Cryosurgery in urology

Androgenic steroids and their non-androgenic 5beta-H metabolites enhance the number of colonies of hemoglobin synthesizing cells grown from rat bone marrow in response to a standard (0.25 unit/ml) concentration of erythropoietin. The target cells for two steroids were found to be different. Cells influenced by the androgen, fluoxymesterone (fluoxy), resembled cells responding to erythropoietin in their cycle characteristics, as measured by tritiated thymidine suicide, and in their physical characteristics, as determined by velocity sedimentation gradient separation. Cells responding to etiocholanolone (etio) had a much lower tritiated thymidine suicide rate and different sedimentation velocities. Preincubation of marrow cells with etio for two hours was sufficient to enhance erythroid colony growth by 84%, whereas a similar incubation with fluoxy produced no increment. These studies demonstrate that different classes of steroids may influence in vitro erythropoiesis by acting on distinct populations of marrow cells. Fluoxymesterone appears to act through cells already committed to respond to erythropoietin, while etiocholanolone appears to act on a separate, perhaps more primitive population of marrow cells.     

Colony formation by human haemopoietic precursor cells cultured in semi-solid agar in diffusion chambers

A technique which allows colony growth of haematologically normal human bone marrow cells is described. The cells are supported by semi-solid-agar-medium inside modified Millipore diffusion chambers implanted in the peritoneal cavity of irradiated mice. After 9 days incubation colonies containing up to 1000 cells are found in these Agar Diffusion Chambers. All haematologically normal patients studied so far produced colonies, the majority with between 10 and 40 colonies per 2 X 10(5) bone marrow cells inoculated. This culture system therefore provides a convenient and reliable clonal assay for human bone marrow cells which, in contrast to the agar colony assay in vitro, does not require a source of Colony Stimulating Factor (CSF).     

Circulating erythropoietic precursors assessed in culture: characterization in normal men and patients with hemoglobinopathies

Circulating erythropoietic precursors in normal men and patients with hemoglobinopathies were characterized in culture. Blood mononuclear cells harvested with a modification of the Ficoll-Isopaque technique were cultured in methylcellulose for 14 days. The majority of erythropoietic colonies consisted of several subcolonies assuming the morphology of erythropoietic "bursts" described in murine marrow cultures. Time course studied of colony formation from marrow and blood nucleated cells confirmed that the circulating erythropoietic precursors represented only early stages of development. Peak sedimentation velocity of the circulating precursors analyzed using a Staput apparatus averaged 5.31 mm/hr and corresponded with that of the early erythropoietic precursors in human marrow. One ml of blood yielded an average of 153 colonies in normal men and 785 colonies in patients with hemoglobinopathies. No correlation was observed between colony formation and reticulocyte indices of individual patients. Examination of the proliferative state of the erythropoietic precursors using high specific activity tritium-labeled thymidine revealed that almost none of the cells in normal men or patients with hemoglobinopathies were in the DNA synthetic phase.     

Effect of ESF preparations on different types of erythroblasts

A new method of quantitative (14)C-autoradiography was applied for evaluating possible effects of erythropoietin(ESF) on the DNA synthesis rate of differentiated erythroid murine bone marrow cells identified as proerythroblasts, basophilic and polychromatic erythroblasts...     

Nonuniform biosynthesis of multiple hemoglobins in the adult rat and guinea pig

Separation of adult rat bone marrow cells by the method of thin layer countercurrent distribution permits the analyses of 59Fe-tagged erythroid cells for the various multiple hemoglobins and the assignment of such hemoglobins to erythroid cells at different stages of their development. Of the six adult red cell hemoglobins, hemoglobin 5 is synthesized most actively in the earliest erythroid cell whereas hemoglobin 4 (the major hemoglobin of the red cell) is synthesized most actively in the latest erythroid cells, e.g. the reticulocyte. Experimental evidence also indicates that maturation of the erythroid cell is accompanied by a decreased rate of synthesis of hemoglobin 5. The earliest erythroid cells of the marrow contain two hemoglobins, 7 and 8, which are absent in the adult red cell. Similar studies with the guinea pig confirm the nonuniform biosynthesis of its two hemoglobins and suggest that the phenomenon may be a general one among mammalian multiple hemoglobins.     

