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.

Glucan: mechanisms involved in its "radioprotective" effect.

It has generally been accepted that most biologically derived agents that are radioprotective in the hemopoietic-syndrome dose range (eg, endotoxin, Bacillus Calmette Guerin, Corynebacterium parvum, etc) exert their beneficial properties by enhancing hemopoietic recovery and hence, by regenerating the host's ability to resist life-threatening opportunistic infections. However, using glucan as a hemopoietic stimulant/radioprotectant, we have demonstrated that host resistance to opportunistic infection is enhanced in these mice even prior to the detection of significant hemopoietic regeneration. This early enhanced resistance to microbial invasion in glucan-treated irradiated mice could be correlated with enhanced and/or prolonged macrophage (but not granulocyte) function. These results suggest that early after irradiation glucan may mediate its radioprotection by enhancing resistance to microbial invasion via mechanisms not necessarily predicated on hemopoietic recovery. In addition, preliminary evidence suggests that glucan can also function as an effective free-radical scavenger. Because macrophages have been shown to selectively phagocytize and sequester glucan, the possibility that these specific cells may be protected by virtue of glucan's scavenging ability is also suggested.

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.

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.

The rhesus monkey: a primate model for hemopoietic stem cell studies.

Two heterogeneous cell populations (CP 1-7 and CP 8-10) were separated from rhesus monkey bone marrow using counterflow centrifugation-elutriation (CCE). These two cell populations were distinct with respect to morphological composition, expression of cell surface antigens, hemopoietic progenitor cell activity, and concentration of hemopoietic stem cells (HSC). The hemopoietic progenitor cell activity and HSC were concentrated in CP 8-10. In autologous transplantation studies, CP 8-10 reconstituted the lymphohemopoietic system of lethally irradiated monkeys in a manner similar to that of monkeys transplanted with unfractionated bone marrow cells. CP 1-7 was lymphocyte rich and depleted of progenitor cell activity. Transplantation of CP 1-7 led to eventual lymphohemopoietic reconstitution of irradiated monkeys; however, complete engraftment was delayed by as much as 14 days compared to either the transplantation of CP 8-10 or to unfractionated bone marrow. Thus, a presence of the HSC in the lymphocyte-rich progenitor-cell-depleted population can be detected in the rhesus model.

Survival enhancement and hemopoietic regeneration following radiation exposure: therapeutic approach using glucan and granulocyte colony-stimulating factor.

C3H/HeN female mice were exposed to wholebody cobalt-60 radiation and administered soluble glucan (5 mg i.v. at 1 h following exposure), recombinant human granulocyte colony-stimulating factor (G-CSF; 2.5 micrograms/day s.c., days 3-12 following exposure), or both agents. Treatments were evaluated for their ability to enhance hemopoietic regeneration, and to increase survival after radiation-induced myelosuppression. Both glucan and G-CSF enhanced hemopoietic regeneration alone; however, greater effects were observed in mice receiving both agents. For example, on day 17 following a sublethal 6.5-Gy radiation exposure, mice treated with saline, G-CSF, glucan, or both agents, respectively, exhibited 36%, 65%, 50%, and 78% of normal bone marrow cellularity, and 84%, 175%, 152%, and 212% of normal splenic cellularity. At this same time, granulocyte-macrophage colony-forming cell (GM-CFC) values in saline, G-CSF, glucan, or combination-treated mice, respectively, were 9%, 46%, 26%, and 57% of normal bone marrow values, and 57%, 937%, 364%, and 1477% of normal splenic values. Endogenous spleen colony formation was also increased in all treatment groups, with combination-treated mice exhibiting the greatest effects. Likewise, although both glucan and G-CSF alone enhanced survival following an 8-Gy radiation exposure, greatest survival was observed in mice treated with both agents. These studies suggest that glucan, a macrophage activator, can synergize with G-CSF to further accelerate hemopoietic regeneration and increase survival following radiation-induced myelosuppression.

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.

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.

Recovery of hematopoietic colony-forming cells in irradiated mice pretreated with interleukin 1 (IL-1).

