Murine leukocyte dysfunction in response to a non-lethal flame burn.

Long-lasting neurobehavioral alterations in burn-injured mice resembling post-traumatic stress disorder in humans

Burn injury induces bacterial translocation (BT) from the gut in multiple animal models. Etiologic factors contributing to BT may be an ischemia-reperfusion injury to the gut, the release of inflammatory cytokines, oxygen metabolites and other mediators, and cytotoxic effects mediated by endotoxin (lipopolysaccharide). Bactericidal, permeability-increasing protein is a neutrophil granule protein with potent bactericidal and lipopolysaccharide-neutralizing activities. The use of this protein has not been previously reported in a burn-injury model. The purpose of this study was to determine whether recombinant bactericidal, permeability-increasing protein (rBPI23) affects the incidence of BT and myeloperoxidase content in lung tissue (a measure of leukocyte sequestration) in a burn-injury model. Mice received a 32% total body surface area, full-thickness, scald burn, and 10 mg/kg body weight rBPI23 in saline solution was given by intraperitoneal injection at 0, 3, and 6 hours after the burn. Control animals received intraperitoneal saline solution only. All animals received a total of 1 ml saline solution intraperitoneally immediately after burn injury for fluid resuscitation. At 24 hours after burn injury, mesenteric lymph nodes (MLN) were harvested, homogenized, and plated. Lung tissue was harvested and assayed for myeloperoxidase. Burned mice treated with rBPI23 had significantly (p = 0.005, Fisher's Exact Test, two-tailed) decreased incidence of BT, compared to burned mouse controls. Leukosequestration into lung tissues was not affected by rBPI23. Postburn administration of rBPI23 reduces but does not abolish the incidence of BT after burn injury in mice, perhaps by reducing intestinal injury during burn shock and the ischemia-reperfusion period by inhibiting the effects of lipopolysaccharide. An alternate explanation may be that rBPI23 could increase clearance and killing of bacteria by host defenses.

Burn wounds are prone to infection by Pseudomonas aeruginosa, which is an opportunistic pathogen causing various human diseases. During infection, the bacterium senses environmental changes and regulates the expression of genes appropriate for survival. A purine-auxotrophic mutant of P. aeruginosa was unable to replicate efficiently on burn wounds, suggesting that burn wounds are purine-deficient environments. An in vivo expression technology based on purEK gene expression was applied to the burned mouse infection model to isolate P. aeruginosa genes that are specifically induced during infection. Four such in vivo-inducible (ivi) genetic loci were identified, including the gene for a superoxide response regulator (soxR), the gene for a malate synthase G homologue (glcG), an antisense transcript of a putative regulator responding to copper (copR), and an uncharacterized genetic locus. SoxR of Escherichia coli is known to regulate genes involved in protecting the bacterium against oxidative stress. The expression of soxR was proven to be highly inducible during the infection of burned mice and also inducible by treatment with paraquat, which is a redox-cycling reagent generating intracellular superoxide. The SoxR protein functions as an autorepressor in the absence of paraquat, whereas in the presence of paraquat, this autorepression is diminished. Furthermore, a soxR null mutant was shown to be much more sensitive than wild-type P. aeruginosa to macrophage-mediated killing. In support of this observation, a soxR null mutant exhibited a significant delay in causing systemic infections in the burned mice. Since most mortality in burn patients is caused by systemic infection, the defect in the ability to cause efficient bacteremia in burned mice suggests an important role of the soxR gene in the infection of burn wounds.