Erythropoiesis inhibiting factor(s) (EIF). The specificity and toxicity of urinary EIF studied in vivo and in vitro

The specificity and toxicity of the urinary erythropoiesis inhibiting factor (EIF) has been tested both in vivo and in vitro. When EIF was given to ESF stimulated erythropoietically suppressed polycythaemic mice and to mice at maximal endogenous erythropoietic stimulation, a reduction of the erythroid bone marrow cells, the erythropoietic 3H-TdR L.I. and the total number of bone marrow cells were observed. No effect was seen on the myelopoietic bone marrow cells. An unspecific toxic effect was unlikely, since addition of EIF did not alter the proliferation of lymphoblastic cells nor change the glucose utilization of bone marrow cells in vitro. Neither did the amount of dead bone marrow cells increase after being incubated with EIF for 72 h. The results indicate that the urinary EIF is a non-toxic, cell specific inhibitor on erythropoiesis.     

Decline in the growth potential of spleen-colonizing bone marrow stem cells of long-lived aging mice

The growth capacity of femoral bone marrow stem cells from young and old long-lived mice was assessed in the spleen of X-irradiated young and old syngeneic recpients by determining: (a) the number of stem cells colonizing the spleen, (b) the rate of incorporation of 125I-labeled iododeoxyuridine by proliferating colony cells, and (c) the number of cells present in the largest colonies at the end of the growth phase.We found that the growth capacity of stem cells declined with age. We further found that the spleen-seeking and spleen colony growth capacities of old stem cells remained characteristically old even after they were allowed to self-replicate in the bone marrow of young recipients for an extended period of time. On the other hand, the spleen colony growth capacity of young stem cells could be reduced by allowing them to self-replicate in old recipients. These results suggest that the growth capacity of old stem cells is an intrinsic characteristic which cannot be readily altered, but that of young stem cells can be aged in an accelerated manner by allowing them to self-replicate in old recipients. An additional reduction was noted in the frequency of both young and old stem cells colonizing the spleen of old recipients and in the cell density of the largest colonies produced. These results indicate that factors extrinsic to the stem cells are also responsible for the decline with age in their spleen colony growth capacity.Thus, the growth capacity of old stem cells in old recipients could be as low as 10% that of young stem cells in young recipients.     

Effects of stem cell factor (SCF) on human marrow neutrophil, neutrophil/macrophage mixed, macrophage and eosinophil progenitor cell growth

The effects of stem cell factor (SCF) on the subpopulations of granulocyte/macrophage colony-forming units (CFU-GM) were examined. Hematopoietic progenitor cells were enriched from normal adult bone marrow specimens by immunomagnetic beads using an anti-CD34 antibody and lineage marker antibodies for positive selection and negative selection, respectively. SCF enabled neutrophil and neutrophil/macrophage mixed progenitors to respond to granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3) and to develop the colony and further cluster formation. The neutrophil colonies stimulated by GM-CSF or IL-3 consisted mainly of immature cells, while the colonies stimulated by granulocyte colony-stimulating factor (G-CSF) consisted of mature neutrophils irrespective of the addition of SCF. In macrophage and eosinophil lineages, SCF augmented the colony formation in the presence of GM-CSF or IL-3, whereas the enhancement of total progenitor cell growth (colonies plus clusters) was not so marked as compared with the neutrophil lineage. Time-course observation revealed that SCF could stimulate macrophage and eosinophil progenitors to form colonies rapidly. These findings indicate that SCF acts on late myeloid progenitor cells in manners different from the lineages of commitment.     

Cause analysis of pain following vitreous surgery and nursing strategies]

OBJECTIVE: To assess the efficacy of "method of activating blood circulation and removing stasis (ABCRS)" in treating polycythemia vera. METHODS: 28 cases of polycythemia vera were treated with ABCRS and observed with bone marrow colonies. RESULTS: 15 casses were responsibe, of them 4 cases reached clinical remission, 11 cases were improved. Both bone marrow colonies of colony forming unit-erythroid dependent of erythropoietin (EPO + CFU-E) and colonies of CFU-E independent of EPO. (EPO-CFU-E) from these cases decreased significantly, especially the latter. ABCRS mainly suppressed the proliferation and differentiation of EPO-CFU-E. In addition, bone marrow colonies of colony formng unit-fibroblastoids (CFU-F) from these cases also changed obviously. CONCLUSION: ABCRS is effective in treating polycythemia vera.     

