Candidate hematopoietic stem cells from fetal tissues, umbilical cord blood vs. adult bone marrow and mobilized peripheral blood

BACKGROUND: The eosinophil granulocyte is an inflammatory cell that plays an active part in diseases such as asthma and rhinitis. This study aimed to investigate oxidative metabolism by blood eosinophils taken from allergic rhinitis patients, asthmatics, and nonallergic controls before and during the birch-pollen season. METHODS: Twenty patients with allergy to birch pollen and seasonal symptoms of rhinitis, some of whom were also asthmatic, were followed before and during the birch-pollen season in Sweden. The cells were purified using a Percoll gradient and the MACS system. Eosinophil purity in all samples was > 95%. Oxidative metabolism was measured by a chemiluminescence (CL) assay, with luminol and lucigenin acting as enhancers, and PMA, serum-treated zymosan (STZ), interleukin (IL)-5, or RANTES as stimuli. RESULTS: The allergic subjects showed reduced luminol CL when activated before the season with PMA (P = 0.040) or STZ (P = 0.0055). This was not seen during pollen exposure. STZ-activated lucigenin CL was also reduced before the season (P = 0.0027). The reduction was most evident in the group with asymptomatic rhinitis. In terms of eosinophil stimulation, IL-5 and RANTES were equally effective in allergic and nonallergic subjects, both before and during the pollen season. CONCLUSIONS: Blood eosinophils from asymptomatic allergics may have a lower capacity to produce oxygen-free radicals than eosinophils from nonallergics.     

Effect of a vitamin D3 analog, EB1089, on hematopoietic stem cells from normal and myeloid leukemic blasts

The seco-steroid 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induces differentiation and inhibits clonal proliferation of HL-60 cells. We analyzed the effect of a novel vitamin D3 analog, EB1089, on normal myeloid and leukemic cells as well as CD34+ cells. EB1089 showed an extraordinary inhibition of clonal growth of HL-60 cells (ED50 = 5 x 10(-11) M) and AML blast cells (ED50 = 9 x 10(-10) M) compared to 1,25(OH)2D3 without suppression of growth of normal human bone marrow CFU-GM. The CD34+ cells from acute myeloid leukemia (AML) blasts were inhibited in a dose-dependent fashion by 1,25(OH)2D3 with an ED50 of 1.2 x 10(-9) M; and even more strikingly, 10(-10) M of EB1089 inhibited all clonal growth of human CD34+ leukemic colony-forming cells. In contrast, both EB1089 and 1,25(OH)2D3 (10(-8) M) showed little or only mild inhibition of CD34+ clongenic hematopoietic cells from normal human peripheral blood (PB); and in liquid culture, EB1089 stimulated the proliferation of normal human CD34+ cells about 2.5 times as compared to control cultures. In order to evaluate the potential use of EB1089 for purging leukemic cells from normal CD34+ progenitor cells for PB stem cell transplantation (PBSCT), normal human PB mononuclear cells (PBMNC) were contaminated with HL-60 cells, and then CD34+ cells purified and treated with EB1089. We found that CD34+ purification and EB1089 purging was able to eliminate approximately 100% of HL-60 leukemic cells with no toxicity to normal CD34+ hematopoietic progenitor cells. These data suggested that purification of CD34+ cells and ex vivo treatment with EB1089 might provide an effective therapeutic approach for PBSCT.     

Changes in the number of stem cells during mouse bone marrow cultivation on a sublayer of fibroblast-like cells

A determination was made of the number of colonies in the spleen of irradiated mice after administration to them of a culture of mouse bone marrow cells. Colony-forming units proved to persist in the culture only for a short time. Use of preliminarily grown underlayer of fibroblasts of bone marrow origin had no effect on the perservation and dynamics of the changes in the number oc colony-forming units.     

