William E. Jackson

Androstenediol stimulates myelopoiesis and enhances resistance to infection in gamma-irradiated mice.

The ionizing radiation-induced hemopoietic syndrome is characterized by defects in immune function and increased mortality due to infections and hemorrhage. Since the steroid 5-androstene-3beta, 17beta-diol (5-androstenediol, AED) modulates cytokine expression and increases resistance to bacterial and viral infections in rodents, we tested its ability to promote survival after whole-body ionizing radiation in mice. In unirradiated female B6D2F1 mice, sc AED elevated numbers of circulating neutrophils and platelets and induced proliferation of neutrophil progenitors in bone marrow. In mice exposed to whole-body (60)Co gamma-radiation (3 Gy), AED injected 1 h later ameliorated radiation-induced decreases in circulating neutrophils and platelets and marrow granulocyte-macrophage colony-forming cells, but had no effect on total numbers of circulating lymphocytes or erythrocytes. In mice irradiated (0, 1 or 3 Gy) and inoculated four days later with Klebsiella pneumoniae, AED injected 2 h after irradiation enhanced 30-d survival. Injecting AED 24 h before irradiation or 2 h after irradiation increased survival to approximately the same extent. In K. pneumoniae-inoculated mice (irradiated at 3-7 Gy) and uninoculated mice (irradiated at 8-12 Gy), AED (160 mg/kg) injected 24 h before irradiation significantly promoted survival with dose reduction factors (DRFs) of 1.18 and 1.26, respectively. 5-Androstene-3beta-ol-17-one (dehydroepiandrosterone, DHEA) was markedly less efficacious than AED in augmenting survival, indicating specificity. These results demonstrate for the first time that a DHEA-related steroid stimulates myelopoiesis, and ameliorates neutropenia and thrombocytopenia and enhances resistance to infection after exposure of animals to ionizing radiation.

Development and Assessment of a Quantitative Reverse Transcription-PCR Assay for Simultaneous Measurement of Four Amplicons

BACKGROUND High-throughput and forward-deployable biological dosimetry capabilities are required for tactical and medical decisions after radiologic events. We previously reported a quantitative reverse transcription (QRT)-PCR assay for human radiation-responsive gene targets using a whole-blood ex vivo irradiation model, but we needed a multitarget assay on a smaller, less costly, real-time PCR detection system. METHODS We developed a quadruplex QRT-PCR assay in a 96-well, closed-plate format suitable for use with RNA extracted from whole blood. Four cDNA targets were simultaneously amplified in a sealed tube by hybridization to exonuclease probes, each conjugated to distinct fluorogenic reporters. A novel primer-limited 18S rRNA reference target was validated from serial dilutions of human total RNA. To test assay precision, we incorporated a positive-control cDNA mimic into duplex and quadruplex PCR reactions. The master mixture was supplemented with more enzyme, MgCl(2), and deoxyribonucleotides. Simultaneous detection of four targets was evaluated in comparison with respective duplex QRT-PCR assays. RESULTS The simultaneous detection of three radiation-responsive genes by quadruplex QRT-PCR was quantitative, with gene expression changes similar to those observed with optimized duplex and triplex QRT-PCR assays. The 18S rRNA and GADD45 calibration curves (threshold cycle vs log(10) cDNA) were linear and reproducible and showed optimal PCR efficiencies as indicated by slopes statistically equivalent to the theoretical value of -3.322. CONCLUSIONS This is the first study of a quadruplex QRT-PCR assay. Our approach has diagnostic utility in the detection of biomarkers, biological and toxicologic agents, and genes of inherited diseases and cancer.

In vivo radioprotection by 5-androstenediol: stimulation of the innate immune system.

