Alterations to maternal cortical and trabecular bone in multiparous middle-aged mice.

OBJECTIVES: During the reproductive cycle, altered calcium homeostasis is observed due to variable demand for mineral requirements. This results in increased bone resorption during the time period leading up to parturition and subsequent lactation. During lactation, women will lose 1-3% of bone mineral density per month, which is comparable to the loss experienced on an annual basis post-menopausal. The purpose of this study was to determine the effect of parity on bone formation in middle-aged mice. METHODS: Mice were mated and grouped by number of parity and compared with age matched nulliparous controls. Measurements were taken of femoral trabecular and cortical bone. Calcium, protein and alkaline phosphatase levels were also measured. RESULTS: An increase in trabecular bone mineral density was observed when comparing mice that had undergone parity once to the nulliparous control. An overall decrease in trabecular bone mineral density was observed as parity increased from 1 to 5 pregnancies. No alteration was seen in cortical bone formation. No difference was observed when calcium, protein and alkaline phosphatase levels were assessed. CONCLUSIONS: This study demonstrates that number of parity has an impact on trabecular bone formation in middle-aged mice, with substantial changes in bone density seen among the parous groups.

Which plants are needed for a French vertical-flow constructed wetland under a tropical climate?

Plants are essential in the functioning of constructed wetlands. When setting up systems in tropical areas, Phragmites australis is not always a good choice because of its invasiveness. In vertical-flow constructed wetlands (VFCWs) fed with raw wastewater, the main role of plants is their mechanical action, which helps prevent clogging of the deposited organic matter. Various species have already been used in some tropical climate studies, but generally not for such systems, and no attempt has been made to screen large numbers of alternative species. Here we describe a method to select species among a hundred studied, along with promising plants tested in batches, and at full scale. Species of the order Zingiberales showed good adaptation to the main stresses generated by VFCWs. They have long vegetative cycles, which may require weed growth control after plantation, but low harvesting frequency. Root systems with long rhizomes such as Heliconia psittacorum should take priority to ensure even growth and avoid clumps. To limit the phytosanitary risk with Musaceæ (banana tree), Canna indica or Canna glauca are preferable. Species of the genus Cyperus also demonstrate good adaptation, and could be of interest, especially when a high stem density is required (e.g. planted sludge drying beds).

Regulation of macrophage and granulocyte proliferation. Specificities of prostaglandin E and lactoferrin.

Hemopoietic colony-forming cells committed to macrophage differentiation (M-CFC) are selectively and differentially inhibited by prostaglandin E (PGE). A hierarchy of sensitivity was observed among murine CFC stimulated by colony-stimulating factors (CSF) which differ in their ability to initiate proliferation of morphologically distinct colony types, or stimulated by CSF provided by macrophage feeder layers. Inhibition of macrophage colony formation to 50 percent levels occurred with PGE concentrations between 10(-8) and 10(-9) M, and was still evident at 10(-10) -10(-11) M PGE concentrations. The growth of mixed colonies containing both macrophages and neutrophils was less sensitive to the inhibitory effects of PGE, however, the monocytoid component of these colonies was reduced in the presence of PGE. Neutrophil progenitor cell proliferation was not influenced by PGE concentrations below 10(-6) M, regardless of time of addition of PGE, whereas clonal macrophage expansion, as well as clone size, was sensitive to inhibition by PGE when added as late as 3 d after culture initiation. Prostaglandin F(2alpha), was not inhibitory to colony formation. Experimental evidence for a selective role of macrophage PGE in the regulation of macrophage colony formation was directly provided by utilizing resident peritoneal macrophages as a source of CSF for bone marrow target cell overlays. Simultaneous morphological analysis of colonies proliferating in bilayer culture in response to increasing concentrations of macrophages, and direct measurements of PGE synthesized by an identical number of macrophages maintained in liquid culture demonstrate that a specific decline in macrophage colony formation occurs coincident with a linear increase in macrophage PGE synthesis. Inhibition of macrophage PGE synthesis by indomethacin results in the specific enhancement of macrophage colony formation. Furthermore, macrophage PGE synthesis is induced by CSF preparations with the selective capacity to differentially stimulate macrophage proliferation, but not by those which preferentially stimulate granulocyte colony formation. In comparison to the effects of PGE on M-CFC, polymorphonuclear granulocyte-derived lactoferrin (LF) reduces macrophage production of colony-stimulating activities for macrophage, mixed macrophage- neutrophil and neutrophil colony formation. The ability of LF to reduce macrophage PGE synthesis, presumably by decreasing CSF production, suggests that LF and PGE can interact in the control of macrophage and granulocyte proliferation.

