Eschar dissolution and the immunoregulator effect of keratinase on burn wounds.

At present, enzyme debridement preparation has shown a good curative effect on eschar removal of burn wounds. Keratinase has shown great potential in enzymatic debridement because of its good fibrin-degrading ability. In this study, the debridement of keratinase was examined by using a third degree burn wound model in rats. We observed the wound, and keratinase shortened the time of eschar dissolution after debridement. Histopathology and immunofluorescence staining showed that the eschar in the keratinase group became thinner, inflammatory cell infiltration in the wound increased, the fluorescence intensity of the macrophage surface marker CD68 increased, and the CD163/CD86 ratio increased. In bone marrow-derived macrophages (BMDMs), there was no significant difference in the activity of CCK-8 in cells in the keratinase group compared with the control group. The fluorescence intensity of the keratinase group was higher than that of the control group. At 12 h, the cell scratches were obviously closed. The number of migrated Transwell cells increased. Flow cytometry and immunofluorescence analysis showed increased expression of CD206 and Arg-1 and decreased expression of CD86 and iNOS. The gene expression of the Arg-1, iNOS and IL-10 was increased, as shown by qPCR. The secretion of IL-10 was increased and TNF-α was decreased, as shown by ELISA. We concluded that keratinase dissolution of eschar not only has a hydrolytic effect on eschar but may also affect immune regulation to enhance the migration and phagocytosis of macrophages, promote the polarization of macrophages, and further enhance the effect of eschar dissolution. Therefore, keratinase may have good prospects for the debridement of burn wounds.

Fluorimetric ex vivo quantification of protease debriding efficacy on natural substrate.

Debridement is the process of removal of necrotic and infected tissue to clean a wound or burn and expedite healing. Proteases such as papain, bromelain, and collagenase that promote debridement by degrading proteins in the dead tissue are in use today. However, the only method to measure debriding efficacy in vitro is the fluorescent monitoring of the digestion of an Artificial Wound Eschar (AWE) substrate. This AWE substrate contains a pellet of only three eschar matrix proteins collagen, elastin, and fibrin which do not account for the complexity and the composition of necrotic tissue. Here, we describe an ex vivo method using dry necrotic full thickness human skin and ortho-phthalaldehyde (OPA), a molecule commonly used for sensitive fluorimetric protein detection to monitor debridement activity. We advocate this simple yet sensitive approach to detect debridement efficacy that can readily be used commercially to benchmark products prior to in vivo testing.

Protease levels are significantly altered in pediatric burn wounds.

Burn wounds contain high levels of protease activity due to the need to remodel the damaged extracellular matrix proteins. While necessary, excessive protease activity can lead to improper wound healing and is associated with increased contraction and fibrosis. No studies to date have investigated the expression changes of all the collagenases and elastases in burn wounds. The present study compares gene expression changes and changes in collagenase and elastase activity between burn wound eschar and normal skin in a pediatric population. Deidentified pediatric tissues were used for these experiments. Burn wound tissue was excised as part of normal standard care within a week from injury; normal skin was removed during elective plastic surgery procedures. RNA-sequencing was performed and significant results were confirmed with qRT-PCR. Activity assays showed a significant increase in both collagenase and elastase activity in the burn wound tissue compared to the normal skin. Western blotting and substrate zymography of tissue homogenates evaluated the results at the protein levels. Four elastases and three collagenases were determined to be significantly upregulated in the wound tissues by both RNA-sequencing and qRT-PCR. Cathepsin V was the only protease that was significantly downregulated. All but one metalloproteinase studied was significantly upregulated. None of the serine proteases were significantly altered in the wound tissues. In conclusion, matrix metalloproteinases appear to be the most highly elevated proteases after a pediatric burn wound injury, at least within the first 3-7 days. The data warrant further investigation into the effects of MMPs on burn wound healing.

Dysregulation of the actin scavenging system and inhibition of DNase activity following severe thermal injury.

BACKGROUND: Circulating cell-free DNA (cfDNA) is not found in healthy subjects, but is readily detected after thermal injury and may contribute to the risk of multiple organ failure. The hypothesis was that a postburn reduction in DNase protein/enzyme activity could contribute to the increase in cfDNA following thermal injury. METHODS: Patients with severe burns covering at least 15 per cent of total body surface area were recruited to a prospective cohort study within 24 h of injury. Blood samples were collected from the day of injury for 12 months. RESULTS: Analysis of blood samples from 64 patients revealed a significant reduction in DNase activity on days 1-28 after injury, compared with healthy controls. DNase protein levels were not affected, suggesting the presence of an enzyme inhibitor. Further analysis revealed that actin (an inhibitor of DNase) was present in serum samples from patients but not those from controls, and concentrations of the actin scavenging proteins gelsolin and vitamin D-binding protein were significantly reduced after burn injury. In a pilot study of ten military patients with polytrauma, administration of blood products resulted in an increase in DNase activity and gelsolin levels. CONCLUSION: The results of this study suggest a novel biological mechanism for the accumulation of cfDNA following thermal injury by which high levels of actin released by damaged tissue cause a reduction in DNase activity. Restoration of the actin scavenging system could therefore restore DNase activity, and reduce the risk of cfDNA-induced host tissue damage and thrombosis.

