A comparative study of the kinetic changes of hemopoietic stem cells in mice infected with lethal and non-lethal malaria.

The kinetic changes of hemopoietic stem cells in bone marrow and spleen were compared between lethal Plasmodium berghei- and non-lethal P. yoelii 17x-infected mice. P. yoelii 17x-infected mice showed more severe splenomegaly than those infected with P. berghei. P. yoelii 17x-infected mice also showed a greater degree of sustained increase in number of multipotent hemopoietic stem cells (colony-forming units in spleen: CFU-S) and committed stem cells for granulocytes and macrophages (CFU-GM) and for erythrocytes (CFU-E) than P. berghei-infected mice. Such an increase was predominantly seen in the spleen of P. yoelii 17x-infected mice. In P. berghei-infected mice, the number of CFU-S, CFU-GM and also CFU-E only transiently increased and then decreased to a subnormal level at the late stage of infection. The proportion of cycling CFU-S was higher in P. berghei-infected mice than in P. yoelii 17x-infected mice. The IL-3 producing activity per spleen was much higher in P. yoelii 17x-infected than in P. berghei-infected mice at any point in time during the infection. Thus, hemopoietic changes seen after malaria infection seem to be closely related to the pathogenicity of the malaria parasite.     

Effects of okadaic acid on mouse hemopoietic cells

Effects of okadaic acid, a potent non-12-O-tetradecanoyl-phorbol-13-acetate(TPA)-type tumor promoter, on mouse hemopoietic cells were investigated. Okadaic acid stimulated mouse bone marrow cells to form granulocyte-macrophage colony-forming unit (CFU-GM) colonies without added colony stimulating factors(CSFs). At the concentration of 1.82 x 10(-8) M, colony formation of 77 +/- 14 colonies/1 x 10(5) bone marrow cells was observed. Observations on the effects of other cells on the CSF induction suggested that okadaic acid primarily stimulated the functions of macrophages, and the CSF production from macrophages might be attributed to the CFU-GM colony formation. On the other hand, the erythroid colony-forming unit(CFU-E) colony formation stimulated by     

造血细胞活力冷冻损伤的可恢复性

人骨髓冻存后其造血祖细胞活力有一定程度下降,本研究对这种下降的可逆性作了初步观察。结果发现,用双层法和单层法作CFU-GM培养时,未冻存骨髓集落产率相近,冻存骨髓双层法的CFU-GM产率高于单层法。骨髓细胞用20％FM-CM、PHA-LYCM、PHA-PMCM预孵育2h后,分别测定其CFU-GM、BFU-E与CFU-Mix,发现这种孵育过程对未冻存骨髓的集落产率无明显影响,而冻存骨髓的集落产率在孵育后可升高(GEMmeg除外)。说明骨髓造血祖细胞对冻存的损伤反应不均一,部分受损细胞在一定条件下可以恢复其增殖活力。这对于用冻存骨髓作骨髓移植可能有一定意义。     

Role of humoral factors in the regulation of hemopoiesis in stress]

The change of 11-1, IL-3, CSA concentrations in adherent and nonadherent bone marrow cells condition medium at stress were investigated. The activation of bone marrow hemopoiesis was registered at mice after immobilization stress. The number of CFU-GM increased on 1, 4 and 5 day after stress. Maximum of CSA in adherent and nonadherent cells conditioned medium was observed on day 4, 6 or 2, 5 respectively. The increasing of 11-3 activity in culture of nonadherent bone marrow cells was registered from day 1 and mount to maximum at 4-5 days. The increasing of 11-1 level in culture of adherent bone marrow cells was found at 1 and 4 days.     