A small, nonpeptidyl mimic of granulocyte-colony-stimulating factor [see commetns].

A nonpeptidyl small molecule SB 247464, capable of activating granulocyte-colony-stimulating factor (G-CSF) signal transduction pathways, was identified in a high-throughput assay in cultured cells. Like G-CSF, SB 247464 induced tyrosine phosphorylation of multiple signaling proteins and stimulated primary murine bone marrow cells to form granulocytic colonies in vitro. It also elevated peripheral blood neutrophil counts in mice. The extracellular domain of the murine G-CSF receptor was required for the activity of SB 247464, suggesting that the compound acts by oligomerizing receptor chains. The results indicate that a small molecule can activate a receptor that normally binds a relatively large protein ligand.

Resilience and reliability of compact vertical-flow treatment wetlands designed for tropical climates.

Most of the tropical areas have sanitation problems to contend with. The French system of vertical-flow treatment wetlands (FS-VFTW) fed with raw wastewater could be a good water and sludge management solution. The purpose-adapted tropical design can reduce area requirement to below 1 m2/population equivalents (p.e.). The Taupinière FS-VFTW on Martinique Island was built according to this design, with one stage but with a saturated layer at the bottom of the filter and a simplified trickling filter (TF) added for further treatment to meet the high performances targeted. Unsaturated/saturated vertical-flow filters (US/S FS-VFTW) have shown improved performances on total nitrogen, carbon and suspended solids removal in temperate climates, but the performances in tropical conditions remain unknown. Here, we report on real-world-operation in the French Overseas Territories (FOT), the reliability and performances of this VFCW tropical-design. The system experienced loading conditions ranging from 30% to 165% of nominal carbonaceous biological oxygen demand (BOD5), as well as tropical rainstorms that brought over 7 times the nominal hydraulic load. Over a period of 3 years, 29 campaigns collected 24-h flow-proportional samples at each treatment stage (raw wastewater, FS-VFTW outlet, TF outlet). When applied loads were close to nominal values, the US/S FS-VFTW itself guarantees 85/90/60/50% removal and 125/25/40/50 mg/L at the outlet for chemical oxygen demand (COD)/total suspended solids (TSS)/total Kjeldahl nitrogen (TKN)/total nitrogen (TN), respectively. By comparison with US/S systems in mainland France, it appears that the warmer tropical-climate temperatures facilitate both nitrification and denitrification kinetics. Performances in overload conditions confirm that the US/S FS-VFTW remains robust and reliable although COD and TKN removal are impacted, especially after strong tropical rain events. By adding a simple compact trickling filter to a US/S FS-VFTW, the treatment system delivers high-level performances (>95% removal for BOD5, COD, TSS and TKN) at less than 1 m2/p.e.

Association between colony forming units-granulocyte macrophage expression of Ia-like (HLA-DR) antigen and control of granulocyte and macrophage production. A new role for prostaglandin E.

The expression of Ia-like antigens on human colony forming units-granulocyte macrophage (CFU-GM) is related to S-phase of the cell cycle, and associated with the control of normal granulocyte and macrophage production by prostaglandin E and acidic isoferritins in vitro. Ia-antigen expression by CFU-GM is lost within 3-6 h of culture at 37 degrees C and occurs simultaneously with loss of responsiveness to inhibition by these factors. Culture of bone marrow CFU-GM in a limited exposure suspension culture with 1 microM-1pM prostaglandin E (PGE1 or PGE2), but not prostaglandin F2 alpha or dibutyryl-cyclic-3'-5'-AMP results in the detection of CFU-GM Ia-antigen after 24 h. Antigen expression is associated with an absolute increase in total and S-phase CFU-GM, and restoration of responsiveness to inhibition by prostaglandin E and acidic isoferritins. The detection of Ia-antigen on CFU-GM after suspension culture with prostaglandin E results both from Ia-antigen reexpression as well as stimulation of noncycling cells to enter S-phase, express Ia-antigen and give rise to CFU-GM sensitive to inhibition by prostaglandin E and acidic isoferritins. The sensitivity of CFU-GM to inhibition by these factors after suspension culture with prostaglandin E is identical to that of the same cells tested prior to the suspension culture. These studies provide evidence for a direct regulatory association between Ia-antigen expression and control of myeloid progenitor cell differentiation, and suggest a role for prostaglandin E in the control of CFU-GM cell cycle, Ia-antigen expression, and growth regulation.

