The human gene encoding GM-CSF is at 5q21-q32, the chromosome region deleted in the 5q- anomaly.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 22,000-dalton glycoprotein that stimulates the growth of myeloid progenitor cells and acts directly on mature neutrophils. A full-length complementary DNA clone encoding human GM-CSF was used as a probe to screen a human genomic library and isolate the gene encoding human GM-CSF. The human GM-CSF gene is approximately 2.5 kilobase pairs in length with at least three intervening sequences. The GM-CSF gene was localized by somatic cell hybrid analysis and in situ hybridization to human chromosome region 5q21-5q32, which is involved in interstitial deletions in the 5q- syndrome and acute myelogenous leukemia. An established, human promyelocytic leukemia cell line, HL60, contains a rearranged, partially deleted GM-CSF allele and a candidate 5q- marker chromosome, indicating that the truncated GM-CSF allele may reside at the rejoining point for the interstitial deletion on the HL60 marker chromosome.

Purified human granulocyte-macrophage colony-stimulating factor: direct action on neutrophils.

Neutrophil migration inhibition factor from T lymphocytes (NIF-T) is a lymphokine that acts to localize granulocytes. Medium conditioned by the Mo human T-lymphoblast cell line was used to purify NIF-T, a glycoprotein with a molecular weight of 22,000. The NIF-T was found to potently stimulate the growth of granulocyte and macrophage colonies from human bone marrow and colony formation by the KG-1 myeloid leukemia cell line. Thus a human lymphokine (NIF-T) that modulates the activities of mature neutrophilic granulocytes is also a colony-stimulating factor acting on precursors to induce growth and differentiation of new effector cells.

Granulocyte-macrophage colony-stimulating factor and tetradecanoyl phorbol acetate induce a distinct, restricted subset of primary-response TIS genes in both proliferating and terminally differentiated myeloid cells.

Induction of early-response genes (tetradecanoyl phorbol acetate [TPA]-induced sequences, or TIS genes; R.W. Lim, B.C. Varnum, and H.R. Herschman, Oncogene 1:263-270, 1987) by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPA was examined both in a factor-dependent murine cell line, 32D clone 3, and in mature human neutrophils. When GM-CSF-deprived 32D clone 3 cells were exposed to GM-CSF or to TPA, four TIS mRNAs (TIS7, TIS8, TIS10, and TIS11) were rapidly and transiently induced. However, neither GM-CSF nor TPA could induce accumulation of TIS1 mRNA in 32D clone 3 cells, even under superinducing conditions. Both GM-CSF and TPA also elicited rapid, transient expression of TIS8 and TIS11 mRNA in postmitotic human neutrophils. However, neither agent could induce accumulation of TIS1 mRNA in human neutrophils. TIS1 is a member of the nuclear receptor supergene family that codes for ligand-dependent transcription factors. Cell-type restriction of inducible transcription factors may contribute to developmental specification.

Effects of human GM-CSF on neutrophil degranulation in vitro.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates multiple differentiated functions of mature neutrophils. Increased expression of leukocyte adhesion molecules and chemotactic receptors on GM-CSF-treated neutrophils suggested that GM-CSF may stimulate neutrophil degranulation. were assessed by quantitating the release of an exclusive component of the specific granules, vitamin B12 binding protein. Incubation of neutrophils with GM-CSF alone resulted in a significant release of [57Co]-vitamin B12 binding protein quantitatively similar to that elicited by cytochalasin B or N-formyl-methionyl-leucylphenylalanine (f-Met-Leu-Phe) alone. In addition, cells preincubated with GM-CSF and subsequently stimulated with f-Met-Leu-Phe, platelet-activating factor, or the calcium ionophore, A23187, demonstrated enhanced degranulation, which greatly exceeded that produced by GM-CSF alone. These results demonstrate a small direct effect of GM-CSF on neutrophil degranulation, as well as enhanced degranulation in cells stimulated by chemotactic agents and calcium ionophore. Neutrophil degranulation in response to GM-CSF may be involved in the phlebitis associated with therapeutic administration of GM-CSF.

Radiorecovery activity of manganese(III)2(II)(mu 3-O)-(mu-3,5-diisopropylsalicylate)6.

