Protective effect of the immunomodulator AS101 against cyclophosphamide-induced testicular damage in mice.

BACKGROUND: Cyclophosphamide (Cy), a widely used anticancer drug, is associated with significant testicular damage and sterility. Co-administration of the immunomodulating compound AS101 during chemotherapy treatments was previously shown to protect organs against cytotoxic damage, without attenuating the drug's anticancer effect. In this animal study, we investigated the effect of AS101 on testicular damage, sperm DNA damage and infertility induced by Cy. Akt and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation were investigated as a possible chemoprotective mechanism. METHODS: Mature male mice, 10 in each group, were injected intraperitoneally with 200 mg/kg Cy once a week for 5 weeks, with or without concurrent treatment with 10 microg per mouse AS101 three times per week. Damage to testicular tubules and sperm production was determined, sperm chromatin damage was analyzed and fertility was gauged. Akt and GSK-3beta phosphorylation were evaluated. RESULTS: Co-treatment with AS101 during the course of Cy administration significantly reduced the percentage of damaged seminiferous tubules (76.0 +/- 10.8% versus 40.3 +/- 2.6%), and reduced sperm DNA fragmentation (%DFI) from 44.7 +/- 1.0% to 25 +/- 6.5%. Co-treatment with AS101 also partially protected against the decrease in numbers of impregnated females and litter size. AS101 increased Akt and GSK-3beta phosphorylation. CONCLUSIONS: Our results indicate that AS101 can significantly protect against Cy-induced testicular damage and sperm DNA damage, probably by acting through Akt/GSK-3beta phosphorylation.

Topical treatment for human papillomavirus-associated genital warts in humans with the novel tellurium immunomodulator AS101: assessment of its safety and efficacy.

BACKGROUND: Various methods are currently used for the treatment of anogenital warts. However, a complete cure is unlikely, and the rate of recurrence is high. OBJECTIVES: The purpose of this open-label, multicentre trial was to evaluate the safety and clinical efficacy of a new treatment using the immunomodulator ammonium trichloro (dioxoethylene-O,O') tellurate (AS101; Biomas Ltd, Kefar Saba, Israel) 15% w/w cream to clear vulval/perianal condylomata acuminata. METHODS: Study participants comprised 48 women and 26 men, age range 18-62 years. Of the 48 woman, 44 were diagnosed with vulval condylomata and four with perianal condylomata. All 26 men were diagnosed with perianal condylomata. All the patients in the study received AS101 15% w/w cream twice a day. Maximal treatment duration was 16 weeks. To evaluate the safety and clinical efficacy, patients were examined and lesional areas photographed on a biweekly basis. RESULTS: By the end of the treatment, 56 of 74 (76%) patients were considered completely cleared. Complete cure was achieved in 35 of 44 (80%) patients with vulval condylomata and in 21 of 30 (70%) patients with perianal condylomata. No scarring of treated areas was observed. Complete cure was achieved within a time range of 10-109 days. The most frequent side-effects observed were mild-to-moderate itching, soreness, burning and erythema. In post-treatment follow up of up to 6 months, disease recurrence was observed in two patients (4%), at 105 and 144 days following completion of treatment. CONCLUSIONS: AS101 15% w/w cream is an effective and safe, self-administered therapy used for the treatment of external vulval and perianal warts. The cream is applied topically twice daily for up to 16 weeks. A very low recurrence rate was reported.

Synergistic effect of AS101 and Bryostatin-1 on myeloid leukemia cell differentiation in vitro and in an animal model.

