Pseudothrombocytosis in a patient with severe burns.

Burned patients with detectable blood alcohol levels (BAL) show an elevated mortality rate. Interleukin (IL)-6 and reactive oxygen species (ROS) production is stimulated independently by alcohol and burn injury. The aim of the study was to determine whether increasing levels of alcohol differentially enhance the hepatic production of IL-6 and ROS after burn in a murine model of dorsal scald injury. Groups of mice received either saline or alcohol intraperitoneally to reach a BAL of 100 mg/dl or 300 mg/dl at the time of burn (15% total body surface scald) or sham injury. Burn injury alone resulted in a low mortality rate at 24 hr after injury as did the burn group with a BAL of 100 mg/dl (15%), whereas 57% of the mice burned with a BAL of 300 mg/dl did not survive (p = 0.02). Twenty-four hours after burn or sham injury, IL-6 levels were measured by enzyme-linked immunosorbent assay in serum and liver. In the saline-treated group, IL-6 circulating and hepatic levels rose after burn injury (p < 0.03). Circulating IL-6 levels in sham mice increased 1.5-fold in the group with a BAL of 100 mg/dl and 3-fold in those with a BAL of 300 mg/ml (p = 0.005 versus burn-injured, saline-treated). IL-6 hepatic production after burn injury was higher in the mice with a BAL of 300 mg/dl than in those with a BAL of 100 mg/dl and the saline-treated group (p = 0.001). Among the burned mice, alcohol exposure increased hepatic ROS production, measured by lipid peroxidation and protein oxidation, in a dose-dependent manner. Alcohol enhances in a dose-dependent manner the hepatic production of IL-6 induced by burn injury through the modulation of oxidative stress. The increased mortality rate of mice exposed to alcohol and burn injury may be due to the adverse effect on immune function induced by IL-6 elevation.

The morbidity and mortality of burn victims increase when burn injury is combined with smoke inhalation. The goal of the present study was to develop a murine model of burn and smoke inhalation injury to more precisely reveal the mechanistic aspects of these pathological changes. The burn injury mouse group received a 40% total body surface area third-degree burn alone, the smoke inhalation injury mouse group received two 30-s exposures of cotton smoke alone, and the combined burn and smoke inhalation injury mouse group received both the burn and the smoke inhalation injury. Animal survival was monitored for 120 h. Survival rates in the burn injury group, the smoke inhalation injury group, and the combined injury group were 70%, 60%, and 30%, respectively. Mice that received combined burn and smoke injury developed greater lung damage as evidenced by histological changes (septal thickening and interstitial edema) and higher lung water content. These mice also displayed more severely impaired pulmonary gas exchange [arterial PO2 (PaO2)/inspired O2 fraction (FiO2)<200]. Lung myeloperoxidase activity was significantly higher in burn and smoke-injured animals compared with the other three experimental groups. Plasma NO2-/NO3-, lung inducible nitric oxide synthase (iNOS) activity, and iNOS mRNA increased with injury; however, the burn and smoke injury group exhibited a higher response. Severity of burn and smoke inhalation injury was associated with more pronounced production of nitric oxide and accumulation of activated leukocytes in lung tissue. The murine model of burn and smoke inhalation injury allows us to better understand pathophysiological mechanisms underlying cardiopulmonary morbidity secondary to burn and smoke inhalation injury.

Delayed and impaired wound healing in burn and incisional wound can lead to scarring and may cause considerable distress to patients. In line with Sustainable Development Goal (SDG) particularly SDG no 3, the study of finding materials that accelerate skin wound healing and minimize the scarring of the wound became important field of study. In some regions in Indonesia, porcupine quills are used as a traditional medicine to heal wound and relieve pain by the traditional community. Therefore, this study aims to evaluate the comparison between burn and incisional wound healing in rat using crude extract of porcupine quills as the natural compound. The results showed that crude extract of porcupine quills in gel base had positive effect on burn and incisional wound healing compared to those without treatment or in cream base. In addition, the crude extract of porcupine quills in gel base decreased the mortality rate in burn wounded rats, increased body weight, and contributed in quicker wound closure in burn and incisional wounded rats. Incisional wound closed quicker than in burn wound indicated by skin closure and full re-epithelization histologically. The burn wound healed more than 21 days, however, the administration crude extract of porcupine quills in gel base results in comparable rate of wound closure compared to commercial product. These findings indicated that crude extract of porcupine quill in gel base can improve the burn and incisional wound healing with less mortality.

