A randomized, open-label, multicenter, phase II study evaluating the efficacy and safety of BTH1677 (1,3-1,6 beta glucan; Imprime PGG) in combination with cetuximab and chemotherapy in patients with advanced non-small cell lung cancer.

Introduction BTH1677, a 1,3-1,6 beta-glucan immunomodulator, stimulates a coordinated anti-cancer immune response in combination with anti-tumor antibody therapies. This phase II study explored the efficacy, pharmacokinetics (PK), and safety of BTH1677 combined with cetuximab/carboplatin/paclitaxel in untreated stage IIIB/IV non-small cell lung cancer (NSCLC) patients. Methods Patients were randomized 2:1 to the BTH1677 arm (N=60; BTH1677, 4 mg/kg, weekly; cetuximab, initial dose 400 mg/m2 and subsequent doses 250 mg/m2, weekly; carboplatin, 6 mg/mL/min AUC (area-under-the-curve) by Calvert formula, once each 3-week cycle [Q3W]); and paclitaxel, 200 mg/m2, Q3W) or Control arm (N=30; cetuximab/carboplatin/paclitaxel as above). Carboplatin/paclitaxel was discontinued after 4-6 cycles; patients who responded or remained stable received maintenance therapy with BTH1677/cetuximab (BTH1677 arm) or cetuximab (Control arm). Investigator and blinded central radiology reviews were conducted. Efficacy assessments included objective response rate (ORR; primary endpoint), disease control rate, duration of objective response, time-to-progression and overall survival (OS); safety was assessed by adverse events (AEs). Potential biomarker analysis for BTH1677 response was also conducted. Results Compared to control treatment, the addition of BTH1677 numerically increased ORR by both investigator (47.8% vs 23.1%; p=0.0468) and central (36.6% vs 23.1%; p=0.2895) reviews. No other endpoints differed between arms. PK was consistent with previous studies. BTH1677 was well tolerated, with AEs expected of the backbone therapy predominating. Biomarker-positive patients displayed better ORR and OS than negative patients. Conclusions BTH1677 combined with cetuximab/carboplatin/paclitaxel was well tolerated and improved ORR as first-line treatment in patients with advanced NSCLC. Future patient selection by biomarker status may further improve efficacy ClinicalTrials.gov Identifier: NCT00874848.

Two randomized, double-blind, placebo-controlled, dose-escalation phase 1 studies evaluating BTH1677, a 1, 3-1,6 beta glucan pathogen associated molecular pattern, in healthy volunteer subjects.

BACKGROUND: BTH1677 is a beta glucan pathogen associated molecular pattern (PAMP) currently being investigated as a novel cancer therapy. Here, the initial safety and pharmacokinetic (PK) results of BTH1677 in healthy subjects are reported. SUBJECTS AND METHODS: In the Phase 1a single-dosing study, subjects were randomized (3:1 per cohort) to a single intravenous (i.v.) infusion of BTH1677 at 0.5, 1, 2, 4, or 6 mg/kg or placebo, respectively. In the Phase 1b multi-dosing study, subjects were randomized (3:1 per cohort) to 7 daily i.v. infusions of BTH1677 at 1, 2, or 4 mg/kg or placebo, respectively. Safety and PK non-compartmental analyses were performed. RESULTS: Thirty-six subjects (N = 24 Phase 1a; N = 12 Phase 1b) were randomized to treatment. No deaths or serious adverse events occurred in either study. Mild or moderate adverse events (AEs) occurred in 67% of BTH1677-treated subjects in both studies. Treatment-related AEs (occurring in ≥10% of subjects) included dyspnea, flushing, headache, nausea, paraesthesia, and rash in Phase 1a and conjunctivitis and headache in Phase 1b. BTH1677 serum concentration was linear with dose. Clearance, serum elimination half-life (t1/2) and volume of distribution (Vss) were BTH1677 dose-independent. In Phase 1b, area under the curve, t1/2, and Vss values were larger at steady state on days 6-30 versus day 0. CONCLUSIONS: BTH1677 was well tolerated after single doses up to 6 mg/kg and after 7 daily doses up to 4 mg/kg.

Kinetics of cellular and cytokine responses in a chimeric mouse model for the study of staphylococcal enterotoxin B pathogenesis.