We evaluated the synergistic activity of AS101 (ammonium trichloro-(dioxoethylene-0-0')-tellurate) with the protein kinase C (PKC) activators, Bryostatin-1 and phorbol-12-myristate-13-acetate (PMA), on human myelocytic leukemia cell differentiation in vitro, and in a mouse model. Use of AS101 with Bryostatin-1 or with a low concentration of PMA resulted in the differentiation of HL-60 cell line to cells with characteristics of macrophages. A similar synergistic effect was found in vivo. Compared with mice treated with AS101 alone or with Bryostatin-1 alone, the infiltration of leukemic cells into the spleen and the peritoneum of mice treated with both compounds, as well as the number of the HL-60 colonies extracted from those organs, were markedly reduced. The antitumor effects were associated with significantly prolonged survival (100% for 125 days) of the treated mice. Finally, the mechanism of action of this antitumor effect was explored, and was found to involve the Ras/extracellular signal-regulated kinase signaling pathway. Combined treatment with AS101 and Bryostatin-1 synergistically increased p21(waf1) expression levels independently of p53. Upregulation of p21(waf1) was necessary for HL-60 cell differentiation, which was found to be both c-raf-1 and mitogen-activated protein kinase dependent. This study may have implications for the development of strategies to induce differentiation in myeloid leukemias, myelodysplasias and possibly in other malignancies.

Predominance of TH1 response in tumor-bearing mice and cancer patients treated with AS101.

BACKGROUND: Several studies have recently suggested that the immune response to malignant growths is regulated by distinct patterns of type 2 cytokine production. These cytokines, regulating the cytotoxic T-lymphocyte response in patients with advanced cancers, may be associated with disease progression. Evidence suggests that the T Helper 1 (TH1) and T Helper 2 (TH2) types of reaction are reciprocally regulated in vivo. The immunomodulator AS101 (ammonium trichloro[dioxoethylene-O,O']tellurate) was found to stimulate mouse and human cells to proliferate and secrete a variety of cytokines. Clinical trials using AS101 on cancer patients are now in progress. PURPOSE: The aim of this study was to evaluate the ability of AS101 to modulate TH1 and TH2 responses in tumor-bearing mice and in patients with advanced cancer. In addition, we investigated the association between the predominance of each type of response with the antitumoral effects of AS101. METHODS: Mice into which Lewis lung carcinoma (3LL) had been transplanted (n = 221) and cancer patients (n = 13) were treated with AS101 on alternate days, at 10 micrograms/mouse intraperitoneally, or for the patients, at 3 mg/m2 intravenously. The types were sarcoma, melanoma, and colon, lung, ovarian, and renal cancers. Cytokine levels were determined by immunoassay kits and compared with the paired Student's t test: in mice, they were tested in spleen cell supernatants; in humans, in sera and mononuclear cell supernatants. The chi-squared test was used to compare tumor volumes. All P values represent two-sided tests of statistical significance. RESULTS: Our results show that treatment of mice and patients with AS101 results in a clear predominance in TH1 responses, with a concomitant decrease in the TH2-type response. This was reflected by a significant enhancement in interleukin 2 (IL-2) and interferon gamma (IFN gamma) levels (P < .01) paralleled by a substantial decrease in IL-4 and IL-10 (P < .01). Moreover, the concentration of IL-12 was significantly increased (P < .01) in AS101-treated patients who also showed enhanced levels of natural and lymphokine-activated killer cell-mediated cytotoxicity. The statistically significant increases in IL-2 and IFN gamma levels, paralleled by the pronounced decrease in IL-4 and IL-10 in the AS101-treated mice, were associated with its antitumoral effects. In addition, systemic cotreatment of 3LL-transplanted mice with AS101 and anti-IL-12 antibodies partly abrogated the antitumoral effect of AS101. CONCLUSIONS: Immunotherapy with AS101 enhances TH1 function while interfering with the TH2 response. This TH1 trend may be related to the antitumor effects of AS101. IMPLICATIONS: Isolation and characterization of a distinct cytokine pattern in patients with advanced cancer treated with AS101 may contribute to the development of intervention strategies using this compound.

Protection of bone marrow stromal cells from the toxic effects of cyclophosphamide in vivo and of ASTA-Z 7557 and etoposide in vitro by ammonium trichloro(dioxyethylene-O-O')tellurate (AS101).