Toll-like receptors (TLR) 2 and TLR4 expressed on innate immune cells are important mediators of the immune response to pathogens. In this study, we hypothesized that burn injury results in altered cytokine secretion profiles after TLR2 or TLR4 ligation that is associated with altered TLR expression on innate immune cells. Female C56BL/6 mice were subjected to 20% full thickness burn or sham injury. Three or 14 days after injury whole splenocytes or purified splenic macrophages were cultured with TLR2 ligand peptidoglycan or TLR4 ligand lipopolysaccharide. Supernatants were assayed for TNF-alpha, MCP-1, IL-6 and IL-10. Cell death was assessed using flow cytometry. Innate CD11b F4/80 macrophages were sorted 14 days after burn injury and TLR2 and 4 expression was determined by quantitative reverse-transcriptase polymerase chain reaction and flow cytometry. Burn injury results in a steady accumulation in the periphery of CD11bF4/80 macrophages. Macrophages purified early after burn injury upregulated TLR2 and 4, followed by a decrease of TLR2 and TLR4 expression late after burn injury. TLR2 and TLR4 ligation of an equivalent number of purified macrophages 3 days after burn injury revealed no significant differences in cytokine secretion compared with sham. Stimulation 14 days after burn injury revealed a significant reduction in tumor necrosis factor-alpha secretion by macrophages compared with sham mice. In contrast, interleukin-10 was significantly increased (mean, approximately 1.8-fold) late after burn injury after either TLR2 or TLR4 stimulation. Interleukin-6 and monocyte chemotactic protein-1 secretion was unchanged from sham levels. In contrast, whole splenocyte stimulation resulted in increased cytokine 3 days and 14 days after burn injury. This effect is likely caused by the accumulation of TLR macrophages, which are resistant to TLR-induced cell death. Cytokine secretion profiles after TLR2 and TLR4 ligation after burn injury are altered in a manner not clearly reflective of an anti-inflammatory or proinflammatory state and are associated with unique changes in the macrophage population. TLR2 and TLR4 ligation have complex and varied roles in mediating the immune response to burn injury.

Previous studies showed that alcohol (EtOH) intoxication before burn injury suppresses mesenteric lymph node (MLN) T cell functions and increases gut bacterial translocation. In this study, we examined whether corticosterone (Cort) plays any role in suppressing MLN T cell function and bacterial accumulation after EtOH intoxication and burn injury. Rats were gavaged with EtOH to achieve a blood EtOH level of approximately 100 mg/dl before receiving 25% total body surface area burn or sham injury. A group of rats was treated with the Cort synthesis inhibitor metyrapone (25 mg/kg) at the time of injury and on day 1 after injury. Two days after injury, a significant increase in blood Cort levels and suppression of MLN T cell proliferation and IL-2 production was observed in rats receiving combined insult of EtOH intoxication and burn injury compared with rats receiving EtOH intoxication or burn injury alone. There was no change in T cell apoptosis after combined insult of EtOH and burn injury. Furthermore, T cell suppression was accompanied by a significant decrease in p38 and ERK1/2 activation (phosphorylation). There was no difference in JNK activation after EtOH and burn injury. Treatment of rats with metyrapone prevented the suppression of MLN T cell proliferation, IL-2 production, and p38 and ERK1/2 phosphorylation. Restoration of T cell function in metyrapone-treated animals was also associated with the decrease in bacterial accumulation in MLN. These findings suggest that EtOH intoxication before burn injury augments Cort release, which suppresses MLN T cell function by inhibiting p38 and ERK1/2 activation and promotes bacterial accumulation in MLN after EtOH and burn injury.

