Myelo-erythroid commitment after burn injury is under ß-adrenergic control via MafB regulation.

Severely injured burn patients receive multiple blood transfusions for anemia of critical illness despite the adverse consequences. One limiting factor to consider alternate treatment strategies is the lack of a reliable test platform to study molecular mechanisms of impaired erythropoiesis. This study illustrates how conditions resulting in a high catecholamine microenvironment such as burns can instigate myelo-erythroid reprioritization influenced by ß-adrenergic stimulation leading to anemia. In a mouse model of scald burn injury, we observed, along with a threefold increase in bone marrow LSK cells (linneg Sca1+cKit+), that the myeloid shift is accompanied with a significant reduction in megakaryocyte erythrocyte progenitors (MEPs). ß-Blocker administration (propranolol) for 6 days after burn, not only reduced the number of LSKs and MafB+ cells in multipotent progenitors, but also influenced myelo-erythroid bifurcation by increasing the MEPs and reducing the granulocyte monocyte progenitors in the bone marrow of burn mice. Furthermore, similar results were observed in burn patients' peripheral blood mononuclear cell-derived ex vivo culture system, demonstrating that commitment stage of erythropoiesis is impaired in burn patients and intervention with propranolol (nonselective ß1,2-adrenergic blocker) increases MEPs. Also, MafB+ cells that were significantly increased following standard burn care could be mitigated when propranolol was administered to burn patients, establishing the mechanistic regulation of erythroid commitment by myeloid regulatory transcription factor MafB. Overall, results demonstrate that ß-adrenergic blockers following burn injury can redirect the hematopoietic commitment toward erythroid lineage by lowering MafB expression in multipotent progenitors and be of potential therapeutic value to increase erythropoietin responsiveness in burn patients.

Nonviral human beta defensin-3 expression in a bioengineered human skin tissue: a therapeutic alternative for infected wounds.

The innate immune system differentially regulates the expression of host defense peptides to combat infection during wound healing. We enhanced the expression of a host defense peptide, human beta defensin-3 (hBD-3), in keratinocytes to generate a three-dimensional biologic dressing to improve healing of infected wounds. The NIKS human keratinocyte cell line was stably transfected ex vivo with a construct containing an epidermis-specific promoter driving hBD-3 (NIKS(hBD) (-3) ) using nonviral methods. Levels of hBD-3 mRNA and protein in three-dimensional skin tissue produced from NIKS(hBD) (-3) were determined using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Tissue architecture was characterized by hematoxylin and eosin staining and by indirect immunofluorescence using proliferation and keratinocyte differentiation markers. Antimicrobial activity was assessed using an in vitro bacterial growth assay and in vivo using a murine burn infection model. Three-dimensional full thickness skin tissues containing epidermal NIKS(hBD) (-3) or control NIKS possessed histologic features of interfollicular epidermis and exhibited normal tissue growth and differentiation. NIKS(hBD) (-3) tissue contained approximately fivefold more hBD-3 protein than tissue containing unmodified control NIKS. In vitro studies showed that NIKS(hBD) (-3) tissue produced a significant reduction in the growth of Staphylococcus aureus multiple peptide resistance factor (mprF) compared with control tissue. In an in vivo infected murine burn model, NIKS(hBD) (-3) tissue resulted in a 90% reduction in bacterial growth. These results demonstrate that sustained delivery of hBD-3 by a bioengineered skin tissue results in a therapeutically relevant reduction in growth of a S. aureus strain in an animal model of infected third-degree burn wounds.

Consumers in the Circular Economy: A Path Analysis of the Underlying Factors of Purchasing Behaviour.

