In vivo studies with chimeric toxins. Interleukin-2 fusion toxins as immunosuppressive agents.
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Animals↗
Biomedical subjects
J C Nichols
Publications and source records attributed to J C Nichols.
Protein engineering of DAB-IL-2 fusion toxins to increase biologic potency.
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Amino Acid Sequence↗
DAB486IL-2 (IL-2 toxin) selectively inactivates high-affinity IL-2 receptor-bearing human peripheral blood mononuclear cells.
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Diphtheria Toxin↗
Cytotoxic properties of DAB486EGF and DAB389EGF, epidermal growth factor (EGF) receptor-targeted fusion toxins.
Elevated expression of the receptor for epidermal growth factor (EGF) is a characteristic of several malignancies including those of the breast, bladder, prostate, lung, and neuroglia. To therapeutically target the cytotoxic action of diphtheria toxin to EGF receptor-expressing tumor cells, we have constructed a hybrid gene in which the sequences for the binding domain of diphtheria toxin have been replaced by those for human EGF. The resulting fusion toxins, DAB486EGF and DAB389EGF, bind specifically to the EGF receptor and inhibit protein synthesis in a variety of EGF receptor expressing human tumor cell lines with an IC50 as low as 0.1 pM. Comparisons of DAB486EGF and DAB389EGF showed that DAB389EGF was consistently 10- to 100-fold more cytotoxic than DAB486EGF. Like diphtheria toxin, the cytotoxic action of DAB389EGF results from ADP-ribosylation of elongation factor-2 and is sensitive to the action of chloroquine. Studies of the kinetics of cellular intoxication showed that a 15-min exposure of EGF receptor-expressing A431 cells to DAB389EGF results in complete protein synthesis inhibition within 4 h. Furthermore, inhibition of protein synthesis results in elimination of human tumor cell colonies. These findings show that DAB389EGF is a potential therapeutic agent for a wide variety of EGF receptor-expressing solid tumors.
Adenosine Diphosphate Ribose↗
Impact of interleukin-2-receptor-targeted cytotoxins on a unique model of murine interleukin-2-receptor-expressing malignancy.
DAB486IL-2 is a genetically engineered fusion protein consisting of a portion of diphtheria toxin fused to human IL-2. It is specifically cytotoxic for tumor cells which bear high-affinity IL-2 receptors (IL-2R). DAB389IL-2 is a similarly constructed hybrid protein which is smaller than DAB486IL-2 and is slightly more potent in vitro. We have developed a murine model of IL-2R-expressing malignancy to study the in vivo efficacy of these genetically engineered cytotoxins. Following intravenous administration of CP3 cells, C57BL/6 mice develop tumors which are lymphatic in distribution. When mice are injected i.v. with 10(6) CP3 cells, 90% of the animals show signs of observable tumor by day 10 to 20; death occurs in 50% of untreated animals by day 30. Intravenous treatment of mice with DAB486IL-2 (10 micrograms daily for 10 days), beginning 24 hr after administration of CP3 cells, increases mean survival time by approximately 50%. In comparative studies, DAB389IL-2 is more potent in vivo than DAB486IL-2, with approximately 90% of treated animals with no evidence of tumor at 60 days. The mechanism of action of tumor inhibition by DAB486IL-2 is specific, since treatment of animals which have IL-2R-negative EL4 tumors has not resulted in increased survival time. In addition, treatment of such tumors with DAglu53B486IL-2, a fusion protein which can bind to the IL-2R but is incapable of inhibiting protein synthesis, is ineffective.
Animals↗
Selective elimination of HIV-1-infected cells with an interleukin-2 receptor-specific cytotoxin.
Infection by human immunodeficiency virus type 1 (HIV-1) is associated with cellular activation and expression of the interleukin-2 (IL-2) receptor. A genetically engineered fusion toxin, DAB486 IL-2, that contains the enzymatic site and translocation domain of diphtheria toxin and the receptor binding domain of IL-2 specifically kills cells that express high-affinity IL-2 receptors. This toxin selectively eliminated the HIV-1-infected cells from mixed cultures of infected and uninfected cells and inhibited production of viral proteins and infectious virus. Thus, cellular activation antigens present a target for early antiviral intervention.
Cell Survival↗
Inhibition of human antigen-specific memory B cell response in vitro by a diphtheria toxin-related interleukin 2 fusion protein.
