β−/− MKs significantly suppressed HPC colony formation, which was completely extinguished in vitro by neutralizing anti–TGF-β antibody, and treatment of pitpα−/−/β−/− mice in vivo with anti–TGF-β antibodies completely reverted their defects in BM HSC and HPC numbers. TGF-β and IL-4 synergized to inhibit HPC colony formation in vitro. Electron microscopy analysis of pitpα−/−/β−/− MKs revealed ultrastructural defects with depleted α-granules and large, misshaped multivesicular bodies. Von Willebrand factor and thrombospondin-1, like TGF-β, are stored in MK α-granules and were also elevated in CM of cultured pitpα−/−/β−/− MKs. Altogether, these data show that ablating PITPs in MKs indirectly dysregulates hematopoiesis in the BM by disrupting α-granule physiology and secretion of TGF-β1.

A Pilot Trial of Unmatched Human Placenta-Derived Stem Cells (HPDSCs) in Conjunction with Unrelated Cord Blood Transplantation (UCBT) in Children and Young Adults with Malignant and Non-Malignant Disease (IND 14949)

Background: Myeloablative conditioning (MAC) and reduced toxicity conditioning (RTC) followed by UCBT (matched at 4-6/6 HLA loci) in children with malignant and non-malignant diseases is safe and effective (Wagner/Cairo et al, Blood, 1996; Geyer/Cairo et al, BJH, 2011). However, there is a low concentration of CD34+ hematopoietic progenitor cells (HPCs) in banked UCB (Cairo/Kurtzberg et al, Transfusion, 2005) compared to bone marrow or peripheral blood, leading to delayed hematopoietic reconstitution and 10-20% incidence of graft failure (Cairo et al, Blood, 1997; Cairo et al, Transfusion, 2005; Satwani/Cairo et al, BBMT, 2013). Human placenta-derived stem cells (HPDSCs) are rich in HPCs and HPC colony forming unit capabilities, low in HLA Class I and II expression, low in T-cell content, and have regenerative, anti-inflammatory, and immunosuppressive properties (Cairo et al, BMT, 2015). Mendez-Ferrer et al have demonstrated that HPDSCs enhance in-vivo engraftment when combined with UCBT in NOD-SCID animals (Mendez-Ferrero et al, Nature, 2010).Objective: To determine the safety of administering unrelated donor HPDSC in conjunction with unrelated single or double UCBT following MAC or RTC in children and adults with malignant and non-malignant diseases and to determine the time to hematopoietic engraftment, immune reconstitution and probability of donor chimerism.Methods: Patients ≤55 years of age with malignant and non-malignant diseases with a single UCB 5-6/6 HLA match and TNC ≥ 3.5x 107/kg, or double UCB 4-6/6 HLA match and combined TNC >5.0 x 107/kg are eligible. HLA typing was performed by serology for Class I A and B and by high resolution DNA typing of DRB1. Patients received either fully myeloablative or reduced toxicity pre-UCBT conditioning as indicated for the targeted disease followed by single or