The immunomodulator AS101 has previously been shown to protect mice from lethal and sublethal doses of cyclophosphamide (CYP). AS101 was also shown to protect BM granulocyte-macrophage colony-forming cells from the toxic effects of ASTA-Z 7557. In the present study we examined the ability of AS101 to protect functional properties of BM stromal cells from the toxic effects of CYP in vivo or ASTA-Z in vitro. The functional properties of stromal cells from CYP-injected mice were tested with respect to stromal cell number and viability as reflected by the number of colony-forming unit fibroblasts, the ability of established stromal layers to secrete colony-stimulating factor and interleukin 6, as well as the capacity to support hemopoietic cells. All of these parameters were tested from day 1 to day 7 after CYP treatment. We demonstrate that all stromal functions are severely damaged following CYP treatment. Pretreatment of mice with 10 micrograms AS101 24 h before injection of 250 mg/kg CYP resulted in a significant amelioration of stromal cell functions as early as 24 h following CYP treatment. In addition we show that prior incubation of BM cells with AS101 protects the development of stromal colony-forming unit fibroblasts from the toxic effects of ASTA-Z, a potent derivative of CYP, and etoposide, a derivative of podophyllotoxin. These results strongly suggest the usefulness of AS101 in counteracting chemotherapy-induced BM microenvironmental suppression and the important role of the compound as an adjunct treatment of cancer when used in combination with CYP. The data also suggest the effectiveness of AS101 in purging bone marrow when used concomitantly with ASTA-Z or etoposide.

The protective role of ammonium trichloro(dioxoethylene-O,O')tellurate in combination with several cytotoxic drugs acting by different mechanisms of action.

We have demonstrated previously that the immunomodulator AS101 can protect mice from acute lethal toxicity mediated by high doses of radiation or chemotherapy. The compound was shown to rescue mice from toxic effects of cyclophosphamide or 5-fluorouracil. The results presented herein demonstrate that pretreatment of mice with AS101 protects them from lethal effects of several chemotherapeutic drugs acting by distinct mechanisms. At sublethal doses, AS101 could prevent hemopoietic damage caused by the drugs. A significantly higher proportion of colony forming cells granulocyte-macrophage as well as a higher level of colony stimulating factor secretion by bone marrow (BM) cells was observed in mice pretreated with AS101 before injection of doxorubicin or cyclohexylchloroethylnitrosourea. Moreover, a significantly higher rate of survival was observed in mice injected with AS101 before treatment with lethal doses of these drugs. AS101 could also rescue BM stromal cells from damages caused by doxorubicin. We show that injection of mice with AS101 or pretreatment of BM cells with AS101 protects BM-colony forming cells granulocyte-macrophage from toxic effects of etoposide. We suggest that the protective effects of AS101 against damages caused by a variety of cytotoxic drugs may be attributed to the ability of the compound to expand the colony forming unit spleen subpopulation of early progenitors, those cells that are resistant to several DNA damaging agents and are the precursor cells essential for reconstitution of the hemopoietic system. It seems that AS101, by minimizing adverse cytotoxicity resulting from a variety of drugs, is a promising candidate for chemoimmunotherapy with cancer patients.

Use and mechanism of action of AS101 in protecting bone marrow colony forming units-granulocyte-macrophage following purging with ASTA-Z 7557.

