CBLB613: a TLR 2/6 agonist, natural lipopeptide of Mycoplasma arginini , as a novel radiation countermeasure

To date, there are no safe and effective drugs available for protection against ionizing radiation damage. Therefore, a great need exists to identify and develop non-toxic agents that will be useful as radioprotectors or postirradiation therapies under a variety of operational scenarios. We have developed a new pharmacological agent, CBLB613 (a naturally occurring Mycoplasma-derived lipopeptide ligand for Toll-like receptor 2/6), as a novel radiation countermeasure. Using CD2F1 mice, we investigated CBLB613 for toxicity, immunogenicity, radioprotection, radiomitigation and pharmacokinetics. We also evaluated CBLB613 for its effects on cytokine induction and radiation-induced cytopenia in unirradiated and irradiated mice. The no-observable-adverse-effect level of CBLB613 was 1.79 mg/kg and 1 mg/kg for single and repeated doses, respectively. CBLB613 significantly protected mice against a lethal dose of (60)Co γ radiation. The dose reduction factor of CBLB613 as a radioprotector was 1.25. CBLB613 also mitigated the effects of (60)Co γ radiation on survival in mice. In both irradiated and unirradiated mice, the drug stimulated induction of interleukin-1β (IL-1β), IL-6, IL-10, IL-12, keratinocyte-derived chemokine, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-1α. CBLB613 also reduced radiation-induced cytopenia and increased bone marrow cellularity in irradiated mice. Our immunogenicity study demonstrated that CBLB613 is not immunogenic in mice, indicating that it could be developed as a radioprotector and radiomitigator for humans against the potentially lethal effects of radiation exposure.     

Inhibitory effect of memantine, an NMDA-receptor antagonist, on electroporation-induced inward currents in pituitary GH3 cells

Experimental and clinical data support the notion that peroxisome proliferator-activated receptor γ (PPARγ) activation is associated with anti-atherosclerosis as well as anti-diabetic effect. Telmisartan, an angiotensin receptor blocker (ARB), acts as a partial PPARγ agonist. We hypothesized that telmisartan protects against diabetic vascular complications, through PPARγ activation. We compared the effects of telmisartan, telmisartan combined with GW9662 (a PPARγ antagonist), and losartan with no PPARγ activity on vascular injury in obese type 2 diabetic db/db mice. Compared to losartan, telmisartan significantly ameliorated vascular endothelial dysfunction, downregulation of phospho-eNOS, and coronary arterial remodeling in db/db mice. More vascular protective effects of telmisartan than losartan were associated with greater anti-inflammatory effects of telmisartan, as shown by attenuation of vascular nuclear factor kappa B (NFκB) activation and tumor necrosis factor α. Coadministration of GW9662 with telmisartan abolished the above mentioned greater protective effects of telmisartan against vascular injury than losartan in db/db mice. Thus, PPARγ activity appears to be involved in the vascular protective effects of telmisartan in db/db mice. Moreover, telmisartan, but not losartan, prevented the downregulation of vascular PPARγ in db/db mice and this effect of telmisartan was cancelled by the coadministration of GW9662. Our data provided the first evidence indicating that PPARγ activity of telmisartan contributed to the protective effects of telmisartan against diabetic vascular complication. PPARγ activity of telmisartan was involved in the normalization of vascular PPARγ downregulation in diabetic mice. Thus, telmisartan seems to exert vascular protective effects in hypertensive patients with diabetes.     

Myeloid progenitors: a radiation countermeasure that is effective when initiated days after irradiation

