Apoptosis Of Marrow Cells Research Articles

Study on the combined toxicity of DEHP and lead on the blood system of rats

Di(2-ethylhexyl) phthalate (DEHP) is a commonly used phthalate ester compound, while lead is a persistent and bioaccumulative heavy metal. Both can be exposed to the body through a variety of ways, which may have an impact on the blood system. In this study, we examined the impact of co-exposure to DEHP (0, 10, 100 mg/kg) and Pb (0, 5, 50 mg/kg) on the blood system of male SD rats. The study revealed that continuous exposure to DEHP and Pb for 20 days resulted in a decrease in leukocytes and lymphocytes, while an increase in neutrophils and monocytes. Co-exposure led to a significant decrease in the spleen coefficients. Furthermore, the combined exposure could increase the ratio of bone marrow cells in G1 phase, and decrease the ratio of cells in S phase and G2 phase. Cytokine testing showed that combined exposure affects the secretion of hematopoietic factors and may cause bone marrow cell apoptosis. Single or combined exposure to DEHP and Pb can cause oxidative stress in serum and bone marrow. Overall, these results indicate that the co-exposure of DEHP and Pb adversely affected the blood system of rats, mainly due to the induction of oxidative stress and ultimately affects the secretion of cytokines. The combined effect of the two substances is primarily antagonistic. These results have important implications for the risk assessment of combined pollution and provide valuable theoretical guidance.

The effect of platelet infusion on the repair of bone marrow hematopoietic niche damage in mice induced by (60)Co radiation and hematopoietic reconstruction after bone marrow transplantation

Objective: To observe the effect of platelets on hematopoietic stem cell (HSCs) implantation in mice with radiation-induced bone marrow injury and bone marrow transplantation models. Methods: ①Male C57BL/6 mice were divided into a single irradiation group and a radiation infusion group after receiving (60)Co semimyeloablative irradiation for 18-10 weeks. The irradiation infusion group received 1×10(8) platelets expressing GFP fluorescent protein. ② The allogeneic bone marrow transplantation model was established. The experimental groups included the simple transplantation group (BMT) and the transplantation infusion group (BMT+PLT). The BMT group was infused through the tail vein only 5 × 10(6) bone marrow cells, the BMT+PLT group needs to be infused with bone marrow cells at the same time 1× 10(8) platelets. ③ Test indicators included peripheral blood cell and bone marrow cell counts, flow cytometry to detect the proportion of hematopoietic stem cell (HSC) and hematopoietic progenitor cells, bone marrow cell proliferation and apoptosis, and pathological observation of vascular niche damage and repair. Results: ①On the 3rd, 7th, 14(th), and 21st days after irradiation, the bone marrow cell count of the infusion group was higher than that in the single irradiation group (P<0.05), and the peripheral blood cell count was also higher. A statistically significant difference was found between the white blood cell count on the 21st day and the platelet count on the 7th day (P<0.05). In the observation cycle, the percentage of bone marrow cell proliferation in the infusion group was higher, while the percentage of apoptosis was lower. ② The results of bone tissue immunofluorescence after irradiation showed that the continuity of hematopoietic niche with red fluorescence was better in the irradiation infusion group. ③The chimerism percentage in the BMT+PLT group was always higher than that in the BMT group after transplantation.④ The BMT+PLT group had higher bone marrow cell count and percentage of bone marrow cell proliferation on the 7th and 28th day after transplantation than that in the BMT group, and the percentage of bone marrow cell apoptosis on the 14th day was lower than that in the BMT group (P<0.05). After the 14th day, the percentage of stem progenitor cells in the bone marrow cells of mice was higher than that in the BMT group (P<0.05). ⑤The immunohistochemical results of bone marrow tissue showed that the continuity of vascular endothelium in the BMT+PLT group was better than that in the BMT group. Conclusion: Platelet transfusion can alleviate the injury of vascular niche, promotes HSC homing, and is beneficial to hematopoietic reconstruction.

