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The effect of aging on the myelopoietic progenitor cell (CFU-GM) numbers in mice was studied with special emphasis on biological rhythms in hemopoiesis. Female C3H mice, 16-, 21- and 26-month-old, were investigated versus 3-month-old controls every 3 hours during the 24-h period at three different times of the year. Strong circadian rhythms were observed in both CFU-GM concentration and content in the femur of all age-groups. The amplitudes and the 24-h mean values were declining in mice aged 21 and 26 months. From 16 months of age, a significant advance of circadian peak phases was observed. The overlapping was variable during the 24-h period. The present results may explain previous inconsistencies regarding myelopoietic progenitor cell numbers in aging mice. Some seasonal differences in rhythmicity patterns were also observed. Fitting of original data to single sinus functions was highly significant, although important details sometimes were obscured.

The density distribution in Percoll gradients of clonogenic cells forming colonies in diffusion chambers (CFU-D) or in agar culture (CFU-GM) was studied in chronic myeloid leukaemia (CML). The density distribution of CFU-GM in CML was homogeneous with peaks within 1.058-1.061 g/ml, which is slightly lower than normal. CFU-D, on the other hand, showed heterogeneous distributions both in CML and in normal controls. Two separable populations of CFU-D were recognized, one with the same or lower density than CFU-GM that formed almost exclusively neutrophilic colonies in diffusion chambers, and a second population concentrating in the density range 1.068-1.075 g/ml which primarily formed macrophage colonies. The second population contained colony-forming cells derived from both Fc-receptor positive and Fc-receptor negative precursor cells, suggesting that at least some colonies in diffusion chambers arise from Fc-receptor positive granulopoietic cells of intermediate maturity and/or monocytes. The concentration of CFU-D in peripheral blood was increased 40- to 100-fold in 2 patients currently off treatment who had increased WBC counts.

This paper reports on the production of tumor necrosis factor (TNF) and granulocyte macrophage colony-stimulating factor (GM-CSF) by cultured mononuclear adherent cells derived from bone marrow of 25 patients affected by myelodysplastic syndrome (MDS) of different FAB subtypes. Mean production of GM-CSF was much lower than in controls, without significant differences among different subtypes. Mean production of TNF was similar in MDS patients and in controls, but noteworthy differences were observed between patients with RA, RAEB and RAEB-t and patients with RARS and CMML. Growth of bone marrow granulocyte macrophage and erythroid progenitors did not correlate with TNF and GM-CSF production, although in MDS subtypes with higher GM-CSF levels, colony growth was slightly higher than in subtypes with lower GM-CSF production.

The radiosensitivity of human cord blood haematopoietic stem cells was measured by assessing colony formation in methylcellulose cultures and diffusion chambers. The results show that fetal colony forming units, granulocytic, monocytic (CFU-GM), are radiosensitive: colonies formed by electron-irradiated CFU-GM, both in spontaneous and in colony stimulating factor (CSF) supplemented cultures decrease in number in an exponential fashion with increasing doses of irradiation. Do values up to 800 rads were, for highly purified fetal lymphoid cells, 160 +/- 88 rads; for whole buffy coat cells, 155 +/- 75 rads; and for buffy coat cells plus exogenous CSF: 155 +/- 77 rads. Extrapolation numbers (N) approached the value of 1. Among cord blood diffusion chambers progenitor cells (DCPC), radiosensitive and radioresistant subpopulations of myelopoietic stem cells exist, as tested over a range up to 1500 rads. Monocytopoietic CFU-D are radioresistant.

A synthetic analog of a hemoregulatory peptide associated with mature human granulocyte (HP5B) has been investigated for inhibitory effects on human myelopoietic stem cells in vitro. In addition, it has been tested for effects on phagocytosis by human granulocytes and monocytes by use of an automatic flow cytometric method. A dose-dependent inhibition of colony formation was found after preincubation of bone marrow cells for 1 h at 37 degrees C in the range 10(7) -10(-11) mol/l. Above or below these concentrations, no inhibitory effects were seen. The degree of inhibition varied from experiment to experiment, indicating variable responsiveness of the donor cells. Maximal effect was of magnitude 90% inhibition, and the optimal dose was 10(7) mol/l. The peptide had no effect on the kinetics of phagocytosis by measurements of the uptake of fluorescent Zymosan particles or Staphylococcus aureus. This may indicate a selective effect on the precursor cells, with no effect on the functional state of their progeny, the granulocytes and monocytes.

The mechanism for agranulocytosis induced by antithyroid drugs is not established. The few available studies have proposed an immune-mediated process against mature granulocytes. We investigated the effect of methimazole and propylthiouracil and serum from a patient with methimazole-induced agranulocytosis on marrow myeloid colony growth. In the presence of normal serum or patient's recovery serum, antithyroid drugs had no effect on the growth of CFU-GM colonies from normal or patient's marrow. However, the patient's serum obtained during agranulocytosis inhibited the in vitro myeloid colony growth from both autologous and allogeneic bone marrow. These results are compatible with an immune-mediated mechanism for methimazole-induced agranulocytosis rather than a direct toxic effect of the drug on abnormally sensitive cells.