One of the major obstacles to the adoption of the circular economy is the lack of consumer interest and awareness. Despite this, the unique role of consumers in the circular economy is an understudied topic, as the literature tends to focus rather on the application of circular practices in the organizational and industrial sectors. This paper aims to examine the individual-level factors that have an influence over circular purchasing behavior. Specifically, this paper elaborates an explicative path model of purchasing circular products that takes into account environmental concern, climate skepticism, and the attitudinal factor towards circular products. The final sample consisted of 566 respondents from Romania. Our findings showed that environmental concern has a positive significant impact on circular purchase behaviour and this relationship is mediated by the attitude towards circular products. Moreover, those with high levels of climate skepticism showed an increased level of perceived greenwashing among organizations.

How Should We Measure? A Review of Circular Cities Indicators.

As the world continues to urbanize, it is necessary to identify and implement new urban development models and strategies in order to meet the challenges of sustainable development. As cities continue to face challenges in becoming fully circular, the need to establish a framework to measure the circular economy in urban areas grows. Many definitions for circular cities have been developed and addressed in recent years, as have numerous indicators. To make the transition to a circular city, we must integrate the findings and develop a general definition and measurement framework. This article aims at outlining a framework for circular cities indicators based on their key characteristics, as well providing directions for fostering circularity at the city level. To accomplish this goal, we conducted a systematic review and analyzed key papers published in the field of circular economy to determine how circular cities are measured. Choosing the right indicators to use for developing, monitoring, and evaluating circular cities is a difficult task for urban policymakers, managers, and planners. This highlights the significance of standardized frameworks for urban indicators. As a result, the authors propose a framework and highlight some key points about circular cities and smart urban metabolism.

Enabling the Circular Economy Transition in Organizations: A Moderated Mediation Model.

The notion of Corporate Environmental Responsibility has been extensively researched in the literature so far, but less is known about how this concept fits into the circular economy paradigm. We performed a moderated mediation analysis in order to identify the mechanism that links corporate environmental responsibility with readiness for change towards a circular economy business model. The findings from 311 respondents show that there is a positive association between corporate environmental responsibility and the readiness for change to a circular model, mediated by perceived circular economy drivers. In addition, perceived circular economy barriers hinder this positive relationship, acting as a buffer. These findings can further contribute to the elaboration of a conceptual framework for embedding circular economy in the corporate social responsibility strategies of organizations.

Burn injury dampens erythroid cell production through reprioritizing bone marrow hematopoietic response.

BACKGROUND: Anemia in burn patients is due to surgical blood loss and anemia of critical illness. Because the commitment paradigm of common bone marrow progenitors dictates the production of erythroid, myeloid, and lymphoid cells, we hypothesized that skewed bone marrow lineage commitment decreases red cell production and causes anemia after a burn injury. METHODS: After anesthesia, B(6)D(2)F(1) mice received a 15% total body surface area dorsal scald burn. The sham group did not receive scald burn. Femoral bone marrow was harvested on 2, 5, 7, 14, and 21 postburn days (PBD). Total bone marrow cells were labeled with specific antibodies to erythroid (CD71/Ter119), myeloid (CD11b), and lymphoid (CD19) lineages and analyzed by flow cytometry. To test whether erythropoietin (EPO) could increase red blood cell production, EPO was administered to sham and burn animals and their reticulocyte response was measured on PBD 2 and PBD 7. RESULTS: Burn injury reduced the erythroid cells of the bone marrow from 35% in sham to 17% by PBD 5 and remained at similar level until PBD 21. Myeloid cells, however, increased from 42% in sham to 60% on PBD 5 and 77% on PBD 21. Burn injury reduced reticulocyte counts on PBD 2 and PBD 7 indicating that the erythroid compartment is severely depleted. This depleted compartment, however, responded to EPO but was not sufficient to change red cell production. CONCLUSION: Burn injury skews the bone marrow hematopoietic commitment away from erythroid and toward myeloid cells. Shrinkage of the erythroid compartment contributes to resistance to EPO and the anemia of critical illness.

Inhibition of multidrug-resistant Acinetobacter baumannii by nonviral expression of hCAP-18 in a bioengineered human skin tissue.

