GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF.

We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.

Recombinant human platelet-derived growth factor-BB for the treatment of chronic pressure ulcers.

A randomized phase I/II double-blind, placebo-controlled study was designed to evaluate 1, 10, and 100 micrograms/ml (0.01, 0.1, and 1.0 micrograms/cm2) recombinant human BB homodimeric platelet-derived growth factor (rPDGF-BB) applied topically to chronic pressure ulcers for 28 days. Twenty patients were enrolled and completed the trial. No toxicities were associated with rPDGF-BB treatment. Patients treated with 100 micrograms/ml of rPDGF-BB had a pronounced healing response compared with placebo-treated patients. By day 29, ulcers treated with 100 micrograms/ml of rPDGF-BB were smaller in remaining size compared with those of placebo-treated patients when the following specific parameters were measured: percentage of initial depth (14.1 +/- 7.4 vs. 34.9 +/- 6.7) and percentage of initial volume (6.4 +/- 4.0 vs. 21.8 +/- 5.6). Histological analyses of biopsies revealed active wound healing processes in all groups with no disruption in the normal healing sequence in rPDGF-BB-treated wounds. The results of this small, descriptive study suggest rPDGF-BB is a potent vulnerary agent for accelerating soft-tissue repair, warranting further study.

Role of platelet-derived growth factor in wound healing.

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and fibroblasts in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as 20-200 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and fibroblasts and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other growth factors, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide growth factors may be the critical regulators of extracellular matrix deposition within healing wounds.

A pilot study of intralymphatic interleukin-2. II. Clinical and biological effects.

Interleukin-2 (recombinant methionyl human interleukin-2 alanine 125; IL-2) was administered intralymphatically to 12 patients with advanced cancer in a phase I trial. Doses were administered once a week for 6 weeks in a dosage escalation schedule; patients were entered in four groups at successively higher starting dosages. Toxicity occurred in a profile similar to that seen with intravenous IL-2. The maximum tolerated dose with this route/schedule was 275,000 units/kg, a figure not higher than expected with intravenous administration. T1/2 alpha was prolonged to 54 min from the 13 min figure we obtained with IL-2 given intravenously. Granulocytosis and eosinophilia were seen, along with lymphocytosis following initial lymphopenia. Anti-IL-2 antibodies were seen in 42% of patients (compared to 16% with this agent given intravenously), suggesting increased immunogenicity of this route/schedule. No clinical response was achieved. Immunologic effects will be reported separately but are summarized.

Interleukin-6 is not involved in the interleukin-1-induced production of colony-stimulating factors by human bone marrow stromal cells and fibroblasts.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays a role in regulation of hematopoiesis. Because IL-6 is coinduced with colony-stimulating factors (CSFs) by various cell types in response to stimulation with IL-1, we investigated whether IL-6 is involved in the IL-1-induced production of CSF by human bone marrow (BM) cells in long-term culture or human fibroblasts. We showed that IL-6 does not induce CSF production by these cells. Neither addition of exogenous IL-6 nor neutralization of endogenous production of IL-6 by an anti-IL-6 monoclonal antibody (MoAb) diminished the IL-1-induced colony-stimulating activity (CSA), indicating that IL-6 did not act synergistically with IL-1. Finally, IL-6 did not influence the kinetics of IL-1-induced CSA production by human fibroblasts. We conclude that IL-6, either alone or in combination with IL-1, does not induce CSF production by human BM stromal cells or fibroblasts.

Interleukin-1 synergizes with granulocyte-macrophage colony-stimulating factor on granulocytic colony formation by intermediate production of granulocyte colony-stimulating factor.

Interleukin-1 (IL-1) was found to act synergistically with granulocyte-macrophage colony-stimulating factor (GM-CSF) on granulocytic colony growth of normal human bone marrow cells, depleted of mononuclear phagocytes and T lymphocytes. Using CD34/HLA-DR-enriched bone marrow cells we demonstrated that this activity of IL-1 was not a direct action on hematopoietic progenitor cells, but an effect of an intermediate factor produced by residual accessory cells in response to IL-1. Neutralization experiments using an anti-IL-6 antiserum showed that IL-1-induced IL-6 did not contribute to the observed synergy. Furthermore, IL-6 by itself had neither a direct stimulatory effect on CFU-GM colony growth, nor did it act synergistically with GM-CSF on granulocytic or monocytic colony formation. Neutralization experiments with an anti-G-CSF monoclonal antibody showed that IL-1-induced G-CSF production was responsible for the synergy with GM-CSF. Using combinations of G-CSF and GM-CSF this synergistic activity could be detected at concentrations of G-CSF as low as 0.1 ng/mL (10 U/mL). Our results indicate that IL-1, but not IL-6, stimulates the GM-CSF-dependent proliferation of relatively mature myeloid progenitor cells in the presence of small numbers of accessory cells.