ANTECEDENTES: El ADN libre de las células circulantes (circulating cell-free DNA, cfDNA) no se encuentra en sujetos sanos, pero se detecta fácilmente después de una lesión térmica y puede contribuir al riesgo de fallo multiorgánico. La hipótesis fue que una disminución en la actividad de la proteína/enzima ADNasa tras la lesión térmica podría contribuir a la elevación del cfDNA que ocurre tras la misma. MÉTODOS: Los pacientes con quemaduras graves con una extensión ≥ 15% del área de superficie corporal total (total body surface area, TBSA) se incluyeron en un estudio prospectivo de cohortes durante las primeras 24 horas posteriores a la lesión. Se recogieron muestras de sangre desde el día de la lesión hasta los 12 meses posteriores a la misma. RESULTADOS: El análisis de muestras de sangre de 64 pacientes reveló una reducción significativa de la actividad de la ADNasa en los días 1 a 28 después de la lesión, en comparación con los controles sanos. Los niveles de proteína ADNasa no se vieron afectados, lo que sugiere la presencia de un inhibidor enzimático. Un análisis adicional reveló que la actina (un inhibidor de la ADNasa) estaba presente en las muestras de suero de los pacientes, pero no en los controles, y las concentraciones de la gelsolina, proteína que causa la disociación de la actina, y la proteína de unión a la vitamina D se redujeron significativamente después de la lesión térmica. En un estudio piloto de 10 pacientes con politrauma por lesiones militares, la administración de hemoderivados produjo un aumento en la actividad de la ADNasa y de los niveles de gelsolina. CONCLUSIÓN: Este estudio sugiere un nuevo mecanismo biológico para la acumulación de cfDNA después de una lesión térmica, por el cual los altos niveles de actina liberada por el tejido dañado causarían una reducción en la actividad de la ADNasa. La restauración del sistema eliminador de actina podría, por lo tanto, restaurar la actividad de la ADNasa y reducir el riesgo de daño tisular y trombosis en el huésped inducido por el cfDNA.

Cold atmospheric plasma modulates endothelial nitric oxide synthase signalling and enhances burn wound neovascularisation.

Treatment with cold atmospheric plasma (CAP) has been reported to promote wound healing in animals. However, how this process is mediated remains unclear. In this study we examined the mechanisms which underlie the improved wound healing effects of CAP and the roles of associated reactive oxygen and nitrogen species (RONS), which are generated by plasma. By using in vitro models which mimicked various steps of angiogenesis, we demonstrated that CAP triggered the production of nitric oxide (NO), and enhanced cell migration and the assembly of endothelial cells into vessel-like structures. These are both hallmarks of the proliferative phase of wound healing. Using a mouse model of a third-degree burn wound, we went on to show that CAP treatment was associated with enhanced angiogenesis, characterised by accelerated in vivo wound healing and increased cellular proliferation. Here, CAP significantly increased the in vivo production of endothelial NO synthase (eNOS), an enzyme that catalyses NO synthesis in endothelial cells, and significantly increased the expression of pro-angiogenic PDGFRß and CD31 markers in mouse wounds. Mechanistically, we showed that CAP induced eNOS phosphorylation and activation, thereby increasing the levels of endogenous NO in endothelial cells. Increased NO generation facilitated by CAP further stimulated important pro-angiogenic VEGFA/VEGFR2 signalling in vitro. This proof-of-concept study may guide future efforts aimed at addressing the use of physical plasma and its therapeutic applications in a variety of pathological scenarios. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

[Alteration of oxidative stress and expression of antioxidases in diaphragm of severely burned rats].