The mechanisms by which superantigens, such as staphylococcal enterotoxin B (SEB), contribute to microbial pathogenicity have been poorly defined. The study of such pathogenic processes has been hampered by the lack of an adequate animal model. We utilized a previously described murine chimeric model to determine the cytokines and cell populations that might be involved in SEB toxicity. In the absence of bone marrow transplantation (BMT), all total body irradiated (TBI) mice died, while all transplanted mice survived up to 6 months. Compared with non-TBI and non-BMT mice, chimeric mice had an increased percentage of CD11b (Mac-1)-positive splenocytes (17 vs. 59%, P < 0.05) and decreased CD45R-positive (B) cells (33 vs. 6%, P < 0.05) at 6 weeks after BMT. The relative numbers of splenocyte CD4 and CD8 cells were similar in chimeric and normal mice. Susceptibility of chimeric animals to 10 or 100 microg SEB was time-dependent: no mice challenged at 2 weeks post-BMT died, but 15% of mice challenged at 4 weeks and 50% of those challenged at 6-8 weeks died. Compared with TBI and non-BMT C3H/HeJ mice, SEB-challenged chimeric mice at 6-8 weeks had (1) increased splenocyte mRNA expression for: IFN-gamma (3.5 x optimally at 1 h), TNF-alpha (6.5 x at 2 h), IL-6 (4.8 x at 4 h), IL-1beta (8.4 x at 4 h), IL-2 (4.7 x at 4 h), and IL-10 (3 x at 16 h), and (2) increased and earlier peak serum levels of IFN-gamma, IL-6, IL-1beta and IL-2, but no increase in serum TNF-alpha or IL-4. These data support the hypothesis that the decreased percentage of B cells and increased macrophages in chimeric mice lead to enhanced T cell-macrophage interactions after SEB administration and a lethal burst of T cell and macrophage cytokine release. This model will provide insight into cell populations and mechanisms that mediate superantigen-induced toxicity.

The polysaccharide, PGG-glucan, enhances human myelopoiesis by direct action independent of and additive to early-acting cytokines.

beta-Glucans stimulate leukocyte anti-infective activity, enhance murine hematopoietic recovery following bone marrow injury and mobilize murine progenitor cells from bone marrow. This study evaluated the in vitro hematopoietic potential of the beta-glucan, PGG-glucan, on human bone marrow mononuclear cells (BMMC) and CD34+ BMMC compared with protein cytokines. In the presence of submaximal concentrations of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 0.5 ng/ml), PGG-glucan significantly increased BMMC myeloid colony formation comparable to the increase observed with either interleukin-3 (rhIL-3) or stem cell factor (rhSCF). Moreover, the addition of PGG-glucan to cultures containing GM-CSF + IL-3 or GM-CSF + SCF significantly augmented granulocyte-macrophage colony production above baseline, demonstrating that PGG-glucan acts independently of those early-acting cytokines and can enhance their activity in an additive manner. Anti-PGG-glucan monoclonal antibody specifically abrogated the growth-enhancing effect of added PGG-glucan in a saturable manner and other control carbohydrate polymers failed to affect colony formation. Further, PGG-glucan was not associated with induction of IL-6, GM-CSF production and removal of accessory cells by CD34+ cell isolation did not alter the PGG-glucan effect. These data demonstrate that PGG-glucan acts on committed myeloid progenitors to enhance human hematopoietic activity by a mechanism of direct action independent of IL-3 or SCF and independent of secondary cytokine stimulation.

In vitro and in vivo hematopoietic activities of Betafectin PGG-glucan.

