The novel antibacterial drug XF-70 is a potent inhibitor of Staphylococcus aureus infection of the burn wound.

The authors report the findings of in vivo studies of XF-70 (a novel, dicationic porphyrin) against Staphylococcus aureus in a murine model of a burn wound infection. Mice received a 15% total body scald burn wound, which were inoculated with S. aureus (1.8 x 10 CFU). After 24 hours, escharectomies were performed and groups (n = 8) received single or two doses (6 hours apart) of XF-70* (100 microg/wound) or silver sulfadiazine, Acticoat, or saline applied topically. Viable bacteria were quantified from homogenized burn tissue biopsies and the spleen by plating dilutions onto agar plates and CFU determination. A single dose of XF-70 reduced bacterial burden by 98.77% (untreated: 2.78 +/- 2.96 x 10 CFU/g vs XF-70 treated: 3.4 +/- 0.19 x 10 CFU/g, P < .01). Two XF-70 doses reduced the growth of S. aureus by 99.96% (1.2 +/- 0.6 x 10 CFU/g, P < .01). These results were similar to the results obtained from commonly used topical antibacterials silver sulfadiazine and Acticoat. The spleens of mice treated with saline had a robust growth of S. aureus (7.0 +/- 1.97 x 10 CFU/g) whereas those treated with one or two XF-70 doses grew only 3.5 +/- 0.002 x 10 CFU/g and 5.7 +/- 0.002 x 10 CFU/g, respectively, a significant (P < .001) reduction in S. aureus dissemination. Single and multiple doses of XF-70 were effective in controlling S. aureus growth in burn wounds and inhibited systemic dissemination of S. aureus. Early treatment of burn wounds with XF-70 may be effective in slowing bacterial dissemination to other tissues.

Perturbed bone marrow monocyte development following burn injury and sepsis promote hyporesponsive monocytes.

The mechanism of monocyte deactivation in critically injured burn patients remains unresolved. Two functionally distinct F4/80+Gr-1+ and F4/80+Gr-1- monocyte subsets have been characterized based on their homing to inflammatory or noninflammatory tissues, respectively. We hypothesized that the posttraumatic milieu in the bone marrow (BM) blunts the production of "inflammatory" monocytes. C57Blk/J male mice were divided into sham (S), burn (B), and burn sepsis (BS) groups. B and BS received a 15% dorsal scald burn and BS was inoculated with 15K colony forming units Pseudomonas aeruginosa at the burn site. Animals were killed and blood and femoral BM were collected 48, 72, and 96 hours after injury. ER-MP20 monocyte progenitors were isolated from BM and differentiated into macrophage (MØ) or dendritic cells (DCs) and characterized by the cell surface expression of F4/80 and CD11c, respectively. In both cell types, TLR-4 agonist induced cytokine levels were determined. Results showed a 2-fold increase in the F4/80+Gr-1+ subset at 48 hours in BS that started to decline at 72 hours and remained low at 96 hours. ER-MP20 progenitors isolated at 48 hours exhibited robust MØ differentiation potential but a significant decline in the percentage of the F4/80+Gr-1+ subset (P < .05 vs S) with a concomitant decrease in tumor necrosis factor alpha production. DC development from ER-MP20 progenitors and LPS-stimulated tumor necrosis factor alpha production were impaired. Therefore, BM progenitor derived MØ will replace the transient hyper-responsive circulating monocytes later during the course of the septic insult. Hypo-reactivity of the developing monocytes and DC in the BM and their subsequent egress to the periphery provide a plausible explanation for the immunosuppression that ensues a critical burn injury and sepsis.

Murine hematopoietic stem cells and progenitors express adrenergic receptors.

Association between the nervous and immune system is well documented. Immune cells originate within the bone marrow that is innervated. Thermal injury induces adrenergic stimulation, augments monocytopoiesis and alters the beta-adrenergic receptor (AR) profile of bone marrow monocyte committed progenitors. This provides an impetus to study AR expression in hematopoietic progenitors along myeloid lineage. Using FACS analysis and confocal microscopy, we report the expression of alpha1-, alpha2- and beta(2)-AR in enriched populations of ER-MP209(+) and ER-MP12(+) myeloid progenitors, CD117(+) and CD34(+) multi-potential progenitors and more importantly pluripotent stem cells suggesting a plausible role for catecholamine in hematopoietic development.

