Steven Gillis

CD30 antigen, a marker for Hodgkin's lymphoma, is a receptor whose ligand defines an emerging family of cytokines with homology to TNF

CD30 is a surface marker for neoplastic cells of Hodgkins lymphoma and shows sequence homology to members of the tumor necrosis factor (TNF) receptor superfamily. Using a chimeric probe consisting of the extracellular domain of CD30 fused to truncated immunoglobulin heavy chains, we expression cloned the cDNA cognate from the murine T cell clone 7B9. The encoded protein is a 239 amino acid type II membrane protein whose C-terminal domain shows significant homology to TNF alpha, TNF beta, and the CD40L. Cross-hybridization to an induced peripheral blood T cell cDNA library yielded the human homolog, which is 72% identical at the amino acid level. The recombinant human ligand enhances the proliferation of CD3-activated T cells yet induces differential responses, including cell death, in several CD30+ lymphoma-derived clones. The human and murine genes map to 9q33 and the proximal region of chromosome 4, respectively.

Recombinant granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for lymphoid cancer

Background. The period of neutropenia after autologous bone marrow transplantation results in substantial morbidity and mortality. The results of previous phase I-II clinical trials suggest that recombinant human granulocytemacrophage colony-stimulating factor (rhGM-CSF) may accelerate neutrophil recovery and thereby reduce complications in patients after autologous bone marrow transplantation. Methods. We conducted a randomized, double-blind, placebo-controlled trial at three institutions. The study design and treatment schedules were identical, and the results were pooled for analysis. One hundred twenty-eight patients were enrolled. Sixty-five patients received rhGM-CSF in a two-hour intravenous infusion daily for 21 days, starting within four hours of the marrow infusion, and 63 patients received placebo. Results. No toxic effects specifically ascribed to rhGM-CSF were observed. The patients given rhGM-CSF had a recovery of the neutrophil count to 500×106 per liter 7 days earlier than the patients who...

Stimulation of Myelopoiesis in Patients with Aplastic Anemia by Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor

Aplastic anemia is a syndrome in which pancytopenia occurs in the presence of hypocellularity of the bone marrow. To assess the biologic activities of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) in aplastic anemia, we gave GM-CSF (60 to 500 micrograms per square meter of body-surface area) to 10 patients with moderate or severe disease, by continuous intravenous infusion daily for two weeks, and repeated the treatment after a two-week rest period. The treatment increased the white-cell count (1.6- to 10-fold) in all patients, primarily because of an increase in the numbers of neutrophils (1.5 to 20-fold), eosinophils (12- to greater than 70-fold), and monocytes (2- to 32-fold). Rates of hydrogen peroxide production in purified granulocyte fractions increased during GM-CSF treatment. Increases in bone marrow cellularity, myeloid precursor cells, and myeloid:erythroid cell ratios accompanied the white-cell response. Despite the in vivo response of the white-cells, the concentration of colony-forming cells remained the same. Measurable concentrations of interleukin-2 (2 to 15 units per milliliter) were found in the serum of 8 patients, and high levels of erythropoietin (81 to 1200 IU per liter) were found in 10 patients. The predominant side effects were constitutional symptoms. These results indicate that recombinant human GM-CSF is effective in stimulating myelopoiesis in patients with severe aplastic anemia and may benefit some patients in whom the disorder is refractory to standard forms of therapy.

Molecular Characterization of Interleukin 2

The isolation of specific T cell growth factors termed interleukin 2 (IL 2) is changing the approach to understanding T cell function. This class of growth factors has allowed te establishment of a number of human and murine T cell lines. We summarize the biochemical properties of human and murine IL 2. Studies have been initiated to isolate mRNA encoding for IL 2. Such RNA can be translated in rabbit reticulocyte lysates yielding IL. 2. This RNA may be useful for the development of probes to isolate lymphokine genes.

Human macrophage-colony stimulating factor: Alternative RNA and protein processing from a single gene

Macrophage-colony stimulating factor (M-CSF, CSF-1) has been reported to be required for the proliferation and differentiation of macrophages from hematopoietic progenitor cells. Recently, two human M-CSF cDNA clones were isolated encoding proteins of 256 and 554 amino acids. We report here the isolation of a third M-CSF cDNA that encodes a protein of 438 amino acids. The coding regions for the three cDNA clones share a common amino-terminus of 149 amino acids and a common carboxyl-terminus of 75 amino acids including a membrane spanning region. In addition, we isolated a genomic clone of human M-CSF. When each of the cDNA clones or the genomic clone were transfected into COS-7 monkey kidney cells, biologically active M-CSF was expressed as judged by the ability of transfected cell supernatants to stimulate proliferation and colony formation of murine bone marrow cells, as well as formation of monocytic colonies from human bone marrow cells. Surprisingly, proliferation of human bone marrow cells was not induced by recombinant human M-CSF. Analysis of the M-CSF proteins released by COS-7 cells revealed that monomer subunit proteins of 44 or 28 kDa were produced. In addition, we found that the membrane spanning region, present in all three forms of M-CSF cDNA, was not required for the synthesis of a biologically active protein. However, when the membrane spanning region was present in the three M-CSF cDNAs, cell surface associated forms of M-CSF could be readily detected.