Ammonium trichloro(dioxoethylene-O,O')tellurate (AS101) has been shown previously to provide radioprotective effects when given to mice 24 h prior to irradiation and to protect mice from lethal and sublethal doses of cyclophosphamide (CTX). In this study we examined the ability of AS101 to protect mice bone marrow colony forming units-granulocyte-macrophage treated in vitro with various doses of ASTA-Z 7557, a potent derivative of cyclophosphamide. We demonstrate that prior incubation with AS101 protects colony forming units-granulocyte-macrophage from toxic effects of ASTA-Z. This protection can also be conferred by injection of mice with AS101 prior to incubation of their bone marrow in vitro with ASTA-Z. Prior incubation with AS101 was shown not to protect K562 leukemic cells or HL-60 cells from the toxic effects of ASTA-Z. We show that AS101 protection from the toxic effects of ASTA-Z in vitro and CTX in vivo can be partially ascribed to increased aldehyde dehydrogenase (ALDH) activity induced by AS101. This was shown directly by measuring cellular ALDH activity and indirectly by measuring the toxicity of ASTA-Z and CTX in the presence of cyanamide, an inhibitor of ALDH. AS101 is also demonstrated in this study to protect spleen cells from the toxic effects of 5-fluorouracil, probably through a different mechanism. These properties of AS101 make it a useful candidate for increasing the qualitative potential of bone marrow used for autologous transplantation after purging with ASTA-Z. In addition, the results suggest an increase in ALDH activity by AS101 as one of the mechanisms of protection from the toxic effects of ASTA-Z and CTX. However, the chemoprotectiveness of AS101 was found not to be restricted to cyclophosphamide, since as shown in this study, AS101 helped by other mechanisms to reconstitute the number of spleen cells after 5-fluorouracil treatment.

Protective and restorative role of AS101 in combination with chemotherapy.

The immunomodulator AS101 has been found previously by us to stimulate the secretion of high levels of interleukin 1 and colony stimulating factor (CSF) in vitro, as well as the production of CSF in vivo in mice models. These cytokines are known to induce proliferation and differentiation of hematopoietic progenitor cells from the spleen and bone marrow (BM) and to protect mice from DNA-damaging agents. The present studies were designed to evaluate the effects of prolonged treatment with AS101 on myelopoiesis, BM cellularity, and CSF secretion in mice treated with a sublethal dose of cyclophosphamide (CYP) and on the survival of mice undergoing treatment with lethal doses of this compound. In this model, the hematopoietic progenitors were suppressed during the overbound phase of myelopoiesis resulting from the cytotoxic effects of CYP. This allowed the detection of a significant proliferative effect of AS101 in vivo on BM colony-forming units granulocyte-macrophage progenitor cells, BM cellularity, and the secretion of CSF. Moreover, AS101 protected these animals from the lethal effects of high doses of CYP. These protective effects were demonstrable only when AS101 was administered to mice prior to CYP treatment. The only exception was CSF secretion by spleen cells that had been reconstituted when AS101 was administered both prior to and following CYP treatment. AS101 was found to have a synergistic effect with CYP in the treatment of tumor-bearing mice, suggesting that the combination of these two modalities provides a more effective treatment of their tumors. These results strongly suggest an immunoregulatory role for AS101 in counteracting the chemotherapy-induced hematopoietic suppression as well as usefulness as adjunct treatment of cancer when used in combination with CYP.

Bone marrow-sparing and prevention of alopecia by AS101 in non-small-cell lung cancer patients treated with carboplatin and etoposide.