Thermal injury and sepsis modulates beta-adrenergic receptors and cAMP responses in monocyte-committed bone marrow cells.

We have previously reported that adrenergic stimulation enhances monocytopoiesis following experimental burn injury and sepsis (BI/S). In the present work we measured beta-adrenergic receptor number and affinity in bone marrow committed monocyte progenitor cells (CD59(+)) following BI/S. We find that BI/S treatment significantly decreased monocyte progenitor cell beta-adrenergic receptors but significantly increased receptor binding affinity and isoproterenol-stimulated cAMP production. CD14 expression in macrophages derived in vitro from CD59(+) cells following BI/S was significantly increased by epinephrine and this change was blocked by beta(2)-adrenergic receptor antagonist. PCR analysis suggests the presence of beta(2)- but not beta(1)-adrenergic receptors. Enhanced adrenergic receptor signaling in CD59(+) bone marrow cells following BI/S may be important in macrophage development.

The expression of cyclooxygenase and the production of prostaglandin E2 in neutrophils after burn injury and infection.

Recent studies have demonstrated that neutrophils have the capacity to produce a variety of cytokines after stimulation. The synthesis and release of prostaglandin E2 (PGE2) via the cyclooxygenase (COX) pathway has been reported to occur in activated neutrophils. In the present study, we sought to determine the status of COX protein synthesis and PGE2 production in murine neutrophils after burn injury. The effect of burn injury on neutrophil COX and PGE2 response to infection or lipopolysaccharide (LPS) was also examined. Peritoneal neutrophils were obtained from BDF1 mice at 4, 18, 24, and 36 hours after a 15% TBSA full-thickness scald burn or sham burn. We found that neutrophils from healthy mice express a low level of COX-2 protein. Neutrophil COX-2 protein expression in burn animals was significantly increased at 4 hours and dramatically decreased at 36 hours after burn injury. Animals 36 hours after burn and topically infected with Pseudomonas Aeruginosa had neutrophil COX-2 expression almost identical to burn injury only. Neutrophils harvested from healthy mice cocultured with LPS (1 microg/ml) had a marked induction of COX-2 protein. Neutrophils 24 hours after burn were unresponsive to LPS-stimulated COX-2 enhancement. COX-1 protein was strongly expressed constitutively and not affected further by burn injury or LPS. The production of PGE2 corresponded with the changes in COX-2 expression for all groups of mice. Our data suggested that neutrophils express both COX-1 and COX-2 and produce PGE2. The effects of burn injury on neutrophil COX-2 protein synthesis and PGE2 production suggest that after burn there is a time-dependent response. Insights into not only the global cellular response to injury and infection but also temporal nature of the response are important in the development of the therapeutic treatment strategies for burn patients.

Macrophage mediated suppression of granulocyte and macrophage growth after burn wound infection reversal by means of anti-PGE2.

The production and release of granulocytes and macrophages, crucial elements of the host defense system, are significantly impaired after burn injury and sepsis. Prostaglandin E2 (PGE2) is known to be myelosuppressive. We hypothesized that the macrophages contributed to myelopoietic suppression by means of increased PGE2 production, which is induced by thermal injury and sepsis. In this study, peritoneal macrophages were elicited at day 3 from normal mice and from mice who underwent a 15% total body surface area dorsal scald burn with or without Pseudomonas aeruginosa burn wound infection. The macrophages were incubated with or without endotoxin and with or without PGE2 polyclonal antiserum (anti-PGE2) for 18 hours. Macrophage supernatants were then used in co-cultures of bone marrow cells in a clonogenic assay of granulocyte-macrophage colony-forming cells (GM-CFCs) to determine the effect of burn wound infection on the alteration of the proliferative status of the GM-CFCs. Burn wound infection and endotoxin both caused marked reductions in GM-CFC growth in culture (20%-40% as compared with normal, P < .05-.01). The inhibition of GM-CFC growth induced by burn, burn plus infection, or endotoxin was significantly reversed by the addition of anti-PGE2 to the cultures (30%-40% increase in GM-CFC colony growth as compared with cultures without anti-PGE2). These results suggest that PGE2 is a key mediator in the gram-negative sepsis-induced macrophage suppression of granulocyte and macrophage production. The ability of anti-PGE2 to neutralize PGE2 activity may provide a useful means of mitigating myeloid depression that follows postburn sepsis.