Some characteristics of both erythroid and granulocyte monocyte progenitors in human cord blood were compared to those in adult blood and bone marrow. The number of progenitors in cord blood was higher than that in adult blood and bone marrow. Most colonies in cord blood culture were monocyte-macrophage, whereas those from adult blood were largely eosinophilic. Cord blood progenitors had a slower sedimentation velocity than that reported for marrow, but sedimented faster than that for adult blood. A significant proportion of progenitors in cord blood as well as adult marrow was found to be in the DNA synthetic phase of the cell cycle whereas progenitors in adult blood were not. Cord blood BFU-E were more resistant than adult blood BFU-E but cord blood CFU-GM were not different from adult blood CFU-GM with regard to radiation sensitivity. Cord blood CFU-GM appeared to be more radio-resistant than adult marrow GFU-GM. From these results is seems clear that progenitors in cord blood differ in some aspects from those in adult blood and bone marrow.

We report a case of dyskeratosis congenita ( DCG ) with neutropenia, lymphocytopenia and thrombocytopenia. Peripheral blood T lymphocytes (T cells) were proved to have a suppressive effect on the colony forming unit granulocyte-macrophage (CFU-GM). Splenectomy caused a transient increase of neutrophil count with the disappearance of the suppressive T cell activity. However, pancytopenia recurred without re-appearance of suppressive T cell activity.

The expression of CD33 and CD34 antigens on human bone marrow cells was examined by fluorescence-activated cell sorting and colony assays. A marked difference of antigen expression was observed on sorted progenitors of the granulocyte/macrophage lineage (CFU-GM) with respect to enrichment and maturation status. Single-color cell sorting revealed no difference of enrichment by anti-CD33 antibody between day-7 and d-14 progenitors, while anti-CD34 antibody preferentially enriched d-14 colony-forming units (CFU). By two-color cell sorting it could be shown that enrichment of d-14 CFU-GM occurred mostly in the CD34+CD33- and to a lesser extent in the double-positive fraction. In contrast, there was no difference in degree of enrichment between d-7 and d-14 CD34-CD33+ CFU-GM. From these data we conclude that, during early myelopoiesis, CD34 antigens are gradually lost while CD33 antigens are acquired.

Changes occurring in human haematopoiesis with advancing age were studied using an in vitro haematopoietic colony assay in 22 elderly subjects with unexplained anaemia, and in 15 elderly and 15 young subjects without anaemia. Both elderly groups were found to have significantly lower numbers of bone marrow early erythroid-committed progenitors (BFU-E) than the young controls. The elderly anaemic group also showed significantly lower numbers of granulocyte/macrophage progenitors (CFU-GM) than the young controls. However, the responses of the erythroid-committed progenitors in the elderly groups to erythropoietin and burst-promoting activity were similar to those observed in the young controls. Therefore, it is probable that anaemia tends to occur easily in elderly individuals as a result of reduction of reserves of haematopoietic progenitors with advancing age.

Human osteoclasts can be efficiently generated in vitro from cord blood mononuclear cells and derived CFU-GM colonies. However, CFU-M colonies are poorly osteoclastogenic. Short-term (2–48 h) treatment with GM-CSF stimulates osteoclast formation by proliferating precursors, whereas longer exposure favors dendritic cell formation. Introduction: Osteoclasts (OC) differentiate from cells of the myelomonocytic lineage under the influence of macrophage-colony stimulating factor (M-CSF) and RANKL. However, cells of this lineage can also differentiate to macrophages and dendritic cells (DC) depending on the cytokine environment. The aims of this study were to develop an efficient human osteoclastogenesis model and to investigate the roles of granulocyte macrophage-colony stimulating factor (GM-CSF) and M-CSF in human OC differentiation. Materials and Methods: A human osteoclastogenesis model, using as precursors colony forming unit-granulocyte macrophage (CFU-GM) colonies generated from umbilical cord mononuclear cells cultured in methylcellulose with GM-CSF, interleukin (IL)–3 and stem cell factor (SCF), has been developed. CFU-GM, colony forming unit-macrophage (CFU-M), or mixed colonies were cultured on dentine with soluble RANKL (sRANKL) and human M-CSF with and without GM-CSF. Major endpoints were OC number, dentine resorption, and CD1a+ DC clusters. Results: Osteoclast generation from CFU-GM and mixed colonies treated with M-CSF and sRANKL for 7–14 days was highly efficient, but CFU-M colonies were poorly osteoclastogenic under these conditions. Pretreatment of precursors with M-CSF for 7 or 14 days maintained the precursor pool, but OCs were smaller and resorption was reduced. The effect of GM-CSF treatment was biphasic, depending on the timing and duration of exposure. Short-term treatment (2–48 h) at the beginning of the culture stimulated cell proliferation and enhanced OC formation up to 100%, independent of sRANKL. Longer-term GM-CSF treatment in the presence of sRANKL, however, inhibited OC generation with the formation of extensive CD1a+ DC clusters, accompanied by downregulation of c-Fos mRNA. Delaying the addition of GM-CSF resulted in progressively less inhibition of osteoclastogenesis. Conclusions: Human CFU-GM, but not CFU-M, progenitors have high osteoclastogenic potential. GM-CSF plays an important role in osteoclastogenesis and has a biphasic effect: Short-term treatment potentiates OC differentiation by proliferating precursors, but persistent exposure favors DC formation.

