Biotechnology therapeutics最新文献

The immunomodulating properties of arglabin, a sesquiterpene lactone isolated from Artemisia myriantha Wall. (Asteraceae) were investigated using the murine macrophage tumor line J774.1. Arglabin-stimulated macrophages displayed a strong cytotoxic activity and the lowest doses (1.25 micrograms/mL and 0.125 micrograms/mL) induced a significant stimulation of cell mitochondrial metabolism, which correlated with [3H]TdR uptake by J774.1 cells under the same experimental conditions. In addition, the secretion of cytokines involved in host defence mechanisms--IL-1, TNF-alpha, and IL-2--was investigated upon incubation of J774-1 cells with arglabin. Arglabin triggered the production of the three cytokines from J774-1 cells. However, the pattern of cytokine secretion differed to some extent, according to the methodology used for cytokine measurement: either traditional bioassay or specific immunoassay (ELISA). Our data emphasize a possible proliferative effect of arglabin in the traditional bioassays, at least for the highest concentrations used. The results were verified with specific ELISA immunoassays. Using either method, lower concentrations of arglabin (ranging from 12.5 micrograms/mL to 0.125 micrograms/mL) were the most effective in inducing IL-1, TNF-alpha, or IL-2 secretion. In addition, preliminary data on phagocytosis showed that arglabin enhanced the uptake of fluorescent latex beads by J774.1 cells.

In previous experiments gp160 incorporated into iscom was shown to induce neutralizing antibodies to the homologous as well as the heterologous isolates of HIV-1 (Akerblom et al., AIDS Res., 1991). In the present work we have incorporated into iscoms three defined recombinant DNA products of HIV-1. The carboxy-terminal part of gp120 expressed in E. Coli-PB-1; a chimera containing parts of both p24 and p15 expressed in E. coli-GAG; and baculovirus gp160 cloned in baculovirus and produced in insect cells. Immune responses were induced by the iscom preparations to the homologous antigen as well as to defined recombinant products and to the synthetic peptide RP135 (aa 304-328) harboring a neutralizing epitope. Sera from mice immunized with PB1-iscoms and gp160 (baculo) iscoms were tested in a syncytie inhibition assay. The serum from a mouse immunized with PB1 iscoms reacted strongly with the synthetic peptide RP135 and also neutralized the homologous isolate HIV-1/IIIB with a neutralization titer of 1/64. Three gp160 (baculo) iscom antisera were tested, of which two reacted strongly with the synthetic peptide RP135 but did not neutralize the homologous isolate HIV-1/IIIB. High serum titers were induced in mice by the gp160 iscoms (2 micrograms) to homologous antigen and the recombinant DNA E. coli construct p121 covering part of gp41. The ceilings of the antibody responses were reached after two immunizations. The PB1- and GAG-iscoms required three immunizations to reach the ceiling of the antibody response.

In this paper we show the presence of 2-[125I]melatonin binding sites in human neutrophils (hN). The specific binding of melatonin to hN cells and hN membranes was dependent on time and temperature, stable, saturable, and reversible. In competition studies, the specific binding of radioactive melatonin to hN cells or hN membranes was inhibited by increasing concentrations of native melatonin. Scatchard analysis showed the existence of a single class of binding sites with a Kd of 2.1 and 7.1 microM for hN cells and hN membranes, respectively. The binding capacity was of 84 and 132 pM for hN cells and hN membranes, respectively. The affinity of the binding sites for melatonin suggests that they may be relevant in studies on the pharmacological properties of melatonin in regulating human neutrophil activity.

Aggregation studies have become a useful criterion for analyzing leukocyte motility and activation in vitro. The T-cell-derived lymphokine human leukocyte inhibitory factor (LIF) is a modulator of many important polymorphonuclear (PMN) functions in addition to aggregation such as chemotaxis, lysosomal degranulation, phagocytosis, bactericidal killing, augmented antibody-dependent cellular cytotoxicity (ADCC), induction of neutrophil Fc-gamma, complement type-1 and FMLP receptors, and production of superoxide and H2O2. Our investigations focused on the ability of LIF to modulate the aggregation of macrophages (MO) induced by calcium ionophore A23187. The ionophore A23187 directly induced potent aggregation of macrophages, which was markedly enhanced when the cells were pretreated with LIF. However, the addition of LIF in the absence of other costimuli did not directly induce MO aggregation. LIF was shown to enhance PMN aggregation induced by N-formyl-Met-Leu-Phe (FMLP), but did not augment the aggregation of FMLP-stimulated macrophages, indicating a cellular specificity of aggregation-inducing costimuli following LIF priming. Additional cytokines examined for possibly inducing MO aggregation were interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF), and interleukin-6 (IL-6); all proved to be incapable of inducing aggregation directly, nor did they enhance the effect of A23187 ionophore on macrophage aggregation. Additionally, we found that LIF can directly stimulate MO to activate specific pathways of the arachidonic acid cascade, inducing the synthesis and release of thromboxanes and leukotriene B4. LIF did not augment the potent ability of A23187 to induce increased production of LTB4 or TxA2 by human MO. These new results coupled with our previously published data indicate that LIF can enhance the activation of both MO and PMN leukocytes when exposed to either A23187 or FMLP, respectively. Moreover, these data suggest that LIF can contribute directly to monocyte-macrophage leukocyte activation, in addition to PMN activation, during inflammatory responses, resulting in greater cell aggregation, activation, and specific proinflammatory arachidonic acid product release.

