Bone Marrow Hemopoietic Cells Research Articles

The Role of Reactive Oxygen Species in Tumor Treatment and its Impact on Bone Marrow Hematopoiesis.

Reactive oxygen species (ROS), an important molecule inducing oxidative stress in organisms, play a key role in tumorigenesis, tumor progression and recurrence. Recent findings on ROS have shown that ROS can be used to treat cancer as they accelerate the death of tumor cells. At present, pro-oxidant drugs that are intended to increase ROS levels of the tumor cells have been widely used in the clinic. However, ROS are a double-edged sword in the treatment of tumors. High levels of ROS induce not only the death of tumor cells but also oxidative damage to normal cells, especially bone marrow hemopoietic cells, which leads to bone marrow suppression and (or) other side effects, weak efficacy of tumor treatment and even threatening patients' life. How to enhance the killing effect of ROS on tumor cells while avoiding oxidative damage to the normal cells has become an urgent issue. This study is a review of the latest progress in the role of ROS-mediated programmed death in tumor treatment and prevention and treatment of oxidative damage in bone marrow induced by ROS.

Roles of Notch Glycoslation in Signaling

Notch receptors are single pass transmembrane glycoproteins with an extracellular domain (ECD) comprised of many epidermal growth factor‐like (EGF) repeats. Specific Ser and Thr residues in numerous EGF repeats have an attached O‐glycan initiated by fucose, glucose, or GlcNAc, and elongated with 1–3 more sugars. Notch receptors signal in response to the binding of Notch ligands to Notch ECD. Notch signaling regulates cell fate decisions in many organs during development and differentiation, and its dysregulation leads to developmental disorders and cancer. A point mutation that precludes the addition of a single O‐fucose glycan to EGF12 in the NOTCH1 ligand binding domain alters Notch signaling differently depending on genetic background. In a mixed genetic background a mild effect on T and B cell development is observed, whereas in the C57BL/6J background, lack of the EGF12 O‐fucose glycan is embryonic lethal. Lunatic (LFNG), Manic (MFNG) and Radical (RFNG) Fringe are GlcNAc transferases that modify Notch EGF repeats which already carry a fucose. We are investigating roles for each Fringe in T and B cell development using mice lacking two or three Fng genes. We are also identifying individual roles for Fng genes using a co‐culture assay containing OP9 stromal cells expressing one of the four Notch ligands, DLL1, DLL4, JAG1 or JAG2 and bone marrow hemopoietic cells from mice expressing all Fng genes, no Fng genes (tKO) or a single Fng gene. The combined results show that the O‐fucose glycans on NOTCH1 are key regulators of the strength of NOTCH1 signaling induced by canonical Notch ligands.Support or Funding InformationNational Institutes of Health grant RO1 GM106417National Institutes of Health grant PO1 CA13330

Inhibitory effects of vitamin E on osteocyte apoptosis and DNA oxidative damage in bone marrow hemopoietic cells at early stage of steroid-induced femoral head necrosis.

