Spleen Colony Research Articles

Megakaryocytic TGFβ1 Partitions Hematopoiesis into Amplifying Stem and Progenitor Cells and Maturing Effector Cells

Background: Chronic anemia is a significant problem affecting over 3 million Americans annually. Therapies are restricted to transfusion and Erythropoietin Stimulating Agents (ESA). There is a need for new approaches to treat chronic anemia. Immature erythroid progenitors are thought to be continuously produced and then permitted to survive and mature if there is sufficient erythropoietin (Epo) available. This model is elegant in that oxygen sensing within the kidney triggers Epo production so anemia can increase Epo and promote erythroid output. However, during homeostasis this model suggests that considerable energy is used to produce unneeded erythroid progenitors. We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation.Methods: We previously found the proportion of bone marrow megakaryocytes (MKs) staining for active, signaling-competent TGFβ transiently increases during bone marrow regeneration after chemotherapy. To assess the functional role of Mk-TGFβ, we crossed murine strains harboring a floxed allele of TGFβ1 (TGFβ1Flox/Flox) littermate with a Mk-specific Cre deleter to generate mice with Mk-specific deletion of TGFβ1 (TGFβ1ΔMk/ΔMk). We analyzed hematopoiesis of these mice using high-dimensional flow cytometry, confocal immunofluorescent microscopy and in vitro and in vivo assays of hematopoietic function (Colony forming assays, and in vivo transplantation).Results: Using validated, 9-color flow cytometry panels capable of quantifying hematopoietic stem cells (HSCs) and six other hematopoietic progenitor populations, we found that Mk-specific deletion of TGFβ1 leads to expansion of immature hematopoietic stem and progenitor cells (HSPCs) (Fig1A&B). Functional assays confirmed a more than three-fold increase in hematopoietic stem cells (HSCs) capable of serially-transplanting syngeneic recipients in the bone marrow (BM) of TGFβ1ΔMk/ΔMk mice compared to their TGFβ1Flox/Flox littermates. Expansion was associated with less quiescent (Go) HSCs implicating Mk-TGFβ in the control of HSC cell cycle entry. Similarly, in vitro colony forming cell assays and in vivo spleen colony forming assays confirmed expansion of functional progenitor cells in TGFβ1ΔMk/ΔMk mice. These results place Mk-TGFβ as a critical regulator of the size of the pool of immature HSPCs.We found that the blood counts and total BM cellularity of TGFβ1ΔMk/ΔMk mice was normal despite the dramatic expansion of immature HSPCs. Using a combination of confocal immunofluorescence microscopy (cleaved caspase 3) (Fig1C) and flow cytometry (Annexin V and cleaved caspase 3) (Fig1D), we found ~10-fold greater apoptosis of mature precursor cells in TGFβ1ΔMk/ΔMk BM and spleens. Coincident with this, we found the number of Epo receptor (EpoR) expressing erythroid precursors to be dramatically increased. Indeed, apoptosis of erythroid precursors peaked as they transitioned from dual positive Kit+EpoR+ precursors to single positive cells expressing EpoR alone. Epo levels were normal in the serum of these mice. We reasoned that the excess, unneeded EpoR+ cells were not supported physiologic Epo levels but might respond to even small doses of exogenous Epo. Indeed, we found that the excess erythroid apoptosis could be rescued by administration of very low doses of Epo (Fig1E). Whereas TGFβ1Flox/Flox mice showed minimal reticulocytosis and no change in blood counts, TGFβ1ΔMk/ΔMk mice responded with exuberant reticulocytosis and raised RBC counts almost 10% within 6 days (Fig. 1F). Low dose Epo also rescued survival of Epo receptor positive erythroid precursors in the bone marrow, spleen and blood of TGFβ1ΔMk/ΔMk mice. TGFβ1ΔMk/ΔMk mice showed a similarly brisk and robust erythropoietic response during recovery from phenylhydrazine-induced hemolysis (Fig.1G). Exogenous TGFβ worsened BM apoptosis and caused anemia in treated mice. Pre-treatment of wild-type mice with a TGFβ signaling inhibitor sensitized mice to low dose Epo.Conclusion: These results place megakaryocytic TGFβ1 as a gate-keeper that restricts the pool of immature HSPCs and couples immature hematopoiesis to the production of mature effector cells. This work promises new therapies for chronic anemias by combining TGFβ inhibitors to increase the outflow of immature progenitors with ESAs to support erythroid maturation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Roundabout ways to lineage commitment

The lineage potential, heterogeneity, and relationships of hematopoietic stem and progenitor cells (HSPCs) are currently unclear. To gain new insights into these questions, we performed quantitative analyses of mature cell production by transplanted HSPCs. Assessment of the absolute numbers of mature cell types produced by each progenitor cell revealed a striking erythroid bias of all myeloid-competent progenitors assessed, accompanied by strong platelet production that far exceeded generation of myelomonocytic, B and T cells. Clonal analysis by single cell transplantation and spleen colony assays revealed that a significant fraction of HSCs and multipotent progenitors have multilineage potential at the single-cell level. The physiological relevance of this multipotency was underscored by lineage tracing analysis that demonstrated that all hematopoietic lineages develop through a shared stage in situ. Collectively, these new insights prompt an erythroid-biased model of hematopoietic differentiation. We acknowledge funding from NHLBI, NIDDK, NIAID, the American Cancer Society and the American Asthma Foundation.