Extracellular vesicles from human urine-derived stem cells inhibit glucocorticoid-induced osteonecrosis of the femoral head by transporting and releasing pro-angiogenic DMBT1 and anti-apoptotic TIMP1

Osteonecrosis of the femoral head (ONFH) frequently occurs after glucocorticoid (GC) treatment. Extracellular vesicles (EVs) are important nano-sized paracrine mediators of intercellular crosstalk. This study aimed to determine whether EVs from human urine-derived stem cells (USC-EVs) could protect against GC-induced ONFH and focused on the impacts of USC-EVs on angiogenesis and apoptosis to explore the mechanism by which USC-EVs attenuated GC-induced ONFH. The results in vivo showed that the intravenous administration of USC-EVs at the early stage of GC exposure could rescue angiogenesis impairment, reduce apoptosis of trabecular bone and marrow cells, prevent trabecular bone destruction and improve bone microarchitecture in the femoral heads of rats. In vitro, USC-EVs reversed the GC-induced suppression of endothelial angiogenesis and activation of apoptosis. Deleted in malignant brain tumors 1 (DMBT1) and tissue inhibitor of metalloproteinases 1 (TIMP1) proteins were enriched in USC-EVs and essential for the USC-EVs-induced pro-angiogenic and anti-apoptotic effects in GC-treated cells, respectively. Knockdown of TIMP1 attenuated the protective effects of USC-EVs against GC-induced ONFH. Our study suggests that USC-EVs are a promising nano-sized agent for the prevention of GC-induced ONFH by delivering pro-angiogenic DMBT1 and anti-apoptotic TIMP1. Statement of SignificanceThis study demonstrates that the intravenous injection of extracellular vesicles from human urine-derived stem cells (USC-EVs) at the early stage of glucocorticoid (GC) exposure efficiently protects the rats from the GC-induced osteonecrosis of the femoral head (ONFH). Moreover, this study identifies that the promotion of angiogenesis and inhibition of apoptosis by transferring pro-angiogenic DMBT1 and anti-apoptotic TIMP1 proteins contribute importantly to the USC-EVs-induced protective effects against GC-induced ONFH. This study suggests the promising prospect of USC-EVs as a new nano-sized agent for protecting against GC-induced ONFH, and the potential of DMBT1 and TIMP1 as the molecular targets for further augmenting the protective function of USC-EVs.

Effect of α2‑macroglobulin in the early stage of jaw osteoradionecrosis.

Advanced osteoradionecrosis (ORN) is one of the most serious complications in patients with head and neck cancer, resulting in poor prognosis. Numerous studies have therefore focused on the pathogenesis and interventions of ORN early stage. The present study aimed to investigate whether α2-macroglobulin (α2M) could prevent early-stage jaw osteoradionecrosis caused by radiotherapy (RT). Following local injection of α2M, a single dose of 30 Gy was delivered to rats for pathological exploration. For 28 days, the irradiated mandible and soft tissues were examined for potential changes. Furthermore, primary human bone marrow mesenchymal stem cells pretreated with α2M followed by 8 Gy irradiation (IR) were also used. Tartrate-resistant acid phosphatase assay, terminal uridine deoxynucleotidyl nick end labeling assay and immunohistochemical staining were performed on irradiated mandibular bone, tongue or buccal mucosa tissues from rats. Cell proliferation was assessed by evaluating the cell morphology by microscopy and by using the cell counting kit-8. Fluorescence staining, flow cytometry and western blotting were conducted to detect the reactive oxygen species level, cell apoptosis and protein expression of superoxide dismutase 2 (SOD2), heme oxygenase-1 (HO-1) and phosphorylated Akt following irradiation. The results demonstrated that α2M attenuated physical inflammation, osteoclasts number and fat vacuole accumulation in mandibular bone marrow and bone marrow cell apoptosis following IR in vivo. Furthermore, α2M pretreatment suppressed the expression of 8-hydroxy-2′-deoxyguanosine in mandibular bone and tongue paraffin embedded sections, which is a marker of oxidative damage, and increased SOD2 expression in mucosa and tongue paraffin embedded sections. The present study demonstrated the efficient regulation of antioxidative enzymes, including SOD2 and heme oxygenase-1, and reduction in oxidative damage by α2M. In addition, in vitro results confirmed that α2M may protect cells from apoptosis and suppress reactive oxygen species accumulation. Overall, the present study demonstrated that α2M treatment may exert some radioprotective effects in early-stage ORN via antioxidant mechanisms, and may therefore be considered as a potential alternative molecule in clinical prophylactic treatments.