Objective: To investigate the effects of hydrogen on the lung damage of mice at early stage of severe burn. Methods: One hundred and sixty ICR mice were divided into sham injury, hydrogen, pure burn, and burn+ hydrogen groups according to the random number table, with 40 mice in each group. Mice in pure burn group and burn+ hydrogen group were inflicted with 40% total body surface area full-thickness scald (hereafter referred to as burn) on the back, while mice in sham injury group and hydrogen group were sham injured. Mice in hydrogen group and burn+ hydrogen group inhaled 2% hydrogen for 1 h at post injury hour (PIH) 1 and 6, respectively, while mice in sham injury group and pure burn group inhaled air for 1 h. At PIH 24, lung tissue of six mice in each group was harvested, and then pathological changes of lung tissue were observed by HE staining and the lung tissue injury pathological score was calculated. Inferior vena cava blood and lung tissue of other eight mice in each group were obtained, and then content of high mobility group box 1 (HMGB1) and interleukin-6 (IL-6) in serum and lung tissue was determined by enzyme-linked immunosorbent assay. Activity of superoxide dismutase (SOD) in serum and lung tissue was detected by spectrophotometry. After arterial blood of other six mice in each group was collected for detection of arterial partial pressure of oxygen (PaO(2)), the wet and dry weight of lung tissue were weighted to calculate lung wet to dry weight ratio. The survival rates of the other twenty mice in each group during post injury days 7 were calculated. Data were processed with one-way analysis of variance, LSD test and log-rank test. Results: (1) At PIH 24, lung tissue of mice in sham injury group and hydrogen group showed no abnormality. Mice in pure burn group were with pulmonary interstitial edema, serious rupture of alveolar capillary wall, and infiltration of a large number of inflammatory cells. Mice in burn+ hydrogen group were with mild pulmonary interstitial edema, alveolar capillary congestion accompanied by slight rupture and bleeding, and the number of infiltration of inflammatory cells was smaller than that in pure burn group. The lung tissue injury pathological scores of mice in sham injury group, hydrogen group, pure burn group, and burn+ hydrogen group were (0.7±0.5), (0.8±0.5), (6.1±1.0), and (2.8±0.8) points, respectively. The lung tissue injury pathological score of mice in pure burn group was significantly higher than that in sham injury group (P 0.05). (5) At PIH 24, the ratios of lung wet to dry weight of mice in sham injury, hydrogen, pure burn, and burn+ hydrogen groups were 3.52±0.22, 3.61±0.24, 7.24±0.32, and 5.21±0.41, respectively. The ratio of lung wet to dry weight of mice in pure burn group was significantly higher than that in sham injury group (P<0.001). The ratio of lung wet to dry weight of mice in burn+ hydrogen group was significantly lower than that in pure burn group (P<0.001). (6) The survival rates of mice in sham injury group and hydrogen group during post injury days 7 were 100%. Compared with those in sham injury group, survival rates of mice in pure burn group from post injury days 3 to 7 were significantly decreased (with P values below 0.05). Compared with those in pure burn group, survival rates of mice in burn+ hydrogen group from post injury days 5 to 7 were significantly increased (with P values below 0.05). Conclusions: Hydrogen can significantly alleviate the infiltration of inflammatory cells and improve the pathological lesions of lung tissue of mice with severe burn. It has the effects of reducing inflammatory reaction and inhibiting oxidative stress, further showing the protective effect on the lung of burn mice.

Cytokine expression profile over time in burned mice

Vitamin E improves cell-mediated immunity in the burned mouse: A preliminary study

Exoenzyme S is an extracellular ADPR transferase produced by P. aeruginosa. Forms of this enzyme that have thus far been purified are not toxic, however, exoenzyme S clearly contributes to the virulence of strain 388. Thus, a Tn1 mutant deficient in exoenzyme S was found to be markedly less virulent than its exoenzyme S-producing parental strain in both a burned mouse infection model and a rat chronic lung infection model. Exoenzyme S does not appear to contribute to initial colonization of the rat lung or the burned mouse skin. Exoenzyme S does, however, appear to contribute to local tissue damage in the rat lung, and to dissemination of P. aeruginosa from the skin into the blood and distant organs of the burned mouse. Perhaps our most important observation is that specific antibody against exoenzyme S confers a high level of protection against subsequent infection of burned mice. While these results must be extended to include additional strains they are encouraging, and they underscore the relative importance of exoenzyme S in the pathogenesis of P. aeruginosa infections.