Betafectin PGG-glucan is a novel beta-(1,3)glucan that has broad-spectrum anti-infective activities without cytokine induction. Here we report that PGG-glucan also has both in vitro and in vivo hematopoietic activities. In vitro studies with bone marrow target cells from the C3H/HeN mouse revealed that although PGG-glucan alone had no direct effect on hematopoietic colony-forming cell (CFC) growth, when combined with granulocyte colony-stimulating factor (CSF) or granulocyte-macrophage CSF, it increased CFC numbers 1.5- to 2.0-fold over those obtained with CSFs alone. Bone marrow cells cultured for high-proliferative-potential CFCs in the presence of interleukin (IL)-1, IL-3, macrophage CSF, and stem cell factor (SCF), or cultured for erythroid burst-forming units in the presence of IL-3, SCF, and erythropoietin, also exhibited enhanced growth in the presence of PGG-glucan. The synergistic effect of PGG-glucan was specific and could be abrogated by anti-PGG-glucan antibody. The ability of PGG-glucan to modulate hematopoiesis in vivo was evaluated in myelosuppressed rodents and primates. C3H/HeN female mice were intravenously administered saline solution or PGG-glucan (0.5 mg/kg) 24 hours before the intraperitoneal administration of cyclophosphamide (200 mg/kg), and the recovery of bone marrow cellularity and granulocyte-macrophage progenitor cells was evaluated on days 4 and 8 after cyclophosphamide treatment. At both time points, enhanced hematopoietic recovery was observed in PGG-glucan-treated mice compared with saline-treated control mice. In a final series of in vivo experiments, we evaluated the ability of therapeutically administered PGG-glucan to enhance hematopoietic recovery in cyclophosphamide-treated cynomolgus monkeys. Monkeys received intravenous infusions of cyclophosphamide (55 mg/kg) on days 1 and 2, followed on days 3 and 10 by intravenous infusion of PGG-glucan (0.5, 1.0, or 2.0 mg/kg). Compared with those in saline-treated monkeys, accelerated white blood cell recovery and a reduction in the median duration of neutropenia were observed in PGG-glucan-treated monkeys. These studies illustrate that PGG-glucan has both in vitro and in vivo hematopoietic activities and that this agent may be useful in the prevention and/or treatment of chemotherapy-associated myelosuppression.

Enhanced clearance of a multiple antibiotic resistant Staphylococcus aureus in rats treated with PGG-glucan is associated with increased leukocyte counts and increased neutrophil oxidative burst activity.

PGG-Glucan [Betafectin], a highly purified soluble beta-(1-6)-branched beta-(1 3)-linked glucan isolated from Saccharomyces cerevisiae, has broad in vitro and in vivo anti-infective activities unrelated to cytokine induction. Here we present in vivo results on the anti-infective activity of PGG-Glucan against a multiple antibiotic resistant Staphylococcus aureus. PGG-Glucan (0.25-4 mg/kg) was administered intramuscularly to male Wistar rats 48 h, 24 h, and 4 h before and 4 h after intraperitoneal implantation of a gelatin capsule containing 10(8)S. aureus colony forming units (CFU). Blood samples were collected at various times after challenge to determine CFU levels, leukocyte counts and neutrophil oxidative burst activity; serum TNF-alpha, and IL-1beta levels were also evaluated. The 0.25 mg/kg PGG-Glucan dose had no effect on reducing blood CFU levels; however, PGG-Glucan doses of 0.5 mg/kg, 1 mg/kg, 2 mg/kg or 4 mg/kg significantly reduced blood CFU levels by 48 h after challenge. Reduced CFU levels correlated with significantly elevated absolute monocyte counts, absolute neutrophil counts, and neutrophil oxidative burst activity in the absence of any effect on TNF-alpha or on IL-1beta levels. In additional studies, effects on mortality and blood CFU levels were evaluated in rats treated with ampicillin (an antibiotic to which the S. aureus was resistant), PGG-Glucan, or both agents. Mortality and blood CFU levels were reduced most in combination-treated rats compared to saline control rats or rats treated with either ampicillin alone or PGG-Glucan alone. We conclude that in vivo (1) PGG-Glucan can enhance clearance of an antibiotic resistant S. aureus, (2) that this clearance is accompanied by an increase in monocytes and neutrophils as well as a potentiation of neutrophil oxidative microbiocidal activity without alteration of the proinflammatory cytokine response, and (3) PGG-Glucan can enhance the effectiveness of traditional antibiotic treatment.

Effects of particulate and soluble (1-3)-beta-glucans on Ca2+ influx in NR8383 alveolar macrophages.