Objective: To explore the occurrence of oxidative stress and antioxidases expression in diaphragm of severely burned rats, so that the mechanism of respiratory muscle atrophy and dysfunction post-burn injury will be further clarified. Methods: Eighty male Wistar rats (aged 7 to 8 weeks) were divided into sham injury group and burn injury group according to the random number table, with 40 rats in each group. Rats in burn injury group were inflicted with 50% total body surface area full-thickness scald (hereinafter referred to as burn) on the back and abdomen by immersing into 80 ℃ water for 15 s and 8 s respectively. Immediately after injury, 40 mL/kg normal saline was injected through abdomen for resuscitation, and the wounds were treated with iodine. Except for immersing into 37 ℃ warm water and no resuscitation, the other treatments of rats in sham injury group were the same as those of burn injury group. Whole diaphragms of 8 rats per time point per group were collected after anesthesia at post injury hour (PIH) 2 and on post injury day (PID) 1, 3, 7, and 14, and muscle mass was determined. The protein carbonyl content was determined by microplate reader. The protein expressions of catalase, superoxide dismutase 2 (SOD2), and glutathione peroxidase 1 were determined by Western blotting. Data were processed with analysis of variance of factorial design, t test, and Bonferroni correction. Results: (1) There were no statistically significant differences in the diaphragm mass of rats between the 2 groups at PIH 2 and on PID 1 (t=0.453, 0.755, P>0.05). The diaphragm mass of rats in burn injury group started to decrease from PID 3, which was significantly lower than that of sham injury group (t=3.321, P<0.01). The diaphragm mass of rats in burn injury group started to increase from PID 7 to PID 14, which was significantly lower than that of sham injury group (t=4.622, 4.380, P<0.01). (2) Protein carbonyl content in diaphragm of rats in burn injury group at PIH 2, and on PID 1, 3, 7, and 14 [(2.7±0.3), (2.5±0.5), (2.4±0.4), (2.5±0.4), (3.2±0.6) pg/mL] was significantly higher than that of sham injury group respectively [(1.2±0.4), (1.6±0.3), (1.5±0.7), (1.7±0.3), (1.8±0.4) pg/mL, t=5.994, 3.263, 3.666, 3.158, 5.763, P<0.05 or P<0.01]. (3) Protein expressions of catalase in diaphragm of rats in burn injury group on PID 1 and 3 were close to those of sham injury group (t=0.339, 0.324, P>0.05). There were no statistically significant differences in protein expressions of SOD2 in diaphragm of rats between the 2 groups at PIH 2 and on PID 1, 3, 7, and 14 (t=1.446, 1.385, 0.757, 1.561, 0.531, P>0.05). There were no statistically significant differences in protein expressions of glutathione peroxidase 1 in diaphragm of rats in the 2 groups at PIH 2 and on PID 1, 3, and 7 (t=0.200, 0.729, 0.385, 1.559, P>0.05). Conclusions: Continuous oxidative stress and relatively insufficient expression of antioxidases in diaphragm induced by burn injury could be a contributor to diaphragm atrophy.

Utilizing Plasma Composition Data to Help Determine Procoagulant Dynamics in Patients with Thermal Injury: A Computational Assessment.

INTRODUCTION: The development of methods that generate individualized assessments of the procoagulant potential of burn patients could improve their treatment. Beyond its role as an essential intermediate in the formation of thrombin, factor (F)Xa has systemic effects as an agonist to inflammatory processes. In this study, we use a computational model to study the FXa dynamics underlying tissue factor-initiated thrombin generation in a small cohort of burn patients. MATERIALS AND METHODS: Plasma samples were collected upon admission (Hour 0) from nine subjects (five non-survivors) with major burn injuries and then at 48 hours. Coagulation factor concentrations (II, V, VII, VIII, IX, X, TFPI, antithrombin (AT), protein C (PC)) were measured and used in a computational model to generate time course profiles for thrombin (IIa), FXa, extrinsic tenase, intrinsic tenase and prothrombinase complexes upon a 5 pM tissue factor stimulus in the presence of 1 nM thrombomodulin. Parameters were extracted from the thrombin and FXa profiles (including max rate (MaxRIIa and MaxRFXa) and peak level (MaxLIIa and MaxLFXa)). Procoagulant potential was also evaluated by determining the concentration of the complexes at select times. Parameter values were compared between survivors and non-survivors in the burn cohort and between the burn cohort and a simulation based on the mean physiological (100%) concentration for all factor levels. RESULTS: Burn patients differed at Hour 0 (p < 0.05) from 100% mean physiological levels for all coagulation factor levels except FV and FVII. The concentration of FX, FII, TFPI, AT and PC was lower; FIX and FVIII were increased. The composition differences resulted in all nine burn patients at Hour 0 displaying a procoagulant phenotype relative to 100% mean physiological simulation (MaxLIIa (306 ± 90 nM vs. 52 nM), MaxRIIa (2.9 ± 1.1 nM/s vs. 0.3 nM/s), respectively p < 0.001); MaxRFXa and MaxLFXa were also an order of magnitude greater than 100% mean physiological simulation (p < 0.001). When grouped by survival status and compared at the time of admission, non-survivors had lower PC levels (56 ± 18% vs. 82 ± 9%, p < 0.05), and faster MaxRFXa (29 ± 6 pM/s vs. 18 ± 6 pM/s, p < 0.05) than those that survived; similar trends were observed for all other procoagulant parameters. At 48 hours when comparing non-survivors to survivors, TFPI levels were higher (108 ± 18% vs. 59 ± 18%, p < 0.05), and MaxRIIa (1.5 ± 1.4 nM/s vs. 3.6 ± 0.7 nM/s, p < 0.05) and MaxRFXa (13 ± 12 pM/s vs. 35 ± 4 pM/s, p < 0.05) were lower; similar trends were observed with all other procoagulant parameters. Overall, between admission and 48 hours, procoagulant potential, as represented by MaxR and MaxL parameters for thrombin and FXa, in non-survivors decreased while in survivors they increased (p < 0.05). In patients that survived, there was a positive correlation between FX levels and MaxLFXa (r = 0.96) and reversed in mortality (r= -0.91). CONCLUSIONS: Thrombin and FXa generation are increased in burn patients at admission compared to mean physiological simulations. Over the first 48 hours, burn survivors became more procoagulant while non-survivors became less procoagulant. Differences between survivors and non-survivors appear to be present in the underlying dynamics that contribute to FXa dynamics. Understanding how the individual specific balance of procoagulant and anticoagulant proteins contributes to thrombin and FXa generation could ultimately guide therapy and potentially reduce burn injury-related morbidity and mortality.