double UCB plus HPDSC infusion. All patients received filgrastim (G-CSF) starting at least 24 hours after the HPDSC infusion until myeloid engraftment and GVHD prophylaxis with tacrolimus or cyclosporine plus mycophenolate mofetil.Results: Twenty one patients have been enrolled to date: mean age 11 (0.31-34.9) years, 11 males, 10 females. Fourteen subjects had malignancies (relapsed/refractory or hypodiploid precursor B-ALL (n=7), relapsed/refractory or high risk AML (n=5), T-cell ALL induction failure (n=1), and T-cell lymphoblastic lymphoma (n=1)) and seven had non-malignant disorders (adrenoleukodystrophy (n=1), chronic granulomatous disease (n=1), congenital amegakaryocytic thrombocytopenia (n=1), bone marrow failure (n=1), SCID (n=1), severe aplastic anemia (n=1), and dyskeratosis congenita (n=1)). Fourteen patients received single UCBT followed by HPDSC infusion, and seven patients received double UCBT followed by HPDSC infusion. There were no severe adverse events associated with the HPDSC infusions. Median time to neutrophil and platelet engraftment were 22 (13-53) and 45 (20-98) days post UCBT, respectively. Mean percent of whole blood donor chimerism at days 30, 60, 100 and 180 were 97% (SEM 1.25), 99%, (SEM 0.36), 99% (SEM 0.39), and 97.9 (SEM 1.31), respectively. Average percent of whole blood HPDSC chimerism was <1% at day 30. Average absolute CD3/4/8 at days 100, 180, 270, and 365 were 150/107/43, 411/ 256/154, 1020/630/371, and 1207/728/458, respectively, per mcl. Average absolute CD56 and CD19 at days 100, 180, 270 and 365 were 478/654, 190/713, 308/2225 and 361/1215 respectively. The probability of NRM at day 100 was 0%. Of patients with malignant disease, the incidence of relapse was 0/14 (0%) at day 100 and 1/14 (7.1%) at day 180 post-transplant. For the full cohort, the probability of Grade II-IV aGVHD was 14.8% (CI95 0.0-28.9) (Figure 1) and the probability of 12 month overall survival was78.6% (CI95 62.0-99.6) (Figure 2). OS at day 100 and day 180 are 19/19 (100%) and 15/19 (78.9%) respectively. 2/21 patients have not yet reached day 100. Sixteen of twenty-one patients are alive at a median of 535 (53-1175) days post UCBT.Conclusions: These preliminary results suggest that single or double UCBT combined with unrelated HPDSC is safe, well tolerated, and has a lower than expected probability of Grade II-IV aGVHD. A larger cohort and longer follow-up is required to determine the safety and clinical significance of these early findings. [Display omitted] [Display omitted] DisclosuresPulsipher:Novartis: Consultancy, Other: Study Steering Committee; Jazz Pharmaceutical: Consultancy; Chimerix: Consultancy; Medac: Other: Housing support for conference. Zhang:Celgene: Employment, Equity Ownership, Patents & Royalties. Gurney:Celgene: Employment, Equity Ownership. Cairo:Celgene: Research Funding.