Particulate and soluble (1-3)-beta-glucans are effective in preventing infections by enhancing macrophage and neutrophil functions. However, the mechanisms triggering these enhanced cellular responses are essentially unknown. We recently demonstrated that zymosan, a particulate (1-3)-beta-glucan receptor agonist, caused an influx of Ca2+ in NR8383 rat alveolar macrophages (AMs) and a resulting increase in intracellular Ca2+ (Zhang et al., J. Leukoc. Biol. 62 (1997) 341-348). Since Ca2+ is important in mediating leukocyte responses, we investigated whether other (1-3)-beta-glucans also alter Ca2+ mobilization in AMs. Particulate and soluble (1-3)-beta-glucans derived from Saccharomyces cerevisiae were used in these studies. Like zymosan, particulate (1-3)-beta-glucan (WGPs) caused a concentration-dependent increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+ and by SKF96365, an inhibitor of receptor-operated Ca2+ channels. When three different soluble (1-3)-beta-glucans, with molecular weights of approximately 11,000, 150,000, and 1,000,000 Da, were tested alone for effects on Ca2+ responses, the low molecular weight (1-3)-beta-glucan produced no effect and the intermediate and high molecular weight (1-3)-beta-glucans caused only a small increase in [Ca2+]i. Interestingly, however, all three soluble (1-3)-beta-glucans could significantly reduce the Ca2+ responses induced by a subsequent exposure to either WGPs or zymosan. These results demonstrate that: 1) particulate (1-3)-beta-glucan activates Ca2+ influx in NR8383 macrophages through receptor-operated Ca2+ channels; 2) soluble (1-3)-beta-glucans do not strongly activate Ca2+ influx in these cells; and 3) soluble (1-3)-beta-glucans significantly inhibit Ca2+ influx induced by WGPs or zymosan. Soluble (1-3)-beta-glucans are likely to prevent Ca2+ influx by competitively binding to the (1-3)-beta-glucan receptors recognizing zymosan and WGPs. The smaller Ca2+ influx induced by soluble (1-3)-beta-glucans may represent only a partial activation of post-receptor signal transduction pathways necessary for inducing Ca2+ influx.

Mobilization of peripheral blood progenitor cells by Betafectin PGG-Glucan alone and in combination with granulocyte colony-stimulating factor.

Betafectin PGG-Glucan, a novel beta-(1,6) branched beta-(1,3) glucan purified from the cell walls of Saccharomyces cerevisiae, has been shown to synergize with myeloid growth factors in vitro and to enhance hematopoietic recovery in myelosuppressed mice and primates. Here we report that PGG-Glucan is also capable of mobilizing peripheral blood progenitor cells (PBPC). PGG-Glucan (0.5 mg/kg to 16 mg/kg) was administered intravenously to C3H/HeN male mice and blood collected at times ranging from 30 min to seven days after injection. Based on granulocyte-macrophage colony-forming cell (GM-CFC) levels, peak mobilization occurred 30 min after a 2 mg/kg PGG-Glucan dose. At this time GM-CFC numbers in PGG-Glucan-treated mice were approximately fourfold greater than in saline-treated control mice. A second, smaller wave of GM-CFC mobilization (approximately twofold increase) also occurred on days 4 and 5 after PGG-Glucan treatment. Mobilization was not associated with the induction of alpha-chemokines, which have recently been reported to induce rapid progenitor cell mobilization. Competitive repopulation experiments performed in irradiated female C3H/HeN mice revealed that, at three months after transplantation, more male DNA was present in bone marrow, splenic, and thymic tissues from animals transplanted with cells obtained from mice 30 min after a 2 mg/kg PGG-Glucan dose than in tissues from animals transplanted with cells obtained from saline-treated mice. Additional experiments evaluated the mobilization effects of PGG-Glucan (2 mg/kg) administered to mice which had been pretreated for three consecutive days with G-CSF (125 microg/kg/day). When blood was collected 30 min after PGG-Glucan treatment, the number of GM-CFC mobilized in combination-treated mice was additive between the number mobilized in mice treated with G-CSF alone and the number mobilized in mice treated with PGG-Glucan alone. These studies demonstrate that: A) PGG-Glucan can rapidly mobilize PBPC; B) the kinetic pattern of PGG-Glucan-induced mobilization is different from that of the CSFs; C) the reconstitutional potential of PGG-Glucan mobilized cells is greater than that of steady-state PBPC, and D) PGG-Glucan can enhance G-CSF-mediated PBPC mobilization.

Sublethal gamma irradiation increases IL-1alpha, IL-6, and TNF-alpha mRNA levels in murine hematopoietic tissues.