Data in this report determined the effect of a single injection of recombinant interleukin 1 alpha (rIL-1) prior to irradiation of B6D2F1 mice on the recovery of colony-forming cells (CFC) at early and late times after sublethal and lethal doses of radiation. Injection of rIL-1 promoted an earlier recovery of mature cells in the blood and CFC in the bone marrow and spleen. For example, 8 days after 6.5 Gy irradiation, the number of CFU-E (colony-forming units-erythroid), BFU-E (burst-forming units-erythroid), and GM-CFC (granulocyte-macrophage colony-forming cells) per femur was approximately 1.5-fold higher in rIL-1-injected mice than in saline-injected mice. Also, 5, 9, and 12 days after irradiation, the number of both day 8 and day 12 CFU-S (colony-forming units-spleen) was almost twofold greater in bone marrow from rIL-1-injected mice. The earlier recovery of CFU-S in rIL-1-injected mice was not associated with an increase in the number of CFU-S that survived immediately after irradiation. Also, 7 months after irradiation, the number of CFU-S per femur of both saline- and rIL-1-injected mice was still less than 50% of normal values. Data in this report demonstrate that a single injection of rIL-1 prior to irradiation accelerates early hematopoietic recovery in irradiated mice, but does not prevent expression of radiation-induced frontend damage or long-term damage to hematopoietic tissues.

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.

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

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.

Combined modality radioprotection: the use of glucan and selenium with WR-2721.

Glucan, WR-2721, and selenium, three agents with distinct radioprotective mechanisms, were evaluated in C3H/HeN mice for survival-enhancing and hemopoietic-regenerating effects when administered alone or in combinations before exposure to 60Co radiation. At LD50/30 radiation doses (radiation doses lethal for 50% of mice within 30 days postexposure), dose reduction factors of 1.21, 1.02, 1.37, 1.51, and 1.66 were obtained following glucan (75 mg/kg i.v., -20 hr), selenium (0.8 mg/kg, i.p., -20 hr), WR-2721 (200 mg/kg, i.p., -30 min), glucan + WR-2721, and glucan + selenium + WR-2721 treatments, respectively. All treatments increased numbers of hemopoietic stem cells as measured by the day 12 endogenous spleen colony-forming unit (E-CFU) assay; the most significant E-CFU effects, however, were observed following glucan + WR-2721 and glucan + selenium + WR-2721 treatments. Combined modality treatments were also more effective than single-agent treatments at accelerating bone marrow and splenic granulocyte-macrophage colony-forming cell (GM-CFC) regeneration. These results demonstrate the value of multiple-agent radioprotectants.

Postirradiation treatment with granulocyte colony-stimulating factor and preirradiation WR-2721 administration synergize to enhance hemopoietic reconstitution and increase survival.

These studies tested whether WR-2721 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 WR-2721 (4 mg/mouse, i.p.) 30 min before 60Co irradiation and G-CSF (2.5 micrograms/mouse/day, s.c.) from days 1-16 after irradiation. In survival studies, saline, G-CSF, WR-2721, and WR-2721 + 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 (DRF) of 1.64 obtained in combination-treated mice was more than additive between the DRF's of G-CSF-treated mice (1.06) and WR-2721-treated mice (1.44). Bone marrow and splenic multipotent hemopoietic stem cell (CFU-s) and granulocyte-macrophage progenitor cell (GM-CFC) recoveries were also accelerated most in mice treated with WR-2721 + G-CSF. In addition, mice treated with WR-2721 + G-CSF exhibited the most accelerated peripheral blood white cell, platelet, and red cell recoveries. These studies (a) demonstrate that therapeutically administered G-CSF accelerates hemopoietic reconstitution from WR-2721-protected stem and progenitor cells, increasing the survival-enhancing effects of WR-2721 and (b) suggest that classic radioprotectants and recombinant hemopoietic growth factors can be used in combination to reduce risks associated with myelosuppression induced by radiation or radiomimetic drugs.

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.

Leukotriene-induced radioprotection of hematopoietic stem cells in mice.

Leukotrienes (LTs), known primarily for their pathological roles, may also be capable of exerting "cytoprotection" against toxic agents in a manner similar to that of prostaglandins. In this report, it is shown that treatment of mice with leukotrienes C4, D4, E4, or B4 prior to sublethal irradiation increased the number of endogenous hematopoietic stem cells (E-CFU), with LTC4 producing the greatest response (LTC4 much greater than B4 greater than E4 greater than D4). LTC4-induced hematopoietic radioprotection was examined in greater detail using the exogenous spleen colony (CFU-S) and granulocyte/macrophage progenitor cell (GM-CFC) assays. The dose reduction factors for these cells in LTC4-treated mice at radiation doses resulting in 37% cell survival were 1.65 and 2.01, respectively.