Apoptosis and DNA oxidative damage serve significant roles in the pathogenesis of steroid-induced femoral head necrosis. Vitamin E demonstrates anti-apoptotic and anti-oxidant properties. Therefore, the present study investigated the effects of vitamin E on osteocyte apoptosis and DNA oxidative damage in bone marrow hemopoietic cells at an early stage of steroid-induced femoral head osteonecrosis. Japanese white rabbits were randomly divided into three groups (steroid, vitamin E-treated, and control groups), each comprising 12 rabbits. Those in the steroid group (group S) were initially injected twice with an intravenous dose of 100 µg/kg Escherichia coli endotoxin, with a 24 h interval between the two injections, and then with an intramuscular dose of 20 mg/kg methylprednisolone, three times at intervals of 24 h in order to establish a rabbit model of osteonecrosis. The vitamin E treated group (group E) received the same treatment as group S, and were administered 0.6 g/kg/d vitamin E daily from the beginning of modeling. The control group (group C) was injected with normal saline at the equivalent dosage and times as the aforementioned two groups. Two time points, weeks 4 and 6 following the completion of modeling, were selected. Osteonecrosis was verified by histopathology with hematoxylin-eosin staining. The apoptosis rate of osteonecrosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The apoptosis expression levels of caspase-3 and B-cell lymphoma 2 (Bcl-2), and DNA oxidative damage of bone marrow hematopoietic cells were analyzed by immunohistochemistry. At weeks 4 and 6 following the completion of modeling, the vacant bone lacunae rates of group E were 15.87±1.97 and 25.09±2.67%, respectively, lower than the results of 20.02±2.21 and 27.79±1.39% for group S; and the osteocyte apoptosis indexes of group E were 20.99±2.95 and 33.93±1.62%, respectively, lower than the results of 26.46±3.37 and 39.90±3.74% from group S. In addition, the Bcl-2 expression at week 4 in the femoral head tissues of group E was higher compared with group S; and the proportion of Bcl-2-positive cells of group E was 9.81±1.01%, higher compared with group S at 8.26±1.13%. The caspase-3 staining data at week 4 in femoral head tissues demonstrated that in the 12 femoral heads of group S, four were negative (32%) and eight were positive (68%); in group E, five were negative (45%) and seven were positive (55%); and in group C, 11 were negative (95%) and one was positive (5%). In addition, the DNA oxidative damage rate at week 4 in the bone marrow hemopoietic cells of group E was (7.24±1.44%), lower compared with group S (11.80±1.26%), and higher compared with group C (5.75±1.47%). Vitamin E is effective in intervening in apoptosis through decreasing caspase-3 expression and upregulating Bcl-2 expression, and by alleviating DNA oxidative damage in bone marrow hemopoietic cells at the early stage of steroid-induced femoral head necrosis in rabbit models.

IgG autoantibody subclasses altered in immuno-related hemocytopenia

Recently, we have detected autoantibodies to bone marrow (BM) hemopoietic cells in some idiopathic cytopenia of undetermined significance (ICUS), terming as immunorelated hemocytopenia. Immunoglobulin G consists of 4 subclasses which have different biological functions in many diseases. In this study, we analyzed the alterations of IgG subclasses in 27 IRH patients compared with 20 ICUS patients and 20 normal controls. The results showed that IgG1 and IgG3 levels were increased in the bone marrow supernatant in IRH patients, and had inverse correlations with hematopoietic function. Meanwhile IgG1 level had a positive correlation with the proportion of CD5+ B lymphocytes. Using immunohistochemical staining, IgG1 were also detected on bone marrow nucleated cells in IRH patients. All these results indicated that IgG1 on bone marrow cells in some IRH patients might be involved in the destruction of hematopoietic cells leading to cytopenia and might be a novel therapeutic target in future.

Erythropoietin Receptors and IgG Autoantibody Expression on Nucleated Erythrocytes in Some Cases of Immuno-Related Pancytopenia.

In recent years, we have detected autoantibodies in bone marrow (BM) hemopoietic cells in some patients with idiopathic cytopenia of undetermined significance (ICUS), termed immunorelated pancytopenia (IRP). However, we know little about the targets of these autoantibodies. Twenty-six newly diagnosed IRP patients with IgG autoantibody on nucleated erythrocytes and 20 healthy donors as controls were enrolled in this study. The serum erythropoietin (EPO) level was examined by ELISA. Expression of EPO receptor (EPOR) and IgG autoantibody on the membrane of nucleated erythrocytes were detected by flow cytometry before and after autoantibody stripping. The serum EPO level of the untreated patients was 199.9 ± 106.4 mIU/mL, which was significantly higher than that of normal controls (13.2 ± 8.41 mIU/mL, p < 0.01). EPOR expression on nucleated erythrocytes in the patients was 1.38 ± 0.73%, lower than that of normal controls (2.33 ± 1.73%), but there was no significant difference; EPOR in these patients was inversely correlated with IgG autoantibody on erythrocytes (r = -0.479, p = 0.013). The regression equation was Y = 0.116-0.479X; EPOR expression on the membrane increased significiantly (5.63 ± 4.99%, p < 0.01) after stripping the autoantibodies. After immunosuppressive treatment, median hemoglobin increased from 72 g/L to 98 g/L, and median reticulocytes increased from 1.46% to 3.56%. IgG autoantibodies might block or competitively inhibit EPOR on nucleated erythrocytes in some cases of ICUS.