Instantaneous Specific Burn Debridement With an Enzymatic Debriding Agent: A New Resource for the Treatment of Burns.

Nexobrid is a new resource for debridement that has emerged in recent years and is gaining relevance in the treatment of all kinds of thermal injuries. This product is an ointment (formed with a mixture of pineapple-derived enzymes enriched with bromelain) that is directly applied over the burn. With a single application, it performs a burned tissue-specific debridement in less than 4 hr, leaving a vital and completely debrided wound bed. In this article, we describe our experience with this product, and through a representative case, we explain the management of these patients in our Burns unit in consonance with national and international consensus.

Effect of 3-mercaptopyruvate Sulfurtransferase Deficiency on the Development of Multiorgan Failure, Inflammation, and Wound Healing in Mice Subjected to Burn Injury.

The gaseous transmitter hydrogen sulfide (H2S) has been implicated in various forms of critical illness. Here, we have compared the outcome of scald burn injury in wild-type mice and in mice deficient in 3-mercaptopyruvate sulfurtransferase (3-MST), a mammalian H2S-generating enzyme. Outcome variables included indices of organ injury, clinical chemistry parameters, and plasma levels of inflammatory mediators. Plasma levels of H2S significantly increased in response to burn in wild-type mice, but remained unchanged in 3-MST-/- mice. The capacity of tissue homogenates to produce H2S from 3-mercaptopyruvate was unaffected by burn injury. In 3-MST-/- mice, compared to wild-type controls, there was a significant enhancement in the accumulation of polymorphonuclear cells (as assessed by the quantification of myeloperoxidase) in the liver (but not heart, lung, or skin) at 7 days postburn. Oxidative tissue damage (as assessed by malon dialdehyde content) was comparable between wild-type and 3-MST-deficient mice in all tissues studied. 3-MST-/- and wild-type mice exhibited comparable burn-induced elevations in circulating plasma levels of hepatic injury; however, 3-MST-/- mice exhibited a higher degree of renal injury (as reflected by elevated blood urea nitrogen levels) at 7 days postburn. Inflammatory mediators (eg, TNF-α, IL-1ß, IL-2, IL-6, IL-10, and IL-12) increased in burn injury, but without significant differences between the 3-MST-/- and wild-type groups. The healing of the burn wound was also unaffected by 3-MST deficiency. In conclusion, the absence of the H2S-producing enzyme 3-MST slightly exacerbates the development of multiorgan dysfunction but does not affect inflammatory mediator production or wound healing in a murine model of burn injury.

Insulin promotes macrophage phenotype transition through PI3K/Akt and PPAR-γ signaling during diabetic wound healing.

