Antibabesial effect of the immunomodulator AS101 in mice: role of increased production of nitric oxide.

The immunomodulator AS101 has been shown to induce cell proliferation and to increase the secretion of a variety of cytokines. In the present study we evaluated the effect of AS101 on the pathogenicity of B. rodhaini infected mice. In order to clarify its mechanism of action we studied the ability of AS101 to activate neutrophils and macrophages, both of which inhibit parasite growth. More specifically, we studied the ability of AS101 to induce secretion of nitric oxide (NO). We found that AS101 protects mice from babesiosis in a time and dose dependent manner. At 10 and 20 micrograms/injection, two weeks prior to parasites, AS101 significantly increased the number of neutrophils and more than doubled the survival rate of infected mice. Similarly, at these concentrations when injected one month, or at 20 micrograms, injected 24 h before parasites. AS101 mitigated the course of infection and reduced by half the peak of parasitaemia. At 0.1 microgram/ml AS101 induced the secretion of significantly higher levels of NO in vitro than control. This was abrogated by adding the NO synthase inhibitor. NG-monomethyl-L-arginine. In vivo the antiparasitic protection of AS101 was abrogated by another NO synthase inhibitor, aminoguanidine. We found that AS101, partly by elevating levels of NO, can significantly mitigate the course of infection and thus increase survival, and may therefore be proven as an effective antiparasitic drug.

Photofrin II induces cytokine secretion by mouse spleen cells and human peripheral mononuclear cells.

The aim of our study was to find out if Photofrin II, a cytotoxic drug used routinely in photodynamic therapy (PDT), can induce immune responses in vitro, and to compare its effects with those of the protoporphyrin 9, hemin, which also has antitumor properties. We tested the effect of these porphyrins on lymphocyte proliferation and secretion of interleukin-2, interleukin-3, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma), by human or murine mononuclear cells (MNC) without an activating light. Both the Photofrin II- and hemin-treated cells showed a significant increase in cytokine secretion in the presence of suboptimal concentrations of mitogen. Moreover, Photofrin II and hemin significantly increased production of TNFalpha and IFNgamma even in the absence of mitogen. The cellular binding sites of Photofrin II and hemin to MNC were localized by electromicroscopy or fluorescence. Combined stimulation of cells by mitogens and porphyrins maintained optimal vital ionic balance of potassium, sodium and chlorine in the lymphocytes. In the cells thus treated there was a significant increase in intracellular calcium, a vital second messenger for lymphokine secretion. We demonstrate that the effect of Photofrin II on the immune system involves enhanced cytokine secretion which may account for the subsequent tumor eradication by PDT.

Effect of the immunomodulator AS101 on chemotherapy-induced multilineage myelosuppression, thrombocytopenia, and anemia in mice.

The immunomodulator AS101 has previously been found to induce mouse and human hematopoietic cells to secrete cytokines such as interleukin-1 alpha (IL-1 alpha), IL-2, tumor necrosis factor-alpha (TNF-alpha), and gamma interferon (IFN-gamma). The compound was shown to protect mice from lethal and sublethal effects of chemotherapy and irradiation. AS101 prevented the decrease in the number of bone marrow (BM) and spleen myeloid progenitor cells, and increased the survival of lethally treated mice. In this study, we show a dose-dependent response of AS101 in the induction of high secretion levels of IL-6, IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF). Since these growth factors are known to induce the proliferation and differentiation of multilineage progenitors, including megakaryocytic and erythroid progenitors, we designed this study to evaluate the role of AS101 in attenuating thrombocytopenia, anemia, and multilineage myelosuppression associated with chemotherapy. We demonstrate that pretreatment of mice with AS101 24 hours before intraperitoneal injection of 250 mg/kg cyclophosphamide (CYP) or intravenous injection of 150 mg/kg 5-fluorouracil (5-FU) significantly increased the number of circulating white blood cells (WBC) and platelets. The numbers of both neutrophils and lymphocytes were significantly increased in AS101-treated mice subjected to chemotherapy. In addition, AS101 attenuated erythropenia caused by 5-FU. It could also increase megakaryocyte and erythroid progenitor cells (CFU-MK and CFU-E) in the BM of treated mice severely affected by chemotherapy. We demonstrate that the protective effect of AS101 could be abrogated by treatment with anti-IL-1R or anti-SCF antibodies. We suggest that the endogenous production of cytokines such as IL-1, IL-6, IL-3, SCF, and GM-CSF in mice treated with AS101 offers protection to circulating blood elements and ameliorates the reconstitution of megakaryocytic and erythroid progenitors. The simultaneous protection by AS101 of multilineage cell compartments is probably due to stimulation by AS101 of a selective subpopulation of primitive stem cells resistant to chemotherapy. On the basis of these studies, phase II clinical trials with patients treated with chemotherapy in combination with AS101 have been initiated.