Genomic insult oriented mitochondrial instability and proliferative hindrance in the bone marrow of aplastic mice including stem/progenitor population

Aplastic anemia is the bone marrow failure condition characterized by the development of hypocellularity in both marrow and peripheral blood compartments. Anti-tumor chemotherapeutic agents often exert secondary effect on hematopoietic system leading to aplastic anemia by marrow failure. The precise mechanisms behind the marrow ablative effects of the drugs remain yet to be established. The present study holds a mechanistic approach to unveil the mystery. Aplastic anemia was generated in mice with the administration of busulfan and cyclophosphamide followed by the characterization of the disease with peripheral blood hemogram, histopathological and cytochemical examinations of bone marrow. To gain deep knowledge about the molecular mechanisms of the hematopoietic disruption, cytotoxicity assay, DNA damage measurement, apoptosis study, replicative senescence analysis, redox balance study, mitochondrial membrane potential change assessment, flowcytometric expressional analysis of p21, p53, ATM, Chk-2, Necdin, Gfi-1, c-myc, KU-80 and Sod-2 were done with marrow hematopoietic stem/ progenitor cells (HSPCs). Severe blood pancytopenia and marrow hypocellularity was found in aplastic mice. Proliferative hindrance and apoptosis of marrow cells were identified as the cause behind the hematopoietic catastrophe. The genotoxic effects of the drugs triggered chromatin damage and induced replicative senescence in aplastic HSPCs by upregulating p21 in a p53 independent manner. Moreover, accumulation of genomic insults also caused apoptotic elimination of marrow cells due to disruption of mitochondrial membrane potential by generating redox imbalance. The study established the underlying mechanisms behind hematopoietic disruption during drug induced marrow aplasia. Outcome of the study may be helpful in successful designing of therapeutic strategies for the disease concerned.

Mitochondrial protein OPA mediates osteoporosis induced by radiation through the P38 signaling pathway.

Bone marrow is full of mitochondria. However, the role of bone marrow mitochondrial protein in bone marrow damage and related signal transduction mechanism remains to be further studied. OPA is a newly discovered mitochondrial transmembrane protein. Its expression pattern and function in the physiological and pathological conditions of bone marrow are still elusive. The purpose of this study is to investigate the potential role of OPA in osteoporosis. A mouse osteoporosis model was established by radiation. The OPA expression was tested by Western blot and qRT-PCR. The P38 signaling activity was evaluated by enzymatic activity kit. The mitochondrial ATP production was determined by flow cytometry. The bone marrow cell apoptosis was detected by flow cytometry. U0126 was used to pretreat mouse before modeling. Bone marrow tissue was collected from patients who received osteoporosis surgery to test the OPA expression, P38 activation and cell apoptosis. The OPA and P38 levels were analyzed by correlation. The mouse osteoporosis model was successfully established by radiation induction. In this osteoporosis model, the expression of OPA was increased. The P38 signaling was activated while the mitochondrial ATP production was reduced, with the increase of apoptosis of bone marrow cells. By contrast, U0126 pretreatment markedly inhibited the OPA expression, restrained the P38 signaling pathway, enhanced mitochondrial ATP production and suppressed the bone marrow cell apoptosis in mouse osteoporosis model. A significantly positive correlation was found between OPA and P38. The down-regulation of OPA inhibits cell apoptosis and improves osteoporosis via inducing mitochondrial ATP production and suppressing the P38 signaling pathway.

