Interleukin 2 regulates the expression of Tac antigen on peripheral blood T lymphocytes.

We investigated the effect of OKT3 antibody and interleukin 2 (IL-2) on Tac antigen expression and the proliferation of human peripheral blood mononuclear leukocytes. OKT3 monoclonal antibody at low, nonmitogenic concentrations (25 pg/ml) or IL-2 alone at optimal concentrations (20 U/ml) did not induce IL-2 receptor expression, as measured by Tac antibody or by T cell proliferation. However, costimulation with these concentrations of OKT3 antibody and IL-2 led to Tac antigen expression and T cell proliferation. These data suggest that the T cells are activated in two steps: OKT3 antibody at 25 pg/ml does not induce Tac antigen expression, but preactivates T cells to become responsive to IL-2. The addition of exogenous IL-2 then leads to expression of the IL-2 receptor, as recognized by Tac antibody, and to subsequent proliferation.

Biological activities of a human pluripotent hemopoietic colony stimulating factor on normal and leukemic cells.

We studied the biological effects of pluripoietin, a human pluripotent hemopoietic colony-stimulating factor (CSF) purified from the 5637 bladder carcinoma cell line. We found that this human CSF appears to be a unique hemopoietic growth factor, differing from interleukin 3 (IL-3) by virtue of its leukemia differentiating activity in mouse and man, and from mouse granulocyte CSF, which does have differentiation-inducing activity, but lacks pluripoietic activity. In addition, differences from IL-3 were observed in cross-species activity on normal and leukemic cells.

Activation of human macrophages. Comparison of other cytokines with interferon-gamma.

Cytokines affecting mononuclear phagocytes were screened for activation of human macrophages to secrete H2O2 and kill toxoplasmas. In contrast to recombinant interferon-gamma (rIFN gamma), the following factors, tested in partially or highly purified form and over a wide range of concentrations, did not augment these functions: native interferon-alpha (nIFN alpha), rIFN alpha A, rIFN alpha D, rIFN beta, colony stimulating factor (type 1) (CSF-1), CSF for granulocytes and macrophages (GM-CSF), pluripotent CSF (p-CSF), tumor necrosis factor (TNF), native interleukin 2 (nIL-2), and rIL-2. Partially purified migration inhibitory factor (MIF) enhanced H2O2-releasing capacity submaximally without inducing antitoxoplasma activity, and warrants further study.

Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells.

Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition, recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL, classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore, the murine WEHI-3B(D+) cells and human leukemic cells classified as M2, M3, and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600.

HLA-DR human histocompatibility leukocyte antigens-restricted lymphocyte-monocyte interactions in the release from monocytes of acidic isoferritins that suppress hematopoietic progenitor cells.

Acidic isoferritins, which under normal conditions are released from monocytes and macrophages, have a suppressive effect in vitro on granulocyte-macrophage, erythroid, and multipotential hematopoietic progenitor cells. Cell interactions modulating the release of acidic isoferritin-inhibitory activity (AIFIA) from human monocytes were investigated using the bone marrow granulocyte-macrophage progenitor cells as a target cell assay for assessing AIFIA. Monocytes, in the absence of T lymphocytes, released AIFIA when allowed to condition culture medium at 10(4) or higher concentrations of monocytes/ml. However, subpopulations of T lymphocytes modulated the release of AIFIA from monocytes. OKT8+- and OKT4+-T lymphocytes were obtained from E-rosette-positive lymphocytes by using T lymphocyte subset-specific monoclonal antibodies in either a complement-dependent cytotoxicity test to select negatively for the cells or by selection using a "panning" procedure. OKT8+-T lymphocytes suppressed completely and OKT4+-T lymphocytes enhanced the constitutive release of AIFIA from monocytes. OKT4+ lymphocytes also induced the release of AIFIA from concentrations of 10(3) monocytes/ml which did not release measurable amounts of AIFIA by themselves. The release of AIFIA from monocytes involved HLA-DR+-monocytes and -T lymphocytes. Pulsing monocytes with monoclonal antibodies to framework determinants on HLA-DR molecules, in the absence of complement, did not influence the constitutive release of AIFIA. Pulsing monocytes or T lymphocyte subpopulations with such antibodies, in the absence of complement, blocked the suppressing and inducing activities of the appropriate subpopulations of T lymphocytes. Monoclonal antibodies to common determinants shared by HLA-A, B, and C molecules did not block these cellular interactions. Treating monocytes and T lymphocytes in a complement-dependent cytotoxicity test with dilutions of the anti-HLA-DR antibodies that did not block the cellular interactions removed the populations of monocytes constitutively releasing AIFIA and the T lymphocyte subsets modulating this release. Modulation of the release of AIFIA from monocytes by T lymphocyte subpopulations required the use of autologous cells, cells from HLA-identical siblings, or unrelated donors matched for HLA-DR. Matching for only one HLA haplotype gave partial responses and this was seen in testing cells from related individuals as well as among unrelated test combinations. These cellular interactions were not detected with HLA-DR-incompatible cells differing for two HLA-DR antigens. Admixture of such HLA-DR- incompatible allogeneic cells did not interfere with the regulation of AIFIA release in the autologous cell interactions. Thus, release of AIFIA from monocytes is restricted genetically by HLA-DR at the level of T lymphocyte-monocyte interactions. The genetic determinants on the HLA-class II molecules that induce stimulation in vitro in mixed lymphocyte culture (i.e., HLA-D), however, were not involved in this effort.