BackgroundIn cystic fibrosis (CF), impaired immune cell responses, driven by the dysfunctional CF transmembrane conductance regulator (CFTR) gene, may determine the disease severity but clinical heterogeneity remains a major therapeutic challenge. The characterization of molecular mechanisms underlying impaired immune responses in CF may reveal novel targets with therapeutic potential. Therefore, we utilized simultaneous RNA sequencing targeted at identifying differentially expressed genes, transcripts, and miRNAs that characterize impaired immune responses triggered by CF and its phenotypes.MethodsPeripheral blood mononuclear cells (PBMCs) extracted from a healthy donor were stimulated with plasma from CF patients (n = 9) and healthy controls (n = 3). The PBMCs were cultured (1 × 105 cells/well) for 9 h at 37 ° C in 5% CO2. After culture, total RNA was extracted from each sample and used for simultaneous total RNA and miRNA sequencing.ResultsAnalysis of expression signatures from peripheral blood mononuclear cells induced by plasma of CF patients and healthy controls identified 151 genes, 154 individual transcripts, and 41 miRNAs differentially expressed in CF compared to HC while the expression signatures of 285 genes, 241 individual transcripts, and seven miRNAs differed due to CF phenotypes. Top immune pathways influenced by CF included agranulocyte adhesion, diapedesis signaling, and IL17 signaling, while those influenced by CF phenotypes included natural killer cell signaling and PI3K signaling in B lymphocytes. Upstream regulator analysis indicated dysregulation of CCL5, NF-κB and IL1A due to CF while dysregulation of TREM1 and TP53 regulators were associated with CF phenotype. Five miRNAs showed inverse expression patterns with three target genes relevant in CF-associated impaired immune pathways while two miRNAs showed inverse expression patterns with two target genes relevant to a dysregulated immune pathway associated with CF phenotypes.ConclusionsOur results indicate that miRNAs and individual transcript variants are relevant molecular targets contributing to impaired immune cell responses in CF.

Effect of neutralizing transforming growth factor B1 on murine Pseudomonas aeruginosa clearance after thermal injury

‘Metabolic’ factors in thermal injury: role of nucleic acids

In this study, intradermal MRSA infection in burn mice was pathogenically analyzed. Burn mice (3rd degree, 25% TBSA flame burn) and normal mice were infected i.d. with 108 CFU/mouse of MRSA, and abscesses formed in the infection sites and mortality rates of these mice were examined. Also, the abscess formation was examined in burn and normal mice after inoculation with F4/80+Mϕ isolated from tissues surrounding the infection sites of normal (N-F4/80 Mϕ) or burn mice (B-F4/80 Mϕ). Finally, these Mϕ were biophysically characterized. In the results, the abscess was formed in normal mice infected with MRSA and all of them survived. However, the abscess was not formed in burn mice infected with MRSA, and all of them died. The abscess was formed and sepsis was not developed in burn mice inoculated with N-F4/80 Mϕ. However, the abscess was not formed and sepsis was developed in normal mice inoculated with B-F4/80 Mϕ. N-F4/80 Mϕ produced IL-12 (but not IL-10) and were characterized as M1Mϕ, whereas B-F4/80 Mϕ produced IL-10 (but not IL-12) and were characterized as M2Mϕ. These results indicate that the abscess formation is a major host defense against MRSA i.d. infection. M1Mϕ at tissues surrounding the infection site play a pivotal role in the abscess formation. An abscess is not formed in burn mice predominated with M2Mϕ, which are inhibitory on the M1Mϕ generation. This study was supported by SHC NA #8840. A.A was supported by The James W. McLaughlin Fellowship Fund.