The effects of sublethal (7.75 Gy) 60Co gamma radiation exposure on endogenous bone marrow and splenic interleukin-1alpha (IL-1alpha), IL-6, and tumor necrosis factor-alpha (TNF-alpha) mRNA and protein levels were assayed in B6D2F1 female mice. Bone marrow and spleen were harvested from normal and irradiated mice on days 2, 4, 7, 10, and 14 postexposure, and cytokine mRNA levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and Southern blot analysis. IL-1alpha mRNA levels were significantly increased in bone marrow at days 2 and 4 postirradiation and at day 7 in spleen compared with controls. Postirradiation IL-6 mRNA levels showed a significant increase at day 2 in bone marrow and at days 7 and 10 in spleen. TNF-alpha mRNA levels exhibited a significant increase at day 2 postirradiation in bone marrow, but in spleen no difference between control and irradiated samples was observed on any day postirradiation. Interestingly, there were no significant differences in the cytokine protein levels in postirradiation bone marrow, spleen, or serum when compared with normal controls.

Bone marrow and splenic granulocyte-macrophage colony-stimulating factor and transforming growth factor-beta mRNA levels in irradiated mice.

The effects of a myeloablative sublethal 775 cGy 60C gamma radiation exposure on endogenous bone marrow (BM) and splenic granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta (TGF-beta) mRNA levels were assayed in B6D2F1 female mice. BM and spleen were harvested from normal mice and irradiated mice on days 2, 4, 7, 10, and 14 after exposure. Cytokine mRNA levels were determined using reverse transcription-polymerase chain reaction. After irradiation, GM-CSF mRNA levels were significantly increased in the BM from days 2 to 10 and in the spleen from days 4 to 10. However, when BM and splenic GM-CSF protein levels were measured using Western dot blot, no increased protein levels were detected. Serum GM-CSF levels were likewise unchanged. Radiation exposure did not affect BM or splenic TGF-beta mRNA levels and this cytokine is known to be produced by cell populations similar to those that produce GM-CSF. These data suggest that radiation injury to hemopoietic tissues results in differential effects on GM-CSF and TGF-beta mRNA levels and that, in the case of GM-CSF, increased mRNA levels are not matched by increased protein production.

Comparison of interleukin-1 alpha gene expression and protein levels in the murine spleen after lethal and sublethal total-body irradiation.

To understand the effects of ionizing radiation on the production of IL-1 alpha in vivo within a hematopoietic organ, we evaluated acute changes in splenic IL-1 alpha mRNA and IL-1 alpha protein after exposing B6D2F1 mice to lethal and sublethal 60Co radiation. Results suggest that in vivo, ionizing radiation induces a time- and dose-dependent accumulation of IL-1 alpha mRNA in the mouse spleen after exposure to gamma radiation. Time-dependent increases in the level of IL-1 alpha protein were also observed, although the magnitude of increased protein expression did not complement the magnitude of the accumulation of the message. Selective concentration of cells producing IL-1 alpha does not appear to account completely for the increase in splenic IL-1 alpha mRNA observed in this in vivo system.

c-kit ligand gene expression in normal and sublethally irradiated mice.

The c-kit ligand (KL; Steel factor, mast cell growth factor, stem cell factor) is a hematopoietic factor that has been shown to act as a potent cofactor for hematopoietic growth and differentiation in vitro. The in vivo effects of KL, however, have been variable. To study the hematopoietic role of KL in vivo, we evaluated KL gene expression in both normal mice and mice recovering from myelosuppressive radiation exposure using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. In a single RNA sample, we found that the RT-PCR technique has high precision (co-efficient of variation, 15.7%). Amplifications of serial 1:2 dilutions of template RNA precisely correlated with starting RNA concentrations at 20 cycles or at 25 cycles, depending on the level of expression. Amplification of individual normal bone marrow and spleen cell RNA showed basal expression in all normal bone marrows but irregular expression in normal spleens. On day 2 after a sublethal 7.75-Gy (0.4 Gy/min) 60Co irradiation, splenic KL gene expression increased approximately 2.5-fold (P = .011), and bone marrow expression increased 15-fold (P = .004). During a 28-day postirradiation recovery period, KL expression increased in bone marrow on days 2 through 7. Splenic expression during the same period was more variable. In conclusion, the KL gene is invariably expressed in normal murine spleens. Postirradiation, recovering bone marrow and spleen both express increased levels of KL mRNA at day 2 and continue to express increased levels for several days postexposure. These data support a role for KL in the endogenous recovery of hematopoiesis after hypoplastic injury.