The aim of this study was to elucidate the potential of mouse myeloid progenitor cells (mMPC) to mitigate lethal doses of (60)Co γ radiation and X rays in various strains of mice. Different cell doses of pooled allogeneic mMPC generated ex vivo from AKR, C57Bl/6, and FVB mice were transfused intravenously into haplotype-mismatched recipient Balb/c or CD2F1 mice at various times after irradiation to assess their effect on 30-day survival. Our results show that cryopreserved allogeneic mMPC significantly improve survival in both strains of mice irradiated with lethal doses of (60)Co γ radiation (CD2F1, 9.2 Gy) and X-ray exposures (Balb/c, 9 Gy) that are known to cause acute radiation syndrome in hematopoietic tissues. Survival benefit was mMPC-dose dependent and significant even when mMPC administration was delayed up to 7 days after irradiation. We further show that mMPC administration mitigates death from acute radiation syndrome at radiation doses of up to 15 Gy ((60)Co γ radiation, CD2F1), which are radiation exposure levels that cause mice to succumb to multi-organ failure, and determined that the dose-reduction factor of 5 million mMPC administered 24 h after irradiation of CD2F1 mice is 1.73. Even at high doses of up to 14 Gy (60)Co γ radiation, mMPC administration could be delayed up to 5 days in CD2F1 mice and still provide significant benefit to 30-day survival. These results demonstrate that mMPC are a promising radiation countermeasure with the potential to mitigate radiation injury in unmatched recipients across a broad range of lethal radiation doses, even when administration is delayed days after radiation exposure. With respect to efficacy, timing, and practicality of administration, mMPC appear to be a very promising radiation countermeasure for acute radiation syndrome among all candidate therapeutics currently under development.     

Relative biological effectiveness of 12C and 28Si radiation in C57BL/6J mice

Study of heavy ion radiation-induced effects on mice could provide insight into the human health risks of space radiation exposure. The purpose of the present study is to assess the relative biological effectiveness (RBE) of (12)C and (28)Si ion radiation, which has not been reported previously in the literature. Female C57BL/6J mice (n = 15) were irradiated using 4-8 Gy of (28)Si (300 MeV/nucleon energy; LET 70 keV/μm) and 5-8 Gy of (12)C (290 MeV/nucleon energy; LET 13 keV/μm) ions. Post-exposure, mice were monitored regularly, and their survival observed for 30 days. The LD(50/30) dose (the dose at which 50 % lethality occurred by 30-day post-exposure) was calculated from the survival curve and was used to determine the RBE of (28)Si and (12)C in relation to γ radiation. The LD(50/30) for (28)Si and (12)C ion is 5.17 and 7.34 Gy, respectively, and the RBE in relation to γ radiation (LD(50/30)-7.25 Gy) is 1.4 for (28)Si and 0.99 for (12)C. Determination of RBE of (28)Si and (12)C for survival in mice is not only important for space radiation risk estimate studies, but it also has implications for HZE radiation in cancer therapy.     

A TPO receptor agonist, ALXN4100TPO, mitigates radiation-induced lethality and stimulates hematopoiesis in CD2F1 mice

Thrombopoietin (TPO) receptor agonists lacking sequence homology to TPO were designed by grafting a known peptide sequence into the hinge and/or kappa constant regions of a human anti-anthrax antibody. Some of these proteins were equipotent to TPO in stimulating cMpl-r activity in vitro and in increasing platelet levels in vivo. ALXN4100TPO (4100TPO), the best agonist in this series with a K(d) of 30 nM for cMpl-r, exhibited potent activity as a radiation countermeasure in CD2F1 mice exposed to lethal total-body radiation from a cobalt-60 γ-ray source. 4100TPO (2 mg/kg, s.c.) administered once either 24 h before or 6 h after TBI showed superior protection to five daily doses given before or after TBI. Prophylactic administration (69 to 94% survival) was superior to therapeutic schedules (60% survival). 4100TPO conferred a significant survival benefit (P < 0.01) when administered 4 days before or even 12 h after exposure and across a dose range of 0.1 to 8 mg/kg. The dose reduction factors (DRFs) with a single dose of 1 mg/kg 4100TPO 24 h before or 12 h after TBI were 1.32 and 1.11, respectively (P < 0.0001). Furthermore, 4100TPO increased bone marrow cellularity and megakaryocytic development and accelerated multi-lineage hematopoietic recovery in irradiated mice, demonstrating the potential of 4100TPO as both a protector and a mitigator in the event of a radiological incident.     