Multiple lymphokine production by a phorbol ester-stimulated mouse thymoma: relationship to cell cycle events.

Interleukin 2 (IL-2) production was studied in a subclone of the murine thymoma EL 4. Phenotypic characterization revealed the EL 4-17-2 line to be Thy-1.2+, Lyt-1.2+, and Lyt-2.2-. Costimulation with 500 ng 12-O-tetradecanoylphorbol 13-acetate (TPA)/ml and 5 micrograms concanavalin A (Con A)/ml induced optimal levels of IL-2. Three related phorbol esters stimulated comparable levels of IL-2 when used in conjunction with Con A. Kinetic experiments indicated that IL-2 first became detectable at 2 hours in TPA-treated cultures, whereas in cultures stimulated with Con A alone IL-2 production was not evident until 8 hours. Flow cytometry indicated that TPA and its related phorbol esters cause a perturbation in the cycling of the cell which may be related to increased IL-2 production. Under the conditions examined, no interferon-gamma (IFN-gamma) was detectable. Conversely, both granulocyte-macrophage colony-stimulating factor (CSF-GM) and interleukin-3 (IL-3) were found under conditions that led to stimulation of IL-2 synthesis. CSF-GM was produced in cultures treated singly with 500 ng TPA/ml or with Con A. IL-3 production was similar to IL-2 production, because optimal levels were found in cultures after combined treatment with phorbol ester and mitogen.

Cell cycle specific effects of deferoxamine on human and murine hematopoietic progenitor cells.

The effect of the iron chelator deferoxamine (DSF) on the proliferation of normal erythroid and granulocyte-macrophage progenitor cells from human and murine bone marrow was examined. The addition of DSF at a concentration equivalent to the concentration of iron present in the culture system resulted in dose dependent inhibition of colony formation by human and murine granulocyte-macrophage progenitor cells and human normal erythroid progenitor cells. The addition of FeCl3 at culture initiation completely reversed the effects of DSF. Furthermore, significant numbers of progenitor cells could be rescued from the effects of DSF by iron added back as late as 24-48 h after exposure to DSF. The cell cycle specificity of DSF was also examined using bone marrow cells treated with high specific activity tritiated thymidine. Kinetic experiments demonstrated that in the presence of DSF the number of erythroid or granulocyte-macrophage colonies that could be rescued was dependent on the length of exposure to DSF. Comparisons between control and tritiated thymidine treated cells indicated that the proliferation of progenitor cells in S phase of the cell cycle was inhibited if iron was withheld until 6 and 24 h after exposure to DSF for murine and human cells, respectively, with little to no effect observed on progenitor cells not in S phase during this time period. These results confirm the importance of iron for hematopoietic progenitor cell proliferation and represent a new method by which the proliferation of cycling cells may be investigated in situ in semisolid culture systems.

Abnormal responsiveness of granulocyte-macrophage committed colony-forming cells from patients with chronic myeloid leukemia to inhibition by prostaglandin E1.

The formation of myeloid colonies in soft-agar cultures of normal human marrow was markedly inhibited by prostaglandin E. Morphological characterization of colonies in the presence or absence of prostaglandin E1 showed that inhibition was restricted to monocytoid rather than neutrophil differentiation. Myeloid colony formation by granulocyte-macrophage-commited colony-forming cells from patients with chronic myeloid leukemia was not inhibited even by high concentrations of prostaglandin E and was independent of colony morphology. The altered sensitivity of leukemic colony-forming cells to prostaglandin E was observed at all stages of the disease and persisted following chemotherapy-induced reversion to a partial or complete Philadelphia chromosome-negative bone marrow status. This evidence suggests that altered myeloid stem cell sensitivity to a normal regulatory factor may play a role in the pathophysiology of chronic myeloid leukemia.

Phase I trial of recombinant interferon gamma in cancer patients.