Protective effect of sugar polyphenols A on radiation injury in mice

Objective To study the radiation protection effect of sugar polyphenols A obtained by structured modification using mouse model of radiation injury induced by 137Cs γ-ray. Methods Total 42 male Kunming mice aged 4-6 weeks were randomly divided into six groups: negative control group (NCG), positive control group (PCG), irradiation control group (ICG) as well as three irradiation experimental groups. The radiation injury model was developed by once whole-body irradiation with 6.0 Gy 137Cs γ-ray. The mice in three experimental groups were fed sugar polyphenols A respectively with doses of 0.05, 0.10 and 0.20 g/kg, for 14 d before irradiation, and continuously fed with the drug for 7 d after the irradiation. After that the mice were sacrificed, and the organ index, hematological parameters, spleen colony number, bone marrow DNA content and nucleated cells in bone-marrow were tested to evaluate the effect of sugar polyphenols A. Results Compared with the ICG, the irradiation experimental groups with the sugar polyphenols A doses of 0.05, 0.10, 0.20 g/kg showed reductions in irradiation injuries of immune system and hematopoietic system, which makes the significant increases of the spleen indexes (Z=-2.364, -3.003 and -2.747, P<0.05) and the number of endogenous spleen colony (Z=-2.458, -2.141 and -2.206, P<0.05). In the groups with the doses of 0.05 and 0.2 g/kg, the number of white blood cells were significantly increased (Z=-2.652 and-2.139, P<0.05). Besides, in the group with the dose of 0.2 g/kg, the level of red blood cells (Z=-2.364, P<0.05), bone marrow DNA content (Z=-2.492, P<0.05) and nucleated cells in bone-marrow (Z=-2.492, P<0.05) were also increased compared with the ICG. Conclusions Sugar polyphenols A has the positive effects on the recovery of immune and hematopoietic function of mice through improving the organ index, endogenous spleen colony number, peripheral white blood cell number, bone marrow DNA content and nucleated cells in bone-marrow, which can reduce the injury induced by ionizing radiation. Key words: Structural modification; Sugar polyphenols A; Ionizing radiation; Hematological parameters; Endogenous spleen colony; Bone marrow DNA

Therapeutic effects of traditional Chinese medicine Niubeixiaohe in mouse tuberculosis models

Ethnopharmacological relevanceThe traditional Chinese medicine Niubeixiaohe (NBXH) is an effective anti-tuberculosis prescription, which is made up of Bulbus Fritillariae Cirrhosae, Rhizoma Bletillae, Radix Platycodonis, Fructus Arctii, Herba Houttuyniae and Glutinous rice. In this study, NBXH powder (I) and three kinds of NBXH extracts (II, III, and IV) were prepared. The water decoction of NBXH had been used to treat TB in clinic sixteen years suggested that it was effective to treat TB. Aim of the studyThis study evaluated the effects of different processing products of NBXH on mouse TB model in vivo and provide a new Chinese medicine for the clinical treatment of TB. Materials and methodsIn this study, 120 female BALB/c mice infected with Mycobacterium tuberculosis H37Rv, were treated with distilled water, M. vaccae vaccine, the low, middle and high doses of NBXH I, the low, middle and high doses of NBXH II, the low, middle and high doses of NBXH III, the low, middle and high doses of NBXH IV for 12 weeks, respectively. ResultsThe body weights of mice in all NBXH groups were higher than that in the water group. The weight indexes of the spleens in M. vaccae group, the middle dose of NBXH II group, the low dose of NBXH IV group and in the high dose of NBXH IV group were significantly lower than that in the water group(P<0.05). Compared with the water group, the spleen colony counts in the low dose of NBXH I group, the high dose of NBXH II group, the low dose of NBXH III group and the high dose of NBXH IV group reduced by 0.43, 0.46, 0.73, 0.58 logs (P<0.05), respectively. But the lung colony counts had no significant difference between each group. Pulmonary general pathology and histopathology displayed that the lung lesions in treatment groups were improved at certain degree, especially in the low dose of NBXH IIIand IV groups, in which their areas of the lesions were less than 50%, and the half normal lung structure in half of the mice could be observed. ConclusionPowder and three extracts of traditional Chinese medicine NBXH all had anti-tuberculosis therapeutic effects on mouse tuberculosis model, and this study provided a base for the further development of Chinese patent medicine NBXH. Also, this is the first report on comprehensive experimental research of NBXH extracts coming from six kinds of traditional Chinese medicine.

