Combined immunomodulator and antimicrobial therapy eliminates polymicrobial sepsis and modulates cytokine production in combined injured mice.

PURPOSE: A combination therapy for combined injury (CI) using a non-specific immunomodulator, synthetic trehalose dicorynomycolate and monophosphoryl lipid A (STDCM-MPL), was evaluated to augment oral antimicrobial agents, levofloxacin (LVX) and amoxicillin (AMX), to eliminate endogenous sepsis and modulate cytokine production. MATERIALS AND METHODS: Female B6D2F(1)/J mice received 9.75 Gy cobalt-60 gamma-radiation and wound. Bacteria were isolated and identified in three tissues. Incidence of bacteria and cytokines were compared between treatment groups. RESULTS: Results demonstrated that the lethal dose for 50% at 30 days (LD(50/30)) of B6D2F(1)/J mice was 9.42 Gy. Antimicrobial therapy increased survival in radiation-injured (RI) mice. Combination therapy increased survival after RI and extended survival time but did not increase survival after CI. Sepsis began five days earlier in CI mice than RI mice with Gram-negative species predominating early and Gram-positive species increasing later. LVX plus AMX eliminated sepsis in CI and RI mice. STDCM-MPL eliminated Gram-positive bacteria in CI and most RI mice but not Gram-negative. Treatments significantly modulated 12 cytokines tested, which pertain to wound healing or elimination of infection. CONCLUSIONS: Combination therapy eliminates infection and prolongs survival time but does not assure CI mouse survival, suggesting that additional treatment for proliferative-cell recovery is required.

Application of multivariate modeling for radiation injury assessment: a proof of concept.

Multivariate radiation injury estimation algorithms were formulated for estimating severe hematopoietic acute radiation syndrome (H-ARS) injury (i.e., response category three or RC3) in a rhesus monkey total-body irradiation (TBI) model. Classical CBC and serum chemistry blood parameters were examined prior to irradiation (d 0) and on d 7, 10, 14, 21, and 25 after irradiation involving 24 nonhuman primates (NHP) (Macaca mulatta) given 6.5-Gy (60)Co Υ-rays (0.4 Gy min(-1)) TBI. A correlation matrix was formulated with the RC3 severity level designated as the "dependent variable" and independent variables down selected based on their radioresponsiveness and relatively low multicollinearity using stepwise-linear regression analyses. Final candidate independent variables included CBC counts (absolute number of neutrophils, lymphocytes, and platelets) in formulating the "CBC" RC3 estimation algorithm. Additionally, the formulation of a diagnostic CBC and serum chemistry "CBC-SCHEM" RC3 algorithm expanded upon the CBC algorithm model with the addition of hematocrit and the serum enzyme levels of aspartate aminotransferase, creatine kinase, and lactate dehydrogenase. Both algorithms estimated RC3 with over 90% predictive power. Only the CBC-SCHEM RC3 algorithm, however, met the critical three assumptions of linear least squares demonstrating slightly greater precision for radiation injury estimation, but with significantly decreased prediction error indicating increased statistical robustness.

Skin injuries reduce survival and modulate corticosterone, C-reactive protein, complement component 3, IgM, and prostaglandin E 2 after whole-body reactor-produced mixed field (n + γ-photons) irradiation.

Skin injuries such as wounds or burns following whole-body γ-irradiation (radiation combined injury (RCI)) increase mortality more than whole-body γ-irradiation alone. Wound-induced decreases in survival after irradiation are triggered by sustained activation of inducible nitric oxide synthase pathways, persistent alteration of cytokine homeostasis, and increased susceptibility to systemic bacterial infection. Among these factors, radiation-induced increases in interleukin-6 (IL-6) concentrations in serum were amplified by skin wound trauma. Herein, the IL-6-induced stress proteins including C-reactive protein (CRP), complement 3 (C3), immunoglobulin M (IgM), and prostaglandin E2 (PGE2) were evaluated after skin injuries given following a mixed radiation environment that might be found after a nuclear incident. In this report, mice received 3 Gy of reactor-produced mixed field (n + γ-photons) radiations at 0.38 Gy/min followed by nonlethal skin wounding or burning. Both wounds and burns reduced survival and increased CRP, C3, and PGE2 in serum after radiation. Decreased IgM production along with an early rise in corticosterone followed by a subsequent decrease was noted for each RCI situation. These results suggest that RCI-induced alterations of corticosterone, CRP, C3, IgM, and PGE2 cause homeostatic imbalance and may contribute to reduced survival. Agents inhibiting these responses may prove to be therapeutic for RCI and improve related survival.

