Colony-forming Unit Granulocyte-macrophage Colonies Research Articles

Adenovirus-mediated delivery of p53 results in substantial apoptosis to myeloma cells and is not cytotoxic to flow-sorted CD34(+) hematopoietic progenitor cells and normal lymphocytes.

Multiple myeloma (MM) is an incurable disease; therefore, there is a need for new modalities of treatment for this disease. We designed a study to test the sensitivity of MM cell lines, freshly isolated myeloma cells, and CD34(+) hematopoietic progenitor cells to adenovirus-mediated delivery of wild-type p53 (Ad-p53). Replication-deficient Ad-p53, previously used in phase I-II clinical trial for treatment of patients with solid tumors, was used in this study. Myeloma cells from seven MM cell lines with mutated or w.t. p53 and varying expression of bcl-2 were used. Fresh myeloma cells (CD38(bright)CD45(-)) and fresh CD34(+) hematopoietic stem cells and CD34(-) cells were purified by flow sorting of apheresis collections of MM patients undergoing high-dose chemotherapy and stem cell rescue. The effect of Ad-p53 on colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit erythroid (BFU-E) colony formation in methylcellulose was tested on purified CD34(+) and CD34(-) cells to evaluate bone marrow toxicity. Myeloma cells from cell lines, or freshly isolated myeloma cells, were sensitive to Ad-p53 only if they had mutated p53 and had low expression of bcl-2. CD34(+) cells were resistant to Ad-p53-mediated apoptosis, and CFU-GM and BFU-E colony formation was not affected by treatment with Ad-p53.Ad-p53 is a potent inducer of apoptosis in MM cell lines and in freshly isolated myeloma cells expressing low levels of bcl-2. Ad-p53 is not overtly cytotoxic to normal hematopoietic stem cells or normal lymphocytes; therefore, it could be considered for a phase I clinical trial of MM patients with mutated p53.

Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells

AbstractThe ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)–derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34+cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34+ cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34+population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400 000 cord blood CD34+ cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP+ lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells.

Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells

The ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)-derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34(+) cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34(+) cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34(+) population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400,000 cord blood CD34(+) cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP(+) lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells. (Blood. 2000;96:3725-3733)

Amifostine (WR-2721), a cytoprotective agent during high-dose cyclophosphamide treatment of non-Hodgkin's lymphomas: a phase II study

Clinical trials indicate that amifostine may confer protection on various normal tissues without attenuating anti-tumor response. When administered prior to chemotherapy or radiotherapy, it may provide a broad spectrum of cytoprotection including against alkylating drugs. The mechanism of protection resides in the metabolism at normal tissue site by membrane-bound alkaline phosphatase. Toxicity of this drug is moderate with hypotension, nausea and vomiting, and hypocalcemia being observed. We report a phase II study using amifostine as a protective drug against high-dose cyclophosphamide (HDCY) (7 g/m2), used to mobilize peripheral blood progenitor cells (PBPC) and to reduce tumor burden. We enrolled 29 patients, 22 (75. 9%) affected by aggressive and 7 (24.1%) by indolent non-Hodgkin's lymphoma (NHL), who were submitted to 58 infusions of amifostine and compared them with a historical group (33 patients) affected by aggressive NHL and treated with VACOP-B followed by HDCY. The most important results in favor of amifostine were the reduction of intensity of cardiac, pulmonary and hepatic toxicity, and a significant reduction of frequency and severity of mucositis (P = 0. 04). None of the 29 patients died in the protected group, while in the historical group 2/33 patients died because of cardiac or pulmonary toxicity and 2 patients stopped therapy due to toxicity. Amifostine did not prevent the aplastic phase following HDCY. PBPC collection and hematological recovery were adequate in both groups. The number of CFU-GM (colony-forming units-granulocyte/macrophage) colonies and mononuclear cells in the apheresis products was significantly higher in the amifostine group (P = 0.02 and 0.01, respectively). Side effects were mild and easily controlled. We conclude that amifostine protection should be useful in HDCY to protect normal tissues, with acceptable side effects.