Interferon gamma (IFN-gamma) is a lymphokine with potent in vitro effects on cell growth and immune function. We have investigated the effects of rIFN-gamma (sp act approximately 2 X 10(7) U/mg, purity greater than 99%) in 16 evaluable patients with advanced malignancy in a phase 1 trial. Patients were treated with six-hour intravenous (IV) infusions daily, five days a week for 2 weeks. After a 2-week rest period, the IV treatment cycle was repeated. Responders were maintained on repeated IV treatment cycles or daily intramuscular (IM) injections. Patients were entered at fixed dose levels of 0.1, 0.5, or 1.0 mg/m2/d. The maximum safely tolerated dose was 0.5 mg/m2. The most common side effects were constitutional symptoms, including fever, chills, fatigue, and myalgias. Reversible and transient increases in hepatic transaminase and decrease in granulocyte counts were seen. Treatment was associated with a dose-dependent increase in serum levels of beta 2 microglobulin. Partial responses (PRs) were observed in one patient with Hodgkin's disease and one patient with chronic lymphocytic leukemia. Fairly constant levels of serum IFN were found at four and six hours during infusion, followed by a rapid decline within one to two hours. We conclude that rIFN-gamma can be safely administered by a six-hour IV infusion and that it can induce in vivo some of the biologic effects reported in in vitro studies.

Marrow cytogenetic and cell-culture analyses of the myelodysplastic syndromes: insights to pathophysiology and prognosis.

Marrow cytogenetic and granulocyte-macrophage colony formation (CFU-GM) studies were performed on 34 previously untreated patients with documented myelodysplastic syndromes seen between January 1978 and June 1982. All patients were managed without chemotherapy until progression to acute leukemia was observed. All 10 patients with exclusively abnormal marrow metaphases developed acute leukemia (100%) while only one (7%) of 14 patients with solely normal marrow metaphases subsequently developed leukemia (p less than 0.001). Three (42%) of the seven patients with both normal and abnormal marrow metaphases developed acute leukemia. Fifteen (86%) of the 19 patients with either large cluster or no growth patterns developed acute leukemia while only two (13%) of 15 patients with either small cluster or colony forming growth patterns developed acute leukemia (p less than 0.001). Abnormal marrow cytogenetic status correlated with abnormal marrow CFU-GM growth pattern (p less than 0.05). Analysis of CFU-GM sensitivity to inhibition by prostaglandin E was performed in 12 patients. Nine patients showed CFU-GM refractoriness to inhibition by prostaglandin E. Seven of these patients eventually developed leukemia. Three patients had CFU-GMs which were initially sensitive to prostaglandin E inhibition. In these three patients, a loss of CFU-GM sensitivity to prostaglandin E was observed prior to their progression to morphologically identifiable acute leukemia.

Differential expression of class II MHC antigens in subpopulations of human hematopoietic progenitor cells.

Expression of major histocompatibility complex class II Ags HLA-DR, HLA-DP, and HLA-DQ on human BM granulocyte-erythroid-macrophage-megakaryocyte CFU (CFU-GEMM), BFU-E, and CFU-GM was examined by indirect immunofluorescence, cell sorting, and complement-mediated cytotoxicity. BM, highly enriched for progenitor cells by depletion of mature hematopoietic elements, was further separated by sterile sorting into HLA-DR (-), low, intermediate, and high intensity HLA-DR (+), as well as HLA-DP (+) and HLA-DP (-) cell fractions and assayed for progenitor cell content. In addition, in the case of HLA-DR, CFU-GM response to inhibition by prostaglandin E was determined. Cell sorting and cytotoxicity data confirm that approximately 95% of assayable erythroid, myeloid, and multipotential progenitor cells expressed HLA-DR, whereas HLA-DQ Ags were undetectable. HLA-DR and HLA-DP Ags were co-expressed on 61% of these progenitor cells, predominantly those expressing HLA-DR at high intensity. Day 7 and 14 CFU-GM showed a trend toward segregation to the high HLA-DR (+) cell fractions, especially when recombinant human G-CSF was used to stimulate clone formation. Both day 7 and day 14 CFU-GMs were found predominantly in the HLA-DP (+) cell fraction. In contrast, BFU-E and CFU-GEMM were found in the low intensity HLA-DR cell fraction and predominantly in the HLA-DP (-) fraction. Both eosinophil CFU and cells giving rise to basophil/mast cells in suspension culture were found in the low and intermediate intensity HLA-DR fractions, but could be segregated into HLA-DP (+) and HLA-DP (-) cell fractions, respectively. Functional analysis of day 7 CFU-GM segregated, based upon HLA-DR intensity, indicated a positive correlation between increasing HLA-DR intensity and responsiveness to inhibition by prostaglandin E. Furthermore, only those CFU-GM expressing HLA-DR at high intensity could be removed by cytolytic treatment using a mAb anti-HLA-DR previously shown to be selective for CFU-GM responsive to PGE and in S phase of the cell cycle.