Wound trauma alters ionizing radiation dose assessment.

BACKGROUND: Wounding following whole-body γ-irradiation (radiation combined injury, RCI) increases mortality. Wounding-induced increases in radiation mortality are triggered by sustained activation of inducible nitric oxide synthase pathways, persistent alteration of cytokine homeostasis, and increased susceptibility to bacterial infection. Among these factors, cytokines along with other biomarkers have been adopted for biodosimetric evaluation and assessment of radiation dose and injury. Therefore, wounding could complicate biodosimetric assessments. RESULTS: In this report, such confounding effects were addressed. Mice were given 60Co γ-photon radiation followed by skin wounding. Wound trauma exacerbated radiation-induced mortality, body-weight loss, and wound healing. Analyses of DNA damage in bone-marrow cells and peripheral blood mononuclear cells (PBMCs), changes in hematology and cytokine profiles, and fundamental clinical signs were evaluated. Early biomarkers (1 d after RCI) vs. irradiation alone included significant decreases in survivin expression in bone marrow cells, enhanced increases in γ-H2AX formation in Lin+ bone marrow cells, enhanced increases in IL-1ß, IL-6, IL-8, and G-CSF concentrations in blood, and concomitant decreases in γ-H2AX formation in PBMCs and decreases in numbers of splenocytes, lymphocytes, and neutrophils. Intermediate biomarkers (7 - 10 d after RCI) included continuously decreased γ-H2AX formation in PBMC and enhanced increases in IL-1ß, IL-6, IL-8, and G-CSF concentrations in blood. The clinical signs evaluated after RCI were increased water consumption, decreased body weight, and decreased wound healing rate and survival rate. Late clinical signs (30 d after RCI) included poor survival and wound healing. CONCLUSION: Results suggest that confounding factors such as wounding alters ionizing radiation dose assessment and agents inhibiting these responses may prove therapeutic for radiation combined injury and reduce related mortality.

Efficacy of radiation countermeasures depends on radiation quality.

The detonation of a nuclear weapon or a nuclear accident represent possible events with significant exposure to mixed neutron/γ-radiation fields. Although radiation countermeasures generally have been studied in subjects exposed to pure photons (γ or X rays), the mechanisms of injury of these low linear energy transfer (LET) radiations are different from those of high-LET radiation such as neutrons, and these differences may affect countermeasure efficacy. We compared 30-day survival in mice after varying doses of pure γ and mixed neutron/γ (mixed field) radiation (MF, Dn/Dt = 0.65), and also examined peripheral blood cells, bone marrow cell reconstitution, and cytokine expression. Mixed-field-irradiated mice displayed prolonged defects in T-cell populations compared to mice irradiated with pure γ photons. In mouse survival assays, the growth factor granulocyte colony-stimulating factor (G-CSF) was effective as a (post-irradiation) mitigator against both γ-photons and mixed-field radiation, while the thrombopoietin (TPO) mimetic ALXN4100TPO was effective only against γ irradiation. The results indicate that radiation countermeasures should be tested against radiation qualities appropriate for specific scenarios before inclusion in response plans.

Wound trauma increases radiation-induced mortality by activation of iNOS pathway and elevation of cytokine concentrations and bacterial infection.

Abstract Although it is documented that concurrent wounding increases mortality from radiation injury, the molecular mechanism of combined injury is unknown. In this study, mice were exposed to gamma radiation followed by skin wounding. Wound trauma exacerbated radiation-induced mortality, reducing the LD(50/30) from 9.65 Gy to 8.95 Gy. Analyses of histopathology, inducible nitric oxide synthase (iNOS), and serum cytokines were performed on mouse ileum and skin at various times after 9.75 Gy and/or wounding. In the ileum, the villi were significantly shortened 3 days postirradiation but not after wounding; combined injury resulted in decreased villus width and tunica muscularis thickness. The skin of mice subjected to combined injury was less cellular and had a smaller healing bud than the skin of mice subjected to wounding alone. Combined injury significantly delayed wound closure times; it also prolonged the increased levels of iNOS protein in the skin and ileum. iNOS up-regulation was correlated with increases in transcription factors, including NF-kappaB and NF-IL6. The increase in NF-IL6 may be due to increases in cytokines, including IL-1beta, -6, -8, -9, -10 and -13, G-CSF, eotaxin, INF-gamma, MCP-1, MIP-1alpha and MIP-1beta. Combined injury resulted in early detection of bacteria in the blood of the heart and liver, whereas radiation alone resulted in later detection of bacteria; only a transient bacteremia occurred after wounding alone. Results suggest that enhancement of iNOS, cytokines and bacterial infection triggered by combined injury may contribute to mortality. Agents that inhibit these responses may prove to be therapeutic for combined injury and may reduce related mortality.