Physiologic Hematopoiesis Is Dependent on Stromal Expression of Laminin-γ1

OBJECTIVE: The microenvironment of the bone marrow hematopoietic niche includes: 1) blood vessels; 2) adjacent stromal cells; 3) hematopoietic cells; and 4) cytokines, growth factors, and structural support molecules. Injury to the bone marrow niche affects hematopoietic cells through loss of either physical contact with stromal cells or niche-derived growth factors. Extracellular matrix factors required to maintain the steady-state bone marrow perivascular niche and hematopoiesis have not been fully identified. Here, we examine the role of the extracellular matrix protein laminin-γ1 in adult bone marrow for maintaining the perivascular hematopoietic niche and hematopoiesis.METHODS: A global and inducible laminin-γ1 deficient mouse, hereafter referred to as mutant, was generated in which LAMC1 gene recombination could be monitored by a fluorescent reporter transgene. Tamoxifen was used to induce LAMC1 gene recombination and knock-down of laminin-γ1 protein expression in 8-12 week old mice. Tamoxifen-treated mice lacking the inducible Cre transgene were used as controls for all experiments. Analysis of mutant mice was performed 17-24 days following the first dose of tamoxifen. Bone marrow samples were examined by immunohistochemistry for the presence of laminin-1 protein, and hematopoietic tissues, including bone marrow, peripheral blood, spleen, thymus and lymph node were analyzed by flow cytometry for hematopoietic stem and progenitor cells, and mature hematopoietic cell populations, and by ex vivo hematopoietic progenitor cell colony forming assays. To examine the stromal cell contribution to laminin-γ1 mediated hematopoietic dysfunction, control and mutant mice that had not yet undergone LAMC1 gene recombination were then transplanted with wild type bone marrow. After full hematopoietic reconstitution, chimeric mice were induced with tamoxifen and hematopoietic stem and progenitor cells and mature hematopoietic cell populations were examined.RESULTS: LAMC1 gene recombination in the bone marrow of tamoxifen-treated mutant animals varied between 20-45%. With LAMC1 gene recombination, laminin-protein in the bone marrow is rapidly depleted. Bone marrow blood vessels dilate, and the bone marrow becomes hypocellular. Hematopoietic stem and progenitor cells are reduced in number, as are bone marrow B-cell progenitors, and thymic double-positive T-cells. Erythrocyte and thrombocyte numbers are not changed, nor were NK cell populations, the majority of myeloid cell subtypes, or mature B cells in the bone marrow and blood. CD8+ T-cells were increased within the bone marrow, as were CD11b+, Ly6Clo Ly6G+ myeloid cells in the peripheral blood. Examination of gene recombination within each of these cell types was not consistent with cell-intrinsic mechanism of hematopoietic alterations. Analysis of bone marrow chimeric animals identified loss of stromal cell-produced laminin-γ1 as a significant mediator of these hematopoietic alterations.CONCLUSIONS: The rapid depletion of laminin-γ1 from the bone marrow indicates a high basal turnover rate for the extracellular matrix in the bone marrow. The attendant hematopoietic dysfunction that follows loss of laminin-γ1 production in the bone marrow is specific for immature hematopoietic cell populations, and is dependent on stromal cell production of laminin-γ1. This study identifies laminin-γ1 as a niche-dependent regulator of hematopoietic stem and progenitor cell populations, and suggests that that regulation of its production and degradation may be new targets for the study of the hematopoietic niche. DisclosuresNo relevant conflicts of interest to declare.

Recombinant DEK Enhances Ex Vivo expansion of Human Cord Blood and Mouse Bone Marrow Hematopoietic Stem Cells in a CXCR2- and Hspg-Dependent Manner