Trehalose dimycolate enhances survival of fission neutron-irradiated mice and Klebsiella pneumoniae-challenged irradiated mice

The survival of B6D2F1 female mice exposed to lethal doses of fission neutron radiation is increased when trehalose dimycolate (TDM) preparations are given either 1 h after exposure or 1 day before exposure to radiation. TDM in an emulsion of squalene, Tween 80, and saline was the most effective formulation for increasing the 30-day survival of mice when given 1 day before (90%) or 1 h after (88%) exposure to radiation. An aqueous suspension of a synthetic analog of TDM was less effective at increasing 30-day survival (60%) when given 1 day prior to radiation exposure and not effective when given 1 h after radiation. Mice receiving a sublethal dose (3.5 Gy) of fission neutron radiation and either the TDM emulsion or synthetic TDM 1 h after irradiation were substantially more resistant to challenge with 10, 100, 1000, or 5000 times the LD50/30 dose of Klebsiella pneumoniae than untreated mice.     

Expression of genes involved in mouse lung cell differentiation/regulation after acute exposure to photons and protons with or without low-dose preirradiation

The goal of this study was to compare the effects of acute 2 Gy irradiation with photons (0.8 Gy/min) or protons (0.9 Gy/min), both with and without pre-exposure to low-dose/low-dose-rate γ rays (0.01 Gy at 0.03 cGy/h), on 84 genes involved in stem cell differentiation or regulation in mouse lungs on days 21 and 56. Genes with a ≥1.5-fold difference in expression and P < 0.05 compared to 0 Gy controls are emphasized. Two proteins specific for lung stem cells/progenitors responsible for local tissue repair were also compared. Overall, striking differences were present between protons and photons in modulating the genes. More genes were affected by protons than by photons (22 compared to 2 and 6 compared to 2 on day 21 and day 56, respectively) compared to 0 Gy. Preirradiation with low-dose-rate γ rays enhanced the acute photon-induced gene modulation on day 21 (11 compared to 2), and all 11 genes were significantly downregulated on day 56. On day 21, seven genes (aldh2, bmp2, cdc2a, col1a1, dll1, foxa2 and notch1) were upregulated in response to most of the radiation regimens. Immunoreactivity of Clara cell secretory protein was enhanced by all radiation regimens. The number of alveolar type 2 cells positive for prosurfactant protein C in irradiated groups was higher on day 56 (12.4-14.6 cells/100) than on day 21 (8.5-11.2 cells/100) (P < 0.05). Taken together, these results showed that acute photons and protons induced different gene expression profiles in the lungs and that pre-exposure to low-dose-rate γ rays sometimes had modulatory effects. In addition, proteins associated with lung-specific stem cells/progenitors were highly sensitive to radiation.     

Effective dose of A-bomb radiation in Hiroshima and Nagasaki as assessed by chromosomal effectiveness of spectrum energy photons and neutrons

The effective dose of combined spectrum energy neutrons and high energy spectrum γ-rays in A-bomb survivors in Hiroshima and Nagasaki has long been a matter of discussion. The reason is largely due to the paucity of biological data for high energy photons, particularly for those with an energy of tens of MeV. To circumvent this problem, a mathematical formalism was developed for the photon energy dependency of chromosomal effectiveness by reviewing a large number of data sets published in the literature on dicentric chromosome formation in human lymphocytes. The chromosomal effectiveness was expressed by a simple multiparametric function of photon energy, which made it possible to estimate the effective dose of spectrum energy photons and differential evaluation in the field of mixed neutron and γ-ray exposure with an internal reference radiation. The effective dose of reactor-produced spectrum energy neutrons was insensitive to the fine structure of the energy distribution and was accessible by a generalized formula applicable to the A-bomb neutrons. Energy spectra of all sources of A-bomb γ-rays at different tissue depths were simulated by a Monte Carlo calculation applied on an ICRU sphere. Using kerma-weighted chromosomal effectiveness of A-bomb spectrum energy photons, the effective dose of A-bomb neutrons was determined, where the relative biological effectiveness (RBE) of neutrons was expressed by a dose-dependent variable RBE, RBE(γ, D _n), against A-bomb γ-rays as an internal reference radiation. When the newly estimated variable RBE(γ, D _n) was applied to the chromosome data of A-bomb survivors in Hiroshima and Nagasaki, the city difference was completely eliminated. The revised effective dose was about 35% larger in Hiroshima, 19% larger in Nagasaki and 26% larger for the combined cohort compared with that based on a constant RBE of 10. Since the differences are significantly large, the proposed effective dose might have an impact on the magnitude of the risk estimates deduced from the A-bomb survivor cohort.     