PURPOSE: The aim of this study was to evaluate the ability of the immunomodulator AS101 to prevent chemotherapy-induced neutropenia and thrombocytopenia and thus allow patients to receive full-dose antineoplastic agents according to protocol design. We also aimed to determine the production level of various hematopoietic growth factors in treated patients. PATIENTS AND METHODS: This study of 44 unresectable or metastatic non-small-cell lung cancer (NSCLC) patients was an open-label prospective randomized study of standard chemotherapy alone versus chemotherapy plus AS101. Each patient received carboplatin (300 mg/m2 intravenously [IV] on day 1 of a 28-day cycle, and etoposide (VP-16) (200 mg/m2 orally) on days 3, 5, and 7 of each cycle. AS101 was administered at 3 mg/m2 three times per week starting 2 weeks before chemotherapy. RESULTS: AS101, which manifested no major toxicity, significantly reduced neutropenia and thrombocytopenia and thus allowed all treated patients to receive full-dose antineoplastic agents, in contrast to only 28.5% of the control group. Continuous treatment with AS101 significantly reduced the number of days per patient of thrombocytopenia and neutropenia and did not provide protection to tumor cells as reflected by the higher overall response rate compared with the chemotherapy-alone arm. Interestingly, AS101 treatment also significantly prevented chemotherapy-induced alopecia. These effects correlate with the ability of AS101-treated patients to increase significantly the production of colony-stimulating factors (CSFs) interleukin-1 alpha (IL-1 alpha) and IL-6. CONCLUSION: AS101 has significant bone marrow (BM)-sparing effects and prevents hair loss in chemotherapy-treated patients, with minimal overall toxicity. These effects are probably due to increased production of IL-1 alpha, IL-6, and granulocyte-macrophage (GM)-CSF.

The effect of AS101 on the reconstitution of T-cell reactivity following irradiation or cyclophosphamide treatment.

AS101 (ammonium trichloro[dioxyethylene-O-O']tellurate) is a new synthetic compound previously described by us as having immunomodulating properties and minimal toxicity. Phase II clinical trials are currently in progress with AS101 on cancer patients. AS101 has been recently found to have both radioprotective and chemoprotective effects on hemopoiesis of irradiated mice or mice treated with cyclophosphamide (CYP). In this study the effect of AS101 on the recovery of the immune system from sublethal irradiation or CYP treatment was assessed. Mice were injected once with AS101 24 h before being irradiated with 450 cGy or treated with 250 mg/kg body weight CYP. At various time points after treatment the functional capacity of the immune system was determined. It was found that AS101 could significantly reduce the decrease in the number of spleen cells and thymocytes, the decrease in the proliferation rate of these cells to the T-cell mitogen concanavalin A, and the decrease of interleukin 2 secretion by spleen cells. AS101 could initially protect these functions because they were increased over control levels immediately 24 h after treatment. AS101 was also shown to normalize the distribution of T-cell subsets that was impaired following both treatments. These results suggest an immunoregulatory role for AS101 in counteracting chemotherapy and radiation-induced immunological suppression as well as its usefulness as an adjunct treatment of cancer when used in combination with CYP or irradiation.

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.

Mechanism of radioprotection conferred by the immunomodulator AS101.

AS101 [ammonium trichloro (dioxyethylene-0-0') tellurate] is a new synthetic compound previously described by us as having immunomodulating properties and minimal toxicity. Clinical trials are currently in progress with AS101 in AIDS and cancer patients. AS101 has recently been found to have radioprotective effects on hemopoiesis in irradiated mice when administered prior to irradiation. Since the early progenitors, spleen colony-forming units (CFU-S), are the critical cells needed for the reconstitution of the hemopoietic system, the mechanisms of action of AS101 were explored in this study by examining the compound's effect on the recovery of CFU-S, its protective effect on endogenous CFU-S and its effect on self-renewal of CFU-S. We also studied the effect of AS101 on the induction of progenitor cells into the radioresistant S-phase of the cell cycle. On days 1 and 5 after irradiation, the number of CFU-S in the bone marrow and spleen of AS101-treated mice was significantly higher than that of PBS-injected mice. Nine days after sublethal doses of irradiation, the number of endogenous spleen colonies was highest in mice given AS101 every 24 hours or every other day for 1 week prior to irradiation. AS101 administered immediately after irradiation, however, also resulted in an increase in the endogenous CFU-S. The higher number of CFU-S found in each 9-day endogenous spleen colony suggests increased self-renewal of CFU-S in AS101-treated mice. Moreover, we found that AS101 induced a higher number of progenitor cells in the S-phase of the cell cycle. These findings suggest that the radioprotection conferred by AS101 results from induction of progenitor cells in DNA synthesis (S-phase) and from the enhanced stimulation of CFU-S, not only toward proliferation but also toward CFU-S self-renewal.