Effect of moisture content of the medium on colony morphology of Campylobacter fetus subsp. jejuni

Reduction in the moisture content of the medium produced a profound effect on the colony morphology of Campylobacter fetus subsp. jejuni. Fresh medium produced flat, grayfish, spreading colonies with an irregular shape and variety appearance. Plates that were incubated at 30 degrees C for 48 h produced round, convex, butyrous colonies with an entire edge. Plates incubated at 30 degrees C for 24 h before inoculation produced colonies of an intermediate nature; they were round and raised, but not convex, and slightly water or mucoid in nature. This marked effect produced by moisture content of the medium was reproducible and may account for the variation in colonies observed by other investigators.     

A complement independent erythropoietic inhibitor acting on the progenitor cell in refractory anemia

An erythropoietic inhibitor was detected in the serum of a patient with refractory anemia. Using an in vitro heme synthesis method, the patient's serum produced tenfold inhibition of erythropoietin-stimulated radioactive iron (Fe59) incorporation into heme of normal human marrow at 72 hours, as compared with AB serum. In a separate experiment the patient's serum produced threefold inhibition, whereas immunoglobulin G (IgG) prepared from the same serum sample produced 12-fold inhibition. To identify the site of action of the inhibitor, serum was tested in a cell culture system whereby human marrow cells, grown in a plasma clot, respond to exogenous erythropoietin with the appearance of nucleated erythroid colonies. Each colony arises from a committed erythroid progenitor. The patient's serum produced a two- or tenfold reduction in the number of colonies from normal human marrow. The effect was also demonstrated on autologous marrow obtained when the patient was in "partial clinical remission". Serum samples obtained at various times during the course of the patient's illness all demonstrated a suppressive effect on colony growth. All serums were heat-inactivated, and total hemolytic complement could not be detected in either culture system. It is concluded that the anemia is due to an inhibitor, probably of IgG class, that acts on the erythroid progenitor cell. The absence of heat-labile complement components in the culture systems suggests that the mechanism is not due to immune cytolysis.     

Leptin stimulates the proliferation of murine myelocytic and primitive hematopoietic progenitor cells

Leptin, the product of obese gene, was originally identified as a factor regulating body-weight homeostasis and energy balance. The present study has shown that leptin acts on murine hematopoiesis in vitro. In the culture of bone marrow cells (BMC) of normal mice, leptin induced only granulocyte-macrophage (GM) colony formation in a dose-dependent manner, and no other types of colonies were detected even in the presence of erythropoietin (Epo). Leptin also induced GM colony formation from BMC of db/db mutant mice whose leptin receptors were incomplete, but the responsiveness was significantly reduced. The effect of leptin on GM colony formation from BMC of normal mice was also observed in serum-free culture, and comparable with that of GM-colony-stimulating factor (CSF ). Although leptin alone supported few colonies from BMC of 5-fluorouracil (5-FU)-treated mice in serum-free culture, remarkable synergism between leptin and stem cell factor (SCF ) was obtained in the colony formation. The addition of leptin to SCF enhanced the SCF-dependent GM colony formation and induced the generation of a number of multilineage colonies in the presence of Epo. When lineage (Lin)-Sca-1(+) cells sorted from BMC of 5-FU-treated mice were incubated in serum-free culture, leptin synergized with SCF in the formation of blast cell colonies, which efficiently produced secondary colonies including a large proportion of multilineage colonies in the replating experiment. In serum-free cultures of clone-sorted Lin-c-Kit+Sca-1(+) and Lin-c-Kit+Sca-1(-) cells, although synergism of leptin and SCF was observed in the colony formation from both cells, leptin alone induced the colony formation from Lin-c-Kit+Sca-1(-), but not Lin-c-Kit+Sca-1(+) cells. These results have shown that leptin stimulates the proliferation of murine myelocytic progenitor cells and synergizes with SCF in the proliferation of primitive hematopoietic progenitors in vitro.     