The effects of a human GM-CSF/IL-3 fusion protein (PIXY321) were examined in a primate model of rebound hematopoiesis following sublethal irradiation. The results demonstrated an enhanced rate of both neutrophil and platelet regeneration, as well as functional activation of circulating neutrophils.

Biodistribution of the photosensitizer benzoporphyrin derivative monoacid ring A (BPD) was compared in DBA/2 mice bearing the syngeneic M-1 tumor and nude mice bearing the A549 human squamous cell carcinoma. These studies, using internally labeled 14C-BPD showed that in general, biodistribution between the two strains was equivalent with the exception of two tissues; lymph nodes (BPD levels were higher in nude mice) and tumor (BPD levels were lower in nude mice). Further studies were carried out in A549-tumor-bearing nude mice in which the biodistribution of BPD conjugated to a monoclonal antibody (5E8) with specificity for an antigen on A549 cells was compared to a conjugate prepared with an irrelevant monoclonal antibody (T48). These studies showed that both conjugates had biodistribution characteristics which distinguished them from free BPD in that they remained in the circulation and most tissues for significantly longer times than did free BPD. Also, with the exception of the 5E8-BPD conjugate and A549 tumor tissue, levels in all tissues were highest at the 3-h time point following injection of conjugates. In the case of A549 tumor and the 5E8-BPD conjugate the highest concentration of 14C-labeled material was observed at the 14-h time point following injection. The results reported herein show that the conjugates tested behaved differently from free BPD, indicating that the materials did not become dissociated in vivo and that the specific conjugate (5E8-BPD) demonstrated specificity for the A549 tumor in terms of the kinetics of its accumulation in tumor tissue.

Immunomodulatory action of nuclear proteins derived from spleen and brain of immunized rats was found in ConA-stimulated mouse spleen lymphocyte cultures. In vitro experiments on 32P- or Dig-labeled promoter-enhancer region (-548 bp) of the human interleukin-2 (IL-2) gene showed specific DNA-nuclear protein complex formation for spleen (6-14 kD) and brain (21 kD) proteins. The functional influence of these trans-factors on the IL-2 promoter region was studied in the model system of Jurkat cells with the firefly luciferase (Luc) reporter gene. The spleen and brain proteins were shown to influence the membrane surface properties of thymic and spleen cells as revealed in a two-phase aqueous system. We conclude that both spleen and brain from immunized rats contain certain protein trans-factors which can be active in T cells and display a common mode of regulatory action the level of IL-2 gene.

The in vitro effects of the bioflavonoid antioxidant silymarin on the expression and activity of superoxide dismutase (SOD) enzyme was studied in erythrocytes and lymphocytes from patients with chronic alcoholic liver disease. In vitro incubation with the agent in a concentration corresponding to the usual therapeutic dosage markedly increased the SOD expression of lymphocytes as measured by flow-cytofluorimetry following staining with monoclonal anti-Cu/Zn-SOD antibody and FITC-conjugated anti-mouse Ig, as well as erythrocyte and lymphocyte SOD activities. The data indirectly suggest that antioxidant activity might be one of the important factors in the hepatoprotective action of this bioflavonoid.

Substantial increases in dose-intensity of chemotherapy yield a severalfold increase in complete remission rates and durable responses in several types of malignant disease. Hematopoietic colony-stimulating factors decrease the duration of the resultant severe neutropenia but optimal dosing regimens of these cytokines have not yet been determined. This study was designed to explore the use of both yeast-derived and E. coli-derived GM-CSF given pre- and postchemotherapy with an intensive combination chemotherapy regimen. The chemotherapeutic regimen consisted of cyclophosphamide 5000 mg/m2, etoposide 1500 mg/m2, and cisplatin 150 mg/m2 (DICEP). Patients receiving either yeast-derived GM-CSF (6.5 days) or E. coli-derived GM-CSF (6.0 days) had a shorter duration of severe granulocytopenia with an absolute granulocyte count below 300/microL than patients receiving no GM-CSF (11.0 days, p = 0.0001). Administration of GM-CSF for 6 days immediately preceding DICEP did not further shorten the duration of cytopenia. E. coli-derived GM-CSF given at doses above 5 micrograms/kg was poorly tolerated and offered no hematologic advantage. Lower doses (3 micrograms/kg) of the E. coli product were better tolerated but still produced more toxicities than yeast-derived GM-CSF. The yeast-derived product produced no local skin reactions and decreased the incidence of nonhematologic and all grade 3 or 4 toxicities compared to the control group.