Interaction of anthelmintic benzimidazoles and benzimidazole derivatives with bovine brain tubulin.

The binding and inhibitory properties of 11 benzimidazoles for bovine brain tubulin were investigated. The effects of the benzimidazoles on the initial rates of microtubule polymerization were determined by a turbidimetric assay. The median inhibitory concentrations (I50) for nocodazole, oxibendazole, parbendazole, mebendazole and fenbendazole ranged from 1.97 . 10(-6) to 6.32 . 10(-6) M. Benomyl, cambendazole and carbendazim had I50 values from 5.83 . 10(-5) to 9.01 .10(-5) M. Thiabendazole had an I50 value of 5.49 . 10(-4) M. Inhibitor constants (Ki) were determined by the colchicine binding assay. Oxibendazole, fenbendazole, and cambendazole had Ki values of 3.20 . 10(-5), 1.73 . 10(-5) and 1.10 . 10(-4) M, respectively. Oxibendazole and fenbendazole were competitive inhibitors of colchicine. In contrast, cambendazole was a noncompetitive inhibitor of colchicine. The ability of these benzimidazoles to inhibit microtubule polymerization and the mode of action for the anthelmintic benzimidazoles is discussed.

Interaction of anthelmintic benzimidazoles with Ascaris suum embryonic tubulin.

The ability of mebendazole and fenbendazole to bind to tubulin in cytosolic fractions from 8-day Ascaris suum embryos was determined by inhibition studies with [3H]colchicine. Colchicine binding in the presence of 1 . 10(-6) M mebendazole was completely inhibited during a 6 h incubation period at 37 degrees C. Inhibition of colchicine binding to A. suum embryonic tubulin by mebendazole and fenbendazole appeared to be noncompetitive. The inhibition constants of mebendazole and fenbendazole for A. suum embryonic tubulin were 1.9 . 10(-8) M and 6.5 . 10(-8) M, respectively. Mebendazole and fenbendazole appeared to be competitive inhibitors of colchicine binding to bovine brain tubulin. The inhibition constants of mebendazole and fenbendazole for bovine brain tubulin were 7.3 . 10(-6) M and 1.7 . 10(-5) M, respectively. These values are 250-400 times greater than the inhibition constants of fenbendazole and mebendazole for A. suum embryonic tubulin. Differential binding affinities between nematode tubulin and mammalian tubulin for benzimidazoles may explain the selective toxicity. The importance of tubulin as a receptor for anthelmintic benzimidazoles in animal parasitic nematodes is discussed.

Randomized study of recombinant human granulocyte colony-stimulating factor after high-dose chemotherapy and autologous bone marrow transplantation for high-risk lymphoid malignancies.

PURPOSE: The aim of this prospective randomized trial was to examine the efficacy and safety of filgrastim after high-dose chemotherapy and autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS: Patients with poor-risk non-Hodgkin's lymphoma or relapsed Hodgkin's disease were treated in a randomized, open-label trial to study the use of filgrastim as an adjunct to high-dose chemotherapy and ABMT. Of 43 assessable patients, 19 were randomized to receive filgrastim by continuous subcutaneous infusion at a dose of 10 micrograms/kg/d, 10 to filgrastim 20 micrograms/kg/d, and 14 to a parallel control group that received no filgrastim after ABMT. RESULTS: For all filgrastim-treated patients analyzed together, the median time to neutrophil recovery > or = 0.5 x 10(9)/L after the day of ABMT was significantly accelerated to 10 days compared with 18 days in control patients (P = .0001). The median number of platelet transfusions was identical in both groups. Clinical parameters, including the median number of days with fever (1 v 4, P = .0418) and neutropenic fever (5 v 13.5, P = .0001) were significantly shorter in the filgrastim than in the control group. The number of days on intravenous antibiotics and duration of hospitalization were also shorter in the treated groups; however, the differences did not reach statistical significance. For patients treated with the two different dose levels of filgrastim, the neutrophil recovery and clinical results were similar. Filgrastim-associated toxicity appeared to be minimal, with five adverse events considered at least possibly related to filgrastim: two in the higher-dose group and three in the lower-dose group. All of these were rated moderate, except one case of severe bone pain that did not preclude continued filgrastim treatment at a lower dose. Survival and relapse-free survival were similar for control and filgrastim-treated patients. CONCLUSION: Taken together, the results of this first randomized study support the role of filgrastim given as an adjunct to ABMT in accelerating neutrophil recovery, as well as in reducing treatment-related morbidity and overall duration of the treatment procedure.