DEK is a nuclear DNA-binding protein that has been implicated in the regulation of transcription, chromatin remodeling, and mRNA processing. Endogenous DEK regulates hematopoiesis, as BM from DEK-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term and secondary hematopoietic stem cell (HSC) engrafting capability (Broxmeyer et al., 2012, Stem Cells Dev., 21: 1449; 2013, Stem Cells, 31: 1447). Moreover, recombinant mouse (rm) DEK inhibits HPC colony formation in vitro. We now show that rmDEK is myelosuppressive in vitro in an S-phase specific manner and reversibly decreases numbers (~2 fold) and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo, with DEK-/- mice being more sensitive than control mice to this suppression. In contrast, in vivo administration of rmDEK to wild type and DEK-/- mice enhanced numbers of phenotypic LT-HSC. This suggests that DEK may enhance HSC numbers by blocking production of HPCs. We thus assessed effects of DEK on ex vivo expansion of human CD34+ cord blood (CB) and mouse Lin- BM cells stimulated with SCF, Flt3 ligand, and TPO. DEK significantly enhanced ex vivo expansion of rigorously-defined HSC by ~3 fold both on day 4 (~15 fold increase from day 0) and 7 (~29 fold increase from day 0) when compared to cells expanded without DEK. Expanding HSC with DEK also resulted in a decrease in the percentage of apoptotic HSC.Further studies were done to better define how DEK works on HSC and HPC. As extracellular DEK can bind to heparan sulfate proteoglycans (HSPG), become internalized, and then remodel chromatin in non-hematopoietic cells in vitro (Kappes et al., 2011, Genes Dev., 673; Saha et al., 2013, PNAS, 110: 6847), we assessed effects of DEK on the heterochromatin marker H3K9He3 in the nucleus of purified mouse lineage negative, Sca-1 positive, c-Kit positive (LSK) BM cells by imaging flow cytometry. DEK enhanced the presence of H3K9Me3 in the nucleus of DEK-/- LSK cells, indicating that rmDEK can be internalized by LSK cells and mediate heterochromatin formation. We also investigated whether inhibiting DEK's ability to bind to HSPG would block the inhibitory function of DEK in HPC. Blocking the synthesis of, the surface expression of, and the binding capability of HSPG blocked the inhibitory effect of DEK on colony formation. Blocking the ability of DEK to bind to HSPG also blocks the expansion of HSC in ex vivo expansion assays, suggesting that DEK mediates its function in both HSC and HPC by binding to HSPG but with opposing effects.To further evaluate the biological role of rmDEK, we utilized single-stranded anti-DEK aptamers that inactivate its function. These aptamers, but not their control, neutralized the inhibitory effect of rmDEK on HPC colony formation. Moreover, treating BM cells in vitro with truncated rmDEK created by incubating DEK with the enzyme DPP4 (DEK has targeted truncation sites for DPP4) eliminated the inhibitory effects of DEK, suggesting that DEK must be in its full- length form in order to perform its function. Upon finding that DEK has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, and as DEK is a chemoattractant for mature white blood cells, we hypothesized that DEK may manifest at least some of its actions through CXCR2, the receptor known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case, we first confirmed expression of CXCR2 on the surface of HSC and HPC and then determined if neutralizing CXCR2 could block DEK's inhibitory function in HPC. BM treated in vitro with rmDEK, rhIL-8, or rmMIP-2 inhibited colony formation; pretreating BM with neutralizing CXCR2 antibodies blocked the inhibitory effect of these proteins. DEK inhibition of CFU-GM colony formation is dependent on Gai-protein-coupled receptor signaling as determined through the use of pertussis toxin, which is a mechanism unique to DEK, as we have previously reported that IL-8 and MIP-1a are insensitive to the inhibitory effects of pertussis toxin. Blocking the ability of DEK to bind to CXCR2 also inhibited the expansion of HSC in an ex vivo expansion assay. This suggests that DEK binds to CXCR2, HSPG or both to mediate its function on HPC and HSC, enhancing HSC but decreasing HPC numbers. Therefore, DEK may be a crucial regulatory determinant of HSC/HPC function and fate decision that is utilized to enhance ex vivo expansion of HSC. DisclosuresNo relevant conflicts of interest to declare.

Effects of HS 6101 and rhG-CSF on Hematopoiesis Recovery of ICR Mice Injured by Cyclophosphamide

To investigate the effects of HS 6101 and recombinant human granulocyte colony stimulating factor (rhG-CSF) on hematopoiesis recovery of ICR mice injured by cyclophosphamide (CTX). A total of 103 ICR mice were divided into 4 groups, including CTX control (46 mice), HS 6101 (21 mice), rhG-CSF (18 mice) and HS 6101+rhG-CSF (18 mice), respectively. The mouse model of chemotherapy-induced haematopoietic injury was established by CTX intraperitoneal injection at a dose of 100 mg/kg once a day for 3 consecutive days. Single dose of HS 6101 (27 µg/mouse) was injected subcutaneously at 1 hour before the first administration of CTX. One day after the last CTX treatment, 2 µg/mouse of rhG-CSF was injected subcutaneously once a day for 5 consecutive days. The peripheral blood cell counts of the mice were observed once every 2 days. Hematopoietic progenitor cell colony counting and histopathological assessment of bone marrow cells were evaluated in the mice at days 4 and 9 after the first administration of CTX. Both HS 6101 and rhG-CSF alone or in combination, significantly elevated the nadirs of peripheral blood leukocytes and neutrophils, increased the number of bone marrow hematopoietic progenitor cells, and stimulated the hematopoietic cell hyperplasia of bone marrow in the mice treated with CTX. The effect of HS 6101 combined with rhG-CSF was better than that of the drugs used alone. The HS 6101 at 27 µg/mouse can significantly promote the recovery of hematopoiesis in ICR mice treated with CTX chemotherapy, and its combination with rhG-CSF shows synergistic effects.