Alcoholic Extract of Wrightia tinctoria leaves and Isolates there of afford DNA Protection in Post Irradiated Sprague Dawley Rats

Objective: The current study investigates the potential of alcoholic extract of Wrightia tinctoria leaves and its isolates in repairing the DNA damage induced by gamma radiation. Methodology: The ethanolic extract of Wrightia tinctoria ( EEWT ) the leaves was subjected to in vitro anti oxidant assay by DPPH radical scavenging and hydrogen peroxide scavenging methods. He EEWT was investigated to the Micronucleus assay, spleen colony assay and comet assay in Sprague Dawley rats wherein DNA damage had already been induced by irradiation. Results: The ethanolic extract of Wrightia tinctoria leaves having good antioxidant activity. In the micronucleus assay, a 2 Gy quantum of whole-body gamma radiation significantly enhanced the levels of micronucleated reticulocytes and this induction was substantially reversed in the extract treatment group. Results of the spleen colony assay pointed to the extract’s ability to enhance the recovery of the hematopoietic system, in a dose-dependent manner, following 6 Gy gamma irradiation. The decrease in cellular repair index due to enhanced DNA repair was evident from the comet assay parameters. Moreover, the isolates from ethanolic extract of Wrightia tinctoria, when imperiled to DNA protection by in vitro gamma radiation method in plasmid pBR322 DNA, abetted DNA repair at higher dose. Conclusion: The DNA protection afforded is likely attributable to free radical scavenging properties along with a potential for regenerating the immune system through repair of post irradiated DNA. Key words: Comet assay, DNA damage, Micronucleus assay, Radioprotective, Spleen colony assay, Wrightia tinctoria.

생약조성물(MH-30)의 면역조혈계 및 재생조직 방사선 손상에 대한 방호 효과

본 연구팀에서는 방사선에 의한 면역조혈계 및 위장관계 손상에 대한 방호를 위하여 당귀, 천궁, 작약 혼합물의 열수추출물로부터 그 다당체 함량을 강화한 생약조성물 Hemo HIM을 개발한 바 있다. 본 연구에서는 열수 추출물에 기반을 두어 제조된 HemoHIM에 비하여 지용성 폴리페놀 성분을 강화함으로써 더 뛰어난 생리활성을 갖는 생약조성물을 개발하고자 30% 에탄올 추출과 열수 추출을 함께 시행하여 얻은 추출물을 기반으로 생약조성물 MH-30을 제조하였다. MH-30과 HemoHIM의 에탄올 분획 내의 성분을 HPLC로 비교 분석한 결과 수용성 성분에는 큰 차이가 없었으나 여러가지 지용성 성분이 MH-30에서 크게 증가하는 것을 확인하였으며, 특히 decursin의 함량은 8.7배로 크게 증가하였다. 다음으로 MH-30의 시험관 내 항산화 및 면역세포 활성화 효과와 방사선 조사 마우스에서 면역조혈계 및 재생조직의 방호 효과를 HemoHIM과 비교하여 관찰하였다. 항산화 활성을 비교하기 위한 시험관 내 hydroxyl radical 및 superoxide anion 소거 활성 평가에서 MH-30이 HemoHIM 보다 높은 항산화 활성을 보였다. 림프구 증식능을 이용한 시험관 내 면역세포 활성화 시험에서는 MH-30과 Hemo HIM은 거의 비슷한 활성을 나타내었다. 방사선 조사 마우스에서 MH-30은 내재성 비장 조혈세포 집락수를 증가시키고 골수조직 내 세포사멸을 줄였으며, 위장관 재생조직인 소장움의 생존율을 증가시키는 등 방사선에 의한 면역조혈계와 재생조직 손상을 방호하는 효과를 보여주었다. 또한, 종합적인 방사선 방호 효과를 평가하는 지표인 방사선 조사 마우스의 30일 생존율도 MH-30 투여로 유의하게 증가함을 확인하였다. 특히 상기한 모든 마우스 실험에서 MH-30은 Hemo HIM보다 더 뛰어난 방호 효과가 있음을 관찰할 수 있었다. 이상의 결과들은 새롭게 개발한 생약조성물 MH-30이 면역조혈계와 재생조직의 방사선 손상을 줄여주는 효과가 있으며 기존에 개발된 HemoHIM에 비해 더 뛰어난 활성을 갖고 있음을 보여주었다. 따라서 MH-30은 방사선 사고 또는 암환자의 방사선치료 시 발생할 수 있는 면역조혈계 및 재생조직의 손상을 경감시킬 수 있는 방사선 방호 물질로서 유용하게 활용될 수 있을 것으로 생각한다.

Abstract 3346: Testing the cancer stem cell hypothesis using the hybrid spheroid assay and Koch's postulates