NK cells differentiated from bone marrow, cord blood and peripheral blood stem cells exhibit similar phenotype and functions

In the present study, we investigated the differentiation of human NK cells from bone marrow, cord blood and mobilized peripheral blood purified CD34+ stem cells using a potent culture system. Elutriated CD34+ stem cells were grown for several weeks in medium supplemented with stem cell factor (SCF) and IL-15 in the presence or absence of a murine stromal cell line (MS-5). Our data indicate that IL-15 induced the proliferation and maturation of highly positive CD56+ NK cells in both types of culture, although murine stromal cells slightly increased the proliferation of NK cells. NK cells differentiated in the presence of MS-5 were mostly CD56+ CD7 and a small subset expressed CD16. These in vitro differentiated CD56+ NK cells displayed cytolytic activity against the HLA class I- target K562. The CD56+ CD16+ subset also lysed NK-resistant Daudi cells. Neither of these NK subsets were shown to express Fas ligand. Total CD56+ cells expressed high amounts of transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor, but no IFN-gamma. Investigation of NK receptor expression showed that most CD56+ cells expressed membrane CD94 and NKG2-A mRNA. PCR analysis revealed that p58 was also expressed in these cells. The role of CD94 in NK cell-mediated cytotoxicity was assessed on human HLA-B7-transfected murine L cells. While a low cytotoxic activity towards HLA-B7 cells was observed, the HLA-DR4 control cells were killed with high efficiency. These studies demonstrate that cytolytic and cytokine-producing NK cells may be derived from adult and fetal precursors by IL-15 and that these cells express a CD94 receptor which may influence their lytic potential.     

Growth and differentiation of bone marrow hematopoietic cells in mice bearing Ehrlich ascite tumor and treated with Dicyclopentadienildichlorotitanium (IV)

In this work we have investigated the growth and differentiation of bone marrow stem cells in mice bearing Ehrlich ascites tumor and treated with three dose-regimens of Dicyclopentadienyldichlorotitanium (IV) (DDCT). We also studied the presence of colony stimulating factors in the serum of DDCT-treated animals, as well as the effects of the drug on the survival of the tumor-bearing mice. The results demonstrated that the myelosuppression developed in the tumor-bearing animals is prevented by the administration of 1, 2 or 3 doses of 15 mg/kg DDCT. In the treatment with three doses, however, 23% of the animals died. Moreover, DDCT treatment in normal animals resulted in increased numbers of CFU-GM. We observed the presence of stimulating factors in the serum of drug-treated animals which induced the growth and differentiation of bone marrow progenitor cells from normal animals in vitro. On the other hand, in vitro addition of the drug to these cultures had no effect. Thus, we conclude that the drug protects against the myelosuppression induced by the tumor and that this protection may be related to an indirect action of the drug.     

Effects of La~（3＋） on osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells

In order to elucidate the action of La3+ on bone metabolism,effects of La3+ on the osteogenic and adipogenic differentiation of pri-mary mouse bone marrow stromal cells(BMSCs) were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) test,alkaline phosphatase(ALP) activity measurement,mineralized function,oil red O stain and measurement.The results showed that La3+ pro-moted the proliferation of BMSCs except at 1×10-10 and 1×10-6 mol/L.The effect of La3+ on the osteogenic differentiation depended on con-centrations at the 7th day,but the osteogenic differentiation was inhibited at any concentration at the 14th day.La3+ promoted the formation of mineralized matrix nodules except at 1×10-8 and 1×10-5 mol/L.La3+ inhibited adipogenic differentiation except at 1×10-10 and 1×10-7 mol/L at the 10th day,and inhibited adipogenic differentiation except at 1×10-9 mol/L at the 16th day.These findings suggested that La3+ might have protective effect on bone at appropriate dose and time.This would be valuable for better understanding the mechanism of the effect of La3+ on bone metabolism.     

Serum interleukin-3 levels following autologous or allogeneic bone marrow transplantation: effects of T-cell depletion, blood stem cell infusion, and hematopoietic growth factor treatment