TLR4在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达

目的：检测Toll样受体4（TLR4）在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达,为进一步研究肝硬化时骨髓损伤的发生机制提供实验依据。方法：选择Wistar大鼠给予腹腔注射CCl4,一周两次,建立肝硬化大鼠模型。分别于建模8周和12周检测大鼠血浆内毒素的水平,免疫组化检测大鼠骨髓血窦内皮细胞上TLR4的表达情况,RT-PCR测定骨髓组织中TLR4mRNA的表达,分析TLR4的表达与内毒素血症间的关系。结果：给予CCl4 8周和12周时,对照组大鼠血浆内毒素水平分别为（0．216±0．024）Eu／ml和（0．133±0．022）Eu／ml,模型组大鼠血浆内毒素水平分别为（0．626±0．021）Eu／ml和（0．725±0．031）Eu／ml,分别较对照组显著升高,差异均有统计学意义（P〈0．001）;骨髓血窦内皮细胞TLR4蛋白表达及骨髓组织中TLR4mRNA的表达均显著高于对照组,差异均有统计学意义（P〈0．05）。大鼠骨髓TLR4蛋白和mRNA表达与血浆内毒素水平均呈显著正相关（r=0．841,0．803,P均〈0．001）。结论：CCl4诱导的肝硬化大鼠骨髓血窦内皮细胞TLR4表达升高,并伴随大鼠内毒素血症的发生,提示肝硬化时肠源性内毒素血症可能参与了骨髓的造血功能的损害和病变。     

TLR4 在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达

目的：检测Toll样受体4(TLR4)在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达,为进一步研究肝硬化时骨髓损伤 的发生机制提供实验依据。方法：选择Wistar大鼠给予腹腔注射CCl4,一周两次,建立肝硬化大鼠模型。分别于建模8 周和12 周 检测大鼠血浆内毒素的水平,免疫组化检测大鼠骨髓血窦内皮细胞上TLR4 的表达情况,RT-PCR 测定骨髓组织中TLR4 mRNA 的表达,分析TLR4 的表达与内毒素血症间的关系。结果：给予CCl4 8 周和12 周时,对照组大鼠血浆内毒素水平分别为(0.216± 0.024) Eu/ml 和(0.133± 0.022) Eu/ml,模型组大鼠血浆内毒素水平分别为(0.626± 0.021) Eu/ml 和(0.725± 0.031) Eu/ml,分别较对 照组显著升高,差异均有统计学意义(P<0.001);骨髓血窦内皮细胞TLR4蛋白表达及骨髓组织中TLR4 mRNA的表达均显著高于 对照组,差异均有统计学意义(P<0.05)。大鼠骨髓TLR4 蛋白和mRNA表达与血浆内毒素水平均呈显著正相关（r=0.841,0.803,P 均<0.001）。结论：CCl4 诱导的肝硬化大鼠骨髓血窦内皮细胞TLR4 表达升高,并伴随大鼠内毒素血症的发生,提示肝硬化时肠源 性内毒素血症可能参与了骨髓的造血功能的损害和病变。     

Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and long-term cultures.

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.     

Long-term maintenance of multilineal hemopoiesis in collagen gel culture.

We examined the long-term maintenance of multilineal hemopoiesis in a collagen gel culture of mouse bone marrow cells. When cells were inoculated into the gel, stromal cells formed foci that were composed of sinusoidlike capillary structures, fibroblastic cells, adipocytes and macrophages. Many small hemopoietic foci similar to granulocyte-macrophage colonies (CFU-GM) appeared within a week and disappeared after two weeks. Several large hemopoietic foci appeared after two to three weeks of culture, without a second challenge of marrow cells. These large hemopoietic foci were composed mainly of myeloid cells. Megakaryocytes and mast cells were also observed. When erythropoietin (EPO) was added to the culture at the beginning, the erythroid focus appeared after 3 weeks and the number of megakaryocytes was greater than that in the culture without EPO. However, when EPO was added to the cultures after 6 or 12 weeks, erythroid cells appeared after 1 week and the number of megakaryocytes increased. This hemopoiesis lasted more than 6 months.     

小鼠胎肝和骨髓造血基质细胞贴壁层对红系祖细胞生长的影响

本实验以Dexter培养体系作小鼠胎肝和骨髓造血基质细胞贴壁培养。在所获的基质细胞贴壁层上作红系造血祖细胞集落培养,观察两种来源造血基质细胞对红系集落生长的影响。实验结果表明,胎肝造血基质细胞贴壁层能明显促进早期红系造血祖细胞(BFU-E)形成集落,却不明显影响晚期红系造血祖细胞(CFU-E)的生长。成年小鼠骨髓造血基质细胞贴壁层对BFU-E和CFU-E均有刺激生长的作用;但对前者生长的刺激性影响较胎肝造血基质细胞贴壁层为弱。造血基质细胞贴壁层对红系集落生长的促进作用主要是通过体液因子实现的,细胞间短距离调节的影响亦不能除外。     