Interleukin-1 stimulates proliferation of acute myeloblastic leukemia cells by induction of granulocyte-macrophage colony-stimulating factor release.

In this study, we further established the role of interleukin-1 (IL-1) alpha and IL-1 beta as regulators of proliferation of acute myeloid leukemia (AML) cells. IL-1 stimulated tritiated thymidine (3H-TdR) uptake of AML cells in 13 of 28 cases. Cytogenetic analysis confirmed the leukemic clonality of the IL-1-stimulated cells. Most likely, IL-1 exerted these stimulative effects directly on AML blast cells because IL-1 effectively induced 3H-TdR uptake of CD34-positive AML blasts (separated following cell sorting). Furthermore, adherent cell-depleted AML samples of three patients were more effectively stimulated than nondepleted AML fractions. Cluster and colony formation from adherent cell depleted AML samples could also be stimulated with IL-1, ie, in seven of ten cases analyzed. Subsequent experiments indicated that IL-1 stimulation depended on the release of GM-CSF because (1) induction of DNA synthesis of AML cells by IL-1 could be abrogated with antigranulocyte-macrophage colony-stimulating factor (GM-CSF) antibody, (2) conditioned media (CM) prepared from IL-1 stimulated AML blasts (adherent cell depleted) could stimulate the proliferation of purified normal bone marrow progenitors whereas supernatants from nonstimulated AML blasts did not, and (3) GM-CSF was demonstrated in IL-1/AML-CM with a specific radioimmunoassay, while GM-CSF was not detectable in nonstimulated supernatants. In one case of AML showing significant 3H-TdR uptake in the absence of CSFs, this spontaneous DNA synthesis was found to depend on autocrine IL-1 beta release as it could be suppressed with anti-IL-1 beta antibody or anti-GM-CSF. The blockade by anti-IL-1 beta could be overcome by the addition of high concentrations of IL-1 beta as well as GM-CSF. Thus, in this particular case, endogenously produced IL-1 beta had stimulated the release of GM-CSF which resulted in GM-CSF-dependent proliferation. The results indicate that GM-CSF production by AML blasts is often regulated by IL-1 rather than being constitutive.

Interleukin 1 and poly(rI).poly(rC) induce production of granulocyte CSF, macrophage CSF, and granulocyte-macrophage CSF by human endothelial cells.

Electrophoretically pure human interleukin 1 (IL-1) beta was found to stimulate human endothelial cells in monolayer culture to elaborate colony-stimulating activity (CSA). Supernatant fluids from cultures stimulated with increasing concentrations of IL-1 were found to stimulate colony formation of myeloid (CFU-GM), erythroid (BFU-E), and multipotent (CFU-GEMM) progenitor cells in a dose-dependent fashion. The effect on mixed colony formation, however, was less than on CFU-GM and BFU-E growth. Similar to IL-1, the double-stranded RNA polyriboinosinic-polyribocytidilic acid (poly[rI].poly[rC]) also stimulated release of CSA by endothelial cells in a dose-dependent manner. The kinetics of IL-1-induced CSA release as opposed to poly(rI).poly(rC)-induced release were found to be different, in that poly(rI).poly(rC)-induced CSA production occurred more slowly. An anti-IL-1 beta antiserum was able to completely neutralize the IL-1-induced CSA release, but had no effect on poly(rI).poly(rC)-dependent CSA production, indicating that the latter effect was mediated by other mechanisms than intermediate production of IL-1 beta. Using specific immunologic assays, IL-1- as well as poly(rI).poly(rC)-inducible production of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF, and macrophage CSF was found. The release of CSF from endothelial cells in response to IL-1 may be a mechanism for stimulating production of neutrophils and mononuclear phagocytes, and for attracting and activating these cells at sites of inflammation.

Systemic administration of recombinant methionyl human interleukin-2 (Ala 125) to cancer patients: clinical results.