Engraftment of marrow following autologous or allogeneic bone marrow transplantation (BMT) may be influenced by quantity and function of stem cells. T lymphocytes, supporting microenvironmental cells, and hematopoietic growth factors (HGF). To elucidate the physiologic role of interleukin-3 (IL-3) in the engraftment process, serum IL-3 levels were measured in over 400 samples from 77 transplant recipients before and for up to 3 weeks following transplantation using a novel enzyme-linked immunoabsorbent assay (ELISA) with a sensitivity of > or = 78 pg/mL. Thirty-seven patients received two to three log T-cell-depleted allografts. In the remaining 40 patients (18 autologous marrow, 12 allogeneic marrow, and 10 autologous peripheral blood [PB] stem cell), T cells were not depleted (non-TCD) from the grafts. A burst of IL-3 (peak levels, 1,500 to 6,000 pg/mL) was detected in the immediate posttransplant period between day 0 and day 14 in all non-TCD recipients and in 21 of 37 (57%) of TCD recipients. A strong inverse relationship between IL-3 levels and absolute neutrophil count (ANC) was observed in both non-TCD recipients (r = -.796) and in TCD recipients (r = -.897). However, both peak IL-3 levels and mean IL-3 levels from day 0 through 14 were significantly lower in TCD recipients compared with either autologous or unmodified allogeneic marrow recipients (P < .01). The lowest peak or mean day 0 through 14 IL-3 levels were observed in matched related recipients undergoing the most aggressive (2.5 to 3.0 log) T-cell-depleted BMT. Autografted patients receiving blood stem cell transplants alone or posttransplant granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) also had significantly lower peak IL-3 levels (P < .01). In patients receiving TCD grafts, administration of antithymocyte globulin (ATG) posttransplant significantly increased peak IL-3 levels compared with patients not treated with ATG (P < .04). This study shows that endogenous release of IL-3 is strongly associated with myeloid engraftment and inversely related to ANC. Removal of T lymphocytes from donor marrow or acceleration of engraftment by use of stem cells or growth factors appears to blunt the endogenous release of IL-3 whereas use of ATG posttransplant increases IL-3 release.     

Differential inhibition of telomerase activity during induction of differentiation in hematopoietic, melanoma, and glioma cells in culture

Accumulating evidence indicates that telomerase activity is stringently repressed in normal human somatic cells but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity may play a role in carcinogenesis and immortalization. Recently, down-regulation of telomerase activity by induction of differentiation has been reported for cells of pre-myelocytic and myelocytic leukemia as well as embryonic carcinoma. To gain further insight about the regulation of telomerase activity following induction of differentiation, telomerase activity was examined in a human hematopoietic progenitor cell line (D2), a melanoma cell line (CM73-36) and a glioma cell line (Ast812) before and after addition of differentiation inducing agents. The state of differentiation was assessed by growth inhibition and cell morphological maturation. Telomerase activity was assayed by a PCR-based telomeric repeat amplification protocol (TRAP). Our data show that telomerase activity was inhibited only in differentiation-induced D2 cells but not in differentiation-induced melanoma and glioma cells. A model for the differential inhibition of telomerase activity following induction of differentiation in different cancer cells will be presented.     

Effect of recombinant canine stem cell factor, a c-kit ligand, on hematopoietic recovery after DLA-identical littermate marrow transplants in dogs

We studied the effect of recombinant canine stem cell factor (rcSCF) on hematopoietic recovery, incidence of graft failure, graft-vs.-host disease (GVHD), and survival after marrow transplantation from dog leukocyte antigen (DLA)-identical canine littermates. Ten animals received 100 microg rcSCF/kg/day b.i.d. by subcutaneous injection on days 1 through 10 after 920 cGy total body irradiation and transplantation of a mean of 3.7x10(8) marrow cells/kg body weight. None of the dogs received GVHD prophylaxis. All animals showed hematopoietic engraftment. The median number of days to achieve 1000 neutrophils/mm3 was 9; 100 monocytes/mm3 were reached after 15 days, 500 lymphocytes/mm3 after 21 days, and 20,000 platelets/mm3 after 16 days. One animal developed GVHD involving skin, gut, and liver and died of bacterial pneumonia 21 days after transplantation. The remaining nine dogs were observed for a median of 37 days (range 29-84 days) posttransplantation until they were killed. Facial edema was seen in three dogs during the first 2-3 days of rcSCF administration. These results show that within the limits of this study it appears to be safe to administer SCF after DLA-identical littermate marrow transplants in dogs. Comparison with previously published data in the same model showed that neutrophil and monocyte recovery was significantly faster in dogs receiving SCF treatment compared with dogs without growth factor treatment (recovery to achieve 1000 neutrophils/mm3: median 9 days vs. 13 days, p = 0.002; recovery to 100 monocytes/mm3: median 15 days vs. 105 days, p = 0.0002). Otherwise, no significant differences were seen. Results obtained with SCF treatment were similar to those previously obtained in the same model with recombinant human granulocyte colony-stimulating factor (rhG-CSF) treatment except that recovery of lymphocytes to 500/mm3 appeared to be more rapid in G-CSF-treated dogs (median 15 days vs. 21 days, p = 0.03).     

