Cellular immune function monitoring after allogeneic haematopoietic cell transplantation: evaluation of a new assay.

Managing the patient's immune system after haematopoietic cell transplantation (HCT) is a challenge, mainly in the unstable period immediately after the transplant. Currently there is no standardized non-invasive diagnostic tool for the evaluation of immunological complications such as graft-versus-host disease (GVHD) and for managing the cellular immune function of the transplant recipient. The ImmuKnow assay for cellular immune function monitoring has been incorporated successfully into the clinical follow-up routine of solid organ transplant recipients. This study aims to explore the relevance and potential contribution of immune monitoring using the assay in the setting of HCT. We found that ImmuKnow-level measurement can distinguish between states of immune function quiescence and between events of acute GVHD. ImmuKnow levels were significantly higher in patients going through GVHD than the levels measured for the same patients during immunological stability. Moreover, we demonstrate a patient case where longitudinal monitoring using the ImmuKnow assay provided a trustworthy depiction of the patient's cellular immune function post-HCT. In conclusion, we provide evidence for the potential contribution of the ImmuKnow assay for longitudinal individualized cellular immune function monitoring of patients following HCT. Further studies are necessary in order to establish the optimal practice for utilizing the assay for this purpose.

Long-term culture and cloning of nontransformed human B lymphocytes.

B lymphocyte-enriched cell populations cultured with mitogens in initial suspension cultures formed colonies in soft agar when the same mitogenic agent was present in the lower layer of a two-layer soft agar system. Colony formation depended upon the presence of T cells in the initial culture, and was optimal after an initial 72-h culture with phytohemagglutinin (PHA; 12.5 microliters/ml), pokeweed mitogen (PWM; 2.5 micrograms/ml), or protein A (10 micrograms/ml). The colonies could be picked from the agar and propagated by feeding every 3 d with medium supplemented with a growth factor-containing tissue culture supernate. The growth factor-containing supernate was prepared by stimulating pools of human peripheral blood mononuclear cells for 72 h with PHA or PWM. The lines propagated in this manner were membrane Ig+, lacked sheep erythrocyte rosette-forming ability, and did not ingest latex. They lacked the Epstein-Barr nuclear antigen (EBNA) and had 46 chromosomes. Such lines have been propagated for over 1 yr. One line (BL1) was subjected to limiting dilution cloning and a line, BL1.1, was prepared that contained 96% lambda-bearing cells and no kappa-bearing cells. This line was also EBNA negative. This procedure can thus be used to prepare and clone long-term lines of nontransformed human B lymphocytes.

Antigen-specific T cell clones restricted to unique F1 major histocompatibility complex determinants. Inhibition of proliferation with monoclonal anti-Ia antibody.

The existence of T cells specific for soluble antigens in association with unique F(1) or recombinant major histocompatibility complex (MHC) gene products was first postulated from studies on the proliferative response of whole T cell populations to the antigen poly(Glu(55)Lys(36)Phe(9))(n) (GLphi). In this paper we use the newly developed technology of T lymphocyte cloning to establish unequivocally the existence of such cells specific for GLphi and to generalize their existence by showing that F(1)- specific cells can be isolated from T cell populations primed to poly(Glu(60)Ala(30)Tyr(10))(n) (GAT) where such clones represent only a minor subpopulation of cells. Gl.4b-primed B10.A(5R) and GAT-primed (B10.A x B10)F(1) lymph node T cells were cloned in soft agar, and the colonies that developed were picked and expanded in liquid culture. The GLphi-specific T cells were then recloned under conditions of high-plating efficiency to ensure that the final colonies originated from single cells. T cells from such rigorously cloned populations responded to stimulation with GILphi but only in the presence of nonimmune, irradiated spleen cells bearing (B10.A x B10)F(1) or the syngeneic B 10.A(5R) recombinant MHC haplotype. Spleen cells from either the B10 or B 10.A parental strains failed to support a proliferative response, even when added together. (B10 x B10.D2)F(1) and (B10 x B10.RIII)F(1) spleen cells also supported a proliferative response but (B10 x B10.Q)F(1) and (B10 X B10.S)F(1) spleen cells did not. These results suggested that the T cell clones were specific for GL[phi} in association with the beta(AE)(b)-alpha(E) (k,d,r,) Ia molecule and that recognition required both gene products to be expressed in the same antigen-presenting cells. Support for this interpretation was obtained from inhibition experiments using the monoclonal antibody Y-17 specific for a determinant on the beta(AE)(b)-alphaE Ia molecule. Y-17 completely inhibited the proliferative response of a GLphi-specific clone but had no effect on the response of either a PPD-specific or GAT-specific clone, both of which required the beta(A)-alpha(A) Ia molecule as their restriction element. No evidence could be found for the involvement of suppressor T cells in this inhibition. We therefore conclude that the phenomenon of F(1)-restricted recognition by proliferating T cells results from the presence of antigen- specific clones that must recognize unique F(1) or recombinant Ia molecules on the surface of antigen-presenting cells in addition to antigen in order to be stimulated.

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.

Induction of primary antiphospholipid syndrome in mice by immunization with a human monoclonal anticardiolipin antibody (H-3).