Abstract We tested the Cancer Stem Cell (CSC) hypothesis using the patented Hybrid Spheroid (HS) Assay (HSA) and by applying Koch's postulates to test its validity. The HSA is an in vitro assay that enables one to take a viable sample of an individual patient's tumor, make a single cell suspension, mix it in known proportions with human fibroblasts (AG1522) and dispense a known number of cells of the mixture into each well of Ultra Low Attachment (ULA) 96-well plates to agglomerate into 1 HS/well, each containing an average of &amp;lt;1 CSC. The HS provides an analog of the CSC niche, enabling the CSC to proliferate (with some daughters differentiating into Amplifying Transit Cells (ATCs)) and undergo 10-15 symmetric divisions before exhausting the nutrients. This satisfies the McCulloch and Till (spleen colony assay) requirements for a stem cell. Applying Koch's postulates: (1) Does the patient's tumor contain cells with CSC properties? Yes: (a) The HSs grow to a size consistent with containing a CSC, (b) those that grew contain cells expressing ≥ 1 OKSM factor(s), and (c) such HSs contain differentiated progeny of the initial CSC. (2) Can we isolate and propagate these cells? In progress: testing HS serial growth, dissociation and reculture. (3) Can these cells induce the tumor in vivo? In progress: testing by xenografting individual CSC-containing HSs in NSG mice. (4) Do these cells contain and express specific gene products that give them CSC properties? Yes: Demonstrated for Oct4 and Nanog. (5) If we disrupt these genes, do the cells lose their CSC properties? In progress: (shRNA). (6) If we eliminate the CSCs do we eliminate the cancer? Yes: tested by (a) mathematical modeling of single CSC growth, differentiation of some progeny into ATCs capable of a limited number of divisions, that then become terminal non-proliferative tumor cells in the HSA; if a single CSC survives/remains, the HS will grow, i.e., the cancer will recur (63% for an average of 1 CSC in a HS, with a Poisson distribution, assuming that niche sites remain available), and (b) by measuring surviving fractions after exposure to radiation and/or chemotherapy agents (where sterilization of the CSC prevents the HS from growing ≥ 10 divisions. The HSA was applied to tumor samples taken from individual endometrial adenocarcinoma patients and correctly predicted, based on CSC radioresistance, which patients would fail their standard-of-care treatments. Conclusion: The CSC hypothesis is validated in the HSA. Citation Format: Christopher S. Lange, Talal Syed, Mike Zhang, Christian Sabalvaro, Hana I. Lim, Ghadir Salame, Bhargava Chitti, Ifeanyi Ilonzo, Arsalaan Ansari, Michelle Chan, Catherine Li, Jing Xu, Xiaotong Chen. Testing the cancer stem cell hypothesis using the hybrid spheroid assay and Koch's postulates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3346.

Protective effects of rosmarinic acid against radiation-induced damage to the hematopoietic system in mice.

Rosmarinic acid (RA) is an ester of caffeic acid and 3, 4-dihydroxyphenyl lactic acid. It is a potent antioxidant that functions by scavenging free radicals. Here, we used a 30-day survival assay to investigate the radioprotective effects of RA. Mice were treated with RA once per day for 10 consecutive days starting at 3 days before gamma irradiation at 7.5 Gy until 7 days post irradiation. Mice treated with 100 and 200 mg/kg body weight (bw) of RA had 30-day survival rates of 89% and 72%, respectively, compared with 32% in the control group, and the differences were statistically significant (P = 0.0008 and 0.0421, respectively). Spleen colony–forming units (CFU-S), the number of nucleated cells in the bone marrow (BMNC), bone marrow DNA content, and hematological parameters of the peripheral blood were measured to investigate the radioprotective effect of RA on the hematopoietic system. The treatment groups that received RA at 50, 100 and 150 mg/kg bw and whole-body exposure to 5.5 Gy of 137Cs γ- radiation had significantly higher CFU-S, BMNC and DNA content than the irradiation-only group. Assessment of hematological parameters in the peripheral blood showed that the treatment groups receiving RA at doses of 50, 100 and 150 mg/kg bw had higher white blood cell counts, hemoglobin and platelets than the radiation-only group. These results suggested that the administration of RA promoted the recovery of peripheral blood cells in irradiated mice.

Влияние криоконсервирования на функциональный статус стволовых кроветворных и мезенхимальных клеток костного мозга животных с аутоиммунной патологией

The application of cryopreserved autologous bone marrow (BM) is the reconstructive therapy tool for autoimmune diseases (AIDs). The BM use efficiency is determined by preservation rate of its hematopoietic and mesenchymal stem cells (HSCs and MSCs, respectively). This fact determines the need to design the optimal cryopreservation regimens for the BM of AIDs donors. Here we assessed a functional status of bone marrow HSCs and MSCs of mice with adjuvant arthritis (AA), initiated by Freund's complete adjuvant administration. The bone marrow of CBA/H mice to day 14 of AA (chronic stage) and that of healthy animals were cryopreserved under dimethyl sulfoxide protection. The content of HSCs of various differentiation extent was determined by spleen colony formation in vivo to days 8 (CFU-S-8) and 14 (CFU-S-14). Functional activity of MSCs in BM was studied in vitro by the content of fibroblast colony forming units (CFU-F). There was established the fact of altering the functional potential of hematopoietic progenitor cells of BM of various differentiation levels (CFU-S-8, CFU-S-14) and their stromal microenvironment (CFU-F) during clinical manifestation of AA. After cryopreservation of AA animals' BM cells with 7% DMSO a functional potential of CFU-S and CFU-F was established to be similar to the healthy animals' BM, cryopreserved with 10% DMSO. The cryopreserved with the proposed regimen autologous BM may be used for a targeted recovery of lymphohemopoietic system in AA animals. Probl Cryobiol Cryomed 2016; 26(1):63-72.

Biochemical and Molecular Mechanisms of Radioprotective Effects of Naringenin, a Phytochemical from Citrus Fruits.

The present study was aimed to evaluate the radioprotective effects of naringenin in vivo using Swiss albino mice as a model system. Oral administration of 50 mg/kg body weight of naringenin for 7 days prior to radiation exposure protected mice against radiation-induced DNA, chromosomal and membrane damage. Naringenin pretreatment also increased the antioxidant status of irradiated mice. Multiple factors operating at cellular and molecular levels led to increased endogenous spleen colonies and survival of mice. Although naringenin induces apoptosis in cancer cells we found that it can protect against radiation-induced apoptosis in normal cells by modulating the expression of p53, Bax, and Bcl-2. The results from the present study indicate that naringenin inhibits the NF-kB pathway and down regulates radiation-induced apoptotic proteins resulting in radioprotection at the cellular, tissue and organism levels.