Nineteen evaluable patients with advanced malignancy were treated with recombinant methionyl human interleukin-2 (Ala 125), 5 days per week by intravenous bolus. Patients were entered in five groups at starting doses ranging from 0.05 to 2.56 x 10(6) U/m2. Doses were escalated weekly as tolerated toward a potential maximal dose of 11.6 x 10(6) U/m2. Maximal tolerated dose was 3.84 x 10(6) U/m2. Dose-limiting toxicity included fatigue, rigors, nausea/vomiting, fever, and diarrhea. Other toxicities included hyperesthesias, arthralgias/myalgias, rash, fluid retention, balanitis, and mild confusion. Leukocytosis, including granulocytosis, eosinophilia, and mild lymphocytosis, was observed, as was rare mild thrombocytopenia. No partial or complete response occurred. T1/2 alpha averaged 13.4 min, with interleukin-2 detectable 2 h after doses of greater than or equal to 2.56 x 10(6) U/m2. Three patients developed anti-IL-2 antibodies without demonstrable clinical significance.

Control of misincorporation of de novo synthesized norleucine into recombinant interleukin-2 in E. coli.

Interleukin-2 produced from a recombinant E. coli was found to contain as much as 19% norleucine in place of methionine in a minimal medium fermentation. Medium supplementation experiments and use of a leucine-requiring mutant host strain indicated the origin of norleucine to be de novo biosynthesis by reactions involving the enzymes of the leucine biosynthetic pathway. The misincorporation was highly suppressed by addition of either L-leucine or L-methionine to the fermentation and completely suppressed by adding both amino acids.

Human fibroblasts produce granulocyte-CSF, macrophage-CSF, and granulocyte-macrophage-CSF following stimulation by interleukin-1 and poly(rI).poly(rC).

Electrophoretically pure human interleukin-1 (IL-1) beta was found to stimulate human fibroblasts in a monolayer culture to elaborate colony-stimulating activity (CSA). Supernatant fluids from cultures induced with increasing concentrations of IL-1 were found to stimulate colony formation of myeloid (CFU-GM), erythroid (BFU-E), and multipotent (CFU-GEMM) progenitor cells in a dose-dependent fashion. The effect on mixed colony formation, however, was less than on CFU-GM and BFU-E growth. Similar to IL-1, the synthetical double-stranded RNA poly(rI).poly(rC) also stimulated release of CSA by fibroblasts. The kinetics of IL-1- and poly(rI).poly(rC)-induced CSA release were found to be different, in that poly(rI).poly(rC)-induced CSA production occurred more slowly. Anti-IL-1 antiserum was able to completely neutralize the IL-1-induced CSA release, but had no effect on poly(rI).poly(rC)-induced CSF production, suggesting that the latter effect was mediated by other mechanisms than IL-1 in supernatant. By the use of specific immunologic assays, G-CSF, M-CSF, and GM-CSF could be identified in media conditioned by fibroblasts treated with IL-1 or poly(rI).poly(rC). Poly(rI).poly(rC) appeared to be a better inducer for M-CSF than IL-1.

Interleukin 1 induces human marrow stromal cells in long-term culture to produce granulocyte colony-stimulating factor and macrophage colony-stimulating factor.

Pure interleukin 1 (IL 1) was found to stimulate established human bone marrow stromal layers in long-term culture to produce colony-stimulating activity (CSA). Maximal concentrations in the culture medium were reached 24 hours after a single IL 1 pulse. The effect could be neutralized by a specific rabbit anti-IL 1 antiserum. Stromal layers, once stimulated by IL 1, continued to release CSA into the culture medium in the absence of exogenous IL 1. A second IL 1 pulse induced CSA release in an identical manner, as did the primary stimulation, indicating that the CSA released was actively produced. Using specific immunologic assays, both granulocyte colony-stimulating factor (G-CSF) and macrophage CSF (M-CSF) could be identified in the culture supernatants, and production of both factors was inducible by IL 1. Shortly after initiation of the long-term marrow cultures "spontaneous" G-CSF and M-CSF release occurred. The release of G-CSF diminished following addition of the anti-IL 1 antiserum, indicating that endogenous production of IL 1 by stromal cells had contributed to this effect. These results further support the role of IL 1 as an important modulator of CSF production by cells of the hematopoietic microenvironment.

Vascular endothelial cells and granulopoiesis: interleukin-1 stimulates release of G-CSF and GM-CSF.