Randomized, controlled, dose-range study of Ro 25-8315 given before and after a high-dose combination chemotherapy regimen in patients with metastatic or recurrent breast cancer patients.

PURPOSE: To evaluate the safety, pharmacokinetics, and efficacy of three different dose levels of pegylated granulocyte colony-stimulating factor (Ro 25-8315) on progenitor cell mobilization and hematologic recovery in cancer patients. PATIENTS AND METHODS: Breast cancer patients (n = 36) were randomly assigned to receive before (part I) and after (part II) chemotherapy either a single-dose injection of Ro 25-8315 (20 microg/kg, n = 9; 60 microg/kg, n = 9; 100 microg/kg, n = 10) or a standard daily dose of filgrastim (part I, 10 microg/kg/d; part II, 5 microg/kg/d) (control group, n = 8). RESULTS: Overall, Ro 25-8315 was well tolerated. In part I, more progenitor cell mobilization was observed with Ro 25-8315 100 microg/kg. The peak of circulating CD34(+) cells was obtained at day +5 in the four groups, and the absolute neutrophil count (ANC) returned to less than 20 x 10(9)/L by day +15. In part II, high levels of circulating CD34(+) cells (> 20 cells/microL) were obtained in all four groups. The chemotherapy-induced neutropenia (< 1 x 10(9)/L) was similar in the four groups. Ro 25-8315 100 microg/kg was more effective than filgrastim in reducing the number of patients with an ANC less than 0.5 x 10(9)/L on day +12 after chemotherapy. CONCLUSION: A single injection of Ro 25-8315 100 microg/kg might be the optimal dose for steady-state peripheral-blood progenitor cell mobilization. A single injection of 20, 60, or 100 microg/kg could be as efficient as daily administration of filgrastim to correct chemotherapy-induced cytopenia. The optimal dose of Ro 25-8315 should be determined according to the planned chemotherapy regimen.

Effect of interleukin 3 on cytosine arabinoside-mediated cytotoxicity of leukemic myeloblasts.

The concept of biologic modification of proliferation and differentiation of myeloid leukemia cells has attracted much attention over the past years. One promising strategy involves the recruitment of leukemic cells into the cell cycle by hematopoietic growth factors in combination with cycle-specific cytotoxic drugs. Because cytosine arabinoside (Ara-C), which targets only cells in S-phase of the mitotic cell cycle, is included in most chemotherapeutic regimens for the treatment of acute myelogenous leukemia, we explored the hypothesis that the recruitment of quiescent immature leukemic blasts into the cell cycle by the early acting growth factor interleukin 3 (IL-3) can increase the efficacy of Ara-C for kill of leukemic stem cells. We show that IL-3 increases the fraction of blasts in S-phase, as assessed by DNA histogram analysis with propidium iodide staining, leading to an enhancement of kill of clonogenic blast cells when combined with Ara-C. Expression of the protooncogenes c-myc, c-fms, and c-fos, known to be linked to cellular proliferation and differentiation, was also altered by IL-3 in Ara-C-treated cultures, further substantiating the role that IL-3 plays as an enhancer of the cytotoxicity of Ara-C.

Gamma interferon induces colony-forming cells of the human monoblast cell line U937 to respond to inhibition by lactoferrin, transferrin, and acidic isoferritins.