Overactivation and persistent chronic inflammation are the major pathogenic characteristics of diabetic-impaired healing, and diabetic wound healing can be promoted by stimulating the transition of macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2). Our previous studies found that the application of insulin induced an advanced initiation and resolution of inflammatory response. To further explore the mechanism, we have investigated the effect of insulin on macrophage phenotype switch utilizing a diabetic rat model and a human monocytic THP-1 cell. We have utilized the high glucose (HG) and HG plus insulin to stimulate the M1 macrophages derived from lipopolysaccharide-treated THP-1 cells. We studied the secretion of inflammatory mediator and related signaling pathways by using western blot test, immunofluorescence, and Rac1 pull-down assay. We have found that the production of pro-inflammatory mediators, which thereafter induced macrophage polarization toward M1 phenotype, has been elevated due to consistent HG exposure. HG plus insulin stimulation, on the other hand, promoted anti-inflammatory effects. Experiments performed on diabetic burn wounds indicated that the insulin modulated macrophages transition from M1 to M2 phenotype. We found that PI3K/Akt/Rac-1 and PPAR-γ signaling pathways are involved in the anti-inflammatory effect of insulin. Insulin inhibited HG-induced activation of p38, NF-κB, and STAT1 transcriptional activity by activating Akt-Rac-1 signaling. Moreover, insulin performs anti-inflammatory effects through upregulation of PPAR-γ expression and induced P38-mediated dephosphorylation of PPAR-γ (Ser112). In conclusion, insulin downregulates inflammatory response, regulates M1 macrophage transition in response to HG, and thus improves chronic wound healing.

Phosphodiesterase 4B knockout prevents skeletal muscle atrophy in rats with burn injury.

The phosphodiesterase 4 (PDE4)-cAMP pathway plays a predominant role in mediating skeletal muscle proteolysis in burn injury. The present investigations to determine the PDE4 isoform(s) involved in this action revealed that burn injury increased the expression of rat skeletal muscle PDE4B mRNA by sixfold but had little or no effect on expression of other PDE4 isoforms. These observations led us to study the effects of burn in PDE4B knockout (KO) rats. As reported by us previously, burn injury significantly increased extensor digitorum longus (EDL) muscle total and myofibrillar proteolysis in wild-type (WT) rats, but there were no significant effects on either total or myofibrillar protein breakdown in EDL muscle of PDE4B KO rats with burn injury. Moreover, burn injury increased PDE4 activity in the skeletal muscle of WT rats, but this was reduced by >80% in PDE4B KO rats. Also, burn injury decreased skeletal muscle cAMP concentration in WT rats but had no significant effects in the muscles of PDE4B KO rats. Incubation of the EDL muscle of burn-PDE4B KO rats with an inhibitor of the exchange factor directly activated by cAMP, but not with a protein kinase A inhibitor, eliminated the protective effects of PDE4B KO on EDL muscle proteolysis and increased muscle proteolysis to the same extent as in the EDL of burn-WT rats. These novel findings confirm a major role for PDE4B in skeletal muscle proteolysis in burn injury and suggest that an innovative therapy based on PDE4B-selective inhibitors could be developed to treat skeletal muscle cachexia in burn injury without the fear of causing emesis, which is associated with PDE4D inhibition.

ER stress and subsequent activated calpain play a pivotal role in skeletal muscle wasting after severe burn injury.

Severe burns are typically followed by hypermetabolism characterized by significant muscle wasting, which causes considerable morbidity and mortality. The aim of the present study was to explore the underlying mechanisms of skeletal muscle damage/wasting post-burn. Rats were randomized to the sham, sham+4-phenylbutyrate (4-PBA, a pharmacological chaperone promoting endoplasmic reticulum (ER) folding/trafficking, commonly considered as an inhibitor of ER), burn (30% total body surface area), and burn+4-PBA groups; and sacrificed at 1, 4, 7, 14 days after the burn injury. Tibial anterior muscle was harvested for transmission electron microscopy, calcium imaging, gene expression and protein analysis of ER stress / ubiquitin-proteasome system / autophagy, and calpain activity measurement. The results showed that ER stress markers were increased in the burn group compared with the sham group, especially at post-burn days 4 and 7, which might consequently elevate cytoplasmic calcium concentration, promote calpain production as well as activation, and cause skeletal muscle damage/wasting of TA muscle after severe burn injury. Interestingly, treatment with 4-PBA prevented burn-induced ER swelling and altered protein expression of ER stress markers and calcium release, attenuating calpain activation and skeletal muscle damage/wasting after severe burn injury. Atrogin-1 and LC3-II/LC3-I ratio were also increased in the burn group compared with the sham group, while MuRF-1 remained unchanged; 4-PBA decreased atrogin-1 in the burn group. Taken together, these findings suggested that severe burn injury induces ER stress, which in turns causes calpain activation. ER stress and subsequent activated calpain play a critical role in skeletal muscle damage/wasting in burned rats.

