Background: Acinetobacter baumannii infections have been noted to occur more frequently in battlefield casualties involving wounds to the extremities. It has been suggested that the organism is present in the soil and contaminates the wounds. This explanation would provide a source, but not necessarily explain why soldiers become septic. The hypothesis we are advancing is that morphine, which would probably have been given to the injured service person, acts as a cofactor in sensitizing to sepsis. If such a causal relationship were to be established, it would be highly significant. A. baumannii, in settings other than the military, is usually characterized as a nosocomial infection, with a predilection for the immunosuppressed. Work from our laboratories and others shows that morphine is immunosuppressive, as is withdrawal from morphine. It is noteworthy that some of these observations have been confirmed in humans. Further, morphine has been shown to sensitize to other infections in murine models and to induce sepsis in the mouse. Maintenance of immune integrity early after trauma may be important for preventing an infection such as A. baumannii from becoming systemic, and morphine may compromise immune status. Our studies propose a unique hypothesis that morphine sensitizes to sepsis, particularly in conjunction with trauma.

Objective/Hypothesis: We propose to test the hypothesis that morphine and trauma each sensitize to Gram-negative infection with A. baumannii and that when both conditions are present, an additive or synergistic susceptibility to this infection results.

Specific Aims: (1) To test whether morphine will increase susceptibility to Gram-negative infection with A. baumannii. (2) To test whether a mouse model of trauma, in the form of a femur break with 40% hemorrhage, will result in increased susceptibility to A. baumannii infection. (3) To test whether the combination of morphine with trauma (femur break and 40% hemorrhage) will have an additive or synergistic effect on infection with A. baumannii. (4) To determine whether morphine and/or trauma, in combination with A. baumannii infection, alters levels of cytokines and other molecules and pathways that mediate systemic inflammatory response syndrome (SIRS) and septic shock.

Study Design: A mouse model of systemic infection with A. baumanni will be developed. Animals will be given morphine acutely, subacutely, chronically, or they will be withdrawn from the drug after they develop tolerance. Alternatively, they will be subjected to trauma by femur break and hemorrhage, or a combination of morphine plus trauma. The effect of these treatments on sensitization to infection will be assessed by survival rates of animals exposed to morphine versus those given a placebo or a morphine antagonist, and by quantitative necropsy, in which the number of viable A. baumannii in experimental versus control groups will be determined using plate counts of organisms in blood, peritoneal fluid, spleen, liver, or lung. Results will be verified using mu opioid receptor knockout mice. Experiments will be carried out to determine the mechanisms by which these treatments alter susceptibility to infection. Proinflammatory cytokines, nitric oxide, and prostaglandin E2 will be measured in blood and/or in culture supernatants of spleen cells or splenic macrophages, with or without stimulation with bacterial lipopolysaccharide. Levels of mRNA for cytokines, inducible nitric oxide synthase, and cyclooxygenase will be determined. Pathways of macrophage activation under the different conditions will be investigated by measurement of transcription factors including NF-kB and by examining activation of the MAP kinase pathway.

Relevance: If the hypothesis that morphine alone or in combination with trauma sensitizes to this Gram negative infection is borne out by the experimental results, the implications would be profound. They would suggest that morphine, one of the most widely used analgesics in the military, might actually predispose to sepsis, especially in conjunction with a traumatic injury. The implications of such a conclusion would be an imperative to consider use of alternative analgesics that are currently available, as well as to provide an impetus to pursue development of new analgesics to replace morphine. Delineation of the mechanisms underlying the deleterious effects of morphine that result in sepsis could lead to other modalities of treatment to be used in conjunction with morphine to counteract the sepsis promoting pathways. Knowledge gained from these experiments would be applicable to both treatment on the battlefield and to treatment of veterans in V.A. hospitals. If morphine is be shown to be a factor in sepsis in soldiers experiencing trauma or in postoperative patients, it would affect our current strategies for pain treatment, especially in immunocompromised hosts, including those with trauma sustained on the battlefield.