NOS2 deficiency has no influence on the radiosensitivity of the hematopoietic system

ObjectivePrevious studies have shown that inhibition of inducible NO synthase (NOS2 or iNOS) with an inhibitor can selectively protect several normal tissues against radiation during radiotherapy. However, the role of NOS2 in ionizing radiation (IR)-induced bone marrow (BM) suppression is unknown and thus was investigated in the present study using NOS2−/− and wild-type mice 14 days after they were exposed to a sublethal dose of total body irradiation (TBI).MethodsThe effects of different doses of IR (1, 2 and 4 Gy) on the apoptosis and colony-forming ability of bone marrow cells from wild-type (WT) and NOS2−/− mice were investigated in vitro. In addition, we exposed NOS2−/− mice and WT mice to 6-Gy TBI or sham irradiation. They were euthanized 14 days after TBI for analysis of peripheral blood cell counts and bone marrow cellularity. Colony-forming unit-granulocyte and macrophage, burst-forming unit-erythroid and CFU-granulocyte, erythroid, macrophage in bone marrow cells from the mice were determined to evaluate the function of hematopoietic progenitor cells (HPCs), and the ability of hematopoietic stem cells (HSCs) to self-renew was analysed by the cobblestone area forming cell assay. The cell cycling of HPCs and HSCs were measured by flow cytometry.ResultsExposure to 2 and 4 Gy IR induced bone marrow cell apoptosis and inhibited the proliferation of HPCs in vitro. However, there was no difference between the cells from WT mice and NOS2−/− mice in response to IR exposure in vitro. Exposure of WT mice and NOS2−/− mice to 6 Gy TBI decreased the white blood cell, red blood cell, and platelet counts in the peripheral blood and bone marrow mononuclear cells, and reduced the colony-forming ability of HPCs (P < 0.05), damaged the clonogenic function of HSCs. However, these changes were not significantly different in WT and NOS2−/− mice.ConclusionThese data suggest that IR induces BM suppression in a NOS2-independent manner.

Effect of the traditional Chinese medicine compound Yisui Lixue decoction on apoptosis of marrow cells in rats with myelodysplastic syndrome induced by dimethyl benzanthracene

To investigate the effect of the traditional Chinese medicine compound Yisui Lixue decoction on apoptosis of marrow cells in rats with myelodysplastic syndrome (MDS) induced by dimethyl benzanthracene (DMBA). Methods: The rats with MDS induced by the chemical mutagens DMBA were divided into 5 groups (12 rats in each group): a control group, a model+PBS group, a model+compound Zaofan pill group, a model+low dose of Yisui Lixue decoction group and a model+high dose of Yisui Lixue decoction group. After DMBA treatment for 14 days, rats were treated with different drugs for 1 month and executed on the 31 day. The peripheral blood cells, expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax), apoptosis of bone marrow cells were measured. Results: The counts of peripheral leukocyte, erythrocyte, hemoglobin and platelet in the model+PBS group were significantly decreased compared with those in the control group (P<0.01), while the peripheral hemograms of the treatment groups were improved significantly compared with those in the model+PBS group (P<0.05 or P<0.01), especially in the model+high dose group (P<0.01). In the model+PBS group, the expressions of Bax and Bcl-2 was significantly increased (P<0.01) or decreased (P<0.01), respectively, which were reversed by treatment of Yisui Lixue decoction (P<0.05 or P<0.01), especially in the model+high dose group (P<0.01). Compared with control group, the total apoptosis rate, earlier apoptosis and later apoptosis rate of bone marrow cells in the model+PBS group were increased (P<0.01), which were blocked by treatment of Yisui Lixue decoction (P<0.05 or P<0.01), and the model+high dose group was better than the model+low dose group. Conclusion: The traditional Chinese medicine compound Yisui Lixue decoction can improve peripheral hemogram, decrease the expression of Bax, increase the expression of Bcl-2, and reduce the apoptosis of bone marrow cells.

MicroRNA-146a Induces Lineage-Negative Bone Marrow Cell Apoptosis and Senescence by Targeting Polo-Like Kinase 2 Expression.