Effect of spiperone on mesenchymal multipotent stromal and hemopoietic stem cells under conditions of pulmonary fibrosis.

The antifibrotic properties of spiperone and its effect on stem and progenitor cells were studied on the model of reversible bleomycin-induced pulmonary fibrosis in C57Bl/6 mice. Spiperone reduced infiltration of the alveolar interstitium and alveolar ducts with inflammatory cells and prevented the growth of the connective tissue in the parenchyma of bleomycin lungs. Apart from anti-inflammatory effect, spiperone suppressed bone marrow hemopoietic cells (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b (Mac1), TER-119)-, Sca-1+, c-Kit+, CD34- and progenitor hemopoietic cells (granulocyte-erythroid-macrophage-megakaryocytic and granulocyte CFU). Spiperone-induced disturbances of fi brogenesis were paralleled by restoration of endothelial cells in the lung parenchyma, reduction of the number of circulating bone marrow cells and lung mesenchymopoietic cells (mesenchymal multipotent stromal cells (CD31-, CD34-, CD45-, CD44+, CD73+, CD90+, CD106+) and progenitor fi broblast cells), and suppression of multilineage differentiation of multipotent mesenchymal stromal cells (including fi broblast-lineage cells).

Detection and analysis of autoantigens targeted by autoantibodies in immunorelated pancytopenia.

Previously, we described a group of patients with hemocytopenia who did not conform to diagnostic criteria of known hematological and nonhematological diseases. Most patients responded well to adrenocortical hormone and/or high-dose intravenous immunoglobulin treatment, indicating that cytopenia might be mediated by autoantibodies. Autoantibodies were detected on the membrane of various bone marrow (BM) hemopoietic cells by bone marrow mononuclear-cell-Coombs test or flow cytometric analysis. Thus, the hemocytopenia was termed “Immunorelated Pancytopenia” (IRP) to distinguish it from other pancytopenias. Autoantigens in IRP were investigated by membrane protein extraction from BM hemopoietic cells and BM supernatant from IRP patients. Autoantibody IgG was detected in the BM supernatant of 75% of patients (15/20), which was significantly higher than that in aplastic anemia, myelodysplastic syndrome, or autoimmune hemolytic anemia patients (0%) and normal healthy controls (0%) (P < 0.01). Autoantigens had approximate molecular weights of 25, 30, 47.5, 60, 65, 70, and 80 kDa, some of which were further identified by mass fingerprinting. This study identified that a G-protein-coupled receptor 156 variant and chain P, a crystal structure of the cytoplasmic domain of human erythrocyte band-3 protein, were autoantigens in IRP. Further studies are needed to confirm the antigenicity of these autoantigens.

Increased frequency of bone marrow T follicular helper cells in patients with immune-related pancytopenia.

Immune-related pancytopenia (IRP) is one kind of bone marrow failure diseases which is related to autoantibodies. Autoantibodies have been detected on the membrane of various bone marrow (BM) hemopoietic cells by BM mononuclear-cell-Coombs test or flow cytometric analysis. There are autoantibodies in the BM supernatant of IRP patients, which can target several antigens on hematopoietic cells membranes by western blot. T follicular helper (Tfh) cells are the true helper cells for Ab responses, which represent one of the most numerous and important subsets of effector T cells. Dysregulation of Tfh cell function or expression of Tfh cell-associated molecules could contribute to the pathogenesis of autoimmune diseases. Currently, there are no studies regarding the role of Tfh cells in IRP patients. The percentages of Tfh cells, Tfh-related molecules ICOS, CD40L, IL-21, and Bcl-6 in BM were investigated in 90 patients with IRP, and 25 healthy controls. We observed that there exist increased quantity and hyperfunction of Tfh cells in IRP, and the results were correlated with patient characteristics. It was indicated that dysregulated Tfh cells might be involved in the pathogenesis of IRP and that inhibition of Tfh cells effector molecules might provide opportunities for new therapeutic approaches to IRP and even other human autoimmune diseases.