When skin is compromised, a cascade of signals initiates the rapid repair of the epidermis to prevent fluid loss and provide defense against invading microbes. During this response, keratinocytes produce host defense peptides (HDPs) that have antimicrobial activity against a diverse set of pathogens. Using nonviral vectors we have genetically modified the novel, nontumorigenic, pathogen-free human keratinocyte progenitor cell line (NIKS) to express the human cathelicidin HDP in a tissue-specific manner. NIKS skin tissue that expresses elevated levels of cathelicidin possesses key histological features of normal epidermis and displays enhanced antimicrobial activity against bacteria in vitro. Moreover, in an in vivo infected burn wound model, this tissue results in a two log reduction in a clinical isolate of multidrug-resistant Acinetobacter baumannii. Taken together, these results suggest that this genetically engineered human tissue could be applied to burns and ulcers to counteract bacterial contamination and prevent infection.

Terminal Maturation of Orthochromatic Erythroblasts Is Impaired in Burn Patients.

Mechanisms of erythropoietin (Epo)-resistant anemia in burn patients are poorly understood. We have recently found that administering a nonselective beta 1,2-adrenergic blocker propranolol (PR) was effective in reversing myelo-erythroid commitment through MafB regulation and increase megakaryocyte erythrocyte progenitors in burn patients' peripheral blood mononuclear cell (PBMC)-derived ex vivo culture system. Having known that Epo-dependent proliferation of early erythroblasts is intact after burn injury, here we inquired whether or not Epo-independent maturation stage of erythropoiesis is affected by burn injury and the relative role of PR on late-stage erythropoiesis. While majority of erythropoiesis occurs in the bone marrow, maturation into reticulocytes is crucial for their release into sinusoids to occupy the peripheral circulation for which enucleation is vital. peripheral blood mononuclear cells (PBMCs) from burn patients were extended beyond commitment and proliferation stages to late maturation stage in ex vivo culture to understand the role of PR in burn patients. Burn impedes late maturation of orthochromatic erythroblasts into reticulocytes by restricting the enucleation step. Late-stage erythropoiesis is impaired in burn patients irrespective of PR treatment. Further, substituting the microenvironment with control plasma (homologous) in place of autologous plasma rescues the conversion of orthochromatic erythroblasts to reticulocytes. Results show promise in formulating interventions to regulate late-stage erythropoiesis, which can be used in combination with PR to reduce the number of transfusions.

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.

Discrete ß-adrenergic mechanisms regulate early and late erythropoiesis in erythropoietin-resistant anemia.

BACKGROUND: Anemia of critical illness is resistant to exogenous erythropoietin. Packed red blood cells transfusions is the only treatment option, and despite related cost and morbidity, there is a need for alternate strategies. Erythrocyte development can be divided into erythropoietin-dependent and erythropoietin-independent stages. We have shown previously that erythropoietin-dependent development is intact in burn patients and the erythropoietin-independent early commitment stage, which is regulated by ß1/ß2-adrenergic mechanisms, is compromised. Utilizing the scald burn injury model, we studied erythropoietin-independent late maturation stages and the effect of ß1/ß2, ß-2, or ß-3 blockade in burn mediated erythropoietin-resistant anemia. METHODS: Burn mice were randomized to receive daily injections of propranolol (nonselective ß1/ß2 antagonist), nadolol (long-acting ß1/ß2 antagonist), butoxamine (selective ß2 antagonist), or SR59230A (selective ß3 antagonist) for 6 days after burn. Total bone marrow cells were characterized as nonerythroid cells, early and late erythroblasts, nucleated orthochromatic erythroblasts and enucleated reticulocyte subsets using CD71, Ter119, and Syto-16 by flow cytometry. Multipotential progenitors were probed for MafB expressing cells. RESULTS: Although propranolol improved early and late erythroblasts, only butoxamine and selective ß3-antagonist administrations were positively reflected in the peripheral blood hemoglobin and red blood cells count. While burn impeded early commitment and late maturation stages, ß1/ß2 antagonism increased the early erythroblasts through commitment stages via ß2 specific MafB regulation. ß3 antagonism was more effective in improving overall red blood cells through late maturation stages. CONCLUSION: The study unfolds novel ß2 and ß3 adrenergic mechanisms orchestrating erythropoietin resistant anemia after burn, which impedes both the early commitment stage and the late maturation stages, respectively.