Abstract Whitnall, M. H., Inal, C. E., Jackson, W. E., III, Miner, V. L., Villa, V. and Seed, T. M. In Vivo Radioprotection by 5-Androstenediol: Stimulation of the Innate Immune System. Radiat. Res. 156, 283–293 (2001). We showed previously that 5-androstenediol stimulates myelopoiesis, increases the numbers of circulating neutrophils and platelets, and enhances resistance to infection in γ-irradiated mice. We have extended those studies to include monocytes, natural killer (NK) cells, eosinophils and basophils, and we have measured the activation marker CD11b using flow cytometry. Androstenediol (160 mg/kg) was administered subcutaneously to female B6D2F1 mice 24 h before whole-body γ irradiation. Androstenediol treatments increased the blood levels of neutrophils, monocytes and NK cells in unirradiated animals; decreased the numbers of circulating eosinophils; and ameliorated radiation-induced decreases in neutrophils, monocytes, NK cells, erythrocytes and platelets. The androstenediol treatments had no significant effect on the numbers of circulating B cells or T cells. CD11b labeling intensity on monocytes was decreased slightly after androstenediol treatment. In contrast, radiation or androstenediol alone caused increases in CD11b labeling intensity on NK cells. Androstenediol and radiation combined caused a marked increase in NK cell CD11b. The results indicate that androstenediol increases the numbers of the three major cell types of the innate immune system (neutrophils, monocytes and NK cells), that androstenediol-induced changes in blood elements in irradiated animals persist for at least several weeks, and that there is a significant positive interaction between radiation and administration of androstenediol in the activation of NK cells.

RADIOPROTECTIVE EFFICACY AND ACUTE TOXICITY OF 5-ANDROSTENEDIOL AFTER SUBCUTANEOUS OR ORAL ADMINISTRATION IN MICE

ABSTRACT We previously showed that one subcutaneous (sc) injection of 5-androstene-3beta,17beta-diol (AED) stimulated the innate immune system in mice and prevented mortality due to hemopoietic suppression after whole-body ionizing irradiation with gamma rays. In the present study, we tested whether there was any significant toxicity in mice that might hinder development of this steroid for human use. There were no indications of toxicity in chemical analyses of serum after sc doses as high as 4000 mg/kg. At this dose, 2 of 54 mice died when given AED alone. When 4800 mg/kg was given orally, no deaths resulted. The only adverse findings attributed to AED administration were 1) a moderate elevation of granulocytes in abdominal organs and fat after sc injections of 320 mg/kg; and 2) occasional wasting of skin over the injection site in female B6D2F1 but not male C3H/HeN mice. Significant weight loss (6%) was observed after sc injections of 320 mg/kg but not 160 or 80 mg/kg. When male C3H/HeN mice were injected sc with AED at doses of 0–200 mg/kg 24 h before whole body gamma-irradiation (9 Gy), a significant improvement in survival was observed at doses as low as 5 mg/kg. Oral administration of AED produced significant survival enhancement at a dose of 1600 mg/kg. We conclude that the radioprotective efficacy of AED is accompanied by low toxicity.Androst-5-ene-3 beta, 17 beta-diol; Ionizing radiation; Experimental radiation injuries; Toxicity; Clinical chemistry; Histopathology

Effects of Whole-Body Gamma Irradiation and 5-Androstenediol Administration on Serum G-CSF

5-Androstenediol (5-AED) is a natural circulating adrenocortical steroid hormone that interconverts in vivo with other members of the 5-androstene family of steroids: dehydroepiandrosterone and 5-androstenetriol. These steroids stimulate immune responses and resistance to infection. 5-AED has been identified as a systemic radiation countermeasure that enhances survival in mice exposed to gamma irradiation and ameliorates radiation-induced neutropenia in mice and nonhuman primates. 5-AED mitigates radiation-induced decreases in platelets, natural killer (NK) cells, red blood cells, and monocytes. Administration of 5-AED causes functional activation of circulating granulocytes (phagocytic ability), monocytes (oxidative burst), and NK cells (surface CD11b expression). The effects of 5-AED on survival and hematological parameters are consistent with induction of hematopoietic cytokines. To test this hypothesis, we measured serum cytokines by ELISA, Luminex, and a cytokine array. A cytokine array was used for 62 different cytokines, chemokines, growth factors, and soluble receptors. 5-AED caused significant increases in circulating granulocyte colony-stimulating factor (G-CSF) in irradiated and unirradiated animals as observed with ELISA and Luminex. The cytokine array results suggest induction of G-CSF and additional cytokines, and related molecules. Since G-CSF is an important hematopoietic cytokine, the results support our hypothesis that the previously observed increases in numbers of hematopoietic progenitors, circulating innate immune cells and platelets, and functional activation of granulocytes, monocytes, and NK cells result from a cytokine cascade induced by 5-AED.