Mesenchymal stem cells reverse bone marrow dysfunction following injury and stress

Bone marrow (BM) dysfunction following experimental lung contusion (LC) resolves in 7 days; however, if followed by chronic stress (CS) following, BM dysfunction is persistent. Mesenchymal stem cells (MSCs) have protective immunomodulatory effects. We hypothesize that MSC can protect the BM against the deleterious effect of CS following LC. Male Sprague-Dawley rats (n = 6-7 per group) underwent LC or LC/CS ± MSC injection. CS consisted of a daily 2-hour period of restraint with repositioning and alarming every 30 minutes to prevent habituation. A single intravenous dose of 5 × 10 MSCs was given within 10 minutes following LC. Animals were sacrificed at Day 7, and peripheral blood (PB) and BM were collected. Flow cytometry was used to assess hematopoietic progenitor cells (HPCs) mobilized to PB. Plasma granulocyte colony-stimulating factor (G-CSF) levels were measured by enzyme-linked immunosorbent assay. BM cellularity and growth of BM HPC colonies (colony-forming unit-erythroid [CFU-E], burst-forming unit-erythroid [BFU-E], colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte [CFU-GEMM]) were also evaluated. As previously reported, the addition of CS to LC resulted in a 32% decrease in BM cellularity; significant decreases in CFU-GEMM, BFU-E, and CFU-E; and marked increase in HPC in the PB as compared with the naive animals. The addition of MSC to LC/CS resulted in a 22% increase in BM cellularity and significant increases in CFU-GEMM, BFU-E, and CFU-E cultured from the BM. MSCs additionally reduced plasma G-CSF, prevented prolonged mobilization of HPC to PB, and restored colony growth to naive levels. CS following LC results in persistent BM dysfunction manifested by a significant decrease in cellularity, HPC colony growth, and increased G-CSF levels and HPC mobilization to the PB at 7 days following injury. The addition of a single dose of MSCs following acute traumatic injury reverses the deleterious effects of CS on BM function. Further study is warranted to better understand the mechanisms behind MSC-mediated protection of BM function in the setting of CS.

Mesenchymal stem cells reverse trauma and hemorrhagic shock-induced bone marrow dysfunction

BackgroundLung contusion (LC) followed by hemorrhagic shock (HS) causes persistent bone marrow (BM) dysfunction lasting up to 7 d after injury. Mesenchymal stem cells (MSCs) are multipotent cells that can hasten healing and exert protective immunomodulatory effects. We hypothesize that MSCs can attenuate BM dysfunction after combined LCHS. Materials and methodsMale Sprague–Dawley rats (n = 5–6 per group) underwent LC plus 45 min of HS (mean arterial pressure of 30–35). Allogeneic MSCs (5 × 106 cells) were injected intravenously after resuscitation. At 7 d, BM was analyzed for cellularity and growth of hematopoietic progenitor cell (HPC) colonies (colony-forming unit–erythroid; burst-forming unit–erythroid; and colony-forming unit–granulocyte, erythrocyte, monocyte, megakaryocyte). Flow cytometry measured %HPCs in peripheral blood; plasma granulocyte colony-stimulating factor (G-CSF) levels were measured via enzyme-linked immunosorbent assay. Data were analyzed by one-way analysis of variance followed by the Tukey multiple comparison test. ResultsAs previously shown, at 7 d, LCHS resulted in 22%, 30%, and 24% decreases in colony-forming unit–granulocyte, erythrocyte, monocyte, megakaryocyte, burst-forming unit–erythroid, and colony-forming unit–erythroid colony growth, respectively, versus naive. Treatment with MSCs returned all BM parameters to naive levels. There was no difference in %HPCs in peripheral blood between groups; however, G-CSF remained increased up to 7 d after LCHS. MSCs returned G-CSF to naive levels. Plasma from animals receiving MSCs was not suppressive to the BM. ConclusionsOne week after injury, the persistent BM dysfunction observed in animals undergoing LCHS is reversed by treatment with MSCs with an associated return of plasma G-CSF levels to normal. Plasma from animals undergoing LCHS plus MSCs was not suppressive to BM cells in vitro. Treatment with MSCs after injury and shock reverses BM suppression and returns plasma G-CSF levels to normal.