Burn injury with infection alters prostaglandin E2 synthesis and metabolism.

OBJECTIVE: The aim of this study was to examine the relationship between prostaglandin synthesis and prostaglandin degradation in a model of burn injury with infection. METHODS: Male B2D6F1 mice were assigned to control, burn (16% dorsal scald burn), or burn with infection (burn with topical application of 1,000 colony forming units of Pseudomonas aeruginosa) groups. Lung tissue was harvested at 1, 2, and 3 days after burn injury and subsequently processed for total RNA and protein. Northern and Western blot analyses were used to examine differences in cyclooxygenase 2 (COX-2) and prostaglandin 15-OH dehydrogenase (PGDH) protein and mRNA expression. Total RNA was probed with the riboprobe for murine PGDH and COX-2 and the 100,000g protein fraction was immunoblotted by using an rabbit anti-murine PGDH and anti-murine COX-2 antibody. RESULTS: COX-2 expression was elevated in the burn with infection animals on day 1 and day 2 after burn injury. At these time points in the burn + infection group, PGDH was significantly depressed. Burn injury increased COX-2 expression on day 1, but by day 2, COX-2 expression had decreased to control values. A corresponding increase in PGDH expression was observed on day 2 in the burned mice. The mRNA expression of COX-2 was followed by a similar increase in COX-2 protein expression at all time points in the injured animals. This was not the case with PGDH expression. On day 1, PGDH mRNA expression was depressed in the burn with infection mice with no change in PGDH protein expression. This finding indicates that PGDH is subject to regulation at both the transcriptional and posttranscriptional levels. CONCLUSION: Burn wound infection depressed both PGDH mRNA and protein expression and increased COX-2 mRNA and protein expression. Therefore, increases in circulating prostaglandin E2 levels during septic injury are derived from alterations in synthesis and degradation of prostaglandin E2.

Improvement in survival with peptidyl membrane interactive molecule D4B treatment after burn wound infection.

OBJECTIVE: To examine the effects of peptidyl membrane interactive molecule D4B in a murine model of lethal burn wound infection. EXPERIMENTAL DESIGN: Four experiments were performed: (1) growth inhibition assays of Pseudomonas aeruginosa treated with D4B, 0 to 100 micromol/L; (2) in vitro coculture of bone marrow cells with D4B, 0 to 100 micromol/L; (3) D4B treatment survival studies after burn injury only or burn wound infection in mice; and (4) peripheral white blood cell count, burn wound tissue bacterial culture, and burn wound morphological analysis at days 1, 2, and 3 after injury. SETTING: University medical center laboratory. SUBJECTS: Groups of B6D2F1 male mice (20 each) were studied. INTERVENTIONS: Full-thickness scald burn, 15% of total body surface area, with P aeruginosa topical infection, and subeschar injections of D4B at 200 microg or 0.25 mL of placebo per mouse at 2 and 24 hours after injury. MAIN OUTCOME MEASURES: Animal survival after thermal burn wound bacterial infection, circulating leukocyte numbers, in vitro clonal cell culture of granulocyte-macrophage progenitor cells, and wound histopathological analysis. RESULTS: The survival rate in the D4B-treated group was nearly 2-fold greater than that in controls (P<.01) during 14 days of study. Bacterial quantitative wound cultures disclosed significant reductions in bacterial numbers at days 1, 2, and 3 in D4B-treated animals as compared with controls (P<.05 to <.01). D4B induced a dose-dependent inhibition of bacterial cell growth when added to in vitro P aeruginosa cultures (P<.01). Granulocyte-macrophage progenitor cell growth in culture was not altered by D4B treatment. D4B-treated animals displayed no signs of toxic effects or impairment in wound healing. CONCLUSIONS: The peptidyl membrane interactive molecule D4B had the ability to improve survival after gram-negative burn wound sepsis via direct antimicrobial effects. Peptidyl membrane interactive molecules may offer the potential of alternative treatments to standard topical agents or in patients with drug-resistant microbes.

Cyclooxygenase-2 inhibitor NS-398 improves survival and restores leukocyte counts in burn infection.