Perturbed MafB/GATA1 axis after burn trauma bares the potential mechanism for immune suppression and anemia of critical illness.

Patients who survive initial burn injury are susceptible to nosocomial infections. Anemia of critical illness is a compounding factor in burn patients that necessitates repeated transfusions, which further increase their susceptibility to infections and sepsis. Robust host response is dependent on an adequate number and function of monocytes/macrophages and dendritic cells. In addition to impaired RBC production, burn patients are prone to depletion of dendritic cells and an increase in deactivated monocytes. In steady-state hematopoiesis, RBCs, macrophages, and dendritic cells are all generated from a common myeloid progenitor within the bone marrow. We hypothesized in a mouse model of burn injury that an increase in myeloid-specific transcription factor V-maf musculoaponeurotic fibrosarcoma oncogene homolog B at the common myeloid progenitor stage steers their lineage potential away from the megakaryocyte erythrocyte progenitor production and drives the terminal fate of common myeloid progenitors to form macrophages vs. dendritic cells, with the consequences being anemia, monocytosis, and dendritic cell deficits. Results indicate that, even though burn injury stimulated bone marrow hematopoiesis by increasing multipotential stem cell production (LinnegSca1poscKitpos), the bone marrow commitment is shifted away from the megakaryocyte erythrocyte progenitor and toward granulocyte monocyte progenitors with corresponding alterations in peripheral blood components, such as hemoglobin, hematocrit, RBCs, monocytes, and granulocytes. Furthermore, burn-induced V-maf musculoaponeurotic fibrosarcoma oncogene homolog B in common myeloid progenitors acts as a transcriptional activator of M-CSFR and a repressor of transferrin receptors, promoting macrophages and inhibiting erythroid differentiations while dictating a plasmacytoid dendritic cell phenotype. Results from small interfering RNA and gain-of-function (gfp-globin transcription factor 1 retrovirus) studies indicate that targeted interventions to restore V-maf musculoaponeurotic fibrosarcoma oncogene homolog B/globin transcription factor 1 balance can mitigate both immune imbalance and anemia of critical illness.

Correlation between conformational stability of the ternary enzyme-substrate complex and domain closure of 3-phosphoglycerate kinase.

3-phosphoglycerate kinase (PGK) is a typical two-domain hinge-bending enzyme with a well-structured interdomain region. The mechanism of domain-domain interaction and its regulation by substrate binding is not yet fully understood. Here the existence of strong cooperativity between the two domains was demonstrated by following heat transitions of pig muscle and yeast PGKs using differential scanning microcalorimetry and fluorimetry. Two mutants of yeast PGK containing a single tryptophan fluorophore either in the N- or in the C-terminal domain were also studied. The coincidence of the calorimetric and fluorimetric heat transitions in all cases indicated simultaneous, highly cooperative unfolding of the two domains. This cooperativity is preserved in the presence of substrates: 3-phosphoglycerate bound to the N domain or the nucleotide (MgADP, MgATP) bound to the C domain increased the structural stability of the whole molecule. A structural explanation of domain-domain interaction is suggested by analysis of the atomic contacts in 12 different PGK crystal structures. Well-defined backbone and side-chain H bonds, and hydrophobic and electrostatic interactions between side chains of conserved residues are proposed to be responsible for domain-domain communication. Upon binding of each substrate newly formed molecular contacts are identified that firstly explain the order of the increased heat stability in the various binary complexes, and secondly describe the possible route of transmission of the substrate-induced conformational effects from one domain to the other. The largest stability is characteristic of the native ternary complex and is abolished in the case of a chemically modified inactive form of PGK, the domain closure of which was previously shown to be prevented [Sinev MA, Razgulyaev OI, Vas M, Timchenko AA & Ptitsyn OB (1989) Eur J Biochem180, 61-66]. Thus, conformational stability correlates with domain closure that requires simultaneous binding of both substrates.