Altered bone marrow cell sensitivity in the lupus-prone NZB/W mouse: regulation of CFU-GM colony formation by estrogen, tamoxifen and thrombopoietin.

Estrogen is thought to contribute to the onset of systemic lupus erythematosus (SLE) in women through mechanisms that are not completely understood. Although estrogen serves as a negative regulator in normal hematopoietic development, little research has been conducted examining alteration in hematopoietic development triggered by estrogen in lupus-susceptible individuals. We examined whether estrogen and other factors could influence colony formation of bone marrow cells obtained from normal and lupus-susceptible mice. Bone marrow cells isolated from New Zealand Black (NZB) and lupus-prone New Zealand Black and New Zealand White cross (NZB/W) mice were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) alone or in combination with estrogen, thrombopoietin (TPO), tamoxifen, estrogen and TPO. or estrogen and tamoxifen, and plated in methylcellulose culture medium. Plates were scored for the number of CFU-GM (colony forming unit granulocyte-macrophage) colonies after 6d in culture. For females of both mouse strains, estrogen significantly decreased (P < 0.05) the number of GM colonies. Co-treatment of NZB/W cells, but not NZB cells, with TPO or tamoxifen reversed the suppressive action of estrogen (P < 0.05). In contrast, while estrogen did suppress colony formation from cells of NZB/W males (P < 0.05), neither TPO nor tamoxifen reversed this effect. Our results indicate that the sensitivity of bone marrow cells isolated from both female and male NZB/W lupus-prone mice to hormones/growth factors is qualitatively different from cells of NZB mice, and suggest that hematopoietic alterations at the level of the bone marrow may be related to the pathogenesis of SLE.

Stem Cell Factor as a Single Agent Induces Selective Proliferation of the Philadelphia Chromosome Positive Fraction of Chronic Myeloid Leukemia CD34+ Cells

The interaction between p145c-KIT and p210bcr-abl in transduced cell lines, and the selective outgrowth of normal progenitors during long-term culture of chronic myeloid leukemia (CML) cells on stroma deficient in stem-cell factor (SCF) suggests that the response of CML cells to SCF may be abnormal. We examined the proliferative effect of SCF(100 ng/mL), provided as the sole stimulus, on individual CD34+ cells from five normal donors and five chronic-phase CML patients. Forty-eight percent of isolated single CML CD34+ cells proliferated after 6 days of culture to a mean of 18 cells, whereas only 8% of normal CD34+ cells proliferated (mean number of cells generated was 4). SCF, as a single agent, supported the survival and expansion of colony-forming unit–granulocyte-macrophage (CFU-GM) from CML CD34+CD38+ cells and the more primitive CML CD34+CD38− cells. These CFU-GM colonies were all bcr-abl positive, showing the specificity of SCF stimulation for the leukemic cell population. Coculture of CML and normal CD34+ cells showed exclusive growth of Ph+cells, suggesting that growth in SCF alone is not dependent on secretion of cytokines by CML cells. SCF augmentation of β1-integrin–mediated adhesion of CML CD34+cells to fibronectin was not increased when compared with the effect on normal CD34+ cells, suggesting that the proliferative and adhesive responses resulting from SCF stimulation are uncoupled. The increased proliferation may contribute to the accumulation of leukemic progenitors, which is a feature of CML.

Stem Cell Factor as a Single Agent Induces Selective Proliferation of the Philadelphia Chromosome Positive Fraction of Chronic Myeloid Leukemia CD34+ Cells

The interaction between p145c-KIT and p210bcr-abl in transduced cell lines, and the selective outgrowth of normal progenitors during long-term culture of chronic myeloid leukemia (CML) cells on stroma deficient in stem-cell factor (SCF) suggests that the response of CML cells to SCF may be abnormal. We examined the proliferative effect of SCF(100 ng/mL), provided as the sole stimulus, on individual CD34+ cells from five normal donors and five chronic-phase CML patients. Forty-eight percent of isolated single CML CD34+ cells proliferated after 6 days of culture to a mean of 18 cells, whereas only 8% of normal CD34+ cells proliferated (mean number of cells generated was 4). SCF, as a single agent, supported the survival and expansion of colony-forming unit–granulocyte-macrophage (CFU-GM) from CML CD34+CD38+ cells and the more primitive CML CD34+CD38− cells. These CFU-GM colonies were all bcr-abl positive, showing the specificity of SCF stimulation for the leukemic cell population. Coculture of CML and normal CD34+ cells showed exclusive growth of Ph+cells, suggesting that growth in SCF alone is not dependent on secretion of cytokines by CML cells. SCF augmentation of β1-integrin–mediated adhesion of CML CD34+cells to fibronectin was not increased when compared with the effect on normal CD34+ cells, suggesting that the proliferative and adhesive responses resulting from SCF stimulation are uncoupled. The increased proliferation may contribute to the accumulation of leukemic progenitors, which is a feature of CML.