Manganese(III)2(II)(mu 3-O)(mu-3,5-diisopropylsalicylate)6 [Mn3(O)(3,5-DIPS)6] was used to treat female C57BL/6 mice irradiated with LD50/30 doses of gamma rays and examine the possibility that treatment after irradiation increases survival. Female C57BL/6 mice were treated with 0, 10, 20, or 40 mumol Mn3(O)(3,5-DIPS)6/kg of body mass 1 or 3 h after irradiation. Treatment with 40 mumol/kg 1 or 3 h after irradiation produced survivals of 72 or 92%, respectively, increases of 29 or 130% in comparison with 56 or 40% survivals in the respective vehicle-treated groups. These data support the hypothesis that Mn3(O)(3,5-DIPS)6 is an effective radiorecovery agent.

Effect of ethanol upon gastric emptying.

The effect of ethanol upon gastric emptying in healthy human subjects was studied by measuring the gastric emptying rates of three 750 ml meals, the osmolalities, energy densities, and pH of which were similar. Meal A, which contained 80 ml alcohol, emptied more rapidly than meal B, which contained 40 ml ethanol and 63.3 g dextrose; and meal B emptied more rapidly than meal C, which contained 126.6 g dextrose but no ethanol. The slower rate of emptying of the dextrose meal (C) was not due to an increased gastric secretory rate, as serial measurements of gastric pH were substantially and significantly higher with this than with the other two meals; nor was it due to a greater degree of duodenogastric reflux, as serial measurements of gastric bile acid concentrations were similar for the three meals. We conclude that the duodenal osmoreceptor mechanism is relatively insensitive to ethanol; that the relationship between energy density and gastric emptying rate does not hold in the case of ethanol; and that the gastro-oesophageal reflux which occurs in response to ethanol is not due to impairment of gastric emptying.

Induction of gastro-oesophageal reflux by alcohol.

In order to establish whether alcohol in amounts in amounts customarily imbibed during social drinking causes gastro-oesophageal reflux, 12 healthy young individuals, without symptoms of gastro-oesophageal reflux, were studied twice. Each time, distal oesophageal pH was monitored continuously for three hours after a standard meal which included either 180 ml 100 proof vodka or 180 ml water. The order of studies with and without alcohol was random. Peak blood alcohol concentrations ranged between 0.63 and 1.29 g/l. Eleven of the 12 subjects refluxed more after alcohol; and the difference in mean reflux scores for studies with and without alcohol was highly significant. We conclude that relatively modest quanttities of alcohol induce gastro-oesophageal reflux in healthy people.

Characterization of ligand binding to immobilized biotinylated extracellular domains of three growth factor receptors.

For the purpose of developing a cell-free binding assay suitable for the large scale screening of receptor agonists and antagonists, the extracellular ligand binding domains of the low affinity NGF receptor (p75NGFR), a splicing variant of the fibroblast growth factor receptor (FGFR), and the PDGF beta-receptor (PDGFR) were modified by the biotinylation of amino groups. After immobilization in streptavidin-coated microtiter wells, it was determined by ligand binding analysis that all three receptors retained high affinities (0.7-1.8 nM) for their respective ligands. Two receptors, p75NGFR and FGFR, also displayed low affinity ligand binding (45-85 nM) which is likely a result of differential biotinylation. The three assay systems displayed excellent tolerance to several solution variables including organic solvents and high salt, with the exception of the FGF assay's sensitivity to thiols. The consistency obtained by using a coated well protocol, coupled with elimination of cell culture while maintaining natural binding affinities, demonstrates that the immobilized biotinylated extracellular domain creates an excellent system for pharmaceutical screening.

Electronic collection of health-related quality of life data: validity, time benefits, and patient preference.

This study sought to validate World Wide Web-compliant software tools used to collect health-related quality of life (HRQOL) data, relative to pencil-and-paper collection. The RAND-36 general health survey and the Seattle Angina questionnaire (SAQ), a disease-specific functional status measure for patients with coronary artery disease, were each administered in paper and electronic format to 55 consecutive patients visiting the cardiology outpatient clinic of a public hospital. All eight sub-scale scores of the RAND-36 (interclass correlation coefficient range = 0.54-0.75, p < 0.01) and all five domains of the SAQ (interclass correlation coefficient range = 0.84-0.90, p < 0.01) collected using the software were significantly correlated with those collected using the paper version of questionnaires. Computer literacy, educational level, age, sex, and race were not significantly associated with the ability to successfully complete the computer-assisted questionnaire. Eighty-two percent of patients preferred the computer-assisted administration to paper, and 89% reported that they would feel comfortable using the software in the future without any technical assistance. This pilot study suggests that HRQOL measures can be reliably collected using software operating over the World Wide Web. Data collected in this manner are valid and of comparable quality to self-reported, HRQOL data obtained via paper survey.

