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.

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 60Co 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 such as wounds or burns following whole-bodyγ-irradiation (radiation combined injury (RCI)) increase mortality more than whole-bodyγ-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 E 2 (PGE 2 ) 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 PGE 2 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 PGE 2 cause homeostatic imbalance and may contribute to reduced survival. Agents inhibiting these responses may prove to be therapeutic for RCI and improve related survival.

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.

: Symbiotic microorganisms are spatially separated from their animal host, e.g., in the intestine and skin, in a manner enabling nutrient metabolism as well as evolutionary development of protective physiologic features in the host such as innate and adaptive immunity, immune tolerance, and function of tissue barriers . The major interface barrier between the microbiota and host tissue is constituted by epithelium reticuloendothelial tissue, and mucosa-associated lymphoid tissue (MALT) . Traumatic damage to skin and the internal epithelium in soft tissues can cause infections that account for 7% to 10% of hospitalizations in the United States (4). Moreover, wound infections and sepsis are an increasing cause of death in severely ill patients, especially those with immunosupression due to exposure to cytotoxic agents and chronic inflammation (4). It is well accepted that breakdown of the host-bacterial symbiotic homeostasis and associated infections are the major consequences of impairment of the first line of antimicrobial defense barriers such as the mucosal layers, MALT and reticuloendothelium. Under these impairment conditions of particular interest then is the role of sub-mucosal structures, such as connective tissue stroma, in the innate defense compensatory responses to infections. The mesenchymal connective tissue of different origins is a major source of multipotent mesenchymal stromal cells (i.e., colony-forming-unit fibroblasts). Recent discovery of immunomodulatory function of mesenchymal stromal cells (MSCs) suggests that they are essential constituents that control inflammatory responses.

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.

Background  Animals undergoing experimental manipulations, such as exposure to radiation, may exhibit physiologic and behavioral signs of pain and distress. Telemetry permits close monitoring of these parameters for early and effective management during procedures.

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-kappa B and NF-IL6. The increase in NF-IL6 may be due to increases in cytokines, including IL-1 beta, -6, -8, -9, -10 and -13, G-CSF, eotaxin, INF-gamma, MCP-1, MIP-1 alpha and MIP-1 beta. 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. (C) 2010 by Radiation Research Society

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 60Co 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 + γ) = 0.95] configuration at 4.2 kW or a mixed-field [n/(n + γ) = 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.

BackgroundMicroflora populations residing in oropharyngeal and gastrointestinal sites defend against pathogenic bacterial colonization. Perturbations in these microbial communities may allow opportunistic pathogenic bacteria to establish themselves and cause morbidity and mortality from sepsis particularly after stressful experimental procedures. This study determined the prevalent facultative bacteria in a resident population of Macaca mulatta prior to use in experimentally induced immunosuppressive radiation studies.MethodsStandard microbiological methods were used to assess prevalent facultative bacteria in the oropharynx and rectum of 24 male M. mulatta.ResultsThe majority of the bacteria isolated from the oropharyngeal and rectal sites were gram-positive cocci. Species of Staphylococcus and Streptococcus predominated in all samples. Few gram-negative bacteria were isolated.ConclusionsBacteriological assessment is recommended to identify predominant bacterial species to be prepared to provide appropriate antimicrobial therapy in non-human primates that are expected to undergo stressful immunocompromising procedures.

Radiation combined injury (CI) results in mortality greater than that following radiation injury (RI) or skin‐wound trauma. The molecular mechanism for this increase is not clear. In this study, mice were given 9.75 Gy r‐radiation followed by a 15% total body skin surface wound. On 1d, 3d, and 7d after RI or CI, sections of ileum and skin were removed and histologically examined. Also, iNOS in ileum and skin and serum cytokines were determined. RI but not wounding vastly shortened and widened villi 3 d post‐RI, whereas CI resulted in smaller alterations in villus. However, the serosal layer was thinner after CI than after RI. The skin in CI mice was less cellular with a smaller healing bud than in wounded mice resulting in significantly delayed wound closure times after CI from about 14 d to 28 d. CI enhanced the levels of iNOS in both tissues that were upregulated by increases in transcriptional factors KLF6, NF‐kB, and NF‐IL6. The latter was related to a burst in cytokine concentrations including IL‐1b, ‐6, ‐8, ‐9, ‐10, ‐13, and c‐GSF. Bacteria were isolated from heart blood and liver, indicating translocation from intestinal tract and wound site may have induced increases in these cytokines. The data are consistent with the idea that increases in iNOS and cytokines by CI may contribute to the CI‐induced mortality. Agents that inhibit iNOS and the cytokine cascade may ameliorate CI‐induced sequelae including death.

