Cercopithecine Herpesvirus 9 (Simian Varicella Virus) Infection after Total-Body Irradiation in a Rhesus Macaque (Macaca mulatta).

This case report describes a rhesus macaque (Macaca mulatta; male; age, 5 y; weight, 6.7 kg) with anorexia, dehydration, lethargy, ataxia, and generalized skin rashes that occurred 30 d after total-body irradiation at 6.5 Gy ((60)Co γ-rays). Physical examination revealed pale mucus membranes, a capillary refill time of 4 s, heart rate of 180 bpm. and respirations at 50 breaths per minute. Diffuse multifocal maculopapulovesicular rashes were present on the body, including mucocutaneous junctions. The CBC analysis revealed a Hct of 48%, RBC count of 6.2 × 10(6)/µL, platelet count of 44 × 10(3)/µL, and WBC count of 25 × 10(3)/µL of WBC. The macaque was euthanized in light of a grave prognosis. Gross examination revealed white foci on the liver, multifocal generalized petechiation on serosal and mucosal surfaces of the gastrointestinal tract, hemorrhagic lymph nodes, and hemorrhagic fluid in the thoracic cavity. Microscopic examination revealed cutaneous vesicular lesions with intranuclear eosinophilic viral inclusions within the epithelial cells, consistent with herpesvirus. Immunohistochemistry was positive for herpesvirus. The serum sample was negative for antibodies against Macacine herpesvirus 1 and Cercopithecine herpesvirus 9 (simian varicella virus, SVV). Samples submitted for PCR-based identification of the etiologic agent confirmed the presence of SVV DNA. PCR analysis, immunohistochemistry, and histology confirmed that lesions were attributed to an active SVV infection in this macaque. This case illustrates the importance of screening for SVV in rhesus macaques, especially those used in studies that involve immunosuppressive procedures.

Circulating interleukin-18 as a biomarker of total-body radiation exposure in mice, minipigs, and nonhuman primates (NHP).

We aim to develop a rapid, easy-to-use, inexpensive and accurate radiation dose-assessment assay that tests easily obtained samples (e.g., blood) to triage and track radiological casualties, and to evaluate the radioprotective and therapeutic effects of radiation countermeasures. In the present study, we evaluated the interleukin (IL)-1 family of cytokines, IL-1ß, IL-18 and IL-33, as well as their secondary cytokines' expression and secretion in CD2F1 mouse bone marrow (BM), spleen, thymus and serum in response to γ-radiation from sublethal to lethal doses (5, 7, 8, 9, 10, or 12 Gy) at different time points using the enzyme-linked immune sorbent assay (ELISA), immunoblotting, and cytokine antibody array. Our data identified increases of IL-1ß, IL-18, and/or IL-33 in mouse thymus, spleen and BM cells after total-body irradiation (TBI). However, levels of these cytokines varied in different tissues. Interestingly, IL-18 but not IL-1ß or IL-33 increased significantly (2.5-24 fold) and stably in mouse serum from day 1 after TBI up to 13 days in a radiation dose-dependent manner. We further confirmed our finding in total-body γ-irradiated nonhuman primates (NHPs) and minipigs, and demonstrated that radiation significantly enhanced IL-18 in serum from NHPs 2-4 days post-irradiation and in minipig plasma 1-3 days post-irradiation. Finally, we compared circulating IL-18 with the well known hematological radiation biomarkers lymphocyte and neutrophil counts in blood of mouse, minipigs and NHPs and demonstrated close correlations between these biomarkers in response to radiation. Our results suggest that the elevated levels of circulating IL-18 after radiation proportionally reflect radiation dose and severity of radiation injury and may be used both as a potential biomarker for triage and also to track casualties after radiological accidents as well as for therapeutic radiation exposure.

Preliminary nonclinical toxicity, pharmacokinetics, and pharmacodynamics of ALXN4100TPO, a thrombopoietin receptor agonist, in CD2F1 mice.