An in vivo/in vitro method for assessing micronucleus and chromosome aberration induction in rat bone marrow and spleen. 2. Studies with chlorambucil and mitomycin C

An in vivo/in vitro system using rat bone marrow cells and spleen cells to assess micronucleus (MN) and structural chromosome aberrations (SCA) simultaneously (Moore et al., 1995) was further developed. In two separate experiments, two rats/dose/experiment were treated i.p. with 0, 5, 10 and 15 mg chlorambucil (CA)/kg or with mitomycin C (MMC) at 0, 1, 2, 4 mg/kg (experiment 1) or 0, 4, 6, and 8 mg/kg (experiment 2) and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested 24 h after establishment of cultures, while spleen cells were harvested at 48 h. In addition, spleen cells were concurrently assayed for chromosome aberrations. With the MN endpoint, spleen cells appeared more sensitive than bone marrow cells to the effects of CA due both to a lower background and an increased response. For MMC, bone marrow cells exhibited both a higher background of MN and a greater numerical response than did spleen cells. However, on the basis of a fold-increase over control values, spleen cells were more sensitive than bone marrow cells. In general, the MN endpoint appeared more sensitive than the SCA in spleen cells after treatment with CA or MMC. Thus, the approach described here shows greater potential in detecting genotoxicity.     

Endotoxin-induced size change in bone marrow progenitors of granulocytes and macrophages

Injection of 5 mug endotoxin to adult C57BL mice caused a marked increase in the sedimentation velocity of granulocytic and macrophage progenitor (colony-forming) cells in the bone marrow. This change was maximal two days after injection and was not accompanied by corresponding changes in total marrow nucleated cell populations. The endotoxin-induced shift was not dependent on the presence of the thymus but did not occur in mice challenged after preinjection with endotoxin. No changes in buoyant density, cell cycle status, pattern of differentiation and responsiveness of granulocytic and macrophage progenitor cells were observed after the injection of endotoxin. The increased sedimentation velocity of progenitor cells appears to indicate an increase in cell volume but the mechanisms involved have not been identified.     

Ultrastructural characteristics of Fc epsilon R-positive basophils in the spleen and bone marrow of mice immunized with goat anti-mouse IgD antibody

BACKGROUND: Previous work showed that injection of mice with goat anti-mouse IgD antibodies results in increased numbers of Fc epsilon R-positive, non-B, non-T cells in the spleen and Fc epsilon R-positive cells in the bone marrow, and that some of these cells had ultrastructural features of basophils. Fc epsilon R-positive, non-B, non-T cells express virtually all of the capacity of mouse splenic "non-B, non-T cells" to produce interleukin-4 in response to stimulation by cross-linking of Fc epsilon R or Fc gamma R, or by the calcium ionophore, ionomycin. EXPERIMENTAL DESIGN: The present study is a detailed ultrastructural analysis of Fc epsilon R-positive bone marrow cells or Fc epsilon R-positive splenic non-B, non-T cells sorted from mice injected with goat anti-mouse IgD antibody and of Fc epsilon R-positive bone marrow cells or spleen cells pooled from normal mice not injected with goat anti-IgD. RESULTS: Basophils represented the majority (90%) of the granulated cells present in the Fc epsilon R-positive splenic non-B, non-T cells or Fc epsilon R-positive bone marrow cells of goat anti-IgD-injected mice. In contrast, the cytoplasmic granule-containing Fc epsilon R-negative cells sorted from spleen or bone marrow of goat anti-IgD-injected animals contained predominantly a mixture of neutrophils, eosinophils, monocytes and their precursors. Both the Fc epsilon R-positive and -negative preparations contained rare (< 5%) cells with ultrastructural features of very immature mast cells. Basophils were also identified in Fc epsilon R-positive cells sorted from total bone marrow cells or spleen cells of normal mice not injected with goat anti-IgD. CONCLUSIONS: Taken together with data concerning the numbers of Fc epsilon R-positive, non-B, non-T cells in the spleen, and Fc epsilon R-positive B220-negative cells in the bone marrow, these ultrastructural findings indicate that injection of mice with goat anti-IgD results in increased numbers of basophils, particularly in the spleen, that exhibit an 8-fold increase in basophils as a result of injection of goat anti-IgD.     