The immunomodulator, ammonium trichloro[1,2-ethanediolato-O,O']-tellurate, suppresses the propagation of herpes simplex virus 2 by reducing the infectivity of the virus progeny.

Persistent investigations for the identification of novel anti-herpetic drugs are being conducted worldwide, as current treatment options are sometimes insufficient. The immunomodulator, ammonium trichloro[1,2­ethanediolato­O,O']­tellurate (AS101), a non­toxic tellurium (Ⅳ) compound, has been shown to exhibit anti­viral activity against a variety of viruses in cell cultures and in animal models. In the present study, the anti­viral activity of AS101 against herpes simplex virus (HSV)­1 and 2 was investigated in vitro. The results demonstrated that AS101 significantly restricted HSV­2-induced plaque formation and reduced the infectivity of the HSV­2 yield, while HSV­1 was affected to a lesser extent. The incubation of mature HSV­1 and HSV­2 viruses with AS101 had no effect on viral infectivity, indicating that the compound interrupts de novo viral synthesis. The addition of AS101 at up to 9 h post­infection had almost the same effect as did the addition of the drug together with the virus (it maintained 80% of its total anti­viral capacity). Quantitative PCR and immunofluoresence staining of viral structural proteins revealed that the viral DNA and protein synthesis stages were not interrupted by the administration of AS101. By contrast, in the presence of the compound, significantly fewer viable viruses (≥2 log reduction) were recovered from the AS10­treated cell cultures. Of note, when we determined the viability of the intracellular virus, formed in the presence of the compound, a less severe (≤1 log) effect was observed. Taken together, these data strongly suggest that AS101 primarily interferes with late stages of viral replication, such as viral particle envelopment or egress, leading to the production of a defective virus progeny.

Inhibition of the reverse transcriptase activity and replication of human immunodeficiency virus type 1 by AS 101 in vitro.

In a search for compounds active against human immunodeficiency virus type 1 (HIV-1), it was found that the novel low-molecular weight immunoenhancer ammonium trichloro(dioxyethylene-O,O'-) tellurate (AS101) suppresses production of HIV-1 in vitro. Treatment of HIV-1-infected peripheral blood mononuclear cells (PBMC) with increasing concentrations of AS101 resulted in substantial inhibition of virus production as measured by both reverse transcriptase (RT) activity and antigen presence in supernatants of treated cells. AS101 had no effect on PBMC viability, growth, or morphology up to a concentration of 15 microM for 14 days. To elucidate a possible mechanism for the inhibition of AS101, we have analyzed the effect of the drug on the catalytic functions associated with HIV RT, namely the RDDP, DDDP, and RNase H activities. RDDP and DDDP activities were impaired by the drug with calculated IC50 value of about 4 microM. On the other hand, the RNase H activity was less sensitive to AS101, with an apparent IC50 value of about 30 microM. The anti-HIV-1 activity of AS101 as reflected by inhibition of the different catalytic functions associated with viral RT, in the absence of drug-related toxicity to lymphocytes, together with its immunomodulating activity strongly argues in favor of its evaluation, as a therapeutic agent for patients with HIV infection.

Inhibition of B16 melanoma metastasis by the immunomodulator AS101.