In vivo synergistic effect of the immunomodulator AS101 and the PKC inducer bryostatin.

The immunomodulator AS101 has recently been found to have radioprotective properties when injected prior to sublethal and lethal doses of irradiation. In addition, this compound was found to protect mice from hemopoietic damage caused by sublethal doses of cyclophosphamide (CYP) and to increase the rate of survival of mice treated with lethal doses of CYP. AS101 was previously shown to exert a synergistic effect with the PKC-inducer bryostatin in cytokine secretion in vitro. The present studies were designed to evaluate the effects of in vivo combined treatment with AS101 and bryostatin on bone marrow and spleen cellularity and on the number of committed progenitors in the bone marrow at various points of time after their treatment with a sublethal dose of CYP or irradiation. In addition, the combined effect was tested on the survival of mice irradiated with a lethal dose of irradiation. Our data show the presence of synergism which greatly enhances the number of bone marrow and spleen cells 48 hr and 9 days after CYP treatment or irradiation. The combined effect was also demonstrated when bone marrow colony-forming units granulocyte-macrophage (CFU-GM) progenitor cells were evaluated. Moreover, AS101 and bryostatin synergized in their protective effects against lethal damages of irradiation. These results strongly suggest that bryostatin, which lacks tumor-promoting activity, is a particularly good candidate in combination with AS101 for treatment in vivo in counteracting chemotherapy- or radiation-induced hematopoietic suppression or in generally improving the restoration of immune response under conditions involving immune or hemopoietic damage.

Toxicity study in rats of a tellurium based immunomodulating drug, AS-101: a potential drug for AIDS and cancer patients.

Male and female Sprague Dawley rats were injected intraperitoneally for 4 weeks with ammonium trichloro (dioxyethylene-0-0'-) tellurate, an immunomodulating drug at doses ranging from 3 to 24 mg/kg/week. Routine laboratory examinations included body weight, food consumption, clinical chemistry and hematological examinations. At termination of the experiment, all rats were sacrificed and subjected to a detailed necropsy. Few mortalities were recorded during the course of the study. Clinical signs included hind limb paresis and paraphimosis. A garlic odor pervaded the room. Body weight and food consumption were adversely affected in a dose-related manner. Effects were elicited on the hematological system; changes being noted in the platelet and leukocyte counts as well. Clinical chemistry evaluation revealed signs of hepatoxicity, especially in the female treated groups. The level of beta-globulin was increased. At necropsy organs were found to have a grayish-blue discoloration. Tellurium related histopathological changes were observed in the eyes, liver, thymus, bone marrow, heart and kidneys. An attempt has been made to compare the toxicity of this drug with other tellurium-containing compounds. A good correlation was found. Novel effects of the drug were retinopathy and replacement of bone marrow by bony or fibrous tissue. The possibility that some of the effects may have been elicited due to selenium-vitamin E deficiency has been considered.

A new immunomodulating compound (AS-101) with potential therapeutic application.