Differential sensitivity of human myeloma cell lines and normal bone marrow colony forming cells to a recombinant diphtheria toxin-interleukin 6 fusion protein

The cytotoxicity of a recombinant interleukin 6 (IL-6)-diphtheria toxin (DT) fusion protein towards human myeloma cell lines was investigated. DAB389-IL-6 inhibited protein synthesis and methylcellulose colony formation by U266 myeloma cells. In the clonogenic assay, the fusion protein approached the level of cytotoxicity achieved by native DT. The specificity of killing by DAB389-IL-6 was demonstrated by inhibition of cytotoxicity by a molar excess of free rhIL-6. The effect of DAB389-IL-6 on colony formation by six OCI-My cell lines was assessed. Similar to U266 cells, colony growth by the OCI-My 5 and -My 2 cell lines was inhibited in a simple dose dependent manner. However, a biphasic effect was observed for the IL-6 dependent OCI-My 4 cells; DAB389-IL-6 stimulated colony formation at low (< or = 10(-11) M) concentrations, yet was inhibitory at higher doses. Three other cell lines whose growth was not altered by IL-6 were relatively unaffected by DAB389-IL-6, despite their sensitivity to native DT. Flow cytometric analysis for IL-6 receptor expression using phycoerythrin-conjugated IL-6 demonstrated specific binding sites on both DAB389-IL-6 sensitive and certain insensitive cell lines, suggesting that other factors in addition to the expression of IL-6 receptors are involved in killing by the fusion toxin. Despite evidence for a role of IL-6 in myeloid cell development, normal bone marrow was insensitive to the IL-6 fusion toxin. In cultures containing both normal bone marrow and U266 cells DAB389-IL-6 effectively inhibited the growth of U266 myeloma colonies but had little effect on normal bone marrow erythroid, granulocyte and mixed erythroid/granulocyte colony growth. From these experiments we conclude that DAB389-IL-6 is specifically cytotoxic towards a subset of IL-6-responsive human myeloma cell lines and may be useful, in some cases, in the selective elimination of tumour cells from mixed populations of normal and malignant cells.     

The risk of osteoarthritis with running and aging: a 5-year longitudinal study

Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free Dulbecco's modified Eagle's medium containing 10 mM nicotinamide and 10 ng/ml of epidermal growth factor (EGF), acidic and basic fibroblast growth factors (FGF), hepatocyte growth factor (HGF), or transforming growth factor-alpha (TGF-alpha). Every colony consisted of cells that each had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically the cells induced by any mitogen were stained with albumin, transferrin, cytokeratin-8 and -18. But these cells expressed neither cytokeratin-7 nor -19. When 6 x 10(5) cells were plated on 35-mm dishes, about 15 colonies per 1,000 attached cells were observed in the cultures treated with EGF, HGF, and TGF-alpha. Although FGFs could also induce colonies, their number was less than half of the number induced by EGF. Furthermore, the numbers of colonies induced by the combinations of EGF+HGF, EGF+TGF-alpha, and HGF+TGF-alpha were not different from those of the colonies induced by each mitogen alone. To examine the ability of co-mitogenic factors to induce small-cell colonies, angiotensin-II, insulin-like growth factor-I, norepinephrine, tumor necrosis factor, and vasopressin were used. In the cells cultured without EGF, these co-mitogens neither stimulated DNA synthesis nor induced colonies. On the other hand, in cells cultured with both EGF and each co-mitogen, although the DNA synthesis of the hepatocytes was enhanced, the number of colonies detected was not significantly different from the number which EGF alone could induce. These results showed that the small-cell colonies in primary cultures of rat hepatocytes were inducible by EGF, HGF, TGF-alpha, or FGFs and that the co-mitogens did not influence the formation of the small-cell colonies.