Phenotype of Peripheral Blood Cells Mobilized by Granulocyte Colony-Stimulating Factor in Patients with Chronic Heart Failure

Administration of granulocyte CSF preparation to patients with chronic heart failure produced a hemostimulating effect and increased the content of leukocytes and neutrophils in the peripheral blood. Granulocyte CSF induced mobilization of bone marrow progenitor cells into the peripheral blood. The content of hemopoietic CD34(+)progenitor cells, which attained 0.42% (0.25-0.64) by the end of mobilization, inversely correlated with the number of myocardial infarctions. Administration of granulocyte CSF not only led to mobilization of bone marrow hemopoietic cells, but also increased the pool of endothelial progenitor cells in the peripheral blood: the content of CD34(+)/CD133(+)and CD34(+)/KDR(+)attained 0.02% (0.013-0.075) and 0.1% (0.05-0.20), respectively. Peripheral blood is an available source of progenitor cells, while mononuclear cells after administration of granulocyte CSF can produce a reparative effect on ischemic myocardium.

Computerized texture analysis of atypical immature myeloid precursors in patients with myelodysplastic syndromes: an entity between blasts and promyelocytes

BackgroundBone marrow (BM) blast count is an essential parameter for classification and prognosis of myelodysplastic syndromes (MDS). However, a high degree of cell atypias in bone marrow hemopoietic cells may be found in this group of clonal disorders, making it difficult to quantify precisely myeloblasts, and to distinguish them from promyelocytes and atypical immature myeloid precursors. Our aim was to investigate whether computerized image analysis of routine cytology would help to characterize these cells.MethodsIn May-Grünwald-Giemsa stained BM smears of 30 newly diagnosed MDS patients and 19 cases of normal BM, nuclei of blasts and promyelocytes were digitalized and interactively segmented. The morphological classification of the cells was done by consensus of two observers. Immature granulocytic precursors, which could not be clearly classified either as blasts or promyelocytes, were called "atypic myeloid precursors". Nuclear morphometry and texture features derived from the co-occurrence matrix and fractal dimension (FD) were calculated.ResultsIn normal BM, when compared to myeloblasts, nuclei of promyelocytes showed significant increase in perimeter and local texture homogeneity and a decrease in form factor, chromatin gray levels, Haralick's entropy, inertia, energy, contrast, diagonal moment, cluster prominence, the fractal dimension according to Minkowski and its goodness-of-fit. Compared to normal myeloblast nuclei, the chromatin texture of MDS myeloblasts revealed higher local homogeneity and goodness-of-fit of the FD, but lower values of entropy, contrast, diagonal moment, and fractal dimension. The same differences were found between nuclei of normal promyelocytes and those of MDS. Nuclei of atypical myeloid precursors showed intermediate characteristics between those of blasts and promyelocytes according to the quantitative features (perimeter, form factor, gray level and its standard deviation), but were similar to promyelocytes according to the texture variables inertia, energy, contrast, diagonal moment, cluster prominence, and Minkowski's fractal dimension.ConclusionBM atypical immature myeloid precursors are difficult to be correctly classified in routine cytology. Although their cytoplasm is more similar to that of myeloblasts, computerized texture analysis indicates a nuclear chromatin remodeling more close to the promyelocyte, thus indicating an asynchronous intermediate maturation stage between blast and promyelocyte.