BACKGROUND: Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandin E2 (PGE2) from activated macrophages. PGE2 is increased during trauma and sepsis and has been implicated as a negative immunomodulator. The objective of this study was to determine the therapeutic benefits of a COX-2 inhibitor (NS-398) on survival and leukocyte production in a murine model of burn sepsis. METHODS: To determine the in vitro ability of NS-398 to inhibit macrophage production of PGE2, peritoneal elicited macrophages were stimulated for 18 hours with medium alone, endotoxin (ETX) (1 mumol/L), or ETX plus NS-398 (0.3 mumol/L). Macrophage supernatant PGE2 levels were determined by an enzyme immunoassay. To test the in vivo efficacy of NS-398, mice subjected to a 15% dorsal scald burn plus 1,000 colony-forming units of topical Pseudomonas aeruginosa received either 10 mg/kg NS-398 intraperitoneally or placebo 4 to 6 hours after infection and twice daily for 3 days. Survival was measured up to 14 days, and circulating white blood cell (WBC) count and absolute neutrophil count (ANC) were determined 3 days after injury. RESULTS: Macrophage PGE2 production was significantly increased in the ETX-treated group compared with the medium-alone group, and this increase was completely normalized with the addition of NS-398. NS-398 also augmented WBC count (4,288 +/- 649 vs. 7,866 +/- 435 per mm3; p < 0.01) and ANC (1,068 +/- 255 vs. 3,663 +/- 474 per mm3) after burn infection and attenuated macrophage depression of hematopoietic proliferation. Finally, NS-398 treatment significantly improved survival after burn infection, from 0 to 45.5%. CONCLUSION: Inhibition of the COX-2 isoform of cyclooxygenase with NS-398 inhibited macrophage PGE2 production, restored ANC, and improved survival during burn infection. NS-398, therefore, has potential therapeutic benefits in septic patients who have developed neutropenia.

Prostaglandin E2 alterations during sepsis are partially mediated by endotoxin-induced inhibition of prostaglandin 15-hydroxydehydrogenase.

Prostaglandin E2 (PGE2) is significantly elevated in the plasma of septic or injured patients and is thought to be a component of the resultant immune suppression associated with augmented rates of infection and mortality. Many studies have examined the effect of burn injury and sepsis on PGE2 synthesis. However, the effect of sepsis or burn injury on the expression of prostaglandin 15-hydroxydehydrogenase (PGDH), the key enzyme responsible for PGE2 degradation, has not been explored. The aim of this study was to examine the effect of endotoxin treatment and/or burn injury on the expression of PGDH. Male BDF1 mice were assigned to four groups (n = 4/group): sham, lipopolysaccharide (LPS) (2.5 mg/kg, Escherichia coli LPS, i.p.), burn (15% body surface area scald injury), and burn + LPS (15% body surface area + 2.5 mg/kg LPS, i.p.). Lung tissue was harvested at specific time points after treatment and subsequently was processed for total RNA and protein. Northern and Western blot analyses were used to examine differences in PGDH protein and mRNA expression. Total RNA was probed with the riboprobe for murine PGDH, and the 100,000 g protein fraction was immunoblotted using an rabbit antimurine PGDH antibody. PGDH was expressed in lung at t = 0 in both the saline and LPS-treated animals. A decrease in mRNA expression was initially observed at 2 hours after LPS treatment. The decrease was also significant (p < 0.05) at 3 hours after LPS and maximal decrease in mRNA and protein expression was observed at 6 hours. At 24 hours after LPS administration, the PGDH mRNA and protein expression was still significantly depressed to 49% of control expression. PGDH expression was similar and not statistically different in both burn and burn + LPS treatment at t = 0. At 2 hours after LPS, PGDH mRNA expression in the burn + LPS treatment group had significantly decreased to 47% in comparison with the burn alone group. Maximal decrease in PGDH mRNA and protein expression in lung from burn + LPS was observed at 6 hours after LPS treatment. This change represents a 73% decrease in mRNA in comparison with the time-matched burn control. At 24 hours after LPS administration, PGDH mRNA but not protein expression in the lung from burn + LPS treated mice was still significantly decreased. In summary, LPS treatment alters PGDH mRNA expression at the transcriptional and protein levels. Consequently, sepsis-induced increases in PGE2 levels may not be only due to increased PGE2 synthesis but also due to decreased PGDH expression and, hence, PGE2 degradation.