The survival of mice and the metallothionein content of their livers and kidneys as criteria for assessing exposure to pulsed neutron radiation

The effect of pulsed neutron radiation was studied in comparison with continuous neutron radiation and continuous gamma-radiation. Animal survival and induction of metallothionein (MT) synthesis in liver and kidney of mice exposed to equivalent doses were chosen as criteria for evaluation of radiation effects. It was found that the level of MT in liver and kidney of mice exposed to neutron radiation decreased 24 hours after irradiation and then continued decreasing in kidney for 48 hours after irradiation. This is evidence of more intensive free-radical processes initiated by pulsed neutron radiation. At the same time, RBE values of pulsed neutrons did not differ significantly from that of continuous neutron radiation.     

Effect of Pseudomonas contamination or antibiotic decontamination of the GI tract on acute radiation lethality after neutron or gamma irradiation

The influence of antibiotic decontamination of Pseudomonas contamination of the GI tract prior to whole-body neutron or gamma irradiation was studied. It was observed that for fission neutron doses greater than 5.5 Gy, cyclotron-produced neutron doses greater than 6.7 Gy, and 137Cs gamma-ray doses greater than 14.4 Gy, the median survival time of untreated rats was relatively constant at 4.2 to 4.5 days, indicating death was due to intestinal injury. Within the dose range of 3.5 to 5.5 Gy of fission neutrons, 4.9 to 6.7 Gy of cyclotron-produced neutrons, and 9.6 to 14.4 Gy of gamma rays, median survival time of these animals was inversely related to dose and varied from 12 to 4.6 days. This change in survival time with dose reflects a transition in the mechanisms of acute radiation death from pure hematopoietic, to a combination of intestinal and hematopoietic, to pure intestinal death. Decontamination of the GI tract with antibiotics prior to irradiation increased median survival time 1 to 5 days in this transitional dose range. Contamination of the intestinal flora with Pseudomonas aeruginosa prior to irradiation reduced median survival time 1 to 5 days in the same radiation dose range. Pseudomonas-contaminated animals irradiated within this transitional dose range had maximum concentrations of total bacteria and Pseudomonas in their livers at the time of death. However, liver bacteria concentration was usually higher in gamma-irradiated animals, due to a smaller contribution of hematopoietic injury in neutron-irradiated animals. The effects of both decontamination of the GI tract and Pseudomonas contamination of the GI tract were negligible in the range of doses in which median survival time was dose independent, i.e., in the pure "intestinal death" dose range. Finally, despite the marked changes in survival time produced by decontamination or Pseudomonas contamination in the "transitional dose range," these treatments had little effect on ultimate survival after irradiation as measured by the LD50/5 day and the LD50/30 day end points. The implications of these results with respect to treatment of acute radiation injury after whole-body irradiation are discussed.     

HemaMax™, a recombinant human interleukin-12, is a potent mitigator of acute radiation injury in mice and non-human primates