Mobilization of hematopoietic progenitors in patients with chronic myeloid leukemia.

Although the mobilization and harvesting of Philadelphia chromosome-negative progenitors has been proven feasible by a number of groups, it is not clear which techniques should be used with regard to the monitoring of purging and evaluation of stem cell yield. Therefore, we isolated CD34-positive cells from leukapheresis samples of seven CML patients after induction therapy. These cells were analyzed using the colony-forming unit granulocyte-macrophage (CFU-GM) and long-term culture-initiating cell (LTCIC) assays. In addition, we analyzed frequency and clonogenicity of single stem cells using the LTCIC assay at limiting dilution. Individual colonies derived from these assays were subsequently analyzed for the presence of the bcr-abl gene with interphase fluorescence in situ hybridization (FISH) and for the presence of bcr-abl mRNA with RT-PCR. We compared these results with the cytogenetic analysis of the leukapheresis material. Molecular analysis of individual colonies correlated well with the results of cytogenetic analysis. However, in nine out of 18 samples, cytogenetic analysis exclusively demonstrated the presence of either Philadelphia chromosome-positive or -negative cells whereas with the molecular analysis of individual colonies tumor contamination or the presence of residual normal cells could be substantiated. We concluded that molecular analysis of individual colonies should be employed to further optimize induction protocols. With regard to stem cell mobilization we demonstrated that about 67 CFU-GM colonies and clusters were generated by one single LTCIC both for the CML samples and the samples obtained from patients with non-hematologic malignancy. This number is important since until now most centres use a number of four colonies and clusters generated by one LTCIC. Dividing the CFU-GM output in the LTCIC assay by four to determine LTCIC frequency could thus lead to an almost 20-fold overestimation of the LTCIC frequency. It is concluded that stem cell frequency analysis is an important tool with regard to the interpretation of mobilization protocols.

Imprecision of counting CFU-GM colonies and CD34-expressing cells.

Determinations of committed haemopoietic progenitor cells, namely CFU-GM (colony-forming unit-granulocyte/macrophage) and of CD34-expression haemopoietic cells as assessed by multiparameter flow cytometry are routine diagnostic tools in haemopoietic cell therapy. Generally, the tests are used to optimise the timing and management of cytapheresis and to assess the engraftment potential of the harvested cells. Both measurements, however, are at best surrogate markers, as an adequate routine test which effectively assesses the short- and long-term repopulating haemopoietic cell is not available. Nonetheless, cell threshold doses have been established. Above these thresholds rapid engraftment is almost invariable but below these thresholds the outcome is variable. In this study we have focussed on the imprecision in counting haemopoietic cells, as assessed as CFU-GM and as CD34-expressing cells. The data on both tests have been analysed from six European institutions. The coefficient of variation in CFU-GM colony counting was about 30%, whereas the coefficient of variation in flow cytometric counting of CD34-expressing cells was about 10%. These data suggest that the technical imprecision in enumerating progenitor cells, particularly CFU-GM, at low levels, might make a major contribution to the clinical variability observed after transplantation of sub-threshold progenitor cell dose.

Molecular status of individual CFU-GM colonies derived from chemotherapy-mobilised peripheral blood stem cells in chronic myeloid leukaemia

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 selective uptake of benzoporphyrin derivative mono-acid ring A results in differential cell kill of multiple myeloma cells in vitro.