Activities of nonlysosomal proteolytic systems in skeletal and cardiac muscle during burn-induced hypermetabolism.

The aim of this study was to assess the activity of nonlysosomal proteolytic systems in skeletal and cardiac muscle during burn-induced hypermetabolism (BHM) in rats. Rats underwent 30% TBSA scald burn or sham injury and were observed for up to 42 days. Body weights and resting energy expenditures were determined weekly. Skeletal (soleus/pectoral) muscle and hearts were harvested on days 0 (=control), 7, 14, 21, and 42 after burn. Calpain, caspase-1, caspase-3/7, caspase-6, caspase-8, caspase-9, and proteasome peptidase activities were measured in tissue extracts. Hypermetabolism developed within 3 weeks after burns, as documented by increased resting energy expenditures and decreased body weights on postburn days 21 to 42 (P < 0.05 vs control). Calpain activities did not show significant alterations. Pan caspase activities increased by time and were significantly increased in skeletal and cardiac muscle extracts during hypermetabolism. Although increases in caspase-1, caspase-8, and caspase-9 activities were predominantly responsible for elevated pan caspase activities in skeletal muscle, increases in caspase-6 activities dominated in the heart. Proteasome peptidase activities in skeletal muscle extracts were not significantly altered. Proteasome peptidase activities in heart extracts increased time dependently and were significantly increased during BHM. Activation of caspase cascades during BHM constitutes a uniform response in skeletal and cardiac muscle and may contribute to enhanced metabolic protein turnover. Activation of myocardial proteasome activities may reflect persistent cardiac stress. Further exploration of caspase cascades and the proteasome as therapeutic targets to influence long-term consequences of BHM appears justified.

High MafB expression following burn augments monocyte commitment and inhibits DC differentiation in hemopoietic progenitors.

We have previously shown that perturbed bone marrow progenitor development promotes hyporesponsive monocytes following experimental burn sepsis. Clinical and experimental sepsis is associated with monocyte deactivation and depletion of mDCs. Decrease in circulating DCs is reported in burn patients who develop sepsis. In our 15% TBSA scald burn model, we demonstrate a significant reduction in the circulating MHC-II(+) population and mDCs (Gr1(neg)CD11b(+)CD11c(+)) with a corresponding decrease in bone marrow MHC-II(+) cells and mDCs for up to 14 days following burn. We explored the underlying mechanism(s) that regulate bone marrow development of monocytes and DCs following burn injury. We found a robust bone marrow response with a significant increase in multipotential HSCs (LSK) and bipotential GMPs following burn injury. GMPs from burn mice exhibit a significant reduction in GATA-1, which is essential for DC development, but express high levels of MafB and M-CSFRs, both associated with monocyte production. GMPs obtained from burn mice differentiated 1.7 times more into Mϕ and 1.6-fold less into DCs compared with sham. Monocytes and DCs expressed 50% less MHC-II in burn versus sham. Increased monocyte commitment in burn GMPs was a result of high MafB and M-CSFR expressions. Transient silencing of MafB (siRNA) in GMP-derived monocytes from burn mice partially restored DC differentiation deficits and increased GATA-1 expression. We provide evidence that high MafB following burn plays an inhibitory role in monocyte-derived DC differentiation by regulating M-CSFR and GATA-1 expressions.

Dendritic cell depletion in burn patients is regulated by MafB expression.