Jiao, W., Kiang, J.G., Cary, L., Elliott, T. B., Pellmar, T. C. and Ledney, G. D. COX-2 Inhibitors Are Contraindicated for Treatment of Combined Injury. Radiat. Res. 172, 686-697 (2009).Casualties of radiation dispersal devices, nuclear detonation or major ionizing radiation accidents, in addition to radiation exposure, may sustain physical and/or thermal trauma. Radiation exposure plus additional tissue trauma is known as combined injury. There are no definitive therapeutic agents. Cyclooxygenase-2 (COX-2), an inducible enzyme expressed in pathological disorders and radiation injury, plays an important role in inflammation and the production of cytokines and prostaglandin E-2 (PGE(2)) and could therefore affect the outcome for victims of combined injury. The COX-2 inhibitors celecoxib and meloxicam were evaluated for their therapeutic value against combined injury in mice. In survival studies, the COX-2 inhibitors had no beneficial effect on 30-day survival, wound healing or body weight gain after radiation injury alone or after combined injury. Meloxicam accelerated death in both wounded and combined injury mice. These drugs also induced severe hepatic toxicity, exaggerated inflammatory processes, and did not enhance hematopoietie cell regeneration. This study points to potential contraindications for use of COX-2 inhibitors in patients undergoing therapy for radiation injury and combined injury. (C) 2009 by Radiation Research Society

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.

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.

: The unique capabilities of the Armed Forces Radiobiology Research Institute's (AFRRI) cobalt-60 (Co60) gamma-photon irradiator were used for bilateral whole-body irradiation of the nonhuman primate (NHP) Macaca mulatta. In this study, 6.5 Gy radiation was delivered at 0.4 Gy/min to 24 male NHPs 7-10 years of age weighing 7-14 kg in order to evaluate the (a) pharmacokinetics and pharmacodynamics (PK/PD) of ciprofloxacin (CIP) in an irradiated non-rodent large-animal species phylogenetically close to man, (b) utility of CIP in managing postirradiation infection related to bacterial translocation from the alimentary canal, and (c) the side effects of CIP in acutely irradiated NHPs. This dose was chosen from published and unpublished studies indicating 6.5 Gy (a) approximated the LD50/30 for NHPs and (b) resulted in severe hematopoietic system injury with increased risk for death from sepsis. For irradiation, an acrylic plastic container was constructed to comfortably restrain large NHPs that were 60-80 cm in length. Three acrylic plastic cylinders (7, 10.1, and 12.6 cm in diameter) were used in conjunction with alanine pellets to estimate the dose to the mid-abdominal target area of various sized NHPs.

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 mu M genistein. However, L. reuteri, E. coli, S. sonnei, and K. pneumoniae were not altered by in vitro culturing in the presence of 100 mu M 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.

Ionizing radiation depresses host defenses and enhances susceptibility to local and systemic infection due to endogenous or exogenous microorganisms. Giving mice a lethal dose of ionizing 60 Co-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 four days. Although the number of anaerobic bacteria remains low, measured by per gram of stool bacteria increases significantly, up to 10 9 per gram of stool by the twelfth day following irradiation. This increase is associated with the bacterial translocation of Enterobacteriaceae and fatal bacteremia. Treatment with metronidazole suppressed the anaerobic stool flora and facilitated the increase in the number of Enterobacteriaceae, thus promoting earlier fatal bacteremia. The use of quinolones in the animals was effective in controlling systemic endogenous Gram-negative infection following irradiation. Supplementation with penicillin prevented treatment failures caused by Streptococcus spp. and increased survival. Quinolones given for 21 consecutive days also was effective in managing systemic exogenous infections due to orally ingested Klebsiella pneumoniae and Pseudomonas aeruginosa. The effectiveness of quinolones may be attributed to the inhibition of exogenous microbial growth within the gut lumen while preserving the anaerobic gut flora, and to their systemic antibacterial activity. Based on these findings, the antimicrobial agents recommended for therapy of infection following radiation exposure are ciprofloxacin, levofloxacin, ceftriaxone, cefepime, or gentamicin with or without amoxicillin or vancomycin (to cover gram-positive bacteria), given for 21 days.

ABSTRACT

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.