High-dose chemotherapy (HDCT) and autologous bone marrow/blood stem cell transplantation are an effective combination for treating a number of malignant disorders. The contamination of the autograft by malignant cells may be a reason for recurrences in spite of this treatment, for instance, in multiple myeloma. Therefore, we evaluated the use of photodynamic therapy (PDT) using the photosensitizer benzoporphyrin derivative mono-acid ring A (BPD-MA) on multiple myeloma cells in comparison to the components of the normal bone marrow (NBM) and peripheral blood apheresis product. Flow cytometry was used to measure differential BPDMA uptake of NBM components: namely lymphocytes, monocytes, granulocytes and enriched hematopoietic stem cell (CD34+) populations and also the multiple myeloma cell lines OCI-MY7 and OCI-MY4. When each population was measured individually, the order of uptake was [OCI-MY7/MY4] > [CD34+] > [granulocytes] = [monocytes] >> [lymphocytes]. Further, clonogenic assay was used to demonstrate surviving fractions for OCI-MY7, OCI-MY4 and NBM in vitro. The LD90 for OCI-MY7 and OCI-MY4 was between 10 and 20 ng/mL BPD-MA whereas this concentration did not show any significant cell kill for the colony-forming units-granulocyte/macrophage (CFU-GM) and burst-forming units-erythrocyte (BFU-E). When the NBM was "contaminated" with multiple myeloma cells in vitro, the LD90 for OCI-MY7 in this cell mixture was shifted to between 40 and 80 ng/mL BPD-MA. However, at 40 ng/mL BPD-MA at least 50% of normal CFU-GM and BFU-E colonies survived. For CFU-GM and BFU-E derived from the enriched CD34+ cell population, BPDMA up to a concentration of 80 ng/mL did not significantly reduce the surviving fractions. We have observed a 3-4 log therapeutic window with differential cell kill when comparing multiple myeloma cell lines to the components of the NBM and apheresis product in vitro. We conclude, that BPD-MA is a molecule potentially useful as an ex vivo purging agent.

A Functional Analysis of Protooncogene Vav's Role in Adult Human Hematopoiesis

The Vav protooncogene is expressed almost exclusively in hematopoietic cells, but its role in regulating adult human hematopoietic cell development remains uncertain. To analyze Vav function in adult blood cell formation, we used antisense (AS) oligodeoxynucleotides (ODN) to disrupt its expression in normal and malignant human hematopoietic cells. Bone marrow or peripheral blood mononuclear cells (MNC) were obtained from consenting normal donors and patients with acute or chronic myelogenous leukemia (AML and CML, respectively) and polycythemia vera (PV). Adherent and T-cell-depleted (A-T-) MNC or CD34+ MNC were exposed to unmodified sense, antisense, or scrambled sequence ODN corresponding to codons 2-7 of Vav's mRNA sequence. Cells were then assayed for Vav mRNA expression by reverse transcription-polymerase chain reaction and Vav protein expression by Western binding. After showing that Vav-targeted AS ODN could specifically diminish Vav mRNA and protein expression, we assessed the ability of Vav-deficient cells to form myeloid and erythroid colonies in methyl-cellulose cultures. When normal CD34+ MNC were exposed to Vav AS ODN, no effect on colony-forming unit-granulocyte-macrophage (CFU-GM) or CFU-megakaryocyte colony formation was observed. In contrast erythroid colony growth was inhibited by a mean +/- SD of 62% +/- 16%. In patients with hematopoietic malignancies. Vav-targeted AS ODN inhibited CFU-GM colony formation in a sequence-specific and dose-dependent manner in 1 of 3 AML, 13 of 17 CML, and 2 of 2 PV patients. At the highest concentration used, the Vav AS ODN inhibited CFU-GM colony formation from 66% to 81% when compared with control cell colony growth. Burst-forming unit-erythroid (BFU-E) colony-formation was also assessed in 7 PV patients. The Vav-targeted AS ODN inhibited BFU-E colony formation in all by a mean +/- SD of 81% +/- 4%. These findings suggest that Vav function may not be easily complemented in a significant subset of normal adult erythroid progenitor cells and may also be necessary for myeloid progenitor cell growth in a variety of hematopoietic malignancies.

