Oxidative stress and anti-oxidative mobilization in burn injury.

A severe burn is associated with release of inflammatory mediators which ultimately cause local and distant pathophysiological effects. Mediators including Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) are increased in affected tissue, which are implicated in pathophysiological events observed in burn patients. The purpose of this article is to understand the role of oxidative stress in burns, in order to develop therapeutic strategies. All peer-reviewed, original and review articles published in the English language literature relevant to the topic of oxidative stress in burns in animals and human subjects were selected for this review and the possible roles of ROS and RNS in the pathophysiology of burns are discussed. Both increased xanthine oxidase and neutrophil activation appear to be the oxidant sources in burns. Free radicals have been found to have beneficial effects on antimicrobial action and wound healing. However following a burn, there is an enormous production of ROS which is harmful and implicated in inflammation, systemic inflammatory response syndrome, immunosuppression, infection and sepsis, tissue damage and multiple organ failure. Thus clinical response to burn is dependent on the balance between production of free radicals and its detoxification. Supplementation of antioxidants in human and animal models has proven benefit in decreasing distant organ failure suggesting a cause and effect relationship. We conclude that oxidative damage is one of the mechanisms responsible for the local and distant pathophysiological events observed after burn, and therefore anti-oxidant therapy might be beneficial in minimizing injury in burned patients.

Antimicrobial peptides in burns and wounds.

Burn-induced immunosuppression not only increases susceptibility to infection, but also predisposes burn patients to related adverse sequelae, including systemic inflammatory response syndrome and sepsis. Although burn-related immunosuppression is not fully understood, it is characterized by decreased T- and B-lymphocyte function and by impaired functions of circulating leukocytes and complement. Alterations in defensins, a family of cationic, naturally occurring antimicrobial peptides, may underlie these immune deficiency patterns. Defensins are considered important components of the innate immune system, as they inhibit bacterial, fungal, and viral colonization. They also chemoattract immature dendritic cells and T lymphocytes, recruit neutrophils, macrophages, and monocytes, modulate complement and adjuvant activity, and promote inflammation and wound healing. Infectious states are associated with upregulation of circulating defensins, which suggests an underlying antimicrobial role. In addition, data from our laboratory demonstrated diminished levels of certain defensins in burned tissue. The inference is that decreased defensin levels in burn injury may facilitate infection and subsequent sepsis. It may also alter functions of T- and B-lymphocytes, neutrophils, macrophages, and complement, thereby contributing to the pathophysiology of burn-related systemic inflammatory responses. This article is a comprehensive review on the role of antimicrobial peptides in burns and wounds.

Localization of antimicrobial peptides in normal and burned skin.

Severe burn causes immunosuppression, and the eschar remains a perfect culture medium for microbial growth. The resulting sepsis is a common complication of burns with a high mortality. The skin produces a number of molecules including antimicrobial peptides (AMPs) that act in the first line of host defense. Previous studies from our laboratory suggested decreased expression of human beta-defensin-2 (HBD-2) in burned wounds. Here, we have expanded our work by identifying HBD-1, HBD-2, HBD-3 and human neutrophil peptide (HNP) in normal and burn skin samples using fluorescence deconvolution microscopy. In normal skin, HBD-1 was localized to the perinuclear region of keratinocytes, while HBD-2 was seen primarily in the stratum basale of the epidermis. HBD-3 was found in dendritic cells of the stratum spinosum. HNP was distributed in a somewhat random pattern in the papillary dermis. In burned skin, in which the epidermis had been destroyed or disrupted, the presence of HBD-1 was localized to dermal glandular structures and hair shafts. HBD-2 was found in both the upper portions of the remaining keratin layers, and localized to lower, f-actin containing, acini-like structures, a pattern also evident with HBD-3. We conclude that although the upper layers of skin are destroyed and disrupted by burn, cells in the lower portions of the skin demonstrate an ability to synthesize most of the AMPs, thereby maintaining some barrier against infection. The results of these studies further contribute to an understanding of the role of AMPs in the pathophysiology of cutaneous burn and the possibility of using these sites for upregulation of AMP synthesis in the prevention of burn sepsis.

Sensitization and translocation of TRPV1 by insulin and IGF-I.