Lineage-negative bone marrow cells (lin- BMCs) are enriched in endothelial progenitor cells and mediate vascular repair. Aging-associated senescence and apoptosis result in reduced number and functionality of lin- BMCs, impairing their prorepair capacity. The molecular mechanisms underlying lin- BMC senescence and apoptosis are poorly understood. MicroRNAs (miRNAs) regulate many important biological processes. The identification of miRNA-mRNA networks that modulate the health and functionality of lin- BMCs is a critical step in understanding the process of vascular repair. The aim of this study was to characterize the role of the miR-146a-Polo-like kinase 2 (Plk2) network in regulating lin- BMC senescence, apoptosis, and their angiogenic capability. Transcriptome analysis in lin- BMCs isolated from young and aged wild-type and ApoE-/- (apolipoprotein E) mice showed a significant age-associated increase in miR-146a expression. In silico analysis, expression study and Luciferase reporter assay established Plk2 as a direct target of miR-146a. miR-146a overexpression in young lin- BMCs inhibited Plk2 expression, resulting in increased senescence and apoptosis, via p16Ink4a/p19Arf and p53, respectively, as well as impaired angiogenic capacity in vitro and in vivo. Conversely, suppression of miR-146a in aged lin- BMCs increased Plk2 expression and rejuvenated lin- BMCs, resulting in decreased senescence and apoptosis, leading to improved angiogenesis. (1) miR-146a regulates lin- BMC senescence and apoptosis by suppressing Plk2 expression that, in turn, activates p16Ink4a/p19Arf and p53 and (2) modulation of miR-146a or its target Plk2 may represent a potential therapeutic intervention to improve lin- BMC-mediated angiogenesis and vascular repair.

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.

Allogeneic Mesenchymal Stem Cell Therapy Promotes Osteoblastogenesis and Prevents Glucocorticoid-Induced Osteoporosis

: Gene-modified mesenchymal stem cell (MSC)-like cells with enhanced bone marrow homing and osteogenesis have been used in treating glucocorticoid-induced murine osteoporosis (GIOP). Recent preclinical studies have further demonstrated the immunomodulatory and anticatabolic potential of allogeneic MSCs in treating osteoporosis under inflammatory and autoimmune conditions. In this study, we investigated whether systemic infusion of allogeneic MSCs without genetic manipulation could prevent GIOP, whether anabolic and anticatabolic effects existed, and whether homing or immunomodulation underlay the putative therapeutic effects. Allogeneic bone marrow-derived MSCs (BMMSCs) were isolated, identified, and systemically infused into mice treated with excessive dexamethasone. We revealed that allogeneic MSC transplantation prevented the reduction of bone mass and strength in GIOP. Bone histomorphometric analyses of bone remodeling demonstrated the maintenance of bone formation and osteoblast survival after MSC therapy. Using green fluorescent protein (GFP)-labeled BMMSCs, we showed that donor BMMSCs(GFP) homed and inhabited recipient bone marrow for at least 4 weeks and prevented recipient bone marrow cell apoptosis, as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Furthermore, donor BMMSCs(GFP) committed to Osterix (Osx)(+) osteoblast progenitors and induced recipient osteoblastogenesis, as exhibited by GFP-Osx double-labeling immunofluorescence analysis. No anticatabolic effects or systemic immunomodulatory effects of infused BMMSCs were detected. These findings demonstrated that allogeneic MSC therapy prevented GIOP by inhabiting and functioning in recipient bone marrow, which promoted osteoblastogenesis, which in turn maintained bone formation. Our findings provide important information regarding cell-based anabolic therapy for GIOP and uncover MSC behaviors following the homing event. This study revealed the therapeutic potential of systemically infused, genetically unmodified allogeneic MSCs in glucocorticoid-induced osteoporosis. The donor MSCs inhabited recipient bone marrow and promoted osteoblastogenesis. The therapeutic effects were based on maintenance of bone formation. These results provide important information regarding cell-based anabolic therapy for glucocorticoid-induced osteoporosis and uncover previously unrecognized mesenchymal stem cell behaviors following a homing event. The current study also indicates that minimizing the time of cell culture confers an advantage for increasing transplanted mesenchymal stem cells to the targeted organ to promote therapeutic effects.

The rate of in vitro fludarabine-induced peripheral blood and bone marrow cell apoptosis may predict the chemotherapy outcome in patients with chronic lymphocytic leukemia.