Amifostine plus granulocyte colony-stimulating factor therapy enhances recovery from supralethal radiation exposures: preclinical experience in animals models.

A murine model was used to explore whether the cytoprotective agent amifostine (WR-2721) can be used to protect a critical fraction of haemopoietic stem cells against radiation, and whether granulocyte colony-stimulating factor (G-CSF) can then be used to stimulate the protected cells to proliferate and reconstitute the haematopoietic system. Groups of C3H/HeN mice treated with 200 mg/kg amifostine i.p. 30 min before 60Co irradiation and/or 125 micrograms/kg G-CSF subcutaneously from days 1-16 post irradiation were compared. The dose reduction factor (DRF) of the combination of amifostine and G-CSF from LD50/30 values was greater than the sum of the DRFs for amifostine and G-CSF individually. Acceleration of recovery bone marrow and splenic multipotent stem cells (CFU-s) and granulocyte-macrophage progenitor cells (GM-CFC), as well as of peripheral blood red and white cells and platelets, was greatest in mice treated with amifostine plus G-CSF. These studies suggest that amifostine and recombinant haematopoietic growth factors can be used in combination to reduce myelosuppression and lethality associated with radiation or radiomimetic drugs

Therapeutic efficacy of recombinant interleukin-6 (IL-6) alone and combined with recombinant human IL-3 in a nonhuman primate model of high-dose, sublethal radiation-induced marrow aplasia.

Using a nonhuman-primate model of radiation-induced bone marrow aplasia, we examined whether the single, concomitant, or sequential administration of recombinant human interleukin-3 (IL-3) and IL-6 would promote bone marrow regeneration measured by an increase in circulating platelets (PLT) and neutrophils (PMN). Rhesus monkeys were irradiated at 450 cGy and were randomly assigned to one of five treatment protocols, receiving IL-6; IL-3; combined IL-6 and IL-3; sequential IL-3 and IL-6; or human serum albumin (HSA) as a control. Cytokines or HSA were administered at total dosages of 15 micrograms/kg/day. Complete blood counts and white blood cell differentials were monitored for 60 days postirradiation. Both IL-3 and IL-6 significantly enhanced the regeneration of PLTs and decreased the duration of thrombocytopenia (P = .005) without affecting PMN recovery. The radiation-induced anemia that was observed in the HSA-treated controls was less severe and resolved more quickly in the IL-6 treated animals. Sequential IL-3/IL-6 significantly increased the production of PLTs when compared with the HSA-treated controls (P = .003) and monkeys receiving concomitant IL-3/IL-6 (P = .041) but did not alter PMN levels significantly (P = .80). Coadministration of IL-6 and IL-3 did not enhance PLT but improved PMN recovery over IL-6 alone. In this primate model of marrow aplasia, IL-6 significantly enhanced the regeneration of PLTs but had no significant effect on PMN production, and did not exacerbate radiation-induced anemia. Furthermore, the use of sequentially administered IL-3 and IL-6 may improve PLT recovery as compared with concurrent IL-3/IL-6 administration, although this protocol is not significantly different in effect than either cytokine alone.

Granulocyte colony-stimulating factor and amifostine (Ethyol) synergize to enhance hemopoietic reconstitution and increase survival in irradiated animals.

The reported studies tested whether amifostine could be used to protect hemopoietic stem cells, which, after irradiation, could be stimulated by granulocyte colony-stimulating factor (G-CSF) to proliferate and reconstitute the hemopoietic system. Female C3H/HeN mice were administered amifostine (Ethyol, US Bioscience, Inc, West Conshohocken, PA) (200 mg/kg intraperitoneally 30 minutes before cobalt-60 irradiation and G-CSF (125 micrograms/kg/d subcutaneously from days 1 to 16 after irradiation. Saline, G-CSF, amifostine, and amifostine plus G-CSF treatments resulted in LD50/30 values of 7.85 Gy, 8.30 Gy, 11.30 Gy, and 12.85 Gy, respectively. At these LD50/30 values, the dose reduction factor of 1.64 obtained in combination-treated mice was more than additive between the dose reduction factors of G-CSF-treated mice (1.06) and amifostine-treated mice (1.44). Bone marrow and splenic multipotent hemopoietic stem cell and granulocyte-macrophage progenitor cell recoveries, as well as peripheral white blood cell, platelet, and red blood cell recoveries were also accelerated most in mice treated with amifostine plus G-CSF. These studies demonstrate that therapeutically administered G-CSF accelerates hemopoietic reconstitution from amifostine-protected stem and progenitor cells, increasing the survival-enhancing effects of amifostine, and suggest that classic radioprotectants and recombinant hemopoietic growth factors can be used in combination to reduce the risks associated with myelosuppression induced by radiation or radiomimetic drugs.