Combined injury: factors with potential to impact radiation dose assessments.

Combined injuries, which are expected after a radiation dispersal device release or nuclear weapon detonation, are the combination of radiation exposure and tissue injuries from blast and thermal energy. To determine the impact of such trauma, mice were used to (1) evaluate the consequences of skin tissue injuries after various qualities and doses of radiation and (2) document substances that increase survival from radiation injury. Female 12- to 20-wk-old mice weighing 23 +/- 3 g received dorsal skin burns or wounds (15% total body skin surface) under methoxyflurane anesthesia before or after irradiation in this study approved by the Armed Forces Radiobiology Research Institute (AFRRI) Institutional Animal Care and Use Committee. Methoxyflurane is analgesic up to 48 h after injury. The radiations used in these studies included Co gamma photons (1.25 MeV) and research-reactor-produced neutrons with an average energy of 0.96 MeV in either an enriched-field [n/(n + gamma) = 0.95] configuration at 4.2 kW or a mixed-field [n/(n + gamma) = 0.67] configuration operated at 45 kW. Dose rates averaged 0.4 Gy/min. Endpoints included survival, LD50/30s (lethal dose to produce 50% mortality in 30 d), dose modifying factors, relative biological effectiveness values, tissue alterations, susceptibility to bacterial challenge, and countermeasure efficacies. Countermeasures evaluated included S-3-(3-methylaminopropylamino) propylthiophosphorothioic acid (WR-151327), antibiotics, immune modulators, and bone marrow transplantation. Of these treatments, survival was improved by WR-151327, antibiotics, synthetic trehalose discorynomycolate, and bone marrow transplantation. Because trauma to irradiated personnel and medical countermeasures may affect biodosimetric measurements, it will be necessary to quickly determine radiation dose in order to implement appropriate therapy.

Medical countermeasures for radiation combined injury: radiation with burn, blast, trauma and/or sepsis. report of an NIAID Workshop, March 26-27, 2007.

Non-clinical human radiation exposure events such as the Hiroshima and Nagasaki bombings or the Chernobyl accident are often coupled with other forms of injury, such as wounds, burns, blunt trauma, and infection. Radiation combined injury would also be expected after a radiological or nuclear attack. Few animal models of radiation combined injury exist, and mechanisms underlying the high mortality associated with complex radiation injuries are poorly understood. Medical countermeasures are currently available for management of the non-radiation components of radiation combined injury, but it is not known whether treatments for other insults will be effective when the injury is combined with radiation exposure. Further research is needed to elucidate mechanisms behind the synergistic lethality of radiation combined injury and to identify targets for medical countermeasures. To address these issues, the National Institute of Allergy and Infectious Diseases convened a workshop to make recommendations on the development of animal models of radiation combined injury, possible mechanisms of radiation combined injury, and future directions for countermeasure research, including target identification and end points to evaluate treatment efficacy.

Retrospective analyses of serum lipids and lipoproteins and severity of disease in 60Co-irradiated Sus scrofa domestica and Macaca mulatta.

Profound neutropenia that provides an opportunity for infections to develop into sepsis remains an important cause of morbidity and mortality in patients after irradiation. Human clinical studies find extremely low concentrations of cholesterol (less than 120 mg/dl) associated with high risk of death in critically ill adult patients admitted to intensive care units. This retrospective study was initiated as part of separate investigations of radiation-induced acquired infections in 2 large animal species receiving high-dose whole-body irradiation from a 60Co gamma-photon source. Nine Yucatan minipigs (Sus scrofa domestica) and 16 rhesus macaques (Macaca mulatta) were evaluated for sepsis, serum lipid and lipoprotein concentrations, and other blood parameters. For each species, animals were grouped into 2 categories--septic and nonseptic--and severity of disease was quantified by use of a scoring system. Significantly lower high-density lipoprotein (HDL) concentrations were found in the septic pigs at 24 and 48 h as compared with nonseptic pigs. HDL was significantly decreased in septic macaques within 24 h and 3 to 4, 6 to 7, and 9 to 10 d after diagnosis of sepsis, compared with that in nonseptic macaques. Coupled with hypocholesterolemia, decreased serum HDL was the parameter that was associated with disease severity at the time of sampling. Our data indicate that HDL is a reliable marker for severity of disease in these 2 preclinical models of irradiation-induced sepsis.

