Abrogating chemotherapy-induced myelosuppression by recombinant granulocyte-macrophage colony-stimulating factor in patients with sarcoma: protection at the progenitor cell level.

Purpose: The purpose of this study was to optimize the dose, schedule, and timing of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) administration that would best abrogate myelosuppression in patients with sarcoma.

Patients And Methods: Sarcoma patients who had experienced severe myelosuppression after chemotherapy with Cytoxan (cyclophosphamide; Bristol-Myers Squibb Co, Evansville, IN), Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), and dacarbazine ([CyADIC], cycle 1) were eligible. GM-CSF was administered during a 14-day period until 1 week before cycle 2 of CyADIC and was resumed 2 days after cycle 2 completion.

Myelosuppressive effects in vivo of purified recombinant murine macrophage inflammatory protein-1 alpha.

Purified recombinant murine macrophage inflammatory protein-1 alpha (rmuMIP-1 alpha), a cytokine with myelopoietic activity in vitro, was assessed in vivo by injection into C3H/HeJ mice for effects on proliferation (percentage of cells in S phase DNA synthesis of the cell cycle) and absolute numbers of granulocyte-macrophage, erythroid, and multipotential progenitor cells in the femur and spleen, and on nucleated cellularity in the bone marrow, spleen, and blood. rmuMIP-1 alpha rapidly decreased cycling rates (at 2 to 10 micrograms/mouse i.v.

Increased osteoclast development after estrogen loss: mediation by interleukin-6.

Osteoclasts, the cells that resorb bone, develop from hematopoietic precursors of the bone marrow under the control of factors produced in their microenvironment. The cytokine interleukin-6 can promote hematopoiesis and osteoclastogenesis. Interleukin-6 production by bone and marrow stromal cells is suppressed by 17 beta-estradiol in vitro.

Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults.

We estimated whether single collections of cord blood contained sufficient cells for hematopoietic engraftment of adults by evaluating numbers of cord blood and adult bone marrow myeloid progenitor cells (MPCs) as detected in vitro with steel factor (SLF) and hematopoietic colony-stimulating factors (CSFs). SLF plus granulocyte-macrophage (GM)-CSF detected 8- to 11-fold more cord blood GM progenitors [colony-forming units (CFU)-GM] than cells stimulated with GM-CSF or 5637 conditioned medium (CM), growth factors previously used to estimate cord blood CFU-GM numbers. SLF plus erythropoietin (Epo) plus interleukin 3 (IL-3) enhanced detection of cord blood multipotential (CFU-GEMM) progenitors 15-fold compared to stimulation with Epo plus IL-3.

Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment.

The possibility that umbilical cord and placental blood from an HLA-identical sibling might produce stable donor-derived lymphohematopoietic engraftment was tested in a patient with juvenile chronic myelogenous leukemia (JCML). After conditioning with high-dose busulfan and cyclophosphamide, cryopreserved umbilical cord blood, containing 0.5 x 10(8) nucleated cells/kg and 2.

Influence of combinations of cytokines on proliferation of isolated single cell-sorted human bone marrow hematopoietic progenitor cells in the absence and presence of serum.

Human mast cell growth factor (MGF, a c-kit ligand) and colony stimulating factors (Epo, GM-CSF, G-CSF, IL-3) were assessed in the absence or presence of serum for stimulation in semi-solid medium of single CD34 , CD34 HLA-DR+, or CD34 HLA-DR+CD33- cells sorted per microtiter well. The % of wells containing CFU-GM and erythroid containing (BFU-E and CFU-GEMM) colonies increased in proportion to the number of cytokines added. In the presence of serum, 1, to 4 cytokine combinations resulted in respective increases in cloning efficiencies of 10 to 21.

Suppressor cytokines and regulation of myelopoiesis. Biology and possible clinical uses.

A number of biologically active and biochemically well-characterized cytokines have been shown to have either direct or indirect suppressive activities on the proliferation/differentiation of myeloid stem/progenitor cells. This article is a brief review of the actions of some of these molecules. These molecules include H-subunit ferritin, macrophage inflammatory protein-1 alpha, the interferons-alpha, -beta, and -gamma, the tumor necrosis factors-alpha and -beta (lymphotoxin), prostaglandins, E1 and E2, inhibin, transforming growth factor-beta, and lactoferrin.

Umbilical cord and placental blood hematopoietic stem cells: collection, cryopreservation, and storage.