Cultured mononuclear phagocytes produce soluble factors that stimulate endothelial cells to release GM-colony-stimulating activity (GM-CSA). One such factor was recently identified as interleukin 1 (IL 1). Studies were designed to determine which types of granulopoietic factors are released by IL 1-stimulated endothelial cells. Supernatants from endothelial cells cultured for 3 days in medium containing IL 1 alpha and beta were tested in both murine and human CFU-GM colony growth assays. The effect of conditioned media on differentiation of WEHI-3B myelomonocytic leukemic cells was also examined. Control media containing IL 1 alone or unstimulated endothelial cell-conditioned media contained no detectable CSA in any bioassay. Medium conditioned by IL 1-stimulated endothelial cells stimulated the clonal growth of both human and murine CFU-GM and induced macrophage differentiation of WEHI-3B cells. Treatment of these conditioned media with a highly specific neutralizing monoclonal G-CSF antibody completely inhibited their activity in the murine CFU-GM assay, but only partially inhibited GM colony growth by human marrow. Treatment of the active conditioned media with a neutralizing rabbit anti-human GM-CSF antibody partially reduced the activity of the media in the human GM-colony growth assay. G-CSF radioimmunoassay of endothelial cell culture supernatants and Northern blot analysis of endothelial cell cytoplasmic RNA for GM-CSF gene transcripts confirmed that IL 1 induced expression of both G-CSF and GM-CSF genes. Because treatment of media with both antibodies abrogated all activity in the human GM colony growth assay, we conclude that IL 1-stimulated endothelial cells release both G and GM-CSF and that these are the only granulopoietic factors detectable in clonogenic assays released by these cells in vitro.

Construction, purification and biological activities of recombinant human interleukin-2 analogs.

Fourteen human interleukin-2 (IL-2) analogs have been cloned and expressed in E. coli, starting from a chemically synthesized gene for human IL-2 optimized for expression in E. coli. These analogs were purified to greater than 95% purity as determined by SDS-PAGE, and were measured for biological activity in a 3H-thymidine incorporation assay using an IL-2 dependent murine T-cell line (CTLL). One analog was made which eliminated the N-terminal 23 amino acids from the protein by replacing one restriction endonuclease fragment with another. This analog, which begins at an internal methionine, had no detectable CTLL activity. Thirteen analogs were constructed using oligonucleotide site-directed mutagenesis. Four of these analogs were truncated at various residues near the C-terminus (residues 106, 116, 121 and 126). These analogs had at least 500-fold lower CTLL activities than the natural recombinant IL-2. The remaining nine analogs had substitutions at 1, 2, or all 3 of the three cysteine residues in the protein (residues 58, 105 and 125). Substituting an alanine, asparagine, aspartic acid, or serine at residue 125 resulted in highly active molecules with CTLL activities similar to that of the natural recombinant IL-2. The analogs with alanine and serine substitutions at residue 125 actually had slightly higher CTLL activities than the natural recombinant IL-2. Substituting alanine for cysteine at position 125 and serine for cysteine at either position 58 or 105 yielded analogs with about 150-fold lower CTLL activities than natural recombinant IL-2. Substituting an alanine for the cysteine at position 125 and serines for cysteines at both positions 58 and 105 resulted in an analog with 30-fold lower CTLL activity than the natural recombinant IL-2. The ten analogs with less than 1.0% of the CTLL activity of natural recombinant IL-2 were tested for competition with the natural recombinant IL-2 by mixing a 10-to 100- fold excess of the analog with the natural recombinant IL-2 and assaying the mixture in the CTLL assay. None of these analog mixtures resulted in a lower activity than mixing the natural recombinant IL-2 with buffer alone, implying that none of these analogs effectively competes with the natural recombinant IL-2 for binding to IL-2 receptors during incubation with the CTLL cells. If reduced binding does occur, it may be the direct cause of their lower activities.

Structure and activity of glycosylated human interferon-gamma.

Structural properties and activity of recombinant human interferon-gamma (IFN-gamma) purified from Chinese hamster ovary (CHO) cells or a natural source were determined and compared with those of Escherichia coli-derived IFN-gamma. One preparation of CHO-derived IFN-gamma showed three bands, with the middle band being a doublet, in a SDS-polyacrylamide gel. The two higher-molecular-weight bands were shown to be glycosylated. Western blot analysis indicated that the three bands are IFN-gamma and lack an intact carboxyl terminus. The circular dichroic (CD) spectra showed that conformation of the CHO-derived IFN-gamma is similar in the native state, in acid, and after renaturation from acid to the E. coli-derived IFN-gamma. These results indicate that neither glycosylation nor carboxy-terminal processing affects conformational properties of the protein, as detected by CD spectroscopy. However, the antiviral activity was fourfold lower for the preparation of CHO-derived IFN-gamma than for the E. coli-derived IFN-gamma. A different preparation or a natural IFN-gamma preparation with less extensive carboxy-terminal processing showed similar conformational properties and antiviral activity to the E. coli-derived IFN-gamma. These results indicate that the carboxyl terminus, but not glycosylation, plays an important role in the antiviral activity of IFN-gamma.

Antiviral and antitumor effects of a human interferon analog, IFN-alpha Con 1, assessed in hamsters.