Human gamma interferon (HuIFN gamma) was assessed for its capacities to induce MHC class-II antigens on U937 cells and to induce responsiveness of U937 colony-forming cells (CFC) to the suppressive influences of lactoferrin (LF), transferrin (TF), and acidic isoferritins (AIF). U937 cells grown in suspension culture for many years demonstrated variable percentages of MHC class-II antigen+ cells (6%-42%) as determined by analysis with monoclonal anti-MHC class-II and the FACS IV when checked at different times. The percentage of U937 cells positive for MHC class-II antigens, as well as the density distribution of MHC class-II antigens on these cells, was increased by preincubating the cells for 72 h in the presence of 10(-6) M indomethacin and increasing concentrations of natural HuIFN gamma up to 20-40 U/ml. Colony formation by cells preincubated in control medium plus indomethacin for 72 h was not decreased by treating cells with monoclonal anti-MHC class-II plus complement (C'), high specific activity tritiated thymidine (3HTdr), LF, TF, or AIF. After preincubation of U937 cells with natural HuIFN gamma plus indomethacin in suspension culture for 72 h, colony formation in semisolid medium was reduced 40%-50% by treating the cells with anti-MHC class-II plus C', 3HTdr, LF, TF, or AIF. Colony formation was not reduced further by LF, TF, or AIF, after cells were pretreated with anti-MHC class-II (1:200 dilution) plus C' or 3HTdr. Increasing concentrations of HuIFN gamma up to 20 U/ml increased the percentage of MHC class-II antigen+ U937 CFC as well as the sensitivity of U937 CFC to suppression by LF, TF, and AIF. The inducing activities of natural HuIFN gamma were due to the IFN gamma itself since the inducing activity of natural HuIFN gamma was inactivated by pretreatment with a monoclonal antibody against natural HuIFN gamma. Also the inducing effects were mimicked by recombinant HuIFN gamma. The suppressive effects of LF, TF, and AIF on colony formation were blocked by treating the cells with monoclonal anti-MHC class-II (1:50 dilution, but not 1:200 dilution) in the absence of C'. The suppressive effect of TF only was blocked by pretreating cells with a monoclonal antibody against the TF receptor. U937 cells can be used as a model to study the regulatory mechanisms of action of HuIFN gamma, LF, TF, and AIF.

In vivo 31P NMR spectrum of Hymenolepis diminuta and its change on short-term exposure to mebendazole.

The 31P NMR spectrum of the adult tapeworm, Hymenolepis diminuta, at 37 degrees C during perfusion with physiological saline was composed of 10 peaks. Based on chemical shifts and analysis of worm extracts, the phosphorus components included glucose-6-phosphate, fructose-6-phosphate, phosphorylcholine, glycerophosphoryl choline and -ethanolamine, nucleotide monophosphate-diphosphate and -triphosphate, nicotinamide adenine dinucleotide and uridine diphosphate glucose. The mean level of nucleotide triphosphate was 0.86 nmol (mg fresh weight)-1 and the nucleotide triphosphate/-diphosphate ratio 3.9. Based on the nucleotide triphosphate level, worms were viable for at least 3 h and the intracellular pH was maintained constant at approximately 6.7. Short-term exposure to mebendazole perfused at 11 or 27 microM solubilized in physiological saline containing 0.5% Tween 80 or 0.1% dimethyl sulphoxide had little effect on the nucleotide triphosphate level. Some cytological changes, however, were evident following perfusion of mebendazole. In contrast, exposure to 2,4-dinitrophenol caused a rapid decline in nucleotide triphosphate level. It was concluded that mebendazole does not exert its primary effect on oxidative phosphorylation.

A simple, inexpensive electroelution device for the recovery of nucleic acid fragments from agarose gels.

A simple, inexpensive apparatus for the electroelution of nucleic acids is described. It is constructed from disposable supplies commonly found in molecular biology laboratories.

A simple assay for quantifying the inducible adherence of neutrophils.

By making use of the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) reducing ability of stimulated human neutrophils, we developed an alternative method to quantify the adherence of neutrophils in vitro. After discarding non-adherent neutrophils, the adherent cells were exposed to MTT solution containing 10 ng/ml phorbol 12-myristate 13-acetone (PMA). Subsequently, MTT reduced by this simultaneous stimulation was measured optically and used to calculate percent of adhesion. In these experiments, heat-inactivated autologous serum and tumor necrosis factor alpha (rhTNF-alpha) were observed to promote the adherence to polystyrene surfaces. In contrast, minimal or no effects on neutrophil adherence were achieved in case of treatment with native autologous serum, recombinant human granulocyte colony stimulating factor (rhG-CSF), recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), or recombinant human interferon gamma (rhIFN-gamma).

Human pluripotent hemopoietic colony stimulating factor: activities on human and murine cells.