BACKGROUND: Ex vivo expansion strategies with different cytokine combinations are currently used by several groups as a means of increasing the number of HPCs for a variety of special clinical applications. Because there is little information on the potential role of IL-10 in such ex vivo expansion models, the effect of this cytokine on the generation of myeloid progenitor cells in suspension cultures was investigated. STUDY DESIGN AND METHODS: On the basis of data from the literature and from new experiments, the combination of SCF and IL-3 at concentrations of 100 ng per mL and 100 U per mL, respectively, was chosen as the standard cocktail. The addition of IL-10 to such cultures resulted in a marked and dose-dependent potentiation of myeloid progenitor cell production. RESULTS: Using unmanipulated leukapheresis components from 13 individuals (including lymphoma and cancer patients and normal donors), the expansion multiple of CFU–GM after 14 days as compared with pre-expansion values was 9.54 ± 2.31 times by SCF/IL-3 and 46.38 ± 7.37 times by the combination of SCF/IL-3 and 100 ng per mL of IL-10 (p

The pattern of emergence of multipotential (CFU-A) and committed (CFU-GM and BFU-E) progenitor cells in peripheral blood has been examined in patients with Hodgkin's disease and non-Hodgkin's lymphoma. Mobilization protocols used chemotherapy with or without granulocyte colony-stimulating factor (n = 8 and n = 5, respectively). In all patients, the numbers of CFU-A, CFU-GM and BFU-E peaked simultaneously, rather than sequentially, suggesting that marrow regeneration after these mobilization protocols occurred from progenitors at all stages of differentiation. We conclude that peripheral blood stem cell harvest strategies based on peak values for total progenitor numbers will also capture maximum numbers of multipotential progenitors. However, the variable relationship between CFU-A and CFU-GM numbers suggests that overall progenitor cell numbers can give only a broad estimate of the absolute numbers of multipotential progenitors in an individual harvest.

The aim of the study was to investigate the influence of gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) used alone and in combination with 2-chlorodeoxyadenosine (2-CdA) on the colony growth of normal granulocyte-macrophage progenitor cells (CFU-GM) from 15 hematologically healthy donors as well as on CFU-GM from 15 chronic myelogenous leukemia (CML) patients in semisolid cultures in vitro. dFdC and 2-CdA were used either separately or in the following combinations of concentrations: (1) 0.25 nM of dFdC and 2.5 nM of 2-CdA; (2) 0.5 nM of dFdC and 5 nM of 2-CdA; (3) 1 nM of dFdC and 10 nM of 2-CdA; (4) 2 nM of dFdC and 20 nM of 2-CdA. We observed that both dFdC and 2-CdA used separately inhibited the growth of colonies formed by normal and CML CFU-GM cells in a dose-dependent manner. Moreover, the combined therapy with dFdC and 2-CdA caused statistically significant inhibition of the colony growth in both normal and CML CFU-GM cultures. In addition, the inhibition of CML CFU-GM colony formation was greater and statistically significant in the case of the combined therapy using higher concentrations of these drugs as compared with inhibition of the growth of normal CFU-GM colonies. These observations were the basis for the evaluation of the interaction type between dFdC and 2-CdA. We have shown that dFdC used in a combination with 2-CdA acts in an additive way on normal and CML CFU-GM cells.

Peripheral blood CD34/4 5 + cell (CD34/45) enumeration is an expensive and labour-intensive investigation but remains the standard assay for optimizing yield and timing of peripheral blood stem cell harvesting (PBSCH). The present study examined the value of the Sysmex SE9000 parameters (WBC, neutrophil count, and immature myeloid index (IMI)) and Sysmex R2000 reticulocyte parameters (absolute, high and medium fluorescence reticulocytes) in predicting the optimum timing of PBSCH in comparison to peripheral blood CD34/45. Sixty-four PBSCH from 23 patients with haematological malignancies were assessed with a variety of mobilization regimes used. Reticulocyte parameters showed high interpatient variability and did not prove clinically useful. IMI did not consistently predict satisfactory PBSCH yield except when > 1000 × 10 6 /l. Peripheral blood CD34/45 was the most useful predictor of yield. IMI > 20 × 10 6 /l was, however, a useful surrogate for predicting a rise in peripheral blood CD34/45 from nadir and proved to be superior to WBC or neutrophil count. A rising IMI is a marker of early regeneration and has a role in determining when to initiate enumeration of peripheral blood CD34/45.