Prostaglandin-E-mediated modulation of human marrow CFU-GM Ia-antigen expression: kinetics and specificity.

In suspension culture, human granulocyte-macrophage colony-forming cells CFU-GM lose the ability to express Ia antigen within 3-6 h and coincidently become insensitive to inhibition by prostaglandin E (PGE) and acidic isoferritins (AIF). However, when cultured in the presence of PGE, Ia+ CFU-GM sensitive to inhibition by these regulatory factors can be detected after 24 h. Kinetic analysis indicates that the effects of PGE on CFU-GM Ia-antigen expression and growth response detected after 24 h in culture are (a) initiated within the first 3 h in suspension phase, (b) are essentially irreversible under the culture conditions employed, and (c) persist for up to seven days provided PGE is added at culture initiation. The demonstrated capacity of PGE to modulate day-7 CFU-GM Ia antigen and consequently responsiveness to inhibition extends also to day 14 CFU-GM. The effects of PGE on progenitor cell Ia-antigen expression appear specific for prostaglandins of the E series (PGE1, PGE2) and do not appear to be mediated via cyclic AMP. These studies provide additional evidence for a direct regulatory association between Ia antigens and control of hemopoietic cell proliferation, and support and further define a new role for PGE in the positive regulation of myeloid cell differentiation.

In vivo modulation of myelopoiesis by prostaglandin E2. II. Inhibition of granulocyte-monocyte progenitor cell (CFU-GM) cell-cycle rate.

The effects of in vivo administration of native prostaglandin E2 (PGE) on the cycling status of the granulocyte-monocyte progenitor cell (CFU-GM) were examined in a mouse model. In mice hematopoietically rebounding from a sublethal dose of cyclophosphamide, intravenous injection of PGE in doses ranging from 100 to 10(-4) micrograms/mouse/day over three days, significantly decreased the absolute number of CFU-GM per femur and the percentage of CFU-GM in the S-phase of the cell cycle. Administration of PGE2, 10 micrograms/mouse/day, over three days to steady-state mice resulted in significant suppression of the absolute number of CFU-GM per femur, but was without effect on CFU-GM cycling. However, when an additional PGE2 injection was included on day 4 and mice killed 6 h later, inhibition of both parameters was observed. Single bolus injection of PGE2 into steady-state mice was found to have the identical effect. Inhibition of the absolute number of CFU-GM per femur and percentage of CFU-GM in S-phase were apparent 6 h after injection of a single dose of 10 micrograms PGE, with a loss of effect on CFU-GM cycling status 12-14 h after injection. The inhibitory effect of PGE2 on the absolute number of CFU-GM per femur persisted for two days after injection. These studies confirmed the cell-cycle effects of PGE, heretofore demonstrated only in vitro; illustrated the importance of timing and dose analysis when examining the effects of hematopoietic inhibitors in vivo; and suggested that PGE2 may be useful as an adjunct to chemotherapy in ameliorating myelotoxicity. Furthermore, dose-response analysis revealed an enhanced in vivo sensitivity of macrophage progenitor cells (M-CFC) to the inhibitory effects of injected PGE2. Inhibition of in vivo incorporation of tritiated thymidine (3HTdr) into bone marrow CFU-GM by PGE could also be demonstrated.

Modulation of the expression of HLA-DR (Ia) antigens and the proliferation of human erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells by prostaglandin E.