The immunomodulator ammonium trichloro (dioxyethylene-O-O')tellurate (AS101) has previously been found to induce secretion of various cytokines in mouse and human, which include interleukin-l, interleukin-2, colony-stimulating factor, interferon-gamma, tumor necrosis factor, etc. It also protects mice from lethal and sublethal effects of chemotherapy and irradiation. The present studies were designed to evaluate its effect on pulmonary metastasis following intravenous (i.v.) injection of mouse B16 melanoma cells on day 0 of the experiment. AS101, given 10 mu g/mouse intraperitoneally in 7 daily injections starting the day before B16 cell infusion (day -1) led to a significant inhibition by 60%. When B16 cells were pretreated with AS101 for 24 h before injection, the lung metastases were further reduced by subsequent AS101 treatment of the tumor-loaded mice. In mice that had been depleted of natural killer (NK) cells using anti-asialo-GM1 antisera, AS101 was deprived of its stimulatory effect on the NK activity. The inhibition by AS101 on the B16 lung metastasis was also profoundly reduced by the antisera. Moreover, in vitro treatment of B16 cells with AS101 resulted in suppression of the cell growth in a semisolid culture. This was accompanied by an inhibition of the DNA synthesis and a dephosphorylation/activation of the retinoblastoma susceptibility protein (RE), a tumor suppressor gene product, in the B16 cells. Taken together, these data suggest that AS101 possesses an anti-metastatic activity, which probably involves two mechanisms: the stimulation of the host NK cell activity and the inhibition of the tumor cell proliferation.

Increased DNA repair ability after irradiation following treatment with the immunomodulator AS101.

Ammonium trichloro(dioxoethylene-o-o')tellurate (AS101) is a new synthetic compound previously described by us as being able to modulate the immune system and having minimal toxicity. Clinical trials are currently in progress with AS101 on cancer patients. AS101 has recently been found to have radioprotective effects on hemopoiesis and survival of irradiated mice when administered prior to irradiation. Radioprotection conferred by AS101 has recently been demonstrated by us to result partly from induction of progenitor cells to enter into S phase, which is assumed to be a more radioresistant phase of the cell cycle, and partly from the enhanced stimulation of CFU-S not only toward proliferation but also toward self-renewal. In the present study we demonstrate that the DNA repair processes expressing the cellular reponses associated with the restoration of the normal nucleotide sequence after damage caused to the DNA were also increased significantly after treatment with AS101. Unscheduled DNA repair synthesis was found to be significantly higher in both spleen and bone marrow cells from mice injected with AS101 compared to mice injected with PBS. DNA repair synthesis in spleen cells incubated with AS101 in vitro was also higher than that of PBS-treated cells. This was demonstrated by equilibrium alkaline cesium chloride density gradient of DNA from irradiated and nonirradiated spleen cells in the presence of hydroxyurea. In addition, using the neutral filter elution technique, we show that AS101 can both protect cells from DNA double-strand breaks (DSBs) induced by irradiation and enhance the ability of the affected cells to rejoin the DSBs. We show that extracts of splenocytes, either incubated with AS101 in vitro or obtained from mice injected with AS101, contain substantial DNA polymerase activity which is significantly higher compared to that of control treated cells. Aphidicolin, an inhibitor of DNA polymerases alpha and delta, and dideoxy-thymidine, an inhibitor of DNA polymerase beta, inhibited DNA repair synthesis of irradiated splenocytes stimulated with AS101. These results collectively indicate that AS101 confers its radioprotective effects partly by preventing the induction of DSBs induced by irradiation and partly by enhancing the ability of irradiated cells to repair their damaged DNA, probably by increasing mainly DNA polymerase activity. The understanding of the mechanism of radioprotection conferred by AS101 will enable us to use AS101 more effectively for the restoration of hemopoiesis in patients after radiation therapy or in patients suffering from overdose or accidental irradiation.

Tellurium compound AS101 induces PC12 differentiation and rescues the neurons from apoptotic death.