There are considerable differences in haemopoietic activity between young children and adults on the one hand, and between adults and the elderly on the other. A fundamental unanswered question is whether these differences relate to discrete stages or are part of a continuous process. We have sought to define aspects of the haematological ageing process, and have found that results from children with bone marrow failure syndromes differ from age-matched reference values. Haemopoietic cells were obtained from umbilical cord blood, from blood and bone marrow of healthy individuals and from the blood of young patients with bone marrow failure syndromes. Clonogenic myeloid progenitors (CFU-GM) were grown in semi-solid medium to measure their frequency; the proliferative capacity of myeloid progenitors was measured by replating colonies and observing secondary colony formation. We found that the frequency of CFU-GM in normal marrow increased and their proliferative capacity decreased exponentially with age. The proliferative capacity of CFU-GM in normal blood also decreased exponentially with age. This relationship extrapolated back to the levels of proliferation measured for cord blood CFU-GM (age = 0). The proliferative capacities of CFU-GM from children with bone marrow failure syndromes were severely reduced compared with age-matched reference values. These results indicate that a decline in haemopoietic progenitor cell function begins at birth and continues throughout life. This decline may occur prematurely in childhood marrow failure syndromes with a predisposition to leukaemia.

Semisolid agar cultures of committed granulocyte monocyte precursors (CFU-GM) make it possible to study the regulation of normal and abnormal stem cells. In vitro growth of CFU-GM is dependent on the presence of specific stimulators, called CSA (colony stimulating activity) (Metcalf, 1977; Broxmeyer & Moore, 1978). The influence of CSA on CFU-GM may be inhibited by prostaglandins of the E series (Broxmeyer & Moore, 1978; Kurland et al, 1978). Recently it has been demonstrated that chronic granulocytic leukaemia (CGL) CFU-GM are insensitive to prostaglandin E1 (PGE1) over a wide range of concentrations that are inhibitory to normal CFU-GM development (Aglietta et al, 1980; Pelus et al, 1980; Taetle & Koessler, 1980). We report the effect of PGE1 on CFU-GM from patients with idiopathic myelofibrosis (MF).

A class of primitive progenitor cells with high proliferative potential in vitro (HPP-CFC), has been identified in fetal liver and adult bone marrow both in murine and human systems. The kinetic properties of HPP-CFC2 and the more mature granulocyte-macrophage colony forming units (CFU-GM) derived from murine fetal liver on d13, d15 and d19 of gestation, newborn liver and neonatal liver on d3 and d8 postpartum have been evaluated and compared with the kinetic properties of these progenitor cell populations derived from adult bone marrow. The frequency of HPP-CFC2 in fetal liver was found to be greatest on d15 of gestation then subsequently declined in newborn and neonatal liver. Similarly, the highest proportion of HPP-CFC2 engaged in DNA synthesis (53+/-3%) was detected in d15 fetal liver. This proportion decreased to 13+/-2% in the liver 1 wk after birth, which is comparable to the number of HPP-CFC2 derived from adult BM which were in S-phase (10+/-1%). Production of CFU-GM was found to be greater in adult bone marrow than in either fetal or newborn liver. While the proportion of CFU-GM in S-phase was high all 3 tissue samples, the greatest proportion of cycling CFU-GM (50+/-2%) was detected in d15 fetal liver. These results suggest that HPP-CFC2 derived from fetal liver are actively cycling while HPP-CFC2 derived from adult bone marrow are relatively quiescent. In contrast, a high proportion of CFU-GM derived from fetal, newborn liver and adult bone marrow are engaged in DNA synthesis.

Autologous peripheral blood haemopoietic stem cells (PBSC) were harvested from 30 patients with de novo acute leukaemia, 29 of whom had entered remission following standard chemotherapy. Correlation of CD34+ cells/kg to CFU-GM/kg in the harvests was good (correlation coefficient=0.72, P 50×109/l post final induction course (P 20×109/l at time of harvest (P=0.02), time to WBC >1.0×109/l post intensification/mobilization (correlation coefficient=−0.70, P 0.5×109/l rs=−0.72, P=0.001; platelets >20×109/l unsupported rs=−0.71, P=0.001). Our results show that the number of induction courses received, and thus exposure to cytotoxic agents received, made no significant difference to subsequent CFU-GM harvest content. We collected superior harvests from those patients with faster platelet recovery following mobilization therapy. We also found that faster platelet recovery following the final induction therapy was a better predictor of the CFU-GM harvest following mobilization than was the neutrophil recovery following final induction.