Radioprotection of mice with interleukin-1: relationship to the number of spleen colony-forming units.

Compared to saline-injected mice 9 days after 6.5 Gy irradiation, there were twofold more Day 8 spleen colony-forming units (CFU-S) per femur and per spleen from B6D2F1 mice administered a radioprotective dose of human recombinant interleukin-1-alpha (rIL-1) 20 h prior to their irradiation. Studies in the present report compared the numbers of CFU-S in nonirradiated mice 20 h after saline or rIL-1 injection. Prior to irradiation, the number of Day 8 CFU-S was not significantly different in the bone marrow or spleens from saline-injected mice and rIL-1-injected mice. Also, in the bone marrow, the number of Day 12 CFU-S was similar for both groups of mice. Similar seeding efficiencies for CFU-S and percentage of CFU-S in S phase of the cell cycle provided further evidence that rIL-1 injection did not increase the number of CFU-S prior to irradiation. In a marrow repopulation assay, cellularity as well as the number of erythroid colony-forming units, erythroid burst-forming units, and granulocyte-macrophage colony-forming cells per femur of lethally irradiated mice were not increased in recipient mice of donor cells from rIL-1-injected mice. These results demonstrated that a twofold increase in the number of CFU-S at the time of irradiation was not necessary for the earlier recovery of CFU-S observed in mice irradiated with sublethal doses of radiation 20 h after rIL-1 injection.

Radioprotection of mice with interleukin-1: relationship to the number of erythroid and granulocyte-macrophage colony-forming cells.

This report presents the results of an investigation of changes in the number of erythroid and granulocyte-macrophage colony-forming cells (GM-CFC) that had occurred in tissues of normal B6D2F1 mice 20 h after administration of a radioprotective dose (150 ng) of human recombinant interleukin-1 (rIL-1). Neutrophilia in the peripheral blood and changes in the tissue distribution of GM-CFC demonstrated that cells were mobilized from the bone marrow in response to rIL-1 injection. For example, 20 h after rIL-1 injection marrow GM-CFC numbers were 80% of the numbers in bone marrow from saline-injected mice. Associated with this decrease there was a twofold increase in the number of peripheral blood and splenic GM-CFC. Also, as determined by hydroxyurea injection, there was an increase in the number of GM-CFC in S phase of the cell cycle in the spleen, but not in the bone marrow. Data in this report suggest that when compared to the spleen, stimulation of granulopoiesis after rIL-1 injection is delayed in the bone marrow. Also, the earlier recovery of GM-CFC in the bone marrow of irradiated mice is not dependent upon an increase in the number of GM-CFC at the time of irradiation.

Postirradiation glucan administration enhances the radioprotective effects of WR-2721.

Based on murine survival studies, endogenous hemopoietic spleen colony formation (E-CFU), and recovery of bone marrow and splenic granulocyte-macrophage colony-forming cells (GM-CFC), it was demonstrated that the postirradiation administration of glucan, an immunomodulator and hemopoietic stimulant, enhances the radioprotective effects of WR-2721. LD50/30 dose reduction factors for mice treated with WR-2721 (200 mg/kg approximately 30 min before irradiation), glucan (250 mg/kg approximately 1 h after irradiation), or both agents were 1.37, 1.08, and 1.52, respectively. Enhanced survival in mice treated with both agents appeared to be due in part to glucan's ability to accelerate hemopoietic regeneration from stem cells initially protected from radiation-induced lethality by WR-2721. Following a 10-Gy radiation exposure, E-CFU numbers in mice treated with saline, WR-2721, glucan, or both WR-2721 and glucan were 0.05 +/- 0.03, 6.70 +/- 1.05, 0.95 +/- 0.24, and 33.90 +/- 2.96, respectively. Similarly, bone marrow and splenic GM-CFC numbers were greater in mice treated with both WR-2721 and glucan than in mice treated with either agent alone. These results demonstrated at least additive radioprotective effects when mice were given WR-2721 prior to irradiation and glucan following irradiation. These effects appeared to depend on the sequential cell protection mediated by WR-2721 and hemopoietic repopulation mediated by glucan.