In vitro and in vivo myelotoxicity of CAI to human and murine hematopoietic progenitor cells.

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.

Is Nitrous Oxide Safe for Bone Marrow Harvest?

Patients with non-Hodgkins lymphoma undergoing autologous bone marrow harvest were studied in a prospective, randomized fashion. All patients received a general anesthetic consisting of intravenous thiopental, fentanyl, and vecuronium and were ventilated with oxygen and isoflurane. Group I (19) patients also were ventilated with nitrous oxide (70%) whereas patients in Group II (19) did not receive nitrous oxide. Bone marrow samples were obtained at the beginning and end of the harvest. Viability of bone marrow mononuclear cells was assessed with a colony-forming unit-granulocyte macrophage (CFU-GM) assay, CFU-GM growth is a marker for myeloid progenitor cells and is dependent on intact deoxyribonucleic acid synthesis. Rate of neutrophil engraftment after autologous bone marrow transplantation was also studied. Both groups of patients were statistically similar in age, weight, anesthetic duration, CFU-GM counts at both sample draws, and the time for successful engraftment. There appears to be no difference in bone marrow viability as assayed by both CFU-GM colony growth and engraftment in human bone marrow exposed to a general anesthetic with nitrous oxide.

Growth factor stimulation of cryopreserved CD34+ bone marrow cells intended for transplant: an in vitro study to determine optimal timing of exposure to early acting cytokines.

Stimulating CD34+ hematopoietic cells with growth factors prior to transplantation may decrease the duration of post-transplant aplasia. The optimal time in which to deliver this stimulus remains unclear. We therefore sought to determine if progenitor cell cloning efficiency, as reflected by colony forming units-granulocyte-macrophage (CFU-GM) colony formation, differed when growth factor stimulation was carried out prior to freezing or after thawing. To address this issue, CD34+ marrow cells were suspended in Iscove's medium with 20% bovine calf serum. They were then stimulated with kit ligand (KL), interleukin-3 (IL-3), and interleukin-1 beta (IL-1 beta) for 36 h either prior to cryopreservation or immediately after thawing. We observed that cells stimulated prior to freezing formed more CFU-GM colonies than cells stimulated after thawing. We also found that CD34+ cells stimulated with growth factors either before or after freezing gave rise to more CFU-GM colonies than thawed cells plated without prestimulation. Thus, the cloning efficiency of stored marrow, as reflected by CFU-GM colony formation, may be effectively enhanced by pretreating cells with hematopoietic growth factors. Our data further suggest that the optimal time for this treatment is before cryopreservation as opposed to immediately after thawing.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor Expression by Oncostatin M

AbstractOncostatin M (OM) is structurally and functionally related to a subclass of hematopoietic cytokines including leukemia-inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), granulocyte colony-stimulating factor (G-CSF), and interleukin-6 (IL-6). Using human endothelial cells (HEC) as a model for cytokine regulation of hematopoietic growth factor expression, we tested OM as an inducer of colony-stimulating activity. Colony-forming cell assays supplemented with culture supernatants from OM-treated HEC contained a threefold increase in colony-forming unit granulocyte-macrophage colonies. Specific immunoassay (enzyme-linked immunosorbent assay) of culture supernatants indicated that OM treatment of HEC resulted in a dose- and time-dependent increase in the accumulation of G-CSF and granulocyte-macrophage CSF (GM-CSF) (> 28-fold). The ED50 for OM induction of G-CSF and GM-CSF protein expression was 17 and 7 pmol/L, respectively. Increased protein expression was associated with a similar increase in steady-state expression of G-CSF and GM-CSF mRNA. Furthermore, a period of 12 to 24 hours elapsed before there were measurable increases in CSF expression, suggesting that OM may stimulate CSF production through a mechanism requiring the synthesis or activation of a secondary mediating factor or pathway. These findings provide the first evidence that OM may regulate myelopoiesis by inducing the cellular expression of hematopoietic growth factors.