The Bone Marrow "Mystery Population" of Stem Cells 20 Years Later - a Puzzle Solved?

Background. Almost 20 years ago, a "mystery" population of small stem cells with many of the phenotypic characteristics attributed to resting hematopoietic stem cells was identified in murine bone marrow (BM) (Stem Cells 1998, 16, 38-48). These cells expressed high levels of Sca-1, H-2K, and CD38 and low levels of Thy-1.1; they expressed CD45 antigen but were lineage-negative (lin-) for other hematopoietic markers. These cells incorporated only low levels of Rh123 and were resistant to the cytotoxic effects of 5-fluorouracil. The only phenotypic characteristic that distinguishes these cells from Sca-1+, Lin-, CD45+ Thy-1.1low long-term-reconstituting hematopoietic stem cell population is the lack of c-kit expression. In sum, this "mystery" population of small Sca-1+, lin-, c-kit- but CD45+ stem cells do not respond to hematopoietic growth factors in vitro, form in vivo spleen colonies, or reconstitute lethally irradiated mice. With our discovery of Sca-1+ Lin- CD45- very small embryonic-like stem cells (VSELs) in murine bone marrow (BM) (Leukemia 2006, 20, 857-869), we became interested in this "mystery" population of stem cells. VSELs, like the "mystery" population, are c-kit - and, if freshly isolated from BM, do not show any hematopoietic activity in standard in vitro and in vivo assays. In order to become specified to hematopoiesis, they need to be expanded over an OP-9 stromal support (Exp Hematol 2011;39:225-237).Hypothesis. Since (1) very small CD45- VSELs can be specified in OP-9 co-cultures into long-term reconstituting CD45+ HSCs, (2) the size of the "mystery" population is intermediate between VSELs and HSCs, and (3) VSELs and HSCs differ in cell surface receptor expression, we hypothesized that the "mystery" population is a missing developmental intermediate between VSELs and HSCs.Materials and Methods. Multicolor FACS analysis was employed to compare size and expression of surface markers between murine BM HSCs, the unknown population of stem cells, and VSELs. Next, the populations of small Sca-1+ H2-K+ lin- c-kit+ CD38+/- CD45+ cells (HSCs), smaller Sca-1+ H-2K+ lin- c-kit- CD38+ CD45+ cells (the "mystery" population), and very small in size Sca-1+ H-2K+ lin- c-kit- CD38+/- CD45- cells (VSELs) were purified by FACS from BM (Figure 1) and tested for in vitro colony formation. All these cell populations were primed/expanded over OP-9 support and subsequently evaluated for their hematopoietic potential after passaging in consecutive methylocellulose cultures (passages 1-4). RQ-PCR analysis was employed for detection of pluripotency marker expression as well as hematopoietic gene expression.Results. We found that, in contrast to HSCs, neither freshly sorted stem cells from the "mystery" BM population nor, as expected, VSELs grew hematopoietic colonies in standard methylcellulose cultures. This was also an important step in excluding contamination of our sorted populations with clonogenic cells. We also found that, while VSELs highly expressed Oct-4, this transcription factor was expressed at very low levels in the "mystery" population and was not detectable in HSCs. The most important observation was that the "mystery" population of stem cells became specified in OP-9-supported cultures into clonogenic HSPCs, and this specification occurred faster than the delayed specification of VSELs. VSELs first became enriched for HSPCs after acquiring CD45 antigen expression.Conclusions. Based on the results presented, we propose that the "mystery" population in murine BM is a population of stem cells intermediate between the most primitive population of BM-residing stem cells (VSELs) and the population of stem cells already specified to lympho-hematopoietic development (HSCs). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Homeobox Gene VENTX Collaborates with AML1-ETO in Inducing an Acute Leukemia in Mice

Homeobox genes are key factors in the development of acute leukemias. So far, little is known about the role of non-clustered homeobox genes such as VENTX. The Vent-like homeobox gene VENTX is a member of the Vent gene family in mammals and is the mammalian homolog of the Xenopus xvent gene. Our group has previously shown that VENTX promotes myeloid differentiation when overexpressed in normal CD34+ human stem - and progenitor cells and shows high and aberrant expression in human acute myeloid leukemia (AML) characterized by the AML1-ETO (AE) fusion (Rawat et al., PNAS 2010). To prove the functional relevance of aberrant VENTX expression in this AML genotype, we set up in vitro assays and a murine bone marrow transplantation model, mimicking high expression of VENTX by retrovirally engineered expression of the gene in murine bone marrow progenitor cells. In the CFU-S assay coexpression of both genes significantly increased the frequency of spleen colonies 14-fold and 7-fold compared to VENTX and AE, respectively (p<0.02), and more than 7-fold compared to the control (p<0.001). Most importantly and in contrast to AE alone, coexpression of AE and VENTX induced an acute leukemia after a median latency of 11.3 months post transplant. All leukemias evaluated were rapidly transplantable with a median latency until leukemia induced death of 40 days. The median blast percentage in primary recipient mice was 66% (range 35-100%, n=24). Of note, massive expansion of the red pulpa was documented with the appearance of erythroblasts in the spleen. Furthermore, blasts in the bone marrow expressed CD19, partly in combination with Sca1 and Gr1, pointing to an aberrant expression of this antigen in the AE/VENTX leukemias. Ex vivo, leukemic cells grew permanently generating AE/VENTX positive cell lines. RNA-Seq analyses from CD34+ cord blood transduced with VENTX documented 279 differentially expressed genes compared to the GFP control, hitting 7 pathways (Hematopoietic cell lineage, Cytokine-cytokine receptor pathway, Jak-STAT signaling pathway, Porphyrin and Chlorophyll Metabolism, Asthma, Thyroid cancer and Endocytosis) in the KEGG-Analysis and showed a downregulation of genes necessary for terminal erythroid differentiation such as the EPO-receptor and GATA1. Taken together, these data characterize VENTX as an important contributing factor to AE positive leukemias. Importantly, coexpression of AE and VENTX allows the generation of a murine AE model, which in contrast to all other published mouse models mimics aberrant expression of CD19 in human AE positive AML, a hallmark of this AML genotype. DisclosuresBuske:CELLTRION, Inc.: Consultancy, Honoraria.