An analog of human alpha-and beta-interferons (IFN-alpha and -beta) (generally consisting of the most frequently observed amino acid residue at each position in the molecule) has pronounced antiviral and antiproliferative activity in human and hamster cells. Intraperitoneal administration of this analog (designated IFN-alpha Con 1) to hamsters at 10(6) to 10(8) U/kg resulted in proportional increases in plasma concentrations through 6 h of monitoring. IFN-alpha Con 1 at these doses effectively limited encephalomyocarditis virus (EMCV) infections of hamsters. A natural human IFN-alpha preparation was also active against virus infections in hamsters. The antitumor activity of IFN-alpha Con 1 and natural human IFN-alpha was assessed in hamsters inoculated with lethal TBD932 lymphosarcoma. Various IFN treatment schedules resulted in prolonged survival following tumor challenge. IFN-alpha Con 1 administered at 10(5) to 10(6) U/hamster daily for 9-12 days following tumor challenge was effective in delaying tumor development, as was a natural human IFN-alpha preparation. The efficacies of combined IFN and cyclophosphamide therapies were determined. Unlike the natural human subtype IFN-alpha A, IFN-alpha Con 1 did not diminish the efficacy of cyclophosphamide (2.5 mg/hamster for 3 days) against the lymphosarcoma. However, an ineffective dose of cyclophosphamide (0.05 mg/hamster for 3 days) when combined with IFN-alpha Con 1 treatment showed enhanced antitumor activity. Combinations of cimetidine (16 mg/hamster for 4 days) and IFN-alpha Con 1 treatment did not prolong survival in this model system.

Parameters for the evaluation of IL-2 stability.

Recombinant DNA derived interleukin-2 stability in accelerated and long term studies was evaluated using biological and protein chemical methods. Various biophysical parameters were investigated for their correlation with bioassay results and for their accuracy and utility as quantitative indicators of change. Biochemically distinguishable forms of the molecule exhibited different levels of IL-2 activity in vitro. Of the methods evaluated, SDS-PAGE and, to a greater extent, reverse phase and TSK based HPLC analyses have been found to measure relevant changes in the structure of human interleukin-2. Those test systems detect covalent and non-covalent aggregates, degradation products, and inappropriately oxidized forms of human interleukin-2 all of which contribute to an overall loss of IL-2 biological activity. Such measures provide sensitive and reproducible indications of changes relative to the standard bioassay. These studies have led to the development of an analog of interleukin-2 which is biologically active and shows improved long term stability.

Detection of acquired provirus sequences in mammary tumors from low-expressor, low-risk mice.

Murine mammary tumor virus (MuMTV) provirus sequences in the DNA from early-occurring (average age 10 mo) and late occurring (age greater than 20 mo) tumors in BALB/cfC3H mice were analyzed by Eco RI restriction endonuclease mapping procedures. All early tumors were MuMTV antigen-positive mammary adenocarcinomas that contained the 0.92- and 4.0-kilo base (kb) exogenous C3H MuMTV-specific Pst I restriction endonuclease fragments. All but 1 of the late mammary adenocarcinomas had MuMTV antigens detected by peroxidase antiperoxidase staining, and all contained the 0.92- and 4.0-kb exogenous virus Pst I fragments. Three late nonmammary tumors lacked both MuMTV antigens and acquired provirus sequences. Greater numbers of MuMTV sequences were detected in both early and late-arising mammary tumors by Eco RI restriction endonuclease mapping than were detected in tissues from uninfected BALB/c mice. However, neither the number nor the location of MuMTV proviruses correlated with tumor latent period.

Murine mammary tumor virus seroepidemiology in BALB/cfC3H mice: correlation with tumor development.

Serum reactivity to murine mammary tumor virus (MuMTV) was inversely related to mammary tumor risk in 8-to 22-week-old BALB/cfC3H breeding females. Mice at low tumor risk exhibited high-titered serum reactivity to MuMTV (50% end point, greater than or equal to 1:40 by radioimmunoassay) approximately 3-6 months earlier than did the mice at high tumor risk. Maternal MuMTV antibody levels were correlated with the serum anti-MuMTV reactivity of their neonatal offspring (2 wk of age). Serologic antiviral reactivity in infected mice did not change during periods of pregnancy and lactation. All infected animals had detectable serum MuMTV reactivity by 33 weeks of age. The virus-neutralizing capabilities of some of these sera were tested, Sera from some of the young, low-tumor-risk animals that had MuMTV-precipitating antibodies also had virus-neutralizing activity. Conversely, none of the sera from the high-tumor-risk animals had detectable MuMTV-neutralizing antibodies.