Antibacterial activity of the soy isoflavone genistein.

Genistein, a radioprotective soy isoflavone and protein kinase inhibitor, blocks the invasion of pathogenic bacteria in mammalian epithelial cells. The purpose of this study was to evaluate the direct effect of genistein on the survival and growth of the probiotic Lactobacillus reuteri and selected opportunistic bacteria in vitro as a prelude to in vivo use for managing postirradiation sepsis. We evaluated the opportunistic bacterial enteropathogens Escherichia coli, Shigella sonnei, and Staphylococcus aureus as well as Klebsiella pneumoniae and the non-pathogenic organism, Bacillus anthracis (Sterne). The latter two bacteria are found in the environment and may be of concern in irradiated individuals. A standard in vitro test was employed to evaluate the direct effect of genistein on the bacteria. This test involved determining bacterial colony forming unit (CFU) counts at a single concentration of genistein. In the CFU assays, significant reductions in CFUs were found for S. aureus and B. anthracis when cultured in the presence of 100 muM genistein. However, L. reuteri, E. coli, S. sonnei, and K. pneumoniae were not altered by in vitro culturing in the presence of 100 muM genistein. These results demonstrate the in vitro antimicrobial activity of genistein. Furthermore, the use of genistein in combination with probiotics may augment the effectiveness of antimicrobial therapies currently used in the management of infections, including those induced by ionizing irradiation.

Comparison of clarithromycin and ciprofloxacin therapy for Bacillus anthracis Sterne infection in mice with or without (60)Co gamma-photon irradiation.

Biological agents and ionizing radiation lead to more severe clinical outcomes than either insult alone. This study investigated the survival of non-irradiated and (60)Co-gamma-irradiated mice given therapy for inhalation anthrax with ciprofloxacin (CIP) or a clinically relevant mixture of clarithromycin (CLR) and its major human microbiologically important metabolite 14-hydroxy clarithromycin (14-OH CLR). All B6D2F1/J 10-week-old female mice were inoculated intratracheally with 3 x 10(8) c.f.u. of Bacillus anthracis Sterne spores 4 days after the non-lethal 7 Gy dose of (60)Co gamma radiation. Twenty-one days of treatment with CLR/14-OH CLR, 150 mg kg(-1) twice daily, or CIP, 16.5 mg kg(-1) twice daily, began 24 h after inoculation. Pharmacokinetics indicate that the area under the curve (AUC) for 14-OH CLR on the concentration-versus-time graph was slightly higher in gamma-irradiated than non-irradiated animals. Neither drug was able to increase survival in gamma-irradiated animals. CIP and CLR/14-OH CLR therapies in non-irradiated animals increased survival from 49 % (17/35 mice) in buffer-treated animals to 94 % (33/35) and 100 %, respectively (P < 0.001). B. anthracis Sterne only was isolated from 25-50 % of treated mice with or without irradiation. Mixed infections with B. anthracis Sterne were present in 50-71 % of gamma-irradiated mice but only in 5-10 % of mice without irradiation.

Management of postirradiation infection: lessons learned from animal models.

Ionizing radiation depresses host defenses and enhances susceptibility to local and systemic infection due to endogenous or exogenous microorganisms. Exposure of mice to a lethal dose of ionizing 60Co-gamma radiation induces a dose-related reduction in the number of both aerobic and anaerobic bacteria from 10(10-12) to 10(4-6) per gram of stool within 4 days. The number of anaerobic bacteria stays low, but the number of Enterobacteriaceae per gram of stool increases significantly up to 10(9) by the 12th day after irradiation. This increase is associated with bacterial translocation of these organisms and fatal bacteremia. The use of quinolones in the irradiated animals was effective in controlling systemic endogenous Gram-negative infection after irradiation. Supplementation with penicillin prevented treatment failures due to Streptococcus spp. and increased survival. Quinolones given for 21 days also were effective in management of systemic exogenous infections due to orally ingested Klebsiella pneumoniae and Pseudomonas aeruginosa. Effectiveness of quinolones may be attributed to inhibition of exogenous organism growth within the gut lumen while preserving the anaerobic gut flora as well as their systemic antibacterial activity. Based on these findings, antimicrobial agents recommended for therapy of infection after exposure to irradiation are: ciprofloxacin, levofloxacin, ceftriaxone, cefepime, gentamicin +/- amoxicillin, or vancomycin.