ALXN4100TPO, a thrombopoietin (TPO) receptor agonist, increases platelets, abrogates radiation-induced thrombocytopenia and affords significant survival benefit to lethally irradiated mice. This preliminary nonclinical safety study assessed effects of a single subcutaneous (sc) administration of ALXN4100TPO in CD2F1 mice randomized into naïve, control antibody (ALXN4200, 100 mg/kg), low (1 mg/kg), medium (10 mg/kg), or high (100 mg/kg) ALXN4100TPO doses. End points included clinical observations, body weight changes, hematology, histopathology, pharmacokinetics, pharmacodynamics by measuring platelet counts, and endogenous TPO (eTPO) levels. Salient findings were prominent increase in platelet counts and end cells of myeloid and lymphoid lineages; elevated megakaryopoiesis in bone marrow; and extramedullary hematopoiesis in spleen and liver. Serum ALXN4100TPO levels were maximum 24 hours after administration, with a half-life of 13 days. Endogenous TPO levels were elevated in 10 and 100 mg/kg ALXN4100TPO-treated groups. In conclusion, ALXN4100TPO (1-100 mg/kg, sc) treatment in CD2F1 mice resulted in profound pharmacological changes in the hematopoietic tissue; however, no life-threatening adverse events were observed.

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.

Hematopoietic Recovery and Amelioration of Radiation-Induced Lethality by the Vitamin E Isoform δ-Tocotrienol.

δ-Tocotrienol (DT3), a vitamin E isoform, is associated with strong antioxidant and immunomodulatory properties. We confirmed the potent antioxidant activity in membrane systems and showed that DT3 is an effective radiation protector and mitigator. DT3 (4 µM, P < 0.001) inhibited lipid peroxidation in mouse liver microsomes and nitric oxide (NO) formation (20 µM DT3, P < 0.01) in RAW264.7 cells, a murine alveolar macrophage line. In CD2F1 mice exposed to lethal total-body radiation from a (60)Co γ-radiation source, a single subcutaneous (s.c.) injection of DT3 before or after irradiation produced a significant increase in 30-day survival. DT3 was effective from 18.75 to 300 mg/kg (--24 h, P < 0.001). A single dose of 150 or 300 mg/kg DT3 given 24 h before irradiation (radioprotection) resulted in dose reduction factors (DRFs) of 1.19 and 1.27, respectively (P < 0.001). Further, DT3 reduced radiation lethality when administered 2, 6 or 12 h after irradiation, and 150 mg/kg DT3 administered 2 h after exposure conferred a DRF of 1.1 (mitigation). The optimum schedule of 300 mg/kg DT3 24 h prior to 7 Gy significantly reduced pancytopenia compared to irradiated controls (P < 0.05). The large therapeutic potential of and multi-lineage hematopoietic recovery for DT3 warrants further studies.

Delta-tocotrienol protects mouse and human hematopoietic progenitors from gamma-irradiation through extracellular signal-regulated kinase/mammalian target of rapamycin signaling.

BACKGROUND: Exposure to γ-radiation causes rapid hematopoietic cell apoptosis and bone marrow suppression. However, there are no approved radiation countermeasures for the acute radiation syndrome. In this study, we demonstrated that natural δ-tocotrienol, one of the isomers of vitamin E, significantly enhanced survival in total body lethally irradiated mice. We explored the effects and mechanisms of δ-tocotrienol on hematopoietic progenitor cell survival after γ-irradiation in both in vivo and in vitro experiments. DESIGN AND METHODS: CD2F1 mice and human hematopoietic progenitor CD34(+) cells were treated with δ-tocotrienol or vehicle control 24 h before or 6 h after γ-irradiation. Effects of δ-tocotrienol on hematopoietic progenitor cell survival and regeneration were evaluated by clonogenicity studies, flow cytometry, and bone marrow histochemical staining. δ-tocotrienol and γ-irradiation-induced signal regulatory activities were assessed by immunofluorescence staining, immunoblotting and short-interfering RNA assay. RESULTS: δ-tocotrienol displayed significant radioprotective effects. A single injection of δ-tocotrienol protected 100% of CD2F1 mice from total body irradiation-induced death as measured by 30-day post-irradiation survival. δ-tocotrienol increased cell survival, and regeneration of hematopoietic microfoci and lineage(-)/Sca-1(+)/ckit(+) stem and progenitor cells in irradiated mouse bone marrow, and protected human CD34(+) cells from radiation-induced damage. δ-tocotrienol activated extracellular signal-related kinase 1/2 phosphorylation and significantly inhibited formation of DNA-damage marker γ-H2AX foci. In addition, δ-tocotrienol up-regulated mammalian target of rapamycin and phosphorylation of its downstream effector 4EBP-1. These alterations were associated with activation of mRNA translation regulator eIF4E and ribosomal protein S6, which is responsible for cell survival and growth. Inhibition of extracellular signal-related kinase 1/2 expression by short interfering RNA abrogated δ-tocotrienol-induced mammalian target of rapamycin phosphorylation and clonogenicity, and increased γ-H2AX foci formation in irradiated CD34(+) cells. CONCLUSIONS: Our data indicate that δ-tocotrienol protects mouse bone marrow and human CD34(+) cells from radiation-induced damage through extracellular signal-related kinase activation-associated mammalian target of rapamycin survival pathways.