HemaMax, a recombinant human interleukin-12 (IL-12), is under development to address an unmet medical need for effective treatments against acute radiation syndrome due to radiological terrorism or accident when administered at least 24 hours after radiation exposure. This study investigated pharmacokinetics, pharmacodynamics, and efficacy of m-HemaMax (recombinant murine IL-12), and HemaMax to increase survival after total body irradiation (TBI) in mice and rhesus monkeys, respectively, with no supportive care. In mice, m-HemaMax at an optimal 20 ng/mouse dose significantly increased percent survival and survival time when administered 24 hours after TBI between 8-9 Gy (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by increases in plasma interferon-γ (IFN-γ) and erythropoietin levels, recovery of femoral bone hematopoiesis characterized with the presence of IL-12 receptor β2 subunit-expressing myeloid progenitors, megakaryocytes, and osteoblasts. Mitigation of jejunal radiation damage was also examined. At allometrically equivalent doses, HemaMax showed similar pharmacokinetics in rhesus monkeys compared to m-HemaMax in mice, but more robustly increased plasma IFN-γ levels. HemaMax also increased plasma erythropoietin, IL-15, IL-18, and neopterin levels. At non-human primate doses pharmacologically equivalent to murine doses, HemaMax (100 ng/Kg and 250 ng/Kg) administered at 24 hours after TBI (6.7 Gy/LD(50/30)) significantly increased percent survival of HemaMax groups compared to vehicle (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by a significantly higher leukocyte (neutrophils and lymphocytes), thrombocyte, and reticulocyte counts during nadir (days 12-14) and significantly less weight loss at day 12 compared to vehicle. These findings indicate successful interspecies dose conversion and provide proof of concept that HemaMax increases survival in irradiated rhesus monkeys by promoting hematopoiesis and recovery of immune functions and possibly gastrointestinal functions, likely through a network of interactions involving dendritic cells, osteoblasts, and soluble factors such as IL-12, IFN-γ, and cytoprotectant erythropoietin.     

Administration of recombinant human IL11 after supralethal radiation exposure promotes survival in mice: interactive effect with thrombopoietin

In the present study, we evaluated the therapeutic potential of recombinant human IL11 in lethally irradiated C57BL6/J mice exposed to gamma rays. IL11 administered for 5 consecutive days beginning 2 h after total-body irradiation with 8 or 9 Gy 60Co gamma rays resulted in a significant increase in 30-day survival. When IL11 was administered, only a slight improvement in the hematopoietic status (both blood cell counts and progenitor cells) was observed after an 8-Gy exposure, and no improvement in hematopoietic reconstitution was observed after 9 Gy total-body irradiation. The enhancement of fibrinogen in the plasma of irradiated animals suggested the importance of infections in the death of animals. IL11 was able to limit the increase in fibrinogen levels. However, prevention of bacterial infections by antibiotic treatment, although it delayed death, was ineffective in promoting survival either in placebo-treated and IL11-treated mice. IL11 was administered along with thrombopoietin (TPO) or bone marrow transplantation to limit the hematopoietic syndrome, in addition to antibiotic treatment. When IL11 was combined with TPO, a potent stimulator of hematopoiesis, the survival of animals which had been irradiated with 10 Gy 137Cs gamma rays was increased significantly compared to those treated with IL11 or TPO alone. Furthermore, an interactive effect of TPO and IL11 on hematopoietic reconstitution was observed. Similarly, IL11 in combination with bone marrow transplantation enhanced survival after 15 Gy 137Cs gamma rays. These data suggest that the effect of IL11 on the hematopoietic system is only moderate when it is used alone in supralethally irradiated mice but that the effect is improved in the presence of a hematopoietic growth factor or bone marrow transplantation.     

Antagonist of peroxisome proliferator-activated receptor γ induces cerebellar amyloid-β levels and motor dysfunction in APP/PS1 transgenic mice

Recent evidences show that peroxisome proliferator-activated receptor γ (PPARγ) is involved in the modulation of the amyloid-β (Aβ) cascade causing Alzheimer’s disease (AD) and treatment with PPARγ agonists protects against AD pathology. However, the function of PPARγ steady-state activity in Aβ cascade and AD pathology remains unclear. In this study, an antagonist of PPARγ, GW9662, was injected into the fourth ventricle of APP/PS1 transgenic mice to inhibit PPARγ activity in cerebellum. The results show that inhibition of PPARγ significantly induced Aβ levels in cerebellum and caused cerebellar motor dysfunction in APP/PS1 transgenic mice. Moreover, GW9662 treatment markedly decreased the cerebellar levels of insulin-degrading enzyme (IDE), which is responsible for the cellular degradation of Aβ. Since cerebellum is spared from significant Aβ accumulation and neurotoxicity in AD patients and animal models, these findings suggest a crucial role of PPARγ steady-state activity in protection of cerebellum against AD pathology.     

Thrombopoietin regulates Bcl-xL gene expression through Stat5 and phosphatidylinositol 3-kinase activation pathways.