Burn wound infection-induced myeloid suppression: the role of prostaglandin E2, elevated adenylate cyclase, and cyclic adenosine monophosphate.

Suppressed granulocyte and macrophage growth after burn infection or endotoxicosis appears to be mediated by macrophage-derived products. In this study, we found that after burn, burn plus infection, or endotoxicosis, peritoneal-elicited macrophages or bone marrow cells released increased amounts of prostaglandin E2 (PGE2) and inhibited growth of granulocyte-macrophage progenitor cells (GM-CFC). PGE2, when added in culture, inhibited in vitro GM-CFC growth in a dose-dependent manner. Pretreatment of bone marrow cells with either dibutyryl cyclic adenosine monophosphate or Forskolin in vitro mimicked the PGE2 inhibition, further aggravated the inhibition induced by burn, burn plus infection, or endotoxicosis, and was not blocked by co-culture with indomethacin. Pretreatment of bone marrow cells with SQ22536, an adenylate cyclase inhibitor, significantly restored the suppressed GM-CFC growth found after burn, burn plus infection, or endotoxicosis. Alterations in myeloid production after burn infection appear to be related in part to the level of intracellular cyclic adenosine monophosphate for the GM-CFC and are responsive to PGE2.

Augmentations of glucose uptake and glucose transporter-1 in macrophages following thermal injury and sepsis in mice.

Glucose is the primary metabolic substrate of macrophages, which are critical components of the host response to injury and infection. We have carried out a series of studies to examine macrophage glucose uptake and the status of glucose transporter 1 (GLUT1) at both the mRNA and protein level. Peritoneal macrophages that were obtained from mice undergoing sham burned (S), 15%TBSA burn (B) +/- Pseudomonas aeruginosa burn infection (B + I) and lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) administration. [3H]deoxyglucose uptake was significantly increased (B, 157 +/- 9%; B + I, 243 +/- 19%; S + LPS, 231 +/- 24%; S + TNF-alpha, 379 +/- 18%; B + LPS, 230 +/- 13%; and B + TNF, 305 +/- 23%, P< 0.01 vs. sham). GLUT1 mRNA and protein levels were increased as well (mRNA: B, 135 +/- 13%; B + I, 250 +/- 33%; S + LPS, 282 +/- 29%; S + TNF-alpha, 193 +/- 19%; B + LPS, 378 +/- 20%; and B + TNF-alpha, 204 +/- 16%; protein: B, 159 +/- 27%; B + I, 181 +/- 17%; S + LPS, 219 +/- 26%; S + TNF-alpha, 343 +/- 51%; B + LPS, 366 +/- 41%; and B + TNF-alpha, 415 +/- 44, P< 0.01 vs. sham). Macrophages co-cultured with LPS or TNF-alpha in vitro demonstrated a similar response pattern. Following burn injury and infection, macrophages augment their cellular glucose uptake, which is facilitated by an increased GLUT1 mRNA and protein levels. TNF-alpha elicited by LPS may mediate this enhanced carbohydrate metabolism at the point of glucose entry into the cell.

Recombinant human granulocyte colony-stimulating factor treatment improves macrophage suppression of granulocyte and macrophage growth after burn and burn wound infection.

Granulocyte and macrophage production after burn injury or burn wound infection is significantly reduced and further compromised by endotoxin (ET). Moreover, the macrophage seems to be the major source of this bone marrow suppression. We sought to determine if recombinant human granulocyte colony-stimulating factor (rhG-CSF), a hematopoietic growth factor that is capable of improving survival after experimental burn wound sepsis, altered postburn macrophage-mediated marrow suppression. Groups of male BDF1 mice (n = 6 to 10) receiving a 15% total body surface area burn +/- infection (B or B + I) with Pseudomonas aeruginosa were injected with 100 ng rhG-CSF twice daily. On day 3, peritoneal-elicited macrophages (5 x 10(6) cells/mL) from either rhG-CSF-treated or control (5% dextrose in water) mice were incubated +/- ET (300 ng/mL). The resultant macrophage supernatant was added to cultures of target marrow granulocyte-macrophage progenitor cells (GM-CFC) at a volume of 1:10. The GM-CFC growth as a percentage of cultures not containing macrophage supernatant were compared and reductions in the number of GM-CFC taken as an index of marrow suppression. Macrophages obtained from B and B + I animals reduced target GM-CFC growth, compared with macrophages from normal animals (B vs. normal animals p < 0.05). In addition, ET-stimulated macrophages induced further bone marrow suppression for all three groups (p < 0.01). Macrophages from granulocyte colony-stimulating factor-treated animals caused significantly less bone marrow suppression, compared with untreated animals for all groups (p < 0.05 to 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage suppression of granulocyte and macrophage growth following burn wound infection.