Effects of radiation on survival and recovery of T lymphocyte subsets in C3H/HeN mice.

The aims of this study were to determine the radiosensitivities of murine thymic and splenic CD4+ and CD8+ lymphocytes and to evaluate the regeneration of these cells in a model of radiation-induced hematopoietic and immune suppression. CD4+ and CD8+ cells were quantitated using two-color flow-cytometric analysis. Cells obtained from C3H/HeN mice 24 hours after exposure to 0.25-8.0 Gy (0.4 Gy/min) 60Co were used to determine D0 values. Thymic CD4+ cells contained a radiosensitive subpopulation with a D0 of 0.97 +/- 0.05 Gy and a radioresistant subpopulation that survived exposures up to 8.0 Gy. CD8+ cells also contained a radiosensitive subpopulation with a D0 of 1.24 +/- 0.05 Gy and a radioresistant subpopulation with a D0 of 3.93 +/- 2.01 Gy. Double-positive thymic CD4+/CD8+ cells were uniformly radiosensitive, with a D0 of 1.03 +/- 0.28 Gy. Multiple T lymphocyte subpopulations based on radiosensitivity and CD4/CD8 antigen expression were also observed in the spleen. When mice were exposed to a sublethal 6.5-Gy radiation dose and recovery of T lymphocyte subsets was monitored, the relative radioresistance of CD4+ cells resulted in a selective enrichment of these cells among the surviving thymocytes and splenic lymphocytes. This relative enrichment of CD4+ cells became even more prominent 7 days after irradiation, when atrophy of the organs was greatest. Similar, although less dramatic, effects were observed for CD8+ cells. These studies demonstrate that (1) multiple T lymphocyte subpopulations can be identified based on radiosensitivity and CD4/CD8 antigen expression; (2) both CD4+ and CD8+ cells contain radioresistant subpopulations, with the CD4+ subpopulation being more resistant than the CD8+ subpopulation; and (3) although the number of radioresistant CD4+ cells is quite small, they persist in increased proportions during the periods preceding and corresponding to postirradiation hematopoietic recovery.

Ultrastructural localization of tumour necrosis factor-alpha.

The application of an antibody against tumour necrosis factor-alpha (TNF) to thin sections of plastic-embedded mouse tissue has identified sites of TNF activity in normal and endotoxin-treated C3N/HeN mice. Prior to endotoxin treatment, TNF was observed in the secretory granules of the antibacterial Paneth cell and one type of crypt endocrine cell. Four hours after endotoxin treatment, these two types of intestinal cell were found to have degranulated. In addition, endotoxin treatment resulted in the appearance of TNF in the secretory granules of all eosinophils, neutrophils and monocytes in the bone marrow, spleen, lung and the proximal intestine. TNF was also observed in the internal elastic lamina (IEL) of arterioles. These results suggest that the process of TNF induction specifically targets the immune system and the vasculature. An invasive stimulus, such as circulating endotoxin, can provoke the immune cells to be armed with TNF. That same stimulus may cause arteriole smooth muscle cells to secrete TNF. TNF secretion in the presence of arteriole smooth muscle cells may play a role in the adjustment of arteriole tone. In the venules, TNF may be responsible for platelet and neutrophil accumulation which leads to embolism formation.

Mast cell growth factor enhances multilineage hematopoietic recovery in vivo following radiation-induced aplasia.