Thrombopoietin (TPO), an essential factor for megakaryopoiesis and thrombopoiesis, works as a survival factor for megakaryocytic lineage cells. However, little is known about the molecular mechanism in detail. We show here that TPO supports the survival of TPO-dependent leukemia cell line UT-7/TPO and normal megakaryocytic progenitors via the induction of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. We further analyzed the signal transduction pathways required for TPO-induced Bcl-xL gene expression. A reporter assay with various lengths of Bcl-x gene promoter revealed that both Stat- and nuclear factor kappa B-binding sites are prerequisites for TPO-induced promoter activity. Consistent with these results, TPO induced the binding of Stat5 and subunits of nuclear factor kappa B, p50, and c-Rel to the Bcl-x gene promoter. AG490, a specific inhibitor for Jak2, and LY294002, a specific inhibitor for phosphatidylinositol (PI) 3-kinase, reduced the protein level of Bcl-xL in UT-7/TPO cells, accompanied by an increase in the ratio of apoptotic cells. Interestingly, LY294002 enhanced the TPO-induced DNA binding activity of Stat5 without affecting the Jak2 activation and tyrosine phosphorylation of Stat5. Concomitantly, confocal microscopy revealed that LY294002 clearly inhibited the nuclear export of Stat5, suggesting that PI 3-kinase regulates the subcellular localization of Stat5. Taken together, our results suggest that both Jak-Stat and PI 3-kinase activation pathways regulate the TPO-induced survival of megakaryocytic cells via Bcl-xL gene expression. In addition, our data suggest possible cross-talk between these two signaling pathways.     

Podophyllotoxin and rutin in combination prevents oxidative stress mediated cell death and advances revival of mice gastrointestine following lethal radiation injury

Intestinal injury is inevitable during exposure to high radiation doses and is a common side effect observed during abdominal/pelvic radiotherapy. Yet, no radiation countermeasures are available for gastrointestine (GI) injury management. The aim of this study is to determine the effects of podophyllotoxin and rutin in combination (G-003M) on ionising radiation induced GI injury. We prophylactically administered G-003M to C57BL/6J mice exposed to 9?Gy total body radiation (TBI) and assessed for morphological changes, loss in absorption, fluid retention, biochemical alterations, immunohistochemical analysis to study cPARP, caspase-3, PCNA expression, and TUNEL staining. The irradiated intestine demonstrated extensive loss in crypts and villi, disrupted mucosal lining with reduced xylose uptake and enhanced fluid level post 7-day radiation. Mice receiving G-003M before radiation showed significant protection to intestinal epithelium, better allocation of secretory goblet cells, recovery in absorption, and reduced intestinal oedema. Additionally, G-003M administration also prevented radiation induced ROS generation, lipid peroxidation (MDA levels) and maintained the intestinal glutathione pool compared to the irradiated animals. G-003M supplementation also resulted in restoration of intestinal mitochondrial membrane potential, which was otherwise depolarised by radiation treatment. Immunohistochemical analysis demonstrated decrease in c-PARP and caspase-3 expression in jejuna cross sections and upregulation of PCNA in G-003M treated crypt cells as compared to 9?Gy irradiated mice. Our findings show that G-003M augment survival of mice against lethal radiation by promoting structural and functional regeneration in intestinal tissue. This combination therefore can be effectively explored for preventing radiation induced GI toxicity.     

Sperm shape abnormalities in mice exposed to californium-252 radiation

Male mice of the B6C3F1 hybrid strain were whole-body irradiated with different doses of 252Cf/60Co. They were killed 35 days later and spermatozoa from cauda epididymides were stained with eosin-Y. The air-dried smears were examined under light microscope for sperm shape abnormalities. There was an increase in the frequency of abnormal sperm in all the treated groups compared to controls. The RBE for the mixed neutron and gamma radiation of 252Cf was 2.6. The RBE for the neutron component was 3.4. The increased frequency of abnormal sperm was associated with a concomitant decrease in testis weight in the irradiated animals.     