Following chemotherapy in chronic myeloid leukaemia (CML), some peripheral blood (PB) cells may be Philadelphia (Ph) chromosome negative. The BCR-ABL mRNA status of residual Ph+ progenitors is not known. We examined the BCR-ABL mRNA status of individual colony-forming-unit granulocyte-macrophage (CFU-GM) colonies derived from PB harvested following chemotherapy. Seven patients were treated with 200 mg/m2/day cytarabine and 20 mg/m2/day Idarubicin and followed by Lenograstim. PB collections commenced daily when the white blood cell count reached 0.6 x 10(9)/l and continued until at least 5 x 10(8)/kg nucleated cells were obtained. CD34+ cells, Ph status, and CFU-GM were estimated at each harvest. For each patient, up to 24 individual CFU-GM colonies were analysed for BCR-ABL status. Two cases were BCR-ABL negative on all colonies and completely Ph-, and another case was BCR-ABL positive in all colonies and completely Ph+. In contrast, in two patients all colonies were BCR-ABL negative, despite virtually complete Ph+ metaphases. The final assessible case had five of nine BCR-ABL negative colonies, despite 94% Ph+ metaphases. After chemotherapy priming, the PB may contain Ph+ CFU-GM that do not express BCR-ABL.

The 'discordant maturation hypothesis' proposes that the most mature proliferating cells in chronic-phase chronic myeloid leukaemia (CML) are responsible for the expansion of the Ph-positive population. To evaluate this hypothesis we used a delta assay for primitive haemopoietic cells (P delta assay for P delta cells) which allows investigation of the kinetics of granulocyte-macrophage progenitor (CFU-GM) production. The frequencies of these primitive (P delta) cells were similar in CML blood (14.5/10(5) mononuclear cells), CML marrow (17.3/10(5)) and normal marrow (11.6/10(5)) The average frequency in normal blood is only 0.58/10(6). The absolute numbers of P delta cells in CML patients are therefore greatly increased. The average numbers of CFU-GM produced by individual P delta cells were reduced in CML blood (8.1) and marrow (11.6) compared with normal marrow (28.5). This is consistent with a reduced probability of differentiation at the single cell level in CML. Although the absolute number of CFU-GM produced by individual CML P delta cells was subnormal there was a relative increase in the number of day 7 CFU-GM compared with the number of day 14 and 21 CFU-GM, which agrees with the 'discordant maturation hypothesis'. This bias towards day 7 colony formation could reflect accelerated maturation by the CFU-GM produced by P delta cells or, alternatively, the production of CFU-GM with shorter than normal maturation pathways. Overall, these results suggest that discordant maturation does not by itself account for myeloid expansion in CML. It is more likely that myeloid expansion in CML is due mainly to an increase in the number of primitive haemopoietic progenitor cells.

We previously reported a successful peripheral blood stem cell harvest by co-administration of recombinant human (rh) interleukin-6 (IL-6) and rh granulocyte colony-stimulating factor (G-CSF) in normal mice. In the present study, to evaluate further the utility of this observation for autologous peripheral blood stem cell transplantation, we examined the effects of rhIL-6 and rhG-CSF on peripheral blood granulocyte-macrophage colony-forming units (CFU-GM) in carboplatin (CBDCA)-induced and irradiation-induced myelosuppressive mouse models. After CBDCA administration, blood cell counts decreased to the nadir, and then recovered to a normal level. In this recovery phase, the peripheral CFU-GM level increased to 3.8-fold higher than the pretreatment level. Administration of rhIL-6 (10 microgram/day) alone induced a 40-fold increase in peripheral CFU-GM from the normal level at day 14. In combination with rhG-CSF (0.35 microgram/day), which alone induced a 74-fold increase, rhIL-6 synergistically increased the CFU-GM level by 1200-fold. In irradiated mice, similar results were observed. Administration of rhIL-6 at 3 and 10 microgram/day significantly increased CFU-GM. Interestingly, in combination with rhG-CSF, a lower dose of rhIL-6 (1 microg/day) could induce CFU-GM increase. We also examined CFU-GM distribution in bone marrow, spleen and peripheral blood. Cytokine administration induced not only a change of CFU-GM distribution, but also an increase in total CFU-GM counts per mouse. These results suggest that co-administration of rhIL-6 and rhG-CSF may be useful for autologous peripheral blood stem cell transplantation.

Carboxyamido-triazole (CAI), an agent that targets calcium-sensitive signal transduction pathways, has both antiproliferative and antimetastatic properties. The objective of this study was to evaluate the myelotoxicity of CAI to normal human and murine hematopoietic cells. In vitro toxicity of CAI was determined by inhibition of myeloid [colony-forming unit-granulocyte/macrophage (CFU-gm)] and erythroid [burst-forming unit-erythroid (BFU-e)] colony formation in clonal assays. The effects of oral CAI on CD2F1 mouse marrow and splenic cellularity, marrow progenitor content, and peripheral blood cell counts were assessed in relation to plasma CAI levels. In vitro, CAI caused a concentration-dependent inhibition of CFU-gm and BFU-e colonies following continuous drug exposure. Murine CFU-gm and BFU-e were inhibited > 90% by 10 and 15 micrograms/mL CAI, respectively. However, suppression of human CFU-gm and BFU-e did not exceed 65% at the same concentrations. In vivo, CAI reduced the number of CFU-gm and BFU-e per femur after the initial dose and through day 4. Variations in colony inhibition paralleled changes in CAI plasma concentrations. While colony inhibition increased in vitro with escalating drug concentrations, this was not observed in vivo with additional CAI doses. The low toxicity of CAI in vivo combined with the significant difference between toxicity for human and mouse progenitors in vitro suggests a relatively low adverse potential to the bone marrow for this new signal transduction inhibitory agent.