Effects of lactational exposure of olanzapine and risperidone on hematology and lymphoid organs histopathology: A comparative study in mice neonates

Body weight gain, sexual/reproductive dysfunction and hematological abnormalities are serious consequences of atypical antipsychotics treatment. No attempts however have been made preclinically to elucidate the adverse hematological impacts. Presently, effects of lactational exposure of olanzapine (4, 8 and 10 mg/kg) and risperidone (1 and 2 mg/kg) on hematology as well as lymphoid organ histopathology of mice neonates were investigated. Both olanzapine and risperidone transfers through milk and make the neonates susceptible to their adverse side effects. Corticosterone elevation tendency of both the drugs further enhance the susceptibility for immune dysfunction. Analysis of total and differential leukocytes counts revealed neutropenia with all the doses of olanzapine but only with risperidone 2 mg/kg. Weight analysis and histopathology of thymus and spleen indicated a state of suppression; less in the risperidone-exposed groups. Significant plasma corticosterone elevation occurred on 4 and 8 mg/kg olanzapine exposures but not with 10 mg/kg as well as with both the risperidone doses. Elevation of plasma prolactin levels occurred dose-dependently for both the drugs. Hematological toxicity (neutropenia) might be the direct toxic effects of the drugs/unstable metabolites on circulating neutrophils and/or on the bone marrow hemopoietic cells. Direct toxicity of the drugs might also have suppressed the lymphoid organs thymus and spleen. Further, it could be associated to hormonal imbalance induced by adverse pharmacological effects of the drugs on the endocrine system. Suppression of lymphoid organs in olanzapine groups might have resulted because of corticosteronemia and hyperprolactinemia, while in risperidone it could be mediated by pronounced hyperprolactinemic effect alone.

Effect of ionizing radiation on the synthetic activity of blood system cells in ground squirrels in different physiological states

The functional (synthetic) activity of blood lymphocytes and bone marrow haemopoietic cells in ground squirrels during the annual cycle as well as in hibernating and awaken animals in winter have been studied by fluorescent microspectrometry. The effect of ionizing radiation on animals in different functional states of the hibernation-arousal bout was investigated too. It was shown that the synthetic activity (parameter alpha) in blood lymphocytes was minimal in hibernating state in winter and maximal in active euthermic spring animals, then slightly decreased in June and more considerably decreased in the prehibernating autumn period. In awake animals in winter, the values of parameter alpha reached the same values as in summer. The changes of parameter alpha in bone marrow haemopoietic cells were essentially the same: the minimal values were observed in the prehibernation autumn period and in awake animals in winter the alpha values were slightly higher than in active euthermic animals in summer. The maximal synthetic activity in bone marrow haemopoietic cells in active euthermic spring animals is due mainly to cells in G1-G2 phases of the cell cycle. The decrease of the synthetic activity in summer is a result of the cell transition from G2 to mitosis and transition of a part of cells to G0 When investigating the hibernation-arousal bout in ground squirrels in winter, during arousal, we found two stages considerably differing in both the values of parameter alpha in bone marrow haemopoietic cells and the number of blood cells. The synthetic activity and the total number of blood and bone marrow cells in ground squirrels irradiated in the state of deep hibernation did not differ significantly from the state of non-irradiated hibernating animals. The negative effect of radiation appeared upon the arousal of these animals but it was expressed to a lesser degree in comparison with the animals irradiated in the active state. It was found that the acute irradiation of animals during arousal from hibernation in the second stage caused the most pronounced functional inactivation and cell death. The physiological state of ground squirrels subjected to ionizing irradiation at different phases of the hibernation-arousal bout plays a determining role in the changes of the qualitative and quantitative characteristics of blood system cells. Thus, the hypometabolic state of ground squirrels in hibernation is a factor of protection from the action of ionizing radiation on the organism and the immune system.

Lentivirus transduction of bone marrow hemopoietic precursor cells with Lin-c-kit+ phenotype Ex Vivo using a genetic construct containing green fluorescent protein gene

We developed a technology of labeling bone marrow precursor cells with the Lin-c-kit+ phenotype in culture by green fluorescent protein gene using a lentivirus vector. The proposed system provides effective transduction of bone marrow precursor cells and high transgene expression level in vitro (27%). The integration of the transgene into the transduced cell genome in vivo was verified by the method of splenic colonies.