Based on in vitro studies, mast cell growth factor (MGF; also known as steel factor, stem cell factor, and c-kit ligand) has been implicated as an important hematopoietic regulator, especially in the presence of additional hematopoietic cytokines. Since hematopoietic regeneration follows sublethal radiation-induced hematopoietic injury and is thought to be mediated by endogenously produced cytokines, the ability to accelerate recovery from radiation-induced hematopoietic hypoplasia was used to evaluate in vivo effects of MGF administration. Female B6D2F1 mice were exposed to a sublethal 7.75-Gy dose of 60Co radiation followed by subcutaneous administration of either saline or 100, 200, or 400 micrograms/kg/d recombinant murine MGF on days 1 to 17 postirradiation. Recoveries of bone marrow and splenic spleen colony-forming units (CFU-S), granulocyte-macrophage colony-forming cells (GM-CFC), and peripheral white blood cells (WBC), red blood cells (RBC), and platelets (PLT) were determined on days 14 and 17 during the postirradiation recovery period. MGF accelerated hematopoietic recovery at the 100 and 200 micrograms/kg/d doses. The 100 micrograms/kg/d dose accelerated recovery of only GM-CFC, while the 200 micrograms/kg/d dose accelerated CFU-S, GM-CFC, WBC, and PLT recoveries. In contrast, hematopoietic recovery was delayed in mice receiving the 400 micrograms/kg/d dose. These studies demonstrate the in vivo dose-dependent ability of MGF to accelerate multilineage hematopoietic regeneration following radiation-induced hematopoietic hypoplasia. They also document detrimental effects of providing "supraoptimal" doses of this growth factor and suggest caution in dose-escalation trials in humans.

Adverse effects of pefloxacin in irradiated C3H/HeN mice: correction with glucan therapy.

Opportunistic bacterial infections are the predominant cause of death following myelosuppressive radiation exposure. When used alone, a variety of immunomodulators and antibiotics have been reported to reduce radiation-induced death. In these studies, the combined therapeutic effects of the immunomodulator glucan and the quinolone antibiotic pefloxacin were evaluated for survival-enhancing effects in myelosuppressed C3H/HeN mice. Mice were exposed to 7.9 Gy of whole-body 60Co radiation and treated with saline, glucan (250 mg/kg of body weight intravenously, 1 h after irradiation), pefloxacin (64 mg/kg/day orally, days 3 to 24 after irradiation), or glucan plus pefloxacin. Survival 30 days after irradiation in mice receiving these respective treatments was 25, 48, 7, and 85%. Evaluation of granulocyte-macrophage progenitor cell (GM-CFC) recovery in mice receiving these treatments revealed that, compared with recovery in saline-treated mice, glucan stimulated GM-CFC recovery, pefloxacin suppressed GM-CFC recovery, and glucan administered in combination with pefloxacin could override pefloxacin's hemopoietic suppressive effect.

Effects of combined administration of interleukin-6 and granulocyte colony-stimulating factor on recovery from radiation-induced hemopoietic aplasia.

Hemopoietic aplasia is the primary limitation of drug and radiation cancer therapies. We have previously demonstrated that, individually, both interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) can accelerate recovery from radiation-induced hemopoietic aplasia. In vitro studies suggest that IL-6 affects cells early in the hemopoietic hierarchy, while G-CSF affects more committed progenitor cells. Because these cytokines may also affect different cell populations in vivo, we hypothesized that the use of these agents in combination may further enhance recovery from hemopoietic aplasia. Female B6D2F1 mice were exposed to a high sublethal 7.75 Gy dose of 60Co radiation. Following irradiation, mice were administered subcutaneous injections of either saline, 500 micrograms/kg of recombinant human IL-6 once daily on days 1-6, 125 micrograms/kg of recombinant human G-CSF once daily on days 1-17, or both cytokines as described. Peripheral white blood cell (WBC), red blood cell (RBC), and platelet (PLT) counts, as well as femoral and splenic granulocyte-macrophage colony-forming cell (GM-CFC) and day-12 spleen colony-forming unit (CFU-S) contents were evaluated on days 7, 10, 14, 17 and 21 postirradiation. IL-6 treatment alone slightly accelerated postirradiation recovery of most hemopoietic parameters, while G-CSF treatment dramatically enhanced recovery of all hemopoietic parameters evaluated. Co-administration of IL-6 and G-CSF further enhanced the hemopoietic recovery. The most notable effects in combination-treated mice were on recoveries of bone marrow and splenic CFU-S, which were significantly enhanced above those in G-CSF-treated irradiated mice as early as day 10 postirradiation. Although by day 14 postirradiation, splenic GM-CFC and CFU-S recoveries in both G-CSF- and combination-treated mice had surpassed unirradiated control values, combination-treated mice exhibited a greater overshoot. These studies demonstrate the ability of IL-6 treatment to enhance G-CSF-mediated acceleration of multilineage recovery following radiation-induced hemopoietic aplasia.