Recombinant diphtheria toxin-related interleukin-2 fusion protein (DAB486IL-2) is specifically cytotoxic for cells bearing the high-affinity IL-2 receptor (p55/75). We evaluated the effects of DAB486IL-2 on the generation of tetanus toxoid (TT)-specific IgG antibody-forming cells in 6-day cocultures of human splenocytes and TT-coupled Sepharose beads. The results indicate that a significant portion (30-75%) of the anti-tetanus toxoid IgG response in vitro was susceptible to inhibition by 10(-10) M DAB486IL-2. The inhibition required both the IL-2 portion of the fusion protein and an active toxin moiety and was greater when the IL-2 toxin was added on Day 3 as compared with Day 0 of culture. The induction of the p55 (Tac) subunit of the IL-2R was demonstrable by two-color flow cytometry on a small percentage (5%) of B cells and on a higher percentage (10%) of non-B cells 3 days after exposure to TT-coupled Sepharose. Short-term (2 hr) treatment of T and B cell subpopulations separated on Day 3 of culture followed by remixing indicated that while activated T helper cells were most strongly inhibited by DAB486IL-2, up to 50% of the TT-specific IgG response was inhibited by treatment of B cells alone with DAB486IL-2. Our results suggest that a strategy of eliminating human memory B cells by a combination of antigen activation and properly timed administration of a recombinant lymphokine-toxin fusion protein is feasible.
Animals↗
Suppression of immune responses to acetylcholine receptor by interleukin 2-fusion toxin: in vivo and in vitro studies.
The pathogenesis of the autoimmune disease, myasthenia gravis (MG), involves an antibody-mediated attack against acetylcholine receptors (AChRs). Since the relevant antibody response is T cell dependent, a therapeutic strategy aimed at T lymphocytes actively participating in the immune reaction to AChR should result in relatively selective suppression of AChR antibody. During an active immune response, T cells express receptors for interleukin 2 (IL2). In this study, we have used a genetically engineered fusion protein comprised of the binding region of IL2 and the toxic portion of diphtheria toxin (DAB486-IL2), to attempt to treat an experimental animal model of MG in rodents. We examined the effects of treatment with DAB486-IL2 in vivo on primary, ongoing, and secondary antibody responses to purified Torpedo AChR. Treatment of mice with intraperitoneal injections of DAB486-IL2 beginning at the time of immunization inhibited the primary AChR antibody response by 50% during the treatment period. Ongoing and secondary antibody responses to AChR were not suppressed in vivo by treatment with DAB486-IL2. In comparison, DAB486-IL2 was far more potent in suppressing antibody responses and lymphoproliferation in cell culture. At a dose comparable to that given in vivo, cellular proliferation and antibody production were virtually eliminated in a secondary response in vitro. The suppressive effect of DAB486-IL2 was much more pronounced when it was given at the time of initial antigen stimulation, as compared with its effect when given during an already established antibody response. These findings suggest that the effect of the fusion toxin on AChR antibody production was due predominantly to inhibition of T cells rather than B cells.
Animals↗
Interleukin 2 receptor-targeted cytotoxicity. Receptor binding requirements for entry of a diphtheria toxin-related interleukin 2 fusion protein into cells.
The receptor binding requirements for entry of the NAD+ ADP-ribosyltransferase component of DAB486-IL 2 into target cells were examined. Experiments utilizing cell lines bearing either high-affinity or individual subunits of the interleukin 2 receptor (IL 2R) as well as human peripheral blood mononuclear cells with natural killer activity demonstrate that the high-affinity receptor facilitates delivery of fragment A from DAB486-IL 2 to the cytosol approximately 1000 times more efficiently than either the intermediate-(p75) or low-affinity (p55) forms of the IL 2R. We show that elongation factor 2 (EF-2) in these cells is not quantitatively or qualitatively altered indicating that the relative resistance to intoxication displayed by IL 2R variant cell lines cannot be attributed to an altered intracellular target of the hybrid toxin. We also demonstrate that an alteration in the binding of DAB486-IL 2 to the p75 subunit of the IL 2R may account for the selective cytotoxicity of DAB486-IL 2 for cells bearing the heterodimeric high-affinity IL 2R.
Amino Acid Sequence↗
The impact of PROs on orthopaedic practice.
The practicing orthopaedic surgeon is beginning to feel the impact of the Medicare review process as conducted by the PRO system. Retrospective review, payment denials, preauthorization, substandard care review and similar activities by the PRO will have a direct effect on how orthopaedic surgeons care for patients and document their practices. The following is a brief description of some of these activities and suggestions as to how the practicing orthopaedist can respond to these changes.
Humans↗
Cytotoxicity of interleukin 2-toxin toward lymphocytes from patients with adult T-cell leukemia.