MHC class II and c-kit expression allows rapid enrichment of T-cell progenitors from total bone marrow cells

T-cell progenitors in the embryonic bone marrow express the tyrosine kinase receptor c-kit. RR5, an anti-MHC class II β chain monoclonal antibody, subdivides this c-kit positive population. Intrathymic transfer experiments showed that most of the T-cell progenitors belong to the MHC class II+/c-kit+ bone marrow population in the embryo and young adult. On transplantation, these bone marrow progenitors lose this expression and differentiate into CD4 CD8 T lymphocytes. In contrast, erythroid progenitors are restricted to the MHC class II−/c-kit+ population. The MHC class II+/c-kit+ pro-T cells are metabolically active, because they stain brightly with rhodamin 123. Their cyclin A and B expression level suggests that they are in the mitotic phase of the cell cycle. Thus, we define an easy sorting protocol, which allows enrichment of T-cell progenitors from total bone marrow hemopoietic cells.

Effects of adrenal glands on bone marrow hemopoietic microenvironment

Effects of bilateral adrenalectomy on the hemopoiesis-inducing microenvironment and bone marrow hemopoietic cells were studied. It was shown that adrenal hormones regulate secretory activity of the hemopoiesis-inducing microenvironment, in particular its nonadherent fraction. Adrenalectomy did not change the content of hemopoietic islets, number of erythro- and granulocytopoietic precursors, and blood indexes. The data suggest that structural and functional properties of the hemopoiesis-inducing microenvironment determined by the great variety and high plasticity of organ and intersystem regulatory interactions for a long time compensate the effects of hypocorticism on the requirements for mature blood cells.

Implication of expression of GDNF/Ret signalling components in differentiation of bone marrow haemopoietic cells

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) mediate their actions through a unique multicomponent receptor system composed of Ret receptor tyrosine kinase and glycosyl-phosphatidylinositol-linked cell surface proteins (designated GFRalpha-1 and GFRalpha-2). In the present study, expression of these signalling components in the process of differentiation of haemopoietic cells was investigated. Ret was expressed at variable levels in normal and malignant cells of the myelomonocyte lineage. Immunohistochemical analysis of human and mouse tissues revealed that Ret expression was increased in intermediate mature myeloid cells such as promyelocytes and myelocytes and decreased in mature granulocytes and monocytes. Consistent with this observation, when THP-1 monocytic and HL-60 promyelocytic leukaemia cells expressing Ret were differentiated toward macrophages or granulocytes by treatment of 12-O-tetradecanoylphorbol-13-acetate (TPA) or all-trans retinoic acid (RA), Ret expression strikingly decreased during differentiation. Expression of GDNF, NTN, GFRalpha-1 and GFRalpha-2 was undetectable in THP-1 and HL-60 cells as well as in bone marrow haemopoietic cells. In contrast, bone marrow stromal cells appeared to express GDNF, GFRalpha-1 and GFRalpha-2 but not Ret. These findings suggested that the interaction between stromal cells and Ret-expressing haemopoietic cells in the bone marrow microenvironment may play a role in the differentiation of myelomonocyte-lineage cells through activation of the GDNF/Ret signalling pathway.

Implication of expression of GDNF/Ret signalling components in differentiation of bone marrow haemopoietic cells

Implication of expression of GDNF/Ret signalling components in differentiation of bone marrow haemopoietic cells

A novel surface marker (B203.13) of human haemopoietic progenitors, preferentially expressed along the B and myeloid lineages.