Effect of Danggui Buxue Tang on immune-mediated aplastic anemia bone marrow proliferation mice

To investigate the pharmacological effects of Danggui Buxue Tang (DBT) on immune-mediated aplasia anemia mice. The model of immune-mediated aplasia anemia mice was induced by means of ⁶⁰Co γ-ray irradiation and mixed cells of thymus and lymphnode of DBA/2 mice infusion through tail vein, the parameters tested indices were as following: blood picture, bone marrow nucleated cell count (BMNC), murine colony-forming unit-megakaryocytes (CFU-GM) of bone marrow cells, murine colony-forming unit-erthroid (CFU-E) and burst forming unit-erythroid (BFU-E). The results showed that DBT could not only withstand significantly decreation of blood cells by immune-mediated, but also stimulate on the growth of bone marrow colony cell and increase the weight of hemopoietic progenitor of bone marrow. Therefore, DBT had an obvious treat effect on immune-mediated aplasia anemia models mice.     

Retrovirus-induced feline pure red cell aplasia: the kinetics of erythroid marrow failure

Cats viremic with feline leukemia virus subgroup C (FeLV-C) develop pure red cell aplasia (PRCA) characterized by the loss of detectable late erythroid progenitors (CFU-E) in marrow culture. Normal numbers of early erythroid progenitors (BFU-E) and granulocyte-macrophage progenitors (CFU-GM) remain, suggesting that the maturation of BFU-E to CFU-E is impaired in vivo. We have examined the cell cycle kinetics of BFU-E and their response to hematopoietic growth factor(s) to better characterize erythropoiesis as anemia develops. Within 3 weeks of FeLV-C infection, yet 6-42 weeks before anemia, the traction of BFU-E in DNA synthesis as determined by tritiated thymidine suicide increased to 43 +/- 4% (normal 23 +/- 2%) while there was no change in the cell cycle kinetics of CFU-GM. In additional studies, we evaluated the response of marrow to the hematopoietic growth factor(s) present in medium conditioned by FeLV-infected feline embryonic fibroblasts (FEA/FeLV CM). With cells from normal cats or cats viremic with FeLV-C but not anemic, a 4-fold increase in erythroid bursts was seen in cultures with 5% FEA/FeLV CM when compared to cultures without CM. However, just prior to the onset of anemia, when the numbers of detectable CFU-E decreased, BFU-E no longer responded to FEA/FeLV CM in vitro. BFU-E from anemic cats also required 10% cat or human serum for optimal in vitro growth. These altered kinetics and in vitro growth characteristics may relate to the in vivo block of BFU-E differentiation and PRCA. Finally, when marrow from cats with PRCA was placed in suspension culture for 2 to 4 days in the presence of cat serum and CM, the numbers of BFU-E increased 2- to 4-fold although no CFU-E were generated. By 4 to 7 days, CFU-E were detected, suggesting that conditions contributing to the block of erythroid maturation did not persist. The suspension culture technique provides an approach to study further the defect in erythroid differentiation characteristic of feline PRCA.     

In vivo effects of interleukin-17 on haematopoietic cells and cytokine release in normal mice

In order to gain more insight into mechanisms operating on the haematopoietic activity of the T-cell-derived cytokine, interleukin-17 (IL-17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL-17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL-6, IL-10, IGF-I, IFN-gamma and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL-17 did not affect granulocyte-macrophage (CFU-GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU-E) progenitors was increased at 48 h, while the number of mature erythroid (CFU-E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL-17 also increased IL-6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin- progenitor cells, demonstrated a slightly enhancing effect of IL-17 on CFU-GM and no influence on BFU-E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL-17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL-17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU-E found at the same time indicate that IL-17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.     