Recombinant DEK Regulates Hematopoietic Stem and Progenitor Cells in a CXCR2- GαI Protein-Dependent Manner

DEK, a non-histone nuclear phosphoprotein involved in heterochromatin remodeling, is released from cells and can regulate hematopoiesis (Broxmeyer et al., 2012, Stem Cells Dev., 21: 1449; 2013, Stem Cells, 31: 1447). Marrow from DEK-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term and secondary hematopoietic stem cells (HSC) engrafting capability. Moreover, recombinant mouse (rmu) DEK inhibited HPC colony formation in vitro. We now show that rmuDEK is myelosuppressive in vitro in an S-phase specific manner and reversibly decreases numbers and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo, with DEK-/- mice being much more sensitive than control mice to this suppression. In addition, rmuDEK treatment of WT mice in vivo reversibly reduces the phenotypic number of ST-HSC, MPP, CMP, GMP, and MEP in the marrow. In vivo administration of rmuDEK to DEK-/- mice greatly enhanced the number of phenotypic LT-HSC and functional HSC in these mice (competitive HSC repopulation in lethally irradiated mice). DEK-/- mice are also more sensitive to 5-FU than control mice resulting in slower hematopoietic cell recovery, possibly due to the enhanced cycling status of HPC in DEK-/- mice. To determine the biological role of extracellular DEK, we utilized a reagent that could inactivate its function. This inactivating agent, but not its control, neutralized the inhibitory effect of rmuDEK. In addition, treating marrow cells in vitro with truncated rmuDEK by pretreating the DEK with the enzyme DPP4 (which DEK has targeted truncation sites for) also blocked the inhibitory effects of DEK suggesting that DEK must be in its full length form in order to perform its functionUpon our discovery that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, we hypothesized that DEK may manifest at least some of its actions through CXCR2, known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case we first confirmed expression of CXCR2 on the surface of HSC and HPC. To determine whether rmuDEK’s inhibitory function is mediated through the CXCR2 receptor, a neutralizing monoclonal antibody against CXCR2 was utilized. Marrow treated in vitro with rmuDEK, rhIL-8, or rmuMIP-2 inhibited colony formation; however pretreating marrow cells with the neutralizing CXCR2 antibody blocked the inhibitory effect of these proteins. Marrow treated with rmuMIP-1α (a chemokine that does not bind to CXCR2) also inhibited colony formation; however neutralizing CXCR2 antibody had no effect on the ability of MIP-1α to inhibit colony formation. Neutralizing CXCR4, a chemokine receptor that binds SDF-1 but not IL-8, MIP-2 or MIP-1α, had no effect on the inhibition of colony formation. DEK inhibition of CFU-GM colony formation is dependent on Gαi-protein-coupled receptor signaling as determined through the use of pertussis toxin. This is a unique mechanism since IL-8 and MIP-1α had been previously reported by us to be insensitive to the inhibitory effects of pertussis toxin.As extracellular DEK can remodel chromatin in non-hematopoietic cells in vitro (Kappes et al., 2011, Genes Dev., 25: 673; Saha et al., 2013, PNAS, 110: 6847), we next assessed the effects of DEK on the heterchromatin marker H3K9Me3 in the nucleus of purified mouse Lineage negative, Sca-1 positive, c-kit positive (LSK) marrow cells by imaging flow cytometry. RmuDEK enhanced the presence of H3K9Me3 in the nucleus of DEK-/- LSK marrow cells indicating that rmuDEK can be internalized by LSK cells and mediate heterchromatin formation. These results add to emerging evidence that DEK, a unique nuclear protein, plays a role in regulating hematopoiesis through a CXCR2/Gαi protein signaling pathway. Its modulation may be of practical use. DisclosuresNo relevant conflicts of interest to declare.