Evaluation of EUK-189, a synthetic superoxide dismutase/catalase mimetic as a radiation countermeasure.

EUK-189, a salen-manganese complex and superoxide dismutase/catalase mimetic, was administered subcutaneously (sc; 30 or 70 mg/kg) to mice at - 24, - 1, +1, or +6 h relative to whole-body cobalt-60 gamma irradiation (LD(90/30) dose), and survival was monitored for 30 days. Cell counts and cytokines in circulation were measured in sublethally irradiated mice treated with EUK-189. EUK-189 (70 mg/kg, - 24 h) enhanced 30-day survival with a dose reduction factor (DRF) of 1.15 (p = 0.047, 95% confidence limits: 1.053, 1.244). LD(50/30)s were 7.96 and 9.13 Gy for saline- and EUK-189-treated groups, respectively. Drug treatment was associated with elevations in numbers of total white blood cells, eosinophils, lymphocytes, and platelets in irradiated mice, compared to vehicle-injected, irradiated controls. EUK-189 did not stimulate production of any cytokine or chemokine tested.

Radioprotection by N-palmitoylated nonapeptide of human interleukin-1beta.

Interleukin-1beta (IL-1beta) is a cytokine involved in homeostatic processes of the immune system and specifically in inflammatory reactions. The nonapeptide of human IL-1beta (VQGEESNDK, position 163-171) has been shown to retain adjuvant and immunostimulatory activities of the native molecule without any inflammatory and pyrogenic properties. A lipophilic derivative of IL-1beta nonapeptide having a palmitoyl residue at the amino terminus was synthesized in order to determine the effects of such structural modification on its bioactivities. The structurally modified peptide derivative, palmitoylated peptide, significantly protected C3H/HeN mice against potentially lethal doses of ionizing radiation. The dose reduction factor was found to be 1.07. Hematological studies show improved recovery of red blood cells and platelets in irradiated and palmitoylated peptide treated mice as compared with the untreated and irradiated group. These results suggest the importance of the derivatization of small peptides of radioprotective, but toxic cytokines in order to enhance radioprotective activity while reducing unwanted toxic side effects.

Oral delivery of spray dried PLGA/amifostine nanoparticles.

Amifostine (Ethyol, WR-2721) is a cytoprotective drug approved by the US Food & Drug Administration for intravenous administration in cancer patients receiving radiation therapy and certain forms of chemotherapy. The primary objective of this project was to develop orally active amifostine nanoparticles using spray drying technique. Two different nanoparticle formulations (Amifostine-PLGA (0.4:1.0 and 1.0:1.0)) were prepared using a Buchi B191 Mini Spray Dryer. A water-in-oil emulsion of amifostine and PLGA (RG 502) was spray dried using an airflow of 600 L h(-1) and input temperature of 55 degrees C. A tissue distribution study in mice was conducted following oral administration of the formulation containing drug-polymer (0.4:1.0). The efficiency of encapsulation was 90% and 100%, respectively, for the two formulations while the median particle sizes were 257 and 240 nm, with 90% confidence between 182 and 417 nm. Since amifostine is metabolized to its active form, WR-1065, by intracellular alkaline phosphatase, the tissue levels of WR-1065 were measured, instead of WR-2721. WR-1065 was detected in significant amounts in all tissues, including bone marrow, jejunum and the kidneys, and there was some degree of selectivity in its distribution in various tissues. This work demonstrates the feasibility of developing an orally effective formulation of amifostine that can be used clinically.

Genistein treatment protects mice from ionizing radiation injury.

The radioprotective and behavioral effects of an acute administration of the isoflavone genistein (4',5,7-trihydroxyflavone) were investigated in adult CD2F1 male mice. Mice were administered a single subcutaneous (s.c.) dose of genistein either 24 h or 1 h before a lethal dose of gamma radiation (9.5-Gy of cobalt-60 at 0.6 Gy min(-1)). Mice received saline, PEG-400 vehicle or genistein at 3.125, 6.25, 12.5, 25, 50, 100, 200, or 400 mg kg(-1) body weight. For mice treated 24 h before irradiation there was a significant increase in 30-day survival for animals receiving genistein doses of 25 to 400 mg kg(-1) (p<0.001). In contrast, the 30-day survival rates of mice treated with genistein 1 h before irradiation were not significantly different from those of the vehicle control group. Additionally, the acute toxicity of genistein was evaluated in non-irradiated male mice administered a single s.c. injection of saline, vehicle, or genistein at 100, 200 or 400 mg kg(-1). At these genistein doses there were no adverse effects, compared with controls, on locomotor activity, grip strength, motor coordination, body weight, testes weight, or histopathology. These results demonstrate that a single s.c. administration of the flavonoid genistein at non-toxic doses provides protection against acute radiation injury.