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 glucans 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.

Molecular Specificity of 5-Androstenediol as a Systemic Radioprotectant in Mice

We compared in vivo radioprotective efficacy of 5-androstenediol (5-AED) to that of ten other steroids: 17α-androstenediol, dehydroepiandrosterone, 5-androstenetriol (AET), 4-androstenedione (AND), testosterone, estradiol, fluasterone, 16α-bromoepiandrosterone, 16α-fluoro-androst-5-en-17α-ol (α-fluorohydrin, AFH), and 16α-fluoro-androst-5-en-17β-ol (β-fluorohydrin). Steroids were administered 24 or 48 hr before, or 1 hr after, whole-body γ-irradiation. Two days after irradiation at 3 Gy, blood elements were counted. In addition, after irradiation at 9–12.5 Gy, survival was recorded for 30 days. The results showed radioprotective efficacy was specific for 5-AED. One other steroid, AFH, demonstrated appreciable survival effects but was less efficacious than 5-AED. AND and AET produced slight enhancement of survival in some experiments. This is the first demonstration that the prophylactic window for survival enhancement by 1 subcutaneous (sc) injection of 5-AED is as long as 48 hr in mice. Moreover, the results indicate that 1 sc injection of 5-AED 1 hr after irradiation is much less effective than 1 injection 24–48 hr before irradiation. Comparing the molecular features of steroids with radioprotective efficacy leads to the following conclusions: 1) these effects are due to interaction with specific receptors, since sc injection of extremely similar molecules with the same physicochemical properties as 5-AED were not radioprotective; 2) the17-hydroxyl group is essential; 3) this group must be in the β configuration in the absence of nearby side groups; 4) a halogen atom at 16 changes the 17-hydroxyl specificity to α; 5) the 3β-hydroxyl group is not essential; 6) addition of a 7β-hydroxyl group is deleterious; and 7) the effects are not due to activation of sex steroid receptors.

Immune Response to Prevotella Intermedia in Patients with Recurrent Nonstreptococcal Tonsillitis

The role of three oral flora organisms (Prevotella intermedia, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) was investigated in 31 children with recurrent nonstreptococcal tonsillitis. Antibody titers to the three organisms were measured by enzyme-linked immunosorbent assay in the 31 patients, as well as in 32 control patients who had not suffered from recurrent tonsillitis. None of the individuals in either group suffered from periodontal or dental illness. Significantly higher antibody levels to P intermedia were found in the study group as compared to controls (median 91.0 versus 72.5; p = .02). In contrast, the antibody titers to the other two organisms were generally low (less than 0.30), and no difference was found among the two study groups. The elevated antibody levels to P intermedia, a known oral pathogen that is also isolated from most recurrently inflamed tonsils, suggest a pathogenic role for this organism in recurrent tonsillitis.