Antiphospholipid syndrome (APLS) is characterized by thrombocytopenia, thromboembolic phenomena, and recurrent fetal loss, associated with anticardiolipin antibodies (ACA) and/or lupus anticoagulant. The syndrome may be primary or may be associated with other conditions such as systemic lupus erythematosus. We have previously shown the ability to induce APLS in naive mice following passive transfer of serum and monoclonal ACAs. Similarly we generated the secondary APLS in BALB/c mice following immunization with a pathogenic anti-DNA antibody. In the current study we report on the induction of primary APLS following immunization of BALB/c mice with a human monoclonal ACA (H-3). The mice developed high persistent titers of ACA. The APLS was characterized by prolonged activated partial thromboplastin time, low fecundity rate (21% vs. 48% of control immunized mice), high resorption index of fetuses (25% vs. 3%), and low weights of embryos and placentae. Our study points to the ability of inducing primary APLS in naive mice. The induction of various presentations of APLS by different ACA may explain the diversity of clinical manifestations seen in patients with APLS.

Prevention of fetal loss in experimental antiphospholipid syndrome by in vivo administration of recombinant interleukin-3.

Antiphospholipid antibodies are strongly associated with arterial and venous thrombosis and with fetal loss. Recently an experimental model for antiphospholipid syndrome (APLS) was established in our laboratory. In this model, mice are immunized passively or actively with anticardiolipin antibodies and acquire the syndrome, which is characterized by prolonged activated partial thromboplastin time (APTT), thrombocytopenia, low fecundity rate, and fetal loss. In a normal process of pregnancy, lymphokines affect fetal implantation and development. Cytokines from the colony stimulating factor family, like GM-CSF and IL-3, were shown to be positive signals for implantation and to promote placental development and fetal growth. Given our preliminary findings of low IL-3 in mice with APLS and the efficacy of IL-3 in preventing fetal loss in a strain of mice prone to fetal resorption, our aim in the present study was to examine the effect of murine recombinant IL-3 (mrIL-3) on pregnant mice induced with experimental APLS. Mice were passively transfused to the tail vein, 24 h following mating, with anticardiolipin antibodies. The mice were divided into two groups: one group was injected intraperitoneally with mrIL-3 on days 6.5, 8.5, and 10.5 after mating, while the control group was injected with PBS. When the mice were killed on day 15 of pregnancy a 32% +/- 4.2 resorption rate was observed in the anti-cardiolipin-immunized group, which was reduced to 4% +/- 0.3 following treatment with mrIL-3. The thrombocytopenia associated with the experimental APLS was also corrected following lymphokine administration. IL-3 may be effective in prevention of recurrent fetal loss in APLS.

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.

Double-stranded RNA-dependent protein kinase, PKR, down-regulates CDC2/cyclin B1 and induces apoptosis in non-transformed but not in v-mos transformed cells.

The interferon (IFN)-induced, double stranded RNA (dsRNA)-activated serine/threonine kinase, PKR, is a potent negative regulator of cell growth when overexpressed in yeast or mammalian cells. Paradoxically, while it can function as a tumor suppressor and inducer of apoptosis, it is overexpressed in a variety of human cancers. To resolve this enigma, we established cell-lines that overexpress PKR in non-transformed and in v-mos transformed CHO cells. Overexpression of PKR suppressed the proliferation of CHO cells by inducing a transient G0/G1 arrest, followed by a delayed G2/M arrest, which attenuated cell cycle progression. These effects were accompanied by early induction of p21/WAF-1 and delayed downregulation of CDC2 and cyclin B1. Induction of proapoptotic activity of the ectopic PKR paralleled the onset of G2/M arrest in CHO cells. However, while transiently inducing p21/WAF-1, PKR did not impose G2/M arrest or apoptosis in v-mos-transformed cells, nor was CDC2 or cyclin B1 down-regulated in those cells. These findings link the proapoptotic activity of PKR to the arrest of cell cycle at the G2/M phase. Consequently, the apoptotic activity of PKR could be counter-acted by an oncogene-like v-mos that overrides the G2/M arrest induced by PKR.

Rat testis lipoxygenase-like enzyme. Characterization of products from linoleic acid.

The linoleate oxidation products of the affinity chromatography-purified lipoxygenase-like enzyme isolated from rat testes microsomes were characterized. Three types of reaction products separated by thin-layer chromatography were generally present: polar byproducts (A and B) and hydroperoxides. The methyl hydroxystearates obtained from the enzymically produced hydroperoxides were analysed by gas-liquid chromatography and showed a ratio of 67% 13-hydroxy isomer to 33% 9-hydroxy isomer. The major polar byproduct was analysed by infrared spectra, nuclear magnetic resonance and mass spectrometry (of the toluene-p-sulphonyl derivative) and its structure was established as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid. The possibility of the existence of a linoleate hydroperoxide isomerase in the affinity-purified preparation is discussed.

Lipoxygenase-like enzyme in rat testis microsomes.

Microsomes, separated from rat testes, were found capable of oxidizing linoleate and arachidonate. The enzyme activity was solubilized with 1% Triton X-100 in acetate buffer (pH 5.0) and purified by affinity chromatography. The overall purification from the starting preparation was approx. 40-fold. The affinity-purified enzyme was almost homogeneous as determined by electrophoresis in polyacrylamide gel. The enzyme was characterized as lipoxygenase-like from its spectrum, specificity, effect of linoleate on its fluorescence and linoleate oxidation products. Three types of compounds separated by thin-layer chromatography were generally present in the lipoxygenase-like enzyme reaction on linoleic acid: substrate fatty acid, polar by-products and hydroperoxides. The hydroperoxides were analyzed by infrared spectra and mass spectrometry and showed the presence of both 9- and 13-hydroxy isomers.

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.

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.

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.

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.