Evaluación de tiempos quirúrgicos y transfusiones en pacientes quemados tratados con desbridamiento enzimático / Evaluation of surgical times and transfusions in burn patients treated with enzymatic debridement

Introducción y Objetivo. En los últimos años se han publicado resultados que demuestran un desbridamiento enzimático rápido, eficaz y específico de tejidos quemados con el uso de Nexobrid(R) (Mediwound, Alemania), producto compuesto por un concentrado de enzimas proteolíticas enriquecidas con bromelaína. En este estudio comparamos el uso de Nexobrid(R) como desbridante enzimático frente a un Grupo Control con tratamiento estándar, con el objetivo de evaluar; en función de la superficie corporal quemada (SCQ), la cantidad de intervenciones quirúrgicas realizadas, la colonización del lecho quirúrgico, la necesidad de escarotomías, así como los requerimientos transfusionales de los pacientes tratados. Material y Método. Estudio retrospectivo con 65 pacientes, edad media de 46.87 años, tratados con Nexobrid(R) entre septiembre de 2015 y diciembre de 2016. Comparamos con un grupo control de 177 pacientes, edad media de 48.24 años, intervenidos mediante desbridamiento tangencial desde enero a diciembre de 2014. El Grupo Control presenta unas características homogéneas a las del Grupo Nexobrid(R). Ambos grupos fueron estratificados en función de su SCQ mayor o menor del 15%. Resultados. Encontramos diferencias estadísticamente significativas (p <0.01) con un menor número de días desde la quemadura y el ingreso hasta la cirugía para el Grupo Nexobrid(R) independientemente de la SCQ. Obtuvimos diferencias estadísticamente significativas para la cantidad de cirugías (p <0.01) en el subgrupo de SCQ <15%. Evaluamos la colonización sin obtener diferencias estadísticamente significativas entre los grupos. El número de escarotomías en el Grupo Nexobrid(R) fue significativamente menor que en el Grupo Control para las poblaciones con SCQ ≥15%. Los requerimientos transfusionales fueron menores en el Grupo Nexobrid(R) frente al Grupo Control en los pacientes con SCQ ≥ 15% (p <0.05). Conclusiones. Nexobrid(R) permite reducir el número de cirugías y el tiempo hasta el primer desbridamiento sin aumentar la tasa de colonización respecto al Grupo Control. El desbridamiento enzimático precoz reduce la necesidad de escarotomías en pacientes con SCQ ≥15%. Finalmente, reduce la necesidad de transfusión sanguínea en pacientes con SCQ mayor del 15% (AU)

Background and Objective. During the last years, results have been published demonstrating fast, efficient and specific enzymatic debridement of burned tissues with the use of a concentrate of proteolytic enzymes enriched with bromelain (Nexobrid(R), Mediwound, Germany). In this study we compare the use of Nexobrid(R) against a Control Group with standard treatment, in order to evaluate; the number of surgical procedures performed, the colonization of the surgical bed, the need for scarotomies, as well as the number of transfusions of the treated patients according to the total burned surface area (TBSA). Methods. We conduct a retrospective study evaluating 65 patients (mean age 46.87 years) treated with Nexobrid(R) between September 2015 and December 2016. We compare this population with a control group of 177 patients (mean age 48.24 years) treated with tangential excision from January to December 2014. The Control Group was homogeneous to Nexobrid(R) Group. Both groups were stratified according to their TBSA in 2 groups: more or less than 15%. Results. We found lesser number of days from burn and hospital admission to the first debridement for the Nexobrid(R) Group regardless of the burned body surface. Statistically significant differences were obtained for the number of surgeries (p <0.01) in the subgroup with <15% TBSA. We evaluated the colonization without obtaining statistically significant differences between the groups. The number of scarotomies carried out in the Nexobrid(R) Group was significantly lower than in the Control Group when comparing the populations with ≥15% TSBA. The transfusion requirements were lower in the Nexobrid(R) Group compared to the Control Group in patients with ≥15% TBSA (p <0.05). Conclusions. The use of Nexobrid(R) allows reducing the number of surgeries and the time elapsed to first debridement without increasing the rate of colonization when compared to the Control Group. Early enzymatic debridement reduces the need for scarotomies in ≥15% TBSA patients. The use of Nexobrid (R) reduces the need for blood transfusion in patients with ≥15% TBSA (AU)

Targeting palmitoyl acyltransferase ZDHHC21 improves gut epithelial barrier dysfunction resulting from burn-induced systemic inflammation.