Protective effect of HS-6101 on rhesus monkeys with severe hematopoietic acute radiation sickness

This study was purposed to investigate the protective effects of lipoprotein HS-6101(6101) on rhesus monkey total body irradiated with 7.0 Gy ⁶⁰Coγ-ray. A total of 30 health adult rhesus monkeys were randomly divided into symptomatic therapy (ST), WR2721 and HS-6101 30, 90 and 270 mg/kg groups (n = 6), the rhesus monkeys of each groups were injected with physiological saline 0.3 ml/kg, WR-2721 30 mg/kg, or HS-6101 30, 90 and 270 µg/kg, respectively. All agents were once intramuscularly injected at 1 hr prior irradiation. General observation, peripheral blood cell counts, colony forming unite assay of bone marrow hemopoietic progenitor cells, and histopathological examination were performed. The results showed that animals in symptomatic therapy group begin to die on the 13(th) day and 4 animals died within 24 days, the average survival time was 18.2 ± 4.3 days; 2 animals in WR-2717 groups died on day 15.8 and day 18.5 post irradiation respectively. 1 animal in HS-6101 270 mg/kg group died on day 35.8, all other animals survived. Nadirs of peripheral blood white blood cells, neutrophils and platelets of animals in HS-6101 treatment groups were significantly higher than those in other 2 groups including ST and WR-2721 groups, and the hemopoietic recovery were also significantly speeding up(P < 0.05 and 0.01). In vitro results showed that HS-6101 obviously promoted 7.0 Gy ⁶⁰Coγ irradiated monkey's bone marrow mononuclear cells to form various hematopoietic progenitor cell colonies (P < 0.05 and 0.01) . Compared with symptomatic therapy and WR-2717 groups, bone marrow histopathological changes in HS-6101 treatment groups showed more active hemopoietic cell proliferation and higher density structure. It is concluded that HS-6101 90 µg/kg treatment can promote the bone marrow recovery of 7.0 Gy ⁶⁰Coγ irradiated monkey, alleviate their animal symptom, simplify the treatment measures and improve the animal survival rate. The HS-6101 shows remarkable radioprotective effects as compared with the currently internationally acknowledged radioprotectant of WR-2721.

Radiation protection effect of rhIL-12 on monkey hematopoietic system

This study was aimed to investigate the radioprotective effects of recombinant human interleukin-12 (rhIL-12) on monkey hematopoietic system, and to provide experimental evidence for future clinical prophylaxis and treatment for patients who suffered from acute radiation syndrome. In in vitro study, the effect of rhIL-12 in different concentrations (0, 1, 5, 25, 125 and 625 ng/ml) on colony forming capacity of human or monkey bone marrow-derived mononuclear cells was examined in methylcellulose H4434 medium. In in vivo study, the acute radiation syndrome model was established in 11 Rhesus monkeys which received lethal total body irradiation by 6 Gy (60)Co γ in single time irradiation. The irradiated monkeys were randomly divided into 3 subgroups: control group (n = 4) which received subcutaneous PBS injection, rhIL-12 single-dose group (n = 3) which received subcutaneous single injection of rhIL-12 (4 µg/kg) at 2 h after irradiation, and multiple-dose group (n = 4) which received subcutaneous injection of rhIL-12 (1 µg/kg per injection) at 2 h, day 3, 6 and 9 after irradiation respectively. Peripheral blood cells were counted before and after irradiation every other day. The survival status of animals were observed daily. In vitro test results showed that different concentrations of rhIL-12 obviously promoted human and healthy monkeys' bone marrow mononuclear cells to form various hematopoietic progenitor cell colonies, especial CFU-E and CFU-GM. All animals in control group died within 22 d after lethal total body irradiation, average survival time was (20.3 ± 1.2) d. Only one monkey in multiple-dose group died due to anemia on day 17. All monkeys in single-dose group survived. Compared with control group, rhIL-12-administrated monkeys' white blood cell count, hemoglobin level, platelet and reticulocyte counts showed faster recovery from high dose radiation. It is concluded that the rhIL-12 treatment can promote the bone marrow hematopoietic stem/progenitor cell colony formation in vitro and protect lethally-irradiated monkeys. There is an obvious therapeutic effect of rhIL-12 on monkeys suffered from bone marrow failure caused by severe acute radiation exposure.