Cyclosporin A (CyA) overcomes P-glycoprotein (P-gp) associated multidrug resistance (MDR). P-gp expression is frequently observed among, not only various cancer cells, but also several normal tissues including bone marrow progenitor cells. These findings lead us to examine whether CyA enhances the myelotoxicity of anti-cancer agents. Bone marrow mononuclear cells were incubated with anti-cancer agents (vincristine, VCR; doxorubicin, ADM; etoposide, VP-16; cytarabine, Ara-C; methotrexate, MTX) and a concentration of CyA (0.5, 5.0 micrograms/mL). The methylcellulose assay for granulocyte-macrophage progenitors (CFU-GM) was conducted using the post-treated cells. There was no significant toxicity for marrow CFU-GM formation after 72 h incubation with CyA (84-108% of control). The inhibitory concentration that reduced colonies by 50% (IC50) was 12 nmol/L for VCR, 6 nmol/L for ADM, 220 nmol/L for VP-16, 15 nmol/L for Ara-C and 35 nmol/L for MTX, respectively. For VCR, ADM and VP-16, the number of CFU-GM was unchanged with the addition of CyA at 0.5 microgram/mL concentration. In contrast at 5 micrograms/mL CyA, the number of CFU-GM (% of control) was reduced significantly (P < 0.05 or P < 0.01). With MTX and Ara-C, the number of CFU-GM was unchanged after addition of CyA, even at 5 micrograms/mL concentration. We conclude CyA may therefore enhance cytotoxic drug sensitivity in MDR tumor cells at a clinically achievable concentration (0.5 microgram/mL) without marrow toxicity.

Hematologic abnormalities in the peripheral blood and bone marrow are associated with human immunodeficiency and simian immunodeficiency virus (HIV, SIV) infection. The reasons for these abnormalities remain unclear. Bone marrow specimens collected from uninfected animals (Group A, Controls) and from rhesus macaques experimentally infected with SIVsmm9 during the asymptomatic stage (Group B, SIV+ "well") and during the clinically ill stage (Group C, SIV+ "sick"), underwent a variety of in vitro assays of hematopoiesis. Colony forming unit-granulocyte/macrophage (CFU-GM) per plate growth was 46.7 +/- 7.7, 31.9 +/- 8.4 and 6.5 +/- 5.0 (mean +/- sd, P < .02 each mean compared to the others) in the 3 groups respectively. Burst forming unit-erythroid (BFU-E) growth was similarly decreased in bone marrow samples from the SIV+ animals. There was no change in the number of CFU-GM per plate with the removal of plastic adherent or T-cell mononuclear cell fractions. There was no increase in CFU-GM per plate growth with the addition of low dose GM-CSF (1 or 5 ng/mL) though there was a near 67% increase (48 to 80 CFU-GM per plate) with the addition of 100 ng/mL recombinant rhesus IL-3 and 100 ng/mL GM-CSF in SIV+ "sick" animals. Variation in frequency of CD34+ progenitor cells in SIV+ animals was seen, with 3.0% CD34+ cells in SIV- controls, 4.9% CD34+ cells in SIV+ "well" animals and 0.5% CD34+ progenitor cells in SIV+ "sick" monkeys (P < .01, each mean compared to the others). Finally, there was minimal evidence of SIV sequences by polymerase chain reaction in pooled cultured CFU-GM, and no evidence in flowcytometrically sorted CD34+ progenitor cells from selected animals. Thus, the SIV seropositive rhesus monkey appears to have similar hematopoietic aberrations as are found in HIV infected human subjects and may be an excellent model for studying the interaction of lentiviruses on the kinetics of blood formation.

Rhesus progenitor-enriched BM was exposed overnight to SIV and cultured in a limiting dilution assay where the potential for progenitor interaction with lymphocytes or macrophages was low. Virus was consistently isolated late in culture, detection being aided by coculture with CEM174 lymphoblasts. Although infected cells had reduced clonogenic activity, colonies were indistinguishable from those derived from uninfected BM with respect to proliferative potential, morphology, and longevity in culture. Primate immunodeficiency viruses, therefore, may infect immature BM populations, directly affecting hematopoietic activity.