A scanning and transmission electron microscopic study on rat bone marrow sinuses and transmural migration of blood cells

Bone marrow sinuses of young rats were examined under the scanning (SEM) and transmission electron microscopes (TEM). Marrow sinus wall was composed of three layers: an inner or luminal endothelium, an outer or adventitial cell layer, and a basal lamina in between. The luminal surface of the endothelial cells was quite smooth and showed some fenestrations, which could be divided into two types according to their size. One was represented by larger fenestrations (1-3 mum in diameter) which were presumed to be formed transiently at the site of blood cell migration, while the other by small pores (0.1 mum) grouped into a cribriform area. The adventitial cells showed a discontinuous layer in the TEM. Under the SEM, the discontinuity corresponded to the spaces formed between the cytoplasmic attenuations of the cells. Blood cell migration from the extravascular hemopoietic tissue into the sinus lumen was numerously observed. The migration occurred not through an intercellular gap, but through the larger intracellular fenestration of the endothelial lining mentioned above. A number of megakaryocytes were identified by their bulky cytoplasm in the parenchyme. Figures suggesting the sequence of platelet liberation from this cell could be demonstrated. First, the megakaryocyte extended its peripheral cytoplasmic processes into the sinus through endothelial fenestrations. The processes, being conspicuously extended, became periodically constricted. Finally, platelets were believed to be produced by separation at the constricted portions and liberated to circulation. The occurrence of a few endothelial fenestrations apparently unassociated with blood cell migration may possibly be ascribed to detachment of a blood cells due to vascular perfusion. The functional significance of the adventitial cell was discussed in association with blood cell migration.     

The response of IL-3 dependent B6SUtA bone marrow cells to both erythropoietin and chemical inducers of differentiation

To develop cell lines which respond to both a physiological cytokine and chemical agents by the induction of differentiation pathway, factor dependent B6SUtA murine bone marrow cells were transfected with the erythropoietin receptor (EpoR). Clones were obtained that exhibited different sensitivities to erythopoietin (Epo), with one clone exhibiting erythroid differentiation in response to Epo, while in another Epo acted as a proliferation stimulus. Moreover, parental B6SUtA cells were sensitive to the initiation of differentiation by butyrate, diazepam and hemin. Thus, B6SUtA cells appear to represent a unique model to dissect the signaling molecules involved in the growth and differentiation pathways employed by Epo and non-physiological chemicals.     

Studies of the hemopoietic microenvironments. V. Changes in murine splenic and bone marrow glycosaminoglycans during post irradiation hemopoietic regeneration

Changes in the amount of sulphated and unsulphated glycosaminoglycans in the spleen and the bone marrow of lethally irradiated mice were followed up to 11 days after irradiation. One day after irradiation, a sharp decrease in the amount of glycosaminoglycans was observed. In the absence of hemopoietic cells, the remaining stromal elements of spleen and bone marrow underwent subsequently marked changes in the amount of the sulphated and unsulphated components of the glycosaminoglycans in both organs. Reconstitution of irradiated mice with bone marrow cells affected the pattern of changes in the amount of glycosaminoglycans. Although no linear correlation could be observed between the amount of glycosaminoglycans and the number of hemopoietic cells present in the hemopoietic organs, these results suggest an interaction between hemopoietic cells and stromal cells in the hemopoietic organs with regard to the glycosaminoglycan metabolism.     

Transfer of human adenine deaminase gene into murine hematopoietic stem cells: sequential study of spleen colony-forming units from bone marrow of living mice and the requirement of the microenvironment

PURPOSE: To explore relationships between pregnant women's childhood experiences with discipline and 1) prenatal plans for disciplining own child and 2) later maternal-infant interactions DESIGN: Prospective correlation study. SETTING: Prenatal clinics and home visits PARTICIPANTS: Low-income pregnant women (N = 205) with a mean educational level of 12 years MAIN OUTCOME MEASURES: Adapted Ways of Handling Irritating Behaviors (Mother and You) Scales, NCAST Teaching Scale, Home Observation for Measurement of the Environment Scale RESULTS: Correlations between childhood experiences with discipline and 1) plans for later discipline strategies with own child and 2) infant components of maternal-infant interactions, including clarity of cues and responsiveness to the mother CONCLUSIONS: Prenatal assessment for childhood experiences with discipline may uncover factors associated with a break in the trajectory for optimal maternal-child interactions described by Barnard's theoretical framework.     