Human pluripotent colony stimulating factor (Pluripoietin) was shown to act synergistically with human pluripotent alpha-like colony-stimulating activity (Pluripoietin-alpha) supporting the proliferation and differentiation of human CFU-GM progenitor cells in vitro, increasing colony size and numbers. In addition, Pluripoietin enhanced cytotoxic activity of mature human neutrophil granulocytes in an antibody-dependent cellular cytotoxicity assay. Biological activities of Pluripoietin known so far suggest great potentials for clinical use. Preclinical in vivo studies of Pluripoietin in different disease situations may be feasible in mice, because Pluripoietin is active on granulocyte precursors and on a variety of other murine cells.

Rapid miniprep isolation of mitochondrial DNA from metacestodes, and free-living and parasitic nematodes.

A method, based on one to isolate supercoiled plasmid DNA from bacterial cells, has been developed to purify mitochondrial DNA (mtDNA) from cestode and nematode tissue easily and efficiently. Starting with as little as 100 mg of helminth tissue, sufficient mtDNA for electrophoretic analysis was extracted. This DNA was essentially free of nuclear DNA and readily digested by restriction endonucleases. Approximately 20% of the mtDNA in helminth tissue was recovered, which is a significant improvement over previously available techniques.

Phosphoglycerides and derivatives in Taenia crassiceps examined by 31P NMR spectroscopy.

Examination of the larval stage of the tapeworm, Taenia crassiceps, by 31P NMR spectroscopy revealed the presence of a major phosphoglyceride component. However, using saturation transfer, no exchange between glycerophosphorylcholine and phosphoglyceride or any other NMR-detectable phosphorus metabolites was detected.

Arginine kinase and phosphoarginine, a functional phosphagen, in the rhabditoid nematode steinernema carpocapsae.

Moderate activity of arginine kinase was found in Steinernema carpocapsae, an entomopathogenic nematode. In the forward reaction, 4.60 and 3.12 micromol ATP/min/mg protein was produced in infectious third-stage juveniles (J3s) and adult nematodes, respectively. For the reverse reaction, 3.20 and 2.27 micromol phosphoarginine/min/mg protein was produced by J3s and adults, respectively. The K(m)s for phosphoarginine and ADP were 0.73 and 0.42 mM, respectively, in the forward reaction, whereas in the reverse reaction, the K(m)s were 0.37 and 2.35 mM for arginine and ATP, respectively, for the enzyme from J3s. The pH optimum for the forward reaction was 7.2 and 7.3 in J3s and adults, respectively. The pH optimum was elevated for the reverse reaction, 7.8 and 7.9-8.5 in J3s and adults, respectively. In the J3s, the in vitro optima for arginine kinase activity was correlated with the in vivo tissue pH in hypoxic (6.9) and aerobic (7.5) J3s estimated by in vivo flow 31P-NMR.

Characterization of Otostrongylus circumlitus from Pacific harbor and northern elephant seals.

Otostrongylus circumlitus (Railliet, 1899) from Pacific harbor seals (Phoca vitulina richardsi) and northern elephant seals (Mirounga angustirostris) were examined using morphological and molecular methods to determine whether northern elephant seals along the central California coast are infected by the same species of Otostrongylus as are Pacific harbor seals in the same area. Fixed nematodes were examined and measured using light microscopy. The polymerase chain reaction (PCR) was used to amplify and sequence the second internal transcribed spacer (ITS-2) and D3 expansion (26S) regions of ribosomal DNA of O. circumlitus from Pacific harbor and northern elephant seals. The ITS-2 region was also amplified from Parafilaroides sp. from the Pacific harbor seal, northern elephant seal, and California sea lion (Zalophus californianus) and used for restriction fragment length polymorphism (RFLP) analysis. Morphologically, it was not possible to distinguish O. circumlitus from Pacific harbor and northern elephant seals, and over a consensus length of 443 base pairs (bp) for ITS-2 and 321 bp for D3 the sequences of O. circumlitus from both hosts were identical. With the PCR-RFLP assay, it was possible to distinguish O. circumlitus from Parafilaroides sp. The results suggest that O. circumlitus is the same species in Pacific harbor and northern elephant seals, and molecular methods make it possible to distinguish this nematode from related nematodes.

High energy phosphate metabolites observed by NMR in infective larvae of Haemonchus contortus.

Phosphorus resonances consistent with phosphoarginine and ATP were observed in the in vivo 31P-nuclear magnetic resonance (NMR) spectra of infective larvae of Haemonchus contortus. The level of phosphoarginine quickly declined when nematode suspensions were purged with nitrogen and was restored upon return to aerobic conditions. Saturation transfer NMR demonstrated forward and reverse exchange of phosphorus between phosphoarginine and ATP.