The inhibitory effect of a diphtheria toxin-related interleukin 2 fusion protein, IL-2-toxin, on protein synthesis in adult T-cell leukemia/lymphoma (ATL) cells was examined in vitro. Peripheral blood ATL cells from 12 patients (six acute type, four chronic type, and two smoldering type ATL) and the lymph node cells from three ATL patients (two acute type and one lymphoma type ATL) were examined. At a concentration of 10(-8) M, IL-2-toxin inhibited protein synthesis by 60 to 98% in lymph node ATL cells, whereas protein synthesis in peripheral blood ATL cells was inhibited from 20 to 57% in acute type, and from 3 to 13% in chronic type. In contrast, IL-2-toxin had no measurable effect on T-cells from either patients with smoldering type ATL or normal controls. The cytopathic effects of IL-2-toxin were blocked by the addition of anti-CD25 monoclonal antibody, suggesting that the inhibition of protein synthesis in target cells was mediated by the IL-2 receptor (IL-2R). The degree of inhibition of protein synthesis, however, was not closely correlated with expression of CD25 antigen (low-affinity Mr 55,000 glycoprotein, IL-2R, Tac antigen) on ATL cells. There was an apparent correlation between the degree of inhibition and the rate of protein synthesis in ATL cells. We demonstrate that ATL cells from patients with acute or lymphoma type disease were more sensitive to IL-2-toxin than cells from chronic or smoldering disease. These findings suggest that the high affinity IL-2R present on acute and lymphoma type ATL cells may serve as a target for therapy with this recombinant chimeric toxin.
Adult↗
Interleukin 2-diphtheria toxin fusion protein can abolish cell-mediated immunity in vivo.
De novo expression of the interleukin 2 receptor (IL-2R) is a critical and pivotal event in initiation of an immune response. Targeting the low-affinity IL-2-binding p55 subunit of the high-affinity IL-2R with the rat anti-mouse IgM monoclonal antibody M7/20 suppresses a variety of T-cell-mediated reactions, including transplant rejection, autoimmunity, and delayed-type hypersensitivity (DTH). A hybrid IL-2-toxin gene was constructed from the diphtheria toxin gene by replacing the DNA encoding the diphtheria toxin receptor-binding domain with the DNA encoding the receptor-binding domain of IL-2, and the fusion protein encoded by the hybrid gene was expressed in Escherichia coli [Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F., Strom, T.B. & Murphy, J.R. (1987) Protein Eng. 1, 493-498]. We examined the action of the chimeric IL-2-toxin fusion protein on an in vivo T-cell mediated response, DTH. The IL-2-toxin fusion protein was found to be a potent immunosuppressive agent. Treatment of mice with the IL-2-toxin blocks DTH and prevents expansion of IL-2R+ T cells. Indeed, IL-2-toxin treatment targets IL-2R+ T cells in vivo and is shown to selectively eliminate their appearance in draining lymph nodes. DTH suppression was observed even in mice possessing high titers of antibodies to diphtheria toxoid.
Animals↗
Propranolol and depression: a reevaluation based on a pilot clinical trial.
A wide range of neuropsychiatric side effects are attributed to propranolol including visual hallucinations, somnulence, memory impairment, decrease in response time, dizziness, confusional states, insomnia, nightmares, fatigue, sedation and depression. Benson et al., in a summary review of several clinical studies of 5,846 patients being treated with a variety of beta adrenergic blocking agents, listed depression as a rare side effect of propranolol that was usually reported only after long term treatment at high doses. Despite the widely circulated attribution that depression is a side effect of propranolol, there is a paucity of evidence to directly link this drug with clinically significant mood disturbance. For example, the most widely quoted reference attributing propranolol as a depressogenic agent was a "letter to the editor" which was a retrospective, uncontrolled, unblinded study that did not use a standardized depression rating scale. Most of the evidence linking propranolol to depressive symptoms have derived from scattered case reports in which the onset of depressive symptoms were attributed to this agent. Given the well known cyclic onset and remissions of affective disorders, and the prevalence of depression in the general medical population as a whole, the role of propranolol in these cases is debatable.
Aged↗
Isolation of hybridoma cell lines and characterization of monoclonal antibodies against cholera enterotoxin and its subunits.
Hybridoma cell lines which produced monoclonal antibodies against cholera toxin were isolated. These cell lines were detected with an enzyme-linked immunosorbent assay screening procedure with purified cholera toxin or subunit A of cholera toxin. Seven cell lines were characterized with respect to their reactivity with cholera toxin subunits by Western blot analysis. Five clones produced antibodies which were directed against subunit A, and two clones produced antibodies which reacted with subunit B. These antibodies were also characterized by Western blot analysis for reactivity with the heat-labile enterotoxin produced by porcine and human enterotoxinogenic strains of Escherichia coli. Monoclonal antibodies which reacted with subunit A of cholera toxin also reacted with subunit A of both porcine and human heat-labile enterotoxins. In contrast, monoclonal antibodies to subunit B of cholera toxin did not react with subunit B of the heat-labile enterotoxin. Antibodies directed against subunit B neutralized the biological activity of cholera toxin in vitro in the S49 mouse lymphosarcoma assay. In contrast to polyclonal anti-subunit A antisera, monoclonal anti-subunit A from four of five clones had small but measurable neutralizing capacities in vitro.