Comparison of retroviral-mediated gene transfer into cultured human CD34+ hematopoietic progenitor cells derived from peripheral blood, bone marrow, and fetal umbilical cord blood

Genetic alteration of stem cells ex vivo followed by bone marrow transplantation could potentially be used in the treatment of numerous diseases and malignancies. However, there are many unanswered questions as to the best source of hematopoietic cells for long-term reengraftment and the most effective way to introduce foreign genes into this target cell. We have compared retroviral-mediated gene transfer into CD34+-enriched cells derived from peripheral blood (PB), bone marrow (BM), or fetal umbilical cord blood (CB). Cells from all three sources that had been expanded ex vivo in the presence of stem cell factor (SCF), interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF) showed transduction efficiencies ranging from 5-45%, as measured by acquisition of G418 resistance. The average efficiencies of gene transfer from multiple experiments for PB, BM, and CB were not statistically different. To determine the effect of ex vivo expansion on gene transfer into CB CD34+ cells, we compared the transduction efficiencies of cells exposed to virus immediately after harvest and CD34 selection or after 6 days of culture CD34+ CB cells were more effectively transduced after expansion in culture, showing gene transfer efficiencies 3- to 5-fold higher on day 6 compared with day 0. Last, we examined retroviral transduction via spinoculation of CB CD34+ cells and found it to be approximately as effective as our standard transduction with no significant loss of cell viability as measured by colony formation in semi-solid medium.     

Effects of vesnarinone on the bone marrow stromal cell-dependent proliferation and differentiation of HL60 cells in vitro

There is no simple and efficient method for assaying phage isolated from libraries without having to resort to PEG purification of the phage, or to the biotinylation or other labelling of the target molecule. We report here a method for producing 'bifunctional' phage that express two types of peptide; one peptide, fused to pVIII, will bind to immobilized fibrinogen, allowing capture of the phage out of culture supernatants; this allows the other peptide, fused to pIII or pVIII to be assayed by simple ELISA. This system has also been developed for the capture of phage bearing a streptavidin-binding peptide. The bifunctional phage are produced by bacterial cells bearing a plasmid that expresses pVIII fused either to the fibrinogen-binding peptide or to the streptavidin-binding one. Thus, when these cells are infected with a phage clone or pool to be assayed, phage will be produced whose 'capture-peptide' is produced from the plasmid and whose 'assay-peptide' is produced from the phage genome. We show here that, by this method, bifunctional phage can be produced that will bind to immobilized streptavidin or fibrinogen.     

Effect of time on migration of Oesophagostomum spp. and Hyostrongylus rubidus out of agar-gel

CAP affects all ages but predominantly the elderly. The microbial aetiology is diverse and rarely established at the time of admission. Initial management includes assessment of severity, correction of dehydration and imbalances of gas exchange, and prompt administration of antibiotic. The regimens will vary by risk factor and severity assessment. Mortality remains high, especially in those requiring intensive care. Prevention includes control of underlying disease, smoking and ethanol abuse, and the appropriate use of influenza and pneumococcal vaccines.     

Myelosuppressive changes from single or repeated doses of radioantibody therapy: effect of bone marrow transplantation, cytokines, and hematopoietic suppression

Apolipoprotein E phenotype (APOE phenotype) has been demonstrated to be a genetic determinant of cardiovascular disease. This atherogenicity may be a reflection of the association of APOE phenotype and plasma lipoprotein concentrations. The Coronary Artery Risk Development in Young Adults (CARDIA) Study affords the opportunity to assess the frequency of apolipoprotein E alleles in population-based samples of African Americans and whites in the United States and to compare the associations of APOE phenotype with lipoprotein and apoprotein concentrations. Data from 3,485 African-American and white men and women between the ages of 25 and 37 years who attended the fourth CARDIA Study examination in 1992-1993 were used in this analysis. African-American men and women had significantly higher frequencies of E2 and E4 phenotype and thus higher frequencies of *epsilon2 and *epsilon4 alleles (p < 0.005). Men and women of both races with APOE4 phenotype generally had higher low density lipoprotein cholesterol, apolipoprotein B, and lipoprotein(a) concentrations and lower high density lipoprotein cholesterol concentration, and individuals with APOE3 phenotype had the lowest triglyceride concentration. Major differences between African Americans and whites were observed in the distribution of APOE phenotypes and *epsilon alleles, but APOE phenotype was associated with similar differences in lipoprotein and apoprotein concentrations in both races. The data suggest that APOE phenotype may be a risk factor for cardiovascular disease in both African Americans and whites because it is associated similarly with an adverse lipoprotein profile.