PurposeThe problem of drug sensitivity and predicting the outcome of chemotherapy seems to be of great importance in hemato-oncological disorders. There are some factors that can help to predict effects of chemotherapy in chronic lymphocytic leukemia (CLL), such as presence of del17p, del11q, or TP53 gene mutations, which result in resistance to purine analogues and alkylating drugs. Despite the new therapeutic options introduced recently, purine analogues in combination with cyclophosphamide and the monoclonal antibody rituximab is still the gold standard for the first-line treatment of fit patients with CLL. The aim of this study was to assess whether the rate of apoptosis caused by one of purine analogues—fludarabine in cell cultures differs between patients who clinically respond to fludarabine-based chemotherapy and those who do not respond.MethodsCLL leukemic cells, obtained from peripheral blood and bone marrow of 23 patients, were cultured in the presence of fludarabine. After 24 h of incubation, the rate of apoptosis, indicated by the expression of active caspase-3, was assessed with flow cytometry and then analyzed regarding clinical response to fludarabine-based regimens.ResultsThe percentage of apoptotic cells induced by fludarabine was significantly higher in the group of patients who achieved remission in comparison to the group with no response to purine analogues therapy. Interestingly, we observed that among the patients who did not respond to chemotherapy, the presence of del17p and del11q was detected only once. Other non-responders had no detectable genetic abnormalities.ConclusionsBased on these results, it can be presumed that in vitro drug sensitivity test, which is easy to perform, may predict the outcome of fludarabine-based chemotherapy in CLL patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s00228-015-1893-0) contains supplementary material, which is available to authorized users.

Effect of Norcantharidin on Hematopoietic Function in Leucopenia Model Rat Induced by Cyclophosphamide

To explore the effect and mechanism of norcantharidin (NCTD) on hematopoiesis function in leucopenia model rat induced by cyclophosphamide (CTX). Leucopenia model was replicated in SD rat with cyclophosphamide(CTX) and model animal was treated with NCTD. Peripheral blood and bone marrow tissue samples were collected from the rats in each experimental group. Peripheral white blood cells (WBC) were counted and analyzed by automatic blood cell analyzer. Histopathologic changes of the biopsied bone marrow tissues were observed by histopathological techniques. The cell cycle and apoptosis rate of bone marrow cells were detected by flow cytometry. Immunohistochemical method was applied to observe the expression of apoptosis-related proteins BCL-2 and BAX in bone marrow. After NCTD treatment in model rats, the WBC count of peripheral blood obviously increased, the cell structure of bone tissue significantly recovered, NCTD could promote the cell proliferation and cycle changes of bone marrow cells, inhibit the bone marrow cell apoptosis and necrosis induced with CTX, up-regulate the expression of apoptosis-related protein BCL-2 and downregulated the BAX. NCTD can stimulate the bone marrow hematopoiesis and promote recovery of peripheral white blood cell level in the leukopenia model induced by CTX, and its mechanism may be related with NCTD regulating bone marrow cell cycle and with NCTD inhibiting cell apoptosis.

Modulation of Inflammasome-Mediated Pulmonary Immune Activation by Type I IFNs Protects Bone Marrow Homeostasis during Systemic Responses to Pneumocystis Lung Infection

Although acquired bone marrow failure (BMF) is considered a T cell-mediated autoimmune disease, possible innate immune defects as a cause for systemic immune deviations in response to otherwise innocuous infections have not been extensively explored. In this regard, we recently demonstrated an important role of type I IFNs in protecting hematopoiesis during systemic stress responses to the opportunistic fungal pathogen Pneumocystis in lymphocyte-deficient mice. Mice deficient in both lymphocytes and type I IFN receptor (IFrag(-/-) mice) develop rapidly progressing BMF due to accelerated bone marrow (BM) cell apoptosis associated with innate immune deviations in the BM in response to Pneumocystis lung infection. However, the communication pathway between lung and BM eliciting the induction of BMF in response to this strictly pulmonary infection has been unclear. In this study, we report that absence of an intact type I IFN system during Pneumocystis lung infection not only causes BMF in lymphocyte-deficient mice but also transient BM stress in lymphocyte-competent mice. This is associated with an exuberant systemic IFN-γ response. IFN-γ neutralization prevented Pneumocystis lung infection-induced BM depression in type I IFN receptor-deficient mice and prolonged neutrophil survival time in BM from IFrag(-/-) mice. IL-1β and upstream regulators of IFN-γ, IL-12, and IL-18 were also upregulated in lung and serum of IFrag(-/-) mice. In conjunction, there was exuberant inflammasome-mediated caspase-1 activation in pulmonary innate immune cells required for processing of IL-18 and IL-1β. Thus, absence of type I IFN signaling during Pneumocystis lung infection may result in deregulation of inflammasome-mediated pulmonary immune activation, causing systemic immune deviations triggering BMF in this model.