Insulin and insulin-like growth factors (IGFs) maintain vital neuronal functions. Absolute or functional deficiencies of insulin or IGF-I may contribute to neuronal and vascular complications associated with diabetes. Vanilloid receptor 1 (also called TRPV1) is an ion channel that mediates inflammatory thermal nociception and is present on sensory neurons. Here we demonstrate that both insulin and IGF-I enhance TRPV1-mediated membrane currents in heterologous expression systems and cultured dorsal root ganglion neurons. Enhancement of membrane current results from both increased sensitivity of the receptor and translocation of TRPV1 from cytosol to plasma membrane. Receptor tyrosine kinases trigger a signaling cascade leading to activation of phosphatidylinositol 3-kinase (PI(3)K) and protein kinase C (PKC)-mediated phosphorylation of TRPV1, which is found to be essential for the potentiation. These findings establish a link between the insulin family of trophic factors and vanilloid receptors.

Observations on the microcirculation of the human burn wound using orthogonal polarization spectral imaging.

Orthogonal polarization spectral imaging (OPS) utilizes the illumination of the tissue with polarized light within the haemoglobin spectrum. We report here on OPS for the assessment of the skin microcirculation non-invasively through the surface of the human burn wound. This allows inspection of individual capillaries of the cutaneous microcirculation and flow through these vessels in real time. Two distinct microcirculatory patterns were seen. Superficial burns had small visible dermal capillaries studied throughout the field of view. The flow of individual erythrocytes through these capillaries was clearly visible in real-time. Conversely, deep burns showed large thrombosed vessel coursing in a criss-cross fashion. There was marked difference between the mean optical densities for normal skin and superficial burns (65.8+/-15.6 and 64+/-14.6, respectively) and deep burns (131.2+/-31.1). These findings indicate that OPS may have utility in the assessment of cutaneous microcirculation in burns.

Cisplatin up-regulates the adenosine A(1) receptor in the rat kidney.

Cisplatin, a widely used anticancer drug, produces significant oto- and nephrotoxicity. Previous data from our laboratory, using cultured cell lines, indicated that cisplatin increases the expression of the adenosine A(1) receptor subtype through generation of reactive oxygen species and activation of nuclear factor-kappa B (NF-kappa B). Since the adenosine A(1) receptor plays an important role in normal renal physiology, this study was performed to determine whether cisplatin modulates adenosine A(1) receptor expression in vivo and whether these receptors play a role in the nephrotoxicity. Male Sprague-Dawley rats, treated with cisplatin (8 mg/kg), developed nephrotoxicity within 3 days, as demonstrated by increased serum creatinine and blood urea nitrogen. Cisplatin also produced a significant increase in malondialdehyde, apoptosis and necrosis in the kidney. The above changes were associated with a time-dependent increase in the expression of adenosine A(1) receptor, as determined by radioligand binding assays, Western blotting and immunocytochemistry, and an increase in adenosine A(1) receptor transcripts. Administration of selective and nonselective antagonists of the adenosine A(1) receptor produced either no change or exacerbated the nephrotoxicity produced by cisplatin. These data indicate that cisplatin can regulate the adenosine A(1) receptor in the kidney and suggest a cytoprotective role of this receptor subtype against cisplatin-induced nephrotoxicity.

Effects of cytokines and heat shock on defensin levels of cultured keratinocytes.

Burns have been associated with high levels of circulating pro-inflammatory cytokines which promote systemic inflammatory response syndrome (SIRS), immunosuppression and sepsis for which no effective treatment is currently available. Defensins, a family of cationic naturally occurring antimicrobial peptides, are considered important components of the innate immune system and enhance adaptive immunity. This study examines the effects of pro-inflammatory cytokines, interleukin-1beta (IL-1beta), gamma-interferon (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) on human beta-defensin-2 (HBD-2) levels in cultured keratinocytes. We also examined the effects of heat shock at 42 degrees C. The results demonstrate that only TNFalpha shows significant induction of HBD-2 but this induction was not sustained in the long-term. In addition, endogenous levels of defensin were significantly reduced by exposure to heat shock. The keratinocytes also responded to IL-1beta by becoming hypertrophic. These results indicate that stress-related, pro-inflammatory cytokines can induce keratinocytes to synthesize HBD-2, while heat shock appears to reduce its production. These experiments give us further insight into the role of natural antimicrobial peptides under conditions of stress.

Expression of human beta defensin 2 in thermal injury.