Hoechst 33342 staining of mouse bone marrow: effects on colony-forming cells

We have systematically studied the effect on hemopoietic colony-forming cells of staining cellular DNA with the bisbenzimidazole dye, Hoechst 33342. Mouse bone marrow cells could be adequately stained in a 30-60 min incubation with a 5 microM concentration of stain. Flow-cytometric analysis of stained cells provided cell distributions with coefficients of variation for the G1 peaks of 6% or less under these conditions. We found considerable heterogeneity among hemopoietic colony-forming cells with respect to the toxicity of the dye. Toxicity in the proliferatively quiescent stem cell population was not changed when the population became proliferatively active. In the sequence of most sensitive to least sensitive, the five progenitors studied could be arranged as follows: CFU-M, a megakaryocyte colony-forming cell; CFU-E, a relatively differentiated erythroid precursor; BFU-E, a primitive erythroid precursor; CFU-GM, a granulocyte-macrophage precursor; and CFU-S, the spleen colony-forming cell or hemopoietic stem cell. A staining procedure involving a 30-min exposure to 5 microM Hoechst 33342 provided optimal staining and no loss in four of the five progenitor populations; the CFU-M population was diminished by about 50%. We conclude that Hoechst can be regarded as a vital DNA stain for most bone marrow precursor populations, including the hemopoietic stem cell.     

小鼠胎肝基质细胞造血刺激因子体外生物活性研究

本实验对基质细胞造血刺激因子－１（ＳＨＦ－１）的体外生物活性进行了研究。结果表明,ＳＨＦ－１可刺激小鼠骨髓ＣＦＵ－Ｅ、ＢＦＵ－Ｅ、ＣＦＵ－ＧＭ、ＣＦＵ－Ｍｉｘ集落的形成,它产生的这些广泛造血刺激作用是其自身所具活性的直接影响。正常小鼠骨髓细胞与ＳＨＦ－１在体外孵育４ｈ,其中ＣＦＵ－Ｓ的自杀率可提高约１０％,显示它对造血干细胞也有诱导增殖作用。     

Collection and freezing of blood stem cells in acute nonlymphoblastic leukemia

Blood-derived hemopoietic stem cells were collected using a continuous (10 patients) or a semi-continuous flow separator (2 patients) in some patients with acute non-lymphocytic leukemia. For ten out of those patients, five to seven leukaphereses were performed during a short period (median = 11 days) of marrow recovery following severe aplasia induced by an intensive chemotherapy. The mean number of CFU-GM cells collected per leukapheresis and per patient respectively was 6.7 X 10(4)/kg and 38.5 X 10(4)/kg. This latter number was similar to that obtained during a marrow harvest performed for a bone marrow transplantation, suggesting that high numbers of hemopoietic stem cells can be collected from the peripheral blood in leukemic patients and used for autologous transplantation.     

Increased therapeutic efficacy of zidovudine in combination with vitamin E

Antiviral activity and bone marrow toxicity of 3'-azido-3'deoxythymidine (Zidovudine; AZT) was evaluated in the presence of alpha-D-tocopherol acid succinate (ATS) in the MT4 cell line and in murine hematopoietic progenitor cells, respectively. At varying concentrations (.016 to .125 microM) of AZT, addition of ATS (5 to 15 micrograms/ml) showed a dose-dependent increase in anti-HIV activity. The ED90 of AZT in this test system was 0.37 microM, whereas in the presence of ATS (15 micrograms/ml) it was 0.06 microM, thus producing an approximately 6-fold increase in anti-HIV activity. In contrast, in murine bone marrow cells, ATS (4 micrograms/ml) showed significant protection (p less than 0.05) against AZT-induced toxicity as measured by CFU-E and CFU-GM assays. The IC50 values in the presence and absence of ATS for CFU-E were 3.7 and 1.5 microM, whereas for CFU-GM were 6.0 and 2.7 microM, respectively. Overall, these data suggest that AZT in combination with ATS has greater therapeutic efficacy against HIV-1.     

The effect of interleukin-17 on hematopoietic cells and cytokine release in mouse spleen

To evaluate whether the response of hematopoietic cells to interleukin-17 (IL-17) depends on the tissue microenvironment in which hematopoiesis occurs, the influence of recombinant mouse IL-17 on spleen hematopoietic cells and cytokine release was assessed in normal mice in vitro and in vivo. In vitro, IL-17 did not significantly affect the growth of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) derived colonies. A single injection of IL-17 in vivo exhibited stimulatory effects on hematopoietic cells from both granulocytic and erythroid lineages. The increased number of metamyelocytes 48 h after treatment imply to the IL-17-induced stimulation of granulopoiesis. The number of BFU-E was increased at 24 h, while the number of CFU-E increased 6 h and 24 h after treatment. Since the same treatment in the bone marrow decreased the number of CFU-E, it may be concluded that the local microenvironment plays an important role in IL-17-mediated effects on CFU-E. IL-17 increased the release of IL-6 both in vitro and in vivo, but showed tendency to suppress the constitutive secretion of IL-10 by spleen cells. Our results suggest the complexity of target cell response and interplay of secondary induced cytokines by IL-17 in different hematopoietic organs.     