Background Although glucagon-like peptide 1- (GLP-1-) based therapy of hyperglycemia in burn injury has shown great potential in clinical trials, its safety is seldom evaluated. We hypothesize that exendin-4, a GLP-1 analogue, might affect the immune response via the activation of the sympathetic nervous system in burn injury. Methods Male Balb/c mice were subjected to sham or thermal injury of 15% total body surface area. Exendin-4 on T cell function in vitro was examined in cultured splenocytes in the presence of β-adrenoceptor antagonist propranolol (1 nmol/L) or GLP-1R antagonist exendin (9-39) (1 μmol/L), whereas its in vivo effect was determined by i.p. injection of exendin-4 (2.4 nmol/kg) in mice. To further elucidate the sympathetic mechanism, propranolol (30 mg/kg) or vehicle was applied 30 min prior to injury. Results Although the exacerbated burn-induced mortality by exendin-4 was worsened by propranolol pretreatment, the inhibition of T cell proliferation by exendin-4 in vitro could be restored by propranolol instead of exendin (9-39). However, a Th2 switch by exendin-4 in vitro could only be reversed by exendin (9-39). Likewise, the inhibition of splenic T cell function and NFAT activity by exendin-4 in vivo was restored by propranolol. By contrast, the increased splenic NF-κB translocation by exendin-4 in vivo was potentiated by propranolol in sham mice but suppressed in burn mice. Accordingly, propranolol abrogated the heightened inflammatory response in the lung and the accelerated organ injuries by exendin-4 in burn mice. On the contrary, a Th2 switch and higher serum levels of inflammatory mediators by exendin-4 were potentiated by propranolol in burn mice. Lastly, exendin-4 raised serum stress hormones which could be remarkably augmented by propranolol. Conclusions Exendin-4 suppresses T cell function and promotes organ inflammation through the activation of the sympathetic nervous system, while elicits Th2 switch via GLP-1R in burn injury.

Study on the effect and mechanism of recombinant human intestinal trefoil factor on intestinal mucosal injury and repair in burned mice

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that causes severe infections in patients with burns. The P aeruginosa regulatory gene, lasR, regulates the expression of several virulence factors. The specific lasR isogenic mutant, PAO-R1, is defective in the synthesis of the 2 elastases (LasB and LasA) and produces low levels of exotoxin A and alkaline proteases. In this study, we used a burned mouse model to examine the role of lasR in the pathogenesis of P aeruginosa infections. We have examined the following aspects of P aeruginosa infections: 1) lethality to the burned mouse, 2) the dissemination within the body of the burned mouse, and 3) the local spread within the burned skin. In comparison with its parent strain, PAO1, PAO-R1 was less lethal. In addition, the numbers of PAO-R1 microorganisms recovered from the livers and spleens of the burned mice were less than those of PAO1. Furthermore, at 8 hours postinfection, equivalent numbers of PAO1 and PAO-R1 were detected at the inoculation site of the burned skin. However, only PAO1 microorganisms were detected at other sites of the burned skin. These results suggest that the lasR gene contributes (directly and indirectly) to the dissemination of P aeruginosa within the body of burned mice and its horizontal spread within the burned skin.

Gram-negative sepsis causes morbidity and mortality in burned patients. To determine whether immunization with core endotoxin (lipopolysaccharide) via one of two routes could protect burned mice from septic death, mice were immunized either three times subcutaneously (SC) or one time intramuscularly (IM) then two times intraperitoneally (IP) with a core-lipopolysaccharide vaccine. Control mice were immunized with either saline or an irrelevant antigen. Postimmunization, mice were immunocompromised with a 15% TBSA flame burn and challenged subeschar with Klebsiella pneumoniae or Escherichia coli. Vaccine immunization improved the survival of both E. coli- and K. pneumoniae-challenged mice when given SC but not when given IM, IP. Postimmunization, total immunoglobulin titers were elevated over preimmune titers, but titers in IM, IP-immunized mice were higher than those in SC-immunized mice. Both isotyping and flow cytometry studies indicated that sera from mice immunized via IM, IP opsonized better than sera from mice immunized via SC. Hence, this vaccine provided route-specific protection of burned mice against gram-negative sepsis; its mechanism of action was not solely dependent upon increased immunoglobulin titers or phagocytosis.