Sepsis is a common and serious complication of major burn injury and accounts for over 54% of deaths in burn patients. Burns are associated with high levels of circulating pro-inflammatory cytokines and immunosuppression, promoting systemic inflammatory response syndrome (SIRS) and sepsis, for which no effective treatment is currently available. Defensins, a family of cationic, naturally occurring, antimicrobial peptides are important components of the innate immune system, playing a major role in the body's defence by inhibiting activities of bacteria, fungi and enveloped viruses. These natural antimicrobials also chemoattract immature dendritic cells, some types of T and B-lymphocytes, neutrophils and macrophages, and act as an adjuvant, enhancing adaptive immunity. Our prior studies suggested a decreased expression of human beta defensin 2 (HBD2) in burn wounds. Here we have identified HBD2 protein in skin samples of partial and full thickness burns and in normal skin using fluorescence deconvolution microscopy. Images showed that in normal skin the majority of HBD2 is located in the Malpighian layer and, in smaller amounts, in the more superficial layers, a pattern that is absent in burned skin in which the epidermis is destroyed or damaged. However, surviving dermal and subcutaneous layers revealed the presence of HBD2 in a number of other cell types and structures, such as hair follicles and sweat gland acini, but not in vascular endothelium and fat cells. The results of these studies further contribute to an understanding of the role of antimicrobial peptides in the pathophysiology of burn injury, associated immunosuppression and sepsis and the possibility of using these other sites of HBD2 deposition for upregulation of antimicrobial synthesis in the treatment of burns.

The pathogenesis of burn wound conversion.

OBJECTIVE: Burn wound progression is a poorly understood process by which certain superficial partial-thickness burns spontaneously advance into deep partial-thickness or full-thickness wounds. Progression of an injury into deeper tissue is an important phenomenon in the treatment of thermal injury due to the fact that burn wound depth may be a significant determinant of morbidity and treatment. This article reviews current knowledge of the pathogenesis, molecular and cellular mechanisms, local and systemic factors, and treatment modalities related to wound conversion. DATA SOURCES AND STUDY SELECTION: All peer-reviewed, original, and review articles published in English-language literature relevant to the topic of burn wound conversion on animals and human subjects were selected for this review. DATA EXTRACTION AND SYNTHESIS: After assessing data relevance, independent extraction by a sole reviewer was performed. Data were tabulated according to the following categories: pathogenesis, mechanisms, local and systemic factors, and treatment. CONCLUSIONS: Burn wound progression is complex and caused by additive effects of inadequate tissue perfusion, free radical damage, and systemic alterations in the cytokine milieu of burn patients, leading to protein denaturation and necrosis. Even though insufficient evidence exists for causal inferences, infection, tissue desiccation, edema, circumferential eschar, impaired wound perfusion, metabolic derangements, advanced age, and poor general health play important roles. Although consensus-building research is ongoing, current mainstays of treatment include adequate fluid resuscitation, nutritional support, and local wound care, with an emphasis on topical antimicrobial agents and biosynthetic dressings. Identifying early indicators by elucidating possible interacting or synergistic mechanisms and by developing preventative strategies will enhance prevention and treatment.

Modulation of the complement system by human beta-defensin 2.

OBJECTIVE: Human beta-defensin (HBD) and the complement system are two important innate immune mechanisms active against a broad range of burn and wound pathogens. However, excessive or uncontrolled complement activation, following thermal injury, contributes to tissue damage. Previous studies from our laboratory suggested a decreased expression of HBD-2 in burn wounds and its absence in burn blister fluid. Prior studies have demonstrated that human neutrophil peptide can bind to the C1q component of the complement system and prevent complement activation. The objective of this study was to determine whether HBD-1 and HBD-2 can also bind to the C1q component and modulate complement activity. METHODS: The binding efficiency of HBD-1 and HBD-2 to the C1q component was determined by utilizing dot blot hybridization. The effect of HBD-2 on the activation of the complement system by the classical and alternative pathways was determined by CH50 and AP50 assays. In addition, the ability of HBD-1 and HBD-2 to inhibit C1q activity was predicted by a comparison with known C1q inhibitor peptide 2J in a DNAStar computer modeling study. RESULTS: C1q binding to HBD-2 was strong, whereas C1q binding to HBD-1 was weak. HBD-2 inhibits the classical pathway significantly without affecting the alternative pathway. In addition, a computer modeling study also revealed structural homology of HBD-2 with known C1q inhibitory sequences of HBD-2. CONCLUSION: HBD-2 inhibits the classical pathway. The replacement of missing defensin, a natural inhibitor of the complement system, may have a dual protective role not only as an antimicrobial agent but also in providing protection against uncontrolled activation of the complement system.

Nuclear localization of HBD-1 in human keratinocytes.