Inhibition of Multidrug-resistant Acinetobacter baumannii by Nonviral Expression of hCAP-18 in a Bioengineered Human Skin Tissue

Impact of Thermal Injury on Wound Infiltration and the Dermal Inflammatory Response

Previous studies have shown that alcohol (EtOH) ingestion before burn injury impaired intestinal barrier and immune function. This study determined whether EtOH and burn injury up-regulate interleukin (IL)-18 and whether IL-18 up-regulation following EtOH and burn injury is a cause for neutrophil recruitment and increased intestinal edema. Rats (250 g) were gavaged with EtOH to achieve a blood EtOH level in the range of 100 mg/dL prior to burn or sham injury (25% total body surface area). A group of rats was treated with Ac-YVAD-CHO (5 mg/kg), an inhibitor of caspase-1 (an enzyme that converts pro-IL-18, an inactive form of IL-18, to mature IL-18), at the time of injury. One day after injury, rats were killed. IL-18 production was determined in circulation and in the supernatants harvested from spleen, mesenteric lymph nodes, and Peyer's patch cell cultures as well as in intestinal tissue homogenates. Neutrophil accumulation in intestine was determined by measuring myeloperoxidase (MPO) activity. We found a significant increase in IL-18 levels in the lymphoid cell supernatants and intestinal tissue homogenates obtained from EtOH and burn-injured rats compared with the rats receiving burn or sham injury. This was accompanied by an increase in intestinal MPO and edema. No demonstrable change in intestinal morphology was observed in any group. Treatment of rats with caspase-1 inhibitor significantly attenuated the increase in IL-18 levels and intestinal MPO activity in EtOH and burn-injured rats. Inhibition of IL-18 also prevented an increase in intestinal tissue water content. As MPO is considered an index of neutrophil infiltration, results presented in this manuscript collectively suggest that IL-18 up-regulation is likely to contribute to the increased neutrophil infiltration and edema in intestinal tissue observed following EtOH and burn injury.

Introduction Accurate analysis of injuries and illnesses is paramount when allocating resources for education, prevention, recovery, and research. Inconsistencies in public health reports and increased lobbying/marketing by medical nonprofits has obscured the perception of burn injury (BI) leading to inequities in healthcare access and funding of reconstruction and research. A primary example of these developments is the frequently referenced and inaccurate BI statistics. Our study aims to compare national references reporting the incidence of BI and the related sequela in the U.S. Methods The American Burn Association Burn (ABA) Injury Summary Report (BISR), ABA Fact Sheet, Centers for Disease Control and Prevention (CDC) Web-based Injury Statistics Query and Reporting (WISQARS) database, the CDC National Center for Health Statistics’ National Hospital Ambulatory Medical Care Survey (NHAMCS), and the commercially available claims databases were queried for 2020 burn admissions and emergency department (ED) visits. Non-U.S. reports, claims, or analyses were excluded from our review. The costs of BI were reported in U.S. dollars where available. Claims analyses were conducted using ICD-10 BI codes coupled with burn DRG for admission, and CPT for surgical intervention from a commercially available database incorporating managed care, Medicare, and Medicaid payors. Results The ABA Fact Sheet reported 486,000 BI annually. The 2021 ABA BISR data represented burn admissions at approximately 100 burn centers (BC) excluding non-BC, ED, and BC not participating in BCQP. CDC’s WISQARS database reported 3,529 burn-related deaths resulting in 45,135 years of potential years of life lost, 287,926 non-fatal BI from fire/flame and contact with 76.1% of patients treated in ED only and total costs of $42.4B. Mechanisms such as scald, chemical, electrical, radiation, and inhalation injury were not included. The CDC NHAMCS reported 359,000 BI presenting to ED (23% increase from CDC WISQARS). Our analysis from claims databases demonstrated over 698,555 BI as the primary code likely underreporting self-pay/uninsured, military facilities, and endowment or community-based hospitals not submitting claims data. Conclusions Our study demonstrates a large variability in the incidence of BI. Given the increased population noted in the U.S. 2020 census, the true number of BI is likely closer to the claims database suggesting a substantial misunderstanding in the burden of burns. We additionally noted no ICD-10 codes to characterize the sequela of BI limiting our assessment of post-acute burn care challenges. Future efforts to improve CDC reporting policies and new ICD-10 codes capturing BI sequela are essential to improve advocacy and understanding of our patients and their healthcare needs. Applicability of Research to Practice This abstract will help clinicians, patients, and investigators better advocate for population health research in burn injury.