Clinical studies in burn patients demonstrate a close association between leaky guts and increased incidence or severity of sepsis and other complications. Severe thermal injury triggers intestinal inflammation that contributes to intestinal epithelial hyperpermeability, which exacerbates systemic response leading to multiple organ failure and sepsis. In this study, we identified a significant function of a particular palmitoyl acyltransferase, zinc finger DHHC domain-containing protein-21 (ZDHHC21), in mediating signaling events required for gut hyperpermeability induced by inflammation. Using quantitative PCR, we show that ZDHHC21 mRNA production was enhanced twofold when intestinal epithelial cells were treated with TNF-α-IFN-γ in vitro. In addition, pharmacological targeting of palmitoyl acyltransferases with 2-bromopalmitate (2-BP) showed significant improvement in TNF-α-IFN-γ-mediated epithelial barrier dysfunction by using electric cell-substrate impedance-sensing assays, as well as FITC-labeled dextran permeability assays. Using acyl-biotin exchange assay and click chemistry, we show that TNF-α-IFN-γ treatment of intestinal epithelial cells results in enhanced detection of total palmitoylated proteins and this response is inhibited by 2-BP. Using ZDHHC21-deficient mice or wild-type mice treated with 2-BP, we showed that mice with impaired ZDHHC21 expression or pharmacological inhibition resulted in attenuated intestinal barrier dysfunction caused by thermal injury. Moreover, hematoxylin and eosin staining of the small intestine, as well as transmission electron microscopy, showed that mice with genetic interruption of ZDHHC21 had attenuated villus structure disorganization associated with thermal injury-induced intestinal barrier damage. Taken together, these results suggest an important role of ZDHHC21 in mediating gut hyperpermeability resulting from thermal injury.NEW & NOTEWORTHY Increased mucosal permeability in the gut is one of the major complications following severe burn. Here we report the novel finding that zinc finger DHHC domain-containing protein-21 (ZDHHC21) mediates gut epithelial hyperpermeability resulting from an experimental model of thermal injury. The hyperpermeability response was significantly attenuated with a pharmacological inhibitor of palmitoyl acyltransferases and in mice with genetic ablation of ZDHHC21. These findings suggest that ZDHHC21 may serve as a novel therapeutic target for treating burn-induced intestinal barrier dysfunction.

Overexpression of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in serum of children after thermal injury.

PURPOSE: The study aims to determinate concentrations of ubiquitin C-terminal hydrolase 1 (UCHL1), which hydrolyzes amino acids from ubiquitin and cleave di-ubiquitins, in serum of children after thermal injury. PATIENTS/METHODS: 42 children scalded by hot water, managed at the Department of Pediatric Surgery, with burns in 4-20% TBSA were included into the study (age 9 months up to 14 years, mean age 2.5±1 years). Blood plasma UCHL1 concentration was assessed in 2-6h, 12-16h, 3d, 5d, and 7d after injury using surface plasmon resonance imaging biosensor. 18 healthy subjects admitted for planned surgeries served as controls. RESULTS: The UCHL1 concentration in the blood plasma of patients with thermal injuries reached its peak 12-16h after thermal injury and slowly decreased over time, and still did not reach the normal range on the 7th day after thermal injury. Mean concentrations of UCHL1 after thermal injury were above the range measured in controls (0.12ng/ml): 2-6h after injury - 5.59ng/dl, 12-16h after injury - 9.16ng/dl, 3 days after injury - 6.94ng/dl, 5 days after 5.41ng/dl, 7 days after injury - 4.09ng/dl. CONCLUSIONS: We observed sudden increase in the concentration of UCHL1 2-16h after thermal injury with the slow decrease in the UCHL1 concentration over the time. UCHL1 concentration was proportional to the severity of the burn. Further studies are needed to determine the mechanisms by which UCHL1 contributes to metabolic response following thermal injury.

Modification of the Catalytic Properties of Erythrocyte Aldehyde Dehydrogenase in Rats after Nitric Oxide Inhalation.

We studied aldehyde dehydrogenase activity in erythrocytes from healthy rats and animals with thermal trauma after NO inhalation. NO had an activating effect on catalytic properties of aldehyde dehydrogenase in healthy rats and burned animals. The effect of NO was more pronounced during burn disease.

NADPH oxidase 2 plays a role in experimental corneal neovascularization.

Corneal neovascularization, the growth of new blood vessels in the cornea, is a leading cause of vision impairment after corneal injury. Neovascularization typically occurs in response to corneal injury such as that caused by infection, physical trauma, chemical burns or in the setting of corneal transplant rejection. The NADPH oxidase enzyme complex is involved in cell signalling for wound-healing angiogenesis, but its role in corneal neovascularization has not been studied. We have now analysed the role of the Nox2 isoform of NADPH oxidase in corneal neovascularization in mice following chemical injury. C57BL/6 mice aged 8-14 weeks were cauterized with an applicator coated with 75% silver nitrate and 25% potassium nitrate for 8 s. Neovascularization extending radially from limbal vessels was observed in corneal whole-mounts from cauterized wild type mice and CD31+ vessels were identified in cauterized corneal sections at day 7. In contrast, in Nox2 knockout (Nox2 KO) mice vascular endothelial growth factor-A (Vegf-A), Flt1 mRNA expression, and the extent of corneal neovascularization were all markedly reduced compared with their wild type controls. The accumulation of Iba-1+ microglia and macrophages in the cornea was significantly less in Nox2 KO than in wild type mice. In conclusion, we have demonstrated that Nox2 is implicated in the inflammatory and neovascular response to corneal chemical injury in mice and clearly VEGF is a mediator of this effect. This work raises the possibility that therapies targeting Nox2 may have potential for suppressing corneal neovascularization and inflammation in humans.