Exercise promotes bone marrow cell survival and recipient reconstitution post-bone marrow transplantation, which is associated with increased survival

Bone marrow transplantation (BMT) is associated with a high risk of mortality, partially because of the harmful effects of the preconditioning myeloablative regimens. We have recently demonstrated increased bone marrow cell survival and proliferation in response to exercise training, which may be attributable to increased quality of the niche. The purpose of the present study was to determine the extent to which exercise preconditioning of recipients could increase the success of BMT. Recipient mice remained sedentary (SED) or were exercise-trained (EX) on a treadmill (3 d/wk for 8 weeks) before reconstitution with green fluorescent protein (GFP)-labeled donor marrow. Recipient survival, both donor-derived and total (donor- and recipient-derived) blood reconstitution were measured by flow cytometry. The first and fourth day after BMT apoptosis, cellularity and donor cell homing were determined in the recipients' bone marrow cavity by flow cytometry. Whereas only 25% of SED mice survived, 82% of EX recipients survived the BMT. Homing of donor-derived marrow cells to the recipients' marrow cavity acutely after BMT was not altered in EX, but EX mice displayed decreased levels (10%; p < 0.05) of activated caspase-3/-7 one day after BMT, leading to a maintenance of marrow cellularity in mice preconditioned with exercise. The acute inhibition of marrow cell apoptosis in EX mice resulted in increased total blood cell reconstitution at 1 and 3.5 months after BMT in EX mice (42% and 43%, respectively; both p < 0.05). Short- and long-term donor-derived engraftment was not different between EX and SED recipients. Exercise training increases recipient survival after BMT with increased total blood cell reconstitution.

A systematic modeling study on the pathogenic role of p38 MAPK activation in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell diseases. In addition to intrinsic genetic alterations, the effects of the extrinsic microenvironment also play a pathological role in MDS development. The presence of increased inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), in marrow and abnormal activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in hematopoietic cells are associated with the ineffective hematopoiesis in MDS. However, the molecular mechanism of p38 MAPK activation triggered by microenvironment cytokines remains poorly understood. To address this question, we combined computational modeling analysis and molecular biology studies to perform a systematic investigation of signaling events regulated by microenvironment cytokines in hematopoietic cells from MDS patients. We examined dynamic changes of key signaling events, including the p38 MAPK and the c-Jun N-terminal kinase (JNK) pathway in bone marrow mononuclear cells from MDS patients or normal donors in response to TNF-α stimulation using reverse phase protein array technology. The results were analyzed by a novel computational model and preliminarily validated by immunohistochemistry analysis of the bone marrow tissues from twelve MDS patients and normal donors. Our systematic model revealed that the dynamic response patterns of p38 MAPK and JNK to TNF-α stimulation in MDS were different from that observed in normal marrow cells. Particularly, B-cell lymphoma-X (BCL-XL) protein degradation was regulated by the JNK pathway in normal cells, but by p38 MAPK in MDS cells. By immunohistochemistry, BCL-XL was highly expressed in hematopoietic cells from normal marrow, but was minimally expressed in MDS marrow. Additionally, immunostaining for phosphorylated p38 MAPKα showed much higher p38 MAPK activation in MDS marrows, supporting over-activation of p38 MAPK-enhanced degradation of BCL-XL in MDS. The degradation of BCL-XL triggered by p38 MAPK over-activation may contribute to the increasing apoptosis of marrow cells, a phenomenon commonly observed in MDS, and lead to ineffective hematopoiesis. Our study suggests that the combination of molecular biological studies and systematic modeling is a powerful tool for comprehensive investigation of the complex cellular mechanisms involved in MDS pathogenesis.