We used a monoclonal antibody (mAb) (B203.13, IgM) generated from a mouse immunized with the human B/myeloid bi-phenotypic B1b cell line, to analyse haemopoietic cells. The antigen recognized by this mAb is expressed on most adult and umbilical cord blood CD21+ B cells, at minimal density on mature monocytes, and is undetectable on granulocytes, T, natural killer (NK) cells, and erythrocytes. Within umbilical cord blood and adult bone marrow haemopoietic progenitor cells, the B203.13 mAb recognized a surface marker, present on progenitor cells of several haemopoietic lineages, that was transiently expressed on early erythroid and T/NK progenitors, and was preferentially maintained on cells of the B and myeloid lineages. Within the CD34+ cells, B203.13 was expressed on early committed myeloid (CD33+) and erythroid (CD71dim) progenitor cells, as confirmed in colony formation assays. The mAb also reacted with cells of B and myeloid chronic leukaemias and cell lines. These data define B203.13 mAb as a novel reagent useful for the characterization of haemopoietic progenitors and leukaemias.

Lymphoid Hyperplasia, Autoimmunity, and Compromised Intestinal Intraepithelial Lymphocyte Development in Colitis-Free Gnotobiotic IL-2-Deficient Mice

IL-2-deficient (IL-2(-/-)) mice develop disorders of the hemopoietic and immune systems characterized by anemia, lymphocytic hyperplasia, and colitis. The mechanisms responsible for these abnormalities remain unclear. To investigate the underlying basis of autoimmunity, the particular role of commensal gut flora in the initiation of colitis, and the role of IL-2 in the development of intestinal intraepithelial lymphocytes (iIEL), we evaluated IL-2(-/-) mice reared and maintained under gnotobiotic (germfree) conditions. By 8 wk of age, 80% (20 of 25) of germfree IL-2(-/-) mice show signs of disease, including anemia, disturbances in bone marrow hemopoietic cells, lymphocytic hyperplasia, and generalized autoimmunity, similar to those seen in specific pathogen-free (SPF) IL-2(-/-) mice. In striking contrast to SPF IL-2(-/-) mice, germfree IL-2(-/-) mice do not develop colitis. However, the numbers of gammadelta+ and TCR alphabeta+ CD8 alphaalpha+ iIELs are reduced, and in lethally irradiated SPF IL-2(+/+) mice, reconstituted with IL-2(-/-) bone marrow TCR gammadelta+ iIELs fail to develop, consistent with an important role of IL-2/IL-2R signaling in the development of gammadelta iIELs. Consequently, our findings demonstrate that the colitis seen in SPF IL-2(-/-) mice depends upon the presence of intestinal bacterial flora and that environmental Ags are not responsible for the anemia and extraintestinal lymphoid hyperplasia that occur in IL-2(-/-) mice. Thus, germfree IL-2(-/-) mice represent a unique system in which the role of IL-2 deficiency in hemopoietic and immune system disorders can be investigated in dissociation from complications that may arise due to colitis.

Amount of mpl on bone marrow haemopoietic precursor cells from healthy volunteers and patients with refractory anaemia.

Using a non-isotopic ligand binding assay using multi-colour flow cytometry, we quantitatively examined the amount of mpl in megakaryocyte-platelet lineage cells. Firstly, we quantified the amount of mpl on cell lines. Mpl gene-transfected BaF3 cells expressed a large amount of mpl, whereas original BaF3, K562, HL-60 and NOMO-1 cells showed no mpl. In bone marrow cells from healthy volunteers, mpl was expressed on CD34+ cells from the very early stage of differentiation when they had no CD38 antigen. The amount of mpl increased with differentiation to CD34+ CD41+ cells, but decreased with further differentiation to CD34- CD41+ cells. In CD34+ CD41+ cells the amount of mpl varied according to cell size: abundant in large cells, moderate in medium-size cells and a little in small cells. In bone marrow cells from patients with refractory anaemia (RA), the amount of mpl was decreased compared with that in bone marrow cells from healthy volunteers. When analysed by the same CD phenotype and same cell size, the amount of mpl was less in RA patients compared with that in healthy volunteers in all phenotypes and sizes tested. The proportion of large CD34+ CD41+ cells was less in RA patients than in normal volunteers.