A 45-year-old Caucasian female with seropositive rheumatoid arthritis was found coincidentally to have a circulating lymphocytosis (6.4 x 10(9)/l) and neutropenia (0.1 x 10(9)/l). Initial presentation was with mouth ulceration and recurrent infections. A spleen scan showed no evidence of splenomegaly and serum titres against EBV, CMV and toxoplasmosis were negative. No anti-neutrophil antibodies were found. Marrow aspiration demonstrated a lymphocytosis of 60% with reduced numbers of granular precursors. Lymphocytes in both blood and bone marrow were CD3, CD8, CD16 and HLA-DR positive. Lymphocyte conditioned medium (LCM) generated from the patient's blood lymphocytes (without phytohaemagglutinin) was found to inhibit allogeneic colony-forming unit, granulocyte-macrophage (CFU-GM) stem cells in semi-solid culture compared with control LCM. This inhibitory activity was abrogated by the cytolytic removal of CD8 cells prior to LCM production and was significantly reduced by co-culture with indomethacin. Culture of the patient's marrow at autostimulatory light density marrow cell concentrations showed poor spontaneous CFU-GM colony formation until marrow CD8 lymphocytes were removed cytolytically. Prednisolone was used therapeutically (40 mg/d) and resulted in the patient's neutrophil count rising from 0.06 x 10(9)/l to 1.1 x 10(9)/l and a fall in the total lymphocyte count to 1.9 x 10(9)/l. Reevaluation of the patient's LCM post steroid therapy showed loss of the previous inhibitory effect. The patient's neutrophil count is maintained on oral azathioprine and indomethacin.

A patient with Philadelphia chromosome (Ph) chronic myelogenous leukemia (CML), in chronic phase, was treated with recombinant gamma-interferon (r gamma-IFN) in a phase I clinical trial. Prior to treatment, analysis of in vitro agar culture parameters indicated hyporesponsiveness of granulocyte-macrophage colony-forming cells (CFU-GM) to inhibition by prostaglandin E and acidic isoferritins and diminished expression of class II major histocompatibility complex (MHC) antigens (HLA-DR). Treatment was associated with no change in bone marrow cellularity or in the percentage of Ph cells. However, in vitro cultures of bone marrow cells showed a return to normal levels of both expression of CFU-GM class II antigen and of sensitivity to inhibition by prostaglandin E and acidic isoferritins which predicted and/or confirmed clinical response. Throughout the course of interferon therapy, white blood cell counts (WBC) and the percentage of bone marrow blast cells were maintained at normal levels. Onset of aggressive-phase disease was associated with increased WBC, an increase in bone marrow blast cells, a secondary chromosomal abnormality, loss of CFU-GM sensitivity to inhibition by putative negative growth regulators, and markedly diminished MHC class II antigen expression. Following a bone marrow transplant from a matched sibling, all hematologic parameters studied were found to be normal. These findings indicate that treatment with r gamma-IFN can modulate some of the abnormal growth characteristics of CFU-GM observed in CML.

In order to investigate collection of committed hematopoietic progenitor cells (CFU-GM) by hemapheresis, four protocols using a discontinuous cell separator (Haemonetics) were evaluated. The combined platelet and granulocytapheresis procedure using hydroxyethyl starch (HES) but no steroids was significantly more efficient (75%) than a standard plateletapheresis (40%, p less than 0.001) or a plateletapheresis modified by either using HES during the collection (49%, p less than 0.05) or pretreating the donors with steroids in an attempt to raise circulating stem cell levels prior to harvesting (46%, p less than 0.001). Donor basal CFU-GM levels were very variable but could be predicted by the circulating lymphocyte levels. In all four protocols, there was a direct correlation between the number of stem cells circulating at the start of the procedure and the final harvest. With this equipment, an appropriate transplant dose of CFU-GM could be provided for a child under 30 kg with less than seven procedures.

In order to determine whether the rise in blood circulating committed hematopoeitic progenitor cells (CFU-GM) was sustained long enough after exercise to be a practical adjunct to increasing stem cell yields by hemapheresis, the authors evaluated two exercise protocols in 15 normal donors. Immediately after a brief period of intense exercise, there was a significant increase in the absolute number of blood CFU-GM from 164 +/- 27 to 240 +/- 46 per ml. However, all values returned to baseline within 15 minutes. Prolonging the exercise did not enhance either the circulating CFU-GM concentration or the duration of the rise. If blood hematopoietic stem cells respond to exercise similarly, such a transient increase would not appear to be of use in stem cell harvesting.

Summary. Five retinoid analogues differ in their effects on the growth of CFU-GM from normal human marrow. At concentrations above 10−6 M all have inhibitory activity. This is maximal with 13-cis-retinoic acid. At lower concentrations the effect of retinoids is usually to reduce clone size rather than clone numbers though 10−8-10−7 M etretinate increases clone size. The incubation of marrow cultures in the presence of 10−6 M all-trans retinoic acid showed that in normal subjects colony counts are never reduced to less than 45% of the control value and total clone counts to less than 70% of the control value. In cultures from patients with myelodysplastic syndrome 20 out of 34 cases showed a greater inhibition of colony numbers than normal and 14 out of 35 cases showed a greater reduction in total clone numbers than normal. The results suggest that CFU-GM from some patients with myelodysplastic syndrome may have a greater sensitivity than normal to the inhibitory effect of retinoic acid.