Human umbilical cord and placental blood is a rich source of hematopoietic stem cells that can be used to reconstitute hematopoiesis after intensive myeloablative therapy. Therefore, it has become necessary to develop techniques for (i) collecting large volumes of umbilical cord aseptically, (ii) optimizing the yield of viable cells after cryopreservation and thawing, and (iii) minimizing the risk of maternal T-cell contamination. Our protocol for the collection, cryopreservation, storage, and transport of human umbilical cord and placental blood is described in detail.

Clinical and biological aspects of human umbilical cord blood as a source of transplantable hematopoietic stem and progenitor cells.

Presented is a short review of the current status fo the clinical and biological aspects of using human umbilical cord blood as a source of transplantable hematopoietic stem and progenitor cells.

Effects in vivo of purified recombinant human activin and erythropoietin in mice.

Activin has been shown to act in vitro as an erythroid specific enhancing activity for erythropoietin (epo)-stimulated erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells. To evaluate effects in vivo, purified recombinant activin-A and epo were administered s.c.

The kit receptor and its ligand, steel factor, as regulators of hemopoiesis.

Mouse strains carrying mutations at the Dominant White Spotting (W) locus or the Steel (Sl) locus are anemic and display defects in pigmentation and gametogenesis. In W mutants the anemia is due to a deficiency of hemopoietic stem cells and, in Sl mutants, to a deficiency of supporting stromal cells in the bone marrow. The W locus encodes the c-kit proto-oncogene product, a cell surface receptor with protein-tyrosine kinase activity, and the Sl locus encodes its ligand, a hemopoietic cytokine known variously as Steel factor (SLF), mast cell growth factor, stem cell factor, and Kit ligand.

Comparative analysis of signaling pathways between mast cell growth factor (c-kit ligand) and granulocyte-macrophage colony-stimulating factor in a human factor-dependent myeloid cell line involves phosphorylation of Raf-1, GTPase-activating protein and mitogen-activated protein kinase.

Mast cell growth factor (MGF, the ligand for c-kit receptor) can stimulate proliferation of factor dependent myeloid cell line, M07e, and MGF synergizes with granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3 in this effect. The effect of MGF on protein tyrosine kinase activity in M07e cells was investigated by immunoblotting with anti-phosphotyrosine mAb and this was compared with effects of GM-CSF. MGF stimulation rapidly induced or enhanced at least 12 tyrosine phosphorylated bands.

Hybrid cytokines as hematopoietic growth factors.

A large body of in vitro and in vivo data suggests that combinations of cytokines provide the most effective mechanism for stimulating multilineage acceleration of hematopoiesis. Creation of a granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin 3 (IL-3) fusion protein has yielded a single therapeutic which has enhanced biological activity in comparison to the individual cytokines from which it is composed. In vivo studies with this fusion protein (PIXY321) suggest that it may provide a means to accelerate both neutrophil and platelet recovery in clinical settings in which hematopoiesis is suppressed.

Mast cell growth factor (c-kit ligand) supports the growth of human multipotential progenitor cells with a high replating potential.

The replating capability of human multipotential (colony-forming unit-granulocyte-erythrocyte-macrophage-megakaryocyte [CFU-GEMM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitors was assessed in vitro as a potential measure of self-renewal using purified, recombinant (r) human (hu) or murine (mu) mast cell growth factor (MGF), a ligand for the c-kit proto-oncogene receptor. Primary cultures of human umbilical cord blood or adult human bone marrow cells were initiated in methylcellulose with erythropoietin (Epo) alone or in combination with rhu interleukin-3 (IL-3) or MGF. Individual day 14 to 18 CFU-GEMM or BFU-E colonies were removed from primary cultures and reseeded into secondary methylcellulose cultures containing a combination of Epo, MGF, and rhu granulocyte-macrophage colony-stimulating factor (GM-CSF).

Mast cell growth factor (c-kit ligand) enhances cytokine stimulation of proliferation of the human factor-dependent cell line, M07e.

Murine mast cell growth factor (muMGF), a c-kit ligand, has additive to greater-than-additive effects on in vitro colony formation of murine and human myeloid progenitor cells stimulated with erythropoietin, granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or interleukin (IL)-3. To confirm direct-acting effects on responding cells, MGF was assessed alone and in combination with other cytokines for effects on the proliferation of the human factor-dependent cell line, M07e. Proliferation was assayed in liquid culture by [3H]thymidine uptake and in semisolid medium by colony formation.

Identification of cell surface receptors for murine macrophage inflammatory protein-1 alpha.

We have produced recombinant proteins for a cytokine, L2G25BP (macrophage inflammatory protein-1 alpha) (MIP-1 alpha). By using the recombinant protein (rMIP-1 alpha), receptors for MIP-1 alpha were identified on Con A-stimulated and unstimulated CTLL-R8, a T cell line, and LPS-stimulated RAW 264.7, a macrophage cell line.