Radioprotective activity of Polyalthia longifolia standardized extract against X-ray radiation injury in mice

The radioprotective effect of Polyalthia longifolia was studied in mice. P. longifolia treatment showed improvement in mice survival compared to 100% mortality in the irradiated mice. Significant increases in hemoglobin concentration, and red blood cell, white blood cell and platelet counts were observed in the animals pretreated with leaf extract. Pre-irradiation administration of P. longifolia leaf extract also increased the CFU counts of the spleen colony and increased the relative spleen size. A dose-dependent decrease in lipid peroxidation levels was observed in the animals pretreated with P. longifolia. However, although the animals pretreated with P. longifolia exhibited a significant increase in superoxide dismutase and catalase activity, the values remained below normal in both liver and the intestine. Pre-irradiation administration of P. longifolia also resulted in the regeneration of the mucosal crypts and villi of the intestine. Moreover, pretreatment with P. longifolia leaf extract also showed restoration of the normal liver cell structure and a significant reduction in the elevated levels of ALT, AST and bilirubin. These results suggested the radioprotective ability of P. longifolia leaf extract, which is significant for future investigation for human applications in developing efficient, economically viable, non-toxic natural and clinically acceptable novel radioprotectors.

Lin-Sca-1+c-KitlowCD48+CD71+ Cells Are the Engine of Bone Marrow Regeneration

The hematopoietic tissue is the most suitable tissue for studies into the biology of regeneration. We examined bone marrow regeneration starting from a very low number of repopulating cells.Mice were irradiated by a sublethal dose of 6 Gy and recovery of hematopoiesis was examined in its three basic compartments: stem and progenitor cells, precursors of blood cells, and peripheral blood between days 8 – 90 following irradiation. The irradiation reduced existing pools of progenitors and stem cells to ̴0.1%. We focused on the pools of stem cells and progenitors that were evaluated by several assays.A novel assay based on engraftment of a defined number of transplanted competitors revealed delayed filling of the niches for long-term repopulating cells (LTRCs; stem cells) as compared to filling of the niches for short-term repopulating cells (STRCs; progenitors) by endogenous STRCs and LTRCs generated by cells surviving irradiation. This indicated a very slow recovery of the pool of stem cells, lagging after recovery of the pool of progenitors. This was confirmed by a traditional competitive transplantation assay, as well as by another novel assay that measured the capacity of bone marrow to establish pools of stem cells and progenitors in lethally irradiated mice. Direct assays of progenitors by the spleen colony (CFU-S) and in vitro CFC-GM assays indicated a low frequency of the clonogenic cells in regenerating bone marrow. Productive hematopoiesis was re-established about 10 days after irradiation in presence of very low pools of progenitors and stem cells.Next we examined numbers of immature hematopoietic cells by flow cytometry focused at lineage negative (Lin-), Sca-1 positive (Sca-1+), c-Kit+ cells (LSK cells), characterized further by means of the CD150, CD48 and CD71 antigenic markers. In absolute terms, only the population of LSK CD150+CD48+ recovered and overshot normal values after day 12 following irradiation. In relative terms, the LSK CD48+ cells (CD150+ and CD150-) represented more than 99% of all LSK cells two weeks after irradiation. Also in relative terms, the LSK CD150+CD48- cells, which are highly enriched for hematopoietic stem cells in normal bone marrow, recovered by day 30 and became relatively abundant later on. Another striking change consisted in initial lack and later severe scarcity of cells with the phenotype of LSK CD150-CD48-. These cells, serving as multipotent progenitors in normal bone marrow thus remained severely depleted in regenerating bone marrow during the whole follow-up period. LSK cells were mostly negative for CD71 (transferrin receptor) in normal bone marrow but became highly positive for this marker between days 10 – 20 after irradiation.In conclusion, our semi-quantitative evaluation of regenerating hematopoiesis reveals its highly peculiar features. Those include an early recovery of blood cell production, followed by significant expansion of the Lin-Sca-1+c-KitlowCD48+CD71+ cells in presence of a highly reduced pool of hematopoietic stem cells. DisclosuresNo relevant conflicts of interest to declare.