Burn injury induces immunosuppression, which is associated with an increased susceptibility to infection. Our laboratory has demonstrated that burn injury also impairs humoral immunity. We reported that burn injury enhanced expression of transforming growth factor-beta (TGF-beta) mRNA and that exogenous TGF-beta further impaired humoral immunity. The objective of this study was to clarify the role of TGF-beta on humoral immunity after burn injury with a neutralizing experiment. Twelve BALB/c mice were randomly divided into two groups: sham and burn. Anesthetized mice received a 20% full-thickness burn or sham injury. The murine splenocytes containing 1.5 x 10 cells/mL were cultured with 2.5 microg/mL of lipopolysaccharide with or without 0.5 ng/mL of TGF-beta or 1 microg/mL of anti-TGF-beta neutralizing antibody, if necessary. Concentrations of immunoglobulin (Ig) M in the cell culture supernatant were determined by enzyme-linked immunosorbent assay and the number of IgM-secreting cells in the culture was measured by enzyme-linked immunospot assay. After 2-day culture, neutralization of TGF-beta dramatically restored IgM synthesis after burn injury. After 5-day culture, however, it restored IgM concentration but failed to restore a number of IgM-secreting cells. This neutralizing experiment demonstrated that TGF-beta is one of the inhibitors of IgM synthesis after burn injury. However, neutralization of TGF-beta was not enough to completely restore humoral immunity after burn injury. Investigation of the mechanism of impaired IgM synthesis after burn injury should be continued.

Burn injury is associated with a dynamic T cell response. We have previously reported an enhanced functional T cell response 14 days after burn injury. Toll-like receptors (TLR), primarily expressed on innate immune cells, have recently been identified on certain T cell subsets, including activated and memory T cells. Our hypothesis is that increased TLR4 expression on memory T cells may be a mechanism for enhanced T cell response 14 days after burn injury. Splenocytes from wild-type C57Bl/6 mice were harvested 14 days after a 20% total body surface area (TBSA) scald burn or sham injury. Splenocytes ex vivo were surface stained either with monoclonal anti-CD3, anti-CD4, anti-CD8, or anti-CD44 antibodies or a two-step biotin-TLR4 monoclonal antibody-streptavidin-FITC surface stain and results analyzed by flow cytometry. TLR4 expression is successfully detected on CD4 and CD8 T cells. TLR4 expression is significantly (p < 0.05) increased on CD4 T cells and CD8 T cells 14 days after burn injury. There is a significant (p < 0.05) increase in CD44 (memory) CD4 and CD44 (memory) CD8 T cells 14 days after burn injury and this is associated with a significant (p < 0.05) increase of TLR4 expression in both T cell populations. This study demonstrates for the first time the potential role of TLR4 expression on memory T cells generated late after burn injury. Although further analysis is required, these data reiterate the importance of adaptive immunity and the complexity of the immune response to burn injury.

Alteration in intestine tight junction protein phosphorylation and apoptosis is associated with increase in IL-18 levels following alcohol intoxication and burn injury

In this study, we examined whether IL-18 plays a role in lung inflammation following alcohol (EtOH) and burn injury. Male rats ( approximately 250 g) were gavaged with EtOH to achieve a blood EtOH level of approximately 100 mg/dl before burn or sham injury ( approximately 12.5% total body surface area). Immediately after injury, rats were treated with vehicle, caspase-1 inhibitor AC-YVAD-CHO to block IL-18 production or with IL-18 neutralizing anti-IL-18 antibodies. In another group, rats were treated with anti-neutrophil antiserum approximately 16 h before injury to deplete neutrophils. On day 1 after injury, lung tissue IL-18, neutrophil chemokines (CINC-1/CINC-3), ICAM-1, neutrophil infiltration, MPO activity, and water content (i.e., edema) were significantly increased in rats receiving a combined insult of EtOH and burn injury compared with rats receiving either EtOH intoxication or burn injury alone. Treatment of rats with caspase-1 inhibitor prevented the increase in lung tissue IL-18, CINC-1, CINC-3, ICAM-1, MPO activity, and edema following EtOH and burn injury. The increase in lung IL-18, MPO, and edema was also prevented in rats treated with anti-IL-18 antibodies. Furthermore, administration of anti-neutrophil antiserum also attenuated the increase in lung MPO activity and edema, but did not prevent the increase in IL-18 levels following EtOH and burn injury. These findings suggest that acute EtOH intoxication before burn injury upregulates IL-18, which in turn contributes to increased neutrophil infiltration. Furthermore, the presence of neutrophils appears to be critical for IL-18-meditaed increased lung tissue edema following a combined insult of EtOH and burn injury.

(134) - Evaluating the impact of high fat diet on pain behaviors in mice with acute burn injury

Induction of apoptosis in intestine following ethanol intoxication and burn injury