Evidence for similarities between radiation damage expressed by beta-araA and damage involved in the interaction effect observed after exposure of V79 cells to mixed neutrons and gamma radiation

Plateau-phase V79 cells were exposed sequentially to fast neutrons and gamma rays. A dose-dependent reduction in the shoulder width of the gamma-ray survival curve was observed after preexposure of cells to neutrons. A similar effect was demonstrated on the neutron survival curve when cells were preirradiated with gamma rays. Treatment of cells with 150 microM beta-araA after either gamma or neutron irradiation reduced primarily the shoulder of the survival curve. When beta-araA was given to the cells after exposure to mixed radiation modalities, survival curves similar to those observed after exposure to a single radiation modality and treatment with beta-araA were obtained. The kinetics of loss of the interaction observed after exposure of cells to gamma rays following neutron irradiation was similar to the kinetics of loss of sensitivity to beta-araA (T1/2 = 1 h) measured by delaying drug administration after exposure to gamma rays. The results suggest that the PLD expressed by beta-araA is at least partly involved in the interactive effect observed after combined exposure of plateau-phase V79 cells to neutrons and gamma rays.     

Metallothionein induction as a potent means of radiation protection in mice

A striking resistance to lethal damage from a single dose of 6-8 Gy of X rays has been found in mice which had received various pretreatments to induce metallothionein (MT) synthesis in the liver prior to irradiation. Mice were injected with manganese (10 mg Mn/kg) or cadmium (3 mg Cd/kg) salt subcutaneously, or a patch of dorsal skin (2 X 2 cm2) was excised 1 or 2 days prior to irradiation. The increased tolerance of these mice to radiation was established by a marked decrease of mortality rate, an increase of mean survival time, a reduction of weight loss, and a smaller decrease in the number of leukocytes as compared with the control group. The LD50/30 for control mice was 6.3 Gy, while the corresponding values for the groups pretreated with Mn, Cd, and skin excision were 7.5, 7.7, and 7.9 Gy, respectively. The normal level of MT in mouse liver was approximately 25 micrograms/g tissue. This level increased 2.5- to 3-fold 24 h after 6.3 Gy irradiation. The MT levels of mice pretreated with Cd, Mn, and skin excision were increased 8-, 5-, and 7-fold, respectively, prior to irradiation as compared with the preirradiation control. These results indicate that the induction of MT in mouse liver is a significant factor in the mechanism of protection against radiation.     

Prevention and mitigation of acute radiation syndrome in mice by synthetic lipopeptide agonists of Toll-like receptor 2 (TLR2)

Bacterial lipoproteins (BLP) induce innate immune responses in mammals by activating heterodimeric receptor complexes containing Toll-like receptor 2 (TLR2). TLR2 signaling results in nuclear factor-kappaB (NF-κB)-dependent upregulation of anti-apoptotic factors, anti-oxidants and cytokines, all of which have been implicated in radiation protection. Here we demonstrate that synthetic lipopeptides (sLP) that mimic the structure of naturally occurring mycoplasmal BLP significantly increase mouse survival following lethal total body irradiation (TBI) when administered between 48 hours before and 24 hours after irradiation. The TBI dose ranges against which sLP are effective indicate that sLP primarily impact the hematopoietic (HP) component of acute radiation syndrome. Indeed, sLP treatment accelerated recovery of bone marrow (BM) and spleen cellularity and ameliorated thrombocytopenia of irradiated mice. sLP did not improve survival of irradiated TLR2-knockout mice, confirming that sLP-mediated radioprotection requires TLR2. However, sLP was radioprotective in chimeric mice containing TLR2-null BM on a wild type background, indicating that radioprotection of the HP system by sLP is, at least in part, indirect and initiated in non-BM cells. sLP injection resulted in strong transient induction of multiple cytokines with known roles in hematopoiesis, including granulocyte colony-stimulating factor (G-CSF), keratinocyte chemoattractant (KC) and interleukin-6 (IL-6). sLP-induced cytokines, particularly G-CSF, are likely mediators of the radioprotective/mitigative activity of sLP. This study illustrates the strong potential of LP-based TLR2 agonists for anti-radiation prophylaxis and therapy in defense and medical scenarios.