Animals↗
Cholera toxin is synthesized in precursor form on free polysomes in Vibrio cholerae 569B.
Membrane-bound and free polysomes have been isolated from Vibrio cholerae 569B. Nacent polypeptide chains were completed in a cell-free translation mixture containing Escherichia coli S-300 extracts and [3H]leucine or [35S]methionine. Cholera toxin-related polypeptides synthesized in vitro were immunologically detected after treatment with either anti-subunit A or anti-subunit B serum. Immunoreactive translation products were removed from reaction mixtures with formalinized Cowan's strain of Staphylococcus aureus, electrophoresed on sodium dodecyl sulfate-polyacrylamide gels, and visualized by fluorography. Anti-subunit A serum precipitated two major polypeptide species (molecular weights 52,000 and 45,000) from translation mixtures programed with free polysomes, whereas anti-subunit B serum precipitated only the 45,000-molecular-weight polypeptide. No cholera toxin-related polypeptides were detectable in translation mixtures programed with membrane-bound polysomes. Purified subunit A and cholera toxin competed for anti-subunit A binding sites and blocked the immunoprecipitation of the 35S-labeled 52,000- and 45,000-dalton polypeptides from in vitro translation mixtures. The data presented suggest that cholera toxin is synthesized in the cytoplasm in a precursor form on free polysomes and is secreted post-translationally.
Cholera Toxin↗
Capacity of virulent Treponema pallidum (Nichols) for deoxyribonucleic acid synthesis.
Treponema pallidum (Nichols) was extracted from infected rabbit tissue, and cell lysates were prepared for monitoring thymidine kinase and deoxyribonucleic acid polymerase activities. No thymidine kinase could be demonstrated in preparations of T. pallidum or the cultivable T. phagedenis biotype Reiter. Significant levels of deoxyribonucleic acid polymerase were detected in both treponemal samples. Interestingly, comparisons of polymerase activity among a spectrum of bacterial genera revealed a direct correlation between enzyme concentrations and estimated generation time. Incorporation of [3H]uridine and [3H]thymidine into macromolecules by intact T. pallidum and the Reiter treponeme was examined. Selective ribonuclease-deoxyribonuclease digestion and cesium chloride gradient banding demonstrated that T. pallidum, independent of the host, and T. phagedenis were capable of synthesizing deoxyribonucleic acid only from the [3H]-uridine precursor.
DNA, Bacterial↗
Ribosomal ribonucleic acid synthesis by virulent Treponema pallidum.
Ribosomal ribonucleic acid (rRNA) synthesis by virulent Treponema pallidum was monitored by incorporation of [(3)H]uridine into trichloroacetic acid-precipitable counts and examination of radiolabeled rRNA on polyacrylamide gels. Verification that rRNA synthesis originated with T. pallidum was based upon co-electrophoresis with Escherichia coli rRNA, proportionate reductions in the amount of rRNA synthesized when numbers of treponemes were decreased, and inclusion of appropriate animal cell controls. The rate of treponemal rRNA synthesis was greater at temperatures of 37 and 39 degrees C than at 33 degrees C; rRNA synthesis was inhibited at 4 and 42 degrees C and was effectively inhibited by actinomycin D. Kinetic experiments indicated that the majority of rRNA synthesis occurred early after extraction of treponemes from infected rabbit testicular tissue. Polyacrylamide gel profiles demonstrated the capacity of virulent T. pallidum to synthesize and process RNA to 23s, 16s, and 4 to 5s classes. Although motility of T. pallidum appeared unaffected during longer periods of incubation, pulselabeling experiments confirmed significant reductions in the rate of rRNA synthesis. When the effect of various environmental conditions upon rRNA synthesis was investigated, optimal synthesis was found to occur in an atmosphere of 20% oxygen whereas virtually no synthesis was observed under anaerobic or low-oxygen conditions.
Animals↗
Virulent Treponema pallidum: aerobe or anaerobe.
Substrate degradation and protein synthesis served as indicators of metabolism in virulent Treponema pallidum. Opitmal metabolic activity in these spirochetes was observed at 10 to 20% O2 concentrations, with markedly reduced activity at higher or lower O2 levels or under anaerobiosis; alternate functioning electron acceptors that might substitute for O2 were not found. Carbon monoxide and cyanide at concentrations that inactivate cytochrome oxidase were not effective metabolic poisons for T. pallidum, although Micrococcus lutea, a strict aerobe with cytochrome-dependent respiration, was inhibited under similar experimental conditions. Motility of virulent T. pallidum was vigorous in the presence of O2 and sluggish or inhibited in its absence, reinforcing the role of O2 in T. pallidum metabolism.
Aerobiosis↗