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra (SN). Studies show that anti-apoptotic and neurotrophic agents are suitable candidates to prevent delayed cell death and/or restore neural function. Here we present the nontoxic immunomodulating compound AS101, which has the ability to induce neurite outgrowth and neural differentiation in PC12 cells. The present study shows that components of the ras signaling pathway are crucial for AS101-induced PC12 differentiation. These include p21ras and its downstream effectors, c-raf-1 and MEK, as well as PI3K. Moreover, these components mediate AS101-induced upregulation of p21waf, which is obligatory for AS101-induced PC12 differentiation. Furthermore, nitric oxide plays a significant role in these AS101 activities. Finally, we show that AS101 prevents apoptosis of NGF-differentiated PC12 cells after NGF withdrawal. Taken together, these results suggest that AS101 induces PC12 cell differentiation and survival by activating the ras-ERK1/2 and ras-PI3K signal transduction pathways, as well as inducing NO production. Our findings may be important in understanding the regulation of survival/apoptosis of neurons deprived of neurotropic support. Futhermore the data propose that AS101 may have clinical potential in the treatment of neurodegenerative disorders like Parkinson's disease.

Intracranial pressure and cerebrospinal fluid outflow conductance in healthy subjects.

Conductance of cerebrospinal fluid (CSF) outflow (Cout) is an important parameter to be considered in patients with CSF circulation abnormalities. In patients with normal-pressure hydrocephalus it is the single most important parameter in determining if the patient needs CSF shunting. The lower normal limit for Cout has been estimated from the effect of shunting in patients with normal-pressure hydrocephalus, from patients retrospectively reevaluated after recovering from illness, and from patients with known abnormalities in the brain or the CSF system. The true value of Cout in normal individuals, however, has hitherto not been reported. In the present study, Cout has been measured by a lumbar infusion test in eight young volunteers with no suspicion of disease. The mean intracranial pressure (ICP) was 11 mm Hg and a linear relationship was found between CSF absorption and ICP. The mean Cout was 0.11 ml/min/mm Hg and the lower 95% confidence level was 0.10 ml/min/mm Hg. These values are in accordance with those obtained from previous studies.

Age dependency of resistance to cerebrospinal fluid outflow.

OBJECT: Resistance to cerebrospinal fluid (CSF) outflow (Rout)is an important parameter in assessing the need for CSF shunt placement in patients with hydrocephalus. The normal lower limit of Rout has been estimated on the basis of the clinical effect of shunt placement in patients with varying values of Rout and in young healthy volunteers. The lack of clinical effect from CSF shunts in some elderly patients, despite elevated Rout,suggests that the normal value of Rout increases with age and may be higher in elderly persons. The aim of the present study was to examine the relationship between Rout and age in patients without known CSF dynamic disturbances. METHOD: Fifty-two patients ranging from 20 to 88 years of age and with no known CSF dynamic disorders were examined. The Rout was measured using a lumbar computerized infusion test. The correlation between Rout and age was analyzed by performing linear regression. The Rout increased significantly with patient age. The Rout in a patient in the eighth decade will be approximately 5 mm Hg/ml/minute higher than in a young patient. CONCLUSIONS: The present study shows a small but critical increase in Rout with increased patient age. A notable residual variation was present and borderline values of Rout should be regarded and used with caution.

A controlled comparison of myelography, computed tomography, and magnetic resonance imaging in clinically suspected lumbar disc herniation.

STUDY DESIGN: A controlled prospective blinded study. OBJECTIVES: To compare the diagnostic power of myelography, computed tomography and magnetic resonance imaging in the diagnosis of low lumbar disc herniation. METHODS: Eighty patients with monoradicular sciatica were examined by myelography, computed tomography, and magnetic resonance imaging, and all underwent subsequent surgery. The images were evaluated twice in a blinded fashion, and the diagnostic power of the modalities was expressed by a decision-analytic regret function. RESULTS: In 57 patients (71%) a disc herniation at the expected level was disclosed at surgery. The largest amount of diagnostic information was gained from computed tomography, followed by magnetic resonance imaging and myelography. Both computed tomography and magnetic resonance imaging were significantly informative, whereas this was not the case for myelography. CONCLUSION: The results indicate that computed tomography or magnetic resonance imaging should be the first choice for imaging in patients with suspected lumbar disc herniation.