Down-Regulation of c-Kit Receptor on Hematopoietic Cells By Stem Cell Factor (SCF; c-Kit-ligand): No Impact on Transplantation

Presence of the c-Kit tyrosine kinase receptor is a hallmark of the mouse hematopoietic stem cells (HSCs) and progenitors routinely used for their identification and separation. c-Kit is activated after binding of its ligand, the stem cell factor (SCF; c-Kit-ligand). c-Kit receptors with bound SCF form dimers that are rapidly internalized and degraded. This activates the c-Kit signaling pathways supporting cell survival, proliferation or quiescence and self-renewal. Although there is a consensus that c-Kit signaling is important for functioning of HSCs, published results are partly controversial.We have defined HSCs and progenitors as Lineage- Sca-1+c-Kit+ cells (LSK cells) and characterized them further by means of the CD150 and CD48 markers. We used anti-c-Kit antibody minus (FMO; Fluorescence Minus One) samples to distinguish between c-Kit+ and c-Kit- bone marrow cells and analyzed the distribution of c-Kit on the immature hematopoietic cells carrying different phenotypes. Further, we exposed bone marrow cells to a wide range of concentration of a recombinant mouse SCF in vitro and measured a change in c-Kit presence and distribution on these different cell types. Also, SCF was injected to mice in vivo and their bone marrow was similarly analyzed for a change in c-Kit expression. Bone marrow cells exposed to SCF concentrations that deeply down-regulated c-Kit receptors were transplanted to recipient mice, and their transplantation efficiency was compared to that of normal bone marrow.c-Kit was unevenly but characteristically distributed on different types of LSK CD150/CD48 cells, showing the highest and the most homogeneous density on cells with the LSK CD150+CD48- phenotype. Exposure of bone marrow cells to SCF in ranges of concentrations from 0.3-2000 ng/ml induced progressive down-regulation of c-Kit. However, the cells mostly remained c-Kit+(low). The response to SCF was the most prominent in a range of SCF concentrations between 1-100 ng/ml. Cells with the phenotype LSK CD150+CD48+were relative low-responders. In vivo administration of SCF to mice in doses exceeding 300 ng/mouse, either intraperitoneally or intravenously, had similar effect on c-Kit expression by bone marrow cells as their incubation with SCF in vitro.Next we investigated correlation of the intensity of c-Kit receptor expression on bone marrow cells with their repopulating capacity after transplantation. A significantly decreased c-Kit expression on transplanted cells, induced by exposure of the cells to SCF, did not decrease contribution of the cells to chimeric hematopoiesis in competitive transplantation assays. Formation of spleen colonies was also not affected in the CFU-S assay.Experiments which measured the effect of SCF administered to normal mice in vivo demonstrated an effect that lasted for less than 12 hours. c-Kit turnover on hematopoietic cells is thus rapid, and this fact may explain why down-regulation of c-Kit, on otherwise normal bone marrow cells, does not affect their capacity to be transplanted.In conclusion, c-Kit receptor density on hematopoietic cells does not appear to be a critical factor for the homing of transplanted hematopoietic stem and progenitor cells into the blood-forming tissues and their engraftment into specific niches. Also their performance in establishing productive hematopoiesis is not altered by the SCF-induced down-regulation of the c-Kit receptor density in time of their transplantation. DisclosuresNo relevant conflicts of interest to declare.

Radioprotective effect of Rapana thomasiana hemocyanin in gamma induced acute radiation syndrome

The radioprotective effect of Rapana thomasiana hemocyanin (RtH) against radiation-induced injuries (stomach ulcers, survival time and endogenous haemopoiesis) and post-radiation recovery was investigated in male albino mice (C3H strain). Radiation course was in a dose of 7.5 Gy (LD 100/30 – dose that kills 100% of the mice at 30 days) from 137Cs with a dose of 2.05 Gy/min. Radiation injuries were manifested by inducing а hematopoietic form of acute radiation syndrome. RtH was administered intraperitoneally in a single dose of 50, 100, 150 and 200 mg/kg body weight (b. w.) once a day for five consecutive days before irradiation. The results obtained showed that radiation exposure led to (1) 100% mortality rate, (2) ulceration in the stomach mucosa and (3) decrease formation of spleen colonies as a marker of endogenous haemopoiesis. Administration of RtH at a dose of 200 mg/kg provided better protection against radiation-induced stomach ulceration, mitigated the lethal effects of radiation exposure and recovered endogenous haemopoiesis versus irradiated but not supplemented mice. It could be expected that RtH will find a use in mitigating radiation induced injury and enhanced radiorecovery.

Metformin exhibits radiation countermeasures efficacy when used alone or in combination with sulfhydryl containing drugs.