There has been interest in the potential of synthetic compounds to modify immune responses by imitation of cytokine action. Direct administration of interleukin 2 (IL-2) in conjunction with adoptive transfer of lymphokine activated killer cells has been used in the treatment of cancer, but there are toxic effects resulting from the high doses of IL-2 required. We have developed a new synthetic compound, ammonium tri-chloro(dioxoethylene-O,O'-)tellurate (AS-101), which has immunomodulating properties and minimal toxicity. The effects of AS-101 on the activation and function of immunocompetent cells have been assessed. We have found that AS-101 induces proliferation and IL-2 production by human lymphocytes in vitro, and enhances the production of IL-2 and colony-stimulating factor by mouse spleen cells. Splenocytes of BALB/c mice injected with AS-101 increased production of IL-2 and CSF in vitro in the presence of mitogen. Mononuclear cells of normal donors acquired responsiveness to recombinant IL-2 and bound monoclonal antibody to IL-2 receptor after incubation with AS-101. Splenocytes of mice treated in vivo with AS-101 expressed high levels of IL-2 receptor. The stimulation of lymphocytes by AS-101 apparently involves an increase in intracellular free calcium. AS-101 administered systemically to mice mediated antitumour effects which could be attributable to its immunomodulatory properties. In addition, AS-101 could directly enhance the ratio of OKT4 to OKT8-positive cells in cultured mononuclear cells from AIDS (acquired immune deficiency syndrome) patients. These results indicate that AS-101 is potentially useful in the treatment of clinical conditions involving immunosuppression.

Growth of human basophil lines derived from chronic myelocytic leukaemia cells in vitro: ultrastructure and X-ray microanalysis studies.

Mononuclear cells from the peripheral blood of patients with chronic myelocytic leukaemia were grown and expanded in liquid culture in the presence of Con A-conditioned medium. An accelerated development of cells of the basophilic lineage was observed and resulted in the appearance of 85% mature basophils after 14 days of incubation. Transmission electron microscopy of developing basophils showed changes in the nucleus and active granule formation in the cytoplasm. By scanning electron microscopy, the immature cells were relatively smooth in comparison with the mature basophils which showed membranous microvilli. The chemical content of the cells at different days of culture was detected by X-ray microanalysis. Immature cells were characterized by a high level of phosphorus with a low level of sulphur. As maturation progressed, the amount of phosphorus decreased, while the level of the sulphur increased, reaching its highest peak in the mature basophils. The different amount of sulphur found in the cells during the maturational process most probably represents the amount of heparin located in the cell granules. This finding may be useful for studying the influence of growth factors on the development and differentiation of human basophils.

Elemental X-ray microanalysis of mouse peritoneal and bone marrow derived cultured mast cells.

Elemental X-ray microanalysis of intact mouse peritoneal cells revealed significantly high K alpha sulfur and chloride peaks, while phosphorus, sodium and calcium displayed relatively low K alpha peaks. Bone marrow derived cultured mast cells exhibited a low K alpha peak for sulfur and a high K alpha peak for chloride. Peritoneal macrophages which served as controls differed from peritoneal mast cells by their high phosphorus and chloride content and a relatively low sulfur peak. The finding of two different spectra for the mast cells may provide additional parameters for discrimination of mast cell subsets.

In vitro growth of colonies of mitogen-stimulated mouse T lymphocytes: I. Conditions affecting colony formation; II. Structure of colonies and component cells.

Mouse lymph node cells sensitized with PHA or Con A in liquid phase grew into T-cell colonies when seeded in a two-layer soft agar culture system containing the mitogen. The colony cells were of T-cell lineage. This was deduced from their morphology, ultrastructure, positive strain for theta-isoantigen and the fact that no colonies were formed by lymphoid cells from congenitally athymic nude mice. The architecture of the colonies and their component cells was studied by scanning electron microscopy. Clonogenic assay indicated that macrophages are active modulators of T cell proliferation. Colony formation was markedly enhanced by hemolysate and/or amino acid, L-glutamine or L-cystine, added to the culture medium. The largest number of colonies grew when both the liquid and soft agar media were supplemented with hemolysate and one of the amino acids. Under these conditions the minimal seeding level for colony formation could be reduced from 2.0 X 10(5) to 1.6 X 10(4) cells/culture.