Effect of stem cell factor on colony growth from acquired and constitutional (Fanconi) aplastic anemia

The aim of this study was to evaluate the effect of stem cell factor (SCF) on the in vitro growth of bone marrow hematopoietic progenitors from patients with acquired severe aplastic anemia (AA) or Fanconi's anemia (FA). For this purpose, we studied 11 patients with acquired AA (5 at diagnosis, 6 after ALG treatment), 12 patients with FA, and nine normal controls. Bone marrow cells were plated in vitro for colony- forming unit granulocyte-macrophage (CFU-GM) (in the presence of granulocyte-macrophage colony-stimulating factor [GM-CSF]), and for burst-forming unit-erythroid (BFU-E) and CFU-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) colonies (in the presence of erythropoietin and interleukin-3 [IL-3]), with or without 20 ng/mL of SCF. In normal controls, SCF enhanced the growth of CFU-GM colonies from 103 to 263 (median), of BFU-E from 168 to 352, and of GEMM colonies from 6 to 38/10(5) cells plated. In patients with acquired AA, SCF induced a significant enhancement of BFU-E growth (8 to 29; P = .01) and allowed the formation of GEMM colonies that were not scored in baseline culture conditions (0 to 8; P = .01). CFU-GM growth was enhanced (4 to 20), but not significantly (P = .3). This was true both for patients at diagnosis and after antilymphocyte globulin treatment. By contrast, 10 of 12 FA patients grew no CFU-GM, BFU-E, or CFU-GEMM colonies, with or without SCF. In two FA patients (one transfusion- dependent and one transfusion-independent), an enhancement of CFU-GM and/or BFU-E was observed. The lack of response of hematopoietic progenitor cells from FA patients to GM-CSF+SCF or IL-3+SCF was not dependent on a defective expression of cytokine receptor messenger RNAs. Northern blot analysis showed in marrow cells from acquired AA and FA patients the presence of normal transcripts for alpha- and beta- chains of GM-CSF/IL-3 receptor and for c-kit protein. In conclusion, SCF promotes the in vitro growth of hematopoietic progenitors in patients with acquired AA, but not in patients with FA, pointing out the intrinsic nature of the defect in the latter disorder.

Effect of Stem Cell Factor on Colony Growth From Acquired and Constitutional (Fanconi) Aplastic Anemia

AbstractThe aim of this study was to evaluate the effect of stem cell factor (SCF) on the in vitro growth of bone marrow hematopoietic progenitors from patients with acquired severe aplastic anemia (AA) or Fanconi's anemia (FA). For this purpose, we studied 11 patients with acquired AA (5 at diagnosis, 6 after ALG treatment), 12 patients with FA, and nine normal controls. Bone marrow cells were plated in vitro for colony-forming unit granulocyte-macrophage (CFU-GM) (in the presence of granulocyte-macrophage colony-stimulating factor [GM-CSF]), and for burst-forming unit-erythroid (BFU-E) and CFU-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) colonies (in the presence of erythropoietin and interleukin-3 [IL-3]), with or without 20 ng/mL of SCF. In normal controls, SCF enhanced the growth of CFU-GM colonies from 103 to 263 (median), of BFU-E from 168 to 352, and of GEMM colonies from 6 to 38/105 cells plated. In patients with acquired AA, SCF induced a significant enhancement of BFU-E growth (8 to 29; P =. 01) and allowed the formation of GEMM colonies that were not scored in baseline culture conditions (0 to 8; P =. 01). CFU-GM growth was enhanced (4 to 20), but not significantly (P =. 3). This was true both for patients at diagnosis and after antilymphocyte globulin treatment. By contrast, 10 of 12 FA patients grew no CFU-GM, BFU-E, or CFU-GEMM colonies, with or without SCF. In two FA patients (one transfusion-dependent and one transfusion-independent), an enhancement of CFU-GM and/or BFU-E was observed. The lack of response of hematopoietic progenitor cells from FA patients to GM-CSF + SCF or IL-3 + SCF was not dependent on a defective expression of cytokine receptor messenger RNAs. Northern blot analysis showed in marrow cells from acquired AA and FA patients the presence of normal transcripts for a- and β-chains of GM-CSF/IL-3 receptor and for c-kit protein. In conclusion, SCF promotes the in vitro growth of hematopoietic progenitors in patients with acquired AA, but not in patients with FA, pointing out the intrinsic nature of the defect in the latter disorder.