Beta Blockade Protection of Bone Marrow Following Injury: A Critical Link between Heart Rate and Immunomodulation.

IntroductionSevere trauma induces a profound elevation of catecholamines that is associated with bone marrow (BM) hematopoietic progenitor cell (HPC) colony growth suppression, excessive BM HPC mobilization, and a persistent anemia. Previously, propranolol (BB) use after injury and shock has been shown to prevent this BM dysfunction and improve hemoglobin levels. This study seeks to further investigate the optimal therapeutic dose and timing of BB administration following injury and shock.MethodsMale Sprague-Dawley rats were subjected to a combined lung contusion (LC), hemorrhagic shock (HS) model ± BB. In our dose response experiments, animals received BB at 1, 2.5, 5, or 10 mg/kg immediately following resuscitation. In our therapeutic window experiments, following LCHS rats were given BB immediately, 1 hour, or 3 hours following resuscitation. BM and peripheral blood (PB) were collected in all animals to measure cellularity, BM HPC growth, circulating HPCs, and plasma G-CSF levels.ResultsPropranolol at 5 and 10 mg/kg significantly reduced HPC mobilization, restored BM cellularity and BM HPC growth, and decreased plasma G-CSF levels. Propranolol at 5 and 10 mg/kg also significantly decreased heart rate. When BB was administered beyond 1 hour after LCHS, its protective effects on cellularity, BM HPC growth, HPC mobilization, and plasma G-CSF levels were greatly diminished.ConclusionEarly Buse following injury and shock at a dose of at least 5mg/kg is required to maintain BM cellularity and HPC growth, prevent HPC mobilization, and reduce plasma G-CSF levels. This suggests that propranolol exerts its BM protective effect in a dose and time dependent fashion in a rodent model. Finally, heart rate may be a valuable clinical marker to assess effective dosing of propranolol.

Does selective beta-1 blockade provide bone marrow protection after trauma/hemorrhagic shock?

Previously, nonselective beta-blockade (BB) with propranolol demonstrated protection of the bone marrow (BM) after trauma and hemorrhagic shock (HS). Because selective beta-1 blockers are used commonly for their cardiac protection, the aim of this study was to more clearly define the role of specific beta adrenergic receptors in BM protection after trauma and HS. Male Sprague-Dawley rats underwent unilateral lung contusion (LC) followed by HS for 45minutes. After resuscitation, animals were injected with a selective beta-blocker, atenolol (B1B), butoxamine (B2B), or SR59230A (B3B). Animals were killed at 3 hours or 7 days. Heart rate and blood pressure were measured throughout the study period. BM cellularity, growth of hematopoietic progenitor cells (HPCs) in BM, and hemoglobin levels (Hb) were assessed. Treatment with a B2B or B3B after LCHS restored both BM cellularity and BM HPC colony growth at 3 hours and 7 days. In contrast, treatment with a B1B had no effect on BM cellularity or HPC growth but did decrease heart effectively rate throughout the study. Treatment with a B3B after LCHS increased Hb as compared with LCHS alone. After trauma and HS, protection of BM for 7 days was seen with use of either a selective beta-2 or beta-3 blocker. Use of a selective beta-1 blocker was ineffective in protecting the BM despite a physiologic decrease in heart rate. Therefore, the protection of BM is via the beta-2 and beta-3 receptors and it is not via a direct cardiovascular effect.