Interleukin-1 is released at sites of human cutaneous allergic reactions

Interleukin-1 (IL-1) promotes cell recruitment and influences allergic mediator release. We analyzed histamine, prostaglandin D2, IL-1, and leukocytes accumulating hourly for 12 hours at skin-chamber sites after local ragweed challenge in eight allergic subjects with cutaneous late-phase reactions. Ragweed induced a peak of histamine at 1 hour (p less than 0.02), which diminished, and then steadily increased (p less than 0.02). Prostaglandin D2 levels peaked by the second hour (p less than 0.02) and then decreased, approaching prechallenge levels by 12 hours. Leukocyte infiltration (predominantly neutrophils) was detectable 3 to 4 hours after challenge, although selective enrichment of mononuclear cells, eosinophils, and basophils ws observed at later hours (p less than 0.02). IL-1 bioactivity was detected in fluids 10 to 12 hours after challenge but not at control sites (p less than 0.05). Analysis of IL-1 beta levels by RIA revealed an initial peak at 1 hour of 0.90 ng/ml (p less than 0.02) and a second elevation of up to 0.75 ng/ml during the later hours (p less than 0.04). Ragweed challenge of three nonatopic subjects did not change levels of the above-mentioned mediators or cells. Bioactivity in chamber fluids from antigen-challenged sites of atopic subjects was significantly neutralized by an anti-IL-1 beta antiserum, although treatment with anti-IL-1 alpha and anti-IL-1 beta was needed for complete neutralization, IL-1 released locally during cutaneous allergic reactions may contribute to IgE-dependent cutaneous inflammation.

Expression, purification and characterization of recombinant marine granulocyte-macrophage colony-stimulating factor and bovine interleukin-2 from yeast

Expression and secretion of two lymphokines, murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and bovine interleukin-2 (BoIL-2), to levels of 50-60 mg per liter were achieved by placing these cDNAs in a Saccharomyces cerevisiae expression vector that utilized the yeast alcohol dehydrogenase-2 promoter and alpha-factor leader peptide. These lymphokines were purified to homogeneity by direct application of the crude yeast medium to reversed-phase high-performance liquid chromatography. Despite the fact that both lymphokines contain at least one N-glycosylation site and have identical N-terminal residues (Ala-Pro-Thr), recombinant (R) GM-CSF was found to be heterogeneously glycosylated by yeast while RBoIL-2 was secreted without glycosylation. Additionally, approximately 40% of the RGM-CSF was found to be proteolytically cleaved after the second amino acid residue, while RBoIL-2 was found to be intact.

Cloning, sequence and expression of bovine interleukin 1α and interleukin 1β complementary DNAs

Abstract Interleukin 1 (IL-1) is a cytokine which mediates a variety of immunoregulatory and inflammatory activities. Using human IL-1α and IL-1β probes, cDNAs for the corresponding bovine genes were isolated from an alveolar macrophage library. The open reading frames of the bovine IL-1α and IL-1β cDNAs encode proteins of 268 and 266 amino acids, respectively, each with a predicted mol. wt of approx. 31,000. Both forms of bovine IL-1 exhibit a high degree of sequence homology with IL-1 gene products from other mammalian species. Based upon comparisons with human IL-1 amino acid sequences, the post-translationally processed, mature forms of bovine IL-1 would occur as 17–18,000 mol. wt proteins. Sequences encoding mature bovine IL-1α and IL-1β were inserted into E. coli expression plasmids and biologically active proteins were synthesized as judged by the ability of the recombinant proteins to induce proliferation of bovine thymocytes. Both IL-1α and IL-1β exist as single genomic copies. In addition, bovine IL-1β mRNA is approx. 10-fold more abundant than IL-1α mRNA in stimulated alveolar macrophages.

Bovine GM-CSF: molecular cloning and biological activity of the recombinant protein.

The lymphokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages from bone marrow progenitors, and regulates biological functions expressed by mature cells of these lineages. In order to isolate a bovine GM-CSF cDNA, a cDNA library, generated from the BT2 bovine T cell line, was screened with a human GM-CSF cDNA probe. A cDNA clone was isolated with an insert of 783 bp, that would encode a protein of 143 amino acids, with a predicted mol. wt of 16,160. Bovine GM-CSF exhibits a high degree of sequence homology with mouse and human GM-CSF at both the nucleotide and amino acid levels. Comparison of GM-CSF amino acid sequences from the three species indicates that the bovine GM-CSF precursor contains a putative 17 amino acid signal sequence, cleavage of which would yield a 14,250 mol. wt protein. The cDNA was inserted into a mammalian expression vector and transfected into COS-7 monkey kidney cells. Biologically active bovine GM-CSF was secreted as judged by a bovine bone marrow proliferation assay. Bovine GM-CSF was weakly active in both human and mouse bone marrow proliferation assays. In contrast, human GM-CSF was weakly active on bovine but not murine mouse bone marrow cells and mouse GM-CSF was only active on murine bone marrow cells.

DISPLAY OF THE NEUTRAL GLYCOLIPID GANGLIO-N-TETRAOSYLCERAMIDE (ASIALO GM1) ON CELLS OF THE NATURAL KILLER AND T LINEAGES

Analysis was made of the display of the neutral glycolipid asialo GM1 on cells involved in the differentiation and expression of natural killer (NK) and T cell-mediated cytotoxicity in vitro. Removal of asialo GM1-bearing cells from CBA spleens, by treatment with a specific rabbit antibody in the presence of complement, led not only to the abrogation of NK cell activity but also to the lack of responsiveness of such populations to polyinosinic: polycytidylic acid (poly I:C) and to interferon, indicating that both NK cells and interferon-responsive cells of the NK cell lineage bear asialo GM1. Cytotoxic T lymphocytes (CTLs) induced by mixed lymphocyte culture in vitro were unaffected by treatment with antiasialo GM1 serum in the presence of complement, but normal spleen cells subjected to this treatment failed to mount CTL responses to alloantigen, even in the presence of an exogenous source of Interleukin-2 (IL-2). Furthermore, spleen cell populations depleted of asialo GM1-bearing cells showed a decreased ability to produce IL-2 in response to mitogenic stimulation.