Determination of WR-1065 and WR-33278 by liquid chromatography with electrochemical detection.

A liquid chromatographic method using electrochemical detection is presented for measuring the thiol WR-1065 12-(3-aminopropylamino) ethanethiol] and its symmetrical disulfide WR-33278 [NH2(CH2)3NHCH2CH2S]2. WR- 1065 is the active, radioprotective drug derived from the phosphorothioate pro-drug WR-2721 (amifostine). External standard curves for both compounds were linear over the range of 40-200 pmol injected (r2 = 0.999 and 0.996 for the thiol and disulfide, respectively). The detection and quantitation limits for WR-1065 were 9 and 18 pmol, respectively, whereas the corresponding values for WR-33278 were 30 and 59 pmol, respectively. Within- and between-day determinations of measurement Vision and accuracy for both compounds validated the suitability of this assay method.

Nutritional approaches to radioprotection: vitamin E.

Low-level radiation injury is dependent on the radiation dose and dose rate. The major military use of any potential radioprotectant is to prevent the short-term effects of lethality and the long-term effects of cancer and other pathologies from radiation exposure that may occur in a nuclear battlefield or in a nuclear material contaminated field of operation. Therefore, a radioprotectant should not affect the ability of military personnel to perform tasks. Because exposure to ionizing radiation induces free radical species, effective antioxidants, either alone or in combination with other agents, can be used as potential radioprotectors. To test this hypothesis, we studied vitamin E for its radioprotective efficacy. Using CD2F1 male mice as the model system, we observed that vitamin E at a dose of 400 IU/kg acts as a good radioprotectant against lethal doses of cobalt-60 radiation. Vitamin E was more efficacious when given subcutaneously than when given orally.

Protection against gamma-irradiation with 5-androstenediol.

We showed previously that treatment of gamma-irradiated female B6D2F1 mice with 5-androstenediol (AED) enhanced survival, stimulated myelopoiesis, and ameliorated radiation-induced decreases in circulating neutrophils and platelets. We have now tested survival in male CD2F1 mice, and we have investigated molecular and functional effects on neutrophils and bone marrow stromal cells and screened for toxicity in female B6D2F1 mice. AED (160 mg/kg, subcutaneously, 24 hours before irradiation) enhanced survival in male CD2F1 mice with a dose-reduction factor of 1.23, similar to the dose-reduction factor of 1.26 found previously for female B6D2F1 mice. Expression of CD11b, a developmental marker, was reduced on circulating neutrophils after either in vivo AED administration or whole-body gamma-irradiation (3 Gy), but neutrophil peroxidase activity was unchanged. Stromal cell progenitors (fibroblastoid colony-forming units) were reduced in marrow 5 days after AED injection in nonirradiated mice. Clinical chemistry, histopathology, and behavioral assays showed no evidence of toxicity. We conclude that AED and related steroids are attractive candidates to explore as countermeasures to high- and low-level ionizing radiation.

New strategies for the prevention of radiation injury: possible implications for countering radiation hazards of long-term space travel.

New strategies for the prevention of radiation injuries are currently being explored with the ultimate aim of developing globally radioprotective, nontoxic pharmacologics. The prophylactic treatments under review encompass such diverse pharmacologic classes as novel immunomodulators, nutritional antioxidants, and cytokines. An immunomodulator that shows promise is 5-androstenediol (AED), a well-tolerated, long-acting androstene steroid with broad-spectrum radioprotective attributes that include not only protection against acute tissue injury, but also reduced susceptibility to infectious agents, as well as reduced rates of neoplastic transformation. Other potentially useful radioprotectants currently under study include the nutraceutical vitamin E and analogs, a chemically-engineered cytokine, interleukin-1beta, and a sustained-release formulation of an aminothiol, amifostine. Results suggest that a new paradigm is evolving for the prophylaxes of radiation injuries, based on use of newly identified, nontoxic, broad-spectrum prophylactic agents whose protective action may be leveraged by subsequent postexposure use of cytokines with organ-specific reparative functions.