O-linked oligosaccharide on the 75-kDa neurotrophin receptor.

Four neurotrophic factors, important for survival and function of neurons, bind a common receptor, the 75-kDa neurotrophin receptor (NTR). An O-glycosylated peptide connects the ligand-binding domain of NTR to its transmembrane helix. This peptide, the transmembrane helix, and intracellular sequences are highly conserved in vertebrate evolution. To investigate the structure and function of O-glycosylation on NTR, we produced the extracellular domains by expression in mammalian cells. Addition during biosynthesis of O-linked glycans was evaluated, and structures were characterized by lectin blotting and glycosidase digestion. Effects of disialylation, deglycosylation, and lectin attachment on the equilibrium binding constant were measured. Addition of O-linked glycans during biosynthesis was found to have a large effect on NTR structure assessed by mobility in polyacrylamide gels. NTR O-linked glycans synthesized by cultured cells had the structure (NeuNAc)(1-2-) Gal beta 1-3GalNAc. Modification of the O-linked oligosaccharide produced small, possibly significant effects on the binding constant of NTR for nerve growth factor. The results are discussed in reference to a potential role for the stalk region in ligand binding and signaling.

High-affinity binding of granulocyte-macrophage colony-stimulating factor to normal and leukemic human myeloid cells.

Purified natural and biosynthetic (recombinant) human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate colony formation by myeloid progenitor cells and enhance the function of mature neutrophils. Both of these actions occur at concentrations between 1 and 100 pM, with half-maximal stimulation at 10-20 pM. We have examined specific binding of 125I-labeled GM-CSF to responsive target cells in this range of concentrations. The results show a low number (50-250) of high-affinity (15-30 pM) binding sites on GM-CSF-responsive leukemic cells (KG-1, HL-60), as well as on peripheral blood neutrophils from normal donors. This high-affinity binding component was absent from unresponsive cell lines (KG-1a, K562). These results suggest that this binding site mediates the biological activities of GM-CSF on both proliferation and function of myeloid cells.

High-affinity urokinase receptor antagonists identified with bacteriophage peptide display.

Affinity selection of a 15-mer random peptide library displayed on bacteriophage M13 has been used to identify potent ligands for the human urokinase receptor, a key mediator of tumor cell invasion. A family of receptor binding bacteriophage ligands was obtained by sequentially and alternately selecting the peptide library on COS-7 monkey kidney cells and baculovirus-infected Sf9 insect cells overexpressing the human urokinase receptor. Nineteen peptides encoded by the random DNA regions of the selected bacteriophage were synthesized and tested in a urokinase receptor binding assay, where they competed with the labeled N-terminal fragment of urokinase with IC50 values ranging from 10 nM to 10 microM. All of the isolated peptides were linear and showed two relatively short conserved subsequences: LWXXAr (Ar = Y, W, F, or H) and XFXXYLW, neither of which is found in urokinase or its receptor. Competition experiments demonstrated that the most potent peptide, clone 20, prevented binding of bacteriophage displaying the urokinase receptor binding sequence (urokinase residues 13-32). In addition, this peptide blocked other apparently unrelated receptor binding bacteriophage, suggesting overlapping receptor interaction sites for all of these sequences. These results provide a demonstration of bacteriophage display identifying peptide ligands for a receptor expressed on cells and yield leads for the development of urokinase receptor antagonists.

K562 cells produce and respond to human erythroid-potentiating activity.

Human erythroid-potentiating activity (EPA) is a 28,000 mol wt glycoprotein that stimulates the growth of erythroid progenitors in vitro and enhances colony formation by the K562 human erythroleukemia cell line. EPA has potent protease inhibitory activity, and is also referred to as tissue inhibitor of metalloproteinases (TIMP). We observed that colony formation by K562 cells in semi-solid medium containing reduced fetal calf serum (FCS) is not directly proportional to the number of cells plated, suggesting production of autostimulatory factors by K562 cells. Using radioimmunoprecipitation and a bioassay for EPA, medium conditioned by K562 cells was found to contain high levels of biologically active EPA; Northern hybridization analysis confirmed the expression of EPA mRNA. Radiolabeled EPA was used to identify cell surface receptors on K562 cells. Together, these results suggest that EPA may act as an autocrine growth factor for K562 cells.

Purification and characterization of a human T-lymphocyte-derived glial growth-promoting factor.