Granulocyte-macrophage colony-forming (CFU-GM) cells from peripheral blood of normal subjects and patients with chronic granulocytic leukemia (CGL) were cultured in soft agar. Drugs under study were added in a liquid overlay 2 days after initiation of cultures, providing prolonged exposure to these agents thereafter. Dose-dependent inhibition of colony growth was recorded with each of eleven agents examined, and at the higher concentrations tested, colony formation was often completely suppressed. Cytarabine showed selectivity against CFU-GM from patients in the chronic stage of CGL (P = 0.006); the median 50% inhibitory concentration for 12 such patients was 3.4 ng/ml versus 11.8 ng/ml for 15 healthy subjects. Such selectivity was not found with busulfan, hydroxyurea, mercaptopurine, thioguanine, daunorubicin, vincristine, vinblastine, methotrexate, desacetylmethylcolchicine, and trimethylcolchicinic acid. One other group has also reported a preferential effect of cytarabine against colony-forming cells from patients with CGL, and this appears to be the only drug for which such selective activity has been recorded to date.

Mouse bone marrow cells were cultured in vitro in semisolid medium containing various concentrations of beta-lactam antibiotics, moxalactam (LMOX), cefotetan (CTT), flomoxef (FMOX), ceftizoxime (CTZ), cefmenoxime (CMX), cefotasime (CTX), cefamandole (CMD), cefotiam (CTM), carbenicillin (CBPC) or sulbenicillin (SBPC). All of these antibiotics dose-dependently suppressed colony formation from granulocyte or macrophage progenitors (CFU-GM) and from megakaryocyte progenitors (CFU-M). The suppressive potencies of these antibiotics for both progenitors, as evaluated from the drug concentrations that induced 50% suppression of colony formation (IC50), were ranked as the weakest for CBPC and SBPC, moderate for LMOX, CTT, CMX, FMOX and CTZ, and strongest for CMD, CTX and CTM. LMOX, FMOX, CTX, CBPC and SBPC also showed dose-dependent inhibition of colony formation from erythroid progenitors (CFU-E and BFU-E). In the ranking of suppression of colony formation from CFU-E, CMD and CTX showed comparatively strong inhibition but others showed a weak effect, and in that from BFU-E, the effect was weakest for CBPC and SBPC, moderate for LMOX and FMOX, and strongest for CMD and CTX. The suppressive effect of LMOX and CMD on colony formation from each progenitor could almost be prevented by washing the cells after incubation with these antibiotics for 24 hr.

Summary Peripheral blood lymphocytes from 20 patients with neutropenia not consistent with aplastic anaemia were tested for their ability to inhibit the proliferation of normal granulopoietic precursor cells (CFU–GM) in agar culture. Two patients, both with features of an autoimmune disorder, had lymphocytes which were more inhibitory than normal lymphocytes to both normal and their own CFU–GM. Two other patients had lymphaocytes which were more inhibitory than normal lymphocytes to either their own CFU-GM or normal CFU–GM but not both. Eight patients had lymphocytes which were significantly less inhibitory than normal lymphocytes to either normal or their own CFU-GM, but only one showed this feature against both normal and their own CFU-GM. One patient had a highly potent plasma inhibitor of CFU-GM — this patient had received multiple transfusions and had a leucocyte antibody of a broad specificity. No clinical or haematological features were common to any of these groups of patients which reflects the heterogeneity of patients studied and stresses the importance of controls.

Progesterone inhibited proliferation of granulocyte/macrophage progenitor cells (CFU-GM) in human bone marrow cultured in methylcellulose; the inhibition ranged from 8% at 3 X 10(-9) M, 22% at 3 X 10(-8) M to 38% at 3 X 10(-7) M progesterone in 14 non-malignant marrows. Depletion of marrow monocytes/macrophages with a monoclonal antibody MO2, abolished the inhibitory effect of progesterone. However, production of colony stimulating activity (CSA) did not appear to be affected, either in PHA stimulated peripheral blood or in bone marrow. Indomethacin, a prostaglandin synthetase inhibitor, greatly reduced the degree of inhibition and the addition of prostaglandin E1 (PGE1) to culture in concentrations as low as 10(-12) M completely removed any additional inhibitory effect of progesterone. PGE1 is known to inhibit only those CFU-GM bearing the Ia antigen; we observed that depletion of Ia positive CFU-GM by either 24 h incubation or by monoclonal (I2) treatment, reduced or abolished progesterone mediated inhibition. This confirms that the mechanism of progesterone inhibition of CFU-GM is mediated through increased PGE production by marrow macrophages, and subsequent inhibition by PGE of Ia positive CFU-GM.

Il faut etre tres prudent dans la prediction de la capacite de reconstitution du sang peripherique au cours de la determination isolee du taux de CFU-GM