Macrophage inflammatory protein (MIP)-1 beta abrogates the capacity of MIP-1 alpha to suppress myeloid progenitor cell growth.

The effects of recombinant murine macrophage inflammatory protein (MIP)-1 beta and MIP-2 on the suppressive activity of MIP-1 alpha were tested using colony formation by human and murine bone marrow burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte erythroid macrophage, megakaryocyte (CFU-GEMM), and colony-forming unit-granulocyte macrophage (CFU-GM) progenitor cells. MIP-1 beta, but not MIP-2, when added with MIP-1 alpha to cells, blocked the suppressive effects of MIP-1 alpha on both human and murine BFU-E, CFU-GEMM, and CFU-GM colony formation. Similar results were observed regardless of the early acting cytokines used: human rGM-CSF plus human rIL-3, and two recently described potent cytokines, a genetically engineered human rGM-CSF/IL-3 fusion protein and MGF, a c-kit ligand.

In vivo effects of recombinant human interleukin 6, alone or in combination with local irradiation, on tumor growth in Lewis lung carcinoma-bearing mice.

Lewis lung carcinoma (LLC)-bearing mice were used as a mouse model to evaluate effects of recombinant human (rh) interleukin (IL) 6 and local X-irradiation (LR) on the growth of primary tumors and lung metastases. Mice were inoculated s.c.

Efficacy of recombinant human macrophage colony-stimulating factor in combination with whole-body hyperthermia in the treatment of mice infected with the polycythemia-inducing strain of the Friend virus complex.

Macrophage colony-stimulating factor (M-CSF, CSF-1) and whole-body hyperthermia (WBH) were evaluated, alone or in combination, for their capability to influence disease progression in mice inoculated with the polycythemia-inducing strain of the Friend virus complex (FVC-P). DBA/2 mice were injected i.v.

Influence of T-lymphocytes and lactoferrin on the survival-promoting effects of IL-1 and IL-6 on human bone marrow granulocyte-macrophage and erythroid progenitor cells.

Purified recombinant human (rhu) interleukin (IL)-1 alpha, rhuIL-6, iron saturated lactoferrin (LF), and T-lymphocytes were assessed for their effects on the survival of granulocyte-macrophage (granulocyte-macrophage colony-forming units, CFU-GM) and erythroid (erythroid burst-forming units, BFU-E) progenitor cells from human low-density (LD) and nonadherent LD T-lymphocyte-depleted (NALT-) bone marrow (BM) cells. Colony-stimulating factor (CSF) deprivation studies showed that 10 ng/ml IL-1 alpha could promote the survival of CFU-GM and BFU-E from NALT- BM cells. Concentrations of 1 ng/ml IL-1 alpha and 1-100 ng/ml IL-6 alone could not promote progenitor cell survival from NALT- BM cells; however, concentrations of 1 ng/ml each of IL-1 alpha and IL-6 could synergize to promote the survival of CFU-GM but not of BFU-E.

Differential transcriptional and posttranscriptional regulation of gene expression of the colony-stimulating factors by interleukin-1 and fetal bovine serum in murine fibroblasts.

Colony-stimulating factors (CSF) are important factors in the proliferation and differentiation of hematopoietic progenitor cells (HPC), and in the survival and activation of mature blood cells. Interleukin-1 (IL-1) combined with fetal bovine serum (FBS) strongly induces the expression of macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), and granulocyte-macrophage-CSF (GM-CSF) in fibroblasts. Here, we report on the regulation of CSF gene expression in murine fibroblasts following IL-1 and FBS stimulation.

CD45 cell surface antigens are linked to stimulation of early human myeloid progenitor cells by interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (a c-kit ligand).

CD45 antigens are protein tyrosine phosphatases. A possible link was evaluated between expression of CD45 antigens on human myeloid progenitor cells (MPC) (colony-forming unit-granulocyte/macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], and colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte [CFU-GEMM]) and regulation of MPC by colony-stimulating factors (CSF) (interleukin 3 [IL-3], GM-CSF, G-CSF, M-CSF, and erythropoietin [Epo]), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (MGF; a c-kit ligand). Treatment of cells with antisense oligodeoxynucleotides (oligos) to exons 1 and 2, but not 4, 5, or 6, of the CD45 gene, or with monoclonal anti-CD45, significantly decreased CFU-GM colony formation stimulated with GM-CSF, IL-3, fusion protein, and GM-CSF + MGF, but not with G-CSF or M-CSF.