Antigen- or lectin-stimulated T lymphocytes and human T-cell leukemia virus (HTLV)-infected cell lines secrete lymphokines that can influence the growth and function of a variety of cell types. We recently demonstrated that supernatants from the HTLV-II-infected Mo-T-cell line stimulate the proliferation of rat brain oligodendrocytes and astrocytes. We have now purified a glial growth-promoting factor (GGPF) from these supernatants. Purification from serum-free conditioned medium was accomplished by sequential concentration, ammonium sulfate precipitation, lentillectin affinity chromatography, gel filtration, and reversed-phase high-performance liquid chromatography. GGPF is assayed by its ability to stimulate DNA synthesis in oligodendrocytes, as measured by [3H]thymidine uptake. The purified GGPF has an apparent Mr of 30,000 when analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, however, GGPF appears as a single band of Mr 18,000. Both reduced and unreduced forms have biological activity, suggesting that GGPF exists in both a functional monomeric and dimeric form. Purified GGPF appears to be a biochemically and functionally distinct lymphokine.

Yeast expression and phagemid display of the human urokinase plasminogen activator epidermal growth factor-like domain.

The human urokinase plasminogen activator (uPA) epidermal growth factor-like domain (residues 1-48) and a variant with a C-terminal epitope tag have been secreted from recombinant yeast. Purified human uPA 1-48 and uPA 1-48glu complete for binding to the human uPA receptor with Kds of 180 and 400 pM respectively, in an in vitro assay using an immobilized recombinant uPA receptor. A synthetic gene encoding human uPA 1-48 with an N-terminal epitope tag was inserted into a phagemid expression vector as a fusion with residues 249-406 of the M13 pIII protein with an intervening amber codon (TAG). Phagemid production led to infectious particles which were selectively bound and eluted from both epitope tag antibody and urokinase receptor. Sequential binding to this antibody and receptor demonstrated a substantial enrichment, where up to 10% of the infectious particles were then retained on urokinase receptor-coated plates. A PCR strategy was used to convert previously described peptide bacteriophage ligands for the urokinase receptor to phagemid display. The yields of these peptide phagemids and the uPA 1-48 phagemid showed a correlation with peptide affinity, in contrast to when the peptides are multivalently displayed on a bacteriophage.

Nonhematopoietic tumor cells express functional GM-CSF receptors.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the colony growth of myeloid progenitors in semisolid media, and enhances the function of mature effector cells, including neutrophils, monocytes, and eosinophils. Small cell carcinoma lines (SCCL) have properties of amine precursor uptake and decarboxylation (APUD) cells and express high levels of the enzyme, L-aromatic amino acid decarboxylase. We looked for possible expression of GM-CSF receptors on nonhematopoietic cells and found specific high-affinity binding of human GM-CSF to SCCL and to the SV40-transformed African green monkey kidney cell line, COS. The small cell carcinoma lines responded to GM-CSF with enhanced proliferation, and both small cells and COS cells were found to express authentic 84,000 dalton GM-CSF receptor protein. These findings indicate that nonhematopoietic cells can bind and respond to GM-CSF, suggesting additional biological activities as well as the possibility of tumor responses when GM-CSF is used therapeutically in humans. Since preliminary clinical trials using CSFs as adjunctive treatment in patients with solid tumors are underway, it will be important to consider the possible responsiveness of nonhematopoietic tumor cells to CSFs.

Molecular characterization and expression of the gene encoding human erythroid-potentiating activity.

Erythropoietin is the primary physiological regulator of erythropoiesis; however, in vitro studies have identified another class of mediators which appear to be important in stimulating erythroid progenitors. These factors have generally been referred to as burst-promoting activities (BPA), because they stimulate the growth of early erythroid progenitors referred to as burst-forming units-erythroid (BFU-E) which give rise to colonies of up to thousands of haemoglobinized cells. We recently reported purification of a burst-promoting activity from medium conditioned by the Mo T-lymphoblast cell line infected with human T-cell lymphotropic virus type II (HTLV-II). This purified glycoprotein of relative molecular mass (Mr) 28,000 also stimulates colony formation by more mature erythroid precursors (CFU-E) and is therefore referred to as erythroid-potentiating activity (EPA). Purified EPA specifically stimulates human and murine cells of the erythroid lineage, unlike murine interleukin-3 (IL-3) which stimulates precursor cells from all haematopoietic lineages. We report here the isolation of a complementary DNA molecular clone encoding EPA and its use in producing EPA in COS (monkey) cells and CHO (Chinese hamster ovary) cells. We also define the organization of the EPA gene in human DNA.