The relationship between the presence of Ia-like antigens on human CFU-GEMM and BFU-E, and their responsiveness to the regulatory effects of AIF and PGE have been studied using normal human bone marrow cells. In primary methylcellulose culture the addition of 10(-6)-10(-9) M PGE1 results in the enhancement of the total number of BFU-E detected, with no observed effect on the number of CFU-GEMM. Addition of acidic isoferritins to primary cultures results in an approximately 50% inhibition of both BFU-E and CFU-GEMM proliferation. Removal of Ia+ cells by cytotoxic treatment with monoclonal antihuman HLA-DR (Ia) antibody plus C' resulted in: (a) reduction of total CFU-GEMM and BFU-E by approximately 50%, (b) abrogation of the enhancing effect of PGE on BFU-E, and (c) detection of populations of CFU-GEMM and BFU-E that are no longer sensitive to inhibition by AIF. Culture of marrow cells in suspension culture at 37 degrees C for 24 h prior to methylcellulose culture resulted in the loss of detectable Ia antigen on BFU-E and CFU-GEMM, loss of their responsiveness to AIF, loss of the enhancing effect of PGE on BFU-E, and the inability to detect cycling cells. Exposure of marrow cells to PGE, however, during the suspension phase augmented the total number of BFU-E, and CFU-GEMM detected and resulted in the detection of S-phase cells, expression of Ia antigens of both BFU-E and CFU-GEMM, and restoration of the ability to detect BFU-E and CFU-GEMM sensitivity to inhibition by AIF. After suspension culture with PGE, no further enhancement of BFU-E by PGE was observed. These results indicate that the expression of Ia antigens is important in the regulation of BFU-E and CFU-GEMM proliferation and add further evidence for a role for PGE in controlling progenitor cell Ia-antigen expression, cell cycle and, as a consequence, their proliferative capacity.

Stimulation of human hematopoietic progenitor cell proliferation and differentiation by recombinant human interleukin 3. Comparison and interactions with recombinant human granulocyte-macrophage and granulocyte colony-stimulating factors.

Recombinant human interleukin 3 (IL3) produced in Escherichia coli was purified and its activities examined in cultures of highly enriched human bone marrow progenitor cells. Human IL3 stimulated multipotential (CFU-GEMM) and erythroid (BFU-E) progenitor cells, generating 95% more BFU-E than recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). No further enhancement of BFU-E or CFU-GEMM occurred when IL3 and GM-CSF were used in combination. Human IL3 was more effective than GM-CSF in stimulating granulocyte-macrophage colony-forming cells (CFU-GM) in short-term suspension cultures, but did not induce an increase of CFU-GM, BFU-E, or CFU-GEMM above input levels. IL3 was more active on day-14 (d14) than on d7 CFU-GM, similar to GM-CSF, but generated fewer and smaller CFU-GM-derived clones than either GM-CSF or granulocyte CSF (CI-CSF). The simultaneous addition of plateau levels of IL3 and GM-CSF resulted in an infra-additive augmentation of d7 and d14 CFU-GM-derived clones, whereas IL3 and G-CSF enhanced the number and cellularity predominantly of d14 CFU-GM. In liquid cultures, IL3 induced a greater than 100-fold increase in the number of basophil-mast-like cells and eosinophils and allowed maintenance of these cultures for up to 7 weeks. Human GM-CSF was an almost equally potent, stimulus of eosinophil development but had only a marginal effect on basophilic precursors, whereas G-CSF lacked both activities. Therefore, human IL3 is a multilineage hemopoietic growth factor whose activities appear to encompass and extend beyond those of GM-CSF.

Monocyte-conditioned medium and interleukin 1 induce granulocyte-macrophage colony-stimulating factor production in the adherent cell layer of murine bone marrow cultures.

We have studied how production of colony-stimulating factors (CSF) can be induced in murine long-term bone marrow cultures (LTBMC). We found that the adherent cells, but not the nonadherent cells, of LTBMC synthesized and secreted large amounts of CSF upon stimulation with monocyte-conditioned medium (MCM) from the early phase of monocyte culturing. This CSF induced both granulocyte- and macrophage-containing colonies. Interleukin 1 (IL-1) also induced CSF production by the adherent cells, although not to the same extent as MCM. Medium conditioned by E-rosette-positive lymphocytes could not substitute for MCM. CSF production varied in long-term bone marrow cultures less than two weeks old, but thereafter the amount of CSF obtained was relatively independent of the age of the cultures (2-26 weeks). No correlation was found between the content of granulocyte-macrophage colony-forming cells (GM-CFC) in the nonadherent cell fraction of LTBMC and the ability of the adherent cell layer to produce CSF. These results suggest a two-stage process for CSF synthesis. Monocytes produce a factor(s) that, in a second step, leads to bioassayable levels of CSF in the supernatant of adherent cells in LTBMC.