A model of hemopoietic stress in a lactate dehydrogenase mouse mutant with hemolytic anemia

A codominantly inherited mutation of the lactate dehydrogenase (LDH) in the C3H mouse causes a severe hemolytic anemia in homozygous mutants, whereas viability and fertility are close to normal. Investigation of multipotent hemopoietic stem cells (CFU-S), myeloid (GM-CFC) and erythroid progenitors (BFU-E, CFU-E) in femur and spleen indicates a general shift from bone marrow to splenic hemopoiesis. In terms of total body hemopoiesis, however, the BFU-E pool is 1.4- and the CFU-E pool 19-fold enlarged, whereas CFU-S and GM-CFC show little or no deviation from normal. It is concluded that this mouse mutant is an appropriate model of long-term hemopoietic stress showing that compensation in this severe hemolytic anemia is achieved primarily by an increase of the number of the most mature erythroid progenitors.     

Thy-1 is a differentiation antigen that characterizes immature murine erythroid and myeloid hematopoietic progenitors

To determine the role of Thy-1 antigen in murine hematopoietic differentiation, bone marrow was treated with anti-Thy-1.2 antibody and complement or complement alone. Growth of immature hematopoietic progenitors, erythroid burst-forming units (BFU-E), and granulocyte/macrophage colony-forming units (CFU-GM) was greatly reduced following antibody and complement treatment and was not restored by mitogen-stimulated spleen cell supernatants. In contrast, more mature erythroid and myeloid progenitors, the erythroid colony-forming unit (CFU-E) and the macrophage progenitor stimulated by L-cell-conditioned media (LCM), were spared by anti-Thy-1.2 antibody and complement treatment. Here, to separate the effects of anti-Thy-1.2 antibody treatment on accessory cells from those on progenitors, splenic T cells and thymocytes were added to treated marrow at ratios of up to 200%. Growth of BFU-E and CFU-GM was not restored. To more precisely replace required accessory cells, male complement-treated marrow was cocultured with female anti-Thy-1.2 antibody and complement-treated marrow. Even marrow cells failed to restore female BFU-E and CFU-GM growth. Fluorescent-activated cell sorting (FACS) and immune sheep red cell rosetting with anti-Thy-1.2-labeled marrow were then performed to determine if immature hematopoietic progenitors bear Thy-1.2. These techniques revealed enrichment of BFU-E and CFU-GM in the Thy-1.2-positive fraction, demonstrating the presence of Thy-1.2 on early murine hematopoietic progenitors. CFU-E and CFU-M were present in the Thy-1.2-negative fraction following FACS separation. These data demonstrate that Thy-1.2 is a differentiation antigen, present on at least some murine BFU-E and CFU-GM and lost as they mature to CFU-E and CFU-M.     

Characterization of erythropoietin-like activity from mouse spleen cells

Supernatants from mouse spleen cell cultures contain a factor which acts in a similar manner to erythropoietin (Ep) to stimulate the formation of 2-day erythroid (CFU-E) colonies in vitro from bone marrow or fetal liver cells. Analysis of conditioned media by high performance liquid chromatography (HPLC) on anion exchange, reverse phase, molecular size exclusion, and hydroxyapatite columns demonstrated that the erythropoietin-like activity (EpLA) has different biochemical characteristics to mouse Ep from anemic mouse serum. In addition, EpLA has a molecular weight (Mr), of 20,000 daltons determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), compared to 42,000 for mouse Ep. Partially purified EpLA was found to be active in vivo as well as in vitro. Highly purified preparations of gamma-interferon, Multilineage hemopoietic growth factor (Multi HGF), Interleukin-2 (IL-2), IL-1, and colony stimulating factor 1 (CSF-1) did not support CFU-E colony formation. Thus, it was established that EpLA could not be attributed to other known components of spleen cell conditioned medium. Titration of mouse Ep and EpLA suggests that only a portion of the Ep-responsive CFU-E population in fetal liver is sensitive to EpLA.