Tissue inhibitor of metalloproteinase-2 inhibits burn-induced derangements and hyperpermeability in microvascular endothelial cells.

BACKGROUND: Burns induce microvascular hyperpermeability. We hypothesize that this occurs partly through an imbalance between matrix metalloproteinases (MMPs) and endogenous MMP inhibitors such as tissue inhibitors of metalloproteinases (TIMPs), and that such derangements can be attenuated with the use of TIMP-2. METHOD: Rats underwent either sham or burn: serum and tissue were collected. Western blot was used to examine MMP-9 and TIMP-2 levels and MMP activity was assayed from lung tissue. Rat lung microvascular endothelial cells were used to assess monolayer permeability and evaluate the adherens junction proteins ß-catenin, vascular endothelial cadherin and filamentous actin after exposure to burn serum ± TIMP-2. RESULTS: Lung tissue from burn animals showed increased MMP activity, decreased levels of TIMP-2, and no difference in levels of active MMP-9 in burn vs control groups. Burn serum increased monolayer permeability, damaged adherens junction proteins, and incited actin stress fiber formation; TIMP-2 attenuated these derangements. CONCLUSIONS: Burns may lower TIMP-2 levels and increase MMP activity and that TIMP-2 application in vitro may attenuate burn-induced hyperpermeability and decreases damage to endothelial structural proteins. These links warrant further investigation.

The extracellular matrix protein matrilin-2 induces post-burn inflammatory responses as an endogenous danger signal.

OBJECTIVE AND DESIGN: This prospective experimental study aims to investigate whether matrilin-2 is released from burn injury and induces post-burn inflammatory responses as an endogenous danger signal. SUBJECTS: Fifteen burn patients, 15 volunteers, 12 matrilin-2-deficient mice, 36 C57BL/6 mice and raw 264.7 cells. METHODS: Matrilin-2 levels were detected by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction. The inflammatory cytokines production in Matn2 deficient mice and wide type mice were detected by ELISA. Macrophages were activated by recombinant mouse MATN2 with or without adding anti-Toll-like receptor (TLR) 4 antibody. Student's t test and one-way analysis of variance were used for statistical analysis. RESULTS: The matrilin-2 levels in serum of burned patients were drastically elevated as compared to those of healthy controls. The matrilin-2 levels in burned mice were significantly increased than those of non-burned controls, whereas the matrilin-2 mRNA expression was not significantly changed after burn. In addition, Matn2 deficient mice showed remarkably less inflammatory cytokines production and less neutrophil infiltration in lung. Exogenous MATN2 induced potent expression of proinflammatory cytokines production in macrophages, which was inhibited by anti-TLR4 antibody. CONCLUSION: Matrilin-2 induces post-burn inflammatory responses as an endogenous danger signal, partly through a TLR4-mediated mechanism.

Dynamic changes of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 after burn injury.

PURPOSE: Severe burn is a life-threatening condition. Many trials discuss the role of matrix metalloproteinases and tissue inhibitor of metalloproteinases in diseases generating systemic inflammatory response syndrome, and in some, their prognostic importance has been established. We aimed to describe the time courses of the aforementioned system and to evaluate the difference between survivors and nonsurvivors in burns. MATERIALS: Thirty-one patients were enrolled. Blood samples were collected on admission and on the 5 consecutive days. Circulating matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) have been measured. Healthy individuals were invited as controls. RESULTS: Tissue inhibitor of metalloproteinase 1 increased in the burn group (P < .001) by day 2 and remained elevated thereafter. Plasma MMP-9 and MMP-9/TIMP-1 were already elevated on admission (P < .001) and decreased in tendency thereafter. In burned patients, significantly lower MMP-9 were noted on days 4 to 6 as MMP-9/TIMP-1 were also lower on days 3 to 6 (P < .01) compared with controls. We experienced difference regarding survival on days 5 and 6 by TIMP-1 (P < .05). CONCLUSIONS: Our research is the first follow-up study elucidating the dynamic changes of MMP-9-TIMP-1 system in severe burns. Alteration of MMP-9-TIMP-1 balance might influence systemic inflammatory response and related mortality. Matrix metalloproteinase 9 might be a good injury marker in burns after an extensive trial.