Prostaglandin E acts at two levels to enhance colony formation in vitro by erythroid (BFU-E) progenitor cells.

The prostaglandin E (PGE) enhancement of erythroid colony formation by human bone marrow erythroid progenitor cells (BFU-E) is mediated by a T8+ subset of lymphocytes. Medium was conditioned by bone marrow and blood T-lymphocytes and T-lymphocyte subsets (T8+, T8-, T4+, and T4- cells) in the absence or presence of PGE1 in order to determine if the cells could release a cell-free source of erythroid colony enhancing activity and what the conditions for this release would be. The T-lymphocyte conditioned medium was assayed for its effects on erythroid colony formation by nonadherent low-density T-lymphocyte depleted (NALT-) bone marrow cells plated in the presence of erythropoietin, hemin, phytohemagglutinin-stimulated leukocyte conditioned medium, or medium conditioned by 5637 cells, in the absence or presence of PGE1 and in the presence or absence of serum. PGE1 induced the release of an erythroid colony enhancing activity from the T8+ and T4-, but not from the T8- and T4+ subsets of lymphocytes, but this cell-free source of activity was only apparent if it was tested for colony formation in the presence of added PGE1. The release and action of the PGE1 induced T-lymphocyte erythroid enhancing activity did not require the presence of serum. Erythroid colony formation by NALT- bone marrow cells was not enhanced by PGE1 alone, by medium conditioned by T-lymphocytes in the absence of PGE1, or by PGE1 plus medium conditioned by T-lymphocytes in the absence of PGE1. The results suggest that the PGE1 enhancement of erythroid colony formation occurs by an apparently synergistic action on non-T-lymphocytes by PGE1 itself and by a factor or factors released from T8+ lymphocytes in response to PGE1.

Regulation of colony-stimulating activity production from bone marrow stromal cells by the hematoregulatory peptide, HP-5.

It has been reported that the granulocyte-derived hematoregulatory pentapeptide, HP-5, and its dimer (HP-5b) have potent hematoregulatory properties. The proposed mechanism of action for HP-5b is synergy with colony-stimulating activity (CSA) resulting in enhanced myeloid colony formation in vitro. We now demonstrate that the effects of HP-5b on enhanced colony formation are indirect and mediated by an effect on CSA production by bone marrow stromal cells. Bone marrow stromal cell culture systems from mice, rats, and humans were used as target cells for the action of HP-5 monomer and dimer. Cell-free supernatants from these cultures were assayed for CSA in a murine granulocyte-macrophage colony-forming unit (CFU-GM) assay. Supernatants from stromal cell cultures pulsed for 1 h with HP-5b resulted in increased murine CFU-GM colony proliferation with an estimated half-maximal effective concentration (EC50) of 1-5 ng/ml. This increase in CFU-GM proliferation was neutralized by anti-macrophage colony-stimulating factor (anti-M-CSF) antibodies. The HP-5 monomer was without effect on constitutive CSA production by stromal cells, but it antagonized HP-5b-induced CSA production in a dose-responsive manner with an estimated half-maximal inhibitory concentration (IC50) of 0.2-0.4 ng/ml. The ability of HP-5 monomer to antagonize HP-5b induction of CSA appears specific in that HP-5 monomer failed to alter interleukin 1 (IL-1) or lipopolysaccharide (LPS)-induced stromal cell CSA production.

Detection of luxol-fast-blue positive cells in human promyelocytic leukemia cell line HL-60.

Differentiation of HL-60 cells toward the eosinophilic series has not been reported previously. Eosinophil granule specific staining with Luxol-fast-blue was used to determine if HL-60 cells could differentiate into the eosinophilic lineage. The specificity of the Luxol-fast-blue stain for cells of the eosinophilic series was substantiated by comparison of the staining of cells from a patient with an eosinophilic syndrome by Wright-Giemsa and Luxol-fast-blue. Luxol-fast-blue positivity was most notable in cells found in colonies formed from HL-60 clonogenic cells in semisolid agar medium. Colony and cluster formation was spontaneous but in the presence of medium conditioned by either human placental cells or the human monocyte-like cell line, GCT, Luxol-fast-blue positive colonies and clusters were detected at a higher frequency.