Studies have shown that monocytes are hyporesponsive and that dendritic cells (DCs) are depleted in burn patients. We have recently shown in a mouse model that burn injury alters the transcriptional regulation in bone marrow progenitors and inhibits myeloid-derived DC (mDC) production. In the present study, using human burn patient peripheral blood mononuclear cells, we have shown an overexpression of MafB with a corresponding reduction in peripheral blood mononuclear cell-derived mDCs. We isolated mononuclear cells from burn patient (23­68% TBSA) and control volunteer peripheral blood samples by Ficoll gradient centrifugation and cultured mDCs by using a standard ex vivo culture system. Fluorescence-activated cell sorter analysis was used to select myeloid cells based on the cell surface expression of CD45+. The mDC fraction was identified by the expression of human leukocyte antigen (HLA)-DR+CD11c+, and we found a significant reduction in HLA-DR+ leukocytes for up to 4 weeks postburn. MafB expression was then examined in HLA-DR+CD14+ monocytes. Burn injury alters the phenotype of CD14+ monocytes augmenting MafB expression and reducing their differentiation into mDCs. MafB was then silenced in ex vivo culture prior to DC differentiation by using small interfering RNA technique. MafB gene silencing improved the differentiation potential of CD14+ cells into mDCs, increasing the percentage of mDCs by >75%. Furthermore, GATA-1+ and HLA-DR+ mDCs were increased following MafB silencing. Although burn injury augments the number of peripheral blood monocytes, the frequency of mDC is reduced. This impairment is likely secondary to the down-regulation of mDC differentiation by high MafB-expressing monocytes following burn injury.

ß-adrenergic stimulation increases macrophage CD14 expression and E. coli phagocytosis through PKA signaling mechanisms.

CD14 is a glycoprotein that binds bacterial LPS in MØ. It is an essential component of the phagocytic system and is increased in septic shock. Critical injury and sepsis result in elevated endogenous CA levels. CAs have a significant impact on MØ inflammatory functions. We tested the hypothesis that ß-adrenergic stimulation regulates CD14 expression and bacterial phagocytosis in BMØ. Murine BMØ stimulated with isoproterenol (>8 h) induced a dose-dependent increase in cell surface CD14 expression. Specific PKA inhibitor (H-89) and gene-silencing (siRNA) studies demonstrated the role of cAMP-dependent PKA in mediating this response. In addition, we observed a correlation between an isoproterenol-mediated increase in CD14 expression and live Escherichia coli uptake in BMØ. Further, the essential role of CD14 in an isoproterenol-mediated increase in E. coli uptake was highlighted from experiments using CD14(-/-) mice. Moreover, the dose response of isoproterenol stimulation to CD14 expression and E. coli phagocytosis overlapped with similar EC50. Additionally, isoproterenol-mediated E. coli phagocytosis was prevented by H-89, suggesting that ß-adrenergic stimulus in BMØ increases CD14 expression and live E. coli phagocytosis through a common signaling pathway. Our studies indicate the potential impact of ß-adrenergic agents on important innate immune functions.

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.

Propranolol restores the tumor necrosis factor-alpha response of circulating inflammatory monocytes and granulocytes after burn injury and sepsis.