Bystander-Type Effects Mediated by Long-Lived Inflammatory Signaling in Irradiated Bone Marrow

Radiation-induced bystander and abscopal effects, in which DNA damage is produced in nonirradiated cells as a consequence of communication with irradiated cells, indicate mechanisms of inducing damage and cell death additional to the conventional model of deposition of energy in the cell nucleus at the time of irradiation. In this study we show that signals generated in vivo in the bone marrow of mice irradiated with 4 Gy γ rays 18 h to 15 months previously are able to induce DNA damage and apoptosis in nonirradiated bone marrow cells but that comparable signals are not detected at earlier times postirradiation or at doses below 100 mGy. Bone marrow cells of both CBA/Ca and C57BL/6 genotypes exhibit responses to signals produced by either irradiated CBA/Ca or C57BL/6 mice, and the responses are mediated by the cytokines FasL and TNF-α converging on a COX-2-dependent pathway. The findings are consistent with indirect inflammatory signaling induced as a response to the initial radiation damage rather than to direct signaling between irradiated and nonirradiated cells. The findings also demonstrate the importance of studying tissue responses when considering the mechanisms underlying the consequences of radiation exposures.

Overexpression of MCM2 in myelodysplastic syndromes: Association with bone marrow cell apoptosis and peripheral cytopenia

Myelodysplastic syndromes (MDS) are characterized by proliferation and apoptosis of bone marrow cells. Minichromosome maintenance protein (MCM) 2, which is known to be essential for regulating DNA replication, has proven to have a pro-apoptotic effect in our recent study. Thus, to determine the role of MCM2 in MDS, real-time PCR, immunohistochemistry and in vitro analysis were performed. Our results showed higher MCM2 expression in MDS than in control and AML. Notably, there was no correlation between MCM2 and Ki67-labeling indices (LIs) in MDS, while MCM2 LIs were significantly correlated with cleaved caspase 3 LIs in MDS. In vitro analysis revealed that MCM2 overexpression induced apoptosis in HL60 cells. Furthermore, MDS bone marrow exhibited higher ratio of MCM2 and cleaved caspase 3 double-positive cells and the ratio was correlated with the degree of leukocytopenia. These results suggest that the up-regulated expression of MCM2 is associated with frequent apoptosis in MDS and may have an important role in the pathogenesis of MDS.

Prevention of Bone Marrow Cell Apoptosis and Regulation of Hematopoiesis by Type I IFNs during Systemic Responses to Pneumocystis Lung Infection

We recently demonstrated that lack of type I IFN signaling (IFNAR knockout) in lymphocyte-deficient mice (IFrag(-/-)) results in bone marrow (BM) failure after Pneumocystis lung infection, whereas lymphocyte-deficient mice with intact IFNAR (RAG(-/-)) had normal hematopoiesis. In the current work, we performed studies to define further the mechanisms involved in the induction of BM failure in this system. BM chimera experiments revealed that IFNAR expression was required on BM-derived but not stroma-derived cells to prevent BM failure. Signals elicited after day 7 postinfection appeared critical in determining BM cell fate. We observed caspase-8- and caspase-9-mediated apoptotic cell death, beginning with neutrophils. Death of myeloid precursors was associated with secondary oxidative stress, and decreasing colony-forming activity in BM cell cultures. Treatment with N-acetylcysteine could slow the progression of, but not prevent, BM failure. Type I IFN signaling has previously been shown to expand the neutrophil life span and regulate the expression of some antiapoptotic factors. Quantitative RT-PCR demonstrated reduced mRNA abundance for the antiapoptotic factors BCL-2, IAP2, MCL-1, and others in BM cells from IFrag(-/-) compared with that in BM cells from RAG(-/-) mice at day 7. mRNA and protein for the proapoptotic cytokine TNF-α was increased, whereas mRNA for the growth factors G-CSF and GM-CSF was reduced. In vivo anti-TNF-α treatment improved precursor cell survival and activity in culture. Thus, we propose that lack of type I IFN signaling results in decreased resistance to inflammation-induced proapoptotic stressors and impaired replenishment by precursors after systemic responses to Pneumocystis lung infection. Our finding may have implications in understanding mechanisms underlying regenerative BM depression/failure during complex immune deficiencies such as AIDS.

Myelodysplastic Syndromes: Dissecting the Heterogeneity

Myelodysplastic Syndromes: Dissecting the Heterogeneity