Modulation of myeloid proliferation and differentiation by monoclonal antibodies directed against a protein that interacts with the interleukin-3 receptor

Hematopoietic cells can be transformed through the acquisition of autocrine growth factor production. Because of their ability to inhibit autocrine growth, antibodies directed against the growth factor or its receptor may have therapeutic potential. However, these agents may also inhibit normal cell development. We have developed two monoclonal antibodies, 4G8 and 2F2, directed against a protein of 110 to 150 Kd that interacts with the interleukin-3 (IL-3) receptor (R) complex. These antibodies inhibit IL-3-induced proliferation of nonleukemic and leukemic IL-3-dependent cell lines, as well as the autonomous growth of WEHI-3B in vitro and in vivo. These results suggest the possibility that anti-IL-3R antibodies may be useful in the treatment of some leukemias. However, the effect of anti-IL-3R antibodies on normal myeloid development in vitro has not been examined. We examined the effect of 4G8 and 2F2 on the growth in vitro of colony-forming unit granulocyte-macrophage (CFU-GM) colonies induced by IL-3, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and macrophage-CSF (M-CSF). Our results show that while 4G8 and 2F2 inhibited CFU-GM colony formation induced by IL-3, they augmented colony formation induced by the other hematopoietins. 4G8 and 2F2 also enhanced G-CSF-induced proliferation of 32Dc13 and GM-CSF-induced proliferation of PT18, confirming that the effect on CFU-GM was a direct effect. Finally, 4G8 and 2F2 inhibited G-CSF-induced differentiation of 32Dc13, similar to low levels of IL-3; yet, neither 4G8 nor 2F2 blocked binding of G-CSF to its receptor. These results indicate that, in the absence of IL-3 and in the presence of other hematopoietins, 4G8 and 2F2 can function as weak IL-3 agonists. These studies suggest that antibodies such as 4G8 and 2F2, directed against components of the IL-3R, could potentially augment myeloid growth in vivo, rather than inhibit myeloid growth.

Modulation of Myeloid Proliferation and Differentiation by Monoclonal Antibodies Directed Against a Protein That Interacts With the Interleukin-3 Receptor

Abstract Hematopoietic cells can be transformed through the acquisition of autocrine growth factor production. Because of their ability to inhibit autocrine growth, antibodies directed against the growth factor or its receptor may have therapeutic potential. However, these agents may also inhibit normal cell development. We have developed two monoclonal antibodies, 4G8 and 2F2, directed against a protein of 110 to 150 Kd that interacts with the interleukin-3 (IL-3) receptor (R) complex. These antibodies inhibit IL-3-induced proliferation of nonleukemic and leukemic IL-3-dependent cell lines, as well as the autonomous growth of WEHI-3B in vitro and in vivo. These results suggest the possibility that anti-IL-3R antibodies may be useful in the treatment of some leukemias. However, the effect of anti-IL-3R antibodies on normal myeloid development in vitro has not been examined. We examined the effect of 4G8 and 2F2 on the growth in vitro of colony-forming unit granulocyte-macrophage (CFU-GM) colonies induced by IL-3, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and macrophage-CSF (M-CSF). Our results show that while 4G8 and 2F2 inhibited CFU-GM colony formation induced by IL-3, they augmented colony formation induced by the other hematopoietins. 4G8 and 2F2 also enhanced G-CSF-induced proliferation of 32Dc13 and GM-CSF-induced proliferation of PT18, confirming that the effect on CFU-GM was a direct effect. Finally, 4G8 and 2F2 inhibited G-CSF-induced differentiation of 32Dc13, similar to low levels of IL-3; yet, neither 4G8 nor 2F2 blocked binding of G-CSF to its receptor. These results indicate that, in the absence of IL-3 and in the presence of other hematopoietins, 4G8 and 2F2 can function as weak IL-3 agonists. These studies suggest that antibodies such as 4G8 and 2F2, directed against components of the IL-3R, could potentially augment myeloid growth in vivo, rather than inhibit myeloid growth.