Management of postirradiation sepsis.

Ionizing radiation depresses the immune defenses and enhances susceptibility to local and systemic infection due to endogenous or exogenous microorganisms. Exposure to a lethal dose of ionizing cobalt-60 gamma radiation induces a dose-related reduction in the number of both aerobic and anaerobic bacteria from 10(10-12) to 10(4-6) per gram of stool within 4 days. Whereas the number of anaerobic bacteria stays low, the number of Enterobacteriaceae per gram of stool increases significantly--up to 10(9) by the 12th day following irradiation. This increase is associated with bacterial translocation of these organisms and fatal bacteremia. The use of quinolones was effective in controlling systemic endogenous Gram-negative infection following irradiation. Supplementation with penicillin prevented treatment failures due to Streptococci and increased survival. Quinolones given for 21 days were also effective in the management of systemic exogenous infections due to orally ingested Klebsiella pneumoniae and Pseudomonas aeruginosa. Quinolones may be effective because they inhibit growth of the exogenous organism within the gut lumen while preserving the anaerobic gut flora and their systemic antibacterial activity. Coadministration of antimicrobials effective against anaerobes may be required for the management of polymicrobial infections. The availability of both oral and parenteral routes of administration, the advantage of achieving selective inhibition of potential pathogens in the gut, and the ability to treat systemic infection make the quinolones promising agents for the therapy of endogenous and exogenous infections after irradiation.

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.

Nuclear, biological, and chemical combined injuries and countermeasures on the battlefield.

The Armed Forces Radiobiological Research Institute (AFRRI) has developed a research program to determine the major health risks from exposure to ionizing radiation in combination with biological and chemical warfare agents and to assess the extent to which exposure to ionizing radiation compromises the effectiveness of protective drugs, vaccines, and other biological and chemical warfare prophylactic and treatment strategies. AFRRI's Defense Technology Objective MD22 supports the development of treatment modalities and studies to assess the mortality rates for combined injuries from exposure to ionizing radiation and Bacillus anthracis, and research to provide data for casualty prediction models that assess the health consequences of combined exposures. In conjunction with the Defense Threat Reduction Agency, our research data are contributing to the development of casualty prediction models that estimate mortality and incapacitation in an environment of radiation exposure plus other weapons of mass destruction. Specifically, the AFFRI research program assesses the effects of ionizing radiation exposure in combination with B. anthracis, Venezuelan equine encephalomyelitis virus, Shigella sonnei, nerve agents, and mustard as well as their associated treatments and vaccines. In addition, the long-term psychological effects of radiation combined with nuclear, biological, and chemical (NBC) injuries are being evaluated. We are also assessing the effectiveness of gamma photons and high-speed neutrons and electrons for neutralizing biological and chemical warfare agents. New protocols based on our NBC bioeffects experiments will enable U.S. armed forces to accomplish military operations in NBC environments while optimizing both survival and military performance. Preserving combatants' health in an NBC environment will improve warfighting operations and mission capabilities.

Susceptibility of irradiated mice to Bacillus anthracis sterne by the intratracheal route of infection.

The susceptibility of sublethally irradiated mice to pulmonary infection with Bacillus anthracis was investigated in a mouse model. Female B6D2F1/J mice were challenged intratracheally with 4.3 x 10(6), 3.7 x 10(7) and 4.4 x 10(8) cfu of B. anthracis Sterne spores 4 days after 60Co gamma-radiation at a dose of 0, 1, 2, 3, 4, 5, 6 or 7 Gy. Bacterial cultures were obtained from lung, spleen homogenates and heart blood. A biphasic mode of mortality was observed, with a constant response of up to 3 or 4 Gy (up to 18% mortality), after which a sharp increase in mortality occurred (up to 100%). When irradiation was delayed beyond 15 days after inoculation, the susceptibility to B. anthracis infection and subsequent mortality disappeared. B. anthracis was recovered from the organs and blood of up to 89% of the animals. However, organisms of enteric origin were also isolated mixed with B. anthracis from up to 36% of the animals exposed to 3, 5 or 7 Gy. Inoculation of B. anthracis delta-Sterne-1 that lacks lethal toxin and oedema toxin also induced infection with B. anthracis, but not translocation of enteric micro-organisms. The synergic adverse effect of exposure to gamma-radiation followed by intratracheal challenge with B. anthracis was observed above 4 Gy. The lethal toxin of B. anthracis may enhance the emergence of polymicrobial infection with B. anthracis and enteric micro-organisms.