Beta-adrenergic blockade ameliorates the hypermetabolism and catabolism in severe burn injury. Despite the salutary effects of beta-adrenergic blockade, the immunologic responses that accompany beta-blockade are not known. We have shown that burn sepsis is associated with increased sympathetic activation leading to altered monocytopoiesis and cytokine release in macrophages (MØ). Recent evidence suggests that murine MØ expressing F4/80+Gr1+ are the inflammatory phenotype. Here, we report that propranolol given after burn sepsis modulates the number and function of myeloid cells in circulation. B6D2F1 male mice were divided into sham (S), burn (B), and burn sepsis (BS) groups. Dorsal hair was shaved from S, B, and BS; B and BS received 15% scald burn; BS was inoculated with Pseudomonas Aeruginosa (PA 14, 4000-5000 colony-forming units) at the burn site. Mice from each group were then subjected to two different treatment regimens. One set received subcutaneous injections of propranolol (5 mg/kg body weight) at 24 and 48 hours after the injury while the control groups received saline. Blood was collected by cardiac puncture at 72 hours. The distribution of total F4/80+ monocyte population was determined by flow cytometry. Inflammatory monocyte subset was gated on Gr1+ expression in the F4/80+ fraction. Lipopolysaccharide-stimulated intracellular tumor necrosis factor (TNF)-alpha (ic-TNF) was also measured as an indicator of inflammatory response. The total F4/80+ monocyte fraction was significantly increased in BS (45 +/- 0.8%) vs S and B (10 +/- 0.8%; 9.5 +/- 0.6%). Propranolol treatment for 2 days reduced the number of circulating monocytes by 60% in BS. The mean fluorescent intensity (MFI) of ic-TNF produced per cell (F4/80+Gr1+ MØ) was significantly decreased in B and BS (S: 3043 +/- 213, B: 1638 +/- 343, BS: 1463 +/- 67). Of importance, propranolol treatment partially restored the MFI of ic-TNF (2177 +/- 114) and increased the percentage of inflammatory monocyte subset (F4/80+Gr1+) in BS by 70% compared with saline treatment. In contrast, beta-blockade after BS increased the percentage of granulocytes in circulation (28.4 +/- 3.6% in BS propranolol vs 15.4 +/- 0.3% in BS saline; P < .05) and augmented their TNF production (MFI = 903 +/- 102 in BS propranolol vs 644 +/- 5 in BS saline; P < .05). Propranolol reverses burn sepsis-induced monocytosis and simultaneously increases the number of granulocytes and enhances the inflammatory potential of the granulocytes and inflammatory monocyte subsets in circulation suggesting that monitoring MØ subsets and granulocytes in blood is a reliable biomarker to predict the efficacy of beta-blockade.

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.

A novel antibacterial gene transfer treatment for multidrug-resistant Acinetobacter baumannii-induced burn sepsis.

Sepsis caused by multidrug-resistant bacterial infections in critically injured patients has become a major clinical problem. Recently, Acinetobacter baumannii (AB) wound infections, especially in our critically injured soldiers fighting in Iraq and Afghanistan, is posing a major clinical problem and an economic burden. ConjuGon, Inc., has developed a novel antibacterial therapeutic technology using bacterial conjugation. The donor cells are attenuated Escherichia coli carrying a conjugative plasmid. The expression of bactericidal genes cloned on the plasmid is tightly repressed in the donor cells but becomes de-repressed once mobilized into a pathogen and disrupts protein synthesis. Here, we tested the efficacy of this novel conjugation technology to control and eradicate a drug-resistant clinical isolate of AB wound infection both in vitro and in a murine burn sepsis model. C57Blk/6J mice were divided into burn (B) and burn sepsis (BS) groups. All animals received a 12% TBSA dorsal scald full-thickness burn. The BS group was inoculated with multidrug-resistant AB (1 x 10(5) colony-forming units [CFU]) at the burn wound site. BS animals were either untreated or treated with increasing concentrations (10(3) - 19(10) CFU) of attenuated donor E. coli encoding bactericidal proteins. The survival rate was monitored for 10 days. The ability of donor cells to significantly diminish AB levels in the burn wound 24 hours after injury was determined by quantitative cultures. Donor cells were highly effective in killing AB in vitro. In the burn sepsis model, 90% B group animals survived, and 40% to 50% BS animals survived with no treatment in 5 to 6 days. Treatment with donor cells at 10(10) to 10(6) provided significant survival advantage (P < .05). Quantitative cultures of burn wounds revealed that AB numbers increased from 3 x 10(4) CFU to 7.8 +/- 4.4 x 10(9) CFU in 24 hours in the untreated group. Single treatment with donor cells (10(10) CFU) significantly reduced AB in the burn wound to less than the levels seeded into the wound (1.23 +/- 0.5 x 10(4) CFU; P < .05). Taken together, these results indicate that this novel technology is an efficient method to control drug-resistant AB burn wound infections and prevent their systemic spread.