Metformin, a biguanide drug used in the treatment of type II diabetes, was evaluated alone and in combination with amifostine, captopril, MESNA or N-acetyl-cysteine (NAC) for its ability to protect when administered 24 h after irradiation. Mouse embryo fibroblasts (MEF), human microvascular endothelial cells (HMEC) and SA-NH mouse sarcoma cells were exposed to 4 Gy in vitro. C3H mice were exposed to 7 Gy and evaluated utilizing an endogenous spleen colony assay system. Amifostine and WR1065, administered 30 min prior to irradiation, were used as positive controls. Treatment of MEF, HMEC and SA-NH cells with metformin elevated survival levels by 1.4-, 1.5- and 1.3-fold compared to 1.9-, 1.8- and 1.6-fold for these same cells treated with WR1065, respectively. Metformin (250 mg/kg) was effective in protecting splenic cells from a 7 Gy dose in vivo (protection factor = 1.8). Amifostine (400 mg/kg), administered 30 min prior to irradiation resulted in a 2.6-fold survival elevation, while metformin administered 24 h after irradiation in combination with NAC (400 mg/kg), MESNA (300 mg/kg) or captopril (200 mg/kg) enhanced survival by 2.6-, 2.8- and 2.4-fold, respectively. Each of these agents has been approved by the FDA for human use and each has a well characterized human safety profile. Metformin alone or in combination with selected sulfhydryl agents possesses radioprotective properties when administered 24 h after radiation exposure comparable to that observed for amifostine administered 30 min prior to irradiation making it a potentially useful agent for radiation countermeasures use.

Polysaccharide isolated from Parmelia tinctorum ameliorates ionizing irradiation-induced damage in mice.

In this study, WPT-A, a type of water-soluble homogeneous lichen polysaccharide, was isolated and purified from Parmelia tinctorum. We investigated whether WPT-A has radioprotective effects when administered before total-body irradiation (TBI). Mice were treated with WPT-A via intraperitoneal injection (i.p.) once per day for three consecutive days prior to 7, 7.5, 8.5, 10 or 10.5-Gy TBI. Our results indicated that the survival rate was enhanced at a range of levels of TBI. The calculated dose reduction factor (DRF) was 1.2. White blood cell (WBC) counts, spleen colony forming units (CFU-S) and bone marrow nucleated cell (BMNC) counts were used to investigate the radioprotective effects of WPT-A on the hematopoietic system. The treatment groups received WPT-A at 20, 50 and 80 mg/kg b.w. doses before 6.5-Gy TBI and showed significantly higher BMNC and WBC counts compared with the radiation-only group. The groups administered 50 and 80 mg/kg b.w. WPT-A showed a significant increase in CFU-S compared with the radiation-only group. We also carried out a single cell gel electrophoresis assay to explore the radioprotective effects of WPT-A on DNA damage. The results from single-cell gel electrophoresis of peripheral blood leukocytes showed that WPT-A attenuated radiation-induced DNA damage. These results indicate a potential use for WPT-A as a radioprotector.

Gamma-tocopherol-N,N-dimethylglycine ester as a potent post-irradiation mitigator against whole body X-irradiation-induced bone marrow death in mice

We examined the radioprotective and mitigative effects of gamma-tocopherol-N,N-dimethylglycine ester (GTDMG), a novel water-soluble gamma-tocopherol derivative, against X-irradiation-induced bone marrow death in mice. Mice (C3H, 10 weeks, male) were injected intraperitoneally with GTDMG suspended in a 0.5% methyl cellulose solution before or after receiving of 7.5-Gy whole body X-irradiation. GTDMG significantly enhanced the 30-day survival rate when given 30 min before or immediately after the irradiation. Its mitigative activity (administered after exposure) was examined further in detail. The optimal concentration of GTDMG given immediately after irradiation was around 100 mg/kg body weight (bw) and the 30-day survival rate was 97.6 ± 2.4%. When GTDMG was administered 1, 10 and 24 h post-irradiation, the survival rate was 85.7 ± 7.6, 75.0 ± 9.7 and 36.7 ± 8.8%, respectively, showing significant mitigation even at 24 h after irradiation (P < 0.05). The value of the dose reduction factor (100 mg/kg bw, given intraperitoneally (i.p.) immediately after irradiation) was 1.25. GTDMG enhanced the recovery of red blood cell-, white blood cell-, and platelet-counts after irradiation and significantly increased the number of endogenous spleen colonies (P < 0.05). Subcutaneous (s.c.) administration also had mitigative effects. In conclusion, GTDMG is a potent radiation mitigator.

Garlic Oil Suppressed the Hematological Disorders Induced by Chemotherapy and Radiotherapy in Tumor‐Bearing Mice

Although the anticancer effects of garlic and its products have been demonstrated by a variety of studies; however, few studies were conducted to investigate the effects of garlic on the adverse effects of chemo/radiotherapy. In order to clarify the above question and make a more comprehensive understanding of the anticancer effects of garlic, tumor xenograft mice model was established by subcutaneous injection of H22 tumor cells, and was used for the investigation of effects of garlic oil (GO) on the chemo/radiotherapy. In the chemotherapy test, tumor-bearing mice were treated with cyclophosphamide (CTX) or CTX plus GO (25 or 50 mg/kg bw) for 14 d, while the mice received a single 5 Gy total body radiation or radiation plus GO (25 or 50 mg/kg bw) in radiotherapy test. The results showed that GO did not increase the tumor inhibitory rate of CTX/radiation, which indicated that GO could not enhance the chemo/radiosensitivity of cancer cells. However, the decrease of the peripheral total white blood cells (WBCs) count induced by CTX/radiation was significantly suppressed by GO cotreatment. Furthermore, GO cotreatment significantly inhibited the decrease of the DNA contents and the micronuclei ratio of the bone marrow. Lastly, the reduction of the endogenous spleen colonies induced by CTX/radiation was significantly suppressed by GO cotreatment. These findings support the idea that GO consumption may benefit for the cancer patients receiving chemotherapy or radiotherapy.