OBJECTIVE: Human defensins and cathelicidins are a family of cationic antimicrobial peptides (AMPs), which play multiple roles in both innate and adaptive immune systems. They have direct antimicrobial activity against several microorganisms including burn pathogens. The majority of components of innate and adaptive immunity either express naturally occurring defensins or are otherwise chemoattracted or functionally affected by them. They also enhance adaptive immunity and wound healing and alter antibody production. All mechanisms to explain multiple functions of AMPs are not clearly understood. Prior studies to localize defensins in normal and burned skin using deconvolution fluorescence scanning microscopy indicate localization of defensins in the nucleus, perinuclear regions, and cytoplasm. The objective of this study is to further confirm the identification of HBD-1 in the nucleus by deconvolution microscopic studies involving image reconstruction and wire frame modeling. RESULTS: Our study demonstrated the presence of intranuclear HBD-1 in keratinocytes throughout the stratum spinosum by costaining with the nuclear probe DAPI. In addition, HBD-1 sequence does show some homology with known cationic nuclear localization signal sequences. CONCLUSION: To our knowledge, this is the first report to localize HBD-1 in the nuclear region, suggesting a role for this peptide in gene expression and providing new data that may help determine mechanisms of defensin functions.

Modalities for the assessment of burn wound depth.

OBJECTIVE: Burn wound depth is a significant determinant of patient treatment and morbidity. While superficial partial-thickness burns generally heal by re-epithelialization with minimal scarring, deeper wounds can form hypertrophic or contracted scars, often requiring surgical excision and grafting to prevent a suboptimal result. In addition, without timely intervention, more superficial burn wounds can convert to deeper wounds. As such, the rapid and accurate assessment of burn wound depth is a priority in treating burn-injured patients. The object of this article is to review current research on modalities useful in the assessment of burn wound depth with emphasis on the relative costs and benefits of each technique. METHODS: PubMed and Cochrane computerized databases were used for data retrieval, using the search terms "burns," "burn wounds," "burn depth," "burn depth measurement," and "burn depth progression." In addition, bibliographic references from prior reviews of burn depth were reviewed. All peer-reviewed, English-language articles relevant to the topic of burn depth measurement were reviewed, including those focusing on animal and human populations. Where appropriate, conclusions drawn from review articles and expert analyses were included. RESULTS: Although bedside evaluation remains the most common modality of diagnosing the depth of burn wounds, recent technological advances have broadened the scope of depth assessment modalities available to clinicians. Other depth assessment techniques include biopsy and histology, and perfusion measurements techniques such as thermography, vital dyes, indocyanine green video angiography, and laser Doppler techniques. CONCLUSION: Of the depth assessment modalities currently used in clinical practice, LDI and ICG video angiography offer the best data-supported estimates of accuracy. Until the future of new modalities unfolds, a combination of clinical evaluation and another modality--thermography, biopsy, or, ideally, ICG video angiography or LDI--is advised to best assess the depth of acute burn wounds.

Osmotic diuretics induce adenosine A1 receptor expression and protect renal proximal tubular epithelial cells against cisplatin-mediated apoptosis.

Osmotic diuretics are used successfully to alleviate acute tubular necrosis (ATN) produced by chemotherapeutic agents and aminoglycoside antibiotics. The beneficial action of these agents likely involves rapid elimination of the nephrotoxic agents from the kidney by promoting diuresis. Adenosine A1 receptor (A1AR) subtype present on renal proximal tubular epithelial and cortical collecting duct cells mediates the antidiuretic and cytoprotective actions of adenosine. These receptors are induced by activation of nuclear factor (NF)-kappaB, a transcription factor reported to mediate hyperosmotic stress-induced cytoprotection in renal medullary cells. In this study, we tested the hypothesis that induction of the A1AR in renal proximal tubular cells by NF-kappaB contributes to the cytoprotection afforded by osmotic diuretics. Exposure of porcine renal proximal tubular epithelial (LLC-PK1) cells to mannitol or NaCl produced a significant increase in A1AR. This increase was preceded by adenosine release and NF-kappaB activation. Expression of an IkappaB-alpha mutant, which acts as a superrepressor of NF-kappaB, abrogated the increase in A1AR. Cells exposed to mannitol demonstrated increased reactive oxygen species (ROS) generation, which was attenuated by inhibiting xanthine oxidase with allopurinol. Allopurinol attenuated both the increase in A1AR expression and NF-kappaB activation produced by osmotic diuretics, indicating a role of adenosine metabolites in these processes. Treatment of LLC-PK1 cells with cisplatin (8 microm) resulted in apoptosis, which was attenuated by mannitol but exacerbated by selective A1AR blockade. Administration of mannitol to mice increases A1AR expression and activation of NF-kappaB in renal cortical sections. Taken together, these data provide novel mechanisms of nephroprotection by osmotic diuretics, involving both activation and induction of the A1AR, the latter mediated through activation of a xanthine oxidase pathway leading to ROS generation and promoting activation of NF-kappaB.