Burn injury results in alterations in granulocyte and macrophage production. Since macrophages may mediate these alterations we determined the effects of macrophages obtained from animals with burn injury with and without infection on the growth of marrow granulocyte macrophage progenitor cells (GM-CFCs). The in vitro GM-CFC growth response for maximally stimulated cultures was reduced by 25% to 30% (p < 0.01) for burned and infected (B + I) animal macrophages compared with burned (B) or sham (S) animals. Macrophages stimulated with endotoxin caused a further reduction for all groups in GM-CFC growth, most notably so for B + I macrophages. Burned + infected animal macrophages or all-endotoxin macrophages cocultured with indomethacin did not suppress GM-CFC growth. Following burn injury and infection, macrophages spontaneously elaborate negative regulators of myeloid growth that is further increased by endotoxin. It is likely that PGE2, a known negative regulator of granulocyte macrophage growth, is largely responsible for this suppressive effect.

Marrow granulocyte-macrophage progenitor cell response to burn injury as modified by endotoxin and indomethacin.

The production and release of granulocytes and macrophages are significantly impaired following burn injury and infection. In an attempt to determine the factors responsible for these adverse effects and their potential treatments, we performed a series of studies in mice analyzing the bone marrow response to burn wound infection. The proliferative response of the marrow granulocyte-macrophage progenitor cell (GM-CFC) in male BDF1 mice undergoing a dorsal scald burn or burn wound seeding with 1000 colony forming units of Pseudomonas aeruginosa was determined on day 3 postburn using a clonal culture of GM-CFC. Mice with infected burn wounds had a rate of GM-CFC proliferation that was 50% that of noninfected animals and levels of circulating colony stimulating activity (CSA) 30% those of controls (p = 0.006). Similar suppression of marrow proliferative status could be replicated with the administration of endotoxin to normal or burned animals as had been observed for burn-infected animals. The administration of indomethacin (5 mg/kg.day) substantially restored the GM-CFC proliferation in mice with infected burns as well as in animals given endotoxin. Indomethacin-treated animals had CSA values 244% those of untreated burn-infected animals (p = 0.016). We take these observations to suggest that suppression of myelopoiesis in burn-infected animals is related in part to endotoxin-stimulated production of prostaglandin mediators that altered myeloid proliferation and was responsive to cyclooxygenase blockade.

Alterations of glucose transporter mRNA and protein levels in brain following thermal injury and sepsis in mice.

Since glucose transport and utilization are profoundly influenced by injury and infection, and the brain is an organ which primarily utilizes glucose as its energy source, we examined the status of the facilitative glucose transporters GLUT1 and GLUT3 in brain following thermal injury and infection. BDF1 mice underwent a 15% total body surface area burn with or without Pseudomonas aeruginosa infection. At 4 and 72 h post injury +/- infection, GLUT1 and GLUT3 mRNA abundance was measured by Northern blotting, and the correlative proteins determined using Western blotting. At 4 h, both brain GLUT1 mRNA and protein abundance were significantly increased in burned (mRNA 150 +/- 12%, protein 122 +/- 6%) and burn/infected (mRNA 165 +/- 11%, protein 119 +/- 5%) animals. At 72 h, GLUT1 mRNA and protein levels were also significantly increased in burn (mRNA: 139 +/- 11%, protein: 120 +/- 7%) and burn/infected (mRNA: 145 +/- 14%, protein: 138 +/- 8%) animals. Our studies suggest that alterations of GLUT1 mRNA and protein abundance were primary responses to the burn injury and were not further altered by burn wound infection.