Pegfilgrastim Administered in an Abbreviated Schedule, Significantly Improved Neutrophil Recovery after High-Dose Radiation-Induced Myelosuppression in Rhesus Macaques

Conventional daily administration of filgrastim is effective in reducing the duration of severe neutropenia and enhancing survival following lethal radiation, myelosuppressive cytotoxic therapy or myeloablation and stem cell transplantation. A sustained-duration form of filgrastim, pegfilgrastim has significantly simplified scheduling protocols after chemotherapy-induced neutropenia to a single injection while maintaining the therapeutic effectiveness of daily administration of filgrastim. We examined the ability of a single or double (weekly) administration of pegfilgrastim to significantly improve neutrophil recovery in a rhesus macaque model of severe radiation-induced myelosuppression. Animals were exposed to potentially lethal 6 Gy total-body X radiation. After irradiation all animals received supportive care and were administered either pegfilgrastim at 300 μg/kg on day 1 or day 1 and day 7 post exposure, or filgrastim at 10 μg/kg/day initiated on day 1 post exposure and continued daily through neutrophil recovery. Pharmacokinetic parameters and neutrophil-related values for duration of neutropenia, neutrophil nadir, time to recovery to an absolute neutrophil count ≥500/μL or ≥2000/μL, and days of antibiotic support were determined. Effective plasma concentrations of pegfilgrastim were maintained in neutropenic animals until after the onset of hematopoietic recovery, which is consistent with neutrophil-dependent properties of elimination. Administration of pegfilgrastim at day 1 and day 7 was most effective at improving neutrophil recovery compared to daily administration of filgrastim or a single injection of pegfilgrastim on day 1, after severe, radiation-induced myelosuppression in rhesus macaques.

Administration of 5-androstenediol to mice : Pharmacokinetics and cytokine gene expression

The development of an effective pharmacological countermeasure is needed to reduce the morbidity and mortality in military and civilian populations associated with possible exposure to ionizing radiation. Previous studies in mice have shown that a single subcutaneous (sc) injection of the natural steroid androst-5-ene-3beta,17beta-diol (5-androstenediol, 5-AED), 24-48 h prior to a lethal dose of whole-body (60)Co gamma radiation, stimulated hematopoiesis and enhanced survival. These effects are consistent with our previous observation of 5-AED-induced elevations in circulating G-CSF in normal and irradiated mice. The purpose of this study was to obtain data on the pharmacokinetics of 5-AED after sc and buccal administration to mice, and to determine whether cytokine genes are induced by sc 5-AED in hematopoietic tissues (bone marrow, spleen). We studied effects on serum cytokines and chemokines, and also analyzed the pharmacokinetics of 5-AED after sc administration and compared it with buccal delivery. 5-AED was administered 24 h before irradiation or sham-irradiation. Cytokine mRNAs were quantified by quantitative real-time PCR (QRT-PCR), and cytokine levels in serum by multiplex Luminex. 5-AED administration was associated with elevation of message for GM-CSF, IL-2, IL-3, IL-6, and IL-10 in spleen, and GM-CSF and IL-2 in bone marrow. Irradiation enhanced G-CSF, GM-CSF, IFN-gamma, TPO, IL-2, IL-3, IL-6, IL-10, and IL-12 in spleen, and GM-CSF, IFN-gamma, TPO, IL-3, and IL-10 in bone marrow. Serum levels of G-CSF were significantly elevated in 5-AED-treated mice 4 h after irradiation or sham-irradiation. Serum macrophage inflammatory protein-1gamma (MIP-1gamma) was significantly elevated 4 h after irradiation in 5-AED-treated mice. Plasma 5-AED peaked 2 h after sc injection (30 mg/kg), and remained significantly above control after 4 days, but not 8 days. The time course of plasma 5-AED after buccal delivery (60 mg/kg) was similar, but levels were significantly lower compared to sc delivery. Plasma 5-AED 24 h after administration was not significantly different between sc and buccal delivery. However, in contrast to many studies showing enhanced survival after sc administration of 5-AED, we found no effect on survival of buccal 5-AED. The results suggest that radioprotection is not dependent on the 5-AED concentration at the time of irradiation, but rather on events triggered during the first few hours after administration. The current results suggest that further studies are warranted to directly test the roles of cytokines in the radioprotective effects of 5-AED.